Indian Journal of Experimental Biology
Vol. 42, October 2004, pp. 989-992

Antioxidant effect of Boerhavia diffusa L. in tissues of alloxan induced
diabetic rats

M Amarnath Satheesh & L Pari*

Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608 002, India

Received 6 February 2004; revised 27 May 2004

Administration of B. diffusa leaf extract (BLEt; 200 mg/kg) for 4 weeks resulted in a significant reduction in thiobarbutric acid reactive substances and hydroperoxides, with a significant increase in reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase and glutathione –S- transferase in liver and kidney of alloxan induced diabetic rats. The results suggest that BLEt has remarkable antidiabetic activity and can improve antioxidant status in alloxan induced diabetic rats.

Keywords: Boerhavia diffusa, Alloxan diabetes, Lipid peroxidation, Enzymic antioxidants

IPC Code: Int Cl7 A61P

 

The harmful influence of diabetes mellitus on metabolism of tissues and organ is well known. Insulin is a major anabolic hormone in the body, and therefore, derangement of insulin function affects not only glucose metabolism but also fat and protein metabolism in the majority of tissues1. Glucose control plays an important role in the pro-oxidant /antioxidant balance. Macromolecules such as molecules of extra cellular matrix, lipoproteins and deoxy ribonucleic acid are also damaged by free radicals in diabetes mellitus2.

    The roots of Boerhavia diffusa L. possess diuretic action3, anti-inflammatory4, antifibrinolytic5, anti­convul­sant6 and hepatoprotective activities7,8. Its leaf extract has hypoglycemic effects9. In the present communication, the effects of B. diffusa leaf extract (BLEt) on antioxidant status in liver and kidney of alloxan diabetic rats are reported.

 

Materials and Methods

    Plant material¾Boerhavia diffusa leaves were collected freshly from Chidambaram, Cuddalore district. The plant was identified at the herbarium of Botany Department of the Annamalai University. A voucher specimen (No. 2865) was deposited.

    Preparation of plant extract¾B. diffusa leaves (500g) were chopped into small pieces, extracted with 1500 ml water by the method of continuous hot extraction at 60°C for 6 hr and evaporated. A dark semi-solid (greenish–black) material was obtained (22.5 g). It was stored at 4°C until used. When needed, the residual extract was suspended in distilled water and used in the study10.

    Animals¾Albino rats weighing 160–200g body weight were obtained from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College, Annamalai University. All animal experiments were approved by the ethical committee (Vide. No: 64, 2002), Annamalai University and were in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. Before and during the experiment, rats were fed with normal laboratory pellet diet (Lipton India Ltd, India) and water ad libitum. After randomization into various groups, the rats were acclimatized for a period of 2-3 days in the new environment before initiation of experiment.

    Chemicals¾Alloxan monohydrate was purchased from BDH Chemicals Limited, Poole, England. Boehringer Mannheim GmbH Kit (ELISA-Principle) was used for insulin assay. All the biochemicals and chemicals used in the experiment were of analytical grade and purchased locally.

    Induction of experimental diabetes¾The rats were injected with alloxan monohydrate dissolved in sterile normal saline at a dose of 140 mg/kg body weight, ip11. After 2 weeks, rats with moderate diabetes having glycosuria (indicated by Benedict’s qualitative test) and moderate hyperglycemia (200 – 280 mg/dl) were used for the experiment.

    Experimental design¾In the experiment, a total of 30 rats (18 diabetic surviving rats, 12 normal rats) were used. The rats were divided into following 5 groups of 6 each after the induction of alloxan diabetes:

Group 1:       Normal untreated rats.

Group 2:       Normal rats given BLEt 200 mg/kg body weight in aqueous solution daily using an    intragastric tube for 4 weeks.

Group 3:       Diabetic control.

Group 4:       Diabetic rats given BLEt 200 mg/kg body weight 9 in aqueous solution daily using an   intragastric tube for 4 weeks.

Group 5:       Diabetic rats given glibenclamide 600 µg/kg body weight12 in aqueous solution daily   using an intragastric tube for 4 weeks.

   

    Sample collection¾At the end of 4 weeks, the animals were deprived of food overnight and sacrificed by decapitation. Fasting blood samples were collected in fresh vials containing sodium fluoride and potassium oxalate (anticoagulant agent) for the estimation of glucose. Plasma was separated for the estimation of insulin. Liver and kidney were dissected out, washed in ice-cold saline, patted dry and weighed.

    Biochemical measurements¾Fasting blood glucose13, thiobarbituric acid reactive substances (TBARS)14, hydroperoxides15, reduced glutathione (GSH)16, superoxide dismutase (SOD)17, catalase18, glutathione peroxidase (GPx)19 and glutathione-S-transferase (GST)20 were determined.

    Statistical analysis¾Statistical analysis was done by analysis of variance (ANOVA) followed by Duncans Multiple Range Test (DMRT).

 

Results and Discussion

    The results are shown in Tables 1-3. The BLEt leaves extract produced a marked decrease in blood glucose at 200mg/kg body weight in normal as well as in alloxan diabetic rats after 4 weeks treatment. These findings are in agreement with those reported by Chude et al9. The antidiabetic effect of BLEt may be due to increased release of insulin from the existing b cells of pancreas similar to that observed after glibenclamide administration.

 

 

Table 1¾Changes in levels of blood glucose and plasma insulin of normal and experimental animals
[Values are given as mean
± SD for 6 rats in each group]

 

 

Groups

Fasting blood glucose (mg/dl)

Plasma insulin (mU/ml)

Normal

91.99 ± 6.28a

17.18 ± 0.84a

 

Normal + BLEt

81.01 ± 5.83b

19.76 ± 1.18b

 

Diabetic control

257.18 ± 12.54c

4.92 ±0.30c

 

Diabetic +  BLEt

129.92 ± 8.03d

10.40 ±0.63d

 

Diabetic + glibenclamide

135.70 ± 9.91d

9.74 ±0.57d

 

Values not sharing a common superscript letter differ significantly at P<0.05 (DMRT).

Duncan procedure, Range for the level 2.91, 3.06, 3.16, 3.22.

 

 

 

Table 2¾Changes in levels of Tbars, hydroperoxides and reduced glutathione in liver and kidney of normal and experimental animals
[Values are given as mean
± SD for 6 rats in each group]

 

Groups

 

TBARS

Hydroperoxide

Reduced glutathione

(mM/100g tissue)

(mg / 100 mg tissue)

Normal

Liver

0.87± 0.03a

76.17 ± 2.79a

46.91±2.08a

Kidney

1.59± 0.07ab

56.35 ± 2.23a

31.08 ± 2.15a

Normal +BLEt

Liver

0.82±0.02 a

72.07±3.25 a

49.95± 2.91a

Kidney

1.51±0.06a

52.09±2.17 b

34.83 ± 2.48a

Diabetic control

Liver

2.04 ± 0.11b

101.70 ±6.05b

23.35±0.80b

Kidney

2.25 ± 0.19c

79.14 ± 4.49c

20.53 ± 0.90b

Diabetic + BLEt

Liver

1.36 ± 0.05c

84.18 ± 4.83c

41.29± 1.86c

Kidney

1.73± 0.11bd

62.75 ± 3.07d

26.83 ± 1.46c

Diabetic + Glibenclamide

Liver

1.59 ± 0.06d

90.03 ± 4.32d

39.66 ± 1.43c

Kidney

1.86 ±0.12d

66.94 ± 3.24d

25.08 ± 1.27c

Values not sharing a common superscript letter differ significantly at P<0.05 (DMRT).

Duncan procedure, Range for the level  2.91, 3.06, 3.16, 3.22.

 

 

Table 3¾Changes in activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione–S–transferase (GST) in liver of normal and experimental animals
[Values are given as mean
± S D for 6 rats in each group]

 

Groups

 

CatalaseA

Superoxide dismutaseB

Glutathione peroxidaseC

Glutathione–S–transferaseD

Normal

Liver

72.03 ± 4.39a

6.33 ± 0.30a

6.57 ± 0.32a

6.19 ± 0.44a

Kidney

33.78 ± 2.00a

14.53 ± 0.72a

4.66 ± 0.27a

5.50 ± 0.23a

Normal + BLEt

Liver

73.63±4.87a

6.52±0.39 a

6.82±0.41 a

6.65±0.48 b

Kidney

35.11±2.35 a

15.78±0.82 b

5.26±0.32 b

5.89± 3.35 b

Diabetic control

Liver

44.97 ± 2.00b

4.16 ± 0.13b

4.45 ± 0.17b

3.24 ± 0.18c

Kidney

23.19 ± 0.75b

9.86 ± 0.44c

2.56 ± 0.11c

2.63 ± 0.12c

Diabetic + BLEt

Liver

66.37 ± 3.02c

5.85 ± 0.20c

5.68 ± 0.23c

5.64 ± 0.30d

Kidney

28.40 ± 1.61c

13.80 ± 0.64a

4.00 ± 0.20d

4.83 ± 0.20d

Diabetic + glibenclamide

Liver

62.71 ± 2.80c

5.63 ± 0.18c

5.12± 0.20d

4.96 ± 0.25d

Kidney

25.57 ± 1.35d

11.57 ± 0.52d

3.55 ± 0.19e

3.77 ± 0.13e

Values not sharing a common superscript letter differ significantly at P<0.05 (DMRT).

Duncan procedure, Range for the level  2.91,3.06, 3.16, 3.22.

A = m mole of H2O2 consumed / min/mg protein

B = One unit of activity was taken as the enzyme reaction which gave 50% inhibition of NBT reduction in one min

C = mg of GSH consumed / min/mg protein

D = m moles of CDNB – GSH conjugate formed / min/mg protein

 

 

 

    Lipid peroxidation is one of the characteristic features of chronic diabetes. It has been observed that insulin secretion is closely associated with lipoxygenase derived peroxides21,22. The reduction of two electrons from alloxan gives dialuric acid, which undergoes oxidation and leads to generation of O2, H2O2 and OH•23. Dialuric acid has been observed to stimulate lipid peroxidation in vitro. In this context, a marked increase in the concentration of TBARS and hydroperoxides were observed in liver and kidney of diabetic rats. Increased lipid peroxide concentration in the liver and kidney of diabetic animals has already been reported24. Administration of BLEt and glibenclamide significantly decreased the levels of TBARS and hydroperoxides in diabetic rats.

     Glutathione (GSH), a tripeptide present in all the cells is an important antioxidant25. Decreased glutathione levels in diabetes has been considered to be an indicator of increased oxidative stress26. GSH also functions as free radical scavenger and in the repair of radical caused biological damage27,28. A decrease was observed in GSH in liver and kidney during diabetes. The decrease in GSH level represents increased utilization due to oxidative stress29. Administration of BLEt and glibenclamide increased the content of GSH in liver and kidney of diabetic rats.

     SOD is an important defense enzyme which catalyses the dismutation of superoxide radicals30. Catalase is a hemeprotein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals31. Therefore, reduction in the activity of these enzymes (SOD, CAT) result in a number of deleterious effects due to the accumulation of superoxide anion radicals and hydrogen peroxide. Administration of BLEt and glibenclamide increased the activities of SOD and catalase in diabetic rats.

    The activities of GPx and GST were observed to decrease significantly in diabetic rats. Both GPx, an enzyme with selenium, and GST catalyse the reduction of hydrogen peroxide and hydroperoxides to non-toxic products32. The decreased activities of these enzymes result in the involvement of deleterious oxidative changes due to the accumulation of toxic products. Administration of BLEt and glibenclamide increased the activities of GPx and GST in diabetic liver and kidney.

 

    The B. diffusa leaves are rich in alkaloids and sterols including ursolic acid, hypoxanthine-9-L-arabinofuranoside, punarnavine 1 and 2, myricyl alcohol, myristic acid and quinolizidine alkaloids33. These compounds may be responsible for the antioxidant and antidiabetic activity of B. diffusa leaves, which may be attributed to its protective action on lipid peroxidation and to the enhancing effect on cellular antioxidant defense contributing to the protection against oxidative damage in alloxanized diabetes.

 

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