In Vitro and in Silico Analysis on the Bone Formation Activity of N-Hexane Fraction of Semanggi (Marsilea crenata Presl.)

Abstract

Agnis Pondineka Ria Aditama, Burhan Ma'arif, Denis Mery Mirza, Hening Laswati, Mangestuti Agil.

Background: Estrogen deficiency in postmenopausal women causes various health problems, including osteoporosis. Osteoporosis is a result of imbalances between new bone formation and old bone resorption. Phytoestrogens can be used as an alternative to increase bone formation and overcome estrogen deficiency.  Semanggi (Marsilea crenata Presl.) is a plant that contain phytoestrogens. The aim of this research was to investigate the bone formation activity of n-hexane fraction of Marsilea crenata Presl. leaves against hFOB 1.19 cells by observing the expression of osteocalcin and predicting the phytoestrogen contents of the fraction through metabolite profiling and in silico studies.
Methods:  hFOB 1.19 cells were cultured in 24 well microplates and added the n-hexane fraction of Marsilea crenata Presl. leaves at doses of 62.5, 125, and 250 ppm. Genistein 1 µM was used as a positive control. Analysis of osteocalcin expression was conducted using immunocytochemistry with CLSM. Metabolite profiling was conducted using UPLC-QToF-MS/MS. In silico study of the compounds found in metabolite profiling was conducted using molecular docking with PyRx 0.8 software and 1ERE protein.
Results: n-hexane fraction of Marsilea crenata Presl. leaves increased osteocalcin expression with the optimum dose of 62.5 ppm and a value of 457.35 AU at p <0.05. Metabolite profiling of n-hexane fraction found 26 known compounds, 14 unknown compounds. 10 of the identified compounds showed ER-β agonists activity.
Conclusions: The n-hexane fraction of Marsilea crenata Presl. leaves increased bone formation activity. Compounds of the fraction that showed ER-β agonists activity might be phytoestrogens.