Article

Protective effects of ethyl acetate fraction of Lawsonia inermis fruits extract against carbon tetrachlorideinduced oxidative damage in rat liver

Toxicology and Industrial Health 2016, Vol. 32(4) 694–706 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713502839 tih.sagepub.com

Anis Ben Hsouna1, Saoudi Mongi2, Ge´rald Culioli3, Yves Blache3, Zohra Ghlissi4, Rim Chaabane5, Abdelfattah El Feki2, Samir Jaoua1,6 and Mohamed Trigui1 Abstract This study aimed to investigate the antioxidant properties of different fractions obtained from the fruits of Lawsonia inermis, a widely used medicinal plant, against carbon tetrachloride (CCl4)-induced oxidative stress in rat liver. The results show that several fractions obtained from L. inermis fruits possessed important antioxidant activity. Among them, the ethyl acetate (EA) fraction showed the highest antioxidant activity. Then, EA fraction was selected for the purification of potential antioxidant compounds. The hepatoprotective effects of EA fraction and its most active constituent, gallic acid (GA), were evaluated against CCl4-induced hepatotoxicity in rats. CCl4 induced oxidative stress by a significant rise in serum marker enzymes. However, pretreatment of rats with EA fraction of fruits of L. inermis at a dose of 250 mg kg1 body weight and GA significantly lowered some serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase) in treated rats. A significant reduction in hepatic thiobarbituric acid reactive substances and an increase in antioxidant enzymes namely superoxide dismutase, catalase, and glutathione peroxidase by treatment with plant extract and GA, against CCl4-treated rats, were observed. Histopathological examinations showed extensive liver injuries, characterized by extensive hepatocellular necrosis, vacuolization, and inflammatory cell infiltration. This potential antioxidant activity is comparable to those of the major purified antioxidant compound, GA. Based on these results, it was observed that fruits of L. inermis protect liver from oxidative stress induced by CCl4 and thus help in evaluation of traditional claim on this plant. Keywords Lawsonia inermis, hepatotoxicity, carbon tetrachloride, oxidative stress, beneficial effect

Introduction Reactive oxygen species (ROS) including superoxide anions (O2 -), hydrogen peroxide (H2O2), hydroxyl radicals (OH), and peroxyl radical (ROO) are particularly reactive and have long been recognized to contribute to oxidative damages and the development of pathologies, such as atherosclerosis, diabetes, cancers, neurodegenerative diseases, liver cirrhosis, and ageing processes. In previous works (e.g. Kevin and Hannah, 2007), they were also reported to be caused by ROS. Liver damages constitute widespread pathologies in most cases involving oxidative stress. They are characterized by a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular

1

Biopesticides Team (LPIP), Center of Biotechnology of Sfax, Sfax, Tunisia 2 Laboratory of Animal Ecophysiology, Faculty of Science, University of Sfax, Sfax, Tunisia 3 Laboratoire MAPIEM (EA 4323), Equipe Biofouling and Substances Naturelles Marines, Universite´ du Sud Toulon-Var, Toulon, France 4 Laboratory of Pharmacology, School of Medicine, University of Sfax, Sfax, Tunisia 5 Biochemistry Laboratory, CHU Hedi Chaker of Sfax, Sfax, Tunisia 6 College of Arts and Sciences, University of Qatar, Doha, Qatar Corresponding author: Saoudi Mongi, Laboratory of Animal Ecophysiology, Faculty of Science, University of Sfax, BP 1171, Sfax 3000, Tunisia. Email: [email protected]

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carcinoma. Oxidative stress in the body can result from either excess ROS production and/or deficient antioxidant capacity. Several chemical drugs have been implicated in the etiology of liver diseases. Carbon tetrachloride (CCl4) catabolizes radical-induced lipid peroxidation and damage of liver cell membranes and organelles and causes swelling and necrosis of hepatocytes. The result of such phenomena consists of an increment in cytosolic enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) into the circulating blood (Mustafa et al., 2008). Exogenous antioxidative compounds must be delivered to maintain balance between oxidants and antioxidants and to prevent pathologies. Nevertheless, conventional and synthetic drugs used in the treatment of liver diseases are sometimes inadequate and can show serious adverse effects (Adom et al., 2003; Botterweck et al., 2000; Wolfe et al., 2003). However, most consumers prefer a safer approach like the utilization of more effective antioxidants of natural origin. Accordingly, plant extracts and their derived metabolites, such as phenolic components, offer the opportunity in this regard (Selles et al., 2012; Wallace, 2004). The use of natural antioxidants has been proposed as therapeutic agents as well as drug co-adjuvants in order to counteract liver damages. Previous studies have shown that plant extracts rich in phenolic compounds provide an effective protection against CCl4 hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity (Sheweita et al., 2001). Therefore, several works show that hepatoprotective effects have been associated with plant extracts containing high levels of phenolic compounds (Huang et al., 2010; Jain et al., 2008). Lawsonia inermis L. (synonym Lawsonia Alba; also known as henna) is used in traditional medicine to treat various diseases such as menstrual disorder, edema, rheumatism, bronchitis, and hemorrhoids and is used as cosmetic agent (Bich et al., 2004). The chemical composition of this plant widely present in North Africa, India, and Middle East has been extensively studied; terpenoids and aliphatic hydrocarbons, mainly phenolic compounds, such as coumarins, flavonoids, naphthalene, and gallic acid (GA) derivatives, obtained from this plant have been described in various studies. Henna has been reported to have many different healing effects, antibacterial, antitumoral, and antifungal activities (Berenji et al., 2010). The decoction of bark and leaves has been found to inhibit trypsin enzymes and showed anti-inflammatory activity (Adesuyi et al.,

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2012). The bark was also considered in alleviating serum transaminase levels and in restoring normal bile flow (Girish and Pradhan, 2012). The hepatoprotective effects of an extract of L. inermis fruits were studied in rats intoxicated with CCl4 in the present study. Our work aimed to (i) investigate the antioxidant and radical scavenging activities of n-hexane, chloroform, ethyl acetate (EA), n-butanol, and water fractions obtained from the hydroalcoholic extract of L. inermis fruits, (ii) determine the amounts of total phenolics and flavonoids in these fractions, (iii) analyze the chemical composition of the most active fractions by high-performance liquid chromatography (HPLC) and to identify the chemical structure of their main components, and (iv) evaluate the potential protective effects of the most active fractions and compounds in CCl4-induced damages of rat liver.

Materials and methods Preparation of plant extracts Fresh fruits of L. inermis were collected from Sfax, Tunisia, and a voucher specimen (LBPes C.S. 15.04) was deposited in the herbarium of Biopesticides Team (LPIP) of the Center of Biotechnology of Sfax, Sfax, Tunisia. The dried fruits were ground to fine powder using a grinder and the resulted material (500 g of powder) was extracted at room temperature by maceration into 1.5 L of aqueous ethanol (ethanol–water of 4:1, v/ v) with occasional shaking. After 3 days, the extracts were filtered (45 mm) and concentrated under vacuum. The concentrated extracts (100 g) were suspended in 500 mL of distilled water and sequentially partitioned into n-hexane, chloroform, EA, and then n-butanol (3  350 mL for each solvent). After solvent removal, the resulting residues gave n-hexane (1.4 g), chloroform (2.0 g), EA (21.0 g), n-butanol (32.0 g), and water (43.0 g) soluble fractions and were kept at 4 C in the dark.

In vitro antioxidant activities Determination of total phenolics and flavonoids content. Total phenolics content was determined using the Folin–Ciocalteu method (Waterman and Mole, 1994) adapted to a microscale sample. GA was used as standard. The absorbance of all the samples was measured at 760 nm using a Bio-Rad SmartSpec™ plus UV–Vis spectrophotometer (SmartSpecTm3000; Bio-Rad; Hercules, CA). The results are expressed in milligrams of GA equivalent per gram of dry plant extract (mg GAE

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g1). The flavonoids content in extracts was determined spectrophotometrically according to the method given by Quettier-Deleu et al. (2000). The flavonoids content was expressed in milligram of quercetin equivalent (QE) per gram of dry plant extract (mg QE g1).

where A0 and A0 0 are absorbances of the sample and the blank, respectively, measured at zero time, and At and A0 t are absorbances of the sample and the blank, respectively, measured after 2 h. All tests were carried out in triplicate.

Determination of DPPH radical scavenging capacity. Radical scavenging activity of the different fractions was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as a reagent according to the method of Kirby and Schmidt (1997) with some modifications. Briefly, 1 mL of a 4% (w/v) solution of DPPH radical in ethanol was mixed with 500 mL of sample solutions in ethanol (different concentrations). The mixture was incubated for 20 min in the dark at room temperature. Scavenging capacity was read spectrophotometrically by monitoring the decrease in the absorbance at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. Ascorbic acid was used as standard. The percent DPPH scavenging effect was calculated using the following equation

Purification and structural elucidation of the active compounds of the EA fraction of L. inermis. The first stage of this process was conducted on the EA fraction, which was subjected to fractionation by flash chromatography (FC) on a Spot Flash (Armen Instruments, Saint-Ave´, France) chromatographic system. For this purpose, a part of the EA fraction was solubilized in 15 mL of methanol (MeOH) and mixed with 20 g of C18-bonded silica gel (Sepra C18e, 50 mm; Phenomenex, Torrance, California, USA). The dry extract obtained after removal of the solvent was loaded in an empty cartridge and placed before a reversedphase FC column (SuperVarioFlash D26-RP18 model, 40–63 mm, 37 g; Merck, Darmstadt, Germany). The fractionation was monitored by UV at 254 nm and a ternary solvent gradient composed by water (A), MeOH (B), and dichloromethane (C) was used. The elution began with an initial isocratic step with 100% A from 0 to 3 min, and was followed by a linear gradient up to 100% B from 3 to 10 min, an isocratic step with 100% B from 10 to 12 min, and a linear gradient up to 100% C from 12 to 19 min, and a final isocratic step with 100% C from 19 to 28 min. The flow rate was initially fixed at 2 mL min1, then a linear ramp from 2 to 20 mL min1 in 2 min was applied, and this final value of the flow rate was kept during all the remaining of the process. After FC fractionation, the obtained subfractions were combined according to their HPLC profiles, yielding several pools of subfractions. The selected pool was subjected to a second stage of purification using a reversed-phase semi-preparative column (Gemini C6-Phenyl, 250  10 mm, 5 mm, Phenomenex) on a Biotek-Kontron (Montigny-leBretonneux, France) HPLC system equipped with a ternary pump (525 model), an auto sampler (560 model), a column oven (582 model), and a UV detection (LDC analytical, 3100 model) fixed at 280 nm. A MeOH–H2O binary gradient was employed with an initial isocratic step with MeOH–H2O 20:80 (v/v) during 2.5 min, a linear ramp to MeOH–H2O 45:55 (v/v) from 2.5 to 20 min, and then a second linear ramp to 100% MeOH in 0.1 min. This composition was maintained up to 25 min until re-equilibration of the system to the initial conditions (5 min). The flow rate was fixed at 3 mL min1.

DPPH scavenging effectð%Þ ¼

Acontrol  Asample  100 Acontrol

where Acontrol is the absorbance of the control reaction were the sample is replaced by 500 mL ethanol. Tests were carried out in triplicate. b-Carotene bleaching assay. The antioxidant activity was determined according to the b-carotene bleaching method described by Pratt (1980). A stock solution of b-carotene–linoleic acid mixture was prepared as follows: 0.5 mg of b-carotene was dissolved in 1 mL of chloroform with 25 mL of linoleic acid and 200 mg of Tween-20. Chloroform was completely evaporated, using vacuum evaporator. Then, 100 mL of distilled water, saturated with oxygen (30 min), was added and the obtained solution was vigorously shaken. A 4-mL of this reaction mixture was dispensed into the test tubes, and 200 mL of each sample prepared at different concentrations were added. The emulsion system was incubated for 2 h at 50 C. The same procedure was repeated with butylated hydroxytoluene (BHT) as positive control and a blank as a negative control. After this incubation period, the absorbance of each mixture was measured at 490 nm. Antioxidant activity in b-carotene bleaching model in percentage (A%) was calculated with the following equation   A0  At  100 A% ¼ 1  A00  A0t

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The chemical structures of the compounds purified by HPLC were assessed through extensive spectrometric analysis (electrospray ionization (ESI)–mass spectrometry (MS), one-, and two-dimensional nuclear magnetic resonance (NMR)) and by comparison with previously published data. (þ)-ESI-MS mass spectra were measured on an ion trap mass spectrometer fitted with an ESI interface (Esquire 6000; Bruker Daltoniks, Bremen, Germany). NMR spectra were obtained at 400 and 100 MHz for 1H and 13C, respectively, on an Avance 400 MHz NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) in deuterated methanol (CD3OD). All chemical shifts were referenced to the solvent peaks (H 3.31 and C 49.0 for CD3OD).

In vivo antioxidant properties Animal. Male albino Wistar rats (n ¼ 48 rats; weighing 200–220 g) were used in this study. The animals were purchased from the Central Pharmacy of Tunisia (SIPHAT, Tunisia). They were housed at 22 + 3 C with light–dark periods of 12 h and minimum relative humidity of 40%. The rats were fed with a commercial balanced diet (SICO, Sfax, Tunisia) and drinking water ad libitum. All animal experiments were conducted without anesthesia and according to the Ethical Committee Guidelines for the care and use of laboratory animals of our institution. Experimental design. After 2 weeks of acclimatization, the rats were allocated randomly to four experimental groups of eight animals each (n ¼ 8) with free access to food and water. Based on the preliminary experiments, the hepatoprotective dose of the EA extract of L. inermis and its major compound, GA, was decided. In multiple dose pretreatment experiment, EA extract was administered at 250 mg kg1 body weight (bw) by intraperitoneal injection.  Group I: control rats received the vehicle (olive oil, 1 mL kg1 orally) at day 8.  Group II: received CCl4 in olive oil (1 mL kg1, intraperitoneally (i.p.)) at day 8.  Group III: received the EA fraction (250 mg kg1 bw) daily by i.p. injection for 8 days followed by a single dose of CCl4 in olive oil at a dose of 1 mL kg1 using an intragastric tube 24 h after the last dose (Girish et al., 2009).  Group IV: received EA fraction (250 mg kg1 bw) daily by i.p. injection for 8 days.

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Group V: received GA (50 mg kg1 bw) daily by i.p. injection for 8 days. Group VI: received GA (50 mg kg1 bw) daily by i.p. injection for 8 days followed by a single dose of CCl4 in olive oil at a dose of 1 mL kg1 using an intragastric tube 24 h after the last dose.

The animals were killed on day 9 by cervical decapitation. Blood samples were collected, allowed to clot at room temperature, and serum separated by centrifuging at 2700g for 15 min for various biochemical parameters. The liver was quickly excised, minced with ice cold saline, and blotted on filter paper. Homogenates were centrifuged at 10,000g for 15 min at 4 C (Ultra Turrax T25, Germany) (1:2, w/v) in 50 mmol L1 phosphate buffer (pH 7.4). The supernatant and serum were frozen at 30 C in aliquots until analysis. Serum parameters. Serum samples were obtained by the centrifugation of blood at 2700g for 15 min at 4 C and were then divided into eppendorf tubes. Isolated sera were stored at 30 C until used for further analyses. The levels of serum ALT, AST, ALP, and lactate dehydrogenase (LDH) were measured using commercial kits according to the manufacturer’s directions.

Oxidative stress analysis TBARS measurements. Lipid peroxidation in the tissue homogenate was estimated by measuring thiobarbituric acid reactive substances (TBARS) and was expressed in terms of malondialdehyde (MDA) content, which is the end product of lipid peroxidation, according to Buege and Aust (1978). In brief, 125 mL of supernatants were homogenized by sonication with 50 mL of Tris-buffered saline and 125 mL of trichloroacetic acid–butylated hydroxytoluene (BHT) in order to precipitate proteins and centrifuged (1000g, 10 min, 4 C). The obtained supernatant (200 mL) was mixed with 40 mL of hydrochloric acid (0.6 M) and 160 mL of TBA dissolved in Tris and the mixture was heated at 80 C for 10 min. The absorbance of the resultant supernatant was read at 530 nm. The amount of TBARS was calculated using an extinction coefficient of 156  105 mM1 cm1. Anitoxidant enzymes studies. In liver tissues, superoxide dismutase (SOD) activity was determined according to the colorimetric method of Beyer and Fridovich

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Table 1. The yield extraction, total phenolic, flavonoid contents, IC50 values of the DPPH free radical scavenging, and the b-carotene linoleate assays of Lawsonia inermis fruit fractions.a

Extracts

Yield extract (%)

n-Hexane Chloroform EA n-Butanol Water Ascorbic acid BHT

0.28 0.40 4.20 6.40 8.60 – –

Total phenolics (mg GAE g1) 23 + 244 + 374 + 338 + 53 + – –

1 6 2 3 1

Total flavonoids (mg QE g1)

DPPHb IC50 (mg mL1)

b-Caroteneb bleaching assay IC50 (mg mL1)

15 + 2 21 + 3 60 + 6 13 + 4 21 + 1 – –

>100 18.0 + 0.9 3.5 + 0.1 5.0 + 1.4 7.5 + 0.3 3.5 + 0.2 –

>50 + 0.1 + 0.1 + 0.9 + 0.2 – 1.4 + 0.1 5.0 1.6 4.0 6.0

DPPH: 1,1-diphenyl-2-picrylhydrazyl; IC50: half maximal inhibitory concentration; DPPH: 1,1-diphenyl-2-picrylhydrazyl; GA: gallic acid. a Ascorbic acid and BHT were used as standards. Each value represents the mean + SD of three experiments. b Each value represents the mean + SD of three experiments.

(1987) using the oxidizing reaction of nitroblue tetrazolium; catalase (CAT) activity was measured by the UV colorimetric method of Aebi (1974) using H2O2 as substrate; glutathione peroxidase (GPx) activity was measured by a modification of the colorimetric method of Flohe and Gu¨nzler (1984) using H2O2 as substrate and the reduced glutathione (GSH).

Histopathological studies Pieces of liver tissues were excised, washed with normal saline, and processed separately for histopathological observation. The liver and kidney tissues were fixed in Bouin’s solution, dehydrated in graded (50–100%) alcohol, and embedded in paraffin. Thin sections (4–5 mm) were cut and stained with routine hematoxylin–eosin stain. The sections were examined microscopically for histopathology changes, including cell necrosis, fatty change, and ballooning degeneration (Gabe, 1968).

Statistical analysis All values are expressed as mean + SEM. The results were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test using Statistical Package for the Social Sciences (version 11) for Windows. Differences were considered significant at p < 0.05.

Results Total phenolic and flavonoid contents The extraction yield varied from 0.28 to 8.6% depending on the used extraction solvent in the following order: n-hexane > chloroform > EA > n-butanol >

water (Table 1). Naturally occurring phenolic compounds, including phenolic acids, flavonoids, tannins, coumarins, and others, have been reported to be significantly associated with the biological activities of plant extracts (Gupta and Prakash, 2009). In the present study, the total phenolic and flavonoid contents of L. inermis fruits extracts were examined and are presented in Table 1. These compounds showed differences in their total contents depending on solvents polarities. The highest content of total phenolics was found in extracts obtained with EA fraction (374 mg GAE g1) followed by n-butanol and chloroform fractions with 338 and 244 mg GAE g1, respectively. The contents obtained with n-hexane and water extracts were much smaller. Total flavonoid content varied from 13 to 60 mg QE g1. EA fraction showed the highest levels with 60 mg QE g1. Chloroform and water fractions had moderate levels (21 mg QE g1 for each fraction), while n-butanol and n-hexane have the lowest amounts (12 and 15 mg QE g1).

Antioxidant capacities of fruits of L. inermis The DPPH radical scavenging activities of L. inermis fruits extracts were investigated using the reduction capacity of DPPH to diphenylpicrylhydrazine derivative detected at 517 nm (Duh, 1998). A high percentage of radical scavenging indicated a strong antioxidant activity in the tested sample. As shown in Figure 1, DPPH test revealed that increase in extracts concentration resulted in increase in free radical-scavenging activity in a dose dependent manner. Then, this low concentration and high inhibition showed a high scavenging activity. EA, n-butanol, and water fractions were considerably less effective than DPPH radicals’

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Figure 1. Scavenger effect of Lawsonia inermis fruit fractions at different concentrations, 2, 4, 5, 10, 30, 50, and 100 mg mL1, on the stable 1,1-diphenyl-2-picrylhydrazyl radical. Results are expressed as percentage decrement in absorbance at 517 nm with respect to control. Ascorbic acid was used as a standard. Each value represents the mean + SEM of three experiments.

scavenger. The antioxidant activity of the extracts can also be evaluated by the determination of the half maximal inhibitory concentration (IC50) values corresponding to the amount of extract required to scavenge 50% of DPPH radicals present in the reaction mixture. High IC50 values indicate low antioxidant activity. Based on the IC50 values, the best activities were obtained with EA fraction (3.5 mg mL1), followed by n-butanol (5.0 mg mL1) and water fraction (7.5 mg mL1). It is clear that EA fraction exhibited a potent antioxidant activity nearly equal to those of ascorbic acid used as standard (3.5 mg mL1), but much better than the reported IC50 values obtained for usual references such as GA (64 mg mL1) and BHA (114 mg mL1) (Ozsoy et al., 2008). n-Hexane and chloroform extracts showed higher IC50 values and consequently a low antioxidant activities. Additionally, the inhibitory effect of the different L. inermis fruit fractions on lipid peroxidation was determined by the b-carotene–linoleic acid bleaching test. Figure 2 shows various degrees of linoleic acid oxidation and subsequently the b-carotene bleaching after addition of the L. inermis fruits organic extracts and the positive control BHT used as at different concentrations. This antioxidant activity was dose-dependent as found in the DPPH test. The EA fraction exhibited a high antioxidant activity nearly equal to that of the BHT standard with IC50 values of 1.6 mg mL1. The antioxidant activities of this fraction are probably due to the occurrence of active phenolic compounds.

Isolation and identification of phenolic compounds in the active EA fraction of L. inermis fruits Based on our in vitro antioxidant test, the EA fraction obtained from L. inermis fruits was chosen for in vivo model. In this study, a part (1.11 g) of this fraction was subjected to FC fractionation and yielded 36 subfractions, which were gathered into 12 groups (F-1 to F-12). As F-2 was found by analytical HPLC to contain the major components of the EA fraction, this sub-fraction was further purified by semi-preparative HPLC and afforded three pure compounds 1 (60.0 mg), 2 (3.0 mg), and 3 (5.0 mg). Compound 1, a white powder, showed a molecular formula C7H6O5 which was deduced from (þ)-ESI– MS (m/z 171.0 [MþH]þ) and 13C NMR data. These spectroscopic data together with a complete NMR structural analysis and comparison with literature values allowed the unambiguous identification of compound 1 as GA. This molecule has been already reported in several parts of L. inermis. Compound 2 was isolated as a white solid and its molecular formula C15H14O6 was deduced from its (þ)-ESIMS (m/z 291.1 [MþH]þ) and 13C NMR spectra. The comparison of all the MS and NMR data set with those described in the literature confirmed that compound 2 was (þ)-catechin. Compound 3 was obtained as a white powder with a molecular formula of C8H8O5 [(þ)-ESIMS; m/z

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Figure 2. Antioxidant activities of Lawsonia inermis fruit fractions at different concentrations measured by b-carotene bleaching method. butylated hydroxytoluene was used as reference antioxidant. OH

O

RO

O

HO HO

OH OH 1 R=H 3 R=CH3

OH

were significantly reversed on treatment with L. inermis fruits extract. The activity of L. inermis fruits extract was comparable to that of the reference antioxidant GA.

OH

OH

Effects on lipid peroxidation 2 (+)-catechin

Figure 3. Chemical structures of the phenolic compounds isolated from the ethyl acetate soluble fraction of Lawsonia inermis fruits extract.

185.0 [MþH]þ] and NMR data consistent with those of methyl gallate. The chemical structures of compounds 1–3 isolated from the EA fraction of the hydroalcoholic extract of henna fruits are depicted in Figure 3. To our knowledge, this is the first report of (þ)-catechin and methyl gallate in L. inermis.

TBARS level is widely used as a marker of free radical-mediated lipid peroxidation injury. We determined TBARS levels in the liver, and the results are shown in Figure 4. TBARS levels in the CCl4-treated group (0.57 + 0.02 nmol MDA mg1 protein) were significantly higher than that of the control group (0.19 + 0.03 nmol MDA mg1 protein, p < 0.001). TBARS levels in the L. inermis fruits extract-treated group (0.26 + 0.01 nmol MDA mg1 protein) were significantly lower than that in the CCl4-treated group (p < 0.001). GA also inhibited the elevating TBARS levels upon CCl4 administration.

Effects on antioxidant enzymes Serum biochemical parameters The activities of various biochemical enzymes in normal, control, CCl4-, and other treated groups are presented in Table 2. The activities of AST, ALT, ALP, and LDH were significantly increased by 37%, 45%, 41% and 33%, respectively (p < 0.001; p < 0.01; p < 0.001; and p < 0.001, respectively), in CCl4 compared to normal control. The levels of the above enzymes

SOD, CAT, and GPx were measured as an index of antioxidant status of tissues. Significantly lower activities hepatic enzymes SOD, CAT, and GPx by 53%, 35%, and 69%, respectively (p < 0.01; p < 0.001 and p < 0.001, respectively), were observed in rats from the CCl4-treated group as compared to the normal control group (Table 3). There was a significant increase in SOD, CAT, and GPx activities in L. inermis fruits extract-treated group as compared

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Table 2. Effects of CCl4 and EA soluble fraction of Lawsonia inermis fruits extract, GA, and their combination (EA–CCl4 and GA–CCl4) on hepatic markers in serum of control (C) and experimental rats.a Treatment

AST

C CCl4 EA–CCl4 GA–CCl4 EA GA

155 + 4 246 + 11b 157 + 4d 152 + 7d 159 + 7 154 + 3

ALT 48 + 88 + 45 + 46 + 48 + 47 +

ALP

5 8c 8d 4d 3 3

58 + 98 + 61 + 61 + 67 + 57 +

8 10c 5e 3e 3 2

LDH 898 + 89 1345 + 132b 906 + 112e 887 + 145e 911 + 132 898 + 112

CCl4: carbon tetrachloride; EA: ethyl acetate; GA: gallic acid; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; LDH: lactate dehydrogenase. a Values are mean + SEM for eight rats in each group. b CCl4-, EA-, EA–CCl4-, GA-, and GA–CCl4-treated groups versus control group, p < 0.001. c CCl4-, EA-, EA–CCl4-, GA-, and GA–CCl4-treated groups versus control group, p < 0.01. d CCl4 group versus (EA–CCl4 and GA–CCl4) group, p < 0.01. e CCl4 group versus (EA–CCl4 and GA–CCl4) group, p < 0.001.

Table 3. Effects of CCl4, EA soluble fraction of Lawsonia inermis fruits extract, GA and their combination (EA–CCl4 and GA–CCl4) on the activities of enzymatic antioxidants in liver of control (C) and experimental rats.a Treatment C CCl4 EA–CCl4 GA–CCl4 EA GA

SOD (Units mg1 protein) 21 9.9 20 17.7 19.3 18.1

+1 + 0.2b + 1d + 0.8d + 0.4 + 0.3

CAT (mmol H2O2 mg1 protein) 445 + 288 + 432 + 403 + 427 + 413 +

15 12c 12d 5d 8 8

GPx (mmol GSH min1 mg1 protein) 9.35 + 2.87 + 8.76 + 9.11 + 8.95 + 8.85 +

0.24 0.11c 0.28e 0.25e 0.16 0.30

CCl4: carbon tetrachloride; EA: ethyl acetate; GA: gallic acid; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GSH: reduced glutathione; H2O2: hydrogen peroxide. a Values are mean + SEM for eight rats in each group. b CCl4-, EA-, EA–CCl4-, GA-, and GA–CCl4-treated groups versus control group, p < 0.01. c CCl4-, EA-, EA–CCl4-, GA-, and GA–CCl4-treated groups versus control group, p < 0.001. d CCl4 group versus (EA–CCl4 and GA–CCl4) group, p < 0.01. e CCl4 group versus (EA–CCl4 and GA–CCl4) group, p < 0.001.

to the CCl4-treated group. Treatment with CCl4 significantly decreased the SOD, CAT, and GPx levels in the liver of treated group (9.93 + 0.24 Units mg1 protein, 288 + 12 mmol H2O2 mg1 protein, and 2.9 + 0.1 mmol GSH min1 mg1 protein, respectively) as compared to the liver of normal control group (21 + 1 Units mg1 protein, 446 + 15 mmol H2O2 mg1 protein, and 9.35 + 0.24 mmol GSH min1 mg1 protein, respectively). The levels were similar at animals treated with GA and those treated with L. inermis fruits extract (250 mg kg1 bw).

cells as well as neutrophilic infiltration (arrow) and several ballooning degenerations (asterisk) of hepatocytes (Figure 5). However, according to the microscopic examinations, the severe hepatic lesions induced by CCl4 were considerably reduced by the administration of EA (250 mg kg1 bw) and GA as the major component of the EA fraction of L. inermis fruits extract (Figure 4), with the results being well correlated with those of serum biochemical parameters and oxidative stress markers. Necrosis was markedly reduced and minimized by the pretreatment with 250 mg kg1 of EA fraction of extract of L. inermis fruits.

Histopathological findings The treatment with CCl4 caused excessive necrosis associated with neutrophilic infiltration, which is frequently observed in the case of swelling of the liver

Discussion In this study, the quantification of the phenolic and flavonoid content of L. inermis fruits extract revealed a

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Figure 4. Effects of CCl4, EA soluble fraction of Lawsonia inermis fruits extract, GA, and their combinations (EA– CCl4 and GA–CCl4) on hepatic TBARS of control (C) and experimental rats. Values are mean + SEM for eight rats in each group. CCl4, EA, EA–CCl4, GA, and GA–CCl4-treated groups versus control group; ***p < 0.001, CCl4 group versus (EA–CCl4 and GA–CCl4) group; ##p < 0.01. CCl4: carbon tetrachloride; EA: ethyl acetate; GA: gallic acid; TBARS: thiobarbituric acid reactive substances.

high level of phenolics in the EA and n-butanol. Due to their suitable polarities, these solvents were the best solvents to use for the extraction of phenolics compounds compared to the other solvents. Our results revealed that L. inermis fruit fractions contain more significant phenolics and flavonoids levels than those of other plants, commonly used as antioxidant and antimicrobial agents (Aziz et al., 1998). The high level of the total phenolic content in our case may be related to the hard climate conditions (hot temperatures, high solar exposure, dryness, and short growing season). Phytochemical investigations of L. inermis have shown predominantly the presence of phenolic compounds (coumarins, flavonoids, naphthalene, and GA derivatives) that could be glycosylated (Ben Hsouna et al., 2011). Other compounds such as triterpenoids, steroids, and aliphatic hydrocarbons have also been isolated from this plant (Siddiqui et al., 2003). Interestingly, the phenolic compounds of fruits of L. inermis were characterized and well investigated for the first time. Several studies have been reported on the beneficial effects of these phenolics, because of some interesting new findings regarding their biological activities (Ow and Stupans, 2003). The above data indicated that EA fraction of extract of L. inermis fruits contained phenolic acids and flavonoids that appeared to be responsible for its antioxidant activity. Free radicals and other ROS are known to take part in lipid peroxidation, cause food deterioration, and are

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reported to be a causative agent of various diseases (Nichenametla et al., 2006). Antioxidants act as radical-scavengers and inhibit lipid peroxidation and other free radical-mediated processes. Actually, there is a growing interest in the substitution of synthetic antioxidants used in food preservation, which involve toxic side effects, with natural ones. Polyphenols with their redox properties can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Galato et al., 2001). In the present study, the antioxidant potential of L. inermis fruits was measured by two different biochemical assays: scavenging activity on DPPH radicals and lipid peroxidation inhibition by the b-carotene–linoleate system. Based on the susceptibility of the different fractions to the free radicals, it is possible to conclude that the EA fraction of L. inermis fruits extract has a stronger and broader spectrum of antioxidant activity as compared to the other fractions. In fact, the present investigation showed that the EA fraction exhibited significantly higher DPPH scavenging activity. It was found that EA fraction exhibited strong hydrogen donating and hydroxyl radical scavenging ability. Furthermore, this fraction could protect polyunsaturated fatty acids in food from lipid peroxidation. Our results show a good correlation between the antioxidant activity and the phenolic contents in the EA fraction. Therefore, phenolic compounds seems to be responsible for the antioxidant activity by its ‘chain-breaking’ property that can counteract peroxyl and alkoxyl radical generated during lipid peroxidation to prevent continual hydrogen abstraction and thus inhibiting chain propagation step. Based on our in vitro antioxidant assays, EA was chosen as the most potent antioxidant extract and subsequently used to estimate the antioxidant activity of L. inermis in vivo. The CCl4-induced hepatotoxicity model is extensively used for the evaluation of antioxidant effects of drugs and plant extracts (Avijeet et al., 2008). CCl4, a well-known model compound for producing chemical hepatic injury, requires biotransformation by hepatic microsomal cytochrome P450 (CYP) to produce toxic metabolites, namely trichloromethyl free radicals (Brautbar and Williams, 2002). CCl4 is accumulated in hepatic parenchyma cells and metabolized to the CCl3 radical by liver CYP-dependent monooxygenases (Recknagel, 1967). Free radicals are well known to induce cellular and tissue pathogenesis leading to oxidative processes (Halliwell et al., 1992). CCl3 radical rapidly reacts with oxygen to yield a highly reactive trichloromethyl peroxy radical, CCl3OOc_ . These

Hsouna et al.

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Figure 5. Effect of EA soluble fraction of Lawsonia inermis fruits extract on CCl4-induced liver damage. (a) Control group; (b) animals treated with CCl4 showing spotty liver cell death with neutrophilic infiltrates (arrow) and several ballooning degenerations of hepatocytes (asterisk); (c) animals pretreated with EA and then with CCl4; (d) animals pretreated with GA and then with CCl4; (e) and (f) animals pretreated with EA soluble fraction of L. inermis fruits extract and GA, respectively; hematoxylin–eosin staining; magnification 400. CCl4: carbon tetrachloride; EA: ethyl acetate; GA: gallic acid.

radicals react with proteins and lipids. They remove hydrogen atoms from unsaturated lipids, thus initiating lipid peroxidation (Amani et al., 2006). Hence, free radical scavengings provide important ways to protect against CCl4-induced oxidative injury. In the present study, the capability of L. inermis to protect against CCl4-induced hepatotoxicity and oxidative stress were investigated. We found that treatment with L. inermis fruits significantly extract prevented CCl4-induced liver damage as evidenced by decreased serum activities of

AST, ALT, ALP, and LDH. The elevated activities of these enzymes are indicative of cellular leakage and loss of the functional integrity of the cell membranes in liver (Rajesh and Latha, 2004). The stabilization of these enzyme levels by the L. inermis fruits extract and its active principle is a clear indication of the improvement of the functional status of the liver. Our results clearly indicated that CCl4 induced an increase in hepatic LPO in treated animals. Enhanced lipid peroxidation expressed in terms of TBARS contents in CCl4-treated

704

rats as observed in our study indicates the damage to the hepatic cells, which is confirmed by the earlier reports (Bhandarkar and Khan, 2004). L. inermis fruits extract showed ability to prevent CCl4-induced increment in TBARS content, suggesting that extract inhibit lipid peroxidation and its propagation in the liver. This result is in agreement with the previous findings (Dasgupta et al., 2003). GA may protect the liver by preventing LPO because it scavenges the superoxide and OH that are involved in the production of the free radicals. These results suggest that binding of the gallate compounds to lipid membrane is a principal determining factor of the antioxidant action (Shahrzad et al., 2001). ROS, such as O2 .- and H2O2, are produced throughout the cells during normal aerobic metabolism. The intracellular concentration of ROS is a consequence of both their production and their removal by various antioxidants. A major component of the antioxidant system in mammalian cells consists of three enzymes, namely, SOD, CAT, and GPx. These enzymes work in concert to detoxify superoxide anion and H2O2 in cells. Our results indicated that pretreatment of L. inermis fruits extract caused an increase in the activity of antioxidant enzymes. The antioxidant enzyme system plays an important role in the defense of cells against oxidative insults. The study examined the ameliorating effect of the extracts from L. inermis, on oxidative stress induced by CCl4. GA being an antioxidant reduces the stress to a considerable extent by restoration of the antioxidant enzymes to normal control. This evidenced that the administration of EA fraction and GA showed hepatoprotective effect under CCl4-induced oxidative stress. These were in agreement with previous findings that followed that phenolic acids were important antioxidant components in Acacia confusa, among which GA had the highest antioxidant activities (Tung et al., 2009). Chaudhary et al. (2010) mentioned that L. inermis has been used in traditional medicine that contains carbohydrates, proteins, flavonoids, tannins, and phenolic compounds. This plant is currently considered as a valuable source of unique natural products for the development of medicines against various diseases and also for the development of industrial products. GA is well known for its antioxidant properties in biomembranes, where it scavenges and destroys free radicals and prevents lipid peroxidation, it also protects against CCl4-induced hepatotoxicity. The hepatoprotective activity of the EA fraction of L. inermis may probably be due to its antioxidant property. Significant prophylactic activity of the isolated constituents indicates that these compounds are solely responsible for hepatoprotective activity of L. inermis fruits

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extract. Being antioxidants, they may act as scavengers of free radicals, such as trichloromethyl and superoxide radicals, and protect the liver against liver plasma membrane peroxidative degradation or promote cellular mitosis for the repair of damaged liver cells. The histological observations basically supported the results obtained from serum enzyme assays. The liver of CCl4-intoxicated rats showed massive fatty changes such necrosis, infiltration of lymphocyte, and several ballooning degenerations of hepatocytes. The histopathological observations of the liver of rats pretreated with L. inermis fruit fractions and subsequently given CCl4 showed a more or less normal architecture. In conclusion, it may be mentioned that the altered biochemical and oxidative stress profiles because of exposure to CCl4 is reversed toward normalization by L. inermis fruits extract. The contents of the extract not only protect the integrity of plasma membrane but, at the same time, increased the regenerative and reparative capacity of the liver. Beneficial effect of the L. inermis fruits extract may be due to the presence of some phenolic components such as GA that have membrane stabilizing effects. These results suggest that the compound present in the plant extract efficiently works on the liver to keep it normally functioning and minimizing cell membrane disturbances. Acknowledgments The authors thank the volunteers for their cooperation in the present study.

Authors’ Note A.B.H. and S.M. contributed equally to this work.

Funding This work was supported by grants from the Tunisian Ministry of Higher Education.

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