GJRMI - Volume 3, Issue 2, February 2014 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI - Volume 3, Issue 2, February 2014 MEDICINAL PLANTS RESEARCH Bio-technology – Short communication CONVENTIONAL METHOD FOR BORIVILIANUM Sant. et Fernand

SAPONIN

EXTRACTION

FROM

CHLOROPHYTUM

Sharma Rohit, Saxena Nidhi, Thakur Gulab S, Sanodiya Bhagwan S, Jaiswal Pallavi

33–39

Pharmacology HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDROALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA Roy Deb Saumendu, Karmakar Prithivi Raj, Dash Suvakanta, Chakraborty Jashabir, Das Biswajit

40–47

Phytochemistry IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING ELICITORS Tamandani Ehsan Kordi, Valizadeh Jafar, Valizadeh Moharam

48–56

Review INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS OF NANDA DEVI BIOSPHERE RESERVE – A REVIEW BASED ON PREVIOUS STUDIES Singh Rahul Vikram

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF CONVOLVULUS QUAMOCLIT L., OF THE FAMILY CONVOLVULACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

57–66

Short communication CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM CHLOROPHYTUM BORIVILIANUM Sant. et Fernand Sharma Rohit1*, Saxena Nidhi2, Thakur Gulab S3, Sanodiya Bhagwan S4, Jaiswal Pallavi 5 1, 2, 3, 4, 5

Plant Biotechnology Laboratory, R&D Division, Tropilite Foods Pvt. Ltd., Davars Campus, Tansen Road, Gwalior-474002 (M.P.), India. *Corresponding Author: accessrohit25@gmail.com; Mob: +919755594040

Received: 07/12/2013; Revised: 25/01/2014; Accepted: 31/01/2014

ABSTRACT Saponins are imperative non-volatile chemical compounds valued for several medicinal properties. The pharmaceutical use of saponins for semi-synthesis of steroidal drugs makes it an essential element of life with a diverse range of properties including antimicrobial, insecticidal, haemolytic, aphrodisiac, foaming and emulsification. The tuberous roots of Chlorophytum borivilianum always remains a major source for isolation of saponin. A conventional efficient method was developed for saponin isolation from in-vivo and in-vitro samples of C. borivilianum by delipidization and deproteinization with petroleum ether and chloroform leading to development of a whole new process for saponin isolation. Protocol was tested with saponin confirmatory test followed by thin layer chromatography. KEY WORDS: Saponin, Chlorophytum borivilianum, delipidization, steroid.

Cite this article: Sharma Rohit, Saxena Nidhi, Thakur Gulab S, Sanodiya Bhagwan S, Jaiswal Pallavi (2014), CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM CHLOROPHYTUM BORIVILIANUM Sant. et Fernand, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 33â&#x20AC;&#x201C;39

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

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39

INTRODUCTION Saponins are generally known as nonvolatile, surface-active compounds that are widely distributed in nature, occurring primarily in the plant kingdom (Hostettmann et al., 2005). The name ‘saponin’ is derived from the Latin word sapo, which means ‘soap’, because saponin molecules form soap-like foams when shaken with water. They are structurally diverse molecules that are chemically referred to as triterpene and steroid glycosides. They consist of nonpolar aglycones coupled with one or more monosaccharide moieties (Oleszek, 2002). This combination of polar and non-polar structural elements in their molecules explains their soap-like behaviour in aqueous solutions. Saponins are the important chemical compounds from tubers of C. borivilianum. They are used in the indigenous systems of medicine as a well known health tonic, aphrodisiac and galactogogue (Chopra et al., 1956; Marais et al., 1978; Nadkarni, 1996; Oudhia, 2001). Pharmaceutical industries buy saponins in large quantities because of their use for the semi-synthesis of steroidal drugs for phyto-therapy and in cosmetic industry (Sharma et al., 2012; Haque et al., 2011; Ksouri et al., 2011). They are believed to form the main constituents of many plant drugs and folk medicines responsible for numerous pharmacological properties (Marais et al., 1978; Estrada et al., 2000; Debnath et al., 2006; Katoch et al., 2010). Therefore, it is a category of phyto-nutrients (plant nutrients) found abundantly in many beans, and other plants such as Ginseng, Alfalfa, Yucca, Aloe, Quinoa seed and also in Safed Musli (Chopra et al., 1956; Nadkarni, 1996). Saponins have a diverse range of properties from sweetness to bitterness (Grenby, 1991; Kitagawa, 2002; Heng et al., 2006; Thakur et al., 2009), foaming and emulsification (Price et al., 1987), pharmacological and medicinal (Attele et al., 1999; Debnath et al., 2007), haemolytic (Oda et al., 2000; Sparg et al., 2004), and antimicrobial, insecticidal, and molluscicidal activities (Sparg et al., 2004; Sundaram et al., 2011) and finds some place in

beverages, confectionery and cosmetic industry (Price et al., 1987; Petit et al., 1995; Uematsu et al., 2000). Saponins consist of a sugar moiety, usually containing glucose, galactose, glucuronic acid, xylose, rhamnose or methylpentose, glycosidically linked to a hydrophobic aglycone (sapogenin) which may be triterpenoid or steroid (Abe et al., 1993; Haralampidis et al., 2002); derived from the 30 carbon atoms containing precursor oxidosqualene (Haralampidis et al., 2002). The difference between the two classes lies in the fact that the steroidal saponins have three methyl groups removed (i.e. they are molecules with 27 C-atoms), whereas in the triterpenoid saponins all 30 C-atoms are retained. Saponins were classified into three classes, namely, the triterpenoid saponins, the spirostanol saponins and the furostanol saponins. However, due to secondary biotransformation such a classification emphasizes incidental structural elements and does not reflect the main biosynthetic pathways (Sparg et al., 2004). There are some other classes of compounds that have been considered as saponins, such as the glycosteroid alkaloids (Haralampidis et al., 2002). Baumann et al., (2000) reported that saponins have hemolytic properties that generally are attributed to the interaction between the saponins and the sterols of the erythrocyte membrane. As a result erythrocyte membrane bursts, causing an increase in permeability and a loss of haemoglobin. A study was made to establish the relationship between the adjuvant and haemolytic activity of saponins derived and purified from 47 different food and medicinal plants. However, the results indicated that the adjuvant activity does not relate with haemolytic activity (Oda et al., 2000). Chlorophytum borivilianum Sant. et Fernand commonly known, as Safed Musli is a traditional rare Indian medicinal herb having many therapeutic applications in Ayurvedic, Unani, Homeopathic and Allopathic medicine system. It is an herbaceous plant with fasciculated tuberous root found naturally in forests and its shoots can be seen during the rainy seasons (Kothari et al., 2003). Research

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studies on Chlorophytum conducted in India and elsewhere indicate that saponins (viz. neohecogenin, neotigogenin, stigmasterol and tokorogenin) are responsible for medicinal properties (Jat et al., 1990). Safed musli is among the few medicinal plants witnessing steady growth in pharmaceutical, phytopharmaceutical and nutraceutical products (Debnath et al., 2006, 2007; Thakur et al., 2009). Due to the many therapeutic applications and several bioactive compounds, C. borivilianum is also called ‘The white gold for biopharmaceuticals and neutraceuticals’ (Thakur et al., 2009). It contains steroidal and triterpenoidal saponins, sapogenins, fructans and flavonone glycosides, which are powerful uterine stimulants. Dried roots of Chlorophytum contain 42% carbohydrate, 80–89% protein, 3– 4% fiber and 2–17% saponin (Wagle et al., 2000). It is useful in curing impotency with spermatogenic property and is considered as an alternative to ‘Viagra. It is a rich source of over 25 alkaloids, vitamins, proteins, carbohydrates, steroids, saponins, potassium, calcium, magnesium, phenol, resins, mucilage and polysaccharides with high content of simple sugars mainly sucrose, glucose, fructose, galactose, mannose and xylose (Ramawat et al., 2000; Debnath et al., 2006, 2007; Thakur et al., 2009). Due to their high medicinal value, several medicinal herbs are being indiscriminately collected before they could reach phenological maturity and vegetative regeneration capacity (Biswas et al., 2003). This has led to the depletion of natural source of several valuable plants like Safed musli. The restricted distribution and indiscriminate overexploitation of this plant coupled with low seed set and viability and poor seed germination rates has made its status rare in the wild (Debnath et al., 2006). Among all the species of Chlorophytum present in India, C. borivilianum produces the maximum root tuber along with the highest saponin content (Attele et al., 1999). Traditionally, roots of these species are reputed to posses various pharmacological utilities having saponins as one of the important phyto-chemical

constituents (Marais et al., 1978). The objective of the manuscript is to develop a brisk protocol for extraction of saponin from tubers of C. borivilianum with special attention on the screening of extracted metabolite. MATERIAL AND METHODS 1. Plant Material: Plants and roots of Chlorophytum borivilianum were collected from plant herbarium, Plant biotechnology laboratory at Tropilite foods Pvt. Ltd., Gwalior, India. Plants were available in vitro (in test tubes) and in vivo (in pots) conditions in laboratory. Plants (both roots and shoots) were washed thoroughly and were sliced into pieces followed by drying in hot air oven at 100°C for 4–5 days. On complete drying, the plant material was grinded uniformly with the help of mortar-pestle and stored in an airtight container. 2. Chemicals used: Chemicals used for isolation purposes were 95% Ethanol (Merck Millipore), Petroleum ether (Sigma-Aldrich), Ethyl acetate (SigmaAldrich), Chloroform (Ultra pure, HiMedia), Methanol (Merck Millipore), Acetone (LR grade, HiMedia), Distilled water. Quality of isolated saponin was tested on TLC plates Silica gel 60 F254 plates (Merck) with Sulphuric acid (Rankem) as spraying agent. 3. Saponin Extraction Procedure: The extraction process was carried out with both in vivo and in vitro samples by soaking the dried plant material in ethanol 95% overnight. The extraction was done with Petroleum ether, Ethyl acetate, Chloroform, Methanol and Acetone. Petroleum ether was used for delipidization and chloroform for deproteinization of dried mixture. On extraction of crude saponin, methanol was used to mellow the developing mixture followed by drop wise addition into acetone solution leading to precipitation. The precipitated material was extracted and dried in hot air oven

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39

leading to formation of whitish brown crystals (Lakshmi et al., 2012). 4. Saponin Confirmatory Test Froth test: 0.5 gm of the alcoholic extract was dissolved in 10 ml of distilled water in a test tube. The test tube was shaken vigorously for about 30 seconds .The test tube was allowed to stand in vertical position and was observed over a 30 min period of time. Thick persistent froth was observed on the surface of the liquid indicating presence on saponin. 5. Thin layer Chromatography TLC technique was used for purification of saponins isolated from C. borivilianum.

Samples (crude saponin) and the reference standards (Saponin, Sigma) were loaded on the pre-coated TLC plates silica gel 60 F254 plates. Mobile phase chloroform: methanol: water (65:35:10 v/v/v) was used for the separation. Two drops of standard and sample were loaded up on TLC plates with the help of a micropipette. The loaded plates were placed in the TLC jar which contained the solvent system. After the completion of the run the plates were taken out and kept at room temperature to get dried for 10 minutes. The plates were developed with the spraying reagent (5%, H2SO4). After spraying the reagents, the plates were kept at 110ºC for 10– 15 minutes in hot air oven and results were observed later (Fig 1).

Fig 1: Thin layer chromatography results of In vivo, in vitro and standard samples developed in mobile phase of chloroform: methanol: water (65:35:10 v/v/v)

RESULT AND DISCUSSION The phytochemical extraction of in vivo root tubers and in vitro plant body of Chlorophytum borivilianum was carried out

using six different solvent systems (Ethanol, petroleum ether, ethyl acetate, chloroform, methanol and acetone). Whitish brown crystals were obtained as end product of the process.

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39

Experimental procedure: 1. Powder Soaking: In vivo and in vitro dried plant samples with a quantity of 30 gm each were mixed in 95% ethanol (180 ml) solution separately in conical flasks. After uniform mixing the solutions were placed in orbital shaker for stirring at 100 RPM for 12 hours. The supernatant was collected by filtration and the process was repeated 2–3 times. 2. Delipidization: Ethanol was evaporated by heating the collected supernatant at 45– 55oC in hot water bath to concentrate the solution. Petroleum ether was added to the concentrated solution and heated for around 30 minutes. After complete evaporation of the solvent, the residue was collected on a filter paper. Petroleum ether was used to remove lipid and fatty acids from plant and tuber of C. borivilianum. 3. Deproteinization: Residue was treated with equal ratio of Ethyl acetateChloroform and stirred the mixture for 15 minutes. Chloroform is deproteinizing agent used to remove proteins from plant and tuber of C. borivilianum. 4. Precipitation: In this step, ethyl acetatechloroform was evaporated by heating the

mixture at 45–55oC in hot water bath and leading to formation of a crude residue. The residue was again dissolved in methanol and heated at 45–55oC. The remaining warm residue was dropped in acetone solution drop by drop. White colored powder was obtained as precipitate in acetone. The precipitate was filtered and oven dried to obtain white crystals. Saponin in form of small crystals was collected on filter paper and preserved in air tight container for further testing. CONCLUSION The commercial promotion of saponin as dietary and nutraceutical supplement and evidences of presence of saponins in traditional medicine preparations also propagating a need for efficient method saponin isolation. The developed protocol is economic and less time consuming as well which only includes soaking, delipidization and deproteinization and avoiding the steps of water as mixing solvent and overnight stirring in water bath followed by dipping in organic solvent. The final quantity of product obtained depends upon the quality of ex-plant cultured. The final product obtained from the protocol was tested on froth confirmatory test and on thin layer chromatographic against the standard saponin.

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Baumann, E., Stoya, G., Volkner, A., Richter, W., Lemke, C., Linss, W. (2000). Hemolysis of human erythrocytes with saponin affects the membrane structure. Acta Histochemica, 102, 21–35. Biswas, S., Jain, S., Pal, M. (2003). Research needs and priorities for conservation of Indian medicinal flora. Indian Forest, 129, 85–92.

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Chopra, R.N., Nayar, S.L., Chopra, I.C. (1956). Glossary of Indian medicinal plants. CSIR, New Delhi. Debnath, M., Malik, C.P., Bisen, P.S. (2007). Clonal Propagation of Chlorophytum borivilianum, An Endangered Medicinal Plant. Phytomorphology, 57, 117–121. Debnath, M., Malik, C.P., Bisen, P.S. (2006). Micropropagation: A Tool for the production of high quality plantbased medicines. Current Pharmaceutical Biotechnology, 7, 33–49. Estrada, A., Katselis, G.S., Laarveld, B., Barl, B. (2000). Isolation and evaluation of immunological adjuvant activities of saponins from Polygala senega L. Comparative Immunology, Microbiology and Infectious Diseases, 23, 27–43. Grenby, T.H. (1991). Intense sweeteners for the food industry: an overview. Trends in Food Science and Technology, 2, 2–6. Haque, R., Saha, S., Bera, T. (2011). A Peer Reviewed of General Literature on Chlorophytum borivilianum Commercial Medicinal Plant. International Journal of Drug Development and Research, 3, 140– 155. Haralampidis, K., Trojanowska, M., Osbourn, A.E. (2002). Biosynthesis of triterpenoid saponins in plants. Advances in Biochemical Engineering, 75, 31–49. Heng, L., Vincken, J.P., Hoppe, K., van Koningsveld, G.A., Decroos, K., Gruppen, H., van Boekel, M.A.J.S., Voragen, A.G.J. (2006). Stability of pea DDMP saponin and the mechanism of its decomposition. Food Chemistry, 99, 326–334.

Hostettmann, K., Marston, A. (2005). Saponins: Chemistry and pharmacology of natural products. Cambridge University Press, Cambridge, isbn-10: 0521020174. Jat, R.D., Bordia, P.C. (1990). Propagation studies in Safed Musli (Chlorophytum species) Proc. of National symposium on Adv. In Plant Sci.: Current Status and Emerging Challenges. Deptt. of Botany, Sukhadia Univ., Udaipur, 46. Katoch, M., Kumar, R., Pal, S., Ahuja, A. (2010). Identification of Chlorophytum species (C. borivilianum, C. arundinaceum, C. laxum, C. capense and C. comosum) using molecular markers. Industrial Crops and Products, 32, 389–393. Kitagawa, I. (2002). Licorice root: A natural sweetener and an important ingredient in Chinese medicine. Pure and Applied Chemistry, 74, 1189–1198. Kothari, S.K., Singh, K. (2003). Production techniques for the cultivation of safed musli (Chlorophytum borivilianum). The Journal of Horticulture science and Biotechnology, 78, 261–264. Ksouri, R., Ksouri, W.M., Jallali, I. (2011). Medicinal halophytes: potent source of health promoting biomolecules with medical, nutraceutical and food applications. Critical Review in Biotechnology. doi:10.3109/07388551. Lakshmi, V., Mahdi, A.A., Agarwal, S.K., Khanna, A.K. (2012). Sterodal saponin of Chlorophytum nimonii (Grah) with lipid lowering and antioxidant activity. Chronicles of young scientists, 3(3), 227–232. Marais, W., Reilly, J. (1978). Chlorophytum and Its Related Genera (Liliaceae). Kew Bulletin., 32(3), 653–63.

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Nadkarni, K.M. (1996). Indian Materia Medica. 3rd ed. Popular Book Depot, Bombay. Oda,

K., Matsuda, H., Murakami, T., Katayama, S., Ohgitani, T., Yoshikawa, M., (2000). Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biological Chemistry, 381, 67–74.

Oleszek, W.A, (2002). Chromatographic determination of plant saponins. Journal of Chromatography A, 967, 147–162. Oudhia, P. (2001). Problems perceived by Safed Moosli (Chlorophytum borivilianum) growers of Chhattisgarh (India) region: A study. Journal of Medicinal and Aromatic Plant Sciences, 22, 396–399. Petit, P.R., Sauvaire, Y.D., Hillaire-Buys, D.M., Leconte, O.M., Baissac, Y.G., Posin, G.R., Ribes, G.R. (1995). Steroid saponins from fenugreek seeds: extraction, purification, and pharmacological investigation on feeding behavior and plasma cholesterol. Steroids., 60, 674–80. Price, K.R., Johnson, I.T., Fenwick, G.R. (1987). The chemistry and biological significance of saponins in foods and feedstuffs. Critical Review in Food Science and Nutrition, 26, 27–135. Ramawat, K.G., Sharma, R., Soni, S.S. (2000). Medicinal plants. In: Ramawat KG, Merillon JM (Eds.), Biotechnology: secondary metabolites, Oxford and IBH Publishing Co, Pvt. Ltd. New Delhi. pp. 356

Source of Support: NIL

Sharma, R.S., Thakur, G.S., Sanodiya, B.S., Pandey, M., Bisen, P.S. (2012). Saponin: A wonder drug from chlorophytum species. Global Journal of Research on Medicinal Plants and indigenous medicine, 1(10), 503–515. Sparg, S.G., Light, M.E., van Staden, J. (2004). Biological activities and distribution of plant saponins. Journal of Ethnopharmacology, 94, 219–43. Sundaram, S., Dwivedi, P., Purwar, S. (2011) Antibacterial activities of crude extracts of Chlorophytum borivilianum to bacterial pathogens. Research Journal of Medicinal Plant, 5, 343–347. Thakur, G.S., Bag, M., Sanodiya, B.S., Debnath, M., Bhadouriya, P., Prasad, G.B.K.S., Bisen P.S. (2009). Chlorophytum borivilianum: A White Gold for Biopharmaceuticals and Neutraceuticals. Current Pharmaceutical Biotechnology, 10, 650–666. Uematsu, Y., Hirata, K., Saito, K. (2000). Spectrophotometric determination of saponin in Yucca extract used as food additive. Journal of AOAC International, 83, 1451–54. Wagle, A., Kelkar, G.D., Heble, M.R. (2000). Biotechnology: Secondary Metabolites. Oxford and IBH Publishing Co, Pvt.Ltd., New Delhi, pp. 219–220.

Conflict of Interest: None Declared

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Research Article HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA Deb Roy Saumendu1*, Karmakar Prithivi Raj2, Dash Suvakanta3, Chakraborty Jashabir4, Das Biswajit5 1,2

Deptt. of Pharmacognosy, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017, Assam, India. 3 Deptt. of Pharmaceutics, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017, Assam, India. 4 Deptt. of Pharmacology, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017, Assam, India. 5 Deptt. of Quality Assurance, Hetero Labs Ltd. Unit-II, Baddi, Himachal Pradesh, India. *Corresponding Author: E-mail: baharu@rediffmail.com; Mobile: +91-9435071898

Received: 07/01/2014; Revised: 25/01/2014; Accepted: 31/01/2014

ABSTRACT In this particular Study Hydro-alcoholic extract of Liquorice was evaluated for its use in Alopecia, where the extracts were compared with the activity of Marketed drug Minoxidil and the tests were carried out on Female Albino Rat. The Results were very much encouraging as 2% Hydroalcoholic extract has shown a better hair growth than that of the marketed drug Minoxidil. The Extract was also subjected to preliminary Phytochemical Evaluations whereby it has shown the presence of Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch and Fixed Oils. TLC of the extract using Pre Coated Silica gel GF Plate while detected under UV have shown 1 Spot (Rf:0.67) with Ethyl acetate as Mobile Phase, 1 Spot (Rf:0.44) with Benzene: Toluene (4:6) as Mobile Phase and 1 Spot (Rf:0.96) with Benzene: Chloroform (3:7) as Mobile Phase. KEY WORDS: Hair Growth, Glycyrrhiza glabra, Liquorice, TLC, Hydro-alcoholic extract.

Cite this article: Deb Roy Saumendu, Karmakar Prithivi Raj, Dash Suvakanta, Chakraborty Jashabir, Das Biswajit (2014), HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 40–47

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INTRODUCTION Hair follicle growth occurs in cycles. Each cycle consists of a long growing phase (anagen), a short transitional phase (catagen) and a short resting phase (telogen). At the end of the resting phase, the hair falls out (exogen) and a new hair starts growing in the follicle beginning the cycle again. Normally, about 40 (0â&#x20AC;&#x201C;78 in men) hairs reach the end of their resting phase each day and fall out. When more than 100 hairs fallout per day, clinical hair loss (telogen effluvium) may occur. A disruption of the growing phase causes abnormal loss of anagen hairs (anagen effluvium) (Rudnicka L et al., 2008; Zhou Z.Y et al., 2007). Alopecia means loss of hair from the head or body. Alopecia can mean baldness, a term generally reserved for pattern alopecia. Compulsive pulling of hair can also induce hair loss. Hairstyling routines such as tight ponytails or braids may cause traction alopecia. Both hair relaxer solutions, and hot hair irons can also induce hair loss. In some cases, alopecia is due to underlying medical conditions, such as iron deficiency (Rudnicka L et al., 2008). Generally, hair loss in patches signifies Alopecia areata. Alopecia areata typically presents with sudden hair loss causing patches to appear on the scalp or other areas of the body. If left untreated, or if the disease does not respond to treatment, complete baldness can result in the affected area, which is referred to as Alopecia totalis. When the entire body suffers from complete hair loss, it is referred to as Alopecia universalis. It is similar to the effects that occur with chemotherapy (Zhou Z.Y et al., 2007). The nature remains as the potential source of organic structures of unparalleled diversity. The therapeutic use of Medicinal Plants has gained considerable momentum in the world during the past decade. The overuse of synthetic drugs with impurities, resulting in higher incidence of adverse drug reactions in more advanced communities, has motivated

mankind to go back to nature for safer remedies. The selected plant Glycyrrhiza glabra was reported to have wide ethnomedical use (Shibata S. et al., 2000). Minerva med reported that metabolic and toxic effects caused by prolonged daily ingestion of Liquorice are well known. Such acquisition doesn't seem to be known enough by practitioners and by common people. Besides it contains active substances such as Glycyrrhizin, steroids similar to the adrenocortical ones; among these the most important is Beta-Glycyrrhetinic acid. This, in vivo and in vitro, produces salt and water retention by means of a mineral-corticoid mechanism, and clear suppression of the ReninAngiotensin-Aldosterone axis. A low plasmatic level of Renin and Aldosterone is a common feature. The clinical picture in many ways is similar to the primary Aldosteronism and for this reason the above mentioned syndrome is usually called "Pseudoaldosteronism" (Colloredo G et al., 1987). Saeedi .M et al. reported that Creams containing whole licorice (often combined with extract of chamomile) are in wide use as "natural hydrocortisone creams." However, there is only preliminary supporting evidence for this use. In one double-blind, placebo-controlled trial of 30 people, licorice gel at 2% was more effective than placebo or 1% gel for reducing symptoms of eczema (Saeedi M et al., 2003). Besides there were some ethno-medicinal claims regarding the use of Liquorice in Alopecia, which encouraged us to go for the study, and we have tried here to establish the ethnomedicinal claim regarding its use in Alopecia. METHODS: Place of Collection: The roots were collected from an established Crude Drug dealer from Kolkata and have been identified by Deptt. of Botany, Guwahati University vide Specimen No: 11746 and a Sample voucher has been deposited for further reference.

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47 pH

1 gm. of the accurately weighed drug was treated with 100 ml. of distilled water and filtered. pH of the filtrate was checked with a pH meter having standardized glass electrode.

minutes at a temperature not exceeding 450ºC. The weight of the insoluble matter was subtracted from the weight of the ash, the difference in weight represents the water soluble ash. The percentage of water soluble ash with reference to the air dried drug was then calculated.

b) pH 10% Solution :

c) Acid Insoluble Ash :

10 gm. of the accurately weighed drug was treated with 100 ml. of water and filtered. pH of the filtrate was checked with a pH meter having standardized glass electrode.

The ash was boiled for 5 minutes with 25 ml of 2M hydrochloric acid and the insoluble matter was collected in a Silica crucible or on an ash less filter paper, washed with hot water, incinerated, cooled in a desiccator and weighed. The percentage of acid insoluble ash with reference to the air dried drug was then calculated.

Determination: a) pH of 1% Solution :

Loss on drying: A glass stoppered shallow weighing bottle was dried and weighed and 3 gm of the powdered drug was transferred to the bottle. The bottle was then stoppered and the bottle along with the contents was weighed. The sample was then distributed as evenly as practicable by gentle side wise shaking to a depth not exceeding 10 mm. The loaded bottle was then placed in the hot air oven; the stopper was removed and left it also in the oven. The powdered drug was then dried to constant weight or for 30 mm and at a temperature of 105°C. After drying was completed the hot air oven was opened and the bottle was closed promptly and allowed to cool at room temperature. The bottle and the contents were then weighed. The procedure was continued until a constant weight was obtained. Ash value: (Pharmacopoeia of India) a) Total Ash : 2 gm of air dried drug was weighed accurately in a tared silica crucible and incinerated at a temperature not exceeding 450°C until free from carbon, cooled and weighed. The percentage of ash with reference to the air dried drug was calculated. b) Water Soluble Ash : The ash was boiled for 5 minutes with 25 ml of distilled water and the insoluble matter was collected on an ash less filter paper, washed with hot water, and incinerated for 15

Extractive value: a) Ethanol Soluble Extractive : 5 gm of the air dried drug was coarsely powdered, taken in a stoppered conical flask and macerated with 50 ml of ethanol (90%) for 24 hrs shaking frequently during the first 6 hrs and allowing standing for 18 hrs. Thereafter, it was filtered rapidly taking precautions against loss of ethanol, and then the filtrate was evaporated to dryness in a tared flat bottom shallow dish, dried at 105°C and weighed. The percentage of ethanol – soluble extractive was calculated with reference to the air dried drug. b) Chloroform Soluble Extractive : 5 gm of air dried drug was coarsely powdered, taken in stoppered conical flask and macerated with 25 ml of chloroform for 24 hrs shaking frequently during the first 6 hrs and allowed standing for 18 hrs. Thereafter, it was filtered rapidly taking precaution against loss of petroleum ether, and then the filtrate was evaporated to dryness in a tared flat bottomed shallow dish, divides at 105°C and weighed. The percentage of petroleum ether soluble extractive was calculated with reference to the air dried drug.

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Water Soluble Extractive:

5 gm of the air dried drug was coarsely powdered, taken in a stoppered conical flask and macerated with 50 ml of distilled water for 24 hrs, shaking frequently during the first 6hrs and allowing standing for 18 hrs. Thereafter it was filtered rapidly taking precautions against loss of chloroform water, and then the filtrate was evaporated to dryness in a tared flat bottom shallow dish, dried at 105°C and weighed. The percentage of water soluble extractive was calculated with reference to the air dried drug. Drying and pulverization: The collected plant material (Roots) was shade dried and then they were pulverized to coarse powder and passed through mesh size 40. Preparation of extract by continuous hot extraction: The roots were dried in shade and powdered to get a coarse powder. About 75 gm of dry coarse powder was extracted with waterethanol (1:1) by continuous hot percolation using soxhlet apparatus (40–60°c). The extraction was continued for 7 days. The hydro-alcoholic extract then filtered and concentrated by vacuum distillation. A brown colour shiny residue was obtained. Qualitative chemical evaluation: Extract was subjected to various qualitative chemical tests for detecting the presence of various Phytoconstituents (Table No. 6). The procedures were followed as per the procedures laid down by Kokate C.K et al., 2008 and Khandelwal K.R, 2012. Thin layer chromatographic separation: For TLC, Precoated Silica Gel GF plates were used, by trial and error method various solvent systems were selected and spot visualization was done in UV chamber (Mukharjee P.K, 2010).

Hair growth stimulating activity: Approval of the study: The research protocol of the animal experimentation was approved by the „Institutional Animal Ethical Committee‟ of Girijananda Chowdhury Institute Of Pharmaceutical Science, Azara, Guwahati-17, Assam. GIPS/IAEC No. : GIPS BPH/2013/6 Collection of animals: Animals (Wister Albino Rats) weighing 120– 150 g and aged 3–4 months were collected from Animal House of GIPS, Azara and used for Hair growth stimulatory study. The animals were handled according to CPCSEA Guidelines of Good Laboratory Practice. Animals were kept overnight in laboratory conditions acclimatize with the Laboratory environment. Preparation of test sample: The test sample was prepared by preparing suspension of the dried hydro-alcoholic extract. 0.5gm & 1gm extract were dissolved in 50ml ethanol (90%) each, which gave concentration of 1% & 2% solution respectively. Hair growth stimulatory study: A 4 cm2 area of the dorsal skin of the rats were shaved off using a marketed hair removal cream. The extract solution and Minoxidil (0.4 ml) was applied to the denuded area of the rat once a day. This treatment was continued for 10 days during and after which hair growth pattern was observed visually and recorded (Adhirajan N et al., 2003; Dattaa K et al., 2009). RESULTS AND DISCUSSION: Pharmacognostic Evaluations: (Table I) pH of 1% solution was found to be 6 and that of 10% solution was found to be 5, which suggests the drug to be an acidic. Moisture Content was found to be 8%w/w. Total Ash, Acid insoluble ash and Water Soluble ash were found to be 7.9%, 2.07% and 5.67%

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47

respectively, when calculated with reference to the air dried drug. Water Soluble Extractive value, Ethanol Soluble extractive value and Chloroform Soluble extractive values were found to be 12.2%, 4.04% and 1.28% respectively, when calculated with reference to the air dried drug. These parameters help, in identification of the pure crude drug, while checking for adulterants. The results are shown

in Table I. Phytochemical Evaluation: (Table II) The extract when screened for various Phytoconstituents and has shown the presence of, Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch and Fixed Oils, and are shown in Table II.

TABLE I: Pharmacognostic Evaluation Sl. No. Parameter 1. pH of 1% Solution

Values (w/w) 6

pH of 1% Solution

Loss on Drying

8.0 %

ASH VALUES

A. Total Ash Values

7.9 %

B. Acid insoluble ash

2.07 %

C. Water soluble ash

5.67 %

EXTRACTIVE VALUES A. Water soluble extractive

12.2 %

B. Ethanol soluble

4.04 %

extractive

C. Chloroform soluble Extractive 1.28 % Table II: Phytochemical Evaluations Sl. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Phytoconstituent Carbohydrate Gums and Muscilage Lipids Alkaloids Anthraquinone Glycoside Cardiac Glycoside Coumarine Saponins Phytosterol Flavonoids Tannins and Phenolic Compounds

Result +ve −ve +ve −ve −ve −ve +ve +ve +ve +ve -ve

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Thin layer Chromatography: (Table III) When the extract was subjected to Thin Layer Chromatography, using Precoated Silica Gel GF plates as Stationary Phase and Choosing various mobile phases based upon the various Phytoconstituents found through phytochemical screening on a trial and error method, the extract has shown Single spots with, Chloroform, Benzene, Ethyl Acetate, Benzene :Toluene (4 :6) and Benzene : Chloroform (3 : 7) as Mobile Phases, while UV Detector was used for detection. The Results were recorded and Shown in Table III.

Hair growth stimulatory activity: (TABLE IV) (Fig.I) After carrying out the study it was found that the hydro-alcoholic extract of liquorice showed a profound hair growth activity. Further it was also found that 2% concentration of the extract as compared with the standard drug used (Minoxidil 2%), has shown better hair growth activity. The Findings are shown in Table IV. and a graphical representation is also provided. Fig.I below which shows the Hair Growth observed after 10 days.

Table III: Thin Layer Chromatography SOLVENT SYSTEM

HYDRO-ALCOHOLIC EXTRACT

DETECTION SYSTEM

Chloroform

1 spot,

Rf:- 0.38

UV Chamber

Benzene

1 spot,

Rf:- 0.84

UV Chamber

Ethyl acetate

1 spot,

Rf:- 0.67

UV Chamber

Benzene : Toluene (4:6)

1 spot,

Rf:- 0.44

UV Chamber

Benzene : Chloroform (3:7)

1spot,

Rf:- 0.96

UV Chamber

Table IV: Hair Growth Stimulatory Activity

Sl.No

Sample Applied

Animals Used

1. 2. 3. 4.

Extract (1%) Extract (2%) Standard (2%) Control

06 06 06 06

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No. of hairs (±SEM) After 10 days. 474 ± 2.55 894.67 ± 6.94 620 ± 10.36 211± 4.38

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FIG. I: Showing Hair Growth after 10 days

Graphical Representation I: Hair Growth Stimulatory Activity

HAIR GROWTH STIMULATORY ACTIVITY FOLLICLE(NO.) PER 4 CM²

1000 900 800 700 600 500 400 300 200

100 0 CONTROL

STANDARD 1% EXTRACT DRUG SAMPLE

2% EXTRACT

CONCLUSION:

ACKNOWLEDGEMENT:

The results indicate that Liquorices (Glycyrrhiza glabra) has a potent hair growth activity and after careful checking of other safety parameters it can be used in herbal formulations to treat various types of Alopecia.

The authors are very much grateful to the Management of Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati, Assam, India, for providing the facilities needed to carry out this Study.

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REFERENCES: Adhirajan N, Ravi Kumar T, Shanmugasundaram N, Babu M (2003) In vivo and in vitro evaluation of hair growth potential of Hibiscus rosasinensis Linn. J Ethnopharmacol; 88: 235–239. Colloredo G, Bertone V, Peci P, Locatelli A, Brembilla G, Angeli G., (1987 ) Pseudoaldosteronism caused by licorice. Review of the literature and description of 4 clinical cases., Minerva Med. Jan 31;78 (2);93-101. Dattaa K, Singha TA, Mukherjee A, Bhata B, Ramesh B, Burmana AC.( 2009) Eclipta alba extract with potential for hair growth promoting activity. J Ethnopharmacol; 124: 450–456.

Mukharjee P.K. (2010) “Quality control of herbal drugs”, Business Horizone, New Delhi-110048, 4th reprint.;452–456. Pharmacopoeia of India (1996), The Controller of Publications, Delhi-110054, Vol-II, p.A-54. Ramar PS, Peter NP, Ponnampalam G. (2008) “A compilation of bioactive compounds from Ayurveda.”; Biomedical informatics publishing; 3(3); 100-110, Rudnicka L, Olszewska M, Rakowska A, Kowalska-Oledzka E, Slowinska M. (2008) "Trichoscopy: a new method for diagnosing hair loss". J Drugs Dermatol; 7 (7); 651–654.

Khandelwal K.R. (2012) “Practical Pharmacognosy”, Nirali Prakashan, 22nd edition.; 18.15–18.18.

Saeedi M, Morteza-Semnani K, Ghoreishi MR. (2003) “The treatment of atopic dermatitis with licorice gel.” J DermatologTreat . Sep;14;153–157.

Kokate C.K. Purohit, A.P. & Gokhale, S.B. (2008) “Text book of Pharmacognosy”, Nirali Prakashan, Pune-411005, 41st edition, 8.52-8.56./A-1 to A-6

Shibata S. (2000) "A drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice." J. Pharm sci., Oct; 120 (10); 849–62.

Minerva Med. (1987) Jan 31;78(2);93–101.

Source of Support: NIL

Zhou Z.Y, Jin H.D. (2007) “Clinical manual of Chinese herbal medicine and acupuncture.”

Conflict of Interest: None Declared

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Research Article IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING ELICITORS Tamandani Ehsan Kordi1*, Valizadeh Jafar2, Valizadeh Moharam3 1

M.sc Graduate of Phytochemistry, Department of Chemistry, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran. 2 Department of biology, University of sistan and baluchistan, Zahedan, iran. 3 Research center of Medicinal and Aromatic Plants, University of Sistan and Baluchestan. *Corresponding Author: kordi3236@yahoo.com; Tel: +98-541-2452335, Fax: +98-541-2446565

Received: 10/01/2014; Revised: 05/02/2014; Accepted: 09/02/2014

ABSTRACT Elicitors are compounds with highly specific structures, that at low quantities, induce plants defense responses and subsequently, increasing of anti oxidant activity and secondary metabolites production. The elicitors such as CuSo4 (0.05 g/l), yeast extract (2 g/l), arachidonic acid (50 mg/l) and AlCl3 (0.026 g/l) were added to callus cultured of Cicer spiroceras (wild chickpea) in MS medium. After two month the calli were harvested and dried in the room temperature. About 0.1 g well powdered callus were used for each measurement of protein, total phenol, carbohydrate contents and antioxidant activity. Types various elicitors could induce diversely accumulating effects. The addition of Cu+2 had not the positive effect in the higher accumulation of primary, secondary metabolites and antioxidant activity than control in leaf and root calli. Yeast extract (YE) promoted antioxidant activity and total phenolic content in leaf callus. Arachidonic acid induced significantly to promote carbohydrate and protein contents in both root and leaf, but had a negative effect on anti oxidant activity and phenolic components accumulation in compare control. The highest protein, carbohydrate, total phenolic contents and antioxidant activity was obtained in culture treated with AlCl3, so induced to increase 98 % antioxidant activity and 74 % phenolic components. The effect CuSO4 and YE on calli growth was as well as (or more than) control, whereas AlCl3 and arachidonic acid suppressed the growth of calli. The outcomes of this study have been highlighted that using elicitors may increase the primary, secondary metabolites and antioxidant activity in C. spiroceras callus cultured. KEYWORDS: elicitor, Cicer spiroceras, callus culture

Cite this article: Tamandani Ehsan Kordi, Valizadeh Jafar, Valizadeh Moharam (2014), IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING ELICITORS, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 40â&#x20AC;&#x201C;47

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INTRODUCTION

MATERIAL AND METHODS

One of the important components of our dietary is vegetarian that can provided more necessaries body with their beneficial ingredients. In paste, for health disorder, infection and illness peoples has been provided major drugs and body's useful substances from the plants (Schneider, 1993) whereas, nowadays by increasing population the consumption of industrial drugs are increasing in spite of theirs side effect (Zhang et al., 2011). So, medicinal plants (or materials) can be suitable candidate to replace with the industrial drags (Wojcik et al., 2010, Raskin et al., 2002). Some of the plants produce various components such as alkaloids, anti-cancer, anti bacterial and anti oxidant (Kawai et al., 1987). But generally, plants cannot produce many quantities of secondary metabolites (less than 1 % weight dry) use as drug (Oksman-Caldentey and Inze, 2004). Thus applied substances such as elicitor on plants can cause to stimulate in order that more secondary and primary metabolites production using tissue culture (Poulev et al., 2003). So far a vast wide of elicitors has been used to modify cell metabolisms to raise the desirable secondary metabolites. in certain studies methyljasmonate, YE, chitosan and specially heavy metals such as Hg, Pb, Cr, Ag, V (as salt) and recently, Cu and Al have been test as elicitor (Hanson and Howell, 2004), for example both YE and Cadmiumchloride enhance Sesquiterpenes (from 1µmg/mg to 87 µgm/gm and) in N.tabacum (Chintapakorn and Hamill, 2007) and Thorn apple (from 0 to 140 nmol /gm) (Kawauchi et al., 2010) which show the plants power in secondary metabolites production. Moreover, elicitors can also increase carbohydrate and protein (primary metabolites) accumulation in plants (Graham and Graham, 1996).

Chemical

The purpose of this study was to estimate the effect of elicitors as a inducer to produce primary and secondary metabolites in comparison industrial drugs.

Methanol, ethanol, AlCl3, CuSO4, Gallic acid, Folin silicato and all of MS medium constituents were purchased from Merck Co and used DPPH (2, 2-diphenyl-1picrylhydrazyl) were from sigma chemical Co. (Germany). YE were obtained from Scharlau Co. (Spain). Collection of seed and preparation of MS medium The seeds of C. spiroceras were collected from 2497 m altitude Taftan (N28º 36, 25.9 and E61º 04, 36.8) in Sistan and Baluchestan province, Iran. After translating to laboratory and disinfected with Ethanol and NaClO3 3%, the seeds were taken into MS medium with 0.7 % agar and 3 % sucrose (w/v) without hormone to achieve to root and leaf at 25º C. The PH was adjusted to 5.8 with NaOH(Fernandez et al., 2008). After 25 days root and leaf grown were fragmented and explants were shift to new MS medium having hormone (2, 4 D: 2 mg / l, BA: 0.5 mg/l) for affording callus. In addition, in this stage 200 µM CuSo4 (0.05 g / l), 2 g / lit YE, 50 mg / l arachidonic acid and 200 µM AlCl3 (0.026 g / l) were added to MS medium. After 40 days, once time these calli were sub cultured for more grow them. Determination of total phenol To measure the total phenol content in the samples, 0.1 g of the callus was added to 1.5 ml ethanol 80% and this solution was shaken. After 1 day they were centrifuged at 16000 rpm in 15 min then 500 µl extracts filtered were added to 2 cc Na2Co3 5 % and 2.5 ml Folin silicato reagent 10 %, and were placed in dark for 25 min. the samples absorbance were read with UV-Vis spectrophotometer in 765 nm wavelength. Gallic acid was tested as standard (Giorgi et al., 2013).

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Anti oxidant activity evaluation

RESULTS AND DISCUSSION

0.01 g of methanolic extracts was added to 5 ml methanol. By 50 µl of these solutions methanolic, 5 different concentrations were prepared and were added to 1.95 ml DPPH solution and were being remain in the dark for 30 min. the decrease in the absorbance against blank sample was determined using UV-Vis spectrophotometer of Varian and the remaining DPPH concentration in reaction with extracts were calculated from the calibration curve. The percentage of DPPH radical scavenging activity was calculated as follows:

The effect of four elicitors on calli cultured of Cicer spiroceras have been shown in table 1. Cu+2 was only elicitor that raised weight of calli more than controls in root and leaf calli so it caused that the calli weight of root and leaf to be 5.2 % and 54 % more than Control whereas showed the inverse impact on secondary, primary metabolites and Antioxidant activity accumulation. YE just was able to rise total phenol content from 20.7 ± 1.6 to 24.1 ± 1.9 and Antioxidant activity from 68 ± 3.2 to 59.8 ± 2.9 in leaf and did not induce to raise of carbohydrate and protein amount in root and leaf calli. Calli undergo with arachidonic acid had weight minimum amongst calli (leaf: 0.027 ± 0.005 and root: 0.027 ± 0.003) toward control (leaf: 0.104 ± 0.02 and root: 0.114 ± 0.03). Also, Applying of arachidonic acid increased significantly carbohydrate content from 1.81 ± 0.4 to 5.05 ± 0.2 in leaf and 1.65 ± 0.1 to 2.47 ± 0.06 in root and protein content from 2.04 ± 0.1 to 3.41 ± 0.2 in leaf and 1.19 ± 0.1 to 5.37 ± 0.1 in root, although it had a negative influence on raising of total phenol content and anti oxidant activity. The highest positive jump in total phenol and antioxidant activity in leaf (phenol: 36.1 ± 3.3, antioxidant activity: 34.3 ± 3.7) belonged to Al+3 that caused to increase 98 % antioxidant activity and 74 % phenolic compositions, but it was no effect on production of secondary metabolites and antioxidant activity in root. Also the presence of Al+3 as elicitor caused to a wide impact in carbohydrate and protein contents of calli cultured, increased dramatically the carbohydrate content in leaf from 1.81 ± 0.4 to 10.13 ± 0.7, in root from 1.65 ± 0.1 to 4.1 ± 0.1 and protein content from 2.04 ± 0.1 to 7.74 ± 0.3 in leaf and in root from 1.9 ± 0.1 to 11.5 ± 1.0. To estimate of amounts of protein, carbohydrates and total phenol and antioxidant activity (mg/g) and (µg/g) have been used respectively.

% RSA = (AB – AS) / AB × 100 Where AB was absorbance of blank sample and AS was sample absorbance (Ananthi et al., 2010) Determination of protein and carbohydrate contents The protein content was measured by the Bradford method. 5 cc of extraction buffer was added to 0.1 g to samples powdered and were being maintained for 24 h at 4°C. Then they were centrifuged at 20000 rpm for 30 min. After filtration, 2 ml water was added to extract and 10 µl of it were mixed with 1990 µl of Bradford reagent. The absorbance of samples was read with UV-Vis spectrophotometer in wavelength of 595 nm. Bovine serum albumin (BSA) was tested as standard (Mattarozzi et al., 2012). To determine the carbohydrates content, 0.1 g of the callus was added to 2.5 ml of ethanol 80 %, was maintained in water batch at 95°C for 1 h and after that was centrifuged at 20000 rpm. 2.5 ml water were added to extracts and 200 µl of it was added to 5 cc of Antron reagent and was placed in the water batch at 95 for 10 min. after cooling, the absorbance of the samples were read in wavelength at 620 nm with UV-Vis spectrophotometer. Glucose was tested as standard (Roe, 1955).

As given in table 1, any extracted elicitors had an independent influence on plant and there was not any significant relation between synthesize extent of phenolic components and antioxidant activity in comparison primary

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metabolites ,whereas generally primary metabolites can synthesize a wide variety of low molecular weight components as preface to produce secondary metabolites (Dixon, 2001). The Phenolic components and the anti oxidant activity were been enhanced only in leaf with AlCl3 and YE, furthermore any one of elicitors unable to stimulate of calli to more secondary metabolites production toward Control in root. It seems to root's cells had no more ability to synthesize phenolic components and antioxidant activity even in presence of elicitors. Similarly, the literature reports in Eschscholtzia and Datura strumonium cells culture with YE and AlCl3, Sanguinarine (from 20 to 60 mg/l) and Sesquiterpenoids production increased, respectively (Schluepmann and Paul, 2009; Byun and Pedersen, 1994). YE are known as triggering agent to stimulate of poly phenol. The addition YE to Medicago truncatula cell culture is a way to Shikimic acid accumulation, a precursor of poly phenolic compounds pathway (Broeckling et al., 2005). The primary metabolites were been increased just by AlCl3 and arachidonic acid elicitors so that AlCl3 had the highest effect on protein and carbohydrate quantities accumulation in root and leaf, respectively. AlCl3 has been just

elicitor which its stimulating caused to more synthesize of all carbohydrate, protein, total phenol contents and antioxidant activity. In the few quantities, AlCl3 as toxic molecule can regulate genes involved in the defense responses of plant (Eswaranandam et al., 2012). It has been previously established that CuSO4 and YE has above stimulating power in primary, secondary metabolites production in many cell culture (Kim et al., 2007), but in this study the existence of CuSO4 and YE, and more calli growth, did not caused to a positive impact on primary and secondary metabolites accumulation. The result of present study highlighted that there is association between growth suppression and biochemical activity in presence of Alcl3 and arachidonic acid. This relates may have been caused by triggering of phytoalexin components, biosynthesized in cell after applying elicitors (Chong et al., 2004). Similar results are shown a relationship between growth suppression of calli and biochemical activity in vanila, salvia and morina cell cultures (Chavan et al., 2011). Figure 1 shows Influence of different elicitors on Antioxidant activity, primary, secondary metabolites contents of C. spiroceras calli cultured in MS medium.

Table 1: Summary of effect elicitors on secondary, primary metabolites content and weight of callus in cell cultured of Cicer spiroceras (root and leaf) in MS medium. Elicitors

Weight (mg/g D.W)

Antioxidantactivity(µg/g)

Phenol (mg/g D.W)

Protein (mg/g D.W)

Carbohydrate (mg/g D.W)

34.3 ± 3.7 87.8 ± 4.9 137.2 ± 7.2

36.1 ± 3.3 16.7 ± 1.4 17.1 ± 2.3

7.74 ± 0.3 1.29 ± 0.05 3.41 ± 0.2

10.13 ± 0.7 1.54 ± 0.09 5.05 ± 0.2

59.8 ± 2.9 68.1 ± 3.2

24.1 ± 1.9 20.7 ± 1.6

1.35 ± 0.2 2.04 ± 0.1

1.63 ± 0.3 1.81 ± 0.4

Leaf 0.058 ± 0.005 AlCl3 0.161 ± 0.05 CuSO4 Arachidonic 0.027 ± 0.005 0.124 ± 0.02 YE 0.104 ± 0.02 Control

Root 0.052 ± 0.005 161.7 ± 9.4 15.1 ± 1.1 11.5 ± 1.0 4.1 ± 0.2 AlCl3 0.12 ± 0.02 92.1 ± 4.0 16.4 ± 1.0 1.4 ± 0.2 0.82 ± 0.3 CuSO4 181.5 ± 10.8 14.8 ± 0.8 5.37 ± 0.1 2.47 ± 0.06 Arachidonic 0.027 ± 0.003 0.1 ± 0.02 82.5 ± 4.1 16.8 ± 2.9 1.61 ± 0.1 0.64 ± 0.1 YE 0.114 ± 0.03 72.1 ± 4.8 17.0 ± 1.6 1.19 ± 0.1 1.65 ± 0.1 Control Datas are significant at P<0.05 toward control. Each amount is the means of 3 replicate ± SD.

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Figure 1: Influence of different elicitors on primary (B) and secondary (A) metabolites content of Cicer spiroceras cell cultured in MS medium. 250

Antioxidant-activity Phenol

protein carbohydrate

150

mg/g D.W

A-a: IC50 Ph: mg/g D.W

200

100

50 2

0 0

(A)

(B)

Datas are means Âą SD of triplicates. Abbreviations: r: root; l: leaf; 1: Control r; 2: control l, 3: Cu+2 r, 4: Cu+2 l; 5: Yeast extract r, 6: Yeast extract l; 7: Al+3 r; 8: Al+3 l; 9: Arachidonic acid r; 10: Arachidonic acid l.

Correlation between Antioxidant-activity and phenolic compositions As shown in figure 2. There was a link (R2 = 0.664) between total Phenolic content and antioxidant activity because they are able scavenging the free radicals and operation as antioxidant (Rajkumar et al., 2011). This experiment shows that 66 % antioxidant activity has belonged to phenolic compound and 44 % else could be another parts of secondary metabolites. Low (R2 = 0.38) and high (R2 = 0.97) correlation coefficient between antioxidant activity and total phenolic content have been reported for sweet potato (Sikora and

Bodziarczyk, 2012) and sorgom (Rabah et al., 2004) respectively. Chickpea plants are an important part of food's south Asia people especially India and Pakistan (Verma et al., 2012). The present study is the endeavor to increase the defense mechanism, primary (protein and carbohydrate), secondary metabolites and antioxidant activity of C. spiroceras (wild chickpea) by the application of biotic and abiotic elicitors. With applying elicitor as valuable biotechnological strategy can produce foods with more beneficial substances, especially in fabaceae species consumed as basic part of our dietary.

Figure 2: Correlation between Antioxidant-activity and phenol composition that shows 66% amount of Antioxidant-activity belong to phenol composition.

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CONCLUSION Of four elicitors used, AlCl3 had the highest positive effect in production of parameters measured. Arachidonic acid raised dramatically the primary metabolites content in leaf and root. However, dry weigh, fresh weight and growth ratio of calli was strongly inhibited by ALCl3 and arachidonic acid. The addition YE increased only anti oxidant activity and total phenolic compounds in leaf callus. CuSO4 caused that plant had the most weight of calli against control, although did not any affect on more production of primary and secondary metabolites toward control. In This results The synthesize of primary and secondary

metabolites depended to elicitor type used ,although generally primary metabolites can synthesize a wide variety of low molecular weight components as preface to produce secondary metabolites (Dixon, 2001), But in presence of elicitor did not occur this topic and so Antioxidant activity and phenolic components behaved independently from the primary metabolites. ACKNOWLEDGMENTS The authors wish to thank the University of Sistan and Baluchestan, Zahedan, Iran for financially supporting this project through grants to JV.

REFERENCES ANANTHI, S., RAGHAVENDRAN, H. R., SUNIL, A. G., GAYATHRI, V., RAMAKRISHNAN, G. & VASANTHI, H. R. (2010). In vitro antioxidant and in vivo antiinflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga). Food Chem Toxicol, 48, 187–92. BROECKLING, C. D., HUHMAN, D. V., FARAG, M. A., SMITH, J. T., MAY, G. D., MENDES, P., DIXON, R. A. & SUMNER, L. W. (2005). Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot, 56, 323–36. BYUN, S. Y. & PEDERSEN, H. (1994.) Twophase airlift fermentor operation with elicitation for the enhanced production of enzophenanthridine alkaloids in cell suspensions of Escherichia californica. Biotechnol Bioeng, 44, 14–20.

CHAVAN, S. P., LOKHANDE, V. H., NITNAWARE, K. M. & NIKAM, T. D. (2011). Influence of growth regulators and elicitors on cell growth and alphatocopherol and pigment productions in cell cultures of Carthamus tinctorius L. Appl Microbiol Biotechnol, 89, 1701–7. CHINTAPAKORN, Y. & HAMILL, J. D. (2007). Antisense-mediated reduction in ADC activity causes minor alterations in the alkaloid profile of cultured hairy roots and regenerated transgenic plants of Nicotiana tabacum. Phytochemistry, 68, 2465–79. CHONG, T. M., ABDULLAH, M. A., FADZILLAH, N. M., LAI, O. M. & LAJIS, N. H. 2004. Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies. Plant Cell Rep, 22, 951–8. DIXON, R. A. (2001). Natural products and plant disease resistance. Nature, 411, 843–7.

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ESWARANANDAM, S., SALYER, J., CHEN, P. & LEE, S. O. (2012). Effect of elicitor spray at different reproductive stages on saponin content of soybean. J Food Sci, 77, H81–6. FERNANDEZ, R., BERTRAND, A., CASARES, A., GARCIA, R., GONZALEZ, A. & TAMES, R. S. (2008). Cadmium accumulation and its effect on the in vitro growth of woody fleabane and mycorrhized white birch. Environ Pollut, 152, 522–9. GIORGI, A., PANSERI, S., MATTARA, M. S., ANDREIS, C. & CHIESA, L. M. (2013). Secondary metabolites and antioxidant capacities of Waldheimia glabra (Decne.) Regel from Nepal. J Sci Food Agric, 93, 1026–34. GRAHAM, T. L. & GRAHAM, M. Y. (1996). Signaling in Soybean Phenylpropanoid Responses (Dissection of Primary, Secondary, and Conditioning Effects of Light, Wounding, and Elicitor Treatments). Plant Physiol, 110, 1123– 1133.

KIM, O. T., BANG, K. H., SHIN, Y. S., LEE, M. J., JUNG, S. J., HYUN, D. Y., KIM, Y. C., SEONG, N. S., CHA, S. W. & HWANG, B. (2007). Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate. Plant Cell Rep, 26, 1941–9. MATTAROZZI, M., MILIOLI, M., CAVALIERI, C., BIANCHI, F. & CARERI, M. (2012). Rapid desorption electrospray ionization-high resolution mass spectrometry method for the analysis of melamine migration from melamine tableware. Talanta, 101, 453– 9. OKSMAN-CALDENTEY, K. M. & INZE, D. (2004). Plant cell factories in the postgenomic era: new ways to produce designer secondary metabolites. Trends Plant Sci, 9, 433–40.

HANSON, L. E. & HOWELL, C. R. (2004). Elicitors of Plant Defense Responses from Biocontrol Strains of Trichoderma viren. Phytopathology, 94, 171–6.

POULEV, A., O'NEAL, J. M., LOGENDRA, S., POULEVA, R. B., TIMEVA, V., GARVEY, A. S., GLEBA, D., JENKINS, I. S., HALPERN, B. T., KNEER, R., CRAGG, G. M. & RASKIN, I. (2003). Elicitation, a new window into plant chemodiversity and phytochemical drug discovery. J Med Chem, 46, 2542–7.

KAWAI, A., GOTO, S., MATSUMOTO, Y. & MATSUSHITA, H. (1987). [Mutagenicity of aliphatic and aromatic nitro compounds. Industrial materials and related compounds]. Sangyo Igaku, 29, 34–54.

RABAH, I. O., HOU, D. X., KOMINE, S. & FUJII, M. (2004). Potential chemopreventive properties of extract from baked sweet potato (Ipomoea batatas Lam. Cv. Koganesengan). J Agric Food Chem, 52, 7152–7.

KAWAUCHI, M., ARIMA, T., SHIROTA, O., SEKITA, S., NAKANE, T., TAKASE, Y. & KUROYANAGI, M. (2010). Production of sesquiterpene-type phytoalexins by hairy roots of Hyoscyamus albus co-treated with cupper sulfate and methyl jasmonate. Chem Pharm Bull (Tokyo), 58, 934–8.

RAJKUMAR, V., GUHA, G. & KUMAR, R. A. (2011). Antioxidant and antineoplastic activities of Picrorhiza kurroa extracts. Food Chem Toxicol, 49, 363–9.

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RASKIN, I., RIBNICKY, D. M., KOMARNYTSKY, S., ILIC, N., POULEV, A., BORISJUK, N., BRINKER, A., MORENO, D. A., RIPOLL, C., YAKOBY, N., O'NEAL, J. M., CORNWELL, T., PASTOR, I. & FRIDLENDER, B. (2002). Plants and human health in the twenty-first century. Trends Biotechnol, 20, 522–31. ROE, J. H. (1955). The determination of sugar in blood and spinal fluid with anthrone reagent. J Biol Chem, 212, 335–43. SCHLUEPMANN, H. & PAUL, M. (2009). Trehalose Metabolites in Arabidopsiselusive, active and central. Arabidopsis Book, 7, e0122. SCHNEIDER, E. (1993). [Medicinal plants of the New World--500 years of discovery in the Americas]. Pharm Unserer Zeit, 22, 15–24.

Source of Support: University of Sistan and Baluchestan, Zahedan, IRAN

SIKORA, E. & BODZIARCZYK, I. (2012). Composition and antioxidant activity of kale (Brassica oleracea L. var. acephala) raw and cooked. Acta Sci Pol Technol Aliment, 11, 239–48. VERMA, A. K., KUMAR, S., TRIPATHI, A., CHAUDHARI, B. P., DAS, M. & DWIVEDI, P. D. (2012). Chickpea (Cicer arietinum) proteins induce allergic responses in nasobronchial allergic patients and BALB/c mice. Toxicol Lett, 210, 24–33. WOJCIK, M., BURZYNSKA-PEDZIWIATR, I. & WOZNIAK, L. A. (2010). A review of natural and synthetic antioxidants important for health and longevity. Curr Med Chem, 17, 3262– 88. ZHANG, J., YUAN, K., ZHOU, W. L., ZHOU, J. & YANG, P. (2011). Studies on the active components and antioxidant activities of the extracts of Mimosa pudica Linn. from southern China. Pharmacogn Mag, 7, 35–9.

Conflict of Interest: None Declared

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Review Article INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS OF NANDA DEVI BIOSPHERE RESERVE – A REVIEW BASED ON PREVIOUS STUDIES Singh Rahul Vikram1* 1

Department of Biotechnology Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun, Uttarakhand, India -248002 *Corresponding author: rahul.negi121@gmail.com; Tel.-+918791649600

Received: 21/11/2013; Revised: 20/01/2014; Accepted: 05/02/2014

ABSTRACT Indian Himalayan Region is considered as a store house of many medicinal and aromatic plants which are being used to cure many diseases since centuries. Due to habitat degradation and over exploration, some of the plant species have been recorded in the Red Data Book of Indian plants and their importance in day to day life has gone undocumented. Keeping in view to document such valuable information on these plant species, this article was aimed to document the indigenous uses of medicinal and edible plants grown in Nanda Devi Biosphere Reserve (Uttarakhand), India along with their cultivation, growth, trade & economic values. Ethno-medicinal information on 80 plant species belonging to 53 families has been compiled in this paper, which is based on various previous studies. KEYWORDS: Aromatic & Medicinal plants, Edible plants, Red Data Book, Nanda Devi Biosphere reserve, cutivation

Cite this article: Singh Rahul Vikram (2014), INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS OF NANDA DEVI BIOSPHERE RESERVE – A REVIEW BASED ON PREVIOUS STUDIES, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 57–66

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INTRODUCTION: The Indian Himalaya is a home for biological and cultural diversity. It supports about 18,440 species of plants, of which 25.3% are endemic to Himalaya (Singh & Hajra 1997, Samant et al., 1998a). 1748 species of medicinal plants & 675 wild edible species are reported (Samant et al., 1998b; Samant & Dhar, 1997) in the Indian Himalayan region. The Nanda Devi Biosphere Reserve (70° 40’ to 80° 05’ E longitude and 30° 17’ to 30° 41’ N latitudes) is situated in the northern part of west Himalayas including Chamoli District (Gharwal), Bageshwer District (Kumaun) and Pithoraghar (Kumaun), Uttarakhand, India. It has an area of 624.6 sq. km. and has an average altitude exceeding 4500 m AMSL surrounded by high mountain ridges and peaks on all sides. The buffer zone of Nanda Devi Biosphere Reserve covers twelve villages in Chamoli District (Badola, 1998). There are two groups namely Indo-Mongoloid (Bhotia) and IndoAryans, which use plant resources as medicine, food, fodder, fuel, timber and various other purposes (Samant, 1996). The present review was aimed at documenting the medicinally important plants and their indigenous uses, their cultivation, growth, trade & economic values, grown in Nanda Devi Biosphere reserve, Uttarakhand, India, based on various previous studies. Climate and vegetation: The Nanda Devi Biosphere Reserve consists of four geological formations, Lata, Ramni, Kharapatal and Martoli (MaruoYugi, 1979). Climatically the area is dry with annual precipitation. The core zone of reserve remains snow covered almost throughout the year except mid-May to October. Most suitable visiting time is March to September. The vegetation in any given area is indicator of prevailing climatic conditions. The information compiled from publication of Govind Ballabh Pant Institute of Himalayan Environment & Development Kosi-Katarmal, Almora Uttarakhand (GBPHIED) and Botanical survey of India from the expedition reports reveal the presence of approximately 800 species of

plants, and several species being rare to very rare, and of medicinal importance. (Hajra and Batodi, 1995, Coordinating Unit of Survey of Medicinal Plants of Western Ghats of India, Final Report, 2005–2008) (Gaur and Tiwari, 1987; Uniyal 1977). Medicinal plants wealth of NDBR: The Nanda Devi Biosphere Reserve is well known for its rich biodiversity. The inhabitants of the area largely depend on plants for food, dye, medicine, beverage, woodwork and various religious and cultural needs. Information on the utilization of plant species of NDBR has been provided by Uniyal (1977), Negi et al., (1985), Tiwari (1986), Gaur et al., (1983), Samant (1993), Gaur (1999), Samant and Palni (2003) and Tiwari et al., (2010). However, there exists wealth of information with the medicine men (Vaidyas), peasants, shepherds, priests and village headmen. Table 1 provides a list of various medicinal plants with their altitudinal distribution, plant parts used and ethno-botanical uses based on the literature review of NDBR (Joshi et al., 1999). In the NDBR, of the seventy six rare endangered species reported, three species are restricted to the western Himalaya (Kumaun, Garhwal) and narrow range endemics, three species are adjacent areas of the Himalaya are near endemic (Samant et al., 1995). Dependence of human beings on plants of NDBR for various uses i.e., medicine, food, fodder, fuel, timber, agriculture tools, religious purpose etc. putting them in conservation threat is given in Table 1. Economic Values of medicinal plants In the NDBR buffer zone, villagers cultivate some medicinal plants for their own use and for local sale occasionally. In the buffer zone of NDBR Bhotiya people practice seasonal and altitudinal migration and stay inside the buffer zone of NDBR for six months (May–October). A survey conducted in five villages in the buffer zone of NDBR falling in Pithoragarh district (Uttarakhand) indicated

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that a total of seventy one families cultivated medicinal plants on 78% of the total cultivated area (Bosak, 2008). On average, a family earns about Rs. 2423 ± 376.95 per season from the sale of medicinal plants (Silori and Badola, 2000). In another study (Ramakrishnan et al., 1996), production of Aconitum heterophyllum and Picrorhiza kurroa has been worked out as

1100 kg/ha while it is 4410 kg/ha for Aconitum balfourii and Rheum emodi from their mature natural stand. Production of Podophyllum hexandrum and Nardostachys jatamansi was approximately 3938 kg/ha and 1764 kg/ha (Table 2). In this region, many species of medicinal plants are marketed by the State Government (Table 3).

Table 1: Medicinal plants of NDBR according to their Family/Taxa, Local name, Altitude Range (m), Endemism, Parts Used, Indigenous uses (Source: Joshi et al., 1999). Family/Taxa Achyranthaceae Achyranthes aspera L. A. bidentata Bl. Adiantaceae Adiantum venustum Don Apiaceae Angelica glauca Edgew.

Local name

Altitude Range (m)

Latjira

2000–3000

Adhajhar

2000–2200

Sun raj

200–2600

Frd.

Gandhrayan, Chhipi

3200–4000

Rh,rt

3500–4500

Kala jeera

2500–4000

3300–4900

Gandrajan

2500–2870

Rt, fr

2500–3000

2400–3000

Chhipi

2800–3500

Bhutkesh

3000–3500

Bhutkesh

3000–4000

Incens, insecticidal, nervine sedative Nervine sedative

Takkar

3000–5000

Lf, rt

Mentel disorder

Bang

3500–4000

Khanbankh,jinjok

2000–3000

2300–3500

Lf, fr

Bupleurum falcatum L. Carum carvi L. Cortia depressa (Don ) Norm Heracleum candicans wall. Ex. Pimpinella acuminata (Edgew) Cl P. diversifolia DC Pleurospermum angelicodes (DC.) Cl Selinum tenuifolium Wall. S. vaginatum (Edgew.) Selei sibirieum (L.)Boss. Araceae Arisaema flavum (Forsk.) Schott. A. jacquemontii Bl. Araliaceae Hedera nepalensis

Endemism Parts Used

Indigenous uses Antifertility in women, dysentery, ear and eyes complaints, pains in body Fever, whooping cough, jaundice Fever Dysentery, gastric, menorrhea, stomach disorder, vomiting; Abdominal inflammation,, Abdominal inflammation fever fever, liver complaints Carminative, cold. cough, fever, stomach disorder, ediblesedative, Rheumatism, stomachache Leucoderma, menstrual disorder Stomach disorder, gastric Carminative, stomach disorder Antithelmic, gastric

Skin diseases

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Ringworm, skin disease, edible Stimulant, Diaphoretic, cathartic, rheumatism,

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stimulant

Koch. Asclepiadaceae Marsdenia roylei Wt. Asteraceae Adenostemna lavenia (L.) Kuntze A. triplinervis

Cold, cough, edible Dudh bel

2500–2800

Fr, ft

2200–2800

Antiseptic, insect bite, cuts, wounds

Bukki

2300–3000

Diuretic

Samsa, Araka-jhar

2000–2500

Gutti

2000–2500

Cough, cuts, diarrhea, leprosy, skin disorders, edible Aromatic

Namchoo

2300–2800

Kilmora

2200–3000

Rt, br, fr

B. pseudumbellata Parker B. jaeschkeana Sch.

Kilmor

2700–3500

Rt, lf,

Rat and snake bite, boil, eye complaints, anticancer and blood pressure, edible Intestinal disorders

3000–3500

Rt,fl

Betulaceae Betula utilis D.Don

Bhojpatra

3500–4000

Bidens pilosa L.

Tagetus minuta L. Balsaminaceae Impatienens scabrida DC. Berberidaceae Berberis aristata DC.

Boraginaceae Arnebia benthamii Wall. Ex Maharanga emodi (Wall.)DC. Brassicaceae Arabidopsis thaliana (L.) Heynh Capsella bursa-pastoris (L.) M edic. Thlaspi arvense L. Cannabinaceae Cannabis sativa L.

Caprifoliaceae Viburnum erubescens Wlll.ex DC

Br, res -

Ratanjot

3300–3800

Shankhuli

3700–3800

3000–3600

Wp -

2000–4000 3200–4200

2000–3000 Bhang

2700–3600 Asara

Wp Lf, br, sd, fr, fl

Fr -

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Astringent, blood purifier, eye disorder, jaundice, skin disease, edible Antiseptic, burns, cuts, contraceptic, ear complaints, hysteria, jaundice, wounds Antiseptic, cuts, wounds, hair tonic, fungal hair infection Skin disorders, rheumatism, urinary disorder Treatment of sores in mouth

Blood pressure, diarrhea, dropsy Wounds, cuts, pulmonary infection, swelling Anthelmintic, appetite, bronchitis, cuts, dyspepsia, gonorrhea, narcotic, piles, skin disorders, cold cough, epilepsy, laxative, stimulant, paralysis of tongue, sleep piles, sores, edible Edible

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V. ctinifolium Don Caryophyllaceae Cerastium cerastoides (L.) Britt. Chenopodiaceae Chenopodium foliolosum (Moench .) Asch Commelinaceae Commelina benghalensis L. Cornaceae Cornus macrophylla Wall. Corylaceae Corylus jacquemontii Dcne Crassulaceae Sedum ewersii Ledeb. Cucurbitaceae Cucumis melo L. Cucurbita maxima Cupressaceae Juniperus indica Bertol. J. communis L. Dioscoreaceae Dioscorea deltoid Kunth. Elaeaginaceae Elaeagnus parviflora Wall. Ex royle Eriaceae Gaultheria fragrantissima Wall. Euphorbiaceae Euphorbia stracheyi Boiss Fabaceae Parochetus communis Don Fumariaceae Corydalis govanaina Wall Gentianaceae Swertia angustifolia Buch-Ham.

Ghinua

2300–3000 2000–4000

Br, fr Wp

Menorehoea, edible Backache, bodyache, headache, renal pain, cough

Lf,

Edible

Lf, rt

Fever, diarrhea, liver disorders, edible

Pangein 3000–4000 2200–2500 2200–2600

Edible

Pamakhor

2300–2700

Tonic, edible

Churappa

3000–4000

Lf, st

Toothache, apetite

Cooling, stomach disorder Kharbooza Kaddu

2000–2700 2000–3000

Fr, st Fr, sd

Chila Pallas

3200–4200 3500–4500

Fr Fr, lf

Intestinal worms, edible Incense Aromatic, incense Edible

Gun

2400–2800

Gewai

2200–3000

Br,fr

Cuts, ulcer, wound, edible

Cough, cold, edible Jalan-thrit

3000–4000

Lf, fr

Dudhibish

3500–4500

Latex

Khia-knoi

2100–2800

Butkeshi

3300–4000

Rheumatism

Stomach disorder

Chiraitu

2200–3600

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Antipyretic, diuretic, eye and ear disorder, gastric pain, ,muscles pain, skin disorder Malaria, fever

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Geraniaceae Geranium wallichianum D.Don ex Sw. Helvellaceae Morchella esculenta (L) Pers. Hypoxidaceae Curculigo orchioides Gaertn Iridaceae Iris kumaunensis D.Don ex Juglandaceae Juglans regia L Lamiaceae Ajuga parviflora Benth

2500–4000

Guchhi

2400–3800

Fr, body

Talmuli, Turum -

2400–3000

3000–4200

Br, lf, fr

Akhrot

2200–3000

Br, lf, fr

Titpati

2000–3500

Lf, sd

stracheyi Baker Moraceae Ficus palmata Forsk Morinaceae Morina longifolia Wall. Ex DC. Oleaceae Jasminum humile L Orchidaceae Dactylorrhiza hatagirea Don Oxalidaceae Oxalis corniculata L Paeoniaceae Paeomia emodi Wall. Papaveraceae Meconopsis aculeata Royle Parnassiaceaer Parnssia nubicola Hk.f. Pinaceae Abies pindrow Spach.

Edible Stomach disorder, scorpion and snake bite, wounds, skin diseases, cough and cold Fever Anthelmintic, astringent, frost bite, rheumatism, toothache, edible Ascariasis, stomachache, fever

Ban ajwain

2500–4000

Piyaj

2000–2500

Bb, lf

Jambu

3000–4500

Bedu

2000–2200

Eye complaints, liver and skin disorder, edible Anthelmintic, asthma, nose bleeding, boils bronchitis, diuretic, ear complaints, itching, piles, and ringworm. Edible Dysentery, indigestion, laxative, edible

Biskandara

3700–4000

Boils

Thymus linearis Benth Liliaceae Allium cepa L

Astringent, ear & eye disorder, toothache

Sinus, skin disorder Sungli

2500–3500

Br, rt.

Hattazari

3000–4000

Khata-mitha

2000–2500

Chandra

2300–2700

Rt, lf, st.

3500–4500

Nirbis

3000–4000

Raga

2300–3000

Res,br.

Astringent, bone fracture, tonic, wounds Appetite, corns, cuts, dysentery, fever, jaundice, edible Blood purifier, cuts, ulcer, wound, colic, dropsy, epilepsy Backache, colic, renal pain, tonic. Food poisoning, snake bite Rheumatism, ulcer

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Polygonacaeae Fagopyrum tataricum (L.) Ranuculaceae A.heterophyllum Wall Rhamnaceae Rhamnus purpureus Edgew. Rosaceae Prunus armeniaca L. Pyrus lantana Don Rubiaceae Galium actum Edgew Rutaceae Skimmia laureola (DC.) Zucc. Taxaceae Taxus baccata subsp. Wallichiana Valerinaceae Nardostachys grandiflora DC Valeriana jatamansi

Phaphar

2000–3500

Edible

Atis

2500–3500

Anthelmintic, cough, fever

Bakauro

2100–2500

Purgative

Chuli Moul

2000–3800 2200–2700

Fr Fr

Edible Edible

Kura

2500–4000

Antiscorb, skin disorder

Narr.

2500–4000

Lf, fr

Antiseptic, boils, gastric pain, smallpox

Thuner

2400–3500

Br, lf, fr.

Swelling, anticancer, edible

Jattamansi

3500–4200

Mushkbala

1500–2500

Cooling, cough, snake bite, blood purifier, ulcer Used as stimulant and carminative

Abbreviations used: H – Herbs; Sh –Shrub; T – Tree; Fn – Fungus; Lf – Leaf; Frd – Frond; Bb – Bulb; Br – Bark; Wp – Whole plant; AP – Arial part; Fl – Flower; Fr – Fruit; St – Stem; Tw – Twig; E – Endemic; NE – Near Endemic.

Table 2: Economics of cultivation of some medicinal plants (from mature plant after 8–9 years of growth) (Source: Ramakrishan et al.,, 1996). Species

Estimated yield Present market Total income (kg/ha) rate (Rs./kg) (Rs/ha) 4410 80 352000 Aconitum balfourii 1100 500 550000 Aconitum heterophyllum 4410 26 114660 Rheum emodi 1100 65 71500 Picrorhiza kurroa 1764 80 141120 Nardostachys jatamansi 3938 60 236280 Podophyllum hexandrum Wild Fruits with Economic Potential in NDBR: Despite abundant wild edible plant resources with immense potential for economic development, Uttarakhand remains underdeveloped (Phondani et al., 2011), owing primarily to inaccessibility and poor

infrastructure. Development initiatives show little concern for mountain perspectives. Yet the region is rich in resources and underutilized plant species with potential food value, about which there is little knowledge. Wild species such as Aegle marmelos (bael or Bengal quince), Berberis asiatica (berberry), Hippophae rhamnoides (seabuckthorn), Myrica

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nagi (kafal), Rubus ellipticus (yellow Himalayan raspberry), and Prunus armeniaca (apricot) has good economic potential (Table 3). A variety of value-added edible products

such as jam, jelly, juice, and squash could be made to generate income from these wild fruits, particularly for poor rural people (Maikhuri et al., 2004).

Table 3: Trade value of medicinal plants found in Uttarakhand Botanical Name

Local name

Rate/kg

Euophia dabia Pleurospermum angelicodes Podophyllum hexandrum Castanea sativa Zanthoxyllum armatum Paris polyphylla Sm. Rhododendron anthopogon Lichens (Parmelia sp. Usena sp.) Myrica esculenta Syzygium venosum Taxus baccata Valerina wallichii Aconitum heterophyllum Dactyloriza hatagirea Nordostachys grandiflora Picrorhiza kurroa Allium humile

Salam misri Choru Ban kakri Khan panger Temmor Bankh Takkar Safedjhula Kafal jamun Thuner Samewa Atis Hathajari Jatamansi Katuki Faran

35–40 30–50 50–100 35–40 20–25 18–22 30–60 25–40 50–60 25–30 30–50 30–60 180–250 900–1400 100–200 70–100 75–90

(Source: District Drug Cooperative Limited Almora 1992–93; Samant et al., 1996) Status: Among the recorded species Nardostachys grandiflora (Vulnerable), Picrorhiza kurroa (Vulnerable), Saussurea costus (Endangered) have been recorded in Red Data Book of Indian Plants (Samant et al., 1996). New IUCN Red list, categorizes these species as critically rare Aconitum heterophyllum, Aconitum balfourii, Podophyllum hexandrum, Valerina wallichii, Nordostachys grandiflora, Taxus baccata etc. Endangered - Saussurea obvallata, Berberis aristata, Picrorhiza kurroa. Near threatened Jurinella macrocephala. CONCLUSION: There are a number of medicinal plants found in NDBR, which have high medicinal

values, Due to habitat degradation and over exploration/anthropogenic activities, some species are declining, which seems to be a critical issue. There is a need to continue conservation of these medicinal plants. Documentation of the uses of these plant species may draw the attention of the researchers to conserve these plants. This article might be helpful for future references on the species grown in Nanda Devi Biosphere Reserve. ACKNOWLEDGMENT: The author is specially thankful to Dr. G.C.S. Negi and G.B. Pant Institute of Himalayan Environment & Development KosiKatarmal, Almora (Uttarakhand) for providing all facilities and support.

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