Phoenix dactylifera protects against oxidative stress and hepatic injury induced by paracetamol intoxication in rats

Biomedicine & Pharmacotherapy 104 (2018) 366–374

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Phoenix dactylifera protects against oxidative stress and hepatic injury induced by paracetamol intoxication in rats

T

⁎⁎

Gamal A. Salema,b, , Ahmed Shabana, Hussain A. Diabc, Wesam A. Elsaghayerd, ⁎ Manal D. Mjedibc, Aomassad M. Hneshc, Ravi P. Sahue, a

Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Misurata University, Misurata, Libya c Department of Drug Technology, Faculty of Medical Technology, Misurata University, Misurata, Libya d Department of Pathology, Faculty of Medicine, Misurata University, Misurata, Libya e Department of Pharmacology and Toxicology, Wright State University, Dayton, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Phoenix dactylifera Leaves Paracetamol Hepatoprotective Antioxidant Free radicals Phytochemicals

The current studies were sought to determine effects of antioxidant potential of aqueous and methanolic extracts of Phoenix dactylifera leaves (PLAE and PLME) against the widely-used analgesic paracetamol (PCM) induced hepatotoxicity. Groups of rats were treated with or without PCM (1500 mg/kg), PLAE and PLME (300 mg/kg) and n-acetylcysteine (NAC, 50 mg/kg) followed by assessments of liver function tests, oxidative stress, antioxidant defenses, and hepatotoxicity. We observed that PCM significantly elevated serum liver markers, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), and bilirubin compared to control (untreated) group. These PCM-induced effects were associated with oxidative stress as demonstrated by increased levels of malondialdehyde (MDA) and reduced levels of hepatic antioxidant enzymes, glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). Pretreatment of PLME decreased ALT and AST by 78.2% and tissue MDA by 54.1%, and increased hepatic GPx (3.5 folds), CAT (7 folds) and SOD (2.5 folds) compared to PCM group. These PLME-mediated effects were comparable to NAC pretreatment. Histological analysis demonstrates that PLME conserved hepatic tissues against lesions such as inflammation, centrilobular necrosis, and hemorrhages induced by PCM. In contrast, PLAE-mediated effects were less effective in reducing levels of liver function enzymes, oxidative stress, and liver histopathological profiles, and restoring antioxidant defenses against PCM-induced intoxication. These findings indicate that PLME exerts protective effects against PCM-induced hepatotoxicity via scavenging free radicals and restoring hepatic antioxidant enzymes. Thus, PLME and its bioactive components could further be evaluated for their pharmacological properties against drug-induced deleterious effects.

1. Introduction Reactive oxygen species (ROS) are free radicals which affect various biological and pathophysiological processes including liver ailments, diabetes, heart diseases, cancer, and aging [1]. Molecular redox processes including thiol and NAD/NADP systems inside the cell cause

production of superoxide anions (O2%−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH%−) which are components of ROS [2]. These free radicals can have physiologically beneficial effects when produced at low levels, but at high levels, they can lead to oxidative stress and cellular damage [3].The latter effect occurs due to electron pairing of ROS with cellular macromolecules such as lipids, leading to a chain

Abbreviations: AAP, 4-aminophenazone; ALP, alkaline phphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAT, catalase; CYPs, cytochrome P 450s; DHBS, 3,5Dichloro -2-hydroxybenzene sulfonic acid; EDTA, ethylenediaminetetraacetic acid; GGT, gamma glutamyl transferase; GSH, glutathione; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde; NAC, n-acetylcysteine; NAPQI, N-acetyl-p-benzoquinoneimine; NO, nitric oxide; O2%–, superoxide anions; OH%–, hydroxyl radicals; ONOO–, peroxynitrite; PBS, phosphate buffer saline; PCM, paracetamol; PLAE, Phoenix dactylifera leaves aqueous extract; PLME, Phoenix dactylifera leaves methanolic extract; ROS, Reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid ⁎ Corresponding author at: Department of Pharmacology and Toxicology, 230 Health Sciences Building, Boonshoft School of Medicine at Wright State University, 3640 Col. Glenn Hwy, Dayton, OH, 45435-0001, USA. ⁎⁎ Corresponding author at: Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, P.O. Box 44511, Egypt. E-mail addresses: [email protected], [email protected] (G.A. Salem), [email protected], [email protected] (A. Shaban), [email protected] (H.A. Diab), [email protected] (W.A. Elsaghayer), [email protected] (M.D. Mjedib), [email protected] (A.M. Hnesh), [email protected] (R.P. Sahu). https://doi.org/10.1016/j.biopha.2018.05.049 Received 11 March 2018; Received in revised form 8 May 2018; Accepted 9 May 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.

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transferase (GGT), and total bilirubin were purchased from Biomaghreb Laboratories. The kits for GPx, CAT, SOD, and MDA were purchased from Biodiagnostics Co. (Cairo, Egypt). All other chemicals were purchased from standard commercial suppliers and were of analytical grade. All solutions were prepared immediately before use.

reaction of lipid peroxidation, DNA damage and dysfunction of proteins and enzymes, especially containing sulfur moieties [2]. The imbalance between the levels of pro-oxidants and cellular antioxidant defenses including SOD, CAT, and GPx enzymes contribute to an increase in cellular oxidative stress [4]. Paracetamol (PCM) is a widely-used antipyretic and analgesic drug for people of all ages [5,6]. PCM undergoes biotransformation via cytochrome P 450s (CYPs) including CYP2E1, CYP3A4 and CYP1 A2 into a highly reactive radical, N-acetyl-p-benzoquinoneimine (NAPQI), which can be detoxified with glutathione (GSH) conjugation when produced at low levels [7,8]. Accumulating evidences indicate that PCM at large doses can produce high levels of NAPQI that exceed the amount of GSH needed to metabolize it, which can cause enhanced ROS generation, lipid peroxidation, mitochondrial dysfunction, depletion of ATP, DNA breakdown and apoptosis [8]. These events could lead to oxidative damage of hepatic cells (i.e. hepatotoxicity) leading to subsequent elevation of bilirubin and liver enzymes in the serum such as AST and ALT [9]. Supplementation of antioxidants has been shown to attenuate oxidative stress-induced deleterious effects. Notably, n-acetylcysteine (NAC), the SH-containing antioxidant compound serves as a clinical antidote to PCM-induced toxicity [10]. NAC treatment has been shown to exert protective effects against PCM-induced hepatotoxicity via its ability to scavenge free radicals and restore depleted GSH [11]. However, there are limitations on its use due to the associated side effects such as nausea, diarrhea, headache and anaphylactic reactions [12]. In recent years, the synthetic medical compounds being practiced to treat or manage liver diseases have exerted various adverse effects [13]. To that end, natural antioxidants from plant sources have been shown to exert preventive effects against oxidative stress compared to chemical compounds [13,14,15]. As large population in developing countries of Asia and Africa still use traditional medicines for various ailments [16], bioactive compounds of natural resources could offer the safe and effective alternative means to manage drug-induced adverse effects [17]. Among these natural resources, date palm tree (Phoenix dactylifera L.) is a member of the family Arecaceae [18]. The production of dates in Arab countries constitutes about 80% of the total world production [19], which are considered as one of the most important traditional foods for Arabian people. The different parts of P. dactylifera tree are used in traditional medicine in Arab countries, especially in Egypt, as a remedy for diabetes, gastrointestinal problems, liver diseases, pharyngitis, fevers, and venereal diseases like gonorrhea [20]. The phytochemical analysis has revealed its richness in phenolics, carbohydrates, sterols, carotenoids, anthocyanins, procyanidins, flavonoids, vitamins and tannins [18,21,22,23]. Notably, P. dactylifera leaves are rich in total polyphenols, flavanoids, and flavonols, which possess potent antioxidant properties [24]. These phytoconstituents properties of P. dactylifera leaves have been shown to exert beneficial biological and pharmacological effects including the potent antioxidant [19], hepatoprotective [25], anti-hyperlipidemic [26] and antiviral [27] activities. Importantly, treatment of P. dactylifera leaves have been shown to improve the lipid profiles in alloxan-induced diabetes in rats [28]. However, to the best of our knowledge, the in vivo antioxidant, and hepatoprotective potentials of P. dactylifera leaves have not been studied. The current study sought to determine effects of methanolic (PLME) and aqueous (PLAE) extracts of P. dactylifera leaves against the hepatotoxic effect and oxidative stress induced by PCM in a rat model.

2.2. Preparation of PLAE and PLME Leaflets of Phoenix dactylifera L. Hammory cultivar (family Arecaceae) were collected from different areas in Misurata, Libya. A voucher specimen (ph-d.3) was deposited in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Misurata University, Misurata, Libya. These leaflets were cleaned, dried sun and cut into tiny pieces. A watery extract was prepared by boiling 200 g m of date leaflets in 500 ml distilled water then filtering twice. The filtrate was lyophilized using a freeze dryer. For the preparation of the methanolic extract, the small pieces of date leaflets were crushed into powder. After that, 200 g m of this powder was macerated into 700 ml methanol (1:3.5 w/v) for 48 h at room temperature away from the light, then the mixture was filtered three times using filter paper to obtain a clear filtrate. The filtrate was concentrated in a rotary evaporator in a vacuum at 60 °C and dried further at 45 °C. The blackish brown lyophilized watery extract was stored at room temperature away from the air, and the blackish green methanolic extract was stored in a refrigerator. Sterilized distilled water was used as a solvent in order for the two extracts to be administered orally to mice for experimental purposes. 2.3. Preliminary phytochemical analysis PLME and PLAE were subjected to preliminary phytochemical analysis for the detection of the following compounds; alkaloids, steroids, terpenoids, saponins, tannins and polyphenolic compounds and flavonoids as per the published reports [29,30]. 2.4. Acute toxicity study of PLME and PLAE Thirty five albino mice were used for the acute toxicity study. These mice were divided into 3 groups of 5 animals each to receive PLAE, PLME, and normal saline. These animals were fasted for 3 h before the administration of PLAE and PLME at 100, 300 and 2000 mg/kg doses by oral gavage to determine the safe doses of these extracts. Each individual animal was observed every 30 min for the first 4 h, then every hour for 24 h, then twice a day for the next 2 weeks. The observational parameters included behavioral analysis such as mood and alertness; neurological analysis such as central excitation and inhibition, muscle tone and coordination, reflexes, body temperature, and pain as well as autonomic analysis such as pupil size, heart rate, secretions, excretions, rate and depth of respiration. The mouse number and their time of mortality were recorded. All the procedures were in accordance with Organization for Economic Co-operation and Development (OECD) guideline 423 [31]. 2.5. Animal care and monitoring Male Sprague-Dawley rats of 4–5 months old weighing 250–300 g m, and albino mice of 2-3 months old weighing 25-30 gm were randomly allocated to polypropylene cages having autoclaved wooden shaving beddings maintained under standard controlled conditions of 12 h light/dark cycle with an ambient room temperature of 25 ± 2 °C and relative humidity of 55 ± 5. These animals were fed with commercial pellet diet and water ad libitum. All treatments were in accordance with the animal care guidelines of the Institutional Animal Ethics Committee, Misurata University, Faculty of Pharmacy.

2. Materials and methods 2.1. Chemicals Paracetamol (PCM) was purchased from Almaya Co. (Libya), and nacetylcysteine (NAC) from Labortoires Galpharma (Tunisia). Kits for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl 367

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malondialdehyde (MDA) and thiobarbituric acid (TBA) in an acidic medium [37].

2.6. Acute toxicity study of PCM Twenty-five rats were randomly distributed into 5 groups with 5 rats in each group, and given 0.5 ml of 20% propylene glycol p.o daily for 8 days. The first group served as a control, and the other 4 groups were orally administered PCM at the dose of 250, 500, 750 or 1500 mg/ kg body weight on the 8th day. These rats were examined twice daily for signs of toxicity, morbidity, and mortality. After 48 h, rats were euthanized using sevoflurane, and serum samples were collected from the blood for the examination of liver function parameters including AST, ALT, ALP and GGT enzymes and total bilirubin.

2.11. Determination of serum liver function Alanine aminotransferase (ALT), alkaline phosphate (ALP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT) and total bilirubin were measured according to the published studies [38–42]. 2.12. Histological procedures A part of the liver tissues from these studies were fixed in 10% formalin for at least 24 h followed by the preparation of paraffin sections using an automated tissue processor. The 5 microns thick sections cut through a rotary microtome were then stained with hematoxylin and eosin (H&E) dye for the assessment of histopathological changes. Histological damages were scored as follows: absent (-); mild (+); moderate (++) and severe (+++).

2.7. Experimental design Experimental animals were randomly divided into 5 groups with 5 rats in each group. These rats were treated for 8 days as follows: Group I: control negative: received 0.5 ml of 20% propylene glycol, per Os (p.o) daily; Group II: received PLAE 300 mg/kg, p.o daily; Group III: received PLME 300 mg/kg, p.o daily; Group IV: received NAC 50 mg/kg p.o daily and; Group V: control positive: received 0.5 ml of 20% propylene glycol p.o daily. We used the control and PCM 1500 mg/kg treated groups of the acute toxicity study as a control negative and positive groups in this experiment. We took advantage of Mard et al.,2010 studies to select the optimum dose of PLAE and PLME, where different doses of Phoenix dactylifera leaves extracts (100, 200 and 400 mg/kg) were tested for the evaluation of their anti-diabetic and anti-lipemic activities [32]. We choose a moderate dose of 300 mg/kg from our acute toxicity study. On the 8th day and after the last treatment, the rats of groups II, III, IV and V were given PCM dissolved in 20% propylene glycolate at the dose of 1500 mg/kg p.o. After 48 h of PCM administration, the animals were euthanized using sevoflurane and serum samples were collected from the blood for the evaluation of liver enzymes.

2.13. Statistical analysis The statistical significant differences between various groups were analyzed by One Way Analysis of Variance (ANOVA) followed by LSD as a post hoc test using SPSS 22. The figures were generated using Graph Pad Prism 7 software, and the data were expressed as mean ± SE. The level of significance was set as p ≤ 0.05. 3. Results 3.1. Phytochemical analysis of compounds present in PLME and PLAE Our first studies determined the phytochemical constituents of PLME and PLAE. We observed that both these extracts possess various, yet similar components (Table 1).

2.8. Sample preparation for oxidative stress markers assay After dissection, the liver tissues were washed with PBS (phosphate buffer saline) solution containing 0.16 mg/ml heparin to avoid any RBC clots. The liver tissues were homogenized in 7 volumes (per tissue weight) of cold buffer containing 50 mM potassium phosphate pH 7.0 and 5 mM EDTA. The homogenates were subjected to cold (4 °C) centrifugation at 4000 rpm for 15 min, and supernatants were collected and stored at -80 °C until assayed.

3.2. Acute toxicity study of PLME and PLAE The acute toxicity study was performed in albino mice to determine the optimal dose of PLME and PLAE extracts. These mice treated with PLME and PLAE extracts at the dose of 100, 300 and even 2000 mg/kg were observed for 14 days. Our findings demonstrate that these extracts did not induce any behavioral, neurological and autonomic abnormalities in any of the tested parameters or mortality. This indicates thattheLD50of tested extracts could be more than 2000 mg/kg body weight, thus, we did not test the higher doses of these extracts.

2.9. Determination of GPx, SOD, andCAT enzymatic assays The GPx was indirectly assayed according to the published report [30]. In this assay, oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by GPx, is recycled to its reduced state by the enzyme glutathione reductase (GR). The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm (A340) [33]. The SOD assay was performed as reported [34], which is based on the ability of the enzyme to hinder the reducing power of phenazine methosulphate to nitroblue tetrazolium dye. The CAT assay is based on the reaction with a known quantity of H2O2, the reaction was stopped after exactly one minute with catalase inhibitor. In the presence of peroxidase (HRP) remaining H2O2 reacts with 3,5-Dichloro -2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP) to form a chromophore with a color intensity inversely proportional to the amount of catalase in the original sample [35].

3.3. Serum liver function markers We observed that the rats treated with PCM at 1500 mg/kg dose after 48 h resulted in significant elevation of serum ALT (80%), AST (88.4%), ALP (67.8%), GGT (55.1%) and total bilirubin (59.2%) levels as compared to the control group. In addition, PCM treatment Table 1 Phytochemical analysis of PLME and PLAE.

2.10. Determination of lipid peroxide (malondialdehyde) The lipid peroxidation was measured using an indirect assay as per Sorg studies [36]. This colorimetric assay involves the measurement of the absorbance at 534 nm resulting from the reaction between the tissue

Phytochemical constituents

PLME

PLAE

Alkaloids Saponins Flavonoids Steroids Tannins and Polyphenolic compounds Terpenoids

+ + + + + +

+ + + + + +

The sign (+) denotes that these constituents were present in both PLME and PLAE extracts. 368

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Fig. 1. Effects of various PCM doses on liver function tests. Sprague Dawley rats were treated with or without various indicated doses of PCM, and its effects on the assessments of liver function tests: A) AST; B) ALT; C) GGT; D) ALP and; E) T. bilirubin concentrations in serum. Each value represents a mean ± SE (n = 5/group). * indicates significance from the control group (p ≤ 0.05) and #, $, @, α, & and β signs indicate significance between different treatment groups (p ≤ 0.05).

Fig. 2. Effects of PLME, PLAE and NAC on liver function tests against PCM induced hepatotoxicity in rats. Sprague Dawley rats were treated with or without PLME (300 mg/kg), PLAE (300 mg/kg), NAC (50 mg/kg) and PCM (1500 mg/kg) followed by evaluation of various doses of PCM: A) ALT; B) AST; C) ALP; D) GGT; and; E) T. bilirubin concentrations in serum of rats. Each value represents a mean ± SE (n = 5/group). * indicates significance from the control group (p ≤ 0.05) and #, $, @ , α, & and β signs indicate significance between different treatment groups (p ≤ 0.05).

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Fig. 3. Effect of PLME, PLAE and NAC on oxidative stress markers against PCM induced intoxication in rats. Sprague Dawley rats were treated with or without PLME (300 mg/kg), PLAE (300 mg/kg), NAC (50 mg/kg) and PCM (1500 mg/kg), and analysis of antioxidant enzymes and oxidative stress related marker were done: A) GPx; B) CAT; C) SOD; D) MDA concentrations in liver tissues of rats. Each value represents a mean ± SE (n = 5/group). * indicates significance from the control group (p ≤ 0.05) and #, $, @, α, & and β signs indicate significance between different treatment groups (p ≤ 0.05).

3.5. Histopathological evaluation

(1500 mg/kg) exhibited a marked elevation in these enzymes and total bilirubin level when compared with 750, 500 and 250 mg/kg PCMtreated groups (Fig. 1). Based on these findings, we choose 1500 mg/kg dose to induce hepatic injury, and to test the protective effects of PLME and PLAE extracts. Our studies demonstrate that pretreatment of PLME and PLAE at the dose of 300 mg/kg significantly attenuate PCM-induced increase in the serum concentrations of liver ALT, AST, ALP, GGT and total bilirubin compared to the PCM-alone treated group (Fig. 2). These PLME-induced reduction in serum levels of AST, ALT and GGT were significantly higher compared to the PLAE-pretreated group. Moreover, all liver function indices with PLME group reached comparable levels to those of the NAC treated and untreated control groups (Fig. 2).

In addition to the above evidences, the histopathological analysis of liver sections from different groups confirmed the hepatoprotective effects of PLME and PLAE which were similar to the standard NAC. Microscopic examination of H&E stained sections of the control group revealed normal liver structure, where hepatocytes were arranged in cords radiating from the central veins leaving sinusoids in between (Fig. 4a). Hepatic sections of the PCM-intoxicated group showed focal necrosis, hemorrhage, degeneration, and infiltration of lymphocytes around the central veins (Fig. 4b). Systemic administration of PLAE prior to PCM conserved the general architecture of the liver almost completely, and also moderately ameliorated the inflammatory infiltrates and necrosis (Fig. 4c). In addition, pretreatment with PLME similar to NAC prevented necrosis, hemorrhage or lymphocytic infiltration, and fully restored the normal classical architecture of the liver tissues (Figs. 4d and 4e). The H&E staining of liver sections demonstrating increased hemorrhage and lymphocytic infiltration induced by PCM, and decreased levels of these events by PLME treatment at higher magnification are shown in supplementary Fig. 1A summary of all the histopathological results is presented in Table 2.

3.4. Tissue oxidative stress indices In order to determine if the PCM-induced increase in serum liver function parameters are due to enhanced production of free radicals leading to oxidative stress, we measured liver antioxidants enzymes. Our studies demonstrate that the levels of antioxidant enzymes SOD, CAT and GPx were significantly reduced in liver tissues in response to PCM treatment as compared to the control group (Fig. 3). In contrast, pre-treatment of PLME and PLAE at a dose 300 mg/kg exerted potent antioxidant effect in attenuating oxidative stress response as manifested by significantly increased levels of SOD, CAT and GPx in the liver homogenate compared to the PCM-intoxicated and control group (Fig. 3). Both extracts restored the antioxidant enzyme levels similar to as seen in NAC and normal control groups. In particular, PLME group exhibited slightly improved response than PLAE group as observed by 3.5, 7 and 2.5 folds increase in GPx, CAT and SOD levels by PLME compared to PCM-alone treated group, whereas PLAE restored the levels of these enzymes by 2.5, 6 and 2 folds(Fig. 3). The oxidation of polyunsaturated fatty acids in the cell membrane results in increased formation of MDA as an end product and is considered to be an indicator of lipid peroxidation. To that end, we observed that treatment of PCM resulted in a prominent increase (4-fold) in MDA level in liver tissue as compared to normal control group (Fig. 3). In contrast, pretreatment of PLAE and PLME extracts (300 mg/ kg) counterbalanced the lipid peroxidation caused by the PCM, and reduced the concentration of MDA by 37.4% and 46% respectively (Fig. 3). Overall, PLME treatment exhibited better antioxidant effects than PLAE in restoring the levels of GPx, CAT and SOD and reducing MDA concentrations in the liver tissue homogenate, and these effects were similar to as observed in the control and NAC-treated groups (Fig. 3).

4. Discussion Hepatocytes are the major reservoir for metabolic reactions, thus, any damage in hepatocytes will lead to disturbances in body metabolism [43]. Hepatic cells develop antioxidant defenses by enzymatic and non-enzymatic mechanisms to scavenge ROS and other free radicals. However, a state of oxidative stress can arise in which such protective capacities become inadequate, leading to abnormalities such as liver injury, fibrosis, cirrhosis, and even hepatocellular carcinoma when such injuries persist fora longer time [44,45]. Therefore, the clinical intervention of hepatic injury is required to protect against oxidative stressinduced liver diseases [46]. The current study aimed to evaluate the phytochemical composition, safety profiles of PLME and PLAE and determined the possible mechanism underlying their hepatoprotective effects against PCM-induced hepatotoxicity. PCM overdose is one of the most prevalent drug-induced toxicity reported worldwide, where PCM metabolites can cause liver and kidney damage in the form of centrilobular hepatic and renal cortical necrosis [47,48]. PCM-induced hepatotoxicity results in the release of several liver enzymes and metabolites such as ALT, AST, LDH and bilirubin in the serum [47,49]. The increased levels of these soluble products can be used as a quantitative markers to access the degree of hepatic necrosis. The published reports indicate some variabilities in the levels of PCM doses that induce hepatoxicity ranging from lower doses of 300 mg/kg 370

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Fig. 4. Histological analysis of liver tissues. Liver sections from various groups of rats were stained with H&E (100X) for the overall analysis of liver pathology as follows: a) Control group: normal hepatic tissue structure. b) PCM intoxicated: H; Hemorrhage, CN; Centrilobular Necrosis and LI: Lymphocytic Infiltration. c) PLAE treated: LI and N; Necrotic areas and; d–e PLME and NAC treated groups respectively: complete restoration of normal structure.

and disrupt the electron transport chain to form ROS and RNS in the mitochondria. The essential ROS formed is superoxide (O2·), which is either dismutated into hydrogen peroxide and hydroxyl free radicals or reacts with nitric oxide (NO) to form peroxynitrite (ONOO−) as RNS [60,61]. ROS can cause further DNA/RNA breakage and lipid peroxidation leading to liver damage [62,63]. Liver cells contain a natural defense mechanism against oxidative free radicals in the form of antioxidant enzymes. SODs are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide, and thus form a crucial part of the cellular antioxidant defense mechanism [64]. Cellular GPx is a member of a family of GPx enzymes whose function is to detoxify peroxides in the cell. Because peroxides can decompose to form highly reactive radicals, the GPx enzymes play a critical role in protecting cells from free radical damage, particularly the lipid peroxidation [65]. CAT is an antioxidant enzyme that is present in most aerobic cells and serves as one of the body's defense systems against H2O2, a strong oxidant that causes intracellular damage [33,64]. In our study, PCM-intoxication caused a severe depletion in the amount of SOD, CAT and GPx in the liver tissue homogenate. Our studies are in line with the notion that formation of NAPQI by PCM generates free radicals (ROS and RNS), which exhausts the antioxidant enzyme stores in hepatic cells leading to hepatocellular necrosis [66]. Hepatic dysfunction due to PCM was also confirmed by the histopathological findings, where inflammatory changes including hemorrhages, lymphocytic infiltration, and necrotic changes of liver parenchyma were found. These results are consistent with the previous studies, which documented the presence of extensive coagulative necrosis around the central vein to periportal sparing, congestion of sinusoids, huge lymphocytic and neutrophil infiltration, and degenerative changes including pyknosis of nuclei and vascular and fatty degeneration [55,62]. PLME and PLAE exhibited, to different degrees, a potential hepatoprotective effects, where these extracts reduced the elevated levels of serum hepatic enzymes and bilirubin. These findings indicate their

Table 2 Effects of PLME, PLAE and NAC on histopathological liver damages induced by PCM toxicity. Groups

Necrosis

Hemorrhage

Infiltration of lymphocytes

Control PCM, 1500 mg/kg NAC, 50 mg/kg PLAE, 300 mg/kg PLME, 300 mg/kg

– +++

– +++

– +++

–

–

–

+

–

++

–

–

–

Absent: −Mild: +; Moderate: ++; Severe: +++.

[46,50], 800 mg/kg [51,52] and 1000 mg/kg [53] to even higher doses of 3 g m/kg [49,54,55] and 5 g m/kg [56]. We therefore tested different and increasing doses of PCM in order to detect the standard hepatotoxic dose and observed a dose dependent elevation in serum levels of ALT, AST, ALP, GGT and total bilirubin. The 1500 mg/kg dose of PCM was found to be adequate in inducing marked acute hepatotoxicity, which was also confirmed in other studies [57]. We therefore collected the blood samples 48 h after PCM-intoxication (1500 mg/kg) similar to as previously reported [57,58], where maximum levels of hepatic enzymes were detected in the serum after 48 h, but not at 24 or 72 h. The hepatic damage induced by PCM overdose can be attributed to saturation of glucuronide and sulphate conjugation, shifting the metabolism toward N-hydroxylation by the CYP enzyme leading to excessive production of NAPQI [7,8]. In addition, PCM overdose can deplete GSH reserve and NAPQI-free radicals can induce oxidative stress through oxidizing cell membrane phospholipids, which can be assessed by elevated levels of MDA, a marker of lipid peroxidation [46,59]. Similar to these reports, we also observed a significant increase in MDA level in the liver tissue. The NAPQI radicals can bind to mitochondrial enzymes

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Conflict of interests

capabilities to prevent hepatic cell necrosis and the leakage of cell constituents. Mechanistically, the hepatoprotective effects of these extracts could be attributed to their ability to scavenge NAPQI, ROS and RNS generated by PCM-intoxication as shown by elevated levels of antioxidant enzymes (GPx, SOD, and CAT) and the decrease in the amount of oxidative stress marker, MDA in hepatic tissues. These hepatoprotective effects of PLME and PLAE are further supported by histological analysis demonstrating the normal hepatic tissue architecture, and protection of liver from the necrotic and inflammatory changes caused by PCM. These PLME and PLAE-induced protective effects were comparable to NAC pretreated group which showed a significant reduction in serum markers and elevation in tissue antioxidant enzymes as well as protecting the histological alterations of the liver. Oral supplementation of NAC has been shown to be concentrated in hepatic cells in higher amount compared to when given via intravenously [67]. The NAC is subjected to high first pass metabolism where the acetyl group is removed to generate free reduced cysteine that is incorporated into the abundant intracellular antioxidant tripeptide, glutathione (GSH) [68]. NAC remains the drug of choice to ameliorate PCM-induced toxicities because of its ability to restore the depleted GSH after a PCM overdose [69]. GSH plays various antioxidant roles where its reducing efficiency is important for the activity of GPx to detoxify peroxides [70] as well as assisting SOD to deactivate superoxide radicals [71]. All these reports support our findings in explaining the hepatoprotective and antioxidant potentials of oral NAC, whereas the beneficial effects of PLME and PLAE could be attributed to the presence of their phytoconstituents. Phytochemical analysis of PLME and PLAE revealed the presence of alkaloids, carbohydrates, steroids, saponins, and terpenoids in addition to flavonoids, tannins and polyphenolic compounds. Several other reports have also confirmed the presence of alkaloids, steroids, tannins, terpenoids, carbohydrates, amino acids, and flavonoids in the methanolic extract of date palm leaves [72,73]. Our results are consistent with Biglari et al. [74] in that the date palm contains these constituents in addition to vitamins. In addition, studies by Laouini et al. [24] also confirmed the presence of total polyphenol (215.24 to 156.46 mg GAE/ g DW), flavonoid (90.79 to 101.09 mg RE/g DW) and flavonol (24.58 to 39.21 mg QE/g DW) in methanolic leaves extract of Phoenix dactylifera L. Notably, flavonoids, saponins, and tannins can exert various pharmacological properties, especially antioxidant [75–77], anti-inflammatory[78] and hepatoprotective [75,77,79] activities. Phenolic compounds constitute the cornerstone of the antioxidant potential of date leaves extract, which is attributed to the OH group in their structure that can detoxify the peroxy radicals and charged hydroxyl radicals, thus protect the polyunsaturated fatty acids in cell membrane from peroxidation [80]. Based on the aforementioned reports, it is reasonable to suggest that the antioxidant and hepatoprotective effects of PLME and PLAE could be attributed to the synergistic activity of total phenolics, flavonoids, and saponins. The difference in the hepatoprotective and antioxidant capabilities of PLME and PLAE may be related to differences in their individual bioactive phytoconstituents. Therefore, further detailed phytochemical tests are needed to be carried out to isolate the active compounds responsible for the preceding pharmacological effects to delineate the exact mechanism(s) by which PLME and PLAE exert hepatoprotective activity.

Authors declare no conflict of interests. Authors' contribution Gamal Amer Salem and Ahmed Shaban Abdelaziz designed the research work, carried out the major experiments, performed statistical analysis of the data and wrote the manuscript. Hissein Diab participated in the design and executing experiments. Wesam A. Elsaghayer performed histopathological studies. Manal D. Mjedib and Aomassad M. Hnesh assisted in experiments. Ravi P. Sahu was involved in scientific discussions, data analysis and revising the manuscript. All the authors approved the final version of the manuscript. Acknowledgment The authors would like to thank Dr. AssemHalfawy and Dr. Sarfaraj Hussein for helping in phytochemical tests. We are also grateful to Mr. Omar Elatrash Mr. Ali Elwarfally for their technical assistance. Finally, we would like to express deep appreciation to the administration of Faculty of Pharmacy and Faculty of Medical Technology, Misurata University for allowing us to use all laboratories and facilities needed for the accomplishment of this work. This work did not receive any grant from any commercial, public or not-for-profit sectors. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.05.049. References [1] P. Vitaglione, F. Morisco, N. Caporaso, V. Fogliano, Dietary antioxidant compounds and liver health, Crit. Rev. Food Sci. Nutr. 44 (2004) 575–586 (Accessed 9 June 2017), http://www.ncbi.nlm.nih.gov/pubmed/15969329. [2] B. Hazra, S. Biswas, N. Mandal, Antioxidant and free radical scavenging activity of Spondias pinnata, BMC Complement. Altern. Med. 8 (63) (2008), http://dx.doi.org/ 10.1186/1472-6882-8-63. [3] L.A. Pham-Huy, H. He, C. Pham-Huy, Free radicals, antioxidants in disease and health, Int. J. Biomed. Sci. 4 (2008) 89–96 (Accessed 9 June 2017), http://www. ncbi.nlm.nih.gov/pubmed/23675073. [4] D.P. Jones, Radical-free biology of oxidative stress, Am. J. Physiol. Cell Physiol. 295 (2008) C849–C868, http://dx.doi.org/10.1152/ajpcell.00283.2008. [5] N. Kaplowitz, Acetaminophen hepatoxicity: what do we know, what don't we know, and what do we do next? Hepatology 40 (2004) 23–26, http://dx.doi.org/10.1002/ hep.20312. [6] A. Davie, Acetaminophen poisoning and liver function, N. Engl. J. Med. 331 (1994) 1311 author reply 1311-2 http://www.ncbi.nlm.nih.gov/pubmed/7935695 (Accessed 9 June 2017). [7] A.M.L. Slitt, P.K. Dominick, J.C. Roberts, S.D. Cohen, Effect of ribose cysteine pretreatment on hepatic and renal acetaminophen metabolite formation and glutathione depletion, Basic Clin. Pharmacol. Toxicol. 96 (2005) 487–494, http://dx. doi.org/10.1111/j.1742-7843.2005.pto_13.x. [8] E. Song, J. Fu, X. Xia, C. Su, Y. Song, Bazhen decoction protects against acetaminophen induced acute liver injury by inhibiting oxidative stress, inflammation and apoptosis in mice, PLoS One 9 (2014) e107405, http://dx.doi.org/10.1371/ journal.pone.0107405. [9] J.-R. Noh, Y.-H. Kim, J.H. Hwang, D.-H. Choi, K.-S. Kim, W.-K. Oh, C.-H. Lee, Sulforaphane protects against acetaminophen-induced hepatotoxicity, Food Chem. Toxicol. 80 (2015) 193–200, http://dx.doi.org/10.1016/j.fct.2015.03.020. [10] N.A. Buckley, I.M. Whyte, D.L. O’Connell, A.H. Dawson, Oral or intravenous Nacetylcysteine: which is the treatment of choice for acetaminophen (paracetamol) poisoning? J. Toxicol. Clin. Toxicol. 37 (1999) 759–767 (Accessed 9 June 2017), http://www.ncbi.nlm.nih.gov/pubmed/10584588. [11] P. Astaraki, G.A. Mahmoudi, A. Zafar Mohtashami, M. Ahadi, N-acetylcysteine overdose after acetaminophen poisoning, Int. Med. Case Rep. J. 8 (2015) 65, http:// dx.doi.org/10.2147/IMCRJ.S74563. [12] E.A. Sandilands, D.N. Bateman, Adverse reactions associated with acetylcysteine, Clin. Toxicol. 47 (2009) 81–88, http://dx.doi.org/10.1080/15563650802665587. [13] S. Lotito, B. Frei, Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radic. Biol. Med. 41 (2006) 1727–1746, http://dx.doi.org/10.1016/j.freeradbiomed. 2006.04.033. [14] T.M. Rababah, N.S. Hettiarachchy, R. Horax, Total phenolics and antioxidant

Conclusion These studies demonstrate that PLME and PLAE can protect against the hepatic damage induced by PCM in rats. As the activity of PLAE was lower than PLME, this suggests a need of future phytopharmacological studies to categorize the active phytochemicals responsible for hepatoprotective and antioxidant activities. The underlying mechanism of the protective effects of PLME and PLAE is the amelioration of the oxidative stress and enhancement of liver antioxidant enzymes. 372

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