## 4 Discussion  
NO is essentially a free radical involved in the immune system and inflammation. Furthermore, NO is commonly expressed with and binds with thiol-contained proteins in rheumatoid arthritis patients [33]. In recent studies, SKE has been investigated for possible anti-inflammatory properties, and it is believed that SKE decreases NO levels due to components such as spinasterol-Glc contained within SKE [34; 35]. In addition, xanthone and flavonoids included in CTE have been used to have anti-inflammatory and antioxidant effects for a long time [36; 37]. Previously our study has shown that SKE strongly inhibits (Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-induced MAPKs phosphorylation, activation of NFATc1, and expression of c-FOS in Bone Marrow Macrophage (BMM). Moreover, we confirmed that SKE (5 \(\upmu\)g/kg of body weight, intraperitoneally injection) increased bone density loss by LPS in the LPS-induced bone loss animal model [23]. Ethyl acetate _Cudrantia_ extracts are known to inhibit the proliferation of rheumatoid synovial fibroblast and the production of MMPs, COX-2, and PGE2 by induced IL-18. This extract strongly inhibited the differentiation of osteoclasts via downregulation of MAPKs/c-FOS/NFATc1 signaling pathways [31; 32]. As such, we designed this study to assess the synergy of different components thought to be effective in improving inflammatory diseases such as rheumatoid arthritis. We designed an in vitro model of LPS-induced inflammation in RAW264.7 mouse macrophages, where it was confirmed that it had a significant inhibitory effect on NO levels. Based on the in vitro results, more diverse inflammatory cytokines such as TNF-\(\upalpha\), IL-6, and IL-18 were considered in an in vivo model. Among the various cytokines expressed in the synovial membrane, TNF-\(\upalpha\) and IL-18, which are produced by macrophages and are known as upper cytokines that induce lower inflammatory mediators such as IL-6, various chemokines, and proteases amplify inflammation and cause damage to joints by promoting the proliferation of synovial cells [12; 13; 14; 15; 16; 17]. TNF-\(\upalpha\) and IL-18 cytokines can be observed at high concentrations in the synovial fluid of rheumatoid arthritis patients and promote the production and secretion of metalloproteinase, prostaglandin, and NO in various cells while inhibiting the production of matrix components, thereby reducing inflammation and damage in joints [7; 8; 9]. In addition to stimulating the secretion of inflammatory mediators from synovial cells, it triggers systemic effects such as fever, muscle wasting, and loss of appetite. The expression levels of IL-6, TNF-\(\upalpha\), and IL-18 among inflammatory cytokines induced by LPS were significantly decreased with the administration of the sample [10; 11; 12; 13]. Among the study samples, it was found that the CTE/SKE 2:1 ratio displayed the best anti-rheumatoid arthritis effects both in vitro and in vivo. In the mice treated with this ratio of CTE/SKE, the spleen increased by about 2.1 times, and the lymph nodes decreased by about 2.5 times relative to the vehicle group. During ELISA, TNF-\(\upalpha\), IL-1 \(\upbeta\), and IL-6 levels were decreased by about three, eight, and three times, respectively, while PRG4 expression was increased by about 2.5 times. These values quantify the greatest effect among all samples. In addition, PRG4, bound with LPS receptors including TLR2 and TLR4, increases the expression of both receptors and is considered a key factor in regulating chronic inflammatory inhibitory activity in joints by operating as an antagonist during the TLR signaling process. In this study, we couldn't reveal the cause of the difference in activity according to the CTE/SKE mixing ratio. However, the CTE/SKE 2:1 ratio mixture has decreased the expression of inflammatory cytokines such as TNF-\(\upalpha\), IL-18, and IL-6 in a CIA-induced animal model. We suggest that the combination of these extracts has potential as a therapeutic agent for rheumatoid arthritis.