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 -5- 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 antioxjdants

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 tissues I. Glucose control plays an important role in the pro-oxidant lantioxidant 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-inflammatorl, antifibrinolytic5, anti­

convulsant6 and hepatoprotective activities 7,8. 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 material--Boerhavia 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 extract-B. diffusa leaves (500g) were chopped into small pieces, extracted with 1500 ml water by the method of continuous hot

*Correspondent author Phone: +91-4144-238343 Fax: +91-4144-238145 E-mail : paribalaji@rediffmail.com

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 studylO.

Animals-Albino 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.

Chemicals-Alloxan 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 diabetes-The rats were injected with alloxan monohydrate dissolved in sterile normal saline at a dose of 140 mglkg body weight, ipll. After 2 weeks, rats with moderate diabetes having glycosuria (indicated by Benedict's qualitative

990 INDIAN J EXP BIOL, OCTOBER 2004

test) and moderate hyperglycemia (200 - 280 mg/dl) were used for the experiment.

Experimental design----1n 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 Ilg/kg body weight '2 in aqueous solution daily using an intragastric tube for 4 weeks.

Sample collection--At 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 measurements-Fasting blood glucoselJ, thiobarbituric acid reactive substances (TBARS)14, hydroperoxides ls, reduced glutathione (GSH)16, superoxide dismutase (SOD)I7, catalase 's, glutathione peroxidase (GPX)19 and glutathione-S­transferase (GSTio were determined.

Statistical analysis-Statistical analysis was done by analysis of variance (ANOY A) 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 a19

• The antidiabetic effect of BLEt may be due to increased release of insulin from the existing ~ cells of pancreas similar to that observed after glibenclamide administration.

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',

Table I-Changes in levels of blood glucose and plasma insulin 0

normal and experimental animals (Values are given as mean ± SD for 6 rats in each group]

Groups

Normal Normal + BLEt Diabetic control Diabetic + BLEt Diabetic + glibenclamide

Fasting blood glucose (mg/dl)

91. 99 ± 6.28" 81.0 I ± 5.83b

257_18 ± 12.54c

129.92 ± 8.03d

135.70±9.9I d

Plasma insulin (JlUlml)

17.18±0.84" 19.76 ± 1.18b

4.92 ±0.30e

10040 ±0.63u

9.74 ±0.57d

Values not sharing a common superscript letter differ significantl at P<0.05 (DMRT). Duncan procedure, Range for the level 2.91, 3.06, 3.16, 3.22.

Table 2-Changes 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

(mM/IOOg tissue)

Normal Liver 0.87± 0.03a

Kidney 1.59± 0.07ab

Normal +BLEt Liver 0.82±0.02 a

Kidney 1.51±0.06a

Diabetic control Liver 2.04 ± O.ll b

Kidney 2.25 ± 0.19c

Diabetic + BLEt Liver 1.36 ± 0.05c

Kidney 1.73±0.llbd Diabetic + Glibenclamide Liver 1.59 ± O.06d

Kidney 1.86 ±0.12d

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.

76.17 ± 2.79a

56.35 ± 2.23a

n.07±3.25 a

52.09±2.17 b

101.70 ±6.05b

79.14±4A9c

84.18 ± 4.83c

62.75 ± 3.07d

90.03 ± 4.32d

66.94 ± 3.24d

Reduced glutathione

(mg / 100 mg tissue)

46.91±2.08' 31.08 ± 2.15' 49.95± 2.91' 34.83 ± 2.48" 23.35±0.80b

20.53 ± 0.90b

41.29± 1.86c

26.83 ± 1.46c

39.66 ± 1.43c

25.08 ± 1.27c

SATHEESH & PARI: ANTIOXIDANT EFFECT OF BOERHA VIA DIFFUSA 991

Table 3------(:hanges in activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S­transferase (GST) in liver of normal and experimental animals

[Values are given as mean ± S D for 6 rats in each group]

Groups CatalaseA Superoxide Glutathione Glutathione-S-dismutaseB peroxidasec transferaseD

Normal Liver 72.03 ± 4.39· 6.33 ± 0.30' 6.57 ± 0.32" 6.19 ± 0.44" Kidney 33.78 ± 2.00" 14.53 ± o.n" 4.66 ± 0.27" 5.50 ± 0.23"

Normal + BLEt Liver 73.63±4.87" 6.52±0.39" 6.82±O.41 " 6.65±0.48 b

Kidney 35.11±2.35 " 15.78±O.82 b 5.26±0.32 b 5.89± 3.35 b

Diabetic control Liver 44.97 ± 2.oob 4.16 ± O.13b 4.45 ± 0.17b 3.24 ± 0.18e

Kidney 23.19 ± 0.75b 9.86 ± O.44e 2.56 ± O.lle 2.63 ± 0.12e Diabetic + BLEt Liver 66.37 ± 3.02c 5.85 ± 0.20e 5.68 ± 0.23c 5.64 ± 0.30d

Kidney 28.40± 1.61c 13.80 ± 0.64" 4 .oo± 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 ± l.35d 11 .57 ± 0.52d 3.55 ± 0.1ge 3.77 ± O.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 = ~ mole of H20 2 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 = ~g of GSH consumed / min/mg protein D = ~ moles of CDNB - GSH conjugate formed / min/mg protein

H20 2 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 radicals3o•

Catalase is a hemeprotein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals3J

•

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-L­arabinofuranoside, 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 III

alloxanized diabetes.

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