## 4 Discussion
### Antioxidant and Anti-Inflammatory Biomarkers  
In our study, we performed the collagenase-induced osteoarthritic experimental model in rats to evaluate the anti-inflammatory and antioxidant effect of JR extract compared to EA as a compound with beneficial results demonstrated in OA. The oxidative stress was evaluated by MDA level and by the enzymatic activity of CAT and SOD in blood. MDA is a well-known marker for the assessment of redox imbalance which leads to an increase level of the reactive oxygen species (ROS). Catalase is an antioxidant enzyme responsible for the conversion of hydrogen peroxide in water and oxygen, while SOD is an important enzymatic scavenger of oxygen radicals. The results of our work showed that there were no changes in MDA concentrations across all treated groups in comparison with the control. After 2 weeks of EA administration, SOD activity had significantly increased. Additionally, a favorable effect of antioxidant defense was also shown in SOD activity in JR-treated animals with OA. Inflammatory cytokines, such as TNF-\(\alpha\), IL-1B and IL-6, besides exhibiting their pro-inflammatory activity, also proved to promote osteoclast activity leading to bone loss and  
Figure 3: RANKL (**a,b**) and hydroxyproline (**c,d**) levels in serum at 2 weeks and 4 weeks of treatments’ administration with EA, JR, AIN and vehicle in animals. The statistical significance of the difference between treated and control groups was evaluated with one-way ANOVA, followed by Tukey posttest, * \(p<0.05\) vs. AIN or EA groups; ** \(p<0.01\) vs. AIN or vehicle groups; *** \(p<0.001\) vs. AIN group. resorption. TNF-\(\alpha\) also suppressed osteoblast function, while IL-6 boosted RANKL synthesis by osteoblasts [38]. Concerning the effects on pro-inflammatory cytokines, JR and EA diminished the secretion of TNF-\(\alpha\) and IL-6 compared to the control group at 2 weeks of treatments. The effect of _Bixa orellana_ ethyl acetate fraction (BoEA) and ellagic acid was evaluated by [PERSON] et al. on rats with monosodium iodoacetate-induced OA, leading to a reduction in inflammation. The levels of the proinflammatory cytokine TNF-\(\alpha\) were suppressed in the groups treated with BoEA and EA, similarly to the indomethacin-treated group. The levels of the anti-inflammatory cytokine IL-10 was increased in the groups treated with BoEA, EA and indomethacin, illustrating better inflammation control in those groups [39]. [PERSON] et al. demonstrated the anti-inflammatory effect of JR methanolic extract and of EA on endothelial cells by decreasing the TNF-a concentration, as well as the mineralization effect on osteoblasts by inducing nodule formation [40]. In comparison, in our study, TNF-\(\alpha\) levels were decreased in the JR and EA groups only at 2 weeks of treatment. Meanwhile, in our previous work, the mineralization effect was observed in EA and JR groups due to accumulation of calcium and phosphorus, respectively [24]. Guava leaf extract rich in EA administered to rats with OA through diet suppressed the development of OA [41]. EA treatment improved arthritis-associated pathology in an experiment on rats with induced-arthritis, serum TNF-\(\alpha\) levels decreased in the EA-treated group than in the control [42]. In a clinical study conducted on women with knee OA, treatment with pomegranate peel extract rich in EA, demonstrated an anti-inflammatory effect by reducing serum levels of inflammatory markers [43]. Epimedin C, a natural flavonoid compound found in Epimedium genus, markedly reduced the levels of TNF-\(\alpha\), IL-6, NO and PGE2 and effectively attenuated iNOS and COX-2. The antioxidant activity was also recorded by notable reduction in ROS production; meanwhile, MDA was significantly decreased and SOD expression was increased [44]. The same results concerning the suppression of inflammatory factors and the inhibition of ROS production were obtained by using pretreatment with _Oldenlandia diffusa_ extracts [45].