Abstract: Background and Objective: Carbon tetrachloride (CCl4), a carcinogenic compound commonly used in household cleaning agents and spot removers can cause free radical-mediated liver damage. Recent research shows polyphenols in pomegranate (Punica granatum L.), rich in exogenous antioxidants, may protect the liver from hepatotoxic damage. This study aimed to investigate the effect of pomegranate juice on CCl4-induced liver damage in a rat model. Materials and Methods: A total of 25 rats, aged 8 weeks and weighing an average of 200 g were used. They were divided into 5 groups as follows: (1) Normal, (2) Hepatotoxic rats and (3), Hepatotoxic rats with pomegranate juice 0.4 mL/200 g body weight (BW) (4) Hepatotoxic rats with pomegranate juice 0.8 mL/200 g BW and (5) Hepatotoxic rats with pomegranate juice 1.2 mL/200 g BW, respectively. Hepatotoxicity was done by intraperitoneal injection of 0.5 mL–1 CCl4 kg–1 BW for 5 days. Before and after 2 weeks administration of treatment, blood samples were collected for assessment of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT) and malondialdehyde (MDA). At the end of the study, the animals were euthanized and the livers were used for histopathological assessment. Data were analyzed by paired t-test and one-way ANOVA followed by the Games-Howell test. Results: Supplementation with three different doses of pomegranate juices in CCl4-induced rats significantly reduced the serum levels of ALT, AST, ALP, γ-GT and MDA (p<0.05) and pomegranate juice as much as 0.8 and 1.2 mL/200 g BW reduced liver damage in CCl4-induced rats. Conclusion: According to findings of this study supplementation with pomegranate juice can improve liver damage induced by CCl4 in rats.
INTRODUCTION
Most chemicals induce free radical-mediated lipid peroxidation leading to disruption of biomembranes and dysfunction of cells and tissues1. Carbon tetrachloride (CCl4), a common household compound is a carcinogenic agent that causes hepatic injury. Biotransformation of CCl4 by hepatic microsomal cytochrome P-450 produces toxic metabolites2. According to Adewale et al.2 the toxic effect of various chemicals can be prevented by antioxidants. Some studies showed that antioxidants prevent CCl4 toxicity, particularly hepatotoxicity, by inhibiting lipid peroxidation3, suppressing alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities4 and increasing antioxidant enzyme activity5.
Phenolic compounds in vegetables and fruits have antioxidant properties and are important to investigate because these compounds have strong antioxidant activity but low toxicity compared to the synthetic phenolic antioxidants, such as butylated hydroxytoluene6. One of the most powerful antioxidant-containing fruits is pomegranate which contains flavonoids, anthocyanins and ellagitanin7. These substances have been known to protect pancreatic β-cells from damage due to free radicals8. Flavonoids are powerful antioxidants with free radical scavenging properties and can reduce free radicals formation9. In comparison, antioxidant activity of pomegranate juices is 3 times higher than red wine and green tea infusion10. According to Anoosh et al.11 pomegranate juice has anti-atherogenic action in hypercholesterolemia patients. Considering these benefits, this study evaluated the effect of pomegranate juice on carbon tetrachloride-induced liver damage in rat.
MATERIALS AND METHODS
This study was conducted at Universitas Gadjah Mada from June, 2016 until January, 2017. Pomegranate (Punica granatum L.) fruit was purchased from the local market in Jakarta, Indonesia. Twenty-five male test rats (Rattus norvegicus), 8 weeks old, weighing an average of 200 g were obtained from the Food and Nutrition Department/PAU (Pangan dan Gizi), Universitas Gadjah Mada, Yogyakarta, Indonesia.
Reagents: Assay kits for measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) were purchased from Dyasis® (Holzheim, Germany) and diagnostics for gamma-glutamyl transferase (γ-GT) from Human Gesellschaft for Biochemica Und Diagnostica mbH (Wiesbaden, Germany), while malondialdehyde (MDA) level was measured using TBAR methods. All other chemicals were of analytical grade.
Experimental procedure: The rats were housed in individual cages and acclimatized to the laboratory condition (22-25°C room temperature and 12 h daylight cycle) for 7 days with free access to food and water during the experimental period. The standard diet was AIN 93 M consisting of (g kg–1 mix): Cornstarch (465.692), casein (140), dextrinized cornstarch (155), sucrose (100), soybean oil (40), alphacel (50), AIN-93-M-MX (35), L-cysteine 1.8, AIN-93-VM (10), choline bitartrate (2.5) and tert-butylhydroquinone (0.008). The present study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Gadjah Mada. Twenty-five rats were divided into 5 Groups: (1) Normal, (2) Hepatotoxic rats, (3) Hepatotoxic rats with pomegranate juice 0.4 mL/200 g BW, (4) Hepatotoxic rats with pomegranate juice 0.8 mL/200 g BW and (5) Hepatotoxic rats with pomegranate juice 1.2 mL/200 g BW. Hepatotoxicity of rats was done by intraperitoneal injection of CCl4 at dose of 0.5 mL kg–1 BW for 5 days according to the method described by Hewawasam et al.12. The pomegranate juice was given orally to groups 3, 4 and 5 for 2 weeks. Before and after treatment, fasting blood samples were collected from the retro orbitalis plexus for analysis of ALT, AST, ALP, γ-GT and MDA. Histopathological assessment of liver damage was done by haematoxylin and eosin (HE) staining.
Data analysis: All results were expressed as Mean±SE. Differences in blood ALT, AST, ALP, γ-GT and MDA were compared by one-way ANOVA followed by the Games-Howell test. Paired t-test was used to analyze the level of ALT, AST, ALP, γ-GT and MDA before and after treatment. The statistical analysis was performed using the SPSS program (Version 18; SPSS Inc., USA) with significance considered at p<0.05.
RESULTS
Administration of pomegranate juice significantly decreased serum MDA and ALT levels. The greatest decline in serum MDA and ALT levels were seen in the group with 1.2 mL/200 g BW of pomegranate juice. Compared to the hepatotoxic and normal groups, the effects of the administration of 0.4, 0.8 and 1.2 mL/200 g BW pomegranate juice were significantly different (Table 1 and 2). After 2 weeks administration of pomegranate juice serum ALP levels were significantly p<0.05 decreased.
| Table 1: | Serum malondialdehyde (MDA) levels in rats |
Values are presented as Mean±SE (n = 5). p-value in column indicates p<0.05 according to one-way ANOVA test. a,b,c,d,e indicated significant difference between groups according to one-way ANOVA test followed by the Games-Howell test. p-value in bottom row indicates p<0.05 according to paired t-test | |
| Table 2: | Serum alanine aminotransferase (ALT) levels in rats |
Values are presented as Mean±SE (n = 5). p-value in column indicates p<0.05 according to one-way ANOVA test. a,b,c,d,e indicated significant difference between groups according to one-way ANOVA test followed by the Games-Howell test. p-value in bottom row indicates p<0.05 according to paired t-test |
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| Table 3: | Serum ALP levels in rats |
Values are presented as Mean±SE (n = 5). p-value in column indicates p<0.05 according to one-way ANOVA test. a,b,c,d indicated significant difference between groups according to one-way ANOVA test followed by the Games-Howell test. p-value in bottom row indicates p<0.05 according to paired t-test |
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| Table 4: | Serum AST levels in rats |
Values are presented as Mean±SE (n = 5). p-value in column indicates p<0.05 according to one-way ANOVA test. a,b,c,d indicated significant difference between groups according to one-way ANOVA test followed by the Games-Howell test. p-value in bottom row indicates p<0.05 according to paired t-test | |
The greatest decline was seen in the hepatotoxic group with 1.2 mL/200 g BW. Compared to the hepatotoxic group, the hepatotoxic with pomegranate juice groups were significantly p<0.05 different. However, administration of 0.4 mL/200 g BW pomegranate juice gave similar effects with the normal group (Table 3). Decreased serum AST levels in all of the hepatotoxic groups with pomegranate juice were observed, with the greatest decline in the hepatotoxic group with 1.2 mL/200 g BW pomegranate juice. It is found that administration 1.2 mL/200 g BW had similar effects with the normal group (Table 4). Reduced serum gamma-GT levels were also observed in all hepatotoxic groups with pomegranate juice (Table 5). The greatest decline was found in the hepatotoxic group with 1.2 mL/200 g BW. Compared with the normal group, administration of 0.8 mL/200 g BW pomegranate juice had similar effects.
Histological studies also provided supporting evidence for the results of the biochemical analysis. The HE staining revealed normal rat’s hepatocyte (Fig. 1a) with level of damage less than pathological control (Fig. 1b). Hemorrhage, widening sinusoid, hepatocyte necrosis and hepatocyte karyolysis were observed in the livers of the rats treated with CCl4 (Fig. 1b). A similar pattern was observed in rat liver within 2 weeks after treatment of 0.4 mL/200 g BW pomegranate juice compared to the pathologic control. Interestingly, the necrotic area was less observed in the group 32 weeks after treatment of 0.4 mL/200 g BW (Fig. 1c).
| Fig. 1(a-e): | Light microphotographs of HE-stained sections of the formalin-fixed livers. The effect of pomegranate juice on histopathological damages induced by CCL4 in rat liver, (a) Normal control group (b) Carbon tetrachloride group (c) Carbon tetrachloride received 0.4 mL/200 g BW pomegranate juice group (d) Received 0.8 mL/200 g BW pomegranate juice group and (e) Received 1.2 mL/200 g BW pomegranate juice group. A: Vena centralis, B: Hemorrhage, C: Sinusoid, D: Hepatocytes, E: Karyolysis and F: Cloudy cytoplasma |
| Table 5: | Serum gamma-GT level in rats |
Values are presented as Mean±SE (n = 5). p-value in column indicates p<0.05 according to one-way ANOVA test. a,b,c,d indicated significant difference between groups according to one-way ANOVA test followed by the Games-Howell test. p-value in bottom row indicates p<0.05 according to paired t-test | |
In the rat liver treated with 0.8 mL/200 g BW pomegranate juice (Group 4), hemorrhage with multinucleated normochromatic hepatocytes were observed (Fig. 1d) and compared to CCl4-induced rats the extent of damage was less. The group treated with 1.2 mL/200 g BW pomegranate juice, compared to the pathologic control group (Group 2), showed induced recovery faster as seen by the absence of hemorrhage and necrosis (Fig. 1e).
DISCUSSION
The present study revealed that CCl4 induction in rats increased ALT, AST, ALP, γ-GT and MDA levels, signaling hepatotoxic liver damage. CCl4 is one of the xenobiotics reported to induce acute and chronic tissue injuries13,14. CCl4 causes acute hepatocyte injuries and membrane integrity alteration, which leads to hepatocyte enzymes leak out15. However, after treatment with pomegranate juice the increase of ALT, AST, ALP and γ-GT were significantly reduced. These results indicated that pomegranate juice has the ability to protect the liver from CCl4-induced hepatocyte injury. This finding is in agreement with one study16 that showed polyphenolic compounds protect liver from CCl4-induced liver cirrhosis. According to other research4 the liver protective and antioxidative effects of phenolic containing plant extracts in CCl4-induced liver injury were related to free radical scavenging effects.
The decrease of these enzymes after treatment with pomegranate juice correlates with the antioxidant and anti-inflammatory potential in pomegranate juice. Pomegranate juice contains flavonoid compounds which have antioxidant and anti-inflammation properties7,10. Flavonoid compounds are known to decrease hepatotoxicity caused by xenobiotic chemicals and are protective against damage caused by oxidative stress17. The antioxidant effect in pomegranate juice stops radical trichloromethyl formation from CCl4 metabolism. The protective factor in pomegranate can be due to its action as an electron donor to free radicals thus stopping the radical reaction damage. As seen in our results, damage that was caused by the radical reaction stopped18. Balancing of enzymes against free radicals such as superoxide dismutase, catalase and peroxidase was maintained by antioxidants from pomegranate. This result is because the antioxidant potential of pomegranate minimized hydrogen peroxide and lipid peroxidation thus preventing membrane damage. Similar research results have shown that the antioxidant effect of pomegranate decreased lipid peroxidation and increased plasma antioxidants19. The results in this study showed that the antioxidant effect of pomegranate also reduced free radicals and decreased oxidative stress in macrophage activity. There are three indicators for decreased oxidative stress. They are decreased cell and DNA damage, decreased glutathione and oxidized glutathione and decreased liver antioxidant enzyme levels.
CONCLUSION
The present study concluded that administration of pomegranate juice with three different doses decreased serum ALT, AST, ALP, gamma-GT and MDA. Administration of 0.8 and 1.2 mL/200 g BW of pomegranate juice effectively reduced liver damage in carbon tetrachloride-induced rats.
SIGNIFICANCE STATEMENTS
This study examines the benefits of pomegranate juice in improving liver damage induced by oxidative stress. This study will help researchers to uncover critical areas of liver disease that may have previously not been available. Thus a more comprehensive understanding about antioxidants in pomegranate juice, may be applied in health sciences and nutrition.
ACKNOWLEDGMENT
This research was supported by the Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.