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Proceedings of the NationalAcademy of Sciences, India Section B:Biological Sciences ISSN 0369-8211 Proc. Natl. Acad. Sci., India, Sect. B Biol.Sci.DOI 10.1007/s40011-012-0088-z

A Comparative Pharmacognostic Study onthe Leaf, Stem and Root Components ofGymnema sylvestre (Retz) R. Br. ex. Sm

Ganesh C. Nikalje, Saurabha B. Zimare& Nutan P. Malpathak

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RESEARCH ARTICLE

A Comparative Pharmacognostic Study on the Leaf, Stemand Root Components of Gymnema sylvestre (Retz) R. Br. ex. Sm

Ganesh C. Nikalje • Saurabha B. Zimare •

Nutan P. Malpathak

Received: 22 May 2012 / Revised: 25 August 2012 / Accepted: 27 August 2012

� The National Academy of Sciences, India 2012

Abstract Gymnema sylvestre is commonly used in

indigenous system of medicine to control diabetes mellitus

and is a source of various chemical constituents which are

medicinally important. There is much scope to study phar-

macognosy of this potent antidiabetic plant. Pharmacog-

nostic study of leaf, stem and roots was performed. For

quantitative microscopy of leaf the authors observed sto-

matal index 12 per mm2 area. The studied plant material

showed the presence of saponin, proteins, sugars, alkaloids

while starch is absent in leaf, stem and root. Phytochemical

analysis of plant material showed saponins, proteins,

reducing sugars and fats present in all samples. For percent

extractive values methanolic extract showed highest

extractive value in leaf (6.25), stem (4.9) and root (1.75)

while benzene showed lowest extractive values in leaf (1.2),

stem (0.5) and root (0.5). The distilled water, acetone,

absolute alcohol, petroleum ether and chloroform showed

moderate percentage extractive value. The total ash content

of leaf (0.083) was higher than that of stem (0.047) and root

(0.036), and acid insoluble ash of stem (0.02) was higher

than that of leaf (0.01) and root (0.001). The authors

observed quantity of gymnemic acid for leaf, stem and root

as 33.52, 22.20 and 13.00 mg/g DW respectively by high

performance thin layer chromatography.

Keywords Gymnema sylvestre � Pharmacognosy �Histochemistry � Phytochemistry �High performance thin layer chromatography

Introduction

Gymnema sylvestre (Retz) R. Br. ex Sm. belongs to family

Apocynaceae (previously Asclepiadaceae) [1], a vulnerable

slow growing species, perennial woody climber of tropical

and subtropical regions [2], having well known antidiabetic

property. Leaves of G. sylvestre are commonly used in

indigenous system of medicine to control diabetes mellitus

[3–5]. Besides these, leaves of G. sylvestre also have

hypoglycaemic [6], stomachic [4, 7], stimulant [8], laxa-

tive, diuretic, anti-sweetener [4], anti-bacterial [9–11], anti-

viral [4, 9] and anti-inflammatory [12] activity. It is a good

source of various secondary metabolites such as tannins,

alkaloids, saponins etc. Twenty different saponins and

glycosides have been reported in G. sylvestre [5]. Some

important saponins are gymnemic acid, deacyl gymnemic

acid, gymnemagenin and gymnestogenin [4]. The medici-

nal properties of G. sylvestre are mainly due to the pres-

ence of gymnemic acid.

Over the last decade, diabetes mellitus has emerged as

an important clinical and public health problem throughout

the world. The world prevalence of diabetes among adults

(aged 20–79 years) has been 6.4 %, affecting 285 million

adults in 2010 and will increase to 7.7 % affecting

439 million adults by 2030. Between 2010 and 2030, there

will be a 69 % increase in number of adults with diabetes

in developing countries and a 20 % increase in developed

countries [13].

Of all various chemical constituents found in G. sylves-

tre, gymnemic acid is the most important compound which

G. C. Nikalje � S. B. Zimare � N. P. Malpathak (&)

Department of Botany, University of Pune, Pune 411 007,

Maharashtra, India

e-mail: mpathak@unipune.ac.in

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-012-0088-z

Author's personal copy

is present in all plant parts [4]. There is much scope to

study of pharmacognosy of this potent antidiabetic plant as

pharmacognosy helps in standardization of herbal drugs.

Material and Methods

Plant Material

Fresh leaves, stem and roots were collected from Path-

argaon, Lonavala, District Pune for pharmacognostic

study. The geographical location of collection site is

18�45015.3100 N. 73�31039.8100 E with elevation of 676 m.

Microscopic Studies

Thin transverse sections (TS) were taken from leaf

(through midrib), stem and root, double stained with

alcoholic (70 %) safranin and light green and mounted in

Canada balsam by following the micro technique method

of Johanson [14]. Microscopic characters were studied as

per Wallis [15] and Trease and Evans [16]. Microphotog-

raphy was performed for all preparations.

Quantitative Microscopy

Leaves were studied for their quantitative microscopy.

It includes stomatal number and stomatal index.

Stomatal Number and Stomatal Index (SI)

For the study of stomata and epidermis of leaf, upper and

lower epidermis was peeled off. It was then immersed into

hot alcohol to remove adhering tissues and pigments and

then stained with aqueous safranin and mounted in glyc-

erine. Stomatal index was calculated using following for-

mula [16]:

SI per mm2 area� �

% ¼ S

Eþ S� 100

where SI is the stomatal index, S is the no. of stomata per

unit area, and E is the no. of ordinary epidermal cells per

unit area.

Histochemical Tests

Free hand sections of the leaf, stem and root were done and

tested with the respective reagents. The detection and

localization of alkaloids and saponins were determined

using methods given by Johanson [14] and Krishnamurthy

[17].

1) Test for Alkaloids Sections were treated with the

following reagent for detecting alkaloids.

– Wagner’s Reagent Iodine 1 g and potassium

iodide 2 g were dissolved in 50 ml of distilled

water. Reddish brown precipitate shows presence

of alkaloids.

– Dragendroff’s Reagent (a) Solution A: basic

bismuth nitrate 0.85 g was dissolved in mixture

of 10 ml of acetic acid and 40 ml of distilled

water. (B) Solution B: 8 g of KI was dissolved in

20 ml of distilled water. Stock solution: equal

volumes of solution A and B were mixed together.

The formation of precipitate or development of

turbidity in the sections clearly indicated presence

of alkaloids.

– Mayer’s Reagent Potassium mercuric iodide solu-

tion: about 13.55 g of mercuric chloride (HgCl2)

and 50 g potassium iodide (KI) were dissolved in

one litre of distilled water. Cream or pale yellow

colour indicated presence of alkaloids.

– Hager’s Reagent Saturated aqueous solution of

picric acid was diluted in equal amount of distilled

water. Yellow precipitate in section indicates pres-

ence of alkaloids.

2) Test for Saponins Sections were placed directly in 1

drop of H2SO4 on a slide, which gives a characteristic

sequence of colour reactions, beginning with yellow,

changing to red in 30 min and finally becoming violet

or blue green in a short time. To determine localization

of saponins, Sections were put in saturated barium

hydroxide solution for about 24 h. Sections were

washed with calcium chloride then placed in potas-

sium dichromate. Yellow colour indicated presence of

saponins.

Phytochemical Tests

Qualitative tests for the presence of starch, proteins, tan-

nins, saponins, reducing sugars, anthroquinones, flavonoids

and glycosides were carried out was carried out by standard

methods of Harborne [18] and Trease and Evans [19].

Determination of Percentage Extractives

The percentage extractives determined by the method used

in Indian pharmacopoeia by Anonymous [20]. About 5 gm

of air dried powder was macerated with 100 ml of desired

solvent such as water, alcohol, petroleum ether and sol-

vent ether of specified strength, in closed flask of for 24 h.

The mixture was vigorously shaken at intervals. After

24 h the solution was rapidly filtered without any loss of

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solvent. Them from the filtrate about 20 cc of the solution

was evaporated of dryness in a flat bottomed shallow

porcelain dish, dried at 100 �C and weighed. Then the

percentage of solvent was calculated with reference to the

air dried drug.

Ash Analysis

About 1 gm of powder was taken into porcelain crucible

previously heated and weighed. The powder was evenly

scattered in fine layer on bottom of the crucible. Then the

crucible was heated in furnace at a temperature not exceed-

ing 450 �C until free from carbon, cooled and weighed. The

percentage of total carbon free ash was calculated with

reference to air dried powder.

Acid Insoluble Ash

Carbon free ash was boiled with 25 ml of 2 M hydro-

chloric acid for 5 min, filtered through ash less filter

paper, was washed with hot water, and then the filter paper

was dried in oven, ignited in crucible previously weighed,

cooled and weighed. The percentage of the acid insoluble

ash was calculated with reference to the air dried powder

[20].

Fluorescence Analysis

The fluorescence analysis of powdered plant parts were

studied as per the methods followed by Chase and Pratt

[21]. The source of radiation was mercury, provided with a

Corning ultra-type filter which transmits the near ultravi-

olet (above 3,100 A) and absorbs wavelength in the visible

portions of the spectrum.

Quantification of Gymnemic Acid Using

Gymnemagenin by HPTLC

Extraction of Gymnemagenin

Air dried plant samples were grounded to powder. An

aliquot (1 g) accurately weighed was cold macerated in

50 ml of mixture of 95 % ethanol:water (1:1 v/v) for ca

48 h. The obtained solution was filtered and concentrated

under vacuum, redissolved in methanol (25 ml) in a water

bath at 55 �C. The final volume was made up to 50 ml.

This extract was further used for analysis with the help of

high performance thin layer chromatography (HPTLC).

Standard gymnemagenin was obtained from Natural

Remedies, Bangalore. All other chemicals used were of

analytical grade (Merck, India).

HPTLC Analysis of Samples

Sample solutions were applied onto the plates (10 9

10 cm) with automated TLC sampler Linomat V (Camag,

Muttenz, Switzerland) and were controlled by Win CATS

software 1.3.3 (Camag, Muttenz, Switzerland). Plates were

developed in 10 9 10 cm twin trough glass chambers

(Camag, Muttenz, Switzerland). A TLC scanner III with

Win CATS software was used for scanning the TLC plates.

Pre-coated silica gel aluminium plates 60 F254 (10 9

10 cm) (Merck, Darmstadt, Germany) were used. A con-

stant application rate of 0.2 ll/s1 was employed with a

bandwidth of 6 mm. The slit dimension was kept at 4.0 9

0.30 mm and scanning speed of 50 mm/s1 was employed.

The mobile phase consisted of chloroform:methanol (8:2)

v/v. The length of chromatogram run was 90 mm. After

development, chromatographic plates were dipped into

derivatization reagent (5 % modified vanillin-sulphuric

acid reagent) and then the derivatized plates were heated at

110 �C for 15 min in a pre-heated oven. The bluish-black

coloured spot corresponding to gymnemagenin was

observed. The plates were scanned at 610 nm.

Conversion of Gymnemagenin to Gymnemic Acid

The conversion of gymnemagenin to gymnemic acid was

done using the equation as follows:

C = X (809.0/506.7) where, C is the content of gym-

nemic acid in the sample, X is the content of gymne-

magenin present in the sample, 506.7 is the molecular

weight of gymnemagenin and 809.0 is the molecular

weight of gymnemic acid [4].

Results and Discussion

Microscopic Studies

TS of Stem

The TS of stem shows circular outline with entire or

somewhat wavy margin. Trichomes are multicellular, un-

iserate [22]. Epidermis forms a single outermost layer

consisting of barrel to rectangular cells. A thick cuticle

covers the epidermis. The outer region of cortex is made up

of 3–4 rows of polygonal or oval parenchymatous cells.

A distinct endodermis is absent. Pericycle is represented by

scattered groups of thick walled stone cells, forming a large

and complete ring. The vascular tissues occur in following

sequence—primary phloem, secondary phloem, cambium,

secondary xylem, primary xylem, interaxillary phloem and

pith (Fig. 1a).

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TS of Leaf

Leaf is dorsiventral and shows the presence of midrib and

lamina (Fig. 1b).

Midrib

The midrib is broadly hemispherical on the abaxial side

with short lump on the adaxial side. Multicellular

unbranched trichomes consist of single rows of cells on

both sides of midrib. The ground tissue is parenchymatous

and compact. Vascular strand of midrib occurs as discon-

tinuous wide arc on the abaxial side. Xylem elements are in

radial rows, multiples of phloem patches both in upper and

lower sides of xylem strands.

Lamina

The epidermal cells of the lamina are square shaped with

outer convex wall and thin cuticle. Palisade tissue is single

layered, cylindrical, compact and occupies one-third

thickness of lamina. The spongy parenchyma cells are

lobed, four to five layered and loosely arranged. The lateral

vascular bundles of vein are small, collateral with thin

bundle sheath.

TS of Root

The TS of root shows circular outline. It is entire or wavy

in nature. Epidermis is multilayered with parenchymatous

cells and covered with thick layer of cuticle. Parenchy-

matous cortex was 6–7 cells in thickness with some glands

present in the cortex, presence of endodermis and pericy-

cle, xylem followed by phloem and pith (Fig. 1c).

Quantitative Microscopy

Stomatal Number and Stomatal Index Paracytic or aniso-

cytic type stomata were observed, which coincided with the

observations of Madhurima et al. [7]. Stomata were con-

fined to the lower epidermis and absent in upper epidermis.

Stomatal index (12 per mm2) was recorded which differed

with the findings of Madhurima et al. [7] who has reported

SI (20–30 per mm2) on abaxial surface, whereas coincides

with the observations made by Najafi and Deokule [23]

who reported SI 12.04/mm2 (Fig. 2; Table 1).

Histochemical Tests

Gymnema sylvestre is rich sources of saponin and alkaloids

[11] so in the present study histochemical test are restricted

Fig. 1 Anatomy (TS) of different plant parts of G. sylvestre

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to only histochemistry of saponin and alkaloids of different

plant parts. The free hand sections of leaf, stem and root of

G. sylvestre were treated with different reagents to analyze

histochemistry. For confirming presence of saponins, sec-

tions were treated with dilute H2SO4 which gave charac-

teristic sequence of colour reactions beginning with

immediate occurrence of yellow, followed by red within

30 min and finally to bluish green. This was observed in all

the sections which concluded that saponin was present in

all studied plant parts. Alkaloids were detected using dif-

ferent reagents such as Mayer’s, which gave reddish brown

precipitate showing presence of alkaloids. For Dragendr-

off’s reagent, formation of precipitate or development

of turbidity in the sections clearly indicated presence of

alkaloids. Hager’s reagent produced yellow precipitate in

section indicating presence of alkaloids, while Wagner’s

reagent gave cream or pale yellow colour which showed

presence of alkaloids (Figs. 3, 4, 5). All the above men-

tioned tests were positive for alkaloids for all studied plant

parts.

Phytochemical Tests Alkaloids, glycosides and flavo-

noids were detected in alcoholic extracts and tannins,

saponins and anthroquinones were detected in water

extracts of leaf and/or stem and/or root powders. They are

in agreement with Kalidass and Mohan [24] who per-

formed phytochemical screening of the powdered aerial

plant parts of G. sylvestre. Contrasting results were

obtained for steroids in leaf, stem and root extracts when

compared with Kalidass and Mohan [24]. Alkaloids,

flavonoids and glycosides were detected in alcoholic

extracts of leaf, stem and roots using different reagents.

Alkaloids were detected using Mayer’s, Wagner’s, Drag-

endroff and Hager’s Reagent and it was present in all

studied plant parts. Flavonoids were confirmed in all

studied plant parts where Mg turnings were added to conc.

hydrochloric acid and positive test was indicated by

occurrence of pink colour. Glycosides were present only in

stem extract which gave white precipitation in benzene and

absent in leaf and root extract which did not give any

precipitation. Water extract of leaf, stem and root was used

to detect starch, saponins, tannins, proteins, reducing sug-

ars, anthroquinones, fats and steroids. I2KI reagent gave

bluish black precipitate to the extract which confirmed

presence of starch in leaf and stem extract. Vigorous

shaking of the extract in distilled water produced foam

confirming presence of saponins in all studied plant parts.

Leaf, stem and root extract when added with acidic FeCl3and MBB reagent gave a red and orange precipitation resp.

to confirm presence of tannins and proteins resp. in all

studied plant parts. Benedict’s reagent when added to the

extracts did not show any change which showed absence of

reducing sugars in all studied plant parts. Anthroquinones

were detected in leaf extract only, which gave orange

precipitation in benzene-ammonium hydroxide reagent.

Sudan III dye confirmed presence of fats in all studied plant

parts which gave orange precipitation. Chloroform–conc.

Sulphuric acid reagent when added to water extracts did

not show any change in colour which indicates absence of

steroids (Tables 2, 3).

Percentage Extractive Value

The selection of the solvents was done on the basis of

findings of Madhurima et al. [7] where five solvents were

used such as hexane, water, acetone, alcohol and chloro-

form. In addition to these solvents methanol, benzene,

petroleum ether and chloroform were also used. Hexane

was excluded from the experiment as hexane showed

Fig. 2 Quantitative microscopy

of leaves of G. sylvestre(Stomata)

Table 1 Quantitative microscopy of G. sylvestre leaves

No. Parameter Result

1. Stomatal index 12/mm2 area

2. Stomatal number 11/unit area

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lowest percent extractive value in previous findings [7].

Maximum percent extractive value (6.25 %) was obtained

for methanolic extract of leaves followed by distilled

water, absolute alcohol, chloroform, acetone, petroleum

ether and benzene, whereas highest percent extractive

value for methanolic extract of leaves reported by Mad-

hurima et al. [7] was recorded in alcohol followed by

water, chloroform, acetone and hexane. Present investiga-

tion shows agreement with Madhurima et al. [7] where

chloroform extract showed better results than acetone and

use of methanol, benzene and petroleum ether also proved

better than hexane. In the present study leaf, stem and roots

were used for determining the percent extractive value and

a comparison is also done (Fig. 6), whereas use of only

leaves has been reported [7]. Highest and lowest percent

extractive value in leaf (6.25, 1.2), stem (4.9, 0.5) and roots

(1.75, 0.5) was recorded in methanolic extract and benzene

extract, respectively (Fig. 6). The percent extractive values

reported by Madhurima et al. [7] does not correspond to the

values reported in the present investigation because, the

plant material used in both studies differ in their ecological

conditions. This could be one of the reasons that may affect

the percent extractive values.

Ash and Acid Insoluble Ash Analysis The total ash

content of leaf (0.083) was higher than that of stem (0.047)

and root (0.036), and acid insoluble ash of stem (0.02) was

higher than that of leaf (0.01) and root (0.001) (Table 4).

Fluorescence Analysis

Experiment was performed for leaf, stem and root powder

under three wavelengths namely, visible light and UV light

(254, 365 nm). To 1 N NaOH in methanol, powder was

added and fluorescence was analysed at the above men-

tioned wavelengths. Powder in 1 N NaOH in methanol was

incubated for 30 min. The results obtained in the present

investigation correlates with Kalidass and Mohan [24]

where green colour was emitted in stem and leaf (aerial

parts) in visible light and shades of green (light or dark)

were obtained in UV light. In addition to the aerial parts,

root powder was also subjected for fluorescence analysis

where powder emitted brown colour in visible light and

dark brown to black in UV light (Table 5).

Results indicated that plants growing in different regions

might have difference in phytochemical analysis. Present

results showed concurrence with Najafi and Deokule [23]

that different results for phytochemical analysis for same

studied plant are possible because of the different ecolog-

ical conditions in which plant had grown.

Fig. 3 Histochemical tests of leaves of G. sylvestre.a Dragendroff (ppt./ turbidity); b Mayer (cream/ paleyellow colour); c Hager (yellow ppt.);

d Wagner(reddish brown ppt.); e H2SO4 (Yellow colour)

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Fig. 4 Histochemical tests of stem of G. sylvestre. a Dragendroff (ppt./ turbidity); b Hager (yellow ppt.); c Wagner (reddish brown ppt.) ;

d Mayer (cream/ paleyellow colour); e H2SO4 (Yellow colour)

Fig. 5 Histochemical tests of root of G. sylvestre. a Dragendroff (ppt./ turbidity); b Mayer (cream/ paleyellow colour); c Hager (yellow ppt.);

d Wagner(reddish brown ppt.); e H2SO4 (Yellow colour)

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Quantification of Gymnemic Acid Using Gymnemagenin

by HPTLC

Quantification of gymnemagenin was carried out by

HPTLC and conversion of gymnemagenin to gymnemic

acid was done by method established by Manohar et al. [4],

Rf value for present experiment is 0.52 ± 0.05. The

authors observed quantity of gymnemic acid for leaf, stem

and root as 33.52, 22.20 and 13.00 mg/g DW respectively

which showed similarities with the results of Manohar et al.

[4] (Fig. 7; Table 6).

Conclusion

From the present investigation it can be concluded that

paracytic or anisocytic type stomata were restricted to the

lower epidermis with a stomatal index of 12/mm2. Histo-

chemical analysis confirmed the presence of saponins and

alkaloids in leaf, stem and root. Phytochemistry revealed

alkaloids, glycosides and flavonoids were present in alco-

holic extracts and tannins, saponins and anthroquinones

were present in water extracts of leaf, stem and root

powders. Maximum percent extractive value was recorded

for methanolic extract of leaves followed by distilled

water, absolute alcohol, chloroform, acetone, petroleum

ether and benzene. The total ash content of leaf was higher

than that of stem and root. The data for fluorescence

analysis exemplified that stem and leaf powders emitted

green colour in visible light and shades of green (light or

dark) in UV light. Root powder emitted brown colour in

visible light and dark brown to black in UV light. HPTLC

Table 2 Preliminary phytochemical screening of alcoholic extract of leaf, stem and root sample of G. sylvestre

Test Reagents Leaf extract Stem extract Root extract

Observation Results Observation Results Observation Results

Alkaloids Mayer’s reagent Pale yellow ppt. ?ve Pale yellow ppt. ?ve Pale yellow ppt. ?ve

Wagner’s reagent Brownish ppt. ?ve Brownish ppt. ?ve Brownish ppt. ?ve

Dragendroff reagent Orange ppt. ?ve Orange ppt. ?ve Orange ppt. ?ve

Hager’s reagent Yellow ppt. ?ve Yellow ppt. ?ve Yellow ppt. ?ve

Flavonoids Conc. HCl ? Mg turnings Pink colour ?ve Pink colour ?ve Pink colour ?ve

Glycosides Benzene No change -ve White ppt. ?ve No change -ve

Table 3 Preliminary phytochemical screening of water extract of leaf, stem and root extract of G. sylvestre

Phyto-constituents Reagents used Leaf extract Stem extract Root extract

Observation Results Observation Results Observation Results

Starch I2KI Bluish black ppt. ?ve Bluish black ppt. ?ve No change -ve

Saponins Distilled water Foam ?ve Foam ?ve Foam ?ve

Tanins Acidic FeCl3 Red ppt. ?ve Red ppt. ?ve Red ppt. ?ve

Proteins MBB reagent Orange ppt. ?ve Orange ppt. ?ve Orange ppt. ?ve

Reducing sugars Benedict’s reagent No change -ve No change -ve No change -ve

Anthro-quinones Benzene ? 10 % NH4OH Orange ppt. ?ve No change -ve No change -ve

Fats Sudan III dye Orange ppt. ?ve Orange ppt. ?ve Orange ppt. ?ve

Steroids Chloroform ? conc. H2SO4 No change -ve No change -ve No change -ve

Fig. 6 Determination of percent extractive values by using different

solvents for different samples of G. sylvestre

Table 4 Determination of ash values for different samples of

G. sylvestre

No. Parameters Ash value (% w/v)

Leaf sample Stem sample Root sample

1 Total ash value 0.083 0.047 0.036

2 Acid insoluble ash 0.01 0.002 0.001

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analysis revealed that gymnemic acid content was highest

in leaf followed by stem and root. In present investigation

it is also concluded that plants growing in different regions

might have difference in phytochemical contents due to the

variability in ecological conditions.

As G. sylvestre has various traditional, therapeutic and

pharmacological uses, the present study may be useful to

provide information with respect to its identification,

authentication and standardization. Pharmacognostic and

phytochemical analysis can serve as a tool for accurate

identification.

References

1. Rapini A, Chase MW, Goyder DJ, Griffiths J (2003) Asclepiad-

aceae classification: evaluating the phylogenetic relationships of

New World Asclepiadaceae (Apocynaceae). Taxon 52:33–50

Table 5 Fluorescence analysis of leaf, stem and root powder of G. sylvestre

Experiment Leaf powder Stem powder Root powder

Visible

light

UV light (nm) Visible

light

UV light (nm) Visible light UV light (nm)

254 365 254 365 254 365

Powder Green Dark

green

Light green Green Green Light green Brown Brown Dark

brown

Powder ? 1 N NaOH

in methanol

Light

green

Dark

green

Greenish

brown

Light

brown

Green Greenish

brown

Yellowish

brown

Light

brown

Brown

Powder in 1 N NaOH

in methanol (30 min)

Green Dark

green

Blackish

green

Dark

green

Green Blackish

green

Dark brown Dark

brown

Black

Fig. 7 Quantification of gymnemagenin using HPTLC. a Standard gymnemagenin, b leaf, c stem, d root

Table 6 Conversion of gymnemagenin to gymnemic acid

Extract Gymnemagenin

content (mg/g DW)

Gymnemic

acid (mg/g DW)

Leaf 21.00 33.52

Stem 13.91 22.20

Root 08.70 13.00

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2. Anonymous (2001) Use of marker compounds in manufacturing

and labelling botanically derive dietary supplements. American

Herbal Products Associations, Silver Spring

3. Mall GK, Mishra PK, Prakash V (2009) Antidiabetic and hypo-

lipidemic activity of Gymnema sylvestre in alloxan induced dia-

betic rats. GJBBR 4(1):37–42

4. Manohar SH, Naik PM, Praveen N, Murthy HN (2009) Distri-

bution of gymnemic acid in various organs of Gymnema sylves-tre. J For Res 20(3):268–270

5. Trivedi PD, Pundarikakshudu K (2008) A validated high per-

formance thin-layer chromatographic method for the estimation

of gymnemic acids through gymnemagenin in Gymnema sylves-tre, materials, extracts and formulations. Int J Appl Sci 6:1–19

6. Spasov A, Samokhina MP, Bulanov AE (2008) Antidiabetic

properties of Gymnema sylvestre. Pharm Chem J 42(11):626–629

7. Madhurima, Ansari SH, Prawez A, Sayeed A, Akhtar MS (2009)

Parmacognostic and phytochemical analysis of Gymnema syl-vestre R. Br. leaves. J Herb Med Toxicol 3(1):73–80

8. Yoshikawa K, Ahihira S, Matsura K, Miyase T (1992) Dammarane

saponin from Gymnema sylvestre. Phytochemistry 31:237–241

9. Satdive RK, Abhilash P, Fulzele DP (2003) Antimicrobial

activity of Gymnema sylvestre leaf extract. Fitoterapia 74(7–8):

699–701

10. Shivanna Y, Raveesha KA (2009) In vitro antibacterial effect of

selected medicinal plant extracts. J Nat Prod 2:64–69

11. Khanna VG, Kannabiran K (2008) Antimicrobial activity of

saponin fractions of the leaves of Gymnema sylvestre and Eclipta

prostrate. World J Microbiol Biotechnol 24:2737–2740

12. Malik JK, Manvi FV, Alagawadi KR, Noolvi M (2008) Evalution

of antiinflammatory activity of Gymnema sylvestre leaves extract

in rats. Int J Green Pharm 2:114–115

13. Shaw JE, Sicree RA, Zimmet PZ (2010) Global estimates of the

prevalence of diabetes for 2010 and 2030. Diabetes Res Clin

Pract 87:4–14

14. Johanson DA (1940) Plant microtechnique. McGraw-Hill Book

co. Inc., New York

15. Wallis TE (1967) A textbook of pharmacognosy. J and A

Churchill Ltd, London

16. Trease G, Evans W (1972) Pharmacognosy. University press

Aberdeen, Great Britain, pp 161–163

17. Krishnamurthy KV (1988) Methods in plant histochemistry.

Vishvanandan Pvt. Ltd, Madras

18. Harborne JB (1973) Phytochemical methods, 2nd edn. Chapman

and Hall International edition, London, pp 5–8

19. Trease GE, Evans WC (eds) (2002) Trease and Evans pharma-

cognosy, 15th edn. W. B. Saunders, Edinburgh/London/New

York/Philadelphia/St. Louis/Sydney/Toranto, pp 3–4, 528–533,

538–547

20. Anonymous (1955) Pharmacopoeia of India, 1st edn. Govern-

ment of India, Ministry of Health Manager Publication, Delhi,

pp 370–864

21. Chase C, Pratt R (1949) Fluorescence of powdered vegetable

drugs with particular reference to development of system of

identification. J Am Pharm Assoc 38:324

22. Saneja A, Sharma C, Aneja KR, Pahwa R (2010) Gymnemasylvestre (Gurmar): a review. Der Pharm Lett 2(1):275–284

23. Najafi S, Deokule SS (2011) Studies on Gymnema sylvestre—a

medicinally important plant of the family Asclepiadaceae. TJS

9(2):26–32

24. Kalidass C, Mohan VR (2010) Pharmacognostical and phyto-

chemical investigation studies on Gymnema sylvestre R.Br. Int J

Biol Technol 1(1):8–11

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

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