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 ( < 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 ( < 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 ( < 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.

中文翻译：

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从 Clinacanthus nutans Lindau 中分离的羽扇豆醇的成骨活性：活性和作用方式

Clinacanthus nutans Lindau 传统上用于治疗骨脆性，但作用机制尚未阐明。在这项研究中，使用成骨细胞 MC3T3-E1 的体外模型评估了来自C. nutans的羽扇豆醇的骨再生活性。研究结果表明，该化合物在≤40 μ g/mL 的浓度下对成骨细胞没有显着毒性 。Lupeol 通过将成骨细胞的碱性磷酸酶 (ALP) 分别在 5、10 和 20 µg /mL 的浓度下提高至 31.2%、21% 和 12% 来证明其具有成骨活性。  < 0.05)。此外，在 5、10、20 和 40 μg /mL 的浓度下，矿化活性分别增加了 170、230、185 和 117%  （ <0.05）。在羽扇豆醇存在下，在表达水平上评估的与成骨细胞分化相关的标记基因，包括胶原蛋白 I (col 1)、骨桥蛋白 (opn)、osterix (osx) 和 runx2，在所有测试基因中都显示出上调的表达 (  < 0.05 ）。蛋白质印迹分析表明羽扇豆醇对参与 MAPK 信号通路的 p38/p-p38 和 ERK/p-ERK 蛋白的表达有明显影响。因此，从C. nutans 中分离的羽扇豆醇通过增强重要的成骨标志物的表达表现出成骨活性，并影响与成骨细胞分化相关的 MAPK 信号通路。这是关于羽扇豆醇对骨再生作用的详细机制的第一份报告，也解释了这种药用植物用于骨愈合的传统用途。