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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.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.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.(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).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.

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.

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.

Graphical abstract, Highlights • The contents of glabridin and medical component glycyrrhizin were analyzed in herbal medicines and health foods containing licorice. • A small amount of glycyrrhizin was detected in some of health foods. • Estrogenic activity was detected in some of health food ingredients and health foods. • Antioxidant activity was detected in all the materials. • Hepatic CYP activity, liver and uterine weights in estrogen deficient mice are also valuable for the safety evaluation of estrogenic ingredients., Focusing on licorice, a highly used raw material in health foods, quantitative analysis of functional/medicinal components and a safety and functional evaluation was carried out for herbal medicines, health food ingredients, and so-called health foods. A functional component, glabridin, was detected in herbal medicines from Glycyrrhiza glabra and G. inflata, health food ingredients, and in commercially available health foods that contain licorice. Likewise, glycyrrhizin, a medicinal component, was detected in these sources, except in licorice oil extract. Estrogen activity in vitro was detected in some of the herbal medicines, health food ingredients, and in health foods containing licorice. In the in vivo study, liver weight in ovariectomized (OVX) mice treated with licorice oil extract was significantly higher than that in OVX and sham mice in a dose dependent manner. These results suggest that excessive intake of licorice oil extract from health foods should be avoided, even though these ingredients might be beneficial for medical use in order to maintain bone health in postmenopausal women. Measurement of hepatic cytochrome P-450 (CYP) activity, reproductive organ weight, and fat and bone mass in OVX mice was considered useful for evaluating the safety and efficacy of estrogenic health food ingredients derived from herbal medicines.

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.

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.

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.

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.

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.

OBJECTIVES: Osteoporosis is a major health problem in the elderly characterized by bone loss and micro-architectural deterioration of bone tissue and associated with an increased risk of fracture. Prolonged bed rest, or physical inactivity during space flight causes rapid and marked bone loss. The effects of catechin, the main ingredient of Japanese green tea, on the bone are currently under study. It has been shown that green tea catechin modulates bone resorption in osteoclasts. However, there is no evidence supporting its inhibitory effect on bone loss during physical inactivity. In the present study, we investigated whether green tea catechin prevented bone loss through skeletal hindlimb-unloading in mice. METHODS: Female 8-week-old ddY mice were divided into five groups (n = 6–8 each) and subjected to: (1) normal housing fed a control diet, (2) sham unloading fed a control diet, (3) hind limb-unloading fed a control diet, (4) hind limb-unloading fed a 0.05% epigallocatechin gallate (EGCG)-containing diet, and (5) hind limb-unloading fed a 0.25% EGCG-containing diet for three weeks. Purified EGCG (97%) was used for green tea catechin. RESULTS: Bone mineral density of the tibia significantly decreased in hind limb-unloading mice. Treatment with 0.25% EGCG prevented bone loss and maintained trabecular bone mineral density more significantly than in cortical bones. The 0.25% EGCG diet inhibited decrease in the gene expression of alkaline phosphatase, a marker of bone formation, in the bone marrow in hind limb-unloading mice. CONCLUSIONS: These results suggest that EGCG has ability to prevent bone loss induced by hindlimb-unloading in mice. These osteoprotective effects of EGCG may result from the inhibition of unloading-induced decrease in bone formation. FUNDING SOURCES: This work was supported by the Honjo International Scholarship Foundation of Japan.

OBJECTIVES: Dish based nutrient profile analyses are essential in setting goals to achieve a balanced diet. The Japanese enjoy longevity, but their current diet is still high in salt, which was 10.1g/day among adults in the 2019 National Health and Nutrition Survey. In 2013, the Japanese government's “Health Meal” proposed the salt content of 3g/650 kcal per meal. However, studies are scarce regarding the nutrient profiles of the dishes eaten by the general population, especially who exceed the current standard. In order to examine the current achievement status and related nutritional problems among a nationally representative sample, we conducted the secondary analyses of the 2013–2018 National Health and Nutrition Survey data. METHODS: Dietary data from 35,915 participants in the 2013–2018 National Health and Nutrition Survey aged 18–74 years who ate three meals on the survey day, were applied for analyses. We extracted 554,257 dishes from each meal, and categorized them according to the definition of “staple dish” (grain dish), “main dish”(meat, fish, eggs, or beans), and “side dish”(vegetables, seaweeds, potatoes, or mushrooms) shown in the 2005 “Japanese Food Guide Spinning Top”. Participants were grouped as “high salt” consumers if their intake were 3g/650kcal or higher, and “adequate” consumers if they consumed less. The nutrient profile of each dish category was compared between the two groups by using the Wilcoxon's signed rank test. RESULTS: 22 300 participants were identified as “high salt” and 13 615 as “adequate”. The mean age of the “high salt” group (53.2 years) was higher than the “adequate” group (50.5 years), but their mean energy intake was similar (1901 and 2081 kcal/day, respectively). The mean salt intake in the “high salt” group was 11.8 g/day and the “adequate” group was 7.7 g/day. Among the dishes, 59 936 were categorized as “staple dish”, 47 081 as “main dish”, 45 828 as “side dish”, 30 151 as “combined dish”, and 371 261 as “miscellaneous”. Except for “staple dish”, dishes consumed by the “high salt” group were significantly low in energy and fat but high in salt, compared to the “adequate”. CONCLUSIONS: Older participants were more likely to consume dishes high in salt, but low in fat. Examples of balanced dishes low in both salt and fat could be proposed for further diet modification. FUNDING SOURCES: Ministry of Health, Labour and Welfare research grant.

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.

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.

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 17β-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.

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.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.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% (p0.05) after the kiwifruit and isoflavone treatment. There were no changes in serum CTx or in the diversity of the gut microflora.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.

Few studies have examined the effects of isoflavones and particularly equol, a metabolite of the isoflavone daidzein, on bone formation during the growth period in animals. The present study investigated the effects of orally administered daidzein or equol on bone formation and bone mineral density in growing female rats. Female Sprague-Dawley rats, aged 3 weeks, were divided into four groups (n = 8 per group) as follows: rats were orally administered a corn oil, 8 mg/day of daidzein, 4 mg/day of equol or 8 mg/day of equol in corn oil for 4 weeks. Daidzein and equol increased the bone mineral density of growing female rats by stimulating bone formation without exhibiting a substantial effect on the weight of their reproductive organs. Bone growth caused by increased bone mineralizing surface and bone formation rate in rats administered with equol was approximately twice that of rats administered with daidzein. These results suggest that equol might be more efficient than daidzein for bone formation in growing female rats. Copyright © 2015 John Wiley & Sons, Ltd.

Highlights • We assessed perinatal or peripubertal exposure to daidzein in rats. • Perinatal exposure to daidzein does not confer a positive effect on BMD. • Peripubertal exposure to daidzein protects against a decline in BMD. • Exposure to daidzein does not have serious adverse effects on sexual development., Neonatal exposure to isoflavones improved bone health in thereafter in previous animal studies. However, since isoflavones possess hormonal activity, it may interfere with reproductive development. In the present study, we assessed the safety and efficiency of perinatal or peripubertal exposure to daidzein on bone and reproductive organ development at early adulthood in rats. Sprague-Dawley pregnant rats (n = 18) were divided into 3 groups: (1) dams and their offspring were fed the control diet. (2) Dams were fed the daidzein diet (0.5 g daidzein/kg diet) during pregnancy and then the control diet at postnatal day 13 and their offspring were fed the control diet. (3) Dams and their offspring were fed the daidzein diet through the experiment. While perinatal exposure to daidzein did not confer a positive effect on bone mineral density on postnatal day 35, peripubertal exposure to daidzein protected against a decline in bone mineral density. Meanwhile, exposure to daidzein during the perinatal or peripubertal period did not affect reproductive organ weights at early adulthood in rats. Further investigations should assess the mechanisms underlying these responses of bone metabolism to daidzein, as well as the safety of daidzein exposure during the perinatal period and throughout life.

Low circulating 25-hydroxyvitamin D [25(OH)D] concentration has been linked to a high prevalence of cardiovascular disease. One explanation for this phenomenon is that there is an association between the serum 25(OH)D level and lipid profiles. However, studies examining this relationship are limited and have yielded inconsistent results. We thus aimed to evaluate the association between the serum 25(OH)D concentration and lipid profiles in Japanese men taking into consideration confounding factors, including the visceral fat area (VFA) and cardiorespiratory fitness.A total of 136 men (age range: 20-79 years) participated in our study. Fasting blood samples were analyzed to determine the 25(OH)D, oxidized low-density lipoprotein (oxLDL), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), apolipoprotein (Apo)A-1 and ApoB levels. The VFA was evaluated on magnetic resonance imaging (MRI), and cardiorespiratory fitness was assessed by measuring the peak oxygen uptake (VO2 peak).The median 25(OH)D concentration was 35.6 nmol/L, and the prevalence of 25(OH)D deficiency was 78.7%. A multiple linear regression analysis revealed that the serum 25(OH)D concentration was inversely related to the LDL-C/HDL-C, TG, ApoB and ApoB/ApoA-1 values, even after adjusting for age, season, smoking status, alcohol consumption, medication use, vitamin D intake, calcium intake, VFA and cardiorespiratory fitness.Serum 25(OH)D level is inversely correlated with the LDL-C/HDL-C, TG, ApoB and ApoB/ApoA-1 values in Japanese men, independent of the VFA and cardiorespiratory fitness.

β-Carotene has been reported to be useful to maintain a positive balance of bone turnover. However, the effects of β-carotene on bone loss remain to be elucidated in mice with hind limb unloading. Therefore, we investigated whether β-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% β-carotene-containing diet, (5) hind limb unloading fed a 0.05% β-carotene-containing diet, and (6) hind limb unloading fed a 0.25% β-carotene-containing diet. After 3 weeks, bone mineral density of the tibia was markedly reduced by unloading, which was prevented by 0.025% β-carotene. Histological analysis revealed a hind limb unloading-induced decrease in the calcified bone of the femur, which was slightly prevented by 0.025% β-carotene. The 0.025% β-carotene-containing diet increased the gene expression of osteoprotegerin in the bone marrow cells in unloading mice. These results suggest that a β-carotene-containing diet may preserve bone health in subjects with disabilities as well as in astronauts.

Equol is produced from daidzein, a soy isoflavone, through metabolism by intestinal bacteria, and benefits health. This cross-sectional study aimed to investigate its physiological range, using 24 h urinary equol excretion as an indicator, in Japanese people who consume soybeans in their usual diet. The 24 h urine of 1345 subjects (545 men and 800 women) was collected, and equol, daidzein, and genistein concentrations were measured. Subjects with detectable equol in their urine (⩾0.27 nmol/ml) were considered equol excretors. Among men, 36.3% were equol excretors; among women, 40.8%. The rate of equol excretors and urinary equol excretion differed significantly by age, but not by gender. In all equol excretors, the range of 24 h urinary equol excretion was 0.4–318.0 μmol/day; the median was 12.5 μmol/day; and the 95th percentile was 119.2 μmol/day. This range appears to be the physiological range of equol excretion in Japanese people who consume soy food regularly.

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.

In this study, we investigated the synergistic effects of daidzein (Dz) and kiwifruit on bone and equol production in ovariectomised (OVX) rats. Female Sprague-Dawley rats were randomly assigned to one of five groups: sham operated, OVX control, OVX fed 0.1% Dz-supplemented diet (OVX + Dz), OVX fed 0.1% Dz and green kiwifruit (GRK)-supplemented diet (OVX + Dz + GRK) and OVX fed 0.1% Dz and gold kiwifruit (GOK)-supplemented diet (OVX + Dz + GOK). There were no significant differences in whole body and femur bone mineral density (BMD) among groups at week 8. BMD in the OVX group significantly decreased at week 8; however, BMD in the OVX + Dz + GRK was not significantly different from baseline in the end of the study. However, supplementation with kiwifruit did not affect urinary equol concentrations, urinary ratios of equol to Dz and the composition of caecal microbiota. These results suggest that the combination of Dz and GRK may slightly reduce bone loss caused by oestrogen deficiency but does not affect equol production.

The purpose of this study was to examine whether hesperidin inhibits bone loss in androgen-deficient male mice. Male ddY mice aged 7 weeks underwent either a sham operation or orchidectomy (ORX) and were divided into five groups: a sham-operated group fed a control diet (Sham) based on AIN-93G formulation with corn oil instead of soy bean oil, an ORX group fed the control diet (ORX), a group fed the control diet containing 0.5% hesperidin (ORX + H), a group fed the control diet containing 0.7% α-glucosylhesperidin (ORX + αG), and a group fed the control diet containing 0.013% simvastatin (ORX + St). Four weeks after intervention, ORX mice showed a striking decrease in seminal vesicle weight, which was not affected by the administration of hesperidin, α-glucosylhesperidin, or simvastatin. Femoral BMD was significantly reduced by ORX, and bone loss was inhibited by the administration of hesperidin, α-glucosylhesperidin or simvastatin. Histomorphometric analysis showed that the bone volume and trabecular thickness were significantly lower, and the osteoclast number was higher in the distal femoral cancellous bone in the ORX group than in the Sham group, and these were normalized in the ORX + H, ORX + αG and ORX + St groups. These results indicate that hesperidin inhibited bone resorption and hyperlipidemia, in ORX mice, and the preventive effect was stronger than that observed in ovariectomized mice in our previous study. Copyright © 2013 John Wiley & Sons, Ltd.

The sum of hydrophilic antioxidants of 23 vegetables and 13 fruits commonly consumed in Japan was evaluated by a modified hydrophilic-oxygen radical absorbance capacity (H-ORAC) method. The “typical vegetable” and “typical fruit” in Japan contained hydrophilic antioxidants that are equivalent to 6.95 and 12.23 μmol of Trolox per g of the edible portion, respectively, on average. Hence, the daily intake of hydrophilic antioxidants from vegetables and fruits was estimated to be 4423 μmol Trolox equivalent (TE)/d based on data of the National Health and Nutrition Survey in Japan. However, the biological significance of these antioxidant values has not yet been clarified. To address this issue, our data will provide a foundation for high-quality epidemiological studies aimed at elucidating the relationship between daily intake of antioxidants and health. In addition, the comparison of the results of the H-ORAC assay with those of polyphenol content and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was also studied. The H-ORAC values had a strong positive correlation with polyphenol contents ( r = 0.956), and were 1.0–18.2-times higher than the antioxidant capacities evaluated by the DPPH assay.

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.

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.