ABSTRACT
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.
Subject(s)
Cell Differentiation/drug effects , Citrus/chemistry , Flavonoids/chemistry , Osteogenesis/drug effects , RANK Ligand/pharmacology , Signal Transduction/drug effects , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Citrus/metabolism , Down-Regulation/drug effects , Flavonoids/isolation & purification , Flavonoids/pharmacology , Fruit/chemistry , Fruit/metabolism , Mice , Molecular Conformation , Osteoclasts/cytology , Osteoclasts/metabolism , Plant Extracts/chemistry , Proto-Oncogene Proteins c-fos/metabolism , RAW 264.7 Cells , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Bone Resorption/drug therapy , Osteoclasts/drug effects , Proto-Oncogene Proteins c-fos/metabolism , RANK Ligand/antagonists & inhibitors , Triterpenes/pharmacology , Animals , Bone Resorption/metabolism , Bone Resorption/pathology , Celastrus/chemistry , Cell Differentiation/drug effects , Male , Mice , Mice, Inbred ICR , Osteoclasts/metabolism , Osteoclasts/pathology , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , RANK Ligand/metabolism , Signal Transduction/drug effects , Triterpenes/isolation & purificationABSTRACT
Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F-actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.