P. umbrosa was purchased from Jungdo Herb, Inc. (Guri, Korea). A total of 100 g P. umbrosa was extracted with 1 liter distilled water for 24 h at room temperature (RT) with shaking. Following filtration, the extract was concentrated under decreased pressure with a rotary evaporator and lyophilized (yield=31.44%). The obtained powder was termed ‘PU’. A voucher specimen was deposited at the College of Korean Medicine of Kyung Hee University (Seoul, Korea).

PU was identified on the basis of its loganin and sweroside content by high-performance liquid chromatography (HPLC) with diode-array detection. The extract was dissolved in 70% methanol and sonicated for 30 min. Following filtration through a 0.2 µm filter membrane, 10 µl of aliquot was subjected to HPLC Agilent 1100 series (Agilent Technologies, Inc., Santa Clara, CA, USA). Chromatographic separation was achieved using a C18 column (250×4.6 mm, 5 µm; Shiseido, Osaka, Japan). Mobile phase A involved water with 0.1% formic acid, and mobile phase B consisted of acetonitrile with 0.1% formic acid. The separation temperature was set at 30°C and a flow rate of 0.45 ml/min. The peak on PU was synchronized with loganin and sweroside. The concentration of loganin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and sweroside (Sigma-Aldrich) in PU was 82.32 and 237.65 µg/ml, respectively.

ICR mice were purchased from Raonbio, Inc. (Yongin, Korea). Female 6-week-old ICR mice were housed at 22±1°C in an atmosphere with 55±10% humidity in a 12 h light: dark cycle with ad libitum access to a standard chow diet (Orient Bio., Inc., Seongnam, Korea) and water. The animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University Laboratory Animal Center [approval no. KHUASP (SE)-15-079].

The mice were randomized into 7 groups (n=7; total=49 mice): Sham-operated mice (Sham group); OVX mice treated orally with vehicle (OVX group); OVX mice injected intraperitoneally with 10 µg/kg 17β-estradiol (E2 group); OVX mice treated orally with 150 mg/kg calcium chloride (Ca group); OVX mice treated orally with 1 mg/kg PU (PU1 group); OVX mice treated orally with 10 mg/kg PU (PU10 group); and OVX mice treated orally with 100 mg/kg PU (PU100 group). E2 and Ca were used as positive controls. All treatments started at 7 weeks following OVX surgery, and lasted for 6 weeks. At 13 weeks after the experiment began, the animals were sacrificed, and blood was collected by cardiac puncture. The right and left femurs were obtained.

The right femur was fixed in 10% neutralized formalin for 18 h at RT and demineralized with 0.1 M ethylenediaminetetraacetic acid aqueous solution for 1 month. Following demineralization, femur samples were dehydrated by using xylene and consecutive ethanol concentrations (70, 80, 90, 95 and 100%) at 10 min each. Sagittal sections of the paraffin-embedded tissues were sliced at a 7 µm thickness. The slides were stained with hematoxylin for 5 min and eosin solution for 5 sec at RT according to kit instructions (Sigma-Aldrich). Histological changes were monitored using the Leica Microscope DML B2/11888111 equipped with a Leica camera DFC450 (Leica Microsystems, Buffalo Grove, IL, USA) at ×100 magnification.

Following sacrifice, the left femur was collected and cleaned by removing the attached muscles and connective tissues. The sample was stored in 10% neutralized formalin until use. The levels of BMD and BMC in the left femur were determined by dual-energy X-ray absorptiometry with an InAlyzer instrument (Medikors, Seongnam, Korea).

Samples were prepared from blood collected by cardiac puncture in heparinized tubes. The collected blood was centrifuged at 27,000 × g and 22°C for 30 min, and then the supernatant was stored at −80°C until use. The concentration of serum calcium was measured using the Calcium Colorimetric Assay kit (AdipoGen Life Sciences, Shizuoka, Japan) according to the manufacturer's protocol. The concentration of calcium in the serum was measured at 570 nm absorbance using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The human osteoblastic Saos-2 cell line (Korean Cell Line Bank, Seoul, Korea) was routinely grown in Dulbecco's modified minimal essential medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% penicillin and 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂ and 95% humidity. The culture medium was changed every 3–4 days. To confirm the cytotoxicity of PU, Saos-2 cells were incubated with culture medium containing different concentrations of PU extract (0.01, 0.1 and 1 µg/ml) for 10 days. Subsequently, 2 mg/ml MTT solution was added for 4 h. Dimethyl sulfoxide was then added, and cell viability was measured at an absorbance of 570 nm.

The cells were seeded in 6-well plates at density of 0.8×10⁵ cells/well and stabilized for 24 h. To induce osteoblast differentiation, 50 µg/ml L-ascorbic acid (AA; Thermo Fisher Scientific, Inc.) and 10 mM β-glycerophosphate (β-GP; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were added into osteogenic culture medium for 10 days. The culture medium was changed every 3–4 days. Then, the cells were fixed in 10% formalin for 10 min and stained with the 40 mM Alizarin Red-S (pH 4.2; Sigma-Aldrich; Merck KGaA) for 15 min, all at RT. The plates were observed under the Leica Microscope DML B2/11888111 equipped with Leica camera DFC450 at ×100 magnification. For quantification of Alizarin red S, 500 µl citrate solution containing 20% methanol and 10% acetic acid was added for 20 min at RT, and the absorbance of supernatants was measured at 570 nm using an ELISA reader (Molecular Devices, LLC., Downingtown, PA, USA).

The Saos-2 cells were lysed with radioimmunoprecipitation assay lysis buffer (BioPrince, Seoul, Korea) containing protease inhibitors (Sigma-Aldrich). The Bradford method was used for quantification of total protein. Subsequently, 20 µg of each sample was resolved using 10% SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) for 1 h at RT and then incubated with primary antibodies against runt-related transcription factor 2 (Runx2; 1:700 dilution; cat. no. 12556; Cell Signaling Technology, Inc., Danvers, MA, USA), transcription factor Sp7 (osterix; 1:1,000 dilution; cat. no. ab22552; Abcam, Cambridge, MA, USA) and β-actin (1:1,000 dilution; cat. no. sc-69879; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) diluted in TBS containing 0.1% Tween-20 (TBS-T) overnight at 4°C. The membrane was washed and incubated with m-IgG BP-HRP (1:2,000 dilution; cat. no. sc-516102; Santa Cruz Biotechnology, Inc.) and mouse anti-rabbit immunoglobulin G-horseradish peroxidase (1:2,000 dilution; cat. no. sc-2357, Santa Cruz Biotechnology, Inc.) diluted in TBS-T for 2 h at RT. Following washing, the bands were visualized with enhanced chemiluminescence (ECL) reagent (Amersham; GE Healthcare, Chicago, IL, USA). β-actin was used as an internal loading control for Runx2 and osterix. The band intensity was quantified using Image J software version 1.38e (National Institutes of Health, Bethesda, MD, USA). All experiments were performed in triplicate.

Significance was determined by one-way analysis of variance and followed by Dunnett's post-hoc test, using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. All values are expressed as the mean ± standard error of the mean.

The thickness of the epiphyseal plate was significantly increased in the OVX group compared with the Sham group. Administration of E2 and Ca decreased the growth plate thickness compared with OVX group. Similarly, PU-treated mice (1, 10 and 100 mg/kg) exhibited an amelioration of growth plate hyperplasia compared with the OVX-induced osteoporotic mice (Fig. 1).

The BMD level of the OVX group (0.115±0.004 g/cm²) was significantly decreased by 0.025 g/cm² compared with the Sham group (0.140±0.006 g/cm²). E2 injection and Ca administration as positive controls significantly increased the level of BMD (0.130±0.005 and 0.133±0.006 g/cm², respectively). Similar to the results from the positive controls, there were significant increases in BMC level in 10 and 100 mg/kg PU-treated femurs (0.123±0.005 and 0.127±0.002 g/cm², respectively; Fig. 2A) compared with in the OVX group.

The BMC level of the OVX mice (0.038±0.001 g) was 19.15% decreased compared with the Sham group (0.047±0.002 g). E2 injection and Ca administration as positive controls significantly increased BMC (0.043±0.001 and 0.044±0.001 g, respectively). PU treatment with 1, 10 and 100 mg/kg induced significant increases in BMC levels at all concentrations to 0.040±0.001; 0.041±0.002 and 0.043±0.002 g, respectively, compared with in OVX mice (Fig. 2B).

The serum calcium level was significantly decreased in OVX group (8.96±0.24 mg/dl) compared with the Sham group (10.70±0.52 mg/dl). E2 and Ca treatment significantly increased the calcium level compared with the OVX group (9.83±0.14 and 9.80±0.63 mg/dl, respectively). Similarly, administration of 1, 10 and 100 mg/kg PU markedly increased the calcium levels to 9.77±0.49, 9.78±0.50 and 9.84±0.55 mg/dl, respectively (Fig. 3).

The cells in the presence of AA and β-GP exhibited intense red coloring. Addition of 0.1 and 1 µg/ml PU markedly increased the intensities of Alizarin Red S staining compared with control cells in the absence of PU. The percent of calcification was 12.32±3.80 and 25.62±0.68%, respectively (Fig. 4). No cytotoxic effect was observed in Saos-2 cells at any concentration of PU.

Runx2 expression levels were significantly increased by 0.01, 0.1 and 1 µg/ml PU treatment as compared with differentiated Saos-2 osteoblast cells (39.04±13.03, 46.73±7.32 and 63.06±6.92%, respectively; Fig. 5A). However, osterix expression was not significantly altered compared with the differentiated cells. To confirm the osteogenic effects of PU on Runx2 expression, cells were treated with PU in the absence of differentiation medium for 10 days. Consistent with the result from differentiated osteoblasts, Runx2 expression was significantly increased by 0.1 and 1 μg/ml PU treatment (41.62±14.31 and 69.81±11.00%, respectively; Fig. 5B) in undifferentiated Saos-2 osteoblast cells. By contrast, osterix expression was not increased in PU-treated cells.