## 3 Results  
### MLD Synergistically Protects the Liver in CCl\({}_{4}\)-Treated Mice  
To investigate the hepatotoxicity effects of MLD, CCl\({}_{4}\) (0.5 mL/kg in olive oil; i.p.) was treated once into mice that had been administered with silymarin (200 mg/kg), LB (100 mg/kg), DL (100 mg/kg), or MLD (200, 100, or 50 mg/kg) for 7 days. On day 0, body weight of vehicle-treated group was 30.15 \(\pm\) 0.84 \(g\), and there were no differences among experimental groups. Body weight on day 7 after treatment with CCl\({}_{4}\), CCl\({}_{4}\) + silymarin, CCl\({}_{4}\) + LB, CCl\({}_{4}\) + DL, CCl\({}_{4}\) + MLD 200, CCl\({}_{4}\) + MLD 100, and CCl\({}_{4}\) + MLD 50 was 88.65 \(\pm\) 4.49, 95.66 \(\pm\) 4.80, 95.83 \(\pm\) 6.02, 93.91 \(\pm\) 4.75, 101.39 \(\pm\) 5.17, 100.44 \(\pm\) 3.26, and 101.15 \(\pm\) 5.75% of vehicle-treated group, respectively. Compared to the vehicle, CCl\({}_{4}\) injection significantly decreased body weight gain (\(p\) < 0.01; Figure 1a), and this was parallel with previous observation that CCl\({}_{4}\) causes anorexia [15; 20]. However, MLD (50-200 mg/kg) significantly inhibited the CCl\({}_{4}\)-mediated reduction in body weight gain (\(p\) < 0.01). In addition, three doses of MLD tended to further increase the body weight gain compared to silymarin, LB, or DL. But body weight gains due to silymarin versus 100-200 mg/kg MLD, LB versus 200 mg/kg MLD, and DL versus 100 mg/kg MLD were only statistically significant (\(p\) < 0.05; Figure 1a). Furthermore, CCl\({}_{4}\)-mediated increases in relative liver weight were significantly attenuated by the three doses of MLD, silymarin, LB, and DL (\(p\) < 0.01; Figure 1b). The magnitude of the MLD-mediated reduction in relative liver weight was greater than that associated with silymarin or either of the herbal extracts alone (\(p\) < 0.01; Figure 1b). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Gene** & **Forward Primer** & **Backward Primer** & **GenBank** & **Amplicon** \\
**Name** & & **ID** & **Size (bp)** \\ \hline Nrf2 & 5\({}^{\prime}\)-CGAGATATACGCAGGA & 5\({}^{\prime}\)-GCTCGCAAATGTTTCTC & AH006764.2 & 79 \\  & GAGGTAAGA-3\({}^{\prime}\) & CAGCTT-3\({}^{\prime}\) & \\ TNF-\(\alpha\) & 5\({}^{\prime}\)-GATCCGGAA & 5\({}^{\prime}\)-AGTTCAGACAGA & \\  & TGCG-3\({}^{\prime}\) & AGAGC-3\({}^{\prime}\) & AB039227.1 & 140 \\ IL-1\(\beta\) & 5\({}^{\prime}\)-ATGCAAACTGTTTCCTGA & 5\({}^{\prime}\)-CAGGACGGATATAGA & NM\_008361.3 & 563 \\  & ACT-3\({}^{\prime}\) & TTCTT-3\({}^{\prime}\) & \\ IL-6 & 5\({}^{\prime}\)-TTCCATCCAGTTGCCTT & 5\({}^{\prime}\)-ATTCCACGATTTTCCA & DQ788722.1 & 170 \\  & CTT-3\({}^{\prime}\) & GAG-3\({}^{\prime}\) & \\ \(\beta\)-actin & 5\({}^{\prime}\)-GCTGAGAGGGAAACTG & 5\({}^{\prime}\)-GAAGCATTTGCGGGTGC & NM\_007393.5 & 516 \\  & TGCGT-3\({}^{\prime}\) & ACGATG-3\({}^{\prime}\) & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Primer sequences for amplifying specific genes. Histopathological analyses using hematoxylin and eosin-stained tissue sections indicated that CCl4 increased modified HAI scores as a result of hepatocyte degeneration and infiltration of inflammatory cells into the hepatic parenchyma (\(p<0.01\)). These CCl4-induced hepatic histopathological changes were significantly prevented by the administration of 50-200 mg/kg MLD (\(p<0.01\); Figure 2a,b). These three different doses of MLD yielded a greater reduction in CCl4-induced acute hepatic damage relative to silymarin or either of the herbal extracts alone, though statistically significant differences in the number of degenerated hepatocytes was only seen in the 200 mg/kg MLD-administered group (\(p<0.01\); Figure 2b, upper). Although the magnitude of the decrease in the number of degenerated hepatocytes seen in the 100 mg/kg MLD group was greater than that seen in either the LB (\(p<0.05\)) or DL (\(p<0.05\)) groups, no differences were evident between the 100 mg/kg MLD and silymarin groups.