Improvement of Menopausal Symptoms by Oat Hull Extracts in Ovariectomized Animal Model

Improvement of Menopausal Symptoms by Oat Hull Extracts in Ovariectomized Animal Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improvement of Menopausal Symptoms by Oat Hull Extracts in Ovariectomized Animal Model Mi-Ja Lee, Yongjin Lee, Han Gyeol Lee, Holim Jin, Yoon-A Shin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5382261/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Female menopause is a disease characterized by the gradual loss of ovarian function that occurs before and after menopause. Female menopausal symptoms include osteoporosis, facial flushing, and dyslipidemia. Oats are known to be rich in protein and lipids, and their hulls are used as feed. In this study, we sought to explore the potential of oat hull extract as a treatment for menopause by analyzing its estrogen-like activity and menopause-related biomarkers in ovariectomized (OVX) animals. We investigated changes in mRNA expression and estrogen activity of several menopause-related genes induced by oat hull extract in vitro using MCF-7 breast cancer cells. Additionally, we used an ovariectomized mouse model to investigate the effects of oat hull extract on menopause in vivo through changes in body weight, tissue morphology, and bone mineral density. Therefore, this study suggests that oat hull extract may be a promising alternative for treating female menopause. Oat hull menopausal symptoms ovariectomized (OVX) bone density Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Menopause is defined by the World Health Organization (WHO) as the absence of menstrual periods for 12 consecutive months without any physiological or pathological cause, indicating that natural menopause has occurred. Menopause most often occurs between ages 45 and 55, and some women experience early menopause before age 40 [ 1 ]. As of the "World Population Prospects 2022" report released by the United Nations (UN), the global average life expectancy in 2021 was 71 years [ 2 ]. With increasing life expectancy, a significant number of women are expected to spend more than a third of their lives in a state of menopause. Moreover, as the global population ages, the proportion of women experiencing perimenopause is also on the rise [ 3 ]. Therefore, interest in menopause is increasing all over the world. Menopause in women refers to the gradual loss of ovarian function occurring before and after menopause, defined as the cessation of menstruation for one year after the final menstrual period [ 4 ]. With the advancement of medicine and improvement in living conditions, life expectancy has continued to increase, and most women now spend more than one-third of their lives in menopausal status [ 5 ]. Menopause, a natural phenomenon occurring due to aging, is characterized by sudden hormonal changes, which can disrupt daily life and diminish quality of life. Menopausal and postmenopausal women experience various menopausal symptoms due to decreased estrogen levels, including osteoporosis, hot flashes, dyslipidemia, and other conditions [ 6 , 7 ]. To alleviate these symptoms, hormone therapy has traditionally been used; however, concerns about side effects such as endometrial cancer, cardiovascular disease, and breast cancer due to excessive estrogen have led to research on plant-derived estrogen-like substances or natural menopause relief agents [ 8 – 10 ]. However, side effects of its use as a therapeutic agent include breast pain, fluid retention, nausea, leg cramps, and headaches Fait [ 11 ]. Estrogen plays a crucial role in the function and growth of the human reproductive system. While primarily secreted by the ovaries, estrogen is also produced by other tissues such as adipose tissue, adrenal glands, pancreas, and the brain. Additionally, E2 (17β-estradiol), the primary estrogen in humans, can be converted to E1 (estrone), and both E1 and E2 can be converted to E3 (estriol) [ 12 ]. When estrogen binds to the estrogen receptor (ER), the receptor dimerizes, subsequently binding to the estrogen response element (ERE). This forms the ER-ERE complex, which regulates estrogen-regulated target genes such as the progesterone receptor (PR) and prezenelin-2 (pS2), thereby stimulating cell proliferation and differentiation [ 13 ]. Plant estrogens are natural compounds obtained from plants, abundant in flowers, stems, roots, and seeds rich in polyphenolic components, classified broadly into flavonoids, coumestans, lignans, stilbenes, etc. [ 14 ]. Representative isoflavonoids include genistein, daidzein, formononetin, and biochanin A found in soybeans. Flavones include chrysin, apigenin, naringenin, kaempferol, and quercetin, while coumestans contain coumestrol. Lignans are found in fiber-rich plants such as flaxseed, seed oils, and green tea, including secoisolariciresinol, matairesinol, and syringaresinol [ 15 , 16 ]. The structural characteristics of plant estrogens, containing phenolic rings and hydroxyl groups, are similar to estrogen, allowing them to bind to estrogen receptors (ER) α and β and exert estrogenic effects [ 17 ]. Oats (Avena sativa L.), a member of the Gramineae family, have been consumed in the form of oatmeal for a long time, although consumption is lower compared to rice and wheat. According to the dehulling process, oats are divided into hulled oats with husks attached and hull-less oats obtained after dehulling [ 18 ]. Known for being rich in protein and lipids, oats contain balanced essential amino acids and 2–6% β-glucan, a type of dietary fiber, making them highly valued in food science [ 19 , 20 ]. With increasing interest in oat-based functional foods and consumer health, various studies on oats have been conducted, leading to increased consumption [ 21 , 22 ]. As oat consumption increases, so does the production of oat hulls, which are mostly used as feed. Oat grains have hulls called lemma and palea. During processing, these hulls are removed, producing oat hulls as a byproduct [ 23 , 24 ]. In particular, the insoluble dietary fiber derived from the outer layer of oat hulls was studied for its effects on colonic function and serum lipids. The results indicated an increase in fecal weight without impacting transit time or serum lipids and demonstrated resistance to fermentation in the colon. Additionally, research on oat groats and hulls revealed that phenolic compounds such as caffeic acid and avenanthramides were predominantly present in groats, while other phenolic compounds were more concentrated in the hulls [ 25 , 26 ]. Previous research results have shown that oat hull extract has an effect on improving osteoporosis, one of the symptoms of menopause. Therefore, in this study, we investigated the potential of oat hull extracts as a menopausal relief agent by analyzing estrogen-like activity and menopause-related biomarkers in an ovariectomized animal model. Materials and methods Preparation of Oat Hull Water (OBW) Extract The oat hull used in this study was grounded in a laboratory test mill (Brabender Technologies, Germany). The flour (100 g) was extracted with water (1.2 L) for 24 h at room temperature and the supernatant was dried with a freeze dryer. Cell Culture MCF-7 cells, a breast cancer cell line used for in vitro cell analysis related to menopause, were purchased from the Korean Cell Line Bank. All cells were cultured at 37°C with less than 5% CO. MCF-7 cells were cultured in medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL) in Dulbecco's Modified Eagle's (DMEM). Cytotoxicity Analysis of OBW To investigate the effect of OBW on cell viability, MCF-7 cells were seeded at 1 × 10 4 cells/well in a 96-well plate. The next day, OBW was treated at 0, 1, 3, 10, 30, and 100 (µg/mL). Additionally, estradiol (100 µM) was treated as a positive control. Cell viability was determined according to the manufacturer's protocol of Cell Counting kit-8 (CCK-8; Tomado Molecular Technology, Kumamoto, Japan). Confirmation of mRNA expression of genes involved in menopausal improvement To evaluate the estrogenic activity of OBW, we performed a real time quantitative PCR (RT-qPCR) using estrogen receptor-dependent MCF-7 cells. MCF-7 cells were seeded in a 6-well plate at 3×10 5 cells/well. The next day, the samples were added to a DMEM medium containing 2.5% charcoal stripes and collected 48 hours later. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA), and cDNA was synthesized using the M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Korea). RT-qPCR was performed using TOPreal qPCR 2x PreMIX (BioRad, Hercules, CA, USA) in a real-time PCR detection system (Bio-Rad). The mRNA levels of the genes were determined using the 2 − ΔΔCT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard [ 27 ]. Confirmation of protein expression of genes related to menopausal improvement The protein expression of AKT and ERK, biomarkers related to estrogen signaling, was investigated. Proteins were extracted from OBW-treated cells using lysis buffer (10 µg/mL aprotinin, 1 mM DTT, 2 mM EDTA, 5 µg/mL leupeptin, 150 mM NaCl, 1% v/v Igepal CA-630, 1 mM sodium orthovanadate, 2 µg/mL pepstatin, 1 mM sodium fluoride and pH 7.5 20 mM Tris-HCl). 20 µg of the extracted protein were subjected to electrophoresis on 10% SDS-PAGE, and the proteins on the PAGE were transferred to a PVDF membrane (Amersham Biosciences, NJ, USA). The primary antibody was incubated on the membrane at 4°C for 16 hours, and then the secondary antibody coupled to HRP (horseradish peroxidase) was incubated at room temperature for 2 hours. After washing with TBS containing 1% Tween 20 and incubating with Super-Signal West Pico Chemiluminescent Substrate (Pierce Chemical, Rockford, IL, USA), the intensity of the protein bands was confirmed using MicroChemi 4.2 (DNR Bio-imaging System, Jerusalem, IL, USA). Ovariectomized Model As experimental animals, 4-week-old female ICR mice were purchased from GBIO (Seongnam, South Korea). Thereafter, the mice were allowed to adapt to the laboratory environment for 7 days before the start of the experiment and then used. All mice had easy access to food and water in an animal room where the temperature was 22 ± 3°C, and the day and night cycles were constantly maintained at 12 hours each. Animal experiments were conducted in accordance with the guidelines of the Suncheon University Animal Experiment Ethics Committee (SCNU IACUC-2023-9). Five groups of eight 5-week-old ICR female mice were designated: a control group (Sham), OVX (Ovariectomized), OVX + positive control substance (Estradiol, 0.5 mg/kg BW), OVX + low concentration oat extract (100 mg/kg BW), and OVX + high concentration oat extract (400 mg/kg BW). Mice were dorsally opened, and the ovaries were removed in the OVX, OVX + estradiol, OVX + low-concentration extract, and OVX + high-concentration extract groups; the sham group was dorsally opened without removing the ovaries. The samples were prepared and incorporated into the diet at concentrations of 100 and 400 mg per kg of body weight. The samples were prepared and fed into the diet at concentrations of 100 and 400 mg per kg of body weight, and the control group was fed a normal diet without the sample for 7 weeks. Body weight was measured once each week at the same time, and food intake and body temperature changes were measured once each day at the same time. After 7 weeks, sacrifice was performed. The weights of the animal's body, uterus, uterine fat, abdominal fat, subcutaneous fat, kidney, and liver were measured. Blood was centrifuged at 5000 RPM for 5 minutes to collect serum, which was then immediately stored at -80°C. The femur was preserved in 10% formalin and 70% alcohol. Measurement of Bone Loss in OVX Mice To analyze the trabecular bone within the femur, it was scanned and assessed using a high-resolution micro-computed tomography (CT) scanner (SkyScan 1173, Skyscan NV, Kontich, Belgium) and SkyScan DataViewer software (Skyscan NV, Kontich, Belgium). The total volume of the area within the cortical bone was measured, and the trabecular bone volume was assessed within the total volume. The percentage of bone volume was calculated by dividing the trabecular bone volume by the total volume. Several cortical bone parameters, such as bone mineral density (BMD) were measured. Serum analysis Mouse serum was used to analyze blood lipid levels, alkaline phosphatase, calcium, and osteocalcin, which are associated with cardiovascular health. Blood lipids were analyzed using triglyceride (Asan pharm, Korea), total cholesterol (Asan pharm, Korea), and HDL-cholesterol (Asan pharm, Korea) kits. Alkaline phosphatase and Calcium were analyzed using a dry-type clinical chemistry automatic analyzer (Fuji Drichem 4000i). Osteocalcin was analyzed using a mouse osteoprotegerin (OPG) (CUSABIO, Wuhan, Hubei, China) ELISA kit. Statistical analysis The results of this study were repeated three times, and the statistical differences were analyzed using Student's t-tests. It was considered significant when the probability value ( p -value) was 0.05 or less ( p -value: *<0.05, **<0.01, ***<0.001). Results and Discussion Confirmation of Cytotoxicity of OBW Oat hulls are rich in functional components and phytochemicals, notably phenolic compounds such as ferulic acid, chlorogenic acid, p-hydroxybenzoic acid, rutin, apigenin, vanillin, and luteolin. These compounds make oat hull a significant resource for natural antioxidants [ 28 ]. To confirm OBW cytotoxicity, a CCK-8 experiment was performed 48 hours after treating MCF-7 cells, a breast cancer cell line, with OBW (1,3,10,30,100 µg/mL) or E2 (100 µM). As a result of the experiment, no cytotoxicity of OBW (1,3,10,30,100 µg/mL) or E2 (100 µM) was observed (Fig. 1 A). Plants defend against infection by accumulating phytoalexins, toxic secondary metabolites that help reject pathogens. These compounds can build up in plant tissues under certain conditions but may decrease due to plant-catalyzed transformations. While phytoalexin biosynthesis is well-studied, their metabolic fate is less understood. Turnover of phytoalexins is believed to regulate their levels in fresh plant tissues like pepper and soybean [ 29 ]. Effect of OBW on mRNA Expression of Genes related to Female Menopause To analyze the effect of OBW-treated cells on the menopausal mechanism, we analyzed the expression of menopause-related genes, including ERα, ERβ, pS2, and ESR1, at the transcriptional level. Following treatment with OBW (0, 1, 3, 10, 30, 100 µg/mL) and additional treatment with estradiol (100 µM) as a positive control, the cells were cultured for 48 hours. Subsequently, cells were collected, mRNA was extracted, cDNA was synthesized, and real-time qPCR was performed. As a result, with the increasing concentration of OBW, the expression of ERα and ESR1 decreased, while the expression of ERβ and pS2 increased, indicating the suppression of menopause-related genes (Fig. 1 B). In addition, as osteoporosis is induced by an inflammatory response among various factors during menopause, the expression of genes related to the inflammatory response, IL-6 and TNF-α, was analyzed at the transcriptional level. As a result, the expression of IL-6 decreased as the concentration of OBW increased, but TNF-α showed no significant changes (Fig. 1 C). Effect of OBW on Protein Expression of Genes related to Female Menopause We investigated the signal transduction that occurs when OBW is treated with E2, a positive control substance, using AKT and ERK, which are essential indicators of estrogen signaling. The cells were divided into an experimental group that was not treated with OBW and E2, an experimental group that was treated with OBW (100 µg/mL), and an experimental group that was treated with E2 (100 µM). They were then collected and analyzed at 0, 5, 15, and 30 minutes. As a result, an increase was observed when processing E2 and OBW compared to the control. That is, estrogen activity increases when OBW is processed (Fig. 2 ). Osborne MCF-7 cells, a breast cancer cell line, are widely used in studies of tumor biology and hormone action mechanisms, especially as a model for studying estrogen responses [ 30 ]. There are estrogen receptors in MCF-7 cells, which belong to a genus of vertebrates. There are two main types: ERα and ERβ. The expression of pS2 and ESR1 is regulated by the signaling proteins AKT and ERK via the estrogen receptor. Therefore, in this study, after treating MCF-7 cells with E2 a positive control and OBW the improvement effect on female menopause was confirmed through menopause-related biomarkers and inflammation-related biomarkers. Food Intake, Body Weight, and Body Temperature Changes in OVX Model Mice due to OBW To examine the suppressive effect of OBW on weight gain in mice induced to undergo menopause by OVX, daily food intake and weekly body weight were measured for 7 weeks, and the rate of change was calculated. Then, the change in body temperature was measured to assess the effect on body temperature during menopause induction. Looking at the results of measuring body temperature in each group, changes in body temperature were observed in the OBW400 group (Fig. 3 C), and there was no significant difference in dietary intake between each group. However, body weight increased significantly in the OVX group compared to the Sham group from the 5th week after dietary intake, and an inhibitory effect on weight gain was observed in the E2, OBW100, and OBW400 groups compared to the OVX group. Therefore, consuming OBW has an effect of suppressing weight gain (Fig. 3 A). Uterus, liver weight, and eWAT, scWAT, prWAT, and mWAT were measured. We confirmed that the uterus, which had been reduced by OVX, significantly recovered in mice fed the OBW400 diet, and we observed a tendency for OBW to suppress adipose tissues such as eWAT and scWAT (Fig. 3 B). The ovariectomized mouse model is a typical experimental model for studying menopausal osteoporosis resulting from estrogen deficiency in women. In this study, when mice whose menopause was induced with OVX were treated with E2 a positive control and OBW, for 7 weeks, changes in body weight, food intake, tissue weight, cardiovascular index, bone metabolic index, and femur were confirmed. There are reports that decreased estrogen secretion due to oophorectomy causes weight gain, and like this study, OVX mice had higher body fat and body weight than Sham [ 31 ] (Bracht et al, 2020). Both OBW and E2 equally suppressed weight gain and fat accumulation. Analysis of Changes in Cardiovascular Indicators by OBW Mouse serum was used to measure blood lipid levels related to cardiovascular health. After separating serum from each group, total cholesterol, HDL-cholesterol, and triglyceride content were measured. The levels of total cholesterol, HDL-cholesterol, and triglycerides increased in the OVX group but decreased in the OBW group (Fig. 4 A). As estrogen deficiency is implicated in the development of cardiovascular disease (CVD), early and active prevention and response for the management of cardiovascular risk factors are important [ 32 ] (Mendelsohn et al, 2005). In this study, we identified triglyceride (TG), total cholesterol (TC), and high-density lipoprotein (HDL) cholesterol levels associated with cardiovascular disease. As a result, OBW suppressed these levels compared to OVX. Because estrogen plays a vital role in skeletal homeostasis, menopause causes osteoporosis [ 33 ]. Analysis of Changes in Bone Metabolism Indicators by OBW Osteoblasts produce alkaline phosphatase, an enzyme involved in bone mineralization. Osteoblasts also absorb acids produced by mineral deposition by depositing calcium through mechanisms involving phosphate and calcium transport along with alkalization. Because osteocalcin has a high affinity for calcium, calcium deficiency may reduce the formation of hydroxyapatite crystals in osteoporotic women. Therefore, in conditions of low inorganic nitridation, osteocalcin can be advantageously used in the circulatory system. The results of measuring the amount of alkaline phosphatase present in mouse serum show a tendency to increase at a high OBW concentration (OBW400), but no significant changes are observed in calcium. Additionally, the amount of osteocalcin was decreased in the high OBW concentration (OBW400) group compared to the OVX group, in which osteoporosis and female menopause were induced by oophorectomy surgery. In other words, the decrease in the amount of osteocalcin, a marker of bone metabolism in female menopause, suggests that OBW is effective in improving female menopause (Fig. 4 B). Menopausal Improvement Effect of OBW in Ovariectomized Mouse Model (OVX) Micro-CT analysis showed that trabecular bone damage in the femur caused by ovarian removal surgery was recovered in the femur of mice injected with estradiol (E2), which is a positive control group, and mice fed low and high concentrations of OBW. Additionally, when measuring bone density in each group, bone density, which was reduced to 17% based on Sham in OVX, improved to 63.8%, 54.0%, and 90.6% in the E2, ObW100, and ObW400 groups, respectively. Therefore, OBW suppresses the induction of osteoporosis caused by ovarian removal in a concentration-dependent manner (Fig. 5 ). Estrogen intake has been reported to increase bone mineral content and collagen fiber composition and is effective in preventing bone fractures. Estrogen is also known to affect bone resorption. Therefore, in this study, we confirmed osteocalcin, blood ALP, and Ca, which are indicators of osteoblast activity. As a result, it was confirmed that at high concentrations, ALP tended to increase in OBW, and osteocalcin decreased in OBW compared to OVX. Additionally, micro-CT analysis confirmed that OBW improved bone density compared to OVX. Conclusions In conclusion, the effect of OBW on improving female menopausal symptoms was confirmed both in vitro using menopause-related biomarkers and in vivo using a menopausal animal model induced through oophorectomy. Therefore, oat husk water extract is expected to be useful as a new alternative in treating female menopause. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Yongjin Lee, Holim Jin, and Yoon-A Shin prepared figures 1-5. Han Gyeol Lee, Seung Yeob Song, and Woo Duck Seo prepared oat hull extract, which was the experimental material. 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Ann N Y Acad Sci 1461(1):127–143. https://doi.org/10.1111/nyas.14281 Mendelsohn ME, Karas RH (2005) Molecular and cellular basis of cardiovascular gender differences. Science 308(5728):1583–1587. https://doi.org/10.1126/science.1112062 Clark AP, Schuttinga JA (1992) Targeted estrogen/progesterone replacement therapy for osteoporosis: calculation of health care cost savings. Osteoporos Int 2(4):195–200. https://doi.org/10.1007/BF01623926 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5382261","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374670192,"identity":"80dc54ec-93d3-4afb-a987-429d0cad3da8","order_by":0,"name":"Mi-Ja Lee","email":"","orcid":"","institution":"National Institute of Crop Science, Rural Development Administration (RDA)","correspondingAuthor":false,"prefix":"","firstName":"Mi-Ja","middleName":"","lastName":"Lee","suffix":""},{"id":374670194,"identity":"741f231a-3fb0-43c3-b508-d86caa5e06d1","order_by":1,"name":"Yongjin Lee","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Yongjin","middleName":"","lastName":"Lee","suffix":""},{"id":374670195,"identity":"07d1e66a-98ab-4378-8298-04217c0eb99c","order_by":2,"name":"Han Gyeol Lee","email":"","orcid":"","institution":"National Institute of Crop Science, Rural Development Administration (RDA)","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"Gyeol","lastName":"Lee","suffix":""},{"id":374670196,"identity":"3ae4eb78-e1a6-4ce7-a5ba-4e621a41a17f","order_by":3,"name":"Holim Jin","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"prefix":"","firstName":"Holim","middleName":"","lastName":"Jin","suffix":""},{"id":374670197,"identity":"cc2c7e09-92e1-4bec-907e-414560cab657","order_by":4,"name":"Yoon-A Shin","email":"","orcid":"","institution":"Sunchon National University","correspondingAuthor":false,"prefix":"","firstName":"Yoon-A","middleName":"","lastName":"Shin","suffix":""},{"id":374670198,"identity":"50b2b2be-df48-4891-a32d-9438389bc117","order_by":5,"name":"Seung Yeob Song","email":"","orcid":"","institution":"National Institute of Crop Science, Rural Development Administration (RDA)","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Yeob","lastName":"Song","suffix":""},{"id":374670199,"identity":"39ef9abf-2ec4-4d7b-9798-4bd4b1fe014c","order_by":6,"name":"Woo Duck Seo","email":"","orcid":"","institution":"National Institute of Crop Science, Rural Development Administration (RDA)","correspondingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Duck","lastName":"Seo","suffix":""},{"id":374670200,"identity":"5c7afd0a-e080-44b6-8157-329fb5ca9a46","order_by":7,"name":"Young-Jin Son","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYLCCDyBCAsqRwKMQDhhnkKyFmYckLfLtvcekbf4czuOf3XzsAUONHYPk7AP4tRicOZcmndt2uFjizrF0A4ZjyQzSfAkEtEjkmEnnNhxO3ABkSDCwHWCQ4yHksBlALRZ/YFr+EaGF4QZQCwMbVAtj2wEGaUJaDM6cMbbsbUtPnHEjLU0isS+ZR7KHkMPaewxv/Phjndg/I/mYxIdvdnISZwg5jIGBBRETCQwMBH0CAswfiFE1CkbBKBgFIxgAAP+WOsUtDAYUAAAAAElFTkSuQmCC","orcid":"","institution":"Sunchon National University","correspondingAuthor":true,"prefix":"","firstName":"Young-Jin","middleName":"","lastName":"Son","suffix":""}],"badges":[],"createdAt":"2024-11-03 13:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5382261/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5382261/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69000369,"identity":"dae79d63-bf03-42e0-8a8e-54c242644d84","added_by":"auto","created_at":"2024-11-14 11:28:57","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":435714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of OBW on mRNA expression of genes related to female menopause. \u003c/strong\u003e(A) CCK-8 assay assessing the cytotoxicity of the extracts on MCF-7 cells. (B) OBW extract inhibited the female menopause-induced mRNA expression of ERα, ERβ, ESR1, and pS2 in MCF-7 cells, as determined by RT-PCR. (C) OBW extract inhibited the inflammatory marker genes expression of IL-6 and TNF-α in MCF-7 cells, as determined by RT-PCR. Representative images are displayed. Data are presented as means ± SEM of three independent replicates. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the non-treated OVX group by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/dbcabf8e13dd3b190a39b88f.jpeg"},{"id":68999155,"identity":"f9efc269-e719-4e45-87cd-8ccfd71484d8","added_by":"auto","created_at":"2024-11-14 11:20:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":415777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of female menopause-specific protein expression by OBW extract.\u003c/strong\u003e Anticipated mechanism of OBW extract’s action in female menopause. The cells were divided into an experimental group that was not treated with OBW and E2, an experimental group that was treated with OBW (100 μg/mL), and an experimental group that was treated with E2 (100 μM). They were then collected and analyzed at 0, 5, 15, and 30 minutes.Representative images are displayed. Data are presented as means ± SEM of three independent replicates.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/ed3760fc02eb5340b9306b3d.jpeg"},{"id":68999024,"identity":"8e8bd199-c3e8-47ea-bc8d-47402536cf9a","added_by":"auto","created_at":"2024-11-14 11:12:57","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":484925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological changes in OVX mice treated with OBW extract. \u003c/strong\u003e(A) Food intake, body weight, and percentage of boy weight gain, (B) Uterus, eWAT, scWAT, prWAT, mWAT, and liver tissues. (C) Body temperature. Data are presented as means SEM (n = 6). \u003csup\u003e# \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e## \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e### \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the sham group; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,\u003csup\u003e ***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the non-treated OVX group by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/c7f7f8b28a4251dba1a8b88f.jpeg"},{"id":68999027,"identity":"4f93d5ea-4d4b-4e05-9eae-b346c9b5d2db","added_by":"auto","created_at":"2024-11-14 11:12:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of OBW extract on cardiovascular indicators and biomarker expression levels in OVX mice. \u003c/strong\u003e(A) Serum levels of cardiovascular maker were detected using kits. (B) Serum levels of ALP, Ca, Osteocalcin using ELISA kits.\u003cstrong\u003e \u003c/strong\u003eData are presented as means SEM (n = 6). \u003csup\u003e# \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e### \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the sham group; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 compared to the non-treated OVX group by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/bb8ecae47a1ab72d16f944b8.jpeg"},{"id":68999157,"identity":"b027cf36-7ab8-48c9-a246-986955b7a8a3","added_by":"auto","created_at":"2024-11-14 11:20:57","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":291737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicro-CT analysis of bone structure changes in OVX and OBW-treated OVX mice. \u003c/strong\u003e(A) Representative Micro-CT images. Quantification of microstructural parameters including (B) BMD. Scale var, 1 mm. Data are presented as means SEM (n = 6). \u003csup\u003e### \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the sham group; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the non-treated OVX group by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/26c940cd58dab593f71c1aa8.jpeg"},{"id":73413275,"identity":"7c780de7-40a2-4f61-b1f0-f9d77f365afe","added_by":"auto","created_at":"2025-01-09 16:32:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2858763,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5382261/v1/4c90419a-416f-44cd-a09c-4ab6af38fccc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improvement of Menopausal Symptoms by Oat Hull Extracts in Ovariectomized Animal Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMenopause is defined by the World Health Organization (WHO) as the absence of menstrual periods for 12 consecutive months without any physiological or pathological cause, indicating that natural menopause has occurred. Menopause most often occurs between ages 45 and 55, and some women experience early menopause before age 40 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As of the \"World Population Prospects 2022\" report released by the United Nations (UN), the global average life expectancy in 2021 was 71 years [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With increasing life expectancy, a significant number of women are expected to spend more than a third of their lives in a state of menopause. Moreover, as the global population ages, the proportion of women experiencing perimenopause is also on the rise [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, interest in menopause is increasing all over the world.\u003c/p\u003e \u003cp\u003eMenopause in women refers to the gradual loss of ovarian function occurring before and after menopause, defined as the cessation of menstruation for one year after the final menstrual period [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. With the advancement of medicine and improvement in living conditions, life expectancy has continued to increase, and most women now spend more than one-third of their lives in menopausal status [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Menopause, a natural phenomenon occurring due to aging, is characterized by sudden hormonal changes, which can disrupt daily life and diminish quality of life. Menopausal and postmenopausal women experience various menopausal symptoms due to decreased estrogen levels, including osteoporosis, hot flashes, dyslipidemia, and other conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To alleviate these symptoms, hormone therapy has traditionally been used; however, concerns about side effects such as endometrial cancer, cardiovascular disease, and breast cancer due to excessive estrogen have led to research on plant-derived estrogen-like substances or natural menopause relief agents [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, side effects of its use as a therapeutic agent include breast pain, fluid retention, nausea, leg cramps, and headaches Fait [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Estrogen plays a crucial role in the function and growth of the human reproductive system. While primarily secreted by the ovaries, estrogen is also produced by other tissues such as adipose tissue, adrenal glands, pancreas, and the brain. Additionally, E2 (17β-estradiol), the primary estrogen in humans, can be converted to E1 (estrone), and both E1 and E2 can be converted to E3 (estriol) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. When estrogen binds to the estrogen receptor (ER), the receptor dimerizes, subsequently binding to the estrogen response element (ERE). This forms the ER-ERE complex, which regulates estrogen-regulated target genes such as the progesterone receptor (PR) and prezenelin-2 (pS2), thereby stimulating cell proliferation and differentiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlant estrogens are natural compounds obtained from plants, abundant in flowers, stems, roots, and seeds rich in polyphenolic components, classified broadly into flavonoids, coumestans, lignans, stilbenes, etc. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Representative isoflavonoids include genistein, daidzein, formononetin, and biochanin A found in soybeans. Flavones include chrysin, apigenin, naringenin, kaempferol, and quercetin, while coumestans contain coumestrol. Lignans are found in fiber-rich plants such as flaxseed, seed oils, and green tea, including secoisolariciresinol, matairesinol, and syringaresinol [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The structural characteristics of plant estrogens, containing phenolic rings and hydroxyl groups, are similar to estrogen, allowing them to bind to estrogen receptors (ER) α and β and exert estrogenic effects [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Oats (Avena sativa L.), a member of the Gramineae family, have been consumed in the form of oatmeal for a long time, although consumption is lower compared to rice and wheat. According to the dehulling process, oats are divided into hulled oats with husks attached and hull-less oats obtained after dehulling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Known for being rich in protein and lipids, oats contain balanced essential amino acids and 2\u0026ndash;6% β-glucan, a type of dietary fiber, making them highly valued in food science [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. With increasing interest in oat-based functional foods and consumer health, various studies on oats have been conducted, leading to increased consumption [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. As oat consumption increases, so does the production of oat hulls, which are mostly used as feed. Oat grains have hulls called lemma and palea. During processing, these hulls are removed, producing oat hulls as a byproduct [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In particular, the insoluble dietary fiber derived from the outer layer of oat hulls was studied for its effects on colonic function and serum lipids. The results indicated an increase in fecal weight without impacting transit time or serum lipids and demonstrated resistance to fermentation in the colon. Additionally, research on oat groats and hulls revealed that phenolic compounds such as caffeic acid and avenanthramides were predominantly present in groats, while other phenolic compounds were more concentrated in the hulls [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious research results have shown that oat hull extract has an effect on improving osteoporosis, one of the symptoms of menopause. Therefore, in this study, we investigated the potential of oat hull extracts as a menopausal relief agent by analyzing estrogen-like activity and menopause-related biomarkers in an ovariectomized animal model.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Oat Hull Water (OBW) Extract\u003c/h2\u003e \u003cp\u003eThe oat hull used in this study was grounded in a laboratory test mill (Brabender Technologies, Germany). The flour (100 g) was extracted with water (1.2 L) for 24 h at room temperature and the supernatant was dried with a freeze dryer.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eMCF-7 cells, a breast cancer cell line used for in vitro cell analysis related to menopause, were purchased from the Korean Cell Line Bank. All cells were cultured at 37\u0026deg;C with less than 5% CO. MCF-7 cells were cultured in medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 \u0026micro;g/mL) in Dulbecco's Modified Eagle's (DMEM).\u003c/p\u003e\n\u003ch3\u003eCytotoxicity Analysis of OBW\u003c/h3\u003e\n\u003cp\u003eTo investigate the effect of OBW on cell viability, MCF-7 cells were seeded at 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate. The next day, OBW was treated at 0, 1, 3, 10, 30, and 100 (\u0026micro;g/mL). Additionally, estradiol (100 \u0026micro;M) was treated as a positive control. Cell viability was determined according to the manufacturer's protocol of Cell Counting kit-8 (CCK-8; Tomado Molecular Technology, Kumamoto, Japan).\u003c/p\u003e\n\u003ch3\u003eConfirmation of mRNA expression of genes involved in menopausal improvement\u003c/h3\u003e\n\u003cp\u003eTo evaluate the estrogenic activity of OBW, we performed a real time quantitative PCR (RT-qPCR) using estrogen receptor-dependent MCF-7 cells. MCF-7 cells were seeded in a 6-well plate at 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. The next day, the samples were added to a DMEM medium containing 2.5% charcoal stripes and collected 48 hours later. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA), and cDNA was synthesized using the M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Korea). RT-qPCR was performed using TOPreal qPCR 2x PreMIX (BioRad, Hercules, CA, USA) in a real-time PCR detection system (Bio-Rad). The mRNA levels of the genes were determined using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eConfirmation of protein expression of genes related to menopausal improvement\u003c/h3\u003e\n\u003cp\u003eThe protein expression of AKT and ERK, biomarkers related to estrogen signaling, was investigated. Proteins were extracted from OBW-treated cells using lysis buffer (10 \u0026micro;g/mL aprotinin, 1 mM DTT, 2 mM EDTA, 5 \u0026micro;g/mL leupeptin, 150 mM NaCl, 1% v/v Igepal CA-630, 1 mM sodium orthovanadate, 2 \u0026micro;g/mL pepstatin, 1 mM sodium fluoride and pH 7.5 20 mM Tris-HCl). 20 \u0026micro;g of the extracted protein were subjected to electrophoresis on 10% SDS-PAGE, and the proteins on the PAGE were transferred to a PVDF membrane (Amersham Biosciences, NJ, USA). The primary antibody was incubated on the membrane at 4\u0026deg;C for 16 hours, and then the secondary antibody coupled to HRP (horseradish peroxidase) was incubated at room temperature for 2 hours. After washing with TBS containing 1% Tween 20 and incubating with Super-Signal West Pico Chemiluminescent Substrate (Pierce Chemical, Rockford, IL, USA), the intensity of the protein bands was confirmed using MicroChemi 4.2 (DNR Bio-imaging System, Jerusalem, IL, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOvariectomized Model\u003c/h2\u003e \u003cp\u003eAs experimental animals, 4-week-old female ICR mice were purchased from GBIO (Seongnam, South Korea). Thereafter, the mice were allowed to adapt to the laboratory environment for 7 days before the start of the experiment and then used. All mice had easy access to food and water in an animal room where the temperature was 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, and the day and night cycles were constantly maintained at 12 hours each. Animal experiments were conducted in accordance with the guidelines of the Suncheon University Animal Experiment Ethics Committee (SCNU IACUC-2023-9). Five groups of eight 5-week-old ICR female mice were designated: a control group (Sham), OVX (Ovariectomized), OVX\u0026thinsp;+\u0026thinsp;positive control substance (Estradiol, 0.5 mg/kg BW), OVX\u0026thinsp;+\u0026thinsp;low concentration oat extract (100 mg/kg BW), and OVX\u0026thinsp;+\u0026thinsp;high concentration oat extract (400 mg/kg BW). Mice were dorsally opened, and the ovaries were removed in the OVX, OVX\u0026thinsp;+\u0026thinsp;estradiol, OVX\u0026thinsp;+\u0026thinsp;low-concentration extract, and OVX\u0026thinsp;+\u0026thinsp;high-concentration extract groups; the sham group was dorsally opened without removing the ovaries. The samples were prepared and incorporated into the diet at concentrations of 100 and 400 mg per kg of body weight. The samples were prepared and fed into the diet at concentrations of 100 and 400 mg per kg of body weight, and the control group was fed a normal diet without the sample for 7 weeks. Body weight was measured once each week at the same time, and food intake and body temperature changes were measured once each day at the same time. After 7 weeks, sacrifice was performed. The weights of the animal's body, uterus, uterine fat, abdominal fat, subcutaneous fat, kidney, and liver were measured. Blood was centrifuged at 5000 RPM for 5 minutes to collect serum, which was then immediately stored at -80\u0026deg;C. The femur was preserved in 10% formalin and 70% alcohol.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of Bone Loss in OVX Mice\u003c/h3\u003e\n\u003cp\u003eTo analyze the trabecular bone within the femur, it was scanned and assessed using a high-resolution micro-computed tomography (CT) scanner (SkyScan 1173, Skyscan NV, Kontich, Belgium) and SkyScan DataViewer software (Skyscan NV, Kontich, Belgium). The total volume of the area within the cortical bone was measured, and the trabecular bone volume was assessed within the total volume. The percentage of bone volume was calculated by dividing the trabecular bone volume by the total volume. Several cortical bone parameters, such as bone mineral density (BMD) were measured.\u003c/p\u003e\n\u003ch3\u003eSerum analysis\u003c/h3\u003e\n\u003cp\u003eMouse serum was used to analyze blood lipid levels, alkaline phosphatase, calcium, and osteocalcin, which are associated with cardiovascular health. Blood lipids were analyzed using triglyceride (Asan pharm, Korea), total cholesterol (Asan pharm, Korea), and HDL-cholesterol (Asan pharm, Korea) kits. Alkaline phosphatase and Calcium were analyzed using a dry-type clinical chemistry automatic analyzer (Fuji Drichem 4000i). Osteocalcin was analyzed using a mouse osteoprotegerin (OPG) (CUSABIO, Wuhan, Hubei, China) ELISA kit.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results of this study were repeated three times, and the statistical differences were analyzed using Student's t-tests. It was considered significant when the probability value (\u003cem\u003ep\u003c/em\u003e-value) was 0.05 or less (\u003cem\u003ep\u003c/em\u003e-value: *\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of Cytotoxicity of OBW\u003c/h2\u003e \u003cp\u003eOat hulls are rich in functional components and phytochemicals, notably phenolic compounds such as ferulic acid, chlorogenic acid, p-hydroxybenzoic acid, rutin, apigenin, vanillin, and luteolin. These compounds make oat hull a significant resource for natural antioxidants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo confirm OBW cytotoxicity, a CCK-8 experiment was performed 48 hours after treating MCF-7 cells, a breast cancer cell line, with OBW (1,3,10,30,100 \u0026micro;g/mL) or E2 (100 \u0026micro;M). As a result of the experiment, no cytotoxicity of OBW (1,3,10,30,100 \u0026micro;g/mL) or E2 (100 \u0026micro;M) was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlants defend against infection by accumulating phytoalexins, toxic secondary metabolites that help reject pathogens. These compounds can build up in plant tissues under certain conditions but may decrease due to plant-catalyzed transformations. While phytoalexin biosynthesis is well-studied, their metabolic fate is less understood. Turnover of phytoalexins is believed to regulate their levels in fresh plant tissues like pepper and soybean [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of OBW on mRNA Expression of Genes related to Female Menopause\u003c/h2\u003e \u003cp\u003eTo analyze the effect of OBW-treated cells on the menopausal mechanism, we analyzed the expression of menopause-related genes, including ERα, ERβ, pS2, and ESR1, at the transcriptional level. Following treatment with OBW (0, 1, 3, 10, 30, 100 \u0026micro;g/mL) and additional treatment with estradiol (100 \u0026micro;M) as a positive control, the cells were cultured for 48 hours. Subsequently, cells were collected, mRNA was extracted, cDNA was synthesized, and real-time qPCR was performed. As a result, with the increasing concentration of OBW, the expression of ERα and ESR1 decreased, while the expression of ERβ and pS2 increased, indicating the suppression of menopause-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn addition, as osteoporosis is induced by an inflammatory response among various factors during menopause, the expression of genes related to the inflammatory response, IL-6 and TNF-α, was analyzed at the transcriptional level. As a result, the expression of IL-6 decreased as the concentration of OBW increased, but TNF-α showed no significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of OBW on Protein Expression of Genes related to Female Menopause\u003c/h2\u003e \u003cp\u003eWe investigated the signal transduction that occurs when OBW is treated with E2, a positive control substance, using AKT and ERK, which are essential indicators of estrogen signaling. The cells were divided into an experimental group that was not treated with OBW and E2, an experimental group that was treated with OBW (100 \u0026micro;g/mL), and an experimental group that was treated with E2 (100 \u0026micro;M). They were then collected and analyzed at 0, 5, 15, and 30 minutes. As a result, an increase was observed when processing E2 and OBW compared to the control. That is, estrogen activity increases when OBW is processed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOsborne MCF-7 cells, a breast cancer cell line, are widely used in studies of tumor biology and hormone action mechanisms, especially as a model for studying estrogen responses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. There are estrogen receptors in MCF-7 cells, which belong to a genus of vertebrates. There are two main types: ERα and ERβ. The expression of pS2 and ESR1 is regulated by the signaling proteins AKT and ERK via the estrogen receptor. Therefore, in this study, after treating MCF-7 cells with E2 a positive control and OBW the improvement effect on female menopause was confirmed through menopause-related biomarkers and inflammation-related biomarkers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFood Intake, Body Weight, and Body Temperature Changes in OVX Model Mice due to OBW\u003c/h2\u003e \u003cp\u003eTo examine the suppressive effect of OBW on weight gain in mice induced to undergo menopause by OVX, daily food intake and weekly body weight were measured for 7 weeks, and the rate of change was calculated. Then, the change in body temperature was measured to assess the effect on body temperature during menopause induction. Looking at the results of measuring body temperature in each group, changes in body temperature were observed in the OBW400 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and there was no significant difference in dietary intake between each group. However, body weight increased significantly in the OVX group compared to the Sham group from the 5th week after dietary intake, and an inhibitory effect on weight gain was observed in the E2, OBW100, and OBW400 groups compared to the OVX group. Therefore, consuming OBW has an effect of suppressing weight gain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUterus, liver weight, and eWAT, scWAT, prWAT, and mWAT were measured. We confirmed that the uterus, which had been reduced by OVX, significantly recovered in mice fed the OBW400 diet, and we observed a tendency for OBW to suppress adipose tissues such as eWAT and scWAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe ovariectomized mouse model is a typical experimental model for studying menopausal osteoporosis resulting from estrogen deficiency in women. In this study, when mice whose menopause was induced with OVX were treated with E2 a positive control and OBW, for 7 weeks, changes in body weight, food intake, tissue weight, cardiovascular index, bone metabolic index, and femur were confirmed. There are reports that decreased estrogen secretion due to oophorectomy causes weight gain, and like this study, OVX mice had higher body fat and body weight than Sham [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] (Bracht et al, 2020). Both OBW and E2 equally suppressed weight gain and fat accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Changes in Cardiovascular Indicators by OBW\u003c/h2\u003e \u003cp\u003eMouse serum was used to measure blood lipid levels related to cardiovascular health. After separating serum from each group, total cholesterol, HDL-cholesterol, and triglyceride content were measured. The levels of total cholesterol, HDL-cholesterol, and triglycerides increased in the OVX group but decreased in the OBW group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs estrogen deficiency is implicated in the development of cardiovascular disease (CVD), early and active prevention and response for the management of cardiovascular risk factors are important [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] (Mendelsohn et al, 2005). In this study, we identified triglyceride (TG), total cholesterol (TC), and high-density lipoprotein (HDL) cholesterol levels associated with cardiovascular disease. As a result, OBW suppressed these levels compared to OVX. Because estrogen plays a vital role in skeletal homeostasis, menopause causes osteoporosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of Changes in Bone Metabolism Indicators by OBW\u003c/h2\u003e \u003cp\u003eOsteoblasts produce alkaline phosphatase, an enzyme involved in bone mineralization. Osteoblasts also absorb acids produced by mineral deposition by depositing calcium through mechanisms involving phosphate and calcium transport along with alkalization. Because osteocalcin has a high affinity for calcium, calcium deficiency may reduce the formation of hydroxyapatite crystals in osteoporotic women. Therefore, in conditions of low inorganic nitridation, osteocalcin can be advantageously used in the circulatory system.\u003c/p\u003e \u003cp\u003eThe results of measuring the amount of alkaline phosphatase present in mouse serum show a tendency to increase at a high OBW concentration (OBW400), but no significant changes are observed in calcium. Additionally, the amount of osteocalcin was decreased in the high OBW concentration (OBW400) group compared to the OVX group, in which osteoporosis and female menopause were induced by oophorectomy surgery. In other words, the decrease in the amount of osteocalcin, a marker of bone metabolism in female menopause, suggests that OBW is effective in improving female menopause (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMenopausal Improvement Effect of OBW in Ovariectomized Mouse Model (OVX)\u003c/h2\u003e \u003cp\u003eMicro-CT analysis showed that trabecular bone damage in the femur caused by ovarian removal surgery was recovered in the femur of mice injected with estradiol (E2), which is a positive control group, and mice fed low and high concentrations of OBW. Additionally, when measuring bone density in each group, bone density, which was reduced to 17% based on Sham in OVX, improved to 63.8%, 54.0%, and 90.6% in the E2, ObW100, and ObW400 groups, respectively. Therefore, OBW suppresses the induction of osteoporosis caused by ovarian removal in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEstrogen intake has been reported to increase bone mineral content and collagen fiber composition and is effective in preventing bone fractures. Estrogen is also known to affect bone resorption. Therefore, in this study, we confirmed osteocalcin, blood ALP, and Ca, which are indicators of osteoblast activity. As a result, it was confirmed that at high concentrations, ALP tended to increase in OBW, and osteocalcin decreased in OBW compared to OVX. Additionally, micro-CT analysis confirmed that OBW improved bone density compared to OVX.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the effect of OBW on improving female menopausal symptoms was confirmed both in vitro using menopause-related biomarkers and in vivo using a menopausal animal model induced through oophorectomy. Therefore, oat husk water extract is expected to be useful as a new alternative in treating female menopause.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYongjin Lee, Holim Jin, and Yoon-A Shin prepared figures 1-5. Han Gyeol Lee, Seung Yeob Song, and Woo Duck Seo prepared oat hull extract, which was the experimental material. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eWe are grateful to the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ013524012022). The funder had no role in the experiment, the data analysis, the decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSochocka M, Karska J, Pszczołowska M (2023) Cognitive Decline in Early and Premature Menopause. 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Osteoporos Int 2(4):195\u0026ndash;200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF01623926\u003c/span\u003e\u003cspan address=\"10.1007/BF01623926\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Oat hull, menopausal symptoms, ovariectomized (OVX), bone density","lastPublishedDoi":"10.21203/rs.3.rs-5382261/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5382261/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFemale menopause is a disease characterized by the gradual loss of ovarian function that occurs before and after menopause. Female menopausal symptoms include osteoporosis, facial flushing, and dyslipidemia. Oats are known to be rich in protein and lipids, and their hulls are used as feed. In this study, we sought to explore the potential of oat hull extract as a treatment for menopause by analyzing its estrogen-like activity and menopause-related biomarkers in ovariectomized (OVX) animals. We investigated changes in mRNA expression and estrogen activity of several menopause-related genes induced by oat hull extract in vitro using MCF-7 breast cancer cells. Additionally, we used an ovariectomized mouse model to investigate the effects of oat hull extract on menopause in vivo through changes in body weight, tissue morphology, and bone mineral density. Therefore, this study suggests that oat hull extract may be a promising alternative for treating female menopause.\u003c/p\u003e","manuscriptTitle":"Improvement of Menopausal Symptoms by Oat Hull Extracts in Ovariectomized Animal Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 11:12:52","doi":"10.21203/rs.3.rs-5382261/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d89782dd-b744-473e-8bad-8895d0d90258","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-09T16:24:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-14 11:12:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5382261","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5382261","identity":"rs-5382261","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}