Bone sparing effect of a novel phytoestrogen diarylheptanoid from Curcuma comosa Roxb. in ovariectomized rats.

Phytoestrogens have been implicated in the prevention of bone loss in postmenopausal osteoporosis. Recently, an active phytoestrogen from Curcuma comosa Roxb, diarylheptanoid (DPHD), (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, was found to strongly promote human osteoblast function in vitro. In the present study, we demonstrated the protective effect of DPHD on ovariectomy-induced bone loss (OVX) in adult female Sprague-Dawley rats with 17β-estradiol (E2, 10 µg/kg Bw) as a positive control. Treatment of OVX animals with DPHD at 25, 50, and 100 mg/kg Bw for 12 weeks markedly increased bone mineral density (BMD) of tibial metaphysis as measured by peripheral Quantitative Computed Tomography (pQCT). Histomorphometric analysis of bone structure indicated that DPHD treatment retarded the ovariectomy-induced deterioration of bone microstructure. Ovariectomy resulted in a marked decrease in trabecular bone volume, number and thickness and these changes were inhibited by DPHD treatment, similar to that seen with E2. Moreover, DPHD decreased markers of bone turnover, including osteocalcin and tartrate resistant acid phosphatase (TRAP) activity. These results suggest that DPHD has a bone sparing effect in ovariectomy-induced trabecular bone loss and prevents deterioration of bone microarchitecture by suppressing the rate of bone turnover. Therefore, DPHD appears to be a promising candidate for preserving bone mass and structure in the estrogen deficient women with a potential role in reducing postmenopausal osteoporosis

Estrogenic activity of DPHD compared to E₂.

<p>Structure of the phytoestrogen diarylheptanoid DPHD, (3<i>R</i>)-1,7-diphenyl-(4<i>E</i>,6<i>E</i>)-4,6-heptadien-3-ol, isolated from the rhizome of <i>C. comosa</i> (A). Effects of DPHD on body weight (B) and uterine weight (C) of sham-operated and ovariectomized (OVX) rats receiving vehicle and various doses of DPHD (25, 50 and 100 mg/kg Bw) or 17β-estradiol (E₂, 10 µg/kg Bw) for 12 weeks. Results are expressed as the mean ± SEM, n = 6–8. **p<0.01, significantly different from sham rats. ^†p<0.05 and <b>^††</b>p<0.01, significantly different from OVX rats.</p

DPHD increases ex vivo bone mineral density (BMD), as measured by pQCT.

<p>Total (A), trabecular (B), and cortical (C) BMD of tibial metaphysis from sham-operated and ovariectomized (OVX) rats receiving vehicle, DPHD (25, 50 and 100 mg/kg Bw) or 17β-estradiol (E₂, 10 µg/kg Bw) for 12 weeks. Results are expressed as mean ± SEM, n = 6−8. **p<0.01, significantly different from sham rats. ^†p<0.05 and ^††p<0.01, significantly different from OVX rats.</p

Effects of DPHD on biochemical bone turnover markers.

<p>Serum osteocalcin levels (A) and TRAP activity (B) of sham-operated and ovariectomized (OVX) rats receiving the indicated doses of DPHD (25, 50 and 100 mg/kg Bw) or 17β-estradiol (E₂, 10 µg/kg Bw) for 12 weeks. Results are expressed as the mean ± SEM, n = 6−8. **p<0.01 significantly different from sham rats and ^†p<0.05 and ^††p<0.01, significantly different from OVX rats.</p

Reversal of OVX induced bone microarchitectural changes by DPHD treatment.

<p>Representative 2D images of the proximal tibial metaphysic (trabecular structure) of sham operated and OVX rats receiving vehicle, DPHD (DPHD 100 mg/kg Bw and 17β-estradiol (E₂, 10 µg/kg Bw) for 12 weeks. Samples were stained with Goldner’s trichrome for bright-field microscopy at a magnification of 2X showing the following: epiphysis, epiphyseal plate (EP), trabecular bone (Tb), and cortical bone (Cor) (A). Fluorescence micrographs (calcein labeling) (B). Static parameters: trabecular bone volume normalized by tissue volume (BV/TV, %) (C), trabecular number (Tb.N, mm⁻¹) (D), trabecular thickness (Tb.Th, µm) (E), and trabecular separation (Tb.Sp, µm) (F). Dynamic parameters: mineral apposition rate (MAR) (G) and bone formation rate (BFR) (H). Data are expressed as the mean ± SEM, n = 6−8. **p<0.01 significantly different from sham rats and ^†p<0.05 and ^††p<0.01, significantly different from OVX rats.</p

Effect of DPHD on bone area and thickness of OVX rats.

<p>Total, trabecular, and cortical cross sectional area (CSA) and cortical thickness were measured from sham-operated and ovariectomized (OVX) rats receiving vehicle, DPHD (25, 50 and 100 mg/kg Bw) or 17β-estradiol (E₂, 10 µg/kg Bw) for 12 weeks. Data are expressed as mean ± SEM, n = 6−8.</p>*<p>p<0.05, significantly different from sham rats.</p>†<p>p<0.05, significantly different from OVX rats.</p