## 4 Discussion
Since _Lizhong-tang_ displayed the highest anti-inflammatory effect among the 10 drugs, we investigated the inhibition of NO production by the different active compounds of _Lizhong-tang_ further to determine the mechanism of inhibition of inflammation. Among the active compounds of _Lizhong-tang_, liquiritin and 6-gingerol had the highest NO inhibitory effect, and upon simultaneous treatment with liquiritin and 6-gingerol, they inhibited NO production as much as the sum of the NO inhibitory effects of each sample (Figures 3 and 4). When an intracellular inflammatory response occurs, NF-\(\kappa\)B is activated, which affects various cellular signaling molecules. Representative signals that affect its activation include JNK, ERK, and p38 MAPK [42]. They are unphosphorylated in the cytoplasm but are phosphorylated and translocated to the nucleus upon stimulation by LPS. In addition, NF-\(\kappa\)B is known to be a transcriptional regulator that induces the expression of mediators related to inflammation. When activated by LPS, NF-\(\kappa\)B is involved in the expression of genes such as _iNOS_, _COX-2_, and pro-inflammatory cytokines [43, 44, 45]. Therefore, to investigate whether liquiritin and 6-gingerol affected the activation of phosphorylated iNOS, COX-2 MAPKs, an increase in the phosphorylation of iNOS, COX-2 MAPKs was confirmed by Western blotting following LPS stimulation. In addition, the effects of liquiritin and 6-gingerol treatment on the expression of iNOS and COX-2 in the cytoplasm of RAW 264.7 cells were also analyzed (Figure 5). The active compounds of _Lizhong-tang_, liquiritin and 6-gingerol, were found to inhibit LPS-activated JNK, ERK, and p38 MAPK expression, and inhibiting iNOS and COX-2 expression. Mice are the most widely used animal species for the modeling of acute and chronic diseases. Additionally, as in humans, the mouse gut microbiome is stable and resilient. Therefore, mouse model studies are often used to investigate the relationship between gut microbiota and disease [40,41,46,47]. In the present study, animal experiments were performed to investigate the effects and control of intestinal microflora induced by _Lizhong-tang_ and _M. officinalis_ extracts in AAD. The effect of herbal medicines in a mouse model induced by antibiotic administration was investigated by measuring weight change, water intake, and diarrhea status score of the mice. In the control group, which was a model of AAD, sustained weight loss was observed while water intake increased. Similar to the untreated group (normal), the _Lizhong-tang_-treated group showed almost no weight change, with the greatest improvement in diarrheal symptoms (Table 2). Although _Lizhong-tang_ relieved AAD symptoms through gastrointestinal function control, body weight initially reduced due to the decrease in diarrhea but recovered rapidly thereafter. Thus, it can be concluded that _Lizhong-tang_ was effective in improving AAD. This suggests that _Lizhong-tang_ can alleviate diarrheal symptoms by controlling the composition and diversity of intestinal microbes induced by antibiotics. The mammalian gut microbiota consists of a complex bacterial community of at least 1000 species, most of which belong to the phyla Bacteroidetes and Firmicutes. The phylum Bacteroidetes is Gram-negative and includes _Bacteroides_, _Cytophaga_, _Flavobacterium_, whereas the phylum Firmicutes has Gram-positive bacteria, which includes Enterococcuscenceae, Lactobacillusace, and Clostridia [49; 50]. Macrolides have been reported to decrease _Actinobacteria_ and increase _Bacteroides_ and _Proteobacteria_ by altering intestinal microflora. In addition, vancomycin has been shown to decrease _Firmicutes_ in feces, increase _Proteobacteria_, and decrease microbial diversity [51]. Although this study is aimed at screening the efficacy of herbal preparations, there are some limitations in animal experiments that need to be considered in the future studies.