MATERIALS AND METHODS

Animals: Wistar albino rats (156-182 g) were procured from Centre for Toxicology and Developmental Research, Sri Ramachandra University. The animals had free access to standard rat pellet with water supplied ad libitum under strict hygienic conditions. Animals were habituated to laboratory conditions (i.e. room temperature of 25±1°C; relative humidity 45-55% and a 12:12 light/dark cycle) for 48 h prior to experimental protocol to minimize non-specific stress if any. The approval of the Institutional Animal Ethical Committee (IAEC) of Sri Ramachandra University (IAEC/XII/SRU/75/2008) was taken prior to the experiments. All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) from 28-6-2008 to 5-9-2008.

Plant materials: The hydroalcoholic extracts of Fruits of Solanum nigrum and Roots of Cichorium intybus were used in the study (Obtained from pharmacognocy Department, Sri Ramachandra University, Chennai).

Silymarin suspension: The stock solution was prepared by dissolving 100 mg of Silymarin (Micro labs, Bangalore) in 5 mL aqueous suspension of Carboxymethyl cellulose.

Induction of hepatic injury: CCL₄ (E.Merck, Mumbai) was used as hepatotoxine. Liver damage was induced in these rats with 1:1(v/v) mixture of CCL₄ in olive oil, administered at the dose of 1.5 mL kg^-1 i.p on 7th day.

The parameters assessed are:

Experimental design: The experiment was carried out for a period of 14 days with 30 healthy albino rats. Before starting the experiment, the animals were allowed to acclimatize to the laboratory environment for 1 week. Then animals were randomly divided into 5 groups of 6 animals each.

Animals were treated as mention above, food was withdrawn 12 h before carbon tetrachloride administration to enhance the acute liver damage in animals of Groups 2, 3, 4 and 5 and received a single dose of CCl₄ (1.5 mL kg^-1, i.p.) diluted with olive oil (1:1) on 7th day (1 h after treatment).

Assessment of hepatoprotective activity: Blood was withdrawn directly from the heart on 8th and 15th day under light ether anaesthesia. Then, the blood was kept for 30 min without disturbing. The clot was dispersed with a glass rod and then centrifuged for 15 and 20 min at 2000 rpm to separate the serum. Biochemical investigations were carried out to assess liver function viz., total protein, albumin globulin ratio (Kingsley and Frankel, 1939), Bilirubin level, serum alkaline phosphatise (Gowenlock et al., 1981) and serum transaminases (Reitman and Frankel, 1957). Liver homogenate was collected for estimation of markers of oxidative stress viz. GSH, SOD and LPO.

Histopathology: After collection of blood, liver samples were excised and washed with normal saline. A careful record of each liver was made, regarding the size and shape, colour and presence or absence of any nodule. Then, the liver were taken and fixed immediately in 10% formalin solution. Paraffin section were taken at 5 mm thickness processed in alcohol-xylene series and were stained with haematoxylin and eosin (Culling, 1974). The sections were examined microscopically for histopathological changes.

Statistical analysis: Mean, standard error of mean were calculated for each parameters in each group. One way ANOVA was used for multiple group comparisons followed by Post- Hoc Tukey HSD (Honestly Significant Difference) test for inter group comparisons of all the parameters. p-values less than <0.05 was taken to be significant.

RESULTS

Solanum nigrum and CCL₄ group: Levels of ALT, AST and S. Bilirubin were significantly less in Solanum nigrum group as compared with group treated with CCL₄ alone, Mean difference of -92.92 U L^-1 (p<0.0001), -133.37 U L^-1 (p<0.0001), -0.71 mg dL^-1 (p<0.0001), respectively. Levels of total protein were significantly high in Solanum nigrum group as compared to group treated with CCl₄ alone Mean difference+0.60 mg dL^-1 (p<0001) (Table 1).

Levels of SOD and GSH were significantly more in Solanum nigrum group as compared with group treated with CCL₄ alone, Mean difference of +2.23 U mg^-1 protein (p<0.0001) and 3.35 μmol mg^-1 protein (p<0.004), respectively. Levels of LPO were significantly low in Solanum nigrum group as compared to group treated with CCL₄ alone, Mean difference of -0.168 μmol mg^-1 protein (p<0.0001) (Table 1).

Cichorium intybus and CCL₄ group: Levels of ALT, AST and S.Bilirubin were significantly less in Cichorium intybus group as compared with group treated with CCl₄ alone. Mean difference of -114.44 U L^-1 (p<0.01), -163.38 U L^-1 (p<0.0001), -0.91 mg dL^-1 (p<0.0001) respectively. Levels of Total protein were significantly high in Cichorium intybus group as compared to group treated with CCL₄ alone. Mean difference of +0.99 mg dL^-1 (p<0.0001) (Table 1).

Levels of SOD and GSH were significantly more in Cichorium intybus group as compared with group treated with CCL₄ alone. Mean difference of +3.54 U mg^-1 protein (p<0.0001) and +5.82 μmol mg^-1 protein (p<0.0001), respectively. Levels of LPO were significantly low in Cichorium intybus group as compared to group treated with CCL₄ alone. Mean difference -0.251 μmol mg^-1 protein (p<0.0001) (Table 1).

Solanum nigrum and Silymarin group: Levels of ALT, AST and S. Bilirubin were significantly less in silymarin group as compared with group treated with Solanum nigrum alone, Mean difference of ALT, AST, S.Bilirubin and Total protein 44.73 U L^-1, 62.19 U L^-1, 0.4166 mg dL^-1 and -0.768 mg dL^-1 respectively. Compared to Silymarin, Solanum nigrum has got significantly less antioxidant activity. Mean difference of SOD, GSH and LPO was -2.59 U mg^-1 protein, -5.53 μmol mg^-1 protein and +0.161 μmol mg^-1 protein, respectively.

Cichorium intybus and Silymarin group: Levels of ALT, AST and S.Bilirubin were significantly less in silymarin group as compared with group treated with Cichorium intybus alone, Mean difference of ALT, AST, S. Bilirubin and Total protein +23.21 U L^-1, +32.18 U L^-1, +0.22 mg dL^-1 and -0.37 mg dL^-1 respectively. Compared to Silymarin, Cichorium intybus has got significantly less antioxidant activity. Mean difference of SOD, GSH and LPO was -1.28 U mg^-1 protein, -3.06 μmol mg^-1 protein and +0.078 μmol mg^-1 protein respectively.

Cichorium intybus and Solanum nigrum group: Levels of ALT, AST and S.Bilirubin were significantly less in Cichorium intybus group as compared with group treated with Solanum nigrum, Mean difference of -21.52 U L^-1 (p<0.02), -30.01 U L^-1 (p<0.008), -0.191 mg dL^-1 (p<0.044) respectively. Levels of Total protein were significantly high in Cichorium intybus group as compared to group treated with Solanum nigrum. Mean difference +0.396 mg dL^-1 ( p<05) (Table 1).

Levels of SOD and GSH were significantly more in Cichorium intybus group as compared with group treated with Solanum nigrum, Mean difference of -1.30 U mg^-1 protein (p<0.039) and -2.46 μmol mg^-1 protein (p<0.048), respectively). Levels of LPO were significantly less in Cichorium intybus group as compared to group treated with Solanum nigrum, Mean difference -0.0833 (p<0.031) (Table 1).

Table 1:	Results of liver function test in-group 1 to 5
*Results are expressed as Mean±SEM (n = 6). **p values of<0.05 (statistically significant). ALT: Alanine transaminase, AST: Aspartate aminotransferase, CCL4: Carbon tetrachloride, CMC: Carboxymethyl Cellulose GSH: Glutathione, SOD: Superoxide Dismutase, LPO: Lipid peroxidase

Fig. 1(a-d):

Photomicrographs of rat liver (HPE). (a) Normal control (CMC) Positive control (CCL4) (Normal arrangements of hepatocytes with nuclei) (Intense centrilobular necrosis) (b) Silymarin (100 mg kg-1, p.o.) (Scanty degeneration of hepatocytes without any necrotic changes) (c) Solanum nigrum (250 mg/kg/day, p.o.) and (d) Cichorium intybus (75 mg/kg/day, p.o. (Moderate centrilobular hepatocytes degeneration) (Mild centrilobular hepatocytes degeneration)

Histopathological study
Control group: Histology of liver from control group showed portal triad, rows of hepatocytes and normal arrangements of hepatocytes with nuclei.

CCl₄ group: Histology of CCl₄ treated group rat liver showed intense centrilobular necrosis, bridging type of necrosis, congestion of sinusoids with small lipid globules and hydropic vacuolation Fig. 1a.

Silymarin+CCl₄ group: Histology of liver section from Silymarin+CCl₄ treated rats showed marked decrease in inflammation, very scanty degeneration of hepatocytes without any necrotic changes and mild fatty degeneration Fig. 1b.

Solanum nigrum+CCl₄ group: Histology of liver sections from Solanum nigrum+CCl₄ treated rats revealed moderate centrilobular hepatocytes degeneration, few areas of congestion with mild fatty changes Fig. 1c.

Cichorium intybus+CCl₄ group: Histology of liver sections from Cichorium intybus+CCl₄ treated rats revealed mild centrilobular hepatocytes degeneration, few areas of congestion with mild fatty changes Fig. 1d.

DISCUSSION

CCl₄ mediated acute hepatotoxicity was taken here as the experimental model for liver injury. It has been established that CCl₄ is accumulated in hepatic parenchymal cells and metabolically activated by cytochrome p-450 dependent monoxygenases to form a trichloromethyl free radical (CCl^*₃) which alkylates cellular proteins and other macromolecules with simultaneous attack on polyunsaturated fatty acids in the presence of oxygen to produce lipid peroxidases leading to liver damage (Recknagel et al., 1989).

The Mean serum AST, ALT and Bilirubin levels in control group rats were 45.90 U L^-1, 81.27 U L^-1 and 0.29 mg dL^-1 respectively, whereas in CCl₄ treated rats, the levels significantly increased to 217.70 U L^-1, 327.37 U L^-1 and 1.62 mg dL^-1 respectively, similar observations of increased AST, ALT and Bilurubin levels are seen in ethanol induced hepatotoxicity (Baranisrinivasan et al., 2009). Solanum nigrum and Cichorium intybus significantly reduced the serum AST, ALT and Bilirubin levels to 124.77 U L^-1and 103.25 U L^-1, 194.00 U L^-1and 163.99 U L^-1 and 0.89 mg dL^-1and 0.70 mg dL^-1 respectively. Silymarin significantly reduced the serum AST, ALT and Bilirubin levels to 80.04 U L^-1, 131.80 U L^-1 and 0.48 mg dL^-1 respectively. There was significant decrease in serum protein levels after CCl₄, similar observations were made by others (Samudram et al., 2008). In the present study serum protein levels was reversed by Solanum nigrum, Cichorium intybus and Silymarin with statistical significance.

The Mean levels of SOD and GSH content of liver in control group rats were 12.84 U mg^-1 protein and 22.40 μmol mg^-1 protein respectively, whereas in CCl₄ treated rats, the levels significantly decreased to 5.33 U mg^-1 protein and 9.51 μmol mg^-1 protein ,respectively similar parameter are used to assess antioxidant properties against paracetamol induced hepatotoxicity (Malathi and Gomez, 2007). Solanum nigrum and Cichorium intybus significantly increased the SOD and GSH levels to 7.57 U mg^-1 protein and 12.86 U mg^-1 protein and 8.87 μmol mg^-1 protein and 15.33 μmol mg^-1 protein respectively. Silymarin significantly increased the SOD and GSH content of liver to 10.16 U mg^-1 protein and 18.40 μmol mg^-1 protein, respectively. There was significant increase in LPO levels after CCl₄ which was reversed by Solanum nigrum, Cichorium intybus and Silymarin. Similar studies against CCl₄-induced rat liver injury through potentiating antioxidant capacity of liver cells and prevention of oxidative stress that accompanied with CCl₄ hepatotoxicity was established (Khadr et al., 2007).

Histology of control group rat liver showed normal architecture of hepatic lobule. Histology of CCl₄ treated group rat liver showed intense centrilobular necrosis, bridging type of necrosis, congestion of sinusoids with small lipid globules and hydropic vacuolation which proves successful development of liver injury model. Similar histopathological observations were made by other researchers (Prakash et al., 2008; Turel et al., 2009; Chavda et al., 2010). Histology of Solanum nigrum and CCl₄ treated group rats showed moderate centrilobular degeneration of hepatocytes and mild congestion. Histology of Cichorium intybus and CCl₄ treated group rats showed mild centrilobular degeneration of hepatocytes and mild congestion. Histology of Silymarin and CCl₄ treated group rats showed almost normal lobular architecture with mild congestion.

The extract of Solanum nigrum and Cichorium intybus confers significant protection against CCL₄ induced liver injury and probable mechanism is by maintenance of structural integrity of hepatocyte cell membrane and may be due to their ability to suppress the oxidative degradation of DNA (Sultana et al., 1995). Histological examination of the liver also confirms these findings by showing moderate centrilobular hepatocytes degeneration, few areas of congestion with mild fatty changes.

CONCLUSION

The results indicate that the extract of Cichorium intybus and Solanum nigrum has significant hepatoprotective activity in albino rats with CCL₄ liver injury. Cichorium intybus has got significantly better hepatoprotective activity as compared to Solanum nigrum. Both are commonly used traditional herbs which are safe, cost effective and grow widely in India and used by different ethnic groups for various diseases. Further studies are necessary to evaluate the molecular and biochemical basis of its pharmacological action as a hepatoprotective agent.