Journal of Ethnopharmacology 73 (2000) 461 – 470

www.elsevier.com/locate/jethpharm

Anti-hyperglycemic effect of Eugenia jambolana and

Tinospora cordifolia in experimental diabetes and their
effects on key metabolic enzymes involved in carbohydrate
metabolism
J.K. Grover a,*, V. Vats a, S.S. Rathi b
a
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110049, India
b
St Boniface Institute of Cardio6ascular Sciences, Winnipeg, Canada
Received 15 December 1999; received in revised form 10 July 2000; accepted 29 July 2000

Abstract

In India, the decoction of kernels of Eugenia jambolana (EJ) and extracts of Tinospora cordifolia (TC) are used as
a household remedy for diabetes. These also form constituents of many herbal formulations for diabetes that are
marketed in this country. The anti-hyperglycemic effect of aqueous and alcoholic extracts as well as lyophilized
powder of these two plants was evaluated in diabetic animals using different doses of diabetogenic agents for varying
duration (21–120 days) so as to assess their effect in mild (plasma sugar \ 180 mg/dl, duration 21 days), moderate
(plasma sugar \ 280 mg/dl, duration 120 days) and severe (plasma sugar \ 400 mg/dl, duration 60 days) diabetes
mellitus. In the pilot study (mild diabetes), maximum reduction of 73.51 and 70.37% in glucose levels was seen in
animals receiving 200 mg/kg per day of lyophilized powder of EJ and 400 mg/kg per day of aqueous extract of TC
after 3 and 15 weeks of treatment, respectively. There percent reduction in glucose decreased significantly in the
moderate and severe diabetes; 55.62 and 17.72% for EJ and 48.81 and 0% for TC at the similar time intervals. The
alteration in hepatic and skeletal muscle glycogen content and hepatic glucokinase, hexokinase, glucose-6-phosphate
and phosphofructokinase levels in diabetic mice were partially restored by EJ but not by TC. The mechanism of
action of EJ and TC is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Eugenia jambolana; Tinospora cordifolia; Jamun; Ambervel; Alloxan diabetic rats; Streptozotocin rats

1. Introduction

Diabetes mellitus (DM) is a major cause of

* Corresponding author. Tel.: +91-11-6594897 (office), +
91-11-4615315 (home); fax: + 91-11-6862663.
E-mail address: jkgrover@hotmail.com (J.K. Grover).
disability and hospitalization and it results in
significant financial burden ($92 billion per year in
US) (Foster, 1994). Though a subsection of
NIDDM patients can be managed by diet alone,

0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 3 1 9 - 6
462 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470
most patients require oral hypoglycemic and/or
insulin. Insulin therapy affords effective glycemic
control, yet its short comings such as ineffective-
ness on oral administration, short shelf life, re-
quirement of constant refrigeration, and in the
event of excess dosage — fatal hypoglycemia —
limits its usage. Treatment with sulfonylureas and
biguanides is also associated with side effects
(Rang and Dale, 1991).
For various reasons in recent years, the popu-
larity of complementary medicine has increased.
Dietary measures and traditional plant therapies
as prescribed by Ayurvedic and other indigenous
systems of medicine were used commonly in India
(Warier, 1995). Surveys conducted in Australia
and US indicate that almost 48.5 and 34% respon-
dents had used at least one form of unconven-
tional therapy including herbal medicine
(Eisenberg et al., 1993; Maclennan et al.,
1996). Indian figures are not available. WHO
(1980) has also recommended the evaluation of
the plants effective and in conditions where we
lack safe modern drugs (Upadhayay and Pandey,
1984).
Eugenia jambolana (EJ) belongs to the Myrat-
ace family and is commonly called Jamun or
Jambul in Hindi, Black Plum or Black Berry in
English. Various medicinal properties of EJ in-
cluding its astringent, stomachic, astringent, di-
uretic and anti-diabetic properties have been
described in traditional medicine (Nadkarni,
1992). The jamun tree is a large evergreen and is
native to India but is also found in other parts of
the world especially tropical countries. Jamun
seeds have been used by the natives in the treat-
ment of diabetes (Chopra et al., 1958).
Tinospora cordifolia (TC) belongs to the Menis-
permaceae family and is commonly known as
Glunchanb or Tinospora in English and Giloe or
Ambervel in Hindi. It has been indicated in
Ayurvedic treatment as tonic, vitalizer and as a
remedy for diabetes and metabolic disorders
(Nadkarni, 1954; Chopra et al., 1958). It caught
the notice of European physicians in India for its
tonic and diuretic property (Pendse and Dutta,
1932) and the drug became an official preparation
in Indian Pharmacopoeia in 1932. Gulancho was
also included in Bengal Pharmacopoeia in 1844
and in Indian Pharmacopoeia in 1868. Scientific
reports describing anti-diabetic (Gupta et al.,
1967), immunomodulatory (Atal et al., 1986),
anti-hepatoxic (Peer and Sharma, 1989),
anti-pyretic (Vedavathy and Rao, 1991) and
anti-stress activity (Sarma et al., 1995) are avail-
able.
A literature survey showed that the anti-hyper-
glycemic activity of EJ (Shrotri et al., 1963;
Bansal et al., 1981; Achrekar et al., 1991) and TC
(Gupta et al., 1967; Wadood et al., 1991) has been
demonstrated in many experimental studies. The
duration of these studies however was not long
enough to comment on the effect of the plant
extracts on the course of a chronic disorder —
diabetes mellitus. Moreover the anti-hyper-
glycemic activity of these plants has also not been
assessed against different intensities of hyper-
glycemia. Therefore, the primary objectives of this
study were to assess the anti-hyperglycemic effi-
cacy of plants against different intensities of hy-
perglycemia (in models utilizing different
diabetogenic agents in different doses) labeled as
mild, moderate and severe diabetes for acute and
chronic duration.
2. Materials and methods
2.1. Preparation of extracts
2.1.1. Aqueous extract of EJ
Kernels of the fruit of EJ were purchased from
the local market in June 1997 and were authenti-
cated by Dr Manasi Ram, Head, Department of
Botany, Miranda House, University of Delhi (In-
dia) (Voucher number 163¯97). After grinding
them in an electric grinder, the powder was
soaked in an equal amount of water and stirred
intermittently and then left overnight. The macer-
ated pulp was then filtered through a coarse sieve
and the filtrate was dried at reduced temperature.
This dry mass (yield 69 g/kg of powdered kernel)
served as aqueous extract of EJ for experimenta-
tion. To increase the shelf life and uniformity, this
extract was completely lyophilized by continuous
freeze drying operation of 54 h (Christ freeze
 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470
dryer, alpha 1-4, Germany), yielding 46.6 g/100 g
of aqueous extract.
2.1.2. Alcoholic extract of EJ
For the preparation of alcoholic extract of EJ,
1 kg of kernels was powdered in an electric
grinder and then mixed with 500 ml of alcohol
(Glaxo Chemicals Laboratories, Bombay) and
kept at room temperature for 36 h. The slurry was
stirred intermittently for 2 h and left overnight.
The mixture was then filtered and the filtrate was
freed from solvent under partial vacuum (71
mmHg) at 35–45°C to yield 23 g/kg of the pulp.
A few drops of silicon emulsion were added near
the end of distillation to avoid frothing. The
residue collected (yield 83.2 g/kg of powdered
kernel) was a thick paste, green in color and
gummaceous in nature.
2.1.3. Extracts of TC
Alcoholic and aqueous extracts of TC were
received as a gift from Brawn Pharmaceuticals
Ltd., Faridabad (India). All the extracts were
dissolved in 1% carboxy methylcellulose (Central
Drug House, New Delhi) and given orally.
2.2. Animals
Albino rats (150– 200 g) and albino mice (30 –
50 g) of both sexes were obtained from the exper-
imental animal facility of the All India Institute of
Medical Sciences. Before the start and during the
experiment, rats were fed a standard chow diet.
After randomization into various groups, the rats
were acclimatized for a period of 2 – 3 days in the
new environment before initiation of experiment.
Animals described as fasting had been deprived of
food for at least 16 h but had been allowed free
access to drinking water.
2.3. Sample collection
Blood was collected retro-orbitally from the
inner canthus of the eye under light ether anesthe-
sia using capillary tubes (Micro Hematocrit Capil-
laries, Mucaps). Blood was collected in fresh vials
containing sodium fluoride and sodium oxalate as
463
anti-coagulant/anti-glycolytic agents and plasma
was separated in a T8 electric centrifuge (Remi
Udyog, New Delhi) at 2000 rev./min for 2 min.
2.4. Experimental design
2.4.1. Anti-hyperglycemic acti6ity
2.4.1.1. Pilot studies (mild diabetes). In the pilot
study lasting for 3 weeks, albino Wistar rats were
made diabetic by a single i.v. injection of alloxan
monohydrate (Loba Chemie, Bombay) (32 mg/kg)
given in the tail vein. Alloxan was first weighed
individually in Eppendorf tubes for each animal
according to the weight and then solubilized with
0.2 ml saline (154 mM NaCl) just prior to injec-
tion. After 2 days of alloxan injection, rats with
plasma glucose levels \ 175 mg/dl were included
in the study. For EJ, three doses of alcoholic and
aqueous extracts (50, 100 and 200 mg/kg per day)
and two doses of lyophilized powder (100 and 200
mg/kg per day) were tested. For TC, three doses
of aqueous extract only were tested (100, 200 and
400 mg/kg per day). All doses were started 48 h
after alloxan injection. Blood samples were drawn
at weekly intervals till the end of study (i.e. 21
days for EJ and 15 weeks for TC).
2.4.1.2. Moderate diabetes. Overnight fasted rats
were injected with alloxan (120 mg/kg) in the
manner described above. Forty-eight hours after
administration of alloxan, rats with plasma glu-
cose levels \ 250 mg/dl were included in the
study. Blood samples were drawn at monthly
intervals till the end of study (i.e. 120 days).
2.4.1.3. Se6ere diabetes. Albino mice were injected
a bolus of streptozotocin (STZ) (Sigma, St Louis,
MO) (150 mg/kg dissolved in 3 mM citrate buffer
pH 4.5) intraperitoneally. After 10 days of STZ
injection, mice exhibiting blood glucose levels \
300 mg/dl were included in the study. The selected
doses of the plant extracts (which proved effective
in pilot study) were given everyday till the com-
pletion of the experiment (i.e. 50 days). Blood
samples were drawn on every 10th day and
assessed for plasma glucose levels till the 50th
day.
464 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470
2.5. Effect of plant extracts on glycogen content and
different enzymes in6ol6ed in carbohydrate
metabolism
Overnight fasted mice were made diabetic by a
single i.v. injection of alloxan (32 mg/kg) as de-
scribed previously. Mice with plasma glucose B
175 mg/dl were rejected for the study and selected
doses of the plant extracts were administered orally
everyday till the end of the experiment to the
selected animals. The animals were killed by decap-
itation on the 10th day and blood as well as tissue
samples were collected for the assessment of plasma
glucose, hepatic glucokinase, hexokinase, glucose-
6-phosphate and phosphofructokinase content
along with glycogen content in different tissues, i.e.
liver, brain, heart and skeletal muscle.
2.6. Sub-acute toxicity studies
Albino mice were given 200 mg/kg of lyophilized
powder of aqueous extract of EJ and 400 mg/kg of
aqueous extract of TC every day (starting from day
one) up to the 60th day. At the end of experiment,
blood samples were collected as described previ-
ously for assessment of white and red blood cells
counts (WBC and RBC), hemoglobin (Hb), mean
corpuscular volume (MCV), hematocrit (HCT),
mean corpuscular hemoglobin (MCH) and mean
plasma glucose levels.
2.7. Biochemical analysis
2.7.1. Plasma glucose
Glucose levels were estimated by commercially
available glucose kits based on glucose oxidase
method (Trinder, 1969) (Autopak®, Bayer Diag-
nostics, Baroda).
2.7.2. Hepatic glucokinase and hexokinase
acti6ity
The liver was perfused with ice cold 0.15 M KCl
and 1 mM EDTA solution and homogenized with
twice its weight of ice cold buffer (0.01 cysteine and
1 mM EDTA in 0.1 ml Tris – HCl, pH 7.4) and
centrifuged (at 20 000×g) for 20 min at 4°C.
Glucose phosphorylation was assayed by means of
the glucose-6-phosphate dependent spectrophoto-
metric method (Crane and Sols, 1955).
2.7.3. Glucose-6 -phosphate
The liver was homogenized with 40 times its
weight of ice cold buffer (0.1 citrate–KOH, pH 6.5)
and filtered through cheese cloth. Glucose-6-phos-
phatase activity was measured by phosphate release
by the method of Marjorie (1950). The calorimetric
method for determination of phosphoric acid con-
centration in the supernatant of the assay mixture
was employed (Fiske and Subbaraw, 1925).
2.7.4. Hepatic phosphofructokinase acti6ity
The liver was homogenized with 10 times its
weight of ice cold buffer (100 mM KF, 15 mM
EGTA and 50 mM Hepes–KOH, pH 7.4) and
centrifuged (at 60 000× g) for 15 min at 4°C. The
phosphofructokinase activity was assayed spec-
trophotometrically (Castano et al., 1925).
2.7.5. Glycogen content
The tissue sample was digested by hot concen-
trated 30% KOH and treated with anthrone
reagent. Glycogen content was determined colori-
metrically as glucose (Morales et al., 1973).
2.7.6. Hematological and biochemical parameters
Blood samples were assessed for RBC, WBC,
HB, HCT, MCV and MCH with an auto-analyzer
(Alpha 4, Beckman, UK).
2.8. Statistical analysis
The results were analysed for statistical signifi-
cance by one-way ANOVA test using Microcal
Origin version 2.9 (Northampton, USA).
3. Results
In all groups prior to alloxan administration, the
basal levels of plasma glucose of the rats were not
significantly different. However, 48 h after alloxan
administration, plasma glucose levels were signifi-
cantly higher in the rats selected for the study. In
contrast, non-diabetic controls remained persis-
 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470 465

tently euglycemic throughout the course of the
study.
3.1. Mild diabetes
The pilot study was aimed at selecting the most
effective dose of plant. Tables 1 and 2 show the
effect of treatment with extracts of EJ and TC,
respectively on plasma glucose levels. In the EJ
treated groups (all doses), although a significant
anti-hyperglycemic (P B0.01) effect was evident
from the first week onwards, the decrease in
plasma sugar was maximum on completion of the
third week (73.51%) (P B 0.001) in the group re-
ceiving 200 mg/kg per day of lyophilized powder
of EJ. On the other hand, TC-treated groups
showed a anti-hyperglycemic response much later
(i.e. on completion of the third week). Therefore,
the study was extended further and a significant
decrease in glucose levels was observed from the
sixth week onwards. Maximum anti-hyper-
glycemic effect (70.37%) was seen in the group

Table 1
Pilot study: the effect of 3-week treatment with various doses of aqueous, alcoholic and lyophilized powder extract of EJ on glucose
levels (mg %) in alloxan (32 mg/kg) diabetic ratsa

Group 0 day 48 h 1st week 2nd week 3rd week

NC 56.01 9 1.09 53.219 1.31 55.09 9 3.2 60.07 9 3.8 56.31 94.31
DC 52.33 9 3.83 198.739 4.9** 188.859 8.84** 178.63 9 9.33** 190.23 913.1**
EJ-Aq-50 52.19 6.69 181.049 9.01 148.04 9 8.8* (22.5) 124.07 9 8.17* (35.05) 108.55 97.58** (43.17)
EJ-Aq-100 53.71 9 5.4 205.529 9.01 148.269 13.9* (27.86) 125.33 9 9.84* (39.01) 109.85 97.46** (46.55)
EJ-Aq-200 51.92 9 3.59 208.35910.4 136.939 12.3* (34.27) 103.49 99.35* (50.32) 58.84 96.02** (71.75)
EJ-Alc-50 54.55 9 6.20 181.439 8.43 151.98 9 3.83* (24.54) 120.02 9 9.36** (40.41) 82.31 911.35** (59.13)
EJ-Alc-100 52.45 9 6.47 210.0799.88 148.83 9 3.83* (29.15) 117.06 98.14** (44.27) 75.21 911.04** (64.19)
EJ-Alc-200 50.93 9 7.63 214.969 10.4 140.4912.07* (34.67) 103.98 9 8.42** (51.62) 58.7 97.14** (72.69)
EJ-Lyp-100 50.82 9 7.12 219.959 9.36 145.07915.8* (34.04) 112.21 911.3** (48.98) 67.55 93.69** (69.28)
EJ-Lyp-200 47.83 9 3.37 221.959 11.8 136.359 6.58* (38.56) 100.87 9 5.04** (54.55) 58.79 93.89** (73.51)

a
Values are given as mean9 S.D. for groups of eight animals each; values in parentheses indicate the percentage lowering of
plasma sugar in comparison to basal reading after alloxan administration at 48 h. Diabetic control was compared with normal
control. Experimental groups were compared with the corresponding values at 48 h. NC, non-diabetic control; DC, diabetic control;
Aq, aqueous extract; Alc, alcoholic extract.
* PB0.01.
** PB0.001.

Table 2
Pilot study: the effect of 15 week treatment with various doses of aqueous extract of TC on glucose levels (mg %) in alloxan (32
mg/kg) diabetic ratsa

Group 0 day 48 h 3rd week 6th week 9th week 12th week 15th week

DC 44.39 2.1 192.997.02 171.49 5.2 132.19 6.04 129.5 94.08 127.6 93.6 125.4 9 4.1
TC-Aq-100 41.9 9 2.2 183.397.1 175.99 8.2 141.89 7.6* 128.1 95.4** 118.8 94.6** 114.3 9 5.5**
(4.02) (22.6) (30.07) (35.16) (37.60)
TC-Aq-200 44.1 9 1.9 196.697.9 159.79 10.2* 119.8 95.7** 111.3 93.8** 103.1 94.3** 102.6 9 3.8**
(18.78) (39.07) (43.38) (47.56) (47.80)
TC-Aq-400 44.8 9 2.9 198.299.09 151.19 8.4** 112.2 99.9** 94.3 93.8** 61.6 96.3** 58.7 9 4.2**
(23.74) (43.36) (52.42) (68.89) (70.37)

a
Values are given as mean9 S.D. for groups of eight animals each; values in parentheses indicate the percentage lowering of
plasma sugar in comparison to basal reading after alloxan administration at 48 h. Experimental groups were compared with the
corresponding values at 48 h. DC, diabetic control; Aq: aqueous extract; Alc: alcoholic extract.
* PB0.01.
** PB0.001.
466 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470

Table 3
Moderate diabetes: effect of administration (4 months) of selected doses of EJ (200 mg/kg per day of lyophilized powder of aqueous
extract) and TC (400 mg/kg per day of aqueous extract) on glucose levels (mg %) in alloxan (120 mg/kg) diabetic ratsa

Group 0 day 48 h 1 month 2 month 3 month 4 month

NC 57.3 91.7 54.29 1.5 56.039 0.9 52.6 9 2.3 55.1 91 58.2 94.1
DC 54.6 9 3.3 282.3**9 5.4 300.9**9 11.5 309.4** 911.8 298.4** 9 12.6 290.5** 912.1
EJ-Lyp-200 53.01 9 4.7 295.69 11.7 131.1**9 9.5 118.7** 9 5.2 110.5** 99.5 101.9** 95.6
(55.62) (59.85) (62.60) (65.52)
TC-Aq-400 52.48 93.1 289.196.6 179.23**9 7.3 172.2** 98.3 168** 9 8.6 148** 9 9.3
(38.01) (40.41) (41.90) (48.81)

a
Values are given as mean 9 S.D. for groups of eight animals each; values in parentheses indicates the percentage lowering of
plasma sugar in comparison to basal reading after alloxan administration at 48 h. Diabetic control was compared with the normal.
Experimental groups were compared with the corresponding values at 48 h. NC, non-diabetic control; DC, diabetic control; Aq,
aqueous extract; Alc: alcoholic extract.
** PB0.001.

Table 4
Severe diabetes: effect of administration (60 days) of selected doses of EJ (200 mg/kg per day of lyophilized powder of aqueous
extract) and TC (400 mg/kg per day of aqueous extract) on glucose (mg %) levels in streptozotocin (150 mg/kg) diabetic micea

Group 0 day 10th day 20th day 30th day 40th day 50th day 60th day

NC 53.3 9 1.9 59.39 0.08 55.4 9 1.7 58.3 91.2 55 93.2 53.1 9 4.1 56.09 9 2
DC 60.3 91.1 437.05**9 12.8 441.1** 9 19.9 471.3** 919.3 470.5** 9 12.3 450.5** 9 10.03 439.5** 9 14.2
EJ-Lyp-200 58.9 9 2.4 439.8 97.9 456.5 9 22.9 466.6 915.4 430.6 9 18.8 399.3 9 29.4 361.8* 9 31.1
TC-Aq-400 59.06 9 2.6 454.79 11.3 462.8 9 14.4 482.3 98.5 462.8 9 6.8 455.8 9 12.9 420.8 9 13.3

a
Values are given as mean 9 S.D. for groups of eight animals each; diabetic control was compared with the normal. Experimental
groups were compared with the corresponding values on 10th day. NC, non-diabetic control; DC, diabetic control; Aq: aqueous
extract; Alc: alcoholic extract.
* PB0.01.
** PB0.001.

receiving 400 mg/kg per day of aqueous extract
after 15-weeks of treatment as compared to the
groups receiving 100 and 200 mg/kg (37 and 47%,
respectively). On the basis of these studies, doses of
200 mg/kg per day of lyophilized powder of
aqueous extract of EJ and 400 mg/kg per day of
aqueous extract of TC were selected for further
evaluation.
significant decrease of 55.62, 59.85, 62.6 and
65.52% in plasma sugar levels at 1-, 2-, 3- and
4-month intervals, respectively. On the other hand,
aqueous extract of TC (400 mg/day) at the same
time interval caused decreases in plasma sugar of
38.01, 40.41, 41.9 and 48.81%, respectively.
3.3. Se6ere diabetes

3.2. Moderate diabetes The effect of administration of selected doses of

Table 3 shows the effect of oral administration
of 200 mg/kg per day of lyophilized powder of EJ
and aqueous extract of TC for 4 months. During
the treatment period, administration of lyophilized
powder of EJ (200 mg/kg per day) caused a
EJ (200 mg/kg per day of lyophilized powder) and
TC (400 mg/kg per day of aqueous extract) for 60
days in STZ diabetic mice is shown in Table 4.
Treatment with lyophilized extract of EJ caused a
total reduction of 17.72% at the end of study. On
the other hand, TC had no significant effect on
 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470 467

the plasma glucose levels throughout the duration
of study.
3.4. Glycogen content
Glycogen content of various tissues (liver,
skeletal muscle, heart, brain and kidneys) was
estimated on the 10th day in non-diabetic control,
diabetic control, EJ (200 mg/kg per day of
lyophilized powder) and TC (200 mg/kg per day
of alcoholic extract) treated groups, as shown in
Table 5. In diabetic controls, hepatic and skeletal
muscle glycogen content decreased significantly by
79.66 and 18%, respectively as compared to non-
diabetic controls. On the other hand, renal glyco-
gen content increased by 7% in diabetic animals
as against non-diabetic animals. The glycogen
content of brain and heart remained unaltered in
all the groups.
Treatment with EJ extract led to 35.65 and
35.9% increase in hepatic and skeletal muscle
glycogen content, respectively and a 5% decrease
in renal glycogen content in comparison to dia-
betic controls. Respective percentage alterations
in hepatic, skeletal muscle and renal glycogen
content in TC treated mice were 20.56, 25.65 and
6.72%.
3.5. Hepatic enzymes
To establish diabetes, plasma glucose was deter-
mined 48 h after alloxan administration. Only
those mice with over 180 mg% were included in
the study. On the 10th day, hepatic enzymes
(hexokinase, glucokinase and phosphofructoki-
nase) and substrate (glucose-6-phosphate) were
estimated in saline controls, diabetic controls, EJ
(200 mg/day of lyophilized powder, orally) and

Table 5
Effect of administration (10 days) of selected doses of EJ (200 mg/kg per day of lyophilized powder of aqueous extract) and TC (400
mg/kg per day of aqueous extract) on glycogen content (mg/g tissue) of various tissues of micea

Group Brain Kidney Heart Muscle Liver

NC 1.969 0.13 1.96 9 0.36 2.13 90.05 4.81 90.31 43.31 9 1.3
DC 1.9590.100 2.19 0.23 1.91 90.08 3.95 90.55* 8.81 9 1.41**
EJ-Lyp-200 2.1090.24 2.289 0.348 1.89 90.662 6.28 90.796** 22.60 9 4.71**
TC-Aq-400 1.9990.100 2.07 9 0.0 73 1.9790.067 4.166 90.711 8.92 9 2.08

a
Values are given as mean 9 S.D. for groups of eight animals each. Diabetic control was compared with the normal. Experimental
groups were compared with diabetic control; NC, non-diabetic control; DC, diabetic control; Aq: aqueous extract; Alc: alcoholic
extract.
* PB0.01.
** PB0.001.

Table 6
Effect of administration (10 days) of selected doses of EJ (200 mg/kg per day of lyophilized powder of aqueous extract) and TC (400
mg/kg per day of aqueous extract) on enzymes in carbohydrate metabolism in micea

Group Hexokinase (mg/mg) G-6-P (mg/mg) Glucokinase (mg/mg) Phosphofructokinase activity (% age)

NC 0.181 9 0.013 0.381 9 0.063 23.0591.68 99.16 9 0.983

DC 0.087 9 0.007** 0.151 9 0.006** 5.05 90.804** 79.16 9 9.725*
EJ-Lyp-200 0.139 9 0.007 0.224 9 0.0182** 7.983 92.898* 113.3 9 11.69**
TC-Aq-400 0.077 9 0.012 0.1559 0.017 5.45 90.768 82.339 14.41

a
Values are given as mean 9 S.D. for groups of eight animals each. Diabetic control was compared with the normal. Experimental
groups were compared with diabetic control; NC, non-diabetic control; DC, diabetic control; Aq, aqueous extract; Alc, alcoholic
extract.
* PB0.01.
** PB0.001.
468 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470

Table 7 4. Discussion and conclusion

Results of sub-acute toxicity studies of feeding EJ (200 mg/kg
per day of lyophilized powder of aqueous extract) and TC (400
mg/kg per day of aqueous extract) to mice for 60 daysa Currently available drug regimens for manage-
ment of DM have certain drawbacks (University
Parameter Control EJ-Lyp-200 TC-Aq-400 Group Diabetes Program, 1974; Knatterud et al.,
1978) and therefore, there is a need for safer and
W.B.C. 7.466 8.466 7.38 92.97
more effective anti-diabetic drugs. This study was
(×103/ml) 91.508 9 1.613
R.B.C. 8.326 8.845 8.52 9 0.95
therefore undertaken to assess anti-hyperglycemic
(×106/ml) 91.558 9 0.819 properties of two plants, which have been re-
Hb% (g/dl) 12.316 13.066 13.18 9 1.50 ported in Ayurveda to be useful in diabetes
91.979 9 1.727 mellitus.
HCT (%) 34.583 41.100 40.60 9 5.29
In the present study, treatment with extracts of
95.717 93.989
MCV (fl) 42.198 45.783 45.889 3.77 plants (EJ and TC) showed significant anti-hyper-
9 6.518 9 3.758 glycemic activity in mild to moderate degree of
MCH (pg) 14.893 14.750 14.58 9 0.98 hyperglycemia. In mild diabetes, the maximum
90.990 9 0.791 percent reduction in glucose levels was seen in
groups receiving 200 mg/kg per day of lyophilized
a
Values are given as mean 9 S.D. for groups of eight ani-
mals each.
powder of EJ and 400 mg/kg per day of aqueous
extract of TC (73.51 and 70.37%, respectively)
TC (400 mg/day of aqueous extract) treated ani- and therefore, further work was carried with these
mals. The results have been compiled in Table 6. doses. In moderate diabetes (blood sugar\ 280
As compared to non-diabetic control values, mg/dl), 4 months of EJ and TC treatment resulted
mean levels of enzymes (hexokinase, glucokinase in a moderate reduction in plasma glucose levels
and phosphofructokinase) and substrate (G-6-p) (65.52 and 48.81%). Respective percentage reduc-
values decreased in the diabetic controls. The tion in severe diabetes was considerably less
respective percentage decrease was 51.93, 78.08, (17.72% with EJ and no reduction with TC treat-
20.16 and 60.36% in diabetic controls. Treatment ment). Since the percentage fall in plasma glucose
with EJ (200 mg/kg for 10 days) led to a rise in levels was different in models with varying inten-
percentage of these parameters by 59.77, 58.07, sity of hyperglycemia, it implies that the anti-hy-
43.12 and 48.34% respectively (P B0.05, 0.001, perglycemic effect of these plants is dependent
0.001, 0.001 as compared to diabetic controls). On upon the dose of diabetogenic agent and therefore
the other hand, treatment with TC extracts re- on the degree of b-cell destruction. This is proba-
sulted in an increase in glucokinase, glucose-6- bly indicative of efficacy of the plants only in mild
phosphate and phosphofructokinase values by to moderate degree of diabetes and this fact has
7.92, 2.64 and 4.00 only (statistically insignificant) been hinted earlier (Pabrai and Sehra, 1962).
and with no effect on hexokinase activity. Moreover, it indirectly indicates that part of the
anti-hyperglycemic activity of these plants is
through release of insulin from the pancreas. In
addition, since EJ showed some anti-hyper-
3.6. Sub-acute toxicity studies of EJ and TC glycemic effect even in severely diabetic mice
(plasma glucose \ 400 mg/dl), there is a possibil-
Results of sub-acute toxicity studies of EJ and ity that it also exerts a direct insulinomimetic
TC to establish their safety profiles are shown in effect.
Table 7. At the end of 2 months of the study As reported earlier (Welihinda and
period, no statistically significant differences were Karunanayake, 1986), in the present study also,
seen in the mean WBC and RBC counts, HB, hepatic and skeletal-muscle glycogen content was
HCT, MCV and MCH values as compared to the reduced significantly in diabetic controls as com-
normal. pared to non-diabetic controls. Treatment with EJ
 J.K. Gro6er et al. / Journal of Ethnopharmacology 73 (2000) 461–470
prevented this alteration in glycogen content but
could not normalize it and the content of glyco-
gen remained 47.81 and 30.56% of the non-dia-
betic controls. On the other hand, treatment with
TC failed to exert any statistically significant
changes in glycogen content. This prevention of
depletion of glycogen in the liver and muscle is
possibly due to either stimulation of insulin re-
lease from b cells (Lolitkar and Rao, 1966) or due
to insulinomimetic activity of some component of
the fruit resulting in direct peripheral glucose
uptake or due to a combination of the two.
Anderson and Stowring (1973) showed an in-
crease in renal glycogen content in diabetic ani-
mals. Similar results were obtained in the present
study. In spite of the reduction in glucose levels,
EJ failed to affect renal glycogen content. Thus,
EJ did not affect the mechanisms responsible for
increase in renal glycogen. Brain glycogen levels
were not affected either in diabetic controls or in
the treated groups. These results are in contradic-
tion with results of the study by Prasannan (1973)
who reported a rise in brain glycogen content of
alloxan diabetic rats. This can be due to the use of
a higher dose of alloxan (70 mg/kg) by Prasannan
as compared to the present study (32 mg/kg). The
rise in glucose levels in the study by Prasannan
was 449 mg/dl versus 180 mg/dl in the present
case.
Decreased enzymatic activity of glucokinase,
hexokinase and phosphofructokinase has been re-
ported in diabetic animals resulting in depletion
of liver and muscle glycogen (Hikino et al., 1989).
The present study also had similar results. Treat-
ment with EJ and TC increased the G-6-P content
in the liver, indicating an overall increase in glu-
cose influx; the effect of TC extract was consider-
ably less. The content of phosphofructokinase was
increased significantly in both treatment groups
while the content of glucokinase and hexokinase
was increased only in the EJ treated group. Thus,
EJ seems to have an overall effect in increasing
glucose utilization.
Studies done to assess the safety profile of these
plant extracts showed no adverse effect on hema-
tological parameters including WBC and RBC
count, HB, HCT, MCV and MCH. Thus, these
plant extracts can be presumed to be free from
469
toxicological effects on these parameters at the
doses used in the study.
In conclusion, EJ exerts a dual effect, namely a
combination of mechanism of action of sulfony-
lureas and biguanides. It can best be used for
controlling hyperglycemia in mild to moderate
non-insulin dependent diabetics only. Its onset of
action is late as compared to that seen with
insulin or sulfonylureas clinically and chronic
treatment did not result in tachyphylaxis.
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