Reza Mohebbati1, Maryam Paseban1, Farimah Beheshti1, Mohammad Soukhtanloo2, Mohammad Naser Shafei3, Hasan Rakhshandeh4, Abolfazl Khajavi Rad1,3. 1. Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. 4. Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract
OBJECTIVES: The hepatotoxicity induced by Acetaminophen (AAP) mostly mediated by effect on oxidative stress parameters. The Zataria multiflora (Z.M) is an herbal medicine with well-known antioxidant effect. The aim of this study is investigation of preventive effects of Z.M and Carvacrol (CAR) on AAP-induced hepatotoxicity in rats. METHODS: Rats were randomly divided into four groups including: 1) Control, 2) Acetaminophen (AAP), 3) and 4) CAR. The saline, Z.M (200 mg/kg) and CAR (20 mg/kg) were administrated orally for 6 days, after that AAP (600 mg/kg) was administrated in the 7th day. Blood sampling was performed on the first and last days. Also, the liver tissue was removed for evaluation of Malondyaldehide (MDA), Thiol content, Superoxide dismutase (SOD) and Catalase (CAT). Total Protein (tPro), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT) and Alkaline Phosphatase (ALP) in liver tissue were evaluated. The changes (Δ) of enzymes activities were presented. RESULTS: The Δ GOT, Δ GPT and Δ ALP in CAR group significantly decreased compared to AAP group (P < 0.01 to P < 0.001) and Δ GPT in Z.M group was significantly reduced in comparison with AAP group (P < 0.05). Also, MDA, Thiol, SOD and CAT levels in treated groups were attenuated compared to AAP group (P < 0.05 to P < 0.001). CONCLUSION: Z.M and CAR have a powerful hepatoprotective effect. CAR is more effective than Z.M. Based on the results. Z.M and CAR could be potent supplementary agents against hepatotoxicity of AAP in patients.
OBJECTIVES: The hepatotoxicity induced by Acetaminophen (AAP) mostly mediated by effect on oxidative stress parameters. The Zataria multiflora (Z.M) is an herbal medicine with well-known antioxidant effect. The aim of this study is investigation of preventive effects of Z.M and Carvacrol (CAR) on AAP-induced hepatotoxicity in rats. METHODS: Rats were randomly divided into four groups including: 1) Control, 2) Acetaminophen (AAP), 3) and 4) CAR. The saline, Z.M (200 mg/kg) and CAR (20 mg/kg) were administrated orally for 6 days, after that AAP (600 mg/kg) was administrated in the 7th day. Blood sampling was performed on the first and last days. Also, the liver tissue was removed for evaluation of Malondyaldehide (MDA), Thiol content, Superoxide dismutase (SOD) and Catalase (CAT). Total Protein (tPro), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT) and Alkaline Phosphatase (ALP) in liver tissue were evaluated. The changes (Δ) of enzymes activities were presented. RESULTS: The Δ GOT, Δ GPT and Δ ALP in CAR group significantly decreased compared to AAP group (P < 0.01 to P < 0.001) and Δ GPT in Z.M group was significantly reduced in comparison with AAP group (P < 0.05). Also, MDA, Thiol, SOD and CAT levels in treated groups were attenuated compared to AAP group (P < 0.05 to P < 0.001). CONCLUSION: Z.M and CAR have a powerful hepatoprotective effect. CAR is more effective than Z.M. Based on the results. Z.M and CAR could be potent supplementary agents against hepatotoxicity of AAP in patients.
The liver has an important role in the nutrients metabolism such as proteins, carbohydrates and lipids, and also detoxification of powerful toxic drugs and metabolites, chemicals and environmental pathogens [1]. Drugs and alcohol is one of the most important causes of liver diseases [2]. One of this drugs is Acetaminophen (AAP) (Figure 1) which is commonly used as an antipyretic and analgesic drug for many years, has several side effect especially hepatotoxicity. Epidemiologically, in the USA 400 deaths each year occurs [3] due to AAP usage. Therefore, AAP is now the second leading cause of toxic drug ingestions in the USA [4]. Overdose of this drug in many countries is common [5]. Pharmacologically, AAP is metabolized to N-acetyl-p-benzoquinone imine (NAPQI) via cytochrome P450 enzymes in the liver that is generally produced in little amounts at therapeutic doses of this drug but in overdosing, production of NAPQI vigorously increased [6]. Glutathione depletion is reason of severe damages to the liver when it conjugates with NAPQI. Depletion of glutathione is the main reason of hepatotoxicity and necrosis [7]. The metabolic pathway of AAP is shown in Figure 2. Hepatotoxicity induced by AAP is common in people with alcohol abuse, unintentional overdoses and underlying hepatic disease [8] and N-acetylcysteine treatment was used in clinic for AAP overdose cases [9].
Figure 1
Acetaminophen structure [28]
Figure 2
Metabolic and hepatotoxicity pathways of AAP [28]
Zataria multiflora (Z.M) which is also known as Avishan Shirazi in Iran, is one of the important herbal plants belonging to the Lamiaceae family [10]. This plant has several components such as multiflorol, multiflotriol, aromatic ester of p-hydroxy benzoic acid, dihydroxyaromadendrane, luteolin and α-tocopherolquinone [11]. In addition, the essential oils such as Carvacrol (CAR) (52%), thymol (16%) and p-cymene (10%) are more effective substances of [12, 13]. CAR (2-methyl-5-(1-methylethyl)-phenol) (Figure 3) is a dominant monoterpenic phenol that is present in many essential oils of the Labiatae family including Zataria and Thymus species [14]. CAR is the effective ingredient that has significant effects on the systemic anti-oxidant and tissue inflammation [12, 15]. Also, the therapeutic effects of Z.M and CAR include antinociceptive [16], antiulcer [17], antimicrobial [18], antiasthma [19, 20], improvement of the chronic obstructive pulmonary disease (COPD) [19, 21], anti-proliferative in cancers [22], anti-diabetic [23], antidepressant [24], etc. Some features of this plant such as antioxidant [25, 26] and anti-inflammatory [27] effects have already been studied. The aim of this study is to investigate the preventive effects of Zataria multiflora and Carvacrol and comparative effect of them on AAP-induced hepatotoxicity in rats.
Figure 3
Carvacrol structure [29]
2. Material and Methods
2.1. Plant material and preparation of the extract
Zataria multiflora was purchased from local herbal shop in Mashhad, Khorasan province, Iran and identified by botanists in the herbarium of Ferdowsi University of Mashhad (ID:35314).The aerial parts of Zataria multiflora (100 gr) were cleaned, dried, ground, weighed, and homogenized in 70% ethanol at a ratio of 1:10 of plant to ethanol and macerated for 3 days at 37°C with occasional shaking. The mixture was then filtered and the resulting liquid was concentrated under decreased pressure at 45°C in an EYELA rotary evaporator. The concentrated extract was then kept in the incubator at 45°C for 3 days to evaporate the ethanol residue yielding the crude extract. Finally, the extract was dissolved in saline before being gavaged to animals.
2.2. Extract standardization
A The polyphenols content of the ZM extract evaluated based on the Folin–Ciocalteu method in which ZM extract has 37.1 mg gallic acid/g crude extract. Also, the flavonoids content of the ZM extract estimated based on the aluminum chloride colorimetric assay in which ZM extract has 13.7 mg quercetin/g crude extract. Finally, the anthocyanin content of the ZM extract evaluated based on the pH-differential method described by Rodriguez-Saona in which ZM extract has 1.9 mg/g crude extract. The line equations for standard curves of gallic acid and quercetin were Y = 0.0669X + 0.0116 and Y = 0.06632X - 0.01448, respectively.
2.3. Chemicals and drugs
The ether and CAR were purchased from Merck and Sigma companies. AAP was obtained from Tehran Daru, Iran. Also, biochemical assay kits were provided by Pars Azmoun, Tehran, Iran.
2.4. Animals and treatment
Twenty four male Wistar rats (220 – 240 gr) were housed in a room with standard condition and free access to food and water. The ethical code for current study is IR.MUMS.fm.REC.1396.581.Rats were randomly divided into four groups including: Control (Co), Acetaminophen (AAP), Z.M and CAR. The saline, Z.M (200 mg/kg) [30] and CAR (20 mg/kg) [31] were administrated orally by gavage for 6 days in different groups, after that AAP (600 mg/kg) [32] was administrated orally by gavage in the 7th day in all groups except Co group. The rats were weighed on the first and last days. Blood samples were collected from the orbital sinus on the first and at the end of the experiment. Also, the liver tissue samples were removed for oxidative markers evaluation.
2.5. Biochemical assessment
Blood samples were used to determine the levels of some enzymes including total Protein (tPro), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvic Transaminase (GPT) and Alkaline Phosphatase (ALP) with the relevant kits. Finally, the changes (Δ) of enzymes activities were presented.Total thiol contents were evaluated using DTNB (2, 2′-dinitro- 5, 5′-dithiodibenzoic acid), a reagent which reacts with the SH groups and produces a yellow colored complex that has a peak absorbance at 412 nm [33]. Generally, 1 ml Tris-EDTA buffer (pH=8.6) was added to 50 μl liver homogenate in 1 ml cuvettes and the absorbance was read at 412 nm versus Tris-EDTA buffer (A1). Then 20 μl DTNB reagents (10 mM in methanol) were added to the mixture and after 15 minutes incubation in room temperature, the absorbance was read again (A2). Also, the absorbance of DTNB reagent was read as a blank (B). A total thiol content (mM) was calculated based on an equation described by Hosseini et al [34, 35].Malondialdehyde (MDA) concentrations as an index of lipid peroxidation were evaluated in the liver tissue. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) and produces a red colored complex which has a peak absorbance at 535 nm. Briefly, 2 ml TBA/ trichlorooacetic acid (TCA)/ hydrochloric acid (HCL) reagent was added to 1 ml homogenate and the solution was incubated in a boiling water bath for 40 minutes. After cooling, the whole solutions were centrifuged at 1000 g for 10 minutes. The absorbance of supernatant was evaluated at 535 nm. The MDA concentration (C) was calculated as follows [34, 35].Catalase (CAT) activity was estimated using the Aebi method [36]. The principle of the assay is based on determination of the rate constant, k, (dimension: s-1, k) of hydrogen peroxide decomposition. By measuring the reduction in the absorbance at 240 nm per minute, the rate constant of the enzyme was evaluated. Activities were expressed as k (rate constant) per liter.Superoxide dismutase (SOD) activity was evaluated by the procedure explained by Madesh and Balasubramanian [37]. A colorimetric assay involving production of superoxide by pyrogallol auto-oxidation and the inhibition of superoxide-dependent reduction of the tetrazolium dye, MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) to its formazan by SOD was determined at 570 nm. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition in the MTT reduction rate.
2.6. Data analysis
The data were expressed as mean ± SEM. The maximal changes (Δ) of data calculated by last data-first data, then Statistical comparisons were done by one-way ANOVA followed by the Tukey’s post hoc test. A value of P < 0.05 was used to indicate statistical significance.
3. Results
The results showed that ΔGOT, ΔGPT and ΔALP in AAP group significantly increased compared to Co group (P < 0.05 to P < 0.01; Figure 4) while the tPro did not have any significant effects. Also, the results indicated that ΔGOT, ΔGPT and ΔALP in CAR group significantly decreased compared to AAP group (P < 0.01 to P < 0.001; Figures 5,6 and 7). The ΔGPT in Z.M group was significantly reduced in comparison with AAP group (P < 0.05; Figure 6). The ΔtPro did not have any difference between four groups (Figure 8).
Figure 4
Changes (Δ) of different parameters induced by AAP group in comparison with Co group (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
*:P < 0.05, **:P < 0.01, compared to Co group.
Co: Control, AAP: Acetaminophen, GOT: Glutamic Oxaloacetic Transaminase, GPT: Glutamic Pyruvic Transaminase, ALP: Alkaline Phosphatase, tPro: total Protein
Figure 5
Comparison the changes (Δ) of Glutamic Oxaloacetic Transaminase (GOT) in four experimental groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Comparison the changes (Δ) of Glutamic Pyruvic Transaminase (GPT) between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Comparison the changes (Δ) of Alkaline Phosphatase (ALP) concentration between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Comparison the changes (Δ) of tPro between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
In the liver tissue, MDA level in AAP group was significantly enhanced compared to Co group, (P < 0.05). In CAR group, level of MDA was significantly reduced compared to AAP group, (P < 0.01) (Figure 9).
Figure 9
Comparison of Malondyaldehide (MDA) concentration between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Level of Thiol in AAP group was reduced compared to Co group (P < 0.001). Thiol level in ZM group significantly increased compared to AAP group (P < 0.05) (Figure 10).
Figure 10
Comparison of Thiol concentration between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
The CAT activity in liver of AAP group was not significantly lower than Co group and increased in CAR group in comparison with AAP group (P < 0.01) (Figure 11).
Figure 11
Comparison of CAT activity between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Also, the results showed that SOD level in AAP group compared to Co group was significantly decreased (P < 0.05) while this level in ZM and CAR groups significantly increased (P < 0.001) (Figure 12). Also, significant decrease of MDA concentration (P < 0.05) and significant increase of CAT activity (P < 0.001) in Z.M group compared to the AAP group was observed.
Figure 12
Comparison of Superoxide dismutase (SOD) activity between four groups (n=6 in each group). Values are the Mean ± SEM. The data were analyzed using One-Way ANOVA with tukey post hoc.
Acetaminophen (AAP) which is broadly used as an antipyretic and analgesic drug in higher doses could induce hepatotoxicity in humans and experimental animals [38]. The NAPQI is a toxic metabolite of APP that is produced in the liver by the cytochrome P450 pathway [6]. This agent conjugates with glutathione and excretes in the urine. The AAP overdose causes glutathione depletion that leads to NAPQI accumulation and mitochondrial dysfunction [39]. Glutathione depletion promotes the tumor necrosis factor alpha (TNFα) that leads to production of oxygen free radicals from NADPH oxidase and hepatotoxicity finally [40]. The ample studies have been shown that AAP overdose (600 mg) causes hepatotoxicity in rats [32, 41].The AAP is a potential trigger of cytochrome P450 that induces the high reactive quinone-imine production. This matches with sulphahydryl groups in proteins and results in rapid depletion of intracellular glutathione [42]. Generally, one part of the potential intracellular antioxidant defensive system is glutathione that consumes the singlet oxygen, superoxide and hydroxyl radicals [43]. Enhancing the intracellular flux of oxygen free radicals resulting from glutathione depletion leads to oxidative stress in hepatocytes [43]. The increased serum levels of GOT, GPT and ALP have been attributed to the structural integrity of hepatic damage [44]. In the liver tissue, the high concentration of GOT and GPT are located in cytosol and mitochondria. In following of liver damage, hepatocyte transport function disrupted and results in the plasma membrane leakage then GOT and GPT were released into blood circulation [45, 46]. In consistent with previous studies [47] in this study also GOT, GPT, ALP and tPro in AAP group, significantly increased compared to Co group. Excretion pathway of ALP is normally via bile. In liver damage due to hepatotoxin, the excretion of bile by the liver is defective that is promoted their serum levels [46]. The tPro is performed as a common test to determinate the toxicity of many chemicals [48]. In liver injury because of reduction in liver function, the protein synthesis decreased and its serum levels reduced. In recent study because of short toxicity induction time, the tPro has a mild fall. Nevertheless, because the half span of albumin is more than two week, we did not expect to change in albumin concentration in serum. Pretreatment of animals with Z.M and specially CAR attenuated the serum GOT, GPT and ALP enzymes that increased by AAP. Probably, these effects of Z.M and CAR refer to functional improvement of hepatocytes. CAR in comparison with Z.M has a better effect on hepatotoxicity induced by AAP because the hepatic enzymes in this group have a significantly reduction. CAR indicated that has a significant antioxidant effect against hepatotoxicity induced by D-Galactosamine [14] and methotrexate [49] via attenuation of liver markers such as ALT, AST and ALP. In one study has been shown that administration of Z.M with four doses orally for a week balanced the liver marker. In this study Z.M with dose 200 as an effective dose, has a significant effect on hepatotoxicity compared to other doses [10]. The pharmacochemistry of Z.M components indicated that it has several phenolic components. Flavonoids and CAR as the effective constituent of Z.M extract have an important role as a powerful antioxidant. Probably, their positive effect of Z.M comes from their ability to lipid peroxidation inhibition and balancing other procedure involving ROS [11].Increasing of MDA and reduction of total thiol (tT), SOD and CAT confirm the hepatotoxicity induced by AAP. Main compounds of the enzymatic antioxidant system are three, namely, SOD, CAT and tT which have an important role in detoxifying of H2O2 and superoxide anion in cells. Ample of hepatotoxic drugs induces the liver damage by lipid peroxidation indirectly or directly. The proxy radicals are main factors that mediate lipid peroxidation leading to liver injury and kidney damage [50]. MDA as a main reactive aldehyde appears during polyunsaturated fatty acid peroxidation in the biological membranes [51]. The antioxidant effect of CAR has been shown in previous study. For example Nafees et al, reported the protective effect of CAR in doses 25 and 50 mg/kg against thioacetamide (TAA)-induced oxidative stress and inflammation [52]. In other study also, the protective effects of CAR against oxidative stress induced by chronic stress in liver of rat has been shown by Samarghandian et al. suggests that CAR can prevent restraint stress induced oxidative stress in liver [53]. In this studies antioxidant markers such as SOD, CAT, glutathione peroxidase (GPx) and MAD were evaluated and all of these ameliorated by CAR. Our results also are in this direction and confirm other results. Decreased activities of antioxidant enzymes will result in the gathering of high reactive free radicals, leading to worse effects. These effects are loss of cell membrane function and integrity that increases the releasing of GOT and GPT from cell to out [14, 54]. A minor source of oxidant after AAP overdose in liver may be Kupffer cells so that using of Kupffer cells inhibitors completely protects liver against AAP-induced hepatotoxicity [9]. Also, because of glutathione depletion following of AAP overdoses, AAP metabolites bind to membrane and mitochondrial proteins and lead to increasing of cytosolic calcium and reduction of ATP synthesis. This mitochondrial dysfunction incases the superoxide radicals generation [9]. The superoxide radicals can react with nitric oxide and generates the peroxynitrite or increases the lipid peroxidation [9]. CAR has a scavenger role for hydrogen peroxide and superoxide radicals [14]. These antioxidant properties of CAR may reduce the free-radical-mediated inactivation of enzyme proteins and thereby maintaining the enzymatic antioxidants activities [14].
5.Conclusion
Our results indicated that Z.M and CAR have a powerful protective effect against hepatoprotective induced by APP. However, the effect of CAR was higher than Z.M. Therefore, Z.M and CAR could be used as a supplement agent against hepatotoxicity induced by APP.
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