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Tropical Journal of Pharmaceutical Research
Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
ISSN: 1596-5996 EISSN: 1596-9827
Vol. 8, Num. 5, 2009, pp. 393-398
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Tropical Journal of Pharmaceutical Research, June 2002; 1 (1): 63-22
Tropical Journal of Pharmaceutical Research, Vol. 8, No. 5, October, 2009, pp. 393-398
Research Article
Hypoglycemic
Effects of Clitoria ternatea Linn. (Fabaceae) in Alloxan-induced Diabetes in Rats
P
Daisy1 and M Rajathi2
1Department of Biotechnology, Holy Cross College, Tiruchirappalli-620002, Tamilnadu,
2Department of Biotechnology, MVJ College of Engineering,
Near ITPL, Channasandra, Bangalore-560067. Karnataka, India
*Corresponding author: E-mail: santhanamaryrajathi@yahoo.co.in; Tel: +91-080-28452324; Fax: +91-080- 28452443
Received: 24 February 2009
Revised
accepted: 17 May 2009
Code Number: pr09051
Abstract
Purpose: This study aims
to investigate the therapeutic effects of the aqueous extract of Clitoria
ternatea Linn. Fabaceae leaves and flowers on alloxan-induced diabetes in rats.
Methods: The effect of
orally administered aqueous extracts (400 mg/kg body weight) of Clitoria
ternatea leaves and flowers on serum glucose, glycosylated hemoglobin, and
insulin were examined in control and extract-treated diabetic rats. The glycogen
content of the liver and skeletal muscles of the rats was evaluated while the
activities of the glycolytic enzyme, glucokinase, and the gluconeogenic enzyme,
glucose-6-phosphatase in the liver were assessed. The extracts were
administered over a period of 84 days.
Results: The aqueous
extracts of Clitoria ternatea leaves and flowers significantly (P<0.05)
reduced serum glucose, glycosylated hemoglobin and the activities of
gluconeogenic enzyme, glucose-6-phosphatase, but increased serum insulin, liver
and skeletal muscle glycogen and the activity of the glycolytic enzyme,
glucokinase. For all the biochemical tests performed, the leaf extract-treated
rat showed essentially the same profile as those treated with the flower
extract.
Conclusion: The present
investigation suggests that Clitoria ternatea leaf and flower extracts exhibit
antihyperglycaemic effect in rats with alloxan-induced diabetes mellitus.
Keywords: Alloxan,
Diabetes mellitus, Clitoria ternatea, Blood glucose, Glucose-6-phosphatase,
Glucokinase, Glycogen
INTRODUCTION
Diabetes
mellitus is a syndrome characterized by chronic hyperglycaemia and disturbances
of carbohydrate, fat and protein metabolism associated with absolute or
relative deficiency in insulin secretion and/or action [1]. Insulin therapy and
oral hypoglycaemic agents offer effective glycaemic control, but Insulin
therapy has shortcomings such as ineffectiveness following oral administration,
short shelf life, of the need for constant refrigeration, and fatal
hypoglycaemia, in the event of excess dosage [2]. As a result, there is a need
to search for compounds with effective antidiabetic activity when taken orally
taken orally. The oral hypoglycemic agents that are capable of reducing blood
sugar level belong to two chemical classes - sulfonylureas and biguanides [3].
However, the use of oral antidiabetics is limited due to their adverse side
effects including hematological, cutaneous and gastrointestinal reactions,
hypoglycaemic coma and disturbances of liver and kidney functions. In addition,
they are not suitable for use during pregnancy [4].
Plants
are well known in traditional herbal medicine for their hypoglycaemic
activities, and available literature indicate that there are more than 800
plant species showing hypoglycaemic activity [5]. The World Health Organization
has recommended the evaluation of the effectiveness of plants in conditions
where safe orthodox drugs are scarce [6]. Studies have shown that
phytochemicals isolated from plant sources have been used for the prevention
and treatment of cancer, heart disease, diabetes mellitus, and high blood
pressure [7].
Clitoria
ternatea Linn, belonging to the
family Fabaceae, is a perennial twining herb found in India, China, Philippines and Madagascar. Since the flowers of the plant resemble a conch shell, it
is commonly called Shankpushpi in the Sanskrit language of India where it is reported to be a good Medhya (brain tonic) drug and, therefore, mainly used in
the treatment of Masasika roga (mental illness). It is also useful in the
treatment of severe fever, bronchitis and asthma[8]. It has been used too as
an antidote for snake bite and scorpion sting [9]. The root has been used
traditionally to induce abortion and its paste for curing abdominal swellings,
sore throat, mucous disorders and fever [10]. A preliminary study of the fresh
flowers of Clitoria ternatea showed hypoglycaemic and hypolipidaemic
effects [11]. The present study was undertaken to evaluate the effectiveness of
the leaf and flower extracts of Clitoria ternatea as antihyperglycaemic
agents in alloxan-induced diabetic rats.
EXPERIMENTAL
Plant
material
Alloxan
monohydrate was obtained from Sigma Chemical Company, St. Louis, Mo, USA. All the other chemicals used were of analytical grade and were acquired from
commercial sources. The leaves and flowers of Clitoria ternatea were
collected from Thirumayam, Pudukkottai District, Tamilnadu, India. They were carefully identified and authenticated by Dr. Annie Xavier, Professor of Botany,
Holy Cross College, Tiruchirappali 620 002, India. The shade-dried leaves
and flowers were powdered and boiled in water (100 g/L distilled water). The
decoction was filtered through nitrocellulose filter and the filtrates were
evaporated to dryness under vacuum at 50 °C in a rotary
evaporator. The dried residue was stored in airtight containers pending
further use.
Animal
studies
Male
adult Wistar strain albino rats (100-150 g) were used for the studies. Ethical
approval was obtained from the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA, approval no.585/05/A/CPCSEA),
the institutional ethical review committee. The animals were obtained from
Tamilnadu Veterinary and Animal Science University, Chennai, India, and fed on a standard feed (Sai Durga Feeds and Foods, Bangalore, India) and water ad
libitum. The animals described as fasted were deprived of food for 16 h
but were allowed free access to water. After randomization into groups, the
rats were acclimatised to the laboratory conditions of temperature and
photoperiod for a period of 1-2 weeks prior to commencement of the experiment.
Diabetes mellitus was induced in a batch of normoglycaemic albino rats starved
for 16 h by injecting intraperitoneally 150 mg/kg body weight of alloxan
monohydrate dissolved in physiological saline. Since alloxan is capable of
producing fatal hypoglycaemia as a result of massive pancreatic insulin
release, rats were treated with 20 % glucose solution intraperitoneally after 6
h. For the next 24 h, the rats were kept on 5 % glucose solution in their
cages to prevent hypoglycaemia. Seven days after alloxan injection, rats with
blood glucose > 300 mg/dl were considered as diabetic and included in the
study. They were divided into different groups, with five rats in each group.
Aqueous extracts of the leaf (CTL) and flower (CTF) in doses ranging from 50
mg/kg body weight to 500 mg/kg body weight, at incremental doses of 50 mg/kg
body weight, were administered by oral intubation to the animals, and blood
glucose was estimated 5 h after. The lowest dose that brought about the maximum
antihyperglycaemic effect for each extract (400 mg/kg body weight for both CTL
and CTF) was selected for further studies.
In
the further studies that followed, rats in which diabetes was induced as
described above, were used. They were divided into three groups with ten rats
in each group. One group received only distilled water while two other groups
received 400 mg/kg of CTL and CTF, respectively. A fourth group (control)
consisted of normal rats that received distilled water only.
The
treatments were continued daily for 84 days while normal control and diabetic
control groups were given distilled water everyday for 84 days. All the
treatments were by oral intubation.
At
the end of the experiment, the animals were sacrificed by cervical dislocation.
Blood was collected from the heart using a syringe, transferred to sodium
fluoride bottles bottles, allowed to
clot and the serum was separated by centrifugation at 3500 r.p.m for 10 min.
The serum was assayed either immediately or stored at 20 °C pending assay. Commercial diagnostic kits were used to assay serum
glucose (using a kit supplied by Reddys Laboratories, Hyderabad, India), glycosylated hemoglobin (using a kit obtained from Bio Systems, Costa Brava, Spain), and insulin (using a radioimmunoassay kit from Diasorin, Italy). Tissues from the liver and
skeletal muscle were collected. The glycogen contents of both the liver and
skeletal muscle were estimated by the method described by Plummer[12].
Glucokinase and glucose -6-phosphatase were assayed by the method of Brandstrup
et al [13]. and Baginsky et al [14], respectively.
Data
analysis
The
group data were statistically evaluated using the Statistical Package for
Social Sciences (SPSS) version 7.5. Hypothesis testing was by one-way analysis
of variance (ANOVA) followed by least significant difference test. P-values of
less than 0.05 were considered statistically significant. All the results were
expressed as mean ± SD (n = 10).
RESULTS
As
shown in Table 1, a significant increase in blood glucose and glycosylated
hemoglobin and a significant decrease in serum insulin were observed in
diabetic control rats when compared to normal control rats (P < 0.05).
Administration of CTL and CTF to diabetic rats significantly decreased the
levels of blood glucose and glycosylated hemoglobin and at the same time
increased serum insulin
Table
1: Effect of treatment with leaf
extract (CTL, 400 mg/kg) and flower extract (CTF, 400 mg/kg) for 84 days on
serum parameters of control and diabetic rats. (Values are mean ± SD, n = 10).
Parameter |
Normal rats (Control) |
Diabetic rats (Control) |
Diabetic rats treated
with CTL |
Diabetic rats treated
with CTF |
Glucose (mg/dl) |
74.8±3.1 |
361.0±10.4 |
102.4±4.8 * |
107.6±4.9 |
Glycosylated hemoglobin
(%) |
2.44±0.29 |
4.86±0.68 |
2.81±0.76 * |
2.92±0.38 |
Insulin (mU/ml) |
38.6±4.5 |
8.2±1.3 |
30.6±2.1 * |
29.3±2.2 * |
*
Statistically significant when compared with diabetic controls (P< 0.05).
Table
2: Effect of treatment with CTL (400
mg/kg) and CTF (400 mg/kg) for 84 days on liver and skeletal muscle glycogen,
and on glucokinase and glucose-6-phosphatase activities of control and diabetic
rats. (Values are mean ± SD, n = 10)
Parameter |
Normal rats (Control) |
Diabetic rats (Control) |
Diabetic rats treated
with CTL |
Diabetic rats treated
with CTF |
Liver glycogen (mg/g) |
49.0±1.3 |
9.0±2.5 |
35.0±1.0 * |
34.0±1.9 * |
Skeletal muscle
glycogen (mg/g) |
9.8±1.8 |
1.8±0.6 |
6.2±0.4 * |
5.8±0.5 * |
Glucokinase (m mol of glucose-6-Po4 formed /min/mg protein ) |
207.5±6.4 |
115.4±8.9 |
163.7±4.2 * |
161.0±3.2 * |
Glucose-6-phosphatase (m mol of Pi liberated/min/mg protein) |
0.150±0.021 |
0.252±0.028 |
0.199±0.006 * |
0.201±0.036 * |
* Statistically
significant when compared with diabetic controls (P< 0.05).
to
near normal control levels. Table 2 shows the content of liver glycogen and
skeletal muscle glycogen in control and diabetic rats. The glycogen contents
of rat liver and skeletal muscle decreased in diabetic animals when compared to
normal control animals but these levels increased to near normal after CTL and
CTF treatment. As Table 2 also shows, the activity of glucokinase in the liver
of diabetic control animals decreased while that of glucose-6-phosphatase
increased, compared to normal control (P < 0.05). However, oral
administration of CTL and CTF for 84 days to the diabetic rats resulted in an
increase in glucokinase activity and a decrease in glucose-6-phosphatase.
However, there was no significant difference (P < 0.05) between the
biochemical data obtained for the leaf and flower extract-treated rats.
DISCUSSION
Diabetes
mellitus of long duration is associated with several complications such as
atherosclerosis, myocardial infarction, nephropathy, etc [15]. These
complications are usually related to chronically elevated blood glucose level.
Alloxan causes a massive reduction in insulin release by the destruction of b-cells of the islets of Langerhans, thereby inducing hyperglycaemia[16].
Daily administration of the aqueous extracts of Clitoria ternatea (CTL
and CTF) for 84 days resulted in decrease in the blood glucose levels of
alloxan-induced diabetic rats. The possible hypoglycaemic mechanism of CTL and
CTF may be through potentiation of pancreatic secretion of insulin from b-cell of islets or due to enhanced transport of blood glucose to the
peripheral tissues.
Glycosylated
hemoglobin is produced by glycosylation of hemoglobin. Glycosylated
hemogolobin is formed progressively and irreversibly over a period of time and
is stable over the life span of the red blood cells. It is unaffected by diet,
insulin or exercise, even on the day of test. Therefore, glycosylated
hemoglobin can be used as an excellent marker of overall glycaemic control.
Since it is formed slowly and does not dissociate easily, it reflects the real
blood glucose level[17,18]. In this study, the diabetic rats had elevated
levels of glycosylated hemoglobin, and therefore, the significant decrease in
the level of glycosylated hemoglobin in alloxan-induced diabetic rats following
CTL and CTF therapy indicates that the overall blood glucose level was
controlled, probably due to improvement in insulin secretion. It noteworthy
that the serum insulin level in diabetic animals treated with CTL and CTF also
increased when compared to the diabetic control animals. Thus, it seems that
CTL and CTF stimulated increased insulin secretion in alloxan-induced diabetic
rats. In this respect, the mode of action of these extracts is similar to
those reported for extracts of Gymnema sylvestre[19], Momordica
charantia[20] and Enicostemma littorale[21].
The
glycogen content of skeletal muscles and liver markedly decreases in diabetes[22].
The decrease in glycogen content of liver and skeletal muscle observed in
diabetic rats is probably due to lack of insulin in the diabetic state.
Prevention of glycogen depletion in the liver and muscles, following the
administration of the extracts, could therefore have been achieved by
stimulation of insulin release[23]. Administration of CTL and CTF to the
diabetic animals increased the activity of glucokinase in liver. The extract-induced
decrease in the concentration of blood glucose in alloxan-treated rats may be
the result of improved glucose uptake. Similar observations have been made in
respect of the extracts of Catharanthus roseus[24], Tinospora
cordifolia[25] and Gymnema sylvestre [26]. The
activity of the gluconeogenic enzyme, glucose-6-phosphatase, is usually
enhanced during diabetes[27]. Following extract administration, blood glucose
level fell while liver glycogen content rose. This may be due to the
mobilization of blood glucose into the liver glycogen reserve. In this context,
a number of plants have been reported to decreased the activity of
glucose-6-phosphatase in the liver of diabetic rats [28].
CONCLUSION
The
results of this investigation indicate that the leaf and flower extracts of Clitoria
ternatea have a hypoglycaemic effect on alloxan-induced diabetes in rats.
One possible mechanism of action is increased insulin secretion and enhancement
of the glycogenesis process. The extracts were effective in regulating the
biochemical indices associated with diabetes mellitus such as glycogen content
and the activities of glucokinase and glucose-6-phosphatase. Further studies
are in progress to isolate the active principle(s) of the extracts as well as
to elucidate their exact mechanism(s) of action.
ACKNOWLEDGEMENT
Financial
assistance from the University Grants Commission, New Delhi, to carry out this
work is acknowledged.
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© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
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