**5. Conclusions**

We report here for the first time that AyuFlex® inhibits oxidative stress, inflammation, and cartilage degradation and improves cartilage regeneration through suppressing the phosphorylation of the ERK and NF-κB p65 signaling pathways triggered by IL-1β in human chondrocytes. Moreover, we discovered the anti-osteoarthritic effects of AyuFlex® in rats with MIA-incurred OA by reducing the levels of oxidative stress, pro-inflammatory mediators, and pro-inflammatory cytokines. Additionally, AyuFlex® decreased the expression of MMPs and suppressed the destruction of synovial membrane and arthrodial cartilage, thereby suppressing the progression of OA. Furthermore, AyuFlex® revealed collagen preserving properties as shown by the significantly upregulated collagen synthesis-related proteins. These results from the in vitro and in vivo studies demonstrated that the biochemical actions of AyuFlex® relieve pain by reducing oxidative stress, inflammation, and cartilage breakdown in arthritis. These results from our study sugges<sup>t</sup> that AyuFlex® could be used as a safe and effective herbal formulation in the treatment of OA among the natural products.

**Author Contributions:** H.L.K. and H.J.L. performed the experiments and wrote the original draft preparation. D.-R.L. and B.-K.C. conceptualized and discussed the conclusions and edited the manuscript. S.H.Y. undertook the manuscript editing and supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors thank Natreon Inc., New Brunswick, NJ, USA and Maypro Industries LLC, Purchase, NY, USA for providing samples, encouragement, and generous support.

**Conflicts of Interest:** The authors declare no conflict of interest.
