## 3 Results and Discussion
### Evaluation of Anti-Inflammatory Activity of Limonoids  
The anti-inflammatory activity of the seven obtained limonoid derivatives, together with another eight natural limonoid compounds [18], shown in Figure 6, was evaluated and compared with the use of cell experiments. The results of the anti-inflammatory activity bioassay of mouse macrophage cells RAW264.7 are listed in Table 1. The natural compounds 30-O-Acetylhainangranatumin E (**9**) and Xylgranatin A (**13**) obviously inhibited LPS-induced RAW264.7 cells from producing and releasing inflammatory factors, such as TNF-\(\alpha\). Compound **13** was particularly prominent, of which the inhibition ratio reached 90.0%. The inhibition ratio of compound **9** was 55.3%. Other natural limonoid derivatives possessed a relatively low inhibition ratio, especially for compounds **11**, **12**, and **15**, which did not show any inhibitory effects. This result was consistent with the results of [PERSON]'s work, in which the studied natural limonoid derivatives also showed vastly different inhibition ratios, ranging from 10.5 to nearly 100%, at the same concentration of 20 uM [19]. In contrast, the seven compounds yielded by the hydrogenation of limonin all showed decent anti-inflammatory activity. Their inhibition ratio of TNF-\(\alpha\), an inflammatory factor released by LPS-induced RAW264.7 cells, ranged from 26.9 to 37.8%. Among them, compound **3**, which had a high degree of hydrogenation, presented the highest inhibition ratio of 37.8%, while compound **2**, which was only slightly hydrogenated, presented the lowest inhibition ratio of 26.9%. The result suggested that the anti-inflammatory activities of hydrogenation products of limonin were related to the degree of hydrogenation. Figure 6: Eight Natural Limonoid Compounds Studied in This Work. Moreover, the BV2 cell model was employed to further investigate the ameliorating effects and cytotoxicity of limonoid compounds on LPS-induced BV2 cell inflammation. The results are shown in Table 2. The natural limonoid derivatives **9**, **10**, **13**, **14**, and **16** presented inhibitory ratios of NO greater than 30% on LPS-induced BV2 cell inflammation at a concentration of 10 \(\upmu\)M. Among them, compound **13** had the best inhibitory activity on LPS-induced BV2 cell inflammation, corresponding to an inhibitory ratio of 63.77%. The better bioactivity of **13** might be related to the unique 2,3-dimethacrylate group. Similar results were observed in [PERSON]'s study, which showed that natural limonoid derivatives with 2,3-dimethacrylate group generally presented higher anti-inflammatory activity [20]. Among the seven compounds obtained by the hydrogenation of limonin, compound **3** offered the best result at 1 \(\upmu\)M concentration, and the inhibitory ratio of NO was 10.27%, which was comparable to that of natural derivatives **9**, **15**, and **16**. At 10 \(\upmu\)M concentration, compound **8** gave the best result of a 16.16% NO inhibitory ratio. In addition to anti-inflammation activity, the cytotoxicity of limonoid derivatives were also evaluated (Table 3). At the concentration of 1 \(\upmu\)M, all compounds discussed in this study showed low cytotoxicity, with cell viability in the range of 93-100%. However, at the concentration of 10 \(\upmu\)M, compound **11** showed slightly more cytotoxicity with a cell viability of 88.1%. Compared to natural limonoid derivatives, the seven synthesized limonoid derivatives were less cytotoxic, and the corresponding cell viabilities were all higher than 90% at 10 \(\upmu\)M. The better safety and gentleness of the synthesized compounds should be ascribed to the very low cytotoxicity of limonin, which has been widely demonstrated in BV2 cells and other cell models [21, 22].