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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

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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: accessrohit25@gmail.com; Mob: +919755594040

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

ABSTRACT

Saponins are imperative non-volatile chemical compounds valued for several medicinal

properties. The pharmaceutical use of saponins for semi-synthesis of steroidal drugs makes it an

essential element of life with a diverse range of properties including antimicrobial, insecticidal,

haemolytic, aphrodisiac, foaming and emulsification. The tuberous roots of Chlorophytum

borivilianum always remains a major source for isolation of saponin. A conventional efficient

method was developed for saponin isolation from in-vivo and in-vitro samples of C. borivilianum by

delipidization and deproteinization with petroleum ether and chloroform leading to development of a

whole new process for saponin isolation. Protocol was tested with saponin confirmatory test

followed by thin layer chromatography.

KEY WORDS: Saponin, Chlorophytum borivilianum, delipidization, steroid.

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: baharu@rediffmail.com; Mobile: +91-9435071898

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

ABSTRACT

In this particular Study Hydro-alcoholic extract of Liquorice was evaluated for its use in

Alopecia, where the extracts were compared with the activity of Marketed drug Minoxidil and the

tests were carried out on Female Albino Rat. The Results were very much encouraging as 2% 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.

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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%

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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

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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

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REFERENCES:

Adhirajan N, Ravi Kumar T,

Shanmugasundaram N, Babu M (2003)

In vivo and in vitro evaluation of hair

growth potential of Hibiscus

rosasinensis Linn. J Ethnopharmacol;

88: 235–239.

Colloredo G, Bertone V, Peci P, Locatelli

A, Brembilla G, Angeli G., (1987 )

Pseudoaldosteronism caused by

licorice. Review of the literature and

description of 4 clinical cases., Minerva

Med. Jan 31;78 (2);93-101.

Dattaa K, Singha TA, Mukherjee A, Bhata B,

Ramesh B, Burmana AC.( 2009) Eclipta

alba extract with potential for hair

growth promoting activity. J

Ethnopharmacol; 124: 450–456.

Khandelwal K.R. (2012) “Practical

Pharmacognosy”, Nirali Prakashan, 22nd

edition.; 18.15–18.18.

Kokate C.K. Purohit, A.P. & Gokhale, S.B.

(2008) “Text book of Pharmacognosy”,

Nirali Prakashan, Pune-411005, 41st

edition, 8.52-8.56./A-1 to A-6

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

Chinese herbal medicine and

acupuncture.”

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: kordi3236@yahoo.com; Tel: +98-541-2452335, Fax: +98-541-2446565

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

ABSTRACT

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

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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).

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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: rahul.negi121@gmail.com; Tel.-+918791649600

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

ABSTRACT

Indian Himalayan Region is considered as a store house of many medicinal and aromatic plants

which are being used to cure many diseases since centuries. Due to habitat degradation and over

exploration, some of the plant species have been recorded in the Red Data Book of Indian plants and

their importance in day to day life has gone undocumented. Keeping in view to document such

valuable information on these plant species, this article was aimed to document the indigenous uses

of medicinal and edible plants grown in Nanda Devi Biosphere Reserve (Uttarakhand), India along

with their cultivation, growth, trade & economic values. Ethno-medicinal information on 80 plant

species belonging to 53 families has been compiled in this paper, which is based on various previous

studies.

KEYWORDS: Aromatic & Medicinal plants, Edible plants, Red Data Book, Nanda Devi Biosphere

reserve, cutivation

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

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INTRODUCTION:

The Indian Himalaya is a home for

biological and cultural diversity. It supports

about 18,440 species of plants, of which 25.3%

are endemic to Himalaya (Singh & Hajra 1997,

Samant et al., 1998a). 1748 species of

medicinal plants & 675 wild edible species are

reported (Samant et al., 1998b; Samant &

Dhar, 1997) in the Indian Himalayan region.

The Nanda Devi Biosphere Reserve (70° 40’ to

80° 05’ E longitude and 30° 17’ to 30° 41’ N

latitudes) is situated in the northern part of west

Himalayas including Chamoli District

(Gharwal), Bageshwer District (Kumaun) and

Pithoraghar (Kumaun), Uttarakhand, India. It

has an area of 624.6 sq. km. and has an average

altitude exceeding 4500 m AMSL surrounded

by high mountain ridges and peaks on all sides.

The buffer zone of Nanda Devi Biosphere

Reserve covers twelve villages in Chamoli

District (Badola, 1998). There are two groups

namely Indo-Mongoloid (Bhotia) and 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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