{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T13:38:07Z","timestamp":1767706687155,"version":"build-2065373602"},"reference-count":25,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2023,3,14]],"date-time":"2023-03-14T00:00:00Z","timestamp":1678752000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Society for the Promotion of Science","award":["22K05490"],"award-info":[{"award-number":["22K05490"]}]},{"name":"Institute of Global Innovation Research","award":["22K05490"],"award-info":[{"award-number":["22K05490"]}]},{"name":"Support for Pioneering Research Initiated by the Next Generation of FLOuRISH Institute","award":["22K05490"],"award-info":[{"award-number":["22K05490"]}]},{"name":"Tokyo University of Agriculture and Technology","award":["22K05490"],"award-info":[{"award-number":["22K05490"]}]},{"name":"Ministry of Education, Culture, Sports, Science and Technology (MEXT)","award":["22K05490"],"award-info":[{"award-number":["22K05490"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Nutrients"],"abstract":"<jats:p>Citrus nobiletin (NOB) and tangeretin (TAN) show protective effects against disease-related bone destruction. We achieved demethylation of NOB and TAN into 4\u2032-demethylnobiletin (4\u2032-DN) and 4\u2032-demethyltangeretin (4\u2032-DT) using enzyme-manufacturing methods. In this study, we examined the effects of 4\u2032-DN and 4\u2032-DT on in vitro osteoclast differentiation, and on in vivo osteoporotic bone loss in ovariectomized (OVX) mice. 4\u2032-DN and 4\u2032-DT clearly suppressed the osteoclast differentiation induced by interleukin IL-1 or RANKL treatment. 4\u2032-DN and 4\u2032-DT treatments resulted in higher inhibitory activity in osteoclasts in comparison to NOB or TAN treatments. RANKL induced the increased expression of its marker genes and the degradation of I\u03baB\u03b1 in osteoclasts, while these were perfectly attenuated by the treatment with 4\u2032-MIX: a mixture of 4\u2032-DN and 4\u2032-DT. In an in silico docking analysis, 4\u2032-DN and 4\u2032-DT directly bound to the ATP-binding pocket of IKK\u03b2 for functional inhibition. Finally, the intraperitoneal administration of 4\u2032-MIX significantly protected against bone loss in OVX mice. In conclusion, 4\u2032-DN, 4\u2032-DT and 4\u2032-MIX inhibited the differentiation and function of bone-resorbing osteoclasts via suppression of the NF-\u03baB pathway. Novel 4\u2032-DN, 4\u2032-DT and 4\u2032-MIX are candidates for maintaining bone health, which may be applied in the prevention of metabolic bone diseases, such as osteoporosis.<\/jats:p>","DOI":"10.3390\/nu15061403","type":"journal-article","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T04:39:45Z","timestamp":1678855185000},"page":"1403","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Effects of 4\u2032-Demethylnobiletin and 4\u2032-Demethyltangeretin on Osteoclast Differentiation In Vitro and in a Mouse Model of Estrogen-Deficient Bone Resorption"],"prefix":"10.3390","volume":"15","author":[{"given":"Michiko","family":"Hirata","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5683-8951","authenticated-orcid":false,"given":"Tsukasa","family":"Tominari","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryota","family":"Ichimaru","sequence":"additional","affiliation":[{"name":"Cooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Naruhiko","family":"Takiguchi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuki","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Cooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masaru","family":"Takatoya","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daichi","family":"Arai","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shosei","family":"Yoshinouchi","sequence":"additional","affiliation":[{"name":"Cooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chisato","family":"Miyaura","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chiho","family":"Matsumoto","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0606-5759","authenticated-orcid":false,"given":"Sihui","family":"Ma","sequence":"additional","affiliation":[{"name":"Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Tokyo 359-1192, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6572-5809","authenticated-orcid":false,"given":"Katsuhiko","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Tokyo 359-1192, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8101-0558","authenticated-orcid":false,"given":"Florian M. W.","family":"Grundler","sequence":"additional","affiliation":[{"name":"Institute of Crop Science and Resource Conservation, University of Bonn, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany"},{"name":"Life Science Inada Unit, Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masaki","family":"Inada","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"},{"name":"Life Science Inada Unit, Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3597","DOI":"10.1073\/pnas.95.7.3597","article-title":"Osteoclast Differentiation Factor Is a Ligand for Osteoprotegerin\/Osteoclastogenesis-Inhibitory Factor and Is Identical to TRANCE\/RANKL","volume":"95","author":"Yasuda","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1080\/14397595.2017.1369491","article-title":"Rankl: A Therapeutic Target for Bone Destruction in Rheumatoid Arthritis","volume":"28","author":"Tanaka","year":"2017","journal-title":"Mod. Rheumatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1879","DOI":"10.4049\/jimmunol.177.3.1879","article-title":"Membrane-Bound Prostaglandin E Synthase-1-Mediated Prostaglandin E2 Production by Osteoblast Plays a Critical Role in Lipopolysaccharide-Induced Bone Loss Associated with Inflammation","volume":"177","author":"Inada","year":"2006","journal-title":"J. Immunol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1016\/j.lfs.2004.10.079","article-title":"IL-1\u03b1 Stimulates the Formation of Osteoclast-like Cells by Increasing M-CSF and PGE2 Production and Decreasing OPG Production by Osteoblasts","volume":"77","author":"Tanabe","year":"2005","journal-title":"Life Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1530\/eje.0.1510503","article-title":"Dose-Response of Estrogen on Bone versus the Uterus in Ovariectomized Mice","volume":"151","author":"Modder","year":"2004","journal-title":"Eur. J. Endocrinol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.bbrc.2016.07.027","article-title":"BA321, a Novel Carborane Analog That Binds to Androgen and Estrogen Receptors, Acts as a New Selective Androgen Receptor Modulator of Bone in Male Mice","volume":"478","author":"Watanabe","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"100289","DOI":"10.1016\/j.bonr.2020.100289","article-title":"Sustained Anti-Osteoporotic Action of Risedronate Compared to Anti-RANKL Antibody Following Discontinuation in Ovariectomized Mice","volume":"13","author":"Omiya","year":"2020","journal-title":"Bone Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1254\/jphs.10193SC","article-title":"Nobiletin, a Polymethoxy Flavonoid, Suppresses Bone Resorption by Inhibiting NF\u03baB-Dependent Prostaglandin E Synthesis in Osteoblasts and Prevents Bone Loss Due to Estrogen Deficiency","volume":"115","author":"Harada","year":"2011","journal-title":"J. Pharmacol. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1254\/jphs.11188SC","article-title":"Polymethoxy Flavonoids, Nobiletin and Tangeretin, Prevent Lipopolysaccharide-Induced Inflammatory Bone Loss in an Experimental Model for Periodontitis","volume":"119","author":"Tominari","year":"2012","journal-title":"J. Pharmacol. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1080\/09168451.2014.952616","article-title":"Heptamethoxyflavone, a Citrus Flavonoid, Suppresses Inflammatory Osteoclastogenesis and Alveolar Bone Resorption","volume":"79","author":"Matsumoto","year":"2014","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Matsumoto, S., Tominari, T., Matsumoto, C., Yoshinouchi, S., Ichimaru, R., Watanabe, K., Hirata, M., Grundler, F., Miyaura, C., and Inada, M. (2018). Effects of Polymethoxyflavonoids on Bone Loss Induced by Estrogen Deficiency and by LPS-Dependent Inflammation in Mice. Pharmaceuticals, 11.","DOI":"10.3390\/ph11010007"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Ohyama, Y., Ito, J., Kitano, V.J., Shimada, J., and Hakeda, Y. (2018). The Polymethoxy Flavonoid Sudachitin Suppresses Inflammatory Bone Destruction by Directly Inhibiting Osteoclastogenesis Due to Reduced ROS Production and MAPK Activation in Osteoclast Precursors. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0191192"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5177","DOI":"10.1016\/j.bmcl.2007.06.096","article-title":"Anti-Inflammatory Property of the Urinary Metabolites of Nobiletin in Mouse","volume":"17","author":"Li","year":"2007","journal-title":"Bioorganic Med. Chem. Lett."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4381","DOI":"10.1021\/acs.jafc.0c06334","article-title":"Identification of 4\u2032-Demethyltangeretin as a Major Urinary Metabolite of Tangeretin in Mice and Its Anti-Inflammatory Activities","volume":"69","author":"Guo","year":"2021","journal-title":"J. Agric. Food Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1007\/s11418-017-1091-8","article-title":"Fermented Citrus Reticulata (Ponkan) Fruit Squeezed Draff That Contains a Large Amount of 4\u2032-Demethylnobiletin Prevents MK801-Induced Memory Impairment","volume":"71","author":"Kawahata","year":"2017","journal-title":"J. Nat. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1016\/j.jff.2015.09.035","article-title":"Anti-Inflammatory Effects of 4\u2032-Demethylnobiletin, a Major Metabolite of Nobiletin","volume":"19","author":"Wu","year":"2015","journal-title":"J. Funct. Foods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"22758","DOI":"10.1074\/jbc.M113.482596","article-title":"Crystal Structure of a Human I\u03baB Kinase \u03b2 Asymmetric Dimer","volume":"288","author":"Liu","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/1758-2946-3-33","article-title":"Open Babel: An Open Chemical Toolbox","volume":"3","author":"Banck","year":"2011","journal-title":"J. Cheminform."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2377","DOI":"10.1007\/s00198-013-2313-x","article-title":"NF-\u039aB Signaling and Bone Resorption","volume":"24","year":"2013","journal-title":"Osteoporos. Int."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3155","DOI":"10.1021\/acs.jafc.8b00509","article-title":"Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin, and 5-Demethyltangeretin from Citrus Peel in Saccharomyces Cerevisiae","volume":"66","author":"Wang","year":"2018","journal-title":"J. Agric. Food Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1248\/bpbreports.2.1_1","article-title":"Structure-Activity Relationship of Anthocyanidins as an Inhibitory Effect on Osteoclast Differentiation","volume":"2","author":"Hirata","year":"2019","journal-title":"BPB Rep."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Hirata, N., Ichimaru, R., Tominari, T., Matsumoto, C., Watanabe, K., Taniguchi, K., Hirata, M., Ma, S., Suzuki, K., and Grundler, F. (2019). Beta-Cryptoxanthin Inhibits Lipopolysaccharide-Induced Osteoclast Differentiation and Bone Resorption via the Suppression of Inhibitor of NF-\u039aB Kinase Activity. Nutrients, 11.","DOI":"10.3390\/nu11020368"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1002\/biof.5520300305","article-title":"Citrus Nobiletin Suppresses Bone Loss in Ovariectomized DdY Mice and Collagen-Induced Arthritis in DBA\/1J Mice: Possible Involvement of Receptor Activator of NF-KappaB Ligand (RANKL)-Induced Osteoclastogenesis Regulation","volume":"30","author":"Murakami","year":"2007","journal-title":"BioFactors"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"7839","DOI":"10.2147\/IJN.S213724","article-title":"Nobiletin-Loaded Micelles Reduce Ovariectomy-Induced Bone Loss by Suppressing Osteoclastogenesis","volume":"14","author":"Wang","year":"2019","journal-title":"Int. J. Nanomed."}],"container-title":["Nutrients"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6643\/15\/6\/1403\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:55:05Z","timestamp":1760122505000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6643\/15\/6\/1403"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,14]]},"references-count":25,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["nu15061403"],"URL":"https:\/\/doi.org\/10.3390\/nu15061403","relation":{},"ISSN":["2072-6643"],"issn-type":[{"type":"electronic","value":"2072-6643"}],"subject":[],"published":{"date-parts":[[2023,3,14]]}}}