Chemical constituents in safflower leaves and inhibitory effects of quercetin and luteolin derivatives on osteoclast differentiation

Context Safflower (Carthamus tinctorius L.) seeds have been reported to exert bone protective effects, but research on safflower leaves remains limited.Objective This study aimed to evaluate the inhibitory effects of safflower leaf and seed extracts on osteoclast differentiation, compare their chemical compositions, and identify anti-osteoclastogenic compounds from safflower leaves.Materials and methods The inhibitory effects on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation were evaluated in bone marrow-derived macrophages (BMDMs). Compositional differences between safflower seeds and leaves were determined using liquid chromatography-mass spectrometry (LC-MS) analysis. Compounds from safflower leaves were isolated by medium-pressure liquid chromatography (MPLC) and identified using MS and nuclear magnetic resonance (NMR) analyses.Results Hot water and ethanol extracts of safflower leaves suppressed RANKL-induced osteoclast differentiation in BMDMs, with effects comparable to those of safflower seed extracts. These extracts also downregulated the expression of osteoclast-related genes, including nuclear factor of activated T cells 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP). LC-MS metabolite analysis detected quercetin and luteolin derivatives in safflower leaves, whose chemical composition differs from that of safflower seeds. Fourteen compounds, including a novel compound, 4-(2-nitroethyl)phenyl-O-rutinoside, were isolated and identified from safflower leaves. Seven quercetin and luteolin derivatives inhibited osteoclast differentiation and downregulated osteoclast-related gene expression.Discussion and conclusions These findings provide in vitro evidence supporting further in vivo investigation of safflower leaves as promising candidates for suppressing osteoclast differentiation, with quercetin and luteolin derivatives identified as potential bioactive markers.