PHARMACODYNAMIC INTERACTION OF FENUGREEK LEAVES EXTRACT WITH FLUVASTATIN IN DIABETES INDUCED VASCULAR DYSFUNCTION

About Authors:
ATUL KABRA^* , PREMAL KAPADIA
Department of Pharmacology,
G.H.B Pharmacy college,
Aniyad, Gujarat
* atul.kbr@gmail.com

ABSTRACT:
The cardiovascular risk factors were significantly abnormally altered in diabetic rats when compared to normal control. The present study FLEt with low dose of OHA’s indicates the significant improvement in endothelial dependent vasodilatation because the percentage relaxation produced by acetylcholine was more significant when these groups were compared with diabetic control.However all the treated groups restored these changes significantly to normal conditions in diabetic rats when compared to diabetic control. The combined therapy of FLEt and Fluvastatin was found to be significantly effective. Moreover, this combination was also found to demonstrate synergistic behavior than pioglitazone plus fluvastatin.
We conclude that of preventive therapy with combination of FLEt with OHA’s (low dose) may prevent the endothelial dysfunction in diabetic patients (prediabetic state) due to free radical scavenging activity, metabolic control.

Reference Id: PHARMATUTOR-ART-1340

INTRODUCTION:
Obesity is usually the result of the combination of genetic factors with an inappropriate lifestyle, characterized by inadequate nutrition and lack of regular physical activity and is associated with development of diabetes, dyslipidemia hypertension and other medical problems¹.

Diabetes is of two types:
1. Insulin dependent diabetes(Type-I) 
2. Non-insulin dependent diabetes(Type-II)

Type-I diabetes is  a chronic disease characterized by hyperglycaemia secondary to inadequat production of insulin by the pancreas. It is due to complete destruction of β cells. It may be
1.Immune mediate
2.Idiopathic^2.                 

Type II diabetes is a result from a defect of both insulin secretion and insulin sensitivity. Other factors responsible for
Pathogenesis of diabetes II may be
1. Genetics and/or environment in the early life
2.β cell dysfunction^3.

Diabetes is a multisystem disorder that brings about microvascular and macrovascular complications^4.
The pharmacodynamic interaction of Fenugreek leaves extract (Rich in polyphenol) and fluvastatin in diabetic induced vascular dysfunction is not studied until now.  Hence an attempt is made to study the effect of Fenugreek leaves extract with fluvastatin in diabetes induced vascular dysfunction in rats.

MATERIAL AND METHODS:
Experimental Animals:
Male Sprague dawley Rats weighing 150-180 g were housed at 25° ± 5°C in a well-ventilated animal house under 12:12 h light dark cycle. The animals were maintained under standard conditions in an animal house as per the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

Extraction of polyphenolic rich Fenugreek leaves:
Preparation of Fenugreek Leaves Extract:
Fenugreek Leaves (500 g) were finely powdered, mixed with 80% methanol and kept at room temperature for 5 days. After 5 days it was filtered and the solvent was evaporated. The residue was dissolved in water and the aqueous layer was washed with petroleum ether several times until a clear upper layer of petroleum ether was obtained. The lower layer was then treated with ethyl acetate containing glacial acetic acid (10 ml/l).Extraction of polyphenols was carried out for 36 h at room temperature and the combined ethyl acetate layer was concentrated). The residue was lyophilized and stored. This yielded about 6–8 g per 500 g of leaves powder. An Methanolic extract was prepared and used for the studies.

INDUCTION OF DIABETES:
Development of high fat diet (HFD) fed / Low dose streptozotocin Treated type 2 diabetic rats:
The animals were fed with HFD once a day for two weeks followed by I.P injection of   streptozotocin (35mg/kg) dissolved in 0.5M/l citrate buffer ( pH: 4.4) after over night fasting. STZ injected animals were then given 5% w/v glucose solution for 5-6 hours following the injection to prevent initial drug induced hypoglycemic mortality. The rats with non fasted plasma level ≥ 300mg/dl were considered diabetic. The blood Sample was collected from tail vein and blood glucose was checked using glucose diagnostic kit (accucheck) .

STANDARDISATION AND SELECTION OF LOW DOSE FOR STANDARD DRUGS:
Oral Glucose Tolerance Test: After overnight fasting(18hrs), a 0-min blood sample was taken from the tip of the tail of each rat of different groups. Glucose solution (2 g/kg P.O) was given after 30 min after the administration of the drug . Four more samples were taken at 30, 60, 90 and 120 min after glucose administration . All blood samples were checked with the help of  accucheck glucometer.
The oral glucose tolerance test will be performed on overnight fasted Sprague dawley rats. Rats will be divided into different groups as follows:
Group 1: Normal control, rats receive saline/vehicle.
Group 2: Diabetic control.
Group 3:Fenugreek leaves extract(200 mg/kg)
Group 4: Pioglitazone (10 mg/kg)
Group 5: Glipizide (5 mg/kg)

Effect of fenugreek leaves extract alone and in combination with oral hypoglycemic agents in streptozotocin induced diabetic vascular dysfunction:
Rats was divided into different groups (n=6) as follows and the treatment with the respective drugs was given for 8 weeks. The estimations was carried out on the 15^th, 30^th, 45^th and 60^th days of the treatment.
Group 1: Normal control, rats receive saline/vehicle.
Group 2: Diabetic control
Group3: FSEt (200 mg/kg p.o)⁵
Group 4: Pioglitazone (10 mg/kg)⁶
Group 5: Glipizide (5mg/kg)⁷
Group 6: Pioglitazone (7.5mg/kg) plus Glipizide (3.75mg/kg)
Group 7: pioglitasone + Fluvastatin (10MG/KG)⁸
Group 7 : Pioglitazone(7.5 mg/kg )+ FSPE (200mg/kg)
Group 10: Glipizide (3.75 mg/kg) + FLEt (200 mg/kg)
Group 11: Glipizide (3.75 mg/kg) + FLEt (200 mg/kg)
Group12: FLEt (200 mg/kg) + Fluvastatin

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EXPERIMENTAL ANIMAL MODELS:
Isolated Rat Aortic ring preparation:
The rat aortic preparations were isolated, mounted in a temperature controlled tissue/organ bath and suspended in K-H solution that was aerated with carbogen gas so as to maintain the isolated tissues in a physiological state i.e. in a viable state during the experimental period.Thus using the tissue /organ bath the necessary physiological warmth is provided to the tissue even after  isolation from the animal while the K-H solution is used to provide the essential medium containing the various ions, buffer, energy source etc. but also to supply oxygen requirements to the isolated tissue.

RESULTS: 
Table-1.Effect of FLEt and its combination with Oral Hypoglycemic Agents on OGTT in Normal rats:

All values are mean ± SEM, n=6,^a P<0.001, ^bP<0.01, ^cP<0.05 when compared to normal control group.

Table-2. Effect of FLEt and its combination with Oral Hypoglycemic Agents on OGTT in Diabetic rats:

All values are mean ± SEM, n=6,^a P<0.001, ^bP<0.01, ^cP<0.05 when compared to diabetic control group.

Table 3: Effect of FLEt and its combination with Oral Hypoglycemics on Blood Glucose (mg/dl)

FLEt:- Fenugreek leaves exteact.
All values are mean ± SEM, n=6,^a P<0.001, ^bP<0.01, ^cP<0.05 when compared to    diabetic control group

Table:4  Comparison of relaxant responses in treated groups:

All values are mean ± SEM, n=6,^a P<0.001, ^bP<0.01, ^cP<0.05 when compared to diabetic control group

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FIGURE 1:GRAPHS OF TREATED GROUPS:

CONCLUSION:
Endothelial dysfunction associated with insulin resistance appears to precede the development of overt hyperglycemia in patients with type 2 diabetes mellitus. Therefore, endothelial dysfunction may be a critical early target for the prevention of atherosclerosis and CVD in patients with diabetes mellitus or insulin resistance. Therefore preventive therapy with combination of FLEt with OHA’s (low dose) may prevent the endothelial dysfunction in diabetic patients (prediabetic state) due to free radical scavenging activity, metabolic control.

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