The roots of C. wilfordii Hemsley was obtained from Jung-do Herb, Co. Ltd. (Guri, Korea). A total of 20 g of C. wilfordii were extracted with 200 ml distilled water for 24 h at room temperature (RT). The extract was concentrated in a rotary vacuum evaporator, and designated CW (yield: 13.7%). A voucher specimen (CW-W100) was deposited at the department of Convergence Korean Medical Science, Kyung Hee University.

For quantification of CW, 2,4-dihydroxyacetophenone was used as a standard. The content of 2,4-dihydroxyacetophenone was measured using a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD; Agilent 1100 series; Agilent Technologies, Inc., Santa Clara, CA, USA). The extract was dissolved in 70% methanol and sonicated for 30 min. After filtering through a 0.45 µm filter membrane, an aliquot was injected in HPLC analysis. The column used was a Shiseido Capcell Pak C18 (250×4.6 mm, 5 µm; Shiseido Co., Ltd., Tokyo, Japan). The mobile phase consisted of 0.05% acetic acid in water and acetonitrile with 1.0 ml/min of flow rate at 30°C. As demonstrated in Fig. 1, the concentration of 2,4-dihydroxyacetophenone in CW was 13.689 µg/ml (0.091%).

A total of 36 female ICR mice aged 6 weeks (Raon Bio Animal, Inc., Yong-in, Korea) were provided free access to a standard chow diet (Orient Co. Ltd., Seongnam, Korea) and tap water. They were housed in a controlled environment (22±2°C, a relative humidity of 50±5% and a 12 h light:dark cycle). The animal studies were conducted in accordance with the rules and regulations established by the Institutional Animal Ethics Committee of the Kyung Hee University [KHUASP (SE) −15-079].

After acclimatization for 1 week, the mice, with the exception of the normal group, were surgically ovariectomized (OVX), then recovered and osteoporosis induced for 9 weeks. They were randomly divided into three groups (OVX + vehicle, OVX + E2, and OVX + CW). 17β-estradiol (E2; 10 µg/kg/day) was injected intraperitoneally to the OVX + E2 group as a positive control and 1 mg/kg/day CW orally administrated to OVX + CW group. Normal and OVX + vehicle mice were orally administrated vehicle (in PBS containing 1% DMSO). All mice were treated 5 times per week for 3 weeks, then sacrificed. The body weight was measured weekly. The blood sample was collected by cardiac puncture.

The epicondyles were removed and immediately fixed in 10% formalin for 18 h. Prior to dehydration, bone tissues were demineralized in 0.1 M ethylenediaminetetraacetic acid for 1 month. The sections of epicondyle were cut at a 5 µm thickness and stained with H&E or an Acid Phosphatase, Leukocyte TRAP kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Stained tissues were observed using Leica Application Suite microscope software (version 3.2.276.2; Leica Microsystems, Buffalo Grove, IL, USA).

Subsequent to sacrifice, the proximal femur was collected and cleaned without attached muscles and connective tissue. The sample was stored at −80°C in PBS until analysis. Dual-energy X-ray absorptiometry with a PIXImus instrument (Lunar Corp., Madison, WI, USA) was used for the determination of BMC and BMD.

The collected blood was centrifuged at 12,000 × g for 30 min at RT and the supernatant stored at −80°C until use. The concentration of serum osteocalcin was measured using Mouse Gla-osteocalcin high sensitive EIA kit (TaKaRa Bio, Inc., Otsu, Japan) according to the manufacturer's protocol.

Saos-2 cells (human osteosarcoma cell line; Korean Cell Line Bank, Seoul, Korea) were cultured with Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% humidified CO₂ atmosphere. Saos-2 cells were plated in 6-well culture plates at 0.8×10⁵cells/well. CW (1, 10 and 100 µg/ml) in FBS-free DMEM medium was administered for 24 h.

RIPA buffer (50 mM Tris-HCl; pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) was used for uterus and Saos-2 cell protein extraction. The lysate (30 µg) was denatured with 2X loading buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then electrotransferred onto a PVDF membrane (Bio-Rad Laboratories, Inc.). Primary antibodies targeting osterix (cat. no. Ab94744; Abcam, Cambridge, UK), osteoprotegerin (cat. no. sc-11383; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) in TBS-T (1:1,000 dilution) were incubated overnight at 4°C and secondary antibody anti-mouse IgG (1:2,000 dilution; Cell Signaling Technology, Inc.) in TBS-T was incubated for 1 h at RT. The proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Uppsala, Sweden). Visualized bands were quantified using a computerized densitometry system ImageJ (version 1.38e; National Institutes of Health, Bethesda, MD, USA). All samples were analyzed in triplicate.

Significance was determined by one-way analysis of variance and Dunnett's multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference.

The BMC level in the OVX + vehicle group was significantly decreased to 0.034±0.003 g compared with the normal group (0.045±0.004 g). CW treatment significantly increased the level of BMC ~32% (0.045±0.004 g; Fig. 2). In addition, the BMD level of the OVX + vehicle group (0.074±0.001 g/cm²) was significantly lowered ~10.8% compared with the normal group (0.083±0.008 g/cm²). There was a 10.5% increase in BMD level following treatment of CW (0.082±0.006 g/cm²; Fig. 2). Taken together, CW treatment demonstrated recoveries of BMC in addition to BMD levels.

In the OVX + vehicle group, the pores within interstitial cells filling the lateral and medial epicondyles were markedly increased in comparison with the normal group. E2 injection as a positive control drug ameliorated histopathological changes of epicondyles. As recoveries in the E2 injected group, CW-treated mice demonstrated dense and well-formed bone marrow cells. Additionally, CW treatment reduced the bone marrow pores in the lateral and medial epicondyles (Fig. 3A).

The OVX + vehicle group demonstrated substantial increases of TRAP-stained multinucleated osteoclasts compared with normal mice. In lateral and medial epicondyles of the CW-administrated group, stained TRAP-positive cells were fewer compared with the OVX + E2 group (Fig. 3B).

The value of serum osteocalcin concentration was significantly lower in the OVX + vehicle group compared with the normal group. While the serum concentration of osteocalcin in normal group was 56.26±2.6 pg/ml, that in the OVX + vehicle group was 39.91±2.69 pg/ml; a significant ~29.06% decrease. Following treatment by CW, serum osteocalcin level demonstrated a 25.93% recovery (50.26±0.7 pg/ml; Fig. 4).

When the cells were treated with various concentrations of CW, the expression of OPG was significantly increased (Fig. 5). In addition, the expression of osterix in Saos-2 cells was increased by CW treatment compared with non-treated cells (Fig. 5).