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ISSN 0973 – 9874 Evaluation of Antioxidant Activities of Aqueous Extract of Stem Bark of Boswellia Ovalifoliolata in Streptozotocin Induced Diabetic Rats Y.K. PRABHAKAR1, MD. SUBHAN ALI1, M.V. JYOTHI KUMAR2, T. KRISHNA TILAK1 AND CH. APPA RAO1* 1 Department of Biochemistry, Sri Venkateswara University, Tirupati, AP, India. Department of Biotechnology, Sri Venkateswara University, Tirupati, AP, India. 2 ABSTRACT This study was designed to investigate the antioxidant activity of the aqueous extract of stem bark of Boswellia ovalifoliolata (AESBBO) in streptozotocin (STZ) induced diabetic rats. Oral administration of aqueous extract at a dose of 200 mg /kg bw/day/ for 40 days significantly decreased hepatic and renal thiobarbituric acid reactive substances (TBARS) and activity of catalase (CAT). There was a significant improvement in the activities of superoxide dismutase (SOD), glutathione peroxidise (GPx) and glutathione-s-transferase (GST) in liver and kidney of STZ induced diabetic rats after treatment with AESBBO when compared with untreated diabetic rats. These results clearly indicate that aqueous extract of Boswellia ovalifoliolata (AESBBO) possess significant antioxidant effect in diabetic rats. Keywords: Antioxidant activity, Boswellia ovalifoliolata, Glibenclamide, Streptozotocin. and treatment [6]. In the natural system of medicine many plants have been claimed to be useful for the treatment of diabetes mellitus. The dependence of large rural population on medicinal plants for treatment of diabetes is because of its availability and affordability [7]. In recent years, several authors evaluated and identified the antidiabetic potential of traditionally used Indian medicinal plants using experimental animals. Although a large number of medicinal plants have been tested for their antidiabetic effects, it remains to be investigated in several other Indian medicinal plants. Introduction Herbal medicines are the oldest remedies known to mankind. In the present scenario, the demand for herbal products is growing exponentially throughout the world and major pharmaceutical companies are currently conducting extensive research on plant materials for their potential medicinal value. In many journals, national and international, we find an increasing number of research publications based on herbal drugs. Many analysis-based studies regarding pharmacological research in India have been conducted in the past [1]. Excessive oxidative stress is observed in the diabetes [8]. Oxidative stress is currently suggested as mechanism underlying diabetes and diabetic complication [9]. During diabetes, persistent hyperglycemia causes increased production of free radicals, especially reactive oxygen species (ROS) for all tissues from glucose auto-oxidation and protein glycosylation. The increase in the level of ROS in diabetes could be due to their increased production and or decreased destruction by non enzymatic and enzymatic antioxidants. The level of these antioxidants critically influences the susceptibility of various tissues to oxidative stress and is associated with the development of complications in diabetes [10]. Oxidants are generated as a result of normal intracellular metabolism in mitochondria and peroxisomes, as well as from a variety of cytosolic enzyme systems. In addition, a number of external agents Diabetes is a metabolic disorder characterized by hyper glycemia resulting due to deficiency of insulin secretion by pancreas, ineffectiveness of produced insulin, or both [2]. It is the most important non-infective epidemic to hit the globe in the present millennium. The number of people suffering from diabetes worldwide is increasing at an alarming rate. It is predicated that about 366 million people are likely to be diabetic by the year 2030 [3]. It causes number of complications like retinopathy, neuropathy, and peripheral vascular insufficiencies [4]. Hyperglycemia can be handled initially with oral synthetic agents and insulin therapy. However, these synthetic agents produce some serious side effects and are relatively expensive for developing countries [5]. The toxicity of oral antidiabetic agents differs widely in clinical manifestations, severity, October - December 2013 19 Journal of Pharmacy and Chemistry • Vol.7 • Issue.4 can trigger ROS production. A sophisticated enzymatic and nonenzymatic antioxidant defence system including catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) counteracts and regulates overall ROS levels to maintain physiological homeostasis. Lowering ROS levels below the homeostatic set point may interrupt the physiological role of oxidants in cellular proliferation and host defence. Similarly, increase in ROS may also be detrimental and lead to cell death or to acceleration in ageing and age-related diseases. Traditionally, the impairment caused by increased ROS is thought to result from random damage to proteins, lipids and DNA. In addition to these effects, a rise in ROS levels may also constitute a stress signal that activates specific redox sensitive signalling pathways. Once activated, these diverse signalling pathways may have either damaging or potentially protective functions [11]. by the Taxonomist of the Herbarium, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. A voucher specimen (Herbarium Accession Number: 516) has been kept in our library herbarium for future reference. The stem bark of Boswellia ovalifoliolata was dried in shade, powdered and the powder was used for the preparation of different solvent extracts. Preparation of Aqueous extract To prepare aqueous extract the SBBO powder (1 kg) was soaked in distilled water (3 volumes) in a glass jar for 2 days at room temperature and the solvent was filtered. This was repeated 3 to 4 times until the filtrate gave no coloration. The filtrate was concentrated under reduced pressure in the Buchi rotavapour R-200 and finally freeze dried. The yield of the extract was 26% w/w. Phytochemical analysis of the aqueous extract Aqueous extract of SBBO was screened for the presence of various phytochemical constituents using standard methods of phytochemical analysis [24]. In, India Ayurvedic medicine has great importance to treat the diabetes and its complications. Since ancient period, it gains more popularity due to its less toxic effects and more efficacious. Many herbs have been shown to have antidiabetic action in both human and animals [12]. Induction of diabetes Diabetes was induced in male Wistar albino rats aged 2-3 months (180-200 g body weight) by intraperitoneal administration of STZ (single dose of 50 mg/kg b.w.) dissolved in freshly prepared 0.01M citrate buffer, pH 4.5. After 72 h rats with marked hyperglycemia (FBG 250 mg/ dI) were selected and used for the study [25]. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages, as per the guidelines of Institute Animals Ethics committee. This study was approved by institutional animal ethics committee vide Resolution no: 31/2012-2013/(i)/a/CPCSEA/ IAEC/SVU/ CAR-YKP dt. 01-07-2012. The ethnobotanical information reports about 800 plants that may possess antidiabetic potential [13], Folk medicine for diabetes from Rayalaseema reports 26 plants with antidiabetic activity, one such plant is Boswellia sarrata, which showed antidiabetic effect in diabetic rats [14,15], other species of this genus Boswellia ovalifoliolata has wide range of medicinal uses [16], along with antidiabetic activity [17]. Boswellia ovalifoliolata Bal and Henry, a member of Burseraceae, is an endemic species [18], which occurs at an altitudinal range of 250 - 600 m on Seshachalam hill ranges of Palakonda region of Eastern Ghats of India. This plant is vernacularly known as Konda guggilum, Adavi sambrani. The plant is used by tribals to treat number of medicinal ailments. The plant is over exploited for its medicinal uses; especially the leaf juice is used to prevent throat ulcers [19], the gum is used to cure amoebic dysentery and hydroceal [20]. Stem bark is used to cure rheumatic pains [21]. Equal mixture of Gum and stem bark one tea spoon full is given daily with sour milk on empty stomach for a month to cure stomach ulcers [22]. It is used in synthesizing silver nanoparticles, which can provide a new platform to this plant making it’s a value added tree for nanotechnology based medicine in future [23]. But there are no significant reports on antioxidant activity of the stem bark of Boswellia ovalifoliolata, hence this study was taken up for the evaluation of antioxidant effect of SBBO in STZ induced diabetic rats. Experimental design The rats were divided into 5 groups and each group consisted of 6 rats as given below. Group 1. Normal untreated rats Group 2. Normal rats treated with 200mg of AESBBO / kg bw/ day/ for 40 days Group 3. Diabetic untreated rats Group 4. Diabetic rats treated with 200mg of AESBBO / kg bw/ day/ for 40 days Group 5. Diabetic rats treated with 20mg glibenclamide / kg bw/ day / for 40 days AESBBO or glibenclamide was administered to the rats every day morning for 40 days by gastric intubation using oral gavage. All the five groups of rats were sacrificed on the 41th day after an overnight fast, by anesthetizing with anaesthetic ether and further by cervical dislocation. Different tissues including liver and kidney were collected and immediately frozen until the use, for measurement of lipid peroxides and activities of antioxidant enzymes. Materials and Methods Collection of Plant material The stems bark of Boswellia ovalifoliolata (SBBO) Bal and Henry was collected from Tirumala hills, Tirupati, Andhra Pradesh, India. It was identified and authenticated October - December 2013 20 Journal of Pharmacy and Chemistry • Vol.7 • Issue.4 being quenched by the antioxidant systems [32]. There are convincing experimental and clinical evidences that the generation of reactive oxygen species is increased in both types of diabetes and that the onset of diabetes is closely associated with oxidative stress [33,34]. Free radicals are formed disproportionately in diabetes by glucose auto oxidation, polyol pathway and non-enzymatic glycation of proteins [35]. Analytical procedures The levels of TBARS in tissues were estimated by the method of Fraga et al., 1988[26]. CAT activity was assayed following the method of Sinha., 1972 [27]. SOD activity was assessed according to the method of Kakkar et al., 1984 [28]. GPx activity was measured as described by Rotruck et al., 1973 [29]. GST activity was estimated according to the method of Habig et al. 1974[30]. STZ - induced hyperglycemia induces free radical generation which there by leads to DNA damage, protein degradation, lipid peroxidation and finally culminating into damage to various organs of the body like liver, kidney, brain, eyes, enzymes and development of complications of diabetes mellitus [36]. Implication of oxidative stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free radical generation but also due to nonenzymatic protein glycosylation, auto-oxidation of glucose, impaired antioxidant enzyme, and formation of peroxides [37,38]. Increased oxidative stress as measured by the index of lipid peroxidation has been shown to be increased in both insulin-dependent (IDDM), and non-insulindependent diabetes mellitus (NIDDM) [39] and it could cause initial ²-cell damage in type I diabetes or impaired insulin production, release, or function in type II diabetes [40,41]. The increased lipid peroxidation in the diabetic animals may be due to the observed remarkable increase in the concentration of TBARS and hydroperoxides (lipid peroxidative markers) in the liver and kidney of diabetic rats (Stanely et al,. 2001) [42]. Statistical analysis The results were expressed as mean ± S.D. The statistical analysis of results was carried out using one-way analysis (ANOVA) followed by DMRT. Results Phytochemical analysis of Aqueous extract of SBBO Phytochemical analysis of the aqueous extract of stem bark of Boswellia ovalifoliolata has shown the presence of Flavonoids, Saponins, Carbohydrates and Tannins. Table.1 and table.2 Show the effect of long term treatment with the AESBBO on lipid peroxides, activities of CAT, SOD, GPX and GST. Table.1 and Table.2 show the liver and kidney (respectively) levels of TBARS, activities of SOD, CAT, GPx and GST in the normal and experimental groups of rats. There was a significant increase in the levels of TBARS, CAT activity and a significant decrease in the activities of SOD, GPx and GST in both tissues of diabetic rats compared to those in normals. The treatment with AESBBO decreased the levels of TBARS, CAT activity and significantly increased activities of SOD, GPx and GST in liver and kidney of diabetic rats. Treatment of the 5th group of rats with glibenclamide resulted in similar changes in the levels of lipid peroxides and antioxidant enzyme activities. In our study STZ was used to induce DM in rats rather than alloxan. At low dose, STZ (50 mg/kg bw) partially destructs the ²-cells, which secreted insufficient insulin causing type 2 diabetes [43]. It is widely accepted animal model and reported to resemble human hyperglycaemic non ketotic diabetes mellitus [44], is often associated with kidney hypertrophy which may contribute to end stage renal damage, hepatotoxicity, oxidative stress and hypercholesterolemia [45,46]. Discussion The use of plant products in the treatment of diabetes mellitus is becoming advantageous due to the presence of several bioactive compounds with therapeutic potential. In recent years, several researchers have studied the worth of different medicinal plants in controlling DM and delaying the long term effects of DM. Boswellia ovalifoliolata is one of the herbs mentioned in all ancient scriptures of Ayurved. The present study was conducted to evaluate the beneficial effects of Aqueous extract of stem bark of Boswellia ovalifoliolata (AESBBO) on lipid peroxidation and antioxidant status in STZ induced diabetic rats. It has been stated that STZ diabetic animals may exhibit most of the diabetic complications mediated through oxidative stress (Ozturia et al., 1996) [31]. Oxidative stress depicts the existence of products called free radicals and reactive oxygen species (ROS) which are formed under normal physiological conditions but become deleterious when not October - December 2013 Hypoinsulinaemia in diabetes increases the activity of the enzyme fatty acyl coenzyme A oxidase, which intiates ²-oxidation of fatty acids, resulting in lipid peroxidation [47]. Increased lipid peroxidation impairs membrane function by decreasing membrane fluidity and changing the activity of membrane-bound enzymes and receptors. The products of lipid peroxidation are harmful to most cells in the body and are associated with a variety of diseases, such as atherosclerosis and brain damage [48]. In the present study, we observed a significant increase in lipid peroxide levels (TBARS) in the liver and kidney of diabetic rats compared to normal rats. Administration of AESBBO or glibenclamide decreased the levels of TBARS in the liver and kidney of diabetic rats. This shows that AESBBO might protect the tissues (liver and kidney) against the cytotoxic action and oxidative stress of streptozotocin. 21 Journal of Pharmacy and Chemistry • Vol.7 • Issue.4 and glycation of the enzymes in diabetic state [53]. On long-term treatment of diabetic rats AESBBO had reversed the activities of these enzymatic antioxidants, This means that the extracts can reduce the potential glycation of enzymes or they may reduce the production of reactive oxygen free radicals and improve the activities of antioxidant enzymes. DM is associated with increased formation of free radicals and decrease in antioxidant potential. Due to these events, the balance normally present in cells between radical formation and the protection against them is disturbed [49]. An imbalance of the oxidant / antioxidant defence systems results in alterations in the activity of antioxidant enzymes, such as SOD, CAT,GR, GPx, and impaired glutathione metabolism [50]. The present data indicates that STZinduced diabetes disrupts actions of liver and kidney antioxidant enzymes. The decreased activities of these result in accumulation of superoxide (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH) that reduce the activity of these enzymes [51,52]. In our study the activity of CAT was significantly increased in liver and kidney of diabetic untreated rats. The possible explanation for the increases in catalase activity is that it could be a compensatory mechanism to prevent tissue damage by the increased levels of H2O2 and decreased levels of GPx,. In diabetes,it is thought that hypoinsulinemia increases the activity of the enzyme, fatty acyl coenzyme A oxidase, which initiates ± oxidation of fatty acids, resulting in increased levels of H2O2. The CAT activity was restored In our study, the activities of SOD, GPx and GST were decreased in diabetic rats compared to normal rats, which could be due to free radical-induced inactivation Table - 1 Effect of long term treatment with the AESBBO on TBARS levels and antioxidant enzyme activities in the livers of different experimental animals Group 1 2 3 4 5 F value Significance Lipid Peroxides (nmoles MDA/ml) 0.123±0.001 0.130±0.001 0.232±0.007 0.157±0.005 0.146±0.004 500.906 0 a b e d c Catalase (U/mg Protein) 19.1±0.52 20.4±0.25 46.4±0.30 21.8±0.59 23.3±0.28 452.1 0 Glutathione Peroxidase (U/ mg protein) a 0.257±0.018 0.232±0.017 0.072±0.014 0.165±0.012 0.128±0.011 150.843 0 b e c d e d a c b Superoxide Dismutase (U/ mg protein) Glutathione-STransferase (U/ mg protein) 16.0±0.68 c 17.4±0.43 d 6.4±0.33 a 13.1±0.59 b 12.0±0.55 b 362.044 0 21.4±0.33 d 24.0±0.40 e 8.7±0.45 a 18.3±0.27 c 16.0±0.55 b 119.478 0 Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p< 0.01 (DMRT). Table – 2 Effect of long term treatment with AESBBO on TBARS levels and antioxidant enzyme activities in the Kidneys of different experimental animals. Group 1 2 3 4 5 F value Significance Lipid Peroxides (nmoles MDA/ml) 0.134±0.003 0.135±0.002 0.253±0.002 0.163±0.002 0.174±0.003 179.95 0 a a d b c Catalase (U/mg Protein) 34.4±0.35 32.1±0.74 60.3±0.30 38.5±0.40 40.3±0.33 356.9 0 Glutathione Peroxidase (U/ mg protein) b 0.229±0.008 0.235±0.012 0.128±0.007 0.198±0.039 0.171±0.012 28.64 0 a e c d d d a c b Superoxide Dismutase (U/ mg protein) 34.03±0.73 35.95±0.76 14.98±0.65 31.05±0.61 26.88±0.45 97.55 0 d e a c b Glutathione-STransferase (U/ mg protein) 26.58±0.42 d 25.06±0.69 c 9.56±0.48 a 22.86±0.69 b 28.33±0.68 e 90.97 0 Values are given as mean ± S.D from six rats in each group. Values not sharing a common superscript letter differ significantly at p< 0.01 (DMRT). October - December 2013 22 Journal of Pharmacy and Chemistry • Vol.7 • Issue.4 H. Pharmacokinetics and Bioavailability of herbal medicinal products. Phytomed 2002; 9: 1-36. to near normal in diabetic rats treated with AESBBO, which might be due to decreased LPO levels and increased insulin secretion. [13] Alarcon Aguilara FJ, Roman Ramos R, Perez Gutierrez S, A Aguilar Contreras CC. Contreras Weber JL. Flores Saenz, Study of the antihyperglycaemic effect of plants used as antidiabetics, Journal of Ethanopharmacology 61 (1998) 101–110. Various studies in the past reported conflicting results regarding the status of antioxidant enzymes in diabetes [54,55]. Majority of authors reported the decreased enzymatic antioxidant activites (SOD,CAT,GPx and GST) in tissues of diabetic rats. [56,57]. 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