gjrmi - volume 3, issue 2, february 2014
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Global Journal of Research on Medicinal plants & Indigenous medicine - February 2014TRANSCRIPT
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INDEX – GJRMI - Volume 3, Issue 2, February 2014
MEDICINAL PLANTS RESEARCH
Bio-technology – Short communication
CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM CHLOROPHYTUM
BORIVILIANUM Sant. et Fernand
Sharma Rohit, Saxena Nidhi, Thakur Gulab S, Sanodiya Bhagwan S, Jaiswal Pallavi 33–39
Pharmacology
HAIR GROWTH STIMULATING EFFECT AND PHYTOCHEMICAL EVALUATION OF HYDRO-
ALCOHOLIC EXTRACT OF GLYCYRRHIZA GLABRA
Roy Deb Saumendu, Karmakar Prithivi Raj, Dash Suvakanta, Chakraborty Jashabir, Das Biswajit 40–47
Phytochemistry
IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING
ELICITORS
Tamandani Ehsan Kordi, Valizadeh Jafar, Valizadeh Moharam
48–56
Review
INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS OF NANDA DEVI BIOSPHERE
RESERVE – A REVIEW BASED ON PREVIOUS STUDIES
Singh Rahul Vikram 57–66
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF CONVOLVULUS QUAMOCLIT L., OF THE
FAMILY CONVOLVULACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT,
KARNATAKA, INDIA
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM
CHLOROPHYTUM BORIVILIANUM Sant. et Fernand
Sharma Rohit1*
, Saxena Nidhi2, Thakur Gulab S
3,
Sanodiya Bhagwan S4, Jaiswal Pallavi
5
1, 2, 3, 4, 5 Plant Biotechnology Laboratory, R&D Division, Tropilite Foods Pvt. Ltd., Davars Campus, Tansen
Road, Gwalior-474002 (M.P.), India.
*Corresponding Author: [email protected]; Mob: +919755594040
Received: 07/12/2013; Revised: 25/01/2014; Accepted: 31/01/2014
ABSTRACT
Saponins are imperative non-volatile chemical compounds valued for several medicinal
properties. The pharmaceutical use of saponins for semi-synthesis of steroidal drugs makes it an
essential element of life with a diverse range of properties including antimicrobial, insecticidal,
haemolytic, aphrodisiac, foaming and emulsification. The tuberous roots of Chlorophytum
borivilianum always remains a major source for isolation of saponin. A conventional efficient
method was developed for saponin isolation from in-vivo and in-vitro samples of C. borivilianum by
delipidization and deproteinization with petroleum ether and chloroform leading to development of a
whole new process for saponin isolation. Protocol was tested with saponin confirmatory test
followed by thin layer chromatography.
KEY WORDS: Saponin, Chlorophytum borivilianum, delipidization, steroid.
Short communication
Cite this article:
Sharma Rohit, Saxena Nidhi, Thakur Gulab S,
Sanodiya Bhagwan S, Jaiswal Pallavi (2014), CONVENTIONAL METHOD FOR
SAPONIN EXTRACTION FROM CHLOROPHYTUM BORIVILIANUM Sant. et
Fernand, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 33–39
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Saponins are generally known as non-
volatile, surface-active compounds that are
widely distributed in nature, occurring
primarily in the plant kingdom (Hostettmann et
al., 2005). The name ‘saponin’ is derived from
the Latin word sapo, which means ‘soap’,
because saponin molecules form soap-like
foams when shaken with water. They are
structurally diverse molecules that are
chemically referred to as triterpene and steroid
glycosides. They consist of nonpolar aglycones
coupled with one or more monosaccharide
moieties (Oleszek, 2002). This combination of
polar and non-polar structural elements in their
molecules explains their soap-like behaviour in
aqueous solutions. Saponins are the important
chemical compounds from tubers of C.
borivilianum. They are used in the indigenous
systems of medicine as a well known health
tonic, aphrodisiac and galactogogue (Chopra et
al., 1956; Marais et al., 1978; Nadkarni, 1996;
Oudhia, 2001). Pharmaceutical industries buy
saponins in large quantities because of their use
for the semi-synthesis of steroidal drugs for
phyto-therapy and in cosmetic industry
(Sharma et al., 2012; Haque et al., 2011;
Ksouri et al., 2011). They are believed to form
the main constituents of many plant drugs and
folk medicines responsible for numerous
pharmacological properties (Marais et al.,
1978; Estrada et al., 2000; Debnath et al.,
2006; Katoch et al., 2010). Therefore, it is a
category of phyto-nutrients (plant nutrients)
found abundantly in many beans, and other
plants such as Ginseng, Alfalfa, Yucca, Aloe,
Quinoa seed and also in Safed Musli (Chopra et
al., 1956; Nadkarni, 1996).
Saponins have a diverse range of properties
from sweetness to bitterness (Grenby, 1991;
Kitagawa, 2002; Heng et al., 2006; Thakur et
al., 2009), foaming and emulsification (Price et
al., 1987), pharmacological and medicinal
(Attele et al., 1999; Debnath et al., 2007),
haemolytic (Oda et al., 2000; Sparg et al.,
2004), and antimicrobial, insecticidal, and
molluscicidal activities (Sparg et al., 2004;
Sundaram et al., 2011) and finds some place in
beverages, confectionery and cosmetic industry
(Price et al., 1987; Petit et al., 1995; Uematsu
et al., 2000). Saponins consist of a sugar
moiety, usually containing glucose, galactose,
glucuronic acid, xylose, rhamnose or
methylpentose, glycosidically linked to a
hydrophobic aglycone (sapogenin) which may
be triterpenoid or steroid (Abe et al., 1993;
Haralampidis et al., 2002); derived from the 30
carbon atoms containing precursor
oxidosqualene (Haralampidis et al., 2002). The
difference between the two classes lies in the
fact that the steroidal saponins have three
methyl groups removed (i.e. they are molecules
with 27 C-atoms), whereas in the triterpenoid
saponins all 30 C-atoms are retained. Saponins
were classified into three classes, namely, the
triterpenoid saponins, the spirostanol saponins
and the furostanol saponins. However, due to
secondary biotransformation such a
classification emphasizes incidental structural
elements and does not reflect the main
biosynthetic pathways (Sparg et al., 2004).
There are some other classes of compounds that
have been considered as saponins, such as the
glycosteroid alkaloids (Haralampidis et al.,
2002). Baumann et al., (2000) reported that
saponins have hemolytic properties that
generally are attributed to the interaction
between the saponins and the sterols of the
erythrocyte membrane. As a result erythrocyte
membrane bursts, causing an increase in
permeability and a loss of haemoglobin. A
study was made to establish the relationship
between the adjuvant and haemolytic activity
of saponins derived and purified from 47
different food and medicinal plants. However,
the results indicated that the adjuvant activity
does not relate with haemolytic activity (Oda et
al., 2000).
Chlorophytum borivilianum Sant. et
Fernand commonly known, as Safed Musli is a
traditional rare Indian medicinal herb having
many therapeutic applications in Ayurvedic,
Unani, Homeopathic and Allopathic medicine
system. It is an herbaceous plant with
fasciculated tuberous root found naturally in
forests and its shoots can be seen during the
rainy seasons (Kothari et al., 2003). Research
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
studies on Chlorophytum conducted in India
and elsewhere indicate that saponins (viz.
neohecogenin, neotigogenin, stigmasterol and
tokorogenin) are responsible for medicinal
properties (Jat et al., 1990). Safed musli is
among the few medicinal plants witnessing
steady growth in pharmaceutical,
phytopharmaceutical and nutraceutical products
(Debnath et al., 2006, 2007; Thakur et al.,
2009). Due to the many therapeutic
applications and several bioactive compounds,
C. borivilianum is also called ‘The white gold
for biopharmaceuticals and neutraceuticals’
(Thakur et al., 2009).
It contains steroidal and triterpenoidal
saponins, sapogenins, fructans and flavonone
glycosides, which are powerful uterine
stimulants. Dried roots of Chlorophytum
contain 42% carbohydrate, 80–89% protein, 3–
4% fiber and 2–17% saponin (Wagle et al.,
2000). It is useful in curing impotency with
spermatogenic property and is considered as an
alternative to ‘Viagra. It is a rich source of over
25 alkaloids, vitamins, proteins, carbohydrates,
steroids, saponins, potassium, calcium,
magnesium, phenol, resins, mucilage and
polysaccharides with high content of simple
sugars mainly sucrose, glucose, fructose,
galactose, mannose and xylose (Ramawat et al.,
2000; Debnath et al., 2006, 2007; Thakur et al.,
2009). Due to their high medicinal value,
several medicinal herbs are being
indiscriminately collected before they could
reach phenological maturity and vegetative
regeneration capacity (Biswas et al., 2003).
This has led to the depletion of natural source
of several valuable plants like Safed musli. The
restricted distribution and indiscriminate over-
exploitation of this plant coupled with low seed
set and viability and poor seed germination
rates has made its status rare in the wild
(Debnath et al., 2006). Among all the species
of Chlorophytum present in India, C.
borivilianum produces the maximum root tuber
along with the highest saponin content (Attele
et al., 1999). Traditionally, roots of these
species are reputed to posses various
pharmacological utilities having saponins as
one of the important phyto-chemical
constituents (Marais et al., 1978). The objective
of the manuscript is to develop a brisk protocol
for extraction of saponin from tubers of C.
borivilianum with special attention on the
screening of extracted metabolite.
MATERIAL AND METHODS
1. Plant Material:
Plants and roots of Chlorophytum
borivilianum were collected from plant
herbarium, Plant biotechnology laboratory at
Tropilite foods Pvt. Ltd., Gwalior, India. Plants
were available in vitro (in test tubes) and in
vivo (in pots) conditions in laboratory. Plants
(both roots and shoots) were washed
thoroughly and were sliced into pieces
followed by drying in hot air oven at 100°C for
4–5 days. On complete drying, the plant
material was grinded uniformly with the help of
mortar-pestle and stored in an airtight
container.
2. Chemicals used:
Chemicals used for isolation purposes were
95% Ethanol (Merck Millipore), Petroleum
ether (Sigma-Aldrich), Ethyl acetate (Sigma-
Aldrich), Chloroform (Ultra pure, HiMedia),
Methanol (Merck Millipore), Acetone (LR
grade, HiMedia), Distilled water. Quality of
isolated saponin was tested on TLC plates
Silica gel 60 F254 plates (Merck) with Sulphuric
acid (Rankem) as spraying agent.
3. Saponin Extraction Procedure:
The extraction process was carried out with
both in vivo and in vitro samples by soaking the
dried plant material in ethanol 95% overnight.
The extraction was done with Petroleum ether,
Ethyl acetate, Chloroform, Methanol and
Acetone. Petroleum ether was used for
delipidization and chloroform for
deproteinization of dried mixture. On
extraction of crude saponin, methanol was used
to mellow the developing mixture followed by
drop wise addition into acetone solution
leading to precipitation. The precipitated
material was extracted and dried in hot air oven
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
leading to formation of whitish brown crystals
(Lakshmi et al., 2012).
4. Saponin Confirmatory Test
Froth test: 0.5 gm of the alcoholic extract was
dissolved in 10 ml of distilled water in a test
tube. The test tube was shaken vigorously for
about 30 seconds .The test tube was allowed to
stand in vertical position and was observed
over a 30 min period of time. Thick persistent
froth was observed on the surface of the liquid
indicating presence on saponin.
5. Thin layer Chromatography
TLC technique was used for purification of
saponins isolated from C. borivilianum.
Samples (crude saponin) and the reference
standards (Saponin, Sigma) were loaded on the
pre-coated TLC plates silica gel 60 F254 plates.
Mobile phase chloroform: methanol: water
(65:35:10 v/v/v) was used for the separation.
Two drops of standard and sample were loaded
up on TLC plates with the help of a
micropipette. The loaded plates were placed in
the TLC jar which contained the solvent
system. After the completion of the run the
plates were taken out and kept at room
temperature to get dried for 10 minutes. The
plates were developed with the spraying
reagent (5%, H2SO4). After spraying the
reagents, the plates were kept at 110ºC for 10–
15 minutes in hot air oven and results were
observed later (Fig 1).
Fig 1: Thin layer chromatography results of In vivo, in vitro and standard samples developed
in mobile phase of chloroform: methanol: water (65:35:10 v/v/v)
RESULT AND DISCUSSION
The phytochemical extraction of in vivo
root tubers and in vitro plant body of
Chlorophytum borivilianum was carried out
using six different solvent systems (Ethanol,
petroleum ether, ethyl acetate, chloroform,
methanol and acetone). Whitish brown crystals
were obtained as end product of the process.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 33–39
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Experimental procedure:
1. Powder Soaking: In vivo and in vitro dried
plant samples with a quantity of 30 gm each
were mixed in 95% ethanol (180 ml)
solution separately in conical flasks. After
uniform mixing the solutions were placed
in orbital shaker for stirring at 100 RPM for
12 hours. The supernatant was collected by
filtration and the process was repeated 2–3
times.
2. Delipidization: Ethanol was evaporated by
heating the collected supernatant at 45–
55oC in hot water bath to concentrate the
solution. Petroleum ether was added to the
concentrated solution and heated for around
30 minutes. After complete evaporation of
the solvent, the residue was collected on a
filter paper. Petroleum ether was used to
remove lipid and fatty acids from plant and
tuber of C. borivilianum.
3. Deproteinization: Residue was treated
with equal ratio of Ethyl acetate-
Chloroform and stirred the mixture for 15
minutes. Chloroform is deproteinizing
agent used to remove proteins from plant
and tuber of C. borivilianum.
4. Precipitation: In this step, ethyl acetate-
chloroform was evaporated by heating the
mixture at 45–55oC in hot water bath and
leading to formation of a crude residue. The
residue was again dissolved in methanol
and heated at 45–55oC. The remaining
warm residue was dropped in acetone
solution drop by drop. White colored
powder was obtained as precipitate in
acetone. The precipitate was filtered and
oven dried to obtain white crystals. Saponin
in form of small crystals was collected on
filter paper and preserved in air tight
container for further testing.
CONCLUSION
The commercial promotion of saponin as
dietary and nutraceutical supplement and
evidences of presence of saponins in traditional
medicine preparations also propagating a need
for efficient method saponin isolation. The
developed protocol is economic and less time
consuming as well which only includes
soaking, delipidization and deproteinization
and avoiding the steps of water as mixing
solvent and overnight stirring in water bath
followed by dipping in organic solvent. The
final quantity of product obtained depends
upon the quality of ex-plant cultured. The final
product obtained from the protocol was tested
on froth confirmatory test and on thin layer
chromatographic against the standard saponin.
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Source of Support: NIL Conflict of Interest: None Declared
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ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
HAIR GROWTH STIMULATING EFFECT AND
PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC
EXTRACT OF GLYCYRRHIZA GLABRA
Deb Roy Saumendu1*, Karmakar Prithivi Raj
2, Dash Suvakanta
3,
Chakraborty Jashabir4, Das Biswajit
5
1,2Deptt. of Pharmacognosy, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017,
Assam, India. 3Deptt. of Pharmaceutics, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017,
Assam, India. 4Deptt. of Pharmacology, Girijananda Chowdhury Institute of Pharmaceutical Science, Guwahati-781017,
Assam, India. 5Deptt. of Quality Assurance, Hetero Labs Ltd. Unit-II, Baddi, Himachal Pradesh, India.
*Corresponding Author: E-mail: [email protected]; Mobile: +91-9435071898
Received: 07/01/2014; Revised: 25/01/2014; Accepted: 31/01/2014
ABSTRACT
In this particular Study Hydro-alcoholic extract of Liquorice was evaluated for its use in
Alopecia, where the extracts were compared with the activity of Marketed drug Minoxidil and the
tests were carried out on Female Albino Rat. The Results were very much encouraging as 2% Hydro-
alcoholic extract has shown a better hair growth than that of the marketed drug Minoxidil. The
Extract was also subjected to preliminary Phytochemical Evaluations whereby it has shown the
presence of Coumarines, Saponins, Phytosterols and Flavonoids along with Carbohydrates, Starch
and Fixed Oils. TLC of the extract using Pre Coated Silica gel GF Plate while detected under UV
have shown 1 Spot (Rf:0.67) with Ethyl acetate as Mobile Phase, 1 Spot (Rf:0.44) with Benzene:
Toluene (4:6) as Mobile Phase and 1 Spot (Rf:0.96) with Benzene: Chloroform (3:7) as Mobile
Phase.
KEY WORDS: Hair Growth, Glycyrrhiza glabra, Liquorice, TLC, Hydro-alcoholic extract.
Research Article
Cite this article:
Deb Roy Saumendu, Karmakar Prithivi Raj, Dash Suvakanta,
Chakraborty Jashabir, Das Biswajit (2014), HAIR GROWTH STIMULATING EFFECT AND
PHYTOCHEMICAL EVALUATION OF HYDRO-ALCOHOLIC EXTRACT OF
GLYCYRRHIZA GLABRA, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 40–47
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Hair follicle growth occurs in cycles. Each
cycle consists of a long growing phase
(anagen), a short transitional phase (catagen)
and a short resting phase (telogen). At the end
of the resting phase, the hair falls out (exogen)
and a new hair starts growing in the follicle
beginning the cycle again. Normally, about 40
(0–78 in men) hairs reach the end of their
resting phase each day and fall out. When more
than 100 hairs fallout per day, clinical hair loss
(telogen effluvium) may occur. A disruption of
the growing phase causes abnormal loss of
anagen hairs (anagen effluvium) (Rudnicka L
et al., 2008; Zhou Z.Y et al., 2007).
Alopecia means loss of hair from the head
or body. Alopecia can mean baldness, a term
generally reserved for pattern alopecia.
Compulsive pulling of hair can also induce hair
loss. Hairstyling routines such as
tight ponytails or braids may cause traction
alopecia. Both hair relaxer solutions, and hot
hair irons can also induce hair loss. In some
cases, alopecia is due to underlying medical
conditions, such as iron deficiency (Rudnicka L
et al., 2008). Generally, hair loss in patches
signifies Alopecia areata. Alopecia areata
typically presents with sudden hair loss causing
patches to appear on the scalp or other areas of
the body. If left untreated, or if the disease does
not respond to treatment, complete baldness
can result in the affected area, which is referred
to as Alopecia totalis. When the entire body
suffers from complete hair loss, it is referred to
as Alopecia universalis. It is similar to the
effects that occur with chemotherapy (Zhou
Z.Y et al., 2007).
The nature remains as the potential source
of organic structures of unparalleled diversity.
The therapeutic use of Medicinal Plants has
gained considerable momentum in the world
during the past decade. The overuse of
synthetic drugs with impurities, resulting in
higher incidence of adverse drug reactions in
more advanced communities, has motivated
mankind to go back to nature for safer
remedies. The selected plant Glycyrrhiza
glabra was reported to have wide ethnomedical
use (Shibata S. et al., 2000).
Minerva med reported that metabolic and
toxic effects caused by prolonged daily
ingestion of Liquorice are well known. Such
acquisition doesn't seem to be known enough
by practitioners and by common people.
Besides it contains active substances such as
Glycyrrhizin, steroids similar to the
adrenocortical ones; among these the most
important is Beta-Glycyrrhetinic acid. This, in
vivo and in vitro, produces salt and water
retention by means of a mineral-corticoid
mechanism, and clear suppression of the Renin-
Angiotensin-Aldosterone axis. A low plasmatic
level of Renin and Aldosterone is a common
feature. The clinical picture in many ways is
similar to the primary Aldosteronism and for
this reason the above mentioned syndrome is
usually called "Pseudoaldosteronism"
(Colloredo G et al., 1987). Saeedi .M et al.
reported that Creams containing whole licorice
(often combined with extract of chamomile) are
in wide use as "natural hydrocortisone creams."
However, there is only preliminary supporting
evidence for this use. In one double-blind,
placebo-controlled trial of 30 people, licorice
gel at 2% was more effective than placebo or
1% gel for reducing symptoms
of eczema (Saeedi M et al., 2003). Besides
there were some ethno-medicinal claims
regarding the use of Liquorice in Alopecia,
which encouraged us to go for the study, and
we have tried here to establish the ethno-
medicinal claim regarding its use in Alopecia.
METHODS:
Place of Collection: The roots were collected
from an established Crude Drug dealer from
Kolkata and have been identified by Deptt. of
Botany, Guwahati University vide Specimen
No: 11746 and a Sample voucher has been
deposited for further reference.
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pH Determination:
a) pH of 1% Solution :
1 gm. of the accurately weighed drug was
treated with 100 ml. of distilled water and
filtered. pH of the filtrate was checked with a
pH meter having standardized glass electrode.
b) pH 10% Solution :
10 gm. of the accurately weighed drug was
treated with 100 ml. of water and filtered. pH
of the filtrate was checked with a pH meter
having standardized glass electrode.
Loss on drying:
A glass stoppered shallow weighing bottle
was dried and weighed and 3 gm of the
powdered drug was transferred to the bottle.
The bottle was then stoppered and the bottle
along with the contents was weighed. The
sample was then distributed as evenly as
practicable by gentle side wise shaking to a
depth not exceeding 10 mm. The loaded bottle
was then placed in the hot air oven; the stopper
was removed and left it also in the oven. The
powdered drug was then dried to constant
weight or for 30 mm and at a temperature of
105°C. After drying was completed the hot air
oven was opened and the bottle was closed
promptly and allowed to cool at room
temperature. The bottle and the contents were
then weighed. The procedure was continued
until a constant weight was obtained.
Ash value: (Pharmacopoeia of India)
a) Total Ash :
2 gm of air dried drug was weighed
accurately in a tared silica crucible and
incinerated at a temperature not exceeding
450°C until free from carbon, cooled and
weighed. The percentage of ash with reference
to the air dried drug was calculated.
b) Water Soluble Ash :
The ash was boiled for 5 minutes with
25 ml of distilled water and the insoluble
matter was collected on an ash less filter paper,
washed with hot water, and incinerated for 15
minutes at a temperature not exceeding 450ºC.
The weight of the insoluble matter was
subtracted from the weight of the ash, the
difference in weight represents the water
soluble ash. The percentage of water soluble
ash with reference to the air dried drug was
then calculated.
c) Acid Insoluble Ash :
The ash was boiled for 5 minutes with
25 ml of 2M hydrochloric acid and the
insoluble matter was collected in a Silica
crucible or on an ash less filter paper, washed
with hot water, incinerated, cooled in a
desiccator and weighed. The percentage of acid
insoluble ash with reference to the air dried
drug was then calculated.
Extractive value:
a) Ethanol Soluble Extractive :
5 gm of the air dried drug was coarsely
powdered, taken in a stoppered conical flask
and macerated with 50 ml of ethanol (90%) for
24 hrs shaking frequently during the first 6 hrs
and allowing standing for 18 hrs. Thereafter, it
was filtered rapidly taking precautions against
loss of ethanol, and then the filtrate was
evaporated to dryness in a tared flat bottom
shallow dish, dried at 105°C and weighed. The
percentage of ethanol – soluble extractive was
calculated with reference to the air dried drug.
b) Chloroform Soluble Extractive :
5 gm of air dried drug was coarsely
powdered, taken in stoppered conical flask and
macerated with 25 ml of chloroform for 24 hrs
shaking frequently during the first 6 hrs and
allowed standing for 18 hrs. Thereafter, it was
filtered rapidly taking precaution against loss of
petroleum ether, and then the filtrate was
evaporated to dryness in a tared flat bottomed
shallow dish, divides at 105°C and weighed.
The percentage of petroleum ether soluble
extractive was calculated with reference to the
air dried drug.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
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C) Water Soluble Extractive:
5 gm of the air dried drug was coarsely
powdered, taken in a stoppered conical flask
and macerated with 50 ml of distilled water for
24 hrs, shaking frequently during the first 6hrs
and allowing standing for 18 hrs. Thereafter it
was filtered rapidly taking precautions against
loss of chloroform water, and then the filtrate
was evaporated to dryness in a tared flat bottom
shallow dish, dried at 105°C and weighed. The
percentage of water soluble extractive was
calculated with reference to the air dried drug.
Drying and pulverization:
The collected plant material (Roots) was
shade dried and then they were pulverized to
coarse powder and passed through mesh size
40.
Preparation of extract by continuous hot
extraction:
The roots were dried in shade and
powdered to get a coarse powder. About 75 gm
of dry coarse powder was extracted with water-
ethanol (1:1) by continuous hot percolation
using soxhlet apparatus (40–60°c). The
extraction was continued for 7 days. The
hydro-alcoholic extract then filtered and
concentrated by vacuum distillation. A brown
colour shiny residue was obtained.
Qualitative chemical evaluation:
Extract was subjected to various qualitative
chemical tests for detecting the presence of
various Phytoconstituents (Table No. 6). The
procedures were followed as per the procedures
laid down by Kokate C.K et al., 2008 and
Khandelwal K.R, 2012.
Thin layer chromatographic separation:
For TLC, Precoated Silica Gel GF plates
were used, by trial and error method various
solvent systems were selected and spot
visualization was done in UV chamber
(Mukharjee P.K, 2010).
Hair growth stimulating activity:
Approval of the study:
The research protocol of the animal
experimentation was approved by the
„Institutional Animal Ethical Committee‟ of
Girijananda Chowdhury Institute Of
Pharmaceutical Science, Azara, Guwahati-17,
Assam. GIPS/IAEC No. : GIPS BPH/2013/6
Collection of animals:
Animals (Wister Albino Rats) weighing 120–
150 g and aged 3–4 months were collected
from Animal House of GIPS, Azara and used
for Hair growth stimulatory study. The animals
were handled according to CPCSEA Guidelines
of Good Laboratory Practice. Animals were
kept overnight in laboratory conditions
acclimatize with the Laboratory environment.
Preparation of test sample:
The test sample was prepared by preparing
suspension of the dried hydro-alcoholic extract.
0.5gm & 1gm extract were dissolved in 50ml
ethanol (90%) each, which gave concentration
of 1% & 2% solution respectively.
Hair growth stimulatory study:
A 4 cm2 area of the dorsal skin of the rats were
shaved off using a marketed hair removal
cream. The extract solution and Minoxidil (0.4
ml) was applied to the denuded area of the rat
once a day. This treatment was continued for
10 days during and after which hair growth
pattern was observed visually and recorded
(Adhirajan N et al., 2003; Dattaa K et al.,
2009).
RESULTS AND DISCUSSION:
Pharmacognostic Evaluations: (Table I)
pH of 1% solution was found to be 6 and
that of 10% solution was found to be 5, which
suggests the drug to be an acidic. Moisture
Content was found to be 8%w/w. Total Ash,
Acid insoluble ash and Water Soluble ash were
found to be 7.9%, 2.07% and 5.67%
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
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respectively, when calculated with reference to
the air dried drug. Water Soluble Extractive
value, Ethanol Soluble extractive value and
Chloroform Soluble extractive values were
found to be 12.2%, 4.04% and 1.28%
respectively, when calculated with reference to
the air dried drug. These parameters help, in
identification of the pure crude drug, while
checking for adulterants. The results are shown
in Table I.
Phytochemical Evaluation: (Table II)
The extract when screened for various
Phytoconstituents and has shown the presence
of, Coumarines, Saponins, Phytosterols and
Flavonoids along with Carbohydrates, Starch
and Fixed Oils, and are shown in Table II.
TABLE I: Pharmacognostic Evaluation
Sl. No. Parameter Values (w/w)
1. pH of 1% Solution 6
2. pH of 1% Solution 5
3. Loss on Drying 8.0 %
4. ASH VALUES
A. Total Ash Values 7.9 %
B. Acid insoluble ash 2.07 %
C. Water soluble ash 5.67 %
5. EXTRACTIVE VALUES
A. Water soluble extractive 12.2 %
B. Ethanol soluble extractive 4.04 %
C. Chloroform soluble Extractive 1.28 %
Table II: Phytochemical Evaluations
Sl. No. Phytoconstituent Result
1. Carbohydrate +ve
2. Gums and Muscilage −ve
3. Lipids +ve
4. Alkaloids −ve
5. Anthraquinone Glycoside −ve
6. Cardiac Glycoside −ve
7. Coumarine +ve
8. Saponins +ve
9. Phytosterol +ve
10. Flavonoids +ve
11. Tannins and Phenolic Compounds -ve
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
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Thin layer Chromatography: (Table III)
When the extract was subjected to Thin
Layer Chromatography, using Precoated Silica
Gel GF plates as Stationary Phase and
Choosing various mobile phases based upon
the various Phytoconstituents found through
phytochemical screening on a trial and error
method, the extract has shown Single spots
with, Chloroform, Benzene, Ethyl Acetate,
Benzene :Toluene (4 :6) and Benzene :
Chloroform (3 : 7) as Mobile Phases, while UV
Detector was used for detection. The Results
were recorded and Shown in Table III.
Hair growth stimulatory activity: (TABLE
IV) (Fig.I)
After carrying out the study it was found
that the hydro-alcoholic extract of liquorice
showed a profound hair growth activity.
Further it was also found that 2% concentration
of the extract as compared with the standard
drug used (Minoxidil 2%), has shown better
hair growth activity. The Findings are shown in
Table IV. and a graphical representation is also
provided. Fig.I below which shows the Hair
Growth observed after 10 days.
Table III: Thin Layer Chromatography
Table IV: Hair Growth Stimulatory Activity
SOLVENT SYSTEM HYDRO-ALCOHOLIC
EXTRACT
DETECTION
SYSTEM
Chloroform 1 spot, Rf:- 0.38 UV Chamber
Benzene 1 spot, Rf:- 0.84 UV Chamber
Ethyl acetate 1 spot, Rf:- 0.67 UV Chamber
Benzene : Toluene (4:6) 1 spot, Rf:- 0.44 UV Chamber
Benzene : Chloroform (3:7) 1spot, Rf:- 0.96 UV Chamber
Sl.No Sample Applied Animals Used No. of hairs
(±SEM)
After 10 days.
1. Extract (1%) 06 474 ± 2.55
2. Extract (2%) 06 894.67 ± 6.94
3. Standard (2%) 06 620 ± 10.36
4. Control 06 211± 4.38
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
FIG. I: Showing Hair Growth after 10 days
Graphical Representation I: Hair Growth Stimulatory Activity
CONCLUSION:
The results indicate that Liquorices
(Glycyrrhiza glabra) has a potent hair growth
activity and after careful checking of other
safety parameters it can be used in herbal
formulations to treat various types of Alopecia.
ACKNOWLEDGEMENT:
The authors are very much grateful to the
Management of Girijananda Chowdhury
Institute of Pharmaceutical Science, Guwahati,
Assam, India, for providing the facilities
needed to carry out this Study.
0
100
200
300
400
500
600
700
800
900
1000
CONTROL STANDARD 1% EXTRACT 2% EXTRACT
FO
LL
ICL
E(N
O.)
PE
R 4
CM
²
DRUG SAMPLE
HAIR GROWTH STIMULATORY ACTIVITY
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 40–47
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REFERENCES:
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Colloredo G, Bertone V, Peci P, Locatelli
A, Brembilla G, Angeli G., (1987 )
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licorice. Review of the literature and
description of 4 clinical cases., Minerva
Med. Jan 31;78 (2);93-101.
Dattaa K, Singha TA, Mukherjee A, Bhata B,
Ramesh B, Burmana AC.( 2009) Eclipta
alba extract with potential for hair
growth promoting activity. J
Ethnopharmacol; 124: 450–456.
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Pharmacognosy”, Nirali Prakashan, 22nd
edition.; 18.15–18.18.
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(2008) “Text book of Pharmacognosy”,
Nirali Prakashan, Pune-411005, 41st
edition, 8.52-8.56./A-1 to A-6
Minerva Med. (1987) Jan 31;78(2);93–101.
Mukharjee P.K. (2010) “Quality control of
herbal drugs”, Business Horizone, New
Delhi-110048, 4th
reprint.;452–456.
Pharmacopoeia of India (1996), The Controller
of Publications, Delhi-110054, Vol-II,
p.A-54.
Ramar PS, Peter NP, Ponnampalam G. (2008)
“A compilation of bioactive compounds
from Ayurveda.”; Biomedical
informatics publishing; 3(3); 100-110,
Rudnicka L, Olszewska M, Rakowska A,
Kowalska-Oledzka E, Slowinska M.
(2008) "Trichoscopy: a new method for
diagnosing hair loss". J Drugs
Dermatol; 7 (7); 651–654.
Saeedi M, Morteza-Semnani K, Ghoreishi MR.
(2003) “The treatment of atopic
dermatitis with licorice gel.” J
DermatologTreat . Sep;14;153–157.
Shibata S. (2000) "A drug over the millennia:
pharmacognosy, chemistry, and
pharmacology of licorice." J. Pharm
sci., Oct; 120 (10); 849–62.
Zhou Z.Y, Jin H.D. (2007) “Clinical manual of
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Source of Support: NIL Conflict of Interest: None Declared
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 48–56
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING ELICITORS
Tamandani Ehsan Kordi1*, Valizadeh Jafar2, Valizadeh Moharam3
1M.sc Graduate of Phytochemistry, Department of Chemistry, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.2Department of biology, University of sistan and baluchistan, Zahedan, iran.3Research center of Medicinal and Aromatic Plants, University of Sistan and Baluchestan.*Corresponding Author: [email protected]; Tel: +98-541-2452335, Fax: +98-541-2446565
Received: 10/01/2014; Revised: 05/02/2014; Accepted: 09/02/2014
ABSTRACT
Elicitors are compounds with highly specific structures, that at low quantities, induce plants defense responses and subsequently, increasing of anti oxidant activity and secondary metabolites production. The elicitors such as CuSo4 (0.05 g/l), yeast extract (2 g/l), arachidonic acid (50 mg/l) and AlCl3 (0.026 g/l) were added to callus cultured of Cicer spiroceras (wild chickpea) in MS medium. After two month the calli were harvested and dried in the room temperature. About 0.1 g well powdered callus were used for each measurement of protein, total phenol, carbohydrate contents and antioxidant activity. Types various elicitors could induce diversely accumulating effects. The addition of Cu+2 had not the positive effect in the higher accumulation of primary, secondary metabolites and antioxidant activity than control in leaf and root calli. Yeast extract (YE) promoted antioxidant activity and total phenolic content in leaf callus. Arachidonic acid induced significantly to promote carbohydrate and protein contents in both root and leaf, but had a negative effect on anti oxidant activity and phenolic components accumulation in compare control. The highest protein, carbohydrate, total phenolic contents and antioxidant activity was obtained in culture treated with AlCl3, so induced to increase 98 % antioxidant activity and 74 % phenolic components. The effect CuSO4 and YE on calli growth was as well as (or more than) control, whereas AlCl3 and arachidonic acid suppressed the growth of calli. The outcomes of this study have been highlighted that using elicitors may increase the primary, secondary metabolites and antioxidant activity in C. spirocerascallus cultured.
KEYWORDS: elicitor, Cicer spiroceras, callus culture
Research Article
Cite this article:Tamandani Ehsan Kordi, Valizadeh Jafar, Valizadeh Moharam (2014),
IN VITRO PRODUCTION OF SECONDARY METABOLITES IN CICER SPIROCERAS USING ELICITORS,
Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 40–47
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 48–56
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
One of the important components of our dietary is vegetarian that can provided more necessaries body with their beneficial ingredients. In paste, for health disorder, infection and illness peoples has been provided major drugs and body's useful substances from the plants (Schneider, 1993) whereas, nowadays by increasing population the consumption of industrial drugs are increasing in spite of theirs side effect (Zhang et al.,2011). So, medicinal plants (or materials) can be suitable candidate to replace with the industrial drags (Wojcik et al., 2010, Raskin et al., 2002). Some of the plants produce various components such as alkaloids, anti-cancer, anti bacterial and anti oxidant (Kawai et al., 1987). But generally, plants cannot produce many quantities of secondary metabolites (less than 1 % weight dry) use as drug (Oksman-Caldentey and Inze, 2004). Thus applied substances such as elicitor on plants can cause to stimulate in order that more secondary and primary metabolites production using tissue culture (Poulev et al., 2003). So far a vast wide of elicitors has been used to modify cell metabolisms to raise the desirable secondary metabolites. in certain studies methyljasmonate, YE, chitosan and specially heavy metals such as Hg, Pb, Cr, Ag, V (as salt) and recently, Cu and Al have been test as elicitor (Hanson and Howell, 2004), for example both YE and Cadmiumchloride enhance Sesquiterpenes (from 1µmg/mg to 87 µgm/gm and) in N.tabacum (Chintapakorn and Hamill, 2007) and Thorn apple (from 0 to 140 nmol /gm) (Kawauchi et al., 2010) which show the plants power in secondary metabolites production. Moreover, elicitors can also increase carbohydrate and protein (primary metabolites) accumulation in plants (Graham and Graham, 1996).
The purpose of this study was to estimate the effect of elicitors as a inducer to produce primary and secondary metabolites in comparison industrial drugs.
MATERIAL AND METHODS
Chemical
Methanol, ethanol, AlCl3, CuSO4, Gallic acid, Folin silicato and all of MS medium constituents were purchased from Merck Co and used DPPH (2, 2-diphenyl-1-picrylhydrazyl) were from sigma chemical Co. (Germany). YE were obtained from Scharlau Co. (Spain).
Collection of seed and preparation of MS medium
The seeds of C. spiroceras were collected from 2497 m altitude Taftan (N28º 36, 25.9 and E61º 04, 36.8) in Sistan and Baluchestan province, Iran. After translating to laboratory and disinfected with Ethanol and NaClO3 3%, the seeds were taken into MS medium with 0.7 % agar and 3 % sucrose (w/v) without hormone to achieve to root and leaf at 25º C. The PH was adjusted to 5.8 with NaOH(Fernandez et al., 2008). After 25 days root and leaf grown were fragmented and explants were shift to new MS medium having hormone (2, 4 D: 2 mg / l, BA: 0.5 mg/l) for affording callus. In addition, in this stage 200 µM CuSo4 (0.05 g / l), 2 g / lit YE, 50 mg / l arachidonic acid and 200 µM AlCl3 (0.026 g / l) were added to MS medium. After 40 days, once time these calli were sub cultured for more grow them.
Determination of total phenol
To measure the total phenol content in the samples, 0.1 g of the callus was added to 1.5 ml ethanol 80% and this solution was shaken. After 1 day they were centrifuged at 16000 rpm in 15 min then 500 µl extracts filtered were added to 2 cc Na2Co3 5 % and 2.5 ml Folin silicato reagent 10 %, and were placed in dark for 25 min. the samples absorbance were read with UV-Vis spectrophotometer in 765 nm wavelength. Gallic acid was tested as standard(Giorgi et al., 2013).
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 48–56
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Anti oxidant activity evaluation
0.01 g of methanolic extracts was added to 5 ml methanol. By 50 µl of these solutions methanolic, 5 different concentrations were prepared and were added to 1.95 ml DPPH solution and were being remain in the dark for 30 min. the decrease in the absorbance against blank sample was determined using UV-Vis spectrophotometer of Varian and the remaining DPPH concentration in reaction with extracts were calculated from the calibration curve. The percentage of DPPH radical scavenging activity was calculated as follows:
% RSA = (AB – AS) / AB × 100
Where AB was absorbance of blank sample and AS was sample absorbance (Ananthi et al.,2010)
Determination of protein and carbohydrate contents
The protein content was measured by the Bradford method. 5 cc of extraction buffer was added to 0.1 g to samples powdered and were being maintained for 24 h at 4°C. Then they were centrifuged at 20000 rpm for 30 min. After filtration, 2 ml water was added to extract and 10 µl of it were mixed with 1990 µl of Bradford reagent. The absorbance of samples was read with UV-Vis spectrophotometer in wavelength of 595 nm. Bovine serum albumin(BSA) was tested as standard (Mattarozzi et al.,2012).
To determine the carbohydrates content, 0.1 g of the callus was added to 2.5 ml of ethanol 80 %, was maintained in water batch at 95°C for 1 h and after that was centrifuged at 20000 rpm. 2.5 ml water were added to extracts and 200 µl of it was added to 5 cc of Antron reagent and was placed in the water batch at 95 for 10 min. after cooling, the absorbance of the samples were read in wavelength at 620 nm with UV-Vis spectrophotometer. Glucose was tested as standard (Roe, 1955).
RESULTS AND DISCUSSION
The effect of four elicitors on calli cultured of Cicer spiroceras have been shown in table 1. Cu+2 was only elicitor that raised weight of calli more than controls in root and leaf calli so it caused that the calli weight of root and leaf to be 5.2 % and 54 % more than Control whereas showed the inverse impact on secondary, primary metabolites and Antioxidant activity accumulation. YE just was able to rise total phenol content from 20.7 ± 1.6 to 24.1 ± 1.9 and Antioxidant activity from 68 ± 3.2 to 59.8 ± 2.9 in leaf and did not induce to raise of carbohydrate and protein amount in root and leaf calli. Calli undergo with arachidonic acid had weight minimum amongst calli (leaf: 0.027 ± 0.005 and root: 0.027 ± 0.003) toward control (leaf: 0.104 ± 0.02 and root: 0.114 ± 0.03). Also, Applying of arachidonic acid increased significantly carbohydrate content from 1.81 ± 0.4 to 5.05 ± 0.2 in leaf and 1.65 ± 0.1 to 2.47 ± 0.06 in root and protein content from 2.04 ± 0.1 to 3.41 ± 0.2 in leaf and 1.19 ± 0.1 to 5.37 ± 0.1 in root, although it had a negative influence on raising of total phenol content and anti oxidant activity. The highest positive jump in total phenol and antioxidant activity in leaf (phenol: 36.1 ± 3.3, antioxidant activity: 34.3 ± 3.7) belonged to Al+3 that caused to increase 98 % antioxidant activity and 74 % phenoliccompositions, but it was no effect on production of secondary metabolites and antioxidant activity in root. Also the presence of Al+3 as elicitor caused to a wide impact in carbohydrate and protein contents of calli cultured, increased dramatically the carbohydrate content in leaf from 1.81 ± 0.4 to 10.13 ± 0.7, in root from 1.65 ± 0.1 to 4.1 ± 0.1 and protein content from 2.04 ± 0.1 to 7.74 ± 0.3 in leaf and in root from 1.9 ± 0.1 to 11.5 ± 1.0. To estimate of amounts of protein, carbohydrates and total phenol and antioxidant activity (mg/g) and (µg/g) have been used respectively.
As given in table 1, any extracted elicitors had an independent influence on plant and there was not any significant relation between synthesize extent of phenolic components andantioxidant activity in comparison primary
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metabolites ,whereas generally primary metabolites can synthesize a wide variety of low molecular weight components as preface to produce secondary metabolites (Dixon, 2001). The Phenolic components and the anti oxidant activity were been enhanced only in leaf with AlCl3 and YE, furthermore any one of elicitors unable to stimulate of calli to more secondary metabolites production toward Control in root. It seems to root's cells had no more ability to synthesize phenolic components and antioxidant activity even in presence of elicitors. Similarly, the literature reports in Eschscholtzia and Datura strumonium cells culture with YE and AlCl3, Sanguinarine (from 20 to 60 mg/l) and Sesquiterpenoids production increased, respectively (Schluepmann and Paul, 2009; Byun and Pedersen, 1994). YE are known as triggering agent to stimulate of poly phenol. The addition YE to Medicago truncatula cell culture is a way to Shikimic acid accumulation, a precursor of poly phenoliccompounds pathway (Broeckling et al., 2005). The primary metabolites were been increased just by AlCl3 and arachidonic acid elicitors so that AlCl3 had the highest effect on protein and carbohydrate quantities accumulation in root and leaf, respectively. AlCl3 has been just
elicitor which its stimulating caused to more synthesize of all carbohydrate, protein, total phenol contents and antioxidant activity. In the few quantities, AlCl3 as toxic molecule can regulate genes involved in the defense responses of plant (Eswaranandam et al., 2012). It has been previously established that CuSO4 and YE has above stimulating power in primary, secondary metabolites production in many cell culture (Kim et al., 2007), but in this study the existence of CuSO4 and YE, and more calli growth, did not caused to a positive impact on primary and secondary metabolites accumulation. The result of present study highlighted that there is association between growth suppression and biochemical activity in presence of Alcl3 and arachidonic acid. This relates may have been caused by triggering of phytoalexin components, biosynthesized in cell after applying elicitors (Chong et al., 2004). Similar results are shown a relationship between growth suppression of calli and biochemical activity in vanila, salvia and morina cell cultures (Chavan et al., 2011). Figure 1 shows Influence of different elicitors on Antioxidant activity, primary, secondary metabolites contents of C. spiroceras calli cultured in MS medium.
Table 1: Summary of effect elicitors on secondary, primary metabolites content and weight of callus in cell cultured of Cicer spiroceras (root and leaf) in MS medium.
Carbohydrate(mg/g D.W)
Protein(mg/g D.W)
Phenol(mg/g D.W)
Antioxidant-activity(µg/g)
Weight(mg/g D.W)
Elicitors
Leaf10.13 ± 0.77.74 ± 0.336.1 ± 3.334.3 ± 3.70.058 ± 0.005AlCl3
1.54 ± 0.091.29 ± 0.0516.7 ± 1.487.8 ± 4.90.161 ± 0.05CuSO4
5.05 ± 0.23.41 ± 0.217.1 ± 2.3137.2 ± 7.20.027 ± 0.005Arachidonic1.63 ± 0.31.35 ± 0.224.1 ± 1.959.8 ± 2.90.124 ± 0.02YE1.81 ± 0.42.04 ± 0.120.7 ± 1.668.1 ± 3.20.104 ± 0.02Control
Root4.1 ± 0.211.5 ± 1.015.1 ± 1.1161.7 ± 9.40.052 ± 0.005AlCl3
0.82 ± 0.31.4 ± 0.216.4 ± 1.092.1 ± 4.00.12 ± 0.02CuSO4
2.47 ± 0.065.37 ± 0.114.8 ± 0.8181.5 ± 10.80.027 ± 0.003Arachidonic0.64 ± 0.11.61 ± 0.116.8 ± 2.982.5 ± 4.10.1 ± 0.02YE1.65 ± 0.11.19 ± 0.117.0 ± 1.672.1 ± 4.80.114 ± 0.03Control
Datas are significant at P<0.05 toward control. Each amount is the means of 3 replicate ± SD.
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Figure 1: Influence of different elicitors on primary (B) and secondary (A) metabolites content of Cicer spiroceras cell cultured in MS medium.
(A) (B)Datas are means ± SD of triplicates. Abbreviations: r: root; l: leaf; 1: Control r; 2: control l, 3: Cu+2 r, 4: Cu+2 l;
5: Yeast extract r, 6: Yeast extract l; 7: Al+3 r; 8: Al+3 l; 9: Arachidonic acid r; 10: Arachidonic acid l.
Correlation between Antioxidant-activity and phenolic compositions
As shown in figure 2. There was a link (R2
= 0.664) between total Phenolic content and antioxidant activity because they are able scavenging the free radicals and operation as antioxidant (Rajkumar et al., 2011). This experiment shows that 66 % antioxidant activity has belonged to phenolic compound and 44 % else could be another parts of secondary metabolites. Low (R2 = 0.38) and high (R2 = 0.97) correlation coefficient between antioxidant activity and total phenolic content have been reported for sweet potato (Sikora and
Bodziarczyk, 2012) and sorgom (Rabah et al.,2004) respectively. Chickpea plants are an important part of food's south Asia people especially India and Pakistan (Verma et al.,2012). The present study is the endeavor to increase the defense mechanism, primary (protein and carbohydrate), secondary metabolites and antioxidant activity of C.spiroceras (wild chickpea) by the application of biotic and abiotic elicitors. With applying elicitor as valuable biotechnological strategy can produce foods with more beneficial substances, especially in fabaceae species consumed as basic part of our dietary.
Figure 2: Correlation between Antioxidant-activity and phenol composition that shows 66% amount of Antioxidant-activity belong to phenol composition.
0 1 2 3 4 5 6 7 8 9 10 11
A-a
: IC
50P
h: m
g/g
D.W
0
50
100
150
200
250
Antioxidant-activity Phenol
0 1 2 3 4 5 6 7 8 9 10 11
mg/
g D
.W
0
2
4
6
8
10
12
14
protein carbohydrate
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CONCLUSION
Of four elicitors used, AlCl3 had the highest positive effect in production of parameters measured. Arachidonic acid raised dramatically the primary metabolites content in leaf and root. However, dry weigh, fresh weight and growth ratio of calli was strongly inhibited by ALCl3 and arachidonic acid. The addition YE increased only anti oxidant activity and total phenolic compounds in leaf callus. CuSO4
caused that plant had the most weight of calli against control, although did not any affect on more production of primary and secondary metabolites toward control. In This results The synthesize of primary and secondary
metabolites depended to elicitor type used ,although generally primary metabolites can synthesize a wide variety of low molecular weight components as preface to produce secondary metabolites (Dixon, 2001), But in presence of elicitor did not occur this topic and so Antioxidant activity and phenoliccomponents behaved independently from the primary metabolites.
ACKNOWLEDGMENTS
The authors wish to thank the University of Sistan and Baluchestan, Zahedan, Iran for financially supporting this project through grants to JV.
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Source of Support: University of Sistan and Baluchestan, Zahedan, IRAN
Conflict of Interest: None Declared
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ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS OF NANDA
DEVI BIOSPHERE RESERVE – A REVIEW BASED ON PREVIOUS
STUDIES
Singh Rahul Vikram1*
1Department of Biotechnology Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun,
Uttarakhand, India -248002
*Corresponding author: [email protected]; Tel.-+918791649600
Received: 21/11/2013; Revised: 20/01/2014; Accepted: 05/02/2014
ABSTRACT
Indian Himalayan Region is considered as a store house of many medicinal and aromatic plants
which are being used to cure many diseases since centuries. Due to habitat degradation and over
exploration, some of the plant species have been recorded in the Red Data Book of Indian plants and
their importance in day to day life has gone undocumented. Keeping in view to document such
valuable information on these plant species, this article was aimed to document the indigenous uses
of medicinal and edible plants grown in Nanda Devi Biosphere Reserve (Uttarakhand), India along
with their cultivation, growth, trade & economic values. Ethno-medicinal information on 80 plant
species belonging to 53 families has been compiled in this paper, which is based on various previous
studies.
KEYWORDS: Aromatic & Medicinal plants, Edible plants, Red Data Book, Nanda Devi Biosphere
reserve, cutivation
Review Article
Cite this article:
Singh Rahul Vikram (2014), INDIGENOUS USES OF MEDICINAL AND EDIBLE PLANTS
OF NANDA DEVI BIOSPHERE RESERVE – A REVIEW BASED ON PREVIOUS
STUDIES, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 57–66
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION:
The Indian Himalaya is a home for
biological and cultural diversity. It supports
about 18,440 species of plants, of which 25.3%
are endemic to Himalaya (Singh & Hajra 1997,
Samant et al., 1998a). 1748 species of
medicinal plants & 675 wild edible species are
reported (Samant et al., 1998b; Samant &
Dhar, 1997) in the Indian Himalayan region.
The Nanda Devi Biosphere Reserve (70° 40’ to
80° 05’ E longitude and 30° 17’ to 30° 41’ N
latitudes) is situated in the northern part of west
Himalayas including Chamoli District
(Gharwal), Bageshwer District (Kumaun) and
Pithoraghar (Kumaun), Uttarakhand, India. It
has an area of 624.6 sq. km. and has an average
altitude exceeding 4500 m AMSL surrounded
by high mountain ridges and peaks on all sides.
The buffer zone of Nanda Devi Biosphere
Reserve covers twelve villages in Chamoli
District (Badola, 1998). There are two groups
namely Indo-Mongoloid (Bhotia) and Indo-
Aryans, which use plant resources as medicine,
food, fodder, fuel, timber and various other
purposes (Samant, 1996). The present review
was aimed at documenting the medicinally
important plants and their indigenous uses,
their cultivation, growth, trade & economic
values, grown in Nanda Devi Biosphere
reserve, Uttarakhand, India, based on various
previous studies.
Climate and vegetation:
The Nanda Devi Biosphere Reserve
consists of four geological formations, Lata,
Ramni, Kharapatal and Martoli (MaruoYugi,
1979). Climatically the area is dry with annual
precipitation. The core zone of reserve remains
snow covered almost throughout the year
except mid-May to October. Most suitable
visiting time is March to September. The
vegetation in any given area is indicator of
prevailing climatic conditions. The information
compiled from publication of Govind Ballabh
Pant Institute of Himalayan Environment &
Development Kosi-Katarmal, Almora
Uttarakhand (GBPHIED) and Botanical survey
of India from the expedition reports reveal the
presence of approximately 800 species of
plants, and several species being rare to very
rare, and of medicinal importance. (Hajra and
Batodi, 1995, Coordinating Unit of Survey of
Medicinal Plants of Western Ghats of India,
Final Report, 2005–2008) (Gaur and Tiwari,
1987; Uniyal 1977).
Medicinal plants wealth of NDBR:
The Nanda Devi Biosphere Reserve is well
known for its rich biodiversity. The inhabitants
of the area largely depend on plants for food,
dye, medicine, beverage, woodwork and
various religious and cultural needs.
Information on the utilization of plant species
of NDBR has been provided by Uniyal (1977),
Negi et al., (1985), Tiwari (1986), Gaur et al.,
(1983), Samant (1993), Gaur (1999), Samant
and Palni (2003) and Tiwari et al., (2010).
However, there exists wealth of information
with the medicine men (Vaidyas), peasants,
shepherds, priests and village headmen. Table 1
provides a list of various medicinal plants with
their altitudinal distribution, plant parts used
and ethno-botanical uses based on the literature
review of NDBR (Joshi et al., 1999).
In the NDBR, of the seventy six rare
endangered species reported, three species are
restricted to the western Himalaya (Kumaun,
Garhwal) and narrow range endemics, three
species are adjacent areas of the Himalaya are
near endemic (Samant et al., 1995).
Dependence of human beings on plants of
NDBR for various uses i.e., medicine, food,
fodder, fuel, timber, agriculture tools, religious
purpose etc. putting them in conservation threat
is given in Table 1.
Economic Values of medicinal plants
In the NDBR buffer zone, villagers
cultivate some medicinal plants for their own
use and for local sale occasionally. In the buffer
zone of NDBR Bhotiya people practice
seasonal and altitudinal migration and stay
inside the buffer zone of NDBR for six months
(May–October). A survey conducted in five
villages in the buffer zone of NDBR falling in
Pithoragarh district (Uttarakhand) indicated
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that a total of seventy one families cultivated
medicinal plants on 78% of the total cultivated
area (Bosak, 2008). On average, a family earns
about Rs. 2423 ± 376.95 per season from the
sale of medicinal plants (Silori and Badola,
2000). In another study (Ramakrishnan et al.,
1996), production of Aconitum heterophyllum
and Picrorhiza kurroa has been worked out as
1100 kg/ha while it is 4410 kg/ha for Aconitum
balfourii and Rheum emodi from their mature
natural stand. Production of Podophyllum
hexandrum and Nardostachys jatamansi was
approximately 3938 kg/ha and 1764 kg/ha
(Table 2). In this region, many species of
medicinal plants are marketed by the State
Government (Table 3).
Table 1: Medicinal plants of NDBR according to their Family/Taxa, Local name, Altitude
Range (m), Endemism, Parts Used, Indigenous uses (Source: Joshi et al., 1999).
Family/Taxa Local name Altitude
Range (m)
Endemism Parts
Used
Indigenous uses
Achyranthaceae
Achyranthes aspera L.
Latjira
2000–3000
-
Wp
Antifertility in women,
dysentery, ear and eyes
complaints, pains in body
A. bidentata Bl. Adhajhar 2000–2200 - Wp Fever, whooping cough,
jaundice Adiantaceae
Adiantum venustum Don
Sun raj
200–2600
-
Frd.
Fever
Apiaceae
Angelica glauca Edgew.
Gandhrayan,
Chhipi
3200–4000
E
Rh,rt
Dysentery, gastric,
menorrhea, stomach
disorder, vomiting;
Abdominal inflammation,
fever Bupleurum falcatum L.
-
3500–4500 - Rt Abdominal inflammation ,
fever, liver complaints
Carum carvi L. Kala jeera 2500–4000 - Sd Carminative, cold. cough,
fever, stomach disorder,
edible Cortia depressa (Don )
Norm
- 3300–4900 Ne Sd Rheumatism, sedative,
stomachache
Heracleum candicans
wall. Ex.
Gandrajan 2500–2870 - Rt, fr Leucoderma, menstrual
disorder
Pimpinella acuminata
(Edgew) Cl
- 2500–3000 E Rt Stomach disorder, gastric
P. diversifolia DC - 2400–3000 - Wp Carminative, stomach
disorder
Pleurospermum
angelicodes (DC.) Cl
Chhipi 2800–3500 - Rt Antithelmic, gastric
Selinum tenuifolium
Wall.
Bhutkesh 3000–3500 Ne Rt Incens, insecticidal, nervine
sedative
S. vaginatum (Edgew.) Bhutkesh 3000–4000 - Rt Nervine sedative
Selei sibirieum (L.)Boss. Takkar 3000–5000 - Lf, rt Mentel disorder
Araceae
Arisaema flavum (Forsk.)
Schott.
Bang
3500–4000
-
Bb
Skin diseases
A. jacquemontii Bl. Khan-
bankh,jinjok
2000–3000 Ne Bb Ringworm, skin disease,
edible
Araliaceae
Hedera nepalensis
-
2300–3500
-
Lf, fr
Stimulant, Diaphoretic,
cathartic, rheumatism,
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Koch. stimulant
Asclepiadaceae
Marsdenia roylei Wt.
Dudh bel
2500–2800
-
Fr, ft
Cold, cough, edible
Asteraceae
Adenostemna lavenia (L.)
Kuntze
-
2200–2800
-
Lf
Antiseptic, insect bite, cuts,
wounds
A. triplinervis Bukki 2300–3000 - Wp Diuretic
Bidens pilosa L. Samsa,
Araka-jhar
2000–2500 - Wp Cough, cuts, diarrhea,
leprosy, skin disorders,
edible
Tagetus minuta L. Gutti 2000–2500 - Wp Aromatic
Balsaminaceae
Impatienens scabrida
DC.
Namchoo
2300–2800
- Wp -
Berberidaceae
Berberis aristata DC.
Kilmora
2200–3000
Ne
Rt, br,
fr
Rat and snake bite, boil, eye
complaints, anticancer and
blood pressure, edible
B. pseudumbellata
Parker
Kilmor 2700–3500 E Rt, lf, Intestinal disorders
B. jaeschkeana Sch. - 3000–3500 Ne Rt,fl Astringent, blood purifier,
eye disorder, jaundice, skin
disease, edible
Betulaceae
Betula utilis D.Don
Bhojpatra 3500–4000
-
Br, res Antiseptic, burns, cuts,
contraceptic, ear complaints,
hysteria, jaundice, wounds
Boraginaceae
Arnebia benthamii Wall.
Ex
Ratanjot 3300–3800 Ne Wp Antiseptic, cuts, wounds,
hair tonic, fungal hair
infection
Maharanga emodi
(Wall.)DC.
Shankhuli 3700–3800 Ne Wp Skin disorders, rheumatism,
urinary disorder
Brassicaceae
Arabidopsis thaliana (L.)
Heynh
-
3000–3600
-
Wp Treatment of sores in mouth
Capsella bursa-pastoris
(L.) M edic.
-
2000–4000
-
Wp Blood pressure, diarrhea,
dropsy
Thlaspi arvense L. 3200–4200 - Wp Wounds, cuts, pulmonary
infection, swelling
Cannabinaceae
Cannabis sativa L.
Bhang
2000–3000
-
Lf, br,
sd, fr,
fl
Anthelmintic, appetite,
bronchitis, cuts, dyspepsia,
gonorrhea, narcotic, piles,
skin disorders, cold cough,
epilepsy, laxative, stimulant,
paralysis of tongue, sleep
piles, sores, edible
Caprifoliaceae
Viburnum erubescens
Wlll.ex DC
Asara
2700–3600
-
Fr Edible
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
V. ctinifolium Don Ghinua 2300–3000 - Br, fr Menorehoea, edible
Caryophyllaceae
Cerastium cerastoides
(L.) Britt.
Pangein
2000–4000 Ne Wp Backache, bodyache,
headache, renal pain, cough
Chenopodiaceae
Chenopodium foliolosum
(Moench .) Asch
3000–4000
-
Lf, Edible
Commelinaceae
Commelina benghalensis
L.
2200–2500
-
Lf, rt Fever, diarrhea, liver
disorders, edible
Cornaceae
Cornus macrophylla
Wall.
2200–2600 - Fr Edible
Corylaceae
Corylus jacquemontii
Dcne
Pamakhor 2300–2700 Ne Sd Tonic, edible
Crassulaceae
Sedum ewersii Ledeb.
Churappa 3000–4000
-
Lf, st Toothache, apetite
Cucurbitaceae
Cucumis melo L.
Kharbooza
2000–2700
-
Fr, st
Cooling, stomach disorder
Cucurbita maxima Kaddu 2000–3000 - Fr, sd Intestinal worms, edible
Cupressaceae
Juniperus indica Bertol.
Chila
3200–4200
-
Fr
Incense
J. communis L. Pallas 3500–4500 - Fr, lf Aromatic, incense
Dioscoreaceae
Dioscorea deltoid Kunth.
Gun
2400–2800
-
Tu
Edible
Elaeaginaceae
Elaeagnus parviflora
Wall. Ex royle
Gewai
2200–3000
-
Br,fr
Cuts, ulcer, wound, edible
Eriaceae
Gaultheria
fragrantissima Wall.
Jalan-thrit
3000–4000
-
Lf, fr
Cough, cold, edible
Euphorbiaceae
Euphorbia stracheyi
Boiss
Dudhibish
3500–4500
-
Latex
Rheumatism
Fabaceae
Parochetus communis
Don
Khia-knoi
2100–2800
-
Fl
Stomach disorder
Fumariaceae
Corydalis govanaina
Wall
Butkeshi
3300–4000
Ne
Wp
Antipyretic, diuretic, eye and
ear disorder, gastric pain,
,muscles pain, skin disorder
Gentianaceae
Swertia angustifolia
Buch-Ham.
Chiraitu
2200–3600
-
Wp
Malaria, fever
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Geraniaceae
Geranium wallichianum
D.Don ex Sw.
-
2500–4000
Ne
Rt
Astringent, ear & eye
disorder, toothache
Helvellaceae
Morchella esculenta (L)
Pers.
Guchhi
2400–3800
-
Fr,
body
Edible
Hypoxidaceae
Curculigo orchioides
Gaertn
Talmuli,
Turum
2400–3000
-
Wp
Stomach disorder, scorpion
and snake bite, wounds, skin
diseases, cough and cold
Iridaceae
Iris kumaunensis D.Don
ex
-
3000–4200
Ne
Br, lf,
fr
Fever
Juglandaceae
Juglans regia L
Akhrot
2200–3000
Ne
Br, lf,
fr
Anthelmintic, astringent,
frost bite, rheumatism,
toothache, edible
Lamiaceae
Ajuga parviflora Benth
Thymus linearis Benth
Titpati
Ban ajwain
2000–3500
2500–4000
Ne
-
Lf, sd
Wp
Ascariasis, stomachache,
fever
Eye complaints, liver and
skin disorder, edible
Liliaceae
Allium cepa L
Piyaj
2000–2500
-
Bb, lf
Anthelmintic, asthma, nose
bleeding, boils bronchitis,
diuretic, ear complaints,
itching, piles, and ringworm.
stracheyi Baker Jambu 3000–4500 E Lf Edible
Moraceae
Ficus palmata Forsk
Bedu
2000–2200
-
Fr
Dysentery, indigestion,
laxative, edible
Morinaceae
Morina longifolia Wall.
Ex DC.
Biskandara
3700–4000
Ne
Rt
Boils
Oleaceae
Jasminum humile L
Sungli
2500–3500
-
Br, rt.
Sinus, skin disorder
Orchidaceae
Dactylorrhiza hatagirea
Don
Hattazari
3000–4000
Ne
Tu
Astringent, bone fracture,
tonic, wounds
Oxalidaceae
Oxalis corniculata L
Khata-mitha
2000–2500
-
Wp
Appetite, corns, cuts,
dysentery, fever, jaundice,
edible
Paeoniaceae
Paeomia emodi Wall.
Chandra
2300–2700
-
Rt, lf,
st.
Blood purifier, cuts, ulcer,
wound, colic, dropsy,
epilepsy
Papaveraceae
Meconopsis aculeata
Royle
-
3500–4500
Ne
Wp
Backache, colic, renal pain,
tonic.
Parnassiaceaer
Parnssia nubicola Hk.f.
Nirbis
3000–4000
Ne
Wp
Food poisoning, snake bite
Pinaceae
Abies pindrow Spach.
Raga
2300–3000
Ne
Res,br.
Rheumatism, ulcer
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Polygonacaeae
Fagopyrum tataricum
(L.)
Phaphar
2000–3500
-
Lf
Edible
Ranuculaceae
A.heterophyllum Wall
Atis
2500–3500
Ne
Rt
Anthelmintic, cough, fever
Rhamnaceae
Rhamnus purpureus
Edgew.
Bakauro
2100–2500
Ne
Fr
Purgative
Rosaceae
Prunus armeniaca L.
Chuli
2000–3800
-
Fr
Edible
Pyrus lantana Don Moul 2200–2700 - Fr Edible
Rubiaceae
Galium actum Edgew
Kura
2500–4000
Ne
Wp
Antiscorb, skin disorder
Rutaceae
Skimmia laureola (DC.)
Zucc.
Narr.
2500–4000
Ne
Lf, fr
Antiseptic, boils, gastric
pain, smallpox
Taxaceae
Taxus baccata subsp.
Wallichiana
Thuner
2400–3500
-
Br, lf,
fr.
Swelling, anticancer, edible
Valerinaceae
Nardostachys grandiflora
DC
Jattamansi
3500–4200
-
Rt
Cooling, cough, snake bite,
blood purifier, ulcer
Valeriana jatamansi Mushkbala 1500–2500 Rt Used as stimulant and
carminative Abbreviations used: H – Herbs; Sh –Shrub; T – Tree; Fn – Fungus; Lf – Leaf; Frd – Frond; Bb – Bulb; Br – Bark; Wp –
Whole plant; AP – Arial part; Fl – Flower; Fr – Fruit; St – Stem; Tw – Twig; E – Endemic; NE – Near Endemic.
Table 2: Economics of cultivation of some medicinal plants (from mature plant after 8–9 years
of growth) (Source: Ramakrishan et al.,, 1996).
Species Estimated yield
(kg/ha)
Present market
rate (Rs./kg)
Total income
(Rs/ha)
Aconitum balfourii 4410 80 352000
Aconitum heterophyllum 1100 500 550000
Rheum emodi 4410 26 114660
Picrorhiza kurroa 1100 65 71500
Nardostachys jatamansi 1764 80 141120
Podophyllum hexandrum 3938 60 236280
Wild Fruits with Economic Potential in
NDBR:
Despite abundant wild edible plant
resources with immense potential for economic
development, Uttarakhand remains
underdeveloped (Phondani et al., 2011), owing
primarily to inaccessibility and poor
infrastructure. Development initiatives show
little concern for mountain perspectives. Yet
the region is rich in resources and underutilized
plant species with potential food value, about
which there is little knowledge. Wild species
such as Aegle marmelos (bael or Bengal
quince), Berberis asiatica (berberry),
Hippophae rhamnoides (seabuckthorn), Myrica
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
nagi (kafal), Rubus ellipticus (yellow
Himalayan raspberry), and Prunus armeniaca
(apricot) has good economic potential (Table
3). A variety of value-added edible products
such as jam, jelly, juice, and squash could be
made to generate income from these wild fruits,
particularly for poor rural people (Maikhuri et
al., 2004).
Table 3: Trade value of medicinal plants found in Uttarakhand
(Source: District Drug Cooperative Limited Almora 1992–93; Samant et al., 1996)
Status:
Among the recorded species Nardostachys
grandiflora (Vulnerable), Picrorhiza kurroa
(Vulnerable), Saussurea costus (Endangered)
have been recorded in Red Data Book of Indian
Plants (Samant et al., 1996). New IUCN Red
list, categorizes these species as critically rare -
Aconitum heterophyllum, Aconitum balfourii,
Podophyllum hexandrum, Valerina wallichii,
Nordostachys grandiflora, Taxus baccata etc.
Endangered - Saussurea obvallata, Berberis
aristata, Picrorhiza kurroa. Near threatened -
Jurinella macrocephala.
CONCLUSION:
There are a number of medicinal plants
found in NDBR, which have high medicinal
values, Due to habitat degradation and over
exploration/anthropogenic activities, some
species are declining, which seems to be a
critical issue. There is a need to continue
conservation of these medicinal plants.
Documentation of the uses of these plant
species may draw the attention of the
researchers to conserve these plants. This
article might be helpful for future references on
the species grown in Nanda Devi Biosphere
Reserve.
ACKNOWLEDGMENT:
The author is specially thankful to Dr.
G.C.S. Negi and G.B. Pant Institute of
Himalayan Environment & Development Kosi-
Katarmal, Almora (Uttarakhand) for providing
all facilities and support.
Botanical Name
Local name
Rate/kg
Euophia dabia Salam misri 35–40
Pleurospermum angelicodes Choru 30–50
Podophyllum hexandrum Ban kakri 50–100
Castanea sativa Khan panger 35–40
Zanthoxyllum armatum Temmor 20–25
Paris polyphylla Sm. Bankh 18–22
Rhododendron anthopogon Takkar 30–60
Lichens (Parmelia sp. Usena sp.) Safedjhula 25–40
Myrica esculenta Kafal 50–60
Syzygium venosum jamun 25–30
Taxus baccata Thuner 30–50
Valerina wallichii Samewa 30–60
Aconitum heterophyllum Atis 180–250
Dactyloriza hatagirea Hathajari 900–1400
Nordostachys grandiflora Jatamansi 100–200
Picrorhiza kurroa Katuki 70–100
Allium humile Faran 75–90
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 2 | February 2014 | 57–66
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
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