Hepatic damage and dysfunction have been considered among the most severe issues impacting human health and have become an epidemic worldwide. Despite of the vigorous efforts to establish therapies, there are still very less effective hepatoprotective drugs available (38). Thus, the quest for new types of compounds with hepatoprotective and antihepatotoxic properties are important. One approach for treating liver damage is to find natural compounds with new scaffolds that are distinct from current conventional hepatoprotective agents.
CCl4 is a well-known hepatotoxin that results in marked degeneration of centrilobular fat and liver damage induced by extensively utilized as an experimental model for the evaluation of potent hepatoprotective agents (39). The findings revealed that the levels of liver serum transaminases i.e. AST, ALT, ALP in rat serum were substantially elevated after CCl4 exposure, which suggested hepatocellular injury as these are adopted as non-invasive hepatic markers. Damage to liver cells altered the integrity of the membrane changes its functional transition, and contributes to enzyme leakage into extracellular spaces. On the other hand, pre-treatment with all the doses (40, 80, and 160 mg kg− 1 b.wt.) of trans-Anethole alleviates the activities of AST, ALT, ALP suggesting that trans-Anethole had beneficial effects against hepatic injury caused by CCl4. In a study, eugenol caused a decrease in the SGOT, SGPT, and ALP serum marker enzyme levels triggered by metanil yellow (My1) in albino Wistar rats (40). It was stated that combination of Curcumin with Sulfamethoxazole decreased the elevated levels of hepatic markers (AST and ALT) against carbon tetrachloride induced liver injury in Swiss albino mice (41). Also, the intoxication of rats with CCl4 caused a substantial rise in TG, Chol, TB, and DB serum levels relative to the control group. trans-Anethole pre-treatment at the maximum dose (160 mg kg− 1 b.wt.) substantially decreased the levels of all measured liver markers as compared to CCl4 treated group, results are as good as those of silymarin. From 3D contour plots it is clear that ALP is positively correlated with SGPT and SGOT, suggesting that ALP increases with increase in SGPT and SGOT (Fig. 6A). Similar results have been found by Das et al., (42) in which MSGPT is positively associated with ALP and SGOT. It was evident from literature that increased levels of SGOT, SGPT, and ALP in animals reflect cellular damage and diminution of cell membrane function (43). SGPT is associated positively with direct bilirubin and total bilirubin, suggesting that with increase in direct bilirubin and total bilirubin, SGPT increases (Fig. 6B). Further, Pearson’s Correlation analysis was performed on different serum marker. The SGOT, SGPT and ALP are positively correlated with direct bilirubin, total bilirubin, urea, creatinine, triglycerides and cholesterol, while negatively related with protein and albumin (Fig. 7). In a study, it was reported that the mean SGPT is positively related with TB (42). This indicates that serum marker enzymes correlated with the non-enzymatic serum markers.
Lipid peroxidation is another significant indicator of liver injury. It contributes to oxidative destruction of cellular membranes which causes the excessive production of free radicals and eventually leads to cell death. MDA content is an intracellular end result that is used to measure the amount of lipid peroxidation (44). Results indicated that there was a significant increase in the lipid peroxidation marker MDA content in CCl4 administrated group and this increase was attenuated due to the application of trans-Anethole for all doses tested but the most significant effect was found at 160 mg kg− 1 b.wt. Ojeaburu and Oriakhi (45) demonstrated that Gallic acid lowered the level of serum markers (AST/ALT) and also decreased the levels of malondialdehyde (MDA) that were raised in rats treated with CCl4. Chebulinic acid significantly reduced the toxicity of CCl4, as the application of chebulinic acid decreased the level of MDA in male ICR mice (46). In the animals of group II, the protein and reduced glutathione content were also found to be reduced. A significant lowering of the protein content and the amount of GSH in the liver specifically reflects a decline in the antioxidant levels. Nonetheless, the pre-treatment of trans-Anethole normalized the level of these biochemical parameters at all tested doses.
The antioxidant enzymes, viz. GR, CAT, and LDH are the main components of the endogenous antioxidant mechanism and perform a vital function in the free radical scavenging activity. The pre-treatment of trans-Anethole in the present study moderates the toxic CCl4 level through the regulation of antioxidative enzyme activity (GR, CAT, and LDH) as in control group I. The data indicates that trans-Anethole can boost the antioxidant defense ability of rat liver. The positive effects of p-coumaric acid were reported against HFD-induced hyperlipidemia in mice models by increasing the level of antioxidative enzymes viz. CAT, SOD, and GSH-Px (47). Also, cinnamaldehyde demonstrated restoration of SOD, CAT, and GSH levels in liver tissues of albino Wistar rats exposed to metanil yellow (MY1) (48).
The elevation of phase I enzymes (mixed-function oxidases) in the CCl4 treated group is a signal of a transformation of CCl4 into hydrophilic electrophile. The biotransformation of CCl4 by CYP2E1 generates more toxic and reactive trichloromethyl free radical (CCl3.) and trichloromethylperoxy radical (CCl3OO.) than the parent compound. These free radicals can bind to biological macromolecules which ultimately result in lipid peroxidation and cause damage to the liver (49). CCl4 treatment was shown to increase the amount of Cyt b5 and NADH Cyt b5 enzymes while the pre-treatment of trans-Anethole helped in the reduction of these enzymes level. Inhibition of CYP2E1 expression is a key pathway for many drugs to combat hepatic injury. However, in all treatment groups, increase in the NADPH cytochrome P450 content, cytochrome P450, and P420 was found with respect to CCl4-treated rats. By significantly decreasing the CYP2E1 enzyme level, curcumin exhibited a protective effect against chronic alcohol administration (50). trans-Anethole treatment (160 mg kg− 1 b.wt.) exerted more protection against CCl4-induced hepatic injury as compared with treatment with trans-Anethole at 40 mg kg− 1 b.wt. and 80 mg kg− 1 b.wt. By ploting data into 3D-graphs it was found that Cyt P420 and Cyt P450 are positively associated, suggesting that with increase in the concentration of Cyt P420, the content of Cyt P450 also increases, on the other hand the concentration of Cyt P420 decreases with increase in the concentration of Cyt b5 (Fig. 6C). Pearson’s correlations analysis was conducted to find the relationship among different liver biochemical parameters in male Wistar rats treated with CCl4. The results indicated that TBARS is negatively correlated with protein, Glu, CAT, GR, Cyt P450, Cyt P420, NADPH cyt P450 and GST, whereas it is positively correlated with LDH, Cyt b5 and NADH cyt b5 reductase. Protein, Glu, CAT and GR showed positive correlation with all studied parameters except LDH, Cytb5 and NADPH Cyt b5 reductase (Fig. 8).
The decreased amount of GST in rats treated with CCl4 resulted in the accumulation of free radicals inside the body that are known to cause several deleterious effects. trans-Anethole and silymarin pre-treatment significantly increases the enzyme level that contributed to the removal of toxic metabolites. Glutathione-S-Transferase performs a crucial function in the detoxification of many endogenous and xenobiotic compounds (51). In a report, it was revealed that 6-gingerol recovered the activities of GST, SOD, and GSH in diethylnitrosamine (DEN) intoxicated in adult male albino rats (52).
The influence of trans-Anethole was examined on the histopathology, expression level of p53, and cyclin D in liver tissue. Hematoxylin and Eosin (H&E) staining findings revealed the histopathological disorders such as degenerated hepatic parenchyma, central dilated vein, fibrosis, hepatocellular ballooning, and hepatic steatosis in CCl4-treated group II. Histopathological findings indicated that trans-Anethole treatment restored the normal architecture of the liver intoxicated by CCl4. Group I, III, and IV showed a zero pathological score indicating its non-toxic effects. The highest damage showed in CCl4 treated group (7/18) (Table 7) (53). In a study p-coumaric acid administration enhanced the appearance of liver histopathology in adult male Wistar rats as indicated by Hematoxylin and Eosin (H&E) staining (54). It was demonstrated that rats treated with eugenol had shown improvement in liver function and structure which was altered by Ischemia/reperfusion (I/R) injury. Eugenol has also inhibited the rise in MDA levels and loss in GSH levels in rats (55).
Pro-apoptotic protein (p53) functions as a tumor-suppressor, nuclear transcription factor, and regulates the transcription of proteins that are involved in DNA repair, cell differentiation, cell cycle, and apoptosis. p53 is maintained at a low level in normal cells but during stress and DNA damage p53 is triggered and induces fibrosis which leads to hepatocyte apoptosis (56). In the present study, the CCl4 treated group II has confirmed a higher expression of p53 as compared to control. With the application of trans-Anethole along with CCl4, the p53 expression level has been significantly reduced in groups V, VI, and VII, confirmed the presence of oxidative DNA damage induced by ROS generated by CCl4. Oncogene Cyclin D is a crucial regulator for cell cycle progression. Overexpression and magnification of cyclin D have been related to aggressive forms of human hepatocellular carcinoma (HCC). Also, increased levels of Cyclin D cause early-onset and development of tumors (57). The current research showed that the CCl4-treated group has more expression of Cyclin D with respect to the control group I, while Cyclin D expression was substantially decreased with the application of trans-Anethole. Expression of p53 and Cyclin D were low in the controlled liver samples. So, the decreasing expressions of p53 and Cyclin D in the current study reveal the hepatic protection by using trans-Anethole treatment. Thus, trans-Anethole greatly decreased oxidative stress and tissue injuries caused by CCl4, and also down-regulated p53 and Cyclin D expression.