Introduction

Liver diseases are considered as one of the serious health problems. Steroids, vaccines and antiviral drugs that are employed as therapy for liver diseases have potential adverse effects especially when administered for long periods (Sehrawat et al., 2006). There is a worldwide trend for use of traditional herbal drugs for the treatment of liver diseases. Several leads from plant sources have been found as potential hepatoprotective agents with diverse chemical structures. Although, a big list of hepatoprotective phytomolecules are reported in the scientific literature, only few were potent against various types of liver damages. Of these, silymarin, andrographolide, neoandrographolide, curcumin, picroside, kutkoside, phyllanthin, hypophyllanthin, and glycyrrhizin have largely attracted the scientific community (Rajesh and Latha, 2004).

Zizyphus jujuba (Rhamnaceae) fruit commonly known as jujube or Chinese date is used widely for the treatment of different diseases such as treatment of chronic fatigue, loss of appetite, diarrhoea, anaemia, irritability and hysteria. The fruits are also believed to possess activities such as anodyne, anticancer, refrigerant, sedative, stomachic, styptic and tonic. The principle chemical constituents of Zizyphus jujuba fruit are flavonoids, saponins, tannins, vitamins A, B₂, and C, sugars, mucilage, calcium, phosphorus, and iron (www.ibiblio.org, 2009). Zizyphus jujuba is reported for anti-complementary (Lee et al., 2004), anti-cancer (Huang et al., 2007), hypoglycemic (Iganacimuthu and Amalraj, 1998) and anxiolytic activities (Peng et al., 2000)7. Furthermore, traditionally, jujube is used prophylactically for liver diseases (Khare, 1995). The fruits are also used in Chinese medicine to strengthen liver function (www.ibiblio.org, 2009).

In view of the firm belief of Chinese and Indian system of medicine, for hepatoprotective role of Zizyphus jujuba in treating hepatic diseases, we thought it is imperative to evaluate the hepatoprotective activity of Zizyphus jujuba fruits using modern scientific techniques in experimental models.

Materials and Methods

Experimental animals – Laboratory bred female Sprague-Dawley (SD) rats weighing 175-250 g were housed at 25° ± 5°C in a well-ventilated animal house under 12:12 h light dark cycle. Institutional Animal Ethics Committee approved the experimental protocol. The animals were maintained under standard conditions in an animal house as per the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).

Plant material- The shade dried fruits of Zizyphus jujuba were purchased from local market in Bangalore (India) and Regional Research Institute (Ay), Bangalore authenticated the fruits. A specimen (RRCBI-Ap2901) has been preserved for future reference. The fruits were mechanically powdered and subjected to exhaustive extraction in a soxhlet apparatus-using methanol. The extract was concentrated under reduced pressure and stored in a desicator until further use.

Phytochemical estimations of the extract- The methanolic extracts of Zizyphus jujuba fruits (MEZJ) were subjected to qualitative analysis for various phytoconstituents such as alkaloids, carbohydrates, glycosides, phytosterols, saponins, tannins, proteins, amino acids and flavonoids

Acute toxicity study - The acute oral toxicity study was performed according to the OPPTS guidelines (Office of Prevention, Pesticide and Toxic Substance) following the limit test procedure (www.epa.gov/opptsfrs, 2008). The animals were fasted over night prior to the experiment. Test dose of 2 g/kg and 5 g/kg were given orally to mice. Both doses were found to be safe. Hence, 1/5th, 1/10th and 1/20th of the maximum safe dose corresponding to 1000, 500 and 250 mg/kg orally were selected as high, medium and low doses respectively.

Paracetamol (PCM) induced hepatic damage (Chattopadhyay, 2003)- The animals were divided into six groups (n=6) and treated for 10 days orally. The animals of Group I and II were treated with vehicle for 10 days. Group III animals received silymarin (100 mg/kg, p.o) as standard drug. The groups IV, V and VI animals were given low (250 mg/kg, p.o), medium (500 mg/kg, p.o) and high dose (1000 mg/kg, p.o) of MEZJ respectively. On 11th day, PCM (2 g/Kg, p.o.) suspended in sucrose solution (40% w/v) was administered in 3 divided doses to animals of groups II, III, IV, V and VI. Food was withdrawn12 hr before PCM administration to enhance the acute liver toxicity. Animals were sacrificed 48 hr after the administration of PCM. Blood samples were collected and the serum was used for determinations of activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and serum bilirubin levels (Total and direct). The liver was immediately isolated and washed with normal saline, blotted with filter paper and weighed. Liver tissue homogenate (LTH) was prepared in sucrose solution (0.25 M) and used for estimation of endogenous marker enzymes and biological antioxidants viz., superoxide dismutase (SOD) (Erich and Elastner, 1976) and catalase (Eva, 1988) activities. The liver was then subjected to histopathological examination.

Thioacetamide (TAA) induced hepatic toxicity (Roy et al., 2006) - The animal groupings and treatment was similar to that used in PCM induced liver damage. Single dose of TAA (100 mg/kg, s.c) diluted with distilled water (2% solution) was administered on 10th day to animals of groups II, III, IV, V and VI and the animals were sacrificed 48 hr after administration of TAA. Biochemical estimations were carried out in serum and LTH as mentioned above in PCM model.

Histological analysis- Liver sections were prepared, stained with hematoxylin and Eosin (H&E) and change in histology were recorded.

Statistical analysis- Results are expressed as mean  SEM. Statistical significance was assessed using One-way Analysis of variance (ANOVA) followed by Tukey-Karmer multiple comparison tests. p<0.05 was considered significant.

Results

Preliminary phytochemical investigation - The preliminary phytochemical investigation of the MEZJ extract showed that it contains carbohydrates, tannins, flavanoids and saponins. The percentage yield of MEZJ was found to be 17%.

Paracetamol induced liver toxicity – After 48 hr of administration of PCM, the serum levels of ALT, AST, ALP and bilirubin (total and direct) were markedly increased when compared to normal control. Pretreatment with low dose of MEZJ (250 mg/kg, p.o), medium dose of MEZJ (500 mg/kg, p.o) and silymarin significantly reduced the levels of these biochemical markers compared to PCM control (P0.001) (Table-1). Similarly, there was significantly high level of these biomarker enzymes in LTH of animals pretreated with low and medium dose of MEZJ (250 and 500 mg/kg, p.o) and silymarin compared to PCM control (Table-2). Pretreatment with low and medium MEZJ (250 and 500 mg/kg, p.o) and silymarin also attenuated the increase in the liver weight observed after PCM intoxication (Table-3). The activities of SOD and catalase in LTH were significantly increased in animals pretreated with all the three doses of MEZJ (250, 500 and 1000 mg/kg) and silymarin when compared to PCM control (Table-3). Histopathological studies revealed that PCM induced centrilobular necrosis and mild hydropic degeneration (Figure-2). Prophylactic administration of low and medium doses of MEZJ (250 and 500 mg/kg, p.o) and silymarin reduced hydropic degeneration of liver cells when compared to PCM control (Figure-3). The high dose of MEZJ (1000 mg/kg, p.o) was less effective in reducing PCM induced hepatotoxicity.

Figure 1

Table 1-Effects on serum ALT, AST, ALP and Bilirubin (Total and Direct) level in paracetamol (PCM) induced acute hepatotoxicity in rats

Figure 2

Table 2- Effect on ALT, AST, ALP and Bilirubin (Total and Direct) level in Liver tissue homogenate in paracetamol (PCM) induced acute hepatotoxicity in rats

Figure 3

Table 3- Effect on liver weight, SOD and Catalase in paracetamol (PCM) induced acute hepatotoxicity in rats

Values are mean ± SEM, n = 6, a P <0.05, b P <0.01, c P <0.001 Vs vehicle control. *P<0.05, ** P <0.01, *** P <0.001 Vs PCM control. MEZJ= Methanolic extract of Zizyphus jujuba fruits.

Figure 4

Figure 1- The representative microphotographs of H & E (200 X) stained histological section of liver from rat treated with Vehicle control showing normal architecture

Figure 5

Figure 2- Microphotographs of H & E (200 X) stained histological section of liver from rat treated with paracetamol (2 g/kg ) showing centrilobular necrosis with hydropathic changes

Figure 6

Figure 3- Microphotographs of H & E (200 X) stained histological section of liver from rat treated with paracetamol (2 g/kg ) and MEZJ (250 mg/kg) showing partially preserved hepatocytes and architecture with small areas of necrosis and mild hydropathic necrosis

Thioacetamide induced liver injury- The effects observed in this model were almost similar to that observed in PCM induced hepatotoxicity. The low dose of MEZJ (250 mg/kg, p.o) and medium dose of MEZJ (500 mg/kg, p.o) were effective in reducing TAA induced damage while the high dose of MEZJ (1000 mg/kg, p.o) was less effective in reducing TAA induced hepatic damage (Table-4, 5 & 6). Histopathological studies revealed that TAA produced cloudy swelling surrounding central vein, hydropic degeneration and coagulative necrosis (Figure-4). The hydropic degeneration was reduced by silymarin and low and medium dose of MEZJ (250 and 500 mg/kg, p.o) (Figure-5).

Figure 7

Table 4-Effects on serum ALT, AST, ALP and Bilirubin (Total and Direct) level in thioacetamide (TAA) induced acute hepatic necrosis in rats

Values are mean ± SEM, n = 6, a P <0.05, b P <0.01, c P <0.001 Vs vehicle control. *P<0.05, ** P <0.01, *** P <0.001 Vs TAA control. MEZJ= Methanolic extract of Zizyphus jujuba fruits.

Figure 8

Table 5-Effect on ALT, AST, ALP and Bilirubin (Total and Direct) level in Liver tissue homogenate in Thioacetamide (TAA) induced acute hepatic necrosis in rats

Figure 9

Table 6-Effect on liver weight, SOD and Catalase in Liver tissue homogenate in Thioacetamide (TAA) induced acute hepatic necrosis in rats

Values are mean ± SEM, n = 6, a P <0.05, b P <0.01, c P <0.001 Vs vehicle control. *P<0.05, ** P <0.01, *** P <0.001 Vs TAA control. MEZJ= Methanolic extract of Zizyphus jujuba fruits.

Figure 10

Figure 4- Microphotographs of H & E (200 X) stained histological section of liver from rat treated with thioacetamide (100 mg/kg ) showing cloudy swelling of hepatocytes around central vein, hydropic degeneration and mild coagulative necrosis

Figure 11

Figure 5- Microphotographs of H & E (200 X) stained histological section of liver from rat treated with thioacetamide (100 mg/kg ) and MEZJ (250 mg/kg) showing mild protection of hepatocytes

Discussion

The present study reveals the hepatoprotective activity of MEZJ against PCM and TAA induced hepatic damage in rats. The results show that MEZJ is effective in low and medium doses (250 mg/kg, p.o and 500 mg/kg, p.o) while the effect is much less with higher dose (1000 mg/kg, p.o).

PCM is a commonly used as analgesic and antipyretic drug and is safe in therapeutic doses but produces fatal hepatic necrosis with toxic doses (Mitchell et al., 1973). The toxic effect of PCM is due to oxidative damage induced by its metabolite, N-acetyl-p-benzoquinoneimine, produced by the action of cytochrome P-450 in the liver. This metabolite reacts with reduced glutathione (GSH) to yield non-toxic 3-GS-yl-PCM. Depletion of GSH causes the remaining quinone and other natural endogenous oxygen species to bind to cellular macromolecules leading to cell death (Udem et al., 1997). Damage induced by PCM is accompanied by an increase in the levels of serum biomarker enzymes of liver damage and decreased levels of the same in LTH. Low and medium doses of MEZJ (250 mg/kg and 500 mg/kg) significantly reduced the levels of these biomarkers in serum and there was a corresponding increased level of these biomarkers in LTH when compared to PCM control. The low and medium dose of MEZJ also increased the activities of SOD and catalase in the LTH indicating that they have good antioxidant effect. Drugs having antioxidant activity are also effective in treating PCM induced hepatotoxicity by scavenging the free radicals produced by PCM metabolism, thereby preventing the liver damage induced by both PCM metabolite as well as subsequent depletion of glutathione. Inclination in endogenous antioxidant activities in LTH is an indication of structural integrity and protection to the hepatic cells by prior administration of MEZJ. Elevated activity of catalase in LTH is more beneficial than increase in SOD activity alone because without a simultaneous increase in catalase activity, increased SOD activity may lead to intracellular accumulation of H₂O₂ with detrimental effects (Das et al., 1995). The PCM induced a significant increase in liver weight, which is due to the blocking of secretion of hepatic triglycerides into the plasma (Yoko et al., 2005).

Toxicity experienced by the liver during TAA poisoning results from the production of a metabolite, thioacetamide s-oxide, which is a direct hepatotoxin responsible for change in cell permeability and inhibition of mitochondrial activity followed by cell death (Kumar et al., 2004). Pretreatment with low and medium doses of MEZJ (250 and 500 mg/kg p.o) significantly reversed the elevated serum enzyme markers in animals treated with TAA. This effect may also be due to antioxidant effect of MEZJ, which may neutralize the reactive metabolite of TAA.

The results of the present study support the traditional claim in both Indian and Chinese medicine that fruits of Zizyphus jujuba possess hepatoprotective effect. The exact constituent(s) responsible for this effect cannot be explained with the present data. It is speculated that antioxidant and hepatoprotective effects of MEZJ may be due to the presence of flavonoids (Raj and Kapoor, 1999).

Conclusion

The low and medium doses of MEZJ (250 mg/kg and 500 mg/kg) possess good hepatoprotective activity against PCM and TAA induce liver damage, whereas high dose (1000 mg/kg, p.o) of MEZJ failed to produce similar effect. The protective effect of the extract may be due to its antioxidant activity.

Acknowledgement