Antihyperlipidemic effect of Stevia rebaudiana on Alloxan Induced Diabetic Rats

 

T. Sudha¹*, D. Akila Devi², L. Kaviarasan³

¹Associate Professor, Department of Pharmaceutical Analysis, Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram Dist, Tamilnadu

²Department of Pharmaceutics, Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram Dist, Tamilnadu

³ Department of Pharmaceutical Chemistry, Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram Dist, Tamilnadu

 

ABSTRACT:

Stevia rebaudiana is one of the main constituent of various herbal formulations available for diabetes. The present study was aimed at assessing the antihyperlipidemic (Total cholesterol and triglycerides) effect of the aqueous extract of Stevia rebaudiana in alloxan induced diabetic rats. The powdered leaf was successfully extracted with water using maceration process. The wistar strains of male albino rats were used for the present study. The aqueous extract of Stevia Rebaudiana was administered intraperitoneally injection as a dose of 60mg/kg body weight to alloxan induced diabetic rats and it was found to reduce blood lipid level significantly (p<0.05). The plant extraction also exhibit correction of altered biochemical parameters like SGOT and SGPT levels in diabetic rats. The study concluded that Stevia Rebaudiana possess hypolipidemic effect in experimental diabetic rats.

Keywords:

 

 

 

INTRODUCTION:

Hyperlipidemia is an elevation of lipids in the blood stream. These lipids include cholesterol esters phospholipids and triglycerides¹. They are transported in the blood as a part of large molecules called lipoproteins. The predictor of coronary artery disease is hyperlipidemia, which is mainly an increased level of total cholesterol along with a decrease in high density lipoprotein cholesterol². It is an important risk factor in initiation and progression of atherosclerotic impasse³.

 

Hypercholesterolemia is common complications of diabetes mellitus in addition to hyperglycemia. The frequency of hyperlipidemia with diabetes is high depending upon type of diabetes and its degree of control. Stevia a sweet herb is used as non-caloric sweetener⁴. It is safe for diabetics and replaces the chemical sweetener like table sugar. The sweetness in leaf is due to presence of sweetening agent called stevioside. Stevia is helpful in weight and blood pressure management. The plant Stevia finds variety of usage in antioxidant activity, anti-inflammatory activity, bactericidal activity, antihypertensive activity, antiviral and gastroprotective activity^5-13. The rationale behind the present study is to evaluate the antihyperlipidemic activity in aqueous extract of Stevia rebaudiana on normal and alloxan induced diabetic rats.

MATERIALS AND METHODS:

Plant Materials:

Fresh leaves of Stevia rebaudiana were collected from Salem and Dharmapuri district of Tamilnadu. The plants were authenticated by Dr. P. Jayaraman, Director, Plant Anatomy Research Centre (PADC), Sakthinagar, West Tambaram, and Chennai. After thorough washing the leaves were dried completely under mild sun and ground in electric grinder into a coarse powder.

 

Extraction of the plant material:

The dried coarsely powdered leaves of Stevia rebaudiana were extracted by cold maceration process. The aqueous extract of Stevia rebaudiana was prepared by weighed quantity of powder was placed into the maceration tank and 10 volumes of water was added. This content was macerated for 24 hours, first six with occasional shaking and allowed to stand for remaining hours. After 24 hours of maceration the extract was filtered and solvent evaporated to get dried extract. This dried extract was used for various experimental purposes.

 

Selection of Experimental Animals:

In the present study, healthy adult male albino rats were used as experimental animal. A total number of 18 long-Evans male rats weighing about 120-240gm, age 1 month were selected and housed five in a polypropylene cage.  Prior to commencement of the experiment all the rats were acclimatized to the new environmental condition for a period of two weeks. During the experimental period the rats were kept in ventilated animal house at room temperature of 25˚C.

 

Grouping of experimental rats:

18 long –Evans male rats were randomly assigned into 3 groups, 6 rats in each group.

Group 1   Normal control

Group 2   Diabetic control

Group 3   Diabetic + Aqueous fraction SR (100mg/kg)

 

Experimental induction of diabetes:

Group 1 animals were used for normal control receives only vehicles (normal Saline) Group 2 animals were allowed to fast for 12 hrs and were induced diabetic by injection intraperitoneally a freshly prepared solution of alloxan (60mg/kg) in normal saline after base line glucose estimation was done. The alloxan treated animals were allowed to feed over night to overcome drug induced hyper glycemia. Group 3 animals were treated intraperitoneally with single dose of alloxan and co-administrated with aqueous extract of stevia rebaudiana (100mg/kg).

 

Test of Anti hyperlipidemic effect of plant extracts:

The blood was collected from hearts after sacrificing rats. Then the serum was separated by centrifugation at 2500 rpm. The serum was then stored in the refrigerator for 48 hours. It was used for the analysis of estimation of glucose, estimation of protein, determination of Albumin and determination of Globulin. The concentrations were absorbance by UV spectrophotometer (Shimadzu UV-1200, Tokyo, Japan).

 

Determination of Total Serum Cholesterol (ZAK’s method):

About 0.1 ml of serum was added to 4.9 ml of ferric chloride precipitating agent and centrifuged for about 15 minutes. Then the solution was filtered. From the above solution 2.5 ml of filtrate was taken in a series of test tube and 2.5 ml of ferric chloride diluting agent and 4 ml of concentrated sulphuric acid were added and mixed well. The serial dilutions were made 0.5 to 2.5ml from working standard corresponding to concentration of 5-25mg and 4ml of concentrated sulphuric acid was added and made upto 5ml of ferric chloride diluting agent. 5ml of ferric chloride solution was used as blank. The colour developed in the solution was measured at 560nm.

 

Estimation of Serum Triglycerides:

About 1ml of working reagent (Buffer pH 7.5-50mM/lit,-Chlorophenol-5mM/lit, 4-Aminoantipyrine-0.25mM/lit, Magnesium ions 4.5mM/lit, Adenosine triphosphate-2 mM/lit, lipase-1.34µg/ml, Peroxidase-0.54 µg/ml, glycerol kinase-0.4 µg/ml, glycerol -3-phosphate- 1.54 µg/ml) was added in three test tubes marked as blank, standard (10µl of Triglycerides 200mg/dl) and sample containing 10µl of plasma. The contents were mixed well and incubated for 5 min at 37˚C and the absorbance was measured at 500nm.

 

Estimation of HDL Cholesterol:

About 0.5ml of plasma solution was taken in a centrifuge tube and 50ml of ferric chloride precipitating reagent was added. It was mixed thoroughly in a vortex mixer for 30sec. The test tubes were allowed to stand for 10 minutes at 15 -25˚C. Then the tubes were centrifuged at 2000rpm for 15 minutes and supernatant solution was used for high density lipoprotein (HDL) cholesterol estimation.

 

About 1ml of working reagent taken in a test tube and 10ml of supernatant solution was added and mixed well. Then the solution was incubated for 10 min at 37˚C before taking the absorbance or readings at 500nm.

 

Determination of LDL and VLDL Cholesterol:

Low density lipoprotein (LDL) cholesterol and very low density lipoprotein (VLDL) cholesterol levels were calculated from the estimated total cholesterol triglycerides and high density lipoprotein cholesterol. The following formula was used to calculate LDL and VLDL Cholesterol.

Triglycerides

VLDL = ------------------------------------------ 

                                            5         

LDL = Total Cholesterol – (HDL+VLDL)

 

Estimation of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT):

About 0.5 to 2.5ml of working standard was taken in a series of test tube and made upto 2.5ml with distilled water. The concentration of solution was found to be 0.05-0.25moles. The blank test tube containing 2.5ml of distilled water. 1ml of DNPH (Dinitrophenyl hydrazine) reagent and 5ml 0.4N sodium hydroxide solution were transferred into all the test tubes. The optical density was measured at 520nm and a standard graph was plotted. The sample concentration was estimated by following the above procedure using standard calibration curve. The difference between the test and control was plotted in the standard curve and from the curve the activity of SGPT were determined.

 

RESULTS AND DISCUSSION

Lipid profile

Biochemical analysis on lipid profile was carried out in the serum of control alloxan induced diabetic rats treated with the stevia plant and the results are tabulated in table No 1 and illustrated in figure 1. Alloxan induced diabetic rats showed sharp and significant elevation of cholesterol when compared to control rats was due to increase in mobilization of free fatty acids from the peripheral fat depots to meet the energy demands carbohydrate metabolism impaired by alloxan treatment¹⁴. The diabetic rats treated with Stevia extract showed decrease in total serum cholesterol by 20% as compared to diabetic rats. It showed the hypocholestremic effect of stevia plant. A similar observation has been reported earlier^15.

 

Table -1 Determination of Total cholesterol

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	98.50 ± 2.10 150.68 ± 28.37	Group-I Vs Group-II	52.98(↑)	P˂0.01
Group-1 Group- III	98.50 ± 2.10 120.33 ± 6.33	Group-I Vs Group-III	22.17(↑)	P˂0.01
Group-1I Group- III	150.68 ± 28.37 120.33 ±6.33	Group-II Vs Group-III	20.14 (↓)	P >0.05

 

 

Figure-1 Estimation of Serum Cholesterol

 

Serum triglycerides:

The triglycerides (TG) level in rats diabetic male rats was higher than the control as represented in table No 2 and illustrated in figure.2. But it was found to be decreased by 20%. In diabetic rats treated with Stevia plant increased in TG level in alloxan induced diabetic rats could be due to the rate of mobilization of free fatty acid in order to meet the energy requirements carbhohydrate metabolism was impaired in diabetic rat. The decrease in level of TG in Stevia treated rats indicated not only in antidiabetic effect of the plant and also its hypolipidemic effect Hypertriacylglycerolimia found in uncontrolled diabetes mellitus is due to increased mobilization of adipose tissue fat¹⁶. The hypolipidemic effect of Stevia may be directly connected to the improvement in insulin levels.

 

Table -2 Determination of Serum Triglycerides (TG)

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	110.48 ± 6.82 250.33 ± 17.88	Group-I Vs Group-II	126.58(↑)	P˂0.01
Group-1 Group- III	110.48 ± 6.82  200.23 ± 17.81	Group-I Vs Group-III	81.23(↑)	P˂0.01
Group-1I Group- III	250.33 ± 17.88  200.23 ±17.81	Group-II Vs Group-III	20.01 (↓)	P˂0.01

 

Figure  2 Estimation of Serum Triglycerides

 

High density lipoprotein cholesterol (HDLc):

HDL cholesterol is also known as reverse cholesterol as its transporting cholesterol from various hepatic tissues to liver for its final catabolism or disposal. So the amount of cholesterol is degraded. Alloxan induced diabetic rats decrease in level of HDL- cholesterol when compared to control rats but the diabetic rats treated with stevia plant showed increase in level of HDL-cholesterol by 21 % when compared diabetic rats. It showed the cardioprotective effect of plants as indicated in table No 3 and figure. 3.

Table -3 Determination of Serum HDL Cholesterol

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	45.22 ±11.14 30.55 ± 4.73	Group-I Vs Group-II	32.44(↓)	P˂0.01
Group-1 Group- III	45.22 ±11.14 40.02 ± 7.75	Group-I Vs Group-III	11.50 (↓)	P>0.05
Group-1I Group- III	30.55 ± 4.73 40.02 ± 7.75	Group-II Vs Group-III	30.99 (↑)	P>0.05

 

 

Figure  3 Estimation of serum HDL Cholesterol

 

Very low density lipoprotein cholesterol:

The VLDL cholesterol was increased in alloxan induced diabetic rats by 26% when compared to that control group. The concentration of total cholesterol VLDL and LDL in the serum were significantly increased alloxan induced diabetic rats. The rats treated with stevia showed mild increase in VLDL cholesterol as compared to control rats but the cholesterol level is increased by 8 % in diabetic rats. VLDL derived from the liver for the export of triacylglycerol. VLDL is vehicles of transport of tricaylgycerol from the liver to the extrahepatic tissues¹⁷. The reports of analysis were showed in table No 4 and figure. 4.

 

Table -4 Determination of Serum VLDL Cholesterol

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	20.05 ±3.59 25.25 ± 4.22	Group-I Vs Group-II	25.94(↑)	P˂0.05
Group-1 Group- III	20.05 ±3.59 23.33 ± 3.48	Group-I Vs Group-III	16.38 (↑)	P>0.05
Group-1I Group- III	25.25 ± 4.22 23.33 ± 3.48	Group-II Vs Group-III	7.59 (↓)	P>0.05

 

 

Figure  4 Estimation of serum VLDL Cholesterol

 

 

Low Density lipoprotein cholesterol:

LDL cholesterol level in alloxan induced diabetic rat was higher than normal rat. The increase in level of LDL cholesterol noticed in diabetic rats was very high (189%) when compared to control rats. But the rats treated with stevia showed decreased 25 % in LDL cholesterol level as compared to diabetic rats though it was higher when compare to diabetic rats. LDL regulates the rate of cholesterol biosynthesis in extrahepatic tissues by delivering hepatic cholesterol to those tissues¹⁸.The greater the mobilization of free fatty acid greater the cholesterol ester synthesized in liver with greater LDL. This was one of cause of LDL cholesterol in uncontrolled diabetes.¹⁹. The results were shown in Table No 5 and figure. 5.

 

Table -5 Determination of Serum LDL Cholesterol

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	55.58 ±4.73 160.60 ± 21.12	Group-I Vs Group-II	188.94(↑)	P˂0.01
Group-1 Group- III	55.58 ±4.73 120.98 ± 6.17	Group-I Vs Group-III	117.66 (↑)	P˂0.01
Group-1I Group- III	160.60 ± 21.12 120.33 ± 6.17	Group-II Vs Group-III	24.67 (↓)	P˂0.01

 

Figure  5 Estimation of LDL Cholesterol

 

Serum glutamate oxaloacetate transferrase:

SGOT is the market enzyme of impairment of liver function. It is found to be elevated by 18% in alloxan induced diabetic rats. But it was decreased by 11% in the rats treated with stevia plant. It showed that stevia plant. It was proved that stevia plant possess hypoglycemic effect and improved liver function. The concentration of enzyme is very high myocardium. In acute myocardial infarction, serum activity rises rapidly. The elevation of SGOT is seen in muscle and hepatic diseases²⁰. Male albino rats receiving leaf extracts exhibit only moderate changes are evident in all groups. The results were shown table 6 and figure 6.

 

 

Table -6 Determination of SGOT

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	25.53 ±3.15 30.25 ± 1.42	Group-I Vs Group-II	18.47(↑)	P˂0.05
Group-1 Group- III	25.53 ±3.15 27.02 ± 3.68	Group-I Vs Group-III	5.81 (↑)	P>0.05
Group-1I Group- III	30.25 ± 1.42 27.02 ± 3.68	Group-II Vs Group-III	10.69 (↓)	P>0.05

 

 

Figure  6 Estimation of SGOT

 

Serum glutamate pyruvate transferrase (SGPT):

This enzyme also shows significant elevation (69%) after alloxan administration. SGPT level in alloxan treated group was significantly elevated as compared to control rats. This enzyme showed marked increase in viral hepatitis in cirrhosis liver and in case of skeletal muscle disease²¹. However the increase was marginal and it was less than 23% in stevia treated rats when compared to alloxan treated diabetic rats within the control in these groups that receive leaf extracts. The SGPT enzyme is more specific to liver disease than SGOT. So increase in level of SGPT absorbed in alloxan treated rats indicates impairment of liver function but it is reversible when the animals are treated with stevia plant. The report analyses were shown in table 7 and figure 7.

Table -7 Determination of SGPT

Group of Animals	Mean value ± SD	Group Compared	% Increase (↑) % Decrease (↓)	t-Value
Group-1 Group- II	12.02 ±2.65 20.30 ± 1.90	Group-I Vs Group-II	68.93(↑)	P˂0.01
Group-1 Group- III	12.02 ±2.65 15.53 ± 0.70	Group-I Vs Group-III	29.26 (↑)	P˂0.05
Group-1I Group- III	20.30 ± 1.90 15.53 ± 0.70	Group-II Vs Group-III	23.48(↓)	P˂0.01

 

Figure  7 Estimation of SGPT

 

REFERENCES:

1.     Roberts CK, Barnard RJ, Sindhu RK, et al. Oxidative stress and dysregulation of NADPH oxidase and antioxidant enzymes in diet induced metabolic syndrome. Metabolism. 55; 2006: 928-934.

2.     Deng R. Food and food suppliments with hypercholesterolemic effects. Recent patents on food. Nutrition and Agriculture. 1; 2009:15-24.

3.     Harrison D, Kathy KG, Hornig B,Drexler H. Role of oxidative stress in atherosclerosis. Am.j.Card. 91; 2003: 7A-11A.

4.     Cardello HM, Da Silva MA and Damasio MH. Measurement of the relative sweetness of stevia extract, aspartame and cyclamate/ saccharin blend as compared to sucrose at different concentrations, Plant foods Hum Nutr. 54; 1994:119-130.

5.     Bracht AK, Alvarez M and Bracht A. Effects of Stevia rebaudiana natural products on rat liver mitochondria. Biochem Pharmacol. 1985; 34: 873-882.

6.     Yasukawa K, Kitanaka S and Seo S. Inhibitory effect of stevioside on tumour promotion by 12-O- tetradecanoylphorbol, 13-acetate in two –stage carcinogenesis in mouse skin. Bio Pharm Bull. 2002; 25: 1488-1490.

7.     Boonkaewwan C, Toskulkao C and Vongasakul MJ. Antiinflammatory and immunomodulatory activities of stevioside and its metabolite steviol on THP-1 cells. J Agric food Chem.54; 2006:785-9.

8.     Sekhar I, Kaul A, Bani S, Pal HC et al. Immune up regulatory response of a non-caloric natural sweetener, stevioside. Chemico-Biological Interactions. 173; 2008: 115-121.

9.     Brusick J. A critical review of the genetic toxicity of steviol and steviol glycosides, Food Chem Toxicol. 46(7); 2008: S83-S91.

10.   Melis MS. Chronic administration of aqueous extract of Stevia rebaudiana in rats: renal effects, renal effects. J Ethnopharmacol. 47; 1995: 129-134.

11.   Melis MS. A crude extract of Stevia rebaudiana increases the renal plasma flow of normal and hypertensive rats. Braz J Med Biol Res. 29; 1996: 669-675.

12.   Takahashi K, Mori S, Sato N and Shigeta S. Extracts of Stevia rebaudiana is a potent anti-rotavirus inhibitor in vitro and in vivo. Antiviral Res. 46; 2000: A67.

13.   Shiozaki K, Fujji A, Nakano T et al. Inhibitory effects of hot water extract of the Stevia stem on the contractile response of the smooth muscle of the guinea pig ileum, Biosci Biotechnol Biochem. 70; 2006: 489-494.

14.   Kole GW, Headley J, Skowskey R, et al. Scleredema diabetic corum A common and district cutaneous manifestation of diabetes mellitus. Diabetes Care. 6;1983:189-192.

15.   Brownlee M, Vlasasara H, Kooney A et al. Aminiguanidine prevents diabetes- induced arterial wall protein cross-linking. Science 232; 1986: 1629-1632.

16.   Rosenb loom AL, Frais Jl. Diabetes mellitus, short stature and joint stiffness –a new syndrome, Clin Res 22, 1974: 92A.

17.   Rahber S. An abnormal haemoglobin in red cells of diabetes. clin chim Acts, 22; 1968: 296-298.

18.   Creutz feldt W, frenichs H, Sickinger K. Liver diseases ad diabetes mellitus. Prog Liver Dis. 13; 1970:371-407.

19.   Chatilia R, West AB. Hepatoegaly and abnormal liver tests due to glycogenesis with diabetes. Med Balt 75; 1996: 327-333.

20.   Katz LD, Glickman NMG, Repaports et al. Splanchnic and peripheral disposal of oral glucose in man. Diabetes 32;1983; 675-79.

21.   Mann FC, Magath TB. Studies on the physiology of the liver II. The effect of the removal of the liver on the blood sugar level. Arch Intem Med 30; 1992: 73-84.

 

 

 

 

 

 

Received on 15.09.2017                Accepted on 19.10.2017               

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