Isolation and in vitro investigation of osteogeneic activity of a natural compound in Clinacanthus nutans Lindau to clarify the effect of the plant in treatment of osteoporis and the related diseases.

Theoretical results:
-The ethanol, n-hexane, ethyl acetate, and butanol extracts from Clinacanthus nutans Lindau were obtained. All extracts showed osteogeneic activity, especially the ethyl acetate fraction through: i) enhancement of ALP enzyme activity in MC3T3-E1 osteoblasts up to >30% at the concentration of 25 µg/mL; ii) enhancement of mineralization activity up to >135%, 185%, 125% and 140% at concentrations of 5, 10, 25, 50 µg/mL, respectively.
- The major natural substances isolated from the ethyl acetate extract of C. nutans were successfully isolated and their chemical structures wể elucidated as lupeol with the chemical formula C30H50O with molecular weight of 426.7 g/mol and -sistosterol with the chemical formula C29H50O with the molecular weight of 414.7 g/mol.
- Osteogeneic activity of lupeol was evaluated on osteoblasts MC3T3-E1. It exhibited osteogeneic activity through: i) enhancement of the alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21% and 12% at concentrations of 5, 10, respectively ( <0.05); ii) enhancement of cell mineralization activity up to 170, 230, 185, and 117%  at concentrations of 5, 10, 20 and 40 μg/mL, respectively (p < 0.05); iii) upregulation of expression levels of marker genes involved in osteoblast differentiation, including osteopontin (opn), osteorix (osx), collagen I (col 1), and runx2 up to 2,2, 5,4, 2,8 và 4,1 folds at concentrations of 2.5, 5,10, 20 µg/mL (p<0,05), respectively; iv) enhancement of protein expression of the p38/p-p38 and ERK/p-ERK in the MAPK signaling pathway related to differentiation and mineralization of osteoblasts; iv) Cytotoxicity data of lupeol on the two normal cell lines (BHK, MC3T3-E1) and the four cancer cell lines (Hela, SW1353, A549, và HepG2) were obtained. The data showed low toxicity effects of lupeol on  all the tested cell lines.

The osteogeneic effect of lupeol on osteoblats MC3T3-E1 was reported in this study for the first time. This substance enhanced the activity of ALP by interacting with key amino acids in the active site of the enzyme. It also strongly increased mineralization in the osteoblasts. At the molecular level, it affected the expression of genes involving in osteoblast differentiation and the MAPK signaling pathway related to cell mineralization and differentiation. These are new scientific data, as the basis for further applied research.

-    Scientific papers in referred journals (list):
i. Minh T. H. Nguyen, Quang V. Ngo, Huyen T. T. Nguyen, Quan M. Pham, Trang H. Dinh, Huong T. T. Nguyen, Nguyen V. Tinh, and Phuong T. M. Nguyen* (2021) Osteogeneic Activity of lupeol isolated from Clinacanthus nutans Lindau: Activity and mode of action. Journal of Chemistry. https://doi.org/10.1155/2021/6704999. (Web of Science, Q2)
ii. Pham Thi Thu Phuong, Nguyen Thi Hong Minh, Quach Thi Lien, Nguyen Thi Thanh Huong, Nguyen Thi Mai Phuong* (2020) Osteogenesis activity of fraction extracted from Clinacanthus nutans (Burm. F.) Lindau. Acad J Biol.42(2): 85–91.
iii. Dinh Ha Trang, Nguyen Thi Mai Phuong, Nguyen Thi Hong Minh (2020) Evaluation of osteogenesis activity of natural compounds isolated from Clinacanthus nutans (Burm. F.) Lindau. International conference in Quy Nhơn 12/2020 (Poster/Abstract).
iv. A procedure for isolation of the osteogeneic agents and 03 scientific reports were archived in the Department of Plant Biochemistry, Institute of Biotechnology. The purified compounds (100 mg) and the extract fraction (500 mg) were used up for this study.
v. 01 trained Master Science student