{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T21:52:46Z","timestamp":1785793966005,"version":"3.56.0"},"reference-count":52,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T00:00:00Z","timestamp":1780099200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004442","name":"National Science Centre in Poland","doi-asserted-by":"publisher","award":["2023\/51\/D\/NZ9\/01445"],"award-info":[{"award-number":["2023\/51\/D\/NZ9\/01445"]}],"id":[{"id":"10.13039\/501100004442","id-type":"DOI","asserted-by":"publisher"}]},{"award":["2023\/51\/D\/NZ9\/01445"],"award-info":[{"award-number":["2023\/51\/D\/NZ9\/01445"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Flavonols are dietary polyphenols whose biological activity is influenced by structural modifications such as O-methylation. This study compared two quercetin derivatives, isorhamnetin (3\u2032-O-methylquercetin) and rhamnetin (7-O-methylquercetin). Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2\u2032-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays. Cyclooxygenase-2 (COX-2) inhibitory activity was assessed in vitro and supported by molecular docking simulations. In vivo effects included developmental toxicity, behavioral assessment, and locomotor responses in a 6-hydroxydopamine (6-OHDA) model. The results demonstrated that rhamnetin exhibited significantly stronger radical-scavenging and reducing activity in DPPH, ABTS, and FRAP assays, whereas no significant differences were observed in the CUPRAC assay. Isorhamnetin showed stronger COX-2 inhibition, with docking results suggesting a different mode of binding when analyzing possible interactions with enzyme active site. In zebrafish larvae, rhamnetin showed lower observable developmental toxicity within the tested concentration range, whereas isorhamnetin induced developmental abnormalities at higher concentrations. Both flavonols attenuated 6-OHDA-associated locomotor deficits and modulated antioxidant enzyme activity under oxidative stress conditions. In conclusion, our findings indicate that the position of O-methylation influences flavonol antioxidant properties, COX-2 interactions, and organism-level responses.<\/jats:p>","DOI":"10.3390\/ijms27114988","type":"journal-article","created":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T12:45:08Z","timestamp":1780404308000},"page":"4988","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Structure\u2013Activity Relationship of Flavonol O-Methylation Revealed by In Vitro, In Silico and Zebrafish Neurodegeneration Models"],"prefix":"10.3390","volume":"27","author":[{"given":"Kamila","family":"Borowiec","sequence":"first","affiliation":[{"name":"Department of Biotechnology, Microbiology and Human Nutrition, Faculty of Food Science and Biotechnology, University of Life Sciences in Lublin, 20-950 Lublin, Poland"},{"name":"Independent Unit for Behavioral Studies, Faculty of Medical Sciences, Medical University of Lublin, 20-059 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agnieszka","family":"Michalak","sequence":"additional","affiliation":[{"name":"Independent Unit for Behavioral Studies, Faculty of Medical Sciences, Medical University of Lublin, 20-059 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Targowska-Duda","sequence":"additional","affiliation":[{"name":"Department of Biopharmacy, Faculty of Pharmacy, Medical University of Lublin, 20-059 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"5377","DOI":"10.1021\/acsomega.2c06451","article-title":"Attenuation of Lipopolysaccharide-Induced Inflammatory Responses through Inhibition of the NF-\u039aB Pathway and the Increased NRF2 Level by a Flavonol-Enriched n-Butanol Fraction from Uvaria Alba","volume":"8","author":"Notarte","year":"2023","journal-title":"ACS Omega"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1007\/s12640-019-00147-2","article-title":"NF-\u039aB-Mediated Neuroinflammation in Parkinson\u2019s Disease and Potential Therapeutic Effect of Polyphenols","volume":"37","author":"Singh","year":"2020","journal-title":"Neurotox. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1080\/00207454.2016.1212854","article-title":"Neuroinflammation Pathways: A General Review","volume":"127","author":"Shabab","year":"2017","journal-title":"Int. J. Neurosci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.arr.2012.01.006","article-title":"Natural Polyphenols against Neurodegenerative Disorders: Potentials and Pitfalls","volume":"11","author":"Ebrahimi","year":"2012","journal-title":"Ageing Res. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Gervasi, T., Calderaro, A., Barreca, D., Tellone, E., Trombetta, D., Ficarra, S., Smeriglio, A., Mandalari, G., and Gattuso, G. (2022). Biotechnological Applications and Health-Promoting Properties of Flavonols: An Updated View. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23031710"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6742","DOI":"10.1080\/10408398.2021.1905604","article-title":"Flavonoids from Edible Fruits as Therapeutic Agents in Neuroinflammation\u2013a Comprehensive Review and Update","volume":"62","author":"Borowiec","year":"2022","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1093\/toxsci\/kfw139","article-title":"Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System","volume":"154","author":"Bugel","year":"2016","journal-title":"Toxicol. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1016\/j.tifs.2021.03.030","article-title":"Food Flavonols: Nutraceuticals with Complex Health Benefits and Functionalities","volume":"117","author":"Barreca","year":"2021","journal-title":"Trends Food Sci. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1282","DOI":"10.1016\/j.biopha.2018.07.079","article-title":"Emerging Novel Drug Delivery Strategies for Bioactive Flavonol Fisetin in Biomedicine","volume":"106","author":"Mehta","year":"2018","journal-title":"Biomed. Pharmacother."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"24269","DOI":"10.1038\/s41598-021-03804-9","article-title":"Etazene Induces Developmental Toxicity in Vivo Danio Rerio and in Silico Studies of New Synthetic Opioid Derivative","volume":"11","author":"Kurach","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"172828","DOI":"10.1016\/j.pbb.2019.172828","article-title":"Identification of Antiparkinsonian Drugs in the 6-Hydroxydopamine Zebrafish Model","volume":"189","author":"Vaz","year":"2020","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"123470","DOI":"10.1016\/j.ijbiomac.2023.123470","article-title":"Statistical Evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu Assays to Assess the Antioxidant Capacity of Lignins","volume":"233","author":"Rumpf","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Shamsudin, N.F., Ahmed, Q.U., Mahmood, S., Shah, S.A.A., Sarian, M.N., Khattak, M.M.A.K., Khatib, A., Sabere, A.S.M., Yusoff, Y.M., and Latip, J. (2022). Flavonoids as Antidiabetic and Anti-Inflammatory Agents: A Review on Structural Activity Relationship-Based Studies and Meta-Analysis. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232012605"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"537","DOI":"10.18433\/J3KW2Z","article-title":"Antioxidant and Chemosensitizing Effects of Flavonoids with Hydroxy and\/or Methoxy Groups and Structure-Activity Relationship","volume":"10","author":"Jeong","year":"2007","journal-title":"J. Pharm. Pharm. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1042\/bj20021972","article-title":"Intracellular Metabolism and Bioactivity of Quercetin and Its in Vivo Metabolites","volume":"372","author":"Spencer","year":"2003","journal-title":"Biochem. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2439","DOI":"10.1039\/c1ay05320e","article-title":"A Comprehensive Review of CUPRAC Methodology","volume":"3","author":"Bakan","year":"2011","journal-title":"Anal. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1017\/S000711450999170X","article-title":"The Relationship between Fasting Plasma Concentrations of Selected Flavonoids and Their Ordinary Dietary Intake","volume":"103","author":"Cao","year":"2010","journal-title":"Br. J. Nutr."},{"key":"ref_18","first-page":"15\u221222","article-title":"Investigation of the Bioavailability of Hypericin, Pseudohypericin, Hyperforin and the Flavonoids Quercetin and Isorhamnetin Following Single and Multiple Oral Dosing of a Hypericum Extract Containing Tablet","volume":"55","author":"Schulz","year":"2005","journal-title":"Arzneim.-Forsch.\/Drug Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1007\/s11101-021-09763-3","article-title":"Flavonoid Delivery by Solid Dispersion: A Systematic Review","volume":"21","author":"Colombo","year":"2022","journal-title":"Phytochem. Rev."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1017\/S0007114508966083","article-title":"A Comparison of the Effects of Kaempferol and Quercetin on Cytokine-Induced pro-Inflammatory Status of Cultured Human Endothelial Cells","volume":"100","author":"Crespo","year":"2008","journal-title":"Br. J. Nutr."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1016\/j.jnutbio.2014.04.006","article-title":"Plant Flavonol Isorhamnetin Attenuates Chemically Induced Inflammatory Bowel Disease via a PXR-Dependent Pathway","volume":"25","author":"Dou","year":"2014","journal-title":"J. Nutr. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Acosta-Quiroga, K., Rocha-Valderrama, E., Z\u00fa\u00f1iga-Bustos, M., Mera-Adasme, R., Cabrera-Barjas, G., Olea-Azar, C., and Moncada-Basualto, M. (2025). Gross Antioxidant Capacity and Anti-Inflammatory Potential of Flavonol Oxidation Products: A Combined Experimental and Theoretical Study. Antioxidants, 14.","DOI":"10.3390\/antiox14040479"},{"key":"ref_23","first-page":"675","article-title":"Exploring the Interactions of Natural Flavonoids with COX-2: Insights from Molecular Docking, DFT Analysis, and ADME Calculations","volume":"12","author":"Lachi","year":"2024","journal-title":"Phys. Chem. Res."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Liu, Y., Fernie, A.R., and Tohge, T. (2022). Diversification of Chemical Structures of Methoxylated Flavonoids and Genes Encoding Flavonoid-O-Methyltransferases. Plants, 11.","DOI":"10.3390\/plants11040564"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"42717","DOI":"10.1038\/srep42717","article-title":"SwissADME: A Free Web Tool to Evaluate Pharmacokinetics, Drug-Likeness and Medicinal Chemistry Friendliness of Small Molecules","volume":"7","author":"Daina","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Ami\u0107, A., and Masti\u013e\u00e1k Cagardov\u00e1, D. (2022). DFT Study of the Direct Radical Scavenging Potency of Two Natural Catecholic Compounds. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232214497"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Berga, M., Logviss, K., Lauberte, L., Paulausks, A., and Mohylyuk, V. (2023). Flavonoids in the Spotlight: Bridging the Gap between Physicochemical Properties and Formulation Strategies. Pharmaceuticals, 16.","DOI":"10.3390\/ph16101407"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Taldaev, A., Svotin, A.A., Obukhov, S.I., Terekhov, R.P., and Selivanova, I.A. (2025). Modification of Biopharmaceutical Parameters of Flavonoids: A Review. Front. Chem., 13.","DOI":"10.3389\/fchem.2025.1602967"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Vedder, V.L., Reinberger, T., Haider, S.M.I., Eichelmann, L., Odenthal, N., Abdelilah-Seyfried, S., Aherrahrou, Z., Breuer, M., and Erdmann, J. (2023). PyHeart4Fish: Chamber-Specific Heart Phenotype Quantification of Zebrafish in High-Content Screens. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1143852"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.bbr.2011.11.041","article-title":"On the Edge: Pharmacological Evidence for Anxiety-Related Behavior in Zebrafish Larvae","volume":"228","author":"Richendrfer","year":"2012","journal-title":"Behav. Brain Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"113718","DOI":"10.1016\/j.biopha.2022.113718","article-title":"Molecular Understanding of the Translational Models and the Therapeutic Potential Natural Products of Parkinson\u2019s Disease","volume":"155","author":"Pang","year":"2022","journal-title":"Biomed. Pharmacother."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"110301","DOI":"10.1016\/j.biopha.2020.110301","article-title":"Isorhamnetin: A Review of Pharmacological Effects","volume":"128","author":"Gong","year":"2020","journal-title":"Biomed. Pharmacother."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Rana, J.N., Gul, K., and Mumtaz, S. (2025). Isorhamnetin: Reviewing Recent Developments in Anticancer Mechanisms and Nanoformulation-Driven Delivery. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26157381"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1323","DOI":"10.1007\/s00204-024-03696-4","article-title":"Several Lines of Antioxidant Defense against Oxidative Stress: Antioxidant Enzymes, Nanomaterials with Multiple Enzyme-Mimicking Activities, and Low-Molecular-Weight Antioxidants","volume":"98","author":"Jomova","year":"2024","journal-title":"Arch. Toxicol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"904","DOI":"10.1007\/s11033-024-09814-w","article-title":"Zebrafish as an Innovative Model for Exploring Cardiovascular Disease Induction Mechanisms and Novel Therapeutic Interventions: A Molecular Insight","volume":"51","author":"Ali","year":"2024","journal-title":"Mol. Biol. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1194\/jlr.D032698","article-title":"A Specific, Accurate, and Sensitive Measure of Total Plasma Malondialdehyde by HPLC","volume":"54","author":"Moselhy","year":"2013","journal-title":"J. Lipid Res."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Nitti, M., Marengo, B., Furfaro, A.L., Pronzato, M.A., Marinari, U.M., Domenicotti, C., and Traverso, N. (2022). Hormesis and Oxidative Distress: Pathophysiology of Reactive Oxygen Species and the Open Question of Antioxidant Modulation and Supplementation. Antioxidants, 11.","DOI":"10.3390\/antiox11081613"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"150004","DOI":"10.1016\/j.brainres.2025.150004","article-title":"Isoquercetin Mitigates Neuroinflammation and Oxidative Stress by Targeting the NF-\u039aB Pathway","volume":"1869","author":"Kaur","year":"2025","journal-title":"Brain Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"101901","DOI":"10.1016\/j.mito.2024.101901","article-title":"The Tissue-Specific Nature of Physiological Zebrafish Mitochondrial Bioenergetics","volume":"77","author":"Leite","year":"2024","journal-title":"Mitochondrion"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Du, Y., Guo, Q., Shan, M., Wu, Y., Huang, S., Zhao, H., Hong, H., Yang, M., Yang, X., and Ren, L. (2016). Spatial and Temporal Distribution of Dopaminergic Neurons during Development in Zebrafish. Front. Neuroanat., 10.","DOI":"10.3389\/fnana.2016.00115"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Cellini, B.R., Edachola, S.V., Faw, T.D., and Cigliola, V. (2025). Blueprints for Healing: Central Nervous System Regeneration in Zebrafish and Neonatal Mice. BMC Biol., 23.","DOI":"10.1186\/s12915-025-02203-0"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/S0023-6438(95)80008-5","article-title":"Use of a Free Radical Method to Evaluate Antioxidant Activity","volume":"28","author":"Cuvelier","year":"1995","journal-title":"LWT-Food Sci. Technol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.foodchem.2018.09.040","article-title":"In Vitro Antioxidant Activity of Kyoho Grape Extracts in DPPH [Rad] and ABTS [Rad] Assays: Estimation Methods for EC 50 Using Advanced Statistical Programs","volume":"275","author":"Sridhar","year":"2019","journal-title":"Food Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1353","DOI":"10.1016\/j.fct.2011.03.019","article-title":"In Vitro Antioxidant, Anticholinesterase and Antimicrobial Activity Studies on Three Agaricus Species with Fatty Acid Compositions and Iron Contents: A Comparative Study on the Three Most Edible Mushrooms","volume":"49","author":"Duru","year":"2011","journal-title":"Food Chem. Toxicol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/j.foodchem.2014.11.009","article-title":"A Universally Calibrated Microplate Ferric Reducing Antioxidant Power (FRAP) Assay for Foods and Applications to Manuka Honey","volume":"174","author":"Henderson","year":"2015","journal-title":"Food Chem."},{"key":"ref_46","first-page":"772","article-title":"Crystal Structure of Rofecoxib Bound to Human Cyclooxygenase-2","volume":"72","author":"Orlando","year":"2016","journal-title":"Acta Cryst."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Gawel, K., Kukula-Koch, W., Banono, N.S., Nieoczym, D., Targowska-Duda, K.M., Czernicka, L., Parada-Turska, J., and Esguerra, C.V. (2021). 6-Gingerol, a Major Constituent of Zingiber Officinale Rhizoma, Exerts Anticonvulsant Activity in the Pentylenetetrazole-Induced Seizure Model in Larval Zebrafish. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22147745"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1080\/07391102.2018.1427634","article-title":"Molecular Interactions of Type I and Type II Positive Allosteric Modulators with the Human A7 Nicotinic Acetylcholine Receptor: An in Silico Study","volume":"37","author":"Kaczor","year":"2019","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"115338","DOI":"10.1016\/j.indcrop.2022.115338","article-title":"Carlina Oxide Inhibits the Interaction of SARS-CoV-2 S Glycoprotein with Angiotensin-Converting Enzyme 2","volume":"187","author":"Wnorowska","year":"2022","journal-title":"Ind. Crops Prod."},{"key":"ref_50","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_51","unstructured":"OECD (2013). Test No. 236: Fish Embryo Acute Toxicity (FET) Test, OECD."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"113461","DOI":"10.1016\/j.ygcen.2020.113461","article-title":"Profound Effects of Glucocorticoid Resistance on Anxiety-Related Behavior in Zebrafish Adults but Not in Larvae","volume":"292","author":"Sireeni","year":"2020","journal-title":"Gen. Comp. Endocrinol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4988\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T13:04:09Z","timestamp":1780405449000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4988"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,30]]},"references-count":52,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ijms27114988"],"URL":"https:\/\/doi.org\/10.3390\/ijms27114988","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,30]]}}}