In Vitro and In Silico Studies of the Ethyl Acetate Fraction of Semanggi (Marsilea crenata Presl.) Leaves on Bone Formation in hFOB 1.19 Cells

Agnis Pondineka Ria Aditama

Phytoestrogen is an estrogenic compound of plant origin that has been used in estrogen replacement therapy. It helps in overcoming estrogen deficiency in menopausal women that causes an imbalance in bone remodelling. Such an imbalance is one of the causes of osteoporosis. Previous research found out, that phytoestrogens showed estrogen receptor-β (ER-β) agonist activity to exert their action in bone formation. Semanggi (Marsilea crenata Presl.) leaves have been used for generations in East Java Province, Indonesia, to control osteoporosis among menopausal women. Research identified some phytoestrogen compounds of the leaves.

This research aimed to investigate the activity of the ethyl acetate fraction of Marsilea crenata Presl. leaves on bone formation in estrogen-deficient hFOB 1.19 cells in vitro. Metabolite profiling to investigate the phytoestrogen contents and in silico study to investigate the interaction between phytoestrogen compounds in the fraction and the ER-β receptor were conducted.

Activity of bone formation was conducted using immunocytochemical method to measure expressions of ER-β, osterix, and osteocalcin. Metabolite profiling was carried out with an ultra-performance liquid chromatography quadrupole time of flight – mass spectrometry (UPLC-QToF-MS/MS) instrument and MassLynx 4.1 software. Molecular docking was performed using the ER-β protein (PDBid. 1ERE). Autodock vina embedded with PyRx software was used to study molecular docking in silico.

Result of in vitro study showed a significant enhancement of osterix and osteocalcin expressions at fraction concentrations of 62.5; 125 and 250 µg/ml. This might indicate a possibility of differentiation process in bone formation. Osteocalcin is a late marker of bone formation, and osterix is an osteoblastogenesis-specific transcription factor. As ER-β expression did not decrease, there might a possibility of activities on ERs other than ER-β to promote bone formation. Metabolite profiling identified 20 compounds that showed affinity towards ER-β in the in silico study.

These results suggest the phytoestrogen content of the ethyl acetate fraction of Marsilea crenata Presl. might be able to overcome the imbalance of bone remodelling caused by estrogen deficiency that causes osteoporosis. Further research is needed to do experiments on animal models.

Keywords: Bone formation, ER-β, Marsilea crenata Presl., phytoestrogen