Comparative Hypoglycemic Effects of Cassia Glauca Lam. in Streptozotocin- Induced Diabetic Rats
Abstract
The aim of this study was to evaluate comparative hypoglycemic activity of aqueous extract of Cassia glauca leaf and bark. The various parameters studied included effect on fasting blood glucose levels of normoglycemic rats, oral glucose tolerance test and hypoglycemic activity in streptozotocin induced diabetic rats. On oral administration , aqueous extract showed statistically significant (P < 0.001) effect by reducing the effect of external glucose load in diabetic rats. In chronic model of treatment, the difference between the experimental (Cassia glauca bark extract) rats in lowering the fasting plasma glucose levels was statistically significant (P < 0.001) as compared in diabetic rats on day 21. These findings suggest the significant hypoglycemic potential of the Cassia glauca bark extracts in ameliorating the diabetic conditions in diabetic rats. No significant effects were found in the normal rats.
Source of Support: The authors sincerely thank Dr. F. V. Manvi, Principal, KLES’s College of Pharmacy for providing the necessary facilities for this work.
Introduction
Diabetes is a serious illness with multiple complications and premature mortality, accounting for at least 10% of total health care expenditure in many countries (King et al., 1998) and is likely to remain a significant threat to public health in the years to come (Marx, 2002).The prevalence of diabetes for all age-groups worldwide is projected to rise from 171 million in 2000 to 366 million in 2030 (Amos et al., 1997). Despite the considerable progress in the understanding and management of diabetes, the disease and the complications related to it continue to increase (Tiwari and Madhusudana, 2002). The ethnopharmacological use of herbal remedies for the treatment of diabetes mellitus is an area of study ripe with potential as a starting point in the development of alternative inexpensive therapies for treating the disease (Marles and Farnsworth, 1995; Eddouks et al., 2002).
Cassia glauca Lam is a glabrous tree belonging to family Caesalpiniacea, found throughout India, tropical Asia and Australia. The leaves are long linear, acute, curved in shape. The flower is yellow in colour and shorter than the leaves (Kirtikar and Basu, 1935). Phytochemical study of Cassia glauca stem has indicated the presence of chrysophenol, physcion, stearic acid, -sitosterol and -D glucoside (Hemlata and Kalidhar, 1994). In folk medicine bark and leaves of Cassia glauca are used for the treatment of diabetes and gonorrhea. The leaves are used to treat blennorrhagia (Kirtikar and Basu, 1935). Here we report hypoglycemic activity of the aqueous extract from leaf and bark of Cassia glauca, with a view to provide scientific evidence of modern lines.
Materials And Methods
Collection of plant material
The leaves and bark of Cassia glauca was collected during November 2007 from the Western ghats, Karnataka, India. The plant was identified by Dr. Harsha Hegde, Research Officer, RMRC, ICMR, Belgaum, Karnataka, India. A voucher specimen (SA-01) has been kept in herbarium of Department of Pharmacognosy, KLES College of Pharmacy, Belgaum, Karnataka, India.
Preparation of the test samples
Cassia glauca leaves and bark was air dried in the shade and cut into small pieces. Hundred grams each of leaves and bark was extracted with 1000 ml of water by the method of hot extraction at 60ºC for 6 h with occasional shaking and the extracts were filtered and filtrate were evaporated to dryness under vacuum to yield dry residue. Animals in the control group received only the vehicle (Normal saline 5 ml/kg, b.w.) (Jain, 1968; Latha and Pari, 2004; Aslan et al. 2007).
Animals
Wistar rats (180-200 g) were obtained from animal house, Department of Livestock Production, Government Veterinary College, Hebbal, Bangalore, India. Rats were kept in groups of six in plastic cages at controlled temperature (25°C) and humidity (about 40%) with a 12 h light cycle beginning at 7 am. Rats were provided with standard diet and tap water ad libitum. All experiments were carried out between 8 and 12 am at an ambient temperature of 23-25°C. The study was approved by Institutional Animal Ethical Committee of Jawaharlal Nehru Medical College, Belgaum, India (Resolution No. 1/16/2007).
Acute toxicity studies
The acute oral toxicity study was carried out as per the guidelines set by OECD. The animals (n=3 in each group) were observed continuously for 2 h under the following profiles:
(i) Behavioral profile. Alertness, restlessness, irritability and fearfulness.
(ii) Neurological profile. Spontaneous activity, reactivity, touch response and pain response.
(iii) Autonomic profile. Defecation and urination.
After a period of 24 and 72 h animals were observed for any lethality or death.
Evaluation of hypoglycemic activity
Effect of aqueous extract in normoglycemic rats (NG)
Fasting blood sugar level of each animal was determined, after overnight fasting for 16 h. Rats divided in to three groups (n=6) were administered saline to control group and 500 mg/kg aqueous extract of Cassia glauca leaves and bark to second and third group respectively. Blood samples were collected at 30, 60 and 120 min after the oral administration of aqueous extract (Shirwaikar and Rajendran, 2006; Aslan et al., 2007).
Oral glucose tolerance test in normal rats (OGTT)
The oral glucose tolerance test was performed in overnight fasted (18 h) normal rats. Rats divided in to three groups (n=6) were administered saline to control group and 500 mg/kg aqueous extract of Cassia glauca leaves and bark second and third group respectively. Glucose (2 g/kg) was fed 30 min after the administration of extract. Blood samples were withdrawn from sinus retrorbital at 30, 60 and 120 min of glucose administration (Shirwaikar and Rajendran, 2006; Aslan et al., 2007). and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and a glucometer (Sugarcheck, Wockhardt Ltd., Mumbai, India).
Induction of hypeglycemia
Rats were fasted for 16 h prior to STZ injection. Diabetes was induced in rats by intraperitoneal (i.p.) injection of streptozotocin (STZ) (Spectrochem Pvt.Ltd. Mumbai, India) at a dose of 70 mg/kg b.w., (Brain et al., 1997; Kadnur et al., 2005) dissolved in 0.1 M cold citrate buffer (pH= 4.5). Seven days after the injection, the blood glucose concentration level above 200 mg/dl was considered to be diabetic and used in the experiments. To prevent the hypoglycemia 5% glucose solution was orally given to the diabetic rats. Only rats found with permanent diabetes were used for the antidiabetic study.
Determination of hypoglycemic activity on acute administration
Diabetic rats were divided into 3 groups as follows:
Group 1: Diabetic control. Received only saline.
Group 2: Diabetic rats given (500 mg/kg b.w. p.o.) aqueous extract of leaves.
Group 3: Diabetic rats given (500 mg/kg b.w. p.o.) aqueous extract of bark.
The oral glucose tolerance test was performed in overnight fasted (18 h) diabetic rats. The blood glucose concentrations of the animals were measured at the beginning of the study and Glucose (2 g/kg p.o.) was fed 30 min after the administration of extracts of Cassia glauca leaves and bark (500 mg/kg p.o.). Blood samples were withdrawn at 30, 60 and 120 min of glucose administration and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and a glucometer (Sugarcheck, Wockhardt Ltd., Mumbai, India).
Chronic treatment model
Rats were divided in to five groups of six rats (n=6) each. Groups 1 served as normal healthy control group and group 2 as diabetic untreated control and saline was given to both groups. Group 3 served as standard and was treated with glibenclamide (Sun Pharmaceuticals Ltd, India) (0.25 mg/kg/day p. o.) (Shirwaikar et al., 2004; Bairy et al., 2005; Shirwaikar and Rajendran, 2006). Group 4 and 5 was treated with dose of 500 mg/kg p.o. per day aqueous extract of Cassia glauca leaves and bark respectively for 21 day. Blood glucose levels of animals were measured on day 1, 7, 14 and 21 day of the study (Latha and Pari 2004; Chakrabarti et al., 2005)
Statistical analysis
Values are presented as mean ± S.E.M. Statistical difference between the treatment and the controls were tested by using one-way analysis of variance (ANOVA) followed by Student's 't ' test (500 mg/kg drug treated group was compared with diabetic control). A difference in the mean P value <0.05 was considered significant.
Results
Acute toxicity study
Experiments were carried out on normal healthy rats. The behavior of the rats treated with aqueous extract of Cassia glauca leaves and bark appeared normal. No toxic effect was seen even with the dose of 5 g/kg b.w. and there were no lethality in any of the group.
Hypoglycemic effect of aqueous extract on acute administration
Since no toxicity was observed with the extract in toxicity studies, the aqueous extract of Cassia glauca leaves and bark was prepared and administered to normal, glucose loaded model and STZ-induced diabetic rats at a dose of 250 and 500 mg/kg b.w. (p.o.) to determine the effect of aqueous extract on the blood concentrations upon acute administration. Changes in the blood glucose level of each group of animal were followed during a 2 h period (Table 1). It was observed that the aqueous extract of Cassia glauca leaves and bark at a dose of 250 and 500 mg/kg p.o. failed to show any hypoglycemic activity in normal and OGTT. However, aqueous extract of Cassia glauca leaves and bark at a dose of 500 mg/kg p.o. showed significant hypoglycemic activity in acute model of STZ-diabetic rats, while no remarkable effect was observed on normoglycemic rats. The improvement in diabetic-glucose tolerance in aqueous extract of bark treated (500 mg/kg) group was brought by lowering the initial blood glucose level of 437.2± 4.19 mg/dl significantly (p< 0.001) to 288.7±9.95 mg/dl at 2 h, where as the leaves extract showed significant (p< 0.001) hypoglycemic effect by lowering the initial fasting blood glucose level of 430.2 ± 31.9 mg/dl to 241.5 ± 2.0 mg/dl at 2 h. While in the untreated diabetic group the fasting blood glucose increased from the initial value of 380.5 ± 5.3 mg/dl to 399.2 ± 6.9 mg/dl. From the result it is clear that the aqueous extract of Cassia glauca leaves and bark (250 and 500 mg/kg b.w. p.o.) did not showed any significant effect on normoglycemic rats (Table 1).
Legends of Table 1: Values are given as mean ± S.E M from six rats in each group
***P < 0.001 highly significant from diabetic control animals and initial values.
Aq. E: Aqueous extract
Table 1: Hypoglycemic effect of aqueous extract in different models of rats
Hypoglycemic effect on chronic administration
Since significant hypoglycemic effect was observed at 500 mg/kg in diabetic acute model, it was decided to determine the chronic effect of the extract of Cassia glauca leaves and bark by using 500 mg/kg/day for 21 days. From the results of chronic model study (21 days), it was observed that there was significant decrease (P<0.001) of serum glucose level on day 21 (260.3 ± 12.4 mg/dl)) from day 1 (354.5 ± 7.3 mg/dl) with the treatment of aqueous extract of Cassia glauca bark, which is comparable to the effect produced by standard drug glibenclamide (Table 2). Glibenclamide lowered the serum glucose level significantly to day 21 (212.2 ± 6.8 mg/dl) from day1 (294.5 ± 11.0 mg/dl). The aqueous extract of leaves failed to produce any significant changes in fasting blood glucose levels on day 21 from day1. However the aqueous extract of leaves produced significant effect by lowering the fasting blood glucose level of day 7 (305.3 ± 13.5 mg/dl) as compared to fasting blood glucose level of day 21 (284.5 ± 10.5 mg/dl).These finding suggest that the aqueous extract of Cassia glauca bark possessed a significant hypoglycemic effect at 500 mg/kg dose in diabetic rats.
Legends of Table 2: Values are given as mean ± S.E M from six rats in each group
***P < 0.001 highly significant from diabetic control animals and initial values.
Aq. E: Aqueous extract
Table 2: Effect of aqueous extracts on serum glucose level on chronic administration
Discussion And Conclusions
Ayurvedic system of medicine relies on the administration of crude extract or concentrated extract of the plant material for the treatment of diabetes mellitus. Preliminary studies demand further research so that their novel possibilities as a source of oral hypoglycemic agents could be investigated [Eshrat and Hussain, 2002; Sabu et al., 2002]. Literature survey indicates that there is no scientific evidence to support the antidiabetic effect of Cassia glauca Lam. Therefore the present study is undertaken to investigate the action of aqueous extract of Cassia glauca leaves and bark in different models of rats to ascertain the scientific basis for the use of these plants in the treatment of diabetes.
In the present study, aqueous extracts were screened for hypoglycemic activity by using different animal models such as effect on normoglycemic rats, OGGT in normal and STZ induced diabetic rats and chronic diabetic model of treatment. The hypoglycemic activity was compared with respective control (Normal and Diabetic Control) and standard drug Glibenclamide. Acute toxicity studies revealed the non toxic nature of the aqueous leaves and bark extract of Cassia glauca. There was no lethality or any toxic reactions found with selected doses until the end of the study period.
Since no toxicity or lethality was found with any extract even at higher dose of 5g/kg body weight, there fore two scalar doses were selected for preliminary hypoglycemic activity by administering (orally) single dose of 250 and 500 mg/kg b.w. to different model of normoglycemic, OGTT and STZ-induced hyperglycemic rats. The fasting blood glucose levels were recorded during 2 h. The results of study have showed that aqueous extract of selected plant material.
The results of study have showed that aqueous extract of selected plant material failed to show any hypoglycemic effect at a dose of 250 mg/kg b.w. Whereas, administration (oral) of 500 mg/kg b.w. has showed some interesting results. Treatment of fasting STZ hyperglycemic rats with a single dose of aqueous extract of Cassia glauca leaves and bark at 500 mg/kg showed marked hypoglycemic activity by improvement of glucose tolerance test by lowering the blood glucose levels in STZ-induced diabetic rats at 2 h (Table ). The fasting blood glucose level did not change in the saline treated normal control group, but in the aqueous extract of Cassia glauca leaves and bark treated groups, the fasting blood glucose level changed markedly 1 h after the oral administration. These results indicates that effectiveness of aqueous extract of Cassia glauca leaves and bark in reducing the glucose level in fasting diabetic rats only after 1 h of administration and the significant reduction was observed during 2 h. The extracts did not produce any significant effect with both the doses on normal rats. The reduction of fasting blood glucose by aqueous extract raised the possibility that the aqueous extract might have stimulated the release of insulin in the pancreas or reduced in insulin resistance in peripheral tissues (Mai and Chuyen, 2007).
After the initial studies with acute treatment of aqueous extract of Cassia glauca leaves and bark in normoglycemic rats, glucose loaded and diabetic models it was felt worthwhile to evaluate further efficacies of aqueous extracts in chronic diabetic model. It was observed that there was significant decrease (P<0.001) of serum glucose level on day 21 from day 1 with the treatment of aqueous extract of Cassia glauca l bark, which is comparable to the effect produced by standard drug glibenclamide. Glibenclamide lowered the serum glucose level significantly to day 21 from day1. The aqueous extract of Cassia glauca leaves failed to produce any significant changes in fasting blood glucose levels on day 21 from day 1. These finding suggest that the aqueous extract of Cassia glauca bark possessed a significant hypoglycemic effect at 500 mg/kg dose in diabetic rats (Table 2).
This was considered to be the first study which reports about the hypoglycemic effects Cassia glauca leaves and bark extract. Earlier, antidiabetic effects of Cassia species such as Cassia auriculata (Pari and Latha, 2002; Latha and Pari, 2003; Jalalpure et al., 2004) and Cassia kleini (Babu et al., 2003) were reported.
In conclusion, the exact mechanism of action of the plant extract either alone or in combination cannot be stated. However it may be possible that extract might have insulin secretagogue activity, which in turn controls the hyperglycemic state of diabetes.
Acknowledgements
The authors sincerely thank Dr. F. V. Manvi, Principal, KLES’s College of Pharmacy for providing the necessary facilities for this work.