Hepatoprotective and Antioxidant Effects of Cymbopogon Citratus Stapf (Lemon Grass) Extract in Paracetamol- Induced Hepatotoxicity in Rats
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Saenthaweesuk et al Tropical Journal of Pharmaceutical Research January 2017; 16 (1): 101-107 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v16i1.13 Original Research Article Hepatoprotective and antioxidant effects of Cymbopogon citratus Stapf (Lemon grass) extract in paracetamol- induced hepatotoxicity in rats Suphaket Saenthaweesuk1, Narongsuk Munkong2, Wason Parklak1, Atcharaporn Thaeomor3, Janeyuth Chaisakul4 and Nuntiya Somparn1* 1Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Pathum Thani, 2Department of Medicine, School of Medicine, University of Phayao, Phayao, 3School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 4Department of Pharmacology, Phramongkutklao College of Medicine, Bangkok, Thailand *For correspondence: Email: [email protected]; Tel/Fax: +66-2926-9710 Received: 4 July 2016 Revised accepted: 5 December 2016 Abstract Purpose: To investigate the protective effect of Cymbopogon citrates Stapf. (CS, lemongrass) extract on paracetamol (PCM)-induced hepatotoxicity in rats. Methods: The rats were orally administered CS extract (1000 mg/kg/day) for 30 days prior to induction of hepatotoxicity by a single oral administration of PCM (3 g/kg). Hepatoprotection was assessed by measuring the level of hepatic markers including aspartate transaminase (AST), alanine transaminase (ALT) and oxidant/antioxidant markers including Malondialdehyde (MDA), protein carbonyl, and glutathione (GSH) in liver homogenate and serum. Phytochemical screening of the CS extract was also performed. Results: Phytochemical screening of the extract indicate the presence of tannins, flavonoids, and phenolic compounds. Elevation of serum AST, ALT, and MDA levels along with depletion GSH in the liver were observed in rats treated with PCM alone compared with control (p < 0.05). Pre-treatment of the animal with CS extract reduced the levels of hepatic markers (AST and ALT). Pre-treatment with CS extract also significantly reduced oxidative stress induced by PCM as shown by an increase in GSH level and reduction of MDA compared to rats treated with PCM alone (p<0.05). Conclusion: The results indicate that CS possesses antioxidant activity and it exerts its effect by reducing lipid peroxidation and restoring GSH. Pre-treatment with CS extract reduces oxidative stress and ameliorates hepatic injury induced by PCM. Keywords: Cymbopogon citratus, Antioxidant, Oxidative stress, Hepatotoxicity Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION toxic metabolite that can be detoxified by glutathione (GSH), which results in extensive Paracetamol (PCM) is an over-the-counter drug hepatic GSH depletion and oxidative stress [1]. commonly used for its analgesic and antipyretic GSH depletion leads to lipid peroxidation, which activities. At therapeutic doses, PCM is further leads to the initiation of liver damage. considered safe. However, PCM overdose can Several studies have demonstrated that the cause severe hepatotoxicity and nephrotoxicity. antioxidant properties of certain medicinal plants, PCM is activated and converted by liver enzymes such as ginseng, soybean, and pomegranates, to N-acetyl-p-benzoquinoneimine (NAPQI), a Trop J Pharm Res, January 2017; 16(1): 101 Saenthaweesuk et al can help provide substantial protective and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical health benefits against oxidative stress [2,3]. scavenging assay Cymbopogon citrates Stapf. (CS), commonly The DPPH radical-scavenging activity was known as lemongrass, is a widely used herb in determined as per the previously described Southeast Asia. Studies have reported the method [7]. Briefly, 0.08 mM DPPH was pharmacological properties, including free radical prepared in 100 % ethanol. DPPH solution, Tris scavengers, anti-oxidant, anti-inflammatory and buffer, and 80 % ethanol were mixed in order to anti-mutagenicity properties, of specific parts of obtain a 1:1:1 ratio for 1.8 mL. Then the plant CS [4]. Moreover, the hypoglycemic and extract (0.6 mL) was added and incubated for 30 hypolipidemic effects of fresh CS leaf extracts min in the dark. The absorbance was read at 525 have been demonstrated [5]. Therefore, the nm. present study investigates the antioxidant potential of CS against PCM-induced Animals and experimental design hepatotoxicity in rats. The animals were divided into 6 groups, each of EXPERIMENTAL which consisted of 6 rats that were treated as follows: Chemicals and drugs Group I: (Control) distilled water (DI) Paracetamol, 2, 2-dipheneyl-1-picrylhydrazil Group II: (PCM only) DI + PCM (DPPH), thiobarbituric acid (TBA) were Group III: (positive control) DI + PCM followed purchased from Sigma Chemical Company (St. by N-acetylcysteine (NAC). Louis, MO, USA). All other chemicals and Group IV: DI + PCM followed by CS extract reagents used were of analytical grade. Group V : CS extract Group VI: CS extract followed by PCM Plant material and identification DI (1 mL/kg) and CS (1,000 mg/kg) were Fresh CS was procured from a local agricultural administered orally once daily for 30 consecutive field in Khon Kaen Province, Thailand in days, and then, the animals were administered November 2014. The plant was authenticated by with a single oral dose of PCM (3 g/kg) on the st Ms. Dujhathai Anekchai, a taxonomist from the 31 day. Groups III and IV received NAC (1 g/kg) Faculty of Pharmaceutical Sciences, Prince of or CS (1,000 mg/kg) orally after the single oral Songkla University, Songkla, Thailand. A dose of PCM. voucher specimen (no. SKP 081030301) was deposited in the herbarium of the same At 24 h after PCM administration, all the animals institution. were anesthetized and euthanized. Blood was collected by cardiac puncture, and the liver tissue Extraction and phytochemical screening was dissected for biological and histological evaluation. The protocol for the present study Whole plants were washed thoroughly in water, was approved by the Animal Research cut into small pieces, soaked in 50 % ethanol (1 Committee of Thammasat University, Thailand part of plant material: 2 parts of 50 % ethanol) for (license no. AE 005/2015). All institutional and 72 h, and then filtered. The filtrate was national guidelines for the care and use of concentrated using rotary vacuum evaporation laboratory animals were followed in this study [8]. and then lyophilized with a freeze dryer. The yield of the extract was 1.86 %. The extract was A single dose of PCM was selected according to kept at −80 °C before its screening for the the previous reports in the hepatoxicity-induced presence of tannins, flavonoids, and phenolic rat model [9,10]. The dose of extract (1000 compounds [6]. mg/kg) used in the present study was selected based on our preliminary study on serum total Total phenolic content assay antioxidant capacity (TAC). Serum TAC, which is considered as the cumulative action of all the The CS extract was assayed for total phenolic antioxidants, was demonstrated to be content using the Folin-Ciocalteu reagent significantly elevated only in 1000 mg/kg CS according to a previously described method [6]. treated rat (1.74 ± 0.45 mM trolox equivalent) Gallic acid was used as a standard, and the compared to the control group (1.05 ± 0.75 mM results were expressed as gallic acid equivalent trolox equivalent). However, the doses of 250 per gram CS extract. mg/kg and 500 mg/kg (1.15 ± 0.77 and 1.10 ± 0.87 mM trolox equivalent, respectively) did not Trop J Pharm Res, January 2017; 16(1): 102 Saenthaweesuk et al demonstrate the significant changes in the serum glutathione assay kit (CS0260, Sigma Chemical TAC. Company (St Louis, MO, USA). Acute toxicity studies Measurement of liver and kidney function markers Thirty-six male Sprague-Dawley rats, weighing 200 - 250 g, were used for the acute toxicity The blood of animals from both acute toxicity test study. They were allowed to acclimatize for and hepatoprotective study was collected by seven days to the laboratory condition before the cardiac puncture. 10 mL of whole blood was experiment. The procedure was performed allowed to clot in a conical tube at room according to OECD 425 guidelines [11], with temperature. After coagulation, the blood was respect to the guidelines that the volume of centrifuged at 2,000 rpm 4 °C for 10 min to administration should not excess 1 mL/100 g separate the serum. The levels of liver function body weight. The maximum concentration of CS dissolved in aqueous solution is 1000 mg/mL. markers, including kinetic assay of enzyme Therefore, our limit test dose is 2000 mg. The aspartate aminotransferase (AST) and alanine rats received 2,000 mg/kg CS extract orally. On aminotransferase (ALT), and kidney function testing the first dose with one animal, we found markers, including blood urea nitrogen (BUN) that the animal survived. Thus, the remaining and serum creatinine (Cr), were measured using animals (n = 4) were administered with 2,000 automated machine analyzer at The Clinical mg/kg CS