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methodology (RSM), with the primary goal of maximizing the extraction of bioactive compounds from Thymus vulgaris L. Eight phenolic compounds were identified in the extracts, with rosmarinic acid being the most abundant. Depending on the selected conditions in each optimization, the concentration of this compound varied from 2.04 to 72.6 mg\/g of extract for HAE and 0.73 to 73.5 mg\/g of extract for UAE. The optimal conditions for each method that yielded the highest rosmarinic acid levels were as follows: for HAE, 27% ethanol at 91 \u00b0C for 121 min; and for UAE, 60 min at 500 W using 50% ethanol. The UAE-produced extract demonstrated superior antioxidant performance, particularly in inhibiting the formation of thiobarbituric acid reactive substances and oxidative haemolysis. Additionally, the extracts obtained using both optimized methodologies exhibited cytotoxic activity against five tumor cell lines and showed significant bactericidal and fungicidal effects against six bacterial and fungal strains. Overall, UAE proved more efficient as it enables the production of rosmarinic-acid-enriched extracts in less time, making them suitable as natural ingredients for industrial applications.<\/jats:p>","DOI":"10.3390\/antiox14050567","type":"journal-article","created":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T08:20:23Z","timestamp":1746778823000},"page":"567","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Effect of Heat-Assisted and Ultrasound-Assisted Extraction Methods on the Phenolic Profile and Biological Activity of Thymus vulgaris L. Extracts"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2017-6401","authenticated-orcid":false,"given":"Rafael Mascoloti","family":"Spr\u00e9a","sequence":"first","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"},{"name":"Nutrition and Food Group (NuFoG), Department of Analytical Chemistry and Food Science, Instituto de Agroecolox\u00eda e Alimentaci\u00f3n (IAA)-CITEXVI, Universidade de Vigo, 36310 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5769-8484","authenticated-orcid":false,"given":"Cristina","family":"Caleja","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2854-6745","authenticated-orcid":false,"given":"Eliana","family":"Pereira","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8978-4547","authenticated-orcid":false,"given":"M\u00e1rcio","family":"Carocho","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7523-1637","authenticated-orcid":false,"given":"Jos\u00e9","family":"Pinela","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"},{"name":"National Institute for Agricultural and Veterinary Research (INIAV), I.P., Rua dos L\u00e1gidos, Lugar da Madalena, 4485-655 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3516-0795","authenticated-orcid":false,"given":"Tiane C.","family":"Finimundy","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6801-4578","authenticated-orcid":false,"given":"Ricardo C.","family":"Calhelha","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8717-6495","authenticated-orcid":false,"given":"Marina","family":"Kosti\u0107","sequence":"additional","affiliation":[{"name":"Institute for Biological Research \u201cSini\u0161a Stankovi\u0107\u201d-National Institute of Republic of Serbia, University of Belgrade, 11000 Belgrade, Serbia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7381-756X","authenticated-orcid":false,"given":"Marina","family":"Sokovi\u0107","sequence":"additional","affiliation":[{"name":"Institute for Biological Research \u201cSini\u0161a Stankovi\u0107\u201d-National Institute of Republic of Serbia, University of Belgrade, 11000 Belgrade, Serbia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3513-0054","authenticated-orcid":false,"given":"Miguel A.","family":"Prieto","sequence":"additional","affiliation":[{"name":"Nutrition and Food Group (NuFoG), Department of Analytical Chemistry and Food Science, Instituto de Agroecolox\u00eda e Alimentaci\u00f3n (IAA)-CITEXVI, Universidade de Vigo, 36310 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3648-7303","authenticated-orcid":false,"given":"Joana S.","family":"Amaral","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9050-5189","authenticated-orcid":false,"given":"Lillian","family":"Barros","sequence":"additional","affiliation":[{"name":"CIMO, LA SusTEC, Instituto Polit\u00e9cnico de Bragan\u00e7a, Campus de Santa Apol\u00f3nia, 5300-253 Bragan\u00e7a, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,9]]},"reference":[{"key":"ref_1","unstructured":"Florou-Paneri, P., Christaki, E., and Giannenas, I. 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