Abstract: The hepatoprotective effects of garlic (Allium sativum), ginger (Zingiber officinale) and vitamin E pre-treatment against carbon tetrachloride (CCl4)-induced liver damage in male wistar albino rats were investigated. Carbon tetrachloride (0.5 mL kg-1 body weight) was administered after 28 days of feeding animals with diets containing ginger, garlic, vitamin E and various mixtures of ginger and garlic. Serum alanine amino transferase, aspartate amino transferase and alkaline phosphatase levels, 24 h after CCl4 administration, decreased significantly (p≤0.05) in rats pre-treated with garlic, ginger, vitamin E and various mixtures of garlic and ginger than in CCl4-treated rats only. Lipid peroxidation expressed by serum malondialdehyde (MDA) concentration was assayed to assess the extent of liver damage by CCl4; including the extent of hepatoprotection by garlic, ginger and vitamin E. MDA concentration was significantly decreased (p≤0.05) in rats pretreated with garlic, ginger, vitamin E and various mixtures of garlic and ginger than in rats administered CCl4-alone. Histological examination of the liver revealed severe infiltration of inflammatory cells in rats treated with CCl4 alone. However, the observed alteration in the normal architecture of the hepatic cells decreased remarkably in pre-treated rats.
INTRODUCTION
Carbon tetrachloride (CCl4) is a highly toxic chemical agent. The toxic effects of CCl4 on the liver have been known for years and studied extensively (Recknagel, 1967; Dianzani, 1991). The effects of CCl4 on hepatocytes, depending on dose and exposure time, are manifested histologically as hepatic steatotis (e.g., fatty infiltration), centrilobular necrosis and ultimately cirrhosis (Junnila et al., 2000). The damage or death of tissue usually results in the leakage of the enzymes in the affected tissue(s) into the blood stream (Sieger et al., 1985; Obi et al., 2001).
Serum or plasma enzyme levels have been used as markers for monitoring chemically induced tissue damages (Lin and Wang, 1986; Ngaha et al., 1989; Hukkeri et al., 2002). The enzymes L-alanine aminotransferase (L-ALT) (E.C. 2.6.1.2), L-aspartate amino transferase (L-AST) (E.C.2.6.1.1) and alkaline phosphatase (ALP) (E.C. 3.1.3.1) are important enzymes that are often employed in assessing liver injury (Ngaha et al., 1989; Obi et al., 1998; Hukkeri et al., 2002).
The biotransformation of CCl4 to metabolites is a cytochrome P450 mediated reaction that initiate lipid peroxidation and attendant tissue damage. The metabolic effects of CCl4 inside mitochondria have been described (Brattin et al., 1984) and it has been found that damage to the calcium pump in mitochondria (Albano et al., 1985) is dependent upon haloalkylation. However, the profound accumulation of fat following CCl4 poisoning is considered to be independent of mitochondrial damage (Artizzu and Dianzani, 1962; Reynolds et al., 1962). The fatty infiltration of the liver is thought to develop as a result of the action of free alkyl radicals on biomembranes that in turn cause halo alkylation-dependent blocking at the exit of the lipoprotein micelles from the golgi apparatus (Poli et al., 1987).
Some plants have been shown to have protective antioxidant effects and are therefore hepatoprotective. Such plants would include H. rosasinensis (Obi et al., 1998), Celosia argentea (Koji et al., 1996), Ginkgo biloba (Shenoy et al., 2001) among others. A combination of garlic and ginger has been reported by Ahmed and Sharma (1997) to be much more effective in reducing blood glucose and serum lipids while Mustafa and Srivastava (1990) have shown that administration of ginger may exert abortive and prophylactic effects in migraine headache without any side effects.
This study was therefore designed to evaluate the anti-oxidant protective effects of Allium sativum, zingiber officinale and vitamin E in rats exposed to carbon tetrachloride (CCl4).
MATERIALS AND METHODS
Plant material: Ginger (Zingiber officinale) and garlic (Allium sativum) were purchased from Fruit Garden Market D/Line, Port Harcourt, Nigeria.
Preparation of ginger and garlic: The samples were sun-dried for 4 days, ground into powdery form using an electric blender (Moulinex) and stored in air-tight containers.
Animals: Forty five adult male wister albino rats (150-170 g) used in this study were obtained from the Animal House, Department of Biochemistry, University of Port Harcourt, Port Harcourt, Nigeria. They were housed in standard cages (Griffin and George Modular Cage System) and left to acclimatize for 7 days to laboratory conditions before the commencement of the experiment. During the acclimatization, the animals were fed with pelleted rat chew and water ad libitum.
The forty five rats were arranged into nine groups of 5 animals each.
Group 1:This group was given normal feed and CCl4
Group 2:Received feed, ginger and 0.5 mL CCl4/kg BW
Group 3:Received feed, garlic and 0.5 mL CCl4/kg BW
Group 4:Received feed, vitamin E and 0.5 mL CCl4/kg BW
Group 5:Feed, garlic: ginger (1:1) and 0.5 mL CCl4/kg BW
Group 6:Received feed, garlic: ginger (2:1) and 0.5 mL CCl4/kg BW
Group 7:Received feed, garlic: ginger (1:2) and 0.5 mL CCl4/kg BW
Group 8:Served as control I
Group 9:Served as control II
Induction of hepatic injury: Carbon tetrachloride (CCl4) induced liver damage was achieved by injecting 0.5 mL kg-1 CCl4 intraperitonealy after 28 days of feeding animals of groups 1-7 with commercial feed, ginger, garlic and vitamin E as stated above.
Preparation of samples: Twenty four hour after the administration of CCl4, the rats were anaesthetized in a chloroform-saturated chamber. The animals were sacrificed by cervical dislocation and blood was collected by cardiac puncture using a 5 mL hypodermic syringe and needle and introduced into an anticoagulant free bottle. Serum was separated by centrifugation and stored in a refrigerator. The measurement of different biochemical parameters (L-AST, L-ALT and ALP) was performed using the Humazym M UV-text kits. The liver was excised and fixed in formalin for histological assessment of hepatic damage. Lipid peroxidation was also determined by estimating the malondialdehyde levels, using the method of Hunter et al. (1963) modified by Gutteridge and Wilkins (1980).
Analysis of data: The mean values of the control and test serum activities of a given enzyme were compared using the student`s t-test (Zar, 1984). The significance level was set at p≤0.05.
RESULTS
Figure 1a-d indicate that treatment
of rats with single dose of CCl4 (0.5 mL kg-1 body
wt.) led to the development of severe hepatic injuries as compared to
rats pretreated with garlic, ginger, vitamin E; including various mixtures
of the drugs. As shown in the figures, marked increase in the concentrations
of ALT, AST, ALP and MDA were evident in CCl4-treated rats
alone when compared with those that were pre-treated with ginger, garlic
and the control (1). In order words the rats in groups 2 to 9 showed significant
improvement in the altered AST, ALT, ALP levels and lipid peroxides. The
rats fed with a combination of garlic and ginger (1:1) alone (control
II) did not show toxicity in the liver tissue as levels of the biochemical
parameters and lipid peroxides gave values that were close to those of
control 1 (Group 8). Histopathological examinations (Fig.
2a-c) revealed defects ranging from congested central vein, centrilobular
necrosis, fatty degeneration and infiltration by inflammatory cells in
rats treated with CCl4 alone. However, reductions in the observed
defects were evident in the pretreated rats.
| Fig. 1a: | Influence of ginger, garlic and vit. E on serum ALP level in CCl4-hepatotoxicity |
| Fig. 1b: | Influence of ginger, garlic and vit. E on serum AST level in CCl4-hepatotoxicity |
| Fig. 1c: | Influence of ginger, garlic and vit. E on serum ALT level in CCl4-hepatotoxicity |
| Fig. 1d: | The Malondialdehyde (MDA) concentration of liver homogenate in CCl4 hepatotoxicity. 1 = Feed + CCl4; 2 = Feed + Ginger + CCl4; 3= Feed + garlic + CCl4; 4 = Feed + Vit. E + CCl4; 5 = Feed + Garlic + Ginger (1:1) + CCl4; 6 = Feed + Garlic + Ginger (2:1) + CCl4; 7 = Feed + Garlic + Ginger (1:2) + CCl4; 8 = Feed only (Control 1); 9 = Feed + Garlic + Ginger (1:1) (Control II) |
| Fig. 2a: | A section of rat liver showing normal architecture |
| Fig. 2b: | A section of rat liver treated with CCl4 only, showing vacuolar degeneration of centrilobular hepatocytes and infiltration by mixed inflammatory cells |
| Fig. 2c: | A section of rat liver pre-treated with drugs prior to CCl4 administration showing a pattern of reduced inflammatory infiltration |
DISCUSSION
Carbon tetrachloride (CCl4) is one common hepatotoxin used in the experimental study of liver diseases (Koji et al., 1996; Obi et al., 1998; Shenoy et al., 2001).
The serum levels of these marker enzymes: AST, ALT and ALP reflect the physiological state of the liver. The levels of these enzymes change according to distortion of the liver, resulting from cellular injury of the organ caused by toxic metabolites and diseases.
The efficacy of any hepatoprotective drug is essentially dependent on its capability of either reducing the harmful effects or in maintaining the normal hepatic physiological mechanism which have been altered by a hepatotoxin (Raghavendran et al., 2003).
The results in this study indicate that CCl4 caused an increase in the serum levels of marker enzymes (AST, ALT and ALP) in group 1 rats that received CCl4 alone. Such elevation is indicative of liver injury, especially the rise in L-ALT level (Lin and Wang, 1986). The toxicity of CCl4 to the liver of mammals is largely as a result of the active metabolite, trichloromethyl radical (Johnson and Kruening, 1988). The trichloromethyl radical binds to tissue macromolecule and thus induce peroxidative degradation of membrane lipids of the endoplasmic reticulum which are rich in polyunsaturated fatty acids. Shenoy et al. (2001) postulated that such development would ultimately lead to the formation of lipid peroxides that in turn yield other products, among which is malondialdehyde (MDA).
The increased enzyme levels in the plasma of CCl4-treated rats suggests the toxicant was able to reach the liver and induce a detectable damage within 24 h.
The result in this study suggest that feeding rats with vitamin E, garlic (Allium sativum) and ginger (Zingiber officinale), singly or as mixtures in different ratios prior to CCl4 administration caused a decline in hepatotoxicity of rats treated with CCl4. This is evidenced in the marked decrease in serum L-AST and L-ALT level relative to the group treated with CCl4 alone. Comparatively, ginger appear to exhibit higher protection of liver than garlic and vitamin E as the levels of all the enzymes studied were lower in rats treated with ginger only than those treated with garlic and vitamin E. The decrease in enzyme level in rats treated with ginger, garlic, combination of garlic and ginger and vitamin E suggest that garlic and ginger contain antioxidant properties with ginger having greater active constituent.
Hukkeri et al. (2002) reported that the elevation in the plasma level of cytoplasmic and mitochondrial enzymes accurately reflects liver injury induced by CCl4. This increase in the levels of the enzymes (i.e., ALT, AST and ALP) under the influence of CCl4 has been attributed to the damaged structural integrity of the liver. Significant decrease in the CCl4 - Induced elevation of ALT, AST and ALP level by ginger, garlic and vitamin E is almost definitely suggestive of protection of the structural integrity of the hepatocyte membrane or regeneration of damaged liver cells by test samples. The histopathological observations as well have demonstrated the protective action of the drugs against CCl4 induced liver damage. Thus, from the result of the current investigation it may be inferred that ginger and garlic possess potent hepatoprotective activity.
ACKNOWLEDGMENTS
The authors are grateful to Dr. S.O. Nwosu (Pathology Department, UPTH) for his assistance in carrying out histological studies. Mr Uche Ezeigbo and Miss Ezinne Nwoke (NNPC Medical Clinic) for their timely assistance in carrying out the biochemical parameters.