Antihyperlipidemic and Antiperoxidative Effect of Dibola A Polyherbal Formulation in Alloxan Induced Diabetic Rats

B. C. Koti and R. A. Patil*

Tatyasaheb Kore College of Pharmacy, Warananagar.

ABSTRACT:

This study was undertaken to investigate the effect of Dibola, a polyherbal formulation composed of medicinal plants on blood glucose, plasma insulin, serum lipid profile and lipidperoxidation in alloxan induced diabetes rats. Dibola, was administered orally (242 mg/kg body weight) for 21 days. The effect of Dibola on blood glucose and plasma insulin in diabetic rats were studied and the levels of lipid peroxides [TBARS and Hydroperoxides] and serum lipids [cholesterol, triglyceride, LDL, HDL and VLDL] were also estimated in alloxan induced diabetic rats. The effects were compared with standard drug Gilbenclamide. Treatment with Dibola and Gilbenclamide resulted in a significant reduction of blood glucose and increase in plasma insulin. Dibola also resulted in a significant decrease in serum cholesterol, increase HDL and reduction in tissue lipid peroxide formation. The effect produced by Dibola was comparable with that of Gilbenclamide.

The decreased lipid peroxides, serum cholesterol, increase HDL levels clearly showed the antihyperlipidemic and antiperoxidative effect of Dibola apart from its antidiabetic effect.

KEYWORDS: Dibola, Alloxan, Antihyperlipidemic, Antiperoxidative.

INTRODUCTION:

Diabetes mellitus (DM), long considered a disease of minor significance to world health, is now taking its place as one of the main threats to human health in the 21st century. It is the most common non-communicable disease worldwide and the fourth to fifth leading cause of death in developed countries. The current prevalence of type 2 diabetes is 2.4% in the rural population and 11.6% in the urban population of India. It has been estimated that by the year 2025, India will have the largest number of diabetic subjects in the world¹. Diabetes is defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by insulin. This results primarily in elevated fasting and postprandial blood glucose levels. If this imbalanced homeostasis does not return to normalcy and continues for a protracted period of time, it leads to hyperglycemia that in due course turns into a syndrome called diabetes mellitus. Besides hyperglycemia, several other factors including dyslipidemia or hyperlipidemia are involved in the development of micro and macrovascular complications of diabetes which are the major causes of morbidity and death². Significant changes in lipid metabolism and structure also occur in diabetes. In these cases the structural changes are clearly oxidative in nature and are associated with development of vascular disease in diabetes³. In diabetic rats increased lipidperoxidation is also associated with hyperlipidemia⁴. Inspite of the presence of known antidiabetic medicine in the pharmaceutical market, remedies from medicinal plants is used with success to treat this disease. Many traditional plant treatments for diabetes are used throughout the world. Plant drugs and herbal formulation are frequently considered to be less toxic and more free from side effects than synthetic one^5,
6

Moreover, continuous use of the synthetic antidiabetic drugs cause side effects and toxicity. Therefore, seeking natural and non-toxic antidiabetic drugs is necessary for diabetic therapy⁷. Dibola, a polyherbal formulation and principal ingredients are powders of mainly, Curcuma Longa, Tinospora cordifolia, Azadirachta Indica, Momordia Charantia, Syzium Cumini, Berberis Aristata, Sympolocos Racemosa, Trignetia Foeumgraecum, Swertia Chirata, Trikatu, Pichrohiza Kurroa, Trikatu, Shilajit.

MATERIALS AND METHODS:

Healthy male Wistar rats weighing 150-200 gm were used and are procured from college animal house. They were housed in a group of six under environmentally controlled room with 12-h light/dark cycle and had free access to food and water. After seven days of acclimatization period, they were randomly selected for different experimental groups.

All the experimental procedures were carried out accordance with committee for the purpose of control and supervision of experiments on animal (CPCSEA) guidelines. All the experimental procedures were approved by the institutional animal ethical committee (IAEC).

Chemicals and drugs:

“Dibola” (Sampurna Jeevan Pharma Chem Pvt. Ltd, Ichalkaranji, India.) Alloxan (Sd.Fine-Chem. Ltd, Mumbai, India) Gilbenclamide (Sun Pharmaceuticals Ltd, Jammu, India.) Glucose, Cholesterol, HDL, Total Protein and Triglycerides kits were procured from Transasia Bio-medicals Ltd, Daman, India. Trichloroacetic acid (Sd.Fine-Chem. Ltd, Mumbai, India), Thiobarbituric acid (Himedia lab), Butylated hydroxyl toluene (Sd.Fine-Chem. Ltd, Mumbai, India) Ammonium ion sulphate (Sd.Fine-Chem. Ltd, Mumbai, India) Xylenol orange indicator (Merck specialties private ltd, Mumbai.)

a) Preparation of Dibola suspension:

Suspension of finely powdered Dibola (242 mg/kg) was prepared in 1% (w/v) sodium carboxy methyl cellulose (CMC) suspension and administered using an intragastric tube daily for 21 days.

b) Preparation of Gilbenclamide drug suspension:

Gilbenclamide in its pure form was obtained from Sun Pharmaceuticals Jammu, India. Was prepared in 1% (w/v) sodium carboxy methyl cellulose (CMC) suspension and administered using an intragastric tube daily for 21 days.

c) Procedure for alloxan administration:⁸

The animals were selected and weighed then marked for individual identification. The rats were injected with alloxan in saline (0.9% NaCl) at a dose of 150 mg/kg body weight intrperitonally. Normal control rats were injected with saline only. Prior to this, the rats were fasted for 16 hours.

After one hour of alloxan administration the animals were given feed ad libitum. A 5% dextrose solution was given in feeding bottle for a day to over come the early hypoglycemic phase.

After 72 hours blood glucose was measured by Blood Chemistry – semiauto analyzer (Erba Mennheim). Blood was collected retro–orbital from the inner canthus of the eye under light ether anesthesia using capillary tubes and centrifuged at 2000 rpm to separate blood serum.The diabetic rats (glucose level 200- 300 mg/dl) were separated and divided into different experimental groups, each group contain six animals.

d) Dose selection:

Three different doses were used by therapeutic equivalent dose; that is 81 mg/kg, 162 mg/kg, 242 mg/kg that is as per M. N. Ghosh (practical pharmacology). More effective dose was selected for further study.

e) Experimental design:

The rats were divided into 7 groups of six animals each.

They were grouped as:

1) Group I: Normal control will receive (saline) vehicle.

2) Group II: Animals were administered suspension of Dibola.

3) Group III: Animals were administered Alloxan 150 mg/kg body weight an intraperitonally at once.

4) Group IV: Diabetic rats were administered suspension of Dibola daily using an intragastric tube for 21 days.

5) Group VI: Diabetic rats were administered suspension of Dibola daily using

an intragastric tube for 21 days.

6) Group VI: Diabetic rats were administered suspension of Dibola daily using

an intragastric tube for 21 days.

7) Group VII: Diabetic rats were administered suspension of gilbenclamide (600 μg/kg

body weight) daily using an intragastric tube for 21 days.

Biochemical estimation:

Estimation of blood glucose and insulin:

Blood glucose was determined by Glucose Oxidase / Peroxidase (GOD/POD) method . The insulin estimation was done by using ImmuChem Radioimmunoassay method using a standard kit obtained from BI-INSULIN IRMA, Cisbio, France.

Methods for estimation of oxidative stress:

Estimation of lipid peroxidation (MDA):⁹

Lipid peroxidation was estimated in terms of thiobarbituric acid reactive species (TBARS), using malondialdehyde (MDA) as standard. 1.0 ml of the sample extract was added with 2.0 ml of the TCA- TBA- HCl reagent (15%w/v TCA, 0.375% w/v TBA and 0.25 N HCl). The contents were boiled for 15 minutes, cooled and centrifuged at 10000 rpm to remove the precipitate. The absorbance was read at 535 nm and malondialdehyde concentration of the sample was calculated using extinction coefficient of 1.56 x 10⁵M^-1cm^-1.

Estimation of Hydroperoxides:¹⁰

0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg Butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37°C for 30 min. The color developed was read at 560 nm calorimetrically, concentration of the sample was calculated using extinction coefficient of 4.3 x 10⁴M^-1cm^-1.

Estimation of lipids:

For estimation of lipid profile, serum was isolated from the blood collected by retro-orbital puncture under mild ether anesthesia from fasted rats on 21 days of treatment. Serum total cholesterol , triglyceride, HDL- cholesterol were estimated by using respective diagnostic kits. (Erba Mannheim Cholesterol Kit) VLDL and LDL- cholesterol were calculated as per Friedevald’s equation:

Body Weight Measurement:

Body weight of all the experimental animals was recorded on zero day and final day using a digital weighing scale. The percentage change in body weight was calculated using the formula -

(Final weight-Initial weight/ Final weight) X 100

Statistical Analysis:

All data are presented as Mean ± S.E. The statistical analyses were performed using One-Way ANOVA followed by Dunnett’s Multiple Comparison test. Statistical significance was assumed if p<0.05.

RESULTS:

Table-: 1. Effect of Dibola in level on blood glucose and plasma insulin of experimental rats.

Sr. no.	Treatment (n=6) P.O	Fasting blood glucose mg/dl	Plasma insulin μIU/ml
I	Normal Control (NC)	105.6±4.445	0.3650±0.01821
II	Dibola 242mg/kg	115.1±6.587^ns	0.3567±0.02140^ns
III	Diabetic Control (DC)	249.5±15.49^###	0.1350±0.01803^###
IV	Alloxan+ Dibola 242 mg/kg	175.6±19.45^**	0.2317±0.02442^*
V	Alloxan + Gilbenclamide 600 μg/kg	158.5±9.328^***	0.2400±0.02620^**

One-way ANOVA followed by Dunnett’s Multiple Comparison test. Values are expressed as mean  SEM; n=6. p<0.05 is considered as significant, ^nsnon-significant,^###p< 0.001 compared to normal control, ^*p< 0.05 ^**p< 0.01 ^***p<0.001 compared to diabetic control.

Table-: 2. Effect of Dibola on serum lipid profile on experimental rats.

Groups	Treatment (n=6) P.O	Triglycerides (mg/dl)	Total cholesterol (mg/dl)	HDL cholesterol (mg/dl)	LDL cholesterol (mg/dl)	VLDL cholesterol (mg/dl)
I	Normal Control	87.86±1.677	84.30±3.010	53.43±1.994	13.30±3.108	17.57±0.3353
II	Dibola 242 mg/kg	90.84±3.114^ns	88.93±2.461^ns	49.38±2.654^ns	21.75±2.573^ns	18.29±0.5955^ns
III	Diabetic Control	176.4±3.941^###	141.5±3.537^###	38.40±3.387^###	67.78±6.855^###	35.48±0.8639^###
IV	Alloxan + Dibola 242 mg/kg	147.5±7.922^**	102.8±4.055^***	48.47±1.240^*	24.62±3.280^***	29.49±1.584^***
V	Alloxan + Gilbenclamide 600 μg/kg	139.5±5.124^***	106.4±4.481^***	51.08±0.9015^**	27.38±4.490^***	27.91±1.025^***

Table-:18. Effect of Dibola in level of TBARS and Hydroperoxides in liver and kidney of experimental rats.

Groups	Treatment (n=6)	TBARS μm/gm of tissue		Hydroperoxides μm/gm of tissue
Groups	Treatment (n=6)	Liver	Kidney	Liver	Kidney
I	Normal Control	2.495±0.2545	3.162±0.3213	12.93±0.7924	11.43±1.138
II	Dibola 242 mg/kg	2.317±0.3005^ns	3.150±0.3640^ns	13.19±1.067^ns	11.07±3.215^ns
III	Diabetic Control	4.078±0.4517^#	5.672±0.3666^###	34.66±7.345^##	30.03±3.936^#
IV	Alloxan + Dibola 242 mg/kg	2.357±0.1571^**	3.528±0.2048^***	17.80±2.237^*	19.48±2.063^*
V	Gilbenclamide 600 μg/kg	2.490±0.4589^*	3.472±0.2048^***	17.93±2.402^*	18.43±2.577^*

One-way ANOVA followed by Dunnett’s Multiple Comparison test. Values are expressed as mean  SEM; n=6. p<0.05 is considered as significant, ^nsnon-significant,^#p< 0.05, ^##p< 0.01,^###p< 0.001 compared to normal control, ^*p<0.05, ^**p< 0.01,^***p<0.001 compared to diabetic control.

Table –:15. The effect of 21 days treatment of Dibola on body weight after alloxan induced diabetes in rats.

Groups	Treatment	Average body weight (g) ± SEM		% Change in body weight
Groups	Treatment	Initial value (0 day)	Final value (42^nd day)	% Change in body weight
I	Normal Control (NC)	175.4±5.837	190.4±2.430	7.8781
II	Dibola 242 mg/kg	177.5±4.423	194.6±4.9760	8.7872
III	Diabetic Control (DC)	179.5±4.847	151.1±5.839	-18.79
IV	Alloxan+ Dibola 242 mg/kg (DD)	169.0±4.225	178.5±2.582	5.3221
V	Alloxan + Gilbenclamide 600 μg/kg (DGLIB)	173.9±6.605	180.5±2.131	3.6550

One-way ANOVA followed by Dunnett’s Multiple Comparison test. Values are expressed as meanSEM; n= 6.

Histopathology of liver:

Fig.1: Normal

Fig. 2: Diabetic control

Fig. 3: Alloxan + Diabola 242mg/kg treated rat.

Fig. 4: Alloxan + Gilbenclamide μg /kg treated rat.

Histopathology of Kidney:

Fig. 5: Normal

Fig. 6: Diabetic control

Fig. 7: Alloxan + Diabola 242mg/kg treated rat.

Fig. 8: Alloxan + Gilbenclamide μg /kg treated rat.

DISCUSSION:

Alloxan has been widely used for the induction of diabetes mellitus in various experimental animals, it produce diabetes mellitus by cytotoxic action on pancreatic β- cells results in insulin deficiency¹¹. Many reports have shown that progression of diabetes mellitus is also due to the generation of reactive oxygen species along with decrease in endogenous antioxidant defence against elevated free radical attack in pancreatic β- cells of alloxan induced diabetic rats.

The study reports show that, antihyperglycemic, antihyperlipidemic, antiperoxidative effect offered by Dibola 242 mg/kg was found to be significant as compaired to diabetic animals. The possible mechanism underlying these action is due to the active principles present in individual herb such as Curcuma Longa, Tinospora cordifolia, Azadirachta Indica, Momordia Charantia, Syzium Cumini, Berberis Aristata, Sympolocos Racemosa, Trignetia Foeumgraecum, Swertia Chirata, Trikatu, Pichrohiza Kurroa, Trikatu, Shilajit.

In this study we observed that Dibola decreases blood glucose level and increases plasma insulin level in alloxan diabetic rats. Diabetes mellitus is a metabolic disorder showing significant impact on lipid metabolism with alteration in blood lipids and lipoprotein profile. The deficiency of insulin alters the entire metabolism in the body including lipid metabolism. The abnormal high level of blood lipids in diabetes is mainly due to the increase in mobilization of free fatty acid from peripheral depots, increased lipolysis, as hormone sensitive lipase is not inhibited in diabetes due to the insulin deficiency¹². In present study, serum total cholesterol, triglyceride, VLDL, LDL cholesterol levels were elevated in untreated diabetic rats. Dibola treatment for 21 days in diabetic rats showed significant reduction in all these lipid profiles. The observation indicates that Dibola is beneficial in enhancing HDL cholesterol and lowering LDL, VLDL cholesterol, thereby reveals its usefulness therapeutic value.

It is evidenced that glucose lowering and insulin enhancing activity of Dibola is responsible for controlling and correcting the altered lipid profile; this effect of Dibola may be due to the effect of active constituents of different plants. Accumulation of triglycerides is one of the risk factors in Coronary Heart Disease (CHD). The significant increase in the level of triglycerides in blood serum of diabetic control rats may be due to the lack of insulin. Since under normal condition, insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides¹³. Dibola reduces triglycerides in blood serum of alloxan-induced diabetic rats and may prevent the progression of CHD. Increased lipid peroxidation impairs membrane functions by decreasing membrane fluidity and changing the activity of membrane-bound enzymes. Its products (lipid radicals and lipid peroxide) are harmful to the cells in the body and are associated with atherosclerosis and brain damage¹⁴. Administration of Dibola and Gilbenclamide reduced the lipid peroxidative markers in liver and kidney tissues of diabetic rats. This indicates that Dibola inhibit oxidative damage due to the antiperoxidative effect of ingredients present in Dibola.

Induction of diabetes with alloxan is associated with a characteristic loss of body weight, which is due to increased muscle wasting, loss of tissue proteins. The differences in the body weights observed during the period of treatment of the rats treated with Dibola and Gilbenclamide were less as compared to the diabetic control, which may be due to its protective effect in controlling muscle wasting, i.e. reversal of gluconeogenesis and may also be due to proper glycemic control¹⁵ . Diabetic treatment significantly increases the insulin level this is due to the regeneration or increased stimulation of insulin secretion from remnant pancreatic β cells. Many plants polyphenol such as flavonoids, condensed tannins, saponin and coumarins had shown antioxidant and lipid peroxidation inhibitory activity.

The reports of the study suggest that, the Dibola having antihyperglycemic antihyperlipidemic and antiperoxidative activity in alloxan induced diabetes model. So it can be considered as safe supplementary in management of diabetes mellitus and related complications. On the basis of above results, it could be concluded that Dibola a combination of herbal plants exert a significant antihyperlipidemic and antiperoxidative effect. This could be due to different types of active principles, each with a single or a diverse range of biological activities, which serves as a good adjuvant in the presence of antidiabetic drug.

CONCLUSION:

The liver and kidney exhibits numerous morphological and functional alterations during diabetes. Since both diabetes and hyperlipidemia are considered to be major risk factors for the premature atherosclerosis and essentially all the cholesterol in atherosclerotic plaques is derived from that of circulatory cholesterol. The antihyperlipidemic and antiperoxidative effect of Dibola in particular could be considered as of possible therapeutic value. The present study shows that the Dibola not only posses antihyperlipidemic properties but also reduces oxidative stress in diabetic rats. The effect produced by Dibola was comparable with that of Gilbenclamide.

ACKNOWLEDGEMENT:

The authors thankful to Sampurna Jeevan Pharmachem Pvt. Ltd. Ichalkaranji provided grants for this scientific work.

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Received on 16.12.2011

Modified on 12.01.2012

Accepted on 24.01.2012

Research J. Pharmacology and Pharmacodynamics. 4(2): March - April, 2012, 102-107