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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.

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.

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.

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.

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.

New Zealand is a rich source of food components that may have bioactivity on bone. Docosahexaenoic acid (DHA) from fish oil has been shown to maintain bone in ovariectomised (OVX) rats. Kiwifruit, a source of fibre and carotenoids, may also affect bone via a prebiotic as well as direct cell-based mechanisms. We aimed to 1) ascertain the effects of DHA on two cell models, including interactions with soy isoflavones; 2) and investigate the specific effects of carotenoids from kiwifruit as well as whole kiwifruit in cell-based and rodent models as well as in a human study. RAW 264.7 mouse monocytes or mouse bone marrow was used to generate osteoclasts (OC). Cells were exposed to the agents between 5 and 21 d and formation and activity of OC measured, including molecular markers. DHA inhibited OC formation in both cell models, including expression of cathepsin K, NFATc1 as well as actin ring formation. Combination with isoflavones enhanced these effects. In OVX rats and mice fed with kiwifruit for 8 wk, green kiwifruit reduced the rate of bone loss after OVX, and in mice it reduced C-telopeptide of Type 1 collagen (CTX) levels and RANKL expression while in menopausal women, green kiwifruit affected blood lipids and bone markers positively.

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.

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.

OxHLIA is a cell-based antioxidant assay for evaluating inhibition of free radical-induced haemolysis in sheep erythrocytes by antioxidants. The original OxHLIA was performed in a test-tube format, and the degree of haemolysis was determined by absorbance of the supernatant after centrifugation. This made it difficult to evaluate large numbers of samples at the same time. Thus, we first showed here that the degree of haemolysis can be determined by turbidity of the erythrocyte suspension without centrifugation. We then developed OxHLIA in a 96-well plate format by adapting the turbidity method. There have already been some methodological studies on cell-based antioxidant assays using erythrocytes, and the method described here was comprised of the best mix of these earlier works with ingenious modifications. This plate method enables evaluation of large numbers of samples in small quantity at the same time with satisfactory precision and reproducibility.

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.

Fructooligosaccharides stimulate the growth of Bifidobacteria, which cleave isoflavone glycosides to yield corresponding aglycones, and convert metabolites by enhancing enterohepatic recirculation of isoflavones in rats. In the present study, we determined the synergistic effect of dietary isoflavone glycosides and fructooligosaccharides on postgastrectomy osteopenia in rats. Nine-week-old male Sprague-Dawley rats were gastrectomized (n = 20) or sham operated, (control, n = 5) and then randomly assigned to 5 diet groups: sham-a purified diet control, gastrectomized-control, gastrectomized-isoflavone (0.2% isoflavone glycosides), gastrectomized-fructooligosaccharides (7.5% fructooligosaccharides), and isoflavone and fructooligosaccharides (0.2% isoflavone glycosides + 7.5% fructooligosaccharides). After 6 weeks, the rats were killed and biological samples were collected. In gastrectomized rats, fructooligosaccharides prevented femoral bone fragility, but isoflavone without fructooligosaccharides did not inhibit postgastrectomy osteopenia. Isoflavone and fructooligosaccharides exhibited a synergistic in the distal metaphyseal trabecular bone, indicated by peripheral quantitative computed tomography. Moreover, fructooligosaccharides increased calcium absorption and equol production from daidzein in gastrectomized rats. These results indicate that isoflavone alone did not inhibit postgastrectomy osteopenia, but the combination of isoflavone and fructooligosaccharides improved the inhibition of trabecular bone loss by increasing calcium absorption and equol production through fructooligosaccharides supplementation.

The female athlete triad is a syndrome that leads to disordered eating, amenorrhea, and osteoporosis. The pathophysiologic mechanisms that lead to reduced bone mass in female athletes are low energy and functional hypothalamic amenorrhea. The aim of the present study was to establish an animal model of osteoporosis, which is similar to the osteoporosis seen in female athletes. We investigated the effects of long-term energy restriction on bone mineral density (BMD), and the levels of 17β-estradiol (E2) and luteinizing hormone (LH) in voluntary wheel-running female rats. Fourteen female Sprague-Dawley rats (8 weeks old) were randomly categorized into 2 groups: running (RUN) group and sedentary (SED) group. At 18 weeks of age, the rats in the RUN group were further randomly divided into 2 groups: running-ad libitum feeding group (RC group) or running-restricted feeding group (RR group). Twenty-four weeks after the experimental period, the RR group showed significantly lower BMD and plasma LH levels than the RC and SED groups (p < 0.05). Plasma E2 levels in the RR group were significantly lower than that in the SED group (p < 0.05). The present study indicated that long-term energy restriction with voluntary wheel running exhibited low bone mass in female rats with intact ovaries. Furthermore, the distinct endocrine profiles observed in this model suggest that energy restriction with voluntary exercise disturbs the hypothalamic-pituitary-ovarian axis. This may establish a model for the development of osteoporosis in the exercising human female with energy restriction, caused by dysfunction of the reproductive system.

Prevention is the most important measure against osteoporosis, since bone mass, once it is lost, cannot be recovered. Bone mass, in both men and women, reaches a maximum level in the 20s to 30s age range, and maintains this level or slightly increases toward the 40s, and gradually decreases thereafter. In particular, bone mass dramatically decreases in postmenopausal women because of a decrease in serum estrogen level. Bone mass is influenced by genetic and environmental factors such as mechanical loading, nutrition, and lifestyle, besides gender, age, and metabolic factors. Among these factors, mechanical loading, i.e. exercise, and nutrition seem to be important in maintaining bone health, since they are expected to improve with changes in lifestyle. Routine exercise and a diet rich in minerals and vitamins, throughout life, both contribute to maintaining bone health.

Objectives: If and to what extent soy protein, soy isoflavones, and their metabolites, including S(Y)-equol, have beneficial effects on women_s health is currently unclear. The North American Menopause Society (NAMS)/Utian Translational Science Symposium on Soy and Soy Isoflavones convened October 9-10, 2010, to clarify basic and clinical research findings as they relate to the risk and benefits of soy products for peri- and postmenopausal women. Methods: A working group of faculty and panelists composed of clinical and research experts in the fields of women_s health and botanicals met during a 2-day translational symposium to cover the latest evidence-based science on isoflavones as they affect menopausal symptoms, breast and endometrial cancer, atherosclerosis, bone loss, and cognition. Full descriptions of the bioavailability and pharmacokinetics of isoflavones were also presented. Subspecialty groups then broke off with the goal of translating the information into a report for general medical practice and identifying further research areas. All faculty and panelists reviewed the final report, which was then approved by the NAMS Board of Trustees. Results: From the hundreds of studies reviewed in this report, there are mixed results of the effects on midlife women. Soy-based isoflavones are modestly effective in relieving menopausal symptoms; supplements providing higher proportions of genistein or increased in S(Y)-equol may provide more benefits. Soy food consumption is associated with lower risk of breast and endometrial cancer in observational studies. The efficacy of isoflavones on bone has not been proven, and the clinical picture of whether soy has cardiovascular benefits is still evolving. Preliminary findings on cognitive benefit from isoflavone therapy support a Bcritical window[ hypothesis wherein younger postmenopausal women derive more than older women. Conclusions: Several areas for further research have been identified on soy and midlife women. More clinical studies are needed that compare outcomes among women whose intestinal bacteria have the ability to convert daidzein to equol (equol producers) with those that lack that ability (equol nonproducers) in order to determine if equol producers derive greater benefits from soy supplementation. Larger studies are needed in younger postmenopausal women, and more research is needed to understand the modes of use of soy isoflavone supplements in women. The interrelations of other dietary components on soy isoflavones consumed as a part of diet or by supplement on equol production also require further study, as do potential interactions with prescription and over-thecounter medications. And finally, greater standardization and documentation of clinical trial data of soy are needed.

Both soy isoflavone and n-3 polyunsaturatedfatty acids are known to reduce the levels of bone-resorbingcytokines; however, the synergistic effects of these foodingredients have not been examined yet. This study wasperformed to elucidate the effect of concomitant intake ofsoy isoflavone and fish oil on bone mass in ovariectomizedmice. Eight-week-old ddY female mice were subjectedto ovariectomy (OVX) or sham surgery, and then fed anAIN-93G with safflower oil (So) as a control lipid source,isoflavone-supplemented safflower oil (So ? I), fish oilinstead of safflower oil (Fo) or isoflavone-supplemented fishoil (Fo ? I) for 4 weeks. Femoral bone mineral density wassignificantly decreased by OVX; however, this decrease wasinhibited by the intake of isoflavone and/or fish oil. Histomorphometricanalyses showed that bone volume and trabecularthickness in the distal femoral trabecular bone weresignificantly lower in the So group than in the sham group,but those were restored in the Fo ? I groups. The number ofosteoclasts was significantly decreased by isoflavone intake, Original article. オンライン版はSpringerLinkで The Japanese Society for Bone and Mineral Research and Springer 2010. 45p.

Introduction Effects of soy isoflavone supplements on bone turnover markers remain unclear. This up-to-date systematic review and meta-analysis of randomized controlled trials (RCTs) was performed primarily to more completely and precisely clarify the effects on urinary deoxypyridinoline (DPD) and serum bone alkaline phosphatase (BAP) and secondarily to evaluate the effects on other bone turnover markers, compared with placebo in menopausal women. Methods PubMed, CENTRAL, ICHUSHI, and CNKI were searched in June 2009 for relevant studies of RCTs. Data on study design, participants, interventions, and outcomes were extracted and methodological quality of each included trial was assessed. Results From 3740 identified relevant articles, 10 (887 participants), 10 (1210 participants), and 8 (380 participants) RCTs were selected for meta-analysis of effects on DPD, BAP, and serum osteocalcin (OC), respectively, using Review Manager 5.0.22. Daily ingestion of an average 56 mg soy isoflavones (aglycone equivalents) for 10 weeks to 12 months significantly decreased DPD by 14.1% (95% CI: − 26.8% to − 1.5%; P = 0.03) compared to baseline (heterogeneity: P I 2 = 93%; random effects model). The overall effect of soy isoflavones on DPD compared with placebo was a significant decrease of − 18.0% (95% CI: − 28.4% to − 7.7%, P = 0.0007; heterogeneity: P = 0.0001; I 2 = 73%; random effects model). Subgroup analyses and meta-regressions revealed that isoflavone dose and intervention duration did not significantly relate to the variable effects on DPD. Daily supplementation of about 84 mg and 73 mg of soy isoflavones for up to 12 months insignificantly increased BAP by 8.0% (95% CI: − 4.2% to 20.2%, P = 0.20; heterogeneity: P I 2 = 98%) and OC by 10.3% (95% CI: − 3.1% to 23.7%, P = 0.13; heterogeneity: P = 0.002; I 2 = 69%) compared with placebo (random effects model), respectively. Conclusions Soy isoflavone supplements moderately decreased the bone resorption marker DPD, but did not affect bone formation markers BAP and OC in menopausal women. The effects varied between studies, and further studies are needed to address factors relating to the observed effects of soy isoflavones on DPD and to verify effects on other bone turnover markers.

Equol binds to the estrogen receptor with greater affinity than its precursor, daidzein, an isoflavone found in soybeans. Inter-individual differences in the ability to produce equol may lead to differential effects of isoflavone intervention on human health. Here, we review previously published work from our laboratory on equol producer status and bone health in humans and in a mouse model of osteoporosis. We performed a 1-y, double-blind, randomized trial to compare the effects of isoflavone (75 mg of isoflavone conjugates/d; equivalent to 47 mg/d of the aglycone form) with those of placebo on bone mineral density (BMD), fat mass, and serum isoflavone concentrations in 54 early postmenopausal Japanese women classified by their equol-producer phenotype. Isoflavone intervention increased the serum equol concentration in equol producers but not in nonproducers (P < 0.04). The annualized changes in BMD in the total hip and intertrochanteric regions in the isoflavone-treated equol producers (-0.46 and -0.04%, respectively) were less than in the nonproducers (-2.28 and -2.61%, respectively). The annualized change in fat mass was lower in the equol producers compared with the nonproducers in the isoflavone group. The annualized changes in BMD and fat mass did not differ between the equol producers and nonproducers in the placebo group. Equol also inhibited bone loss and fat accumulation in estrogen-deficient osteoporotic mice. Our data suggest that prevention of bone loss and fat accumulation in early postmenopausal women by isoflavones may depend on an individual's equol-producing capacity.

It has been reported that treatment with a pharmacological dose (45 mg/d) of menaquinone-4 (MK-4) prevents bone loss in postmenopausal women. However, it is not known whether supplementation with low dose MK-4 has beneficial effects on bone metabolism in healthy women. The aim of this study is to examine the effects of the supplementation of 1.5 mg/d MK-4 for 4 wk on bone and lipid metabolism in healthy postmenopausal Japanese women. The study was performed as a randomized double blind placebo-controlled trial. The participants aged 53-65 y were randomly assigned to 2 groups and supplemented with 1.5 mg/d of MK-4 or a placebo for 4 wk (n=20 for each group). The most marked effects of MK-4 intake were observed on serum osteocalcin (OC) concentrations. Serum undercarboxylated OC (ucOC) concentration decreased, and the gamma-carboxylated OC (GlaOC) and GlaOC/GlaOC+ucOC ratio that indicates the degree of OC gamma-carboxylation increased significantly at 2 and 4 wk compared with that at baseline in the MK-4 group. The serum ucOC and GlaOC concentrations in the MK-4 group were significantly different from those in the placebo group at 2 wk. These results suggest that supplementation with 1.5 mg/d MK-4 accelerated the degree of OC gamma-carboxylation. The concentrations of serum lipids and other indices were not different between the groups at either intervention period. Thus, the additional intake of MK-4 might be beneficial in the maintenance of bone health in postmenopausal Japanese women.

Kyoko Taku1, Keizo Umegaki1, Yoshiko Ishimi2, Shaw Watanabe31Information Center, National Institute of Health and Nutrition, Tokyo, Japan; 2Nutritional Epidemiology Program, National Institute of Health and Nutrition, Tokyo, Japan; 3Nutritional Education Program, National Institute of Health and Nutrition, Tokyo, JapanAbstract: When provided concurrently with soy protein for 1–3 months, soy isoflavones exert synergistic or additive cholesterol-lowering effects. This meta-analysis was performed to evaluate the effects of extracted soy isoflavones alone (not ingested concurrently with soy protein) on total and low density lipoprotein (LDL) cholesterol. MEDLINE (1966–2007), EMBASE (1966–2007), CENTRAL (1966–2007), ICHUSHI (1983–2008), and CNKI (1979–2007) were searched for randomized placebo-controlled trials published in English, Japanese, and Chinese, describing the changes in lipid profiles in adult humans resulting from ingestion of extracted soy isoflavones for 1–3 months. Reference lists of relevant systematic reviews and meta-analyses were hand-searched. Meta-analysis of 10 and 9 trials with usable information using REVMAN found that an average of 70 mg soy isoflavones/day (27–132 mg, as the aglycone form) alone had a nonsignificant effect on total (0.01 mmol/L [95% CI: –0.12, 0.14]; P = 0.86) and LDL (0.03 mmol/L [95% CI: –0.11, 0.16]; P = 0.71) cholesterol in menopausal women, respectively. It is concluded that ingestion of about 70 mg extracted soy isoflavones/day alone for 1–3 months does not improve total and LDL cholesterol levels in normocholesterolemic menopausal women; further studies are needed to verify the effects of extracted soy isoflavones.Keywords: extracted soy isoflavones, lipid, total cholesterol, LDL cholesterol

Objective: Equol is more biologically active than its precursor daidzein, which is the principal isoflavone found in soybean. There are interindividual differences in the ability to produce equol; these may lead to differences in the effects of isoflavone intervention on human health. This study aimed to investigate whether the effects of soy isoflavones on bone and fat mass are related to an individual's equol status. Design: We performed a 1-year double-blind, randomized trial to compare the effects of isoflavone (75 mg of isoflavone conjugates/day) with those of placebo on bone mineral density, fat mass, and serum isoflavone concentrations in early postmenopausal Japanese women who were classified based on their equol-producer phenotype. Results: After 1 year, the isoflavone intervention significantly increased the serum equol concentration in the equol producers but not in the nonproducers. In the isoflavone group, the annualized changes in the bone mineral density of the total hip and intertrochanteric regions were -0.46% and -0.04%, respectively, in the equol producers and -2.28% and -2.61%, respectively, in the nonproducers; these values were significantly different (P < 0.05 for both the regions). Significant differences were observed between the equol producers and nonproducers in the isoflavone group with regard to the annualized changes in the fat mass. No significant difference in the annualized changes in bone mineral density and fat mass was observed between the equol producers and nonproducers in the placebo group. Conclusions: Our data suggest that the preventive effects of isoflavones on bone loss and fat accumulation in early postmenopausal women depend on an individual's equol-producing capacity.

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.

Kiwifruit is a good source of dietary components and has beneficial effects for health. In this study, we investigated the effects of two types of kiwifruit, green kiwifruit (GRK) and gold kiwifruit (GOK), on bone metabolism in ovariectomized (OVX) mice. Seven-week-old female Balb/c-strain mice were divided into four groups: sham-operated (sham) group, OVX group, and OVX mice that were fed a GRK-supplemented diet or GOK-supplemented diet. Freeze-dried GRK and GOK were prepared and added in the diet at a concentration of 3 g/100 g. After 9 wk, the mice were sacrificed, and the serum, uterus, and femurs were obtained. Final body weight did not differ significantly among the four groups. Compared to the sham group, uterine weight was significantly lower and serum C-terminal telopeptide of type I collagen (CTx) levels and receptor activator of NF-κB ligand (RANKL) mRNA expression of the whole femur were significantly higher in the OVX group. Compared to the OVX group, GRK, but not GOK, reduced serum CTx concentrations and RANKL mRNA expression of the whole femur without changes in uterine weight. These results suggest that the GRK inhibited bone resorption, which might be due to a decrease in RANKL mRNA expression in OVX mice.

Prevention is the most important measure that can be taken to avoid osteoporosis, because bone mass, once it is lost, cannot be recovered. Both men and women reach their maximum bone mass when they are in their 20s and 30s; bone mass is maintained or may even increase slightly as people reach their 40s, and gradually decreases thereafter. In particular, bone mass dramatically decreases in postmenopausal women because of a decrease in serum estrogen level. Bone mass is influenced by genetic and environmental factors such as mechanical loading, nutrition, and lifestyle, along with gender, age, and metabolic factors. Among these factors, mechanical loading, i.e. exercise, and nutrition seem to be important in maintaining bone health, and lifestyle choices, including following a regular exercise program and consuming a diet rich in minerals and vitamins, when maintained throughout life, can be expected to preserve bone health. In contrast, excessive exercise with energy restriction induces osteoporosis in female athletes. This section will discuss the importance of appropriate exercise and diet on bone health in women, and some insights gained from studies using a rat model.

It is well known that skeletal unloading induces bone loss. It has been shown that soybean isoflavones are effective in preventing bone loss in osteoporotic animals. We investigated the ability of isoflavones to protect bone loss induced by hindlimb unloading by using a tail-suspension mouse model. Eight-week-old female mice were divided into five groups: (1) normal housed group (Normal), (2) sham unloaded group fed a control diet (Sham-UL), (3) hindlimb unloaded group fed a control diet (UL-C), (4) hindlimb unloaded group fed a 0.25% isoflavone conjugates diet (UL-ISO 0.25), and (5) hindlimb unloaded group fed a 0.5% isoflavone conjugates diet (UL-ISO 0.5). After 3 weeks, bone mineral density (BMD) of the femur was significantly decreased in UL-C, and this bone loss was prevented by isoflavone treatment. Histomorphometric analysis revealed a decrease in the cancellous bone of the distal femur in the UL-C group, and isoflavone prevented this change. Serum corticosterone increased in the UL-C group, and isoflavones inhibited the elevation. These results suggest that isoflavones might be promising food components that provide protection from bone loss and normalize stress-induced serum corticosterone during skeletal unloading.

The combined intervention of isoflavone intake and walking exercise over 1 year in postmenopausal Japanese women exhibited a trend for a greater effect on prevention of bone loss at the total hip and Ward's triangle regions. Introduction: The additive effects of isoflavones and exercise on bone and lipid metabolism have been shown in estrogen-deficient animals. In this study, we determined the effects of isoflavone intake, walking exercise, and their interaction on bone, fat mass, and lipid metabolism over 1 year in postmenopausal Japanese women. Materials and Methods: A total of 136 postmenopausal women at

本研究は母親ラットとその母親から産まれた仔ラットに対して, 大豆イソフラボンの主成分であるゲニステイン, ダイゼイン (0.50g/kg diet) を含む飼料の投与による生体影響の相違を比較検討し, それらの安全性を確認することを目的として実験を行った。母親ラットではゲニステイン, ダイゼイン投与による妊娠, 出産に対する影響を, 仔ラットでは成長への影響をそれぞれ観察した。さらに母乳のみを摂取している仔ラットの胃内容物および血清中のゲニステイン, ダイゼイン, エクオール濃度を測定した。1. 母親ラットへの飼料からのダイゼイン投与により母親ラットの飼料摂取量, 飼料効率が低下し, 体重増加が抑制された。さらに1匹の母親ラットが出産した仔ラット全体重量でもD群が低値を示した。ゲニステイン投与ではダイゼイン投与と同様の影響は観察されず, ゲニステインとダイゼインでは生体影響が異なることが示された。2. 母乳を介してダイゼインを摂取した仔ラットで体重増加が抑制された。仔ラットの臓器重量, AGD, 大腿骨乾燥重量では授乳期から成長期において一貫した影響は観察されなかった。3. 母親ラット, 仔ラットへのゲニステイン, ダイゼイン投与は甲状腺ホルモン濃度に明らかな影響を示さなかった。4. 母乳を介してダイゼインを摂取した仔ラットの胃内容物および血清中に, ダイゼインの代謝産物であるエクオールが多量に検出された。これにより, ダイゼインの作用の一部はエクオールの強い生理作用を含む可能性が示唆された。5. ダイゼイン投与により観察された体重増加抑制を除いて, 母親ラット, 仔ラットのどちらにおいても特に健康上の不都合は観察されなかった。

There is general agreement that soy isoflavones can be beneficial to health in adults. However, isoflavones are well known as endocrine-disrupting chemicals. The possibility that soy foods might adversely affect the reproductive system of mothers and infants should be considered. The aim of this study was to evaluate the adverse effects of a soy isoflavone mixture on rat dams and their offspring. The rat dams were fed diets containing the isoflavone mixture (commercial name: Soyact, aglycone type) at 0, 0.25, 0.5 and 1.0 g/kg as total isoflavones from pregnancy day 5 to postnatal day 13. We found that the dietary soy isoflavone mixture decreased the dam body weights in a dose-dependent manner and that the number of pups also tended to decrease. Genistein and daidzein were detected in the blood of the dams and the stomach contents of the suckling pups. The concentrations of daidzein were higher than those of genistein. We also found the transfer of isoflavones to the fetus. The reproductive output and fetus numbers were not significantly different in the isoflavone groups compared with the control group. The number of absorbed fetuses tended to increase though not significant. Our experiments suggested that soy isoflavones have the possibility of inducing adverse effects on endocrinic functions and others in animal studies at very high doses.