Medicinal Plants in Farwest Nepal: Indigenous Uses ...

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Editor Dr. Amjad Masood Husaini Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, India

Cover photos/figures: Left plate: Musical instrument of local deity Jamlu, Malana village (top), Angelica glauca (center left), Hedychium spicatum (center right), Udithach (largest alpine pasture in the valley) (bottom) (Sharma et al., pp 47-63). Top right row: Some Himalayan medicinal plants. (Left) Rhus parviflora. Fruits are indigenously used for diarrhea and dysentery. (Center) Urtica dioica. Stem juice is valued for sprain and fractures. (Right) Euphorbia royleana. Plant is kept in roof of house for protecting from evil. (Kunwar et al., pp 28-42). Center plate and gel: Agrobacterium rhizogenes-mediated genetic transformation in Rauwolfia serpentina with rolA (top gel) and virD1 (top gel) PCR detection (Goel et al., pp 8-14). Center right: Mature rhizomes of different genotypes of Picrorhiza scrophulariiflora. (Top) Bhutan, (Center) North Sikkim, (Bottom) East Sikkim (Bantawa et al., pp 1-7). Bottom right: In vitro propagation and acclimatization of Cichorium intybus through indirect callus culture on MS + 10 µM IBA (Hamid et al., pp 84-86). Disclaimers: All comments, conclusions, opinions, and recommendations are those of the author(s), and do not necessarily reflect the views of the publisher, or the Editor(s). GSB does not specifically endorse any product mentioned in any manuscript, and accepts product descriptions and details to be an integral part of the scientific content. Printed in Japan on acid-free paper. Published: December, 2010.

The Editor

Dr. Amjad Masood Husaini Dr. Amjad Masood Husaini, a young Scientist working as Assistant Professor in Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir (India) holds a Ph.D. in Biotechnology and PG Diploma in Bioinformatics (Jamia Hamdard, New Delhi), besides certificates in Intellectual Property Rights (Indian Law Institute, New Delhi) and Remote Sensing Applications in Agriculture (Indian Agricultural Research Institute-Indian Space Research Organization). Recipient of Young Scientist Award-2009 in Agriculture (Jammu & Kashmir State Council for Science & Technology, Government of J&K), Jawahar Lal Nehru Award for Agricultural Research-2008 (Indian Council of Agricultural Research, Government of India), Junior Scientist of the Year Award-2007 (National Environmental Sciences Academy, New Delhi), he is listed among Top 100 Scientists of 2010 by the International Biographical Centre (IBC, Cambridge), and his biography included in 27th edition of Marquis Who’s Who in the World. With an illustrious academic career Dr. Husaini holds the distinction of being top position holder in National Eligibility Tests for Life Sciences and Agricultural Biotechnology in India. His publications include book entitled ‘Strawberry- Transgenics for stresses’ and more than two dozen research/ review papers in National and International journals of repute, discussing different aspects of agricultural research and technology. Dr. Husaini serves as member of professional associations like World Association of Young Scientists, New York Academy of Sciences, The Indian Science Congress Association, Biotechnology Society of India, National Environmental Science Academy (India), Young Professionals’ Platform for Agricultural Research for Development, Scientists Without Borders, International Association of Computer Science and Information Technology, Royal Society of Crop Science, International Society for Biosafety Research, and serves in the capacity of editor/ associate editor etc. in editorial boards of various International journals of repute.

Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK

Foreword Amjad Masood Husaini Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, J&K 191121, India E-mail: [email protected] “Man, ever desirous of knowledge, has already explored many things, but more and greater still remains concealed; perhaps reserved for far distant generations, who shall prosecute the examination of their Creator’s work in remote countries, and make many discoveries for the pleasure and convenience of life” (Linnaeus, 1754). One such vast unexplored region and a biodiversity hot spot, lies between two great ancient civilizations of India and China and is famous as “The Great Himalayan Region”. The main Himalaya range runs west to east, from the Indus river valley to the Brahmaputra river valley, forming an arc 2,400 km long, which varies in width from 400 km in the western Kashmir-Xinjiang region to 150 km in the eastern Tibet-Arunachal Pradesh region. The range consists of three coextensive sub-ranges, with the northernmost, and highest, known as the Great or Inner Himalayas. The ancient religious scripture of Hindus, Atharvaveda is the earliest celebrated treatise mentioning the use of medicinal plants of the region. Atharvaveda contains 114 hymns or formulations for the treatment of diseases. Ayurveda, a system of traditional medicine native to the Indian subcontinent, originated in and developed from these hymns. The Suśruta Saṃhitā and the Charaka Saṃhitā are two important works on this traditional system of medicine. In addition there is a famous reference in Valmiki’s Ramayana, a religious scripture of Hindus, about the existence of rare medicinal plant Sanjivani (Selaginella bryopteris) in Himalayas, which saved the life of Lakshmana (brother of the Hindu god Lord Rama). Over the centuries people have depended on these medicinal plants for treating daily ailments like cough, colds, indigestion, ulcers, sore eyes etc. In fact Sir Lawrence, a British Settlement Commissioner in his book, ‘The Valley of Kashmir’ (1895) refers to this point as, “when I have made inquiries as to various herbs which I have seen in the valley and on hillsides, I am always told that they are hot and good for cold humours, cold and good for hot humours, dry and beneficial to damp humours, damp and beneficial to dry humours.” In this Special Issue (SI) on Himalayan MAPS, an attempt has been made to present various issues pertaining to conservation, documentation, biotechnological applications and medicinal uses of plants of Himalayan region. The SI comprises of 13 research articles related to different areas of plant biotechnology. In the first paper Bantawa et al. take-up an important highly valued endangered medicinal plant of Indo-China Himalayas viz. Picrorhiza scrophulariiflora Pennell and describe in detail its micropropagation. This study is first such report on this plant and illustrates the usefulness of additives for mass propagation and germplasm conservation. In a similar study Hamid et al. describe a method for in vitro shoot organogenesis of Cichorium intybus using shoot tips as explants. Cichorium intybus is known for its anti-cancerous and anti-hepatotoxic properties and their successful transfer to pots with 60% survival percentage is a step forward towards its ex situ conservation. The potential of Agrobacterium rhizogenes-mediated genetic transformation for the synthesis of phytomolecules of high pharmaceutical value is well established. Goel et al. present the first report of reserpine production in quantifiable amounts from the Agrobacterium rhizogenes-generated transgenic hairy roots of Rauwolfia serpentine, whose root-extracts have been used for centuries in Ayurvedic medicine. In one clone the reserpine level was found to be 23 times that of field grown roots, which is quite encouraging. Supply of authentic medicinal plants to herbal drug industry is an important requisite for enabling their commercial use in production of genuine phytoceuticals. An authentic identification system based on amplified fragment length polymorphism (AFLP) for Aconitum heterophyllum, A. violaceum, A. balfourii and A. ferox has been reported in an original research paper by Misra et al., which could be used for checking adulteration-related problems faced by commercial users

of the herb. Rasool et al. compared antioxidant and antimicrobial properties of wild and in vitro-regenerated plants of a Kashmir Himalayan perennial medicinal herb, Prunella vulgaris. Their study is probably the first report giving evidence that in vitro grown P. vulgaris has antioxidant and antibacterial activities similar to that of wild, suggesting the substitution of wild P. vulgaris with tissue culture raised plants for use in pharmaceutical industry. In another study on antibacterial activity, the potential of methanolic extract of seeds and leaves of Euryale ferox was tested against nine clinically isolated bacterial strains by Parray et al. The broad spectrum activity displayed by these extracts appears to provide logic for the use of E. ferox as ethno-medicine in urinary tract infections. The issues related to ethno-medicinal uses and overexploitation of medicinal plants of Haigad watershed of Kumaun Himalaya have been discussed by Joshi et al. They argue that for sustainable use, in addition to rapid conservation efforts, farmers should be involved in the cultivation of medicinal plants. An exhaustive ethno-botanical survey on phyto-diversity, spanning over more than 250 species, of Parvati Valley in Northwestern Himalayas described by Sharma et al. is highly informative. They stress the involvement of local inhabitants for conservation of indigenous knowledge and traditional practices. In a similar study on medicinal plants of west Nepal, Kunwar et al. compare indigenous knowledge of therapies of 48 medicinal plants with the latest common pharmacological findings, suggesting complementarities and thus forming base for use in modern therapeutic medicine. Similar correlation was reported by Ryakala et al. while studying the ethnobotany of 52 plant species used to cure diabetes by the inhabitants of north eastern India. Raj et al. have screened phytochemical constituents of 21 medicinal plants used in traditional Amchi system of medicine in the Ladakh region of India. The significance of these plants is discussed in the context of their role in ethnomedicine All these studies have generated the possibilities of using the unexplored plants as potential sources of future drugs. Verma et al. have contributed an informative paper describing the chemical composition of leaf and flower essential oils of Thymus serpyllum and T. linearis from Western Himalaya, while Hamid et al. discuss the impact of chromium on the oxidative defense system of Brassica juncea L., a medicinally important plant commonly used as a diuretic and stimulant. I hope that the scientists working on medicinal plants will find this Special Issue helpful in moving forward in their important quest of contributing in the area of medicine, drug discovery, and conservation of medicinal plants etc. I would like to thank Dr. Jaime A. Teixeira da Silva and Ms. Kasumi Shima at Global Science Books Ltd., UK for their cooperation and helpful suggestions; and my family for their understanding and support during the prolonged and time-consuming work on this volume.

December, 2010 Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK

Muhammad Iqbal, PhD, FNASc Professor

Department of Botany

JAMIA HAMDARD (Declared as Deemed-to-be University under Section 3 of the UGC Act, 1956 vide Notification No. F.9-18/85-U.3 dated 10.5.1989 of the Government of India)

December 24, 2010

The tradition of herbal treatment for curing the human ailments is pretty old in India. Formal accounts of medicinal plants appeared as early as during the Vedic period in the VedicoBrahminic treatises like Atharvaveda (2000 BC), Sushruta Samhita (1300 BC) and Charaka Samhita (300 BC). Of the tradional Indian systems of medicine, the Ayurveda (science of life) and the Unani (Greeco-Arabian) systems are based largely on medicinal plants, whereas Siddha depends mainly on minerals. Over the centuries, the traditional practitioners have developed a number of herbal formulations for the treatment of various ailments with special emphasis on sexual debility, liver disorders and kidney problems. As the popular alternative medicine, these preparations now constitute an important segment of the integrated health management all over the world. The Himalayas, often called "The Roof of the World", encompass a number of biodiversity hot spots and repositories of medicinal plants. The whole Himalayan range is envisaged as a trove of medicinal herbs, offering refuge to a variety of rare plants in its varied mountain ecosystems. The research work carried out in the recent past has accumulated enough scientific information on a variety of medicinal plants inhabiting the various zones of the Himalayan range with diverse climatic conditions. Given the above, the document in hand is a commendable effort that duly elucidates the various aspects of the medicinal-plant research that have a potential promise for a safe herbal medication without the much talked about adverse after-effects. The information covered by this issue is comprehensive and most of the plants mentioned are well known for their therapeutic efficacy. Information on the traditional knowledge, describing the ethno-medicinal uses of plants is also included. I heartily appreciate Dr. Amjad Masood Husaini of the Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, India, for editing this useful volume that focuses on the medicinal plants of the Himalayas, and also the galaxy of distinguished scientists and researchers who have contributed for this special issue of the Medicinal and Aromatic Plant Science and Biotechnology, an emerging research journal of the Global Science Books (GSB), UK. This document must prove a useful guide to botanists, cultivators and collectors of medicinal plants and a pride possession of all those who are keen on the Himalayan vegetation.

(Muhammad Iqbal)

Hamdard Nagar, New Delhi – 110 062, INDIA Phone: +91-11-2605 9688, Extn.: 5530 (O), 5531 (R); Fax: +91-11-2605 9663 E-mail: [email protected]

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CONTENTS Pranay Bantawa, Swapan Kumar Ghosh, Pamita Bhandari, Bikram Singh, Partha Deb Ghosh, Paramvir Singh Ahuja, Tapan Kumar Mondal (India) Micropropagation of an Elite Line of Picrorhiza scrophulariiflora, Pennell, an Endangered High Valued Medicinal Plant of the Indo-China Himalayan Region

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Manoj Kumar Goel, Shilpa Goel, Suchitra Banerjee, Karuna Shanker, Arun Kumar Kukreja (India) Agrobacterium rhizogenes-Mediated Transformed Roots of Rauwolfia serpentina for Reserpine Biosynthesis

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Amita Misra, Ashutosh K. Shukla, Ajit K. Shasany, V. Sundaresan, Shital P. Jain, Subhash C. Singh, Guru D. Bagchi, Suman P. S. Khanuja (India) AFLP Markers for Identification of Aconitum Species

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Rafia Rasool, Bashir Ahmad Ganai, Azra Nahaid Kamili, Seema Akbar, Akbar Masood (India) Antioxidant and Antibacterial Activities of Extracts from Wild and in Vitro-Raised Cultures of Prunella vulgaris L.

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Ripu M. Kunwar (Nepal/USA), Chundamani Burlakoti (USA), Chhote L. Chowdhary (Nepal), Rainer W. Bussmann (USA) Medicinal Plants in Farwest Nepal: Indigenous Uses and Pharmacological Validity

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Mukesh Joshi, Munesh Kumar (India), Rainer W. Bussmann (USA) Ethnomedicinal Uses of Plant Resources of the Haigad Watershed in Kumaun Himalaya, India

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Parveen Kumar Sharma, N. S. Chauhan, Brij Lal, Amjad M. Husaini (India), Jaime A. Teixeira da Silva (Japan), Punam (India) Conservation of Phyto-diversity of Parvati Valley in Northwestern Himalayas of Himachal Pradesh, India

47

Venkat Kishore Ryakala (India), Shahin Sharif Ali (Ireland/India), Hallihosur Sharanabasava, Naushaba Hasin, Pragya Sharma, Utpal Bora (India) Ethnobotany of Plants Used to Cure Diabetes by the People of North East India

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Ram Swaroop Verma, Rajendra Chandra Padalia, Amit Chauhan, Ajai Kumar Yadav (India) Chemical Composition of Leaf and Flower Essential Oils of Two Thymus spp. from Western Himalaya

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Ram Swaroop Verma, Rajendra Chandra Padalia, Amit Chauhan (India) Chemical Profiling of Mentha spicata L. var. ‘viridis’ and Mentha citrata L. Cultivars at Different Stages from the Kumaon Region of Western Himalaya

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Amit Chauhan, Ram Swaroop Verma (India) Cultivation Potential of Three Rose-scented Geranium (Pelargonium graveolens) Cultivars in the Kumaon Region of Western Himalayas

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Javid Ahmad Parray, Azra N. Kamilli, Raies Qadri, Rehana Hamid (India), Jaime A. Teixeira da Silva (Japan) Evaluation of Antibacterial Activity of Euryale ferox Salisb., a Threatened Aquatic Plant of Kashmir Himalaya

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Rehana Hamid, Azra N. Kamili, Mahmood uz Zaffar (India), Jaime A. Teixeira da Silva (Japan), A. Mujib, Javid Ahmad Parray (India) Callus-Mediated Shoot Organogenesis from Shoot Tips of Cichorium intybus

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Rehana Hamid, Mahmood uz Zaffar (India), Jaime A. Teixeira da Silva (Japan), Azra N. Kamili, Javid Ahmad Parray (India) Impact of Chromium on the Oxidative Defense System of Brassica juncea L. cv. ‘Pusa Jai Kissan’ under Hydroponic Culture

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Janifer Raj, Ballabh Basanth, Pal M. Murugan (India), Jaime A. Teixeira da Silva (Japan), Kumar Saurav, Om P. Chaurasia, Shashi Bala Singh (India) Screening Phytochemical Constituents of 21 Medicinal Plants of Trans-Himalayan Region

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How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

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Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books

Micropropagation of an Elite Line of Picrorhiza scrophulariiflora, Pennell, an Endangered High Valued Medicinal Plant of the Indo-China Himalayan Region Pranay Bantawa1 • Swapan Kumar Ghosh1 • Pamita Bhandari2 • Bikram Singh2 • Partha Deb Ghosh3 • Paramvir Singh Ahuja2 • Tapan Kumar Mondal1* 1 Biotechnology Laboratory, Faculty of Horticulture, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar, West Bengal, 736165 India 2 Institute of Himalayan Bioresource Technology, Palampur, Kangra, Himachal Pradesh, India 3 Department of Botany, Kalyani University, Kalyani, Nadia, West Bengal, India Corresponding author: * [email protected]

ABSTRACT An elite genotype of Picrorhiza scrophulariiflora Pennell was multiplied in vitro for its conservation. Rhizomes of mature plants collected from various locations of the eastern Himalayan region of Indo-China border were characterized morphologically and analyzed by HPLC to determine the content of marker compounds, namely picroside I and II. Amidst the genotypes, one from Ha, Bhutan was found to contain the highest amount of total picroside (7.33% dw). Subsequently, a rapid and highly reproducible method of micropropagation from rhizome or shoot tips was developed. While 100% bud break from rhizomes was achieved on Woody Plant Medium (WPM) containing 0.44 PM BAP (6 benzyl amino purine), 40-fold multiplication was achieved on WPM fortified with 2.3 PM Kn (kinetin) within 12 weeks. The multiplied shoots were elongated on WPM supplemented with 0.44 PM BAP. Around 90% of in vitro shoots were rooted without basal callus formation on WPM supplemented with 5.3 PM NAA (-naphthalene acetic acid) within 4 weeks. Following this protocol, 1100 micropropagated plantlets of an elite line (Ha, Bhutan) were hardened in their natural habitat. The present study illustrates the usefulness of additives for mass propagation and germplasm conservation and is, to the best of our knowledge, the first report of in vitro propagation of P. scrophulariiflora.

_____________________________________________________________________________________________________________ Keywords: in vitro regeneration, herb, HPLC, herbal medicine, picroside, Scrophulariacea

INTRODUCTION Picrorhiza scrophulariiflora (Pennell), Scrophulariacea, is an endangered small herbaceous plant found in the subalpine as well as alpine zone of the eastern Himalayas comprising Sikkim, Nepal and China (Hara et al. 1982). The rhizomes are used in Tibetan and Chinese traditional medicines to treat various ailments such as liver disorders, fever, asthma, jaundice and have pharmaceutical value for hepatoprotective, immunomodulator and antiasthamatic activities (Ghisalberti 1998; Smit et al. 2000). Though both Picrorhiza species i.e. P. kurroa and P. scrophulariiflora, are a rich source of irridoid glycosides such as picroside I and II, and kutkoside (Rastogi et al. 1949; Kitagawa et al. 1969; Weinges et al. 1972; Jia et al. 1999), P. scrophulariiflora contains an additional phenylethanoid glycoside and plantamajoside which are absent in P. kurroa (Li et al. 1998). Thus P. scrophulariiflora is a better substitute for P. kurroa (Smit et al. 2000). Several reports indicate the need for its conservation, sustainable utilization and cultivation (Ohba and Akiyama 1992; Olsen 1998; Manandhar 1999; Subedi 2000). This plant is not only heavily exported by local traders but also natural regeneration is hampered due to intentional fires set by local shepherds for making grazing area for their yaks which ultimately leads to unsustainable management and depletion of the species (Bantawa et al. 2009). As a result, this species was enlisted in a red data book around 20 years ago (Anon. 1987). Additionally, seed setting and seedling survival has been reported to be generally poor in alpine plants (Pandey 2000). An extensive literature survey revealed that though the genus Picrorhiza is well characterized chemically as well as

pharmacologically, except for few reports of the micropropagation of P. kurroa (Lal et al. 1988; Upadhyay et al. 1989; Chandra et al. 2006), no attempts either to identify elite lines of any kind or in vitro culture of this species have been made. Thus the present study was undertaken to identify chemically superior plants among the existing population and mass scale propagation of this line through tissue culture for sustainable management. MATERIALS AND METHODS Plant material Detailed accounts of plant material (Fig. 1A) collected from different locations of the eastern Himalayas during September-November are given in Table 1. Morphological parameters of 10 dried rhizomes per ecotype in three independent experiments were recorded which were then used for chemical analysis.

Thin Layer Chromatography (TLC) A Camag HPTLC system equipped with an automatic TLC sampler ATS4, TLC scanner 3 and an integrated software Win-CATS version 1.2.3 was used for the analysis. The entire matured rhizome (~6 cm long) of an individual plant was oven-dried, powdered and out of that, 100 mg was extracted with water: ethanol (50: 50) 2-3 times. The combined percolations were dried under vacuum at 45°C and dissolved in 2 ml of HPLC grade methanol. Samples and standards were applied to a pre-coated silica gel 60 F254 TLC plate (Merck, Darmstadt, Germany) as 10 mm bands, 10 mm from the bottom, 10 mm from the side, 6 mm between two spots with a Camag automatic TLC applicator (ATS4), equipped with a 25 μl syringe under N2 gas flow. Ascending development of

Received: 22 January, 2009. Accepted: 25 December, 2009.


Original Research Paper

Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 1-7 ©2010 Global Science Books How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

to appropriate concentrations (0.8, 1.6, 2.4, 3.2 μg/ml) and injected with 7725i rheodyne injector in triplicate.

Explant preparation for micropropagation For standardization of the micropropagation protocol, explants of Kuppup origin were used due to the availability of a large number of plants, after which the protocol was employed for mass-scale micropropagation using the elite lines i.e., from Ha in Bhutan. Two different types of explants were used in this study, 1) shoot tips and rhizomes immediately after collection, 2) two-weeks old sprouted buds that emerged from collected rhizomes, kept in the laboratory (Fig. 1B) under moist-dark conditions with fungicide Bavistin (BASF India Pvt. Ltd., India) solution (0.2% w/v) at room temperature. For inoculation, explants were washed thoroughly under running tap water for 10-15 min to reduce the surface dirt. Thereafter, the terminal or single nodes of the rhizomes were cut into small pieces (1-1.5 cm), washed with Tween-80 (Himedia) for 10 min followed by a wash under running tap water for 30 min. Immediately after the wash, once again they were treated together with a mixture of fungicide Bavistin (0.5% w/v) and Master (Tata Chemicals Ltd., India) (0.2%) as well as rifampicin (Himedia) (50 mg/l) for 4 h. Subsequently they were placed under a laminar hood and treated with mercuric chloride (Himedia) (0.1% w/v) for 3 min and washed 3 times with autoclaved sterile water each with 10 min. All explants, one per test tube (25 × 200 mm) were inoculated in an upright position with the 5 mm basal portion embedded in 5 ml of MS (Murashige and Skoog 1962) medium solidified with 0.8% (w/v) agar (Himedia) in the presence of activated charcoal (AC, Himedia) (0.2% w/v) and fortified with 3% (w/v) sucrose (Himedia). The pH of the medium was adjusted with 0.1 N KOH to 5.8 ± 0.1 before autoclaving the medium at 15 psi for 15 min. Cultures were then kept at 24 ± 2°C under a 12 h photoperiod at a light intensity of 2000 lux from cool florescent light tubes (Model LIFEMAX-A 73, Phillips India Ltd., India). Sub-culturing was done at 4-week intervals. Subsequently for various experiments, the basal medium, either MS or WPM (Woody Plant Medium; Lloyd and McCown 1980) were used along with different combinations of cytokinin such as Kn (kinetin, Himedia), BAP (6 benzyl amino purine, Himedia) and TDZ (thidiazuron, Sigma-Aldrich) (alone or in combination with NAA (-naphthalene acetic acid). After bud break, the shoots were cut at the base and subcultured onto multiplication medium consisting of WPM with various concentration of either Kn or BAP with different auxins such as IAA (indole-3-acetic acid, Himedia), NAA (Himedia) and subsequently onto elongation medium (WPM with various concentration of 0.44 PM BAP). For rooting, the individual shoots of 3-4 cm length were segregated from the clumps and subcultured on media containing various concentrations of NAA, IBA (indole-3-butyric acid, Himedia and IAA.

Fig. 1 In vitro propagation of P. scrophulariiflora. (A) The plants are in its natural habitat of Sikkim (mature spike). (B) Adventitious buds induced at laboratory. (C) Aseptic culture initiation from apical shoots WPM with 0.44 M BAP. (D) Germinated seeds on MS. (E) Multiple shoot formation at WPM with 2.3 M Kn at initial stage after 4 weeks. (F) After 8 weeks. (G) After 16 weeks. (H) The in vitro multiplied shoots rooted on WPM with 5.3 M NAA. (I) Transferred plantlets on plastic pots containing 9: 1 (virgin soil: sand). (J) Well hardened plants after 6 month. (K) Closed up view of acclimatized plantlets after 1 year and (L) Before field transfer.

the plate, migration distance 90 mm, was performed at 25 ± 2°C in choloroform: methanol (82: 18) as the mobile phase in a saturated Camag twin-trough chamber. After development, TLC plates were dried with the help of an air drier in a wooden chamber of appropriate ventilation. Densitometric scanning was performed at O = 270 nm with Wincats Software, using the deuterium light source with a slit width of 6 × 0.4 mm, scanning speed of 20 mm/s, and data resolution with 100 μl/step.

Effect of activated charcoal (AC) on multiplication To check the effect of AC on the multiple shoot formation, two combinations that induced high multiplication i.e. Kn at 1.8 and 2.3 μM in MS were fortified with 0.2% AC. The data were recorded after 4 weeks of subculture.

High performance liquid chromatography (HPLC) For quantifying the picrosides of different genotypes, we adopted the HPLC protocol of Singh et al. (2005). Briefly, 100 mg of the same sample which was prepared for TLC analysis from dried rhizome was used for HPLC analysis on a Shimadzu Prominence HPLC system, equipped with an LC-20AT quaternary gradient pump, dual wavelength SPD-20A UV-VIS detector, CBM-20A communication bus module, CTO-10AS VP column oven, 7725i rheodyne injector and Shimadzu CLASS-VP software. The chromatography was carried out on a Luna C18 (2) column (250 mm × 4.6 mm, 5 m particle size) from Phenomenex (Torrance, CA, USA). Desired resolution of picroside I and II with symmetrical and reproducible peaks was achieved using isocratic elution of water: acetonitrile (70: 30) as mobile phase with a 0.7 ml min-1 flow rate, for a run time of 30 min and detection wavelength of 270 nm. For preparation of a calibration curve, standard stock solution prepared in methanol of picroside I and II (Life Technologies India Pvt. Ltd., India, 99.90% purity). (0.2 mg/ml) was serially diluted

Hardening The rooted explants (about 3 cm) from any treatment were transferred directly to potting mixture containing virgin soil (top layer of black jungle soil collected from deep forest area) and sand (9: 1) in Hikko trays under a poly-shade house at Kyungnosla nursery, Department of Forest and Wild Life, Govt of Sikkim, Changhu (3758 msl), Sikkim, India. The survival percentage was recorded after 60 d of transfer.

Statistical analysis The experiments were set up in a randomized block design. Data were analyzed using analysis of variance (ANOVA) to detect significant differences between means (Sokal and Rohlf 1987). Means differing significantly were compared using Duncan’s Mul-


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Micropropagation of an elite line of Picrorhiza scrophulariiflora. Bantawa et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 1 Morphological descriptions and Picroside I and II contents in different Picrorhiza rhizomes. Rhizome Picrorhiza sp. Altitude Diameter Length Dry weight (cm) (cm) (g/rhizomes) P. kurroa Palampur, Himachal Pradesh (3000 m) 0.45 ± 04 d 7 ± 0.23 c 1.45 ± 0.16 c P. scrophulariiflora Thangu, North Sikkim (4000 m) 0.67 ± 0.05 b 9.74 ± 0.52 b 1.56 ± 0.14 b P. scrophulariiflora Kuppup, East Sikkim (4200 m) 0.64 ± 0.05 c 6.43 ± 0.41 d 1.41 ± 0.05 d P. scrophulariiflora Ha, Bhutan (4200 m) 0.7 ± 0.12 a 11.89 ± 0.4 a 2.12 ± 0.31 a

Picroside I (%)

Picroside II (%)

Total (%)

0.55 ± 0.23 d 0.95 ± 0.05 c 2.99 ± 0.12 a 2.21 ± 0.56 b

1.34 ± 0.24 d 5.40 ± 0.56 a 4.17 ± 1.02 c 5.12 ± 0.12 b

1.89 ± 0.47 c 6.35 ± 0.61 b 7.16 ± 1.14 a 7.33 ± 0.68 a

*Data (mean ± SE) pooled from three independent experiments; Means followed by the same letter does not differ significantly according to Duncan’s Multiple Range Test

tiple Range Test (DMRT) at P 0.05 with STATISTICA software ver. 5.0 (INC StatSoft 1995). Data is expressed as the mean ± standard error (SE).

A

B

C

RESULTS AND DISCUSSION Rhizome morphology In general, the rhizomes of Ha (Bhutan) plants are found to be the thickest (Fig. 2) and the longest (Table 1). This is very important as rhizomes are the only economic part of this species, thus higher biomass production has a direct link with the profitability for a commercial cultivation. However, no colour or texture differences were noticed among the collected rhizomes.

Fig. 2 Mature rhizomes of different genotypes of P. scrophulariiflora. (A) Bhutan, (B) North Sikkim, (C) East Sikkim.

P-I (Rf=0.75)

Quantification of picrosides The identification of picrosides was confirmed by TLC (Fig. 3) and later by comparison of their retention times, UV spectrum with standard compounds and by spiking the samples with standard stock solution (Fig. 4). Although different techniques such as spectrophotometry (Narayanan and Akamanchi 2003) and HPTLC (Sharma and Ramamurthy 2000) have been standardized for determining picroside content, HPLC has been the most successful (Dwivedi et al. 1997; Sturm and Stuppner 2000, 2001; Drasar and Moravcova 2004). The analytical data revealed that percentage mean values of total picroside content varied from a minimum of 6.35% (dw) of Thangu, North Sikkim, to a maximum of 7.33% (dw) of the plants from Ha, Bhutan (Table 1), which is higher than an earlier report of Smit et al. (2000), who also found that the total picroside content of P. scrophulariiflora was 6.2% (dw). Under the same experimental conditions, we also compared and found that the total picroside content of P. kurroa was 1.89%. Thus in the present study, picroside content is clearly higher in P. scrophulariiflora than in P. kurroa, which is in agreement with an earlier report of Singh et al. (2005) who found that total picroside content of P. kurroa varied from 0.021 to 3.1% among the different genotypes collected from the western Himalayas. In the present study, a wide range of variability in terms of rhizome morphology and picroside content has been detected and the best genotype was subsequently used for micropropagation using a range of explants. Such variability in chemical content has already been reported in a number of other medicinal plants (Hisiger and Jolicoeur 2007), including Picrorhiza (Singh et al. 2005). The difference in chemical contents among the accessions of P. scrophulariiflora could be explained by abiotic and biotic factors (Echeverrigaray et al. 2003; Kamarainen et al. 2003; Jayram and Prasad 2008). The chemical diversity determined in the present investigation will open further opportunities for varietal improvement through conventional breeding.

P-II (Rf=0.5)

)

Fig. 3 TLC plate of picrosides I and II. Arrow indicates the migration of the sample.

types of available explants were tested. Plants with forcibly sprouted buds under laboratory conditions registered a low (30%) level of contamination whereas these levels in shoot tips as well as rhizomes used as explants immediately after collection reached as much as 60%. Some contaminations, even after 4-5 months of sub-culture, were noticed. Of importance, buds that emerged from shoot tips were stronger and healthier (diameter of the shoots < 5 mm) (Fig. 1C) than those that emerged from rhizomes. Evaluation of basal medium, growth regulators on establishment and bud break A different degree of bud break occurred between MS and WPM medium under a wide range of PGR concentrations. In general, though, bud break was achieved within 4 weeks in all combinations, the lowest being 50 to 55% on basal media and highest 100% with BAP (0.44 μM) alone in both media (Table 2). However, increasing or decreasing the concentration of either BAP alone or in combination with NAA not only did not improve the percentage of bud break but also a tendency to produce long comparatively thinner (< 2 mm diameter) shoots was observed (Fig. 1D). Occasional rooting and lower bud break (60%) were also observed with 0.26-0.53 M NAA alone, a typical effect of NAA normally found during in vitro rooting. Although both media resulted in 100% bud break, subsequent sub-culturing on MS medium lead to sudden death of explants due to the secretion of some unknown, jelly-like substances at the basal portion of the explants. Therefore we decided to restrict all subsequent experiments to WPM medium only. This may have been caused by a high salt concentration in MS more than in WPM resulting in a stress which led to exudation of some secondary metabolites from the explants.

Micropropagation Being a high altitude plant, the climate was wet due to high rainfall and humidity which favour microbial and algal growth (Martin and Pradeep 2003) and thus establishment of an aseptic culture was a big challenge due to high percentage of microbial contamination. To avoid this, different


3


Picroside-I Std

Picroside-I (Kuppup) 1 .8 0

281.7 0.08 0

2 0 0 .7

1 .6 0

0.07 0

1 .4 0

203.0 0.06 0

1 .2 0

0.05 0

1 .0 0 AU

AU

2 8 1 .7

0.04 0

0 .8 0

0.03 0

0 .6 0

0.02 0

0 .4 0

0.01 0

0 .2 0

374.1 389.7 407.7 428.2 442.6

0.00 0

0 .0 0

200.00

250.00

300.00

350.00

400.00

2 0 0 .0 0

2 5 0 .0 0

3 0 0 .0 0

nm

3 5 0.0 0

4 0 0 .0 0

3 5 0.0 0

4 0 0 .0 0

nm

Picroside-II Std

Picroside-II (Kuppup) 2 .4 0

201.9

2 0 1 .9

2 .2 0

0.12 2 .0 0

2 2 0 .6

1 .8 0

0.10

1 .6 0 1 .4 0

264.0

AU

AU

0.08

0.06

2 6 4 .0

1 .2 0 1 .0 0

294.7

2 9 4 .7

0 .8 0

0.04 0 .6 0 0 .4 0

0.02

0 .2 0

0.00 200.00

0 .0 0

250.00

300.00

350.00

400.00

2 0 0 .0 0

2 5 0 .0 0

nm

3 0 0 .0 0 n m

Fig. 4 Comparison of UV spectrum of picrosides extracted from P. scrophulariiflora and standard.

Effect of PGRs on multiplication and elongation

subsequent sub-cultures (Table 3). Thus, we decided to use 0.44 μM BAP for elongating individual shoots. In contrast, Upadhyay et al. (1989) found that BAP (0.88 μM) was best for shoot multiplication for P. kurroa. Although the reason is not clear at present, this observation may be attributed to the difference in species, a phenomenon which often occurs in plant tissue culture. Additionally, Kn, being a mild cytokinin, is perhaps suitable for tender herbs such as Stevia rebaudiana (Ahmed et al. 2007), Alpinia galangal (Borthakur et al. 1999) and Asparagus adscendens (Mehta and Subramanian 2005).

Further for multiplication, a wide range of PGR combinations was used (Table 3). All combinations of Kn alone (0.46-9.2 μM) induced shoot multiplication but the maximum of 33 shoots per explant was observed only at 2.3 μM Kn (Fig. 1E-G). Multiple-shoot induction (i.e. >1) was observed within 12-15 days of incubation at all concentrations of PGRs tested. However, when Kn was used in combination with either IAA (0.28-5.7 μM) or NAA (0.28-5.3 μM), the multiplication rate did not improve as the maximum of 21% only of the multiplication rate was achieved in 0.46 μM Kn- and 0.26 μM NAA-containing media (Table 3). BAP alone (0.44-8.8 μM) or in combination with IAA (0.28 and 0.57 μM) or NAA (0.26 and 5.3 μM), produced multiple shoots from a minimum of 1/explant (8.8 μM, BAP) to a maximum of 13/explant (0.88 μM BAP). Shoots at lower concentrations of BAP (0.88–1.76 μM) were normal and had the tendency to elongate while at a higher concentration (2.2-8.8 μM), they became thinner and weaker in

Effect of AC on multiplication and elongation Further, to improve the multiplication rate, the effect of AC was evaluated in two ideal media formulations. Although we found that AC was required for initial bud break, later it hindered shoot bud multiplication significantly. In contrast, AC-free media enhanced shoot bud multiplication (Fig. 5). Therefore, in all subsequent multiplication and elongation steps, media was devoid of charcoal. Ebert et al. (2005) de-


4


Table 2 Effect of BAP and NAA in MS/WPM on bud break response. Percentage of bud break BAP (PM) NAA (PM) 0 0.13 0.26 0.53

0 50 ± 0.6 m 55 ± 1.2 l 60 ± 0.9 k 60 ± 1.3 k

0.44 100 ± 1.2 a 98 ± 0.4 abc 92 ± 0.2 e 95 ± 0.8 d

MS 1.33 98 ± 0.9 ab 88 ± 1.05 f 96 ± 0.2 d 92 ± 0.6 e

2.22 60 ± 0.85 k 60 ± 1.2 k 68 ± 0.6 i 64 ± 0.6 j#

0 55 ± 0.78 l 57 ± 0.89 l 61 ± 1.12 k 60 ± 0.7 k

0.44 100 ± 0.55 a 97 ± 1.56 cd 96 ± 0.56 cd 95 ± 0.12 d

WPM 1.33 100 ± 0.5 a 92 ± 0.8 e 98 ± 0.64 abc 98 ± 0.78 abc

2.22 76 ± 0.56 h 80 ± 0.23 g 88 ± 0.52 f 88 ± 0.56 f#

All values represents the mean ± SE. Means followed by the same letter does not differ significantly according to Duncan’s Multiple Range Test (P < 0.05) *= thin shoots; # = stunted growth

Table 3 Effect of different PGRs in the shoot multiplication of Picrorhiza scrophulariiflora. PGR name and concentration (PM) of shoots/explant* of leaves/shoot* 0 (control) 1.2 ± 0.22 j 8.0 ± 0.26 j 0.46 Kn 2.1 ± 0.65 j 13.43 ± 0.12 a 0.93 Kn 5.1 ± 0.30 i 11.36 ± 0.21 e 1.4 Kn 7.26 ± 0.20 h 9.9 ± 0.15 fg 1.8 Kn 21.1 ± 1.23 c 8.56 ± 0.23 ij 2.3 Kn 33.13 ± 1.13 a 12.56 ± 0.08 bc 4.6 Kn 20.76 ± 0.32 c 11.76 ± 0.14 de 9.2 Kn 11.16 ± 0.31 f 9.76 ± 0.08 fg 0.46 Kn + 0.28 IAA 1.7 ± 0.26 j 12.56 ± 0.37 bc 0.93 Kn + 0.28 IAA 3.33 ± 0.49 j 12.26 ± 0.26 bcd 1.4 Kn + 0.28IAA 6.6 ± 0.20 h 11.33 ± 0.13 e 1.8 Kn + 0.28 IAA 13.83 ± 1.01 e 9.6 ± 0.20 g 2.3 Kn + 0.57 IAA 19.76 ± 0.68 b 9 ± 0.47 hi 0.46 Kn + 0.26 NAA 21.06 ± 0.62 c 8.7 ± 0.5 ij 0.93 Kn + 0.26 NAA 17.06 ± 0.18 d 8.16 ± 0.2 j 1.4 Kn + 0.26 NAA 2.7 ± 0.49 j 7.6 ± 0.44 j 1.8 Kn + 0.26 NAA 1.16 ± 0.08 j 9.56 ± 0.26 gh 2.3 Kn + 0.53 NAA 2.13 ± 0.27 j 13.3 ± 0.11 a 0.44 BAP 11.83 ± 0.95 f 9.46 ± 0.13 a 0.88 BAP 13.63 ± 0.78 e 12 ± 0.30 d 1.32 BAP 2.60 ± 0.97 j 7.9 ± 0.20 j 1.76 BAP 2.2 ± 0.05 j 6.96 ± 0.16 j 2.2 BAP 1.8 ± 0.24 j 6.6 ± 0.21 k 4.4 BAP 1.6 ± 0.41 j 6.0 ± 0.45 k 8.8 BAP 1.15 ± 0.36 j 4.2 ± 0.22 k 0.44 BAP + 0.28 IAA 1.63 ± 0.16 j 13.43 ± 0.12 ij 0.88 BAP + 0.28 IAA 10.8 ± 0.36 f 10.2 ± 0.23 f 1.32 BAP + 0.28 IAA 5.9 ± 0.20 hi 9.9 ± 0.10 fg 1.76 BAP + 0.28 IAA 2.73 ± 0.44 j 10.03 ± 0.12 fg 2.2 BAP + 0.57 IAA 2.1 ± 0.55 j 8.53 ± 0.17 ij 0.44 BAP + 0.26 NAA 1.63 ± 0.16 j 13.43 ± 0.12 a 0.88 BAP + 0.26NAA 1.77 ± 0.44 j 13.22 ± 0.20 a 1.32 BAP + 0.26NAA 2.08 ± 0.42 j 13.40 ± 0.38 a 1.76 BAP + 0.26 NAA 2.26 ± 0.37 j 13.63 ± 0.23 a 2.2 BAP + 0.53 NAA 9.06 ± 0.38 g 12.6 ± 0.20 b

Shoot length (in mm)* 5.2 ± 0.19 jkl 7.4 ± 0.15 a 6.56 ± 0.17 de 6.46 ± 0.23 ef 6.13 ± 0.08 ef 6 ± 0.15 fg 5.7 ± 0.15 gh 5.06 ± 0.06 kl 7 ± 0.15 fg 6.2 ± 0.10 fg 5.56 ± 0.17 hij 4.96 ± 0.03 kl 4.86 ± 0.14 kl 4.63 ± 0.18 l 4.16 ± 0.14 l 3.56 ± 0.08 l 5.1 ± 0.11 jkl 6.03 ± 0.14 fg 7.96 ± 0.12 d 4.93 ± 0.14 kl 4.93 ± 0.13 kl 4.73 ± 0.08 l 3.3 ± 0.20 l 3.0 ± 0.08 l 1.8 ± 0.28 l 6.8 ± 0.20 abc 5.83 ± 0.06 g 5.23 ± 0.06 ijkl 5.06 ± 0.18 kl 4.73 ± 0.13 l 6.8 ± 0.20 d 6.84 ± 0.18 bcd 6.98 ± 0.42 abc 7.3 ± 0.22 ab 6.6 ± 0.20 de

*Each value represents the mean ± SE. Each mean value followed by the same letter does not differ significantly according to Duncan’s Multiple Range Test (P 0.05)

40

35 No. of shoots

concentration of those PGRs in the medium and subsequently reduced the multiplication rate. Similarly, Sharma and Ramamurthy (2000) as well as Chagas et al. (2003) found that AC inhibited the multiplication rate of Eucalyptus tereticornis and sweet orange (Citrus sinensis) in in vitro cultures.

Without activated charcoal With activated charcoal

30

25 20

b

b

15

Rooting and hardening of the plantlets

10

Well developed shoots (3 cm) from in vitro culture growing on WPM with 0.44 PM BAP were excised and transferred to rooting medium containing WPM salt augmented with NAA (0.53-10.6 μM), IAA (0.51-10.2 μM) or IBA (0.499.8 μM) alone. While basal medium induced minimal in vitro rooting, a maximum of 97% shoots formed an average of 7 roots/shoot on WPM with 5.3 μM NAA. Roots were also found to be longest at this concentration (Table 4). Rooting was, however, also observed at all the concentrations of IAA and IBA but the maximum only of 1-2 and 4-5 roots/shoot were produced by 56% (11.4 μM IAA) and 75% (10.7 μM IBA) of shoots, respectively. Both increasing or decreasing the concentration of either IAA or IBA did

5 0 Kn (2.3 μM)

Kn (1.8 μM)

Media formulation

Fig. 5 Effect of shoot multiplication rate of AC in MS media. Bar represent mean ± SE. Means followed by the same letter does not differ significantly according to Duncan’s Multiple Range Test.

monstrated that activated AC absorbs plant growth regulators (PGRs) such as BAP and 2,4-D, which lowered the


5


Table 4 Rooting response of micropropagated shoots of Picrorhiza scrophulariiflora. Response PGRs (PM ) 30 days No. of roots Root length (cm) 0 (control) 0.77 ± 4.22 g 0.7 ± 6.21 g IAA 0.5 1.66 ± 1.00 cdefg 1.8 ± 1.22 ef IAA 2.8 1.8 ± 0.86 cdefg 2.0 ± 1.42 de IAA 5.7 1.89 ± 1.26 cdefg 2.2 ± 1.22 de IAA 11.4 1.45 ± 1.68 fg 0.9 ± 1.26 fg NAA 0.5 3.43 ± 1.0 bcd 4.1 ± 0.34 c NAA2.6 5.07 ± 1.34 b 4.3 ± 0.12 bc NAA 5.3 7.0 ± 2.96 a 5.1 ± 0.71 ab NAA 10.7 7.1 ± 1.56 a 5.8 ± 0.33 a IBA 0.4 3.34 ± 0.26 bcde 2.2 ± 1.33 de IBA 2.6 3.68 ± 1.46 b 2.9 ± 0.96 d IBA 5.3 3.92 ± 1.86 b 4.2 ± 0.88 bc IBA 10.7 4.55 ± 2.22 b 4.4 ± 1.94 c

Rooting % after 30 days 24.02 ± 4.88 i 25.28 ± 1.88 hi 28.08 ± 1.08 h 34.74 ± 1.42 g 56.24 ± 2.22 d 54.60 ± 1.80 d 68.88 ± 2.08 c 97.28 ± 2.22 a 75.06 ± 1.66 b 38.22 ± 1.88 f 47.22 ± 1.66 e 68.28 ± 2.02 c 75.08 ± 0.98 b

*Each value represents the mean ± SE. Each mean value followed by the same letter does not differ significantly according to Duncan’s Multiple Range Test (P < 0.05). MS was used as basal media

not improve in vitro rooting. However, NAA as a better choice for in vitro rooting has been well reported in a number of plants such as in Picrorhiza kurroa (Upadhyay et al. 1989), Vitis labrusca (Lewandowski 1991), Berberis trifoliate (Mackay et al. 1996), Actinidia polygama (Tanaka et al. 1997), Stevia rebaudiana (Ahmed et al. 2007), and Dioscorea oppositifolia (Poornima and Ravishankar 2007). Although root initiation started within 15-18 d, maximum rooting occurred at 30 d (Fig. 1H). Following this protocol, ~1100 in vitro-rooted shoots were transferred from culture tubes into plastic cups (Fig. 1I-J) containing virgin soil: sand (9: 1) with a 90% survival after 60 d. The acclimatized, well-rooted plantlets (Fig. 1K-L) were successfully established in the field after 12 weeks.

tween ‘perario’ sweet orange and ‘ponca’ mandarin. Revista Brasileira de Fruticultura 25, 483-488 Chandra B, Palni LMS, Nandi SK (2006) Propagation and conservation of Picrorhiza kurroa Royal ex Benth.: An endangered medicinal herb of high commercial value. Biodiversity Conservation 15, 2325-2338 Drasar P, Moravcova J (2004) Recent advances in analysis of Chinese medical plants and traditional medicines. Journal of Chromatography B 812, 3-21 Dwivedi AK, Kulkarni D, Singh S (1997) Sensitive high performance liquid chromatography assay method for determination of picroside–I in plasma. Journal of Chromatography B 698, 317-320 Ebert A, Taylor F, Blake J (2005) Changes of 6-benzyl aminopurine and 2,4Dichlorophenoxyacetic acid concentration in plant tissue culture media in the presence of activated charcoal. Plant Cell, Tissue and Organ Culture 3, 157162 Echeverrigaray S, Fracaro F, Atti dos Santos AC, Paroul N, Wasum R, Atti Serafini LK (2003) Essential oil composition of South Brazilian population of C. galioide Benth and its relation with the geographic distribution. Biochemistry and Systematic Ecology 31, 467-475 Ghisalberti EL (1998) Biological and pharmacogical activity of naturally occurring iridoids and secoiridoids. Phytomedicine 5, 147-163 Hara H, Chater AO, Williams LHJ (1982) An Enumeration of the Flowering Plants of Nepal, Trustees of British Museum, London, 126 pp Hisiger S, Jolicoeur M (2007) Analysis of Catharanthus roseus alkaloids by HPLC. Phytochemical Review 6, 207-234 Jia Q, Hong MF, Minter D (1999) Picroside: A novel iridoid from Picrorhiza kurroa. Journal of Natural Products 62, 901-903 Jayaram K, Prasad MNV (2008) Genetic diversity in Oroxylum indicum (L.) Vent. (Bignoniaceae), a vulnerable medicinal plant by random amplified polymorphic DNA marker. African Journal of Biotechnology 7, 254-262 Kitagawa I, Hino K, Nishimura T, Mukai E, Yosioak I, Inouye H, Yoshida I (1969) Picroside I: A bitter principle of Picrorhiza kurroa. Tetrahedron Letter 43, 3837-3840 Kamarainen T, Laine K, Hohtola A, Uusitalo J, Jalone J (2003) Regional and habitat differences in 7-methyljuglone content of Finnish Drosera rotundifolia. Phytochemistry 63, 309-314 Lal N, Ahuja PS, Kukreja AK, Pandey B (1988) Clonal propagation of Picrorhiza kurroa Royle ex Benth. by shoot tip culture. Plant Cell Reports 7, 202-205 Lewandowski V (1991) Rooting and acclimatization of micropropagated Vitis labrusca ‘Delaware’. Horticultural Science 26, 586-589 Li JX, Li P, Tezuka Y, Namba T, Kadota S (1998) Three phenylethanoid glycosides and an iridoid glycoside from Picrorhiza scrophulariiflora. Phytochemistry 48, 537-542 Lloyd G, McCown W (1980) Commercially feasible micropropagation of mountain laurel Kalmia latifolia by use of shoot tips culture. Proceeding of the International Plant Propagators’ Society 30, 421-427 Mackay WA, Molinar F, Wall MM, Cardennas M (1996) Micropropagation of Agarita, Berberis trifoliata Moric. Horticultural Science 31, 1030-1032 Manandhar N (1999) Conservation of medicinal plants in Nepalese forests: Problems and perspectives. Medicinal Plant Conservation 5, 3-4 Martin KP, Pradeep AK (2003) Simple strategy for the in vitro conservation of Ipsea malabarica an endemic and endangered orchid of the Western Ghats of Kerala, India. Plant Cell, Tissue and Organ Culture 74, 197-200 Metha SR, Subramanian RB (2005) Direct in vitro propagation of Asparagus adscendens Roxb. Plant Cell, Tissue and Organ Culture 15, 25-32 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiology Plantarum 15, 473-497 Narayanan P, Akamanchi KG (2003) Colorimetric estimation of total iridoid content of Picrorhiza kurroa. Journal of Asian Natural Product Research 5, 105-111

CONCLUSION In the present study, we have identified a genotype of P. scrophulariiflora which is superior to any other inter and intra species genotypes for picroside content. Additionally, a highly reproducible micropropagation protocol has been developed that will be an immense help for producing large number of plantlets. Presently works are also in progress (1) to develop a composite cultivation package for large scale cultivation at their natural habitat, (2) to know the reason for variation of picroside content in different genotypes through molecular markers. Thus demonstration of propagation techniques and distribution of elite plantlets among the interested farmers for large scale cultivation will pave the way for in situ conservation of this endangered species. ACKNOWLEDGEMENTS The authors are thankful to Department of Biotechnology and Department of Science and Technology, Govt. of India for financial assistance, Mr. Bijoy Gurung, Department of Wildlife and Forest, Govt. of Sikkim, India for his help to conduct the survey, Mr. Kamal Das of this laboratory for his assistance.

REFERENCES Ahmed MB, Salahin M, Karim RR, Hannan MM, Sultana R, Hossain M, Islam R (2007) An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni) in Bangladesh. American Journal of Scientific Research 2, 121-125 Anonymous (1987) Rare and Endangered Plants of China, Shanghai Educational Press, Shanghai, China, 54 pp Bantawa P, Ghosh SK, Maitra S, Ghosh PD, Mondal TK (2009) Status and conservation threats of Picrorhiza scrophulariiflora Pennell. (Scrophulariaceae): An endangered high valued medicinal plant of Indo-China Himalayan region. Bioremediation, Biodiversity, Bioavailability 3, 15-22 Borthakur M, Hazarika J, Singh RS (1999) A protocol for micropropagation of Alpina galangal. Plant Cell, Tissue and Organ Culture 55, 231-233 Chagas EA, Pasqual M, Ramos JD, Cardoso P, Cazetta JO, DeFigueiredo MA (2003) Development of globular embryos from the hybridization be-


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Ethnopharmacology 73, 101-109 Sokal R, Rohlf FJ (1987) Introduction to Biostatistics (2nd Edn), Freeman WH and Co., New York, 363 pp Sturm S, Stuppner H (2001) Analysis of iridoids glycosides from P. kurroa by capillary electrophoresis and high performance liquid chromatography-Mass spectrometry. Chromatographia 53, 612-618 Sturm S, Stuppner H (2000) Analysis of cucurbitacins in medicinal plants by high pressure liquid chromatography-mass spectrometry. Phytochemical Analysis 11, 121-127 Subedi BP (2000) Plant profile: Kutki (Picrorhiza scrophulariiflora). Himalayan Bioresource 4, 4-8 Tanaka H, Shoyama Y, Sasaki Y, Sashida Y (1997) Micropropagation of Actinidia polygama from fruit galls. Plant Cell, Tissue and Organ Culture 48, 135-137 Upadhyay R, Arumugam N, Bhojwani SS (1989) In vitro propagation of Picrorhiza kurroa Royle Ex Benth: an endangered species of medicinal importance. Phytomorphology 39, 235-242 Weinges K, Kloss P, Henkels WD (1972) Natural products from medicinal plants. XVII, Picroside II, a new 6-vanilloyl catapol from Picrorhiza kurroa. Liebigs. Analytical Chemistry 759, 173-182

Ohba H, Akiyama SF (1992) The Alpine Flora of the Jaljale Himal, East Nepal, University of Tokyo, Tokyo, Japan, 59 pp Olsen CS (1998) The trade in medicinal and aromatic plants from central Nepal to northern India. Economic Botany 52, 279-292 Pandey H, Nandi SK, Nadeem M, Palni LMS (2000) Chemical stimulation of seed germination in Aconitum heterophylum Wall and A. balfourii Stapf.: important Himalayan species of medicinal of medicinal value. Seed Science and Technology 28, 39-48 Poornima GN, Ravishankar RV (2007) In vitro propagation of wild yams, Dioscorea oppositifolia L. and Dioscorea pentaphylla L. African Journal of Biotechnology 6, 2348-2352 Rastogi RP, Sharma VN, Siddiqui S (1949) Chemical examination of Picrorhiza kurroa Benth. Indian Journal Science and Research 8B, 172 Sharma SK, Ramamurthy V (2000) Micropropagation of 4 years old elite Eucalyptus tereticornis tree. Plant Cell Reports 19, 511-518 Singh N, Gupta AP, Singh B, Kaul VK (2005) Quantification of Picroside-I and Picroside-II in Picrorhiza kurroa by HPTLC. Journal of Liquid Chromatography and Related Techniques 28, 1679-1691 Smit HF, Kroes BH, Van den Berg AJJ, Van der Wa D, Van den Worm E, Beukelman CJ, Van Dijk H, Labadie RP (2000) Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora Pennell. Journal of


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®


Agrobacterium rhizogenes-Mediated Transformed Roots of Rauwolfia serpentina for Reserpine Biosynthesis Manoj Kumar Goel1,3* • Shilpa Goel4 • Suchitra Banerjee1 • Karuna Shanker2 • Arun Kumar Kukreja1 1 Plant Tissue Culture Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow-226015, U.P., India 2 Analytical Chemistry Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow-226015, U.P., India 3 Bio Agiculture Unit, Avesthagen Limited, Bangalore, 560066, Karnartaka, India 4 Department of Statistics, J.V. College Baraut, Bagpat, 250811, U.P., India Corresponding author: * [email protected]

ABSTRACT Root extracts of Rauwolfia serpentina have been used for centuries in Ayurvedic medicine as a panacea for a wide variety of physical as well as mental disorders. The potential of Agrobacterium rhizogenes-mediated genetic transformation for the synthesis of phytomolecules of high pharmaceutical value is now well established and documented. Transgenic roots were induced from R. serpentina leaf explants in response to A. rhizogenes A4 strain on semi-solid ½-strength MS medium. Amongst 200 hairy root clones developed, 27 showing persistent and incessant growth over several generations were selected. Transformed roots grew vigorously and branched profusely on hormone-free liquid B5 medium with 3% sucrose with higher biomass yields compared to the control and showed two stable and distinct morphotypes. Medium devoid of any carbon source served as the control. The transformed nature of the roots was confirmed by PCR amplification with rolA primers. Growth kinetic studies exhibited the highest growth index (58.57 ± 1.92) at the 10th week followed by slow growth in the subsequent period up to 14 weeks. Reserpine content increased with root growth and was highest in 10-weeks-old cultures. Hairy root clones showed a wide array of variation in relative reserpine content, varying from 0.0064 to 0.0858% dry weight (DW). On the basis of relative reserpine content, these hairy root clones were classified into 5 different groups. SM12 clone had the highest reserpine level (0.0858% DW) producing 2-3 times more than the content of field-grown roots harvested after 18-24 months. A distinct relationship between root morphology and reserpine content was observed. The present study is the first report of reserpine production in quantifiable amounts from the hairy roots of any Rauwolfia species.

_____________________________________________________________________________________________________________ Keywords: genetic transformation, hairy roots, HPLC

INTRODUCTION Roots of Rauwolfia serpentina are the principal natural source of the alkaloid ‘reserpine’ known for various pharmacological activities (Muller et al. 1952). The extracts of roots and total alkaloids R. serpentina are highly effective in hypertension, insomnia, giddiness, anxiety states, maniacal behavior, psychosis, schizophrenia and hyperglycemia (Duke 1985; Trivedi 1995; Bhattacharjee 1998). Reserpine depletes catecholamines (epinephrine and norepinephrine) and serotonin (5-hydroxytryptamine) from central and peripheral neurons by interfering with the uptake of these amines from the cytosol into vesicles and granules. The domestic demand for R. serpentina roots is continuously increasing. Long duration required for root harvesting (1824 months) and poor seed germination in the crop has restricted its commercial cultivation. Plant tissue culture techniques could be helpful in circumventing these problems. In vitro clonal propagation and indole alkaloids from multiple shoots of R. serpentina (Roja et al. 1985; Mathur et al. 1987; Roja and Heble 1996; Goel 2007) and isolation of alkaloids along with enzymes involved in their biosynthesis in cell suspension cultures (Ohta and Yatazawa 1979; Stockigt et al. 1981, 1983; Schubel and Stockigt 1984; Shimolina et al. 1986; Yamamato and Yamada 1986; Roja et al. 1987; Yamamato and Yamada 1987; Molokhova et al. 1988; Kunakh and Alkhimova 1989; Schuebel et al. 1989; Obitz et al. 1995; Kirilova et al. 2001) have been reported. However, the problem of genetic and biosynthetic instability of cell cultures and resurgence of interest in the potential of Agrobacterium rhizogenes-mediated hairy roots has

opened up a new area for enhanced secondary metabolite production (Benjamin et al. 1993; Falkenhagen et al. 1993; Sheludko and Kostenyuk, 1994; Klushychenko et al. 1995; Sheludko et al. 2002). However, commercial production of reserpine through hairy root cultures in R. serpentina has not been achieved so far. Therefore, the present study was aimed to enhance A. rhizogenes-mediated hairy root biomass and select high reserpine-yielding hairy root clones of R. serpentina as a potential alternative source. MATERIALS AND METHODS Hairy root induction In vitro cultures of R. serpentina maintained on MS (Murashige and Skoog 1962) medium with 1.0 mgl-1 BAP (6-benzylaminopurine, Sigma-Aldrich, India) and 0.1 mgl-1 NAA (-naphthelene acetic acid, Sigma-Aldrich) served as explant source for hairy root induction (Goel et al. 2007). Two wild type strains of A. rhizogenes viz. A4 (pRiA4) and LBA 9402 were used for transformation events and were grown at 28°C for 48 h in YMB (yeast mannitol broth, Hi-Media, India) medium. Fresh suspension was prepared by inoculating a single bacterial colony in 10 ml YMB medium and incubating for 48 h at 28°C at 100 rpm. Bacterial growth was estimated by optical density at 660 nm using a Nanodrop (ND-1000) spectrophotometer.

Bacterial inoculation in the explants and cocultivation In vitro leaf explants were pricked with a sterile needle using the

Received: 3 July, 2009. Accepted: 23 October, 2010.




bacterial suspension for induction of hairy roots. Leaf explants pricked using sterile distilled water served as the control. Pricks were made on the upper surface of the leaves so as to only cause sub-lethal injury. Explants were co-cultured on hormone-free MS basal medium with 3% sucrose and 0.8% agar at 25 ± 2°C and 40 μmol m-2 s-1 light intensity. After 2-3 days the explants were transferred to MS medium supplemented with 1 μg/μl of antibiotic “Sporidex” (Ranbaxy) to eliminate vestigial bacteria. The explants were repeatedly cultured on antibiotic supplemented medium until bacterium completely disappeared.

optimized hormone-free liquid basal medium. A minimum of three replicates were maintained for each treatment.

Qualitative analysis of the selected fast-growing hairy root clones Dried hairy roots of R. serpentina were extracted as per the protocol described earlier (Goel et al. 2009). The vacuum-dried extracts were checked on a TLC plate 20 × 20 cm silica gel 60 F254 (Merck Darmstadt, Germany). The plate was run in chloroform: methanol (95: 5) and visualized under UV light at 254 nm. In order to isolate reserpine, preparative TLC was carried out and the spot corresponding to authentic reserpine (Sigma-Aldrich) was eluted and redissolved in chloroform: methanol (3: 1). It was filtered and concentrated and run three times in the same mobile phase followed by an ethyl acetate: hexane: methanol (65: 25: 10) mixture. Finally, the plate was developed with Dragondorff’s reagent.

Effect of co-cultivation medium on hairy root induction After one week of incubation on MS medium, half of the disinfected explants were transferred to hormone-free ½-strength MS antibiotic medium and the remaining half were left on the same medium. Transformation frequency (TF %) was recorded up to the 6th week after root induction by the following formula

Growth kinetic studies in R. serpentina hairy roots and reserpine biosynthesis

Transformation Frequency (TF %) =

Growth kinetic studies were carried out to assess the optimum growth period, higher biomass and reserpine production in five (SM14, SM19, SM21, SM28, and SM30) randomly selected fastgrowing hairy root clones. Initially, about 150 mg of roots were inoculated in 50 ml medium. A minimum of three replicates were harvested at 2-week intervals from the 4th week onwards up to the 12th week. Different parameters i.e. dry matter (DM) %, growth index (GI) and reserpine content (% DW) were recorded using the following formula:

No. of explants showing hairy root emergence

X 100 Total No. of explants infected

Disinfection and maintenance of transformed hairy roots Putatively transformed roots 1.0-1.5 cm in length were excised from the explants and were transferred to hormone-free liquid MS medium containing antibiotic. Normal (non-transformed) roots obtained from in vitro shoot cultures were also maintained under same culture conditions.

Biomass dry weight Dry matter (%) = Biomass fresh weight

X 100

Quantitation of reserpine through HPLC

Confirmation of transformed nature of hairy roots

Quantitative estimation of reserpine in hairy root clones was carried out by reverse-phase high-performance liquid chromatography (RP-HPLC) using photodiode array (PDA) detection (Srivastava et al. 2006). An analytical HPLC system consisted of LC20AD solvent delivery pumps, a DGU-20A5 degasser, a CTO-20A column oven, 10AF auto-sampler and a SPD-M 20A photodiode array detector was used. Data acquisition was performed on lab Solution 3.21. Separation was achieved with a binary gradient program for pump A (acetonitile), and pump B (0.01 M phosphate buffer (NaH2PO4)) containing 0.5% glacial acetic acid at pH 3.5. A chromolith RP-18e HPLC column, 4.6 × 100 mm ID was used for all analyses. Column temperature was maintained at 26 ± 2°C and analysis was performed at a flow rate of 0.1 ml/min throughout the gradient run and data acquisition was performed at = 254 nm. Solvents were of HPLC grade (Merck, Darmstadt, Germany). Dried extracts from 10-week-old hairy root samples were sonicated and dissolved in methanol (methanol-HCl 98: 2, v/v) at 1 mg/μl on a dry weight (dw) basis. The reserpine (Sigma) standard was prepared in methanol (1 mg/ml). Reserpine content (0.0300.034% dw) in var. ‘CIM-Sheel’ developed at CIMAP (Gupta et al. 2005) was used as the benchmark to categorize the hairy roots.

In order to confirm the transformed nature of the hairy roots the putatively transformed and non-transformed roots were subjected to polymerase chain reaction (PCR) with primers for universal wild type A. rhizogenes A4 strain specific rolA gene harbored within the T-DNA. Forward (5-GGAATTAGCCGGACTAAACG3) and reverse (3-CCGGCGTGGAAATGAATCG-5) primers for rolA were procured from Genie Bangalore (India). Primers for the VirD1 gene (forward 5-ATGTCGCAAGGCAGTAAGC-3 and reverse 3-CGACGGTTGCTCCTGCTGA-5), coding for DNA outside the T-DNA of the Ri plasmid, were also used to rule out the possibility of A. rhizogenes contamination in hairy roots. This involved isolation of DNA (Khanuja et al. 1999) from roots and A. rhizogenes (Sambrook et al. 1989) followed by PCR amplification, which was carried out in a total volume of 25 μl in a Bio-Rad icycler version 4.006. The reaction comprised of 25-30 ng of template DNA, 0.3 U of Taq DNA polymerase, 0.25 μl of each dNTP, 1.5 mM MgCl2 buffer and 5 pmol of each primer. After initial denaturation at 94°C (5 min), the program was run for 35 cycles consisting of 94°C denaturation step (1 min), 60°C primer annealing step (1 min) and 72°C amplification step (1 min), at the end of the run a final amplification period (5 min; 72°C) was appended. Amplified DNA was loaded onto 1.2% agarose gel in TAE buffer stained with 0.5 μg/ml ethidium bromide and photographed on a polaroid gel documentation system.

Statistical analysis The results from growth kinetics experiments were analyzed by two-way ANOVA. The results were interpreted as statistically significant at P >0.01. This was computed as the ratio of mean square corresponding to the treatment to the mean square value representing the error variability from entire samples as opposed to using the value corresponding to the error variability computed by twoway ANOVA in the denominator of the ratio used to calculate the F-value.

Media optimization for hairy root growth at shake flask level MS, LS (Linsmaier and Skoog 1965), B5 (Gamborg et al. 1968), and NB (Nistch and Nistch 1969) basal culture media at ½ and ¼ strengths with 3% sucrose were examined to try and obtain higher biomass yield. The pH of the medium was adjusted to 5.86 ± 0.02 prior to autoclaving. A single root with lateral branches weighing approx. 20 mg was inoculated in 20 ml medium and growth was recorded after 6 weeks of inoculation. In another experiment, different pH levels (3.86, 4.86, 5.86, 6.86 and 7.86) and different levels of sucrose (0, 1.5, 3.0, 4.5 and 6.0%) were tested in the

RESULTS Hairy root induction Agrobacterium A4 strain was capable of inducing transgenic roots in R. serpentina leaf explants after 6 weeks with


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Agrobacterium-mediated transformed roots of Rauwolfia serpentina. Goel et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Maintenance of transformed hairy roots cultures

A

Emergence of root(s) from each needle prick on the in vitro leaf explants was considered as a distinct transformation event and was maintained as an individual root clone. More than 200 different hairy root clones were initially induced and the majority of these root clones formed callus and slow growth in hormone-free medium and therefore were discarded leaving behind only 40 fast-growing hairy root clones. These clones were named SM1, SM2, and SM3, etc. Some of the clones lost their growth potential during the course of subculture. Finally, 27 hairy root clones which grew vigorously with profuse branching on hormone- and antibiotic-free medium and showed persistent and incessant growth even after three years were selected (Fig. 1C). These roots showed two distinct morphological phenotypes that remained stable over subsequent generations. Of the 27 clones, four were morphologically different. These four root clones were thin, up to 15 cm long, less branched, soft, flexible, greenish white in color and were able to survive up to 7-8 months without further sub-culture, whereas remaining clones were highly branched, only 5-6 cm long, creamish in color, brittle and turned reddish on maturity and could survive up to 16-20 weeks without sub-culture. Nontransformed roots exhibited very slow growth in hormonefree medium.

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Confirmation of transformed nature of hairy roots

Fig. 1 A. rhizogenes mediated genetic transformation in R. serpentina. Hairy root induction in leaf explant (A) emergence of root bunches (inset); profuse hairy root growth in liquid B5 medium (B); maintenance of various hairy root clones at shake flask level (C).

PCR analysis of the DNA with rolA primers exhibited the amplification of the TL-DNA fragment (600 bp) in transformed roots (Fig. 2). Non-amplification of DNA from transformed root with the virD1 primers (Fig. 3) confirmed the lack of Agrobacterium contamination in hairy root clones. The expected amplification (650 bp) was obtained with A. rhizogenes A4 DNA (positive control). Non-transformed roots did not show any amplification either with rol A or vir D1 primers.

70% transformation frequency (TF) vs. 45% in LBA 9402. The average number of roots produced by A4 and LBA 9402 strains were 6 and 4, respectively. LBA 9402 induced callus before the onset of root emergence. Upon transfer of agro-infected leaf explants from MS to ½-MS medium, roots emerged from leaf explants on the 19th day of co-cultivation compared to hormone-free MS medium where roots were visible on the 27th day. Relative TF% on ½- and fullMS medium was 85.93 and 70.27, respectively after 6 weeks of culture. The transformed roots exhibited typical features of fast growth, profuse branching and negative geotropism (Fig. 1A, 1B).

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Effect of nutrient medium composition, pH and sucrose concentration Amongst various media (MS, LS, B5 and N6) tested, liquid basal B5 medium at pH 5.86 with 3% sucrose supported fast growth and highest biomass production of hairy root clones. Hairy root growth decreased as the strength of the culture medium decreased. There was a consistent increase in the growth of hairy roots with an increase in media pH from 3.86 to 5.86 followed by a decrease at higher pH levels i.e. 6.86 and 7.86, respectively. Highest root biomass (3.26 ± 15

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Fig. 2 PCR with rol A primers and hairy root clones. M = marker DNA; 1-27 = hairy root clones; C = control (non-transformed root); A = DNA from A. rhizogenes A4 strain. Arrow indicated 600-bp fragment.

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Fig. 3 PCR with vir D1 primers and hairy root clones. M = marker DNA; 1-27 = hairy root clones; C = control (non-transformed root); A = DNA from A. rhizogenes A4 strain. Arrow indicates 650-bp fragment. Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK

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Table 1 Growth kinetics and reserpine content of hairy root clone(s) at different growth periods. Hairy root clones Growth (weeks) Growth parameters SM14 SM19 SM21 4 Dry wt. (g) 0.26 ± 0.033 0.44 ± 0.082 0.43 ± 0.025 Dry matter % 10.92 8.99 8.5 Growth index 15.18 ± 1.77 31.62 ± 5.55 32.44 ± 0.81 Reserpine (% dw) 0.0131 0.0213 0.0332 6 Dry wt. (g) 0.50 ± 0.057 0.73 ± 0.023 0.66 ± 0.12 Dry matter % 9.14 11.93 10.05 Growth index 35.8 ± 2.23 39.95 ± 8.0 42.75 ± 1.81 Reserpine (% dw) 0.0162 0.0263 0.0388 8 Dry wt. (g) 0.62 ± 0.01 0.67 ± 0.023 0.68 ± 0.026 Dry matter % 8.86 9.49 8.92 Growth index 45.74 ± 2.06 46.06 ± 7.02 49.78 ± 2.09 Reserpine (% dw) 0.0201 0.0339 0.0320 10 Dry wt. (g) 0.68 ± 0.006 0.73 ± 0.03 0.73 ± 0.046 Dry matter % 7.55 7.77 8.34 Growth index 58.97 ± 0.26 61.62 ± 2.13 57.31 ± 4.11 Reserpine (% dw) 0.0230 0.0426 0.0422 12 Dry wt. (g) 0.65 ± 0.031 0.66 ± 0.038 0.78 ± 0.006 Dry matter % 7.07 7.26 9.1 Growth index 60.7 ± 1.1 59.93 ± 2.62 56.2 ± 3.67 Reserpine (% dw) 0.0139 0.0414 0.0365

SM28 0.24 ± 0.058 11.61 12.78 ± 3.53 0.0293 0.63 ± 0.075 10.84 37.98 ± 2.29 0.0323 0.44 ± 0.031 8.40 34.11 ± 2.08 0.0458 0.68 ± 0.012 7.91 56.67 ± 0.63 0.0546 0.66 ± 0.014 7.96 54.00 ± 0.30 0.0472

Table 2 ANOVA table for the study of effect of hairy root lines and culture period on the growth index of R. serpentina. Source of variation Df S.S M.S = S.S/ df Fcal Hairy root lines (L) 4 685983.3 (±2643.76) MSL= 171495.8 (±660.94) MSL/MSE= 3.2113 (±3.1799) Culture period (T) 4 683644.3 (±2666.98) MST= 170911.1 (±666.74) MST/MSE= 3.2003 (±3.2078) L*T 16 854466.4 (±3325.56) MSE= 53404.15 (±207.85) Total 24 2224094 (±8636.3) -

SM30 0.47 ± 0.036 7.3 41.99 ± 4.2 0.0411 0.73 ± 0.053 9.27 51.82 ± 4.56 0.0345 0.67 ± 0.026 8.29 52.86 ± 3.64 0.0506 0.66 ± 0.006 7.45 58.29 ± 2.05 0.0562 0.68 ± 0.039 7.38 60.6 ± 3.2 0.0528

Ftab F4,16 = 4.7726 F4,16 = 4.7726 -

Since F values for hairy root lines (L) and growth period (T) are less than the tabulated value, therefore H0 is accepted at 1% level of significance. Values in bracket are the ANOVA of respective standard deviation

70 Growth Index

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Fig. 4 Average growth indices (AGI) of five hairy root clones at different growth periods.

0.15) was obtained at 3% sucrose. Sucrose concentration beyond 3% inhibited growth. Roots exhibited mortality within 5-6 weeks in medium devoid of a carbon source (data not shown).

decline in subsequent weeks, irrespective of their relative reserpine content (Table 1, Fig. 5). As is evident from the ANOVA (Table 2), since F values for hairy root lines (L) and growth period (T) are less than the tabulated value therefore the observations were accepted at P = 0.01.

Growth kinetics studies in R. serpentina hairy root clones and reserpine analysis

Qualitative analysis of the selected fast growing hairy root clones

Growth kinetic studies firmly revealed a continued increase in root growth in clones SM19, SM21 and SM28 until the 10th week of culture followed by a marginal decline. Although clones SM14 and SM30 exhibited a continuous increase in biomass up to the 12th week of culture, a subsequent increase was not significant during the 10-12th week culture period (Table 1). Growth index (GI) of each of the 5 hairy root clones exhibited a definite sigmoid growth pattern. A continuous increase in root biomass was recorded during first 10 weeks. Highest GI (58.57 ± 1.92) was recorded at the 10th after which there was no significant increase in biomass (GI = 58.29 ± 3.02) up to the 12th week of culture (Fig. 4). In all 5 hairy root clones, reserpine content also reached the highest level after 10 weeks of growth followed by a

Reserpine was detected at Rf = 0.5 in chloroform and methanol (95: 5) and at Rf = 0.75 when the same TLC plate was run in ethyl acetate: hexane: methanol (65: 25: 10). A single spot was detected at Rf = 0.5 when the same fraction was run anew in ethyl acetate: hexane: methanol (65: 25: 10). Selection of high reserpine-producing hairy root clones in R. serpentina Hairy root clones exhibited a wide range (0.0064-0.0858% dw) of reserpine content (Fig. 6). Compared to the reserpine content (0.03-0.034) of var. ‘CIM-Sheel’, all root clones


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Reserpine content (% DW)

0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01

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Fig. 7 HPLC chromatogram of reserpine standard (A) and hairy root clone SM12 (B). Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK

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Fig. 6 Variation in reserpine content in different hairy root clones after 10 weeks of growth.

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fate of rol-induced meristem depends upon the local hormonal balance of a cell/tissue (Arroo et al. 1995; Baumann et al. 1999). Putatively transformed roots of R. serpentina demonstrated amplification of rolA. In earlier reports the transformed nature of hairy roots in R. serpentina was confirmed by opine analysis (Falkenhaegen et al. 1993; Benjamin et al. 1994). The literature so far does not support any evidence of genetic transformation at the molecular level in this species; this is the first report of molecular evidence of genetic transformation in R. serpentina. Stable integration of Ri T-DNA into the host plant genome accounts for the genetic stability of transformed root cultures. Their biochemical stability leads to a high growth rate with a stable and high level of production of secondary metabolites (Kamada et al. 1986). Secondary metabolite biosynthesis in transformed roots is genetically controlled (Hamill and Rhodes 1988) but it is also strongly influenced by nutritional and environmental factors (De-Eknamkul and Ellis 1984; Hilton and Rhodes 1993). These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. The rol genes in Ri T-DNA induce changes in sensitivity to plant hormones and/or in the metabolism of plant hormones (Akutsu et al. 2004). Owing to the random integration of T-DNA into the host plant genome, the resulting hairy roots often show variable patterns of secondary metabolite accumulation. Due to a certain amount of heterogeneity, repeated selection seems to be an important approach to obtain high-yielding hairy root lines (Yukimune et al. 1994). To the best of our knowledge this is the first report of reserpine synthesis in the hairy roots of R. serpentina.

Table 3 Reserpine content in 10-weeks-old R. serpentina hairy root clones. Category Hairy root Tissue DW Extract wt. Reserpine clone (g) (mg) (% DW) SM12 0.66 92.1 0.0858 Group 1 SM22 1.33 192.4 0.0799 SM36 1.37 250.6 0.0729 SM4 1.26 175.4 0.0709 1.41 207.0 0.0701 SM1 1.54 218.2 0.0665 SM5 Group 2 SM2 1.17 178.2 0.0574 SM30 1.52 250.6 0.0562 SM28 1.30 231.2 0.0546 SM3 1.96 243.1 0.0533 SM26 1.35 225.3 0.0519 1.31 170.4 0.0502 SM8 1.07 173.3 0.0473 SM13 Group 3 SM6 1.35 195.7 0.0467 SM16 1.25 149.6 0.0466 SM32 1.04 163.6 0.0465 SM11 1.38 208.3 0.0458 SM19 1.36 110.1 0.0426 0.94 152.9 0.0422 SM21 Group 4 1.39 97.2 0.0345 SM15 SM9 1.43 171.5 0.0326 Group 5 1.55 186.4 0.0230 SM14 SM29 1.49 114.8 0.0139 SM33 1.50 114.8 0.0109 SM7 1.06 51.4 0.0089 SM40 2.08 117.1 0.0065 SM17 3.07 354.5 0.0064

ACKNOWLEDGEMENTS Authors are thankful to the Director, Central Institute of Medicinal and Aromatic Plants, Lucknow, for providing facilities. A fellowship provided by Council of Scientific and Industrial Research, Govt. of India to M.K. Goel is gratefully acknowledged.

were grouped into 5 different categories (Table 3). All 5 clones SM1, SM4, SM12, SM22 and SM36 synthesized most reserpine, with SM12 containing the highest reserpine content (0.0858% dw) (Fig. 7B, Table 3). Most of the hairy root clones exhibited a relatively higher reserpine content (0.0422-0.0665% dw). Reserpine content (0.0326-0.0345% dw) in clones SM9 and SM15 was almost equivalent to ‘CIM-Sheel’ and 6 clones recorded lower reserpine content than the control. Reserpine content in SM12 was about 14 times higher than that produced by SM17, which revealed the variable nature of hairy roots for alkaloid production. A fair relationship between root morphology and reserpine content was also observed. As already mentioned, four clones were morphologically distinct from others: they produced a very low amount (0.0064-0.0139% dw) of reserpine. On the other hand, other clones produced a higher amount of reserpine.

REFERENCES Akutsu M, Ishizaki T, Sato H (2004) Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes. Molecular Breeding 13, 69-78 Akramian M, Tabatabaei SMF, Mirmasoumi M (2008) Virulence of different strains of Agrobacterium rhizogenes on genetic transformation of four Hyoscyamus species. American Eurasian Journal of Agricultural and Environmental Sciences 3, 759-763 Arroo RRJ, Develi A, Meijers H, Van de WE, Kemp AK, Croes AF, Williams GJ (1995) Effects of exogenous auxin on root morphology and secondary metabolism in Tagetes patula hairy root cultures. Physiologia Plantarum 93, 233-240 Baumann K, De Paolis A, Costantino P, Gualberti G (1999) The DNA binding site of the Dof protein NtBBF1 is essential for tissue specific and auxinregulated expression of the rolB oncogene in plants. The Plant Cell 11, 323333 Benjamin BD, Roja G, Heble MR (1993) Agrobacterium rhizogenes mediated transformation of Rauwolfia serpentina: Regeneration and alkaloid synthesis. Plant Cell, Tissue and Organ Culture 35, 253-257 Benjamin BD, Roja G, Heble MR (1994) Alkaloid synthesis by root cultures of Rauwolfia serpentina transformed by Agrobacterium rhizogenes. Phytochemistry 35, 381-383 Bhattacharjee SK (1998) Handbook of Medicinal Plants, Pointer Publishers, Jaipur, India, 293 pp Bulgakov VP (2008) Functions of rol genes in plant secondary metabolism. Biotechnology Advances 26, 318-324 De-Eknamkul W, Ellis BE (1984) Rosmarinic acid production and growth characteristics of Anchusa officinalis cell suspension cultures. Planta Medica 51, 346-350 Duke JA (1985) Handbook of Medicinal Herbs, CRC Press Inc., Boca Raton, Florida, 401 pp Falkenhagen H, Stockigt J, Kuzovkina IN, Alterman IE, Kolshorn H (1993) Indole alkaloids from the hairy roots of Rauwolfia serpentina. Canadian Journal of Chemistry 71, 2201-2203 Flores HE, Vivanco JM, Loyola-Vargas VM (1999) ‘Radicle’ biochemistry: the biology of root-specific metabolism. Trends in Plant Science 4, 220-226 Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soya bean root callus. Experimental Cell Research 50, 151158 Giri A, Narasu LM (2000) Transgenic hairy roots: Recent trends and applica-

DISCUSSION The differences in virulence and morphology may be attributed to the different Ri-plasmids harbored by the strains (Nguyen et al. 1992; Akramian et al. 2008). The plagiotropic characteristic of hairy roots is advantageous as it increases aeration in liquid medium and roots grown in air have an elevated accumulation of biomass. The faster growth of transformed roots may be attributed to their extensive branching, resulting in many meristems (Flores et al. 1999; Giri and Narasu 2000; Srivastava and Srivastava 2007). The hairy roots are usually non-chimeric because they originate from single cells and each hairy root clone consists of uniformly transformed cells (Ohara et al. 2000). Hormone autonomy is due to the capability of endogenous synthesis of auxin in hairy roots. Genes present on A. rhizogenes TDNA are involved in overproduction of plant hormones at the infection site, causing the hairy root diseases (Machado et al. 1997; Bulgakov 2008). The difference in morphology and branching and growth pattern may be due to the difference in endogenous hormone level of target cells because rol expression is largely an auxin-inducible system and the


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Rauwolfia serpentina. Experimentia 8, 338 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473-497 Nguyen C, Bourgaud F, Forlot P, Guckert A (1992) Establishment of hairy root cultures of Psoralea species. Plant Cell Reports 11, 424-427 Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 167, 8587 Obitz P, Endreb S, Stockigt J (1995) Enzymatic biosynthesis of raumacline. Phytochemistry 40, 1407-1417 Ohara A, Akasaka Y, Daimon H, Mii M (2000) Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L. Plant Cell Reports 19, 563-568 Ohta S, Yatazawa M (1979) Growth and alkaloid production in callus tissue of Rauwolfia serpentina. Agricultural and Biological Chemistry 43, 2297-2303 Roja PC, Benjamin BD, Heble MR, Chadha MS (1985) Indole alkaloids from multiple shoot cultures of Rauwolfia serpentina. Planta Medica 50, 7374 Roja PC, Sipahimalani AT, Heble MR, Chadha MS (1987) Multiple shoot cultures of Rauwolfia serpentina: Growth and alkaloid production. Journal of Natural Products 50, 872-875 Roja G, Heble MR (1996) Indole alkaloids in clonal propagules of Rauwolfia serpentina Benth ex Kurz. Plant Cell Tissue and Organ Culture 44, 111-115 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual (2nd Edn), Cold Spring Harbor Laboratory Press, New York, pp 4758 Schübel H, Stöckigt J (1984) RLCC-isolation of raucaffricine from its most efficient source - cell suspension cultures of Rauwolfia serpentina Benth. Plant Cell Reports 3, 72-74 Schubel H, Ruyler CM, Stockigt J (1989) Improved production of raucaffricine by cultivated Rauwolfia cells. Phytochemistry 28, 491-494 Sheludko Y, Gerasimenko I, Kolshorn H, Stockigt J (2002) New alkaloid of the sarpagine group from Rauwolfia serpentina hairy root cultures. Journal of Natural Products 65, 1006-1010 Sheludko Y, Kostenyuk V (1994) Growth dynamics and total alkaloid content in the transgenic root culture of Rauwolfia serpentina Benth. Tsitologia Genetica 28, 35-38 Shimolina LL, Minina SA, Derbina GA (1986) Alkaloids of Rauwolfia serpentina cell cultures. Kultura Kletok Rast. J. Bioteknol. M. 58-60 (Russian). Chemical Abstracts 1987, 106, 135321h Srivastava A, Tripathi AK, Pandey R, Verma RK, Gupta MM (2006) Quantitative determination of reserpine, ajmaline, and ajmalicine in Rauwolfia serpentina by reversed-phase high-performance liquid chromatography. Journal of Chromatographic Science 44, 557-560 Srivastava S, Srivastava AK (2007) Hairy root cultures for mass production of high value secondary metabolites. Critical Reviews in Biotechnology 27, 2943 Stockigt J, Pfitzner A, Keller PI (1983) Enzymatic formation of ajmaline. Tetrahedron Letters 24, 2485-2486 Stöckigt J, Pfitzner A, Firl J (1981) Indole alkaloids from cell suspension cultures of Rauwolfia serpentina Benth. Plant Cell Reports 1, 36-39 Trivedi KC (1995) Sarpagandha. In: Chadha KL Gupta R (Eds) Advances in Horticulture (Vol 11) Medicinal and Aromatic Plants, Malhotra Publishing House, New Delhi, pp 453-466 Vakil RJ (1949) A clinical trial of Rauwolfia serpentina in essential hypertension. British Heart Journal 11, 350-355 Yamamoto O, Yamada Y (1987) Selection of a reserpine-producing cell strain using UV-light and optimization of reserpine production in the selected cell strain. Plant Cell, Tissue and Organ Culture 8, 125-133 Yamamoto O, Yamada Y (1986) Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. cells. Plant Cell Reports 5, 50-53 Yukimune Y, Hara Y, Yamada Y (1994) Tropane alkaloid production in root cultures of Duboisia myoporoides obtained by repeated selection. Bioscience, Biotechnology and Biochemistry 58, 1443-1446

tions. Biotechnology Advances 18, 1-22 Goel MK (2007) Application of bioreactors for micropropagation and secondary metabolite production in Rauwolfia serpentina Benth. Ex. Kurz. Ph.D. thesis, Hemvati Nandan Bahuguna University, Garhwal, Uttarakhand, India, 280 pp Goel MK, Kukreja AK, Khanuja SPS (2007) Cost-effective approaches for in vitro mass propagation of Rauwolfia serpentina Benth. Ex. Kurz. Asian Journal of Plant Sciences 27, 957-961 Goel MK, Mehrotra S, Kukreja AK, Shanker K, Khanuja SPS (2009) In vitro propagation of Rauwolfia serpentina using liquid medium, assessment of genetic fidelity of micropropagated plants, and simultaneous quantitation of reserpine, ajmaline and ajmalacine. In: Jain SM, Saxena PK (Eds) Methods in Molecular Biology, Protocols For in vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants (Vol 547), Humana Press Inc., Springer, USA, pp 17-34 Guillon S, Guiller JT, Pati PK, Rideau M, Gantet P (2006) Hairy root research: Recent scenario and exciting prospects. Current Opinion in Plant Biology 9, 341-346 Gupta AK, Khanuja SPS, Gupta MM, Darokar MP, Singh S, Chauhan HS, Mathavan RE, Saikia D, Shasany AK, Verma RK, Kukreja AK, Dhawan OP, Kalra A, Singh HN, Roy SK, Bahl JR, Singh A, Singh SP (2005) A genetically improved variety of Rauwolfia serpentina with high alkaloid yield named “CIM -Sheel. Journal of Medicinal and Aromatic Plant Sciences 27, 532-534 Hamill JD, Rhodes MJC (1988) A spontaneous light independent and prolific plant regeneration response from hairy roots of Nicotiana besperis transformed by Agrobacterium rhizogenes. Journal of Plant Physiology 133, 506509 Hilton MG, Rhodes MJC (1993) Factors affecting the growth and hyosyamine production during batch culture of transformed roots of Datura stramonium. Planta Medica 59, 340-344 Kamada H, Okamura N, Satake M, Harada H, Shimomura K (1986) Alkaloid production by hairy root cultures in Atropa belladonna. Plant Cell Reports 5, 239-242 Khanuja SPS, Shasany AK, Darokar MP, Kumar S (1999) Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Molecular Biology Reporter 17, 74-80 Kirillova NV, Smirnova MG, Komov VP (2001) Sequential isolation of superoxide dismutase and ajmaline from tissue cultures of Rauwolfia serpentina Benth. Applied Biochemistry and Microbiology 37, 160-163 Klyushnichenko VE, Yakimov SA, Tuzova TP, Syagailo IN, Kuzovkina AN, Wulfson AN, Miroshnikov AI (1995) Determination of indole alkaloids from Rauwolfia serpentina and Rauwolfia vomitoria by high performance liquid chromatography and high performance thin layer chromatography. Journal of Chromatography A 704, 357-362 Kunakh VA, Alkhimova EG (1989) Rauwolfia serpentina: In vitro culture and the production of ajmaline. In: Bajaj YPS (Ed) Biotechnology in Agriculture and Forestry (Vol. 7) Medicinal and Aromatic Plants II, Springer-Verlag, Berlin, pp 398-416 Linsmaier EM, Skoog F (1965) Organic growth factors requirements of tobacco tissue cultures. Physiologia Plantarum 18, 100-127 Machado LOR, Andrade GM, Cid LPB, Penchel RM, Brasileiro ACM (1997) Agrobacterium strain specificity and shooty tumour formation in eucalyptus (Eucalyptus grandis x E. urophylla). Plant Cell Reports 16, 299-303 Madhusudan KP, Banerjee S, Khanuja SPS, Chattopadhyay SK (2008) Analysis of hairy root cultures of Rauwolfia serpentina using direct analysis in real time mass spectrometric techniques. Biomedical Chromatography 22, 596-600 Molokhova EI, Astakhova TV, Shimolina LL, Minina SA (1988) Chromatophotocolorimetric determination of indole alkaloids in a culture of Rauwolfia serpentina tissue. Chemistry of Natural Compounds 23, 773-774 Muller JM, Schllitller E, Bein HJ (1952) Reserpine the sedative principle of


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AFLP Markers for Identification of Aconitum Species Amita Misra1 • Ashutosh K. Shukla1* • Ajit K. Shasany1 • V. Sundaresan2 • Shital P. Jain3 • Subhash C. Singh3 • Guru D. Bagchi3 • Suman P. S. Khanuja1 1Genetic Resources and Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR), P.O. CIMAP, Lucknow 226015, Uttar Pradesh, India 2CIMAP Resource Centre (CSIR), P.O. Dairy Farm, Nagla, Pantnagar 263149, Uttarakhand, India 3 Botany and Pharmacognosy Division, Central Institute of Medicinal and Aromatic Plants (CSIR), P.O. CIMAP, Lucknow 226015, Uttar Pradesh, India Corresponding author: * [email protected]

ABSTRACT The genus Aconitum is highly complex and its taxonomy has been traditionally difficult due to the high level of variation among the various species. The Aconitum species are known for their highly toxic diterpenoid alkaloids but have been described in traditional medicine systems as high-value medicine after proper and prescribed detoxification. In India, A. heterophyllum, A. balfourii and A. violaceum are found mainly in the North-Western Himalayas whereas A. ferox is found in the North-Eastern Himalayan region. Among these species, A. heterophyllum is the most significant in terms of therapeutic importance and herbal drug market value. It has become critically endangered due to high demand of the herb and indiscriminate overexploitation. There is an existing demand in the bulk herbal drug industry to have an authentic identification system for the Aconitum species in order to enable their commercial use as genuine phytoceuticals. In the present study we have used Amplified Fragment Length Polymorphism (AFLP) for developing DNA fingerprints for 4 Aconitum species. A total of 10 accessions (4 of A. heterophyllum, 3 of A. violaceum, 2 of A. balfourii and 1 of A. ferox) from the 4 species were used in the study, which employed 64 AFLP selective primer pairs. Only 26 selective primer pairs were found to respond with all the accessions and generated a total of 4112 fragments. A number of species-specific markers were identified for all the 4 Aconitum species (16 for A. heterophyllum, 125 for A. violaceum, 79 for A. balfourii, and 226 for A. ferox). These AFLP fingerprints of the Aconitum species could be used in future for authentication of the drug and checking the adulteration-related problems faced by the commercial users of the herb.

_____________________________________________________________________________________________________________ Keywords: adulteration, DNA fingerprinting, crude drug, rare plant

INTRODUCTION Aconitum L. (belonging to the buttercup family Ranunculaceae), which is also known as aconite or monkshood, is a diverse genus with nearly 300 species worldwide, primarily in the temperate regions of the northern hemisphere (Zhang et al. 2005). The genus is represented by around 26 species in India, mainly distributed in the sub-alpine and alpine zones of the Himalayas. Interestingly, the Aconitum species of North-Western Himalayas are not found in North-Eastern Himalaya and vice versa (Chaudhary and Rao 1998). A. heterophyllum, A. balfourii and A. violaceum are found mainly in the North-Western Himalayas whereas A. ferox is found in the North-Eastern Himalayan region. Among these species, A. heterophyllum is the most significant in terms of therapeutic importance and herbal drug market value. Its importance has further grown due to its critically endangered status and lowest density (1 individual/m2) among all the threatened plants in the Himalayan region (Singh et al. 2008). Illegal and over-exploitation of Aconitum species pose a threat to their existence (Nautiyal et al. 2002) and the problem has been further complicated by destructive harvesting of root/rhizome of the plants (Pradhan and Badola 2008). Besides, regeneration of A. heterophyllum under natural conditions is low due to poor seed germination and low seedling survival and being endemic to the North-Western Himalayas, the species grows only in localized, restricted ecological niches (2500-5000 m above sea level) that have only a few, thin-scattered populations (Beigh et al. 2006). The stringent and critical ecological requirements for A. heterophyllum have ensured that it neither invades newer areas nor survives at lower altitudes with comparatively higher temperatures. However, ex situ conservation of A. heterophyllum has been attempted and it has

been found that there is some possibility of successful adaptation of the plant in conditions other than its natural habitat (Pandey et al. 2005). Aconitum species are known for their highly toxic diterpenoid alkaloids (Chan 2009), which, have been used as a source of arrow poisons (Fico et al. 2003). The pharmacological effects of preparations of Aconitum roots are attributed to these diterpenoid alkaloids. The main alkaloid of these plants is aconitine, which is known to suppress the inactivation of voltage-dependent Na+ channels by binding to neurotoxin binding site 2 of the alpha-subunit of the channel protein (Ameri 1998). For therapeutic use, Aconitum has to be processed and combined with specifically matching herbs to reduce its toxicity (Wang et al. 2009). Quantitative structure–activity relationship (QSAR) analyses have been performed to study the mechanism of action of Aconitum alkaloids and to provide a rational for their chemical manipulation to reduce their toxicity (Bello-Ramirez and NavaOcampo 2004). A. heterophyllum finds a mention in Ayurveda for curing stomach ache and fever. It is one of the main ingredients of Ayurvedic medicines, “Ativishadi churna”, “Chandraprabha vati” and “Amritarishta” whereas in the Unani system of medicine it is an important ingredient of “Sufuf habib”, which is used for curing piles and “Majun jograj guggal” that is used against arthiritis (Uniyal et al. 2006). A. ferox has traditional use for curing fever, skin diseases, cough and gout (Pradhan and Badola 2008). In recent years, Aconitum has been compared with other similar genera like Delphinium that produce similar type of alkaloids (Lin et al. 2010). Earlier, Aconitum was considered to be an antidote of malaria and a substitute of quinine (Chakrabarti 2010). A high demand for the drug and its endangered status has raised other concerns like adulteration of the authentic

Received: 28 July, 2009. Accepted: 21 April, 2010.




Table 1 Aconitum germplasm collection details. Name of species and accession number A. heterophyllum 1 A. heterophyllum 2 A. heterophyllum 3 A. heterophyllum 4 A. ferox A. balfourii 1 A. balfourii 2 A. violaceum 1 A. violaceum 2 A. violaceum 3

Place of collection Uttarkashi, Uttarakhand Uttarkashi, Uttarakhand Chamba, Himachal Pradesh Chamba, Himachal Pradesh Darjeeling, West Bengal Chamba, Tehri Garhwal, Uttarakhand Uttarkashi, Uttarakhand Chamba, Himachal Pradesh Rohtang Pass, Lahaul-Spiti, Himachal Pradesh Chamba, Himachal Pradesh

drug with substitutes (that are less effective and often harmful) that could not be identified when the herb is present in the crude drug form. It is therefore an absolute necessity for the herbal drug industry to have stable molecular markers (like DNA markers) for various Aconitum species so as to differentiate and authenticate the herbal material when it is present in the form of a crude drug. In the past many molecular marker-based studies have been carried out to analyze Aconitum species. Isozyme and random amplification of polymorphic DNA (RAPD) analyses have been quite popular for such studies (Cole and Kuchenreuther 2001). Polymorphic microsatellites (Le Cadre et al. 2005) and RAPD (Fico et al. 2003) have been used to analyse some European species of Aconitum and nuclear intergenic transcribed spacer (ITS) sequences have been used to study the phylogeny of Aconitum (Kita and Ito 2000; Luo et al. 2005). A Chinese group carried out ISSR-based genetic diversity analysis in Aconitum carmichaeli (Luo et al. 2006). Since, DNA marker-based studies have not been carried out on the species of Aconitum found in India, the present study was undertaken to generate AFLP-based DNA markers for 4 such species (A. heterophyllum, A. balfourii, A. violaceum and A. ferox) that are most commonly used in the herbal trade.

Voucher specimen number 7781 7780 9114 8503 9125 7778 7774 8510 8507 9113

DNA was isolated from the plant leaf samples using the protocol described by Khanuja et al. (1999) and its quality and quantity were analysed using agarose gel electrophoresis and ND-1000 spectrophotometer (NanoDropTechnologies, USA).

1 l 0.5 M NaCl, 0.5 l 1 mg/ml BSA, 1 l MseI adapter (Applied Biosystems, USA), 1 l EcoRI adapters (Applied Biosystems, USA) and 1 l enzyme master mix, as described above. The reaction was then incubated overnight at room temperature and subsequently diluted 20-fold with T10E0.1 buffer. The ligated adaptors served as primer binding sites for low-level selection in the preselective amplification of restriction fragments. The MseI complementary primer had a 3-C and the EcoRI complementary primer a 3-A. Only the genomic fragments having an adapter on each end amplified exponentially during the PCR. The preselective amplification mix was prepared by adding 4 l of 20-fold diluted DNA from the restriction ligation reaction, 0.5 l AFLP preselective primer (EcoRI, Applied Biosystems), 0.5 l AFLP preselective primer (MseI, Applied Biosystems) and 15 l AFLP core mix. The preselective amplification was carried out in a thermal cycler programmed as: 72°C for 2 min; 20 cycles of 94°C for 20 sec, 56°C for 30 sec and 72°C for 2 min; 60°C for 30 min and 4°C to infinity. The preamplified DNA was diluted 20-fold with T10E0.1 buffer and selective amplifications were carried out using different MseI and EcoRI primer combinations (Applied Biosystems). Primers chosen for the amplification were from 16 available AFLP selective primers (8 fluorescently tagged EcoRI and 8 untagged MseI primers). The EcoRI primers contained 3 selective nucleotides with the sequence 5 [Dye-Primer-Axx]-3, while the MseI primers had the 3 selective nucleotides starting with C i.e. 5 [Primer-Cxx]3. Selective amplification of each sample was done with all 64 (8x8)-primer combinations (MseI/EcoRI) using multiplex-PCR reactions. For selective amplification the reaction were set up as follows: 3 l of 20-fold diluted preselective amplification product, 15 l AFLP core mix, 1 l MseI primer 5-[Primer-Cxx]-3, 1.5 l EcoRI primers 5-[Dye-Primer-Axx]-3 {0.5 l of 3 EcoRI primers each were pooled here}. Selective amplification was carried out in a thermal cycler programmed as 94°C for 2 min; 10 cycles of 94°C for 20 sec, 66°C (-1°C/ cycle) for 30 sec, 72°C for 2 min; 20 cycles of 94°C for 20 sec, 56°C for 30 sec, 72°C for 2 min; 60°C for 30 min; and 4°C to infinity. The samples were loaded onto a 5% (29:1) polyacrylamide gel on an ABI Prism 377 DNA Sequencer (Applied Biosystems, USA). For gel electrophoresis, 3 l of the selective amplification reaction product was mixed with 4 l of loading buffer {ROX500 size standard (10%), blue dextran (10%), deionised formamide (80%)}, and 1.5 l of this mix was finally loaded on the gel. The AFLP amplification modules and the guidelines supplied by Applied Biosystems, USA were used for setting up the reactions as described above.

AFLP

Data analysis

For AFLP analysis, DNA was restricted using two restriction endonucleases EcoRI and Tru9I (an isoschizomer of MseI) and double stranded adapters were ligated to the ends of DNA fragments, generating template for subsequent PCR amplification (preselective followed by selective). Restriction and ligation reactions were carried out simultaneously in a single reaction (Vos et al. 1995). To carry out the reaction, an enzyme master mix for 10 reactions was prepared containing 1 l 10X T4 DNA ligase buffer, 1 l 0.5 M NaCl, 0.5 l 1 mg/ml BSA, 1 l Tru9I (10 U/l), 4.25 l EcoRI (12 U/l), 0.5 l T4 DNA ligase (20 U/l, high concentration) and 1.75 l water. The restriction ligation reaction consisted of 300 ng of DNA (5.5 l), 1 l 10X T4 DNA ligase buffer,

Fragment analysis was carried out for bands in the range 35-400 bp. For diversity analysis, bands were scored as present (1) or absent (0) to form a raw data matrix. A square symmetric matrix of similarity was then obtained using Jaccard similarity coefficient (Jaccard 1908) by SPSS v 7.5 software. The average similarity matrix was used to generate a tree for cluster analyses by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method using NTSYSpc version 2.02j (Applied Biostatistics Inc.).

MATERIALS AND METHODS Plant material The plant material used in this study was collected from the Himalayan region falling in the Indian states of West Bengal, Uttarakhand and Himachal Pradesh and the herbarium was submitted to the National Gene Bank for Medicinal and Aromatic Plants at CIMAP, Lucknow (Table 1). Leaf samples from the selected plants were used for DNA isolation. The samples consisted of four accessions of A. heterophyllum, three accessions of A. violaceum, two accessions of A. balfourii and one accession of A. ferox.

DNA isolation

RESULTS AND DISCUSSION In the AFLP analysis, of the 64 primer pairs used, only 26


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AFLP markers for identification of Aconitum. Misra et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 2 Unique AFLP marker fragments for the 4 Aconitum species. Primer Unique bands of Unique bands of A. ferox combination A. heterophyllum (size in bp) MseI/EcoRI (size in bp) CAA/ACG 43 67, 124 CAA/AGC CAC/ACT 122, 205 349

Unique bands of A. balfourii (size in bp) -

CAT/ACG

256

77, 87

220

CAT/AGC CTG/AGC

163, 208 -

72, 209 102, 127, 144

CTC/ACA

195

70, 78

130, 203, 314

CTC/ACG CTG/ACT

379 190 -

49, 92, 142, 150, 182 35, 63, 101, 174, 175, 184, 197, 317 62, 290

60, 81, 111 48, 122, 222

CTG/AGG CAT/ACC

179, 363

42, 57, 60, 64, 101, 108, 116 82, 91, 107, 121, 133, 154, 160, 163, 168, 173, 177, 181, 189, 191, 234, 255, 257, 262, 267, 306, 390, 395 45, 48, 53, 58, 87, 98, 105, 110, 126, 131, 140, 151, 170, 185, 202, 205, 220, 227, 242, 248, 266, 285, 316, 325, 326, 329, 335, 345, 348, 359, 366, 381, 389 68, 77, 88, 89, 109, 171, 185, 198, 207, 244, 306, 342, 383 57, 66, 68, 82, 115, 135, 195, 198, 199, 205, 269, 278, 338, 350, 354, 392, 398 50, 65, 85, 89, 218, 237, 239, 250, 272, 285, 291, 308, 313, 349 46, 55, 135, 263

Unique bands of A. violaceum (size in bp) 52, 71, 102, 116, 286 41, 88 133, 149, 199, 220, 234, 243, 283, 318, 353, 392 57, 65, 86, 104, 117, 146, 161, 162, 192, 210, 235, 283, 295, 296, 332, 356 69, 70, 156, 169, 250 54, 97

CTA/AAC CTG/ACA

-

CAA/ACA

-

68, 106, 155 67, 69, 71, 74, 90, 91, 101, 125, 134, 140, 150, 153, 176, 186, 196, 199, 207, 210, 213, 223, 225, 230, 233, 240, 261, 262, 265, 267, 283, 285, 288, 304, 328, 329, 344, 349, 359, 374, 384, 398 83, 353

CAA/AGG CAA/ACC CAC/ACA

-

189 276, 289 243, 251, 266, 270, 285, 286, 325, 327

45, 46, 47, 58, 60, 68, 74, 128, 129 167 61, 71, 107, 210, 211 49, 217, 278

CAG/AGC CAT/ACT CAT/AAC CTA/ACA CTA/AGG

242, 356

63, 65, 73, 78, 94, 95, 105, 117, 128, 159, 181, 219, 269 81, 85 64, 67, 80, 89, 244 111, 124, 141, 351

76 72 71, 74, 98, 104 76, 134, 136

CTA/ACC

160

37, 66

64, 68, 152

CTA/ACG CTA/AGC

280

35, 38, 109, 193, 196 35, 85, 169, 313

CTC/AAG

94

Total

16

47, 64, 88, 97, 117, 154, 161, 166, 168, 174, 183, 196, 203, 206, 211, 228, 291, 292, 336, 367 226

44, 69, 79, 81, 131 120, 122, 128, 144, 162, 187, 314 72, 120, 124, 189

61, 78, 137 49, 110 48, 86, 102, 191, 209

79

40, 43, 94, 118, 172, 219, 233, 235, 283, 297, 315, 344 47, 58, 59, 98, 145, 148, 168, 221, 240, 324, 351 82, 329 54

101, 197, 215, 251 204, 230, 345 64, 89, 109, 201, 238, 240, 265, 275, 297 47, 80, 128 46 177, 209, 317 68, 96, 182, 197, 201, 240, 268, 286, 324 40, 88, 129, 206, 249, 305, 311, 369 55, 165, 229 47, 82 81, 199, 278, 338, 355 125

contributes to its safety and efficacy (Joshi et al. 2004). This identification requires the use of molecular markers that are unique to the relevant plant and are stable under different conditions (plant age, environment, etc.). Although, chromatographic fingerprinting combined with similarity and hierarchical clustering analysis has been recently applied to distinguish closely related Aconitum species (Zhao et al. 2009), DNA markers are best suited in terms of stability to serve this purpose. In an earlier study, for clarification of the circumscription and relationships among the six species within the Aconitum delavayi complex that is distributed mainly in the Hengduan Mountains of China, RAPD markers were employed to examine the differentiation of the populations representing the species (Zhang et al. 2005). The AFLP markers (unique bands) for Aconitum species generated in the present study (Table 2) would provide a useful reference tool to identify the herbal material when present in the form of crude drug and circumvent the problems associated with morphological, chemotypic and isozyme markers. The occurrence of these unique bands in the analysis of the DNA isolated from the crude drug preparation could be used as an assay for the presence of a specific species popu-

responded positively and generated discrete bands with all the plant samples. From a total of 4112 bands, 4 were monomorphic and 4108 were polymorphic. A polymorphism of 99.9% was detected among the species tested. A total of 446 bands were found to be unique for various Aconitum species. In this analysis, species-specific markers were identified for the 4 Aconitum species (16 for A. heterophyllum, 125 for A. violaceum, 79 for A. balfourii, and 226 for A. ferox) (Table 2). In the cluster diagram obtained after analysis (Fig. 1) accessions of each of the 4 Aconitum species grouped separately. The single accession of A. ferox was found to be closest to A. balfourii. The four A. heterophyllum accessions grouped together with a similarity of 28%. Similarly, the two A. balfourii accessions had 49% similarity and the three A. violaceum had 74% similarity. Possibly many ecotypes and/or chemotypes of even a single species like A. heterophyllum exist in nature. While using the plant commercially as a herbal drug it is very important to identify the correct chemotype having the maximal content of the therapeutically useful secondary metabolites. Besides, correct identification and quality assurance of the starting plant material is an essential prerequisite for ensuring reproducible quality of herbal medicine and also


17


Ah1 Ah2 Ah3 Ah4 Af Ab1 Ab2 Av1 Av2 Av3 0.00

0.25

0.50

0.75

1.00

Coefficient Fig. 1 Cluster diagram showing the relationship among various accessions of the 4 Aconitum species. A. heterophyllum (Ah), A. ferox (Af), A. balfourii (Ab) and A. violaceum (Av).

lation in it. Previously also AFLP and other DNA markers have been used to resolve complex polyherbal mixtures and identify specific species present therein. In a previous study, AFLP markers have been used to resolve the “Safed Musli” complex and detect the presence of adulterants in crude drug preparations of the herb that is commonly known to contain Chlorophytum species along with Asparagus adscendens (Misra et al. 2007). Species-specific sequence characterized amplified region (SCAR) markers have been used to tag Phyllanthus species that are used in herbal drug trade (Jain et al. 2008). AFLP has been particularly useful for discriminating closely related species and authentication of herbs as exemplified in an earlier study for Plectranthus genus (Passinho-Soares et al. 2006). AFLP has also been used for determining the levels of genetic diversity of other critically endangered herbs like Dendrobium officinale (Li et al. 2008) and Primulina tabacum (Ni et al. 2006). The present study also provides a well defined grouping pattern for all the 4 Aconitum species analysed. The significance of this study lies in the fact that it has provided an authentic tool to detect adulterants in the crude drug preparations of Aconitum and help the herbal drug industry in maintaining the quality standards.

Himalayas. Journal of Herbs, Spices and Medicinal Plants 11, 47-56 Bello-Ramirez AM, Nava-Ocampo AA (2004) The local anesthetic activity of Aconitum alkaloids can be explained by their structural properties: a QSAR analysis. Fundamental and Clinical Pharmacology 18, 157-161 Chakrabarti P (2010) Empire and alternatives: Swietenia febrifuga and the Cinchona substitutes. Medical History 54, 75-94 Chan TY (2009) Aconite poisoning. Clinical Toxicology 47, 279-285 Chaudhary LB, Rao RR (1998) Notes on the genus Aconitum L. (Ranunculaceae) in North-West Himalaya (India). Feddes Repertorium 109, 527-537 Cole CT, Kuchenreuther MA (2001) Molecular markers reveal little genetic differentiation among Aconitum noveboracense and A. columbianum (Ranunculaceae) populations. American Journal of Botany 88, 337-347 Fico G, Spada A, Braca A, Agradi E, Morelli I, Tome F (2003) RAPD analysis and flavonoid composition of Aconitum as an aid for taxonomic discrimination. Biochemical Systematics and Ecology 31, 293-301 Jaccard P (1908) Nouvelles recherches sur la distribution florale. Bulletin de la Societe Vaudoise des Sciences Naturelles 44, 223-270 Jain N, Shasany AK, Singh S, Khanuja SPS, Kumar S (2008) SCAR markers for correct identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in scientific investigations and dry leaf bulk herb trade. Planta Medica 74, 296-301 Joshi K, Chavan P, Warude D, Patwardhan B (2004) Molecular markers in herbal drug technology. Current Science 87, 159-165 Khanuja SPS, Shasany AK, Darokar MP, Kumar S (1999) Rapid isolation of PCR amplifiable DNA from the dry and fresh sample of plants producing large amounts of secondary metabolites and essential oils by modified CTAB procedure. Plant Molecular Biology Reporter 17, 74 Kita Y, Ito M (2000) Nuclear ribosomal ITS sequences and phylogeny in East Asian Aconitum subgenus Aconitum (Ranunculaceae), with special reference to extensive polymorphism in individual plants. Plant Systematics and Evolution 225, 1-13 Le Cadre S, Boisselier-Dubayle M-C, Lambourdière J, Machon N, Moret J, Samadi S (2005) Polymorphic microsatellites for the study of Aconitum napellus L. (Ranunculaceae), a rare species in France. Molecular Ecology Notes 5, 358-360 Li X, Ding X, Chu B, Zhou Q, Ding G, Gu S (2008) Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP. Genetica 133, 159166 Lin L, Chen D-L, Liu X-Y, Chen Q-H, Wang F-P (2010) Trichocarpinine, a novel hetidine-hetisine type bisditerpenoid alkaloid from Aconitum tanguti-

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial help provided by ICMR and CSIR, India. The authors also acknowledge the help of Dr Anil K. Gupta, Curator, National Gene Bank for Medicinal and Aromatic Plants, CIMAP, Lucknow.

REFERENCES Ameri A (1998) The effects of Aconitum alkaloids on the central nervous system. Progress in Neurobiology 56, 211-235 Beigh SY, Nawchoo IA, Iqbal M (2006) Cultivation and conservation of Aconitum heterophyllum: a critically endangered medicinal herb of the northwest


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AFLP markers for identification of Aconitum. Misra et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Plectranthus genus. Planta Medica 72, 929-931 Pradhan BK, Badola HK (2008) Ethnomedicinal plant use by Lepcha tribe of Dzongu valley, bordering Khangchendzonga Biosphere Reserve, in North Sikkim, India. Journal of Ethnobiology and Ethnomedicine 4, 22 Singh KN, Gopichand, Kumar A, Lal B, Todaria NP (2008) Species diversity and population status of threatened plants in different landscape elements of the Rohtang Pass, Western Himalaya. Journal of Mountain Science 5, 73-83 Uniyal SK, Singh KN, Jamwal P, Lal B (2006) Traditional use of medicinal plants among the tribal communities of Chhota Bhangal, Western Himalaya. Journal of Ethnobiology and Ethnomedicine 2, 14 Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Paleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23, 4407-4414 Wang J, van der Heijden R, Spruit S, Hankermeier T, Chan K, van der Greef J, Xu G, Wang M (2009) Quality and safety of Chinese herbal medicines guided by a systems biology perspective. Journal of Ethnopharmacology 126, 31-41 Zhang F-M, Chen W-L, Yang Q-E, Ge S (2005) Genetic differentiation and relationship of populations in the Aconitum delavayi complex (Ranunculaceae) and their taxonomic implications. Plant Systematics and Evolution 254, 39-48 Zhao Y-Y, Zhang Y, Lin R-C, Sun W-J (2009) An expeditious HPLC method to distinguish Aconitum kusnezoffii from related species. Fitoterapia 80, 333338

cum var. trichocarpum. Helvetica Chimica Acta 93, 118-122 Luo Q, Ma D-W, Wang Y-H (2006) ISSR identification of genetic diversity in Aconitum carmichaeli. Chinese Traditional and Herbal Drugs 37, 1554-1557 Luo Y, Zhang F-M, Yang Q-E (2005) Phylogeny of Aconitum subgenus Aconitum (Ranunculaceae) inferred from ITS sequences. Plant Systematics and Evolution 252, 11-25 Misra A, Shasany AK, Shukla AK, Sundaresan V, Jain SP, Bagchi GD, Singh J, Khanuja SPS (2007) AFLP-based detection of adulterants in crude drug preparations of the ‘Safed Musli’ complex. Natural Product Communications 2, 93-97 Nautiyal BP, Prakash V, Bahuguna R, Maithani U, Bisht H, Nautiyal MC (2002) Population study for monitoring the status of rarity of three Aconite species in Garhwal Himalaya. Tropical Ecology 43, 297-303 Ni X, Huang Y, Wu L, Zhou R, Deng S, Wu D, Wang B, Su G, Tang T, Shi S (2006) Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP). Genetica 127, 177-183 Pandey S, Kushwaha R, Prakash O, Bhattacharya A, Ahuja PS (2005) Ex situ conservation of Aconitum heterophyllum Wall. – an endangered medicinal plant of the Himalaya through mass propagation and its effect on growth and alkaloid content. Plant Genetic Resources 3, 127-135 Passinho-Soares H, Felix D, Kaplan MA, Margis-Pinheiro M, Margis R (2006) Authentication of medicinal plant botanical identity by amplified fragmented length polymorphism dominant DNA marker: Inferences from the


19


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Antioxidant and Antibacterial Activities of Extracts from Wild and in Vitro-Raised Cultures of Prunella vulgaris L. Rafia Rasool1* • Bashir Ahmad Ganai1 • Azra Nahaid Kamili2 • Seema Akbar3 • Akbar Masood1 1 Department of Biochemistry, University of Kashmir, Srinagar-190006, Jammu and Kashmir, India 2 Plant Tissue Culture Laboratory, Centre of Research for Development, University of Kashmir, Srinagar, 190006, Jammu and Kashmir, India 3 Laboratory of the Central Council for Research in Unani Medicine, University of Kashmir, Srinagar, 190006, Jammu and Kashmir, India Corresponding author: * [email protected]

ABSTRACT MeOH, EtOH, CHCl3 and aqueous extracts from the whole plant of wild Prunella vulgaris, a Kashmir Himalayan perennial medicinal herb, as well as from in vitro-regenerated plants were evaluated and compared for their antioxidant and antimicrobial properties. Antioxidant activity was screened by using various in vitro models: scavenging of the free radicals using DPPH, riboflavin photo oxidation, DNA damage, inhibition of lipid oxidation via PMS, FTC and TBA assay. The MeOH and CHCl3 extract from wild and in vitro-regenerated plants possessed an almost equal radical scavenging effect. In vitro and wild grown plant extracts in different solvent systems were also screened for antimicrobial activity against medically important bacterial strains by the agar well diffusion method. The MeOH extract of both (wild and in vitro) plants extracts were almost equally effective against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Kleibsella pneumonae. Both in vitro and wild dried plant extracts showed an almost similar concentrationdependent antioxidant and antimicrobial inhibition. Therefore, the commercial manufacture of active constituents from these improved elite lines would be useful and profitable. The present study provides first evidence that in vitro grown P. vulgaris has antioxidant and antibacterial activities, suggesting the potential of the tissue culture technique to substitute wild P. vulgaris in the pharmaceutical industry.

_____________________________________________________________________________________________________________ Keywords: antimicrobial, extracts, medicinal plant, radicals, scavenging Abbreviations: Aq, aqueous; BAP, 6-benzylamino purine; CHCl3, chloroform; DNA, deoxy ribose nucleic acid; DPPH, di-phenylpicryl hydrazyl; EtOH, ethanol; FTC, ferric thiocyanate assay; MeOH, methanol; MS, Murashige and Skoog medium; NAA, naphthalene acetic acid; PMS, post mitochondrial supernatant; RPO, riboflavin photo oxidation; TBA, thiobarbituric acid; TRIS, trishydroxylmethyl amino methane

INTRODUCTION Prunella vulgaris (Lamiaceae), a Kashmir Himalayan perennial medicinal herb, also known as self-heal, popular in Europe and China, is inching towards extinction due to tremendous medicinal use. In China it has been used as an astringent and an anti-pyretic agent (Pinkas et al. 1994). It is used in the treatment of fever, sore throat and ulcers (Markova 1997). In India and China, P. vulgaris has been used against pulmonary disease, jaundice and liver inflammations. Moreover, it has been used as a laxative, anticough, antiparasitic, antirheumatic, against vertigo and hemorrhoid as well as for eye and ear diseases (Ahmed et al. 2008). The whole herb is used for medicinal purpose (Phytomania French 2000). The organic fraction of P. vulgaris was found to exhibit: DPPH scavenging activity, inhibition against in vitro human LDL copper mediated oxidation (Psotova et al. 2003). The aqueous extract of P. vulgaris contains an antiHIV-1 active compound named Prunellin which is chemically a polysaccharide (Tabba et al. 1989). Remarkable antiHIV-1 activity was also confirmed by Yamasaki et al. (1993). The antiviral action of P. vulgaris was also reported against the herpes simplex virus type 1 and type 2 (Zheng 1990). The aqueous extract of this herb inhibits anaphylactic shock and immediate-type allergic reactions (Shin et al. 2001). It protects rat RBC against haemolysis and kidney and brain homogenates against lipid peroxidation (Liu and Ng 2000). An immune modulator effect of P. vulgaris was carried on monocytes (Xuya et al. 2005). It contains a high content of rosmarinic acid which makes plant more usable as far as its therapeutic applications are concerned

(Markova et al. 1997). The aqueous extract of this herb is recently used in clinical treatment of herpetic keratitis (Xu et al. 1999). The wild sources of P. vulgaris will decrease dramatically due to the exhaustive collection for use in pharmaceutical preparations. To conserve the natural sources of P. vulgaris, tissue culture is being developed, which might be used as a potential substitute for wild P. vulgaris in the pharmaceutical industry. In the present study, therefore the antioxidant and antimicrobial activities of the product of tissue culture of P. vulgaris were investigated. MATERIALS AND METHODS Antibiotics (Himedia, Mumbai, India), ascorbic acid (SRL, Mumbai, India), calf thymus DNA (SRL), disodium hydrogen phosphate (Loba Chemie, Mumbai, India), TRIS-buffer (SISCO, Mumbai, India), DPPH (HIMEDIA), ferric nitrate (CDH, New Delhi, India), thiobarbituric acid (CDH), ammonium thiocyanate (Qualigens, Mumbai, India), NBT (BDH, Poole, UK) and EDTA (SISCO), sodium dihydrogen monophosphate (Loba Chemie), riboflavin (SISCO), trichloroacetic acid (Suvidnath Lab, Baroda, India), dimethyl sulfoxide (SISCO), linoleic acid (SRL), nutrient agar (CDH) and Mueller-Hinton agar (Micromaster, Mahrashtra, India). All other chemicals used in this study were either of analytical grade or of the highest purity grade available commercially.

Wild plant material The wild plants of P. vulgaris used in this study were identified in the herbarium of department of Botany, University of Kashmir and

Received: 23 May, 2009. Accepted: 19 April, 2010.




mixture was made by adding 500 μl of DNA (1 mg/1 ml), 100 μl of different extracts, 100 μl of ascorbic acid (500 mM), 100 μl ferric nitrate (20 mM), 30 μl of H2O2 and final volume was made to 1 ml by Tris-HCL buffer (0.001 M, pH 7.5). The mixture was incubated at 37°C for 20 hrs. The reaction was terminated by adding 1 ml TCA (25%) and in case of any precipitation, tubes were centrifuged at 3000 × g. To the supernatant 1 ml of TBA (1.68%) was added. Tubes were kept in boiling water bath for 10 min and then cooled in an ice bath followed by centrifugation at 10000 rpm. TBARS (thiobarbituric acid reactive species) formation was estimated at 535 nm by spectrophotometer. The percentage of hydroxyl radical scavenging was estimated using the following equation:

collected from Naranag area of district Ganderbal, Kashmir. The plant material (prior to flowering stage) was shade dried and ground. The ground material (20 g) was extracted using different solvent systems (MeOH, EtOH, CHCl3 and aqueous (Aq.)) using a Soxhlet extractor. The extract was collected and the solvent was evaporated. The filtrate was concentrated on a hot water bath at 35°C, then dried and weighed.

Tissue culture material Explants (shoot tips, nodal buds) obtained from wild plants of P. vulgaris were cultured on both full and half-strength MS medium (Murashige and Skoog 1962) on different phytohormonal regimes i.e., BAP (5 to 20 μM) and NAA (2.5 to 15 μM). Cultures were kept for incubation under cool fluorescent tubes in a 16-hr photoperiod with light intensity of 21-42 μmol/m2/s1 at a constant temperature of 25 ± 3°C. Relative humidity between 60 and 70% was maintained. The in vitro raised plant material was shade dried and ground. The ground material was extracted using different solvent systems (MeOH, EtOH, CHCl3 and Aq.). The extract was collected and the solvent was evaporated. The filtrate was concentrated on a hot water bath at 35°C and dried. Then the dried extract was weighed and stored at 4°C in airtight bottles for further studies. Both types of extracts (four in vitro and four wild) were redissolved in 30% DMSO with a concentration of 50 mg of extract per 50 ml of 30% DMSO. Different dilutions were also prepared from it.

controlabsorbance - testsample absorbance ½ % inhibition = ® ¾ ×100 controlabsorbance ¯ ¿

4. Ferric thiocyanate (FTC) method The method was described previously by Kikuzaki and Nakatani (1993). 2 ml of extract (1 mg/1 ml) was mixed with 2.88 ml of linoleic acid (2.51%, v/v in 4 ml of 99.5% (w/v) EtOH), 0.05 M phosphate buffer pH 7.0 (8 ml), and distilled water (3.9 ml) and incubated at 40°C for 96 hrs. To 100, 200 and 300 μl of this solution, 9.7, 9.6, 9.5 ml of 75% (v/v) EtOH was added, respectively followed by 0.1 ml of 30% (w/v) ammonium thiocyanate. Precisely after 3 min, 0.1 ml of 20 mM ferrous chloride in 3.5% (v/v) hydrochloric acid was added to the reaction mixture, the absorbance at 500 nm of the resulting red solution was measured, and it was recorded again every 24 hrs until the day when the absorbance of the control reached the maximum value. Vitamin C was used as positive control. The percentage inhibition of linoleic acid peroxidation was calculated by using the following formula:

Anti-oxidant studies 1. General free radical scavenging - DPPH assay This is the primary method, in which the stable free radical i.e., DPPH (1, 1-diphenyl-2-picryl hydrazyl) which is purple in color is reduced to di-phenyl-picryl hydrazine (yellow color) based on the efficacy of the antioxidant. The method was done as described by (Kring and Berger 2001). 2 ml reaction mixture was prepared by adding 1 ml DPPH (500 μM) to different volumes (200, 300 and 400 μl) of crude extracts followed by TRIS buffer (100 mM, pH 7.4). The mixture was incubated at room temperature for 30 min. Absorbance of yellow colored complex was read at (517 nm) spectrophotometrically. Ascorbic acid was taken as positive control and reaction mixture without extract as negative control. The percentage inhibition was calculated from the following equation:


5. Thiobarbituric acid assay Thiobarbituric acid was added to the reaction mixture which interacts with malionaldehyde (MDA) (end product of LPO) and TBARS produced was measured spectrophotometrically according to Kishida et al. (1993). To 2 ml of the reaction mixture of ferric thiocyanate assay, 2 ml of trichloroacetic acid (20%) and 2 ml thiobarbituric acid (0.67%) was added and kept in boiling water for 10 min. It was cooled under tap water, centrifuged at 3000 rpm for 20 min and the supernatant was read at 500 nm. Reaction mixture without extract was taken as negative control and ascorbic acid as positive control. The percentage inhibition was calculated by using the following formula:

control absorbance - testsample absorbance ½ % inhibition = ® ¾ ×100 controlabsorbance ¯ ¿

2. Superoxide anion scavenging – Riboflavin photo-oxidation method


In this method, the photo-oxidation of riboflavin leads to the generation of riboflavin radical which then auto oxidizes and generates superoxide radical. NBT i.e. nitro blue tetrazolium is a dye which is reduced by superoxide radical to diformazan and is detected by change in color of NBT in presence of extract. The method was taken from (Tevfik and Kadir 2008). 1.7 ml reaction mixture was made by adding 300 μl EDTA (0.1 M), 500 μl NBT (1.5 mM), phosphate buffer (0.067 M, pH 8) and 200 μl of wild and in vitro plant extract. The tubes were incubated at 37°C for 5-8 min. Finally 200 μl of riboflavin (0.12 mM) was added and then the tubes were kept in sunlight for 10-12 min until color change was observed (purple). The absorbance was then read at 560 nm. The percentage inhibition of superoxide anion generation was calculated using the following equation:

6. PMS preparation and lipid peroxidation assay a) Liver from the freshly sacrificed sheep was perfused in icecold 0.9% (w/v) NaCl followed by removal of extraneous materials. After this it was weighed and minced, the pieces of liver were homogenized with 4 volumes of ice-cold 0.1 M potassium phosphate buffer (pH 7.4) containing 1.15% (w/v) KCl. The homogenate was centrifuged at 6000 rpm for 10 min. The supernatant was collected and further centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant obtained was PMS (post mitochondrial supernatant). b) Lipid peroxidation was measured as described by Halliwell (1990). Peroxidation was induced by 5 mM FeSO4 and 500 mM ascorbate. In this assay to 1 ml of supernatant obtained above (PMS), 0.2 ml of ferric nitrate, 0.2 ml of ascorbic acid was added to 100, 150 and 250 μl of plant extracts and total volume was made to 2 ml with phosphate buffer. Then the solutions were incubated at 37°C for 1 hr. The reaction was then stopped by adding 1 ml of TCA (20%) followed by addition of 1 ml of 1.67% TBA. The mixture was then heated at 100°C for 10 to 20 min. After the addition of TCA, precipitation of proteins was removed by centri-


3. Hydroxyl radical scavenging – deoxyribose assay The method used was that of Halliwell and Gutteridge (1981). The highly reactive radical i.e. hydroxyl radical was generated by using ferric nitrate (Fe3+), ascorbic acid, 30 mM H2O2. Reaction


21

Antioxidant and antibacterial activities of wild and in vitro Prunella vulgaris L. Rasool et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 1 Zones of inhibition (mm) of wild and in-vitro 10% (w/v) plant extracts of Prunella vulgaris L. Methanol extract Ethanol extract Chloroform extract Type of strain Wild In-vitro Cn* Wild In-vitro Cn* Wild In-vitro Cn* Echerchia coli 20 18 0 25 20 0 13 12 0

Aqueous extract Wild In-vitro Cn* 0 0 0

Proteus vulgaris

0

0

0

0

0

0

10

10

0

0

0

0

Staphylococcus aureus

30

26

0

23

20

0

30

27

0

0

0

0

Salmonella typhimurium Kliebsella Pneumonae

10 18

13 14

0 0

0 17

0 12

0 0

15 14

25 12

0 0

0 0

0 0

0 0

Ant** CD30=45 NA30=0 C30=10 C30=10 NA=0 Az=35 C=40 NA=18 CD=45 CD=15 CD=0 Az=14

Data represents mean of 3 replicates/culture; 70 μl used in one well *Control samples (MeOH, EtOH, CHCl3, and water). **Antibiotics (Ant**) used cefadroxyl 30 (CD 30); naldixic acid 30 (NA 30); chloramphenicol 30 (C 30); azithromycin 5 (Az 15)

Antioxidant activity

fugation. Then the absorbance of the MDA-TBA complex in the supernatant was detected at 535 nm. The percentage inhibition of lipid peroxidation was calculated by using the following formula:

Deoxy ribose assay: The scavenging of the hydroxyl radicals generated by Fenton’s reaction by P. vulgaris extract is shown in (Fig. 1). It shows that MeOH and CHCl3 extracts exhibit maximum activity. The activity of both in-vitro and wild grown plants are almost in same range. The amount of the extracts used per ml was 100, 50 and 25 μg.


Antimicrobial studies

Riboflavin photo-oxidation method: The scavenging of superoxide radicals by the different extracts are shown in Fig. 2. MeOH extract exhibits maximum activity in both the wild and in vitro grown plants. The amount of the extracts used per ml was 117.6, 58.8 and 29.41 μg. Reference antioxidant used were ascorbic acid and thiourea.

To assess the antimicrobial activity the concentration of extracts was 10% (w/v) prepared in the same solvent in which extractions were made. The studies were done according to method of (Lansing et al. 2006; Debnath 2008). Five pathogenic bacterial strains were used to find the antibacterial activity of P. vulgaris extracts. Certified strains of bacteria i.e. Escherichia coli, Staphylococcus aureus, Kleibsella pneumonae, Salmonella typhimurium and Proteus vulgaris were obtained from the Microbiology Lab, Sheri Kashmir Institute of Medical Sciences. Various media viz. nutrient agar (E. coli, S. aureus and S. typhimurium), Mueller-Hinton agar (K. Pneumonae and P. vulgaris) were used for culture maintenance. Stock cultures were maintained on respective agar slants. Subculturing was done once a month to maintain purity and viability. Experiments carried were done when the microbes were in the log phase. Overnight cultures were prepared by transferring a loop-full of stock cultures to tube having media and incubating at 37°C for 24 hrs. These cultures were then used as inocula for culturing pathogenic strains on Petri dishes for the antimicrobial activity using the agar diffusion method. The micro-organisms were used to inoculate different media agar plates; one strain per plate, wells were made on the plates with a sterile cork borer of 4 mm diameter for the different extracts and the plates were incubated at 37°C for 24 hrs. The same procedure was repeated with all extracts and strains as well with commercial antibiotics. Antimicrobial activity of both wild and in vitro grown plant extracts was determined by measuring the diameter of the zone of inhibition and the mean values (presented in Table 1) were compared with standard antibiotics like ampicillin, cefadroxyl, naldixic acid and chloramphenicol and azithromycin (Himedia). The effect of the extraction solvents alone (without extract) on the growth of microorganisms was also measured (Table 1).

General free radical scavenging - DPPH assay: The general free radical scavenging assay shown in Fig. 3 illustrates the anti-oxidant activity of EtOH, MeOH and Aq. extracts of both wild and in vitro plants. The amount of the extracts used per ml of reaction mixture was 100, 150 and 200 μg. Reference antioxidant used were ascorbic acid and thiourea. Post mitochondrial supernatant assay: Here PMS and positive control ascorbic acid were used as a model to study peroxidation and inhibition of peroxidation by plant extracts. The amount of extract used was 50, 75 and 125 μg. Results were simply expressed by following the formation of MDA. The effect of in-vitro and wild extract of P. vulgaris on Fe3+-Ascorbic acid/H2O2-mediated PMS lipid peroxidation is shown in Fig. 4. In both cases CHCl3 and MeOH extracts showed maximum activity; in vitro CHCl3 extract had more activity than the wild extract. Ferric thiocyanate assay: This method evaluates the effect of extracts and reference antioxidant on preventing peroxidation of linoleic acid (Fig. 5). MeOH followed by EtOH extract is having high antioxidant activity in both in-vitro grown and wild collected plants. The amount of extract used is 30 μg/ml of reaction mixture. Reference antioxidants used were ascorbic acid and thiourea.

RESULTS AND DISCUSSION Thiobarbituric acid assay: In TBA method, formation of MDA is the basis for evaluating the extent of lipid peroxidation. At low pH and high temperature (100°C), MDA binds TBA to form a red complex. The amount of extract used is 10, 20 and 30 μg. MeOH extract (wild and in-vitro) had showed highest antioxidant activity (Fig. 6). The FTC method was used to measure amount of peroxide at the beginning of lipid peroxidation and the TBA method measures free radicals present after peroxide oxidation. The antioxidant activity detected with TBA method was higher than that detected with the FTC method. This might suggest that the amount of peroxide in initial stage of lipid peroxidation was less than the amount of peroxide in

In vitro raised plantlets Out of number of trials the most successful concentration was BAP (15 μM) that yielded the highest number (30 ± 0.6) of shoots per shoot tip (Rasool et al. 2009). MS(x) + NAA (2.5μM) + BAP (15 μM) showed best proliferation potential in nodal bud culture (Rasool et al. 2008). By repeated sub culturing a better frequency multiplication rate was created for production of elite plants of P. vulgaris. The shoots obtained rooted well on half-strength MS basal medium with a mean of 8 roots per shoot. Plantlets transferred to open lab conditions showed 70% survival.


22


C1 = 100 μg

C2 = 50 μg

C3 = 25 μg

60%

A

Inhibition

50% 40% 30% 20% 10% 0%

Control (-)

MeOH

EtOH

CHCL3

Aq.

Control (-)

MeOH*

EtOH*

CHCL3*

Aq.*

60%

B

Inhibition

50% 40% 30% 20% 10% 0%

Concentration (μg/ml)

Fig. 1 Deoxyribose assay of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represents the mean ± standard deviation of three independent experiments (n = 3). C1=117.6 μg

C2=58.8 μg

C3=29.41 μg

80% 70%

A

Inhibition

60% 50% 40% 30% 20% 10% 0% Cn(-)

Cn(+)

MeOH

EtOH

Cn(-)

Cn(+)

MeOH*

EtOH*

CHCl3

Aq.

80% 70%

B

Inhibition

60% 50% 40% 30% 20% 10% 0% CHCl3*

Aq.*

Concentration (μg/ml)

Fig. 2 Riboflavin photo-oxidation method of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represents the mean ± standard deviation of three independent experiments (n = 3).

the second stage. Furthermore secondary product was much more stable for a period of time.

extract does not show any zone of inhibition against the five types of strains. CHCl3 extract is very effective against all types of strains, particularly S. aureus (Fig. 7). It seems to be a good solvent for secondary metabolites. Besides CHCl3, MeOH extract had also proven to be effective antimicrobial agent. Cefadroxyl 30 (CD 30) has proven ineffective against K. pneumonae and naldixic acid 30 (NA 30) is ineffective against P. vulgaris. Plants are a tremendous source for the discovery of new products of medicinal value for drug development. Today several distinct chemicals derived from plants are important drugs currently used in one or more countries in the world.

Antibacterial activity The results depicted in Table 1 show that secondary metabolites present in in-vitro grown plants are in same range and of same type as found in wild plant extracts. The antimicrobial properties of the in vitro regenerated plantlets establish a fact, that these can be a source of elite plantlets. In some cases the activity of the in vitro extract was even more potent and effective than wild grown plant extract. Aq.


23


C1=100 μg

C2=150 μg

C3=200 μg

70% 60%

A

Inhibition

50% 40% 30% 20% 10% 0% Cn(-)

Cn(+)

Cn(-)

Cn(+)

MeOH

EtOH

CHCl3

Aq.

70% 60%

B

Inhibition

50% 40% 30% 20% 10% 0% MeOH* EtOH* CHCL3* Aq.* Concentration (μg/ml) Fig. 3 DPPH assay of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represents the mean ± standard deviation of three independent experiments (n=3).

C1=50 μg 70%

Inhibition

60%

A

C2=75 μg

C3=125 μg

A

50% 40% 30% 20% 10% 0% Cn(-)

MeOH

Cn(-)

MeOH*

EtOH

CHCL3

Aq.

70%

Inhibition

60%

B

50% 40% 30% 20% 10% 0% EtOH*

CHCL3*

Aq.*

Concentration (μg/ml ) Fig. 4 PMS assay of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represents the mean ± standard deviation of three independent experiments (n = 3).

ferent strategies, using an in vitro system, have been extensively studied to improve the production of plant chemicals (Vanisree et al. 2004). The medicinal properties are attributed to the primary and secondary metabolites synthesized by the plants (Faizi et al. 2003). In our studies we have compared the secondary metabolite production of wild and in vitro grown plants of P. vulgaris by exploiting their two medicinal attributes, antioxidant nature and antibacterial activity. Results suggest that the plant grown using tissue

Many of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The evolving commercial importance of secondary metabolites in recent years has resulted in a great interest in secondary metabolism, particularly in the possibility of altering the production of bioactive plant metabolites by means of tissue culture technology. Plant cell culture technologies were introduced at the end of the 1960’s as a possible tool for both studying and producing plant secondary metabolites. Dif-


24


Inhibition

Inhibition

Cn (+) 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

1 = MeOH

2 = EtOH

3 = Aq.

4 = CHCL3

A

Ist day

2nd day

Ist day

2nd day

3rd day

4th day

B

3rd day

4th day

Days of incubation Fig. 5 Ferric thiocyanate assay of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represent the mean ± standard deviation of three independent experiments (n = 3).

Inhibition

Inhibition

C1=10 μg 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

C2=20 μg

C3=30μg

A

Cn(-)

Cn(+)

MeOH

Cn(-)

Cn(+)

MeOH*

EtOH

CHCL3

Aq.

B

EtOH*

CHCL3*

Aq.*

Concentration (μg/ml) Fig. 6 TBA assay of wild (A) and in-vitro (B) plant extracts of Prunella vulgaris L. Values represents the mean ± standard deviation of three independent experiments (n = 3).

culture technology do contain the secondary metabolites in almost same range as in wild. Percentage inhibition against free radicals and zone of inhibition values against different bacteria are in same range, also in some cases higher than wild plant confessing that the in vitro grown plants are the elite clones of the parental stock and can be substituted against the wild plant so that further exploitation of medicinal plants can be curbed. Such findings are also reported by some authors (Jia et al. 2005; Landa et al. 2006; Deb-

nath 2008) otherwise overall reports on such type of work is rare. Crude methanol extracts from callus cultures of Nigella species were investigated for their antimicrobial activity. Results showed that the extracts of all calli tested exhibited significant antimicrobial activity, especially against Bacillus cereus, S. aureus and Staphylococcus epidermidis (Landa et al. 2006). Our results also confirm the antibacterial activity of tissue culture grown plant especially in S. aureus. Similar procedure has been outlined for plant regeneration and anti-


25


A

for acetone extracts of shoots obtained from in vitro culture followed by the extracts of shoots of intact plants grown in the field (Grzegorczyk et al. 2007). In contrast, our results showed methanol and chloroform extracts to be good antioxidants except in the DPPH assay where results showed variations possibly due to different reaction mechanisms.

B

CONCLUSIONS

C

Reports suggest good similarities in medicinal properties between micropropagated plants and wild grown plants of P. vulgaris L. Therefore, commercial manufacture of active constituents from these improved elite lines would be useful and profitable without any loss of biodiversity.

D

REFERENCES

E

Ahmed JH, Ezer N (2008) Anatomical features of genus Prunella L. growing in Turkey. Turkish Journal of Pharmaceutical Sciences 5, 17-27 Debnath M (2008) Clonal propagation and antimicrobial activity of an endemic medicinal plant Stevia rebaudiana. Journal of Medicinal Plants Research 2, 45-51 Faizi S, Rasool N, Rashid M, Ali Khan RA, Ahmed S, Khan SA, Aqeel AA, Bibi N, Ahmed SA (2003) Evaluation of the antimicrobial property of Polyalthia longifolia var pendula: isolation of a lactone as the active antibacterial agent from the ethanol extract of the stem. Phytotherapy Research 17, 11771181 Grzegorczy I, Matkowski A, Wysokiska H (2007) Antioxidant activity of extracts from in vitro cultures of Salvia officinalis L. Food Chemistry 104, 536-541 Halliwell B, Gutteridge JMC (1981) Formation of a thiobarbituric acid reactive substance from deoxyribose in presence of iron salts. FEBS Letters 128, 347-352 Halliwell B (1990) How to characterize a biological antioxidant. Free Radical Research Communication 9, 1-32 Jia JM, Wu CF, Liu W, Yu H, Hao Y, Zheng JH, Ji YR (2005) Anti inflammatory and analgesic activities of the tissue culture of Saussurea involucrate. Biological and Pharmaceutical Bulletin 28, 1612-1614 Krings U, Berger R G (2001) Antioxidant activity of roasted foods. Food Chemistry 72, 223 Kishida E, Tokumaru S, Ishitani Y, Yamamoto M, Oribe M, Iguchi H, Kojo S (1993) Comparison of the formation of malondialdehyde and thiobarbituric acid reactive substances from auto-oxidized fatty acids based on oxygen consumption. Journal of Agricultural and Food Chemistry 41, 1598-1600 Kikuzaki H, Nakatani N (1993) Antioxidant effects of some ginger constituents. Journal of Food Science 58, 1407-1410 Landa P, Marsik P, Vanek T, Rada V, Kokoska L (2006) In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species. Biologia 61, 285-288 Lansing MP, John PH, Donald AK (2006) Microbiology (6th Edn), McGraw Hill, Boston, 783 pp Liu F, Ng TB (2000) Anti oxidative and free radical scavenging activities of selected medicinal herbs. Life Sciences 66, 725-735 Markova H, Sousek J, Ulrichova J (1997) Prunella vulgaris L. – a rediscovered medicinal plant. Ceska a Solvenska Farmacie 46, 58-63 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Plant Physiology 15, 473-497 Phytomania French (2000) Pharmacopoeia. Available online: www.phytomania.com Pinkas M, Trotin F, Peng M, Torck M (1994) Use, chemistry and pharmacology of the Chinese medicinal plants. Fitoterapia 55, 343-353 Psotova J, Kolar M, Sousek J, Svagera Z, Vicar J, Ulrichova J (2007) Biological activities of Prunella vulgaris extract. Phytotherapy Research 17, 10821087 Rasool R, Kamili AN, Ganai BA, Akbar S (2009) Effect of BAP and NAA on shoot regeneration in Prunella vulgaris. Journal of Natural Sciences and Mathematics 3, 20-26 Rasool R, Kamili AN, Ganai BA, Akbar S (2008) In vitro nodal bud culture of Prunella vulgaris. Journal of Himalayan Ecology and Sustainable Development 3, 116-123 Shahzad A, Faisal M, Anis M (2007) Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro regenerated plants and seedlings. Annals of Applied Biology 150, 341-349 Shin TY, Kim YK, Kim HM (2001) Inhibition of immediate type allergic reaction by Prunella vulgaris in a murine model. Immunopharmacology and Immunotoxicology 23, 423-435 Tabba HD, Chang RS, Smith KM (1989) Isolation purification and partial characterization of prunellin an anti–HIV compound from aqueous extract of Prunella vulgaris. Antiviral Research 11, 263-273 Tevfik O, Kadir K (2008) Determination of antioxidant activity of various extracts of Parmelia saxatalis. Biologia 63, 211-216

F

Fig. 7 Zones of inhibitions of in-vitro and wild plant extracts of Prunella vulgaris L. (A) Effect of CHCl3 extract against E. coli; (B) Effect of MeOH extract against E. coli; (C) Effect of CHCl3 extract against K. peumonae; (D) Effect of MeOH extract against K. peumonae; (E) Effect of CHCl3 extract against S. aureus; (F) Effect of MeOH extract against S. aureus.

microbial screening of a medicinal herb, Stevia rebaudiana Bertoni, through in vitro culture of nodal segments with axillary buds on MS medium. In vitro and wild grown leaf extracts in different solvent system showed that the chloroform and methanol extract exhibited a concentration dependent antibacterial and antifungal inhibition. Both in vitro and wild dried leaf extract showed similar antimicrobial activity, which are in concordance with our results. Therefore, commercial manufacture of active constituents from these improved elite lines would be useful and profitable (Debnath 2008). The tissue culture of Saussurea involucrata was studied to determine its anti inflammatory and analgesic activities in experimental animals and study provided evidence that tissue culture raised plant has anti inflammatory and analgesic activities, suggesting the potential of the tissue culture technique to substitute for wild S. involucrata in the pharmaceutical industry (Jia et al. 2005). Same observations are recorded in this study which indicates the antioxidant and antibacterial activities of in vitrocultured P. vulgaris. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile of in vitro grown and wild plants of Clitoria ternatea was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro regenerated and wild grown plants was almost the same (Shahzad et al. 2007). Methanolic and acetone extracts from Salvia officinalis, as well as from shoots and roots of in vitro regenerated plants were evaluated for their antioxidant properties. The methanolic hairy root and roots of regenerated plant extracts possessed the strongest effects on reducing molybdenum. DPPH radical scavenging and protective effect against linoleic acid oxidation was observed


26


latory effects of Prunella vulgaris on monocytes. International Journal of Molecular Medicine 16, 1109 Yamasaki K, Otake T, Mori H, Morimoto M, Ueba N, Kurokawa Y, Shiota K, Yuge T (1993) Screening test of crude drug extract on anti-HIV activity. Yakugaku Zasshi 113, 818-824 Zheng M (1990) Experimental study of 472 herbs with antiviral action against the herpes simplex virus. Zhong Xi Yi Jie He Za Zhi 10, 39-41

Vanisree M, Lee CY, Lo SF, Nalawade SM, Lin CY, Shengtsay H (2004) Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Botanical Bulletin of Academia Sinica 45, 1-22 Xu HX, Lee SH, Lee SF, White RL, Blay J (1999) Isolation and characterization of an anti-HSV polysaccharide from Prunella vulgaris. Antiviral Research 44, 43-54 Xuya F, Mabel MS, Wai HS, Yuen YZ, Raymond CC (2005) Immune modu-


27


®


Medicinal Plants in Farwest Nepal: Indigenous Uses and Pharmacological Validity Ripu M. Kunwar1,2* • Chundamani Burlakoti2 • Chhote L. Chowdhary2 • Rainer W. Bussmann3 1 Ethnobotanical Society of Nepal, GPO Box 5220, Kathmandu, Nepal 2 Center for Biological Conservation, GPO Box 19225, Kathmandu, Nepal 3 William L. Brown Center, Missouri Botanical Garden, St. Louis, USA Corresponding author: * [email protected]

ABSTRACT Medicinal plants have been used indigenously since ancient past as medicines for the treatment of various ailments. However, the knowledge of indigenous therapies have been distorting to these days due to changing perception, acculturation, commercialization and socio-economic transformations. The present study compares indigenous knowledge of therapies of 48 medicinal plants with the latest common pharmacological findings. Traditional indigenous plant knowledge and phytomedicine are consistently gaining acceptance in global society. The present study found that over two-thirds of traditionally used plants in the region show clear pharmacological efficacy. Total 23 species possessed strong resemblances and the species Euphorbia royleana, Ricinus communis, Plantago major, Chenopodium album, Cordyceps sinensis, etc. contributed the most. The complementarity of indigenous therapies and pharmacological uses is obvious and it is base of the modern therapeutic medicine. The increasing use of indigenous therapies demands more scientifically sound evidence, therefore further investigation and phytochemical screening of ethnopharmacologically used plants and assessment of the validity to the indigenous uses is worthwhile.

_____________________________________________________________________________________________________________ Keywords: Baitadi district, Chenopodium album, coumarin, pharmacology, traditional therapy

INTRODUCTION Archaeological discoveries of 60,000 year-old Neanderthal burial grounds in Shanidar, Iraq, pointed to the use of several plants like Marshmallow, Yarrow and Groundsel that are still used in contemporary folk medicine (Lietava 1992). Evidence for the medicinal use of Papaver somniferum, the opium poppy, dates back to 8,000 years (Stockwell 1989; Lewington 1990). Concomitantly, the earliest written record of plants used as medicine in the Himalayas is found in the Rigveda in about 6,500 years ago (Malla and Shakya 1984), in the Atharvaveda in about 4,000 year ago (Nambier 2002) and in the Ayurveda in about 2,500 year ago (Kunwar et al. 2006). Hippocrates (460–377 B.C.) described the usage of leaves and bark of willow tree to treat fever and pain (Julkunen-Tuto and Tahvanainen 1989). According to Schmid and Heide (1995), there is a report of preparation of salicylate pain remedies for indigenous uses from Birch bark in North America in 200 B.C. Therefore, until the 19th century,

plants were the main therapeutic agents used by humans, and even today their role in medicine is immense (Bhattarai et al. 2009; Uprety et al. 2010). The first medically useful alkaloid was morphine isolated from Opium poppy Papaver somniferum (Solanaceae) in 1805 (Fessenden and Fessenden 1982); the name morphine comes from the Greek Morpheus, god of dreams. A drug used in indigenous culture transformed into a medication and research tool since 1864 after the first systematic studies of Claude Bernard (Bernard 1966) on physiologicpharmacological effects. Therefore, the essence of phytomedicine recounts prehistoric and isolation of useful plant constituents and researches are imminent. Scientific study of traditional medicines and research of drug discovery through traditional medicines is designated as ethnopharmacology (Bussmann 2002) was first used in 1967 by Efron et al. (1970) in a book, Ethnopharmacological Search for Psyactive Drugs (Heinrich and Gibbons 2001). Tubocurarine was the first ethnophar-

Fig. 1 Some Himalayan medicinal plants. (Left) Rhus parviflora. Fruits are indigenously used for diarrhea and dysentery. (Center) Urtica dioica. Stem juice is valued for sprain and fractures. (Right) Euphorbia royleana. Plant is kept in roof of house for protecting from evil.

Received: 23 July, 2009. Accepted: 25 October, 2010.




MATERIALS AND METHODS

macological drug, derives from Menispermaceae (Chondrodendron spp.) and Loganiaceae (Strychnos spp.), researched and medicated extensively (Bisset 1991). There are many other examples (quinine from Cinchona succirubra, colchicine from Colchicum autumnale, etc.) of pharmaceutical relevant substances, which were developed based on observations of indigenous drugs during the last century (Heinrich 2001). Quinine, the cure for malaria, was originally the ritual medicine of Incas of Peru (Osujih 1993). The phytocompound used for medication and entered into the international market was ephedrine, an amphetamine like stimulant from Ephedra sinica (Patwardhan et al. 2005). Numerous other traditional therapy base phyto-drugs artimisinin from Artemisia annua as a potent antimalarial drug, alkaloids of Rauvolfia serpentina as hypertension, phyllanthin of Phyllanthus emblica as antiviral, etc. deserve special interest. Some other plants and their compounds worth from traditional therapy to modern medicine are Holarrhena for amoebiasis, Mucuna pruriens for Parkinson’s disease, Commiphora as hypolipidaemic, Asclepias as cardiotonic, psoralens for vitiligo, curcumines for inflammation, baccoside for mental retention, picrosides for hepatoprotective, indirubin for cancer, diosgenin for the synthesis of steroidal hormones, guggulsterons as hypolipidemic, piperidine as bioavailability enhancers, asarone as hallucinogenic, withanolides and many other steroidal lactones and their glycosides as immunomodulators, etc. (Jain 1994; Patwardhan 2000). Till 2002, 1141 different traditional plant drugs were registered for their therapeutic activities (Patwardhan et al. 2005) and it is estimated that about 25% of the prescription drugs contain active principles of higher plants (Farnsworth and Morris 1976; Tiwari and Joshi 1990; Cox 1994), and most are entrenched from traditional therapies. In some cases, about 60% of the antitumoral and antimicrobial medicines currently available in the markets are derived mainly from the higher plants (Cragg et al. 1997). Therefore, global demand of herbal medicine is accelerating and its worth was US $ 19.4 billion in 1999 (Laird and Pierce 2002). Herbal trade of over US $ 60 billion per year and its 7% annual increment was estimated (Nagpal and Karki 2004). Its market was valued for 2.3 and 2.1 billion in 1994 respectively in Asia and Japan (Grunwald 1995). The worth annual growth rate about 20% was reported in India (Srivastava 2000; Subrat 2002). Interest of phytomedicine is gradually renewed (Bhattarai et al. 2010) or increased and numerous medicinal plant based drugs have spread into the international market through exploration of ethnopharmacology and indigenous therapies (Bussmann 2002). The search for pharmacological principles from existing indigenous therapies is encouraging and complemented the achievements of modern medicine. With increasing use of traditional therapies of plant resource base (Acharya and Acharya 2010), a verification of efficacy by western scientific means would be interesting, because the traditional health system adopt customized and multi-pronged strategies in treatment involving drug, diet and therapy (Patwardhan et al. 2005). Moreover, the indigenous therapies have been criticized due to inadequate research, critical evaluation, in vivo studies and validations (Houghton 1995; Fong 2002). Despite growing interest in assessing phytochemical constituents of plants with pharmacological activities and modern medicine (Dalvi et al. 1994; Gupta 1994; Vaz et al. 1998; Dahanukar et al. 2000), to date only about 5% of the total plant species have been thoroughly investigated (Goswani et al. 2002; Patwardhan et al. 2005; Palombo 2006) to ascertain safety and efficacy of traditional remedies. Moreover, the current species extinction rate (the world is losing one major drug every two years) (Groombridge and Jenkins 2002) and distortion and percolation of indigenous knowledge, use and ethnopharmacology (Bussmann et al. 2007) aggravating the situation further. In this connection, present study aimed at surveying and assessing indigenous knowledge of uses and therapies of medicinal plants and their pharmacological validity.

Field study for primary data collection was carried out in Baitadi, Dadeldhura and Darchula districts of West Nepal in May-June, December 2006 and Jan-Feb 2007, March-April 2008. Study sites Anarkholi, Dasharathchand, Jhulaghat, Khodpe, Kulau, Pancheswor, Patan, Salena, and Sera from Baitadi; Brikham, Jakh, Jogbudha, Patram and Rupal from Dadeldhura and Dumling, Gokule, Joljibi, Khalanga, Lali, and Uku from Darchula district were visited. All three districts are delineated as western borders to the country and adjacent to India. Dadeldhura district ranges with 29°–29°30N latitude, 80°03–80°50E longitude and altitude 3902950 m; Baitadi district with 29°22–29°57N latitude, 80°05– 80°57E longitude and altitude 390-2950 m; and Darchula district lies within 29°26-30°15N latitude, 80°22-81°9E longitude and 357-7132 m altitude. Owing to varied topography, bioclimate and elevation, the districts harbor diversity of forest products (Devkota and Karmacharya 2003, Pant and Panta 2004), and the products have been collecting by local ethnic groups since time immemorial for both the subsistence and commercial purposes, however the subsistence use is profound particularly for home herbal healing (Burlakoti and Kunwar 2008; Kunwar et al. 2009). Primary data collection was facilitated by ten local assistants. Group discussions, informal meetings, questionnaire surveys and field observations were made for primary data collection. Group discussions, as informal interactions and meetings were held at the immediate spot and they were managed within the community forest user groups. Altogether 172 questionnaires were asked to the particular respondents representing ethnic groups: Badi, Bijale, Chanda, Chuhar, Dadal, Dhami, Hodke, Lawad, Lohar, Pali, Pariyar, Parki, Sitoli, Tamata, Uud, etc; age groups (25-74 year), sexes (both male and female), and occupations (collectors, cultivators, traders, herders, traditional healers). Information was validated by common responses (at least by three responses) and responses from less than three respondents were considered as insignificant. Species with common responses were preceded for crosschecking and key informant survey. Elders, traditional healers - Baidhyas, medicinal plant cultivators and collectors were individually asked for detail analysis. The species possessed highest common responses were considered for the present assessment. The assessment was made with comparing the present observations and latest and common phytochemical findings.

RESULTS Observations (*significant and # partial affinities) #Adiantum capillus-veneris L. Maidenhair fern (English), Gophale (Nepali), Hansapadi, Nilkanthasikha (Sanskrit), Adiantaceae. Indigenous uses: Root juice is applied for snake bite, migraine, and scorpion sting. Principal chemical compounds: Adiantone, carotenoid, filicene, flavonoides, kaemferol, leucopelarcogonidin, mollugogenol, quercetin, tannins (CSIR 1988). Pharmacological uses: Whole plant extract possess hypoglycaemic activity (Jain and Sharma 1967). It showed potent antimicrobial activity against Escherichia coli, Trichophyton rubrum and Aspergillus terreus (Singh et al. 2008). Plant extract is potential elicitor of phytoalexins in sorghum and soybean (Meinerz et al. 2008). *Rhus parviflora Roxb. (Fig. 1) Nepal Sumac (English), Bewoti (Local), Satibayer (Nepali), Tintideek (Sanskrit), Anacardiaceae. Indigenous uses: Fruit decoction is taken for diarrhoea and dysentery. Principal chemical compounds: Abinoside, biflavonoides, hetriocontane, kaemferol, lignoceric acid, myricetin, quercetin, rhamnoside, sitosterol (Husain et al. 1992). Pharmacological uses: Methanolic extracts of the ripen fruits possess antidiarrhoeal effect (Thangpu and Yadav 2004). Rhus species have reactive oxygen (RO) which can damage DNA resulting in mutagenesis, aging, carcinogenesis, and antimicrobial effect (Lin et al. 2008). Plant ex-


29

Medicinal plants in farwest Nepal. Kunwar et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

tract is also antibacterial (Mahato 2006) in effect.

Principal chemical compounds: Cicin, monogalactosyldiacyl glycerol, sterols, terpenes, etc. (Lee et al. 2002). Pharmacological uses: Methanolic extract of whole plant juice is antimicrobial (Lee et al. 2002; Barbour et al. 2004).

*Angelica archangelica L. Angelica (English), Gannano (Nepali), Apiaceae. Indigenous uses: Dried roots are anthelmintic and useful in gastric, and stomachache. Principal chemical compounds: Angelicin, coumarin, furocoumarin, isoimperatorin, pinene, prangolarin, umbelliferene (Anonymous 1948; Kaul 1997). Pharmacological uses: Ethanol extract of root of this plant shows anti-trypanosomal activity (Schinella et al. 2002).

#Inula racemosa Hook.f. Elecampane (English), Rithaula (Local), Puskarmul (Nepali), Puskaram (Sanskrit), Asteraceae. Indigenous uses: Root extract is useful in severe stomacheache, dysentery and blood pressure. Principal chemical compounds: Alantolactone, aplotaxene, curcumine, elemene, inunolide, ionone, tetraene (Husain et al. 1992). Pharmacological uses: Methanol extract of root exhibited antimycobacterial activity (Cantrell et al. 1999) and its alcoholic extract enhanced liver glycogen and lowered blood glucose level (Tripathi and Chaturvedi 1995). Lung fibrosis (Thresiamma et al. 1996), blood pressure control (Dikshit et al. 1995) and anti-inflammatory properties (Kohli et al. 2005) are due to curcumine of the plant.

#Pleumeria rubra L. Pagoda tree (English), Choya phool (Local), Galaincha phool (Nepali), Kshirchampaka, Swetachampa (Sanskrit), Apocynaceae. Indigenous uses: Flowers are useful in indigestion and cholera. Principal chemical compounds: Acetonine, amyrin, bornesitol, farnesol, fluroplumierin, kaemferol, lignan, lupeol, melilotic acid, oleanic acid, para-coumaric acid, plemeride, plumeric acid, plumerinine, quercetin, rubrinol, syringic acid, vanilic acid (Cambie and Ash 1994; Coppen and Cobb 1983). Pharmacological uses: Plant extract is antibiotic, antitumour, antiviral, analgesic, antispasmodic, etc. and fluroplumierin inhibits mycobacteria (Sundarrao 1993; Cambie and Ash 1994).

*Xanthium strumarium L. Sheep burr, Bur weed (English), Musekuro (Local), Bhede kuro (Nepali), Sankesvara, Arista (Sanskrit), Asteraceae. Indigenous uses: Seed powder is useful in earache, dysentery and skin diseases. Principal chemical compounds: Atractyloside, caffeyolquinic acid, carboxyatractyloside, caffeoylquinic acid, glycosides, hydroquinone, isoxanthanol, oxalic acid, strumaroside, thiazinedine, xanthanol, xanthin, xanthostrumarin, xanthanolide (Badam et al. 1988; Joshi 2004). Pharmacological uses: Plant extract is antitussive, antibacterial, antifungal, antimalarial, hypoglycemic, stomachic, cytotoxic (Kupiecki et al. 1974; Gautam et al. 2007). Fruits are anti-inflammatory in effect (Han et al. 2007).

Ageratum conyzoides L. Goat weed (English), Nilgandhe (Local), Kalo jhar (Nepali), Visamusti, Osari (Sanskrit), Asteraceae. Indigenous uses: Stem juice is useful in bleeding control. Principal chemical compounds: Ageratochromene derivatives, caffeic acid, chromenes, conyzorigun, coumarin, echinatine, eupalestin, friedelin, fumaric acid, kaemferol, lycopsamine, quercetin, rhamnoside, scutellarein, sitosterol, stigmasterol (Cambie and Ash 1994; Ayyanar and Ignacimuthu 2005). Pharmacological uses: Embryotoxic, tannin is insecticidal, antidiarrhoeal, anti-inflammatory, anticoagulant, muscle relaxant, analgesic (Sharma et al. 1978; Cambie and Ash 1994). Fumaric acid shows hepatoprotective properties (Sharma et al. 1995). Caffeic acid is effective against viruses, bacteria and fungi (Brantner et al. 1996).

*Drymaria cordata (L.) Willd. ex Roem. & Schult. Lightening weed (English), Abijalo (Nepali), Caryophyllaceae. Indigenous uses: Leaf is used as calmness, fresh and for cough. Principal chemical compounds: Plant contains methoxycanthin, starch, etc. Pharmacological uses: The methanolic extract of Drymaria was active against Gram-positive bacteria (Taylor et al. 1995). The extract of the plant has been reported to be useful in sinusitis, cold attack, burns and skin diseases (Mukherjee et al. 1995) which could suggest anti-inflammatory and antitussive activities (Mukherjee et al. 1997). The pounded leaf is applied to snake bites in China (Duke and Ayensu 1985). Uses of plant extract as emollient, febrifuge, laxative and stimulant have also been reported (Chopra et al. 1986).

Ainslea latifolia (D.Don) Sch. Bippekuro (Local), Asteraceae. Indigenous uses: Root juice is taken for stomach pain. Principal chemical compounds: Plant contains flavonoids (Chandel et al. 1996). Pharmacological uses: Ethanolic extract plant roots is diuretic (Chandel et al. 1996). Flavonoides are anti-inflammatory and anti-aggregant in properties (Mekhfi et al. 2004; Sharma 2004).

*Chenopodium album L. Goose foot, Pigweed (English), Bethe (Local, Nepali), Vastukah (Sanskrit), Chenopodiaceae. Indigenous uses: Whole plant is useful in constipation and indigestion. Principal chemical compounds: Ascariodes, beta-carotene, catechin, caffeic acid, ecdysteroides, ethereal oil, ferulic acid, furanocoumarins, linolenic acid, oxalic acid, oleanic acid, phenolic acid, polypodine, sitosterol, vitamin C (CSIR 1988; Joshi 2004). Pharmacological uses: Oil, leaf infusion and whole plant parts possess anthelmintic activity against sheep gastrointestinal nematodes (MacDonald et al. 2004; Jabbar et al. 2007). The compounds like betain, oxalic acid, oleanolic acid and furanocoumarins (Nicholas et al. 1955; Hegnauer 1989) may be responsible for anthelmintic activity. The ethanolic extract reveals anti-inflammatory (Matsuda et al. 1997) and antipruritic effects (Dai et al. 2002).

Artemisia indica Willd. Mug wort (English), Kurje pati (Local), Titepati (Nepali), Surparnaa, Nakuli, Nagadamni, Damanaka (Sanskrit), Asteraceae. Indigenous uses: Plant is used in headache, fever and it is also used as insecticide. Leaves are used in skin itching and scabies. Principal chemical compounds: Artemisin, exiguaflavonone, maackiain, sesquiterpene, thujone. Pharmacological uses: Root extracts possessed insignificant hypoglycaemic effects (Villasenor and Lamadrid 2006). Plant infusion is used to reduce the post operative blood loss and relieve purulent inflammation (Davidov et al. 1995). Artemisin and its derivative -arteether are used as antimalarial (Vishwakarma 1990). *Cirsium verutum (D.Don) Spreng. Creeping thistle (English), Thakil, Dhande kanda (Local), Thakailo (Nepali), Asteraceae. Indigenous uses: Root is used as refresher and for calmness. It is also applied for stomachache and abdominal pain.

*Cordyceps sinensis (Berk.) Sacc. Caterpillar fungus (English), Jara (Local), Yarsagumba (Nepali), Sanjiwani (Sanskrit), Clavicipitaceae.


30


Fabaceae. Indigenous uses: Bark is used in cuts, wounds, sprain and fracture. Root is tonic. Principal chemical compounds: Agathisflavone, betulinic acid, campesterol, kaemferol, quercetin, sitosterol, stigmasterol (Husain et al. 1992). Pharmacological uses: Methanolic extract of the plant possesses activity against herpes simplex virus (Taylor et al. 1996). Quercetin is effective in reducing infectivity (Cowan 1999). Betulinic acid is anti-inflammatory (Mukherjee et al. 1997).

Indigenous uses: Whole plant is tonic and aphrodisiac and useful to increase memory and immune system. Principal chemical compounds: Adenosine, cadoverin, campesterol, cerevisterol, cordycepic acid, cordycepin, daucosterol, ergesterol, guanosine, mycosporin, quinic acid, spermidine, uracil, uridine (Halpern 1999; Watanabe et al. 2005). Pharmacological uses: Cordyceps has been used as an anti-tumor herb and an adjuvant of chemo and radiotherapy for various cancers (Bok et al. 1999; Huang et al. 2000; Wu et al. 2007). It is also used as haemostatic, mycolytic, antiasthmatic, expectorant and tonic (Wang and Shiao 2000; Kunwar 2002). Cordycepin and polysaccharides are most widely detected cytotoxic, antibiotic, antitumor (Chen et al. 1997; Kodama et al. 2000), anti-oxidation (Li et al. 2001), and potentiating the immune system (Liu et al. 1992).

Caesalpinia decapetala (Roth.) Alston. Black bonduc, Fever nut (English), Ulto Kanda (Nepali), Lata karanja (Sanskrit), Fabaceae. Indigenous uses: Bark is poisonous and used in fish poisoning. Principal chemical compounds: Braziline, caesalpine, heptocosan, sitosteroide, etc. (Datte et al. 2004) Pharmacological uses: Fruit extract shows inhibitory effect against Candida albicans (Kumar et al. 2006) and anthelmintic effect (Datte et al. 2004), however failure reports on inhibition had also been noted (Rai 1996).

*Coriaria napalensis Wall. Musoorie berry (English), Dahikamlo, Bhojinsi (Local), Machhaino (Nepali), Masuri (Sanskrit), Coriariaceae. Indigenous uses: Bark paste is applied on burns and scalds. Principal chemical compounds: Coreolic acid, coriamyrtin, heptulose, naringenin, tannin, ursolic acid (Buckingham 1994). Pharmacological uses: Methanolic extract of plants and fruits showed significant antimicrobial activity on Escherichia and Staphylococcus bacteria (Joshi and Bhatta 1999). Ursolic acid shows hepatoprotective (Saraswat et al. 1996) and antitumor properties (Bilia et al. 2004).

*Cassia tora (L.) Roxb. Sickle pod (English), Tinkosi, Chakramandi (Local), Tapre (Nepali), Ayadham, Chakramardha (Sanskrit), Fabaceae. Indigenous uses: Plant relieves bronchitis and its juice is anthelmintic and antiseptic. Principal chemical compounds: Anthraquinones, cassiaside, chrysophanol, emodin, obtusifolin, rubrofusarin, toralactone, torachrysone, toralactone (Buckingham 1994). Pharmacological uses: Plant seed extract is antibacterial, anticoagulant, antifungal, hepatoprotective (Mukherjee et al. 1995). Alcoholic extract of seeds exhibited hypoglycemic effect (Simon et al. 1987; Rao et al. 1994). Methanolic extract of seeds insignificantly inhibits leukotriene, which causes pain, inflammation and broncho-muscular constriction (Kumar and Muller 1999). Anthraquinones contracts intestinal walls and stimulate bowel movement and make stool loose (Sharma 2004).

Dioscorea deltoidea Wall. Deltoid yam (English), Vyakur (Local), Gittha (Nepali), Brahmakanda, Varahi (Sanskrit), Dioscoreaceae. Indigenous uses: Yam is used as pesticide and anthelmintic. Principal chemical compounds: Diosgenin, epismilagenin, kryptogenin, nitrogenin, rhamnopyranoside, smilagenin, yamogenin (Husain et al. 1992; Sharma 2004). Pharmacological uses: Diosgenin is used as anabolic, antiarthritic, antinflammatory, antiinfertility (Sharma 2004). Rhizome extract reveals cytotoxic activity against human cancer (Hu and Yao 2002). *Euphorbia royleana Bioss. (Fig. 1) Cactus spurge (English), Siudi (Local, Nepali), Snuhi (Sanskrit), Euphorbiaceae. Indigenous uses: Stem latex is used in joint pain/leg pain. Principal chemical compounds: Amyrin, campesterol, cycloroylenol, diterpene, ellagic acid, ingenol, luepol, octacosanol, phenolics, sitosterol, stigmasterol, succinic acid, taraxerol, terpenes, tetracosanol (Husain et al. 1992). Pharmacological uses: Ethanolic plant extract shows antiinflammatory (Amatya 1994) and latex reveals anti-arthritic activities (Bani et al. 1996).

Entada pursaetha DC. Mackay bean, Ladynut (English), Pangar (Local, Nepali), Kakavali, Gilagaccha (Sanskrit), Fabaceae. Indigenous uses: Fruits are used in cuts and wounds, and body pain. Principal chemical compounds: Entadamide, entanin, myristic acid, palmitic acid, phaseoloidin, phenylacetic acid, prosapognine, thionine, threonine, tryptophan (Buckingham 1994; Joshi 2004). Pharmacological uses: Seed saponin is spasmolytic and central nervous system active (Chandel et al. 1996). Entanin is an antitumor saponin. Saponins have strong haemolytic action and depressant effect (Joshi 2004).

*Ricinus communis L. Castor bean (English), Indeya (Local), Arandi (Nepali), Eranda (Sanskrit), Euphorbiaceae. Indigenous uses: Root juice is analgesic and seed is used in constipation. Principal chemical compounds: Avenasterol, avercetin, amarin, brassicastrol, campesterol, carotene, casbene, chlorogenic acid, coumarin, ellagic acid, haemaglutinin, lupeol, lectin, linolenic, palmitic acid, phenolics, quinic acid, ricinin, ricin, ricinoleic acid, stearic acid, sitosterol, stigmasterol, tannins, terpene, vitamins B6, B1 (Cambie and Ash 1994; Singh 1986). Pharmacological uses: Plant is diuretic, larvicidal, anticholestatic, antiamoebic, analgesic, estrogenic, laxative, cytotoxic, arbortifacient (Singh 1986; Desta 1993) and antimycotic (Rai 1996) and its seed is hepatoprotective (Reddy et al. 1993) and antidote for scorpion sting. Phenolics are antiseptic and anti-inflammatory when taken internally (Banerjee et al. 1991; Sharma 2004).

Milletia extensa (Benth.) Baker Milletia (English), Gaujo (Nepali), Fabaceae. Indigenous uses: Root is useful as insecticide and piscicide. Principal chemical compounds: Auriculatin, aurimillone, iso-flavones, miletin, sumatrol (Husain et al. 1992). Pharmacological uses: Milletia have chemoprotective (Shirwaikar et al. 2003), antipyretic (Srinivasan et al. 2003), anti-inflammatory (Yankep et al. 2003) and cytotoxic properties (Ito et al. 2004). Leaf methanolic extract showed antimycobacterial activity (Taylor et al. 1996). Mimosa pudica L. Sensitive plant (English), Lajjabati (Nepali), Lajja, Saptaparni (Sanskrit), Fabaceae. Indigenous uses: Leaves are used in skin diseases. Principal chemical compounds: Amino acid, amyrin, crocetin, -sitosterol, friedelin, gentisic acid, jasmenic acid, mimosine, nor-epinephrine, pinitol, sitosterol (Husain et al. 1992; Cambie and Ash 1994; Joshi 2004). Pharmacological uses: Plant juice is used as antiviral, anti-

#Bauhinia vahlii Wight & Arn. Camel’s foot climber (English), Malu (Local), Bhorla (Nepali), Murva (Sanskrit),


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trum of antibacterial activity (Khan et al. 2001). Meliacine can be used as a therapeutic agent against HSV-1 ocular infection (Petrera and Coto 2003).

bacterial, anti-inflammatory, antispasmodic, diuretic (Singh 1986). #Sophora mollis (Grah. ex Royle) Himalayan laburnum (English), Chunnjado (Nepali), Fabaceae. Indigenous uses: Roots are taken for rheumatism, and cold. Principal chemical compounds: Cystine, matrine, rutin, etc. Pharmacological uses: Matrine is anti-inflammatory, antidiarrhoeal, analgesic and antotumorous, and it inhibits liver fibrosis (Tan and Zhang 1985; Zhang et al. 2001) and reduces body weight (Cheng et al. 2006). Rutin, a flavonoid protects heart (Chopra and Singh 1994), relieves acute and chronic inflammations (Lee et al. 2000) and strengthens capillary walls (Sharma 2004).

*Psidium guajava L. Guava (English), Ambak (Local), Amba, Belauti (Nepali), Amratphala, Peruk, Mamsala (Sanskrit), Myrtaceae. Indigenous uses: Fruit is laxative, colic, astringent to bowls and beneficial to constipation. Principal chemical compounds: Amritoside, arjunolic acid, asiatic acid, brahmic acid, daucosterol, ellagic acid, eugenol, gallic acid, guavin, isostrictin, latechin, lupol, maslinic acid, pedunculagin, procyanidin, quaverin, quercetin, oleanolic acid, strictinin trans-cinnamic acid, ursolic acid, zeatin (Buckingham 1994; Cambie and Ash 1994). Pharmacological uses: Leaves are antidiabetic due to pedunculagin, and are antibacterial, antimycobacterial, antifungal, antimalarial, analgesic, anti-inflammatory (Suksamrarn et al. 2002), antidiarrhoeal, anticough, antiamoebic, muscle relaxant, hypoglycaemic (Cambie and Ash 1994; Lozoya et al. 1994; Tona et al. 1999; Antoun et al. 2001).

#Didymocarpus villosa D.Don. Kumkum dhup (Nepali), Gesneriaceae. Indigenous uses: Leaf infusion and dust are useful in respiratory problem of children and chronic asthma. Principal chemical compounds: Anthraquinone, chalcone, didymocalyxin, isoflavone, onyselin, pedicinin (Segaw et al. 1999). Pharmacological uses: Plant oil is weak antimicrobial (Chandel et al. 1996). Plant is also affirmative in body weight reduction (Rao et al. 1999).

#Dactylorhiza hatagirea (D.Don) Soo. Marsh orchid, Salep (English), Hathajadi (Local), Panchaunle (Nepali), Salammisri, Munjatak (Sanskrit), Orchidaceae. Indigenous uses: Root juice is taken in cuts and wounds. Principal chemical compounds: Albumin, butanedic acid, dactylorhizin, hydroquinone, lesoglossin, militarrin, pyranoside, pyrocatechol, volatile oil (Kizu et al. 1999). Pharmacological uses: The decoction and plant extract with sugar are useful in pierce, cuttings, wounds, and the plant is tonic and aphrodisiac (Thakur and Dixit 2007).

*Morchella esculenta (L.) Pers. Morel mushroom (English), Mathyaura (Local), Guchhi chyau (Nepali), Helvellaceae. Indigenous uses: Plant stalk and cap are aphrodisiac in properties and used as tonic and immunostimulant. Principal chemical compounds: Amino acid, carotene, protein, saponins (Zheng et al. 1998). Pharmacological uses: Methanolic extract of plants inhibits leukotriene, which causes pain, inflammation and broncho-muscular constriction (Kumar et al. 2000).

*Oxalis corniculata L. Creeping sorrel (English), Chalmaro (Local), Chari amilo (Nepali), Changeri, Amla patrika (Sanskrit), Oxalidaceae. Indigenous uses: Leaves are stomachic and useful for throat pain. Principal chemical compounds: Carotene, citric acid, eugenol, glycoxylic acid, malic acid, pentylfuran, pyruvic acid, tartaric acid, tocopherols, votexin, etc. (Ayyanar and Ignacimuthu 2005). Pharmacological uses: The plant is antihypertensive, hypoglycemic, uterine relaxant, muscle relaxant and rich source of Vitamin B (Cambie and Ash 1994). Eugenol is considered a bacteriostatic and fungistatic (Duke 1985). Alcoholic leaf extract is antibacterial (Joshi 2004).

Colebrookea oppositifolia Sm. Bedmauri (Local), Dhursool (Nepali), Lamiaceae. Indigenous uses: Leaf juice is taken for skin disease. Principal chemical compounds: Chrysin, flavonene, ladanein, negletein, sitosterol, triacontane, triacontalol (Husain et al. 1992; Yang et al. 1996). Pharmacological uses: Ethanolic root extract is central nervous system active (Chandel et al. 1996). #Leea indica (Burm. f.) Merr. Galeno (Nepali), Kakanasika (Sanskrit), Leeaceae. Indigenous uses: Leaf is useful in spleen problems. Young leaves are digestive. Principal chemical compounds: Eicosanol, farnesol, gallic acid, leeaoside, lupeol, palmitic acid, phthalic acid, sitosterol, solanesol, ursolic acid (Srinivasan et al. 2008). Pharmacological uses: The methanolic extract of L. indica was reported to possess strong antioxidant and nitric oxide inhibitory activities (Saha et al. 2004) and it was due to gallic acid, a well known antioxidant compound (Srinivasan et al. 2008). Plant extract is antiviral and anticancer in properties (Jain et al. 1991).

*Plantago major L. Blond psyllium (English), Ishabgol (Nepali), Ashvagola, Snigdhabija (Sanskrit), Plantaginaceae. Indigenous uses: Plant seeds are useful in diarrhea, dysentery and indigestion. Principal chemical compounds: Apigenin, ascorbic acid, aucubin, baicalein, benzoic acid, caffeic acid, catalpol, chlorogenic acid, cinnamic acid, papa-coumaric acid, ferulic acid, hispidulin, loliolide, luteolin, majoroside, nepetin, plantagonine, planteose, scutellarein, syringic acid, vannillic acid, vitamin A (McCutcheon et al. 1992). Pharmacological uses: Root and seed extract is antibacterial, anti-inflammatory, antiviral, antitumor, hypotensive, oestrogenic, wound healer, kidney stone disintegration, diuretic (McCutcheon et al. 1992). Ethanolic root extract show little inhibitory effect of human tumor cell growth (Whelan and Ryan 2003). Caffeic acid is effective against viruses, bacteria and fungi (Brantner et al. 1996). Seeds are useful in diarrhea and amoebic dysentery (Sharma 2004).

#Melia azedarach L. Bead tree, Persean lilac (English), Bakaino (Local, Nepali), Mahanimba (Sanskrit), Meliaceae. Indigenous uses: Bark and leaf juice is useful in spleen disorders. Principal chemical compounds: Azaridin, azadirachtin, bakalactone, bakayanin, benzoic acid, deacetylsalanin, dihydronimocinol, fraxinellone, quercetin, meliacarpinin, meliacine, meliotannic acid, melazolide, nimbolinin, rutin, salanin, salannal, vilasinin (Husasain et al. 1992; Watanabe et al. 2005). Pharmacological uses: The extract of leaf suppresses nitric oxide (NO) synthesis, since increased NO production is associated with acute and chronic inflammation (Lee et al. 2000) and it is antioxidant (Virgili et al. 1998). Methanol extract of root, stem bark and leaves showed a broad spec-

Cynodon dactylon (L.) Pers. Bermuda, Dog’s teeth grass (English), Dubi (Local), Dubo (Nepali), Durva (Sanskrit), Poaceae. Indigenous uses: Plant paste is effective on sprain. Inflorescence is grinded with water and applied for earache. Principal chemical compounds: Coumarin, ferulic acid, phytol, stigmasterol, syringic acid, tricin, vanilic acid


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(Husain et al. 1992). Pharmacological uses: Rhizome juice possesses antiviral property (Foster and Duke 2000). The aqueous extract of Cynodon dactylon has high antidiabetic potential along with significant hypoglycemic and hypolipidemic effects (Singh et al. 2007). The aqueous plant extract is used as antiinflammatory, diuretic, anti-emetic and purifying agent (Ahmed et al. 1994) and used in treating dysentery, dropsy and secondary syphilis (Chopra and Handa 1982). The ethanolic extracts of the plant showed antioxidant activity (Auddy et al. 2003).

1994).

*Imperata cylindrica (L.) Beauvois. Cogon grass (English), Siru (Local, Nepali), Sarba (Sanskrit). Poaceae. Indigenous uses: Rhizome paste is applied for urinary problems. Principal chemical compounds: Arundoin, chromone, cylindrene, cylindol, fernenol, flidersiachromone, graminone, imperanene (Matsunaga et al. 1995; Yoon et al. 2006). Pharmacological uses: Rhizome extracts possessed insignificant hypoglycaemic effect (Villasenor and Lamadrid 2006), weak antibacterial activity (Risal 1994) and decreased the urine volume (Kanchanapee 1966; Sripanidkulchai et al. 2001). Imperanene showed inhibitory activity on platelet aggregation (Matsunaga et al. 1995) and chromone is neuroprotective (Yoon et al. 2006).

Anthocephalus chinensis (Lam.) A. Rich. ex Walp. Wild cinchona (English), Kadam (Nepali), Kadamba (Sanskrit), Rubiaceae. Indigenous uses: Fruits are used in urinary problems. Principal chemical compounds: Cadambine, dihydrocadambine, geraniol, linalool, linalylacetate, nonanol, phellandrene, saponins, sitosterol, selinine (Husain et al. 1992). Pharmacological uses: Bark extract is astringent and useful in snake bite poison (Yusuf et al. 1994). Linalool exhibits significant antimutagenic and antioxidative properties (Deans et al. 1993; Stevic et al. 2004).

Rubus ellipticus Sm. Golden raspberry (English), Ainselu (Nepali), Gauriphala (Sanskrit), Rosaceae. Indigenous uses: Root juice is given for relieving fever and diarrhoea and dysentery. Principal chemical compounds: Amyrin, arjunetin, rosamultin (Bilia et al. 1994) Pharmacological uses: Antiimplantation and early abortifacient activities of Rubus ellipticus were denoted (Dhanabal et al. 2000).

*Citrus medica L. Adam’s apple, Citron (English), Bimiro (Nepali), Mahulunga (Sanskrit), Rutaceae. Indigenous uses: Leaf is antipyretic and used as insect or pest repellant. Principal chemical compounds: Aureusilin, bergamotene, caffeine, grandmarin, hesperidine, kinocoumarin, limonene, lumbelliferone, nomilinic acid, resveratrol, rutaevin, theophylline, xanthyletin (Buckingham 1994; Kretschmar and Baumann 1999; Govindachari et al. 2000). Pharmacological uses: Leaf extract is useful in fever and febrile illnesses (Ajaiyeoba et al. 2003). Peel is aromatic and tonic (Font Quer 1992). Seeds, leaves and fruit pulp have anticancer property due to their limonin content (Tian et al. 2001; Arias and Laca 2005). Oil from leaves possesses antibacterial property (Limyati and Juniar 1998).

*Rumex nepalensis Spreng. Sheep sorrel (English), Ban haldi (Local), Halhale (Nepali), Amlavetasa (Sanskrit), Polygonaceae. Indigenous uses: Root extract is applied in joint pain and paralysis. Principal chemical compounds: Anthraquinones, chrysophanol, emodin, lupeol, musizin (nepodin), orientalone, physcion, sitosterol, tannins (Husain et al. 1992). Pharmacological uses: Methanol extract significantly possesses the hypotensive effect and shows the property of muscle relaxant and tranquilizer (Murugesan et al. 1999; Ghosh et al. 2002). Tannins draw the tissues closer and improve the resistance to infection (Sharma 2004).

Osyris wightiana Wall. Wild tea (English), Nundhikya (Local), Jhuri, Nundhiki (Nepali), Santalaceae. Indigenous uses: Bark infusion is given to stop bleeding. Leaf and bark decoction is used in sprains and fractures. Principal chemical compounds: Lanceol, proline, tannins, etc. (Chandel et al. 1996) Pharmacological uses: Leaf extracts possess antiviral activity (Chandel et al. 1996). Tea made from the leaves of O. wightiana stimulated the flow of breast milk and also acted as a labor-inducing agent (Osujih 1993).

*Thalictrum cultratum Wall. Meadow rue (English), Peljadi (Local), Dampate (Nepali), Peet ranga (Sanskrit), Ranunculaceae. Indigenous uses: Root juice is commonly used in stomacheache and dysentery. Principal chemical compounds: Berberine, diterpene, jatrorhijine, magnoflorine, palmatine, thalictrine (Husain et al. 1992). Pharmacological uses: Root extract is antiperiodic, diuretic, purgative (Chauhan 1999) and antimicrobial (Omulokoli et al. 1997; Schmeller et al. 1997; Iwasa et al. 1998). Berberine is antibacterial and antimalarial (Yamamoto et al. 1993) and Thalictrine has inhibitory effect on lymphoma, sarcolymphoma and hepatoma (Jain et al. 1991).

Aesandra butyracea (Roxb.) Baehni. Butter tree (English), Chiura (Local), Chiuri (Nepali), Sapotaceae. Indigenous uses: Oil cake is used to escape out snake, and it can be used as fish poisoning. Oil or ghee is taken to cure cracked heels and lips. Root juice is useful in dysentery. Principal chemical compounds: Betulinic acid, friedelin, hentriacontane, linoleic acid, oleanic acid, palmitine, protobasic acid, quercetin, rhamnoside, stearic acid, sitosterol (Husain et al. 1992; Bhattacharjee et al. 2002). Pharmacological uses: Betulin and quercetin of Butter tree are anti-infectivity (Cowan 1999) and anti-inflammatory in properties (Mukherjee et al. 1997).

*Agrimonia pilosa (D.Don) Nakai. Hairy agrimony, Couch grass (English), Kathlange (Nepali), Rosaceae. Indigenous uses: Plant is used to cure dysentery and root juice is used as antidote for snake bite. Principal chemical compounds: Agrimonolides, agrimophol, apigenin, coumarins, ellagic acid, flavonoides, luteolin, phenylpropanoides, quercetin, pilosanol, pyranoside, triterpenes, tormentic acid (Kimura et al. 1995). Pharmacological uses: Antitumor, bacteriostatic, antiyeast, antidysenteric (Kimura et al. 1996, Peter 1969). Triterpenes show antitumor and expectorant properties (Sharma 2004). Ellagic acid is antimutagenic (Kaur et al. 1997) and antimicrobial (Gyamfi and Aniya 2002). Luteolin has better antiviral activity against Respiratory syncytial virus (RSV) (Ma et al. 2002). RSV is a major cause of pneumonia and bronchiolitis in infants, in young children, and even in adults. Luteolin demonstrates anti-inflammatory effect (Park et al. 2001; Panthong et al. 2007). Luteolin and quercetin inhibit proliferation of cancer cells (Elangovan et al.

*Astilbe rivularis Buch.-Ham. ex D.Don. Astilbe (English), Sutkeribelo (Local), Thulo okhati, Budho okhato (Nepali), Saxifragaceae. Indigenous uses: Root juice is used for easy delivery and control bleeding during child birth. It is valued for diarrhoea, dysentery and hemorrhage. Principal chemical compounds: Aesculatin, astilbic acid, astilbin, aticoside, bergenin, dimethylaesculatin, daucosterol, eucryphin, palmitine, peltoboykinoleic acid, scopoletin, sitosterol, stilbene (Jain et al. 1991; Buckingham 1994). Pharmacological uses: Pharmacological experiments indi-


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cated the extracts from Astilbe chinensis had antineoplastic and immunopotentiating activities (Chen et al. 1996). Dried rhizome is used as substitute drug for Shengma (Han et al. 1998). Astilbic acid is beneficial in regulating various inflammatory processes (Moon et al. 2005).

Traditional medicines are conferred in ancient, natural health care practices such as folk/tribal practices, home herbal remedy, Baidhya, Ayurveda and Amchi healing systems. Folk-lore medicine, home herbal remedy and Baidhya practices are indigenous to farwest Nepal and are partly influenced by the Ayurveda (Kunwar and Bussmann 2008). Baidhyas are traditional herbalists of far western Nepal (Bhattarai 1992) and adjoining areas of India (Kala 2005) and they pursue their remedies to cure diseases and aliments, taking advantage of the abundance of nearby medicinal plants. Amchi healing system is widely accepted and practiced throughout high altitude areas (Kunwar et al. 2006) and the Darchula district is particularly influenced, albeit with varying degrees of modifications (Lama et al. 2001). All these traditional medicinal systems are popular with a long tradition in the use of medicinal plants (Uprety et al. 2010) and they are due to easy and open access, availability and cheaper in use (Shale et al. 1999; Kunwar and Bussmann 2008). Ayurveda is most important in bio-prospecting of new medicines (Patwardhan et al. 2005) in among. Consequently, acceptance of the Ayurveda is gearing up (Kunwar et al. 2009). The traditional therapies have played vital roles in health care delivery systems especially in high hills and remote areas of study districts where clinics and hospitals are absent or sparsely located. Moreover the extensive usage of traditional therapies is due to high cost of western pharmaceuticals and healthcare. Inadequate modern medical facilities (Sherpa 2001) and government subsidies, and intensive uses of plants (Bussmann and Sharon 2006) also made home herbal remedies pertinent in the Himalayas. Modern medicines are also difficult to find (Manandhar 2002) when needed particularly in the Himalayas due to complex geomorphology. Such situation consents to the data where there is one traditional healer for less than 100 people (Gillam 1989) and one physician for 6,000-20,000 people (WRI 2005, Pradhan 2007). Since the apposite of traditional therapies, the role of natural products and herbal medicine is being increasingly appreciated (Cragg et al. 1997) in recent years. The therapies mostly using plants and plant products of western Nepal incorporate ancient beliefs and are passed down from generations to generations by oral tradition and/or guarded literatures (Bhattarai 1997; Kunwar and Bussmann 2008). This study shows that information obtained from traditional healers and local herbal medicine practitioners can support to renew and increase in use of herbal medicines and discovery of therapeutically useful agents and vice versa. However, changing perception of local people, acculturation, commercialization and socio-economic transformations have jeopardized the indigenous knowledge of phytotherapies. Furthermore, some tribal therapies were not supported by systematic ethnopharmacological findings. Therefore validity assessment of indigenous therapies of plant resources base received greater attention.

*Urtica dioica L. (Fig. 1) Stinging nettle (English), Sisnu (Local, Nepali), Agni damani (Sanskrit), Urticaceae. Indigenous uses: Stem is valued for sprain and fractures. Root juice is given for gastric problems and maintaining blood pressure. Principal chemical compounds: Acetylcholine, betaine, choline, flavonoides, histamine, linoleic acid, oleic acid, palmitic acid, plastoquinone (Husain et al. 1992). Pharmacological uses: The aqueous extract has antihyperglycaemic effect (Bnouham et al. 2003; Farzami et al. 2003), and it is also a good antioxidant (Pieroni et al. 2002), hepatoprotective (Lebedev et al. 2001), analgesic (Gulcin et al. 2004), antiviral (Manganelli et al. 2005), diuretic and hypotensive in properties (Tahri et al. 2000; Testai et al. 2002). Flavonoides shows the anti-aggregant property (Mekhfi et al. 2004). #Callicarpa arborea Roxb. Urn fruit, Beauty berry (English), Gotmelo (Local), Dahikamlo (Nepali), Gandhaphali (Sanskrit), Verbenaceae. Indigenous uses: Fruits are edible and help in indigestion. Principal chemical compounds: Amyrin, apigenin, astilbin, beta sitosterol, calliterpenone, cartegolic acid, luteolin, maslinic acid, oleanoic acid, oleanolic acid, sitosterol, ursoleic acid (Husain et al. 1992). Pharmacological uses: Luteolin has antiviral (Cheng Ma et al. 2002) and anti-inflammatory effects (Park et al. 2001; Panthong et al. 2007). Along with quercetin, luteolin inhibits cancer cell proliferation (Elangovan et al. 1994). *Viscum album L. Mistletoe, Devil’s fuge (English), Hadchur (Local), Ainjeru (Nepali), Viscaceae. Indigenous uses: Plant is used in fractures and sprains. Principal chemical compounds: -sitosterol, caffeic acid, dimethoxyflavone, eleutheroside, flavonoides, glycoproteins, kaemferol, lectin, oleanic acid, pectin, quercetin, syringin, triterpene, ursolic acid (Husain et al. 1992; Ergun and Deliorman 1995; Lyu et al. 2000; Deliorman et al. 2005). Pharmacological uses: Immuno-regulatory, diuretic, antibacterial, antiviral, inhibits cell proliferation (Yoon et al. 1999), diuretic, anti-inflammatory as well as immunostimulant effects (Yesilada et al. 1998). The extract produces antihypertensive (Ofem et al. 2007) and antioxidant effect (Ucar et al. 2006). Cissus repens Lam. Wild grape (English), Pureni (Nepali), Asthisamharaka (Sanskrit), Vitaceae. Indigenous uses: Stem juice is useful in eye redness. Principal chemical compounds: -sitosterol, luteolin, piceatannol, pallidol perthenocissin, resveratrol (Adesanya et al. 1999; Gupta and Verma 1991). Pharmacological uses: Pharmacological studies revealed the bone fracture healing property (Chopra et al. 1976; Deka et al. 1994) and antiosteoporotic effect (Shirwaikar et al. 2003). Murthy et al. (2003) reported the antibacterial and antioxidant activities of the extract. Plant demonstrates anti-inflammatory effect (Singh et al. 1984) due to -sitosterol and luteolin of the plant (Park et al. 2001; Panthong et al. 2007).

Validity analysis We compared the traditional and modern pharmacological uses of 48 medicinal plant species commonly used in folklore of farwest Nepal. The species represented from 34 families and 34 genera. Families Fabaceae and Asteraceae contributed the most and provided 7 and 6 species respectively. Euphorbiaceae and Rutaceae families possessed the most contribution in earlier study (Kunwar et al. 2009) and moderate contribution in present study, rendered two and one species respectively. Among the surveyed 48 species in the present study, 15 species possessed weak analogy or their indigenous uses were differed to the pharmacological findings. It was may be due to knowledge distortion. Changing perception of local people, commercialization and socio-economic transformations are prevalent in study area (Kunwar et al. 2010) and they contributed misleading situations to the traditional therapies. Moreover, younger generations were uninterested on traditional thera-

DISCUSSION Traditional medical systems Prehistoric uses of medicinal plants as therapy for illness in farwest Nepal has been investigated in present study. Traditional therapies abound in nearby medicinal plants (Bhattarai et al. 2010), and the tribal people/ethnic groups, wherever they exist, chiefly rely on herbal medicines.


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pies. The situation was also provoked due to research limitations and diverse resource users. As a result, essence of ethnopharmacological surveys and cross-referencing approaches on those species revealing trivial affinities is warranted. Misled of indigenous knowledge and use of ethnomedicine out of the experience or ignorance and willful deception may deviate knowledge out of standard and ultimate cause illness and even fatal (Zhao et al. 2006; Kumar 2007). Approximately 68% (33) species used in indigenous medicine of the present study demonstrated some analogous effects and the 23 species (48%) bestowed the strong supports. This fair corroboration of pharmacological activity gives the claims by traditional healers a significantly high credibility and such similar conceivable remarks were also observed in abroad by Marles and Farnsworth (1995), Chandel et al. (1996), Hamza et al. (2006) and Gautam et al. (2007). These results substantiated the importance of surveys of indigenous knowledge of utilization plant resources for screening plants as a potential source for bioactive compounds. Hence ethnomedicine and ethnopharmacology could result in discovery of novel constituents because they are developed through long trial and error operations (Rijal 2008).

teria are multilayered in structure and more resistant (Yao and Moellering 1995). Diterpenoid alkaloids, commonly isolated from the plants of Ranunculaceae family, are commonly found to have antimicrobial properties (Omulokoli et al. 1997). Root juice of Thalictrum cultratum (Ranunculaceae) commonly used in stomachache and dysentery in study area is affirmative to the in vitro antimicrobial properties. Berberine, a benzylisoquinoline alkaloid, acted as an antibacterial and antimalarial drug (Yamamoto et al. 1993), is a principal chemical constituent of T. cultratum. Berberine shows strong antimicrobial activity to both Gram-positive and -negative bacteria as well as to other microorganisms (Schmeller et al. 1997; Iwasa et al. 1998). It is potentially effective against trypanosomes (Frieburghaus et al. 1996) and plasmodia (Omulokoli et al. 1997). Ethanol extract of root of Angelica archangelica (Apiaceae) also shows resistant to the trypanosomes (Schinella et al. 2002). Dried roots of Angelica are anthelminthic and useful in gastritis and stomacheache in the study area. Root juice or raw roots of Astilbe rivularis (Rosaceae) are consumed for easy delivery and control bleeding during child birth. Because of its effects, it is called as sutkeribelo in local dialect i.e. plant is useful in parturition for easy delivery and controlling bleed. Because of its astilbic acid, it is beneficial in regulating various inflammatory processes (Moon et al. 2005). Stilbene and asiaticoside from Astilbe rhizomes have wound healing properties (Gomathi et al. 2003; Kapoor et al. 2004) and accentuate burn and wound healing. Furthermore, astilbin and bergenin are effective in treatment of obesity (Han et al. 1998). Astilbin has antiarthritic and antiallergy effects (Cai et al. 2003) and Bergenin, an isocoumarin prevents arrhythmia, liver injury (Pu et al. 2002), and gastric troubles (Goel et al. 1997). Scopolamine (hyosine) of Astilbe rhizomes is used as analgesic (Yamamoto et al. 1993; Iwasa et al. 1998) and is tranquilizer in property (Duke 1992). We observed anti-arthritic and anti-paralytic effects of plant juice of Rumex nepalensis (Polygonaceae). Tannin from Rumex nepalensis (Polygonaceae) draws tissues together and improves their resistance to infections (Sharma 2004). Polygonaceae is also widely used as anthelmintic due to its anthraquinones (Midiwo et al. 1994). R. nepalensis is also persuaded as antipyretic (Suresh et al. 1994) and its lupeol and its derivatives regulate genito-urinary systems (Anand et al. 1995). Coriaria nepalensis (Coriariaceae) contains tannins and ursolic acid as main constituents. Tannin is antinflammatory, muscle relaxant, analgesic, etc. (Sharma et al. 1978; Cambie and Ash 1994) and ursolic acid shows hepatoprotective (Saraswat et al. 1996) and antitumor properties (Bilia et al. 2004). Tannin cures and prevents variety of illness (Scortichini and Rossi 1991; Haslam 1996). In folklore, Coriaria bark is applied on burns and scalds and it is coincided to its anti-inflammatory, analgesic, antibacterial, muscle relaxant and antimicrobial properties (Joshi and Bhatta 1999). It is well known that Plantago major (Plantaginaceae) has demonstrated antineoplastic activity against cancer of the breast, anus, stomach, eye, foot, intestine and liver, and against neuroblastoma cancer (Duke 1985). P. major contains caffeic acid which is effective against viruses, bacteria and fungi (Brantner et al. 1996). Plant seeds are used in indigestion and dysentery as ethnomedicine. Ethnomedicinal use was beneficial due to its antibacterial and antiviral properties of caffeic acid. Ageratum conyzoides and Viscum album also contain caffeic acid. Caffeic acid, coumarins and tannins of A. conyzoides (Asteraceae) possess antibacterial (Mahato and Chaudhary 2005), anthelmintic, anti-inflammatory, analgesic (Hedberg et al. 1983; Namba et al. 1988; Tandon et al. 1994) and anticoagulant and muscle relaxant (Cambie and Ash 1994) effects. Anti-inflammatory activity was also shown by sterols, especially stigmasterol (Garcia et al. 1999). Coumarin of A. conyzoides is a potential insecticide (Kamboj and Saluja 2008). Folk use of stem juice of A. conyzoides as bleeding control was supported by haemo-

Strong affinities between indigenous and pharmacological findings There were two species: Euphorbia royleana and Ricinus communis from Euphorbiaceae exhibited strong ethnopharmacological properties in present study. Ethnopharmacological usage of latex of Euphorbia royleana for joint/leg pain is supported by phytochemical investigations: ethanolic extracts of plant latex has anti-arthritic activities (Bani et al. 1996). Root juice of Ricinus communis is indigenously taken as analgesic and antidiarrhoeic in study area resembled to the findings of pharmacology where plant possessed anticholestatic, antiamoebic, analgesic, arbortifacient, estrogenic (Singh 1986; Desta 1993), antiseptic and anti-inflammatory effects when taken internally; are due to phenolics (Sharma 2004). The phenolic acid of the plant acts as cholagogues, stomach refresher, and immuno-stimulants, as well as anti-tumor, antioxidant, antibacterial, and antifungal agents (Hamauzu et al. 2005; Mishima et al. 2005). Ricinoleic acid, an active component of castor oil causes irritation and inflammation to the intestinal mucosa, results an increase in the net secretion of water and electrolytes into the small intestine (Pierce et al. 1971; Luderer et al. 1980) and induces diarrhea (Gaginella et al. 1975). Euphorbiaceae that is rich in active compounds including terpenoids, alkaloids, phenolics and fatty acids, having various ethnopharmaceutical uses (Rizk 1987). Terpenenes are active against bacteria (Kubo et al. 1992; Habtemarium et al. 1993), fungi (Taylor et al. 1996; Rana et al. 1997), viruses (Fujioka and Kashiwada 1994), and protozoa (Vishawakarma 1990). Root juice of Cirsium verutum (Asteraceae) is ethnopharmacologically applied for stomachache and abdominal pain, and the use is coincided to biological activity of terpenes. Plant is rich in cicin, glycerol, sterols and terpenes (Lee et al. 2002) and its uses as antimicrobial (Lee et al. 2002; Barbour et al. 2004) supports the folklore. Topical anti-inflammatory properties of Xanthium strumarium (Asteraceae) fruits (Han et al. 2007) supports the use of plants’ seeds and fruits for treatment of inflammatory diseases in folk medicine. The natural xanthones showed good inhibitory activity against pathogenic fungi (Gopalakrishnan 1997). Juice from the plant Drymaria diandra is used to treat coughs, fever and eye disease (conjunctivitis) (Manandhar 1990), which could all possibly be caused by bacteriostatic properties (Mukherjee et al. 1997). The methanolic extract of Drymaria diandra was active against Grampositive bacteria. Various researches have already shown that Gram positive bacteria are more susceptible towards plant extracts as compared to Gram negative bacteria (Lin et al. 1999; Parekh and Chanda 2006). Gram-negative bac-


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yan et al. 2008) and have shown to increase mucus secretion, prostaglandin synthesis and blood flow (Singh et al. 1998). Urtica stem is indigenously valued for sprain and fractures and its root juice is valued for gastric and blood pressure problems. Aqueous U. dioica plant extract control blood sugar level (Bnouham et al. 2003; Farzami et al. 2003), and it is a good antioxidant (Pieroni et al. 2002) and hypotensive (Tahri et al. 2000; Testai et al. 2002) due to flavonoids (Galvez et al. 1993). Antiviral, anti-inflammatory and anti-aggregant properties of flavonoides (Farnsworth 1966; Su et al. 2000; Mekhfi et al. 2004; Sharma 2004) of U. dioica are consistent to the folk uses. Polysaccharide is one of the active components in Cordyceps sinensis (Clavicipitaceae) that has multiple pharmacological activities. It has high concentrations of adenosine, guanosine and uridine (Li et al. 2001) among these; adenosine is most worth in pharmacology. Adenosine has widespread effects on circulation of blood, cerebral and coronary (Berne 1980; Toda et al. 1982), prevention of cardiac arrhythmias (Pelleg and Porter 1990), inhibition of neurotransmitter release and the modulation of adenylate cyclase activity (Ribeiro 1995), potentiating immune system (Liu et al. 1992; Xu et al. 1992) and antitumor activity (Chen et al. 1997). The indigenous uses of plants as tonic, aphrodisiac, immuo-stimulative and useful in memory longetivity throughout Nepal (Uprety et al. 2010) are justifiable to the pharmacological observations. Inhibition of LTB4 biosynthesis and lipoxygenase activity by the Morchella esculenta (Helvellaceae) extracts supports their indigenous uses in various diseases known to be mediated by 5-lipoxygenase products, i.e. leukotrienes. Plant stalk and cap are considered as aphrodisiac (Kunwar 2006) and are used as tonic and immunostimulant in folklore. Methanolic extract of plants facilitates healing and soothing (Kumar et al. 2000). Lipoxygenase induces inflammation and the activity of lipoxygenase can also be inhibited by the rhizome extract of Imperata cylindrica (Matsunga et al. 1995). Rhizome extracts of I. cylindrica (Poaceae) decreased the urine volume (Kanchanapee 1966). Alike to pharmacological findings, ethnomedicinal use of the plant rhizome paste was for urinary complaints. Pharmacological literatures reveal antipyretic, digestive and tonic properties of Citrus fruits and leaves (Font Quer 1992; Ajaiyeoba et al. 2003) since the antipyretic effect of Citrus (Rutaceae) is recognized by folklore in Nepal Western Himalaya. Anticancer properties have been associated with the components of various natural products including polyphenols, resveratrol, and limonene (Kaegi 1998). Resveratrol and limonene of Citrus fruits have multiple biological activities including vasodilatory (Duarte et al. 1993), anticarcinogenic, anti-inflammatory, antibacterial, antiviral effects, etc. (Brown 1980; Middleton and Kandaswami 1992). Cassia tora (Fabaceae) is used for bronchitis and its juice is applied as anthelmintic and antiseptic in study area argued with the antibacterial, antifungal (Mukherjee et al. 1995), anti-inflammatory and broncho-dilator efficacies of the plant (Kumar and Muller 1999). Seed extracts is anticoagulant (Mukherjee et al. 1995) and hypoglycaemic (Simon et al. 1987; Rao et al. 1994). Plant anthraquinones placate intestinal walls and stimulate bowel movement and make stool loose (Sharma 2004).

static (Akah 1988) and antibacterial (Mahato and Chaudhary 2005) effects. Plants’ use as bleeding control could be a part of further research because the juice of plant is extensively used in cuts, wounds and bleeding control in western Nepal (Bhattarai 1993; Manandhar 1998; Joshi and Joshi 2000). Agrimonia pilosa (Rosaceae) is indigenously used to cure dysentery and its root juice is taken as antidote for snake bite. The purport of indigenous uses was substantiated by pharmacological findings, A. pilosa plant extract and its active constituent the coumarin act as bacteriostatic, antiyeast and antidysenteric, etc. (Peter 1969; Kimura et al. 1996). Coumarin also act as antithrombotic (Thastrup et al. 1985), anti-inflammatory (Piller 1975), and vasodilatory (Namba et al. 1988). Ellagic acid of the plant is antimicrobial (Gyamfi and Aniya 2002) and supports ethnopharmacology. Antibacterial and antiviral properties of caffeic acid of Viscum album (Viscaceae) (Yoon et al. 1999) support its indigenous use for sprain and fracture. Leaf and fruit extracts of V. album possesses immunostimulant effects (Yesilada et al. 1998). Viscum album, Psidium guajava and Coriaria nepalensis species of present survey contains ursolic acid. Ursolic acid and its derivatives have shown a significant activity against P-388 and L-12 10 lymphocytic leukemia cells as well as human lung carcinoma (Bilia et al. 2004). These biological studies indicate that the antitumor activity of the plant could be due to presence of triterpenes. Eugenol, available in plant extract of Psidium guajava (Myrtaceae) and Oxalis corniculata (Oxalidaceae), was found as bacteriostatic and fungicidal (Thomson 1978) corroborates ethnopharmacological uses of Psidium fruits for constipation and colic. Gallic acid derivatives from Psidium fruits are more effective against both types of Staphylococcus aureus (Sato et al. 1997) and they show potent antimicrobial properties (Gyamfi and Aniya 2002). Pedunculagin of P. guajava is anti-inflammatory (Suksamrarn et al. 2002) in effects. In our observation, O. corniculata has been used to cure throat pain and mouth problems. The cure of aphthae might be due to eugenol and supplement of Vitamin B complex to quick healing and there by relieving of pain. The mechanism of action of these plants on aphthae is worth for further investigation. The compounds like betalain alkaloids, phenolic acids, betain, oxalic acid, oleanolic acid, sitosterol, furanocoumarins and saponins may be responsible for anthelmintic activity of Chenopodium album (Chenopodiaceae) (Nicholas et al. 1955; Hegnauer 1989). The oil and infusion of plant leaves possess worth anthelmintic activity against gastrointestinal nematodes (MacDonald et al. 2004; Jabbar et al. 2007). Catechin, a flavonoid of C. album also exhibited antibacterial, antiviral and antimicrobial properties (Sakanaka et al. 1992; Vijaya et al. 1995; Borris 1996). The indigenous use of C. album species for constipation and indigestion is rational to its antibacterial, antiviral and antimicrobial properties. Indigenous use of Rhus fruits decoction for diarrhea and dysentery concurred its antidiarrhoeal properties (Galvez et al. 1993; Su et al. 2000). Because, most naturally occurring flavonoids of plant show an antioxidant and antidiarrhoeal effects (Galvez et al. 1993; Thangpu and Yadav 2004), but some flavonoids are mutagenic in bacterial and mammalian systems (Mdee et al. 2003) and have antiviral and anti-inflammatory activities (Farnsworth 1966; Sharma 2004). Flavonoides, essential constituents of the cells of all higher plants (Brouillard and Cheminat 1988), play a major role in successful medical treatment of ancient times and their use has preserved till date (Dixon et al. 1998). Rhus species, widely distributed in the subtropical regions of the world and used medicinally in various ways, are rich in biflavonoids. Flavonoides along with sterols work as bioactive for diabetes (Rhemann and Zaman 1989; Patil et al. 2005). Plant extract of Urtica dioica (Urticaceae) also contains active flavonoides. Flavonoides pose anti-inflammatory, antibacterial and wound healing properties (Afolo-

Moderate affinities between indigenous and pharmacological findings Alcoholic extract of Inula racemosa (Asteraceae) enhanced liver glycogen and lowered blood glucose level (Tripathi and Chaturvedi 1995). Lung fibrosis (Thresiamma et al. 1996), blood pressure control (Dikshit et al. 1995) and antiinflammatory properties (Kohli et al. 2005) are due to curcumine of the plant. Root extract of the plant is useful in stomachache, dysentery and blood pressure in study area. Indigenous use of plant for stomachache and dysentery infers connotation of antibacterial and antiviral properties. Antibacterial (Negi et al. 1999) and antiviral (Bourne et al.


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1999) properties of curcumin are suggested. Curcumin, a yellow colored phenolic pigment, is found to inhibit arachidonic acid metabolism, cytokines, and release of steroidal hormones. It has strong oxygen radical scavenging activity which is responsible for anti-inflammatory property (Kohli et al. 2005; Singh et al. 2008). Rutin, a flavonoid from Melia azedarach (Meliaceae) strengthens capillary walls (Sharma 2004), relieves acute and chronic inflammations (Lee et al. 2000) and protects heart (Chopra and Singh 1994). Methanol extract of plant root, stem bark and leaves showed a broad spectrum of antibacterial activity (Khan et al. 2001) and it is partially consented to the indigenous usage as bark and leaf juice is therapeutically used for spleen disorders. Rutin from Sophora mollis (Fabaceae) protects heart (Chopra and Singh 1994), and relieves acute and chronic inflammations (Lee et al. 2000) and capillary wall infections (Sharma 2004). The cardioprotective action of the plant is traditional therapy base where the plant roots are taken for rheumatism, and cold. Antiviral property of Leea indica (Leeaceae) (Jain et al. 1991) and indigenous use of plant leaves as digestive are partially justified. Leaves of Artemisia are used in skin itching and scabies in ethnopharmacology, and in phytochemical studies plant leaf extract possessed activities against bacteria (Bhattarai et al. 2009) which produce malodors in skin surface (Moulari et al. 2004). Pleumeria rubra (Apocynaceae) is antibiotic, antiviral, etc. and fluroplumierin of the plant inhibits mycobacteria (Sundarrao 1993; Cambie and Ash 1994) which consented to the indigenous uses as digestive and anticholeric. Fruit of Callicarpa arborea (Verbenaceae), considered as edible and digestive in study area, has antiviral property because of its luteolin (Cheng Ma et al. 2002). Bark of Bauhinia vahlii (Fabaceae) is used in cuts, wounds, and fractures and this is substantiated by quercetin and betulin of the plant which are respectively anti-infectivity (Cowan 1999) and anti-inflammatory in properties (Mukherjee et al. 1997).

which plants are most likely to be useful in treatment of diseases. Despite the high potential plants have as sources of new antimicrobial agents, they may soon disappear because of over-population, indiscriminate exploitation and irrational managements (Fabry et al. 1998). The environment where people learnt and experienced folklore is imperiled on account of deforestation and overexploitation (Bhattarai 1997) and acculturation and social transformation of aboriginal life (Kunwar and Bussmann 2008). It is therefore important that the age-old plant based indigenous therapy to be explored and documented properly for future uses before it is lost. Significant corroboration of pharmacological activity gives the claims by traditional healers a significantly high credibility albeit with varying degrees of modifications. Some plants that were thought to be effective in ethnopharmacology were ineffective while pursuing their comparative assessment with phytochemical findings, as a result. Several instances are rational behind a certain function of a phytomolecule. Such species can be reevaluated in the fields for their effect therefore further research is imperative. REFERENCES Acharya KP, Acharya M (2010) Traditional knowledge on medicinal plants used for the treatment of livestock diseases in Sardikhola VDC, Kaski, Pokhara, Nepal. Journal of Medicinal Plant Research 4 (2), 235-239 Ahmed S, Reza MS, Jabbar A (1994) Antimicrobial activity of Cynodon dactylon. Fitoterapia 65, 463-464 Ajaiyeoba EO, Oladepo O, Fawole OI, Bolaji OM, Akinboye DO, Ogundahunsi OAT, Falade CO, Gbotosho GO, Itiola OA, Happi TC, Ebong OO, Ononiwu IM, Osowole OS, Oduola OO, Ashidi JS, Odoula AMJ (2003) Cultural categorization of febrile illness in correlation with herbal remedies used for treatment in southwestern Nigeria. Journal of Ethnopharmacology 85, 179-185 Akah PA (1988) Haemostatic activity of aqueous leaf extracts of Ageratum conyzoides L. International Journal of Crude Drug Research 26, 97 Amatya MP (1994) Anti-inflammatory activity of Euphorbia royleana. In: Proceeding of II National Conference on Science and Technology, National Academy of Science and Technology, Kathmandu, Nepal, pp 813-815 Anand R, Patnaik GK, Roy K, Bhaduri AP (1995) Antioxaluric and anticalciuric activity of lupeol derivatives. Indian Journal of Pharmacology 27, 265268 Anonymous (1948) Report of the Committee on Indigenous System of Medicine (Vol I), Ministry of Health, Government of India, New Delhi, 254 pp Antoun MD, Ramos Z, Vazques J, Oquendo I, Proctor GR, Gerena L, Franzblau SG (2001) Evaluation of the flora of Puerto Rico for in vitro antiplasmodial and antimycobacterial activities. Phytotherapy Research 15, 638642 Arias BA, Laca LR (2005) Pharmacological properties of citrus and their ancient and medieval uses in the Mediterranean region. Journal of Ethnopharmacology 97, 89-95 Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, Tripathi PC, Seal T, Mukherjee B (2003) Screening of antioxidant activity of three Indian medicinal plants; traditionally used for management of neurodegenerative diseases. Journal of Ethnopharmacology 84, 131-138 Ayyanar M, Ignacimuthu S (2005) Traditional knowledge of Kani tribals in Kouthalai of Tirunelvelli hills, Tamilnadu, India. Journal of Ethnopharmacology 102, 246-255 Badam L, Bedekar SS, Sonawane KB, Joshi SP (2002) In vitro antiviral activity of bael (Aegle marmelos Corr) upon human coxsackieviruses B1-B6. Journal of Communicable Diseases 34, 88-99 Banerjee S, Bandhopadhyay SK (1991) Further studies on the anti-inflammatory activities of Ricinus communis in albino rats. Indian Journal of Pharmacology 22, 149-152 Bani S, Chand D, Suri KA, Suri OP, Sharma OP (1996) Anti-inflammatory effects of an ethyl acetate extract of Euphorbia royleana. Phytotherapy Research 10, 285-291 Barbour EK, Sharif MA, Sagherian VK, Habre AN, Talhouk RS, Talhouk SN (2004) Screening of selected indigenous plants of Lebanon for antimicrobial activity. Journal of Ethnopharmacology 93, 1-7 Bernard C (1966) Physiologische Untersuchungen uber einige amerikanische Gifte. In: Das C, Bernard C, Mani N (Eds) Ausgewahlte Physiologische Schriften, Huber Verlag. Bern. [frz. orig. 1864], pp 84-133 Berne RM (1980) The role of adenosine in the regulation of coronary blood flow. Cancer Research 47, 807-813 Bhattarai NK (1992) Medical ethnobotany in the Karnali zone, western Nepal. Economic Botany 46, 257-261 Bhattarai NK (1993) Medical ethnobotany in the Rapti zone, western Nepal. Fitoterapia 64, 483-493 Bhattarai NK (1997) Folk medicinal uses of indigenous aromatic plants of

Discrepancies between indigenous and pharmacological uses Antiimplantation and early abortifacient activities of Rubus species were denoted (Dhanabal et al. 2000). The results are in agreement with the traditional use of this plant as abortifacient by the tribal of Nilgiri, India but such folklore was not observed in study area. Rubus root juice was given for relieving fever, diarrhea and dysentery in far-western Nepal. Caesalpinia bonduc has been cited as a cure for cutaneous eruptions and stomachache (Kerharo and Adams 1974) but we observed only its usage as fish stupefying. Rhizome juice of Cynodon dactylon (Poaceae) possesses antiviral pro-perty (Foster and Duke 2000) and its aqueous extract has anti-inflammatory, diuretic, anti-emetic effects (Ahmed et al. 1994) and is useful in treating dysentery, dropsy and secondary syphilis (Chopra and Handa 1982). The ethanolic extracts of the plant showed antioxidant activity (Auddy et al. 2003). However, the plant rhizome paste was recognized only for sprain and its grinded inflorescence was applied for earache by local people in farwest Nepal. Several instances were rational behind a certain function of a chemical constituents of a species therefore further research is imperative to delve the actual medicinal effect of a species against particular diseases. CONCLUSION Plant resources have been used as immediate and ultimate ingredients for therapies and the indigenous therapies have been employed and appreciated by local populace for centuries. Because of prolong existence and uses, the therapies have become an integral part of the culture. Knowledge base for therapies were also stemmed from customs, livelihood strategies and available nearby resources. The ethnomedicinal and ethnopharmacological information gleaned from the present research provided the potential to identify


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Ethnomedicinal Uses of Plant Resources of the Haigad Watershed in Kumaun Himalaya, India Mukesh Joshi1 • Munesh Kumar1* • Rainer W. Bussmann2 1 Department of Forestry, HNB Garhwal University, Srinagar Garhwal, Uttarakhand, India 2 William L. Brown Center, Missouri Botanical Garden, P.O. Box 299, Saint Louis, MO 63166-0299, USA Corresponding author: * [email protected]

ABSTRACT The present study was carried out in the Haigad watershed of Kumaun Himalaya. A total of 32 medicinal plant species belonging to 26 families were recorded. A major proportion of species were in forested landscape (62%) and the rest in cultural landscape (38%) of the watershed. The plants used for medicinal purposes in the local health traditions are gradually becoming extinct due to developmental activities, population explosion and other anthropogenic reasons. To avoid overexploitation and promote sustainable use, rapid conservation efforts are needed. Farmers should be involved in the cultivation of medicinal plants emphasizing suitable production methods, especially on barren and fallow land.

_____________________________________________________________________________________________________________ Keywords: ethnomedicine, plant resources, watershed, Himalaya

INTRODUCTION There are over 400 different tribal and other ethnic groups in India (Jain 1991) constituting about 7.5% of India’s population. Plants have been used in traditional medicine for several thousands of years (Abu-Rabia 2005). 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 and there are many reports on the use of plants in traditional healing by either tribal people or indigenous communities of India (Maruthi 2000; Chhetri et al. 2005). The knowledge of medicinal plants has accumulated over the course of many centuries and has been documented in different medicinal systems such as Ayurveda, Unani and Siddha. In India, it is reported that traditional healers use 2500 plant species while 100 species of plants serve as regular sources of medicine (Pei 2001). Documenting the indigenous knowledge through ethnobotanical studies is important for the conservation and utilization of biological resources. The Himalayan regions are particularly rich in biodiversity because of their varied geographical, physiographical, topographical, climatic and ecological zones (Khoshoo 1992). Plant resources have been in use by different communities for various purposes such as food, fodder, fuel, medicine, religious and other purposes (Badhwar and Fernandez 1964; Pangtey et al. 1982; Negi 1988; Negi and Gaur 1994). Many plants have become associated with environments close to human dwellings, such as homes or kitchen gardens (Borthakur et al. 1998). Due to cultural and ethnic diversity in different biogeographic provinces of the region the traditional knowledge base varies considerably. Based on the use of local natural resources such knowledge/ practices are closely linked to the ecological and socioeconomic conditions of the region. The Indian Central Himalaya covers an area of 51,125 km2. The indigenous knowledge of the region is unique. Such knowledge is widely followed and relied upon throughout this region, particularly by people of remote areas. Increasing population pressure, and the spread of global market economics and consumerism have already

brought profound changes to the region, and its inhabitants are gradually changing their traditional way of life (Rawat et al. 2000). However, with renewed global interest in traditional medicine and the increasing demand for plant products, the documentation of such knowledge is necessary to maintain the cultural view point as well as to establish a sound scientific basis of the efficacy of traditional medicine, and for the conservation of important species. This study attempts to identify and document the existing important ethnomedicinal plants used by the people of the Haigad watershed in Kumaun Himalaya (Fig. 1). MATERIALS AND METHODS The study was carried out in the Haigad watershed, which is located in the Lesser Himalayan belt. The area of the watershed (9.5 km2) includes four villages, Hawil-Kulwan, Jyuna Estate, Laskar Khet and Pinglon. The watershed represents a typical, densely populated mountainous ecosystem. With an altitudinal range of 1160 to 2338 m, this watershed can serve as an interesting example for a large part of the Central Himalayan Range, because most of the rural population is concentrated in this altitudinal zone of the Central Himalaya. Due to the high anthropogenic impacts, this altitudinal zone is popularly referred as the “problem zone”. About 47.3% of the area of the watershed is under forest and administered by the State Forest Department, 1.0% under community forest while 51.7% are agricultural land (Joshi et al. 2009). Extensive field surveys were conducted in and around the Haigad watershed to collect ethnomedicinal information and indigenous knowledge on plants from natural habitats (forest) as well as from the home gardens (cultivated landscape). The survey involved collection of plant specimens during the different seasons of a year. Ethnomedicinal information of plants on the villages at different altitudes was collected using direct interviews with the adult laypeople (men and women), as well as local vaidyas (healers) of the villages who were randomly selected and interviewed after obtaining prior informed consent. Of total existing households of the villages, the 10% households sampling survey was done randomly using well structured questionnaire. Each selected household was personally interviewed to collect information which was also

Received: 25 February, 2009. Accepted: 15 April, 2010.


Research Note


Location of Haigad Watershed

Alder forest

Fig. 1 Location of the study area.

from forested landscape (62%) and the rest from the cultural landscape (38%). Interestingly, the majority of tree species (67%) were recorded from the cultural areas, while a higher proportion of shrubs and herbs were found in the forest (88 and 75% for shrubs and herbs, respectively). The traditional home gardens harbor a rich mixture of often otherwise uncommon, annual or perennial species grown in association (Agnihotri et al. 2004). Along an altitudinal gradient (300 to 2400 m asl) in Garhwal of Central Himalaya, Kumar et al. (2008) recorded a total of 61 plants species used by the local inhabitants for curing various diseases (e.g., dysentery, cold, scabies, rheumatism, cholera, malarial fever, etc.). Similar studies on ethnomedicinal plants of Uttarakhand have been carried out for the Jaunsari tribals and a total of 66 plant species were recorded, including 9 trees, 11 shrubs and 46 herbs (Bhatt and Negi 2006). In the urban environment of Varanasi, Uttar Pradesh, 72 ethnomedicinal plants were recorded (Verma et al. 2007). Acharya and Rokaya (2005) conducted a study in Nepal and concluded that in spite of the establishment of modern western styled medical centers, traditional practices on the uses of medicinal plants will continue to play a significant role in the socio-cultural life of people. The research in ethnomedical practices can lead to add the knowledge on new and less known medicinal plants. Therefore, it is essential to conserve such knowledge hidden in the different parts of the country and people should be encouraged to use herbal medicines for the ever increasing requirements of human health care which has less or no side effects. The medicinal plant resources used in the local health

verified with relevant existing ethnobotanical literature. The information was collected from both male and female adults approximated uniform ratio of male and female were taken to avoid error between the opinion. The youth have not given relevant information of the ethnomedicinal plants therefore only opinions of adult peoples have been considered. Personal field observations of ethnomedicinal uses of plants for curing particular diseases were carried out in each village and the results were discussed with the villagers involved. The gained information was compared between the villages and to available scientific literature. A survey of the vegetation was also conducted as part of an ecological study of the region.

RESULTS AND DISCUSSION The survey of the available literature reveals that about 2500 species from the Indian sub-continent have local medicinal use for commerce and trade, especially for the pharmaceutical industry (Singh et al. 2005). Out of these, 1745 species are from the Indian Himalayan region and most of these are found in Uttarakhand (Kirtikar and Basu 1933; Nadkarni 1954; Chopra et al. 1956). The state of Uttarakhand is a part of north-western Himalaya and has a dense vegetation cover (65%) harboring a vast range of important medicinal plants (Singh et al. 2005). People in this region are partially or completely dependent on forest resources e.g. for medicine, food, and fuel. In the present study a total of 32 medicinally important plant species from 26 families in the watershed area were found (Table 1). 12 species each were trees and herbs and 8 shrubs. A major proportion of the species were recorded


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Ethnomedicinal uses of plant resources in Kumaun Himalaya. Joshi et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 1 Ethnomedicinal uses of plant resources of Haigad watershed in Kumaun Himalaya Botanical Name Local name Family Habitat Part used Trees Rhododendron arboreum Burans Ericaceae F Flower Sm. Bauhinia variegata L. Quairal Fabaceae HG Bark Cinnamomum tamala Nees Tejpat Lauraceae F Bark, leaves ex Eberm. Ficus palmata Forsk Bedu Moraceae F Fruit, twigs

Uses Flower juice used for heart patients as tonic, in diarrhea and dysentery. Astringent, tonic useful in ulcers and skin diseases. Leaves are carminative and are used in colic and diarrhea. Leaves and bark are also used as condiment. Used in the treatment of lung and bladder diseases, milky juice used in skin diseases Fruit used for the treatment of afts. The juice of the root is used in bladder ailments Used as laxative.

Ficus semicordata Buch.Ham ex J.E Smith Ficus roxburghii Wallich ex Miq. Myrica esculenta Buch.Ham.

Kheun

Moraceae

HG

Fruit, Root

Timil

Moraceae

HG

Fruit

Kaphal

Myricaeae

F

Fruit, bark

Pinus roxburghii Sargent Punica granatum L. Pyrus pashia Buch.-Ham. ex D.Don Sapindus mukorossi Gaertner

Chir Anar Mehal

Pinaceae Punicaeae Rosaceae

F HG HG

Aerial parts Fruit, flower Bark, fruit

Ritha

Sapindaceae

HG

Fruit, seed

Tusar

Urticacea

F

Bark

Bark is used in the treatment of bone fractures.

Kilmore

Berberidaceae

F

Fruit, root

Berberis lyceum Royle Tinospora cordifolia (Willd.).f. & Thomson

Kilmore Giloy

Berberidaceae F Menispermaceae HG

Root, stem Aerial parts

Crataegus crenulata Roxb. Solanum indicum L.

Ghingaroo Banbhatuja

Rosaceae Solanaceae

F

Fruit, leave Root, fruit

Datura stramonium L

Dhatura

Solanaceae

F

Leaves

Urtica dioica L.

Bichhu

Urticaceae

F

Whole plant

Fruits are a mild laxative for children. Root and bark used as astringent, stomatic, diaphoretic, and curative of piles. Used in the treatment of eye problems and piles. Used for the treatment of debility, dyspepsia, fevers and urinary disease. Leaf decoction is used for the treatment of gout. Dried powered fruit used for jaundice and rheumatism. Used as heart tonic. Root is used for cough, catarrhal affections, colic and nasal ulcers. Fruits are laxative. Leaves are applied to boils and sores. Flower is used for earache. Fruit juice used for dandruff control and hair loss. Plant is diuretic, anti-rheumatic, astringent, anthelmintic, used for Jaundice, hemorrhages form the kidney, nephritic troubles and sciatica.

Brahmi

Apiaceae

HG

Leaves

Shrubs Debregeasia longifolia (Burm. f.) Wedd. Berberis asiatica L.

Herbs Centella asiatica L.

Fruit edible used as a source of vitamin C. Bark decoction used for asthma, chronic bronchitis, diarrhea. Bark chewed to relive toothache. Resin used for treatment of cracked toes. Dried rind is chewed in cough, fruit juice as tonic. Fruit is astringent, laxative. Bark is anthelmintic, febrifuge. Flower to stop nosebleeds. Fruit is expectorant, antiepileptic, emetic. Seed is febrifuge and used in dental caries.

Plant infusion is used in the treatment of leprosy, as alterative tonic, to increase memory, healing of wounds. Plant extract has laxative and anti-febrile properties.

Drymaria cordata (L.) Willd. Pithpapra ex Roemer & Schultes Diosorea bulbifera L. Gethi

Caryophyllaceae

F

Aerial parts

Dioscoreaceae

HG

Ocimum sanctinum L.

Tulsi

Lamiaceae

HG

Thymus serphyllum L.

Banajwain

Lamiaceae

F

Oxalis corniculata L.

Chalmori

Oxalidaceae

F

Thalictrum foliosum DC.

Mamira

Ranunculaceae

F

Fragaria vesca L.

Gand-kaphal

Rosaceae

F

Potentilla fulgens L. Berginia ligulata Wall. Engl

Bajradanti Pasanbhed

Rosaceae Saxifragaceae

F F

Valeriana wallichii (DC.) Wall. Viola conescense (Wall.) Roxb.

Shameo

Valerianaceae

F

Banafsha

Violaceae

F

Tuber, leaves Tuber is expectorant, useful in asthma, bronchitis, anti-diarrheic, for dyspepsia, urinary discharge, leucoderma, bronchitis. Leaves are febrifuge. Whole Plant Leave juice is used in catarrh, bronchitis, as expectorant and diaphoretic, and anti-periodic. Leave infusion used as a stomatic in gastric disorders of children and in hepatic affections. Root used to treat malaria. Aerial parts Anti-asthmatic, expectorant, carminative, antiseptic, anti-convulsive, for whooping cough, kidney and eye troubles, bronchitis. Aerial parts Good appetizer, astringent, cures dysentery and diarrhea, skin disease, scurvy, as diuretic, refrigerant and astringent. Whole plant Diarrhea, purgative, diuretic, febrifuge, discoloration of the skin, eye problems. Fruit, leaves Fruit is astringent and diuretic. Leave infusion is given in diarrhea and problems of urinary organs. Root Diarrhea, strengthens the gums and teeth, spasmolytic, anticancer Leaves, root Leaves are used in earache. Root is astringent, diuretic, anti-diarrheal, febrifuge, cures pulmonary affection, dissolve kidney stones. Rhizomes Used in hysteria, hypochondriasis, nervous affections, itch fever and as an incense. Whole plant Expectorant, antipyretic, diaphoretic, blood purifier, catarrhal and pulmonary troubles, treatment of skin diseases and relief of ear pain.

HG=Home Garden, F=Forest

traditions are gradually destroyed by developmental activities, population explosion and other anthropogenic impacts. In order to reverse this trend, the domestication of wild medicinal species is of high importance. Farmers should be

involved in the cultivation of medicinal plants at least on their barren and fallow land. This would augment their income and in turn help in the conservation of the species. Appropriate research should be carried out in institutions in


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the hills to develop agro-techniques for the cultivation of medicinal plants on priority basis (Chettri et al. 2005).

of Agroforestry in Central Himalaya: A case study from Haigad watershed, India. Trees, Forests, Livelihoods (Submitted for publication) Khoshoo TN (1992) Plant diversity in Himalaya. Conservation and utilization. GB. Pant memorial Lecture II, GB Pant Institute of Himalayan Environment and Development, Koshi-Katarmal, Almora, India, 129 pp Kirtikar KR, Basu BD (1933) Indian Medicinal Plants (Vols II, 2nd Edn), Lalit Mohan Basu, Allahabad, India, pp 1478-1480 Kumar M, Bussmann RW, Joshi M, Gusain M (2008) Ethnomedicinal uses of plants close to rural habitation in Garhwal Himalaya. Ethnobotany Research Application in press Maruthi KR, Krishna V, Manjunatha BK, Nagaraja VP (2000) Traditional medicinal plants of Davanagere district, Karnataka with reference to cure skin diseases. Environment and Ecology 18, 441-446 Nadkarni AK (1954) Indian Materea Medica (Vol 1, 3rd Edn), Popular Book Depot, Bombay, pp 1292-1294 Negi KS (1988) Some little known wild edible plants of U.P hills. Journal of Economic and Taxonomic Botany 12, 345-360 Negi KS, Gaur RD (1994) Principle wild food plants of western Himalaya, Uttar Pradesh, India. In: Gupta BK (Ed) Hishar Plants of Indian Subcontinent, Bishan Singh Mahendra Pal Singh, Dehradun, India, pp 1-78 Pangtey YPS, Rawat GS, Kalkoti BS (1982) Unusual and supplement and food plants of Kumaun Himalayan. Himalaya. Himalayan Research and Development 1 (1), 35-40 Pei SJ (2001) Ethnobotanical approaches of traditional medicine studies: Some experiences from Asia. Pharmaceutical Biology 39, 74-79 Rapoport EH, Raffaele E, Ghermandi L, Morgutti L (1995) Edible weeds: a scarcely used resource. Bulletin of the Ecological Society of America 76 (3), 163-166 Rawat DS, Joshi R, Joshi M (2000) Indigenous methods of hill farmers for bioresoruce utilization. Ambio 29 (6), 365-358 Singh D, Srivastava R, Khanduri VP (2005) Marketing strategies and trade of medicinal plants in Uttaranchal: Present and future prospects 131 (3), 330340 Singh HB, Arora RK (1978) Wild Edible Plants of India, ICAR, New Delhi, 88 pp Verma KA, Kumar M, Bussmann RW (2007) Medicinal plants in an urban environment: the medicinal flora of Banares Hindu University, Varanasi, Uttar Pradesh. Journal of Ethnobiology and Ethnomedicine 3, 35

ACKNOWLEDGEMENTS The authors are thankful to Dr. S.S. Samant and Dr. D.S. Rawat, GBPHIED, Kosi-Katarmal, Almora for identification of plants and valuable suggestions, respectively.

REFERENCES Abu-Rabia A (2005) Urinary diseases and ethnobotany among pastoral nomads in the Middle East. Journal of Ethnobiology and Ethnomedicine 1, 4 Acharya KP, Rokaya MB (2005) Ethnobotanical survey of medicinal plants traded in the streets of Kathmandu valley. Scientific World 3 (3), 44-48 Agnihotri R, Sharma S, Joshi M, Palni LMS (2004) Crop diversity in home gardens of the Central Himalaya, India. Plant Genetic Resources Newsletter 138, 23-28 Alcorn JB (1981) Huastec noncrop resource management. Human Ecology 9, 395-417 Badhwar RL, Fernandez RR (1964) Edible Wild Plants of Himalaya, Government publication, Delhi, 462 pp Bhatt VP, Negi GCS (2006) Ethnomedicinal plant resources of Jaunsari tribe of Garhwal Himalaya, Uttaranchal. Indian Journal of Traditional Knowledge 5 (3), 331-335 Borthakur SK, Sharma TR, Natha KK, Deka P (1998) The house gardens of Assam: a traditional Indian experience of management and conservation of biodiversity. Journal of Ethnobotany 10, 32-37 Bye RA (1979) Incipient domestication of mustards in north-west Mexico. Kira 44, 237-256 Chhetri DR, Parajuli P, Subba GC (2005) Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India. Journal of Ethnopharmacology 99, 199-202 Chopra RN, Nayar SL, Chopra IC (1956) Glossary of Indian Medicinal Plants, Publication and Information Department, CSIR, New Delhi, 329 pp Jain SK (1991) Dictionary of Indian Folk Medicine and Ethnobotany, Deep Publications, Paschim Vihar, New Delhi, 311 pp Joshi M, Sharma S, Rawat DS, Palni LMS (2009) Structure and functioning


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Conservation of Phyto-diversity of Parvati Valley in Northwestern Himalayas of Himachal Pradesh-India Parveen Kumar Sharma1* • N. S. Chauhan2 • Brij Lal3 • Amjad M. Husaini4 • Jaime A. Teixeira da Silva5 • Punam1 1 KVK, Lahaul and Spiti, Camp Office at CSKHPKV, Palampur, Himachal Pradesh-176062, India 2 Department of Forest Products, Dr. Y. S. Parmar University of Horticulture and Forestry Nauni, Solan, Himachal Pradesh-173230, India 3 Institute of Himalayan Bioresource and Technology, Palampur, Himachal Pradesh- 176 062, India 4 Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry Nauni, Solan, Himachal Pradesh-173230, India 5 Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan Corresponding author: * [email protected]

ABSTRACT This study provides information about the traditional indigenous uses of plants by the inhabitants of the Parvati Valley of Kullu district in the western Himalayas of India. Since no published literature from the past 10 years exists, an ethnobotanical survey was conducted among the ethnic groups of the Parvati valley and first hand information on these plant species was recorded. A total of 266 species belonging to 180 genera and 71 families (including 44 species as recorded for the first time in the area) were collected. Out of these, 223 species within 152 genera of 61 families belong to dicots; 31 species and 22 genera under 7 families belong to monocots and 10 species with 6 genera in 3 families belong to gymnosperms.

_____________________________________________________________________________________________________________ Keywords: biodiversity, Himalayan medicinal plants, plant resources

INTRODUCTION The Himalayas is a biodiversity hotspot and a storehouse of endemic medicinal plants, which grow in valleys, hills, terraces and on the exposed flat mountain tops and valleys (Myers et. al. 2000). The famous valleys like Kashmir in Jammu and Kashmir, and Lahaul-Spiti, Kinnaur, Kangra, and Kullu valleys of Himachal Pradesh are located in the western Himalayan region and are well-known for their scenic beauty. Parvati valley is among such beautiful but lesser known valleys and falls under the geographical jurisdiction of Kullu district (31°2021-32°250 N and 76°563077°5220 E) in the state of Himachal Pradesh, India. The valley is situated in the south-east of Kullu district within the Sino-Himalayan subzone of the Boreal biogeographic zone (Khoshoo 1993). The valley is rich in natural resources like flora, fauna, minerals, perennial sources of water and many hot springs. Due to a wide range in altitudinal variations (1100-5500 m), the Parvati valley harbors a variety of natural flora comprising subtropical to temperate alpine floral elements. The climate of the study area is generally cool and dry. Snowfall is generally received during the period from November to March on the higher reaches. Forests occupy a prominent place in the economy of the Kullu district and extensive tracts of forests exist throughout the district. The reserve forests spread over an area of 15618 ha, while the protected forests constitute 193,495 ha of land. Unclassified forests account for 146,580 ha. The valley is also rich in wildlife. A sanctuary named Kanawar has been established in the valley for the protection and conservation of wildlife (Anonymous 1992). The forests of Kullu district are rich in various kinds of medicinal herbs like Karu, Dhoop, Muskwala and Kakarsingi. Mushrooms, especially ‘guchi’, are also readily available and extracted in large quantity. Deodar attains considerable dimensions in the upper Beas and Parvati valleys. The Kullu district in its present form constitutes the central part of Himachal Pradesh. Lahaul and Spiti district

surrounds it from north and east, while Shimla and Kinnaur districts from the south and southeast, and Kangra and Mandi districts on the west and southwest. Parvati valley starts from Bhuntar where Beas and Parvati rivers have their confluence, and stretches from Bhuntar to Mantalai in an area of 80 km (Janartha 2000). Due to the remoteness of the area and lack of modern medical facilities, the local people still depend upon local traditional healers, called Vaids, who are considered as experts in medicinal uses of plants. Parvati valley is inhabited by different communities i.e. native people like Malanis, Kulluvis and migratory people like Gaddis and Gujjars, who supplement their earning by selling medicinal and aromatic plants. Because of the richness in plant resources, there is a need to harness their potential for life-saving drugs and day to day medicines, so that the raw material is available on a sustainable basis for the service of mankind. The history of plant exploration of the Parvati valley is quite old and several teams have made significant contributions to the botany of the Parvati valley (Jain and Bhardwaj 1951; Puri 1952; Uniyal and Chauhan 1972; Chowdhery and Wadhwa 1984; Badola 1998; Singh 1999; Dhaliwal and Sharma 1999; Singh and Rawat 2000). However, the valley remained unexplored from the point of view of ethnobotanical studies and could not be ignored any further as the rapid increase in anthropogenic activities like the construction of a hydroelectric project, roads, tunnels, housing colonies, etc. is causing an unquantifiable loss of genetic resources. With this in mind, the present investigation was carried out in the Parvati valley of Kullu district, Himachal Pradesh. The inventorization and documentation of these plant resources will be useful in establishing future strategies for their conservation and management. MATERIALS AND METHODS Extensive field surveys were carried out in various parts of the study area. Starting from the lower elevation i.e. Bhunter, Jari,

Received: 8 January, 2010. Accepted: 15 November, 2010.


Research Note


Table 1A Medicinal and aromatic plants of commercial importance from the study area (Parvati Valley) and their economic uses. Grey indicates medicinally important plants. Underlined indicates plants with ethnobotanical importance. Name of species Common names Official parts used Economic uses (based on their commercial importance) Abies spectabilis Tosh Leaves Considered carminative used for cough and phthisis; cones yield a violet coloured dye. Tree yields a white resin. Acer capadcocicum Dhadonga Leaves, bark Leaves are used to raise blisters. Bark is used as an astringent. Achillea millefolium Biranjasif, Whole herb Bitterish, pungent and aromatic aerial parts are used as a flavouring agent. The Millefoeil herb is also substituted for hops in the preparation of beer. Decoction of leaves is carminative and stimulant. Herb is considered astringent, tonic, diaphoretic, vulnerary and styptic. Achyranthes bidentata Puthkanda Whole herb From the seeds, two saponins; saponin A and saponin B have been isolated, which have shown cardiotonic activity. Decoction of the entire plant, Panchang is used in asthma and the root of the plant is used in snakebites. Aconitum Atish, Patish Roots The alkaloids isolated from the roots include astine, heteratisine, heterophyllistine, heterophyllum heterophylline, atidine and hetidine. The alkaloid content is 0.79%. Roots used for hysteria, throat infections, dyspepsia and vomiting, abdominal pain and diabetes. Aconitum violaceum Kali-Patish Roots Roots are reported to contain the alkaloid Indaconitine and used as a tonic. Also used as a substitute for Aconitum heterophyllum. Acorus calamus Bach Rhizomes The dry rhizomes contain 2-3% of yellow bitter aromatic volatile oil. The roots also contain a glucoside, acorin, calamene, tannin, mucilage, starch, vitamin C, fatty acids, sugar and calcium oxalate. Essential oils finds used in insecticides/pesticide, cosmetics and perfumery industry. Aesculus indica Bankhor, Indian Seeds The seeds contain a mixture of saponins, one of, which is described as aescine, horse-chestnut which easily crystallizes. Also contains flavonoid glycosides, aesculine, albumin and fatty oils. Ajuga bracteosa Nilkandhi Whole herb Contains glycosides and tanins. Herb is astringent febrifuge, apparent, tonic and diuretic. Used in gout, rheumatism, palsy and amenorrhoea. Anaphalis contorta Rui-Ghass Whole herb Herb yields an essential oil having anti-bacterial properties. Anemone obtusiloba Laljari Rootstocks Rootstock of the plant is used for concussions. The oil extracted from the seeds is used in rheumatism. Anemone rivularis Laljari Rootstocks Extract gave positive test for saponin. Arctium lappa Jungli-kuth, Roots It has diuretic properties and has been used for cutaneous eruptions, rheumatism, burdock cytitis, gout and specifically for eczema and psoriasis. The plant extract has been found to cause sharp, long lasting reduction of blood sugar within increase in carbohydrate tolerance and less toxicity. Arisaema tortuosum Samp-ki-Kumb Tubers Tubers are used as insecticides. Seeds are cooked like vegetable and eaten. Arnebia benthami Ratanjot Roots The roots are considered expectorant and used for cardiac disorders. Aqueous extracts, syrup and jam prepared from the flowering shoots are considered useful in disease of the tongue, throat and are also useful in fever. Used as a colouring matter in hair oils, cookeries and for dyeing of silk. Root is frequently used as an antiseptic and antibiotic. Artemisia roxburghiana Kundia Whole plant Leaves and flowering tops yield an essential oil having thujone-like flavour. A. vulgaris Nagdana Whole plant Leaves contain essential oil up to 0.35%. Infusion of leaves is given in asthma, nervous and spasmodic affections. Roots are used as tonic and antiseptic. Asclepias curassavica Kaktundi Roots, leaves Roots are emetic and cathartic. Used in piles and gonorrhoea. Juice from the leaves is anthelmintic, antidysentric and also used against cancer. Latex is used to remove warts and corns. Plant is used as substitute/adulterant for Ipecac (Cephaelis ipecacuanna Tussac) and as a fish poison. Asparagus filicinus Satavar Tuberous roots Root contains asparagine, saponin. Fruits contain diosgenin. Root is used as appetizing, diuretic, aphrodisiac, laxative, astringent and is useful in dysentery, diarrhoea; throat complaints and leprosy. It is an ingredient of GERIFORTE used against fatigue and senile pruritus. Also used as demulcent in veterinary medicines. Aster mollusculus Roots Used for cough and pulmonary affections. Also used in malarial fever and haemorrhage. Atropa acuminata Indian-belladona Roots, leaves Indian belladona is used in India for the manufacture of tinctures, plasters etc. Ethyl alcohol extract (50%) of leaves is antiprotozoal, antiviral and hypoglycaemic. Atropince, hyocyamine, hyoscine are the most important alkaloids present in leaves and roots. Seeds contain fatty oil (25%) and also essential oil. Berberis aristata Daru-haldi Roots, fruits, stem Dried stems are used as bitter tonic for intermittent fevers. The dried fruits are edible. Root-bark contains principle alkaloid berberine. Roots and stems yield a yellow die. The fruits contain malic acid, citric acid and tannins. The extract from root-bark is known as Rasount. Bergenia ciliata Pashan-bhed Rhizomes Rhizomes are astringent, diuretic, antiscorbutic, and laxative, used in diarrhea, spleen enlargement, renal and pulmonary affections. Rhizomes yield tannin. Bergenia stracheyi Gatikpa Rhizomes Rhizomes and roots are bitter, astringent, diuretic aphrodisiac, tonic, also used in fever and applied to boils and ophthalmia. Rhizomes contain gallic acid, tannic acid, glucoside, mucilage, wax, starch, calcium oxalate and mineral salts. Betula utilis Bhoj-patra Papery bark (Bhojpatra) The plant contains betulin, lupeol, olenolic acid and acetyle oleolic acid in addition and fungal growth to leucocyanadin in the outer bark and polymeric anthocyanidins in the inner bark. (Bhurja-granthi) Infusion of the bark is aromatic, antiseptic and used as a carminative. Bistorta affinis Sarbguni Whole plant Plant is used as an astringent and is useful in curing diarrhoea. Bistorta amplexicaulis Sarbguni Root stock Rootstock constitutes a drug Anjubar, used medicinally both in Unani and Ayurvedic system of medicine. Also contains tannin.


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Traditional therapeutic uses of plant diversity of Parvati Valley. Sharma et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 1A (Cont.) Name of species Boenninghausenia albiflora Bupleurum candolli Caltha palustris

Common names Pissu-mar-buti

Official parts used Whole plant

Kaligewar Marsh-marigold

Whole plant Whole plant

Cannabis sativa

Chara, Bhang, Ganja

Whole plant

Cedrus deodara

Devdar

Wood and oil

Chenopodium album

Bathu

Whole herb

Chenopodium foliolosum

Bathu

Whole herb

Cissampelos pareira

Batindu, Patindu

Stems, roots, leaves

Codonopsis ovata Corydalis govaniana

SardandaSardandi Bhutjata

Roots and leaves Root and juice

Corylus colurna Cotoneaster microphylla Cyathula capitata

Bhutia-badam Riu

Nuts (seeds) Stolons

Silath

Whole plant

Cyathula tomentosa Dactylorhiza hatagirea

Silath Salam-panja, Hatpanja

Roots The roots

Delphinium denudatum

Salyan

Leaves

Delphinium vestitum

Nirbishi

Whole plant

Desmodium tiliaefolium Kathi

Roots and leaves

Dicliptera bupleuroides Ludra-buti Dioscorea deltoidea Singli-mingli

Whole herb Rhizomes

Echinops niveus

Oont-kandara

Roots

Elscholtzia fruticosa Elscholtzia strobilifera Euphorbia cognata Fagopyrum esculentum

Pothi Rangchari Dudhla Buckwheat

Leaves and fruits Leaves Roots Whole plant

Fragaria vesca

Wild strawberry

Roots

Fritillaria cirrhosa Hadjod Geranium wallichianum Ratanjot

Corms Rootstock

Geum elatum Habenaria intermedia Habenaria pectinata Hedychium acuminatum

Masreen Ridhi-Vridhi Ridhi-Vridhi Kapur-Kachri

Whole herb Tubers Tubers Rhizomes

Heracleum candicans

Patrala

Roots

Economic uses (based on their commercial importance) Leaves have insect repellent properties. Extract from the herb has shown Chemosterilant against harmful insects. Plant is a source of rutin, which is used as an anticoagulant. Plant is considered poisonous. Root contains important Helleborin and Veratrin contents. The flowering buds are also kept in vinegar and used as cappers. Source of hemp fibre and also of narcotics bhang, charas and ganja. Dried flowering, tops of female plants are used as sedative and analgesic and narcotic. Seeds are source of hemp seed oil, used in paints, varnishes and soaps. Wood oil contains oleo-resin and essential oil while the needles contain ascorbic acid. The wood is carminative, diaphoretic and diuretic. The tar is used as alterative and given in chronic skin diseases. In large doses, it is used in leprosy. Also applied externally to ulcers. Used as a pot-herb and accredited in the laxative and anthelmintic properties. Also yields an essential oil. The plant is an anthelmintic and its oil is used in medicines. The oil is effective against many forms of intestinal parasites. Shoots and roots extract has shown nematicidal properties. The alkaloids isoquinoline, pelosine and berberine are present in roots. Also contains reserpine and cissampeline. The root is regarded as anthelmintic and antidote to poison. Useful in asthma, cold and cough and inflammation of kidney and bladder. Roots and leaves are used for ulcers, bruises and wounds. The root is considered tonic, diuretic, alterative and antiperiodic. It is prescribed in syphilitic, scrofulose and cutaneous infections. Nuts (seeds) are edible and regarded as tonic. Used as an astringent. Twigs used for making baskets. Plant is a source of asterone and showed moulting hormone activity in Calliphor bioassay. Decoction used in dysentery. Also used for skin complaints. The roots are used as a farinaceous food, nervine tonic and aphrodisiac, Mucilage jelly is nutritious and useful in diarhoea, dysentery and chronic fevers. In Unani system of medicines, it is used in seminal debility, chronic diarrhoea and general weakness in debilitated women after delivery. Juice of leaves used to destroy ticks, regarded as cardiac and respiratory depressant. Plant is used for cardiac ailments and as a respiratory depressant. Leaves are poisonous to goats. Leaves lopped for fodder. Roots carminative, tonic and diuretic, used in bilious complaints. Used as a tonic. A rhizome of good quality is reported to contain from 4-8% of diosgenin content, which is used in the partial synthesis of modern drugs like cortisone and other steroids. Being rich in saponin, the rhizome are used for washing silk, wool and hair and also in dyeing. They are reported to kill lice. Plant contents are used in manufacturing tablets and injections for the uses in modern medicines including birth control pills. Plant is diuretic, nerve tonic, and used in cough, indigestion and ophthalmia. Powdered roots are applied to wounds in cattle to destroy maggots. Fruiting tops and leaves yield essential oil. Used for choleric diarrhoea, contains an essential oil. Juice is acidic and irritant. Roots are used for fistular sores. Important source of glucoside – rutin used in the modern medicines as an anticoagulant. Procynadins extracted from roots showed anti-bacterial and angioprotective properties. Fruit esteemed as a dessert. Used to prepare jams, jellies and syrups. Also used in ice creams, soda, beverages and strawberry wine. Leaves yield an essential oil. Leaves are also used as an astringent and diuretic. Dried corms are used in asthma, bronchitis and tuberculosis. Used as an astringent, in toothaches and eye troubles. Rootstock is sometimes substituted with those of Coptis teeta (Wallich). Roots are also used as a tanning material. Used as an astringent, in diarrhoea and dysentery. An ingredient of Ashtawarga, regarded as tonic. Tubers are regarded as tonic. Aromatic rhizomes are employed in the preparation of Abir, a fragrant; coloured powder used during holy festivals and in religious ceremonies. They are considered stomachache, carminative, stimulant and tonic. Used in dyspepsia. Yields an essential oil used in soaps, hair oils and face powders. Leaves woven into mats. Plant yields xanthotoxin, useful in the treatment of leucoderma and psoriasis. Also used in the preparations of sun tan lotions.


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Table 1A (Cont.) Name of species Hypericum choisianum

Common names Bassant

Official parts used Flowers

Impatiens urticifolia

-

Whole plant

Inula grandiflora Jasminum officinale

Poshkar White jasmine

Roots Flowers

Juglans regia

Akhrot

Leaves

Juniperus communis

Bethar, Haubar

Fruits

Juniperus macropoda

Indian juniper, Dhoop Weeping-bluejuniper, Dhoop

Wood

Jurinea dolomiaea

Dhoop

Roots

Lactuca lessertiana

-

Leaves

Leonurus cardiaca

-

Flowering- tops

Leucas lanata Litsea consimilis

Dhorighas -

Tender shoots Seeds

Malaxis muscifera Malva verticillata Meconopsis aculeata

Jeevak Laffa Himalayan-bluepoppy Bish-kandara

Tuberous roots Leaves, roots Roots

Juniperus recurva

Morina longifolia Nardostachys grandiflora

Jatamansi

Nasturtium officinale

Chuuch

Nepeta linearis Nicandra physaloides

Catmint Apple-of-Peru

Nicotiana tabacum

Ban-tambaku

Olea ferruginea

Kau

Origanum vulgare

Sathra

Orobanche cernua Osyris arborea

Ban-chai

Oxyria digyna

Amlu

Parnassia rubicola

-

Pedicularis siphponantha Phytolacca acinosa

Jharka

Wood, leaves, twigs

Economic uses (based on their commercial importance) Astringent, expectorant and diuretic, used in diarrhoea, pulmonary and urinary troubles. An oil is prepared by infusing fresh flowers which is used externally for wounds, sores, ulcers, swellings and sometimes against rheumatism and lumbago. An alcoholic extract of flower is reported to possess marked antibiotic activity against some pathogenic fungi and bacteria. Aromatic roots employed as an adulterant of kuth. Flowers are known to yield an essential oil used in the perfumery. Root extracts of the plant yield a dye. Used for ringworm. Leaves are effective to cure stomachache and toothache, when chewed. Leaves are valued for alternative properties and given in scrofula, leucorrhoea and rickers. Oil is used as a mild laxative and given in torpid lever. Decoction of the bark is used to stop mammary secretions. Also used as an astringent to check diarrhoea and mennorrhagia and as a gargle in sore throat. The dried kernel is valued in confectionery and ice cream, as an article of food. Bark is used as a dye and also for cleaning teeth. Sweet, aromatic fruits are used for flavouring gin, liqueurs and cordials; contain an essential oil fermentable sugar and fatty oil. Bark contains tannin. Needles are rich in vitamin-C. Fruits and roots yield dyes. Wood is used for making pencils, pen-holders and walking stick. Volatile oil from fruits has been used as a substitute for oil of J. communis. Wood is locally used as fuel; suitable for pencils. Wood, leaves and twigs are used as incense; smoke from green wood, however is said to be emetic. Fruit yield an essential oil. The aromatic roots are used as incense and form a chief ingredient of dhoop industry. The roots are considered stimulant and given in fever after child birth. A decoction of the root is given in colic. Aromatic oil from the roots is useful in gout and rheumatism. Leaves possess tonic and having digestive properties. Dried latex is reported to be used as substitute for opium. Flowering tops are used in medicines as diaphoretic, stomatchic, tonic and antispasmodic. Tender shoots used as a vegetable, also given for cough after frying. Seeds yield an aromatic wax, which is used for preparing candles and soap. Refined fat is a rich source of lauric acid, which may be utilized for making detergents. Fat is also used in medicines for curing rheumatism and bark is used in diarrhoea and dysentery. The leaves are used as fodder. Used as tonic and lactagogue. Roots used for whooping cough and the ash of dried leaves are used in scabies. Roots are used as narcotic.

Roots

Used as incense in the preparation of dhoop and agarbatties etc. Yields an essential oil. Roots The hairy roots contain essential oil having jatamansone, jatamansinol and jatamansin. The roots are considered as tonic, stimulant, anti-spasmodic and laxative. The roots remarkable properties to tone up the brain. Entire plant Consumed as salad. Chopped leaves incorporated in fruit and vegetable juice, cocktails, soups and biscuits. Plant also used in asthma and tuberculosis. Leaves, flowering tops The dried leaves and flowering tops yield an essential oil. Whole plant The plant possesses diuretic, anthelmintic and insecticidal properties. Used as a fly-poison. A decoction of the leaves is used for killing head lice. Leaves The leaves are used for smoking and also contain alkaloids, which are used as insecticides. The oil, obtained from the seeds, is used as an illuminant, and is also used in the manufacture of paints and varnishes. Entire plant The timber is used chiefly for tool-handles, walking sticks, toys, ploughs and boatbuildings. The fruits are edible. Leaves and bark are used as antiperiodic in fever and debility. Leaves Leaves and tops cut prior to blooming are used as a flavouring agent, origanum oil is carminative, stomachache, diuretic, diaphoretic and emmenagogue, used as a stimulant and tonic in diarrhoea and earache. Given in whooping cough and bronchitis because of its spasmolytic action. Also employed in cosmetics and soaps. Entire plant Plant is used as cure for boils in the throat of cattle. Leaves An infusion of the leaves has powerful emetic properties; the wood is used for making walking-sticks and reported to be used for adulterating sandalwood. Leaves Leaves have sorrel-like pleasantly acidic taste and consumed as a vegetable or used in salads and chutneys. Herb is regarded as antiscrobutic and refrigerant. Entire plant Decoction of plant is used as sedative in nervous palpitation and epileptic convulsions. Flowers yield a dye. Whole plant Plant is used as diuretic. Entire plant

Herb has narcotic effect. Fruits are occasionally used as a flavouring agent. Seeds yield fatty oil.


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Table 1A (Cont.) Name of species Picrorrhiza kurrooa

Common names Karu, Kutki

Official parts used Roots

Economic uses (based on their commercial importance) Constitute the drug picrorhiza, used as a substitute of Indian gentian (Gentiana kurroo) in liver problems. Contains picrorhizin, kutkin and other compounds. Pimpinella diversifolia Entire plant Carminative, roots yield essential oil. Pinus roxburghii Chirpine Oleo-resin/ Turpentine Tree is an important source of oleo-resin, which yields turpentine oil and rosin. oil Turpentine oil contains 20-30% -pinene, The turpentine oil is used in pharmaceutical preparations, perfumery industry, synthetic pine oils, disinfectants, insecticides and denaturants. The oil is valued in medicines. It is especially recommended in the treatment of gangrene of the lungs and has been found beneficial as a carminative. Pinus wallichiana Kail, blue pine Resin The yield is oleo-resin and turpentine oil is about half than that of chir pine, but the oil is of superior quality and has high -pinene contents. Plantago depressa Isabgol Herb Leaves and roots are astringent and vulnerary. Used in cough, asthma and other pulmonary diseases. Plantago major Isabgol Herb In homoeopathy, it is used in disorders of epidermis, headache, earache and toothache. Leaves and roots are also used for dyeing cotton. Pleurospermum Nesar, Lossar Whole plant The dried herb is used as a preserving agent against the attack of moth, silver fish brunonis etc. to protect woolen garments. Essential oil is of great value in perfumery industry. Podophyllum Bankakri Roots, rhizomes Constitute a compound, podophyllin, which is commonly used as a purgative; hexandrum Podophyllotoxin is the active principle. Podophyllin is an effective vermifuge. Recently it has acquired importance because of its possible use in controlling some forms of cancer. Fruits are edible. Polygonatum Salam-misri Rhizomes Valued as a salep, a strength-giving food; plant is diuretic and contain a glucoside cirrhifolium of the digitalis group. P. multiflorum Salam-misri Rhizomes Rhizomes are edible and in the powdered form, it is used for piles, tumours and inflammations. P. verticillatum Mahameda, Salam- Rhizomes Physical tonic and under the name of Mahameda, it is an ingredient of Ashtawarga, misri a principle constituent of Chyavan-prash. Polygonum plebeium Leaves Leaves are applied to swellings. Potentilla Ratanjot Rootstock Rootstock is depurative. Ash of the plant mixed with oil is applied to burns. Root atrosanghuinea yields red dye. Primula denticulata Roots Powdered roots are used for killing leeches. Also used as substitute for Senega. Prinsepia utilis Bhekhal Seed oil Oil from the seed (35-40%) pale yellow fatty oil) is used for the hydrogenation and soap making. Also possesses rubefacient properties and is applied externally in rheumatism and pains resulting from fatigue. Prunella vulgaris Ustakha-ddus Whole herb Herb is considered antiseptic, anti-rheumatic, expectorant, alternative, tonic, astringent, carminative, anti-spasmodic and stimulant. Useful in fevers and cough. Infusion is effective in haemorrhages, diarrhoea and bleeding piles. Used as a mouthwash. Applying the juice of plant, mixed with rose oil cures headache. Prunus cornuta Jamun Fruits Fruits are edible and used for brewing liquors. Kernels yield oil, used as a substitute for oil of bitter almond. Punica granatum Daru Anardana, The rind contains about 28% of gallotannic acid together with a yellow colouring pomegranate rind matter. Useful in brain affection, coughs, colds, diarrhoea and dysentery, heart tonic, stops bleeding from the nose. The fruit is a good source of sugar and vitamin C. Quercus semecarpifolia Kharsu Wood Wood is source of good charcoal Ranunculus arvensis Buttercup Whole plant Plant is used for its acrid and toxic properties. Rhamnus virgatus Pajji Fruit Fruit is valued as emetic, purgative and also used in spleen affections. Rheum australe Chuchi, Chukri Roots Used as astringent, laxative and also as tonic. The extract made out from the roots known as USHARE-REVAND is used in Unani medicines. Rheum moorcroftianum Rhubarb, Roots Roots are valued as purgative. Roots are used for dying woolen clothes (since it Revadchini contains tannins). Rhododendron Talispatra Leaves Leaves possess stimulant properties, these are aromatic and administered as an anthopogon errhine to produce sneezing. Rhododendron Buras Flowers A sub-acidic jelly or preserve is made from the petals, used in diarrhoea and arboreum dysentery. Rhododendron Kashmiri-patha Leaves, flowers Leaves are used as a nervine sedative. Also employed as incense; yield an essential companulatum oil with hypotensive, sedative and analgesic properties. Rhododendron Simrish Leaves Leaves are stimulant and yield essential oil. Used in perfume and incense. lepidotum Ricinus communis Arandi, Erand Seeds Seeds contains about 45-40% of fixed oil known is castor oil. Castor seed is poisonous and two or three seeds have been known to prove fatal. Castor oil is used as purgative. Rosa macrophylla Jungli-gulab Fruits Fruit is rich in Vitamin C. Flowers yield essential oil, used in the manufacture of perfumes. Roscoea alpina Kakoli Roots Root is used as a tonic in general debility. Beneficial in impotency, diabetes, leucorrhoea, diarrhoea and dysentery. Plant also finds use in veterinary medicines. Roscoea capitata Kakoli Roots A substitute of Safed musli in Ashtavarga and used in Chyavanprash-avleha. Roscoea purpurea Kakoli Roots Used as substitute for Safed musli. Rumex hastatus Khatti-imli Whole plant The bark of the roots is used to cure fire burns. Leaves are acidic in taste.


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Table 1A (Cont.) Name of species Rumex nepalensis

Common names Albare, Junglipalak

Official parts used Leaves

Salvia nubicola Sarcococca saligna

-

Leaves and flowers Leaves

Scutellaria angulosa

-

Entire plant

Sedum ewersii

Hiunshai

Whole plant

Selinum tenuifolium

Muramansi

Roots

Selinum vaginatum

Bhutkesi

Roots

Senecio chrysanthemoides Silene edgeworthii

-

Whole plant

-

Whole plant

Skimmia laureola

Dhoop

Leaves

Solidago virga-aurea

-

Whole plant

Sorbus mycrophylla

-

Leaves, fruits

Swertia angustifolia

Chirata

Whole plant

Swertia chirata

Chirayita

Whole herb

Swertia paniculata Swertia purpurascens Swertia racemosa Tanacetum longifolium

Chirata Chirata Chirata Langri

Whole plant Whole plant Whole plant Roots

Taraxacum officinale

Kanphool

Rhizomes

Taxus wallichiana

Talispatra, Rakhal

Entire plant

Thalictum foliolosum

Pilijari

Roots

Thymus serpyllum

Banjwain

Entire plant

Trifolium pratense

Red clover

Flowers

Trillidium govanianum

-

Roots

Valeriana hardwickii

Nihani, Tagar

Roots

Valeriana jatamansi

Nihani, Muskbala, Tagar Ban-tambaku

Roots

Verbascum thapsus

Entire plant

Economic uses (based on their commercial importance) Infusion of leaves is given in colic and applied to syphilitic ulcers. Leaves are rubbed on the affected parts for the relief from irritation caused by stinging nettle (Urtica dioica). Leaves and flowers are very aromatic and yield essential oil. Several alkaloids, isolated from the leaves induce a non-recoverable fall in blood pressure in dogs, and are toxic to paramoecia. Used as laxative, febrifuge, antispasmodic, astringent, nervine, anodyne and stomachic. Important glucosides rutin, quercitin and asbatin have been isolated from the plant. Rutin is used as anticoagulant. Roots are employed as incense. Also used as sedative. Oil from the roots showed anti-bacterial properties. Roots are used as a nervine sedative. Roots yield an essential oil having hypotensive, sedative and analgesic properties. Also employed as incense. Plant is toxic to cattle’s. Yield an essential oil, which may be found suitable as a perfumery material. Plant is used as an emollient and as fumigant. Juice of the plant is used in opthalmia. Contains saponins. Leaves are aromatic and used as an incense and flavouring agent. Yields an essential oil, a source of potential linalyl acetate and is used in the perfumery as a substitute for Petit grain oil (Citrus aurantium Linn.) Leocarpozide at 0.1 g/kg showed anti-phylogistic and analgesic activities in rats for inflammation and pains. Fruits are edible and are considered to be a very rich source of Pro-vitamin A and vitamin C. An infusion of the leaves is used as pectoral in cough and is given in diarrhoea. Infusion of plant is used as tonic and febrifuge. Plant is also used as a substitute for S. chirata, but exhibit inferior bitter tonic properties. Ophelic acid (yellowish and bitter), two bitter glucosides (chiratin and amarogentin), gentiopicrin, two yellow crystalline phenols and a new xanthone, swerchirin have been isolated from the plant. In Indian medicines, chirata is prescribed in a variety of forms and combinations in chronic fevers and anaemia. It has got the special reputation as a remedy for bronchial asthma and liver disorders. Chirata is said to be used for dyeing cotton cloth yellow and is used in the liquor industry as a bitter ingredient. Plant is used as substitute for Swertia chirata. Plant is used as substitute for Swertia chirata. Plant is used as substitute for Swertia chirata. Roots are used as incense. A gum resin Gogul is obtained, which is used as incense. Resh and dried rhizomes constitute the drug, Taraxacum, which is used as a mild laxative. Also used as a diuretic, stomachic, hepatic, stimulant and tonic. The roots and leaves are eaten as salad, used in soups and cooked as vegetable. Leaves and open flowers are used in the manufacture of beer, wines and other dietary drinks. Leaves are antipasmodic and emmenagogue, used for nervousness, hysteria and as a tithontriptic. An extract of various parts of the tree is added to hair lotions, beauty and shaving creams and dentifrices. Roots are much valued for opthalmia used as extract, decoction or powder. Also used as diuretic, purgative and bitter tonic during convalescence and atonic dyspepsia. Plant is used both for culinary and medicinal purposes. Shoots are used for flavouring. Leaves are used for the preparation of non-alcoholic beverages. Plant is bitter and posses anti-spasmodic, antiseptic, expectorant, carminative, anthelmintic and stimulant properties. Infusion of the plant is useful in the treatment of itch and eruptions of skin. Thyme oil is used in toothaches. Ethanolic extracts of the herbaceous plant are used in hair lotions. Flowers exhibit depurative, alterative and sedative properties. An extract of the flowers is used as a remedy for cancerous ulcers and corns. Roots contain Trilarin, which on hydrolysis yields 2.5% diosgenin – a corticosteroidal hormone. This hormone is used in preparations like sex hormones, birth control and regularization of menstrual flow. Same properties and uses at those of Valeriana jatamansi and are therefore a good substitute of the drug valeriana. Roots are known as Indian valerian, yields an essential oil, used as an adjunct to certain flavours in tobacco, honey etc. also used as heart tonic and stimulant. Leaves and fruits are used in diarrhoea and pulmonary disease of cattle. Leaves are also used as demulcent, in pectoral complaints and as local application in piles, sunburns and inflammation of mucus membrane. Dried leaves are smoked, relieve irritation. Decoction of the leaves is used as a heart stimulant. Roots show febrifuge properties. Seeds are narcotic. The herb yields oil used as a bactericide. The oil is used as a suitable remedy for frostbite, piles and bruises in Europe.


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Table 1A (Cont.) Name of species Viola biflora

Common names Pila-banaksha

Official parts used Whole herb

Viola serpens

Banafsha, Banaksha

Flowers, roots

Vitex negundo

Bana

Leaves

Withania somnifera

Ashwa-gandha, Ashgandh

Roots

Woodfordia fruticosa

Dhai

Flowers

Xanthium strumarium

Banokra

Entire plant

Zehneria umbellata

Jungli-kheera

Fruits

Economic uses (based on their commercial importance) Herb is used as one of the adulterant of Viola odorata. Roots are emetic. Flowers are known to posses emollient, pectoral and diaphoretic properties. Leaves are used as an emollient and laxative. Constitute a part of commercial Banafsha and is considered to be posses medicinal properties more or less similar of V. odorata. A decoction of flowers is given for improvement in general complications. Herb is the main ingredient of joshanda – a Unani medicine used in the form of decoction for cough and colds. Leaves are used as tonic and vermifuge, smoked for relief in catarrh and headache. Leaves also yield an essential oil (0.05%). The roots are aphrodisiac, tonic, deobstruent, diuretic, narcotic, hynotic, sedative, restorative and abortifacient. These are used in rheumatism, cough, debility from old age, dropsy, emaciation of children, consumption and general weakness. Flowers as well as practically the whole plant yields tannin up to 20%. Flowers are valued for dyeing. Also used against diarrhoea and dysentery, complaints of the liver; stimulant in pregnancy and for skin diseases. The seeds on solvent extraction yield 30-35 per cent of semi drying oil, resembling sunflower oil. The herb is reputed as a medicine in Europe, China, Indo-China, Malaysia and America. The drug is credited with powerful diaphoretic properties. The dose of half to one ounce is recommended in chronic malaria, leucorhoea and urinary diseases. The ripe fruits are edible for their sweet taste. The root extract is useful to cure seminal debility, spermatorrhoea and also improves vitality.

Sources: Trease 1952; Chopra et al. 1958; Anonymous 1986; Schultes 1987; Paroda and Mal 1989; Chauhan 1995; Natarajan et al. 2000.

aceae (4 genera, 7 species), Gentianaceae (3 genera, 11 species), Boraginaceae (3 genera, 5 species), Asclepiadaceae (3 genera, 3 species), Oleaceae (3 genera, 3 species), Ericaceae (2 genera, 5 species), Valerianaceae (2 genera, 3 species), Saxifragaceae (3 genera, 6 species), Primulaceae (2 genera, 3 species), Crassulaceae (2 genera, 4 species), Amaranthaceae (2 genera, 3 species), Euphorbiaceae (2 genera, 3 species), Urticaceae (2 genera, 2 species), Berberidaceae (2 genera, 3 species), Rutaceae (2 genera, 2 species), Companulaceae (2 genera, 2 species), Geraniaceae (1 genus, 4 species), Violaceae (1 genus, 4 species), Chenopodiaceae (1 genus, 2 species), Hypericaceae (1 genus, 2 species), Plantaginaceae (1 genus, 2 species), Betulaceae (1 genus, 2 species), Fumariaceae (1 genus, 2 species). Menispermae, Papaveraceae, Cruciferae, Caryophyllaceae, Malvaceae, Balsaminaceae, Rhamnaceae, Hippocastanaceae, Aceraceae, Parnassiaceae, Lythraceae, Punicaceae, Onagraceae, Cucurbitaceae, Cornaceae, Caprifoliaceae, Dipsaceae, Orobanchaceae, Acanthaceae, Verbenaceae, Phytolaccaceae, Lauraceae, Thymelaeaceae, Santalaceae, Buxaceae, Salicaceae, Cannabinaceae, Juglandaceae, Corylaceae and Fagaceae all contained 1 genus and 1 species each and were among the least represented families among the dicots. Among the monocots, the dominating families are Orchidaceae (9 genera, 10 species), Liliaceae (5 genera, 9 species), Araceae (3 genera, 4 species), Zingiberaceae (2 genera, 4 species), Iridaceae (1 genus, 2 species). Dioscoriaceae and Juncaceae contained 1 genus and 1 species each and were the least represented families among the monocots. The gymnosperms were represented by the following families: Pinaceae (4 genera, 5 species), Cupressaceae (1 genus, 4 species), Taxaceae (1 genus, 1 species). Out of the total of 266 species, 157 were classified as medicinal and aromatic plant on the basis of their economic importance for the pharmaceutical (Ayurveda, Sidha, Unani) and perfumery industries (highlighted in grey in Table 1). Being very near to nature and having daily encounters with plant life, there is an incarnate relationship between herbs and the people of Parvati valley. Of the 266 plant species collected around 100 have ethnobotanical importance (species names underlined in Table 1A). Local inhabitants use these herbs in their daily life for the remedy of various diseases. Many of these plant species exhibit high medicinal and aromatic properties and need extensive screening for clinical use. Only after proper elucidation and authentication can such claims be accepted for human welfare.

Kasol Manikaran and moving up to the higher elevation i.e. Chanderkhani jot, Tosh nalah, Khirganga, Tunta bhoj, Pandu pul and Mantalai the range of elevation is approximately 1400 to 5400 m amsl (Janartha 2000). The information regarding traditional knowledge, local uses of the plants of the study area, local names of the plants, parts used, purpose of use, mode of administration and curative properties were recorded through interviews and informal discussion with elderly people, herbal healers, local Vaids and rural women. These are documented in the results. Voucher specimens were collected in the flowering/fruiting period to facilitate the process of identification. Specimens of angiosperms and woody plants were collected and identified according to Bentham and Hooker’s system of classification. These were then processed and deposited in the Herbarium of Dr. Y. S. Parmar University of Horticulture and Forestry, Solan.

RESULTS AND DISCUSSION The Western Himalayas, of which Himachal Pradesh forms a central part, is a vast repository of healing herbs (Chauhan 1999). The age-old practice of plant use as medicine forms a part of culture of this hilly state. The tribal ways of life, adherence to the primitive myths and legends, custom and beliefs, nearness to forests and daily encounters with wild plants seem to be the basic reasons for the persisting herbal lores and mores in the state. The area still maintains rich biodiversity, in addition to rich cultural heritage. Surveys were conducted during the flowering and fruiting period of plants from April-May to SeptemberOctober in 2000, 2001 and 2002. During the study, a total of 266 species belonging to 180 genera and 71 families (including 44 species as first time record from the area) were collected from different areas and locations of the study area of the Parvati valley. The species surveyed included: 223 species in 152 genera from 61 dicot families; 31 species in 22 genera from 7 monocot families; 10 species in 6 genera from 3 gymnosperm families. Thus, it is clear from these figures that high species diversity is exhibited by dicots in this area. Two species of ferns namely, Dryopteris barbigera and Diplazium esculentum were also collected from the area (Table 1). Among the dicots, the dominant families include: Asteraceae (19 genera, 32 species), Labiatae (12 genera, 15 species), Rosaceae (10 genera, 15 species), Leguminosae (9 genera, 9 species), Ranunculaceae (7 genera, 14 species), Polygonaceae (8 genera, 13 species), Umbeliferae (7 genera, 9 species), Solanaceae (5 genera, 5 species), Scrophulari-


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Table 1B Plants of traditional importance from the study area (Parvati Valley). Name of species Local name(s) Uses Achillea millefolium Chuabu, Saijum The leaves and the flowering tops are used to cure gastric problems and fever. Leaves are chewed in the severe toothache to relief pain. A decoction of whole plant is employed for bleeding piles and is good for kidney diseases. Achyranthes bidentata Umblakanta, About 3-mashes of root powder is mixed with equal quantity of smoked tobacco from Hooka and is Puthkanda applied as a paste to the snake bitten organ after giving a proper cross cut. Sufficient ghee is given to the patient orally. The patients are not allowed to sleep at least for 12 hours. Aconitum heterophyllum Atish, Patish, Pongar Root of the herb is used to cure diarrhoea, fever and abdominal pains. Aconogonum rumicifolium Nyelo, Choarh The leaves are cooked and squeezed. The water is thrown away and the cooked leaves are prepared as vegetable by the fuals (shepherds). The paste of the leaves is applied locally in abscesses and boils. Acorus calamus Bach, Bare Locally, the root paste is applied on chest to treat pneumonia in children. A small piece of rhizomes is rubbed over stone together with fruit of Jaiphal (Myrstica fragrance) and Rada (Calunarejan spinosa) and given with mother’s milk to children suffering from cold, cough and fever. Aesculus indica Khanor The fruits are dried and beaten into flour, washed several times in water to remove the bitter taste, dried and kept for use as tonic for ladies. Leaves are used as dried fodder. The oil extract from the fruits is used in healing of wounds and the bark is applied in the form of a paste in dislocated joints. Ajuga bracteosa Nilkanthi, Ratpacho The leaves are used to erase deposition on tongues of children suffering form stomach complaints and fever. The pounded leaves are given in pneumonia and typhoid fever. Androsace rotundifolia Nirodhak buti The leaves of the herb along with needles of deodar are powdered. With some amount of ghee and Gur it is given to the women’s from the first day onset of menses for affecting birth control. Angelica glauca Chora Used as condiment in cookery also used in dyspepsia and stomachache. Small quantity is also collected and sold in the market. Arctium lappa Jangli-kuth Root extract is used as diuretic, diaphoretic, in gout and skin affections. Tincture of the seeds is used for psoriasis and toothache. Arnebia benthami Ratanjot Red dye from the roots is used for dyeing silk and wool. Roots are used against toothache, earache and the paste is applied to cuts and wounds and also in fire burns. Artemisia vulgaris Chhambar The leaves of the plant are crushed and the paste is applied on the cuts and wounds, to check bleeding. The wound is fastened with a cloth, after few minutes, bleeding stops. Asparagus filicinus Sansbai The roots are used to increase the milk yield in cattle and also to get the milk germ-free. Berberis aristata Kashmal The roots are used as fuel wood, while the extract of the root bark called as Rasount is used to cure eye diseases, skin diseases, jaundice, piles and malaria. Fruits are eaten as laxative and antiscorbutic. Bergenia ciliata Pashanbhed, Takli The crushed roots mixed with milk are given in backache. On burns, it is applied after mixing with curd. Rhizomes are also used against kidney stone, piles, diabetes and heart diseases. The paste of fresh rhizome is very effective in the treatment of swellings etc. in livestock. Betula utilis Takpa, Bhojpatra The bark of the tree is burnt and the bhasma is used to cure rheumatic pains. It is also used as healing agents against deep cuts. Bhujera, a fungal formation on the tree is used for alimentary disorders in animals. Bistorta affinis Chunru Plant is used as a medicine for cough and diarrhoea. Roots are chewed to relieve irritation of throat. Bistorta amplexicaulis Sarbguni The root paste is applied on sores and wounds. The roots are also given with the milk to the women to check extract bleeding during menstruation period. Boehmeria platyphylla Samrala Fibres from the stem are used for making fishing-nets. Boenninghausenia Pisumar-buti The entire aerial part is used to repel lice, fleas and other insects. albiflora Bupleurum candolli Kaligewar Herb is used to induce perspiration and for stomach and liver complaints. Caltha palustris Horgul The herb is used for the treatment of leprosy and rheumatism. Young flower tops are pickled in the vinegar and used as capers. The herb is poisonous. Cannabis sativa Bhang, Charas The paste of fresh leaves is used to resolve tumors. Leaf powder is useful for dressing wounds and sores. Seeds are roasted and eaten as culinary by the local people. The resinous exudation commonly known as charas is also taken with tobacco as a sedative. Cassiope fastigata Hieunshelo The leafy twigs are ground into a paste and applied to fire burns. It exerts immediate cooling effect and is effective in healing the wound also. It is kept in houses as an emergency medicine for this purpose. Cedrus deodara Diar, Devdar The oil is used as an effective insect repellant in cattle wounds, especially in sheep and goats. Chenopodium album Bathu The young leaves are used in vegetable and given to patients suffering from leucoderma. The decoction of seeds is given in large doses to induce abortion in women. Cissampelos pareira Patindu The leaves are crushed and then given to the children in case of heat with milk of honey. Codonopsis ovata Sardanda, Sardandi The roots are considered as a good physical and sexual tonic. Corydalis govaniana Inder-jata The decoction of the whole plant is given in chronic fevers and liver complaints. Cotoneaster microphylla Ruins The bright-red fruits are eaten. The pulp is used to prepare chutney and jams. Cyathula tomentosa Silath Locally, flowering spike is used to repel away the mouse. Cynoglossum denticulatum Kumbru, Kuri The juice of the leaves is applied like eye drops in conjunctivitis and reddening of the eyes. The crushed leaves are also effective on cut wounds. Locally, the tubers are used in general weakness or loss of sexual power and nerve debility. Dactylorhiza hatagirea Panja, Hathpanja Delphinium vestitum Changuthpa, Salyan Leaves of the plant are poisonous to goats. Root powder is also helpful in healing of ulcers and wounds in cattle. Dicliptera bupleuroides Ludra-buti Paste of leaves and new shoots is applied against the wounds of snakebite and yellow secretion is reported to ooze out the poison. Dioscorea deltoidea Singli-mingli Locally the tubers are used to kill lice’s and to poison fish. It is also used for washing the woolen clothes. Echinops niveus Oontkatara Root bark is powdered and mixes with honey, taken internally to cure cough and asthma. Fragaria vesca, F. indica Wild strawberry The local people eat fruits.


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Table 1B (Cont.) Name of species Fritillaria cirrhosa

Local name(s) Hadjod

Geranium wallichianum Geum elatum Girardinia diversifolia

Ratanjot Masreen Bara-bichua

Habenaria pectinata Heracleum candicans

Meda Rasal

Indigofera heterantha Inula grandiflora Iris hookerana Juniperus communis

Surmai Poshkar Iris Bethar

Juniperus recurva

Dhoop, Shur

Jurinea dolomiaea

Dhoop

Leucas lanata

Dhurlughas

Morina longifolia

Bishkandara, Chow

Nardostachys grandiflora

Nihani

Nasturtium officinale

Chuuch

Origanum vulgare

Sathra, Banajwain, Baslughas

Oxyria digyna

Suma, chucha

Phytolacca acinosa

Jharka

Picrorrhiza kurrooa

Karu, Kutki

Pinus wallichiana

Kail

Plantago depressa

Musalniani

Plantago major Pleurospermum brononis

Luhuriya, isabgol Nesar, Losar

Podophyllum hexandrum

Shathjalari, Bankakri, Rodhari

Polygonatum cirrhifolium

Salam-misri

Polygonatum verticillatum Potentilla atrosanghuinea Primula denticulata Prinsepia utilis

Salam-misri Larsu Keecha Bhekhal

Punica granatum

Daru

Rheum australe, R. moorcroftianum

Chukri, Leechu

Rhododendron anthopogon Tali, Tama

Rhododendron arboreum

Buras

Uses The paste of the bulbs is applied on fractured bones and it is reported that minor fractures are fully recovered in 15-20 days. Herb is used against toothache and eye troubles. Roots yield tanning material and red dye. It is a very useful drug for wounds and cuts. It induces quick healing of the wounds. Leaves are used in headache and swollen joints, to activate blood circulation. Its decoction is given in fever. The bark forms a very good fibre for making ropes and cordage’s. Its tubers is mixed with Khoya and greater cardamom and eaten, to get relief from joint pains. Roots are considered poisonous. The powder of the plant is given in giddiness. Leaves and shoots are often used as fodder. The plant is considered to be a good fodder, and also used as green-manure. The roots are aromatic and are used to cure cough, cold and throat irritations. Paste of flowers and leaves is given to a person suffering from fever. Twigs are used as incense and in oracle rites in driving away the evil spirits. Also known to be a useful remedy in joint pains (rheumatic arthritis). People regard this plant as a repellent of evil spirits. The twigs are used as incense and commonly used in Havans. Used in the preparation of Dhoop, which is used to purify the air and employed in worships and prayers. The roasted leaves in ghee have been successfully tried as a remedy to expel the placenta as after delivery in cattle’s. The plant is also used in the remedy of diarrhoea and dysentery in cattle’s. Root powder is applied as poultice in boils for sucking the puss out of it and facilitates healing of wounds. The flowers eaten by the shepherds during their visit to high altitude areas so that they are protected from high altitude problems. A small piece of the root is powdered and mixed with tobacco and smoked in cases of palpitation of heart and mental tension. The young shoots are used as leafy vegetables and its use is said to act as appetizer, laxative and diuretic. It is believed to increase blood circulation. The plant is regarded an important house hold remedy for various purposes. The paste of leaves and terminal shoots is applied to boils, ulcers, wounds, cuts and weeping eczema. The paste of leaves is reported to be highly useful in healing the wounds caused by fire-burns. The root pieces of plant is bound in a cloth piece and tied to the necks of infants as a protective measure against conjunctivitis. The leaves are considered as carminative and digestive. Useful in abdominal problems. Leaves are also used as vegetable and for making chutney. The tender leaves and twigs are cooked as vegetable. The herb is believed to have narcotic effect, which is destroyed on boiling. The roots are used in abdominal pains and as a purgative too. In case of nose bleeding, leaves are crushed and 1-2 drops of the juice are put in the nose to stop bleeding. The bark taken out in the form of long circular cylinders is used to bandage the dislocated/fractured/fractured organs both in cattle, sheep and goats and on human beings after setting the dislocated or broken bone in proper place. The paste of the roots and leaves is applied to skin eruptions, boils and rashes. Seeds are used to cure dysentery. The root of the plant is tied to the button hole of the infants as a protective measure against infection of stomach disease. Whole plant also used to cure tail gangrene of cattle. Seeds are used in gastric complaints, burning sensation in stomach and dysentery. The powder of the flowering shoots is mixed in cow’s fresh butter and massaged over the entire body to allay fevers. The dried herb or the dried garland is kept in the boxes containing clothes as a preservative against the attack of moths and silver fish. The root powder is administered internally for gastric ulcers. It is applied as a paste on cuts and wounds for regeneration of the tissues. Decoction of roots is used to cure liver problems. Shepherds eat fruits. The local people eat rhizomes to cure blood pressure problem. It is believed that this application keeps the blood pressure in equilibrium. Rhizomes are used to cure kidney problems. The local people eat the rhizomes being sweet in taste. The decoction of root is used as gargle to cure toothache. The flowering tops are used to cure cough and paralysis. The oil extracted from the seeds is taken internally as tonic and is considered useful in general debility and rheumatism. The oil is also massaged on rheumatic joints for relief. Children’s make the hollow branches into flutes. The long hollow tubes are also used as pipes for smoking tobacco in hookas. The fruit rinds are dried and powdered and taken with cold water to relieve cough. When children (new borne) start cutting out teeth, the peels are powdered mixed with Kashmal (Berberis sp.) roots, made into a paste and applied on the palate to case the process of emergence of teeth. The paste of the root in water is applied externally in muscular injury, cuts, wounds and mumps and to forehead in headache. The watery extract is given orally in stomach pains, constipation dysentery, swelling of the throat and tonsillitis. Lotion is dropped in ears in earache. It purifies the blood and being astringent, reduces the swellings and rheumatic paints quite effectively. The decoction of the leaves in used in cold, cough and chronic bronchitis. People prepared tea from the leaves. Excess dose is regarded poisonous. The powder of the dried flowers mixed in bland oil is used as massage over the entire body in post delivery complications like, fevers, cough and cold. The plant is also used as incense. The fresh petals are used in chutneys. The powder of the dried flowers is used as an efficacious drug to check bloody and chronic dysentery.


55


Table 1B (Cont.) Name of species Rhododendron companulatum Rosa macrophylla Rumex hastatus

Local name(s) Shargar

Uses Roots are used to cure boils. Buds are considered poisonous for sheep and goats.

Jungli-gulab Khatti

Flowers are used by local vaids to make medicines to cure stomachaches. The bark of the root is used to cure fire burns and is applied as ‘lep’ (paste). Leaves are used in chutneys, being acidic in taste. Locally, leaves are commonly used to cure constipation. Also used as vegetable. The plant is regarded as sacred. Its fumigation is employed to allay the affects of evil spirits. The wooly hairs mixed with ghee are given to the asthma patients. The root paste is applied in cuts and bruises. Also offered as worship to the deities. The chutney of the whole plant is useful in acute gastric problems. Paste of the plant is also used to heal burns. The smoke produce from the roots is used for killing and repelling the insects, for purifying the atmospheric air. Roots are also used as a substitute for ‘Bhutkesi’ and used an ingredient in Dhoop preparation. Rhizomes are used to prepare local liquors and for treating the patient with mental disorder. Decoction of the whole plant is used to cure fever and abdominal pains. Essential oil obtained from the plant is used as perfumery material. The herb is toxic to animals. The leaves of the plant are used to produce aroma. Dried leaves are used in havens, while worshiping deities. The plant is a bitter tonic used to cure fevers, stomachache, febrifuge and laxative.

Rumex nepalensis Saussurea gossypiphora

Palak Gugghi-badshah

Saussurea obvallata Sedum ewersii

Dodaphool Hiunsheli

Selinum tenuifolium

Mathosal

Selinum vaginatum Senecio chrysanthemoides

Bhutkesi, Bhutjata Semgebala

Skimmia laureola

Dhoop

Swertia chirata and other Chirayita Swertia species Tanacetum longifolium Langri Taraxaxum officinale

Aachak

Taxus wallichiana

Rhakhal

Thalictum foliolosum

Pilijari

Thymus serpyllum

Ban-ajwain

Valeriana jatamansi

Nihani

Verbascum thapsus

Kolomasta

Viola serpens and other Viola species Vitex negundo

Banaksha Bana

Withania somnifera

Ashganth

Woodfordia fruticosa

Dhai

Zehneria umbellata

Jangli-kakri

Its aroma, especially when found bruised and crushed by sheep is said to cause giddiness. The leaf juice is useful as an antispasmodic, carminative and antipyretic. The sheep and goats browse it as a potent fodder. The whole plant is crushed into a mesh and given internally in snakebites. The paste is applied externally on the wound. Leaves are effectively used for fomentation in swollen parts, boils and sprains. Leaves are used as sedatives, antiseptic and emmenagogue; its tea is used to cure asthma, bronchitis, epilepsy and cough, etc. Used internally in abdominal pains and as a blood purifier. The paste of the roots is applied on the eyelids to cure eye diseases. The poultice of the root is applied to cure the boils and ulcers. Also beneficial to cure foot and mouth disease of animals. The decoction of the plant is an effective home remedy for colds, cough, fever and stomach problems. The local people drinks tea with this herb regularly to cure from common colds and different stomach ailments. Locally, used as antispasmodic, carminative and in acute stomachaches. Decoction is a beneficial remedy in insomnia and nervous exhaustion due to heavy mental work. The crushed leaves are given in constipation and allied stomach pains. Dhuni (smoke) of the plant is used by the tantrics to drive away the ghostly instincts, especially in the children’s, where bad spirit is evolved. The decoction prepared is given for expulsion of phlegm. It is a good cure for sore throat. The leaves are boiled in cow’s urine and when half the quantity is left, a paste is made and applied on wounds and painful body organs to get relief. Boil leaves in water and apply with the help of cloth on swellings to get relief. Young shoots are also used in tantra-mantra. Root powdered is given with milk/water to cure sexual weakness, loss of appetite, cough, dropsy and general debility. The young leaves are cooked as vegetable and regarded as blood purifier. It is said to be a cure for skin diseases. The fine paste of the plant, especially of the root part, is applied in hidden muscular pains. The shocks of the stings of its twigs two to three times are applied in swollen, rheumatic joints for relief from pain and swelling. The ripe fruits are edible for their sweet taste. Dried powdered and taken with milk in the dose of about two grams twice a day as a cure for seminal debility, spermatorrhoea and its use improves vitality.

The predominant woody tree species include Abies spectablis, Acer pictum, Betula alnoides, B. utilis, Cedrus deodara, Corylus colurna, Juglans regia, Picea smithiana, Pinus roxburghii, Prunus cornuta, Quercus semecarpifolia, Rhododendron arboreum and Taxus baccata ssp. wallichiana. Among the cultivated plants, apple, apricots, plums, cherries and japaniphal grow abundantly in the lower valley and are a good source of revenue. The total plant species collected were further classified as medicinal and aromatic (184), fodder (53), fuel wood (45), timber (21), fibre (9), yielding tans and dyes (27), gums and resins (4), bee flora (31), edible (43), ornamental (123), use in tantra-mantra i.e. ethno botanical uses (24) and oil yielding (essential 32; others 9) (Table 2). As a result of continuous and relentless extraction over many decades, many valuable species are facing danger to their survival in their natural habitats. Some of the threat-

ened species are Aconitum heterophyllum, Atropa acuminata, Dioscorea deltoidea, Dactylorhiza hatagirea, Jurinea dolomiaea, Nardostachys grandiflora, Picrorrhiza kurrooa, Podophyllum hexandrum, Rheum australe, Swertia chirayita, Valeriana hardwickii, Saussurea royleii, Saussurea gossypiphora, Saussurea obvallata, Pleurospermum brunonis, Polygonatum cirrhifolium, Fritillaria cirrhosa, and Codonopsis ovata. Unregulated exploitation and disorganized trades are responsible for the sharp decline in the herbal wealth of the area. During our investigation, we found that a wealth of knowledge regarding the ethno botanical and medicinal uses of plant species lies with shepherds (Gaddies, Gujjars), healers (Vaids) and the old people living in the area. However, these people seldom agree on revealing the information and only through persistent requests and motivation do they share their knowledge about the use of herbs. One


56

Traditional therapeutic uses of plant diversity of Parvati Valley. Sharma et al.

Abies spectablis Acer pictum Achillea millefolium Achyranthes bidentata Aconitum heterophyllum Aconitum violaceum Aconogonum rumicifolium Acorus calamus Actaea spicata Aesculus indica Ajuga bracteosa Anaphalis busua Anaphalis contorta Anaphalis triplinervis Androsace lanuginosa Androsace rotundifolia Anemone obtusiloba Anemone rivularis Anemone tetrasepala Angelica glauca Arctium lappa Arisaema tortuosum Arisaema wallichianum Arnebia benthami Artemisia dubia Artemisia gmelinii Artemisia roxburghiana Artemisia vulgaris Asclepias curassavica Asparagus filicinus Aster himalaicus Aster trinervius Astragalus concretus Atropa acuminata Benthamida capitata Berberis aristata Berberis edgeworthiana Bergenia ciliata Bergenia stracheyi Betula alnoides Betula utilis Bistorta affinis Bistorta amplexicaulis Bistorta vivipara Boehmeria platyphylla Boenninghausenia albiflora Bupleurum candolli Calanthe tricarinata Caltha palustris Campanula pallida Cannabis sativa Cassiope fastigata Cedrus deodara Cephalanthera longifolia Chaerophyllum reflexum Chenopodium album Chenopodium foliosum Cicer microphylla Cissampelos pareira Codonopsis ovata Colutea multiflora Corydalis flabellata Corydalis govanianum Corylus colurna Cotoneaster acuminatus

+ + + + + + + +

+ + + + + + + + + + + +

+ + + + + + + + + + + + + + +

+ + + + + + -

+ -

-

+ + + + + + -


57

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

+ + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + -

+ + + + + + + + -

Oil

Tantra- Mantra/ Ethnobotanical importance

Edible

Medicinal and Aromatic

Bee flora

Tans and dyes

Gums and resins

Fibre and flosses

Fodder

Fuel wood

Timber/ Furniture

Table 2 Economic importance of different plant species in the Parvati valley. Latin name

Ornamental landscape


+ + + + + + + + + + + -

Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©2010 Global Science Books

Cotoneaster microphyllus Cremanthodium arnicoides Cryptolepis buchanani Cyathula capitata Cyathula tomentosa Cynoglossum denticutatum Cynoglossum wallichii Cynoglossum zeylanicum Dactylorhi hatagirea Daphne canabina Delphinium denudatum Delphinium vestitum Desmodium tiliaefolium Dicliptera bupleuroides Dioscorea deltoidea Ehinops niveus Elsholtzia fruticosa Elsholtzia strobilifera Epilobium cylindricum Epipactis royleana Euphorbia cognata Euphorbia wallichii Fagopyrum esculentum Fragaria indica Fragaria vesca Fritillaria cirrhosa Fritillaria roylei Gentiana venusta Gentianella falcata Geranium donianum Geranium himalayense Geranium refractum Geranium wallichianum Gerardinia diversifolia Geum alatum Habenaria intermedia Habenaria pectinata Hackelia uncinata Hedychium acuminatum Heracleum candicans Herminium lanceum Hypericum choisianum Hypericum elodeoides Impatiens urticifolia Indigofera heterantha Inula grandiflora Iris hookerana Iris kemaonensis Jasminum officinale Juglans regia Juncus leucanthus Juniperus communis Juniperus indica Juniperus macropoda Juniperus recurva Jurinea dolomiaea Lactuca bracteata Lactuca decipines Lactuca lessertiana Lathyrus humilis Leonurus cardiaca Leucas lanata Litsea umbrosa Lonicera spinosa Meconopsis aculeata

+ + + + + -

+ + + + + + + + + + + + + -

+ + + + + + + + + + + -

+ + -

-

+ + + + -


58

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + -

+ + + + + + -

Oil


Edible


Bee flora

Tans and dyes

Gums and resins

Fibre and flosses

Fodder

Fuel wood

Timber/ Furniture

Table 2 (Cont.) Latin name



+ + + + + + + + -


Malaxis muscifera Malva verticillata Morina longifolia Myriactis wallichii Nardostachys grandiflora Nasturtium officinale Nepeta govaniana Nepeta leucocephalla Nepeta linearis Nicandra physalodes Nicotiana tabacum Olea ferruginea Origanum vulgare Orobanche cernua Osyris arborea Oxyria digyna Oxytropis cachemiriana Parnassia nubicola Pedicularis longifolia Pedicularis oederi Pedicularis pyramidata Pedicularis siphonantha Persicaria polystachya Phlomis bracteosa Phytolacca acinosa Picea simithiana Picrorrhiza kurrooa Pimpinella diversifolia Pinus roxburghii Pinus wallichiana Plantago dipressa Plantago major Pleurospermum brunonis Pleurospermum govanianum Podophyllum hexandrum Polygonatum cirrhifolium Polygonatum hookeri Polygonatum multiflorum Polygonatum verticillatum Polygonum plebeium Potentilla atrosanghuinea Potentilla cuneata Potentilla polyphylla Potentilla sibbaldi Prenanthes brunoniana Primula denticulata Prinsepia utilis Prunella vulgaris Prunus curnuta Punica granatum Quercus semecarpifolia Rabdosia rugosa Ranunculus arvensis Ranunculus diffusus Rhamnus virgatus Rheum australe Rheum moorcroftianum Rhlodiola bupleuroides Rhlodiola himalensis Rhlodiola imbricata Rhododendron anthopogon Rhododendron arborium Rhododendron campanutatum Rhododendron lepidotum

+ + + + + + + -

+ + + + + + + + + + + + +

+ + + + + + + + + + + + + + + -

+ + -

+ + + -

+ + + + + + + -


59

+ + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + -

+ + + -

Oil


Edible


Bee flora

Tans and dyes

Gums and resins

Fibre and flosses

Fodder

Fuel wood

Timber/ Furniture




+ + + + + + + + + + + + + -

Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©2010 Global Science Books

Rhynchospermum verticillatum Ribes himalyense Ricinus communis Rosa macrophylla Roscoea alpina Roscoea capitata Roscoea purpurea Rumex acetosa Rumex hastatus Rumex nepalensis Salix lindleyana Salvia nubicola Sarcococca saligna Satyrium nepalense Sauromatum venosum Saussurea auriculata Saussurea gossypiphora Saussurea obvallata Saussurea roylei Saxifraga diversifolia Saxifraga moorcroftiana Saxifraga parnassifolia Scutellaria angulosa Sedum ewersii Selinum tenuifolium Selinum vaginatum Senecio cappa Senecio chrysanthemoides Senecio rufinervis Silene edgeworthii Skimmia laureola Smilacina purpurea Solanum pseudo-capsicum Solidago virga-aurea Sorbaria tomentosa Sorbus macrophylla Spiranthes sinensis Spirarea bella Swertia alternifolia Swertia angustifolia Swertia chirata Swertia cordata Swertia paniculata Swertia petiolata Swertia purpurascens Swertia racemosa Swertia speciosa Syringa emodi Tanacetum longifolium Taraxacum officinale Taxus wallichiana Thermopsis inflata Thalictrum alpinum Thalictrum foliolosum Thalictrum javanicum Thymus serpyllum Trifolium pretens Trillidium govanianum Valeriana hardwickii Valeriana Jatamansii Verbascum thapsus Vincetoxicum hirundinaria Viola biflora

+ -

+ + + + + + -

+ + + + + + + + + + + + -

-

-

+ + + + + + + -


60

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + -

+ + + + + -

Oil


Edible


Bee flora

Tans and dyes

Gums and resins

Fibre and flosses

Fodder

Fuel wood

Timber/ Furniture




+ + + + + -


Viola canescens Viola kunawarensis Viola serpens Vitex negundo Withania somnifera Woodfordia fruticosa Wulfenia amherstiana Xanthium strumarium Zehneria umbellata

-

+ + -

-

-

-

+ -

+ + + + + -

+ + -

+ + + + + + + +

+ +

+ -

Oil


Edible


Bee flora

Tans and dyes

Gums and resins

Fibre and flosses

Fodder

Fuel wood

Timber/ Furniture




+ + -

REFERENCES

superstitious belief is that the herbs loose healing power if their ‘secret’ is shared with ‘outsiders’ and another reason they cite is that herbs are useful only when used in combination with ‘tantra-mantra’ (i.e., occult practices).

Anonymous (1992) Wildlife of Himachal, Department of Forest Farming and Conservation, Himachal Pradesh. Shimla, 34 pp Badola HK (1998) Biodiversity Conservation study of Kanawar wildlife sanctuary in Himachal Pradesh, In: Research for Mountain Development: Some Initiatives and Accomplishments, Gyanadoya Prakashan, Nanital, pp 407-430 Chauhan NS (1999) Medicinal and Aromatic Plants of Himachal Pradesh, Indus Publishing Company, New Delhi, 500 pp Chowdhery HJ, Wadhwa BM (1984) Flora of Himachal Pradesh (Vol 3), Botanical Survey of India, Howrah, pp 276-278 Dhaliwal DS, Sharma M (1999) Flora of Kullu District, Himachal Pradesh. In: Singh B, Singh MP (Eds) Survey of Flora, Jaipal Publications, Dehradun, 221 pp Jain SK, Bhardawaja RC (1951) On a botanical trip to Parvati valley. Indian Forester 75, 302-315 Janartha TC (2000) Himachal Pradesh District Gazetteer, Kullu, Shimla, 621 pp Khoshoo TN (1993) Himalayan biodiversity conservation – an overview. In: Dhar U (Ed) Himalayan Biodiversity Conservation Strategies, Gyanodaya Parakashan, Nanital, pp 5-39 Myer N, Muttermeier RA, Muttermeier CA, da Fonseca ABG, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403, 853-858 Puri GS (1952) The distribution of conifers in Kullu, Himalayas, with special reference to geology. Indian Forester 76, 144-153 Singh SK (1999) Ethnobotanical study of useful plants of Kullu district in north western Himalaya, India. Journal of Economic and Taxonomic Botany 23, 185-198 Singh SK, Rawat GS (2000) Flora of Great Himalayan National Park, Himachal Pradesh. In: Singh B, Singh MP (Eds) The Great Himalayas, Jaipal Publications, Dehradun, pp 105-109 Uniyal MR, Chauhan NS (1972) Commercially Important Medicinal Plants of Kullu, Forest Division of Himachal Pradesh, Nagarjuna, New Delhi, 154 pp

CONCLUSION The Parvati valley is very rich in plants with medicinal value and a concerted effort is needed for their conservation. To check the loss of biodiversity owing to overexploitation and habitat degradation, effective measures for conservation and management need to be put in place. Priority should be given for conservation of high-value species listed in this study (Figs. 1, 2). The involvement of local inhabitants with their local tradition and culture (Fig. 3) is very important for conservation of indigenous knowledge and traditional practices. The present study will serve as baseline information for planning and policy regarding the Parvati valley, rich in aesthetic and historically important places (Fig. 4). ACKNOWLEDGEMENTS The authors thank Dr. K. K. Katoch DEE, CSKHPKV Palampur and Dr. S. K. Thakur Programme Coordinator, KVK Kukumseri for providing necessary facility. Help receive from the Faculty of Solan University, Sh. Amar Nath Sharma; DFO Parvati is acknowledged providing necessary help during the study. Prof. Atul, Dr. S. S. Samant, Dr. R. D. Singh and Dr. H. C. Sharma are acknowledged for their valuable suggestions and occasional guidance. The valuable information received from the local inhabitants and migratory nomads is greatly acknowledged.


61


A

B

F

C

D

G

J

H

E

I

K

L

M

Fig. 1 Threatened species with high commercial value. (A) Aconitum violaceum; (B) Angelica glauca; (C) Arnebia benthamii; (D) Dactylorhiza hatagirea; (E) Dioscorea deltoidea; (F) Jurinella macrocephala; (G) Nardostachys grandiflora; (H) Picrorhiza kurrooa; (I) Podophyllum hexandrum; (J) Saussurea roylei; (K) Saussurea obvallata; (L) Saussurea gossypiphora; (M) Selinum vaginatum.

A

E

B

C

F

G

D

H

Fig. 2 Other high value economical important species. (A) Acorus calamus; (B) Betula utilis; (C) Fritillaria cirrihosa; (D) Cannabis sativa (Malana Village); (E) Hedychium spicatum; (F) Pleurospermum candolii; (G) Polygonatum cirrihifolium; (H) Rheum webbianum.


62


A

B

Fig. 3 Traditional culture and custom. (A) Musical instrument of local deity Jamlu, Malana village; (B) Local festival Phagli in Malana Village.

A

B

C

D

Fig. 4 Aesthetic and historically important places. (A) Dibinallah (proposed hydroeclectric project site); (B) Khirganga (hot water spring); (C) Mantallai lake (source of Parvati river); (D) Udithach (largest alpine pasture in the valley).


63


®


Ethnobotany of Plants Used to Cure Diabetes by the People of North East India Venkat Kishore Ryakala1† • Shahin Sharif Ali1,2† • Hallihosur Sharanabasava1 • Naushaba Hasin3 • Pragya Sharma4 • Utpal Bora1* 1 Department of Biotechnology, Indian Institute of Technology, Guwahati-781039, India 2 School of Biology and Environmental Science, University College Dublin, Dublin-4, Ireland 3 Indian Council of Medical Research (NE Region), PB No. 105, Dibrugarh-786001, India 4 Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India Corresponding author: * [email protected] or [email protected]

† These authors contributed equally

ABSTRACT Northeast India is considered as an ecological hot spot and has a wide variety of flora and fauna. Diverse ethnic communities inhabit the area, each having their own traditional medical cures for different diseases. During the course of present studies it was found that 52 species of plants belonging to 36 families are used as antidiabetic agents in folk medicinal practice. Leaves and bark were found to be the two major plant parts used for making hypoglycemic herbal preparations. Around 26 treatments involve administration of decoction to the diabetic patient. These decoctions are either prepared from leaves, bark, fruit, root, seeds or from whole plants. Out of the 52 plants 12 are also reported to have antidiabetic properties in the Diabetes Medicinal Plant Database. The remaining plants could be a potential source of new and efficient cures for diabetes.

_____________________________________________________________________________________________________________ Keywords: medicinal plant, traditional medicine

INTRODUCTION Diabetes is a major metabolic disorder responsible for 9% of the total number of deaths in the world. At present around 171 million people are affected by this disorder and the number is likely to be doubled by 2030 (WHO 2008). As a very common chronic health problem, diabetes is the third “major killer” after cancer and cardiovascular diseases because of its high prevalence, morbidity and mortality (Li et al. 2004). According to the WHO (2008) India has the highest number of diabetics in the world with more then 31.7 million followed by 20.8 million in China and 17.7 million in US. With transition to the more sedentary lifestyle of industrialized nations, the prevalence of diabetes is expected to increase among all age groups. Due to the chronic nature of diabetes and the complications related to it treatment for the disorder has become very costly. A lowincome family in India spends as much as 25% of the family earnings for taking care of an adult with diabetes thereby causing grave socioeconomic imbalances as well. Therefore cheaper remedies are needed for developing countries like India. The available synthetic drugs for treating diabetes also have many limitations and undesirable side effects like hepatotoxicity, cardiomegaly, hemotoxicity (Akhtar and Iqbal 1991; Watkins and Whitcomb 1998) and have high rates of secondary failures (Chang et al. 2006). Medicinal herbs with anti-hyperglycemic activities are being increasingly used as an alternative approach in the treatment of diabetes due to their low cost, effectiveness and little or no adverse effects (Xie et al. 2003). The modern drug metformin (a biguanide) is a derivate of an active natural product galegine which was used in medieval times to relieve the intense urination in diabetic people (AndradeCetto and Heinrich 2005); galegine is a guanidine isolated from the plant Galega officinalis L. (Witters 2001). Northeast India consists of the states of, Assam, Arunachal Pradesh, Meghalaya, Manipur, Mizoram, Nagaland and Tripura. The study area extends between 21.57-29.30°

Fig. 1 Different locations of NE India (as mentioned in Table 1 with Site No.) where traditional healers ware interviewed

N latitude and 88-97.30° E longitude, is mostly covered by the hills of Himalayan range with the valley of Brahmaputra River running through the middle. The region is rich in floral diversity and many endemic elements (Myers et al. 2000) and the people of this region have rich ethnomedicinal traditions. In the present study we report 52 species of plants used as anti diabetic agents in the traditional treatment of diabetes in Northeast India and some of the diabetic symptoms known to common people of this region.

Received: 18 January, 2009. Accepted: 30 November, 2009.


Research Note


EXPERIMENTAL

Table 1 Number of traditional healers interviewed in different places of NE India. Site No. in Fig. 1 Place name No of traditional healers interviewed 1 Guwahati 6 2 Tezpur 4 3 Lakhimpur 8 4 Jorhat 20 5 Golaghat 24 6 Mariani 3 7 Goalpara 12 8 Mayang 2 9 Sunapur 2 10 Barpeta 8 11 Nalbari 10 12 Diphu 13 13 Haflong 22 14 Hojai 1 15 Demaji 9 16 Dibrughar 12 17 Tinsukia 13 18 Majuli 3 19 Shillong 20 20 Tura 4 21 Aizwal 7 22 Champhai 2 23 Lunglei 3 24 Mamit 4 25 Kolasib 8 26 Imphal 6 27 Senapati 6 28 Ukhrul 9 29 Chandel 8 30 Bishnupur 3 31 Itanagar 9 32 Bomdila 10 33 Tawang 12 34 Ziro 14 35 Roing 10 36 Tezu 9 37 Pasighat 20 38 Seppa 4 Total 340

Method of database preparation The study was carried out by regular field trips to different locations of the Himalayan hilly regions (Fig. 1, Table 1) conducted between July 2004 and February 2008 to gather data. Various traditional healers, village elders and diabetic patients were interviewed, in different locations in the study area. Age, experience and reputation were taken into consideration while selecting the interviewers. Structured forms in local languages were prepared and filled in with the information received from the respondent (both healer and patients). The interviewees were aware about the objective of the study, which was to compile traditional knowledge of ethnomedicine among the people of North East India. The collected plant species were submitted to taxonomists in Assam State Zoo cum Botanical Garden Guwahati, for botanical identification. The data obtained was tabulated to include the botanical name, local name, family name, plant part(s) used, followed by the mode of preparation and administration.

RESULTS AND DISCUSSION Northeast India is considered as an ecological hot spot and is inhabited by diverse tribal communities each having its own traditional medicinal cure for different diseases. Our group has been involved in documenting the richness of this folklore medicine as modernity is slowly wiping out this knowledge from public memory. Earlier we have reported the use of different herbal remedy by the people of Assam and Northeast India for treating skin infections (Saikia et al. 2006) and malaria (Bora et al. 2007). In the present study, 52 species of plant belonging to 36 families were found to be used as anti-diabetic agents by the people of Northeast India (Table 2). The plant parts used range from roots, shoots, leaves, stems, barks, seeds, flowers to fruits and in some cases the whole plant (Fig. 2). It has been observed that water is used as the medium in most preparations. Around 26 treatments involve administration of a decoction to the diabetic patient. These decoctions are either prepared from leaves, bark, fruit, root, seeds or from whole plants. Other preparations are administered in the form of soup, infusion, juice, powder or whole plant extract. Some of the plant parts are eaten either raw or cooked. We found that leaves and bark were used more frequently for making anti-diabetic preparations (Table 2). In the case of the preparation from Inula cappa, two other herbs, Plantago asiatica and Lobelia angulata were combined to form a multi-herbal formulation. The efficacy of these ethno-medicinal plants needs to be subjected to pharmacological validation. Some antidiabetic plants may exert their action by stimulating the function or number of -cells of pancreas and thus increasing insulin release (Persaud et al. 1999). In some other plants, the effect is due to decreased blood glucose synthesis due to decreased activity of enzymes like glucose-6-phosphatase and fructose 1,6-bisphosphatase (Chhetri et al. 2005). In many other plants, the activity is due to slow absorption of carbohydrate and inhibition of glucose transport (Madar 1984). However, these products may interact with conventional medicines for diabetes (Shane-McWhorter 2001). Therefore a cautious approach should be adopted before administering these drugs. Out of the 52 plants 12 are also reported to have antidiabetic properties in the Diabetes Medicinal Plant Database (2008). These plants are Allium sativum, Catharanthus roseus, Emblica officinalis, Ficus benghalensis, Mangifera indica, Scoparia dulcis, Syzygium cumini, Tamarindus indica, Terminalia chebula, Tinospora cordifolia, Tragia involucrata, and Trigonella foenum-graecum. The remaining plants are a potential source of investigation for novel therapies. In this study we observed that most preparations are derived from a single plant source suggesting the presence of potential anti-diabetic compounds in them. Isolation of

these compounds will lead to the development of clinically useful medicines and especially phytomedicines or adequate nutritional supplements, which would be of direct benefit to patients. On the other hand, the ever-increasing demand, particularly in view of world-wide shift for drugs of herbal origin over synthetic counterparts, has led to overexploitation of medicinal plants. In addition, the lack of organized cultivation has resulted in many of these plants finding place in the list of vulnerable, endangered or threatened categories. Thus, there is an immediate need for mass multiplication of many of these species to make available the planting material for taking up organized cultivation. ACKNOWLEDGEMENTS We thank Mr. N.C. Das, Botanist, Assam State Zoo cum Botanical Garden, Guwahati for botanical identification. We also thank Dr. D. K. Hore, NBPGR, Barapani and Dr. C. S. Rao, BRDC, Shillong for their valuable suggestions.

REFERENCES Akhtar MS, Iqbal J (1991) Evaluation of the hypoglycemic effect of Achyranthes aspera in normal and alloxan diabetic rabbits. Journal of Ethnopharmacology 31, 49-57 Andrade-Cetto A, Heinrich M (2005) Mexican plants with hypoglycaemic effect used in the treatment of diabetes. Journal of Ethnopharmacology 99, 25-348 Bora U, Sahu A, Saikia AP, Ryakala VK, Goswami P (2007) Medicinal plants used by the people of Northeast India for curing malaria. Phytotherapy Research 21, 800-804


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Medicinal plants used for curing Diabetes in Northeast India. Ryakala et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Table 2 Medicinal plants used for curing Diabetes in Northeast India. Botanical name Local name Family Plant part used State found Mode of use and administration Allium sativum L. Naharu Alliaceae Bulbs Assam 3-4 cloves of a bulb is fried with mustard oil and consumed with the normal diet, daily twice or thrice for a period of 3-6 months. Ardisia colorata Roxb. U-thum Myrsinaceae Leaves Manipur 10-12 leaves are cooked and eaten daily twice for at least 2 months. Barleria albostellata C.B. Hanu-khulam Acanthaceae Leaves Manipur Soup is prepared by boiling 15-20 fresh leaves in Clarke 50 ml and 20 ml of soup is taken orally, once daily for a period of 4 to 5 months. Bryphytum sensativum (L.) Zarero Oxalidaceae Leaves Manipur, A saline extract of 10-20 ml from matured leaves DC. Arunachal is consumed daily twice for two months. Pradesh, Mizoram Cassia alata L. Mongrangiangtong Caesalpiniaceae Leaves Manipur, Decoction of leaves taken orally twice for 6-8 Arunachal weeks. Pradesh, Mizoram Cassia bicapsularis L. ThaonamCaesalpiniaceae Shoot Manipur Tender shoot of about 100 g per day is cooked and nashangbi eaten for 6 weeks. Cassia occidentalis L. Hant thenga Caesalpiniaceae Bark Manipur, 50 g bark is used to make infusion and is given Arunachal orally daily, for 8-10 weeks. Pradesh, Mizoram Catharanthus roseus (L.) Nayantara Apocynaceae Roots, leaves, Assam, The decoction is prepared from leaves and roots G. Don whole plant Manipur, and 20 ml is taken orally once a day, for eight to Mizoram ten weeks. Cinnamomum tamala T. Lappyrring Lauraceae Leaves Manipur, The powder is made from dried leaves and 5 g per Nees & Eberm. Arunachal day is taken orally for 5-6 weeks. Pradesh, Mizoram Cissampelos pareira L. Tubukilota Menispermaceae Whole plant Assam Whole plant can be used to make decoction and is orally taken daily once for 2-3 months. Clerodendrum viscosum Kuthab-ukabi Lamiaceae Leaf Manipur 50 g of tender leaves is cooked and eaten daily Vent. once for 1/2 months. Coccinia grandis (L.) Kunduli Cucurbitaceae Fruits Assam 1/2 raw fruits are eaten daily and fruits are cooked Voigt and taken orally daily, for 2-3 months. Dillenia pentagyna Roxb. Kaihzawl Dilleniaceae Bark Mizoram The decoction is prepared from the 100g bark, is taken orally once a day for 6-8 weeks. Diospyros malabarica Kendu Ebenaceae Bark Assam Decoction is made by using 50 g of bark, and is Kostel. taken orally at bedtime daily, for 4-6 weeks. Emblica officinalis Gaertn. Amlokhi Euphorbiaceae Leaves Assam Water boiled leaf extract, 40 to 50 ml is taken orally, twice a day for 4 weeks. Thangjing Nymphaeaceae Fruit Manipur 2-3 raw fruits are eaten daily for 2-3 months. Euryale ferox Salisb. Fagopyrum esculentum Wakha-yendem Polygonaceae Shoot Manipur Tender shoot about 100 g is cooked and eaten, Moench once a day, for 6-7 weeks. Ficus auriculata Lour. Hei-it Moraceae Fruit, bark Manipur The decoction is prepared from fruit and bark and 50 ml taken orally once a day for 3-4 months. Ficus benghalensis L. Bot Moraceae Bark Assam Infusion is made by using 100 g bark is taken orally, regularly once a day for 3 months. Ficus semicordata Miq. Theipui Moraceae Bark Mizoram The decoction is prepared from the bark and 2030 ml is taken orally once a day, for 5-6 weeks. Flacourtia jangomas Heitroi Flacourtiaceae Fruit Manipur 2-3 raw fruits are taken orally per day, for 4-5 (Lour.) Raeusch. weeks. Girardinia palmata Saru sorat Urticaceae Young Assam Young inflorescence is boiled in water and taken Gaudich. inflorescence as nettle soup alternate day for 3 months. Hibiscus mutabilis L. Sthalpadma Malvaceae Bark and leaves Assam Decoction is made by using stem, bark and leaves is orally taken daily morning before food for 4 weeks. Hibiscus rosa-sinensis L. Jaba Malvaceae Flowers Assam The flower infusion is taken orally, once a day for 2 months. Ichnocarpus frutescens Dudhkuri lota Apocynaceae Root Assam, The root powder 1 or 2 gm is administered along (L.) R.Br. Manipur, with milk and also; root decoction is taken orally, Arunachal daily once, for 4-6 weeks. Pradesh, Mizoram Inula cappa (Buch.Buarthau Asteraceae Leaves Mizoram Leaves are crushed with Plantago asiatica and Ham.ex D. Don) DC Lobelia angulata and the 20 to 30 ml juice is taken orally once a day, for 6-8 weeks. Kyllinga nemoralis Keya bon Cyperaceae Tuber Assam Decoction is prepared from the roots and 20 ml (Forster) Dandy ex Hutch. per day taken orally for 2-4 weeks. Lepionurus sylvestris Anpangthuam Opiliaceae Leaves Mizoram Leaves are boiled and the water is taken ½ cup (50 Blume ml) once a day, for 6-8 weeks.


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Table 2 (Cont.) Botanical name Mallotus roxburghianus Müll.Arg.

Local name Family Zawngtenawh-lung Euphorbiaceae

Mangifera indica L.

Am

Anacardiaceae

Melothria heterophylla Cogn. Momordica charantia L.

Kabomako

Cucurbitaceae

Tita kerela

Cucurbitaceae

Musa glauca Roxb.

Saisu

Musaceae

Paederia foetida L.

Bhedai lata

Rubiaceae

Phragmites karka (Retz.) Stend. Picrasma javanica Blume

Nalkhagari

Poaceae

Thingdamdawi

Simaroubaceae

Pistia stratiotes L.

Borpuni

Araceae

Portulaca oleracea L.

Leibak-kundo

Portulacaceae

Primula L.

Kengoi

Primulaceae

Punica granatum L.

Dalim

Lythraceae

Saraca asoca (Roxb.) De Wilde Scoparia dulcis L.

Asok

Leguminosae

Seni bon

Scrophulariaceae

Sesbania sesban (L.) Merr. Chuchu-rangmei

Fabaceae

Solena amplexicaulis (Lam.) Gandhi Syzygium cumini (L.) Skeels Tamarindus indica L.

Belipoka

Cucurbitaceae

Kala Jamu

Myrtaceae

Teteli

Caesalpiniaceae

Tectona grandis L. f.

Sagun

Lamiaceae

Terminalia chebula Retz.

Silikha

Combretaceae

Tinospora cordifolia (Willd.) Hook.f. & Thomson Tragia involucrata L.

Sagunilota

Menispermaceae

Dumuni

Euphorbiaceae

Trigonella foenumgraecum L.

Methi

Fabaceae

Vitex peduncularis Wall.

Thingkhawilu

Lamiaceae

Plant part used State found Mode of use and administration Leaves Mizoram The decoction is prepared from leaves and is taken orally ¼ cup (25 ml) twice daily as tea, for 3-4 months. Leaves Assam Decoction is prepared from the leaves is taken orally for 4-6 weeks. Roots Arunachal The decoction is prepared from roots and Pradesh consumed orally once daily, for 6-8 weeks. Fruit Assam, 2-3 fruits are cooked and consumed, and also raw Manipur, fruit juice of 50 ml is taken orally once a day for Arunachal 5-6 weeks. Pradesh, Mizoram Seeds Mizoram The seeds are powdered and 5 to 10 g of powder is taken orally twice a day, for 6-8 weeks. Whole plant Assam The decoction is made from whole plant and is taken orally for 3-4 weeks. Roots, rhizomes Assam Infusion of roots & rhizomes are used once a day for 4 weeks. Bark Mizoram Decoction is prepared from bark, and two tablespoonfuls (15 ml) of decoction are taken orally twice a day, for 6-8 weeks. Whole plant Assam The decoction is prepared from whole plant, and is taken orally once a day, for a period of 4-6 weeks. Whole plant Manipur Boiled soup is prepared from shoot and is taken orally once a day, for 6-8 weeks. Whole plant Manipur 50-100 g plant parts is cooked and eaten daily once, for 8-10 weeks. Seeds Assam Decoction is prepared from seeds and is mixed with honey and taken orally, daily once for 4-6 weeks. Flowers Assam Infusion is made from the dried flowers is taken orally once a day, for 8-10 weeks. Leaves, stems Assam The decoction is prepared from leaves and stems and is taken orally once a day, for 6 weeks. Whole plant Manipur Whole plant part is used to make the decoction and is taken orally once a day, for 2-3 months. Leaves Assam Decoction is prepared from leaves and is orally taken daily for 2 months. Bark Assam The decoction is prepared from bark and is orally taken once a day, for 6 weeks. Leaves Assam The raw leaves are orally taken once or twice daily, and also decoction of the leaves taken orally once a day, for a period of 6-8 weeks. Bark Assam Decoction is prepared from 15g bark and is orally taken once a day, for 3-4 months. Fruits Assam 3-4 raw fruits or cooked with normal daily food are consumed thrice a week, for 6 months. Stem Assam and Aqueous and alcoholic extract of dry stem is Arunachal orally taken and also decoction of stem is taken Pradesh orally once a day for 45-50 days. Roots Assam Decoction is prepared from 40-50 g of root and is taken orally once a day, for 30 to 40 days. Seeds Assam 5-10 g of seeds and also seed powder is added with food and consumed daily twice or thrice, for four to six months. Bark Mizoram The decoction is prepared from bark and orally taken ½ cup (50 ml) twice a day, for 2-3 months.

ducing postprandial glucose levels in diabetic rats. Nutrition Report International 29, 1267-1273 Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403, 853-858 Persaud SJ, A1-Majed H, Raman A, Jones PM (1999) Gymnemasylvestre stimulates insulin release in vitro by increased membrane permeability. Journal of Endocrinology 163, 207-212 Saikia AP, Ryakala VK, Sharma P, Goswami P, Bora U (2006) Ethnobotany of medicinal plants used by Assamese people for various skin ailments and cosmetics. Journal of Ethnopharmacology 106, 149-157 Shane-McWhorter L (2001) Biological complimentary therapies: A focus on botanical products in diabetes. Diabetes Spectrum 14, 199-208 Watkins PB, Whitcomb RW (1998) Hepatic dysfunction associated with trog-

Chang MS, Oh MS, Kim DR, Jung KJ, Park S, Choi SB, Ko B, Park SK (2006) Effects of Okchun-San, a herbal formulation, on blood glucose levels and body weight in a model of Type 2 diabetes mellitus. Journal of Ethnopharmacology 103, 491-495 Chhetri DR, Parajuli P, Subba GC (2005) Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India. Journal of Ehnopharmocology 99, 199-202 Diabetes Medicinal Plant Data-base (2008) Available online: http://www.progenebio.in/dmp/listz.htm Li WL, Zheng HC, Bukuru J (2004) Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. Journal of Ethnopharmacology 92, 1-21 Madar Z (1984) Fenugreek (Trigonella foenum-graecum) as a means of re-


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Medicinal plants used for curing Diabetes in Northeast India. Ryakala et al. How to reference: Author name(s) (2010) Title of chapter. In: Husaini AM (Ed). Medicinal Plants of the Himalayas: Advances and Insights. Global Science Books, UK, pp. X-XX

Young Inflorescence Whole Plant Tuber Stems Shoot Seeds Roots Leaves Fruit Flow er Bulbs Bark 0

2

4

6

8

10

12

14

16

18

Num ber of preparations

Fig. 2 Number of preparations obtained from various plant parts.

Witters L (2001) The blooming of the French lilac. The Journal of Clinical Investigation 108, 1105-1107 Xie JT, Wang A, Mehendale S, Wu J, Aung HH, Dey L, Qiu S, Yuan CS (2003) Anti-diabetic effects of Gymnema yunnanense extract. Pharmacological Research 47, 323-329

litazone. New England Journal of Medicine 338, 916-917 WHO (2008) Diabetes: the cost of diabetes. Available online: http://www.who.int/mediacentre/factsheets/fs312/en/index.html WHO (2008) Title required. Available online: http://www.who.int/diabetes/actionnow/en/mapdiabprev.pdf


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Chemical Composition of Leaf and Flower Essential Oils of Two Thymus spp. from Western Himalaya Ram Swaroop Verma* • Rajendra Chandra Padalia • Amit Chauhan • Ajai Kumar Yadav Central Institute of Medicinal and Aromatic Plants (CIMAP, CSIR), Research Centre, Pantnagar, P.O. - Dairy farm Nagla, Udham Singh Nagar, Uttarakhand - 263149, India Corresponding author: * [email protected]

ABSTRACT Thymus species (Lamiaceae) are considered to be very beneficial whether used as food or as a medicament. Essential oils (EOs) derived from leaves and flowers of Thymus serpyllum and Thymus linearis grown in northern India were analyzed by GC and GC-MS. A total of 37 components forming 94.8-98.4% of EO composition were identified. The EOs of both species were rich in thymol, p-cymene and Jterpinene. Thymol was higher in the EO of T. linearis (74.6-75.8%) compared to T. serpyllum (51.9-70.1%). The amount of thymol methyl ether, p-cymene, 1-octen-3-ol, camphor and borneol was relatively higher in T. serpyllum EO. Further, phenolic monoterpenes were higher in flower EOs of both species than in leaf EOs.

_____________________________________________________________________________________________________________ Keywords: Thymus serpyllum, T. linearis, Hydrodistillation, Essential oil, GC-MS, thymol Abbreviations: GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry

INTRODUCTION The genus Thymus L. (Lamiaceae), commonly known as thyme in English consists of about 215 species of herbaceous perennial and sub-shrubs that have achieved great commercial importance. The Mediterranean region can be described as the centre of the genus (Stahl-Biskup 2002). In India the genus is represented by two species viz. T. linearis (native) and T. serpyllum (exotic) (Jalas 1973). Thyme is one of the most widely used culinary herbs. The dried leaves are used for food flavouring and the source of essential oil (EO) in pharmaceutical and cosmetic industries. A number of benefits in human and animal wellbeing have been associated with the use of thyme EO by the industry (Youdim et al. 2002). At this point, this plant can be considered as a potential impulse of new trends in food, pharmaceutical and cosmetic industries (Echeverrigaray et al. 2001). Recent studies have showed that thyme EOs have strong antibacterial, antifungal, antiviral, antiparasitic and antioxidant activities (Davidson and Naidu 2000; Stahl-Biskup 2002; Parajuli et al. 2005; Dababneh 2007; Al-Fatimi et al. 2010). The antiseptic, antioxidative, insecticidal, preservative and anesthetic properties of thyme EO are owed mainly to the presence of thymol, carvacrol, geraniol and other volatile components (Van-Den Broucke and Lemli 1981). The antioxidant potential of thyme EO has shown the uses of this product by the food industry and its effectiveness as a dietetic supplement (Youdim and Deans 1999). The chemical polymorphism of thyme EO has been reviewed by Stahl-Biskup (1991). The most important components found in this genus are thymol and carvacrol followed by linalool, p-cymene, J-terpinene, borneol, terpinen4-ol and 1,8-cineole (Sfaei-Ghomi et al. 2009). In India, the EO composition of this genus has also been studied on a few occasions (Mathela et al. 1980; Verma et al. 2009a, 2010a). However, detailed research work has not been undertaken so far from this region. The chemical composition of aromatic plants is significantly influenced by the plant part (Wang and Liu 2010), season and plant ontogeny (Hudaib et al. 2002; Jordan et al. 2006; Ebrahimi et al. 2008; Verma et al. 2009b), location of

growing (Cabo et al. 1986), and drying (Venskutonis 1997; Verma et al. 2010b). A literature survey revealed that the EO of leaves and flowers of T. serpyllum and T. linearis have not been studied separately to date, therefore the present paper deals with the detailed analysis of the oils by GC and GC-MS. MATERIALS AND METHODS Plant material The fresh flowering herbs of T. linearis and T. serpyllum were collected during summer (21st April and 25th June, respectively) from the experimental farm of the Central Institute of Medicinal and Aromatic Plants, Research Centre, Purara, Uttarakhand, India. The reproductive (flowers) and vegetative (leaves) parts of both species were separated and dried under shade. The site is located at an altitude of 1250 m in the Kattyur valley, western Himalayas. Climatologically, it is categorized as a temperate zone. The monsoon usually breaks in June and continues up to September.

Extraction of EOs The EO of leaf and flower of both Thymus spp. was extracted separately by hydrodistillation for 3 hrs using a Clevenger-type apparatus (Clevenger 1928). The percentage EO content (v/w) was estimated on a dry weight basis. The oil samples obtained were dehydrated over anhydrous sodium sulphate and kept in a cool and dark place before analyses.

Gas chromatography (GC) The GC analyses of the oil samples was carried out on a PerkinElmer Auto XL GC and Nucon gas chromatograph model 5765 equipped with a FID using two different stationary phases PE-5 (60 m × 0.32 mm; 0.25 μm film coating) and CP-Wax 52 CB (30 m × 0.32 mm × 0.25 μm film thickness) fused silica columns, respectively. Hydrogen was the carrier gas at 1.0 mL/min. Oven temperature programming was done from 70-250°C at 3°C/min for PE-5 and from 70-230°C at 4°C/min for CP-Wax 52 CB. The injector and detector temperatures were 210 and 230°C, respectively.

Received: 5 April, 2010. Accepted: 20 September, 2010.




Table 1 Essential oil composition of leaf and flower of Thymus serpyllum and T. linearis. Compound* RIa RIb

D-Pinene Camphene E-Pinene E-Myrcene D-Terpinene Limonene 1,8 Cineole J-Terpinene p-Cymene D-Terpinolene 3-Octanol (Z)-Linalool oxide 1-Octen-3-ol (E)-Sabinene hydrate D-Copaene Camphor Linalool Bornyl acetate E-Caryophyllene Thymol methyl ether Carvacrol methyl ether Terpinen-4-ol D-Humulene D-Terpineol Borneol Geranial Bicyclogermacrene Geranyl acetate p-Cymen-8-ol Thymyl acetate Carvacryl acetate Caryophyllene oxide Spathulenol Eugenol epi-D-Cadinol Thymol Carvacrol Class composition Monoterpene hydrocarbons Oxygenated monoterpenes Phenolic monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Aliphatic Total identified Essential oil (%) #

1026 1065 1105 1158 1177 1185 1196 1240 1271 1278 1394 1435 1448 1463 1481 1507 1536 1585 1594 1594 1604 1606 1660 1682 1695 1740 1747 1750 1846 1867 1880 2004 2143 2161 2178 2196 2233

941 954 982 994 1019 1034 1038 1065 1029 1089 1001 1070 986 1069 1374 1147 1103 1285 1418 1220 1230 1180 1457 1192 1167 1277 1495 1373 1185 1350 1368 1584 1579 1362 1643 1306 1320

A 1.1 1.1 t 0.1 0.1 6.9 9.4 t t 2.6 0.6 3.5 0.1 1.1 10.4 0.1 2.5 0.1 2.5 0.1 0.8 0.9 0.1 0.1 51.9 t

Peak area (%) Thymus serpyllum B C D 0.4 0.8 1.1 0.4 0.1 0.2 T t t 0.2 0.8 0.6 0.4 0.9 0.8 t t t t t t 6.2 9.2 6.8 7.0 3.1 3.5 t t t 0.1 t t t 3.1 1.3 1.3 0.6 0.4 0.6 t 2.5 0.2 0.4 t t 0.1 t 1.5 1.2 1.5 5.8 2.3 2.6 0.1 0.1 0.1 2.8 2.9 3.0 t t 0.2 0.5 0.5 2.8 1.2 1.4 t t 1.1 0.1 0.7 1.5 1.2 1.6 t 0.3 0.2 t t t t t t t t t t 59.5 70.1 66.8 0.6 0.8 1.1

Thymus linearis B D 0.3 0.3 t t t t 0.2 0.2 0.4 0.4 t t t t 9.5 8.2 5.1 3.2 t t t t t 0.4 0.5 t t t t t 0.6 1.0 t t t 2.0 2.3 t t 0.8 0.8 1.8 1.9 0.9 2.5 t 0.1 t t t t 0.1 0.1 0.1 74.6 75.8 1.3 1.0

18.7 10.4 62.5 1.9 2.6 96.1 0.80

14.6 10.5 66.0 2.6 t 3.2 96.9 0.94

15.5 4.1 75.9 2.4 0.1 t 98.0 2.50

14.9 6.7 73.3 1.3 t 1.3 97.5 0.39

13.0 7.7 70.6 2.2 t 1.3 94.8 1.30

12.3 6.1 77.0 2.9 0.1 t 98.4 3.33

*

Mode of identification: Retention Index (RI), MS (GC-MS); a Retention indices on CP-WAX 52 CB column; Retention indices on PE-5 column; A: Shade dry leaves (vegetative stage); B: Shade dry leaves (flowering stage); C: Fresh flower; D: Shade dry flower; # Dry weight basis except ‘C’ which is calculated on fresh weight basis; t: Trace (