GJRMI - Volume 1, Issue 3 - March 2012

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

Join Bodhi GJRMI, Volume 1, Issue 3,Nighantu…

March 2012, 62 - 68

An E- Herbal Encyclopedia, to share and contribute about Medicinal plants. Post a plant’s Photo and description directly from your mail and you will find people replying you with what they know about the plant & its Herbal uses in various systems of Medicine. It’s a free google group with more than 300 members & 3000 Active discussions & 2 years of hardship from scholars all over the World on Medicinal plants and its uses Bodhi nighantu helps you Share your knowledge, Learn new things about different plant species, Get new plants identified & discussed elaborately…

Just click the link and you are in the Google group to share and learn for free…

Bodhi Nighantu

To Advertise in the Flip book Cover page write to chiefeditor@gjrmi.com or Call - +919590574495 Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

An International, Peer Reviewed, Open access, Monthly E-Journal

ISSN 2277 – 4289 www.gjrmi.com Editor in chief Dr Hari Venkatesh K Rajaraman M.D.(Ay) P.G.D.H.M

Advisory Board

Prof. Rabinarayan Acharya M.D., Ph.D (Ay) Dr. Mathew Dan M.Sc., Ph.D (Botany) Dr. Tanay Bose M.Sc., (Ph.D) (Botany) Dr. Nagaraja T.M M.Pharm.,Ph.D (Pharm) Dr. Narappa Reddy M.D. (Ay)

Editorial board

Dr. Kumaraswamy

M.Tech. Ph.D (Bio-Tech)

Dr. Madhu .K.P M.D. (Ay) Dr. Sushrutha . C.K

M.D.(Ay)

Dr. Ashok B.K Dr. Janardhana.V.Hebbar M.D. (Ay) Dr. Shwetha Hari Venkatesh

B.A.M.S.

Dr. Vidhya Priya Dharshini. K.R. Mr. R. Giridharan Miss. Shyamala Rupavahini

B.N.Y.S.

M.Tech (Bio-tech) M.Sc., M.Phil (Bio-Chem)

Honarary Members - Editorial Board Dr. Shubha Ganguly M.V.Sc.,Ph.D Dr Farhad Mirzaei, M.Sc., Ph.D.,

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

Original Research Article MALARIA: NOVEL PLANT REMEDIES SHOW GREAT PROMISE IN TREATING THE DEADLY DISEASE

Taba K.M.*†, Paulus J. **, Kayembe J.S.* *

University of Kinshasa, Faculty of Sciences, Dept of Chemistry P.O. Box 190 Kinshasa XI, Democratic Republic of Congo **

University of Kinshasa, Faculty of Sciences, Dept of Biology P.O. Box 190 Kinshasa XI, Democratic Republic of Congo †

Corresponding Author - Tel. +243 81 333 02 42 tabakalulu@yahoo.fr

Received : 13/01/2012;

Revised : 17/02/2012;

Accepted : 27/02/2012;

ABSTRACT Clinical investigation of eight plant remedies used as traditional medicines in Kinshasa, D.R. Congo, to treat Malaria patients showed significant removal of parasites in the blood, as well as elimination of clinical detection of disease. The percentage recovery from Malaria depends on the type of remedy chosen: Cassia occidentalis Linn. (97%), Carica Papaya Linn. (94%), Cymbopogon citratus (DC.) Staff, 1906 (93%), Garcinia kola Heckel. (94%), Lantana camara L. (90%), Ocimum gratissimum L. (86%), Phyllanthus niruri L. (93%) and Vernonia amygdalina Delile. (67%). No identifiable side effects were noticed during and after treatment. In vitro study of alcohol extracts of these remedies showed an inhibition concentration of Plasmodium growth of 83% (12.5 µg/ml) for C. occidentalis Linn., 91% (25 µg/ml) for C. Papaya Linn., 93% (25 µg/ml) for C. citratus (DC.) Staff, 1906, 93% (25 µg/ml) for L. camara L., 100% (12.5 µg/ml) for O. gratissimum L., 100% (12.5µg/ml) for P. niruri L. and 0% (25 µg/ml) for V. amygdalina Delile. Secondary metabolites were isolated from some plant remedies and their IC50 determined in vitro on P. falciparum. The highest IC50 was observed in alkaloid extract of P. niruri L. (8 µg/ml); terpenes extract of O. gratissimum L. (0.27µg/ml); quinone extract of G. kola Heckel. (1.02 µg/ml) and flavonoid extract of G. kola Heckel. (1.5µg/ml).

Keywords: Malaria, Plant Remedies, Cassia occidentalis Linn.,Carica Papaya Linn., Cymbopogon citratus (DC.) Staff, 1906, Garcinia kola Heckel., Lantana camara L., Ocimum gratissimum L., Phyllanthus niruri L., Vernonia amygdalina Delile.

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

INTRODUCTION Malaria constitutes a major health problem to wide populations across the tropical and subtropical areas of the world. Due to increasing levels of drug resistance and unaffordable prices, many poor areas in Africa cannot access the newer chemotherapeutic pharmaceuticals (Jurg et al., 1991). For thousands of years, traditional medicines have been used to treat Malaria. Some herbal remedies have been the sources of two main groups of modern Anti-malarial drugs. Quinine from Cinchona species (Andrade-Neto et al., 2003) and Artemisinin from Artemisia species (Anonym, 1982; Li and Rieckmann, 1992; Wang and Xu, 1985). Quinine and Artemisinin have also been used as template molecules in the synthesis of other Anti-malarial drugs such as Amodiaquine from Quinine (Chen et al., 1987; O’Neil et al., 2003) as well as Artemether and Artesunate from Artemisinin (Pe et al., 1989; Yang, Ski, Li, 1982).

Collaborations with traditional healers are needed so that we may understand the use of ancestral medicines in modern practice.

Knowledge about efficacy and safety of herbal treatments provides the possibility of combating these diseases at primary healthcare level, and will also provide new leads for research on new Malaria therapies. Based on this, it was of high importance to access the efficacy, both clinically and in-vitro, of several antimalarial remedies used by traditional healers (Jurg et al., 1991).

An ethno-botanic survey conducted in gardens in the suburb of Kinshasa, the capital of D.R. Congo, led to identification of 58 species of plants used for treatment of fever (Malaria) (Ngalamulume et al., 1982; Kasuku et al., 1999). The area surveyed is the poorest urban environment of the city where most people are inclined to use traditional remedies to treat diseases. The eight most used remedies are: Cassia occidentalis Linn., Carica papaya Linn., Cymbopogon citratus (DC.) Staff, 1906, Garcinia kola Heckel., Lantana camara L., Ocimum gratissimum L., Phyllanthus niruri and Vernonia amygdalina Delile.

The current work focuses on the Anti-malarial activity of these remedies clinically by replicating the traditional healer’s therapeutic procedure in clinical trials. Furthermore, the in -vitro inhibition of Plasmodium falciparum growth with crude extracts is also being evaluated and documented.

Experimental Preparation of materials Eight plant materials were collected by traditional healers in the suburb of Kinshasa.

These were authenticated at Herbarium of the University of Kinshasa, where specimens are deposited. The decoction of each plant (except G. kola Heckel. of which seed is chewed) was prepared according to the traditional healer protocol. The dose and the mode of administration proposed by traditional healer are reported in Table 3.

200g of dried plant material was taken up with 1 liter of ethanol and refluxed for 6 hours.

Upon cooling, ethanol was removed under reduced pressure. The secondary metabolites, Alkaloids, Terpenes, Quinones and Flavonoids were obtained according to the classical procedure described by Bruneton (Bruneton, 1993). Thin layer chromatography analysis / preparation of extracts and fractions were performed on pre-coated plates (Silica gel 60 F254, MERCK). The spots were observed under UV (254 and 366 nm).

Clinical investigation Patients were treated with each traditional remedy according to the protocol of traditional healers at modern health centre. Patients’ selection was based on: (1) indications of malaria and of parasites P. falciparum trophozoites in Giemsa stained film prepared from capillary blood; (2) the absence of Antimalarial drug use; (3) age between 1 and 60 years (Pregnant women and severe sick patients were excluded) (Jurg et al., 1991).

All external signs of malaria including fever, Sweating, Vomiting, Nausea, Headache, Muscle and Joint pains, were recorded before

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

and during treatment. Blood samples were also taken at the starting and at days 5, 7, 9 and 11 of treatment. Asexual parasitaemia was quantified by counting against white blood

cells (number of asexual parasites/500 leucocytes × 8000 leucocytes/µl), (Jurg et al., 1991). The results of clinical investigation are reported in Table 1.

Table 1: Results of clinical investigation and in-vitro inhibition of Plasmodium’s growth by total alcohol extracts Clinical investigation Plants

No. of Patients 56

% of recovery 93

C. occidentalis Linn.

62

97

C. papaya Linn.

46

94

G. kola Heckel.

L. camara L.

39 50

94 90

O. gratissimum L. P. niruri L. V. amygdalina Delile.

Chloroquine ND*: Not determined

69 83 40

86 93 67

C. citratus (DC.) Staff, 1906

Test for in-vitro Anti-malarial activity The in-vitro assays were conducted by using the micro dilution technique of Desjardin (Desjardin et al., 1979). The P. falciparum parasites were derived by direct visualization and micro manipulation from fresh patient isolates. The test compounds were initially dissolved in ethanol: water mixture (1:3) or in Dimethylsulfoxide and diluted 100- fold in Roswell Park Memorial Institute 1640 (RPMI, Sigma Aldrich) culture medium, supplemented with 25mM Hepes and 32mM NaHCO3. These solutions were diluted in 10 different concentrations. The parasites were exposed to different dilutions of each compound for 48h and incubated at 37°C. Direct estimation of parasite growth inhibition was used and it was based on direct reading of smears made in 24well, flat-bottomed plates to estimate growth and evolution stages of the parasites (Benoit et al., 1996). Parasitaemia and parasite stage were

Alcohol extracts Conc (µg/ml)

% of inhibition

25 12.5 25 12.5 25 12.5 ND* 25 12.5 12.5 12.5 25 0.1

93 53 83 24 91 20 ND* 93 0 100 100 0 100

determined after 48h of contact between extracts and parasites. Concentration-response data was analyzed by nonlinear regression logistic dose response model and IC50 values for each compound were calculated.

RESULTS AND DISCUSSION Patients diagnosed with Malaria were treated at modern health centre via the technique of traditional healer for a period of 7 days. By the fifth day, all symptoms of Malaria disappeared in all cases. The level of Malaria parasite in the blood however did not completely disappear.

The percentage recovery was calculated based on both the amount of residual parasite in the blood, and the elimination of symptoms. The percentage of recovery ranged from 97% for C. occidentalis Linn. to 67% for V. amygdalina Delile. The observed high level of recovery showed that plant remedies are potential

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

candidates in the search of new molecules with potent biological activities (Jurg et al., 1991).

Further, it is necessary to look into classes of compounds responsible for Anti-plasmodial

activities observed during clinical investigation and in-vitro studies of these plant remedies.

The most likely classes are Alkaloids, Terpenes, Quinones and Flavonoids.

Table 2: IC50 values of isolated compounds Plant species C. papaya Linn.

C. papaya Linn.

G. kola Heckel.

G. kola Heckel.

G. kola Heckel.

G. kola Heckel.

G. kola Heckel.

G. kola Heckel.

G. kola Heckel.

O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. O. gratissimum L. P. niruri L. P. niruri L. EtOAc: Ethyl Acetate

Isolated compounds Total alkaloids Alkaloid Quinone Quinone Quinone Quinone Flavonoid Flavonoid Flavonoid Total alkaloids Alkaloid Alkaloid Alkaloid Terpene Terpene Terpene Terpene Terpene Terpene Terpene Total alkaloid Alkaloid PE: Petroleum Ether

Eluents

RF

EtOAc / n -hexane 1:1 PE/ EtOAc 1.2:1 PE/ EtOAc 1.2:1 PE/ EtOAc 1.2:1 PE/ EtOAc 1.2:1 CCl4/EtOH 1.8:1 CCl4/EtOH 1.8:1 CCl4/EtOH 1.8:1 EtOAc / n- hexane 1:1 EtOAc / n- hexane 1:1 EtOAc / n- hexane 1:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc/PE 4:1 EtOAc / n- hexane 1:1

0.5 0.17 0.25 0.42 0.54 0.15 0.45 0.64 0.20 0.70 0.90 0.06 0.14 0.21 0.37 0.47 0.59 0.87 0.23

IC50 value µg/ml 185 50 1.02 2.0 16.9 15.75 <1.5 3 10 190 <65 <75 250 0.32 0.27 1.41 3.96 0.44 0.65 0.52 100 <8

EtOH: Ethanol

Alkaloids: Alkaloids are one of the major classes of compounds possessing Anti-malarial activity (Ancolo et al., 2002; Kenny-Ang et al., 2000; Bringmann et al., 2000). One of the oldest and most important Anti-malarial drugs, Quinine belongs to this class of compounds and is still a very relevant therapy in the fight against malaria. A number of naturally occurring Alkaloids are reported to possess Anti-malarial activity against different malarial models. Crude Alkaloid extracts of C. papaya Linn., O. gratissimum L. and P. nururi L. were found to inhibit the growth of P. falciparum

with IC50 of 185µg/ml, 190µg/ml and 100µg/ml, respectively. The highest IC50 for alkaloids (8µg/ml) was obtained for the purified extract of P. nururi L. with an RF 0.23 (Ethyl acetate/Petroleum Ether, 4:1).

Terpenes: Artemisinin which is isolated from Artemisia annua, a sesquiterpene lactone endoperoxide, has been shown to possess strong Anti-malarial activity. It has prompted the investigation of some other naturally occurring terpenoids for their schizonticidal activity. Many were shown to affect the growth

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

of P. falciparum (Lin et al., 1987; Chukwejeku et al., 2005; Uys et al., 2002). Ethanolic extract of O. gratissimum L. revealed seven terpenes, three of which possessed strong Anti-malarial activity with RF of 0.06, 0.14, 0.47 and IC50 in µg/ml of 0.32, 0.27 and 0.44 respectively. The Ocimum species, commonly used in traditional medicine in many parts of the world contain Eugenol, Thymol and Geraniol as major volatile oil constituents, whereas the major flavones are Xanthomicrol and Cirsimaritin (Vieira R.F. et al 2001).

furnished four Quinones which were separated on Silica gel plates with RF of 0.17, 0.25, 0.42 and 0.54 and values of IC50 in µg/ml of 1.02, 2.0, 16.9 and 15.75 respectively. It will be of great interest to determine structure of at least the Quinone with the lowest IC50 value.

Flavonoids: The Anti-malarial activity from this class of compounds is not very common.

Nevertheless, Brandao et al. report that the presence of Flavonoids in Bidens pilosa explains its Anti-malarial activity (Brandao et al., 2004). Ethanolic extract of G. kola Heckel. gave three Flavonoids by preparative TLC which showed remarkable inhibition on the growth of P. falciparum with IC50 in µg/ml of 1.5, 3 and 10 respectively for RF of 0.15, 0.45 and 0.64.

Quinones: The structure of many naturally occurring Quinones is based on Benzoquinone, Naphtoquinone and Anthraquinone ring systems. Naphtoquinone has been highly active against P. falciparum in-vitro (Kapadia et al., 2001). Ethanolic extract of G. kola Heckel.,

Table 3: Traditional healer decoctions preparation and administered dosages Plants

Part

Daily dosage for adults

Treatment duration (days)

Leaves

Quantity used for decoction; 30min of boiling 10g in 1 l

C. citratus

1×2C

4

C. occidentalis

Aerial part without seed

100g in 1 l

1×1C

5

C. papaya Linn.

Leaves

200g in 1l

2×1C

5

G. kola Heckel.

Seeds

2×1S to chew

5

L. camara L.

Leaves

100g in 1 l

1×1C

5

O. gratissimum.

Leaves

250g in 1 l

1×2C

6

P. niruri L.

Aerial part

100g in 1 l

1×1C

5

V. amygdalina

Leaves

100g in 1 l

2×1C

5

C = 145ml;

1× or 2× = one or two times a day

S = Seed

CONCLUSION In the present study we showed that eight remedies used by traditional healers to treat

malaria possess significant Anti-plasmodial activity that was confirmed by inhibition of P. falciparum growth in-vitro of crude alcoholic extracts of these remedies. Some purified

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68

extracts of Alkaloids, Terpenes, Quinones and Flavonoids of the remedies are shown to inhibit P. falciparum growth in-vitro and should be responsible for the observed Anti-malarial activity. It is necessary to determine structures of these constituents in order to determine efficiently in-vivo therapeutic dose for human

therapy. We will be working with companies such as Cambridge Major Laboratories (Germantown, WI, USA) on full structural elucidation of the active constituents, and possible synthesis of the active ingredients for further testing.

REFERENCES

Bruneton J. (1993) : Pharmacognosie, Phytochimie, Plantes médicinales. Seconde Edition. Tec & Doc, Lavoisier Paris, 623–642.

(ISBN 2–85206–911–3).

Ancolo N., Azas V., Mahiou E., Ollivier C., Di Giorgio A., Keita P., Timonè-David G., Ballansard G. (2002): Antimalarial activity of extracts and alkaloids isolated from six plants used in traditional medicine in Mali and Sao Tomé; Phytotherapia Research, 16(7): 642– 649.

Andrade-Neto.

V.F., Brandao M.G.L,.

Stehmann J.A, Oliveira L.A. and Krettli A.U. (2003): Antimalarial Activity of Cinchonalike species plants used to treat fever and malaria in Brazil: J. Ethnopharmacol. 87 (2–3): 253–256.

Anonym (1982): China Cooperative group on Qinghaosu and its derivatives as antimalarials Antimalarial efficacy and Mode of action of Qinghaosu and its derivatives in experimental models. J. Tradit. Chin. Med. 2: 17.

Benoit F., Valentin A., Pelissier Y., Diafouka F., Marion C., Kone - Bamba D., Kone M., Mallie M., Yapo A., and Bastide J.M. (1996) : In vitro antimalarial activity of vegetal extracts used in West African Traditional Medicine; Am. J. Trop. Med. Hyg. 54(1): 67–71.

Bringmann G., Hamm A., Gunther C. Michel M., Brun R., and Mudogo V. (2000): Ancistrocalaine A and B, Two New Bioactive Naphtylisoquinolines and Related Naphtoic Acids from Ancistrocladus ealaensis. J. Nat. Prod., 63 : 1465–1470.

Chen E.H., Tanabe K., Saggio A.J., Nodiff E.A., (1987): Modification of Primaquine as Antimalarial. 4,5-Alkoxy Derivatives of Primaquine; J.Med. Chem. 30(7): 1193–1199.

Chukwejeku J.C., Smith P.S., Combes P.H., Mulholland D.A. ans Van Staden J.(2005): Antiplasmodial Diterpenoid from leaves of Hyptis suaveolens. J. Ethnopharmacol. 102(2): , 295–297.

Desjardins R.E., Canfield C.J., Haynes, Chulay J.D. (1979): Quantitative Assessment of Malarial activity in vitro by a semiautomatic Micro Dilution Technique. Antimicrob. Agents Chemother. 16: 710–718.

Jurg; T. Tomas and J. Pividal (1991): Antimalarial activity of some plant remedies in use in Marracuene, Southern Mozambique; J. Ethnopharmacol. 33: 79–83.

Kapadia G.J., Azuine M.A., Balasubramanian V., Sridhar R. (2001): Aminonaphthoquinones - a novel class of compounds with potent antimalarial activity against Plasmodium falciparum Pharmacol. Res. 43(4): 363–367.

Kasuku W.; Lula F.; Paulus J.; Ngiefu N.; Kaluila K. (1999) (in French) Contribution à l’Inventaire des Plantes utilisées pour le traitement du Paludisme à Kinshasa ; Rev. Pharm. Afr. 13 (1): 95–105.

Kenny- Ang K.H., Holmes M.J., Higa T., Hamann M.T. and Kara U.K. (2000): In vivo antimalarial activity of Beta-Carboline alkaloid

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 62 - 68 Chemotherapy.

antimalarials; J. Med. Chem. 46(23): 4933– 4945.

Li X., Rieckmann K. (1992): A bioassay for derivatives of Qinghaosu (artemisinin) Trop.

Med. Parasitol. 43: 195–196.

Pe T.M., Tin S., Lin S., Ya H., Win M. (1989): Clinical study of the treatment of cerebral malaria with Artemether (Qinghaosu derivative): Trans. R. Soc. Trop. Med. Hyg. 83: 72

Manzanine-A. Agents 44(6): 1645–1649.

and

Lin A.J., Klayman D.L., Milhous W.K. (1987): Antimalarial activity of New Water-soluble Dihydroartemisinin Derivatives. J. Med. Chem., 30: 2147–2150.

Ngalamulume T.; Paulus J., Nlandu L. Kizeka K. (1995) (in French) : Plantes Médicinales à usage domestique cultivées Dans deux quartiers Kinshasa. Rev. Pharm. Afr. 9 (1): 9–14.

Oliveira F.Q., Andrade-Neto V., Krettli A.U., Brandao M.G. (2004): New evidences of antimalarial activity of Bidens pilosa roots extract correlated with polyacetylene and flavonoids. J. Ethnopharmacol. 93(1): 39–42.

O’Neil P.M., Mukhtar A., Stocks P.A., Randle L.E., Hindley S., Ward S.A., Storr R.C., Bickley J.T., O’Neil L.A. et al. (2003): Isoquine and related Amodiaquine analogues: a new generation of improved 4- Aminoquinoline

Source of Support: Nil

Uys A.C.U., Malan S.F., Van Dyk S. and Van Zyl R.L. (2002): Antimalarial compounds from Parinari capsensis. Bio-organic. Med. Chem.

Lett. 12(16): 2167–2169.

Vieira R.F., Grayer R.J., Paton A., Simon J.E. (2001): Genetic of Ocimum gratissimum L. based on volatile constituents, flavonoids and RAPD markers. Biochem. System. Ecol., 29: 287–304.

Wang T., Xu R. (1985): Clinical studies of Treatment of Plasmodium falciparum malaria with Artemether: A derivative of Qinghaosu. J. Tradit. Chin. Med., 5: 240–242.

Yang Q., Ski W., Li R. (1982): The antimalarial and toxic effect of Artesunate on animal models. J. Trad. Chin. Med. 2: 99–103.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

Original Research Article PHARMACOGNOSTICAL AND PHYTOCHEMICAL EVALUATION OF VIGNA UNGUICULATA LINN. (KULATTHA) SEED Kolhe Rasika1, Acharya Rabinarayan2 , Bhide Bhargav3, Harisha CR4, Shukla VJ5 1 2 3 4 5

PG Scholar, Dravyaguna Department, IPGT&RA Gujarat Ayurved University, Jamnagar.

Associate Professor, Dravyaguna Department, IPGT&RA Gujarat Ayurved University, Jamnagar.

PhD scholar Dravyaguna Department, IPGT&RA Gujarat Ayurved University, Jamnagar.

Head, Pharmacognosy laboratory, IPGT&RA Gujarat Ayurved University, Jamnagar.

Head, Pharmaceutics laboratory, IPGT&RA Gujarat Ayurved University, Jamnagar. *Corresponding Author - email - dr.rasika_kolhe@yahoo.com mob.no.09374333651

Received: 03/02/2012;

Revised: 17/02/12;

Accepted: 29/02/12;

ABSTRACT Kulattha (Vigna unguiculata Linn, Papilionaceae), one of the seed drugs described under dietetic group, is being used as both drug and diet, in different classical texts of Ayurveda. It is one of the drugs of choice for the management of urinary calculus (Ashmari). Though used as a source of both drug and diet, it is reported as a major causative factor of acid peptic disorder (Amlapitta). Seed of V.unguiculata can be identified microscopically by the presence of rhomboidal crystals, simple starch grains with hilum. Purity test shows loss on drying (91.89% w/w), total ash (4.89% w/w), acid insoluble ash (1.22% w/w), alcohol soluble extractive (1.31% w/w) and Water-soluble extractive (1.94% w/w). Preliminary analysis revealed the presence of starch, tannin and amino acid. HPTLC study of its methanolic extract showed the presence of four and seven spots in short and long UV respectively. The information generated by this study provides relevant Pharmacognostical and Physico-chemical data needed for proper identification and authentication of the seeds of this particular species.

Key words: Kulattha, Pharmacognosy, Phyto-chemistry, HPTLC Abbreviations: Âľm - micrometer

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

INTRODUCTION Kulattha (Vigna unguiculata Linn.

Papilionaceae) is found throughout India from Punjab and Himalaya to Sikkim in the North (where it ascends to over 1500 m), the upper Gangetic plain, Central and South India till Kanyakumari. (Lakshmi1996). It is known as Horse gram In English, Kulathi in Hindi, Mudiraa in Malayalam, Kalathi in Gujarati, Kulitha in Marathi. (Anonymus, Database on Medicinal Plants 2008). It is extensively cultivated in Dehradun, Bengal, Chotanagpur, Deccan, and Coromandel Coast of Kerala as a food crop especially for Horses and Cattles as well as for its seeds which are considered nutritious. (Lakshmi, 1996) Classical text of Ayurveda, Charaka Samhita described it as one of the drug/diet causing Acid Peptic Disorders (Amlapitta) (Charaka Sutrasthana 25/40).

Sushruta Samhita highlights its Anti-lithiatic (Shukrashmari) activity. (Sushruta Sutrasthana 46/37) In recent study V.unguiculata shows to have better results than the use of conventional potassium citrate in recurrence of renal calculus and can be used to reduce the recurrence of calcium oxalate stone. (Singh et al. 2010) Seed’s extract also shows Anti-oxidant and Anti-free radical activities. (Hazra B et al. 2009) Plant morphology It is a Sub-erect downy to rarely glabrescent annual herb growing upto 30–60cm or more in height with a short erect stem and several elongate, diffuse, Sub-erect or at times twining branches. The young shoot usually covered with epidermal hairs, bearing pinnately trifoliate alternate stipulate leaves having ovate oblong or ovate lanceolate entire membraneous stipellate leaflets 2.5 cm or more long, very small pedicelled pale yellowish racemes axillary, with 2–6 flowers clustered at top of rachis and compressed, linear, falcate to much

curved, four to six seeded pods 3.85 cm long and 6–9 mm wide (J.S. Gamble 1997).

Fruit: 4–6 seeded, with trichomes or downy linear, broadly linear falcate or scimitar shaped to much recurved (very slightly so in wild variety) compressed pod. Measuring 3.8–5cm long and 6–9mm broad tipped with the persistent style. Seeds reniform, compressed with a shiny hard testa of various colours, mostly reddish brown, grey black as well as mottled. Cotyledon orbicular to cordate and persists for a long time on seedling.

Though used extensively as a drug and diet, the detailed pharmacognostical and preliminary phytochemical characters of the seeds are not reported anywhere. Hence, in this article an attempt has been made to study the morphological and microscopical characters of the seed and its powder along with preliminary phytochemical characters including HPTLC study.

MATERIAL AND METHODS Kulattha seeds were purchased from the local market in the month of March, authenticated by Pharmacognosy laboratory of IPGT&RA Jamnagar. Voucher herbarium specimen along with crude drug sample was preserved in Pharmacognosy laboratory, vide Ref No. 6036 March 2011. Pharmacognostical evaluation of Kulattha seeds including Histo-chemical studies were carried out by taking free hand sections. Powder microscopy of seeds was carried out following standard procedures (Wallis 1985) and the slides for powder microscopy were separately prepared first with distilled water, then stained with Phloroglucinol and concentrated HCl. Photomicrographs were taken using Carl Zeiss Binocular Microscope attached with Camera.

Histo-chemical tests were carried out by taking thick sections following the standard procedure

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

methods (Krishnamurthy 1988). Physicochemical parameters and chemical screening (Anonymous, Planner Chromatography 1999) studies were carried out following standard procedures. (Anonymous, API 2004) The methanol extract obtained during Physicochemical parameters was used for HPTLC.

Sample was prepared by 30min sonication of drugs with methanolic medium and filtrate was used for experimental task. ‘Benzene’ was selected as the mobile phase. Chromatographic conditions were as follows. (Anonymous, Planner Chromatography 1999)

Chromatographic conditions Application mode Development Chamber Plates Chamber Saturation Development Time Development distance Scanner Detection Data System

: : : : : : : : :

Camag Linomat V Camag Twin trough Chamber.

Precoated Silica Gel GF254 Plates.

30 min.

30 min.

7 cm.

Camag Scanner III. Deuterium lamp, Tungsten lamp Win cats software.

RESULTS AND DISCUSION

Microscopic Character: T.S. of Seed

Macroscopic Characters of seed

Detailed Transverse section passing through the center of the seed shows the testa having three layers, of which first and second layer were single celled, while the third layer consisted of several rows of thin walled narrow cells containing rhomboidal calcium oxalate crystals.

The outermost row namely the epidermis was composed of vertically elongated palisade like cells, each cell with slight constriction nearer its upper end, 45µm or more in height, 12– 15µm in width at their broader part, 3–6µm width at their narrow constricted part and 9– 12µm in width at the extreme tip. [Figure 1.3, 1.4] There was a thin covering of cuticle over the epidermis about 3µm in thickness. The palisade like cells were further characterized by the presence of narrow transverse light line at about two-third of their length from the base.

The second or Sub-epidermal row consisted of shorter and broader column like cells with their outer and inner ends broader and the middle

Seeds shape reniform, 5–6mm long, 3–4mm broad and 2–3 mm in thickness, compressed with a polished or shiny and hard brown coloured testa. The micropyle was situated near the hilum. The hilum was 1–1.5 mm in length.

The seed were exalbuminous. The testa was tough but comparatively thin except at the region of the hilum. The embryo which was exposed after removing the testa, by softening it through emersion of the seed in water, consists of two fleshy cotyledons, 5–6mm long and 4–5mm wide and an incurved radical which was 4mm long. [Figure 1.1] Organoleptic evaluation Organoleptic evaluation of powder of seeds of V. unguiculata revealed astringent (kashaya) taste, buff colour and characteristic odour while the texture was coarse. [Figure 1.2]

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

portion constricted. Cells with inter-cellular spaces had characteristic “hour glass” like shapes. They varied from 18–30µm in height and 12–15µm in width at the base, 6–9µm in width at the narrow middle constricted region and 15–18µm near the top. [Figure 1.5]. Third layer or zone was composed of 8–10 rows of thin walled narrow cells, some of which contained rhomboidal crystals that measured 30–60µm × 18–27µm, these parenchymatous cells were tangentially elongated with more or less oblique radical walls. These cells were

lacking the inter cellular spaces. At the region of the hilum, two rows of palisade like cells were present (instead of one row). Beneath, there was a group of sclerenchyma cells with narrow elongated pits on their walls that appeared as an elongate lanceolate patch in T.S. The Sub-epidermal cells were columnar, expanded beneath the hilum into a cushion in which these groups of sclerenchymatous cells appeared embedded, surrounded by two layers of narrow thin walled elongated parenchymatous cells. [Figure1. 6, 1.7]

Plate 1: Microscopic characters of V.unguiculata

Fig.1.1 Seeds of V.unguiculata Fig. 1.2 Seed Powder of Fig. 1.3 Rhombidal shaped calcium oxalate crystal V.unguiculata

Fig. 1.4 T.S of V.unguiculata Fig. 1.5 T.S. +cotyledon seed with testa

of

testa Fig. 1.6 cotyledon

Global Journal of Research on Medicinal Plants & Indigenous Medicine

T.S.

showing

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

Fig. 1.7 T.S. of cotyledon Fig. 1.8 Protein content stain with iodine

Fig. 1.10 Starch with tannin Fig. 1.11 Simple material grain with hilum Powder Microscopy Diagnostic characters of dried powder of V. unguiculata under the Microscope were prismatic crystals of Calcium oxalate from epidermis, dark brown coloured content; which was confirmed to be Tannin by adding Ferric chloride solution to it, which turned black; from Sub-epidermal region, simple starch

Fig. 1.9 Lignified fiber

starch Fig. 1.12 Crystals

grains with Hilum, Iodine stained starch grains, Loosely arranged parenchymatous cells.

Clumped masses may be protein content, with some Aleurone grains. [Figure 1.8 to1.12] Histochemical Test: The results of the various Histo-chemical tests carried out, to detect Lignin, Calcium, Starch and Tannin are depicted in table no. 1

Table -1 Histochemical evaluation of sections of V. unguiculata seed.

Material Section Section Section Section Section

Prismatic

Reagent Phloroglucinol+Conc HCl Phloroglucinol+Conc HCl KI Ferric chloride solution Conc.H2SO4

Test for Lignin

Result Present

Calcium oxalate Present crystals Starch Present Tannin Present Protein

Global Journal of Research on Medicinal Plants & Indigenous Medicine

Present

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

Phyto-chemical constituents While observing the physicochemical characters for purity test, the loss on drying was not more than 91.89% w/w, ash value was not more than 4.89 % w/w, acid insoluble ash was not more than 58.14% w/w, water soluble extractive was not more than 1.94 % w/w and the methanol soluble extractive was not more than 1.34 % w/w. [Table no.2] Qualitative analysis showed presence of tannin, starch,

amino acid and absence of alkaloids, cyanogenic glycosides and flavonoids. [Table no.3]. The methanol soluble extract was examined for high performance thin layer chromatography profile at 254nm frequency, using the solvent system of Benzen, Under high performance thin layer chromatography profile, at 254nm frequency four peaks were observed and at 366nm frequency seven peaks were observed. [Table no.4] [Plate 2]

Table-2 Showing the physicochemical parameters of V. unguiculata Parameter Observation Loss on drying 91.89 Ash value (%w/w) 4.89 Acid insoluble ash (%w/w) 1.22 Water soluble extract (%w/w) 1.94 Alcohol soluble extract (%w/w) 1.31

Table- 3 Showing the results of qualitative test for various functional groups in V. unguiculata Active constituents Tannin Flavonoids Starch Alkaloid Amino acid Cyanogenetic glycoside

Result Present Absent Present Absent Present Absent

Table- 4 HPTLC Profile of V. unguiculata Short UV 254 Long UV 366

No. of spots 4 7

Rf 0.02,.0.01,0.36,0.44 0.04,0.09,0.14,0.18,0.32,0.46,0.55

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

Plate 2 : Methanol soluble extractive Chromatogram of V.unguiculata

254 nm

366 nm

CONCLUSION Pharmacognostical and Phytochemical evaluation of seed and powder of Kulattha (V.unguiculata) market sample were found to be authentic and meet the standard parameters of API. Further Pharmacognostical findings i.e. rhomboidal calcium oxalate crystals, tannin, lignified fibres were important characters found during the study apart from what was

After spray

mentioned in API. Phytochemical screening and HPTLC results can be considered as standards for further research works.

ACKNOWLEGMENTS Author is thankful to the authorities of IPGT & RA, Jamnagar for providing facilities to carry out the research work.

REFERENCES Anonymous, Ayurvedic Pharmacopoeia of India (2004). Part I, Vol I, Appendix 2.1.4, 2.1.5 and 2.1.7 New Delhi: Government of India publication; Anonymous, Database on Medicinal Plants (2008) , Vol 5, NewDelhi; CCRAS publication ; p.no.123 Anonymous, Planner Chromatography, Modern Thin layer Chromatography, Switzerland (1999), pg. 2â&#x20AC;&#x201C;16 Anonymous, The Ayurvedic Pharmacopoeia of India (2004) Part I, Vol-III, Appendix 2.2.3, New Delhi: Government of India Publication; p.no.234

Gupta Ram Bhagawat (2003) Ayurveda Ka Pramanika Itihas, Chaukhamba Krishnadas Academy, Varanasi, p.no. 265 Gupta Ram Bhagawat, (2003) Ayurveda Ka Pramanika Itihas, Chaukhamba Krishnadas Academy, Varanasi, p.no.247 Hazra B, Sarkar R, Mandal S, Biswas S, and Mandal N, (2009). Studies on antioxidant and antiradical activities of Dolichos biflorus seed extract, African Journal of Biotechnology. 8 (16): 3682â&#x20AC;&#x201C;398 J.S. Gamble, Flora of the Presidency of Madras, Vol.1, 14 old Connaught place, Dehraduna, India; p.no.366

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 69 - 76

Kaviraj Kunjalal Bhishagratna,(2005) Sushruta Samhita, English translation, Vol I, Chaukhamba Sanskrit series office, Varanasi, Sutrasthana 46/36, p.no.455 Krishnamurty, K.V.(1988), Methods in the plant histochemistry, Vishwanadhan Pvt, Limited, Madras, p.no.1â&#x20AC;&#x201C;77.

Lakshmi N (1996) Pharmacognosy of Ayurvedic drugs Kerala, Pharmacognosy Unit, Ayurveda research institute, Poojapura,Thiruvananthapuram. p.no.31 Lakshmi N (1996) Pharmacognosy of Ayurvedic drugs Kerala, Pharmacognosy Unit,

Source of Support: Nil

Ayurveda research institute, Poojapura,Thiruvananthapuram. p.no.32 Rana Gopal Singh, Sanjeev Kumar Behura, Rakesh Kumar (2010), Litholytic Property of Kulattha (Dolichous Biflorus) vs Potassium Citrate in Renal Calculus Disease: A Comparative Study, JAPI May, Vol.58 Sharma P V,(2009) Charaka Samhita, English translation, Vol I, Chaukhamba oriantalia, Sutrasthana 25/40; p.no.169 Wallis TE. (1985) Textbook of Pharmacognosy, London: Churchill Publication ; p.no.572â&#x20AC;&#x201C;82

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Original Research Article RUBIA CORDIFOLIA LINN. (MANJISHTHA) IN PRIMARY DYSMENORRHOEA (KASHTARTAVA) - A CLINICAL STUDY Dhiman Kamini1*, Lata Kusum2, Dhiman K. S3.

1

Department of Stri Roga & Prasooti Tantra, IPGT & RA, Gujarat Ayurved University, Jamnagar Department of Stri Roga & Prasooti Tantra, Ayurvedic College, Hoshiarpur 3 Prof. & Head, Deparment of Shalakya, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: kd44ayu@yahoo.co.in

2

Received: 09/02/2012;

Revised: 21/02/2012;

Accepted: 05/03/2012;

ABSTRACT

Dysmenorrhoea is one of the most common gynaecologic complaints in young women who present to clinicians now a days. There are references that Rubia cordifolia L. (Manjishtha) was in use traditionally for Dysmenorrhoea in India and China since ancient times. On this basis a clinical study to evaluate its efficacy in Dysmenorrhoea (Kashtartava) was undertaken with a total number of 20 patients having been registered for the study. Manjistha Churna orally with luke warm water in a dose of 3 gms twice a day(12 hrly) was given for the duration of 2 months. Statistically highly significant results were observed in the study.

Key Words: Dysmenorrhoea, Kashtartava, Artavadushti, Manjishtha, Rubia Cordifolia L, Raktaprasadana

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

INTRODUCTION Menstruation is a normal physiological process indicating womanhood. It is a cyclical process which repeats every month and becomes an important physiological manifestation in a woman’s reproductive life Menstruation normally flows without pain and burning sensation and the flow is not unctuous.

(Charak 700 BC) When menstruation is associated with pain, it is termed as Dysmenorrhoea; the pain incapacitates day-today activities of a female (Dutta D. C. 2007).

Dysmenorrhoea is a major cause of absenteeism from work amongst women thus decreasing efficiency and quality of life among affected Women (Balbi et al., 2009). When painful menstruation is present in Women with normal pelvic anatomy, is defined as Primary Dysmenorrhoea, usually begins during adolescence (Wentz Anne Colston1985).

Affected women experience sharp, intermittent spasm of pain usually concentrated in the supra pubic area. Pain may radiate to back of the legs (Thigh region) or the lower back. Systemic symptoms include Nausea, Vomiting, Diarrhoea, Fatigue, mild Fever and Headache or Light headedness (Dutta D. C. 2007).

Painful menstruation with pelvic pathology is defined as Secondary Dysmenorrhoea (Shah PK et al., 2011).

Aartava and Menstruation both convey the same meaning. ‘Kashta’ means painful, difficult & ‘Aartava’ means menstruation.

Hence, Kashtartava refers to “Kashtena munchyati iti Kashtartava” i.e. the condition where in Aartava is shed with pain is termed as Kashtartava (Dysmenorrhoea). In Ayurvedic texts, though Kashtartava (Dysmenorrhoea) is not separately described as a disease but there are many diseases in which Kashtartava has been mentioned as a symptom.

Dysmenorrhoea is one of the most common gynaecological complaints in young women who present to clinicians now a days. Modern life style changes, faulty dietary habits and stress seem to be few important causes for Dysmenorrhoea. In this today’s high-tech era, where the females are in par with male in getting education or in their profession, don’t

take chance of skipping their duty in every cycle and also reduced pain threshold in females is one of the reason for increased reporting of incidence of dysmenorrhoea.

Analgesics and NSAIDS which are active inhibitors of PGs synthesis are used to combat with pain during Dysmenorrhoea but these drugs produce side effects on long term use (Campbell, et al., 1997). Hence it is a need of the day to find safe and effective remedy for this problem.

It is a well-known fact that Ayurvedic System of medicine always played an important role in meeting the global health care needs which is true even in the case of management of dysmenorrhoea. In recent times, focus on plant research has got increased all over the world.

Rubia cordifolia Linn. (Rubiaceae), popularly known as Indian Madder or majit or manjishtha is also one of the important drugs which is time tested. It is a perennial, herbaceous climber with very long, cylindrical roots with a thin red bark (Kirtikar & Basu 1980). Stems are long, rough, grooved and become slightly woody at the base. It is found throughout the hilly districts of India from North-west Himalayas Eastwards, ascending to 8000ft. and Southwards to Ceylon. Generally, root, leaves, fruits, stem etc. of the plant Rubia cordifolia are used for their therapeutic properties. The roots of this plant are of high medicinal value and are traditionally used as Anti-inflammatory, Astringent, Tonic, Antiseptic, Diuretic, Anti-dysenteric, Blood purifier, Anti-helminthic, Analgesic, Hepatoprotective etc [(Kirtikar & Basu (1980), Williamson Elizabeth (2002), Khare (2004), Anonymous (2005)]. There are references that Rubia Cordifolia (Manjishtha) was in use traditionally for Dysmenorrhoea in India and China [Guangzhou (1992) Hocking (1997)].

But till date there are no scientific references available of any study using Manjishta as a single drug in Dysmenorrhoea, keeping this fact in mind present clinical study had been undertaken to evaluate and establish its efficacy in Dysmennorhoea (Kashtartava).

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

MATERIAL AND METHODS Materials: Here, for the clinical study dried root of Manjishtha(Rubia cordifolia L.) were collected and identified in the Dravya Guna Department, and then fine powder was done in the Pharmacy of RGGPG Ayurved College, Paprola (HP).

Methods: Clinical Study: Patients attending the OPD of Prasooti Tantra and Stree roga at Rajiv Gandhi Govt. Ayurvedic Hospital, attached with Rajiv Gandhi Govt. Post Graduate Ayurvedic College, Paprola Distt Kangra (H.P.) with characteristic features of Kashtartava (Dysmenorrhoea) were selected for the present study. The patients were selected and registered irrespective of their caste, creed, religion, income, occupation, etc.

Study Design: Single blind, prospective observational study.

Plan of the Study Inclusion Criteria: 1. Patients of the age group of 10â&#x2C6;&#x2019;50 yrs 2. Patients presenting sign and symptoms of Kashtartava(Dysmenorrhoea) Criteria for Exclusion: 1. Patients not willing for trial 2. Patients having irregular periods 3. Patients having heavy and excessive periods 4. Patients having any anatomical anomaly of Reproductive System 5. Patients suffering from any systemic disease 6. Patients having conditions where surgical intervention was needed Method of Study: - This Clinical study was accomplished in three phases: i) Diagnostic Phase, ii) Interventional Phase and iii) Assessment Phase Diagnostic Phase: Total 20 patients were diagnosed on the basis of signs and symptoms (Clinical presentation) of Dysmenorrhoea (Kashtartava)and selected for the study after following inclusion and exclusion criteria. The nature of the study was explained to all the selected patients and their consent (informed consent) was obtained. A special clinical Proforma was prepared incorporating both Ayurvedic (Dashavidha Pareeksha) and Modern parameters. A detailed history was taken and complete physical examination and laboratory investigations were also carried out.

Subjective Criteria adopted for the present study were â&#x20AC;&#x201C; Intensity of Pain Duration of Pain

Nausea Vomiting Breast Tenderness Fever Headache Vertigo Diarrhoea Anorexia & Nervousness

Objective Parameters: For the purpose of assessing the general condition of the patient and to exclude the other pathologies routine Haematological and USG (Abdomen & Pelvis) examination was carried out. After arriving at the diagnosis, clinical Proforma was filled up, which incorporated all the signs and symptoms based on both Ayurvedic as well as Modern Parameters. A detailed clinical history was taken initially and complete physical and Gynaecological examination of each patient

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

was carried out and the same was recorded in the proforma.

Before Treatment (BT) and After Treatment (AT).

Interventional Phase: The study was intervened by the treatment with Powder of Rubia Cordifolia L.( Manjistha Churna).

Clinical Assessment: The criteria adopted for intensity of pain was VAS (Visual Analogue Scale). Visual Analogue Scale (VAS) is a measurement instrument that tries to measure a characteristic or attitude that is believed to range across a continuum of values and which cannot be easily and directly measured.

Drug Schedule: Manjistha Churna (Powder of Rubia cordifolia L) orally with luke warm water in a dose of 3gms twice a day (12 hrly) was given for a duration of 2 months.

Duration of Trial: The total duration of treatment for the subjects was two months.

Follow Up Study: Follow up was conducted after one month during trial and then after the completion of trial.

Assessment Phase: The effect of treatment (results) was assessed regarding the clinical signs and symptoms (on the basis of VAS and grading, scoring system) and the overall improvement was observed and recorded as

Operationally a VAS is usually a horizontal line, 100 mm (10 cm) in length, anchored by word descriptor at each end, as illustrated in figure-1.

The patient marks on the line the point that they feel represent their perception of their current state. The VAS score is determined by measuring in millimetres from the left hand end of the line to the point that the patient marks.

The signs and symptoms were assessed by adopting suitable scoring methods. The details are illustrated in Table 1.

Figure â&#x20AC;&#x201C; 1, Visual Analogue Scale (VAS)

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Table -1 Showing grading of Pain in Patients Symptoms 1. Duration of pain 2. Nausea 3.Vomiting 4.Breast tenderness 5. Fever

0 No pain

1 Upto 24 hr.

2–3 Absent times/day Absent Occasionally No Mild Tenderness Tenderness No fever

Mild fever at night

6. Headache 7. Vertigo

Absent Absent

Mild Occasionally

8. Diarrhoea 9. Anorexia 10. Nervousness

Absent Absent Absent

Occasionally Mild Mild

Statistical Analysis: The obtained data were analyzed statistically and presented as Standard Error of Mean. The observed difference was calculated by adopting student’s “t” test (Significance level: ≥0.05).

Overall Assessment of Therapy: To assess the overall effect of therapy following criteria was laid down.

Completely Cured: More than 90% relief in symptoms and signs as well as one (1) score obtained according to VAS. Markedly Improved: More than 75% and less than 90% relief in symptoms and signs as well as > 1–5 change of score are according to VAS. Moderately Improved: More than 50% and less than 75% relief in symptoms and signs as well as 5–9 change of score according to VAS. No improvement/ Unchanged: Less than 25% relief in signs and symptoms and score greater than 9. The total effect of therapy was assessed on the basis of the above first three criteria.

2 Upto 48 hr.

3 Upto 72 hr.

4–5 times/day

>5 times/day

1–2 times/day Moderate tenderness Moderate fever throughout the day Moderate 2–3 times in 1– 2 days

>2 times/day Severe Tenderness

2–3 times/day Moderate Moderate

Severe fever Severe More than 4 times in 3–4 days >3 times/day Severe Severe

RESULTS Total 20 patients were registered, among them 65.00% patients belonged to age group of 18– 25 years and 70% were unmarried. Most of the patients (70.00%) were students and maximum (75.00%) were of lower middle class. All the patients were of Hindu religion and from rural area. Maximum patients(85.00%) were vegetarian; 80.00% patients had spicy food as their dietary habits and 20.00% were on non spicy diet. 45.00% patients were used to consume Amla rasa and 40.00% were used to consume Lavana rasa dominantly. 85.00% patients had disturbed sleep due to pain and 15.00% had normal sleep pattern. Maximum number (90.00%) of patients had Menarche between 13–15 years. 40% patients had 4–5 days, 35.00% and 25.00% had bleeding P/V for 3 days & more than 5 days respectively.

85.00% patients had interval of 26–30 days, 15.00% patients had 31–35 days interval in between two Menstrual cycles. The amount of blood loss was scanty (75.00%) in maximum number of patients & moderate flow in rest (25.00%) of the patients. Maximum number of patients (07.00%) were having spasmodic pain and 25.00% were having dull ache. Maximum number of patients had radiation of pain to

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

thighs (50.00 %), to abdomen (45.00%) and 05.00% in the back. 60.00% patients were having Nausea, 30.00% patients had Anorexia, 25.00% were having Fever, 10.00% had Headache, and 05.00% had vertigo as

associated Symptoms. Majority were of Vata Pittaja (85.00%), Pitta Kaphaja (10.00%) & Vata kaphaja prakriti (05.00%).

Table -2 STATISTICAL ANALYSIS OF EFFECT OF THERAPY RUBIA CORDIFOLIA L. (MANJISTHA CHURNA) Symptom

N

Relief Diff. In % 6.98 77.82 1.05 80.77

SD+

SE+

t

p

20 20

Mean score B.T. A.T. 8.97 1.99 1.3 0.25

Intensity of pain Duration of pain Associated Symptoms Nausea Vomiting Tenderness Fever Headache Vertigo Diarrhoea Anorexia Nervousness

0.597 0.2236

0.133 0.499

52.48 21.00

<0.001 <0.001

20 20 20 20 20 20 20 20 20

0.7 0.05 0.25 0.1 0.05 0.05 0.3 -

0.7 0.05 0.25 0.1 0.05 0.05 0.3 -

0.6569 0.1468 4.768 <0.001 0.2236 0.05 1 >0.05 0.4442 0.0993 2.51 <0.05 0.3077 0.06882 1.4530 >0.05 0.2236 0.05 1 >0.05 0.2236 0.05 1 >0.05 0.4701 0.105 2.857 =0.01 -

0 0 0 0 0 0 0 -

Intensity of pain: The initial mean score of intensity of pain was 8.97 before the treatment which was reduced to 1.99 after the treatment.

The percentage of relief was 77.82% which is significant statistically at the level of p<0.001 (t=52.48).

Duration of pain: The initial mean score of duration of pain was 1.3 before the treatment which was reduced to 0.25 after the treatment.

The percentage of relief was 80.77% which is significant statistically at the level of p<0.001 (t=21.00) Nausea: The initial mean score was 0.7 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100% which is not significant statistically at the level of p<0.001 (t = 4.768).

Vomiting: Was not reported in any patient.

Breast tenderness: The initial mean score was 0.05 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100% which is not significant statistically at the level of p>0.05 (t=1).

100 100 100 100 100 100 100 -

Fever: The initial mean score was 0.25 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100% which is significant statistically at the level of p<0.05 (t=2.51).

Headache: The initial mean score was 0.1 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100% which is not significant statistically at the level of p>0.05 (t=1.4530).

Vertigo: The initial mean score was 0.05 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100% which is not significant statistically at the level of p>0.05 (t=1).

Diarrhoea: The initial mean score was 0.05 before the treatment which was reduced to 0 after the treatment. The percent of relief was 100% which is not significant statistically at the level of p >0.05 (t=1).

Anorexia: The initial mean score was 0.3 before the treatment which was reduced to 0 after the treatment. The percentage of relief was 100%, which is significant statistically at the level of p=0.01 (t=2.857).

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Nervousness: Was not reported even in a single patient.

Overall Results The result was assessed on the basis of VAS and Grading, Scoring system 14(70%) patients were completely cured, 1(5%) patient markedly improved & 5 (25%) were moderately improved.

DISCUSSION In Ayurvedic classic Kashtartava is not considered as a separate disease however mentioned as a symptom of various diseases (Charak 700 BC) like Vatala, Sannipatika and Udavarta Yonivyapad.

Commentator Chakrapani (Charak 700 BC) says that any symptom may manifest as a separate disease Acharya Sushruta while depicting the importance of Shuddha Artava (Menstrual blood) has assigned a separate chapter in Sharira Sthana and has quoted Kashtartava as a type of Artava Dushti (Menstrual Disorder).

According to Acharya Kashyapa, the blood in adult females during their reproductive period enters into Garbha Kosha (Uterus) every month and Rajovaha Shiras (vessels carrying menstrual blood) in the uterus carries the Artava (Menstrual blood) formed by the action of Artavagni upon the Rakta (Blood) and fill the uterus in one month and after that this Artava (menstrual blood) is expelled out by these Shiras (blood vessels) at the interval of one month (Charak 700BC).

In Sushruta Samhita, (Sushruta 600 BC) characteristics of Shuddha artava (Menstrual blood) is mentioned as â&#x20AC;&#x153;Raktalakshanamâ&#x20AC;?(having characteristics of blood) and it prepares the Garbhashaya (Uterus) to receive the fertilized egg as well as for the growth and development of the foetus21.

All these references reflect that Artava (Menstrual blood) is having similar qualities of Rakta (Blood) hence if Rakta (Blood) is pure and having no any impurities there will not be any disorder related to Artava (Menstruation) but if Rakta vitiates there will be artavadushti (Menstrual disorder) too which results in

Gynaecological disorders like Kashtartava (Dysmenorrhoea).

From the observed results on signs and symptoms, it can be revealed that Rubia Cordifolia L.(Manjishtha), which is Raktaprasadak dravya (Blood purifying agent) eliminates and nullifies all types of impurity, toxicity, contamination and harmful effect of unwanted material from blood and restores its health (Brahmashankara Mishra, 1993). As maximum patients were having a tendency to consume Amla (sour) and Lavana rasa (salty taste) predominantly which are responsible to cause Raktadushti (vitiation of blood).

Furthermore, Artavadushti (Menstrual disorder (Charak 700 BC) itself manifests as Kashtartava (Dysmenorrhoea) and to alleviate dushti (impurities), prasadana (purification) is much supportive.

Raktavardhaka (Blood enhancing) property of Manjishtha (Rubia Cordifolia L.) improves pain threshold by improving the Immunity. The biological investigations have shown that many of the medicinal properties claimed for Rubia Cordifolia (Manjishtha) in the historical texts have sound scientific basis (Singh et al., 2005).

It has a variety of uses such as blood purifying etc. (Joharapurkar et al., 2003). Intensity and duration of pain was observed to be reduced in maximum patients. Anti-inflammatory and Analgesic activity of Rubia cordifolia is quoted by many Researchers. [Mhaskar, Blatter & Caius (2000), Mooradian (1988), Kasture, Deshmukh(2000), Khare (2007)].

Rubia cordifolia L. is said to contain substantial amount of Anthraquinones, especially in the roots which is responsible for its pharmacological activities (e.g. Antiinflammatory and Analgesic). (Meena et al., 2010); (Anar Patel et al., 2010); (Yeungnam 2007), (Gonzalez, et al., 1974); (Schildknecht et al., 1976); (Itokawa et al., 1989); (Ho, et al., 1996); (Hua, et al., 1992); (Kawasaki et al., 1992); (Marec et al., 2001); (Chung et al., 1994); (Antarkar et al., 1983); (Tripathi and Sharma 1998); (Wealth of India 2002).

Psychological factors like Depression, Anxiety and Stress are quoted as the risk factors of Dysmenorrhoea (L Wang et al., 2004) and due to Anti-stress property (Patil, Jagdale et al.,

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

2006) of Rubia cordifolia (Manjishtha) it is more likely to modify the factor like pain in Kashtartava (Dysmenorrhoea).

pain production improves the condition like Dysmenorrhoea.

CONCLUSION

Prostaglandins and Prostinoids are biosynthesized from Arachidonic acid through the COX pathway after production of Arachidonic acid from hydrolysis of Phospholipids by Phospholipase. In excess or imbalanced amount of Prostinoids released from the Endometrium during menstruation induces uterus to contract frequently and dysrhythmically, with increased basal tone and increased active pressure. Uterine hyper contractility, reduced uterine blood flow and increased peripheral nerve hypersensitivity induce pain. Mollugin is one of the major 2H naphtho pyran component isolated from Rubia cordifolia is having strong inhibitory activity on arachidonic acid. Thus Rubia cordifolia L.(Manjishtha) by breaking the pathway of

Analysis of the data of the present study revealed Manjishtha (Rubia Cordifolia L.) churna has significant role in the management of Kashtartava (Dysmenorrhoea). Though the results are good, but further study on large numbers of patients should be done along with some specific investigations like Prostaglandin synthetase evaluation.

ACKNOWLEDGEMENTS The author thank the staff of labs., hospital, Pharmacy of the College for their help and support of patients for participating in this study.

REFERENCES Anar Patel, Timir Patel, Carol Macwan, Mayuree Patel, Khushbu Chauhan, Jatin Patel, “Evaluation of Anti inflammatory and Analgesic activity of roots of Rubia cordifolia in rats. J. Pharm. Sci. & Res. Vol. 2(12), 2010, 809 – 813.

Anonymous, (2005) Quality Standards of Indian Medicinal Plants, Indian council of Medical research, New Delhi, V. 3, p78−87, 307−315

Balbi C, Musone R, Menditto A, Di Prisco L, Cassese E, D Ajello M, Ambrosio D, Cardonel A. Influence of menstrual factors and dietary habits on menstrual pain in adolescence age.

Eur J Obstet Gynecol Reprod Biol 2009, 91: 143–48.

Campbell M, McGrath P., (1997) Use of medication by adolescents for the management of menstrual discomforts Arch Pediatr Adolesc Med;151: 905−912.

Antarkar Antarkar SS, Chinwalla T.,Bhatt N. 1983. Anti inflammatory activity of Rubia cordifolia Linn in rats Indian J. Pharmacology 15(3): 185 – 188.

Chung, M. I. Jou, S. J. Cheng, H. C. Lin, C. N., Ko. F. N. , Teng, C. M. J. Nat Prod. 1994, 57,313 Dutta D. C. (2007) Textbook of Gynaecology, NCBA

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Gonzalez, A.; Cardona, R. J.; Medina, J. M.;Rodriquez Luis, F. Anal. Quimica (1974) 70, 858. (CA 83: 4915)

Kasture S B, Kasture (2001)V S, Chopde C T. Anti-inflammatory activity of Rubia cordifolia roots. J Nat rem. 1: 111.

Guangzhou (1992) Pharmacopeia of the People’s Republic of China; Guangdong Science and Technology Press:, People’s Republic of China, p 179.

Kasture V S, Deshmukh (2000) V K, Chopde C T, Anticonvulsant and behavioral actions of triterpene isolated from rubia cordifolia. Indian J Exp. Biol; 38:675.

http://www.Ayurvedicdietsolutions.com/Manjis htha.php. on 5/2/2011.

Kawasaki, Y. Goda, Y, Yoshihira, K. Chem.

Pharm. Bull. 1992, 40,1504.

Ho; L. – K; Don, M-J; Chen, H-C; Yeh, S-F; Chen, J-M. J. Nat Prod. 1996, 59,330

Khare C.P (2004), Encyclopedia of Indian Medicinal Plants, Rational Western Therapy & other Traditional Usage, Botany, Springer Verlag Berlin Meidelberg, p. 406- 407.

Hocking, G. M. (1997) A Dictionary of Natural Products; Plexus Publishing: Medford, NJ p 679.

Hua, H. M., Wang, S. X. , Wu, L. J. Li, X, Zhu, T. R. Acta Pharm. Sinica 1992, 27, 279.

Itokawa, H., Mihara, K., Takaya, K. Chem.

Pharm. Bull. 1983,31,2353.

Itokawa, H.; Mihara, K.; Takeya, K. (1983) Chem. Pharm. Bull. 31, 2353.

Itokawa, H.; Qiao, Y.; Takeya, K. (1989) Phytochemistry, 28, 3465.

Jamieson D J, Steege J F. The prevalence of dysmenorrhea, dyspareunia, pelvic pain, and irritable bowel syndrome in primary care practices. Obstet Gynecol. Jan 1996; 87(1):55−8. [Medline].

Joharapurkar A A, Zambad S P, Wanjari M M, Umathe S N 2003. In vivo evaluation of antioxidant activity of alcoholic extract of Rubia cordifolia Linn and its influence on ethanol- induced immunosuppression. Indian J Pharmacology 35 : 232– 236.

Khare C P. (2007) Indian Medicinal plants. An lllustrated dictionary. Springer science. 238239.

Kirtikar K.R., Basu B.D (1980), Indian Medicinal Plants vol II, International Book Distributors, Dehradun, 1305−1307.

Meena A. K., Bhavana Pal, P. Panda, R. Sannd, M M Rao. A review on Rubia cordifolia: Its phyto constituents and therapeutic uses : Drug Invention Today 2010, 2(50), 244 –246.

Mhaskar K S, Blatter E & Caius J F. (2000) Indian medicinal plants – their usage in ayurveda and unani medicine.

Mishra Brahmashankara (1993), Bhavaprakasha Nighantu Part 1. Haritakyadi Varga- 64, Chaukhambha Sanskrit Sansthan, Varanasi, p 110 Mooradian A D. (1988) Diabetic complication of central nervous system, endocrinological rev; 9: 346.

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Patil R A, Jagdale S C, Kasture S B, Antihyper glycemic, anti stress and nootropic activity of roots of Rubia cordifolia Linn : Indian J Exp. Biol. 2006 Dec; 44(12): 987–92.

Raja Radhakantade (2003); Shabdakalpadruma, Nag Publishers.

Schildknecht, H.; Straub, F.; Scheidel, V(1976) Liebigs Ann. Chem, 1295.

Shah P K (2011), Sheetal dholakia, Essentials of Gynaecology, Jaypee Brothers.

Sharma R K, Bhagwan Dash (2009) Charaka Samhita, Vol I, II & V Chaukhambha Sanskrit Series Office. Varanasi, Sutra Sthana 26/43, Nidana Sthana 8/40, Shareera Sthana 3/3, Chikitsa Sthana 30/9,15,25, p 366., Vol I p 467, Vol V p. 131,132,135.

Sharma Pandit Hemraj Kashyapa Samhita by Vriddha jivaka revised by Vatsya with Sanskrit introduction by with Vidyotini Hindi Commentary, Chaukhambha Sanskrit Sansthan Khi. Sha. 9/17, P- 287.

Singh R, Jain A, Panwar S. Antimicrobial activity of some natural Dyes and Pigments: 2005, 66: 99 –102

Source of Support: Nil

Tripathi and Sharma (1998) Comparision of antioxidant action of the alcoholic extract of R. Cordifolia with Rubiadin.

Indian J Biochemistry and Biophysics 12:313.

Wealth of India (2002). First Supplementary Series, Vol -3, (D-1), Raw materials. Niscom 130.

Wentz Anne Colston (1985). Novak’s Textbook of Gynaecology. Williams & Wilkins Publications.

Williamson Elizabeth M (2002), Major herbs of Ayurveda, Churchill ivingstone, 13−15, 69−75, 257−260.

Yadavji Trikamji Acharya (1994) Sushruta, Sushruta Samhita with Nibandhasangraha of Dalhanacharya, Chaukhambha Surabharati Prakashana,. Su. Su.15/9, Su. Sha. 3/10, p 59, 265, 266, 272 Yeungnam Uni. 712–749, Korea. Bull. Korean Chem. Soc. (2007), Gyeongsan College of Pharmacy, Vol-28, No-10, 1863.

Conflict of Interest: None Declared

Global Journal of Research on Medicinal Plants & Indigenous Medicine

www.gjrmi.com

GJRMI, Volume 1, Issue 3, March 2012, 77 - 86

Global Journal of Research on Medicinal Plants & Indigenous Medicine

Call for Papers â&#x20AC;&#x201C; fifth Issue www.gjrmi.com Volume 1, Issue 3, March 2012, 77 - 86 Submit your GJRMI, manuscripts (Research articles, Review articles, Short Communications, Letters to the Editor) to Global Journal of Research On Medicinal plants & Indigenous Medicine â&#x20AC;&#x201C; GJRMI

Submit it online through www.gjrmi.com or mail it to chiefeditor@gjrmi.com

To Advertise in the Flip book Cover page write to chiefeditor@gjrmi.com or Call - +919590574495

Global Journal of Research on Medicinal Plants & Indigenous Medicine