(S)-Equol Is More Effective than (R)-Equol in Inhibiting Osteoclast Formation and Enhancing Osteoclast Apoptosis, and Reduces Estrogen Deficiency-Induced Bone Loss in Mice.

BACKGROUND: Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol. OBJECTIVES: We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo. METHODS: Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues. RESULTS: (S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively). CONCLUSIONS: These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

Nutrient Profiles of Dishes Consumed by the Adequate and High-Salt Groups in the 2014-2018 National Health and Nutrition Survey, Japan.

Dish-based nutrient profile analyses are essential for setting goals to achieve a balanced diet. In 2014, the Japanese government proposed the "Healthy Meal" criteria, which requires a salt content of 3 g/650 kcal per meal. To examine the current intake status of a nationally representative sample, we conducted a series of secondary analyses of the 2014-2018 National Health and Nutrition Survey data. Participants (aged 18-74 years) were grouped as "high-salt" consumers if their salt intake was 3 g/650 kcal or higher and "adequate" consumers if they consumed less than 3 g/650 kcal. A total of 13,615 participants were identified as "adequate" consumers and 22,300 as "high-salt" consumers. The median salt intake in the "high-salt" group was 11.3 g/day, while that in the "adequate" group was 7.5 g/day. Almost all dishes consumed by the "adequate" group had significantly high energy and fat content but low salt content, compared with those consumed by the "high-salt" group. For example, the median energy, fat, and salt contents in the main dishes consumed by the "adequate" group were 173 kcal, 10.4 g, and 0.9 g/dish, respectively, while those in the main dishes consumed by the "high-salt" group were 159 kcal, 8.9 g, and 1.1 g/dish, respectively. Examples of balanced dishes that are low in both salt and fat content can be proposed to help improve the Japanese consumers' dietary behavior.

The Combination of Soy Isoflavones and Resveratrol Preserve Bone Mineral Density in Hindlimb-Unloaded Mice.

It is well known that physical inactivity during space flight or prolonged bed rest causes rapid bone loss. Soy isoflavones (ISOs) and resveratrol (RES) have been reported to be useful to maintain a positive balance for bone turnover. Therefore, we examined the combined effects of ISO and RES on bone loss that was induced by hindlimb-unloading in mice. Female eight-week-old ddY mice were divided into the following six groups (n = 6-8 each): normally housed mice, loading mice, hindlimb-unloading (UL) mice fed a control diet, UL mice fed a 0.16% ISO conjugates, UL mice fed a 0.15% RES diet, and UL mice fed a 0.16% ISO and 0.15% RES diet. After three weeks, femoral bone mineral density was markedly decreased in unloading mice. The combination of ISO and RES prevented bone loss and especially maintained the trabecular bone mineral density more effectively compared with cortical bones. ISO and/or RES inhibited the increase in the RANKL/OPG expression ratio in bone marrow cells in UL mice. These results suggest that the combination of ISO and RES had a preventive effect against bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of ISO and RES may result from the inhibition of bone resorption.

Assessment of safety and efficacy of pine bark extract in normal and ovariectomized mice.

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women.

[Proficiency Testing Schemes for Food Nutrition Analysis in Japan (2017-2018)].

This paper deals with proficiency testing schemes for food nutrition analysis in Japan. In schemes in 2017 and 2018, 65 and 73 organizations participated, respectively, and more than 70% of the participants were public organizations responsible for a nutrition-labeling compliance test. The food matrices were pork and chicken sausages, and analytes were protein, fat, ash, moisture, carbohydrate, energy, sodium, salt equivalent, calcium (2018 only), and iron (2018 only). The organizations reporting inadequate laboratory values in one or more nutrients for mandatory declaration (energy, protein, fat, carbohydrate, or salt equivalent) were 11 and 15% of all organizations and 9 and 13% of public organizations in the 2017 and 2018 schemes, respectively. The approximate relative standard deviations for proficiency assessment (RSDr) were as follows: protein, 2%; fat, 3%; ash, 2%; moisture, 0.5%; carbohydrate, 9%; energy, 1%; sodium (salt equivalent), 4%; calcium, 7%; and iron, 7%. Notably, the large RSDr value for carbohydrate may cause inconsistency among laboratories in compliance tests for foods containing several grams or less of carbohydrate per 100 grams.

Activation of peroxisome proliferator-activated receptor gamma/small heterodimer partner pathway prevents high fat diet-induced obesity and hepatic steatosis in Sprague-Dawley rats fed soybean meal.

Soybeans are a complete nutritional resource and soybean proteins are known to affect lipid metabolism via multiple mechanisms. Soybean meal (SBM) is produced after extraction of soybean oil and in this study, we investigated the ability whether the SBM could prevent high fat diet-induced obesity and understand the underlying mechanisms. Male Sprague-Dawley rats, aged 5 weeks, were randomly divided into three groups (n=8 each) and fed one of three diets for 28 days: Cont (AIN-93G), HFD (high fat diet with 40% of calories derived from fat) and HFD+SBM (HFD with 30% SBM). White adipose tissue weight as well as plasma and hepatic triglycerides were significantly decreased in HFD+SBM rats. Expression of hepatic SREBP-1 and its target genes was significantly decreased in HFD+SBM rats. Meanwhile, expression of SHP gene expression was significantly increased in HFD+SBM, and there was a negative correlation between SHP and SREBP-1 expression. Together these results suggest that hepatic SREBP-1 gene expression is negatively regulated by SHP. Expression of PPARG, the transcriptional factor that regulates SHP expression, was increased in HFD+SBM rats. Furthermore, expression of genes controlled by PPARG/SHP, such as those involved in hepatic gluconeogenesis, was also significantly decreased in HFD+SBM rats. Therefore, in addition to the previous findings of SBM on obesity here we show an additional mechanism which by changing the expression of genes involved in lipid metabolism via the PPARG/SHP pathway in the liver.

Anti-Osteoporotic Effect of Soy Isoflavones Intake on Low Bone Mineral Density Caused by Voluntary Exercise and Food Restriction in Mature Female Rats.

Female athlete triad (FAT) is an interrelationship between menstrual dysfunction, low energy availability with or without eating disorder, and decreased bone mineral density (BMD) in female athletes. The purpose of this study was to investigate whether isoflavone intake can prevent bone loss caused by voluntary wheel running under energy-restricted condition. We used a female rat model of osteoporosis for female athletes established previously. Fourteen female Sprague-Dawley rats (8-wk old) were fed ad libitum and had free access to wheels throughout the study. At 18 wk of age, the rats were divided randomly into the following groups: 1) running control (RC), 2) running energy restriction (RR), and 3) running energy restriction and isoflavone-fed (RR+Iso) groups. The RR group was 30% dietary restricted. The RR+Iso group was 30% dietary restricted and fed the diet containing 0.5% isoflavone powder (Fujiflavone P40). The experimental period lasted 31 wk. At the end of this experiment, BMD of the proximal femur in the RR group was significantly lower than that in the RC group. However, the BMD in the RR+Iso group was not significantly different from that in the RC group. Moreover, the plasma estradiol (E2) level in the RR and RR+Iso groups was significantly lower than that in the RC group. These findings suggest that isoflavone intake inhibited bone loss when the E2 level was low in female mature rat model. Our findings may reveal the possible novel role of isoflavone in osteoporosis among female athletes.

Safety and Efficacy Assessment of Isoflavones from Pueraria (Kudzu) Flower Extract in Ovariectomised Mice: A Comparison with Soy Isoflavones.

Numerous Foods with Function Claims that contain the extract of Pueraria flower (kudzu) isoflavones (PFI) are available in the Japanese market. These are labelled with function claims of reducing visceral fat. However, these foods have not undergone proper safety assessment such as the evaluation of their oestrogenic activity and effects on drug-metabolising enzymes (cytochrome P-450: CYP) in the liver. This study evaluated the estrogenic effect and the hepatic CYP activity and mRNA expression in normal female mice as a safety assessment of PFI (Experiment 1). In addition, the bone mineral density and visceral fat weight in ovariectomised mice (OVX) compared to soy isoflavones (SI) was evaluated to assess the efficacy of PFI (Experiment 2). OVX control fed a control diet, OVX fed a PFI diet (the recommended human intake of PFI), OVX fed a PFI20 diet (20- times the recommended PFI), OVX fed an SI diet (the recommended human intake of SI), and OVX fed an SI20 diet (20 -times the recommended intake of SI) for 28 days in Experiment 2. Body, liver, and visceral fat weights were not affected by the PFI, PFI20, SI, or SI20 diets. The hepatic CYP1A and CYP3A activities were elevated by the SI20 treatment. Ovariectomy-induced bone loss was inhibited by the SI20 treatment, but not by the PFI20 treatment. These results suggest that (1) PFI intake in human doses had no oestrogenic properties and did not affect CYP activity in the liver; (2) there was no evidence that PFI affects the amount of visceral fat in OVX mice.

Comparison of Nutrient Reference Values for Food Labeling in Japan with CODEX Recommendations, Based on DRIs and Nutrient Intake in Japan.

To clarify the degree of consistency between the international recommendations and the national Japanese system, the nutrient reference values (NRVs) adopted by the CODEX were compared with current Japanese NRVs 2015, the dietary reference intakes for Japanese (DRIs-J) 2015, and actual nutrient intake levels by the Japanese population. The Japanese NRV for protein was high relative to CODEX NRV-R (i.e., NRV-Requirement). The Japanese NRVs for folate and calcium were low, and vitamin K was high, relative to each CODEX NRV-R. However, it was similar to the DRI-J values, and current intake levels for the Japanese population. For iron, calculation methods were different between the CODEX and Japan. Japanese iron NRV was calculated based on the RDA without menstruatating women, whereas CODEX NRV-R was calculated based on the INL98 of all adult men and women. Actual intake levels of iron for the Japanese population were similarly low. The Japanese NRV for sodium was high and potassium was low based on DRI-J values, relative to the CODEX NRV-NCD. For nutrients that show large discrepancies between the CODEX and Japanese NRVs, the values should be discussed further.

Resistant Starch Attenuates Bone Loss in Ovariectomised Mice by Regulating the Intestinal Microbiota and Bone-Marrow Inflammation.

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.

ß-Carotene prevents bone loss in hind limb unloading mice.

ß-Carotene has been reported to be useful to maintain a positive balance of bone turnover. However, the effects of ß-carotene on bone loss remain to be elucidated in mice with hind limb unloading. Therefore, we investigated whether ß-carotene prevented bone loss induced by skeletal hind limb unloading in mice. Female 8-week-old ddY mice were divided into six groups (n = 6-8 each) and subjected to: (1) normal housing, (2) sham unloading fed a control diet, (3) hind limb unloading fed a control diet, (4) hind limb unloading fed a 0.025% ß-carotene-containing diet, (5) hind limb unloading fed a 0.05% ß-carotene-containing diet, and (6) hind limb unloading fed a 0.25% ß-carotene-containing diet. After 3 weeks, bone mineral density of the tibia was markedly reduced by unloading, which was prevented by 0.025% ß-carotene. Histological analysis revealed a hind limb unloading-induced decrease in the calcified bone of the femur, which was slightly prevented by 0.025% ß-carotene. The 0.025% ß-carotene-containing diet increased the gene expression of osteoprotegerin in the bone marrow cells in unloading mice. These results suggest that a ß-carotene-containing diet may preserve bone health in subjects with disabilities as well as in astronauts.

The effects of green kiwifruit combined with isoflavones on equol production, bone turnover and gut microflora in healthy postmenopausal women.

BACKGROUND AND OBJECTIVES: Isoflavone (daidzein and genistein) interventions in postmenopausal women have produced inconsistent skeletal benefits, partly due to population heterogeneity in daidzein metabolism to equol by enteric bacteria. This study assessed changes in microflora and bone turnover in response to isoflavone and ki-wifruit supplementation in New Zealand postmenopausal women. METHODS AND STUDY DESIGN: Healthy women 1-10 years post-menopause were randomly allocated to group A (n=16) or B (n=17) for a 16-week crossover trial. Two consecutive 6-week treatment periods had a 2-week lead-in period at intervention commencement and a 2-week washout period between treatments. Treatments prescribed either (1) daily isoflavone supplementation (50 mg/day aglycone daidzein and genistein) alone, or (2) with two green kiwifruit. At treatment baseline and end-point (four time points) the serum bone markers C Telopeptide of Type I collagen (CTx), undercarboxylated os-teocalcin (unOC), and serum and urinary daidzein and equol, were measured. Changes in gut microflora were monitored in a subgroup of the women. RESULTS: Equol producers made up 30% of this study population (equol producers n=10; non-equol producers n=23) with serum equol rising significantly in equol producers. Serum ucOC decreased by 15.5% (p<0.05) after the kiwifruit and isoflavone treatment. There were no changes in serum CTx or in the diversity of the gut microflora. CONCLUSIONS: 50 mg/day isoflavones did not reduce bone resorption but kiwifruit and isoflavone consumption decreased serum ucOC levels, possibly due to vitamin K1 and/or other bioactive components of green kiwifruit.

Effect of daidzein and equol on DNA replication in MCF-7 cells.

It has been reported that daidzein and equol stimulate DNA replication and proliferation of MCF-7 cells. However, their molecular mechanisms of action are still unclear. We examined the effects of daidzein and equol on DNA replication of MCF-7 cells, focusing on MCM2-7 proteins, which function as the replicative helicase. In the presence of either 1 µM of daidzein or equol, the number of cells in S-phase, which was determined by detecting bromodeoxyuridine incorporated into replicated DNA, almost doubled. The total amounts of MCM7 protein and chromatin-bound MCM7 protein increased in the presence of daidzein. The data suggest that phytoestrogens facilitate cell cycle progression in G1-phase by increasing the level of MCM proteins. In the presence of phytoestrogens, phosphorylation of Rb and levels of MCM2, 3 and 7 mRNA increased, suggesting that stimulation of MCM2-7 transcription is involved in the cell cycle progression. Under the same conditions, double-stranded DNA breakage in logarithmically growing MCF-7 cells, which was detected using anti-γ-H2AX antibodies, did not increase in the presence of equol.

Kanamycin inhibits daidzein metabolism and abilities of the metabolites to prevent bone loss in ovariectomized mice.

BACKGROUND: Daidzein is an isoflavone derived from soybeans that exerts preventive effects on bone loss in ovariectomized (OVX) animals. These effects have been correlated with increasing serum equol levels. In the present study, we investigated the effects of antibiotic intake on equol metabolism from daidzein, and the corresponding levels of bone loss in OVX mice. METHODS: Eight-week-old female ddY mice (n = 42) were either ovariectomized (OVX) or subjected to a sham operation (sham). OVX mice were then divided into six dietary subgroups: control diet (control), 0.3 % kanamycin diet (KN), 0.1 % daidzein diet (Dz), 0.1 % daidzein and 0.0375 % kanamycin diet (Dz+KN3.75), 0.1 % daidzein and 0.075 % kanamycin diet (Dz+KN7.5), and 0.1 % daidzein and 0.3 % kanamycin diet (Dz+KN30). The mice were fed their respective diets for 4 weeks. RESULTS: Uterine weight and femoral bone mineral density (BMD) were significantly lower in the OVX mice compared in the sham mice. No significant differences in uterine weight were observed among all OVX dietary subgroups. The Dz subgroup was found to exhibit higher plasma equol and O-desmethylangolensin (O-DMA) concentrations, as well as greater femoral BMD, compared to all other OVX subgroups. Furthermore, when compared to the Dz group, kanamycin intake decreased plasma equol and O-DMA concentrations, as well as femoral BMD in the OVX mice. CONCLUSIONS: These results suggest that kanamycin intake inhibited the conversion of daidzein to equol and O-DMA, blocking the preventive effects of daidzein on bone loss in OVX mice. Therefore, the bone-protective effects of daidzein intake may be predominantly associated with increased plasma concentrations of either equol or O-DMA.

A combination of soy isoflavones and cello-oligosaccharides changes equol/O-desmethylangolensin production ratio and attenuates bone fragility in ovariectomized mice.

We examined the cooperative effects of isoflavones and cello-oligosaccharides on daidzein metabolism and bone fragility in ovariectomized mice. Cello-oligosaccharides increased urinary equol and decreased O-desmethylangolensin. A combination of isoflavones and cello-oligosaccharides attenuated decreases in bone breaking force and stiffness caused by ovariectomy. Combination treatment with isofalvones and cello-oligosaccharides increases urinary equol/O-desmethylangolensin production ratio and prevents ovariectomy-induced abnormalities in bone strength.

The combined effects of soya isoflavones and resistant starch on equol production and trabecular bone loss in ovariectomised 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.

Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro.

The present study aimed at investigating the effects of Puerarin (PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized (OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR (2, 4, and 8 mg·d(-1), respectively) or 17ß-estradiol (E2, 0.03 µg·d(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density (BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand (RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin (OPG) and RANKL mRNA expression in osteoblasts. The expression of OPG mRNA and RANKL mRNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG mRNA and reduced the expression of RANKL mRNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts.

Combined effects of soy isoflavones and milk basic protein on bone mineral density in hind-limb unloaded mice.

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.

Osteoporosis and Lifestyle.

Skeletal tissue is formed during the first two decades of life; then a constant bone mass is maintained until 40 y of age. In the case of women, the bone mass is rapidly reduced at menopause at around 50 y of age. After that, bone mass slowly decreases in both men and women who have passed the 70-y-old mark. The National Institute of Health Consensus Conference adopted the definition of osteoporosis as a skeletal disorder that is characterized by compromised bone strength leading to a predisposition for and an increased risk of fracture. Since osteoporotic fractures are the third-highest cause for becoming bedridden, the maintenance of healthy bones is an important factor in extending a person's healthy lifespan. Bone mass is influenced by many factors, such as nutrition, physical activity, smoking and alcohol intake, as well as by genetic factors. Thus, a healthy diet providing balanced nutrients including calcium, vitamin D, vitamin K and protein, regular physical activity, and not smoking help maintain bone health and delay or prevent osteoporosis. Some functional foods containing soy isoflavones, milk basic protein and n-3 fatty acid may help promote bone health.

Combined Effects of Soy Isoflavones and ß-Carotene on Osteoblast Differentiation.

Soy isoflavones, genistein, daidzein and its metabolite equol, as well as ß-carotene have been reported to be effective for maintaining bone health. However, it remains to be elucidated whether combining soy isoflavones with ß-carotene is beneficial to bone formation. This study investigated the combined effect of soy isoflavones and ß-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 ß-carotene resulted in higher ALP activity than treatment with isoflavones or ß-carotene alone. We observed significant main effects of ß-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 ß-carotene affected osteoblast differentiation, MC3T3-E1 cells were treated with retinoic acid receptor (RAR) pan-antagonist combined with ß-carotene. Osteopontin and ALP mRNA expression levels, which were increased following treatment with ß-carotene, were significantly suppressed by the RAR pan-antagonist. This suggests treatment with ß-carotene enhanced early osteoblastic differentiation, at least in part via RAR signaling. These results indicate that a combination of isoflavones and ß-carotene may be useful for maintaining a positive balance of bone turnover by inducing osteoblast differentiation.