The methanol extract of the fruits of Ficus glomerata Linn. (Moraceae) was evaluated for hepatoprotective activity in rats with liver damage induced by carbon tetrachloride. The extract at an oral dose of 500 mg/kg exhibited a significant protective effect by lowering the serum levels of aspartate aminotransferase, alanine aminotransferase, total serum bilirubin, and malondialdehyde equivalent, an index of lipid peroxidation of the liver. These biochemical observations were supplemented by histopathological examination of liver sections. The activity of extract was also comparable to that of Liv-52, a known hepatoprotective.
Liver is the largest organ in the vertebrate body and the site for intense metabolism. Liver diseases remain one of the serious health problems and the Indian traditional system of medicine, especially Ayurveda have put forward a number of medicinal plants and their formulations for liver disorders. In this modern age it is very important to provide scientific proof to justify the various medicinal uses of herbs. Herbal drugs are prescribed widely even when their biologically active components are unknown because of their effectiveness, fewer side effects and relatively low cost (1). However, we are not aware of a satisfactory remedy for serious liver diseases and search for effective and safe drugs for liver disorders continues to be an area of interest. Ficus glomerata Linn. (Moraceae) is a deciduous tree, which grows in tropical and subtropical regions of India and is commonly known as fig tree (2). In traditional medicine the roots are used in treatment of leucoderma and ringworms and its fruits which are sweet, have antipyretic, purgative, aphrodisiac properties and have shown to be useful in inflammations and paralysis (3,4). F. carica is claimed to be useful in liver and spleen disorders, to cure piles and in treatment of gout. Locally the fruits are being used in the treatment of jaundice (personal information from users). Earlier chemical examination of this plant have shown the presence of psoralen, bergapten, umbelliferone (5,6), Î²-sitosterol, campesterol, stigmasterol, fucosterol, fatty acids (7), 6-(2- methoxy-Z-vinyl)-7-methyl-pyranocoumarin and 9,19-cycloarlane triterpenoid as an anticancer (8,9) and antiproliferative agent: 6-O-acyl-Î²-Dglucosyl- Î²-sitosterol (10), calotropenyl acetate, and lupeol acetate (11). Previously it was reported that the fruit extracts of Ficus racemosa (12) and Ficus hispida (13) possess significant hepatoprotective activity against carbon tetrachloride- and paracetamolinduced hepatotoxicity in rats, respectively. In view of the reported hepatoprotective activity of other Ficus species and traditional claims, the fruits of F. carica was evaluated against carbon tetrachloride induced hepatic damage in rats with the aim of developing a natural hepatoprotective drug.
MATERIALS AND METHODS
Collection and Identification of Plant Material
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The branches and fruits of Ficus glomerata Linn were collected from plants growing around Anantapur, Andhra Pradesh, India. The plant was identified and authenticated in Department of Botany, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India. The voucher specimen (number: SKUB 0256) was kept at the Herbarium of the College of Pharmaceutical Sciences, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India.
Carrageenan, Sodium CMC and Diclofenac sodium were gift samples from Waksman and Selman Pharmaceuticals, Anantapur, India. Methanol, 95% Ethanol were procured from SD fine chemicals Mumbai, India. All the chemicals used were of AR grade and deionized water was used throughout the experiment.
Preparation of extract
Fresh fruits were cut, seeds were removed, wash with water and defatted with petroleum ether (60-80oC) and further separately extracted with ethanol and water by soxhlet extractor for 72 hour. The solvent was removed under reduced pressure and semisolid mass obtained dried in vacuum to yield solid residue (5.9 %w/w and 4.9% w/w respectively). The chemical constituents of the extracts were identified by qualitative analysis followed by their confirmation by thin layer chromatography.
The concentrated ethanolic extract (24.8 g) was tested for qualitative phytoconstituents and indicated the presence of steroids/triterpenoids and their glycosides and coumarins. (Trease & Evans, 1994; Tyler, 1948; Chatwal, 1995).
Selection and maintenance of animals
Wistar albino rats (male) weighing 200-250 g were employed for the study (procured from National Institute of Nutrition, Hyderabad, India). The rats were maintained under standard laboratory conditions (temperature of 25 Â± 2oC and relative humidity of 50 Â± 15%) and normal photo period was used for the experiment. Commercial pellet diet (Ratan Brothers, India) and water were provided when desired. The experimental protocol has been approved by the Institutional Animal Ethics committee and by the Regulatory body of the government (Regd no.516/01/A/CPCSEA).
Preparation of drug suspension
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Drug suspensions containing 5 mg/mL of extract was prepared by using 1% Sodium Corboxy Methyl Cellulose as suspending agent. A dose of 300mg/kg was given.
Acute toxicity studies
The acute oral toxicity studies were carried out as per the guidelines of Organization for Economic Co-operation and Development (OECD), draft guidelines 423 received from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Acute toxicity studies on ethanolic and aqueous extracts of Ficus glomerata fruits (EEFG and AEFG respectively) are carried out at dose range of 50mg to 3000mg/kg body weight orally and the number of animals that died in a 7 day period after a single dose was recorded. The animals were also closely examined for signs of intoxication, lethargy, behavioural modification and morbidity
Hepatoprotective effect against CCl4-induced hepatotoxicity in rats
Wistar albino rats were divided into five groups of six animals each. The CCl4 (1ml/kg) was administered to all groups of animals by subcutaneous injection. Group-I served as control received normal saline only (10 ml/kg i.p). Group-II received Liv-52 the reference drug (25mg/kg i.p). Group-III received CCL4 (1ml/kgi.p). Group IV and Group V received aqueous and ethanolic extract respectively in a dose of 200mg/kg daily once for fifteen days after CCl4 administration. All the animals were dissected at the end of 15th day after CCl4 administration. Blood was withdrawn from the carotid artery and serum was separated by centrifugation at 1000 rpm. The separated serum were used for the estimation of the biochemical parameters [Alanine aminotrasferase (ALT/GPT), Aspartase aminotrasferase (AST/GOT), Alkaline phosphatase, Bilirubin (Direct and Total)]. ALT and AST was assayed by using Serum diagnostic kits by Span Diagnostic Ltd.
The tissues of liver were fixed in 10% formalin and embedded in paraffin wax. Sections of 4-5 microns thickness were made using rotary microtome and stained with haematoxylin-eosin and histological observations were made under light microscope (20,21).
The results are expressed as means Â± S.D. The difference between experimental groups were compared by one-way ANOVA (toxic control versus treatment, Bonferrioni's method; using Jandal scientific, Sigmastat statistical software, version 1.0) and were considered statistically significant when p< 0.05.
Both extract of fruits of Ficus glomerata was found to be practically non-toxic since no mortality was observed even at the dose of 3000mg/kg body weight. Hence the biological dose was fixed 200mg/kg for EEFG and AEFG. Rats treated with CCl4 developed a significant liver damage and showed an elevated serum levels of hepato-specific enzymes as well as severe alteration in other biochemical parameters (P<0.001). Values of the biochemical parameters were observed to be increased in the CCl4 intoxicated rats. Treatment with EEFG and AEFG decreased the CCl4 induced alterations in AST, ALT, alkaline phosphatase and total bilirubin. It is found that both the extracts offer protection against toxin as evidenced by remarkable reduction in all biochemical parameter. Histopatholgical studies demonstrated that carbon tetrachloride causes focal necrosis, portal infiltration, fatty change, kupffer cell hyperplasia and hydropic change. In the treated groups, necrosis which is more severe form of injury is markedly prevented; milder form of injury like fatty change and reduced necrosis persisted in both the extracts.
In the assessment of liver damage by CCL4 hepatotoxin, the determination of enzyme levels such as ALT (SGPT) and AST (SGOT) is largely used. Necrosis or membrane damage releases the enzyme in to circulation; therefore, it can be measured in serum. High levels of AST (SGOT) indicate liver damage, such as that due to viral hepatitis as well as cardiac infarction and muscle injury. ALT (SGPT) catalyses the conversion of alanine to pyruvate and glutamate, and is released in a similar manner. Therefore, ALT (SGPT) is more specific to the liver, and is thus a better parameter for detecting liver injury20. Our results using the model of CCL4-induced hepatotoxicity in the rats demonstrated that ethanol and aqueous extracts caused significant inhibition of ALT (SGPT) and AST (SGOT) levels. Serum ALP and bilirubin levels on other hands are related to the function of hepatic cell. Increase in serum levels of ALP is due to the increased synthesis, in presence of increasing biliary pressure21. Our results using the model of CCL4 - induced hepatotoxicity in rats demonstrated that EEFG and AEFG caused significant inhibition of serum ALP and bilirubin levels. Effective control of bilirubin level and alkaline phosphatase activity points towards an early improvement in the secretory mechanism of the hepatic cell.
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It has been reported that Ficus glomerata contain tannins and related polyphenol19. A number of scientific report indicated certain flavonoids, triterpenoids and steroids have protective effect on liver due to its antioxidant properties22,23. Presence of those compounds in EEFG and AEFG may be responsible for the protective effect on CCL4 induced liver damage in rats. In the present experimental conditions, EEFG and AEFG showed protection against toxin, as there is a significant reduction in all biochemical parameters on treatment. EEFG at a dose 200 mg/kg showed the better activity than AEFG in the CCL4 induced rats. The observed protective effect of the plant extract against CCL4 may be attributed to the presence of phenolic compounds like flavonoids, tannins
Table 1: Data for effect of ethanolic and aqueous extracts of acacia ferruginea dc. On CCl4 induced hepatotoxicity in rats
(mg/dL of blood)
39.4 Â± 2.57
27.4 Â± 1.28
11.5 Â± 1.57
7.8 Â± 0.05
0.28 Â± 0.01
85.1** Â± 5.97
42.4** Â± 1.49
19.8** Â± 2.45
9.6* Â± 0.25
0.16* Â± 0.01
168.2 Â± 9.54
163.7 Â± 11.57
91.2 Â± 6.54
51.2 Â± 2.59
0.87 Â± 0.02
114.2** Â± 6.49
75.4** Â± 6.49
30.2** Â± 3.01
11.2* Â± 0.08
0.46 Â± 0.03
89.7 Â± 2.59
57.4 Â± 2.57
23.4 Â± 2.15
9.3 Â± 0.28
0.35 Â± 0.02
Data are expressed as mean + S.E., n =6; *P<0.01 Vs Control; **P<0.001 Vs Control
The methanolic extract of Ficus glomerata fruits, administered prophylactically, exhibited significant protection against CCl4-induced liver injury as manifested by the reduction in toxinmediated rise in serum transaminases, ALP and total bilirubin in rats. Liver damage induced by CCl4 is commonly used model for the screening of hepatoprotective drugs (22). The rise in serum levels of AST, ALT and ALP has been attributed to the damaged structural integrity of the liver, because they are cytoplasmic in location and released into circulation after cellular damages (23). The rise in the levels of serum bilirubin is the most sensitive and confirms the intensity of jaundice (24). The CCl4 is converted into reactive metabolite, halogenated free radical by hepatic cytochrome P450s (25,26), which in turn covalently binds to cell membrane and organelles to elicit lipid peroxidation with subsequent tissue injury. High lipid peroxidation values indicate excessive free radical induced peroxidation. The measurement of lipid peroxide is also a marker of hepatocellular damage (27,28). Pretreatment of animals with methanolic extract of F. carica and Liv-52 prevented the CCl4- induced rise in serum level of transaminases and total serum bilirubin, confirming the protective effects of methanolic extract of F. carica fruits against carbon tetrachloride induced hepatic damage. The hepatoprotective activity of F. carica (500 mg/kg) was comparable with the activity of standard Liv-52 (100 mg/kg). However there was no effect on rise in serum alkaline phosphatase levels by the test extract and Liv-52. A comparative histopathological study of the livers from different groups further corroborated the hepatoprotective potential. In animals treated with methanolic extract and Liv-52, the rise in lipid peroxides in liver tissue homogenate was prevented significantly. The decrease in lipid peroxides may be due to the antioxidant effect of the extract. A possible mechanism of the F. carica extract as hepatoprotective may be due to its anti-oxidant effect or inhibition of cytochrome P450s which impair the bioactivation of CCl4 (29) into their corresponding reactive species. The preliminary phytochemical studies indicated the presence of steroids/triterpenoids and their glycosides and coumarins in the methanolic extract of fruits of F. carica. Since coumarins have hepatoprotective activity (30-32), it may be speculated that these constituents of Ficus are be responsible for the observed protective effects. However the role of steroids/triterpenoids cannot be ruled out.
The authors wish to thank Principal, UCPSC for providing research facilities. The authors also wish to thank Prof. D.R. Krishna, UCPSC, Kakatiya University, Warangal, India for his valuable help and suggestions during the course of work. The All India Council of Technical Education, New Delhi, India financially supported this investigation.
Fig. 4. Histopathological Microphotograph of Rat liver tissue a) Control group b) CCl4 Treated group c) Standard Liv-52 group d) Pet. ether group e) Alcoholic group f) Aqueous group
Figure 1. Microphotograph of normal rat liver section H&E staining (x200)
Figure 2. Microphotograph of rat liver section treated with CCl4 H&E staining (x200)
Figure 3. Microphotograph of rat liver section treated with Liv-52 H&E staining (x200)
Figure 4. Microphotograph of rat liver section treated with Alcoholic extract H&E staining (x200)
Figure 5. Microphotograph of rat liver section treated with aqueous extract H&E staining (x200)
Figure 6. Microphotograph of rat liver section treated with petroleum ether extract H&E staining (x200)
Group-I treated with normal saline (control) when observed under 100 x H.E of magnification showed liver tissue with typical lobular arrangement. Individual lobules consist of hepatocytes arranged as cords radiating around centrally placed terminal hepatic veins. Hepatocytes seen are uniform in size, poly herbal in shape, with centrally located large nuclei. The cytoplasm is strongly eosinophilic with a fine basophilic granularity. Portal tracts containing terminal branches of the hepatic portal vein and hepatic artery at the periphery in fibrous stroma were also seen and this impression for a normal liver tissue.
Group-II treated with CCl4 extract when observed under 100 x H.E of magnification showed liver tissue with disturbances in the lobular arrangement. Degenerative and early necrotic changes extending across lobules. Hepatocytes show ballooning degeneration and steatotic changes. Some amount of fibrosis seen in portal tracts. The net result gave the impression that the Liver with cytotoxic injury showing mild necrosis and fibrotic changes.
Group-III treated with Liv-52 (standard), when observed under 400 x H.E of magnification, showed liver tissue with typical lobular arrangement. Hepatocytes shows variable size. There is a mild increase in fibrous connective tissues. The net result gave an impression that the Liver with mild sign of hepatotoxicity.
Group-IV treated with Petroleum ether extract, when observed under 100 x H.E of magnification showed liver tissue with disturbances in the lobular arrangement. Degenerative and early necrotic changes extending across lobules. Apoptotic cells and Mallory's bodies are seen. PMN's are seen along dilated blood vessels. Some amount of fibrosis along the portal tract and sinusoids are seen. This indicates the Liver with cytotoxic injury.
Group-V treated with ethanolic extract, when observed under 400 x H.E of magnification, showed liver tissue with typical lobular arrangement. Hepatocyte shows variable size. Ballooning changes, steatotic accumulation. There is a mild increase in fibrous connective tissues. Inflammatory cells are seen within the parenchyma. It gave the impression that the Liver with sign of hepatotoxicity
Group-VI treated with aqueous extract, when observed under 100 x H.E of magnification, showed liver tissue with typical lobular arrangement. Few hepatocytes showd steatotic accumulation. It gave the impression that the Liver with minimal sign of hepatotoxicity