Detecting phytoestrogens and mammalian estrogens in blackcurrants (Ribes nigrum L.)

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Naoki Nanashima, Kayo Horie, Hayato Maeda, Yoji Kato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3869797/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 5 You are reading this latest preprint version Abstract Phytoestrogens are estrogen-like molecules found in various plants. Menopausal women experience various menopausal symptoms due to reduced estrogen secretion. The ingestion of phytoestrogens is known to alleviate these symptoms. Blackcurrant ( Ribes nigrum L.) is a fruit rich in phytochemicals, including anthocyanins. Blackcurrant extract (BCE) and anthocyanins have phytoestrogenic effects. Although BCE has stronger phytoestrogenic activity than blackcurrant anthocyanins, this aspect has not been explored. BCE likely contains phytoestrogens other than anthocyanins. In this study, the components of blackcurrant fruits were comprehensively investigated through metabolome analysis. In addition to phytoestrogens, such as naringenin, daizein, and resveratrol, mammalian estrogens, such as estrone and β-estradiol, were detected in blackcurrant fruits. An ultrasensitive estrogen measurement analysis revealed that BCE contains approximately 40.2 and 24.5 pg/g of estrone and β-estradiol, respectively, corresponding to approximately 100 times the estrogen content in blackcurrant fruits. We report for the first time that blackcurrant contains phytoestrogens and mammalian estrogens. blackcurrant estrogen metabolomics phytoestrogen Figures Figure 1 Introduction Estrogen is a steroid hormone and is of three types: estrone, β-estradiol, and estriol. Among these, β-estradiol is the most active (Denver et al. 2019 ). Women experience reduced estrogen levels during menopause, aggravating various menopausal symptoms, such as hot flashes and osteoporosis (Sassarini and Lumsden 2015 ). Many plants contain phytoestrogens, such as isoflavones and resveratrol, which function similarly to estrogen, and their intake is known to reduce menopausal symptoms (Patra et al. 2023 ). In addition, mammalian estrogens have been detected in plants (Janeczko 2021 ; Janeczko and Skoczowski 2005 ). Blackcurrant ( Ribes nigrum L.) is a small fruit grown in cool regions, such as Poland, and is known to improve vision and blood flow (Cortez and Gonzalez de Mejia 2019 ). In addition, blackcurrant extract (BCE) and four anthocyanins exhibit phytoestrogenic activity (Nanashima et al. 2017 ; Nanashima et al. 2015 ). Our previous research indicated that BCE alleviates menopausal symptoms in menopausal model rats (Horie et al. 2021 ; Nanashima et al. 2020 ). Furthermore, it was found that the phytoestrogen effect of blackcurrant is stronger with BCE than with anthocyanin alone (Nanashima et al. 2015 ); however, it is challenging to determine the cause. Therefore, we hypothesized that BCE contains phytoestrogens other than anthocyanins. Metabolome analysis is a tool for comprehensively identifying various molecules in serums, urine, and food. In this study, we investigated the phytoestrogens and estrogen-like molecules other than anthocyanins in BCE using a metabolomic analysis and quantified them using ultrasensitive estrogen measurement analysis. Materials and Methods Materials The Tsema and Aomori cultivars were collected at Fujisaki Farm, Faculty of Agriculture and Life Science, Hirosaki University. The blueberries were collected from YoKa Food Science Laboratory Inc. (Hirosaki, Japan). The blackcurrant extract (BCE) powder, i.e., CaNZac-35, was purchased from Koyo Mercantile Co. (Tokyo, Japan). BCE contains high concentrations of anthocyanins (approximately 38%) (Nanashima et al. 2015 ). Metabolome analysis : capillary electrophoresis (CE)-time-of-flight mass spectrometry (TOFMS) measurement All metabolome analyses were performed at Human Metabolome Technologies (HMT; Tsuruoka, Japan). The blackcurrant fruit sample was added to a methanol solution containing an internal standard substance and crushed under cooling. Subsequently, chloroform and Milli-Q water were added, followed by stirring and centrifugation (2300 × g, 4°C, 5 min). After the centrifugation, the aqueous layer was transferred to an ultrafiltration tube (Ultrafree MC-PLHCC, HMT, centrifugal filter unit, 5 kDa) and centrifuged (9,100 × g, 4°C, 120 min). The filtrate was dried, dissolved in Milli-Q water, and used for measurement. CE-TOFMS measurements in the cation and anion modes were performed under the conditions presented in Table S1 . Metabolome analysis : liquid chromatography (LC)-TOFMS measurement The blackcurrant fruit was crushed at 4°C in a 1% formic acid-acetonitrile solution containing 20 µM of the internal standard substance. Next, Milli-Q water was added, and the mixture was further crushed. Centrifugation was performed (2300 × g, 4°C, 5 min), and the supernatant was collected. Further, 1% formic acid-acetonitrile and Milli-Q water were added to the precipitate and stirred. After centrifugation, the supernatants were collected and mixed, and the mixture was subjected to ultrafiltration using an ultrafiltration tube (Nanosep 3 K Omega; Pall, Port Washington, NY, USA). The phospholipids were removed via solid-phase extraction. The filtrate was dried and dissolved in 50% isopropanol aqueous solution (v/v) for measurement. Positive- and negative-mode measurements were performed under the conditions presented in Table S2 . Data processing The detected peaks were automatically extracted using the automatic integration software MasterHands ver. 2.17.1.11 (developed by Keio University (Sugimoto et al. 2010 )). Peaks with a signal/noise ratio of ≥ 3 were automatically extracted. The mass-to-charge ratio ( m/z ), peak area values, migration time (MT) for CE-TOFMS, and retention time (RT) for LC-TOFMS were recorded. The peak areas were converted into relative areas using the following formula: relative area = area of target peak/area of internal standard or sample Candidate metabolite search The detected peaks were compared and searched against all substances registered in the HMT metabolite library and the known–unknown library according to m/z and MT or RT values. The tolerances for the search were ± 0.5 min for MT, ± 0.3 min for RT, ± 10 ppm (CE-TOFMS), and ± 25 ppm (LC-TOFMS) for m/z . Ultrasensitive estrogen quantitative analysis All ultrasensitive estrogen quantitative analyses were performed using liquid chromatography–mass spectrometry (LC-MS/MS) at ASKA Pharma Medical Co., Ltd. (Fujisawa, Japan) (Ishihara et al. 2017 ). Result and Discussion Phytoestrogens have been detected in various plants, e.g., naringenin and daidzein in soybeans and resveratrol in grape skins (Lephart 2021 ). However, there have been no reports on detecting these phytoestrogens in blackcurrants. In this study, Tsema and Aomori cultivars were examined for the two varieties of blackcurrants via metabolome analysis. Naringenin and resveratrol were detected in both varieties as a molecule known as phytoestrogen (Lephart 2021 ), and daidzein was detected in Aomori (Fig. 1 ) alone. Estriol and β-estradiol, along with the primary metabolite of β-estradiol, i.e., estrone-3-glucuronide, were detected in both varieties (Fig. 1 ). We attempted to quantify these phytoestrogens using high-performance liquid chromatography but were unable to detect them below the detection limit (data not shown). Hence, in the future, they must be quantified using a highly sensitive method. The results also suggested that the type of phytoestrogen depends on the variety of blackcurrant. Furthermore, we compared blueberries and blackcurrant fruits. Naringenin and β-estradiol were detected in blackcurrants and blueberries; however, β-estradiol 17-glucuronide—a metabolic product of β-estradiol—was detected only in blackcurrants (Fig. S1 ). Mammalian estrogen has been detected in plants (Janeczko 2021 ; Janeczko and Skoczowski 2005 ), but its presence in blackcurrants has not been reported. We quantified samples of the blackcurrant fruits and BCE using the ultrasensitive estrogen measurement method. The fruits and BCE could quantify estrone and β-estradiol. The samples were more concentrated in the BCE than in the fruit; 40.2 pg/g dry weight (D.W.) of estrone—approximately 100 times higher than the concentration in the fruit—and 24.5 pg/g D.W. of β-estradiol were detected (Table 1) in the BCE. The blackcurrant estrogen concentration is lower than the estrogen concentration in other plants reported to date (Janeczko 2021 ; Janeczko and Skoczowski 2005 ); however, our research indicated BCE estrogen activity. Molecules other than anthocyanins have also been claimed to exhibit estrogen-like effects. Furthermore, pollens of kiwifruit contain relatively high concentrations of β-estradiol (Janeczko 2021 ). Because the concentration is expected to vary depending on the part or variety of the blackcurrant, it is necessary to investigate each part and variety. Conclusions In summary, blackcurrant anthocyanins exhibited phytoestrogenic activity; however, we could not detect the presence of molecules that show estrogen activity in addition to anthocyanins. This study revealed that in addition to phytoestrogens, blackcurrants contain mammalian estrogens. Declarations Ethics Approval Not applicable Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions N.N., K.H., H.M., and Y.K. designed the study; N.N., K.H., and Y.K. performed the experiments and analyzed the data; N.N. and H.M. wrote the original manuscript. All authors have reviewed the final manuscript and approved it for publication. Funding This research was partially supported by the Japan Society for the Promotion of Science, KAKENHI (grant numbers 20K02402 and 23K02038). Data Availability The data generated in this study are available from the corresponding author upon request. References Cortez RE, Gonzalez de Mejia E (2019) Blackcurrants (Ribes nigrum): A review on chemistry, processing, and health benefits. J Food Sci 84:2387–2401. https://doi.org/10.1111/1750-3841.14781 Denver N, Khan S, Homer NZM, MacLean MR, Andrew R (2019) Current strategies for quantification of estrogens in clinical research. J Steroid Biochem Mol Biol 192:105373. https://doi.org/10.1016/j.jsbmb.2019.04.022 Horie K, Nanashima N, Maeda H, Tomisawa T, Oey I (2021) Blackcurrant ( Ribes nigrum L.) extract exerts potential vasculoprotective effects in ovariectomized rats, including prevention of elastin degradation and pathological vascular remodeling. Nutrients 13. https://doi.org/10.3390/nu13020560 Ishihara Y, Itoh K, Tanaka M, Tsuji M, Kawamoto T, Kawato S, Vogel CFA, Yamazaki T (2017) Potentiation of 17beta-estradiol synthesis in the brain and elongation of seizure latency through dietary supplementation with docosahexaenoic acid. Sci Rep 7:6268. https://doi.org/10.1038/s41598-017-06630-0 Janeczko A (2021) Estrogens and androgens in plants: The last 20 years of studies. Plants (Basel) 10. https://doi.org/10.3390/plants10122783 Janeczko A, Skoczowski A (2005) Mammalian sex hormones in plants. Folia Histochem Cytobiol 43:71–79 Lephart ED (2021) Phytoestrogens (resveratrol and equol) for estrogen-deficient skin-controversies/misinformation versus anti-aging in vitro and clinical evidence via nutraceutical-cosmetics. Int J Mol Sci 22. https://doi.org/10.3390/ijms222011218 Nanashima N, Horie K, Maeda H (2017) Phytoestrogenic activity of blackcurrant anthocyanins is partially mediated through estrogen receptor beta. Molecules 23. https://doi.org/10.3390/molecules23010074 Nanashima N, Horie K, Tomisawa T, Chiba M, Nakano M, Fujita T, Maeda H, Kitajima M, Takamagi S, Uchiyama D, Watanabe J, Nakamura T, Kato Y (2015) Phytoestrogenic activity of blackcurrant ( Ribes nigrum ) anthocyanins is mediated through estrogen receptor alpha. Mol Nutr Food Res 59:2419–2431. https://doi.org/10.1002/mnfr.201500479 Nanashima N, Horie K, Yamanouchi K, Tomisawa T, Kitajima M, Oey I, Maeda H (2020) Blackcurrant ( Ribes nigrum ) Extract Prevents Dyslipidemia and Hepatic Steatosis in Ovariectomized Rats. Nutrients 12. https://doi.org/10.3390/nu12051541 Patra S, Gorai S, Pal S, Ghosh K, Pradhan S, Chakrabarti S (2023) A review on phytoestrogens: Current status and future direction. Phytother Res 37:3097–3120. https://doi.org/10.1002/ptr.7861 Sassarini J, Lumsden MA (2015) Oestrogen replacement in postmenopausal women. Age Ageing 44:551–558. https://doi.org/10.1093/ageing/afv069 Sugimoto M, Wong DT, Hirayama A, Soga T, Tomita M (2010) Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles. Metabolomics 6:78–95. https://doi.org/10.1007/s11306-009-0178-y Tables Table 1 . Quantification of estrogens Sample Estrone β-estradiol Whole fruit (pg/g F.W.) 0.55±0.2 0.25±0.1 BCE powder (pg/g D.W.) 40.2±5.1 24.5±2.0 Data represent the mean± standard deviation of triplicate experiments. F.W.-fresh weight; D.W.-dry weight Supplementary Files GraphicalabstractBJP.pdf SupplementaryMaterialBCEestrogen.docx Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Editorial decision: Major revisions 02 Jun, 2024 Reviewers agreed at journal 06 Feb, 2024 Reviewers invited by journal 06 Feb, 2024 Editor assigned by journal 25 Jan, 2024 First submitted to journal 23 Jan, 2024 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-3869797","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":271486606,"identity":"d12a6b4a-4725-4af8-bacf-e6a7daff2e7e","order_by":0,"name":"Naoki Nanashima","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYBCDBDYGBsYHDAwHIFwJIrUwG5CmBYjZJOBa8AFzBt6Dnwtq6vL4xA4/q/jZdiexgf3wAwbLHbi1WDbwJUvPOHa4mE06zexmb9uzxAaeNAMGyTO4tRgc4DGQ5mE7kNgmnWB2g7ftcG4DQw4Dg2QbXi3Gv3n+1QG1pH8r/AvSwv+GoBYzad42ZqCWHDNmsC0ShG0xs+btOwzSUiwtc+5wfZvEM4MDBPxifJvnW13i/NnpGz++KTtszM+f/PCxJJ4QY5B/gCYAjFOGw5INeLRgBYwfSdYyCkbBKBgFwxgAAOWsT1yp3hTIAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4729-3432","institution":"Aomori University of Health and Welfare: Aomori Kenritsu Hoken Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Naoki","middleName":"","lastName":"Nanashima","suffix":""},{"id":271486607,"identity":"92bc1172-fe16-4ccd-94a7-026ffa0d4515","order_by":1,"name":"Kayo Horie","email":"","orcid":"","institution":"Hirosaki University: Hirosaki Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kayo","middleName":"","lastName":"Horie","suffix":""},{"id":271486608,"identity":"cf0068df-4077-48e5-bd9c-d60f70934199","order_by":2,"name":"Hayato Maeda","email":"","orcid":"","institution":"Hirosaki University: Hirosaki Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hayato","middleName":"","lastName":"Maeda","suffix":""},{"id":271486609,"identity":"4de5bed4-ca24-4c41-88be-9702f2cd5af7","order_by":3,"name":"Yoji Kato","email":"","orcid":"","institution":"Hirosaki University: Hirosaki Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Yoji","middleName":"","lastName":"Kato","suffix":""}],"badges":[],"createdAt":"2024-01-16 12:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3869797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3869797/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-024-00572-1","type":"published","date":"2024-08-05T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50841208,"identity":"7dc07b96-c80b-463d-ab03-8605e9abc961","added_by":"auto","created_at":"2024-02-08 07:47:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":138621,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolome analysis of blackcurrants. The bars indicate the relative area of each molecule in Tsema and Aomori. The steps for calculating the relative area are presented in the Supplementary Information.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3869797/v1/d2614e3c553a1059d0497f72.png"},{"id":62298557,"identity":"687e89c2-21e2-41fc-aea3-cbdcac26c053","added_by":"auto","created_at":"2024-08-12 16:14:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":456229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869797/v1/56f9da1b-6529-4bdf-8e4c-dab5419b0eb4.pdf"},{"id":50841210,"identity":"c6ff2e82-bafa-4508-a6e4-f1a608b1c707","added_by":"auto","created_at":"2024-02-08 07:47:45","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":234762,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalabstractBJP.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3869797/v1/bea1f542c179c0e4148939af.pdf"},{"id":50841209,"identity":"5eae68e6-9a9b-44c7-9a72-e810a077ab5b","added_by":"auto","created_at":"2024-02-08 07:47:45","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":207925,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialBCEestrogen.docx","url":"https://assets-eu.researchsquare.com/files/rs-3869797/v1/7425a2251e9adae1b52064c6.docx"}],"financialInterests":"","formattedTitle":"Detecting phytoestrogens and mammalian estrogens in blackcurrants (Ribes nigrum L.)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEstrogen is a steroid hormone and is of three types: estrone, β-estradiol, and estriol. Among these, β-estradiol is the most active (Denver et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Women experience reduced estrogen levels during menopause, aggravating various menopausal symptoms, such as hot flashes and osteoporosis (Sassarini and Lumsden \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Many plants contain phytoestrogens, such as isoflavones and resveratrol, which function similarly to estrogen, and their intake is known to reduce menopausal symptoms (Patra et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, mammalian estrogens have been detected in plants (Janeczko \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Janeczko and Skoczowski \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBlackcurrant (\u003cem\u003eRibes nigrum\u003c/em\u003e L.) is a small fruit grown in cool regions, such as Poland, and is known to improve vision and blood flow (Cortez and Gonzalez de Mejia \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, blackcurrant extract (BCE) and four anthocyanins exhibit phytoestrogenic activity (Nanashima et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nanashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our previous research indicated that BCE alleviates menopausal symptoms in menopausal model rats (Horie et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Nanashima et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, it was found that the phytoestrogen effect of blackcurrant is stronger with BCE than with anthocyanin alone (Nanashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); however, it is challenging to determine the cause. Therefore, we hypothesized that BCE contains phytoestrogens other than anthocyanins.\u003c/p\u003e \u003cp\u003eMetabolome analysis is a tool for comprehensively identifying various molecules in serums, urine, and food. In this study, we investigated the phytoestrogens and estrogen-like molecules other than anthocyanins in BCE using a metabolomic analysis and quantified them using ultrasensitive estrogen measurement analysis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe Tsema and Aomori cultivars were collected at Fujisaki Farm, Faculty of Agriculture and Life Science, Hirosaki University. The blueberries were collected from YoKa Food Science Laboratory Inc. (Hirosaki, Japan). The blackcurrant extract (BCE) powder, i.e., CaNZac-35, was purchased from Koyo Mercantile Co. (Tokyo, Japan). BCE contains high concentrations of anthocyanins (approximately 38%) (Nanashima et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolome analysis\u003c/b\u003e: \u003cb\u003ecapillary electrophoresis (CE)-time-of-flight mass spectrometry (TOFMS) measurement\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll metabolome analyses were performed at Human Metabolome Technologies (HMT; Tsuruoka, Japan). The blackcurrant fruit sample was added to a methanol solution containing an internal standard substance and crushed under cooling. Subsequently, chloroform and Milli-Q water were added, followed by stirring and centrifugation (2300 \u0026times; g, 4\u0026deg;C, 5 min). After the centrifugation, the aqueous layer was transferred to an ultrafiltration tube (Ultrafree MC-PLHCC, HMT, centrifugal filter unit, 5 kDa) and centrifuged (9,100 \u0026times; g, 4\u0026deg;C, 120 min). The filtrate was dried, dissolved in Milli-Q water, and used for measurement. CE-TOFMS measurements in the cation and anion modes were performed under the conditions presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMetabolome analysis\u003c/b\u003e: liquid chromatography (LC)-TOFMS measurement\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe blackcurrant fruit was crushed at 4\u0026deg;C in a 1% formic acid-acetonitrile solution containing 20 \u0026micro;M of the internal standard substance. Next, Milli-Q water was added, and the mixture was further crushed. Centrifugation was performed (2300 \u0026times; g, 4\u0026deg;C, 5 min), and the supernatant was collected. Further, 1% formic acid-acetonitrile and Milli-Q water were added to the precipitate and stirred. After centrifugation, the supernatants were collected and mixed, and the mixture was subjected to ultrafiltration using an ultrafiltration tube (Nanosep 3 K Omega; Pall, Port Washington, NY, USA). The phospholipids were removed via solid-phase extraction. The filtrate was dried and dissolved in 50% isopropanol aqueous solution (v/v) for measurement. Positive- and negative-mode measurements were performed under the conditions presented in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003eThe detected peaks were automatically extracted using the automatic integration software MasterHands ver. 2.17.1.11 (developed by Keio University (Sugimoto et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)). Peaks with a signal/noise ratio of \u0026ge;\u0026thinsp;3 were automatically extracted. The mass-to-charge ratio (\u003cem\u003em/z\u003c/em\u003e), peak area values, migration time (MT) for CE-TOFMS, and retention time (RT) for LC-TOFMS were recorded. The peak areas were converted into relative areas using the following formula: relative area\u0026thinsp;=\u0026thinsp;area of target peak/area of internal standard or sample\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCandidate metabolite search\u003c/h2\u003e \u003cp\u003eThe detected peaks were compared and searched against all substances registered in the HMT metabolite library and the known\u0026ndash;unknown library according to \u003cem\u003em/z\u003c/em\u003e and MT or RT values. The tolerances for the search were \u0026plusmn;\u0026thinsp;0.5 min for MT, \u0026plusmn;\u0026thinsp;0.3 min for RT, \u0026plusmn;\u0026thinsp;10 ppm (CE-TOFMS), and \u0026plusmn;\u0026thinsp;25 ppm (LC-TOFMS) for \u003cem\u003em/z\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eUltrasensitive estrogen quantitative analysis\u003c/h2\u003e \u003cp\u003eAll ultrasensitive estrogen quantitative analyses were performed using liquid chromatography\u0026ndash;mass spectrometry (LC-MS/MS) at ASKA Pharma Medical Co., Ltd. (Fujisawa, Japan) (Ishihara et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Result and Discussion","content":"\u003cp\u003ePhytoestrogens have been detected in various plants, e.g., naringenin and daidzein in soybeans and resveratrol in grape skins (Lephart \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there have been no reports on detecting these phytoestrogens in blackcurrants. In this study, Tsema and Aomori cultivars were examined for the two varieties of blackcurrants via metabolome analysis. Naringenin and resveratrol were detected in both varieties as a molecule known as phytoestrogen (Lephart \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and daidzein was detected in Aomori (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) alone. Estriol and β-estradiol, along with the primary metabolite of β-estradiol, i.e., estrone-3-glucuronide, were detected in both varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We attempted to quantify these phytoestrogens using high-performance liquid chromatography but were unable to detect them below the detection limit (data not shown). Hence, in the future, they must be quantified using a highly sensitive method. The results also suggested that the type of phytoestrogen depends on the variety of blackcurrant. Furthermore, we compared blueberries and blackcurrant fruits. Naringenin and β-estradiol were detected in blackcurrants and blueberries; however, β-estradiol 17-glucuronide\u0026mdash;a metabolic product of β-estradiol\u0026mdash;was detected only in blackcurrants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMammalian estrogen has been detected in plants (Janeczko \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Janeczko and Skoczowski \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), but its presence in blackcurrants has not been reported. We quantified samples of the blackcurrant fruits and BCE using the ultrasensitive estrogen measurement method. The fruits and BCE could quantify estrone and β-estradiol. The samples were more concentrated in the BCE than in the fruit; 40.2 pg/g dry weight (D.W.) of estrone\u0026mdash;approximately 100 times higher than the concentration in the fruit\u0026mdash;and 24.5 pg/g D.W. of β-estradiol were detected (Table\u0026nbsp;1) in the BCE.\u003c/p\u003e \u003cp\u003eThe blackcurrant estrogen concentration is lower than the estrogen concentration in other plants reported to date (Janeczko \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Janeczko and Skoczowski \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); however, our research indicated BCE estrogen activity. Molecules other than anthocyanins have also been claimed to exhibit estrogen-like effects. Furthermore, pollens of kiwifruit contain relatively high concentrations of β-estradiol (Janeczko \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Because the concentration is expected to vary depending on the part or variety of the blackcurrant, it is necessary to investigate each part and variety.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, blackcurrant anthocyanins exhibited phytoestrogenic activity; however, we could not detect the presence of molecules that show estrogen activity in addition to anthocyanins. This study revealed that in addition to phytoestrogens, blackcurrants contain mammalian estrogens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.N., K.H., H.M., and Y.K. designed the study; N.N., K.H., and Y.K. performed the experiments and analyzed the data; N.N. and H.M. wrote the original manuscript. All authors have reviewed the final manuscript and approved it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by the Japan Society for the Promotion of Science, KAKENHI (grant numbers 20K02402 and 23K02038).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCortez RE, Gonzalez de Mejia E (2019) Blackcurrants (Ribes nigrum): A review on chemistry, processing, and health benefits. 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Metabolomics 6:78\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11306-009-0178-y\u003c/span\u003e\u003cspan address=\"10.1007/s11306-009-0178-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"437\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.267734553775746%\" colspan=\"3\" style=\"width: 99.7712%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Quantification of estrogens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.267734553775746%\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.76430205949657%\"\u003e\n \u003cp\u003eEstrone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.96796338672769%\"\u003e\n \u003cp\u003e\u0026beta;-estradiol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.267734553775746%\"\u003e\n \u003cp\u003eWhole fruit\u0026nbsp;(pg/g F.W.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.76430205949657%\"\u003e\n \u003cp\u003e0.55\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.96796338672769%\"\u003e\n \u003cp\u003e0.25\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.267734553775746%\"\u003e\n \u003cp\u003eBCE powder\u0026nbsp;(pg/g D.W.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.76430205949657%\"\u003e\n \u003cp\u003e40.2\u0026plusmn;5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.96796338672769%\"\u003e\n \u003cp\u003e24.5\u0026plusmn;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData represent the mean\u0026plusmn;\u0026nbsp;standard deviation of triplicate experiments.\u003c/p\u003e\n\u003cp\u003eF.W.-fresh weight; D.W.-dry weight\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"blackcurrant, estrogen, metabolomics, phytoestrogen","lastPublishedDoi":"10.21203/rs.3.rs-3869797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3869797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhytoestrogens are estrogen-like molecules found in various plants. Menopausal women experience various menopausal symptoms due to reduced estrogen secretion. The ingestion of phytoestrogens is known to alleviate these symptoms. Blackcurrant (\u003cem\u003eRibes nigrum\u003c/em\u003e L.) is a fruit rich in phytochemicals, including anthocyanins. Blackcurrant extract (BCE) and anthocyanins have phytoestrogenic effects. Although BCE has stronger phytoestrogenic activity than blackcurrant anthocyanins, this aspect has not been explored. BCE likely contains phytoestrogens other than anthocyanins. In this study, the components of blackcurrant fruits were comprehensively investigated through metabolome analysis. In addition to phytoestrogens, such as naringenin, daizein, and resveratrol, mammalian estrogens, such as estrone and β-estradiol, were detected in blackcurrant fruits. An ultrasensitive estrogen measurement analysis revealed that BCE contains approximately 40.2 and 24.5 pg/g of estrone and β-estradiol, respectively, corresponding to approximately 100 times the estrogen content in blackcurrant fruits. We report for the first time that blackcurrant contains phytoestrogens and mammalian estrogens.\u003c/p\u003e","manuscriptTitle":"Detecting phytoestrogens and mammalian estrogens in blackcurrants (Ribes nigrum L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-08 07:47:40","doi":"10.21203/rs.3.rs-3869797/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-06-02T18:26:10+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-06T07:28:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-06T06:55:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T07:49:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2024-01-23T05:41:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"89e9d618-59cc-45dd-8143-d2937b9a9515","owner":[],"postedDate":"February 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:05:35+00:00","versionOfRecord":{"articleIdentity":"rs-3869797","link":"https://doi.org/10.1007/s43450-024-00572-1","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2024-08-05 15:58:00","publishedOnDateReadable":"August 5th, 2024"},"versionCreatedAt":"2024-02-08 07:47:40","video":"","vorDoi":"10.1007/s43450-024-00572-1","vorDoiUrl":"https://doi.org/10.1007/s43450-024-00572-1","workflowStages":[]},"version":"v1","identity":"rs-3869797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3869797","identity":"rs-3869797","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}