{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T09:30:33Z","timestamp":1774603833741,"version":"3.50.1"},"reference-count":39,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2019,6,3]],"date-time":"2019-06-03T00:00:00Z","timestamp":1559520000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UID\/QUI\/50006\/2013"],"award-info":[{"award-number":["UID\/QUI\/50006\/2013"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["PD\/BD\/113565\/2015"],"award-info":[{"award-number":["PD\/BD\/113565\/2015"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["NORTE-01-0145-FEDER-000024"],"award-info":[{"award-number":["NORTE-01-0145-FEDER-000024"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Marine Drugs"],"abstract":"<jats:p>Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5\u03b1,8\u03b1-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5\u03b1,8\u03b1-epidioxycholest-6-en-3\u03b2-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-\u03b1) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.<\/jats:p>","DOI":"10.3390\/md17060330","type":"journal-article","created":{"date-parts":[[2019,6,3]],"date-time":"2019-06-03T11:31:01Z","timestamp":1559561461000},"page":"330","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Anti-Inflammatory Effects of 5\u03b1,8\u03b1-Epidioxycholest-6-en-3\u03b2-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8500-7364","authenticated-orcid":false,"given":"Renato B.","family":"Pereira","sequence":"first","affiliation":[{"name":"REQUIMTE\/LAQV, Laborat\u00f3rio de Farmacognosia, Departamento de Qu\u00edmica, Faculdade de Farm\u00e1cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n\u00ba 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0384-7592","authenticated-orcid":false,"given":"David M.","family":"Pereira","sequence":"additional","affiliation":[{"name":"REQUIMTE\/LAQV, Laborat\u00f3rio de Farmacognosia, Departamento de Qu\u00edmica, Faculdade de Farm\u00e1cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n\u00ba 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2628-303X","authenticated-orcid":false,"given":"Carlos","family":"Jim\u00e9nez","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica, Facultade de Ciencias e Centro de Investigaci\u00f3ns Cient\u00edficas Avanzadas (CICA), Universidade da Coru\u00f1a, E-15071 A Coru\u00f1a, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5348-6970","authenticated-orcid":false,"given":"Jaime","family":"Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica, Facultade de Ciencias e Centro de Investigaci\u00f3ns Cient\u00edficas Avanzadas (CICA), Universidade da Coru\u00f1a, E-15071 A Coru\u00f1a, Spain"}]},{"given":"Rosa M.","family":"Nieto","sequence":"additional","affiliation":[{"name":"Departamento de Qu\u00edmica, Facultade de Ciencias e Centro de Investigaci\u00f3ns Cient\u00edficas Avanzadas (CICA), Universidade da Coru\u00f1a, E-15071 A Coru\u00f1a, Spain"}]},{"given":"Romeu A.","family":"Videira","sequence":"additional","affiliation":[{"name":"REQUIMTE\/LAQV, Laborat\u00f3rio de Farmacognosia, Departamento de Qu\u00edmica, Faculdade de Farm\u00e1cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n\u00ba 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1555-7883","authenticated-orcid":false,"given":"Olga","family":"Silva","sequence":"additional","affiliation":[{"name":"Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9764-3920","authenticated-orcid":false,"given":"Paula B.","family":"Andrade","sequence":"additional","affiliation":[{"name":"REQUIMTE\/LAQV, Laborat\u00f3rio de Farmacognosia, Departamento de Qu\u00edmica, Faculdade de Farm\u00e1cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n\u00ba 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0740-4396","authenticated-orcid":false,"given":"Patr\u00edcia","family":"Valent\u00e3o","sequence":"additional","affiliation":[{"name":"REQUIMTE\/LAQV, Laborat\u00f3rio de Farmacognosia, Departamento de Qu\u00edmica, Faculdade de Farm\u00e1cia, Universidade do Porto, R. Jorge Viterbo Ferreira, n\u00ba 228, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2019,6,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Pereira, R.B., Andrade, P.B., and Valent\u00e3o, P. (2016). Chemical diversity and biological properties of secondary metabolites from sea hares of Aplysia genus. Mar. Drugs, 14.","DOI":"10.3390\/md14020039"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1111\/j.1469-185X.2010.00124.x","article-title":"Molluscan biological and chemical diversity: Secondary metabolites and medicinal resources produced by marine molluscs","volume":"85","author":"Benkendorff","year":"2010","journal-title":"Biol. Rev. Camb. Philos. Soc."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1007\/BF00349763","article-title":"Sequestration of dietary secondary metabolites by three species of sea hares: Location, specificity and dynamics","volume":"117","author":"Pennings","year":"1993","journal-title":"Mar. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"15766","DOI":"10.3390\/molecules200915766","article-title":"Digestive gland from Aplysia depilans Gmelin: Leads for inflammation treatment","volume":"20","author":"Oliveira","year":"2015","journal-title":"Molecules"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1021\/np50047a023","article-title":"Epidioxy sterols from the tunicates Dendrodoa grossularia and Ascidiella aspersa and the gastropoda Aplysia depilans and Aplysia punctata","volume":"49","author":"Castedo","year":"1986","journal-title":"J. Nat. Prod."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"709","DOI":"10.4172\/2161-0444.1000217","article-title":"Natural bioactive sterol 5\u03b1,8\u03b1-endoperoxides as drug lead compounds","volume":"4","author":"Bu","year":"2014","journal-title":"Med. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1142\/S0192415X13500109","article-title":"Immunomodulatory activities of medicinal mushroom Grifola frondosa extract and its bioactive constituent","volume":"41","author":"Wu","year":"2013","journal-title":"Am. J. Chin. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1002\/jlac.198819880616","article-title":"Studies on the constituents of marine opisthobranchia III. Isolation and structure of two 5,8\u03b1-epidioxysterols and a cholesteryl ester mixture from the albumen gland of Aplysia juliana","volume":"1988","author":"Miyamoto","year":"1988","journal-title":"Liebigs Annalen der Chemie"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1007\/s10600-016-1767-4","article-title":"Secondary metabolites isolated from the sea hare Aplysia pulmonica from the South China Sea","volume":"52","author":"Mou","year":"2016","journal-title":"Chem. Nat. Compd."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3860","DOI":"10.1021\/jo00332a020","article-title":"Minor and trace sterols in marine invertebrates. 26. Isolation and structure elucidation of nine new 5.alpha.,8.alpha.-epidoxy sterols from four marine organisms","volume":"46","author":"Gunatilaka","year":"1981","journal-title":"J. Org. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1679","DOI":"10.1007\/s11746-998-0111-y","article-title":"5\u03b1,8\u03b1-Epidioxycholest-6-en-3-\u03b2-ol from three cone snails of the Indian ocean","volume":"75","author":"Aknin","year":"1998","journal-title":"J. Am. Oil Chem. Soc."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.steroids.2008.09.007","article-title":"5\u03b1,8\u03b1-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: Isolation and evaluation of their antiproliferative activity","volume":"74","author":"Ioannou","year":"2009","journal-title":"Steroids"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1007\/BF02980141","article-title":"Cytotoxic constituents of Diadema setosum","volume":"27","author":"Minh","year":"2004","journal-title":"Arch. Pharm. Res."},{"key":"ref_14","first-page":"1537","article-title":"Ecology- and bioassay-guided drug discovery for treatments of tropical parasitic disease: 5alpha, 8alpha-epidioxycholest-6-en-3beta-ol isolated from the mollusk Dolabrifera dolabrifera shows significant activity against Leishmania donovani","volume":"8","author":"Clark","year":"2013","journal-title":"Nat. Prod. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"8981","DOI":"10.1039\/C4RA14333G","article-title":"Fatty acids from edible sea hares: anti-inflammatory capacity in LPS-stimulated RAW 264.7 cells involves iNOS modulation","volume":"5","author":"Pereira","year":"2015","journal-title":"RSC Adv."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.jff.2017.07.053","article-title":"UHPLC-MS\/MS profiling of Aplysia depilans and assessment of its potential therapeutic use: Interference on iNOS expression in LPS-stimulated RAW 264.7 macrophages and caspase-mediated pro-apoptotic effect on SH-SY5Y cells","volume":"37","author":"Pereira","year":"2017","journal-title":"J. Funct. Foods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1016\/j.chemosphere.2018.06.034","article-title":"Trace elements in wild edible Aplysia species: Relationship with the desaturation\u2013elongation indexes of fatty acids","volume":"208","author":"Pereira","year":"2018","journal-title":"Chemosphere"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1007\/s10787-007-0013-x","article-title":"Role of nitric oxide in inflammatory diseases","volume":"15","author":"Sharma","year":"2007","journal-title":"Inflammopharmacology"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Huang, C.-Y., Chang, C.-W., Tseng, Y.-J., Lee, J., Sung, P.-J., Su, J.-H., Hwang, T.-L., Dai, C.-F., Wang, H.-C., and Sheu, J.-H. (2016). Bioactive steroids from the formosan soft coral Umbellulifera petasites. Mar. Drugs, 14.","DOI":"10.3390\/md14100180"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.jfda.2016.11.006","article-title":"Macrophages in oxidative stress and models to evaluate the antioxidant function of dietary natural compounds","volume":"25","author":"Castaneda","year":"2017","journal-title":"J. Food Drug Anal."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"a001651","DOI":"10.1101\/cshperspect.a001651","article-title":"The nuclear factor NF-kappaB pathway in inflammation","volume":"1","author":"Lawrence","year":"2009","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_22","first-page":"147","article-title":"Inhibition of nitric oxide production, iNOS and COX-2 expression of ergosterol derivatives from Phellinus pini","volume":"18","author":"Hong","year":"2012","journal-title":"Natural Product Sciences"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7129","DOI":"10.1016\/j.tet.2010.06.094","article-title":"Steroid and cembranoids from the Dongsha atoll soft coral Lobophytum sarcophytoides","volume":"66","author":"Lu","year":"2010","journal-title":"Tetrahedron"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"187","DOI":"10.3109\/13880209.2013.821664","article-title":"Bioactivities of six sterols isolated from marine invertebrates","volume":"52","author":"Zhou","year":"2014","journal-title":"Pharm. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.pharmthera.2018.01.004","article-title":"Towards targeting resolution pathways of airway inflammation in asthma","volume":"186","author":"Barnig","year":"2018","journal-title":"Pharmacol. Ther."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1007\/s00044-019-02315-7","article-title":"Developmental perspectives of the drugs targeting enzyme-instigated inflammation: A mini review","volume":"28","author":"Prasher","year":"2019","journal-title":"Med. Chem. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1472-8206.1996.tb00144.x","article-title":"Distinct isoforms (COX-1 and COX-2) of cyclooxygenase: Possible physiological and therapeutic implications","volume":"10","author":"Pairet","year":"1996","journal-title":"Fundam. Clin. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1046\/j.1365-2044.2000.01271.x","article-title":"Cyclo-oxygenase isoenzymes: Physiological and pharmacological role","volume":"55","author":"Kam","year":"2000","journal-title":"Anaesthesia"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1016\/j.phymed.2006.12.006","article-title":"Ergosterol peroxides as phospholipase A2 inhibitors from the fungus Lactarius hatsudake","volume":"14","author":"Gao","year":"2007","journal-title":"Phytomedicine"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.phytol.2018.11.024","article-title":"Anti-inflammatory metabolites from endophytic fungus Fusarium sp.","volume":"29","author":"Khayat","year":"2019","journal-title":"Phytochem. Lett."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1038\/sj.bjp.0707367","article-title":"Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization","volume":"152","author":"Chang","year":"2007","journal-title":"Br. J. Pharmacol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.jmgm.2015.08.010","article-title":"Sterols and triterpenoids as potential anti-inflammatories: Molecular docking studies for binding to some enzymes involved in inflammatory pathways","volume":"62","author":"Salazar","year":"2015","journal-title":"J. Mol. Graph. Model."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1078\/0944-7113-00272","article-title":"Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita","volume":"10","author":"Zhang","year":"2003","journal-title":"Phytomedicine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1820","DOI":"10.1038\/nprot.2008.182","article-title":"Methods for isolation, purification and structural elucidation of bioactive secondary metabolites from marine invertebrates","volume":"3","author":"Ebada","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Silva, C.T., Andrade, P.B., Paiva-Martins, F., Valent\u00e3o, P., and Pereira, D.M. (2017). In Vitro anti-inflammatory and cytotoxic effects of aqueous extracts from the edible sea anemones Anemonia sulcata and Actinia equina. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18030653"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Pereira, D.M., Correia-da-Silva, G., Valent\u00e3o, P., Teixeira, N., and Andrade, P.B. (2014). Anti-inflammatory effect of unsaturated fatty acids and ergosta-7,22-dien-3-ol from Marthasterias glacialis: Prevention of CHOP-mediated ER-stress and NF-\u03baB activation. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0088341"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/S0003-2697(03)00331-2","article-title":"A continuous spectrophotometric assay for phospholipase A2 activity","volume":"319","author":"Cabanes","year":"2003","journal-title":"Anal. Biochem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1016\/j.foodchem.2018.11.082","article-title":"HPLC-DAD-ESI\/MSn phenolic profile and in vitro biological potential of Centaurium erythraea Rafn aqueous extract","volume":"278","author":"Kachmar","year":"2019","journal-title":"Food Chem."}],"container-title":["Marine Drugs"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1660-3397\/17\/6\/330\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T12:55:43Z","timestamp":1760187343000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1660-3397\/17\/6\/330"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,6,3]]},"references-count":39,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2019,6]]}},"alternative-id":["md17060330"],"URL":"https:\/\/doi.org\/10.3390\/md17060330","relation":{},"ISSN":["1660-3397"],"issn-type":[{"value":"1660-3397","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,6,3]]}}}