## 3 Results and Discussion
### Anti-Inflammatory Activity of TAEM and TAEE  
Prior to the anti-inflammatory investigation, the cytotoxicity of TAEM and TAEE on RAW264.7 cells were examined. Figure 6 showed they did not affect the viability of RAW2634.7 cells at a concentration of 12.5-200 ug/mL. The cytotoxicity on RAW264.7 was no more than 3%. The concentration of 100 ug/mL or less was selected to conduct an experimental study on the anti-inflammatory activity.  
NO is a factor synthesized and released by vascular endothelial cells to activate the inflammatory response. The large amount of NO produced by the host in the inflammatory response may provide a defense against the invasion of cells, viruses and parasites, and the non-specific immunity of NO can prevent the invasion of pathogens. To determine the effect of TAEM and TAEE on anti-inflammatory activity, NO production was measured. As shown in Figure 7, a low NO expression (2.46 uM) was exhibited in the control group. After being stimulated by LPS, the NO content was significantly up-regulated to 5.89 uM. In the experimental group, when LPS was present with the co-treatment of different concentrations of tested drugs, the production of NO stimulated by LPS was significantly decreased. More specifically, production of NO was decreased to 4.76, 4.32, 3.98 and 3.26 uM by TAEM, and 4.87, 4.56, 4.31 and 4.07 uM by TAEE at 12.5, 25, 50, 100, 200 ug/mL, respectively. Obviously, TAEM possessed stronger inhibition ability on NO production than TAEE.  
Inducible nitric oxide synthase (iNOS) is an indispensable enzyme for NO synthesis [29]. In activated macrophages, regulation of iNOS levels is critical for NO production. Regulation of NO production or iNOS expression may be an important target for the treatment of inflammation. As shown in Figure 8, the control group was treated without tested drugs or LPS. The basal expression of iNOS protein in RAW264.7 cells was very low. In the LPS group, the expression of iNOS protein in RAW264.7 cells increased significantly after stimulation by LPS, which was 5.24 \(\pm\) 0.16-fold of the control. When TAEM and TAEE (12.5 ug/mL) were given, the expression of iNOS protein decreased significantly to 2.04 \(\pm\) 0.11 and 3.02 \(\pm\) 0.12-fold of the control, TAEM and TAEE both inhibited iNOS expression in a concentration-dependent manner. However, TAEM exhibited a stronger inhibition activity than TAEE especially at a high concentration (50-100 ug/mL).  
Figure 6: Cytotoxicity of TAEM and TAEE in lipopolysaccharide-stimulated RAW264.7 cells. Data are expressed as mean \(\pm\) SD (\(n\) = 3). \({}^{\#\#}\)\(p\) \(<\) 0.05 compared with control group. \({}^{*}\)\(p\)\(<\) 0.05 compared with LPS treatment group.  
The anti-inflammatory activity of components from AC has been reported in many studies. One of the most representative components, Antcin K, plays an important role in the hepatoprotective effect. The mechanism was that Antcin K inhibited the expression of iNOS, thereby exhibiting the anti-inflammatory effect [30]. Besides, the presence of 2,4-dimethoxy-6-methylbenzene-1,3-diol also contributed to its anti-inflammatory activity by reducing NO production [1]. Obviously, at any concentration, there was a big difference between TAEE and TAEM in inhibiting the expression of iNOS, and the difference of Antcin K content was the most critical. The content of Antcin K in TAEM is much higher than that in TAEE, so TAEM has a better anti-inflammatory activity.  
Figure 8: Effects of TAEM and TAEE on the vitality of iNOS. Data are expressed as mean \(\pm\) SD (\(n\) = 3). \({}^{\#\#}\)\(p\) < 0.01 compared with control group (without TAEM or TAEE treatment); \({}^{*}\)\(p\) < 0.05, \({}^{**}\)\(p\) < 0.01 compared with LPS treatment group.  
Figure 7: Effects of TAEM and TAEE on NO production in LPS-stimulated RAW264.7 cells. Data are expressed as mean \(\pm\) SD (\(n\) = 3). \({}^{\#\#}\)\(p\) < 0.01 compared with control group (without TAEM or TAEE treatment); \({}^{*}\)\(p\) < 0.05, \({}^{**}\)\(p\) < 0.01 compared with LPS treatment group.