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ORIGINAL ARTICLE
Chemical constituents and protective effect of Ficus ingens (Miq.) Miq. on carbon tetrachloride-induced acute liver damage in male Wistar albino rats
Abd El Raheim M. Donia a,b *, Gamal A. Soliman c, Ahmed M. Zaghloul a,d, Saleh I. Alqasoumi e, Amani S. Awaad f, Asmaa M. Radwan g, Omer A. Basodan
a Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj, Saudi Arabia b Medicinal and Aromatic Plants Department, Desert Research Center, Cairo, Egypt
c Pharmacology Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj, Saudi Arabia d Pharmacognosy Department, College of Pharmacy, Mansoura University, Egypt e Pharmacognosy Department, College of Pharmacy, King Saud University, Saudi Arabia f Chemistry Department, College of Science, King Saud University, Saudi Arabia
8 Botany and Microbiology Department, College of Science, Girls Branch, Al Azhar University, Cairo, Egypt
Received 30 April 2012; accepted 29 May 2012 Available online 7 June 2012
KEYWORDS
Phytochemical studies; Flavonoids; Antharquinone; Hepatoprotective effect
Abstract The aim of the present study was to investigate the chemical constituents and hepatoprotective effect of Ficus ingens (Miq.) Miq. (Moraceae) extract against carbon tetrachloride-induced acute liver damage in male Wistar albino rats. The ethanol extract of F. ingens, was subjected to phytochemical study. In addition, its acute and sub-chronic toxicities were assessed. Eight compounds were isolated from this plant and identified as b-sitosterol, b-sitosterol glucoside, chrya-sophanol, 7-hydroxy-2,5 dimethyl chromen-4-one, quercetin, Aloe emodin glucoside, rutin and Patuletin-3'-O-methyl-3-O-rutinoside. The structure elucidation was based on and 13C NMR, proton-proton correlation spectroscopy (1H-1H Cosy), distortionless enhancement by polarization transfer (DEPT), Heteronuclear Multiple-Quantum Correlation (HMQC), and heteronuclear
Corresponding author at: Pharmacognosy Department, College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj, Saudi Arabia. Tel.: +966 560019012. E-mail address: donia22276@yahoo.com (A.M. Donia).
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Peer review under responsibility of King Saud University. http://dx.doi.org/10.1016/j.jscs.2012.05.008
multiple bond correlations spectrum (HMBC). Hepatotoxicity induced with CCl4 was evidenced by elevation of liver marker enzymes (ALT, AST, ALP and LDH) and TB content in serum. In addition, antioxidant enzymes were drastically inhibited with significant reduction of GSH and increased LPO in liver homogenate of CCl4-intoxicated rats. Pre-treatment with F. ingens (200 and 400 mg/kg) and silymarin (50 mg/kg) avoided the changes observed in CCl4-intoxicated rats. In conclusion, the ethanol extract of F. ingens showed protective activity against liver injury, which might be developed into a new hepatoprotective agent.
© 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
Liver is a vital organ that has a wide range of functions, including detoxification, plasma protein synthesis, and production of biochemicals necessary for digestion. Damage to the liver inflicted by hepatotoxic agents is of grave consequence. Today, liver damage is one of very common aliment in the world resulting in serious debilities ranging from severe metabolic disorders to even mortality (Akilavalli et al., 2011). Various xenobiotics are known to cause hepatotoxicity; one among them is CCl4 that may cause lipid peroxidation (Kodavanti et al., 1989; Demirdag et al., 2004). Many hepatoprotective herbal preparations have been recommended in alternative medicine for the treatment of liver diseases. Therefore, the search of a new natural hepatoprotective agent is of great interest.
Ficus is a genus belonging to family Moraceae, it comprises about 850 species of woody trees, shrubs, vines, epiphytes, and hemiepiphyte. F. ingens is an evergreen deciduous tree up to 10 m height, occasionally higher, with a rounded or spreading crown and with a spread of up to 30 m wide. The plant grows in various habitats including Saudi Arabia. All the parts have milky latex when broken. Fruits are found on the tree usually throughout the year but peaking in summer (Myburgh et al., 1994).
Many active compounds were isolated from Ficus benghalen-sis bark; 20-tetratriaconthene-2-one, 6 heptatriacontene-10-one, pentatriacontan-5-one, b-sitosterol, b-D-glucoside and meso inositol (Mousa et al., 1994). In addition, the fruit extract of F. benghalensis exhibited antitumor activity (Joy etal., 2001), while the methanol extract of F. benghalensis possesses antioxidant activity (Yadav et al., 2011). Ficus sycomorus extracts are used in Folk medicine in the treatment of infertility and sterility in humans (Malgras, 1992; Pakia et al., 2003; Kone and Atinde-hou, 2008). Ficus capensis extract was used for treatment of azoospermia (Gelfand et al., 1985). Ficus asperifolia extract has been reported to have an estrogenic effect in female rats (Watcho et al., 2009).
2. Materials and methods
2.1. Plant material
The aerial parts of Ficus ingens (Miq.) Miq. were freshly collected from Tabouk area-KSA, during Summer 2010. The collected plant was kindly authenticated by Prof. Dr. Abd El Naser El-Gifri, Prof. of Taxonomy, Salman bin AbdulAziz University, KSA. A voucher specimen has been deposited in the herbarium of the Pharmacognosy department, Salman bin AbdulAziz University. The plant was dried under shade and then ground to fine powder.
2.2. Extraction
One kg of the dried powder was extracted by percolation in 70% aqueous ethanol for 72 h. The combined ethanol extracts were concentrated under reduced pressure at a temperature not exceeding 45 0C. The extract was fractionated using silica gel column chromatography (350 g) and gradiently eluted with chloroform containing increasing proportions of methanol. Fractions (85, 100 mL each) were collected and monitored by TLC (silica gel, chloroform-methanol). Similar fractions were combined together to obtain 5 groups. Each group was reapplied to silica gel column eluted with chloroform containing gradually increasing proportions of methanol. Further purification was carried out using Sephadex LH-20 columns to afford compounds 1-8.
2.3. Acid hydrolysis
Two mg of compounds 2, 6, 7 and 8 was dissolved in 2 mL of methanol: water (1:1, v/v), mixed with 1 mL of 2 N HCl, and refluxed at 60 oC for 3 h. The aglycone moiety was subsequently extracted with ethyl acetate. The aqueous phase was neutralized with silver oxide then filtered. The filtrate was used for identification of the sugar moiety (Stahl, 1969).
2.4. Apparatus
Proton (1H) and carbon 13 (13C NMR) spectra were recorded on Bruker VX500 NMR spectrometer operating at 500 and 125 MHz respectively. *H-13C correlations were established by using HMQC and HMBC pulse sequences respectively. 1H-1H correlations were determined by double quantum filtered COSY.
2.5. Experimental animals
Male Wistar albino rats (160-180 g) and albino mice of both sexes (27-30 g) were maintained in the Laboratory Animal Unit of the College of Pharmacy, Salman Bin Abdulaziz University. They were housed in polypropylene cages and fed with standard chow diet and water ad libitum. The animals were exposed to alternate cycle of 12 h of darkness and light. Male rats were used because of their constant metabolism compared to the variation in the female physiology. Animals were allowed to adapt to the laboratory environment for one week before experimentation. The care and handling of the animals were in accordance with the internationally accepted standard guidelines and were approved by an institutional review board.
2.6. Preparation of the extract for biological studies
The concentrated ethanol extract of F. ingens was suspended in 3% v/v Tween 80 in distilled water (vehicle).
2.7. Acute toxicity experiment
Albino mice were divided into control and test groups (6 animals each). Control group received the vehicle (3% Tween 80) while the test groups got graded doses (1000-4000 mg/kg) of F. ingens ethanol extract orally and were observed for mortality till 48 h and the LD50 was calculated (Ghosh, 1994).
2.8. Doses
The dose selection for the ethanol extract of F. ingens was based on the acute toxicity study, which did not show any adverse effect following oral administration of doses up to 4000 mg/kg. Accordingly, experimental oral doses of 100, 200 and 400 mg/ kg that equal to one-fortieth, one-twentieth and one-tenth of the maximum possible dose of the extract that did not cause mortalities in mice were selected.
2.9. Sub-chronic toxicity
Twenty-four male Wistar albino rats were randomly divided into 4 groups of 6 animals. The 1st group was kept as control (5 mL/kg of 3% Tween 80), while 2nd, 3rd and 4th groups were administered the ethanol extract of F. ingens in doses of 100, 200 and 400 mg/kg, respectively. All medications were administered orally with the aid of an orogastric cannula for 35 consecutive days. Rats were maintained under identical conditions with food and water ad libitum for the entire period with close observation. At the end of the experimental period, blood samples (2 mL) were drawn by puncturing retro-orbital venous sinus of each rat (under ether anesthesia) and centri-fuged at 10,000 rpm for 5 min. Sera were separated to be used for the biochemical estimations.
2.10. Measurement of liver and kidney function markers
Liver functions were evaluated by measuring the serum activity of ALT and AST following the method of Reitman andFrankel (1957) while the activities of ALP and LDH were estimated by the methods of Babson et al. (1966) and King (1965), respectively. The serum concentrations of TB (Walter and Gerarde, 1970), TP (Henary et al., 1974) and Alb (Doumas et al., 1971) were estimated. Serum levels of urea (Wills and Savory, 1981) and creatinine (Kroll et al., 1987) were determined colorimetri-cally as measures of kidney functions.
2.11. Experimental induction of hepatic damage
CCl4 was dissolved in corn oil in the ratio 1:1 v/v. Liver damage was induced in rats following subcutaneous (SC) injection of CCl4 in the lower abdomen at a dose of 3 mL/kg (Theophile et al., 2006).
2.12. Hepatoprotective activity
Thirty-six adult male Wistar albino rats were randomly divided into six groups of six animals, each. Rats of the 1st (nor-
mal control) and 2nd (CCl4-intoxicated control) groups received the vehicle in a dose of 5 mL/kg. Animals of the 3rd group (reference) received silymarin at a dose of 50 mg/kg. The 4th, 5th and 6th groups were treated with the ethanol extract of F. ingens in doses of 100, 200 and 400 mg/kg, respectively. All medications were administered orally by gastric intubation for 7 consecutive days. Two h after the last dose, normal control rats were given a single dose of corn oil (3 mL/kg, SC), while animals of the 2nd to 6th groups received a single dose of CCl4 (3 mL/kg, SC).
After 24 h of corn oil and CCl4 injections, blood sample from each rat (2 mL) was withdrawn by puncturing their retro-orbital plexus of veins and collected in previously labeled centrifuging tubes and allowed to clot for 30 min at room temperature. Serum was separated by centrifugation at 10,000 rpm for 5 min. Livers were dissected out and divided into two parts. One part was kept in liquid nitrogen for determination of antioxidant status and the other part was immediately fixed in buffered formalin 10% and was used for histopathological examination.
2.13. Assessment of liver biochemical markers
The levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total bilirubin (TB), total protein (TP) and albumin (Alb) were determined in serum. glutathione (GSH) and lipid peroxidation (LPO). Hematoxylin and eosin (H&E) staining assays deployed to evaluate liver damage were estimated in serum of rats.
2.14. Assessment of CCl4 mediated oxidative stress
Liver tissue was homogenized in 10 volume of 100 mM KH2PO4 buffer containing 1 mM EDTA (pH 7.4) and centri-fuged at 12,000 rpm for 30 min at 4 0C. The activities of the antioxidant enzymes such as superoxide dismutase (SOD), glu-tathione peroxidase (GPx), catalase (CAT), were assayed in the hepatic tissue homogenate of the control and experimental rats according to the methods of Sun and Zigman(1978), Mohandas etal. (1984) and Chanceand Maehley (1995), respectively. Moreover, the extent of lipid peroxidation (LPO) was estimated in liver homogenate of all rats as the concentration of thiobarbituric acid reactive product (malondialdhyde - MDA) according to (Ohkawa et al., 1979), while GSH tissue content was measured according to the method described by Moron et al. (1979).
2.15. Histopathological study
Liver from each rat was removed after dissection and preserved in 10% formalin. Then representative blocks of liver tissues from each lobe were taken and possessed for paraffin embedding using the standard microtechnique (Galighor and Kozloff, 1971). Sections (5 im) of livers stained with hemot-oxylin and eosin were observed microscopically for the evaluation of histopathological changes.
4. Statistical analysis
The values are expressed as mean ± standard error of six observations in each group. All groups were subjected to one-way analysis of variance (ANOVA), which was followed
by Bonferoni's test to determine the intergroup variability by using SPSS ver. 14.0. A comparison was made with the experimental control (CCl4-intoxicated) group and with the standard (silymarin). We took a P-value of <0.05 as our desired level of significance.
5. Results
5.1. Isolated compounds
Compound (3): colorless needles, 1H NMR (500 MHz, DMSO-d6) d: 2.33 (3H,s, CH3-11), 2.70 (3H, s, CH3-12), 6.04 (1H, s, H-3), 6.67 (1H, d, J = 0.9 Hz, H-6), 6.69 (1H, d, J = 0.9 Hz, H-8) 10.65 (1H, s, OH); 13C NMR (DMSO-d6): 178.26 (C-4), 163.83 (C-2), 160.88 (C-7), 159.09 (C-9), 141.40 (C-5), 116.46 (C-6), 114.0 (C-10), 110.64 (C-3),), 100.47 (C-8), 19.3 (C-11).
Compound (4): obtained as an orange crystal; (6 mg) *H NMR (DMSO, 500 MHz); 8 (ppm) 12.04 (1H, s, OH-8), 11.97 (1H, s, OH-1), 7.80 (1H, dd, J = 7.2, 2.5 Hz, H-5), 7.73 (1H, dd, J = 7.2, 7.5 Hz, H-6), 7.56 (1H, d, 2.2 Hz, H-4), 7.40 (1H, d, J = 8.4 Hz, H-7), 7.23 (1H, s, H-2), 2.44 (3H, s, CH3). 13C NMR: 8 (ppm); 192.42 (C-9), 181.85 (C-10), 162.65 (C-1), 162.36 (C-8), 149.32 (C-3), 136.91 (C-6), 133.56 (C-12), 133.19 (C-14), 124.51 124.32 (C-2), 121.31 (C-4), 119.89 (C-5), (C-7), 115.80 (C-13), 113.66 (C-11), 22.24 (CH3).
Compound (5): yellow needles, *H NMR using CD3OD: 8 7.75 (1H, d, J = 2.5 Hz, H-2'), 7.66 (1H, dd, J = 8.5, 2.5 Hz, H-6'), 6.90 (1H, d, J = 8.5 Hz, H-5'), 6.40 (1H, d, J = 2.5 Hz, H-6) and 6.19 (1H, d, J = 2.5 Hz, H-8). 13C NMR (CD3OD): d ppm 177.32 (c-4), 165.63 (C-7), 162.52 (C-5), 158.22 (C-2),
149.1 (C-9), 148.77 (C-4'), 146.23 (C-3'), 137.26 (C-3), 124.1 (C-6'), 121.66 (C-1'), 116.21 (C-2'), 115.96 (C-5'), 104.5 (C-10), 99.23 and 94.40 (C-6 and C-8, respectively).
Compound (6): yellow powder, *H NMR (500 MHz, dimethyl sulphoxide (dMSO, d6) d: 6.78 (1H, d, J = 2.3 Hz, H-2), 7.19 (1H, d, J = 2.3 Hz, H-4), 7.28 (1H, dd, J = 7.2, 2.5 Hz, H-5), 7.46 (1H, dd, J = 7.5, 8.1 Hz H-6), 7.02 (1H, dd, J = 7.4, 2.5 Hz, H-7), 4.85 (2H, s, CH2), 5.0 (1H, s, OH-1), 5.16 (1H, s, H-1), 3.47 (2H, s, H-2), 3.36 (1H, s, H-3), 3.35 (1H, s, H-4), 3.47 (1H, s, H-5)' 3.45 (2H, s, H-6), 2.0 (4H, s, 4 glu-OH). 13C NMR (125 MHz, DMSOd6) d: 190.1 (c-9), 186.1 (C-10), 162.1 (C-8), 160.8 (C-1), 148.7 (C-3),
145.2 (C-12), 132.5 (C-6), 130.0 (C-13), 123.1 (C-11), 115.6 (C-5), 115.4 (C-4), 102.4 (C-7), 110.2 (C-14), 101.2 (glu-C-1), 100.1 (C-2), 68.8 (C-CH2OH), 67.5 (glu-C-2), 74.1 (glu-C-3), 69.2 (glu-C-4), 75.9 (glu-C-5), 65.2 (glu-C-6).
Compound (7): yellow crystals, UV kmax in MeOH: (nm) 256, 290, 355. (AlCl3): 274, 432. (ALCl3/HCl): 270, 298, 359, 399. (NaOAc): 272, 324, 398, (NaOAc/H3BO3): 263, 292, (NaOMe): 272, 310, 410. *H NMR (DMSO-d6): d 7.71 (1H,
d, J = 2 Hz, H-2'), 7.64 (1H, dd, J =8, 2.1 Hz, H-6'), 6.87 (1H, d, J =8 Hz, H-5'), 6.38 (1H, d, J = 2 Hz, H-8), 6.20 (lH, d, J = 2 Hz, H-6), 5.12 (1H, d, J = 7.50 Hz, H-1''), 4.54 (1H, d, J = 1.3 Hz, H-1'''), 3.82 (1H dd, J = 8.4, 2 Hz, H-6''), 3.62 (1H, dd, J = 3.5 Hz, H-2'''), d 3.47-3.87 (6H m for sugar protons) and d 1.25 (3H d J = 6 Hz, CH3). 13C NMR (DMSO-d6): d ppm 179.34 (C-4) 166. 07 (C-7) 162.90 (C-5) 159.31 (C-2) 149.82 (C-9) 148.1 (C-4') 144.8 (C-3'), 135.65 (C-3), 123.62 (C-6'), 123.07 (C-1'), 117.73 (C-2')
116.07 (C-5'), 104.80 (C-10), 100.0 and 94.93 (C-6 and C-8, respectively), 104.8 (C-1), 75.73 (C-2), 78.15 (C-3), 72 (C-4), 77.2 (C-5), 61.3 (C-6), 102.43 (C-1), 72.09 (C-2), 71.36(C-3), 72.22 (C-4), 69.72 (C-5), 17.93 (C-6).
Compound (8): 1H NMR (MeOD), 8 7.89 (1H, dd, J = 8.5, 2.5 Hz, H-2'), 7.62 (1H, dd, J = 8.5, 2.5 Hz, H-6'), 6.91 (1H, d, J = 8.5 Hz, H-5'), 6.47 (1H, s, H-8). 5.21 (1H, d, J = 7 Hz, H1''), 4.52 (1H, d, J = 2, H1'''), 3.95-3.87 sugar protons, 3.28-3.36 (m, remaining sugar protons) and 1.1 (3H, d, J = 6, CH3 rhamnose. 13C NMR (MeOD): 179.2 (C-4), 158.6 (C-7),
150.8 (C-5), 149.2 (C-2), 148.3 (C-9), 147.3 (C-4'), 135.0 (C-3'), 132.1 (C-3), 123.9 (C-6'), 123.0 (C-1''), 116.1 (C-2'), 114.4 (C-5'), 104.5 (C-10), 102.4 (C-1'''), 101.9 (C-1'''') 98.8 (C-6), 93.7 (C-8), 60.8 and 56.3 (two-OCH3 groups), The remaining sugar carbons appeared at 63.6-76.9, 18.24 C-6''.
5.2. Acute toxicity experiment
All mice treated with different doses (1000-4000 mg/kg) of F. ingens extract survived during the 48 h of observation. The animals did not show visible signs of acute toxicity.
5.3. Sub-chronic toxicity experiment
In the current study, no significant changes were detected in the biochemical parameters of rats after 35 days of treatment with different doses of F. ingens extract. Oral adminstration of the tested extract in doses of 100, 200 and 400 mg/kg to rats for 35 days did not show any significant effect on the levels of ALT, AST, ALP, LDH (Table 1) as well as the mean values of TB, TP, Alb, urea and creatinine (Table 2) in their sera as compared to control animals.
5.4. Hepatoprotective activity
SC injection of CCl4 to rats showed significant elevation of liver marker enzymes (ALT, AST, ALP and LDH) in their serum after 24 h of intoxication. The level of TB in the serum of CCl4-intoxicated control was also significantly increased when compared to the normal control group. Administration of silymarin (50 mg/kg) and the ethanol extract of F. ingens (200 and 400 mg/kg) once daily for 7 days prior to CCl4, exhibited a significant hepatoprotective activity, resulting in reduc-
Table 1 Effect of prolonged oral administration of ethanol extract of F. ingens for 35 days enzymes in rats, (n = 6). on the serum activity of liver marker
Groups Dose (mg/kg) ALT (U/L) AST (U/L) ALP (U/L) LDH (U/L)
Control Ficus ingens 00 100 200 400 61.96 ± 3.22 63.08 ± 2.74 66.36 ± 3.16 67.22 ± 3.50 134.91 ± 5.77 135.87 ± 5.36 137.56 ± 6.15 138.62 ± 6.24 73.52 ± 4.22 71.45 ± 4.47 77.10 ± 4.50 75.78 ± 4.63 55.74 ± 2.45 60.15 ± 2.11 58.22 ± 2.68 61.20 ± 2.40
Table 2 Effect of prolonged oral administration of ethanol extract of F. ingens for 35 days on the serum levels of TB, TP, Alb, urea and creatinine in rats, (n = 6).
Groups Dose (mg/kg) TB (mg/dL) TP (g/dL) Alb (g/dL) Urea (mg/dL) Creatinine (mg/dL)
Control Ficus ingens 00 1.42 ± 0.08 100 1.45 ± 0.09 200 1.45 ± 0.07 400 1.48 ± 0.06 8.12 ± 0.23 8.15 ± 0.27 8.22 ± 0.20 8.20 ± 0.26 3.70 ± 0.16 3.72 ± 0.14 3.79 ± 0.15 3.80 ± 0.10 37.34 ± 1.63 38.58 ± 1.17 38.70 ± 1.20 40.58 ± 1.43 0.34 ± 0.03 0.36 ± 0.02 0.38 ± 0.03 0.38 ± 0.02
Table 3 Effect of the ethanol extract of F. ingens on the hepatotoxicity. serum activity of liver marker enzymes in rats with CCl4 induced -
Croups ALT (U/L) AST (U/L) ALP (U/L) LDH (U/L)
Normal control CCl4-intoxicated control Silymarin (50 mg/kg) + CCl4 F. ingens (100 mg/kg) + CCl4 F. ingens (200 mg/kg) + CCl4 F. ingens (400 mg/kg) + CCl4 68.0 ± 2.11** 378.6 ± 12.95* 154.0 ± 5.07* 355.6 ± 16.19* 284.1 ± 10.43** 200.1 ± 7.50** 140.5 ± 4.18** 436.3 ± 14.91* 264.5 ± 9.40* 418.3 ± 11.61* 363.8 ± 10.57** 318.0 ± 12.88** 72.6 ± 2.65** 131.8 ± 5.43* 79.0 ± 3.45* 123.1 ± 5.48* 95.1 ± 5.88** 84.5 ± 5.01* 52.5 ± 2.44** 142.3 ± 5.78* 68.0 ± 3.27* 130.6 ± 5.22* 94.1 ± 4.79** 76.5 ± 4.95*
The results are expressed as mean ± S.E.M., n = 6 rats/group. indicate significance compared to CCl4 group (p < 0.05). * indicate significance compared to silymarin group (p < 0.05).
Table 4 Effect of the ethanol extract of F. ingens on the serum levels of TB, TP and Alb in rats with CCl4 induced - - hepatotoxicity.
Croups TB (mg/dL) TP (g/dL) Alb (g/dL)
Normal control CCl4-intoxicated control Silymarin (50 mg/kg) + CCl4 F. ingens (100 mg/kg) + CCl4 F. ingens (200 mg/kg) + CCl4 F. ingens (400 mg/kg) + CCl4 1.24 ± 0.06** 3.44 ± 0.13* 1.79 ± 0.12* 3.22 ± 0.17* 2.48 ± 0.13** 2.13 ± 0.13* 8.47 ± 0.39** 5.16 ± 0.25* 7.71 ± 0.21* 5.80 ± 0.23* 6.70 ± 0.42** 7.56 ± 0.34* 3.65 ± 0.18** 2.21 ± 0.16* 3.48 ± 0.15** 2.41 ± 0.15* 2.95 ± 0.11** 3.21 ± 0.17*
The results are expressed as mean ± S.E.M., n = 6 rats/group. indicate significance compared to CCl4 group (p < 0.05), * indicate significance compared to silymarin group (p < 0.05).
tion in the elevated serum activities of liver marker enzymes (Table 3) and level of TB (Table 4) when compared to CCl4-intoxicated rats. Furthermore, pretreatment with the ethanol extract of F. ingens in a dose of 100 mg/kg, did not elicit any significant effect.
The oxidative stress caused by CCl4 in the liver was assessed by measuring the activity of hepatic antioxidant defense enzymes (SOD, GPx, and CAT), GSH and the level of lipid peroxidation product (MDA). Results presented in Table 5 showed that SC injection of CCl4-induced significant reduction in the activities of SOD, GPx and CAT enzymes with a decreased level of GSH content as compared to the normal control group. On the other hand, it increased the MDA level in liver tissues. Pre-administration of silymarin (50 mg/kg) and F. ingens (200 and 400 mg/kg) reduced the severity of CCl4 toxicity, as evident from the non-significant differences observed in the oxidative stress indicators and antioxidant enzyme levels in these groups.
Histopathological examination of the liver sections from normal rats showed hepatocytes with normal parenchymal architecture (Fig. 2A). Liver sections of CCl4-intoxicated animals exhibited diffuse central and peripheral necrosis and destruction of the lobular architecture (Fig. 2B). Liver sections of rats treated with the ethanol extract of F. ingens in a dose
of 400 mg/kg showed normal hepatic cords (Fig. 2C) and absence of severe congestion and pyknosis indicating pronounced protection of hepatocytes against CCl4-induced hepatic damage.
6. Discussion
Preliminary phytochemical study indicates the presence of flavonoids, coumarins, steroids and anthraquinone in the extract of F. ingens.
6.1. Isolated compounds
Eight compounds were isolated from Ficus ingens. Compounds were identified examining their *H NMR, 13C NMR and as well comparison with the published data (Anand et al., 2010). Acid hydrolysis and TLC of the sugar part (ethyl ace-tate-methanol-acetic acid-water (65:15:10:10) revealed that compounds 2 and 6 contain glucose, compounds 7 and 8 contain glucose and rhamnose.
Compounds 1 and 2 were identified as b-sitosterol and b-sitosterol glucoside by comparing their data with pervious published data (Maridass & Ramesh, 2010).
Table 5 Effect of the ethanol extract of F. ingens on hepatic antioxidant profile, glutathione (GSH) and lipid peroxidation (MDA) in
liver homogenate of rats with CCl4 induced - hepatotoxicity.
Groups SOD (U/mg protein) GPx (U/mg protein) CAT (U/mg protein) GSH (umol/g tissue) MDA (nmol/g tissue)
Normal Control 41.5 ± 1.87** 2.71 ± 0.10** 12.4 ± 0.63** 9.73 ± 0.28** 41.1 ± 1.88**
CCl4-intoxicated Control 26.1 ± 1.88* 1.13 ± 0.16* 7.7 ± 0.69* 5.50 ± 0.19* 123.6 ± 5.31*
Silymarin (50 mg/kg) + CCl4 39.8 ± 1.83* 2.28 ± 0.11* 11.6 ± 0.66* 8.68 ± 0.18* 54.6 ± 2.33*
F. ingens (100 mg/kg) + CCl4 30.3 ± 1.30* 1.38 ± 0.10* 9.3 ± 0.61* 6.06 ± 0.26* 114.1 ± 5.56
F. ingens (200 mg/kg) + CCl4 34.8 ± 1.40** 1.81 ± 0.04** 10.1 ± 0.47 7.40 ± 0.20** 85.6 ± 3.56*
F. ingens (400 mg/kg) + CCl4 38.8 ± 1.75* 2.00 ± 0.15* 10.3 ± 0.49* 8.00 ± 0.44* 67.3 ± 3.49*
The results are expressed as mean ± S.E.M., n = 6 rats/group.
indicate significance compared to CCl4 group (p < 0.05).
* indicate significance compared to silymarin group (p < 0.05).
Compound 3 from the given data of 1H NMR, 13C NMR, COSY, HMQC and HMBC (Fig. 1) this compound is identified as 7-hydroxy-2,5-dimethyl-chromen-4-one, also the spectral data were in agreement with the literature reported (Kimura et al., 1992; Gu, 2009 &Konigs et al., 2010).
Compound 4 is identified as chrysophanol as it gave positive test for anthraquinones (Harborne, 1993; Sofowora, 1993 and Trease and Evans, 2002). Its *H NMR spectrum displayed two sharp singlets at 8 11.97 and 12.04 ppm, assigned for two che-lated hydroxyl groups, five signals assigned for aromatic protons at 8 7.23 (1H, s, H-2), 8 7.56 (1H, s, H-4), 8 7.40 (1H, d, J = 8.4 Hz, H-7), 8 7.80 (1H, d, J = 7.5 Hz, H-5), 8 7.73 (1H, m, H-6). Besides, the methyl singlet at 8 2.44 ppm was assigned to the methyl group at C-3. 13C NMR spectrum and DEPT experiment data are almost identical with the reported data for chrysophanol (Rani et al., 2010; Sosa et al., 2006).
Compound 5 is identified as quercetin through comparison of its 1H NMR and 13C NMR spectral data with those reported for quercetin (Mabry et al., 1970).
Compound 6 based on the characterization, the isolated compound was Aloe emodin glucoside (Anand et al., 2010).
Compound 7 is identified as rutin. It gave positive test for flavonol glycosides. m.p. 190°C. Its UV, *H NMR and 13C NMR spectral data were identical with those reported for rutin (Geissman, 1962 and Mabry et al., 1970 & Harborne et al., 1975).
Compound 8 was identified as Patuletin-3'-O-methyl-3-O-rutinoside (5,7,3' trihydroxyl-6,3'-dimethoxy flavone) by com-pairing data with that published by Lin et al. (2002).
In the current study, oral administration of F. ingens extract in doses up to 4000 mg/kg did not produce any symptom of acute toxicity and none of mice died during 48 h of observation. Accordingly, it suggested that oral LD50 of the tested extract was higher than 4000 mg/kg b.wt. Therefore, F. ingens plant can be categorized as highly safe since substances possessing LD50 higher than 50 mg/kg are non toxic (Buck et al., 1976).
The non-toxic nature of F. ingens extract in acute toxicity study in mice is well supported by the normal levels of ALT, AST, ALP, LDH, TB, TP and Alb following 35-days treatment period in rats. Urea and creatinine are the most sensitive biochemical markers employed in the diagnosis of renal damage. In kidney damage, there will be retention of urea and creatinine in the blood (Nwanjo et al., 2005), therefore marked increases in serum urea and creatinine are indications of functional damage to the kidney (Panda, 1999). By these indicators, ethanol extract of F. ingens is therefore, not nephrotoxic in rats.
6.2. Hepatoprotective activity
CCl4 is a well-known hepatotoxic agent. It has been used as a tool induced hepatotoxicity in experimental animals (Okuno et al., 1986). Acute exposure to CCl4 produces rapid cellular injury due to its reductive dehalogenation in the endoplasmic reticulum of hepatocytes to generate an unstable highly reactive complex CCl»3 or trichloroperoxyl free radical (CCl3O3). These radicals attack microsomal lipids causing lipid peroxida-tion. Products of lipid peroxidation may causes damage to the biological membranes leading to serious cellular injury and leakage of liver marker enzymes like ALT, AST, ALP and LDH (Cotran etal., 1994). The prevention of this phenomenon can be considered as hepatoprotective activity (Mak et al., 1996).
Liver marker enzymes are localized in the cytosol of hepatic cells and thus are extruded into the serum when cells are damaged or necrotic. In this study, rats intoxicated with CCl4 developed significant hepatic damage as manifested by a significant increase in the serum activities of ALT, AST, ALP and LDH that are indicators of hepatocyte damage and loss of functional integrity. Pretreatment of rats with the ethanol extract of F. ingens in doses of 200 and 400 mg/kg effectively protected rats against CCl4-induced hepatic damage, resulting in reduction in serum activities of liver marker enzymes when compared to the intoxicated control rats. Decrease in the level of these enzymes with F. ingens is an indication of the stabilization of plasma membrane as well as repair of liver damage caused by CCl4.
Figure 1 (A) 7-Hydroxy-2,5-dimethyl-chromen-4-one HMBC correlation. (B) Isolated compounds from Ficus ingens.
[3] 7-hydroxy-2,5-dimethyl- chromen-4-one
[4] Chrysophanol
[5] Quercetin: R1=R2= H, R3=OH
[7] Rutin R1= rutinoside,R2 =H, R3=OH
[8] Cmpound 8: R1= rutinoside, R2= R3= -OCH3
[6] 8 -Aloe emodin -O-glucoside
Figure 1 (continued)
Figure 2 Photomicrographs of rat liver (H & E stain) under low power (x200), (A) Normal control group showing normal parenchymal architecture; (B) CCl4 (3 mL/kg) showing diffuse central and peripheral necrosis and destruction of the lobular architecture; (C) CCl4 (3 mL/kg) + F. ingens (400 mg/kg) showing repairing of hepatocytes.
Further, the rise in the level of TB in serum following CCl4 intoxication is also a measure of hepatotoxicity and could be attributed to impaired hepatic clearance due to hepatic paren-chymal damage and biliary obstruction (Blanckaert and Schmid, 1982). The ability of the ethanol extract of F. ingens (200 and 400 mg/kg) to reduce the level of TB in the serum of intoxicated rats suggests its potential hepatoprotective effect. The lowered serum levels of TP and Alb due to CCl4-intoxication are attributed to the initial damage of the endoplasmic reticulum which results in the loss of P-450 leading to fatty liver (Recknagel, 1967). Administration of the ethanol extract of F. ingens in doses of 200 and 400 mg/kg remarkably prevented CCl4-induced reduction of TP and Alb in serum. This assures the hepatoprotective activity of this extract against damage by CCl4.
Hepatic antioxidant enzymes (SOD, GPx and CAT) represent one protection against oxidative tissue-injury (Halliwell and Gutteridge, 1990). SOD converted O2 into H2O2 while GPx and CAT metabolize H2O2 to non-toxic products. In
the present investigation SC injection of CCl4 to rats was shown to cause oxidative stress in liver and this damage was manifested by reduced activities of the antioxidant enzymes as well as GSH depletion in the liver homogenate. Depletion of GSH leads to cell death. Accordingly, the possible mechanism of the antihepatotoxic effect of F. ingens extract may be, in part, attributed to its antioxidant activity. This effect was evidenced by the ability of F. ingens to return the reduced activities of SOD, GPx and CAT and GSH level in the liver homogenate back to their control levels.
Since CCl4 induced hepatotoxicity is due mostly to oxida-tive stress (Weber et al., 2003 and Lin et al., 2008), antioxidant mediated protective role of F. ingens extract has been assessed. Oxidative stress was evidenced by reduced activities of the antioxidant enzymes as well as GSH depletion in the liver homogenate. Depletion of GSH leads to cell death. In this work, the possible mechanism of the antihepatotoxic effect of F. ingens extract may be, in part, attributed to its antioxidant activity. This effect was evidenced by the ability
of F. ingens to return the reduced activities of SOD, GPx and CAT and GSH level in the liver homogenate back to their control levels.
In addition, lipid peroxidation is supposed to be a critical factor in the pathogenesis of CCl4-induced hepatic injuries. Pre-treatment of F. ingens extract, on the other hand, prevented the toxic effects of CCl4 by restoring the increased MDA level in the liver homogenate toward the level of control animals, implying that the tested extract may prevent the peroxidation of lipids by CCl4. Increased activities of the antioxidant enzymes with concomitant increase in GSH level and reduced lipid peroxidation product are the indications that F. ingens extract offered significant protection. The hepatoprotective effect of F. ingens (200 and 400 mg/kg), was also supported by histopathological examination which showed recovery of the damaged liver cells. In accordance with these results, the protective effect of F. ingens extract against CCl4 may be attributed to the presence of phyto-constituents such as flavonoids.
7. Conclusion
In the present study, it has been observed that F. ingens offered significant protection against the hepatotoxicant CCl4 in rats, which may be attributed to its phytochemical constituents (which were mainly polyphenolic compounds) with their anti-oxidant and membrane stabilizing properties.
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