Protective Effect of Zataria Multiflora 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 Pesticides, Food Contaminants, and Agricultural Wastes

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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.

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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 cytotoxicity, 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 performance 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- scavenges free radicals.[29] Taken together, MT stimulates ture. The absorbance was recorded at 200–400 nm and a ø the cholinergic system and induces oxidative stress and maximum wavelength of absorbance ( max) was determined inflammation in liver tissue and on the other hand, Z. multi- at 273 nm. Calibration curve of rutin was plotted by measur- flora extract and RA have antioxidant, anti-inflammatory, ing the absorbance of different concentrations of rutin (2.5, and anticholinergic effects. Therefore, the current study 5, 10, 20, 30, and 40 mg/ml) at 273 nm. TFC of the plant aimed to investigate the protective effects of RA and MEZM extract was calculated from slope of the calibration curve against MT-induced toxicity in HepG2 cell line. and was expressed as mg of rutin equivalent/g dried extract (w/w).[30] Materials and methods Chemicals and reagents RA content using high-performance liquid Fetal bovine serum (FBS) and low-glucose Dulbecco’s chromatography (HPLC) Modified Eagle’s Medium were purchased from Biosera (France). All the other chemicals with high purity were One of the most common techniques for quality evaluation purchased from Sigma-Aldrich chemical company (St. and authenticity of the medicinal plants is standardization Louis, MO). or determination of specific bioactive components or marker in the plant. In the present work, RA was used for MEZM standardization using HPLC method. For this, separation Preparation and extraction of herbal sample was carried out using HPLC system (Yang, Souh Korea) and VR m Aerial parts of Z. multiflora were collected from Jupar area a Waters C18 column (250 4.6 mm, 4 m) (USA). The m m of Kerman. After authentication of the plant, a herbarium plant extract (100 g/ml, 10 l) was passed through a syringe voucher number (KF1241) was assigned to it in the filter (0.2 mm) and was injected into HPLC. Elution was set Herbarium Center of Kerman Pharmacy Faculty. For carry- at a flow rate of 1.0 ml/min at 25 C. The mobile phases ing out the extraction, 250 g of the dried plant was carefully were A: 0.1% (v/v) formic acid solution in water, and B: weighed, powdered, and then passed through a sieve with 0.1% (v/v) formic acid solution in methanol. A ratio of 90% mesh 35. The plant powder was macerated in 80% methanol A and 10% B was run for 30 min. The wavelength for UV for 24 h. After 30 min of sonication at 35 C, the extracts detection was set at 330 nm. For calibration curve, RA were concentrated under vacuum on a rotary evaporator (10 mg) was accurately weighed and dissolved in 80% and dried at 40 C. The dried extract was kept in 20 C methanol. A series of standard RA ranging 0.05–10 mg/ml until further use. were used for calibration curve. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 3

Cell culture antioxidant capacity was expressed as nmol of Fe II per mg protein according to the standard curve of ferric sulfate. HepG2 cells were bought from Pasteur Institute (Tehran, Iran) and were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% FBS and 1% LPO assay penicillin and streptomycin. The cells were incubated in a The level of malondialdehyde (MDA), an end product of humidified incubator (New Brunswick, Galaxy 170 R, LPO, was determined according to the thiobarbituric acid Canada) with 5% CO2 and 95% O2 at 37 C. All experi- (TBA) reactive substance assay.[33] First, 1.25 ml of 20% tri- ments were conducted on HepG2 cell line with the passage chloroacetic acid and 1.0 ml of 0.67% TBA (dissolved in 2 M number of 1 to 10. sodium sulfate) were added to 250 ll of cell suspension (1 106 cells). The mixture was kept in a boiling water bath Evaluation of cell viability for 30 min and the resulting chromogen was extracted with 2ml of n-butyl alcohol. Then, the organic phase was trans- To evaluate the IC50 of MT and MEZM in HepG2 Cells, ferred to 96 well plates and the absorbance was measured at 1 104 cells were seeded in each well of 96-well plate and 532 nm. The results were expressed as nmol MDA per mg incubation overnight at 37 C with 5% CO2 and 95% O2. protein according to the standard curve of MDA. Then the cells were treated with MEZM (0.1, 1, 10, 100, and 1000 lg/ml) or MT (12.5, 25, 50, 100, 200, 400, and 800 lM) Protein carbonylation assay for 24 h. MTT assay was carried out to evaluate cell viability. Briefly, 20 ll of filtered MTT [3-(4, 5-Dimethylthiazol-2-yl)- According to the method developed by Levin et al.,[34] the 2, 5-Diphenyltetrazolium Bromide] solution (5 mg/ml) was absorbance of the yellow complex (resulted from the reac- added to each well and media was removed after 4 h incubation of 2,4-di-nitrophenylhydrazine and carbonyl) was meas- tion at room temperature in a dark place. The blue forma- ured at 380 nm. A volume of 100 ll of sample was added to zan crystals were dissolved in 100 ml DMSO. The absorbance 0.8 ml of water and 0.5 ml of 20% trichloroacetic acid and at 570 nm was measured using a plate reader (ELISA incubated at room temperature for 10 min. The sample was Reader, Biotek, USA) and IC50 (50% growth inhibition) was centrifuged (10 min at 3000g) and the supernatant discarded, calculated.[31] then 1 ml of guanidine hydrochloride (6 M) was added to the pellet and incubated in a bath (37 C for 30 min) and ð Þ ðÞ¼ Absorption Test centrifuged again. The absorbance of the supernatant was Viability % of control ð Þ 100 Absorption Control read at 380 nm and the results were expressed as nmol of To evaluate the preventive effects of MEZM on cytotox- protein carbonyls per mg protein according to the extinction 1 1 icity induced by MT, 1 104 cells were seeded in each well coefficient of 22,000 M cm . and incubated overnight at 37 C with 5% CO2 and 95% O2. The cells were incubated with MEZM (1 lg/ml) or RA Evaluation of intracellular ROS (0.1 lg/ml) for 4 h before treatment with MT (100 lM). Intracellular ROS generation was measured using 2,7- After 24 h incubation, the viability of cells was measured by [35] MTT test. dichlorodihydrofluorescein diacetate (H2DCF-DA). Cells were seeded at 2.5 104 cells/well and treated with MEZM (1 lg/ml) or RA (0.1 lg/ml) for 4 h and then exposed to MT Evaluation of oxidative stress biomarkers (100 lM). After 24 h, 200 ml H2DCF-DA (10 mM) was added to each well and incubated for 30 min at 37 C in a dark Throughout the study, HepG2 cells (7 106) were treated room. The fluorescence intensity was measured in a micro- with MEZM (1 lg/ml) or RA (0.1 lg/ml) for 4 h and then l plate reader at 485 nm excitation and 528 nm emission and exposed to MT (100 M). After 24 h, the cells were har- finally appropriate images were taken using a fluores- vested, homogenized, and centrifuged 15 min at 4000g and cence microscopy. 4 C. The supernatant was collected and kept at 80 C until more experiments. Evaluation of apoptosis

Total antioxidant capacity assay (FRAP) assay Apoptosis of HepG2 cells was evaluated using Annexin V- FITC apoptosis detection kit (Miltenyi Biotec, Germany). According to the ferric reducing antioxidant power method, Briefly, the cells (2 106) were treated with MEZM (1 lg/ ferric 2,4,6-Tri (2-pyridyl)-s-triazine (Fe III-TPTZ) complex ml) or RA (0.1 lg/ml) for 4 h and then exposed to MT is reduced to the ferrous (Fe II) and the absorption of blue (100 lM). After 24 h, the cells were harvested, washed with [32] color product is measured at 593 nm. Briefly, 5 ll of the PBS twice, suspended in Annexin-V binding buffer and sample was added to 295 ll FRAP reagent (25 ml acetate finally stained with Annexin V and Propidium Iodide (PI) buffer (300 mM, pH 3.6, 2.5 ml 20 mM ferric chloride and according to the manufacturer’s protocol. The apoptosis was 2.5 ml 10 mM Fe III-TPTZ) and incubated for 10 min at examined using Partec flow cytometry (Germany) and ana- 37 C. Absorbance was read at 593 nm and the total lyzed by FloMax software (version 2.25). 4 S. KARAMI-MOHAJERI ET AL.

Figure 1. Calibration curve of (a) gallic acid (50–400 mg/ml) at 765 nm and (b) rutin (0.25–30 mg/ml at 273 nm.

Figure 2. HPLC chromatogram of (a) rosmarinic acid, (b) Zataria multiflora, and (c) calibration curve of rosmarinic acid (0.05–10 mg/ml). JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 5

Results Extraction yield and phytochemical studies The extraction yield of MEZM was 19.2% w/w. Phytochemical screening indicated the presence of flavonoids, saponins, terpenoids, and tannins in MEZM. TLC screening exhibited rutin and RA as the main phenolic components in the plant.

Total phenolic content, total flavonoid content, and RA content Total phenolic content of the plant was determined 58.09 mg gallic acid equivalent/g dried extract and total flavonoid content was 50.02 mg rutin equivalent flavonoid/g dried extract based on calibration curve of gallic acid and rutin, respectively (Fig. 1(a,b)). Calibration curve of RA was provided using HPLC data. In RA calibration curve, where the x and y were RA concentration and peak area, respectively, RA content was calculated from the regression equation. The calibration curve of RA was linear with correlation coefficients (R) of 0.9996 over the concentration range of 0.05–10 mg/ml. The regression equation was determined to be Y ¼ 52.833 1-3.4786. The intra- and inter-day precision variations (RSD%) of RA was in the range of 3.5–10.6% and 3.2–13.2% at three-level concentrations, respectively. LOD (signal/noise [S/N] ¼ 3.3) and LOQ (S/ N ¼ 10) were 0.000134 and 0.00416 mg/ml, respectively for inter-day (Fig. 2(a–c)). RA content of the plant was 73.48 mg/g dried extract.

Cell viability and oxidative stress findings In this experimental set-up, IC50 values for MEZM and MT was 368.56 ± 86.50 mg/ml and 99.43 ± 1.00 mM, respectively (Fig. 3). The findings of cell viability and oxidative stress biomarkers are summarized in Table 1. Treatment with MEZM (1 lg/ml) or RA (0.1 lg/ml) 4 h before exposure to MT (100 lM, for 24 h) significantly increased viability of cells compared to the group received only MT (100 lM, for 24 h) (P < 0.001). There was no significant difference in the percentage of cell viability between groups received MEZM and RA (p ¼ 0.697). The total antioxidant capacity significantly increased after pretreatment with RA (0.1 lg/ml) compared to the MT-exposed cells Figure 3. Cytotoxicity assessment by MTT assay in HepG2 cells following the ¼ < exposure of various concentrations of (a) malathion, (b) Zataria multiflora meth- (P 0.006) and groups received MEZM (P 0.001). anolic extract, and (c) rosmarinic acid in HepG2 cell for 24 h. However, MEZM (1 lg/ml) did not make a significant difference in total antioxidant capacity compared to the MT- Statistical analysis exposed cells (P ¼ 0.346). Treatment of cells with MT All the experiments were carried out in triplicate and (100 lM) for 24 h led to a significant increase in LPO and data were reported as mean ± SD. The analysis of protein carbonylation compared to the control group data was performed by GraphPad Prism 5 software (P < 0.001). LPO product MDA and protein carbonyl level (GraphPad, La Jolla, CA, USA) using ANOVA and Tukey significantly were decreased after pretreatment of HepG2 post-hoc test. Shapiro–Wilk test was used for normality cells with RA (0.1 lg/ml) and MEZM (1 lg/ml) compared to of the data and Bartlett test was used for homogeneity of MT group (P < 0.001). There was no significant difference variances.Thedifferencelessthan0.05wereconsidered between the RA (0.1 lg/ml) and MEZM (1 lg/ml) effects on significant. LPO (P ¼ 0.302) and protein carbonylation (P ¼ 0.282). 6 S. KARAMI-MOHAJERI ET AL.

Table 1. Cell viability and oxidative stress findings after exposure to malathion (100 mM) in HepG2 cell which pretreated with Zataria multiflora methanolic extract (1 mg/ml) and rosmarinic acid (0.1 mg/ml) for 4 h. Malathion (100 mM) Pretreatment Variables Control Without pretreatment MEZM (1 mg/ml) RA (0.1 mg/ml) Viability (%) 99.1 ± 7.6 42.6 ± 4.4 85.2 ± 10.0 93.2 ± 5.6 Total antioxidant capacity (nmol Fe(II)/mg protein) 297.5 ± 30.65 235.4 ± 32.65 292.1 ± 32.11 382.3 ± 38.94 Lipid peroxidation (nmol/mg protein) 1.38 ± 0.08 2.04 ± 0.07 1.40 ± 0.06 1.31 ± 0.05 Protein carbonylation (nmol/mg protein) 11.38 ± 0.45 20.39 ± 1.81 12.58 ± 1.17 10.58 ± 0.41 Data were expressed as mean ± standard deviation in at least three independent experiments, p < 0.001 compared to the controls. MEZM; methanolic extract Zataria multiflora; RA; rosmarinic acid.

Figure 4. A) Fluorescence microscopy images of HepG2 cells. (a) Control cells, (b) cells exposed to malathion (100 lM, MT 100), and pretreated cells, (c) received methanolic extract Zataria multiflora at the concentration of 1 lg/ml (Pre ZM 1), and (d) rosmarinic acid at concentrations of 0.1 lg/ml (Pre RA 0.1) before administration of malathion. (e) Bar chart represents reactive oxygen species (ROS) generation in the groups. Data were expressed as mean ± standard deviation in at least three independent experiments, p < 0.05 compared to the controls.

Effect of MEZM and RA on ROS generation Effect of MEZM and RA on apoptosis of HepG2 As shown in Figure 4, following treatment of HepG2 cells The results of apoptotic cell death using flow cytometry by MEZM for 24 h, ROS production was detected by stain- have been shown in Figure 5.Inthisscatterplot,the ing the live cells with DCFDA. The cellular DCF fluores- fluorescence generated by Annexin V and PI staining has cence levels were increased after 24 h exposure to MT been characterized. The data show that in control group, (100 lM) and decreased in the cells which were pretreated more than 97% cells were alive with 0.80%, 0.76%, and with MEZM (1 lg/ml) or RA (0.1 lg/ml) compared with the 1.07% cells in the early apoptosis, late apoptosis, and control group. necrosis stages, respectively. However, MT caused cell JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 7

Figure 5. Cell death assayed by flow cytometry in HepG-2 cells after staining with Annexin V-FITC/Propidium Iodide (AV/PI). Representative dot plot diagrams obtained by flow cytometry analysis of (a) Control cells, (b) cells exposed to malathion (100 lM, MT 100), and pretreated cells (c) received methanolic extract Zataria multiflora at the concentration of 1 lg/ml (Pre ZM 1), and (d) rosmarinic acid at concentrations of 0.1 lg/ml (Pre RA 0.1) before administration of malathion. The plots are divided into four quadrants; viable (lower left quadrant, negative, AV˗/PI–), and early apoptosis (upper left quadrant, AVþ/PI–), late apoptosis (upper right quadrant, AVþ/PIþ), and necrosis (lower right quadrant, AV–/PIþ). (e) Bar chart represent mean ± standard deviation of the percent of cell population in at least three independent experiments, p < 0.01, p < 0.001 compared to the controls. death through an increase in the cell count in the early cells treated with MEZM exhibited induction of apoptotic apoptosis (9.81%), late apoptosis (16.88%) and necrosis cell death (30.54% early and 9.7% late) and decrease in (10.77%) stages compared to the control group. HepG2 necrotic cell death (1.52%) compared to the untreated MT 8 S. KARAMI-MOHAJERI ET AL. exposed group. RA induced early apoptosis up 10.67%, cytotoxicity, ROS formation, oxidative stress damages, and and decreased the late apoptosis to 7.62% and necrosis the percentage of cells in both necrotic and late apoptosis to 2.44%. stages, however, the percentage of cells in the early apoptotic stage in MT-treated groups which received MEZM and RA Discussion was significantly higher than other groups. Hence, it seems that MEZM and RA promote apoptosis rather than necrosis This study evaluated the protective effects of MEZM and its in MT-exposed cells. In other words, apoptosis develops in active ingredient, RA, in HepG2 cells against MT-induced tox- malathion-treated cells received MEZM and RA after more icity. The results indicate that both MEZM and RA eliminated than 24 h. However, for more accurate conclusion, it is cytotoxicity, oxidative stress, and the percentage of the cell needed to consider the time effect too. The antioxidant population in late apoptosis and necrosis stages induced by MT. effects of ZM and RA might be involved in the delay of MT at the IC50 concentration, 100 mM, decreased total apoptosis development. In addition in early apoptosis, the antioxidant capacity and caused an increase in production function of the cell membrane and mitochondria are main- of intracellular ROS, end-products of LPO and protein car- tained. Cells in the early apoptosis were not distinguished bonylation in HepG2 cells. These findings are in accordance by MTT test, because MTT is reduced in mitochondria to with the findings of previous studies showing the effects of [36] make formazane dye, and the cell membrane and mitochon- MT in the induction of oxidative damages. MT induces dria have function in the early apoptosis. Apoptosis is a oxidative stress by production of the free radical and subse- physiological process in cells to remove the cells functionally quently over-production of ROS and by disruption in cellu- [4] or genetically damaged, without progressive inflammation lar antioxidant system. Oxidative stress consequently [54] and involvement of any other cells. In the early stage of disrupts cell membrane integrity and mediates cell death. apoptosis, membrane integrity of cells retains, and mito- This study also confirmed the previous reports on death [37,38] chondria are active to produce enough energy for the devel- promoting effects of MT. According to the results – opment of the apoptosis process.[55 57] The elevation in LPO obtained from flow cytometry analysis, apoptosis and necro- by MT has been prevented by MEZM and RA. LPO disrupts sis significantly occurred in 50% of HepG2 cell population cell membrane integrity and induces necrosis. The results of exposed to MT. Analysis of Annexin V- and PI-stained cell by flow cytometry was reported as the percentage of the an in vitro study showed that RA significantly inhibited the l [58] apoptotic and necrotic cell. Annexin V binds to exposed proliferation of rat hepatic stellate cell line 7.1 g/ml. The l phosphatidyl serine on the outside of the membrane outside IC50 of RA was 118.04 and 297.32 g/ml in the human of the apoptotic cell while DNA of necrotic cells binds to bone horning breast cancer cells and rat bone marrow stro- [59] PI.[39] Mitochondria as the main site of cellular metabolism mal cells, respectively. Kavoosi et al. reported that essen- have a pivotal role in the regulation of oxidative stress and tial oil of Z. multiflora, thymol, carvacrol, and p-cymene did cell death.[40] Oxidative stress-induced by MT triggers apop- not affect the viability of macrophages at low concentrations tosis, disturbs the mitochondrial function and integrity and (1, 10, 100 ng/ml). However, at higher concentrations [60] releases cytochrome C.[41] Besides, the mitochondrial anti- (1000 ng/ml), all of them reduce some percent of life. oxidant defense system has a crucial role in the detoxifica- Although the protective effect MEZM can be due to RA, but tion of MT and cell survival. In this regard, in different we cannot ignore the presence of other important phenolic in vitro, in vivo, and clinical studies, antioxidants are used compounds in this extract such as thymol and its flavonoids. – to reduce MT-induced oxidative stress.[1,3,7,42 49] There are At first, RA was expected to act much stronger, but the also significant efforts have been made to find natural com- results of toxicity reduction were almost the same in the pounds containing phenolic compounds for protecting cells two groups. from oxidative damage.[27] Polyphenols and flavonoids directly scavenge free radicals and act as an antioxidant or a modulator of the activity of the antioxidant enzymes.[50] Conclusion Essential oils and extracts of Z. multiflora contain the phenolic compounds such as thymol, flavonoids, and phenolic This study shows the protective effects of Z multiflora and acids.[51] The phytochemical results of the current study RA on the toxicity of HepG2 caused by MT. MEZM and have shown that a large amount of phenolic compounds RA can, through antioxidant activity, inhibit oxidative dam- and RA are present in the MEZM. RA has antioxidant and age, prevent necrosis, and delay apoptosis. To investigate the anti-inflammatory effects on cancer cells and antiapoptotic protective effects of other active ingredients in Z multiflora, activity.[52] RA can eliminate hydrogen peroxide and seques- further studies are required to identify other protective tering free radicals.[53] The percentage of viable cells signifi- mechanisms for MT-induced toxicity. cantly increased by about 45% in the pretreatment with RA (0.1 lg/ml) compared with the MT group. MEZM (1 lg/ml) increased cell viability by 38%. In this study, MEZM and Acknowledgments RA decreased MT-induced cytotoxicity and ROS formation We appreciate Pharmaceutical Research Center and Department of and increased cell population in late apoptosis and necrosis Pharmacology and toxicology of Kerman University of Medical stages. Both MEZM and RA in MT-treated group reduced Sciences for their cooperation. All the research done by the authors. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH, PART B 9

Conflict of interests [13] Saedi Dezaki, E.; Mahmoudvand, H.; Sharififar, F.; Fallahi, S.; Monzote, L.; Ezatkhah, F. Chemical Composition along with All authors declare no competing interest. Anti-Leishmanial and Cytotoxic Activity of Zataria Multiflora. Pharm. Biol. 2016, 54, 752–758. DOI: 10.3109/13880209.2015. 1079223. Funding [14] Gholamhoseinian, A.; Fallah, H.; Sharififar, F.; Mirtajaddini, M. The Inhibitory Effect of Some Iranian Plants Extracts on the This work was supported by Kerman University of Medical Sciences Alpha Glucosidase. Iran. J. Basic Med. Sci. 2008, 11,1–9. (Grant No.: 940659). [15] Arabzadeh, A. M.; Ansari-Dogaheh, M.; Sharififar, F.; Shakibaie, M.; Heidarbeigi, M. Anti Herpes Simplex-1 Activity of a Standard Extract of Zataria Multiflora Boiss. Pak. J. Biol. Sci. ORCID 2013, 16, 180–184. DOI: 10.3923/pjbs.2013.180.184. [16] Mandegary, A.; Sharififar, F.; Abdar, M. 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