Ameliorative potential of Coccinia grandis extract on serum and liver marker enzymes and lipid profile in streptozotocin induced diabetic rats

Plant collection and authentication

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.

Chemicals

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.

Selection of animals

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.

Experimental set up

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.

Sample Collection

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.

Preparation of liver homogenate

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.

Serum and liver marker enzymes (ALP, AST, ALT and LDH)

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.

Table 1

Effect of Coccinia grandis on serum and liver marker enzymes in control and experimental animals

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).

Lipid Profile

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.

Table 2

Effect of Coccinia grandis leaf extract on total cholesterol, phospholipids, triglycerides, free fatty acids and free cholesterol in control and experimental rats

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.

Units:

¥ - μmoles of phenol liberated / L

Ψ - nmoles of phenol liberated / min / mg protein

£ - μmoles of pyruvate liberated / L

ɸ - 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.[28]