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Diabetes mellitus is the most severe metabolic pandemic of the 21st century, affecting essential biochemical activities in almost every cell in the body. Indian literatures have already mentioned herbal remediation for a number of human ailments. The present study was undertaken to evaluate the potential of Coccinia grandis extract on serum and liver marker enzymes (ALP, AST, ALT and LDH) and lipid profile (total cholesterol, phospholipids, triglycerides and free fatty acids in serum and liver) in streptozotocin induced diabetic animals. The experimental animals were treated with methanolic extract of Coccinia grandis and the levels of marker enzymes and lipid profile were estimated. The ALP, AST, ALT and LDH levels were increased in diabetic rats and restored to near normal levels after administration of plant extract. The lipid profile increased in diabetic group and after the treatment with the plant extract the levels were reverted to near normal. Thus the methanolic extract of Coccinia grandis has a potent ability to restore the marker enzymes and the lipid profile was reverted to near normal levels.
Diabetes, a lifelong progressive disease, is the result of body's inability to produce insulin or use insulin to its full potential, and is characterized by high circulating glucose. This disease has reached epidemic proportion and has become one of the most challenging health problems of the 21st century. It is the fourth leading cause of death by disease globally; every 10 seconds a person dies from diabetes - related causes (Kowluru & Chan, 2007).
India faces a grave health care burden due to the high prevalence of type 2 diabetes and its sequalae. Epidemiological data from different parts of the country show a rising prevalence of diabetes in the urban areas. There is a wide urban-rural difference in the prevalence of diabetes indicating a major role for urbanization in the causation of the disease.
Liver is an insulin dependent tissue which plays a pivotal role in glucose and lipid homeostasis. It involves in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol and phospholipids and secretion of specific classes of plasma lipoproteins (Brown et al., 1993). During diabetes, a profound alteration in the concentration and composition of lipids occurs.
The preventing activity of present day drugs against progressive nature of diabetes and its complications was modest and not always effective. The doubts about the efficacy and safety of the oral hypoglycemic agents have prompted a search for safer and more effective drugs in the treatment of diabetes.
The search of new herbal drugs for the treatment of DM is increasing, as they are non-toxic (Momin, 1987). Coccinia grandis distributed in tropical Asia, Africa and is commonly found in Pakistan, India and Srilanka (Cooke, 1903). Coccina is a climber and trailer. Every part of this plant is valuable in medicine and various preparations have been mentioned in indigenous system of medicine for various skin diseases, bronchitis and Unani systems of medicine for ring worm, psoriasis, small pox, scabies (Perry, 1980) and other itchy skin eruptions and ulcers (Behl et al., 1993). The plant is used in decoction for gonnorhoeae (Nadkarni, 1976), diabetes and also useful in dropsical condition, pyelitis, cystitis, strangury, snake bite, urinary gravel and calculi (Jayaweera, 1980; Nadkarni, 1976). In this accord, we have chosen Coccinia grandis leaf extract to treat Diabetes and its complications in streptozotocin induced diabetes.
The fresh leaves of Coccinia grandis (Linn.) Voigt (Family: Cucurbitaceae) Syn. Coccina indica (Wight & Arn) were collected in the month of April 2009 from Karur district, Tamilnadu, India. Taxonomic authentication was done by Dr.V.S.Ramachandran, Taxonomist, Department of Botany, Bharathiar University, Coimbatore, Tamilnadu, India. The leaves were washed with water, shade dried at room temperature and powdered using mixer grinder. The powdered material (10g) was extracted with 100ml of methanol using Soxhlet apparatus and filtered. The filtrate was concentrated and dried under reduced pressure and controlled temperature.
The chemicals and solvents used in the study were of highest purity and analytical reagents grade. They were purchased from SD Fine Chem., Himedia and Qualigens, India.
Female albino rats of Wistar strain weighing 150 - 200g were purchased from animal breeding centre of Kerala Agricultural University, Mannuty, Thrissur, Kerala India. Animals were provided with standard pellet diet (AVM feeds, Coimbatore) and water was provided ad-libitum and maintained under standard laboratory conditions. (Temperature - 24-28°C and relative humidity - 60-70%). The animals were allowed to get acclimatized to the laboratory conditions for one week.
The experiments were conducted according to the ethical norms approved by ministry of social justices and empowerment, Government of India and Institutional Animal Ethics committee Guide (Approval No: 659/02/a/CPCSEA). The place where the experiments were conducted was kept very hygienic by cleaning with antiseptic solutions as the diabetic animals are susceptible to infections.
After one week acclimatization period, the animals were divided into four groups with six animals in each.
Group I: Control rats fed with standard pellet diet and water.
Group II: The rats were made diabetic by an intraperitoneal injection of single dose of 110 mg/kg body weight Nicotinamide followed by 65 mg/kg body weight Streptozotocin. Animals whose blood glucose level exceeded 200 mg/dl at 24 h after treatment were considered diabetic. These animals served as untreated diabetic control.
Group III: The diabetic rats treated with methanolic extract of Coccinia grandis at a dose of 200 mg/kg body weight for 20 days.
Group IV: Control rats were given methanolic extract of Coccinia grandis at a dose of 200 mg/kg body weight for 20 days.
After the experimental regimen, the animals were sacrificed by cervical dislocation under mild chloroform anesthesia. Blood was collected by an incision made in the jugular veins and the serum was separated by centrifugation at 2000 rpm for 20 minutes. The liver was excised immediately and thoroughly washed in ice cold physiological saline.
A 10% homogenate of the washed tissue was prepared in 0.1M Tris HCl buffer (pH 7.4) in a potter homogenizer filled with a Teflon plunger at 600 rpm for 3 mins.
The marker enzymes in serum and liver such as alkaline phosphatase activity was estimated by the method of King and Amstrong (1934), aspartate amino transferase activity was estimated by the method of Reitman & Frankel (1957), alanine amino transferase activity was estimated by the method of Reitman & Frankel (1957) and lactate dehydrogenase activity was estimated by the method of King (1965). Lipid samples were isolated from tissue and serum by Folch method (1957) and the total cholesterol was estimated in serum and liver tissue by the method of Parekh & Jung, (1970), phospholipids content was estimated in serum and liver tissue by the method of Rouser et al (1970), triglycerides were estimated in serum and liver by the method of Rice (1970 and free fatty acids level was estimated in serum and liver tissue by Horn & Mehanan (1981) method.
Table 1 depicts the effect of Coccinia grandis on serum and liver marker enzymes in control and experimental animals. The levels of the marker enzymes (ALP, AST, ALT and LDH) in serum and liver were significantly increased in streptozotocin induced rats when compared with the normal rats. The methanolic extract of Coccinia grandis treatment significantly reduced the increased levels of the enzymes both in serum and liver. The administration of the plant extract to the normal animals showed no significant change in the levels of these markers in serum and liver.
Measurement of enzymic activities of aminotransferases (AST and ALT) and phosphatases (acid and alkaline) is of clinical and toxicological importance as changes in their activities are indicative of tissue damage by toxicants or in disease conditions. Serum enzymes including AST and ALT are used in the evaluation of hepatic disorders. An increase in these enzyme activities reflects active liver damage. Serum levels of AST & ALT were increased than that of normal in diabetic animals and diabetic animals treated with extract show improvement. Recovery of AST, ALT, ALP and LDH levels of diabetic rats towards normal shows that the Coccinia grandis extract has no adverse effect on liver functions.
The increase in the activities of serum AST and ALT indicated that diabetes may be mainly due to leakage of these enzymes from the liver cytosol into the blood stream, which gives an indication on the hepatotoxic effect of Streptozotocin. Severe long-term hyperglycemia resulted in hepatolysis reflected by the increased ALT and AST activities in blood plasma of the diabetic rats (Lapshina et al., 2006).
The restoration of AST, ALT and ALP to their normal levels may be due to the presence of flavonoids in the alcoholic P.rheedii extract, which are reported to be hepatoprotective agents (Mustaq et al., 2000). Thus, the flavonoids present in the plant extract may be responsible for the reduction of serum AST, ALT and ALP. It has been postulated that the ingredients present in the extract of Coccinia indica acts like insulin, correcting the elevated enzymes gluocose-6-phosphatase and lactate dehydrogenase in the glycolytic pathway and restore the lipoprotein lipase activity in the lipolytic pathway with the control of hyperglycemia in diabetes (Kamble et al., 1998).
Diabetes mellitus is known to cause hyperlipidemia through various metabolic derangements and uncontrolled diabetes mellitus is manifested by very high rise in serum triglycerides and free fatty acid levels (Ferrannini et al., 1991).
Table 2 depicts the effect of Coccinia grandis on total cholesterol, phospholipids, triglycerides and free fatty acids in serum and liver of normal and experimental rats. The total cholesterol, phospholipids, triglycerides and free fatty acids were increased significantly in the tissues of the diabetic rats when compared to the normal rats. The diabetic animals treated with the plant extract showed near normal values. The normal animals administered with the plant extract showed no significant change in the lipid parameters. The increased lipid profile in the diabetic animals might be due to the lack of insulin secretion or insulin action.
In diabetic animals, body fat is used as a preferred source of energy because of incomplete utilization of glucose due to lack of insulin secretion or its action which resulted into secondary hyperlipidaemia, in particular, hypertriglyceridaemia as well as hypercholesterolaemia (Ferrnnini et al., 1991). This rise is proportional to the severity of the disease. Moreover, insulin deficiency in diabetes mellitus is known to stimulate lipolysis in the adipose tissue that gives rise to hyperlipidaemia and fatty liver and is also responsible for decreased activity of the enzyme, lipoprotein lipase which is responsible for the hydrolysis of the lipids resulting into the significant increase in serum triglycerides concentration (Brown et al., 1993).
Besides hyperglycemia, significant hypercholesterolaemia and hyper-triglyceridaemia have been reported to occur in STZ and alloxan diabetic rats. The marked hyperlipidaemia that characterizes the diabetic state may be regarded as a consequence of the uninhibited actions of lipolytic hormone on the fat depots (Hardman & Limberd, 2001).
The accompanying increase in the transport of oxidizable compounds such as glucose, amino acids, lipids (Staprans et al., 1993) along with the increased synthesis of cholesterol (Feingold et al., 1982) and triglycerides (Feingold et al., 1990) and decreased utilization of glucose within the erythrocyte (Madsen et al., 1995) can lead to transient increases in the intracellular concentrations of these compounds. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in alloxan induced diabetic rats (Joy & Kuttan, 1999). In this study, the active components of the plant extract reversed these levels to near normalcy. Earlier studies showed a moderate effect, which revealed the hypolipidemic activity of Phyllanthus simplex. These results were in line with the work of Akthar et al (2007) in Cathranthus roseus and Coccinia cordifolia.
¥ - μmoles of phenol liberated / L
Ψ - nmoles of phenol liberated / min / mg protein
£ - μmoles of pyruvate liberated / L
- nmoles of phenol liberated / min / mg protein
$ - μmoles of pyruvate liberated / min / mg protein
Values are expressed as Mean±SD (n=6) (p<0.05)
Statistical comparison: a : Group I and Group II; b : Group II and Group III; c : Group I and Group IV; *P<0.05 ns- non significant.
Statistical comparison: a: Group I and Group II; b:Group II and Group III; c:Group I and Group IV; *P<0.05 ns- non significant.