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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: dr.amjadhusaini@hotmail.com
“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
Muham m ad Iqbal , PhD, FNASc
Professor
Department of Botany
J AM I A H AM DARD
(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: iqbalg5@yahoo.co.in
Create PDF files without this message by purchasing novaPDF printer (http://www.novapdf.com)
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
1
Manoj Kumar Goel, Shilpa Goel, Suchitra Banerjee, Karuna Shanker, Arun Kumar Kukreja (India) Agrobacterium
rhizogenes-Mediated Transformed Roots of Rauwolfia serpentina for Reserpine Biosynthesis
8
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
15
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.
20
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
28
Mukesh Joshi, Munesh Kumar (India), Rainer W. Bussmann (USA) Ethnomedicinal Uses of Plant Resources of the Haigad
Watershed in Kumaun Himalaya, India
43
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
64
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
69
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
73
Amit Chauhan, Ram Swaroop Verma (India) Cultivation Potential of Three Rose-scented Geranium (Pelargonium
graveolens) Cultivars in the Kumaon Region of Western Himalayas
77
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
80
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
84
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
87
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
90
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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
®
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: * mondaltk@yahoo.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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.
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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
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Picroside-I Std
Picroside-I (Kuppup)
2 8 1 .7
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
1 .0 0
AU
AU
0.05 0
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
442.6
428.2
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 2 0 .6
2 .0 0
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.
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).
Effect of PGRs on multiplication and elongation
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-
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Micropropagation of an elite line of Picrorhiza scrophulariiflora. Bantawa et al.
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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
ɚ
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
ɚ
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
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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
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.
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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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * mkgoel20@gmail.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Original Research Paper
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 8-14 ©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
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
X 100
Dry matter (%) =
Biomass fresh weight
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
9
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.
B
C
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).
L
1
2
3
4
5
6
7
8
9 10
11 12 13
14
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
16 17 18 19
20
21 22
23
24 25
26 27
C A
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.
L
1
2
3
4
5
6
7
8
9 10
11 12
13
14
15 16 17 18
19 20 21 22 23 24
25
26 27
C A
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
10
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 8-14 ©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
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
Dry wt. (g)
0.50 ± 0.057
0.73 ± 0.023
0.66 ± 0.12
6
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
60
50
40
30
20
10
0
1
2
3
4
5
4
6
8
10
12
Growth Index
26.802
41.66
45.71
58.572
58.286
SD
3.172
3.778
3.378
1.836
2.178
weeks
Weeks
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
11
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
SM14
SM19
SM21
SM28
SM30
Reserpine content (% DW)
0.06
0.05
0.04
0.03
0.02
0.01
0
4
6
8
10
12
Weeks
Fig. 5 Reserpine content in hairy root clones of R. serpentina at different growth periods.
Reserpine content (% DW)
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
Hairy root clones
Fig. 6 Variation in reserpine content in different hairy root clones after 10 weeks of growth.
A
B
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
12
40
36
SM
33
SM
32
SM
30
SM
29
SM
28
SM
26
SM
22
SM
21
SM
19
SM
17
SM
16
SM
15
SM
14
SM
13
SM
12
SM
SM
9
8
7
6
5
4
11
M
S
SM
SM
SM
SM
SM
SM
3
SM
M
S
S
M
1
2
0
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 8-14 ©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
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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * ashupov@yahoo.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Original Research Paper
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 15-19 ©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
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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.
Unique bands of A. ferox
Unique bands of
Primer
(size in bp)
A. heterophyllum
combination
(size in bp)
MseI/EcoRI
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
17
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 15-19 ©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
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
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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
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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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * rasoolrafiya@gmail.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Original Research Paper
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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
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:
controlabsorbance - testsample absorbance ½
%inhibition = ®
¾ ×100
controlabsorbance
¯
¿
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
controlabsorbance - testsample absorbance ½
%inhibition = ®
¾ ×100
controlabsorbance
¯
¿
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-
controlabsorbance - testsample absorbance ½
%inhibition = ®
¾ ×100
controlabsorbance
¯
¿
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
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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)
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:
Antioxidant activity
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.
controlabsorbance - testsample absorbance ½
%inhibition = ®
¾ ×100
controlabsorbance
¯
¿
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 anti- oxidant 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.
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 20-27 ©2010 Global Science Books
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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).
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.
the second stage. Furthermore secondary product was much
more stable for a period of time.
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.
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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
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).
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-
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
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 20-27 ©2010 Global Science Books
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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
Inhibition
Inhibition
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).
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
C1=10 μg
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).
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-
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-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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
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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
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * ripukunwar@gmail.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Original Research Paper
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 28-42 ©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
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-
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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.
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 28-42 ©2010 Global Science Books
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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).
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).
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),
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
bacterial, anti-inflammatory, antispasmodic, diuretic (Singh
1986).
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).
#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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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 28-42 ©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
(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).
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).
*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).
*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-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 28-42 ©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
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-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
35
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
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-
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).
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
36
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 28-42 ©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
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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * muneshmzu@yahoo.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 43-46 ©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
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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
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
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
45
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medicinal plants on priority basis (Chettri et al. 2005).
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Kirtikar KR, Basu BD (1933) Indian Medicinal Plants (Vols II, 2nd Edn), Lalit
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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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * praveenkumarsharma11@rediffmail.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©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
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
to leucocyanadin in the outer bark and polymeric anthocyanidins in the inner bark.
and fungal growth
Infusion of the bark is aromatic, antiseptic and used as a carminative.
(Bhurja-granthi)
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
48
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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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
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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Traditional therapeutic uses of plant diversity of Parvati Valley. Sharma et al.
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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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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
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©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
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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 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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©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
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
Withania somnifera
Ashganth
Woodfordia fruticosa
Dhai
Zehneria umbellata
Jangli-kakri
Bana
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.
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
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-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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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
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 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
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Timber/ Furniture
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Ornamental landscape
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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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
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Fuel wood
Timber/ Furniture
Table 2 (Cont.)
Latin name
Ornamental landscape
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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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
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Fodder
Fuel wood
Timber/ Furniture
Table 2 (Cont.)
Latin name
Ornamental landscape
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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Viola canescens
Viola kunawarensis
Viola serpens
Vitex negundo
Withania somnifera
Woodfordia fruticosa
Wulfenia amherstiana
Xanthium strumarium
Zehneria umbellata
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Medicinal and Aromatic
Bee flora
Tans and dyes
Gums and resins
Fibre and flosses
Fodder
Fuel wood
Timber/ Furniture
Table 2 (Cont.)
Latin name
Ornamental landscape
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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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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 47-63 ©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
A
B
F
C
D
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J
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K
L
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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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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
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).
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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
®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * ubora@iitg.ernet.in or ubora@rediffmail.com
† 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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 64-68 ©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
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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65
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
The decoction is prepared from leaves and roots
Catharanthus roseus (L.)
Nayantara
Apocynaceae
Roots, leaves,
Assam,
and 20 ml is taken orally once a day, for eight to
G. Don
whole plant
Manipur,
ten weeks.
Mizoram
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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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 (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
orally taken and also decoction of stem is taken
Arunachal
orally once a day for 45-50 days.
Pradesh
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
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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-
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(2006) Effects of Okchun-San, a herbal formulation, on blood glucose levels
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Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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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.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
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: * rswaroop1979@yahoo.com
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.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Original Research Paper
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 69-72 ©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
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 (<0.10%)
b
standards or known EO constituents, MS Library search (NIST
and WILEY), by comparing with the MS literature data (Davies
1990; Adams 1995). The relative amounts of individual components were calculated based on the GC peak area (FID response)
without using a correction factor.
The injection volume was 0.02 μL neat (syringe: Hamilton 1.0 μL
capacity, Alltech USA) and the split ratio was 1: 30.
Gas chromatography-mass spectrometry (GC-MS)
GC-MS analysis of the EO samples was carried out on a Perkin
Elmer AutoSystem XL GC interfaced with a Turbomass Quadrupole Mass Spectrometer fitted with an Equity-5 fused silica capillary column (60 m u 0.32 mm i.d., film thickness 0.25 μm) The
oven temperature was programmed from 60-210RC at 3RC/min
using helium as the carrier gas at 1.0 mL/min. The injector temperature was 210RC, injection volume 0.1 μL prepared in n-hexane,
split ratio 1: 40. MS were taken at 70 eV with a mass scan range of
40-450 amu.
RESULTS AND DISCUSSION
The EOs were obtained by hydro-distillation from shadedried leaves and flowers of T. serpyllum and T. linearis and
subsequently analyzed by GC (RI) and GC-MS. The identified components with their relative percentages are reported in Table 1. The percentage EO content (v/w) in the
shade-dried leaves of T. serpyllum varied from 0.8% (vegetative stage) to 0.94% (flowering stage), but was 0.39% in
fresh and 1.3% in shade-dried flowers. However, the EO
content was higher in the shade-dried leaves (2.5%) and
flowers (3.33%) of T. linearis than of T. serpyllum. In total,
36 constituents representing 94.8-97.5% of T. serpyllum and
33 constituents forming 98.0-98.4% of T. linearis EO were
Identification of components
Constituents were identified on the basis of a Retention Index (RI,
determined with reference to homologous series of n-alkanes, C9C24, under identical experimental conditions, co-injection with
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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GC-MS analysis of leaf and flower volatiles of Thymus spp. Verma 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 Essential oil composition of Thymus spp.
Species
Country
Thymol (%)
Thymus serpyllum
India
60.0
T. serpyllum
India
58.8
T. serpyllum
Pakistan
53.3
T. serpyllum
Iran
18.7
T. vulgaris
Brazil
44.7
T. vulgaris
Turkey
46.2
T. vulgaris
Cuba
36.6
T. vulgaris
Cuba
37.4
T. vulgaris
Spain
36.3
T. linearis
India
52.3-66.6
T. thracicus
Turkey
15.7
T. longicaulis
Turkey
16.7
T. pseudopulegioides
Turkey
22.1
T. kotschyanus
Iran
38.0
T. hyemalis
Spain
26.2
T. munbyanus
Algeria
37.7
T. caramanicus
Iran
5.3
T. numidicus
Algeria
51.0
T. fontanesii
North Africa
67.8
T. pulegioides
Italy
26.3
T. alpestris
Slovakia
41.0
T. alpestris
Slovakia
2.4
Carvacrol (%)
2.0
1.0
10.4
0.4
2.4
2.4
6.5
4.4
2.0
1.0-5.3
18.2
1.9
2.3
14.2
1.0
8.4
68.9
9.4
1.7
4.7
3.6
47.0
identified and quantified. Although, the qualitative composition of both the EOs was almost the same there was considerable variation in the quantitative composition due to
plant part and species.
The major components of the T. serpyllum EO were
thymol (51.9-70.1%), J-terpinene (6.2-9.2%), p-cymene
(3.1-9.4%), thymol methyl ether (2.3-10.4%), camphor
(0.2-3.5%), 1-octen-3-ol (1.3-3.1%), terpinen-4-ol (2.53.0%), borneol (1.2-2.8%), geranyl acetate (0.9-1.6%), caryophyllene (1.1-1.5%), D-pinene (0.4-1.1%), bicyclegermacrene (0.1-1.1%), camphene (0.1-1.1%) and carvacrol
(<0.1-1.1%). However, the amount of thymol, -terpinene,
-terpinene, -myrcene and p-cymen-8-ol were higher in
fresh flowers of T. serpyllum than the dried leaves and
flowers. Furthermore, the percentage of thymol methyl
ether, p-cymene and camphor was higher in the leaves
collected at the vegetative stage. On the other hand, 1octen-3-ol, borneol and bicyclogermacrene were relatively
higher in the leaves collected at the flowering stage of T.
serpyllum. The major components of the EO from leaves
and flowers of T. linearis were almost the same as from T.
serpyllum. Nevertheless, the percentage of thymol was
higher in the leaves (74.6%) and flowers (75.8%) of T. linearis than of T. serpyllum. Other major components of T.
linearis leaves and flowers were J-terpinene (9.5 and 8.2%),
p-cymene (5.1 and 3.2%), terpinen-4-ol (2.0 and 2.3%),
bicyclogermacrene (1.8 and 1.9%), p-cymen-8-ol (0.9 and
2.5%) and carvacrol (1.3 and 1.0%).
It was interesting to note that the phenols and alcohols
(thymol, p-cymen-8-ol and terpinen-4-ol) accumulated
more in the reproductive part, while thymol methyl ether
and p-cymene accumulated more in the vegetative part of
both Thymus species. Thyme EO with high thymol content
strongly inhibited bacterial and fungal growth (Broucke
1983; Davidson and Naidu 2000; Dorman and Deans 2000;
Iten et al. 2009). Thus, considering the compositional variation, it could be said that the flowers would be of more
medicinal worth than leaves because the former possessed
more phenolic monoterpenes than the latter.
The EO composition of genus Thymus has been evaluated from various countries and majority of its members
were dominated by phenolic monoterpene viz. thymol or
carvacrol (Table 2). The present study and earlier reports
from India showed that the EO of Thymus spp contained
thymol as principal component and percentage of this component varied from 51.9 to 75.8% with the maximum in T.
linearis. Further, the Table 2 clearly indicated that the percentage of thymol observed in Indian Thymus was quite
p-Cymene (%)
5.7
8.8
20.7
18.6
9.9
17.6
26.1
27.8
1.8-21.6
25.4
15.0
13.7
2.2
30.4
14.2
6.0
0.5
13.0
19.9
8.8
6.6
Ȗ-Terpinene (%)
3.4
22.7
16.5
14.1
17.6
0.9
13.1
1.9-12.5
11.1
1.8
1.3
0.9
14.3
10.1
4.6
15.9
7.9
7.3
Reference
Mathela et al. 1980
Verma et al. 2009a
Ahmad et al. 2006
Sefidkon et al. 2004
Porte and Godoy 2008
Ozcan and Chalchat 2004
Pino et al. 1997
Perez et al. 2007
Arraiza et al. 2009
Verma et al. 2010a
Akcin 2006
Akcin 2006
Akcin 2006
Rustaiyan et al. 1999
Jordan et al. 2006
Hazzit et al. 2006
Ebrahimi et al. 2008
Saidj et al. 2008
Ghannadi et al. 2004
Martino et al. 2009
Martonfi 1992
Martonfi 1992
higher than that reported from Iran, Brazil, Turkey, Cuba,
Spain, Algeria, Italy and Slovakia. In addition to this, the
chemical profile of our tested EOs was found in good
agreement with the quality standards of European Pharmacopoeia (EP, 2002) for thyme oil. Thymol content (EP limits
36.0-55%) was found either well within the limit or higher
than the limit of EP. Therefore, due to high thymol and EO
content and hence; therapeutic potential, Indian thyme (T.
linearis) can be considered as better option of common
thyme oil (T. vulgaris).
ACKNOWLEDGEMENTS
The authors are thankful to Director, Central Institute of Medicinal
and Aromatic Plants (CIMAP, CSIR), Lucknow, U.P., India for
providing financial support under Young Scientist Project
(MLP09.19).
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Chemical Profiling of Mentha spicata L. var. ‘viridis’
and Mentha citrata L. Cultivars at Different Stages
from the Kumaon Region of Western Himalaya
Ram Swaroop Verma* • Rajendra Chandra Padalia • Amit Chauhan
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: * rswaroop1979@yahoo.com
ABSTRACT
Two spearmint (Mentha spicata L. var. ‘viridis’) cultivars viz. ‘Neerkalka’ and ‘Supriya’ and one bergamot mint (Mentha citrata L.)
cultivar ‘Kiran’ cultivated in the Kumaon region of northern India were investigated for their essential oil content and composition at
different stages of crop growth. Essential oil content and composition were both affected by crop age in all cultivars. All the cultivars
accumulated maximum essential oil at 150 days after transplanting. The percentage carvone in ‘Neerkalka’ was higher at 90 days (67.0%)
followed by the 150-days-old crop (61.68%), while in ‘Supriya’, carvone concentration increased at 150 days (72.47%). In ‘Kiran’,
linalool and linalyl acetate were highest in 150- and 180-days-old crops, respectively.
_____________________________________________________________________________________________________________
Keywords: composition, essential oil content, Lamiaceae, Mentha spp, plant age
Abbreviations: GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry
INTRODUCTION
MATERIALS AND METHODS
Mints comprise a group of species of the genus Mentha
(family Lamiaceae). The members of this genus are widely
distributed in semi-temperate to tropical agroclimates. The
aerial parts of the herb on distillation yields essential oil
(EO) containing a large variety of aroma chemicals in varying amounts, such as menthol, menthone, iso-menthone,
menthofuran, carvone, linalool, linalyl acetate and piperitenone oxide used in pharmaceutical, food, flavour, cosmetics,
beverages and allied industries (Spencer et al. 1990; Sharma and Tyagi 1991; Kokkini et al. 1995; Singh et al. 2005;
Chowdhury et al. 2007; Zheljazkov et al. 2010b).
The spearmint and bergamot mint EOs are obtained
from Mentha spicata L. var. viridis and Mentha citrata L.
plants, respectively and are extensively used in the flavour
and cosmetic industries. These mint species are being cultivated in several countries. The annual world production of
M. spicata and M. citrata EOs are 4500 and 4,000 tonnes,
respectively, while India produces 2000 t/year of each (Bahl
et al. 2000; Khanuja 2007). Large efforts to genetically improve mint have resulted in the development of a number of
superior varieties such as ‘Neerkalka’, ‘Supriya’ and ‘Kiran’
which are mainly cultivated in the north Indian plains (Virmani et al. 1987, 1988; Patra et al. 2001; Patra and Kumar
2005; Chauhan et al. 2009).
To find a new ecological area for cultivation, prevalent
cultivars of spearmint and bergamot mint were introduced
into the Kumaon region of Uttarakhand. It is well known
that yield and composition of EO is strongly influenced by
the development stage of the plant, which further depends
on genotype/chemotype and environmental settings (Shah
and Gupta 1989; Chalchat et al. 1997; Bahl et al. 2000;
Zheljazkov et al. 2010a). Therefore, in this study, the qualitative and quantitative performance of three cultivars belonging to two species viz. M. spicata, and M. citrata have
been examined at different developmental stages from the
hilly region of Uttarakhand.
Plant material and isolation of EO
The origin and special features of the spearmint cultivars (‘Neerkalka’ and ‘Supriya’) and bergamot mint cultivar (‘Kiran’) are
summarized in Table 1. All the cultivars were planted in January
using vegetative propagules at an inter-row spacing of 50 cm. The
crops were raised following normal agricultural practices at the
experimental farm of the Central Institute of Medicinal and Aromatic Plants, Research Centre, Purara, Bageshwar. The experimental site is located at an elevation of 1250 m with a temperate
mild climate. Sampling began at 90 days after planting (DAP) and
took place every month at 30-days intervals up to 180 DAP.
Freshly harvested samples of all cultivars were hydrodistilled
separately in triplicates in a Clevenger-type apparatus for 3 hrs to
extract the EO. The EOs were collected, measured, dehydrated by
anhydrous sodium sulphate and kept in a cool and dark place prior
to analysis.
GC and GC-MS analysis
The GC analysis of the oil samples was carried out on a Nucon
5765 gas chromatograph equipped with an FID using two different
stationary phases, BP-20 (30 m u 0.25 mm i.d., 0.25 μm film
coating) and DB-5 (30 m u 0.32 mm i.d., 0.25 μm film coating)
fused silica capillary columns. Hydrogen was used as the carrier
gas at 1.0 ml/min. The temperature programme was: 70-230°C at
4°C/min (for BP-20) and from 60-210°C at 3°C/ min (for DB-5).
The injector and detector temperatures were 210 and 230°C, respectively. The injection volume was 0.02 μL neat (syringe: Hamilton 1.0 μL capacity, Alltech USA) and the split ratio was 1:40.
GC-MS analysis of the EOs was carried out on a PerkinElmer Turbomass Quadrupole Mass spectrometer fitted with PE-5
fused silica capillary column (50 m × 0.32 mm; 0.25 μm film
coating). The column temperature was programmed from 100 to
280°C at 3°C/min using He as the carrier gas at a flow rate of 1
mL/min. The injector temperature was 220°C and MS conditions
were: EI mode 70 eV, ion source temperature 250°C. Identification
Received: 28 November, 2010. Accepted: 3 December, 2010.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 73-76 ©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
Table 1 Origin and characteristics of different mint cultivars.
Plant
Cultivar
Origin
Mentha spicata L. var. ‘viridis’ ‘Neerkalka’ Interspecific hybridization between
M. arvensis L. cv. ‘Kalka’ (2n=96)
and M. spicata cv. ‘Neera’(2n=24)
Mentha spicata L. var. ‘viridis’ ‘Supriya’
An northern Himalayan accession
Mentha citrata L.
‘Kiran’
Induced mutagenesis
Characteristics
Essential oil (0.8%), carvone (50-55%)
with trace
quantity of menthol
Essential oil (0.35%), carvone (65%)
Essential oil (0.5%), linalool (48%),
linalyl acetate (37%)
Reference
Patra et al. 2001; Patra
and Kumar 2005
Virmani et al. 1987
Virmani et al. 1988
Table 3 Chemical profile of Mentha spicata L. var. ‘viridis’ cultivar
‘Supriya’ at different stages of crop growth.
Age of plant (DAP)
Compound
RI
90
120
150
180
D-Pinene
1026
1.05
0.77
0.92
1.21
E-Pinene
1105
0.84
0.86
0.85
1.02
Sabinene
1119
0.24
0.26
0.28
0.35
E-Myrcene
1158
4.36
2.61
2.21
1.99
D-Terpinene
1177
0.19
0.15
0.19
0.42
Limonene
1194
11.29
12.61
11.72
12.74
1,8 Cineole
1204
1.49
1.58
1.49
2.02
E-Phellandrene
1206
0.29
0.11
0.10
t
(Z)--Ocimene
1210
0.82
0.44
0.37
0.83
(E)--Ocimene
1251
0.44
0.22
0.12
t
p-Cymene
1271
0.23
t
0.60
t
Terpinolene
1279
t
3-Octyl acetate
1345
0.84
0.60
3-Octanol
1394
1.14
1.41
1.67
2.21
(E)-Sabinene hydrate
1463
0.36
1.69
2.65
1.78
-Bourbonene
1515
1.09
0.46
0.47
0.21
Linalool
1530
0.12
0.26
t
t
-Caryophyllene
1589
1.35
0.93
0.1
t
Terpinen-4-ol
1606
3.21
0.59
0.16
0.31
1610
t
2.17
0.16
1.75
(Z)-Dihydrocarvone
(E)-Dihydrocarvone
1624
0.12
t
t
t
(E)-Dihydrocarvyl acetate 1633
0.21
t
t
0.37
(E)--Farnesene
1662
t
t
t
t
D-Terpineol
1701
1.07
1.78
0.64
1.41
Germacrene-D
1721
0.39
0.30
t
t
Carvone
1751
62.92
64.77
72.47
62.55
(Z)-Carvyl acetate
1777
0.24
0.44
t
0.76
(E)-Carveol
1825
1.59
2.00
1.00
0.91
(Z)-Carveol
1866
0.31
0.38
0.57
1.08
(Z)-Jasmone
1968
2.16
0.61
0.86
t
Piperitenone epoxide
2004
t
0.47
t
Viridiflorol
2102
0.72
0.80
0.38
0.42
0.46
0.50
0.72
0.70
Essential oil content (%)*
Table 2 Chemical profile of Mentha spicata L. var. ‘viridis’ cv. ‘Neerkalka’ at different stages of crop growth.
Age of plant (DAP)
Compound (%)
RI
90
120
150
180
D-Pinene
1026
0.50
0.39
0.68
0.77
E-Pinene
1105
0.27
0.26
0.30
0.40
Sabinene
1119
t
t
t
t
E-Myrcene
1158
0.25
0.31
0.61
0.64
Limonene
1194
10.70
14.30
21.01
24.81
1,8 Cineole
1204
0.20
0.24
0.21
0.25
3-Octanol
1394
1.52
1.06
0.88
0.19
Menthone
1460
0.32
0.27
0.20
(E)-Sabinene hydrate
1463
0.69
0.22
0.20
0.41
iso-Menthone
1488
0.74
0.48
0.47
0.89
-Bourbonene
1515
0.60
0.54
0.62
0.89
Linalool
1530 t
t
0.23
t
-Caryophyllene
1589 0.43
t
0.60
0.78
Terpinen-4-ol
1606 0.1
t
(Z)-Dihydrocarvone
1610 0.97
1.87
1.00
1.58
(E)-Dihydrocarvone
1624 0.10
0.10
t
t
(E)-Dihydrocarvyl acetate 1633 0.15
0.25
0.43
0.39
Menthol
1646
0.65
0.74
2.63
0.80
Germacrene-D
1721
t
t
t
t
Carvone
1751 67.00
59.62
61.68
57.49
(Z)-Carvyl acetate
1777
0.55
0.90
0.19
0.12
(E)-Carveol
1825
1.27
1.75
1.15
0.85
(Z)-Carveol
1866
1.77
2.26
3.12
3.32
(Z)-Jasmone
1968 1.03
t
t
t
Piperitenone oxide
2004
2.50
1.21
t
t
0.38
0.60
0.75
0.70
Essential oil content (%)*
RI: retention indices on BP-20 column; t: trace (<0.10%)
DAP: Days after planting; *v/w
was done on the basis of retention index (determined with reference to homologous series of n-alkanes (C9-C24) under identical
experimental conditions), co-injection with known compounds, an
MS Library search (NIST and WILEY), by comparing with the
MS literature data (Davies 1990; Adams 1995). The retention
times of standards/marker constituents of known EOs were also
used to confirm the identities of constituents. The relative amounts
of individual components were calculated based on GC peak area
(FID response) without using a correction factor.
RI: retention indices on BP-20 column; t: trace (<0.10%)
DAP: Days after planting; *v/w
180 DAP. Furthermore, the percentage of -myrcene (0.250.64%), limonene (10.7-24.81%) and (Z)-carveol (1.773.22%) increased while that of 3-octanol (1.52-0.19%) and
menthone (0.32-0%) decreases with advancing crop age.
The menthol concentration in ‘Neerkalka’ ranged from 0.65
to 2.63%, which was not reported in the other cultivars of M.
spicata. The presence of a relatively lower concentration of
carvone (compared to other spearmint cultivars), menthone
and menthol in trace quantities is due to the hybrid nature
(hybrid between M. arvensis L. cv. ‘Kalka’ and M. spicata
cv. ‘Neera’) of ‘Neerkalka’ (Patra et al. 2001). On the other
hand, in ‘Supriya’, the percentage of carvone (62.5572.47%) and (E)-sabinene hydrate (0.36-2.65%) increased
as crop age progressed, becoming highest at 150 DAP; 3octanol and (Z)-carveol also showed a similar trend but
their highest amount was recorded at 180 DAP (2.21 and
1.08%, respectively). A decreasing trend was recorded for
-myrcene (4.36-1.99%), E-bourbonene (1.09-0.21%) and
E-caryophyllene (1.35%-trace). Moreover, germacrene-D,
3-octyl acetate, terpinen-4-ol and (Z)-jasmone reached the
highest levels at 90 DAP, while limonene 1,8 cineole and
(Z)-carvyl acetate were highest at 180 DAP. The major
RESULTS AND DISCUSSION
The EO content and composition of spearmint, and bergamot mint cultivars were found to vary with respect to crop
age (Tables 2-4). The EO content varies from 0.38 to 0.75%,
0.46 to 0.72%, and 0.34 to 0.57% in ‘Neerkalka’, ‘Supriya’
and ‘Kiran’, respectively during different stages. Interestingly, in all the cultivars maximum EO content was obtained when the crop was harvested at 150 DAP; thereafter,
it showed a downward trend. A similar trend of EO accumulation was also reported in these cultivars from Indo-gangetic plains (Bahl et al. 2000).
The EOs obtained from different cultivars at different
crop ages were analyzed by GC and GC-MS. A total of 25,
32 and 25 compounds were identified in the EOs of ‘Neerkalka’, ‘Supriya’ and ‘Kiran’, respectively (Tables 2-4).
‘Neerkalka’ and ‘Supriya’ were mainly composed of carvone and limonene. In ‘Neerkalka’, the carvone content
ranged between 57.49-67.0% with the highest amount at 90
DAP followed by 150 DAP (61.68%), the lowest value at
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Chemical profile of spearmint and bergamot mint. Verma 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
ACKNOWLEDGEMENTS
Table 4 Chemical profile of Mentha citrata L. cultivar ‘Kiran’ at different
stages of crop growth.
Age of plant (DAP)
Compound
RI
90
120
150
180
E-Myrcene
1158
1.68
1.08
1.93
2.57
Limonene
1194
0.34
0.26
0.37
0.74
1,8 Cineole
1204
0.35
0.39
t
t
(Z)-E-Ocimene
1210
1.09
0.57
1.18
0.98
-Terpinene
1239
0.21
t
0.36
0.42
(E)-E-Ocimene
1251
0.10
t
t
t
p-Cymene
1271
t
t
0.36
0.42
Terpinolene
1279
t
0.24
0.23
Menthone
1460
t
t
t
t
(E)-Linalool oxide
1450
t
Linalool
1530
42.04
40.32
46.31
32.86
Linalyl acetate
1546
19.27
29.22
24.51
37.72
E-Caryophyllene
1589
1.53`
1.06
1.14
1.66
Menthol
1646
t
t
0.33
0.45
(E)-E-Farnesene
1662
t
t
t
t
D-Humulene
1675
t
t
0.33
0.45
D-Terpineol
1701
4.08
4.61
3.46
2.90
-Cadinene
1739
1.00
1.15
1.17
0.89
-Cadinene
1742
t
t
Geranyl acetate
1762
2.27
2.83
2.14
2.26
Citronellol
1782
t
0.13
0.12
Nerol
1804
1.09
1.01
1.33
0.76
Geraniol
1858
2.66
3.21
2.36
2.43
Caryophyllene oxide
1980
t
0.12
0.99
0.40
Viridiflorol
2102
1.94
3.46
0.73
0.63
0.34
0.50
0.57
0.52
Essential oil content (%)*
The authors are grateful to the Director, CIMAP, Lucknow (UP),
India, for continuous encouragement and providing necessary facilities.
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Verma RS, Rahman L, Verma RK, Chauhan A, Yadav AK, Singh A (2010c)
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RI: retention indices on BP-20 column; t: trace (<0.10%)
DAP: Days after planting; *v/w
constituents of ‘Kiran’ EO were linalool (32.86-46.31%),
linalyl acetate (19.27-37.72%), D-terpineol (2.90-4.61%),
geranyl acetate (2.14-2.83%), geraniol (2.36-3.21%) and myrcene (1.08-2.57%). Although there was no regular trend
for any component with respect to crop age in ‘Kiran’,
nevertheless linalyl acetate, -myrcene, -caryophyllene,
limonene, -terpinene and D-humulene were highest at 180
DAP, whereas linalool, (Z)--ocimene and terpinolene were
highest at 150 DAP. Furthermore, the amount of D-terpineol,
viridiflorol geraniol, geranyl acetate, 1,8 cineole and citronellol were highest at 120 DAP. The EOs obtained from different crop ages thus showed considerable variation in content and composition of all three cultivars. This could be
due to expression of different genes at different developmental stages of the plant and further by the environmental
factors arising from seasonal variations (Verma et al. 2010a,
2010b, 2010c, 2010d).
Carvone-rich spearmint has been investigated in India
as well as in other countries. In India, under the climatic
conditions of indo-gangetic plains the carvone percentage
was varied from 45.9-71.6% in cultivar ‘Neerkalka’ and
53.3-77.1% in cultivar ‘Supriya’. Interestingly, both cultivars were noted to have higher percentage of carvone at
early stages of crop growth. Similar, pattern was also noted
with cultivar ‘Neerkalka’ in present study, however, the
trend of carvone accumulation in cultivar ‘Supriya’ was
entirely different in present study than that observed in indo
gangetic plains (Bahl et al. 2000). Further, the carvone percentage was varied from 46.4-53.3% in spearmint grown at
different locations in Egypt (El-Wahab and Mohamed 2009).
However, the spearmint grown in Iran was found to contain
relatively lesser amount of carvone (22.4%) (Hadjiakhoondi
et al. 2000). The percentages linalool and linalyl acetate in
bergamot mint was increased towards crop maturity (Bahl
et al. 2000). However, no any regular trend was noticed
with these constituents in present study. Finally, it was concluded that the yield and chemical composition of spearmint and bergamot mint essential oils was strongly dependent on developmental stage of the plant, and therefore harvesting time is one of the most important factors influencing the oil quality.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
75
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 73-76 ©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
Zheljazkov VD, Cantrell CL, Astatkie T, Ebelhar MW (2010b) Productivity,
oil content, and composition of two spearmint species in Mississippi. Agronomy Journal 102 (1), 129-133
Zheljazkov VD, Cantrell CL, Astatkie T, Ebelhar MW (2010a) Peppermint
productivity and oil composition as a function of nitrogen, growth stage, and
harvest time. Agronomy Journal 102 (1), 124-128
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76
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Cultivation Potential of Three Rose-scented
Geranium (Pelargonium graveolens) Cultivars
in the Kumaon Region of Western Himalayas
Amit Chauhan* • Ram Swaroop Verma
Central Institute of Medicinal and Aromatic Plants, Research Centre, Pantnagar, P.O. - Dairy Farm Nagla, Udham Singh Nagar, Uttarakhand 263149, India
Corresponding author: * amitcimap@rediffmail.com
ABSTRACT
A field experiment was conducted to evaluate the production potential of three cultivars of rose-scented geranium viz., ‘Bourbon’, ‘CIMPawan’ and ‘Kelkar’ in the temperate region of Uttarakhand. ‘CIM-Pawan’ had the highest essential oil yield (103.87 g plot-1) followed by
‘Kelkar’ (79.93 g plot-1) and ‘Bourbon’ (72.01 g plot-1). The essential oil profile of ‘Bourbon’ was rich (relative percentages) in citronellol
(29.05), geraniol (24.36), citronellyl formate (5.94), isomenthone (5.82); the oil of ‘CIM-Pawan’ was rich in citronellol (32.60), geraniol
(21.38), 10-epi--eudesmol (6.83), citronellyl formate (6.29) while the essential oil of ‘Kelkar’ showed a different profile with citonellol
(61.48) and isomenthone (10.56) being almost twice that of other cultivars.
_____________________________________________________________________________________________________________
Keywords: composition, cultivars, essential oil yield
Abbreviations: FID, flame ionization detector; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry
INTRODUCTION
Pelargonium graveolens L’Herit ex Ait. (Family Geraniaceae), with the common name rose-scented geranium, is an
important high value perennial, aromatic shrub. The essential oil (EO), which possesses a tenacious rose-like odour, is
the most widely traded product of rose-scented geranium.
The main constituents of the EO are citonellol, geraniol,
isomenthone, citronellyl formate and geranyl formate. The
EO is largely utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is one of the best
skincare oils because it is good for opening skin pores and
cleaning oily complexions (Swamy et al. 1960; Weiss 1997;
Miller 2002; Peterson et al. 2006). The other use of geranium leaves is in the form of herbal tea to treat distress,
fight anxiety, ease tension, improve circulation and cure
tonsillitis (Peterson et al. 2006). A study between the EO of
geranium and tropical capsaicin, a commonly prescribed
conventional remedy for shingles pain, showed that geranium EO was extremely useful in reducing pain due to postherpetic neuralgia followed by shingles (Greenman et al.
2003).
Worldwide, annual geranium EO production is estimated to be worth about US$ 12.5 million (Williams and
Harborne 2002). Trade in EOs is expected to increase in the
future as a result of the growing number and preferences of
consumers, and the continuously widening uses of EO constituents (Sangwan et al. 2001). The current international
demand of about 600 t of geranium oil is being met largely
by China, Egypt, Morocco, Reunion Island and South Africa.
Therefore, most of the 145 t requirement of geranium oil of
the Indian industry is being met through imports (Ram et al.
2004).
In India, geranium is being grown in Nilgiri, the Pulney
hills of Tamil Nadu, and on the plains of Andhra Pradesh,
Karnataka, Maharashtra and Uttar Pradesh. It is cultivated
as a rain-fed perennial crop in hills and an annual crop in
plains of Northern India (Rao et al. 1990; Ram et al. 1995,
1996). The productivity and quality of different geranium
cultivars has also been assessed in the Tarai region of Uttarakhand (Ram et al. 2004) and its possible cultivation in
perennial conditions was exploited by CIMAP in the hills of
Uttarakhand (Anonymous 2003; Verma et al. 2010). However, studies on the comparative performance of different
cultivars of rose-scented geranium are not available from
the hilly regions of Northern India.
Therefore, as part of our institute’s mandate to develop
the agricultural and processing technologies of economically viable crops and to disseminate these technologies to
beneficiaries, the present study was conducted to assess the
production and quality potential of different cultivars of
rose-scented geranium in the valley region of western Himalaya.
MATERIALS AND METHODS
The experiment was performed at the experimental farm of the
Central Institute of Medicinal and Aromatic Plants, Research Centre, Purara, Bageshwar, Uttarakhand, India, during 2008-2009. The
experimental location experiences a temperate (Western Himalayan region of India) climate; the soil is sandy loam with pH 6.8
(soil: water, 1:2.5), 0.40% organic carbon, 145 kg ha-1 available
nitrogen, 11.0 kg ha-1 available P, 130 kg ha-1 exchangeable K
(Verma et al. 2009).
The experiment was initiated on the 15th February 2009. Well
developed one-month-old rooted cuttings (which were taken from
2-year-old mother plants with 2-3 leaflets and were planted in a
polybag containing local farm soil on the 15th January 2009 under
polyhouse conditions) of three cultivars (treatments), namely
‘Boubon’, ‘CIM-Pawan’ and ‘Kelkar’, were transplanted. Transplanting was done in plots of 5 m × 3 m replicated three times in a
completely randomized block design. The spacing was maintained
at 50 cm × 50 cm with the total number of plants per plot = 60.
The recommended dose of fertilizers, i.e., N: P: K at 100: 60: 60
kg ha-1, was applied. Before transplanting, a full dose of P, K and a
half-dose of N together with vermicompost, at the rate of 2.5 t ha-1,
were applied. The remaining N was top dressed in two equal splits
at monthly intervals. The plots were irrigated immediately after
Received: 7 December, 2010. Accepted: 15 December, 2010.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 77-79 ©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
Table 1 Growth and yield performance of rose-scented geranium cultivars in temperate region of Uttarakhand.
Cultivars
Plant height
Canopy
Branches/plant Leaf/stem
Oil content*
(cm)
(cm2)
ratio
(%)
Bourbon
59.56
65.30
5.66
2.06
0.19
CIM-Pawan
85.53
80.66
7.33
1.61
0.22
Kelkar
93.00
69.16
5.66
1.31
0.11
CD 5 %
4.50
2.93
1.41
0.37
0.02
Herb yield
(kg/plot)
42.86
51.70
80.76
2.68
Oil yield
(g/plot)
72.01
103.87
79.93
12.05
*calculated on a laboratory basis
transplanting and further irrigation was provided at monthly intervals in spring (March-April) and at fortnightly intervals in summer
(May-June). Other agronomic practices needed during the cropping period were applied uniformly.
In each plot 20 plants replication-1 were randomly selected for
observations on morphological characters such as plant height,
canopy, number of branches plant-1 and leaf/stem ratio. The crop
was harvested 120 days after planting; herb yield, EO content and
EO yield from harvested biomass was determined. Oil yield was
computed by multiplying herbage yield with oil content.
Table 2 Qualitative performance of three cultivars of rose-scented geranium in temperate region of Uttarakhand.
Relative peak area (%)
Compound
‘Bourbon’
‘CIM-Pawan’ ‘Kelkar’
Menthone
0.23
0.17
0.23
(E)-Linalool oxide
t
t
1.26
Isomenthone
5.82
5.80
10.56
-Bourbonene
0.16
t
t
Linalool
4.83
3.25
0.32
-Caryophyllene
0.15
0.17
0.56
Citronellyl formate
5.94
6.29
1.18
Citronellyl acetate
0.97
0.80
0.22
Geranyl formate
3.70
2.62
t
Geranial
1.03
1.17
1.79
-Cadinene
0.51
0.15
t
Geranyl acetate
t
0.23
t
Citronellol
29.05
32.60
61.48
Nerol
0.29
0.37
t
Citronellyl butyrate
0.32
0.32
Geraniol
24.36
21.38
0.61
Geranyl isovalerate
0.64
0.21
t
Geranyl butyrate
0.44
0.26
t
10-epi--Eudesmol
5.50
6.83
0.27
Geranyl tiglate
1.41
1.85
0.18
2-Phenyl ethyl tiglate
0.29
0.76
0.30
C/G ratio
1.19
1.52
100.78
Essential oil extraction and analysis
The EO was extracted by hydro-distillation for 3 h using a Clevenger-type apparatus. The oil content (w/v %) was estimated on a
fresh weight basis. The oil samples obtained were dehydrated over
anhydrous sodium sulphate and kept in a cool and dark place prior
to GC analysis.
The oil samples were subjected to GC analysis on a Nucon
gas chromatograph model 5765 equipped with an FID using a CPWAX 52CB fused silica capillary column (30 m × 0.32 mm × 0.25
μm film thickness). Hydrogen was used as a carrier gas at the rate
of 1.0 ml min-1. Injector and detector temperatures were 200 and
230°C, respectively. The oven temperature was programmed from
70-230°C at 4°C/min with an initial hold time of 2 min. Identification was done on the basis of retention index (determined with
reference to homologous series of n-alkanes (C9-C24) under identical experimental conditions), co-injection with known compounds, and MS Library search (NIST and WILEY), by comparing with the MS literature data (Davies 1990; Adams 1995).
The retention times of standards/marker constituents of known
EOs were also used to confirm the identities of constituents. The
relative amounts of individual components were calculated based
on GC peak area (FID response) without using a correction factor.
tivar ‘CIM-Pawan’ in the Tarai region when compared with
the herb yield in the valley regions of western Himalaya
(Ram et al. 2004). ‘Bourbon’ had intermediate oil content
(0.19%) and herb yield (42.86 kg plot-1) among the three
cultivars. The oil content of ‘Bourbon’ was higher than in
previous studies from South India (Rao et al. 1990), north
Indian Plains (Jain et al. 2001) and even in the Kashmir
valley (Shawl et al. 2006).
There was an obvious disparity in the percentage of different EO components in all three cultivars (Table 2). The
major components of the EO of ‘Bourbon’ were citronellol
(29.05%), geraniol (24.36%), citronellyl formate (5.94%),
isomenthone (5.82%); the EO of ‘CIM-Pawan’ was represented with major proportions of citronellol (32.60%), geraniol (21.38%), 10-epi--eudesmol (6.83%), citronellyl formate (6.29%). Unlike the EO of the other two cultivars, that
of ‘Kelkar’ showed marked variation in percentage of major
components. The major component, citonellol was almost
twice (61.48%) that of the EOs of ‘Bourbon’ and ‘CIMPawan’. Another major component in the EO of ‘Kelkar’
was isomenthone (10.56%). However, the other major components in the oils of ‘Bourbon’ and ‘CIM-Pawan’ viz.,
linalool, citrnellyl formate, geranyl formate and 10-epi-eudesmol were found in very small quantities in the EO of
‘Kelkar’.
The citronellol to geraniol (C/G) ratio is an important
factor which determines the quality of EO of geranium.
Generally, geranium oil with a C/G ratio 0.5-2.0 possesses a
good odor value and is accepted by the perfume industry
(Saxena et al. 2000). Thus, the present study showed that
the cultivars ‘CIM-Pawan’ and ‘Bourbon’ with a C/G ratio
1.52 and 1.19, respectively were superior to ‘Kelkar’. Furthermore, ‘Kelkar’ may be used as a rich source of natural
citronellol.
In conclusion, ‘CIM-Pawan’ outperformed other culti-
Statistical analysis
The experimental data were statistically analyzed by analysis of
variance. Estimation of the significance of differences between
means was based on a probability of p < 0.05 (Snedecor and Cochran 1989).
RESULTS AND DISCUSSION
The data in Table 1 shows the comparative growth and
yield attributes of the three cultivars of rose-scented geranium. ‘Kelkar’ attained maximum plant height and was significantly superior to the other two cultivars. Maximum
canopy extent was recorded in ‘CIM-Pawan’ (80.66 cm),
significantly higher than ‘Kelkar’ (69.16 cm) and ‘Bourbon’
(65.30 cm). A similar trend was also observed in the Tarai
region of Uttarakhand in the case of ‘CIM-Pawan’ (Ram et
al. 2004). ‘CIM-Pawan’ also recorded the maximum number of branches plant-1 (7.33), while ‘Bourbon’ and ‘Kelkar’
had the same number of branches plant-1 (5.66). ‘Bourbon’
had the maximum leaf/stem ratio (2.06), followed by ‘CIMPawan’ (1.61) and ‘Kelkar’ (1.31).
The highest oil content (0.22%) was recorded in the
fresh biomass of ‘CIM-Pawan’, which was significantly
superior to the other two cultivars. The oil content of ‘Kelkar’ was lowest (0.11%), although it produced the highest
herb yield (80.76 kg plot-1) among the three cultivars. Earlier studies showed a more or less similar trend except that
the herb yield of ‘Kelkar’ was lower than the superior cul-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
78
Performance of rose-scented geranium cultivars. Chauhan and Verma
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
vars in fresh biomass and EO yield followed by ‘Bourbon’
in the valley region of western Himalayas. There was considerable variation in the chemical composition of the EO,
possibly due to the genetic makeup of the cultivars and also
due to geographical regions of cultivation (Ram et al. 2004).
Plant Sciences 19, 3-4
Ram M, Kumar S (1996) Maintenance of geranium Pelargonium graveolens
resource plants over monsoon season in field condition of north Indian plains
for the winter summer cropping. Journal of Medicinal and Aromatic Plant
Sciences 18, 801-802
Ram P, Kumar B, Srivastava N, Verma RS, Sashidhara KV, Patra NK
(2004) Productivity and quality assessment of different chemotypes of geranium under tarai of Uttaranchal. Journal of Medicinal and Aromatic Plants
Sciences 26, 482-485
Rao BRR, Shastry KP, Rao EVS Ramesh S (1990) Variation in yields and
quality of geranium (Pelargonium graveolens L’ Her. ex Aiton) under varied
climatic and fertility conditions. Journal of Essential Oil Research 2, 73-79
Sangwan NS, Farooqi AHA, Shabih F, Sanguan RS (2001) Regulation of
essential oil production in plants. Plant Growth Regulation 34, 3-21
Saxena G, Banerjee S, Rahman L, Sharma S, Kumar S (2000) An efficient
in vitro procedure for micropropagation and generation of somaclones of
rose-scented Pelargonium. Plant Science 155, 133-140
Shawl AS, Kumar T, Chisti N, Shabir S (2006) Cultivation of rose scented
geranium (Pelargonium sp.) as a cash crop in Kashmir Valley. Asian Journal
of Plant Sciences 5 (4), 673-675
Snedecor GW, Cochran WG (1989) Statistical Methods (8th Edn), Iowa State
University Press Ames, Iowa
Swamy AY, Sreshta NJ, Kalyansundaram S (1960) Cultivation of scented
geranium on the Nilgiris. Indian Perfumer 4 (1), 3-9
Verma RK, Rahman L, Verma RS, Yadav A, Mishra S, Chauhan A, Singh
A, Kalra A, Kukreja AK, Khanuja SPS (2009) Biomass yield, essential oil
yield and resource use efficiency in geranium (Pelargonium graveolens L.
Her. ex. Ait) intercropped with fodder crops. Achieves of Agronomy and Soil
Science 55 (5), 557-567
Verma RS, Verma RK, Yadav AK, Chauhan A (2010) Changes in the essential oil composition of rose-scented geranium (Pelargonium graveolens L’
Herit. Ex Ait.) due to date of transplanting under hill conditions of Uttarakhand. Indian Journal of Natural Products and Resources 1 (3), 367-370
Weiss EA (1997) Essential Oil Crops, Center of Agriculture and Biosciences,
CAB International, Wallingford, UK, pp 24-50
Williams CA, Harborne JB (2002) Photochemistry of the genus Pelargonium.
In: Lis-Balchin M (Ed) Geranium and Pelargonium: The Genera Geranium
and Pelargonium, Taylor and Francis, London, 99 pp
ACKNOWLEDGEMENTS
The authors are thankful to Director CIMAP, Lucknow for providing necessary facilities and encouragements.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Evaluation of Antibacterial Activity of Euryale ferox Salisb.,
a Threatened Aquatic Plant of Kashmir Himalaya
Javid Ahmad Parray1* • Azra N. Kamilli1 •
Raies Qadri2 • Rehana Hamid3 • Jaime A. Teixeira da Silva4
1 Department of Environmental Science, University of Kashmir, Srinagar-190006, J and K, India
2 Department of Biotechnology, University of Kashmir, Srinagar-190006, J and K, India
3 Department of Botany, Jammia Hamdard, New Delhi, 1100621 India
4 Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan
Corresponding author: * javid06@gmail.com
ABSTRACT
The antibacterial activity of methanolic extract of seeds and leaves of Euryale ferox was tested against nine clinically isolated bacterial
strains (Staphylococus aureus, Escherichia coli, Pseudomonas aureoginosa, Citrobacter freundi, Shigella flexneri, Klebsiella pneumoniae,
Proteus vulgaris, Salmonella typhi and Salmonella typhimurium) and subsequently was also tested for minimum inhibitory concentration
(MIC) values which ranged from 0.25 to 500 mg/l against six ATCC bacterial strains using the micro broth dilution method. The broad
spectrum activity displayed by the seed and leaf extracts appears to provide a scientific basis for the use of E. ferox in kidney problems
and urinary tract infections in ethnomedicines.
_____________________________________________________________________________________________________________
Keywords: inhibition zones, methanol, MIC, Nymphaceae, seed extract
Abbreviations: CFU, colony forming unit; GNB, Gram-negative bacilli; GPC, Gram-positive cocci; IZD, inhibition zone diameter;
MIC, minimum inhibitory concentration
INTRODUCTION
MATERIALS AND METHODS
Euryale ferox, commonly known as makhana, is an annual
aquatic medicinal plant that belongs to the family Nymphaceae. It is widely distributed throughout Russia, Korea,
China, Japan, Bangladesh and India (Wu and Raven 1994).
It grows wild in Kashmir and is a non-endemic threatened
aquatic species (Khan 2000; Dar et al. 2002; Khan et al.
2004).The plant produces starchy seeds and is edible.
Traditionally the plant has been used throughout the world
to cure many diseases including chronic diarrhhoea, kidney
problem, leucorrehea and spleen hypofunction (Brown
1995). The plant as a whole is used as an analgesic, aphrodisiac, astringent, deobstruent, oxytonic and tonic in China
(Duke 1985). Leaves are used in difficult parturition (Shankar 2010). The plant is internally taken to treat vaginal discharge, impotency, premature and involuntary ejaculation
and nocturnal emission (Brown 1995). In China this plant is
cultivated for its stem, rhizomes and seeds (Sturtevant
1972). In the Indian traditional system of medicines the dry
seeds of the plant are being used as an immunostimulant for
mothers after childbirth and invalids with a relatively poor
immune status (Puri et al. 2000). Various aspects of significant antioxidant activity have been evaluated in total extracts and fractions derived from E. ferox. The plant has
also been shown to enhance the activities of Superoxide dismutase, Catalase and Glutathione peroxidase in V79-4 cells
(Lee et al. 2002). Recent studies on antioxidant activities of
E. ferox and its glycoside composition in vitro reveal that
the plant extracts have potent scavenging activity against
reactive oxygen species suggesting that seed of this plant
have cardioprotective properties which may link with the
ability of makhana to induce TRP32 and TrX-1 protein and
to scavenge ROS (Samarajit et al. 2006; Verma et al. 2010).
In view of its traditional use, we decided to determine
the antibacterial activity of E. ferox extracts for the development of possible new antimicrobial compounds.
Plant material
E. ferox was collected as a whole plant from Manasbal Lake of
Kashmir Himalaya, J&K, India in September 2008. Sampling was
carried out immediately after seed formation and plants were collected manually at the age of 7 months in bulk from the Lake.
After that four parts i.e., leaves, petioles, rhizomes and seeds were
separated and dried separately. The plant was identified by the
HOD, Kashmir University Herbarium (KASH), Centre of Plant
Taxonomy, Department of Botany, University of Kashmir, Srinagar. A sample was deposited in the herbarium as voucher no. 1015.
Preparation of plant extracts (hot process)
Dried powder (50 g) from the seeds, leaves, petioles and rhizomes
of E. ferox were extracted successively with petroleum ether, chloroform and methanol in a Soxhlet extractor. The solvents from all
12 extracts were concentrated under vacuum at 40–50°C using a
rotary flash evaporator (Heidolph, Germany) and crude extracts
were air dried at room temperature in a steady air current. The
weight of the solid residue was recorded and assumed as the yield
of crude extract. The dried extracts were then stored in air-tight
jars at 4°C for microbial analysis.
Microbial strains
All bacterial strains used in this study were clinical strains, namely
Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae,
Salmonella typhi, Salmonella typimurium, Shigella flexneri, Pseudomonas aeruginosa, Citrobacter fruendii, Proteus vulgaris and
were obtained from the Bacteriology Laboratory, Department of
Microbiology, SKIMS, Soura, Srinagar, J&K.
Received: 16 June, 2009. Accepted: 25 October, 2010.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 80-83 ©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
Antibacterial susceptibility tests
A
The disc diffusion method (Bauer et al. 1966) was used to determine the antibacterial activity of all 12 extracts which was determined in a mixed culture of Gram-positive cocci (GPC) and
Gram-negative bacilli (GNB). The extracts which exhibited inhibitory activity were then tested against clinically isolated strains
using Muller Hinton agar (MHA; Hi-Media). The MHA plates
were prepared by pouring 15 ml of MHA into sterile Petri dishes.
The plates were allowed to solidify for 15 min and 0.1 ml (0.5
MacFarland standard) inoculum suspension was swabbed uniformly and the plates were allowed to dry for 5-10 min. The concentrations of extracts (160 and 320 μg/ml/disc) were loaded onto
6 mm sterile discs (Hi Media). The loaded discs were placed on
the surface of the medium and the compound was allowed to diffuse for 5 min and the plates were incubated. Inhibition zones
formed around the discs were measured with a transparent ruler (in
mm). Antibiotic discs and solvents were taken as positive and
negative controls, respectively.
C
B
D
Determination of minimum inhibitory
concentration (MIC)
Minimum inhibitory concentration (MIC) of the extracts was performed by using a broth micro dilution method on 96 micro-well
plates based on recommendations of the National Committee for
Clinical Laboratory Standards (NCCLS 2000). Stored bacteria
were resituated, identified and incubated in 5 ml of Muller Hinton
broth (HMB) in an incubator at 37°C for 24 hrs to a density equal
to that of No.1 McFarland standard. The bacterial suspension was
further diluted with a broth to give a final inoculum of 106 CFU/ml.
To evaluate bacterial sensitivity, serial two-fold dilutions of 500,
256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 mg/l were prepared
with HMB (Phillips et al. 1990). Each micro-well of the 96 microwell plates contained 50 μl diluted agents and 50 μl bacterial
strain suspension. A final test volume of 100 μl was dispensed into
each well. Both methanol and gentamicin (Hi Media) were kept in
one set of wells to act as negative and positive control, respectively. After 24 hrs incubation of the micro-well plate in an incubator at 37°C, bacterial growth was observed by visual inspection.
At the same time, from every micro-well, 50 μl of treated suspension was removed and was inoculated on new MHA plates; bacterial colonies were count after incubation for 48 hrs. The initial
concentration of no bacterial growth on the plates was considered
as the individual agent minimum inhibitory concentration (MIC)
in the micro dilution corresponding to individual strains.
RESULTS AND DISCUSSION
The present investigation of antimicrobial activity of E.
ferox against a mixed culture of GPC and GNB reveals that
the methanolic extracts of seeds and leaves possessed antibacterial activity (Table 1, Fig. 1A). The inhibition zone
diameters (IZD) obtained through the disc diffusion assay
of methanolic extracts of seeds and leaves of E. ferox
against clinically isolated bacterial strains is shown in Table
2. The MIC values of the methanolic extract of seed and
leaf extracts of E. ferox with gentamicin and methanol as
positive and negative control, respectively against six standard strains are shown in Table 2. Among the strains, K.
pneumoniae and C. fruendii were resistant towards the
methanolic extract of both seeds and leaves (Figs. 1B, 1C).
P. aeruoginosa showed the maximum IZD (Fig. 1D) followed by S. typhi (Fig. 1E), E. coli (Fig. 1F) and S. flexnerii (Fig. 1G). S. aureus and S. typhimurium (Figs. 1H, 1I)
were sensitive towards only the methanolic extract of seeds
whereas P. vulgaris (Fig. 1J) was sensitive to higher concentrations of seed and leaf extracts. The antimicrobial activity of crude extracts from numerous plants has been evaluated by the agar disc dif-fusion assay (Guven et al. 2005)
and MIC was determined, based on which anti-infective
lead molecules were isolated or used as a therapeutic agent
(Sawer et al. 2005). The IZD of some Gram-negative strains
like P. aeruoginosa was greater than Gram-positive strains
(Venkata and Venkata 2008), as determined for fruit extracts
E
F
G
H
I
J
Fig. 1 (A) Gram-positive cocii and Gram-negative bacillus. (B) Klebsiella
pneumoniae; (C) Citrobacter fruendi; (D) Pseudomonas aeruginosa; (E)
Salmonella typhii; (F) Escherichia coli; (G) Shigella flexneri; (H) Staphylococcus aureus; (I) Salmonella typhimurium; (J) Proteus vulgaris. M =
methanol: SI = 160 μg/ml, S2 = 320 μg/ml = seed methanol extract. L1 =
160 μg/ml, L2 = 320 μg/ml = leaf methanol extract, Ab = antibiotic; GNB
= Gram-negative bacilli, GPC = Gram-positive cocci; Pt = petroleum
ether; C = choloroform; Sm = seed methanol; SC = seed chloroform; SP =
seed pet ether; LM = leaf methanol; LC = leaf chloroform; LP = leaf pet
ether; PM = petiole methanol; PC = seed chloroform; PP = seed pet ether;
RM = leaf methanol; RC = leaf chloroform; RP = leaf pet ether
of two Syzygium spp. The higher the extract concentration,
the greater the inhibitory effect, supporting similar studies
such as that by Boakye-Yiadom (1977), who discovered
antimicrobial properties of some West African medicinal
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
81
Evaluation of antibacterial activity of Euryale ferox. Parray 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 Antimicrobial activity of Euryale ferox extracts against mixed culture of GPC and GNB bacteria.
Inhibition zone diameter (mm)
Bacterial strains
Rhizome
Petiole
Leaf
Pt
C
M
Pt
C
M
Pt
C
Mixed culture of GPC and GNB NA
NA
NA
NA
NA
NA
NA
IA
M
IA
Pt
NA
Seed
C
IA
M
IA
GPC = Gram-positive cocci, GNB = Gram-negative bacilli, Pt = petroleum ether extract, C = chloroform extract, M = methanol extract, IA = inhibitory activity; NA = no
activity.
Table 2 Anti bacterial activity of methanolic extracts of seeds and leaves of E. ferox against clinically isolated bacterial strains.
Inhibition zone diameter (mm)
Bacterial strains
Antibiotics
Methanolic seed extract
Methanolic leaf extract
160 μg/ml
320 μg/ml
160 μg/ml
320 μg/ml
Staphylococcus aureus
12
23
NA
NA
Erythromycin (25)
Pseudomonasaeruginosa a
18
28
16
22
Amikacin (15)
Shigella flexneri
8
12
10
15
Ciprofloxacin (29)
Klebsiella pneumoniae
NA
NA
NA
NA
Gentamycin (25)
Salmonella typhi
14
19
8
12
Imipenin (28)
Salmonella typhimurium
0
15
0
10
Gatifloxacin (20)
Proteus vulgaris
NA
19
NA
20
Cefixime (0)
Citrobacter fruendii
NA
NA
NA
NA
Vancomycin (22)
Escherchia coli
12
15
8
12
Ceftazidime (30)
Results are mean of three readings; NA = no activity.
plants. Among the solvents, methanol extracts showed a
higher activity than other solvents (petroleum ether and
chloroform). Pavithra et al. (2010) also found that the methanolic extracts of M. cerviana exhibited significant antibacterial activity against Gram-positive and -negative strains
with minimum bactericidal concentration (MBC) ranging
from 1.5 to 100 mg/ml. This may be due to the higher
polarity and maximum number of compounds extracted in
methanol, which has also proved to be the most effective
solvent for extracting a broad spectrum of antimicrobial
compounds from plants (Vlachos et al. 1996).
From our study, the methanolic seed extract exhibited
more inhibitory activity than the leaf extract against all tested bacterial strains and hence showed bactericidal activity
against standard strains with S. aureus ATCC 25923 having
a low MIC value (64 mg/l) and S. flexneri the highest (500
mg/l). The methanolic leaf extract of E. ferox showed bactericidal activity against some standard strains i.e., with an
MIC value of 128 mg/l against S. aureus ATCC 25923, 256
mg/l against Pseudomonas aeruginosa ATCC 27853 and no
activity against a standard P. vulgaris strain. Among the
bacterial strains tested S. aureus and P. aeruginosa showed
higher activity towards both seed and leaf extracts and some
tested strains like Acintobacter sp., K. pneumoniae were
least effective since they are naturally resistant to antibacterial agents (Walker and Edward 1999). The results of MIC
revealed a decreasing trend in activity as the concentrations
of extracts decreased, which implies that the extracts were
more active at higher concentrations. The active results of
most of strains tested were in the range of 256-500 mg/l and
our findings are in agreement with the results of Idu et al.
(2007) for the methanolic extracts of Senna allata flowers.
The methanolic seed extract exhibited more inhibitory activity than the leaf extract against all tested bacterial strains
and hence showed bactericidal activity against standard
strains with S. aureus ATCC 25923 having a low MIC value
of 64 mg/l and S. flexneri having the highest MIC value of
500 mg/l. while S. aureus and E. coli were inhibited by both
seed and leaf extracts. Similarly, Pongpaichit et al. (2005),
for the crude methanolic extract of Acorus calamus seed
and leaf extracts and Panda et al. (2009), for the methanolic
leaf and bark extracts of Vitex negundo, reported significant
antibacterial activity against E. coli and S. aureus, respectively. However, results against P. auruginosa were contradictory to that observed by Pongpaichit et al. (2005). The
antibacterial activity of E. ferox may be due to the presence
of some antimicrobial compounds like cerebrosides and
glycosterols (Zhao et al. 1989, 1994; Lin et al. 2003; Li and
Xu 2007; Row et al. 2007).
Table 3 Minimum inhibitory concentration (MIC) of methanolic extracts
of seed and leaf extracts of E. ferox against Standard strains (mg/l).
MIC (mg/l)
Standard bacterial strains
Seed
Leaf
Gentamycin
extract extract 10 mg/l
Staphylococcus aureus ATCC 25923
64
128
32
Escherichia coli ATCC 25922
128
256
128
Pseudomonas aeruginosa ATCC 27853 64
256
16
Shigella flexneri ATCC 12022
ND
500
ND
Proteus vulgaris*
500
ND
256
Salmonella typhi*
256
500
ND
The seed and leaf extracts exhibited marked inhibitory
action against S. typhi, a virulent strain that causes typhoid
(Prescott 2005). With the increase in resistance to antityphoid drugs, medicinal plants have gained popularity
among both urban and rural dwellers in the treatment of the
ailment. Similarly, Seena siamae leaf extracts have been
shown to have antibacterial activity against S. typhi and S.
typhimurium (Doughari and Okafor 2008).
K. pneumoniae was not inhibited by any of the extracts
tested and this may be due to the fact that it produces a capsule (Sule and Aghabiaka 2008). Methanolic extracts exhibited higher activity, as also concluded by Babayi et al. (2004)
for Eucalyptus camaldulensis and Terminalia catappa. The
crude methanolic extracts of E. ferox extract exhibited
highest activity against P. aeruginosa and E. coli and similar activity has been reported from other medicinal plants,
namely Grewia erythraea, Hymenocrater sessilifolius, Vincetoxicum stocksii, Zygophyllum fabago and Arcenthobium
Oxycedri (Zaidi et al. 2005; Mudassir et al. 2006).
Among the strains, P. aureoginosa, S. aureus and E. coli
exhibited higher zones of inhibition against both methanolic
extracts. The antibacterial activity of seed extracts was also
reported by Murtaza et al. (1994) while the antibacterial activity of the leaf extract may be due to the presence of alkaloids and flavonoids as reported by Ahmad et al. (2001) in
the leaves of Adhantoda vasica Nees. However, the antibacterial activity may be due to the presence of one or more
phytocompounds present in plants as most of them are
known to have antimicrobial activity (Bruenton 1995). The
MIC results depicted that gentamicin exhibited higher activity than the methanolic extracts of both plant parts of E.
ferox (Table 3). This could be attributed to the fact that, unlike conventional antibiotics and other pharmaceutical products which are usually prepared from synthetic materials by
means of reproducible manufacturing techniques and proce-
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
82
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 80-83 ©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
dures, herbal medical products are prepared from materials
of plant origin which may be subjected to contamination
and deterioration (Babu et al. 2002). The literature reveals
that no prior work has been done on evaluating the antimicrobial activity of E. ferox and it is now expected that
screening of this plant for antibacterial activity against a
wide variety of test organisms will be helpful in obtaining a
broad spectrum herbal formulation as well as new antimicrobial substances for the treatment of diseases like kidney problems and urinary tract infections.
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ACKNOWLEDGEMENTS
The Authors are highly thankful to Central Research Institute of
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facilities to carry out these studies.
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Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Callus-Mediated Shoot Organogenesis
from Shoot Tips of Cichorium intybus
Rehana Hamid1 • Azra N. Kamili 2 • Mahmood uz Zaffar1 •
Jaime A. Teixeira da Silva3 • A. Mujib1 • Javid A. Parray2*
1 Department of Botany, Jamia Hamdard New Delhi 1100621, India
2 Plant Tissue Culture Laboratory, Centre of Research for Development, University of Kashmir, Srinagar, 190006, Jammu and Kashmir, India
3 Department of Horticultural Science, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan
Corresponding author: * javid06@gmail.com
ABSTRACT
Cichorium intybus L. is a medicinally important plant with anti-cancerous and anti-hepatotoxic properties. An efficient method for
totipotent callus formation has been developed in C. intybus from the basal portion of shoot tip explants on MS medium supplemented
with different concentrations of plant growth regulators (PGRs) like 6-benzylamino purine (BAP) and kinetin (Kn) with an auxin, indole3-butyric acid (IBA). Cultures growing under the influence of BAP+IBA produced considerably more callus than cytokinins used alone.
Re-differentiation of such callus led to multiple shoot formation on the same medium after 3 weeks. Isolated shoots were individually
rooted in the presence of different concentrations of IBA. Plantlets obtained were transplanted into small pots containing peat, vermiculite,
sand and soil mixture (1:1:1:1), 60% of which survived.
_____________________________________________________________________________________________________________
Keywords: Asteraceae, phytohormones, re-differentiation
Abbreviations: BAP, 6-benzylamino purine; IBA, indole-3-butyric acid; Kn, kinetin; MS, Murashige and Skoog; PGR, plant growth
regulator
INTRODUCTION
Cichorium intybus L. (chicory) is a member of the Asteraceae. It is distributed in Northern and Southern Europe and
Turkey (Bais and Ravishanker 2001). Chicory has been
successfully cultivated in India since 1918 in Coimbatore
and subsequently Nilriris in Tamil Nadu and at Broach,
Amalsad and Jamnanagar in Gujrat (Muthuswami and
Pappiah 1980). Chicory forms flowering shoots and seeds
after overwintering. Initially, chicory seeds were imported
into India but are now successfully produced locally (Anonymous 1992). Phytochemicals or plant constituents are
distributed throughout the entire chicory plant but the main
constituents are present in the roots. The tuberous roots of
this plant contain a number of medicinally important compounds such as inulin, lactones, coumarins, flavonoids and
vitamins (Varotto and Lucchin 2000). Bitter substances
found in this plant are lactucin, lactucopicrin and esculentin
(Chopra et al. 1956). The plant root is used as an antiulcerogenic, anti-inflammatory and appetizer. It is also used
to cure various heart diseases and has anti-hepatotoxic
(Zafar and Ali 1998) and antibacterial (Petrovic et al. 2004)
activity. This plant is used in the treatment of AIDS, colon
cancer and insomnia (Duke 1983). It is useful in vitiated
conditions of cephalalgia, heapatomegaly, inflammation
and asthma (Nadkarni 1976). In vitro regeneration of C.
intybus has been previously reported through shoot organogenesis using different explants (e.g. roots, shoots, leaves,
nodal buds, petioles, etc.) and with various plant growth
regulator (PGR) combinations (Profumo et al. 1985; Pieron
et al. 1993; Kamili et al. 2003; Rehman et al. 2003). This
paper describes the development of an indirect regeneration
protocol from cultured shoot tips of C. intybus.
MATERIALS AND METHODS
Seeds of C. intybus obtained from Pusa, New Delhi, India were
used as experimental material. Seeds soaked overnight were
washed with a few drops of laboratory detergent (Labolene) and 23 drops of Tween-20 (surfactant) after washing under running tap
water. Chemical sterilization of seeds was achieved by treating
them with 0.2% of HgCl2 for 10 min. Finally they were washed
with autoclaved double distilled water 3-4 times to remove all
traces of sterilant and then germinated on Murashige and Skoog
(MS) basal medium (1962) containing 3% (w/v) sucrose (HiMedia, Mumbai, India) and 0.8% (w/v) difcobactoagar (Hi-Media).
Shoot tip explants obtained from aseptically grown seedlings were
inoculated onto MS medium containing different concentrations of
Kinetin (Kn), 6-benzylamino purine (BAP), and BAP+IBA
(indole-3-butyric acid)(all PGRs from Hi-Media). After 4 weeks
shoots were singled out and transferred to rooting medium. The
pH of media used was adjusted to 5.5 using 1N HCl or 1N NaOH
before autoclaving the medium at 121°C for 20 min. After inoculation, all the cultures were incubated under cool fluorescent tubes
in a 16-h photoperiod with a light intensity of 1500–3000 lux at a
constant temperature of 25 ± 3°C; relative humidity of 60–70%
was maintained. For the hardening-off procedure, plantlets were
washed with sterile distilled water to remove traces of medium and
agar and then transferred to plastic pots containing peat, vermiculite, sand and soil mixture (1: 1: 1: 1, v/v). Experiments were set
up in a Randomised Block Design and all the experiments were
repeated three times and 10 replicates were used for each treatment. Observations were recorded for the number of shoots/explant, length of shoots. Mean and standard deviation were calculated for each treatment.
RESULTS AND DISCUSSION
After 2 weeks of incubation on MS medium fortified with
either Kn (2.5-10 μM) or BAP (1-7 μM) alone, shoot tips
started callusing at their cut ends. After 4 weeks both the
cytokinins (CKs) used induced shoots via callus. BAP at 7
μM produced a maximum of 15.2 shoots/explant (Table 1)
with an average length of 3.5 cm (Fig. 1A). As the BAP
Received: 7 August, 2009. Accepted: 21 April, 2010.
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
Research Note
Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 84-86 ©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
Table 1 Response of in vitro raised shoot tips of Cichorium intybus to different concentrations of BAP.
PGR
% Response
Callusing
Shoot no.
(Mean ± SD)
BAP (1 μM)
88
++
4.8 ± 1.55
BAP (2 μM)
85
++
6.4 ± 1.9
BAP (3 μM)
90
+++
8.8 ± 2.8
BAP (4 μM)
90
+++
13.1 ± 2.99
BAP (7 μM)
85
+++
15.2 ± 3.8
Average shoot length
(cm) ± SD
6.5 ± 0.35
6.25 ± 0.25
6 ± 0.70
5.75 ± 0.50
3.5 ± 0.57
+ (minimum callusing), ++ = (moderate callusing), +++ (maximum callusing); Growth period = 6 weeks; Data represents mean ± SD. The experiment was repeated three
times.
Table 2 Response of in vitro raised shoot tips of Cichorium intybus to different concentrations of kinetin.
PGR
% Response
Callusing
Shoot no.
(Mean ± SD)
Kn (2.5 μM)
80
+
4.2 ± 0.77
Kn (5.0 μM)
85
+
6.4 ± 1.0
+
7.6 ± 1.0
Kn (7.5 μM)
85
++
6 ± 1.0
Kn (10 μM)
85
Average shoot length
(cm) ± SD
3.98 ± 0.31
4.1 ± 0.66
4.4 ± 1.31
4.7 ± 1.16
+ (minimum callusing), +++ (maximum callusing); Growth period = 6 weeks. Data represents mean ± SD. The experiment was repeated three times.
Table 3 Response of in vitro raised shoot tips of Cichorium intybus to different concentrations of BAP + IBA.
PGR
% Response
Callusing
Shoot no.
(Mean ± SD)
BAP (4 μM) + IBA (1 μM)
88
+++
19.5 ± 4.135
BAP (4 μM) + IBA (2 μM)
85
++++
22.36 ± 5.24
BAP (4 μM) + IBA (3 μM)
90
++++
24 ± 4.7
BAP (4 μM) + IBA (4 μM)
90
++++
18.4 ± 2.99
Average shoot length
(cm) ± SD
3 ± 0.70
3.2 ± 0.81
3.5 ± 0.54
3.8 ± 0.81
+ (minimum callusing), +++ (moderate callusing), ++++ (maximum callusing) Growth period = 6 weeks. Data represents mean ± SD. The experiment was repeated three
times.
A
B
C
D
E
F
concentration increased, shoot length decreased. Multiple
shoot formation from nodal explants via callus with 15 μM
BAP was reported by Kamili et al. (2003); Veklaylayutham
et al. (2003) also reported multiple shoots of C. intybus
from callus using 4.4 μM BAP. Induction of shoots using
BAP has been documented in other related important medicinal herbs like Niger (Nikame and Shitole 1993), Anageisus (Kaul et al. 1992), Thevetia (Kumar and Kumar 1995),
and Piper spp. (Bhat et al. 1995). Among various concentration of Kn used (2.5-10 μM), the maximum average
number of shoots/explant formed in the presence of 7.5 μM
Kn was 7.6 and average shoot length was 4.4 cm (Table 2;
Fig. 1B). Yucesan et al. (2007) also reported that Kn alone
at 35.36 μM induced most shoots from lamina explants of C.
intybus and also reported BAP to be more effective than Kn
in terms of callus formation and mean number of shoots/explant.
In another trial auxin (IBA) + CK (BAP) and auxin
(IBA) + another CK (kinetin) were used together interactively, enhancing callus formation and shoot multiplication.
Callus was more compact, nodular and green than all other
concentrations and combinations of different PGRs used
and maximum callus was obtained on MS medium supplemented with 4 μM BAP + 2 μM IBA, 4 μM BAP + 3
μM IBA and 4 μM BAP + 4 μM IBA and a maximum
number of 24 shoots/explants were regenerated from 4 μM
BAP + 3 μM IBA acquiring a length of 3.5 cm (Table 3;
Fig. 1C, 1D). However, at 7.5 μM Kn + 3 μM IBA a maximum of 13.0 shoots were recorded with an average length
of 3.8 cm (Table 4; Fig. 1E) Yucesan et al. (2007), Rehman
et al. (2000) and Velayutham et al. (2006) all reported an
increase in shoot multiplication in C. intybus when a combination of auxins and CKs were used.
For rooting, multiple shoots were singled out and cultured on MS medium containing different concentrations of
PGRs; however, 10 μM IBA produced dense and healthy
roots (Fig. 1F). Acclimatized plantlets showed a 60% survival rate. This study resulted in the establishment of a
protocol for callus-mediated organogenesis of C. intybus
through in vitro raised shoot tips. Through callus a number
of shoots can be achieved which could improve the growth
and yield of C. intybus to meet the demand of the pharmaceutical industry.
Fig. 1 In vitro propagation and acclimatization of Cichorium intybus
through indirect callus culture. (A) MS+ BAP (7μM), (B) MS+ Kn (7.5
μM), (C) MS+ BAP (4 μM) + IBA (3 μM), (D) MS+ BAP (4 μM) + IBA
(3 μM), (E) MS+ Kin (7.5 μM) + IBA (3 μM), (F) MS+ IBA (10 μM).
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
85
Shoot organogenesis from shoot tips of C. intybus. Hameed 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 4 Response of in vitro raised shoot tips of Cichorium intybus to different concentrations of Kn + IBA.
PGR
% Response
Callusing
Shoot no.
(Mean ± SD)
Kn (7.5 μM) + IBA (1 μM)
75
++
6.2 ± 0.81
Kn (7.5 μM) + IBA (2 μM)
85
++
9.0 ± 0.77
Kn (7.5 μM) + IBA (3 μM)
85
+++
13 ± 0.77
Kn (7.5 μM) + IBA (4 μM)
80
++
8.2 ± 1.99
Average shoot length
(cm) ± SD
3 ± 0.90
3.8 ± 0.81
3.5 ± 0.74
3.3 ± 0.71
+ (minimum callusing), ++ (moderate callusing), +++ (maximum callusing) Growth period = 6 weeks. Data represents mean ± SD. The experiment was repeated three times.
ACKNOWLEDGEMENTS
Pvt. Ltd., Bombay, India
Nikam TD, Shitole MG (1993) Regeneration of Niger (Guizotia abyssinica
Cass.) cv. Shayadri from seedling explants. Plant Cell, Tissue and Organ
Culture 32, 345-349
Muthuswami S, Pappiah CM (1980) The culture of chicory. Seeds and Farms
6 (4), 34-35
Petrovic J, Stanojkovic A, Comic LJ, Curcic S (2004) Antibacterial activity
of Cichorium intybus. Fitoterapia 75, 737-739
Pieron S, Belaizi M, Boxus P (1993) Nodular culture, a possible morphogenetic pathway in Cichorium intybus propagation Scientia Horticulturae 53, 1-11
Profumo P, Gastaldo P, Cafffaro L, Dameri RM, Michelozzi GR, Bennici A
(1985) Callus induction and plantlet regeneration in Cichorium intybus L. II
effect of different hormonal treatments. Protoplasma 126, 215-220
Rehman RU, Fazili IS, Srivastava PS, Abbdin MZ (2000) Plant regeneration
from leaf sex plants of Cichorium intybus. Journal of Medicinal and Aromatic Plant Sciences 22/4a, 23/IA, 206-211
Rehman RU, Israr M, Srivastava PS, Bansal KC, Abdin MZ (2003) In vitro
regeneration of witloof chicory (Cichorium intybus L.) from leaf explants and
accumulation of esculin. In Vitro Cellular Developmental Biology – Plant 39,
142-146
Sharma N, Chandal KPS, Srivastava VK (1991) In vitro micropropagation of
Coleus foorskohlii Briq, a threatened medicinal plant. Plant Cell Reports 10,
67-70
Veklaylayutham P, Ranjitha Kumari BD (2003) Influence of photoperiod of
in vitro flowering in Cichorium intybus L. India. Journal of Plant Physiology
Special Issue, 447-450
Varotto S, Lucchin PP (2000) Immature embryos culture in Italian red chicory.
Plant Cell, Tissue and Organ Culture 62, 75-77
Wealth of India (Vol 3, revised Edn), (1992) PID/CSIR, New Delhi
Zaffar R, Ali SM (1998) Antihepatotoxic effect of root and callus extracts of
Cichorium intybus L. Journal of Ethnopharmacology 63, 227-231
The authors wish to thank the Director, CORD, Kashmir University for providing laboratory facilities to carry out these investigations.
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utility, value addition and biotechnology with an emphasis on current status
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Buhara Y, Arzu UT, Ekrem G (2007) TDZ–induced high frequency plant regeneration through multiple shoot formation in witloof chicory (Cichorium
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Chopra L, Nagar P (1956) Glossary of Indian Plants, PID, New Delhi, pp 613664
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Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
86
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
®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Impact of Chromium on the Oxidative
Defense System of Brassica juncea L. cv.
‘Pusa Jai Kissan’ under Hydroponic Culture
Rehana Hamid1* • Mahmood uz Zaffar1 • Jaime A. Teixeira da Silva2 •
Azra N. Kamili3 • Javid Ahmad Parray3
1 Department of Botany, Jamia Hamdard New Delhi 1100621, India
2 Department of Horticultural Science, Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan
3 Plant Tissue Culture Laboratory, Centre of Research for Development, University of Kashmir, Srinagar, 190006, J and K, India
Corresponding author: * rehanahameed@gmail.com
ABSTRACT
Brassica juncea is a medicinally important plant and is commonly used as a diuretic, stimulant and to treat arthritis. Seed are used for the
treatment of tumors and stomach disorders. B. juncea can hyperaccumulate cadmium and many other soil trace elements like selenium,
chromium, iron and zinc food supplements. Chromium (Cr)-induced oxidative damage and changes in the contents of proline and
glutathione in leaves of B. juncea. L. cv. ‘Pusa Jai Kissan’ were investigated after 3 and 5 days of treatment under hydroponic culture. Cr
was supplied as K2Cr2O7. The main response was an increase in superoxide dismutase activity and proline content which subsequently
reduced the activity of catalase and glutathione content in plants.
_____________________________________________________________________________________________________________
Keywords: antioxidants, catalase, proline, superoxide dismutase
Abbreviations: CAT, catalase; Cr, chromium; GTH, glutathione content; ROS, reactive oxygen species. SOD superoxide dismutase
INTRODUCTION
Brassica juncea belongs to the Cruciferae family and is
known as Indian mustard (English name). This plant is reported to be an anodyne, aperitif, diuretic, emetic, rubefacient, stimulant and is a folk remedy for arthritis, foot-ache,
lumbago, and rheumatism (Duke and Wain 1981). Seeds
are used to treat tumors in China while its roots are used as
a galactagogue in Africa. Ingestion may impart a body
odour which repels mosquitoes (Burkill 1966). Believed to
be aperient and tonic, its volatile oil is used as a counterirritant and stimulant. Leaves of this plant are said to relieve
headache (Burkill 1966). The seeds are used for abscesses,
colds, lumbago, rheumatism, and stomach disorders in Korea.
Chinese eat the leaves in soups to treat bladder inflammation or hemorrhaging. Mustard oil is used for skin eruptions and ulcers (Perry 1980). In Africa, the leaves are
cooked as a vegetable (Grubben and Denton 2004).
The rapid development and evolution of metal-base
industries have lead to contamination of environment with
heavy metals especially chromium (Cr). Heavy metals cannot be destroyed but can only be transformed from one oxidation state or organic complex to another (Marques et al.
2009). It is easily absorbed by plants from the soil and atmosphere, accumulates in their organs and shows cytotoxic
and phytotoxic effects (Parmar and Chanda 2005). Cr is
toxic to plants and does not play any role in plant metabolism (Dixit et al. 2002). Evidence also indicates that chromosomal abnormalities (micronuclei) and genomic instability (microsatellite instability) are possible by induction of
Cr(VI) (Wise et al. 2008). Metabolic alterations by exposure to Cr have also been described in plants either by a
direct effect on enzymes or other metabolites or by its ability to generate reactive oxygen species (ROS) which may
cause oxidative stress. The potential of plants with the capacity to accumulate or to stabilize Cr compounds for bioremediation of Cr contamination has gained interest in re-
cent years (Shanker et al. 2005). Cr phytotoxicity can induce the production of ROS like superoxide radical (O2-),
OH-, alkoxy radical (RO-), singlet oxygen (1O2-) and toxic
H2O2 (Brensgen et al. 2001) and these ROS produced are
detoxified by both enzymatic catalase (CAT), peroxidase
(POX), superoxidase (SOD) and gluthatione reductase (GR)
and non-enzymatic ascorbate (ASC), glutathione, -tocopherol (-TOC) and carotenoids (CAR). The oxidative system, if not detoxified, causes serious damage to cholorophyll, protein, membrane lipids and nucleic acids (Alcher et
al. 1997). Cr(VI) is a strong oxidant with a high redox
potential in the range of 1.33-1.38 eV accounting for a rapid
and high generation of ROS and its resultant toxicity (Shanker et al. 2004). Cr increases radical growth (Panda et al.
2002) and is reported to affect Hill’s reaction affecting both
dark and light reactions (Krupa and Baszynki 1995; Zeid et
al. 2001). B. juncea has been widely used in phytoremediation because of its capacity to accumulate high levels of Cr
and other metals such as lead (Zaier et al. 2010). The efficiency removal of copper from soil by B. juncea (L.) Czern
and Bidens alba (L.) DC. var radiata was reported (Naiyanan and Winaipanich 2006).
MATERIALS AND METHODS
Mustard seeds were germinated in paper towels and germinated
seedlings of similar size were placed in half-strength Hoagland’s
solution (Hoagland and Arnon 1950) containing (in mM): 2.4 Ca
(NO3)2, 1.0 KH2PO4, 3.0 KNO3, 1.0 MgSO4 and 0.5 NaCl and (in
M) 23.1 H3BO3, 4.6 MnCl2, 0.38 ZnSO4, 0.16 CuSO4, 0.052
H2MoO4 and 44.8 FeSO4 (as ferric sodium-EDTA complex) on
perforated polystyrene floats. The nutrient solution was bubbled
with sterile air and changed on alternate days. The experiment was
conducted in a completely randomized design with five replications. Growth chamber conditions were: photosynthetic photon
flux density of 430 M m2 s2, 14-h photoperiod and 60% relative
humidity. After day 7, plants were subjected to three Cr treatments.
Received: 7 August, 2009. Accepted: 24 November, 2010.
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Table 1 Effect of various concentrations of chromium on SOD activity (EU mg protein h-1), catalase activity (EU mg protein min-1), total glutathione
content (μmol g-1 fw) (GSH) and proline (μg g-1 fw).
TREATMENT (μM)
Parameter
T0
T1
T2
T3
Mean ± SD (PV)
Mean ± SD (PV)
Mean ± SD (PV)
Mean ± SD (PV)
SOD activity after 3 days
1.036 ± 0.115 (0.0)
1.699 ± 0.045 (64.4)
1.238 ± 0. 242 (45.2)
1.36 ± 0.058 (51.4)
SOD activity after 5 days
0.773 ± 0.02 (0.0)
0.951 ± 0.005 (23.1)
0.852 ± 0.011 (10.2)
1.220 ± 0.03 (50.3)
0.47 ± 0.030 (21.2)
Catalase activity after 3 days
0.687 ± 0.067 (0.0)
0.577 ± 0.0092 (8.4)
0.600 ± 0.48 (1.74)
0.310 ± 0.069 (17.5)
0.298 ± 0.043 (22.2)
Catalase activity after 5 days
0.433 ± 0.038 (0.0)
0.391 ± 0.136 (10.3)
880.10 ± 5.99 (15.09)
988.7 ± 4.70 (16. 3)
Glutathione content after 3 days
1061.6 ± 5.02 (0.0)
1046 ± 4.88 (12.08)
701 ± .4.06 ( 20.0)
760 ± 2.25 (18.23)
Glutathione content after 5 days
1008.4 ± 4.08 (0.00)
828.8 ± .5.01 (17.06)
Proline concentration after 3 days
0.320 ± 0.120 (0.0)
0.778 ± 0.04 (60.5)
1.32 ± 0.112 (72.1)
1.29 ± 0.080 (68.9)
Proline concentration after 5 days
0.976 ± 0.289 (0.0)
1.011 ± 0.096 (51.0)
1.532 ± 0.120 (63.0)
2.01 ± 0.0126 (74.5)
Data represents average of three samples analysed r S.D, Values in brackets represent % variation, compared to control. Plants were subjected to three concentrations of Cr
i.e., T1 = 25 M, T2 = 50 M and T3 = 100 M; T0 = control.
regulation of SOD occurred in response to Cr; high SOD
activity might be in direct response to the generation of
super oxide radicals by Cr-induced blockage of the electron
transport chain in the mitochondria. The decrease in the
activity of SOD as the concentration of external Cr increased might be because of the inhibitory effect of Cr ions
on the enzyme itself (Schiavon et al. 2008). One of the strategies that plants have evolved to counteract toxic effects of
heavy metal stress is through accumulation of an organic
solute like Pro which is an imminoacid that accumulates
under stress conditions. Accumulation of Pro in plants
under stress results due to its active synthesis from glutamate. Pro accumulation helps to conserve nitrogenous compounds and protect plant under stress. An observed increase
of Pro in plants treated with Cr is not unexpected, since
metabolism reaction of these compounds used may represent a stress situation comparable to various environmental stresses (Aspinall and Paleg 1981; Sumira et al. 2010).
Under Zn stress a marked decrease in Proline content was
found by Prasad and Saradhi (1995) in Brassica and Cajanus.
Cr was supplied as K2Cr2O7. Plants (15/treatment) were subjected
to three concentrations of Cr (T1 = 25 M, T2 = 50 M, T3 = 100
M). One set of seedlings was kept without Cr i.e. T0 and served
as control. SOD (EC 1.15.1.1) activity was estimated by the
method of Dhindsa et al. (1981), CAT oxididoredutase (EC
1.11.1.6) activity in leaves following the method of Aebi (1984),
Glut (EC 1.8.1.7) content as per Anderson (1985) and proline
content by the method of Bates et al. (1973). All results represent
the mean ± SE of three replicates per treatment.
RESULTS AND DISCUSSION
Proline concentration and SOD activity increased as Cr
concentration increased 3-5 days after treatment (Table 1).
In contrast, Glut content and CAT activity decreased as Cr
concentration increased 3-5 days after treatment (Table 1).
A common feature of different stress factors is their
potential to increase the production of ROS in plant tissues.
To prevent damage, plants possess an antioxidative system
composed of low molecular weight antioxidants like Glut
and Pro and protective antioxidant enzymes like SOD and
CAT (Asada and Takahashi 1987; Aguirre and Borneo
2010). Heavy metals generate toxic ROS such as H2O2, O2-,
OH- and O2- which degrade important cellular components
by inducing oxidative stress (Dietz et al. 1999). CAT is an
important heme-containg enzyme that catalyses the dismutation of H2O2 to H2O and O2 and is localized in peroxisomes. It is an important enzyme required for ROS detoxification of CAT in response to Cr and has been studied in
many crops like rice, wheat, green gram and even in lower
mosses (Choudhury and Panda 2004; Panda and Patra
2004). Sen et al. (1994) observed a decrease in CAT activity
and increase in peroxidase activity at concentrations above
10 μg L-1 Cr(VI). In most cases a decline in CAT activity
was registered (Panda and Patra 1998, 2000, 2002; Panda
2003). In the present study, total CAT content in plants in
response to Cr decreased since Cr is a heme-containing enzyme that affects iron uptake in dicots (Guerinot and Yi
1994). Glut and Glut-reductase are important components
of the ascorbate-Glut cycle, which plays an important role
in detoxification of ROS. In the present study there was a
marked decrease in total Glut content. De Vos et al. (1997)
also reported a decrease in Glut content in Silene cucubalis
after exposure to a heavy metal (copper). Several authors
have observed oxidation of different cellular thiols such as
GSH, glutathione and cysteine by Cr(VI) in in vitro studies.
Dichromate reacts with GSH at the sulphydryl group forming an unstable gluthione-CrO3- complex (Brauer and
Wetterhahn 1991). Thiolate complexes of Cr(VI) with –
glutamylcysteine, N-accetylcysteine and cysteine have also
been described (Brauer et al. 1996). The inter-conversion of
reduced and oxidized forms of Glut to maintain the redox
status of the cells so as to scavenge free radicals could have
caused a decrease in GSH (Shanker et al. 2004b).
SOD converts superoxide and hydroxyl radicals into
H2O2 which is degraded into water and molecular oxygen
by CAT or peroxidase. SOD plays a central role in defense
against oxidative defense (Beyer 1994). In this study up
ACKNOWLEDGEMENTS
The authors are thankful to Departments Botany and Toxicology
of Jamia Hamdard New Delhi India, for providing facilities and
technical help.
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®
Medicinal and Aromatic Plant Science and Biotechnology ©2010 Global Science Books
Screening Phytochemical Constituents of
21 Medicinal Plants of Trans-Himalayan Region
Janifer Raj1* • Ballabh Basant2 • Pal M. Murugan3 • Jaime A. Teixeira da Silva4 •
Kumar Saurav2 • Om P. Chaurasia2 • Shashi Bala Singh1,2,3
1 Division of Biotechnology, Defence Institute of High Altitude Research (DIHAR), Defence Research and Development Organisation (DRDO), C/O 56 APO,
Leh, Ladakh, Jammu and Kashmir, India
2 Division of Medicinal and Aromatic Plants, DIHAR, DRDO, C/O 56 APO, Leh, Ladakh, Jammu and Kashmir, India
3 Division of Agricultural Extension, DIHAR, DRDO, C/O 56 APO, Leh, Ladakh, Jammu and Kashmir, India
4 Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Ikenobe, Miki-cho, 761-0795, Japan
Corresponding author: * jennipal@gmail.com
ABSTRACT
Alkaloids, tannins, flavonoids, saponins, steroids, and cardiac glycoside distribution in 1 high altitude medicinal plants belonging to
different families (Apiaceae, Asteraceae, Crassulaceae, Lamiaceae, Rosaceae, Rubiaceae, Urticaceae, and Zygophyllaceae) were assessed
and compared. The plants investigated were Achillea millefolium, Artemesia dracunculus, Bidens pilosa, Carum carvi, Dracocephalum
heterophyllum, Ferula jaeskiana, Gallium pauciflorum, Heracleum pinnatum, Hippophae rhamnoides, Inula racemosa, Mentha longifolia,
Nepeta podostachys, Origanum vulgare, Peganum harmala, Rhodiola imbricata, Rhodiola heterodenta, Rosa webbiana, Rosa macrophylla, Rubia cordifolia, Tanacetum gracile, and Utrica hyperborea, which have been widely used for time immemorial in the traditional
Amchi system of medicine in the Ladakh region of India. Phytochemicals were qualitatively detected using aqueous extracts and solvent
fractions of plants using various biochemical tests. These plants are a potential source of useful drugs. Future studies will isolate, identify,
characterize and elucidate the structure of novel bioactive compounds. The significance of these plants in traditional medicine and the
importance of the distribution of their chemical constituents are discussed in the context of the role of these plants in ethnomedicine in
Ladakh.
_____________________________________________________________________________________________________________
Keywords: bioactive compounds, MAPs, traditional medicine, scientific investigation
INTRODUCTION
Medicinal plants are of great importance to the health of
individuals and communities. The plant kingdom represents
an enormous reservoir of biologically active compounds
with various chemical structures and protective or diseasepreventive properties (phytochemicals). Among many biological hotspots around the world, the Himalayas and Western Ghats in India are regions of prime biodiversity concern. The Northern part of India harbors a great diversity of
medicinal plants because of the majestic Himalayan range.
The trans-Himalaya sustains about 337 species of medicinal
plants (Kala 2002). This high proportion of medicinal plants
among the existing flora, known for their medical purposes,
is unique to India more than any other country in the world
(Kala et al. 2006). Ladakh, the cold desert located in the
Northernmost part of trans-Himalaya in Jammu and Kashmir State, is well known for its rich ethnobotanical wealth
and the health care of the tribal population is mainly dependent on the traditional Amchi system of medicine. A great
deal of traditional knowledge of the use of various plant
species is still intact among the indigenous people; this is
especially relevant in the mountainous areas such as the
Himalayas due to poor accessibility of terrain and the comparatively slow rate of development (Farooquee 2004).
Therefore, much research is now devoted to the phytochemical investigation of higher plants which have ethnobotanical information associated with them. Achillea millefolium, Artemesia dracunculus, Bidens pilosa, Carum carvi,
Dracocephalum heterophyllum, Ferula jaeskiana, Gallium
pauciflorum, Heracleum pinnatum, Hippophae rhamnoides,
Inula racemosa, Mentha longifolia, Nepeta podostachys,
Origanum vulgare, Peganum harmala, Rhodiola imbricata,
Rhodiola heterodenta, Rosa webbiana, Rosa macrophylla,
Rubia cordifolia, Tanacetum gracile, and Utrica hyperborea are extensively used in the Amchi system of medicine in the Ladakh region of the Himalayas (Chaurasia et al.
2007). Their various uses in traditional medicine are reviewed in Table 1. The present study is a preliminary phytochemical screening of the important high altitude medicinal
plants used in traditional medicine as an investigation to
find a fundamental scientific basis for the use of these
medicinal plants by defining the phytochemical constituents
present in them.
MATERIALS AND METHODS
Collection of plant material
The leaves, stems and roots of each of these plants were collected
from their natural habitat and from the herbal garden, DIHAR, Leh,
Ladakh. All 21 samples were identified by the authors.
Extract preparation
Whole plants were shade dried, separated into leaves, stems and
roots, then powdered using a pestle and mortar. The samples were
extracted at room temperature with absolute ethanol, chloroform
and distilled water (DW) for 24 hrs. Extracts prepared by different
solvents were used to test for different compounds: DW (i.e.,
aqueous extract) for identification of tannins, flavonoids and saponins, chloroform extract for alkaloids and steroid tests, and ethanol
extract for testing the presence of cardiac glycosides. The filtrates
were obtained by using Whatman No. 1 filter paper.
Received: 12 July, 2010. Accepted: 10 December, 2010.
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Table 1 Review of the various medicinal uses of the high altitude medicinal plants.
Botanical name
Family
Vernacular name
Part used
Achillea millefolium
Asteraceae
Yarrow (E), Chuang (L)
Leaves/flowers
Artemesia dracunculus
Asteraceae
Shersing or Burtse (L)
Leaves/roots
Bidens pilosa
Carum carvi
Asteraceae
Apiaceae
Local tea / Gurgur Chai
Konyot
Leaves
Seeds
Zinkzer (L)
Jangli Heeng / Chuklam
(L)
Phomongo (L)
Spru-ma (L)
Leaves/flowers
Stem/roots
Dracocephalum heterophyllum Laminaceae
Ferula jaeskiana
Apiaceae
Galium pauciflorum
Heracleum pinnatum
Rubiaceae
Apiaceae
Hippophae rhamnoides
Elagnaceae
Seabuckthorn (E)
Tsermang (L)
Whole plant
Inula racemosa
Mentha longifolia
Asteraceae
Laminaceae
Puskarmool (H) Manu (L)
Jungli Pudhina (H)
Roots
Whole plant
Nepeta podostachys
Origanum vulgare
Lamiaceae
Lamiaceae
Peganum harmala
Shangukaram
Wild oregano / marjoram
(E)
Zygophyllaceae Wild rue (E) / Sepan (L)
Rhodiola imbricata
Crassulaceae
Rhodiola heterodonta
Crassulaceae
Rosa webbiana
Rosaceae
Rosa macrophylla
Rubia cordifolia
Rosaceae
Rubiaceae
Tanacetum gracile
Utrica hyperborea
Asteraceae
Utricaceae
Stem/leaves
Roots
Aerial parts
Leaves
Traditional medicinal usage
Astringent, stimulant, used against heartburn, cold, colic,
hysteria, epilepsy and rheumatism. Flowers as a tonic.
Roots as diuretic, for treatment of intestinal worms, lung
diseases, mensural problems and toothache.
As a local tea/Namkin chai
Fruits and seeds are used as febrifuge, eye vision and
digestive.
Cold, cough and headache.
Roots used in rheumatism, Stem gum resin used to treat
septic wounds and toothache.
Used to cure intestinal parasites and reduces fever.
Used to treat inflammation and pain caused by
vulnerable fever, checks haemorrhage and abdominal
cramps.
Whole plant is medicinal. Fruits and seeds are used as
blood purifier, against peptic ulcer, lung disorders, cuts
and wounds. Anti inflammatory and improves digestion.
Anti inflammatory, antiseptic, expectorant and diuretic.
Anti-dysenteric, carminative, antiseptic and stimulant.
Used against fever and heat apoplexy.
Aromatic.
Carminative and stimulates the flow of bile.
Seeds
Used against fever, stomach complaints, anthelmintic,
antiseptic, eye disorders, measles, asthma, rheumatism,
joint pains, lactation and mensural problems. Diuretic
and appetiser.
Rose root or stone crop (E) Leaves/roots
Health tonic. Anti inflammatory, used for treatment of
/ Shrolo (L)
lung problems, cold, cough and restoring memory.
Treatment for lung problems, cold, fever and anti
Rose root or stone crop (E) Leaves/roots
/ Shrolo (L)
inflammatory.
Wild rose (E) / Siah (L)
Flower and
Treatment of fever due to poison, food poisoning,
fruits
inflammation of liver, hepatitis and jaundice.
Wild rose (E) / Siah (L)
Flowers
Treatment of stomach pain
Manjith (H), Indian
Stems and roots Effective against blood diseases, chest complaints,
Maddar (E), Btsod (L)
leucoderma, menstrual disorders, ulcers, stomach ache
and urinary complaints.
Khamchu (L)
Leaves/flowers Used against intestinal worm.
Stinging nettle (E),
Leaves
Used against rheumatism and joint pain.
Dzatsutt or Zozot (L)
Source: Chaurasia et al. 2007; E = English; H = Hindi; L = Bodhi/Ladakhi
ble persistent froth, which was mixed with 3 drops of olive oil and
shaken vigorously, then observed for the formation of an emulsion,
which itself was a positive indicator for the presence of saponins.
Phytochemical screening
Chemical tests were carried out on the aqueous and chloroform
extracts and on the powdered specimens using standard procedures
to identify the constituents as described by Harborne (1973),
Trease and Evans (1989) and Sofowara (1993). All the biochemical tests to test the qualitative presence of tannins, flavonoids,
saponins, steroids, alkaloids and cardiac glycosides were carried
out in triplicate; results were repeatable.
4. Test for cardiac glycosides (Keller-Killani test)
5 ml of the ethanol extract was treated with 2 ml of glacial acetic
acid containing one drop of FeCl3 solution. This was underlayed
with 1 ml of concentrated H2SO4 (98%). A brown ring of the interface indicates a deoxy sugar, characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid
layer, a greenish ring may form just gradually throughout thin
layer. The rings of violet or green indicate the presence of cardiac
glycosides and the brown ring indicates the deoxy-ribo sugars.
1. Test for tannins
0.5 g of the dried powdered samples was boiled in 20 ml of DW in
a test tube and then filtered. A few drops of 0.1% FeCl3 was added
and observed for brownish-green or a blue-black colouration.
5. Test for alkaloids (Wagner’s test)
2. Test for flavonoids
1 ml of the chloroform extract was mixed with 1 ml of Wagner’s
reagent. A positive reaction was indicated by a brown precipitate.
5 ml of dilute ammonia solution were added to a portion of the
aqueous filtrate of each plant extract followed by the addition of
concentrated H2S04. Yellow colouration observed in each extract
indicated the presence of flavonoids. This colouration disappeared
when the solution was left to stand.
6. Test for steroids and terpenoids (Salkowski test)
5 ml of chloroform extract was mixed with 3 ml concentrated
H2S04 and shaken. A positive reaction was indicated by a red solution on standing.
3. Test for saponins
About 2 g of the powdered sample was boiled in 20 ml of DW in a
water bath for 3 min and filtered. 10 ml of the filtrate was mixed
with 5 ml of DW and shaken vigorously for 5 min to obtain a sta-
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91
Phytoconstituents of trans-Himalayan plants. Raj et al.
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Table 2 Qualitative analysis of phytochemicals of the high altitude medicinal plants (whole plants including flowers, leaves, stem and roots).
Tannins
Plants
Alkaloids
Flavonoids
Saponins
Cardiac
Steroids
Terpenoids
(chloroform
(aqueous
(aqueous
(aqueous
glycoside
(chloroform
(chloroform
extract)
extract)
extract)
extract)
(ethanol extract) extract)
extract)
Achillea millefolium
+
+
+
+
+
+
+
Artemesia dracunculus
+
+
+
+
+
Bidens pilosa
+
+
+
+
Carum carvi
+
+
+
+
+
Dracocephalum heterophyllum +
+
+
+
+
Ferula jaeskiana
+
+
+
+
+
+
+
Gallium pauciflorum
+
+
+
+
+
+
Heracleum pinnatum
+
+
+
+
+
Hippophae rhamnoides
+
+
+
+
Inula racemosa
+
+
+
+
Mentha longifolia
+
+
+
Nepeta podostachys
+
+
+
+
+
Origanum vulgare
+
+
+
+
+
Peganum harmala
+
+
+
+
Rhodiola imbricata
+
+
+
+
+
Rhodiola heterodenta
+
+
Rosa webbiana
+
+
+
+
+
Rosa macrophylla
+
+
+
+
+
Rubia cordifolia
+
+
+
+
Tanacetum gracile
+
+
+
+
+
+
+
Utrica hyperborea
+
+
+
+
+
+
+ Presence of constituent, - Absence of constituent
plant phenolic compounds including flavones, flavonols,
isoflavones, flavonones and chalcones, possess numerous
biological/pharmacological activities. Recent reports of
antiviral, anti-fungal, antioxidant, anti-inflammatory, antiallergenic, antithrombic, anticarcenogenic, hepatoprotective,
and cytotoxic activities of flavonoids have generated interest in studies of flavonoid-containing plants. Of these biological activities, the anti-inflammatory capacity of flavonoids has long been utilized in Chinese medicine and the
cosmetic industry as a form of crude plant extracts (Kim et
al. 2004; Aguinaldo et al. 2005; Moon et al. 2006; Veitch
2007; Jiang et al. 2008; Wu et al. 2008; Peteros and Mylene
2010). The presence of flavonoids in all crude plant extracts
may confirm their folkloric use in treating rheumatism
(Ferula jaeskiana, roots) and antiinflammatory (Heracleum
pinnatum, roots; Hippophae rhamnoides, fruits) antioxidant
(Hippophae rhamnoides, fruits and leaves).
Isoprenoids, including saponins and steroids, have
expectorant and antidiabetic properties and are precursors
for steroid hormones (Okwu 2001). 17 and 8 plant samples
were positive for saponins and steroids, respectively (Table
2). The presence of saponins and steroids in the crude extracts examined may justify their therapeutic use as treatment of cold and cough as expectorant (Dracocephalum
heterophyllum, flowers and leaves; Inula racemosa, roots)
and treatment of asthma (Peganum harmala, seeds). 13
plant samples tested positive for cardiac glycosides, which
are used in treating congestive heart failure and cardiac
arrhythmia.
Triterpenoids are studied for their anti-inflammatory,
hepatoprotective, analgesic, antimicrobial, antimycotic,
virostatic, immunomodulatory and tonic effects. They are
used in the prevention and treatment of hepatitis, parasitic
and protozoal infections and for their cytostatic effects. The
disadvantage of using triterpenoids is the toxicity associated
with their hemolytic and cytostatic properties (Dzubak et al.
2006). The presence of terpenoids in 4 crude plant extracts
may confirm their traditional use in treatment against intestinal worm (Tanacetum gracile, leaves), stomach complaints (Achillea millefolium, leaves) and treatment of septic
wounds (Ferula jaeskianastem, gum resin).
The medicinal plants studied here may be rich sources
of phytochemicals, particularly alkaloids, tannins, flavonoids, steroids, cardiac glycosides and terpenoids which can
be isolated and further screened for different kinds of biological activities, depending on their reported ethno-botanical and/or therapeutic uses and potential source of useful
RESULTS AND DISCUSSION
The isolation of pure, pharmacologically active constituents
from plants remains a long and tedious process. For this
reason, it is necessary to have methods available which
eliminate unnecessary separation procedures. Chemical
screening is thus performed to allow localization and targeted isolation of new or useful constituents with potential
activities. This procedure enables recognition of known
metabolites in extracts at the earliest stages of separation
and is thus economically very important.
The preliminary investigation conducted on the 21 high
altitude medicinal plants revealed the presence of medicinally active phyto-constituents (Table 2). The results indicate that these plants were rich in alkaloids, tannins, flavonoids, saponins, steroids and cardiac glycosides, all known
to exhibit medicinal as well as physiological activity. All
the 21 plants screened gave a positive reaction for alkaloids.
These are heterocyclic indole compounds which have proven pharmacological properties such as hypotensive, anticonvulsant, antiprotozoal, antimicrobial, and antimalarial
activities (Lacqlercq et al. 1998; Frederich et al. 2002; Lata
et al. 2010).
Acetogenins screened included tannins and flavonoids.
Tannins are polymeric phenolic substances capable of tanning leather or precipitating gelatin from solution, a property known as astringency (Cowan 1999). Twenty one
plants on investigation gave a positive reaction for tannins.
Tannins possess general antimicrobial and antioxidant activities (Rievere et al. 2009). Low concentration of tannins
can inhibit the growth of microorganisms, and act as an
antifungal agent at higher concentrations by coagulating the
microorganism’s protoplasm (Adekunle and Ikumapayi
2006). Tannins may have potential value as cytotoxic and/or
antineoplastic agents (Aguinaldo et al. 2005). Aside from
the use of tannins as antimicrobial agents or in the prevention of dental caries, they are now being used in the manufacture of plastics, paints, ceramics and water softening
agents (Bandarayanake 2002). The presence of tannins in
the crude extracts examined may justify their therapeutic
use to cure menstrual problems and toothache (Artemesia
dracunculus, roots), toothache (Ferula jaeskiana, stem gum
resin), blood diseases (Rubia cordifolia, roots) as well as
use as an astringent (Achillea millefolium, leaves).
Seventeen samples tested positive for flavonoids which
are known to possess anti-viral and anti-inflammatory properties. Flavonoids, a large group of naturally occurring
Medicinal Plants of the Himalayas: Advances and Insights. Husaini AM (Ed). Global Science Books, UK
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Medicinal and Aromatic Plant Science and Biotechnology 4 (Special Issue 1), 90-93 ©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
drugs. Therefore, the data generated from these experiments
provide a basic qualitative chemical basis for the wider use
of these plants as therapeutic agents for treating various
ailments. However, there is a need to carry out further advanced hyphenated spectroscopic studies in order to elucidate the structure of these compounds. Quantitative analyses of these phytochemicals may also be done as a guide as
to which particular bioactive class of compounds may be
subjected to subsequent target isolation. The antimicrobial
activities of these plants for the treatment of diseases, as
claimed by the traditional healers, are also being investigated.
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