全 文 ：Journal of Forestry Research (2009) 20(3): 268−270
DOI 10.1007/s11676-009-0046-7





Distribution of gymnemic acid in various organs of Gymnema sylvestre

Shirugumbi Hanamanthagouda Manohar, Poornananda Madhava Naik, Nagella Praveen, Hosakatte Niranjana Murthy
Department of Botany, Karnatak University, Dharwad-580 003, India

Abstract: The gymnemic acid contents in various organs of Gymnema sylvestre were investigated by High Performance Liquid Chroma-
tography (HPLC) method. The results shows that the content of gymnemic acid in various organs, obviously different, was 54.29, 31.66,
28.82, 27.67, 25.39, 20.56 and 1.31 mg·g-1 DW in shoot tips, flowers, nodes, leaves, internodes, roots and seeds, respectively. The highest
gymnemic acid content (54.29 mg·g-1 DW) was found in shoot tip, 1.96 fold higher than that in leaves (27.67 mg·g-1 DW). Maximum quan-
tity of gymnemic acid (35.39 mg·g-1 DW) was observed in the young leaves, which was 1.52 times higher than that in old leaves (23.07
mg·g-1 DW). The content of gymnemic acid in young, middle and old internodes was 26.47, 25.77 and 23.94 mg·g-1 DW, respectively, all
lower than that in leaves (27.67 mg·g-1 DW), whereas the content of gymnemic acid in young, middle and old nodes was 27.96, 28.81 and
29.66 mg·g-1 DW, respectively, all higher than that in leaves. The study provides the scientific evidences for the rational development
and utilization of Gymnema sylvestre resources, since over exploitation of natural stands has caused depletion of these plants in nature.
Keywords: gymnemic acid; Gymnema sylvestre; high performance liquid chromatography


Introduction

Higher plants accumulate various secondary metabolites to avoid
plant and environment interaction. These secondary metabolites
are extremely useful to human beings as industrial and biomedical
products. Accumulation of secondary metabolite is not uniform
though out the plant body and it is dependent on accessibility of
the sites of precursor molecules. Exposed external tissues are
usually better defended and accumulate higher amounts of sec-
ondary metabolites than internal tissues in roots, stems, leaves,
seeds, bulbs and tubers and in fruits secondary metabolites are
neutralized during maturation so the fruits will become attractive
to dispersers. In order to utilize the plants or plant parts for me-
dicinal purposes, it is essential to know the distribution secondary
metabolites (Zhao et al. 2007), which enables to choose the right
organs and to obtain good resources for extraction.
Gymnema sylvestre belongs to the Asclepiadaceae family and
the plant is considered to be a good source of a large number of
bioactive substances. It is a vulnerable medicinal species, being a
slow growing, perennial, woody climber found in India and the
southwestern region of China. It has a reputation as a traditional

Foundation project: This work was supported by University Grants
Commission, New Delhi (No. 33-88/2007 (SR))
Received: 2008-12-26; Accepted: 2009-01-16
© Northeast Forestry University and Springer-Verlag 2009
The online version is available at http://www.springerlink.com
Biography: Shirugumbi Hanamanthagouda Manohar (1980-), male,
Ph. D. in Department of Botany, Karnatak University, Dharwad-580 003
India. (Email: nmurthy60@hotmail.com)
Responsible editor: Zhu Hong

remedy to control diabetes mellitus (Shimizu et al. 1996; Shimizu
et al.1997). In addition, the leaves of this plant, popularly known
as Madhunashni or Gur-mar in India, are used for inhibiting the
taste of sweetness (Liu et al. 1992). A number of saponins such as
gymnemic acid, deacyl gymnemic acid, gymnemagnenin (Sub-
barao et al. 1971; Gooper 1887), 23-hydroxylnogispinogenin, and
gymnestrogenin have been purified (Sahu et al. 1996; Yoshikawa
et al. 1992; Yoshikawa et al. 1997) from Gymnema sylvestre. The
leaf extract from this plant is used as stomachic, stimulant, laxa-
tive, diuretic, anti-sweetner, antibacterial (Yoshikawa et al. 1992),
antiviral and anti-inflammatory (Satdive et al. 2003) activities.
HPLC methods have already been reported for the estimation of
gymnemic acid in Gymnema sylvestre (Diwan et al. 1995; Yokota
et al. 1994). The quantity of gymnemic acid, the active principle
in Gymnema leaves is, however, variable among accessions from
different eco-climatic regions (Yokota et al. 1994). Considerable
variations also exist among the morphological traits of Gymnema
accessions from Tamil Nadu and Kerala (Thamburaj et al. 1996).
In this study, a detail investigation of the distribution of gymne-
mic acid content in various organs of Gymnema sylvestre was
carried out. In particular, the contents of gymnemic acid in vari-
ous organs like leaves, shoot tips, internodes, nodes, roots, flow-
ers and seeds of Gymnema sylvestre were determined and com-
pared.

Materials and methods

Plant material

Gymnema sylvestre samples (shoot tips, leaves, nodes, internodes,
flowers, roots and seeds) were collected from the Botanical Gar-
den, Karnatak University, Dharwad, India for the determination
of gymnemic acid contents. The second young leaf - 18 mm × 7
SHORT COMMUNICATION
Journal of Forestry Research (2009) 20(3): 268−270

269
mm, petiole- 3 mm, fifth middle leaf- 32 mm ×22 mm, petiole- 4
mm) and ninth old leaf- 49 mm ×28 mm, petiole- 6 mm were
collected. Each of these leaves was cut into four parts (Leaf apex,
leaf middle, leaf base and petiole) and separated. Young inter-
nodes (between the first and second node), middle internodes
(between the fourth and fifth node) and old internodes (between
the eight and ninth node) were excised as shown in Fig.1. The
second, fifth and ninth node were separated. The shoot tip, ma-
ture flowers, roots and seeds were also collected. The above
samples were shadily dried, ground to a fine powder, sieved
through a 20-μm stainless sieve (Sigma, USA) and used for ex-
traction and estimation of gymnemic acid.



Fig. 1 Schematic picture of Gymnema sylvestre organs used for the
analysis of gymnemic acid contents

Extraction of gymnemic acid

We put 500 mg of sample into a 500-mL round bottom flask and
added 50 mL of extraction solvent (volume ratio of methanol to
water is 1:1) and 10 mL of 11% potassium hydroxide solution.
The mixture was refluxed for an hour. The concentrated HCl of 9
mL was added and refluxed again for one hour. The mixture was
cooled to room temperature. The extract was filtered through
0.45-μm nylon filter (Millipore), the volume was made up to 100
mL with extraction solvent, and the clean supernatant was used
for HPLC analysis.

Determination of gymnemic acid II by HPLC

The analytical HPLC experiments were performed with a Shi-
madzu high performance liquid chromatography equipped with
variable wave length detector operating at 210 nm (SPD-10AVP,
LC-10ATVP). Separations were carried out with Luna C-18 (150
mm × 3 mm, 5 µm) column with a column temperature of 26ºC.
The mobile phase was acetonitrile (A) and water (B) (80A:20B)
with elution rate of 1 mL/min. Gymnemagenin standard was
obtained from ChromaDex (Laguna Hills, CA, USA). Validation
of quantitative method was performed with samples consisting of
five replicates of 20 µL each. The chromatogram of gymnemic
acid in Gymnema sylvestre shoot tips was shown in Fig. 2 with a
retention time of 1.83 min. The conversion of gymnemagenin to
gymnemic acid was done using the equation as follows:
C= X (809.0/ 506.7) (1)
where, C is the content of gymnemic acid in the sample; X is the
content of gymnemagenin present in the sample, 506.7 is the
molecular weight of gymnemagenin, and 809.0 is the molecular
weight of gymnemic acid.


Fig. 2 HPLC chromatogram of gymnemic acid contents in
Gymnema sylvestre shoot tip; x-coordinate is Time (minutes) and
y-coordinate is absorption (absorption units)

Results and discussion

The contents of gymnemic acid II were significantly different
from the different organs of Gymnema sylvestre (Fig. 3). The
maximum content of gymnemic acid was obtained in shoot tips
(54.29 mg·g-1 DW), followed by the leaves, flowers, nodes, inter-
nodes and roots (27.67, 31.66, 28.81, 25.39 and 20.56 mg·g-1
DW, respectively). Whereas seeds, accumulated lower concen-
tration of gymnemic acid (1.31 mg·g-1 DW), the content of
gymnemic acid was 1.71, 1.88, 1.96, 2.13, 2.64 and 41.39 times
higher in shoot tips than that in flowers, nodes, leaves, internodes,
roots and seeds, respectively. Many literatures reported that the
leaves of Gymnema sylvestre were the major resource for the
production gymnemic acid (Gooper 1887; Yokota et al. 1994).
The above results show that gymnemic acid was not only pre-
sent in the leaves but also in shoot tips, internodes, nodes, flow-
ers, roots, and seeds. The contents of gymnemic acid in leaves
were significantly different and the young leaves had higher
content (35.39 mg·g-1 DW) than middle (24.55 mg·g-1 DW) and
old leaves (23.07 mg·g-1 DW), thus the young leaves contained
1.52 fold higher gymnemic acid content than that of old leaves.
The content of gymnemic acid in various parts of middle
leaves also varied considerably and leaf apex had higher content
(26.84 mg·g-1 DW) than leaf middle (25.26 mg·g-1 DW), leaf
base (23.7 mg·g-1 DW) and petiole (22.39 mg·g-1 DW). These
values proved that the higher concentration of gymnemic acid
was accumulated in tip of the leaves, compared to other parts of
the leaves. The content of gymnemic acid in young, middle and
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270
old internodes was 26.47, 25.77 and 23.94 mg·g-1 DW, respec-
tively, which were lower than that in leaves (27.67 mg·g-1 DW).
The content of gymnemic acid in young, middle and old node
was 27.96, 28.81 and 29.66 mg·g-1 DW, respectively, which were
higher than that in leaves (27.67 mg·g-1 DW) (Table 1).


Fig. 3 Distribution of gymnemic acid in Gymnema sylvestre (Values
are mean ± SE of 5 replicates; data are pooled results of different
aged organs)

Table 1. Contents of gymnemic acid in various organs and tissues of
Gymnema sylvestre
Samples
Gymnemic acid content
(mg·g-1 DW ± SE)
(a) Shoot tip 54.29 ± 0.39
(b) Leaves
Whole leaf(18 mm×7 mm) 35.39 ± 0.31
Leaf tip 38.25 ± 0.42
Leaf middle 36.96 ± 0.18
Leaf base 35.46 ± 0.33
Young
leaf
Leaf petiole (3mm) 30.94 ± 0.32
Whole leaf (32 mm×22 mm) 24.55 ± 0.27
Leaf tip 26.84 ± 0.13
Leaf middle 25.26 ± 0.36
Leaf base 23.70 ± 0.24
Middle
leaf
Leaf petiole (4mm) 22.39 ± 0.35
Whole leaf (49 mm×28 mm) 23.07 ± 0.40
Leaf tip 24.60 ± 0.20
Leaf middle 23.50 ± 0.32
Leaf base 22.74 ± 0.67
Old lief tip
Leaf petiole (6 mm) 21.45 ± 0.41
(c) Node
Young node 27.96 ± 0.22
Middle node 28.81 ± 0.40
Old node 29.66 ± 0.66
(d) Internode
Young internode 26.47 ± 0.31
Middle internode 25.77 ± 0.23
Old internode 23.94 ± 0.52
(e) Flower 31.66 ± 0.21
(f) Root 20.56 ± 0.16
(g) Seeds 1.31 ± 0.09
Notes: (a) Shoot tip; (b) young leaf - second leaf; middle leaf - fifth leaf; old
Leaf - ninth leaf; (c) young node – second node, middle – fifth node, old node
– ninth node; (d) young internodes - between first and second node; middle
internodes - between fourth and fifth node, old internodes – between eighth
and ninth node. SE- standard error (Values are mean ± SE of 5 replicates).
In the present study, it was observed that the maximum con-
tents of gymnemic acid were accumulated in the shoot tips and
fresh leaves, which are renewable source. The probable reasons
for the enrichment of these metabolites in these parts may be
attributed to environmental factors, genotypes, morphotypes or
cultivation practices.
In conclusions, the content of gymnemic acid in shoot tip was
higher than that in any other organs. In previous study, gymne-
mic acid was mainly produced from the leaves of Gymnema
sylvestre (Gooper 1887; Yokota et al. 1994) and the rest of the
plant was wasted. The present study proved that the internodes,
roots and flowers are also a good resources for production of
gymnemic acid. Thereby, the stalks, especially the top stalks are
worthy to be the new resource for production of gymnemic acid.
The leaves and stalks of Gymnema sylvestre will play an impor-
tant role in the increase of the availability of raw material for
pharmaceutical purpose.

References

Diwan PV, Margaret I, Ramakrishna S. 1995. Influence of Gymnema sylvestre
on inflammation. Inflammopharmacology, 3 (3): 271−277.
Gooper D, 1887. An Examination of the Leaves of Gymnema sylvestre R.Br.
Nature, 35: 565-567.
Liu HM, Kiuchi F, Tsuda Y. 1992. Isolation and structure elucidation of
gymnemic acids, antisweet principles of Gymnema sylvestre R.Br. Chem
Pharm Bull., 40(6): 1366−1375.
Sahu NP, Mahato SB, Sarkar SK, Poddar G. 1996. Triterpenoid saponins from
Gymnema sylvestre. Phytochemistry, 41(5): 1181−1185.
Satdive RK, Abhilash P, Fulzele DP. 2003. Antimicrobial activity of
Gymnema sylvestre leaf extract. Fitoterapia, 74(7−8): 699−701.
Shimizu K, Abe T, Nakajyo S, Urakawa N, Atsuchi M, Yamashita C. 1996.
Inhibitory effect of glucose utilization by gymnemic acids in guinea pig ileal
longitudinal muscle. J Smooth muscle Res, 32(5): 219−228.
Shimizu K, Lino A, Nakajima J, Tanaka K, Nakajyo S, Urakawa N, Atsuchi M,
Wada T, Yamashita C. 1997. Suppression of glucose absorption by some
fractions extracted from Gymnema sylvestre leaves. J Vetr Med Sci, 59(4):
245−251.
Subbarao G, Joseph ES. 1971. Constituents from Gymnema sylvestre leaves
VIII: Isolation, chemistry and derivatives of gymnemagenin and
gymnestrogenin. J Pharm Sci, 60: 190−192.
Thamburaj S, Subbaraj D, Kasturib S, Vijayakumar, M. 1996. Evaluation of
germplasm accessions of Gymnema sylvestre R.Br. S. Indian Hort, 44:
174–176.
Yokota T, Mizutani K, Okada K, and Tanak O. 1994. Quantitative analysis of
gymnemic acids by high performance liquid chromatography. J Jpn Soc.
Food Sci. Technol, 41: 202–205.
Yoshikawa K, Arihara S, Matsuura K, Miyase T. 1992. Dammarane saponins
from Gymnema sylvestre. Phytochemistry, 31(1): 237−241.
Yoshikawa M, Murakami T, Matsuda H. Medicinal foodstuffs. 1997. X.
Structures of new triterpene glycosides, gymnemosides-c, -d, -e, and -f,
from the leaves of Gymnema sylvestre R. Br.: influence of gymnema gly-
cosides on glucose uptake in rat small intestinal fragments. Chem Pharm
Bull, 45(12): 2034−2038.
Zhao Chunjian, Zu Yuangang, Li Chunying, Tian Chengyu. 2007. Distribu-
tion of solanesol in Nicotiana tabacum. Journal of Forestry Research, 18
(1): 69–72.