Flavonoids from the peels of Citrus unshiu Markov. and their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling in vitro.

Phytochemical investigation of Citrus unshiu peels led to the isolation of eight new flavonols (7-9, 11-15) and sixteen known compounds (1-6, 10, 16-24). Their structures were elucidated using spectroscopic analysis (1D, 2D NMR, and HR-MS). Besides, all isolated compounds (1-24) were evaluated for their inhibitory effects on receptor activator of RANKL-induced osteoclastogenesis in BMMs. Among them, dimethylmikanin (1), quercetogetin (2), 3,3',4',5,7,8-hexamethoxyflavone (3), 3-methoxynobiletin (4) showed a significant inhibitory effect on RANKL-induced osteoclast differentiation at a concentration of 10 µM. Moreover, 3-methoxynobiletin (4) suppressed RANKL-induced osteoclastogenesis by decreasing the number of osteoclasts and osteoclast actin-ring formation in a dose-dependent manner without causing any cytotoxic effects on BMMs. At the molecular level, 3-methoxynobiletin (4) inhibited RANKL-induced c-Fos expression and subsequently NFATc1 activation, as well as the expression of osteoclastogenesis-related marker genes c-Src and CtsK. These findings suggested that 3-methoxynobiletin (4) attenuated osteoclast differentiation by inhibiting RANKL-mediated c-Fos signaling and that it may have therapeutic potential for treating or preventing bone resorption-related diseases, such as osteoporosis.

Triterpenoids from Celastrus orbiculatus Thunb. inhibit RANKL-induced osteoclast formation and bone resorption via c-Fos signaling.

Fourteen triterpenes, lup-20(29)-ene-3ß,6ß-diol (1), betulin (2), lupeol caffeate (3), 3ß-caffeoyloxylup-20(29)-en-6α-ol (4), betulin-3ß-yl-caffeate (5), 3ß-trans-feruloylbetulin (6), betulinaldehyde 3-caffeate (7), 3-O-trans-caffeoylbetulinic acid (8), dammarenediol II 3-caffeate (9), 12-oleanene-3ß,6α-diol (10), 11α-hydroxy-3ß-amyrin (11), nivadiol (12), 29-hydroxyfriedelin (13), and celastrusin A (14) were isolated from Celastrus orbiculatus Thunb. and evaluated for their activity on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages (BMMs). Compounds betulin (2), betulin-3ß-yl-caffeate (5), 3ß-trans-feruloylbetulin (6), and 3-O-trans-caffeoylbetulinic acid (8) significantly inhibited osteoclast formation in a dose-dependent manner. Among these, betulin-3ß-yl-caffeate (5) exhibited the most potent inhibitory activity. We demonstrated that betulin-3ß-yl-caffeate (5) suppressed F-actin-ring formation and bone resorption activity. At the molecular level, betulin-3ß-yl-caffeate (5) inhibited RANK-induced expression of c-Fos and the induction of nuclear factor of activated T cells 1 (NFATc1), a key transcription factor for osteoclast formation, and it also downregulated mRNA expression of osteogenesis-associated marker genes including tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and matrix metalloprotein (MMP). These results indicate that betulin-3ß-yl-caffeate (5) may be a promising candidate for the treatment of osteoclast-related diseases such as osteoporosis.