ANTI HYPERGLYCAEMIC AND ANTIOXIDANT ACTIVITY OF CATUNAREGAM SPINOSA (THUNB) AGAINST DEXAMETHASONE INDUCED DIABETES IN RATS

Objective: The study was aimed at evaluating the folkloric use of the Catunaregam spinosa (Thunb) bark for its hypoglycaemic and antioxidant activity against dexamethasone-induced diabetes in wistar rats. Methods: Diabetes was induced in wistar rats by dexamethasone administration 10 mg/kg, b. wt., s. c for 11 consecutive days to all group animals (except Group I). After confirmation of diabetes, the animals were divided into 5 groups (n=6). Group I: Vehicle control treated with normal saline only, Group II: Diabetic control treated with dexamethasone only, Group III: Standard control treated with dexamethasone plus glibenclamide (5 mg/kg, b. wt., p. o ), Group IV and V: Test control received dexamethasone plus ethanolic extract of Catunaregam spinosa (EECS) at graded doses of 200 mg/kg, b. wt. and 400 mg/kg, b. wt., p. o for 21days respectively. On the last day of the experiment, the effect of Catunaregam spinosa bark was measured by estimating the biochemical and antioxidant parameters. Results: There was a significant alteration in the serum glucose, HbA1c, total proteins, serum lipid profile and tissue antioxidant parameters in dexamethasone-treated animals when compared to the normal control rats. In test groups treated with glibenclamide and EECS, it was significantly ameliorated the altered parameters such as serum glucose, HbA1c, total proteins, serum lipid profile and the antioxidant parameters like SOD, CAT, GSH and MDA when compared to dexamethasone-treated animals with evidence of histopathological studies of the pancreas. Conclusion: The results indicated that the ethanolic extract of Catunaregam Spinosa (Thunb) bark has shown the hypoglycemic and antioxidant property.


INTRODUCTION
Diabetes mellitus is a metabolic disorder characterized by an abnormality in carbohydrate, lipid and protein metabolism resulting in chronic hyperglycemia and abnormality of lipid profile. Chronic hyperglycemia of diabetes is associated with long term damage, dysfunction and eventually the failure of organs, especially the eyes, kidneys, nerves, heart and blood vessels [1]. Environmental factors such as diet, obesity, sedentary lifestyle and other factors like high family aggregation, insulin resistance, nutritional status, age increase the risk of diabetes [2]. Diabetes is managed with drugs such as metformin, sulfonylureas, thiazolidinedione's etc and as well as artificial insulin [3,4]. The serious adverse side effects like liver problems, lactic acidosis and diarrhoea [5,6]. Diabetes is currently affecting around 143 million people [7], the number of those affected is increasing day by day by 2030 and it is predicted to reach 366 million populations worldwide [8]. Management of diabetes without any side effects is still a challenge for the medical system. This leads to an increasing search for improved anti-diabetic drugs [9].
Medicinal plants have been used in the Indian traditional system of medicine and have shown anti-diabetic activity [10,11]. The number of active constituents present in medicinal plants is thought to act on a variety of targets by different mechanisms. Catunaregam spinosa (Thunb) belongs to the family Rubiaceae is commonly known as Gedhpal or Emetic nut. This species is common as undergrowth in the sub-Himalayan tract [12]. The raw fruits have a highly astringent taste due to high tannin content. The seeds contain essential oil and organic acid. The dried and powdered fruit pulp is credited with emetic properties [13]. Plant bark is reported for diarrhoea, dysentery, abortifacient, antihelminthic and antipyretic [14]. It is also considered to be a sedative, hypoglycaemic and stomach ache as first aid remedy. Roots are used in the treatment of epilepsy, eye ache and urinary infection. The fruit is used as emetic and the leaves are used in pulmonary infections [15,16]. Catunaregam spinosa also used as carminative, antipyretic, cures abscess, ulcers, inflammations, wounds, tumours and skin diseases [17]. Based on the scientific evidence of the data, the research was carried out to evaluate the hypoglycaemic and antioxidant activity of ethanolic extract of Catunaregam spinosa (Thunb) (EECS) bark against dexamethasone-induced diabetes in wistar rats.

Collection and authentication of plant specimen
The stem barks of Catunaregam spinosa (Thunb) were collected from surrounding areas of Seshachalam hills, Chittoor district and Andhra Pradesh (A. P). The plant material was authenticated by Dr. K. Madhava chetty, Professor, Department of Botany, Sri Venkateswara University, Tirupati, A. P, and India.

Animals
Healthy Adult Male Wistar rats weighing 150-200 gms were procured from Raghavendra enterprises, Bangalore and were maintained under standardized environmental conditions 12-h light/dark cycle, 24 °C and 35 to 60% humidity, provided free access to pellet diet and purified drinking water ad libtium. The experimental protocol was approved by Institutional Animal Ethical Committee of Krishna Teja Pharmacy College, Tirupati, A. P (Registered No. 1521/P0/11/CPCSEA-Committee for the purpose of control and supervision of experimental animals). subjected to continuous hot percolation with solvent 70% ethanol using soxhlet apparatus with 1:4 volumes. The extraction was carried out until the solvent becomes colourless and the solvent was removed from the extract by evaporation. The dried extract thus obtained was preserved in desicator [18]. The EECS was evaluated for phytochemical constituents using standard procedures [19,20].

Experimental design
Experimental rats were taken and divided into five groups each containing six animals.
Group I animals served as vehicle control received 0.5 ml of normal saline orally.
Group IV and V animals served as test control received dexamethasone (10 mg/kg, b. wt., s. c) for 11 d and EECS at graded doses of 200 mg/kg, b. wt., and 400 mg/kg, b. wt., p. o for 21days respectively.

Assessment of the hypoglycemic effect
The blood samples were collected through retro-orbital route from each rat under mild ether anesthesia on 22 nd day and serum was separated by centrifugation of blood at 4000 rpm for 10 min. The serum was used for the estimation biochemical parameters such as Glycosylated haemoglobin (HbA1 Statistical analysis c), serum glucose levels, Total cholesterol (TC), Triglycerides (TGL), High density lipoprotein (HDL), Low density lipoprotein (LDL), Very low density lipoprotein (VLDL) [22][23][24][25][26] and total protein (TP) [27]. From each group, one animal were taken and sacrificed for pancreas isolation and same one part is used for estimation of antioxidant parameters like Superoxide dismutase (SOD), Catalase (CAT), Glutathione reductase (GSH) and Malondialdehyde (MDA) [28][29][30][31] and another part processed for Histopathological studies [32].
The results were expressed as mean±SEM/SD differences in groups for biochemical estimations. Statistical analysis was determined by one way-analysis of variance (ANOVA), individual groups were compared using Dunnett '

RESULTS
s t-test. P value<0.05 has been considered as a statistical significance level.

Acute toxicity studies and test dose selection
The acute toxicity studies were conducted as per OECD (Organization for economic co-operation and development) 423 guidelines (Acute Toxic Class Method). No sign of toxicity and mortality was observed up to the maximum dose administered a dose of 2000 mg/kg, b. wt. p. o of EECS. The dose was selected based on the maximum dose administered with 1/10 th and 1/20 th

Effect of EECS on HbA
as low dose and a high dose of test extract.

Histopathological studies
Dexamethasone administration for 21 d revealed that the pancreas with degeneration of pancreatic cells and inflammation vacuoles may be due to the free radical damage compared to the normal vehicle control with normal parenchymatous cell without any degeneration and inflammation. Treatment with glibenclamide and EECS significantly reduced the marked inflammation and degeneration of pancreas as compared to the diabetic control rats.

DISCUSSION
In the present study Catunaregam spinosa (Thunb) was selected for hypoglycaemic and antioxidant activity evaluation owing to its ethnomedicinal use in curing diabetes. Therefore, the study was undertaken to justify its claimed use. Ethanolic extract of Catunaregam spinosa (Thunb) (EECS) bark was prepared by soxhlet method and stored in the refrigerator at 4 °C. Rats were selected as experimental animals for evaluating anti-hyperglycemic and antioxidant activity. Acute toxicity was conducted as per OECD guidelines and the EECS was not showed any toxicity at the dose of 2000 mg/kg and no death was reported for 72h. Preliminary phytochemical screening of EECS revealed the presence of Carbohydrates, Glycosides, Saponins, Terpenoids, Phenols, Tannins, Flavonoids, Proteins, Amino acids and Phytosterols.
Subcutaneously administration of dexamethasone for 11 d causes significant (P<0.001) elevation of HbA1c and decrease in the serum total protein levels in diabetic control rats as compared to the vehicle control. Glucocorticoids are widely used therapeutic tools particularly in treatment for anti-inflammatory and immunemodulatory purposes. Side effects of glucocorticoid treatment include steroid diabetes [33,34]. It induces hyperglycemia is partially due to increased hepatic glucose production and insulin resistance of peripheral tissues. Moreover, glucocorticoids are known to inhibit insulin secretion [35,36]. The underlying mechanism involves increased α2-adrenoceptor signaling [37], increased Kv channel activity [38] and impaired glucose metabolism [39,40]. They diminish glucose utilization in the peripheral tissues cause an increase in protein breakdown by increasing the synthesis of glutamine and lipolysis, thereby providing amino acids and glycerol for gluconeogenesis [41]. HbA1c as a biomarker for monitoring the levels of glucose among type 2 diabetic patients provides a reliable measure of chronic glycaemia and correlates well with the risk of long-term diabetes complications. So that it is currently considered the test of choice for monitoring and chronic management of diabetes [42]. During diabetes, the excess of glucose present in the blood reacts with hemoglobin to form glycosylated haemoglobin [43]. The rate of glycation is proportional to the concentration of blood glucose [44,45]. Treatment with glibenclamide and EECS 200 mg/kg and 400 mg/kg b. wt, p.o. ameliorated the abnormal levels of HbA1c and TP when compared to the diabetic control rats depicted in table 1.
In the present study, it has been found that the elevation of serum glucose and abnormal changes in the lipid profile in dexamethasonetreated rats indicating that the hyperglycemia and Hyperlipidemia [46,47]. Dexamethasone increases in glucose levels leading to hyperglycemia due to the inhibition of GLUT-4 (glucose transporter) translocation from intracellular compartments to the plasma membrane particularly of skeletal muscles [48,49]. The elevation of serum glucose levels in diabetic control rats were significantly restored upon treatment with the standard glibenclamide and ethanolic extract of Catunaregam spinosa (Thunb) at both dose levels (200 mg/kg, b. wt., and 400 mg/kg, b. wt., p. o.) for 21 d as compared to the vehicle control rats which was depicted in table 2. The possible mechanism by which EECS mediated its antihyperglycemic effect could be by the potentiation of pancreatic secretion of insulin from existing β-cells of islets thereby increase glucose utilization from tissues through GLUT-4.
Pharmacological doses of glucocorticoids induce ob gene expression in rat adipocyte tissues within 24 h which is followed by complex metabolic changes like hyperleptinemia, resulting in decreased in food consumption, with enhanced blood glucose and an imbalance in lipid metabolism leading to Hyperlipidemia [50][51]. On administration of dexamethasone, significantly (P<0.001) elevate the serum lipid parameters such as TC, TGL, LDL, VLDL and decrease in the HDL levels when compared to the vehicle control rats. Treatment with EECS (200 mg/kg, b. wt., and 400 mg/kg, b. wt., p. o.) for 21 d had ameliorated the abnormal lipid profile depicted in table 3. From the results of the present study, it may be suggesting that improvement in food intake and HDL levels and also a reduction in blood glucose. And it confirms that EECS shows protection of vital organs like pancreas, liver, heart, spleen and kidney thereby reducing the causation of diabetes [52].
The antioxidant enzymes such as catalase (CAT), glutathione Stransferase (GST), glutathione peroxidase (GPx) are able to resist oxidative stress by scavenging free radicals, inhibiting lipid peroxidation, increasing glutathione and catalase activity [53]. Oxidative stress can be generated by hyperglycemia and for a long time, it has been accused to cause insulin resistance. Insulin resistance induces the release of cytokines like TNF-alpha, IL-8 which leads to the development of oxidative stress in the liver by reducing the mitochondrial levels of Cu/Zn SOD, glutathione, producing H2O2 radicals and leads to increase in lipid peroxidation [54]. On Administration of dexamethasone induces the reduction of antioxidant enzymes such as SOD, CAT, GSH and increase in the lipid peroxidation when compared to the vehicle control animals. EECS (200 mg/kg and 400 mg/kg) was given orally for 21 d in both doses increases the reduced levels of SOD, CAT and GSH thus protecting the tissues from oxidative stress by inhibit free radical generation and reduce insulin resistance.
Lipid peroxidation is usually measured through its catabolite malondialdehyde (MDA) as a marker of oxidative stress [55,56]. A marked increase in the concentration of MDA in dexamethasoneinduced diabetic rats indicated that enhances lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanism to prevent the formation of excess free radicals. EECS showed the ability to prevent increased MDA levels in group 4 and 5 animals, suggesting that EECS inhibited lipid peroxidation and improved the pathological condition of diabetes. Hence, the possible antioxidant potential of the EECS may be due to the presence of polyphenolic compounds i.e. flavonoids and phytosterols.
EECS significantly reduced the marked inflammation and degeneration of pancreas as compared to the diabetic control rats. While dexamethasone administration revealed that the pancreas with degeneration of pancreatic cells and inflammation vacuoles may be due to the free radical damage compared to the normal vehicle control with normal parenchymatous cell without any degeneration and inflammation.

CONCLUSION
Medicinal plants have continued to be a powerful source for new drugs contributing about 90% of the newly discovered pharmaceuticals. As for the developed countries, the use of herbal medicine for chronic diseases is encouraged because there is concern about the adverse effects of chemical drugs and treatment using medicines of natural origin appears to offer more gentle means of managing such diseases. Herbal drugs are prescribed widely because of their effectiveness, fewer side effects and are relatively low in cost. The above data suggest that the ethanolic extract of Catunaregam spinosa (Thunb) bark possess hypoglycaemic and antioxidant activity in dexamethasone-treated rats as evidenced by the restoration of the abnormal parameter levels. This may be due to the presence of bioactive constituents in the plant such as tannins, flavonoids, saponins, polyphenols etc. Further studies are required to isolate the active principle responsible for hypoglycaemic and antioxidant activity.

ACKNOWLEDGMENT
Authors are thankful to the Department of Microbiology, Sri Venkateswara University, Tirupati, India, for providing analytical support in their laboratory to complete our research work.