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Rhinacanthus Nasutus Ameliorates Cytosolic and Mitochondrial Enzyme Levels in Streptozotocin-Induced Diabetic Rats

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Rhinacanthus Nasutus Ameliorates Cytosolic and Mitochondrial Enzyme Levels in Streptozotocin-Induced Diabetic Rats Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 486047, 6 pages http://dx.doi.org/10.1155/2013/486047 Research Article Rhinacanthus nasutus Ameliorates Cytosolic and Mitochondrial Enzyme Levels in Streptozotocin-Induced Diabetic Rats Pasupuleti Visweswara Rao,1,2 K. Madhavi,3 M. Dhananjaya Naidu,4 and Siew Hua Gan2 1 Department of Biotechnology, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India 2 Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Malaysia 3 Department of Biochemistry, Sri Venkateswara Medical College, Tirupati 517502, Andhra Pradesh, India 4 Department of Zoology, Yogi Vemana University, Kadapa 516003, Andhra Pradesh, India Correspondence should be addressed to Pasupuleti Visweswara Rao; [email protected] Received 13 February 2013; Revised 11 March 2013; Accepted 16 March 2013 AcademicEditor:WilliamC.Cho Copyright © 2013 Pasupuleti Visweswara Rao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The present study was conducted to evaluate the therapeutic efficacy of Rhinacanthus nasutus (R. nasutus) on mitochondrial and cytosolic enzymes in streptozotocin-induced diabetic rats. The rats were divided into five groups with 6 rats in each group. The methanolic extract of R. nasutus was orally administered at a dose of 200 mg/kg/day, and glibenclamide was administered at a dose of 50 mg/kg/day. All animals were treated for 30 days and were sacrificed. The activities of both intra- and extramitochondrial enzymes including glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH) were measured in the livers of the animals. The levels of G6PDH, SDH, and GDH were significantly reduced in the diabetic rats but were significantly increased after 30 days of R. nasutus treatment. The increased LDH level in diabetic rats exhibited a significant reduction after treatment with R. nasutus. These results indicate that the administration of R. nasutus altered the activities of oxidative enzymes in a positive manner, indicating that R. nasutus improves mitochondrial energy production. Our data suggest that R. nasutus should be further explored for its role in the treatment of diabetes mellitus. 1. Introduction impaired oxidative stress in diabetic rats [3, 4]. Generally, herbal products are gaining popularity due to their natural Type 1 diabetes mellitus (T1DM) is caused by the destruction origins, the lower incidence of side effects, and their relatively of insulin-producing cells in the pancreas and is usually the lower costs relative to synthetic drugs [5], although their result of an autoimmune disease. T1DM leads to uncontrolled efficacies may still be inferior when compared to some blood glucose levels, which are associated with long-term current available treatments such as insulin and meformin. damage, including retinopathy, neuropathy, nephropathy, Rhinacanthus nasutus (Linn) is a flowering plant that and damage to the heart and blood vessels and with multiple belongs to the Acanthaceae family and is well recognized organ failures [1]. To date, various types of herbs and other for its remedial uses. This plant is commonly known as plantmaterialsfromallovertheworldhavebeenusedfor Nagamalli in Telugu, Doddapatika in Kannada, Kaligai and the treatment of diabetes mellitus. Medicinal plants are a Anichi in Tamil, Yuthikaparni in Sanskrit, Jupani in Hindi, good source of compounds with hypoglycemic effects and and Gajakarni in Marathi [6]. R. nasutus is also commonly are important for the development of new drugs and as known as Snake Jasmine due to the shape of its flowers. adjuncts to existing therapies [2]. Some of the medicinal In addition, the root of this plant has been reported to plants such as Panax ginseng and Piper longum possess be used in traditional medicine to counter the effects of significant antihyperglycemic and antihyperlipidemic actions snake venom [7].Differentpartsofthisplanthavealso which have been reported to have the potential to alleviate beentraditionallyusedforthetreatmentofvariousdiseases 2 Evidence-Based Complementary and Alternative Medicine such as diabetes, eczema, pulmonary tuberculosis, herpes, Group I. Normal rats (controls: animals receiving hypertension, hepatitis, and several types of skin diseases. only buffer). In Thailand, R. nasutus has been traditionally used for the Group II. R. nasutus-treated normal rats (200 mg/kg/ treatment of various cancers such as colon [8], cervical, and day). liver cancers [9]. Previously, we reported that R. nasutus possesses antimi- Group III. Diabetic rats (untreated). crobialpropertiesandcankillavarietyofinfectingorgan- Group IV. R. nasutus-treated diabetic rats (200 mg/ isms, in addition to exhibiting antidiabetic effects, hypolipi- kg/day). demic activity [10], and significant in vitro and in vivo Group V.Glibenclamide-treated diabetic rats (50 mg/ antioxidant activities [11]. In this study, we hypothesized that kg/day). R. nasutus may exert antidiabetic effects by ameliorating cytosolic and mitochondrial enzymes levels. The dose of 200 mg/kg was selected based on our previous study [13]wherewefoundthat200mg/kgdosegavesimilar 2. Materials and Methods effects to 250 mg/kg dose. The toxicity of the extract has been tested. There were no 2.1. Collection of Plant Material. The fresh leaves of R. nasutus toxic effects observed. There are some previous studies where were collected from Tirumala Hills, Tirupati, and Chittoor it has been reported that the plant extract has no toxic effect district of Andhra Pradesh, in the period of July–October when used in animals at higher doses also 500 mg/kg dose 2009. Seshachala and Tirumala Hills (Rayalaseema region, [8]. Andhra Pradesh, India), areas that are geographically located Diabetes was induced by a single intraperitoneal injection in the South Eastern Ghats, are recognized for their rich flora of a freshly prepared STZ solution (Sigma, no. 242-646-8) and fauna [12]. The plant specimen was verified to be of the (50 mg/kg in citrate buffer 0.01 M, pH 4.5) to overnight-fasted correctspeciesbyDr.MadhavaSetty,abotanistfromthe rats. Diabetes was confirmed by the presence of polydipsia Department of Botany, S. V. University, Tirupati. and polyurea and by measuring the nonfasting plasma glucose levels 48 h after injection of STZ. Only animals that 2.2. Preparation of the Extract. Fresh leaves of R. nasutus were confirmed to have blood glucose levels of greater than (500 g) were shade-dried and milled into fine powder using 250 mg/dL [1] were included. All of the animals were allowed a mechanical grinder (TTK Prestige, Chennai, India). The free access to tap water and pellet show per the guidelines of powdered plant material was macerated and shaken in the Institute Animal Ethics committee. methanol using a bath shaker (Thermo Scientific, Mumbai, India) for 48 h. The extract was then filtered with filter paper 2.6. Phytochemical Analysis. Qualitative tests were con- (Whatman No. 1) and evaporated to dryness under vacuum ∘ ducted on the crude extract using various solvents such as and reduced pressure using a rotary evaporator at 40 C. The hexane, ethyl acetate, methanol, and water for the different concentrate was then placed on aluminum foil before freeze phytochemical constituents present in the plant extract based drying. The residual extract was dissolved in sterile water on the previous method described by Harborne [14]. (1 mL) before use. 2.7. Test for Glycosides (Keller-Kiliani Test). The test for 2.3. Chemicals. Streptozotocin (STZ) was purchased from glycosides was conducted based on the method published Sigma (USA). All other chemicals and reagents used in by Kokate et al. [15]. Briefly, the extracts were dissolved in this study were of analytical grade. Glibenclamide (Sugatrol, glacial acetic acid, and two drops of ferric chloride solution Hyderabad,India)waspurchasedfromalocaldrugstore. (5%, w/v in 90% alcohol) were added. The mixture was then transferred to a test tube containing 2 mL of concentrated 2.4. Experimental Design. Adult male Wistar rats weighing sulfuric acid. The presence of a reddish brown ring between between 150 and 180 g were obtained from Sri Venkateswara thetwolayersconfirmedthepresenceofglycosides. Enterprises, Bangalore. They were individually housed in clean, sterile polypropylene cages under standard conditions 2.8. Test for Flavonoids. The test for flavonoids (lead acetate (12 h light/dark cycles) with free access to standard chow test) was conducted based on the method published by Peach (Hindustan Lever Ltd., Bangalore, India) and water ad libi- and Tracey [16]. Briefly, several drops of lead acetate solution tum. For one week prior to the start of the experiments, (10%) were added to the alcoholic solution of the extract and the animals were acclimatized in the laboratory. The animal a yellow precipitate was formed. experiments were designed and performed in accordance with the ethical norms approved by the local Ministry of Social Justices and Empowerment, Government of India, 2.9. Test for Phenols. The presence of phenols (ferric chloride and the Institutional Animal Ethics Committee Guide- test) was confirmed using the method published by Trease lines (Resolution no. 05/(i)/a/CPCSEA/IAEC/SVU/MDN-
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