Journal of Environmental Science and Health, Part B Pesticides, Food Contaminants, and Agricultural Wastes ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lesb20 Protective effect of Zataria multiflora Boiss. and its main compound, rosmarinic acid, against malathion induced oxidative stress and apoptosis in HepG2 cells Somayyeh Karami-Mohajeri , Amir Najafi , Behzad Behnam , Mitra Sadeghi Meymandi , Zahra kashitarash Ifahani , Elham Jafari , Mohmoudreza Heidari , Neda Mohamadi & Fariba Sharififar To cite this article: Somayyeh Karami-Mohajeri , Amir Najafi , Behzad Behnam , Mitra Sadeghi Meymandi , Zahra kashitarash Ifahani , Elham Jafari , Mohmoudreza Heidari , Neda Mohamadi & Fariba Sharififar (2021): Protective effect of Zatariamultiflora Boiss. and its main compound, rosmarinic acid, against malathion induced oxidative stress and apoptosis in HepG2 cells, Journal of Environmental Science and Health, Part B To link to this article: https://doi.org/10.1080/03601234.2021.1879595 Published online: 09 Feb 2021. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=lesb20 JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B https://doi.org/10.1080/03601234.2021.1879595 Protective effect of Zataria multiflora Boiss. and its main compound, rosmarinic acid, against malathion induced oxidative stress and apoptosis in HepG2 cells Somayyeh Karami-Mohajeria,b , Amir Najafia,b, Behzad Behnama,c, Mitra Sadeghi Meymandia, Zahra kashitarash Ifahanid, Elham Jafarie, Mohmoudreza Heidaria,b, Neda Mohamadid, and Fariba Sharififard aPharmaceutics Research Center, Kerman University of Medical Sciences, Kerman, Iran; bDepartment of Pharmacology and Toxicology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; cDepartment of Biotechnology, School of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran; dFaculty of Pharmacy, Department of Pharmacognosy, Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran; ePathology and Stem Cell Research Center, Kerman University of Medical Science, Kerman, Iran ABSTRACT ARTICLE HISTORY Malathion (MT) is one of the most widely used organophosphorus insecticides which induces tox- Received 1 September 2020 icity through oxidative stress induction, free radical production and acetylcholinesterase inhibition. Accepted 13 January 2021 In this work, HepG2 cells were used to determine the effect of Zataria multiflora methanolic KEYWORDS extract (MEZM) and rosmarinic acid (RA) on MT-induced cytotoxicity, oxidative stress, and apop- Zataria multiflora; tosis. Total phenolic content (TPC) and total flavonoid content (TFC) were determined and plant rosmarinic acid; malathion; was further standardized based on RA content using HPLC method. The cultured HepG2 cells HepG2 cell line; were pretreated with MEZM (1 lg/ml) and RA (0.1 lg/ml) for 4 h and exposed to MT (100 lM). Cell hepatoprotective viability, oxidative stress biomarkers, ROS production, and cell death were examined after 24 h. The amount of RA was determined 73.48 mg/g dried extract. IC50 values of MEZM and MT were 368.56 lg/ml and 99.43 lM, respectively. Pretreatment with MEZM and RA decreased the cytotox- icity, oxidative stress, and cell percentage in the late apoptosis and necrosis stages induced by MT. There was no significant difference between MEZM and RA effects. The present study showed the significant protective effects of MEZM against toxicity induced by MT in hepatocytes which can be attributed to the plant antioxidant constituents including RA. Abbreviations: FBS: Fetal bovine serum; FRAP: Total antioxidant capacity assay; HPLC: High per- formance liquid chromatography; LPO: Lipid peroxidation; MT: Malathion; MDA: malondialdehyde; MEZM: Zataria multiflora methanolic extract; RA: Rosmarinic acid; TBA: thiobarbituric acid; TPC: Total phenolic content; TFC: Total flavonoid content; TLC: Thin layer chromatography; ROS: Reactive Oxygen Species Introduction disrupts the liver detoxification mechanisms and endogen- ous antioxidant status.[4] Animal studies have shown that Malathion (MT) is an organophosphorus insecticide that is MT is responsible for the necrotic changes in the liver tis- widely used in agriculture and pest control throughout the sue, hepatomegaly, as well as an increase in liver enzymes world. MT is metabolized by hepatic cytochrome P450 activity.[5] MT increases the membrane permeability through enzymes into its active metabolite, malaoxon, which is peroxidation of the cell membrane lipids[6] and also induces responsible for MT toxicity and irreversibly inhibits cholin- cytotoxic effects in HepG2 cells which are probably medi- esterase enzyme activity. In addition to the cholinergic ated through induction of oxidative stress and lipid peroxi- effects of MT, production of free radicals and consequently dation (LPO).[7] Currently, medicinal plants have become a reactive oxygen species (ROS) occurs during the metabolism subject of intensive investigations as a natural source of anti- [1] of MT. The consequence of an imbalance between the oxidant compounds, and literature tracking reveals numer- antioxidant defense system and the production of ROS, oxi- ous reports of their antioxidant activity which can help dative stress affects the biological function of essential mole- eliminate pesticide toxicity.[8,9] The main role of phenolic cules, disturbs the integrity of the cell membrane and compounds as free radical scavengers is confirmed in contributes to the cell death. Some studies have shown MT’s numerous studies, especially in medicinal plants, which can – lipoperoxidative effects. Because it has a lipophilic origin, it lead to other biological and pharmacological activities.[9 11] interacts with the cell membrane and thus disturbs the cell Biological compounds with antioxidant properties can pro- membrane lipid bilayer in almost all visceral organs.[2,3] MT tect cells against the harmful effects of ROS and other also induces liver damage by overproduction of ROS and free radicals.[12] CONTACT Fariba Sharififar [email protected]; [email protected] Faculty of Pharmacy, Department of Pharmacognosy, Herbal and Traditional Medicines Research Center, Kerman University of Medical Sciences, Kerman, Iran. ß 2021 Taylor & Francis Group, LLC 2 S. KARAMI-MOHAJERI ET AL. Medicinal plants have been used since the beginning of Phytochemical analysis human existence as herbs and nutrients. Thanks to great The presence of flavonoids, tannins, saponins, alkaloids and effectiveness, antioxidant activity, minimal side effects, and terpenoids was investigated in Z. multiflora as explained nutritional values, these are still the cornerstone of modern [18] [11] previously. drugs. Zataria multiflora Bioss. (Z. multiflora) with the Total phenolic content (TPC) of the plant was determined Persian name of Avishane-Shirazi is a plant growing in Iran and is highly known for its copious benefits. Lately, antioxi- by Folin-Ciocalteu method. In brief, 0.50 ml of different con- dant, antibacterial, anticonvulsant, antiviral, antileishmanial centrations of gallic acid as a reference standard (50, 100, 150, m and anticholinesterase effects have been investigated for this 200, 250, 300, and 400 g/ml) was added to 2.5 ml – – plant.[11,13 19] The plant has been shown alpha glycosiase Folin Ciocalteu reagent (10%) and incubated at room tempera- inhibitory and antihelicobacter pylori effect too.[14,20] Z. ture for 4 min. Saturated sodium carbonate solution (2 ml) was multiflora prevents lipid peroxidation in chemicals-induced added to the mixture, and absorbance was read at 760 nm after hepatotoxicity by increasing the activity of antioxidant 2 h incubation at room temperature. TPC was expressed as mg [10] enzymes, including catalase and superoxide dismutase gallic acid equivalent/g dried extract (w/w). – enzymes.[21 23] Phenolic compounds have protective effects For total flavonoid content, firstly, the major flavonoid of against the damages induced by free radicals and act directly MEZM was determined using thin-layer chromatography as free radical scavengers and metal chelators, or indirectly (TLC). For this, a stock solution of the extract and different by activating important signaling pathways such as tran- reference standards of catechin, rutin, kaempferol, and quer- scription of nuclear factors resulting in the production of cetin (100 mg/ml) was spotted on silica gel GF254 plates and – protective proteins such as antioxidant enzymes.[17,24 26] was developed in chloroform:ethyl acetate:methanol:formic Rosmarinic acid (RA), the main phenolic component of Z. acid (3:1:2:0.5) as the mobile phase. The plates were eval- multiflora, increases glutathione levels and decreases lipid uated visually under UV light at 365 nm after drying and peroxidation in HepG2 cells.[27] This compound is found in the intensity of MEZM spots was compared to the reference a different genus of Boraginaceae and Lamiaceae families standards. Rutin was considered as the major flavonoid of ¼ which go with varying intriguing biological activities, includ- MEZM (Rf 0.27). For TFC determination, 1 ml rutin stock ing anti-microbial, anti-inflammatory, and antioxidant.[28] It solution (50 mg/ml) was added to AlCl3 solution (2% in is generally believed that RA acts as an antioxidant and methanol, 1 ml) and incubated for 30 min at room tempera-