Skip to main content
NCBI home page
Food Science and Biotechnology logoLink to Food Science and Biotechnology
. 2024 Dec 17;34(6):1495–1505. doi: 10.1007/s10068-024-01768-w

Fermented soybean powder containing S-equol alleviates the postmenopausal signs of ovariectomized rats

Byung Hee Choi 1, Kwanyong Choi 2, Soo-Yeon Park 2, Ji Yeon Kim 2,✉
PMCID: PMC11914535  PMID: 40110397

Abstract

Menopausal symptoms, including hot flashes, metabolic changes, and osteoporosis, significantly impact postmenopausal women. Given concerns about hormone replacement therapy, S-equol, a metabolite of the soybean isoflavone daidzein, has emerged as a potential alternative. This study explored the efficacy of S-equol-containing-supplement (SE5-OH), a fermented soy germ containing S-equol, on menopausal symptoms and osteoporosis in 6-month-old and 8-week-old ovariectomized (OVX) rat models. SE5-OH did not affect uterine weight or endometrial thickness but significantly reduced body weight in 8-week-old OVX rats (p < 0.01). SE5-OH also decreased estrogen receptor upregulation in the liver and hypothalamus. In the hypothalamus, SE5-OH downregulated calcitonin gene-related peptide levels, which are linked to hot flashes and osteoporosis (p < 0.05). Additionally, SE5-OH stabilized the bone-formation marker RANKL/OPG, tended to increase estrogen receptor levels in tibial bone tissue and increased serum bone-turnover biomarkers. In conclusion, SE5-OH may alleviate postmenopausal symptoms, including hot flashes, lipid metabolism changes, and osteoporosis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-024-01768-w.

Keywords: S-equol, Menopause, Osteoporosis, Estrogen receptor, Hot flash

Introduction

Menopausal symptoms, including osteoporosis, hot flashes, and metabolic changes, significantly impact the quality of life of postmenopausal women. Osteoporosis is a metabolic disease in which bone integrity and strength are weakened due to qualitative changes in bone microstructure, thereby increasing the risk of fractures of the spine, femur, radius, etc. (Jilka et al., 1992). Additionally, various changes in lipid metabolism occur during the menopausal transition period. Moreover, hot flashes, characterized by sudden feelings of warmth, flushing of the face, and sweating, are among the most common and distressing symptoms of menopause (Santoro, 2008). Hormone replacement therapy has been shown to effectively alleviate the symptoms experienced by women during and after menopause. However, the use of estradiol therapy is associated with an increased risk of developing breast and ovarian cancers, outweighing the benefits of the treatment (Vinogradova et al., 2020). As a result, there is a growing need for alternative approaches or nutritional strategies to address estrogen deficiency during menopause. S-equol, a metabolite of the major soybean isoflavone daidzein produced by the gut microflora, has garnered attention because its health benefits are superior to those of its parent isoflavone. Numerous studies have demonstrated the efficacy of S-equol in alleviating postmenopausal symptoms such as hot flashes and inhibiting postmenopausal bone loss, as shown in both human and animal studies. Evidence from pilot studies, dose-finding studies, and animal research suggests that S-equol may prevent or alleviate postmenopausal symptoms (Aso et al., 2012; Ishiwata et al., 2009). Additionally, natural S-equol supplementation has been shown to contribute to bone health by preventing bone loss and reducing fat accumulation in early postmenopausal women. S-equol appears to ameliorate bone loss caused by estrogen deficiency by regulating hematopoiesis and the production of inflammatory cytokines in bone marrow cells, all of which are mechanisms supported by previous research on isoflavones (Nishide et al., 2013; Tousen et al., 2011). These findings underscore the potential role of S-equol in the management of menopausal symptoms and preservation of bone health. Therefore, a standardized natural S-equol-containing supplement, SE5-OH, which was developed by fermentation of a whole soy germ solution with a strain of the equol-producing lactic acid bacteria Lactococcus garvieae 20–92 isolated from the intestines of healthy equol producers, was developed and assessed for its efficacy and safety (Matulka et al., 2009; Yee et al., 2008). In Korea, a safety evaluation of the equol-producing lactic acid bacteria Lactococcus garvieae 20–92 was performed by the Ministry of Food and Drug Safety (MFDS), and it was temporarily approved as a food ingredient (Approval No. 2023-1). The ovariectomy (OVX) animal model is commonly used to explore the effects of estrogen deficiency and osteoporosis. Additionally, the administration of SE5-OH and S-equol has been shown to lower tail skin temperature and reduce urine deoxypyridinoline (DPD) and plasma total cholesterol levels (Yoneda et al., 2011). However, the direct mechanism by which SE5-OH influences estrogen receptor expression in menopause-related organs and the regulatory effects of estrogen receptors are unclear. In this study, two different rat models were used to address the abovementioned knowledge gaps; a 6-month-old OVX rat model was used to examine the effects of the test substances on symptoms induced by menopause, and an 8-week-old OVX rat model was used to evaluate estrogen receptor expression and biomarkers associated with bone metabolism.

Materials and methods

Materials

2,5-E2-estradiol, hydroxypropyl methylcellulose, and formalin solutions were purchased from Sigma‒Aldrich (St. Louis, MO, USA). SE5-OH natural S-equol supplement was produced from soy germ by fermentation with Lactococcus garvieae 20–92 by Otsuka Pharmaceutical Co., Ltd. (Japan). The supplement formulated in the dried powder contained 5.30 mg/g Equol in Study 1 (Lot No. S1E83-B) and 6.86 mg/g Equol in Study 2 (Lot No. S3K88-B).

Ovariectomized 6-month-old rat model for the evaluation of general menopausal symptoms

To confirm the menopausal health functionality of soy embryo fermentate (SE5-OH) in a menopause-induced animal model, we used an ovariectomized rat model. Twelve sham-operated and 36 ovariectomized six-month-old female Sprague–Dawley rats were provided by SLC, Inc. (Shizuoka, Japan), and housed in a specific pathogen-free animal facility with a 12 h/12 h light/dark cycle. The rats were acclimated for one week. Ovariectomized rats were randomly divided into 3 groups of 12 rats each. Vehicle (OVX; 1% hydroxypropyl methylcellulose in PBS), SE5-OH (SE5; 1000 mg/kg B.W. in vehicle) and E2-estradiol (EST; 0.5 mg/kg B.W. in vehicle) were administered by oral gavage to the rats in the indicated groups once daily for 8 weeks. Detailed information about the administration of the test substances is provided in Fig. S1. All the experimental processes were approved by the NDIC Inc. Center’s Committee (IACUC: P221052).

The animals were weighed regularly and sacrificed on week 8 after the first day of treatment. Uterine weight was measured, and then, the uterus was divided in half: the left portion was stored in a deep freezer at temperatures below − 70 °C, and the right portion was fixed in 10% neutral formalin for hematoxylin and eosin (H&E) tissue staining. Histopathological variation in the uterus was assessed by measuring endometrial thickness. The liver and tibia were excised, separated, and stored in a deep freezer at temperatures below -70 °C.

Ovariectomized 8-week-old rat model for evaluating osteometabolism

To evaluate biomarkers in detail in a menopause-induced animal model, we used a younger OVX rats, which are more sensitive to the expression of hormone receptors and bone metabolism. In total, 48 seven-week-old female Sprague–Dawley rats were provided by Orient Bio, Inc. (Gyeonggi, Korea), and housed in a specific pathogen-free animal facility with a 12 h/12 h light/dark cycle. After an adaptation period of one week, the rats were allocated to a total of 4 groups. The rats were either sham-operated (CON; 1 group) or ovariectomized (OVX; 3 groups). Ovariectomized rats were randomly allocated to 3 groups of 12 rats each. After 3 days of recovery, vehicle (1% hydroxypropyl methylcellulose in PBS) or SE5-OH (2000 mg/kg B.W. in vehicle) was administered orally by oral gavage once daily for a total of 6 weeks to the rats in each group. E2-estradiol (0.03 mg/kg B.W. in 3:97 ethyl alcohol:olive oil) was administered by subcutaneous injection (Figure S2). All the experimental processes were approved by the CORESTEMCHEMON Inc. Center’s Committee (IACUC: 2024-0125). The animals were weighed regularly and sacrificed on week 6 after the first day of treatment, and whole blood was collected via the abdominal vena cava. The hypothalamus and tibia were excised and stored in a deep freezer at temperatures below − 70 °C.

Serum biochemical markers of bone metabolism

For the analysis of serum biochemical markers of bone metabolism, tartrate-resistant acid phosphatase 5b (TRACP-5b) and osteocalcin (OC) enzyme-linked immunosorbent assay (ELISA) kits were purchased from MyBioSource (Vancouver, Canada). The concentrations of receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and N-terminal telopeptide-1 (Ntx-1) were determined via enzyme immunoassay (EIA) kits from Lsbio (Washington, USA). Alkaline phosphatase (ALP) activity was measured with colorimetric enzyme assay kits from Asanphram Inc. (Gyeonggi, Korea). Each biomarker was analyzed according to the protocols provided with the assay or ELISA kits.

Liver biomarker analysis

Lipids were extracted from liver tissue by means of the Folch method. First, 10 mg of homogenized liver was mixed with 20 ml of methanol:chloroform (1:2 ratio) and vortexed vigorously. The homogenate was centrifuged at 3000×g for 10 min, and the homogenate was collected to obtain a crude extract solution. The supernatant was mixed thoroughly to achieve a 20% volume of water, and the mixture was separated into two phases via centrifugation. After the upper layer was aspirated, the lower organic layer was transferred and condensed with a nitrogen gas evaporator. The condensed lipids were dissolved in 5% Triton X-100 (Sigma‒Aldrich) in phosphate-buffered saline. Triglycerides (TG) and total cholesterol (TC) levels were measured via colorimetric enzyme assay kits (Asanphram Inc.).

mRNA extraction and quantitative real-time PCR analysis

Total mRNA was extracted from liver, tibia and hypothalamus tissues with TRIzol reagent (Life Technologies) according to the product protocol. The extracted mRNA was diluted with nuclease-free water (400–800 ng/mL). cDNA was immediately synthesized via a high-capacity RNA-to-cDNA synthesis kit. Quantitative real-time PCR was performed with a StepOnePlus™ Real-Time PCR System (Hoffmann-La Roche Ltd., Basel, Switzerland). The comparative 2.−ΔΔCT method was used to quantify relative mRNA expression, with GAPDH expression used as the internal control. To evaluate the mRNA expression of biomarkers related to menopausal symptoms in animal studies, primer pairs for the indicated rat mRNAs were generated and are presented in Table S1

Statistical analysis

All the data are presented as the mean and standard error of the mean (SE). Statistical analyses were performed via one-way analysis of variance (ANOVA) with normality tests and Duncan’s multiple range tests as high-power post hoc tests to measure differences identified via ANOVA. The general linear model approach of the Statistical Package for the Social Sciences (SPSS 20, SPSS Inc., Chicago, IL) was used, and p < 0.05 indicated a statistically significant difference.

Results

Study 1. Evaluation of menopausal symptoms in 6-month-old ovariectomized rats

Body weight and food intake of 6-month-old OVX rats

After induction of menopause by ovariectomy, body weight was measured regularly during the treatment period, and the results revealed a significant increase (p < 0.01) in body weight in the OVX group compared with the CON group. However, no significant changes in body weight were observed between the SE5 group and the EST group (CON, 342.05 ± 5.33 g; OVX, 376.70 ± 7.49 g; SE5, 385.90 ± 9.63 g; and EST, 382.33 ± 10.59 g in week 8), as shown in Fig. 1A. Additionally, no significant differences in food consumption were observed between any of the test groups (Fig. 1B).

Fig. 1.

Fig. 1

Open in a new tab

The effects of SE5-OH on body weight changes (A) and food consumption (B) in 6-month-old sham- or OVX-operated rats and body weight changes (C) and food consumption (D) in 8-week-old sham- or OVX-operated rats. The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

Influence of uterine weight and endometrial thickness

Next, the histology of the uterus was assessed by measuring tissue weights and endometrial thickness via H&E staining (Fig. 2A). The analysis revealed a significant reduction in ovarian tissue weight in the OVX group compared with the CON group but no significant differences were between the experimental groups receiving the test substances (CON, 0.74 ± 0.04 g; OVX, 0.19 ± 0.01 g; SE5, 0.20 ± 0.01 g; and EST, 0.25 ± 0.01 g; p < 0.05; Fig. 2B). Furthermore, endometrial thickness was lower in the OVX group than in the CON group but the differences among the ovariectomized groups were not significant (CON, 23.86 ± 0.85 mm; OVX, 18.52 ± 0.59 mm; SE5, 17.43 ± 0.70 mm; and EST, 18.38 ± 0.57 mm). p < 0.05, Fig. 2C).

Fig. 2.

Fig. 2

Open in a new tab

The effects of SE5-OH on H&E staining of uterine tissue sections (A) and the effects of SE5 and EST on uterine weight (B) and endometrial thickness (C) in 6-month-old rats. The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

Hepatic lipid concentrations and mRNA expression in 6-month-old OVX rats

To evaluate the degree of hepatic steatosis caused by menopausal symptoms, the concentrations of TG and TC in the lipid fraction of liver tissue were analyzed. The TG levels in liver tissue were significantly higher in the OVX group than in the normal control group (CON, 13.74 ± 2.72 mg/g; OVX, 39.13 ± 6.11 mg/g; SE5, 11.65 ± 0.79 mg/g; and EST, 34.80 ± 5.56 mg/g per wt tissue, Fig. 3A). Post hoc tests revealed that the TG level was significantly lower in the rats in the SE5-OH group than in those in the OVX group (p < 0.05). There were no significant differences in TC levels among the groups (CON, 1.61 ± 0.11 mg/g; OVX, 1.73 ± 0.10 mg/g; SE5, 1.38 ± 0.06 mg/g; and EST, 1.65 ± 0.14 mg/g per tissue wt, Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

The effects of SE5-OH on hepatic total triglyceride levels (A) and total cholesterol levels (B). The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

To explore the underlying mechanism by which SE5-OH downregulated liver fat compared with that in the placebo group, relative mRNA expression in liver tissue was analyzed. Both estrogen receptor α (Estr1) (OVX, 3.44-fold; SE5, 1.13-fold; and EST, 0.92-fold vs. CON) and estrogen receptor β (Estr2) (OVX, 3.71-fold; SE5, 0.51-fold; and EST, 0.44-fold vs. CON) were upregulated in the OVX group compared with the CON group, and their levels were significantly reduced in the SE5 group compared with the OVX group (p < 0.05, Fig. 4A). With respect to lipid metabolism, the levels of peroxisome proliferator-activated receptor alpha (PPARa) (OVX, 3.18-fold; SE5, 2.16-fold; and EST, 12.35-fold vs. CON) and hormone-sensitive lipase (HSL) (OVX, 2.38-fold; SE5, 1.88-fold; and EST, 9.67-fold vs. CON) did not significantly differ between the OVX and CON groups but were significantly higher in the EST group than in the CON group (p < 0.05, Fig. 4B). Fatty acid synthase (FAS), a lipid synthesis enzyme, was significantly increased in the OVX group compared with the CON group (OVX, 1.43-fold; SE5, 1.23-fold; and EST, 1.47-fold vs. CON, p < 0.05; Fig. 4C). However, compared with the OVX group, the SE5 group presented slight downregulation of FAS expression, but the difference was not significant according to the post hoc test.

Fig. 4.

Fig. 4

Open in a new tab

Effects of SE5-OH on mRNA expression in the liver tissue of 6-month-old rats (C–E). mRNA expression in tibia bone tissue (A and B) and the RANKL/OPG ratio (C) of 6-month-old rats. Estr1, estrogen receptor α; Estr2, estrogen receptor β (A and D); PPARα, peroxisome proliferator-activated receptor alpha; HSL, hormone-sensitive lipase; (B), FAS, fatty acid synthase (C); RANKL, receptor activator of nuclear factor kappa-Β ligand; OPG, osteoprotegerin (E). The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

mRNA expression in the tibia bone tissue of 6-month-old OVX rats

To evaluate the degree of bone loss caused by menopausal symptoms, mRNA expression in the tibia bone tissue was analyzed. There were no significant differences in the expression of Estr1 and Estr2 between the groups (OVX, 1.45- and 1.64-fold; SE5, 1.77- and 1.29-fold; and EST, 1.70- and 2.03-fold vs. CON, respectively; Fig. 4D). However, the level of receptor activator of nuclear factor kappa-Β ligand (RANKL), which is associated with bone resorption, was significantly higher in the OVX group than in the normal control group (OVX, 1.82-fold; SE5, 1.18-fold; and EST, 1.83-fold vs. CON). Osteoprotegerin (OPG), which supports bone formation, was significantly decreased in the OVX group (OVX, 0.53-fold; SE5, 1.10-fold; and EST, 1.15-fold vs. CON). Both the RANKL and OPG levels in the SE5 group were significantly higher than those in the CON group (p < 0.05), as shown in Fig. 4E. In addition, the RANKL/OPG ratio, a determinant of bone mass and skeletal integrity, was significantly higher in the SE5-OH and EST groups than in the OVX group (OVX, 510.37%; SE5, 67.09%; and EST, 82.32%. p < 0.05), as shown in Fig. 4F.

Study 2. Evaluation of menopausal symptoms in 8-week-old ovariectomized rats

Body weight and food intake of 8-week-old OVX rats

In Study 2, the SHAM animals gained weight, whereas the SE5 and EST groups gained significantly less weight than the OVX groups did, as shown in Fig. 1C (CON, 277.73 ± 3.86 g; OVX, 333.24 ± 6.08 g; SE5, 317.68 ± 5.17 g; and EST, 253.07 ± 4.31 g in week 6; p < 0.05). No significant differences in food consumption were observed between any of the test groups (Fig. 1D).

mRNA expression in the hypothalamus of 8-week-old OVX rats

The OVX model of 8-week-old rats, in which hormonal sensitivity is increased relative to the OVX model with older rats, was used in the second animal experiment to perform an in-depth analysis of estrogen receptor expression and osteosis metabolism related to menopausal changes. Estr1 and Estr2 gene expression in the hypothalamus was significantly higher in the OVX group than in the CON group (OVX, 2.24- and 2.99-fold; SE5, 1.40- and 1.50-fold; and EST, 1.11- and 1.07-fold vs. CON, respectively), as shown in Fig. 5A. Compared with that in the OVX group, the expression of Estr1 and Estr2 in the SE5 and EST groups was significantly lower (p < 0.05, Fig. 5A). Although there were no significant differences in the expression of the serotonin transporter (SERT) (OVX, 1.28-fold; SE5, 1.18-fold; and EST, 1.14-fold vs. CON), as shown in Fig. 5B, the expression of calcitonin gene-related peptide was significantly downregulated in the SE5 and EST groups compared with the OVX group (CGRP) (OVX, 1.38-fold; SE5, 0.76-fold; and EST, 0.67-fold vs. CON. p < 0.05; Fig. 5C).

Fig. 5.

Fig. 5

Open in a new tab

The effects of SE5-OH on mRNA expression in the hypothalamus (A–C) and tibial bone tissue (D) of 7-week-old rats. Estr1, estrogen receptor α; Estr2, estrogen receptor β (A and D); SERT, serotonin transporter (B); CGRP, calcitonin gene-related peptide (C); The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

mRNA expression in the tibia bone tissue and serum biochemical markers of 8-week-old OVX rats

In the 8-week-old rat model, there were significant differences in the expression of Estr1 and Estr2 among the groups. In particular, Estr1 expression was higher in the OVX group than in the CON group but not significantly different from that in the OVX group (OVX, 1.24-fold; SE5, 1.65-fold; and EST, 0.76-fold vs. CON). Additionally, Estr2 expression was significantly higher in the EST group than in the OVX group; the SE5 group had intermediate values for Estr2, but they were not significantly different from those of the OVX group (OVX, 0.59-fold; SE5, 2.33-fold; and EST, 3.13-fold vs. CON; p < 0.05; Fig. 5D). Furthermore, the values for serum ALP were significantly higher after OVX, but this rise was prevented by treatment with SE5-OH or E2-estradiol (CON, 8.97 ± 0.89; OVX, 11.97 ± 0.95 K.A.U.; SE5, 7.97 ± 0.93 K.A.U.; and EST, 4.78 ± 0.37 K.A.U.; p < 0.001), as shown in Fig. 6A. There was a trend toward an increase in serum osteocalcin (CON, 0.55 ± 0.12 ng/mL; OVX, 0.89 ± 0.13 ng/mL; SE5, 5.89 ± 0.10 ng/mL; and EST, 0.36 ± 0.12 ng/mL) and TRACP-5b (CON, 8.34 ± 2.84 ng/mL; OVX, 10.56 ± 2.17 ng/mL; SE5, 9.14 ± 3.08 ng/mL; and EST, 7.56 ± 2.08 ng/mL) in the OVX group compared with the CON group, and the concentrations of OC and TRACP-5b in the EST group were significantly; however, the SE5 group had intermediate values, but they were not significant (p < 0.05, Fig. 6B and C). Additionally, serum Ntx-1 expression (CON, 5.01 ± 0.76 ng/mL; OVX, 5.79 ± 0.34 ng/mL; SE5, 2.48 ± 0.37 ng/mL; and EST, 1.23 ± 0.50 ng/mL) was significantly lower in the SE5 and EST groups than in the OVX group (p < 0.001, Fig. 6D). The serum RANKL level was significantly lower in the SE5 and EST groups than in the OVX group (CON, 5.01 ± 0.76 ng/mL; OVX, 5.79 ± 0.34 ng/mL; SE5, 2.48 ± 0.37 ng/mL; and EST, 1.23 ± 0.50 ng/mL) p < 0.001, Fig. 6E). The concentration of OPG was significantly higher in the SE5 and EST groups than in the OVX group (CON, 128.89 ± 19.76 ng/mL; OVX, 111.36 ± 10.99 ng/mL; SE5, 166.87 ± 10.53 ng/mL; and EST, 169.03 ± 17.53 ng/mL; p < 0.05, Fig. 6F). The ratio of RANKL to OPG was lower in the SE5 and EST groups than in the OVX group (CON, 38.82%; OVX, 44.42%; SE5, 122.94%; EST. 14.65%. p < 0.001, Fig. 6G).

Fig. 6.

Fig. 6

Open in a new tab

Effects of SE5-OH on serum bone marker levels (A–F) and the serum RANKL/OPG ratio (G) in 8-week-old rats. ALP alkaline phosphatase (A); OC osteocalcin (B); TRACP-5b tartrate-resistant acid phosphatase 5b (C); Ntx-1 N-terminal telopeptide-1 (D); RANKL receptor activator of nuclear factor kappa-Β ligand (E); OPG osteoprotegerin (F). The letters above the bars indicate significant differences (Duncan’s multiple range test; p < 0.05). The order of values from small to large is alphabetical. CON sham-operated, OVX ovariectomized with vehicle, SE5 ovariectomized with SE5-OH treatment, EST ovariectomized with E2-estradiol treatment

Discussion

The effects of S-equol, a metabolite of the soybean isoflavone daidzein, are promising in the promotion of bone health and alleviation of menopausal symptoms, particularly in individuals who can produce it efficiently (Schmitt et al., 2001). Notably, a previous clinical trial demonstrated that S-equol supplementation significantly improved menopausal symptoms, including mood-related symptoms such as tension, anxiety, and depression, especially in equol nonproducers (Ishiwata et al., 2009). The unique ability of equol to selectively bind to estrogen receptor (ER) β endows it with distinct estrogenic properties, which are believed to be crucial for its role in bone metabolism and overall menopausal symptom management. In several reports, the health benefits of S-equol, particularly in relieving menopausal symptoms, have been highlighted, and its role as an receptor agonist is known. S-equol is a nutritional supplement that has demonstrated efficacy in this regard (Jackson et al., 2011; Setchell et al., 2005). Recently, many researchers have hypothesized that the estrogen receptor actions are tissue- or ligand specific. Different estrogen receptors are localized to specific areas within distinct tissues, and both endogenous and exogenous estrogens interact in a complex manner with Estr1 (ERα) and/or Estr2 (ERβ) to stimulate the expression of various target genes (Diel, 2002). This higher receptor affinity allows S-equol to effectively mimic the beneficial effects of estrogen, particularly in tissues such as bone, where estrogen plays a critical role in maintaining bone density (Morito et al., 2001). This makes S-equol a highly attractive natural alternative to hormone replacement therapy, especially for postmenopausal women at risk of osteoporosis. Given the important role of the estrogen receptor in menopausal symptoms, this study was designed to address the hypothesis that S-equol-rich supplementation and SE5-OH may attenuate OVX-induced osteoporosis via the regulation of estrogen receptor expression. The OVX model has been widely used in the study of osteoporosis caused by menopause in animals (Frost and Jee, 1992). Therefore, the results were measured for body weight changes, uterine weight and thickness, and metabolism-related improvements in the natural S-equol-rich supplement SE5-OH. The studies investigating the effects were designed with two different models: a 6-month-old OVX rat model to examine the effects of the test substances on symptoms induced by menopause and an 8-week-old OVX rat model to further identify estrogen receptor expression and biomarkers associated with bone metabolism. Compared with the older rat model, the younger rat model is more sensitive to changes in estrogen receptor levels, making it more suitable for detecting osteoporosis induced by hormonal changes (Gordon et al., 2009).

OVX typically leads to a noticeable increase in body weight in rats. This weight gain is primarily due to the reduction in estrogen levels. Estrogen has a role in regulating energy balance and metabolism, so its absence disrupts this balance, leading to increased food intake and reduced energy expenditure (Wronski et al., 1987). In another study, dietary soy phytoestrogen metabolites, particularly equol lowered food intake in female rats, which can explain why isoflavones might be bound by the estrogen receptor regardless of the serum estrogen conditions and might lower food intake depending on the stringency of estrogen sensitivity (Kishida et al., 2008). In this study, regulation of body weight was higher in 8-week-old rats than in 6-month-old rats. While mature OVX rats are more suitable for studying overall menopausal symptoms, such as histological changes in the uterus and metabolic alterations due to their stable, fully developed stage, metabolic activity is generally higher in younger rats, as reported by (Clark and Tarttelin, 1982), and greater sensitivity to hormonal changes, including those involving estrogen, is higher (Gordon et al., 2009). This heightened sensitivity in younger rats likely contributed to the more pronounced effects on body weight regulation observed in this study. Since 8-week-old rats are at a developmental stage where metabolism and hormone regulation are still highly responsive, the impact of phytoestrogen metabolites, such as equol, on body weight is likely to be more significant. In contrast, older rats may have a more established metabolic baseline and potentially reduced responsiveness to dietary interventions owing to age-related changes in their hormonal and metabolic systems. This difference in sensitivity likely accounts for the more noticeable body weight regulation in the younger rat model. However, the administration of SE5-OH did not affect uterine weight or endometrial thickness in OVX rats, indicating that SE5-OH had no estrogenic effect on this organ. These findings suggest that SE5-OH is safe as a nonhormonal alternative for the management of menopausal symptoms. (Kondo et al., 2020; Tanaka et al., 2021).

To identify the different estrogen receptors localized in various tissues and their roles in stimulating the expression of specific target genes, we analyzed the transcriptomic expression in the liver and tibia of the 6-month-old rat model. Additionally, to investigate hormone-sensitive mechanisms related to osteoporosis, particularly those involving neural pathways and bone metabolism, we analyzed the mRNA expression in the hypothalamus and tibia of the 8-week-old rat model.

In the liver, the expression of estrogen receptors plays a crucial role in regulating lipid metabolism. Previous studies have shown that OVX leads to the upregulation of estrogen receptors 1 and 2 in liver tissue, a compensatory effect caused by the sudden loss of estrogen (Hao et al., 2010). This upregulation can significantly alter hepatic lipid metabolism, often resulting in increased lipid accumulation and changes in fatty acid synthesis. In this study, the administration of SE5-OH significantly mitigated the OVX-induced upregulation of estrogen receptor expression in the liver. Additionally, the sample group exhibited a notable reduction in the expression of FAS genes, which are key regulators of lipogenesis, along with a decrease in hepatic triglyceride levels, a critical biomarker for liver lipid accumulation (Chukijrungroat et al., 2017). These findings suggest that SE5-OH effectively modulates hepatic lipid metabolism and may help prevent the adverse lipid changes associated with estrogen deficiency following OVX.

In bone tissue, especially in the 8-week-old rat model, which is more sensitive to changes in estrogen receptors, SE5-OH treatment tended to upregulate the gene expression of Estr1 and Estr2, which can regulate bone metabolism (Khalid and Krum, 2016). There was no significant change in Estr1 and/or Estr2 expression in the 6-month-old OVX rat model, but there was a significant change in these genes only in the tibia bone tissue of the 8-week-old OVX rats. The estrogen receptors antagonize each other in bone, especially by the estrogen-mediated suppression of osteoclasts, which involves the regulation of the RANKL/OPG ratio (Bord et al., 2003). An increase in RANKL expression by osteoclasts reduces bone calcium levels, whereas an increase in OPG expression by osteoblasts promotes bone formation (Li et al., 2015). In terms of the ratio of RANKL to OPG, which is closely related to bone resorption and formation, a decrease in RANKL and an increase in OPG were observed in the SE5-OH treatment group compared with the OVX group. The significant improvement in the RANKL:OPG ratio in the treatment groups suggests that SE5-OH may reduce the risk of osteoporosis associated with menopausal symptoms. Furthermore, the results of the serum bone marker analysis in the younger rat model in this study provide insights into the potential mechanisms by which SE5-OH may mitigate osteoporosis associated with menopause. Similarly, in previous clinical trials involving SE5-OH supplementation, the levels of bone turnover markers tended to increase in the urine and blood of menopausal women. These findings, combined with the results from both clinical and animal studies, suggest that SE5-OH may have a positive effect on metabolic processes related to osteoporosis during menopause, effectively alleviating the associated risks. OC, ALP, and TRACP-5b are well-established markers of osteoclasts, whereas NTx-1 is a marker of osteoblasts (Coleman et al., 2008). Blood analysis revealed a trend toward a reduction in OC and TRACP-5b levels in the SE5-OH treatment group compared with the OVX group, indicating a potential decrease in bone formation and resorption activities due to estrogenic effects (Tantikanlayaporn et al., 2013). More notably, the levels of ALP and NTx-1 were significantly reduced in the SE5-OH treatment group, suggesting a marked improvement in bone turnover markers associated with menopause. Regulation of the elevated levels of ALP, an enzyme linked to bone mineralization, indicates the potential normalization of bone formation rates (Grigoryan et al., 2017). Similarly, elevated NTX-1 levels are often indicative of increased bone turnover, which is a significant factor in conditions such as osteoporosis, and the decrease in NTx-1 in response to SE5-OH treatment points to a reduction in bone resorption, which is crucial in managing osteoporosis.

Ovariectomy strongly decreases estrogen secretion but slightly increases estrogen sensitivity or estrogen receptor expression, especially in the hypothalamus. The hypothalamus plays a central role in the regulation of the reproductive system through its interaction with the pituitary gland. During menopause, a decrease in estrogen disrupts the normal feedback loop between the ovaries, hypothalamus, and pituitary gland (Rance, 2009). This disruption can lead to several common menopausal symptoms, such as hot flashes, sleep disturbances and weight gain (Weiss et al., 2004). The results of this study suggest that SE5-OH has a significant modulatory effect on the hypothalamic changes induced by OVX, which has been shown to increase the expression of Estr1 and Estr2 in the hypothalamus as a compensatory response to the sharp decline in circulating estrogen levels (Liu and Shi, 2015). Specifically, increased expression of CGRP, a neuropeptide linked to thermoregulation, has been implicated in the development of hot flashes during menopause (Noguchi et al., 2002). Although the mRNA expression of SERT in the hypothalamus was slightly, although nonsignificantly, downregulated by SE5-OH treatment, the expression of CGRP was significantly modulated in this study. SE5-OH supplementation alleviated hot flashes in another previous clinical trial (Aso et al., 2012). Additionally, S-equol and the fermented soy product SE5-OH containing S-equol similarly decreased the OVS-induced increase in rat tail skin temperature in an animal model of hot flashes in a previous study, enhancing the evidence related to the modulation of menopausal hot flashes by SE5-OH supplementation (Yoneda et al., 2011). Interestingly, CGRP and serotonin, both of which are associated with hot flashes, also play a role in bone metabolism. Specifically, increased brain-derived serotonin has been found to inhibit CGRP release in the femur, suggesting a complex interaction between these neuropeptides in the regulation of bone health. An increase in serotonin levels may negatively impact bone metabolism by suppressing CGRP, which is otherwise involved in promoting bone formation (Zhang et al., 2021). While this study highlights the potential of S-equol and soybean isoflavones in mitigating bone loss and alleviating menopausal symptoms, several limitations must be acknowledged. One significant limitation is the lack of direct measurements of bone density, which would provide a more comprehensive understanding of the impact of equol on bone metabolism. Additionally, the signaling pathways through which the biological effects of equol occur remain largely unexplored. Proteomic analysis could provide valuable insights into the molecular mechanisms underlying the estrogenic properties of equol and its role in bone metabolism. By identifying specific signaling pathways and proteins involved, we can better understand how equol influences bone turnover and other estrogen-dependent processes. The incorporation of such advanced methods into future research could help validate the therapeutic potential of equol and soybean isoflavones and might contribute to the development of more targeted interventions for menopausal women at risk of osteoporosis and other estrogen deficiency-related conditions.

In this study, SE5-OH not only mitigated the OVX-induced upregulation of estrogen receptors in the hypothalamus but also potentially influenced menopausal symptoms such as weight gain, lipid accumulation, and neuropeptide-mediated pathways that affect bone metabolism. By attenuating the compensatory increase in hypothalamic estrogen receptor levels, SE5-OH may help reduce menopausal symptoms. The observed improvements in bone markers suggest that SE5-OH may offer a comprehensive approach to managing menopausal symptoms, including those affecting both neurovascular function and bone metabolism. These findings highlight the potential of SE5-OH as a multifaceted therapeutic agent for alleviating menopausal symptoms by addressing both the central nervous system and the skeletal aspects of menopause, ultimately improving the quality of life of women during this transitional period.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 74 KB) (73.8KB, docx)

Acknowledgements

This work was supported by Korea Otsuka Pharmaceutical Co., Ltd.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Aso T, Uchiyama S, Matsumura Y, Taguchi M, Nozaki M, Takamatsu K, Ishizuka B, Kubota T, Mizunuma H, Ohta H. A natural S-equol supplement alleviates hot flushes and other menopausal symptoms in equol nonproducing postmenopausal Japanese women. Journal of women's health 21: 92-100 (2012) [DOI] [PubMed] [Google Scholar]
  2. Bord S, Ireland D, Beavan S, Compston J. The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts. Bone 32: 136-141 (2003) [DOI] [PubMed] [Google Scholar]
  3. Chukijrungroat N, Khamphaya T, Weerachayaphorn J, Songserm T, Saengsirisuwan V. Hepatic FGF21 mediates sex differences in high-fat high-fructose diet-induced fatty liver. American Journal of Physiology-Endocrinology and Metabolism 313: E203-E212 (2017) [DOI] [PubMed] [Google Scholar]
  4. Clark RG, Tarttelin MF. Some effects of ovariectomy and estrogen replacement on body composition in the rat. Physiology & Behavior 28: 963-969 (1982) [DOI] [PubMed] [Google Scholar]
  5. Coleman R, Brown J, Terpos E, Lipton A, Smith MR, Cook R, Major P. Bone markers and their prognostic value in metastatic bone disease: clinical evidence and future directions. Cancer treatment reviews 34: 629-639 (2008) [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Diel P. Tissue-specific estrogenic response and molecular mechanisms. Toxicology letters 127: 217-224 (2002) [DOI] [PubMed] [Google Scholar]
  7. Frost HM, Jee WS. On the rat model of human osteopenias and osteoporoses. Bone and mineral 18: 227-236 (1992) [DOI] [PubMed] [Google Scholar]
  8. Gordon A, Aguilar R, Garrido-Gracia JC, Guil-Luna S, Sanchez-Cespedes R, Millan Y, Martin de las Mulas J, Sanchez-Criado JE. Activation of estrogen receptor-α induces gonadotroph progesterone receptor expression and action differently in young and middle-aged ovariectomized rats. Human Reproduction 24: 2618-2628 (2009) [DOI] [PubMed] [Google Scholar]
  9. Grigoryan AV, Dimitrova AA, Kostov KG, Russeva AL, Atanasova MA, Blagev AB, Betova TM, Trifonov RG. Changes of serum concentrations of alkaline phosphatase and metalloproteinase-9 in an ovariectomized wistar rat model of osteoporosis. Journal of Biomedical and Clinical Research 10: 32-36 (2017) [Google Scholar]
  10. Hao L, Wang Y, Duan Y, Bu S. Effects of treadmill exercise training on liver fat accumulation and estrogen receptor alpha expression in intact and ovariectomized rats with or without estrogen replacement treatment. European Journal of Applied Physiology 109: 879-886 (2010) [DOI] [PubMed] [Google Scholar]
  11. Ishiwata N, Melby MK, Mizuno S, Watanabe S. New equol supplement for relieving menopausal symptoms: randomized, placebo-controlled trial of Japanese women. Menopause 16: 141-8 (2009) [DOI] [PubMed] [Google Scholar]
  12. Jackson RL, Greiwe JS, Schwen RJ. Emerging evidence of the health benefits of S-equol, an estrogen receptor β agonist. Nutrition reviews 69: 432-448 (2011) [DOI] [PubMed] [Google Scholar]
  13. Jilka RL, Hangoc G, Girasole G, Passeri G, Williams DC, Abrams JS, Boyce B, Broxmeyer H, Manolagas SC. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 257: 88-91 (1992) [DOI] [PubMed] [Google Scholar]
  14. Khalid AB, Krum SA. Estrogen receptors alpha and beta in bone. Bone 87: 130-135 (2016) [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kishida T, Mizushige T, Ohtsu Y, Ishikawa S, Nagamoto M, Izumi T, Obata A, Ebihara K. Dietary soy isoflavone-aglycone lowers food intake in female rats with and without ovariectomy. Obesity (Silver Spring) 16: 290-7 (2008) [DOI] [PubMed] [Google Scholar]
  16. Kondo T, Ishimi Y, Takebayashi J, Tousen Y. Assessment of safety and efficacy of pine bark extract in normal and ovariectomized mice. Journal of food science 85: 1956-1962 (2020) [DOI] [PubMed] [Google Scholar]
  17. Li C, Liang B, Shi X, Wang H. Opg/Rankl mRNA dynamic expression in the bone tissue of ovariectomized rats with osteoporosis. Genet Mol Res 14: 9215-24 (2015) [DOI] [PubMed] [Google Scholar]
  18. Liu X, Shi H. Regulation of estrogen receptor α expression in the hypothalamus by sex steroids: implication in the regulation of energy homeostasis. International journal of endocrinology 2015: 949085 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matulka RA, Matsuura I, Uesugi T, Ueno T, Burdock G. Developmental and reproductive effects of SE5‐OH: an equol‐rich soy‐based ingredient. Journal of Toxicology 2009: 307618 (2009) [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morito K, Hirose T, Kinjo J, HIRAKAWA T, OKAWA M, NOHARA T, OGAWA S, INOUE S, MURAMATSU M, MASAMUNE Y. Interaction of phytoestrogens with estrogen receptors α and β. Biological and Pharmaceutical Bulletin 24: 351-356 (2001) [DOI] [PubMed] [Google Scholar]
  21. Nishide Y, Tadaishi M, Kobori M, Tousen Y, Kato M, Inada M, Miyaura C, Ishimi Y. Possible role of S-equol on bone loss via amelioration of inflammatory indices in ovariectomized mice. J Clin Biochem Nutr 53: 41-8 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noguchi M, Ikarashi Y, Yuzurihara M, Mizoguchi K, Kurauchi K, Chen J, Ishige A. Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats. Journal of endocrinology 175: 177-184 (2002) [DOI] [PubMed] [Google Scholar]
  23. Rance NE. Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback. Peptides 30: 111-122 (2009) [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Santoro N. Symptoms of menopause: hot flushes. Clinical obstetrics and gynecology 51: 539-548 (2008) [DOI] [PubMed] [Google Scholar]
  25. Schmitt E, Dekant W, Stopper H. Assaying the estrogenicity of phytoestrogens in cells of different estrogen sensitive tissues. Toxicology in vitro 15: 433-439 (2001) [DOI] [PubMed] [Google Scholar]
  26. Setchell KD, Clerici C, Lephart ED, Cole SJ, Heenan C, Castellani D, Wolfe BE, Nechemias-Zimmer L, Brown NM, Lund TD. S-Equol, a potent ligand for estrogen receptor β, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora1–4. The American journal of clinical nutrition 81: 1072-1079 (2005) [DOI] [PubMed] [Google Scholar]
  27. Tanaka T, Iwamoto K, Wada M, Yano E, Suzuki T, Kawaguchi N, Shirasaka N, Moriyama T, Homma Y. Dietary syringic acid reduces fat mass in an ovariectomy-induced mouse model of obesity. Menopause 28: 1340-1350 (2021) [DOI] [PubMed] [Google Scholar]
  28. Tantikanlayaporn D, Wichit P, Weerachayaphorn J, Chairoungdua A, Chuncharunee A, Suksamrarn A, Piyachaturawat P. Bone sparing effect of a novel phytoestrogen diarylheptanoid from Curcuma comosa Roxb. in ovariectomized rats. PloS one 8: e78739 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tousen Y, Ezaki J, Fujii Y, Ueno T, Nishimuta M, Ishimi Y. Natural S-equol decreases bone resorption in postmenopausal, non-equol-producing Japanese women: a pilot randomized, placebo-controlled trial. Menopause 18: 563-74 (2011) [DOI] [PubMed] [Google Scholar]
  30. Vinogradova Y, Coupland C, Hippisley-Cox J. Use of hormone replacement therapy and risk of breast cancer: nested case-control studies using the QResearch and CPRD databases. Bmj 371 (2020) [DOI] [PMC free article] [PubMed]
  31. Weiss G, Skurnick JH, Goldsmith LT, Santoro NF, Park SJ. Menopause and hypothalamic-pituitary sensitivity to estrogen. Jama 292: 2991-2996 (2004) [DOI] [PubMed] [Google Scholar]
  32. Wronski TJ, Schenck PA, Cintrón M, Walsh CC. Effect of body weight on osteopenia in ovariectomized rats. Calcif Tissue Int 40: 155-9 (1987) [DOI] [PubMed] [Google Scholar]
  33. Yee S, Burdock GA, Kurata Y, Enomoto Y, Narumi K, Hamada S, Itoh T, Shimomura Y, Ueno T. Acute and subchronic toxicity and genotoxicity of SE5-OH, an equol-rich product produced by Lactococcus garvieae. Food and Chemical Toxicology 46: 2713-2720 (2008) [DOI] [PubMed] [Google Scholar]
  34. Yoneda T, Ueno T, Uchiyama S. S-equol and the fermented soy product SE5-OH containing S-equol similarly decrease ovariectomy-induced increase in rat tail skin temperature in an animal model of hot flushes. Menopause 18: 814-820 (2011) [DOI] [PubMed] [Google Scholar]
  35. Zhang R-h, Zhang X-b, Lu Y-b, Hu Y-c, Chen X-y, Yu D-c, Shi J-t, Yuan W-h, Wang J, Zhou H-y. Calcitonin gene-related peptide and brain-derived serotonin are related to bone loss in ovariectomized rats. Brain Research Bulletin 176: 85-92 (2021) [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 74 KB) (73.8KB, docx)

Articles from Food Science and Biotechnology are provided here courtesy of Springer

RESOURCES