Research Journal of Pharmacognosy (RJP) 7(2), 2020: 43-51 Received: 1 Dec 2019 Accepted: 10 Feb 2020 Published online: 18 Feb 2020 DOI: 10.22127/rjp.2020.207706.1533 Original article The Role of Allium saralicum Extract on Prevention of Acetaminophen- Induced Hepatic Failure: an Experimental Study 1,2 1,3 4 1,2* Mahdi Alvandi , Dara Dastan , Sara Soleimani Asl , Amir Nili-Ahmadabadi 1Medicinal Plants and Natural Products Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. 2Department of Pharmacology and Toxicology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran. 3Department of Pharmacognosy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran. 4Anatomy Department, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Abstract Background and objectives: Acetaminophen (APAP) is a common analgesic medicine whose overdose leads to severe hepatic dysfunction. Due to the known antioxidant properties of Allium species, the present study aimed to evaluate the protective effects of Allium saralicum plant on APAP induced liver toxicity. Methods: The hydro-alcoholic extract of A. saralicum was prepared by maceration and ultrasonic methods. Forty-two rats in seven groups were treated by gavage as follows: groups 1 and 2 received normal saline, groups 3 received 400 mg/kg of A. saralicum hydro-alcoholic extract, and the groups 4-7 were treated with 50, 100, 200 and 400 mg/kg of A. saralicum extract, respectively. After two consecutive weeks, the therapeutic groups, as well as the positive control (APAP) group, were administered a single dose of APAP (2 g/kg). After 48 hours, the animals were anesthetized, and blood and liver samples were collected for histological and biochemical examinations. Results: Our findings indicated that APAP caused a significant rise in ALT (p<0.001), AST (p<0.001), ALP (p<0.001) and LDH (p<0.001) serum levels, total and direct bilirubin (p<0.001), hepatic lipid peroxidation (LPO; p<0.001) and nitric oxide (NO; p<0.001). In addition, APAP let to the decreasing of the total antioxidant capacity (TAC; p< 0.001), total thiol molecules (TTM; p<0.001), and structural changes in the hepatic tissue. Following administration of A. saralicum extract, a remarkable improvement was observed in the functional and oxidative stress indices of liver tissue alongside histopathologic alterations. Conclusion: Our results showed that A. saralicum extract significantly improved APAP-induced hepatic failure through inhibition of oxidative/nitrosative stress. Keywords: acetaminophen; Allium saralicum; hepatotoxicity; oxidative stress Citation: Alvandi M, Dastan D, Soleimani Asl S, Nili-Ahmadabadi A. The role of Allium saralicum extract on prevention of acetaminophen-induced hepatic failure: an experimental study. Res J Pharmacogn. 2020; 7(2): 43-51. Introduction Paracetamol (APAP; N-acetyl-p-aminophenol) is if untreated, it may lead to jaundice and severe an analgesic and antipyretic medication that has liver injury [6]. The studies have indicated that been used as an over-the-counter medication annually about 6% of adults are exposed to [1,2]. Despite being safe at 1-4 g daily doses and APAP at the doses of more than 4 g/day; also, widely accessible, APAP intoxication is 30,000 patients are referred to hospitals for prevalent in all ages [2-5]. APAP toxicity is APAP-induced toxic effects [7]. associated with malaise, vomiting, and diarrhea; Therapeutic doses of APAP are mainly *Corresponding author: [email protected] © 2020. Open access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/) Alvandi M. et al. metabolized by the liver through conjugation pyridyl)-s-triazine (TPTZ) and acetaminophen with sulfate and glucuronide conjugates [8]. powder linear formula (CH3CONHC6H4OH) Higher doses of APAP are metabolized by were obtained from Sigma-Aldrich Chemical hepatic cytochrome P450-2E1 leading to the Company (USA). production of a highly reactive and electrophile product known as N-acetyl-p-benzoquinoneimine Plant material (NAPQI) [9]. Oxidative stress-induced by Collection and extraction procedure NAPQI may be partly due to cellular glutathione The aerial parts of Allium saralicum were (GSH) depletion and mitochondrial proteins gathered from Saral, Kurdistan, Iran in May 2018. adduction leading to mitochondrial oxidant stress. It was identified at the Herbarium of School of Several studies on inducers and inhibitors of Pharmacy, Hamadan University of Medical cytochrome P450 have indicated the role of ROS Sciences (HUMS), Hamadan, Iran with the code generated by P450-mediated metabolism in number (NO: 275). Maceration and ultrasonic APAP hepatotoxicity [10,11]. methods were applied for extraction. The Allium saralicum, native to Iran, is a member of coarsely powdered plant (300 g of aerial parts) the Amaryllidaceae family [12]. It contains was kept in contact with 1500 mL of methanol various compounds including hexanedioic acid, and water (1:1) for 72 h at 25 °C. The obtained γ-tocopherol, eicosane, hexatriacontane, vitamin mixture was then filtered using filter paper. A E, 2-phenyl-5-methylindole, phytol, methyl ester vacuumed rotary evaporator with a controlled and linolenic acid possessing various temperature of 50 °C was used for condensation pharmacologic effects [13,14]. Previous studies of the extract. The whole process was repeated have expressed the therapeutic properties of A. for three times. Finally 78 g dark brown extract saralicum on a wide variety of disorders. For was kept in a sterile vial in a dark and cool place instance, this herbal medicine has been consumed 4 °C. for its antibacterial, anti‐inflammatory, In the next stage, different fractions of antioxidant, anti‐hyperlipidemic and hepatorenal hydroalcoholic extract including n-hexane, protective effects in some Middle Eastern chloroform, ethyl acetate, and methanol fractions countries [15,16]. Regarding the role of oxidative were prepared by column chromatography. For stress in the pathophysiology of APAP poisoning this purpose, a column with dimensions of 8 × 17 and also due to the known antioxidant properties cm was used with a silica gel (70-30 mesh) as the of Allium species and using of Allium saralicum solid phase. A portion of hydroalcoholic extract by local people as food, we evaluated the effect (70 g) was fractionated on a silica gel column of A. saralicum hydro-alcoholic extract as a eluted with 500 mL of n-hexane, chloroform, potential antioxidant on APAP induced acute ethyl acetate, and methanol of increasing polarity liver injury. to give four fractions. The fractions were then concentrated in vacuum at 40 °C using a rotary Materials and Methods evaporator to produce n-hexane, chloroform, Ethical considerations ethyl acetate, and methanol fractions Finally, the The animal experiments were approved by the antioxidant properties of each fraction were Ethics Committee of Hamadan University of determined using ferric reducing antioxidant Medical Science (HUMS), Hamadan, Iran with power methods (FRAP) [17]. Also, the thiol ethical code of (ID: IR.UMSHA.REC.1396.364), levels were assessed using DTNB reagent as in accordance with the guideline of the Research described by Hu and Dillard [18]. According to Ethics Committee of the Health and Medical the results, the methanolic fraction as an effective Education, Iran (2019), based on the Helsinki fraction of hydro-alcoholic extract was selected Protocol (Helsinki, Finland, 1975). and maintained at the temperature of 4°C. Chemicals Experiment design All chemicals were purchased from Merck Male Wistar rats (250±25 g) were obtained from (Darmstadt, Germany) unless otherwise stated. the animal house of HUMS, Hamadan, Iran. The 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), N- animals were kept under controlled conditions of (1-naphthyl) ethylenediamine dihydrochloride laboratory (12 h light/dark cycles, 50% humidity (NED), 2-thiobarbituric acid (TBA), 2,4,6-tris (2- and temperature of 22-25 °C) and supplied with 44 Res J Pharmacogn 7(2): 43-51 Allium saralicum in liver failure standard diet and water ad libitum for one week (A2) and DTNB blank (B) was detected again at before the study. 412 nm. The level of thiol molecules was Forty-two rats were divided into seven groups of calculated by reduced glutathione as the standard. six each and treated for two weeks by gavage as follows: groups 1 and 2 received normal saline, Measurement of nitric oxide groups 3 received 400 mg/kg of A. saralicum Hepatic nitric oxide was measured by the extract, groups 4-7 were treated with 50, 100, 200 colorimetric method using Griess reagent (1% and 400 mg/kg of ASHE, respectively. After two sulfanilamide, 0.1% NED, and 2.5% phosphoric consecutive weeks, except for groups 1 and 3, acid) [19]. Briefly, 100 µL of reagent was mixed animals were administered a single dose of with sample (100 µL) and its optimum APAP (2 g/kg). After 48 h, the animals were absorbance was measured at 520 nm. sodium anesthetized with ether and blood samples were nitrite was used as the standard. collected from their carotid artery which were centrifuged at 2500 rpm (for 15 min) to separate Measurement of lipid peroxidation serum for biochemical assays. Furthermore, a The lipid peroxidation was assayed in liver tissue part of hepatic tissue was kept at the temperature using the thiobarbituric acid reactive substances of -80 °C for preparation of liver homogenate (TBARS) method [20]. Briefly, a reagent was (10%, w/v). The other portion of the liver was prepared containing TBA (0.2%) in H2SO4 (0.05 fixed in 5% buffered formalin for M). Then, 100 µL of homogenate sample and 500 histopathological experiments. µL reagent were mixed and heated in boiling water bath for 30 min, the absorbance was Serum biochemical analysis detected at 532 nm. Different concentrations of Glucose, triglyceride, direct and total bilirubin as malondialdehyde (2, 4, 8 and 16 µM) were used well as alanine aminotransferase (ALT), aspartate for plotting the calibration curve.