Antioxidant and hepatoprotective activities of Egyptian moraceous plants against carbon tetrachloride-induced oxidative stress and liver damage in rats

Abstract

Context: In the absence of reliable liver-protective drugs in modern medicine, a large number of medicinal preparations are recommended for treatment of liver disorders.

Objective: The antioxidant, hepatoprotective and kidney protective activities of methanol extracts of Ficus carica Linn. (Moraceae) leaves and fruits and Morus alba Linn. root barks (Moraceae) are evaluated here.

Materials and methods: Liver and kidney damage were induced in rats by carbon tetrachloride in a subcutaneous dose of 1 mL (40% v/v in corn oil)/kg. The extract was given intraperitoneally at doses of 50 mg/kg (F. carica leaf and M. alba root bark) and 150 mg/kg (F. carica fruit). The activity of the extracts was comparable to that of silymarin, a known hepatoprotective agent. Antioxidant activity was evaluated by measuring blood glutathione (GSH) content, superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde equivalent (MDA). Hepatoprotective activity was evaluated by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, and total protein. These biochemical observations were supported by histopathological examination of liver sections. Kidney function was evaluated by measuring plasma urea and creatinine.

Results: Methanol extracts of Ficus carica and Morus alba showed potent antioxidant and hepatoprotective activities; in-depth chromatographic investigation of the most active extract (Ficus carica leaf extract) resulted in identification of umbelliferone, caffeic acid, quercetin-3-O-β-d-glucopyranoside, quercetin-3-O-α-l-rhamnopyranoside, and kaempferol-3-O-α-l-rhamnopyranoside.

Discussion and conclusion: These findings demonstrate that the phenolic constituents of Ficus carica leaf and Morus alba root bark are responsible at least in part for the observed protective effects.

Keywords::

• Moraceae
• silymarin
• flavonoids
• umbelliferone
• caffeic acid

Introduction

Liver intoxication has increased as a result of exposure to high levels of environmental toxins (Lee et al., 2004). Carbon tetrachloride (CCl₄) is a potent hepatotoxin producing centrilobular hepatic necrosis (Recknagel, 1983), developing liver and kidney damage, and finally can result in cancer (Recknagel, 1967; Masuda, 2006). Herbal preparations are recommended for treatment of liver disorders (Chatterjee, 2000). Evidence from literature is the hepatoprotective activity of plants related to family Moraceae such as Cudrania tricuspidata Carr. root bark (Tian et al., 2005), Ficus hispida Linn. leaf (Mandal et al., 2000), Ficus racemosa Linn. leaf (Mandal et al., 1998, 1999), Morus bombycis Koidzumi (Jin et al., 2005) and Morus alba Linn. leaf (Kalantari et al., 2009).

Ficus carica Linn. (Moraceae) is supposed to be native to Carica in Asia Minor (Baily, 1953). In traditional medicine the roots are used in the treatment of leukoderma and ringworm, and its fruits have antipyretic, purgative and aphrodisiac properties, and have been shown to be useful in inflammations and paralysis (Nadkarni & Nadkarni, 1995; Kirthikar & Bzasu, 1996). Earlier chemical examination of this plant has shown the presence of psoralen, bergapten, umbelliferone (Seong-Kuk et al., 1995; Louis et al., 2000), β-sitosterol, campesterol, stigmasterol fucosterol, fatty acids (Jeong & Lachance, 2001), 6-(2-methoxy-Ζ-vinyl)-7-methyl-pyranocoumarin and 9,19-cycloarlane triterpenoid as an anticancer (Hongming et al., 1997; Weiping et al., 1997) and antiproliferative agent; 6-O-acyl-β-d-glucosyl-β-sitosterol (Shai et al., 2001), calotropenyl acetate, and lupeol acetate (Saeed & Sabir, 2002).

The root bark of the mulberry tree (Morus species) has been used by human beings for at least 4,000 years. Recent research has shown that this plant has free radical scavenging activities, hypolipidemic, antioxidant, antibacterial, antiviral, astringent, emollient, and anti-inflammatory properties (Chung et al., 2003; Du et al., 2003; Singab et al., 2006). A piperidine alkaloid (1-deoxynojirimycin) and glycoproteins (Morans A and 20K) were isolated as antidiabetic agents from Morus root bark and/or leaf (Yagi et al., 1976; Hikino et al., 1985; Kim et al., 1999). White mulberry leaf contains triterpenes (lupeol), sterols (β-sitosterol), bioflavonoids (rutin, moracetin, quercetin-3-triglucoside, and isoquercitrin), coumarins, volatile oil, alkaloids, amino acids, and organic acids (Doi et al., 2001). On the other hand, the antioxidant potency of some phenolic compounds (flavonoids, stilbenes, and 2-arylbenzofurans) from Morus alba has been reported (Nomura et al., 1977, 1980; Fukai & Nomura, 1998; Sharma et al., 2001; Jin et al., 2002; Fukai et al., 2003). The present study was therefore undertaken to evaluate the hepatoprotective activity of both Ficus carica (fruit and leaf) and Morus alba (root bark) extracts.

Materials and methods

Chemicals used for biological study

All chemicals were of analytical grade and purchased from Sigma (St. Louis, MO). Carbon tetrachloride was obtained from Fisher Scientific (Fair Lawn, NJ). Silymarin (Legalon^®) was obtained from Chemical Industries Development (CID) (Giza, Egypt). All the commercial kits used for the estimation of biochemical parameters were supplied by BioMed Diagnostics (Cairo, Egypt).

Apparatus and chemicals for phytochemical study

The UV spectra were measured using a Hitachi U–3200 spectrophotometer. The UV spectral data of the isolated compounds were recorded following standard procedures (Mabry et al., 1970). ¹H-NMR and ¹³C-NMR spectra were recorded using a JEOL JNM-LA-500 spectrophotometer. Chemical shifts were given in δ values (ppm) with tetramethyl silane (TMS) as an internal standard. Fast atomic bombardment mass spectrometry (FAB-MS) data were registered in negative and positive ion mode using a JEOL JMS-DX 302 spectrophotometer. For column chromatographic fractionation and purification, ion exchange resin, diaion HP-20 (Nippon, Rensui, Japan), was used as a stationary phase. TLC was performed on silica gel 60 F₂₅₄ sheets, 0.25 mm thickness, 10 × 20 cm (Fluka Chemika, Buchs, Switzerland). Sheets of Whatman paper No.1 MM was used for paper chromatography (PC) and No. 3 MM for preparative paper chromatography (PPC) (Whatman, Maidstone, Kent).

Experimental animals

Adult female Swiss albino rats (100–120 g body weight) were obtained from the Egyptian Organization for Biological Product and Vaccines, Giza, Egypt. The animals were housed in cages with good ventilation and illumination, and had access to unlimited water and standard rodent chow. Animal care and handling was done according to the guidelines set by the World Health Organization, Geneva, Switzerland and approved by the Committee for Animal Care at the National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA).

Authentication of plants

The leaf and fruit of F. carica were collected from the Zoological garden in Giza, Egypt, and were air-dried. The root bark of M. alba was collected from the Delta region, Egypt, and was air-dried. Identification of plants was verified by Abd El Salam Mohamed Al-Nowiahi, Department of Taxonomy, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt. A voucher specimen of authenticated F. carica leaf (FCL-2009), F. carica fruit (FCF-2009) and M. alba root bark (MRB-2009) were deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt.

Preparation of the aqueous methanol extracts of leaf and fruit of F. carica and root bark of M. alba

Each fresh plant (2 kg) was air-dried in shade and reduced to fine powder, or pieces (in the case of Ficus carica fruits). After that, each was exhaustively extracted with water (15 L). The extracts were evaporated in vacuo at 40°C to dryness and residues were extracted with methanol until exhaustion. The combined methanol portions were evaporated in vacuo to dryness. The dried residues were made free from methanol and kept in tightly closed sample tubes for biological investigation.

Induction of liver damage in rats

Animals received a hepatotoxic compound (CCl₄), administered subcutaneously (s.c.) twice a week as a 40% solution in corn oil in a dose of 1 mL/kg b.wt.

Toxicity studies

Acute toxicity study was performed for the methanol extracts in albino rats. The animals were fasted overnight and provided with only water, after which the extracts were administered intraperitoneally at a dose of 50 mg/kg and observed for 14 days. If mortality was observed in two out of three animals, the dose administered was assigned as the toxic dose. If the mortality was observed in one animal, the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for a higher dose. One tenth of the maximum dose of the extract tested for acute toxicity was selected for evaluation of hepatoprotective activity (Sunilson et al., 2009), i.e. 50 mg/kg for both Ficus carica leaf and Morus alba root bark extracts and 150 mg/kg for Ficus carica fruit extract.

Experimental design

In the present study animals were divided into six main groups with six rats in each group:

• Group 1: Control group left without any treatment.

• Group 2: Animals received a hepatotoxic compound (CCl₄), administered s.c. twice weekly as 40% solution in corn oil in a dose of 1 mL/kg bw, for eight weeks.

• Group 3: Animals intraperitoneally (i.p.) administered silymarin three times weekly in a dose of 50 mg/kg bw (dissolved in DMSO), 1 week before and along with CCl₄ (1 mL/kg bw, s.c., twice a week).

• Group 4: Animals i.p. administered Ficus carica leaf (methanol extract) three times weekly in a dose of 50 mg/kg bw (dissolved in a mixture of saline and DMSO), 1 week before and along with CCl₄ (1 mL/kg bw, s.c., twice a week).

• Group 5: Animals i.p. administered Morus alba root bark (methanol extract) three times weekly in a dose of 50 mg/kg bw (dissolved in a mixture of saline and DMSO), along with CCl₄ (1 mL/kg bw, s.c., twice a week).

• Group 6: Animals i.p. administered Ficus carica fruit (methanol extract) three times weekly in a dose of 150 mg/kg bw (dissolved in saline), along with CCl₄ (1 mL/kg bw, s.c., twice a week).

Groups 3-6 include rats subjected to different types of treatments a week before and along with administration of the hepatotoxic compound CCl₄ (1 mL/kg bw, subcutaneously twice a week).

Blood sampling and plasma preparation

The animals were fasted overnight prior to sample collection. Animals were anethetized with diethyl ether then their abdomen were cut open and blood was collected by heart puncture and liver was dissected. The animals were sacrificed all at once after eight weeks from the beginning of hepatotoxicity induction. Whole blood was collected by heart puncture after light anesthesia with diethyl ether. The collected blood in heparinized tubes, was divided into two parts; one used as whole blood for the determination of reduced glutathione content (GSH) and superoxide dismutase (SOD), the other part of the sample was centrifuged at 3,500 rpm for 15 min and the separated plasma was collected in ependorff tubes and stored in a freezer at −20°C to be used for the determination of catalase activity levels, lipid peroxidation concentration measured as malondialdehyde (MDA) end product, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) activity levels, beside total protein, total bilirubin, plasma urea, and creatinine.

Tissue sampling (liver) and histopathological examination of hepatocytes

After animals were anesthetized, rat liver was dissected, washed with distilled water, dried on filter paper, weighed and kept in 10% formalin, then dehydrated with ethanol solution from 50 to 100%, embedded in paraffin and cut into 5 µm sections and stained using hematoxylin-eosin dye for photomicroscopic observation of hepatic cells (Gray, 1964; Mandal et al., 1999, 2000).

Estimation of biochemical parameters

Glutathione content was determined according to the method of Tietze (1969), SOD activity was easily measured according to the method of Minami and Yoshikawa (1979), catalase was measured in plasma according to the colorimetric method of Sinha (1972) and lipid peroxidation was measured colorimetrically as described by Yoshioka et al. (1979), AST and ALT activity in plasma were determined by a colorimetric method as described by Reitman and Frankel (1957), ALP level was determined by a kinetic method according to the method of Henry (1964), serum total protein was determined according to the biuret method described by Henry et al. (1974), plasma total bilirubin was determined according to the method of Walters and Gerarde (1970), urea in plasma was determined by an enzymatic colorimetric method as described by Patton and Crouch (1977) and creatinine in plasma was determined kinetically without deproteinization by a colorimetric method as described by Henry (1974).

Statistical analysis

Statistical analysis was done using SPSS software version 10. The inter-group variation was measured by one-way analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) test. Results were expressed as mean ± SD (standard deviation). For all analysis, the level of statistical significance was set at P < 0.05.

Results

Biological study

Changes in blood glutathione content, superoxide dismutase (SOD) content, plasma catalase activity, and malondialdehyde (MDA) equivalent are presented in Table 1.

Table 1.  Effect of methanol extracts of Ficus carica (F.c.) leaves, fruits and Morus alba (M.a.) root bark on blood glutathione (GSH) content, activity levels of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) content in rats that received CCl₄.

Groups	GSH	SOD	CAT	MDA
	mg/dL	mg/dL	µmol/mL	µmol/L
Control	68.29 ± 1.52†‡	75.43 ± 2.37†	2.11 ± 0.1†	57.05 ± 2.07†
	100%	100%	100%	100%
CCl4	50.6 ± 2.04*‡	54.56 ± 2.74*‡	1.53 ± 0.12*‡	72.55 ± 2.1*‡
	74.09%	72.33%	72.38%	127.17%
Silymarin + CCl4	65.7 ± 2.28*†	73.22 ± 1.86†	2.14 ± 0.15†	57.77 ± 1.51†
	96.21%	97.07%	101.42%	101.27%
F.c. leaf + CCl4	83.31 ± 1.77*†‡	75.3 ± 2.52†	2.08 ± 0.13†	57.99 ± 1.49†
	122%	99.83%	98.58%	101.66%
M.a. root bark+ CCl4	64.98 ± 2.17*†	70.63 ± 1.36*†	1.93 ± 0.13*†‡	56.44 ± 2.94†
	95.16%	93.64%	91.47%	98.94%
F.c. fruit + CCl4	63.32 ± 2.37*†	73.66 ± 2.6†	2.06 ± 0.15†	56.77 ± 0.83†
	92.72%	97.65%	97.63%	99.52%

Values are given as mean ± SD from six rats in each group; CCl₄ group (receiving CCl₄ alone) is compared with control group and silymarin group (receiving silymarin along with CCl₄); experimental groups are compared with normal, CCl₄ and silymarin groups; values are statistically significant at P <0.05, ^*as compared with the control group; ^†as compared with CCl₄ group; ^‡as compared with silymarin group.

After induction of liver damage with CCl₄ in the experimental rats, blood glutathione content, SOD and catalase activities markedly decreased by 25.91, 27.67, and 27.62%, respectively, as compared to control, while MDA equivalent of CCl₄-treated groups markedly increased by 27.17% as compared to control.

When hepatotoxic rats were treated intraperitoneally with F. carica leaf extract at a dose of 50 mg/kg, or fruit extract at a dose of 150 mg/kg, or M. alba root bark extract at a dose of 50 mg/kg for eight weeks, significant increase in glutathione, SOD, and catalase activity were observed, while MDA equivalent was markedly decreased. In comparison with rats with hepatotoxicity receiving silymarin, as a standard hepatoprotective, at a dose of 50 mg/kg, a marked increase in the levels of glutathione, SOD, and catalase and a significant decrease in MDA equivalent parallel to that of the tested extracts were observed when compared to rats with CCl₄ alone.

Rats subjected to CCl₄ regimen alone developed significant hepatocellular damage as evident from a significant elevation in plasma alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin, and significant decrease in total protein by 28, 30.51, 26.79, 64.61, and 23.66%, respectively, as compared to control. Table 2 shows the effect of the different plant extracts on the above liver function parameters. Intraperitoneal administration of one of the tested extracts or silymarin reversed the hepatotoxicity significantly as evident from marked reduction in the CCl₄-induced increase in ALT, AST, ALP and total bilirubin levels, and amelioration in the tremendously decreased level of plasma total protein as a result of CCl₄ administration.

Table 2.  Effect of methanol extracts of Ficus carica (F.c.) leaves, fruits and Morus alba (M.a.) root bark on plasma ALT, AST, ALP activity levels, total protein and total bilirubin in rats that received CCl₄.

Groups	ALT	AST	ALP	Total protein	Total bilirubin
	U/L	U/L	U/L	g/dL	mg/dL
Control	25 ± 1.55†	47.51 ± 2.02†‡	65.67 ± 1.26†	6.95 ± 0.42†	0.3888 ± 0.03†‡
	100%	100%	100%	100%	100%
CCl4	32 ± 1.41*‡	62 ± 0.71*‡	83.27 ± 1.96*‡	5.3 ± 0.38 *‡	0.64 ± 0.05*‡
	128%	130.51%	126.79%	76.34%	164.61%
Silymarin + CCl4	24.76 ± 1.67†	53.95 ± 0.57*†	67.45 ± 1.65†	6.65 ± 0.44†	0.44 ± 0.04*†
	99.04%	113.56%	102.70%	95.75%	113.61%
F.c. leaf + CCl4	22.96 ± 0.89*†‡	45.5 ± 1.9*†‡	70.1 ± 2.97*†	6.98 ± 0.57†	0.39 ± 0.03†‡
	91.84%	95.77%	106.74%	100.42%	100.31%
M.a. root bark + CCl4	24.6 ± 1.6†	55.37 ± 0.91*†	76.36 ± 2.76*†‡	6.8 ± 0.47†	0.3883 ± 0.03†‡
	98.40%	116.54%	116.27%	97.93%	99.87%
F.c. fruit + CCl4	26.2 ± 1.56†	53.5 ± 1.3*†	69.95 ± 2.61*†	6.67 ± 0.44†	0.41 ± 0.04†
	104.80%	112.61%	106.51%	96.04%	105.45%

Values are given as mean ± SD from six rats in each group; CCl₄ group (receiving CCl₄ alone) is compared with control group and silymarin group (receiving silymarin along with CCl₄); experimental groups are compared with normal, CCl₄ and silymarin groups; values are statistically significant at P <0.05, ^*as compared with the control group; ^†as compared with CCl₄ group; ^‡as compared with silymarin group.

Kidney function tests are represented by measuring plasma urea and creatinine in rats as shown in Table 3 and revealed that the CCl₄ receiving group developed significant kidney damage as evident from a significant elevation in plasma urea and creatinine with a change of 28 and 35.77%, respectively, as compared to the control group. Intraperitoneal administration of F. carica leaf extract (50 mg/kg), or fruit extract (150 mg/kg), or M. alba root bark extract (50 mg/kg), reversed the kidney damage markedly and exhibited a significant reduction in the CCl₄-induced elevation of plasma urea and creatinine. The same results were obtained in the silymarin group.

Table 3.  Effect of methanol extracts of Ficus carica (F.c.) leaves, fruits and Morus alba (M.a.) root bark on plasma urea (UR) and creatinine (CR) contents in rats that received CCl₄.

Groups	Control	CCl4	Silymarin + CCl4	F.c. leaf + CCl4	M.a. root bark + CCl4	F.c. fruit + CCl4
UR	49.03 ± 2.68†	61.04 ± 3.41*‡	51.27 ± 3.03†	54.55 ± 3.16*†	35.87 ± 2.14*†‡	51.88 ± 2.67†
mg/dL	100%	124.50%	104.57%	111.25%	73.16%	105.81%
CR	0.81 ± 0.08†	1.1 ± 0.09*‡	0.8 ± 0.07†	0.93 ± 0.08*†‡	0.78 ± 0.07†	0.89 ± 0.08†
mg/dL	100%	135.77%	98.77%	114.81%	96.30%	109.88%

Values are given as mean ± SD from six rats in each group; CCl₄ group (receiving CCl₄ alone) is compared with control group and silymarin group (receiving silymarin along with CCl₄); experimental groups are compared with normal, CCl₄ and silymarin groups; values are statistically significant at P < 0.05, ^*as compared with the control group; ^†as compared with CCl₄ group; ^‡as compared with silymarin group.

At the end of the eight weeks, mortality was observed in the CCl₄-treated group and autopsy showed congested and enlarged liver. However, no mortality was observed in either the control or F. carica or M. alba extract-treated groups.

Histopathological examination of liver sections of the control group showed normal cellular architecture with distinct hepatic cell sinusoidal spaces and central vein (Figure 1). Histopathological profiles of the liver from CCl₄-treated rats revealed disarrangement of normal hepatic cells with intense centrilobular necrosis, vacuolization of cytoplasm with distinct fibrosis (Figure 2). Liver sections of rats treated with silymarin at a dose of 50 mg/kg, i.p. showed significant signs of amelioration of CCl₄-evoked liver injury which was evident from the presence of normal hepatic pods and the absence of necrosis and steatosis (Figure 3). Administration of Ficus carica leaf extract to the experimental animals in a dose of 50 mg/kg, i.p. showed a significant improvement of the hepatocellular architecture over the CCl₄-treated groups, as evident from a considerable reduction in necrosis and fibrosis, also the arrangement of cells in normal architecture (Figure 4). Similar improvement occurred with M. alba root bark extract at a dose of 50 mg/kg i.p. (Figure 5) and F. carica fruit extract at a dose of 150 mg/kg i.p. (Figure 6).

Figure 1.  Microphotograph of liver sections taken from rats of the control group. H and E staining (× 400).

Figure 2.  Microphotograph of liver sections taken from rats of CCl₄ group, (1 mL/kg of 40% solution, s.c.). H and E staining (× 400).

Figure 3.  Microphotograph of liver sections taken from rats of silymarin treated group (50 mg/kg, i.p.). H and E staining (× 400).

Figure 4.  Microphotograph of liver sections taken from rats of F. carica leaves treated group (50 mg/kg, i.p.). H and E staining (× 400).

Figure 5.  Microphotograph of liver sections taken from rats of M. alba root bark treated group (50 mg/kg, i.p.). H and E staining (× 400).

Figure 6.  Microphotograph of liver sections taken from rats of F. carica fruits treated group (150 mg/kg, i.p.). H and E staining (× 400).

Phytochemical study

Ficus carica leaf extract (33.5 g) was fractionated over diaion HP-20 CC (100 L × 4.5 cm internal diameter (i.d.)), eluted with water (F-1), 50% MeOH (F-2), and finally with neat MeOH (F-3). Each fraction was concentrated under reduced pressure at a temperature not exceeding 50°C. F-2 (13.7 g) exhibited four spots on two-dimensional PC under short UV light (254 nm), so it was subjected to preparative paper chromatographic separation, (Whatman paper No.3 MM), using solvent composed of acetic acid:water (6:94). The chromatogram showed two blue bands and two dark purple bands. These bands were cut separately and immersed in methanol. The methanol solution of the separated bands was evaporated in vacuo at a temperature not exceeding 50°C to afford three pure compounds (umbelliferone, caffeic acid, and quercetin-3-O-β-d-glucopyranoside), the fourth dark purple band was then subjected to TLC (silica) chromatographic analysis, where it exhibited two dark purple spots which were isolated and purified using preparative silica TLC with solvent system CHCl₃:MeOH (8:2); this afforded two pure compounds (quercetin-3-O-α-l-rhamnopyranoside and kaempferol-3-O-α-l-rhamnopyranoside). Identification of compounds was done by comparison with literature data.

Discussion

Carbon tetrachloride produces experimental liver damage that histologically resembles viral hepatitis (Laflamme, 2000; Sreelatha et al., 2009). It is accumulated in hepatic parenchyma cells and metabolized to CCl₃ by liver cytochrome P₄₅₀-dependent monooxygenases (Recknagel,1983; Rao et al., 2006). This free radical then combined with cellular lipid and proteins in the presence of oxygen to form a trichloromethyl peroxyl radical, which may attack lipids on the membrane of endoplasmic reticulum, faster than trichloromethyl free radical. Thus trichloromethyl peroxyl free radical leads to lipid peroxidation, the destruction of Ca⁺² homeostasis, and finally results in cell death (Rao et al., 2006).

Hepatic fibrosis is a dynamic process caused by chronic liver injury due to various etiologies, eventually leading to cirrhosis. It is predominantly characterized by excessive accumulation of extracellular matrix caused by both an increased synthesis and decreased or unbalanced degradation of extracellular matrix, CCl₄ is one of the most widely used hepatic toxins for experimental induction of hepatic fibrosis and cirrhosis in laboratory animals (Jiang et al., 2004; Singab et al., 2005). CCl₄-induced fibrosis or cirrhosis in experimental animals resembles human cirrhosis in some aspects of morphology and pathophysiology. In both cases regeneration of hepatocytes occurs after necrosis, and fibrotic infiltration is almost irreversible in the advanced stage of cirrhosis (Beljaars et al., 2002). Thus, CCl₄-induced hepatic fibrosis has also been used to assess the efficacy of anti-fibrotic reagents and to verify correlation between pathophysiological features of the liver and serum marker of fibrosis (WU & Norton, 1996). The increase in the level of serum enzymes is an indication of cellular leakage and loss of functional integrity of the cell membrane in the liver (Meyer & Twedt, 2000). The preventive action of liver damage induced by CCl₄ has widely been used as a marker of the hepatoprotective activity of drugs in general (Wu & Norton, 1996; Noaman, 2007).

Hepatoprotective activity of the extracts was evaluated by their ability to lower the elevated levels of the serum enzymes resulting from CCl₄ administration. The significant reduction in enzyme levels towards respective normal values mediated by the tested extracts is an indication of stabilization of the hepatocyte cell membrane as well as repairing of hepatic tissue damage caused by CCl₄ (Noaman et al., 2006).

Antioxidative action plays an important role in protection against carbon tetrachloride-induced liver injury (Xiong et al., 1998).There is increasing evidence for the hepatoprotective role of hydroxyl- and polyhydroxy organic compounds, particularly from vegetables, fruits and herbs (Bass, 1999).The consumption of fruits and vegetables (Peschel et al., 2006) containing antioxidants has been found to offer protection against these diseases. In spite of the tremendous advances in modern medicine no effective drugs are available which stimulate liver functions and offer protection to the liver from damage or help to regenerate hepatic cells (Chattopadhyay, 2003).

Silymarin is one of the hepatoprotective natural compounds reported to possess many biological properties and perceived as natural remedies. Such compounds show protective effects against agent-induced hepatic injuries (Berger & Kowdley, 2003; Lieber et al., 2003; Hedayat et al., 2004). Other studies state that silymarin exhibited antioxidant action (Soto et al., 2003; Chlopcikova et al., 2004; Lieber, 2004) and showed promising antineoplastic and immunomodulatory effects (Amirghofran et al., 2000; Hannay & Yu, 2003; Schümann et al., 2003; Yoo et al., 2004). Silymarin has significant activation effects upon SOD and glutathione- related enzyme systems in rats (Soto et al., 2003; Hedayat et al., 2004). Silymarin enhanced the activities of glutathione, glutathione reductase and glutathione S-transferase, and inhibited lipid peroxidation in the liver of normal rats (Skottova et al., 2004).

Rats treated with M. alba root bark or F. carica fruit extracts showed similar antioxidant activity as those receiving silymarin, while rats receiving F. carica leaf extract, the was superior to silymarin and showed more potent antioxidant properties. It is clear that Ficus carica leaf extract increased blood glutathione level as compared to control. In this study, methanol extract of F. carica leaf exhibited great hepatoprotective activity. Therefore it was felt important to identify the constituents of this extract. Chromatographic investigation of the methanol extract of Ficus carica leaf resulted in isolation of different compounds namely; umbelliferone (a coumarin), caffeic acid, quercetin-3-O-β-d-glucopyranoside, quercetin-3-O-α-l-rhamnopyranoside, and kaempferol-3-O-α-l-rhamnopyranoside.

The hepatoprotective effect may be attributed to the presence of the isolated compounds, since coumarin compounds have hepatoprotective activity (Matsuda et al., 1998; Lee et al., 2002; Park et al., 2004), and caffeic acid has hepatoprotective activity (Janbaz et al., 2004). Flavonoids (plant phenolic pigment products) such as quercetin (a flavonol compound) may delay oxidant injury and cell death by scavenging oxygen radicals; protecting against lipid peroxidation and thereby terminating the chain–radical reaction, chelating metal ions, to form inert complexes that cannot take part in the conversion of superoxide radicals and hydrogen peroxide into hydroxyl radicals (Dechameux et al., 1992; Ibrahim et al., 2006).

The expected mechanism of the hepatoprotective effect of F. carica leaf, against CCl₄-induced liver damage, is the ability of its flavonol glycoside content to act as strong free radical scavengers intercepting those radicals involved in CCl₄ metabolism by microsomial enzymes. Consequently, by trapping oxygen-related free radicals, the flavonol glycosides of F. carica leaf could hinder the free radical interaction with polyunsaturated fatty acids and would abolish the enhancement of the lipid peroxidative process leading to hepatic cell damage. The present results agree with previously reported data (Hewawasam et al., 2004; Janbaz et al., 2004; Wang et al., 2004; Orhan et al., 2007). In order to verify this finding the antioxidant activity of F. carica leaf was determined. The significant amelioration in the antioxidant parameters might result in the hepatoprotective action of flavonol glycosides against oxidative stress induced by CCl₄. Consequently, the flavonol glycosides content of F. carica leaf are responsible for the abolishment of CCl₄-induced hepatic damage through their strong antioxidant activity. Van Acker and his colleagues discussed the structural aspects of the antioxidant activity of flavonoids. Quercetin appears to be an extremely efficient radical scavenger. The strong inhibitory effect of quercetin was thought to be attributed to its additional phenolic group (3-OH). Also, kaempferol is a very good scavenger, even though it only has one hydroxyl group on the B ring (4′-OH), possibly due to the combination of other characteristics such as, a C2-C3 double bond, 3-OH group, and 4-keto group on ring C (Van Acker et al., 1996, 1998). The mechanism of antiradical action of quercetin and its glycoside was evaluated by Afanas’ev et al. (1989) and Albano et al. (1982) who referred the inhibitory effects of both quercetin and rutin to their ability to ameliorated the damage occurred by CCl₄-dependent lipid peroxidation in rat liver microsomes. Therefore, the data reported herein reveal a protective potential of quercetin and kaempferol glycosides, the main constituents of F. carica leaf, against the acute hepatotoxicity induced by CCl₄ in rats.

Worthy of mentioning, is that both F. carica (leaf and fruit) and M. alba (root bark) extracts were kidney protective against the damage induced by CCl₄, and ameliorated the increase in plasma urea and creatinine levels caused by CCl₄. The marked decrease in plasma urea content, (26.84% less than the control group), caused by Morus alba root bark extract may be attributed to the diuretic effect of M. alba root bark (Singab et al., 2006), and since urea is excreted in urine (Refsum & Stromme, 1974), subsequently a diuretic will decrease urea level in blood.

Conclusion

The results obtained revealed that methanol extract of Ficus carica leaf had the most potent antioxidant activity; these antioxidant properties are attributable to the ability of its phenolic constituents to quench reactive oxygen species. Also, the present study confirmed the kidney and liver protective action of both Ficus carica (leaf and fruit) and Morus alba (root bark) extracts against CCl₄-induced damage in rats, which was comparable to that of a standard hepatoprotective drug, silymarin. The hepatoprotective effect is documented by the biochemical and histopathological data obtained. It may be speculated that the constituents of Ficus carica leaf (quercetin, kaempferol glycosides, caffeic acid, and coumarins such as umbelliferone) are to be responsible, at least in part, for the observed hepatoprotective effect. A possible mechanism of Morus alba extract as hepatoprotective may be due to its antioxidant effect which impairs the activation of carbon tetrachloride into the reactive form, it may be speculated that the high flavonoid content of Morus alba root bark, was responsible for the observed protective effects. Further studies need to be done on Ficus carica fruit extract to identify the active constituent(s) responsible for the hepatoprotective activity and elucidate the mechanism of action.

Acknowledgements

The authors are grateful to the National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA) for hosting the biological studies and providing the necessary facilities.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.