Effect of Garuga pinnata. Roxb Aqueous Bark Extract on Tissue Antioxidant Activity in Streptozotocin-Nicotinamide–Induced Type II Diabetic Rats

Abstract

Oxidative stress plays an important role in chronic complications of diabetes mellitus. In the current study, the antioxidant effect of oral administration of aqueous bark extract of Garuga pinnata. Roxb. (Burseraceae) on tissue antioxidant enzymes and lipid peroxidation in liver and kidney of streptozotocin-nicotinamide–induced type II diabetic rats was evaluated. The diabetic rats showed lower activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione transferase, and reduced glutathione content in liver and kidney, which were restored to near normal levels by treatment with the bark extract. The increased levels of lipid peroxidation (TBARS) in diabetic rats were reverted back to near normal levels after treatment with the bark extract. A two dose level study was conducted. The current study reveals the efficacy of Garuga pinnata. aqueous bark extract in the amelioration of diabetes, which may be attributed to its antioxidant potential.

Keywords:

• Antidiabetic
• antioxidant
• Burseraceae
• free radical
• Garuga pinnata
• oxidative stress
• streptozotocin-nicotinamide-induced type II diabetes

Introduction

Diabetes mellitus is a group of metabolic disorders characterized by chronic hyperglycemia. The metabolic disturbances include alteration in the metabolism of carbohydrates, fats, and proteins reflecting a state of insulin deprivation. This occurs because of deficient insulin secretion or because of factors opposing the tissue effects of insulin or both (Park, 2000). Free radicals are produced in the body because of normal metabolic processes, and interaction with environmental stimuli and have been implicated as one of the major causes of diabetic complications (Menon et al., 2004). These are considered to be of great importance as the cause of many disorders, and of diabetes in particular. Mammalian cells are equipped with both enzymatic and nonenzymatic antioxidant defenses to minimize the cellular damage caused by interaction between cellular constituents and oxygen free radicals (Haliwell & Gutteridge, 1994). In diabetes mellitus, glucose autooxidation and protein glycation may generate more free radicals, which in turn catalyze lipid peroxidation (Wolff & Dean, 1987). Elevated free fatty acids due to increased lipolysis also cause the production of free radicals in diabetes mellitus (Efrat, 2001). In addition to this, impaired generation of naturally occurring antioxidants also results in increased oxidative stress (Giugliano et al., 1996). Attention has been focused on possible interventions to decrease levels of oxidative stress, such as improved glycemic control and drug or antioxidant therapy (Sharma et al., 2000). Hence, hypoglycemic coupled with antioxidative properties would be useful in an antidiabetic agents. In recent decades, a resurgent interest has been observed in traditional plant treatment for diabetes. As plants often contain substantial amounts of antioxidants, it has been suggested that an antioxidant action may be an important property of plant medicines associated with diabetes (Larson, 1988).

Garuga pinnata. Roxb. (Burseraceae) is a medium-sized tree, up to 50 ft high, distributed almost throughout India. It occurs sporadically in mixed deciduous forests and is a common associate of teak and sal trees (The Wealth of India, 1950). The leaves of this tree are used in the treatment of opacities in conjunctivitis. The fruits are used as stomachic and cooling agents (Nadkarni, 1954). The leaf juice mixed with honey is used in the treatment of asthma (Kirtikar & Basu, 1991). The cytotoxic activity of this tree has been reported (Wongsinkongman et al., 2002). The Kol, Kondh, and the Saora communities of tribal Orissa take the bark of this tree with pepper for about 15 days twice daily for the treatment of diabetes (Jain et al., 1991). Though there is no scientific evidence to support the antidiabetic effect of Garuga pinnata. (GP), tribal men continue to use it in the management of diabetes. The objective of this study was to ascertain the efficacy of this plant in the management of oxidative stress in type II diabetic condition.

Materials and Methods

Collection of plant material

The bark of GP was collected during November 2003 from the Chattisgarh forests, Madhya Pradesh, India. Dr. S.C. Jena, Director, Chattisgarh Rajya Van Vikas Nigam Limited, Madhya Pradesh, India, and Mr. A.K. Murthy, Medicinal Plants Survey, Central Ayurvedic Research Institute, Bhubaneswar, Orissa, India, identified the bark. A voucher specimen (PP 530) was-been deposited in the herbarium of the Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, Karnataka, India.

Preliminary phytochemical screening

Preliminary phytochemical screening (Kokate, 1994; Harborne, 1998) revealed the presence of steroids, terpenes, saponins, carbohydrates, tannins, and mucilage.

Preparation of GP aqueous bark extract

The aqueous extract was prepared by cold maceration of 150 g of the shade-dried bark powder in 500 mL of drinking water for 7 days. The extract was filtered, concentrated, dried in vacuo. (yield 65 g), and the residue stored in a refrigerator at 2–8°C for use in subsequent experiments.

Antidiabetic studies

Drugs

Streptozotocin (STZ) used for the induction of diabetes was procured from Sigma-Aldrich (Munich, Germany). Nicotinamide and other reagents used in the experiments were of analytical grade and were procured from Ranbaxy Chemicals (Mumbai, India).

Animals

Healthy adult male Wistar albino rats between 2 and 3 months of age and weighing 200–280 g were used for the study. Housed individually in polypropylene cages, maintained under standard conditions (12-h light and 12-h dark cycle, 25±30°C, 35–60% humidity), the animals were fed with standard rat pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum.. The study was approved by the Institutional Animal Ethical Committee of KMC, Manipal, India (IAEC/KMC/03/2003–2004).

Acute toxicity studies

Healthy adult Wistar mice of either sex, starved overnight, were divided into five groups (n = 6) and were administered through gastric lavage the aqueous extract of GP in increasing dose levels of 100, 500, 1000, 3000, and 5000 mg/kg body weight (Ghosh, 1984). The animals were observed continuously for 2 h for behavioral, neurologic, and autonomic profiles and after a period of 24 and 72 h for any lethality (Turner, 1965).

Induction of non–insulin-dependent diabetes mellitus (NIDDM)

NIDDM was induced (Pellegrino et al., 1998) in overnight fasted adult rats weighing 200–280 g by a single intraperitoneal injection of 60 mg/kg streptozotocin (Sigma Aldrich, Munich, Germany), 15 min after the i.p. administration of 120 mg/kg of nicotinamide (Ranbaxy Chemicals Ltd., Mumbai India). Streptozotocin was dissolved in citrate buffer (pH 4.5), and nicotinamide was dissolved in normal saline. The elevated glucose levels in plasma were determined at 72 h and then on day 7 after injection, to confirm hyperglycemia. The rats found with constant values of elevated plasama glucose levels were used for the study.

Experimental design

Animals were divided into seven groups of six rats each. The extract was administered for 15 days.

Group I: Normal control rats administered gum acacia daily for 15 days. Group II: Normal rats administered GP aqueous extract (250 mg/kg). Group III: Normal rats administered GP aqueous extract (500 mg/kg). Group IV: Diabetic control rats administered gum acacia daily for 15 days. Group V: Diabetic rats administered GP aqueous extract (250 mg/kg). Group VI: Diabetic rats administered GP aqueous extract (500 mg/kg). Group VII: Diabetic rats administered standard drug glibenclamide (0.25 mg/kg) for 15 days.

At the end of the experimental period, the rats were anaesthetized and sacrificed by cervical dislocation. The liver and kidney were excised, rinsed in ice-cold saline, and then homogenised with Tris-HCl buffer (pH 7.4). The tissue homogenates were used for the estimation of thiobarbituric acid reactive substances (TBARS) (Okhawa et al., 1979), reduced glutathione (GSH) (Sedlak & Lindsay, 1968), superoxide dismutase (SOD) (Misra & Fridovich, 1972), glutathione peroxidase (GPx) and glutathione transferase (GST) (Necheles et al., 1968).

Statistical analysis

Data were statistically evaluated using one-way ANOVA, followed by post hoc. Sheffe's test using version 7.5 of SPSS computer software. The values were considered significant when p < 0.05.

Results

The effect of GP aqueous bark extract on the tissue antioxidant markers was studied. Table 1 shows the concentration of TBARS in the liver and kidney of normal and diabetic rats. There was an elevation in the tissue TBARS levels in the diabetic rats, which decreased upon administration of extract. Decrease observed in the concentration of GSH in the liver (Table 2) and kidney (Table 3) of diabetic rats increased in the treated animals. GST also showed a decrease in concentration in the diabetic group, which was increased upon administration of the extract (Tables 2 and 3). Decreased GPx and SOD levels (Tables 2 and 3) observed in diabetic animals were also found to increase upon treatment. Readings obtained from the treated groups were close to those obtained from the normal groups.

Table 1. Effect of aqueous bark extract of Garuga pinnata. (GP) on lipid peroxidation in rats.

		Thiobarbituric acid reactive substances (µM/g)
Group (n = 6)	Treatment	Liver	Kidney
I	Normal control (gum acacia)	1.4±0.5	1.4±1.5
II	Normal + GP (250 mg/kg)	1.4±0.8	1.5±1.1
III	Normal + GP (500 mg/kg)	1.5±0.5	1.5±0.1
IV	Diabetic control (gum acacia)	3.8±0.3	2.9±1.02
V	Diabetic + GP (250 mg/kg)	2.4±0.6^a.	1.8±1.2
VI	Diabetic + GP (500 mg/kg)	1.7±0.2^a.	1.3±0.8^a.
VII	Diabetic + standard (glibenclamide 0.25 mg/kg)	1.4±0.3^a.	1.2±0.1^a.

Readings are values±SE.

^a.p. < 0.05 versus diabetic control.

Table 2. Activity of GSH, GST, GPx, and SOD in liver of normal and diabetic rats.

Group(n = 6)	Treatment	GSH (U/G/min)	GST (U/G/min)	GPx (U/G/min)	SOD (U/G)
I	Normal control (gum acacia)	21.3±1.2	20.3±1.4	48.2±1.6	394.2±10.3
II	Normal + GP (250 mg/kg)	20.5±2.4	20.7±1.6	42.6±1.2	390.3±12.2
III	Normal + GP (500 mg/kg)	19.4±1.7	18.4±1.9	46.3±2.2	390.7±13.4
IV	Diabetic control (gum acacia)	5.5±0.3	8.6±1.8	23.4±1.5	219.2±18.4
V	Diabetic + GP (250 mg/kg)	10.4±1.8	10.2±4.5	38.4±2.6	267.4±12.5
VI	Diabetic + GP (500 mg/kg)	15.9±2.5^a.	18.8±1.2^a.	44.3±1.8^a.	388.7±16.7^a.
VII	Diabetic + standard (glibenclamide 0.25 mg/kg)	16.2±1.3^a.	19.2±2.1^a.	46.5±2.3^a.	392.4±10.3^a.

Readings are values±SE.

^a.p. < 0.05 versus diabetic control.

(U/G/min) = units/gram/min

Table 3. Activity of GSH, GST, GPx, and SOD in kidney of normal and diabetic rats.

Group(n = 6)	Treatment	GSH (U/G/min)	GST (U/G/min)	GPx (U/G/min)	SOD (U/G)
I	Normal control (gum acacia)	10.6±1.9	18.6±1.8	10.6±2.5	382.6±13.4
II	Normal + GP (250 mg/kg)	10.3±2.1	20.3±1.5	12.4±2.1	380.5±12.4
III	Normal + GP (500 mg/kg)	11.5±1.5	20.6±1.8	14.5±1.7	382.2±10.4
IV	Diabetic control (gum acacia)	2.1±0.4	7.5±2.1	7.2±0.4	136.1±14.6
V	Diabetic + GP (250 mg/kg)	10.1±0.8	19.8±1.4^a.	16.2±1.8^a.	300.6±11.4^a.
VI	Diabetic + GP (500 mg/kg)	12.7±1.3^a.	20.6±0.2^a.	18.5±3.4^a.	378.7±10.6^a.
VII	Diabetic + standard (glibenclamide 0.25 mg/kg)	15.1±0.2^a.	22.3±1.4^a.	20.3±2.4^a.	382.3±10.4^a.

Readings are values±SE.

^a. P. < 0.05 versus diabetic control.

Discussion

Oxidative stress has been shown to play a role in the causation of diabetes, and as such, antioxidants may have a role in the alleviation of diabetes (Baynes, 1991).

STZ produces oxygen radicals in the body. Several studies have shown that reactive oxygen metabolites including free radicals like superoxide radical, hydroxyl radical, and hydrogen peroxide are important mediators of tissue injury (Wohaeib & Godin, 1987). The concentration of reactive oxygen species is modulated by scavenging enzymes like GSH. GST, GPx, SOD, and so forth, which are known as the markers of tissue antioxidant system (Kasi Ravi et al., 2004).

Induction of diabetes in rats with STZ uniformly results in an increase in lipid peroxidation. TBARS are considered to be the index of endogenous lipid peroxidation, an indirect evidence of intensified free radical production (Rajasekaran et al., 2005). Increased TBARS in diabetic rats suggests an increase in oxygen radicals that could be due to either their increased production or decreased destruction (Griesmacher et al., 1995; Matkovics et al., 1998). The TBARS levels in our study were found to be elevated in both liver and kidney in diabetic control group, and were significantly reduced upon administration of the extract.

GSH, known to protect the cellular system against the toxic effects of lipid peroxidation acts as a direct free radical scavenger (Meister & Anderson, 1983). The lower levels of GSH seen in the diabetic control group may represent increased utilization due to oxidative stress (Anuradha & Selvam, 1993). Hyperglycemia increases the polyol pathway, advanced glycation end products formation, and free radical generation rates leading to increased GSH oxidation (McLennan et al., 1991). A relative depletion of NADPH due to aldose reductase activation secondary to reduced production through the pentose cycle impairs GSH generation and leads to its depletion (Bruce et al., 1982).

GPx metabolizes hydrogen peroxide to water by using its cosubstrate GSH as a hydrogen donor (Burk, 1983). The diabetic control group showed a lower level of GPx in both liver and kidney. The reduced activity of GPx may cause an accumulation of toxic products due to oxidative damage (Kaplowitz, 1985). GST levels were also found to be low in the diabetic control group. The significant recovery of GSH, GST, and GPx levels on treatment indicates the protective effect of the extract. SOD is an important defense enzyme, which scavenges the superoxide radical by converting it into hydrogen peroxide and molecular oxygen (McCord et al., 1976). Reduced content of SOD observed in the liver and kidney of diabetic control group may be due to increased production of reactive oxygen radicals (McCord et al., 1976). Upon treatment with the extract, SOD levels were significantly increased.

The current study provides some useful insight into the antioxidant potency of Garuga pinnata. bark in streptozotocin–nicotinamide–induced diabetes mellitus. However, further work should be carried out on chronic models at the molecular level to find out the absolute mechanism of action of the plant in experimental diabetes.

Acknowledgment

The authors sincerely thank Manipal Academy of Higher Education, Manipal, India, for providing the necessary facilities to carry out the study.