## 3 Results
### MLD Synergistically Attenates Hepatocyte Apoptosis  
To explore whether MLD protects the liver by reducing hepatocyte damage, we measured the activities of serum biomarkers related to hepatotoxicity. Administration of MLD at three different doses, silymarin, LB, or DL significantly attenuated CCl4-mediated increases in serum AST and ALT activities (\(p<0.01\)). The effects seen in response to the three doses of MLD were more potent than those seen in response to silymarin, LB, or DL (\(p<0.05\), AST activity in the LB versus the 50 mg/kg MLD group; \(p<0.01\), other comparisons; Figure 3a). Next, we quantified the number of apoptotic cells via immunohistochemical staining of hepatic tissues using antibodies against cleaved caspase-3 and cleaved PARP. Compared to the vehicle, CCl4 injection significantly increased the numbers of  
Figure 1: Effects of mixture of lemon balm and dandelion extracts (MLD) on body weight gain and relative liver weight in CCl4-treated mice. Mice were treated with silymarin (Sily), lemon balm leaf extract (LB), dandelion leaf extract (DL) or one of three doses of MLD once daily for 7 days. On day 6, CCl4 was injected at 1 h after the final administration of herbal drugs. Body weight gain (**a**) and relative liver weight (**b**) were measured. All values are expressed as mean \(\pm\) SD of ten mice. **\(p<0.01\) between the vehicle and CCl4 groups; \({}^{\#}p<0.01\), \({}^{\#}p<0.05\) versus CCl4-injected mice; \({}^{++}p<0.01\), \({}^{+}p<0.05\) between Sily- and MLD-treated mice; \({}^{\$58}p<0.01\), \({}^{\$}p<0.05\) between LB- and MLD-treated mice; \({}^{\$6k}p<0.01\), \({}^{\$}p<0.05\) between DL- and MLD-treated mice. cleaved caspase-3- and cleaved PARP-positive cells in hepatic tissue (\(p<0.01\); Figure 3b,c), indicating that CCl4 provokes apoptosis in hepatocytes. By contrast, the three doses of MLD, silymarin, LB, and DL all significantly inhibited the increases in number of cleaved caspase-3- and cleaved PARP-positive cells (\(p<0.05\), number of cleaved PARP-positive cells in the CCl4 versus the DL group; \(p<0.01\), other comparisons; Figure 3b,c). Moreover, the magnitude of the inhibition by MLD was greater than that by silymarin or either of the herbal extracts alone (\(p<0.05\), number of cleaved caspase-3-positive cells in the silymarin versus the 100 mg/kg MLD group and in the LB versus the 50 or 100 mg/kg MLD group; \(p<0.05\), number of cleaved PARP-positive cells in the LB or DL versus the 50 mg/kg MLD group; \(p<0.01\), other comparisons; Figure 3c). Figure 2: MLD protects the liver from CCl4-induced damage. (**a**) Representative hepatic tissues stained with hematoxylin and eosin. Scale bars, 200 \(\upmu\)m. (**b**) The numbers of degenerated hepatocytes (upper) and infiltrated inflammatory cells (middle), and modified HAI scores (lower) were quantified using an image analyzer. All values are expressed as mean \(\pm\) SD of ten mice. **+**\(p<0.01\) between the vehicle and CCl4 groups; **+**\(p<0.01\) versus CCl4-injected mice; **+**\(p<0.01\), **+**\(p<0.05\) between Sily- and MLD-treated mice; **+**\(p<0.01\), **+**\(p<0.05\) between LB- and MLD-treated mice; **+**\(p<0.01\), **+**\(p<0.05\) between DL- and MLD-treated mice. CV, central vein; PT, portal triad. MLD Synergistically Alleviates CCl\({}_{4}\)-Mediated Oxidative Stress by Restoring Antioxidant Activity  
To investigate whether MLD prevents CCl\({}_{4}\)-induced hepatic apoptosis by reducing oxidative stress, hepatic tissues were immunohistochemically stained using a nitrotyrosine (a marker of nitrosative stress) antibody. Compared to the vehicle, CCl\({}_{4}\) significantly  
Figure 3: MLD attenuates apoptosis of hepatocytes. (**a**) Effects of MLD on serum AST and ALT activities. (**b**) Hepatic tissues were immunostained using cleaved caspase-3 or cleaved polymerase (PARP) antibodies. Scale bars, 200 \(\mu\)m. (**c**) Positive cells in the hepatic parenchyma around the central vein were counted. All values are expressed as mean \(\pm\) SD of ten mice.