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

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.


Introduction
An imbalance between osteoclasts and osteoblasts affects bone formation, leading to weakened bone and the development of skeletal diseases such as osteoporosis, rheumatoid arthritis, lytic bone metastases, and chronic obstructive pulmonary disease. The function of osteoclasts, multinucleated giant cells, is bone resorption, but osteoporosis can occur if bone resorption exceeds formation due to an increase in the number of osteoclasts [1]. Most drugs used to treat osteoporosis inhibit osteoclast differentiation to control bone resorption. Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a major osteoclastogenic molecule, is a member of the tumor necrosis factor (TNF) superfamily and is the initial stimulator of osteoclast differentiation, inducing the expression of osteoclast-associated genes, such as tartrate-resistant acid phosphatase (TRAP) [2,3]. Therefore, bisphosphonates and anti-RANKL antibodies that inhibit osteoclast activity are currently used for the treatment of bone resorption diseases.
Scrophulariae Radix, an herbal medicine known as Korean Hyun-Sam, is derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq., plants which are widely distributed throughout the temperate regions of the Northern Hemisphere, including Asia, Europe, and North America [4][5][6]. Scrophulariae Radix has been traditionally used as a therapeutic agent for blood cooling, yin nourishing, fire pursing, and toxin removal, and is widely used to treat rheumatism and arthritis in Southwest Asia [7][8][9][10]. It has also been reported to have neuroprotective, antiinflammatory, anti-allergy, anti-amnesia, antioxidant, and hepatoprotective effects [11][12][13][14][15]. In Korea, S. koraiensis Nakai (Korean: To-Hyun-Sam) has been used as an antipyretic and anti-inflammatory agent in traditional medicine. S. takesimensis Nakai (Korean: Seom-Hyun-Sam) is restricted to Ulleung-do Island [16,17]. Although this species is a valuable endemic resource, its medicinal efficacy has not been assessed to date.
The therapeutic potential of Scrophularia species is associated with the functions of major secondary metabolites, such as phenylpropanoids and iridoid glycosides, which are present in the plant [18,19]. Harpagoside, an iridoid component present in the Scrophularia species, is a bioactive compound of Harpagophytum procumbens DC. (Devil's Claw) and has been used in Southern Africa to treat pain, arthritis, and ulcers. Pharmacological effects of harpagoside on RANKL-induced osteoclast differentiation have also been reported [20,21]. However, there are few reports concerning the pharmacological activity of Scrophularia species on RANKL-induced osteoclast differentiation, and studies on the biological activity of S. koraiensis and S. takesimensis have not been reported.
In the current study, we compared the morphological characteristics and harpagoside content of S. buergeriana, S koraiensis, and S. takesimensis, and compared the effects of Scrophularia species extracts on RANKL-mediated osteoclast differentiation.

Comparative Morphology of Scrophularia Species
The three species can be distinguished on the basis of leaf shape, apex, margins, pubescence of stems, and calyx shape ( Table 1). The leaf blade of S. buergeriana is ovate, with an acute apex ( Figure  1A), and serrate with a spinose tooth marginal shape ( Figure 1D). The stem is glabrous ( Figure 1G), and the calyx is ovate with an obtuse apex ( Figure 1J). S. koraiensis has lanceolate to rarely ovateshaped leaf blades with an acuminate apex ( Figure 1B) and is serrate with a spinose tooth ( Figure  1E). The stem of S. koraiensis is sparsely pubescent with non-glandular trichomes ( Figure 1H), and the calyx is lanceolate with an acute to attenuate apex ( Figure 1K). However, S. takesimensis has ovate leaf blades with an acute apex ( Figure 1C), and has a serrate, almost without spinose tooth, marginal leaf blade ( Figure 1F). The stem surface of S. takesimensis is glabrous ( Figure 1I), and its calyx is semicircular, with a rounded apex ( Figure 1L).

Harpagoside Content of Scrophulariae Species
High-performance liquid chromatography (HPLC) chromatograms of S. buergeriana, S. koraiensis, and S. takesimensis are shown in Figure 2A,B. Figure 2A shows the HPLC chromatograms of the harpagoside standard compound and the three species of Scrophulariae monitored at 280 nm, because the maximum wavelength of harpagoside is 279.5 nm. Harpagoside was detected at approximately 50.1 min. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, 5.50 ± 0.02 mg/g, respectively ( Table 2). The total ion chromatography (TIC) of the mass spectrometry (MS) spectrum was confirmed from 190-850 m/z, and the extracted ion chromatogram (XIC) of harpagoside in the samples was analyzed at 517.11 m/z, because harpagoside was detected at 517.11 m/z [M-H + Na] + ( Figure 2B). Table 2. Harpagoside content of three Scrophularia species (S. buergeriana, S. koraiensis, and S. takesimensis) by high-performance liquid chromatography (HPLC) chromatogram analysis.

Cytotoxic Effects on Primary Murine BMM Growth
The XTT assay was conducted to assess cytotoxicity during osteoclast differentiation. S. buergeriana, S. koraiensis, and S. takesimensis did not reduce cell viability at most of the concentrations tested. Treatment with 200 μg/mL S. koraiensis reduced cell viability from that of the normal control group ( Figure 3A-C), but the difference was not statistically significant.

Effects on Osteoclast Differentiation
To compare the effect of S. buergeriana, S. koraiensis, and S. takesimensis on osteoclast differentiation, mouse BMMs treated with macrophage colony stimulating factor (M-CSF) and RANKL were cultured in the presence or absence of ethanol extracts of the three Scrophularia species. RANKL and M-CSF induced differentiation of BMMs after incubation for 4 days. TRAP-positive osteoclasts were present in higher numbers in the control group, whereas treatment with Scrophularia species extracts inhibited the formation of TRAP-positive cells in a dose-dependent manner ( Figure  4B). The S. koraiensis treatment group showed suppressed formation of RANKL-induced TRAP activity at concentrations of 100 μg/mL and 200 μg/mL.

Effects on Bone Resorption
To evaluate the effects of Scrophularia species on bone resorption, mature osteoclasts and extracts of S. buergeriana, S. koraiensis, and S. takesimensis were applied to plates coated with hydroxyapatite for symbiotic culture of the osteoblasts. Although the area of hydroxyapatite-adsorbed osteoclasts was increased in the control, the resorption area was markedly decreased by treatment with extracts of the three Scrophularia species. The resorption inhibition effect was highest for S. buergeriana, followed by S. koraiensis, and then S. takesimensis. (Figure 5A,B).

Discussion
Osteoporosis, one of the major diseases attracting attention worldwide, is associated with lowered bone mass density as a result of an imbalance between the osteoblasts and osteoclasts that influence bone homeostasis [22,23]. Therapeutic agents such as bisphosphonates are currently used to treat bone resorption diseases, but the long-term use of these drugs leads to the suppression of bone formation and osteonecrosis [24]. Therefore, the beneficial pharmacological effects of plantderived natural compounds have been advocated [10].
In this study, we investigated the inhibitory effects exerted by three Scrophularia species containing harpagoside against osteoclast differentiation and bone resorption without cytotoxic effects in RANKL-induced cells. The cytotoxicity of S. buergeriana, S. koraiensis, and S. takesimensis was evaluated in BMMs and measured by XTT assay during osteoclast differentiation. Treatment with ethanol extracts of the three Scrophularea species did not reveal cytotoxic effects on BMMs up to 200 μg/mL, with more than 90% cell viability being observed.
RANKL, a bone formation biomarker, is associated with stimulation of osteoblasts and osteoclast differentiation [22]. The balance of bone formation is influenced by osteoblasts and bone resorption activity by osteoclastic cells [29]. Previous studies have demonstrated that harpagoside improved bone properties by inhibiting the formation of osteoclasts from BMMs and the maturation of osteoblast cells [21,30].
TRAP expression is associated with osteoclast maturation and differentiation and is a standard approach to the detection of osteoclasts [24]. In our study, TRAP staining indicated that the numbers and areas of TRAP-positive cells increased, whereas the S. buergeriana, S. koraiensis, and S. takesimensis treatment groups exhibited considerably fewer TRAP-stained osteoclasts, without any cytotoxicity. The results suggested that the three Scrophularia species inhibit osteoclast differentiation and formation in BMMs. The inhibitory effect of harpagoside was increased in a dose-dependent manner by downregulating TRAP expression in BMCs [30]. Therefore, it is presumed that the difference in efficacy of the three Scrophularia species in our results would be affected by the content of harpagoside. To study the inhibitory effects of Scrophularia species on bone resorption by osteoclast formation induced by RANKL, we investigated bone resorption pit generation in mature osteoclasts. Although the area of hydroxyapatite-absorbed osteoclasts was increased in DMSO controls, the resorption area was markedly decreased by treatment with the three Scrophularia species. The highest resorption inhibition effect was exerted by S. buergeriana, followed by S. takesimensis, and then S. koraiensis.

BMM (Bone Marrow Macrophage) Isolation and Culture
Five-week-old ICR mice were sacrificed by cervical dislocation, the thigh and shin bones were aseptically excised, and the soft tissue was removed. After cutting both ends of the iliac crest, bone marrow cells were obtained by flushing both ends of the bone material using a 1 mL syringe. The isolated BMMs were incubated in a culture dish for 1 day in α-MEM medium containing 10% FMS and 1% penicillin/streptomycin, and unsaturated cells were collected. After 3 days, attached macrophages were used in this experiment. Macrophages were treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and incubated with S. takesimensis, S. koraiensis, and S. buergeriana extracts at concentrations of 50 μg/mL, 100 μg/mL, and 200 μg/mL. The day following subculture, the cultured cells were stained with a TRAP solution. Cells with three or more nuclei among red-stained cells were considered differentiated osteoclasts, and the degree of differentiation was measured.

Bone Resorption Pit Assay
To obtain mature osteoclasts, BMMs obtained from the shin and thigh bones of 5-week-old ICR mice and osteoclasts isolated from the skull of 1-day-old ICR mice were added to a 90 mm culture plate coated with collagen. To produce a symbiotic culture, 1α,25-Dihydroxyvitamin D3 (VitD3) and prostaglandin E2 were added. After incubation for 6 days, cells were removed by treatment with 0.1% collagenase and added to a 96-well plate coated with hydroxyapatite. At the same time, S. takesimensis, S. koraiensis, and S. buergeriana extracts were added to the hydroxyapatite-coated plates at a concentration of 200 ug/mL and cultured for 24 h. The cells were washed with distilled water and observed through an optical microscope (Nikon, Tokyo, Japan). The hydroxyapatite-absorbed portion was imaged using micrography, and the area was measured using ImageJ software version 1.51 (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed by analysis of variance (ANOVA), followed by a multiple comparison procedure using Dunnett's test. A value of p < 0.05 indicated significant difference.

Conclusions
Our results suggest that the Scrophularia species may prevent bone loss by inhibiting osteoclast differentiation and born resorption without causing cytotoxicity. It was confirmed that similar metabolites were contained in the Scrophularia species, and their extracts exerted similar efficacy. S. buergeriana and S. koraiensis demonstrated inhibitory effects against osteoclast differentiation and bone resorption and had the highest harpagoside content. Therefore, the three Scrophularia species, including S. koraiensis, may be potentially therapeutic for treating bone disorder diseases, but the mechanism underlying their bioactivity requires further study.