## 4 Discussion  
The need of for herbal supplements has been increased because of their merits to control oxidative hepatic damage [36]. Hepatotoxicity induced by CCl4 leads to liver injury, hepatic cell death, and activation of cell necrotic pathways [37]. The long-term exposure of CCl4 leads to chronic liver damage and hepatic carcinoma. Recent strategies focused on usage of plant-derived flavonoids, such as luteolin, as hepato-protective, anti-oxidative, and anti-inflammatory agents [38]. In this study, the effect of orientin on liver protection and anti-inflammatory mechanisms in liver tissues in mice were examined. The pathological score and disease index were significantly reduced by orientin treatment in a concentration of 1 mg/kg bw of mouse. The pathophysiology of liver tissue showed that orientin significantly reduced cirrhosis, necrotic cellular morphology, and inflamed infiltration in hepatic tissue (Figure 1). Some studies showed that the flavonoids improve the hepatic cellular architecture from CCl4-induced hepatic damage [39; 40]. Liver metabolic enzymes play a major role in detoxification, fibrotic steatosis, and inflammatory modifications of the host. Several studies showed plant flavonoids potentially augmented the AST/ALT level in a CCl4-induced mouse model [41; 42; 43]. In this present study, AST/ALT was potentially reduced by orientin 1 mg/kg bw compared to the CCl4-induced mice group. The orientin alone treated mice showed similar levels compared to untreated groups (Figure 2A,B). These data are concurrent with the reported modulatory action of extracts of orientin using acetaminophen-induced liver damage in a mice model showing boosted ALT/AST levels, maintaining a normal liver structure, and moderate activity against the iNOS. The current  
Figure 7: Effect of orientin on hepatic fibrotic signaling markers ZEB-2 and PTEN. Naive mouse-derived HSCs were stimulated with LPS 10 ng for 2 h and treated for 12 h with orientin (30 uM). Treated cells were evaluated for ZEB-2, PTEN, and TNF-\(\alpha\) mRNA and protein were quantified. (**A**) mRNA quantification of ZEB-2, PTEN, and TNF-\(\alpha\) in LPS-stimulated HSCs and LPS with orientin-treated cells was undertaken using SYBR green-based real time PCR assay. (**B,C**) Protein quantification of ZEB-2, PTEN, and TNF-\(\alpha\) in LPS-stimulated HSCs and LPS with orientin (30 uM)-treated cells using immunoblot-based techniques. Values are expressed as three independent experiments and all values are expressed as mean \(\pm\) SD. * Represents the significant comparison with the control and LPS-induced groups. study demonstrated detailed mechanisms of orientin towards reduction of TNF-\(\alpha\), IL-6, IL-8, and IFN-\(\gamma\) in a CCl4-induced mice model and downregulation of ZEB-2 and PTEN fibrotic markers in LPS-induced murine HSCs [44; 45; 46]. The inflammatory cytokines are key players of fibrotic mediators and are associated with worse prognosis of the inflammatory liver disease [26]. The CCl4 administration led to a rise in the level of pro-inflammatory mediators such as TNF-\(\alpha\), IL-6, IL-8, and IFN-\(\gamma\) (Figure 3A-D). However, the orientin treatment showed marked reduction in the inflammatory cytokines compared to the CCl4 control group. Some previous reports showed the similar ameliorative effect of naringenin and orientin on TNF-\(\alpha\), IL-6, and iNOS in mice models [15; 44; 45; 46; 47]. The liver fibrotic markers, ZEB-2 and PTEN, were upregulated in the CCl4-treated group, whereas orientin potentially reduced the expression of ZEB-2 and reached 1.7-fold from a 2.8-fold expression (Figure 3E-G). The PTEN expression was downregulated in the orientin treatment compared to the CCl4 treatment. Protein expression was reduced to 13% compared to the CCl4 group (Figure 3G). The virtual binding of orientin with ZEB-2 and PTEN was achieved and potent binding was observed (Figure 4A,B). The formation of pi-Alkyl hydrophobic bonds between orientin and PTEN changed the structural confirmation and led to less potent receptor molecules.