Daisy & Rajathi

Tropical Journal of Pharmaceutical Research, October 2009; 8 (5): 393-398

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Research Article

Hypoglycemic Effects of Clitoria ternatea Linn.

(Fabaceae) in Alloxan-induced Diabetes in Rats

P Daisy1 and M Rajathi2

1
Department of Biotechnology, Holy Cross College, Tiruchirappalli-620002, Tamilnadu,
2
Department of Biotechnology, MVJ College of Engineering, Near ITPL, Channasandra, Bangalore-560067.
Karnataka, India

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

Received: 24 February 2009 Revised accepted: 17 May 2009

*Corresponding author: E-mail: santhanamaryrajathi@yahoo.co.in; Tel: +91-080-28452324; Fax: +91-080-

28452443

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 Daisy & Rajathi

INTRODUCTION mainly used in the treatment of “Masasika”

roga (mental illness). It is also useful in the
Diabetes mellitus is a syndrome treatment of severe fever, bronchitis and
characterized by chronic hyperglycaemia and asthma[8]. It has been used too as an
disturbances of carbohydrate, fat and protein antidote for snake bite and scorpion sting [9].
metabolism associated with absolute or The root has been used traditionally to induce
relative deficiency in insulin secretion and/or abortion and its paste for curing abdominal
action [1]. Insulin therapy and oral swellings, sore throat, mucous disorders and
hypoglycaemic agents offer effective fever [10]. A preliminary study of the fresh
glycaemic control, but Insulin therapy has flowers of Clitoria ternatea showed
shortcomings such as ineffectiveness hypoglycaemic and hypolipidaemic effects
following oral administration, short shelf life, [11]. The present study was undertaken to
of the need for constant refrigeration, and evaluate the effectiveness of the leaf and
fatal hypoglycaemia, in the event of excess flower extracts of Clitoria ternatea as
dosage [2]. As a result, there is a need to antihyperglycaemic agents in alloxan-induced
search for compounds with effective diabetic rats.
antidiabetic activity when taken orally taken
orally. The oral hypoglycemic agents that are EXPERIMENTAL
capable of reducing blood sugar level belong
to two chemical classes - sulfonylureas and Plant material
biguanides [3]. However, the use of oral
antidiabetics is limited due to their adverse Alloxan monohydrate was obtained from
side effects including hematological, Sigma Chemical Company, St. Louis, Mo,
cutaneous and gastrointestinal reactions, USA. All the other chemicals used were of
hypoglycaemic coma and disturbances of analytical grade and were acquired from
liver and kidney functions. In addition, they commercial sources. The leaves and flowers
are not suitable for use during pregnancy [4]. of Clitoria ternatea were collected from
Thirumayam, Pudukkottai District, Tamilnadu,
Plants are well known in traditional herbal India. They were carefully identified and
medicine for their hypoglycaemic activities, authenticated by Dr. Annie Xavier, Professor
and available literature indicate that there are of Botany, Holy Cross College, Tiruchirappali
more than 800 plant species showing – 620 002, India. The shade-dried leaves
hypoglycaemic activity [5]. The World Health and flowers were powdered and boiled in
Organization has recommended the water (100 g/L distilled water). The decoction
evaluation of the effectiveness of plants in was filtered through nitrocellulose filter and
conditions where safe orthodox drugs are the filtrates were evaporated to dryness
scarce [6]. Studies have shown that under vacuum at 50 °C in a rotary
phytochemicals isolated from plant sources evaporator. The dried residue was stored in
have been used for the prevention and airtight containers pending further use.
treatment of cancer, heart disease, diabetes
mellitus, and high blood pressure [7]. Animal studies

Clitoria ternatea Linn, belonging to the family Male adult Wistar strain albino rats (100-150
Fabaceae, is a perennial twining herb found g) were used for the studies. Ethical approval
in India, China, Philippines and Madagascar. was obtained from the Committee for the
Since the flowers of the plant resemble a Purpose of Control and Supervision of
conch shell, it is commonly called Experiments on Animals (CPCSEA, approval
“Shankpushpi” in the Sanskrit language of no.585/05/A/CPCSEA), the institutional
India where it is reported to be a good ethical review committee. The animals were
“Medhya” (brain tonic) drug and, therefore, obtained from Tamilnadu Veterinary and

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 Daisy & Rajathi

Animal Science University, Chennai, India, groups were given distilled water everyday
and fed on a standard feed (Sai Durga Feeds for 84 days. All the treatments were by oral
and Foods, Bangalore, India) and water ad intubation.
libitum. The animals described as fasted
were deprived of food for 16 h but were At the end of the experiment, the animals
allowed free access to water. After were sacrificed by cervical dislocation. Blood
randomization into groups, the rats were was collected from the heart using a syringe,
acclimatised to the laboratory conditions of transferred to sodium fluoride bottles bottles,
temperature and photoperiod for a period of allowed to clot and the serum was separated
1-2 weeks prior to commencement of the by centrifugation at 3500 r.p.m for 10 min.
experiment. Diabetes mellitus was induced The serum was assayed either immediately
in a batch of normoglycaemic albino rats or stored at – 20 °C pending assay.
starved for 16 h by injecting intraperitoneally Commercial diagnostic kits were used to
150 mg/kg body weight of alloxan assay serum glucose (using a kit supplied by
monohydrate dissolved in physiological Reddy’s Laboratories, Hyderabad, India),
saline. Since alloxan is capable of producing glycosylated hemoglobin (using a kit obtained
fatal hypoglycaemia as a result of massive from Bio Systems, Costa Brava, Spain), and
pancreatic insulin release, rats were treated insulin (using a radioimmunoassay kit from
with 20 % glucose solution intraperitoneally Diasorin, Italy). Tissues from the liver and
after 6 h. For the next 24 h, the rats were skeletal muscle were collected. The
kept on 5 % glucose solution in their cages to glycogen contents of both the liver and
prevent hypoglycaemia. Seven days after skeletal muscle were estimated by the
alloxan injection, rats with blood glucose > method described by Plummer[12].
300 mg/dl were considered as diabetic and Glucokinase and glucose -6-phosphatase
included in the study. They were divided into were assayed by the method of Brandstrup et
different groups, with five rats in each group. al [13]. and Baginsky et al [14], respectively.
Aqueous extracts of the leaf (CTL) and flower
(CTF) in doses ranging from 50 mg/kg body Data analysis
weight to 500 mg/kg body weight, at
incremental doses of 50 mg/kg body weight, The group data were statistically evaluated
were administered by oral intubation to the using the Statistical Package for Social
animals, and blood glucose was estimated 5 Sciences (SPSS) version 7.5. Hypothesis
h after. The lowest dose that brought about testing was by one-way analysis of variance
the maximum antihyperglycaemic effect for (ANOVA) followed by least significant
each extract (400 mg/kg body weight for both difference test. P-values of less than 0.05
CTL and CTF) was selected for further were considered statistically significant. All
studies. the results were expressed as mean ± SD (n
= 10).
In the further studies that followed, rats in
which diabetes was induced as described
above, were used. They were divided into
RESULTS
three groups with ten rats in each group. One
As shown in Table 1, a significant increase in
group received only distilled water while two
blood glucose and glycosylated hemoglobin
other groups received 400 mg/kg of CTL and
and a significant decrease in serum insulin
CTF, respectively. A fourth group (control)
were observed in diabetic control rats when
consisted of normal rats that received distilled
compared to normal control rats (P < 0.05).
water only.
Administration of CTL and CTF to diabetic
rats significantly decreased the levels of
The treatments were continued daily for 84
blood glucose and glycosylated hemoglobin
days while normal control and diabetic control
and at the same time increased serum insulin

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 Daisy & Rajathi

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).

Normal rats Diabetic rats Diabetic rats Diabetic rats

Parameter
(Control) (Control) treated with CTL 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 (µU/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)

Normal rats Diabetic rats Diabetic rats Diabetic rats

Parameter (Control) (Control) treated with CTL 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 9.8±1.8 1.8±0.6 6.2±0.4 * 5.8±0.5 *
(mg/g)
Glucokinase (µ mol of 207.5±6.4 115.4±8.9 163.7±4.2 * 161.0±3.2 *
4
glucose-6-Po formed /min/mg
protein )
Glucose-6-phosphatase (µ 0.150±0.021 0.252±0.028 0.199±0.006 * 0.201±0.036 *
mol of Pi liberated/min/mg
protein)

* Statistically significant when compared with diabetic controls (P< 0.05).

to near normal control levels. Table 2 shows DISCUSSION

the content of liver glycogen and skeletal
muscle glycogen in control and diabetic rats. Diabetes mellitus of long duration is
The glycogen contents of rat liver and associated with several complications such
skeletal muscle decreased in diabetic as atherosclerosis, myocardial infarction,
animals when compared to normal control nephropathy, etc [15]. These complications
animals but these levels increased to near are usually related to chronically elevated
normal after CTL and CTF treatment. As blood glucose level. Alloxan causes a
Table 2 also shows, the activity of massive reduction in insulin release by the
glucokinase in the liver of diabetic control destruction of β-cells of the islets of
animals decreased while that of glucose-6- Langerhans, thereby inducing
phosphatase increased, compared to normal hyperglycaemia[16]. Daily administration of
control (P < 0.05). However, oral the aqueous extracts of Clitoria ternatea (CTL
administration of CTL and CTF for 84 days to and CTF) for 84 days resulted in decrease in
the diabetic rats resulted in an increase in the blood glucose levels of alloxan-induced
glucokinase activity and a decrease in diabetic rats. The possible hypoglycaemic
glucose-6-phosphatase. However, there was mechanism of CTL and CTF may be through
no significant difference (P < 0.05) between potentiation of pancreatic secretion of insulin
the biochemical data obtained for the leaf and from β-cell of islets or due to enhanced
flower extract-treated rats.

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 Daisy & Rajathi

transport of blood glucose to the peripheral the extracts of Catharanthus roseus[24],

tissues. Tinospora cordifolia[25] and Gymnema
sylvestre [26]. The activity of the
Glycosylated hemoglobin is produced by gluconeogenic enzyme, glucose-6-
glycosylation of hemoglobin. Glycosylated phosphatase, is usually enhanced during
hemogolobin is formed progressively and diabetes[27]. Following extract
irreversibly over a period of time and is stable administration, blood glucose level fell while
over the life span of the red blood cells. It is liver glycogen content rose. This may be due
unaffected by diet, insulin or exercise, even to the mobilization of blood glucose into the
on the day of test. Therefore, glycosylated liver glycogen reserve. In this context, a
hemoglobin can be used as an excellent number of plants have been reported to
marker of overall glycaemic control. Since it decreased the activity of glucose-6-
is formed slowly and does not dissociate phosphatase in the liver of diabetic rats [28].
easily, it reflects the real blood glucose
level[17,18]. In this study, the diabetic rats CONCLUSION
had elevated levels of glycosylated
hemoglobin, and therefore, the significant The results of this investigation indicate that
decrease in the level of glycosylated the leaf and flower extracts of Clitoria
hemoglobin in alloxan-induced diabetic rats ternatea have a hypoglycaemic effect on
following CTL and CTF therapy indicates that alloxan-induced diabetes in rats. One
the overall blood glucose level was possible mechanism of action is increased
controlled, probably due to improvement in insulin secretion and enhancement of the
insulin secretion. It noteworthy that the serum glycogenesis process. The extracts were
insulin level in diabetic animals treated with effective in regulating the biochemical indices
CTL and CTF also increased when compared associated with diabetes mellitus such as
to the diabetic control animals. Thus, it glycogen content and the activities of
seems that CTL and CTF stimulated glucokinase and glucose-6-phosphatase.
increased insulin secretion in alloxan-induced Further studies are in progress to isolate the
diabetic rats. In this respect, the mode of active principle(s) of the extracts as well as to
action of these extracts is similar to those elucidate their exact mechanism(s) of action.
reported for extracts of Gymnema
sylvestre[19], Momordica charantia[20] and
Enicostemma littorale[21]. ACKNOWLEDGEMENT

The glycogen content of skeletal muscles and Financial assistance from the University
liver markedly decreases in diabetes[22]. The Grants Commission, New Delhi, to carry out
decrease in glycogen content of liver and this work is acknowledged.
skeletal muscle observed in diabetic rats is
probably due to lack of insulin in the diabetic REFERENCES
state. Prevention of glycogen depletion in the
1 Schoenfelder T, Cirimbelli TM, Citadini-Zanette V.
liver and muscles, following the Acute effect of Trema micrantha on serum
administration of the extracts, could therefore glucose levels in normal and diabetic rats. J
have been achieved by stimulation of insulin Ethnopharmacol 2006; 107: 456-459.
2 Anuradha K, Hota D, Pandhi P. Investigation of
release[23]. Administration of CTL and CTF
central mechanism of insulin-induced
to the diabetic animals increased the activity hypoglycemic convulsions in mice. Indian J
of glucokinase in liver. The extract-induced Exp Biol 2001; 39: 500-502.
decrease in the concentration of blood 3 Trejo-Gonzalez A, Gabriel-Ortiz G, Puebla-Perez
AM, Huizar-Contreras MD, Mungui-
glucose in alloxan-treated rats may be the Mazariegos MR, Mella-Arreguin S, Calva E. A
result of improved glucose uptake. Similar purified extract from prickly pear cactus
observations have been made in respect of (Opuntia fulginosa) controls experimentally

Trop J Pharm Res, October 2009; 8 (5): 397

 Daisy & Rajathi

induced diabetes in rats. J Ethnopharmacol 18 Bunn HF. Evaluation of glycosylated hemoglobin in

1996; 55: 27-33. diabetic patients. Diabetes 1981; 30: 613-617.
4 Alarcon-Aguilara FJ, Jimenez-Estrada M, Reyes- 19 Shanmugasundaram KR, Paneerselvam C,
Chilpa R, Roman-Ramos R. Hypoglycemic Samudram P, Shanmugasundaram ERB. The
effect of extracts and fractions from Psacalium insulinotropic activity of Gymnema sylvestre,
decompositum in healthy and alloxan diabetic an Indian medical herb used in controlling
mice. J Ethnopharmacol 2000; 72: 21-27. diabetes mellitus. Pharmacol Res
5 Rajagopal K, Sasikala K. Antihyperglycaemic and Communications 1981; 13: 475-486.
antihyperlipidaemic effects of Nymphaea 20 Cakici I, Hurmoglu C, Tunctan B, Abacioglu N, Kanzik
stellata in alloxan-induced diabetic rats. I, Sener B. Hypoglycemic effect of Momordica
Singapore Med J 2008; 49: 137-141. charantia extracts in normoglycemic or
6 World Health Organisation. Second Report of the cyprohepatidine-induced hyperglycemic mice.
WHO Expert Committee on Diabetes Mellitus. J Ethnopharmacol 1994; 44: 117-121
Technical Report Series 1980; 646: 66. 21 Maroo J, Vasu VT, Aalinkeel R, Gupta S. Glucose
7 Waltner-Law ME, Wang XL, Law BK. lowering effect of aqueous extract of
Epigallocatechin gallate, a constituent of green Enicostemma littorale Blume in diabetes: a
tea, represses hepatic glucose production. J possible mechanism of action. J
Biol Chem 2002; 277: 34933-34940. Ethnopharmacol 2002; 81: 317-320.
8 Asolkar LV, Kakkar KK, Chakre OJ. Supplement to 22 Vats V, Yadav SP, Grover JK. Effect of Trigonella
glossary of Indian Medicinal Plants with active foenum graecum on glycogen content of
principles, Second edition, Part I (A-K). tissues
Publications and Information Directorate and the key enzymes of carbohydrate
(CSIR), Dr KS Krishnan Marg, New Delhi metabolism. J Ethnopharmacol 2003; 85: 237-
1992: p 217. 242.
9 Chopra RN, Chopra IC, Handa KL, Kapur LD. 23 Chakrabarti S, Biswas TK, Rokeya B, Ali L,
Indigenous drugs of India. Academic Mosihuzzaman M, Nahar N, Khan AKA,
Publishers, Calcutta, India, 1958; p 476. Mukherjee B. Advanced studies on the
10 Devi BP, Boominathan R, Mandal SC. Anti- hypoglycemic effect of Caesalpinia bonducella
inflammatory, analgesic and antipyretic in type 1 and type 2 diabetes in Long Evans
properties of Clitoria ternatea root. Fitoterapia rats. J Ethnopharmacol 2003; 84: 41- 44.
2003; 74: 345-349. 24 Singh SN, Vats P, Suri S, Shyam R, Kumria MML,
11 Rajathi M, Daisy P. Effect of plant extracts on the Ranganathan S, Sridharan K. Effect of an
blood glucose and cholesterol level of alloxan antidiabetic extract of Catharanthus roseus on
diabetic rabbits. Indian J Comp Anim Physiol enzymic activities in streptozotocin-induced
2000; 18: 14-17. diabetic rats. J Ethnopharmacol 2001; 76:
12 Plummer TD. An Introduction to Practical 269-277.
Biochemistry. Tata McGraw Hill Company UK, 25 Prince PSM, Menon VP. Hypoglycemic and other
Whithington, 1987; pp 182-184 related actions of Tinospora cordifolia roots in
13 Brandstrup N, Kirk JE, Bruni C. The hexokinase and alloxan-induced diabetic rats. J
phosphoglucoisomerase activities of aortic and Ethnopharmacol 2000; 70: 9-15.
pulmonary artery tissue in individuals of 26 Shanmugasundaram KR, Paneerselvam C,
various ages. J Gerontol 1957; 12: 166-170. Samudram P, Shanmugasundaram ERB.
14 Baginsky ES, Foa PP, Zak B. Methods of Enzymatic Enzyme changes and glucose utilization in
Analysis, vol 2, 2nd edition, Bergmeyer HU, diabetic rabbits: the effect of Gymnema
Gawehn K, Eds, Academic Press, New sylvestre. J Ethnopharmacol 1983; 7: 205-
York,1992; pp 876-880.. 234.
15 Pushparaj PN, Low HK, Manikandan J, Tan BKH, 27 Babu PS, Ignacimuthu S, Prince PSM. Restoration
Tan CH. Antidiabetic effects of Cichorium of altered carbohydrate and lipid metabolism
intybus in streptozotocin induced diabetic rats. by hyponidd, a herbomineral formulation in
J Ethnopharmacol 2007; 111: 430-434. streptozotocin-induced diabetic rats. Asian J
16 Grover JK, Vats V, Rathi SS. Antihyperglycemic Biochem 2008; 3: 90-98.
effect of Eugenia jambolana and Tinospora 28 Sachdewa A, Khemani LD. Effect of Hibiscus rosa
cordifolia in experimental diabetes and their sinensis L ethanol flower extract on blood
effects on key metabolic enzymes involved in glucose and lipid profile in streptozotocin-
carbohydrate metabolism. J Ethnopharmacol induced diabetes in rats. J Ethnopharmacol
2000; 73: 461-470. 2003; 89: 61-66.
17 Bunn HF, Gabbay KH, Gallop PM. The
glycosylation of hemoglobin: relevance to
diabetes mellitus. Science 1978; 200: 21-27.

Trop J Pharm Res, October 2009; 8 (5): 398