| Summary: | Scrophulariae Radix, derived from the dried roots of <i>Scrophularia ningpoensis</i> Hemsl. or <i>S. buergeriana</i> Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of <i>Scrophularia buergeriana</i>, <i>S. koraeinsis</i>, and <i>S. takesimensis</i> on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of <i>S. buergeriana</i>, <i>S. koraiensis</i>, and <i>S. takesimensis</i>, and compared the effects of ethanol extracts of these species using nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation. The harpagoside content of the three <i>Scrophularia</i> species was analyzed by high-performance liquid chromatography–mass spectrometry (HPLC/MS). Their therapeutic effects were evaluated by tartrate-resistant acid phosphatase (TRAP)-positive cell formation and bone resorption in bone marrow-derived macrophages (BMMs) harvested from ICR mice. We confirmed the presence of harpagoside in the <i>Scrophularia</i> species. The harpagoside content of <i>S. buergeriana</i>, <i>S. koraiensis</i>, and <i>S. takesimensis</i> was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, and 5.50 ± 0.02 mg/g, respectively. Treatment of BMMs with extracts of the three <i>Scrophularia</i> species inhibited TRAP-positive cell formation in a dose-dependent manner. The area of hydroxyapatite-absorbed osteoclasts was markedly decreased after treatment with the three <i>Scrophularia</i> species extracts. Our results indicated that the three species of the genus <i>Scrophularia</i> might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.
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