These findings suggest that 10 mg/day of natural S-equol supplementation contributes to bone health in non-equol-producing postmenopausal women without adverse effects.

No abstract

The intestinal microbiota may regulate bone metabolism by reducing levels of pro-inflammatory cytokines, and T cells in bone tissues of oestrogen-deficient mice have been reported. Resistant starch (RS) is a type of dietary fibre and results in changes in the composition of the gut microbiota. We evaluated the effects of RS supplemented in diets on intestinal microbial composition, bone mineral density, and inflammatory-gene expression in the colon and bone marrow of ovariectomised (OVX) mice. OVX mice were divided randomly into three groups: OVX control, OVX fed a 20% high amylose corn starch (HAS) diet, and OVX fed a 20% acid-hydrolysed HAS (AH-HAS) diet. HAS and AH-HAS diets contained 6.8% and 12% of RS, respectively. After 6 weeks, treatment with HAS or AH-HAS increased the abundance of Bifidobacterium spp. in faeces. The AH-HAS diet tended to upregulate mRNA expression of interleukin (IL)-10 in the colon, and downregulate expression of receptor activator of nuclear factor kappa-B ligand and IL-7 receptor genes in the bone marrow of OVX mice. AH-HAS treatment attenuated ovariectomy-induced bone loss. These findings suggest that AH-HAS might change the microbiota and immune status of the bone marrow, resulting in attenuated bone resorption in OVX mice.

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.

S-equol is a natural metabolite of the soy isoflavone, daidzein, produced by intestinal bacteria. S-equol has been shown to have greater estrogenic activity than other soy isoflavones and prevent bone loss in post-menopausal women. Estrogen regulates both bone remodeling and hemopoiesis in the bone marrow, these processes that communicate closely with each other. In this study, we investigated the effect of S-equol on bone mass and gene expression of bone marrow cells in ovariectomized (OVX) mice. Female ddY strain mice, aged 12 weeks, were either sham operated or OVX. The OVX mice were randomly divided into two groups: (1) OVX control and (2) OVX fed a 0.06% (w/w) S-equol supplemented diet. After 2 weeks, the trabecular bone volume of the femoral distal metaphysis was markedly reduced in OVX mice. However, treatment with equol was observed to ameliorate this. Expression of inflammatory-, osteoclastogenesis- and adipogenesis-related genes was increased in OVX mice compared with sham mice, and equol was observed to suppress their expression. The present study demonstrates that equol might ameliorate bone loss caused by estrogen deficiency through regulating hemopoiesis and production of inflammatory cytokines in bone marrow cells.

Osteoclasts play a major role in bone resorption. Several functional food components, such as soy isoflavones and carotenoids, are reported to inhibit osteoclast formation. However, the cooperative effect of functional foods or their constituents on bone metabolism has not been clarified. This study aimed to investigate the cooperative effect of soy isoflavones and carotenoids on osteoclast formation in vitro using cultures of RAW264 and bone marrow cells in the presence of receptor activator of nuclear factor κ-B ligand. In RAW264 cells, treatment with soy isoflavones (genistein or equol) or carotenoids (β-carotene) suppressed osteoclast formation. At 10 µM, genistein and equol inhibited RAW264 cell proliferation but did not affect cell viability. When 10 µM genistein or equol was combined with 0.1 µM β-carotene, we observed an additive suppressive effect on osteoclast differentiation. Similar results were observed with bone marrow cell cultures. We found that 10 µM of zeaxanthin or lutein suppressed osteoclast formation singly, and further enhanced the suppressive effects of daidzein or genistein when administered in combination. These results suggest that the combination of soy isoflavones and carotenoids have an enhanced suppressive effect on osteoclast formation. This knowledge might be important in planning diet for bone health.

We examined whether the combination of isoflavone and milk basic protein both are reported to be effective for bone metabolism, prevents bone loss induced by skeletal hind-limb unloading in mice. Female ddY strain mice, aged 8 weeks, were divided into six groups (n = 6–8 each): (1) normally housed group, (2) loading group, (3) hind-limb unloading group fed a control diet, (4) hind-limb unloading group fed a 0.2% isoflavone conjugates diet, (5) hind-limb unloading group fed a 1.0% milk basic protein diet, and (6) hind-limb unloading group fed a 0.2% isoflavone conjugates and 1.0% milk basic protein diet. After 3 weeks, femoral bone mineral density was markedly reduced in unloading mice. The combination of isoflavone and milk basic protein showed cooperative effects in preventing bone loss and milk basic protein inhibited the increased expression of osteogenic genes in bone marrow cells in unloading mice. These results suggest that the combination of soy isoflavone and milk basic protein may be useful for bone health in subjects with disabling conditions as well as astronauts.