The effect of sorghum resistance resistant starch‐mediated equol on the histological morphology of the uterus and ovaries of postmenopausal rats

Abstract Equol is a metabolite of daidzein and has a higher biological activity than daidzein. Equol, combined with estrogen receptors, can reduce the incidence of diseases such as cardiovascular disease, osteoporosis, and breast cancer; more effectively alleviate the symptoms of perimenopausal syndrome; and improve age‐related decline of the uterus and ovaries. Research has shown that food composition can greatly affect the formation of equol in the intestinal tract. In the intestines, the content of nonstarch polysaccharides that can stimulate fermentation is high, thereby allowing intestinal bacteria to quickly and completely transform the daidzein into equol. This study used Sprague Dawley (SD) rats as a model, where menopause was established through direct intragastric administration of formistan. In the 6‐week‐long experiment, intragastric administration of RS while feeding bean pulp reduced the body weight of postmenopausal rats, reduced the efficiency of feed utilization of rats, and increased the weight of organs such as the uterus and ovaries. Routine blood indexes showed that no adverse reactions were produced by intragastric administration of RS. 16s rDNA sequencing further verified Lactobacillus and Clostridium XIVa, as the bacteria that converted daidzein into equol.

In the presence of a high content of nonstarch polysaccharides that can stimulate fermentation, intestinal bacteria can quickly and completely convert daidzein into equol, while in cases of low-carbohydrate content, it is almost impossible to produce equol (Rowland, Wiseman, Sanders, Adlercreutz, & Bowey, 2000). That is, dietary fibers or nonstarch polysaccharides can effectively promote the growth or activity of the colonic bacteria that produce equol (Frankenfeld, 2011). Sorghum, as the fifth largest grain crop, is widely cultivated because of its high yield and stress resistance (Wang & Li, 2006). As a type of grass, sorghum has a high-carbohydrate content of up to 80%, making it a rich source of resistant starch (RS), a novel type of dietary fiber. RS has good enzymolysis resistance and cannot be digested and absorbed in the small intestine. It is fermented in the large intestine and produces short-chain fatty acids, which improves the intestinal environment and adjusts the composition of the intestinal flora. At present, it is widely believed that intestinal physiology, host genotype, and dietary composition are the main factors affecting an individual's ability to convert daidzein into equol (Huang, 2010). Therefore, the novelty of this study is in the improvement of the intestinal flora through RS to convert daidzein from bean pulp to equol to ultimately reduce the severity of perimenopausal symptoms in rats.
These experiments showed that when sorghum was naturally fermented for eight days, the content of amylose and the retrogradation value peaked, the RS was easier to prepare, and the fermentation characteristics of the obtained RS were higher. Therefore, natural fermentation of sorghum was carried out in this experiment, and RS was prepared using the pressure-heat compound enzyme method. In vivo experiments were conducted to explore the effect of RS-mediated intestinal flora on the morphology of ovarian and uterine tissues of menopausal rats.

| Experimental material
Sorghum: Sorghum R from Linyi, Shandong. Hydrochloric acid (analytically pure), sodium oxide (analytically pure), potassium bromide (spectrographic grade), high-temperature-resistant α-amylase (1,400 u/g), pullulanase (1,000 ASPU/g), and high-amylose corn starch were made by Damao Chemical Reagent Factory, Tianjin, China. Glucan standards and estradiol standards were purchased from Sigma. Vancomycin, neomycin, metronidazole, and penicillin were made by Qingdao High-Tech Park Haibo Biotechnology Co., Ltd. Sprague Dawley rats were from Changchun Yisi Experimental Animal Technology Co., Ltd. Feed including bean pulp and feed excluding bean pulp was from Biotech HD. Distilled water was produced by the laboratory.

| Preparation of samples
Sorghum was naturally fermented for 8 d. Sorghum was mashed and screened with an 80 sieve and soaked in 0.3 g/100 ml NaOH at 1:3 g/ml for 3 hr followed by centrifuging for 10 min at 3256 g. The supernatant and upper tawny substances in the sediment were removed, washed with clean water four times, and centrifuged until the starch slurry became white. The pH of the starch slurry was adjusted with 1 mol/L HCl to 7.0, centrifuged, dried at 30°C, and screened with a size 80 sieve to obtain fermented sorghum starch (Xu, Cheng, & Zhao, 2007.).
Fermented sorghum starch was used to prepare 10% starch milk.
Starch milk was autoclaved, pressure-heated for 15 min at 115°C, cooled down to 40°C, and pH adjusted to 4.5. Then, 3 U/g pullulanase was added and treated for 8 hr at 40°C, followed by enzyme deactivation for 15 min at 95°C. Samples were cooled to room temperature and kept at 4°C for 12 hr (Bao, 2017). Retrograded starch was removed, dried, and prepared into 20% RS starch milk. The pH was adjusted to 5.4 and 2 ml of 1% high-temperature-resistant α-amylase was added and vibrated in 90°C water for 1.5 hr. The starch paste decomposed into monosaccharides and oligosaccharides through enzymolysis and was then heated in boiling water for 10 min followed by the addition of 4 times the volume of 95% ethyl alcohol to dissolve the monosaccharides and oligosaccharides. The alcohol precipitated for 4 hr before being centrifuged for 20 min at 2442 g.
The supernatant was removed and 10 ml of 95% ethyl alcohol was added to wash the sediment 2-3 times followed by drying at 50°C to a constant weight, mashed, and screened with a size 100 sieve to get fermented sorghum RS (Wang, 2013).

| Diet and animals
Seventy Sprague Dawley rats, three months of age, were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. Each of them weighed 190 ± 10 g and were maintained in the facility at room temperature (24 ± 2°C) with 45%-65% humidity. Animals were divided into eight groups, blank group (NC), model group (MC), RS group (RS), low-dose RS group (RSL), medium-dose RS group (RSM), high-dose RS group (RSH), positive control group (PC), and fecal microbiota transplantation group (FMT), and each rat was fed in single cages. Menopause was established by gavage of Formestane every day at 9 a.m., at a dosage of 50 mg/kg per animal, except for the blank group (NC). During the modeling, the animals had ad-libitum access to water and were fed a bean-free diet. An ELISA kit was used to determine the concentrations of follicle-stimulating hormone and estradiol in the urine of the rats. The aim of this test was to determine the stage of menopause. After the rats were determined to be in the menopausal state, drugs such as resistant starch were administered. The determination of the concentration of resistant starch was based on the recommended daily intake of healthy humans; it was processed and dissolved with an ultrasonic processor. The design of test drug delivery is shown in Table 1. The experimental diet composition was designed with reference to AOAC synthetic feed formula (AIN-93) for experimental rats. Soybean-free diets were prepared by replacing bean pulp with casein (Reeves, Nielsen, & Fahey, 1993). Animal feeding and treatment were in accordance with the provisions of the Chinese Association for Laboratory Animal Sciences (CALAS), and all animals were cared for in strict accordance with animal use guidelines.
Fecal microbiota transplantation (FMT) (Gong et al., 2018): Before the experiment, vancomycin (1 g/L), neomycin (1 g/L), metronidazole (1 g/L), and penicillin (500 g/L) were dissolved in the drinking water of rats. After one week of interference, the intestinal flora of rats was cleared to balance and unify the flora environment in vivo. Fresh feces (100 mg) from the donor rats with the highest content of equol were selected and mixed with 1 ml sterilized normal saline in a centrifuge tube and left standing for 10 min. An aseptic syringe was used to transfer the supernatant to feed the rats through intragastric administration.
During the 6-week-long experiment, urine was collected every week, and the weight and food intake of the rats were recorded. At the end of the experiment, feces samples were collected for immediate freezing and storage in −80°C. After fasting overnight, the rats were euthanized, and the eyes were removed for blood samples. The uterus, ovaries, thymus, and spleen were harvested and weighed.
After weighing, the uterus and ovaries were stored in a formaldehyde solution until histomorphological analysis.

| Body weight of rats: determination of weight gain and efficiency of feed utilization
During the experiment, body weight and food intake of each rat were recorded weekly, and the feed efficiency of the rat was recorded according to the following formula (1): Where: M is the total food intake of the rat during the experiment;M 0 is the initial weight of the rat;M1 is the weight of the rat at the end of the experiment.

| Determination of routine blood indexes
After the rat was weighed and blood was taken from the eye post-removal, the blood was collected into a blood vessel with an EDTA-K2 anticoagulant tube. Blood indexes from freshly collected blood were determined in an immobilized blood cell analyzer.

| Sample pretreatment
A total of 200 mg of feces were added into a centrifuge tube with 1 ml of 70% ethyl alcohol, vibrated to mix, and centrifuged for 3 min at 8000 g at room temperature. The supernatant was removed and 1 ml of phosphate-buffered saline was added, vibrated to mix, and centrifuged for 3 min at 8000 g at room temperature. The supernatant was removed, and the tubes were inverted for 1 min or until all remaining liquid was removed. Sample tubes were dried at 55°C for 10 min to remove residual ethyl alcohol.

| Quantified mixture
The DNA reclaimed by detection kit pairs was accurately quantified to facilitate sequencing after mixing at a 1:1 ratio. From the balanced mix, 10 ng of DNA was taken for each sample and 2 pmoL of DNA was used for sequencing. Sequencing was completed by Sangon Biotech.

| Determination of equol and daidzein contents
Fresh urine was collected, and the equol and daidzein content were determined by ELISA (Shanghai Fanke Industrial Co., Ltd.) The experiment was performed according to manufacturer's protocol.
The log Koc of the equol content:daidzein content, that is log10 (equol/daidzein), was used to represent the equol production capacity of postmenopausal rats (Setchell & Cole, 2006).

| Histological analysis
Uterus and ovary tissues of postmenopausal rats were soaked in formaldehyde solution, fixed with paraffin, and stained with hematoxylin and eosin to make routine sections. The morphology of the liver was observed using a BA210T microscope equipped with a camera.

| statistical analysis
Excel 2016 and SPSS 19.0 software were used for statistical analysis of the data; Origin 8.0 software was used for drawing processing; the data were measured in triplicate to obtain the average value.
The results are presented as the mean ± SD. Group differences were analyzed using Tukey's post hoc test, and the data were measured by two-way repeated-measures analysis of variance (ANOVA). TA B L E 2 Effect of RS-mediated intestinal flora on body weight, feed consumption, and efficiency of feed utilization of postmenopausal rats 5.61 ± 0.046b Note: Data are shown as mean ± SD (n = 7/9). Values with different letters in columns are significantly different (p < .05) from each other.

| Effect of RS-mediated intestinal flora on the body weight, feed consumption, and efficiency of feed utilization of postmenopausal rats
The effect of RS-mediated intestinal flora on the weight and efficiency of feed utilization of postmenopausal rats is shown in (Table 2). Following menopause modeling, the weight of rats in the experimental groups, including the model group, was significantly higher than that of the untreated control group, which sug-  (Zhou & Hu, 2005), thus causing the decrease in rat body weight, as well as weight gain and feed consumption rate.

| Effect of RS-mediated intestinal flora on the viscera weight of postmenopausal rats
Compared with the untreated control group, the weight of the spleen and thymus decreased significantly in the model group

| Effect of RS-mediated intestinal flora on the routine blood indexes of postmenopausal rats
Various diseases and changes in physical conditions can lead to changes in routine blood indexes. Therefore, the number of red blood cells, white blood cells, and blood platelets in the blood of postmenopausal rats and their morphological distribution can be detected through the blood analyzer to detect early signs of various clinical diseases (Lohsoonthorn, Dhanamun, & Williams, 2006).
After the postmenopausal rats were given different doses of RS and fed with bean pulp, most of the indexes in the drug groups were not significantly different from those in the NC blank group, indicating that the rats would not have adverse reactions and effects due to the combined administration of RS and bean pulp (Table 3).

Lymphocytes (LYMs) originate from hematopoietic stem cells,
circulate within the blood to the surrounding lymphoid organs, and then settle and proliferate within tissues. Under stimulation by certain antigens, lymphocytes can differentiate and multiply to produce effector cells, and perform immune functions (Aboulker, Autran, & Beldjord, 1992). Compared with the NC group, the LYMs of groups RSM, RSH, and PC were significantly different, which indicated that the specific immunity was stronger in the case of intragastric administration of the same amount of RS when administered in conjunction with bean pulp feed. MONO, the largest blood cell in the blood, is abundant in nonspecific lipase, which can resist and eliminate invading pathogenic microorganisms by means of phagocytosis and antibody production (Lin, Guo, & Yao, 2003). Compared with the NC group, the percentage of MONO in the PC group was slightly higher, indicating that the nonspecific immunity of the organism was stronger at this time. According to FMT, the immune performance of the body increased after transplanting the dominant bacteria producing equol, which was different from most of the indexes in the RS group, indicating that the immune activity of equol produced by RS-mediated intestinal flora was higher than that of RS of equal dose.

| Effect of RS on the intestinal flora structure of postmenopausal rats
The identification of the strains from the fecal samples of menopausal rats is shown in (Figure 2

Relevant studies have shown that this genus is a biomarker of early
tumor formation (Mangifesta et al., 2018), indicating that postmenopausal rats see an increased risk of colon cancer (Li & He, 2013).
After treatment, the drug group showed a significant decrease in the scope of this bacterial genus, indicating that both RS and equol indirectly produced by RS were associated with a reduced risk of colon and other cancers in postmenopausal rats; in the presence of soybean meal, the higher the concentration of resistant starch, the more significant the decline in this bacterial genus. Lactobacillus genus can synthesize and secrete lactic acid, which plays an important role in maintaining a healthy vaginal environment during childbearing years (Linhares, Summers, Larsen, Giraldo, & Witkin, 2011).
The enrichment and growth of the Lactobacillus genus can form a community with social cohesion, increase the resistance against alien species, and constitute a biological barrier to maintain the health and microecological balance of the host reproductive tract (Huggins & Preti, 1976). After the Formestane-based postmenopausal treatment, the richness of the Lactobacillus genus of the rats was significantly lower than that of the control group, indicating that the immunity of postmenopausal rats was reduced. The results of this study were consistent with the results of the routine blood test. After intragastric administration of RS and feeding of fodder containing bean pulp, the content of the Lactobacillus genus was increased, and the immune barrier of the rats was restored to a certain extent. The richness of the Lactobacillus genus in the intestinal flora of the rats after adding bean pulp was higher than that of the rats only receiving intragastric administration of RS, indicating that the effect of equol on enhancing the immunity of postmenopausal rats was stronger than that of RS. However, the FMT experimental group showed that the intestinal flora had the ability to convert daidzein from bean pulp into equol in the overall experimental process so as to reduce the risk of cancer and improve the recovery function in postmenopausal rats. Research has shown that Lactobacillus and Clostridium XIVa genera play an important role in the metabolism of daidzein in bean pulp (Decroos, Vanhemmens, & Cattoir, 2005;Schoefer, Mohan, Schwiertz, Braune, & Blaut, 2003).
The richness of both genera in the RSL, RSM, and RSH experimental groups was higher than that in the RS group; in the RSL experimental group, the bacterial genera were richer than in the RSM and RSH groups. Therefore, this experiment further verified that Lactobacillus and Clostridium XIVa, as metabolic bacteria of daidzein, can convert daidzein into equol. That is, intestinal flora is the key factor in the regulation of the production of equol by RS.  Note: Data are shown as mean ± SD (n = 7/9). Values with different letters in columns are significantly different (p < .05) from each other. and the logarithmic value of the ratio of equol and daidzein levels, namely log10 (equol/daidzein), was used to characterize the capability of individual rats to produce equol. After the successful establishment of the menopausal model at week zero, there was no significant difference in the capability to produce equol among the groups (Table 4). Since the rats were fed without bean pulp during the modeling period, there was no source of daidzein, or the content of equol was low; therefore, no significant difference between equol production among the groups was detected.

| Effect of RS-mediated intestinal
After treatment of postmenopausal rats in each drug group, the log10 (equol/daidzein) values of all individuals were greater than −1.0, indicating highly efficient equol production. Therefore, it confirms that daidzein in bean pulp was the precursor of equol (Zheng, 2013). The FMT further showed that RS could transform daidzein into equol with higher biological function by regulating the composition of the intestinal flora and increasing the richness of lactobacillus and clostridium, playing an effective role as a phytoestrogen.

| Effect of RS-mediated intestinal flora on the morphology of the uterus and ovary of postmenopausal rats
The pathomorphological observation results of the uterus are shown in (Figure 3): group NC had thicker endometria, the glandular epithelium was of single layer and low columnar, the lamina propria was distributed with metrial glands and blood vessels, and the connective tissues were tight. Compared with the NC blank group, the MC group saw significantly reduced thickness of the uterine tube and the uterine mucosal epithelial cells. That is, the endometrium and muscle layer were significantly thinner, and the muscle fibers

| CON CLUS IONS
In conclusion, this study elucidates the mechanism of RS- XIVa, as metabolic bacteria of daidzein, can convert daidzein into equol. That is, intestinal flora is the key factor in the regulation of the production of equol by RS.