J. Appl. Biomed.

5: 97–104, 2007
ISSN 1214-0287

ORIGINAL ARTICLE

Antihyperglycaemic and antiperoxidative effect of Helicteres

igora L. bark extracts in streptozotocin-induced diabetic rats
Ganesan Kumar1, Gani Sharmila Banu2, Arunachalam Ganesan Murugesan1,
Moses Rajasekara Pandian2
1
Manonmaniam Sundaranar University, Sri Paramakalyani Centre for Environmental Sciences, Alwarkurichi,
Tamilnadu, India 627412
2
Centre for Biotechnology, Muthayammal College of Arts and Sciences, Kakkaveri, Rasipuram, Namakkal,
Tamilnadu, India 637408

Received 21st December 2006.

Revised 22nd January 2007.
Published online 9th March 2007.

Summary
The present investigation shows the antihyperglycaemic activity of aqueous extract of bark of
Helicteres isora L. (100, 200 mg/kg b.w./p.o.) in streptozotocin (STZ) induced diabetic rats. Blood
glucose levels, body weight, food and liquid intake were measured on every 5th day over a period
of 14 days. A single injection of STZ at a dose of 60 mg/kg b.w./i.p. elevated the glucose levels
>240mg/dl after 5 days. Administration of H. isora at a dose of 100, 200 mg/kg/p.o. resulted in a
significant (p<0.05) reduction in blood glucose levels. Body weights were significantly (p<0.05)
reduced in STZ-induced diabetic rats when compared to normal rats while the extract significantly
(p<0.05) prevented a decrease in body weight in the H. isora treated animals. The study also
evaluated the antioxidant potential of H. isora in STZ-induced diabetic rats. Decreased levels of
thiobarbituric acid reactive substances (TBARS), increased levels of reduced glutathione (GSH) and
the activities of superoxide dismutase (SOD) and catalase (CAT) resulted in the reduction of free
radical formation in various tissues such as liver, kidney, and brain of the diabetic rats. Tolbutamide
was used as a standard reference drug. The results clearly indicate that the aqueous extract of bark
of H. isora exhibits significant antihyperglycaemic and in vivo antioxidant activity in STZ-
induced diabetic rats and the results were found to be dose dependent.

Keywords: Helicteres isora – streptozotocin – antihyperglycaemic activity – antioxidant potential

INTRODUCTION of death in the world. About 100 million people

around the world have been diagnosed with
Diabetes mellitus is a non-communicable disease, diabetes and by the year 2010, it is projected that
which is considered one of the five leading causes 215 million people will have the disease (Zimmet
1999). Recently, the search for appropriate
hypoglycaemic agents has been focused on plants
Ganesan Kumar, Faculty of Biochemistry, used in traditional medicine, partly because of
Selvamm Arts and Science College, Namakkal leads provided by traditional medicine to natural
(DT)-637 003, Tamilnadu, India products that may be better treatments than
pgsvedhakumar@yahoo.com currently used drugs (Rates 2001).
+91 4286 244606 The bark of Helicteres isora Linn.
(Sterculiaceae) has been used in the indigenous
 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

systems of medicine in India for the treatment of conducted in accordance with the National Institute
diabetes mellitus since time immemorial. The plant of Health Guide (1985).
is a shrub or small tree available in forests
throughout the Central and Western India. The Induction of experimental diabetes
roots and the bark are expectorant, demulcent and Rats were made diabetic by a single intraperitoneal
useful in the treatment of colic, scabies, administration of streptozotocin (60 mg/kg)
gastropathy, diabetes, diarrhoea and dysentery dissolved in 0.1M citrate buffer, pH 4.5 (Siddique
(Kirtikar and Basu 1995). The fruits are astringent, et al. 1987). Forty-eight hours later, blood samples
refrigerant, stomachic, vermifugal, vulnerary and were collected and glucose levels were determined
useful in bowel gripes, flatulence in children to confirm the development of diabetes. Only those
(Chopra et al. 1956) and has an antispasmodic animals which showed hyperglycaemia (blood
effect (Pohocha and Grampurohit 2001). The glucose levels > 240mg/dl) were used in the
aqueous extract of the bark showed a significant experiment (Ewart et al.1975, Cetto et al. 2000).
hypoglycaemic effect (Kumar et al. 2006a) and a
lowering effect on hepatic enzymes (Kumar et al. Experimental design
2006b). There are no available reports on the In the experiment, a total of 30 rats (24 diabetic
antihyperglycaemic potential of H. isora against surviving rats, 6 normal rats) were used. The rats
diabetes. Therefore, we undertook the present were divided into 5 groups of 6 rats each.
study to determine the antihyperglycaemic Group 1: Normal rats.
efficacy of H. isora in STZ-induced diabetic rats. Group 2: Diabetic control rats
Group 3: Diabetic rats given H. isora bark extract (100
mg/kg b.w.) in an aqueous solution daily for 14
days using an intragastric tube.
MATERIALS AND METHODS Group 4: Diabetic rats given H. isora bark extract (200
mg/kg b.w.) in an aqueous solution daily for 14
Chemicals days using an intragastric tube.
All the drugs and biochemicals used in this Group 5: Diabetic rats given tolbutamide (250mg/kg
experiment were purchased from Sigma b.w.) in an aqueous solution daily for 14 days
Chemical Company Inc., St. Louis, USA. The using an intragastric tube (Kumar et al. 2006).
chemicals were of analytical grade. The effect of H. isora on STZ induced
diabetic rats were determined by measuring blood
Collection and processing of plant material glucose levels, food and fluid intake amount and
The bark of Helicteres isora L. was collected from changes in body weights (Ewart et al. 1975,
Solakkadu, Kollimalai, Namakkal District, Tamil Kamtchouing et al. 1998). After 14 days of
Nadu, India and authenticated by Fr. K.M. treatment, all the rats were decapitated after fasting
Matthew, Director, Rapinat Herbarium, St. for 16 h. The animals were dissected and a drop of
Joseph’s College, Tiruchirapalli. Voucher blood from the heart was used for the estimation of
Herbarium specimens have been deposited in the blood glucose. Tissues (brain, liver, and kidney)
Herbarium (collection number 23644, 27406) for were removed and cleared of blood. They were
future reference. immediately transferred to ice-cold containers
The dried bark of H. isora L. was ground to containing 0.9% NaCl and homogenized in 0.1M
fine powder with an auto-mix blender. Then the Tris-HCl buffer (pH 7.4). After centrifugation at 2000
fine powder was suspended in an equal amount of g for 10 min, the clear supernatant was used to
water, stirred intermittently and left overnight. The measure the assay of enzyme activities.
macerated pulp was then filtered through a coarse
sieve and the filtrate was dried at a reduced Biochemical analysis
temperature. This dry mass (yield 185g/kg of Estimation of blood glucose
powdered bark) served as the aqueous extract of H. Body weight and blood glucose levels were
isora L. for experimentation. measured at the beginning of the experiment and
at 5-day intervals. Blood samples were obtained by
Animals tail-vein puncture of the normal and STZ-induced
Male Wistar albino rats (weighing 160–200 g) were diabetic rats on day zero (0), day 5, day 10 and on
procured from the Animal house, Bharathidasan day 15. Blood glucose levels were determined by the
University, Tiruchirapalli under standard O-toluidine method (Sasaki et al. 1972)
environmental conditions (12 h light/dark cycles at
25–28 °C, relative humidity 60–80 %). They were Acute toxicity study
fed with a standard diet (Hindustan Lever, India) Albino rats of 6 animals per group and weighing
and water ad libitum and allowed to acclimatize for 200-220 g were administered a graded dose (100–
14 days before the procedure. All studies were 5000 mg/kg b.w./p.o.) of the aqueous extract of H.
isora. After administration of the H. isora the rats

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 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

were observed for toxic effects for 48 h. The Statistical analysis

toxicological effects were observed in terms of All data were expressed as Mean ± SD of number
mortality expressed as LD50. The number of of experiments (n=6). The statistical significance
animals dying during a period was noted (Ghosh was evaluated by one-way analysis of variance
1984). (ANOVA) using SPSS version 7.5 (SPSS, Cary,
NC, USA) and the individual comparisons were
Determination of in vivo antioxidants obtained by Duncan's Multiple Range Test
TBARS in tissues was estimated by the method of (DMRT). A value of p<0.05 was considered to
Nichans and Samuelson (1968). GSH was indicate a significant difference between groups
determined by the method of Ellman (1959). SOD and (Duncan 1957).
CAT were assayed utilizing the technique of Kakkar et
al. (1978) and Sinha (1972) respectively.

300
b b b
b b b
b
250
Blood glucose level [mg]

c c
c
d
200
e d
e d
d
150

100 a a a a

50

0
Day 0 Day 5 Day 10 Day 15
Treatment (Days)

Normal Diabetic Diabetic + H. isora (100 mg/kg) Diabetic + H.isora (200 mg/kg) Diabetic + Tolbutamide (250 mg/kg)

Fig. 1. Effect of aqueous extract of bark of Helicteres isora L. on blood glucose levels in STZ induced diabetic rats
Values are given as means ± SD of six animals in each group. Values not sharing a common superscript (a, b, c and d) differ
significantly (Duncan’s Multiple Range Test)

RESULTS
Effect of H. isora on body weight, food and liquid
Effect of H. isora on blood glucose levels intake
Blood glucose levels were measured in normal Body weight, food and liquid intake were
and STZ-induced diabetic rats. The results are measured and are summarized in Table 1. The
shown in Fig. 1. There was a significant increase initial body weights were similar in normal and
in blood glucose levels in STZ-induced diabetic diabetic groups, whereas the final body weights
rats (Group 2). Administration of H.isora at a dose of were significantly decreased in the diabetic control
100 mg and 200 mg/kg b.w. and tolbutamide (Group 2) when compared with the normal control
(250mg/kg b.w.) significantly decreased blood (Group 1). At the same time, there was no
glucose levels in STZ-induced rats (Group 3, 4 significant difference in the body weights in
and 5). The results were found to be dose H. isora and Tolbutamide treated diabetic rats
dependent . (Group 3, 4 and 5). Food and fluid intake amount

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 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

were significantly higher in the diabetic group than in Figs. 2 and 3. There was a significant elevation
the normal (Table 1). of TBARS in STZ-diabetic control rats when
compared to normal rats. There was found to be a
Effect of H. isora on the levels of TBARS and GSH significant decrease in the levels of TBARS in
The concentration of TBARS and the GSH in liver, kidney and brain during diabetes in H. isora
tissues in the experimental diabetic rats are shown and Tolbutamide treated animals.

Table 1. Effect of Helicteres isora L. on body weight, food and liquid intake in rats

Body weight (g) Food intake Liquid intake

Treatment (g/rat/day) (ml/rat/day)
Initial Final

Normal rats 179.2 ± 1.2a 190.6 ± 3.5a 11.12 ± 5.3a 16.33 ± 0.21a

Diabetic control 180.4 ± 4.3a 159.1 ± 2.1b 16.88 ± 2.2b 32.14 ± 0.55b

Diabetic + H.isora
(100mg/kg b.w./p.o.) 164.3 ± 5.8c 191.6 ± 4.3c 14.24 ± 1.6c 27.67 ± 0.11c

Diabetic + H.isora
(200mg/kg b.w./p.o.) 177.3 ± 4.1d 194.1 ± 3.2c 12.14 ± 1.8d 22.81 ± 0.12d

Diabetic + tolbutamide
(250mg/kg b.w./p.o.) 178.5 ± 4.1d 192.2 ± 3.2c 12.32 ± 4.4d 21.10 ± 0.18d

Symbols as in Fig. 1

There was a significant decrease in the

concentration of GSH in the STZ-diabetic control DISCUSSION
group when compared with the normal.
Administration of H. isora at the dose of 100 mg, Hyperglycaemia, the primary clinical
200 mg/kg b.w. reduced the levels of GSH in the manifestation in diabetes, is associated with the
liver and brain during diabetes while the kidneys development of certain complications of diabetes
did not show any significant change when (Brownlee et al. 1981). Streptozotocin causes a
compared with the normal group. significant elevation in the level of blood glucose
in rats. But the administration of H. isora at doses
Effect of H. isora on the activity of SOD and of 100 mg, 200 mg/kg b.w. to diabetic rats caused
CAT a significant decrease in the blood glucose. Thus,
The activities of SOD and CAT in experimental H. isora has an antihyperglycaemic activity.
animal tissues are summarized in Figs. 4 and 5. Changes in body weight show that H. isora has a
There was a significant reduction in the activity of significant effect in controlling the loss of body
SOD in liver kidney and brain during diabetes. weight, which is caused during diabetes. The
Administration of H. isora at 100 mg, 200 mg/kg activities were found to be dose dependent.
b.w. and Tolbutamide 250mg/kg b.w. increased the STZ is toxic to pancreatic β-cells and is thus
activity of SOD in liver kidney and brain to near widely used for induction of experimental diabetes
normal. There was a significant reduction in the mellitus in animals, resulting in the production of
activity of CAT in liver kidney and brain during reactive oxygen species (ROS) (Rakieten et al.
diabetes. Administration of H. isora (100 mg, 200 1963). ROS induced oxidative tissue damage plays
mg/kg b.w.) and Tolbutamide (250 mg/kg b.w.) an important role in many clinical disorders such
increased the activity of CAT in liver kidney and as heart disease, diabetes, gout and cancer (Slater
brain to near normal. 1984, Vuillaume 1987, Meneghini 1988).
Oxidative stress can be associated with the

100
 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

TBARS [mM / 100 g tissue]

3
b
b

a
2 c c
a d d
d
d b

1 a c
d d

0
Liver Kidney Brain
Tissues

Normal Diabetic Diabetic + H. isora (100 mg/kg) Diabetic + H.isora (200 mg/kg) Diabetic + Tolbutamide (250 mg/k

Fig. 2. Effect of aqueous extract of bark of Helicteres isora L. on TBARS levels in rat tissues. Symbols as in Fig. 1.

20 a
d
d
a
d d
GSH [mM / 100 g tissue]

16 c

b c

12
b

4 a
b c c c

0
Liver Kidney Brain
Tissues

Normal Diabetic Diabetic + H. isora (100 mg/kg) Diabetic + H.isora (200 mg/kg) Diabetic + Tolbutamide (250 mg/kg)

Fig. 3. Effect of aqueous extract of bark of Helicteres isora L. on GSH levels in rat tissues. Symbols as in Fig. 1.

101
 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

12 a

d
10 d a
SOD [mM / 100 g tissue]

d
d
8
c

6 c

b b d
a d
4
c
b
2

0
Liver Kidney Brain
Tissues
Normal Diabetic Diabetic + H. isora (100 mg/kg) Diabetic + H.isora (200 mg/kg) Diabetic + Tolbutamide (250 mg/kg)

Fig. 4. Effect of aqueous extract of bark of Helicteres isora L. on SOD levels in rat tissues. Symbols as in Fig. 1.

peroxidation of cellular lipids, which is determined homeostasis, quenching free radicals and by
by measurement of TBARS (Nistico et al. 1992). participating in detoxification reactions (Matcovis
The concentration of lipidperoxidation products et al. 1982). A significant decrease in the levels of
may reflect the degree of oxidative stress in GSH has been observed in H. isora-treated rats
diabetes. It has been reported previously (Baynes when compared to STZ-induced diabetic rats. The
1991, Kakkar et al. 1995) that, in the tissues and decrease in liver GSH levels represents an
blood of rats with STZ-induced diabetes, increased utilization due to oxidative stress
malondialdehyde, the production of (Anuradha et al. 1993). SOD scavenges the
lipidperoxidation, is increased. Further, the superoxide ions produced as cellular byproducts.
increased level of TBARS results in increased SOD is a major defense for aerobic cells in
levels of oxygen free radicals, which attack the combating the toxic effects of superoxide radicals
polyunsaturated fatty acids in cell membranes. (McCrod et al. 1976, Mazumder et al. 2005). CAT
STZ can also give rise to oxygen free radicals reduces the hydrogen peroxide produced by a
because of the increased blood glucose level in dismutation reaction and prevents the generation
diabetes (Rakieten et al. 1963). We evaluated of hydroxyl radicals thereby protecting the cellular
TBARS to determine the activity of H. isora in constituents from oxidative damage in
protection from oxidative damage in diabetes. The peroxisomes (Mazumder et al. 2005). The reduced
level of TBARS in the liver, kidney and brain of activities of SOD and CAT in STZ-treated rats
diabetic rats treated with H. isora was significantly result in the accumulation of superoxide radicals
decreased when compared to the STZ-induced and H2O2 respectively, which produce deleterious
diabetic rats. The decreased level of TBARS effects. H. isora supplementation showed
indicates that H. isora can improve the defective normalization of SOD and CAT in liver, kidney
state of diabetes by means of inhibition of lipid and brain. Hence the present investigation exhibits
peroxidation. strong antihyperglycaemic activity. Experiments
GSH is a major endogenous antioxidant which suggest that H. isora also shows antioxidant
counterbalances free radical mediated damage activity.
(Mazumder et al. 2005). It is well known that GSH Thus, H. isora seems to be effective in
is involved in the protection of normal cell reducing oxidative stress and free radical-related
structure and function by maintaining the redox diseases including diabetes.

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 Kumar et al.: Antihyperglycaemic and antiperoxidative effect

20 a

18
d
CAT [mM / 100 g tissue]
16 d
c a d
14 d

12 b
c
10 b

8 d
d
a
c
6 b

0
Liver Kidney Brain
Tissues
Normal Diabetic Diabetic + H. isora (100 mg/kg) Diabetic + H.isora (200 mg/kg) Diabetic + Tolbutamide (250 mg/kg)

Fig. 5. Effect of aqueous extract of bark of Helicteres isora L. on CAT levels in rat tissues. Symbols as in Fig. 1.

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