Three Scrophularia Species (Scrophularia buergeriana, S. koraiensis, and S. takesimensis) Inhibit RANKL-Induced Osteoclast Differentiation in Bone Marrow-Derived Macrophages

Abstract

Scrophulariae Radix, derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of Scrophularia buergeriana, S. koraeinsis, and S. takesimensis on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis, 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 Scrophularia 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 Scrophularia species. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis 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 Scrophularia 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 Scrophularia species extracts. Our results indicated that the three species of the genus Scrophularia might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.

Keywords: RANKL; S. koraiensis; S. takesimensis; Scrophularia buergeriana; harpagoside; osteoclast differentiation.

Figure 1

Stereomicroscope micrographs showing the morphology of the three Scrophularia species studied. (A–C) Apex of leaf blade. (D–F) Margin of leaf blade. (G–I) Surface of stem. (J–L) Calyx of flower and/or fruit. (A,D,G,J) S. buergeriana. (B,E,H,K) S. koraiensis. (C,F,I,L) S. takesimensis. TR, Trichomes. All scale bars = 1 mm.

Figure 2

(A) Chromatograms of three Scrophularia species (S. buergeriana, S. koraiensis, and S. takesimensis) at 280 nm for harpagoside (λ_max = 279.5 nm) and (B) positive extracted ion chromatogram (XIC) spectrum of harpagoside at 517.11 m/z.

Figure 3

Cell viability affected by ethanol extracts of the three Scrophularia species (A) S. buergeriana (B) S. koraiensis and (C) S. takesimensis. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (dimethyl sulfoxide; DMSO).

Figure 4

Effects on osteoclast differentiation of ethanol extracts of Scrophularia buergeriana, S. koraiensis, and S. takesimensis at concentrations of 50 μg/mL, 100 μg/mL, and 200 μg/mL. (A) Tartrate-resistant acid phosphatase (TRAP)-positive cells photographed (100× magnification) after bone marrow macrophages were cultured with macrophage colony stimulating factor (M-CSF) and nuclear factor (NF)-κB ligand (RANKL) in the presence of ethanol extracts of the three Scrophularia species. (B) TRAP-positive cells were counted as osteoclasts. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (DMSO).

Figure 5

Effects of S. buergeriana, S. koraiensis, and S. takesimensis on bone resorption by mature osteoclasts. (A) Hydroxypatite-adherent cells were collected and imaged under a light microscope. (B) Resorption areas quantified on hydroxyapatite-coated plates. ** p < 0.01 and *** p < 0.001 vs. control (DMSO).