## 3 Results
### Docking of Orientin with Mouse PTEN and ZEB-2  
The docking study demonstrated the virtual interactions of orientin with phosphate membrane domains of mouse PTEN and stem cell membrane protein of mouse ZEB-2 using molecular docking software tools. The binding energy, ligand bonds, bond distance, and amino acid residue interaction were recorded in Tables 1 and 2 and Figure 4A,B. Orientin interacted with PTEN (\(-6.44\)) amino acid residue of the chain A domain (Figure 4A), such as asparagine, threonine, glycine, and arginine forming a hydrogen bond. Interestingly, the strong binding interactions were observed as a carbon hydrogen bond in asparagine and arginine residue of the PTEN protein. The cation and hydrophobic bonds were observed at tyrosine, proline, and arginine positions of 163 to 170 amino acid residues. Totally, ten different bonds were formed between orientin with mouse PTEN protein. The ZEB-2 protein interacted with orientin with a binding energy of (\(-6.17\)) and, in total,  
Figure 1: Morphological and histological evaluation of liver in mice. (**A**) Control group, (**B**) CCl4-treated group, (**C**) orientin (1 mg/kg)-treated group, and (**D**) CCl4 orientin (1 mg/kg)-treated group. Magnification \(\times 200\).  
Figure 2: Effect of orientin on liver. (**A**) The disease pathology scores for liver injury. (**B**) Effect of orientin on CCl4-induced changes in hepatic functional markers of ALT activity and AST activity. Orientin-treated liver tissues were excised after the 7 th day of ip injection and homogenate using ice cold PBS. All values are expressed as mean \(\pm\) SD. * represents the significant comparison with the control. # Represents the significant comparison with control and CCl4-induced groups.  
six different amino acid residues in a position from 19 to 51 interacted with orientin chain B and C. Orientin formed a sigma bond between ZEB-2 at a position of arginine 166 and confirmed the formation of an overlapped covalent bond (Figure 4B). These interactions demonstrated the involvement of orientin in signaling of hepatic cellular fibrosis and inflammatory markers.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Binding** & **Ligand** & **Intermole** & **Ligand Atoms** & **Docked Amino Acid Residue** \\
**Energy** & **Efficiency** & **Energy** & **(Ring)** & **(Bond Length)** \\ \hline \hline \multirow{7}{*}{\(-6.17\)} & \multirow{7}{*}{\(-0.19\)} & \multirow{7}{*}{\(-6.38\)} & \multirow{7}{*}{\(-6.38\)} & \multirow{7}{*}{\(-6.38\)} & \multirow{7}{*}{\(-6.38\)} & \multirow{7}{*}{\(-6.38\)} \\  & & & & & \\ \cline{4-5}  & & & & & & \\ \cline{4-5}  & & & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Interactions of orientin and ZEB2 receptor residues of target protein.  
Figure 3: Effect of orientin on liver inflammatory markers. The CCl4-induced mice were administered for orientin 1 mg/kg body weight of mice for 7 days and liver tissues were excised. Liver homogenate was prepared using ice cold cell lysis buffer with 0.1% DTT. **(A)** The TNF-\(\alpha\) was estimated using a Cayman ELISA kit. **(B)** IL-6 was estimated in liver lysate using a mouse Cayman ELISA kit. **(C)** IFN-\(\gamma\) was estimated in liver lysate using a mouse Cayman ELISA kit. **(D)** IL-8 was estimated in liver lysate using a mouse Cayman ELISA kit. **(E)** mRNA expression of ZEB-2 and PTEN using real time PCR. **(F,G)** Protein quantification of ZEB-2 and PTEN in CCl4-induced and CCl4 with orientin treatment using immunoblot-based techniques. (The data are represented as the mean \(\pm\) SD.) \(p\) \(<\) 0.05. \({}^{*}\) Represents the significant comparison with control and CCl4-induced groups.