Equol is a metabolite of the soya isoflavone (ISO) daidzein that is produced by intestinal microbiota. Equol has greater oestrogenic activity compared with other ISO, and it prevents bone loss in postmenopausal women. Resistant starch (RS), which has a prebiotic activity and is a dietary fibre, was reported to promote equol production. Conversely, the intestinal microbiota is reported to directly regulate bone health by reducing inflammatory cytokine levels and T-lymphocytes in bone. The present study evaluated the combined effects of diet supplemented with ISO and RS on intestinal microbiota, equol production, bone mineral density (BMD) and inflammatory gene expression in the bone marrow of ovariectomised (OVX) mice. Female ddY strain mice, aged 8 weeks, were either sham-operated (Sham, n 7) or OVX. OVX mice were randomly divided into the following four groups (seven per group): OVX control (OVX); OVX fed 0·05 % ISO diet (OVX + ISO); OVX fed 9 % RS diet (OVX + RS); and OVX fed 0·05 % ISO-and 9 % RS diet (OVX + ISO + RS). After 6 weeks, treatment with the combination of ISO and RS increased equol production, prevented the OVX-induced decline in trabecular BMD in the distal femur by modulating the enteric environment and altered OVX-induced inflammation-related gene expression in the bone marrow. However, there were no significant differences in bone parameters between the ISO + RS and ISO-alone groups in OVX mice. Our findings suggest that the combination of ISO and RS might alter intestinal microbiota and immune status in the bone marrow, resulting in attenuated bone resorption in OVX mice.Key words: Soya isoflavones: Resistant starch: Bones: Osteoporosis: Intestinal microbiota Osteoporosis is a chronic disease characterised by low bone strength, predisposing to an increased risk of fracture (1) . Postmenopausal women have a higher risk of developing osteoporosis because of declining oestrogen concentrations associated with menopause (2) . One treatment for osteoporosis is hormone replacement therapy; however, its use can result in adverse effects, such as the induction of hormone-dependent breast and uterine cancers (3) .Epidemiological studies indicate that women with high soya intake have a lower risk for osteoporosis compared with those consuming a typical Western diet (4) . In addition, many studies indicate that soya isoflavone (ISO)-rich extracts have bone-preserving functions in postmenopausal women (5,6) and oestrogen-deficient animals (7) . Soyabean ISO are structurally similar to oestrogen and bind to oestrogen receptors (8) , suggesting that they exhibit weak oestrogenic action in various tissues and therefore might prevent postmenopausal disorders such as osteoporosis (6) . However, the results of randomised-controlled trials on the bone-protective effects of soya ISO in menopausal women are controversial. Some studies have shown that ISO supplements have a modest effect on bone-sparing or on bone metabolism markers (6,9,10) , whereas others considering ISO supplementation with simultaneous...

Soy isoflavones, genistein, daidzein and its metabolite equol, as well as β-carotene have been reported to be effective for maintaining bone health. However, it remains to be elucidated whether combining soy isoflavones with β-carotene is beneficial to bone formation. This study investigated the combined effect of soy isoflavones and β-carotene on the differentiation of MC3T3-E1 preosteoblastic cells. Daidzein and genistein alone did not affect cell growth but increased alkaline phosphatase (ALP) activity. Beta-carotene alone inhibited cell growth and markedly enhanced ALP activity. Soy isoflavones combined with β-carotene resulted in higher ALP activity than treatment with isoflavones or β-carotene alone. We observed significant main effects of β-carotene on the enhanced expression of Runx2, ALP, and ostepontin mRNA, whereas there was a significant main effect of soy isoflavones on the expression of osterix mRNA. To investigate how β-carotene affected osteoblast differentiation, MC3T3-E1 cells were treated with retinoic acid receptor (RAR) pan-antagonist combined with β-carotene. Osteopontin and ALP mRNA expression levels, which were increased following treatment with β-carotene, were significantly suppressed by the RAR pan-antagonist. This suggests treatment with β-carotene enhanced early osteoblastic differentiation, at least in part via RAR signaling. These results indicate that a combination of isoflavones and β-carotene may be useful for maintaining a positive balance of bone turnover by inducing osteoblast differentiation.