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ISSN (Print): 0974-1496 ISSN (Online): 0976-0083 CODEN: RAJCABP Volume-4, Number- 1, January -March, 2011 http://www.rasayanjournal.com

R A S Ā Y AN Journal of Chemistry (An International Quarterly Research Journal of Chemical Sciences)

Editor-in-Chief

Prof. (Dr.) Sanjay K. Sharma STATUTORY WARNING Articles, data, figures, scientific content and its Interpretation and authenticity reported by author(s) and published in RASĀYAN are the exclusive views of author(s). The Editorial board, RASĀYAN is not responsible for any controversies arising out of them. In case of any Plagiarism found, author (s) will be responsible for any legal action.

23, ‘Anukampa’, Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur (India) Phone: 0091-141-2810628, 07597925412, 09414202678 E-mail:rasayanjournal@gmail.com

ISSN: 0974-1496; CODEN: RJCABP Vol.4, No.1 (2011),1-222 http://www.rasayanjournal.com

R A SĀ Y A N Journal of Chemistry [An International Quarterly Research Journal of Chemical Sciences] ISSN: 0974-1496 (Print); ISSN: 0976-0083(Online)

Volume-4, Number-1, January -March, 1-222 (2011)

Contents…

i-iii 1-7

GRAFT COPOLYMERISATION OF ACRYLAMIDE ON CARBOXYMETHYL CELLULOSE (CMC) Abel Tame, Maurice K. Ndikontar, J. Noah Ngamveng, H.N. Ntede ,Richard Mpon and Emmanuel Njungab PHYTOEXTRACTION OF LEAD FROM INDUSTRIAL EFFLUENTS BY SUNFLOWER (HELIANTHUS ANNUUS.L) R. Usha, A. Vasavi, K. Thishya, S.Jhansi Rani and P.Supraja

8-12

ASSAY OF CEFPROZIL IN BULK AND ITS PHARMACEUTICAL FORMULATIONS BY VISIBLE SPECTROPHOTOMETRY C. Ramesh,G. Nagarjuna Reddy,T. V. Narayana , K.V.S. Prasada Rao and B. Ganga Rao

13-16

SYNTHESIS AND BIOLOGICAL ACTIVITY OF SOME 2-AMINO-4,6-SUBSTITUTEDDIARYLPYRIMIDINES: REACTION OF SUBSTITUTED CHALCONES WITH GUANIDINIUM CARBONATE Vandana Sharma and K. V. Sharma

17-23

SYNTHESIS AND ANTIBACTERIAL ACTIVITY BENZOTHIAZOLYL MOIETY T.M.Bhagat, D.K.Swamy ,S.G.Badne and S.V.Kuberkar

CONTAINING

24-28

MALTOSYL

29-35

ISOLATION AND IDENTIFICATION OF ANTIMICROBIAL COMPOUND FROM MENTHA PIPERITA L. Abhishek Mathur, GBKS Prasad, Nageshwar Rao, Pradeep Babu and V.K. Dua

36-42

SURFACE WATER (LAKES) QUALITY ASSESSMENT IN NAGPUR CITY (INDIA) BASED ON WATER QUALITY INDEX (WQI) P. J. Puri, M.K.N. Yenkie, S.P. Sangal , N.V. Gandhare , G. B. Sarote and D. B. Dhanorkar

43-48

SAMPLE PREPARATION FOR FLAME ATOMIC ABSORPTION SPECTROSCOPY: AN OVERVIEW Nabil Ramadan Bader

49-55

BIOSORPTION CHARACTERISTICS OF Cr6+ FROM AQUEOUS SOLUTIONS BY PINUS SYLVESTRIS L. TIMBER FILLINGS Ackmez Mudhoo and Preesena Devi Seenauth

56-65

OF

4-THIAZOLIDINONE

SYNTHESIS AND ANTI-MICROBIAL ACTIVITY OF NOVEL ACETYLATED CARBAMIDES, BENZOTHIAZOLYL CARBAMIDES AND CARBAMATES Sanjay. P. Mote and Shirish. P. Deshmukh

ii ISSN: 0974-1496; CODEN: RJCABP Vol.4, No.1 (2011),1-222

SYNTHESIS AND BIOLOGICAL EVALUATION OF (7-HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID HYDRAZIDE DERIVATIVES USED AS A POTENT BIOLOGICAL AGENTS Deepak P. Kardile, Manchindra R.Holam, Ankit S. Patel and Shailesh B.Ramani

66-72

RECENT DEVELOPMENTS ON BISMUTH (III) IN CARBON-CARBON BOND FORMATION CHEMISTRY Suresh and Jagir S. Sandhu

73-85

OPTICAL AND MECHANICAL CHARACTERIZATION OF SOLUTION GROWN SEMI ORGANIC NLO CRYSTALS. M. N. Ravishankar , R. Chandramani and A.P. Gnanaprakash

86-90

MICROWAVE SYNTHESIS AND ANTI-INFLAMMATORY EVALUATION OF SOME NEW IMIDAZOLO QUINOLINE ANALOGS P.Raghavendra, G.Veena, G.Arun Kumar, E.Raj Kumar, N.Sangeetha, B.Sirivennela, S.Smarani, H.Praneeth Kumar and R.Suthakaran

91-102

SYNTHESIS, CHARACTERIZATION AND ION-EXCHANGING PROPERTIES OF NOVEL IONEXCHANGE RESIN PART-III Sanjay Kumar Saraf and Arun Singh

103-109

POLAROGRAPHIC REDUCTION OF IODATE-STUDY OF KINETIC WAVE G.Sailaja, R.Ramachandramurthy and V. Suryanarayana Rao

110-112

DEVELOPMENT AND VALIDATION OF NEW HPLC METHOD FOR THE ESTIMATION OF PERINDOPRIL IN TABLET DOSAGE FORMS V. Bhaskara Raju and A. Lakshmana Rao

113-116

INFLUENCE OF THE SOLVENT CONDITION FOR THE SYNTHESIS OF THE β-DIKETIMINES LIGANDS Tchirioua Ekou and Lynda Ekou

117-119

SIMULTANEOUS DETERMINATION OF TUNGSTEN (VI) AND MOLYBDENUM (VI) FROM CATALYTIC REDUCTION OF IODATE G.Sailaja, R.Ramachandramurthy and V. Suryanarayana Rao

120-123

POLAROGRAPHIC STUDY OF MIXED LIGAND (CARBOXYMETHYLMERCAPTOSUCCINATEALANINATE OR ASPARTATE OR GLUTAMINATE OR VALINATE WITH CADMIUM (II) LEAD (II) AND THALLIUM (I) IN AQUEOUS ETHANOL MEDIA. Seema Agarwal and S. Kalpana

124-131

ROLE OF ALKALOIDAL PRECIPITANTS FOR THE ASSAY OF IMIPRAMINE HYDROCHLORIDE IN BULK AND PHARMACEUTICAL FORMULATIONS G.Nagarjuna Reddy, C.Ramesh,T.V.Narayana, K.V.S.Prasada Rao and B.Ganaga Rao

132-135

SYNTHESIS, CHARACTERISATION AND BIOLOGICAL EVALUATION OF BIDENTATE LIGANDS (REDUCED SCHIFF’S BASE) WITH METALS OF COPPER, NICKEL AND ZINC COMPLEXES S.Pattanaik, S.S.Rout, J.Panda, P.K.Sahu and M Banerjee

136-141

SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF RHODIUM (I) COMPLEXES WITH MIXED TRIPHENYLPHOSPHINE AND HETEROCYCLIC THIOAMIDE LIGAND R.N. Pandey and A.K.Nag A PRELIMINARY SURVEY OF MERCURY IN FRESH WATER AND FISHES P. J. Puri, M.K.N. Yenkie, S.P. Sangal, N.V. Gandhare, G. B. Sarote and D. B. Dhanorkar

142-146

DEVELOPMENT AND VALIDATION OF REVERSE PHASE LIQUID CHROMATOGRAPHY METHOD FOR ESTIMATION OF ISOTRETINOIN (13-CIS RETINOIC ACID) IN PHARMACEUTICAL DOSAGE

153-158

147-152

iii ISSN: 0974-1496; CODEN: RJCABP Vol.4, No.1 (2011),1-222

FORM Pratik Patel, Ritu Kimbahune, Prachi Kabra, Kuldeep Delvadiyaand L.V.G.Nargund OPTIMIZATION OF PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO BY BACILLUS SUBTILIS NCIM 2724 USING STATISTICAL EXPERIMENTAL DESIGN Haritha Meruvu and Meena Vangalapati

159-164

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF TOLVAPTAN IN BULK AND ITS PHARMACEUTICAL FORMULATION V. Kalyana Chakravarthy and D.Goeri Shankar

165-171

DENSITY, VISCOSITY AND ACTIVATION PARAMETERS OF VISCOUS FLOW FOR CETRIMIDE IN ETHANOL + WATER SYSTEM AT 301.5 K Muktar Shaikh,Mohd. Shafique, B. R. Agrawal and Mazahar Farooqui

172-179

MODELING ISOLATION OF AMARANTH PROTEIN BY ENZYMATIC BREAKDOWN OF POLYSACCHARIDES Pavel Mokrejs, Dagmar Janacova, Karel Kolomaznik and Petr Svoboda

180-188

EFFECT OF MOLECULAR WEIGHT DISTRIBUTION ON CHEMICAL, STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF SODIUM LIGNOSULFONATES Nidal Madad, Latifa Chebil, Christian Sanchez and Mohamed Ghoul

189-202

α,α-DIMETHYL-4-[1-HYDROXY-4-[4 (HYDROXYDIPHENYLMETHYL)-1-PIPERIDINYL]BUTYL] BENZENEACETIC ACID HYDROCHLORIDE AS A CHELATING AGENT IN SOME METAL COMPLEX Jitendra H. Deshmukh and M. N. Deshpande

203-209

STRUCTURE BASED DRUG DESIGNING OF p38 MAP KINASE INHIBITORS FOR THE TREATMENT OF OSTEOARTHRITIS Neelakantan Suresh

210-216

SYNTHESIS AND CHARACTERIZATION OF ZINC OXIDE NANOPARTICLES ANTIMICROBIAL ACTIVITY AGAINST BACILLUS SUBTILIS AND ESCHERICHIA COLI Haritha Meruvu, Meena Vangalapati, Seema Chaitanya Chippada and Srinivasa Rao Bammidi

217-222

INDEX of Contributors of this issue Call for papers for ijCEPr, A New Journal Detailed guidelines to Authors for Manuscript Preparation SUBSCRIPTION FORM

AND

ITS

iii iv v Vi

RASĀYAN widely covers all branches of CHEMISTRY including: Organic, Inorganic, Physical, Analytical, Biological, Pharmaceutical, Industrial, Environmental, Agricultural & Soil, Petroleum, Polymers, Nanotechnology, Green Chemistry, Forensic, Phytochemistry, Synthetic Drugs, Computational, as well as Chemical Physics and Chemical Engineering. Manuscript Categories: Full-length paper, Review Articles, Short/Rapid Communications.

iv ISSN: 0974-1496; CODEN: RJCABP Vol.4, No.1 (2011),1-222

RASĀYAN Journal of Chemistry Volume-4, Number-1, January-March, 1-222, (2011) AUTHOR INDEX OF THIS ISSUE Ackmez Mudhoo, 56 A. Lakshmana Rao,113 A. Vasavi, 8 A.K.Nag,142 A.P. Gnanaprakash,86 Abel Tame, 1 Abhishek Mathur,36 Ankit S. Patel,66 Arun Singh,103 B. Ganga Rao,13 B. R. Agrawal,172 B.Ganaga Rao,132 B.Sirivennela,91 C. Ramesh, 13, 132 Christian Sanchez ,189 D. B. Dhanorkar,43, 147 D.Goeri Shankar,165 D.K.Swamy, 24 Dagmar Janacova,180 Deepak P. Kardile,66 E.Raj Kumar,91 Emmanuel Njungab, 1 G. B. Sarote,43, 147 G. Nagarjuna Reddy, 13 G.Arun Kumar,91 G.Nagarjuna Reddy,132 G.Sailaja,110, 120 G.Veena,91 G.B.K.S. Prasad,36 H.N. Ntede, 1 H.Praneeth Kumar,91 Haritha Meruvu,159, 217 J. Noah Ngamveng, 1 J.Panda,136 Jagir S. Sandhu,73

Jitendra H. Deshmukh,203 K. Thishya, 8 K. V. Sharma, 17 K.V.S. Prasada Rao,13, 132 Karel Kolomaznik,180 Kuldeep Delvadiya,153 L.V.G.Nargund,153 Latifa Chebil,189 Lynda Ekou,117 M Banerjee,136 M. N. Ravishankar,86 M. N. Deshpande,203 M.K.N. Yenkie,43, 147 Manchindra R.Holam,66 Maurice K. Ndikontar, 1 Mazahar Farooqui ,172 Meena Vangalapati,159, 217 Mohamed Ghoul,189 Mohd. Shafique,172 Muktar Shaikh,172 N.Sangeetha,91 N.V. Gandhare,43, 147 Nabil Ramadan Bader,49 Nageshwar Rao,36 Neelakantan Suresh,210 Nidal Madad,189 P. J. Puri,43, 147 P.K.Sahu,136 Preesena Devi Seenauth,56 P.Raghavendra,91 P.Supraja, 8 Pavel Mokrejs,180 Petr Svoboda,180 Prachi Kabra,153 Pradeep Babu,36

Pratik Patel,153 R. Chandramani,86 R. Usha, 8 R.N. Pandey,142 R.Ramachandramurthy,110, 120 R.Suthakaran,91 Richard Mpon, 1 Ritu Kimbahune,153 S. Kalpana,124 S.G.Badne, 24 S.Jhansi Rani, 8 S.P. Sangal,43, 147 S.Pattanaik,136 S.S.Rout,136 S.Smarani,91 S.V.Kuberkar, 24 Sanjay Kumar Saraf,103 Sanjay. P. Mote, 29 Seema Agarwal,124 Seema Chaitanya Chippada,217 Shailesh B.Ramani,66 Shirish. P. Deshmukh, 29 Srinivasa Rao Bammidi,217 Suresh,73 T. V. Narayana, 13 T.M.Bhagat, 24 T.V.Narayana,132 Tchirioua Ekou,117 V. Bhaskara Raju,113 V. Kalyana Chakravarthy,165 V. Suryanarayana Rao,110, 120 V.K. Dua,36 Vandana Sharma, 17

Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: rasayanjournal@gmail.com, drsanjay1973@gmail.com Phone:0141-2810628(O), 09414202678, 07597925412 (M)

ISSN: 0974-1496; CODEN: RJCABP Vol.4, No.1 (2011),1-222 http://www.rasayanjournal.com

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Vol.4, No.1 (2011), 1-7 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

GRAFT COPOLYMERISATION OF ACRYLAMIDE ON CARBOXYMETHYL CELLULOSE (CMC) Abel Tame1, Maurice K. Ndikontar1,*, J. Noah Ngamveng1, H.N. Ntede2; Richard Mpon1 and Emmanuel Njungab1 1

2

Laboratoire Physico-Chimie du Bois, Université de Yaoundé I, Cameroun Laboratoire de Mécanique et de Structure de Matériaux, Ecole Nationale Supérieure Polytechnique, Université de Yaoundé I, Cameroun *Email: mndikontar@yahoo.com

ABSTRACT Graft copolymers of acrylamide (AA) on unmodified holocellulose and holocellulose containing carboxy methyl moieties (of degree of substitution (DS): 0.215; 0.120 and 0.057) were prepared using Ce4+ initiator in aqueous medium at 29°C. The results indicate that carboxymethylation enhances the graftability of the cellulose substrate. The graft levels and molecular weights of grafted polyacrylamide chains on CMC were higher than those obtained on cellulose. Increases in graft levels of up to 70% and 50% for graft copolymer involving CMC with DS 0.215 and 0.120 respectively were observed. The grafting frequencies of polyacrylamide were less on CMC (DS 0.215) than on CMC (DS 0.120) and on cellulose. This study provides information on the grafting characteristics of AA on CMC, a substrate of particular interest because of its wide range of applications. The influence of ceric ion concentration, degree of substitution of substrate, monomer concentration, time of reaction on the extent and rate of graft copolymerisation of AA unto modified and unmodified cellulose were examined. Key words: copolymer, holocellulose, degree of substitution, caboxymethyl cellulose, acrylamide, graft. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION In recent years, the use of ceric ions to initiate the graft copolymerisation of vinyl monomers on cellulose materials has received much attention from the practical and fundamental points of view1-5. Grafting of polysaccharides in general, is of interest because of its potential as a technique for modifying the chemical and physical properties of these polysaccharides. Polyacrylamides, which are especially useful as flocculants for fine solids suspended in water, thickening agents and pigment retainers, can impart these properties onto CMC through grafting, thus enlarging the already remarkable diversity of its practical and potential applications6. Sodium carboxymethyl cellulose (or sodium cellulose glycolate) is the most widely used water-soluble cellulose derivative. Depending on its type of viscosity (grade), it is used in food and pharmaceutical industries, laundering, well drilling, paints, etc.

EXPERIMENTAL Materials Acrylamide reagent (99.9% pure for electrophoresis, Bio Rad Laboratories), chloroacetic acid (from BDH Ltd) and all other regular laboratory reagents were used without further purification. Triplochiton scleroxylon (Obeche) wood meal was obtained from a commercial saw mill in Benin City (Nigeria). It was air-dried and sieved between a 425-µm mesh and 355-µm mesh screen. The portion of the meal retained on the 355-µm mesh was used for the preparation of holocellulosic material.

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Abel Tame et al.

Vol.4, No.1 (2011), 1-7

Preparation of holocellulose The sulphite pulping method7 was used to prepare the holocellulose from the tropical soft wood. A 25.0-g sample of air-dried wood meal was Soxhlet-extracted with 99.7-100% ethanol (v/v) for 6 hours to remove waxes and resinous materials. The resin-free wood meal was then treated with 10% sodium sulphite for eight hours with continuous heating and stirring. The holocellulose obtained was thoroughly washed with water and air dried. Chlorine gas, generated by the reaction of concentrated hydrochloric acid (60 mL) with potassium permanganate (20 g), was bubbled into 180 mL of cold 2-M sodium hydroxide solution. The resulting sodium hypochlorite solution was mixed with 90 mL of 0.5 M nitric acid solution. The holocellulose meal was then steeped in this hypochlorite solution and heated to boil for 15 minutes. The resultant bleached holocellulose was then filtered off , washed with de-ionised water and air-dried. Carboxymethylation of holocellulose 2.00 g of cellulose were added to 39.3 mL isopropanol and 38.5 mL benzene in a mixer. Stirring was commenced and 10.1 mL of aqueous x (= 30, 45, and 60%) sodium hydroxide (w/v) were added in one minute and the mixture was shaken at 30°C for y minutes. z mmol of sodium monochloroacetic acid were added and the reaction mixture was kept at a temperature of 65°C for 70 minutes while shaking8. Excess sodium hydroxide was neutralised by drops of acetic acid and the product pressed to remove the solution and then washed with isopropanol and dried. To determine the degree of substitution9, 0.5 g of dried sodium CMC was ashed gently between 450 and 550°C for 24 hours, and then dissolved in 100 mL of distilled water. 20 mL of this solution were titrated with 0.1 N sulphuric acid, using methyl red as indicator. After the first end point, the solution was boiled and titrated to a sharp end point. The carboxymethyl content was calculated as the degree of substitution:

DS =

0.162B 1 − 0.08B

where B is given by the expression B =

(1)

0 .1 b ; b is the volume (in mL) of 0.1 N sulphuric acid and G is G

the mass of pure sodium CMC in grams. Graft copolymerisation The polymerisation procedure was based on the method described by Lepoutre and Hui10 using variable amounts of ceric ion and monomer and a constant amount of the cellulosic material (1.0 g) dispersed in 100 mL of deionised water. Polymerisation was stopped after 1 hour by adding 2 mL of quinol solution (5% w/v in acetone) to the reaction mixture which was filtered and the residue air-dried and weighed. The ungrafted poly AA was extracted by stirring the residue in 500 mL of deionised water overnight at room temperature. The grafted cellulosic substrate was then air-dried and reweighed. The percentage graft level, Pg and the percent efficiency grafting, Pe were calculated as follows:

Pg =

Pe =

mass of grafted polymer (m g )

× 100 mass of mod ified pulp (m s ) mass of grafted polymer (m g )

mass of hom opolymer (m h ) + mass of grafted polymer (m g )

(2)

× 100

(3)

The average molecular weights of isolated PAA grafts were determined from viscosity measurements in water at 25°C using the relationship11: log η = log 6.31x 10-5 + 0.80 log Mv (4) where η is the viscosity and Mv is the viscosity-average molecular weight. After determination of molecular weights, the frequency of grafting, Fg defined as the number of moles of grafted AA per 104 AGU (anhydrous glucose units) was calculated as follows12:

GRAFT COPOLYMERISATION OF ACRYLAMIDE

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Abel Tame et al.

Vol.4, No.1 (2011), 1-7

Fg =

m g × M AGU Mv

×10 4 = n g × M AGU × 10 4

(5)

and the rate of graft copolymerisation, Rg calculated by the relationship:

Rg =

mg M v × reaction time ( t ) × reaction volume (V )

× 1000

(6)

RESULTS AND DISCUSSION The results in Table 1 show that the degree of substitution increases as the concentration of sodium hydroxide increases. This is probably due to greater swelling and hence easy penetration of carboxymethyl groups in the cellulose structure. The dependence of grafting parameters of AA on ceric ion concentration is shown in Figure 1. The level of incorporation of polyacrylamide graft copolymer on the holocellulose increased by up to 70% and 50% following the modification of the cellulose substrate (DS: 0.215 and 0.120 respectively). These results agree generally with those reported by Kantouch et al.13 who grafted the same monomer on native cotton, alkali-treated cotton and partially carboxymethylated cotton. Okiemen and Eboaye5 reported that during polymerisation, ceric ions are consumed by the initiation process and by adsorption onto the cellulose material; the unreacted or adsorbed ceric ions present in the continuous aqueous phase initiate homopolymerisation by charge transfer to monomer14,15. Thus, the graft level and the efficiency of grafting depend on the relative amounts of ceric ions present. The slight decrease in percent grafting at higher initiator concentration could be due to the fact that at higher ceric ion concentration, an increase is expected in the reaction steps involving ceric ion, leading to an increase in active sites. In fact, this results in termination (combination or disproportionation) prior to monomer addition. Another contributing factor is the increase in homopolymer formation which competes with the grafting reaction for available monomer thus giving the slight decrease observed at high ceric ion concentration. Figure 2 shows the variation of the extent of graft polymer formation on the substrates with monomer concentrations. This suggests that acrylamide was more reactive towards carboxymethylated cellulose than cellulose. This is expected, since the former has a greater accessibility than the latter. During swelling of CMC in the reaction medium, there is a break down of many hydrogen bonds in the amorphous region of the cellulose and the chain molecules are then able to move apart. Hence, there will be a tendency for the reacting molecules of monomer and initiator to approach the crystalline regions better than when the reaction is performed on unmodified cellulose. With a further increase in the concentration of acrylamide, grafting was found to decrease. This is explained by the fact that at higher monomer concentration, the ceric ion preferably enters into complex formation with the monomer, leading to a higher homopolymer formation than grafting, thus decreasing the grafting efficiency. The decrease beyond 43.8 M may also be related to the higher viscosity of the reaction medium, thus limiting monomer diffusion16. The influence of initiation time on the grafting characteristics of acrylamide on cellulose and sodium carboxymethylcellulose using 4.36 mmol of Ce4+ salt, 30 mmol of monomer at a constant polymerisation time of 60 minutes at 29°C is shown in figure 3 below. The result showed an increase in level of grafting with initiation time. The initial increase in graft level may result from an increase in the number of active sites formed on the polymeric substrate. The levelling off around the initial time of 30 minutes may be explained in terms of a limited number of active groups in the polymeric substrate with which the ceric ions would react (formation of a ceric ion complex)17 and a reduction of the ease of reaction of ceric ions with active groups within the polymeric substrate. It can also be attributed to a decrease in monomer concentration (rapid exhaustion) as well as to the retardation of diffusion due to the formation of polymer at the substrate surface. Table 1 presents the influence polymerisation time of graft level of acrylamide on unmodified and partially carboxymethylated cellulose using 4.36 mmol of Ce4+ salt, 30 mmol of monomer at a constant initiation time of 30 minutes at 29°C. The Pg increased with reaction time; the substrate with high DS had

GRAFT COPOLYMERISATION OF ACRYLAMIDE

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Vol.4, No.1 (2011), 1-7

the higher graft level but Pe were almost unchanged. This can be explained by the fact that the longer the reaction time, the greater the number of sites on the CMC activated by ceric ions, followed by greater grafting. This can also result from the difference in intrinsic characteristics: with the increase in the DS, the swellability of the CMC is enhanced, the effect of increasing polymerisation time will accentuate the swelling properties, improve the solubility of acrylamide, accelerate its diffusion from the polymerisation medium into the modified materials thus enhancing the propagation of graft. The molecular weight data of PAA graft on unmodified and carboxymethylated holocellulose and of associated homopolymer using 30 mmol of monomer are shown in table 2. It is observed that the molecular weight of grafted PAA decreased with increase in ceric ion concentration, which indicates a contribution of the excess ceric ion in the termination step. The molecular weight of homopolymer associated with grafting on CMC D.S: 0.120 was slightly higher than that on CMC D.S: 0.215. This suggests that the amount of monomer present in the continuous phase is higher in the former than in the latter polymerisation mixture. It can be seen that the lower number of grafted PAA per gram of substrate lead to a lower frequency of grafting. The number of grafted polymer molecules per 104 AGU varied from two to eleven. Although the values of graft level and molecular weight of grafted polymer were higher for CMC (DS: 0.215), the levels of incorporation of PAA copolymer grafts into the substrate was higher for CMC (DS: 0.120) and unmodified cellulose than CMC (DS: 0.215). This discrepancy can be explained by the suggestion that initiation of graft polymerisation by ceric ion occurs by hydrogen abstraction from the carbon atom carrying hydroxyl groups. The relatively high frequency of grafting of (DS:0.120) may result from the fact that this substrate possesses the double advantage of having a moderate number of -CH2COOH groups which helps in opening its structure compared to that of unmodified cellulose and has a sufficient number of hydroxyl sites where initiation can take place compared to the highly substituted CMC. The values of Rg were generally low and its variation at higher concentration of ceric ion confirms the participation of Ce4+ in the termination of growing grafted chains, resulting in a decrease in the rate of graft copolymerisation. 200

% graft level

180

160

140

120

Ds: 0.215

100

DS: 0.120 DS: 0 80

60

40

20

0 0

2

4

6

8

10

12

Amount of Ce(IV) (mmol)

Fig.-1: Effect of Ce4+ concentration on the graft level of AA on unmodified and CMC using 30 mmol monomer, 1 g substrate at 29째C for 1 hr

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Abel Tame et al.

Vol.4, No.1 (2011), 1-7 Table-1: Variation of the DS of sodium carboxymethyl cellulose with NaOH concentration after soaking for 2 hours Sodium monochloroacetic acid z (mmol) 8.5 5.3 2.1

Sodium hydroxide

x% (w/v)

60 45 30

Reaction time y (hrs) 2 2 2

DS 0.215 0.120 0.057

Table-2: Grafting characteristics of acrylamide on holocellulose with ceric ion concentration, using 30 mmol of monomer

DS of CMC

Fg

Rg (107 mol L-1 min-1)

2.3 2.8 3.5 4.2

N째 of grafted PAA chains (103 ng/100 g substrate) 0.106 0.196 0.292 0.393

1.65 3.05 4.56 6.13

1.7 3.25 4.8 6.5

4.9

0.478

7.45

7.9

0.122

1.9

2.03

0.245 0.432 0.507

3.82 6.74 7.90

3.3 7.2 8.4

11.03 1.85 3.21 4.98 6.68 7.8

11.7 1.9 3.4 5.3 7.1 8.3

Amount of Ce4+ (mmol)

Mv. (10 g)

Mv of homopolymer (10-5 g)

2 4 6 8

7.01 5.9 5.06 4.1

10

3.6

2

5.6

2.5

4 6 8

4.1 3.02 2.8

3.5 4.2 4.5

10 2 4 6 8 10

2.1 4.3 3.2 2.6 2.1 1.9

5.1 2.9 3.5 4.2 4.9 5.2

0.707 0.118 0.206 0.320 0.430 0.500

0.215

0.120

0.00

-5

Table-3: Influence of polymerisation time on graft levels on unmodified and partially CMC DS of CMC

0.215

0.120

0.00

Polymerisation time (mins) 10 30 60 80 100 10 30 60 80 100 10 30 60 80 100

GRAFT COPOLYMERISATION OF ACRYLAMIDE

mg (g)

mh (g)

Pe (%)

Pg (g)

1.19 1.27 1.36 1.35 1.34 0.86 0.90 1.01 1.03 1.05 0.20 0.30 0.41 0.44 0.45

0.033 0.043 0.044 0.052 0.061 0.047 0.050 0.059 0.073 0.047 0.01 0.03 0.09 0.12 0.14

97.2 96.3 96.8 96.4 96 94.7 94.6 94.4 93.3 95.6 95.0 90.0 82.0 78.0 76.0

119 127 136 135 134 86 90 100 103 105 20.5 30 41 44 45

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% graft level

180

160

140

120

100

DS: 0.215 DS: 0.120 80 DS: 0

60

40

20

0 0

10

20

30

40

50

60

amount of AA (mmol)

Fig.-2: Effect of monomer concentration on graft level of AA on unmodified and modified holocellulose using 6 mmol of Ce4+ at 29째C for 1 hour % graft level

200

180

160

140

120

100

DS: 0.215 DS: 0.120 DS: 0

80

60

40

20

0 0

20

40

60

80

100

120

Initiation time (minites)

Fig.-3: Effect of initiation time on graft level of AA on unmodified and modified holocellulose using 4.36 mmol of Ce4+, 30 mmol of monomer at 29째C for 1 hour

CONCLUSION The results from this study are consistent with the view that graft copolymerisation onto cellulose is sensitive to the degree of swelling i.e. accessibility of the substrate.

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ACKNOWLEDGEMENTS This publication was possible with the help of the MANIF Program of ENSP-G.C of the University of Yaoundé I, thanks to the support of the “AIRES-SUD” through grant N° AIRES-SUD 7202.

REFERENCES 1. O. Mansour and A. Nagaty, Progress Polymer Science, 11, 91 (1985). 2. A. Berlin and V.N. Kislenko, Prog. Polym. Sci., 17, 765 (1992). 3. M.K. Ndikontar, Greffage de la cellulose extraite du manioc. Application de la cellulose greffée. Thèse de Doctorat 3ème cycle, Université de Yaoundé, Cameroun, 48 (1990). 4. A. Bayazeed, M.H. El-Rafie and A. Hebeish, Acta Polym.,36, 353 (1986). 5. F.E. Okieimen and J.E. Ebhoaye, J. Appl. Polym. Sci., 31, 1275 (1986). 6. V.S. Ananthanarayanan and M. Santappa, J. Polym. Sci., 9, 2435, (1965). 7. ‘Chemical profile, CMC: Oil, Paint, Drug Repertoire’. 187 (2) 1 (1965). 8. H.O. Paddison and R.W. Sommers, Brit. 772 183 Apr. 10, (CA. 51, p 151 28b) (1957). 9. Sverinsk; Paper Stidn. 59, 218-22, CA 51, p 195 7118d (1956). 10. P. Lepoutre and J.S. Hui, J. Appl. Polym. Sci., A-15 2791 (1967). 11. M. Kurata, Y. Yasunashina, M. Iwana and K. Kamanda. Viscosity-molecular weight relationships and unperturbed dimensions of linear chain molecules, in: Polymer Handbook, J. Brandrup and E.H. Immergut (Eds.), Wiley Publ., New York., p. iv-I (1975). 12. F.E. Okieimen and I.N. Uroghide, Angew Makromol. Chem., 182, 63 (1990). 13. A. Kantouch, A.H. Hebeish and M.H. Rafie, Europ. Polym. J., 6, 1575 (1970). 14. R. Khullar, V.K. Varshney, S. Naithani and P.L. Soni, ePRESS Polymer Letters, 12(1), 12, (2008). 15. A.S. Singha and R.K. Rana, Adv. Mat. Lett., 1(2), 156, (2010). 16. H. Pledger Jr., G.S. Wu, T.S. Young, J.E. Hogen-esch and G.G. Butlet, J. Macromol. Sci. Chem.,A.,22, 1297 (1985). 17. F.E. Okieimen, Europ. Polym. J., 23, 319 (1987). [RJC-576/2010]

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Vol.4, No.1 (2011), 8-12 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

PHYTOEXTRACTION OF LEAD FROM INDUSTRIAL EFFLUENTS BY SUNFLOWER (HELIANTHUS ANNUUS.L) R. Usha*, A. Vasavi, K. Thishya, S.Jhansi Rani and P.Supraja Department of Biotechnology, Sri Padmavathi Mahila Visvavidyalayam, Tirupati – 517 502, Andhra Pradesh, INDIA *E-mail: ushatirupathi@hotmail.com ABSTRACT Phytoextraction is an emerging cost-effective solution for remediation of contaminated soils which involves the removal of toxins especially Heavy metals by roots of the plants with subsequent transport to aerial plant organs. In the present investigation, the phytotoxicity of lead on growing sunflower (Helianthus annuus .L) was studied. The plants were exposed to 5, 10,15,20,25 and 30 ppm of effluent water for a period of 1,2,3,4 & 5 weeks. The extent of damage to chlorophyll, plant height, plant width, dry weight, fresh weight was evaluated. This is a preliminary study to develop a method for treating industrial waters that are contaminated with lead. Lead uptake by sunflower was monitored by Atomic Absorption Spectroscopy (AAS) Maximum accumulation of Heavy metals was found in root than leaf and stem. The Physical ,Biological parameters along with metal accumulation in plants was found to be increased with increase in concentration of effluent water up to 15 ppm followed by decline with high dosage of metal concentration. Key words: Phytoextraction, Phyto toxicity, Heavy metal, Lead and AAS. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION A tremendous increase in human population and the rapid race of industrialization in India have created numerous problems of waste disposal and fresh water contamination mainly in last few decades. According to World Health Organization in developing countries 80% of the total sewage and industrial effluents get mixed untreated into fresh water resources. Pulp and paper, sugar distilleries, fertilizers, tanneries, textiles, petrochemicals and battery based industries are mainly responsible whose effluents contributes to the contamination of fresh water resources in India. Seven crores of people depend upon drinking water with the excess of other metals like Cr, Ni, Pb and Cd etc1-2. During the last decades, heavy metals become common contaminants worldwide. The main metals concerned are cadmium (cd), Lead (pb), Zinc (Zn), Copper (Cu), Nickel (Ni), Mercury (Hg) and metalloid Arsenic (As). In contrast to the organic contaminants which can undergo biodegradation, heavy metals remain in the environment. Long term deposition of metals in soil can lead to accumulation, transport and biotoxicity caused by mobility and bioavailability of significant fraction of the metals. As a result an annual world wide release of heavy metals reached 22,000 t (metric tons) for cadmium, 939000 t for copper, 783000 t for lead and 1,350,000 t for zinc. Heavy metal pollution in the environment shows deleterious effect on human health via food chain. In higher concentration these heavy metal cause severe damage to plants 3-4.Besides conventional site decontamination techniques, phytoremediation is an in-situ emerging technology based on the use of green plants to remove pollutants from the environment. Phytoextraction will be more economically feasible which produce more biomass with an added economical value. Heavy metals like Cu,Zn have known functions as micronutrients in plants 2but metals like lead were not used as micro nutrients .So their presence at low concentrations (or) high concentrations becomes toxic to the environment. High accumulation of metals effect both growth and metabolism of plants. These phytotoxic effects of heavy metals depend on metal concentration, plant species pH and other factors in soil 5-6.

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Lead (Pb) is a major anthropogenic pollutant that has been released to the environment since the industrial revolution and accumulated in different terrestrial and aquatic ecosystems7-8.It is an extremely toxic metal whose effects on human health have been widely described. Lead at 500 ppm in soil (or) solid waste considered the substance as “hazardous waste” .Maximum allowable level of lead in drinking water is 0.05 ppm. Excessive Lead exposure can cause mental retardation and behavioral disorder and its exposure can occur through multiple pathways, through inhalation of air, soil (or) dust, as it is emitted in the environment from vehicles and automobiles. It can also enter the food chain via plants. Thus lead contamination is extremely dangerous, and lead polluted waters need to be cleansed. Thus the sunflower would probably have high tolerance and should be capable of removing large amounts of lead. The present study was aimed to explore the uptake of lead (Pb) in industrial waste water by sunflower plants to find tolerance limit to metal concentration, growth and biochemical changes in sunflower plants.

EXPERIMENTAL The experiment was designed to study the heavy metal accumulation, growth and biochemical responses of sunflower (Helianthus annuus.L) grown in soil contaminated by industrial effluent. The collected soil was air dried, sieved and used for the experiment. The seeds of sunflower were obtained from hybrid seed centers. Seeds were surface sterilized with 0.1-1% sodium hypochlorite solution for 10 minutes and rinsed with double distilled water .Seeds were grown in earthen pots (30x25 cm) containing red soil and sand 3:1 proportion. The pots were kept under natural photo radiation and each pot contained 10 seedlings .The seeds were treated with distilled water up to 7 days. After one week the plants were treated with six different concentrations of effluent water viz.,5,10,15,20,25,30 ppm in separate pots till the growth of crop was observed. Analysis was made after 1, 2, 3, 4, and 5th week of treatment. The solutions used for the treatment were 500ml for each pot. The soil pH was maintained around 6-7.The plants were treated with six different effluent concentrations once in two days with above solutions. The plants were uprooted carefully and were washed with distilled water and then macerated using mortar and pestle for biological and metal analysis. Plant length and Plant width Plant length was measured at weekly intervals. The plants were removed carefully with root system and washed thoroughly. The length of shoot and root was measured by meter scale. The width of plants was also measured by meter scale. Seedlings were measured for plant height every week and visual symptoms of metal toxicity were assessed three times per week. Estimation of Chlorophyll Levels The Chlorophyll content was estimated according to the method of Arnon (1949). About 1 gm of leaf sample was cut into small pieces and homogenized in a pre-cooled mortar and pestle using 80% (v/v) acetone. The extract was centrifuged at 3000 rpm for 15 min and made up to 25 ml with 80% (v/v) acetone.The clear solution was transferred and the optical density was measured at 645 nm and 663 nm against blank in Shimadzu double beam spectrophotometer (UV 240). Total chlorophyll (µg/ml) = (20.2xOD at 645 nm) + (8.02xOD at 663 nm). Plant harvest and metal analysis After every 7 days of effluent treatment, plants were harvested and separated into shoots, roots and leaves and weighed for fresh weight. Then plant material was dried at 80°C for 48 hrs until constant dry weight was obtained, subsequently weighed and ground by mortar and pestle. The powdered plant samples were digested with 5ml di acid mixture (Nitric acid, per chloric acid in the ratio of 3:2 at 110°C for 8 hrs. Metal content was determined by flame atomic absorption spectrometry (Perkin Elmer 2380). Effluent water analysis An effluent sample was collected from outlets of the industry (a battery manufacturing unit). The effluent sample was collected in well cleaned polythene bottle. After filtering the effluent sample, the pH of the sample was immediately measured in the laboratory and later and later the samples were stored at 4°C for further analysis. Phytoextraction of Lead

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RESULTS AND DISCUSSION The primary point of entry for metals into plants is through the roots. However for an efficient metal removal from the soil, the metals must be translocated to the harvestable parts of shoots. The aim was to assess a potentiality of sunflower plants for metal accumulation. Excess of trace elements as well as uptake of non-essential can lead to toxic effects on plant growth. The uptake of metals is frequently related to their concentrations in soil. 9 have investigated the localization of lead in root tissue. Lead was found in the basal part deposited mostly in the cell walls adjacent to the plasma membrane and the endomembrane compartment. There are several possible areas through which lead can penetrate into plants. However, it is understood that roots are the main pathway through which trace metal enter into plant body. It was determined that in plants, metal uptake at first stops on root surface and then a portion of ions which penetrate into roots is bound in cell walls and the rest is accumulated in the intercellular space10.It was determined that in the sunflower plants, roots are the main accumulation on site of lead, because there are severally affected by lead concentration in the medium. The present study mainly focused on the heavy metal toxicity on plants. The effluent was collected from lead-acid battery manufacturing industry; the effluent mainly contains lead at higher concentration. The impact of heavy metal accumulation in sunflower plants were studied by various physical biochemical parameters viz., plant height, plant width, fresh weight, dry weight and chlorophyll content . A series of lab experiments were conducted to assess the toxicity of lead (Pb) and its phytoextraction through sunflower cultivation. From the present investigation it was estimated that all the physical parameters were increased from 5-15 ppm concentration and reduced from 20-30ppm.The chlorophyll content was also reduced from first week to fifth week in all concentrations. This shows that the metal concentration has direct impact on chlorophyll content. The lead removal Study by sunflower for the experimental period of five weeks revealed its potential as an efficient lead accumulator. Visual symptoms of metal toxicity on shoots, roots and leaves of sunflower After 7 days of metal exposure, sunflower seedlings of various concentrations did not show reduced growth compared to the control plants nor any symptoms of metal toxicity. After 14 days of metal exposure, the first visual symptoms of lead toxicity were observed. Sunflowers exposed to concentrations of 25ppm, 30 ppm lost their intensive green pigmentation; the youngest leaves of some plants started to become slightly chlorotic and showed turgor loss. The roots at both 25 and 30 ppm concentrations became brown. After 21 days of metal exposure, the most visible symptoms of metal toxicity showed small circular rust spots, necrosis on leaves and leaf turgor loss at 25 and 30 ppm concentrations. The root system became yellowish brown; on the other hand, the plants showed no symptoms of metal toxicity at lower concentrations of 5, 10 and 15 ppm. After 28 days of metal exposure, the sunflower plants treated with 25, 30 ppm were completely dead. Visible symptoms of metal toxicity were observed on 20 ppm concentration, the plants were lost their green pigmentation, and leaves started curl up where as control plants started to form flower buds. After 35 days of metal exposure, the sunflower seedlings treated with 20 ppm concentration were completely dead. Since the plants showed no symptoms of metal toxicity at lower concentrations of 5,10and 15 ppm. It was observed that sunflower is capable to survive without any apparent negative side effect on its growth up to 15ppm of lead exposure. Beyond these levels chlorosis, necrosis, wilting death decay occurred rapidly. The removal of lead was higher in low concentrations .It was observed that sunflower could accumulate lead up to 129 folds of its dry weight at 15ppm.The accumulation capacity was reduced by increased metal concentration. A high amount of lead accumulation was observed in leaves than roots and stem. Significant increased metal accumulation was observed from from 5-15ppm and decreased from 20-30 ppm because of high dosage effluents applied to plants.

CONCLUSION The absorption and accumulation of lead without any major effect on the normal growth of sunflower was reported at 15 ppm concentration. The easy availability and high mass production of sunflower creates a Phytoextraction of Lead

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great possibility of removal of lead as a phytoremediation agent. Sunflower exhibits a remarkable ability to decrease concentration of lead and other pollutants directly from sewage (or) industrial effluents and has been utilized in phytoremediation.

ACKNOWLEDGEMENTS The authors would like to express their sincere gratitude to Amar raja batteries pvt.ltd.Tirupati, Department of Chemistry, S.V.University, for providing the facilities for this research.

Fig.-1: Irrigational impact of industrial waste water on dry matter production and biochemical responses of sunflower (at 5 weeks after treatment)

Table-1: The physico chemical characteristics of industrial waste water

Physical Examination

Metal Concentration

Phytoextraction of Lead

Colour Density g/cc Odour TS (at 1050C) g/L TDS (at 1050C) g/L TSS (at 1050C) g/L pH value Dissolved Pb mg/L

11

Gray 0.988 (Filtered sample) Muddy 33.3 0.22 3.11 7.2 30.0

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Vol.4, No.1 (2011), 8-12

Fig.-2: Metal accumulation in various parts of plant (after five weeks of treatment) at various concentrations

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

C.P.Sharma, Science publisher, New Hampshire, USA, PP.5-10(2006). S.Singh and P.K.Aggarwal, Ind.J.Agric.Sci.,76, 688 (2006). B.S.Mohan.and B.B.Hosetti, J.Environ.Biol, 29,309 (2006). G.K.Handique. and A.K.Handiwue, J.Environ.Biol.,30, 299 (2009). Sc.Barman, R.K.Sahu, S.K.Bhargava and C.Chatterjee, Bull.Environ.Contam.Toxicol, 64,489 (2000). R. ChandraK.Kumar and J.Singh, J.Environ.Biol, 25,381 (2004). I.H .Wahla and M.B. Kirkham, Environ. Poll. ,155, 271(2008). N.Akguc, I.I. Ozyigit and C. Yarci, Pak. J. Bot., 40, 1767 (2008). G.Kocjan, S.Samardakiewiez, and A. Wozny, Biol.Plant.,38, 107 (1996). S.N.Pandey, J.Appl.BioSci., 34, 79 (2008). [RJC-646/2010]

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Vol.4, No.1 (2011), 13-16 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

ASSAY OF CEFPROZIL IN BULK AND ITS PHARMACEUTICAL FORMULATIONS BY VISIBLE SPECTROPHOTOMETRY C. Ramesh1*,G. Nagarjuna Reddy2,T. V. Narayana3 , K.V.S. Prasada Rao4 and B. Ganga Rao5 1

V.V.Pura Institute of Pharmaceutical Sciences, Bangalore (Karnataka) India 2 A.M.Reddy memorial college of Pharmacy, Narasaraopet(A.P.) India 3 Vikas Institute of Pharmaceutical Sciences, Rajahmundry(A.P.) India 4 Rahul Institute of Pharmaceutical Sciences & Research, Chirala(A.P.) India 5 College of Pharmaceutical Sciences, Andhra University, Visakhapatnam(A.P.) India * E-mail: rameshvips@rediffmail.com ABSTRACT Four simple and sensitive visible spectrophotometric methods (A, B, and C) have been described for the estimation of Cefprozil (CEF). The methods that are based on the formation of radical anion with the involvement of basic nitrogen in CEF (donor) and quinones [2,3-dichloro-5,6-dicyano-p-benzoquinone(DDQ), chloranilic acid (DHQ), 2,3,5,6-tetrachloro-p-benzoquinone (TQ)] (acceptor). The variable parameters in all these methods have been optimized and the chemical reactions involved are presented. The results obtained in the three methods are statistically validated and recoveries range from 99.7 to 101.3%. Common excipients used in additives in pharmaceutical preparations do not interfere in the proposed methods. Keywords: Cefrprozil, DDQ, DHQ, TQ, Spectrophotometric, Phrmaceutical formulations. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Cephalosporins are penicillin- resistant antibiotics with significant activity both gram positive and gram negative bacteria. The key intermediate for semi synthetic production of a large number of Cephalosporins is 7-cephalosporanic acid, which is formed by hydrolysis of cephalosporins C produced by fermentation. Cefprozil (CEF) is a synthetic broad-spectrum 8-methoxyfluoroquinolone antibacterial agent for oral, intravenous administration and chemically known as (6R,7R)-7-[(R)-2-(p-hydroxyphenyl) acetamido]-8-oxo-3-propenyl-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid monohydrate. A number of methods such as spectrophotometric1-10 and HPLC11-21 were reported for the estimation of CEF. Literature survey revealed that only two visible spectrophotometric6,7 method was reported for it quantitative determination in bulk drug and pharmaceutical formulations. Hence there is a need to develop to sensitive and flexible spectrophotometric methods for the assay of CEF. A direct chemical analysis based on the reactivity of the intact molecule with out cleavage is not frequently encountered. The methods that are based on the charge transfer complexation are usually rapid and simple to perform.π-acceptors such as 2, 3-dichloro-5,6-dicyano-p-benzoquinone(DDQ), 2,3,5,6tetrachloro-p-benzoquinone(TQ), chloranilic acid (DHQ) are known to yield charge transfer complexes with a variety of electron donors. The present work describes an improved direct simple analytical procedure that can be applied at quality control laboratories for the analysis of Cefprozil.

EXPERIMENTAL Instrument A Systronics model 117 UV – Visible spectrophotometric with 1cm matched quartz cells was used for spectral and absorbance measurements in the UV and visible regions respectively.

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Vol.4, No.1 (2011), 13-16

Materials All reagents used were of Analytical Grade and freshly prepared solutions were always used. DDQ (Fluka, 4.4 × 10–3M) solution in acetonitrile for Method A, DHQ (Sd-Fine, 0.1%, 4.785× 10–3M) solution in methanol for Method B, TQ (BDH, 0.1%, 4.067 × 10–3M) solution in 1.4-dioxane for Method C were prepared. Standard Drug Solution Stock solution (1mg/1ml) of CEF for method A and B was prepared by dissolving 100 mg of it in 100ml of methanol and for method C was prepared by dissolving 100 mg of it in 100ml of 1,4-dioxane. The working standard solution of CEF of the required strength was prepared by further dilution of stock solution of CEF with Acetonitrile (method A ), Methanol (method B) and 1,4-Dioxane (method C). Recommended Procedures (a) For Bulk Samples Method A: Aliquots of standard drug CEF solution (0.5-2.5 mL, 400 µg/mL) in acetonitrile were delivered into 10ml were delivered into 10 mL graduated tubes. Then 2mL of (4.4 ×10−3M) DDQ in acetonitrile was added and kept aside for 20 min (CEF). The volume was made upto 10 mL with acetonitrile and read at 460 nm against reagent blank during the stability period (15-60min). The amount of drug present was computed from the appropriate calibration curve Method B: Aliquots of standard drug CEF solution (0.5 – 2.5 mL, 500 µg/mL), was transferred into 10mL-graduated tubes. 2.0 mL of (4.785 x 10-3M) DHQ in methanol was added and kept aside for 5 min. Then the volumes of the contents were made upto 10 mL with methanol and read at 540 nm for CEF against a reagent blank within 30 min. The amount of drug was computed from the appropriate calibration curve. Method C: Aliquots of standard drug CEF solution (0.5 – 2.5 mL, 500 µg/mL) in dioxan were delivered into 10 ml graduated tubes. Two mL of (4.067 × 10−3M) TQ in 1, 4-dioxan, followed by dioxan was added for bringing the volume to 7 mL. The final volume was brought to 10 mL with dimethyl formamide and the absorbance was measured against a reagent blank at 580 nm for CEF within the stability period (15-60min). The amount of the drug present was computed from the appropriate calibration graph. (b) For Pharmaceutical Formulations: An accurately weighed amount of tablet powder equivalent to 100 mg of CEF was extracted with isopropanol (4 x 15ml) and filtered. The combined filtrate was evaporated to dryness and the residue was dissolved in Acetonitrile (method A ), Methanol (method B) and 1,4-Dioxane (method C) to get 1mg/mL solution. The working standard solution of CEF of required strength prepared by further dilution of the stock solution of CEF with required solvent in the respective method and analyzed under procedure described for bulk samples.

RESULTS AND DISCUSSION The optimum conditions for the colour development of method were established by varying the parameters one at a time in each method, keeping the others fixed and observing the effect produced on the absorbance of the colored species. The optical characteristics such as Beer’s law limits, molar absorptivity for each method are given in Table -2. The precision of each method was found by measuring absorbances of six replicate samples containing known amounts of drug and the results obtained are incorporated in Table-2. Regression analysis using the method of least squares was made to evaluate the slope (b), intercept (a) and correlation coefficient (r) for each method and is presented in Table 2. The accuracy of each method was ascertained by comparing the results by proposed and reference methods (UV) statistically (Table-3). This comparison shows that there is no significant difference between the results of proposed methods and those of the reference ones. The similarity of the results is obvious evidence that during the application of these methods, the additives and excipients that are usually present in tablets do not interfere in the assay ASSAY OF CEFPROZIL IN BULK

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of proposed methods. As an additional check of accuracy of the proposed methods, recovery experiments were performed by adding a fixed amount of the drug to the pre-analyzed formulations. The amount of drug found, the % recovery was calculated in the usual way. Chemistry The interaction of any of the investigated compounds with poly halo and polycyanoquinone π-acceptors in nonpolar solvents was found to produce colored charge-transfer complexes with low molecular absorptivity values. In polar solvents such acetonitrile or methanol, complete electron transfer from donor to the acceptor moiety takes place with formations of intensely colored radical ions with higher molar absporptivity values according to the following scheme. D.. +A→ (D−A) Polar solvent→A− . +D + . Complex

The dissociation of the D-A Complex is promoted by the high ionizing power of the acetonitrile and the resulting bands of the named drugs with acceptors are similar to the maxima of radical anions of the acceptors obtained by the iodide reduction method. Acetonitrile was considered an ideal solvent as it afforded maximum sensitivity yield of radical anions in addition to its high solvating power of the reagents. Methanol gave maximum sensitivity in case of DHQ and 1,4-dioxane gave maximum sensitivity in case of TQ. The interaction of CEF with TQ, DHQ, DDQ, gave a colored chromogens with a strong absorption maxima in different solvents given in Table-1

CONCLUSION The proposed methods are applicable for the assay of drug (CEF) and have the advantage of wider range under Beer’s law limits. The decreasing order of sensitivity and λmax among the proposed methods are C>B>A respectively. The proposed methods are simple, selective and can be used in the routine determination of CEF in bulk samples and formulations with reasonable precision and accuracy. Table-1: Reaction time and intensity in polar solvent Acceptor

Reaction time

Solvent

TQ DHQ DDQ

10 5 20

1,4-dioxane Methanol Acetonitrile

Absorption Maxima 580 540 460

Table-2: Optical Characteristics, Precision and Accuracy of the Proposed Methods for CEF Parameters

DDQ

DHQ

TQ

λmax (nm) Beer’s Law limits (µg/ml) Molar absorptivity (l mol-1cm-1) Correlation coefficient (r) Sandell’s sensitivity (µg/cm2/ 0.001 absorbance unit) Regression Equation ( y = a + bc) (i)Slope (b) (iii) Intercept (a) Relative Standard Deviation * % Range of error (confidence limits) (i) 0.05 level (ii) 0.01 level *Average of six determinations considered.

460 20-100 3.321×103 0.9999

540 25-125 2.306×103 0.9999

580 25-125 2.025×103 0.9999

0.123

0.177

0.201

0.0081

0.0055

0.0050

-0.0016 0.3474

0.0118 0.2505

-0.0006 0.227

0.290

0.209

0.191

0.430

0.310

0.281

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Table-3: Assay of CEF in pharmaceutical formulations Pharmaceutical formulations#

Labelled amount (mg)

Tablet I

250

Tablet II

250

Tablet III

250

Tablet IV

250

Amount found by Proposed Methods* Method A 250.63±1.28 F=3.36 t=0.49 248.82±1.92 F=1.17 t=0.65 250.90±1.24 F=1.01 t=0.43

Method B 250.56±1.37 F=3.85 t=0.41 249.10±2.27 F=1.64 t=0.23 248.51±1.87 F=2.26 t=1.36

Method C 250.69±1.08 F=2.39 t=1.25 250.85±2.09 F=1.39 t=0.56 249.13±1.67 F=1.81 t=1.24

250.82±2.44 F=2.34 t=0.69

251.67±2.01 F=1.60 t=1.25

249.66±2.39 F=2.25 t=0.32

Reference method##

%Recovery by Proposed methods** Method A 100.25 ±0.51

Method B 100.22 ±0.55

Method C 100.27 ±0.43

249.63 ±1.77

99.52±0.77

99.64±0.91

100.34 ±0.83

250.45 ±1.24

100.36 ±0.50

99.40±0.75

99.65±0.66

249.26 ±1.59

100.32 ±0.97

100.42±0.8 1

99.86±0.96

250.09 ±0.69

#

Four different batches of tablets from a pharmaceutical company. Developed in the laboratory using methanol solvent (λmax 253 nm). *Average ± standard deviation of six determinations; the t- and F- values refer to comparison of the proposed method with the reference method. Theoretical values at 95% confidence limit, t = 2.57, F = 5.05. **After adding 3 different amounts of the pure labelled to the pharmaceutical formulation, each value is an average of 3 determinations. ##

REFERENCES 1. P. Vikas, T. Santosh, B. Santosh, S. Rupali, and G. Lalit, Int. J. Pharm. Pharmaceu. Sci., 2, 82 (2010). 2. A.G. Elrasheed, M.M. Mohammed, E.E.I. Kamal, and A.H. El-Obeid, Int. J. Biomed. Sci., 5, 267 (2009). 3. L. Zhang, Y. Xie, and N. Guo, Zhongguo Yaopin Biaozhun, 9, 291 (2008). 4. D. Gowrisankar, S.S. Prakash, and S.A. Raju, J. Ind. Council. Chemists, 25, 106 (2008). 5. D.R. Kumar, S.V.M. Vardhan, D. Ramachandran, and C. Rambabu, Oriental J. Chem., 24, 617 (2008). 6. D. Gowrisankar, S.S. Prakash, and S.A. Raju, Int. J. Chem. Sci., 5, 2315 (2007). 7. D. Gowrisankar, S.S. Prakash, and S.A. Raju, Int. J. Chem. Sci., 5, 987 (2007). 8. M. S. El-Adl, and M.H. Saleh, Scientia Pharmaceu., 70, 67, (2002). 9. I.O.A. El-Sattar, N.M. El-Abasawy, S.A.A. El-Razeq, M. M. F. Ismail, and N.S. Rashed, Saudi Pharm. J., 9, 186 (2001). 10. H.G. Daabees, M.S. Mahrous, M.M. Abdel-Khalek, Y.A. Beltagy, and K.N. Emil, Analytical Let., 34, 1639 (2001). 11. L. Wen-hua, and Z. Guo-cheng, Yaoxue Jinzhan, 33, 34 (2009). 12. D. Gowrisankar, S.S. Prakash, and S.A. Raju, Int. J. Chem. Sci., 6, 1583 (2008). 13. Z. Chen, Zhongguo Redai Yixue, 7, 2099 (2007). 14. J. Gong, S. Pang, Z. Li, J. Zhou, G. Wang, and Y. Zou, Zhongguo Yaofang, 19, 592 (2008). 15. T. Shi-xin, Z. Li, J. Hong-man, J. Yi-ping, Y. Wu-yun, and H. Jin-hong, Yaoxue Fuwu Yu Yanjiu, 8, 46 (2008). 16. R. Chun-kai, T. Shi-xin, Z. Li, J. Yi-ping, Y. Wu-yun, and W. Fa-cai, Yaoxue Fuwu Yu Yanjiu, 6, 348 (2006). 17. J. Le, and Z. Hong., Yaowu Fenxi Zazhi, 24, 153 (2004). 18. P. Tae-Hwan, K. Jin-Ki, J. Jun-Pil, P. Jeong-Sook, and K. Chong-Kook, J. Pharmaceu. Biomed. Anal., 36, 243 (2004). 19. C. W. Shyu, A. U. Shukla, R. V. Shah, A. E. Papp, and H. R. Barbhaiya, Pharma. Res., 8, 992 (1991). 20. L. Manna, and L. Valvo, Chromatographia, 60, 645 (2004). 21. R. N. Rao, N. Venkateswarlu, and R. Narsimha, J. Chromatogr. A, 1187, 151 (2008). [RJC-656/2010]

ASSAY OF CEFPROZIL IN BULK

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Vol.4, No.1 (2011), 17-23 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND BIOLOGICAL ACTIVITY OF SOME 2-AMINO-4,6-SUBSTITUTED-DIARYLPYRIMIDINES: REACTION OF SUBSTITUTED CHALCONES WITH GUANIDINIUM CARBONATE Vandana Sharma* and K. V. Sharma Department of Engineering Chemistry, Mahakal Institute of Technology, Ujjain 456664 Madhya Pradesh, INDIA *E-mail: vandanak_sharma@yahoo.co.in ABSTRACT A series of substituted 2-Amino-4,6-diarylpyrimidines were synthesized by the reaction of appropriately substituted chalcones and guanidinium carbonate in DMF. The synthesized pyrimidines have been characterized on the basis of their chemical properties and spectroscopic data. These compounds were screened for biological activity against a variety of test organisms. Keywords: Substituted Chalcones, Guanidinium Carbonate, pyrimidines, 2-Amino-4,6- diarylpyrimidines. Š 2011 RASĀYAN. All rights reserved.

INTRODUCTION The nitrogen containing heterocycles are an important class of compounds in the medicinal chemistry and also contributed to the society from biological and industrial point which helps to understand life processes1. The chemistry of pyrimidines and its derivatives have been studied since past century due to their close pharmacological association with diverse pharmacological properties. This seems to be because pyrimidines represents one of the most active class of compounds possessing wide spectrum of biological activity viz. significant in vitro activity against unrelated DNA and RNA, viruses including polio herpes viruses, diuretic, antitumor, anti HIV, cardiovascular2.The biodynamic property of the pyrimidine ring system prompted us to account for their pharmacological properties especially as antiinfective agents3. In view of the significant biological activities4-11 of the compounds having pyrimidine nucleus, and pyrimidines having an amino or a substituted amino group at C-2 and C-4 position12-15 we have synthesized some new substituted 2-Amino-4,6-diarylpyrimidines by action of chalcones with guanidinium carbonate in DMF.

EXPERIMENTAL General Melting points were determined by the open tube capillary method and are uncorrected. The purity of the compounds was controlled by thin layer chromatography (TLC). IR spectra were recorded as KBr pellets on Perkin-Elmer spectrum RX1 spectrophotometer. Carbon, hydrogen, and nitrogen were estimated by Thermo Finnigan FLASH EA 1112 elemental analyzer. 1H-NMR spectra were recorded on Bruker DRX300 spectrometer at room temperature. Mass spectra were measured on JEOL SX 102/DA-6000 mass spectrometer. 2-Amino-4,6-diarylpyrimidines derivatives were prepared according to the reported methods. General procedure for synthesis of 2-amino-4,6-substituted-diphenylpyrimidine(1-24) To a mixture of substituted chalcone and guanidinium carbonate (1:1 molar ratio) in DMF was refluxed for 3 hours. The reaction mixture was poured in cold water. The solid thus separated was filtered, washed with water and dried at 80oC. The product was crystallized from ethanol to afford light yellow crystals.

SOME 2-AMINO-4,6-SUBSTITUTED-DIARYLPYRIMIDINES

Vandana Sharma and K. V. Sharma

Vol.4, No.1 (2011), 17-23

2-Amino-4,6-diphenylpyrimidine(1) Obtained as light yellow crystals in 85% yield, m.p. 164-65◦C; IR(υ) max: 3480, 3300, 1640, 1600, 1585, 1565, 1450, 1370, 1235, 1070, 1025, 930, 840, 770, 710,700, 630, 590 and 440cm-1. MS,m/z: 247, 232, 170, 164, 155, 87. Anal. Calcd. for C16H13N3: C, 77.73; H, 5.26; N, 17.00, Found: C, 77.71; H, 5.27; N, 17.02%. 2-Amino-4-(4′-chlorophenyl)-6-phenylpyrimidine(2) Obtained as light yellow crystals in 95% yield, m.p. 151-52◦C; IR(υ) max: 3490, 3300, 3200, 1630, 1590, 1580, 1560, 1540, 1490, 1460, 1360, 1100, 1020, 830, 815, 775, 700, 650 and 480cm-1. MS,m/z: 281, 266, 204, 189, 168, 153, 121,85. Anal. Calcd. for C16H12ClN3: C,68.32; H, 4.27; N, 14.94, Found: C, 68.34; H, 4.26; N, 14.93%. 2-Amino-4-(4′-methoxyphenyl)-6-phenylpyrimidine(3) Obtain as light yellow crystals in 80% yield, m.p. 159-60◦C; IR(υ) max: 3340, 3200, 1640, 1580, 1560, 1530, 1460, 1360, 1305, 1260, 1180, 1030, 1000, 825, 770, 690, 640, 580, 510 and 460cm-1. MS,m/z: 277, 262, 151, 136, 108, 78. Anal. Calcd. for C17H15N3O: C,73.64; H, 5.41; N, 15.16, Found: C, 73.65; H, 5.38; N, 15.12%. 2-Amino-4-(2′-hydroxyphenyl)-6-phenylpyrimidine(4) Obtain as light yellow crystals in 70% yield, m.p. 169-70◦C; IR(υ) max: 3500, 3400, 1570, 1540, 1500, 1420, 1360, 1300, 1230, 1040, 890, 800, 760, 700, 660 and 550cm-1. MS,m/z: 263, 248, 246, 156, 122, 94,65. Anal. Calcd. for C16H13N3O: C,73.00; H, 4.94; N, 15.96, Found: C, 73.01; H, 4.93; N, 15.92%. 2-Amino-4-(2′-hydroxyphenyl)-6-(2-hydroxyphenyl)pyrimidine(5) Obtain as light yellow crystals in 80% yield, m.p. 169-70◦C; IR(υ) max: 3500, 3360, 1630, 1570, 1540, 1500, 1460, 1420, 1360, 1310, 1230, 1140, 1040, 1000, 890, 860, 840, 800, 760, 700, 640, 550 and 470cm-1. MS,m/z: 279, 264, 262, 186, 118,93, 85. Anal. Calcd. for C16H13N3O2: C,68.81; H, 4.65; N, 15.05, Found: C, 68.78; H, 4.64; N, 15.12%. 2-Amino-4-(2′-hydroxy-5′methylphenyl)-6-phenylpyrimidine(6) Obtain as light yellow crystals in 68% yield, m.p. 170-71◦C; IR(υ) max: 3500, 3300, 3180, 1640, 1580, 1540, 1500, 1440, 1400, 1370, 1300, 1230, 840, 810, 765, 740, 700, 650, 540 and 470cm-1. MS,m/z: 277, 262, 247, 245, 155, 106, 78,49. Anal. Calcd. for C17H15N3O: C,73.64; H, 5.40; N, 15.15, Found: C, 73.65; H, 5.38; N, 15.17%. 2-Amino-4-phenyl-6-(2-hydroxyphenyl)pyrimidine(7) Obtain as light yellow crystals in 65% yield, m.p. 168-69◦C; IR(υ) max: 3480, 3400, 3200, 1640, 1570, 1540, 1500, 1465, 1420, 1360, 1300, 1230, 855, 800, 770, 750, 700 and 650cm-1. MS,m/z: 263, 248, 246, 156, 122, 94,65. Anal. Calcd. for C16H13N3O: C,73.00; H, 4.94; N, 15.95, Found: C, 73.02; H, 4.93; N, 15.97%. 2-Amino-4-(4′-chlorophenyl)-6-(2-hydroxyphenyl)pyrimidine(8) Obtain as light yellow crystals in 71% yield, m.p. 229-30◦C; IR(υ) max: 3500, 3340, 1640, 1580, 1545, 1510, 1400, 1310, 1235, 1090, 1015, 860, 840, 800, 750, 740, 650 and 480cm-1. MS,m/z: 297, 282, 280, 139, 128, 111, 78. Anal. Calcd. for C16H12ClN3O: C,64.64; H, 4.04; N, 14.14, Found: C, 64.65; H, 4.07; N, 14.16%. 2-Amino-4-(4′-methylphenyl)-6-(2-hydroxyphenyl)pyrimidine(9) Obtain as light yellow crystals in 68% yield, m.p. 169-70◦C; IR(υ) max: 3500, 3360, 1630, 1570, 1540, 1500, 1450, 1420, 1300, 1230, 890, 760, 700, 640 and 470cm-1. MS,m/z: 277, 262, 260, 185, 172, 119,

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Vol.4, No.1 (2011), 17-23

117, 100, 92, 91, 78. Anal. Calcd. for C17H15N3O: C,73.64; H, 5.40; N, 15.16, Found: C, 73.65; H, 5.38; N, 15.18%. 2-Amino-4-(4′-methoxyphenyl)-6-(2-hydroxyphenyl)pyrimidine(10) Obtain as light yellow crystals in 66% yield, m.p. 193-94◦C; IR(υ) max: 3480, 3340, 1630, 1600, 1580, 1535, 1515, 1420, 1300, 1230, 1180, 1035, 830, 760 and 590cm-1. MS,m/z: 293, 278, 276, 187, 172, 135, 107, 104, 87, 79, 78. Anal. Calcd. for C17H15N3O2: C,69.62; H, 5.11; N, 14.33, Found: C, 69.65; H, 5.10; N, 14.32%. 2-Amino-4-phenyl-6-(4-chloroyphenyl)pyrimidine(11) Obtain as light yellow crystals in 65% yield, m.p. 152-53◦C; IR(υ) max: 3495, 3305,3200, 1630, 1595, 158, 1560, 1490, 1460, 1360, 1220, 1100, 1020, 830, 815, 775, 700, 645 and 485cm-1. MS,m/z: 281, 266, 246, 204, 136, 111, 101. Anal. Calcd. for C16H12ClN3: C,68.32; H, 4.27; N, 14.94, Found: C, 68.36; H, 4.26; N, 14.93%. 2-Amino-4-(2′-hydroxyphenyl)-6-(4-chlorophenyl)pyrimidine(12) Obtain as light yellow crystals in 61% yield, m.p. 230-31◦C; IR(υ) max: 3500, 3350, 1640, 1580, 1555, 1500, 1420, 1310, 1235, 1090, 1015, 860, 800, 750, 650 and 480cm-1. MS,m/z: 297, 282, 262, 190, 121, 93, 78. Anal. Calcd. for C16H12ClN3O: C,64.64; H, 4.04; N, 14.14, Found: C, 64.65; H, 4.07; N, 14.16%. 2-Amino-4-(4′-chlorophenyl)-6-(4-chlorophenyl)pyrimidine(13) Obtain as light yellow crystals in 62% yield, m.p. 207-08◦C; IR(υ) max: 3480, 3310, 3200, 1640, 1600, 1580, 1530, 1490, 1460, 1370, 1230, 1100, 1015, 810, 730, 590 and 490cm-1. MS,m/z: 215, 300, 280, 204, 190, 139, 111, 101, 78. Anal. Calcd. for C16H11Cl2N3: C,60.95; H, 3.49; N, 13.13, Found: C, 60.92; H, 3.91; N, 13.16%. 2-Amino-4-(4′-methylphenyl)-6-(4-chlorophenyl)pyrimidine(14) Obtain as light yellow crystals in 68% yield, m.p. 149-50◦C; IR(υ) max: 3500, 3320, 3200, 1630, 1595, 1565, 1540, 1490, 1660, 1360, 1300, 1240, 1175, 1100, 1020, 930, 850, 830, 815, 775, 700, 645, 580, 480 and 400cm-1. MS,m/z: 295, 280, 260, 204, 190, 136, 119, 111, 101, 91, 78. Anal. Calcd. for C17H14ClN3: C,69.15; H,4.74 ;N, 14.23, Found: C, 69.18; H, 4.74; N, 14.25%. 2-Amino-4-(4′-methoxyphenyl)-6-(4-chlorophenyl)pyrimidine(15) Obtain as light yellow crystals in 62% yield, m.p. 156-57◦C; IR(υ) max: 3340, 3210, 1640, 1580, 1560, 1530, 1490, 1400, 1360, 1240, 1175, 1090, 1030, 1010, 820, 580 and 510cm-1. MS,m/z: 311, 296, 276, 259, 203, 190, 135, 118, 110, 101, 93, 90,78. Anal. Calcd. for C17H14ClN3O: C,65.59; H,4.50 ;N, 13.50, Found: C, 65.63; H, 4.52; N, 13.45%. 2-Amino-4-phenyl-6-(4-methoxyphenyl) pyrimidine(16) Obtain as light yellow crystals in 72% yield, m.p. 145-46◦C; IR(υ) max: 3340, 3190, 1640, 1600, 1580, 1560, 1530, 1400, 1360, 1260, 1240, 1180, 1030, 820, 770, 690, 580 and 510cm-1. MS,m/z: 277, 262, 243, 169, 101, 78. Anal. Calcd. for C17H15N3O: C,73.64; H,5.41 ;N, 15.61, Found: C, 73.63; H, 5.39; N, 15.20%. 2-Amino-4-(2′-hydroxyphenyl)-6-(4-methoxyphenyl)pyrimidine(17) Obtain as light yellow crystals in 62% yield, m.p. 194-95◦C; 1H NMR(δ): 3.90(3H,s,-OCH3), 5.24(2H,s,NH2), 6.90 to 8.10(9H,m,ArH), 11.80(1H,s,-OH); IR(υ)max: 3500, 3330, 1620, 1575, 1530, 1510, 1415, 1300, 1250, 1230, 1180, 1030, 860, 760, 635 and 590cm-1. MS,m/z: 293, 278, 276, 200, 135, 132, 107, 101, 78. Anal. Calcd. for C17H15N3O2:C,69.62; H,5.11 ;N, 14.33, Found: C, 69.68; H, 5.10; N, 14.30%. 2-Amino-4-(4′-chlorophenyl)-6-(4-methoxyphenyl)pyrimidine(18)

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Vol.4, No.1 (2011), 17-23

Obtain as light yellow crystals in 65% yield, m.p. 155-56◦C; IR(υ)max: 3340, 3210, 1640, 1580, 1560, 1530, 1490, 1400, 1360, 1240, 1175, 1090, 1030, 1010, 820, 580 and 510cm-1. MS,m/z: 311, 296, 276, 259, 203, 190, 135, 118, 110, 101, 93, 90, 78. Anal. Calcd. for C17H14ClN3O: C,65.59; H,4.50 ;N,13.50, Found: C, 65.61; H,4.51; N, 13.49%. 2-Amino-4-(4′-methylphenyl)-6-(4-methoxyphenyl)pyrimidine(19) Obtain as light yellow crystals in 63% yield, m.p. 160-61◦C; IR(υ)max: 3460, 3320, 1640, 1585, 1565, 1535, 1400, 1360, 1260, 1240, 1180, 1030, 825, 770, 690, 580 and 510cm-1. MS,m/z: 291, 276, 199, 168, 135, 107, 101, 78. Anal. Calcd. for C18H17N3O: C,74.22; H,5.84 ;N, 14.43, Found: C,74.20; H,5.83; N,14.46%. 2-Amino-4-(4′-methoxyphenyl)-6-(4-methoxyphenyl)pyrimidine(20) Obtain as light yellow crystals in 70% yield, m.p. 159-60◦C; IR(υ)max: 3360, 3200, 1650, 1600, 1560, 1535, 1510, 1440, 1400, 1365, 1310, 1260, 1240, 1180, 1030, 850, 820, 800, 580 and 520cm-1. MS,m/z: 307, 292, 276, 200, 135, 132, 107, 101. Anal. Calcd. for C18H17N3O2: C,70.35; H,5.53 ;N, 13.68, Found: C, 70.35; H,5.50; N, 13.70%. 2-Amino-4-(2′-hydroxyphenyl)-6-(3,4-dimethoxyphenyl)pyrimidine(21) Obtain as light yellow crystals in 60% yield, m.p. 210-11◦C; IR(υ)max: 3440, 3300, 3180, 1640, 1600, 1570, 1540, 1520, 1460, 1420, 1370, 1310, 1260, 1180, 1140, 1030, 820, 760 and 620cm-1. MS,m/z: 323, 308, 306, 292, 276, 230, 200, 199, 168, 101, 78. Anal. Calcd. for C18H17N3O3:C,66.87; H,5.26 ;N, 13.00, Found: C, 66.84; H,5.27; N, 13.00%. 2-Amino-4-(4′-chlorophenyl)-6-(3,4-dimethoxyphenyl)pyrimidine(22) Obtain as light yellow crystals in 86% yield, m.p. 185-86◦C; IR(υ)max: 3500, 3380, 1620, 1590, 1580, 1530, 1490, 1440, 1360, 1310, 1270, 1140, 1090, 1030, 850, 800, 770 and 600cm-1. MS,m/z: 341, 326, 306, 230, 204, 165, 136, 137, 111, 101. Anal. Calcd. for C18H16ClN3O2:C,63.34; H,4.69 ;N, 12.31, Found: C, 63.32; H,4.67; N, 12.33%. 2-Amino-4-(4′-methylphenyl)-6-(3,4-dimethoxyphenyl)pyrimidine(23) Obtain as light yellow crystals in 66% yield, m.p. 144-45◦C; 1H NMR(δ): 2.40(3H,s,-CH3), 3.904.10(6H,2s,-OCH3), 5.24(2H,s,-NH2), 6.86 to 8.00(9H,m,ArH), 11.80(1H,s,-OH); IR(υ)max: 3440, 3320, 3200, 1630, 1600, 1565, 1530, 1510, 1490, 1450, 1400, 1370, 1325, 1260, 1210, 1180, 1140, 1030, 860, 815, 770, 690, 615, 530, 500 and 460cm-1. MS,m/z: 321, 306, 230, 204, 165, 137, 136, 111, 101. Anal. Calcd. for C19H19N3O2:C,71.02; H,5.91 ;N, 13.08, Found: C, 71.04; H,5.90; N, 13.10%. 2-Amino-4-(4′-methoxyphenyl)-6-(3,4-dimethoxyphenyl)pyrimidine(24) Obtain as light yellow crystals in 65% yield, m.p. 157-58◦C; 1H NMR(δ): 3.80 to 4.10 (9H,3s,-OCH3), 5.25(2H,s,-NH2), 6.90 to 8.10(8H,m,ArH); IR(υ)max: 3420, 3320, 3200, 1630, 1600, 1585, 1570, 1510, 1440, 1375, 1270, 1220, 1185, 1140, 1030, 820, 760, 590 and 510cm-1. MS,m/z: 337, 322, 306, 216, 204, 165, 136, 137, 111, 101. Anal. Calcd. for C19H19N3O3:C,67.65; H,5.63 ;N, 12.46, Found: C, 67.62; H,5.65; N, 112.49%.

RESULTS AND DISCUSSION Substituted 2-Amino-4,6-diarylpyrimidines(1-24) have been prepared by the reaction of variedly substituted chalcones with guanidinium carbonate in DMF. The Structures of all new compounds have been elucidated by elemental analyses, 1H NMR, Mass spectral and IR measurements. IR spectra of substituted 2-Amino-4,6-diarylpyrimidines shows two peaks in the region 3500-3180 cm-1 due to –NH2 and no >C=O stretching. N-H bending vibrations are also observed in the region 1650-1590 cm-1.The stretching vibrations due to intermolecular hydrogen bonded –OH group gives absorption in the region 3100-2700 cm-1. A group of three absorption peaks were found in the region 1600-1500 cm-1. This is due to the absorption of aromatic nucleus and >C=N group.

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Vol.4, No.1 (2011), 17-23

The NMR (CDCl3) spectra of substituted 2-Amino-4,6-diarylpyrimidines(17,23,24) shows multiplates in the range δ 8.1 to 6.9 owing to aromatic protons. Singlets at δ 5.2, δ 4.1 to 3.8 and δ 2.4 due to the –NH2,OCH3 and –CH3 groups observed respectively. R5 R4 R5 R3 R R3

+

R1 R2

NH2

R4

O

. HO

HN

NH2

DMF or NaOH N

OH

R2

N

NH2

O R1

Substituted Chalcones

Guanidinium carbonate

R

2-Amino-4,6-substituted-diarylpyrimidine (1-24)

1: R=R1=R2=R3=R4=R5=H 3: R1=OCH3, R2=R3=R4=R5=H 5: R=R3=OH, R1=R2=R4=R5=H 7: R3=OH, R=R1=R2=R4=R5=H 9: R1=CH3, R3=OH, R=R2=R4=R5=H 11: R=R1=R2=R3=R4=H, R5=Cl 13: R1=Cl, R=R2=R3=R4=H, R5=Cl 15: R1=OCH3, R=R2=R3=R4=H, R5=Cl 17: R=OH, R1=R2=R3=,R4=H, R5= OCH3 19: R1=CH3, R=R2=R3=R4=H, R5= OCH3 21: R=OH, R1=R2=R3=H, R4=R5= OCH3 23: R1=CH3, R=R2=R3=H, R4=R5= OCH3

2: R1=Cl, R=R2=R3=R4=R5=H 4: R=OH, R1=R2=R3=R4=R5=H 6: R=OH, R2=CH3, R1=R3=R4=R5=H 8: R1=Cl, R3=OH, R=R2=R4=R5=H 10: R1=OCH3, R3=OH, R=R2= R4=R5=H 12: R=OH, R1=R2=R3=R4=H, R5=Cl 14: R1=CH3, R=R2=R3=R4=H, R5=Cl 16: R=R1=R2=R3=R4=H, R5=OCH3 18: R1=Cl, R=R2=R3=R4=H, R5= OCH3 20: R1= OCH3, R=R2=R3=R4=H, R5= OCH3 22: R1=Cl, R=R2=R3=H, R4=R5= OCH3 24: R1=OCH3, R=R2=R3=H, R4=R5= OCH3

Scheme-1: Synthesis of 2-Amino-4,6-diarylpyrimidines derivatives (1-24).

Biological activity The synthesized substituted 2-Amino-4,6-diarylpyrimidines (1-24) have been subjected to in vitro antimicrobial activity against various plant and human pathogenic bacteria and fungi. Antimicrobial activity was carried out against gram positive coccus Staphylococcus aureus, Micrococcus luteus, gram positive rod Bacillus megatherium and gram negative rod Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae yeast fungus and Aspergillus niger, Penicillium notatum soil fungi were used for microbial activity. The results are summarized in the Table 1. It can be concluded from the observation that these substituted 2-Amino-4,6– diarylpyrimidines have moderate to high antimicrobial and antifungal activity. Moderate activity was observed when all these synthesized pyrimidines tested against Staphylococcus aureus, Bacillus megatherium, Candida albicans, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Aspergillus niger. All compounds possess wide range of activity against Micrococcus luteus and Penicillium notatum. In conclusion, we have synthesized a systematically substituted series of substituted 2-Amino-4,6– diarylpyrimidines derivatives for structure-activity relationship studies. These substituted derivatives are very stable compounds, which renders them beneficial substances for biological or pharmacological trials.

SOME 2-AMINO-4,6-SUBSTITUTED-DIARYLPYRIMIDINES

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Vandana Sharma and K. V. Sharma

Vol.4, No.1 (2011), 17-23 Table-1: Antimicrobial activity of substituted 2-amino-4,6窶電iarylpyrimidines

Compound No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A

B

C

Culture D

E

F

G

H

8 -

8 5 7 5 8 8 8 9 8 -

4 -

4 4 4 4 4

4 -

4 4 4 4

4 5 5 5 -

4 4 4 5 5 8 8 10 5 5

(Diameter of inhibition zone measured in mm, paper disc 5 mm, inhibition zone measured excluding paper disc diameter) A= Staphylococcus aureus; B= Micrococcus luteus; C= Bacillus megatherium; D= Pseudomonas aeruginosa; E=Candida albicans; F= Saccharomyces cerevisiae; G= Aspergillus niger; H= Penicillium notatum.

ACKNOWLEDGEMENTS Authors are thankful to School of Studies in Chemistry, Vikram University, Ujjain (India), RSIC, CDRI, Lucknow (India), Mahakal Institute of Pharmaceutical Studies, Ujjain (India) for providing spectral and analytical data.

REFERENCES 1. M. Gracia-Valverde and T. Torroba, Molecules, 10, 318 (2005). 2. C. O. Kappe, Tetrahedron, 49, 6937 (1993). 3. M. Kidwai, S. Saxena, S. Rastogi and R. Venkataramanan, Current Medicinal Chemistry- AntiInfective Agents, 2(4), 269 (2003). 4. L. C. Heda, R. Sharma, C. Pareek and P. B. Chaudhari, E-J. Chem., 6(3), 770 (2009). 5. L. Colombeau, K. Teste, A. Hadj-Bouazza, V. Chaleix, R. Zerrouki, M. Kraemer and O. S. Catherine, Nucleosides, Nucleotides and Nucleic Acids, 27(2),110 (2008). 6. M. Watanabe, H. Koike, T. Ishiba, T. Okada, S. Seo and K. Hirai, Bioorg. Med. Chem., 5(2), 437 (1997). 7. D. Bystryakova, O. A. Burova, G. M. Chelysheva, G. V. Zhilinkova, N. M. Smirnova and T. S. Safonova, Pharmaceutical Chemistry Journal, 25(12), 874 (1991).

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Vol.4, No.1 (2011), 17-23

8. R. F. Schinazi, J. Arbiser, J. J. Lee, T. I. Kalman, W. H. Prusoff, J. Med. Chem., 29(7), 1293 (1986). 9. P. J. James, F. Z. Sigmund, H. M. Lawrence, T. H. Maire, J. Med. Chem., 13(6), 1170 (1970). 10. A. V. Kadushkin, I. N. Nesterova, T. V. Golovko, I. S. Nikolaeva, T. V. Pushkina, A. N. Fomina, A. S. Sokolova, V. A. Chernov and V. G. Granik, Pharmaceutical Chemistry Journal, 24(12), 875 (1990). 11. A. Nikitenko, Y. Raifeld, B. Mitsner and H. Newman, Bioorg. Med. Chem. Let., 15(2), 427 (2005). 12. V. J. Ram, Indian J. Chem., 30B, 962 (1991). 13. G. N. Pershin, L. I. Sherbakova, T. N. Jykova and V. N. Sokhova, Farmakol Topsikol., 35, 466 (1972). 14. V. K. Ahluwalia, R. Agarwal, S. Bala and R. Chandra, Indian J. Chem., 28B, 1060 (1989). 15. V. K. Ahluwalia, L. Nayal and S. Bala, Indian J. Chem., 27B, 193 (1988). [RJC-686/2010]

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SOME 2-AMINO-4,6-SUBSTITUTED-DIARYLPYRIMIDINES

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Vandana Sharma and K. V. Sharma

Vol.4, No.1 (2011), 24-28 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF 4THIAZOLIDINONE CONTAINING BENZOTHIAZOLYL MOIETY *,1

T.M.Bhagat*,1, D.K.Swamy2 ,S.G.Badne3 and S.V.Kuberkar4

P.G. Department of Chemistry, G. S. Gawande College, Umarkhed. (M.S) India. 2 Department of Chemistry, P.N.College, Nanded. (M.S) India. 3 P.G. Department of Chemistry, Shri Shivaji College, Akola. (M.S) India. 4 P.G.Chemistry Research Center,Department of Chemistry, Yeshwant College, Nanded. E-mail: bhagat_tm@rediffmail.com ABSTRACT 4-Thiazolidinone has been prepared by the reaction of various substituted Schiff bases (4a-g) with thioglycolic acid. The intermediate schiff bases were synthesized by the condensation of substituted 2-amino benzothiazole with Ovaniline, P-vaniline, salicylialdehyde and N, N-dimethylaminobenzaldehyde. The starting compound substituted 2aminobenzothiazole was prepared from P-toludine and phenetidine. The structure of compounds has been confirmed by elemental and spectral analysis. The antibacterial activities of the synthesized compounds have been screened against Escherichia Coli, Bacillus subtilis, Erwinia Cartovara, and Xanthomonas Citri. Key words: Benzothiazole, Schiff-bases, 4-Thiazolidinone, Antibacterial activity. Š 2011 RASĀYAN. All rights reserved.

INTRODUCTION A survey of literature reveals that large work has been carried out on the synthesis of 4-thiazolidinone and known to exhibits various biological activities as antitubercular1, antiallergic2. Schiff-bases give good antibacterial activity and pharmacological application3. 4-Thiazolidinone ring are reported to possess various biological activities, as antimicrobial, anti-inflammatory, antiviral, antiparasitic and antituberculosis4-18. These Schiff-bases can be prepared by the acid catalysed reaction of amine and aldehyde or ketone which shows good fungicidal acivity19. 4-Thiazolidinone give good pharmacological properties20 are known to exhibit antitubercular21, antibacterial22, anticonvulsant23, antifungal activity24. Large work has been carried out on 4-thiazolidinone but very less information is available about 4-thiazolidinone bearing substituted benzothiazolyl moiety. The starting compound substituted 2-hydrazino benzothizole (1) have been synthesized from substituted amine25. Substituted 2-hydrazino benzothiazole were condensed with various aldehyde to yield Schiffbases (3a-g). The Schiff-bases were further reacted with thioglycolic acid to yield 4-thiazolidinone derivatives (4a-g) Scheme-1.

EXPERIMENTAL All the melting points were determined in open capillary tube and may be uncorrected. The purity of compound was checked by TLC on silica gel coated glass plate. Infra-red spectra were monitored in KBr palates on Bomen 104 FT infra-red spectrophotometer. H1 NMR spectra were obtained on a Gemani 200 Mz spectrometer with tetra methyl silane as an internal standard. Mass spectra were recorded on FTVG7070H mass spectrometer using the EI technique at 70ev. Elemental analysis was performed on a Heraeus CHN-O rapid analyzer. Synthesis of 4-bromo 2-substituted phenyl 6-methyl/ethoxy benzothiazolyl hydrazone (3a-g): The mixture of substituted hydrazino benzothiazole (0.01) was dissolved in ethanol (50ml) and aryl aldehyde (0.01M) was refluxed on water bath for two hours. The reaction mixture was cooled and solid obtained was filtered at pump, washed with ethanol and recrystalised from hot benzene.

4-Thiazolidinone Containing Benzothiazolyl Moiety

T.M.Bhagat et al.

Vol.4, No.1 (2011), 24-28

3a. : Yield: 2.74 gm (70%), M. P. : 175 oC, IR(KBr) : 3160 (N-N) stretch), 1584 (C= N Stretch), 1290 (C-N Stretch), Mass (m/e) : 391 (M+., 30%) and base peak at 55. [Found : C: 47.90 %, H : 10.48%, N : 3.30 %, C16H14BrN3O2S required : C: 48.07%, H : 10.71%, N : 3.57 %.] 3b. : Yield : 3.05 gm (78%), M. P. : 265oC. IR (KBr) : 3200 cm-1 (-OH Stretch), 3167 cm-1 (N-H Stretch), [Found : C: 48.62%; H : 10.50%; N : 3.42%, C16H14BrN3O2S required : C: 48.97%; H : 10.71%; N : 3.57% 3c. : Yield : 2.6 gm (72%), M. P. : 215 oC. IR (KBr):3180 cm-1 (-OH Stretch), 3174 cm-1 (N-N Stretch). [Found : C : 49.44%; H : 3.20%; N : 11.34%, C15H12BrN3OS required : C : 49.72%; H : 3.31%; N : 11.60%]. 3d. : Yield : 2.68 gm (68%), M. P. : 233 oC. I.R. (KBr) : 3389 (N-H stretching) 3053 (= C-H stretch in aromatic ring), 1541 (C=N stretch), 1290 (C-N stretch), [Found : C : 57.32%; H : 3.40% ; N : 10.28%, C19H14BrN3S required C : 57.57%; H : 3.53%; N : 10.60%] 3e. Yield : 3.0 gm (71%), M. P. 245 oC, IR (KBr):3423 cm-1 (O-H) stretching), 3209 cm-1 (N-H stretching), [Found : C : 48.12%; H : 3.62%; N : 9.58%, C17H16BrN3OS required : C : 48.34%; H : 3.79%; N : 9.95%] 3f. Yield : 3.2 gm (75%), M. P. : 214 oC, IR (KBr) : 3448 cm-1 (O-H) stretching), 3200 cm-1 (N-H stretching), [Found : C : 48.00%; H : 3.50%; N : 9.62%, C17H16BrN3O3S required : C : 48.34%; H : 3.79%; N : 9.95%] 3g. : Yield :- 2.6 gm (62%), M. P. :- 138 oC., IR (KBr) : 3302 (N-H stretching) [Found : C : 51.32%; H : 3.62%; N : 12.96%, C18H19BrN4OS required C: 51.55%; H : 3.81%; N : 13.36% ] Synthesis of 2-substituted phenyl 3-substituted benzothiazolyl 4-thiazolidinone (4a-g) : A mixture of hydrazone (Schiff-bases, 3a-g) (0.0025M), DMF (15 ml) and thioglycolic acid (0.005) was taken in round bottom flask. Small amount of fused ZnCl2 (200 mg) was added in reaction mixture. The contents of round bottom flask refluxed for five hours. Cooled and poured on crushed ice. Thus the product obtained was filtered, washed with water and recrystalised from DMF. 4a. : Yield : 0.77 gm (66%), M. P. : 158 oC, I.R. (KBr) : 3400 cm-1 (O-H stretching), 3163 cm-1 (N-H stretching), 1740 cm-1 (C=O) stretching); NMR : δ 2.2 (s, 2H, -CO-CH2), δ 2.5 (s, 3H, Ar- CH3) , δ 3.8 (s, 3H, O-CH3), δ 6.8 (s, 1H, -OH), δ 7.0 (s, 1H, -CH), δ 7.2- 7.6 (m, 3H, Ar-H), δ 8.4 (s, 1H, N-H), δ 9.5 (s, 1H, enolic O-H), Mass : 465(M+) and base peak at 244. [Found : C : 45.96%; H : 3.14%; N : 8.82%, C18H16BrN3O3S2 required : C : 46.35%; H : 3.43%; N : 9.01%. ] 4b. : Yield : 1.0 gm (86%), M. P. : 240 oC, I.R. (KBr) : 3400 cm-1 (O-H stretching), 3151 cm-1 (N-H stretching), 1716 cm-1 (C=O) stretching). [Found : C : 45.86%; H : 2.98%; N : 8.68%, C18H16BrN3O3S2 required : C : 46.35%; H : 3.43%; N : 9.01%. ] 4c. : Yield : 0.72 gm (67%), M. P. : 230 oC, I.R. (KBr) : 3300-3100 cm-1 (broad) due to –OH and N-H stretching, 1701 (C=O stretching). [Found : C : 46.23%; H : 2.94%; N : 9.04%, C17H14BrN3O2S2 required : C : 46.78%; H : 3.21%; N : 9.36%. ] 4d. : Yield : 0.85 gm (73%), M. P.: 94 oC, I.R. (KBr) : 3211 cm-1 due to N-H stretching, 1716 cm-1 (C=O stretching). [Found : C: 46.23%; H : 2.94%; N : 9.04%, C21H16BrN3OS2 required : C: 46.78%; H : 3.21%; N : 9.36%. ] 4e. : Yield : 0.89 (72%), M. P. : 260 oC, I. R. (KBr) : 3400 cm-1 broad (O-H stretching), 3103 cm-1 (N-H stretching), 1716 cm-1 (C=O stretching). [Found : C : 45.38%; H : 3.14%; N : 8.13%, C19H18BrN3O4S2 required : C : 45.96%; H : 3.62% N : 8.46%. ] 4f. : Yield : 0.87 gm (70%), M. P. : 290 oC, I. R. (KBr) : 3448 cm-1 broad (O-H stretching), 3277 cm-1 (N-H stretching), 1734 cm-1 (C=O stretching). [Found : C : 45.52%; H : 3.41%; N : 8.19%, C19H18BrN3O4S2 required : C : 45.96%; H : 3.62%; N : 8.46%. ] 4g. : Yield : 0.94 (77%), M. P. : 276 oC, I. R. (KBR) : 3178 cm-1 (N-H stretching), 1710 cm-1 (C=O stretching), Mass : 493 (M+. 7%). [Found : C : 48.22%; H : 3.965%; N : 11.04%, C20H21BrN4O2S2 required : C : 48.68%; H : 4.25%; N : 11.35%. ]

4-Thiazolidinone Containing Benzothiazolyl Moiety

25

T.M.Bhagat et al.

Vol.4, No.1 (2011), 24-28 O

R

S N H

N H

+

2

H

C

N B r

Ar

2

1

R

S N H

N

CH

Ar

N 3a-g

B r

DMF, ZnCl

2

5 Hrs. SHCH R

(Fused)

COOH

2

S N H

N

HC

Ar

N S

O

B r

4a-g R

S N

B r

Comp.

4a

-CH 3

.

4e HO

4b

-CH 3

N

HC

R

Ar

OC 2 H 5

.

OCH 3

.

HO

OH

4f

OC 2 H 5

.

-CH 3

OCH 3

.

4g

OC 2 H 5

-CH 3

.

CH 3 N CH 3

HO 4d

OCH 3

OH

OCH 3 4c

Ar S

O

Comp.

Ar

R

N H

.

Scheme-1

RESULTS AND DISCUSSION Structures of the compounds synthesized have been confirmed by elemental analysis, IR, 1HNMR and mass spectra. I.R. Spectrum of compound (4a) in (KBr) shows absorption band 3163 cm-l due N-H Stretching and at 1697 cm-1 to five membered cyclic amido C=O Stretching PMR Spectrum of compound (4a) in (dmso d6) shows one singlet δ 2.3 due to –COCH2- δ 2.5 (s) due to Ar-CH3 δ 3.8 due to OCH3, δ 6.7 due to –OH, δ 7.0 due to -CH-, δ 7.2-7.6 (m) due to Ar-H and δ 9.5 due to -NH. Mass spectrum of the same compound (4a) shows peak at 465 (M+.) which corresponds to its molecular weight. 4-Thiazolidinone Containing Benzothiazolyl Moiety

26

T.M.Bhagat et al.

Vol.4, No.1 (2011), 24-28

Similarly I.R. spectra of compounds (4b-4g) exhibit bands in the region 3100-3400 cm-1 and 1600-1800 cm-1 due to N-H stretching and C=O stretching respectively. Mass spectrum of the compound (4g) shows mass peak at 493 (M+) which corresponds to its molecular weight. The PMR spectrum exhibits two singlet peaks for –NH proton (δ 8.4 and δ 9.5) which indicates that, compound (4a) may exists in tautomeric form. Evaluation of antibacterial activity The compound (4a-g) were tested for their antimicrobial activity by cup plate agar diffusion method against E. Coli, Erwinia carotovara, Bacillus subtilis and Xanthomonas citri species using ampicilin, streptomycin penicillin as a standard compound (positive control) for comparison. The antibacterial screening data of the compound are presented in Table-1. Table-1: Antibacterial Activity of Newly Synthesized Compounds. S. No.

Comp.

R

E.coli

1

4a HO

2

Antibacterial activity (zone of inhibition in mm) Erwinia Bacillus cartovara subtilis

Xanthomonas citri

06

07

06

10

14

07

08

12

13

10

09

14

OCH3 OH

4b

OCH3

3

4c HO

4

4d

12

10

08

07

5

4e

10

06

06

08

08

06

00

06

08

06

08

08

16 20 15 00

18 18 20 00

17 22 18 00

15 18 17 00

HO

6

OCH3

4f

OH OCH3

7

CH3

4g

N CH3

Ampicillin Streptomycin Penicillin Control

4-Thiazolidinone Containing Benzothiazolyl Moiety

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Vol.4, No.1 (2011), 24-28

CONCLUSION From the results it is clear that the compounds tested showed variable toxicity against different bacteria. This variation in toxicity can be attributed to different structures and functional groups attached to the basic nucleus. It is also clear from the results presented in table-2 that phenolic –OH and aryl substituted –OCH3 groups in the basic nucleus, the antibacterial activity was increased. This was observed with bacteria that the subsequent addition of phenolic (-OH) and aryl substituted –OCH3 groups antibacterial activity was enhanced.

ACKNOWLEDGEMENTS The authors are grateful to principal, Science College, Nanded for providing laboratory facilities, Director, Indian Institute of Chemical Technology, Hydrabad for providing spectra and Dr. V. N. Kadam, Principal, G. S. Gawande College, Umarkhed, for encouragement.

REFERANCES 1. W. Kasel, M. Dolezal, E. Sidoova, Z. Odlerova and Drasata, J. Chem. Abstr., 110, 128063e (1989). 2. U. Ronssel and Jpn Kokai Tokkyo, Chem. Abst., 106, 156494G (1987). 3. D.U.Warad, C.D. Satish, V.H. Kulkarni and C.S. Bajgur, Indian J. Chem., 39a, 415 (2000). 4. G. Capan, N. Ulusoy, N. Ergenc, M. Kiraz, Monatshefte fur Chemie., 130, 1399 (1999). 5. M.G. Vigorita, R. Ottana, F. Monforte, R. Maccari, A. Trivato, M.T. Monforte, M.F. Zaviano Bio. Org, Med. Chem. Lett., 11, 2791 (2001). 6. R.K. Rawal, Y.S. Prabhakar, S.B. Katti, E. Clercq, Bio.Org. Med. Chem., 13, 6771 (2005). 7. K. Babaoglu, M.A. Page, V.C. Jones, M.R. McNeil, C. Dong, J.H. Naismith, R.E. Lee, Bio. Org. Med. Chem. Lett., 13, 3227 (2003). 8. A.J. Alves, S.V. Ramos, M.J. Silva, P. Fulcrand, A.M. Artis, A.M. Quero, Rev. Farm. Bioquı´m. Univ. Sao Paulo., 34, 77 (1998). 9. A. J. Alves, A.C.L. Leite, D.P. Santana, T.M. Beltrao, M.R.D. Coelho, IL Farmaco 48, 1167 (1993). 10. N. Bharti, K. Husain, M.T.G. Garza, D.E.C. Vega, J.C. Garza, B.D.M. Cardenas, F. Naqvi, Bio.Org. Med. Chem. Lett., 12, 3475 (2002). 11. D. Y. Zhang, H. Xiang, Xu. Y-G., Hua. W.Y., Yaoxue Xuebao, 41 (9), 825, (2006). 12. D. S. Nair and A. C. Shah, Indian Journal of Heterocyclic Chemistry , 16 (3), 231, (2007). 13. P. Mishra, T. Lukose and S. Kashaw, Indian Journal of Pharmaceutical Science., 69 (5),665, (2007). 14. W. Cunico, C.R.B.Gomes, M.de.L.G. Ferrira, L. R. Capri, M. Soares and S.M.S.V. Wardell, Tetrahedron Letters. ,48 (35) ,6217, (2007). 15. W.Cunico, C.R.B. Gomes, M.de.L.G. Ferrira, L. R. Capri, M. R. L. Santos, N. Boachat and L. M. U. Mayer, Letters in Organic Chemistry., 4 (7),505, (2007). 16. S. D. Srivastava, D. K. Shukla, J. Chem. Soc., 85, 306(2008) 17. I. Argyropoulou, A. Geronikaki, P. Vicini, F. Zani, Arkivoc, ,(vi), 89(2009) 18. P. Venkatesh, S. N. Pandeya, Inter. J. Chem. Tech. Res., 1 (4), 1354(2009) 19. B. Dash, P.K. Mahapatra, D.Panda and J.M. Patnaik, Indian Chem. Soc.,61, 1061 (1984). 20. R. Yadav, S. Srivastava, S. K. Srivastava and S.D. Srivastava, Chemistry an Indian Journal., 1, 95 (2003). 21. P.S. Desai and K.S. Desai, J. Indian Chem. Soc., 71, 155 (1994). 22. M. Fadayon, V.D. Kulkarni and S.H, Pakdamana, Asian J. Chem., 5(2), 282 (1993). 23. S.K. Srivastava, S. Srivastava and S.D. Srivastava, Indian J. Chem., 38B, 183 (1999). 24. J.J. Bhatt, B.R. Shah, P.B. Trivedi, N.K. Undavia and N.C. Desai, Indian J. Chem.,33B, 189 (1994). 25. K.G. Ojha, H. Tahiliani and N. Jaisinghani, Chemistry an Indian Journal., 1, 171 (2003). [RJC-698/2010] 4-Thiazolidinone Containing Benzothiazolyl Moiety

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Vol.4, No.1 (2011), 29-35 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND ANTI-MICROBIAL ACTIVITY OF NOVEL ACETYLATED MALTOSYL CARBAMIDES, BENZOTHIAZOLYL CARBAMIDES AND CARBAMATES Sanjay. P. Mote and Shirish. P. Deshmukh* P. G. Department of Chemistry, Shri Shivaji College, Akola-444 001(M.S.), India. E-mail : sanjay.mote2007@gmail.com ABSTRACT As part of ongoing studies in developing new antimicrobials, a class of structurally novel acetylated maltosyl carbamides, benzothiazolyl carbamides and carbamates were synthesized by the interaction of Hepta-O-acetyl-β-Dmaltosyl isocyanate and aryl amines, substituted benzothiazoles and various alcohols. These molecules were evaluated in vitro for their antimicrobial activities. Most of the compounds exhibited significant antibacterial and moderate antifungal activity against all the tested strains. Key words: Maltosyl isocyanate, Aryl amines, Substituted benzothiazoles, Carbamates, Antimicrobial activity. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Compounds containing the urea functionality are biological interest as antimycobacterial1 and as inhibitors of HIV protease2. A series of glucosyl ureas have been shown to be α-glucosidase inhibitors3 and N-acyl-N’-β-D-glucopyranosyl ureas exhibit strong inhibition against glycogen phosphorylase4 and can be used as antidibetic agents5. N-maltosylated compounds and their derivatives have wide applications in industries and medicinal chemistry6. Carbamides and theirderivatives shows strong antibacterial activity and are versatile reagent in organic synthesis7. Benzothiazoles are bicyclic ring system with multiple applications. They have diverse chemical reactivity and broad spectrum of biological activity including antibacterial and antifungal properties8-10, 2-aminobenzothiazoles shows antitumor11 and antimalarial activity12. Bis substituted amidino benzothiazoles act as potential anti HIV agents13. Also schiffbase of benzothiazoles possess antitubercular, anticancer, antitumor, antipyretic and sterase inhibitory activity14,15. Our analogue based design encompasses the synthesis of new 1-hepta-O-acetyl-β-D-maltosyl-3-aryl carbamides 2a-g, 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-substituted benzothiazolyl carbamides 3a-g and N-hepta-O-acetyl-β-D-maltosyl-O-alkyl carbamates 4a-e, to be tested for their in vitro antimicrobial properties against Gram positive and Gram negative bacteria and fungi.

EXPERIMENTAL Melting points were taken in open capillary tube on Mac digital melting point apparatus and were uncorrected. IR spectra were recorded on a Perkin-Elmer Spectum RXI FTIR Spectrometer in solid phase KBr. The 1H NMR spectra were recorded in CDCl3 at 300 MHz on a Bruker DRX- 300 NMR Spectrometer. The Mass spectra were recorded on a Jeol- 102 Mass Spectrometer. Optical rotations [α]30D were measured on Equip-Tronics EQ-800 Digital Polarimeter at 300C in Chloroform. Thin Layer Chromatography [TLC] was performed in E. Merck precoated silica gel plates and detected by exposure under short UV light. The compounds described in this paper were first time synthesized by the multistep reaction protocol. Hepta-O-acetyl-β-D-maltosyl isocyanate (1), synthesized by using hepta-O-acetyl-α-Dmaltosyl bromide was undergone substitution in the presence of lead cyanate in refluxing xylene16-18. The 1-hepta-O-acetyl-β-D-maltosyl-3-aryl carbamides (2), 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-substituted benzothiazolyl carbamides (3) and N-hepta-O-acetyl-β-D-maltosyl-O-alkyl carbamates (4) were obtained

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by refluxing (1) with appropriate aryl amines, 2-aminobenzothiazole / substituted benzothiazoles and alcohols respectively(Scheme- 1). All the new compounds 1, 2a-g, 3a-g and 4a-e were characterized by m.p., elemental analysis and spectroscopic data (IR, 1H NMR and MS). The spectral data and elemental analysis of the new compounds reported in this correlate with the proposed structure. The hepta-O-acetyl-α-D-maltosyl bromide was prepared according to the literature19-21. Synthesis of Hepta-O-acetyl-β-D-maltosyl isocyanate (1) To a solution of hepta-O-acetyl-α-D-maltosyl bromide (3.4g, 0.005mol) in xylene (20ml) and lead cyanate (1.4g, 0.005mol) was added and the resulting mixture was refluxed for 3hr with constant stirring. After the removal of lead bromide the xylene filtrate was evaporated under reduced pressure then triturated with petroleum ether (60-800C) to afford milky white solid. It was purified by dissolving it in chloroform and reprecipitating with petroleum ether (60-800C) to afford a white solid product (1). Yield (71.34%), m.p. 168-170°C. Synthesis of 1-hepta-O-acetyl-β-D-maltosyl-3-aryl carbamides (2a-g) A (0.005mol) solution of aryl amines in benzene was added to a (0.005mol) Hepta-O-acetyl-β-D-maltosyl isocyanate (1) in 15ml benzene and the reaction mixture was refluxed over boiling water bath for 3hr. After refluxing, the solvent was distilled off and the sticky residue obtained was triturated with petroleum ether (60-800C) to afford a white solid (2a-g). The products were purified by recrystalization from ethanol-water (1:3). Synthesis of 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-aryl substituted benzothiazolyl carbamides (3a-g) A (0.005mol) solution of 2-amino benzothiazole / substituted benzothiazoles in 5ml benzene was added to a (0.005mol) Hepta-O-acetyl-β-D-maltosyl isocyanate (1) in 15ml benzene and the reaction mixture was refluxed over boiling water bath for 4hr. After refluxing, the solvent was distilled off and the sticky residue obtained was triturated with petroleum ether (60-800C) to afford a white solid (3a-g). The products were purified by recrystalization from ethanol-water (1:3). Synthesis of N-hepta-O-acetyl-β-D-maltosyl-O-alkyl carbamates (4a-e) A (0.005mol) hepta-O-acetyl-β-D-maltosyl isocyanate (1) was added to various alcohols (25ml) and the reaction mixture was refluxed 3-4hr. After refluxing it was allowed to cool and poured into water with vigorous shaking. A white granular solid separated out (4a-e), products were crystallized from aqueous ethanol. Hepta-O-acetyl-β-D-maltosyl isocyanate (1) IR (KBr, cm-1): 2965 (Ali C-H), 2119 (N=C=O), 1755 (C=O), 1440(C=N), 1228 (C-O), 900 and1038 (maltose unit). 1H NMR (CDCl3) δ: 3.37-5.44 (m, 14H, maltose unit), 1.63-2.18 (m, 21H, 7COCH3). Mass m/z: 661, 619, 331, (Calcd for C27H35O18N; 661.09). Anal. Calcd for C27H35O18N: C, 49.01; H, 5.29; N, 2.11. Found: C, 48.70; H, 5.28; N, 2.15. 1-hepta-O-acetyl-β-D-maltosyl-3-phenyl carbamide (2a) IR (KBr, cm-1): 3465 (N-H), 1752 (C=O), 1376 (C-N), 1232 (C-O), 901 and1039 (maltose unit). 1H NMR (CDCl3) δ: 7.16–7.27 (m, 5H, Ar-H), 6.68–6.70 (s, 2H, NH), 3.67-5.59 (m, 14H, maltose unit), 2.03-2.23 (m, 21H, 7COCH3). Mass m/z: 754, 694, 652 (Calcd for C33H42O18N2; 754.15). Anal. Calcd for C33H42O18 N2: C, 52.51; H, 5.57; N, 3.71. Found: C, 52.07; H, 5.54; N, 3.73. 1-hepta-O-acetyl-β-D-maltosyl-3-o-Cl-phenyl carbamide (2b) IR (KBr, cm-1): 3489 (N-H), 1751 (C=O), 1376 (C-N), 1238 (C-O), 940 and1041 (maltose unit). 1H NMR (CDCl3) δ: 7.19–7.27 (m, 4H, Ar-H), 6.78 (s, 2H, NH), 3.65-5.59 (m, 14H, maltose unit), 2.03-2.23 (m, 21H, 7COCH3). Mass m/z: 787, 753 (Calcd for C33H41O18N2Cl; 788.16). Anal. Calcd for C33H41O18N2Cl: C, 50.25; H, 5.20; N, 3.55. Found: C, 50.23; H, 5.19; N, 3.56. 1-hepta-O-acetyl-β-D-maltosyl-3-m-Cl-phenyl carbamide (2c) IR (KBr, cm-1): 3488 (N-H), 1750 (C=O), 1376 (C-N), 1236 (C-O), 940 and1041 (maltose unit). 1H NMR (CDCl3) δ: 7.20–7.27 (m, 4H, Ar-H), 6.65 (s, 2H, NH), 3.63-5.59 (m, 14H, maltose unit), 2.01-2.23 (m, MALTOSYL CARBAMIDES AND CARBAMATES

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21H, 7COCH3). Mass m/z: 787, 753 (Calcd for C33H41O18N2Cl; 788.60). Anal. Calcd for C33H41O18N2Cl: C, 50.25; H, 5.20; N, 3.55. Found: C, 50.21; H, 5.17; N, 3.56. 1-hepta-O-acetyl-β-D-maltosyl-3-p-Cl-phenyl carbamide (2d) IR (KBr, cm-1): 3489 (N-H), 1750 (C=O), 1375 (C-N), 1236 (C-O), 941 and1041 (maltose unit). 1H NMR (CDCl3) δ: 7.09–7.16 (m, 4H, Ar-H), 6.43 (s, 2H, NH), 3.63-5.60 (m, 14H, maltose unit), 2.00-2.23 (m, 21H, 7COCH3). Mass m/z: 789, 753 (Calcd for C33H41O18N2Cl; 788.60). Anal. Calcd for C33H41O18N2Cl: C, 50.25; H, 5.20; N, 3.55. Found: C, 50.21; H, 5.19; N, 3.55. 1-hepta-O-acetyl-β-D-maltosyl-3-o-tolyl carbamide (2e) IR (KBr, cm-1): 3476 (N-H), 1752 (C=O), 1376 (C-N), 1236 (C-O), 900 and1039 (maltose unit). 1H NMR (CDCl3) δ: 7.06–7.26 (m, 4H, Ar-H), 5.52 (s, 2H, NH), 3.76-5.48 (m, 14H, maltose unit), 2.03-2.25 (m, 21H, 7COCH3). Mass m/z: 768, 708 (Calcd for C34H44O18N2; 768.16). Anal. Calcd for C34H44O18N2: C, 53.12; H, 5.72; N, 3.64. Found: C, 53.12; H, 5.71; N, 3.67. 1-hepta-O-acetyl-β-D-maltosyl-3-m-tolyl carbamide (2f) IR (KBr, cm-1): 3476 (N-H), 1751 (C=O), 1378 (C-N), 1236 (C-O), 901 and1039 (maltose unit). 1H NMR (CDCl3) δ: 7.06–7.27 (m, 4H, Ar-H), 5.65 (s, 2H, NH), 3.56-5.48 (m, 14H, maltose unit), 2.02-2.26 (m, 21H, 7COCH3). Mass m/z: 768, 708 (Calcd for C34H44O18N2; 768.16). Anal. Calcd for C34H44O18N2: C, 53.12; H, 5.72; N, 3.64. Found: C, 53.10; H, 5.70; N, 3.65. 1-hepta-O-acetyl-β-D-maltosyl-3-p-tolyl carbamide (2g) IR (KBr, cm-1): 3478 (N-H), 1752 (C=O), 1376 (C-N), 1238 (C-O), 901 and1039 (maltose unit). 1H NMR (CDCl3) δ: 7.00–7.18 (m, 4H, Ar-H), 5.64 (s, 2H, NH), 3.76-5.53 (m, 14H, maltose unit), 2.03-2.25 (m, 21H, 7COCH3). Mass m/z: 768, 708 (Calcd for C34H44O18N2; 768.16). Anal. Calcd for C34H44O18N2: C, 53.12; H, 5.72; N, 3.64. Found: C, 53.08; H, 5.67; N, 3.67. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)- benzothiazolyl carbamide (3a) IR (KBr, cm-1): 3350 (N-H), 1752 (C=O), 1535 (C=N), 1374 (C-N), 1233 (C-O), 900 and1039 (maltose unit), 769 (C-S). 1H NMR (CDCl3) δ: 7.27–7.69 (m, 4H, Ar-H), 6.25 (s, 2H, NH), 3.76-5.64 (m, 14H, maltose unit), 1.90-2.23 (m, 21H, 7COCH3). Mass m/z: 812, 619 (Calcd for C34H41O18N3S; 811.22). Anal. Calcd for C34H41O18N3S: C, 50.30, H, 5.05; N, 5.17; S, 3.94. Found: C, 50.29; H, 4.98; N, 5.14; S, 3.63. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-4-Cl-benzothiazolyl carbamide (3b) IR (KBr, cm-1): 3471 (N-H), 1751 (C=O), 1599 (C=N), 1376 (C-N), 1237 (C-O), 942 and1041 (maltose unit), 769 (C-S). 1H NMR (CDCl3) δ: 7.26–7.49 (m, 3H, Ar-H), 6.25 (s, 2H, NH), 3.74-5.62 (m, 14H, maltose unit), 2.02-2.25 (m, 21H, 7COCH3). Mass m/z: 845, 725 (Calcd for C34H40O18N3SCl; 845.67). Anal. Calcd for C34H40O18N3SCl: C, 47.28; H, 5.21; N, 5.09; S, 3.87. Found: C, 47.26; H, 5.20; N, 4.98; S, 3.84. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-5-Cl-benzothiazolyl carbamide (3c) IR (KBr, cm-1): 3471 (N-H), 1752 (C=O), 1589 (C=N), 1374 (C-N), 1236 (C-O), 942 and1042 (maltose unit), 769 (C-S). 1H NMR (CDCl3) δ: 7.27–7.50 (m, 3H, Ar-H), 6.22 (s, 2H, NH), 3.73-5.62 (m, 14H, maltose unit), 2.00-2.25 (m, 21H, 7COCH3). Mass m/z: 845, 725 (Calcd for C34H40O18N3SCl; 845.67). Anal. Calcd for C34H40O18N3SCl: C, 47.28; H, 5.21; N, 5.09; S, 3.87. Found: C, 47.20; H, 5.19; N, 4.98; S, 3.83. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-6-Cl-benzothiazolyl carbamide (3d) IR (KBr, cm-1): 3472 (N-H), 1750 (C=O), 1590 (C=N), 1376 (C-N), 1236 (C-O), 942 and1041 (maltose unit), 773 (C-S). 1H NMR (CDCl3) δ: 7.26–7.49 (m, 3H, Ar-H), 6.25 (s, 2H, NH), 3.74-5.62 (m, 14H, maltose unit), 2.02-2.25 (m, 21H, 7COCH3). Mass m/z: 845, 725 (Calcd for C34H40O18N3SCl; 845.67).

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Anal. Calcd for C34H40O18N3SCl: C, 47.28; H, 5.21; N, 5.09; S, 3.87. Found: C, 47.24; H, 5.19; N, 5.02; S, 3.86. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-4-methyl benzothiazolyl carbamide (3e) IR (KBr, cm-1): 3487 (N-H), 1749 (C=O), 1634 (C=N), 1375 (C-N), 1238 (C-O), 941 and1042 (maltose unit), 774 (C-S). 1H NMR (CDCl3) δ: 7.26–7.36 (m, 3H, Ar-H), 5.62 (s, 2H, NH), 3.97-5.59 (m, 14H, maltose unit), 2.37 (s, 3H, Ar-CH3), 2.03-2.23 (m, 21H, 7COCH3). Mass m/z: 825, 705 (Calcd for C35H43O18N3S; 825.23). Anal. Calcd for C35H43O18N3S: C, 50.90; H, 5.21; N, 5.09; S, 3.87. Found: C, 50.89; H, 5.20; N, 5.10; S, 3.86. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-5-methyl benzothiazolyl carbamide (3f) IR (KBr, cm-1): 3485 (N-H), 1750 (C=O), 1636 (C=N), 1376 (C-N), 1238 (C-O), 940 and1043 (maltose unit), 774 (C-S). 1H NMR (CDCl3) δ: 7.25–7.34 (m, 3H, Ar-H), 5.65 (s, 2H, NH), 3.87-5.60 (m, 14H, maltose unit), 2.36 (s, 3H, Ar-CH3), 2.00-2.23 (m, 21H, 7COCH3). Mass m/z: 825, 705 (Calcd for C35H43O18N3S; 825.23). Anal. Calcd for C35H43O18N3S: C, 50.90; H, 5.21; N, 5.09; S, 3.87. Found: C, 50.86; H, 5.18; N, 5.10; S, 3.85. 1-hepta-O-acetyl-β-D-maltosyl-3-(2)-6-methyl benzothiazolyl carbamide (3g) IR (KBr, cm-1): 3492 (N-H), 1750 (C=O), 1634 (C=N), 1375 (C-N), 1238 (C-O), 941 and1042 (maltose unit), 774 (C-S). 1H NMR (CDCl3) δ: 7.26–7.36 (m, 3H, Ar-H), 5.62 (s, 2H, NH), 3.97-5.59 (m, 14H, maltose unit), 2.37 (s, 3H, Ar-CH3), 2.03-2.23 (m, 21H, 7COCH3). Mass m/z: 825, 705 (Calcd for C35H43O18N3S; 825.23). Anal. Calcd for C35H43O18N3S: C, 50.90; H, 5.21; N, 5.09; S, 3.87. Found: C, 50.89; H, 5.20; N, 5.10; S, 3.86. N-hepta-O-acetyl-β-D-maltosyl-O-methyl carbamate (4a) IR (KBr, cm-1): 3487 (N-H), 1749 (C=O), 1375 (C-N), 1237 (C-O), 900 and1040 (maltose unit). 1H NMR (CDCl3) δ: 7.26 (s, 1H, NH), 3.96-5.68 (m, 14H, maltose unit), 3.29 (s, 3H, -CH3), 2.00-2.15 (m, 21H, 7COCH3). Mass m/z: 693, 619 (Calcd for C28H39O19N; 693.09). Anal. Calcd for C28H39O19N: C, 48.48; H, 5.62; N, 2.02. Found: C, 48.37; H, 5.59; N, 1.98. N-hepta-O-acetyl-β-D-maltosyl-O-ethyl carbamate (4b) IR (KBr, cm-1): 3487 (N-H), 1749 (C=O), 1375 (C-N), 1237 (C-O), 900 and1040 (maltose unit). 1H NMR (CDCl3) δ: 7.22 (s, 1H, NH), 3.96-5.68 (m, 14H, maltose unit), 2.00-2.15 (m, 21H, 7COCH3), 1.09 (t, 3H, -CH3). Mass m/z: 707 (Calcd for C29H41O19N; 707.10). Anal. Calcd for C29H41O19N: C, 49.22; H, 5.79; N, 1.98. Found: C, 49.20; H, 5.75; N, 1.97. N-hepta-O-acetyl-β-D-maltosyl-O-isopropyl carbamate (4c) IR (KBr, cm-1): 3487 (N-H), 1749 (C=O), 1375 (C-N), 1237 (C-O), 900 and1040 (maltose unit). 1H NMR (CDCl3) δ: 7.25 (s, 1H, NH), 3.96-5.68 (m, 14H, maltose unit), 2.00-2.15 (m, 21H, 7COCH3), 1.41 (d, 6H, -CH3). Mass m/z: 721 (Calcd for C30H43O19N; 721.11). Anal. Calcd for C30H43O19N: C, 49.93; H, 5.96; N, 1.94. Found: C, 49.89; H, 5.93; N, 1.95. N-hepta-O-acetyl-β-D-maltosyl-O-isoamyl carbamate (4d) IR (KBr, cm-1): 3487 (N-H), 1749 (C=O), 1375 (C-N), 1237 (C-O), 900 and1040 (maltose unit). 1H NMR (CDCl3) δ: 7.25 (s, 1H, NH), 3.96-5.68 (m, 14H, maltose unit), 1.92-2.10 (m, 21H, 7COCH3), 1.10 (d, 6H, -CH3). Mass m/z: 749 (Calcd for C32H47O19N; 749.13). Anal. Calcd for C32H47O19N: C, 51.26; H, 6.27; N, 1.86. Found: C, 50.96; H, 6.21; N, 1.90. N-hepta-O-acetyl-β-D-maltosyl-O-n-butyl carbamate (4e) IR (KBr, cm-1): 3483 (N-H), 1745 (C=O), 1367 (C-N), 1242 (C-O), 941 and1045 (maltose unit). 1H NMR (CDCl3) δ: 7.23 (s, 1H, NH), 3.42-5.43 (m, 14H, maltose unit), 3.02 (t, 2H, O-CH2), 1.94-2.10 (m, 21H, MALTOSYL CARBAMIDES AND CARBAMATES

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7COCH3), 1.30-1.59 (m, 4H, -CH2), 1.01 (t, 3H, -CH3). Mass m/z: 734, 683 (Calcd for C31H43O19N; 733.12). Anal. Calcd for C31H45O19N: C, 50.61; H, 6.12; N, 1.90. Found: C, 50.59; H, 6.09; N, 1.89. Antimicrobial activity The antimicrobial activity of newly synthesized compounds were tested in vitro against a panel of selected Gram positive, Gram negative bacteria and fungi are presented in Table-2 in comparison with those of the references drugs Gentamicin and Fluconazole respectively.The antimicrobial activity was evaluated against different bacterial and fungal strains such as E. coli (MTCC 1680), P. vulgaris (MTCC 1771), P. aeruginosa (MTCC 7191), S. aurues (MTCC 3160) and fungal strains A. niger (clinical isolated), C. albicans (clinical isolated) by using cup plate agar diffusion method. The compounds investigated were dissolved in DMSO [1mg/mL] and filled in 9mm wells in agar media. Inhibition zones read after incubation at 300C for 24 hr for bacterial strains and for fungal strains inhibition zones read after incubation at 350C for 48 hr. OAc O AcO AcO

OAc OAc O

OAc O

Pb(OCN)2

AcO OAc Br

OAc

AcO AcO

O

O

OAc O

N=C=O OAc Hepta-O-acetyl-β-D-maltosyl isocyanate (1) AcO

Lead cyanate

Hepta-O-acetyl-α-D-maltosyl bromide

OAc

(1)

O

A cO A cO

R -N H 2 A ryl am ines

O Ac O

O

OAc O

N H -C -N H -R

A cO OAc

T itle com pounds (2a-g) Where, R = a) Phenyl, b) o-Cl-Phenyl, c) m-Cl-Phenyl, d) p-Cl-phenyl, e) o-tolyl, f) m-tolyl, g) p-tolyl. Ac = COCH3 OAc N

(1)

R

H2N

O

AcO AcO

OAc N R

NH-C-NH

AcO

S

O

O

OAcO

OAc

Substituted benzothiazoles

S

Title compounds (3a-g)

Where, R = a) H, b) 4-Cl, c) 5-Cl, d) 6-Cl, e) 4-CH3, f) 5-CH3, g) 6-CH3. OAc

(1)

A cO A cO

R-O H

O OA c O

OAc O

O

N H -C -O -R

AcO O Ac

Alcohols

Title compounds (4a-e) Where, R = a) methyl, b) ethyl, c) iso-propyl, d) iso-amyl, e) n-butyl. Scheme-1 MALTOSYL CARBAMIDES AND CARBAMATES

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RESULTS AND DISCUSSION Antimicrobial activity Most of the synthesized copounds exhibited mild to moderate anti-microbial activity against the tested microorganisms. Compounds 2b, 2c, 2e, 3b and 3c were found to possess significant antibacterial and antifungal activity when compared to standard drug (Gentamicin and Fluconazol for antibacterial and antifungal respectively). The entire synthesized compounds exhibited mild to moderate activity are shown in Table -2. Table-1: Physical characterization data of the synthesized compounds. Compd 2a 2b 2c 2d 2e 2f 2g 3a 3b 3c 3d 3e 3f 3g 4a 4b 4c 4d 4e

m.p. 0C 113 145 128 118 115 140 136 190-192 186-188 160 153-154 175 183-184 170-173 150 164 170 175-174 153-152

Yield % 75.25 71.69 60.65 71.25 50.68 68.95 70.12 89.30 70.71 65.53 80.36 74.60 62.30 69.70 50.18 49.21 65.17 62.22 73.20

[α]D30 [c, in CHCl3] +180.01°(c, 0.968) +75.04°(c, 0.993) +120.05°(c, 0.932) +212.19°(c, 0.986) +105.88° (c, 0.986) +90.42° (c, 0.993) +150.50° (c,0.994) +99.75° (c, 0.978) + 45.32° (c, 0.987) +54.40° (c, 0.990) +65.70° (c, 0.993) +123.20° (c, 0.991) +141.51° (c, 0.982) -296.43° (c, 0.933) +60.32° (c, 0.667) -40.50° (c, 0.667) -70.35° (c, 0.668) +104.13° (c, 0.674) +64.62° (c, 0.667)

Rf, EtOA:Hexane, 1:1 0.79 0.81 0.83 0.72 0.86 0.82 0.76 0.78 0.90 0.93 0.87 0.79 0.78 0.82 0.65 0.63 0.71 0.77 0.80

Table-2: Anti-microbial activities of the synthesized compounds. (Invitro activity - zone of inhibition measured in mma) Compd E.coli 2a 2b 2c 2d 2e 2f 2g 3a 3b 3c 3d 3e 3f 3g 4a 4b

20 20 24 22 24 20 20 19 20 23 22 17 24 21 20 17

Bacteria P. aeruginosa P.vulgaris 18 19 20 16 21 15 19 13 17 21 16 12 15 13 16 16

MALTOSYL CARBAMIDES AND CARBAMATES

20 20 21 21 21 24 20 18 16 22 12 ------14 18 ---34

S.aureus ---23 20 20 17 23 23 16 19 23 20 24 18 16 14 14

A.niger 12 18 19 18 18 16 18 19 20 18 17 12 12 10 ------

Fungi C. albicans 10 16 17 15 11 17 16 17 18 19 18 17 19 16 10 09 S.P. Mote and S. P. Deshmukh

Vol.4, No.1 (2011), 29-35 4c 21 ---4d 17 ---4e 16 18 Gentamicin 24 20 Fluconazole ---------- No activity was observed. a Values are the average of three readings.

---22 18 23 ----

20 19 17 24 ----

---12 ------20

11 ---15 ----18

ACKNOLEDGEMENTS The authors are thankfull to SAIF, CDRI, Lucknow for providing spectral data. The authors are also thakfull to the Principal Dr. S. G. Bhadange for providing necessary facility. We thanks R. B. Ingle and D. L. Barate for performing antimicrobial studies.

REFERENCES 1. A.Scozzafava, A.Mastrolorenzo and C.T.Supuran, J. Enzyme Inhib., 16, 425 (2001) 2. D.P.Getman, M.L.Decrescenzo, R.M.Heintz, K.L.Reed, J.J.Tally, M.L.Bryant, M.Clare, K.A.Houseman, R.R.Marr, R.A.Muller, M.L.Vazauez, H.S.Shieh, W.C.Stallings and R.A.Stegeman, J. Med. Chem., 36, 288 (1993). 3. N.Tewari, V.K.Tiwari, R.C.Mishra, R.P.Tripathi, A.K.Shrivastava, R.Ahmad, R.Shrivastava and B.S.Shrivastava, Bioorg. Med. Chem., 11, 2911 (2003). 4. N.G.Oikonomakos, M.Kosmopoulou, S.E.Zographos, D.D.Leonidas, E.D.Chrysina, L.Samsak, V.Nagy, J.P.Praly, T.Docsa, B.Toth and P.Gergely, Eur. J. Biochem., 269, 168 (2002). 5. L.Somsak, V.Nagy, Z.Hadady, T.Docsa and P.Gergely, Curr. Pharma. Res., 9, 1177 (2003). 6. I.Goodman, Adv. Carbohydrate Chem. Biochem., Academic Press, New York, 13, 233 (1958). 7. L.H.Cao, C.I.Zhou, H.Y.Gao and Y.T.Liu, J. Chin. Chem. Soc., 48, 207 (2001). 8. M.Lacova, J.Chovaneova, O.Hyblova and S. Varkonda, Chem. Pap., 45, 411 (1991). 9. L.Ballell, R.A.Field, K.Duncan and R.Young, J. Antimicrob. Agents Chemother., 49, 2153 (2005). 10. V.P.Trivedi, N.K.Undavia and P.B.Trivedi, J. Indian Chem. Soc., 81, 506 (2004). 11. T.D.Bradshaw, M.S.Chua, H.L.Browne, V.Tarpan, E.A.Sausville and M.F.G.Stevens, Br. J. Cancer, 86, 1348 (2002). 12. S.Hout, N.Azas, A.Darque, M.Robin, C.Di Giorgio, M.Gasquet, J.Galy and Timan-David, Parasitology, 129, 525 (2004). 13. L.Racane, V.Tralic-Kulenovic, L.Fiseo-Jakie, D.W.Boykin and G.Karmiski-Zamola, Heterocycles, 55, 2085 (2001). 14. M.S.Shingare and D.B.Ingle, J. Indian Chem. Soc., 53, 1036 (1976). 15. B.Dash and M.Patra, Indian J. Chem., 19B, 894 (1980). 16. A.S.Dandale and S.P.Deshmukh, Indian J. Chem., 47B, 613 (2008). 17. A.S.Dandale, D.V.Mangte and S.P.Deshmukh, Carbohydr. Res., 342, 753 (2007). 18. E.Fischer, Chem. Ber., 49, 1377 (1914). 19. Vogel, A Text Book for Practical Organic Chemistry, 4th Ed: Longman (1978). 20. D.V.Mangte and S.P.Deshmukh, Int. J. Chem. Sci., 2(2), 159 (2004). 21. A. G. Sarap and S. P. Deshmukh, Int. J. Chem. Sci., 7(4), 2389 (2009). [RJC-710/2011]

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Vol.4, No.1 (2011), 36-42 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

ISOLATION AND IDENTIFICATION OF ANTIMICROBIAL COMPOUND FROM MENTHA PIPERITA L.

1

Abhishek Mathur*1, G.B.K.S. Prasad2, Nageshwar Rao3, Pradeep Babu3 and V.K. Dua1 Scientist-F and Officer Incharge, National Institute of Malaria Research, Hardwar (U.K), India. 2 Professor, Dept. of Biochemistry, Jiwaji University, Gwalior (M.P), India. 3 Roorkee Research and Analytical Lab Pvt. Ltd., Roorkee (Uttarakhand), India. E-mail: abhishekmthr@gmail.com

ABSTRACT Mentha piperita L. (Lamiaceae) leaves have been traditionally implemented in the treatment of minor sore throat and minor mouth or throat irritation by the indigenous people of India, although the compounds responsible for the medicinal properties have not been identified. In the present study, an antimicrobial compound was isolated and characterized, and its biological activity was assessed. The compound was isolated and characterized from the extracted essential oil using different spectral techniques: TLC, FTIR spectra and HPLC. Antimicrobial activity of the compound was assessed using both well diffusion and microdilution method in 96 multi-well microtiter plates. The isolated compound was investigated for its antimicrobial activity against seven selected pathogenic and nonpathogenic microorganisms: Staphylococcus aureus, Streptococcus mutans, Streptococcus faecalis, Streptococcus pyogenes, Lactobacillus acidophilus, Pseudomonas aeruginosa, E.coli K-12, Bacillus subtilis, Salmonella typhimurium and the fungal strains Candida albicans, Aspergillus niger, Penicillium notatum and Saccharomyces cerevisae. Menthol at different concentrations (1:1, 1:5, 1:10, and 1:20) was active against all tested bacteria except for S.aureus, and the highest inhibitory effect was observed against S. mutans using the well diffusion method. Furthermore, menthol achieved considerable antifungal activity against all the fungal strains except A.niger. The isolation of an antimicrobial compound from M. piperita leaves validates the use of this plant in the treatment of minor sore throat and minor mouth or throat irritation as well as diseases such as typhoid. Key words: M.piperita, antimicrobial compound, menthol, antimicrobial activity Š 2011 RASĀYAN. All rights reserved.

INTRODUCTION Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries. Plants still continue to be almost the exclusive source of drugs for the majority of the world's population. The World Health Organization reported that 80 % of the worlds population rely chiefly on traditional medicine and a major part of the traditional therapies involve the use of plant extracts or their active constituents1. Mentha piperita L. (common name: peppermint ) member of the large mint family Lamiaceae, is a fastgrowing, perennial herb which can reach up to 1.5m high in favorable conditions. M. piperita is an extremely variable species with a widespread distribution in Europe, Mediterranean region, and eastwards into Asia. In Indian folk medicine, the leaves are used for relief of minor sore throat and minor mouth or throat irritation. It is also used in treatments for minor aches and sprains, and in nasal decongestants. In addition to its antiparasitic, carminative, antiseptic and stimulant properties2. Menthol (C10H20O) is a terpenoid, found in the essential oils of the mint family (Mentha sp.). It is a waxy, crystalline substance, clear or white in color, which is solid at room temperature and melts slightly above. Several isomers of menthol exist, some with a menthol smell, others without. In nature it only occurs as (-) menthol, which has the strongest smell and its formal name is (1R, 2S, 5R)-isopropyl 5methylcyclohexanol. The other isomers are known as isomenthol, neomenthol and neoisomenthol (-) menthol can be described as fresh, sweet, minty, cooling, refreshing. The (+) isomer is similar, but less minty, more herby, with musty, bitter,

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phenolic and herbaceous notes, and is less refreshing. (-) menthol has also got about four times the cooling power of the (+) isomer3. In spite of all the information available in literature, no extensive isolation studies of (-) menthol are present. Thus, the aim of this study was to isolate and characterize menthol from M. piperita grown in India using different spectral techniques, and its antimicrobial activity against some selected pathogenic and non pathogenic microorganisms.

EXPERIMENTAL Plant material Mentha piperita leaves were obtained commercially from a local garden in Dehradun, Uttarakhand, India and identified by a botanical taxonomist at Botanical Survey of India Dehradun (Uttarakhand), India. The leaves were washed first under running tap water, followed by sterilized distilled water and dried at room temperature in dark then grinded to powder using an electrical blender. Essential oil extraction and isolation of (-) menthol The dried plant material was submitted to steam distillation in a Clevenger-type apparatus for 3 h. A volume of 1.0 ml (density = 1.04 g/ml) of the resulted plant essential oil was dissolved with 50 ml of hexane and transferred to a 125 ml separatory funnel. 25 ml of methanol was added. The funnel was shaken vigorously and the layers were separated. The hexane phase was dried over anhydrous sodium sulphate to produce menthol4. Characterization of (-) menthol Chemical detection Chemical detection was carried out by adding a small piece of potassium hydroxide into a test tube containing 1 ml of the plant essential oil with heating. The solution was cooled and 1 ml of diethyl ether was added. A few drops of carbon disulfide were added to the solution forming a yellow residue that indicates the presence of menthol. Thin-layer chromatography (TLC) The isolated compound was dissolved in appropriate solvent. 5 µl of reference solution of menthol and 5 µl of investigated wild mint oil were applied to silica gel plates, Merck (Germany) 20 × 20 cm, 0.25 mm in thickness. Plates were developed using the solvent system toluene: diethyl ether: 1.75 M acetic acid (1:1:1) and the separated zones were visualized using iodine chamber. Standard menthol served as positive control. FTIR studies The IR spectrum of menthol was recorded in Roorkee Research and Analytical Laboratory Pvt. Ltd., Roorkee (Uttarakhand), India using a computerized FTIR spectrometer (Perkin Co., Germany) in the range of 400–4000 cm-1 by the KBr pellet technique. High-performance liquid chromatography (HPLC) HPLC analysis was performed in Roorkee Research and Analytical Laboratory Pvt. Ltd., Roorkee(Uttarakhand), India using a Shimadzo LC 2010 HPLC system (Kyoto, Japan), equipped with a Shimadzo LC 2010 UV-VIS detector with a thermostated flow cell and a selectable two wavelengths of 190 - 370 nm or 371–600nm. The detector signal was recorded on a Shimadzo LC2010 integrator. The column used was a C18 block heating-type Shim-pack VP-ODS (4.6 mm interior diameter × 150 mm long) with a particle size of 5 µm. Menthol was separated using a mobile phase of 3% ethyl acetate/isooctane at a flow rate of 3.0 ml/min, column temperature 25°C. Injection volume was 40 µl and detection was carried out at 322 nm. Antimicrobial activity Microbial cultures Nine strains of bacteria and four strains of fungus were used as test microorganisms. All microorganisms were clinical isolates, obtained from the Microbiology Laboratory at Department of Microbiology, Sai Institute of Paramedical & Allied Sciences, Dehradun (Uttarakhand), India. Inoculum preparation Nutrient agar/broth and Sabouraud’s dextrose agar\ broth were used for growing and diluting the microorganism suspensions. Bacterial strains were grown to exponential phase in nutrient broth at 37°C

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for 18 h and adjusted to a final density of 108 cfu /ml by diluting fresh cultures and comparison to McFarland density. Fungal strains were aseptically inoculated on Petri dishes containing autoclaved, cooled, and settled SDA medium. The Petri dishes were incubated at 31°C for 48 h. These were aseptically sub cultured on SDA slants. The fungal colonies from SDA slants were suspended in sterilized 0.9% sodium chloride solution (normal saline), which was compared with McFarland solution. According to the manufacturer's directions, 1 ml of yeast suspension in normal saline was added to 74 ml of sterile medium and kept at 45°C to give a concentration of 2 × 107 cells/ml. Well diffusion assay A modified agar diffusion method5 was used to determine antimicrobial activity. Nutrient agar was inoculated with microbial cell suspension (200 µl in 20 ml medium) and poured into sterile petri dishes. Wells were filled with 20 µl of menthol in different concentrations (1:1, 1:5, 1:10, 1:20 initially prepared by diluting in DMSO and sterilized by filtration through 0.45 µm Millipore filters), and placed on the inoculated agar surface. Standard antibiotics i.e. streptomycin (1 mg/ml) and amphotericin B (1mg/ml) were used as positive controls. The plates were incubated overnight at 37°C for 18–24 h. In contrast, fungal cultures were incubated at 31°C for 48 h. and the diameter of any resulting zones of growth inhibition was measured (mm). Each experiment was tested in triplicate. Micro-well dilution assay Minimal inhibitory concentrations of menthol isolated from M. piperita were determined based on a microdilution method in 96 multi-well microtiter plates as previously described6. Briefly, bacterial strains were cultured overnight at 37°C on nutrient broth and adjusted to a final density of 108 cfu /ml, and used as an inoculum. Menthol was dissolved in DMSO and then in nutrient broth to reach a final concentration of 200 µg/ml. Serial twofold dilutions were made in a concentration range from 7.8 to 200 µg/ml. In each microtiter plate, a column with broad-spectrum antibiotic was used as positive control. As an indicator of bacterial growth, 50 µl of 0.2 mg/ml p-iodonitrotetrazolium chloride (INT) was added to the wells and incubated at 37°C for 30 min. The lowest concentration of compound showing no growth was taken as its minimal inhibitory concentration (MIC). The colourless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms7. Where bacterial growth was inhibited, the solution in the well remained clear after incubation with INT. As for fungal cultures, a simple turbidity test8 was used to determine the MIC value of menthol. A volume of 0.1ml from each serial dilution of menthol concentrations (200-7.8 µg /ml) was added into tubes containing 9.8 ml of sterile nutrient broth, and then the tubes were inoculated with 0.1 ml of specific fungal culture suspension and incubated at 31°C for 48 h. Amphotericin B (1 mg/ml) and Streptomycin (1mg/ml) was used as a positive control. The optical density was determined using a Systronics UV-VIS spectrophotometer at 630 nm. The MIC value was the lowest concentration of compound that showed no growth after 48 h of incubation in comparison with the control tube, which included 9.8 ml of nutrient broth and 0.1 ml of specific fungus suspension in addition to 0.1 ml of each compound concentration.

RESULTS AND DISCUSSION The present study was conducted to isolate the main bioactive compound from M. piperita leaf oil. Menthol was isolated from the extracted essential oil, and then detected on TLC plates in comparison with standard menthol. A purple zone with a retention factor (Rf) value of 0.82 was identified as menthol in comparison with standard menthol that had the same Rf value. The FTIR spectrum confirmed the material isolated from M. piperita leaf oil as menthol (Figure 1). Significant peaks were found at: (3362) cm-1 corresponding to hydroxyl group; (2855, 2924) cm-1 ascribed to methyl group; (1025, 1045) cm-1 attributed to (C-O) bond and (1368) cm-1 corresponding to isopropyl group, all of which confirm the purity of the isolated material. Moreover, menthol was characterized using the HPLC system (Figure 2) and identified by comparing its retention time and UV spectrum with that of the standard compound. The retention time 7–8 min and UV spectra of the isolated compound on HPLC were completely identical to that of standard menthol. The isolated compound was investigated for its antimicrobial activity against nine bacterial species and four fungal strains. The initial screening of antibacterial activity of menthol was assayed in vitro by the agar diffusion method using active against all tested bacterial and fungal strains

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(Table 1). The highest inhibitory effect was observed against S. mutans (zone of inhibition: 25.3 mm) while the weakest activity was demonstrated against S. aureus (zone of inhibition: 15.0 mm). In view of the results obtained by the well diffusion method, the minimal inhibitory concentration MIC of menthol was determined by broth microdilution assay (Table 2). The highest MIC value (15.6 µg/ml) was observed against P.aeruginosa, S. pyogenes, B.subtilis, E.coli K-12, S. mutans and Salmonella typhimurium while S. faecalis, S. aureus and L. acidophilus ranked next (MIC 31.2 µg /ml). Moreover, menthol observed good antifungal activity against the fungal strains i.e. Candida albicans, Saccharomyces cerevisae and Penicillium notatum (zone of inhibition range: 15.0–18.5 mm; MIC: 125.0). The least antifungal activity was observed against Aspergillus niger (zone of inhibition: 15.0 mm).The standard drug streptomycin was active against all reference bacteria (zone of inhibition range: 17.9– 26.2mm; MIC range: 15.6–7.8 µg/ml). In addition, amphotericin B demonstrated good antifungal activity against all the fungal strains (zone of inhibition range: 17.6 -18.5mm; MIC range: 17.4-7.8 µg/ml). The fragrance of plants is carried in the so called quinta essentia, or essential oil (EO) fraction. These oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. They are called terpenes, their general chemical structure is C10H16, and they occur as diterpenes, triterpenes, and tetraterpenes (C20, C30, and C40), as well as hemiterpenes (C5) and sesquiterpenes (C15). When the compounds contain additional elements, usually oxygen, they are termed terpenoids such as menthol and camphor9. In vitro studies in this work showed that menthol inhibited the growth of all tested bacteria except S.aureus and observed good antifungal activity against all the fungal strains except A.niger. The zones of inhibition ranged from 15.0–25.3 mm and 15.0–18.5 mm in diameter against bacterial and fungal strains respectively using the well diffusion method. Furthermore, MIC values ranged from 15.6–31.2 µg/ml against tested bacteria except S.aureus and 125.0 µg/ml against all fungal strains except A.niger. Terpenes or terpenoids have been previously reported to be active against bacteria10, 11 fungi 12, 13, viruses 14, 15 , and protozoa 16, 17. The mechanism of action of terpenes is not fully understood but is speculated to involve membrane disruption by the lipophilic compounds. In view of the results obtained using both disc diffusion and micro-well dilution assays, menthol was found only active against Gram-positive bacteria. It has frequently been reported that Gram-positive bacteria are more susceptible to essential oils than Gram-negative bacteria18. The tolerance of Gram-negative bacteria to essential oils has been ascribed to the presence of a hydrophilic outer membrane that blocks the penetration of hydrophobic essential oils into target cell membrane. According to the previous studies, in particular P. aeruginosa, appear to be least sensitive to the action of essential oils19, 20 but our investigation has revealed that it is showing the significant growth inhibition against the treatment of essential oil. Furthermore, several mechanisms of antimicrobial resistance are readily spread to a variety of bacterial genera. First, the organism may acquire genes encoding enzymes, such as b-lactamases, that destroy the antibacterial agent before it can have an effect. In addition, bacteria may acquire efflux pumps that extrude the antibacterial agent from the cell before it can reach its target site and exert its effect. Finally, bacteria may acquire several genes for a metabolic pathway which ultimately produces altered bacterial cell walls that no longer contain the binding site of the antimicrobial agent, or bacteria may acquire mutations that limit access of antimicrobial agents to the intracellular target site via down-regulation of porin genes21.

CONCLUSION A number of EO components have been registered as flavorings in foodstuffs. The flavorings registered are considered to present no risk to the health of the consumer and include amongst others carvacrol, carvone, cinnamaldehyde, citral, p-cymene, eugenol, limonene, menthol and thymol22. In the present study, the isolated compound demonstrated promising antimicrobial activities against the most prevalent microorganisms in oral infections. The use of this plant in the treatment of sore throat, mouth or throat irritation is validated, scientifically supported by the results obtained in this work.

ACKNOWLEDGEMENTS The authors are thankful to the Research staff of Roorkee research and Analytical Labs, Roorkee (Uttarakhand), India and National Institute of Malaria Research, Hardwar (U.K), India for their valuable

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support. Thanks are due to Mr. Gaurav Verma for the valuable suggestions. Last but not the least special thanks to Mr. Himanshu Gupta and Ms. Sofiya for the technical support. Table-1: Diameter of zone of inhibition (mm) of menthol isolated from Mentha piperita against pathogenic microorganisms Diameter of zone of inhibition (mm) Microorganisms

Menthol (200 Âľg/ml)

Control S

15.0 25.3 22.3 23.5 20.0 24.2 24.3 23.4 24.2 18.5 15.0 17.3 17.5

Staphylococcus aureus Streptococcus mutans Streptococcus faecalis Streptococcus pyogenes Lactobacillus acidophilus Pseudomonas aeruginosa E.coli K-12 Bacillus subtilis Salmonella typhii Candida albicans Aspergillus niger Penicillium notatum S.cerevisae

A

25.6 20.2 18.6 19.8 21.7 19.6 26.2 25.5 24.6 18.6 19.5 18.7 17.9

17.6 18.5 18.5 17.8

S, Streptomycin; A, Amphotericin (1mg ml-1); -, Not tested Table-2: Minimum Inhibitory Concentration (MIC) of menthol isolated from Mentha piperita against pathogenic microorganisms Minimum Inhibitory Concentration (MIC) Microorganisms

Menthol (200 Âľg/ml )

Control S

A

Staphylococcus aureus Streptococcus mutans Streptococcus faecalis Streptococcus pyogenes Lactobacillus acidophilus Pseudomonas aeruginosa

31.2 15.6 31.2 15.6 31.2 15.6

7.8 15.6 15.6 15.6 15.6 15.6

-

E.coli K-12 Bacillus subtilis Salmonella typhii Candida albicans Aspergillus niger Penicillium notatum S.cerevisae

15.6 15.6 15.6 125 125 125 125

7.8 7.9 7.9 15.6 15.6 15.6 15.6

17.4 7.8 7.8 17.4

S, Streptomycin; A,Amphotericin(1mg ml-1); -,Not tested

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Fig.-1: FTIR spectrum of menthol isolated from Mentha piperita leaf oil

Fig.-2: HPLC chromatogram of menthol isolated from Mentha piperita leaf oil

REFERENCES 1. World Health Organization- Summary of WHO guidelines for the assessment of herbal medicines, Herbal Gram, 28, 13 (1993). 2. A. Al-Rawi, H.L. Chakrabarty., National Herbarium of Iraq, Baghdad, 65 (1988).

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

C.S. Sell. Royal Society of Chemistry, 2, 76 (2003). N.D. Cheronis, J.B. Entrikin. Interscience (Wiley), New York, 175 (1963). R.A. Mothana, U. Lindequist. J Ethnopharmacol., 96, 177 (2005). F.A. Al-Bayati. J Ethnopharmacol., 116, 403 (2008). J.N. Eloff, Planta Medica, 64, 711 (1998). F.A. Al-Bayati, K.D. Sulaiman. Turk, J .Biol., 32, 57 (2008) M.M. Cowan, Clin. Microbiol. Rev., 12, 564 (1999). A.A. Ahmed, A.A. Mahmoud, H.J. Williams, A.I. Scott, J.H. Reibenspies, T.J. Mabry,J. Nat. Prod., 56, 1276 (1993). J.A. Amaral, A. Ekins, S.R. Richards, R. Knowles, Appl Environ Microbiol., 64, 520 (1998). G.G. Harrigan, A. Ahmad, N. Baj, T.E. Glass, A.A. Gunatilaka, D.J. Kingston, J .Nat Prod. , 56, 921 (1993). B.K. Rana, U.P. Singh, V. Taneja, J. Ethnopharmacol., 57, 29 (1997). H. Hasegawa, S. Matsumiya, M. Uchiyama, T. Kurokawa, Y. Inouye, R. Kasai, S. Ishibashi, K.Yamasaki, Planta Medica, 6, 240 (1994). H.X. Xu, F.Q. Zeng, M. Wan, K.Y. Sim, J .Nat. Prod., 59, 643 (1996). R.A. Vishwakarma, J .Nat. Prod., 53, 1, 216 (1990). S. Ghoshal, B.N. Krishna Prasad, V. Lakshmi, J. Ethnopharmacol., 50, 1671996). C.M. Mann, S.D. Cox, J.L. Markham, Lett Appl. Microbiol., 30, 294 (2000). H.J. Dorman, S.G. Deans, J. Appl. Microbiol., 88, 308 (2000). G. Pintore, M. Usai, P. Bradesi, C. Juliano, G. Boatto, F. Tomi, M. Chessa, R. Cerri, J. Casanova., Flav. Frag. J., 17, 1, 15 (2002). F. Tenover, Amer. J. Med., 119, 53 (2003). S. Burt, Int. J. Food Microbiol., 94, 223 (2004). [RJC-613/2010]

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SURFACE WATER (LAKES) QUALITY ASSESSMENT IN NAGPUR CITY (INDIA) BASED ON WATER QUALITY INDEX (WQI) P. J. Puri1*, M.K.N. Yenkie1, S.P. Sangal 1, N.V. Gandhare2 , G. B. Sarote3 and D. B. Dhanorkar4 1,*

Department of Chemistry, LIT, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 001, India 1 Department of Chemistry, LIT, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 001, India 2 Department of Chemistry, Nabira Mahavidyalaya, RTM, Nagpur University, Katol - 66302 3 Regional Forensic Science Laboratory, Dhantoli, Nagpur – 440 012 4 Maharashtra State Power Generation Limited, Nagpur- 440 033 * E-mail: puripj@rediffmail.com ABSTRACT This paper is intended to be a study concerning with surface water (lakes) quality in Nagpur city (India) based on water quality index (WQI). In present study, water quality index (WQI) has been calculated for different surface water resources especially lakes, in Nagpur city, Maharashtra, (India), for the session January to December 2008; comprising of three seasons, summer, winter and rainy season. Sampling points were selected on the basis of their importance. Water quality index was calculated using water quality index calculator given by National Sanitation Foundation (NSF) information system. The calculated (WQI) for various studied lakes showed fair water quality in monsoon season which then changed to medium in winter and poor for summer season. Gorewada lake showed medium water quality rating in all season except monsoon season. Futala, Ambazari and Gandhisagar lake has also declined in aesthetic quality over past decade following invasion of aquatic weeds such as hydrilla and water primrose, so the reasons to import water quality change and measures to be taken up in terms of surface water (lakes) quality management are required. Keywords: Surface water, Water quality index, National Sanitation Foundation, Lakes. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Water pollution means contamination of water by foreign matter such as micro-organism, chemicals industrial or other wastes, or sewage. Such matters deteriorate the quality of water and render it unfit for its intended uses. Water pollution is the introduction into fresh or ocean waters of chemical, physical or biological material that degrades the quality of the water and affects the organisms living in it. Although some kinds of water pollution get occur through natural processes, it is mostly a result of human activities. The water we use is taken from lakes and rivers, and from underground (ground water) and after we have used it and contaminated it – most of it returns to these locations. Water pollution also occurs when rain water runoff from urban and industrial area and from agriculture land and mining operations makes its way back to receiving waters (river, lake or ocean) and into the ground.1-3 According to WHO4 organization, about 80% of all diseases in human beings are caused by water. Once the groundwater and surface water quality is contaminated, its quality can’t be restored by stopping the pollutants from the source. It therefore becomes imperative to regularly monitor quality of groundwater and surface water resources and to device ways and mean to protect it. Water quality index (WQI) is one of the most effective tools5-8 to communicate information on the quality of water to the concerned citizens and policy makers. Many civilizations that flourished after developing reliable water supply collapsed when supply was exhausted or its quality deteriorated9. It thus becomes important parameters for the

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assessment and management of ground as well as surface water resources. Water quality index (WQI) is regarded as one of the most effective way to communicate water quality10-13. The (WQI) which was developed in the nearly 1970’s can be used to monitor water quality changes in a particular water supply over time, or it can be used to compare a water supply‘s quality with other water supplies in the region or from around the world. The result can also be used to determine if a particular stretch of water is considered as healthy. Assessments of water quality are very important for knowing it’s suitability for various purposes.14 Lake pollution is one of the serious environment problems in recent years with socio–economics development and pollutant discharge increase from industry, agriculture and domesticity. Nagpur city with coordinates of 21o8’ 55’’ N and 79o 4’46’’E is second capital of Maharashtra state. Nagpur city is popularly known as orange city, also city of lakes. The city had 10 lakes in the past, but unfortunately only 7 of them are there now. The present water supply to city is made from Gorewada lake, Kanhan River and Pench Dam. Futala Lake is used to irrigate 84 acre of cultivated agriculture land. Ambazari lake water is provided to local residence of MIDC colony, Hingna area, Nagpur also it is used for industrial purpose. The present study was completed to obtain a record of seasonal condition in various lakes such as Futala, Ambazari, Gandhisagar and Gorewada Lake are within Nagpur city, Maharashtra, (India). The specific objective of present study is to determine lakes physical and chemical profile over an annual cycle and its overall water quality in terms of water quality index (WQI).

EXPERIMENTAL To characterize water quality throughout the main basin of the lakes, four permanent stations for monthly sampling were established and marked within inflow, mid-lake, outflow and corner regions. Regular samples were collected in sterilized glass bottles for bacteriology and various physicochemical analysis of sample; the precleaned plastic polyethylene bottles were used. Prior to sampling, the entire sampling container’s were washed and rinsed thoroughly with lake water to be taken for analysis. The samples were analyzed for different physical, chemical and bacteriological parameters of water quality index (Electrical conductivity, TDS, Cl-, Total Hardness, BOD, DO, pH, Faecal Coliform) using standard method15 In bacteriological examination, total coliforms and fecal coliforms were determined by Membrane Filtration (MF) technique and average values were recorded. The National Sanitation Foundation water quality index (NSF, WQI) has the following mathematical structure: for n parameters NSF WQI = Where, Li = Sub – index for i th water quality parameter; Wi = Weight (in terms of importance) associated with i th water quality parameter; and n= Number of water quality parameters.

RESULTS AND DISCUSSION A water quality index (WQI) integrates complex analytical raw data and generates a single number (like a grade) that express subjectively the water quality. Such a rating scale allows for simplicity and consumer comprehensibility. A water quality index can be different type depending on its final intended purpose. It can be highly specific for different bodies of water or could be a general one for all types of water meant for human consumption. A WQI can also be based not just on readings at a single point of time but also on readings collected over a period of time (like a year). A WQI may also be arrived at by calculating the number of objective parameters not met, or by calculating the frequency with which they are not met or the amount by which they exceed the norm. The WQI was calculated using NSF information software16 and compared with standard water quality rating, as shown Table 2. The minimum, maximum and average of values obtained for various lakes of WQI rating for monsoon, summer and winter season are represented in Table 2. The graphical representation of WQI rating in different season is given in Fig. 1. Fig. 2 shows location of Futala, Ambazari, Gandhisagar and Gorewada lakes. The contamination of surface water is a significant environmental concern and constitutes a risk to both water quality and aquatic ecosystem. Peoples around SURFACE WATER QUALITY ASSESSMENT IN NAGPUR

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the world have used surface and groundwater as a source of drinking water and even today more than half the world population depends on groundwater and surface water for their survival17. The values of ground and surface water lies not only in its wide spread occurrence and availability but also its consistent which makes it an ideal supply of drinking water. Electrical conductivity of water is a direct function of its dissolved salt18. Hence it is an index to represent total concentration of soluble salts in water19. High values of total dissolved solid in surface water resources (lakes, rives) are generally not harmful to human beings but high concentration of these may affect person who are suffering from kidney and heart diseases20. Water containing high solids may cause laxative or constipation effects21. High values of TDS in Futala and Gandhisagar Lake is due to dense residential area and intensive irrigation.22 The Gandhisagar and Futala lake water samples are poor in quality. In this part, the surface water resources (lakes) may improve due to inflow of fresh water of good quality during rainy season. The low values of WQI at studied lakes have been found to be mainly from the higher values of pH, nitrate, TDS, hardness, bicarbonates and chlorides. The present analysis reveals that surface water resources especially lakes (studied) need some degree of treatment and also need to be protected from perils of contamination. On the basis of the above discussion it may be concluded that the lake water quality at almost all sites at Futala, Ambazari and Gandhisagar Lake are highly polluted. The observed range of average WQI in monsoon was 36, 44, 32 and 70 in Futala, Ambazari, Gandhisagar and Gorewada lakes respectively. In summer WQI values were 27.5, 46, 22 and 56, whereas in winter it was 34, 54, 22, and 54 for Futala, Ambazari, Gandhisagar and Gorewada lake respectively. For Futala, Ambazari and Gandhisagar lake, the water quality at almost all the sites showed the increasing trend of WQI index in monsoon, summer and winter season respectively as shown in Table 3. Table-1: Sub Index Equation for Water Quality Parameter (NSF WQI)16 Water Quality Parameter

Range Applicable

Equation

Percent Saturation D.O.

0-40%

IDO = 0.18 + 0.66 X (% Saturation of DO)

+

40 - 100 %

IDO = - 13.550 + 1.17 X ( %Saturation of DO)

+

BOD(mg/L)

100 - 140 %

IDO = -263.34 – 0.62 X ( %Saturation of DO)

0-10

IBOD = 96.67-1.23 X (BOD)

+

pH

10 - 30

IBOD = 38.90-1.23 X (BOD)

2-5

IpH = 16.10-7.35 X (pH)

5+-7.3

IpH = -142.67+33.50 X (pH)

+

7.3 -10

IpH = 316.96-29.85 X(pH)

+

10 -12 Faecal coliform

IpH = 96.17-8.00X (pH)

1-103 3+

10 -10 10

IColi = 97.20 - 26.80 x log (FC) 5

IColi = 42.23-7.75 x log (FC)

5+

IColi = 2

Where, IDO : Sub index for Dissolved Oxygen IpH : Sub index pH IBOD : Sub index for Biochemical Oxygen Demand IColi : Sub index for coliform

CONCLUSION The result obtained in present study revealed that certain human activities such as immersion of idols of God and Goddess (in large ratio) during festival season, washing activities, recreational activities, surface runoff from resulting rainfall, (poor) sewage have contributed considerable pollution in various lakes within Nagpur city area. Water quality from studied lakes is unsafe for consumption of human use and SURFACE WATER QUALITY ASSESSMENT IN NAGPUR

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therefore need serious attention. The present study revealed that water quality form studied lakes is polluted with reference to almost all the water quality physicochemical parameters studied. The Futala, Ambazari and Gandhisagar lake showed poor water quality in all respect. People dependent on this water may prone to health hazard due to polluted drinking water; therefore some effective measures are urgently required to enhance the lake water quality by delineating an effective water quality management plan for lake system in Nagpur city, Maharashtra, India. Table-2: Description of the Index16 NSF WQI 63-100

Ranking Good to Excellent

Description Drinking water source without conventional treatment but after disinfections

50-62

Medium to Good

38-49

Bad

Outdoor bathing , swimming and water contact sport Drinking water source with conventional treatment : followed by disinfection

0-37

Bad to Worse

Propagation of wildlife fisheries, irrigation, industrial cooling and controlled waste disposal.

The index when used has certain figures, which appears as shown below-

Futala lake

Ambazari lake

Aver.

Max.

Min.

Aver.

Max.

Min.

Aver.

Max.

Min.

Aver.

Max.

80 70 60 50 40 30 20 10 0 Min.

WQI rating

Representation of WQI rating in all season

Gorewada Gandhisagar Lake lake

Lakes resources Rainy season

Summer Season

Winter Season

Figure-1

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Vol.4, No.1 (2011), 43-48 Table-3: Average WQI rating of surface water (lake) sources in different season. Resources Futala lake

Ambazari lake

Gorewada lake

Rainy Season

Summer Season

Winter Season

Min.

30

25

30

Max.

38

30

38

Aver.

34

27.5

34.4

Min. Max. Aver.

42 48 44

40 50 46

38 44 40

Min.

68

50

50

Max. Aver.

75 70

64 56

58 54

Gandhisagar lake

Min. 30 25 Max. 34 30 Aver. 32 22 Note : Max – Maximum, Min – Minimum, Aver – Average

24 28 22

ACKNOWLEDGEMENT The authors hereby acknowledge the kind and wholehearted support of the Dr. S. B. Gholse, Director, LIT, RTMNU, Nagpur, India.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

S. Kulshrestha, S. Sharma and R. V. Singh, Int. J. Chem. Sci. ,2(1) ,27 (2004) J. B. Shukla and B.Dubey India,Ecological Modelling, 86, 91 (1996) V. P. Shukla India, Ecological Modelling, 109(1), 99 (1998) WHO (World Health Organization) , 2nd Ed., Vol. 1, p 188 (1993) P.C. Mishra and R. K. Patel, Indian J Environ Ecoplan ., 5(2),293 (2001) S. Naik and K. M. Purohit , Indian J. Ecoplan ., 5(2), 397 (2001) D. F. Singh , Proc, Acd. Environ. Biol., 1(1) 61 (1992) T. N. Tiwari and M. A. Mishra, Indian J Environ Proc., 5,276 (1985) A. Mohrir, D.S. Ramteke, C. A. Moghe, S. R. Wate and R. Sarin, IJEP, 22 (9), 961 (2002) Proceeding of the international conference on water and environmental (WE-2003). Bhopal India, Allied publishers Pvt., Dec 15-18,2003. K. Kannan , Fundamentals of Environmental pollution , S. Chand & Company Ltd., New Delhi, 1991. D.K. Sinha and A. K. Shriwastava, Indian J Env Prot., 14 (5), 340 (1994) S. K. Pradhan, D. Patnaik and S. P. Raut, Indian J, Environ. Protect. , 21(4), 355 (2001) APHA, Standard Method for the Examination of Water and Wastewater. 17th Ed. American Public Health Association, Washington, DC, 1989. S. Ramkrishnaiah and Sri Y. Babu Rao, Environmental and water quality studies in AP state – A case study, 1991. T.V.Ramachandra, Teri Press, Centre for Ecological Science. IIS, Bangalore (2009). UNESCO, Groundwater UNESCO Environmental and development briefs no.2, 14 p. 1992 C. C. Harilal, A. Hashim, P. R. Arun and S. J. Baji, Ecology, Environment and conservation, 10(2)187 (2004) B. K. Purandara, N. Varadarajan and K.Jayashree , Poll Res., 22 (2), 189 (2003)

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20. 21. 22.

S. Gupta, A. Kumar, C. K. Ojha and G. J. Singh , Environmental Science and Engineering., 46(1), 74 (2004) N. J. Kumaraswamy, Pollut. Res., 10(1), 13 (1999) P.J. Puri, M.K.N. Yenkie, N.V. Gandhare, D.B. Dhanorkar, RASAYAN J. Chem., 3(4), 800 (2010). [RJC-697/2010]

Fig.-2: Map showing Gandhisagar, Ambazari, Gorewada and Futala Lake , Nagpur (MS) India.

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Vol.4, No.1 (2011), 49-55 ISSN: 0974-1496 CODEN: RJCABP

http://www.rasayanjournal.com

SAMPLE PREPARATION FOR FLAME ATOMIC ABSORPTION SPECTROSCOPY: AN OVERVIEW Nabil Ramadan Bader Chemistry department, Faculty of science, Garyounis university, Benghazi, Libya. E-mail: nabil_bader@yahoo.com ABSTRACT Sample preparation is an important step in chemical analysis, from time and reagent consuming point of view and from the probability of errors. The present article gives an overview of recently most used techniques in sample preparation for flame atomic absorption spectroscopy. Wet and dry sample decomposition techniques, separation and pre-concentration methods of the target analyte(s) have been discussed. Keywords: Sample preparation, FAAS, decomposition, separation, pre-concentration Š 2011 RASĀYAN. All rights reserved.

INTRODUCTION Elemental analysis of the majority of organic and inorganic matrices requires the partial or total dissolution of the sample prior to instrumental analysis. Analysis by spectroscopic methods practically always necessitates a simple or more complex preparation of the sample. These steps are generally the most critical part of analysis because they are responsible for the most important errors.1-3 Only a few direct methods allow the introduction of the sample without any preparation. In these cases the lack of reliable calibration is the major problem. On the other hand, sample preparation allows the separation and/or pre-concentration of analytes and makes possible the use of several determination methods. Sample preparations involve digestion, extraction and preparation of the analytes before the analysis, so this step is time limiting, requiring ca. 61% of the total time to perform the complete analysis, and is responsible for 30% of the total analysis error. Nowadays the goals to be reached are the best results, in the shortest time, with minimal contamination, low reagent consumption and generation of minimal residue or waste. 1 Sample preparation was probably the single most neglected area in analytical chemistry relatively to the great interest in instruments. While the level of sophistication of the instrumentation for analysis has increased significantly, a comparatively low technical basis of sample preparation often remains.4 Sample preparation and development of methods have now became a growing field along with instrumental improvements. There are drastic improvements in the detection power of measurement techniques used. Consequently, the analyst also realizes that it is often no longer mandatory to resort to laborious, dubious and time-consuming separation or pre-concentration steps in the sample preparation procedures.2 It is normal in atomic spectroscopy for the sample to be found in one of two forms solid or liquid. The liquid case would seems to be the easiest form in which to handle the sample, with maybe a requirement for filtration being all that is required. However, the inherent lack of sensitivity of many spectroscopic techniques and the need to carry out determinations at lower and lower levels means that invariably some form of pre-concentrations is required. If the sample is in a solid form, the normal requirement is to covert it into the liquid form although it is possible to analyse solids directly by using the atomic spectroscopy, but this is not the preferred approach.4 The principal objectives of sample preparation for residue analysis are; isolation of the analytes of interest from as many interfering compounds as possible, dissolution of the analytes in a suitable solvent and preSAMPLE PREPARATION FOR FAAS

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concentration. In an analytical method sample preparation is followed by a separation and detection procedure.

EXPERIMENTAL The selection of a preparation method is dependent upon: (1) the analyte(s), (2) the analyte concentration level(s), (3) the sample matrix, (4) the instrumental measurement technique, and (5) the required sample size. Matrix The term matrix refers to the collection of all of the various constituents making up an analytical sample. In addition to the analyte, the sample matrix includes all of the other constituents of the sample, which are sometimes referred to as concomitants. Contamination and Losses The major problem in preparing samples for trace analysis is the risk of contamination. Contamination is associated with several probable causes, i.e. the grade of reagents used, sample storage container, steps of digestion or dilution of the sample and their previous history, and human intervention. Losses are a particularly significant problem in trace analysis. Container surfaces, for example, may present a significantly large area on which the analyte can be adsorbed. At higher levels such a small absolute loss would have little effect on the concentration but at trace levels a large proportion of the analyte may be stripped from the solution.5 Samples Samples analyzed may be divided into those which are already in an aqueous solution (e.g. various water samples, beverages, blood, serum, and urine.), in other liquid form (e.g. oils, fuels, and organic solvents.) or in solid form (e.g. soils, sediments, plants, animal tissues, metals, and plastics.). Solid samples may contain a high proportion of organic matter (e.g. plants, animal tissues, and plastics.) or have more inorganic composition (e.g. soils, sediments, and metals.). For routine analysis by atomic spectroscopic techniques, which are all dedicated to work with aqueous samples, the analysis of other liquids must be adapted and the solids are generally converted into a solution by an appropriate dissolution method.2 Liquid Samples Aqueous samples can be generally introduced for analysis directly and without any prior treatment. The only major problem associated with work with solutions is their collection and storage. Concerning atomic spectroscopic analysis itself, no particular precautions have to be taken. If measured concentrations satisfy the principal criteria of the spectroscopic method used (sensitivity, dynamic range) and possible interferences are under control, the analysis of solutions may be performed automatically with all modern atomic spectroscopic systems. Non-aqueous samples can sometimes be run directly, but this depends significantly on their viscosity. In flame atomic absorption spectroscopy (FAAS) analysis, the viscosity should be similar to that of water for which most nebulizers are designed. Only some organic solvents, such as ethanol or methyl isobutyl ketone, fulfill this condition and are consequently often used for dilution of organic liquids, the major drawback, encountered with these techniques is the dilution factor, which reduces the metal content per unit volume.6 Standards can be prepared in the pure solvent. Elements in organic solvents usually give an FAAS analysis response similar to that given by the same element in aqueous solution. Solid Samples In this case, many steps are required, including sampling, sub-sampling, grinding, and dissolution. The risk of contamination is higher than the case of liquid samples. RESULTS AND DISCUSSION Sample preparation techniques A. Decomposition techniques Decomposition involves the libration of the metal of interest from an interfering matrix by using a reagent and/or heat. The utilization of reagents or acids may cause contamination or loss of the analytes. Acid digestion or wet decomposition SAMPLE PREPARATION FOR FAAS

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Acid digestion involves the utilization of mineral or oxidizing acids and an external heat source to decompose the sample matrix. The choice of the acid or the combination of the acids depends upon the nature of the matrix to be decomposed. Wet decomposition has been performed in open systems for many years: Teflon beakers when using HF or glass tubes or beakers on hot plates or aluminum blocks. Open systems are useful for relatively “easy” samples such as food or agricultural, but generally unsuitable for some samples that require lengthy dissolution times of 1–24 h. Other concerns are that they tend to be time consuming and tedious and result in evaporative loss of volatile metals and the risk of contamination.7 In general, nitric acid is used as oxidant alone or in combination with other acids (e.g., sulfuric and hydrochloric acids) or sometimes with hydrogen peroxide.8-10 In addition, hydrofluoric acid can be used in combination with nitric acid for the total decomposition of silica containing organic matrices.11 Nitric acid is popular because of its chemical compatibility, oxidizing ability, availability, purity, and low cost.12 For samples that are not highly aromatic and/or contain a high -OH functionality, it is preferred to use nitric acid followed by perchloric acid. The ability of nitric acid to react with alcohols and aromatic rings forming explosive compounds calls for caution when using nitric acid alone or in combination with other reagents in the decomposition of organic matrices. If the sample contains high -OH functionality it is best to pre-treat the sample with concentrated sulfuric acid which will act as a dehydrating agent. The use of nitric acid is not recommended for digestion of highly aromatic samples.5 Digestion may be performed in open or closed vessels using classical heating blocks or microwave radiation.13-15 The closed vessels have been currently used in order to improve the oxidation efficiency and to reduce the time of digestion, and it has been successfully used for digestion of a variety of samples. Closed systems allow high pressures above atmosphere to be used. This allows boiling at higher temperatures and often leads to complete dissolution of most samples.7, 12 The advantages of the closed system, as compared with the open system are: No volatilization of elements, reduced reagent quantities, and no contamination from external sources.16 Microwave digestion Microwave-assisted sample preparation techniques are becoming widely used in analytical laboratories all over the world.17 Microwave radiation can greatly speed up the extraction and the so-called microwave-assisted extraction (MAE) is thus established.18,19 In principle, only samples or solvents containing dipolar materials or microwave absorbents can be affected by microwaves which heat the extraction body from inside to outside in a very short time, much different from the common heating methods. The acceleration is resulted from the fast and uniform heating feature. MAE can be conducted with an open or closed microwave system. A closed-vessel offers a special way to regulate the extracting temperature by simply adjusting the vessel pressure. Although almost all reported MAE methods were conducted off-line, online approaches have been shown to be possible.20-22 The main advantage of MAE lies in its wide applicability for fast extractions of analytes including some thermally instable substances. The closed digestion technique involves placing the sample in a vial (or bomb), usually constructed of a fluorinated polymer, such as polytetrafluoroethylene (PTFE) or perfluoro alkoxy (PFA). After adding the digestion reagents, the bomb is tightly sealed and placed in the microwave oven for irradiation by microwave energy.23-25 Although digestion using closed vessels may also employ conventional heating, most of the recent applications have been performed using microwave radiation in view of the relatively short time involved and also to allow specific applications where high purity reagents and matrix removal are necessary. Dry ashing Dry ashing or oxidation is usually performed by placing the sample in an open vessel and destroying the combustible (organic) portion of the sample by thermal decomposition, normally in the presence of an ashing aid, using a muffle furnace. Typical ashing temperatures are 450 to 550°C at atmospheric pressure, and the ash residues are dissolved in an appropriate acid. The degree of volatilization loss is a limiting factor and depends on (i) the applied temperature, (ii) the form in which the analyte is present in the SAMPLE PREPARATION FOR FAAS

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sample, and (iii) the chemical environment in the ashing stage. Oxidizing reagents may be used as ashing aids in order to prevent the volatilization of analytes and also to speed up the ashing process. High-purity magnesium nitrate and magnesium oxide are commonly used for that purpose.26 Low-temperature ashing involves treatment of the sample at about 120 °C using activated (singlet state) oxygen. The most important advantages of the ashing are the ability to decompose large sample sizes and dissolving the resulting ash in a small volume of acid, and the need for little or no reagents. A. Trace element separation and pre-concentration The direct determination of metals frequently proves to be insufficiently sensitive therefore, in many cases pre-concentration is necessary.27 Efficient and selective separation of metal ions is gaining more importance because of the increasing demand for high purity products and also for environmental concerns. There are various separation methods such as precipitation, solvent extraction, cloud point extraction, solid phase extraction, and ion-exchange are widely used to solve these problems. Liquid- liquid extraction Solvent extraction separation is based on the differences in the solubility of elements and their compounds in two immiscible liquid phases. Usually, the initial phase is an aqueous solution and the second phase is an organic solvent immiscible with water. Extraction is usually fast and simple process; it demands only very simple equipments. Stripping (re-extraction, back-extraction or scrubbing) involves bringing the element from the organic extract back to the aqueous phase. After extraction the concentration of the metallic complex in the organic phase can then be directly determined by atomic absorption spectrophotometric methods. The use of organic solvents in flame atomic absorption spectrometry (FAAS) increases the efficiency of the atomization, as the viscosity and surface tension of most organic solvents are lower than those of water, resulting in smaller drops and larger volumes of sample entering the flame.28,29 Organic solvents containing chlorine are not recommended in FAAS since they generate toxic products and some times extinguish the air/acetylene flame.30 The most common chelate used in atomic spectroscopy is ammonium pyrrolidine dithiocarbamate (APDC), with methyl isobutyl ketone as the organic solvent.4 Solvent extraction is convenient both for group and selective concentration of heavy metals. 27, 31-34 The main disadvantage of liquid- liquid extraction is the large consumption of solvents which is not environmentally friendly aspect. There is an urgent necessity to evaluate employed analytical procedures not only in respect to the reagents, instrumental costs and analytical parameters but also on the basis of their negative influence on the environment.35 Chromatography The most common type of chromatography for metal separation and pre-concentration is ion exchange chromatography. This process can be achieved in two ways, batch, or column. The term ‘ion chromatography’ does not refer to a specific technique defined by the nature of the stationary and mobile phases, but rather to a group of related liquid chromatographic methods that are applicable to a particular group of analytes. These analytes include inorganic anions and cations, as well as low-molecular-mass organic acids and bases.36 Different types of ligands or groups of compounds are suitable for HPLC such Schiff base chelates, hydrazones, dithizonates, and metal dithiocarbamates. For these groups of substances principally reversed-phase and adsorption systems are suitable; separations by reversed- phase systems are very well reproducible.37-41 Solid phase extraction The solid phase typically consists of small, porous particles of silica with a bonded organic phase or of an organic polymer, such as crosslinked polystyrene. The extraction can take place in a batch mode in which the solid extractant is intimately mixed with liquid sample solution. In chemical analysis it is more common to pack the solid extractant into a small tube and pass the liquid sample through the tube.41

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The nature and properties of the sorbent are of prime importance for effective retention of metallic species. Careful choice of the sorbent is thus crucial to the development of SPE methodology. Two approaches are used for loading the surface with specific organic compounds, chemical immobilization and physical adsorption. In the first case, a chemical bond is formed between the solid support surface groups and those of the organic compound (functionalized sorbent). In the second approach, the organic compound is directly adsorbed on the functional groups of the solid support surface (impregnated or loaded sorbent), either by passing the reagent solution through a column packed with the adsorbent, or by soaking the adsorbent in the reagent solution.42-45 This method is simple and convenient to apply, and has the major advantage of ease of automation. Further, high enrichment factors are achieved and the technique is less prone to sample contamination effects than other methods. A variety of solid materials such as modified polyurethane foam (PUF)46, cellulose47, activated carbon48, silica gel49 and micro crystalline naphthalene50 have been used for pre-concentration of metals. The surface can be modified chemically by chemical reaction or physically by means of adsorption, in order to enhance the selectivity toward certain metal ion or certain group of elements.51-54 Co-precipitation The pre-concentration purpose is achieved by the formation of insoluble compounds. The co-precipitation is used when direct precipitation can not separate the desired metallic species due to its low concentration in sample solution. The co-precipitation can be associated with metal adsorption on the precipitate surface or due to metal incorporation onto the precipitate structures. Inorganic or organic substances can be used as co-precipitation agents. The organic agents usually chosen are those able to originate neutral chelates with metallic species. The carrier element, is precipitated to co-precipitate trace elements in sample solutions, copper and zinc are popular, because of their limited negative effects for environment. After precipitation the precipitate can be removed by centrifugation and dissolved in acids to be measured by FAAS. Many metal ions from water samples have been pre-concentrated by co-precipitation with hydroxides of iron (III)55, indium(III)56, and zirconium57. Organic co-precipitants, generally dithiocarbamates of bismuth and copper have been widely used as efficient collectors of trace elements.58,59 A separation/preconcentration procedure based on the co-precipitation of Pb(II), Fe(III), Co(II), Cr(III) and Zn (II) ions with copper(II)-N-benzoyl-N-phenyl-hydroxylamine complex (Cu BPHA) has been also developed.60 Cloud point extraction Cloud point extraction (CPE) is an attractive technique that reduces the consumption of the solvent, extraction time and the disposal costs. Cloud point methodology has been used for the extraction and preconcentrations of metal ions after the formation of sparingly water soluble complexes.61 The CPE of metal ions involves the formation of sparingly water soluble chelates with suitable reagents. The method is based on the entrapment of uncharged moiety within a micelle formed by heating the surfactant solution above the cloud point temperature. The application of CPE offers an attractive alternative to conventional liquid-liquid extraction by reducing the consumption of and exposure to the solvent, disposal cost, and extraction time. The main parameter to attain a surfactant monomer agglomeration in a micelle-rich phase is the surfactant concentration at the cloud point temperature. The temperature-concentration phase diagram is specific for each surfactant. The effect of additives such as salts and other surfactants also has to be considered. The first application of cloud point extraction for analytical purposes described the micelle aggregation of hydrophobic anionic metal complexes.62,63 The generation of the metal chelates is a main step in the process of CPE. Several ligands, have been listed by Sun et. al. such as 1-(2- thiazolylazo)-2-naphthol (TAN), ammonium pyrrolidinedithiocarbamate (APDC), 8-hydroxyquinoline (Oxine), dithizone, diethyldithiocarbamate (DDTC), 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol (5-Br-PADAP),18 and 1-(2- pyridylazo)-2naphthol (PAN), have been used in cloud point extraction of metal ions.64

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Many metal ions have been determined by atomic absorption spectrometry such as Pb, Mn, Fe, Ag, Ni, Cu, Zn, Pd, Cr and Cd, after CPE in different kinds of samples.65-67

CONCLUSION There are many different techniques in for sample preparation for FAAS. Selection of the sample preparation method depends mainly upon the analyte concentration, matrix, instrument operation conditions, costs and the environmental considerations.

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3. M. J. Cal-Prieto, M. Felipe-Sotelo, A. Carlosena, J. M. Andrade, P. López-Mahía, S. Muniategui and D. Prada, Talanta, 56(1), 41 (2002).

4. J. R. DEAN, Atomic Absorption and Plasma Spectroscopy, Second edition, John Wiley and Sons, Ltd., (1997).

5. A. G. Howard and P. J. Stathm, Inorganic trace analysis, philosophy and practice. John Wiley and sons Ltd. (1997)

6. F. Anwar, T.G. Kazi, R. Saleem and M.I. Bhanger., Grasas y Aceites, 55(2), 160 (2004). 7. J. Sneddon, C. Hardaway, K. K. Bobbadi, and A. K. Reddy, Applied Spectroscopy Reviews, 41, 1 (2006). 8. R. Bock, Handbook of Decomposition Methods in Analytical Chemistry, Willey, New York, (1979), 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

translated and revised by I.L. Marr. F. Barbosa Jr., C. Palmer, F.J. Krug, P.J. Parsons, J. Anal. At. Spectrom. 19, 1000, (2004). J.A. Nóbrega, M.C. Santos, R.A. Sousa, S. Cadore, R.M. Barnes, M. Tatro, Spectrochim. Acta Part B, 61, 465 (2006). R. Anderson, Sample Pretreatment and Separation, John Wiley and Sons, New York, (1987). É. M. Flores, J. S. Barin, M. F. Mesko, G. Knapp, Spectrochimica Acta Part B, 62, 1051 (2007). H. Matusiewicz, Wet digestion methods, in: Z. Mester, R.E. Sturgeon (Eds.), Elsevier, Amsterdam, pp. 193 (2003). J.A. Nóbrega, L.C. Trevizan, G.C.L. Araújo, A.R.A. Nogueira, Spectrochim. Acta Part B, 57, 1855 (2002). D.D. Link, H.M. “Skip” Kingston, Anal. Chem. 72, 2908 (2000). G. Knapp, Mikrochemica Acta, II, 445 (1991) F. E. Smith, E. A. Arsenault, Talanta, 43, 1207 (1996). J.M.R. B´elanger, J.R.J. Pare, Anal. Bioanal. Chem. 386,1049, (2006). J.L. Luque-Garcia, M.D. Luque de Castro, Trends Anal. Chem. 22, 90, (2003). M. Ericsson, A. Colmsjo, Anal. Chem., 75, 1713, (2003). M. Ericsson, A. Colmsjo, J. Chromatogr. A 964 (1-2), 11 (2002). A. Serrano, M. Gallego, J. Chromatogr. A 1104 (1-2), 323 (2006). K. J. Lamble and S. J. Hill, Analyst, 123, 103R (1998). K. I. Mahan, T. A. Foderaro, T. L. Garza, R. M. Martinez, G. A. Maroney, M. R. Trivisonno, E. M. Willging Anal. Chem., 59 (7), 938 (1987). Q. Jin, F. Liang, H. Zhang, L. Zhao, Y. Huan and D. Song, TrAC Trends in Analytical Chemistry., 8(7), 479 (1999) M. Korna, E. Mortea, D. Batista dos Santosa, J. Castroa, J. P. Barbosaa, A. Teixeiraa, A. Fernandesa, B. Welza, W. Carvalho dos Santosab, E. Nunes dos Santosc, M. Korn, Applied Spectroscopy Reviews, 43,67 (2008). Y. A. Zolotov, G.I. Malofeeva, O.M. Petrukhin and A.R. Timerbaev, Pure & Appl. Chem., 59(4), 497 (1987). Hill, S. J. Chem. Soc. Rev., 26, 291, (1997). B. Welz, M. Sperling, M. Atomic Absorption Spectrometry, (Wiley. VCH. Weinheim) 3/e, (1999). R. Laus, A. dos Anjos, R. Osório, A. Neves, M. Laranjeira and V. T. de Fávere, Pak. J. Anal. Environ. Chem. 9(2), 58 (2008).

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S. Abe, T. Sone, K. Fujii and M. Endo. Analytica Chimica Acta, 274(1), 141 (1993) S. Abe, K. Fujii and T. Sono. Analytica Chimica Acta, 293(3), 325 (1994). S. Abe, J. Mochizuki and T. Sone Analytica Chimica Acta, 319(3), 387 (1996) Yi Chena, Zhenpeng Guoa,b, Xiaoyu Wang a,b, Changgui Qiu Journal of Chromatography A, 1184, 191 (2008). J. Curyło, W. Wardencki, J. Namieśnik, J. of Environ. Stud. 16(1), 5 (2007). P. R. Haddad, P. Doble, M. Macka, Journal of Chromatography A, 856, 145 (1999). P. C. Uden, D. M. Parees and F. H. Walters, Anal. Lett. 8, 795 (1975). E. F. Hilder, M. Macka and P. R. Haddad, Analyst, 123, 2865 (1998). J. M. Daud and I. M. Alakili Malaysian Journal of Analytical Sciences, 7(1), 113 (2001). H. S.Rathore, M. Kumar, K. Ishratullah. Indian Journal of Chemical Technology, 13, 84 (2006). Nabil Ramadan Bader, RASAYAN J. Chem., 3(4), 660 (2010). J. S. Fritz, Analytical Solid Phase Extraction, John Wiley and Sons, 8, (1999). M. Ganjali, M. Pourjavid and L. Babaei,.Quim. Nova, 27(29), 213 (2004) F. Shemirani, A. Alsadat, M. Niasiri and R. Kozani,. Journal of Analytical Chemistry, 59(3), 228 (2004). M. H. Mashhadizadeh, A. Moatafavi, H. Allah-Abadi, and M. R. Zadmehr, Bull. Korean Chem. Soc., 25, 9, 1309 (2004). Gama, E. M.; Lima, A. S.; Lemos, V. A. J. Hazard. Mater., 136, 757 (2006). V. Gurnani, A. K. Singh, B. Venkataramani, Anal. Chim. Acta., 485, 221 (2003). S. Xingguang, W. Meijia, Z. Yihua, Z. Jiahua, Z. Hanqi, J. Qinhan, Talanta, 59, 989 (2003). A. Goswami, A. K. Singh, Talanta, 58, 6692002). M. A. Taher, B. K. Puri, R.K. Bansal, Microchem. J., 58, 21 (1998). M. H. Mashhadizadeh, A. Moatafavi, H. Allah-Abadi, and M. R. Zadmehr, Bull. Korean Chem. Soc., 25(9), 1309 (2004). D. Budziak, E. Luiz da silva, S. Denofre da Campos, and Eduardo Carasek Michrocimica Acta, 141, 169 (2003). M. Mashhadizadeh, M. Pesteh, Mahzad Talakesh, I. Sheikhshoaie, M. Ardakani, M.Karimi. Spectrochimica Acta Part B 63, 885 (2008). M. H. Mashhadizade, M. S. Azimi, M. Pesteh, I. Sheikhshoaei,M. M. Ardakani, M. A. Karimi. Spectrochimica Acta Part B 63, 889 (2008). Y. Kashiwagi, E. Kokufuta, Anal Sci. 16, 1215 (2000). M. Hiraide, Z. Chen, H. Kawaguchi, , Anal Sci 7, 65 (1991). Y.Tamari, R. Hirai, H. Tsuji, Y Kusaka, Anal Sci. 3, 313 (1987) H. Sato, J. Ueda, Anal Sci.,17, 461 (2001). S. Tokalıoğlu, T. Oymak, S. Kartal, Microchim Acta, 159:133 (2007). Ş. Saçmacı, and Ş. Kartal, Microchim Acta, 170, 75 (2010). L. M. Jamshid and G. Karim-Nezhad, Iran. J. Chem. Chem. Eng., 24, 4, (2005) M. F. Giné, A. F. Patreze, E. L. Silva, J. S. Sarkis, M. H. Kakazub, J. Braz. Chem. Soc., 19(3), 471 (2008). C. B. Ojeda, F. S. Rojas, J. C. Pavón. American Journal of Analytical Chemistry, 1, 127, (2010). Z. Sun, P. Liang, Q. Ding, J.Cao, ANALYTICAL SCIENCES, 22, 911 (2006). N. Dalali, N. Javadi, Y. Kumar Agrawal, Turk J Chem, 32, 561 (2008). M. Ghaedi, K. Niknam, E. Niknamb and M. Soylak, Journal of the Chinese Chemical Society, 56, 981 (2009). P. Liang, J. Li, and X. Yang, Microchim Acta, 152, 47 (2005) M. Ghaedi, A. Shokrollahi, K. Niknam, E. Niknam, A. Najibi, M. Soylak, Journal of Hazardous Materials, 168, 1022 (2009). G. D. Matos, E. B. dos Reis, A. C.S. Costa, S. L.C. Ferreira, Microchemical Journal, 92, 135 (2009).

[RJC-702/2011]

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Nabil Ramadan Bader

Vol.4, No.1 (2011), 56-65 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

BIOSORPTION CHARACTERISTICS OF Cr6+ FROM AQUEOUS SOLUTIONS BY PINUS SYLVESTRIS L. TIMBER FILLINGS Ackmez Mudhoo* and Preesena Devi Seenauth Department of Chemical & Environmental Engineering, Faculty of Engineering, University of Mauritius, Réduit, Mauritius. * Email: ackmezchem@yahoo.co.uk ABSTRACT Pinus Slyvestris L. timber fillings were boiled and utilized as a biosorbent for the removal of Cr6+ from a synthetic wastewater. The Cr6+ removal increased from 34.8% to 69.41% as biosorbent dosage increased from 3.0 to 8.0 g/L, while the uptake of Cr6+ decreased from 6.09 mg/g to 4.78 mg/g as the biosorbent dosage increased from 3.0 to 8.0 g/L, when the initial Cr6+ concentration was 50 mg/L. The experimental equilibrium data were well described by both the Langmuir and Dubinin-Radushkevich adsorption isotherm models. Based on R2 values, the Langmuir model fitted the equilibrium biosorption data best, confirming monolayer adsorption of Cr6+ onto the biosorbent surface. The biosorption kinetics of Cr6+ was best described by pseudo-second-order kinetics since at all concentrations, the R2 values were higher than the corresponding pseudo-first order values. The overall results indicated that P. Slyvestris L. is a promising biosorbent for Cr6+ removal from dilute aqueous solutions. Keywords: Chromium, biosorption, Pinus Slyvestris L., Langmuir, pseudo-second order kinetics © 2011 RASĀYAN. All rights reserved.

INTRODUCTION The removal of toxic heavy metals from aqueous waste streams is currently one of the most important environmental issues being researched. Among the several heavy metals detected in industrial effluent namely lead, mercury, uranium, selenium, zinc, arsenic, cadmium, gold, silver, copper, nickel, include lead, chromium, mercury, uranium, selenium, zinc, arsenic, cadmium, gold, silver, copper, nickel, found in wastewater streams chromium is one among the most toxic heavy metal contaminant. It exists in two stable oxidation states, Cr3+ and Cr6+. The Cr6+ state is of particular concern because this form is hazardous to health. Cr6+ is introduced into the natural bodies of water from industries like electroplating, leather tanning cement industries, mining, metal finishing industries photography industries dyeing and fertilizer1,2. Cr6+ is highly toxic in nature and in humans it can cause a variety of diseases such as dermatitis, congestion of respiratory tracts and perforations in the nasal septum. It also affects aquatic life as it bio-accumulates in their living tissues throughout the food chain, which has humans at its top, multiplying the danger. The effluent from the industries may contain chromium at concentrations ranging from tenths to hundreds of mg/L3. The tolerance limit for Cr6+ for effluent discharge is 0.05 mg/L (EPA, 2002). In order to comply with this limit, it is essential that industries treat their effluents to reduce the Cr6+ to acceptable levels. Consequently, effluents contaminated with chromium should be treated before discharging into the environment. A number of physical and chemical treatment processes like electrolysis, ion exchange, reverse osmosis, ion-flotation and chemical precipitation have been reported4. Most of these methods suffer from drawbacks such as high capital and operational costs, the disposal of the residual metal sludge and are ineffective for Cr6+ concentrations lower than 100 mg/L5. Hence, in pursuit of novel and ‘green’ heavy metal techniques, biosorption is one of the potential alternatives that can be used to remove chromium from chromium laded waters.

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Biosorption Biosorption describes the removal of heavy metals by the passive binding to non-living biomass from an aqueous solution6. The biosorption process involves a solid phase (biosorbent or biological material) and a liquid phase (solvent, normally water) containing a dissolved species to be sorbed. Due to higher affinity of the biological materials for the chromium ions species, the latter is attracted and bound there by different mechanisms. The process continues till equilibrium is established between the amount metal ions bound and its portion remaining in the solution. The factors affecting the metal binding qualities of biomaterials or their affinity for a metal dissolved in aqueous media include the chemical nature of the metal ions (e.g. size, valence, electron orbital structure, stability of the chemical forms in nature) and that of the biomass (e.g. charge density and structure of the polymer chain, functional groups), and medium conditions (e.g. pH, temperature, ionic strength, presence of competing organic or inorganic metal chelators). Some of the biosorbents that have been tested for Cr sorption are coconut waste7, seeds of Ocimum Basilicum8, defatted rice bran, rice hulls, soybean hulls and cotton seed hulls, waste tea9 and bengal gram husk1. However, these adsorbents do not show high adsorption capacity or need long adsorption equilibrium time. For that reason, the search for new, economical, easily available and effective adsorbent needs to be pursued. Research objectives In this study, the Scots pine (Pinus Slyvestris L.) timber filings were used to adsorb Cr6+ from an aqueous solution. P. Slyvestris L. is the most widely distributed pine in the world. The latter is a wood native over much of Europe, northern and western Asia. It is introduced in many areas in the United States and Canada, and is naturalized in the Northeast and in the Great Lakes states. Scots pine contains various organic compounds such as lignin (with polyphenolic groups), cellulose (with numerous hydroxyl functions) and hemicelluloses (with carboxylic and hydroxyl groups). These functional groups may be useful for binding ions of chromium. It was chosen as it is a cheap and readily available material, basically a waste from the wood industry. The aim of this study has been to analyse the batch biosorption characteristics for Cr6+ ions in synthetic aqueous solutions using treated P. Slyvestris L. biosorbent. The specific objectives were to analyse the effects of pH, concentration of the initial chromium and the dosage of the biosorbent on biosorption of Cr6+, determine the required optimum conditions for maximum adsorption of Cr6+ by P. Slyvestris L., model the batch equilibrium data using Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherm models, and finally study the sorption kinetics using the pseudo first and pseudo second order models.

EXPERIMENTAL Synthetic wastewater stock solution Synthetic wastewater was used and prepared using analytical grade potassium dichromate salt. A stock solution of the synthetic wastewater of 1000 mg/L was prepared according to the ISO 9174. The solution was then stored in borosilicate glass containers at room temperature in the dark. This solution is stable for about a year. The stock solution was then used to prepare standard solutions of concentration 50, 60, 100, 150 and 200 mg/L. Standard solutions from stock solutions The 1000 mg/L stock solution of chromium was used to prepare the standard solutions of different concentrations; 50, 60, 100, 150 and 200 mg/L by adding 25, 30, 50, 75 and 100 mL respectively of the stock solution into 500 mL volumetric flasks. Procurement and pretreatment of biosorbent P. Slyvestris L. is normally used in the manufacture of furniture and the Scots pine timber fillings are generated as a waste product in this course. The timber fillings used in this study was obtained from a local carpenter. P. Slyvestris L. timber fillings were pretreated by boiling it in double-distilled water before being used for the study. The pretreatment was done for the following reasons: removal of inherent colour of the biosorbent as it may interfere with analysis of the Cr6+; functional groups within the

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biosorbent get opened and hence there is more adsorption possibility; the surface area of the Scots pine is increased, thereby liberating more active binding sites; and polymerisation of the biosorbent takes place. P. Slyvestris L. timber fillings were first cut into pieces of 1 to 2 cm long. In the ratio of 1:50 w/v the material was then boiled for 3 h at 105ºC in distilled water. It was then dried in hot air oven at 105ºC for 24 h. Experimental procedure The pH of synthetic wastewater solution, concentration of wastewater solution and dosage of the biosorbent for optimum biosorption of Cr6+ were studied. As mentioned earlier, all the experiments were carried out in batch mode in 500 mL borosilicate conical flasks. For each variable tested, five sets of experiments were carried out using a fixed volume of 400 mL of the synthetic wastewater. An agitation speed of 200 revolutions per minutes (r.p.m) was kept constant throughout the whole experiment. The experiments were run for a contact period of four hours at room temperature. 15 mL of samples from the solution were withdrawn at constant time interval of 40 minutes. The samples were then filtered using Whatman filter paper No. 1 and tested by the AAS at wavelength 357.9 nm. In parallel to the experiments, blank samples were run under similar conditions of concentration, temperature, pH without the biosorbent to correct for any adsorption on the internal surface of bottles. Operating conditions The following operating conditions were kept constant throughout the study: 1. A fixed amount of the synthetic wastewater, 400 mL, was used for all the runs. 2. The study was carried at room temperature or 25 ºC. 3. An agitation speed of 200 r.p.m was kept constant throughout the study. 4. A contact period of four hours was kept constant for all runs. Determination of optimum pH The optimum pH for biosorption of Cr6+ was determined and was kept constant throughout the study. In this process of determination of optimum pH, the concentration of the synthetic wastewater and the dosage of the biosorbent were kept constant at 50 mg/L and 3.5 g/L respectively. The initial pH of the solutions were varied from 2.7, 3.5, 5.0, 7.0, 7.71, 8.04, 8.7 to 9.7 which was adjusted by adding either 0.1 M sulphuric acid (H2SO4) or 0.1 M NaOH. The filtrate was then tested on Atomic Absorption Spectrometric and the results were recorded. The percentage Cr6+ removal (R in %) of the synthetic wastewater at the different pH values were calculated using the following relationship:

R (%) =

Co − CF × 100 Co

where Co is the initial concentration of Cr6+ in the synthetic wastewater and CF is the final concentration of Cr6+ in the synthetic wastewater. Optimum conditions for maximum biosorption of Cr6+ Once the optimum pH determined, it was kept constant throughout the whole study. The effect of dosage of pine wood added to the synthetic wastewater and the concentration of the synthetic wastewater on biosorption of Cr6+ were now studied. To study the effect of dosage on chromium biosorption, 50 mg/L of synthetic wastewater was used and the dosage of pine wood was varied from 3.0, 3.5, 4.0, 6.0 to 8.0 g/L. The same experiment was repeated using different concentrations of the synthetic wastewater: 60, 100, 150 and 200 mg/L. The samples were filtered using Whatman filter paper No. 1 and tested using the Atomic Absorption Spectrometry method. The Cr6+ removal (R in %) of the synthetic wastewater for these sets of experiments were calculated using the following equation

qe =

V (C o − C e ) W

where, qe is the Cr6+ uptake at equilibrium in mg/g. Co is the initial concentration of Cr6+ in the synthetic wastewater in mg/L, Ce is the concentration of Cr6+ at equilibrium in the synthetic wastewater in mg/L, V is the volume of synthetic waster used in mL and W is the mass of Scots pine timber fillings used in g.

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The equilibrium results were used to plot adsorption isotherms to assess the suitability of the most common adsorption isotherms: Langmuir, Freundlich and Dubinin-Radheuskevich equations.

RESULTS AND DISCUSSION Effect of pH on biosorption of Cr6+ The effect of pH biosorption of Cr6+ by the Scots pine timber fillings was studied in the initial pH range of 2 to 10. The experiments were carried out at 50 mg/L initial Cr6+ concentration with 3.5 g/L biosorbent dose, at room temperature for a contact time of four hours at constant agitation speed of 200 r.p.m. Fig. 1 shows the effect of pH on biosorption of Cr6+.

Fig.-1: Effect of pH on biosorption of Cr6+ (Conditions: 50 mg/L, pH = 2.2, 3.5 g/L, 200 r.p.m and 25 ºC)

The results show that the Cr6+ biosorption by P. Slyvestris L. was strongly pH dependent. A decrease in Cr6+ removal efficiency from 34.80 % (initial pH = 2.2) with increasing initial pH was obtained and the removal finally reached 15.24 % at pH 9. The optimum pH was found to be 2.2, where the removal efficiency was 34.80%. Hence, all subsequent experiments were carried out at pH 2.2. Cr6+ may exist in the aqueous phase in different anionic forms such as chromate (CrO2−4), dichromate (Cr2O2−7), or hydrogen chromate (HCrO−4) 10. It is well known that the dominant form of Cr6+ at lower pH is HCrO−4. The increase in Cr6+ sorption with decreasing pH is in agreement with the results of a study on biosorption of Cr6+ using cone biomass of P. Slyvestris L. by Ucun et al. 11. It was reported that the percentage Cr6+ adsorbed increased when pH of the solution was decreased from 7.0 to 1.0. Similar results were found when P. Slyvestris L. was used to adsorb other metal ions. According to a study carried out by Kaczala et al.12 using untreated P. Slyvestris L. sawdust for sorption of lead (II) and vanadium, sorption efficiency increased when initial pH was reduced from 7.4 to 4.0. Effect of initial Cr6+ concentration on biosorption The study was carried out at different Cr6+ concentrations; 50, 60, 100, 150, and 200 mg/L with biosorbent dose 8.0 g/L. The same experiments were carried out using different biosorbent doses: 3.0, 3.5, 4.0, and, 6.0 g/L. All the experiments were run at room temperature for a contact period of four hours, with a constant agitation speed of 200 r.p.m and pH 2.2. The results obtained for the effect of concentration on Cr6+ removal are shown in Fig. 2. It may be inferred that Cr6+ removal decreased from 69.41 % to 30.24 % as Cr6+ concentration increased from 50 to 200 mg/L. However, the actual amount of Cr6+ adsorbed per unit mass of biosorbent increased with the increase in Cr6+ concentration in the test solution. When the biosorbent dosage was 8.0 g/L, the uptake of Cr6+ increased from 4.78 mg/g to 7.71 mg/g as Cr6+ concentration increased from 50 to 200 mg/L. The highest percentage of Cr6+ removed was 69.41 % at 50 mg/L using a biosorbent dose of 8.0 g/L. The effect of initial concentration was studied at

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other biosorbent dosages other than 8.0 g/L, namely at 3.0, 3.5, 4.0, and, 6.0 g/L. The same trend of percentage Cr6+ removal decreasing with increasing Cr6+ concentration was observed. The removal of Cr6+ decreased with an increase in initial Cr6+ concentration. This may be due to an increase in the number of Cr6+ ions competing for the available binding sites for the fixed amount of P.Slyvestris L. biosorbent. At lower concentrations, all Cr6+ ions present in the solution would interact with the binding sites and thus biosorption be more complete for all ions. At higher concentrations, more Cr6+ is left unabsorbed in solution due to the saturation of binding sites. Several researchers have also found that there is a decrease of removal of metal ions with increase of initial metal concentration. Baral et al.13 studied adsorption of Cr6+ by treated weed Salvinia cucullata. They observed that the adsorption decreased from 60.8% to 46.8% and the uptake increased from 121.6 to 163.7 mg/g when the concentration increased from 400 to 700 mg/L. Moreover, Babu and Gupta10 studied adsorption of Cr6+ using activated neem leaves, and also found that the removal of Cr6+ decreased with an increase in initial Cr6+ concentration. The d removal decreased from 99.95% to 89.94% and adsorption capacity increased from 3.98 to 62.9 mg/g when Cr6+ concentration increased from 40 to 700 mg/L. Consequently, it may be inferred that the results obtained for this study are in accordance with diverse previous studies carried. Removal

Fig.-2: Removal of Cr6+ with initial Cr6+ concentration at 8.0 g/L biosorbent dosage. (Conditions: pH = 2.2, 8.0 g/L, 200 r.p.m and 25 ยบC)

Effect of biosorbent dosage The effect of biosorbent dosage on biosorption of Cr6+ by the Scots pine timber fillings was also considered. The experiments were carried out at various biosorbent dosage; 3.0, 3.5, 4.0, 6.0, and 8.0 g/L with Cr6+ concentration of 50 mg/L. The same experiment was run out with diverse Cr6+ concentrations; 50, 60, 100, 150 and 200 mg/L. All the experiments were run at same stated conditions above. The biosorption yields obtained from experimental data for the effect of biosorbent dosage is presented in Fig. 3. It may be observed that the removal of Cr6+ increased with an increase of P. Slyvestris L. dosage. The Cr6+ removal increased from 34.80 to 69.41% as biosorbent dosage increased from 3.0 to 8.0 g/L, when the initial Cr6+ concentration was 50 mg/L. Even though adsorption increased with an increase in biosorbent dosage, the actual amount of Cr6+ adsorbed per unit mass of biosorbent decreased with the increase in the biosorbent dose in the test solution. When the initial Cr6+ concentration was 50 mg/L, the uptake of Cr6+ decreased from 6.09 mg/g to 4.78 mg/g as the biosorbent dosage increased from 3.0 to 8.0 g/L. The results obtained for the analysis of effect of biosorbent dosage was studied at other initial Cr6+ concentrations. Similar trends as that of 50 mg/L were obtained. There was a general decrease in Cr6+ removal with increasing Cr6+ concentration. The observed increase in biosorption with biosorbent dose can be attributed to the increased biosorbent surface area and availability of more adsorption sites. On the other hand, the unit biosorption decreased with increase in biosorbent dose. This may be attributed to overlapping or aggregation of adsorbent surface area available to Cr6+ ions and an increase in the

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diffusion path length. Semerjian14 observed that cadmium uptake decreased from 5.13 to 0.11 mg/g as sawdust concentration increased from 1 to 50 g/L. Baral et al.13 found that the adsorption of Cr6+ by treated weed Salvinia cucullata increased from 31.5 to 66% and the uptake decreased from 196.5 to 137 mg/g when the adsorbent dose increased from 0.8 to 2.4 g/L.

Removal

Fig.-3: Effect of biosorbent dosage on biosorption at 50mg/L (Conditions: pH = 2.2, 50 mg/L, 200 r.p.m and 25 ÂşC)

Adsorption isotherms The equilibrium data for removal of the Cr6+ by P.Slyvestris L. weres analysed using the Langmuir, Freundlich and Dubinin-Radushkevich adsorption models. Langmuir isotherm model: The Langmuir equation was chosen for the estimation of maximum adsorption capacity corresponding to complete monolayer coverage on the adsorbent surface. Application of the Langmuir isotherm equation to analyze the equilibrium isotherms of Cr6+ gave linear plots (Fig. 4) indicating the applicability of this classical adsorption isotherm to this adsorbateâ&#x20AC;&#x201C;adsorbent system. The regression correlation coefficients (R2) generated were 0.970 and 0.975 for initial Cr6+ concentrations of 50 and 60 mg/L, respectively (Table 1) which are high positive values, indicating that the Langmuir model fits the experimental data very well. The high values of correlation confirmed the monolayer adsorption of Cr6+ onto the biosorbent surface. Since there was less than 10% difference between the values obtained for Langmuir constant b, an average value was taken and was found to be 0.127 L/mg. The maximum monolayer coverage capacities qmax was 7.465 mg/g. A further analysis of the Langmuir equation was made on the basis of a dimensionless equilibrium parameter, RL defined by Webber and Chakkravorti15. The dimensionless equilibrium parameter, RL, was calculated for different initial Cr6+ concentrations and the results are tabulated in Table 2. The Langmuir dimensionless parameter remained between 0.038 and 0.136 for concentrations ranging from 50 to 200 mg/L, which were consistent with the requirement 0 < RL < 1 for favorable adsorption. Table-1: Langmuir parameters for the study at varying Cr6+ concentrations Initial Cr6+ concentration (mg/L)

qmax (mg/g)

b ( L/mg)

R2

50

7.41

0.127

0.970

60

7.52

0.126

0.975

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Fig.-4: Langmuir isotherms for biosorption of Cr6+

Fig.-5: Freundlich isotherms for biosorption of Cr6+

Freundlich isotherm model The Freundlich isotherm model was chosen to estimate the adsorption intensity of the sorbent towards the adsorbent. The experimental data of the study was fitted a plot of log qe against log Ce. Fig. 5 shows the Freundlich isotherms at initial Cr6+ concentrations of 50 and 60 mg/L. As clearly illustrated in this plot, the applied Freundlich isotherm equation gave linear plots for P. Slyvestris L. The regression coefficients (R2) generated (Table 2), were found to be 0.768 and 0.700 for 50 and 60 mg/L, respectively. An average value of KF was taken for the KF values found at the two different Cr6+ concentrations. Hence, the value of KF, the binding constant, was taken to be 2.148 mg/g. Values of 1/n less than unity were obtained mostly for the P. Slyvestris L and the exponent n, was 3.279 and 3.367. Hence, the ‘n’ values were within the required range of 2 to 10, thus demonstrating favorable adsorption. Dubinin-Radushkevich isotherm model Dubinin-Radushkevich isotherm model was chosen to estimate the characteristic porosity of the biomass and the apparent energy of adsorption. To analyse the experimental data using this model a plot of ln qe against ε2 for P. Slyvestris L (Fig. 6). The plots yielded straight lines indicating good fit to the experimental data. The R2 values obtained were 0.854 and 0.767 for initial Cr6+ concentrations of 50 and 60 mg/L, respectively (Table 3). The Dubinin-Radushkevich BDR was 0.00005 mol2/kJ2 and qs was 76.86 mg/g. The apparent energy (E) of adsorption from Dubinin-Radushkevich isotherm model was found to be 100.3 kJ/mol.

Fig.-6: Dubinin-Radushkevich isotherms for adsorption of Cr6+

BIOSORPTION CHARACTERISTICS OF Cr6+

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A. Mudhoo and P. D. Seenauth

Vol.4, No.1 (2011), 56-65 Table-2: Freundlich parameters at initial Cr6+ concentrations of 50 and 60 mg/L Initial Cr6+ concentration (mg/L) 50

n 3.279

KF (mg/g) 2.143

R2 0.768

60

3.367

2.153

0.700

Table-3: Dubinin-Radushkevich parameters for the study at varying Cr6+ concentrations Initial Cr6+ concentration (mg/L)

qs (mg/g)

BDR (mol2/kJ2)

R2

50

79.25

0.00005

0.854

60

74.47

0.00005

0.767

Comparison of isotherm models Based on the R2 values, the Langmuir model is the best fitting model followed by the DubininRadushkevich isotherm and finally the Freundlich isotherm for adsorption of Cr6+ by P. Slyvestris L. The present adsorption isotherms model results go in concert with the conclusions of other researchers on the adsorption of Cr6+. Babu and Gupta10 found that the equilibrium binding data for Cr6+ adsorption onto activated neem could be described by the langmuir adsorption models while Ucun et al.16 found that Freundlich isotherm model described best adsorption of Cr6+ onto cone biomass of P.Slyvestris L. Kinetic modeling The biosorption kinetics was investigated for a better understanding of the dynamics of the adsorption of Cr6+ onto P. Slyvestris L and for obtaining predictive models that allow the estimation of the amount adsorbed with the treatment time. The pseudo-first and second-order kinetic modelswere applied to the experimental data obtained. Pseudo first order model From the experimental data of the study, a linear plot between log (qe −qt) and t was obtained (Fig. 7). The k1 and qe values for the initial concentration of 50 mg/L were found to be 0.030 min−1 and 3.373 mg/g, respectively. The R2 value was 0.976 which indicates a very high level of positive correlation of the data to the pseudo-first order model. Linear plots of between log (qe −qt) and t were also drawn at various initial Cr6+ concentrations and the R2 values obtained ranged from 0.966 to 0.957 indicating that this model represented the adsorption kinetic behavior well. Pseudo second order equation To analyse the experimental data using the pseudo second order model, a linear plot between t/qt and t was equally obtained (Fig. 8). In this case, all R2 values were higher than those for the pseudo first order model. The k2 and qe values for the initial concentration of 50 mg/L were 0.042 [min/ (mg/g)] and 4.878 mg/g, respectively. Good agreement has also been observed between the experimental value of qe (4.783 mg/g) and those obtained from the slope (4.878 mg/g). Linear plots of between log (qe −qt) and t were further drawn at various initial Cr6+ concentrations, and the values of the rate constants, k2, were found to decrease from 0.042 to 0.009 [min/(mg/g)]) for an increase in the initial Cr6+ concentration from 50 to 200 mg/L. Since the regression coefficient values (Table 4) are very close to unity, there is thence very good agreement between the true and calculated values of qe at various initial concentrations. It may be hence inferred that that the Cr6+ biosorption on P.Slyvestris L. better followed the pseudo second-order kinetics. The adsorption kinetics model results of this study are also in close agreement with the findings of other workers on the adsorption of Cr6+. Dermibas et al.17 found that adsorption of Cr6+ by activated carbons prepared from agricultural wastes followed pseudo second-order rate equation very well. Baral et al.13 also found that adsorption of Cr6+ by treated weed Salvinia cucullata followed pseudo second order model.

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Fig.-8: Pseudo second order model for adsorption of Cr6+

Fig.-7: Pseudo first order model for adsorption of Cr6+

Table-4: Pseudo second order model parameters for adsorption of Cr6+ Initial Cr6+ concentration (mg/L)

qe (mg/g)

k2 [min/(mg/g)]

R²

50 60

qexp (mg/g) 4.783 4.989

4.878 5.128

0.042 0.024

0.998 0.997

100

5.786

5.988

0.015

0.993

150 200

7.569 7.696

8.333 8.065

0.004 0.009

0.957 0.989

CONCLUSION The highest Cr6+ removal was 69.41% at an initial concentration of 50 mg/L. The effect of pH on the biosorption capacity of P. Slyvestris L. was found to be significant with the equilibrium biosorption capacity increasing from 30.81% to 69.41% when pH was decreased from 10 to 2.0. (optimum pH was 2.2). Cr6+ removal decreased from 69.41 % to 30.24 % while the uptake of Cr6+ increased from 4.78 mg/g to 7.71 mg/g as Cr6+ concentration increased from 50 to 200 mg/L, when the biosorbent dosage was 8.0 g/L. This study confirmed that the effect of the biosorbent dosage is fundamental on the biosorption capacity of P. Slyvestris L. As research sequel, further study on the biosorption of Cr6+ in synthetic competitive and real wastewater samples with P. Slyvestris L. biosorbent under both batch and continuous need to be extensively conducted. Recirculation modes may also be included in the experimental designs to obtain pertinent raw data necessary for continuous adsorber column design.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

N. Ahalya, R.D. Kanamadi, and T.V. Ramachandra, Elect. J. Biotechnol., 8, (2005). Celik, and A. Demirbas, Energy Sour., 27, 1167 (2005). S. Rengaraj, K.H. Yeon, And S.H. Moon, J. Hazard. Mater., B87, 273 (2001). D. Sud, G. Mahajan, and M.P. Kaur, Bioresour. Technol., 99, 6017 (2008). J.T. Matheichal, Q. Yu, and J. Feltham, Environ. Technol, 18, 25 (1997). T.A. Davis, B. Volesky, and A. Muccib, Water Res., 37, 4311 (2003). K. Selvi, S. Pattabhi, and K. Kadirvelu, Bioresour. Technol., 8, 87 (2001). M. Melo, and S.F. Ä&#x17D;'souza, Bioresour. Technol., 92, 151 (2004). H.M. Amir, N. Dariush, V. Forugh, and N. Shahrokh, Am. J. Appl. Sci., 2, 372 (2005). B.V. Babu, and S. Gupta, Adsorption, 14, 85 (2008).

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11. 12. 13. 14. 15. 16. 17.

H. Ucun, Y. Kemal Bayhan, Y. Kaya, A. Cakici, and F. Algur, Bioresour. Technol., 85, 155 (2002). F. Kaczala, M. Marques, and W. Hogland, Bioresour. Technol., 100, 235 (2009). S.S. Baral, S.N. Das, G.R. Chaudhury, and P. Rath, Adsorption, 14, 111 (2008). L. Semerjian, J. Hazard. Mater., 173, 236 (2010). T.W. Webber, and R.K. Chakkravorti, AlChE J., 20, 228 (1974). H. Ucun, O. Aksakal, and E. Yildiz, J. Hazard. Mater., 161, 1040 (2009). E. Demirbas, M. Kobya, and A.E.S. Konukman, J. Hazard. Mater.,154, 787 (2003). [RJC-727/2011]

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Vol.4, No.1 (2011), 66-72 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND BIOLOGICAL EVALUATION OF (7HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID HYDRAZIDE DERIVATIVES USED AS A POTENT BIOLOGICAL AGENTS Deepak P. Kardile*1, Manchindra R.Holam1, Ankit S. Patel1 and Shailesh B.Ramani1 1,*

Department of Pharmaceutical Chemistry, Shree Swaminarayan Pharmacy College, Kevadia Colony, Gujarat-393151, India. *E-mail: Kardile.deepak@rediffmail.com

ABSTRACT The purpose of this research was to development of new potent bioactive molecule with less toxic, safer and easy available. Modern therapeutic is based on scientific observation supported by systematic assessment of activity of drug is simulated and clinical condition. The integrity of the drug molecule, optimization of biological effect, uniform and consistent availability of drug from the dosage. In the present investigation an attempt is carried out for the synthesis of (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazide (V) from (7-hydroxy-2-oxo-2H-chromen-yl) acetic acid ethyl ester (IV) and to carry out their biological activity. Various Phenols like resorcinol (I); m-cresol;has been condensed with ethylacetoacetate (II) in presence of concentrated sulphuric acid to form 7-hydroxy-4-methylcoumarin (III) by pechmann reaction. Further these (7-hydroxy-2-oxo-2H-cromen-4-yl) acetic acid hydrazide (V) condensed with various Schiff’s base. Also ethyl [(8-amino-4-methyl-2-oxo-2H-chromen-7-yl) oxy] acetate have been condensed with fluroaniline, 4chlorodinitrobenzene, 4-chlorobenzonitrile, dibromoethane and dibromopropane. Hydrazide derivatives were synthesized to increase Log P value by increasing microbial intracellular concentration and to decrease microbial resistance. The newly synthesized compounds were tested for its antimicrobial, analgesic and antiinflammatory activity. The structures of newly synthesized compounds were established on the basis ofelemental analysis, IR,1H NMR and mass spectral data. Keywords:Antimicrobial, Analgesic and Antiinflammatory activity, Coumarin and Microbial intracellular concentration. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION A number of natural and synthetic coumarin (2-oxo-2H-chromen) derivatives have been reported to exert notably antimicrobial1-4, analgesic5-6 and anti-inflammatory7-8 activity .Moreover; the antibiotic novobiocin belongs to the hydroxy coumarin series. On the other hand, a large number of hydrazides have been reported to possess antibacterial9, antifungal10,antitumor11,antitubercular12,antiviral13 and other biological activities. Presently there are a number of drugs used clinically. In view of these, a project was undertaken to synthesize a new series of (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazide by conventational method and to evaluate the new compounds for their pharmacological activity. The title compounds were screened for antimicrobial activity by cup plate method14, analgesic activity studies were carried out by acetic acid induced writhing method15and anti-inflammatory ‘Carrageenan induced oedema test16. Synthesis of title compounds was shown in Scheme 1and 2. The physical constants, yield and analytical data of (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazidederivatives.

EXPERIMENTAL The melting points were determined in open capillary tube using Precision melting point apparatus and uncorrected. Thin-layer chromatography was performed with fluorescent silica gel plates HF254 (Merck),

(7-HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID

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Vol.4, No.1 (2011), 66-72

plates were viewed under UV 254 and 265 light. Infrared spectra’s (v-cm-1) were recorded on a Shmadzu FT-IR 4000; using KBr disks.1H-NMR spectra were recorded on Bruker Spectrophotometer at 300MHz frequency in CDCl3 as well as DMSO using TMS as internal standard reference. Peaks are reported in ppm downfield of TMS. Mass spectra were recorded on ‘GCMS-QP2010s’ instrument by direct injection method. 7-Hydroxy-4-methyl coumarin (III) In two necked 500ml round bottom flask take 250 ml conc. H2SO4 and keep into ice bath until the temperature of solution becomes 0-10 0c .after solution becomes ice cold to this add solution of resorcinol (I), 33 gm(0.01moles) and 35 ml (0.01moles) of ethylacetoacetate(II) dropwise for two hrs after completion of addition the solution stirred for one hr at room temperature and this reaction mixture Was stirred for 16 hrs at room temperature after this the reaction mixture was added into crushed ice. The yellowish solid separated out which was filtered off and the solid was dissolved into the 5% sodium hydroxide solution. And made acidic with 2M H2SO4the resultant solid separated was filtered and wash with ice cold water. (7-Hydroxy-2-oxo-2H-chromen-4-yl) acetic acid(IV) In two necked 500 ml R.B.F. take 50-60ml dry DMF. To this add 10gms 7-hydroxy-4-yl coumarin (III), and ethylchloroacetate 6.8 ml (0.056moles) and anhydrous pot. Carbonate 7.7 gms. The resultant mixt was stirred for 9-10 hrs at 80oC .Then after completion of reaction which monitored by taking TLCthe reaction mixture was filtered and pour into large amount of water. The solid separated was filtered and wash with water .the solid was dried and recrystlised from ethanol. The melting point of solid was found to be 212oC-214oC. (7-Hydroxy-2oxo-2H-chromen-yl) acetic acid hydrazide(V) In 500ml R.B.F take 20ml of absolute ethanol to this add 7gms (0.026mole) of (7-hydroxy-2oxo-2Hchromen-4-yl) acetic acid (IV) and 1.3ml(0.026mole) of hydrazine hydrate. The resultant reaction mixture was refluxed for 5-6hrs .after completion of reaction which monitored by taking TLC the reaction mixt was added into ice-cold water. The solid separates out which was filtered and dried and recrystlised from ethanol. General procedure for synthesis of (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazide derivatives (VI a-j) In two necked R.B.F. take 20ml absolute ethanol to this add 1gm (0.00mole) of (7-hydroxy-2oxo-2Hchromen-4-yl)acetic acid hydrazide (V). To this solution add equimolar amount of different aromatic aldehyde and drop glacial acetic acid. The resultant reaction mixt was pour into the ice-cold water. The solid separated was filtered and dried. The solid was recrystlised into ethanol. Table-1:Different aryl aldehyde attaches with the hydrazide-hydrazone. S.No. 1

Compound Code VIa

R-CHO

Sr.No.

CHO

6

Compound Code VIf

R-CHO CHO H 3C

N CH3

2

VIb

H3C

CHO

7

CHO

VIg

F

H3C CH3

(7-HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID

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Vol.4, No.1 (2011), 66-72 3

CHO

VIc

8

CHO

VIh H 3C

O 2N 4

CHO

VId

9

CHO

VIi

CH3

5

O

Br

VIe

10

VIj

CHO O

CHO

Table-2: Some characterizations of the compounds Compound Code VI a

Molecular formula

M.P. (oC)

Rf Value

% Yield

LCMass -

C19H16N2O4

213

0.65

67 377

C22H22N2O4

218

0.45

64

C19H15N3O6

210

0.65

74

C20H18N2O4

198

0.53

71

C17H18N2O5

207

0.42

67

C21H21N3O4

210

0.45

65

C19H15FN2O4

216

0.56

65

IR (KBr cm-1) 3325,3227,3169,2920, 1722,1599,1153 3325,3227,2920, 1722,1650,1599,1153

VI b

VI c VI d VI e

377

3325,3227,3169,1722, 1599,1337,1153 3325,3227,3169,2960, 1722,1620, 1599,1153 3325,3227,3169,1722, 1632,1599,1153 3325,3227,3169,1722, 1599,1153,813

VI f

VI g VI h VI i VI j

C20H18N2O5

214

0.41

78

C19H15BrN2O4

219

0.55

66

C21H18N2O4

212

0.47

NMR) (δ ppm) 2.40(s,3H,,CH3) , 3.81(t,1HCH,benzopyran ring) ,6.11(d,2H ,CH2), 6.20 -7.45(m,8H,ArCH),10.52(s,1H,NH) 2.42 (s,3H,CH3), 4.37(d,1H,CH of ring), 6.22(s,2H ,CH2), 11.36(s,1H,NH), 6.44 8.15(m,7H,Ar-CH) --

-

-

-

-

-

-

-

-

-

-

-

73

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RESULTS AND DISSCUSION From the literature survey it reveals that the coumarin have been reported for number of pharmacological activities and some molecules have shown significant activities and some compounds shows moderate and good activities. Here we have synthesized some novel (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazide derivatives analogues and screened them for their antibacterial, anti-inflammatory, analgesic activities. The purity and homogeneity of the synthesized compounds were preliminary checked by their physical constant. The final compounds were found to be soluble in organic solvents. These compounds were characterized by spectral studies for structural elucidation and studies showed satisfactory results. Biological Agents All the newly synthesized novel (7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid hydrazidewere assayed in vitro for their antibacterial activity against Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria), Amoxicillin was used as the standard antibacterial agents, anti-inflammatory activity by ‘Carrageenan induced oedema test using ibuprofen as a standard and analgesic activities by acetic acid induced writhing using ibuprofen as the standard. The obtained results revealed that the nature of substituent and substitution pattern on thecoumarin ring may have a considerable impact on the antibacterial, anti-inflammatory and analgesic activities of the synthesized compounds. Antibacterial Activity Synthesized compounds were evaluated for antibacterial activity by cup-plate diffusion method using Staphylococcus aureus andEscherichia coli. The amoxicillin were used as standard drug for antibacterial activity. Test compounds and standard drug were used as at the concentration of 100ug/0.01ml.The zones of inhibition of compounds were recorded after incubation of 24 hr at 37oC. Compounds VIa and VIb exhibited good activity against both bacteria Staphylococcus aureusand Escherichia coli, Whereas Compounds VIj and VIk showed moderate activity against both bacteria and remaining compounds displayed weak antibacterial activity. Table-3: Antibacterial activity of newly synthesized compounds [VI a-j]. Note: Standard (S) – Amoxicillin; Control (C) – DMSO

S. No. 1 2 3 4 5 6 7 8 9 10 11 12

Compound VI a VI b VI c VI d VI e VI f VI g VI h VI i VI j S C

E.coli 16 17 11 08 10 11 11 12 13 13 23 08

Zone of inhibition (in mm) S.aureus 17 16 09 10 12 09 13 12 15 14 21 08

Anti-inflammatory activity Synthesized compounds were screened for their anti-inflammatory activity by ‘Carrageenan induced oedema test using ibuprofen as a standard compound.1% Carrageenan produced increase in paw volume (oedema) of all the animals of various groups.

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The onset action was evident from 1 hour in various test groups. The compounds VIc, VIf and VIk showed significant anti-inflammatory activity, other compounds are less active. When compared with standard.

Table-4: Anti-inflammatory activity of newly synthesized compounds [VI a-j]. Note: N=6; One way ANOVA followed by multiple Tukey’s comparison test.* P≤0.05; ** P≤0.01; ***P≤0.001 when compared with control

S. No

Compound

Dosage

The increase in Paw volume (ml)

1

VIa

20 mg/kg

0.32±0.01**

47.54

2

VIb

20 mg/kg

0.37±0.02*

39.34

20 mg/kg

0.25±0.01***

59.01

20 mg/kg

0.47±0.01

22.95

20 mg/kg

0.38±0.02**

37.70 52.45

3

% inhibition (mean)

4

VIc VId

5

VIe

6

VIf

20 mg/kg

0.29±0.01***

7

VIg

20 mg/kg

0.34±0.02**

44.26

8

VIh

20 mg/kg

0.41±0.01

32.78

9

VIi

20 mg/kg

0.39±0.01

36.06

10

VIj

20 mg/kg

0.24±0.02***

60.65

5mg/kg

0.14±0.01***

75.90

11

Ibuprofen

Analgesic activity Synthesized compounds were also screened for their by acetic acid induced writhing using ibuprofen as the standard. The compounds VIa, VIj, VIf and VIe, showed significant analgesic activity while remaining other less active. When their compared with standard. Table-5: Analgesic activity of newly synthesized compounds [VI a-j]. Note: N=6; One way ANOVA followed by multiple Tukey’s comparison test * P≤0.05; ** P≤0.01; ***P≤0.001 when compared with standard. S.No.

Derivatives

Dosage

1 2 3 4 5 6 7 8 9 10 11

VIa VIb VIc VId VIe VIf VIg VIh VIi VIj Pentazocine

20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 20 mg/kg 5 mg/kg

(7-HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID

Number of writhings in10 minutes (mean ± SEM) 32.33 ± 2.30*** 45.5 ± 1.88** 52.66 ±3.77 49.16 ±1.01* 38.33 ± 2.30** 35.5 ± 1.88*** 42.66 ±3.77** 57.50 ± 2.57 33.66 ± 3.47*** 40.51 ± 2.57** 21.66 ± 1.06***

70

% Inhibition 59.68 42.83 33.84 38.24 51.84 55.40 46.40 27.76 57.78 49.10 72.78

Deepak P. Kardile et al.

Vol.4, No.1 (2011), 66-72

CONCLUSION From the data of the Table no. and of antibacterial, anti-inflammatory and analgesic activity, it is clearly concluded that the synthesized compounds are promisingly significant, good antimicrobial agents and anti-fungal agents. The substituted benzofuran moieties are already known for different biological activities. Here we have synthesized some novel benzofuran analogues combining with different substituted aromatic and hetero cyclic amine ring system with view to get a good antibacterial, anti-inflammatory and analgesic activity with less toxic effects. As per the results of screening it is clearly indicated that the compounds of the scheme have shown good antibacterial, anti-inflammatory and analgesic activity equipotent with the standard drugs. From the above results one can establish that the synthesized substituted coumarin can be rich source for the exploitation. Therefore, in search of new generation of the active compounds, it may be worthwhile to explore the possibility in this area or by making or introducing different functional groups or secondary amines or by cyclization as substitutions. Which may results into better pharmacological agents.

R= - Cl, - Br, - F, - NO2, -N (CH3)2, -0CH3, -CH3 Scheme-1

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ACKNOWLEDGEMENTS The authors thank the President Shree swami Harikeshavadasji and Director Rajani Chandarakant, Shree Swaminarayan Pharmacy College, Kevadia colony for providing laboratory facilities and encouragement and Director of Karnataka University, Dharwad helping for studding spectral studies .

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

K.K. Srinivasan, A.J. Sreejith, A.M. Ciraj, V. Rao, Indian J. Heter. Chem., 69,326 (2007). V.V. Mulwad and S. A. Mayekar, Indian J. Heter. Chem., 46B, 1873 (2007). M. Kulkarni, B. Pujar and V. Patil, Indian J. Chem.,38B, 491(1999). D. P. Kardile, N.V.Kalyane. Inter. J. Biopharma.1, 6(2010) R.V. Rao and M.M. Mohan, Indian J. Heter. Chem., 13, 69 (2003). D.N. Nicolaides, K.E. Litinas and K.C. Fylaktakidou, Eur J Med. Chem., 39, 323 (2004). Y.K. Tyagi, H.G.Raj, P. Vohra, G. Gupta, R. Kumari and R.K. Gupta, Eur J Med. Chem., 40, 413(2005). M.D. Braccio, G.Grossi, G.Roma, M.G. Signorello and G. Leancini, Eur J Med. Chem., 39, 337 (2004). E.B. Akerblom, J. Med. Chem.,17, 609 (1974). A.K.S. Gupta, M. Garg and U. Chandra, J. Indian Chem. Soc.,56 1230 (1979). Fung A.K. Mansour, M.M. Eid and N.S. Khalil, Molecules,8, 744 (2003) O.A. Abd Allah, Farmaco., 55, 641 (2000) W.O.Foye and P.Tovivich, J. Pharm. Sci., 66,1607(1977) Indian Pharmacopoeia, controller of publications, Delhi, India, Vol-II., A-91, 100 (1996). C.A.Winter, E.A. Risley and C.W. Nuss, Proc. Soc. Exp. Bio. J., 3, 544(1962). R.A.Turner. Aca. Press New York.1, 125(1965). [RJC-715/2011]

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(7-HYDROXY-2-OXO-2H-CHROMEN-4-YL) ACETIC ACID

72

Deepak P. Kardile et al.

Vol.4, No.1 (2011), 73-85 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

RECENT DEVELOPMENTS ON BISMUTH (III) IN CARBONCARBON BOND FORMATION CHEMISTRY Suresh and Jagir S. Sandhu* Department of Chemistry, Punjabi University, Patiala, Patiala-147 002, Punjab, India *E-mail: j_sandhu2002@yahoo.com ABSTRACT The present mini account overview describes briefly prosperous usage of Bismuth (III) as an attractive, green alternative to strong, toxic and hazardous, catalysts in some selected organic named reactions. Scope of this catalyst system is too broad and developments have been too fast only recent account is presented on very important carbon-carbon bond forming named reactions like Knoevenagel, Michael, Doebnor modification of Knoevenagel, Biginelli and Hanstzch wherein both these reactions are also proven combination of Knoevenagel and Michael reactions. Keywords: bismuth (III) compounds, C-C bond forming name reactions, Bi (OTf)3.xH2O © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Over the past decade synthetic organic chemistry have been focusing on increasing the use of environmentally benign/eco-friendly catalysts and reagents for the use of compounds which follow twelve principle of green chemistry.1-3 Bismuth compounds have been employed as green and effective catalysts in many synthetic reactions to replace toxic catalysts e.g. compounds of mercury, lead, thallium, arsenic and antimony etc. Bismuth: General Information Now it is relevant to discuss certain general aspects of this element i.e. Bismuth is the 83rd element in the periodic table, which is placed in Group V of the Periodic Table with other members like nitrogen, phosphorus, arsenic and antimony and it is a brittle white metal with a pinkish tinge having an atomic mass of 209.980 amu. Bismuth resembles antimony in its mode of occurrence but relatively it is less common.4 Abundance of bismuth in the earth’s crust has been estimated (229) to be 0.00002 weight %, i.e. approximately same as silver and is produced as a by-product of copper and tin refining. Bismuth being a radioactive element, it is stable because of it’s an extremely long half life (t1/2~2×1018years) makes it practically stable.5-7 Despite its heavy metal status, bismuth and its compounds are relatively non-toxic and can be utilized for a variety of different medicinal as well as catalysts purposes.8-14 Lewis Acid and Eco-friendly Nature Several of the heavy metals have a relatively high toxicity and if used in reactions can be present in byproducts. One of the principal factors contributing to the low toxicity of bismuth is the poor solubility of it in aqueous solutions or in bio-fluids at moderate pH values which is found in plasma/fluids of living system.15 From table 1, it is clear that many bismuth compounds are less toxic than common salt (NaCl) and many other metallic salts.16 Since Bi-compounds are comparatively non-toxic and have been employed as eco-friendly mild Lewis acid catalysts system in synthetic green chemistry.17-27 Lewis acidity of bismuth (III) compounds are a result of poor shielding of the f-orbital electrons (electronic configuration of Bi: [Xe]4f145d106s26p3) allowing bismuth to accept an electron pair readily as well as availability of unoccupied orbital’s elevate its affinity to extend coordination. Electron pair demand of Bi further augmented by strong electron withdrawing groups (EWGs) such as halides, triflate etc. Bismuth exists in two oxidation states, +3

BISMUTH (III) IN CARBON-CARBON BOND

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85

[bismuth (IlI) halides, bismuth (lIl) subsalicylate etc.] and +5 [bismuth (V) fluoride, Ph3Bi (OAc)2, Ph3BiCO3 etc.].They are suitable as mild Lewis acid catalysts for organic transformation and synthetic reactions due to their ease of handling and non-corrosive nature. As a result numerous studies have been carried out to establish the effectiveness of Lewis acidity of bismuth compounds and their effects on different synthetic reactions.28-33 Bi (III) catalysts are generally crystalline solids and are commercially available at low cost.34 Because of amplified awareness to green chemistry, bismuth compounds become attractive as green catalysts in place of other, more toxic, metal catalysts derived from elements like indium, nickel, mercury, lead, tin etc. Table-1: LD50 Values of Common Metal Salts from MSDS Data.16 (Species-Rat, Route-oral) HgCl2 NiCl2 SnCl2路2H2O InCl3 CeCl3

0.001 0.105 0.7 1.1 2.1

SmCl3 BiCl3 NaCI ScCl3 Bi (NO3)3.5H2O

2.9 3.3 3.8 3.9 4.4

YbCl3 Bi2O3 BiOCl

4.8 5.0 22

Bi+3 in C-C Bond Formation Named Reaction Synthetic organic chemistry have witnessed extensive use of Lewis acids and there are several reviews and now this scattered information is collected and published in book shape too each chapter in this book is written by prominent workers in the area and there are two chapters on bismuth applications in organic synthesis.23,24 Recent advances in Bismuth (III) chemistry have expanded the versatility and flexibility of modern green/eco-friendly catalysts for carbon-carbon bond formation reaction and functional group transformations.19 This mini-review highlights the considerable progress, which has been made in the last decade to tame the reactivity of Bi+3 salts.17-18

This review focuses on recent advances of bismuth in common useful named reactions leading to the formation of C-C bonds these reactions are tools for production of many pharmacological/biological significant compounds. Named reactions showed in Table 2. are almost discovered a century ago or more by inventors as these are popularaly called after their names, the clasical version of these reactions have undergone a see change but still do require further refinements and renovations according to present day needs like environment etc Evidently old versions had many problems so developments/productive

BISMUTH (III) IN CARBON-CARBON BOND

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Vol.4, No.1 (2011), 73-85

investigations were needed to accomodate modern trends and needs. MSDS data (Table 1) shows that Bi (III) are almost neutral and in recent researsches these salts are widly accepted by different areas of modern green chemistry. Efforts made by authors and other research groups in contributing to develop Bi (III) salts as environment friendly green catalysts in popular C-C bond formation major named reactions of organic chemistry are delineated in following Table 2-9. Aldol Condensation In 1872, C-A. Wurtz and A.P. Borodin discovered most popular carbon-carbon bond forming this condensation reaction in organic synthetic chemistry.45-47 Aldol when dehydrates yields αβ-unsaturated ketones called chalcones and reaction is named as Claisen-Schmidt reaction. Lewis acids play key role in promoting Aldol and Aldol type condensation,48,49 from table 2.it is clear that for this investigation various Bismuth (III) Lewis acids have been extensively used preferentially over the existing one due to their eco-friendly nature, availability and low cost (table 2.).50-62 O R

OH

O

+

C

Cat. H 3C

H

O

Me

R

CH 2

Me

Table-2: Bi+3 Catalysed Aldol and Aldol Type Condensation Bismuth (III) Used BiCl3 BiCl3-Metal Iodides (1:3) BiCl3-NaI (1:3) Bi (OTf)3.4H2O Bi (OTf)3.4H2O MC (microencapsulated)Bi (OTf)3 Bi-ZnF2 Bi (OTf)3·nH2O Bi (OTf)3·4H2O Bi (OTf)3- Chiral Bipyridine Complexes BiC13- ZnI

Reaction Condition

Reactants Silyl enol ethers & Aldehydes Silyl enol ethers & Aldehydes Silyl enol ethers derived from furfural & Aldehydes Silyl enol ethers & Aldehydes/Acetals 2- (trimethylsilyloxy) furan & Aldehydes 4-nitro benzaldehyde & acetone α-bromocarbonyl compounds and aldehydes silyl enolate and aldehyde dioxinone-derived silyl dienol ethers Silyl enol ethers & Aldehydes Ketene Silyl Acetal & Chiral αβ-Epoxyaldehydes

BISMUTH (III) IN CARBON-CARBON BOND

75

References

CH2Cl2, rt

[50-51] ο

CH2Cl2, -30 C, Ultrasound CH2Cl2, rt CH2Cl2, rt to 70οC Diethyl ether, 78οC

[52] [53-54] [55] [56]

Solvent free, rt

[57]

H2O, rt

[58]

[Bmim]BF4, rt

[59]

Diethyl ether, 78οC DME/H2O (9:1), 0 °C CH2Cl2, rt

[60] [61] [62]

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85

Knoevenagel Reaction Emil Knoevenagel63,64 in 1894 invented this reaction as a proficient way of producing carbon-carbon double bonds and it is also a modified version of Aldol condensation.65-66 The conventional Knoevenagel (Scheme 2.) have been reported in several reviews and monographs the use of ecofriendly and green catalysts system in this reaction is a matter of current interest. In the pursuit of developing green chemistry we did make considerable efforts for solvent free green Knoevenagel condensation utilizing 10mol% BiCl3 (table 3.).67-68 O EWG

C H

+

1

CH Cat.

H 2C EWG

EWG

C

-H 2 O

EWG

2

1

2

Table-3: Bi+3 Catalysed Knoevenagel Condenation Bismuth (III) Used

Reactants

Reaction Condition

10mol% BiCl3

Active methylene compds & Aldehydes

Solvent free, 80ÎżC

References [67-68]

Doebner condensation/ modification It is a customized account of Knoevenagel reaction which was given by O. Doebner in 1900.69-70 Always pyridine or piperidine type catalyst systems have been used for this decarboxylative knoevenagel to obtain unsaturated acids from a range of aldehydes and malonic acids.71-77 In this reaction also our group78 have significant contribution for the production highly commercially and otherwise significant (E)cinnamic acids.79-86 In this pursuit cinnamic acids were obtained in good to very good yields employing 510mol% BiCl3 (table 5.).78 In another simplification we were tempted to employ another variant to examine efficiency of bismuth salts in this important reaction of organic chemistry. In this protocol we used bismuth triflate and other bismuth salts in this reaction. We observed bismuth triflates in less catalytic amount gives excellent results as shown in table 4. demonstrating Bi (OTf)3.xH2O proven more economic and efficient catalyst.87 O

O

O

C

C H

+

OH

Cat.

H 2C

CH CH

OH

OH O

Table 4.Comparison of Bismuth (III) catalysed for synthesis of (2E)-3- (4-methoxyphenyl)prop-2-enoic acid.87 Entry Bismuth (III) Salts 1 -------2 Bi (OTf)3.xH2O, PEG 3 BiCl3, Sol.free 4 Bi (NO3)3.5H2O 5 BiCl3, KI, PEG a Isolated yields after recrystallization

BISMUTH (III) IN CARBON-CARBON BOND

Amount (mol %) -------5 mol % 1ml 5 mol % Sol.free 5 mol % Sol.free 5 mol % (1:3) 1ml

76

Time (min) 3.5 3.5 3.5 3.5 3.5

Yield (%)a <10 95 78 82 88

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85 Table-5: Bi+3 Catalysed Doebnor Modification Bismuth (III) Used

Reaction Condition Solvent free, MWI PEG, MWI

Reactants Malonic acid & Aldehydes --do--

BiCl3 Bi (OTf)3.xH2O

References [78] [87]

Michael Reaction Arthur Michael discovered88-90 this reaction which is very similar to Knoevenagel reaction and in conventional chemistry in both the reactions similar solvents and catalysts has been employed and volumous research work is reported and both seem complimentary to each other. Precisely, Knoevenagel is 1-2 and Michael is 1-4 conjugate addition on to carbonyl and electron deficient alkenes respectively. In pursuit of green chemistry here also authors and others did make considerable contribution in developing some environment benign protocols using non-polluting catalysts i.e. bismuth (III) salts and replacing volatile organic solvents (VOCs) under solvent free conditions.91-108 O X

O

O CH

Y

+

H2C

R

Cat.

EWG

X

R

C YOC

CH2

EWG

Table-6: Bi+3 Catalysed Michael and Michael Type Reaction Bismuth (III) Used BiCl3 Bi (NO3)3.5H2O

Bi (NO3)3.5H2O

BiOClO4·xH2O Bi (OTf)3·xH2O Bi (OTf)3·xH2O Bi (OTf)3 Bi (OTf)3 Bi (OTf)3

Bi (OTf)3 BiCl3

Reaction Condition

Reactants 1,3-dicarbonyl compds & α,β-unsaturated carbonyl compds Indoles & 1,2unsaturated ketones trans-1-phenylbut-2-en1-one or 4-methylpent3-en-2-one & 1,3oxazolidin-2-one Indoles & 1,2unsaturated ketones indole or thiol & αβunsaturated carbonyl compds Indoles & p-quinones αβ-ethylenic compds & Aliphatic amines αβ-enones & Indoles 1,2-bis (trimethylsilyloxy)cyclo butene & 1,2-diaza-1,3butadines αβ-unsatd.esters & conjugated amines Pyrroles & Electrondeficient olefines

BISMUTH (III) IN CARBON-CARBON BOND

77

References

Solvent free, MWI

[91]

H2O, 25 °C

[92]

CH2Cl2, rt

[93]

CH3CN, r.t., sonication

[94]

CH3CN, r.t.

[95]

CH3CN, r.t.

[96]

CH3CN, r.t.

[97]

CH3CN, r.t.

[98]

One-pot, r.t.

[99]

Solvent free, MWI Silica gel, Solveni free, MWI

[100] [101-102]

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85

BiCl3

Aliph.Amines & αβunsatd.Esters and nitriles Aryltin compds.& Nitroalkenes

BiCl3

Furan derivatives

Microenccapsulated Bi (OTf)3

α(trimethylsiloxy)styrene / 1(trimethylsiloxy)cycloh exene & adehydes/ Ketones/ acetals/ enones Silyl enol ethers & αβunsatd.ketones

BiCl3

BiCl3

Solvent free, rt

[103]

Palladium (II) salt in acetic acid Metal iodide systems, mild condition

[104-105] [106]

Metalic iodide systems, -30 and 70º

[107]

CH2Cl2, r.t.

[108]

Hanztsch Reaction More than a century ago, in 1881, 1,4-dihydropyridine synthesis was described by Arthur Hantzsch and now produced pyridine is popular as Hantzsch 1,4-dihydropyridine.109,110 Exploration of these pyridines in the beginning were quite slow, later it picked up very fast because of their structural resemblance to reduced nicotinamide adenine dinucleotide (NADH), which is an established hydrogen transferring agent in biological processes.111 Since these molecules had great significance to biologists is as they are taken to be privileged molecule of chemistry (possessing more than one activity such as Nifedipine, Nicardipine, Amlodipine, Nitrendipine, Nimodipine and others have been used as calcium channel blockers, and are used most frequently as cardiovascular agents for the treatment of hypertension) and their use in clinical practice attracted much attention of chemists to develop greener production process. Though, large number of other catalysis in this reaction are also reported112 even then reports are there of uncatalysed reaction113 Present authors also made effective contribution in this direction using bismuth (III) salt mainly for synthesis of Hantzsch pyridines.114 Oxidation of Hantzsch 1,4-dihydropyridine also reported by using bismuth salts which produce oxidized products in excellent yields (table 6.).114-122 R O R

2

1

O

CHO CH2

H 2C

+

C H 3C

R

2

cat.

R

2

C O

O NH

R

O

H 3C

O

CH C

C

C

CH3

4 OAc

1

R

2

C N H

CH3

Table-7: Bi+3 Catalysed Hantzsch Reaction Bismuth (III) Used BiCl3 BiBr3

Reaction Condition

Reactants

Active methylene compds, Ammonium Solvent free, rt acetate & Aldehydes Subs.Anilines & Enol Anhydrous ethers CH3CN, 50οC Hantzsch Oxidation

BISMUTH (III) IN CARBON-CARBON BOND

78

References [114] [115]

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85 R

O R

1

O

O

2

R

2

R

Cat. Oxidation

H 3C

N H

Bi (NO3)3.5H2O

1,4-dihydropyridines

BiCl3,

--do--

BiONO3

isoxazole-substituted pyridines

Bi (NO3)3.5H2O

1,4-dihydropyridines

BiCl3

--do--

1

O

2

R

H 3C

CH 3

R

N

2

CH 3

AcOH, rt BTPPMS, CH3CN, rt Acidic Alumina, CHCl3, rt Silica gel, MWI, Solvent free HZSM-5 zeolite, MWI, Solvent free

[116] [117] [118] [119-120] [121-122]

Biginelli Reaction Italian chemist, Pietro Biginelli about a decade later (after synthesis of Hantzsch 1,4-dihydropyridine) heated same three components acetoactic ester, benzaldehyde in equimolar ratio, urea (slightly in excess) in alcohol and few drops of conc.hydrochloric acid.112,123-126 He isolated a new compound (now called Biginelli compound127,128 and is undoubtedly aza analogue of Hantzsch pyridine) along with small amount of Hantzsch pyridine. Biginelli compounds showed promising antibacterial activity129 and other promising activity is anti-HIV i.e. antiviral activity130 because of the skeletal resemblance with natural batzelladine alkaloids, viz. batzelladine B. These compounds are also important as analgesic,131 antiinflammatory,132 antihypertensive133,134 and specially worth mentioning here is monastrol which has excellent anticancer activity.135 These two reactions Hantzsch and Biginelli are selected since they are now well developed three component reactions and also they are combination of previously discussed two reactions viz. Knoevenagel followed by Michael so both are tandem/cascade reactions. Before presenting work on Bismuth catalysis in this reaction one puzzle is also worth mentioning here i.e. a catalysis is needed or not since detail is irrelevant here only a question is recorded.136 But if one goes by this statement other question arises is it all Knoevenagel reactions and Michael reactions can proceed uncatalyzed obviously it is not possible. For efficient synthesis of Biginelli compounds almost all bismuth salts are reported to enhance reaction rate and yields summary of these are given in table 7.137-149 R

O O R

1

CHO

+

O

X

C H 3C

CH 2

R

2

+

H 2N

Cat. NH 2

X = O; S

R

2

1

CH C

NH

C H 3C

N H

X

Table-8: Bi+3 Catalysed Biginelli Reaction Bismuth (III) Used

Reaction Condition

Reactants

Bi (OTf)3

Active methylene compds, Urea/Thiourea & Aldehydes --do--

BiONO3

--do--

Bi (NO3)3.5H2O

--do--

BiCl3

BISMUTH (III) IN CARBON-CARBON BOND

79

References

CH3CN, reflux

[137]

CH3CN, rt Anhydrous CH3CN, 40ÎżC50ÎżC CH3CN, reflux

[138] [139] [140-142]

Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85 BiOClO4.xH2O Polyanilinebismoclite complex (PANI-BC) Bi (NO3)3.5H2O Bi (NO3)3.5H2O

Bi (NO3)3.5H2O

Bi (TFA)

--do--

CH3CN, 40οC50οC

[143]

--do--

EtOH, Heating

[144]

--do-Primary Alcoholes were used instead of Aldehydes Benzyl halides were used instead of Aldehydes Ketones were used instead of Active methylene compds

MWI

[145-146]

CH3CN /TBAB,

[147]

TBAF, 120οC

[148]

[nbpy]FeCl4, TMSCl, 70οC

[149]

Strecker Reaction Historically, α-amino nitriles is an oldest multi-component reaction reported by Strecker in 1850 and is named after his name which employs aldehydes, amines and sodium cyanide/potassium cyanide to afford α-amino nitriles.150 This involves addition of cyanide to C=N bond is common strategy to obtain α-amino nitriles, which serve as important synthons in organic chemistry for the synthesis of a variety of heterocycles. These nitriles can be conveniently converted into a variety of amino acids151-152 and several nitrogen heterocycles like thiadiazoles,153 imidazoles154 and other biologically significant compounds such as saframycin A.155 In this reaction only BiCl3 was employed by S. K. De et al.156 (table 8.) and use of other salts of bismuth is still remain unexplored. R1 R1 O Catalyst R N Me SiCN N H 3 C r.t. CH R2 H R R2 CN

+

+

Table-9: Bi+3 Catalysed Streacker Reaction Bismuth (III) Used

Reactants

Reaction Condition

References

BiCl3

aldehydes, amines and trimethylsilyl cyanide

CH3CN, rt

[156]

Pechmann Reaction Classically, the Pechmann reaction157 refers to the condensation of β-ketoesters with phenols in the presence of excess of acid catalysts to produce 4-substituted heterocyclic compounds i.e.coumarins, involving tandem hydroxyalkylation, transesterification and dehydration. Esterification/transesterification followed by attack of the activated carbonyl ortho to the oxygen to generate the new ring via dehydration, as in case of aldol condensation.158-159 Under solvent free condition utilizing Bi (NO3)3.5H2O and BiCl3 catalyzed Pechmann reaction came in light during extensive literature survey and both salts provide products in high yields without any side products formation.160-162

BISMUTH (III) IN CARBON-CARBON BOND

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Suresh and Jagir S. Sandhu

Vol.4, No.1 (2011), 73-85

O

O

OH

+

Cat.

C EtO

CH2

R

O

1

C 1 R

O CH

Table-10: Bi+3 Catalysed Pechmann Reaction Bismuth (III) Used

Reactants

Bi (NO3)3.5H2O

phenol & β-ketoesters

BiCl3

--do--

BiCl3

--do--

Reaction Condition Solvent free, 80οC Solvent free, 75οC or 125οC Ultrasound, rt

References [160] [161] [162]

Earliest C-C bond forming reaction seems to be Aldol reaction and others like Knoevenagel and Michael also are old reaction of the past century. Next is discussed Biginelli and Hantzsch dihydropyridine which both have similarity as they involve Knoevenagel and Michael reaction both or involve these reactions concurrently. So they are a mix of both, so here this account primarily is around Aldol, Knoevenagel, Michael and some other reactions are also touched. This selection seemed desirable since we were to punctuate this account with our own work also. From above table it is understandable that various Bismuth salts are employed for same reaction by even same workers or different group of chemists in these reactions. As we know from MSDS data these catalysts are less toxic than common edible salt used by human and every salt of Bismuth (III) gave good results either yields or reaction rate in efficient way in each above discussed reactions. Other metal halide in combination of Bismuth salts further enhanced its catalytic activity to speed-up the reaction but as promoter used metal halide can effect greener nature and other features of these novel Bismuth catalysts. After investigating various trivalent bismuth compounds it is seems that mainly triflates are superior in every way like catalytic activity, moisture tolerance, recycling, inexpensive and eco-friendly etc. In case of multicomponent reactions Knoevenagel, Streacker, Hantzsch synthesis and Doebnor there are only few reports especially with BiCl3 we hope other triflates like bismuth compounds would further accelerate these reactions.

CONCLUSIONS In the beginning of this article we have presented detailed account of bismuth’s properties which have attracted the attention of chemists around the world the result of which very clear from large number of reviews published on this topic not only this fragmented information has come up in chapters written by authorities on this attractive mild Lewis acid Poor solubility of Bismuth salts in bio-fluid indicating that these are green catalysts of present century and widely accepted by research chemists. Other features which make Bi salts popular are their easy availability, inexpensive, non-corrosiveness nature and stability. Role of green and eco-friendly Bi (III) catalysts to speed-up many other C-C bond forming reactions (with various related substrates), asymmetric synthesis and synthetic transformations do still remain unexplored. These Bi catalysts would be a substitute or take position of previously used non-green catalysts systems to protect ecosystem and environment which could be gift to next generation. We do hope this paper would further stimulate researches on bismuth salts.

ACKNOWLEDGEMENTS The authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial assistance and to the Indian National Science Academy (INSA), New Delhi, India for additional financial support for this research project. Also authors thanks Council of Scientific and Industrial Research (CSIR) India for award of fellowship and RSIC (DST) Punjab University Chandigarh for spectral analysis. One of us Jagir S. Sandhu highly appreciates dedicated team of his research associates

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Vol.4, No.1 (2011), 73-85

which was nearly a dozen or so presently occupying responsible positions in CSIR India and Universities in this country who were contributor in bismuth chemistry and their names are in references.

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Vol.4, No.1 (2011), 86-90 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

OPTICAL AND MECHANICAL CHARACTERIZATION OF SOLUTION GROWN SEMI ORGANIC NLO CRYSTALS. M. N. Ravishankar *, R. Chandramani 1 and A.P. Gnanaprakash 1

Department of Physics, PG studies, Manasagangotri, University of Mysore, India. Department of Physics, Dayananda sagar college of Engineering, Bangalore, India. *E-mail: ravibhumi2004@gmail.com

ABSTRACT The present fascinating field of research is to synthesize, grow and characterize semi organic NLO crystals. Here an attempt has been made to synthesize and grow number of NLO semi organic crystals. The semi organic crystals posses both the good qualities of host organic material and additive-inorganic material. The crystals grown are amino acid- γ-Glycine with additives namely, Potassium Nitrate, Sodium Nitrate, barium Nitrate, Ammonium Oxalate and Ammonium Chloride. Good crystals were obtained in a period of 3-4 weeks. Most of the crystals were transparent. The SHG efficiency has been tested by the Kurtz powder technique using Nd: YAG laser, and KDP sample has been used as a reference material. Since hardness play a key role in device fabrication and NLO materials are expected to play a major role in photonics as well as optical information processing, the present work pertains to mechanical characterization and SHG studies. The smooth surfaces of grown crystals were subjected to Vicker’s hardness for duration of 5-10seconds indentation time at room temperature. Load ranging from 5gms to 20gms were applied over a fixed interval of time. The micro hardness H and Mayer’s index n has been estimated. The value of Meyer’s index for various samples investigated falls below 1.6 suggests that crystals are hard or moderately soft. This behavior has increased with additives. Also, SHG efficiency for the grown crystals and laser damage threshold studies have been carried out using Q switched Nd: YAG laser for 10 nano seconds laser pulse width at a wavelength of 1064nm. The value obtained for laser damage threshold is due to lower/ moderate hardness seen by Vicker’s measurements. Plots of Hv, Mayer’s index n, as a function of load P, are shown graphically. PACS: 61.10, 78.30, 42.65 ky, 42.70 mp, 81.10 Dn, 61.72 Ss. Key words: SHG, Laser damage, Semi organic, NLO, Micro hardness. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Non linear optical materials are expected to play a major role or prominent role in the technology of photonics including optical information processing and frequency conversion 1, 2, 3. Most of the organic non linear optical crystals usually have poor mechanical and thermal properties. They are susceptible for damage during the processing even though they have large NLO efficiency. In the present study we have made an attempt to grow number of semi organic non linear crystals of γ-Glycine with Potassium Nitrate, Sodium Nitrate, Barium Nitrate, Ammonium Oxalate and Ammonium Chloride as an additive by aqueous solution method. Mechanical characterization, SHG and laser damage studies have been carried out. Crystal growth and characterization Analytical reagent (AR) grade samples were used. Salts were taken in their molar mass, separately and then mixed together. Supersaturated solutions of the mixed salts were kept for slow evaporation in beakers covered with filter paper at room temperature.

The selected combinations are: Glycine+ Potassium Nitrate (GPN Crystal) Glycine + Sodium Nitrate (GSN Crystal) Glycine + Barium Nitrate (GBN Crystal) Glycine + Ammonium Oxalate (GAO Crystal)

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M. N. Ravishankar et al.

Vol.4, No.1 (2011), 86-90

Glycine + Ammonium Chloride (GAC Crystal) Most of the crystals were transparent. Good transparent crystals were obtained in a matter of 3 to 4 weeks shown in figures 1 to 5. The crystals were characterized on the basis of XRD, FTIR, UV-Vis- IR optical measurements. Also, the above crystals have responded well for second harmonic generation (SHG) efficiency.

Fig.-1: GPN Crystal

Fig.-2: GSN Crystal

Fig.-3: GAOCrystal

Fig.-4: GBN Crystal

Fig.-5: GAC Crystal

RESULTS AND DISCUSSION SHG efficiency Kurtz and Perry 4 powder method is an important tool for researchers searching for organic/semi organic/inorganic NLO material. The experimental setup used in the present investigation was similar to the generic one devised by Kurtz5. It consisted of a Q-switched Nd: YAG laser, the output of which was filtered through 1064nm narrow pass filter. The power of the fundamental beam was monitored by a split beam technique, in one channel of the power meter. The sample was ground in the form of fine powder of known grain size and pressed between two glass plates. The sample size was kept larger than the beam cross section. The generated harmonic was passed through a 532 nm narrow pass filter and fed to other channel of the power meter. The ratio of the fundamental and harmonic intensities determines the efficiency of the sample. To eliminate the experimental error, urea sample of the same size was also tested in the same setup and the efficiency was evaluated as a ratio. The input power of the laser beam was measured to be 16.5 mJ/ pulse. Pure KDP was used as reference sample. Both the reference and test samples had uniform particle size of 130 to 150microns.The experiment was carried out in pure KDP and later in all the samples. Throughout the experiment the laser power was kept constant. Table-1: SHG efficiency Semi organic Crystal γ -Glycine+Potassium Nitrate γ -Glycine + Sodium Nitrate γ -Gycine + Barium Nitrate γ -Gycine + Ammonium oxalate γ -Gycine + Ammonium chloride SEMI ORGANIC NLO CRYSTALS

SHG signal (mV) 6.8 9 0 10.2 6

87

Efficiency with respect to KDP 0.98 1.35 0 1.48 0.85 M. N. Ravishankar et al.

Vol.4, No.1 (2011), 86-90

Results show that SHG efficiency of γ-Gylcine containing alkali nitrates answer for higher efficiency compared to KDP. To exhibit NLO activity, the additives present in the host material (γ-Glycine) have to be macroscopically aligned, then only there can be increase in efficiency, justified experimentally 6. Among the samples studied, crystals namely Glycine with Sodium Nitrate, γ-Glycine with Potassium Nitrate have answered for higher efficiency. Whereas crystal containing ammonium oxalate has resulted in excellent efficiency taking KDP as reference. The best additive to organic material works out to be Ammonium Oxalate. Next in the series happens to be Sodium Nitrate. These additives are favoring SHG. It is surprising that the additives namely Barium Nitrate is acting as poison, not favoring NLO efficiency. In the present study 1% of Barium Nitrate has been used. It is interesting to carry out the work at lower values to know exactly at what amount of the above material starts nullifying the SHG efficiency and making the host crystal lose the NLO property. Definite conclusion regarding the result requires further work on phase matched SHG efficiency of single crystal. Mechanical properties: micro hardness studies One of the methods to determine the mechanical behavior of the grown NLO crystals is micro hardness test. The polished surface of the crystals namely, γ-Glycine with Potassium Nitrate, Sodium Nitrate, Barium Nitrate Ammonium Oxalate and Ammonium Chloride were indented at different sites for the load 5gms to 20gms for 10 seconds and the average value of the hardness were found out using M H – 5 hardness tester. The diagonal lengths of the indented impression were measured using calibrated micrometer attached to the eyepiece of the microscope. The micro hardness is calculated using the expression 7 H= 1.8544P/d2 kg-mm2 Where P is the applied load in grams and d is the average diagonal length of the vicker’s impression in mm after loading. The micro hardness and the diagonal length were calculated from the micro computer attached to the instrument. Plot of H vs P for the investigated samples are shown in figure 6. The non linear variation of H with load implies the presence of imperfection and voids. The imperfections are mainly impurity, dislocation or grain boundary diffusion. The Meyers’ index number n 8 gives the value of work hardening index. Materials are normally characterized by Meyers’ index or work hardening index. The lower the value of the work hardening index better will be the hardness of the material 9, 10, 11. The value of n comes to be 1 or 1.6 for hard material and more than 1.6 for soft material. The log-log plot between d and P yields almost straight line graph. The slope of the line gives the work hardening index n. The n value obtained for different samples are given in Table 2. These values of work hardening index suggest that materials are harder, whereas, in case of γ-Glycine with Ammonium Oxalate crystal work hardening index is 1.8 which implies that the material is softer. Table -2: Meyers’ index number n Grown Crystal

SEMI ORGANIC NLO CRYSTALS

n Value

γ-Glycine+ Potassium Nitrate

0.567

γ-Glycine + Sodium Nitrate

0.653

γ-Glycine + Barium Nitrate

0.702

γ-Glycine + Ammonium Oxalate γ-Glycine + Ammonium Chloride

0.876 1.82

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-G B N C r y s t a l2 52 8 6

-G S N c r y s t a l-

62

-G A O c r y s t a l-

5 5 51 7 2 61

-G P N c r y s t a l-

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5 4 5 6 60 0 2 5 3 5 59 5 19 58 2 54 18 5 57 1 53 1 57 6 50 52 55 1 46 9 5 51 4 1 45 8 0

0

20

50

40

100 60

1 5 08 0

210000

L O A D (g m s ) Fig-6: Plot of H vs P of GBN, GSN, GAO, GPN crystals.

Laser damage The optical damage threshold of an optical crystal is an important factor that hinders its applications. Optical damage threshold studies have been carried out for the solution grown γ-Glycine with Potassium Nitrate single crystal using Q switched Nd: YAG laser of pulse width 10 nano seconds and repetition rate of 10Hz operating in (TEMOO) mode, is used as the source. The laser beam was focused and the sample was moved step by step into the focus along the optical axis of the crystal. The energy density was calculated using the formula, energy density=E/A (GW/cm2), where E is the input energy measured in milli joules and A is the area of the circular spot 12. In the present study the laser damage threshold energy density for γ-Glycine Potassium Nitrate crystals was found to be 12.41 GW/cm2.

CONCLUSIONS Good quality crystals were obtained in a period of 3 to 4 weeks by slow evaporation method. XRD confirms crystalline nature. UV-Vis-IR spectrum confirming the transparent nature in the visible region. FTIR confirms the identity of the grown crystals. All the grown crystals have responded well for second harmonic generation efficiency (SHG). Amino acid- γ -Glycine with additives namely, Potassium Nitrate, Sodium Nitrate, Barium Nitrate, and Ammonium Chloride are hard materials whereas γ –Glycine with Ammonium Oxalate haven proven to be a soft material. Laser damage value reveals that the crystal is having moderate laser damage threshold.

1. 2. 3. 4. 5.

REFERENCES P.N. Prasad and D.J. Williams. Introduction to Nonlinear Optical Effects in Molecules and Polymers; Wiley: New York, 1991. C. Bosshard and K. Sulter, et. al., J. Opt. Soc.Am.,B 10, 186 (1993) D.Xu, M.Jiang and Z.Tan, Acta. Chem. Sin.,41, 570 (1983) S.K. Kurtz and T.T. Perry, J. applied physics, 39, 3798 (1968) M.Esthakupeter and P. Ramaswamy, Journal of Crystal Growth, 312, 1952-1956(2010)

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6. S.R. Marder, J.W. Perry et al ,Mater Res Soc Symp Proc 175, 101(1990). 7. U.V. Subbarao, V. Haribabu, Pramana, 2, 149(1978) 8. E.M.Onitsch, Mikroskopie 2, 131 (1947) 9. H.Li, R.C. Bradt, J. H ard Master. 3, 403-419 (1992) 10. B.Milton Boaz, P. Santana Raman et. al., Meterial Chemistry and Physics, 93,187 (2005) 11. Packiam Julius J, Joseph Arul Pragasam A, Selvakumar S, Sangayaraj P, J Cryst Growth, 267,619, 2004 12. S. Boomadavi, R. Dhanasekaran, J.Cryst. Growth. 261, 70 (2004) [RJC-721/2011]

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MICROWAVE SYNTHESIS AND ANTI-INFLAMMATORY EVALUATION OF SOME NEW IMIDAZOLO QUINOLINE ANALOGS P.Raghavendra, G.Veena, G.Arun Kumar, E.Raj Kumar, N.Sangeetha, B.Sirivennela, S.Smarani, H.Praneeth Kumar and R.Suthakaran* Pharmaceutical Organic Chemistry Laboratory, Department of Pharmaceutical Chemistry, Teegala Ram Reddy College of Pharmacy, Meerpet, Hyderabad-500097, Andhra pradesh (India) Email: sudha_sudhar@rediffmail.com ABSTRACT A Series of 1-(2-((18Z)-4-substituted benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2-dihydro-4methyl-2-oxoquinolin-7-yl AZ1-AZ12 substitutes of imidazolo quinoline analogs were synthesized by condensation of substituted imidazole and substituted quinoline. The title compounds were investigated for anti-inflammatory and its ulecerogenicity activities. All the lead compounds (AZ1-AZ12) were assessed by QSAR and molecular modeling (CADD) studies to predict best physicochemical, pharmacokinetic, toxicological properties and best fit with targets like COX-1 and COX-2. The result indicates that the compounds show convincing activities against inflammation when compared with standard drug (Ibuprofen). Keywords: Imidazolo quinoline, anti-inflammatory, Ibuprofen. Š 2011 RASÄ&#x20AC;YAN. All rights reserved.

INTRODUCTION The chemistry of heterocyclic compounds has been an interesting field of study for a long time. The synthesis of novel imidazolo quinoline analogs and investigation of their chemical and pharmacological behavior have gained more importance in recents decades for medicinal reasons.1,2 Substituted imidazolo quinoline analog have been found to have important activities such as anti-inflammatory, antimicrobial and antioxidant. Microwave technology has been used in inorganic chemistry since 1970s, Giguere and Gedye first implemented it to accelerate the organic reactions in 1986. The slow development of the technique in organic synthesis was principally attributed to the lack of controllability and reproducibility due to using poorly designed domestic microwave ovens as reactors.3,4 However, with the availability of commercial microwave equipment intended for organic synthesis and the development of the solventfree techniques, microwave-assisted organic chemistry has experienced exponential growth since the mid-1990s. Microwaves have been employed in organic chemistry to reduce the reaction times from hours to minutes and, to increase yields and selectivity.

EXPERIMENTAL All chemicals were procured by S.D.Fine Chemicals (India). Melting points of all synthesized compounds were determined in open capillary tubes using Vertigo VMP-1 melting point apparatus and are expressed in c.1H NMR spectra were recorded on Varian 500 MHz NMR spectrophotometer using CDCl3 as a solvent and TMS as an internal standard. The mass spectra were taken on a Jeol SX-102/PA6000(EI) spectrometer. C,H,N estimations were done on Carlo Erba 1108(C H N) Elemental Analyser. Domestic Microwave oven was used to synthesize the all title compounds. 7-Hydroxy-4-methyl-2H-chromen-2-one (1) A solution of resorcinol (0.1 mole) and ethyl acetoacetate (0.1 mole) was mixed with 160 gm of polyphosphoricacid. The reaction mixture was stirred and heated at 75-80 oC for 20 mins and then poured

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into ice-water. The resultant pale yellow solid mixture was collected by suction filtration, washed with a little cold water and dried at 60oC. Recrystallisation from dilute ethanol yields pure and colorless compound. (Yield 76%, m.p.187 0C) IR (cm-1):3350 (Ar-OH), 3052 (Ar), 1643 (C=O); Anal. Calc’d for C10H8O3: C, 68.18; H, 4.58; O, 27.25. Found: C, 668.08; H, 4.63; O, 27.30. 7-Acetyloxy / benzoyloxy /benzyloxy-4-methyl-2H-chromen-2-one (2) 4-Methyl-7-hydroxycoumarin (0.1 mole) in acetic anhydride (0.12 mole) and a few drops of pyridine / benzoyl chloride (0.12 mole) in absolute ethanol (10 mL)/ benzyl chloride (0.12 mole) in absolute ethanol (10 mL) was refluxed for 2 hr, and then poured into ice-water. The resultant product was collected by suction filtration, washed with a little cold water and dried at 60oC and recrystallised from absolute ethanol. [R= COCH3] (Yield 76%, m.p. 176 oC) IR (cm-1):3050 (Ar), 1645 (C=O), 1510 (Lactone); Anal. Calc’d for C12H10O4: C, 66.05; H, 4.62; O, 29.33. Found: C, 66.15; H, 4.47; O, 29.28. [R= COC6H5] (Yield 71%, m.p.172 oC) IR (cm-1):3050 (Ar), 1645 (C=O), 1510 (Lactone); Anal. Calc’d for C17H12O4: C, 72.85; H, 4.32; O, 22.83. Found: C, 72.82; H, 4.31; O, 22.87. [R= CH2C6H5] (Yield 73%, m.p. 181 oC) IR (cm-1):3050 (Ar), 1645 (C=O), 1510 (Lactone); Anal. Calc’d for C17H14O3: C, 76.68; H, 5.30; O, 18.02. Found: C, 76.65; H, 5.37; O, 17.98. 1-(2-Aminoethyl)-7-substituted oxy-4-methylquinolin-2(1H)-one (3)5, 6: Equalent moles of 7-acetyl/ benzoyl / benzyl oxy-4-methyl-2H-chromen-2-ones (0.1mole) with diethyl amine (0.1 mole) in glacial acetic acid was refluxed for 6 hr. The excess solvent was then distilled off under reduced pressure and poured into crushed ice (200 gm) to get the solid. The product so obtained was filtered under suction and dried at room temperature. It was purified by recrystalization from absolute ethanol. [R= COCH3] (Yield 74%, m.p.173oC) IR (cm-1):3410 (Ar-NH2), 3054 (Ar), 1652 (C=O); Anal. Calc’d for C14H16N2O3: C, 64.60; H, 6.20; N, 10.76; O, 18.44. Found: C, 64.56; H, 6.22; N, 10.74; O, 18.46. [R= COC6H5] (Yield 79%, m.p.179oC) IR (cm-1): 3410 (Ar-NH2), 3054 (Ar), 1652 (C=O); Anal. Calc’d for C19H18N2O3: C, 70.79; H, 5.63; N, 8.69; O, 14.89. Found: C, 70.75; H, 5.62; N, 8.72; O, 14.91. [R= CH2C6H5] (Yield 68%, m.p.172oC) IR (cm-1): 3410 (Ar-NH2), 3054 (Ar), 1652 (C=O); Anal. Calc’d for C19H20N2O2: C, 74.00; H, 6.54; N, 9.08; O, 10.38. Found: C, 74.05; H, 6.52; N, 9.11; O, 10.32. Benzoylglycine (4) Dissolve 0.33 mole of glycine in 250 mL 10% sodium hydroxide solution contained in a conical flask. Add 0.385 mole of benzoic chloride in five portions to the solution. Stopper the vessel and shake vigorously after each addition until all the chloride has reacted. Transfer the solution to a beaker and rinse the conical flask with a little water. Place a few grams of crushed ice in the solution and add concentrated hydrochloride acid slowly and with stirring until the mixture is acid to Congo red paper. Collect the resulting crystalline precipitate of benzoylglycine, which is contaminated with a little benzoic acid, upon a Buchner funnel, wash with cold water and drain well. Place the solid in a beaker with 100ml of carbon tetrachloride, cover the break with a watch glass and boil gently for 10mins (fume cup-board);this extracts any benzoic acid which may be present. Allow the mixture to cool slightly, filter under gentle suction and wash the product on the filter with 10-20 ml of carbon tetrachloride. Recrystallise the dried product from boiling water (about 500mL) with the addition of a little decolourising charcoal if necessary, filter

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through a hot-water funnel and allow crystallizing. Collect the benzoylglycine in a buchner funnel and dry it in an oven. The yield is 45gm (76%), m.p. 187oC. Anal. Calc’d for C9H9NO3: C, 60.33; H, 5.06; N, 7.82; O, 26.79. Found: C, 60.32; H, 5.03; N, 7.85; O, 26.8. 4 - (Substituted benzylidene)-2-phenyloxazol-5-one (5) Place a mixture of 0.25 mole of redistilled benzaldehyde/4-Cl benzaldehyde/4-OH benzaldehyde/4-OCH3 benzaldehyde/4-N(CH3)2 benzaldehyde, 0.24 mole of benzoylglycine, 0.75mole of acetic anhydride and 0.25 mole of anhydrous sodium acetate in a 500 mL conical flask and heat on an electric hotplate with constant shaking. As soon as the mixture has liquefied completely, transfer the flask to a water bath and heat for 2 hr. Then add 100 ml of ethanol slowly to the contents of the flask and allow the mixture to stand overnight. Filter the crystalline product with suction, wash with suction, wash with two 25 ml portions of boiling water: dry at 100oC. The yield of almost pure oxazolone is 40gm (64%) and its melting point 164-165 oC. Recrystallisation from benzene raises the m.p. to 167-168 oC. [R1= H] (Yield 79%, m.p.165oC) IR (cm-1):3416 (Ar-NH2), 3056 (Ar), 1655 (C=O); Anal. Calc’d for C16H11NO2: C, 77.10; H, 4.45; N, 5.62; O, 12.84. Found: C, 77.5; H, 4.42; N, 5.62; O, 12.46. [R1= 4-Cl] (Yield 73%, m.p. 177oC) IR (cm-1):3416 (Ar-NH2), 3056 (Ar), 1655 (C=O); Anal. Calc’d forC16H10ClNO2: C, 67.74; H, 3.55; Cl, 12.50; N, 4.94; O, 11.28. Found: C, 67.75; H, 3.58; Cl, 12.48; N, 4.92; O, 11.27. [R1= 4-OH] (Yield 77%, m.p. 182oC) IR (cm-1):3416 (Ar-NH2), 3056 (Ar), 1655 (C=O), 3295(OH); Anal. Calc’d forC16H11NO3: C, 72.45; H, 4.18; N, 5.28; O, 18.09. Found: C, 72.45; H, 4.20; N, 5.22; O, 18.13. R1= 4-OCH3] (Yield 78%, m.p.172oC) IR (cm-1): 3410 (Ar-NH2), 3054 (Ar), 1652 (C=O), 2896(CH3); Anal. Calc’d for C17H13NO3: C, 73.11; H, 4.69; N, 5.02; O, 17.19. Found: C, 73.15; H, 4.68; N, 5.09; O, 17.08. [R1= 4-N (CH3)2] (Yield 82%, m.p.198oC) IR (cm-1): 3416 (Ar-NH2), 3056 (Ar), 1655 (C=O), 3395(NH); Anal. Calc’d for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58; O, 10.95. Found: C, 73.95; H, 5.58; N, 9.59; O, 10.88. [R1= 2-OH] (Yield 83%, m.p. 153oC) IR (cm-1): 3416 (Ar-NH2), 3056 (Ar), 1655 (C=O), 3295(OH); Anal. Calc’d for C16H11NO3: C, 72.45; H, 4.18; N, 5.28; O, 18.09. Found: C, 72.45; H, 4.46; N, 5.29; O, 17.8. 1-(2-((18Z)-4-substituted benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol--1-yl) ethyl)-1, 2dihydro-4-methyl-2-oxoquinolin-7-yl substitute The appropriate 1-(2-aminoethyl)-1, 2-dihydro-4-methyl-2--oxoquinolin-7-yl substitute (0.1 moles) and 4-substituted benzylidene-2-phenyl-oxazol-5-one (0.1 moles) have taken in glacial acetic acid (40 mL) and refluxed for 8 hr. The course of the reaction was monitored every hour with the help of TLC. The excess solvent was then distilled off under reduced pressure and poured into crushed ice to get the solid. The final compounds were filtered, dried and purified by recrystalization from absolute ethanol. 1-(2-((18Z)-4-benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2-dihydro-4-methyl2-oxoquinolin-7-yl acetate (AZ1) (Yield 69 % (0.75 gm), m.p. 148 0C) M.W.491.5; M.F. C30H25N3O4; IR (cm-1):3340 (Ar-NH), 2929(Ar), 1675(C=O), 1528 (CH); Log P: 3.83; MR: 142.39 [cm3/mol]; Anal. Calc’d for C30H25N3O4: C, 73.30; H, 5.13; N, 8.55; O, 13.02. Found: C, 73.28; H, 5.17; N, 8.54; O, 13.01. 1H-NMR: 2.08 CH3 (-C=O), 3.22 CH2-CH2, 7.14-7.30 benzylidene. MS: 59.0133 (C2H3O2): 432.171 (C28H22N3O2) 230.082 (C13H12NO3): 261.103 (C17H13N2O) 401.138 (C23H19N3O4): 90.047 (C7H6)

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1-(2-((18Z)-4-(4-methoxybenzylidene)-4, 5-dihydro-5-oxo--2-phenylimidazol-1-yl) ethyl)-1, 2dihydro-4-methyl-2-oxoquinolin-7-yl acetate (AZ2)7, 8 (Yield 67 % (0.45 gm), m.p. 1530C) M.W. 521.5; M.F. C31H27N3O5; IR (KBr) cm-1: 3348 (Ar-NH), 2912(Ar), 1675(C=O), 1520 (CH); Log P: 3.83, MR: 142.39 [cm3/mol]; Anal. Calc’d for C31H27N3O5: C, 71.39; H, 5.22; N, 8.06; O, 15.34. Found: C, 71.34; H, 5.28; N, 8.13; O, 15.25. 1H-NMR: 2.08 CH3 (-C=O), 3.22 CH2-CH2, 3.73 CH3 (methoxy benzylidene) MS: 59.0133 (C2H3O2): 462.182 (C29H24N3O3): 230.082 (C13H12NO3): 291.113 (C18H15N2O2) 401.138 (C23H19N3O4): 120.058 (C8H8O) 1-(2-((18Z)-4-(4-(dimethylamino)benzylidene)-4,5-dihydro-5-oxo-2-phenylimidazol-1-yl)ethyl)-1,2dihydro-4-methyl-2-oxoquinolin-7-yl acetate (AZ3) (Yield 72 % (1.0 gm), m.p. 1920C) M.W. 534.6; M.F. C32H30N4O4; IR (KBr) cm-1: 3421 (Ar-NH), 2917(Ar), 1646(C=O), 1529(CH), 3390(NH2); Log P: 3.44; MR: 144.21 [cm3/mol]; Anal. Calc’d for C32H30N4O4;: C, 71.89; H, 5.66; N, 10.48; O, 11.97, 15.34. Found: C, 71.84; H, 5.68; N, 10.43; O, 12.05; 1H-NMR: 2.08 CH3 (-C=O), 3.22 CH2-CH2, 2.85 CH3 (dimethylamine). MS: 59.0133 (C2H3O2): 475.213 (C30H27N4O2): 230.082 (C13H12NO3): 304.145 (C19H18N3O) 401.138 (C23H19N3O4): 133.089 (C9H11N) 1-(2-((18Z)-4-(2-hydroxybenzylidene)-4, 5-dihydro-5-oxo--2-phenylimidazol-1-yl) ethyl)-1, 2dihydro-4-methyl-2-oxoquinolin-7-yl acetate (AZ4) (Yield 75 % (0.88 gm), m.p. 1720C) M.W. 507.5; M.F. C30H25N3O5; IR (KBr) cm-1: 3345 (Ar-NH), 2912(Ar), 1675(C=O), 1525 (CH), 3290(OH); Log P: 3.7; MR: 149.64 [cm3/mol]; Anal. Calc’d for C30H25N3O5: C, 70.99; H, 4.96; N, 8.28; O, 15.76. Found: C, 70.98; H, 4.98; N, 8.23; O, 15.81; 1H-NMR: 2.08 CH3 (-C=O), 3.22 CH2 -CH2, 5.0 OH (hydroxybenzylidene) MS: 59.0133 (C2H3O2): 448.166 (C28H22N3O3): 230.082 (C13H12NO3): 277.028 (C17H13N2O2) 401.138 (C23H19N3O4): 106.042 (C7H6O) 1-(2-((18Z)-4-benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2-dihydro-4-methyl2-oxoquinolin-7-yl benzoate (AZ5) (Yield 69 % (0.53 gm), m.p. 1620C) M.W. 553.6; M.F. C35H27N3O4; IR (KBr) cm-1: 3346 (Ar-NH), 2912(Ar), 1675(C=O), 1525 (CH); Log P: 4.12; MR: 157.57 [cm3/mol]; Anal. Calc’d for C35H27N3O4: C, 75.93; H, 4.92; N, 7.59; O, 11.56. Found: C, 75.98; H, 4.95; N, 7.53; O, 11.54. 1H-NMR: 7.41-8.14 Aromatic (benzoate), 7.14-7.30 Aromatic (benzylidene) MS: 121.029 (C7H5O2): 432.171 (C28H22N3O2): 292.097 (C18H14NO3): 261.103 (C17H13N2O) 463.153 (C28H21N3O4): 90.047 (C7H6) 1-(2-((18Z)-4-(4-chlorobenzylidene)-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2-dihydro4-methyl-2-oxoquinolin-7-yl benzoate (AZ6) (Yield 69 % (0.43 gm), m.p. 1920C) M.W. 588; M.F. C35H26ClN3O4; IR (KBr) cm-1: 3442(Ar-NH), 2917(Ar), 1654(C=O), 1588(CH); Log P: 3.44; MR: 144.21 [cm3/mol]; Anal. Calc’d for C35H26ClN3O4: C, 71.49; H, 4.46; Cl, 6.03; N, 7.15; O, 10.88. Found: C, 71.48; H, 4.49; Cl; 6.02, N, 7.13; O, 10.88; 1H-NMR: 7.41-8.14 Aromatic (benzoate), 7.22-7.24 Aromatic (chloro benzylidene) MS: 121.029 (C7H5O2): 466.132 (C28H21ClN3O2):292.097 (C18H14NO3): 295.064 (C17H12ClN2O) 463.153 (C28H21N3O4): 124.0008 (C7H5Cl)

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1-(2-((18Z)-4-(4-methoxybenzylidene)-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2dihydro-4-methyl-2-oxoquinolin-7-yl benzoate (AZ7) (Yield 72 % (0.95 gm), m.p. 1840C) M.W. 583.6; M.F. C36H29N3O5; IR (KBr) cm-1: 3127(Ar-NH), 2915(Ar), 1678(C=O), 1594(CH), 2890(CH3); Log P: 5.73; MR: 162.52 [cm3/mol]; Anal. Calc’d for C36H29N3O5: C, 74.09; H, 5.01; N, 7.20; O, 13.71. Found: C, 74.08; H, 5.03; N, 7.23; O, 13.66; 1H-NMR: 7.41-8.14 Aromatic (benzoate), 3.73 CH3 (methoxy benzylidene), 6.72-7.19 Aromatic (methoxy benzylidene) MS: 121.029 (C7H5O2): 462.182 (C29H24N3O3):292.O97 (C18H14NO3): 291.113 (C18H15N2O2) 463.153 (C28H21N3O4): 120.055 (C8H8O) 1-(2-((18Z)-4-(4-(dimethylamino)benzylidene)-4,5-dihydro-5-oxo-2-phenylimidazol-1-yl)ethyl)-1,2dihydro-4-methyl-2-oxoquinolin-7-yl benzoate (AZ8) (Yield 74 % (0.8 gm), m.p. 2150C) M.W. 596.6; M.F. C37H32N4O4; IR (KBr) cm-1: 3346 (Ar-NH), 2916(Ar), 1675(C=O), 1525(CH), 3395(NH); Log P: 6.29; MR: 167.13 [cm3/mol]; Anal. Calc’d for C37H32N4O4: C, 74.48; H, 5.41; N, 9.39; O, 10.731. Found: C, 74.48; H, 5.43; N, 9.37; O, 10.72; 1H-NMR: 7.41-8.14 Aromatic (benzoate), 2.85 CH3 (dimethylamine), 6.54-7.12 Aromatic (dimethylamino benzylidene) MS: 121.09 (C7H5O2): 475.293 (C30H27N4O2): 292.097 (C18H14NO3): 304.145 (C19H18N3O) 463.153 (C28H21N3O4): 133.089 (C9H11N) 1-(2-((18Z)-4-(2-hydroxybenzylidene)-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2dihydro-4-methyl-2-oxoquinolin-7-yl benzoate (AZ9) (Yield 74 % (0.84 gm), m.p. 1750C) M.W. 569.6; M.F. C35H27N3O5; IR (KBr) cm-1: 3346 (Ar-NH), 2914(Ar), 1675(C=O), 1525 (CH), 3295(OH); Log P: 5.34; MR: 164.34 [cm3/mol]; Anal. Calc’d for C35H27N3O5: C, 73.80; H, 4.78; N, 7.38; O, 14.04. Found: C, 73.88; H, 4.73; N, 7.37; O, 14.02; 1H-NMR: 7.41-8.14 Aromatic (benzoate), 5.0 OH (hydroxy benzylidene), 6.68-7.13 Aromatic (hydroxy benzylidene) MS: 121.029 (C7H5O2): 448.166 (C28H22N3O3):292.097 (C18H14NO3): 277.098 (C17H13N2O2) 463.153 (C28H21N3O4): 106.042 (C7H6O) 1-(2-((18Z)-4-benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-7-(benzyloxy)-4methylquinolin-2(1H)-one (AZ10) (Yield 74 % (0.84 gm), m.p. 1750C) M.W. 539.6; M.F. C35H29N3O3; IR (KBr) cm-1: 3346 (Ar-NH), 2914(Ar), 1675(C=O), 1525(CH); Log P: 5.6; MR: 169.77 [cm3/mol]; Anal. Calc’d for C35H29N3O3: C, 77.90; H, 5.42; N, 7.79; O, 8.89. Found: C, 77.88; H, 5.43; N, 7.77; O, 8.92; 1H-NMR: 7.19 Aromatic (benzyloxy), 5.20 CH3 (benzyloxy), 7.147.30 Aromatic (benzylidene) MS: 107.05 (C7H7O): 432.171 (C28H22N3O2):278.118 (C18H16NO2): 261.103 (C17H13N2O) 449.174 (C28H23N3O3): 90.047 (C7H6) 1-(2-((18Z)-4-(4-(dimethylamino) benzylidene)-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-7(benzyloxy)-4-methylquinolin-2(1H)-one (AZ11) (Yield 74 % (0.84gm), m.p. 1820C) M.W. 582.6; M.F. C37H34N4O3; Rf 0.47; IR (KBr) cm-1: 3346(Ar-NH), 2914(Ar), 1675(C=O), 1525 (CH), 3395(NH); Log P: 6.01; MR: 177.7 [cm3/mol]; Anal. Calc’d for C37H34N4O3 C, 76.27; H, 5.88; N, 9.62; O, 8.24. Found: C, 76.28; H, 5.83; N, 9.67; O, 8.22; 1H-NMR: 7.19 Aromatic (benzyloxy), 5.20 CH3 (benzyloxy), 2.85 CH3 (dimethylamine), 6.54-7.12 Aromatic (dimethyl amino benzylidene) MS: 107.05 (C7H7O): 475.213 (C30H27N4O2): 278.118 (C18H16NO2): 304.145 (C19H18N3O) 449.174 (C28H23N3O3): 133.089 (C9H11N)

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1-(2-((18Z)-4-(2-hydroxybenzylidene)-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-7(benzyloxy)-4-methylquinolin-2(1H)-one (AZ12) (Yield 64 % (0.4 gm), m.p. 1510C) M.W. 555; M.F. C35H29N3O4; IR (KBr) cm-1: 3364 (Ar-NH), 2917(Ar), 1666(C=O), 1534(CH), 3081(OH); Log P: 5.34; MR: 164.34 [cm3/mol]; Anal. Calcâ&#x20AC;&#x2122;d for C35H29N3O4 C, 75.66; H, 5.26; N, 7.56; O, 11.52. Found: C, 75.68; H, 5.23; N, 7.57; O, 11.52; 1H-NMR: 7.19 Aromatic (benzyloxy), 5.20 CH3 (benzyloxy), 5.0 OH (hydroxy benzylidene), 6.68-7.13 Aromatic (hydroxy benzylidene) MS: 107.05 (C7H7O): 448.166 (C28H22N3O3); 278.118 (C18H16NO2): 277.098 (C17H13N2O2) 449.174 (C28H23N3O3): 106.42 (C7H6O) Anti-inflammatory and ulcerogenicity index evaluation Representative compounds were evaluated for their anti-inflammatory and ulcerogenicity index using carageenan induced rat paw edema and pyloric ligation methods respectively.The percent protection was measured and the activity was compared with standard drug ibuprofen.The results of anti-inflammatory screening studies are reported in Table-2.Male albino rats weighing between 100 â&#x20AC;&#x201C; 150 gm were used for the experiment.They were divided into various groups. These animals were used for antiinflammatory studies. Thirteen quinazoline derivatives were screened for antiinflammatory activity. The dose levels were fixed based on an acute toxicity studies. Acute anti-inflammatory model (Carrageenan induced rat hind paw oedema)9, 10 The method of Winter.et.al. (Winter.et.al.1963) was used with slight modification. The animals were divided into fifteen groups of six animals each. One group served as a standard (ibuprofen) and another group served as control (1% CMC) and rest of the groups were used for the test drugs (AZ1, AZ2, AZ3, AZ4, AZ5, AZ6, AZ7, AZ8, AZ9, AZ10, AZ11, and AZ12). The rats were dosed with test drug orally at 200 mg/kg body weight based on the acute toxicity studies by Miller and Tainter method and standard ibuprofen was also given the same dose level. Test compounds and ibuprofen were suspended in 1% CMC which was used as a vehicle for the control group. A solution of 1% carrageenan was used as an inflammatory agent. Food was withdrawn overnight with adequate water before the experiment. The drugs were given orally. After 1 hour, a sub plantar injection of 0.05 mL of 1% carrageenan was administered. The volume of the injected paw was measured at 30 mins, 1hr, 2hr and 3hr intervals with a plethysmograph immediately. The average paw volume in a group of drug treated rats was compared with that of a group with vehicle (control group) and the percentage inhibition of oedema was calculated using the formula. % Inhibition = (1- Vt / Vc) x 100, Vt = Mean volume of the test drug Vc = Mean volume of the control The ulcer index was measured and activity was compared with standard drug aspirin as per standard procedure.11, 12 For estimation of ulcer index, the stomach was cut along the greater curvature and the inner surface was examined for ulcerative with the help of a simple dissecting microscope. Usually circular lesions were observed but sometimes, linear were also seen. The ulcer index was calculated as mentioned below. Ulcer index=10/X Where, X=Total mucosal area/Total ulcerated area

RESULTS AND DISCUSSION The physicochemical properties of the imidazoloquinolines, which were the subject of these biological studies in this report. All the compounds were prepared as shown in scheme 1.2. And 3. The imidazoloquinolines (AZ1-AZ12) made of imidazoline and quinoline through Ethylene Bridge. All the structures were confirmed on the basis of physical and spectral studies viz., IR, 1H-NMR, Mass spectroscopy and elemental analysis.

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It allows preparation of many numbers of compounds at the same time in the microwave cavity. Therefore, it is very useful in parallel synthesis and combinatorial synthesis. Moreover, microwave synthesis can lead to improve isolated yields when compared to conventional technology. While it is still at an early stage of development Microwave heating is a very efficient energy source and can be used significantly to reduce reaction, microwave assisted organic synthesis provides an efficient alternative for the synthetic chemist. All the three general schemes (1, 2 & 3) underwent possible microwave irradiation in dry condition by domestic microwave oven. Anti-inflammatory activity was evaluated by carrageenan induced hind paw oedema tests in rats. The anti-inflammatory activity was observed at intervals of 30 min, 1hr, 2hr and 3hr. The % paw oedema inhibition was found to be high at the 2nd hr of administration and hence the 2nd hr values were discussed with the selected compounds. The anti-inflammatory activity data (Table 4.2.1) indicated the selected test compounds which protected the rats from carrageenan induced inflammation. The compounds AZ1, AZ2, AZ3, AZ4, AZ5, AZ6, AZ7, AZ8, AZ9, AZ10, AZ11, and AZ12 were selected randomly to perform the activity The ulcer index of the test compounds (AZ1, AZ2, AZ3, AZ4, AZ5, AZ6, AZ7, AZ8, AZ9, AZ10, AZ11, and AZ12). The high ulcer index score for these compounds may be due to the suppression of the prostaglandin synthesis. The following compounds exhibited prominent activities, the details are as follows: Anti-inflammatory activity: AZ1, AZ2, AZ3, AZ4, AZ5, AZ6, AZ7, AZ8, AZ9, AZ10, AZ11, AZ12.

CONCLUSION The microwave method for synthesis of the title compounds offers reduction inreaction times from hours to minutes and, to increase yields and selectivity. All the title compounds were evaluated for an in vitro anti-inflammatory activity of by carrageenan paw oedema method. The possible potent anti-inflammatory with less ulcerative effect for the new condensed quinazolines were studied. The data reported in this article may be helpful guide for the medical chemists who are working in this area.

ACKNOWLEDGEMENTS The authors are grateful to the Mr. Dinesh Reddy, Secretary of Teegala Ram Reddy College of Pharmacy, Hyderabad for providing necessary facilities. Table-1: Characterization of the synthesized compounds Compoun d

R

R

AZ1.

CH CO-

H

AZ2.

CH CO-

AZ3.

Mol. formula

1

Mol. wt

m.pt (oc)

Solubility

C H NO

491.5

148

CHCl

4-OCH

C H NO

521.5

150

CHCl

CH CO3

4N(CH )

C H NO

534.6

192

CHCl

AZ4.

CH CO-

2-OH

C H NO

507.5

172

CHCl /CCl

AZ5.

C H CO-

H

C H NO

553.6

160

CHCl /CCl

AZ6.

C H CO-

4-Cl

C H N O Cl

588

190

CHCl /CCl

AZ7.

C H CO-

4-OCH

C H NO

583.6

184

C H OH

3

3

30

3

31

32

25

3

27

4

3

30

5

4

4

Lo g P 3.83

3

3.83

3

3.44

3

3 2

3

6

6

6

5

5

5

30

35

35

3

36

25

3

27

26

29

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5

3

3

4

4

3

5

97

3

3

3

2

5

4

4

4

3.7 4.12 3.44

5.73

MR 3

(cm / mol) 142.3 9 142.3 9 144.2 1 149.6 4 157.5 7 144.2 1 162.5 2

Anal. Calcâ&#x20AC;&#x2122;d found C, 73.28; H, 5.17; N, 8.54; O, 13.01 C, 71.34; H, 5.28; N, 8.13; O, 15.25 C, 71.84; H, 5.68; N, 10.43; O, 12.05 C, 70.98; H, 4.98; N, 8.23; O, 15.81 C,75.98; H, 4.95; N, 7.53; O, 11.54 C, 71.48; H, 4.49; Cl, 6.02; N, 7.13; O, 10.88 C,74.08; H, 5.03; N, 7.23; O, 13.66

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Vol.4, No.1 (2011), 91-102 AZ8.

C H CO6

5

4N(CH )

C H NO 37

32

4

4

596.6

215

CHCl

3

6.29

167.1 3

C, 74.48; H, 5.43; N, 9.37; O, 10.72

5.34

164.3 4 169.7 7 177.7

C, 73.88; H,4.73; N, 7.37; O, 14.02 C, 77.88; H, 5.43; N, 7.77; O, 8.92 C, 76.28; H, 5.83; N, 9.67; O, 8.22

164.3 4

C,75.68; H, 5.23; N, 7.57; O, 11.52

3 2

AZ9.

C H CO-

2-OH

C3 H N O

569.6

175

CHCl /CCl

AZ10.

C H CH -

H

C H NO

539.6

151

CHCl

AZ11.

C H CH -

4N(CH )

C H NO

582.6

182

CHCl

AZ12.

C H CH -

2-OH

C H NO

555.6

149

CHCl /CCl

6

5

6 5

2

6 5

2

5

35

37

27

29

34

3

3

4

5

3

3

3

4

5.6

3

6.01

3

3 2

6 5

2

% Inhibition = (1- Vt / Vc) x 100,

35

29

3

4

3

4

5.34

Vt = Mean volume of the test drug Vc = Mean volume of the control

Table-2: Anti-inflammatory in vitro activity of compounds AZ1-AZ12 % Protection Compound 30 min 1h 2h AZ1 35±1.793 47±1.444 49± 1.876 AZ2 39±1.881 49± 1.643 53± 1.372 AZ3 44±1.079 47± 1.377 52±2.46 AZ4 39±2.198 49± 1.404 57± 1.729 AZ5 35±1.472 47± 2.174 49±1.752 AZ6 39±2.66 48±1.872 54±1.4111 AZ7 39±1.831 42± 1.472 49± 1.876 AZ8 35±1.333 39±2.357 47±1.875 AZ9 36±1.831 39± 2.412 43± 1.163 AZ10 27± 1.362 34±1.522 41± 1.476 AZ11 35±3.214 37±2.327 42± 1.874 AZ12 38± 2.538 41± 1.781 46± 1.323 STD 46±2.429 53± 2.16 65± 1.871 Significant levels p <0.01 as compared with the respective control a Each value represents the means ± SD (n=6)

3h 34±1.414 44± 1.424 43±1.762 39± 2.306 39± 1.861 41± 1.472 35± 1.876 37±3.246 32± 1.159 29± 3.132 36±1.861 32± 1.672 43±1.871

Deterimation of % protection of antiinflammatory av tiv ity 80 70

% P r o t e c t io n

60 50

Series1 Series2

40

Series3 Series4

30 20 10

S T D

A Z 1 1

A Z 9

A Z 7

A Z 5

A Z 3

A Z 1

0

Compounds

Fig.-1: Percent protection antiinflammatory activity of 1-(2-((18Z)-4-substituted benzylidene-4,5-dihydro-5-oxo-2-phenylimidazol-1-yl)ethyl)-1, 2-dihydro-4-methyl2-oxoquinolin-7-yl sub

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Vol.4, No.1 (2011), 91-102 Table-3: Ulcerogenicity index of compounds AZ1-AZ12

Compound

Substituents R R1

Ulcer index

AZ1

CH3CO-

H

0.53± 0.02583

AZ2

CH3CO-

4-OCH3

0.54± 0.01891

AZ3

CH3CO-

4-N(CH3)2

0.56± 0.2668

AZ4

CH3CO-

2-OH

0.61±0.01572

AZ5

C6H5CO-

H

0.65±0.01452

AZ6

C6H5CO-

4-Cl

0.73±0.01881

AZ7

C6H5CO-

4-OCH3

0.71±0.1514

AZ8

C6H5CO-

4-N(CH3)2

0.69±0.02766

AZ9

C6H5CO-

2-OH

0.54±0.01671

AZ10

C6H5CH2-

H

0.71±0.01862

AZ11

C6H5CH2-

4-N(CH3)2

0.68±0.01651

AZ12

C6H5CH2-

2-OH

0.65±0.01572

Control

0.64± 0.01454

Std Asprin

1.7± 0.0216

*Significant levels p <0.01 as compared with the respective control a Each value represents the means ± SD (n=6) Determination of ULCER INDEX 2 1.8 1.6

U lc e rIn d e x

1.4 1.2 1

Series1

0.8 0.6 0.4 0.2

A Z 1 1 C on tr o l

A Z 9

A Z 7

A Z 5

A Z 3

A Z 1

0

Compounds

Fig.-2: Ulcerogenicity index of 1-(2-((18Z)-4-substituted benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol-1-yl) ethyl)-1, 2-dihydro-4-methyl-2-oxoquinolin-7-yl sub.

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Vol.4, No.1 (2011), 91-102

O

O

OH

H3PO4 O HO

Ethyl 3-oxobutanoate

O

O

7-Hydroxy-4-methyl-2H-chromen-2-one

OH

Resorcinol

1 1. (CH3C0)2O / PYRIDINE 2. C6H5COCl /C2H5OH

1 O

3. C6H 5CH2Cl /C2H5OH

O

O

R

7-substituted -4-methyl-2H-chromen-2-one 2 gla. Acetic acid

2

NH2CH2CH2NH2

O

N

O

R

Ethane-1,1,2-triamine

NH 2

1-(2-Aminoethyl)-1,2-dihydro-4-methyl-2-oxoquinolin-7-yl substitute

3 Scheme-1: Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one Table-4: Substituents on coumarins S.NO 1. 2. 3.

Substituent (R) CH3COC6H5COC6H5CH2-

Table-5: Substituents on oxazoles S.No. 1. 2. 3. 4. 5. 6.

R1 H 4-Cl 4-OH 4-OCH3 4-N(CH3)2 2-OH

Table-6: Substituents of imidazolo quinoline derivatives Compounds AZ1

CH3CO-

R H

AZ2. AZ3 AZ4 AZ5

CH3COCH3COCH3COC6H5CO-

4-OCH3 4-N(CH3)2 2-OH H

SOME NEW IMIDAZOLO QUINOLINE ANALOGS

R1

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Vol.4, No.1 (2011), 91-102 AZ6 AZ7. AZ8 AZ9 AZ10 AZ11 AZ12

C6H5COC6H5COC6H5COC6H5COC6H5CH2C6H5CH2C6H5CH2-

4-Cl 4-OCH3 4-N(CH3)2 2-OH H 4-N(CH3)2 2-OH

STEP-1: Synthesis of Benzoylglycine O H N

O O

OH

Cl

1O % NaOH

NH2CH2COOH Benzoyl chloride

2-Aminoacetic acid

2-(Benzamido)acetic acid

4 STEP-2: Synthesis of 4-substituted Benzylidene-2-phenyl-oxazol-5-one O

1. AC2O

N

2. Sod.aceate

R1

R1

4

O

3. C2H5OH

O

substituted Benzaldehyde (4E)-4-substituted Benzylidene-2-phenyloxazol-5(4H)-one

5 Scheme-2: Synthesis of 1-(2-Aminoethyl)-1,2-dihydro-4-methyl-2â&#x20AC;&#x201D;oxoquinolin-7-yl sub.

O O

gla. acetic acid

3

N N

5

O

N

R1

R

1-(2-((18Z)-4-substituted benzylidene-4,5-dihydro-5-oxo-2-phenylimidazol-1-yl)ethyl)-1,2-dihydro-4-methyl-2-oxoquinolin-7-yl substitute

Scheme-3:Synthesis of 1-(2-((18Z)-4-substituted benzylidene-4, 5-dihydro-5-oxo-2-phenylimidazol--1-yl) ethyl)-1, 2-dihydro-4-methyl-2-oxoquinolin-7-yl substitutes

REFERENCES 1. A.I. Vogel, Longmans, Green and Co: London, Text Book of Practical Organic Chemistry., 5th edition, 1156 (1976) 2. M.V. Kulkarni, B.G. Pujar and V.D. Patil, Arch Pharm., (1) 316 (1981) 3. V. Nadaraj and S. Thamarai Selvi,Indian J Chem., 46B,1203 (2007)

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4. G. Bhattacharjee, S. M. Sondhi,Monica Dinodia and Sushanta K Mishra, Indian J Chem Tech., 15,72 (2008) 5. Shashikanth, R.Pattan C K Hariprasad, Nachiket S Dighe, S B Bhawar, S V Hiremath and B N Ingalag, Indian J Pharm Research & Develop., 1(9) (2009) 6. C. Rajveer, B. Stehenrathinaraj, D.Kumaraswamy, S. Sudharshini and C. Swarnalatha, International J Pharm Research., 2(3), 50 (2010) 7. Mohammad Hassan Houshdar Tehrani,Afshin Zarghi and Laleh Erfani Jabarian, Indian J Pharm Research., 1, 37 (2005) 8. Kumari Shalini, Pramod Kumar Sharma and Nitin Kumar, Pelagia Research Library Der Chemica Sinca., 1(3), 36 (2010) 9. Pankaj S Sulunkhe, Harun M Patel, Rahul D. Shimpi, Nikhil N Lalwani,International J Pharm Research and Develop., 2(1) (2010) 10. Y.L.Chem, I.L Chem, Lu CM, E.E. Tzeng, L.T. Tsao and J.P. Wang, Bioorg Med Chem., 12(2), 387 (2004) 11. Ali Khalaj, Mohammad Abdollahi, Abbas Kebbriaeczadeh, Neda Adibpour, Zahra Pandi and Sara Rasoulamini, Indian J Pharmac., 34, 184 (2002) 12. B.Gupta, K.K. Saxena, R.K. Srivastava and D.N. Prasad, Indian J Pharmac.,51 (1985) [RJC-728/2011]

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SYNTHESIS, CHARACTERIZATION AND ION-EXCHANGING PROPERTIES OF NOVEL ION-EXCHANGE RESIN PART-III 1

Sanjay Kumar Saraf1 and Arun Singh2,*

Department of Chemistry, Govt.Geetanjali Girls' College Bhopal, India Joint Director, Rajya Siksha Kendra, Pustak Bhawan, B-wing, Arera Hills, Bhopal, India *E-mail: dr.arunsingh@rediffmail.com

2

ABSTRACT The polyamine (PA) was prepared by condensation of 1,4-bischloromethyl benzene and benzidine. The PA was then treated with cyanuric chloride at 0°C followed by reaction with 5-amino-8-Hydroxyquinoline in THF in conc. NaOH (PH 9-10) at room temperature for 8 hrs. The resultant polymer designated as polyamine-s-triazine-5-amino8-Hydroxyquinoline (PATHQ) was characterized by elemental analysis, IR spectral studies, and thermogravimetry. The PATHQ sample was monitored for its chelating and ion-exchanging properties. The polymeric metal chelates of PATHQ with Cu2+, Zn2+, Mn2+, Ni2+, UO22+ and Co2+ metal ions were prepared and characterised by metal:ligand ratio, IR and reflectance studies, magnetic properties, thermogravimetry and microbicidal activity. Batch equilibration method has been adopted, for evalution of ion-exchange properties. Key words Polyamines, s-triazine, 5-amino-8-Hydroxyquinoline, polymeric metal Chelates, and magnetic properties, IR spectra, ion-exchange properties, Batch equilibrium method, thermogravimetry. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION The effluents from mines and metal industries set up the serious problems in removal of heavy toxic metal ions. The contents of these metals in effluent are almost above the valid limit1-3. The contents of this metal can be reduced by treatment of lime, but result is not satisfactorily. Thus ion-exchange technique has been proved very useful in this context. The ion-exchange resin can be use for metal extraction from ore, analytical reagent, and separation of metal ion and deionization of water4-10. Most of commercial ion-exchange resins are sulfonated polystyrene-divinylbenzene copolymer11-12. The use of complex ion-formation in ion-exchange resin has been prepared to solve the problem11-12.The aim of the present work to prepare and study the novel ion-exchange resin. Thus We reported recently the novel ion-exchange resin13,14. In continuous of this work13,14 the present paper paper comprises the synthesis of novel ion-exchange resin containing a well known metal complexing agent and 8-hydroxyquinoline and its i.e. chelating and ion-exchanging properties  easurements The synthetic route is shown below

EXPERIMENTAL Materials: All the chemicals used were of either pure or analytical grade. Synthesis of polyamines (PA) and PA-triazine (PAT) resin were prepared by method repoted in an earlier communication13-15. 5 – Amino – hydroxyl quinoline was prepared by method reported by method reported in literature16. Synthesis of PAT-5-Amino-8-Hydroxy quinoline (PATHQ) To a mixture of PAT polymer (0.01 mole) and 5-Amino-8-Hydroxy quinoline (0.02 mole) in THF (100 ml), Conc. NaOH was added with maintaining pH 9-10 of the mixture was heated upto 60°C gently for 5 minute and it was stirred at room temperature for 8 hrs. The resulted gel type material was filtered, washed by water and air-dried. It was powdered to 100 mesh size. Yield was 90%. It did not melt up to 300°C and insoluble in water and common organic solvents. Synthesis of polymeric chelates The polymeric metal chelates of PATHQ were synthesized by reaction of PATHQ with corresponding metal acetates. The detail procedure is as follows-

NOVEL ION-EXCHANGE RESIN PART-III

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Vol.4, No.1 (2011), 103-109

A dried PATHQ polymer (0.01 mole) was dispersed in 200 ml aqueous solution of 20% aqueous formic acid and warmed on a water bath for 10 minutes. To this dispersed solution a warm solution of metal acetate (0.01 mole) in 50% aqueous formic acid solution was added drop wise with constant stirring. The reaction mixture as made alkaline with dilute ammonia solution in order to coagulate polymeric chelates. The resultant contents were further digested on water bath for an hour. Finally the solid polymer chelates were filtered off Washed with hot water followed by acetone. DMF and dried in air. The polymer chelates of PATHQ with Cu2+, Co2+, Ni2+, Mn2+, UO22+ and Zn2+ transition metal ions. Measurements Elemental analyses for C,H and N content were carried out on TF 1101 elemental analyzer (Italy). IR spectra of polymer ligand and their metal chelates were scanned on a NICOLET 760 DR FTIR spectrophotometer in KBr phase. The metal content of polychelates was performed by decomposing a weighed amount of each polymer chelate followed by EDTA titration as reported in literature17. Magnetic susceptibility measurements of all the polychelates were carried out at room temperature by the Gouy method using Mercury tetrathiocyanato cobaltate (II) Hg [Co (NCS)4] as a calibrant. The diffuse reflectance spectra of all the solid polychelates were recorded on a Backman DK-2A spectrophotometer with solid reflectance attachments. MgO was employed as the reference attachments. MgO was employed as the reference compound. Thermal behaviour of these metal chelates was studied by TGA performed on thermogravimetric analyzer. The batch equilibration method was adopted for the ion-exchanging properties18,19. The evaluation of the influence of different electrolytes on metal uptake by the polymer, the rate of metal uptake under specified conditions and distribution of various metal ions of different PH values were carried out following the details of the procedures described earlier18,19.

RESULTS AND DISCUSSION The polymer sample PATHQ was in form of dark brown powder and insoluble in common organic solvents. It swells up to some extent in conc. NaOH solution. It did not melt up to 300ºC. The elemental contents in Table-1 are consistents with the predicted structure. The IR spectrum comprises the bands due to secondly NH (3400 cm-1), methylated group (2930, 2850, 1430 cm-1), s-triazine and aromatic 8HQ moieties (3030, 1500, 1600 cm-1). The TGA of PATHQ contains single step degradation. The degradation starts from 280°C, loss rapidly between 300 to 500 and almost lost 85% at 650°C. Characterization of Polymeric Chelates The polymeric chelates of PATHQ with different metal ions such as Cu2+, Ni2+, Co2+, Mn2+ UO22+ and Zn2+ vary in color from dark green to brown. They generally resemble each other. Comparison of IR spectra of the parent ligand with their polymer chelates has revealed certain characteristics differences as mentioned below. One of the significant differences to be expected between IR spectrum of parent ligand and its metal chelates is the absence of a broad band in the region of 3370-3450 cm-1 due to O-H stretching vibration frequencies in IR spectrum of polymer chelates as the oxygen of this O-H of parent ligand has formed a bond with the metal ion. However this band has explicable by the fact that water molecules might have strongly absorbed to the chelates during the formation. Another noticeable difference is that the bands due to C=N stretching vibration of 8-quinolionol at 1606 cm-1 in IR spectrum of PATHQ has assigned to implane O-H deformation and this is shifted towards higher frequency in the spectra of polymer chelates indication the formation of metal-oxygen bonds2022 .This has been further confirmed by a weak band at 1100 cm-1.Corresponding to C-O-M stretching frequency20-22. All these characteristic features of IR suggest the general structure of polymer chelates as shown in Scheme 1. Examination of data about metal content in each polymer chelates (Table- 1 and 2) has revealed a 1:1 metal: ligand stoichiometry in all the polychelates. Magnetic moment (µeff) data of polymer chelates given in Table I has reveals that all metal chelates like Cu2+, Ni2+ and Co2+ are paramagnetic, while that of Zn2+ is diamagnetic in nature. The electronic spectral data assignments are shown in Table-3. The electronic spectra of PATHQ with Cu+ 2 ions show two broad bands at 14950 and 23529 cm-1 due to 2T1g→2Eg NOVEL ION-EXCHANGE RESIN PART-III

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transition and charge transfer spectra respectively suggesting a distorted octahedral structure for PATHQ polymer chelates. The PEAQ with Ni2+ and Co+2 ion polychelates give two absorption bands respectively at 14925, 24096 cm-1 and 14925, 22471 cm-1 corresponding to 4Tg → 2T1g, 4T1g→ 4T1g(P) transition . Thus, absorption band of diffuse reflectance spectral and the values of magnetic moment (µeff) have indicated an octahedral configuration for the Ni2+, and Co2+ polychelates. The spectra of polychelates of Mn2+ ion show two weak bands at 17241 cm-1 and 25031 cm-1 assigned to the transition 6A1g→4T2g (4G) and 6T1g→4T1g(4G) respectively and assigned an octahedral structure for PATHQ chelates. As the spectrum of the Zn2+ chelates is not well resolved it is not interpreted but its µeff value reveals its diamagnetic nature as expected. The TGA data (TG thermograms not shown) of all polymeric chelates are shown in Table-2. The TGA data as thermograms reveals that the rate of decomposition of all polymeric chelates is initially low up to 200ºC temperature and rapidly increases to maximum in the range 400-500 ºC. This might be due to accelerated catalytically by `insitu` formation of metal oxide of thermal stability of all these polychelates is quite similar. Ion-Exchange properties The examination of data presented in Table-2 reveals that the amount of metal ions taken up by a given amount of the PATHQ polymer depends upon the nature and concentration of the electrolyte present in the solution. The amounts of Fe3+, Cu2+ and UO22+ ions taken up by the polymer sample increase with the increase in concentration of ions taken up by the polymer sample increase with the increase in concentration of ions like chloride, chlorate and nitrate but decrease with the increase in concentration of the sulfate ions. The amounts of the remaining three metal ions Co2+ , Mn2+, and Zn2+, taken by the polymer sample decrease with the increase in concentration of chlorate, chloride, nitrate and sulfate ions. Rate of metal uptake The rates of metal absorption by the PATHQ sample were measured for Fe3+, UO22+, Cu2+ and Mn2+ ions presence of 1 M NaHCO3 to know the time required to reach the stage of equilibrium. All experiments were carried out at pH 3. The examination of the results presented in Tab.3 Shows that UO22+ and Fe3+ ions required slightly more than three hours for the establishment of equilibrium and Cu2+ and Mn2+ ions required about five h for the purpose. In the experiments with solution containing UO22+ and Fe3+ ions, more than 70% of equilibrium was established in the first h. This reveals that the rate of uptake of metal ions follows the order UO22+, Fe3+ > Cu2+ > Mn2+. The rates of uptake of Zn2+ and Co2+ ions have been found to be very low at pH 3. Hence the values are no reported. Distribution ratio of metal ions at different pH values The results described in Tab.4 reveal that the amount of metal ions taken up by the polymer sample PATHQ at equilibrium increases with the increase in pH. The selectivity of the polymer sample UO22+ and Fe3+ ions are higher than that for each of the remaining metal ions. The distribution ratio for Fe3+ ions is lower than that for UO22+ by about 1800 units at pH 3. The lower values of the distribution ratio for Fe3+ ions requires its attachment with proper sites on three different polymer chains and that of the UO22+ ion requires such an attachment with sites on two polymer chains . Among the remaining metal ions, Cu2+ has a high value of distribution ration at pH 6 while the other three mental ions Co2+ , Zn2+ , and Mn2+ have a low distribution ration over a pH range from 4 to 6. Further work in the direction of wide range at such polymers and their ion exchanging properties are under progress.

ACKNOWLEDGEMENTS The author thanks to Dr. (Mrs.) Nayantara Pathak Principal of Government Geetanjali P.G. Girls Collage for providing research facility and encouragement. Table-1: Elemental analyses of polymeric metal Chelates of PATHQ and their metal chelates Sample Designation PATHQ

Elemental Analysis N% M% Cal. Found Cal. Found 21.10 21.0 --------

NOVEL ION-EXCHANGE RESIN PART-III

µeff B.M ------

105

Elemental Analysis %Weight loss at different temperature ºC 200

300

400

500

600

S. K. Saraf and A. Singh

700

Vol.4, No.1 (2011), 103-109 PATH (Cu2+) 2H2O 18.83 18.7 7.17 7.1 1.78 0.8 1.4 PATHQ(Ni2+) 2H2O 18.91 18.8 6.75 6.8 3.00 1.0 8.1 PATHQ(UO22) 2H2O 15.34 15.4 24.38 24.3 D 1.8 10.9 PATHQ(CO2+) 2H2O 18.94 18.8 6.65 6.6 4.03 1.6 9.8 PATHQ(Mn2+) 2H2O 19.02 19.0 6.22 6.3 4.68 1.6 11 PATHQ (Zn2+) 2H2O 18.81 18.9 7.27 7.2 D 0.9 9 OH groups: 4 per repeat unit. IR features: 3400 (-NH-), 3600-2200 cm-1 (-OH-) 1604, 1500, 3050 cm-1 (aromatic), 1520, 1260, 860 (S-triazine) 2930, 2850, 1430 cm-1 (-CH2-)

15 23 28 23 24 17

53 56 59 65 63 58

69 72 77 71 76 67

Table-2: Evaluation of the influence of different electrolytes in the uptake of several metal ions ([Mt (NO3)2] = 0.1 mole 路 l -1)a Metal ions

PH

[Electrolyte]

Adsorption of mmol. 路 101 of the metal ion on PATHQ polymerb. NaClO4 NaNO3 NaCl Na2SO4

(mole 路 l -1) Cu2+

5.5

Fe3+

2.75

UO22+

4.0

Co2+

5.5

Mn2+

5.5

Zn2+

5.5

Ni2+

5.5

0.01 0.05 0.1 0.5 1.0 0.01 0.05 0.1 1.0 0.01 0.05 0.1 0.5 1.0 0.01 0.05 0.1 0.5 1.0 0.01 0.05 0.1 0.5 1.0 0.01 0.05 0.1 0.5 1.0 0.01 0.1 0.5 1.0

0.07 0.15 0.11 0.24 0.40 0.08 0.18 0.21 0.31 0.15 0.19 0.14 0.22 0.53 0.11 0.10 0.03 0.02 0.01 0.18 0.15 0.13 0.10 0.08 0.10 0.10 0.08 0.02 0.01 0.06 0.19 0.20 0.31

0.05 0.07 0.12 0.14 0.18 0.10 0.12 0.13 0.21 0.12 0.14 0.23 0.43 0.42 0.13 0.12 0.13 0.04 0.01 0.22 0.20 0.17 0.16 0.17 0.05 0.04 0.06 0.03 0.01 0.10 0.13 0.12 0.20

0.11 0.13 0.14 0.18 0.23 0.01 0.02 0.05 0.21 0.11 0.13 0.17 0.20 0.23 0.07 0.10 0.07 0.05 0.02 0.20 0.16 0.19 0.18 0.13 0.07 0.06 0.02 0.01 0.01 0.01 0.01 0.05 0.20

0.26 0.24 0.23 0.21 0.17 0.18 0.04 0.06 0.04 0.20 0.21 0.24 0.17 0.18 0.04 0.05 0.04 0.03 0.01 0.14 0.10 0.06 0.02 0.01 0.11 0.02 0.05 0.01 0.01 0.16 0.02 0.05 0.03

a. Volume of electrolyte solution 40 ml, time 24h, volume of metal ion solution 1ml, NOVEL ION-EXCHANGE RESIN PART-III

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84 93 92 96 97 90

Vol.4, No.1 (2011), 103-109 temp. 25 ยบC b. Wt. of PATS polymer 25 mg.

Scheme-1

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Vol.4, No.1 (2011), 103-109 Table-3: Comparison of the rates of metal (Mt) ion uptake a Attainment of equilibrium state b.

Time (h) Fe3+ 62.5 71.1 87.3 91.2 90.4 -------

0.5 1 2 3 4 5 6 7

UO22+ 31.2 65.1 83.4 91.1 95.7 -------

Cu2+ 33.7 51.5 63.9 74.3 83.5 89.1 93.6 92.1

Mn2+ 21.7 46.1 62.5 76.3 83.5 85.5 91.5 97.2

a. [Mt (NO3)2] = 0.1 mole · l -1 , volume 1 ml, [NaNO3]=1 mol ·l -1, volume 40 ml pH = 3, temp 25º C, wt of PATS polymer 25 mg. b. Related to the amount of metal ions taken up at the state of equilibrium assumed to be established in 24 h and assumed to be 100%.

Table-4:Distribution rations, D, of different metal ions as a function of the pH Distribution ratioa of metal ionsb

pH Cu2+

Fe3+

UO22+

Co2+

Mn2+

Zn2+

1.5

-----

-----

247

----

-----

-----

1.75

126

123

423

----

-----

-----

2.0

164

162

513

---

----

----

2.5

441

446

542

---

----

----

3.0

949

943

2783

----

----

----

4.0

---

---

----

2

72

75

5.0

----

----

----

75

134

135

6.0

---

---

----

333

255

253

a

mmol of metal ions taken up by 1 g of polymer / mmol of ions present in 1 ml of solution [Mt(NO3)2] = 0.1 mol/l, Volume= 1ml, wt. of polymer=25mg, [NaNO3] = 1mol.l, volume= 44ml, temp.= 250C, time =24hr(equilibrium state). b

Error +/- 5%

REFERENCES 1. R.E.Wing, W.in. Doane, and C.R Runell, J.Appl. Polym. Sci, 19, 847 (1995). 2. A.K.D Metra and A. Karchadhanvi, Ind. J. Chem., 39 B, 311 (2000). 3. L.F. Martin, Industrial water Purification Noyes Data corporation Park Ridge New Jercy, (1974). 4. L.S.M. BentoProc., Sugar process. Res. Conf. ,1990, 99 (1991). 5. M.Shimatani and co.Workeres (to snow brand milk products co,. Ltd.) U.S.Pat., 5,084, 285 (Jan. 28, 1992). 6. O.L.Sprockel, and J.C.Price, Drug Dev. Ind. Pharm., 16 (2), 361 (1990). 7. L.A.Smith(to chemical research & Licensing Co.) U.S.Pat, 4,978,807, (Dec. 18, 1990). 8. F.E.Ahmed, B.D.Young, and A.W. Bryson, Hydrometallurgy, 30 (1-3), 257 (1992). 9. J.A. Ritter and J.P. Bibler, water Sci. Techno. 25(3), 165 (1992). 10. CEP, C.R., 70-77 (Aug.1979) 11. G.F.D’Alelio (to General Electric co.)U.S.Pat 2,366,007 (Dec.26, 1945). 12. G.F.D’Alelio (to General Electric co.)U.S.Pat. 2,596, 417 (Apr.22,1952)., 13. Arun Sing and Varsha parmar, Int. J. of Polymeric Material Sci, 57,1019 (2008). NOVEL ION-EXCHANGE RESIN PART-III

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14. Arun sing, Sanjaykumar Saraf, Int. J. of Polymeric Material Sci.,58,499 (2009). 15. E.Tsuchida, K.Sanada, K.Moribe and I.Shinohara, Markromol.Chem. 151, 207 (1972). 16. Y. T Pratt and N. L. Drake, J. Am. Chem. Soc, 82, 1155 (1960). 17. A. I.Vogel (1978) Textbook of Quantitative Chemical Analysis, (ELBS 4th Edn. London). pp. 317 Co. Bombay(1972). 18. H.P.Greger, M.Tieter, L.Citaval, and E.I. Becker Ind. Elng. Chem. 44, 2834 (1952). 19. R.C.Decoeso, L.G. Donarma and E.A. Tanic, Anal. Chem. 34, 845 (1962). 20. S.Narayan, S.B. Bhave and R.B. Kharat, J. Indian Chem, Soc., 58,1194 (1981). 21. D.R.Agrawal and S.G.Tandon, J. Indian Chem. Soc., 48, 6, (1971). 22. N.R. Gandhi and K.N. Munshi, J. Indian, Chem Soc., 59, 1290 (1982).

[RJC-636/2010]

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Vol.4, No.1 (2011), 110-112 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

POLAROGRAPHIC REDUCTION OF IODATE-STUDY OF KINETIC WAVE G.Sailaja1, R.Ramachandramurthy1 and V. Suryanarayana Rao2,* 1

2,*

Department of Chemistry, Govt. College, Anantapur-515001(A.P.) India Department of Chemistry, S.K. University, Anantapur-515001(A.P.) India E-mail: r.r_murthy@yahoo.co.in

ABSTRACT Polarographic reduction of the conjugate bases of many halogen oxy acids has been of great importance from the kinetic currents owing to their analytical applications. Many of them have been used as catalytic agents for the reductions of many transition mental ions. The polarographic reduction of Iodate has been investigated in the Acetic acid-Sodium acetate bufer solutions of differente pH. A single six electron reduction of Iodate has been reported in the literature. In the present investigations a ssecond wave corresponding to two electron reduction has been reported at 1.62V against SCE. The various studies on the second wave of Iodate and the possible mechanism for it is discussed in the present paper. Keywords: D.C.Polarography, Iodate, Acetic acid –Sodium acetate buffer, d.m.e, height of the mercury column, kinetic wave, SCE. © 2011 RASĀYAN. All rights reserved.

INTRODUCTON The polarographic reduction of Iodae ion at the d.m.e. was investigated by Rylich1 in a detailed fashion. He concluded that the total diffusion current corresponds to the following net electrode reaction. IO3- + 6 H+ + 6 e- → I - + 3 H2O Kolthoff and Orlemann made a more exact investigation and confirmed that the number of electrons involved in the  esponsib of Iodate is equal to 6. Rylich made a systematic study on the reduction of Iodate in acid, neutral and alkaline unbuffered solutions. Kolthoff and Orlemann obtaine two waves of 1 and 5 electron reduction in dilution solutions of strong acids when the hydrogen ion concentration is smaller than two times the concentration of Iodate. The authors of the present investigation reports the presence of two waves of 6 and 2 electron reduction in buffer solutions of certain anions capable of forming complex with Iodate cation. 2

EXPERIMENTAL Reagents All chemicals used were of analytical-reagent grade. Potassium iodate solution (0.1M) prepared in conductivity water with appropriate  esponsi is used. A 0.2 M Sodium acetare and acetic acid buffer solutions of different pH have been employed in the investigations. Apparatus A CL-25 pen recording d.c. polarograph (ELICO, Hyderabad, India), an ELICO Model CL-10 pH meter and a Lingane-type H-cell were employed. The voltages were measured against a saturated calomel electrode. Pure Nitrogen gas is is  espon through the apparatus for about 15 minutes to remove dissolved oxygen.

RESULTS AND DISCUSSION Typical polarogram of Iodate recorded in an acetate-acetic acid buffer solution of pH 5 is presented in Figure 1. The polarogram reveals the presence of two waves of 3:1 ratio under the experimental solutions. Studies on the effect of pH indicated that the polarographic wave has a well defined shape and large currents in solutions of pH 5. The studies on the height of the mercury column on the second wave POLAROGRAPHIC REDUCTION OF IODATE

G.Sailaja et al.

Vol.4, No.1 (2011), 110-112

current indicated its kinetic nature since it remained constant with height of the mercury column from 40 to 80 Cm Table 1. The log plot studies on the kinetic current revealed the six electron reduction of the first wave and the two electron reduction of the second wave. Reduction currents of both the wavs varied linearly with the concentration of Iodate.

Fig.-1: Polarogram

Mechanism of the Electrode Process The wave observed around -0.6 V against SCE pertains to the 6 electron reduction of Iodate to Iodide as reported by the earlier workers. The second wave observed around -1.62 V against SCE of two electron reduction has been attributed to the kinetic wave due to the formation of monovalent Iodine cation formed at the surface of the dropping mercury electrode due to the following chemical reactions. IO3- + 6 H + + 6 e- → I- + 3 H2O IO3- + I- → 3 I2 + 3 H2O IO3 + 2 I2 + 6 H + → 5 I+ + 3 H2O Lang3 proposed that Iodate  esponsib in presence of excess Iodide and hydrogen ions proceed through several stage leading to the formation of Iodine cation. Lang4 has developed methods in which the Iodine cation is stabilized by the formation of the Iodine- cyanide. I+ + HCN → ICN + H +

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Further it is reported that electropositive monovalent Iodine is capable of combining with highly polarizable acid anions such as CN-, CNO-, CNS- and so on. The pKa values of Cyanic acid , Isocyanic acid and Thiocyanic acid are 9.21 , 3.46and 0.85 respectively. The pKa value of acetic acid (4.76) is comparable with the former two acids . Therefore it is probable that acetate ion also combines with Iodine cation forming [ I (CH3COO)] analogous to [I(CN)], [I(CNO)] and [I(CNS)]. This ion is undergoing two electron reduction at the d.m.e yielding Iodide which is  esponsible for the appearance of the second wave at -1.62 V against SCE. Similar results are noticed in presence of cyanic, Isocyanic and Thiocyanic acid buffer solutions confirming the following mechanism. [ I (CH3COO)] + 2 e- + H + â&#x2020;&#x2019; I- + CH3COOH The kinetic reduction of Iodate is dependant on its concentration since polarogram recorded at low concentration lack the two wave reduction of the Iodate reported. This confirms the kinetic nature of the wave and its surface nature. Since the concentration of Iodate as well as Iodide at the d.m.e are very low the kinetic reaction does not take place due to which neither the first wave of 6 electron reduction nor the second wave of 2 electron reduction are noticed. Table-1: Effect of the mercury column height on the kinetic current [IO3-] = 1 X 10-3 M; pH = 5 Mercury column height (cms) 80 70 60 50 40

Kinetic current ( Âľ A)

Half wave potential (- V vs SCE)

10 10 10 10 10

1.62 1.62 1.62 1.62 1.62

ACKNOWLEDGEMENTS The authors express their thanks to the Government College, Anantapur and Department of Chemistry, S.K University, Anantapur for providing necessary facilities.

REFERENCES 1. 2. 3. 4. 5.

A. Rylich , Coll. Czech. Chem. Commun., 7, 288 (1935). E.F. Orlemann and I.M. Kolthoff , J. Am.Chem.Soc., 64 1044, 1970 (1942). R. Lang, Z. Anal. Chem., 106,12 (1936). R. Lang, Z. Inorg.Chem., 122, 322 (1922) H. Remy, Treatise on Inorganic Chemistry, Vol.II, 817 Elsevier, New York (1970) [RJC-592/2010]

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Vol.4, No.1 (2011), 113-116 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

DEVELOPMENT AND VALIDATION OF NEW HPLC METHOD FOR THE ESTIMATION OF PERINDOPRIL IN TABLET DOSAGE FORMS 1

V. Bhaskara Raju1 and A. Lakshmana Rao2,* Sri Vasavi Institute of Pharmaceutical Sciences, Tadepalligudem- 534 101, A.P., India. 2 V.V. Institute of Pharmaceutical Sciences, Gudlavalleru- 521 356, A.P., India. * E-mail: dralrao@gmail.com

ABSTRACT An accurate and precise RP-HPLC method was developed for the determination of perindopril in tablet dosage forms. Separation of the drug was achieved on a reverse phase C18 column using a mobile phase consisting of phosphate buffer and acetonitrile in the ratio of 65:35 v/v. The flow rate was 0.6 ml/min and the detection wavelength was 209 nm. The linearity was observed in the range of 20-100 µg/ml with a correlation coefficient of 0.9997. The proposed method was validated for its linearity, accuracy, precision and robustness. This method can be employed for routine quality control analysis of perindopril in tablet dosage forms. Keywords: Perindopril, Estimation, RP-HPLC, Validation. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Perindopril erbumine1 is the tert-butylamine salt of perindopril, the ethyl ester of a non-sulfhydryl angiotensin-converting enzyme (ACE) inhibitor. It is rapidly metabolized in the liver to perindoprilat, its active metabolite, following oral administration. Perindoprilat is a potent, competitive inhibitor of ACE, the enzyme responsible for the conversion of angiotensin I (ATI) to angiotensin II (ATII). ATII regulates blood pressure and is a key component of the renin-angiotensin-aldosterone system (RAAS). Perindopril may be used to treat mild to moderate essential hypertension, mild to moderate congestive heart failure, and to reduce the cardiovascular risk of individuals with hypertension or post-myocardial infarction and stable coronary disease. Perindopril erbumine is chemically described as (2S, 3αS, 7αS)-1-[(S)-N-[(S)-1carboxy butyl]alanyl]hexahydro-2-indolinecarboxylic acid, 1-ethyl ester, compound with tert-butylamine (1:1) (Fig. 1). A few spectrophotometric2,3, HPLC4 and LC-MS5 methods were reported earlier for the determination of perindopril in bulk and pharmaceutical dosage forms. In the present study the authors report a rapid, sensitive, accurate and precise RP-HPLC method for the estimation of perindopril in bulk samples and in tablet dosage forms.

EXPERIMENTAL Chromatographic conditions The analysis of the drug was carried out on a Waters HPLC system equipped with a reverse phase Xterra C18 column (100 mmx4.6mm; 5 µm), a 2695 binary pump, a 20 µl injection loop and a 2487 dual absorbance detector and running on Waters Empower2 software. Chemicals and solvents The reference sample of perindopril was supplied by Glenmark Pharmaceuticals Ltd, Mumbai. HPLC grade water and acetonitrile were purchased from E. Merck (India) Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR grade were obtained from S.D. Fine Chemicals Ltd., Mumbai. Preparation of phosphate buffer (pH 3.0) Seven grams of KH2PO4 was weighed into a 1000 ml beaker, dissolved and diluted to 1000 ml with HPLC water. 2 ml of Triethyl amine was added and pH adjusted to 3.0 with orthophosporic acid.

ESTIMATION OF PERINDOPRIL

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Vol.4, No.1 (2011), 113-116

Preparation of mobile phase and diluents 650 ml of the phosphate buffer was mixed with 350 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum. Procedure A mixture of buffer and acetonitrile in the ratio of 65:35 v/v was found to be the most suitable mobile phase for ideal separation of perindopril. The solvent mixture was filtered through a 0.45µ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 0.6 ml/min. The column was maintained at ambient temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. The detection of the drug was monitored at 209 nm. The run time was set at 6 min. Under these optimized chromatographic conditions the retention time obtained for the drug was 3.122 min. A typical chromatogram showing the separation of the drug is given in Fig. 2. Calibration plot About 25 mg of perindopril was weighed accurately, transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 65:35 v/v mixture of phosphate buffer and acetonitrile. The solution was sonicated for 15 min and the volume made up to the mark with a further quantity of the diluent to get a 100 µg/ml solution. From this, a working standard solution of the drug (40µg/ml) was prepared by diluting 2 ml of the above solution to 10 ml in a volumetric flask. Further dilutions ranging from 20-100 µg/ml were prepared from the solution in 10 ml volumetric flasks using the above diluent. 20 µl of each dilution was injected six times into the column at a flow rate of 0.6 ml/min and the corresponding chromatograms were obtained. From these chromatograms, the average area under the peak of each dilution was computed. The calibration graph constructed by plotting concentration of the drug against peak area was found to be linear in the concentration range of 20-100 µg/ml of the drug. The relevant data are furnished in Table-1. The regression equation of this curve was computed. This regression equation was later used to estimate the amount of perindopril in tablets dosage forms. Validation of the proposed method The specificity, linearity, precision, accuracy, limit of detection, limit of quantitation, robustness and system suitability parameters were studied systematically to validate the proposed HPLC method for the determination of perindopril. Solution containing 40 µg/ml of perindopril was subjected to the proposed HPLC analysis to check intra-day and inter-day variation of the method and the results are furnished in Table-2. The accuracy of the HPLC method was assessed by analyzing solutions of perindopril at 50, 100 and 150 % concentrated levels by the proposed method. The results are furnished in Table-3. The system suitability parameters are given in Table-4. Estimation of perindopril in tablet dosage forms Two commercial brands of tablets were chosen for testing the suitability of the proposed method to estimate perindopril in tablet formulations. Twenty tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 25 mg of perindopril was transferred into a 100 ml volumetric flask and dissolved in 25 ml of a 65:35 v/v mixture of phosphate buffer and acetonitrile. The contents of the flask were sonicated for 15 min and a further 25 ml of the diluent was added, the flask was shaken continuously for 15 min to ensure complete solubility of the drug. The volume was made up with the diluent and the solution was filtered through a 0.45 µ membrane filter. This solution was injected into the column six times. The average peak area of the drug was computed from the chromatograms and the amount of the drug present in the tablet dosage form was calculated by using the regression equation obtained for the pure drug. The relevant results are furnished in Table-5.

RESULTS AND DISCUSSION In the proposed method, the retention time of perindopril was found to be 3.122 min. Quantification was linear in the concentration range of 20-100 µg/ml. The regression equation of the linearity plot of concentration of perindopril over its peak area was found to be Y=32625.5+38432.49X (r2=0.9997), where X is the concentration of perindopril (µg/ml) and Y is the corresponding peak area. The number of theoretical plates calculated was 3103, which indicates efficient performance of the column. The limit of ESTIMATION OF PERINDOPRIL

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detection and limit of quantitation were found to be 0.03 µg/ml and 0.12 µg/ml respectively, which indicate the sensitivity of the method. The use of phosphate buffer and acetonitrile in the ratio of 65:35 v/v resulted in peak with good shape and resolution. The high percentage of recovery indicates that the proposed method is highly accurate. No interfering peaks were found in the chromatogram of the formulation within the run time indicating that excipients used in tablet formulations did not interfere with the estimation of the drug by the proposed HPLC method.

Fig.-1: Chemical structure of perindopril

Fig.-2: Typical chromatogram of perindopril Table-1: Calibration data of the method Mean peak area (n=5) 781547 1591955 2329492 3138212 3851667

Concentration (µg/ml) 20 40 60 80 100

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Vol.4, No.1 (2011), 113-116 Table-2: Precision of the proposed HPLC method Measured concentration of perindopril (µg/ml) Concentration of perindopril (µg/ml)

Intra-day

Inter-day

Mean (n=5)

% RSD

39.93

0.17

40

Mean (n=5)

% RSD

39.46

0.10

Table-3: Accuracy studies Concentration 50 % 100 % 150 %

Amount added (mg) 5.05 9.95 14.85

Amount found (mg) 4.97 10.0 14.69

% Recovery 98.4 % 100.5 % 98.9 %

% Mean recovery 99.3

Table-4: System suitability parameters

Parameter

Result 20-100

Linearity (µg/ml)

0.9997

Correlation coefficient

3103

Theoretical plates (N)

1.15

Tailing factor

0.03

LOD (µg/ml)

0.12

LOQ (µg/ml) Table-5: Assay and recovery studies Formulation Coversyl Perigard

Label claim (mg) 2 2

Amount found (mg) 2.003 1.992

% Amount found 100.15 99.60

CONCLUSION The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of perindopril and can be reliably adopted for routine quality control analysis of perindopril in its tablet dosage forms.

ACKNOWLEDGEMENTS The authors are thankful to M/s Glenmark Pharmaceuticals Ltd, Mumbai for providing a reference sample of perindopril.

REFERENCES 1. 2. 3. 4. 5.

www.rxlist.com E. Hisham, J. Pharm. Biomed. Anal., 17, 1267 (1998). Z. Simoncic, R. Roskar, A. Gartner, K. Kogej and V. Kmetec, Int. J. Pharm., 356, 200 (2008). M. Medenica, D. Ivanovic, M. Maskovic, B. Jancic and A. Malenovic, J. Pharm. Biomed. Anal., 44, 1087 (2007). D.S. Jain, G. Subbaiah, M. Sanyal, U.C. Pande and P. Shrivastav, J. Chromatogr. B, 837, 92 (2006). [RJC-706/2011]

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Vol.4, No.1 (2011), 117-119 ISSN: 0974-1496 CODEN: RJCABP

http://www.rasayanjournal.com

INFLUENCE OF THE SOLVENT CONDITION FOR THE SYNTHESIS OF THE β-DIKETIMINES LIGANDS 1

Tchirioua Ekou2,* and Lynda Ekou1 Université d’Abobo-Adjamé, Laboratoire de Thermodynamique et de Physico-Chimie du Milieu, 02 BP 801 Abidjan 02, Côte d’Ivoire 2 Université de Montréal, Département de Chimie, Québec, Canada H3C3J7 ∗ E-mail: ekou_tchirioua@yahoo.fr

ABSTRACT A general synthesis for preparation of β-diketimines has been developed. The method reported here demonstrates the use of solvent for conversion of ethylene glycol monoketal to β-diketimines. The reaction can be performed with CH2Cl2 without catalyst providing. O

O

OH OH

O OO

Benzylamine +

H2 O

Toluene, P -TSOH

N OO Solvent, catalyst

Keywords: Synthèses, β-diketimines ligands, Solvent, Catalyst © 2011 RASĀYAN. All rights reserved.

INTRODUCTION In recent years, a significant amount of attention have been concentrated on the design and synthesis of polydentate ligands1,2, attempting to generate novel coordination environments, stabilizing particular oxidation, states and preparing robust catalysts3,4. The chemistry of β-diketimines represents an active investigation area in organic5,6 or organometallic chemistry, due to their high catalytic activity and polymer synthesis7,8. These compounds have been used as ligand for the complex formation with a variety of transition metal9,10 and they have found immense analytical applications, for example in extracting traces of metals. These ligands are particularly useful as they can be prepared in high yields, crystallize easily and offer various coordination modes, thus have the ability to stabilize low oxidation state compounds11.

EXPERIMENTAL All experiments were carried out under a room temperature. All substrates were used without further purification. NMR spectra were recorded on a AMX 300 MHz spectrometer. 2,4-pentanedione-2,2-(ethylene glycol) Monoketal (2) This procedure was slightly modified from that given in the literature. A mixture of 2,4-pentandione (5g, 49.93mmol), ethylene glycol (3.09g, 49.93mmol), and p-toluenesulfonic acid (0.19mg, 0.99mmol) in toluene (15mL) was placed in a three necked round bottom flask equipped with magnetic stirrer and a dream-stark apparatus was used to remove the water continuously from the homogenous reaction mixture. The solution was refluxed in toluene for 2h, then evaporated in vacuo to afford monoketal 2 (yellow dark oil, 85%). 1H NMR, 13C NMR. Benzyl-[1-methyl-2-(2-methyl-[1,3]dioxolan-2-yl)-ethylidene]-amine (3) To a solution of monoketal (0.1g, 0.86mmol) in CH2Cl2 (5mL) was added benzylamine (0.093g, 0.86mmol) at room temperature. The reaction mixture was heated at reflux for 6h after, the solvent was evaporated at reduced pressure to afford 3 (viscous oil, 62%). 1H NMR (CDCl3) δ ppm 7.29 (m, 5H), 3.97

SYNTHESIS OF THE β-DIKETIMINES LIGANDS

Tchirioua Ekou and Lynda Ekou

Vol.4, No.1 (2011), 117-119

(m, 4H), 2.77 (s, 2H), 2.22 (S, 3H), 1.41 (s, 3H). 100.30, 64.42, 52.31, 31.45.

13

C NMR (CDCl3) δ ppm 206.00, 191.09, 107.63,

RESULTS AND DISCUSSION The desired β- diketimines ligand was prepared in two step standard condensation route (scheme1). Method reported here demonstrates the influence of solvent, time and catalyst in the yield of the reaction.

O

O

O

O O

i

N

ii

+

3

2

1

O O

H 2O

Scheme-1: General synthesis of the β-diketimine Reagents : (i) ethylene glycol, toluene, P-TSOH, (ii) benzylamine, solvent, or benzylamine, CH2Cl2, P-TSOH.

First the dean stock reaction of 2,4-pentanedione with substituted ethylene glycol in toluene using PTSOH as catalyst, turned out useful in the synthesis of monoketal ethylene glycol 2 in good yield. The water generated by the reaction was removed by azeotropic distillation promoting the formation product minimizing hydrolysis of the Schiff base. In general, monoketals were prepared through condensation of carbonyl compounds with catechol catalysed by protonic or Lewis acid catalysts such as phosphorus pentoxide, trimethylsilyl chloride12, phosphorus trichloride13, super acid14, p-toluenesulfonic acid15,16. However, some of the previously reported have various disadvantages. For example trimethylsilyl chloride and phosphorus trichloride are poisonous, expensive and unstable. Moreover, they could not be recycled due to the difficulty in purification. The use of p-toluenesulfonic acid as homogenous catalyst in the liquid phase regenerates the catalyst as solid heterogeneous systems when the reaction is carried out. This catalyst exhibits high activities for the reaction. The β-diketimine was prepared by the condensation of the ethylene glycol monoketal of 2,4-pentanedione with 1eq of benzylamine in the presence of solvent. The mixture was refluxed for the specific periods. The reaction process was monitored by NMR analysis. The influence of solvent, the amount of catalyst and reaction time on the yield of product were investigated in order to find out the optimum reaction condition as shown in Table 1. Table-1: Influence of solvent for the β-diketimine preparation Solvent X (h) 1 3 6 21

Toluene

THF

CH2Cl2

(% yield) 30 31 32 41

(% yield) 35 55 55 59

(% yield) 57 57 62 62

CH2Cl2 + toluenesulfonic 2% (% yield) 57 56 56 57

pacid

The experimental observation reported herein shows that a solvent plays an important role in the yield reaction. Whatever the reaction time of monoketal with benzylamine in toluene gave the desired product SYNTHESIS OF THE β-DIKETIMINES LIGANDS

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at low yield. According to THF solvent used, the yields of products obtained were improved with reaction time. The optimum yield reaction was obtained after 21h stirring. The yields are good from the β-diketimine when the CH2Cl2 are used as solvent, the reaction worked very well and the optimum yield was obtained after 6h of mixture. This condensation is preferable to the typical acid- catalysed condensation of monoketal in CH2Cl2. The latter method gave mixtures of product. In conclusion the optimum reaction condition was obtained in the presence of CH2Cl2 at 6 h stirring without catalysts.

CONCLUSION In summary, the reaction of monoketal with benzylamine and CH2Cl2 solvent represents a practical and cheap method for the preparation for the β- diketimines ligand. The reaction procedure is simple and efficient.

ACKNOWLEDGEMENTS The project is financially supported by Abobo-Ajamé University (Ivory cost) and Montréal University (Canada).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

G. Brancatelli, D. Drommi, G. Bruno. and F. Faraone, Inorg Chem Commun., 13, 215 (2010). X.Z. Li and Z.R. Qu, Inorg. Chem. Commun., 13, 220 (2010). K.D. Conroy, W.E. Piers and M. Parvez, J. Organomet.Chem., 693, 834 (2008). Q.Y. Li, G.W. Yang, X.Y. Tang, Y.S. Ma, F. Zhou, W. Liu, J. Chen and H. Zhou, Inorg. Chem. Commun., 13, 254 (2010). L.M. Tang, Y.Q. Duan, X.F. Li and Y.S. Li, J. Org. Chem., 691, 2023 (2006). A.Z. Bradley and D.L.T. Glover, J. Org. Chem., 73, 8673 (2008). V. Daniela, F.H. Vitanova and C.H. Kai, J. Org. Chem., 690, 5182 (2005). C. Fedorchuk, M.C. Sey and T. Chivers, Coord .Chem. Rev., 251, 897 (2007). R.B. Muterle, F. Fbri, R. Buffon, W. Oliveira and U. Schuchardt, Appl. Catal. A Gen., 317, 149 (2007). S. Özkar, D. ÜlkÜ, L.T. Yildirim, N. Biricik and B. Gümgüm, J. Mol. Struct., 688, 207 (2004). A.L. Leslie and F.R. Anne, J. Org. Chem., 691, 4250 (2006). Y. Nishida, H.M. Abe and A. Meguro, Tetrahedron. Asymm., 4 (7), 1431 (1993). R.R. Bikhulatov, T.V. Timofeeva and L.N. Zorina. Zh, Obshch. Khim., 66 (11), 1854 (1996). T.S. Jin, S.L. Zhang and X.F. Wang, J. Chem. Res. Synop., 7, 289 (2001). L.D. Corinne and Z. Mouloungui, Appl. Catal. A, 204 (2), 223 (2000). S. Ficht, L. Roglin, M. Ziche and D. Breyer, Syn. Lett., 14, 2525 (2004). [RJC-712/2011]

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SYNTHESIS OF THE β-DIKETIMINES LIGANDS

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Tchirioua Ekou and Lynda Ekou

Vol.4, No.1 (2011), 120-123 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SIMULTANEOUS DETERMINATION OF TUNGSTEN (VI) AND MOLYBDENUM (VI) FROM CATALYTIC REDUCTION OF IODATE G.Sailaja, R.Ramachandramurthy1 and V. Suryanarayana Rao2,* 1

2,*

Department of Chemistry, Govt. College, Anantpur-515 001(A.P.), India Department of Chemistry, S.K. University, Anantpur-515 001(A.P.), India *E-mail: r.r_murthy@yahoo.co.in

ABSTRACT A simple method for the simultaneous determination of Micro quantities of Tungsten and Molybdenum is developed based on the Catalytic Reduction of Iodate at the d.m.e. The Catalytic wave consists of a peak at -1.65 V vs SCE. Interference of various anions and cations has been investigated. A possible reaction mechanism has been proposed. Keywords: D.C.Polarography, Iodate, Acetic acid-Sodium acetate buffer, d.m.e, height of the mercury column, Catalytic wave, SCE, Tungsten(VI), Molybdenum(VI). Š 2011 RASÄ&#x20AC;YAN. All rights reserved.

INTRODUCTION Many Inorganic redox systems involving Transition Metal ions in higher valent state are known to Catalyse the Polarographic reduction of oxidants such as Hydrogen peroxide, Chlorate, Perchlorate, Bromate and Iodate. These Catalytic waves are exploited to develop sensitive methods for the determination of the Metal ions present in trace quantities1-5. The present paper deals with the simultaneous determination of trace quantities of Tungsen and Molybdenum exploiting the effect of one Metal ion on the other in the Polarographic Catalytic reduction of Iodate.

EXPERIMENTAL Reagents All chemicals used were of analytical-reagent grade. Potassium iodate solution (0.1M) prepared in conductivity water with appropriate dulution is used. A 0.2 M Sodium acetare and acetic acid buffer solutions of different pH have been employed in the investigations. Apparatus A CL-25 pen recording d.c. polarograph (ELICO, Hyderabad, India), an ELICO Model CL-10 pH meter and a Lingane-type H-cell were employed. The voltages were measured against a saturated calomel electrode. Pure Nitrogen gas is is passsed through the apparatus for about 15 minutes to remove dissolved oxygen Recommanded procedure 1-12.5 ml of Tungsten (VI) at 1 X 10-7 M concentration , 1ml of Molybdenum (VI) of 1 X 10-2 M, 2.5 mla of Iodate of 2 X 10-2 M concentration and a required volume of distilled water in a 25 ml standard flask so that the total volume is always constant. The solution is mad upto the mark with a 0.2 M acetate-acetic acid buffer of pH 5.0. The contents of the flask are transferred into the Polarographic cell after thorough shaking. Hydrogen gas is passed for about 15 minutes to remove dissolved oxygen and then the Polarogram is recorded.

RESULTS AND DISCUSSION Addition of Molybdenum(VI) to Tungsten(VI)-Iodate system caused greater enhancement in the Peak Current (Fig1). This effect has been expoited for the simultaneous determination of trace quantities of

DETERMINATION OF W(VI) AND Mo(VI)

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Vol.4, No.1 (2011), 120-123

bothe metal ions. The effect of the concentration of Tungsten(VI), Molybdenum(VI) and height of the mercury column are presented in the tables 1, 2 and 3 respectively. Non-dependance of the Peak current on mercury co lumn height indicates that the wave has kinetic nature. Abnormal currents and kinetic nature of wave suggest the catalytic nature of the wave. Tungsten (VI) is known to exist as hydrogen hexatungstate in weakly acidic solutions6 (between pH 4 and 6). Orlemann and Kolthoff7 reported that the reductio of Iodate in acid solutions (pH 1.0 to 5.5) produces Iodic acid. Tungsten present in the form of hydrogen hexatungstate is known to form polyacids with silicic, boric and phosphoric acids. The reduction of an intermediate species involving hydrogen hexatungstate and iodic acid anlogous to tungstosilicic acid and its regeneration by a fast chemical oxidation bby Iodate is presumed to be responsible for the catalytic wave in the case of Tungsten(VI). It is known that tungsten forms heteropolyacids8 of the type [MoW11O40H2]6- with Molybdenum(VI). It is know that this heteropolyacidundergoes reduction at the d.m.e9. It is suggested that the formation of Molybdotungstate is responsible for the enhancement in the catalytic current. This enhancement in the peak current is found to be linear with the concentration of Tungsten(VI) in the range 4 x 10-9 to 5 x 10-8 M as well as 1 x 10-6 to 1 x 10-5 M of Molybdenum(VI). Interference studies It was found that the common anion such as chloride, bromide,iodide, sulphae, carbonate, ferricyanide, thiocyanate do not interfere in the determination upto to fold excess of Tungsten(VI) concentration. Cu(II), Co(II), Cd (II),Cr(VI), Cr(III), Zn(II), Mn(II), U(VI), Zr(IV), EDTA, tartrate and citrate inerfere if their respective concentartion exceed 10 fold of Tungsten(VI).

ACKNOWLEDGEMENT The authors express their thanks to the Government College, Anantapur and Department of Chemistry, S.K University, Anantapur for providing necessary facilities.

REFERENCES V. Suryanarayana Rao and S. Brahmaji Rao, Z. Anal. Chem., 294(5), 414 (1979) V.S.N. Rao and S.B. Rao, Talanta, 26, 502 (1979) R. Ramachandra Murthy and V. Suryanarayana Rao, Analyst, 109 (8), 1111 (1984). R. Ramachandra Murthy and V. Suryanarayana Rao, Analytical Letters,18 (A12), 1479 (1985). R. Ramachandra Murthy and V. Suryanarayana Rao, Fresenius Z. Anal. Chem, 323, 495 (1986). H. Remy, Treatise on Inorganic Chemistry Vol.II,177 Elsevier, New York (1970) E.F. Orlemann and I.M. Kolthoff , J. Am.Chem.Soc., 64, 1044, 1970 (1942). S.G. Mairanovskii, Catalytic and Kinetic Waves in Polarography, Plenum Press, New York (1968) 9. Myriam Lamache et clarie deguen, J. Electroanl. Chem., 67, 81 (1976)

1. 2. 3. 4. 5. 6. 7. 8.

Table-1: Effect of the concentration of Tungsten(VI) on the peak current in presence of Molybdenum(VI) [Mo(VI)] = 4 x 10-4; [IO3-] = 20 x 10-4; pH = 5.0 Concentration of Tungsten (VI) M 4 x 10-9 1 x 10-8 2 x 10-8 3 x 10-8 4 x 10-8 5 x 10-8

DETERMINATION OF W(VI) AND Mo(VI)

Peak Current ÂľA 26.0 30.2 34.5 38.8 42.3 47.0

121

Peak Potential (-V vs SCE) 1.65 1.65 1.65 1.65 1.65 1.65

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Vol.4, No.1 (2011), 120-123

Table-2: Effect of concentration of Molybdenum (VI) on the peak current in presence of Tungsten (VI) [W(VI)] = 4 x 10-4, [IO3-] = 20 x 10-4 , pH= 5.0 Concentration of Molybdenum (VI) M 1 x 10-6 8 x 10-6 16 x 10-6 24 x 10-6 32 x 10-6 40 x 10-6

Peak Current µA 6.0 12.2 18.5 24.6 31.0 37.5

Peak Potential (-V vs SCE) 1.65 1.65 1.65 1.65 1.65 1.65

Table-3: Effect of the Merucy column height on the peak current in presence of Tungsten(VI) and Molybdenum(VI) [W(VI)] = 4 x 10-8; [IO3-] = 20 x 10-4; pH = 5.0 Height of Mercury Column (Cms) 80 70 60 50 40 30

Peak Current µA 42.0 42.2 42.5 42.0 42.5 42.0

Peak Potential (-V vs SCE) 1.65 1.65 1.65 1.65 1.65 1.65

Peak Current

Effect of concentation of Tungsten(VI) in presence of Molybdenum(VI) 50 45 40 35 30 25 20 15 10 5 0

Series1

0

2 4 6 Concentration of Tungsten(VI) x 10-8

Fig.-1

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Peak current

Effect of concentration of Molybdenum(VI) on the peak current 40 35 30 25 20 15 10 5 0

Y

0

20 40 60 Concentration of Molybdenum X10-6

Fig.-2

Fig.-3: Effect of Molybdenum (VI) on the Tungsten (VI) iodate system. Tungsten to Molybdenum ratio : (A) 1:1, (B) 1:10 and (C) 1:100 [W(VI)] = 4 x 10-6; [IO3-] = 20 x 10-4; pH = 5.0 [RJC-593/2010]

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Vol.4, No.1 (2011), 124-131 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

POLAROGRAPHIC STUDY OF MIXED LIGAND (CARBOXYMETHYLMERCAPTOSUCCINATE-ALANINATE OR ASPARTATE OR GLUTAMINATE OR VALINATE WITH CADMIUM (II) LEAD (II) AND THALLIUM (I) IN AQUEOUS ETHANOL MEDIA. Seema Agarwal and S. Kalpana Electrochemistry Research Laboratory , P.G. Department of Chemistry Govt. College, Kota-324002, Raj. India *E-mail: dr_seema_agarwal@yahoo.co ABSTRACT Mixed ligand systems (carboxymethylmercaptosuccinate – alaninate, carboxymethyl-mercaptosuccinate-aspartate, carboxymethylmercaptosuccinate-glutaminate, carboxymethylmer-captosuccinate-valinate, with Cd(II), Pb(II) and Tl(I) in aqueous-non aqueous (3:2 v/v aqueous ethanol) media at constant ionic strength (KNO3, µ = 1.0 M), pH (6.2 ± 0.02) and temperature (303 ± 2K) have been studied polarographically. Thymol (0.01%) was used as wave maxima suppressor. It was shown that only one mixed ligand entity (MAiXj) is formed in all cases of carboxymethylmercaptosuccinate alaninate/ aspartate/ glutaminate/ valinate systems where 'i' and j are one for all Cd(II) Pb(II), and Tl(I) metal complexes formed. A and X are carboxymethylmercaptosuccinate and alaninate/ aspartate/ glutaminate/ valinate/ respectively. Compositions and stability constants of single mixed ligand species formed have been evaluated employing Souchay and Faucherre's method. Key words: Polarography, mixed complex, stability constant. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION The successful application of the chelating properties of carboxymethylmercaptosuccinic acid referred to herein as R-S-R' where R=CH2 COOH and R'= CH-COOH has been demonstrated by Evan and his 1

group. The sulphur in the R-S-R' and the two adjacent carboxyl groups are ideally arranged with the 2-4

metal ion.

5,6

The use of the polarographic technique for the study of analysis. 18-20

7-17

complexation.

and

18-20

behavioral. of study of organic compound is well known. In view of its importance in diverse disciplines and interesting results obtained by earlier workers, it was considered worthwhile to investigate polarographic study of mixed ligand (carboxymethylmercaptosuccinate- alaninate or aspartate or glutaminate or valinate with cadmium (II) lead (II) and thallium (I) in aqueous ethanol media for which no reference could be traced out in the literature.

EXPERIMENTAL The sodium salts of carboxymethylmercaptosuccinate (95%, Evan's Chemetics, Inc. New York), L-alanine, Laspartic acid, L-glutamine and L-valine (E, Merck India Ltd.) were used as complexing agents. All other reagents used were also of AnalaR grade. Freshly prepared solution of carboxymethylmercaptosuccinate in 50% ethanol was used and other stock solutions were prepared in doubly distilled air free conductivity water. Thymol (0.01%) was used as wave maximum suppressor and potassium nitrate solution (µ = 1.0M) as the supporting electrolyte. An automatic recording polarograph Systronics (India) model 1632, with a saturated calomel electrode as a reference electrode and platinum electrode as auxillary electrode was used for determining current voltage curves. The capillary characteristics in potassium nitrate solution (µ = 1.0 M) at Ed.e = -.07V with respect to a saturated calomel

POLAROGRAPHIC STUDY OF MIXED LIGAND

Seema Agarwal and S. Kalpana

Vol.4, No.1 (2011), 124-131 electrode (SCE) were calculated m2/3 t1/6 = 2.4913 mg2/3 s-½ (h = 45cm). All measurements were made with the cell immersed in a thermostatic bath. Dissolved air was removed by bubbling purified nitrogen through the cell 21

and necessary corrections for the potential drop and charging current were made as usual.

RESULTS AND DISCUSSION The formation of each mixed ligand complex was studied at 303 ± 2K and f6.8 ± 0.02 pH by recording polarograms of Cd (NO3)2 or Pb (NO3)2 or TlNO3 at constant ionic strength KNO3 (µ=1.0 M) for two different sets of different ligand composition in 3:2 v/v aqueous-ethanol and 0.01 % thymol. Metal ligand compositions of two different sets were. SET-1 1.0 mM Cd (NO3)2 or Pb (NO3)2 and a constant alaninate or aspartate or glutaminate or valinate -2

concentration (CX) of 2.0 x 10 M with a carboxymethylmercaptosuccinate concentration (CA) varying -2

-2

between 0.6 x 10 to 4.0 x 10 M. 0.5 mM Tl NO3 and a constant alaninate or aspartate or glutaminate -2

or valinate concentration (Cx) of 2.0 x 10 M with a carboxymethylmercaptosuccinate concentration -2

-2

(CA) varying between 0.6x10 and 4.0 x 10 M. SET-2 1.0 mM Cd (NO3)2 or Pb (NO3)2 and a constant carboxymethyl-mercaptosuccinate concentration (CA) -2

of 2.0 x 10 M with alaninate, aspartate, glutaminate, valinate concentration (Cx) varying between 0.6 x -2

-2

10 M to 4.0 x 10 M. 0.5 mM Tl (NO3)2 and a constant carboxymethylmercaptosuccinate concentration -2

(CA) of 2.0 x 10 M with alaninate, glutaminate, aspartate, valinate concentration (CX) varying between -2

-2

0.6 x 10 M to 4.0 x 10 M. The plots of log i/(id-i) vs Ed.e for all polarograms yielded straight lines with slopes that agreed with the theoretical value corresponding to n=2 for Cd(II) and Pb(II) and n=1 for Tl(I) systems respectively. The values of the slopes for different systems are given in the Table B-IV showing the reversibility of the reduction. Rectilinear plots of id vs h½eff showing constancy of id/√ heff The E½ values evaluated from the log plots of each of the above mentioned current voltage curves and corresponding diffusion current values have been recorded (Table B-1, 2, 3). All the measurements were carried out in well buffered solutions of pH 6.8, which remains almost stable (6.8 ± 0.02) within all concentration ranges of complexing agents used. All the buffer solutions used, were prepared by Clark and Lubs method22. All the E½ values of the metal ion in presence of mixed ligands or single ligand are more negative than that of the free metal ion. Since the ion must be first librated from the complex, this requires certain amount of energy. From the shift in the half wave potential of the complexed metal ion and the concentration of the complex forming agents, both the stability constant and its composition can be calculated. Souchay and Faucherre23 derived an equation where a metal ion complexes with two ligand species simultaneously in solution. If the complexing reaction of the following type is considered: M + iA + j X → MA X i

(1)

j

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Vol.4, No.1 (2011), 124-131

With the restriction that a single mixed-ligand entity MA X is formed, then the shift in the E½ of the i

j

polarographic ware of the metal ion as a function of the concentration of added reagents A and X is given by-

The ratio Dfree/Dcomp may be obtained from the values of the limiting current. From plots of ∆ E½ vs – log CA with Cx kept constant and ∆ E½ vs – log Cx with CA kept constant, values of i and j can be obtained by intersect method because on differentiation. For each of the mixed ligand systems carboxymethylmercaptosuccinatealaninate/aspartate/glutaminate/valinate, plots of ∆E½ vs log CA (with Cx kept constant) and ∆E½ vs log Cx (with CA kept constant) yielded straight [Fig. 1,2] and thus establish the formation of a single mixed ligand entity. The values of coordination numbers 'i' and 'j' of A and X were determined from the graph shown-

∂(∆E½) ∂(logCA) ∂(∆E½) ∂(logCx)

=

i

2.303RT nF

(3)

j

2.303RT nF

(4)

Cx = CA

lines

in (Fig. 1,2) for each system and are given in (Table-4), where A and X are the carboxymethylmercaptosuccinate and alaninate/ aspartate/ glutaminate/ valinate respectively. Integral values of 'i' and 'j' are used in the calculation of stability constants using equation (2) as described in method and are consolidated in Table 4.

CONCLUSION The present investigations suggest the formation of only one kind of mixed ligand species (MA X ) for i

-3

j

each mixed ligand system. The type of mixed ligand species formed are [MAX] with Cd(II) and Pb(II), -4

and [MAX] with Tl(I) metal in carboxymethylmercaptosuccinate - valinate and carboxymethylmercap-4

-5

tosuccinate -alaninate mixed ligand systems [MAX] with Cd(II) and Pb(II) [MAX] with Tl(I) metal in carboxymethylmercaptosuccinate-glutaminate and carboxymethylmercaptosuccinate-aspartate mixed ligand systems.

ACKNOWLEDGEMENT The authors wish to express their sincere thanks to Dr. K. K. Gupta, Head of the Chemistry Department and to Prof. M. L. Meena, Principal, for providing research facilities. POLAROGRAPHIC STUDY OF MIXED LIGAND

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Vol.4, No.1 (2011), 124-131

REFERENCES 1. C.D. Evans, A.W. Schwab and P.M. Conney, J. Am. Oil Chem. Soc., 31, 912, (1954). 2. A.W. Schwab et al., J. Am. Oil Chem. Soc., 30, 177, (1953). 3. A.E. Martell and M. Calvin, Chemistry of the Metal Chelate Compounds, Chapter 4, prentice Hall, New York, (1953). 4. A.W. Schwab et al., J. Amer. Oil Chem. Soc., 30, 10, 413, (1953). 5. S. Sharma, P. Dhingra and R.S. Pandey, J. Indian Chem. Soc., 85, 962 (2008). 6. D.K. Sharma, C. Chauhan, A.Gupta and N Sharma, J. Indian Chem. Soc., 84, 1145 (2007). 7. J.M Diazcurz, C.Arino, M. Esteban and E. Casassas, Electroanal. 5, 677, (1993). 8. R. Jain and Sadhna Srivastav, J. Ind. Chem. Soc., 80, 30 (2003). 9. C. Karadia and O.D. Gupta, Int. J. Chem. Sci., 7, 1531 (2009). 10. F.Khan and Firoz Khan, J. Ind. Chem. Soc., 83, 141 (2006). 11. F.Khan and Kavita Rai, J. Indian Chem. Soc., 87, 971 (2010). 12. H.M. Killa, E.M. Mabrouk, M.M. Moustafa and R.M. Issa, Cratica Chemica Acta, 64,585, (1991). 13. K. Saini, H.P. Gupta and R.S. Pandey, Proceedings on Botanical Products, Seminar and Expo, 227, (2005). 14. L. Tantuvay and F. Khan, Bull. of Electro-Chemistry, 20 ,327 (2004). 15. S. Sharma, A. Garg and R. S. Pandey, J. Indian Chem. Soc., 87, 1205 (2010). 16. S. Kumar and O.D. Gupta, J. of Ultra Chemistry, 5, 289 (2009). 17. S. Agarwal and Kalpana S., J. of Ultra Chemistry, 5 ,349 (2009). 18. M. Singh and F Khan, J. Indian Chem. Soc., 85, 765 (2008). 19. Y.Kumar, Shuda A.Garg and R. Pandey, J. Indian Chem. Soc., 87, 1231 (2010). 20. B. S. Bairwa, M. Gupta Varhney, I. K. Sharma and P. S. Verma, J. Indian Chem. Soc. 87, 609 (2010). 21. J. Heyrovsky, Principles of Polarography, Czechoslovak Academy of Sciences, Prague, 61-63, (1965). 22. W. M. Clark and Lubs H. A., J. Bact. 2, 109, 191, (1917). 23. P. Souchay and J. Faucherre, Bull. Soc. Chem., France, 529, (1947) Table-1:Mixed ligand system with Cadmium (II) [All replicate measurements were made] at (303±2K).E½ (Cd+2 metal ion) = 0.591 volts, id (Cd+2 metal ion) = 4.30 µ A Concentration of

C.M.M.S.

C.M.M.S.

C.M.M.S.

C.M.M.S.

mixed ligands x 10-

Valinate System

Alaninate System

Glutaminate System

Aspartate System

2

CA

M

Log Im/Ic CX

E½(V)

Log Im/Ic

E½(V)

Log Im/Ic

Log Im/Ic

E½(V)

E½(V)

0.6

2.0

1.043

0.050

1.037

0.045

1.075

0.037

1.094

0.029

1.0

2.0

1.061

0.056

1.085

0.051

1.085

0.043

1.102

0.035

2.0

2.0

1.096

0.065

1.096

0.065

1.105

0.052

1.146

0.044

3.0

2.0

1.108

0.070

1.227

0.062

1.113

0.057

1.152

0.048

4.0

2.0

1.128

0.074

1.396

0.065

1.149

0.060

1.165

0.052

2.0

0.6

1.038

0.044

1.072

0.040

1.036

0.033

1.038

0.024

2.0

1.0

1.070

0.052

1.081

0.047

1.038

0.040

1.075

0.032

2.0

3.0

1.105

0.072

1.105

0.067

1.075

0.060

1.128

0.051

2.0

4.0

1.106

0.077

1.128

0.072

1.128

0.063

1.214

0.055

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Vol.4, No.1 (2011), 124-131

Fig.-1: Plots of ∆ E½ as a function of -log C for CMMS + Valinate (A-curve 1-6) and CMMS + alaninate (B- curve 1-6) mixed ligand system.

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Vol.4, No.1 (2011), 124-131

Fig.-2: Plots of ∆ E½ as a function of -log C for CMMS + glutaminate (A-curve 1-6) and CMMS + aspartate (Bcurve 1-6) mixed ligand system.

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Vol.4, No.1 (2011), 124-131 Table-2:Mixed ligand system with Lead Pb (II) [All replicate measurements were made] at (303±2K).E½ (Pb+2 metal ion) = 0.409 volts,id (Pb+2 metal ion) = 3.60 µ A

Concentration of mixed ligands x 102 M CA CX 0.6 2.0 1.0 2.0 2.0 2.0 3.0 2.0 4.0 2.0 2.0 0.6 2.0 1.0 2.0 3.0 2.0 4.0

C.M.M.S. Valinate System Log Im/Ic ∆ E½(V) 0.018 0.078 0.022 0.085 0.036 0.095 0.067 0.100 0.075 0.103 0.057 0.074 0.062 0.083 0.084 0.102 0.101 0.106

C.M.M.S. Alaninate System Log Im/Ic ∆ E½(V) 0.022 0.072 0.036 0.079 0.046 0.089 0.062 0.095 0.067 0.097 0.046 0.068 0.051 0.077 0.090 0.097 0.125 0.100

C.M.M.S. Glutaminate System Log Im/Ic ∆ E½(V) 0.031 0.065 0.051 0.071 0.062 0.081 0.073 0.085 0.084 0.091 0.058 0.060 0.066 0.067 0.101 0.088 0.148 0.092

C.M.M.S. Aspartate System Log Im/Ic ∆ E½(V) 0.036 0.056 0.057 0.063 0.078 0.072 0.090 0.077 0.096 0.081 0.062 0.051 0.067 0.059 0.120 0.077 0.207 0.080

Table-3:Mixed ligand system with Thallium TI (I) [All replicate measurements were made] at (303±2K). E½ (TI+2 metal ion) = 0.457 volts, id (TI+2 metal ion) = 3.14 µ A

Concentration of mixed ligands x 102 M CA CX 0.6 2.0 1.0 2.0 2.0 2.0 3.0 2.0 4.0 2.0 2.0 0.6 2.0 1.0 2.0 3.0 2.0 4.0

C.M.M.S. Valinate System Log Im/Ic ∆ E½(V) 0.980 0.064 1.075 0.071 1.102 0.080 1.117 0.086 1.150 0.090 1.009 0.060 1.029 0.068 1.060 0.088 1.117 0.093

C.M.M.S. Alaninate System Log Im/Ic ∆ E½(V) 1.019 0.061 1.045 0.068 1.073 0.077 1.090 0.082 1.105 0.086 1.009 0.058 1.032 0.066 0.117 0.084 0.133 0.089

C.M.M.S. Glutaminate System Log Im/Ic ∆ E½(V) 1.095 0.056 1.132 0.063 1.182 0.072 1.193 0.078 1.219 0.081 1.060 0.052 1.165 0.061 1.167 0.082 1.203 0.084

C.M.M.S. Aspartate System Log Im/Ic ∆ E½(V) 1.045 0.041 1.060 0.054 1.090 0.064 1.133 0.070 1.150 0.074 1.019 0.044 1.073 0.052 1.117 0.073 1.150 0.078

Table-4 Carboxymethyl Carboxymethyl Carboxymethyl Carboxymethyl Mercapto Succinate Valinate Mercapto Succinate Alaninate Mercapto Succinate Glutaminate Mercapto Succinate Asparatate System with; System with; System with; System with; _____________________________________________________________________________________________ S.No. Cd(II) Pb(II) TI(I) Cd(II) Pb(II) TI(I) Cd(II) Pb(II) TI(I) Cd(II) Pb(II) TI(I) _____________________________________________________________________________________________ 1. Co-ordination 1.006 1.02 1.109 1.031 1.08 1.06 0.981 1.11 1.08 0.978 1.10 1.19 number “i” of ligand A 2. Co-ordination number “j” of ligand X

1.39

1.31

1.396

POLAROGRAPHIC STUDY OF MIXED LIGAND

1.355

1.40

130

1.31

1.27

1.42

1.38

1.35

1.37

1.43

Seema Agarwal and S. Kalpana

Vol.4, No.1 (2011), 124-131 3. Mean log KMA X

9.091 10.058 9.610

8.917

9.920 9.500

8.655

9.655 9.363

8.390 9.367 9.078

4. Standard deviation

± 0.492

± 0.520

± 0.470

± 0.504

± 0.468

± 0.477

± 0.492

± 0.476

± 0.473

± 0.501 ± 0.4770 ± 0.459

5. Slopes of log plots

0.031 0.032

0.062

0.031

0.032 0.061

0.032

0.031 0.061

0.031 0.031 0.062

i j

[RJC-695/2010]

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Vol.4, No.1 (2011), 132-135 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

ROLE OF ALKALOIDAL PRECIPITANTS FOR THE ASSAY OF IMIPRAMINE HYDROCHLORIDE IN BULK AND PHARMACEUTICAL FORMULATIONS

1, 3

G.Nagarjuna Reddy1,∗∗, C.Ramesh2,T.V.Narayana3, K.V.S.Prasada Rao4 and B.Ganaga Rao5

Vikas Institute of Pharmaceutical Sciences, Rajahmundry, E.G. Dist. (A.P.) India. 2 V.V.Pura Institute of Pharmaceutical Sciences, Bangalore, India. 4 Rahul Institute of Pharmaceutical Sciences & Research, Chirala(A.P.) India. 5 College of Pharmaceutical Sciences, Andhra University, Visakhapatnam(A.P.) India. *E-mail: Chem_nag@yahoo.co.in ABSTRACT Simple spectrophotometric methods (A-C) for the assay of Imipramine hydrochloride (IMP) based on the formation of its complexes with alkaloidal precipitants are described. IMP undergo quantitative precipitation in the form of molecular complexes with iodine (I2, method A), ammonium molybdate (AM, method B) or phosphomolybdic acid (PMA, method C) when used in excess. In addition to precipitation reactions, color reactions have also been combined to estimate IMP. They are based on the color formation with either un-reacted precipitant of the filtrate (in I2) or released precipitant from the molecular complex (in AM or PMA) with chromogenic reagent such as P-N-methyl amino phenol sulphate (PMAP)-sulphanilic acid (SAc) (for I2), potassium thiocyanate (PTC) (for AM), cobalt nitrate (Co(II))-disodium salt of ethylene diamine tetra acetic acid (EDTA) complex (for PMA). Keywords: Imipramine hydrochloride, Spectrophotometer, alkaloids, molecular complexes, precipitants, pharmaceutical formulations © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Alkaloids are detected with the aid of group of reactions due to their chemical properties, structure and presence of functional groups. These reactions are based on the ability of the alkaloid to yield insoluble complexes mainly with AM, I2 and PMA and hence these reagents are named as alkaloidal precipitants1. The precipitate is ascribed due to the formation of a molecular complex resulting from the interaction of the unshared electron on nitrogen in amine with an unoccupied molecular orbital of the alkaloidal precipitant molecule. Imipramine hydrochloride (IMP) is an antidepressant agent and chemically it is 5-3(Dimethylamino) propyl-10, 11-dihydro-5H-dibenzazepine hydrochloride. Literature survey reveals that Spectrophotometric1-10 HPLC11-32 and LC-MS 33, 34 methods were reported for the determination of IMP in its formulation and biological fluids. The aim of the present work is to provide simple and sensitive visible spectrophotometric method for the estimation of IMP in bulk and formulations. The effects in this accord resulted to develop the present methods. IMP furnishes precipitates with alkaloidal precipitants given above, since it contains the nitrogen containing groups (tertiary amino groups). In addition to precipitation reactions color reactions have also been combined to estimate IMP. They are based on the chemical reaction with either released alkaloidal precipitant from the precipitate with acetone ( AM) or un-reacted precipitant in the filtrate (I2) with chromogenic reagents such as potassium thiocyanate35 (for AM) PMAP-SAc36 (for I2) or EDTA- Co (II) 37 (for PMA). The results are statistically validated.

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Vol.4, No.1 (2011), 132-135

EXPERIMENTAL Instruments Spectral and absorbance measurements were made on Systronics UV- Visible Spectrophotometer 117 with 10mm matched quartz cells. Reagents All the chemicals & reagents used were analytical grade and the solutions were freshly prepared. Aqueous solution of I2 (0.089%) in 0.83% of potassium iodide (KI), PMAP (2%), SAc (0.4%), hydrochloric acid (HCl) (1M) for method A; AM (2%), PTC (10%), conc.HCl (used as it is) for method B; PMA (4%) Co (II) (3%), EDTA (4%) for method C, 0.01 M HCl for methods B and C were prepared in triple distilled water. A one mg/ml solution of IMP was prepared by dissolving 100 mg of pure IMP in 100 ml of distilled water and this stock solution was diluted stepwise with distilled water to obtain the working standard solution of concentrations of 200 µg / ml for method A and C, 400 µg / ml for method B respectively. Method A Aliquots of working standard solution (0.5-1.5 ml, 200 µg / ml) were delivered into a series of centrifuge tubes and the volume in each tube was adjusted to 3.0 ml with distilled water. Then 2.0 ml each of 1M HCl and I2 were added successively and centrifuged for 5 min. The precipitate was collected by filtration and subsequently washed with 2 ml of distilled water. The filtrate and washings were collected in 25 ml graduated test tubes, then 3.0 ml of PMAP solution and 2.0 ml SAc solution were added successively and the volume was made up to the mark with distilled water. The absorbance was measured during next 30 min. at 520 nm against distilled water. A blank experiment was also carried out omitting the drug. The decrease in absorbance and intern drug concentration was obtained by subtracting the absorbance of the test solution from blank. The amount of drug was calculated from calibration graph. Method B Aliquots of working standard solution (0.5-1.5 ml, 400 µg / ml) were delivered into a series of centrifuge tubes and the volume in each tube was adjusted to 3.0 ml with 0.01M HCl. Then 1.0 ml of AM was added and centrifuged for 5 min. The precipitate was collected by filtration followed by washing with 50% alcohol until it is free from the reagent. The precipitate in each tube was dissolved in 5.0 ml of acetone and transferred into 25.0 ml graduated tube. The 5 ml of conc. HCl and 3 ml PTC solution were successively added and kept aside for 30 min and then volume in each tube was made up to the mark with distilled water. The absorbance was measured at 480 nm against a same blank reagent. The amount of drug IMP was calculated form the calibration graph. Method C Aliquots of working standard solution (0.5-1.5 ml, 200 µg / ml) were delivered into a series of centrifuge tubes and volume in each tube was adjusted to 3.0 ml with 0.01M HCl. The 2.0 ml PMA was added and centrifuged for 5 min. the precipitate was collected by filtration followed by washing with distilled water until it is free from the reagent. The precipitate in each tube was dissolved in 5 ml of acetone and transferred into 25 ml graduated tubes. One ml each of Co (II) and EDTA solution was successively added and the tubes were heated for 15 min. at 60oC in water bath. The tubes were cooled and the solution in each tube was made up to the mark with distilled water. The absorbance was measured at 840 nm against a same blank reagent. The amount of drug was calculated from its calibration graph.

RESUTLS AND DISCUSSION The optimum conditions for the color development of methods (A, B and C) were established by varying the parameters one at a time keeping the others fixed and observing the effect produced on the absorbance of the colored species. The optical characteristics such as Beer’s law limits, molar absorptivity and Sandell’s sensitivity for each method (A-C) are given in table1. The precision of each method to the drug was found by measuring the absorbance of six separate samples containing known amounts of the drug and the results obtained are incorporated in table1. Regression analysis using the method of least squares

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Vol.4, No.1 (2011), 132-135

was made to evaluate the slope (b), intercept (a), correlation coefficient (r) and standard error of estimation (Se) for each system and is presented in table-1. The accuracy of the methods was ascertained by comparing the results by proposed and reference methods (UV) statistically by t- and F-tests (Table 2). The comparison shows that there is no significant difference between the results of studied methods and those of reference ones. The similarity of the results is obvious evidence that during the application of these methods, the excipients that are usually present in pharmaceutical formulations do not interfere in the assay of proposed methods. As an additional check of accuracy of the proposed methods recovery experiments were carried out. The recoveries of the added amounts of standard drug were studied at 3 different levels. Each level was repeated for 6 times and from the amount of drug found, the % recovery was calculated in the usual way.

CONSLUCIONS The higher λmax values of all the proposed methods have a decisive advantage since the interference from the associated ingredients should be generally less at higher wavelengths than at lower wavelengths. Thus the proposed visible spectrophotometric methods are simple and sensitive with reasonable precision, accuracy and constitute better alternatives to the existing ones to the routine determination of IMP in bulk form and pharmaceutical formulations. Table-1:Optical Characteristics, Precision and Accuracy of the Proposed Methods (A, B&C) for IMP Parameters λmax (nm) Beer’s Law limits (µg/ml) Molar absorptivity (l mole-1cm-1) Sandell’s sensitivity (µg/cm2/0.001 absorbance unit)

Regression Equation y = a + bc* Slope (b), Intercept (a)

Method A

Method B

Method C

520 2-18 µg/ml 1.692x104 0.019 0.0541

480 5-35 µg/ml 6.052 x103 0.052 0.0191

840 2-20 µg/ml 1.451 x104 0.022 0.0458

Correlation coefficient (r) 0.9998 0.9998 Relative Standard Deviation (%) ** 0.2651 0.340 % Range of error ** 0.2220 0.340 (0.05 level confidence limit) *Y = a + bc, where c is the concentration in µg/ml. **From six determinations.

0.9998 0.364 0.305

Table-2:Determination of IMP in Pharmaceutical Formulations Sample ∆ (Tablets) T1

Labeled method(mg) 50

UV* Method 49.98± 0.022

T2

50

49.97± 0.006

T3

50

49.98± 0.009

ROLE OF ALKALOIDAL PRECIPITANTS

Amount obtained (mg) Proposed method A B C 49.97± 50.01± 49.97± 0.010 0.026 0.010 F=2.57 F=1.43 F=2.67 t=0.22 t=0.87 t=0.22 50.01± 49.99± 50.00± 0.020 0.024 0.031 F=1.36 F=1.25 F=2.06 t=0.78 t=1.08 t=1.16 49.98± 50.00± 49.98± 0.027 0.032 0.033 F=1.54 F=2.07 F=2.09 t=0.46 t=1.15 t=1.14 134

A 99.97

Recovery(%) B C 100.22 99.97

100.1

99.97

100.13

99.88

99.87

100.13

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Vol.4, No.1 (2011), 132-135 T4

50

49.98± 0.034

50.00± 49.98± 50.01± 0.006 0.027 0.025 F=1.18 F=1.46 F=1.42 t=0.99 t=0.38 t=0.88 ∆ Four different batches of tablets from a Pharmaceutical company.

99.99

99.89

100.22

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

B.Starczewska, P.Halaburda, A.Kojlo, J. Pharm. and Biomed. Anal., 30, 553 (2002). M.Kurzawa, B.Dembinski, A.Szydlowska-Czerniak, Acta Poloniae Pharmaceutical Drug Research , 56, 255(1999). J.M.Garcia-Fraga, A.I.Jimenez-Abizanda, F.Jimenez-Moreno, J.J.Arias-Leon; J.Pharm. and Biomed. Anal. , 9, 109 (1991). S.Gangwal, P.Trivedi, Eastern Pharmacist , 42, 141 (1999) A.C.Minguez, M.M.Velazquez, L.J.Rodriquez; Farmaco Ed. Prat., 42, 165(1987). A.Goldnik, M.Gajewska, E.Dolegowska, B.Pacula, Acta Pol. Pharm., 48, 3 (1991). J.F.Magalhaes, J.L.S.Martins, E.R.M.Hackmann, M.I.R.Santoro, Anais da Farmaciae Quimica de Sao Paulo, 22, 19 (1982). S.L.Bhongade, P.A.Thakurdesai, A.V.Kasture, Indian Drugs, 31, 219 (1994). F.A.El-Yazbi, M.A.Korany, M.Bedair, J. Clin. Hosp. Pharm., 10, 373 (1985) B.Dembinski, Acta Pol. Pharm., 34, 509 (1977). C.Grivel, J.Rocca, D.Guillarme, J.Veuthey, S.Heinisch, J. of Chromatography A, 1217, 459 (2010). E.Choong, S.Rudaz, A.Kottelat, D.Guillarme, J.Veuthey, C.B.Eap, J. of Pharmaceutical and Biomedical Analysis, 50, 1000 (2009). P.Thongnopnua, K.Karnjanaves, Asian Biomedicine, 2, 305(2008). R.Wietecha-Posłuszny, M.Woźniakiewicz, A.Garbacik, P.Kościelniak, Z. Zagadnien Nauk Sadowych, 70, 187(2007). V.F.Samanidou, M.K.Nika, I.N.Papadoyannis, J. of Separation Science, 30, 2391 (2007). G. Zhang, A.V.Terry Jr., M.G.Bartlett, J.of Chromatography B, 856, 20 (2007). H.F.Proelss, H.J.Lohmann, D.G.Miles, Clinical Chemistry, 24, 948 (1978). T.A.Ivandini, B.V.Sarada, C.Terashima, T.N.Rao, D.ATryk, H.Ishiguro, Y.Kubota, A.Fujishima, J. of Electroanalytical Chemistry, 521, 117 (2002). P.A.Reece, R.Zacest, C.G.Barrow, J. of Chromatography, 163, 310 (1979). R.F.Suckow, T.B.Cooper, J. of Pharmaceutical Sciences, 70, 257 (1981). D.H.Mielke, R.P.Koepke, J.H.Phillips, Curr. Ther. Res. Clin. Exp., 25, 738 (1979). S.H.Hansen, J.H.Madsen, Arch. Pharm. Chemi Sci. Ed., 5, 157 (1977). A.Goldnik, M.Gajewska, B.Ostaszewska, Acta Pol. Pharm., 48, 5 (1991). J.P.Foglia, D.Sorisio, J.M.Perel, J. of Chromatography: Biomedical Applications, 572, 247 (1991). S.Sugita, A. Kobayashi, S.Suzuki, T.Yoshida, K.Nakazawa, J. of Chromatography, 421, 412 (1987). A.Kobayashi, S.Sugita, K. Nakazawa, J. of Chromatography, 336, 410 (1984). K.Thoma, K.Albert, Archiv der Pharmazie, 317, 133 (1984). F.Plavsic, Acta Pharm. Jugosl., 32, 67 (1982). R.H.Costa Queiroz, V.L.Lanchote, P.S.Bonato, D.De Carvalho, Pharmaceutica Acta Helvetiae, 70, 181 (1995). S.Hartter, B.Hermes, A.Szegedi, C.Hiemke, Therapeutic Drug Monitoring, 16, 400 (1994). X.H.Yan, H.D.Li, Y.C.Zhang, H.W.Wu, Chinese Journal of Pharmaceutical Analysis, 14, 3 (1994). K.Kramer Nielsen, K.Brosen, J. of Chromatography: Biomedical Applications, 123, 87 (1993). W.Song, D.Pabbisetty, E.A.Groeber, R.C.Steenwyk, D.M.Fast, J. of Pharmaceutical and biomedical analysis, 50, 491 (2009). A.R.Breaud, R.Harlan, M.Kozak, W.Clarke, Clinical Biochemistry, 42, 1300 (2009). Irina Gerasimenko, Yuri Sheludko, Matthiam Unger and Joachim,Stockigt, Phytochem.Anal, 12 ,96 (2000). Sachin Mittal, Kenneths Alexander and David Dollimore, Drug Dev.Ind.Pharm,26, 1059 (2000). W.Golkiewicz, J.Kuczynski, W.Markowski and L.Jusiak, J.Chromatogr., 686, 85 (1994). [RJC-659/2010]

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Vol.4, No.1 (2011), 136-141 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS, CHARACTERISATION AND BIOLOGICAL EVALUATION OF BIDENTATE LIGANDS (REDUCED SCHIFF’S BASE) WITH METALS OF COPPER, NICKEL AND ZINC COMPLEXES S.Pattanaik*,1, S.S.Rout1, J.Panda2, P.K.Sahu1 and M Banerjee1 1

School of Pharmaceutical Sciences, Siksha ‘O’Anusandhan University, Bhubaneswar,Orissa-751003, India. 2 Hi-Tech College of Pharmacy, Rasulgarh,Bhubaneswar,Orissa-751001, India. *E-mail:sovan.sps@gmail.com ABSTRACT Schiff base ligand (L) was synthesized using p-Chlorobenzaldehyde with p-Chloroaniline followed by reduction. The complexes of copper (II), Nickel (II) and Zinc (II) with the bidentate ligand were synthesized having metal: ligand stoichiochemistry 1:2. The ligand and respective complexes were charactersied for their analytical parameters and various spectral features. The structures of these complexes were proposed on the basis of elemental analysis, electronic spectra, molar conductivity, IR spectra, 1HNMR spectra. Electronic spectra suggest that the band was shifted to a shorter wavelength which may be attributed to donation of the lone pairs of the nitrogen atoms. The molar conductance of the complexes indicates that the ligand is coordinated as uninegatively charged ions. The IR spectral data suggest that the coordination of phenolic oxygen to metal ion. The 1HNMR spectral data suggest that the chemical shift observed for the OH protons in the ligand (10.63ppm) was not observed in any of the complexes. This confirms the bonding of oxygen to metal ions (C-O-M). The antibacterial and analgesic activity of the complex and non-complex compounds suggest that metal containing compounds showed good inhibitory activity than noncomplex compounds. Keywords: Cu (II), Ni (II), Zn (II), Schiff base, p-chlorobenzaldehyde and p-chloroaniline © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Inorganic elements play crucial role in biological and biological medical processes, and it is evident that many organic compounds used in medicine do not have a purely organic mode of action, some are activated or biotransformed by metal ions metabolism1. Many drugs possess modified toxicological and pharmacological properties in the form of metal complex and probably schiff bases are versatile C=N (Imine) containing compounds possessing broad spectrum of biological activity2-6 and incorporation of metals in form of complexes showed some degree of antibacterial7, antifungal8, antitumor9 and antiinflammatory activity10,11. In Schiff base azomethane nitrogen and other donor atoms like oxygen play a vital role in co-ordination chemistry12. Hence an attempt is made to study the interaction of reduced Schiff base with transition of metals of biological interest and to investigate the co-ordination chemistry of such interactions. In the present work we described the synthesis and characterization of reduced Schiff base and its metal complexes. Moreover antibacterial and analgesic activity of reduced Schiff base metal complexes is also evaluated and compared with the standards. Keeping in view, the concept of co-ordination of suitable metals as central atoms, in the present work, Schiff base p-chlorobenzaldehyde with p-chloroaniline was synthesized followed by reduction by sodium borohydride, (figure-1 ) and its Cu(II), Ni(II), Zn(II) complexes were prepared. The metal complexes were characterized by elemental analysis, UV visible, Molar conductance, FTIR, 1HNMR. The co-

BIDENTATE LIGANDS

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Vol.4, No.1 (2011), 136-141

ordination of metals to ligand depends on various parameters like temperature of solutions, reagents, pH of solution, solvent media etc.

EXPERIMENTAL All the chemical and solvents used were of A.R. grade. Melting points were determined in open capillaries and were uncorrected by melting point determining apparatus (SISCO). Purity of the compounds were checked by TLC. IR spectra (KBr, cm-1) were recorded on a JASCO FTIR 410 spectrophotometer. 1H NMR (CDCl3) on a Bruker DPX 300-MHz spectrometer using TMS as an internal reference (chemical shifts in ppm). C, H and N analysis were carried out on a Euro EA (Italy) analyzer. Electronic spectrum measurements were carried out by the Jasco V-600 UV/Vis-Spectrophotometer at room temperature. The Conductivity were recorded on ‘Systronics conductimeter System’ at SOA University, Bhubaneswar. The analytical data are presented in table 1.

HN

HN

M O

O

M = CU,Ni & Zn Fig.-1

Synthesis of Reduced Schiff base The ligand (H2L) was synthesized by mixing equimolar quantities of p-chlorobenzaldehyde and pchloroaniline in ethanol, was refluxed for 4hrs with occasional shaking. The excess ethanol was then distilled off under reduced pressure, the resulting solid was washed with ethanol followed by ether and dried in vacuum. Further the synthesized compound (HL) was treated with sodium borohydride using methanol as solvent with continuous stirring in a water bath. The reaction mixture was cooled and a solid precipitated out immediately. Recrystallization of crude product from ethanol gave the desired Schiff reduced base as whitish crystalline solid. The purity was checked by M.P. and TLC technique. Further the structures of the synthesized compound were conformed by subjecting them to IR, 1HNMR and Elemental analysis studies. Their melting points, % yields and molecular formula are given in Table-1 Synthesis of Metal complexes: (H2L) complexes The metal chelates were prepared by mixing methanolic solution of the respective metal (II) chloride and methanolic solution of ligand H2L in a molar ratio 2:1. The pH of the solution was adjusted to pH 8.5 by drop wise addition of alcoholic ammonia. Then it was stirred for 8 hours at temperature 40-60оC. Then the solution was kept in a Petri dish and allowed to dry in open air. Colored crystals appeared after 3-4 days which was filtered, washed respectively repeatedly with methanol and dried in vacuum. The purity was checked by M.P. and TLC technique. Further the structures of the synthesized compound were conformed by subjecting them to IR, 1HNMR and Elemental analysis studies which are given in table-1 and 2.

RESULTS AND DISCUSSION All metal complexes are colored, stable in air. The solids do not melt sharply and undergo decomposition. These are insoluble in water and soluble in organic solvents such as DMF, DMSO giving respective colors to the solutions. All compounds gave satisfactory elemental analysis. Values are in the close agreement with the values calculated for expected molecular formulae assigned to these complexes, BIDENTATE LIGANDS

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Vol.4, No.1 (2011), 136-141

suggesting 1:2 stoichiometries. The physical data of ligand and metal complexes are given in table-1. The molar conductance values of the complexes indicate the non-electrolytic nature of the complexes13. Electronic spectrum of ligand showed two high intensity bands lying at 255 & 291 nm assigned to transition respectively in ligand. The electronic absorption spectrum of Cu (II) complex showed shifting of intense sharp bands at 251 nm and 355 nm, Ni (II) having 258 nm & 317 nm and Zn (II) having 256.1 nm & 305.6 nm respectively. This shift may be attributed to the donation of the lone pairs of the nitrogen atom of Schiff base ligand to the metal14. The co-ordination sites of the ligand have been determined by a careful comparison of the IR spectra complexes with that of the parent ligand. The ligand shows intense absorption at 3300-3394 cm-1 which may be assigned to hydrogen bonded O-H in plane streaching vibration. This sharp band has disappeared in the complexes, indicating its involvement in the bond formation process15. The ligand shows intense absorption at 1281 cm-1 which may be assigned to C-N stretching frequency is lowered by 11-29 cm-1 in the spectra of the complexes, indicating coordination through azomethane nitrogen of the Schiff bases16. The new bands appearing in the region 665-750 cm-1 and 469-579 cm-1 may probably due to coordinated water molecule and the formation of ν(M-N) and ν(M-0) bonds respectively17,18. The proton NMR of ligand and its metal complexes were recorded using TMS as a reference in DMSO solvent. Data related to various protons is summarized in table-2. The spectrum of ligand shows multiple signals in the range of 6.2-7.33 ppm, which are characteristic signal for aromatic ring protons. The corresponding metal chelates also show similar multiple signals with σ value in the range of 7.09-7.9 ppm. Similarly in the spectrum of ligand signal at 8.57 ppm can be assigned to (>NH) proton which was appeared signal at 8.47-8.52 ppm can be assigned to (>NH) proton in the corresponding chelates. However an important feature of the metal complexes spectrum is absence of signal due to phenolic of ligand at 10.64 ppm indicating the coordination through aromatic proton after deprotonation. The molar -3

conductance of 10 M solutions in DMSO at room temperature of the complexes has been measured and the values are reported in table. These values were found to be ranging between 6.01- 10.95 ohm-1 2 -1 cm mol and values indicate that all the complexes are nonelectrolytes. The ligands and their metal chelates were screened for their antibacterial in vitro against Staphylococcus aureus, Enterococcusfaecalis, Pseudomonas aeruginosa, Klebsiella Pneumoniae, using standard agar well diffusion assay method 19. The NI (II), Zn (II) complexes showed moderate antimicrobial activity. The analgesic activity was determined by tail flick method 20. Wistar albino mice of either sex (20-30g) in the groups of six animals each were selected by random sampling technique. Indomethacin at a dose level of 10 mg/kg was administered as a reference drug for comparison. The test compounds at dose level of 100mg/kg were administered orally by intragastric tube. The animals were held in position by a suitable restrained with the tail extending out and the tail (up to 5 cm) was then dipped in a beaker of water maintained at 550C. The time in seconds taken to withdraw the tail clearly out of water was taken as the reaction time. The reading was recorded at 0, 30, 60, 90, 120 and 180 min. after administration of compounds. A cut off point of 10 sec. was observed to prevent the tail damage. The results are presented in Table-3. The Cu (II), NI (II) complexes showed moderate antimicrobial activity. The purpose of the present study was to examine whether molecular modification might result in detection of new potential drugs. A series of compounds were prepared and assayed in a variety of biological test for antimicrobial and analgesic activity. The data reported in table 3 & 4 shows that effect of variation in chemical structure on activity was rather unpredictable. Seldom did a particular structural modification lead to uniform alteration in activity in all tests. However some point of interest did emerge and a few generalizations can be made. The substitution which appeared to be most important for high order of activity in the greatest number of test was the metal chelates. The introduction of Ni (II), Zn (II) in ligand reduced Schiff base produce compounds with potent antimicrobial properties and Cu (II), Ni (II) ligand compounds produce analgesic activity.

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Vol.4, No.1 (2011), 136-141

CONCLUSION From the result one is tempted to conclude that the metal incorporation in reduced Schiff base ligands are more effective as compared to the ligand against these microbes their metal complexes seem to have developed a fair antimicrobial activity. Generally, it can be conclude that synthesized metal complexes a new represent class of analgesic agents with antimicrobial property. Obviously, the comparative evaluation of active compounds will required further studies; the data reported in this article may be helpful guide for the medicinal chemist who is working in this area. Table-1: Colour, elemental analysis, 1HNMR spectral data of schiff base and metal complexes.

Ligand/Complex

Colour

L CuL NiL ZnL

Whitish Crystal Greenish White Bluish White White crystal

1

Elemental analysis (%) : Found (Calcd.) N 5.99 5.21(5.23) 5.39(5.29) 5.27(5.28)

M 5.91 11.95 (12.01) 11.20(11.21) 12.32(12.34)

HNMR Chemical Shift ( ppm)

Ar-H 6.2-7.3 7.08-7.8 7.1-7.7 7.09-7.9

NH 8.57 8.34 8.13 8.47

OH 10.64 -------------

Table-2: IR, Molar conductance and Electronics spectral data of Schiff base and metal complexes. IR Spectral Data (cm-1)

Ligand/ Complex ν(OH)

ν(C-N)

ν(C-N)

ν(M-N)

ν(M-O)

Molar Conductance (S cm2 mol-1)

Electronic Spectra Data Л - Л*

L CuL Nil

1281 1250 1232

1605 1594 1592

1450 1454 1232

-------758 665

-------514 469

6.01 7.78

291 355 317

NЛ* 255 251 258.1

ZnL

1232

1594

1454

758

514

10.95

305.6

256.1

Table-3: Antimicrobial screening of synthesized compounds by Agar diffusion method

Compound code H2L1

Molecular formula

C13H14N2O2Cl

Zone of inhibition of P.a(mm) 100µg/ml* 14

Zone of inhibition of K.p (mm) 100µg/ml* 17

Zone of inhibition of E.c (mm) 100µg/ml*

Zone of inhibition of S.a (mm) 100µg/ml*

19

24

1

C26H22N2O2Cl2Cu

13

11

20

21

1

C26H22N2O2Cl2Ni

22

19

18

23

1

C26H22N2O2Cl2Zn

20

18

14

20

CuL NiL

ZnL

STD

Ampicillin trihydrate

26.4

25.3

26.2

25.6

Solvent

DMF

00

00

00

00

‫٭‬Average of three readings. E. f: Enterococcus faecalis, P.a: Pseudomonas aeruginosa, K.p: Klebsiella.Pneumoniae, E. c: Escherichia coli, S a: Staphylococcus aureus.

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Vol.4, No.1 (2011), 136-141

ACKNOWLEDGMENTS The authors are grateful to the authorities of School of Pharmaceutical Sciences, Siksha ‘O’Anusandhan University, Bhubaneswar Orissa, India for providing the necessary facility to carryout this research work.

Table-4: Analgesic activity of Synthesized compounds by tail immersion method Results are expressed as Mean ± SEM from six observations *P<0.05 **P<0.01 ***P<0.001 Compo und code H2L1

Molecular formula

C13H14N2O2 Cl

Grou p (mg/ kg) 1

Dos e

0

30

Pain reaction time (min) 60 90 120

180

1.26± 0.032 1.33± 0.012 1.34± 0.014 1.24± 0.004

3.37± 0.018* 4.79± 0.015* 3.64± 0.013 5.03± 0.028*

6.43± 0.012* 7.64± 0.040* 5.54± 0.013* 7.23± 0.012**

6.53± 0.009 7.93± 0.010* 6.20± 0.007* 7.45± 0.014*

3.19± 0.014 4.21± 0.03* 3.73± 0.010* 4.43± 0.013**

50

1.32± 0.013

3.33± 0.010*

4.94± 0.013

6.11± 0.023*

100

1.36± 0.009 1.28± 0.013 1.25± 0.004

4.63± 0.010 3.23± 0.013* 4.53± 0.011

7.13± 0.010 4.93± 0.012* 5.94± 0.013*

7.24± 0.015** 5.83± 0.012* 6.33± 0.012***

50 100

CuL1

C26H22N2O2 Cl2Cu

2

50 100

NiL1

ZnL1

C26H22N2O2 Cl2Ni

C26H22N2O2 Cl2Zn

3

4

50 100

5.45± 0.011* 5.61± 0.006* 4.94± 0.013 5.63± 0.010* * 4.33± 0.010* * 5.32± 0.008* 4.44± 0.012 4.72± 0.007*

3.63± 0.016* 4.44± 0.013** 3.24± 0.013 4.11± 0.024***

REFERENCES 1. N. Sultana and M. S. Arayne, Pakistan Journal of Pharmaceutical Sciences, 20, 305 (2007). 2. S.Shrivastava, A. Kumar, Y. Pandey, and S.N. Dikshit, Asian Journal of Chem., 21, 7224 (2009) 3. M. T. H. Tarafder, M. A. Ali, D. J. Nee, K.Azahari, S.Silong and K. A Crouse, Transition Met. Chem., 25, 456 (2000). 4. M. T. H. Tarafder, Teng-Jin Khoo, K. A. Crouse, A. M. Ali, B. M. Yamin and H. –K.Fun, Polyhedron, 21, 2691 (2002). 5. A. Abu-Raqubah, G. Davies, M. A. El-Sayad, A. El-Toukhy, S. N. Shaikh and J.Zubeita, Inorg.Chim.Acta, 193, 43 (1992). 6. S. Ali-Shehri, G.Davies, M. A. El-Sayad and A. El-Toukhy, Inorg. Chem., 29, 1198 (1990). 7. L.K.W. Henri, J.Tagenine and B.M Gupta, Indian J Chem., 404, 999 (2001). 8. N. K. Singh and S. K. Kushawaha, Indian J. Chem., 39A, 1070 (2000). 9. M. H. El-Tabl, A. Fathy, El-Saied and M. I. Ayad, Synth. React. Inorg. Metal_org. Nano_Metal Chem., 32, 1245 (2002). 10. H.C. Zahid, S. I. Mohammad, S. I. Hummara, T.S. Claudiu, Journal of Enzyme Inhibition and Medicinal Chemistry 17(2), 87 (2002). 11. R. K. Parashar, R. C. Sharma and Govind Mohan, Biological Trace Element Research), 145 (1989). 12. K. K. Narang and R.A. Lal, Indian J. Chem., 14A, 442 (1976). 13. S.Chandra and U. Kumar, Spectromchim, Acta, 61A, 219 (2005). BIDENTATE LIGANDS

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14. B. H. Meheta and P.S. More, Asian J. Chem., 19, 4719 (2007). 15. T. R. Rao, M. Sahay and R. C. Aggrawal, Indian J. Chem., 17, 1103 (2005). 16. K. Nakamoto, Infrared Spectra of Inorganic and Coordination compounds, John Wiley, New York, p10, (1970). 17. B. H. Meheta and A. S. Salunke, Asian J. Chem., 17, 1103 (2005). 18. C. N. R. Rao and J.R. Ferraro, Spectroscopy in Inorganic Chemistry, Academics Press, New York, p.10,(1970). 19. D. M. Kar, S. K. Sahu and P.K. Misro, INDIAN DRUG; 40, 261 (2003). 20. Anonymous, British Pharmacopoeia, Voll II, H. M. S.O. Publication Centre, London; A205 (1988). [RJC-676/2010]

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Vol.4, No.1 (2011), 142-146 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF RHODIUM (I) COMPLEXES WITH MIXED TRIPHENYLPHOSPHINE AND HETEROCYCLIC THIOAMIDE LIGAND R.N. Pandey* and A.K.Nag P.G. Centre ofChemistry(M.U.), College of Commerce, Patna-800020. (India). *E-mail: rameshwarnath.pandey@yahoo.com ABSTRACT Synthesis and spectral Characterization of a series of mixed –ligand rhodium (I) complexes with triphenyl phosphine and 1-butyl-tetrazoline-5-thione of the type [Rh(Pф3)(CO)(ligand)X] (X= Cl, Br), [Rh (Pф3)2 (ligand)X] (X=Cl,Br,I,NCS and SnCl3) and [Rh X(Pф3)2 (Py)(ligand)]X (X= BF4and PF6) are reported. The complexes were characterized on the basis of elemental analyses, molar conductance, magnetic susceptibility data, UV-Vis, IR,1H NMR and 31P NMR spectral studies. A square planar configuration has been tentatively proposed for all these complexes. Key words: Mixed-ligand, Rh(I) complexes, thioamide ligand. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Tertiary phosphine complexes of rhodium(I) are versatile catalyst for hydrogenation1-2 and hydroformylation3-6 reactions. Several rhodium(I) complexes with sterically demanding phosphine7 have been reported. Osborn etal8 and Oro and coworkers9 investigated a series of comparable complexes with substituted triphenylphosphine. However, very little attention have been paid to carry out a systematic study on the mixed-ligand phosphine complexes with bulky heterocyclic thioamide. This concomitant paper comprises a resurgence of our interest in the synthesis and spectral characterization of rhodium(I) complexes with mixed-ligand triphenylphosphine and 1-butyltetrazoline-5-thione(I) using wilkinson catalyst 2 and its analogues as precursors.

(I)

EXPERIMENTAL All chemicals used were either of Anal R or Chemically pure grade. The ligand10, 1-butyl tetrazoline-5thione (But5TH) and precursor complexes [RhX(Pф3)3]2 (X= Cl, Br,I,NCS), [Rh(SnCl3) (Pф3)3 ]11 [Rh X (CO) (Pф3)2 ]2 (X= Cl,Br), [Rh(NCS)(CO) (Pф3)2 ]12 were prepared by the methods reported in literature. Preparation of [Rh (Pф3)2 (P4)(But5TH) ]X ; (X= BF4, PF6) A solution of [Rh Cl(Pф3) (But5TH)2] (500mg, 0.910m mol) in methanol(25mL) was stirred until the solid dissolved completely and it was then treated with an excess of pyridine (7.58 m mol) in methanol (10mL) for 1 hr. to afford an orange solution. A solution of NH4BF4 or NH4PF6(1m mol) in methanol (5mL) was added. The solution was concentrated to half of original volume and 10 mL dry ether was added. Orange coloured solid separated which after filtration was washed with methanol, ether and dried in a vacuum. Yield 85%.

RHODIUM (I) COMPLEXES

R.N. Pandey and A.K.Nag

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1. C46H45 N5 F6 SP3 Rh 2. C46H45 N5 BF4 P2S Rh

Calculated(%): Found(%): Calculated(%): Found(%):

C 54.70, H 4.45, N 6.93, Rh 10.2 C 54.79, H 4.55, N 7.11, Rh 10.2 C 58.04, H 4.83, N 7.36, Rh 10.83 C 58.12, H 4.95, N 7.56, Rh 10.85

Preparation of [Rh X(Pф3)2 (But5TH) ] (X= Cl, Br, I, NCS, SnCl3) These complexes were prepared by adopting the similar process as reported by us in the literature13. 1. C41H40 N4 P2 S Cl Rh(Deep Brown) 2. C41H40 N4 P2 S Br Rh(Brown) 3. C41H40 N4 P2S I Rh(Brownish Red) 4. C42H41N5BF4 P2S2Rh(Bright Yellow) 5. C41H40N4P2SCl3 SnRh(Redish Brown)

Calculated(%): Found(%): Calculated(%): Found(%): Calculated(%): Found(%): Calculated(%): Found(%): Calculated(%): Found(%):

C 59.96, H 4.87, N 6.82, Rh 8.07 C 60.11, H 4.89, N 6.92, Rh 8.12 C 56.87, H 4.62, N 6.47, Rh 11.90 C 57.11, H 4.66, N 6.55, Rh 12.01 C 53.94, H 4.49, N 6.14, Rh 11.29 C 54.01, H 5.10, N 6.23, Rh 11.30 C 59.71, H 4.97, N 8.29, Rh 12.20 C 60.11, H 4.98, N 8.35, Rh 12.31 C 48.70, H 4.05, N 5.54, Rh 10.19 C 48.92, H 4.10, N 5.64, Rh 10.25

Preparation of [Rh X(Pф3) (CO)(But5TH) ] (X= Cl, Br) The suspension of freshly prepared precursor complexes of [Rh X(CO)(Pф3)2 ](X= Cl, Br) or [Rh(NCS)(CO)(Pф3)2] in benzene and ethanolic solution of 1-butyl tetrazoline-5-thione were taken in 1:1 molar ratio and stirred on magnetic stirrer at 850C for two hrs. The working mixture was concentrated to 10mL and 5mL, ether was added to the cold solution as a result a solid matter separated out. It was washed with ether and dried over anhydrous CaCl2. 1. C24H25 N4 O Cl P Rh(Yellow) 2. C24H25 N4 OS P Br Rh(Brown) 3. C25H25N5OS2PRh(Brownish Yellow)

Calculated(%): C 49.10, H 4.26, N 9.54, Rh 17.56 Found(%): C 49.16, H 4.28, N 9.68, Rh 17.75 Calculated(%): C 45.64, H 3.96, N 8.87, Rh 16.32 Found(%): C 45.92, H 3.98, N 8.82, Rh 16.50 Calculated(%): C 49.26, H 4.10, N 11.49, Rh 16.91 Found(%): C 50.10, H 4.11, N 11.50, Rh 17.01

Elemental analysis were performed by the micro- analytical section of the Regional Sophisticated Instrumentation centre, Central Drug Research Institute, Lucknow. IR spectra of ligand and complexes were recorded on a Perkin – Elmer 577 spectrophotometer in the range of 4000-200 cm-1 as KBr pellets and electronic spectra on a Beckmann DU-6, Spectrophotometer. The 1H NMR and 31P NMR spectra were recorded on Bruker 400 MHz or Varian FX 90 Q instruments using TMS and ortho phosphoric acid as references respectively. The magnetic measurements were made on a Gouy balance using Hg[Co(SCN)4] as calibrant. The molar conductance of complexes (10-3M) were measured in DMF using Wiss-Wekstatter Weitheim obb Type LBR conductivity meter.

RESULTS AND DISCUSSION The addition of pyridine to methnol solution of [Rh Cl (Pф3)2(But5TH)] followed by metathetical reaction with NH4BF4/ NH4 PF6 yielded the corresponding derivatives [Rh(Pф3)2 (Py)(But5TH)]X (X= BF4/ PF6) (Str. II). The other mononuclear precursors display ligand substitution reaction without change in oxidation state of metal leading to the formation of neutral complexes(Str.III and Str. IV). These isolated products are RHODIUM (I) COMPLEXES

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non hygroscopic, stable, solid and soluble in DMF, DMSO and other coordinating solvents. All complexes were diamagnetic indicating univalent rhodium (Rh+). However, oxidation state of metal in complexes were verified by the titrating the complexes with ceric ammonium sulphate using ferroin as indicator13. The complexes were titrated for two electron charge.

Electronic spectra of complexes display a very broad band of strong intensity between 24680-24000 cm-1 assigned to charge transfer band (T2g → π*). The other ligand field bonds are obscured due to strong reducing character of Rh+ species. These observations are in agreement with our previous work14-16 observed for thioamide ligands. However, electronic spectrum of [RhCl(Pф3)2 (But5TH)] exhibit three bands at 13825, 18320 and 23700 cm-1. The first band(13825 cm-1) is broad and weak while those at 18320 cm-1 and 23700 cm-1 are medium intensity bands.The first band may be due to spin forbidden 1A1g 3 -1 1 1 transition.The → A2g transition. The other band at 18320 cm may be due to spin allowed A1g → B1g -1 ligand (But5TH) absorbs around 23700 cm , so this band could not be assigned. Thus, rhodium(I) complexes are iso-structural with precursors and are four- coordinated square planar17. The ligand (But5TH) contains thioamide group and give rise to four characteristic thioamide bands I(δNH + υ C-N), II (δCH + υ C S + υ C N), III ((υ C-N + υ C-S), IV(υ C S ) in infrared spectra in the region 1500-805 cm-1. Considering normal coordinate analysis (NCA) of thioamides18, strong Rh-S bond is indicated in all complexes. Thioamide band IV of free ligand red shift to 30-40cm-1 on complexation suggesting bonding through thiocabonyl sulphur 19-22. The bonding through imino nitrogen was ruled out considering blue shift(~ 15cm-1) of thioamide band I and υ NH (3055cm-1) of ligand. Bonding through sulphur is further supported by the presence of new bands at 345Cm-1(υasy Rh-S) ,335cm-1(υsym Rh-S) and at 385-390 cm-1 (υRh-P) in far IR spectra of complexes. Moreover, the presence of single Rh-P stretching mode indicate two Pф3 group at trans-disposition in squqre planar structure. All the characteristic bands of Pф3, CO(carbonyl) and pyridine have been observed in the spectra of complexes23-25. The characteristic bands due to counter anions were observed at ca. 1100cm-1 for BF-4and at 1070cm1 26 for PF 6 in the IR spectra of the respective complexes . However, the presence of coordinated anions are

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confirmed at 2110cm-1(υCN), 830 cm-1(υCS), 482cm-1(δNCS) for terminal NCS group27 and at 455 cm-1 and 4435 cm-1 for SnCl-3 group21. 1

H NMR and 31P NMR Spectra 31 The metal-ligand bonding is further substantiated by 1H NMR and P NMR spectra (ii and iii). The complexes (ii and iii) display signals in the δ 8.12-8.89 ppm range due to aromatic protons of Pф328. The signal for methyl protons appear as singlet in the range of δ1.92-2.2 ppm for butyl group. The two middle CH2 signals are complex and are centred at δ2.1 and δ2.3 ppm. The CH2 group attached to the nitrogen atom of the tetrazoline ring is deshielded giving a triplet at δ4.72 ppm. The signal due to imino proton is always diffuclt to identify because of the quadrupole moment of nitrogen and exchange of this proton. However, a peak observed at δ3.26 ppm in the ligand remains almost at the same position in complexes and imino proton is intact on complexation. The protons of coordinated pyridine26 are observed at δ7.42, 8.10, 8.68 ppm. The 31P NMR spectra of [RhCl(Pф3)2 (But5TH)] was recorded in order to confirm the presence of Pф3 group and to determine the geometry of the complex. The appearance of a signal around 23.75- 28.78 in the spectrum of complex confirmed the presence of magnetically equivalent phosphorus atoms and suggesting that the two Pф3 groups are trans to each other29.The 31 P NMR spectrum of the complex [Rh (Pф3) (Py)(But5TH) 2 ]PF6 consisted of singlet δ37.56 ppm corresponding to coordinated phosphine 31P nuclei. A downfield shift as compared with those in the free Pф3 indicate deshielding caused by relatively less donation of electron density from the rhodium(I) centre to phosphorus through back bonding and the high degree of dπ – pπ back bonding influences the chemical shift to the phosphorous atom. The 31P nuclei of the counter ion PF6- resonated at δ ~ 103 ppm in complex in to septet pattern. Thus, 1H NMR and 31P NMR spectral observations are consistent with the conclusions drawn from IR spectral studies. Table-1: characterization Bands(cm-1) of the IR spectra of rhodium(I) complexes. Complex

Thioamide Bands(cm-1) II III 1280(m) 1065(m) 1270(m) 1050(m)

υRh-P

υRh-S

IV 805(m) 775(m)

385(m)

345w 385w 340w 380w 345w 390w 340w 385w 345w 385w 340w 390w 345w 385w 340w 390w 340w 395w

Ligand (But5TH) [Rh (Pф3)2 (P4)(But5TH) ]BF4

I 1500(s) 1515(m)

[Rh (Pф3)2 (P4)(But5TH) ]PF6

1510(m)

1665(m)

1060(m)

770(m)

390(m)

[Rh (Pф3)2 (But5TH)Cl]

1505(s)

1270(m)

1065(m)

765(m)

380(m)

[Rh (Pф3)2(But5TH) Br ]

1510(s)

1272(m)

1062(m)

770(m)

390(m)

[Rh (Pф3)2 (But5TH) I]

1515(m)

1270(m)

1060(m)

775(m)

385(m)

[Rh (Pф3)2 (But5TH) NCS]

1510(m)

1265(m)

1065(m)

770(m)

385(m)

[Rh (Pф3)2 (But5TH)(SnCl3) ]

1504(s)

1270(m)

1070(m)

775(m)

382(m)

[Rh (Pф3) (CO)(But5TH)Cl ]

1505(s)

1275(m)

1075(m)

770(m)

380(m)

[Rh (Pф3) (CO)(But5TH) Br]

1505(s)

1270(m)

1070(m)

775(s)

385(m)

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

J.F. Young, J.A. Osborn, F.H. Jardine and G.Wilkinson, Chem.Comm.,131(1965). J.A. Osborn, F.H. Jardine, J.F.Young and G.Wilkinson, J.Chem. Soc. A, 1711(1966). A.Muller, A.Roodt,S.Otto,A.Oskarsson, S.Young, Acta Crystallogr. Sect E, 58, m715(2002). A. Roodt,S.Otto and G.Steyl, Coord. Chem. Rev. 245, 121(2003). S.Otto and Roodt, Inorg. Chem. Acta, 357, 1(2007). A.C.da Silva, K.C.B. de Oliveira, E.V.Gusevskaya, E.N.da Santo, J.Mol. Catal.(A) 179,133(2002). V.R.Landacta, M.Peruzzini, V.Herrera, C. Bianchini, Roberto A. , Sanchez-Delgado, A.E.Goeta and F.Zanobini, J. Organomet. Chem. 691, 1039(2006). J.R.Shapley, R.R. Schrock and J.A. Osbor, J. Am. Chem. Soc., 91,2816(1969). R.Uson, L.A.Oro and M.J. Fernandez, J. Organomet. Chem. 193, 127(1980). E.Lieber and J. Ramchandran, Can. J. Chem. 37, 101(1959). J.F. Young, R.D.Gillard and G. Wickinson, J. Chem. Soc., 5176(1964). M.A. Jennings and A. Wojcicki, Inorg. Chem. 6,1854(1967). R.N.Pandey and J.N.Das, J. Ind. Chem. Soc.71,187(1994). B.Martin, W.R.Mc Whinnie and G.M. Waind; J.Inorg. Nucl. Chem. 23, 207(1961). R.N.Pandey and Rajnish Kumar, J. Ultra Sc. 21(3), 579(2009). R.N.Pandey, Ashok Kumar and D.P. Singh, Asian J. Chem. 22(3),1661(2010). R.N.Pandey, A.Kumar, R.S.P. Singh, A.N.Sahay and Shashikant Kuma, J. Ind. Chem. Soc. 69,804(1992). I.Suzuki,Bull. Chem. Soc. Jpn., 35,1286,1419,1456(1962). R.N.Pandey, Gunjan Kumari and Rajnish Kumar Singh, Asian J. Chem. ,22(3), 2379(2010). R.N.Pandey, Rajnish Kr.Singh and R.J.Sinha, Acta Ciencia Indica ,35c, 505(2009). R.N.pandey, Gunjan Kumari and Rajnish Kr. Singh, J. Ind. Council Chem., 27(1), 72(2010). R.N.Pandey and Rajnish Kumar Singh, Oriental J. Chem., 25(3),599(2009). R.J.H.Clark and C.S. Williams, Inorg. Chem. , 4, 350(1965). G.B.Deacon and J.H.S.green,; Spectrochim Acta. 24A, 845(1968). E.Sola, J. Navarro, J.A. Lopez; F.J. Lahoz, L.A. Oro and H. Werner, Organometallics, 18, 3534(1999). O.S.Sisodiya, A.N.Sahay and D.S. Pandey, Ind. J. Chem. ,39A, 453(2000). I.Bertini and A.Sabatini, Inorg. Chem. ,5, 837(1966). S.Kanchanadevi, K.P. Balasubramanian, V. Chinnusamy, R. Karvembu and K. Natarajan, Trans. Met. Chem,. 30,330(2005). R.Karvembu and K. Natarajan, Polyhedron, 21, 1721(2002). [RJC-670/2010]]

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Vol.4, No.1 (2011), 147-152 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

A PRELIMINARY SURVEY OF MERCURY IN FRESH WATER AND FISHES P. J. Puri1,*, M.K.N. Yenkie1, S.P. Sangal 1, N.V. Gandhare,2 G. B. Sarote3 and D. B. Dhanorkar4 1*

Department of Chemistry, LIT, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440 001, 2 Department of Chemistry, Nabira Mahavidyalaya, RTM, Nagpur University, Katol - 66302 3 Regional Forensic Science Laboratory, Dhantoli, Nagpur – 440 012 4 Maharashtra State Power Generation Limited, Nagpur- 440 033 * E-mail – puripj@rediffmail.com ABSTRACT The widespread contamination by heavy metals is of major concern because of their toxicity, persistence and bioaccumulative nature. Among heavy metals, mercury is considered to be the most toxic metal. In organic form, it enters human through fish. Hence this investigation of monitoring the levels of Hg in water and fish (muscle, gill, liver and viscera) in different lakes was undertaken within Nagpur city, Maharashtra, India. The concentration of mercury in water and edible portion of fish was below the permissible limit stipulated by W.H.O. and pollution control organizations of other countries. Total mercury Hg content in Futala (0.018 mgL-1 to 0.042 mgL-1), Ambazari (0.019 mgL-1 to 0.044 mgL-1) and Gandhisagar (0.012 mgL-1 to 0.046 mgL-1) lake was recorded. Generally rainy season and summer months showed heavier pollution loads, with Ambazari, Gandhisagar and Futala lake. The study while addressing water quality and interactions due to human activities in shallow lakes, also discusses problems associated with human impacts in selected lake environment. Keywords: Water quality, Lake, Pollution, Fish, Heavy metals, health problems. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Nagpur city is one of India’s fastest growing cosmopolitan city. The city is spread in an area of about 220 Km2. The road length of city under the Nagpur Municipal Corporation (NMC) is 1200 Km. Nagpur city is situated at an altitude of over 290 meters above sea level rising upto 350 meters towards NW, W and SW of the city. In many areas of Nagpur city tap water supply is not available and people are dependent mainly on the ground water sources. In and around Nagpur city (M.S.), there are large numbers of water bodies. Nag River which is a tributary of Kanhan takes its origin from Ambazari and flows towards east through Nagpur city. The Nag river water is completely polluted on account of draining of sewage into it. Lakes are significant resource base of Nagpur city. Some of these are used to supply water for drinking purpose like Gorewada lake and Wena tank. The water from Futala lake is used for irrigation and water from Ambazari lake is used for industrial purpose. The other water bodies in Nagpur city are, namely, Gandhisagar lake, Naik lake, Lendi lake, Sakkardara lake and Khadan lake. In Naik and Lendi lake, the ingress of sewage from nearby locality is rampant. Both of these lakes have been very much encroached by the weeds. This has been resulted into total degradation of Naik and Lendi lake. The major lakes in Nagpur city which once use to be eco-friendly and useful purposes, have lost their grandeur and have rather becomes a source of nuisance. Thus it is quite imperative to know the quality of status of these lakes water with a view to renovate them so that these serve for a useful purpose to the society. All these lakes were used for supply of water for various purposes, but, now due to heavy pollutant level, these are no more suitable for human use. India is a country of festivals, therefore the use of flowers, fruits, leaves are unavoidable. During and after festivals like Ganesh chaturthi, Durga puja, Gouri, Mahalaxmi puja, Holi, Rang Panchami etc. the leaves, fruits, flower, ash and even idols of Gods and Goddess are immersed MERCURY IN FRESH WATER AND FISHES

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in the water bodies (Lakes). During year 2004, about 70 tones of biomass were collected by civic machinery. This material, if not removed, will result in depletion of oxygen due to bio-degradation of waste dumped resulting in anaerobic conditions. If the addition of bio-mass continues, the lake will be converted first into muddy ponds, then to marsh and finally to dry land. The present study was undertaken to study the quality of water of these lakes and to chalk out strategy for their renovation either for drinking purpose or for irrigation, development of fisheries, industrial purpose and also to explore the possibility of recharge of these water bodies (lakes) to the ground water. In order to save these water bodies (Lakes), which would serve us as, reservoirs of fresh water, fishes and other products for hundreds of years, the studies on the level of their pollution have been undertaken throughout the year. Lake ecosystem are increasingly affected by various anthropogenic impacts such as excess of nutrients causing eutrophication, toxic contamination of industrial, agricultural and domestic origin as well as heat pollution reaching the lakes through their catchments area and atmosphere. Typical results of human activities proved to be elevated levels of heavy metals present in fresh water and among these microelement lead (Pb) and mercury (Hg) are most specific. Lead (Pb) and Mercury (Hg) are considered to be one of most important pollutants of aquatic ecosystems due to their environmental persistence and tendency to be concentrated in aquatic organism. In recent years, much attention has been paid to the possible danger of Hg poisoning in human as a result of contaminated fish consumption. There are several reports on Hg content of marine fish from Indian Ocean, Bay of Bengal and Arabian Sea1-4. However, studies on Hg concentration of freshwater and fish in India are scanty5-6. The present investigation deals with Hg concentration in water and fish collected from various lakes in Nagpur city, Maharashtra, India.

EXPERIMENTAL The present investigation deals with Hg concentration in water and fish collected from the various lakes specially Futala, Gorewada, Ambazari and Gandhisagar Lake in Nagpur city (M.S). Sampling Program Monthly samples of water and fish were collected over a period of one year during the session December to January 2008 comprising of four seasons. Fresh fish were collected and brought to the laboratory in icebox. The following species were subject to analysis of mercury concentration in muscle and other organs. 1. Javla 2. Vogte 3. Marvels. Digestion and Pretreatment 7 (a) Water Water sample of 100 ml was treated with 3 ml of concentrated. HNO3, evaporated to dryness and the residue was dissolved in 3ml. of concentrated HNO3 and digested until a clear solution was obtained. The resulting solution was made up to 100 ml. 8 (b) Fish Fish samples were cut open and different organs viz., muscle, gill, liver and viscera were separated. For each organ 5g of the sample was digested with 20ml of 10:1:2 mixture of con. HNO3, H2SO4 and HClO4 at 1050C till a clear solution obtained 9 Estimation of Hg by cold vapor technique using Mercury Analyzer Mercury was estimated using Mercury Analyzer (ECIL 5800 MA) which works on the principle of cold vapor technique.10 The mercury present in the pretreated sample was reduced to elemental state by using stannous salt in an acid solution. The working wavelength was 253.7 nm and the sensitivity 0.001 Âľg Mercury solution of concentration 100mg/l was prepared by dissolving 0.1354 gm of mercuric chloride in 2% HNO3 and made up to 100ml using 2% HNO3. Calibration and estimation The apparatus was set up and connections were checked for leakage. The required aliquot (2-4 ml) of the blank solution (respective solutions used for digesting water, sediment and fish organs) was taken in the reaction vessel. 8ml of 10% HNO3 and 2ml of SnCl2 were added and the stopper replaced immediately. MERCURY IN FRESH WATER AND FISHES

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Magnetic stirrer was switched on and the contents stirred vigorously for about 5 minutes. The pump was started to allow the air to purge through the reaction vessel. Absorbance was recorded as quickly as possible. The above procedure was followed for various concentrations of standard Hg solutions and the respective absorbance recorded. A standard graph was plotted between concentration and absorbance. The above procedure was followed for the various sample solutions and the corresponding absorbance noted (after deducting the absorbance value for the respective blank solution). The Hg concentration was calculated using the standard plot.

RESULT AND DISCUSSION A very interesting source of information about water pollution is that it affects aquatic life. Ten millions of fish are killed each year by a wide variety of different pollutants from many different sources, municipal and industrial. Mercury Hg pollution outside India is also quite alarming, even the US is also not devoid of this scar11,12. Hg has been recognized as a general cellular poison and effective protein precipitant. Hg vapor is almost completely absorbed through the alveolar membrane and is oxidized in blood and tissues before reacting with bimolecular. After acute administration of Hg salts to animals and man, the highest levels of inorganic Hg are found in kidneys and the second highest concentration in liver. Due to their lipid solubility, organomercurials are many times more toxic to man than the metallic form. The earliest cases of poisoning were due to occupational exposure following the introduction of methyl mercury compound as antifungal seed dressing agents. Reports of poisoning from nonoccupational sources appeared with increasing frequency from 1950 onward. The primary signs and symptoms of methyl mercury poisoning results damage in the nervous system. It is characterized by ataxia (loss of coordination), diarrhea (slurred speech), parenthesis (loss of sensation at the extremities of limbs and mouth), tunnel vision (construction of visual field) and loss of hearing. Severe poisoning can cause blindness, coma and death13. It is found reduced activity of the enzyme with a direct exposure of Hg14. The studies have been carried out on the effect of methyl mercury and HgCl2 on binding to the macaronis cholinergic receptor in cellular membrane isolated from the cerebrums of ringed seals15. Mercury is considered to be the most toxic metal. In organic form it enters the human through fish. Hence, this investigation of monitoring the levels of Hg in water and fish (muscle, gill, liver and viscera) was carried out in different lakes in Nagpur city. It may also be expected that the high concentration of metal in water can be gradually accumulated on the sediment and in due course it may get transferred to fish. Fishes being one of the main aquatic organism in food chain may often accumulate large amount of certain metals17,18. Directly acting metals like Fe, Pb, As, Hg and Zn are common toxic pollutants for fish19. In order to judge the fitness of lake water for fisheries point of view, the water quality from different lakes (Fig. 1) viz. Ambazari, Gorewada, Futala and Gandhisagar within Nagpur city (India) was tested. The data on mean mercury content in water (mg/l) and various organs of fish (mg/kg fresh weight) are given in Table 1-4 during different seasons at Gorewada, Futala, Gandhisagar and Ambazari lake for the session January to December 2008. The variation of level of occurrence of heavy metals in water was found different from each other due to variation of solubility of existing forms of metals in water as well as their availability in the immediate environment. The result of present study indicates below detectable limit of mercury content in Gorewada lake. The concentration of mercury in water varied from 0.018 mgL-1 to 0.042 mgL-1, 0.019 mgL-1 to 0.044 mgL-1 and 0.012 mgL-1 to 0.046 mgL-1 for Futala, Ambazari and Gandhisagar lake respectively during different season for the session January to December 2008. The result of present study indicates Hg content in water from Gorewada lake was below detectable limit in all sites during entire period of study. It is clearly evident from result that Gorewada lake water source will not cause any significant heavy metal health hazard to the water consumer; however, periodic monitoring of ground water and surface water (lakes) sources is required in order to check any further increased in heavy metal concentration due to discharge from various industrial effluent, sewage discharge or geochemical alterations. The present result indicates no specific trend is observed in mercury concentrations in different organs of fish in all four studied lakes. The Hg content in fish from all the studied lakes was below detectable limit throughout study period. Mean concentration of Hg in the edible portion of fish was well below stipulated toxic limit (0.5mg/kg) in Futala, Ambazari, Gorewada and Gandhisagar lake and hence fit for consumption.20 MERCURY IN FRESH WATER AND FISHES

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In general mercury (Hg) metal accumulation was found to be maximum at all sites in Futala and Ambazari lake. It is observed that higher concentration of heavy metals in fish organs is attributed to the types of food that fish is feeding on. Highly significant difference was noticed in case of mercury (Hg) in water samples collected from studied lakes. Mercury Hg content was below detectable limits at all sides in Gorewada lakes while in Gandhisagar lake a value in range of 0.012 to 0.046 mgL- was registered. Possible cause of higher values of mercury content in studied lake water environment could be illegal discharge of slaughterhouse waste, immersion of idols, of God and Goddess in large ratio during festival seasons, surface runoff, domestic wastes and washing activities in and around these lakes. Generally, summer and rainy seasonâ&#x20AC;&#x2122;s months showed heavier pollution loads. Seasonal variations show a definite trend in all studied lakes, except Gorewada lake.

CONCLUSION The mercury Hg content in studied lakes revealed highly significant differences between seasons and locations. Generally summer and rainy seasonâ&#x20AC;&#x2122;s months showed heavier pollution loads within Futala, Ambazari and Gandhisagar Lake. There is no specific trend observed in mercury (Hg) content in different tissues (gill, liver, viscera, muscle) of fish in studied area. The results shows low mercury levels in tissues of the species studied and suggest differences due to locality. The Hg content in studied lakes was below permissible limit prescribed by W.H.O. and also level stipulated by pollution control organization of other countries (0.05 mgL-1). It is clearly evident from results that Gorewada, Gandhisagar, Ambazari and Futala lake water source will not cause any significant heavy metal health hazard to the water consumer, however, periodic monitoring of ground water and surface water (lakes) sources is required in order to check any further increased in heavy metal concentration due to discharge from various industrial effluent, sewage discharge or geochemical alterations. Mean concentration of Hg in edible portion of fish was well below the stipulated toxic limit (0.5 mg/kg) in all studied lakes and hence fit for consumption.

ACKNOWLEDGMENT The authors hereby acknowledge the kind and wholehearted support of the Dr. S. B. Gholse, Director, LIT, RTM, Nagpur University, Nagpur. Table-1: Mean Mercury content in water (mg/l) and various organs of fish (mg/kg fresh weight) during different seasons of the year in the FUTALA LAKE Season

Water

Muscle

Gill

Liver

Viscera

Cold Water (Jan-Feb)

0.018

ND

ND

ND

ND

Hot Weather (Mar-May)

0.032

ND

ND

ND

ND

South West Monsoon (June-Sept)

0.042

ND

ND

ND

ND

North East Monsoon (Oct-Dec)

0.028

ND

ND

ND

ND

Mean

0.030

ND

ND

ND

ND

ND = Not detectable; BDL = below detectable limit

Table-2: Mean Mercury content in water (mg/l) and various organs of fish (mg/kg fresh weight) during different seasons of the year in the AMBAZARI LAKE Season

Water

Muscle

Gill

Liver

Viscera

Cold Water (Jan-Feb)

0.019

BDL

BDL

BDL

BDL

Hot Weather (Mar-May)

0.022

BDL

BDL

BDL

BDL

South West Monsoon (June-Sept)

0.044

BDL

BDL

BDL

BDL

North East Monsoon (Oct-Dec)

0.031

BDL

BDL

BDL

BDL

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0.029

BDL

BDL

BDL

BDL

ND = Not detectable; BDL = below detectable limit

Table-3: Mean Mercury content in water (mg/l) and various organs of fish (mg/kg fresh weight) during different seasons of the year in the GOREWADA LAKE Season

Water

Muscle

Gill

Liver

Viscera

Cold Water (Jan-Feb)

BDL

ND

ND

ND

ND

Hot Weather (Mar-May)

BDL

ND

ND

ND

ND

South West Monsoon (June-Sept)

BDL

ND

ND

ND

ND

North East Monsoon (Oct-Dec)

BDL

ND

ND

ND

ND

Mean

BDL

ND

ND

ND

ND

ND = Not detectable; BDL = below detectable limit

Table-4: Mean Mercury content in water (mg/l) and various organs of fish (mg/kg fresh weight) during different seasons of the year in the GANDHISAGAR LAKE Season

Water

Muscle

Gill

Liver

Viscera

Cold Water (Jan-Feb)

0.012

BDL

ND

ND

BDL

Hot Weather (Mar-May)

0.023

BDL

ND

ND

BDL

South West Monsoon (June-Sept)

0.020

BDL

ND

ND

BDL

North East Monsoon (Oct-Dec)

0.018

BDL

ND

ND

BDL

Mean

0.018

BDL

ND

ND

BDL

ND = Not detectable; BDL = below detectable limit

REFERENCES B. L. K. Somayajulu and Rama, Curr. Sci. 41, 207 (1972) B. M. Tejam and B. C. Halder, Indian J. Environ. Hlth., 17, 9 (1979) T. W.Kureishy, M. D. George and R. Sengupta, Marine Poll. Buln., 1, 357 (1979) S.Y.S.Singbal, M. D.George, R. S.Topgi, and R.Naronha, Bull. NIO., 15, 121 (1982) P. Jayachandran and S.P. Raj, Curr. Sci. 44, 828 (1975) K.Ayyadurai, C. S. Swaminathan and V.Krishnasamy, Indian J. Environ. Hlth., 36 (2), 99 (1994) K. Ayyadurai, Swaminathan, 36(2), 99 (1994) E.P. A. Methods for chemical analysis of water and wastes, EPA, 62516-74-003 a (1974) K. Ayyadurai, V. I. Krishnaswamy, J. Environ. Biol., 10 (2-supp.), 165 (1989) K. Ayyadurai, Res. T. Chem. Environ., 8(2), (2004) R. Rossmann, Journal of Great Lakes, Research, 25, 683 (1999) C. H. Marvin, Envir. Res. 95, 3251, (2004) S. Mitra, Mercury Pollution, 195, (1986) M. F.Frasco, D. Fournier, F. Carvalho, Feilhermino L. Biomarkers, 10, 360, (2005) H. Basu, M.Kwan, J. Toxicol Environ. Health, 69, 1133, (2006) P.D.P. Martin and H. T. Khan, E-Journal of Chemistry , 5(1), 16 (2008) S. A. Mansour and M. Sidkey, Food Chem. ,78, 15 (2002) P. V. Hadson, Aquat Toxical; 11, 31989) C.B. Srivastav : A textbook of fishing science and Indian Fisheries, Kitamahal Publications, Allahabad, India, 99, 307, (1995) 20. P.J. Puri, M.K.N. Yenkie, N.V. Gandhare, D.B. Dhanorkar, RASAYAN J. Chem. 801, 800 (2010).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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Fig.-1: Map showing Gandhisagar, Ambazari, Gorewada and Futala Lake , Nagpur (MS) India.

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Vol.4, No.1 (2011), 153-158 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

DEVELOPMENT AND VALIDATION OF REVERSE PHASE LIQUID CHROMATOGRAPHY METHOD FOR ESTIMATION OF ISOTRETINOIN (13-CIS RETINOIC ACID) IN PHARMACEUTICAL DOSAGE FORM Pratik Patel1, Ritu Kimbahune1, Prachi Kabra1,*, Kuldeep Delvadiya1 and L.V.G.Nargund2 1

2

Department of Quality Assurance, Nargund College of Pharmacy, Bangalore, India Department of medicinal chemistry, Nargund College of Pharmacy, Bangalore, India *Email: prachi.v.kabra@gmail.com

ABSTRACT In the present study, a reverse phase high performance liquid chromatographic method was developed and validated for the determination of Isotretinoin in pharmaceutical dosage form. Chromatographic separation was carried out on a C-8 column using a mobile phase consisting of acetonitrile:isopropyl alcohol (50:50, v/v) adjusted at pH 5.0 using 1% ortho phosphoric acid. Flow rate was 1ml min−1 and UV detection was carried at 280 nm. Caffeine was used as an internal standard. The calibration curve was linear over the range 5–600µgml−1. R.S.D. for precision study was found to be <1%. The result of accuracy study was ranged between 98.61% and 101.51% with a R.S.D. lower than 2%. LOD and LOQ were found to be 0.0428µgml−1 and 0.1298µgml−1, respectively. The method was simple, rapid, easy to apply and very suitable for routine analysis of Isotretinoin in pharmaceutical dosage form. Keywords: Isotretinoin, Caffeine, RP-HPLC, Internal Standard,13-Cis Retinoic Acid. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Isotretinion (IST), 13-cis isomer of retinoic acid, is a retinoid classified as vitamin A. Isotretinoin is a topical keratolytic agent which is used in the treatment of skin diseases including acne vulgaris. The mechanism of action is believed to inhibit the secretion of sebum and alter the lipid composition of the skin surface1. Pure crystalline isotretinoin is a yellow-orange powder whose faint odour resembles that of vitamin A. It is soluble in chloroform and methylene chloride, sparingly soluble in ethanol, 2-propanol, polyethylene glycol and very sparingly soluble in water2. Assay of Isotretinoin is performed by titrating with 0.1N sodium methoxide in USP3 whereas potentiometric titration with tetrabutyl ammonium hydroxide is reported in BP4. In previous studies, Isotretinoin was determined by gas chromatography in soft and hard gelatine capsules5. HPLC method for the simultaneous determination of tretinoin and isotretinoin is also reported6. Tretinoin (13-trans retinoic acid) and retinoid were determined in different formulations by reversed-phase HPLC7,8,9. It was revealed that none of RP-HPLC method reported for the determination of IST using an internal standard (IS). The uncertainties introduced by sample injection can be avoided by use of IS in quantitative chromatography. Hence an aim of this work was to develop a rapid, precise, accurate and comparatively economical RPHPLC method with UV detection for quantitative estimation of IST in soft gelatine capsule dosage form. The results obtained have been statistically validated in accordance with the ICH guidelines10.

EXPERIMENTAL Reagents and Chemicals All the reagents like ortho phosphoric acid, acetonitrile (Qualigens fine chemicals, Mumbai) and water used were of HPLC grade. IST and Caffeine (CAF) standards were obtained from Strides Arco

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Laboratories (Bangalore, India) and Juggat Pharma (Bangalore, India) respectively. The marketed formulation (Tufacne-20) was purchased from the local pharmacy. Instrumentation The HPLC system used was Shimadzu LC-20AT pump, Rheodyne injector (20µl), SPD-20A UV detector and the system was controlled through Spinchrome software. Analytical column used for this method was PHENOMENEX Luna C8 (250X4.6mm, 5µ). Sartorius digital Balance was used for weighing and Digital pH meter 7007 for the pH measurement. Chromatographic conditions The composition of the mobile phase was acetonitrile:isopropyl alcohol (50:50, v/v) (adjusted to pH 5.0 with 1% orthophosphoric acid). The mobile phase was vacuum-filtered through 0.2 µm Supor200 membrane and degassed by ultrasonication for 10min before use. The mobile phase flow rate was set at 1 ml min−1. All standard and assay samples were filtered through cellulose acetate (0.45µm) filter before injection. After equilibration of column with the mobile phase indicated by a stable baseline, aliquots of sample (20 µl) were injected and the total run time was kept 20 min. The absorbance of the eluent was monitored at 280nm with a detection sensitivity of 0.1000 aufs. CAF (5µgml−1) was used as an IS. Standards and sample solutions preparation Standard stock solutions of IST (1000µgml−1) and CAF (1000µgml−1) were prepared in HPLC grade methanol. Working standard solutions were freshly prepared daily by appropriate dilution of the stock solutions with mobile phase. Ten soft gelatine capsules were cut with the sharp blade and added in about 50ml of methanol. This was sonicated for 10 to 15 minutes and then filtered by using whatman filter paper no. 41. Extracted solution of soft gelatine capsule was diluted with methanol in 100ml volumetric flask. From the above solution 1 ml was transferred in to 10ml volumetric flask along with 1ml of CAF solution (50 µgml−1) made up to volume with mobile phase (100µgml−1 IST and 5µgml−1 CAF) and results are as given in Table 1. Method validation Analytical method validation was carried out under the guidelines of International Conference on Harmonization (ICH). The assay was validated with respect to linearity, precision, accuracy, sensitivity and robustness. Linearity Calibration curves were obtained from injecting the six sets of eleven serial different drug concentrations (5, 10, 20, 30, 40, 50, 100, 200, 300, 500, 600µgml−1 of IST). The curves were generated by plotting the peak area ratios between IST and CAF against IST concentration. Linearity was evaluated by linear regression equation. Precision The precision of the method was determined by repeatability (intra-day) and intermediate precision (interday) and was expressed as relative standard deviation (R.S.D.). Repeatability was determined by performing nine determinations from triplicate injections of three different concentrations of IST (10, 50 and 100µgml−1) on the same day at different time intervals and on three different days for inter-day precision. Accuracy/recovery In this study, accuracy was determined based on the recovery (percentage) of known amounts of standard IST added in the assay samples. This was performed by analyzing IST at three concentration levels (50, 100 and 150µgml−1), with a constant concentration of 5µgml−1 of internal standard. Samples were prepared in triplicate. The accuracy of the assay was determined by comparing the found concentration with the added concentration. Sensitivity Sensitivity of the method was determined by means of the detection limit (LOD) and quantification limit (LOQ). The LOD and LOQ were measured based on the method described by the International Conference on Harmonization. Calculations for LOD and LOQ were based on the standard deviation of ISOTRETINOIN (13-CIS RETINOIC ACID)

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the calibration curve (σ) and the slope of curve (S), using the equation LOD= 3.3×σ/S and the equation LOQ= 10×σ/S. Robustness Robustness of the method was evaluated by the analysis of IST solution under different experimental conditions such as pH of the mobile phase and flow rate. The flow rate was varied 1±0.1 ml min−1 and pH of the mobile phase was changed 5.0 ±0.2 units. Their effects on the retention time (tR), tailing factor (T) and resolution of the peaks (R) were studied.

RESULTS AND DISCUSSION Optimization of the chromatographic method The chromatographic conditions were adjusted to provide the best performance of the assay. For system optimization the important parameters such as type and concentration of organic solvents, pH and mobile phase flow rate were investigated. Effect of mobile phase composition Different mobile phase composition were tried to achieve better separation and resolution(R) between IST and CAF. It was observed that the acetonitrile:isopropyl alcohol system gave a better resolution and peak symmetry than the methanol:acetonitrile system. Different proportions of acetonitrile:isopropyl alcohol (50:50, 40:60, 30:70, 20:80v/v) were tested and evaluated before the final chromatographic conditions were selected. Finally, acetonitrile:isopropyl alcohol (50:50 v/v) (adjusted to pH 5.0 with1% orthophosphoric acid) was chosen as mobile phase. As a result, the standard solutions of IST and CAF showed symmetric and well-defined peaks, with an average retention time for IST of 3.4 min and 2.9 min for the CAF. Effect of flow rate Different mobile phase flow rates (0.9, 1.0 and 1.1mlmin−1) were investigated. The optimum flow rate for which the column plate number (N) was maximum, with the best resolution between all components and with a short run time (<10min) was found to be 1 mlmin−1. Internal standard Different compounds were tested as IS for the chromatographic procedure. Among them, CAF eluted before 10 min of the analysis and has a better symmetry and resolution with respect to IST. Therefore, CAF has been chosen as an IS. Method validation System suitability System suitability was performed to confirm that the equipment was adequate for the analysis to be performed. The test was carried out by making six replicate injections of a standard solution containing 10.0µgml−1 and 5.0µgml−1 of IST and CAF (IS), respectively, and analyzing each solute for their peak area, theoretical plates (N), resolution(R) and, tailing factor (T). The results of system suitability in comparison with the required limits are shown in table 2. The proposed method fulfils these requirements within the accepted limits. Linearity The standard calibration curve was linear over the concentration range 5–600µgml−1. The correlation coefficient obtained after linear regression analysis was 0.9995. The equation of the calibration curve based on the peak ratio of IST/IS with respect to IST concentration was found to be y = 0.05830x + 0.58306. Precision The R.S.D. of repeatability (intra-day) and intermediate precision (inter-day) ranged between 0.042% and 0.361%. These values show a low variability between the values obtained for each concentration. These values are shown in Table 3. Accuracy The results of the accuracy studies are shown in Table 4. Recovery ranged between 98.67% and 101.51% with R.S.D. less than 2%. The values obtained show a suitable accuracy for the analytical method Sensitivity ISOTRETINOIN (13-CIS RETINOIC ACID)

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LOD and LOQ were 0.042µgml−1 and 0.129µgml−1, respectively. These values are adequate for the detection and quantification of IST. Robustness During the robustness study, peak symmetry (T) was maintained and the retention times were not significantly changed as shown in Table 5. These facts suggest that the method did not change with time and experimental conditions. However, it could be noted that organic composition of the mobile phase can influence the method performance.

CONCLUSIONS In the present research work to achieve highest precision in quantitative chromatography of IST in pharmaceutical dosage form, a RP-HPLC method for IST using IS was developed and validated. The method was validated in terms of linearity, precision, accuracy, detection limit, quantification limit and robustness. It involves a simple procedure for the preparation of the samples and shorter run times for analytical procedure (less than 10 min). Hence the present HPLC method can be considered simple, rapid, suitable and easy to apply for routine analysis of Isotretinoin in pharmaceutical dosage form.

REFERENCES 1. B.R.Simmons, O.Chukwumerije, J.T.Stewart, J. Pharm. Biomed. Anal., 16, 395 (1997). 2. O’Neil, MJ, The Merck index, An Encyclopedia of Chemicals, Drugs and Biological, Merck research laboratories, p.935, 936 (2001). 3. United States of Pharmacopoeia 22 and NF 17, US Pharmacopoeial Convention, Rockville, MD, 22, 742 (1990). 4. British Pharmacopoeia, Her Majesty Stationery Office, London, 1, 755 (1998). 5. M.J.Lucero, J.Vigo, M.J.Leon, Int J Pharm., 110, 241 (1994). 6. B.M.Tashtoush, E.L.Jacobson, M.K.Jacobson, J. Pharm. Biomed. Anal., 43, 859 (2007) 7. C.Leomy, D.Bonhomme, D.Amdidouche, C.Massare, R.Huynh, H.Hagipamtelli, G.Reyes, G.Frej, J.L.Misset, G.Mathe., Int. J. Pharm., 115, 235 (1995). 8. X.Tan, N.Meltzer, S.Lindenbaum, J. Pharm. Biomed. Anal., 11, 817 (1993). 9. C.Lanvers, G.Hempel, G.Blaschke, J.Boos, B.Chromatogr, Biomed. Appl., 685, 233 (1996). 10. International Conference on Harmonization (ICH), Harmonized Tripartite Guidelines on ‘Validation of Analytical Procedure: methodology’, Operational from June 1997, The European Agency for the Evaluation of Medicinal products, Human Medicines Evaluation Unit. Table 1: Assay of Isotretinoin Capsule (IST) Component

IST

Label claim (mg)

Amount found (mg)

Amount found(%)

20

20.58

102.93

20

20.96

104.81

20

19.93

99.66

20

20.96

104.81

20

19.93

99.66

20

20.58

102.93

Mean

20.49

102.45

%RSD

2.2

2.2

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Table 2: System Suitability Results of the Proposed Method. Analyte

R

CAF IST Required limits

2.917 R >2

N

T

4086 3796 N > 2000

1.652 1.400 T < 1.5

RSD (n=3) tR peak area 0.03 0.20 0.01 0.24 R.S.D. < 5%

Table 3: Summary of Precision Determined During Method Validation Concentration (µgml−1)

R.S.D. (%), intra-day

10 50 100

R.S.D. (%),inter-day

0.318 0.113 0.042

0.361 0.008 0.032

Table 4: Accuracy Of The Method Determined According To ICH Q2 Guidelines. Concentration (µgml−1) Recovery (%)a Added Recovered 50 49.35 98.70 50 49.33 98.67 50 50.72 101.45 100 99.88 99.88 100 99.48 99.48 100 100.05 100.05 150 152.27 101.51 150 150.19 101.12 150 152.27 101.51 a( Found concentration/added concentration) ×100.

R.S.D. (%) (n=3)

1.60

0.29

0.22

Table 5: Results of Robustness Study. Parameter

Value

R

T

tR (min)

pH

4.8 5.0 5.2 0.9 1.0 1.1

4.659 3.208 2.494 4.659 3.208 4.347

1.22 1.52 1.48 1.22 1.52 1.31

3.593 3.483 3.610 4.273 3.483 3.900

Flow rate (mlmin−1)

Recovery (%) 99.73 100.00 100.44 103.1 100.0 97.74

R.S.D. (%) 0.02 0.11 0.03 0.02 0.11 0.01

Fig.-1: Chemical structure of isotretinoin (IST) ISOTRETINOIN (13-CIS RETINOIC ACID)

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Fig.-2: Overlay Chromatogram of Blank and IST

Fig.- 3: Calibration Curve Of Isotretinoin

[RJC-703/2011]

International Journal of Chemical, Environmental and Pharmaceutical Research www.ijcepr.com [Abstracted in : Chemical Abstracts Service , American Chemical Society, USA and CAB(I) , UK] _____________________________________________________________________________________ ijCEPr widely covers all fields of Chemical, Environmental and Pharmaceutical Research.

Manuscript Categories: Full-length paper, Review Articles, Short/Rapid Communications. Manuscripts should be addressed to: Prof. (Dr.) Sanjay K. Sharma Editor-in-Chief 23, ‘Anukampa’,Janakpuri, Opp. Heerapura Power Station, Ajmer Road, Jaipur-302024 (India) E-mail: ijcepr@gmail.com Phone:0141-2810628(O), 09414202678, 07597925412(M) ISOTRETINOIN (13-CIS RETINOIC ACID)

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Vol.4, No.1 (2011), 159-164 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

OPTIMIZATION OF PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO BY BACILLUS SUBTILIS NCIM 2724 USING STATISTICAL EXPERIMENTAL DESIGN Haritha Meruvu and Meena Vangalapati* Center for Biotechnology, Department of Chemical Engineering, College of Engineering, Andhra University, Visakhapatnam–530 003, Andhra Pradesh, INDIA. *E-mail: meena_sekhar09@yahoo.co.in ABSTRACT Proteases are industrially important enzymes which are best produced employing microbial species. This research was an attempt to study the effect of nutritional ingredients on protease production from Bacillus subtilis NCIM 2724 in solid state fermentation using black gram husk, (an agricultural waste) as substrate. Moisture content, Carbon supplement (Maltose) concentration and Nitrogen supplement (Ammonium Chloride) concentration which chiefly effected the production of high protease yields were selected for optimization. Response surface methodology using the Box-Behnken design was used in the design of experiments and in the analysis of results. The maximum productivity of protease under optimum conditions was 712.7792 U/ml. Moisture content 40.88327 %v/w, Maltose concentration 1.61499 %w/w and Ammonium Chloride concentration 2.12112 %w/w was found to be optimum for protease production. Key words: Protease, Bacillus subtilis NCIM 2724, Vigna mungo (Black gram) husk, Optimization, Response surface methodology (RSM), Box-Behnken design. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Protease is an enzyme that conducts proteolysis, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain. Proteases are one of the most important commercial enzymes constituting 60-65% of the global enzyme market 6. They are used in food processing, detergents, diary industry and leather making 2. Proteases occur widely in plants and animals, but commercial proteases are produced exclusively from microorganisms like Aspergillus, Penicillium, Bacillus and Rhizopus genera7. Basically proteolytic enzymes are of significant importance in the current biotechnological era in various industries. Proteases have been used in food industry for centuries especially rennet is obtained from fourth stomach of calves in cheese production and papain for tenderizing meats while processing and canning 7.Alkaline proteases are used to remove hair from hides. Proteases can be used in the treatment of diseases and conditions such as cancer and autoimmune diseases and also immunosuppressive agents. Furthermore, proteases may be used as vaccine adjuvants8. Most commercial serine proteases, mainly neutral and alkaline, are produced by organisms belonging to the genus Bacillus 9. Microbial proteases account to approximately 40% of the total worldwide enzymes sale. In addition, proteases from microbial sources are preferred to the enzymes from plant and animal sources since they possess almost all characteristics desired for their biotechnological applications7. Black gram husk is selected as an apt substrate as it is easily available and is of minimal commercial interest being an agricultural waste11. Moreover it has a suitable texture for solid state fermentation12. The present work aims at a better understanding of the relation between the important independent variables (moisture and C/N concentration) and dependent variable (enzyme yield) to determine optimum conditions for the synthesis of protease enzyme from Bacillus subtilis NCIM 2724. The application of RSM and Box-Behnken design which is an efficient statistical technique for optimization of multiple

PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO

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Vol.4, No.1 (2011), 159-164

variables to predict best performance conditions with minimum number of experiments and the results of its application is discussed in the present work.

EXPERIMENTAL Materials and Methods The microorganism used, Bacillus subtilis(NCIM 2724) was procured from NCIM, Pune. The culture was maintained on nutrient agar medium slant and subcultured for every 21 days. The maintenance medium used for sub culturing is nutrient agar medium with composition as follows: Yeast Extract 1.5 g/l, Beef extract 1.5 g/l, NaCl 5.0 g/l, Peptone 15.0 g/l, Agar 5.0 g/l. Preparation of Inoculum Inoculums are prepared by transferring 2ml of suspension from 24 hour old slant culture into 250 ml Erlenmeyer flasks containing production substrate. Substrate used is Black gram husk which is an agricultural waste10. Commercial quality of black gram husk was procured from the local market and used as the solid substrate for the production of protease. It is added with minimal moisture content and autoclaved at 1210C and 12 lb pressure for 20 minutes to sterile it as well as to soften the hard texture so as the microbe digests the husk easily yielding optimal enzyme production. Production medium and conditions Production medium contained black gram husk 10gms, maltose 1.5gms, ammonium chloride 2.0gms and moisture content of 4 ml. The pH of the medium was adjusted to 7.0 and autoclaved. The production medium was inoculated with 2 ml of homogenous spore suspension (108 CFU/ ml). All fermentations were carried out in 250 ml Erlenmeyer flasks and incubated at 270C.The fermented biomass in each case was filtered and centrifuged. The supernatant was ultra-filtered through filter paper and the filtrate was assayed for protease. Fermentation, Enzyme extraction and Assay Buffer was prepared by mixing 25ml of glycine solution (0.8gms glycine/100ml distilled water), 22.7ml of NaOH solution (1.5gms NaOH/100 ml distilled water) and made up to 100ml with distilled water. 50ml of the prepared buffer was added to the contents of the flask and kept for half an hour of shaking. Filtration was done using Whatmann No.1 filter paper, the filtrate was centrifuged for 15 minutes. The supernatant was collected and added with Caesin (0.3mg casein/100ml distilled water) in 1:6 ratio and incubated at 37 ยบC for 10 minutes. Then 6ml of Trichloroacetic acid solution (24.5gm TCA/100ml distilled water) was added and incubated for 30 minutes, again subjected to centrifugation for 10 minutes. The supernatant was collected and FC reagent (3.3ml FC/10ml distilled water) was added incubated for 30 minutes. The OD Values were taken down at 660 nm. One unit of protease is defined as the quantity of enzyme that liberates one micro mole of tyrosine per minute under the assay conditions 8. Effect of additional nutrients on protease production The effects of various additional nutrients (carbon source and nitrogen source) on protease production were studied by adding to Black gram husk. Maltose and ammonium chloride were added as carbon and nitrogen sources13. Optimization of selected nutrients using RSM Box-Behnken design and RSM were used to optimize the concentrations of these factors (Moisture content, Maltose and ammonium chloride) which resulted from the above studies. The lowest and highest concentrations of selected ingredients were Moisture content, 10 and 60 %w/v; Maltose, 0.5 and 3 %w/w; Ammonium chloride, 0.5 and 3.0 %w/w respectively. Design of RSM experiment RSM consists of a group of empirical techniques devoted to the evaluation of relations existing between a cluster of controlled experimental factors and the measured responses, according to one or more selected criteria. Prior knowledge and understanding of the process variables under investigation is necessary for achieving a realistic model. The range and levels of experimental variables investigated in this study were presented in Table 1. The central values (zero level) chosen for experimental design were: 40 %v/wMoisture content (X1) , 1.5%w/w- Maltose (X2), 2.0 %w/w- Ammonium chloride (X3). The production of PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO

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protease was optimized using Box-Behnken design7 when protease production is related to independent variables by a response equation (1) Y = f (x1, x2, x3, …, xk) The true relationship between Y and xk may be complicated and, in most cases, it is unknown; however a second-degree quadratic polynomial can be used to represent the function in the range interest. k

Y= R0+

k

2

∑.. Ri X i + ∑ Rii. X i + i =1

i =1

k −1.

k

∑.∑. Rij X .i X j + €

(2)

i =1,i < j j = 2

Where X1, X2, X3… Xk are the independent variables which affect the response Y, R0, Ri, Rii and Rij (i=1-k, j=1-k) are the known parameters, € is the random error. A second order model is designed such that variance of Y is constant for all points equidistant from the center of the design. The experimental design chosen for the study was a Box-Behnken design that helps in investigating linear, quadratic and crossproduct effects of these factors, each varied at these levels and also includes three center points for replication8.The design is performed because relations for experimental combination of the variables are adequate to estimate potentially complex response functions. The ‘STATISTICA’ software was used for regression and graphical analysis of the data obtained. The optimum values of the selected variables were obtained by solving the regression equation and also by analyzing the response surface plots5.

RESULTS AND DISCUSSION The extra cellular protease enzyme was obtained from the culture filtrate of Bacillus subtilis and the yield of enzyme depended on various growth conditions. The production of protease by Bacillus subtilis was optimized by response surface methodology with middle range parameters, as it is a powerful technique for testing multiple process variables. Experiments were carried out as per the design and the average protease enzyme activity obtained after 24 hours fermentation with 15 experiments in triplicate from the chosen experimental design are shown in Table 2. The application of RSM4 yielded the following regression equation, which is an empirical relationship between the enzyme yield and test variables in coded units. Y = 450.3 + 62.95 X1+82.64 X1X1 + 46.8 X2 + 51.75 X2X2 + 48.18 X3 + 49.85X3X3 - 8X1X2 - 11.2 X1X3 + 9.12 X2X3 (3) Where Y= enzyme yield, X1, X2, X3 are the coded values of the moisture content, maltose and ammonium chloride concentrations respectively. The calculation of regression analysis gives the value of the determination coefficient (R2 = 0.984) indicates that only 1.6% of the total variations are not explained by the model and the F-value of 159.12 indicates that the protease production by Bacillus subtilis has a good model fit due to the high values of R2 and F. The value of adjusted determination coefficient (Adj. R2 = 0.956) is also very high which indicate a high significance of the model.The regression coefficients, along with the corresponding pvalues for the model were given in Table 3. The p-values are used as a tool to check the significance of each coefficient, which also indicate the interaction strength between each independent variable. The smaller the p-values,more the significance of the corresponding coefficient. Table 4 shows the various critical values obtained for the selected factors. Response surface plots as a function of two factors at a time, maintaining all other factors at fixed levels (zero for instance) are more helpful in understanding both the main and the interaction effects of these two factors. These plots can be easily obtained by calculating from the model and the values taken by one factor where the second varies (from -1.0 to +1.0, step 0.5 for instance) with constraint of a given Y value. The yield values for different concentration of the variable can also be predicted from the respective response surface plots between moisture content and maltose concentration; maltose concentration and ammonium chloride concentration; moisture content and ammonium chloride PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO

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Vol.4, No.1 (2011), 159-164

concentration respectively (Figs. 1-3). The results obtained show that the protease activity is increased to 708.39 U/ml from 694.4 U/ml at the same laboratory conditions, due to optimization using Response surface methodology. Table-1:

The range and levels of experimental variables investigated -1

Coded levels 0

+1

35

40

45

1.0

1.5

2.0

1.5

2.0

2.5

Variables Moisture content (% v/w) X1 Maltose (% w/w) X2 Ammonium Chloride(%w/w) X3

Table-2: The average protease enzyme activity obtained after 24 hours fermentation with 15 experiments in triplicate from the chosen experimental design.

Run No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

X1

X2

X3

Observed protease activity(U/ml)

Predicted protease activity(U/ml)

-1 1 -1 1 -1 1 -1 1 0 0 0 0 0 0 0

-1 -1 1 1 0 0 0 0 -1 1 -1 1 0 0 0

0 0 0 0 -1 -1 1 1 -1 -1 1 1 0 0 0

302.4 448.7 421.7 536.0 311.8 455.7 428.7 527.8 410.8 476.5 490.8 593.0 698.2 694.4 695.4

309.4375 451.3375 419.0625 528.9625 308.6625 456.9625 427.4375 530.9375 406.9000 482.2750 485.0250 596.9000 696.0000 696.0000 696.0000

Table -3: The regression coefficients, along with the corresponding p-values for the model are given Factor Mean/ Intercept X1X1 X1Y X2X1 X2Y X3X1 X3Y X1X2 X1X3

Effect

p-value

Coefficient

450.3250

0.000000

450.3250

125.900 165.2875 93.6250 103.5125 96.3750 99.7125 -16.0000 -22.4000

0.000002 0.000000 0.000007 0.000000 0.000006 0.000000 0.068258 0.023250

62.3500 82.3437 46.3215 51.7563 48.1875 49.3562 -8.0000 -11.2000

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Vol.4, No.1 (2011), 159-164 X2X3

18.2500

0.046522

9.1250

Table -4: The various critical values obtained for the three selected factors.

Factor

Critical value

X1

Observed minimum 35.00000

40.88327

Observed maximum 45.00000

X2 X3

1.00000 1.50000

1.61499 2.12112

2.00000 2.00000

Fig 1: Response surface plots between Moisture content and maltose concentration

Fig 2: Response surface plots between Maltose concentration and Ammonium chloride concentration PROTEASE PRODUCTION FROM HUSK OF VIGNA MUNGO

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Vol.4, No.1 (2011), 159-164

Fig 3: Response surface plots between Moisture content and Ammonium chloride concentration

CONCLUSION The work has demonstrated the use of a Box-Behnken design by determining the conditions leading to the optimum yield of enzyme production. This methodology could therefore be successfully employed to any process (especially with three levels), where an analysis of the effects and interactions of many experimental factors are referred. Box-Behnken designs maximize the amount of information that can be obtained, while limiting the number of individual experiments required. Response surface plots are very helpful in visualizing the main effects and interaction of its factors. Thus, smaller and less time consuming experimental designs could generally suffice the optimization of many fermentation processes.

REFERENCES 1. V. Meena ,A. Sumanjali ,K. Dwaraka , K.M. Subburathinam, K.R.S. Sambasiva Rao, Rasayan Journal of Chemistry,3 (2010). 2. D. Usha Priyanka., Ch. Kanakaraju, A. Sumanjali, K. Dwaraka. V. Meena , International Journal of Chemical Sciences, 8 (2010). 3. S. Negi , R. Banerjee , Food Technol. Biotechnol.,44,257 (2006) 4. R.N. Rahman , P.G. Lee , M. Basri, A.B. Salleh , Enzyme Microb Technol,36,749 (2005) 5. S.C.B.A. Gopinath, T. Hilda, Priya Lakshmi , G.Annadurai and P.Anbu, World J. Microbiol. Biotechnol.,19, 681 (2003). 6. Z.Chi, C.Ma, P.Wang, H.F.Li, Bioresource Technology, 98, 534 (2007). 7. T.J.V. Higgins ,Annu Rev Plant Physiol 35,191, (1984) 8. P. Ellaiah , B. Srinivasulu , K. Adinarayana , J Sci Ind Res ,61,690 ( 2002). 9. W. Mitsuhashi , T. Koshiba , T. Minamikawa , Plant Physiol,80, 628 (1986). 10. Ch. Subba Rao , T. , P. Ravichandra and R.S. Prakasham , Process Biochemistry,44, 262 (2009). 11. R.S. Prakasham ,Ch. Subba Rao , R. Sreenivas Rao and P.N. Sarma , Biotechnology Progress, 21,1380 (2005). 12. L.A. De Azeredo , M.B. De Lima , R.R. Coelho , D.M. Freire , J Appl Microbiol.,100, 641 (2006). 13. S.C.B. A. Gopinath , T. Hilda , Priya Lakshmi , G. Annadurai and P. Anbu, Asian J. Microbiol. Biotechnol. Environ.Sci.,5, 327 (2003). [RJC-708/2011]

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Haritha Meruvu and Meena Vangalapati

Vol.4, No.1 (2011), 165-171 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR ESTIMATION OF TOLVAPTAN IN BULK AND ITS PHARMACEUTICAL FORMULATION V. Kalyana Chakravarthy*and D.Gowri Shankar College of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003(A.P.), India * E-mail: Kalyan224@rediffmail.com ABSTRACT An isocratic reverse phase liquid chromatography (RP-LC) method has been developed and subsequently validated for the determination of Tolvaptan in Bulk and its pharmaceutical formulation. Separation was achieved with an AMCHEMTEQ-USA-ACI C18 (150 mmx4.6 mm I.D; particle size 5 µm)Column and Water: Acetonitrile (40:60) as eluent at flow rate 1.0 mL/min. UV detection was performed at 254nm. The method is simple, rapid, and selective. The described method of Tolvaptan is linear over a range of 37.285 µg/mL to 298.282 µg/mL. The method precision for the determination of assay was below 2.0%RSD. The percentage recoveries of active pharmaceutical ingredient (API) from dosage forms ranged from 99.6 to 101.1. The method is useful in the quality control of Bulk and pharmaceutical formulations. Key Words: Tolvaptan, Estimation, RP-HPLC, Validation, Tablets. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Tolvaptan1-2 is indicated for the treatment of clinically significant hypervolemic and euvolemic hyponatremia (serum sodium < 125 mEq/L or less marked hyponatremia that is symptomatic and has resisted correction with fluid restriction), including patients with heart failure, cirrhosis, and Syndrome of Inappropriate Antidiuretic Hormone . Chemically (±)-4'-[(7-chloro-2, 3, 4, 5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl) carbonyl]-o tolu-mtoluidide (Fig-1). Tolvaptan is a white to off white crystalline powder with a Molecular weight 448.94. Tolvaptan is soluble in benzyl alcohol and methanol, practically insoluble in water and hexane. Tolvaptan melting point was approximately 224°c. Its empirical formula is C26H25ClN2O3. It is not official in any pharmacopoeia, few liquid chromatography procedures have been reported for the determination of Tolvaptan3-4 .The author have developed a liquid chromatographic method which would serve as a rapid and reliable method for the determination of Tolvaptan in Bulk and pharmaceutical dosage forms.

EXPERIMENTAL The Experimental5-7 involves the followingsInstrumentation The analysis of the drug was carried out on a waters LC system equipped with 2695pump and 2996 photodiode array detector was used and a Reverse phase HPLC column AMCHEMTEQ-USA-ACI C18 (150 mmx4.6 mm I.D; particle size 5 µm) was used. Chemicals and solvents The HPLC Grade water (Millipore) and Acetonitrile HPLC Grade from E. Merck (India) Ltd., Mumbai. Mobile phase preparation Prepare a filtered and degassed mixture of Water and Acetonitrile in the ratio 400:600 v/v respectively. Standard preparation: (For Tolvaptan tablets 15mg & 30mg) Accurately weigh and transfer about 30.0mg of Tolvaptan working standard into a 200 mL volumetric flask, add 120 mL of mobile phase and sonicate to dissolve. Cool the solution to room temperature and dilute to volume with mobile phase.

ESTIMATION OF TOLVAPTAN IN BULK

V. K. Chakravarthyand D.G. Shankar

Vol.4, No.1 (2011), 165-171

Sample preparation: (For Tolvaptan tablets 15mg) Transfer 5 tablets (equivalent to 75 mg of Tolvaptan) into a 250 mL volumetric flask add about 100 mL of mobile phase sonicate for 20minutes with occasional shakings. Cool the solution to room temperature and dilute to volume with mobile phase. Filter the solution through 0.45um Filter. Transfer 5.0 mL of the filtered solution into a 10 mL volumetric flask and dilute to volume with mobile phase. Sample preparation: (For Tolvaptan tablets 30mg) Transfer 5 tablets (equivalent to 150 mg of Tolvaptan) into a 250 mL volumetric flask add about 100 mL of mobile phase, sonicate for 20minutes with occasional shakings. Cool the solution to room temperature and dilute to volume with mobile phase. Filter the solution through 0.45um Filter. Transfer 5.0 mL of the filtered solution into a 20 mL volumetric flask and dilute to volume with mobile phase. Chromatographic conditions: An AMCHEMTEQ-USA-ACI C18 (150 mmx4.6 mm I.D; particle size 5 µm) column was used for analysis at column temperature 25°C. The mobile phase was pumped through the column at a flow rate of 1.0mL/min. The sample injection volume was 10 µL. The photodiode array detector was set to a wavelength of 254nm for the detection and Chromatographic runtime was 10minutes.

RESULTS AND DISCUSSION Method development5-7 To develop a suitable and robust LC method for the determination of Tolvaptan in different columns and flow rates were employed to achieve the best separation and resolution. The method development was started with AMCHEMTEQ -USA-ACI C18 (250 mmx4.6 mm I.D; particle size 5 µm) column with flow rate of 1.5ml/minute. The retention time of Tolvaptan was 5.224minutes. For further reducing the runtime short column was used with same flow rate AMCHEMTEQ -USA-ACI C18 (150 mmx4.6 mm I.D; particle size 5 µm). The chromatogram of Tolvaptan standard using the proposed method is shown in Fig2. System suitability results of the method are presented in Table-1. Tolvaptan show significant UV absorbance at Wavelength 254nm. Hence this wavelength has been chosen for detection in analysis of Tolvaptan. Column selection Based on the retention and better peak shape of the compound AMCHEMTEQ-USA-ACI C18 (150 mmx4.6 mm I.D; particle size 5 µm) column was selected as suitable column for analysis of Tolvaptan. Method validation6-7 The developed LC method extensively validated for assay of Tolvaptan using the following parameters. Specificity Blank and Placebo interference: A study to establish the interference of placebo was conducted. Assay was performed on placebo in triplicate equivalent to about the weight of placebo in portion of test preparation as per test method. Chromatograms of Blank and Placebo solutions showed no peaks at the retention time of Tolvaptan peak. This indicates that the excipients used in the formulation do not interfere in estimation of Tolvaptan in Tolvaptan tablets. The chromatogram of Tolvaptan Blank and Placebo using the proposed method is shown in Fig- 3 & Fig-4. Linearity of detector response Linearity of detector response was established by plotting a graph to concentration versus average area and determining the correlation coefficient. A series of solutions of Tolvaptan standard were prepared in the concentration range of about 37.285 µg/mL 298.282 µg/ mL. A graph was plotted to concentration in µg/mL on X-axis versus response/Area on Y-axis. The detector response was found to be linear with a correlation coefficient of 0.9999. Linearity graph is shown in Fig-5. Linearity results of the method are presented in Table-2. Precision of test Method The precision of test method was conducted by assay in six samples of Tolvaptan dispersible tablets. The average % assay of Tolvaptan in Tolvaptan tablets was found to be 100.0, 100.2, for 15 & 30 mg tablets respectively and the %RSD is 0.8 and 0.6%. The results were given in Table-3. A typical LC Chromatogram is shown in Fig-6. Accuracy ESTIMATION OF TOLVAPTAN IN BULK

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Vol.4, No.1 (2011), 165-171

A Study of recovery of Tolvaptan from spiked placebo was conducted at six different spike levels i.e.25, 50, 75,100, 125 and 150%. Samples were prepared by mixing placebo with Tolvaptan raw material equivalent to about the target initial concentration of Tolvaptan. Sample solutions were prepared in triplicate for each spike level and assayed as per proposed method. The % recovery was given in Table-4. The mean recoveries of Tolvaptan from spiked were found to be in the range of 99.6-101.1%. Ruggedness A study to establish the stability of Tolvaptan in standard and test solutions were conducted on bench top and refrigerator at Initial, 1 day and 2 day. The assay of Tolvaptan in standard and test solutions were estimated against freshly prepared standard each time. The difference in% assay of standard and test solutions from initial to 1 day and 2 days was calculated and given in Table-5(Bench Top) and Table6(Refrigerator). From the above study, it was established that the Standard and sample preparations are stable for a period of 48hours at room temperature (25°C±2°C) and at refrigerator condition (2°C-8°C). Robustness A study to establish the effect of variation in mobile phase composition, flow, and Temperature was conducted. Standard and test solutions prepared as per proposed method were injected into HPLC system. The system suitability parameters and % assay were evaluated. From the above study the proposed method was found to be robust.

Fig-1: Chemical Structure of Tolvaptan

Fig-2: HPLC Chromatogram of Tolvaptan Standard.

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Vol.4, No.1 (2011), 165-171

Fig-3: HPLC Chromatogram of Tolvaptan Blank

Fig-4: HPLC Chromatogram of Tolvaptan placebo.

Fig-5: Linearity of detector response graph.

ESTIMATION OF TOLVAPTAN IN BULK

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Vol.4, No.1 (2011), 165-171

Fig-6: Typical LC chromatogram of Formulated Tolvaptan tablets 30mg. Table-1: System suitability report Compound

Tolvaptan

Retention Time * (min.) 4.505

Tolvaptan area/response*

USP Tailing*

USP Plate count*

%RSD*

3242491

1.01

6929

0.70

*Number of standard injections analysed are six. Table-2: Linearity Table Report Concentration(mcg/ml) % Level 25 50 75 100 125 150 200

37.285 74.571 111.856 149.141 186.427 223.712 298.282

AREA

y-Best fit

(Difference)2

795865 1633992 2426409 3260276 4073278 4886416 6435768

820884 1628434 2435962 3243490 4051040 4858568 6473625

625950401 30892592 91262101 281758031 494518008 775484949 1433147188

Correlation Coefficient (R)= Regression Coefficient (R2)= y-Intercept= Slope of Regression line= Residual Sum of squares= Minimum = Maximum = y-Intercept at 100%level

0.9999 0.9998 13356 21658 3733013271.0 37.285 298.282 0.4

Table-3: Results for precision of test method Sample No 01

%Assay (15.0mg) 100.1

%Assay (30.0mg) 99.9

02

99.3

101.1

03

99.4

99.8

04

101.5

100.4

05

99.7

99.5

06

100.2

100.6

Average

100.0

100.2

ESTIMATION OF TOLVAPTAN IN BULK

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Vol.4, No.1 (2011), 165-171 SD

0.8042

0.5913

% RSD

0.8

0.6

Table-4: Accuracy in the assay determination of Tolvaptan Sample No.

Spike level

‘mcg/mL’ added

‘mcg/mL’ found (recovered)

% of Recovery

1.

25%

35.8354

36.4123

101.6

2.

25%

35.8196

36.4098

101.6

3.

25%

35.8544

35.9250

100.2

4.

50%

74.2749

74.7582

100.7

5.

50%

74.2759

74.1683

99.9

6.

50%

74.2769

73.6868

99.2

7.

75%

111.2625

111.0127

99.8

8.

75%

111.3614

110.2889

99.0

9.

75%

111.5592

111.4772

99.9

10.

100%

148.3500

149.1637

100.5

11.

100%

148.4489

148.7906

100.2

12.

100%

148.5478

149.6594

100.7

13.

125%

185.4375

186.3601

100.5

14.

125%

185.1408

186.2818

100.6

15.

125%

185.0419

184.4787

99.7

16

150%

222.6239

223.5626

100.4

17

150%

222.7228

223.5033

100.4

18

150%

222.8217

223.5310

100.3

Mean % recovery

101.1

99.9

99.6

100.5

100.3

100.4

Table- 5: Bench top Stability of Tolvaptan Test preparation and Standard Preparation: Time

% Assay of Standard preparation

Difference

% Assay of test preparation

Test-1

Test-2

Difference

Test-1

Test-2

Initial

98.9®

NA*

100.0

101.2

NA*

NA*

After 24 hours

99.3

0.4

101.0

102.0

1.0

1.2

After 48 hours

99.9

1.0

101.5

102.5

1.5

1.3

NA*----Not Applicable, ®--------Potency of Tolvaptan on as is basis.

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Vol.4, No.1 (2011), 165-171 Table- 6: Refrigerator Stability of Tolvaptan Test preparation and Standard Preparation: Time

% Assay of Standard preparation

Difference

% Assay of test preparation

Test-1

Test-2

Difference

Test-1

Test-2

Initial

98.9®

NA*

100.0

101.2

NA*

NA*

After 24 hours

99.1

0.2

100.8

101.7

0.8

0.5

After 48 hours

99.8

0.9

101.2

101.9

1.2

0.7

NA*----Not Applicable, ®--------Potency of Tolvaptan on as is basis.

CONCLUSION The proposed HPLC method is rapid, sensitive, precise and accurate for the determination of Tolvaptan and can be reliably adopted for routine quality control analysis of Tolvaptan in Bulk and its pharmaceutical formulations.

ACKNOWLEDGEMENTS The Authors are thankful to M/s Natco Pharma LTD., Hyderabad for providing a reference sample of Tolvaptan.

REFERENCES 1. www.rxlist.com 2. www.chembink.com 3. Susan E. Shoaf, Zhao Wang, Patricia Bricmont, and Suresh Mallikaarjun, The Journal of Clinical Pharmacology, 2004. (LC/MS/MS) 4. www.ema.europa.eu(Tolvaptan)-Public assessment report, 2009. 5. Practical HPLC Method Development Second Edition Lloyd R. Snyder, Joseph J.Kirkland, Joseph I.Glajch. United States Pharmacopeia USP 34-NF 29, 2011. 6. ICH Guidelines on Validation of Analytical procedure: Text and Methodology Q 2 (R1). [RJC-726/2011]

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ESTIMATION OF TOLVAPTAN IN BULK

171

V. K. Chakravarthyand D.G. Shankar

Vol.4, No.1 (2011), 172-179 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

DENSITY, VISCOSITY AND ACTIVATION PARAMETERS OF VISCOUS FLOW FOR CETRIMIDE IN ETHANOL + WATER SYSTEM AT 301.5 K Muktar Shaikh1,Mohd. Shafique2, B. R. Agrawal3 and Mazahar Farooqui 4,* 1

Shri Anand College, Pathardi, Dist-Ahmednagar (MS)India 2 Milind Science College, Aurangabad( M S) India 3 J.E.S. College, Jalna (M S) India 4 Post Graduate and Research Centre, Maulana Azad College, Aurangabad(MS) India *E-mail: mazahar_64@Rediffmail.com. ABSTRACT Density and Viscosity of drug Cetrimide (CMD) in various aqueous mixtures of ethanol have been determined. These results are further extended for solutes like electrolyte NaCl and non-electrolyte sucrose in the presence of this drug. The density and viscosity data have been analysed for the evaluation of partial molar volume, molar excess volume, Gibbs free energy of viscous flow, excess viscosity and A and B viscosity coefficients using JonesDole equation. It can be inferred from these studies that this drug acts as a structure-making compound due to hydrophobic hydration of drug molecules. B-coefficients values are found to be positive thereby showing drug solvent interactions. Furthermore these results are correlated to understand the solution behavior of drug. Keywords: Cetrimide, Partial molar volume, Gibbs free energy of viscous flow, Excess parameters. Š 2011 RASÄ&#x20AC;YAN. All rights reserved.

INTRODUCTION A systematic knowledge of solution behavior of drugs can be of great importance in order to understand their physiological action1 The thermodynamic properties are the convenient parameter for interpreting solute-solvent interactions in the solution phase, which ultimately explain the excess properties using different interaction parameters.Most of the drugs are organic molecules with both hydrophobic and hydrophilic groups. These molecules often contain certain groups, which are responsible for their acidic, basic or amphoteric properties. Pharmacological properties2,3 of drugs are highly dependent on the solution behavior.In the present communication an attempt has been made to study density and viscosity measurements of Cetrimide in aqueous ethanol to investigate various types of interactions.

EXPERIMENTAL Materials The binary solvent selected for the study was ethanol + water. Commercial ethyl alcohol is refluxed with CaO for six to eight hours and distilled4. Double distilled water is used for preparation of solution mixture. The distillation of water was carried out using a pinch of KMnO4 & KOH in glass quick fit apparatus. The density and viscosity of water and ethanol are measured at 298.15 K and 303.15 K and compared with literature values. Apparatus and procedure Densities of liquids and various solutions were measured at 301.5K by using specific gravity bottle of 10 cm3 capacity. A single pan electronic balance [Sansui; model KD-UBED of capacity 120 gm and with a precision of 0.0001 gm] was used for weighing purpose. The weighing was repeated thrice to ensure the accuracy in weights with a little interval of time. The reproducibility of the result was close to hundred percent.

VISCOUS FLOW FOR CETRIMIDE

Muktar Shaikh et al.

Vol.4, No.1 (2011), 172-179

Viscosity measurements were carried out using Ostwald’s viscometer with precision ± 0.1 %. The viscometer was clamped vertically in a thermostatically controlled water-bath, whose temperature was maintained constant at 301.5K (± 0.02°C). A fixed volume (10ml) of the solution was delivered into the viscometer. The viscometer was kept for 30 minutes in the thermostatically controlled water-bath to achieve constant temperature. The experiments of measurements of flow time of the solution between the two points on the viscometer were performed at least three times for each solution and the average results were noted.

RESULTS AND DISCUSSION

Structure of Cetrimide Cetrimide is chemically Alkyl tri-methyl ammonium bromide. It is white crystalline powder, free flowing hygroscopic powder. It has faint characteristic odour and bitter soapy taste. It is slightly soluble in water. The densities and viscosities of ethanol- water binary mix from 20 % to 100 % range are measured (table 1 and 2) and used for determination of partial molar volume. The partial molar volume Φv was obtained from density results using equation 1.

φV =

1000  d 0 d  M  + c  d  d

(1) Where do is the density of pure solvent & d is the density of solution, c is molar concentration, M is molar mass of drug. Cetrimide is a salt of alkyl ammonium halide which has bulkier positive ion and negative bromine ion. It is observed that the Фv values decreases with concentration of CMD and increases with increase in percentage of alcohol (table3). The density data was also used to evaluate excess molar volumes (table 3) calculated by using the relation (equation 2).

xM +x M  xM x M VE = 1 1 2 2 − 1 1 − 2 2 ρ ρ1 ρ2  

(2)

Where, ρ is the density of mixture, M1, X1, V1 and M2, X2 & V2 are the molecular weight, mole fraction and molar volumes of ethanol & water respectively. The excess molar volumes calculated for CMD in absence and in presence of additives are negative (Fig 2). We observed that VE changes parabolically in negative direction with % of ethanol. Over all range of concentration it was observed that VE values are less in absence of additives and slightly high when NaCl is used. But in presence of sucrose these values are higher in negative direction. This indicates more solubility of it in the mixture. The parabolic shape of VE against % ethanol is characterized by well defined minima which indicate the presence of complex form between mixing components of solution. The partial molar volume of mixture containing CMD with and without additives is shown in table 3. These values are positive and changes in parabolic way with the % of ethanol (Fig.1). The higher values VISCOUS FLOW FOR CETRIMIDE

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of excess viscosities of non-electrolyte in binary system may be due to the presence of larger alkyl chain of CMD. Viscosity is found to maximum at around 50% (V/V) in aqueous mixtures of alcohol. It seems that some kind of structural organization of water surrounding the hydrocarbon chain of alcohol is the most likely explanation of the observed dependence of viscosity on solvent composition. The measured values of viscosities of liquid mixtures and those of pure components were used to calculate the excess viscosity ηE(table 4) in the liquid mixtures using the formula (equation 3), ηE = ηmix –( x1η1 - x2η2)

(3)

Where, ηmix, η1 & η2 are the viscosities of liquid mixtures, component 1 & 2 respectively and x1 & x2 are the mole fractions of component 1 & 2 respectively. The hydrocarbon residue of CMD in alcohol results in a considerable amount of hydrophobic hydration. However the further decrease of excess viscosity with increase in percentage alcohol may result because these hydrophobic groups exerts there effect predominantly with increase in alcohol percent (Fig 3). It appears that above 60 % v/v alcohol concentration a loss of hydrophobic hydration takes place which leads to decrease in excess viscosity5. The excess Gibb’s free energy of activation for viscous flow, ∆GE was calculated (table 5) for all the system under study using the equation6,7: ∆G*E = RT {ln( ηV/η2V2)-x1 ln (η1V1/η2V2)}

(4)

Table-1: Density ρ (g cm–3) of CMD in binary system at 301.5 K. v/v % Et-OH

0.02 M

0.04 M

0.06 M

0.08M

0.10 M

20 40 60 80 100

0.9998 0.9757 0.9492 0.9097 0.8176

1.0066 1.0134 0.9824 0.9891 0.9560 0.9624 0.9169 0.9234 0.8244 0.8313 ρ of CMD +0.01 M NaCl

1.0201 0.9956 0.9694 0.9302 0.8380

1.0266 1.0024 0.9760 0.9366 0.8445

v/v % Et-OH 20 40 60 80 100

v/v % Et-OH 20 40 60 80 100

0.02 M

0.04 M

1.0008 0.9760 0.9496 0.9105 0.8184

0.08M

0.10 M

1.0071 1.0137 0.9828 0.9897 0.9567 0.9630 0.9170 0.9241 0.8253 0.8319 ρ of CMD +0.01 M Sucrose

1.0207 0.9963 0.9697 0.9309 0.8381

1.0271 1.0030 0.9769 0.9370 0.8454

0.02 M

0.04 M

0.06 M

0.08M

0.10 M

1.0034 0.9791 0.9526 0.9134 0.8212

1.0101 0.9856 0.9591 0.9203 0.8278

1.0168 0.9925 0.9662 0.9267 0.8347

1.0236 0.9993 0.9728 0.9334 0.8411

1.0301 1.0058 0.9791 0.9407 0.8482

VISCOUS FLOW FOR CETRIMIDE

0.06 M

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410

390 380

-1.5

Molar excess volume VE

Partial Molar volume (φν)

400

-1.0

0.02 M 0.04 M 0.06 M 0.08 M 0.10 M

370 360 350 340 330

-2.0 -2.5 -3.0 -3.5 -4.0 -4.5

320 -5.0

0.02 M 0.04 M 0.06 M 0.08 M 0.10 M

310 20

40

60

80

100

20

40

%of Et-OH

60

80

100

%of Et-OH

Fig.-1: Variation of Фv with % ethanol

Fig.-2: Variation of VE with % ethanol

1.8 150 140

1.6

130

Gibbs free energy

Excess Viscosity

1.4 1.2 1.0 0.8 0.6 0.4

0.02 M 0.04 M 0.06 M 0.08 M 0.10 M

120

0.02 M 0.04 M 0.06 M 0.08 M 0.10 M

110 100 90 80 70 60 50

20

40

60

80

100

20

%ofEt-OH

60

80

100

%of Et-OH

Fig.-3: Variation of ηE with % ethanol

VISCOUS FLOW FOR CETRIMIDE

40

Fig.-4: Variation of ∆G*E with % ethanol

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Vol.4, No.1 (2011), 172-179 Table-2: Viscosity η (m Pa. s) of CMD in binary system at 301.5 K. v/v % Et-OH 20 40 60 80 100

v/v % Et-OH 20 40 60 80 100

v/v % Et-OH 20 40 60 80 100

0.02 M

0.04 M

1.7138 2.3449 2.4337 2.0475 1.3774

0.06 M

0.08M

0.10 M

1.7186 1.7296 2.3647 2.3852 2.4433 2.4433 2.0861 2.1464 1.3946 1.4138 η of CMD +0.01 M NaCl

1.7368 2.3919 2.4643 2.2158 1.4239

1.7498 2.4126 2.5059 2.3245 1.4566

0.02 M

0.04 M

0.08M

0.10 M

1.7344 2.3542 2.4714 2.0986 1.4017

1.7386 1.7478 2.3871 2.3847 2.4937 2.5021 2.1287 2.2263 1.4166 1.4283 η of CMD +0.01 M Sucrose

1.7588 2.4257 2.5214 2.3382 1.4685

1.7686 2.4437 2.5375 2.3583 1.4877

0.02 M

0.04 M

0.06 M

0.08M

0.10 M

1.7508 2.4101 2.5821 2.1875 1.4318

1.7846 2.4328 2.6041 2.3237 1.4499

1.7953 2.4443 2.6247 2.3637 1.4674

1.8165 2.4784 2.6476 2.4812 1.5654

1.8296 2.5249 2.6682 2.5029 1.5865

0.06 M

Table-3a: Фv in cm3 mol-1 and VE in cm3 mol-1 of CMD in binary mixture at 301.5 K V/V % Et- OH 20 40 60 80 100 V/V % EtOH 20 40 60 80 100

0.02 M (CMD) Фv VE 334.2612 -0.9770 342.3565 -1.6685 351.9199 -2.6479 367.1707 -3.7616 408.1542 -2.5162 0.08 M (CMD) Фv VE 322.0300 -1.3751 329.9654 -2.1348 338.5922 -3.2371 352.3371 -4.5718 390.1187 -3.8858

VISCOUS FLOW FOR CETRIMIDE

0.04 M (CMD) Фv VE 330.6987 -1.1121 338.8955 -1.8276 348.1900 -2.8490 362.7960 -4.0503 402.9985 -2.9803 0.10 M (CMD) Фv VE 318.8008 -1.4992 326.1344 -2.2899 334.7944 -3.4243 348.3971 -4.8175 385.0027 -4.3083

176

0.06 M (CMD) Фv VE 325.5738 -1.2455 333.3433 -1.9845 342.5476 -3.0357 356.6296 -4.3071 395.2026 -3.4434

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Vol.4, No.1 (2011), 172-179 Table-3b: Фv in cm3 mol-1 and VE in cm3 mol-1 of (CMD) +0.01 M NaCl in binary mixture at 301.5 K V/V % Et- OH 20 40 60 80 100 V/V % Et- OH 20 40 60 80 100

0.02 M (CMD) Фv VE 58.3349 -0.9984 59.8586 -1.6761 61.5158 -2.6604 64.1281 -3.7953 71.3378 -2.5729 0.08 M (CMD) Фv VE 57.2212 -1.3898 58.6158 -2.1548 60.2474 -3.2474 62.7308 -4.6034 69.7208 -3.8932

0.04 M (CMD) Фv VE 57.9992 -1.1234 59.4386 -1.8382 61.0403 -2.8717 63.7226 -4.0546 70.7334 -3.0448

0.06 M (CMD) Фv VE 57.6331 -1.2525 59.0124 -2.0010 60.6475 -3.0558 63.1920 -4.3379 70.1982 -3.4869

0.10 M (CMD) Фv VE 56.8704 -1.5120 58.2303 -2.3076 59.7668 -3.4561 62.3426 -4.8359 69.0534 -4.3750

Table-3c: Фv in cm3 mol-1 and VE in cm3 mol-1 of (CMD) +0.01 M sucrose in binary mixture at 301.5 K V/V %

0.02 M (CMD)

Et- OH

Фv

V

20

339.6183

40

0.04 M (CMD) Фv

V

-1.0539

337.4072

348.0874

-1.7545

60

357.7360

80 100 V/V %

0.06 M (CMD) Фv

VE

-1.1906

335.2246

-1.3252

345.8701

-1.9120

343.4039

-2.0778

-2.7541

355.4317

-2.9492

352.5719

-3.1624

372.9085

-3.9168

370.2450

-4.1963

367.7373

-4.4521

414.6380

-2.7704

411.4464

-3.2234

408.0591

-3.6893

E

0.08 M (CMD)

E

0.10 M (CMD) Фv

VE

-1.4607

330.8785

-1.5882

340.9724

-2.2401

338.8784

-2.3899

60

350.3338

-3.3538

348.1944

-3.5337

80

365.1463

-4.7155

361.9745

-5.0052

100

405.1118

-4.1126

401.4479

-4.5816

Et- OH

Фv

V

20

332.9724

40

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E

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Vol.4, No.1 (2011), 172-179 Table-4: Excess Viscosities (ηE) : - (mPas) V/V % Et- OH 0.02 M

20 40 60 80 100

0.9965 1.6029 1.6553 1.2101 0.4278

0.04 M 0.06 M Ethanol-water + (CMD) 1.0013 1.6227 1.6649 1.2487 0.4450

1.0123 1.6432 1.6649 1.3090 0.4642

0.08 M

0.10 M

1.0195 1.6499 1.6859 1.3784 0.4743

1.0325 1.6706 1.7275 1.4871 0.5070

Ethanol-water + (CMD) + 0.01 M Sucrose 1.0673 1.0780 1.0992 1.6908 1.7023 1.7364 1.8257 1.8463 1.8692 1.4863 1.5263 1.6438 0.5003 0.5178 0.6158 Ethanol-water + (CMD) + 0.01 M NaCl 1.0171 1.0213 1.0305 1.0415 1.6122 1.6451 1.6427 1.6837 1.6930 1.7153 1.7237 1.7430 1.2612 1.2913 1.3889 1.5008 0.4521 0.4670 0.4787 0.5189

20 40 60 80 100

1.0335 1.6681 1.8037 1.3501 0.4822

20 40 60 80 100

1.1123 1.7829 1.8898 1.6655 0.6369 1.0513 1.7017 1.7591 1.5209 0.5381

Table-5: Gibbs free energy of viscous flow ∆G*E( kJ mole-1) V/V % Et- OH

0.02 M

0.04 M

0.06 M

0.08 M

0.10 M

71.7183 111.6320 125.9009 128.9671 78.8335

79.4632 123.7697 139.9014 147.3278 88.8280

Ethanol-water + CMD

20 40 60 80 100

49.8205 76.2546 89.0447 88.0297 58.0153

56.9725 88.1738 101.3579 100.1506 64.9408

64.4216 100.1813 113.2090 114.0186 72.3814

Ethanol-water + CMD+ 0.01 M Sucrose 20 40 60 80 100

54.8941 84.1806 100.7296 100.7988 66.9803

63.8249 96.6347 114.5176 119.7808 74.7317

71.8270 108.6055 128.2735 133.8930 82.6047

80.6010 121.8006 142.1723 153.4482 102.7416

89.0034 135.9764 155.9491 166.4854 112.2277

Ethanol-water + CMD + 0.01 M NaCl 20 40 60 80 100

51.2205 77.5489 91.4752 91.6276 61.2662

VISCOUS FLOW FOR CETRIMIDE

58.5486 90.0361 104.5489 103.6846 68.1897

66.0688 101.0942 117.1292 120.2316 74.8514 178

75.6477 117.3757 133.3747 141.6300 87.5429

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Vol.4, No.1 (2011), 172-179

Table-6: A and B coefficient values V/V % t 20

A 2.3836

(CMD) B 0.0727

(CMD) +0.01 Sucrose A B 2.9281 0.1137

(CMD) +0.01 NaCl A B 2.5961 0.0864

40

2.5738

0.0808

3.2013

0.1359

2.7248

0.0962

60

2.7950

0.0912

3.3791

0.1452

3.0465

0.1062

80

3.0238

0.1007

4.3651

0.1714

3.5627

0.1257

100

3.5014

0.1192

4.4359

0.1844

3.6909

0.1341

Where, η , η1 & η2 are the viscosities of liquid mixtures, components 1 & 2 respectively, V1 & V2 are the molar volumes of components 1 & 2 respectively, x1 is the mole fraction of first component, R is the gas constant & T is the absolute temperature. The values are positive and changes parabolically with percentage of ethanol (Fig 4). The maximum Gibbs free energy of viscous flow for ethanol-water is 75.4598 KJ mole-1. The trend in maxima shows that ∆G*E for NaCl is less than ∆G*E for sucrose. This excess free energy increases with increase in concentration of CMD keeping concentration of additive const. The values of ∆G*E is much higher when CMD and additives are present together. The positive values of ∆G*E represent the size effect of mixing components. It is considered that if ∆G*E is positive there are specific interactions like hydrogen bonding which exists between molecules of mixture8. B coefficient values are calculated using Jones-Dole equation (5) ηr =1+A √c + B The values of A & B are determined (table 6) from the intercept & slope of the lines of plots of (η/ ηo -1) verses √c. We observed positive values for all the systems. The B coefficient for CMD in absence of additives is less and in presence of non-electrolyte sucrose is more. This increases with increase in percentage of alcohol which indicates the structural increases of solution from water to alcohol. The B coefficient of CMD solution reflects the net structural effects of polar groups and hydrophobic benzene ring.

REFRENCES 1. S. Chauhan, V.K. Syal, M.S. Chauhan, Poonam Sharma, J. Mol. Liquid, 136, 161 (,2007) 2. K. D. Treepathi, “Essentials of Medical Pharmacology”, 4th ed., Jaypee Brothers Medical Pub (P) Ltd, New Delhi, (1999). 3. V.K. Sayal, S. Chavan and P. Sharma, J. Indian Chem. Soc, 82, 602 (2005). 4. J. D. Lee, ELCH, Publication London 4th edition,(1991). 5. R. L. Bokhra and M. L. Parmar., Aust. J. Chem., 27, 1407, (1974) 6. R. Gopal, D. K. Agarwal and Rajendra Kumar, J. Phy. Chem., 84, 141,(1974) 7. R. J. Bearman and P. F. Jones, J. Chem. Phy. 33, 1432, (1960) 8. M. Javed Iqbal, M. Ahmed Chaudhry, J. Chem Thermodyn. 41, 221(2009) [RJC-701/2011]

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Vol.4, No.1 (2011), 180-188 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

MODELING ISOLATION OF AMARANTH PROTEIN BY ENZYMATIC BREAKDOWN OF POLYSACCHARIDES

1

Pavel Mokrejs1,*, Dagmar Janacova2, Karel Kolomaznik2 and Petr Svoboda1

Tomas Bata University, Faculty of Technology, Department of Polymeric Engineering, nam. TGM 275, 762 72 Zlin, the Czech Republic 2 Tomas Bata University, Faculty of Applied Informatics, Institute of Processing Control and Applied Computer Science, Nad Stranemi 4511, 760 05 Zlin, The Czech Republic *E-mail: mokrejs@ft.utb.cz ABSTRACT This article deals with technology of biochemically separating proteins from amaranth. Technology we examined was enzymatic degradation of polysaccharides from amaranth flour in order to liquefy starch by hydrolysing it into soluble glucose, and to enrich the solid phase with vegetable protein. Three specific enzymes (amylase, glucoamylase and cellulase) were chosen to this purpose. Measured data were mathematically processed applying the mechanism of first-order kinetics in relation to non-decomposed starch. From processed experimental data it followed that the level of activation energy of starch hydrolysis is 9.7 x 104 J/mol and frequency factor is 29 min-1. Separated amaranth components valuable for health (proteins, liquid proteins and sugars, oil, fibre) may serve to prepare functional foodstuffs and quality supplements, cosmetics, as an ingredient in animal feed or as a source of biological fertilisers. Key words: Amaranth, Biochemical isolation, Enzyme; Protein, Starch. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Amaranth grain, depending on strain, has very small dimensions (0.6 to 2.1 mm), low mass (approx. 1,000 seeds / g), and whitish to brown colour. Shape of grain is lenticular, under a tough husk is a sprout curled along the fringe about the plain of greatest circumference, and that takes up a third of grain volume and encircles the perisperm1. Reserve nutrients (lipids, proteins, polysaccharides, organic phosphates as well as a number of inorganic components) are not evenly stored in the grain. Proteins, bound to cells of embryo and endosperm, are stored in a membrane. The lipids are also found there. On the contrary, polysaccharides appear as starch in perisperm (seed) and are not found in endosperm2. Amaranth grain may yield as much as 18 % high-quality protein possessing a very well balanced composition of essential amino acids. Protein content in amaranth grains is higher, as opposed to proteins of current cereals. The higher content of lysine and tryptophane is comparable to that in animal proteins. As much as 65 % protein in amaranth is concentrated in the sprout. Protein content varies depending on amaranth species and cultivation conditions3. Starch content in amaranth ranges from 48 % (Amaranthus cruentus) to approx. 62 % (Amaranthus hypochondriacus). It was microscopically determined that minute grains of starch from amaranth grain are very small, of 1–3 µm diameter, angular polygonal shape. Starch bonds very strongly but is highly sensitive to action by amylases. Compared to wheat starch, it displays lower amylose content, lower swelling, higher solubility, greater water reception, lower and higher gelation temperature range. The very small size of minute starch grains and residual activity of amylases is probably responsible for recorded differences in swelling strength and solubility4. Amaranth grain contains about 3–6 % coarse fibre and as much as 15 % dietetic fibre. Thus, its fibre content is markedly greater as compared to other cereals. Dietetic fibre reduces cholesterol level, reduces the hazard of cancer of the colon and rectum. Coarse fibre is important for preventing and curing constipation. The recommended daily reception of fibre for adults is 30–40 g while the ratio of insoluble and soluble component should be 3:15. Total fat content of cereal amaranth is 5.4–17.0 % containing almost 50 % linolenic acid. Fat contains 6–8 % squalene, which is a natural compound of isoprenoid type, a precursor in the synthesis of

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Vol.4, No.1 (2011), 180-188

steroids and important antioxidant compounds (coenzyme Q10). Squalene inhibits formation of oxygen radicals very efficiently and is a significant means of preventing tumorous diseases, brain damage or atherosclerosis6, 7. Kinetics of enzymatic reactions Usual enzymatic reaction involves a case in which component S (substrate) reacts with component En (enzyme) in a homogeneous environment in such manner that component En is reclaimed, so that on termination of the reaction its concentration e = ei 8:

k1 k-1

S + En

[EnS]

k2

P + En (1)

The rate at which final product (P) originates is expressed by relationship:

r=

dp = k2c dt

(2)

The rate of enzymatic reactions is governed by kinetics of saturation type, that is, rate of action first increases with growing concentration of substrate in almost linear manner, but at higher substrate concentrations the increase in rate is lower and reaction rate slowly approaches a maximum level (Vmax). Such behaviour is brought about by the active centre of enzyme being able to process only a certain maximum quantity of substrate molecules in a unit of time. The rate of forming the product is directly proportional to concentration of complex EnS:

V = K [En S ]

(3)

Concentration of complex EnS is a result of equilibrium, where dissociation constant is:

Km =

[En ][S ] [En S ]

(4)

Dissociation constant Km depends on physico-mechanical parameters of the system, particularly on temperature and pH. At constant concentration of enzyme (as of catalyst that does not â&#x20AC;&#x153;participateâ&#x20AC;? in the reaction nor is consumed therein), the resultant rate of product P yield is described by means of the well-known Michaelis-Menten equation9:

V=

Vmax [S ] K m + [S ]

(5)

Constant Km in this relationship is designated the Michaelis-Menten constant; it is physically equal to substrate concentration at which enzymatic reaction rate V equals just half the maximum rate Vmax which is the rate when enzyme is saturated with substrate. The numerical value of constant Km expresses affinity of enzyme to given substrate. Low values of this constant signify that enzyme is highly specific to the given substrate and is already active at low substrate concentrations. In the opposite case, enzyme exhibits merely very low activity to the substrate. Values of constants Vmax and Km are always specific for a particular enzyme substrate couple and cannot be generalised and applied to other enzymes or substrates without experimental verification. The first function of enzyme is reaction initiation. Molecules of reacting substances must be present in a certain minimum quantity in the medium for the reaction to proceed. When these molecules meet, start of the reaction requires a certain quantity of energy which is called activation energy; its magnitude depends on external conditions, particularly on temperature. The course of biochemical reactions is mostly much limited regarding high temperature because elevated temperatures could cause damage to reacting components. Around 20 oC, activation energy of many reactions is so high that the reaction might not proceed at all. Thanks to the catalytic effect of enzymes, activation energy may be much reduced; enzymes thus enable the reaction to carry on. Their further action, besides reducing activation energy, may be summarised as follows: Enzymes, similar to catalysts, act at minimum concentrations on the limits of threshold concentrations. They come from the reaction unchanged and unconsumed. Which, of course, does not mean enzymes remain in the medium at MODELING ISOLATION OF AMARANTH PROTEIN

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unchanged concentration, because owing to their protein basis they undergo natural breakdown through the activity of bacteria. Enzymes exert no influence on the equilibrium position the chemical reaction arrives at (change in thermodynamic energy G is the same with a non-catalysed as well as catalysed reaction). Hence, enzymes make possible and accelerate a reaction, but do not shift equilibrium condition either to the side of substrate or product. Many enzymes start acting only under the effect of another agent, the so-called activator. Apart from chemical compounds, the role of activator may be played by such factors as temperature, pH or redox potential, that is, physical and chemical parameters of the environment8. Reactions leading to formation of an enzyme-substrate complex may be interfered with various chemical and physico-chemical influences generally contained under the term of inhibition. Prior to potential artificial application of enzymes in a system, a check thus has to be carried out on whether inhibitory influences will act against these enzymatic reactions10. Objective of work Fractionation of amaranth flour has been performed so far by water extraction during which starch and protein are washed out together. Protein may be separated from starch only with great difficulty. However, amaranth protein contains a high proportion of essential amino acids and can be a significant component in functional foodstuffs (as special nutritive dietetic component); it is thus suitable to be concentrated. The aim of tests was to examine possibilities for separating proteins and starch of amaranth flour. The procedure we selected was enzymatic breakdown of polysaccharides to the purpose of liquefying starch by hydrolysing it into soluble glucose, and enriching the solid phase with vegetable protein. Three enzymes were selected to this aim (amylase, glucoamylase and cellulase), and dosed in a conveniently chosen ratio. The reason for applying several enzymes during the reaction was their high specificity, given by the typical three-dimensional structure of enzyme in which the active centre is found.

EXPERIMENTAL Materials Polarimeter Kruss P1000 (Germany) with polarimetric tube 200 mm, drier WTB Binder E/B 28 (Germany), shaft stirrer Heidolph RZR1 (Germany), electronic balance KERN 770/GS/GJ (Germany), water bath HGL W 16 (Germany), centrifuge WERK EBA 20 (Germany), mineralisation apparatus HACH Digesdahl (USA), muffle furnace Nabertherm L 9/S 27 (Germany), Parnas-Wagner distillation apparatus, Soxhlet extractor. Amaranth flour was supplied by the AMR Amaranth Company (Hradec Kralove, The Czech Republic); its composition is presented in Tab. 1. Analytical methods Dry matter was determined by drying a weighed quantity of sample in glass weighing bottle at 103±2 o C for 12 hours and weighing after cooling. Inorganic solid was determined by carefully incinerating a sample of flour in a ceramic crucible over a gas burner and then by annealing at 600 oC in a muffle furnace and weighing after cooling. Total Kjeldahl nitrogen was determined by mineralising a sample of flour by boiling for 30 min (at approx. 440 oC) in sulphuric acid with added catalyst. Nitrogenous substances were thus transformed into ammonium sulphate from which ammonia was released in an alkaline environment, then steam distilled and determined by titration. Coarse proteins were determined by multiplying nitrogen content by conversion factor 5.7011. Fat was extracted from the flour sample with n-hexane in a Soxhlet extraction apparatus for 4 hours. After distilling off the solvent and drying the flask containing fat for 1 hour and cooling, fat content was determined by gravimetry. Starch content was determined according to ISO 10520:199712. This determination is based on transforming starch into soluble starch by action of diluted HCl while warm. After clarification, soluble starch is determined by polarimetry. The method for determining fibre consists in eliminating accompanying substances from the sample by hydrolysis in an acid and alkaline medium; after a 90 min hydrolysis in 1.25 % H2SO4, the undissolved solid fraction was washed with water and hydrolysed for another 90 min in 1.25 % KOH. Non-hydrolysed residue (fibre), after washing with water and drying at 103±2 oC for 6 hours, was weighed13. Enzymatic breakdown of polysaccharides Liquefying starch into soluble glucose used a combination of 3 specific enzymes (from Novozymes A/S, Bagsvaerd, Denmark): BAN 480 L (α-amylase), AMG 300 L (glucoamylase), CELLUCLAST 1.51 FG (cellulase). These were mixed in ratio 4:3:3 in such manner that an enzyme stock solution MODELING ISOLATION OF AMARANTH PROTEIN

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was prepared from concentrated solutions of enzymes: 2 ml BAN + 1.5 ml AMG + 1.5 ml CELLUCLAST and filled to the mark with water in a 500 ml volumetric flask. For enzymatic breakdown of polysaccharides from amaranth flour, the stock solution of enzymes was dosed in a quantity of 5 l / 1,000 kg of dry matter of flour. Amylases break down starch chains and glucoamylases break cross-linked starch segments. Cellulase exerts specific hydrolytic action on cellulose which is present in the shell of amaranth grain. This reaction is a kind of biochemical modification, as such it can lead to many functionalities depending on the extent of enzymatic hydrolysis; thereby various chain lengths that correspond to glucose (dextrose), maltose, oligosaccharides or polysaccharides may be obtained. Kinetics of enzymatic breakdown of polysaccharides from amaranth flour was investigated at temperatures of 60, 70 and 80 oC under conditions that had been proposed already earlier and optimised to this purpose. Amaranth flour was mixed with water (at 22±2 oC) in a ratio of 1:20. Under laboratory conditions, 5 g flour dry matter was dosed into a 250 ml boiling flask and 100 ml distilled water was added. The flask containing mixture was put into a water bath and shaft stirrer set into motion (600 rpm), with simultaneous heating at a rate of 1.5 oC min-1. On attaining the desired temperature, 2.5 ml stock solution of enzyme was added (corresponding to a dose of 5 l per 1,000 kg flour dry matter, or 25 µl enzyme / 5 g flour dry matter). When enzymatic breakdown was over, the mixture was centrifuged (10 min) at a rate of 6,000 rpm. The liquid fraction and solid phase were separated and dried at 103±2 oC to constant mass. Starch content was determined from the dry matter of liquid and solid fraction. A scheme indicating complex processing of amaranth grain and separation of its components is shown in Fig. 1.

RESULTS AND DISCUSSION Enzymatic breakdown of polysaccharides from amaranth flour Measured experimental data were mathematically processed according to mechanics of first-order kinetics related to non-decomposed starch. Assuming the rate of conversion degree (y) is directly proportional to fraction of non-decomposed starch, we may write the differential equation as follows:

dy = k (1 − y ) dτ

(6)

Integration of equation (6) gives: (7) − ln(1 − y ) = kτ Results of experimental data on enzymatic breakdown of polysaccharides from amaranth flour at temperatures of 60, 70 and 80 oC are presented in Tab. 2. Plotting the natural logarithm of unreacted fraction: –ln(1–y) = k τ against time (τ) gives a straight line whose gradient is equal to rate constant of starch hydrolysis (k). Fig. 2 shows experimental data processed in this manner at temperatures of 60, 70 and 80 oC. Evaluation of activation energy (E) and frequency factor (A) assumes validity of the Arrhenius equation:

k = Ae

E RT

(8)

Integrating relationship (8) gives:

ln k = ln A −

E1 RT

(9)

An estimate of activation energy and frequency factor was made from evaluated rate constants (k) at three different temperatures (60, 70 and 80 oC). Rate constants were determined from line gradients. Tab. 3 summarises results of kinetic measurements. Plotting the natural logarithm of rate constant (ln k) against inverse value of absolute temperature (1/T) produces a straight line whose gradient enables to determine activation energy, and section on axis of ordinates to determine the value of frequency factor (see Fig. 3).

E =k R

(10)

From processed experimental data it follows that level of activation energy for hydrolysis of starch E = 9.714 x 104 J/mol, and frequency factor A = 28.967 min-1. MODELING ISOLATION OF AMARANTH PROTEIN

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Proposal of measuring system Enzymatic breakdown of starches may be monitored using a measuring system of fermentors. Studying the process has to focus on determining its optimum conditions for projecting a technological procedure isolating amaranth protein. We propose a measuring system consisting of two identical fermentors with identical system of probes and other auxiliary equipment. Fermentor A serves for reactions of a reference medium, and fermentor B for reactions of the medium under evaluation. Fermentors have to be equipped with stirrers incorporated in a vessel bottom. We propose that heating bodies should be placed along the perimeter in the bottom part of fermentor bodies. An outlet valve is likewise to be situated in the bottom part. Fermentor lids have to be designed so that the vessels can be hermetically closed, with three gas-tight apertures for probes, with a gas exhaust aperture and filling device. A computer-controlled measuring system with further complementary sensors. This system comprises a unit capable of measuring principal physical and chemical quantities. The instrumental part should be equipped with galvanically separated measuring inputs, logical inputs and a continuous control output. A sensor can be attached through every measuring input, and measuring input should enable conversion into analogue inputs. An analogue measuring input may then be set as a voltage input (for example, 0â&#x20AC;&#x201C;10 V) or current intensity input (for example, 0â&#x20AC;&#x201C;20 mA). Proposed connections of measuring instruments are described in Fig. 4.

Amaranth grain

Amaranth oil

Grinding, extraction in CO2

Water extraction, centrifuge

Enzymatic breakdown of polysaccharides

solid residue

drying

Amaranth fibre

drying

Amaranth protein

Liquefied starches

liquid

Fig.-1: Technology for processing amaranth grain.

CONCLUSIONS This study verified a procedure for separating amaranth protein. The selected procedure was biochemical, consisting in employment of enzymes acting specifically on breakdown of polysaccharides into simple water-soluble sugars (glucose). Non-decomposed protein was then separated. Enzymatic hydrolysis of starch was investigated at temperatures of 60, 70 and 80 oC, assuming a first-order mechanism of hydrolysis which is satisfactory up to approx. 50 % conversion. Rate constants were determined by evaluating experimental data. Their numerical values were as follows: k = 1.7 x 10-3 min-1 for 60 oC, k = 1.0 x 10-2 min-1 for 70 oC, and k = 1.3 x 10-2 min-1 for 80

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C. Activation energy E = 9.714 x 104 J/mol was evaluated from the dependency of natural logarithm of rate constant (ln k) on inverse value of absolute temperature (1/T). Corresponding frequency factor was A = 28.967 min-1. The proposed separation procedure, or concentration of proteins through enzymatic breakdown of starch into glucose, sets this technology onto a no-waste level. Separated amaranth protein may be effectively applied in the production of functional foodstuffs and quality supplements. Another field of application is animal feed because it helps, after forbidden use of meatand-bone flour, to overcome the deficit in proteins. In addition, amaranth protein may also be combined with proteins from other sources (for example, collagen) and employed to prepare mixed products for other than food applications. A sugar glucose solution may be utilised for producing yeast biomass. The amaranth plant, due to its composition and qualities, has substantial food potential.

ACKNOWLEDGEMENTS The authors would like to thank to Ministry of Education of The Czech Republic for financial support to this work executed under MSM Grant No. 7088352102. Table-1: Composition of amaranth flour [a. in dry matter] Parameter Dry matter Ash a Total Kjeldahl nitrogen a Coarse proteins (nitrogen x 5.70) a Fat a Starch a Fibre a

Value (%) 86.91 3.57 2.82 16.07 9.81 65.79 4.85

Table-2: Enzymatic breakdown of polysaccharides from amaranth flour. τ (min) 10 20 30 40 50 60 100 1 3 5 10 15 35 60 5 10 20 25 30 40 50 65 100

z (%) y 1–y Enzymatic breakdown of polysaccharides at temperature of 60 oC 2.5 0.025 0.975 4.1 0.041 0.959 5.8 0.058 0.942 7.0 0.070 0.930 8.1 0.081 0.919 13.6 0.136 0.864 15.5 0.155 0.845 Enzymatic breakdown of polysaccharides at temperature of 70 oC 21.9 0.219 0.781 25.6 0.256 0.744 26.2 0.262 0.738 26.6 0.266 0.734 39.6 0.396 0.604 49.0 0.490 0.510 56.3 0.563 0.437 Enzymatic breakdown of polysaccharides at temperature of 80 oC 15.0 0.150 0.850 30.0 0.300 0.700 42.0 0.420 0.580 46.0 0.460 0.540 48.0 0.480 0.520 55.0 0.550 0.450 60.0 0.600 0.400 71.0 0.710 0.290 75.0 0.750 0.250

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–ln (1–y) 0.025317808 0.041864204 0.059750004 0.072570693 0.084469157 0.146182510 0.168418652 0.247180 0.295714 0.303811 0.309246 0.504181 0.673345 0.827822 0.162518929 0.356674944 0.544727175 0.616186139 0.653926467 0.798507696 0.916290732 1.237874356 1.386294361

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line 1 = 60 oC y = 0.0017x + 0.0104

line 3 = 80 oC y = 0.0128x + 0.2517 2 R = 0.9401

line 2 = 70 oC y = 0.0101x + 0.266

2

2

R = 0.9142

R = 0.9447

1.6

3 1.2

–ln (1–y)

2 0.8

0.4

1

0 0

25

50

75

100

τ (min)

Fig.-2: Rate of enzymatic liquefying of starch to soluble glucose at temperatures of 60, 70 and 80 oC. Table-3: Determining activation energy t (o C ) 60 70 80

T (K) 333 343 353

k (min-1) 0.0017 0.0101 0.0128

ln k –6.37713 –4.59522 –4.35831

1/T 0.00300 0.00292 0.00283

Fig.-3: Determining activation energy.

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5

3

4 6

1 2

5

3

4 LAN or WAN

1 2

Legend: 1–fermenting tub, 2–heating control unit, 3–flow recorder, 4–analogue-digital convertor, 5–interface, 6–personal computer Fig.-4: Scheme of measuring system. Nomenclature A c E En [EnS] k k1, k-1, k2 Km p P r R S T t τ V y 1–y z

frequency factor intermediate concentration [EnS] activation energy enzyme reaction intermediate (enzyme-substrate complex) rate constant (min-1) reaction rate constants dissociation constant product concentration product reaction rate of final product molar gas constant (8.314 J/K.mol) substrate absolute temperature (K) reaction temperature (oC) time of enzymatic breakdown (min) reaction rate conversion degree unreacted fraction conversion of starch into glucose (%)

REFERENCES 1. 2. 3. 4. 5. 6. 7.

O.P. Lopez, Amaranth: Biology, Chemistry and Technology, CRC Press, Boca Raton, p. 75-107 (1994). S. Coimbra and R. Salema, Ann. Bot., 74, 373 (1994). A.A. Scilingo, S.E.M. Ortiz, E.N. Martinez and M.C. Anon, Food Res. Int., 35, 855 (2002). R.A. Becker, J. Food Sci., 46, 1175 (1981). T.A.P.C. Ferreira, A.C.G. Matias and J.A.G. Areas, Nutrire, 32, 91 (2007). G.S. Kelly, Altern. Med. Rev., 4, 29 (1999). A. Berger, I. Monnard, F. Dionisi, D. Gumy, K.C. Hayes and P. Lambelet, Food Chem., 81, 119 (2003).

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8. 9. 10. 11.

F. Kastanek, Bioengineering, Academia, Prague, p. 180-240 (2001). Z. Vodrazka, Biochemistry, Academia, Prague, p. 148-151 (1992). A. Blazej, Structure of Fibre Proteins, VEDA, Bratislava, p. 101-128 (1978). J. Davidek, J. Hrdlicka, M. Karvanek, J. Pokorny, J. Seifert and J. Velisek, Handbook of Food Analysis, SNTL, Prague, p. 181-184 (1988). 12. ISO 10520:1997, Native starch - Determination of starch content - Ewers polarimetric method. 13. ISO 5498:1981 Agricultural food products - Determination of crude fibre content - General method. [RJC-713/2011]

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Vol.4, No.1 (2011), 189-202 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

EFFECT OF MOLECULAR WEIGHT DISTRIBUTION ON CHEMICAL, STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF SODIUM LIGNOSULFONATES Nidal Madad, Latifa Chebil, Christian Sanchez and Mohamed Ghoul Laboratoire d’Ingénierie des Biomolécules, ENSAIA-INPL, Vandoeuvre-les-Nancy, France *E-mail: mohamed.ghoul@ensaia.inpl-nancy.fr ABSTRACT Chemical, structural and physicochemical properties of six sodium lignosulfonates (SLS) fractions with high purity and narrow molecular weight distribution (Mw) ranked between 2307 and 19583 g.mol-1 were studied. Structural characterization of these fractions, using 31Phosphor Nuclear Magnetic Resonance (31P NMR) and Fourier Transformed InfraRed (FTIR) analysis, was reported. 31P NMR and FTIR analyses show that these fractions present an important variability of hydroxyl, carboxyl and sulfonic group content. The adsorption isotherms of SLS fractions; fitted by Guggenheim–Andersen–de-Boer model; present different isotherms profile and different values of binding energy and adsorption capacities. SLS fractions were found to be highly charged and present the behaviour of soft particle. Intermediate fractions with Mw of 4297 and 2471 g.mol-1 give the highest surface activity and antioxidant capacity. Moreover, fractions with the highest molecular weight, Mw more than 6953 g.mol-1, present the greatest charge density and apparent viscosity. This data can help to develop new niche applications for the SLS. Keywords: Sodium lignosulfonates, diafiltration, physicochemical properties, antioxidant capacity. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Lignosulfonates (LS) are commercially available lignins obtained from sulphite pulping of wood. More than 1,000,000 tons are annually marketed1. The presence of the sulfonate groups confers to them a high solubility in water. Taking into account to their structure, LS exhibit dispersive, stabilizing, binding, complexing, antioxidant and antifungal properties2-6. These properties open to LS several applications. So, they can be used as concrete admixtures, gelling additive in resins preparation, stabilizing agent of emulsions and foams, raw material in the production of fine chemicals (vanillin, pyrocatechol ...), as well as binders in feed due to their antioxidant and sequestering metal ions capacity7-11. However, their effective industrial use takes place mainly in dispersing and binding applications characterized by very low added values. This limitation is due to the pulping method, the salt used and the origin of wood12, 13. Thus the obtained LS are characterized by a relatively complex chemical composition, a wide range of molecular weight distribution and therefore a high heterogeneity of their physicochemical properties12. Depending on the pulping method, unless four LS (Calcium LS, Sodium LS, Ammonium LS and magnesium LS) are available. The main studied ones are calcium LS (CLS). The obtained results with these biopolymers showed that the structure, the molecular weight distribution and as well as additives affect strongly the physicochemical properties and application performances of LS14, 15. Due to the difficulties of fractionation of LS few papers investigated the effect of molecular weight distribution on the reactivity and physicochemical properties of these biomolecules. For CLS, Ouyang et al.16 studied the dispersive and adsorption capacity of different fractions obtained by ultrafiltration, and observed that, in aqueous suspension, these two properties increase when the molecular weight increases. Moreover, Yang et al.17 reported that for fractions obtained with higher molecular weight the hydrophobic interactions is the main driving forces for adsorption, while for fraction with lower molecular weight the hydrogen–bond and the attractive power of anionic groups are the main driving forces for adsorption. These latter showed also, that the potential zeta is depending on mass concentration and lowest values are reached with

SODIUM LIGNOSULFONATES

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fractions characterized by intermediate molecular weight. For Sodium lignosulfonates (SLS), Li et al.18 showed that the dispersion ability increased with molecular weight and increasing sulfonic groups. Yang et al.19 studied the effects of the molecular weight of SLS on their capacity to reduce the viscosity of the coal water slurry, the adsorption behaviour and the zeta potential. They reported that fractions with molecular weight between (10 kDa and 30 kDa) lead to a better effect on reducing viscosity while fraction greater than 10 kDa is more adsorbed on the coal surface. The potential zeta is more linked to the presence or the absence of the sulfonic and carboxyl groups. As it was mentioned previously the main study was focused on the CLS properties. Few studies were dedicated to SLS where these biopolymers exhibit interesting properties of adsorption and viscosity reducer 20. The aim of this work is to study the effect of molecular weight distribution on the structural and physicochemical properties of different SLS fractions, obtained by diafiltration process using five membranes with a cut off in the range of 5 to 300 kDa. To remove salts and to obtain fractions with a narrow molecular weight distribution, the diafiltration was fed by a five volume of demineralised water/volume of SLS solution. For each fraction chemical and structural characteristics (molecular weight distribution and functional groups) and physicochemical properties (hydration and charge properties, surface activity, antioxidant capacity and rheological property) were investigated and discussed.

EXPERIMENTAL Materials Sodium lignosulfonates (SLS) composed by 90 wt. % of LS, 4 wt. % of reducing sugars and 6 wt% of total impurities and AAPH 2,2’-Azino[2-methyl-propionomidin] dihydrochlorid 97% purity, were furnished by (Aldrich, Deutschland). ABTS 2,2-Azino-bis(3-ethylbenzo-thiozoline-6-sulfonic acid) 98% purity was provided by (Sigma, Deutschland). FL Fluoroscein (free acid) was furnished by (Flucka, Deutschland). Diafiltration Diafiltration (DF) in feed and bleed mode was carried out to remove impurities, like sulphur, ash and salts and to obtain different fractions with a narrow molecular weight distribution. The washing step was realized at a constant volume by adding continuously  demineralised water until 5 volume of SLS solution. Five ceramic tubular membranes (TAMI, France) with successively molecular weight cut-off (MWCO) (300000, 150000, 50000, 15000 and 5000 Da) were used. The retentat was concentrated using a rotary evaporator, freeze-dried and weighed. After each step the obtained permeate was then fractionated using the next MWCO membrane. SLS was separated into six fractions with molecular weight ranges: more than 300000 (F1), 300000~150000 (F2), 150000~50000 (F3), 50000~15000 (F4), 15000~5000 (F5) and less than 5000 (F6). DF was performed under pressure and temperature of 5 bars and 50°C, respectively. SLS determination The SLS content of the commercial sample and the different fractions was determined as reported by Ringena et al.21, at 280 nm, using UV detector 6000LP (Thermo, France). Commercial SLS were used as a calibration standard due that their content of LS is known (90%). Reducing sugar content Reducing sugar was determined using method described by Miller22. Size exclusion chromatography analysis Commercial SLS and their fractions were analysed by Size Exclusion Chromatography (SEC) (HPLC LaChrom Merck, Deutschland). The system consists of a pump L-2130, an autosampler L-2200, and a Superdex 200HR 10/30 column (24ml, 13 µm, dextran/cross linked agarose matrix). Detection was performed using UV detector diode L-2455 at 280 nm. Before analysis, the samples were filtered using regenerated cellulose membrane (0.22 µm) and aliquots of 50 µl were injected to the SEC system. Commercial SLS and their fractions were dissolved in 0.1% solution of Buffer Phosphate pH=7, 0.15 M SODIUM LIGNOSULFONATES

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NaCl. The same solution was used as an eluent. The flow rate was 0.4 ml at 25°C and 11 bars. The calibration was performed using polystyrenes sulfonate (PSS) with weight average molecular weight between 73900 Da and 1100 Da as standard. Fourier transformed infrared analysis Transmission infrared spectra of the SLS fractions were performed using Fourier Transformed InfraRed (FTIR) spectrometer tensor 27 (Bruker, France) equipped with a Platinum ATR optical cell and a deuterated triglycine sulphate (DTGS) detector. Samples (powder) were placed directly on the crystal. The diaphragm was set to 6 mm and the scanning rate to 10 kHz. Each spectrum was recorded 156 scans. The wave number range used is 4000 and 800 cm-1 with resolution of 2 cm-1. The spectra were baseline corrected for further analysis. 31 P NMR NMR experiments were performed on a Bruker Avance-400 spectrometer (Bruker, France), using an inverse-gated decoupling (Waltz-16) pulse sequence with a 30° pulse angle and 25 s pulse delay. The analyses were done by derivatising 30 mg of each fraction with 2-chloro-4,4,5,5-tetramethyl-1-1,3,2dioxaphospholane (TMDP)23. 31P NMR data were processed offline using NUTS NMR data processing software (Acorn NMR Inc.). Commercial SLS and their fractions were phosphitylated with 2-chloro-4,4,5,5-tetramethyl-1,2,3dioxaphospholane in presence of cyclohexanol as an internal standard and analyzed with quantitative 31P NMR according to a method described by Granata and Argyropoulos23. The concentration of each hydroxyl functional group (mmol/g) was calculated using the internal standard (cyclohexanol) with known hydroxyl functional number. Besides, the C9 unit was calculated from the elementary analysis performing for commercial SLS and their fractions. From the concentration of hydroxyl functional group (mmol/g) and the C9 unit formula, the number of functional group per C9 unit (group/C9) was also calculated. Dynamic vapour sorption Water sorption isotherms of commercial SLS and their fractions were determined using dynamic vapour sorption (DVS) technique (Surface Measurement Systems, United Kingdom). The principle of this method is an evaluation of sample weight changes over time at 25°C and at relative humidity (RH) between 0% and 95%. About 40 mg of sample was loaded into the quartz sample pan. A first step (drying phase) consists to control the humidity at 0% for 10 h to obtain internally equilibrates. The sample was then subjected to successive ten steps of 10% RH increase, up to 95%. For each step the mass changes (m) were plotted against time. The equilibrium was considered to be reached when changes in mass with time (dm/dt) was lower than 0.002 %/min. The accuracy of the system was ±1.0% and ±0.2 °C for RH and the temperature respectively. Error for each sample is 2%. The water vapour adsorption isotherms were described by using GAB (Guggenheim–Andersen–de Boer) model, which is the most commonly used isotherm model for moisture sorption isotherms of foods24:

X = Xm

C GAB K GAB a w (1 − K GAB a w )(1 − K GAB a w + C GAB K GAB a w )

(1)

Where Xm is the monolayer moisture content, X is the equilibrium water content, CGAB, is the characteristic energy constant, KGAB is the characteristic constant correcting the properties of the multilayer molecules with respect to the bulk liquid. The parameters were calculated by Origin software25. The electrophoretic mobility investigation The velocity of a particle in a unit electric field is referred to its electrophoretic mobility (µE). Determination of µE values permit to assess the electrical charge of macromolecules. Soft particles are characterized by the presence of an ion penetrable layer at the outer surface exposed to the continuous medium. Ohshima26 proposed a model (equation 2) for µ E that allowing determining 1/λ, which is related to the length of accessible layer and ZN when Z corresponding to the valences of charge density in the SODIUM LIGNOSULFONATES

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polyelectrolytic region and N represents the electrical charge density in the polyelectrolytic region (charges/m3). The µE of soft particles is described in the equation 2:

µE =

ε 0ε r ψ / Km + ψ DON / λ eZN + 2 η 1 / Km + 1 / λ ηλ

(2)

η is the viscosity of the medium (Pa/s)

ε 0 is the absolute permittivity of vacuum ε r is the relative permittivity of the electrolyte solution e is the elementary charge (1.6 10-19 C) ψ DON is the Donnan potential define like : ψ DON

1/ 2 2   KT  ZN   ZN  = ln  +   + 1  ze  2 zn   2 zn    

(3)

ψ0 is the potential at the boundary of the surface region define like : 1/ 2 2    2 zn    ZN  1 / 2  KT   ZN   ZN  1 −   ψ 0= +   + 1  +  + 1  ln     2 zn    ze   2 zn   2 zn  ZN     

(4)

Km is the Debye Hückel parameter in the surface layer that involved the contribution of the fixed-charges

  ZN  2    ZeN Km = k 1 +    2 zn  

1/ 4

(5)

 2z 2e2n    = κ k is the Debye Hückel parameter define like : ε ε kT  0 r 

1/ 2

(6)

z is the valence of the electrolyte solution. T is the thermodynamic temperature. K the Boltzmann constant and n is the bulk concentration of the electrolyte solution. NaCl was the electrolyte used so z=1, e the elementary electric charge. µ E of commercial SLS and their fractions was performed using Zetasizer ZS equipment HPPS 5001 (Malvern Instrument, United Kingdom) by means of laser Doppler electrophoresis. Determination of surface tension Measurements of surface tension of SLS solutions prepared in demineralised water, at concentration ranging from 0 to 10 g.L-1, at 25°C, were made using a tensiometer model K12 (Krüss, deutcshland). The results have been accomplished by the method of Wilhelmy plate. This method based on placement of a sheet over the surface of the solution. The plate is immersed in the solution and the force necessary to return it to its original position equals to the surface tension. Rheological measurements The rheological measurements were carried out in a Stress Tech Rheometer (Reologica AB, Sweden) using a cone and plate geometry (volume sample 1.2 mL, cone angle 4°, diameter 40 mm). A lid was added into the sample to prevent evaporation at high temperatures. The rheometer was connected to a thermostatically controlled bath. Samples at range of concentration from 5% to 20%, dissolved in demineralised water, were allowed to equilibrate at 25 °C and shear rate increasing from 0.03 to 0.2 s-1 were imposed. The GNF (generalized Newtonian fluid) model was used to determine the apparent viscosity of commercial SLS and their fractions; where the shear stress τ is .

proportional to the strain-rate γ SODIUM LIGNOSULFONATES

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τ = ηa γ

(7)

Antioxidant activity determination The antioxidant activity was performed using Xenius multidetection microplate reader (Safas, Monaco). 96-well with black (Trolox equivalent antioxidant capacity assay) and clear (Oxygen radical absorbance capacity assay) polystyrene microplates were used. Trolox equivalent antioxidant capacity The antiradical activity of commercial SLS and their fractions was evaluated by scavenging the radical ABTS ● +. ABTS radical cation (ABTS● +) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12– 16h before being used. Next ABTS● + solution was diluted with PBS (Phosphate Buffer Saline) (pH 7.4) to an absorbance of 0.5 at 734 nm and equilibrated at 30°C. 200 µl of ABTS ● + and (10, 20, 35, 50, 70, 80 µl) of SLS solutions (10 mg.L-1) was mixed and adjusted to 300 µl with buffer solution. After 15 min incubation, the optical density was measured. The percentage inhibition of ABTS ● + is calculated by using equation 8. % inhibition =

DOinitial − DOfinal x 100 DOinitial

(8)

The Trolox equivalent antioxidant capacity TEAC represents the molar concentration of trolox (µM) with the same antioxidant activity of 1 mg.L-1 of SLS solutions. Oxygen radical absorbance capacity Oxygen radical absorbance capacity (ORAC) measures the ability of a molecule to prevent the oxidation of FL by free radicals from the decomposition of AAPH at 37°C. Fluorescence was read with an excitation wavelength of 485 nm and an emission filter of 528 nm. AAPH solution and FL were prepared in a phosphate buffer solution at 75 mM at pH 7.4. The control of this reaction is the trolox which is also prepared in the same buffer. Six different quantities (10, 20, 35, 50, 70, 80 µl) of commercial SLS and their fractions at a concentration of 10 mg.L-1 were placed in the wells of the microplate with FL and adjusted at the same volume with buffer solution (80µl). The mixture was preincubated for 30 min at 37 °C, before rapidly adding the AAPH solution (220µl) using a multichannel pipette. The microplate was immediately placed in the reader and the fluorescence recorded every 6 min for 240 min. A blank with FL and AAPH using sodium phosphate buffer instead of the antioxidant solution was used. The inhibition capacity was expressed as Trolox equivalents (µM), and is quantified by integrating of the area under the curve (AUC). All reaction mixtures were prepared in twenty times. The area under the fluorescence decay curve (AUC) was calculated using the equation 9. i = 240

AUC = 1 +

∑f

i

/ f0

(9)

i =6

where f0 and fi are the initial fluorescence read at 0 min and at I min, respectively. The net AUC corresponding to the sample was calculated by subtracting the AUC corresponding to the blank.

RESULTS AND DISCUSSION Chemical and structural characterization For all fractions of SLS, obtained after each step of diafiltration, the main structural and chemical characteristics were monitored. Composition and SLS recovery of different fractions SODIUM LIGNOSULFONATES

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Recoveries rates, percentage of LS and percentage of reducing sugar of SLS fractions are given in Table 1. The obtained results showed that fraction F1 is the most important one in commercial SLS. It represents more than 48% w/w of the total mass. While for F3 (50 ~ 150 kDa) and F4 (15 ~ 50 kDa) the recoveries rates were only about to 6% and 5%, respectively. Similar results for recovery of intermediaries fractions obtained by ultrafilatration were reported by Ringena et al.21. The percentage of SLS of each fraction was also determined. It is equal to 99%, 97%, 95%, 93% and 75% respectively for F1, F2, F3, F4 and F5. For F6 this percentage is relatively low (19 %) and percentage of reducing sugar is high (19%) compared to the proportion in the other fractions. Therefore, except for F6 and F5, diafiltration allows obtaining high pure fractions. These fractions will be deeply characterized to enhance the development of new niche applications. Determination of molecular weight distribution Figure 1 shows the chromatogram profiles of commercial SLS and their fractions analyzed by SEC-UV. Except for F6, SLS fraction profiles, compared to that of commercial SLS, showed a narrow and symmetrical distribution. The behaviour of F6 can be explained by the high impurities content in this fraction. Moreover, the weight average molecular weight (Mw), the number average molecular weight (Mn) and the polydispersity D (Mw/Mn) were calculated and summarized in Table 2. It appears that the polydispersity decreases progressively with the decrease of the cut off of membrane. It is equal to 6.17, 3.46, 2.15, 1.78, 1.44 and 1.67 for commercial SLS, F1, F2, F3, F4 and F5 respectively. The Mw of the fractions varied from (2307 to 19543 g mol-1) and Mn from (1385 to 5659 g mol-1). Due the high impurities of F6, molecular weight and polydispersity of this fraction were not determined. Similar behaviour was observed by Ringena and al.21. This result confirms, as it was indicated by these authors, that no correlation can be established between the cut off of used membranes and the actual molecular weight of SLS fractions. This is due to the complexity of LS structures compared to the PSS standards used as reference for weight determination. In fact, PSS are rather linear27 while the structure of SLS are highly heterogeneous and complex28. This structural difference can leads to the underestimation of the molecular weight values of SLS. Due to the high heterogeneity of F6 (low LS content and high impurities content), the next analyses were not realized to this fraction. Determination of functional groups FTIR investigation The functional properties of SLS depend strongly on the nature of the different groups forming the skeleton of these polymers. FTIR analysis allows the identification of these groups. The assignments and the intensity (ATR units) of these spectres are given in table 3. These results indicate that several variation of the profile and the intensity of adsorption were occurred in different regions of the spectrum. The band at 3392 cm-1 was assigned to hydroxyl groups. F3 presents the highest adsorption of hydroxyl groups. The bands between 1690 cm-1 and 1650 cm-1 are due to carbonyl/carboxyl groups, F3 and F4 have higher adsorption of carbonyl/ carboxyl groups than the other fractions. Similar results were found for the band at 1040 cm-1 which mean that these fractions (F3 and F4) contain more sulfonic groups than F1, F2, F5 and commercial SLS. The FTIR analyses show that fractions characterized by a low Mw (F3, F4 and F5) absorb at 1125 cm-1 which mean that this fractions contain syringyl units specifically. This observation was confirmed by the highest absorption of these fractions at 1420 cm-1 related to the C-H deformation of OCH3 groups. The variation in the functional groups can be in the origin of new and specific surface properties which can be exploited for the development of new applications to these polymers. 31 P NMR analysis The concentration of hydroxyl functional groups of commercial SLS and their fractions are presented on Table 4. These results show that all fractions contain aliphatic OH, guaiacyl OH and carboxylic acid. For Commercial SLS and their fractions, OH-guaiacyl and OH-syringyl groups were detected. F3 and F4 contain the highest OH-guaiacyl (0.125/C9) and carboxylic acid (0.087/C9), respectively. OH-Syringyl groups (0.013/C9), (0.005/C9) and (0.025/C9) were identified only for F3, F4 and F5, respectively. Observations of 31P NMR spectra confirm FTIR results. Both of them, show that F3 has the highest OH SODIUM LIGNOSULFONATES

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hydroxyl groups content and F4 a highest carboxyl groups content and also confirm the presence of syringyl groups in the lowest fractions. It appears from these results that the fractionation of SLS by diafiltration allows the production of lignosulfonate with a wide range of Mw and structural properties which could in the origin of the development of new applications. Physicochemical properties Chemical and structural investigations revealed that DF can furnish SLS fractions with different molecular weight distribution and functional groups. The variation of molecular weight and the composition can affect the colloidal and physicochemical properties. So, deep investigations of the main colloidal properties of the obtained fractions were performed and compared. Sorption isotherms study The quality of most raw materials depends to a great extent upon their physical and chemical stability. This stability is mainly a consequence of the relationship between the equilibrium moisture content and the corresponding water activity (aw), at a given temperature. These water sorption isotherms are unique for a given composition of the raw materials. Many empirical and semi-empirical equations describing the sorption characteristics of different raw materials have been proposed in the literature. The kinetic models based on a multi-layer and condensed film (GAB model) is considered to be the most versatile sorption model available in the literature. As for several applications the capability of the water retention of LS is an important criterion, thus the sorption isotherm of commercial SLS and their fractions were investigated at 25 °C. The results are summarized in Figure 2 and according to Brunauer29 classification they belong to type II curve shape. The GAB model was used to fit these data. The three parameters of this model Xm (monolayer moisture content), kGAB, and CGAB values are reported in Table 5 together with the mean relative percentage deviation module (E) and R2. Examination of these results indicates that the GAB model fits well the experimental adsorption kinetic for SLS and the different fractions throughout the entire range of water activity. The GAB model, gives E values ranging from 2.64% to 7.26 %, with average value of 4%. The GAB model parameters Xm (monolayer moisture content dry basis), kGAB, and CGAB values provide an indication of the monolayer water adsorption capacity, the binding energy of the water and the monolayer heat of sorption respectively. For several material particularly foodstuff and biopolymers, Xm varies from 0.5 to 15, kGAB between 0.7 and 1 and CGAB is in the range of 1 to 20. It appears that for the three GAB parameters the obtained values for SLS are comparable to those reported for foodstuff. The lowest value of Xm (8.8) was obtained with F1 and the highest value (28.5) with F5. The variation of GAB parameters can be attributed to a combination of factors, which include the conformation and topology of molecule and the hydrophilic/hydrophobic sites adsorbed at the interface. The observed variation of Xm and CGAB confirms the structural and chemical differences occurred between SLS fractions. Despite the importance of knowledge on the mechanism of water–binding of LS the data on isotherm sorption are scarce and no comparison can be made. The results obtained in this study can help to understand the behaviour of the biopolymer when used in different formulation. Electrophoretic measurements The variation of the morphology and the functional groups of the different fractions of SLS can affect their interactions and complex formation with other macromolecules. To quantify these possible effects, the electophoretic mobility (µ E) and conformation of commercial SLS and their fractions were investigated. The obtained µ E profiles for different fractions were reported in Figure 3. Negative µ E were observed due to the negative charge of sulfonic groups. Moreover, the absolute value of µ E across nonzero values and decreases when the NaCl concentration increases. These profiles are characteristic of soft particles. In fact, SLS was previously described like ellipsoid particles would have a dense core surrounded by a less dense surface layer of polymeric chains containing hydroxyl and sulfonic groups20. Table 6 reports the values of charge density (ZN) and the particle softness parameters (1/λ). ZN seems to be molecular weight dependent and present a high value for high molecular weight. As an example, ZN for F1 and F5, is equal -40,5 mM and -27.5 mM respectively. Also, 1/λ values, about 2 nm, doesn’t SODIUM LIGNOSULFONATES

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appeared depending on molecular weight, it was assumed that this parameters depend on the polymer conformation30. Surface tension The surface tension of aqueous solutions of commercial SLS and their fractions is shown in figure 4. It appears that the increase of the concentration in the solution lead to a decrease of the surface tension. However, the commercial SLS and their fractions are not able to form micelle. This behaviour could be attributed to the spherical shape of their hydrophobic skeleton, which hinders the formation of a regular arrangement at the interfacial phase and thus affects their surface activity17. In spite of the no formation of micelle, some of these fractions; particularly F3 and F4 lead to an interesting decrease of surface tension with an increase of the concentration. For 10 g/L these two fractions reduce the surface tension to a value around of 52 mN/m. This behaviour could be attributed to the presence of the hydroxyl and sulfonic groups as it was shown previously by Infrared spectra analysis and 31P NMR. The effect of a high density of sulfonic and hydroxyl groups of a modified LS on the surface properties was reported Pang and al.31. These authors studied the effect of hydroxylation and sulfonation of CLS and they demonstrated that the content of the hydroxyl groups and sulfonic groups are important to enhance the surface activity. Rheological investigation In order to evaluate the effect of the fractionation of LS on their colloidal properties, the apparent viscosity of the different fractions was determined and compared to commercial SLS. The obtained data, for the imposed shear rate, were modelled as a Newtonians fluid. The calculated apparent viscosity (ηa) of SLS solutions at three different concentrations (20%, 10% and 5%) are given in table 7. The highest apparent viscosity 2.19 10-3 and 2.16 10-3 N.m-2.s were obtained respectively with fraction F1 and F2 at 20%. For others fractions ηa was about of 1.5 10-3 N.m-².s. These apparent viscosity decreases as the concentration and the molecular weight of the fraction is decreasing. The lowest value (1.28 10-3 N.m-².s) is obtained with F5 and F4 at 5 %. This difference, observed between ηa values of SLS solutions can be attributed to the presence of more sodium in the fractions characterized by low molecular weight. In fact, as reported by Browning et al.20, the presence of sodium can promote the repulsive forces and therefore reduce the viscosity. The effect of sodium on the viscosity was stated by comparing the viscosity of SLS and CLS. SLS viscosity is lower compared to that of CLS due to the stronger electrokinetic repulsive force of the sodium20. Antioxidant capacity The antioxidant activity of commercial SLS and their fractions, evaluated by TEAC and ORAC assays, are reported in table 8. It indicates that the TEAC values of commercial SLS and their fractions ranked from 1.67 to 2.86 µM and from 1.76 to 2.53 µM for ORAC values. F3 (2.83 µM) and F4 (2.86 µM) showed the greatest TEAC values. ORAC method are also showed that F3 (2.47 µM) and F4 (2.53 µM) are the fraction which have the greatest antioxidant capacity. Compared to the most known antioxidant molecules, such as vitamin C (TEAC, 5.68 µM), vitamin E (TEAC, 2.32 µM) and rutin (TEAC, 3.97 µM)32, SLS fractions exhibit a relatively interesting antioxidant power. The fractionation allows obtaining fractions (F3, F4) with a relatively highest antioxidant activity compared to SLS. Both the used methods (TEAC and ORAC) lead to a similar conclusion. The variation of the antioxidant power between fractions could be attributed to the structural difference observed previously. In fact, Zhou et al.33 studied the antioxidant capacity, using TEAC method, of different phenolic acid (4-OH benzoic, vanillic, and syringyl acids) and they observed that the presence of methoxyl groups (OCH3) in the ortho position to the hydroxyl position on the phenyl ring (syringyl acid) enhances the antioxidant activity. The presence of syringyl units in fractions F3, F4 and F5 may be in the origin of the more important antioxidant capacity. However, values obtained by TEAC are slightly lower compared to ORAC. This difference is attributed to the fact that TEAC assay uses exogenous ABTS●+ radicals, whereas the ORAC assay uses more physiologically relevant peroxyl radicals, and can react with non-lignosulfonates components34. F5 evaluated by TEAC present an antioxidant activity less then commercial SLS due to the presence of impurities. SODIUM LIGNOSULFONATES

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CONCLUSION Diafiltration achieved five SLS fractions with different molecular weight and polydispersity ranking from 1385 g.mol-1 to 19543 g.mol-1 and 1.44 to 3.46, respectively. Structural investigation by FTIR analyses and 31P NMR show a high content on hydroxyl and sulfonic groups for fractions with molecular weight ranking from 2471 g.mol-1 to 4297 g.mol-1 (F3 and F4). The physicochemical properties of SLS fractions were also investigated and compared to the commercial SLS. The adsorption isotherms of commercial SLS and their fractions present different isotherms profiles and well fitted by GAB model. The parameters of this model indicated that the energy binding and adsorption capacities differ from one fraction to another. Thereafter, surface activity were evaluated, results emphasize that fractions with molecular weight with Mw of 4297 and 2471 g.mol-1 (F3 and F4) have a higher surface activity than commercial SLS. These two fractions display also the highest antioxidant activity. In this work, we also demonstrated that LS are soft particle and we determined the charge density. F1 (with Mw of 19543 g.mol1 ) and F2 (with Mw of 6953 g.mol-1) present the highest charge density (-40.5 mM and 41 mM) and the highest apparent viscosity (2.19 10-3 and 2.16 10-3 N.m-².s). The analysis of the whole results indicated that diafiltration permits the obtaining for at least one fraction that shows greater activity in a given property compared to commercial SLS.

Fig.-1: UV detected SEC elution profiles of commercial SLS and their fractions obtained after diafiltration. (

Commercial SLS

F1

F2

F3

F4

F5

F6)

Table-1: Percentage of recovery rate, percentage of SLS content and percentage of reducing sugar of commercial SLS and their fractions. Fraction

Cut-off range (Da)

%Recovery rate

% SLS content

%Reducing sugar

Commercial SLS

----

-----

91.45 ± 1.47

3.68 ± 0.06

F1

More than 300000

48.38±2.66

99.59 ± 0.82

1.33 ± 0.04

F2

150000~300000

14.88 ± 4.96

97.08± 0.27

1.65 ± 0.14

F3

50000~150000

6.55 ± 1.49

95.34 ± 1.91

2.55 ± 0.01

F4

15000~50000

5.31 ± 0.20

93.98 ± 2.36

4.23 ± 0.10

F5

5000~15000

10.48 ± 2.72

75.59 ± 0.52

4.52 ± 0.11

F6

Less than 5000

4.19 ± 0.92

19.81 ± 1.81

19.22 ± 0.28

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Fig.-2: Sorption isotherm profile obtained for commercial SLS and their fractions estimated at 25 °C from 0% to 98% RH and modelled up with the GAB model. (

Commercial SLS

F1

F2

F3

F4

F5

GAB FIT)

Fig.-3: Profile of electrophoretic mobility measurements of commercial SLS and their fractions. Solid lines represent the best-fitted theoretical mobility curves. (

Commercial SLS

F1

F2

F3

F4

F5

FIT)

Table-2: Molecular weight distribution and polydispersity obtained by SEC-UV of commercial SLS and their fractions. Fraction

Cut-off range (Da)

Mn (g.molˉ¹)

Mw (g.molˉ¹)

Polydispersity

Commercial SLS

----

2896 ± 364

17783 ± 1482

6.17 ± 0.33

F1

More than 300000

5659 ± 388

19543 ± 707

3.46 ± 0.11

F2

150000~300000

3236 ± 89

6953± 63

2.15 ± 0.03

F3

50000~150000

2408 ± 153

4297± 430

1.78 ± 0.09

F4

15000~50000

1722 ± 85

2471 ± 48

1.44 ± 0.05

F5

5000~15000

1385 ± 33

2307± 87

1.67± 0.03

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Fig.-4: Surface tension of commercial SLS and their fractions according to concentration (g/L). (

Commercial SLS

F1

F2

F3

F4

F5

FIT)

Table -3: Assignment of FTIR spectra and their intensity (ATR Units) of commercial SLS and their fractions.

Signal Intensity (ATR Units)

Wave number Functional Groups (cm-1)

Commercial SLS OH Stretching 0,151 C=O stretch unconj 0,074 C=O stretch conj 0,097 Aromatic squel vibration 0,145 CH deformations of OCH3 groups 0,074 C-H deformation of Guaiacyl units 0,112 C-H deformation of Syringyl units C-S elongation 0,118

3392 1690 1650 1565 1420 1180 1125 1040

F1

F2

F3

F4

F5

0,070 0,069 0,079 0,153 0,068 0,091 0,103

0,101 0,072 0,082 0,156 0,070 0,132 0,135

0,230 0,086 0,122 0,163 0,84 0,154 0,101 0,154

0,142 0,102 0,117 0,17 0,114 0,152 0,134 0,156

0,082 0,081 0,097 0,111 0,078 0,131 0,097 0,120

Table-4: Commercial SLS and their fraction characteristics calculated from 31P NMR data. (a) Determined by integration with cyclohexanol as an internal standard. (b) Calculated on the basis of C9 units from elemental analysis performed on commercial SLS and their fractions.

Chemical Commercial SLS shift Assignment mmol/g a Group/C9 b range δ31PNMR 145.5– 1-Aliphatic 2,240 0,601 OH 150.0

141.8–

2a-Syringyl

-

SODIUM LIGNOSULFONATES

F1 mmol/g

F2 a

Group/C9

b

mmol/g

F3 a

Group/C9

b

mmol/g

F4 a

Group/C9

b

mmol/g

F5 a

Group/C9

b

mmol/g

a

Group/C9

1,832

0,419

2,432

0,624

1,581

0,426

2,235

0,729

1,619

0,504

-

-

-

-

0,049

0,013

0,015

0,005

0,080

0,025

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Vol.4, No.1 (2011), 189-202 143.5

OH

138.7– 140.1

2bGuaiacyl OH 3Carboxylic acid

134– 135.7

0,314

0,084

0,478

0,109

0,434

0,111

0,465

0,125

0,266

0,087

0,300

0,093

0,070

0,019

0,033

0,008

0,056

0,014

0,057

0,015

0,266

0,087

0,232

0,072

Table-5: Parameters Xm, CGAB and kGAB obtained from the fitted curves with GAB for commercial SLS and their fractions.

Fraction

Xm

CGAB

k GAB

E

R2

Commercial SLS

17.79

0.48

0.90

4.39

0.99

F1

8.79

11.89

0.89

3.62

0.99

F2

11.14

0.72

0.92

4.50

0.99

F3

12.30

1.34

0.92

2.64

0.99

F4

13.31

1.15

0.91

4.87

0.99

F5

28.53

0.36

0.89

7.26

0.99

Table-6: Determination of the surface charge properties (ZN, the spatial charge density in the polyelectrolyte region, and 1/λ, the softness parameter) of commercial SLS and their fractions using the Ohshima’s method.

Fraction Commercial SLS

ZN(mM) -31.0 ± 1.41

1/λ(nm) 1.91 ± 0.01

F1

-40.5 ± 0.28

1.98 ± 0.01

F2

-41.0 ± 0.07

2.10 ± 0.01

F3

-39.5 ± 0.42

2.00 ± 0.01

F4

-31.5± 0.28

1.95 ± 0.01

F5

-27.5 ± 4.41

2.20 ± 0.01

Table-7: Apparent viscosities at different concentration (5%, 10% and 20%) of commercial SLS and their fractions.

-3

N.m-².s)

Fraction

ŋa (10 200g/L

Commercial SLS F1 F2 F3 F4 F5

1.99 ± 0.04 2.19 ± 0.07 2.16 ± 0.01 1.55 ± 0.02 1.53 ± 0.05 1.54 ± 0.05

SODIUM LIGNOSULFONATES

100 g/L

50g/L

1.57 ± 0.09 1.76 ± 0.22 1.74 ± 0.23 1.32 ± 0.01 1.33 ± 0.04 1.37 ± 0.03

1.43 ± 0.06 1.36 ± 0.11 1.30 ± 0.07 1.29 ± 0.03 1.28 ± 0.05 1.28 ± 0.02

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Table-8: Antioxidant capacity evaluated by TEAC and ORAC methods of commercial SLS and their fractions. Fraction Commercial SLS F1 F2 F3 F4 F5

TEAC (µM) 2.52 ± 0.08 2.51 ± 0.21 2.45 ± 0.10 2.83 ± 0.12 2.86 ± 0.18 1.67 ± 0.19

ORAC (µM ) 1.92 ± 0.13 1.91 ± 0.23 1.76 ± 0.13 2.47 ± 0.17 2.53 ± 0.10 2.18 ± 0.06

ACKNOWLEDGEMENTS The authors gratefully acknowledge Doctor Nicolas BROSSE from LERMAB Laboratory for the 31P NMR analysis.

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30. 31. 32. 33. 34.

V. Ducel, P. Saulnier, J. Richard and F. Boury, Colloid Surface B, 41, 95 (2005). Y. X. Pang, X. Q. Qiu, D. J. Yang and H. M. Lou, Colloid Surface A, 312, 154 (2008). P. Pietta, P. Simonetti, C. Gardana and P. Mauri, J. Pharmaceut. Biomed., 23, 223 (2000). K. Zhou, J. J. Yin and L. Yu, Food Chem., 95, 446 (2006). V. Ugartondo, M. Mitjans and M. P. Vinardell, Ind. crop. prod., 30, 184 (2009). [RJC-688/2010]

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α,α α-DIMETHYL-4-[1-HYDROXY-4-[4 (HYDROXYDIPHENYLMETHYL)-1-PIPERIDINYL]BUTYL] BENZENEACETIC ACID HYDROCHLORIDE AS A CHELATING AGENT IN SOME METAL COMPLEX Jitendra H. Deshmukh1 and M. N. Deshpande2 1

2

Department of Chemistry, Yeshwant Mahavidyalaya, Nanded-431602, India P.G. Department of Chemistry, NES, Science College, Nanded-431605, India

ABSTRACT α,α-Dimethyl-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl] benzeneacetic acid hydrochloride treated with metal ion solutions of Fe(II), CO(II), Cu(II) using different experimental conditions, there is formation of metal complexes. After the purification of metal complexes, they are characterized by analytical, thermal, magnetic, Infrared, X-ray diffraction methods. After analysis of the metal complexes, expected geometry and structure of prepared metal complexes were determined. Key Words:- synthesis of Fe (II), Co (II), Cu (II) metal complexes, Analytical, IR, XRD study. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION α, α- Dimethyl-4- [1-hydroxy-4-[4- (hydroxydiphenylmethyl) -1-piperidinyl] butyl]benzeneacetic acid hydrochloride (DHB) having molecular formula C32H39NO4.HCl. OH N . C H 2. C H 2. C H 2. C H

HO

CH3 C

COOH

CH3 .H Cl

Considering the presence of oxygen donor atom we have used α,α-Dimethyl4-[1-hydroxy-4-[4(hydroxydiphenylmethyl)-1-piperidinyl] butyl]benzeneacetic acid hydrochloride (DHB) as a complexing agent to prepare the metal complexes of Fe(II), Co(II), Cu(II). Presence of oxygen donor atom provides a magnitude of bonding possibilities1. The pH of compound is acidic. When the solution of this compound is treated with transition metal ion solution at different pH and different laboratory conditions, the precipitate appears indicating the formation of complex. The prepared complexes are filtered, purified and washed with different solvents. Precipitate is dried in oven at 70 to 800C . The physical parameters of the prepared metal complexes like colour, yield, nature, decomposition point, M:L ratio, conductance, magnetic susceptibility were recorded. Also chemical parameters like estimation of metal ions, estimation of chloride were carried out by standard methods2. The coordinate bonding between donor atom and the metal ion is interpreted by IR spectra3. The electronic spectrum of the complexes also used to find out different transitions. X-ray diffraction method is used to find out the crystal structure of the prepared complexes. After considering physical, chemical, spectral parameters, the proposed structure of prepared complexes is given.

EXPERIMENTAL 0.1 M alcoholic solution (60 ml) of Metal salt were mixed with 0.1 M alcoholic solution (120 ml ) of DHB in round bottom flask. The pH of reaction mixture is adjusted to 7.2 by adding alcoholic ammonia.

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The long vertical water condenser is connected to water bath and the round bottom flask containing reaction mixture kept in water bath for refluxion. It is refluxed for 3 hours. The precipitate appears and it is filtered through ordinary filter paper and is washed with alcohol for three times. Obtained product is dried and stored in sample glass bottle.

RESULTS AND DISCUSSION Characterization of the prepared complexes After preparation of metal complexes yield physical characterization like nature, colour,decomposition point Âľeff of the prepared complexes are measured . The M:L ratio of prepared complexes were determined by heating known weight of complex slowly at beginning later on strong flame. To determine M:L ratio, TGA graphs are also used. Elemental analysis of the prepared complexes was obtained from IICT Hyderabad. The results of elemental analysis are given in the table For Estimation of chloride vollards method is used2. Non coordinated water present in the metal complexes was recorded by keeping the known weight of complex in previously weighed crucible in oven at 800C for one hour. The difference in weight gives the amount of non coordinated water in the complex. Thermal study of complexes Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) techniques are used to determine the decomposition nature of the complex. The samples were heated in the temperature ranges room tempt-10000C. The range of temperature and the experimental and calculated mass losses of the decomposition reactions are given in the Table-2. Thermal study of Fe (II) complex Thermogram of Fe (II) complex shows weight loss corresponds to one mole of hydrated water molecule in the temperature range 45-980C. After this Fe (II) complex shows sharp decrease in weight indicating decomposition of organic part of complex corresponds to 82.88 percent which is comparative with the theoretical value. In the temperature 827-10000c horizontal nature of thermograph indicates stable residual metal oxide as Fe2O34. The percentage weight of metal oxide was found to be 15.08 percent which is close to theoretical value 14.92. Thermal study of Co (II) complex TGA of Co (II) complex shows weight loss corresponding to one mole of lattice water in temperature range 50-960C. Decomposition beyond this temperature in the range 96 â&#x20AC;&#x201C; 2310C corresponds to mass loss of 6.83% in the TG curve assigned to expulsion of two water molecules from the complex5. This temperature range also indicates that they are present in coordination sphere. The decomposition occurs in the temperature 231-8000C indicates the loss of organic part content of complex. This loss is found to be 78.79percent.The end product of decomposition is formation of CoO, weight corresponds to 11.27 percent which is equal to theoretical value 11.50. Thermal study of Cu(II) complex The first step of decomposition in the case of Cu (II) complex occurs in the temperature range 50-890C, Corresponds to mass loss 2.89%. This loss corresponds to loss of one mole of lattice water. The decomposition of organic part of complex occurring with weight loss continues up to 7900C. The total loss in this temperature range is found to be 83.98 percent. It is clear that the final product of decomposition as computed from thermogram corresponds to metal oxide 12.57percent6. It is in agreement with theoretical value. IR spectra of metal complex The IR of ligand and its complexes with Fe(II), Co(II), Cu(II) metal ions were obtained from SAIF, IIT ,Chennai. The IR spectra of ligand is compared with the metal complexes. There are changes in IR spectra of metal complexes gives donor site of ligand. Infra Red spectral data of ligand (DHB) and metal complexes are given in the Table-3.

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IR spectrum of ligand shows strong band at 3297cm-1 attributed to ν(O-H) alcoholic stretching vibration. Intense band at 1705cm-1 attributed to stretching vibrations of ν(C=O). Ligand also exhibits strong bands at 2936cm-1, 1167cm-1 assigned to stretching vibration of ν(C-H) and ν(C–N) groups7 respectively. Study of IR spectra of Fe (II) complex IR spectra of Fe (II) complex reveals that, the band observed in the range 3000-3297cm-1 due to stretching vibration of primary and secondary alcoholic ν(O-H) group in ligand and is shifted in complex towards higher stretching vibration appears at 3407cm-1 attributed to involvement in coordinate bond. The weak band in ligand at 2946 cm-1 due to ν(O-H) of carboxylic group is shifted in complex and merge in the range 3407-2950 cm-1 indicated participation in coordination8. New band in complex observed at 455 cm-1 indicating formation of ν(M-O) bond. If the comparision is made between spectrum of ligand and Co (II) complex gives very useful information. A broad band observed at 3000-3297 cm-1 due to primary and secondary ν(O-H) group shifted to higher frequency appears at 3402 cm-1 indicating coordination of alcoholic (OH). similarly band at 2946 cm-1 in ligand due to carboxylic ν(OH) shifted appear at 3407 cm-1 indicates involvement in coordination. Formation of ν(M-O)9 bond in complex is supported by the appearance of band at 445cm-1. Similarly comparision is made between IR spectra of ligand and Cu (II) complex. Ligand exhibits broad band in the region 3000-3297cm-1 which can be assigned to ν(O-H) stretching vibration which may be due to intermolecular hydrogen bonding. This broad in complex is shifted and appears in at 3640–3400 cm-1 region10. Band at 449 cm-1 due to ν(Cu-O) band11. The stretching vibration in the ligand appears at 1705cm-1 due to carbonyl group12. These vibrating bands were not observed in complex in Fe (II), Co (II) and Cu (II) similarly ν(C-N) band stretching in ligand is at 1167cm-1 and in all complex they observed in the range 1165-1087 cm-1.In case of Co (II) and Cu (II) complexes band observed at 322 and 347 cm-1 respectively in the far infra red spectrum is due to ν(M-Cl) vibration which indicates the presence of chloride bridge13. X-ray diffraction study X-ray diffraction study of Fe (II), Co (II) and Cu (II) were scanned in the range 2θ = 10 – 800C.With the help of X-ray diffraction technique it is possible as certain the special arrangement of the structural units substance in crystalline state and employed in investigating the interior of a crystal. Value ‘d’ is calculated by using Bragg’s equation18 nλ = 2d sin θ The crystal lattice parameters and complete powder diffractogram data Fe (II) complex is given in the Table-4. From the cell data and crystal lattice parameters one can conclude that Fe (II) complex is having monoclinic crystal system. Cell data and crystal lattice parameter of Cu (II) complex attributed to Orthorhombic crystal system14.

CONCLUSION On the basis of crystal lattice parameters, analytical, spectral data of Fe (II), Co (II) and Cu (II) complex, the proposed structure for complexes are as shown in following figure. Table-1: Analytical data and other physical properties of metal complexes.

Metal Complex [Fe (DHB)2 ] H 2O [Co(DHB)Cl 2H2O].H2O

Mol. Wt.

Colour

1074

Orange

647.5

Light green

% of yield

M.L. ration

Molar Cond. (S cm2 mol-1)

247

79

1:1

12.56

4.93

259

87

1:1

14.96

3.85

D.P 0C

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205

µ eff BM

Elemental analysis Found (Calcd) C

H

N

70.64 (70.50) 59.82 (59.30)

7.08 (7.26) 6.83 (6.64)

2.61 (2.60) 2.41 (2.16)

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616

Light green

281

85

1:1

16.30

62.02 (62.33)

2.15

6.73 (6.33)

2.46 (2.27)

OH N

H 3C O H 3C

-

OH

O

Fe

H 2O O

-

O

CH3

OH CH3

N OH

Bis α,α-Dimethyl-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]benzeneacetic acid hydrochloride (DHB) Fe (II) complex [Molecular formula = C64H76N2O9.Fe; Mol. wt. = 1072]

H

H O

Cl OH N H 2O

Co H O OH H

O O

-

CH3 H 3C

Monochloro α,α-Dimethyl-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]benzeneacetic acid hydrochloride (DHB) diaquo Co (II) complex. [Molecular formula C32H43NO7 Cl Co; Mol. wt. = 647.5] Table-2: Thermoanalytical results of Fe (II), Co (II), Cu (II) complexes

Complex

[Fe (DHB)2].H2O

Total loss Theoretic Expt. al 85.08

84.82

[Co (DHB) Cl 2H2O]. H2O

88.50

88.60

[Cu (DHB).Cl] H2O

87.10

86.91

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Tempt. Range 0C 45-98 98-827 827-1000 50-96 96-231 231-800 800-1000 50-89

Loss (%) 1.92 82.88 15.08(residue) 14.92 (calcd.) 2.98 6.83 78.79 11.27 (residue) 11.50 (calcd.) 2.89 (2.93)

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83.98 12.57 (residue) 12.90 (calcd.)

Cl

Cu H 2O O

-

CH3

O

OH

CH3 OH N

Proposed structure of Mono chloro α,α-Dimethyl-4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1piperidinyl]butyl]benzeneacetic acid hydrochloride (DHB) Cu (II) complex, [Molecular formula : C32H39Cl2NO5Cl Cu; Mol. wt. : 616] Table-3: Infra Red spectral data of ligand (DHB) and their Fe (II), Co(II) and Cu(II) metal complexes. ν (O-H) Alco. 3297 3407

Compound FEHC [Fe (DHB)2].H2O [Co (DHB)Cl 2H2O].H2O

3402

[Cu(DHB)Cl].H2O

3400

ν (O-H) (Carb) 2946 3047 34072936 30003297

ν (OCH3)

ν (C=N)

ν (C-H)

ν (M-O)

995 995

1167 1165

2936 2960

455

995

1165

2970

445

995

1087

2961

449

Table-4: Cell data and crystal parameters for [Fe (DHB)2].H2O complex a (A0) = 21.696800 ± 0.045393 b (A0) = 23.311490 ± 0.175393 c (A0) = 27.432940 ± 0.136789 standard deviation = 0.0061989% = 0.61% α= 900 β=800 γ = 900 I/Io 74 100 69 70 62 51 49 42

Dobs 4.157905 4.086196 3.902708 3.682723 3.440960 3.310913 3.152457 2.847374

Volume (A0)3 = 13875.05 Dcal = 1.0260 g/ cm3 Dobs = 1.2613 g /cm3 Z = 8 Crystal system = Monoclinic Porosity (%) = 18.65% Dcal 4.177268 4.066647 3.898463 3.681059 3.453711 3.611283 3.154262 2.843286

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2.758823 2.455439 2.348712 2.179420

2.750708 2.458866 2.351279 2.180199

6 8 9 9

2 4 2 3

6 0 1 4

Table-5: Cell data and crystal parameters for [Co (DHB) Cl 2H2O] complex a (A0) = 21.678130 ± 0.038075 Volume (A0)3 = 13948.54 0 b (A ) = 23.309430 ± 0.069802 Dcal =1.2329 g/ cm3 0 c (A ) = 27.604200 ± 0.265683 Dobs = 1.3869 g/ cm3 standard deviation = 0.0052725 Z =16 = 0.52% Crystal system = Orthorhombic α= 900 β=900 γ = 900 Porosity (%) = 11.10 I/Io 60 100 67 71 82 53 46 39 42 32 30 32 20

Dobs 4.151308 3.961926 3.845215 3.631565 3.396491 3.229337 3.078964 2.847374 2.817214 2.730600 2.251333 2.080324 2.049391

Dcal 4.136347 3.968540 3.867676 3.626518 3.395751 3.231217 3.086347 3.846226 2.816262 2.727429 2.244817 2.082117 2.047135

h 5 4 5 5 3 6 6 7 7 6 8 10 9

k 0 4 1 1 6 2 3 2 3 5 4 3 6

l 2 0 3 4 1 3 3 3 2 3 5 1 0

From above data it is clear that Co (II) complex is having orthorhombic crystal system. Table-6: Cell data and crystal parameters for [Cu (DHB)Cl] H2O complex a (A0) = 21.649080 ± 0.020452 Volume (A0)3 = 14123.17 0 b (A ) = 23.425320 ± 0.046850 Dcal = 1.1584 g/ cm3 Dobs = 1.2738 g/ cm3 c (A0) = 27.848850 ± 0.102685 standard deviation = 0.0040036 Z =16 = 0. 4% Crystal system = Orthorhombic 0 0 0 α= 90 β=90 γ =90 Porosity (%) = 9.05 I/Io Dobs Dcal h k l 52 4.151308 4.134542 5 0 2 93 4.023111 4.018698 5 2 1 100 3.788123 3.781839 3 5 2 62 3.631565 3.632316 5 1 4 56 3.396413 3.403491 2 5 5 49 3.269790 3.275396 6 3 0 63 3.152457 3.159296 5 5 1 34 2.909781 2.911418 7 1 3 32 2.817214 2.815985 7 3 2 32 2.649334 2.656433 8 0 2 72 2.478043 2.476541 6 6 4 32 2.328537 2.330441 7 3 7 19 2.270120 2.271303 7 7 0 16 2.019460 2.019104 8 5 7 16 2.733027 1.728565 7 9 8

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REFERENCES 1. M.Tumer. H. Koksal, S.Serin and S. Patat, Synth. React. Inorg. Met. Org. Chem. ,27, 59 (1997). 2. Vogel; A.I. A. Textbook quantitative chemical analysis, Revised by Bessett, J; Denny R.C. Jeffery, J.H. and Menohan, J. ELBS 5th Edn. London (1996). 3. Koji Nakanshi, Infrared absorption spectroscopy (Practical Nankolo Company, London) (1964) 167. 4. C. Duval , Inorganic Thermogravimetric analysis, 2nd. Edn Elsevier, Amsterdam (1963). 5. A.V. Nikolaev , L.I. Myachina and V.A. Logvienko , 1969, Thermal analysis (New York academic press). 6. P.K. Panchal , D.H. Patel and M.N. Pate , Synth. React Inorg Met. Org. Chem., 34 ,1223(2004). 7. K. Nakamoto, Infrared spectra of Inorganic and co-ordinated compounds John Wiley New York. (1969). 8. Nakanishi Koji 1964 Infrared absorption spectroscopy (Tokyo, Practical Nankolo company Ltd.) 9. K.C. Raju and P.K. Radhakrishanan, Synth. React. Inorg. Met. Org. Chem., 33, 1307 (2003). 10. A.M.Khedry, M.Gaber R.M. Issa, H.Ertn, Dyes Pigments, 67, 859 (2004). 11. H. Temel, S. Ilhan, M. Sekerci and R. Ziyadanogullari, Spectrosc Lett. ,35, 219 (2002). 12. Dhar D.N. and Gupta V.P. 1971 , Indian J. Chem. , 9, 818. 13. O. IBOpishok Singh, A. Bimola. Devi, R.K. Hemakumar Singm and R.M. Kadam, Asian J. Chem. 20(6) (2008) 4397 14. B.H. Mehta and Yogesh A. Swar, Asian J. Chem. ,13, (2001) 928 [RJC-725/2011]

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Vol.4, No.1 (2011), 210-216 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

STRUCTURE BASED DRUG DESIGNING OF p38 MAP KINASE INHIBITORS FOR THE TREATMENT OF OSTEOARTHRITIS Neelakantan Suresh GVK Biosciences Pvt. Ltd., 37, Sterling Road, Nungambakkam, Chennai -600034 (T.N.) India *Email: sura93@gmail.com ABSTRACT p38 MAP kinase is one of the important targets in the treatment of osteoarthritis and inflammation. The best highly active lead compound was docked into the active site of Human p38 MAP Kinase Inhibitor Complex 1IAN using Ligand fit of Cerius 2. The results demonstrate that lead compounds derived in this study could be considered to be a useful and reliable tool in identifying structurally diverse compounds with desired biological activity for the successful treatment of various types of osteoarthritis. Keywords: p38 MAP kinase; Osteoarthritis; Inflammation; Ligand Fit ; Docking © 2011 RASĀYAN. All rights reserved.

INTRODUCTION p38 MAP kinase is a key regulator in stress, inflammation, development, and cell death. Osteoarthritis (OA) is a common rheumatic disease that is characterized by a progressive loss of articular cartilage. Cartilage degeneration results from an imbalance between anabolic and catabolic processes due to the dedifferentiation and apoptosis of chondrocytes and increased synthesis of matrix degrading proteinases. There is increasing evidence that inflammation plays an active role in pathophysiology of osteoarthritis. Proinflammatory cytokines are secreted from the inflamed synovium and from activated chondrocytes. Cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) upregulate numerous cytokines from chondrocytes and synoviocytes as well as prostaglandin E2 and proteinases such as the matrix metalloproteinases (MMPs) and aggrecanases. The aggrecanses and the matrix metalloproteinases are thought to mediate the structural degradation of cartilage in OA1. p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases2.

EXPERIMENTAL Docking studies using Ligand Fit of Cerius 2 (Accelrys) The automatic docking of a flexible ligand into a protein active site is a critical step in the process of structure-based design. Ligand Fit provides structure-based design capabilities including binding site finding and flexible docking and scoring capabilities, allowing evaluation of compounds against a receptor site. Scores from Ligand Fit provide direct insight into the complementary features of ligands and their potential as lead candidates. LigandFit only requires a model or an experimental structure of the protein. No natural ligand or bindingsite information is required. LigandFit simplifies user intervention during docking by automatically moving the ligand, evaluating energies and checking whether the structure is acceptable. Fast flexible docking with Ligand Fit allows quickly evaluating and prioritizing series of compounds with respect to their ability to fit and bind to the receptor3. Steps for docking ligand in active site 1. Active site search by flood filling method.

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Vol.4, No.1 (2011), 210-216

2. 3. 4. 5. 6. 7. 8.

Fast conformational search for ligand in protein cavity. Fast grid method for evaluation of protein-ligand interactions. Scoring with both protein-ligand interaction energy and ligand internal non-bonded energy. Visualization of docked conformations in binding site cavity. Clustering of docked conformers. Multiple scoring functions. Consensus scoring .

Ligand fit is designed to dock a ligand or a series of ligand molecules into a protein binding site. During docking the protein is rigid while the ligand remains flexible allowing different conformations to be searched and docked within the binding site3. The three key steps in ligand fit are1. Site search The aim of the site search is to define the binding site of the protein, the position and shape of which will be used in the docking process. 2. Conformational search The Monte Carlo method is employed in the conformational search of the ligand. During the search, bond lengths and bond angles are untouched but only torsion angles are touched. 3. Ligand fitting After a new conformation is generated, the fitting is carried in two steps. First the non-mass weighted principle moment of inertia (PMI) of the binding site is compared with the non-mass weighted PMI of ligand3. If the fit PMI is above the threshold or not better than fitting results previously saved, no further docking is performed, if the fit PMI is better than previously saved results, the ligand is positioned into the binding site according to the PMI. The docking score is negative value of the non- bonded inter molecular energy between the ligand and protein. After the docking score is calculated for each orientation of the ligand, it is compared with the results saved previously. If the new one is better, it is saved. The process of conformational search and ligand fitting is iterated until maximum number of trials is reached. Finally, rigid body minimization is applied to the saved conformations of the ligand to optimize their positions and docking scores3. Steps followed for docking ligand into active site for ligand fit: 1. Potent molecules which can inhibit the action of p38 MAP kinase were taken. 2. Molecules with diversified similarities and pharmacophore features were selected from the literature. 3. The molecules which are to be docked in a receptor site were created in a SD file so as all the molecules are processed for the docking score at a time. 4. The active site of a protein is identified by the active site viewer which is processed by the flood flow algorithm. 5. The identification of the active site is located by the already docked ligand. 6. The Human p38 Map Kinase Inhibitor Complex IIAN protein molecule obtained from Protein Data Bank is selected, the set of molecules in the SD file are chosen and docking score is calculated. 7. Thus the docking score for a set of molecules are calculated through ligand fit. The training set of 3 active compounds, 3 moderately active compounds and 3 least active compounds were selected for docking into the active site of Human p38 Map Kinase Inhibitor Complex 1IAN obtained from Protein Data Bank4-8.

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The molecular structures of all the training set compounds with their IC50 values and of highly active compound 5 and least active compound 4 are represented in Table 1.

RESULTS AND DISCUSSION The docking score of the molecules show positive values. The Human p38 Map Kinase Inhibitor Complex 1IAN obtained from Protein Data Bank receptor is shown in Fig.-1. Training sets highly active molecule compound 5 of IC50 value of 0.005 uM shows a good dock score of 68.540 as seen in Fig.-2 and Fig.-3. Training sets least active compound 4 of IC50 value of 1000 uM shows low dock score of 31.764 as seen in Fig.-4. About four molecules shows dock score more than 30. Thus, these molecules can be used as the potential ligands for the inhibition of p38 MAP kinase.

CONCLUSION The best highly active lead compound was docked into the active site of Human p38 Map Kinase Inhibitor Complex 1IAN obtained from Protein Data Bank using Ligand fit and a good dock score of 68.540 was obtained when the ligand binded to the active site. The highly active molecules are further used to design more potent lead molecules against p38 MAP kinase inhibitors for the treatment of various types of osteoarthritis. Thus, we hope that the lead molecules generated from this structure based drug designing of p38 MAP kinase inhibitors would be helpful in identifying structurally diverse compounds with desired biological activity for the successful treatment of various types of osteoarthritis.

Fig.-1: Active site of p 38 MAP kinase. The Human P38 Map Kinase Inhibitor Complex 1IAN obtained from Protein Data Bank receptor

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Fig.-2: High active compound docked in active site.Training set highly active molecule compound 5 shows a good dock score of 68.540.

Fig.-3: High active compound 5

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Fig.-4: Low active compound 4. Training set least active compound 4 shows low dock score of 31.764 Table-1

Highly Active Compound used for Docking with High Dock score of 68.540 H N N

N

H N N N

O H

F

Compound 5, IC50 value of 0.005 uM Least Active Compound with Low Dock Score of 31.764 in the Active Site C l

C l

O

N N N N F

F

Compound 4 , IC50 value of 1000 uM

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Three highly active compounds

N S

H

H

N

N

N N

N

N

N

H N

S N

H N

N

N

N

N O

N O

H

H Compound 1, IC50=0.011 uM

H

F Compound 2, IC50=0.002 uM

F Compound 5, IC50=0.005 uM

Three moderately active compounds

N

F

H N H

N

O N

O N

O

O H N

N F

N H H

F Compound 6 , IC50=0.06 uM

S

H N

H N

F N

O

H

Compound 7, IC50=0.03 uM Three least active compounds

Compound 8, IC50=1.5 uM

F

O

O

N

Cl O

N

Cl

N

N

N

N N

N

N

N

N

N O

N

N

N

O F

H

O S

F

Compound 4 , IC50 = 1000 uM

N

N

O

Compound 3, IC50 = 1850 uM

Compound 9, IC50 = 990 uM

ACKNOWLEDGEMENTS All molecular modeling works were performed on a Silicon Graphics Octane R12000 computer running Linux 6.5.12 (SGI, 1600 Amphitheatre Parkway, Mountain View, CA 94043). Ligand Fit of Cerius 2 (Accelrys software) was used for docking of molecules in active site. The author thanks Dr. J.A.R.P. Sarma, Senior Vice President and Dr. S.Vadivelan, Senior Scientist, GVK Biosciences Pvt. Ltd., Chennai for their valuable guidance, providing software facilities and a great chance to work there.

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Vol.4, No.1 (2011), 210-216

REFERENCES 1. K.K. Brown, S.A Heitmeyer, E. B Hookfin, L. Hsieh, M. Buchalova, Y. O Taiwo and M. J Janusz; Journal of Inflammation, 22, (2008). 2. C. Pargellis, L.Tong, L. Churchill, P.F. Cirillo, T. Gilmore, A. G. Graham, P.M. Grob, E. R. Hickey, N.Moss, S.Pav and J.Regan, Nature Structural Biology 9, 268(2002). 3. Accelrys release notes (http://accelrys.com/) 4. B.J. Mavunkel, S. Chakravarty, J.J. Perumattam, G.R. Luedtke, X. Liang, D. Lim, Yong-jin Xu, M. Laney, D.Y. Liu, G.F. Schreiner, J.A. Lewicki and S.Dugar, Bioorg. Med. Chem. Lett., 13 (18), 3087 (2003). 5. J.E. Stelmach, L. Liu, S.B. Patel, J.V. Pivnichny, G.Scapin, S. Singh, C.E.C.A. Hop, Z. Wang, J.R. Strauss, P.M. Cameron, E.A. Nichols, S.J. O’Keefe, E. A. O’Neill and D.M. Schma, Bioorg. Med. Chem. Lett., 13 (2), 277 (2003). 6. L. Li, J.E. Stelmach, S.R.Natarajan, Meng-Hsin Chen, S.B. Singh, C.D. Schwartz, C.E. Fitzgerald, S.J. O’Keefe, D. M. Zaller, D. M. Schmatz and J. B. Doherty, Bioorg. Med. Chem. Lett., 13 (22), 3979 (2003). 7. R. Sarma , S. Sinha, M. Ravikumar, M. K. Kumar and S.K. Mahmood, Eur J Med Chem, 43(12), 2870 (2008). 8. K.K. Brown, S.A. Heitmeyer, E.B. Hookfin, L. Hsieh, M. Buchalova, Y.O. Taiwo and M.J. Janusz, J Inflamm (Lond), 5,22 (2008). [RJC-699/2010]

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Vol.4, No.1 (2011), 217-222 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com

SYNTHESIS AND CHARACTERIZATION OF ZINC OXIDE NANOPARTICLES AND ITS ANTIMICROBIAL ACTIVITY AGAINST BACILLUS SUBTILIS AND ESCHERICHIA COLI Haritha Meruvu, Meena Vangalapati*, Seema Chaitanya Chippada and Srinivasa Rao Bammidi Center for Biotechnology, Department of Chemical Engineering, College of Engineering, Andhra University, Visakhapatnam–530 003 (A.P.) India *E-mail: meena_sekhar09@yahoo.co.in ABSTRACT Metal nanoparticles have been intensively studied within the past decade. Nanosized materials have been an important subject in basic and applied sciences. Zinc oxide nanoparticles have received considerable attention due to their unique antibacterial, antifungal, and UV filtering properties, high catalytic and photochemical activity. The objective of this work is to synthesize Zinc oxide nanoparticles using chemical method and characterize zinc oxide nanoparticles using scanning electron microscope and X-ray diffractometer. Further its antimicrobial activity against Bacillus subtilis and Escherichia coli is studied. Key words: Nanoparticles, Zinc oxide, Bacillus subtilis, Escherichia coli. © 2011 RASĀYAN. All rights reserved.

INTRODUCTION Nanotechnology is the production and use of materials at the smallest possible scale7. Nanotechnology can be useful in diagnostic techniques, drug delivery, sunscreens, antimicrobial bandages, disinfectant, a friendly manufacturing process that reduce waste products (ultimately leading to atomically precise molecular manufacturing with zero waste), as catalyst for greater efficiency in current manufacturing process by minimizing or eliminating the use of toxic materials, to reduce pollution (e.g. Water and air filters) and an alternative energy production (e.g. Solar and fuel cells)1. Bionanotechnology is the integration between biotechnology and nanotechnology for developing biosynthetic and environmental friendly technology for the synthesis of nanomaterials1. Nano scale particles have emerged as novel antimicrobial agents owing to the high surface area to volume ratio, which is coming up as the current interest in the researchers due to the growing microbial resistances against metal ions, antibiotics and the development of resistant strains3.The recent growth in the field of porous and nanometric materials prepared by non-conventional processes has stimulated the search of new applications of ZnO nanoparticulate2. Zinc oxide is an interesting semiconductor material due to its application on solar cells, gas sensors, ceramics, catalysts, cosmetics and varistors4. In this work, the precipitation method was used followed by controlled and freezing drying processes6. The materials obtained were thermally treated at various temperatures. The influence of temperature on structural, textural, and morphological properties of the materials was studied by powder X-ray diffraction, infrared spectroscopy, scanning electron microscopy, nitrogen adsorption, and thermal analysis. Certain chemicals can interfere directly with the proliferation of microorganisms at concentrations that can be tolerated by the host. The antimicrobial activity of zinc oxide nanoparticles is well known. Hence we make use of this property to inhibit growth of Bacillus subtilis, Escherichia coli using disc diffusion method. These two bacterial strains were selected as they are highly contagious; thence we can evaluate the potential antimicrobial activity of zinc oxide nanoparticles11.

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Haritha Meruvu et al.

Vol.4, No.1 (2011), 217-222

EXPERIMENTAL Materials and Methods In this work, precursor of zinc oxide nanoparticles was synthesized by precipitation method. The chemicals used for synthesis are Zinc acetate 2.1g in 100ml, Ammonium carbonate 0.96g in 100ml, Polyethylene glycol (5%) 5g in 100ml. Instruments used for synthesis are Muffle furnace,Magnetic stirrer,scanning electron microscope (JOEL MODEL 6390)and X-ray diffractometer(SHIMADZU-MODEL XRD 6000). Synthesis of Zinc Oxide nanoparticles The zinc oxide nanoparticles were synthesized by precipitation the surfactant Solution (5%PEG) was poured into a three-neck flask, then zinc acetate ,ammonium carbonate were dropped into the flask at same time with vigorous stirring10 .After the reaction, the suspension was kept under stirring for 2 hours at room temperature, precipitate was filtered washed with ammonia solution and absolute ethanol several times, dried under vacuum for 12 hours, and then calcinated in an oven at 450oC for 3 hours. Then zinc oxide nanoparticles were obtained6. Method for Antimicrobial activity Materials used for antimicrobial activity of zinc oxide nanoparticles are Nutrient broth 1.3g,Nutrient agar 5.6g, Agar-agar 0.5g,petriplates ,antibiotic discs , cotton swabs ,zinc oxide nanoparticle sample ,bacillus subtilis,Escherichia coli .Disc diffusion method used for antimicrobial activity of zinc oxide nanoparticles. Preparation of Inolculum Nutrient broth (1.3 g in 100 ml D/W10) was prepared in 2 conical flasks and sterilized. In one conical flask clinically isolated strain of Bacillus subtilis, was inoculated. In the other conical flask clinically isolated strain of Escherichia coli was added. These bacterial cultures inoculated in nutrient broth were kept on rotary shaker for 24 hours at 100 r.p.m. Inoculation of test plate Nutrient agar is prepared(5.6gnutrient agar0.5g Agar Agar in100ml distilled water)9and sterilized.The agar suspension within 15 min is used to inoculate plates by dipping a sterile cotton-wool swab into the suspension and remove the excess by turning the swab against the side of the container. Then we spread the inoculum evenly over the entire surface of the plate by swabbing in three directions. Allow the plate to dry before applying antibiotic to discs. Preparation of Antibiotic discs Discs used for antimicrobial activity are nitrofuration, tetracycline, nalidixicacid, Vancomycin, amoxyclav, gentamycin, ciprofloxarin, erythromycin, ceftazdine and Methicillin. Discs should be firmly applied to the surface of an agar plate that has previously been dried. The contact with the agar should be even. A 60 mm plate will accommodate two discs and ZnO nanoparticles without unacceptable overlapping of zones. Agar plate is divided into 3 sections antibiotic disc, zinc oxide nanoparticles sample, and both antibiotic disc and zinc oxide nanoparticle sample. Disc diffusion method for Antimicrobial activity Antibacterial tests were carried out by the disc diffusion method using the suspension of bacteria spread on nutrient agar11. Dip the swab into the broth culture of the organism. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab to streak agar plate or a nutrient agar plate for a lawn of growth. This is best accomplished by streaking the plate in one direction, then streaking at right angles to the first streaking, and finally streaking diagonally. We end by using the swab to streak the outside diameter of the agar.The inoculated plates were incubated at appropriate temperature for 24hours.Antibiotic discs can be placed on the surface of the agar using a dispenser that dispenses multiple discs at the correct distance apart, or by obtaining individual discs and placing them on the surface of the agar using flame sterilized forceps. The antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms11. Finally we measure (mm) diameters of zones of inhibition of the control strain and test with a ruler, calipers. ZINC OXIDE NANOPARTICLES

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Vol.4, No.1 (2011), 217-222

RESULTS AND DISCUSSION Zinc oxide nanoparticles prepared from the solution of zinc acetate, PEG and ammonium carbonate. Here we get a new line of high purity zinc oxide nanoparticles primarily targeted for antimicrobial activity against Bacillus subtilis and Escherichia coli .as shown in figure 1.Zinc oxide nanoparticles prepared were characterized by scanning electron microscope and X-ray diffraction. Figures 2(a) and (b) shows SEM images Zinc oxide nanoparticles. Scanning electron microscope was used to decide size, location and shape of the Zinc oxide nanoparticles. These images demonstrated that zinc oxide nanoparticles are spherical in shape and their sizes are about 30-63nm. X-ray diffraction studies reveal the characterization through X-ray diffraction graph as shown in Figure 3.Here 12 peaks are noticed in accordance with zincite phase of ZnO.No peaks due to impurity were observed, which suggest that high purity zinc oxide was obtained. In addition the peak was widened implying that the particle size is very small. The average crystallite size D was calculated by the Debye Sherrer formula(D = Kλ/βcosθ) where K is the sherrer constant,λ Is the X-ray wavelength,β is the peak width at half-maximum,θ is the bragg diffraction angle .On substituting the values K =0.90 λ =1.5418A ,β = 0.05, cosθ =0.94; in the debyesherrer formula, ( 0.90×1.5418)÷ ( 0.05×0.94) = 30nm; the crystallite diameter 30 nm was obtained. Disc diffusion method was used for the assessment of antibacterial activity. Antimicrobial activity of ZnO nanoparticles against Bacillus subtilis is shown on basis of the Inhibition zone (mm) size in Table 1.Here the zone of inhibition is more for both Zno nanoparticles and antibiotics like nitrofurantoin, tetracycline, nalidixicacid, gentamicin, methicillin. Erythromycin has no zone of inhibition as Bacillus subtilis is not susceptible to Erythromycin. In table 2 Antimicrobial activity of ZnO nanoparticles against Escherichia coli is shown,here zone of inhibition is more for vancomycin, nalidixicacid as Escherichia coli is susceptible to these Antibiotics. Erythromycin and Amoxyclav has no zone of inhibition as Escherichia coli is not susceptible to these two antibiotics.

CONCLUSION Synthesis of zinc oxide nanoparticles was achieved by using zinc acetate, polyethylene glycol and ammonium carbonate by precipitation method. Detailed structural characterizations demonstrate that the synthesized products are spherical and crystalline in structure and their diameter was about 30nm. These structures clearly evident from SEM and XRD.SEM result were in accordance with X-ray diffraction. Due to the large specific surface Area and high surface energy, some nanoparticles aggregated. The aggregation occurred Probably during the process of drying.XRD Patterns of zinc oxide nanoparticles calcinated at 4500 C. the average particle size increased with the increase of calcinations temperature X-ray diffraction (XRD) with Cu-Kα radiation was used for checking the formation and identification of present compounds in the obtained particles. The average crystallite size D was calculated by Debye-sherrer formula. Microorganisms used for antimicrobial activity are Bacillus subtilis and Escherichia coli. The antibacterial activity performance of ZnO nanoparticles was done by using disc diffusion method. The disc diffusion method for antibiotic susceptibility testing is the Kirby-Bauer method. The agar used is Meuller-Hinton agar that is rigorously tested for composition and pH. Further the depth of the agar in the plate is a factor to be Considered in the disc diffusion method. This method is well documented and standard zones of inhibition have been determined for susceptible and resistant values. There is also a zone of intermediate resistance indicating that some Inhibition occurs using this antimicrobial but it may not be sufficient inhibition to eradicate the organism from the body. The zone of inhibition increases with the increase in Zinc oxide nanoparticle concentration and decrease in particle size.

REFERENCES 1. K. Sobha, K. Surendranath and V. Meena, Biotechnology and Molecular Biology Reviews, 5,01 (2010).

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2. H. Amekura, O.A. Plaksin, N. Umeda, Y. Takeda, N. Kishimoto and Ch. Buchal., Mater.Res.Soc.Symp.Proc ,1, 8.1.1 (2006). 3. Ho Chan and Ming-HsunTsai, Rev.Adv.Mater. Sci,.18,734 (2008). 4. U. Abhulimen, Mater.Res.Soc.Symp.Proc.,1, 27.1 (2005). 5. M. A. Shah and M. Al-Shahry, , JKAU: Sci ,2,61 (2009). 6. Atul Gupta, H.S.Bhatti, D.Kumar, N.K.Vermaa, R.P.Tandonb. , Journal of Nanomaterials and Biostructures,4,1 (2006). 7. Cheng-Hsien Hsieh, Journal of the Chinese Chemical Society,54,31 (2007) 8. Abdolmajid Bayandori Moghaddam,T ayebe Nazari, Jalil Badragh and Mahmood Kazemzad, Int. J. Electrochem. Sci., 4, 247 (2009). . 9. C.P.Rezende, J.B.da Silva, N.D.S. Mohallem, Brazilian Journal of Physics,1,248 (2009). 10. Robert.F.Mulligan, Agis.A.Iliadis and Peter Kofinas, Journal of Applied Polymer Science, 1,1058 (2003). 11. Nagarajan Padmavathy,Rajagopalan Vijayaraghavan,Enhanced Science and Technology of Advanced Material,8, 1 (2008).

Fig.-1: Picture of synthesized Zinc Oxide nanoparticles

Fig.-2(a): SEM images of ZnO nanoparticles ZINC OXIDE NANOPARTICLES

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Vol.4, No.1 (2011), 217-222

Fig.-2(b): SEM images of ZnO nanoparticles

Fig.-3: X-ray diffraction graph Table-1: Inhibition zone(mm) size against Bacillus subtilis by various antibiotics

Antibiotics

ZnO Nanoparticle and antibiotic

Only antibiotic

Organism

Only ZnO nanoparticle

Bacillus subtilis

Nitrofurantoin Tetracycline Nalidixicacid Vancomycin

22 24 20 17

17 20 18 25

7 12 10 6

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Vol.4, No.1 (2011), 217-222 Amoxyclav Gentamicin Ciprofloxacin Erythromycin Ceftazidime Methicillin

15 23 20 19 22

16 21 11 17 -

14 9 8 7 11 5

Table 2: Inhibition zone(mm) size against Escherichia coli by various antibiotics

Organism

Antibiotics

ZnO Nanoparticles And antibiotic

Only antibiotic

Only ZnO Nanoparticles

Escherichia coli

Gentamycin Erythromycin Ceftazidime Nitrofurantoin Vancomycin Methicillin Nalidixicacid Ciprofloxacin Amoxyclav Tetracycline

17 11 17 20 12 23 9 15

16 9 16 23 9 20 8 13

9 6 5 12 14 15 11 7 8 6

[RJC-709/2011]

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