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Clinacanthus nutans Lindau has been traditionally used for healing of bone fragility, but the mechanism of actions has not been clarified yet. In this study, the bone regeneration activity of lupeol derived from C. nutans was assessed using an in vitro model of osteoblast cells MC3T3-E1. The finding revealed that the compound was not significantly toxic to osteoblast cells at concentration of ≤40 μg/mL. Lupeol demonstrated the osteogenic activity through enhancement of alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21%, and 12% at concentrations of 5, 10, and 20 µg/mL, respectively ( p  &lt; 0.05). Besides, the mineralization activity was increased up to 170, 230, 185, and 117% at concentration of 5, 10, 20, and 40 μg/mL, respectively ( p  &lt; 0.05). The marker genes related to osteoblast differentiation evaluated on the expression level in the presence of lupeol, including collagen I (col 1), osteopontin (opn), osterix (osx), and runx2, showed upregulated expression in all the test genes ( p  &lt; 0.05). The Western blot analysis demonstrated a clear effect of lupeol on expression of p38/p-p38, and ERK/p-ERK proteins involved in the MAPK signaling pathway. Thus, lupeol isolated from C. nutans exhibited the osteogenic activity by enhancing expression of important markers of osteogenesis, as well as affected the MAPK signaling pathway relating to osteoblast differentiation. This is the first report on the detailed mechanism of action of lupeol on bone regeneration and also explains for the traditional use of this medicinal plant for bone healing.

Osteogenic Activity of Lupeol Isolated from Clinacanthus nutans Lindau: Activity and Mode of Action