Effect of aqueous extract of Thymus serpyllum on lipid profile and some liver enzymes in alloxan-induced diabetic rabbit

The present study was designed to evaluate the effects of aqueous extract of Thymus serpyllum on lipid profile and some liver enzymes level in the diabetic rabbit. Rabbits were made diabetic with a single intravenous injection of alloxan and were divided into groups. Group 1 and 2 served as normal and diabetic control, respectively. Group 3 and 4 were given standard drugs glibenclamide and acarbose respectively while Group 5 was treated with 500 mg/kg of extract for 30 days. Blood samples were taken on day 0 and 30. The extract significantly reduced the level of serum cholesterol, triglyceride, LDLs, VLDLs, alkaline phosphatase and transaminases without affecting the HDLs level. Total cholesterol/HDL-cholesterol ratio was significantly reduced as compared to diabetic control. It is conceivable that T. serpyllum possesses antihyperlipidemic and hepatoprotective effects in diabetic rabbit. Article Info Received: Accepted: Available Online: 12 September 2016 31 December 2016 3 March 2017 DOI: 10.3329/bjp.v12i1.29606 Cite this article: Alamgeer, Mushtaq MN. Effect of aqueous extract of Thymus serpyllum on lipid profile and some liver enzymes in alloxan-induced diabetic rabbit. Bangladesh J Pharmacol. 2017; 12: 58-62. Effect of aqueous extract of Thymus serpyllum on lipid profile and some liver enzymes in alloxan-induced diabetic rabbit Alamgeer1 and Muhammad Naveed Mushtaq1,2 Department of Pharmacology, Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan; Lahore College of Pharmaceutical Sciences, Raiwind Road, Lahore, Pakistan. from Merck Chemical Co., Germany.


Introduction
Diabetes is usually managed by insulin and oral hypoglycemic drugs but these synthetic agents are associated with serious side effects.Moreover, they fail to significantly control the diabetic complications (Cui et al., 2010;Aslam et al., 2012).This results in exploitation of medicinal plants to treat diabetes and associated complications.
Thymus serpyllum (wild thyme) belongs to the family Labiatae and locally known as Tumuro.In Pakistan, it is found in Gilgit-Baltistan, Kashmir and Jamu.T. serpyllum has been traditionally used in numerous diseases like ailments of the respiratory, digestive and urogenital system.It is given in weak vision, complaints of liver, suppression of urine and menstruation (Qureshi et al., 2007).T. serpyllum has the antidiabetic potential because of its α-glucosidase inhibiting activity (Gholamhoseinian et al., 2008).Moreover, T. serpyllum has been reported to show anti-oxidant activity (Pandey et al., 1996).Many recent studies have proved that anti-oxidants are effective in diabetes and its complications (Naziroglu and Cay, 2001).The plants with both antidiabetic and anti-oxidant activity are considered of better therapeutic value as compared to others for the treatment of diabetes.
In our previous study, the aqueous extract of T. serpyllum showed a hypoglycemic effect in diabetic rabbit (Alamgeer et al., 2012).The current study was conducted to investigate the effects of aqueous extract of T. serpyllum on lipid profile and some liver enzyme levels in diabetic rabbits.
from Merck Chemical Co., Germany.

Plant material
T. serpyllum plant was collected from the mountains of the village Shikiyote; District Gilgit, Gigit-Baltistan, Pakistan.Plant material was shade dried and powered with a Chinese herbal grinder.The powdered material was stored in well-closed cellophane bags at 4ºC in the refrigerator.The powdered plant was extracted by the method of cold maceration.The powder was soaked in distilled water for 48 hours with occasional shaking.It was passed through muslin cloth and then filtered through the filter paper.The extract was dried with the help of rotary evaporator (Mushtaq et al., 2016).

Animals used
Adult rabbits (Oryctolagus cuniculus) of either sex, weighing 1.2-1.5 kg were used.Animals were housed at standard conditions of temperature (23 ± 12ºC), humidity (55 ± 15%) and 12 hours light and dark cycle.Animals were provided with food and water ad libitum.After an overnight fasting, rabbits were made diabetic with a single intravenous injection of alloxan monohydrate (150 mg/kg) dissolved in normal saline (Etuk, 2010).After 72 hours of alloxan administration, induction of diabetes was confirmed by measuring the blood glucose level.Rabbits with blood glucose level between 250-300 mg/dL were considered diabetic and were employed for further studies.

Experimental design
Rabbits were randomly divided into five groups of six animals each.Group 1 and 2 served as untreated control and diabetic control and were administered orally 20 mL of 2% aqueous gum tragacanth solution.Group 3 and 4 received orally 600 µg/kg glibenclamide and 3 mg/kg acarbose, respectively.Group 5 was administered 500 mg/kg aqueous extract of T. serpyllum (Ramesh and Paugalendi, 2006;Maciejewski et al., 2001).Treatment was continued for 30 days on daily basis.Serum liver enzymes (alkaline phosphatase, transaminase) and lipid profile of all the rabbits in each group were estimated on day 0 and 30.

Biochemical analysis
Blood samples were collected from the ear marginal vein.The serum was separated by centrifugation.The separated serum was preserved in Eppendorf tubes and stored in the refrigerator at below 0ºC.The biochemical tests were performed within 24 hours after collecting the blood.
The level of total serum cholesterol, triglyceride, HDLs, alkaline phosphatase and transaminases were estimated by the enzymatic test kits using Microlab 300 (Merck Chemical Co., Germany).LDLs level was calculated by using formula (Fridewald et al., 1979):

Statistical analysis
Values were represented as mean ± SEM and data were analyzed by ANOVA followed by Turkey's test.A value of p<0.05 was considered significant.

Effect on liver enzymes
The aqueous extract of T. serpyllum significantly reduced the level of serum alkaline phosphatase and transaminases in diabetic rabbits treated for 30 days.The standard synthetic drugs, glibenclamide and acarbose also significantly decreased the liver enzyme levels from the start to the end of the month.The levels of serum alkaline phosphatase and transaminases were significantly increased in the rabbits of diabetic control while there was no significant change observed in the normal control rabbits (Table I).Data are expressed as mean ± SEM; Significant at p<0.05, vs control where ( a ) = p<0.05,( b ) = p<0.01 and ( c ) = p<0.001

Effect on lipid profile
Aqueous extract significantly reduced the triglycerides, cholesterol, LDLs and VLDLs levels in diabetic rabbits.HDLs levels of diabetic treated rabbits were not significantly altered.However, total cholesterol/HDL cholesterol ratio was significantly reduced with the treatment of aqueous extract for 30 days.Glibenclamide and acarbose also significantly reduced all the parameters of lipid profile except HDL.However, these synthetic drugs resulted in significant decrease in the ratio of total cholesterol/HDL cholesterol.Lipid profile parameters except HDLs were significantly increased in the diabetic control group of rabbits.There was no significant change in the lipid profile normal control rabbits (Table II).

Discussion
The results of the present study show that the aqueous extract of T. serpyllum possesses antihyperlipidemic and hepatoprotective effects in diabetic rabbits.It is well documented that the lipid profile and liver enzyme levels are abnormally changed in diabetes.The levels of alkaline phosphatase and transaminases have been reported to be increased in alloxan-induced diabetic animals.This increase in liver enzymes is suggested due to the cytotoxic effect of alloxan which causes the leakage of the enzymes from hepatic tissues into the blood circulation (Stanely et al., 1999).During diabetes, the other metabolic processes occurring in the liver are disturbed leading to the adverse effects on liver tissues.This is one of the reasons that liver enzymes are increased during the course of diabetes.The increased blood glucose level is associated with the generation of free radical species which damage the liver tissues.The administration of aqueous extract to alloxanized rabbits significantly reduced the hepatic enzyme levels.
T. serpyllum has been reported to possess anti-oxidant effect (Mihailovic-Stanojevic et al., 2013).The protective effect of the extract on liver could be linked to its antioxidant effect as already reported in a previous study.because anti-oxidants have been reported to possess hepatoprotective effect.Glibenclamide and acarbose reduced the liver enzymes by virtue of their hypoglycemic action.So, tight glycemic control by these synthetic drugs is the cause of lowering of liver enzymes.These results are in agreement with the earlier investigation in which the glibenclamide significantly reduced the liver enzyme level in diabetic animals (Rajasekaran et al., 2006).
The results of the present study also show that the aqueous extract of T. serpyllum produced a protective effect on the lipid profile which is usually altered during alloxan-induced diabetes.The hyperglycemic condition in the body produces reactive oxygen species (ROS) which causes lipid peroxidation.During diabetes, insulin deficiency leads to various metabolic alterations in the animals which result in increased level of cholesterol, alkaline phosphatase and transaminases (Sharma et al., 2010).In the present investigation, the aqueous extract significantly reduced the levels of serum triglyceride, total cholesterol, LDLs and VLDLs.The results are in accordance with the earlier investigations (Akah et al., 2009, Ajiboye et al., 2015).It is also suggested that the anti-oxidant property of the aqueous extract might neutralize the damage produced by the free radicals and reduced the production of lipid peroxidation products.In the present investigation, the  standard drugs glibenclamide and acarbose also produced reduction in the lipid profile of diabetic rabbits.In a previous study, glibenclamide alone and in combination with shilagit reduced lipid profile (Trivedi et al., 2004).It has also been reported that acarbose reduces the some parameters of lipid profile (Mughal et al., 2000).The glycemic control is the major determinant of the total cholesterol and triglyceride concentration.So, the glycemic control in the diabetic treated rabbits by these drugs could be the major cause of the reduction in these parameters.