Phytomedicine 17 (2010) 10331039

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Short communication

In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate -cells
regeneration and anti-diabetic activity in Wistar rats
A. Bakrudeen Ali Ahmed a,c, , A.S. Rao b , M.V. Rao a
a

Department of Plant Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India
c
Department of Chemistry, Pukyong National University, Busan, South Korea
b

a r t i c l e
Keywords:
Gymnema sylvestre
Gymnemic acid
Growth curve
Organ weight
Liver glycogen
-Cell regeneration

i n f o

a b s t r a c t
A methanol extract of Gymnema sylvestre leaf and callus showed anti-diabetic activities through regenerating -cells. Optimum callus was developed under stress conditions of blue light with 2,4-D (1.5 mg/l)
and KN (0.5 mg/l), which induced maximum biomass of green compact callus at 45 days, as determined
by growth curve analysis. Leaf and optimum callus extracts contains gymnemic acid, which was analyzed
using TLC, HPTLC and HPLC methods. The research reported here deals with leaf and callus extracts of G.
sylvestre, which signicantly increase the weight of the whole body, liver, pancreas and liver glycogen
content in alloxan-induced diabetic rats (Wistar rats). The gymnemic acid of leaf and callus extracts signicantly increases the regeneration of -cells in treated rats, when compared with the standard diabetic
rats. It could have potential as a pharmaceutical drug for insulin-dependent diabetes mellitus (IDDM).
2010 Elsevier GmbH. All rights reserved.

Introduction
Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM),
is a common pediatric chronic disease, affecting an increasing
number of children every year. IDDM occurs due to autoimmune
destruction of insulin-producing -cells in the pancreas, resulting
in low or no production of insulin, a hormone necessary for survival
(International Expert Committee 2009).
Gymnema sylvestre (syn. Periploca sylvestris Retz) is a traditional medicinal plant, with reported use as a remedy for diabetes
mellitus, stomachic and diuretic problems. The plant extracts
are also used in folk, ayurvedic and homeopathic systems of
medicine (Mitra et al., 1995). The extract of G. sylvestre plays
a major role in blood glucose homeostasis through increased
serum insulin level through regeneration of the endocrine pancreas
(Shanmugasundaram et al., 1983; Shanmugasundaram et al., 1990).
G. sylvestre occurs mainly in the Deccan peninsula of western India,
Tropical Africa, Vietnam, Malaysia, Srilanka and is widely available
in Japan, Germany and the USA as a health food (Ye et al., 2000).
Within the last 10 years, a number of Gymnema products, including
Gymnema capsules, Gymnema tea, Bioshape , and Diaxinol have
appeared on the world market.

In the past few decades, secondary metabolite production from

plant tissue culture has been identied as a tremendous resource
for new drug development and clinical research in the elds of
pharmacology and medicine. Plant cell culture extracts have also
been used widely in the form of fractions and isolated compounds as potential bioactive molecules (Sokmen et al., 1999).
In vitro developed callus tends to produce various active compounds, including gymnemic acid and gymnemagenin (Kanetkar
et al., 2006). However, external factors like phytohormone, shaking speeds, pH, and medium play an important role in gymnemic
acid production in suspension cultures (Devi et al., 2006). In addition, sucrose, inoculum density, auxins, and aeration also play
a very crucial role in the production of gymnemic acid through
bioreactor-dependent cell growth (Lee et al., 2006). The present
studies were undertaken to identify the G. sylvestre leaf and callus
extract molecules that contribute to or promote -cell regeneration and anti-diabetic effects. In addition, we have characterized the
gymnemic acid role in anti-diabetic experiments; it was found that
pancreas weight and glycogen content were increased in the liver
of alloxan-induced diabetic Wistar rats. Furthermore, an emphasis
is laid on -cell regeneration and the determinants implicated.
Materials and methods
Plant material and sterilization

Corresponding author at: Marine Bioprocess Research Center, Department of

Chemistry, Pukyong National University, Busan, South Korea. Tel.: +82 51 629 7098;
fax: +82 51 629 7099.
E-mail address: [Link]@[Link] (A.B.A. Ahmed).
0944-7113/$ see front matter 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/[Link].2010.03.019

G. sylvestre plants (GS) were collected from the Pachamalai

hills, and maintained in the plant science garden of the Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Leaf explants

1034

A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 10331039

were washed with tap water, Teepol solution, then 70% ethanol for
30 min and 0.1% HgCl2 for 2 min. Prior to inoculation, explants were
washed several times in sterile distilled water.

Pvt. Ltd., India) (200 mg/kg body wt./day/rat) given (P.O.) daily
(Shanmugasundaram et al., 1990; Ahmed et al., 2008).

Callus induction

Experimental animal

Leaf explants of G. sylvestre were grown in MS medium

(Murashige and Skoog, 1962) supplemented with 2,4-D (1.5 mg/l)
and KN (0.5 mg/l). Callus culture was maintained at 25 2 C,
16 h/8 h (light/dark) photoperiod with 25 mol m2 s1 of light
intensity (Ahmed et al., 2009a).

All experiments were approved by Institutional Animal Ethics

Committee (IAEC) procedure guidelines of CPCSEA. Male and
female (89-month old) Wistar rats (body weight 200 25 g each)
were maintained at Bharathidasan University, Tiruchirappalli, on
standard pellet diet (Hindustan Lever Ltd., Bangalore, India) with
free access to tap water.

Callus developed under stress condition

Callus cultures were maintained under different stress conditions for gymnemic acid production (Ahmed et al., 2009a). The
protocol was as follows: different color light (blue, red, green
and white uorescent tubes); temperature (20 C, 25 2 C, 30 C
and 35 C); photoperiod (4 h/20 h, 8 h/16 h, 12 h/12 h, 20 h/4 h
light/dark), sucrose (2%, 4%, 5%, 6%) and ammonium nitrate (1 mM,
2 mM, 3 mM, and 4 mM). Optimum callus biomass was determined
using growth curve analysis, in all treatments.
In vitro callus growth curve
Plant growth regulators (PGRs) with stress treatment callus
fresh and dry weight were determined at 15, 25, 35, 45 and 55
days. Maximum biomass (fresh, dry weight) and green compact
callus were noted for all treatments.
Phytochemical studies on leaf and in vitro callus
G. sylvestre dried leaves and in vitro callus were extracted
with methanol 5 times (Rehman et al., 2003). The collected
methanol extract was centrifuged at 5000 g for 10 min at room
temperature, then the methanol supernatant carefully pipetted
out into fresh eppendorf tubes without disturbing the interphase residues. Green-color supernatant (20 l) was screened
using TLC and HPTLC with standard gymnemic acid (Prof. Kazuko
Yoshikawa, Kyoto Pharmaceutical University, Japan) for gymnemic acid quantication (Ahmed et al., 2009a). HPTLC was
performed using a Camag HPTLC system (CAMAG, Switzerland)
equipped with a sample applicator Linomat IV, TLC scanner III and
integration software CAT 4.0. The mobile phase of Isopropyl alcohol:chloroform:methanol:acetic acid (5:3:1:0.5) was suitable for
separation of gymnemic acid and scanned at 200 nm, respectively.

Chemical preparation and induction of diabetes

Alloxan
monohydrate
(Otta
kemi,
Mumbai,
India)
(100 mg/kg body wt./rat) was dissolved in distilled water and
injected intraperitoneally (i.p.). The experimental rats entered a
diabetic state after 72 h, then the Wistar rats were divided into
groups for treatment with G. sylvestre leaf and callus extracts. Therapeutic human insulin (Torrent Pharmaceuticals Ltd., Mehsana,
India) mixture was shaken well and injected daily (4 U/kg body
wt.) subcutaneously as a diabetic standard (Chattopadhyay 1998).

Instruments and methods

The body weight, liver and pancreas weights of the experimental
Wistar rats were checked in fundamental weight instruments. Liver
glycogen (Raghuramalu et al., 1983) and pancreas were removed
for further analysis.
The removed pancreas was xed in dewaxed and hydrated
parafn for micro section (6 m thickness), then stained with
hematocylin and eosin. In addition, stained pancreas was washed
with Gomoris aldehyde fuchsin for determination of -cells in
Langerhans islets (Gomori, 1950) as described below. For -cells
determination, the 0.5 g basic fuchsin was dissolved in 70% alcohol, to which were added 1.0 ml paraldehyde with 1.5 ml conc.
HCl. Embedding hydrated parafn sections were treated with Lugol
iodine solution for 30 min, then bleached using 5% sodium thiosulfate for 2 min followed by washing of the section for 5 min in
running tap water for oxidation. The -cells were rinsed with 95%
and 70% alcohol for 10 min, and then washed with distilled water
followed by Ehrlichs hematoxylin for 4 min and counter stain for
45 s, respectively. Finally, the stain coupling jar was treated with
aldehyde fuchsin for 210 min. The end point of -cells stood out
clearly in dark purple against a colorless background.

HPLC studies in callus and leaf extracts

Gymnemic acid was screened in leaf and callus (1 g dry wt.)
extracts by above procedure. After centrifugation, an aliquot of
methanol supernatant (4 ml) was evaporated and dried. The residue
(ca. 6 mg) was dissolved in MeOH (5 ml), and injected into an HPLC
column (20 l). For gymnemic acid separation, the following systems and protocols were used: water HPLC system (Shimadzu
model, Japan), 510 pump, 7725 Rheodyne injector, C18 (ODS)
reversed-phase column, water 486 UV detector, with mobile phasewater/methanol (35:65, HPLC grade), 0.1% acetic acid, sample
applied: 20 l, ow rate: 1 ml/min, read at 230 nm, on UV detector.
Preparation of GS leaf and callus extracts
In vivo Gymnema leaves extract (GLE) and in vitro callus
extracts GCE1 GCE6 were prepared by the method reported
previously (Ahmed et al., 2008). The extracted sample was evaporated and dissolved in saline water (Sterile NS saline, Baxter

Treatment groups
Group I (control): free access to tap water.
Groups IIX: diabetes-induced animals were divided into
9 groups administered saline, 4 U/kg body wt. of insulin/day,
200 mg/kg body wt. (rat)/day GLE and GCE1 GCE6 , respectively. The
experimental Wistar rats were sacriced at 10, 20 and 30 days.
The following treatment groups were used in this experiment:
Group II diabetic control (Alloxan); Group III Diabetic standard
(insulin); Group IV diabetic rats + G. sylvestre leaf extract; Group
V (GCE1 ) blue light with MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l);
Group VI (GCE2 ) 5% sucrose with MS + 2,4-D (1.5 mg/l) + KN
(0.5 mg/l); Group VII (GCE3 ) 12 h photoperiod MS + with 2,4-D
(1.5 mg/l) + KN (0.5 mg/l), Group VIII (GCE4 ) 3 mM NH4 NO3 with
MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l), Group IX (GCE5 ) MS + 2,4D (1.5 mg/l) + KN (0.5 mg/l) and Group X (GCE6 ) MS + NAA
(1.0 mg/l) + KN (1.5 mg/l), respectively.

A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 10331039

Statistical analysis

Results and discussion

Only data which showed an advantageous effect were included

in tables and presented in mean SE (replicated 3 times). Experimental design was completely random and factorial with control,
alloxan alone, alloxan with insulin, alloxan with leaf and alloxan
with callus extracts. The data were subjected to analysis of variance
and mean separation was carried out using Duncans multiplerange test (DMRT) at 5% level signicance (Gomez and Gomez,
1976).

Callus initiation under stress conditions

1035

In vitro callus failed without PGRs in MS medium, while different media (MS, B5, SH, WPM) with PGRs affected the callus
initiation in leaf explants (data not shown). Trial of auxins alone,
and auxins with cytokinins combinations showed callus induction, among which 2,4-D (1.5 mg/l) with KN (0.5 mg/l) induced
green compact callus with maximum dry weight at 45 days.

Fig. 1. (A) Effect of different color lights with (OPGRs) optimum plants growth regulators (MS + 2,4-D) + KN (0.5 mg/l). ( ) Blue lights + OPGRs; ( ) green lights + OPGRs; ( )
red lights + OPGRs; ( ) dark (without lights) + OPGRs; ( ) white lights + OPGRs. (B) Effect of different temperature with optimum plant growth regulators (OPGRs) (MS + 2,4D) (1.5 mg/l) + KN (0.5 mg/l); () 20 C + OPGRs; ( ) 30 C + OPGRs; ( ) 35 C + OPGRs; ( ) 25 2 C + OPGRs. (C) Effect of different photoperiod with optimum plant growth
regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 4 h + OPGRs; ( ) 8 h OPGRs; ( ) 12 h OPGRs; ( ) 16 h (control) + OPGRs; ( ) 20 h + OPGRs; ( ) 24 h + OPGRs;
( ) dark + OPGRs. (D) Effect of different concentration carbon source (sucrose) with optimum plant growth regulators (OPGRs) (MS + 2,4-D 1.5 mg/l) + KN (0.5 mg/l); () 2%
sucrose + OPGRs; ( ) 3% sucrose (control) OPGRs; ( ) 4% sucrose + OPGRs; ( ) 5% sucrose + OPGRs; ( ) 6% sucrose + OPGRs. (E) Effect of ammonia nitrate concentration
(NH4 NO3 ) with (OPGRs) optimum plant growth regulators (MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); ( ) 0 mM NH4 NO3 + OPGRs (control) () 1 mM NH4 NO3 + OPGRs; ( ) 2 mM
) 3 mM NH4 NO3 + OPGRs; ( ) 4 mM NH4 NO3 + OPGRs. (For interpretation of the references to color in this gure legend, the reader is referred to the
NH4 NO3 + OPGRs; (
web version of the article.)

Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not signicantly different at 5% probability level according to DMRT.
a
GCE1 blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE4 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN
(0.5 mg/l); GCE5 MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 MS + NAA (1.0 mg/l) + KN (1.5 mg/l).

2.12c
2.26ab
2.35a
1.90bc
1.94b
2.17a
2.00bc
2.17b
2.39a
1.90c
2.04ab
2.15a
1.76a
1.54b
1.35bc
Liver glycogen (mg/g)
10th
2.65c
20th
2.74ab
30th
2.82a

2.15bc
2.24b
2.56a

1.95bc
2.10b
2.32a

2.10c
2.35ab
2.47a

2.16bc
2.25b
2.46a

8.4c
8.7ab
9.0a
8.1bc
8.4b
8.9a
7.9ab
7.5c
8.0a
8.0c
8.4ab
8.7a
7.8a
7.5ab
7.0c
Liver weight (g/animal)
10th
8.0c
20th
8.5ab
30th
9.0a

8.0c
8.2ab
8.4a

8.5bc
8.7b
9.0a

7.6c
8.0ab
8.3a

7.6b
7.2bc
8.2a

1.14c
1.20ab
1.25a
1.30c
1.35ab
1.37a
1.28bc
1.30b
1.39a
1.10c
1.31ab
1.35a
1.35ab
1.20c
1.42a
1.29bc
1.30b
1.37a
1.10c
1.19ab
1.25a
1.28c
1.37ab
1.40a
1.16a
1.05b
0.95bc
Pancreas weight (g/animal)
10th
1.30bc
20th
1.35b
30th
1.42a

208.0bc
199.0fg
205.0f
220.0a
200.0de
196.6fg
205.0c
215.0a
208.0bc
196.0f
195.0fg
215.0a
203.0a
191.6c
172.0e
162.0fg
Body weight (g/animal)
0
200.0d
10th
200.0de
20th
225.0bc
30th
240.0a

223.3c
213.3g
222.5cd
235.0a

226.6e
226.6e
237.5c
240.0a

220.0ed
213.0ef
225.0c
245.0a

206.0cd
200.0e
215.0b
220.0a

Group IX
alloxan + GCE5
Group VIII
alloxan + GCE4
Group VII
alloxan + GCE3
Group VI
alloxan + GCE2
Group V
alloxan + GCE1
Group IV
alloxan + leaf
Group III
alloxan + insulin
Group II
alloxan
Group I
(control)
Days

Table 1
Body weight (g) of Wistar rats with leaf and callus (methanol) extracts (GSC1 GSC6 )a of G. sylvestre.

193.0e
190.0g
205.0b
210.0a

A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 10331039

Group X
Alloxan + GCE6

1036

Kumar et al. (2002) reported MS medium with PGRs were suitable for callus proliferation of G. sylvestre, but callus proliferation
in other auxins with cytokinins concentration was not pursued,
because of their poor quality and production (data not shown).
Recently we have reported on callus production in different media
such as MS, SH, WPM and B5 media, among which MS media
with auxins and cytokinins were suitable for callus production
(Ahmed et al., 2009a). Stress treatment and callus growth curve
were screened at different days (15, 25, 35, 45 and 55 days).
Lag phase (1525 days), callus initiation and proliferation were
observed. 2535 days (exponential phase), biomass and green compact callus increased signicantly. However, stationary phase at 45
days, maximum biomass and green compact callus were shown.
Decline phase, the nature and biomass of callus were signicantly
reduced as compared to other phases. In Asclepiadaceae species,
the stationary phase was observed at 22 days; at this phase cryptosin was isolated from Cryptolepis buchanani (Venkateswara et
al., 1987) and gagaminine from Cynanchum wilfordii (Shin et al.,
2003).
Optimized concentration was maintained under stress conditions; leaf explants under blue light, 5% sucrose induced the
maximum biomass with green compact callus, then photoperiod,
temperature and ammonium nitrate concentrations (Fig. 1AE).
Photoperiod and temperature affected physical appearance, producing white watery and white friable callus (data not shown).
These calluses were stored for a long time and the media
turned brown in color. Blue light revealed green compact callus with maximum biomass at 45 days. Cryptochrome showed
a top peak at 450 nm, a wavelength close to the blue light;
under these conditions, more Pr transformed into Pfr for phenyletanoid glycosides production (Ouyang et al., 2003). Zhao et al.
(2001) reported that blue light stimulates jaceosidin production in
Saussurea medusa.

TLC, HPTLC, HPLC studies

Methanol supernatants of G. sylvestre leaf and in vitro calluses were screened using TLC and HPTLC. After chromatogram,
samples were dried and sprayed with specic reagent (vanillin
sulfuric acid reagent) at room temperature for detection of gymnemic acid. Standard gymnemic acid showed as a single brown
band; however, the callus extract displayed additional brown bands
with Rf value (0.44) greater than that of gymnemic acid (0.43).
In HPTLC analysis, methanol solvent was run up to 80 mm and
scan chromatograms at 200 nm under UV reectance mode. Gymnemic acid content leaf and callus extract data were compared
with standard gymnemic acid. The calibration curve was linear;
the correlation coefcient indicated good linearity between concentration and area. To ascertain peak purity of test sample, we
compared in vivo and in vitro methanol extract reectance spectra with standard, which provides test sample purity (data not
shown). Gymnestrogenin (triterpenoids) determine in G. sylvestre
through HPTLC at 293 nm (Puratchimani and Jha, 2004). For HPLC
analysis, leaf and callus methanol extracts (20 l) were uploaded
in HPLC system to quantify gymnemic acid under retention time.
UV spectrophotometer peak area data were compared with standard gymnemic acid. The gymnemic acid content was increased in
leaf, GSC1 and GSC3 over other callus extracts (data not shown).
Imoto et al., 1991 reported that a methanol extract contains
gymnemic acid through HPLC. Many authors had isolated and
identied gymnemic acid earlier. In 1989, Yoshikawa and coworkers isolated gymnemic acids from a hot water extract of G.
sylvestre, which they named gymnemic acids I, II, III, IV, V, VI
and VII, respectively, and evaluated using HPLC (Sugihara et al.,
2000).

A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 10331039

1037

Fig. 2. Histologic examination of pancreatic -cells in alloxan treated animals. (A) Pancreas of normal rats. Note abundant patches if -cells (arrows) in purple color (200);
(B) pancreas of alloxan treated rats, hardly and -cells noticed, all islets degenerated (200); (C) pancreas of alloxan treated rats, after insulin treatments showed abundant
patches of -cells (arrows) in purple color (200); (D) pancreas treated rats, after G. sylvestre leaf extract (200); (E) pancreas treated rats, after G. sylvestre callus extract
1 (200); (F) G. sylvestre callus extract 2 (200); (G) G. sylvestre callus extract 3 (200); (H) G. sylvestre callus extract 4 (200); (I) G. sylvestre callus extract 5 (200); (J) G.
sylvestre callus extract 6 (200). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

Body, liver, pancreas weight and liver glycogen

As shown in Table 1, the long-term antihyperglycemic effect
was examined for GS leaf and callus extracts. The whole body, liver
and pancreas weights were reduced in the diabetic control group
after 30 days, although oral administration of 200 mg/kg body wt.
GS leaf and callus extracts signicantly increased and maintained
the body liver and pancreas weights in the 4 U/kg body wt. insulin
group Wistar rats. We have recently published a review of the pharmacological activities, a phytochemical investigation and in vitro
studies of G. sylvestre (Ahmed et al., 2009b).
Liver glycogen was signicantly decreased in diabetic control
rats, whereas with leaf and callus extracts of GS, liver glycogen
signicantly increased (Table 1). Restoration of hepatic glycogen
by GS leaf and callus could improve the insulin secretion or inhibition of glucose-6-phosphatase in liver, and has prevented the
conversion of glucose 6-phosphate to glucose (Shanmugasundaram
et al., 1990). Shanmugasundaram et al. (1983) reported that G.
sylvestre restored glycogen and enzymes in diabetic rabbit liver
after 24 weeks of treatment. We earlier reported that leaf and callus

extracts of G. sylvestre reduced blood sugar and lipid proles such

as cholesterol, triglyceride, HDL, LDL, VLDL in alloxan-induced diabetic Wistar rats (Ahmed et al., 2008). Shanmugasundaram et al.
(1990) reported aqueous extract of G. sylvestre leaf (200 mg/kg body
wt.) normalized blood sugar level in STZ-induced diabetic rats.
However, gymnemic acid from G. sylvestre leaf regulated hyperglycemia (Gholap and Kar 2005) and an aqueous extract maintained
the blood glucose level in normal Wistar rats (Raullah et al., 2006).
Histological examinations of pancreas
Fig. 2AJ depicts the histological examination (200) of pancreatic -cells in control, diabetic control, diabetic standard, GS
leaf and callus extracts groups. Histological pancreas islets were
checked separately in hematoxylin and eosin with aldehyde fuchsin
section. In hematoxylin and eosin sections, the degree of atrophy, hydropic degeneration, necrosis, hyalinization, or brosis was
checked (data not shown), while in aldehyde fuchsin sections, the
-cells percentage was recorded based on atrophy cells, and the
-granules were increased or decreased at the 10th, 20th and 30th

1038

A.B.A. Ahmed et al. / Phytomedicine 17 (2010) 10331039

Table 2
Leaf and callus extractsa of Gymnema sylvestre regenerate the pancreatic -cells in alloxan-induced diabetic Wistar rats.
Treatment group

-Cell regeneration frequency (%)

10th day

Group I (normal)
Group II (diabetic control)
Group III (insulin)
Group IV (leaf)
Group V (callus extract 1)
Group VI (callus extract 2)
Group VII (callus extract 3)
Group VIII (callus extract 4)
Group IX (callus extract 5)
Group X (callus extract 6)

81.0
16.0
38.0
49.0
53.3
42.3
51.3
30.3
28.0
27.0

2.82a
2.16j
2.62b
4.54e
8.07c
5.89ef
3.13cd
2.12g
6.01gh
3.77i

20th day
83.6
12.3
41.0
58.6
50.0
47.0
55.0
32.6
31.6
34.0

4.27a
1.44j
1.69ab
1.18c
0.94e
3.85ef
4.54cd
6.15gh
4.11i
3.39g

30th day
80.0
8.6
45.5
66.6
59.6
54.3
57.6
39.0
45.0
37.6

2.62ab
1.65j
2.82a
2.88c
4.09cd
4.57ef
3.47e
4.54gh
1.88g
0.98i

Values are mean of 3 replicates per treatment and repeated thrice. Values with the same letters are not signicantly different at 5% probability level according to DMRT.
a
GCE1 blue light with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE2 5% sucrose with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE3 12 h photoperiod 2,4-D (1.5 mg/l) + KN (0.5 mg/l);
GCE4 3 mM NH4 NO3 with 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE5 MS + 2,4-D (1.5 mg/l) + KN (0.5 mg/l); GCE6 MS + NAA (1.0 mg/l) + KN (1.5 mg/l).

days (Table 2). In electron microscopic analysis, the pancreatic cells islets showed no pathological alternation in control Wistar
rats. The nucleus, nuclear envelope, mitochondria, endoplasmic
reticulum and Golgi complex of -cells were normal (Fig. 2A). However, 80% of -cells contained a number of secretory granules,
which had a space between core and membranes diffusely distributed in cytoplasm at end of experiment. The secretory granules
showed moderate homogenous (or) slightly heterogenous electron
density and single layered membrane (Fig. 2A).
Electron microscope analysis of -cells in diabetic standard
rats (alloxan alone) displayed the loss of nuclear envelope, mitochondrial vacuolization, swelling and dilatation of endoplasmic
reticulum (Fig. 2B). During alloxan treatment, -cytotoxin causes a
massive destruction of -cells in islets of Langerhans, and reduces
the -cells and insulin release (Colca et al., 1983). Diabetic standard (insulin), GS leaf and callus extracts treated groups showed
signicant increase in the secretary granule of -cells and reached
to control level (Fig. 2DJ). The -cells were apparently normal
in the architecture of the nucleus, which revealed that gymnemic
acid prevented the -cells damage as compared to control rats. But
low gymnemic acid content callus extracts decreased the secretory
granules and losses of nuclear envelope, mitochondrial vacuolization, swelling and dilation of the endoplasmic reticulum in diabetic
rats.
Degenerative changes in pancreatic -cells were minimized and
normal morphology maintained in diabetic rats by administration
of gymnemic acid content extracts as evidenced in histopathological examination. Pancreas hematoxylin/eosin with aldehyde
fuchsin stains displayed the ruptured islets and decreased -cells
in diabetic control rats, whereas leaf and callus extracts regenerated the -cells frequency in diabetic standard (Fig. 2C; 45.5%), GS
leaf extract (Fig. 2D; 66.6%), leaf callus extract 1 (Fig. 2E; 59.6%),
callus extract 2 (Fig. 2F; 54.3%), callus extract 3 (Fig. 2G; 57.6%),
callus extract 4 (Fig. 2H; 39.0%), callus extract 5 (Fig. 2I; 45.0%) and
callus extract 6 (Fig. 2J; 37.6%) (Table 2). In treatment pancreatic cells, AMP was activated and the protein kinases showed a unique
function or connecting cellular energy status to the ability of insulin
synthesis and secretion of -cells (Da Silva Xavier et al., 2000). Alcohol extract of G. sylvestre stimulated the insulin secretion in islets
of Langerhans and pancreatic -cell lines (Persaud et al., 1999).
Srivastava et al. (1986) reported that pancreas tissue was completely destroyed in alloxan treatment, after G. sylvestre leaf extract
regenerated the prolonged survival and adaptogenic activity. However, a smaller dose of STZ (45 mg/kg body wt.) led to damage of the
pancreatic acinar cells and reversed by Aegle marmelos (Arumugam
et al., 2008). Successful -cell regeneration was achieved using
diabetic medicinal plants such as Momordica charantia, Beta vulgaris, Catharanthus roseus, and Eugenia jambolana (Das et al., 1996;
Sharma et al., 2006).

In conclusion, the leaf and callus extracts of G. sylvestre have

antihyperglycemic effects as determined using alloxan-induced
diabetic Wistar rats. Our experimental data suggested that alloxan
administered to Wistar rats through intraperitoneal route exhibits
diabetic activity. We conclude that the effect of G. sylvestre leaf and
callus extracts as strong herbal remedies is conrmed in this study,
and suggest that they may be capable of fully restoring pancreatic -cells function and thus curing type I diabetes. Studies of G.
sylvestre callus extract have revealed how -cells may be formed
and regenerated in vitro and could provide further direction for
potential drug development for IDDM treatment.
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