Diminution of oxidative stress in alloxan-induced diabetic rats by Stevia rebaudiana

 

Ena Gupta1, Abubakar Mohammed2, Shalini Purwar1, Syed Ibrahim Rizvi2,

Shanthy Sundaram1

1Centre of Biotechnology, University of Allahabad, Allahabad 211002, India

2Department of Biochemistry, University of Allahabad, Allahabad 211002, India

*Corresponding Author E-mail: enaravish@gmail.com

 

ABSTRACT:

OBJECTIVE: Stevia rebaudiana is one such herb commonly used as natural sweetener and have possible antidiabetic effects. One of the most common symptoms of diabetes is hyperglycemia which is associated with increased generation of free radicals and oxidative damage to tissue compounds. The aim of the present investigation was to evaluate the antihyperglycemic and antioxidative effect of ethanolic leaf extracts of Stevia rebaudiana in normal and alloxan induced diabetic experimental rats for 21 days.

METHODS: In the present investigation the experimental Wistar albino rats were divided into 7 groups containing 6 rats in each group (n=6) viz. non-diabetic (normal control), diabetic control (alloxan-induced), diabetic conventional treated (treated with insulin), normal treated (infused with different doses of Stevia rebaudiana, ethanolic extract) and diabetic treated (infused with different doses of Stevia rebaudiana, ethanolic extract). Diabetes was induced by a single intraperitoneal (i.p) injection of alloxan monohydrate (150 mg/kg body weight). Exposure of alloxan monohydrates elevates the blood glucose, increases the plasma malondialdehyde and sialic acid content which overall disturbed the intracellular antioxidant functions.

RESULTS: The oral administration of ethanolic leaf extracts of Stevia rebaudiana at a dose of 300 and 400 mg/kg body weight showed significant (p˂0.05) reduction in blood glucose and restores the antioxidant power and other biochemical parameters close to normal.

CONCLUSION: Stevia rebaudiana supplementation could minimize hyperglycemia and ameliorate the condition of oxidative stress in diabetes. Thus, it provides a beneficial role in preventing the complications of diabetes by scavenging free radicals in alloxan-induced diabetic rats.

 

KEYWORDS: Medicinal plants, Steviol glycosides, Hyperglycaemia, Antioxidant potential, Oxidative stress.

 

 


 

INTRODUCTION:

Diabetes mellitus (DM) commonly known as diabetes is a metabolic disorder of numerous etiologies, basically characterized by prolonged period of hyperglycaemia with disturbances of carbohydrate, protein and fat metabolism, resulting from defects in pancreas which consequences in inefficient secretion or action of insulin or both1.  The characteristic symptoms include polyuria, polyphagia, polydipsia, blurred vision and weight loss. The long term consequences of diabetes mellitus include body damage, dysfunction and failure of various organs. The untreated most serious forms, include ketoacidosis or a non–ketotic hyperosmolar state leading to coma and finally death.

 

The worldwide estimation as of 2015, the 415 million people have diabetes with 90 percent cases of type 2 DM and by 2040 it will rise to 642 million2. In 2030 it was expected that India, China and the United States will have maximum number of diabetes3.

 

Increased generation of reactive oxygen species (ROS) leads to oxidative damage of cellular structural components (proteins, lipids and DNA) and plays a pivotal role in cumulating pathologies like cancer, diabetes, neurodegreneration, hypertension, atherosclerosis, liver dysfunction and kidney disease. Several evidences suggest that oxidative stress influenced by the generation of excess free radicals or (ROS) weakens the defense mechanisms and further accelerate the oxidation process which increases cell damage, β-cell dysfunction, insulin resistance and impair glucose tolerance which increases the complications of diabetes4.  The reason for the oxidative insult in cells is due to impair functioning of endogenous antioxidant enzymes in neutralizing free radicals, caused by nonenzymatic glycosylation and oxidation. Literatures suggest that researchers are searching for naturally occurring plant derived antioxidants or traditional and alternative medicines to decrease oxidative-stress-induced damage in diabetic pathophysiology.

 

The medicinal plants are capable in combating various diseases. In India (Unani and Ayurveda) and other countries medicinal plants are used in preventing various diseases and are considered to be non-toxic and effective with no serious adverse effects. Numerous plants with their bioactive natural compounds and crude extracts are used in traditional medicine system as they increase the endogenous antioxidants and protect the repair systems and have shown experimental or clinical antidiabetic activity5.

 

From ancient times a number of Indian herbal preparations have been used in the treatment of diabetes. Stevia rebaudiana (sweet leaf) is one such natural herb belonging to Asteraceae family, commonly known as sweet or sugar leaf. In 1930’s scientists isolated the sweet tasting steviol glycosides stevioside and rebaudioside-A present in leaves of S. rebaudiana. These ingredients are 250-300 times sweeter than sucrose along with fewer calories and carbohydrate content. The S. rebaudiana leaf extracts are dense source of alkaloids, glycosides, flavonoids, phenols, chlorophylls, sugars and essential oils. Previously done studies report that leaves of S. rebaudiana appear to have possible antidiabetic effects in stimulating or regenerating pancreatic beta cells which facilitate better insulin secretion. Therefore, the present study focus on studying the in-vivo antidiabetic (hypoglycemic) effect and antioxidant potential of different doses of ethanolic extract of S. rebaudiana leaves on blood glucose, body weight and other biochemical parameters in normal and alloxan-induced diabetic rats.

 

MATERIALS AND METHODS:

Chemicals

All the reagents used to perform the experiment were of analytical grade. Alloxan monohydrate (2,4,4,6-tetra oxo hexahydropyrimidine) was procured from Loba Chemie, Mumbai, India, Insulin, used as a reference antidiabetic drug was purchased from SRL chemicals.  N-Acetylneuraminic acid (NANA) and resorcinol were purchased from Sigma Aldrich, India., ERBA Mannheim (Transasia Bio Medicals Ltd., Daman, India) kits were used for the estimation of total cholesterol, billirubin, alkaline phosphatase, urea, creatinine and uric acid.

 

Plant material

The fresh leaves of S. rebaudiana were collected from Bioved Research Institute of Agriculture and Technology, Allahabad which was taxonomically identified at Department of Botany, University of Allahabad, India. The stems and other unwanted parts were removed and cleaned with warm distilled water to remove the dirt particles, after draining out excess water, the leaves were dried in shade at temperature ranging from 25 - 30º C for 24 to 48 hours. The dried leaves were blended to reduce the size to a coarse powder and were vacuum packed to avoid the decomposition of various bioactive compounds 6.

 

Preparation of crude extracts

The coarsely powdered leaves of S. rebaudiana (250 g) was placed in a stoppered container with the 500 mL of ethanol and kept in a refrigerator for 42 hours with frequent agitation until the soluble matter has dissolved. The obtained mixture was strained and the marc (the damp solid material) was squeezed, and the combined liquids were clarified by filtration using Whatman No. 1 filter paper (W and R Balson Ltd, England) and concentrated to dryness at 40 - 60° C on hot water bath to get the semi solid crude extracts which were stored at 4° C in airtight bottles till further use. The leaf extract suspension was prepared in vehicle solution i.e. 0.1% dimethyl sulfaoxide (DMSO) at a dose of 300 and 400 mg/kg b.wt.

 

Administration of plant extracts

The S. rebaudiana leaves extract suspension was administered to the Wistar rats through force-feeding gastric gavage route. An infant oral feeding tube connected to a syringe containing the extract was inserted into the gastric region of the rat. To ensure maximum bioavailability the animals were fasted 30 min before and after the treatment7.

 

Diabetes induction

Alloxan monohydrate (2, 4, 5, 6-tetraoxyprimidine) dissolved in 0.9 % normal saline at a dose of 150 mg/kg body weight was injected intraperitoneally in overnight fasted Wistar albino rats8. Administration of alloxan in rats destroys insulin producing pancreatic beta cells and induces diabetes mellitus type 1. For the next 24 hours the rats were allowed free access to 50 % glucose solution to drink overnight intended for preventing hypoglycemic shock. After one week of alloxan injection the diabetes was confirmed by determining fasting blood glucose levels by means of a glucometer (Accu-Chek, Roche Diagnostics, USA). The Wistar albino rats showing fasting blood glucose levels (140 - 160 mg/dl) were selected for the study and were considered diabetic.

 

Crude drug dosage determination- Preliminary study

After one week of diabetes induction, the Wistar albino rats with blood glucose levels (140 - 160 mg/dl) were undergone through fasting for 18 h. The rats were randomly divided into different groups, with four rats in each group. Ethanol extract ranging from 100 to 500 mg/kg body weight were orally administered to the animals and they were observed for any changes in behaviour and activity,  after 5 h the blood glucose was determined9. The minimum dose that lowers maximum glucose level in blood was given through oral intubations for the repeated administration. The most effective dose for ethanolic extract was found to be 300 to 400 mg/kg body weight. The selected doses were orally administered daily till end of the experimental period (i.e., 21 days).

 

Experimental design

To carry out the experiment healthy Wistar albino rats of both the sexes (weighing 150-200 gms; 4-5 months old) were used for the study. Under standard laboratory conditions they were housed for one week with temperature controlled facility (25 ± 5° C) with 12 hours light-dark cycle and were fed with nutrient rich diet pellets rich in fat, protein and carbohydrates. After one week of stabilization period, the experimental rats were divided into 7 groups containing 6 rats in each group (n=6).

 

Group I: normal control (NC) receiving no treatment (without alloxan or supplementation ); Group II: diabetic control (DC) injected single dose of alloxan intraperitoneally8 both the groups I and II only receive vehicle (0.1% dimethyl sulfoxide (DMSO); 1 ml/kg body weight); Group III: diabetic insulin group (DI) injected subcutaneously with three units of NPH insulin twice (3-IU/kg)  a day; Group IV: normal with extract (NE, 300 mg/kg b.wt.); Group V: diabetic with extract (DE, 300 mg/kg b.wt.); Group VI: normal with extract (NE, 400 mg/kg b.wt.); Group VII: diabetic with extract (DE, 400 mg/kg b.wt.). Rats were administered ethanolic leaf extracts of S rebaudiana via oral route (gavage technique) which continued up to 21 days. Overnight fasted rats of groups V and VII were made diabetic by injecting alloxan monohydrate with a dose of 150 mg/kg body weight intraperitoneally.

 

Estimation of body weight

On (day 0) before treatment body weight of control group and all the treated groups of rats were recorded which continued during the period of 7th, 14th and 21st day. For taking the body weight of rats (expressed in gram) a calibrated and standardized electronic balance was used.

 

Blood samples collection and blood glucose estimation

The blood samples were collected from the rats tail tip on day 0 (pre-treatment) and during the treatment (7th, 14th and 21st day) for estimating blood glucose parameters (expressed as mg/dl) using glucose test strips of an electronic glucometer (Accu-Chek, Roche Diagnostics, USA).

 

Isolation of plasma and serum

At the end of experimental period, sacrificing of rats was done under the influence of light anesthesia. The cardiac was punctured with 10 unit/ml of heparin rinsed anticoagulant syringes (for serum collection, anticoagulant was not used) and blood samples were collected which were transferred into the test tubes and centrifuged at 800 g for 15 - 20 min at 4°C. The separated plasma was immediately stored at -80° C for further use in biochemical estimations. All the study protocols for the experiments were approved by the Animal Care and Ethical Committee of University of Allahabad.

 

Determination of erythrocyte malondialdehyde (MDA) content

According to the method of Esterbauer and Cheeseman10, erythrocyte MDA was measured with slight modification. 0.2 ml of packed erythrocytes was suspended in 3 ml PBS containing 0.5 mM glucose (pH 7.4). 0.2 ml of the suspension was added to 1 ml of 10% trichloroacetic acid and 2 ml of 0.67% thiobarbituric acid, the obtained solution was boiled for 20 min at 90–100°C and then cooled. Subsequently, the mixture was centrifuged at 1000g for 5 min and the absorbance of supernatant was read at 532 nm. For calculating the concentration of MDA in erythrocytes, extinction coefficient (ε = 31,500) was used and expressed as nmol·mL−1 of packed erythrocytes.

 

Determination of membrane and plasma sialic acid (NANA) levels

It was determined according to the method given by Spyridaki et al, 11.  Sialic acid level was estimated in membrane/plasma. In a glass tube, 0.10 ml of periodic acid-schiff (0.04 M) was added containing 500 μl diluted (20 times) sample solution. It was thoroughly mixed and allowed to stand in ice bath for 30 min. Subsequently, 1.25 ml of resorcinol working solution (5 ml of 6.0% resorcinol solution, 0.125 ml of 0.1 M copper sulphate solution and 19.875 ml of distilled water, and with 50 ml of 10 M HCl final volume was raised), the solution was mixed and heated at 98°C for 5 min. The tubes were allowed to cool in an ice bath for approximately 2 min. Finally 3.25 ml of n-butanol was added. The solutions were mixed vigorously and the tubes were placed in a water bath at 37°C for 3 min (for colour stabilization). Directly after removing the solutions from the water bath their absorbance were measured at 625 nm against a reagent blank set at zero. A calibration graph was prepared in the range of 20–200 μM with standard solutions of NANA and the unknown concentrations of total sialic acid in samples were calculated. Sialic acid level in membrane is calculated in terms of μg/mg membrane protein. Sialic acid in plasma is measured as μM.

 

Measurement of total antioxidant activity by FRAP

The total antioxidant potential of the plasma was estimated using a Ferric reducing ability of plasma (FRAP) assay reported by Benzie and Strain12 with slight modification. Briefly, working FRAP reagent was prepared from 300 mmol/Lacetate buffer (pH 3.6), 20 mmol/L ferric chloride and 10 mmol/L 2,4,6-tripyridyl-s-triazine made up in 40 mmol/L hydrochloric acid. In the ratio 10:1:1 (vol/vol/vol) all the three solutions were mixed together. The prepared FRAP reagent (3 ml) was mixed thoroughly with 100 μl of plasma and the absorbance was recorded at 593 nm at 30-s intervals for 4 min. For calibration aqueous solution of known Fe (II) concentration (100–1000 μmol/L) was used. FRAP values (μmol Fe (II)/L) in plasma was calculated by using regression equation.

 

Biochemical estimations

Earlier isolated heparinised plasma was used for estimating total cholesterol, billirubin, alkaline phosphatase, urea, creatinine and uric acid which was performed by using standard diagnostic kits.

 

Statistical Analysis

The obtained data was demonstrated as mean ± standard deviation. Statistical analysis was performed by using one way analysis of variance followed by Bonferroni test using graph pad prism software package, version 5.0. The values of P˂ 0.05 were considered as statistically significant.

 

RESULTS:

Effects on body weight

One of the major symptoms of diabetes is weight loss which was also seen in this study. In all groups there was a change observed in body weight of each rat noted on days 0, 7th, 14th and 21st. Administration of alloxan increases the reduction in body weight and leads to a condition of wasting as this drug damages the pancreatic beta cells and provoke a condition of insulin deficiency. Reversed condition was seen after seven days in groups treated with ethanolic leaf extracts of S. rebaudiana. There was a significant (P < 0.05) difference observed for the change in body weight of alloxan-induced diabetic control (DC) rats when compared with extract treated diabetic and normal rats (Fig 1).

 


 

 

Fig 1. Graphical representation of ethanolic leaf extract of S. rebaudiana on body weight (gm) of experimental rats. Group 1: normal control (NC); Group II: diabetic control (DC); Group III: diabetic with insulin (DI); Group IV: normal with extract (NE, 300mg/kg b.wt.); Group V: diabetic with extract (DE, 300mg/kg b.wt.); Group VI: normal with extract (NE, 400mg/kg b.wt.); Group VII: diabetic with extract (DE, 400mg/kg b.wt.).


The normal control (NC) group I were found to be stable in their body weights where as in group II (alloxan-induced negative control) rats shows significant decrease in body weight (138.26 g, 125.32 g and 115.11 g) on 7th, 14th and 21st days, respectively. Group III (positive control with standard drug Insulin) rats showed significant increase in the body weights (220.21 g, 225.56 g and 230.31 g) monitored during 7, 14 and 21 days of treatment (Fig 1). Rats in groups IV and V treated with ethanolic leaf extracts of (300 mg/kg) revealed significant increase in body weight (128.12 g and 132.51 g) while groups VI and VII (400 mg/kg) also showed significant increase in body weight (123.42 g and 157.27 g) on day 21st as compared with 0 day value.

Effect on blood glucose

There was a significant (P < 0.05) difference observed for the change in blood glucose of alloxan-induced diabetic control (DC) rats when compared with extract treated diabetic and normal rats. The ethanolic leaf extracts of S. rebaudiana (300 and 400 mg/kg) exhibited a significant (P < 0.05) dose-dependent blood glucose lowering effect in normal and alloxan-induced diabetic rats when compared with diabetic control (DC) (Table 1).


 

 

Table 1. Effect of ethanolic leaf extract of S. rebaudiana on blood glucose concentration

Blood glucose concentration (mg/dl)

Groups

Treatment

Day 0

Day 7

Day 14

Day 21

I

Normal Control

87.47±4.54

89.49±3.72

91.50±5.26

93.55±4.32

II

Diabetic Control

99.01±4.78

385.06±5.32

564.21±6.66

593.06±4.89

III

Diabetic+Insulin

85.12±4.21

109.23±4.24

107.43±5.67

103.06±4.98

IV

Normal + Extract (300mg/kg b.wt)

102.33±3.01

98.01±4.21

92.54±5.68

90.11±6.54

V

Diabetic+ Extract (300mg/kg b.wt)

88.21±5.54

109.34±4.56

107.25±5.78

95.16±4.12

VI

Normal+ Extract (400mg/kg b.wt)

100.15±4.21

91.02±4.89

89.52±4.68

85.79±5.12

VII

Diabetic+ Extract (400mg/kg b.wt)

99.14±4.98

106.09±6.65

101.11±4.12

95.27±5.17

Each value represents mean ± S.D., n = 6, p˂0.05 significant

 

 


Alloxan induced rats (negative control) of group II showed increased blood glucose (hyperglycemia) throughout the study period as they are not receiving any treatment (vehicle) where as alloxanized rats (positive control) of group III treated with antidiabetic reference drug insulin showed significant decline in blood glucose levels and maintained it to normal standard blood glucose level from 7th day to 21st day. Administration of ethanolic leaf extracts of S. rebaudiana at a concentration of (300 mg/kg) in normal and diabetic rats of groups IV and V showed normal blood glucose level (90.11 and 95.16 mg/dl) on 21st day, while normal and diabetic rats of groups VI and VII treated with ethanolic leaf extracts of S. rebaudiana (400 mg/kg) also exhibit remarkable decrease in blood glucose (85.79 mg/dl and 95.27 mg/dl), close to the value of day 0.

 

Erythrocyte malondialdehyde (MDA) content

The level of malondialdehyde content in plasma was significantly (P ˂ 0.05) increased in alloxan induced diabetic control (DC) rats when compared with normal control rats, insulin treated rats and extract treated diabetic and normal rats. Administration of ethanolic leaf extract of S. rebaudiana at a dose of (300 and 400 mg/kg/day) offered significant reduction in the level of malondialdehyde (MDA) content in normal and diabetic rats in groups IV - VII as shown in Figure 2.

 

Fig 2. Levels of malondialdehyde (MDA) content in normal control and experimental groups. The values represent the mean ± SE for six rats per group.  Group 1: normal control (NC); Group II: diabetic control (DC); Group III: diabetic with insulin (DI); Group IV: normal with extract (NE, 300mg/kg b.wt.); Group V: diabetic with extract (DE, 300mg/kg b.wt.); Group VI: normal with extract (NE, 400mg/kg b.wt.); Group VII: diabetic with extract (DE, 400mg/kg b.wt.).

Plasma sialic acid (NANA) levels

A significant (p˂0.05) reduction in plasma sialic acid content was observed in normal control rats in group I as compared to diabetic control group in group II. Administration of ethanolic leaf extracts (300 mg/kg and 400 mg/kg) of S. rebaudiana in normal and alloxan induced rats in groups IV - VII shows that diabetes induced rats retains more content of sialic acid in plasma as compared to normal rats. However, insulin treated diabetic rats in group III also shows a mild sialic acid lowering effect as shown in Figure 3.

 

Fig 3. Levels of plasm sialic acid content in normal, control and experimental groups. The values represent the mean ± SE for six rats per group.  Group 1: normal control (NC); Group II: diabetic control (DC); Group III: diabetic with insulin (DI); Group IV: normal with extract (NE, 300mg/kg b.wt.); Group V: diabetic with extract (DE, 300mg/kg b.wt.); Group VI: normal with extract (NE, 400mg/kg b.wt.); Group VII: diabetic with extract (DE, 400mg/kg b.wt.).

 

Total antioxidant activity by (FRAP) assay

The levels of FRAP content was found to be significantly (p˂0.05) lower in plasma of diabetic control rats as compared to normal control, insulin treated and extract treated groups (Fig. 5). Treatment with ethanolic leaf extract of S. rebaudiana at a dose of (300 and 400 mg/kg/day) improved the antioxidant power in normal and alloxan induced diabetic rats in groups IV – VII as shown in Figure 4.

 

Fig 4.  The total antioxidant activity (FRAP) in normal control and experimental groups. The values represent the mean ± SE for six rats per group.  Group 1: normal control (NC); Group II: diabetic control (DC); Group III: diabetic with insulin (DI); Group IV: normal with extract (NE, 300mg/kg b.wt.); Group V: diabetic with extract (DE, 300mg/kg b.wt.); Group VI: normal with extract (NE, 400mg/kg b.wt.); Group VII: diabetic with extract (DE, 400mg/kg b.wt.).

 

Effect on biochemical parameters

The results of ethanolic leaf extracts of S. rebaudiana at a dose of (300 and 400 mg/kg/day) on plasma total cholesterol, bilirubin (total and direct) urea, creatinine, uric acid and alkaline phosphatase of normal and alloxan induced diabetic rats after 21 days of treatment are presented in Table 2.

 


 

 

 

Table 2. Effect of ethanolic leaf extracts of S. rebaudiana on biochemical parameters in normal and alloxan induced diabetic rats after 21 days of treatment

Biochemical parameters

Groups

Treatment

Total Cholesterol

(mg/dl)

Bilirubin (Direct)

(mg/dl)

Bilirubin (Total)

(mg/dl)

Alkaline Phosphatase

(IU/L)

Urea

(mg/dl)

Creatinine

(mg/dl)

Uric acid

(mg/dl)

I

Normal Control

81.81±

0.59

0.24±

0.13

0.75±

0.08

198.64±

5.72

37.90±

2.15

1.23±

0.21

0.57±

0.04

II

Diabetic Control

98.62±

3.69

0.32±

0.11

3.01±

0.53

421.38±

7.69

61.10±

7.52

2.40±

0.08

0.96±

0.22

III

Diabetic+Insulin

85.55±

0.35

0.25±

0.02

1.61±

0.03

231.10±

15.00

40.21±

5.12

0.85±

0.81

0.37±

0.03

IV

Normal + Extract (300mg/kg b.wt)

88.64±

1.54

0.27±

0.06

1.52±

0.06

205.20±

7.50

39.12±

1.73

1.43±

0.32

0.23±

0.01

V

Diabetic+ Extract (300mg/kg b.wt)

91.41±

0.58

0.32±

0.06

1.58±

0.13

235.38±

17.00

41.72±

2.73

1.51±

0.36

0.43±

0.07

VI

Normal+ Extract (400mg/kg b.wt)

81.30±

0.61

0.24±

0.14

1.43±

0.02

191.71±

12.01

38.81±

4.12

1.39±

0.62

0.22±

0.01

VII

Diabetic+ Extract (400mg/kg b.wt)

86.65±

1.54

0.32±

0.07

1.36±

0.38

215.27±

11.21

37.12±

2.24

0.76±

0.08

0.38±

0.18

Each value represents mean ± S.D., n = 6, p˂0.05 significant.

 


There was significant (p˂0.05) increase in total cholesterol of alloxan-induced diabetic rats (98.62 mg/dl) as compared to normal rats (81.81 mg/dl). Insulin treated diabetic rats of group III, restore normal cholesterol level (85.55 mg/dl). Administration of ethanolic leaf extract of S. rebaudiana (300 mg/kg) in normal and diabetic rats of group IV and V shows significant decline in levels of total cholesterol (88.64 and 91.41 mg/dl) whereas rats in groups VI and VII (400 mg/kg) also showed significant decrease in total cholesterol (81.30 and 86.65 mg/dl) as shown in Table 2.

The levels of bilirubin (direct and total) as shown in table 2, was significantly higher in group II (alloxan induced negative control) rats (0.32 and 3.01 mg/dl) as compared to normal control rats of Group I (0.24 and 0.75 mg/dl). The insulin treated diabetic rats in group III shows normal parameters for direct and total bilirubin (0.25 and 1.61 mg/dl). The oral dose of ethanolic leaf extract of S. rebaudiana (300 mg/kg) in normal and diabetic rats of groups IV and V shows significant decrease in direct (0.27 and 0.32) and total bilirubin (1.52 and 1.58 mg/dl) whereas rats in groups VI and VII (400 mg/kg) also shows decreased direct (0.24 and 0.32) and total bilirubin (1.43 and 1.36 mg/dl).

 

The level of alkaline phosphatase in plasma was significantly increased in alloxan induced diabetic rats in group II (421.38 mg/dl) when compared to normal control rats in group I (198.64 mg/dl). Insulin treated group III also shows decrease in alkaline phosphate value (231.10 IU/L) Oral administration of ethanolic leaf extract of S. rebaudiana (300 mg/kg) in normal and diabetic rats in groups IV and V offered significant decline in the level of alkaline phosphatase (205.20 and 235.38 IU/L), while dose of (400 mg/kg) given to rats in groups VI and VII normal (191.71 and 215.27 IU/L) restored near to the normal level of alkaline phosphatase as compared to normal rats as shown in Table 2.

 

The alloxan induced diabetic rats of group II shows significantly (p˂0.05) higher concentration of urea (61.10 mg/dl), creatinine (2.40 mg/dl) and uric acid (0.96 mg/dl) as compared to normal rats in Group I for urea (37.90 mg/dl), creatinine (1.23 mg/dl) and uric acid (0.57 mg/dl). Administration of ethanolic leaf extract of S. rebaudiana (300 mg/kg) in normal and diabetic rats in groups IV and V significantly reduce the blood urea (39.12 and 41.72 mg/dl), creatinine (1.43 and 1.51) and uric acid (0.23 and 0.43) concentration, where as dose of (400 mg/kg) given to rats in groups VI and VII also showed decreased urea (38.81 and 37.12 mg/dl), creatinine (1.39 and 0.76) and uric acid levels (0.22 and 0.38 mg/dl). However, insulin treated diabetic rats in group III also shows decreased levels of urea (40.21 mg/dl), creatinine (0.85 mg/dl) and uric acid (0.37 mg/dl) as shown in Table 2.

DISCUSSION:

The long term complications of diabetes are related to persistently elevated blood glucose, reduction in body weight and consequent oxidative stress. There are many valuable drugs available in the market are in use for the treatment of diabetes mellitus like sulphonylureas (glibenclamide), biguanides (metformin) and insulin sensitizers (thiazolidinediones), but they have restricted use due to cost, secondary failure rates, limited pharmacokinetic properties and  additional side-effects13.  There are around 1,200 plants species are identified possessing hypoglycemic activity and tend to potentiate better insulin secretion without causing any adverse effects14.

 

The long term 21 day studies shows that there is a difference in initial and final body weight in various groups, showing a significant reduction in body weight of diabetic controls in comparison to normal control, insulin treated and extract treated experimental rats. Similar results were also observed by other researchers15.

 

Administration of ethanolic leaf extract of S. rebaudiana (300 and 400 mg/kg.bwt.) to alloxan induced diabetic rats exert potent in vivo hypoglycaemic or antidiabetic effect  by stabilizing the glucose homeostasis and maintaining the body weight without showing any adverse effect, unlike other synthetic drugs16. Earlier studies on phytochemical screening of ethanolic leaf extracts of S. rebaudiana shows that presence of bioactive plant derived constituents such as flavonoids, phenols, alkaloids, saponins and tannins might be responsible for the anti-diabetic activity which could be due to synergistic effect of these compounds inducing hypoglycaemic effect. They also act as excellent antioxidant and free radical scavengers which can cause inhibition of the cellular oxidative damage and lipid peroxidation16. According to Gregersen et al, 17 reported that supplementation of diterpene glycoside (stevioside) in a test meal in type 2 diabetic patients exert insulinotropic and glucagonostatic actions by reducing the post prandial blood glucose levels and improving the glucose metabolism.

 

Long term increase in blood glucose levels induces carbonyl stress which can further lead to increased lipid peroxidation18 Oxidative damage can occur due to increased concentration of lipid peroxidation which further increases hydroxyl and peroxy radicals leading to chronic uncontrolled diabetes.

 

The most common indicator used in estimating lipid peroxidation is MDA. Its increased concentration is observed in plasma and tissues of diabetic animals due to increased lipid peroxidation. In the present study, significant increase in MDA levels was found in the plasma of diabetic rats as compared to the corresponding control group. Administration of ethanolic leaf extract of S. rebaudiana (300 mg/kg and 400 mg/kg) decreased significantly (P ˂ 0.05) MDA in treated diabetic rats when compared with diabetic control rats. Recently, Shivanna et al, 19 studied that feeding Stevia leaves and its extracted constituents like polyphenols and fiber to streptozotocin induced diabetic rats, helps in reducing the MDA concentration in liver by inhibiting lipid peroxidation and improving its antioxidant status through antioxidant enzymes. Oxidative stress in diabetes has been linked with insulin resistance, which increases the generation of excessive reactive oxygen species (ROS) with a decline in the antioxidant power. According to Cakatay et al, 20 the total antioxidant capacity (FRAP) levels decreased significantly in plasma of chronic diabetic animals as compared to those of control groups. The present work showed significant decrease in plasma FRAP of diabetic rats when compared with normal control rats. Treatment with ethanolic leaf extract of S. rebaudiana (300 mg/kg and 400 mg/kg) significantly (P˂0.05) improve the antioxidant power in treated diabetic rats when compared with untreated alloxan induced diabetic groups. Oxidative stress increases in plasma sialic acid content in diabetes. In this study, a significant (P ˂ 0.05) elevation in levels of plasma sialic acid was observed in diabetic rats, oral administration of ethanolic leaf extract of S. rebaudiana (300 mg/kg and 400 mg/kg) to diabetic treated rats restores the levels of sialic acid in plasma to normal levels which is comparable with insulin.

 

The oral dose of plant extract (300-400 mg/kg) can improve the condition of diabetes mellitus in alloxan induced diabetic rats and maintain the stable condition in normal rats as indicated by the biochemical parameters like total cholesterol, bilirubin, alkaline phosphatise, urea, creatinine and uric acid.

 

The total cholesterol level is generally raised in case of diabetes mellitus which is an indication for hypertension and cardiovascular disease. In diabetes, due to deficiency of insulin the enzyme lipoprotein lipase does not get activated and results in hyper-triglyceridemia21. In case of diabetic dyslipidemia there is a change in lipid content with release of increased flux of free fatty acids from insulin resistant fat cells. Singh et al22, reported that administration  of methanolic leaf extract of S. rebaudiana in diabetic mice significantly reduce the levels of total cholesterol, triglycerides, low density lipoproteins and very low density lipoproteins with enhancement in the level of high density lipoproteins. Increase in levels of hepatic enzyme alkaline phosphatase (ALP) and bilirubin (direct and total) in diabetic controlled rats was related with liver dysfunction and its leakage in blood stream23.

At the beginning of diabetes, the size of kidney enlarges and the glomerular filtration rate (GFR) becomes disturbed with increased tissue proteolysis and decreased protein synthesis contributing to raised serum urea, creatinine and uric acid levels (Table 2). It is often associated with abnormal vasodilatation with the increased generation of reactive oxygen species (ROS) mediated by endothelial derived nitric oxide (NO), signifying a connection between vascular and metabolic abnormalities24. The effects of ethanolic leaf extract of S. rebaudiana at a dose of 300-400 mg kg−1 body weight bring back all the disturbed parameters in alloxan-induced rats to normal levels.

 

CONCLUSION:

From the above results, it can be concluded that ethanolic extract of S. rebaudiana leaves possess antidiabetic and antioxidant property. Analyzed data shows that S. rebaudiana possibly decrease the oxidative stress by reducing the plasma glucose levels in diabetic rats, which prevents excessive generation of reactive oxygen species (ROS) and providing protection against diabetic complications.

 

CONFLICT OF INTEREST STATEMENT:

No conflict of interest was declared by the authors.

 

ACKNOWLEDGEMENTS:

Financial support from the Department of Science and Technology (DST), New Delhi, India under a Women Scientist Project Scheme (WOS-A), vide letter no. [SR/WOS-A/LS-668/2012] is deeply acknowledged.

 

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Received on 29.07.2017          Modified on 13.09.2017

Accepted on 18.09.2017       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2017; 9(3): 158-166.

DOI: 10.5958/0975-4385.2017.00030.9