GJRMI - Volume 1, Issue 12 - December 2012 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI, Vol.1, Iss. 12, December 2012 Medicinal plants Research Biology ANTIMICROBIAL ACTIVITY OF THE EXTRACTS FROM LONICERA HYPOGLAUCA MIQ. ETHNOPHARMACOLOGICAL COMMUNICATION Xiao-Shun Shu, Jin-Hai Lv, Dong-Ming Chen, Yun-Hui Chen 650-655 Ethno-Botany ETHNO--MEDICO BOTANICAL STUDY OF CHONAMPARA TRIBAL SETTLEMENT OF AGASTHYAVANAM BIOLOGICAL PARK, KERALA Samuel A Selvin and Biju C 656-662 Ethno-Botany PLANTS USED FOR NON-MEDICINAL PURPOSES BY MALAYALI TRIBALS IN JAWADHU HILLS OF TAMIL NADU, INDIA Subbaiah Muruganandam, Singaram Rathinakumar and Arunachalam Selvaraju 663-669 Ethno-Botany TRADITIONAL KNOWLEDGE OF MEDICINAL PLANTS AGAINST BIRTH CONTROL BY THE TRIBALS AND OTHER RURAL PEOPLE OF BARGARH DISTRICT IN WESTERN ODISHA, INDIA Kumar Sen Sunil, Pradhan Rabindranath,, Ranjan Pattnaik Mihir, Mohan Behera Lalit 670-677 Biotechnology A STUDY ON ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS OF MEDICINAL PLANTS USING RAPD MARKERS Radhika K, Singh Sumer , Gopinath S M, Ashwini Patil G M 678-686 Review COMPREHENSIVE REVIEW ON HISTORICAL ASPECT GLYCYRRHIZA GLABRA L. Korhalkar Anagha, Deshpande Manasi, Lele Priya, Modak Meera

YASHTIMADHU687-693

Biotechnology MOLECULAR MARKER STUDIES OF SOME MEDICINAL PLANTS FOR ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS Radhika K, Singh Sumer, Gopinath S M, Kumar Pawan 694-704

Indigenous medicine Ayurveda PHARMACOGNOSTICAL EVALUATION OF LEAF OF LIMNOPHILA RUGOSA ROTH. MERR. (SCROPHULARIACEAE) Acharya Rabinarayan, Padiya R H, Patel E D, Harisha C R, Shukla V J, Chauhan MG 705-711

EVALUATION OF DEHA PRAKRITI WITH SPECIAL REFERENCE TO SWETA PRADARA- A CROSS SECTIONAL STUDY Dhiman Kamini, Vardhan Monika, Dhiman K S, Sharma Naresh 712-721

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – PLANT OF HYGROPHYLLA AURICULATA (SCHUMACH) HEINE, ACANTHACEAE PLACE – VRIDDHACHALAM, CUDDALLORE DISTRICT, TAMIL NADU,

Research article ANTIMICROBIAL ACTIVITY OF THE EXTRACTS FROM LONICERA HYPOGLAUCA MIQ. ETHNOPHARMACOLOGICAL COMMUNICATION Xiao-Shun Shu1*, Jin-Hai Lv2, Dong-Ming Chen3, Yun-Hui Chen 4 1, 4

College of Biology & Chemistry Engineering, Changsha University of Science & Technology, Changsha 410114, PR China 2, 3 Department of Biology, Huaihua College, Huaihua 418000, PR China *Corresponding Author: sxs732@yahoo.com.cn; Tel.: +86 1 13723895338

Received: 30/09/2012; Revised: 15/11/2012; Accepted: 20/11/2012

ABSTRACT Lonicera hypoglauca Miq. is used in the traditional medicine for the treatment of sore throat infection, respiratory tract infection and intestinal tract diseases indicating antimicrobial activity. To validate the traditional medicinal claim, in vitro antimicrobial activity of the extracts was screened against eleven human pathogenic bacteria and fungi.The ethyl acetate (EE) and n-butanol (BE) extracts of the flower of Lonicera hypoglauca Miq were tested against five Gram-positive bacteria, five Gram-negative bacteria and five fungi species. Antimicrobial activity was determined by the tube-dilution method. The EE and BE extracts showed antimicrobial activity against all of the tested microorganisms, with minimum inhibitory concentration (MIC) values in the range of 0.32–4.86 mg/ml. The tested microbes Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pneumoniae were highly susceptible to extract BE and antimicrobial activity of extract BE is better than that of extract EE against Staphylococcus aureus and Streptococcus pneumoniae. Both the extracts were found less susceptible against tested fungi. KEYWORDS: Antimicrobial; Extracts; Lonicera hypoglauca Miq.

To Cite this article: Xiao-Shun Shu, Jin-Hai Lv, Dong-Ming Chen, Yun-Hui Chen (2012), ANTIMICROBIAL ACTIVITY OF THE EXTRACTS FROM LONICERA HYPOGLAUCA MIQ. ETHNOPHARMACOLOGICAL COMMUNICATION, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 650–655 Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 650â&#x20AC;&#x201C;655

INTRODUCTION The plants of the genus Lonicera hypoglauca Miq (Caprifoliaceae) wildly grows and is also cultivated in southern area of China, especially in Hunan province. Flos Lonicera of Lonicera hypoglauca Miq is a common Chinese medicinal herb which has a long history of indigenous use in China. Leaves and flower of Lonicera hypoglauca Miq possess antibacterial activity in Chinese medicine, Flos Lonicera of Lonicera hypoglauca Miq Jinyinhua and Lonicera japonica Thunb both called Jinyinhua that is well-known Chinese traditional medicine for the treatment of diverse diseases (Yin et. al., 2007, Su, 2009), especially for the treatment of sore throat infection and respiratory tract infection and intestinal tract infection diseases. In china, the leaves and flowers are boiled and administered to patient, especially Flos Lonicera (flower). It has attracted many interests. Both Lonicera hypoglauca Miq. and Lonicera japonica Thunb are widely used as Jinyinhua in traditional Chinese medicine. Although they have similar geographic distribution, obviously various characteristics are observed (Pu et. al., 2002). Studies of the phytochemistry and bioactivity of Jinyinhua have mostly focused on L. japonica (Japanese honeysuckle) that has been reported to possess properties like anti-inflammatory, antiangiogenic and anti-nociceptive activities (Xu et. al., 2007, Yoo et. al., 2008). Studies have been also reported on the phytochemistry of Lonicera hypoglauca Miq. Ziguglycoside, scopoletin, daucosterol, Î˛-sitosterol have been isolated from ethyl acetate, and macranthoidin A, macranthoidin B, chlorogenic acid have been isolated from n-butanol extracts of rattan of Lonicera hypoglauca Miq (He et. al., 2006). Hexadecanoic acid, Docosane,Linoleic acid

have also been reported from the essential oil of this plant (Guo,et. al.,2005). However, the bioactivity of Lonicera hypoglauca Miq. has barely been studied. The micro-organisms which are often associated with throat, respiratory tract and intestinal tract infectious diseases belong to the genus bacillus and coccus. These organisms are present in water, soil, sewage and in the gastrointestinal tract of animals, included humans (Murray et. al., 1998). As part of our contribution to phytochemical and biological survey and to validation of traditional uses of this medicinal plant we report herein the study on Flos Lonicera of Lonicera hypoglauca Miq antimicrobial activity. In particular, the purpose of this study was to investigate the plant for the potential antimicrobial activity against selected bacterial strains, which may be involved in sore throat diseases and soft tissue infections, especially respiratory tract infection. Further bioassay-guided extractions were carried out in ethyl acetate extract and n-butanol, in order to obtain the most active extract with the final aim to identify the chemical classes responsible for the biological activity. Materials & Methoda Experimental Chemicals and reagents HPLC-grade ethyl acetate and n-butanol were purchased from Changsha Sheng Fan reagents Co., LTD, (Changsha, China); Norfloxacin and Fluconcazole were purchased from Shanghai research born biochemical reagents Co., LTD, China; water was deionized byfiltering through a Direct-Q system (Millipore, Bedford, MA, USA).

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 650–655

Plant material Flos Lonicera (flower) of Lonicera hypoglauca Miq was collected in the month of June 2010 from HuNan, China. The botanical identification of the plant was done at Food and biology Department, Science & Technology University, Chang Sha, HuNan, china. A voucher specimen (no.FLHM01) has also been deposited at Food and biology Department, Science & Technology University, Chang Sha. Flos Lonicera (flower) was dried in the dark, in a ventilate room at 25–30°C, then grounded and the powder stored at −20°C. Extraction Flos Lonicera (flower) of Lonicera hypoglauca Miq was dried and coarsely powdered by grinding. The powdered material (200 g) was extracted with 2 l of ethyl acetate and n-butanol separately. Each filtrate was concentrated under reduced pressure at 50°C. The extracts were further dried at room temperature under reduced pressure. The yield of the extracts of ethyl acetate (EE) and n-butanol (BE) were 1.90 g (w/w) and 3.40 g (w/w), respectively. Antimicrobial activity

aeruginosa (10223), Bacillus subtilis (63501) (Gram-negative bacteria) were obtained from the health ministry identified microbes, Beijin, China. Candida albicans (ATCC 11006), Candida parapsilosis (ATCC22019), Candida tropicalis (ATCC01463), Trichophyton mentagrophytes (ATCC28185), Cryptococcus neoformans (ATCC32609) (Fungi) were obtained from Beijing university fungi and fungal disease research center, China. Preparation of test sample The EE and BE extracts were dissolved in 10% N-N dimethyl formamide (DMF) which is reported to be non-toxic to microorganisms at this percentage (Pujol et. al., 1990). Norfloxacin and Fluconcazole (Shanghai research born biochemical reagents Co., LTD, China) were used as positive reference standards for bacterial and fungal strains, respectively. Preparation of inocula The inocula of microbial strains were prepared from 18 h old culture and suspensions were adjusted to 0.5 McFarland standard turbidity (~104 for bacteria and ~103 for fungi colony forming unit (CFU) per milliliter) (McFarland, 1987).

Micro-organisms The micro-organisms which are often associated with throat, respiratory tract and intestinal tract infectious diseases belong to the genus bacillus and coccus. The following strains were used for testing the antimicrobial activity of the crude extracts: Staphylococcus aureus (26003 ), alpha hemolytic streptococcus (32129), beta hemolytic streptococcus (53214), Streptococcus pneumoniae (32010), Corynebacterium diphtheriae (38101) (Gram-positive bacteria), Shigella flexneri (51236), Salmonellaty phimurium (53185), Escherichia coli (44104), Pseudomonas

Evaluation of minimum concentration (MIC)

inhibitory

Tube-dilution method was used to determine the minimum inhibitory concentration (MIC) of the EE and BE extracts against the microorganisms under study. The EE and BE extracts were dissolved in 10% N-N dimethyl formamide (DMF). The final concentrations of EE and BE extracts for bacteria were 4.0 and 6.0 mg/ml, respectively. The final concentration 5.0 mg/ml was used for EE and BE extracts against fungi. Serial two-fold dilutions were prepared from the stock

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 650–655

solution to give concentration ranging from 4.00–0.006 to 6.0–0.008 mg/ml for EE and BE extracts, respectively against bacterial strains. The concentration range (5.0–0.008 mg/ml) of EE and BE extracts was evaluated against fungal strains. Norfloxacin and Fluconazole were dissolved in sterile distilled water and two-fold dilutions were prepared (1.0–0.002 mg/ml). One ml of each concentration was mixed with 1.0 ml of sterile peptone water (104 CFU/ml for bacteria and 103 CFU/ml for fungal concentration, obtained

from a McFarland turbidity standard no. 0.5). Solvent control was prepared with DMF (10%) and blank control was prepared from virgin media. Tubes were incubated for 24 and 48 h at 37°C for bacteria and fungi, respectively. Assay was performed in replicates and the mean value of three experiments was recorded (n = 3) with standard deviation. MIC was determined as the lowest concentration that inhibits the visible microbial growth (Murthy et. al., 2006; Kuta, 2008).

Table 1: MIC values (mg/ml) of the extracts of Lonicera hypoglauca Miq Microbial strains Gram-positive Staphylococcus aureus alpha hemolytic streptococcus beta hemolytic streptococcus Streptococcus pneumoniae Corynebacterium diphtheriae Gram-negative Shigella flexneri Salmonella typhimurium Escherichia coli Pseudomonas aeruginosa Bacillus subtilis Fungi Candida albicans Candida parapsilosis Candida tropicalis Trichophyton mentagrophytes Cryptococcus neoformans

MIC mean ± standard deviation EE BE Nor 0.46 ± 0.14 0.87 ± 0.26 0.98 ± 0.33 0.53 ± 0.17 0.76 ± 0.26

0.32 ± 0.11 0.65 ± 0.21 0.85 ± 0.24 0.41 ± 0.15 0.61 ± 0.19

0.003 ± 0.001 0.003 ± 0.001 0.004 ± 0.001 0.003 ± 0.001 0.003 ± 0.001

1.32 ± 0.38 1.53 ± 0.53 1.23 ± 0.44 1.88 ± 0.67 1.42 ± 0.41

1.15 ± 0.32 1.35 ± 0.42 1.01 ± 0.35 1.46 ± 0.53 1.25 ± 0.37

2.69 ± 0.86 2.85 ± 0.93 3.05 ± 1.03 4.15 ± 1.42 4.86 ± 1.65

1.96 ± 0.69 2.26 ± 0.78 2.86 ± 0.85 3.18 ± 1.12 3.57 ± 1.33

0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 Flu 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.003 ± 0.001 0.004 ± 0.002

Note: Nor, Flu, standard antibiotics (Norfloxacin for bacteria, fluconazole for fungi). Values are mean ± standard deviation of three experiments in replicate.

RESULTS AND DISCUSSION The antimicrobial activity expressed as mg/ml, of the two extracts of the flower of Lonicera hypoglauca Miq against various

strains of bacteria and fungi are summarized in Table 1. The organisms Staphylococcus aureus and Streptococcus pneumoniae were found to be most susceptible to the extract BE and EE in bacteria strains with MIC values of 0.32 ± 0.11,

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 650–655

0.41 ± 0.19, 0.46 ± 0.14 and 0.53 ± 0.17 respectively. The organisms Corynebacterium diphtheriae, beta hemolytic streptococcus and alpha hemolytic streptococcus was found more susceptible to the extract BE and EE with lower MIC value (<1.0 mg/ml), While the organisms Shigella flexneri, Salmonellaty phimurium, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis were less susceptible and showed higher MIC values (MIC > 1.0 mg/ml). The extract BE was effective against Candida albicans with MIC value of 1.96 ± 0.69. The MIC values more than 2.0 mg/ml were observed against Candida albicans and Candida parapsilosis for extract EE, and Candida parapsilosis for extract BE. The MIC values of more than 3.0 mg/ml were observed against Candida tropicalis, Trichophyton mentagrophytes and Cryptococcus neoformans. for extract BE, and Candida tropicalis for extract EE, while MIC values of the extract EE were found more than 4.0 mg/ml against

Trichophyton mentagrophytes and Cryptococcus neoformans. Both the extracts were found to be less effective than the standard antibiotics used in the present study and their efficacy was least against fungi (Table 1). The experiment results suggested that the extracts have antimicrobial activities. The benefit of local application of the flower of Lonicera hypoglauca Miq as the treatment of sore throat and respiratory tract infection in Chinese could be attributed to their antimicrobial activity as observed in this study. ACKNOWLEDGEMENTS The authors are grateful to Prof. G.M. Li, Department of Food and Biology, College of Chemistry and Biology Chang Sha University of Science & Technology, for identification of the plant. This work was supported by hunan Provincial Education Department (12A008 and 10C0399).

REFERENCES Gou, Z.P.,Wan De-Guang.,(2005), Studies of the chemical constituents of volatile oils from the dry flower buds of Lonicera hypoglauca. Chin JMAP 6;475–476.

McFarland, J., (1987). Standardization of bacterial culture for the disc diffusion assay. Journal of American Medical Association 49, 1176–1178.

He, Q.H. ， Li, H.J. ， Bi, Z.M., Li,S.P. and Ma,S.Z.,2006. Chemical Constituents in the Stem of Lonicera hypoglauca. Chin J Nat Med 5; 385–386．

Murthy, M.M., Subramanyam, M., Giridhar, K.V., Jetty, A., (2006). Antimicrobial activities of bharangin from Premna herbacea Roxb. and bharangin monoactate. Journal of Ethnopharmacology 104, 290–292.

Kuta, F.A., (2008). Antifungal effect of Calotropis procera stem bark on Epidermophyton flocosum and Trichophyton gypseum. African Journal of Biotechnology 7, 2116–2118.

Murray, R.P., Rosenthal, K.S., Kobayashi, S.G., Pfaller, A.M., 1998. Medical Microbiology, vol. 37. Mosby, St. Louis, MO, pp. 296–298.

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Pujol, V., Seux, V., Villard, J., (1990). Recherche de substances Anti-fongiques secretes par les champignons superieurs en culture. Annales Pharmaceutiques Francaises 48, 17–22.

Xu, Y., Oliverson, B.G., Simmons, D.L., (2007), Trifunctional inhibition of COX-2 by extracts of Lonicera japonica: direct inhibition, transcriptional and posttranscriptional down regulation, J. Ethnopharmacol 111,667–670.

Pu, Z., Xing, J., Li, P., Liu, T., Wang, Z., (2002), Studies on the floral morphology of Flos Lonicerae, Zhong Yao Cai 25, 854–859.

Yin, S.G., Xin, N., (2007), General situation of studies on Flos Lonicerae. Li Shi-Zhen Medcine and material medical research 12, 2920–2922.

Su, S.Y., 2009. Progress of studies on Lonicera hypoglauca Miq. Journal of University of South China f Medical Edition 6,744–746.

Yoo, H.J., Kang, H.J., Song, Y.S., Park, E.H., Lim, C.J., (2008), Anti-angiogenic, anti-inoceptive and anti-inflammatory activities of Lonicera japonica extract, J. Pharm. Pharmacol 60,779–786.

Source of Support: Nil

Conflict of Interest: None Declared

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Research article ETHNO--MEDICO BOTANICAL STUDY OF CHONAMPARA TRIBAL SETTLEMENT OF AGASTHYAVANAM BIOLOGICAL PARK, KERALA Samuel A Selvin1 and Biju C2* 1

Associate Professor & Head, Department of Botany, St’ John’s College, Palayamkottai, Tirunelveli District, Tamil Nadu, India 2 Research scholar, Department of Botany, St’ John’s College, Palayamkottai, Tirunelveli District, Tamil Nadu, India *Corresponding Author: bijuc_tvpm@yahoo.co.in

Received: 18/10/2012; Revised: 28/11/2012; Accepted: 30/11/2012

ABSTRACT An ethno-botanical survey was carried out to collect information on the use of medicinal plants in Southern Western Ghats of India (Thiruvananthapuram district, Kerala). Information presented in this paper was gathered from the Kani tribes using an integrated approach of botanical collections, group discussions and interviews in the years 2009–2010. The informants interviewed were 10 among whom 3 were tribal practitioners. A total of 20 ethno-medicinal plant species distributed belongs to 19 families are documented in this study. The medicinal plants used by Kanis are listed with Scientific Name, family, local name, parts used, mode of preparation and medicinal uses. Generally, fresh part of the plant was used for the preparation of medicine. It was observed that the documented ethno-medicinal plants were mostly used to cure skin diseases, poison bites, stomachache and nervous disorders. The results of this study showed that these tribal people still depend on medicinal plants from forest areas for treating ailments. KEY WORDS: Ethno-botany, Kani Tribe, Tribal practitioners.

To Cite this article: Samuel A S and Biju C (2012), ETHNO--MEDICO BOTANICAL STUDY OF CHONAMPARA TRIBAL SETTLEMENT OF AGASTHYAVANAM BIOLOGICAL PARK, KERALA, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 656–662

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 656–662

INTRODUCTION Even from the pre-historic era almost all civilizations have employed plants in the treatment of human ailments. The sages of India had unparalleled knowledge on medicinal plants. “Rig Veda”, has the oldest repository of medical knowledge, which can be traced back to 4500–1600 BCE. “Rig Veda” was followed by “Atharva Veda”, in which, various medicoreligious uses of plants have been cited. Over the years, the traditional Indian medicine has become rather well codified in a holistically oriented practice or system of health care as “Ayurvedic medicine”. (Singh. K.K and Anand Prakash, 1994) There are over 53 million tribal people in India belonging to 550 communities of 227 ethnic groups. They inhabit in about 5000 forested villages or lead a nomadic life in the forest. Each tribal community has a distinct social approach and cultural identity of its own. In Kerala, the tribal communities are nearby 40 in number. Some of them are very primitive and most are advanced. The major groups of tribes in Kerala are Kani, Adiyar, Paniyar, Kurichiar, Koragar, Kurumar, Kattunaykar etc. (Panoor K 1963). Kanis mainly inhabit the forest of Kerala Tamilnadu border which is located in the southern most part of Western Ghats known as ‘Agasthyar Koodam’. It is believed to be the sacred abode of the great saint Agasthya and a heaven of medicinal herbs. (Sunil Anandamangalathu, 2001). It is one of the most diverse biological resources in the region. Indian traditional systems of medicines and their practices have been transmitted to the present society because the medicinal aspects of innumerable number of plants lay hidden in the mind of traditional vaidyars. It should be elicited out for the welfare and well-being of modern society. Indigenous knowledge is the collective wisdom of individuals, families, tribes, communities and societies living in the specific geographic locations on a wide spectrum of human activities in relation to their immediate environment. Herbal drugs obtained

from plants are believed to be much safer; this has been proved in the treatment of various ailments. Rural communities, in particular Kani tribes, depend on plant resources mainly for herbal medicines, food, forage, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dwellings, making household implements, sleeping mats, and for fire and shade. Rural people not only depend on wild plants as sources of food, medicine, fodder and fuel, but have also developed methods of resource management, which may be fundamental to the conservation of some of the world's important habitats. (Jain 1996). The objective of this study was to assess the richness of ethno-medicinal plant species used by the Kani tribes in Chonampara tribal settlement of Agasthyavanam Biological Park forest areas and the traditional medical practices of the people. Similar ethno-botanical studies have been reported in several parts of India to protect the traditional knowledge from disappearing. Documenting the indigenous knowledge through ethno-botanical studies is important for the conservation of biological resources as well as their sustainable utilization. Nature has blessed the Chonampara tribal settlement of Agasthyavanam Biological Park, Kerala with a very rich botanical and ethnomedicinal wealth that has been exploited continuously by the tribals. Kanis are the major inhabitants of this settlement. As far as our knowledge is concerned, there is no perfect ethno-medicinal survey of Chonampara tribal settlement. Hence an attempt has been made in the present investigation to study the Ethnomedico Botanical study of Chonampara tribal settlement of Agasthyavanam Biological Park, Kerala. Setting and the people Kani tribals The Kanikars, who are commonly known as ‘Kanis’ are one of the jungle tribes; mostly depend on medicinal plants growing in the

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 656–662

forests, inhabiting the mountains of southTravancore. The Kanis are in Kerala mainly settled on the Western Ghats region especially Thiruvananthapuram and Kollam Districts. Majority of the Kani settlements are located in Thiruvananthapuram District. The language of the Kanis is a dialect of ‘Malayalam’, with a large admixture of ‘Tamil’ which they call ‘Malampasha’ or language of the hills. Traditional medicines are the primary healthcare resources for the Kani tribes to protect their health. Tribal practitioners are the curators of the tribal society and they have a good knowledge of medicinal plants, diseases and treatment using plants. MATERIAL AND METHODS Description of the study area The study area concentrates in and around the Thiruvananthapuram district forest areas located in Kerala, South India. The area of investigation approximately lies between 85°0' to 89°0' longitude and 28°0' 37° to 0° latitude. Every village has several Kani hamlets. Their hamlets are found in different elevations from 300 m to 2,200 m MSL. There are a number of hill ranges in the study area. The climate is moderately hot and humid. The temperature varies from 16° C to 35° C. The mean annual rain fall is 2,800 mm and the tract receives both South-West and North-East monsoons. Ethno-botanical survey The fieldwork was conducted in several villages around the forest areas of Thiruvananthapuram district during July 2009 to December 2010 as part of the study. Ethnobotanical Wealth of Kani Tribals in Kerala are also obseved. More than 30 families and nearly 175 members of Kanis are found in the study area. During the stay, their daily activities were closely observed and interpersonal contacts were established by participating in several of their social and religious ceremonies such as

marriages, rituals and curing sessions. Various interviews were carried out with several elders and experienced men and women to get information on medicinal plants used by them. Queries were repeatedly made from the head Ramakrishnan Kani (MuttuKani) and different persons (Appukuttan Kani, Kunjukrishnan Kani, Sudarsanan Kani, Pachi, Gopalan Kani, Sundari, Eechan Kani, Mallan Kani, and Kuttappan Kani) inorder to verify the accuracy of the information. Interviews with tribal practitioners The collected plant materials were identified with the help of the Kanis to get more information. During the field trips the medicinal plants were also photographed. The data were recorded in the field note books and the interview and discussions were recorded in audiotapes (Jain,1981). Polythene bags were used to keep the collected materials in fresh condition. Collected plants were identified COrrectly and confirmed by Dr.Selvin Samuel (Head, Dept. of Botany, St.John’s College, Palayamkottai) referring the various Flora viz, Flora of British India by Hooker (1874), The Flora of the Nilgiri and Pulney Hill trops by Fyson (1915, 1921), The Flora of the Presidency of Madras by Gamble and Fischer (1957), Flora of TamilNadu by Nair and Nayar (1986), Excursion Flora of Central Tamil Nadu, India by Matthew (1991), The Flora of Thiruvananthapuram District were referred. The Flora of Presidency of Madras and The Flora of Tamil Nadu Carnatic were used to ascertain the nomenclature. The voucher specimens (No. SJCB 125–145) duplicate were deposited in the herbarium of Department of Botany, St.John’s College, Palayamkottai. The ethno-medico-botanical data include Botanical name of the plant, family, tribal names, medicinal claims / uses and name of the plant collector and field numbers of voucher specimen were also mentioned.

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Table - 1 LIST OF ENTHOMEDICINAL PLANTS AND THEIR USE CATEGORY IN TRIBAL MEDICINE FROM KANI TRIBAL AREAS OF CHONAMPARA SETTLEMENT Sl.No

Botanical Name

Medicinally useful parts

Ailments

Fruits Root, Leaves

Diabetes, Headache, Dysentery. Snake bite.

Leaves

Dysentery, Vomiting

Heartwood, bark

Post-natal-care, gastric complaints, hypertension Diabetes, Cancer, Heart disorders, Leprosy. Blood purification

Bark

Snake bite

Leaves

Delivery pain

Whole plant

Joining the broken bones.

Root

Worms in children

Aegle marmelos, (L.) Correa

2 3

Aristolochia krisagatra, Sivarajan and Pradeep. Biophytum sensitivum, DC.

Capscicum frutescens, L.

Fruits

Leaves, Root

Catharanthus roseus (L.) G.Don Caesalpinia sappan, L..

Couroupita guianensis.Aubl.

Cardiospermum helicacabum,L.. Cissus quadrangularis, L.

9 10

Clerodendron infortunatum, Gearth.

Elephantopus Scaber, L.

Whole plant

Piles.

Fruits Whole plant

Piles, Digestion Diabetes, Asthma

Garcinia gummi-gutta, L. Gymnema sylvestre (Retz.) Schult Kaempferia galanga,L.

Rhizome

Snake bite, Indigestion

Michelia champaka, L.

Seeds

Headache, Hair growth

Myristica fragrans, Houtt.

Fruits

Tooth-ache.

Root

Snake bite

Rauvolfia serpentina, Benth. ex. Kurz. Ruta chalepensis. L.

Leaves

Respiratory problems, Epilepsy, Leukemia.

Saraca indica, L.

Leaves

Menstrual disorders

Trichopus zeylanicus, Gaertn.

Leaves

Obesity, Digestion

RESULTS AND DISCUSSION In the present study, it was recorded that Kanis use about 20 medicinal plants belonging

to 19 angiospermic families and 20 genera to treat various ailments such as diabetes, headache, dysentery, poisonous bites, rheumatism, heart disorders, cough, worms

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problem in children, dental pain, delivery pain, digestive troubles, cancer etc. (Table 1). The present ethno-medicinal study helps to identify new or common medicinal herbs of tribal medicine, provide clue for new leads, for systematic Pharmaco-therapeutic and Clinical research, besides listing the local (tribal) names, for the plants. It is not only essential to conserve such a wealth of information found among the tribals but also to enumerate such details and devise a modern biomedical system to meet the ever increasing clinical requirement of mankind. Rauvolfia serpentina has been described to be used as a popular medicinal herb by different tribes against snakebite and insanity (Rao and Shambhu, (2002). Due to illegal and excessive exploitation, populations of some of the medicinal plants species such as Aegle marmelos, Corr., Aristolochia krisagathra, Sivarajan & Pradeep, Garcinia gummi-gutta,L., Rauvolfia serpentina, Bemth.ex.Kurz., Saraca indica,L., Trichopus zeylanicus,Gaertn., Michelia champaka, L. and Kaempferia galanga,L. became rare, now endangered and threatened (Maikhuri, 1998). Further more, because of ban on legal collection, local medical practitioners are facing problems in getting appropriate plant parts of desired quantity for curing the various ailments. Since the traditional values, culture, faith and indigenous knowledge related to traditional health care system of Kanis of Chonampara tribal pocket are facing serious challenges due to acculturation, brought about by migration of the younger generation to cities and these urban migrants show a gap in the cultural beliefs and practices with those of the local inhabitants, the recording of the indigenous knowledge based on traditional health care system becomes increasingly important. Tribals are dependent on forest for surviving to say that they are lovers of forests. There are 35 tribal communities which are distributed in Kerala. Of these Kanis are the predominant group in Chonampara tribal pocket, Agasthyavanam Biological Park. Kanis live together in small communities under a

Muttukani who wields considerable influence over them and enjoys various perquisites. Their settlements composed of lowly huts built of bamboo. Which are generally situated away from the traces of elephants on steep hill slopes of Chonampara tribal pocket, Agasthyavanam Biological Park, Kerala State. The Kanis collect the honey and wax from the forest for sale thus they achieve the aid for making their livelihood. Kanis of the present study mainly cultivate crops like rice, tapioca, sweet potatoes and banana under the direction of Muttukani.Kanis celebrates several spiritual customs like Karthika koduthi, Koda, Chattu pattu and are preceded under the instruction of Plathi, the Priest of Kanis. In total, 20 numbers of ethno-medicinal plant species were collected from the study area. All are medicinally important plants used in various medicines for curing 26 ailments. Of these 20 medicinal plants, reported from the study area used in human ailments, they used various parts of the plant such as leaves, stem, flower, fruit, bud, inflorescence, underground parts (roots, tubers, corm, and bulb), heartwood, bark, resin/gums etc. The important ethno- medicinal plants are Gymnema sylvestre (Retz.) R. Br. is used to cure Diabetes, Catharanthus roseus (L.) G.Don is used to cure blood cancer and Leprosy, Garcinia gummi-gutta (L.) Roxb. is used to cure Piles, Saraca indica L is used to cure Menstrual problems, Rauvolfia serpentina, Benth. ex. Kurz. is used to cure snake bite and Ruta chalepensis.L. is used to cure Epilepsy and leukemia. Out of the 20 medicinal plants only 8 species such as Aegle marmelos, Corr., Aristolochia krisagatra, Sivarajan and Pradeep., Garcinia gummi-gutta (L.) Roxb., Kaempferia galangal.L, Saraca indica.L, Trichopus zeylanicus, Gaertn., Michelia champaka.L, Rauvolfia serpentina, Benth. ex. Kurz. are enlisted in Red Data list of South Indian medicinal plants (FRLHT- Modified 2006).

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CONCLUSION The present study mainly focused on the medicinal aspects of various plants along with the botanical identity, habit, morphological useful parts used in traditional medicine. In this study 20 medicinal plants were gathered from the study area, which comes under 19 angiospermic families. Medicinal plants from the study area are able to cure 26 diseases such as diabetes, headache, dysentery, poisonous bites, blood impurities, rheumatism, heart disorders, cough, worms problem in children, dental pain, delivery pain, digestive troubles, cancer etc. Most of the people prefer green medicines for curing various diseases without any side effects while using the tonic of allopathic medicines. The tribals gathers different plant parts like leaves, bark, fruit, flowers, tubers, stem, underground parts such as roots, bulb, corm, tubers etc for preparing medicinal forms

like decoction, infusion, mixture, juice extract, powder, fumes etc. and sell it to earn money. Conservation of ethno-botanical resources and wild relatives of crop plants is vital for future breeding programmers. The search for economic and medicinally important plants used by tribes and aboriginals must be continued. Their preservation is necessary to secure supplies of food, fibre, drugs and medicines. The over utilization of medicinal plants will lead to the destruction of the preferable plant materials and will result in endemic condition. It is also necessary to ensure that loss of species does not impair the effective functioning of ecological processes. The over exploitation of medicinal plants should be monitored with the help of Government trainees / NGO’s for providing ideas to the tribal people for the conservation of medicinal plants and their importance. The conservation of the medicinal plants can be enhanced by the cultivation.

REFERENCES Abraham. Z. 1981. Ethno-botany of the Todas, the Kotas and the Irulas of the Nilgiris. In. Glimpses of Indian Ethno-botany. (ed) S.K. Jain. Oxford and IBH, New Delhi. PP: 308–320. Fyson, P.F. 1915, The Flora of the Nilgiri and Palney Hill trops. Delhi. Red Data list of South Indian medicinal plants FRLHT- Modified version 2006 Gamble J.S and C.E.C. Fischer. 1967. (Rep.ed.) Flora of the Presidency of Madras, Vol. IIII. Botanical Survey of India, Calcutta. Jain S.K. 1981. Ethno-botanical Research unfolds New vistar of medicine In Glimpses of Indian Ethno-botanical. Oxford & IBH pub.co. New Delhi. Matthew K.M. 1991. An excursion Flora of Central Tamil Nadu, India, Oxford and IBH pub. Company. Pvt. Ltd, New Delhi.

Mohan.M and A.N Henry. 1994. Flora of Thiruvananthapuram, Botanical Survey of India, Calcutta. (Flora of India series – 3) 1–25. Mohanan. N and Sivadasan. M. 2002. “Flora of Agasthyamala”, Bishen singh Mahendra Pal singh, Dehra Dun. Nagendra Prasad, P.A.William Jebadhas and E.K.Janaki Ammal. 1987. Medicinal plants used by the Kanikars of South India. J.Econ. Taxon. Bot. 11 – 149– 155. Nesamony, S 1985. Oushadha Sasyangal. State Institute of Languages. Kerala, TVM. 1– 48. Pushpangadan. P 1990. Amritha pala (Janakia arayalpatra, Joseph and Chandrasekharan). A new Drug from the Kani tribe of Kerala. Ancient scientific of life. 9: 212–214.

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Pushpangadhan.P, A.K, Sharma and R.Rajasekharan, 1995. Ethnopharmacolgy of Trichopus zeylanicus the Ginseng of Kerala a review. In Glimpses of India Ethno-pharmacology. (ed) Pushpangadhan . P, U.Nyman and V.George. Trop Bot. Garden and Res.Inst.TVM and the Royal Danish school of pharmacy Copenhagen 137–146 Ragupathy. S and A. Mahadevan, 1991. Ethnobotany of Kodiakkarai Reserve Forest,

Source of Support: Nil

Tamil Nadu, South India. Ethno-botany. 3 : 79–82 Rajendran.S.M, K.Chandra Sekhar and V.Sundaresan, 2001, Ethno-medicinal lore of Seithur Hills-Southern Western Ghats-Tamil Nadu – Ethno-botany Vol – 13, 101–109. Singh.

K.K and Anand Prakash, 1994. Indegenous phytotherapy among the Gond tribe of sonbhadre district Uttarpradesh, India. Ethno-botany 6 : 3741.

Conflict of Interest: None Declared

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Research article PLANTS USED FOR NON-MEDICINAL PURPOSES BY MALAYALI TRIBALS IN JAWADHU HILLS OF TAMIL NADU, INDIA Subbaiah Muruganandam1, Singaram Rathinakumar2, Arunachalam Selvaraju3* 1, 2

Department of Plant Biology and Plant Biotechnology, Presidency College, Chennai – 600 005, Tamil Nadu, India 3 Post Graduate and Research Department of Botany, Pachaiyappa’s College, Chennai – 600 030, Tamil Nadu, India *Corresponding author: valarselva84@gmail.com

Received: 02/11/2012; Revised: 29/11/2012; Accepted: 30/11/2012

ABSTRACT An ethno-botanical survey, particularly the plants used other than medicinal purposes was carried out among the Malayali tribals in Thiruvannamalai district of Tamil Nadu, India during June to December 2011. With the help of standardized questionnaires, a number of informants were interviewed on the non-medicinal use of the local flora. A total of 24 plant species belonging to 20 genera were recorded as non-medicinal and food plants in the present study. These plants were used for food, religious purposes, rituals, decorative purposes, as insect repellents, biofertilizers, construction purposes, making household implements and hedge and fuel. The study showed a high degree of ethno-botanical novelty and the use of plants among the Malayali tribals. KEYWORDS: Ethno-botany, Malayali tribals, Non-medicinal uses, Tamil Nadu

To Cite this article: Subbaiah M, Singaram R, Arunachalam S (2012), PLANTS USED FOR NON-MEDICINAL PURPOSES BY MALAYALI TRIBALS IN JAWADHU HILLS OF TAMIL NADU, INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 663–669

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INTRODUCTION

MATERIALS AND METHODS

India is one of the twelve mega-biodiversity countries of the World having rich vegetation with a wide variety of plants with medicinal value. The tribal population of the country, as per the 2001 census, is 8.43 crore, constituting 8.2% of the total population (Ministry of Tribal affairs). With enormously diversified living ethnic groups and rich biological resources, India represents one of the great emporia of ethno-botanical wealth (Pal, 2000). During the last few decades there has been an increasing interest in the study of medicinal plants and their traditional use in different parts of India. Primarily, Janaki Ammal (1956) conceptualized the importance of ethnobotanical studies in India and initiated such studies. After that from the year 1963, Jain extended these studies to the forefront through his pioneering research and publications.

Study area

It is estimated that tribal people of Tamil Nadu occupy 1.05% of the total state population and 0.77% of the total tribal population of the country. Ministry of Tribal affairs presented a list of tribal communities in India for each state and Tamil Nadu contains 36 types of tribal communities and they are distributed in different districts in the forests and adjoining areas. Several studies have revealed that tribal population, not only depend on plant based resources for medicines, food, forage and fuel, but also play a vital role in natural resource management that forms the core aspect of conservation biology (Ministry of Tribal affairs). In the recent years, number of reports on the use of plants in traditional uses by either tribal people or indigenous communities of India is increasing (Ayyanar and Ignacimuthu, 2010, Ayyanar et.al., 2010; Samant and Dhar, 1997; Samant et.al., 1998). The main objective of this study was to assess the diversity of plants used by Malayali tribals in Jawadhu hills of Thiruvannamalai district of Tamil Nadu, India and to document their traditional knowledge.

The study area Kovilur (situated in Eastern Ghats of Tamil Nadu), the Panchayath union of Thiruvannamalai district, Tamil Nadu consists of 52 villages, out of which 10 villages (Kovilur, Thombaretti, Thanchankollai, Athippatti, Perungattur, Mamathoor, Aattaiyanur, Mullippattu, Jamuna marathur and Motlapattu) were selected in the present study for the documentation of ethno-medicine. Most of the hamlets in the villages are occupied by the Malayali tribals. Malayali tribal’s simple food includes Dioscorea tubers. Each village is administrated by a leader (or) headmen called ‘Nattamai’ and he is considered as leader of the village and even during marriages and festivals, he only initiates functions and ceremonies. Occupation being cultivation, other jobs like hunting, honey gathering, fruits gathering and selling also observed in these villages. Ethno-botanical survey Frequent field surveys were carried out in Kovilur Panchayath union in Eastern Ghats of Thiruvannamalai district, Tamil Nadu in different seasons during June to December 2011. The data were collected through general conversations and questionnaires with the elder people (eight informants between the ages of 32–65 years). The information was gathered from them was recorded in field notebooks. The collected plants were identified by their vernacular names through consultations with the local people, photographed and sample specimens were collected for the preparation of herbarium. The Flora of Presidency of Madras (Gamble, 1935) and The Flora of Tamil Nadu Carnatic (Matthew, 1983) were used to ascertain the nomenclature. The voucher specimens were deposited in the herbarium at Department of Botany, Presidency College, Chennai, Tamil Nadu, India. RESULTS AND DISCUSSION The present study focused mainly on the plant species used by the Malayali tribals for various non-medicinal uses. The reported plants were arranged according to their

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scientific name and family, vernacular (local) names as recorded during the field work, habit and uses. During the study period, 24 species of plants belonging to 20 genera and 20 families were identified as non-medicinal and food plants which are used by the Malayali tribals in Eastern Ghats (Thiruvannamalai district) of Tamil Nadu (Table 1). The plants listed mainly

belong to Rutaceae (4) followed by Caesalpinaceae (2) and the remaining with one species each (Fig.1). From the study, it was observed that ten taxa are trees (42%), six are herbs (25%), four are shrubs (12%), three are climbers (13%) and two taxa are small trees (8%) (Fig.2)

Table 1. List of plant species used by Malayali tribal people for non-medicinal purposes in Thiruvannamalai district (Eastern Ghats) of Tamil Nadu Habit Name of the plant Family Local name Non-Medicinal uses Aegle marmelos (L.) Correa

Leaf paste is mixed with Tree goatâ&#x20AC;&#x2122;s milk for the persons of alcohol addiction. Used during festivals as an Herb ornamental plant The leaves are used as a Tree fuel. Ripe fruits are taken to Tree keep the body cool. The mature stem is used as stick for musical Herb instruments. The plant is also used as an ornamental plant. Twigs are used as tooth brush. Tree The leaves are used as green manure The leaves are used to Climber make chutney. Wood is recommended as a valuable timber and also used to make agricultural Tree implements. Leaves are used as cattle feed. Whole plant parts are used Small as bio-pesticide and also tree used as green manure.

Rutaceae

Vilva maram

Amaranthaceae

Poolachedi

Mimosaceae

Thurinji

Annonaceae

Seethamarm

Asclepias curassavica L.

Asclepiadaceae

Sivapupoo chedi

Azadirachta indica A. Juss.

Meliaceae

Veppamaram

Sapindaceae

Mudakkaruthan kodi

Rutaceae

Purusu

Rutaceae

Chavatai

Combretaceae

Karlan kodi

Climber

Stem is used as cordage.

Euphorbiaceae

Yerpoolan poondu

Herb

Leaves are used as manure.

Aerva lanata (L.) Juss. ex. Schult. Albizia amara (Roxb.) Boivin. Annona squamosa L.

Cardiospermum halicacabum L. Chloroxylon swietenia DC.

Clausena dentata (Willd.) Roem. Combretum albidum G. Don. Croton bonplandianum Baillon.

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Name of the plant

Family

Local name

Dalbergia latifolia Roxb.

Fabaceae

Rose wood

Dendrocalamus strictus (Roxb.) Ness.

Grewia orientalis L.

Guizotia abyssinica (L. f.) Cass. Lantana camara L. Limonia acidissima L. Manilkara hexandra (Roxb.) Dubard. Memecylon grande Retz. Nephrolephis sps. Oxalis corniculata L.

Habit

Tree

Non-Medicinal uses Wood is used to make furniture. Stem is used for storing honey; as spatula for stirring, and also as building and roofing material. Leaves are used as manure. The leaves are used as cattle feed

Poaceae

Moongil

Tree

Tiliaceae

Poonaikokukkan chedi

Small tree

Asteraceae

Thattellu

Herb

Flower heads are used to prepare pickles.

Verbenaceae

Randanachedi

Shrub

Fruits are edible.

Rutaceae

Vilamaram

Tree

Leaves are used to make pickles.

Sapotaceae

Paala maram

Tree

Melastomataceae

Sanjeevi sakalathi

Climber

Nephrolepidaceae (Fern)

Vesanaai chedi

Herb

Oxalidaceae

Chootu chedi

Herb

Pterolobium hexapetalum (Roth.) Sant.& Wagh

Caesalpiniaceae

Peenjha

Shrub

Senna montana B. Heyne. ex. Roth.

Caesalpiniaceae

Karungonna maram

Tree

Tarenna asiatica (L.) Kuntze. ex. K.

Rubiaceae

Tharanicheddy

Shrub

Of the plants parts used as food by the Malayali tribals leaves, fruits, seeds and underground parts such as tuber, rhizome, root and corm are mostly used as food. Leafy

Leaves are used as cattle feed. Smoke of dry leaves are released inside the cattle and chicken shed to control mites. Leaf juice is used as a biopesticide in paddy fields. Leaves are used as a bleaching agent for floor. The plant is used for making fence. The young branches are used as cattle feed. Leaves of this plant are used as fodder. Mites in cattle shed can be controlled by smoking the leaves.

vegetables are grown as wild weeds. Kani tribals in Thirunelveli hills eat tubers like Manihot esculenta and Dioscorea oppositifolia (Ayyanar and Ignacimuthu, 2005). Most of the leafy vegetables such as Acacia pennata,

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Commelina ensifolia, Diplocyclos palmatus, Jasminum angustifolium, Premna obtusifolia and Cissus quadrangularis used by the Paliyar tribals of Virudhunagar district in Tamil Nadu have also been used to treat various ailments (Arinathan et.al., 2003). Besides the medicinal and food value, plants also play an important role in the socioeconomic life of Malayalis. Malayali tribals have grown some of the plants as sacred plants in the vicinity of their houses and temples. Malayali tribals also use a number of plants for various economic uses viz., construction

purposes and for hedge; some of the plants for, biofertilizers, fodders, fire wood, construction of household implements etc (Fig.3). Paliyar tribals in Palani hills of Tamil Nadu are using more than 30 plants as commercial non-timber forest products (John Kennedy, 2006). Irulas, Mudugas and Kurumba tribals inhabiting the Attapadi hills of Western Ghats (Nadanakunjidam) use 51 species of plants as timber, fibre, fence, thatching grass, wild food, fodder, manure, insecticide, pesticide, tooth brush, soap, hair and body wash, ornamental, ritual and agricultural implements.

Figure 1. Family wise classification of collected plant species in the study area 5 4 3 2 1 0

Figure 2. Habit wise classification of collected plant species in the study area

Climber 13%

Small tree 8% Tree 42%

Shrub 12%

Herb 25%

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Figure 3. Different non-medicinal uses reported in the present study 6 5 4 3 2 1 0

CONCLUSION Traditional plant use and knowledge thereof is still essential to the Malayali tribals living in the forest areas of Eastern Ghats in Thiruvannamalai district of Tamil Nadu. With changes in lifestyle and associated decline of the use of plants, the ethno-botanical knowledge might continue to decline. The preservation of some rituals especially concerning economically important plants is noteworthy in the area and the study revealed

that the Malayali tribals are using many forest resources for their day-to-day life. ACKNOWLEDGEMENTS The authors are grateful to the Malayali tribals in the study area for revealing their traditional botanical knowledge. The authors gratefully acknowledge Dr. M. Ayyanar, Department of Botany, Pachaiyappa’s College, Chennai for his help in identification of plant species and suggestions on crafting the article.

REFERENCES Arinathan, V., Mohan, V.R., John De Britto, A. and Chelladurai, V. (2003). Studies on food and medicinal plants of Western Ghats. Journal of Economic and Taxonomic Botany 27: 750–753

Ayyanar, M. and Ignacimuthu, S., (2005). Traditional Knowledge of Kani tribals in Kouthalai of Tirunelveli hills, Tamil Nadu, India. Journal of Ethnopharmacology 102: 246–255.

Ayyanar, M. and Ignacimuthu, S., (2010). Plants used for non-medicinal purposes by the tribal people in Kalakad Mundanthurai Tiger Reserve, Southern India. Indian Journal of Traditional Knowledge 9, 515–518.

Ayyanar, M., Sankarasivaraman, K., Ignacimuthu, S. and Sekar, T., (2010). Plants used for non-medicinal purposes by Paliyar tribals in Theni district, Western Ghats of Tamil Nadu, India. Asian Journal of Experimental Biological Sciences 1(4): 765–771.

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Gamble, J.S., The Flora of the Presidency of Madras (Adlard & son, Ltd, London), 1935. Janaki Ammal, E.K., (1956). Introduction to the subsistence economy of India. In: Man’s role in changing face of the earth ( Ed. William LT Jr) University of Chicago Press, Chicago, 324–335. John Kennedy, S.M., (2006). Commercial Nontimber forest products collected by the tribals in the Palni hills. Indian Journal of Traditional Knowledge 5: 212–216 Matthew KM, The Flora of Tamil Nadu Carnatic, Vol I–III (The Rapinat Herbarium, St. Joseph’s College, Tiruchirapalli, India), 1983. Ministry of Tribal Affairs, Annual Report

Source of Support: Nil

(2005–2006).

Nadanakunjidam, S., (2003). Ethno-medicinal observations from Attapadi Hills of Western Ghats. Journal of Economic and Taxonomic Botany 27: 732–740. Pal, D.C., 2000. Ethno-botany in India. In: Flora of India. Introductory volume part II, (Eds. Singh, N.P., Singh, D.K., Hajra, P.K. and Sharma, B.D). Botanical Survey of India, Calcutta, India. pp. 303–320. Samant, S.S. and Dhar, U., (1997). Diversity, endemism and economic potential of wild edible plants of Indian Himalaya. International Journal of Sustainable Development and World Ecology 4: 179–191. Samant, S.S., Dhar, U. and Rawal, R.S., (1998). Biodiversity status of a protected area of West Himalaya-1. Askot Wildlife Sanctuary. International Journal of Sustainable Development and World Ecology 5: 193–203.

Conflict of Interest: None Declared

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Research article TRADITIONAL KNOWLEDGE OF MEDICINAL PLANTS AGAINST BIRTH CONTROL BY THE TRIBALS AND OTHER RURAL PEOPLE OF BARGARH DISTRICT IN WESTERN ODISHA, INDIA Sen Sunil Kumar1*, Pradhan Rabindranath2, Pattnaik Mihir Ranjan3, Behera Lalit Mohan4 1

Demonstrator in Botany, Department of Botany, Panchayat College, Bargarh: 768 028, Odisha, India Ex-Reader in Botany, Panchayat College, Bargarh: 768 028, Odisha, India 3 Forest Range Officer, Range Office, Bargarh: 768 028, Odisha, India 4 Ex-Reader in Botany, Modipara (Near Water Tank), Sambalpur: 768 002, Odisha, India *Corresponding Author: E- Mail: sunilsen06@rediffmail.com; Mobile: 09437159707

Received: 09/10/2012; Revised: 05/12/2012; Accepted: 06/12/2012

ABSTRACT Bargarh is one of the ten districts of Western part of Odisha (previously Orissa) covering a land area of 5837 sq. Kms and is the natural treasure of a large number of plants. Many of these plants have ethnobotanical use. A survey of the medicinal plants at different forest pockets and rural areas of Bargarh district was under taken during 2006–08. This work relates to the study of medicinal plants used by the tribals and other rural people of Bargarh district. The information has been collected by personal contact with these people. It reveals that 20 plant species (belonging to 20 genera and 16 families) are used by them for temporary or permanent birth control either as contraceptive or abortifacient. The plant parts used, mode of drug preparation and their doses are discussed.

KEY WORDS: Traditional use; Medicinal plants; Birth control, Tribals and rural people; Bargarh district

To Cite this article: Sen S K, Pradhan R, Pattnaik M R, Behera L M (2012), TRADITIONAL KNOWLEDGE OF MEDICINAL PLANTS AGAINST BIRTH CONTROL BY THE TRIBALS AND OTHER RURAL PEOPLE OF BARGARH DISTRICT IN WESTERN ODISHA, INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 670–677

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INTRODUCTION The use of wild plants is an integral part of the strong traditional and cultural systems and practice of tribes that have developed and accumulated over generations. The tribes depend predominantly on plants for food, clothing, housing, medicine, oil, agricultural implements, art, crafts and a host of other requirements. The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed (Maheswari, 1995). Population explosion is the greatest problem, India is facing today. Modern medicine has provided several preventive methods of contraception which are not safe and have serious side effect. Many countries have already banned the use of normal contraceptives because of its carcinogenic effects. The discovery of some herbal contraceptives, safe and sure is the very need of the hour. Herbal contraceptives were used even by the primitive people of ancient civilization to control fertility and to prevent pregnancy. Some earlier works from different parts of the country using plant materials relating to birth control, either as contraceptive or abortifacient are Bilore and Audichya (1978), Tewari and Chaturvedi (1981), Kishore and Bhat (1982), Hemadri and Rao (1983), Jain (1986), Murty et al., (1997), Shrivastava et al., (1999), Shah et al., (2009), Ekka (2012). Study area Odisha known as Orissa (prior to 5th Nov 2011), located on the east coast of Indian peninsula ranks second in tribal population with more than 8 million tribal people belonging to 62 different ethnic groups. Kondh, Binjhal, Saora, Santhal, Sabar, Bhatoda Bhumij, Gond, Bhuyan, Mundari, Juang, Koya, Diyayi etc are some of the tribes inhabiting different regions of Odisha (Saxena and Brahmam, 1994 – 96). Bargarh district (20°40’–21°49’N latitude and 82° 45’–83° 48’ E longitude), situated at the

western part of Odisha covers a land area of about 5837 sq Km and is inhabited by 1,478,833 people (GOI, 2011). The average annual rain fall is about 1500 mm. Six types of forests (Tropical Semi-evergreen, Tropical Dry-deciduous, Scrub-woodland, Bamboo, Scrub, Grassland) occur in this district and these diversified forest localities harbor plenty of medicinal plants (Mishra, 1994). Bargarh district offers immense scope for ethno-botanical studies as it possesses rich diversity of flora and a large number of economic and medicinal plants forming an integral part of the culture of the ethnic communities like Sahanra (Soara), Binjhal, Gond, Kondh, Munda, Kuli, Kalanga, Oran, Mirdha, Dharua, Kisan, Kharia and Parja. Earlier many ethnobotanical studies have been carried out in Odisha (Rai Choudhury et al., 1975; Saxena and Dutta, 1975; Saxena et al., 1979; Mudgal and Pal, 1980; Girach, 1992; Mishra, 1992; Satpathy and Panda, 1992; Sahoo and Mudgal, 1995). But medicinal plants available in this region have not yet been explored well except few previous records on taxonomy and ethno-medicinal knowledge published from time to time (Panigrahi, 1963; Brahmam and Saxena, 1990; Misra, 1990; Misra et al., 1994; Saxena and Brahmam, 1995, Misra and Das, 1998; Pradhan et al., 1999; Sen and Pradhan, 1999; Sen and Behera, 2003, 2007, 2008; Sen et al., 2005; Behera and Sen, 2008; Reddy and Pattanaik, 2009). But during the present survey an attempt has been made to explore some useful information on medicinal plants growing in wild and to gather knowledge on new or lesser known ethno-medicinal uses against birth control. METHODOLOGY Regular field trips were made during 2006– 2008 to collect the ethno-botanical information. The ethno-medicinal information was gathered through interviews and discussions with village headmen, traditional healers and elders of the clan. In general, the traditional healers are conservative by nature and do not share all the information regarding the use of plants as

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medicine. Data were recorded in the field notebook relating to the plant parts used, local name, place of collection, process of preparation, mode of administration and dosage. Voucher specimens were collected for authentication of information and for future references and they were identified with the help of Floras (Haines, 1921–25; Saxena and Brahmam, 1994–96). Herbarium specimens are lodged in the herbarium of Botany Department, Panchayat College, Bargarh.

Enumeration Plant species collected and identified during the survey were arranged alphabetically with their botanical name, family name in parentheses, local name, locality and collection number, medicinal uses, dosage and mode of administration (Table -1 and Table -2).

Table 1: List of ethnomedicinal plants used as contraceptive Sl.No.

Botanical name family

Local name, Locality & Voucher No.

Abrus precatorius L. (Fabaceae)

Gunj Barhaguda-55

Azadirachta indica A. Juss (Meliaceae)

Leem Nrusinghnath586

Butea monosperma (Lam.) Taub. (Fbabceae)

Phalsa Ramkhol-378

Crataeva religiosa G. Forst. (Capparaceae)

Barun Khandijharan320

Curcuma longa L. (Zingiberaceae)

Cynodon dactylon (L.) Pers. (Poaceae)

Dubla Barhaguda-98

Embelia ribes Burm.f. (Primulaceae)

Bidang Nrusinghnath171

Haldi

Udepali535

Ethno-medicinal Uses Seed (White variety) paste or powder with honey is taken once in empty stomach on the fourth day of the menstruation. This is used as contraceptive for one month. Before intercourse seed oil (3–4 drops) is applied over male sex organ to check possibilities of pregnancy. Seed ash (1 g) with milk is taken once daily in empty stomach for 3 days from the day of menstrual bath to ensure permanent sterility. Root powder of the plant and fruit powder of Piper longum are mixed together and is taken once daily in empty stomach for 10 days from the 4th day of menstrual cycle to prevent pregnancy for a period of 3 years. Bark paste (5 g) is taken once daily in empty stomach for 3 days from 5th day of menstrual cycle. Repeat it in next 3 years gap for permanent sterility. A piece of rhizome is grinded and taken once daily in empty stomach for 2 weeks from 5th day of menstrual cycle to check possibilities of pregnancy for a period of one year. Whole plant extract (2 teaspoon) with curd (2– 3ml) is taken in its own palm and swallowed on the day of menstrual bath. This is used as contraceptive for at least six months. Fruit of the plant, fruit of Piper longum and gum of Acacia catechu are mixed together in equal amount and the powdered (5 g) is taken once daily in empty stomach for 5 days from the day of menstrual bath to ensure permanent sterility.

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Hibiscus rosesinensis L. (Malvaceae)

Jasminum sambac (L.) Sol. (Oleaceae)

10.

Mentha arvensis L. (Lamiaceae)

Pudina Barhaguda-714 (Planted)

11.

Ricinus communis L. (Euphorbiaceae)

Jada Beherapali-231

12.

Sesamum indicum L. (Pedaliaceae)

Rayesh Ainlapali-114

13.

Tephrosia purpurea (L.) Pers. (Fabaceae)

Jharkulthia Barhaguda-792

Mandar Ainlapali-115

Malli

Bargarh162

Red flower (1 number) crushed with â&#x20AC;&#x2DC;kanjimaniâ&#x20AC;&#x2122; (fermented rice water) and is taken in empty stomach for 3days from the 1st day of menstrual cycle to ensure sterility for 3 years. Flower (red variety) paste with molasses (24 g) is taken for 14 days from the 4th day of the menstrual cycle to ensure sterility for 3 years. Root (6 g of white variety) paste is taken in empty stomach once daily for 7 days from the 4th day of menstrual cycle to ensure sterility for one year. Root paste (1.5 g) of the plant (white variety) is given to woman once daily in empty stomach in the morning of the 5th day of the menstrual cycle to ensure sterility for one year. Root paste (5 g) is taken once daily for 7 days after menstrual cycle for 3 months to induce sterility. Leaf powder (10 g) is taken by woman sometime before sexual relationship to delay conception for at least six months. Seed coat is removed and powdered. It is taken in empty stomach after the menstrual bath to remain sterile for a period of one year. Seed without seed coat (3 numbers) has to swallow after the menstrual bath. Women will not be pregnant at least for a period of 3 years. Immediately after sexual relationship rayesh-tel (1 teaspoon- seed oil of the plant) with a pinch of rock salt is given to woman. It is used as a temporary contraceptive. Root (12 g) paste is taken once daily in empty stomach for 7 days from the day of menstrual bath to prevent pregnancy.

DISCUSSIONS The inhabitants of Bargarh district use a variety of plants for medicinal purposes even today. Local people with all belief and faith consult the herbal medicine practitioner and other traditional healers to remain hale and healthy. However, the fast vanishing forest poses a threat for the sustainable use of the traditional practice. Hence, care must be taken for the conservation of forest, festival and fair in the tribal dominated areas before they go into the pages of history.

Several medicinal plants are used in traditional systems of medicine for birth control (Jain, 1991). The present study provides the information on 20 plant species under 16 families belongs to 20 genera used as birth control. The plant parts are used in the form of paste, powder, oil and ash. Both internal and external applications are involved in the treatment process. Altogether, 18 prescriptions (from 13 plant species) and 8 prescriptions (from 7 plant species) are used against contraception and abortion respectively. Out of

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18 prescriptions used as contraceptive, 6 prescriptions are used from seed followed by 5 prescriptions from root, 2 prescriptions from flower and one prescription each from whole plant, bark, leaf, rhizome and fruit. Again out of 8 prescriptions used as abortive, 4 prescriptions are used from root and one each

from tuber, stem, leaf and seed. These data are also cross-checked and are in agreement with Kirtikar and Basu (1991), Jain (1991), Ambasta et al. (1992), Chopra et al. (1996), Warrier et al. (1997), Paria (2005), Joshi (2006) and Patil (2008).

Table 2: List of ethnomedicinal plants used as Abortive Sl.No.

Botanical name & family

Erythrina suberosa Roxb. (Fabaceae)

Bambusa bambos L. Voss (Poaceae)

3. 4.

Gloriosa superba L. (Colchicaceae) Pergularia daemia (Forssk.) Chiov. (Apocynaceae)

Piper betle L. (Piperaceae)

Sapindus trifoliatus L. (Sapindaceae)

Solanum nigrum L. (Solanaceae)

Local name, Locality & Voucher No.

Ethnomedicinal Uses

A slightly crushed leaf petiole (2 in numbers) is placed on the female sex organ and a piece of Paldhua cloth is wrapped over it. Besides, a small piece of Ramkhol-372 root is crushed and is taken with jaggery to induce abortion. Stem powdered (10 g) is boiled in a glass of water and filtered and half a cup of the filtrate Baunsh and “fitkiri” or alum (1 g) are mixed together and Barhaguda-261 taken in empty stomach regularly for 3–10 days to induce abortion. Puraphul Tuber paste is applied below the naval to induce Nrusinghnath-46 abortion. Root paste (6 g) is taken in empty stomach once Uturli daily for 3–4 days from the 4th day of menstrual Ainlapali-746 cycle to induce abortion. The epidermal layer of the leaf petiole (3 Pan Bargarh- numbers) is removed and inserted into the female 145 (Planted) sex organ during bedtime at night to induce abortion even after 2–3 months of pregnancy. Ritha Seed pulp paste (6 g) is taken in empty stomach Nrusinghnathonce daily for 3–4 days from the 4th day of 484 menstrual cycle to induce abortion. Root paste (10 g) is applied below the naval to Kak-machi induce abortion. Kharmunda-156

CONCLUSION This paper highlights on the use of medicinal plants for birth control by the tribes and other rural people of Bargarh district. But it is not advisable to take any plant or plant part without the supervision of an experienced herbal medicine practitioner as dangerous side

effects in case of overdose. Especially the abortifacient plant should not be used by the pregnant woman in any case. As a recommendation, phytochemical, toxicological and pharmacological studies should be undertaken to show the efficiency level of these plants which are used for the purpose.

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REFERENCES Ambasta SP, Ram Chandran K, Kashyappa K, Chand R (1992). The Useful Plants of India, Publication and Information Directorate, CSIR, New Delhi, pp 1– 918. Behera LM, Sen SK (2008). Ethnobotany of Western Orissa, India. In: D.A. Patil (eds) Herbal Cures: Traditional Approach, Aavishkar Publishers, Distributiors, Jaipur, India, pp 316–331. Bilore KV, Audichya KC (1978). Some oral contraceptives family planning tribal way. J. Res. Ind. Med. Yoga and Homoeo. 13 (2): 104–109.

Jain S K (1991) Dictionary of Indian Folk Medicine and Ethnobotany, Deep publications, New Delhi, pp 1–311. Joshi SG (2006). Medicinal Plants, Oxford and IBH Co. Pvt. Ltd., New Delhi, pp 1– 491. Kirtikar KR, Basu BD (1991). Indian Medicinal Plants. Vol. 1-4. (Repn. Edition). Jayyed Press, Delhi-6, pp 12791. Kishore P, Bhat AV (1982). Oral contraceptives folk claims from Puri district Orissa. Bull. of Med. and Ethnobot. Res. 3(1): 65–67.

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Misra RC, Das P (1998). Inventory of rare and endangered vascular plants of Gandhamardan hill ranges in western Orissa. J. Econ. Tax. Bot. 22(2): 353– 357.

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Panigrahi G (1963). Gandhamardan Parbat, Orissa – A potential source of important indigenous drugs. Bull. Reg. Res. Lab. Jammu. 1: 111–116. Paria ND (2005). Medicinal Plants Resources of South West Bengal. Directorate of Forests, Government of West Bengal, Kolkata, pp 1–198. Patil DA (2008). Herbal Cures: Traditional Approach, Aavishkar Publishers, Distributiors, Jaipur, India, pp 1–396. Pradhan NB, Pradhan RN, Sen SK, Sahu P (1999). Some threatened noteworthy medicinal plants of Bargarh district (Orissa). Neo Botanica. 7(2): 97–100. Rai Choudhury HN, Pal DC, Tarafdar CR (1975). Less known uses of some plants from the tribal areas of Orissa. Bull. Bot. Surv. India. 17: 132–136. Reddy CS, Pattanaik C (2009). An Assessment of floristic diversity of Gandhamardan hill range, Orissa, India. Bangladesh J. Plant Taxon. 16(1): 29–36. Sahoo AK, Mudgal V (1995). Less Known Ethnobotanical uses of plants of Phulbani District, Orissa, India. Ethnobotany. 7: 63–67.

Saxena HO, Brahmam M, Dutta PK (1979). Survey of aromatic and medicinal plants in Orissa. J. Orissa. Bot. Soc. 1: 19–20. Saxena HO, Dutta PK (1975). Studies on ethnobotany of Orissa. Bull. Bot. Surv. India. 17: 124–131. Sen SK, Behera LM (2003). Ethnomedicinal plants used against skin diseases at Bargarh district in Orissa (India). Ethnobotany. 15 (1&2): 90–96. Sen SK, Behera LM (2007). Traditional use of some plants against gynecological disorders by the Tribals pf Ramkhol village forest of Barapahad hill range in Bargarh District (Orissa). Advances in Plant Sciences. 20 (2): 459–462. Sen SK, Behera LM (2008). Ethnomedicinal plants used by the tribals of Bargarh district to cure diarrhoea and dysentery. Indian J. Traditional Knowledge. 7(3): 425–428. Sen SK, Panda HS, Behera LM (2005). Cordia macleodii (Ehretiaceae) – A wonderful wound-healer from Bargarh District, Orissa. Ethnobotany. 17 (1&2): 189– 190. Sen SK, Pradhan NB (1999). Conservation of ethnomedicinal plants of Bargarh district in Orissa. Adv. Plant Sci. 12: 207–213.

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Shah GM, Khan MA, Ahmad M, Zafar M, Khan AA (2009). Observations on antifertility and abortifacient herbal drugs. African Journal of Biotechnology. 8 (9): 1959-1964. Shrivastava JL, Jain Seema, Dubey Abhilasha (1999). Ethno-medicine for antifertility used by the tribals in Bastar District of Madhya Pradesh. J. Eco. Tax. Bot. 23(2): 297–300.

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Tewari PV, Chaturvedi C (1981). Maternity and Child welfare in ayurvedic classics. J. Research in ayurveda and sidha. 11 (2): 107–175. Warrier PK, Nambiar VPK, Ramankutty C (1997). Indian Medicinal Plants (5 vols), Orient Longman Ltd., Chennai.

Conflict of Interest: None Declared

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Research article A STUDY ON ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS OF MEDICINAL PLANTS USING RAPD MARKERS Radhika K1, Singh Sumer 2, Gopinath S M3*, Ashwini Patil G M4 1, 2,

Department of Biotechnology, Singhania university, Pacheri Bari, Jhunjhunu (Rajasathan) Acharya Institute of Technology, Soldevanahalli, Hesaraghatta Road, Bangalore-560090 *Corresponding Author: Email Id: researchgopinath@gmail.com 3, 4

Received: 09/11/2012; Revised: 25/11/2012; Accepted: 30/11/2012

ABSTRACT Molecular genetic fingerprints of medicinal species were developed using Randomly Amplified Polymorphic DNA (RAPD) marker to elucidate the genetic diversity among the 18 species. DNA was isolated using the CTAB method. The amplification was accomplished by using 10 primers and the specific PCR working program. Three decamer-primers generated 250 RAPD fragments, of which 232 fragments were polymorphic with 96.84% of polymorphism. Some of the RAPD markers were useful for species discrimination and identification. Most of the RAPD markers studied showed different level of genetic polymorphism. Amplified fragment sizes ranged from 300 to 5000 bp. Pairwise Nei and Li’s similarity coefficient value ranged from 0.00-0.72 for 18 species of medicinal plants. A dendrogram was constructed based on the Unweighted pair group method using arithmetic averages. Cluster analysis of data using the UPGMA algorithm placed the 18 species of medicinal plants into four groups that are somewhat congruent with classification based on morphological characters proposed by earlier works. This analysis grouped all species into different clusters and clearly differentiated the medicinal plants into separate groups. This method of analysis can be helpful in selecting diverse parents and give broadness to the germplasm base of medicinal plants breeding programs in the future. KEY WORDS: Medicinal plants, RAPD, Genetic Diversity.

To Cite this article: Radhika K, Singh Sumer, Gopinath S M, Ashwini Patil G M, (2012), A STUDY ON ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS OF MEDICINAL PLANTS USING RAPD MARKERS, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 678–686

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INTRODUCTION

Conservation of Medicinal Plants

The world is endowed with a rich wealth of medicinal plants. Herbs have been the principal form of medicine in ancient India. Vital herbs lost their importance due to pharmaceutics-d revolution. They are also becoming popular as people strive to live healthy in the face of chronic stress and pollution and to treat illness with medicines that work in concert with bodyâ&#x20AC;&#x2122;s relied defense. Medicinal plants play a crucial role in the lives of rural people, in remote parts of developing countries with limited facilities for health care (Purohit and Prajapati, 2003).

A taxon is endangered when it is not critically endangered but is facing a very high risk of extinction in the wild in the near future (Ravikumar and Ved, 2000).

Around 70,000 plant species, from lichens to flowering trees have been used for medicinal purposes. Many species are used in herbal medicines and is used in unrefined or semiprocessed form, often as mixtures, which may also contain non-botanical ingredient. A few species are the sources of defined compounds used in the pharmaceutical industry. There is an international trade on medicinal plants used in herbal medicine and in the manufacture of pharmaceuticals. There is also a growing interest in obtaining samples of plant material and traditional knowledge about the uses of plants and also to explore commercial medicinal products. The scale of international trade in medicinal plants is difficult to assess, because of the paucity of reliable statistics and trade secrecy. Ancient Indian literature incorporates a broad definition of medicinal plants and considers "all" plant entities to be potential sources of medicinal substances. While all plant entities are potentially medicinal, only those plants are considered 'medicinal' whose medicinal use has already been discovered for human or animal application. Traditional medicinal plants have been used for human, veterinary and plant health. There are medical texts that deal with the treatment of cows; horses, elephants, and birds, there are also texts on subjects like Vriksh Ayurveda and Krishi Sastra that deal with the use of plants for controlling pests, treating plant diseases and as biofertilizers.

RAPD, markers behaves as dominant genetic markers, meaning that in a segregating population the homozygote of the parental type from which a given RAPD, ISSR and SSR band is amplified cannot be distinguished from the heterozygote, because the heterozygote also produces a RAPD, band. The only unambiguously assigned genotype is the homozygote of the other parental type (no RAPD band). The segregating F2 population may therefore be scored as follows band present: AA or Ab; band absent: bb. Remembering this fact, it is easy to select the populations best suited for the construction of genetic maps with RAPD markers (Banerjee, N.S., Manoj, P. And Das, M.R., 1999). MATERIALS AND METHODS The materials required and methodology of the present work is carried out at the Department of Biotechnology, Acharya Institute of Technology, Karnataka and Plant molecular biology laboratory, Department of Horticulture, Hulimavu Biotechnology Centre, Govt of Karnataka, Bangalore, India. In the year 2010â&#x20AC;&#x201C;2012. The materials used and methods followed in the study are presented here. Materials: Fresh, young, disease free leaves of 18 medicinal plants Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe Vera, Stevia rebaudiana, which were collected from the germplasm maintained at the different regions of Karnataka as medicinal plants germplasm conservation.

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supernatant was transferred to new tubes and repeated the same steps twice. The DNA was then precipitated by adding half volume of 5M NaCl, an equal volume of chilled propanol and incubated at 4ºC over night. DNA was pelleted by centrifuging at 20,000 rpm for 12 min at 4ºC. The pellets were dried after adding 70% ethanol and 1 ml of TE buffer was added to which 20 µl of RNase was added. This was incubated at 37ºC for one hour and added 300 µl of saturated phenol. It was mixed, centrifuged at 8000 rpm for 10 min at 4ºC. The supernatant was transferred to another tube and repeated the same process by adding phenol: chloroform and chloroform respectively. The supernatant was treated with equal volume of isopropanol and incubated at 4ºC for overnight. The DNA was pelleted by centrifuging at 12000 rpm for 20 min. The pellet was washed with 70% ethanol and dried. Around 300 µl of TE buffer was added to dissolve the pellet and stored at 20ºC for further use.

Table.1.Sequence information on RAPD oligonculeotide primers used for amplification and polymorphism study in 18 medicinal plants S.No RAPD Primers Sequence (5’-3”) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

OPC - 7 OPL -11 OPO - 08 OPAH - 15 OPAM - 20 OPAN - 01 OPAO - 01 OPAP - 20 OPAN - 05 OPAP - 10 OPAA - 01 OPAB - 01 OPAB - 05 OPAB - 14 OPAH - 13 OPAF - 02 OPAJ- 19 OPX - 20 OPA - 08 OPD - 13

GTCCCFACGA ACGATGAGCC GCTCCAGTGT CTACAGCGAG ACCAACCAGG ACTCCAGGTC AAGACGACGG CCCGGATACA GGGTGCAGTT TGGGTGATCC AGACGGCTCC CCGTCGGTAG CCCGAAGCGA AAGTGCGACC TGAGTCCGCA CAGCCGAGAA ACAGTGGCC CCCAGCTAGA GTGACGTAGG GGGGTGACGA

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Table 2. Showing the thermal profile used for RAPD Temperature

Time

Steps

93ºC

2 min

Initial denaturation

5 cycles of 93ºC

1 min

Denaturation

35ºC

1 min

Annealing

72ºC

2 min

Extension

72ºC

7 min

Final extension

4ºC

∞

Hold

Data Analysis DNA binding patterns generated by RAPD were scored as '1' for the presence of band and '0' for its absence. All RAPD assays were performed twice and only the reproducible bands were scored. A similarity matrix was generated using a dendrogram based on distance matrix data sets by applying Wards method for cluster analysis using 'STATISTICA' 5.0 computer program. RESULTS The genomic DNA of 18 medicinal plants viz, Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe vera, Stevia rebaudiana were amplified with oligonucleotides primers. RAPD analysis of medicinal plants using Primer OPAB– 05 The genomic DNA of 18 medicinal plants was amplified with decamer oligonucleotide primers such as OPAB-05 and as shown in Fig 1. The distinct and abundant RAPD fragments were recorded. The total numbers of bands generated were 51 RAPD gel profiles. The sizes of the RAPD bands were placed in between 300-5000 bp in length. The primer produced distinct polymorphic banding pattern in all the medicinal plant species, the number of

RAPD bands per primer were 2.8 as expected to sexually reproduce plants. The RAPD bands distributed in the plant is important to know the value of breeding patterns in medicinal plants. (Banerjee, N.S., Manoj, P. and Das, M.R., 1999). The number of RAPD bands was produced to reveal Mendelian inherited character, and number scoring revealed medicinal characters. The banding patterns are important and distinct in medicinal plants. The polymorphism was very high and RAPD values were useful to distinguish between the medicinal plant species, apparently diverse elements such as diploid and other exotic species character. The identification of RAPD is very unique in plants, because medicinal value and coupled with highly cross – pollinated and revealed heterozygous character. In the present data the plants like 1 showed one band and the remaining had revealed 7-3 RAPD bands respectively. Further, 1 and 2 medicinal plants showed 1 and 3 RAPD bands due to amplification of primer with the genomic DNA of these plant species. However, it was observed that some of the plants viz., 1112 have recorded 7 bands indicating diverse character compared to other plants. Polymorphic distribution as far as gene flow is concerned revealed high or low speciation. This has been used for various other calculations of medicinal plant breeding programs. Therefore, amplification of genomic DNA of these medicinal plants revealed moderate diversity among them.

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Fig 1. Gel profiles of 18 medicinal plants amplified with RAPD Primers –OPAB-05

RAPD analysis of 18 medicinal plants using Primer OPAB – 14 The data obtained in the present investigation revealed a total number of 111 RAPD bands. The genomic DNA of 18 medicinal plants amplified with OPAB –14 revealed both monomorphic and polymorphic RAPD bands. The distinct and abundant RAPD fragments were recorded. The total number of bands generated lie in between 300-5000bp in length. The primer produced medium low and high resolution of RAPD bands. The number of bands per primer was recorded at a maximum of 6.1 bands. However four bands were recorded in plants like 1-18 respectively. Despite, the plants revealed a total number of

111 bands, therefore the distribution of banding patterns is common, and one of the plants has revealed 6 RAPD bands due to the amplification of genomic DNA with primer OPAB –14. However, amplification showed very clear and distinct bands, and some of the medicinal plants like 1-18 have revealed four bands respectively. From this data it is possible to identify species specific band for medicinal plants for selection, in turn it helps for the cultivation of medicinal plants. The RAPD banding techniques are useful for selection multiplication and introgression of certain traits for breeding of medicinal plants as shown in Fig2.

Fig 2. Gel profiles of 18 medicinal plants amplified with RAPD Primers –OPAB-14

RAPD analysis of 18 medicinal plants using Primer OPAH -13 The data obtained in the present investigation revealed a total number of 88 RAPD bands. The genomic DNA of 18 medicinal plants amplified by OPAH -13 revealed both monomorphic and polymorphic

RAPD bands. The distinct and abundant RAPD fragments were recorded. The total number of bands generated lie in between 300–5000bp in length. The primer produced medium low and high resolution of RAPD bands. The number of bands per primer was recorded at a maximum of 4.8 bands. However, four bands were recorded in plants like 1–18 respectively.

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bands respectively. From this data it is possible to identify species specific band for medicinal plants for selection, in turn it helps for the cultivation of medicinal plants. The RAPD banding techniques are useful for selection multiplication and introgression of certain traits for breeding of medicinal plants as shown in Fig 3.

Despite, the plants revealed a total number of 88 bands, therefore the distribution of banding patterns is common, and one of the plants as revealed five RAPD bands due to the amplification of genomic DNA with primer OPAH -13. However, amplification showed very clear and distinct bands, and some of the medicinal plants from 1–8 have revealed 8

Fig3. Gel profiles of 18 medicinal plants amplified with RAPD Primers –OPAH-13

Fig 4. Dendrogram of 18 medicinal plants amplified with RAPD primer- OPAB-05 Tree Diagram for 18 Variables of Medicinal Plant Unweighted pair-group average Euclidean distances VAR1 VAR3 VAR2 VAR4 VAR6 VAR7 VAR8 VAR14 VAR15 VAR16 VAR5 VAR9 VAR10 VAR17 VAR18 VAR11 VAR12 VAR13 0

0.5

1.5

2.5

Genetic Distance

Fig 5. Dendrogram of 18 medicinal plants amplified with RAPD primer- OPAB-14 Tree Diagram for 18 Variables of Medicinal Plants Unweighted pair-group average Euclidean distances VAR1 VAR13 VAR7 VAR12 VAR2 VAR14 VAR15 VAR17 VAR18 VAR3 VAR4 VAR16 VAR5 VAR6 VAR10 VAR8 VAR9 VAR11 0

0.5

1.5

2.5

Genetic Distance

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Fig 6. Dendrogram of 18 medicinal plants amplified with RAPD primer- OPAH-13 Tree Diagram for 18 Variables of Medicinal Plants Unweighted pair-group average Euclidean distances VAR1 VAR5 VAR16 VAR6 VAR11 VAR7 VAR8 VAR9 VAR10 VAR12 VAR18 VAR2 VAR15 VAR4 VAR13 VAR3 VAR14 VAR17 0

0.5

1.5

2.5

Genetic Distance

DISCUSSION Genetic resources available for medicinal plant improvement are abundant within plant species. Even though a few species of medicinal plants occur naturally in India, many cultivated medicinal plant species do find their origin within the country especially India. Almost all the cultivated and naturally occurring medicinal plants which are classified under different family and species, cross pollinate with each other and produce fertile offspring showing no signs of sexual incompatibility characteristic of medicinal plant species. This fact suggests a close genetic or non-genetic relationship among the medicinal plants. The present study involves 18 medicinal plants with molecular characterization of RAPD, analysis for further supports this view. RAPD analysis of medicinal plants using Primer OPAB– 05. The results of the present investigation on genomic DNA of 18 medicinal plants viz , Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum ratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus Calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe Vera, Stevia rebaudiana were amplified with oligonucleotides primers OPAB– 05 revealed total of 51 RAPD bands Fig 1 and the Dendrogram of 18 medicinal plants amplified with RAPD primer- OPAB– 05 shown in fig 4 . Similar observations were

recorded by Girish Naik and Dandin 2006, Souframani and Gopalakrishna, 2004. Similar observations have also been made in other species at a cultivars level (Colombo et al., 1998, Banerjee et al., 1999, Das et al., 1998). RAPD analyses of 18 medicinal plants using Primer OPAB –14 were amplified revealed a total of 111 RAPD bands. With an average of 6.1 bands per primer, all the 18 medicinal plants exhibited 10 RAPD bands respectively. Whereas some of the medicinal plants such as Artemisia vulgaris, Mentha citrata and Piper longum have expressed more than 10 RAPD bands per primer as shown in Fig 2 and the Dendrogram of 18 medicinal plants amplified with RAPD primer- OPAB– 14 shown in fig 5. Further, a similar observation was made by Awasthi et al., (2004) Basha, S.D and Sujatha, M. 2007 in mulberry and medicinal plants. RAPD analysis of 18 medicinal plants using Primer OPAH -13. CONCLUSION The genomic DNA of 18 medicinal plants was amplified with decamer oligonucleotide primers In OPAB– 05 primer exhibited 51 bands with distinct and abundant RAPD fragments. Primer OPAB – 14 primer indicates 111bands with an average of 6.1 bands per primer. In the present investigation random polymorphic bands were seen to assess with different polymorphism. The minimum number clusters indicated gene flow in different medicinal plant species belonging to different family is very low.

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REFERENCES Akito Kaga, Norihiko Tomooka, Yoshinobu Egawa, Kazuyoshi Hosaka & Osamu Kamijima.1996.Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis. Euphytica 88:17–24. Banerjee, N.S., Manoj, P. And Das, M.R., 1999. Male sex associated RAPD markers in longum l. Curr. 77 (5): 693– 695. Basha, S.D and Sujatha, M. 2007. Inter and intra-population variability of Jatropa curcas (L.) characterized by RAPD and ISSR markers. Euphytica. 156:375–386. Chikkaswamy B.K, Rabin Chandra Paramik, Nagaraj Varadaraj H.L.Ramesh, M.Shivashankar and V.Sivaram 2007. Determination of genetic variation in Piper species using 4 C Nuclear DNA and RAPD marker. Int J Cytologia 72 (3): 243–349. Darokar.M.P, Rita Rai, Gupta A.K., Shasany A.K.Rajkumar.S.Sundaresan.V. And Khanuja S.P.S., 2003. Molecular assessment of germplasm diversity in Aloe species using RAPD and AFLP analysis. Journal of Medicinal and Aromatic Plant Sciences 25 354–61. Das, A.B., Rai, S and Das, P.1998. Karyotype analysis and 4C DNA content in some species of ginger (Zingiber officinale Rose.). Cytobios 93:175–84. Fabbri .A. Hormaza .J.L and Polito. V.S., 1995. Random amplified Polymorphic DNA analysis of olive (Olea europaea. L.) Cultivars. J. Amer. Soc. Hort. SCI., 120:538–542. Germplasm using RAPD analysis. Indian J. Genet 66: 287–292.

Girish Naik V., Särkar A., Sathyanarayana N. 2002. DNA Fingerprinting of Mysore Local and V-1 cultivars of mulberry (Morus spp) with RAPD markers. Indian J. Genet, 62 (3): 193–196. Hormaza, J.L., Dollo, L., and Polito, V.S, 1995. Determination of relatedness and geographical movement of Pistacia Vera L. (Pistachio\ Anacondisae) germplasm by RAPD analysis. Econ. Bot, 4: 349–358. Koller, B., Lehmann, A., Mc Dermott, J.M. and Gessier, C., 1993. Identification of apple cultivars using RAPD markers. Theor. Appl. Genet, 85: 901–904. Krammer, D., Afza, R., Weising, K., Kahl, G., and Novak, F.G. 1992.Oligonucleotide and amplification fingerprinting of wild species and cultivars of banana (Musa spp.). Bio/Technol. 10: 1030–1035. Nieise, F.P., Hormaza, J.I., and McGranolson, G.H. 1998. Molecular characterization and genetic relatedness among walnut (Jugloss regia L.) genotypes based on RAPD markers. Euphytica. 101:199– 206. Plomion, C., Bahrman,N., Durel,C.E. and Malley, D.M.O.1995. Genome mapping inpinus pinaster (Maritime pine) using RAPD and protein marker. Heredity 74:661–668. Porebski, S., Bailey, G. and Baum, B.R. (1997). Modification of a CTAB DNA extra action protocol for plants containing high polysaccharides and polyphenol components. Plant Molecular Biology Reporter, 15, 8–15. Purohit, S.S. and Prajapati, N.D. 2003. Medicinal Plants: Local Heritage with Global Importance, AGROBIOS News Let, 1 (8) 7–8.

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Souframanien. J and Gopalakrishna. T 2004. A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers. Theor. Appl. Genet. 109:1687–1693.

Source of Support: Nil

Vijayan K. Srivastava P.P. and Awasthi A.K., 2004. AnaIysis of phylogenetic relationship among five mulberry (Monas) species using molecular markers. Genome 47:439–448.

Conflict of Interest: None Declared

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Research article COMPREHENSIVE REVIEW ON HISTORICAL ASPECT OF YASHTIMADHU- GLYCYRRHIZA GLABRA L. Korhalkar Anagha1*, Deshpande Manasi2, Lele Priya3, Modak Meera4 1

Department of Gen.Pathology and Microbiology, Dental College and Hospital, Bharati Vidyapeeth Deemed University, Pune 411043, Maharashtra, India. 2 Department of Dravyagun Vignan, College of Ayurved, Bharati Vidyapeeth University, Pune 411043, Maharashtra, India 3 Department of Periodontology, Dental College and Hospital, Bharati Vidyapeeth Deemed University, Pune 411043, Maharashtra, India. 4 Department of Microbiology, Medical College, Bharati Vidyapeeth Deemed University, Pune 411043, Maharashtra, India. *Corresponding Author: anaghakorhalkar13@gmail.com; Phone: + 91 9823289223; Fax: 020-

24379163 Received: 27/10/2012; Revised: 06/12/2012; Accepted: 10/12/2012

ABSTRACT The knowledge of traditional medicine especially that of herbs and plants, inherited from ancestors is very useful. They are effective and more valuable than the synthetic pharmacological agents. Glycyrrhiza glabra Linn or Licorice is one of the most widely used medicinal herbs and is found in numerous traditional formulas. It has been used in medicine for more than 4000 years. The earliest record of its use in medicine is found in code Humnubari (2100 BC). It was also one of the important plants mentioned in Assyrian herbal documents (2000 BC). Hippocrates (400 BC) mentioned its use as a remedy of ulcers and quenching of thirsts. The drug was also mentioned by Theophrastus and Dioscorides. In traditional Siddha system of medicine licorice is used as a demulcent, expectorant, anti-tussive, laxative and sweetener. Historically, the dried rhizome and root of this plant were employed medicinally by the Egyptian, Chinese, Greek, Indian, and Roman civilizations as an expectorant and carminative. KEY WORDS: Licorice. History, Glycyrrhiza glabra, Yashtimadhu

To Cite this article: Korhalkar A, Deshpande M, Lele P, Modak M (2012), COMPREHENSIVE REVIEW ON HISTORICAL ASPECT OF YASHTIMADHU- GLYCYRRHIZA GLABRA L., Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 687â&#x20AC;&#x201C;693

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INTRODUCTION Glycyrrhiza glabra Linn. [GG] belongs to Fabaceae family and has been used since ancient times as a medicinal herb. It has been referred in Indian traditional medicine some 3,000 years ago. The active components of this plant have extensive therapeutic usage throughout the world and are subjected to enormous works in recent years. Licorice (Glycyrrhiza glabra) is also known as "sweet root". The word “Glycyrrhiza” is made from two Greek words: Glykys, meaning "sweet" and Rhiza, meaning "root" (M. Senthil Raja, et al., 2010) Licorice is one of the most widely used medicinal herbs and is found in numerous traditional formulas. The main active constituent Glycyrrhizic acid is approximately 50 times sweeter than sugar. (Anonymous, Indian Herbal Pharmacopoeia, 2002) Etymology The German name Süßholz sweet wood and its Dutch analogue zoethout are probably simply calqued from liquorice. The characteristic sweet taste of liquorice is also reflected in the Indian names. In Sanskrit, madhu means sweet, pleasant. This element is found in names for licorice not only in Sanskrit (madhuka and yashtimadhu from yashti stem, stalk), but also in modern names of both South and North India, e.g., Marathi jeshthamadha, Bengali yashthimodhu Telugu atimadhuramu and Kannada yashthimadhu. Outside of India, related names are Lithuanian saldymedis and Armenian madudag. The Proto-Indo-European root behind this element is MEDȹU honey, sweet for its linguistic affiliation. (The epicenter spices – Licorice, 2012) The Latin species name ‘glaber’ bald, hairless refers to the seed pods which have a smooth surface in other species of the genus, the fruits are pubescent. (Kumar Anil and Dora Jyotsna, 2012).

Vernacular names (Juniperandsage - Licorice, 2009) Sanskrit: Yashti-madhuh. Madhuka Kannada: Yastimadhuka, atimaddhura Bengali: Jashtimadhu, Jaishbomodhu Gujarat: Jethimadhu Hindi: Jothi-madh, Mulhatti Malayalam: Iratimadhuram Marathi: Jeshtamadha Oriya: Jatimadhu Tamil: Atimaduram Telugu: Atimadhuranu, Yashtimadhukam English: Licorice, Liquorice, Sweet wood Arab: Aslussiesa Persia: Ausareha mahaka France: Boisdoux Germany: Sussholz History of Licorice Egypt The history of Glycyrrhiza in the Western world as medicine and as food flavoring agent can be traced back 3000 years to the Egyptians and Assyrians. It is mentioned by Theophrastus, Pliny the Elder, Hippocrates, Culpepper and numerous other herbalists over the centuries. Glycyrrhiza is highly valued and extensively used in China. They classify it as a superior herb. It is used in small amounts in many formulas to harmonize the action of the other herbs. It is called the “peacemaker.”

(Juniperandsage - Licorice, 2009). Rome Roman legions considered licorice an indispensable ration for their long grueling campaigns. It was said that the soldiers could go up to 10 days without eating or drinking as the licorice properties helped to build stamina and energy, which allayed both hunger and thirst. In the year 1305, King Edward I, placed a duty on licorice sales, which went to help finance the repair of London Bridge. (Steven Foster, 2009). China In the Far East, references to the effectiveness of licorice are contained in the Shen Nong Ben Cao Jing, the first Chinese dispensory. In the Chinese book, 365 crude drugs are classified into three classes (upper:

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plants with lowest side-effects and nontoxic usefulness for health care; middle: plants that are nontoxic or possess only weak toxicity in whose use care must be exercised; lower: toxic and only for clinical use). Licorice is described as belonging to the upper class and is recommended for lengthening one’s life span, for improving health, for injury and swelling, and for its detoxification effect. One hundred and ten prescriptions are recorded in the earlier Chinese medicinal book ‘Shang Han Lun’, where 70 prescriptions include licorice. It is said that licorice is used in as many as half of all traditional Chinese medicine prescriptions. (Taro Nomura, et al., 2002). Japan In Japanese Pharmacopoeia, only G. glabra and G. uralensis are permitted to be used as licorice and licorice powder, and the other Glycyrrhiza species can be used as materials of licorice extract. (Benefits and dangers of Licorice, 2012). United Kingdom In UK, the Benedictine monks who migrated from Spain during the crusades brought the licorice plant to their monastery in ancient West Yorkshire. This was grown in the old town of Pontefract and the extracts were used to flavor drinks. Around 500 years ago, the locals started to make licorice candies known as Pontefract Cakes. While the plants do not exist anymore in Pontefract the candy is still made to this day. Unlike many other 'licorice' candies which are merely aped up by using aniseed, Pontefract cakes still contain pure licorice with molasses. (Benefits and dangers of Licorice, 2012). United state of America G. glabra is native to Eurasia, northern Africa and western Asia, where it grows up to 1,200 m above sea level. It has also been introduced to many countries, for example the USA where it is a weed of moist roadside sites. Liquorice is also cultivated as a crop plant, particularly in Russia, Spain and the Middle East (Kew.org – Glycyrrhiza glabra, 2012).

India In India, the licorice root carries the ancient Sanskrit name of 'Yasthimadhu' (sweet- stalk) and has been a mainstay of Ayurvedic and other traditional medicines. In ancient Ayurvedic system, more than 1250 preparations are described containing Yashtimadhu as one of its constituents. In traditional Ayurvedic medicine, herbs were used as special foods, serving to eliminate the excesses as well as strengthen the deficiencies, restore and rejuvenate. Licorice works on the digestive, respiratory, nervous, reproductive and excretory systems. It is an effective expectorant, often combined with ginger to help liquefy mucus and facilitate its discharge. Licorice is used to calm the mind, nourish the brain and increase the cranial and cerebrospinal fluid, and to benefit vision, voice, hair, complexion and stamina. (Chunekar K C, commentary on Bhavaprakasha Nighantu, 2008). Description of licorice root: Glycyrrhiza is a 4–5 feet shrub that grows in temperate climates. It is a member of the Fabaceae or pea family and looks similar to a large sweet pea plant. Above the ground the foliage forms on upright thin stems, pinnate leaves with 4–8 pairs of dark green elliptic leaflets 2–3 cms long of fern-like appearance. Young leaves feel slightly sticky to touch. Lavender/blue pea flowers 1cm long, form as axil clusters, followed by 2–3 cms long smooth, brown pods containing 1–7 brown kidneyshaped, pinhead-sized seeds. (Anonymous, The Ayurvedic Pharmacopoeia of India, 2007). Roots are 1–5 cms thick, have a brown woody appearance, a yellow colour internally, and with fiber that can be pulled apart like a long string. The mature plants have a long taproot that sends out creeping horizontal rhizomes, creating a tangled mass of underground growth. The rhizomes are harvested during fall of the third or fourth year and are shade dried for six months. To assure high quality of the roots, they are collected with

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a minimum 7 mm diameter and more than 30 cm in length, representing mature roots with high levels of active components. The sliced root is prepared with oblique cuts, with 10 mm diameter slices, having brown bark and yellow cortex and pith. Different Varieties of Glycyrrhiza glabra1. Glycyrrhiza glabra var. typica (spanish licorice) 2. Glycyrrhiza glabra var. glandulifera (Russian Licorice) 3. Glycyrrhiza glabra var. violacea (Persian licorice) Adulterants and SubstitutesManchurian liquorice is obtained from G. uralensis. It is pale chocolate brown in color with exfoliated cork and wavy madullary rays. It is free from sugar but contains glycyrrhizin. Russian loquorice is obtained from G. glabra var. glandulifera. (Kokate C K et al., 2004). Chemical ConstituentsThe chief constituent pf liquorice is a triterpenoid saponin known as glycyrrhizin. Glucyrrhizinic acid is a glycoside and on hydrolysis yields glycyrrhetinic acid, which has a trierpenoid structure. Another important constituent are flavonoides such as liquiritin and isoliquiritin. The Indian loquorice roots have shown the presence of 2methylisoflavones and a coumarin. (Kokate C K et al., 2004) Isoprenoid-substituted phenols of Glycyrrhiza glabra Three varieties of the species have been reported; Spanish licorice and Italian licorice are assigned to G. glabra var. typica, Russian licorice is G. glabra var. glandulifera, and Persian and Turkish licorices are G. glabra var. violacea. About 90 kinds of phenolic compounds have been isolated from the plants. About 50 of them are substituted with isoprenoid group(s), e.g., 3-methyl-2-butenyl (prenyl) group, 2, 2-dimethylpyran ring, etc. These G. glabra could be classified into two groups with the constituents of isoprenoid-

substituted flavonoids. Type I licorice is Spanish and Russian licorices. The main isoprenoid-substituted flavonoid of the plants is a pyranoisoflavan, glabridin. The 5-position of most flavonoids from the type I plants is unsubstituted, e.g., glabrene, glabrol, 3hydroxyglabrol, etc. Type II licorice is Chinese and Kyrghiz G. glabra. From these plants, both 5-unsubstituted flavonoids and 5-oxygenated flavonoids (e.g., 3′, 8-diprenylated dalbergioidin,have been isolated. Nevertheless, most flavonoids from these plants are 5hydroxy- or 5-methoxy-flavonoids. (Taro Nomura, et al., 2002). Traditional uses – (Kumar A and Dora J, 2012). A decoction of Madhuka or its powder was prescribed with honey in anemia. • Yashti mixed with cow‘s milk was prescribed for promoting lactation. • 10 g madhuka powder mixed with 10 g sugar, pounded with rice water was prescribed in metrorrhagia. • A confection of rice-milk, prepared with yashtimadhu was prescribed in hoarseness of voice. • Charaka prescribed 10g madhuka powder mixed with honey, followed by intake of milk, as an aphrodisiac and as an intellectpromoting tonic. • Charaka also prescribed a paste of liquorice and Picrorhiza kurroa with sugar-water as a cardiac tonic. • Charakadatta prescribed Yashtimadhu and Chandan powdered with milk, in haematemesis. • Sushruta prescribed the paste of yashtimadhu 10 g, in intrinsic haemorrhage. • In oedema, the paste of licorice, sesamum indicum and milk mixed with butter was prescribed. • Warm clarified butter mixed with licorice, was applied topically on wounds, bruises and burns. • A decoction of madhuka was applied on erysipelas. • A decoction of the root is a good wash for falling and graying of hair.

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PHOTOPLATES: 1. Licorice plant (G. glabra), flowering; 2. Licorice (G. glabra) flowers

Source: http://www.uni-graz.at/~katzer/engl/spice_phot.html#gly_gla. 3. Root part- Glycyrrhiza Glabra

Source: 101herbs.com

Dosage It is difficult to predict a dose appropriate for all individuals because individual susceptibility to various licorice preparations is vast. Nevertheless a daily oral intake of 1–10 mg of glycyrrhizin, which corresponds to 1–5 g licorice (2% glycyrrhizin), has been estimated to be a safe dose for most healthy adults. (Walker BR and Edwards CR, 1994). Studies of

DGL (Deglycyrrhizinated licorice) for peptic ulcers employed dosages ranging from 760– 2,280 mg DGL daily. (Thorne-Glycyrrhiza glabra monograph, 2005). Pharmacokinetics Research suggests that after oral administration of licorice in humans, the main constituent, glycyrrhizic acid, is hydrolyzed to glycyrrhetic acid by intestinal bacteria

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possessing a specialized ß-glucuronidase. Glycyrrhetic acid is 200–1,000 times more potent an inhibitor of 11-ß-hydroxysteroid dehydrogenase (involved in corticosteroid metabolism) than glycyrrhizic acid; therefore, its pharmacokinetics after oral intake are more relevant. After oral dosing, glycyrrhetic acid is rapidly absorbed and transported via carrier molecules to the liver. In the liver it is metabolized to glucuronide and sulfate conjugates, which are subsequently rehydrolyzed to glycyrrhetic acid. Glycyrrhetic acid is then reabsorbed, resulting in a significant delay in terminal clearance from plasma. After oral administration of 100 mg glycyrrhizin in healthy volunteers, no glycyrrhizin was found in the plasma but glycyrrhetic acid was found at < 200 ng/mL. In the 24 h period after oral administration, glycyrrhizin was found in the urine, suggesting it is partly absorbed as an intact molecule. (Thorne-Glycyrrhiza glabra monograph, 2005). DISCUSSION-

disorders in many part of the world. Conclusive evidence is now available regarding various molecular such as treterpenoids, polyphenols and flavonoids present in G. glabra. Their mechanisms of action on cellular level are also being proved. So it is justified to review the historical aspect in view of its future use to control and monitor various conditions. With vast data base available, it can be safely presumed that G. glabra will be a promising candidate for other yet less understood conditions. CONCLUSIONG. glabra appears to be a potential herbal drug which is used from Vedic era till date. It is widely used in various parts of the world for various disease conditions. More scientific studies are required to explore the medicinal properties of this antique plant to combat with the diseased world and get rid of them. Such a useful plant species has to be preserved and conserved for future to serve the generations to come.

G. glabra [Licorice] has been used for centuries successfully to control various

REFERENCESAnonymous, Indian Herbal Pharmacopoeia Revised New Edition, (2002), Indian Drug Manufacturers Association, Mumbai, pp 206–213.

Kew.org-Glycyrrhiza glabra, (2012), www.kew.org/plants-fungi/Glycyrrhizaglabra.htm. Retrieved in November 2012

Anonymous, The Ayurvedic Pharmacopoeia of India, (2007), Part 1, vol, 1, First edition, Government Of India, Ministry Of Health and Family Welfare, Department Of AYUSH, New Delhi.

Kokate C.K., A.P.Purohit and S.B.Gokhale, (2004), The Text book of Pharmacognosy, Nirali Prakashan, 28th edition, Pune, Maharashtra, India, pp 212–215.

Bhavamishra, Commentary by K.C. Chunekar, (2008), Bhavaprakasha Nighantu, 8th edition, Varanasi, Chaukhambha Bharati Acadamy.

Kumar Anil and Dora Jyotsna, (2012), Review on Glycyrrhiza glabra [Liquorice], Journal of Pharmaceutical and Scientific Innovation, vol. 1 (2), March- April, pp 1–4.

Juniperandsage Licorice, (2009), http://www.juniperandsage.com/materia /licorice.php, retrieved on 14/10/2012

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M. Senthil Raja, Imran Khan, Perumal. P, Surya Rao Srikakolapu and Srujana Divya Gotteti (2010), Quantitative analysis of glycyrrhizic acid in crude drug and its herbal formulation by UV Spectrophotometry, Scholars Research Library Archives of Applied Science Research, vol. 2 (2), pp 184–189. Steven

Foster (2009), www.stevenfoster.com/education/mono graph/licorice.html, retrieved in November 2012.

Taro Nomura, Toshio Fukai and Toshiyuki Akiyama, (2002), Chemistry of phenolic compounds of licorice (Glycyrrhiza species) and their estrogenic and cytotoxic activities, Appl. Chem., vol. 74, no. 7, pp. 1199– 1206.

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The Benefits and Dangers of Liquorice. (2012), http://hubpages.com docmo.hubpages.com, Herbal Remedies, The Benefits and Dangers of Liquorice, retrieved on 6th July 2012. The epicenter spices – Licorice, (2012) http://www.theepicentre.com/Spices/lico rice.html. retrieved on 4th January 2012 Thorne-Glycyrrhiza glabra monograph, (2005). www.thorne.com/altmedrev/.fulltext/10/ 3/230.pdf. Glycyrrhiza glabra Monograph, Alternative Medicine Review, Volume 10, Number 3, September, pp 235 Walker BR and Edwards CR, (1994), Licoriceinduced hypertension and syndromes of apparent mineralocorticoid excess, Endocrinol Metab Clin North Am, vol. 23, pp 359–377.

Conflict of Interest: None Declared

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Research article MOLECULAR MARKER STUDIES OF SOME MEDICINAL PLANTS FOR ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS Radhika K1, Singh Sumer 2, Gopinath S M3*, Kumar Pawan4 1, 2, 4

Received: 09/11/2012; Revised: 25/11/2012; Accepted: 30/11/2012

ABSTRACT Genetic relationships between eighteen medicinal were assayed with Random Amplified Polymorphic DNA (RAPD) markers, which distinguish individuals, as well as reflecting the inherent variation and interrelationships among the medicinal plants. 13 decamer RAPD primers were used in the present study. Over 77.23 reproducible bands were generated by RAPD primers, out of which, 77.23 polymorphic bands were identified, conferring 97.6% polymorphism. All the primers produced typical banding in each of the medicinal plants, suggesting the applicability of this test in medicinal plant identification. Most of the individuals of the test exhibited to have unique molecular genotype. Population genetics structure analysis of these species further revealed high genetic differentiation coefficients (Gst), the heterozygosity among populations (Ht) showed with the low gene flow (Nm) when 1stcluster was paired with other medicinal plants On the basis of these parameters and the results of cluster analysis. A dendrogram was constructed using Euclidean distance methods. Based on the number of bands the of the medicinal plants were grouped to form1-4 clusters. KEY WORDS: RAPD, Genetic Diversity, Medicinal plants.

To Cite this article: Radhika K, Singh Sumer , Gopinath S M , Kumar Pawan (2012), MOLECULAR MARKER STUDIES OF SOME MEDICINAL PLANTS FOR ASSESSMENT OF GENETIC DIVERSITY AND RELATIONSHIPS, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 694â&#x20AC;&#x201C;704

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 694â&#x20AC;&#x201C;704

INTRODUCTION Medicinal plants play a crucial role in the lives of rural people, in remote parts of developing countries with limited facilities for health care (Purohit and Prajapati, 2003). Medicinal plants have curative properties due to complex chemical substances of different composition, found as secondary plant metabolites in one or more parts of these plants. The plant metabolites, according to their composition, are grouped as alkaloids, glycosides, corticosteroids, and essential oils and so on. Medicinal plants are a living and irreparable resource, exhaustible if overused and sustainable if used with care and wisdom. The importance of medicinal plants has been overlooked in the past, however, at present medicinal plants are looked not only as a source of affordable health care but also as a source of income. Around 70,000 plant species, from lichens to flowering trees have been used for medicinal purposes. Many species are used in herbal medicines and is used in unrefined or semi-processed form, often as mixtures, which may also contain non-botanical ingredient. A few species are the sources of defined compounds used in the pharmaceutical industry. There is international trade in medicinal plants used in herbal medicine and in the manufacture of pharmaceuticals. There is also a growing interest in obtaining samples of plant material and traditional knowledge about the uses of plants and also to explore commercial medicinal products. The scale of international trade in medicinal plants is difficult to assess, because of the paucity of reliable statistics and trade secrecy. Molecular markers have particularly been suggested to be useful for confirmation of genetic fidelity in micro propagated tree species, where lifespan is quite long and performance of micro propagated plants could only be ascertained after their long juvenile stage in field conditions. In India also, studies have been conducted and research projects funded for research where lack of polymorphism shown through the use of molecular markers has been used to infer genetic fidelity. Recent advances in molecular genetics techniques and tools over

the past decade have provided considerable impetus to research in this direction. Techniques using molecular markers based on DNA polymorphism are coming up at a rapid rate. However, molecular marker study of these medicinal plants was carried out by previous researchers with sporadic reports. (Basha, S.D and Sujatha, M. 2007). and molecular marker studies in these medicinal plants, revealed a large gap, in view of the above the present investigation was undertaken on some of the medicinal plants such Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe vera, Stevia rebaudiana. However the present investigation was carried out on studies on molecular characterization using molecular RAPD markers in some medicinal plants. MATERIALS AND METHODS The materials and methodology of the present work are carried out at the Department of Biotechnology, Acharya Institute of Technology, Karnataka and Plant molecular biology laboratory, Department of Horticulture, Hulimavu Biotechnology Centre, Govt of Karnataka, Bangalore, India, in the year 2010â&#x20AC;&#x201C; 2012. The materials used and methods followed in the study are presented here. Materials: Fresh, young, disease free leaves of 18 medicinal plants Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe vera, Stevia rebaudiana, were collected from the germplasm maintained at the different regions of Karnataka as medicinal plants germplasm conservation (Darokar. M. P, Rita Rai, Gupta A.K., ShasanyA. K. Rajkumar. S. Sundaresan. V. And Khanuja S.P.S., 2003).

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DNA Extraction Fresh, young and disease free leaves of 6 different medicinal plants were collected and immediately kept on ice to reduce the nuclease activity. It was brought to the laboratory, weighed (2 g each), and frozen in liquid nitrogen and stored at 70ºC till further use. The DNA was extracted using the CTAB method (Porebski et al., 1997) with certain modifications. 2 g of fresh medicinal plants leaf material was ground into a fine powder using liquid nitrogen. The powder was then transferred to sterile centrifuge tubes and 12 ml of extraction buffer was added, mixed thoroughly and incubated 65ºC in a water bath for one hour with intermittent shaking. The tubes were brought to room temperature and centrifuged at 8000 rpm for 10 min at 4ºC. The supernatant was transferred to new tubes, 6 ml of chloroform: isoamyl alcohol (24:1) was added and mixed thoroughly. The tubes were centrifuged at 8000 rpm for 10 min at 4ºC. The supernatant was transferred to new tubes and repeated the same steps twice. The DNA was then precipitated by adding half volume of 5M NaCl, equal volumes of chilled propanol and incubated at 4ºC over night. DNA was pelleted by centrifuging at 20,000 rpm for 12 min at 4ºC. The pellets were dried after adding 70% ethanol and 1 ml of TE buffer was added to which 20µl of RNase was added. This was incubated at 37ºC for one hour and added 300 µl of saturated phenol. It was mixed, centrifuged at 8000 rpm for 10min at 4ºC. The supernatant was transferred to another tube and repeated the same process by adding phenol: chloroform and chloroform respectively. The supernatant was treated with equal volume of isopropanol and incubated at 4ºC for overnight. The DNA was pelleted by centrifuging at 12000 rpm for 20min. The pellet was washed with 70% ethanol and dried. Around 300 µl of TE buffer was added to dissolve the pellet and stored at 20ºC for further use. Data Analysis DNA binding patterns generated by RAPD, were scored as ' 1 ' for the presence of band and

'0' for its absence. All RAPD assays were performed twice and only the reproducible bands were scored. A similarity matrix was generated using adendrogram was constructed based on distance matrix data sets by applying Ward's method for cluster analysis using 'STATISTICA' 5.0 computer program. RESULTS The genomic DNA of 18 medicinal plants viz, Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe vera, Stevia rebaudiana were amplified with oligonucleotides primers OPD-13 revealed total of 172 RAPD bands, however, the medicinal plants such as Artemisia vulgaris, Artemisia pallens, Mentha piparita, Mentha citrata Bacopa moninierii and Stevia rebaudiana have showed 9 RAPD bands respectively. Further, oligonucleotides primers OPD -13 have showed 8-9 RAPD bands in each medicinal plant. The number and size of the amplification products varied depending upon the sequence of random primers and medicinal plants. The size of the amplified products ranged from 300-5000bp with an average of 9.5 bands per primer. The amplified products were generated with primers showing polymorphic bands fig 1 and 14. The genomics were amplified with OPD-07 revealed a total of 104 RAPD bands with an average of 6 bands per primer with the primer sequence of TTGGCACGGG. All the 18 medicinal plants exhibited 10 RAPD bands respectively. Whereas the some of the medicinal plants such as Artemisia vulgaris, Mentha citrata and Piper longum have expressed more than 10 RAPD bands. Shown in Fig 2 and 15. The primer OPL-11 shown 63 RAPD bands. And out of these 63 RAPD bands of the medicinal plants, Acorus calamus has revealed 10 RAPD bands. Whereas other medicinal plants have revealed 7–8 RAPD bands respectively as shown in fig 3 and 16.

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Table.1.Sequence information on RAPD oligonculeotide primers used for amplification and polymorphism study in 18 medicinal plants No

Sequence (5’-3”)

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

RAPD Primers OPC - 7 OPL -11 OPO - 08 OPAH - 15 OPAM - 20 OPAN - 01 OPAO - 01 OPAP - 20 OPAN - 05 OPAP - 10 OPAA - 01 OPAB - 01 OPAB - 05 OPAB - 14 OPAH - 13 OPAF - 02 OPAJ- 19

18 19 20

OPX - 20 OPA - 08 OPD - 13

CCCAGCTAGA GTGACGTAGG GGGGTGACGA

Fig.1: Gel profile 0f RAPDPrimar OPD-13

Fig.3: Gel profile 0f RAPD Primar OPL-11

GTCCCFACGA ACGATGAGCC GCTCCAGTGT CTACAGCGAG ACCAACCAGG ACTCCAGGTC AAGACGACGG CCCGGATACA GGGTGCAGTT TGGGTGATCC AGACGGCTCC CCGTCGGTAG CCCGAAGCGA AAGTGCGACC TGAGTCCGCA CAGCCGAGAA ACAGTGGCC

Fig.2: Gel profile 0f RAPD Primar OPD-07

Fig.4: Gel profile 0f RAPD Primar OPO-08

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 694â&#x20AC;&#x201C;704 Fig.5: Gel profile 0f RAPD Primar OPAH-23

Fig.6: Gel profile 0f RAPD Primar OPAM-20

Fig.7: Gel profile 0f RAPD Primar OPAN-05

Fig.8: Gel profile 0f RAPD Primar OPAO-01

Fig.9: Gel profile 0f RAPD Primar OPAP-20

Fig.10: Gel profile 0f RAPD Primar OPAN-05

Fig.11: Gel profile 0f RAPD Primar OPAP-10

Fig.12: Gel profile 0f RAPD Primar OPAA-01

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 694â&#x20AC;&#x201C;704 Fig.13: Gel profile 0f RAPD Primar OPAB-01

Fig .14.Dendrogram of RAPD primer OPD- 13 Tree Diagram for 18 Variables of Madicine Plants Unweighted pair-group average Euclidean distances VAR1 VAR2 VAR5 VAR9 VAR11 VAR12 VAR14 VAR16 VAR17 VAR4 VAR7 VAR8 VAR10 VAR13 VAR18 VAR3 VAR6 VAR15 0

0.2

0.4

0.6

0.8

1.2

Genetic Distance

Fig.15.Dendrogram of RAPD primer OPD- 07

Fig.16 Dendrogram of RAPD primer OPL-11

Tree Diagram for 18 Variables of Medicinal Plants

Unweighted pair-group average

Unweighted pair-group average Euclidean distances

Euclidean distances VAR1 VAR2 VAR13 VAR14 VAR5 VAR8 VAR18 VAR7 VAR9 VAR12 VAR15 VAR6 VAR3 VAR10 VAR4 VAR16 VAR11 VAR17

VAR1 VAR13 VAR2 VAR14 VAR7 VAR8 VAR18 VAR3 VAR4 VAR16 VAR6 VAR15 VAR5 VAR17 VAR11 VAR12 VAR10 VAR9 0

0.5

1.5

2.5

0.2

0.4

0.6

0.8

1.2

1.4

1.6

Genetic Distance

Linkage Distance

Fig 17.Dendrogram of RAPD primer OP0- 08

Fig 18.Dendrogram of RAPD primer OPAH- 15

Tree Diagram for 18 Variables of Medicinal Plant

Tree Diagram for 18 Variables of Medicinal Plants Unweighted pair-group average

Unweighted pair-group average Euclidean distances

Euclidean distances

VAR1 VAR13 VAR2 VAR7 VAR8 VAR14 VAR9 VAR11 VAR3 VAR15 VAR4 VAR16 VAR12 VAR5 VAR17 VAR6 VAR18 VAR10

VAR1 VAR2 VAR7 VAR8 VAR13 VAR14 VAR3 VAR9 VAR4 VAR10 VAR15 VAR16 VAR5 VAR12 VAR17 VAR18 VAR6 VAR11

0.5

1.5

0.5

Genetic Distance

1.5

2.5

Genetic Distance

Fig 19.Dendrogram of RAPD primer OPAM- 20

Fig 20.Dendrogram of RAPD primer- OPAN-01

Tree Diagram for 18 Variables of Mdicine Plant

Tree Diagram for 18 Variables of Medicinal Plant

Unweighted pair-group average

Unweighted pair-group average Euclidean distances

Euclidean distances VAR1 VAR2 VAR17 VAR18 VAR7 VAR8 VAR15 VAR16 VAR9 VAR10 VAR3 VAR4 VAR5 VAR6 VAR11 VAR12 VAR13 VAR14 0

VAR1 VAR7 VAR9 VAR2 VAR3 VAR4 VAR5 VAR6 VAR8 VAR10 VAR11 VAR12 VAR13 VAR14 VAR15 VAR16 VAR17 VAR18

0.5

Genetic

1.5 Distance

2.5

0.2

0.4

0.6

0.8

Genetic Distance

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1.2

1.4

1.6

1.8

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 694–704 Fig 21. Dendrogram of RAPD primer- OPAO-01

Fig 22. Dendrogram of RAPD primer- 0PAP-20

Tree Diagram for 18 Variables of Medicinal Plants

Unweighted pair-group average

Euclidean distances

VAR1 VAR6 VAR8 VAR3 VAR2 VAR7 VAR9 VAR12 VAR10 VAR15 VAR16 VAR17 VAR18 VAR4 VAR5 VAR11 VAR13 VAR14

VAR1 VAR2 VAR5 VAR8 VAR9 VAR10 VAR3 VAR4 VAR6 VAR7 VAR11 VAR12 VAR13 VAR15 VAR16 VAR17 VAR18 VAR14

0.5

1.5

0.2

Genetic Distance

0.4

0.6

0.8

1.2

Genetic Distance

Fig 23. Dendrogram of RAPD primer- 0PAN-05

Fig 24. Dendrogram of RAPD primer- OPAP-10

Tree Diagram for 18 Variables of Medicinal Plants

Unweighted pair-group average

Euclidean distances

VAR1 VAR2 VAR3 VAR5 VAR11 VAR12 VAR13 VAR14 VAR6 VAR8 VAR9 VAR10 VAR7 VAR15 VAR16 VAR17 VAR18 VAR4

VAR1 VAR14 VAR8 VAR2 VAR3 VAR4 VAR5 VAR7 VAR6 VAR13 VAR10 VAR12 VAR9 VAR11 VAR15 VAR18 VAR17 VAR16

0.5

1.5

0.5

Genetic Distance

1.5

Genetic Distance

Fig 25. Dendrogram of RAPD primer- OPAA-01

Fig 26. Dendrogra m of RAPD primer- OPAB-01

Tree Diagram for 18 Variables of Medicinal Plants

Unweighted pair-group average

Unweighted pair-group average Euclidean distances

Euclidean distances VAR1 VAR4 VAR5 VAR6 VAR8 VAR9 VAR10 VAR11 VAR12 VAR13 VAR14 VAR16 VAR17 VAR18 VAR3 VAR7 VAR15 VAR2 0

VAR1 VAR2 VAR3 VAR4 VAR5 VAR8 VAR6 VAR7 VAR9 VAR10 VAR11 VAR15 VAR16 VAR14 VAR12 VAR13 VAR17 VAR18 0.5

1.5

Genetic Distance

The genomic DNA of medicinal plants was amplified with the primer sequence OPO – 08 revealed a total of 89 RAPD bands. Out of these 89 RAPD bands indicating both monomorphic and polymorphic characters with an average of 4.9 per primer. A total of 89 bands amplified products were generated by random primer OPO – 08, as shown in the fig 4 and 17. The primer OPAH -15 has generated 55 bands shown in the fig. 5 and 18 with an average of 3.05 per primer. Of these 55 bands amplified products showing 99% fragments

0.5

1.5

2.5

Genetic Distance

were polymorphic in pairwise comparison. The highest number of RAPD bands was recorded in plants like 3, 4, 9, 10 and 16 and less bands was recorded in 1 and 2 plants species respectively. Primers such as OPAM– 2 and is shown in fig 6 and 19 were generated 97 RAPD gel profiles. In the present data the plants like 3, 4, 7, 8, 9, 10, 17 and 18 were recorded in 5–9 RAPD bands respectively. These medicinal plants revealed moderate diversity among them. OPAN – 01 primer as shown in fig 7 and 20. Generated a total of 43 RAPD bands.

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However, some of the plant species had shown two bands of RAPD in 10, 11, 12, 13, 14. 15, 16, 17 and 18 respectively. Further, some of the plants showed 3–4 RAPD bands respectively. Primer OPAO – 01 genomic DNA of 18 medicinal plants amplified with OPAO01 showed 45 RAPD bands both monomorphic and polymorphic. The number of bands per primer was recorded as 2.5 However, 2 to 4 bands were recorded in plants like 1 to 18 respectively. Despite, the other plants revealed a total number of 45 bands as shown in fig 8 and 21. The RAPD profile generated by primer OPAP – 20 produced a total number of 41 bands, with 2.2 RAPD bands per primer.. Therefore, the RAPD data was recorded in plants such as 1, 2, 3, 4, 5, 6, 7, 8 and 9 respectively. The genomic DNA of some of the varieties such as 10 to 18 plants was amplified with OPAP – 20 generated one RAPD bands per primer as shown in fig 9 and 22. The RAPD profile generated by primer OPAN – 05 produced a total number of 46 bands. The RAPD data was recorded in plants such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 revealed one band respectively. However the genomic DNA of 4th plant is not amplified with OPAN – 05 as shown in fig 10 and 23. Primer OPAP – 10 revealed a total number of 95 RAPD bands the number of bands per primer was recorded maximum of 5.2 bands. However, amplification showed very clear and distinct bands, and some of the medicinal plants like 7, 12, 15, 16 and 17 have revealed five bands respectively, as shown in Fig 11 and 24. Primer OPAA– 01As shown in fig 12and 25 The distinct and abundant RAPD fragments were recorded. The total numbers of bands were generated 40 RAPD gel profiles. In the present data, the plants like 3, 7, and 15 were recorded with 3 RAPD bands respectively. RAPD analysis of medicinal plants using Primer OPAB – 01 114 RAPD bands. The number of bands per primer was recorded maximum of 12.6 bands.

However, three bands were recorded in plants like 1 to 18 respectively. Despite, the plants revealed a total number of 114 bands, nine bands respectively. From this data it is possible to identify species specific band for medicinal plants for selection. DISCUSSION Genetic resources available for medicinal plant improvement are abundant within plant species. Even though a few species of medicinal plants occur naturally in India, many cultivated medicinal plant species do find their origin within the country especially India. Almost all the cultivated and naturally occurring medicinal plants and which are classified under different family and species, cross pollinate with each other and produce fertile offspring showing no signs of sexual incompatibility characteristic of medicinal plant species. This fact suggests a close genetic or non genetic relationship among the medicinal plants. The present study involving 18 medicinal plants with molecular characterization, of RAPD, analysis for further supports this view. RAPD analysis of 18 medicinal plants using primer OPD-13.The results of the present investigation on genomic DNA of 18 medicinal plants viz, Hemigraphis colorata, Marjorana hortensis, Artemisia vulgaris, Artemisia pallens, Ocimum sanctum, Ocimum basilicum, Ocimum gratissimum, Mentha piparita, Mentha citrata, Mentha spicata, Acorus calamus, Centella asiatica, Bacopa monierii, Piper longum, Piper nigrum, Clitoria ternatea, Aloe vera, Stevia rebaudiana were amplified with oligonucleotides primers OPD-13 revealed total of 172 RAPD bands, (Fig 1–14). Similar observations were recorded by Girish Naik and Dandin 2006, Souframani and Gopalakrishna, 2004. Similar observations have also been made in other species at a cultivars level (Colombo et al., 1998, Banerjee et al., 1999, Das et al., 1998,). RAPD analysis of medicinal plants using Primer OPD-07 were amplified with OPD-07 revealed a total of 104 RAPD bands. With an average of 6 bands per primer, all the 18 medicinal plants exhibited 10 RAPD bands respectively. Whereas the some of the medicinal plants such as Artemisia vulgaris,

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Mentha citrata and Piper longum have expressed more than 10 RAPD bands per primer. as shown in Fig 2–15. Further, similar observation was made by (Awasthi, Basha, S.D and Sujatha, M. 2007) in mulberry and medicinal plants. RAPD analysis of medicinal plants using Primer OPL-11. It is known from the literature that the molecular markers in medicinal plants are important observations (Girish Naik and Dandin 2002). The genomic DNA of 18 medicinal plants was amplified with the primer sequence of OPL –11 produced 3.5 bands per primer. A total of 63 RAPD band discrete amplified products were generated with the primer OPL – 11. Out of these 63 RAPD bands of the medicinal plants, the medicinal plant such as Acorus Calamus is revealed 10 RAPD bands. OPL – 11 have revealed 7–8 RAPD bands respectively as shown in fig 3–16. The Primer OPO – O8 revealed a total of 89 RAPD bands. Out of these 89 RAPD bands indicating both monomorphic and polymorphic characters in the medicinal plants like 1, 7, 8, 13, 15 and 18, significance of cross pollination is slow in these plants and out crossing is less as far as population genetics are considered. This study in accordance with (Balakrishna et al., 2000, Aswathi et al., 2004; Suryanarayan et al., 2002; Chikkaswamy et al., 2007). Fig 4-17. RAPD analysis of medicinal plants using Primer OPAH–15. A total of 13 decamer oligonucleotide primers was examined on the medicinal plants. All the random primers resulted in a distinct polymorphic banding pattern. The results obtained in the primer OPAH – 15 are shown in the fig.5-18. The similar observation made by (Souframanien and Gopalakrishna, 2004, Srivastava et al.,; 2004, Vijayan et al, 2004) with an average of 3.05 per primer with 55 bands. The DNA of the plants revealed some common RAPD bands except species of medicinal plants like 1, 2, 3, 4, 7, 8, 13 and 14 revealed common banding patterns, it clearly indicates that some of the plant species of medicinal plants is deviating from other plants as far as gene flow and elegant factor of RAPD sequence. The highest number of RAPD bands was recorded in plants like 3, 4, 9, 10 and 16 and less bands was

recorded in 1 and 2 plant species respectively. Primer OPAM– 20 and is shown in fig 6–19. The distinct and abundant RAPD fragments were recorded. The total numbers of bands were generated 97 RAPD gel profiles. The number of RAPD bands per primer were 5.3. The RAPD bands distributed in plant is important to know the value of breeding pattern in medicinal plants. The number of RAPD bands was produced to reveals.In the present data the plants like 3, 4, 7, 8, 9, 10, 17 and 18 were recorded with 5–9 RAPD bands respectively. Further, 1 and 2 medicinal plants was showed 5 RAPD bands due to amplification of primer with the genomic DNA of these plant species. The similar observation was made by (Colombo et al., 1998, Das et al., 1998;The Primer OPAN – 01.as shown in fig 7– 20. A total of 43 RAPD bands were generated. With 2.3 markers per primer. However, some of the plant species was shown two bands of RAPD in 10, 11, 12, 13, and 14. 15, 16, 17 and 18 respectively. The distribution of RAPD bands linked and encoding to certain quantitative traits of medicinal plants. This hypothesis has been corroborated from Plomion et al., (1995). Primer OPAO – 01. The data obtained in the present investigation revealed a total number of 45 RAPD bands. The number of bands per primer was recorded as 2.5 However, 2 to 4 bands were recorded in plants like 1–18 respectively. As shown in fig 8–21. Similar observation was made by (Colombo et al., 1998; Das et al., 1998). Primer OPAP – 20. The RAPD profile generated by primer OPAP – 20 produced a total number of 41 bands with 2.2 RAPD bands per primer. Therefore, the RAPD data was recorded in plants such as 1, 2, 3, 4, 5, 6, 7, 8 and 9 respectively. The genomic DNA of some of the varieties such as 10-18 plants was amplified with OPAP – 20 generated one RAPD bands per primer. Medicinal plants using molecular markers as unique powerful tool for taxonomy of medicinal plant identification (Akito Kaga et al., 1996, Bennett 1987, Bennett and Smith 1991). In this direction OPAP – 20 primers as showed low resolution of RAPD banding profiles to distinguish medicinal plants under differential speciation as shown in Fig 9–22. Primer OPAN

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– 05 produced a total number of 46 bands with 2 to 3 RAPD bands per primer. Therefore, the RAPD data was recorded in plants such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 revealed one band respectively. Primers as showed low resolution of RAPD banding profiles to distinguish medicinal plants under differential speciation as shown in Fig 10–23. Similar data have been reported by (Darokar.M.P,1998 and Das et al., 1998). Further, RAPD analysis of medicinal plants using Primer OPAP – 10, revealed a total number of 95 RAPD bands. The banding patterns are common and one of the plants as revealed 5 RAPD bands due to the amplification of genomic DNA with primer OPAP – 10. plants like 7, 12, 15, 16 and 17 have revealed five bands respectively as shown in Fig 11–24. This finding is similar to (Das et al., 1998, Das et al., 2001). The Primer OPAA– 01. The data obtained in the present investigation was similar with respect to the other crop plants reported by (Hormaza 1995), Olive (Fabbri et al., 1995), Walnut (Neieise et

al., 1998). CONCLUSION The genomic DNA of 18 medicinal plants was amplified with decamer oligonucleotide primers such as OPAA– 01 and is shown in Fig 12–25. The distinct and abundant RAPD fragments were recorded. The total numbers of bands were generated 40 RAPD gel profiles.Plants with Primer OPAB – 01 Shown 114 RAPD bands. The number of bands per primer was recorded maximum of 12.6 bands. However, three bands were recorded in plants like 1 to 18 respectively. Plants like 1 to 8 and 11 to 18 have revealed nine bands respectivelyas shown in Fig 13–26. This data is corroborated with the findings of (Krammer et al.,1992) and (Koller et al.,1993) in Apple plants. In the present investigation revealed 1–4 clusters indicated low gene flow in different medicinal plants species belonging to different family as reported by Stebbins evolution group of other plants.

Darokar.M.P, Rita Rai, Gupta A.K., Shasany A.K., Rajkumar.S., Sundaresan.V. and Khanuja S.P.S., 2003. Molecular assessment of germplasm diversity in Aloe species using RAPD and AFLP analysis.Journal of Medicinal and Aromatic Plant Sciences 25 354–361. Das, A.B., Rai, S and Das, P.1998. Karyotype analysis and 4C DNA content in some species of ginger (Zingiberofficinale Rose.).Cytobios 93:175–84. Fabbri .A. Hormaza .J.L and Polito.V.S., 1995. Random amplified Polymorphic DNA analysis of olive (Oleaeuropaea.L.) Cultivars. J. Amer. Soc. Hort. Sci., 120:538–542. Analysis of germplasm using RAPD. Indian J. Genet 66: 287– 292. GirishNaik V., Särkar A., Sathyanarayana N. 2002. DNA Fingerprinting of Mysore Local and V-1 cultivars of mulberry

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(Morusspp) with RAPD markers.IndianJ.Genet, 62(3):193–196.

inpinuspinaster(Maritime pine) using RAPD and protein marker. Heredity 74:661–668.

Hormaza, J.L., Dollo, L., and Polito, V.S, 1995. Determination of relatedness and geographical movement of Pistaciavera L. (Pistachio\ Anacondisae) germplasm by RAPD analysis. Econ.Bot, 4: 349– 358.

Porebski, S., Bailey, G. and Baum, B.R. (1997).Modification of a CTAB DNA extra action protocol for plants containing high polysaccharides and polyphenol components. Plant Molecular Biology Reporter, 15, 8–15.

Koller, B., Lehmann, A., Mc Dermott, J.M. and Gessier, C., 1993. Iden-tification of apple cultivars using RAPD markers.Theor. Appl. Genet, 85: 901– 904.

Purohit, S.S. and Prajapati, N.D. 2003 Medicinal Plants: Local Heritage with Global Importance, AGROBIOS News Let, 1(8) 7–8.

Krammer, D., Afza, R., Weising, K., Kahl, G., and Novak, F.G. 1992.Oligonucleotide and amplification fingerprinting of wild species and cultivars of banana (Musa spp.).Bio/Technol. 10: 1030–1035. Nieise, F.P., Hormaza, J.I., and McGranolson, G.H. 1998. Molecular characterization and genetic relatedness among walnut (JuglossregiaL.) genotypes based on RAPD markers.Euphytica.101:199– 206. Plomion, C., Bahrman,N., Durel C.E. and Malley D.M.O.1995. Genome mapping

Source of Support: Nil

Souframanien.J and Gopalakrishna.T 2004.A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers.Theor. Appl. Genet. 109:1687–1693. Suryanarayana, N. (2002) Indian Silk, 41(7):11–13. Vijayan K .Srivastava P.P. and Awasthi A.K., 2004.AnaIysis of phylogenetic relationship among five mulberry (Monas) species using molecular markers. Genome 47:439–448.

Conflict of Interest: None Declared

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Research article PHARMACOGNOSTICAL EVALUATION OF LEAF OF LIMNOPHILA RUGOSA ROTH. MERR. (SCROPHULARIACEAE) Acharya Rabinarayan1*, Padiya R H2, Patel E D3, Harisha C R4, Shukla V J5, Chauhan MG6 1

Associate Professor, Dept. of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat – 361008. India 2, 3 PhD scholar, Dept. of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat 4 Head, Pharmacognocy laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat 5 Head, Pharmaceutical Laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat 6 Visiting Professor, PGT-SFC-CELL, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat *Corresponding Author: Email: drrnacharya@gmail.com.

Received: 11/10/2012; Revised: 02/12/2012; Accepted: 04/12/2012

ABSTRACT The plant, Limnophila rugosa (Roth.) Merr. (Scrophulariaceae), is being used by the tribal people of Odisha as Bhringaraja, for the preparation of hair oil. Detailed pharmacognostical investigation on any part of this plant is lacking. The present paper deals with the detailed morphological and microscopical characters of its leaves, following standard procedures. The major microscopic characters noticed in the leaf are presence of anomocytic stomata in both the lower and upper epidermis of lamina and simple and glandular trichomes. Cluster and prismatic crystals of calcium oxalate in the parenchymatous cells of the ground tissue and mesophyll cells of the lamina were also noticed. An extra rudimentary vascular bundle is found to be located adjacent to one of the terminal end at the meristele of midrib. This study will be helpful for correct identity of L. rugosa.

KEYWORDS:

Pharmacognosy,

Limnophila

rugosa

Roth.

Merr.,

Bhringaraja,

Gandhamardana hills, Leaf drugs

To Cite this article: Acharya Rabinarayan, Padiya R H, Patel E D, Harisha C R, Shukla V J, Chauhan MG (2012), PHARMACOGNOSTICAL EVALUATION OF LEAF OF LIMNOPHILA RUGOSA ROTH. MERR. (SCROPHULARIACEAE), Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 705–711

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INTRODUCTION The knowledge of therapeutic properties of various herbs is usually found to be transmitted from generation to generation among tribal people. One of such plant, identified as Limnophila rugosa (Roth.) Merr. of the family Scrophulariaceae (Anonymous, The Wealth of India, 1998), is a small, annual, diffuse herb growing in places along streams, under shade across India (Haines. 1988). In Odisha, the traditional practitioners use this plant as a source plant of Bhringaraja (Brahmam et.al., 1990). The classical text of Ayurveda describes three types of Bhringaraja namely sweta (White), Neela (Blue) and Pita (Yellow). The botanical identity of Sweta and pita are reported as Eclipta alba (Linn.) Hask, Wedelia calendulacea (L.) Less. of (Asteraceae) respectively (Bapalal, 2005), where the blue variety is reported as the best among three types of Bhringaraja (Bapalal, 2005). Literary survey does not reveal much information regarding the scientific study on the plant, except its wound healing activity (Panda et. al., 2011). As the detailed pharmacognostical investigation on any part of this plant is lacking, the present study was designed to study the detailed morphological and microscopical characters of its leaves. MATERIALS AND METHODS Collection and authentication Bhringaraja, growing in Gandhamardana hill ranges, Balangir district of Odisha, India (Panda et. al., 2011) was identified as Limnophila rugosa Roth. Merr. of family Scrophulariaceae on the basis of the morphological characters of all the parts of the plant and comparing them with the reported characters mentioned in various floras (Haines, 1988) (Shah, 1978) (Hooker, 1997). The collected plant samples were shaken to remove adherent soil and dirt. The herbarium was prepared (Herbarium No. 6003) and stored in Pharmacognosy department for further documentation. The leaves were separated from the stem, washed with running fresh water and few pieces were stored in solution of FAA

(70% Ethyl alcohol: Glacial acetic acid: Formalin) in the ratio of (90:5:5) (Wallis, 1965) to utilize them for microscopic studies. The remaining leaves were dried under the shade and subjected for 60# powdering. Pharmacognostic studies Morphological characters were studied by observing the leaves as such and also with the help of the dissecting microscope. For detailed microscopical observation, thin transverse sections passing through the midrib, were taken and cleared with chloral hydrate and observed as such for the presence of any crystals, then were stained with Phloroglucinol and Hydrochloric acid to notice the lignified element like fibres, vessels etc. of the meristel and other parts. The figure of the section was drawn with the help of camera lucida. Photographs of the sections were taken with the help of canon Ixus 130 camera. The sections were stained with various reagents like Phloroglucinol followed by HCL for lignified elements (Anonymous, The Ayurvedic Pharmacopoeia of India, 2001). Quantitative microscopy: Determination of certain leaf constants like palisade ratio, stomatal number, stomatal index and vein islet number carried out as per the method described in Ayurvedic Pharmacopoeia of India. (Evans, 2009) RESULTS & DISCUSSION Macroscopical characters (Fig 2.a&b) Leaves simple, thin, oblong, lanceolate, opposite, sub sessile, stipulate, measures about 1.5–4 cm × 1–2 cm, serrate, acute, base symmetrical, petiole winged, surface – glabrous, Upper dark green, lower light green in colour, covered with fine hairs, venation – reticulate, veins – obscure on upper surface, prominent on the lower surface, midrib strong, sub veins 4–5, lateral veins finely divided, astringent bitter in taste with characteristic odour.

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 705â&#x20AC;&#x201C;711

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 12 | December 2012 | 705–711

Table 1 : Quantitative microscopy: Parameter

Mean

Stomatal number

Upper = 6 – 8 Lower = 8 – 10 Upper = 11.66

Stomatal index

Lower = 13.55 Palisade ratio

4.8

Vein islet number

Vein termination

Microscopical characters: (fig 1b & fig 2.c-h) Diagrammatic TS passing through the midrib is convexly protruding at the lower side and flat or slightly depressed on the upper side. It shows an arc of centrally located meristele than band of collenchymatous tissue under both epidermis and wide dorsiventral laminar extension on its lateral side. Detail TS shows an upper and lower epidermis covered with thick cuticle, embedded at places with stomata and bearing simple and glandular trichomes usually located more on the midrib. Cells of upper epidermis are tubular in shape, bigger in size and unlike the cells of lower epidermis which are irregular and smaller in size and shape. Simple trichomes are unicellular to multicellular. Unicellular trichomes are very few, short, conical and with warty cuticle. Multicellular trichomes are short, uniseriate, 2– 3 celled, straight or slightly bent, with warty cuticle, collapsed cell at places or with thread like terminals with number of stomatal openings. Glandular trichomes are sessile with 3–4 celled spherical head, few other trichomes being with unicellular stalk and bulbous spherical head. Transverse section through midrib shows centrally located meristele. Meristele shows rows of radially running vessels alternating

with medullary rays and a narrow band of phloem tissue lying underneath it. A groups of few cells were located underneath the upper epidermis and a layer of collenchymatous cells were located underneath the lower epidermis were embedded in the parenchymatous cells of the ground tissue and mesophyll cells of the lamina. Elevated portion of epidermis seen at some places. Cluster and prismatic crystals of calcium oxalate are embedded in the parenchymatous cells of the ground tissue and mesophyll cells of the lamina. An extra vascular bundle is located adjacent to one of the terminal end at the meristele. Powder characteristics (Fig 1.c) Powder of leaf is greenish in colour having characteristic odour and astringent bitter taste. Diagnostic characters of the powder shows: •

Plenty of glandular trichome, sessile with 3–4 cell head, with short unicellular stalk, unicellular head with stalk, sessile glandular trichome with tri-cellular head, sessile glandular trichome with fourcellular head with stalk, sessile glandular trichome with four celled head, sessile glandular trichome with unicellular head, sessile glandular trichome with collapsed cell and warty head and sessile multicellular glandular trichome

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•

Simple trichome with thread like terminals, very short unicellular warty trichome, bicellular simple warty trichome, simple warty trichome with stalk and multicellular simple warty trichome. Fragments of the upper and lower epidermis of the lamina in surface view, the cells of the upper epidermis somewhat thick walled and bears wavy margin. The lower and upper epidermal cells are embedded with anomocytic stomata. Transversely cut fragments of lamina, showing one to two rows of palisade cells and spongy parenchymatous cells underneath the lower epidermis loaded with prismatic and rosette crystals in the spongy parenchyma. Fragments of upper and lower epidermis in surface view embedded with anomocytic stomata. The cells of the upper epidermis are bigger in size, with faintly sinuous anticinal walls unlike the cells of the lower one which more wavy and smaller in size. Prismatic, rosette and acicular crystals of calcium oxalate scattered throughout the powder. Fragments of annular and spiral vessels.

CONCLUSION Leaf of L. rugosa shows the presence of anomocytic stomata in both the lower and upper epidermis of lamina and simple and glandular trichomes. It has cluster and prismatic crystals of calcium oxalate in the parenchymatous cells of the ground tissue and mesophyll cells of the lamina. An extra rudimentary vascular bundle is found to be located adjacent to one of the terminal end at the meristele of midrib. ACKNOWLEDGEMENTS The authors are thankful to Director IPGT & RA, Gujarat Ayurved University, Jamnagar and Dept. of AYUSH, for providing financial support and other facilities to carry out the research work. We express our thankfulness to Mr B N Hota, Rtd. DFO, Govt. Of Odisha; Mr Govind Baba, traditional practioner; Mr Pareswar Sahu, Pharmacognosy expert; Purrna Chandra Dhal, plant collector; Mr Malaya Das, Forest Range Officer, Govt. of Odisha and other tribal practitioners who helped us during drug collection at Gandhamardan hills, Balangir and Bargarh, Odisha.

REFERENCES Anonymous, The Ayurvedic Pharmacopoeia of India, Part-I, Volume-I, First edition, (2001) Ministry of Health and Family Welfare, Government of India, Department of Indian Systems of Medicine & Homoepathy; p.no. 139. Anonymous, The Wealth of India, Raw Materials (1998). Vol: L-M, CCIR, New Delhi, India; p.no.115, 116. Bapalal, (2005). Some controversial drugs in Indian Medicine, Chaukhambha Orientalia, Varanasi; p.no.155.

Brahmam M & Saxena HO, (1990). Ethnobotany of Gandhmardan Hills – Some Noteworthy Folk-Medicinal Uses. Ethnobotany, Vol.2; p.no. 71–79. G. L. Shah (1978). Flora of Gujarat State, PartI, Shri. K. A. Amin, Registrar, Sardar Patel University, Vallabh Vidyanagar; p.no. 525–527, 549–550. Haines HH, (1988). The Botany of Bihar and Orissa, Part 3-4. Bishansingh Mahendra Palsingh, Dehradun; p.no.625

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Panda Anima & Mishra Malaya (2011) Ethnomedicinal survey of some wetland plants of South Orissa and their conservation, Indian Journal of Traditional Knowledge, Vol. 10 (2); p.no. 296–303. Sir Hooker J. D. (1997). Flora of British India, Vol. IV, B.M. Palsingh, Dehradun,

Source of Support: Nil

p.no.265. Wallis T.E, (1965) Analytical microscopy, Third edition, J. & A. Churchill ltd. (London); p.no. 123–132 & 210–215. Wiliam Charles Evans, (2009) Trease and Evans Pharmacognosy, Sixteenth Edition. Saunders Elsevier, London; p.no. 569, 570.

Conflict of Interest: None Declared

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Research article EVALUATION OF DEHA PRAKRITI WITH SPECIAL REFERENCE TO SWETA PRADARA- A CROSS SECTIONAL STUDY Dhiman Kamini1*, Vardhan Monika2, Dhiman K S3, Sharma Naresh4 1

Reader, Department of Stree Roga & Prasooti Tantra, IPGT & RA, Jamnagar, Gujarat, India Lecturer, Deparment of BP, D. B. Ayurveda College, Mandi Gobindgarh, Punjab, India 3 Professor & HOD, Department of Shalakya, IPGT & RA, Jamnagar, Gujarat, India 4 Prof. and HOD, dept of Basic Principles. R. G. Govt. P.G. Ayu. College. Paprola, Kangra, H. P. *Corresponding Author: kd44ayu@yahoo.co.in 2

Received: 06/11/2012; Revised: 06/12/2012; Accepted: 08/12/2012

ABSTRACT Sweta Pradara (Discharge per vagina) is a common genital health problem of females which affects the efficiency of women. Recurrent nature of disease puts a challenge in front of medical fraternity to develop some extra protocol for proper management of disease. In the description of Deha Prakriti (body constitution), it has been stated that particular doshik prakriti persons are prone to develop that type of doshaj vikaras. The present study was planned in the direction to evaluate Deha Prakriti of patients of Sweta Pradara and their relationship to prevent the occurrence and recurrence of the disease. In the present work, overall 45 patients of Sweta Pradara were selected to study their Deha Prakriti and the relationship of Deha Prakriti and Sweta Pradara was studied. KEY WORDS: Sweta Pradara, Prakriti, Vaginal discharge, Yoni Vyapad.

To cite this article: Dhiman Kamini, Vardhan Monika, Dhiman K S, Sharma Naresh (2012), EVALUATION OF DEHA PRAKRITI WITH SPECIAL REFERENCE TO SWETA PRADARA - A CROSS SECTIONAL STUDY, Global J Res. Med. Plants & Indigen. Med., Volume 1(12), 712–721

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INTRODUCTION Sweta Pradara is a very common but challenging female genital health problem with discharge per vagina. It is a clinical feature of many Yonivyapadas (Gynaecological diorders) (Charaka 700 BC), yet sometimes Sweta Pradara is so much pronounced that it may be labeled as an independent disease. A similar condition of abnormal vaginal discharges has been mentioned in modern literature under the terms Leucorrhoea. Although it may be caused by physiological conditions like pregnancy or intrauterine contraceptive devices, in the vast majority of cases it is caused by Reproductive Tract Infections (RTIs) and very often by Sexually Transmitted Infections (Hawkes S and Santhya KG, 2005) (STIs) and other infective disorders e.g. Trichomoniasis, Candidiasis, Chlamydia etc. Though curative treatment is available, yet these disorders are recurrent and associated with great discomfort to women (Dutta DC 2007). In the last 20 years incidence is doubled. 10% to 41% of women have had Leucorrhoea at least once in their life. 75% of women experience at least one episode of vaginal yeast infection during their life time. Almost 45% of women experience two or more episodes of vaginal yeast infection per year (Hurley R. 1979 & 1981). According to WHO every year 333 million new cases of curable Vulvovaginal infections (VVIs) are registered. A study in India has shown that the prevalence of reproductive tract infections 5â&#x20AC;&#x201C;37.0% based on symptoms, 36.7% - based on laboratory investigations, 31% with Candidiasis, 02% with Trichomoniasis, 45% with Bacterial Vaginosis (BV), 03% with Gonorrhea. (Puri K.J. et al., 2000) Although the reasons are not clear, some of the factors are: lack of awareness, cultural and economic barriers (Mamdani M 1999). Faulty dietary habits & lifestyle, rapid urbanization, excessive work load etc. and individual errors like self negligence, shame, hesitation to submit to doctor, tendency of women to hide the symptoms and bear the agony of infection in silence until it leads to serious complications

like ectopic pregnancy, infertility etc. (Garg S et al., 2000 ) have thus increased the rate of incidence. Keeping these facts in view, there is a need of development of some protocol so as to decrease the incidence as well as recurrence of this disease. Even in Ayurveda, the preventive aspect has been mentioned prior to curative aspect. In the description of Deha Prakriti (body constitution), it has been stated that particular doshik prakriti persons are prone to develop that type of doshaj vikaras (Charaka, 700 BC). Ancient scholars have also described measures for avoiding occurrence of these diseases by modifications in diet and lifestyle (Vagbhatta, 5th A.D). Even if a person is afflicted with particular disease, similar modifications can be added to treatment regimen so as to shorten the duration of treatment and prevention of recurrence (Charaka, 700 BC). Therefore assessment of Deha Prakriti of patients of a particular disease can be a great tool in the hands of physician in preventing the occurrence and recurrence of that disease. Thus the present study was an attempt in this direction to evaluate Deha Prakriti of patients of Sweta Pradara and their relationship. MATERIALS AND METHODS The patients presenting with clinical features of Sweta Pradara were selected for the present study from Stri Roga and Prasuti Tantra O.P.D. of hospital affiliated to R.G.Govt.Post Graduate Ayurveda College, Paprola, Dist. Kangra (H.P) and subjected to meticulous assessment of their Deha Prakriti based on a standardized proforma irrespective of age, caste, religion, socio-economic status etc. The proforma was prepared in the department of Basic Principles of the college, after taking in consideration all features of deha prakriti described in various classics (Charaka, 700 BC). The Proforma consists of three parts as (i) Historical factors (ii) Physical factors (iii) Psychological factors. Besides this, gyaenocological examination and

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investigations have also been made so as to understand the relationship between this disease and their Deha Prakriti. Complete description regarding the details of each research case was recorded in proforma. Inclusion Criteria: • Patients presenting with complaint of abnormal discharges per vaginum. • Patients willing for registration. Exclusion Criteria • Patients having any other systemic disorder, • Patient having physiological discharge, • Patients having vaginal discharge due to foreign body Method of Study: Type of the study: Observational-analyticalcross sectional study Plan of the study: In present study the Deha Prakriti of selected patients was assessed on the basis of prepared exhaustive proforma. This proforma encompasses all of the features detailed in Ayurvedic classics regarding Doshic Prakriti types. Total 45 patients were selected and the nature of the study was explained to them and their consent was obtained. Investigations: For the purpose of assessing the general condition of the patient and to exclude other pathologies as well as to know the cause of vaginal discharges, routine blood examination, routine and microscopic urine examination and microscopic examination of vaginal discharge after Gram Stain were performed in all the selected patients. Observations: In the present work, overall 45 patients of Sweta Pradara were included to study their Deha Prakriti (Physical Constitution). From the observations, it was noticed that maximum number of patients were from the age group of 21−30 years i.e. 48.90%, followed by age group 31−40 years i.e. 35.50%. It is observed here that maximum numbers of patients i.e. 51.10% were educated up to higher secondary level. Maximum numbers of patients i.e. 78.00% were housewives and 8% were office workers. Maximum number of patients i.e. 57.80% were

of lower socioeconomic status. Most women i.e. 93.30% were resident of rural area. Sleep pattern was normal in maximum number of (73.30%) patients. Maximum number of patients (93.30%) had regular sleep followed by 06.70% patients having irregular sleep. Most of the patients (95.60%) registered were doing heavy exertional work. Majority of the patients (86.70%) had prakrita malapravritti (Daefecation), though 13.30% patients had Vibandha (Constipation). Majority of the patients (55.60%) experienced low abdominal pain, followed by general weakness (28.90%). In this study maximum of the patients (51.10%) had complaints since 2−5 years. Majority of the patients (68.90%) had normal age of menarche followed by 26.70% patients having menarche of more than 14 years. Majority of the patients (80.00%) had normal duration of menstruation. In this study most of the patients (71.10%) had normal inter- menstrual period followed by 22.20% patients having 24–26 days interval. Maximum patients (66.70%) had moderate amount of blood loss, followed by scanty menstruation in 22.20%. Majority of patients (71.10%) had regular menstruation, while 28.90% had irregular menstruation, 86.70% patients were multiparous. Majority of the patients (64.40%) had tube ligation whereas 11.10% had not used any contraception. 55.60% patients had no history of garbhasrava or garbhapata (Abortion or miscarriage), while 44.40% had history of garbhasrava and garbhapata. 51.10% of patients had retroverted uterus, while 48.90% had anteverted uterus. Most of the patients (93.30%) had normal uterine size. In 60.00% of patients uterus was freely mobile. The fornices were non tender in 55.60% patients, 57.80% patients had white discharge, while 42.20% patients had yellowish white discharge (Figure 2). Majority of the patients (26.70%) had pichchhila (mucoid) and puyasama (muco-purulent) type (26.70%) of discharge (Figure 3). Majority of the patients (66.70%) had Kashtaartava (Dysmenorrhoea), 40.00% had Maithuna asahishnuta (Dyspareunia), and 28.90% had Yathochita Kala Adarshan (Menstrual irregularities). According to calculation of Prakriti it was

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observed that maximum patients (75.6%) Prakriti. (Table -1) included under study were of Kapha Pittaja Table:1. Calculation of Deha Prakriti Sr.

Name

(Initial of the name of the patient) 1. K 2. P 3. T 4. R 5. M 6. S 7. M 8. S 9. M 10. J 11. S 12. A 13. R 14. P 15. N 16. N 17. A 18. S 19. V 20. S 21. K 22. P 23. S 24. K 25. N 26. S 27. R 28. A 29. K 30. V 31. S 32. M 33. S 34. L 35. S 36. U 37. M 38. M 39. S 40. R 41. R 42. S 43. A 44. V 45. V

Individual Scores Vata

Pitta

10.4 7.5 9.6 17.4 8.9 5.4 13.8 10.7 6.5 13.8 6.9 10.5 2.3 5.5 7.8 5.8 5.4 13.8 6.6 13.3 7.3 12.9 10.5 5.9 10.4 12.5 11.5 7.8 5.2 9.5 3.9 6.3 6.5 9.6 7.4 7.7 5.9 5.5 8.4 5.7 7.5 6.7 9.9 5.8 5.2

6 12 8.2 8.2 10.2 9.7 11.7 11.7 7 11 7.3 7 9.2 5.7 9 10.5 9.4 10.5 13.3 10.4 18.7 10.4 11.8 11.2 12.9 11 10 11 11.9 13.6 10.7 7.5 7.8 14 12 9.8 13.1 9.8 10.5 10.4 10.2 14.4 13.4 11.5 11

Kapha

20.8 24.3 25.7 23.2 22.5 30.8 18.5 26.6 23.5 19.2 21.4 26.2 35 31.1 20.9 29.4 28.2 22 26.4 17.5 26.2 24.3 29.7 18.7 29.5 24.9 22.9 25.7 26.6 21 24.7 31.3 27.9 18.9 24.1 21.3 27.9 24 23.6 30 28.2 25.8 17.5 21.4 26.5

Individual Percentage Vata

23.1 16.7 21.3 38.7 19.8 12 30.7 23.8 14.4 30.7 15.3 23.3 5.1 12.2 17.3 12.9 12 30.7 14.7 29.5 16.2 28.7 23.3 13.1 23.1 27.8 25.5 17.3 11.5 21.1 8.7 14 14.4 21.3 16.4 17.1 13.1 12.2 18.7 12.7 16.7 14.9 22 12.9 11.5

Pitta

16.7 33.3 22.7 22.7 28.3 26.9 32.5 32.5 19.4 30.5 20.3 19.4 25.6 15.8 25 29.2 26.1 29.2 36.9 28.9 51.9 28.9 32.8 31.1 35.8 30.5 27.8 30.5 33 37.8 29.7 20.8 21.7 38.9 33.3 27.2 36.4 27.2 29.2 28.9 28.3 40 37.2 31.9 30.5

Relative Percentage

Kapha

Total

Vata

43.3 50.6 53.5 48.3 46.9 64.2 38.5 55.4 48.9 40 44.6 54.6 72.9 64.8 43.5 61.2 58.7 45.8 30.6 36.4 54.6 50.6 61.9 38.9 61.4 51.9 47.7 53.5 55.4 43.7 51.4 65.2 58.1 39.4 50.2 44.4 58.1 50 49.2 62.5 58.7 53.7 36.4 44.6 55.2

83.1 100.6 97.5 109.7 95 103.1 101.7 111.7 82.7 101.2 80.2 97.3 103.6 92.8 85.8 103.3 96.8 105.7 82.2 94.8 122.7 108.2 118 83.1 120.3 110.2 101 101.3 99.9 102.6 89.8 100 94.2 99.6 99.9 88.7 107.6 89.4 97.1 104.1 103.7 108.6 95.6 89.4 97.2

27.8 16.6 21.8 35.3 20.8 11.6 30.2 21.3 17.4 30.3 19.1 23.9 4.9 13.1 20.2 12.5 12.4 29. 17.9 31.1 13.2 26.5 19.7 15.8 19.2 25.2 25.2 17.1 11.5 20.6 9.7 14 15.3 21.4 16.4 19.3 12.2 13.6 19.2 12.2 16.1 13.7 23 14.4 11.8

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Pitta

20.1 33.1 23.3 20.7 29.8 26.1 32 29.1 23.5 30.1 25.3 19.9 24.7 17 29.1 28.3 27 27.6 44.9 30.5 42.3 26.7 27.8 37.4 29.8 27.7 27.5 30.1 33 36.4 33.1 20.8 23 39.1 33.3 30.7 33.8 30.4 30.1 27.8 27.3 36.8 38.9 35.7 31.4

Kapha

52.1 50.3 54.9 44 49.4 62.3 37.8 49.6 59.1 39.5 55.6 56.1 70.4 69.8 50.7 59.2 60.4 43.3 37.2 38.4 44.5 46.8 52.5 46.8 51 47.1 47.2 52.8 55.4 42.6 57.2 65.2 61.7 39.6 50.3 50. 54 55.9 50.7 60 56.6 49.4 38.1 49.9 56.8

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Table 2. Distribution of 45 patients of Sweta Pradara according to Grading of Signs and Symptoms Symptom/Sign

Volume

Offensive smell

Itching

Dyspareunia

Dysuria

Vaginitis

Cervicitis

Grade 0 I II III 0 I II III 0 I II III 0 I II III 0 I II III 0 I II III 0 I II III

Kaphaja (K) Kapha Vataja (KV) Kapha Pittaja (KP) No. of Pt. Percentage No. of Pt. Percentage No. of Pt. Percentage 0 0 0 0 2 4.4 2 4.4 3 6.7 17 37.8 4 8.9 1 2.2 13 28.9 0 0 1 2.2 2 4.4 2 4.4 0 0 13 28.9 4 8.9 3 6.7 14 31.1 0 0 1 2.2 6 13.3 0 0 1 2.2 1 2.2 1 2.2 4 8.9 22 48.9 4 8.9 1 2.2 8 17.8 1 2.2 0 0 4 8.9 0 0 0 0 0 0 2 4.4 0 0 22 48.9 1 2.2 2 4.4 10 22.2 3 6.7 3 6.7 2 4.4 0 0 0 0 0 0 3 6.7 3 6.7 27 60 2 4.4 1 2.2 6 13.3 1 2.2 1 2.2 1 2.2 0 0 0 0 0 0 3 6.7 5 11.1 28 62.2 2 4.4 0 0 6 13.3 1 2.2 0 0 0 0 0 0 0 0 0 0 1 2.2 1 2.2 11 22.2 1 2.2 1 2.2 15 33.3 3 6.7 3 6.7 7 15.5 1 2.2 0 0 1 2.2

Table 3. Distribution of 45 patients of Sweta Pradara according to Microscopic examination of stained vaginal discharge Vaginal discharge microscopy No. of Patients Percentage Staphylococci/ 26 Bacteria 57.80% Streptococci Diplococci Bacilli Candida

Fungus Pus cells Clue cells Epithelial cells

9 7 10 34 20 45

20.00% 15.50% 22.20% 75.60% 44.40% 100%

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According to the obtained data: (Table -2) 1. Regarding volume of discharge, maximum number of patients (37.80%) were having grade II and 28.90% grade III discharge respectively were of Kapha Pittaja Prakriti. 2. Regarding offensive smell of discharge 31.10% of Kapha Pittaja Prakriti had grade I offensive smell. 3. 17.80% patients had grade I itching and 8.90% patients of Kapha Pittaja Prakriti had grade II itching. In Kaphaja Prakriti 8.9% patients had grade I itching. 4. 13.30% patients of kapha pitttaj prakriti had grade I dysuria and 2.20% had grade II dysuria. 5. 22.20% patients of kapha pitttaj prakriti had grade I and 4.40% had grade II dyspareunia. In Kapha Vataja Prakriti, 6.70% patients had grade II dyspareunia 6. 13.30% patients of kapha pitttaj prakriti had grade I vaginitis. 7. In Kapha Pittaja Prakriti 33.30% patients had grade I cervicitis.

patients (100%) had epithelial cells, 75.60% patients had pus cells. Maximum patients (42.20%) had cervicitis, 35.50% patients had bacterial vaginosis, 22.20% had candidiasis, and 6.70% patients had physiological discharge. In the table -1. 1.To maintain maintain anonymity of the patients, only initial is used (e.g. M for Mamta). 2. Individual scores indicate the presence of vataja, paittika and kaphaja characters present in the patient out of total 45 vataja, 36 paittika and 48 kaphaja characters mentioned in samhitas (Ayurvedic literature). 3. Individual percentages have been calculated from individual scores asIndividual score for particular dosha x 100 Total characters for that particular dosha 4. Relative percentage have been calculated as â&#x20AC;&#x201C; Individual % for particular dosha x 100 Total % of characters of tridoshas

According to Microscopic examination of stained vaginal discharge (Table-3), all the Figure no. 1 Prakriti Prakriti

11.1 13.3 Kapha Pittaja Kapha Vataja Kaphaja 75.6

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Figure no. 2 Colour of Discharges Colour of discharges

42.2 White Yellowish white 57.8

Figure no. 3 Consistency of Discharge Consistency of discharge

11.1

26.7

13.3

Pichchila Puyasama Tanu Dadhisama Ghana

15.5 26.7

DISCUSSION The prakriti (constitution) of a person generated by prevailing state of doshas in shukra (semen) and artava (ovum) at the time of conception (Charaka, 700 BC ). In spite of fundamental similarities in the mankind, we find dissimilitude from individual to individual. The factors responsible for these differences are multifarious and they together exert effect on constitutional, temperamental, psychological and spiritual make up of each individual. How far and in what way the metaphysical (atman

and purava-janmakrita karma), psychological (sattava and emotional state of mother), constitutional and environmental factors (both intra-uterine and external) influence the development of human prakriti can be understood only by deliberating on the essence of determinants of human organism. In the description of Deha Prakriti, it has been stated that particular doshic prakriti persons are prone to develop that type of doshaj vikaras (Charaka, 700 BC). Acharyas have also described measures for avoiding

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occurrence of these diseases by modifications in diet and lifestyle. Sweta Pradara (Abnormal vaginal discharge) is exclusively a disease of female reproductive tract. The state of woman health is indeed completely tied up with the culture in which she lives in, as well as the way she orchestrate her life as an individual. Owing to the complicated structure of the female body, women are subjected to a large number of problems connected with genital system. Sweta pradara is a common condition and it affects the efficiency of women. It sometimes leads to infertility and ectopic pregnancy. Recurrent nature of disease puts a challenge in front of medical fraternity to develop some extra protocol for proper management of disease. In present study analysis of prakriti of patients of sweta pradara was done, so that with the help of prakriti assessment, physician may be able to direct the patient about prevention of the disease, avoid its recurrence and if disease is already existing; help in shortening the cause of treatment by following indicated dietetic regimen and conduct for specific prakriti. In present study, it was observed that most of the patients (48.90%) were between the age of 21â&#x2C6;&#x2019;30 years, thus incidence of Sweta Pradara was maximum in active reproductive age group. Various diseases included under Sweta Pradara are sexually transmitted as males harbor pathogens in their urethra and prostate. Thus during child birth, foreign organisms may be found in the lower part of vagina which produce a low grade vaginitis and give rise to discharge. Incidence of septic abortion and puerperal sepsis is also maximum in this age group, as a result of which acidity of the vagina is reduced and foreign organisms such as coli form bacilli and other pathogens grow. Laceration of cervix during childbirth leads to some degree of chromic cervicitis and hence vaginal discharge. According to Ayurveda, it is the period of Pitta dominance and also of the regression of Kapha i.e. borderline period of Kapha regression and Pitta

dominance may be responsible for discharging and inflammatory conditions because Srava is a pittaja-nanatamaja disease/symptom. Various Yonivyapadas having Sweta Pradara as a clinical feature e.g. Acharana, Aticharana, Atayannda, Upapluta, Karnini belong to this reproductive age group as excessive sexual act has also been mentioned as main etiological factor of these Yonivyapadas. Data from observation revealed that majority of patients (57.80%) were from lower socioeconomic status, the fact clearly highlights the causative factor of disease as lack of proper hygiene, plays major role in provoking the disease. Low backache was observed in 53% patients, which indicates that when patient is suffering from white discharge the same aama dosha induced inflammatory pathology may be affecting the pelvic muscles and joints. Majority of the patients (55.60%) experienced low abdominal pain; pain in lower abdomen, urinary complaints and menstrual irregularity are due to associated pelvic inflammations/ infections, while generalized weakness is itself a causative factor for occurrence of sweta pradara. In this study maximum of the patients (51.10%) had complaints since 2â&#x2C6;&#x2019;5 years, this indicates that sweta pradara is a chronic disease. Shwetapradara neither causes mortality nor morbidity in affected women therefore patients do not consult the physician until it creates major vaginal discomfort or other associated problems like severe backache, body ache etc and this negligence converts the disease to chronic state. Majority of the patients (68.90%) had normal age of menarche. Majority of the patients (80.00%) had normal duration of menstruation, most of the patients (71.10%) had normal inter menstrual period, 88.90% patients had normal fresh menstrual blood. Majority of patients (71.10%) had regular menstruation. It reveals that menstrual cycle is not affected until the infection reaches up to uterus and creates chronic PID. These types of

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menstrual patterns indicate that kapha when dominating does not alter menstrual pattern. 86.70% patients registered were multiparous which indicates that improper management of puerperal period leads to invasion of various pathogens and cervical trauma due to repeated deliveries results in sweta pradara. In such conditions vata gets vitiated due to repeated deliveries and excessive strain or trauma to cervix during labour leads to development of various Yonivyapadas (gyneacological disorders) of which sweta pradara is a clinical feature. Cervicitis is also an important cause of vaginal discharge. It was observed significantly that in early stage cervicitis does not show change in normality of the menstrual cycle. But it presents symptoms that may be due to chronic nature of the disease. Maximum of the patients (42.20%) had cervicitis, 35.50% patients had bacterial vaginosis, 22.20% had candidiasis, and 6.7% patients had physiological discharge. 82.08% patients had mandagni i.e. reduced digestive fire. In Ayurvedic classics, it is mentioned that “rogaha sarve api mandagno”(Generally all diseases are produced by weak digestive activity) which lead to aama formation and Shwetapradara may have occurred due to vitiated/ saama doshas. From the calculation of prakriti it was analyzed that maximum patients (75.6%) included under study were of Kapha Pittaja Prakriti followed by 13.3% of Kapha Vataja Prakriti and 11.1% of Kaphaj Prakriti (Figure 1). It signifies that Kapha was dominant dosha in the Prakriti of patients suffering from Sweta Pradara, supporting the dictum that person of particular doshik deha prakriti are more prone to suffer from diseases originating from vitiation of that particular dosha. Vaginal discharge observed was mostly of sweta varna with picchila guna (kaphaja sava). Besides, Kandu (itching) was also a prominent feature in the patients of Sweta Pradara. Kandu has been described as a feature of Vikrita Kapha (Vagbhatta, 5th A.D) as well as of Vikrita Pitta.

Another feature of the disease, Gandha (Offensive smell) has also been mentioned as a characteristic feature of Vikrita Pitta (Vagbhatta, 5th A.D.) While exploring the various conditions in which Shwetapradara is described, kapha is considered as main causative dosha by its vitiated Snigdha and Pichchhila properties. Aacharya Sushruta has stated that Pooya or suppuration is not possible without Kapha (Vagbhatta, 5th A.D). Aacharya Vagbhata has also considered Kapha as a main dosha responsible for Shopha or inflammation. CONCLUSION Sweta Pradara (kaphaj disorder) appeared more prevalent in dvandwaj prakriti having kapha as a part i.e. Kapha pittaja, supporting the hypothesis that particular doshaj prakriti is prone to that type of doshik illness. Thus it can be concluded that disease and prakriti are interrelated. Symptoms alone should not be used to direct treatment in instances. The importance of determination of prakriti cannot be underestimated in the management of a disease. As a matter of fact this is an advantage with Ayurvedic fraternity. Treatment failures should prompt assessment of Prakriti of a particular patient to have better results. Further studies are required to establish this cost effective modality for the development of preventive aspect of Ayurveda, with following suggestions and modifications; • Large sample study should be done with addition of objective parameters i.e. blood grouping etc. • Clinical studies with modifications in ahara vihara to passify the Kapha & pitta in the patients of sweta pradara should be done to view effect on duration of course of treatment. ACKNOWLEDGEMENT The authors wish to gratefully acknowledge the active support and co-operation Dept. of Basic Principles and other staff in the College.

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REFERENCES Charaka, Commented by Sharma RK, Bhagwan Dash (2009) Charaka Samhita Chaukhambha Sanskrit Series Office, Varanasi. Chikitsa Sthana, Vimana thana Dutta D C (2007) Pelvic Infection, Textbook of Gynaecology. NCBA, Calcutta (India); 116Garg S, Sharma N, Saha R, Sahay R, Gulati N. (2000) Sociological and epidemiological study of symptomatic and asymptomatic RTIs/STIs amongst women in an urban slum. New delhi, Department of Preventive and Social Medicine, Maulana Azad Medical College, (Draft report) Hawkes S and Santhya KG. (2005). Diverse realities:sexually transmitted infections and HIV inIndia. Sexually Transmitted Infections suppl_1 (78), 131.

Hurley R, De Louvois J. (1979). Candida vaginitis. Postgraduate Med J . 55 (1), 645–7. Hurley R. (1981), Recurrent Candida infection. Clin Obstet Gynecol; 8:208–13 Mamdani M. (1999) Management of reproductive tract infections in women: lessons from the field . In: Pachauri S (ed.) Implementing a reproductive health agenda in India: the beginning. Population Council, regional office, South and East Asia region, New Delhi, India pp 435–506. Puri KJ, Madan A, Bajaj K. (2003) Incidence of various causes of vaginal discharge among sexually active females in age group 20–40 years. Indian J Dermatol Venereol Leprol; 69:122–5. [PUBMED] Vagbhatta (2008). Ashtanga Sangraha. 2nd ed. Varanasi: Chukhambha Sanskrit Series A. Sa. Su., Ni. Sthana.

Source of Support: Nil

Conflict of Interest: None Declared

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