## 3 Results
### Effect of Apigenin and Hesperidin on PGE2 Signaling Pathways  
Apigenin and hesperidin both targeted the PGE2 signaling pathway and lowered both biomarkers--TRPV1 and CGRP. The results are shown in Figure 5. Panels A and B represent the individual effects of apigenin and hesperidin on TRPV1, respectively, and results from their combination are shown in panel C. Panels D and E represent the individual effects of apigenin and hesperidin on CGRP, respectively, and results from their combination are shown in panel F. TRPV1 and CGRP levels were compared over a period of 2 days in the presence and absence of apigenin and hesperidin. Figure 5: Individual (**A**,**B**) and combination effect (**C**) of apigenin and hesperidin on TRPV1 production in chondrocytes over simulations periods of 2 days. Apigenin at 30 mg dose (**A**), hesperidin at 1000 mg dose, (**B**) and their combination at 1030 mg dose (**C**) reduced the levels of TRPV1 over a period of 2 days. Individual (panels D and E) and combination effect (panel F) of apigenin and hesperidin on CGRP production in chondrocytes over simulation periods of 2 days. Apigenin at 30 mg dose (**D**), hesperidin at 1000 mg dose, (**E**) and their combination at 1030 mg dose (**F**) reduced the levels of CGRP over a period of 2 days. TRPV1—transient receptor potential cation channel subfamily V member 1. CGRP—calcitonin gene-related peptide. The levels of TRPV1 were estimated to be 0.039 nM for the control condition. At the end of a 2-day period, TRPV1 concentrations reduced to 0.014 nM in the presence of 30 mg of apigenin, as shown in Figure 5A. In the presence of 1000 mg of hesperidin, TRPV1 concentrations reduced to 0.0007 nM over a period of 2 days, as shown in Figure 5B. To determine whether the apigenin and hesperidin combination value of 0.00019 nM reflects a synergistic effect, the additive effect range is calculated for the biomarker TRPV1 by a sequential (forward and reverse) administration of apigenin and hesperidin. As shown in Table 3, the additive effect range for the combination of 30 mg apigenin and 1000 mg hesperidin is 0.0027-0.0325 nM when apigenin and hesperidin were added sequentially. When combined simultaneously, apigenin and hesperidin lowered TRPV1 levels to 0.00019 (Figure 5C). This value is lower than the additive effect range of 1.54-1.93 nM for TRPV1, indicating that this specific combination has a synergistic effect. Specifically, the TRPV1 level with the combination is >99% lower than the additive range. These results indicate that apigenin and hesperidin act in synergy to reduce TRPV1, which is associated with joint pain. The levels of CGRP were estimated to be 0.045 nM for the control condition. At the end of a 2-day period, CGRP concentrations reduced to 0.0004 nM in the presence of 30 mg of apigenin, as shown in Figure 5D. In the presence of 1000 mg of hesperidin, CGRP levels reduced to \(6.82\times 10^{-9}\) nM over a period of 2 days, as shown in Figure 5E. When combined, apigenin and hesperidin lowered CGRP levels even further to 2.65 \(\times\) 10\({}^{-10}\) nM (Figure 5F). To determine whether the apigenin and hesperidin combination value of \(2.65\times 10^{-10}\) nM reflects a synergistic effect, the additive effect range was calculated for the biomarker CGRP by a sequential (forward and reverse) administration of apigenin and hesperidin. As shown in Table 4, the additive effect range for the combination of 30 mg apigenin and 1000 mg hesperidin is 0.066-1.17 \(\times\) 10\({}^{-6}\) nM when apigenin and hesperidin were added sequentially. When combined simultaneously, apigenin and hesperidin lowered CGRP levels to 2.65 \(\times\) 10\({}^{-10}\) nM nM (Figure 5F). This value is lower than the additive effect range of 0.066-1.17 \(\times\) 10\({}^{-6}\) nM for CGRP, indicating that this specific combination has a synergistic effect. Specifically, the CGRP level with the combination is >99% lower than the additive range. These results indicate that apigenin and hesperidin act in synergy to reduce CGRP, which is associated with joint pain.