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Treatment with Rhus Tripartita Extract Curtails Isoproterenol-Elicited Cardiotoxicity and Oxidative Stress in Rats Abdelaaty A

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Treatment with Rhus Tripartita Extract Curtails Isoproterenol-Elicited Cardiotoxicity and Oxidative Stress in Rats Abdelaaty A Shahat et al. BMC Complementary and Alternative Medicine (2016) 16:351 DOI 10.1186/s12906-016-1318-3 RESEARCH ARTICLE Open Access Treatment with Rhus tripartita extract curtails isoproterenol-elicited cardiotoxicity and oxidative stress in rats Abdelaaty A. Shahat1,2*, Mansour S. Alsaid1, Syed Rafatullah1, Mohammed O. Al-Sohaibani3, Mohammad K. Parvez1, Mohammed S. Al-Dosari1, Vassiliki Exarchou4 and Luc Pieters4 Abstract Background: Consumption of plant-derived nutraceuticals and crude drugs in traditional medicine is widely believed to confer beneficial effects in thwarting the progression of cardiovascular diseases. Rhus tripartita (family Anacardiaceae) has been traditionally used to treat a wide range of ailments. Methods: In the present study we investigated the protective effects of an alcoholic extract of the stem part of Rhus tripartita male genotype (RTSM) on experimentally induced myocardial injury in rats. To this end, cardiac injury was induced by administration of isoproterenol (ISO) and serum enzyme markers, lipid profiles and cardiac tissue redox status were determined following RTSM treatment (250 and 500 mg/kg). Results: As a result, RTSM treatment significantly mitigated ISO-triggered upregulation of cardiac-specific markers of injury creatine kinase and lactate dehydrogenase. RTSM treatment significantly attenuated ISO-induced increase in serum cholesterol and triglycerides as well alterations in serum lipoproteins. Determination of oxidative balance showed that RTSM treatment significantly blunted ISO-induced increase in malondialdehyde and decrease in nonprotein sulfhydryl in cardiac tissue. Six compounds were isolated and identified as gallocatechin 1, taxifolin 2, myricetin-3-O-β-glucoside 3, catechin 4, epicatechin 5, and 3′,8-binaringenin 6. Compound 6 was isolated for the first time from the stem part of Rhus tripartita. Furthermore, RTSM treatment enhanced the survival fraction of cardiac cells exposed to oxidative stress in vitro. Conclusion: We conclude that the antioxidant properties of RTSM treatment underpin its cardioprotective pharmacological effects, thus, providing biological evidence for the treatment of cardiovascular diseases using Rhus tripartita in indigenous medicine. Keywords: Rhus tripartita, Anacardiaceae, Cardiovascular diseases, Isoproterenol, Oxidative stress, Chemical components Abbreviations: NP-SH, Nnonprotein Sulfhydryl; LPO, Lipid Peroxidation; TC, Total Cholesterol; TG, Ttriglycerides; HDL-C, High-Density Lipoproteins; MI, Myocardial infarction; CVD, Cardiovascular disease; DMEM, Dulbecco’s Modified Eagle’s medium; DCFH, 2,7-Dichlorodihydrofluorescein diacetate; OD, Optical Density; TCA, Trichloroacetic acid; EDTA, Ethylene diaminetetraacetic acid; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); VLDL, Very-low-density lipoprotein; LDL, Low Density Lipoprotein Cholesterol * Correspondence: [email protected] 1Pharmacognosy Department (Medicinal Aromatic & Poisonous Plants Research Centre), College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2Phytochemistry Department, National Research Centre, 33 El Bohouth St. (former El Tahrir st.) 12622, Dokki, Giza, Egypt Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shahat et al. BMC Complementary and Alternative Medicine (2016) 16:351 Page 2 of 11 Background Methods Cardiovascular disease (CVD) is a major cause of Plant material morbidity and mortality in the modern era and includes Fresh sample of stem bark of Rhus tripartita was conditions such as myocardial infarction, angina pectoris, collected in April 2013 at Hail area in the northwestern and atherosclerosis [1]. Although several mechanisms are part of Saudi Arabia. The plants were identified by an speculated to participate in the pathogenesis of myocardial expert Taxonomist at the Herbarium Unit. The voucher insufficiency, the putative roles of these processes remain specimens have been deposited (SY 202/2013) at the elusive. Mounting evidence suggests that deranged Herbarium of the Faculty of Pharmacy, King Saud myocardial redox balance, enhanced inflammation and University, Riyadh, Saudi Arabia. reduced cell survival influence normal heart function in CVD [2]. Herbal remedies have been utilized for various Extraction and isolation diseases including heart diseases [3, 4], as these herbs The stem of the plant was collected and air-dried at constitute a rich source of bioactive phytochemicals such as room temperature. The dried sample was powdered; flavonoids, polyphenols and other constituents which have 1100 g of dried sample was extracted with 4000 ml of been shown to possess beneficial effects in CVD [5, 6]. The 80 % aqueous methanol three times. The extracts were Anacardiaceae family of plants consists of trees, shrubs filtrated through Whatmann No. 1 and combined and/or woody vines belonging mainly to the genus Rhus, followed by concentration using a rotary evaporator with about 250 species which occurs mostly in the tropics under reduced pressure at 40 °C to yield a dry extract of and subtropics and various temperate zones of the world 231 g (21 %). The percentage yield was expressed in [7, 8]. A number of Rhus species are used in folkloric and terms of air dried weight of plant materials. traditional medicine of many countries including Saudi The dry extract (100 g) was diffused in 400 ml of Arabia. Rhus tripartita (Ucria) Grande is found wildly in distilled water and extracted successively with dichloro- the North-Eastern part of Saudi Arabia. In Arabian methane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol traditional medicine, different parts of Rhus tripartita plant (n-BuOH) (3 x 300 ml) respectively. Each extract was have been used for centuries to treat inflammatory condi- dried over anhydrous sodium sulfate. The organic tions as well as gastrointestinal and cardiovascular disorders fractions were concentrated under reduced pressure at [9]. The fruits of this plant are consumed freshly and in temperature not exceeding 35 °C and the residual decoction to treat diarrhea and ulcers [10]. Previously, ex- aqueous layer was lyophilized named (RTSM1), tracts and pure isolates of Rhus tripartita were isolated and (RTSM2) (RTSM3) and (RTSM4) respectively. The shown to possess robust anti-inflammatory and antioxidant EtOAc fraction (RTSM2) (20 g) was subjected to a properties [11]. Rhus species were documented to possess a Sephadex LH-20 column (Pharmacia) (90 x 4 cm), EtOH wide array of pharmacological activities such as anti- was used as a mobile phase. The fractions 50 ml each inflammatory, antinephritic, antimicrobial [12], antioxidant are collected and examined on TLC (Silica gel 60 F254, and breast cancer preventive properties [13, 14]. Rhus layer thickness 0.2 mm, Merck), the upper layer of mix- coriara (Sumac) was reported to be used for the prevention ture of EtOAc-HOAc-HCOOH-H2O (30/0.8/1.2/8) was and treatment of atherosclerosis [15]. Recently, an attempt used as a mobile phase. The TLC were viewed under UV has been made to intensify the solvent extraction of total (254 and 366 nm) before and after spraying with Neu’s phenols and tannins from the bark of Rhus tripartita.From spray reagent (reagent a) and vanillin H2SO4 (reagent b). the same species of Rhus, flavonoids and isoflavonoids Fractions 16-20, 21-35, 36-45, 46-60, 61-74 and 75-86 have been isolated from its aerial parts and character- were combined together in one group and named as ized. Furthermore, butein (a polyphenolic component) (RTSM2-I), (RTSM2-II), (RTSM2-III) and (RTSM2-IV) was extracted from another species, Rhus verniciflua. and (RTSM2-V) respectively. In our previous study on crateagus species, the phyto- The sub-fractions RTM2-II was subjected to Sephadex chemical constituents (flavonoids and proanthocyani- LH-20 column chromatography using methanol as dins) and the therapeutic nature such as cardiovascular eluent to give compounds 1 and 2. Subfraction RTM2- effects and antioxidant activity were conducted [16–18]. III was subjected to Sephadex LH-20 using methanol as Our current research on Saudi Arabian Rhus tripartita mobile phase to give compounds 3, 4 and 5. The sub showed that it is rich in flavonoids and proanthocyani- fraction RTSM2-IV was subjected to column chroma- dins compounds. tography C18-reversed-phase with MeOH-H2O (6:4) as On account of its wide use in Arabian traditional medi- eluent to give compound 6. cine for the prevention of CVD, the present study, thus, ex- plored the possible protective effect of an alcoholic extract NMR spectroscopy of the stem part of Rhus tripartita male genotype (RTSM) NMR spectra were recorded in deuterated methanol against isoproterenol (ISO)-induced cardiac injury in rats. (CD3OD) on a Bruker DRX-400 spectrometer (Bruker Shahat et al. BMC Complementary and Alternative Medicine (2016) 16:351 Page 3 of 11 Biospin GmbH, Rheinstetten, Germany) operating at In vivo cardioprotective assay
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