Abstract: In this study, hepatoprotective effect of aqueous and methanolic extracts of Capparis decidua stems were evaluated against carbon tetrachloride induced liver damage in rats. Simultaneous oral administration of both extracts (200, 400 mg kg-1) with CCl4 in paraffin oil (1:9 v/v) at a dose of 0.2 mL kg-1 for 10 days covered the liver fatty changes induced by the hepatotoxic compound observed in the intoxicated control rats. Slight to mild changes in hepatocytes were observed in rats dosed by aqueous extract of C. decidua stems and higher dose of methanolic extract, whereas the lower dose of methanolic extract revealed more severe lesions than the higher dose. The results were compared with the hepatoprotective effect of the standard drug silymarin.
INTRODUCTION
Capparis decidua belongs to the family Capparaceae and locally known as Altoundob (Elamin, 1990). This plant is widely used in the treatment of various ailments mainly among the local population as anthelmintic, analgesic, aphrodisiac, carminative, diaphoretic, emmenagogue and laxative (Rahman et al., 2004). The bark of C. decidua has been used by people in the treatment of cough, asthma and inflammation; roots are used in fever and buds in the treatment of boils (Satyanarayana et al., 2008). The water extract of the green stems of C. decidua is used as a bathe for jaundice (El-Ghazali, 1986).
A large number of plants and formulations have been claimed to have hepatoprotective activity. Nearly 160 phytoconstituents from 101 plants have been claimed to possess liver protecting activity. In India, more than 87 plants are used in 33 patented and proprietary multi-ingredient plant formulations (Handa et al., 1986).
In an earlier study, Ali et al. (2009) investigated the hepatoprotective effect of the aqueous and methanolic extracts of C. decidua in rats against CCl4 induced hepatotoxicity and the results indicated that the plant can be exploited for future hepatoprotective drug. In this respect, previous studies also showed that other species of the genus Capparis, e.g., C. spinosa have potential protective activity against liver damage (Gadgoli and Mishra, 1995).
Pharmacological studies of the extracts of C. decidua were found to have anti-inflammatory and antipyretic effects (Ageel et al., 1986), antioxidant (Yadav et al., 1997), hypoglycemic and hypolipidemic (Modak et al., 2007; Chahlia, 2009), anthelmintic, antibacterial and antifungal activities (Satyanarayana et al., 2008) and CNS depressant and anticonvulsant activities (Goyal et al., 2009).
Various substances are known to cause liver and kidney damage and one of them is carbon tetrachloride (CCl4), which is a well known hepato and nephrotoxin. Within the body, CCl4 breaks down to highly toxic trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2) free radicals by cytochrome P450 enzyme and causes damage to hepatocytes (Abraham et al., 1999).
The aim of the present study was to investigate the protective effect of aqueous and methanolic extracts of C. decidua stems against carbon tetrachloride induced liver damage in rats.
MATERIALS AND METHODS
Collection and identification of the plant: Capparis decidua stems were collected from Arkaweit area in southern Khartoum, during May 2004 and dried at room temperature. The plant was identified by the botanists in Medicinal and Aromatic Plants Research Institute.
Preparation of the extract: Aqueous extract was prepared by infusion method and dried using freeze drier apparatus and the yield percentage was 6.94%. Methanolic extract was also prepared by using soxhlet extractor and the yield percentage was 12.61% (Harborne, 1973).
Experimental procedure: Thirty five adult male Wister albino rats were used; the animals were grouped randomly into 7 groups of 5 rats each. Group I served as normal control and received only the vehicle liquid paraffin at a dose rate of 0.2 mL/kg /day i.p. for 10 days. Group II served as intoxicated control given CCl4 at a dose rate of 0.2 mL/kg /day i.p. in liquid paraffin (1:9) for 10 days. Group III served as hepatoprotective drug control when the rats were given CCl4 at a dose rate of 0.2 mL/kg /day i.p. in liquid paraffin (1:9) for 10 days and at the same time received orally silymarin suspended in 5% Acacia mucilage at a dose of 100 mg/kg /day. Group IV, V, VI and VII rats were given CCl4 at a dose rate of 0.2 mL/kg /day i.p. in liquid paraffin (1:9) for 10 days. In addition group IV received simultaneous oral administration of aqueous extract of C. decidua stems at a dose of 200 mg kg-1, group V was given 400 mg kg-1 of aqueous extract of C. decidua stems orally, group VI received 200 mg kg-1 of methanolic extract of C. decidua stems orally and group VII rats were dosed with 400 mg kg-1 of methanolic extract of C. decidua stems orally.
Histopathological studies: The animals were sacrificed by decapitation under anaesthesia and post-mortem examination was performed. Small pieces of livers were collected in 10% neutral buffered formal saline for histopathological study. Section 5-6 μ were cut and stained by haematoxyline and eosine.
RESULTS
Post-mortem investigations of CCl4 group showed severe generalized fatty change, petechial haemorrhages and week adhesions in all lobes of the liver. Petechial haemorrhages and slight fatty change in the livers were seen in standard drug group and rats dosed with 200 and 400 mg kg-1 aqueous extract of C. decidua stems. In rats that received 200 mg kg-1 of methanolic extract also showed fatty change and petechial haemorrhages, similarly, petechial haemorrhages and slight fatty change were observed in the liver of rats receiving the higher dose.
| Fig. 1: | (a, b) Liver section, CCl4 group, showing diffuse areas of vacuolar degeneration and centrilobular necrosis with mononuclear cell infiltration |
| Fig. 2: | (a, b) Liver section, standard drug group, showing mild hepatocyte vacuolation |
No significant changes were observed in control group except slight congestion.
Histopathological examination of CCl4 group (II) demonstrated significant changes in the livers which included diffuse vacuolar degeneration, mainly centrilobular, swelling of hepatocytes and congestion of hepatic veins and sinusoids (Fig. 1a, b).
In standard drug group (III) mild to moderate changes were noticed. Hepatocytes contained small single or multiple cytoplasmic vacuoles, mainly at the periphery of the lobules. Hepatocyte swelling and congestion were also observed (Fig. 2a, b).
Group IV (200 mg kg-1 aqueous extract of C. decidua stems) showed very little changes. However some hepatocytes appeared swollen with fragmentation or dissolution of cytoplasm. The mild vacuolations had a patchy distribution (Fig. 3a, b).
Group V (400 mg kg-1 aqueous extract of C. decidua stems) showed mild to moderate vacuolar changes with hepatocyte swelling and fragmentation or lysis of cytoplasm. These vacuolar changes appeared more peripherally in the lobules (Fig. 4a, b).
In group VI (200 mg kg-1 methanolic extract of C. decidua stems) mild to moderate vacuolations were also observed in hepatocytes. Distinct interlobular tissue and mononuclear cell infiltration in parenchyma and portal areas were observed. Most cells showed small vacuolations (Fig. 5a, b).
In group VII (400 mg kg-1 methanolic extract of C. decidua stems), mild to moderate vacuolar degeneration was observed with no definite pattern. Some hepatocytes exhibited cytoplasmolysis. Others appeared swollen with pyknotic nuclei. Distortion of lobular pattern, areas of sinusoidal congestion and small aggregates of mononuclear cells in parenchyma were also seen (Fig. 6a, b).
| Fig. 3: | (a, b) Liver section, rats treated with 200 mg kg-1 C. decidua stems aqueous extract showing mild hepatocyte swelling |
| Fig. 4: | (a, b) Liver section, rats received 400 mg kg-1 C. decidua stems aqueous extract showing mild vacuolar degeneration |
| Fig. 5: | (a, b) Liver section of 200 mg kg-1 C. decidua stems methanolic extract showing vacuolar degeneration and mononuclear cell infiltration in parenchyma and portal areas |
| Fig. 6: | (a, b) Liver section of 400 mg kg-1 C. decidua stems methanolic extract showing mild vacuolar degeneration and mild hepatocyte swelling |
DISCUSSION
The present study was performed to identify the hepatoprotective activity of C. decidua stems in rats against CCl4 induced liver damage. The plant has been used in different traditional medical practices (Rahman et al., 2004; Al-Yahya, 1986; El-Ghazali et al., 1997).
Many folk remedies from plant origin are tested for its potential antioxidant and hepatoprotective liver damage in experimental animal model. Carbon tetrachloride induced hepatotoxicity model is widely used for the study of hepatoprotective effects of drugs and plant extracts (Ahsan et al., 2009).
In this study, histopathological examination demonstrated that CCl4 induces diffuse centrilobular degeneration and congestion of hepatic veins and sinusoids and this is in agreement with the results of Ozbek et al. (2004) and Gupta et al. (2004).
Administration of aqueous and methanolic extracts of C. decidua stems with CCl4 remarkably masked severe hepatic lesions induced by hepatotoxic compound. The activity of the tested extracts was comparable to that of silymarin used as a reference drug.
Furthermore, the results of the present study indicated that the aqueous extract of C. decidua stems possesses higher activity than the methanolic extract, especially in the lower dose, probably related to the more polar phytoconstituents.
However, CCl4 hepatotoxicity depends on the reductive dehalogenation of CCl4 catalysed by the cytochrome P-450 in the liver cells endoplasmic reticulum leading to the generation of an unstable complex CCl•3 radical (Gupta et al., 2004), which is reported as a highly reactive species. These free radicals attack microsomal lipids leading to its peroxidation and also covalently bind to microsomal lipids and proteins. These results in changes of structures of the endoplasmic reticulum and other membrane, loss of metabolic enzyme activation, reduction of protein synthesis and loss of glucose-6-phosphatase activation, leading to liver injury (Azri et al., 1992).
Concurrent treatment with aqueous and methanolic extracts of C. decidua stems and CCl4 minimized the liver damage induced by CCl4 which indicates that this plant has antihepatotoxic effect, probably due to its antioxidant properties (Gupta et al., 2004). The plant contains high concentration of vitamin C (Duhan et al., 1992; AL-Yahya, 1986), which is an important dietary, water soluble antioxidant and has been reported to decrease the adverse effect of Reactive Oxygen Species (ROS) and Nitrogen Species generated in vivo in animals (Halliwell and Gutteridge, 1999; Adams, 2001).
The preliminary phytochemical screening of the powdered plant showed positive results for the presence of flavonoids, cyanogenic glycosides and triterpenes which are antioxidants and may be responsible of the hepatoprotective property of the plant as suggested by earlier studies (AL-Yahya, 1986; Evans, 2002; Duhan et al., 1992; Pattanayak and Priyashree, 2008).
In conclusion, these results have clearly indicated potential effect of Capparis decidua stems as a hepatoprotective against carbon tetrachloride-induced hepatic damage in rats.
Moreover, our results indicate that the aqueous extract of C. decidua had higher activity than the methanolic extract and this is probably related to the more polar phytoconstituents.
Further investigations are required to determine the exact phytoconstituent(s) responsible for hepatoprotective effect C. decidua.