Small black bean (Rhynchosia volubilis) extract ameliorates gut microbial and metabolic perturbation in ovariectomized mice

Journal of Functional Foods 60 (2019) 103415

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Small black bean (Rhynchosia volubilis) extract ameliorates gut microbial and metabolic perturbation in ovariectomized mice

T

Kwang Hyun Chaa, Kyung-A Kimb, Suk Woo Kangb, Seemi Tasnim Alama,d, Jong Beom Jina, ⁎ Gyhye Yooc, Sang Hoon Jungb,d, Cheol-Ho Pana,d, Kyungsu Kanga,d, a

Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 25451, Republic of Korea Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 25451, Republic of Korea c Smart Farm Research Center, Korea Institute of Science and Technology, Gangneung, Gangwon-do 25451, Republic of Korea d Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Republic of Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Gut microbiome Intestinal metabolite Ovariectomy Phytoestrogen Rhynchosia volubilis

Phytoestrogen is considered a promising natural remedy for various postmenopausal symptoms. However, most studies have focused on common soybean, Glycine max. Here, for the first time, we evaluated the in vivo effect of a distinct species, small black bean (Rhynchosia volubilis), on ovariectomized mice and investigated its impact on the intestinal microbiota and metabolic status. Ovariectomized mice exhibited significant body weight gain, a typical postmenopausal symptom, and microbial changes, such as decreased α-diversity; changes in microbial composition, especially abundances of the families Desulfovibrionaceae and Mogibacteriaceae and genus Dorea; and decreased amino acid and short-chain fatty acid levels. Administration of small black bean extract restores body weight and gut microbial perturbation to levels similar to those observed under normal conditions. Our data suggest that small black bean extract is a potential candidate functional food for treating postmenopausal symptoms via remodeling of the intestinal microbiome.

1. Introduction

women (Beck et al., 2005; Messina, 2016). More than 15 years of emerging research has revealed that the gut microbiota and the associated metabolic activity are crucial regulators of human health and many diseases, such as inflammatory bowel diseases, autoimmune diseases, obesity and diabetes, cardiovascular diseases, and mental disorders (Durack & Lynch, 2019). The gut microbiome plays a pivotal role in the alteration of estrogen levels in women during development, maturation, and menopause (Baker, Al-Nakkash, & Herbst-Kralovetz, 2017; Vieira, Castelo, Ribeiro, & Ferreira, 2017). The relationship between pre- and postmenopause is associated with a wide range of changes in gut microbial composition (Santos-Marcos et al., 2018). Postmenopausal women lack many important bacterial classes compared to premenopausal women, leading to an imbalance in the conditions and hampering the continued production of necessary metabolites for daily life (Baker et al., 2017; Vieira et al., 2017). The imbalance in gut microbial composition can affect and lead to many postmenopausal symptoms, including metabolic and immunological disorders. Therefore, remodeling of the gut microbiome can be a major influential factor for improvement of postmenopausal symptoms (Baker et al., 2017; Vieira et al., 2017). In a previous study, the overall gut

Menopause is a normal phenomenon in women’s lives and occurs when the regular menstruation cycle ends permanently at ages ranging from approximately 40–55, resulting from a loss of ovarian follicles (Valera et al., 2017). The importance of menopause and its symptoms has received significant attention because there have been a large number of clinical reports regarding this phenomenon, and women experience problems caused by menopause (Agarwal, Alzahrani, & Ahmed, 2018). Hot flashes, night sweats, vaginal dryness, osteoporosis, depression, and body weight gain are representative symptoms that are observed in postmenopausal women (Agarwal et al., 2018; Davis et al., 2015; Duffy, Iversen, & Hannaford, 2013). Some therapies have been introduced to address these problems, including hormone replacement therapy (Agarwal et al., 2018; Ellis, Hendrick, Williams, & Komm, 2015). However, synthetic hormone-based or chemical drug-based treatments have undesirable side effects, including increasing the risk of endometrial and breast cancer (Beck, Rohr, & Jungbauer, 2005). Therefore, natural remedies, such as soy-based foods, can be the first choice for treatment, providing a healthy lifestyle for postmenopausal

⁎ Corresponding author at: Natural Product Informatics Research Center, Korea Institute of Science and Technology, 679 Saimdang-ro, Gangneung, Gangwon-do 25451, Republic of Korea. E-mail address: [email protected] (K. Kang).

https://doi.org/10.1016/j.jff.2019.103415 Received 12 April 2019; Received in revised form 11 June 2019; Accepted 11 June 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

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2. Material and methods

microbial composition was similar in ovariectomy-induced obesity and diet-induced obesity, but Bifidobacterium animalis was exclusively observed in ovariectomized mice (Choi, Hwang, Shin, & Yi, 2017). Probiotic supplementation was reported to change the gut microbiota and ameliorate postmenopausal symptoms such as bone density loss and asthma exacerbation (Mendes et al., 2017; Parvaneh et al., 2015). Supplementation with dietary phytochemicals can be a favorable approach for remodeling the gut microbiota of postmenopausal women. A recent in vivo animal study reported that curcumin (Zhang et al., 2017), proanthocyanidin (Jin et al., 2018), and soy isoflavone changed the gut microbial composition in ovariectomized mice (Vieira-Potter et al., 2018). Phytoestrogens are plant-derived compounds that are structurally and functionally similar to 17β-estradiol. Phytoestrogens can bind to the estrogen receptors (ERs) ER-α and ER-β and trigger estrogenic effects (Sirotkin & Harrath, 2014). Plant sources of phytoestrogens include food plants, such as soybean, flaxseed, hops, and medicinal plants (Kang et al., 2009; Sirotkin & Harrath, 2014). Most well-characterized phytoestrogens are soy isoflavones, and these phytoestrogens were found to be effective in treating postmenopausal symptoms in women. Soybean is rich in phytoestrogens, including daidzein, genistein, and glycitein (Fang et al., 2016; Li, Lv, Xu, & Zheng, 2015), and these soy isoflavones have a high affinity for ER-β. Soybean supplementation alleviates hot flashes, renal dysfunction, and depressive symptoms; improves skin health; and reduces body weight in postmenopausal women (Baker et al., 2017; Messina, 2016). In addition, soy isoflavones also affected the gut microbiota in human trials as well as in vivo animal studies, and gut microbial changes are correlated with the beneficial effects of soy isoflavones on postmenopausal symptoms (Huang, Krishnan, Pham, Yu, & Wang, 2016; Vieira-Potter et al., 2018). Despite the great potential of leguminous plants as alternative hormone replacement therapies, most phytoestrogen studies have been limited to common soybean, Glycine max. Small black bean, Rhynchosia volubilis Lour., has small black seeds and is distinct from common soybean, G. max. R. volubilis grows in China, Korea, and Japan, and this leguminous plant was reported to have an abundance of the anthocyanin components cyanidin-3-O-glucoside and delphinidin. These compounds, which originate from the black seed coat (Kang et al., 2018), are known to have various pharmacological effects, such as antioxidant (Jeon, Kang, Um, & Kim, 2014) and antiadipogenic effects (Kim, Bae, Ahn, Lee, & Lee, 2007), can inhibit cancer cell proliferation (Kinjo, Nagao, Tanaka, Nonaka, & Okabe, 2001) and can be used to treat dry eye (Kang et al., 2018). A very limited in vitro study suggested the potential of R. volubilis as a phytoestrogen source; a methanolic extract of this plant activated estrogenresponsive promoters in cultured MG-63 human osteosarcoma cells (Kim et al., 2005). However, the in vivo efficacy of small black bean on ovariectomized animals and the effects of this plant on the gut microbiome have never been determined. In the present study, we first evaluated the in vivo effect of the ethanolic extract of R. volubilis (EERV) in ovariectomized mice. We also examined the effect of EERV on the gut microbial composition as well as metabolite changes. The high anthocyanin content of R. volubilis differs from that of common soybean, and the relationship between phytoestrogen consumption and changes in intestinal microbial metabolites has been poorly investigated in an animal model of postmenopause. For these reasons, we performed ovariectomy surgery in female mice and then observed the body weight gain to evaluate the effects of EERV in vivo. In addition, we analyzed the compositional changes in the gut microbiota as well as gut metabolites using 16S rRNA amplicon sequencing, 1H NMR spectroscopy, and integrated bioinformatics analysis.

2.1. Preparation of small black bean extract Seeds of small black bean, R. volubilis, were obtained from the Highland Agriculture Research Center (Pyeongchang, South Korea). A voucher specimen (KISTGN-RN-2016-003) was deposited at the Korea Institute of Science and Technology (KIST, Gangneung, South Korea). The standardized ethanolic extract of R. volubilis (EERV) was prepared as described previously (Kang et al., 2018). Briefly, seeds of small black bean were extracted with 70% ethanol in an ultrasonic cleaning bath. The liquid extracts were concentrated by a rotary evaporator to obtain EERV. HPLC-diode array detector-MS analysis was used for the compound determination in EERV. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B) with a YMC pack pro C18 column (3 μm particle size, 150 × 4.6 mm I.D., YMC Co., Japan). A linear gradient (5% sol. A for 0–1 min, 5–40% sol. A for 1–26 min, 40–95% sol. A for 26–30 min) at a flow rate of 1 mL/min was used, and absorbance at 260, 280, and 520 nm was monitored. The mass spectrometry conditions were as follows: positive mode; mass range, m/z 200–800; capillary voltage, 10 V; tube lens, 45 V; sheath gas flow rate (N2), 50 arbitrary units; auxiliary gas flow rate (N2), 12 arbitrary units; capillary temperature, 350 °C. EERV contains cyanidin 3-O-glucoside (1.14 mg/g), (-)-epicatechin (3.11 mg/g), daidzin (1.99 mg/g), genistin (3.69 mg/g), 6″-Omalonyldaidzin (4.84 mg/g), and 6″-O-malonylgenistin (8.95 mg/g) (Kang et al., 2018). 2.2. Animals and experimental design Female BALB/c mice (7 weeks old, 22–28 g) were purchased from Central Lab. Animal Inc. (Seoul, South Korea). These mice were housed at 23 ± 0.5 °C and 10% humidity under a 12 h light-dark cycle. All animals were acclimated for at least 1 week, caged, and fed animal chow and water ad libitum. All animal studies were performed in a pathogen-free barrier zone at the Korea Institute of Science and Technology (Gangneung, South Korea). The procedures used in this study were approved by the Animal Care and Use Committee of KIST (no. 2016-083). Thirty-six mice were randomly divided into six groups (n = 6 mice per group): a nonovariectomized group (sham), an ovariectomized group (OVX), a 10 mg/kg EERV-treated OVX group (EERV10), a 50 mg/kg EERV-treated OVX group (EERV50), a 100 mg/ kg EERV-treated OVX group (EERV100), and a 17β-estradiol (E2, 36 µg/ kg) plus progesterone (P4, 360 μg/kg)-treated OVX group (E + P, a positive control). In previous studies, E + P was administered mostly via subcutaneous or intraperitoneal injection in rats and mice. The ratio of E to P was 1:10–10,000, and the estradiol dose was variable from 5 μg/kg to 1 mg/kg (Choi, Shin, Song, & Lim, 2014; Ghazvini et al., 2016; Paris, Fenwick, & McLaughlin, 2014). We decided to use the dose of 36 µg/kg estradiol plus 360 µg/kg progesterone as the positive control, and the E + P was administered orally because different administration methods, such as subcutaneous injection, could trigger unintended stress responses and affect the final efficacy of E + P and EERV. We chose EERV doses ranging from 10 to 100 mg/kg for this study because the oral administration of 50 mg/kg EERV showed protective effects against dry eye disease (Kang et al., 2018) and the oral LD50 of a similar plant species, black soybean, is greater than 2.5 g/kg body weight in rats and mice (Fukuda et al., 2011). 2.3. Ovariectomy surgery and sample treatment Mice were anesthetized with 1.5% isoflurane (4% for induction) in a 30%/70% oxygen/nitrous oxide mixture as previously described (Strom, Theodorsson, Ingberg, Isaksson, & Theodorsson, 2012), and the dorsal surfaces of the mice were shaved. In the OVX groups, a 1-cm skin incision was made on one of the dorsolateral surfaces to expose the 2

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abdominal muscles. Following muscle dissection, the ovary was gently removed under aseptic conditions, and the second ovary was removed from the opposite side in the same manner. The skin incisions were closed with metal clips. The sham group was subjected to surgery without removal of the ovaries (Sophocleous & Idris, 2014). Antibiotics were immediately applied to the surgical sites to prevent bacterial infections. The experiment was performed after one week of recovery (Diaz Brinton, 2012). All samples (EERV or E + P) were dissolved in vehicle solution (0.5% carboxymethyl cellulose with sesame oil in distilled water) and were administered daily via oral gavage for 8 weeks. The EERV-treated groups received 100, 50, or 10 mg EERV/kg body weight per day. The sham and OVX groups received an equal volume of vehicle solution. Body weight was measured weekly, and treatment dosages were adjusted accordingly.

from downstream analyses prior to the generation of phylogenic trees or OTU tables using the ChimeraSlayer algorithm. The resulting tables of OTU counts were subsequently converted to relative abundance tables. Samples were resampled to the minimum sequencing depth (10,000 reads). The observed OTUs, Chao1, Simpson, and Shannon index (indicators of α-diversity) were calculated using the web-based MicrobiomeAnalyst tool with the default parameters (Dhariwal et al., 2017). The same tool was used to generate principal coordinates analysis (PCoA) plots of unweighted UniFrac distances between communities. To determine taxonomic features that were differentially abundant in specific experimental groups, LEfSe (linear discriminant analysis [LDA] effect size) was applied under the condition α = 0.01, with an LDA score of at least 3, and a taxonomic cladogram was generated to highlight significant differences in taxa (Segata et al., 2011).

2.4. Tissue harvesting

2.6. 1H NMR spectroscopy for cecal metabolite analysis

After 8 weeks of sample administration, mice were anesthetized with intraperitoneal injection of a mixture of 16 mg/kg Zoletil (Virbac Laboratories, Fort Worth, TX, USA) and 0.05 mL/kg Rompun (Newbury, UK). Blood samples were collected from the vena cava and immediately placed on ice. After each mouse was sacrificed by cervical dislocation, the uterus was carefully dissected from the surrounding fat and connective tissue and weighed. The cecum was cut rapidly, trimmed, washed in PBS and then stored at −70 °C.

For NMR spectroscopic analysis of the metabolome, cecal samples were prepared as described previously with some modification (Lamichhane et al., 2015). Briefly, cecal tissues (∼130 mg) were mixed with 600 μL of distilled water, vortexed for 30 s and homogenized by tissue homogenizer. After centrifugation (14,000g, 4 °C) for 10 min, 60 μL of deuterium oxide (D2O) containing 0.025 mg/mL 3-(trimethylsilyl) propionic acid-d4 sodium salt, 60 μL of 1 mM imidazole, 60 μL of 2 mM NaN3, and 120 μL of 0.5 M KH2PO4 were added to 300 μL of the supernatant. The mixtures were vortexed for 1 min and centrifuged at 14,000g for 10 min. The clear supernatant was then transferred to a 5 mm NMR tube (Wilmad-Lab glass, Vineland, NJ, USA). All 1 H NMR spectra were acquired using a Varian 500 MHz NMR system (Varian, Palo Alto, CA, USA) spectrometer equipped with a cold flow probe. 1H NMR spectra were collected at 25 °C using the water presaturation pulse sequence. Spectra were collected with 64 transients using a 4 s acquisition time and a 2 s recycle delay. Tentative assignments of 1H NMR signals were carried out using Chenomx NMR Suite 8.3 (Alberta, Canada). A total of 54 metabolites were assigned for analysis, namely, acetate, acetoin, alanine, anserine, aspartate, betaine, butyrate, caffeine, carnitine, cholate, choline, creatine, ferulate, formate, fumarate, glucose, glutamate, glycine, glycocholate, guanidoacetate, hypoxanthine, isobutyrate, isoleucine, isovalerate, lactate, leucine, lysine, malate, methanol, methionine, N-acetylglucosamine, Nacetylglutamate, N-acetyltyrosine, O-phosphocholine, p-cresol, phenylalanine, proline, propionate, putrescine, pyruvate, sarcosine, succinate, taurine, threonine, thymine, trimethylamine, trimethylamine Noxide, tryptophan, tyrosine, uracil, urocanate, valerate, valine, and xanthine. Cecal metabolite data were normalized and uploaded into MetaboAnalyst 3.0 for modeling using sparse partial least squares discriminant analysis (sPLS-DA) (Xia & Wishart, 2011). Metabolites with high loading values were assessed to rank each experimental group for degree of discrimination within the model.

2.5. 16S rRNA sequencing for analysis of the gut microbiota Frozen cecal samples (n = 5 per group) were thawed, completely homogenized in sterile phosphate-buffered saline using TissueLyser II (Qiagen, USA) and centrifuged at 14,000g for 5 min to acquire the bacterial precipitate. Genomic DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen, USA) with the additional bead-beating procedure to improve the DNA recovery for gram-positive bacteria. The 16S rRNA genes were amplified using the Illumina-adapted universal primers 341F/805R for the V3-V4 region (Herlemann et al., 2011). Samples were indexed with a Nextera XT index kit following the manufacturer’s instructions (Illumina, San Diego, CA, USA). The amplicons obtained were cleaned, quantitated, and paired-end sequenced on the Illumina MiSeq platform using v3 reagents (Illumina). The 16S rRNA sequences in the present study were deposited in the Sequence Read Archive (Accession No. PRJNA544903). Sequences generated from the MiSeq run were analyzed using QIIME software (V1.8.0). Briefly, high-quality (quality value > 25) sequence data derived from the sequencing process were demultiplexed and quality filtered using split_libraries_fastq.py. Sequences were then clustered into operational taxonomic units (OTUs) using closed reference OTU picking against the Greengenes 13_5 reference OTU database with a 97% similarity threshold (Caporaso et al., 2010). Chimera sequences were excluded

Fig. 1. Effect of EERV on body weight gain (A) and uterine weight (B) in ovariectomized (OVX) mice. Positive control (17β-estradiol, 36 µg/kg, plus progesterone, 360 μg/kg; E + P) or EERV (10, 50, and 100 mg/kg) were administered daily via oral gavage for 8 weeks. Data are shown as the mean ± SEM (n = 6), and differences were assessed by one-way ANOVA followed by Tukey’s multiple comparison test. Different letters indicate significant differences (p < 0.05). Diagonal lines indicate ovariectomized mice.

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Sirotkin & Harrath, 2014). EERV also contains phytoestrogens such as genistin, 6″-O-malonylgenistin, daidzin, and 6″-O-malonyldaidzin as major compounds (Kang et al., 2018). Therefore, we speculated that these phytoestrogenic compounds contained in EERV may contribute to the decrease in body weight gain in mice administered EERV. Given these results, we conclude that EERV ameliorates the abnormal body weight gain induced by ovariectomy in mice.

2.7. Statistical analysis The obtained data are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. A statistical probability of p < 0.05 was considered significant using GraphPad Prism version 7.0. Permutational multivariate analysis of variance (PERMANOVA) was performed to evaluate the βdiversity difference of microbial composition between groups (sham, OVX, and treatment groups) in PCoA plots.

3.2. Effect of EERV on gut microbial diversity in ovariectomized mice Next, we elucidated the effect of EERV on the gut microbiota because the intestinal microbiota, overall metabolism, and physiology of postmenopausal women are closely correlated (Baker et al., 2017; Vieira et al., 2017). In addition, the restoration of gut microbial perturbation in ovariectomized mice by EERV supplementation was the main goal of the present study. We first evaluated the effect of EERV on the gut microbial diversity index because low microbial diversity is generally considered to be associated with unhealthy conditions (Kriss, Hazleton, Nusbacher, Martin, & Lozupone, 2018). We calculated both the α-diversity (Fig. 2A–D) and β-diversity (Fig. 2E–H) indexes, which demonstrate the microbial diversity within groups and differences in diversity between groups, respectively. The αdiversity is a simple and straightforward index for rough estimation of gut microbial conditions. The Simpson and Shannon indexes, both of which indicate the richness and evenness of microbial α-diversity, of each group did not differ (Fig. 2C and D). However, the observed OTUs and Chao1 index, which reflect the species richness of microbial diversity, of the sham, OVX, positive control (E + P), and EERV-treated groups differed significantly. These observed OTUs and the Chao1 index of the OVX group were significantly lower than those of the sham group, and those of the E + P replacement group and EERV-treated groups were similarly restored to those of the sham group (Fig. 2A and B). These data suggested that EERV supplementation could change the α-diversity of the gut microbiota, especially species richness, which was significantly decreased by ovariectomy. This recovery of gut microbial α-diversity by EERV administration in the ovariectomized model is similar to that observed with supplementation with curcumin (Zhang et al., 2017). In a previous soy-rich diet supplementation study, soy isoflavone did not increase the α-diversity of the gut microbiota in

3. Results and discussion 3.1. Effect of EERV on body weight gain and uterine weight in ovariectomized mice First, we observed body weight gain in OVX mice fed E + P (the positive control) or EERV (10, 50, and 100 mg/kg) to evaluate the in vivo effect of EERV. Body weight was significantly increased in OVX mice compared with the sham control (Fig. 1A). This abnormal increase in body weight gain is a typical postmenopausal symptom in old women (Agarwal et al., 2018; Duffy et al., 2013), and an abnormal increase in body weight was also reported in many animal studies that conducted ovariectomies (Cross et al., 2017; Zhang et al., 2017). Estrogen and progesterone supplementation (E + P, the positive control) significantly decreased the body weight gain compared to that in the OVX group. EERV supplementation also restored the body weight gain in a dose-dependent manner (Fig. 1A). The uterine weights of OVX mice were significantly decreased compared to those of the sham group. These data indicate that ovariectomy resulted in a decrease in uterine weight. The E + P treatment slightly increased the uterine weight compared with the OVX with no supplementation (Fig. 1B). Similarly, EERV (10–100 mg/kg) supplementation did not increase the uterine weight (Fig. 1B), and these observations are similar to those of previous studies in which curcumin and soy extract did not affect uterine weight in ovariectomized mice (Zhang et al., 2017; Zhang, Li, Wan, Helferich, & Wong, 2009). Phytoestrogens such as soy isoflavones are known to have a therapeutic effect on menopause-induced obesity (Baker et al., 2017;

Fig. 2. Effect of EERV on gut microbial diversity in ovariectomized (OVX) mice. Observed OTUs (A), Chao1 index (B), Simpson index (C), and Shannon index (D) were calculated to investigate the α-diversity of the microbial community. Data are shown as the mean ± SEM, and one-way ANOVA followed by Tukey’s multiple comparison test was used to determine differences between groups. Different letters indicate significant differences (p < 0.05) between groups. Unweighted PCoA score plots were analyzed to investigate the β-diversity of the microbial community among the sham group, OVX group, and each treatment group: estrogen plus progesterone (E + P, positive control; E), EERV100 (F), EERV50 (G), and EERV10 (H). 4

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sham group by administration of E + P or EERV treatment, although the effect of EERV on the recovery of three microbial taxa, especially the Mogibacteriaceae family, was not dose dependent. The relative abundances of the Desulfovibrionaceae and Mogibacteriaceae families were significantly decreased in the OVX group and were significantly increased by treatment with E + P and EERV. The relative abundance of the genus Dorea was significantly increased in the OVX group compared to the sham group and restored to levels similar to that in the sham group upon EERV administration at all doses (10, 50, and 100 mg/kg) (Fig. 3C). Our observation is consistent with those of a previous study that described an abnormal increase in the abundance of the genus Dorea in an OVX model and recovery of the levels by administration of genistein, a well-known soybean phytoestrogen (Lee et al., 2017). The similar results observed here can probably be attributed to EERV containing isoflavones similar to genistein. Our chemical profiling analysis showed that isoflavonoid compounds, including genistin and daidzin, could be isolated from EERV (Kang et al., 2018). Based on these data, we concluded that EERV supplementation alleviated the intestinal microbial changes induced by ovariectomy in an experimental model that simulated postmenopausal conditions.

ovariectomized rats (Cross et al., 2017). The β-diversity analysis showed a similar trend as the α-diversity data. In Fig. 2E–H, the unweighted UniFrac PCoA score plot showed that the OVX and sham groups were clearly grouped in a separate cluster (PERMANOVA, p = 0.009). The positive control (E + P)-treated group and the EERV50- and EERV100-treated groups were clearly separated from the OVX group (PERMANOVA, p = 0.023, 0.027, and 0.009, respectively). The EERV10 group was not clearly grouped in a separate cluster (PERMANOVA, p = 0.088), probably due to the low concentration of EERV (Fig. 2H). These data suggest that E + P hormone replacement or EERV consumption could change microbial diversity levels to normal levels, similar to those seen under the sham condition, which were dramatically altered by ovariectomy (OVX), a hormonal depletion condition. Based on these data, we concluded that EERV supplementation could ameliorate the gut microbial perturbation induced in the ovariectomized mouse model. 3.3. Effect of EERV on the gut microbial profiles of ovariectomized mice LEfSe analysis showed the differences in microbial composition between the sham and OVX groups. The abundances of the phylum Bacteroidetes and genus Dorea were apparently increased in the OVX group compared to the sham group (Fig. 3A and B). This result is consistent with previous reports (Cox-York et al., 2015; Lee et al., 2017). As shown in Fig. 3A, the number of microbial taxa in the sham group was higher than that in the OVX group (11 vs 4). Most microbial taxa from the sham group with high LDA scores were minor microbes present in the gut, with relatively low abundances. The decrease in the abundances of these minor bacteria may have resulted in a decrease in the microbial richness of the OVX group, which was observed in the OTUs and Chao1 index (Fig. 2A and B). As shown in Fig. 3C, the levels of 3 microbial taxa were significantly altered in OVX mice and were restored to levels similar to those in the

3.4. Effect of EERV on the cecal metabolite profiles of ovariectomized mice Finally, we analyzed 54 major metabolites from the mouse cecum using 1H NMR spectroscopy to elucidate the effects of EERV on the gut microbiota and metabolic outcome. A sPLS-DA score plot showed that the OVX group was separated from other groups, namely, the sham, E + P, and EERV-treated groups (Fig. 4A). These data suggested that ovariectomy affected not only the intestinal microbiota but also the microbial metabolic status. As shown in Fig. 4B, the levels of 10 major metabolites were decreased in the OVX group, and the 10 decreased metabolites observed in the OVX group were increased by the administration of E + P and EERV, although there were no significant

Fig. 3. Effects of EERV on the gut microbial composition in ovariectomized (OVX) mice. Positive control (E + P) or EERV (10, 50, and 100 mg/kg) was administered daily via oral gavage for 8 weeks. Histogram (A) and cladogram (B) of the linear discriminant analysis (LDA) scores for differentially abundant taxa following ovariectomy. Gray and red colors indicate the taxa overrepresented without and with ovariectomy, respectively. Features with LDA scores > 3 are presented. (C) Three taxa that showed significant recovery of microbial abundance after treatment were selected and are represented as box plots. Different letters indicate significant differences (one-way ANOVA followed by Tukey’s multiple comparison test, p < 0.05) between groups. Diagonal lines indicate ovariectomized rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 5

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Fig. 4. Effect of EERV on the gut metabolite profiles in ovariectomized (OVX) mice. Positive control (E + P) or EERV (10, 50, and 100 mg/kg) was administered daily via oral gavage for 8 weeks. Sparse partial least squares discriminant analysis (sPLS-DA) of the NMR-based metabolomic dataset. The sPLS-DA score plot (A) and loading plot (B) are presented. Light gray and light red ellipses indicate 95% confidence intervals for sham and OVX, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

extract is a promising functional food material for the prevention and treatment of postmenopausal symptoms in women via remodeling of the gut microbiota and microbial metabolic status.

differences (Supplementary Fig. S1). The most strongly affected metabolites were amino acids (tryptophan, tyrosine, leucine, aspartate, and phenylalanine) and short-chain fatty acids (butyrate and acetate). The present observations were similar to those of a previous report, in which butyrate and acetate levels in fecal samples from ovariectomized rats were lower than those of sham rats (Cox-York et al., 2015). The observed abnormal decrease in the levels of amino acids and shortchain fatty acids might be used as metabolite biomarkers to demonstrate intestinal metabolic disorder triggered by ovariectomy. However, further in-depth studies using LC-MS/MS are required for a full evaluation of the biomarker-level metabolites. High doses (50 and 100 mg/ kg) of EERV supplementation changed the levels of these metabolites to normal levels in the sham group. EERV10 could not restore the major metabolites to levels similar to those in the sham group, most likely due to the low dose of EERV. These data suggest that EERV supplementation changes the intestinal metabolic properties, which are perturbed by ovariectomy. This is the first report of the remodeling of intestinal metabolites by small black bean administration in ovariectomized animals. Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jff.2019.103415.

Ethics statement This study contains animal experiments, including mouse ovariectomy surgery, and all procedures were approved by the Animal Care and Use Committee of Korea Institute of Science and Technology (no. 2016-083, Gangneung, South Korea). Acknowledgments We would like to thank Professor Young-Dong Kim (Hallym University, South Korea) for valuable advice relating to plant taxonomy. This work was supported by the Korea Institute of Science and Technology intramural research grant (2Z05620). Declaration of Competing Interest The authors declare no conflict of interest. References

4. Conclusions Agarwal, S., Alzahrani, F., & Ahmed, A. (2018). Hormone replacement therapy: Would it be possible to replicate a functional ovary? International Journal of Molecular Sciences, 19(10), 3160. Baker, J. M., Al-Nakkash, L., & Herbst-Kralovetz, M. M. (2017). Estrogen-gut microbiome axis: Physiological and clinical implications. Maturitas, 103, 45–53. Beck, V. B., Rohr, U., & Jungbauer, A. (2005). Phytoestrogens derived from red clover: An alternative to estrogen replacement therapy? The Journal of Steroid Biochemistry and Molecular Biology, 94(5), 499–518. Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., ... Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5), 335–336. Choi, S., Hwang, Y. J., Shin, M. J., & Yi, H. (2017). Difference in the gut microbiome between ovariectomy-induced obesity and diet-induced obesity. Journal of Microbiology and Biotechnology, 27(12), 2228–2236. Choi, S., Shin, H., Song, H., & Lim, H. J. (2014). Suppression of autophagic activation in the mouse uterus by estrogen and progesterone. Journal of Endocrinology, 221(1), 39–50. Cox-York, K. A., Sheflin, A. M., Foster, M. T., Gentile, C. L., Kahl, A., Koch, L. G., ... Weir, T. L. (2015). Ovariectomy results in differential shifts in gut microbiota in low versus

Here, we evaluate for the first time the effect of small black bean extract on the abnormal body weight gain induced by the experimental postmenopausal model in mice. Small black bean supplementation restored the body weight gain of ovariectomized mice to levels similar to those of the sham group. In addition, we described that supplementation with small black bean extract could ameliorate intestinal microbial perturbation, including alteration of the microbial composition and metabolite profile induced by ovariectomy. Ovariectomy surgery altered the intestinal microbial diversity and the relative abundances of the Desulfovibrionaceae and Mogibacteriaceae families and the genus Dorea and the amino acid and short-chain fatty acid metabolite levels. Eight weeks of oral administration of small black bean extract successfully ameliorated this gut microbial and metabolic perturbation in ovariectomized mice. Therefore, we suggest that small black bean 6

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