PHARMBIT, XXIII & XXIV (1 & 2), Jan - Dec, 2011

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Chief Editor : Dr. R. N. Gupta Associate Editor : Dr. Manik Ghosh

Editorial Advisory Board Prof. B. K. Gupta Chairman, Gluconate Ltd., Kolkata Dr. Somlak Kongmuang Silpakorn University, Thailand Dr. Roop K. Khar Jamia Hamdard, New Delhi Dr. Sunil K. Gupta, Impex Pharmaceuticals Hayward, USA Dr. Malay Chatterjee, Jadavpur University, Kolkata Dr. P. Ramkumar School of Pharmacy, Malaysia Dr. U. K. Mazumdar Jadavpur University, Kolkata Dr. Susmita Chanda Roche, San Fransisco, USA Dr. T. R. Krishanan FDA, Canada Dr. Suresh K. Saravdekar Health Dept., Maharashtra Dr. Sampad Bhattacharya Sun Pharma Ltd., Baroda Dr. Maya Prakash Singh Wyeth Research, Newyork Mr. Anjani Kumar Cipla Ltd., Mumbai Dr. Shivaji Singh Navtech LLC, Atlanta, USA Dr. P. R. Vavia, ICT, Matunga, Mumbai Dr. G. N. Singh, Director, CIPL, Ghaziabad Dr. P. H. Rao ASCI, Hyderabad Prof. B. G. Shivananda, Principal, Al-ameen College of Pharmacy, Bangalore

Contents z Oral Pre Cancerous Lesions (OPL) : A Clinicopathological and

5

Molecular Assessment for Risk Categorization and Treatment Asoke Roy, M. Chatterjee, S. P. Bhatnagar z A Review on Biological Synthesis of Gold Nanoparticles

11

and its Application Sharmistha Banerjee, Sneha Singh, Swati Tikariha, A.S. Vidyarthi z Surfactant Properties of Non Aqueous Microemulsions

17

Shekhar Verma, J.S. Dangi z Design, Optimization and Evaluation of Nanoemulsion

26

Formulations by Ultrasonication as Vehicle for Transdermal Delivery of Diclofenac Diethylamine Praveen Kumar Gupta, Dr. J.K. Pandit, Dr. P. J. Narain, Sanjiv Kumar Gupta, Akash Chaurasya z Using Albino Rats in Your Experiment?

40

Make Sure They Are Healthy Saibal Das, Ankur Datta1, Suvajit Das, Patralekha Roychowdhury, Anuradha De (Pati), S.K. Tripathi z Comparative Hepatoprotective Activity of the Two Varieties

43

of Cajanus cajan (L.) Millsp. Leaf Suman Goswami, S. Jha, A. K. Pattanayak z Structural Optimization of New Class of Anticonvulsants :

48

QSAR Approach Lata K Bisht, Subir S. Samanta, Saumya P Basu z 5HODWLYH (IĂ€FLHQFLHV RI WKH 'LHWDU\ 3URWHLQV RI 3ODQW DQG

Animal Origin : A Comparative Study Mukul Chandra Gope, Ratna Ghosh, Rupali Patra, Manik Ghosh z Isolation and Characterization of a Novel Smart

57

Mucoadhesive Biopolymer from Euryale ferox Seed Coat M.S. Uma Shankar & N. V. Satheesh Madhav z Evaluation and Characterization of Solid-Self Nanoemulsifying

62

Drug Delivery Systems (S-SNEDDS) of Lovastatin Samridhi and Dr. R.N. Gupta z Estimation of Heavy Metals in Locally Available

68

Vegetables Collected from Road Side Market Sites (1-4) of Different Areas of Ranchi City Ratna Ghosh, Reshma Xalxo, Mukul Chandra Gope, Sougata Mishra, Bindu Kumari, Manik Ghosh z Development of Lornoxicam Transdermal Patch:

74

Effect of Natural Penetration Enhancers Dipti Srivastava, Nishant Kumar Verma, Nimisha, Wasim Ahmed z Instructions to Authors

79

Editorial Board Members Abhimanyu Dev

Dr. Uma Ranjan Lal

Sugandha Kumari

Ankita Gupta

Akshay Shrimal

Subham Sonu

Vibha Sharma

Sougata Misra


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

EXECUTIVE COMMITTEE OF PHARMACEUTICAL SOCIETY Dept. of Pharmaceutical Sciences, B.I.T., Mesra, Ranchi Patron President Prof. In-charge Co-Prof. In-charge Treasurer Joint Treasurer Chief Editor-PHARMBIT Associate Editor Cultural Incharge Co-Cultural Incharge Vice President Secretary Jt. Secretary

: : : : : : : : : : : : :

Dr. Ajay Chakrabarty, Vice Chancellor Dr. P. R. P. Verma, HOD, Pharmaceutical Sciences Dr. S. Samanta Dr. (Mrs.) S. M. Verma Dr. S. Jha Dr. (Mrs.) A. Pandey Dr. R. N. Gupta Dr. Manik Ghosh Prof. (Mrs.) S.G. Panpalia Dr. (Mrs.) Trishna Bal Jyotsna Singh Saurav Ganguli, Mayuri Swati Bansal, Amit Kumar

ADVISORY COMMITTEE Dr. D. Sasmal Dr. (Ms.) S. Ganguly Dr. J. Venkatesh Dr. S. P. Pattanayak Mrs. Nibha Mishra Dr. Animesh Ghosh Dr. Uma Ranjan Lal

Dr. B. N. Sinha Prof. (Mrs.) S. G. Panpalia Dr. (Mrs.) P. M. Mazumdar Sri A. K. Patnaik Sri B. Sarkar Sri Dinesh M. Biyani Dr. K. K. Pradhan

Dr. (Mrs.) M. Mukherjee Dr. Jayaram Kumar Sri A Basu Sri Abhimanyu Dev Dr. Sandeep K. Singh Dr. (Mrs.) Neelima Sharma Dr. S. Banerjee

Kahkashan Resham Md. Javed Aktar Abhijeet Mihir Pallavi Gupta

Tripta Kumari Vikash Kumar Swati Anand Prateek Mathur

CLASS REPRESENTATIVES Surya Prakash Samridhi Akshay Shrimal Nalini Singh

REVIEWERS PANEL Dr. B. Mishra, Varanasi Dr. Biswajeet Mukherjee, Kolkata Dr. Javed Ali, Delhi Dr. S. K. Kaushal, Kolkata Dr. P. Suresh, Visakapatnam Dr. T. K. Ravi, Coimbatore Dr. S. C. Mondal, Kolkata Dr. P. N. Murthy, Behrampur Dr. S. K. Sharma, Hisar Dr. D. N. Misra, Hisar

Dr. G. Vidyasagar, Bhuj Dr. (Mrs.) Sarsija Suresh, Bangalore Dr. T. V. Narayana, Bangalore Dr. M. P. Rana, Kolkata Dr. Suresh K. Saravdekar, Mumbai Dr. L. K. Ghosh, Kolkata Dr. B. B. Barik, Bhubneshwar Dr. P. K. Manna, Annamalainagar Dr. G. Parthasarthi, Mysore Dr. S. Madhav, Dehradun


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

From the Editor’s Desk It is a matter of great pleasure for us to bring out the combined YROXPH QR ;;,,, ;;,9 RI 3+$50%,7 VFLHQWLÀF -RXUQDO 7KH MRXUQDO has been accepted for indexing in Chemical abstract published by American Chemical Society from Jan-June 2007 and Natural Science database (www.csa.com) from Jan-June 2008. In this issue 12 papers (3 Review and 9 Research) have been included covering development of formulations, Standardisation, Drug Design, pharmacological and Pharmacognostical evaluation of plant drugs etc. Our country is VHOI VXIÀFLHQW LQ PDQXIDFWXUH RI EXON GUXJV DQG IRUPXODWLRQV GXH WR advancement of pharmaceutical education and technology both during last 20 years. Since as pharmacist our intention is to serve the masses for better health care hence the Pharmaceutical research should be oriented to masses for the treatment of diseases at affordable prices. It LV QRWHG WKDW VHYHUDO WKRXVDQG RI 3DWHQW DSSOLFDWLRQV DUH EHLQJ ÀOHG RQ Pharmaceuticals month wise to get it commercialise after sealing of it. Recently the historical decision by Dr. P.H. Kurian, Controller General of Patent, Government of India to grant Compulsory License to Natco Pharma, Hyderabad to manufacture and Sale the anti cancer drug‘ Nexaver (Sorafenib Tosylate)’ at a price of Rs. 8000.00 only for one month dose where as the same drug was available at Rs.2,88,000.00 per month dose by the Patentee in our Country. It is desired that the Pharmaceutical Patentee/Researchers will work IRU WKH EHQHÀW RI PDVVHV EXW QRW IRU H[SORLWDWLRQ RI VXIIHULQJ PDVVHV through new drugs and advanced drug delivery system. Hope our readers will motivate and inspire others too. As an editor of this journal, I am thankful to Authors, Reviewers, Editorial Board Members, Advertisers, well wishers, subscribers, Faculty members and HOD of department, Vice-Chancellor, BIT, Mesra for their support and encouragement in bringing out this edition of PHARMBIT journal. Lastly I invite suggestions from readers and well wishers to further improve journal. Dr R. N. Gupta


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Oral Pre Cancerous Lesions (OPL) : A Clinicopathological and Molecular Assessment for Risk Categorization and Treatment 1

Asoke Roy1, M. Chatterjee2, S. P. Bhatnagar3 Department of Pathology and Cancer Screening, Chittaranjan National Cancer institute, Kolkata, India 2 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India 3 Department of Pharmaceutical Sciences, BIT, Mesra, Ranchi, India

ABSTRACT In India, oral cancer is the most common cancer in men. Besides smoking, use of smokeless tobacco, betel quid chewing and use of alcohol are the common habits in this part of the world and regarded as the causative agents for oral cancer. Several micro organisms such as Human Papilloma Virus (HPV) are related to the development of this type of malignancy. The concept of the oral pre-cancer now clearly DVVXPHV WKDW D VLJQLÀFDQW SHUFHQWDJH RI RUDO SUH PDOLJQDQW OHVLRQV DUH OLQNHG WR WKH FHOOXODU FKDQJHV OLNH dysplasia and transformation into malignancy. Mainly two types of Pre-malignant oral lesions leukoplakia and erythroplakia are linked to the development of oral malignancy. No western study has determined the incidence of oral leukoplakia, although in India new lesions have shown to occur in 0.8/1,000,000 persons annual oral cancer in the same population is about 5/1,000,000 (Pindborg, 1980). However another study by Li Mao et al, 1999, indicated the incidence ranges from 1% to 10% in adults 40-50 years of age. Out of many risk factors considered for the development of oral malignancy, including other Head and Neck Squamous Cell Carcinoma (HNSCC) the synergistic effect of tobacco and alcohol in oral tumerogenesis has been suggested by many epidemiological studies (Deeker 1982; Nam, 1992). However, only some smokers and alcohol consumers and other addicts develop oral cancer. Therefore, it is desirable to develop novel screening and detection strategies to identify individuals with the highest risk of developing oral malignancy. Under such circumstances histopathological assessment along with cytogenetic and molecular alterations has been proposed. Oral retinoids have been tried to treat OPL; however, recurrence is common after cessation of treatment. Key words : Cancer, leukoplakia, erythroplakia, p53, ki67, PCNA, AgNORs, retinoic acids INTRODUCTION Once upon a time heart disease was considered as the number one killer disease, but in 2011, cancer is considered as the world’s top killer. According to the WHO estimate both the cancer incidence and death will be doubled by 2030. Cancer diagnoses around the world have steadily been rising and expected to hit 12 million this year as well as 7.9 million deaths will occur according to the report by WHO. Annually, over 3,00,000 new cases of oral cancer are diagnosed all over the world and majorities are diagnosed in the advanced stage (Stage III or IV). Approximately 90% of oral cancers are detected above the age of 40, and more than 50% of all cancers occur in persons older than 65 (Edwards BK, 2002). However, recent evidence indicates oral cancers are more common at or around the age of 40 years (Ries et al, 2003). Oral cancer is dominated by squamous cell carcinoma and accounts 90 to 95% of all oral cancers (Sham et al, 2003). Often, these malignancies begin as pre-neoplastic lesions with or without LQà DPPDWRU\ FKDQJH VXFK DV OHXNRSODNLD HU\WKURSODVLD DQG HU\WKURSODNLD 2UDO SUHPDOLJQDQW OHVLRQV DQG RUDO FDUFLQRPD DUH FRQVLGHUHG WKH UHVXOW RI D ÀHOG FKDQJH LQ WKH RUDO FDYLW\ $ SUHFDQFHURXV OHVLRQ 5


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

is histologically altered epithelial tissue that may progress to invasive squamous cell carcinoma. The two clinicopathological conditions are leukoplakia, the most common form, and erythroplakia. Both the lesions present either homogenous or non-homogeneous or as nodular form. /HXNRSODNLD DFFRUGLQJ WR :+2 GHÀQHG DV ZKLWH SDWFK RU SODTXH WKDW FDQQRW EH FKDUDFWHUL]HG FOLQLFDOO\ RU SDWKRORJLFDOO\ DV DQ\ RWKHU GLVHDVH 7KLV GHÀQLWLRQ KDV QR KLVWRORJLFDO FRQQRWDWLRQ EXW WKH lesion is considered by pathologists to be parallel to hyperkeratosis. But the new concept of oral leukoplakia, by Warnakulasuriya et al, shall acknowledge white lesions with questionable risk of being an oral leukoplakia excluding any other pathologies and diseases such candidiasis, lypus erythematosis, lichenplannus, hairy leukoplakia, frictional keratosis, nicotin stomatitis and leukodarma which do not carry any risk for malignancy (Warnakulasuriya et al, 2007). Oral OHXNRSODNLD HQFRPSDVHV WZR EURDG FDWHJRULHV DV OHXNRSODNLD ZLWKRXW DQ\ GHÀQLWH HWLRSDWKRJHQHVLV and oral leukoplakia with tobacco use and use of other forum of addictions (Axcell et al, 1996). Besides smoking for the development of oral premalignant lesions, use of smokeless tobacco is widely prevalent. Betel quid (Pan) has been directly linked to the development of oral premalignant lesions. Gutka in recent years commercially available sachets, khaini, kharra, zarda etc. all contribute to the development of pre-malignant oral lesions and subsequently malignancy. No western study has determined the incidence of oral leukoplakia adequately although in India new lesions have been shown to occur in 0.8/1000000 persons annually as mentioned earlier but it is assumed that all oral cancers in India do not arise from pre-malignant oral lesions, but a large portion of them probably do so. The majority of the oral leukoplakic lesions are caused by tobacco although variation in location IRUP SHUVRQ WR SHUVRQ KDV EHHQ REVHUYHG ZKLFK DOVR KDV EHHQ LQà XHQFHG E\ JHRJUDSKLFDO YDULDWLRQ also (Banoczy, 1982). In India, anatomatically, 88% of lesions were located in the labial commissures DQG EXFFDO PXFRVD DQG UHVW DUH ORFDWHG LQ WKH WRQJXH ZKHUHDV à RRU RI WKH PRQWK LV IDLUO\ FRPPRQ for pre-malignant oral lesion in Danish and US populations (Mehfa, 1969). The multiple genetic abnormalities occurred in the upper aero digestive tract were generally due to the constant carcinogenetic exposure of this area (Slaughter et al, 1953). As a result multiple squamous FHOO OHVLRQV RI RUDO ÀHOG FDQFHUL]DWLRQ SURFHVV GHYHORSHG LQGHSHQGHQWO\ DQG SURFHHG IXUWKHU RISK FACTORS Tobacco and Alcohol use : Tobacco use is known risk factors for the development of oral premalignant lesions and cancer of the oral cavity. In India 57% of all men and 11% of women between 15.49 years of age use some form of tobacco (Sankaranarayan et al, 1998). The majority of oral leukoplakic lesions are caused by tobacco. A special type of homogeneous leukoplakia, a lesion with pumice like appearance, is associated with tobacco use. Acquisition of the tobacco habit occurs early in life through imitation of the family members of peers. In India both form of tobacco such smoking as well as use of smokeless tobacco is widely prevalent. The association of the development leukoplakia and use of tobacco is strong and dose-response relationship. A high frequency of leukoplakia is observed in population with a high prevalence of tobacco habits, particularly chewing, such as those in South and South East Asia (Ramdas, 2009). It is more frequently observed in men, due to their high prevalence of tobacco habits. 6


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A study by Gavarasana et al. in East Godawari, A P. India found that in reverse smokers (smoking with lighted end inside the mouth) had 13.84 times higher incidence of leukoplakia than that of normal smokers. Interestingly, in that area reverse smoking is most common among females. In a ten year VWXG\ RI SURIHVVLRQDO EDVHEDOO SOD\HUV LQ 86$ OHXNRSODNLD ZDV LGHQWLĂ€HG LQ DSSUR[LPDWHO\ RI \HDU round smokeless tobacco users (Sinasun et al, 1990) Both the tobacco and alcohol have suggested as the primary risk factors for oral pre malignant lesions. The role of alcohol in risk of oral leukoplakia has been poorly studied and the results are inconsistent (Graham et al 1977, Jaber et al 1998). However a study on a large scale of population shows that 22% higher risk in those cases additional alcohol beverage consumed daily. Chewing betel quid : The use of betel quid (Pan consisting pieces of areca nut along with processed or unprocessed tobacco and aqueous calcium hydroxide (slaked lime) rapped in the leaf of piper betel vine leaf is very common in India. Typically it is kept in the check and chewed 10-15 minutes or kept it LQ RYHU QLJKW 7KH XVH RI EHWHO TXLG VLJQLĂ€FDQWO\ DVVRFLDWHG ZLWK LQFUHDVH WKH ULVN IRU RUDO SUHPDOLJQDQW lesions. Even when tobacco is not combined with it (Jacob et al, 2004) Viral infection : High risk Human Papilloma Virus (HPV 16 and 18) are found in 15-42% of oral leukoplakia, in 50% of erythorplakia and in 50-100% of oral squamous cell carcinoma (Gassenmaier HW DO 7KH SURJQRVWLF VLJQLĂ€FDQFH RI +39 SUHVHQFH LQ RUDO SUH FDQFHU LV \HW WR EH GHWHUPLQHG by large follow up investigations. However, a study by Nielsen et al for 6.3 years for 49 cases of OPL found that 3 cases of oral premalignant lesions were positive for HPV and subsequently these cases turned into malignancy. Mouth Wash : The United States FDA has approved the inclusion of “Bloodrootâ€? (Sanguinaria canadensis) in toothpastes, mouthwash and dental wash products as an antibacterial or anti-plaque agent. However, this is controversial. Currently, it is believed that this use may cause leukoplakia, a premalignant oral lesion (American Academy of Oral and Maxillofacial Pathology, 2006). CYTOGENETIC ALTERATIONS Genetic mutational changes : It has been widely accepted that a group of oral premalignant lesions often converted into malignancy. One long term study observing the outcome of oral dysplasias concluded that 6 to 36% of oral pre-malignant cell carcinoma (Lumerman et al, 1995) erythroplakia and dysplastic leukoplakia are two premalignant lesions associated with a overall 17.5% rate of malignant transformation at a period of eight years (Silverman et al, 1984). In oral pre-malignancy, dysplastic tissue has been found to have cytogenetic alterations in 9p, 3p and 17p indicating that these are early events in carcinogenesis (Califeno, 1996). Many studies also have observed that P53 is the most mutated gene across all cancer type and pre-malignant lesions (Hollstein, 1991). The risk of progression to cancer is low when nongenetic change is seen, intermediate risk consists of genetic loss on the short arm of chromosome 3 and 6 (3p and 9p) and the risk is high if there is 3p and 9p loss accompanied by genetic loss on additional chromosome arms including 4q, 8q,11q, 13q and 17p (Mao L,1992). Micronuclei assay : Micronuclei are extra nuclear cytoplasmic bodies. They are induced in cells by numerous genotoxic agents, as for example tobacco use, which damages chromosomes. It has been increasingly used in molecular epidemiological study to assess the impact of nutrition, lifestyle factor, genotoxin exposure and geno type on DNA damage and chromosomal malsegrigation, cell death .Micronuclei in exfoliated cell are widely used as biomarkers of cancer risk in humans. 7


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TUMOUR GROWTH MARKERS Wild p53 is a phosphoprotein of 53 KDA comprising 393 amino acids was discovered in 1970. It plays an important role in cell cycle control, acting as a transcription factor (White, 2002). It has a short half life is usually undetected by immunohistochemistry. Wild type P53 gene plays a major role in cell cycle check point specially G1to S phase trasition. Mutant p53 protein occurred due to mutation of p53 tumour suppressor gene, cannot execute these normal functions, leading to a progressive accumulation of genetic alternations and perhaps neoplastic development. A study by Kikegawa showed that mutant p53 protein was over expressed in premalignant lesions, especially in the cases with moderate and severe epithelial dysplasia, since in cancer development cases p53 staining was detected even before malignant transformation of oral leukoplakia to squamous cell carcinoma, it is indicated that p53 accumulation occurred at a early stage of cancer development. Therefore p53 immunohistochemical analysis has been considered as a useful prognostic marker for oral leukoplakia, which may progress to malignancy. PCNA or proliferating cell nuclear antigen is an acidic intra nuclear 36 KDA polypeptide linked to cell cycle. It is detected in G1 phase and peaks in G1/S phase, decreased in G2 phase and undetected in M-phase. PCNA is a useful marker of proliferating cells (Kurkip, 1988) and can be very useful diagnostic marker in association with the process of malignancy, since there is gradual increase in terms of number of PCNA positive cells with the progress from normal epithelium to malignancy. Furthermore, the distribution of PCNA positive cells in the basal and parabasal layers in normal and premalignant lesions and distribution of PCNA positive cells in suprabasal layers of dysplasias, carcinoma in situ and invasive cancer demonstrate that early dysplastic alterations many occur without the histopathological signs seen by immunohistochemical methods (Shim, 1994). Ki 67 DQWLJHQ LV FODVVLF PDUNHU RI FHOOXODU SUROLIHUDWLRQ DQG ZDV RULJLQDOO\ GHÀQHG E\ WKH PRQRFORQDO antibody Ki-67, the name being derived from the city of origin (Kiel) and the number of original cline in 96 well plate. The Ki67 is located on the long arm of chromosome 10 (10q25). It is an acknowledged marker that indicates proliferative capacity of cells, and many studies demonstrated that it was over H[SUHVVHG LQ RUDO SUH PDOLJQDQF\ DQG RUDO FDQFHU &DUORV GH 9LFHQWH DQG PDQ\ VWXGLHV FRQÀUPHG that expression of Ki67 in resting cells has rarely been reported. Retinoic Acid Receptors are required for normal growth and differentiation. The pharmacologic roles of retinoids are carried out mainly through RAR (_, ` and a) and RXR (_, ` and a) receptors. A study by Lotan et al. on oral –leukoplakia found that 60% of the cases lacked detectable RAR m RNA by in situ hybridization. DNA ploidy analysis or quantitative measurement of deoxyribomecleic acid has been shown to be prognostic ally useful in a number of tumours. Many studies related to DNA content and progression to malignancy have shown a close relation between DNA content and future malignancy states in premalignant oral lesions Sudbo et al. have found that dysplastic lesions. DNA content of diploid lesions have only 13% cancer conversion rate and aneuploid lesions have cancer conversion rate of about 60% (Sudbo et al,2001). AgNOR’s or Silver binding nuclear organizer regions (NOR’s) are proteins that are associated ZLWK WKH ÀEULOODU FHQWUHV DQG GHQVH ÀEULOV RI WKH FHOO QXFOHDV GXULQJ LQWHU SKDVH DQG DUH UHVSRQVLEOH IRU 8


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replication of RNA. AgNOR’s are expressed within the nucleas as clear black dots and progressively increase from normal to hyperplasia to dysplasia and cancer and are very good proliferative marker for cancer prediction. TREATMENT Retinoids have been used as chemopreventive agents in a number of cancer prevention trials. These include trials wherein patients with oral premalignant lesions were study subjects (Lippmann 1995). The use of beta-carotene has been recommended in order to prevent oral leukoplakia and possibly oral Cancer (Sankaranarayanan et al, 1997). It has been found that 360mg beta- carotene per week during 12 months presented a complete resolution of oral leukoplakia. However, more than 50% cases recur after cessation of treatment. In another study 90 mg/day beta-carotene for three cycles of 3 months each, 33% cases showed complete clinical response and in another study 30 mg/day for six months only 8.3% presented complete clinical response (Toma et al, 1992; Garewal et al, 1990). However, acute toxicity occurs in the dose of 150 mg and chronic toxicity occurs in adults at a dose of 15 mg/day for several months. Toxicity includes dry skin, celosias, glossitis, vomiting, hyperlipidemia, bone demineralization, birth detects etc. Under these circumstances local application of Vitamin A (all trans and cis- retinoic acid) for the treatment of oral leukoplakia is under consideration. REFERENCES 1.

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Silverman S, Gorsky M. Proliferative verrucous leukoplakia. Oral Surg Med Oral Pathol 1984; 57:379-82.

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Sinusan K, Corso JG. A 10 years study of smokeless tobacco use in a professional baseball organization Med Sci Sports Excerc 2006; 38:1204-1207.

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Toma S, Benso S, Albanese E, et al.� Treatment of oral leukoplakia with beta- carotene. Oncology 1992; 49:77-81.

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:DUQD .6 -RKQVRQ 1: 9DQ GHU :DDO , ´1RPHQFODWXUH DQG FODVVLĂ€FDWLRQ RI SRWHQWLDOO\ PDOLJQDQW GLVRUGHUV RI the oral mucosa.â€? Journal of oral Pathology and Medicine 2007; 36: 575-580. 32.

Whyte DA, Broton CE, Shillitoe EJ. The en explained survival of cells in oral cancer : whatis the role of BP53? J Oral Pathol Med 2002; 31:25-33.

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A Review on Biological Synthesis of Gold Nanoparticles and its Application Sharmistha Banerjee, Sneha Singh, Swati Tikariha, A.S. Vidyarthi Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi, India

ABSTRACT The synthesis of gold nanoparticles has received considerable attention with their potential applications in various life sciences related applications. Because of the photo-optical distinctiveness and biocompatibility, gold nanoparticles have proven to be powerful tools in various nanomedicinal applications. A Variety of chemical and physical procedures could be used for synthesis of metallic nanoparticles. However, these methods are fraught with many problems including use of toxic solvents, generation of hazardous byproducts, and high energy consumption. Accordingly, there is an essential need to develop environmentally benign procedures for synthesis of metallic nanoparticles. A promising approach to achieve this objective is to exploit the array of biological resources in nature. Indeed, over the past several years, plants, algae, IXQJL EDFWHULD DQG YLUXVHV KDYH EHHQ XVHG IRU SURGXFWLRQ RI ORZ FRVW HQHUJ\ HIĂ€FLHQW DQG QRQWR[LF metallic nanoparticles. In this review, we provide an overview on biosynthesis of gold nanoparticles and its certain applications. Keywords : Gold Nanoparticles, Photo-optical, Biocompatibility, Biosynthesis INTRODUCTION Nanoparticles seem to be a promising option when compared to the conventional materials used, ZLWK WKH UDQJH RI DSSOLFDWLRQV WKDW QDQRSDUWLFOHV Ă€QG LQ YDULHG Ă€HOGV RI HQJLQHHULQJ DQG VFLHQFH 7KHLU XQLTXHQHVV DULVHV VSHFLĂ€FDOO\ IURP KLJKHU VXUIDFH WR YROXPH UDWLR DQG LQFUHDVHG SHUFHQWDJH RI DWRPV at the grain boundaries. Nanoparticles usually possess a wavelength below the critical wavelength of light. This renders them transparent, a property that makes them very useful for application in cosmetics, coatings, and packaging. They represent an important class of materials in the development of novel devices that can be used in various physical, biological, biomedical and pharmaceutical applications1-4. There is a growing interest in inorganic nanoparticles as they provide superior material properties with functional versatility. Inorganic nanomaterials have been widely used for cellular delivery due to their versatile features like wide availability, rich functionality, and good biocompatibility, capability of targeted drug delivery and controlled release of drugs5. They can be attached to single strands of Deoxyribonucleic acid non-destructively. This opens up avenues for medical diagnostic applications. Based on their wider application, the synthesis of metallic nanoparticles is an active area of academic and, more importantly, “application researchâ€? in nanotechnology6. Noble metals, especially gold and silver nanoparticles exhibit unique and tunable optical properties on account of their Surface Plasmon Resonance. Gold Nanoparticles (GNPs) have a wide range of potent applications in areas such as catalysis7-9, medical, diagnostics, biomedicine10-13, Photo-electronic materials14 and biological imaging15. The extraordinary properties of nanoparticles largely depend on their particle size16. There are many subtypes of GNPs based on their size, shape and physical properties. CONVENTIONAL SYNTHESIS APPROACHES There are two general strategies to obtain materials on the nano scale : Top down and Bottom up17. In Top down method, nano materials are synthesized from the bulk material by size reducing mechanisms. 11


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Size reduction is usually achieved by techniques like attrition and pyrolysis. Attrition involves size reduction by grinding microscale and macroscale particles whereas in pyrolysis, an organic precursor is forced WKURXJK DQ RULÀFH DW KLJK SUHVVXUH DQG EXUQHG6. These procedures come with various drawbacks like low production rate, enormous energy consumption and importantly, the expense is very high18. In Bottom up, or self assembly method, structures are constructed by the assembly of atom-by-atom, PROHFXOH E\ PROHFXOH RU FOXVWHU E\ FOXVWHU +HUH WKH ÀQDO PDWHULDO LV XVXDOO\ DVVHPEOHG E\ FKHPLFDO or biological procedure(s). The chemical procedure involves growing nanoparticles in a liquid medium containing various reactants, in particular reducing agents (Potassium Bitartrate19 and Hydrazine18). The most popular method of GNPs synthesis is by chemical reduction of gold salts such as Hydrogen Tetra chloroaurate (HAuCl4) using citrate as the reducing agent20. Inspite of the fact that the physical DQG FKHPLFDO PHWKRGV DUH DEOH WR SURGXFH ODUJH TXDQWLWLHV RI QDQRSDUWLFOHV ZLWK D GHÀQHG VL]H DQG shape in a relatively short time, these methods are not preferred for synthesis and are considered as complicated as they are forfeited with production of hazardous toxic wastes that are harmful, not only to the environment but also to human health6. BIOSYNTHESIS APPROACH There is an increasing need to develop high–yield, low–cost, non toxic and environmentally benign procedures for synthesis of metallic nanoparticles. Synthesis and assembly of nanoscale materials using biological systems is considered as a relatively clean, nontoxic and environmental friendly method, namely green chemistry procedure when compared with conventional synthesis techniques. Therefore, the use of biological routes in the synthesis of nanoparticles is emerging as an exciting approach and hence they are gaining importance18. Biosynthesis of nanoparticles is a kind of bottom up approach where the main reaction occurring is reduction/oxidation. A vast array of biological resources available in nature including plants, plant products, algae, fungi, yeast, bacteria and viruses could be employed for synthesis of nanoparticles6. Table 1: List of Biological Resources that Synthesize GNPs Microorganism Fungus Verticillium species21 Trichothecium species22 Fusarium oxysporum23 Colletotrichum species24 Bacterium Bacillus subtilis 16826 Escherichia coli DH5_26 Pseudomonas aeruginosa27 Rhodopseudomonas capsulate28,29 Algae Sargassum wightii30 Chlorella vulgaris31 Plant Lemongrass32 Avena sativa (Oat)33 Aloe vera34

Localization

Size nm

Intracellular Intracellular Extracellular Extracellular

20 ¹ 8 ND 20–40 20–40

Intracellular Intracellular Extracellular Extracellular

5–25 ND 15–30 10-20 & 50-400

Extracellular ND

8–12 9–20

ND Extracellular Extracellular

200–500 5–85 50–350

BIOSYNTHESIS OF GNPS USING BACTERIA Among microorganisms, prokaryotic bacteria have received most attention in the area of biosynthesis of nanoparticles. Early studies reveal that Bacillus subtilis 168 is able to reduce Au3+ ions to produce octahedral gold particles of nanoscale dimensions (5–25 nm) within bacterial cells by incubation of the cells with gold chloride25, 35, 36 under ambient temperature and pressure conditions. Iron (III) reducing bacteria Shewanella algae37 can reduce gold (III) ions in anaerobic environments. In the presence of Shewanella algae and hydrogen gas, the gold ions are completely reduced, which results in the formation of 10-20 nm GNPs38. Advantages of using bacterial biomass are that they are easy to handle and can also be manipulated genetically without much difficulty 39-40. But, the 12


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bacterial synthesis comes with several challenges which are yet to be explored and solved such as reduction in time of synthesis, better control over size and shape, industrial scaling up of the synthesis to get large amount of nanomaterials41. BIOSYNTHESIS OF GNPS USING FUNGI The use of fungi in the synthesis of nanoparticles is a relatively recent addition to the list of microorganisms. The use of fungi is potentially exciting since they secrete large amounts of enzymes and are simpler to deal with in the laboratory. However, the genetic manipulation of eukaryotic organisms DV D PHDQV RI RYHUH[SUHVVLQJ VSHFLÀF HQ]\PHV LGHQWLÀHG LQ QDQR PDWHULDO V\QWKHVLV ZRXOG EH PXFK PRUH GLIÀFXOW WKDQ WKDW LQ SURNDU\RWHV38. Literature reveals the appearance of a distinctive purple colour in the biomass of Verticillium on being its exposure to the 10ï M HAuCl4 solution which indicates the formation of GNPs intracellularly22. Keeping application in view, it would be imperative to harvest the metal nanoparticles formed within the fungal biomass. Quite surprisingly, the plant pathogenic fungal strain Fusarium oxysporum behaved considerably differently; the reduction of the metal ions occurred extracellularly, resulting in the rapid formation of highly stable gold25 nanoparticles of 2-50 nm. The extracellular and intracellular biosynthesis of GNPs by fungus Trichothecium species was reported by Absar and co-workers42. It was observed that when the gold ions reacted with the biomass of Trichothecium species under stationary conditions results in the rapid extracellular formation of GNPs of spherical rod-like and triangular morphology whereas reaction of the biomass under shaking conditions resulted in intracellular growth of the GNPs. Microbiological methods generate nanoparticles at a much slower rate than that observed when plant extracts are used. This is one of the major drawbacks of synthesis of nanoparticles using microorganisms and has to be overcome if it competes Fig 1: Extracellular and Intracellular Synthesis by Tricothecium species42 with other methods43. BIOSYNTHESIS OF GNPS USING PLANTS Use of plants for synthesis of nanoparticles could be advantageous over other environmentally benign biological processes as they eliminate the elaborate process of maintaining cell cultures. Plants can also EH VXLWDEO\ VFDOHG XS IRU ODUJH VFDOH V\QWKHVLV RI QDQRSDUWLFOHV 6RPH RWKHU EHQHÀWV RI V\QWKHVLV XVLQJ plants are they are safe to handle as well as they possess a broad variability of metabolites that may – aid in reduction. Synthesis of GNPs have been shown by the reduction of aqueous AuCl4 ions using H[WUDFWV IURP (PEOLFD RIÀFLQDOLV ,QGLDQ *RRVHEHUU\ IUXLW 7DPDULQGXV LQGLFD OHDI44-45 and lemongrass32. GNPs with a size range of 2- 20 nm have also been synthesized using the live alfa alfa plants46 in solid media. While fungi and bacteria require a comparatively longer incubation time for the reduction of metal ions, water soluble phytochemicals do it in a much lesser time. Therefore compared to bacteria and fungi, plants are better candidates for the synthesis of nanoparticles. Taking use of plant tissue culture techniques and downstream processing procedures, it is possible to synthesize metallic as well as oxide nanoparticles on an industrial scale, once the issues like metabolic status of the plant etc. are properly addressed43. 13


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BIOSYNTHESIS OF GNPS USING ALGAE: The synthesis of nanoparticles using algae as source has been less explored. In an isolated report, Singaravelu et al.30 LPSOHPHQWHG DQ HIÀFLHQW DSSURDFK IRU V\QWKHVLV RI VWDEOH *13V E\ WKH UHGXFWLRQ RI aqueous AuCl4- ions by using marine algae Sargassum wightii. Gold (III) was also successfully recovered as metallic GNPs using dead biomass of the brown alga F. Vesiculosus47. APPLICATIONS OF GNPS GNPs are excellent labels for biosensors because they can be detected by numerous techniques, such as optic absorption à XRUHVFHQFH DQG HOHFWULF FRQGXFWLYLW\ *13V KDYH EHHQ SULPDULO\ used for labelling application and are a very attractive contrast agent. The interaction of GNPs with light can be used for the visualization of particles48. In this way, the therapeutic application of metallic nanoparticles is also possible40. Generally, GNPs provide non-toxic Fig 2 : Application of Gold Nanoparticles routes to drug and gene delivery application. GNPs are capable of delivering large bio molecules (peptides, proteins, or nucleic acids). Gold nanoparticles can also be used as a heat source48. The heat from the gold particles can be used for manipulating the surrounding tissues, e.g., hyperthermia, optically triggered opening of bonds (e.g., melting double stranded DNA and disassembling protein aggregates), and opening of containers. Cells are sensitive to small increases in temperature and an increase of a few degrees can lead to cell death. Gold nanoparticle are engineered not only to identify, target, and kill tumors but also to carry the additional drug to slow down the growth of cancer cells or kill the cancer cells. CONCLUSION Living organisms have huge potential for the production of nanoparticles of wider applications. 1DQRPHGLFLQH LV D EXUJHRQLQJ ÀHOG RI UHVHDUFK ZLWK WUHPHQGRXV SURVSHFWV IRU WKH LPSURYHPHQW RI the diagnosis and treatment of human diseases. With the recent progress and the ongoing efforts in LPSURYLQJ SDUWLFOH V\QWKHVLV HIÀFLHQF\ DQG H[SORULQJ WKHLU ELRPHGLFDO DSSOLFDWLRQV LW LV KRSHIXO WKDW biological resources that are capable of synthesizing nanoparticles would be manipulated at genetic level on a large scale and their commercial applications in medicine and health care will take place in the coming years. REFERENCES 1.

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Shenhar R and Rotello VM. Nanoparticles: Scaffolds and Building Blocks. Acc. Chem. Res. 2003; 36 (7): 549–561.

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Frens G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature Phys Sci. 1973; 241: 20–22.

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Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, Khan MI, Ramani R, Parischa R, Ajaykumar PV, Alam M, Sastry M and Kumar R. Bioreduction of AUC14- ions by the fungus, Verticillium sp, and surface trapping of the gold nanoparticles formed. Angew Chem Int Ed. 2001; 40: 3585-3588.

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Ahmad A, Senapati S, Khan MI, Kumar R and Sastry M. Extra-/Intracellular Biosynthesis of Gold Nanoparticles by an Alkalotolerant Fungus, Trichothecium sp. J Biomed Nanotechnol 2005; 1: 47-53.

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Shankar SS, Ahmad A, Pasricha R and Sastry M. Bioreduction of chloroaurate ions by geranium leaves and its endophytic fungus yields gold nanoparticles of different shapes. J Mat Chem. 2003; 13: 1822-1826.

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Beveridge TJ and Murray RGE. Site of metal deposition in the cell wall of Bacillus subtilis. J Bacteriol 1980; 141: 876–887.

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Husseiny MI, El-Aziz MA, Badr Y and Mahmoud MA. Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa. Spectrochim Acta A: Molecular and Biomolecular Spectroscopy 2007; 67: 3-4 1003-1006.

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He S, Guo Z, Zhang Y, Zhang S, J. Wang and Gu N. Biosynthesis of gold nanoparticles using the bacteria Rhodopseudomonas capsulate. Mater Lett 2007; 61: 3984-3987.

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29.

He S, Zhang Y, Guo Z and Gu N. Biological synthesis of gold nanowires using extract of Rhodopseudomonas capsulate. Biotechnol Prog 2008; 24: 476-480.

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Singaravelu G, Arockiamary SJ, Kumar GV and Govindaraju K. A novel extracellular synthesis of monodisperse gold nanoparticles using marine alga Sargassum wightii. Colloids Surfaces, B. Biointerfaces 2007; 57: 97-101.

-LDQSLQJ ; -LP </ 'DQLHO ,&: DQG <HQ 37 ,GHQWLĂ€FDWLRQ RI DFWLYH ELRPROHFXOHV LQ WKH KLJK \LHOG V\QWKHVLV RI VLQJOH crystalline gold nanoplates in algal solutions. Small 2007; 3(4): 668-72. 32.

Shankar S, Rai A, Ankamwar B, Singh A, Ahmad A and Sastry M. Biological synthesis of triangular gold nanoprisms. Nat Mater 2004; 3: 482-88.

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Parikh RY, Singh S, Prasad BLV, Patole MS, Sastry M and Shouche YS. Extracellular Synthesis of Crystalline Silver Nanoparticles and Molecular Evidence of Silver Resistance from Morganella sp.: Towards Understanding Biochemical Synthesis Mechanism. ChemBioChem 2008; 9: 1415–1422.

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Kalishwaralal K, Gopalram S, Vaidyanathan R, Deepak V, Pandian SRK and Gurunathan S. Optimization of -amylase production for the green synthesis of gold nanoparticles. Colloids and Surfaces B: Biointerfaces 2010; 77 (2): 174-180.

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$QNDPZDU % 'DPOH & $KPDG $ DQG 6DVWU\ 0 %LRV\QWKHVLV RI JROG DQG VLOYHU QDQRSDUWLFOHV XVLQJ (PEOLFD RIĂ€FLQDOLV fruit extract, their phase transfer and transmetallation in an organic solution. J Nanosci Nanotech 2005; 5: 16651671. 45.

Ankamwar B, Chaudhary M and Sastry M. Gold Nanotriangles biologically synthesized using tamarind leaf extract and potential application in vapour sensing. Syn React Inorg Metal Org Nano-Metal Chem 2005; 35: 19–26.

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Surfactant Properties of Non Aqueous Microemulsions Shekhar Verma*, J.S. Dangi Institute of Pharmaceutical Sciences, Gurughasidas Vishwavidyalya, Bilaspur, India

ABSTRACT Two basics strategies could be considered when searching for stable non-aqueous emulsions. One is to design surfactants having two incompatible blocks, each of which is selectively soluble in either of the immiscible liquids. The other approach is to search for a suitable oil immiscible polar liquid that FDQ VXEVWDQWLDOO\ UHSODFH ZDWHU XVLQJ HPXOVLÀHUV $ OLTXLG FDSDEOH RI UHSODFLQJ ZDWHU LQ DQ HPXOVLRQ should have an appreciable polarity to make it immiscible with oils and to make it a good solvent for the solvophilic part of the surfactant molecules. Hydrogen bonding in the polar liquid is expected to play a role in solvating both ionic and non ionic surfactants and in the formation of a hydrogen bonded network in the liquid itself.There are many advantages of non aqueous microemulsions like avoidance of phase separations, increased drug loading capacity, carrying capacity of both lipophilic and hydrophilic drugs, Protection against intestinal proteases, increases the bioavailability of the drug, non aqueous PLFURHPXOVLRQV GHOLYHU\ V\VWHP FDQ LPSURYHG WKH HIÀFDF\ RI GUXJ DOORZLQJ WRWDO GRVH WR EH UHGXFHG DQG thus minimizing side effect. Because of thermodynamic stability non aqueous microemulsion are easy to SUHSDUH DQG UHTXLUH QR VLJQLÀFDQW HQHUJ\ FRQWULEXWLRQ GXULQJ SUHSDUDWLRQ INTRODUCTION The words “surface active agents” are combined to form surfactant. Surfactant is characterized by its tendency to adsorb at surfaces and interfaces. It is an organic compound and widely used in Pharmaceuticals. It exists in both natural and synthetic forms. Surfactants such as phospholipids are the main components of the cell membranes and sustain life by organizing the order of chemical reactions. For a compound to be a surfactant, it should possess three characteristics: the molecular structure should be composed of polar and non-polar groups, it should exhibit surface activity and it should form self-assembled aggregates (micelles, vesicles, liquid crystalline, etc.) in liquids. A surfactant molecule consists of two structures: polar (hydrophilic, lipophobic or oleophobic) head groups and non-polar (hydrophobic, lipophilic or oleophilic) tail groups. The hydrophilic group makes the surfactant soluble in polar solvents such as water. The hydrophobic group makes the surfactant soluble in non-polar solvents and oil. The relative sizes and shapes of the hydrophobic and hydrophilic parts of the surfactant molecule determine many of its properties. Surfactant molecules can have one hydrophilic head and one hydrophobic tail; one hydrophilic head and two hydrophobic; or one hydrophobic tail terminated at both ends by hydrophilic groups (bolaform surfactants or _, t surfactants); hydrophilic heads of two surfactants are combined with a rigid spacer, which is a linear or ring organic structure (gemini surfactants), and a number of hydrophilic (more than two) hydrophobic groups, with both groups linked in the same molecule by covalent bonds (polymeric surfactants) . Hydrophilic molecules are composed of ions (such as sulphonate, sulphate, carboxylate, phosphate and quaternary ammonium), polar groups (such as primary amines, amine oxides, sulphoxides and phosphine oxide) and non-polar groupswith electronegative atoms (such as oxygen atom in ethers, aldehydes, amides, esters and ketones and nitrogen atoms in amides, nitroalkanes and amines). These molecules associate with the hydrogen bonding network in water.1-3 17


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'HSHQGLQJ RQ WKH K\GURSKLOLF JURXSV VXUIDFWDQWV DUH FODVVLĂ€HG DV DQLRQLF FDWLRQLF QRQLRQLF RU amphoteric. Anionic surfactants dissociate in water into a negatively charged ion and a positively charged ion and the hydrophilic head is negatively charged (anion). Anionic surfactants are the most common and inexpensive surfactant. They are sold as alkali metal salts or ammonium salts and are mainly used in detergent formulations and personal care products. Cationic surfactants also dissociate in water into a negatively charged ion and a positively charged ion and the hydrophilic head is positively charged (cation). Due to the positive charge of the head group, cationic surfactants strongly adsorb onto negatively charged surfaces such as fabric, hair and cell membrane of bacteria. Therefore, they are used as fabric softeners, hair conditioners and antibacterial agents. Non-ionic surfactants, on the other hand, do not dissociate in water and the hydrophilic head has a neutral charge. Nonionic surfactants DUH FRPPRQO\ XVHG LQ WKH IRUPXODWLRQ RI HPXOVLĂ€HU GLVSHUVDQW DQG ORZ WHPSHUDWXUH GHWHUJHQWV Depending on pH, the hydrophilic head of amphoteric surfactants in water has a positive, negative or both positive and negative charges. They are cations in acidic solutions, anions in alkaline solutions and zwitterions (both ionic groups show equal ionisation and behave uncharged) in an intermediate pHrange. They are commonly used in toiletries, baby shampoos, daily cleaners and detergents. For further description of surfactant hydrophilic groups and their detail applications, the reader is referred to. The major cost of the surfactant comes from the hydrophobic group because the hydrophobic group except for high ethylene oxide non-ionics is the largest part of the surfactant molecule.The hydrophobic JURXS LQ WKH VXUIDFWDQW VWUXFWXUH LV PDGH XS RI K\GURFDUERQ FKDLQV Ă XRURFDUERQ FKDLQV FRPELQDWLRQ RI Ă XRURFDUERQ DQG K\GURFDUERQ FKDLQV RU VLOLFRQH FKDLQV 7KH PDMRULWLHV RI WKH FRPPHUFLDOO\ DYDLODEOH surfactants (99%) have hydrocarbon chains and are synthesised from natural animal fats, natural vegetable oils or petrochemicals. Hydrocarbons synthesised from natural sources exclusively contain even number of hydrocarbon chains because their structures are built up from ethylene . On the other hand, hydrocarbons derived from petrochemicals contain mixtures of odd and even carbon chains because they are synthesised by cracking higher hydrocarbons. The hydrocarbon chains can be linear or branched and include polycyclic, saturated, unsaturated or polyoxypropylene structures. The linear structure is desirable due to its biodegradability. Fluorocarbon and silicone chain surfactants in water and non-aqueous systems reducethe surface tension lower than the hydrocarbon chain surfactants. Both Ă XRURFDUERQ DQG VLOLFRQH FKDLQ VXUIDFWDQWV KDYH EHWWHU WKHUPDO DQG FKHPLFDO VWDELOLW\ WKDQ K\GURFDUERQV and provide excellent wetting for low-energy surfaces. Due to their costs, these surfactants are used in limited applications.3-7 VITAL ROLE OF NON AQUEOUS MICROEMULSIONS Conventional emulsions are heterogeneous system in which one immiscible liquid is dispersed as droplets in another liquid. Such a thermodynamically unstable system is kinetically stabilized by addition of one further components that exhibit emulsify properties. In emulsion water is an internal phase dispersed in oil are termed as water-in-oil, where as, emulsion in which the oil is dispersed and water forms the continuous phase are known as oil-in-water emulsions. Emulsion is one of the most convenient and advantageous formulation in which one of the liquid phases is water. However emulsion can be formulated without an aqueous phase to produce anhydrous, non-aqueous or oil in oil microemulsions. Such systems, which can replace conventional emulsions where the presence of water to be avoided.7-15 18


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Non aqueous microemulsion have attracted a great deal of attention not only because of there importance in industrial application but also there intrinsic interest. They optimize the performance of a wide spectrum of products and processes. Non aqueous microemulsions are suitable for poorly aqueous VROXEOH GUXJV DQG WKHUPRG\QDPLFDOO\ VWDEOH PXOWLFRPSRQHQW à XLGV FRPSRVHG RI SRODU VROYHQW RLO DQG mixture of a liquid administration of drugs is one of the convient and often-advantageous delivery, HVSHFLDOO\ ZKHQ GHDOLQJ ZLWK FKLOGUHQ RU WKH HOGHUO\ IRU ZKRP SLOO VZDOORZLQJ FDQ EH GLIÀFXOW RU HYHQ hazardous. Unfortunately many drugs are not soluble in water, while water solution of it may have an unpleasant taste. Some drugs are either unstable in the presence of water or are insoluble in water and therefore cannot be incorporated into aqueous formulations. To overcome these various problems a water free liquid preparation of a number of drugs would be desirable. Several novel pharmaceutical formulation approaches have been taken to increase the gastrointestinal absorption and bioavailability and to decrease the intra-and inter-subject variability in the plasma FRQFHQWUDWLRQ WLPH SURÀOHV The formulation approaches can be divided into main two categories: Solid dosage forms developed with the purpose of increasing dissolution rate and liquid dosage forms containing the compound in Solution, often in a lipid phase.15-20 The use of natural and synthetic lipids has generated much academic and commercial interest as a potential formulation strategy for improving the oral bioavailability of poorly water-soluble drugs which administered in traditional solid formulation, these compounds often exhibit low bioavailability as their absorption can be kinetically limited by low rates of dissolution and capacity-limited by poor solubility. The mechanism behind the solubilization and absorption of poorly soluble compounds is not completely understood. In the fasted state, poorly soluble drugs are believed to be solubilized in a mixed bile salt micelle prior to absorption. When entering the unstirred water layer lining the small intestinal epithelium, the mixed micelle disintegrates and the drug diffuses to the small intestinal epithelium and is absorbed. When introducing the fed state, the processes in the gastrointestinal tract become even more complex. Dietary lipids are hydrolyzed by pancreatic lipase into surface active lipid digestion products that will participate in the formation of complex colloid phased, including vesicles, micelles and liquid laminar phases. In general co-administration with food, increase the bioavailability of poorly soluble drugs, partly due to the solubilizing effects of lipid hydrolysis products, but also due to an increased retention time in the stomach. The anhydrous microemulsion useful for drug delivery which largely overcomes the problem mentioned above with water unstable drug. A very palatable formulation can be made in which only one teaspoon contains a normal child’s dose. The emulsion contains a drug dissolved in a suitable non-aqueous internal phase solvent. By drug is meant any therapeutic agents such as vitamins, enzymes, drugs, etc. typical drugs which are suitable include aspirin, indomethacin, ibuprofen, salicylic acid, phenylbutazone, diclofenace, piroxicam, cimetidine, fat soluble vitamins, steroids, vitamins A, D and E and large no. of water unstable compounds such as aspartame. The non aqueous, internal phase of the emulsion is a polar, pharmaceutically acceptable oxygen containing liquid such as polyhydric solvents, polyglycols, etc. The continuous phase of the emulsion is fatty acids (medium chain triglycerides or ethyl palmitate.) etc. The emulsion also FRQWDLQV HPXOVLÀHU WKDW PD\ EH VROLG OLTXLG RU FRPELQDWLRQ UDWLR RI LQGLYLGXDO ZLWK DQRWKHU HPXOVLÀHU or may be a novel gum.20-25 19


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SURFACTANT DESIGN FOR STABLE NON AQUEOUS MICRŒMULSIONS Two basics strategies could be considered when searching for stable non-aqueous emulsions. One is to design surfactants having two incompatible blocks, each of which is selectively soluble in either of the immiscible liquids. The other approach is to search for a suitable oil immiscible polar liquid that FDQ VXEVWDQWLDOO\ UHSODFH ZDWHU XVLQJ HPXOVLÀHUV A liquid capable of replacing water in an emulsion should have an appreciable polarity to make it immiscible with oils and to make it a good solvent for the solvophilic part of the surfactant molecules. Hydrogen bonding in the polar liquid is expected to play a role in solvating both ionic and non ionic surfactants and in the formation of a hydrogen bonded network in the liquid itself. There are many advantages of non aqueous microemulsions like avoidance of phase separations, increased drug loading capacity, carrying capacity of both lipophilic and hydrophilic drugs, Protection against intestinal proteases, increases the bioavailability of the drug, non aqueous microemulsions GHOLYHU\ V\VWHP FDQ LPSURYHG WKH HIÀFDF\ RI GUXJ DOORZLQJ WRWDO GRVH WR EH UHGXFHG DQG WKXV PLQLPL]LQJ side effect. Because of thermodynamic stability non aqueous microemulsion are easy to prepare and UHTXLUH QR VLJQLÀFDQW HQHUJ\ FRQWULEXWLRQ GXULQJ SUHSDUDWLRQ SURFACTANT STABILIZED DISPERSION Most substances acquire a surface electric charge when brought into contact with a polar medium such as water. One mechanism for this involves the adsorption of ionic surfactants. In general, the nature and degree of surface charging is more complicated than this and surfactant adsorption may FDXVH D VXUIDFH HOHFWULF FKDUJH WR LQFUHDVH GHFUHDVH RU QRW VLJQLÀFDQWO\ FKDQJH DW DOO )RU H[DPSOH an oil–aqueous interface can become negatively charged in alkaline aqueous solutions due to the ionization of surface carboxylic acid groups, the adsorption of natural surfactants present in the oil, and the adsorption of charged mineral solids. 7KH SUHVHQFH RI D VXUIDFH FKDUJH LQà XHQFHV WKH GLVWULEXWLRQ RI QHDUE\ LRQV LQ D SRODU PHGLXP DQG an electric double layer (EDL) is formed, consisting of the charged surface and a neutralizing excess of counter-ions over co-ions, distributed near the surface. Most colloidal dispersions, including emulsions, suspensions and foams are not thermodynamically stable, but may possess some degree of kinetic stability. Encounters between dispersed species can occur due to Brownian motion, sedimentation, and/ or stirring. The stability of the dispersion depends upon how the particles interact when this happens. More details are given in reference. Surfactants are frequently involved in the stabilization of colloidal dispersions of droplets, particles or bubbles by increasing the electrostatic repulsive forces. An emulsion, foam or suspension can be made by simply mixing oil, gas or solids into another SKDVH RIWHQ ZDWHU RU DQ DTXHRXV VROXWLRQ ZLWK VXIÀFLHQW PHFKDQLFDO VKHDU 7KH DGGLWLRQDO LQWHUIDFLDO area created between the two phases is important because, as shown by the Laplace equation, even a modest interfacial energy per unit area can become a considerable total interfacial energy requirement if many small droplets, bubbles or particles are formed. In practise, the energy requirement is even greater due to the need for droplets, bubbles or particle aggregates to deform before being disrupted. If this energy requirement cannot be provided, say, by mechanical shear, then another alternative is to use surfactant chemistry to lower the interfacial free energy, or interfacial tension. This can lower the amount of mechanical energy needed for dispersion by several orders of magnitude. Every metastable 20


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emulsion or foam, and many of the suspensions, that will be encountered in practise contain a surfaceactive stabilizing agent. The stabilizing surfactant makes the dispersion easier to form and may create DQ LQWHUIDFLDO ÀOP WKDW KHOSV NHHS WKH V\VWHP IURP breaking or separating. Although surfactants and surface free energies are very important to the stability of dispersions, there are a considerable number of factors involved in determining their stability.25-27

Table 1 : Surface tension of some liquids and solids and interfacial tension of some immiscible Liquids Substance

Temperature ($C)

Liquid Diethyl ether n-octane Ethyl alcohol Methyl alcohol Chloroform Benzene Phenol Water Solid Copper Iron Liquid–liquid interface Diethylether–water n-octane–water Benzene–water

20 20 20 20 20 20 20 20

Surface tension (dyne cmï ) 17.01 21.8 22.3 22.6 27.14 28.88 40.9 72.8

In measuring surface tension, one is measuring the free energy per unit area of the surface between liquid and the air (erg cmï or J mï ). Surface tension LV DOVR TXDQWLÀHG DV WKH IRUFH DFWLQJ QRUPDO WR WKH interface per unit length of the surface at equilibrium (dyne cmï or mN mï ). Due to this force, liquid surface has a propensity to occupy minimum surface area. Therefore, a liquid drop in a gas phase and 1050 1430-1670 bubbles in a liquid phase adopt a spherical shape. 1400 1670 The surface tension of some liquid and solids are shown in Table 1. Surface tension of the substances 20 10.7 decreases with increasing temperature because 20 50.8 increasing temperature reduces the cohesive energy 20 35 between molecules. At the critical temperature, surface tension becomes zero. For example, the critical temperature for chloroform is 280°C. The surface tension of water at 20°C (72.8 dyne cmï ) is higher than the surface tension of chloroform (27.14 dyne cmï ) but lower than the surface tension of mercury (476 dyne cmï ). This indicates that the attractive forces between the water molecules are stronger than the attractive forces between the chloroform molecules but weaker than the attractive forces. INTERFACIAL TENSION Interfacial tension is the tension that is present at the interface of two immiscible phases and it has the same units as surface tension. The value of interfacial tension generally lies between the surface tension of two immiscible liquids although it could also be lower than the surface tension of both liquids (water–diethyl ether). The interfacial tension between phases A and B, aAB, is expressed by: aAB = aA + a% ï sAB where aA, aB and sAB are surface tension of A, surface tension of B, and interaction energy between A and B per unit area, respectively. The value of aAB also shows how similar the molecules at the interface are. The interfacial tension (aAB) will be small if the molecules of the two phases are similar (large sAB).The greater the similarity, the larger the sAB and smaller the aAB. For example, the interfacial tension between water and ethanol (short chain alcohol) is almost zero because the OH group of ethanol orients itself towards the water phase and interacts with water molecules via hydrogen bonding (2sAB = aA + aB). As a result, the 21


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interface disappears (aAB = 0) and the two phases form a single phase. If one phase (phase B) is gas, the interface forms at the surface of the condensed phase (phase A) and the interfacial tension is equivalent to the surface tension of the condensed phase (aAB = aA). It means that molecules in the gas phase are widely spaced, so the tension produced by molecular interaction in the gas phase and gas phase–condensed molecules phase is negligible. SURFACE AND INTERFACIAL TENSION REDUCTION Expansion of the interface by unit area can be achieved by the movement of enough molecules from bulk to the interface. However, the potential energy difference between the interface molecules and bulk molecules hinders this move. A minimum amount of work is required to overcome this potential energy difference between the molecules. The interface free energy per unit area or interfacial tension is a measure of this minimum work. When surfactant is added in such a system, surfactant molecules move towards the interface and the hydrophobic tail of the molecule either lies flat on the surface (few surfactant molecules at the interface) or aligns itself to the less polar liquid (sufficient number of surfactant molecules at the interface) while the hydrophilic head orientates itself towards the polar phase. The surfactant molecules destroy the cohesive forces between polar and non-polar molecules and replace the polar and nonpolar molecules at the interface. The molecular interaction at the interface occurs between the hydrophilic head of the surfactant and the polar phase molecules and between the hydrophobic tail of surfactant and the non-polar phase molecules. This phenomenon lowers the tension across the interface because the newly developed interactions are stronger than the interaction between the non-polar and polar molecules. If one of the phases is gas or air, tension reduction at the interface is named as surface tension reduction since gas or air molecules are mainly non-polar. Surfactant at low concentration has a tendency to adsorb at the surface or interface and significantly reduce the amount of work required to expand those interfaces .The stronger the tendency, the better the surfactant and the denser the surfactant packing at the interface, the larger the reduction in surface tension between the mercury molecules. USE OF SURFACTANT COMPOSITION FOR STABLE NON AQUEOUS MICROEMULSIONS $ VXUIDFWDQW FDQ EH FODVVLÀHG E\ WKH SUHVHQFH RI IRUPDOO\ FKDUJHG JURXSV LQ LWV KHDG $ QRQ LRQLF surfactant has no charge groups in its head. The head of an ionic surfactant carries a net charge. If the FKDUJH LV QHJDWLYH WKH VXUIDFWDQW LV PRUH VSHFLÀFDOO\ FDOOHG DQLRQLF LI WKH FKDUJH LV SRVLWLYH LW LV FDOOHG cationic. If a surfactant contains a head with two oppositely charged groups, it is termed zwitterionic. Anionic : based on permanent anions (sulfate, sulfonate, phosphate) or pH-dependent anions (carboxylate): i)

ii)

Sulfates: (1)

Alkyl sulfates: ammonium lauryl sulfate, sodium lauryl sulfate (SDS);

(2)

Alkyl ether sulfates: sodium laureth sulfate, also known as sodium lauryl ether sulfate (SLES), sodium myreth sulfate;

Sulfonates: (1)

Docusates: dioctyl sodium sulfosuccinate; 22


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6XOIRQDWH Ă XRURVXUIDFWDQWV SHUĂ XRURRFWDQHVXOIRQDWH 3)26 SHUĂ XRUREXWDQHVXOIRQDWH (3)

iii)

Phosphates: (1)

iv)

Alkyl benzene sulfonates; Alkyl aryl ether phosphate; (2) Alkyl ether phosphate

Carboxylates: (1)

Alkyl carboxylates: Fatty acid salts (soaps): sodium stearate;

(2)

Sodium lauroyl sarcosinate;

&DUER[\ODWH Ă XRURVXUIDFWDQWV SHUĂ XRURQRQDQRDWH SHUĂ XRURRFWDQRDWH 3)2$ RU 3)2

Cationic : based on: i)

pH-dependent primary, secondary or tertiary amines: primary amines become positively charged at pH < 10, secondary amines become charged at pH < 4: (1) Octenidine dihydrochloride;

ii)

Permanently charged quaternary ammonium cation: (1) Alkyltrimethylammonium salts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC); (2) Cetylpyridinium chloride (CPC); (3) Polyethoxylated tallow amine (POEA); (4) Benzalkonium chloride (BAC); (5) Benzethonium chloride (BZT); (6) 5-Bromo-5-nitro-1,3dioxane; (7) Dimethyldioctadecylammonium chloride (8) Dioctadecyldimethylammonium bromide (DODAB)

Zwitterionic (amphoteric) : based on primary, secondary or tertiary amines or quaternary ammonium cation with: i)

Sulfonates: (1) CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate); (2) Sultaines: cocamidopropyl hydroxysultaine;

ii)

Carboxylates : (1) Amino acids; (2) Imino acids; (3) Betaines: cocamidopropyl betaine;

iii)

Phosphates : lecithin

Nonionic a)

Fatty alcohols : i) Cetyl alcohol, ii) Stearyl alcohol, iii) Cetostearyl alcohol, iv) Oleyl alcohol;

b)

Polyoxyethylene glycol alkyl ethers (Brij): CH3–(CH2)10–16–(O-C2H4)1–25–OH: i) Octaethylene glycol monododecyl ether, ii) Pentaethylene glycol monododecyl ether;

c)

Polyoxypropylene glycol alkyl ethers: CH3–(CH2)10–16–(O-C3H6)1–25–OH;

d)

Glucoside alkyl ethers: CH3–(CH2)10–16–(O-Glucoside)1–3–OH: i) Decyl glucoside, ii) Lauryl glucoside, iii) Octyl glucoside;

e)

Polyoxyethylene glycol octylphenol ethers: C8H17–(C6H4)–(O-C2H4)1–25–OH: i) Triton X-100; 23


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

f)

Polyoxyethylene glycol alkylphenol ethers: C9H19–(C6H4)–(O-C2H4)1–25–OH: i) Nonoxynol-9;

g)

Glycerol alkyl esters: i) Glyceryl laurate

h)

Polyoxyethylene glycol sorbitan alkyl esters: Polysorbates; i) Sorbitan alkyl esters: Spans;

j)

Cocamide MEA, cocamide DEA;

k)

Dodecyl dimethylamine oxide;

l)

Block copolymers of polyethylene glycol and polypropylene glycol: Poloxamers...

According to the composition of their counter-ion In the case of ionic surfactants, the counter-ion can be: 1)

2)

Monoatomic / Inorganic: a)

Cations: metals : alkali metal, alkaline earth metal, transition metal;

b)

Anions: halides: chloride (Cl ), bromide (Br ), iodide (I );

ĂŻ

ĂŻ

ĂŻ

Polyatomic / Organic: a)

Cations: ammonium, pyridinium; b) Anions: tosyls, trifluoromethanesulfonates, methylsulfate.

REFERENCES

+D - : <DQJ 6 0 5KHRORJLFDO UHVSRQVHV RI RLO LQ RLO HPXOVLRQV LQ DQ HOHFWULF Ă€HOG - 5KHRO ²

2.

Jaitely, V., Sakthivel, T., Magee, G., Florence, A.T., 2004. Formulation of oil in oil emulsions: potential drug reservoirs for slow release. J. Drug Del. Sci. Tech. 14, 113–117.

3.

C.G. Wilsona, M. McJuryb, B. O, Mahony, M. Frier, A.C. Perkins., 1997., Imaging of oily formulations in the gastrointestinal tract., Advanced Drug Delivery Reviews 25., 91- 101.

4.

Andrew J. Humberstone, William N. Charman, 1997, Lipid-based vehicles for the oral delivery of poorly water, Advanced Drug Delivery Reviews 25 103- 128

5.

Brodin, A.F., Kavaliunas, D.R., Frank, S.G., 1978. Prolonged drug release from multiple emulsions. Acta Pharm. Suec. 15 (1), 1–12.

6.

Cameron, N.R., Sherrington, D.C., 1996. Non-aqueous high internal phase emulsions-preparation and stability. J. Chem. Soc., Faraday Trans. 92, 1543–1547.

&ROH 0 / :KDWHOH\ 7 / 5HOHDVH UDWH SURÀOHV IURP WKHRSK\OOLQH DQG LQVXOLQ IURP VWDEOH PXOWLSOH Z R Z emulsions. J. Controlled Release 49, 51–58.

8.

Davis, S.S., Hadgraft, J., Palin, J.K., 1985. Medical and pharmaceutical applications of emulsions. In: Becher, P. (Ed.), Encyclopedia of Emulsion Technology, vol. 2.Marcel Dekker, New York, pp. 159–238.

9.

Kumbaradzi, E.F., Pavlovska, E.P., Simov, A., Coracinova, K., 1996. Phase inversion of multiple water:oil:water into a water:oil emulsion caused by osmotic pressure gradients.Acta Pharm. 46, 177–185.

' +LMUWHU - % 'UHVVPDQÂľ ,QĂ XHQFH RI SK\VLFRFKHPLFDO SURSHUWLHV RQ GLVVROXWLRQ RI GUXJV LQ WKH JDVWURLQWHVWLQDO tract Advanced Drug Delivery Reviews, 25 , 3-14 11.

Florence, A.T., Whitehill, D., 1981. Some features in water-in oil-in water multiple emulsions. J. Colloid Interface Sci. 79 (1), 243–256.

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Florence, A.T., Whitehill, D., 1982. Formulation and stability of multiple emulsions. Int. J. Pharm. 11, 277–308.

13.

Florence, A.T., Law, T.K., Whateley, T.L., 1985. Nonaqueous foam structures from osmotically swollen w:o:w emulsion droplets. J. Colloid Interface Sci. 107, 584–588.

24


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14.

Hamill, R.D., Olson, F.A., Petersen, R.V., 1965. Some interfacial properties of a nonaqueous emulsion. J. Pharm. Sci. 54, 537–540.

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Hamill, R.D., Petersen, V., 1966a. Effect of surfactant concentration on the interfacial viscosity of a nonaqueous system. J. Pharm. Sci. 55, 1274–1277.

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Hamill, R.D., Petersen, V., 1966 b. Effects of ageing and surfactant concentration on the rheology and droplet size distribution of a nonaqueous emulsion. J. Pharm. Sci. 55, 1269–1277.

17.

Hino, T., Takeuchi, H., Niwa, T., Kitagawa, M., Kawashima, Y., 1995. Analysis of drug release from diluted water:oil:water emulsions by a model of the rupture of oil membrane. J. Pharm. Pharmacol. 47, 1–7.

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Imhof, A., Pine, D.J., 1997a. Stability of nonaqueous emulsions. J. Colloid Interface Sci. 192, 368–374.

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Koizumi, T., Higuchi, W.I., 1968a. Analysis of data on drug release from emulsions II. Pyridine release from waterinoil emulsions as a function of pH. J. Pharm. Sci. 57,87–92.

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Koizumi, T., Higuchi, W.I., 1968b. Analysis of data from emulsions III. Boundary effect on pyridine release from water-in-oil emulsions as a function of pH. J. Pharm. Sci. 57, 93–97.

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Laugel, C., Chaminade, P., Baillet, A., Seiller, M., Ferrier, D.,1996. Moisturizing substances entrapped in w:o:w emulsions:analytical methodology for formulation, stability and release studies. J. Controlled Release 38, 59–67.

22.

Magdassi, S., Garti, N., 1986. A kinetic model for release of electrolytes from w:o:w multiple emulsions. J. Controlled Release 3, 273–277.

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Omotosho, J.A., Whateley, T.L., Law, T.K., Florence, A.T.,1986. Nature of the oil phase and the release of solutes from multiple (w:o:w) emulsions. J. Pharm. Pharmacol. 38, 865–870.

2SDZDOH ) 2 %XUJHVV ' - ,Qà XHQFH RI LQWHUIDFLDO SURSHUWLHV RI OLSRSKLOLF VXUIDFWDQWV RQ ZDWHU LQ RLO emulsion stability. J. Colloid Interface Sci. 197, 142–150. 25.

Reichmann, K.W., Petersen, R.V., 1973. Temperature studies with nonaqueous emulsions. J. Pharm. Sci. 62, 1850–1856.

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Roger R.C. New*, Christopher J. Kirby, 1997, Solubilisation of hydrophilic drugs in oily formulations, Advanced Drug Delivery Reviews, 25 , 59-69.

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Sakthivel, T., Jaitely, V., Patel, N.V., Florence, A.T., 2001. Nonaqueous emulsions: hydrocarbon-formamide systems. Int. J.Pharm. 214, 43–48.

‰ ‰ ‰

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Design, Optimization and Evaluation of Nanoemulsion Formulations by Ultrasonication as Vehicle for Transdermal Delivery of Diclofenac Diethylamine Praveen Kumar Gupta1, Dr. J.K. Pandit2, Dr. P. J. Narain3, Sanjiv Kumar Gupta3, Akash Chaurasya4 1 National Institute of Medical Sciences University, Jaipur, India 2 Department of Pharmaceutics IIT- B.H.U. Varanasi, India 3 Agra Public Institute of Technology & Computer Education, Agra, India 4 Department of Pharmaceutics, Jamia Hamdard, New Delhi, India

ABSTRACT The present study aimed at developing an optimal nanoemulsion of Diclofenac Diethylamine by ultrasonication method, its characterization and evaluation of its potential as vehicle for transdermal delivery system. On the basis of solubility of Diclofenac Diethylamine in oils, surfactants and cosurfactants, oil in water nanoemulsions were prepared.The nanoemulsion formulations were characterized for physicochemical evaluation.The optimized nanoemulsion formulation, has composition of 1.16% w/w of Diclofenac Diethylamine 10% w/w of isopropyl myristate ( as oil ) 7.5% w/w of tween-80 (as surfactant) 22.5% w/w of transcutol-P (as co-surfactant) and 60% w/w double distilled water.The in-vitro VNLQ SHUPHDWLRQ SURĂ€OH RI RSWLPL]HG QDQRHPXOVLRQ IRUPXODWLRQ WKURXJK UDW DEGRPLQDO VNLQ ZDV GHWHUPLQHG E\ )UDQ] GLIIXVLRQ FHOO DQG ZDV FRPSDUHG WR WKDW RI FRQYHQWLRQDO JHO QDQRHPXOVLRQ JHO $ VLJQLĂ€FDQW LQFUHDVH LQ SHUPHDELOLW\ SDUDPHWHUV VXFK DV VWHDG\ VWDWH Ă X[ SHUPHDELOLW\ FRHIĂ€FLHQW DQG HQKDQFHPHQW UDWLR ZDV REVHUYHG LQ RSWLPL]HG QDQRHPXOVLRQ IRUPXODWLRQ 7KH UHVXOWV DUH VWHDG\ VWDWH Ă X[ “ PJ FPò K SHUPDELOLW\ FRHIĂ€FLHQW “ [ òFP K DQG HQKDQFHPHQW UDWLR 7KH DQWL LQĂ DPPDWRU\ HIIHFW RI IRUPXODWLRQ VKRZHG D VLJQLĂ€FDQW LQFUHDVH 3 LQ SHUFHQW LQKLELWLRQ YDOXH after 24hours when compared with conventional gel (53.2%) nanoemulusion NE1 (84%) and nanogel NG1 (70.3%) on carrageenan induced paw edema in rats. These results suggested that nanoemulsion developed is potential vehicle for improved transdermal drug delivery system of Diclofenac Diethylamine. Keywords 'LFORIHQDF 'LHWK\ODPLQH 1DQRHPXOVLRQ 1DQRHPXOVLRQ JHO $QWL LQĂ DPPDWRU\ DFWLYLW\ INTRODUCTION Diethylamine salt of Diclofenac is reportedly used for topical application. When given orally drug XQGHUJRHV VXEVWDQWLDO KHSDWLF Ă€UVW SDVV PHWDEROLVP DQG WKXV RQO\ DERXW RI WKH DGPLQLVWHUHG GRVH reaches systemic circulation, this originates the need of an alternative route of administration which FDQ E\SDVV WKH KHSDWLF Ă€UVW SDVV PHWDEROLVP 7KH WUDQVGHUPDO URXWH LV DQ DOWHUQDWH FKRLFH RI URXWH of administration of the drugs. The drug Diclofenac Diethylamine also posses the ideal characteristic such as poor bioavailability (40-60%), short biological half life (2-3 hrs) smaller dose (50-100 mg) etc. to be formulated into a transdermal patch. Transdermal patches offer added advantages such as maintenance of constant and prolonged drug level, reduced frequency of dosing, minimization of intra-patient variability, self administration, and easy termination of medication, leading to improved patient compliance.(1) Therefore, an improved Diclofenac Diethylamine nanoemulsion formulation ZLWK D KLJK GHJUHH RI SHUPHDWLRQ FRXOG EH XVHIXO LQ WKH WUHDWPHQW RI ORFDOO\ LQĂ DPPHG VNLQ DQG LQĂ DPPDWRU\ DQG SDLQIXO VWDWHV RI VXSSRUWLQJ VWUXFWXUHV RI WKH ERG\ VXFK DV ERQHV OLJDPHQWV MRLQWV tendons, and muscles. Many of the dermal vehicles contain chemical enhancers and solvents to achieve better penetration. But use of these chemical enhancers may be harmful, especially in prolong use, 26


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as many of them are irritants. One of the most promising techniques for enhancement of transdermal SHUPHDWLRQ RI GUXJV LV QDQRHPXOVLĂ€FDWLRQ 2, 3 Nanoemulsions are thermodynamically stable transparent (translucent) dispersions of oil and water, VWDELOL]HG E\ DQ LQWHUIDFLDO Ă€OP RI VXUIDFWDQW DQG FRVXUIDFWDQW PROHFXOHV KDYLQJ D GURSOHW VL]H RI WR 200 nm.4,5 Many studies have shown that nanoemulsion formulations possess improved transdermal and dermal delivery properties in-vitro,6,7 as well as in-vivo.8 Nanoemulsions have improved transdermal permeation of many drugs over the conventional topical formulations such as emulsions9 and gels.10 MATERIALS AND METHODS Materials Diclofenac Diethylamine (Pulverised) was a gift sample from Pee-Medica (Agra, India). Isopropyl P\ULVWDWH ,30 /LTXLG 3DUDIĂ€Q &RWWRQVHHG 2LO 7ZHHQ 7ZHHQ 6SDQ 3URS\OHQH *O\FRO DQG methanol were purchased from E-Merck (Mumbai, India). Diethylene glycol monoethyl ether (Transcutol P), were purchased from CDH. All chemicals used in the study were of highest purity grade. Screening of Excipients 7KH VROXELOLW\ RI 'LFORIHQDF 'LHWK\ODPLQH LQ YDULRXV RLOV /LTXLG SDUDIĂ€Q ,VRSURS\O P\ULVWDWH cottonseed oil), surfactants (Tween 80, Tween 20, and span 80), and co-surfactants(Transcutol-P and PEG400) was determined by dissolving an excess amount of Diclofenac Diethylamine in 2 ml of the selected oils, surfactants, and co surfactants in 5 ml capacity stoppered vials separately. An excess amount of Diclofenac Diethylamine was added to each 5 ml and mixed using a vortex mixer (NickelElectro Ltd., Oldmix on Crescent, UK). The mixture vials were then kept at 37°C Âą 1.0°C in an isothermal shaker for 72 hours to get to equilibrium. The equilibrated samples were removed from the shaker and FHQWULIXJHG DW USP IRU PLQXWHV 7KH VXSHUQDWDQW OLTXLG ZDV WDNHQ RXW DQG Ă€OWHUHG WKURXJK D —P PHPEUDQH Ă€OWHU 7KH FRQFHQWUDWLRQ RI 'LFORIHQDF 'LHWK\ODPLQH ZDV GHWHUPLQHG LQ HDFK RLO surfactant and co-surfactant by UV spectrophotometer at its respective ÂŻmax=275nm. PSEUDOTERNARY PHASE DIAGRAM STUDY On the basis of the solubility studies, Isopropyl myristate was selected as the oil phase. Tween 80 and Transcutol P were selected as surfactant and co-surfactant, respectively. Distilled water was used as an aqueous phase. Surfactant and co-surfactant (Smix) were mixed in different weight ratios (1:4, 1:3, 1:2, 1:1, 2:1, 3:1). These Smix ratios were chosen in increasing concentration of surfactant with respect to co-surfactant and increasing concentration of co-surfactant with respect to surfactant for detailed study of the phase diagrams needed for nanoemulsion formation. For each phase diagram, oil and Smix were combined in different weight ratios from 1:9 to 9:1 in different glass vials. Different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3,1:3.5, 1:2, 1:1.5, 1:1) were made so that maximum ratios were covered for the study to delineate the boundaries of phases precisely formed in the phase diagrams. From phase diagram constructed, different formulae were selected from the nanoemulsion region, 1.16% w/w Diclofenac Diethylamine which was kept constant in all the selected formulations, Selected formulations were subjected to different thermodynamic stability tests. THERMODYNAMIC STABILITY STUDIES To overcome the problem of metastable formulation, thermodynamic stability tests were performed.11 27


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Selected formulations were centrifuged for 30 minutes. The formulations that did not show any phase separations were taken for the heating and cooling cycle. Six cycles between refrigerator temperature (4°C) and 45°C with storage at each temperature of not less than 48 hours were done. The formulations, which were stable at these temperatures, were subjected to a freeze-thaw cycle test. Three freeze-thaw done for the formulation between –21°C and 25°C.The formulations that survived thermodynamic stability tests were selected for further study. ULTRASONICATION PROCESS Ultrasonication Process was performed under optimized operating conditions of power intensity, homogenization cycle, temperature, total time and reaction time (On time and off time). 50ml dilution formulation mixes were taken into reaction vessel, the horn probe of ultrasonicator was directly immersed into the reaction vessel at a depth of about 1cm for every running. CHARACTERIZATION OF NANOEMULSIONS TRANSMISSION ELECTRON MICROSCOPY Morphology and structure of the nanoemulsions were studied using transmission electron microscopy (TEM), with Topcon 002B operating at 200 kV (Topcon, Paramus, NJ) and capable of point-to-point resolution. To perform the TEM observations, a drop of the nanoemulsion was directly deposited on WKH KROH\ Ă€OP JULG DQG REVHUYHG DIWHU GU\LQJ NANOEMULSION DROPLET SIZE ANALYSIS Droplet size distribution of the nanoemulsion was determined by photon correlation spectroscopy WKDW DQDO\]HV WKH Ă XFWXDWLRQV LQ OLJKW VFDWWHULQJ GXH WR %URZQLDQ PRWLRQ RI WKH SDUWLFOHV XVLQJ D Zetasizer Nano ZS (Malvern Instruments, Worchestershire, UK). Light scattering was monitored at 25°C at a 90° angle. VISCOSITY DETERMINATION 7KH YLVFRVLW\ RI WKH IRUPXODWLRQV ZDV GHWHUPLQHG XVLQJ D %URRNĂ€HOG '9 ,,, XOWUD 9 59 FRQH DQG SODWH UKHRPHWHU %URRNĂ€HOG (QJLQHHULQJ /DERUDWRULHV 0LGGOHERUR 0$ XVLQJ VSLQGOH &3( DW ƒ& Âą 0.3°C. The software used for the calculations was Rheocalc V2.6. REFRACTIVE INDEX The refractive index of drug loaded formulations was determined using an Abbe-type refractometer (Nirmal International). PREPARATION OF CONVENTIONAL DICLOFENAC FORMULA FOR PREPARATION OF DIETHYLAMINE GEL DICLOFENAC DIETHYLAMINE GEL Conventional Diclofenac Diethylamine gel (CG) was Diclofenac Diethylamine 1.2%w/w prepared by dispersing the 1.2g of the Carbopol 940 in Carbopol 940 1.2%w/w D VXIĂ€FLHQW TXDQWLW\ RI GLVWLOOHG ZDWHU DQG NHSW KUV IRU Iso Propyl Alcohol 20%w/w soaking. Then 1.2g of Diclofenac Diethylamine was dissolved Propylene Glycol 5%w/w LQ VSHFLĂ€HG TXDQWLW\ RI ,VR 3URS\O $OFRKRO 7KLV VROXWLRQ RI GUXJ was added slowly to the aqueous dispersion of Carbopol 940 +HDY\ /LTXLG 3DUDIĂ€Q 2.5%w/w and mixed. Then other ingredients like propylene glycol, heavy Stearic acid 1.07%w/w OLTXLG SDUDIĂ€Q VWHDULF DFLG FHWRPDFURJRO ZHUH KHDWHG Cetomacrogol-1000 2.14%w/w cooled and added to obtain a homogeneous dispersion. The Di-ethanolamine 1.4%w/w pH was adjusted up to 7 by diethanolamine. The weight was Distilled water (q.s.) 100%w/w adjusted to 100g by distilled water. 28


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IN VITRO SKIN PERMEATION STUDIES In vitro skin permeation studies were performed on a Franz diffusion cell with an effective diffusional area of 1.13 cm² and 15 ml of receiver chamber capacity using rat abdominal skin. The full-thickness rat skin was excised from the abdominal region, and hair was removed with an electric clipper. The subcutaneous tissue was removed surgically, and the dermis side was wiped with isopropyl alcohol to remove adhering fat. The cleaned skin was washed with distilled water and mounted between the donor and receiver compartment of the Franz diffusion cell, where the stratum corneum side faced the donor compartment and the dermal side faced the receiver compartment. Initially the donor compartment ZDV HPSW\ DQG WKH UHFHLYHU FKDPEHU ZDV Ă€OOHG ZLWK EXIIHU S+ 7KH UHFHLYHU Ă XLG ZDV VWLUUHG ZLWK a magnetic rotor at a speed of 600 rpm, and the assembled apparatus was placed in the transdermal permeation apparatus and the temperature maintained at 32°C Âą 1°C. All buffers were replaced every PLQXWHV WR VWDELOL]H WKH VNLQ ,W ZDV IRXQG WKDW WKH UHFHLYHU Ă XLG VKRZHG QHJOLJLEOH DEVRUEDQFH DIWHU 4.5 hours and beyond, indicating complete stabilization of the skin. After complete stabilization of the skin, 1 ml of nanoemulsion formulation (1.16% of Diclofenac Diethylamine or 1 g of CG (11.6 mg/g) ZDV SODFHG LQWR HDFK GRQRU FRPSDUWPHQW DQG VHDOHG ZLWK SDUDIĂ€Q Ă€OP WR SURYLGH RFFOXVLYH FRQGLWLRQV Samples were withdrawn at regular intervals (1, 2, 3, 4, 5, 6, 9, 12, 18, 21, and 24 hours), Ă€OWHUHG WKURXJK D PHPEUDQH Ă€OWHU DQG DQDO\]HG IRU GUXJ FRQWHQW E\ 89 VSHFWURSKRWRPHWHU 7KH formulation NE1 provided the highest release as compared with the other nanoemulsion formulations. The formulation NE1 was also converted into nanoemulsion gel formulations by adding 1% w/w Carbopol DQG ZDV FRGHG DV 1* 7KH VNLQ SHUPHDWLRQ SURĂ€OH RI WKH RSWLPL]HG QDQRHPXOVLRQ IRUPXODWLRQ was compared with nanoemulsion gel (NG1) and Conventional Gel (CG) using the Dunnett test of 1-way analysis of variance (ANOVA). PERMEATION DATA ANALYSIS The cumulative amount of drug permeated through the skin (mg/cm²) was plotted as a function of WLPH W IRU HDFK IRUPXODWLRQ 'UXJ Ă X[ SHUPHDWLRQ UDWH DW VWHDG\ VWDWH -VV ZDV FDOFXODWHG E\ GLYLGLQJ WKH VORSH RI WKH OLQHDU SRUWLRQ RI WKH JUDSK E\ WKH DUHD RI WKH GLIIXVLRQ FHOO 7KH SHUPHDELOLW\ FRHIĂ€FLHQW (Kp) was calculated by dividing Jss by the initial concentration of drug in the donor cell (C0): ; Enhancement ratio (Er) was calculated by dividing the Jss of the respective formulation by the Jss of the control formulation: SKIN IRRITATION TEST The skin irritation test was carried out on male Swiss albino mice weighing 20 to 25 g. The animals were kept under standard laboratory conditions. A single dose of 10 ÎźL of the nanoemulsion was applied to the left ear of the mice, with the right ear as a control. The development of erythema was monitored for 6 days using the method of Van- Abbe et al 13. IN VIVO EFFICACY STUDY $SSURYDO WR FDUU\ RXW LQ YLYR HIĂ€FDF\ VWXGLHV ZDV REWDLQHG IURP WKH ,QVWLWXWLRQDO $QLPDO (WKLFV Committee Garhwal University (Uttaranchal, India), and the committee’s guidelines were followed for WKH VWXGLHV 7KH DQWL LQĂ DPPDWRU\ DQG VXVWDLQLQJ DFWLRQ RI WKH RSWLPL]HG IRUPXODWLRQ 1( ZDV HYDOXDWHG by the carrageenan-induced hind paw edema method developed by Winter et al in Wistar rats.14 Young Wistar rats weighing 120 to 150 g were randomly divided into 4 groups: control (1% w/w Carageenan 29


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preparation), nanoemulsion (NE1), nanoemulsion gel (NG1), and Conventional Gel (CG), each group containing 6 rats. The animals were kept under standard laboratory conditions. The dose for the rats was calculated based on the weight of the rats according to the surface area ratio.15 The abdominal region of the rats was shaved 12 hours before the experiments started, except in the control group. NE1, NG1, and CG were applied on the shaved abdominal region of all animals (except in control group) half an hour before sub planter injection of carrageenan in right paws. Paw edema was induced by injecting 0.1 ml of the 1% w/w homogeneous suspension of carrageenan in distilled water. The volume of paw was measured at 1, 2, 3, 6, 12, and 24 hours after injection using a plethysmometer. The amount of paw swelling was determined for 24 hours and expressed as percent edema relative to the initial hind paw volume. Percent inhibition of edema produced by each formulation-treated group ZDV FDOFXODWHG DJDLQVW WKH UHVSHFWLYH FRQWURO JURXS 5HVXOWV RI DQWL LQÁDPPDWRU\ DFWLYLW\ ZHUH FRPSDUHG using the Dunnett test of 1-way ANOVA. RESULTS AND DISCUSSION Excipient Selection The excipients selected needed to be pharmaceutically acceptable, nonirritating, and non-sensitizing to the skin and to fall into the GRAS (generally regarded as safe) category. Higher solubility of the drug in the oil phase was another important criterion, as it would help the nanoemulsion to maintain the drug in solubilized form. Safety is a major determining factor in choosing a surfactant, as a large amount of surfactants may cause skin irritation. Non-ionic surfactants are less toxic than ionic surfactants. An important criterion for selection of the surfactants is that the required hydrophilic lipophilic balance (HLB) value to form the o/w nanoemulsion be greater than 10. The right blend of low and high HLB surfactants leads to the formation of a stable nanoemulsion formulation.16 In this study, we selected Tween 80 as a surfactant with an HLB value of 15. Transient negative interfacial tension and ÁXLG LQWHUIDFLDO ÀOP DUH UDUHO\ DFKLHYHG E\ WKH XVH RI VLQJOH Table 1: Solubility of Diclofenac surfactant; usually, addition of a co-surfactant is necessary. Diethylamine in Various The presence of co-surfactant decreases the bending stress of Excipients (n=3) LQWHUIDFH DQG DOORZV WKH LQWHUIDFLDO ÀOP VXIÀFLHQW ÁH[LELOLW\ WR Solubility take up different curvatures required to form nanoemulsions Excipients Mean ± SD over a wide range of composition.17 Thus, the co-surfactant (mg/ml) selected for the study was Transcutol P, which has an HLB 6.2 ± 0.49 value of 4.2. Diclofenac Diethylamine is a lipophilic drug, /LTXLG 3DUDIÀQ and its physicochemical properties suggest that it has good Cottonseed oil 12.8 ± 1.31 potential for transdermal drug delivery. Therefore, in the present Iso propyl myristate 33.64 ± 2.41 study different nanoemulsions were prepared for transdermal Tween 80 14.6 ± 1.42 delivery. Screening of Excipients The most important criterion for screening of excipients is the solubility of the poorly soluble drug in oil, surfactants, and co-surfactants. Since the aim of this study is to develop a transdermal formulation, it is important to determine the solubility of the drug in oils, surfactants, and co-surfactants. The 30

Tween 20

8.45 ± 1.02

Span 80

4.2 ± 0.41

Transcutol-P

322 ± 3.17

PEG-400

84 ± 2.89

Water

19.1 ± 0.98

Phosphate buffer pH-7.4

15.6 ± 1.12


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solubility of Diclofenac Diethylamine was found to be highest in isopropyl myristate (33.64Âą2.41mg/ ml)as compared with other oils. Thus, it was selected as the oil phase for the development of the optimal formulation. The highest solubility of the drug was seen in Tween 80 (14.6Âą 1.42 mg/ml) and Transcutol P (322Âą 3.17 mg/ml). Therefore, Tween-80 and Transcutol P were selected as surfactant and co-surfactant, respectively, as shown in Table 1. PSEUDOTERNARY PHASE DIAGRAM STUDY Constructing phase diagrams is time-consuming; particularly when the aim is to accurately delineate a phase boundary. Care was taken to ensure that observations were not made on metastable systems. Although the energy required to form an emulsion is low, the formation is thermodynamically approaching to stable. The relationship between the phase behavior of a mixture and its composition can be captured with the aid of a phase diagram.18 Pseudoternary phase diagrams were constructed VHSDUDWHO\ IRU HDFK 6PL[ UDWLR VR WKDW R Z QDQRHPXOVLRQ UHJLRQV FRXOG EH LGHQWLĂ€HG DQG QDQRHPXOVLRQ formulations could be optimized. In the Smix ratio 3:1 has a low nanoemulsion area. Different ratios of o/w nanoemulsion region were represented in Figure 1.

Figure 1 : Pseudoternary Phase Diagrams

Towards the water-rich apex of the phase diagram the maximum concentration of oil that could be solubilized in the phase diagram was only 10% w/w using 20% w/w of 1:2 Smix. As the co-surfactant concentration was increased in the Smix ratio 1:3 a higher nanoemulsion region was observed, SHUKDSV EHFDXVH RI IXUWKHU UHGXFWLRQ RI WKH LQWHUIDFLDO WHQVLRQ LQFUHDVLQJ WKH Ă XLGLW\ RI WKH LQWHUIDFH 31


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thereby increasing the entropy of the system. There may be greater penetration of the oil phase in the hydrophobic region of the co-surfactant monomers. The maximum concentration of oil that could be solubilized in the phase diagram was 12% w/w using 36% w/w of 1:4 Smix and 10% w/w using 30% w/w of 1:3Smix. As we further increased co-surfactant concentration, Smix 1:4 the nanoemulsion regions increased as compared to the region in 1:1. The maximum concentration of oil that could be solubilized by this ratio was 10% w/w using 30% w/w of Smix. When the Smix ratio of 1:1 was studied the nanoemulsion region decreased slightly as compared with1:4, which may have been due to the increased concentration of the surfactant, although the maximum oil that could be solubilized by this ratio of Smix was 10% w/w with 20% w/w of Smix. Similarly, when the Smix ratio of 1:4 was studied the nanoemulsion area further decreased as compared with 1:2 and 1:3. The maximum concentration of oil that could be solubilized by this ratio of Smix was 10% w/w with 30% w/w of Smix. When surfactant concentration increased as compared with co-surfactant, the nanoemulsion area decreased in the 2:1 ratio, but in the 3:1 ratio the nanoemulsion region more decreased again, so there was no need to try a Smix ratio of 4:1. Thus, in the phase diagrams, it can be seen that the free energy of nanoemulsion formation can be considered to depend on the extent to which the surfactant lowers the surface tension of the oil-water interface and the change in dispersion entropy. Thus, a negative free energy of formation is achieved ZKHQ D ODUJH UHGXFWLRQ LQ VXUIDFH WHQVLRQ LV DFFRPSDQLHG E\ VLJQLĂ€FDQW IDYRUDEOH HQWURSLF FKDQJHV In such cases, nanoemulsion dispersions are thermodynamically stable. The surfactant or Smix, which Table 2 : Composition of Selected Nanoemulsion are able to increase the dispersion entropy, reduce the interfacial tension, increase the Formulations interfacial area, and thus lower the free Oil/Smix Smix energy of the system to a very low value Code Oil Smix Water Ratio Ratio with the minimum concentration (weight NE-1 10 30 60 1:3 1:3 ratio), which is thermodynamically stable. NE-2 12 36 52 1:3 1:4 From pseudoternary phase diagrams, the NE-3 12 36 52 1:3 1:3 formulations in which the amount of oil phase completely solubilized the drug and NE-4 12 36 52 1:3 1:2 which could accommodate the optimum NE-5 10 30 60 1:3 1:2 quantity of Smix and distilled water were NE-6 10 30 60 1:3 1:4 selected for the study. The composition of NE-7 10 20 70 1:2 1:1 selected nanoemulsion formulations are NE-8 12 48 40 1:4 2:1 given in Table 2. THERMODYNAMIC STABILITY STUDIES Nanoemulsions are thermodynamically stable systems and are formed at a particular concentration of oil, surfactant, and water, making them stable and not subject to phase separation, creaming, or cracking. It is the thermostability that differentiates nano- or microemulsions from emulsions that have kinetic stability and eventually phase-separate.19 Thus, the formulations were tested for their thermodynamic stability by using centrifugation, a heating-cooling cycle, and a freeze-thaw cycle. Only formulations that survived the thermodynamic stability tests were selected for further study. The compositions and tests of selected formulations are given in Table 3. 32


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Table 3 : Smix ratio (S: CoS)

Thermodynamic stability and dispersibility test of different formulation Percentage w/w of different components in formulation Oil

Smix

Observations of thermodynamic stability studies and dispersible test

Aqueous Heating Cooling Cycle

Centrifugation

Freeze thaw Cycle

Visual Observation

Inference

1:1

1.0 1.2 1.5

1.0 1.2 1.5

1.0 1.2 1.5

7.0 6.4 5.5

P P P

P P F

P P -

Grade A Grade A Grade C

Passed Passed Failed

1:2

1.0 1.2 1.5 1.8

1.0 1.2 1.5 1.8

2.0 2.4 3.0 3.6

6.0 5.2 4.0 4.6

P P P F

P P P -

P P P -

Grade A Grade A Grade B Grade C

Passed Passed Passed Failed

1:3

1.0 1.2 1.5 1.8

0.75 0.90 1.12 1.35

2.25 2.70 3.38 4.05

6.0 5.2 4.0 2.8

P P P P

P P P P

P P P P

Grade A Grade A Grade B Grade B

Passed Passed Passed Passed

1:4

1.0 1.2 1.5 1.8

0.8 3.2 0.96 3.84 1.2 4.8 1.44 5.76

5.0 4.0 2.5 1.0

P P P F

P P F -

P P -

Grade A Grade A Grade C Grade D

Passed Passed Failed Failed

2:1

1.0 1.2 1.5 1.8

2.67 1.33 3.2 1.6 4.0 2.0 4.8 2.4

5.0 4.0 2.5 1.0

P P F F

P P -

P F -

Grade A Grade C Grade D Grade D

Passed Failed Failed Failed

3:1

1.0 1.2 1.5 1.8

2.25 2.70 3.37 4.05

6.0 5.2 4.0 2.8

P P F F

P P -

P P -

Grade A Grade A Grade C Grade C

Passed Passed Failed Failed

0.75 0.90 1.13 1.35

(P=Passed), (F= Failed)

ULTRASONICATION PROCESS 20, 21, 22 The size of a namoemulsion droplet formed by ultrasonication is controlled by the interplay between droplet breakup and droplet coalescence. Droplet breakup is controlled by the type and amount of shear (150watts/cm2) applied to droplets as well as the droplets resistance to deformation (Laplace pressure) which is determined by the surfactant. The rate of droplet coalescence (related to droplet stability) is determined by the ability of the surfactant to rapidly adsorb to the surface of newly formed droplets; this is controlled by surface activity and concentration of surfactant. The total sonication time was 60 min with 3 homogenization cycle of 20 minutes on three consecutive days. The pulse duration was adjusted of 9 sec on 3 sec off every cycle of 12 sec. Energy control can be obtained through pulse mode operation. Ultrasonic probe system, the instrument often allows ultrasonic irradiation to be applied in different pulse modes which enables the sound waves to be delivered intermittently, allowing for periods of cooling. The droplet size passes through a minimum size at an intermediate power application (150 watts/ cm ). At the high shear rates described as “over processing� which is caused by an increase in emulsion droplet coalescence. 2

33


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The optimized temperature of Ultrasonication is 400C; increasing temperature is believed to possible drug decomposition. The optimized parameters of ultrasonication process is shown in Table 4. Table 4 : Optimized parameters of Ultrasonication process Temperature (ºC)

Pressure (watts/cm2)

Homenization Cycle

Off Time (sec)

On Time (sec)

Total Time (min)

40

150

03

03

09

20

TRANSMISSION ELECTRON MICROSCOPE In the TEM positive image, the nanoemulsion appeared dark and the surroundings were bright. Some droplet sizes were measured, as TEM is capable of point-to-point resolution. Particle size TEM as shown in Figure 2. NANOEMULSION DROPLET SIZE ANALYSIS The droplet size increased with the increase in concentration of oil in the formulations. The droplet size of formulation NE1, containing 10% oil, was lowest (56.95 ±1.46 nm). The droplet size of formulation NE8 was highest (68.3 ± 5.26 nm). All the formulations had droplets in the Figure 2 : Transmission Electron Microscope nano range, which is very well evident from the low polydispersity values. Polydispersity is the ratio of standard deviation to mean droplet size, so it indicates the uniformity of droplet size within the formulation. The higher the polydispersity, the lower the uniformity of the droplet size in the formulation. Although the polydispersity values of all formulations were low, indicating uniformity of droplet size within each formulation, the polydispersity of formulation NE1 was lowest (0.315). Different optimized nanoemulsions particle size analysis and zeta potential as shown in Figure 3. (i-a) Particle Size Analysis of DDEA nanoemulsion Oil:Smix (1:3),Smix(1:3)

(i-b) Zeta Potential Report of DDEA nanoemulsion Oil:Smix (1:3),Smix(1:3)

34

(ii-a) Particle Size Analysis of DDEA nanoemulsion Oil:Smix (1:3),Smix(1:4)


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Figure 3 : Particle Size Analysis and Zeta Potential of Nanoemulsions

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VISCOSITY DETERMINATION Table 5 : Droplet size, Polydispersity values, The viscosities of the selected formulations Viscosity and pH of the Nanoemulsion were determined. The viscosity of formulation Formulations (n=3) NE1 (26.28 ± 1.22cP) was lower than that Viscosity Polyof any other formulation, and this difference Code Droplet Size mean ± pH ± SD (nm) dispersity ZDV VLJQLÀFDQW 3 7KH YLVFRVLW\ RI SD(cP) NE-1 56.95 ± 1.46 0.315 26.28 ± 1.22 5.52 formulation NE 8 was highest (60.52 ± NE-2 58.93 ± 1.5 0.302 28.52 ± 1.68 5.48 2.91cP), but it was observed that the viscosity of NE-3 57.31 ± 1.48 0.247 34.46 ± 1.82 5.36 the nanoemulsion formulations generally was very low. This was expected, because one of the NE-4 58.11 ± 1.53 0.370 38.22 ± 1.70 5.32 characteristics of nanoemulsion formulations NE-5 53.58 ± 1.57 0.380 44.12 ± 1.70 5.40 is lower viscosity. The optimized formulations NE-6 65.55 ± 1.62 0.435 46.34 ± 1.86 5.38 values are given in Table 5. NE-7 67.55 ± 1.65 0.351 52.62 ± 1.94 5.28 NE-8

Table 6 :

Code NE-1 NE-2 NE-3 NE-4 NE-5 NE-6 NE-7 NE-8

65.96 ± 1.72

0.456

60.52 ± 2.91

5.22

Zeta Potential, Conductivity and ZETA POTENTIAL 7KH VLJQLÀFDQFH RI ]HWD SRWHQWLDO LV WKDW Refractive Index of selected Nanoits value can be related to the stability of emulsions nanoemulsions. A value of 25 mV (positive ZP(mV) Conductivity Refractive Index or negative) can be taken as the arbitrary Mean ± SD value that showed moderate stability. As per -41.9 0.0655 1.432 ± 0.009 Table 6, Zeta potential values of optimized -37.6 0.0606 1.43 ± 0.005 nanoemulsions were more than 25 mV which -36.5 0.0674 1.429 ± 0.008 -35.3 0.117 1.428 ± 0.007 indicates that the nanoemulsions were fairly -34.8 0.0658 1.427 ± 0.006 stable. -31.9 -31.1 -30.6

0.0803 0.0854 0.0885

1.424 ± 0.003 1.422 ± 0.005 1.423 ± 0.004

REFRACTIVE INDEX The refractive index values for formulations were compared to the Refractive Indices of blank, it was found that there were no significant differences between the values. Therefore, it can be concluded that the nanoemulsion formulations were not only thermodynamically stable but also chemically stable and remained isotropic; thus, there were no interactions between nanoemulsion excipients and drug. The values are given in Table 7. SCANNING ELECTRON MICROSCOPE Scanning Electron microscope image of Diclofenac Diethylamine nano gel showed uniform 36

Table 7 :

Sample Code

Refractive Index of Selected Nano-emulsions and Blank Nanoemulsion Formulations (n=3) Refractive Index ± SD of Nanoemulsion Fresh

Blank

NE1

1.432 ± 0.009

1.439 ± 0.004

NE2

1.43 ± 0.005

1.436 ± 0.008

NE3

1.429 ± 0.008

1.437 ± 0.005

NE4

1.428 ± 0.007

1.435 ± 0.003

NE5

1.427 ± 0.006

1.433 ± 0.007

NE6

1.424 ± 0.003

1.432 ± 0.006

NE7

1.422 ± 0.005

1.430 ± 0.004

NE8

1.423 ± 0.004

1.432 ± 0.002


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distribution of drug particles in the gel matrix and all the particles are in the nanometer range as shown in Figure 4.

Figure 4 : Scanning Electron microscope (SEM) positive image of Diclofenac Diethylamine Nano gel (NG1)

IN VITRO SKIN PERMEATION STUDIES In vitro skin permeation studies were performed to compare the release of drug from 8 different nanoemulsion formulations (NE1NE8), as shown in Figure 5. In vitro skin permeation comparative studies on Nanoemulsion (NE1), Nanoemulsion Gel (NG1), and Conventional Gel (CG) as shown in Figure 6 all having

the same quantity (1.16% w/w) of Diclofenac Diethylamine. In vitro skin permeation was highest in formulation NE1 and lowest for CG. The formulation NG1 showed an intermediate skin SHUPHDWLRQ SURĂ€OH 7KH VNLQ Figure 5 : Permeation Study of Figure 6 : Permeation Study of Nanoemulsion SHUPHDWLRQ SURĂ€OH RI 1( ZDV (NE1), Nanoemulsion Gel (NG1), & Nanoemulsions significantly different when Conventional Gel (CG) compared with that of CG and 1* 3 7KH VLJQLĂ€FDQW GLIIHUHQFH LQ 'LFORIHQDF 'LHWK\ODPLQH SHUPHDWLRQ EHWZHHQ QDQRHPXOVLRQ formulations, NG1, and CG was probably due to the mean size of internal phase droplets, which were VLJQLĂ€FDQWO\ VPDOOHU LQ QDQRHPXOVLRQV 7KH PD[LPXP UHOHDVH LQ 1( FRXOG EH GXH WR KDYLQJ WKH ORZHVW droplet size and lowest viscosity of all the nanoemulsions. Table 8 :

Permeability Parameters of Different Formulations

Formulations CG

Jss Âą SD (mg/ cm2h) 0.0883 Âą 0.015

Kp Âą SD (cm/h) x 10ÂŻ2 0.7613 Âą 0.082

NE1

0.2871 Âą 0.084

2.475 Âą 0.118

3.25

NE2

0.2856 Âą 0.086

2.462 Âą 0.109

3.23

NE3

0.2353 Âą 0.092

2.029 Âą 0.112

2.66

NE4 NE5 NE6

0.1886 Âą 0.088 0.1584 Âą 0.076 0.1392 Âą 0.080

1.626 Âą 0.11 1.365 Âą 0.014 1.2 Âą 0.010

2.13 1.79 1.57

NE7

0.1323 Âą 0.078

1.14 Âą 0.016

1.49

NE8

0.1158 Âą 0.080

0.998 Âą 0.112

1.31

NG1

0.2 Âą 0.06

1.724 Âą 0.102

2.26

37

Er –

PERMEATION DATA ANALYSIS Permeability parameters like VWHDG\ VWDWH Ă X[ -VV SHUPHDELOLW\ FRHIĂ€FLHQW .S DQG HQKDQFHPHQW ratio (Er) were significantly increased in nanoemulsions and the NG1 formulation as compared with CG (P<0.05). This is because nanoemulsions and NG1 excipients contain permeation enhancers like Tween 80, and Transcutol-P. The permeability parameters of different formulations are given in Table 8.


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

SKIN IRRITATION TEST 7KH VNLQ LUULWDWLRQ WHVW ZDV SHUIRUPHG WR FRQÀUP WKH VDIHW\ RI WKH RSWLPL]HG QDQRHPXOVLRQ IRUPXODWLRQ The value between 0 and 7 indicates that the applied formulation is generally not an irritant to human skin13. The mean skin irritation score for formulation NE1 was 1.75 ¹ 0.48. From this it was concluded that the optimized nanoemulsion formulation was safe to be used for transdermal drug delivery. IN VIVO EFFICACY STUDY Based on higher drug permeation, lowest droplet size, lowest viscosity, and lowest polydispersity index, formulation NE1 was VHOHFWHG IRU WKH VWXG\ RI LQ YLYR DQWL LQà DPPDWRU\ HIIHFWV 7KH DQWL LQà DPPDWRU\ DQG VXVWDLQLQJ action of the optimized formulation was evaluated by the carrageenan induced hind paw edema method developed by Winter.et.al. The percent inhibition value after 24 hours of administration was found to be high for NE1 that is, 84% and for NG1 was 70.3%, as compared to 53.2% for CG as shown in Figure 7 this difference was extremely Figure 7 $QWL LQÀDPPDWRU\ HIIHFWV RI 'LFORIHQDF 'LHWK\ODPLQH Nanoemulsion (NE1), Nanoemulsion Gel (NG1), & VLJQLÀFDQW 3 7KH SHUFHQW LQKLELWLRQ YDOXH Conventional Gel (CG) formulations. for formulation between NE1’s and NG1’s was VLJQLÀFDQW 3 7KH HQKDQFHG DQWL LQà DPPDWRU\ HIIHFWV RI IRUPXODWLRQ 1( FRXOG EH GXH WR WKH enhanced permeation of Diclofenac Diethylamine through the skin. CONCLUSION On the basis of highest drug permeation, lowest droplet size, lowest polydispersity, lowest viscosity, and optimum surfactant and co-surfactant concentration, we selected formulation NE1 of Diclofenac Diethylamine which contained isopropyl myristate (10% w/w) Tween 80 (7.5% w/w), TranscutolP (22.5% Z Z DQG GLVWLOOHG ZDWHU Z Z IRU XVH LQ YLYR VWXGLHV 7KH LQ YLYR VWXGLHV UHYHDOHG D VLJQLÀFDQW LQFUHDVH LQ WKH DQWL LQà DPPDWRU\ HIIHFWV DV FRPSDUHG WR 'LFORIHQDF 'LHWK\ODPLQH JHO DQG QDQRHPXOVLRQ gel. From in vitro and in vivo data it can be concluded that the developed nanoemulsions have great potential for transdermal drug delivery. REFERENCES 1.

Arora, P. and Mukherjee, B. Design, development, physicochemical, and in vitro and in vivo evaluation of transdermal patches containing Diclofenac Diethylamine salt. J. Pharm. Sci. 2002, 91: 2076-2089.

) 6KDNHHO 6 %DERRWD $ $KXMD - $OL0 $TLO DQG 6 6KDĂ€T 1DQRHPXOVLRQ DV 9HKLFOHV IRU 7UDQVGHUPDO GHOLYHU\ RI Aceclofenac : AAPS Pharm Sci Tech 2007;104:E1YE9.

6 %DERRWD ) 6KDNHHO $ $KXMD - $OL 6 6KDĂ€T 'HVLJQ GHYHORIPHQW DQG HYDOXDWLRQ RI QRYHO QDQRHPXOVLRQ formulations for transdermal potential of celecoxib. Acta Pharm. 2007; 57:315-332.

6KDĂ€T 6 )DL\D] 6 6XVKPD 7 $KPDG )- .KDU 5. $OL 0 'HVLJQ DQG GHYHORSPHQW RI RUDO RLO LQ ZDWHU UDPLSULO nanoemulsion formulation: in vitro and in vivo evaluation. J Biomed Nanotech. 2007; 3:28Y44.

6KDĂ€T 6 )DL\D] 6 6XVKPD 7 $KPDG )- .KDU 5. $OL 0 'HYHORSPHQW DQG ELRDYDLODELOLW\ DVVHVVPHQW RI UDPLSULO nanoemulsion formulation. Eur J Pharm Biopharm. 2007; 66:227Y243.

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

6.

Osborne DW, Ward AJ, Neil KJ. Microemulsions as topical delivery vehicles: in-vitro transdermal studies of a model hydrophilic drug. J Pharm Pharmacol. 1991; 43:450Y454.

7.

Rhee YS, Choi JG, Park ES, Chi SC. Transdermal delivery of ketoprofen using microemulsions. Int J Pharm. 2001; 228:161Y170.

.HPNHQ - =LHJOHU $ 0XOOHU %: ,QĂ XHQFH RI VXSHUVDWXUDWLRQ RQ WKH SKDUPDFRG\QDPLF HIIHFW RI EXSUDQRORO DIWHU dermal administration using microemulsions as vehicle. Pharm Res. 1992; 9:554Y558.

9.

Ktistis G, Niopas I. A study on the in-vitro percutaneous absorption of propranolol from disperse systems.J Pharm Pharmacol. 1998; 50:413Y419.

10.

Kriwet K, Muller-Goymann CC. Diclofenac release from phospholipiddrug systems and permeation through excised human stratum corneum.Int J Pharm. 1995; 125:231Y242.

6KDĂ€T XQ 1DEL 6 6KDNHHO ) 7DOHJDRQNDU 6 HW DO )RUPXODWLRQ GHYHORSPHQW DQG RSWLPL]DWLRQ XVLQJ QDQRHPXOVLRQ technique: a technical note. AAPS Pharm SciTech [serial online]. 2007; 8:E28. $WWZRRG ' 0DOORQ & .WLVWLV * 7D\ORU &- $ VWXG\ RQ IDFWRUV LQĂ XHQFLQJ WKH GURSOHW VL]H LQ QRQLRQLF RLO LQ ZDWHU microemulsions. Int J Pharm. 1992; 88:417Y422. 13.

Van-Abbe NJ, Nicholas P, Boon E. Exaggerated exposure in topical irritancy and sensitization testing. J Soc Cosmet Chem. 1975; 26:173Y187.

:LQWHU &$ $QWL LQĂ DPPDWRU\ WHVWLQJ PHWKRGV FRPSDUDWLYH HYDOXDWLRQ RI LQGRPHWKDFLQ DQG RWKHU DJHQWV 1RQVWHURLG $QWL LQĂ DPPDWRU\ 'UXJV < 15.

Gosh MN. Fundamentals of Experimental Pharmacology. Kolkata, India: Hilton and Company; 2005:192.

&UDLJ '40 %DUNHU 6$ %DQQLQJ ' %RRWK 6: $Q LQYHVWLJDWLRQ LQWR WKH PHFKDQLVPV RI VHOI HPXOVLĂ€FDWLRQ XVLQJ particle size analysis and low frequency dielectric spectroscopy.Int J Pharm. 1995; 114:103Y110. 17.

Eccleston J. Microemulsions. In: Swarbrick J, Boylan JC, eds. Encyclopedia of Pharmaceutical Technology. vol. 9. New York, NY:Marcel Dekker; 1995:375Y421.

18.

Lawrence MJ, Rees GD. Microemulsion-based media as novel drugdelivery systems. Adv Drug Deliv Rev. 2000; 45:89Y121.

19.

Shinoda K, Kunieda H. Phase properties of emulsions: PIT and HLB. In: Schuster D, ed. Encyclopedia of Emulsion Technology. New York, NY: Marcel Dekker; 1983:337Y367.

6 -DIDUL ( $VVDGSRR < +H % %KDQGDUL 5H &RDOHVFHQFH RI HPXOVLRQ GURSOHWV GXULQJ KLJK HQHUJ\ HPXOVLĂ€FDWLRQ Food Hydrocolloids 2008; 22:1191Y1202. 7 6 + OHRQJ 7 - :RRVWHU 6 ( .HQWLVK 0 $VKRN .XPDU 0LQLPLVLQJ RLO GURSOHW VL]H XVLQJ XOWUDVRQLF HPXOVLĂ€FDWLRQ Ultrasonics sonochemistry 2009; 16:721-727. 22.

S. Kentish, T.J. Wooster, M. Ashok Kumar, S. Balachandran, R.Mawson, L.Simons The use of ultrasonics for nanoemulsion preparation Ultrasonics sonochemistry 2007; 07: 170-175.

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Using Albino Rats in Your Experiment? Make Sure They Are Healthy Saibal Das1, Ankur Datta1, Suvajit Das1, Patralekha Roychowdhury1, Anuradha De (Pati)2, S.K. Tripathi1* 1 Department of Clinical & Experimental Pharmacology, & 2 Department of Pathology, Calcutta School of Tropical Medicine, Kolkata

ABSTRACT Silent presence of cestode infection in experimental rats may prove a hidden confounder and contaminate the outcome of a planned experiment. We present here a chance observation highlighting such infection. :KLOH FRQGXFWLQJ DQ DQWLK\SHUOLSLGHPLF VFUHHQLQJ VWXG\ DOELQR UDWV ZHUH VDFULÀFHG DV D SDUW RI safety assessment, per protocol. In 3 animals, macroscopic examination of liver revealed cysts suggestive of cestode infection. Therefore, histopathological examination of the liver, lung and brain tissues were SHUIRUPHG +LVWRSDWKRORJLFDO H[DPLQDWLRQ FRQÀUPHG LQIHFWLRQ ZLWK Taenia taeniaeformis in the 3 animals. ,Qà DPPDWRU\ FKDQJHV ZHUH DOVR QRWHG LQ OXQJ WLVVXHV RI DQRWKHU DQLPDOV +RZHYHU EUDLQ WLVVXHV showed no abnormality. The chance detection of cestode infection in the otherwise asymptomatic rats reiterated importance of compliance to CPCSEA guidelines with reference to maintaining good laboratory hygiene and use of standard laboratory diet. This may avoid erroneous outcomes of experiment because of such infections. INTRODUCTION Albino rats are most commonly used experimental animals. They are vulnerable to asymptomatic cestode infection with Taenia taeniaeformis (Cysticercus fasciolaris). Rodents, mostly rats and mice are intermediate host and domestic cat is GHÀQLWLYH KRVW in the life cycle of this cestode. The route of entry of the parasite in rodents may be through contaminated food and bedding materials. These infections are not apparently harmful to the host, but may affect the experimental results.1-3 Infection of rats with Taenia taeniaeformis increases hepatic cytochrome P 450 and induces the activities of other cytochrome enzymes thereby altering the metabolism of many experimental drugs using cytochrome P 450 for metabolism.4 MATERIALS AND METHODS We conducted an antihyperlipidemic screening study with 35 albino rats procured from a recognized ORFDO ODE DQLPDO EUHHGHU $V UHTXLUHG E\ WKH SURWRFRO RI WKH VDLG VWXG\ RQO\ UDWV ZHUH VDFULÀFHG as a part of safety assessment of the candidate drug exposed to screening, and livers from each animal were dissected for histopathological examinations. In 3 out of the 10 specimens, macroscopic examination revealed presence of whitish nodular areas on the surface of the liver tissue. We suspected the nodular areas to be cysticercosis, and accordingly focused attention was paid while examining the histology slides under microscope. We also dissected and isolated lung and brain tissues from all WKH VDFULÀFHG DQLPDOV IRU IXUWKHU KLVWRSDWKRORJLFDO H[DPLQDWLRQV +RZHYHU WKHUH ZDV QR REYLRXV PDFURVFRSLF ÀQGLQJV LQ WKH OXQJ RU EUDLQ WLVVXHV Standard procedure was followed5 for histopathological examination of liver, lung and brain specimens. Microscopy was done after haematoxylin and eosin (H & E) staining. 40


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

RESULTS The overall histopathological features has been summarized in Table 1. 7DEOH +LVWRSDWKRORJLFDO ÀQGLQJV RI /LYHU OXQJ DQG EUDLQ WLVVXHV Tissue examined

Number of specimens

Sections

Normal architecture

,QĂ DP mation

Larval stage of Taenia taeniaeformis

The detailed features were as follows:

A. Liver : In 3 specimens, inflammation Liver 10 Through hilum 4 3 3 around portal vessels Lung 10 Longitudinally 7 3 nil and central vein were down the main observed. In another 3 bronchus specimens, cross section Brain 10 Coronally and 10 nil nil of larval stage of cestode saggitally (Taenia taeniaeformis) ZLWK WKLFN ]RQH RI LQà DPPDWRU\ UHDFWLRQ LQ WKH IRUP RI O\PSKRF\WHV SODVPD FHOOV PDFURSKDJHV neutrophils and eosinophils along with encapsulation of the matured larva were found. The wall of WKH ODUYD VKRZ RXWHU ZDY\ WHJXPHQW FRYHUHG ZLWK ÀQH ÀODPHQWV DQG QXFOHL EHQHDWK WKH WHJXPHQW Calcareous corpuscles and loose reticular parenchyma were also seen. (Fig. 1a and b).

B. L u n g : O n h i s t o p a t h o l o g i c a l examination, 3 lung specimens showed lymphocytic cuffing around major airways and around the blood vessels especially in the periphery of the lung tissues. (Fig. 2).

Histological picture

C. Brain : Normal histological picture in all the 10 specimens were observed.

Fig. 2 /XQJ VKRZLQJ O\PSKRF\WLF FXIÂżQJ around bronchioles (H & E x10).

Fig. 1 (a) - Liver with Taenia taeniaeformis in the cyst (H & E x10), Fig 1 (b) - Larval form of Taenia taeniaeformis within liver tissue (H & E x10).

DISCUSSION 6HFWLRQV RI WKH FHVWRGH 7DHQLD WDHQLD HIRUPLV ZLWK WKHLU GLIIHUHQW OD\HUV ZHUH LGHQWLÀHG LQ OLYHU specimens. Cysticercus is the resting stage of the larva in the intermediate host forming a bladder like VWUXFWXUH ZLWK LQYDJLQDWHG VFROH[ RQ LWV ZDOO DQG D FHQWUDO FDYLW\ FRQWDLQLQJ à XLG ORRNLQJ OLNH DQ RYDO or elongated milky white cyst. Cyst wall is multilayered and covered with microvilli. Outer cuticular layer is smooth, hyalinised and wavy (tegument) with a row of tegumental cells beneath it. The inner parenchymatous layer is loose and reticular containing mesenchymal cells and calcareous corpuscles taking bluish purple colour in H & E stain – which is the unique feature of the cestode tissue. 7KH LQà DPPDWRU\ FKDQJHV IRXQG LQ WKUHH OXQJ VSHFLPHQV PD\ EH GXH WR VRPH SQHXPRWURSKLF YLUXV infection, some chronic respiratory diseases6 or due to some non-infective antigenic stimuli from diet. These are however not related with cestode infection. The albino rats employed in our study were procured from external source – a recognized commercial breeder of laboratory animals. After procurement they were maintained in our institute animal house where handling, care and feeding were done in compliance with the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals). However, on enquiry it was revealed that 3 weeks prior to the conduct of this experiment, due to some logistic problems the rats were 41


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

put on some indigenous diet instead of standard laboratory diet, for a couple of weeks. The parasites might have been ingested by the animals with contaminated food either in our animal house or even before we procured them from the commercial supplier. It is important to appreciate that asymptomatic cysticercosis can occur in experimental animals and that can contaminate the outcome of a planned research study1-3. Ultrasonographic image analysis and serologic studies can detect such infection7, but it is not practically feasible to use these screening tests routinely. Therefore, strict compliance of CPCSEA guidelines, with particular reference to maintaining good laboratory hygiene, use of standard, non contaminated and nutritious laboratory diet and proper facilities for hygienic food storage are absolutely essential to avoid unexpected erroneous outcomes of experiments using albino rats because of such infections. REFERENCES 1)

Sengupta P, Sharma A, Mazumdar G, Tripathi SK. Asymptomatic cysticercosis in wistar albino rats: A note of caution to all biomedical researchers. Indian J Pharmacol. 2011 Apr; 43(2): 222–3.

2)

Hanes MA, Stribling LJ. Fibrosarcomas in two rats arising from hepatic cysts of Cysticercus fasciolaris. Vet Pathol. 1995; 32: 441-4.

3)

Kumar JM, Reddy Pl, Aparnav , Srinivas G, Nagarajan P, R Venkatesan R, Sreekumar C et al. Strobilocercus fasciolaris infection with hepatic sarcoma and gastroenteropathy in a Wistar colony. Vet Parasitol, 2006 Nov 5; 141 (3-4): 362-7.

4)

Montero R, Serrano L, DĂĄvila VM, Ito A, Plancarte A. Infection of rats with Taenia taeniformis metacestodes increases hepatic CYP450, induces the activity of CYP1A1, CYP2B1 and COH isoforms and increases the genotoxicity of the SURFDUFLQRJHQV EHQ]R>D@S\UHQH F\FORSKRVSKDPLGH DQG DĂ DWR[LQ % 0XWDJHQHVLV 0DU

5)

Chakraborty P, Chakraborty G. Practical Pathology. Kolkata: New Central Book Agency (P) Ltd; 2003. 33: 293-300.

6)

Lamb D. Rat lung pathology and quality of laboratory animals: the users view. Laboratory animals. 1975 Jan; 9: 1-8.

7)

Ito A, Sakakibara Y, Ma L, Asano K, Takiguchi M, Yasuda J, et al. Ultrasonographic and serologic studies of experimental cysticercosis in rats infected with Taenia taeniaeformis. Parasite Immunol. 1998 Mar; 20(3): 105–10.

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Comparative Hepatoprotective Activity of the Two Varieties of Cajanus cajan (L.) Millsp. Leaf Suman Goswami1*, S. Jha2, A. K. Pattanayak2 1 Sun Pharma Sikkim, Gangtok, India 2 Birla Institute of Technology, Mesra, Ranchi, India

ABSTRACT Herbal plants have been recently popularized in modern medicine, since many therapeutically compounds are derived from them. Extracts prepared from these plants are used in the treatment of liver diseases like hepatitis, chirrhosis etc. Cajanus cajan is one of such plant, which is used extensively in folklore medicine to treat hepatic disorder. In Indian reality, the aqueous leaf extract of Cajanus cajan is popularly known to have hepatoprotective activity and widely used for different hepatic disorders. Seventeen species of Cajanus RFFXU LQ ,QGLD EXW LQVXIÀFLHQW GDWD ZDV IRXQG LQ WKH OLWHUDWXUH DERXW WKH comparative hepatoprotective activity of the leaf of Cajanus cajan (L.) Millsp. and their varieties. So the present study was undertaken to validate the traditional value claimed by tribes of North India. In this study we compared the methanolic extract and aqueous extract of the leaf of Cajanus cajan and their two varieties for carbontetrachloride induced hepatotoxicity studies. Keywords : Cajanus cajan, Carbontetrachloride, methanolic and aqueous extract, SGOT, SGPT, ALP, Total bilirubin. INTRODUCTION Studies are going on throughout the world for the search of protective molecules that would provide maximum protection of liver as well as other organs and practically very little or no side effects would be exerted during their function in the body. A number of herbs are traditionally used in different countries during drug or toxin induced hepatic disorders1. Cajanus cajan is one such plant, which is used extensively in folklore medicine to treat hepatic disorder2. Liver diseases remain a serious health problem. It is well known that free radicals cause cell damage through mechanisms of covalent binding and lipid peroxidation with subsequent tissue injury. Antioxidant agents of natural origin have attracted special interest because they can protect human body from free radicals. Numerous medicinal plants and their formulations are used for liver disorders in ethnomedical practices as well as in traditional systems of medicine in India3. Cajanus cajan (L.) Millsp. (Leguminosae: Papilionoideae) is sixth most important IRRG OHJXPH JURZQ DV D ÀHOG DQG RU EDFN\DUG FURS LQ RYHU FRXQWULHV DFURVV WKH JOREH +RZHYHU it is grown only in 19 countries as regular annual crop in 4.4 million hectare producing 3 million tons. India (3.2 m ha), Mayanmar (0.48 m ha), Kenya (0.15 m ha), and Malawi (0.12 m ha) are the major Cajanus cajan growing countries4. About 90% of the world’s supply is produced in Indian subcontinent, where it is the second most important pulse crop5. It is popularized by the name of pigeonpea. Genus Cajanus is comprised of 32 species, Cajanus cajan is the only cultivated IRUP $FFRUGLQJ WR WKH ,QWHUQDWLRQDO 5XOHV RI %RWDQLFDO 1RPHQFODWXUH WKH QDPH ÀQDOO\ DGRSWHG for pigeonpea is Cajanus cajan (L.) Millspaug6. Leaves of this plant have been traditionally used by village people for liver injury and a number of other clinical disorders, such as diabetes, febrifuge, stabilization of menstrual period and dysentery in South America, and for treatment of hepatitis and measles in Africa. In the folk medicine of China, pigeonpea leaves are used to arrest blood, relieve pain and kill worms. The young leaves of pigeonpea can be chewed for treating aphtha, 43


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

and the decoction of the leaves has been proved to treat cough and diarrohea effectively. In recent years, pigeonpea leaves have been used to treat traumatism, burnt infection, bedsore etc. It was DOVR IRXQG WKDW SLJHRQSHD H[KLELW QRWDEOH DQWL LQĂ DPPDWRU\ DQWLELRWLF DQG DELUULWDWLRQ HIIHFWV DQG inhibit capillary permeability7. The aim of the present study was to make a comparative hepatoprotectve activity of the leaf of Cajanus cajan (L.) Millsp. and their varieties. MATERIALS AND METHODS Plant Material The leaves of Cajanus cajan were collected during the month of January, 2009 from Purulia, West Bengal (Variety-1), and Ranchi, Jharkhand (Variety-2) in India. The varieties (both 1 and 2) of Cajanus cajan OHDYHV >5HI 1R &1+ , , 7HFK ,, @ ZHUH LGHQWLĂ€HG DQG DXWKHQWLFDWHG E\ 'U 0 6 Mondal, the Additional Director of Botanical Survey of India, Central National Herbarium, Howrah. 7KH YDULHWLHV ZHUH IXUWKHU LGHQWLĂ€HG E\ 'U * 9 6 0XUWK\ -RLQW 'LUHFWRU RI %RWDQLFDO 6XUYH\ RI ,QGLD Coimbatore. And the Reference No. is: BSI/SC/5/23/09-10/Tech/202. Animals Male Wistar albino rats of 160 – 200g body weight were used in the study. Animals were procured from Laboratory Animal house of Birla Institute of Technology, Mesra, Ranchi, India. All animal experiments strictly complied with the approval of institutional ethical committee. The animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature 24-27°C and humidity 60-65% with 12:12 light: dark cycles). They were acclimatized for seven days before experiment. Food was provided in the form of dry pellets (Hindustan Lever Ltd.) and water ad libitum. Preparation of Extract Methanolic extract and aqueous extracts of both varieties of Cajanus cajan leaf were suspended in 2% tween 80 in distilled water and administered at a dose of 5 ml/kg body weight orally. Dose 200 mg/kg body weight and 400 mg/kg body weight of methanolic leaf extracts of both varieties were given orally. 25 mg/kg body weight of silymarin and 200 mg/kg body weight of aqueous extract of both varieties were given orally. While carbontetrachloride was given intraperitonially at the dose of 1 ml/kg body weight. Drugs And Chemicals Silymarin (Unichem pharmaceuticals, Baddi, India), Carbon tetrachloride (E. Merck, (India) Ltd., Mumbai), SGPT test kit (Span diagnostics Ltd. Surat, India), SGOT test kit (Span diagnostics Ltd. Surat, India), Bilirubin test kit (Bio in vitro diagnostics Pvt. Ltd. Gujarat, India), Alkaline phosphates test kit (Bio in vitro diagnostics Pvt. Ltd. Gujarat, India), Olive oil (E. Merck, India, Ltd. Mumbai), Haematoxylineosin (H & E) dye (Central Drug House Pvt. Ltd. Gujarat, India). Instruments used Clinical Analyzer (Systronics, Model no – 635), Microtome (LYNX, Lawrence & May (I) Pvt. Ltd.), Microscope (Leica). Experimental Design The animals were numbered, weighed and then divided into nine groups of six animals each. 44


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

On day 7 and 9, Carbon tetra chloride 1 ml/kg/day of body weight i.p was given to all rats except the rats in group I. After 24 hours of the second dose of CCl4 blood was collected from all the animals by retro-orbital plexus and the serum was separated by centrifugation at 5000 rpm for 20 minutes, since many of the biochemical and histological changes are known to manifest after 24 hours of CCl4 administration. The serum was separated and used for the estimation of various serum biochemical parameters such as SGOT, SGPT, ALP and serum bilirubin by using respective test kit. The OLYHU ZDV LPPHGLDWHO\ LVRODWHG VOLFHG DQG Ă€[HG LQ IRUPDOLQ IRU KLVWRSDWKRORJLFDO H[DPLQDWLRQ 7KH SLHFHV RI OLYHU ZHUH SURFHVVHG DQG HPEHGGHG LQ SDUDIĂ€Q ZD[ 6HFWLRQV ZHUH PDGH DERXW ² Îźm in thickness, stained with hematoxylin and eosin. They were mounted and observed under light microscope for histological changes. Assesment of Biochemical Parameters Estimation of SGOT and SGPT were based on standard method11 - 12 and the reagent supplied in the kits (Span Diagonostic Ltd.) were reconstituted, mixed with serum as directed. ALP was estimated by mixing with the reagent (p-nitro phenyl phosphate, magnesium acetate and buffers) with serum as prescribed method12 - 14. Total bilirubin was estimated by Jendrassik methods15. Statistical Analysis The values were calculated as mean “ 6(0 7KH VLJQLĂ€FDQFH RI WKH GLIIHUHQFH RI PHDQ YDOXH ZLWK respect to control group was analyzed by one way ANOVA followed by Ducan’s t-test and P value of RU OHVV ZDV FRQVLGHUHG WR EH VLJQLĂ€FDQW TABLE 1:

Effect of methanolic extract and aqueous extract of Cajanus varieties on serum enzyme and serum biochemical parameters in rats. Study period 9 days

Gr. Dose ml/kg

Serum biochemical parameters

For 9 days

Drug

SGOT (IU/L)

SGPT (IU/L)

ALP (IU/L)

Total Bilirubin (mg/dl)

136.29Âą1.88

66.94Âą2.41

192.69Âą2.89

0.53Âą0.09

I

5

Normal Saline

II

1

Carbon tetra Chloride 662.23Âą11.31 D 350.58Âą5.67 D 455.35Âą4.52 D

1.41Âą0.62 D

III

25

Silymarin

178.30Âą4.11*a

113.11Âą6.29*a 207.32Âą4.79*a

0.96Âą0.16*a

IV

200

MV1

334.06Âą3.86*a

229.40Âą4.02*a 291.76Âą2.11*a

1.26Âą0.11*b

V

200

MV2

384.93Âą1.91*a

250.03Âą3.27*b 300.04Âą6.22*a

1.31Âą0.04*b

VI

200

Aq V.1

312.27Âą2.39*a

217.76Âą4.96*a 279.34Âą3.17*a

1.23Âą0.10*a

VII

200

Aq.V2

334.61Âą1.76*a

228.44Âą2.91*a 287.19Âą4.37*a

1.26Âą0.07*b

VIII

400

MV1

227.52Âą1.84*a

122.79Âą2.73*a 232.33Âą1.16*a

1.14Âą0.15*a

IX

400

MV2

253.08Âą3.20*a

139.83Âą1.46*a 246.54Âą3.88*a

1.19Âą0.12*a

Values are mean ¹ S.E.M, (n= 6). # compared with vehicle control (Group I), * compared with carbon tetrachloride control (Group II), aP DQG E3 DUH FRQVLGHUHG VWDWLVWLFDOO\ VLJQL¿FDQW MV1- Methanolic extracts of variety – 1, MV2- Methanolic extracts of variety – 2, Aq.1-Aqueous extracts of variety – 1,

Aq.2-Aqueous extracts of variety – 2

45


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

FIGURE 1 : SGOT, SGPT and ALP of different groups during hepatotoxicity studies. FIGURE 2 : Photos of rat liver. A: Liver of control group showing the normal histological structure of the hepatocytes (Group I), B: Liver of CCl4 control, only showing severe degenerated and necrobiotic changes in the hepatocytes (Group II), C: Liver protected by silymarin (Group III), D: Liver protected by methanolic extract of 200mg of Variety – 1(Group IV), E: Liver protected by methanol extract of 200mg of Variety – 2 (Group V), F: Liver protected by aqueous extract of 200mg of Variety – 1 (Group VI), G: Liver protected by aqueous extract of 200mg of Variety – 2 (Group VII), H: Liver protected by methanolic extract of 400mg of Variety – 1 (Group VIII), I: Liver protected by methanolic extract of 400mg of Variety – 2 (Group IX)

RESULRS AND DISCUSSION This study examined the hepatoprotective effects of methanol extract and aqueous extract of both varieties of Cajanus cajan in CCl4 induced liver toxicity. Acute administration of CCl4 (1 ml/kg i.p.) leads to increase in SGOT, SGPT, ALP, serum bilirubin concentration, which are used as reliable markers of hepatotoxicity. Oral administration of the leaf extract of both varieties at a dose of 200mg/kg and 400mg/kg body weight, markedly prevented the CCl4 induced elevation of SGOT, SGPT, ALP and VHUXP ELOLUXELQ 7KHUH ZHUH VLJQLÀFDQW UHVWRUDWLRQ RI HQ]\PH OHYHOV RQ DGPLQLVWUDWLRQ RI PHWKDQRO extract and aqueous extract at both doses (Group IV to IX) and silymarin also (Group III). The abnormal high level of serum biomarker enzymes and bilirubin observed in this study are the consequence of CCl4 induced liver dysfunction and denotes the damage to the hepatic cells. Oral administration of both varieties of Cajanus cajan H[KLELWHG D VLJQLÀFDQW UHGXFWLRQ LQ &&O4 induced levels of serum GOT, GPT, ALP and bilirubin value remarkably to the normal group that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage. This is further evidence for the protective effect of Cajanus cajan leaf extract of both varieties as well as both methanolic and aqueous extract and maintains the functional integrity of hepatic cells. The above alterations can be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated regeneration of parenchyma cells1. Effective control of bilirubin level and alkaline phosphatase activity points towards an early improvement in the secretary mechanism of the hepatic cell. The silymarin with a dose of 25mg/kg, body weight has provided a better inhibition of the elevated level of SGOT, SGPT, ALP and serum bilirubin. Traditionally in different folklore medicine the aqueous juice of the Cajanus leaf is used for treatment of hepatic disorders. In this study we also compare the methanolic extract of the leaf and aqueous 46


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

H[WUDFW IRU KHSDWRWR[LFLW\ VWXGLHV )URP WKH DERYH UHVXOW ZH DOVR ÀQG WKDW WKH DTXHRXV H[WUDFW RI WKH OHDI give better result than the methanolic extract in the case of both varieties, when it applied at a same dose on the rats. So from this study, it also proved that the claim made by traditional practitioners is DSSURSULDWH DQG WKH DTXHRXV H[WUDFW RI WKH OHDI LV PRUH HIÀFLHQW IRU KHSDWRSURWHFWLYH DFWLYLW\ WKDQ WKH methanolic extract. REFERENCES 1.

A Ghosh, PC Sil. Anti-oxidative effect of a protein from Cajanus indicus L against acetaminophen-induced hepatonephro toxicity. Journal of Biochemistry and Molecular Biology 2007; 40: 1039 – 1049.

2.

A Ghosh, K Sarkar, and PC Sil. Protective effect of a 43 kD protein from the leaves of the herb, Cajanus indicus L on chloroform induced hepatic-disorder. Journal of Biochemistry and Molecular Biology 2006; 39: 197 – 207.

3.

A Durairaj, ST Vaiyapuri, UK Mazumder, and M Gupta. Protective activity and antioxidant Potential of Lippia QRGLÁRUD extract in paracetamol induced hepatotoxicity in rats. Iranian Journal of Pharmacology & Therapeutics 2008; 7: 83 – 89.

4.

DH Upadhyaya, NK Reddy, LLC Gowda, and S Sing. Phenotypic diversity in the pigeonpea (Cajanus cajan) core collection. Genet Resour Crop Evolution 2007; 54: 1167 – 1184.

5.

Smartt J. Grain Legumes: Evolution and Genetic Resources, Cambridge University Press; 1990. p. 278-280.

6.

Sing JR. and Jauhar PP. Grain Legumes: Genetic Resources, Chromosome Engineering, and Crop Improvement. Vol. I. CRC Press; 2005. p. 87.

7.

A Egunyomi, JO Moody, and OM Eleuta. Antisickling activities of two ethnomedicinal plant recipes used for the management of sickle cell anaemia in Ibadan, Nigeria. African Journal of Biotechnology 2009; 8: 20 – 25.

8.

R Vadivu, JA Suresh, K Girinath, BP Kannan, R Vimala, and NMS Kumar. Evaluation of hepatoprotective and in vitro F\WRWR[LF DFWLYLW\ RI OHDYHV RI 3UHPQD VUUDWLIROLD /LQQ -RXUQDO RI 6FLHQWLÀF 5HVHDUFK ²

9.

S Reitman, and S Frankel. American journal of pathology 1957; 28: 56.

10.

Toro G. and Ackermann PG. Practical clinical chemistry, 1st edition Little, Brown & Company; 1975. p. 484.

11.

Tietz NW. Fundamentals of clinical chemistry. W. B. Saunders company, U. S. A. 1970. p. 447.

12.

Kim HJ. Glucocorticoids suppress bone formation via the osteoclast. Journal of Clinical Investigation 2006; 116: 2152 – 2160.

$ %KDWWDFKDU\D (IIHFW RI ÀVK RLO RQ ERQH PLQHUDO GHQVLW\ LQ DJLQJ & %/ IHPDOH PLFH -RXUQDO RI 1XWULWLRQDO Biochemistry 2006; 18: 372 – 379. 14.

Y Wan, LW Chong, and RM Evans. PPAR-a regulates osteoclastogenesis in mice. Nature Medicine 2007; 13: 1496 – 1503.

47


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Structural Optimization of New Class of Anticonvulsants : QSAR Approach Lata K Bisht1, Subir S. Samanta2, Saumya P Basu3 1 T John college of Pharmacy, Bangalore, India 2 Birla Institute of Technology, Mesra, Ranchi, India 3 NIET, Noida, India

ABSTRACT A three-dimensional quantitative structure activity relationship studies have been conducted on a series of 6-aryl-3-(hydroxypolymethyleneamino) pyridazines. The study was carried out on 26 compounds showing anticonvulsant activity. The models based on 3D-QSAR showed a good ability to predict the activity (r2=0.819, q2=0.68). The equations selected emphasized the importance of Henry’s law constant (H), RFWDQRO ZDWHU SDUWLWLRQ FRHIÀFLHQW &ORJ 3 WRWDO YDOHQFH FRQQHFWLYLW\ 79 &RQ :LHQHU ,QGH[ :,QG[ Keywords : Anticonvulsant, 3D-QSAR, Pyridazine derivatives. INTRODUCTION In recent years, few new compounds have been developed as antiepileptic drugs yet the therapeutic HIÀFDF\ RI DYDLODEOH DQWLHSLOHSWLF GUXJV FDQQRW EH GHÀQHG DV WRWDOO\ VDWLVIDFWRU\ VLQFH RI SDWLHQWV still experience inadequate seizure control. Moreover, antiepileptic drugs may cause burdening adverse effects, such as drowsiness, ataxia, GIT disturbances, hepatotoxicity, gingival hyperplasia and hirsutism. This warrants the continuing search for antiepileptic drugs with more selective anticonvulsant activity and lower toxicity1. The precise mechanisms by which clinically useful antiepileptic drugs exert their anticonvulsant activity is yet poorly understood2. Some of the MOA include: potentiation of GABA-ergic transmission, blockade of voltage gated dependent sodium channels, attenuation of excitatory neurotransmissions and/or modulation of voltage sensitive calcium channels3. Therefore, in the present study, quantitative structure activity relationship studies were performed in order to correlate thus structural requirements which may be useful in designing new molecules with anticonvulsant activity. MATERIALS AND MEHTODS &RPSDT 3UHVDULR & UXQQLQJ RQ ,QWHO 3HQWLXP 'XDO FRUH SURFHVVRU XVLQJ FKHPRIÀFH YHUVLRQ 8.0 was used. For the present study Valstat, a PC based programme developed using C++ language is used. The programme is validated for its accuracy and reproducibility. The anticonvulsant activity data was taken from the reported work of Hallot et al4 and is given in Table1. OZ1

N

R1 N N

R2

(a) (a) = S. No. 1-14

(b) (b) = S. No. 15-17

(c) = S. No. 18-22

(c) (d) = S. No. 23-26

48

(d)


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Table 1: Reported anticonvulsant activity data S. No.

R1

R2

electroshock

tonic seizures

lethality

rotorod

1

H

H

86

83

41

0%

2

2-Cl

H

16

38

34

138

3

2-Cl

4-Cl

14

8.5

8.5

34

4

2-Cl

4-CH3

21

17

16

48

5

2-Cl

5-Cl

30

131

101

10%

6

3-Cl

H

45

10%

40%

40%

7

2-F

H

115

30

34

0%

8

2-F

4-F

60%

10%

10%

30%

9

2-F

4-Cl

10%

60%

29

20%

10

2-NO2

H

27

81

57

0%

11

4-NO2

H

40%

54

57

ND

12

2-CH3

4-Cl

10

10

10

64

13

3-CH3

H

36

30%

50%

0%

38

40%

0%

0%

14

1-naphthyl

S.No.

R1

R2

N

Y

electroshock

tonic seizures

lethality

rotorod

15

2-Cl

H

2

4,4-(-OCH2CH2O)

57

40%

30%

0%

16

2-Cl

H

2

4-O

40

35

25

40%

17

2-Cl

4-Cl

2

4-O

5

7.3

7

50%

S.No.

Z

R1

R2

electroshock

tonic seizures

lethality

rotorod

18

CO(CH2)2CH3

2-Cl

H

28

39

30

0%

19

CHO

2-Cl

4-Cl

24

50%

50%

69

20

COCH3

2-Cl

4-Cl

14

9.8

8.3

70

21

COCH2CH3

2-Cl

4-Cl

50

16

13

71

22

CO(CH2)2CH3

2-Cl

4-Cl

50

31

30

30%

S.No.

Z1

R1

R2

electroshock

tonic seizures

lethality

rotorod

23

CONHCH3

2-Cl

H

10

13

9

37

24

CON(CH3)2

2-Cl

H

2

1.6

1.1

8.4

25

CONHCH3

2-Cl

4-Cl

5.5

1.4

1.4

20%

26

CONHCH3

4-F

H

40

40%

30%

20%

The molecular structures of all 26 compounds were sketched using chem. Draw ultra (version 8.0) software5 RI FKHPRIÀFH DQG VXEMHFWHG WR HQHUJ\ PLQLPL]DWLRQ WHFKQLTXH XVLQJ $OOLQJHUV molecular mechanics (MM2 IRUFH ÀHOG IROORZHG E\ WKH RSWLPL]DWLRQ RI JHRPHWU\ E\ VHPL HPSLULFDO Quantum Mechanics based on Austin model-1. Hamiltonian approximations method and closed VKHOO ZDYH IXQFWLRQ DYDLODEOH LQ 023$& PRGXOH E\ À[LQJ 506 JUDGLHQW DV DQG .FDO mol-A° respectively used in calculating the partial atomic charges and electron density on various 49


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

atoms. Charges kept as mulliken and maximum number of iterations was set to 1000. The energy minimized structures were added to a molecular database to compute various physico-chemical properties using ‘compute properties’ module of software. Generation of equation utilized descriptor values as given in Table 2. Table 2: S. No.

The physico-chemical descriptors were taken Chem 3D descriptors used in as independent variable and biological activity as QSAR equations dependent variable. The sequential multiple regression analysis method was employed for generation of H ClogP TV con Windx equations using VALSTAT programme6.

1

2.8523

1.4605

1.54321

774

2

2.9825

1.9366

1.5154

878

3

3.1127

2.65215

1.4881

1012

4

3.2987

2.4356

1.31238

1012

5

3.1127

2.65215

1.4881

1000

6

2.9825

2.1866

1.5154

891

7

2.9825

2.1866

1.5154

904

8

2.7852

1.61659

5.05134

878

9

2.7852

1.61659

5.05134

904

10

1.1146

1.23156

9.96138

1128

11

0.5276

1.23156

9.96138

1167

12

3.1685

2.36642

3.6081

1308

13

3.2987

2.3856

1.31238

1012

14

3.1685

1.9595

1.34E-05

891

15

3.1685

1.9595

1.34E-05

904

16

3.6885

2.6345

1.29E-06

1316

17

2.9825

2.5659

2.45E-06

1309

18

2.9825

2.42259

1.20E-05

878

19

3.1127

3.13815

1.18E-05

1012

20

2.5686

3.9406

1.41E-06

1778

21

2.5686

3.15215

3.20E-06

1358

22

3.1127

3.59815

2.77E-06

1544

23

3.1127

4.12715

1.96E-06

1754

24

3.1127

4.65615

1.39E-06

1989

25

2.9825

2.5426

1.41E-06

1560

26

2.9825

3.1386

1.26E-06

1756

27

3.1127

3.25815

1.39E-06

1754

28

2.7852

2.2226

4.71E-07

594

In sequential multiple linear regression, the programme searches were made for all permutations and combinations sequentially for the data set. The auto-correlated parameters were eliminated depending on their individual correlation with biological activity to avoid simple collinearity problem. All possible combinations of descriptors were considered for the QSAR study. The predictive powers of the equations were validated by Leave-one-out (LOO) cross – validation equations7, 8. Predicted residual sum of VTXDUH 35(66 FURVV YDOLGDWHG FRUUHODWLRQ FRHIÀFLHQW (Q2) and standard deviation error of prediction (SDEP) were considered for the validation of these equations. The results from cross-validated analysis were expressed DV WKH FURVV YDOLGDWHG VTXDUHG FRUUHODWLRQ FRHIÀFLHQW (Q2 ZKLFK LV GHÀQHG DV

Where Y pred, Y act, Y mean are predicted, actual and mean values of the target property respectively. is the Predictive Residual Error Sum of Squares (PRESS). PRESS is an important cross-validation parameter as it is a good approximation of the real predictive error of the equations. The r2bs is an average squared FRUUHODWLRQ FRHIĂ€FLHQW FDOFXODWHG GXULQJ YDOLGDWLRQ which is computed from a subset of variables used one at a time for validation. A data point is considered as an outlier if it has a large magnitude (when the residual value exceeds twice the standard error of estimate 50


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Table 3 : Observed activity, calculated activity, of the equation). To validate the derived Predicted activity and Z value equation, the overall predictive ability of that S. Comp Obs Cal Pre analysis was evaluated by the terms r2pred, and Z value No. No. activity activity activity calculated using the formula: 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

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

1.934498 1.20412 1.146128 1.322219 1.477121 1.653213 1.462398 2.060698 1.681241 1.431364 1.897627 1 1.556303 1.579784 1.755875 1.60206 0.69897 1.447158 1.380211 1.146128 1.69897 1.69897 1 0.30103 0.740363 1.60206

78.1722 22.729 10.5451 22.6904 22.9434 28.5419 20.7985 115.964 35.0517 21.0532 78.5884 22.7153 27.0794 25.115 57.0053 23.9301 24.7061 25.0484 22.7001 26.1988 56.8095 59.0081 23.0553 23.0028 23.062 22.9857

68.357 23.0973 10.2352 22.7831 22.5617 27.296 20.3264 147.402 34.4499 20.6987 52.8974 23.4119 26.6675 24.4766 65.7808 23.1024 25.6972 24.9042 22.6288 26.7754 58.5916 61.6922 23.764 24.1467 24.0129 22.0629

0.685128 -0.58896 0.302387 -0.14795 0.617629 1.4405 0.717837 -0.0844 1.1333 0.52049 0.036022 -1.1129 0.780775 1.12776 -0.00046 1.40652 -1.72478 0.25834 0.113776 -1.0677 -0.596 -0.78843 -1.14267 -1.83827 -1.53711 1.48917

where SD is the sum of the squared deviation between the biological activities of the test set molecules and the mean activity value of the training set molecules. PRESS is the predictive error sum of squares derived from the leaveone-out method. RESULTS & DISCUSSION The data set comprised of 26 compounds. Among the several generated QSAR equations; the best equation was selected on the basis of REVHUYHG VTXDUHG FRUUHODWLRQ FRHIĂ€FLHQW U2), percent explained variance (%EV), standard deviation (s), sequential Fischer test (F), and validation parameters viz; bootstrapping r2, FKDQFH FURVV YDOLGDWHG FRUUHODWLRQ FRHIĂ€FLHQW (Q2) value, SPRESS, standard deviation of error prediction (SDEP) and predictive squared FRUUHODWLRQ FRHIĂ€FLHQW RI WKH WHVW VHW U2pred). QSAR equation obtained:

BA = [22.6888( Âą 5.92951)] +H [0.00061043( Âą 0.000171694)] +CLogP [0.000177326( Âą 8.96047e-005)] +TVCon [0.0046318( Âą 0.00175518)] +Windx [6.69357e-005( Âą 5.42248e-005)] Statistical parameters: n=26, r=0.90504, r2=0.819, variance=0.155, STD=0.124, F=23.77, FIT=236.683 Validation parameters: Q2=0.681, r2bs= 0.828, chance SPRESS= 0.165, SDEP= 0.148 2Q WKH EDVLV RI YDULRXV VLJQLĂ€FDQW VWDWLVWLFDO and validation parameters best equation was selected to explore the factors responsible for anticonvulsant activity (Table no. 3, Fig. 1). The intercorrelation among the parameters 51

Fig 1: Plot between observed activity and calculated activity


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

is less than 0.3, which indicates orthogonality among the descriptors used for deriving the equation. (Table no. 4) The equations by the Z- score method were Table 4 : Interco-relation between descriptors tested for the outlier, no compound was found to used be outlier; which suggest that equations are able H CLogP TVCon Windx to explain the structurally diverse analogs and H 1.000000 very helpful in designing newer potent compounds CLogP 0.011458 1.000000 using physico-chemical parameters. TVCon

0.081521

0.073366

1.000000

Windx

0.040840

0.040829

0.064246

1.000000

The cross validated squared correlation FRHIĂ€FLHQW 42) (Fig 1); predictive residual sum

of square (SPRESS) and standard deviation of error of prediction (SDEP), all indicate a good internal consistency as well as predictive ability of equations. The boot strapping r2 is at par with conventional VTXDUHG FRUUHODWLRQ FRHIĂ€FLHQW U2). The robustness and applicability for further optimization of the PROHFXOHV ZDV H[SODLQHG E\ VLJQLĂ€FDQW U2pred value. (Table no. 3, Fig 2) The selected equations fulfill the statistical YDOLGDWLRQ FULWHULD WR D VLJQLĂ€FDQW H[WHQW WR EH XVHIXO as theoretical basis for proposing more potent compounds.

Fig. 2: Plot between observed activity and predicted activity

ACKNOWLEDGEMENTS The author wishes to thank Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi, India for providing computational facility. REFERENCES 1.

M.J. Eadie, J.H. Tyrer, The treatment of epilepsy. Ed., MTP Press: Lancaster, 1980; p.129-160.

2.

B.B. Gallagher, J.A. Vida, Anticonvulsants , Ed.; Academic Press, New York, 1977; 11-55.

/XFLDQD *DYHUQHW 0 -RVHĂ€QD 'RPLQJXH] &DEUHUD /XLV ( %UXQR %ODQFK *XLOOHUPLQD / (VWLX ' 46$5 GHVLJQ of novel antiepileptic sulfamides. Bioorganic & Medicinal Chemistry, 15(2007), 1556-67.

4.

A. Hallot, Roger Brodin, Josiane merlier, Joelle Brochard, Jean Pierre chamber and Kathleen Biziere. Synthesis & activity of 6-aryl-3-(hydroxypolymethyleneamino) pyridazines in animal models of epilepsy. J. Med. Chem., 1986, 29, 369-75.

&6 &KHP 2IĂ€FH PROHFXODU PRGHOLQJ VRIWZDUH YHUVLRQ &DPEULGJH VRIW FRUSRUDWLRQ 6RIWZDUH 3XEOLVKHUV association, 1730 M street, NW, suite 700, Washington D.C. 20036(202), 452-1600, USA.

6.

Gupta AK, Babu MA & Kaskhedikar SG, Indian J Pharm Sci, 66, 2004,396.

7.

Schaper KJ, Quant Struct Act Relat, 18, 1999,354.

8.

Wold S & Eriksson L, QSAR: Chemometric methods in molecular design, 1995, Vol. 2, 321.

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Relative Efficiencies of the Dietary Proteins of Plant and Animal Origin : A Comparative Study Mukul Chandra Gope1*, Ratna Ghosh2, Rupali Patra3, Manik Ghosh4 Department of Physiology, Rajendra Institute of Medical Sciences, Ranchi, India 2 Department of Home Science (Division of Nutrition), Ranchi University, Ranchi, India 3 Department of Botany, Jamshedpur Cooperative College, Jamshedpur, India 4 Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi, India 1*

ABSTRACT ,Q WKLV SDSHU DQ HIIRUW KDV EHHQ PDGH WR FRPSDUH WKH UHODWLYH HIĂ€FLHQF\ RI SURWHLQV RI WKH YHJHWDEOH RULJLQ with that of the animal origin. It always has been our tendency to rank proteins of animal origin superior to that of vegetable origin. The qualitative and quantitative assessment of plasma protein including percent protein in body in this study does not allow ranking proteins like that. These data indicate that when taken in recommended quantity the protein status of healthy vegetarian & Non-vegetarian subjects do QRW VKRZ DQ\ VWDWLVWLFDOO\ VLJQLĂ€FDQW GLIIHUHQFH ,Q IDFW YHJHWDEOH SURWHLQV DSSHDU WR EH PRUH HIĂ€FLHQW economical, and easy to digest & assimilate than the proteins of animal origin. After all we do not forget that many animals build up their muscles from the proteins of humble grass. Key words : Vegetable Protein, Animal Protein, A: G ratio, Protein Intake Index INTRODUCTION Although all human beings have common nutritional needs, there are variations from one section of the community to another. Requirements change from infancy through childhood to adolescence and adulthood. The needs of the pregnant women (‘feeding two’) are not the same as those of non-pregnant women. The concept is of the ‘physiological group’, each group needing special consideration. The group implied above must be added two others – aging and aged, and ill a convalescent 1. Protein malnutrition being a widespread problem in developing & under developed countries has DWWUDFWHG PXFK PRUH DWWHQWLRQ WR WKH UHVHDUFK ZRUNHUV LQ WKH PHGLFDO Ă€HOG ,Q FKLOGUHQ DV ZHOO DV LQ adults (especially pregnant females) such studies are very informative. Studies involving comparison of dietary sources to prevent protein malnutrition were the key concern of such research works. Protein is an indispensable constituent of the diet because it is the only source of amino acids, including essential amino acids, which cannot be synthesized in the body 2. 7KH EHVW JXLGHV WR PHDVXUH UHODWLYH HIĂ€FLHQF\ of dietary protein are plasma protein level and percent protein in the body. In this paper an attempt has been made to compare the relative usefulness of proteins obtained from vegetable sources to that from animal sources. Plasma concentration of total plasma protein, DOEXPLQ JOREXOLQV Ă€EULQRJHQ ZHUH FRPSDUHG EHWZHHQ YHJHWDULDQ DQG QRQ YHJHWDULDQ VXEMHFWV Percent protein in the body has been calculated in both sub-groups. MATERIALS & METHODS All the subjects (n=108) have been examined clinically to rule out any acute disease condition. They were divided into two groups viz. Vegetarian (n=54) and Non-vegetarian (n=54). Through dietary history has been taken from them using a “dietary habitâ€? chart, which includes commonly 53


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

taken foodstuffs. The quality, amount & frequency of intake were noted down. Average daily intake of protein was calculated. Protein intake index has been obtained by dividing average daily intake of mixed proteins (in gm) with body weight (in Kg) 3ODVPD SURWHLQ OHYHO LQFOXGLQJ DOEXPLQ JOREXOLQ Ă€EULQRJHQ ZHUH GHWHUPLQHG DIWHU obtaining venous blood. Few precautions have been taken during collection of blood since standing for more than 2 h and short period vigorous exercise may decrease the plasma volume up to 25% 3. This may cause false impression of elevated plasma protein. Subjects were well examined for dehydration since it might reveal greater plasma protein concentration. MRGLĂ€HG ELXUHW PHWKRG ZDV DSSOLHG WR GHWHUPLQH WRWDO VHUXP SURWHLQ4. Albumin was estimated by Bromocresol green method5 )RU Ă€EULQRJHQ HVWLPDWLRQ &ORWWLQJ PHWKRG RI &ODXVV ZDV FDUULHG RXW6. %\ DGGLQJ WRWDO VHUXP SURWHLQ DQG WKH SODVPD Ă€EULQRJHQ WKH WRWDO SODVPD SURWHLQ OHYHO ZDV REWDLQHG Percent protein in the body was determined. The A : G ratio has been calculated. The two groups were compared using the above parameters. RESULTS The comparison of plasma protein level between vegetarians & non-vegetarians shows that there is QR VWDWLVWLFDOO\ VLJQLĂ€FDQW GLIIHUHQFH +RZHYHU WKH DOEXPLQ OHYHO ZDV VOLJKWO\ HOHYDWHG LQ QRQ²YHJHWDULDQ subjects but on the other hand globulins have been found to be raised among vegetarians, making the total plasma protein level almost equal in both subgroups. The plasma protein level corresponds well with the PII (Protein Intake Index). All subject with PII>0.6 had satisfactory plasma protein level. After analyzing dietary history it was found that subjects taking groundnut & dry fruits regularly had more plasma protein level than subjects who does not take them. Similarly subjects taking cheese & food made from soybean had elevated plasma protein level than others. Non-vegetarians who take PXWWRQ SRUN Ă€VK HJJ KDG VDWLVIDFWRU\ SODVPD SURWHLQ OHYHO EXW WKH TXDQWLW\ RI LQWDNH ZDV DOVR D factor. Milk & milk products also affect the plasma protein level positively. Table 1 : Protein Intake Index, in relation with the Protein status Protein Intake Index (gm/kg B.W.)

Plasma Protein Level

0.83 Âą 0.11

6.96 Âą 0.50

Percent Protein in Body

A:G Ratio

17.42 Âą 1.27

1.41 Âą 0.29

(g/dL)

Values reported as Mean Âą SD (n=108; veg. = 54; non-veg. = 54)

Table 2 : Effect of the dietary source of protein on different plasma proteins (g/dL) Dietary Habit

Albumin

Globulins

Fibrinogen

A:G Ratio

Vegetarian

3.86 Âą 0.39

2.94 Âą 0.33

0.25 Âą 0.06

1.33 Âą 0.25

Non-vegetarian

3.93 Âą 0.25ns

2.70 Âą 0.50ns

0.24 Âą 0.05ns

1.50 Âą 0.31ns

Values reported as Mean Âą SD (n=108; veg. = 54; non-veg. = 54). The data were analyzed by one way ANOVA followed by %RQIHUURQLÂśV 7HVW ÂľQVÂś LQGLFDWHG VWDWLVWLFDOO\ QRQ VLJQLÂżFDQW YDOXHV IURP YHJHWDULDQ

54


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Table 3 : Comparison of protein status in vegetarian and non-vegetarian subjects Dietary Habit

Protein Intake Index

Total Plasma Protein Level (g/dL)

Percent Protein In Body

Vegetarian

0.84 ± 0.12

7.06 ± 0.42

17.65 ± 1.05

Non-vegetarian

0.82 ± 0.10

6.87 ± 0.57

17.17 ± 1.40ns

ns

ns

Values reported as Mean ± SD (n=108; veg. = 54; non-veg. = 54). The data were analyzed by one way ANOVA followed by %RQIHUURQL¶V 7HVW µQV¶ LQGLFDWHG VWDWLVWLFDOO\ QRQ VLJQL¿FDQW YDOXHV IURP YHJHWDULDQ

DISCUSSION The protein status of the body depends on the PII. Type of food either vegetarian or non-vegetarian does not alter the protein status 7. 3URWHLQ RI KLJK ORZ ELRORJLFDO YDOXHV DUH VRPHWLPHV FDOOHG ´ÀUVW class” and “second class” protein respectively. As the best-known individual proteins of low biological YDOXH DUH GHULYHG IURP YHJHWDEOH IRRG WKHUH KDV EHHQ UHJUHWWDEOH WHQGHQF\ WR HTXDWH WKH WHUP ¶ÀUVW· and ‘second’ class generally with animal & vegetable protein respectively. Such misuse of term should be strenuously avoided. Before vegetable proteins in general are dismissed as second class it should be remembered that many animals build up their muscles from the protein of humble grass 7. Vegetable protein from various sources, enriched with vitamin B12 can be directly utilized by human EHLQJV :KHQ JLYHQ LQ WKLV ZD\ LW LV FKHDSHU PRUH HIÀFLHQW WKDQ ZKHQ XVHG WR PDNH DQLPDO SURWHLQ 7KH recommendation for adults is that the protein intake should not be less than 1 gram per Kg body weight. Some studies even advocate giving 1.5 gm – 2 gm per Kg body weight per day during adolescence & childhood 8. But in some experiments nitrogenous equilibrium was maintained on 30 gm – 40 gm of protein derived exclusively from vegetable sources e.g. cereals, potatoes, other vegetarian & fruits 9. Studies involving protein status of the body shows that “Protein index” i.e. measured total body protein divided by predicted total body protein can be a useful tool in determining physiological impairments. When the Protein index is < 0.8 it clearly indicates negative nitrogenous equilibrium 10. Experimental evidence gained by feeding one protein at a time in calculated amounts to experimental animals including humans shows that for the great majority of proteins, part only can be utilized by the body for protein synthesis while the remainder is broken down and its nitrogen excreted as urea. This is the basis of expressing protein quality as a percentage. Plasma proteins are normally formed from food proteins but that in protein starvation they may be formed form tissue protein. As albumin and globulins have distinctly different amino acid patterns, some protein favour albumin formation e.g. proteins of animal origin, while other favour globulin formation e.g. proteins of vegetable origin. Many problems of nutrition remain – the positive health effects of food, the hazards of excess, the EDVLV RI XQXVXDO UHVSRQVH RU GHPDQG RI VSHFLÀF IRRGV %XW WKHVH VHHP RI OLWWOH FRQVHTXHQFH EHVLGH the economic and political problems of putting into practice on a world scale what is already known. While the western peoples are better fed than ever before, the emergent countries of the world are falling yet nearer to the starvation mark. Combinations of plant protein that complement one another in biologic value or combinations of animal & plant proteins can increase biologic value & lower total protein requirements. 55


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Protein needs increase during growth, pregnancy lactation & rehabilitation during treatment of malnutrition. For adults, the recommended dietary allowance (RDA) for protein is about 1.0 gm per Kg body weight per day, assuming that energy needs are met and that the protein is of relatively high biological value 9,11. ,Q VXPPDU\ WKHUH LV QR VLJQLĂ€FDQW HIIHFW RI WKH W\SH RI GLHW OLNH YHJHWDULDQ RU QRQ YHJHWDULDQ RQ the protein status of the body. Furthermore proteins obtained from plant sources appear to be cheap PRUH HIĂ€FLHQW RQ D PL[HG GLHW 7KXV ZLWK SURSHU SODQQLQJ ,W LV SRVVLEOH IRU D YHJHWDULDQ WR REWDLQHG a high grade protein, at low cost, from mixed diet of cereals, pulses and vegetables. REFERENCES 1.

Davidsion S, Passmore R, Brock JF, Truswell RS. Human nutrition & dietetics. 3rd ed. Edinburg: Churchill Livingstone; 1975.

2.

MC Latham. Protein-calorie malnutrition in children and its relation to psychological development and behavior. Physiol. Rev. 1974; 54: 541-565.

3.

Varley H, Practical clinical biochemistry. London: William Hienman Medical Books Ltd.; 1980.

4.

AG Gornall, CJ Bardawill, MM David. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949; 177: 751-766.

5.

H Yatzidis. An improved biuret reagent. Clin. Chem. 1977; 23: 908.

6.

RM Corcoran. Clin. Chem. 1977; 23: 663.

7.

A Clauss. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Hematol. 1957; 17: 237-246.

8.

Gopalan C, Rama SBV, Balasubramanian SC. Nutritive value of indian foods, Hyderabad: NIN, ICMR; 2004.

9.

Antia FP, Abraham P. Clinical dietetics and nutrition, 4th ed., New Delhi: Oxford University Press; 2002.

10.

Wright S. Applied Physiology. 13th ed., Oxford: Oxford University Press; 1988.

11.

Hill GL. Body composition research: Implications for the practice of clinical nutrition. J. Parent Ent. Nutr. 1992; 16: 197.

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PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Isolation and Characterization of a Novel Smart Mucoadhesive Biopolymer from Euryale ferox Seed Coat M.S. Uma Shankar1 & N. V. Satheesh Madhav2 1 Krupanidhi College of Pharmacy, Bangalore, India 2 Dehradun Institute of Technology, Dehradun, India

ABSTRACT The aim of our research work was to isolate a novel mucoadhesive biopolymer from Euryale ferox seed coat (Family: Nymphaeaceae). The vernacular name is Makhana. The plant produces white edible seed coated with mucilaginous material, roasted like popcorn, eaten as kheer and soup. Biochemical DQDO\VLV RI WKH VHHGV UHYHDOHG FDUERK\GUDWH SURWHLQ PRLVWXUH ÀEUH ash, and 1.35% fat. The seed coat was isolated by non-solvent addition method and evaluated for its physicochemical characteristics like color, texture, nature, solubility and chemical tests, IR spectra and further for swelling index, color change point and acute animal toxicity study. The mucoadhesivity of the biopolymer was determined by shear stress method, and rotating cylinder method by comparison with standard polymers like sodium carboxymethyl cellulose and hydroxypropylmethyl cellulose. The research revealed the biopolymer of Euryale ferox seed coat possessing inbuilt mucoadhesion and promising PXFRUHWHQWDELOLW\ 7KH PXFRDGKHVLRQ RI WKH ELRSRO\PHU DOVR FRQÀUPHG E\ ,5 VSHFWUD VKRZLQJ FDUER[\O and hydroxyl groups responsible for its inbuilt mucoadhesivity. Hence the isolated biopolymer from the Euryale ferox seed coat can serve as a novel smart natural mucoadhesant and used for formulating various biomucoadhesive drug delivery systems. Key words : Euryale ferox; seed coat; mucilage; biopolymer. INTRODUCTION Mucoadhesive material from natural sources gaining popularity day by day in the global pharma industry and a burning area of further research and development for spatial placement of mucoadhesive dosage devices, if they are biocompatible and biodegradable provides added advantage for formulating various controlled release pharmaceutical formulations and avoids patient noncompliance especially IRU FKURQLFDOO\ LOO SDWLHQWV %LRDGKHVLRQ FDQ EH GHÀQHG DV D SKHQRPHQRQ RI LQWHUIDFLDO PROHFXODU attractive forces amongst the surfaces of the biological substrate and the natural or synthetic polymers, which allows the polymer to adhere to the biological surface for an extended period of time1-3. Primarily, because of their natural origin, ready availability, relatively inexpensive, biodegradable and capable RI PXOWLWXGH RI FKHPLFDO PRGLÀFDWLRQV LV DQ REYLRXV DGYDQWDJH RI WKLV PDWHULDO 0DMRULW\ RI QDWXUDO mucoadhesive agent falls under the category of cellulose, polysaccharides and proteins etc. The ideal characteristics of a mucoadhesive polymer include the rapid adherence to the mucosal layer without any change in the physical property of the polymer matrix, minimum interference to the release of the active agent, biodegradable without producing any toxic byproducts, inhibit the enzymes present at the delivery site and enhance the penetration of the active agent4. Mucoadhesive delivery systems are being explored for the localization of the active agents to a particular location/site. Polymers have played an important role in designing such systems so as to increase the residence time of the active agent at the desired location. Polymers used in mucosal delivery system may be of natural or synthetic origin. Euryale ferox LV DQ DTXDWLF SHUHQQLDO à RDWLQJ OHDYHG KHUE WKH SODQW LV FXOWLYDWHG IRU LWV VHHGV LQ ,QGLD China and Japan. Euryale ferox seed contains the biopolymer coat surrounding the outer surface of the 57


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

seed, which when soaked in water swells, becomes sticky and forms gel 5, 6. The seeds were found to contain 12 amino acids, which are histidine, leucine, isoleucine, glutamic acid, lysine, tyrosine, valine, aspartic, threonine, alanine, methionine and arginine 7. Seeds are believed to strengthen male potency and retard aging 8. The current objective of our research work is to isolate the biopolymer from the seed coat and to screen for its physicochemical characteristics and inbuilt mucoadhesivity and mucoretentivity. MATERIALS AND METHODS Materials Euryale Ferox seed was obtained from the local market. acetone, sodium dihydrogen orthophosphate, potassium dihydrogen orthophosphate, sodium hydroxide were purchased from Qualigen Chemicals Pvt. Ltd., double distilled water was prepared from the institutional laboratory. All Chemicals used were of analytical grade. METHODS Extraction of Mucoadhesive Biopolymer from Euryale Ferox Extraction of the biopolymer was performed by soaking 100 g of seeds with a 500 ml of water for 8 h and kept in a refrigerator. The mixture was subjected for mechanical stirring at the force of 1252g to shred seed coat and get dissolved in the distilled water. The mixture was strained through muslin FORWK 7KH VHHG ZHUH ZDVKHG ZLWK ZDWHU DQG WKH ZDVKLQJV ZHUH PL[HG ZLWK ÀOWUDWH 7KH ELRSRO\PHU was recovered from the extract via precipitation with 3 volumes of acetone of 100 ml each. The SUHFLSLWDWHG ELRSRO\PHU ZDV ZDVKHG UHSHDWHGO\ ZLWK DFHWRQH FROOHFWHG SXULÀHG E\ GLDO\VLV DQG GULHG at a temperature 50-600C under vacuum for 12 h. The dried biopolymer was pulverized and passed through 100 mesh sieve and stored in a desiccator. Physicochemical Characterizations The biopolymer was subjected for physicochemical tests like color, texture, nature, solubility, chemical test, pH of 1% biopolymer solution and test for functional groups. Swelling factor 9-11, viscosity, color changing point, ultraviolet spectra (UV), infrared spectra (IR) interpretation, elemental analysis and thin layer chromatography (TLC) reports were depicted. Acute toxicity study of the E. Ferox seed coat extract The biopolymer was evaluated for acute toxicity study. The study protocol was approved by the Institutional Animal Ethical Committee. The procedure followed as per OECD 423 guidelines. Two groups of albino rats, one for test and other for control, each group containing six unisex animals were used for the study. The animals were accessed with free water ad libitum. The study was performed by administering the dried biopolymer 2g/kg body weight for the test group animals and the acute toxicity study was evaluated for a period of 14 days by observing parameters like body weight, changes in the VNLQ FRUQHDO UHà H[ UHVSLUDWRU\ UDWH VDOLYDWLRQ GLDUUKHD OHWKDUJ\ VOHHS VRPDWRPRWRU DXWRQRPLF symptoms, behavioral pattern and convulsions. Assessment of mucoadhesive properties The mucoadhesive property of E. ferox seed coat extract was determined in vitro by the shear stress method12, park and robinson method13 and rotating cylinder method14. 58


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

SHEAR STRESS STUDY The biopolymer was subjected for shear stress study for in vitro assessment of its adhesive strength in WHUPV RI ZHLJKW UHTXLUHG IRU EUHDNLQJ DGKHVLYH ERQGV EHWZHHQ SRO\PHU DQG JODVV SODWH LQ D VSHFLÀHG contact time like 5, 10, 20, 30 min period with various concentrations of 0.5%, 1%, 2%, 3%, & 5% w/v of the natural mucoadhesive extract and compared with the standard polymer NaCMC 1% w/v. PARK AND ROBINSON METHOD The ex vivo mucoadhesivity of the extracted biopolymer of E. ferox by Park and Robinson method was studied in comparison with NaCMC and HPMC. The average of 6 reading was registered. Rotating Cylinder Method In rotating cylinder method, the stainless steel rotating baskets were covered with a thin layer of aluminium foil. The freshly excised goat soft palate tissue kept stuck securely onto the aluminum foil surface around the rotating cylindrical basket. The compressed bioplate was placed on the mucosal VXUIDFH ,W LV WKHQ LPPHUVHG LQWR WKH à DVN FRQWDLQLQJ PO RI SKRVSKDWH EXIIHU DW S+ DW “ OC. The cylindrical baskets were allowed to rotate at a speed of 100 rpm. Every 30 mins, the machine was stopped and noted for dislodgement or disintegration of the bioplate from the mucosal surface. The results were compared with the standard polymers sodium carboxymethly cellulose (NaCMC) and hydroxypropyl methyl cellulose (HPMC). The average of 6 reading was registered. RESULTS AND DISCUSSIONS Biomaterial extraction and physicochemical evaluations The biopolymer was isolated by a simple extraction procedure and 0.9g of biopolymer was obtained for 100 g of E. ferox seeds. Physicochemical characterization of the E. ferox seed coat extract showed white color, smooth texture, amorphous nature, partially soluble and swellable in water and insoluble in alcohol, pH (1%) is ~6.8, positive molisch’s test for carbohydrate nature of the extract, functional groups test showed the presence Fig 1. IR spectra of the isolated biomaterial from of ketone, aldehyde and alcoholic (OH). Swelling factor Euryale ferox. O (1g) was 2 ml; color changing point was at 200 C. UV spectra showed 240hmax, IR spectroscopy (Fig-1) revealed 3448 cm-1 OH, 2928cm-1 C-H (str), 2363.4 cm-1 C=C (str), 1654.2 cm-1C=O (str), 1421.2 cm-1C-H (def), 1049.8 cm-1C-O(str), 771 cm-1 C-H (def). Elemental analysis showed the presence of carbon 32.95%, hydrogen 5.37% and nitrogen 4.55%. Thin layer chromatography showed the presence of rhamnose. NMR studies revealed multiplets at 3.772 showing methyl groups and M+ 14030 m/z which reveals the isolated biomaterial is protein polymeric in nature. Acute Toxicity Study of the Euryale ferox seed coat extract The acute toxicity study of the biopolymer showed devoid of toxicity sign in animals tested. This may be due to its edibility nature of the E. ferox seeds. 59


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

ASSESSMENT OF MUCOADHESIVE PROPERTY Shear stress method The result of the mucoadhesive property by shear stress method was recorded for various concentration of the biopolymer in comparison with 1% NaCMC was showed in Fig.2 with different contact time. A 2% w/v Fig. 2 : Bioadhesive property of the natural mucoadhesive extract and the synthetic polymer 1% NaCMC by shear solution of the natural mucoadhesive extract of E. ferox stress method Experimental temperature was maintained at exhibited excellent bioadhesive strength with increase 37±1°C. Values are expressed as a mean of 6 observations in time. Further increment in the concentration of the ELRSRO\PHU KDG QR VLJQLÀFDQFH LQ LWV ELRDGKHVLYHQHVV ZLWK LQFUHDVH LQ WLPH. This is probably due to loss of hydration by evaporation had increased the mucoadhesive strength. The macromolecules containing numerous hydrogen bond forming groups e.g. hydroxyl, carboxyl groups show maximum promising mucoadhesivity. Park and Robinson method The ex vivo mucoadhesivity of the extracted biopolymer of E. ferox by Park and Robinson method displayed that the biopolymer possesses promising mucoadhesivity in comparison with NaCMC and found similar to that with HPMC depicted in Fig. 3. Makhana

HPMC

NaCMC

ROTATING CYLINDER METHOD Fig. 3 : Bioadhesive property of the natural Rotating basket method revealed that the biopolymer had a mucoadhesive agent and the synthetic promising mucoretentability which was found to be more than polymers by park and robinson method. Experimental temperature was maintained HPMC & NaCMC showed in Fig 4. This is due to the fact that at 37±1°C. Values are expressed as a mean E. ferox having high molecular weight exhibited higher adhesion of 6 observations

and better mucoadhesive property in comparison to the synthetic polymers (HPMC and NaCMC) at the same concentration. This may be due to the presence of numerous disulphide bridges and carboxyl and hydroxyl groups, which adopt more favorable macromolecular conformation, and accessibility of its hydrogen-binding groups, when compared with other Makhana HPMC NaCMC polymers like HPMC and NaCMC being a cellulose derivatives, Fig 4. : Bioadhesive property of the natural formed weaker bonds with mucus, which may be due to either mucoadhesive and the synthetic polymers by rotating a decrease in available hydrogen binding sites or unfavorable basket method. Experimental temperature was maintained at 37±1 °C. Values are expressed as a entanglement with the mucus. mean of 6 observations

CONCLUSIONS The present study revealed that E. ferox seed coat extract is a better mucoadhesive agent than HPMC and NaCMC with respect to inbuilt mucoadhesive and mucoretentive properties. The process of mucoadhesion has been proposed to begin with the establishment of an intimate contact between the mucoadhesive polymer and mucus gel15, 16. The plausible mechanism of its novel mucoadhesive property may be due to the interaction of mucus with carboxyl or hydroxyl groups of the biomaterial. The role of surface energy thermodynamics in mucoadhesion has been considered vital17, 18 for the mucoadhesive strength exhibited by the biomaterial extract of E. ferox. Since this natural mucoadhesive agent is edible, 60


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it is easily biodegradable and non-allergic and may provide an alternative to the conventional synthetic and natural mucoadhesive agent. Mucoadhesive polymers may provide an important tool to improve the bioavailability of the active agent by improving the residence time at the delivery sites which includes sublingual, buccal cavity, soft palate, nasal cavity, rectal lumen, vaginal lumen and gastrointestinal tract. This work provides a novel natural mucoadhesive agent to the scientists who are at the quest of a smart mucoadhesive agent for the development of novel mucoadhesive delivery systems. Many potential mucoadhesives in the area of novel drug delivery research are being investigated so as to understand the various mechanism of mucoadhesion and improved permeation of active agents to ÀQG WKHLU ZD\ LQWR WKH PDUNHW LQ QHDU IXWXUH ACKNOWLEDGEMENT Thanks are due to SAIF, Lucknow for providing IR data for the extracted biopolymer. We are also gratefully acknowledged Mr Bajrang Tripathi, Manager, Pharmacy College, Azamgarh, India for SURYLGLQJ WKH ÀQDQFLDO VXSSRUW REFERENCES 1. 2. 3. 4. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Kaelbe DH, Moacanin J. A surface energy analysis of bioadhesion. Polym. 1977, 18: 475-481. Gu JM, Robinson JR, Leung S. Binding of acrylic polymers to mucin/epithelial surfaces; Structure-property-relationship. Crit. Rev. Ther. Drug Car. Sys. 1998, 5: 21-67. Duchene D, Touchard F, Peppas NA. Pharmaceutical and medical aspects of Bioadhesive system for drug administration. Drug Dev. Ind. Pharm. 1998, 14: 283-381. Sudhakar Y, Kuotsu K, Bandyopadhyay AK. Buccal bioadhesive drug delivery - A promising option for orally less HIĂ€FLHQW GUXJV - &RQWURO 5HOHDVH ´KWWS ZZZ HĂ RUDV RUJ Ă RUDWD[RQ DVS[ Âľ(XU\DOH IHUR[Âľ The Wealth of India, Raw materials, Niscom and CSIR Publishing, New Delhi 2000. Nadkami KM. India Materia Medica. In Medicinal Plants of India (Satyavati, G.V. Eds.), Bombay Popular Prakashan Pvt. Ltd, India 1976. Alfasane, Md. A. Fruit production and biochemical aspects of seeds of Euryale ferox Salisb: under ex-situ conditions. Bangladesh J. Bot. 2008, 37: 179-181. Findly A. Practical Physical Chemistry. In Longans 6th Eds, London Publishers, London. 1963, 1040. Alfred Martin. Physical Pharmacy. In Physical Chemical Principals in the Pharmaceutical Sciences 4th Eds, B.I. Waverly Pvt. Ltd. New Delhi, 2001, 453-587. Subrahmanyam CVS, Thimma Setty J. Text book of physical pharmaceutics. In Colloids 2nd Eds, Vallabh Prakashan Publishers, New Delhi, 2002, 351-481. Rao YM, Vani G, Bala Ramesha Chary R. Design and evaluation of mucoadhesive drug delivery systems. Ind. Drugs. 1998, 35: 558-65. Park K & Robinson R. Bioadhesive platforms for oral-controlled drug delivery: method to study Bioadhesion. Int. J. Pharm. 1984, 19:107-27. Chen JL and CYR GN. Composition producing hydration through hydration in Adhesion in biological systems. Manley RS, Eds. Adhesion in Biological Systems. Academic Press, London, New York, 1970, 163-181. Andreas, Krajicek ME. Composition of mucoadhesive properties of various polymers. Advanced drug delivery review. 2005, 57: 1713-1723. Mikos AG, Peppas NA. Scaling concepts and molecular theories of adhesion of synthetic polymers to glycoproteinic networks. In Lenaerts, V, Gurny, R., Eds. Biodhesive drug delivery systems. CRC Press, Boca Ration, Florida, 1990, 25-42. Peppas NA. Surface, interfacial and molecular aspects of polymer bioadhesion on soft tissues. J. Controlled Release. 1985, 2:257-275. Lehr CM, Bouwstra JA, Bodde HE, Junginger HE. A surface energy analysis of mucoadhesion contact angle measurements RQ 3RO\FDUERSKLO DQG 3LJ ,QWHVWLQDO PXFRVD LQ SK\VLRORJLFDOO\ UHOHYDQW Ă XLGV 3KDUP 5HV

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Evaluation and Characterization of Solid-Self Nanoemulsifying Drug Delivery Systems (S-SNEDDS) of Lovastatin Samridhi and Dr. R.N. Gupta* Department of Pharmaceutical Sciences Birla Institute of Technology, Mesra, Ranchi, India

ABSTRACT Solid Self-nanoemulsifying drug delivery system (S-SNEDDS) of the poorly water soluble drug, lovastatin (LVS) was developed, evaluated and characterized with varying ratios of Capmul MCM and Cremophor (/ DQG À[HG OHYHOV RI *HOXFLUH $HURVLO DQG 2OH\ODPLQH 7KH SUHSDUHG 61(''6 DQG S-SNEDDS formulations were assessed for drug content, particle size, zeta potential and dissolution studies. The possible drug-excipient compatibility was studied by Fourier Transform Infrared (FTIR), Differential Scanning Calorimetry (DSC) of the pure drug and the formulations. The selected S-SNEDD was evaluated for its surface morphology by using Scanning Electron Microscopy (SEM). The mean particle size of SNEDD and S-SNEDDS formulation of lovastatin was found in the range of 12.70-15.70 nm. The polydispersibility index of the S-SNEDD formulation was found in the range of 0.055-0.221. Zeta potential of SNEDD formulations ranges between +31.5 and +35.2 mV. S-SNEDDS appeared as rough porous surface indicating that the liquid SNEDDS are adsorbed on the surface of Aerosil 200. DE (%) of the S-SNEDDS varied within 87.22 – 92.99 %, whereas, t85% of these formulations varied between 15.38 and 64.89 min. The results of this study indicate that the S-SNEDDS of Lovastatin, owing to nanosize, has potential to enhance its absorption and without interaction or incompatibility between the ingredients. Key words : Selfnanoemulsifying Drug Delivery System, Lovastatin, Zeta Potential, Differential Scanning Calorimeter, Scanning Electron microscopy INTRODUCTION The oral route is still the preferred route for drug administration. However, most of the NCEs discovered and many existing drugs are poorly soluble or lipophilic compounds which lead to poor bioavailability, high intra- and inter-subject variability and lack of dose proportionality1. Recently, lipidbased formulations with particular emphasis on self-emulsifying drug delivery systems (SEDDS) and solid self emulsifying drug delivery systems (S-SEDDS) are emphasized to improve the oral bioavailability of lipophilic drugs2. SEDDS are isotropic mixtures of natural or synthetic oils, solid or liquid surfactants, or alternatively, one or more hydrophilic solvents and co-solvents/surfactants. Upon mild agitation, IROORZHG E\ GLOXWLRQ LQ DTXHRXV PHGLD VXFK DV JDVWURLQWHVWLQDO à XLGV WKHVH V\VWHPV FDQ IRUP ÀQH R Z emulsions or microemulsions3,4. With the advent of nanotechnology in medicine, it is recognized that as particles get smaller, the surface area increases which may render them more biologically active. Self-nanoemulsifying drug delivery system (SNEDDS), typically produce emulsions with a droplet size less than 50 nm. Given the advantages of solid dosage forms, solid-SNEDDS frequently represent more effective alternatives to conventional liquid SNEDDS7,8. Lovastatin, a hypolipidemic agent, was selected as the model drug owing to its poor bioavailability (< 5%), rapid metabolism in gut and liver, low oral dose (10-40 mg), suitable log P (octanol/water) of 4.3, and being a BCS class II (low solubility, high permeability) drug4,6. 62


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EXPERIMENTAL Materials Lovastatin (LVS) was received as a gift from Torrent Pharmaceutical Ltd., (India). Gelucire 50/13 (G 50/13) was generous gift from Gattefosse (Saint-Priest Cedex, France). Capmul MCM (CMCM) was gifted from Abitec Corporation (Janesville, Germany). Cremophor EL (Cr-EL) was generous gift from BASF (Ludwigshafen, Germany). Aerosil 200 was received as gift from Degussa (Germany). Oleylamine (OA) was procured from Fluka (Steinheim, The Netherlands). All solvents and reagents used were of AnalaR grade. Preparation of SNEDDS and S-SNEDDS SNEDDS were prepared using varying ratios of Capmul MCM and Cremophor EL. The levels of Lovastatin (LVS) (1.25 % w/w), Gelucire 50/13 (6.25 % w/w) and OA (0.5 % w/w) were constant as reported by Singh et al.4. The prepared SNEDD formulation was dispersed in 5 mL of distilled water. Next, 1000 mg of solidifying agent, Aerosil 200 was added progressively with continuous stirring until VROLGLÀHG PDVV 6 61('' ZDV REWDLQHG5. Evaluation of SNEDD and S-SNEDD Formulation A. Drug Content Assay of weighed amount of SNEDD and S-SNEDD formulations were carried out to determine the GUXJ FRQWHQW 7KH ZHLJKHG VDPSOHV ZHUH VXLWDEO\ GLOXWHG LQ PHWKDQRO VWLUUHG E\ YRUWH[ PL[HU ÀOWHUHG and estimated spectrophotometrically at 237.8 nm (methanol). B. Determination of Particle Size and Zeta Potential4,8 61('' DQG 6 61('' IRUPXODWLRQ PJ ZHUH GLOXWHG ZLWK 0LOOLSRUH ZDWHU P/ LQ D à DVN DQG ZDV PL[HG JHQWO\ E\ LQYHUWLQJ WKH à DVN 7KH SDUWLFOH VL]H VR IRUPHG DQG LWV ]HWD SRWHQWLDO ZDV GHWHUPLQHG E\ G\QDPLF OLJKW VFDWWHULQJ '/6 WHFKQLTXH XVLQJ =HWDVL]HU DIWHU ÀOWUDWLRQ 1DQR =6 0DOYHUQ Instruments, UK). The zeta potential was ranged from -120 to 120 V. All measurements were done in triplicate using disposable polystyrene cuvettes. C. Fourier Transform Infra-red Spectroscopic Studies (FTIR) FTIR of pure LVS, and S-SNEDD formulations were carried out at a resolution of 4 cm-1 from 4000-600 cm-1 using FTIR- spectrophotometer with KBr disc (FTIR-8400S, Shimadzu, Tokyo, -DSDQ HTXLSSHG ZLWK D GLIIXVH UHà HFWDQFH DFFHVVRU\ '56 6KLPDG]X -DSDQ DQG D GDWD VWDWLRQ D. Differential Scanning Calorimetry Studies (DSC) Thermal analysis was carried out using differential scanning calorimeter (Q10,TA instruments). The DQDO\VLV ZDV SHUIRUPHG XQGHU SXUJH RI GU\ QLWURJHQ JDV FF PLQ 6DPSOH PJ SODFHG LQ à DW ERWWRP DOXPLQXP SDQ ZDV ÀUPO\ FULPSHG ZLWK OLG WR SURYLGH DQ DGHTXDWH VHDO 6DPSOH ZDV KHDWHG from ambient temperature to 200 ºC at preprogrammed heating rate of 10ºC min-1. All the samples viz., pure lovastatin and prepared S-SNEDD formulation were analyzed in the similar manner. E. Scanning Electron Microscopic Studies (SEM)7,8 To observe the surface morphology of solidifying agent, Aerosil 200 and selected S-SNEDD, SEM was performed using JEOL JSM -6390LV (Tokyo, Japan). Particles were coated with thin gold particles layers by a sputter coater unit and the surface topography was analyzed. The images were captured at an H[FLWDWLRQ YROWDJH RI N9 DW YDU\LQJ PDJQLÀFDWLRQV IURP RULJLQDO PDJQLÀFDWLRQ [ WR [ 63


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F. In-Vitro Dissolution Studies6 The in-vitro dissolution study of each LVS-loaded SNEDD was carried out on USP dissolution apparatus equipped with a fractional collector (TDT-08L, Electrolab Lab, India). The dissolution vessels contained 1000 mL of phosphate buffer of pH 6.8 maintained at 37 ¹ 0.5 ºC and paddle speed set at 75 rpm. The withdrawn samples were analyzed for LVS content spectrophotometerically at 238.8 nm. The inYLWUR GLVVROXWLRQ SURÀOHV QDPHO\ FXPXODWLYH SHU FHQW GUXJ UHOHDVH GLVVROXWLRQ UDWH FRQVWDQW GLVVROXWLRQ half-life (t85% DQG SHU FHQW GLVVROXWLRQ HIÀFLHQF\ '( ZHUH FDOFXODWHG 7KH FRQFHQWUDWLRQ RI /RYDVWDWLQ in the test samples was calculated using the regression equation of the calibration curve. The studies were done in triplicate and represented graphically. RESULTS AND DISCUSSSION Preparation of SNEDDS and S-SNEDDS of Lovastatin Self-nanoemulsifying drug delivery system of Lovsatatin were prepared using varying ratios of Capmul 0&0 DQG &UHPRSKRU (/ ZLWK À[HG OHYHOV RI *HOXFLUH DQG 2OH\ODPLQH 7KH SUHSDUHG 61(''6 IRUPXODWLRQV ZHUH VROLGLÀHG E\ DGGLQJ À[HG TXDQWLW\ RI $HURVLO LQ WKH DTXHRXV GLVSHUVLRQ RI HDFK of the SNEDD formulation. Evaluation of SNEDD formulation of Lovastatin Drug Content Irrespective of the ratios of oil and surfactant used, the drug content in all the three formulations (F1F3) was found in the range of 100.3-101.2 %, indicative of uniform dispersion of drug in formulations. Particle Size and Zeta Potential The mean particle size of SNEDD formulation of lovastatin was found in the range of 12.7014.48 nm (Figure 1 A). Furthermore, the percent intensity versus mean particle size of all the SNEDD formulation was found to be monomodal indicating uniform distribution of particles after aqueous dispersion. Moreover, the polydispersibility index of the SNEDD formulation (F1-F3) was found in the range of 0.055-0.119, suggesting uniform distribution of particles in the nano range.

Figure 1 : 3DUWLFOH VL]H GLVWULEXWLRQ SURÂżOH $ DQG =HWD SRWHQWLDO % RI 61('' IRUPXODWLRQ ) ) RI /RYDVWDWLQ LQ GLVWLOOHG ZDWHU

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Zeta potential ranges between +31.5 and +35.2 mV for the formulations F1-F3, suggesting increased adhesion of the droplets to the cell surface because of electrostatic attraction (Figure 1B). The positive zeta potential of the SNEDD formulation could be because of orientation of oleylamine on the surface of emulsion droplets. Evaluation of S-SNEDD formulation of Lovastatin Drug Content 7KH GUXJ FRQWHQW LQ DOO WKH WKUHH VROLGLĂ€HG 61('' IRUPXODWLRQV ) ) ZDV IRXQG LQ WKH UDQJH RI 99.22-101.45 %, indicative of uniform dispersion of drug in S-SNEDD formulations. Furthermore, it can EH LQIHUUHG WKDW WKH DGRSWHG VROLGLĂ€FDWLRQ WHFKQLTXH KDYH QRW DOWHUHG WKH HQWUDSPHQW RI WKH ORYDVWDWLQ in the S-SNEDD formulation. Particle Size The mean particle size of S-SNEDD formulation of lovastatin was found in the range of 13.87-15.70 nm. The percent intensity versus mean particle size of all the S-SNEDD formulation was found to be monomodal indicating uniform distribution of particles after aqueous dispersion. The polydispersibility index of the S-SNEDD formulation (F1-F3) was found in the range of 0.129-0.221, suggesting uniform distribution of particles in the nano range. Fourier Transform Infra-red Spectroscopic Studies (FTIR) and Differential Scanning Calorimetry (DSC) The spectral data shown in Figure 2A showed the retention of the characteristic absorption of the lovastatin in the S-SNEDD formulation (F1-F3) at 3546.35 cm-1(alchohol O-H stretch), 3017.12 cm-1 ROHĂ€QLF & + VWUHWFK FP-1 (methyl C-H asymmetric stretch), 1726.12cm-1 (lactone and ester carbonyl stretch), 1464 cm-1 (methyl asymmetric bend), 1265.52cm-1 (lactone C-O-C asymmetric bend) and 973.88 cm-1 DOFRKRO & 2+ VWUHWFK 7KH ,5 VSHFWUXP GDWD FRQĂ€UPV WKDW WKH H[FLSLHQWV GR QRW DOWHU the performance characteristic of the drug, indicating their compatibility. 100

F1

LVS 60

50

40

60 Transmission (%)

Transmission (%)

80

40

30

DSC mW

20

50.0

10

20

Lovastatin

0

0 4000

3500

3000

2500

2000

1500

1000

4000

500

3500

3000

2500

2000

1500

1000

174.42 C

500

-1

Wavenumber (cm )

-1

Wavenumber (cm ) F2

70

F3

70

F1

0.0 60

60

50

40 30 20

F2

40

Transmission (%)

Transmission (%)

50

30

F3

20

-50.0 10

50.00

10 0

100.00

Temp [C]

0 4000

3500

3000

2500

2000

1500

1000

500

-10 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

-1

Wavenumber (cm )

(A) Figure 2 : IR spectra (A) and DSC (B) of S-SNEDD formulation (F1-F3) of Lovastatin

65

(B)

150.00

200.00


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

The thermal analysis of pure lovastatin, S-SNEDD formulation (F1-F3) is shown in Figure 2B. The sharp endothermic peak of lovastatin appeared at 174.42°C which corresponds to the drug melting point. Thermograms of S-SNEDD formulation (F1-F3) showed the absence of lovastatin peak, suggesting that lovastatin is present as amorphous or as solid solution. Scanning Electron Microscopy 7KH VFDQQLQJ HOHFWURQ PLFURJUDSKV RI VROLGLĂ€HG $HURVLO DQG VHOHFWHG 6 61('' IRUPXODWLRQ ) DUH VKRZQ LQ )LJXUH D G DQG )LJXUH H K UHVSHFWLYHO\ DW YDULRXV PDJQLĂ€FDWLRQV [ WR [ 6ROLGLĂ€HG $HURVLO DSSHDUHG DV VPRRWK VXUIDFH +RZHYHU 6 61(''6 ) DSSHDUHG DV URXJK SRURXV surface indicating that the liquid SNEDDS are adsorbed or coated on the surface of Aerosil 2008. No crystalline structures of lovatatin was apparent on the surface of Aerosil 200 indicating the presence of DPRUSKRXV VWDWH RI ORYDVWDWLQ DV GULHG HPXOVLRQ RQ WKH VXUIDFH RI $HURVLO 7KH UHVXOW IXUWKHU DIĂ€UPV WKH Ă€QGLQJV RI GLIIHUHQWLDO VFDQQLQJ VWXGLHV ZKHUH GUXJ ZDV SUHVHQW DV DPRUSKRXV VWDWH $OVR LW FDQ EH LQIHUUHG WKDW WKH DGRSWHG VROLGLĂ€FDWLRQ WHFKQLTXH GLG QRW DOWHUHG WKH SHUIRUPDQFH FKDUDFWHULVWLFV RI lovastatin in terms of drug recrystallization. (b)

(a)

(e)

(f)

(c)

(d)

(g)

(h)

Figure 3 : 6FDQQLQJ HOHFWURQ PLFURJUDSKV RI $HURVLO DW WKH PDJQLÂżFDWLRQ RI [ D [ E [ F [ G DQG S-SNEDD (F1), x500 (e); x1000 (f); x3000 (g); x5000 (h). 100

Dissolution Studies Dissolution studies were performed to compare the release of lovastatin from three different S-SNEDD IRUPXODWLRQV ) ) 7KH GLVVROXWLRQ SURĂ€OHV RI 6 61('' formulations of lovastatin are presented in Figure 4. The UHOHDVH SURĂ€OHV ZHUH FKDUDFWHUL]HG E\ W85% and percent GLVVROXWLRQ HIĂ€FLHQF\ '( 7DEOH

Pecent Drug Released

75

F1 F2 F3

50

25

0 0

50

100 Time (Min)

150

200

% DE of the S-SNEDDS varied within 87.22 – 92.99 %

Figure 4 : 'LVVROXWLRQ SURÂżOH RI /96 ORDGHG 6 61('' whereas, t85% of these formulations varied between 15.38 formulations. and 64.89 min. t85% decreases while DE values increases All data points represents the mean Âą SD (n=3).

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Table 1. Dissolution characteristic of lovastatin S-SNEDD formulations Formulation F1

F2

F3

DE (%)

87.22

88.90

92.99

t85% (min)

64.89

32.96

15.38

with increase in Cremophor EL content as we go from formulation F1 to F3 (Table 1). Low t85% was observed with those formulations, which consists of higher amount of cremophor EL and lower amount of capmul MCM.

CONCLUSION The prepared S-SNEDDS lovastatin will provide simpler and more robust system which ensures the reliable and reproducible release of the drug in colloidal state leading to rapid onset of action with improved absorption. The results of this study indicate that the S-SNEDDS of Lovastatin, owing to nanosize, has potential to enhance its absorption and without interaction or incompatibility between the ingredients. REFERENCES 1.

Charman, S.A., Charman, W.N., Rogge, M.C., Wilson, T.D., Dutko, F.J., and Pouton, C.W., 1992. Self-emulsifying drug delivery systems, formulation and biopharmaceutic evaluation of an investigational lipophilic compound. Pharm. Res. 9, 87-93.

2.

Gershanik, T., and Benita, S., 2000. Self-dispersing lipid formulations for lipophilic drugs. Eur. J. Pharm. Biopharm. 50, 179-188.

3.

Pouton, C.W., and Porter, C.J.H., 2008. Formulation of lipid-based drug delivery systems for oral administration, Materials, methods and strategies. Adv. Drug Deliv. Rev. 60, 625-637.

4.

Singh, S.K., Verma, P.R.P., and Razdan, B.K., 2009. Development and characterization of lovastatin-loaded selfmicroemulsifying drug delivery system. Pharm Dev Tech. (DOI, 10.3109/10837450903286537).

5.

Singh, S.K., Verma, P.R.P., and Razdan, B.K., 2010. Glibenclamide-loaded self-nanoemulsifying drug delivery system: development and characterization. Drug Dev. Ind. Pharm. (DOI, 10.3109/03639040903585143).

6.

Indian Pharmacopoeia,2007; Controller of publications, New Delhi, vol.1, 477-478

7.

Balakrishnan, P., Lee, B.J., Oh, D.H., Kim, J.O., Hong, M.J., Jee, J.P., Kim, J.A., Yoo, B.K., Woo, J.S., Yong, C.S. and Choi, H.G., 2009. Enhanced oral bioavailability of dexibuprofen by a novel solid Self-emulsifying drug delivery system (SEDDS). Eur. J. Pharm. Biopharm. 72, 539-545.

8.

Kang, J.H., Oh, D H., Oh, Y.K., Yong, C.S. and Choi, H.G., 2012. Effect of solid carriers on the crystalline properties, GLVVROXWLRQ DQG ELRDYDLODELOLW\ RI ÁXUELSURIHQ LQ VROLG VHOI QDQRHPXVLI\LQJ GUXJ GHOLYHU\ V\VWHP VROLG 61(''6 (XU J. Pharm. Biopharm. 80, 289-297.

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Estimation of Heavy Metals in Locally Available Vegetables Collected from Road Side Market Sites (1-4) of Different Areas of Ranchi City Ratna Ghosh1*, Reshma Xalxo1, Mukul Chandra Gope2, Sougata Mishra3, Bindu Kumari3, Manik Ghosh3 1* Department of Home Science, Ranchi University, Ranchi, India 2 Department of Physiology, Rajendra Institute of Medical Sciences, Ranchi, India 3 Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, India

ABSTRACT Heavy metals are among the major contaminants of food supply and may considered the most important problem to our environment. The hypothesis behind the present study is that the irrigation with waste water, transportation and marketing site of vegetables in contaminated environment may elevate the levels of heavy metals in vegetables through surface deposition. Eight Road side Markets and two organised Markets were demarcated for vegetable purchasing. The present study was focused on Site-1 to Site-4 only. Six vegetables out of thirteen showed higher Metal Pollution Index in Site-3 and Site-4. All sites showed several fold higher concentrations of Lead (Pb), than the permissible PFA limit. Site-4 contains VLJQLÀFDQWO\ KLJKHU FRQFHQWUDWLRQ RI 3E 3 WKDQ DOO RWKHU VLWHV 7KH SUHVHQW VWXG\ KDV JHQHUDWHG data on heavy metal pollution in and around Ranchi City, Capital of Jharkhand and associated risk assessment for consumer’s exposure to the heavy metals. Key words : Heavy metals, vegetables, PFA, MPI, ICP-OES INTRODUCTION )RRG VDIHW\ LV D PDMRU SXEOLF FRQFHUQ ZRUOGZLGH DQG IRRG FRQVXPSWLRQ KDV EHHQ LGHQWLÀHG DV WKH major pathway for human exposure to certain environmental contaminants, accounting for >90% of intake compared to inhalation or dermal routes of exposure. About 30% of human cancers are caused by low exposure to initiating carcinogenic contaminants in the diet1. During the last decades, the increasing demand of food safety has stimulated research regarding the risk associated with consumption of foods contaminated by pesticides, heavy metals and/or toxins2. Heavy metals (Lead, Arsenic, Cadmium, Copper, Chromium and Nickel) contamination of vegetables cannot be underestimated as these foodstuffs are important components of human diet. Vegetables are ULFK VRXUFHV RI YLWDPLQV PLQHUDOV DQG ÀEHUV DQG DOVR KDYH EHQHÀFLDO DQWLR[LGDWLYH HIIHFWV +RZHYHU intake of heavy metal-contaminated vegetables may pose a risk to the human health. Heavy metal contamination of the food items is one of the most important aspects of food quality assurance3-6. International and national regulations on food quality have lowered the maximum permissible levels of toxic metals in food items due to an increased awareness of the risk these metals pose to food chain contamination4. Heavy metals are among the major contaminants of food supply and may considered the most important problem to our environment7. Heavy metals, in general, are not biodegradable, have long biological half-lives and have the potential for accumulation in the different body organs leading to unwanted side effects8-9. Lead and cadmium are among the most abundant heavy metals and are particularly toxic. The excessive content of these metals in food is associated with etiology of a number of diseases, especially with cardiovascular, kidney, nervous as well as bone diseases8, 10-12. In addition, they are also implicated in causing carcinogenesis, mutagenesis and teratogenesis13-14. 68


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Heavy metal contamination may occur due to irrigation with contaminated water, addition of fertilizers and metal-based pesticides, industrial emissions, transportation, harvesting process, storage and/or sale. Emissions of heavy metals from the industries and vehicles may be deposited on the vegetable surfaces during their production, transport and marketing. It is well known that plants take up metals by absorbing them from contaminated soils as well as from deposits on parts of the plants exposed to the air from polluted environments15-16. Heavy metals are non-biodegradable and persistent environmental contaminants, which may be deposited on the surfaces and then absorbed into the tissues of vegetables. Plants take up heavy metals by absorbing them from deposits on the parts of the plants exposed to the air from polluted environments as well as from contaminated soils. A number of studies have shown heavy metals as important contaminants of the vegetables. Heavy metal contamination of vegetables may also occur due to irrigation with contaminated water. The potential toxicity, persistent nature and cumulative behavior as well as the consumption of vegetables and fruits, there is necessary to test and analyze these food items to ensure that the levels of these contaminants meet the agreed international requirements. Regular survey and monitoring programmes of heavy metal contents in foodstuffs have been carried out for decades in most developed countries. But, in developing countries limited data are available on heavy metals. Therefore this study will present data on the level of heavy metals in selected vegetables and this study will be also dealing with the daily intake of these metals through consumption of vegetables. MATERIALS AND METHODS Study Areas The study was conducted around Ranchi city (23°21’ N latitude 85°20’ E longitude and 729 m (2,392 ft) above the sea level) in Jharkhand eastern plains of India during July 2010 to February 2012. Various small scale industries situated in this city. A large area around industries have no access to clean water resources, so farmers use treated and untreated wastewater for irrigation. The hypothesis behind the present study is that the irrigation with waste water, transportation and marketing site of vegetables in contaminated environment may elevate the levels of heavy metals in vegetables through surface deposition. Eight Road side Markets viz. Site-1 (Lalpur Market), Market Site-2 (BIT More Market), Site-3 (Daily Market), Site-4 (Kanke Road Market), Site-5 (Booti More Market), Site-6 (RIMS Market), Site-7 (Morabadi Stadium Market), Site-8 (Bahu Bazar Market) and two organised Markets i.e. Site-9 (Reliance Fresh) & Site-10 (Big Bazar) were demarcated for vegetable purchasing. The present study was focused on Site-1 to Site-4 only. Sampling The edible portions of Vegetables were collected from different markets during July 2010 to June 2011. Samples were brought back to the laboratory and washed with clean tap water to remove the soil particles and dusts of the vegetables. After removing the extra water from the surface of vegetables with blotting paper, samples were cut into pieces, packed into separate bags, and kept in an oven until a constant weight was achieved. The dried samples were grinded and passed through a sieve of 2 mm size and then kept at room temperature for further analysis. Digestion of Plant Samples 0.5 gm of the dried powdered sample was digested in Microwave Digester 3000 SOLV at 1400 69


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watt for 3 hours in the solvent system of HNO3 : H2O2, in 7:0.5 ratio until a transparent solution was REWDLQHG $IWHU FRROLQJ WKH GLJHVWHG VDPSOH ZDV ÀOWHUHG XVLQJ :KDWPDQ *UDGH 1R 4XDQWLWDWLYH )LOWHU 3DSHU $VKOHVV DQG WKH ÀOWUDWH ZDV ÀQDOO\ PDLQWDLQHG WR PO ZLWK GLVWLOOHG ZDWHU Analysis of Heavy Metals &RQFHQWUDWLRQV RI $V &G &R &U &X 0Q 1L DQG 3E LQ WKH ÀOWUDWH RI GLJHVWHG SODQW VDPSOHV ZHUH estimated by using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) (Model Optical 2100DV ICP-OES, Perkin Elmer, USA) with argon laser. The Spectral range was of 160 nm WR QP DQG UHVROXWXLRQ RI QP DW QP 7KH LQVWUXPHQW ZDV ÀWWHG ZLWK 89 VHQVLWLYH GXDO backside – illuminated CCD array detector. DATA ANALYSIS Metal Pollution Index (MPI) To examine the overall heavy metal concentrations in all vegetables, metal pollution index (MPI) was computed. This index was obtained by calculating the geometrical mean of concentrations of all the metals in the vegetables17. MPI (Οg/g) = (Cf1 x Cf2 x . . . x Cfn)1/n Where, Cfn = concentration of metal n in the sample. Statistical Analysis The data of heavy metal concentrations in the vegetables of different sites (Site-1 to Site-4) were VXEMHFWHG WR WZR ZD\ DQDO\VLV RI YDULDQFH $129$ WHVW IRU DVVHVVLQJ WKH VLJQLÀFDQFH RI GLIIHUHQFHV LQ heavy metal concentrations due to different irrigation practices, environmental pollutants, etc followed by Bonferroni’s multiple comparison test. All the statistical tests were performed using GraphPad Prism. RESULTS Metal Pollution Index Metal Pollution Index (MPI) is suggested to be a reliable and precise method for metal pollution monitoring of wastewater irrigated areas17. Among different vegetables in Site-1, pea showed highest value of MPI followed by spinach. In Site-2 pea, spinach and beans showed higher MPI. Six vegetables out of thirteen showed higher MPI i.e. more than 2, in Site-3 and Site-4. These were pea, spinach, WRPDWR FXFXPEHU ODG\ ÀQJHU DQG EHDQV +LJKHU 03, VXJJHVWV WKDW WKHVH YHJHWDEOHV PD\ FDXVH PRUH human health risk due to higher accumulation of heavy metals in the edible portion. Concentration of Heavy Metals Heavy metal concentrations showed variations among different vegetables collected from different market sites (Figure 1 to Figure 6). All sites showed several fold higher concentrations of Lead (Pb), than the permissible PFA limit, in cucumber (range, 7.10 ¹ 1.52 ppm to 12.67 ¹ 1.52 ppm), pea (range, 6.17 ¹ 1.51 ppm to 20.67 ¹ 3.29 ppm) and tomato (range, 5.00 ¹ 1.97 ppm to 9.67 ¹ 0.33 ppm). 6LWH DQG 6LWH DOVR VKRZHG KLJKHU FRQFHQWUDWLRQ RI 3E LQ ODG\ ÀQJHU “ SSP DQG “ 1.51 ppm, respectively). Site-2 and Site-4 showed higher concentration of Pb in beet, beans and spinach. Apart from these Site-3 showed higher concentration of Pb in cabbage and coriander. Nickel (Ni) was found to be higher in pea (4.33 ¹ 0.56 ppm to 4.83 ¹ 0.60 ppm) and beans (3.83 ¹ 0.48 ppm to 4.67 ¹ 0.42 ppm) than PFA permissible limit in all sites. Cucumber (2.93 ¹ 0.31 ppm) of Site-3 and tomato 70


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Figure 1 : Concentration of Cadmium (Cd) in vegetables of all four sites

Figure 2 : Concentration of Cobalt (Co) in vegetables of all four sites

Figure 3 : Concentration of Chromium (Cr) in vegetables of all four sites

Figure 4 : Concentration of Copper (Cu) in vegetables of all four sites

Figure 5 : Concentration of Nickel (Ni) in vegetables of all four sites

Figure 6 : Concentration of Lead (Pb) in vegetables of all four sites

(4.33 Âą 0.67 ppm) of Site-4 were found to contain high amount of Ni than the permissible PFA limit. 6LWH FRQWDLQV VLJQLĂ€FDQWO\ KLJKHU FRQFHQWUDWLRQ RI 3E 3 ) than all other sites. Concentration of 1LFNHO LQ FXFXPEHU RI 6LWH DQG WRPDWR RI 6LWH ZHUH VLJQLĂ€FDQWO\ KLJKHU 3 ) in comparison to all RWKHU VLWHV &DGPLXP ZDV IRXQG WR EH VLJQLĂ€FDQWO\ KLJKHU &XFXPEHU 3 ) and Coriander (3 ) of Site-4 in comparison of Site-1. Rest all heavy metals were found to be below the PFA permissible limit in all vegetables collected from different sites. Results of two way ANOVA test showed that variations in WKH KHDY\ PHWDO FRQFHQWUDWLRQV ZHUH VLJQLĂ€FDQW IRU VRPH KHDY\ PHWDOV GXH WR VLWH YHJHWDEOH DQG VLWH plant interaction (Table 1). The variations in heavy metal concentrations in vegetables of the same site may be ascribed to the differences in their morphology and physiology for heavy metal uptake, exclusion, 71


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accumulation and retention18-19. The Table 1. Variation in heavy metal concentration vegetables of different sites use of contaminated irrigation water may also increase the uptake and Concentration Level of S. VegetaHeavy Site accumulation of the heavy metals No. (ppm) 6LJQLÀFDQFH ble metal in the vegetables. 1 Pea Lead Site-1 13.733 ± 2.733** Site-4 vs All CONCLUSION Site-2 12.000 ± 1.932*** **P<0.01 The variations in the Site-3 06.167 ± 1.515*** ***P<0.001 concentrations of the heavy metals Site-4 20.667 ± 3.293 in vegetables observed during the 2 Tomato Nickel Site-1 0.333± 0.076*** Site-4 vs All present study may be ascribed to Site-2 0.317± 0.054*** ***P<0.001 the physical and chemical nature Site-3 0.433± 0.076*** of the soil of the production sites, Site-4 4.333± 0.667 absorption capacities of heavy 3 Cucumber Nickel Site-1 1.167± 0.167** Site-3 vs All metals by vegetables, atmospheric **P<0.01 Site-2 1.333± 0.211*** deposition of heavy metals, which ***P<0.001 Site-3 2.933± 0.981 PD\ EH LQÁXHQFHG E\ LQQXPHUDEOH Site-4 1.167± 0.211*** environmental factors such as Cadmium Site-1 0.100 ± 0.068** Site-4 vs All temperature, moisture and wind Site-2 0.200± 0.045* **P<0.01 velocity, and the nature of the Site-3 0.383 ± 0.083ns *P<0.05 vegetables, i.e. leafy, root, fruit, Site-4 0.800± 0.073 ns: Non exposed surface area, hairy or 20 VLJQLÀFDQW smoothness of the exposed parts . 4 Coriander Cadmium Site-1 0.267± 0.123* Site-4 vs All The variations in the concentrations Site-2 0.333 ± 0.120ns *P<0.05 of heavy metals in the vegetables Site-3 0.517± 0.122ns ns: Non tested may also be ascribed to the VLJQLÀFDQW Site-4 0.867 ± 0.152 variations in the anthropogenic activities such as heavy traffic, Results represented as Mean ± SEM (n=6). Two way analysis of variance (ANOVA) addition of phosphate fertilizers followed by Bonferroni’s multiple comparison test. or use of metal-based pesticides around production sites and urban industrial activities at market sites. The present study has generated data on heavy metal pollution in and around Ranchi City, Capital of Jharkhand and associated risk assessment for consumer’s exposure to the heavy metals. The proposed hypothesis that the transportation and marketing of vegetables in contaminated environment may elevate the levels of heavy metals in vegetables through surface deposition has been proved through this study. Appropriate precautions should also be taken at the time of transportation and marketing of vegetables. Heavy metals have a toxic impact, but detrimental impacts become apparent only when long-term consumption of contaminated vegetables occurs. It is therefore suggested that regular monitoring of heavy metals in vegetables and other food items should be performed in order to prevent excessive build-up of these heavy metals in the human food chain.

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ACKNOWLEDGEMENT The authors are thankful to the Head, Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra for providing the facilities to carry out this work and Mr. Sanjay Swain (Technician), Central Instrumentation Facility, Birla Institute of Technology, Mesra Ranchi for his constant help during the analytical work. REFERENCES 1.

Tricker, A.R., Preussmann, R., 1990. Chemical food contaminants in the initiation of cancer. Proc. Nutr. Soc. 49, 133–144.

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D’Mello, J.P.F., 2003. Food safety: Contaminants and Toxins. CABI Publishing, Wallingford, Oxon, UK, Cambridge, MA, p. 480.

3.

Marshall, 2004. Enhancing food chain integrity: quality assurance mechanism for air pollution impacts on fruits DQG YHJHWDEOHV V\VWHPV &URS 3RVW +DUYHVW 3URJUDP )LQDO 7HFKQLFDO 5HSRUW 5 KWWS ZZZ VXVVH[ DF XN spru/1-4-7-1-11-1.html>.

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Radwan, M.A., Salama, A.K., 2006. Market basket survey for some heavy metals in Egyptian fruits and vegetables. Food Chem. Toxicol. 44, 1273–1278.

5.

Wang, X., Sato, T., Xing, B., Tao, S., 2005. Health risk of heavy metals to the general public in Tianjan, China via FRQVXPSWLRQ RI YHJHWDEOHV DQG Ă€VK 6FL 7RW (QYLURQ ² ²

6.

Khan, S., Cao, Q., Zheng, Y.M., Huang, Y.Z., Zhu, Y.G., 2008. Health risk of heavy metals in contaminated soils and food crops irrigated with waste water in Beijing, China. Environ. Pollut. 152 (3), 686–692.

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Zaidi, M.I., Asrar, A., Mansoor, A., Farooqui, M..A., 2005. The heavy metal concentrations along roadside trees of Quetta and its effects on public health. J. Appl. Sci. 5 (4), 708–711.

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Jarup, L., 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68, 167–182.

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Sathawara, N.G., Parikh, D.J., Agarwal, Y.K., 2004. Essential heavy metals in environmental samples from western India. Bull. Environ. Contam. Toxicol. 73, 756–761.

10.

Steenland, K., Boffetta, P., 2000. Lead and cancer in humans: where are we now? Am. J. Ind. Med. 38, 295–299.

11.

WHO, 1992. Cadmium. Environmental Health Criteria, Geneva. Vol. 134.

12.

WHO, 1995. Lead. Environmental Health Criteria, Geneva. Vol. 165.

13.

International Accreditation Criteria for Laboratories Performing Food Chemistry Testing, 1999. AOAC International, Gaithersburg, MD. pp. 1–33.

3LWRW & + 'UDJDQ 3 < &KHPLFDO FDUFLQRJHQHVLV ÀIWK HG ,Q &DVDUHWW 'RXOOV (GV 7R[LFRORJ\ ,QWHUQDWLRQDO Edition, McGraw Hill, New York, pp. 201–260. 15.

Khairiah, T., Zalifah, M.K., Yin, Y.H., Aminah, A., 2004. The uptake of heavy metals by fruit type vegetables grown in selected agricultural areas. Pak. J. Biol. Sci. 7 (8), 1438–2.

16.

Chojnacha, K., Chojnacki, A., Gorecka, H., Gorecki, H., 2005. Bioavailability of heavy metals from polluted soils to plants. Sci. Total Environ. 337 (1–3), 175–182.

17.

Usero, J., Gonzalez-Regalado, E., Gracia, I., 1997. Trace metals in the bivalve mollusks Ruditapes decussates and Ruditapes phillippinarum from the Atlantic Coast of Southern Spain. Environ. Int. 23 (3), 291–298.

18.

Carlton-Smith, C.H., Davis, R.D., 1983. Comparative uptake of heavy metals by forage crops grown on sludgetreated soils. In: Proceeding of International Conference on Heavy metals in the Environment. CEP Consultants Ltd., Edinburg, UK, pp. 3933–3940.

19.

Kumar, A., Sharma, I.K., Sharma, A., Varshney, S., Verma, P.S., 2009. Heavy metals contamination of vegetable foodstuffs in Jaipur (India). Electron. J. Environ. Agri. Food Chem. 8 (2), 96–101.

20.

Zurera, G., Moreno, R., Salmeron, J., Pozo, R., 1989. Heavy metal uptake from greenhouse border soils for edible vegetables. J. Sci. Food Agric. 49, 307–314.

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Development of Lornoxicam Transdermal Patch: Effect of Natural Penetration Enhancers Dipti Srivastava*, Nishant Kumar Verma, Nimisha, Wasim Ahmed Amity Institute of Pharmacy, Amity University, Lucknow Campus.

ABSTRACT The feasibility of development of transdermal delivery system of Lornoxicam utilizing naturaloils as permeation enhancers was investigated. Penetration enhancing potential of Linseed oils,Fennel oil and Rosemary oil on in vitro permeation of Lornoxicam across rat skin was studied.The physicochemical compatibility of the drug, polymers and oil was studied.All formulations carried 10% w/v of Polyethylene glycol as plasticizer and dichloromethane and methanol (4:1) as solvent system. The prepared transdermal patches were evaluated for in vitro release, moisture absorption, moisture loss and mechanical properties. 7KH GLIIXVLRQ VWXGLHV ZHUH SHUIRUPHG E\ XVLQJ PRGLÀHG )UDQ] GLIIXVLRQ FHOOV 7KH IRUPXODWLRQ FRQWDLQLQJ 5%w/v Fennel oil showed maximum % drug release of 89.33%. which was higher than the percentage drug release 45.21% observed for the transdermal patch where penetration enhancer was not used. The % drug release of the control batch was statistically analysed using independent t test. The p value at FRQÀGHQFH LQWHUYDO ZDV IRXQG WR EH S 2Q WKH EDVLV RI WKH S YDOXH DQG I YDOXH LW ZDV FRQFOXGHG WKDW WKHUH ZDV VLJQLÀFDQW GLIIHUHQFH LQ WKH GUXJ UHOHDVH RI WUDQVGHUPDO SDWFKHV without oil and batch F2. Amongst the three natural oils used, Fennel oil was found to be most effective DV SHQHWUDWLRQ HQKDQFHU 7KH SDWFKHV WKXV SUHSDUHG PD\ LQFUHDVH WKH HIÀFDF\ RI /RUQR[LFDP IRU WKH therapy of arthritis and other painful muscular conditions. INTRODUCTION: Transdermal drug delivery systems (patches) are dosage forms designed to deliver a therapeutically effective amount of drug across a patient’s skin1 DOVR GHÀQHG DV PHGLFDWHG DGKHVLYH SDWFK WKDW LV SODFHG RQ WKH VNLQ WR GHOLYHU D VSHFLÀF GRVH RI PHGLFDWLRQ WKURXJK WKH VNLQ DQG LQWR WKH EORRGVWUHDP The stratum corneum provides the greatest resistance to penetration, and it is the rate-limiting step in percutaneous absorption. Penetration enhancers are the substances that facilitate the absorption of penetrant through the skin by temporarily diminishing the impermeability of the skin.corneum which is the main barrier2,3. Though there are a number of chemical penetration enhancers, the potential of natural penetration enhancers are being explored now a days because of their less side effects. Newer 16$,' OLNH /RUQR[LFDP LV ZLGHO\ XVHG IRU WKH WUHDWPHQW RI 5KHXPDWRLG DUWKULWLV DQG RWKHU LQà DPPDWRU\ and painful conditions. In the present work, the feasibility of development of transdermal delivery system of Lornoxicam utilizing naturaloils as permeation enhancers was investigated using Linseed oil, Rosemary oil and Fennel oil. MATERIALS AND METHOD Lornoxicam was received as a gift sample from Glenmark Pharmaceuticals Pvt. Ltd, Mumbai. HPMC E-5 procured from Ozone International, Mumbai, and Natural oils was as a gift sample from Lala Jagdish Prasad Pvt. Ltd, Kanpur. All other laboratory chemical used in the study was analytical reagents grade. Double distilled was used throughout the study. Drug, polymer and oil interaction study 4 : 7KH SK\VLFRFKHPLFDO FRPSDWLELOLW\ EHWZHHQ /RUQR[LFDP RLO DQG SRO\PHU XVHG LQ WKH ÀOPV ZDV VWXGLHG 74


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by using Fourier transform-infrared (FTIR-8300, Shimadzu Co,Kyoto, Japan) spectroscopy. The FT-IR spectra were recorded in the wavelength region between 4000 and 400 cm-1. The spectra Obtained for Lornoxicam and physical mixtures of Lornoxicam with polymers were compared. Preparation of transdermal patch5,6 : Transdermal patches of Lornoxicam were prepared by solvent evaporation technique. The formulations have been shown in Table.1. Solutions of HPMC E -5 and Fennel oil, Rosemary and Linseed oil solution were prepared separately in dichloromethane: methanol (4:1) mixture. The two solutions were mixed to which weighed amount of Lornoxicam was added slowly. To the mixture, PEG PO ZDV DGGHG DQG PL[HG 7KH GUXJ SRO\PHU VROXWLRQ ZDV FDVWHG LQ 7HĂ RQ SODWH ZLWK DUHD of 3 cm2 which is wrapped by aluminium foil. The plate was kept aside for drying at room temperature IRU KUV ,QYHUWHG IXQQHO ZDV SODFHG RYHU WKH 7HĂ RQ SODWHV WR SUHYHQW WKH FXUUHQW RI DLU $IWHU GU\LQJ WKH SDWFKHV ZHUH SHHOHG IURP 7HĂ RQ SODWHV ZUDSSHG LQ DOXPLQLXP IRLO DQG SUHVHUYHG LQ GHVLFFDWRUV IRU IXUWKHU VWXGLHV $IWHU GU\LQJ WKH SDWFKHV ZHUH SHHOHG IURP 7HĂ RQ SODWHV ZUDSSHG LQ DOXPLQLXP foil, and preserved in desiccators for further studies. A control batch was also prepared that didnot contain any oil. Table1: composition of various batches of transdermal patches of Lornoxicam Formulations

F0

F-1

F-2

F-3

F-4

F-5

F-6

F-7

F-8

F-9

Lornoxicam(mg)

8

8

8

8

8

8

8

8

8

8

HPMCE5 ( mg)

300

300

300

300

300

300

300

300

300

300

Fennel oil (%w/v)

-

3

5

7

-

-

-

-

-

-

Rosemary oil (%w/v)

-

-

-

-

3

5

7

-

-

-

Linseed oil (%w/v)

-

-

-

-

-

-

-

3

5

7

Dichloromethane: Methanol (4:1)(ml)

7

7

7

7

7

7

7

7

7

7

PEG 400 (ml)

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

Glycerol (ml)

1.6

1.6

1.6

1.6

1.6

1.6

1.6

1.6

1.6

1.6

Evaluation of transdermal patches of Lornoxicam: (a) Physical appearance: The prepared patches were physically examined for colour, clarity and surface texture. (b) Thickness uniformity7 : The thickness of patches was measured by using electronic caliper, with D OHDVW FRXQW RI PP 7KLFNQHVV ZDV PHDVXUHG DW WKUHH GLIIHUHQW SRLQWV RQ WKH ÀOP DQG average readings were taken. (c)

Uniformity of weight :The patch of size 1x1 cm2 was cut and weight of each patch was taken individually, the averageweight of the patch was then calculated.

(d) Tensile strength :Tensile strength of the patches was determined with the apparatus fabricated according to the description given in various articles. It consisted of two load cell grips. The ORZHU RQH ZDV À[HG DQG XSSHU RQH ZDV PRYDEOH 7KH WHVW ÀOP RI VL]H ð FP2 ZDV À[HG 75


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

EHWZHHQ WKHVH FHOO JULSV DQG IRUFH ZDV JUDGXDOO\ DSSOLHG WLOO WKH ÀOP EURNH 7KH WHQVLOH VWUHQJWK RI WKH ÀOP ZDV WDNHQ GLUHFWO\ IURP WKH GLDO UHDGLQJ LQ NJ (e) Folding endurance7 : The folding endurance was measured manually for the prepared patches. A strip of patch (2 x 2 cm2) was cut and repeatedly folded at the same place till it broke. The QXPEHU RI WLPHV WKH ÀOP FRXOG EH IROGHG DW WKH VDPH SODFH ZLWKRXW EUHDNLQJ JDYH WKH YDOXH of folding endurance. (f)

Percentage moisture loss7 :The patches were weighed individually and kept in a desiccators FRQWDLQLQJ FDOFLXP FKORULGH 7KH ÀQDO ZHLJKW ZDV QRWHG ZKHQ WKHUH ZDV QR FKDQJH LQ WKH weight of individual patch.

(g) Percentage moisture uptake8 :The patches were weighed accurately and placed in a desiccator where a humidity condition of 80-90 % RH was maintained by using saturated solution of potassium chloride. The patches were kept until uniform weight is obtained, then taken out and weighed. The percentage of moisture uptake was calculated as the difference between ÀQDO DQG LQLWLDO ZHLJKW ZLWK UHVSHFW WR LQLWLDO ZHLJKW (h) Drug content uniformity : The patches were tested for the content uniformity. The patches of size 1 cm2 ZDV FXW DQG SODFHG LQ D PO YROXPHWULF à DVN 7KH FRQWHQWV ZHUH VWLUUHG XVLQJ D PDJQHWLF bead for 24 hrs to dissolve the patches. Subsequent dilutions were made with phosphate buffer (pH 7.4). The absorbance of the solution was measured against the corresponding blank solution at 376 nm using UV-visible spectrophotometer. The experiment was repeated three more time to validate the result. (i)

In vitro release studies8 : The effect of different penetration enhancers on the permeation of /RUQR[LFDP DFURVV UDW VNLQ ZDV VWXGLHG XVLQJ 0RGLĂ€HG )UDQ]¡V GLIIXVLRQ FHOO 7KH IDEULFDWHG patches were cut into 1 cm2 SODFHG RQ WKH UDW VNLQ DQG DWWDFKHG WR WKH 0RGLĂ€HG GLIIXVLRQ FHOO VXFK WKDW WKH FHOO¡V GUXJ UHOHDVLQJ VXUIDFH WRZDUGV WKH UHFHSWRU FRPSDUWPHQW ZDV Ă€OOHG ZLWK 200 ml of phosphate buffer solution of pH 7.4 at 37Âą10 0C. The elution medium was stirred magnetically. The aliquots (5 ml) was withdrawn at predetermined time intervals and replaced with same volume of phosphate buffer of pH 7.4. The samples were analysed for drug content using UVspectrophotometer at 376 nm.

Results and discussions: Drug-excipient compatibility studies : As described previously,the Drug-polymer-and oil interaction studies was carried out using FTIR studies.The results have been given in table 1.0-1.9.From the IR spectra it was observed that the characteristic peaks of Lornoxicam were not affected in presence of the oils . Physical Parameters : $V HYLGHQW IURP 7DEOH QR WKH SUHSDUHG ÀOPV ZHUH VPRRWK DQG WUDQVSDUHQW translucent in appearance. There was not much difference observed in the thickness of the formulations. It was inferred that the concentration of the oils didnot have any effect on the thickness of the patches. The results of uniformity of weight, tensile strength and folding endurance has also been depicted in Table 2. From the readings of the folding endurance, it was evident that all the formulations have JRRG ÀOP SURSHUWLHV 7KH UHVXOWV RI WKH SHUFHQWDJH PRLVWXUH DEVRUSWLRQ VKRZHG WKDW DOO WKH SDWFKHV are within acceptable limit.

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7DEOH (YDOXDWLRQ RI GLIIHUHQW EDWFKHV RI /RUQR[LFDP WUDQVGHUPDO ÀOPV Batch no

Thickness (mm) ±SD

Wt variation (g) ±SD

% Drug content ±SD

Folding endurance ±SD

Tensile str. (kg) dyne/cm2

% Elongation

Absorbed

F-1

0.48±0.031

0.071 ± 0.006

94.24 ± 0.2

88 ± 12.6

0.76

12.33%

0.33%

F-2

0.39±0.023

0.073 ± 0.002

95.45 ±.0.5

70 ± 11.3

0.33

16.02%

0.39%

F-3

0.46±0.072

0.074± 0.003

94.33 ± 0.1

87 ± 12

0.63

14.74%

0.37%

F-4

0.44±0.082

0.074± 0.002

93.62 ± 0.4

91 ± 7.6

0.36

11.75%

0.39%

F-5

0.43±0.060

0.071± 0.004

96.19 ± 0.3

95 ± 1.50

0.75

16.03%

0.29%

F-6

0.38±0.023

0.076 ± 0.003

96.65 ±.0.5

77 ± 12.20

0.46

14.82%

0.34%

F-7

0.43±0.075

0.074± 0.005

96.74 ± 0.2

96 ± 0. 09

0.64

13.56%

0.39%

F-8

0.45±0.069

0.074± 0.006

93.58 ± 0.5

74 ± 5.60

0.46

13.35%

0.29%

F-9

0.42±0.070

0.076± 0.005

95.55 ± 0.4

86 ± 5.50

0.45

14.44%

0.35%

Fig 1: IR spectra of pure drug Lornoxicam

Moisture

Fig 1.1: IR spectra of Lornoxicam and Fennel oil

Fig 1.2: IR Spectrum of Lornoxicam and Rosemary Oil

Fig 1.3: IR Spectrum of Lornoxicam and Linseed Oil

Drug content uniformity: The drug content from the transdermal patches was determined from the calibration curve of Lornoxicam in Phosphate buffer pH 7.4. Drug content of the patch was carried out to ascertain that the 77


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

drug is uniformly distributed into the formulation. The results obtained have been depicted in Table 2. From the results obtained (i.e., lower S.D. values), it was clear that there was uniform distribution of /RUQR[LFDP LQ WKH ÀOP IRUPXODWLRQV +HQFH LW ZDV concluded that drug was uniformly distributed in all the formulation. In vitro release studies: The in vitro release VWXGLHV ÀJ VKRZHG WKDW %DWFK ) FRQWDLQLQJ 5%w/v fennel oil) showed the maximum percentage GUXJ UHOHDVH RI ZKLFK ZDV VLJQLÀFDQWO\ )LJ 'LVVROXWLRQ SUR¿OH RI /RUQR[LFDP WUDQVGHUPDO 3DWFKHV higher than the batch F0(without any oil) as shown (Batches F0-F9) in Fig 2. The maximum percentage drug release shown by rosemary and linseed oil was 78.93 and 67.93 respectively. It can be observed that. for both Fennel oil and Rosemary oil the maximum drug release was at 5%w/v where as for Linseed oil, the maximum drug release was at the concentration of 3% w/v. The percentage drug release of the Batch F2 which showed maximum drug release and batch F0 was statistically analysed using independent t WHVW DW FRQÀGHQFH LQWHUYDO 7KH S YDOXHV REWDLQHG ZDV S DQG I YDOXH 2Q WKH EDVLV RI S DQG I YDOXHV LW ZDV REVHUYHG WKDW WKHUH ZDV VLJQLÀFDQW GLIIHUHQFH EHWZHHQ WKH SHUFHQWDJH drug release from batch F2 and batch F0. among them. CONCLUSION 7KH DLP RI WKH SUHVHQW ZRUN ZDV WR LQYHVWLJDWH WKH SHQHWUDWLRQ HIÀFLHQF\ RI IHQQHO URVHPDU\ DQG linseed oil on the transdermal patches of Lornoxicam. In the recent years the chemical penetration enhancers have been replaced by penetration enhancers of natural origin. The patches thus prepared PD\ LQFUHDVH WKH HIÀFDF\ RI /RUQR[LFDP IRU WKH WKHUDS\ RI DUWKULWLV DQG RWKHU SDLQIXO PXVFXODU FRQGLWLRQV REFRENCES

$QQD :RNRYLFK 0 7UDQVGHUPDO GUXJ GHOLYHU\ V\VWHP 7''6 DGKHVLRQ DV D FULWLFDO VDIHW\ HIĂ€FDF\ DQG TXDOLW\ attribute, European Journal of Pharmaceutics and Biopharmaceutics, 2006; Vol.64, No.1, 1-8.

2.

Cleek R.L, Bunge A.L, A new method for estimating dermal absorption from chemical exposure. General approach, Pharma Res, 1993;Vol.10, 497–506.

3.

Jain, N. K., Controlled and Novel Drug Delivery, CBS Publishers, and Distributors, edition 107, 2002.

4.

Shivakumar HN et al, Formulation characterization and evaluation of matrix-type transdermal patches of a model antihypertensive drug�. Asian J Pharm 2002; 3:59-64.

5

Ubaidulla U et al,Transdermal therapeutic system of carvedilol: effect of hydrophilic and hydrophobic matrix on in vitro and in vivo characteristics. AAPS PharmSciTech 2002; x; 8(1):E13-15.

6.

Rao YM et al, Development of nitrendipine transdermal patches for in vitro and ex vivo characterization�. Current Drug Delivery, 2007; volume-4:69-76.

7.

Ubaidulla U et al,Transdermal therapeutic system of carvedilol: effect of hydrophilic and hydrophobic matrix on in vitro and in vivo characteristics. AAPS PharmSciTech 2002; x; 8(1):E 15-20.

8.

Devi KV et al, Design and evaluation of matrix diffusion controlled transdermal patches of verapamil hydrochloride. Drug Dev Ind Pharm x, 2008;29(5):495-503

‰ ‰ ‰ 78


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

Instructions to Authors PHARMBIT LV DQ RIĂ€FLDO VFLHQWLĂ€F MRXUQDO DQG ELDQQXDO SXEOLFDWLRQ RI 3KDUPDFHXWLFDO 6RFLHW\ RI Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi. The journal is devoted to publish review and research articles in pharmacy and the related disciplines of pharmaceutical education. PHARMBIT is abstracted in Chemical Abstract, USA and Natural Science Database, USA since 2008; making our publication global. Manuscripts will be subjected to peer review process to determine their suitability for publication SURYLGHG WKH\ IXOĂ€OO WKH UHTXLUHPHQWV RI WKH MRXUQDO $IWHU UHYLHZHU¡V FRPPHQWV WKH UHYLVHG PDQXVFULSW should be submitted by e-mail or in CD prepared in MS Word. Submission of a manuscript to PHARMBIT for publication implies that the same has not been either SXEOLVKHG RU XQGHU FRQVLGHUDWLRQ IRU SXEOLFDWLRQ LQ DQRWKHU MRXUQDO 7KH DXWKRU VKRXOG FRQĂ€UP GXULQJ submission of manuscript. PREPARATION OF MANUSCRIPTS : RESEARCH PAPERS Manuscripts should be concisely written and conform to the following general requirements: Manuscripts should be typewritten in 1.5 space in A4 sized sheets, only on one side, with a 1.0 inch margin on both sides. Research Papers, should not exceed 8-10 pages, Review Articles, 12-15 pages and Short Communications, 4-5 pages. Pages should be numbered consecutively, starting with the title page and the matter arranged in the following order: Title, Name and Address, Abstract, Keywords, Introduction, Materials and Methods, Results, Discussion or Results and Discussion, Acknowledgements and References. All other section titles should be in capital letters while subtitles in each section shall be in bold face lower case. TITLE PAGE - Title page should contain title of the paper in bold face, title case (font size 14), names of the authors in normal face, upper case (font size 12) followed by the address(es) in normal face lower case. The author to whom all correspondence be addressed should be denoted by an asterisk mark. ABSTRACT - Start on a new page after the title page and should be typed in single-space to distinguish LW IURP WKH ,QWURGXFWLRQ $EVWUDFWV VKRXOG EULHĂ \ UHĂ HFW DOO DVSHFWV RI WKH VWXG\ DV PRVW GDWDEDVHV OLVW mainly abstracts. Short Communications as well as Review Articles should have an Abstract. KEYWORDS – 4 to 5 Keywords related to topic INTRODUCTION - Start immediately after the Abstract, as the next paragraph, but should be typed in double-space. The Introduction should lead the reader to the importance of the study; tie-up published literature with the aims of the study and clearly states the rationale behind the investigation. MATERIALS AND METHODS - Start as a continuation to introduction on the same page. All important materials used along with their source shall be mentioned. RESULTS ² $OO Ă€QGLQJV SUHVHQWHG LQ WDEXODU RU JUDSKLFDO IRUP VKDOO EH GHVFULEHG LQ WKLV VHFWLRQ 7KH GDWD VKRXOG EH VWDWLVWLFDOO\ DQDO\]HG DQG WKH OHYHO RI VLJQLĂ€FDQFH VWDWHG 5HVXOWV VHFWLRQ VKDOO VWDUW DIWHU materials and methods section on the same page. DISCUSSION – This section should follow results, deal with the interpretation of results, convey how they help increase current understanding of the problem and should be logical. Results and discussion of results can also be combined under one section, Results and Discussion. 79


PHARMBIT Vol. XXIII & XXIV, No. 1 & 2, Jan - Dec, 2011

ACKNOWLEDGEMENTS - Should be given after the text and not in the form of foot-notes. REFERENCES 5HIHUHQFHV VKRXOG EH QXPEHUHG FRQVHFXWLYHO\ LQ WKH RUGHU LQ ZKLFK WKH\ DUH ÀUVW mentioned in the text (not in alphabetic order). Identify references in text, tables, and legends by Arabic numerals in superscript. ARTICLES IN JOURNALS „

KV Devi, RS Pai. Antiretrovirals: Need for an Effective Drug Delivery. Indian J Pharm Sci. 2006; 68:1-6.

BOOKS AND OTHER MONOGRAPHS „

Personal author(s): Ringsven MK, Bond D. Gerontology and leadership skills for nurses. 2nd ed. Albany (NY): Delmar Publishers; 1996.

„

Editor(s), compiler(s) as author: Norman IJ, Redfern SJ, editors. Mental health care for elderly people. New York: Churchill Livingstone; 1996.

„

Chapter in a book: Phillips SJ, Whisnant JP. Hypertension and stroke. In: Laragh JH, Brenner BM, editors. Hypertension: pathophysiology, diagnosis, and management. 2 ed. New York: Raven Press; 1995. p. 465-78.

ILLUSTRATIONS Tables and Figures - They should be inserted within the text. Tables should not be very large that they run more than one A4 sized page. Tables should be numbered consecutively in Arabic numerals and bear a brief title in lower case bold face letters above the table. Figures should be numbered FRQVHFXWLYHO\ LQ $UDELF QXPHUDOV DQG EHDU D EULHI WLWOH LQ ORZHU FDVH EROG IDFH OHWWHUV EHORZ WKH ÀJXUH PREPARATION OF MANUSCRIPTS: REVIEW ARTICLES If should be about 15 pages long, contain up-to-date information, comprehensively cover relevant literature and preferably be written by scientists who have in-depth knowledge on the topic. All format requirements are same as those applicable to full papers. Review articles need not be divided into VHFWLRQV VXFK DV PDWHULDOV DQG 0HWKRGV DQG UHVXOWV DQG 'LVFXVVLRQ EXW VKRXOG GHÀQLWHO\ KDYH DQ abstract and introduction, if necessary. PREPARATION OF MANUSCRIPTS: SHORT COMMUNICATIONS 7KH MRXUQDO SXEOLVKHV H[FLWLQJ ÀQGLQJV SUHOLPLQDU\ GDWD RU VWXGLHV WKDW GLG QRW \LHOG HQRXJK information to make a full paper as short communications. These have the same format requirements as full papers but are only up to 5 pages in length. Short Communications should not have subtitles such as Introduction, Materials and Methods, Results and Discussion – all these have to be merged into the running text. Short Communications preferably should have only 1-2 illustrations. Submission : Authors are required to submit their manuscript by post or by e-mail (rngupta@ bitmesra.ac.in and manik@bitmesra.ac.in). Note : The Editor does not claim any responsibility, liability for statements made and opinion expressed by authors. 80