{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:39:26Z","timestamp":1760146766387,"version":"build-2065373602"},"reference-count":38,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2024,12,10]],"date-time":"2024-12-10T00:00:00Z","timestamp":1733788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science, Technological Development and Innovation of the Republic of Serbia","award":["451-03-66\/2024-03\/200134","451-03-65\/2024-03\/200161","451-03-66\/2024-03\/200161"],"award-info":[{"award-number":["451-03-66\/2024-03\/200134","451-03-65\/2024-03\/200161","451-03-66\/2024-03\/200161"]}]},{"name":"two Grant Agreements between the Ministry of Science, Technological Development, and Innovation of the Republic of Serbia and the University of Belgrade, Faculty of Pharmacy","award":["451-03-66\/2024-03\/200134","451-03-65\/2024-03\/200161","451-03-66\/2024-03\/200161"],"award-info":[{"award-number":["451-03-66\/2024-03\/200134","451-03-65\/2024-03\/200161","451-03-66\/2024-03\/200161"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Since ancient times, many plant species within the Thymus genus have been used due to their numerous health benefits, such as antimicrobial, anti-inflammatory, antiseptic, or diuretic activity. While many of the species within this genus were well known and described, Thymus pannonicus All. or Pannonian thyme remains relatively unexplored despite its unique chemical composition and activity. T. pannonicus is a small shrub, spread over central and eastern Europe, with a diverse, location-dependent chemical composition. Within this study, the citral chemotype of T. pannonicus was used, which up to our knowledge was found and described only in Northern Serbia. Therefore, in order to explore the composition of T. pannonicus low-polar extracts and to present a preliminary insight into their bioactivity, supercritical carbon dioxide (ScCO2) extraction was applied (with and without pre-treatments), followed by GC-MS analysis. Different ScCO2 extraction pressures were tested (100, 150, 200, 250, and 300 bar) in order to determine the most suitable conditions for the extraction of T. pannonicus\u2019s low-polar fraction. Additionally, in order to maximize the yield of the extraction, two types of ScCO2 extraction pre-treatments were investigated (enzymatic and microwave). The highest extraction yield obtained from untreated plant material was 3.01% (w\/w), and it was obtained at conditions of 150 bar and 40 \u00b0C, while the 4% (v\/w) enzymatic pre-treatment, with the same conditions, provided a yield of 3.89%. For all of the obtained extracts, the GC-MS analysis showed that oxygenated monoterpenes and sesquiterpenes were the two most dominant groups with principal bioactive compounds such as (E)-citral (18.95\u201338.17%), (Z)-citral (6.68\u201314.66%), \u03b2-bisabolene (8.2\u201314.4%), and nerol (6.08\u20139.67%). The extracts that exhibited the highest concentration of principal bioactive compounds ((Z)-citral, and (E)-citral) were further analyzed for anticancer potential, using short- and long-term cell viability observations on liver cancer cells.<\/jats:p>","DOI":"10.3390\/plants13243457","type":"journal-article","created":{"date-parts":[[2024,12,10]],"date-time":"2024-12-10T04:15:03Z","timestamp":1733804103000},"page":"3457","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Unlocking the Unique Potential of Thymus pannonicus: Exploring the Efficacy of Supercritical CO2 Extraction, with and Without Pre-Treatments"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7394-5222","authenticated-orcid":false,"given":"Sini\u0161a","family":"Simi\u0107","sequence":"first","affiliation":[{"name":"Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Senka","family":"Vidovi\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0678-7660","authenticated-orcid":false,"given":"Stela","family":"Joki\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuha\u010da 18, 31000 Osijek, Croatia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8595-6065","authenticated-orcid":false,"given":"Nata\u0161a","family":"Mili\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Krunoslav","family":"Aladi\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuha\u010da 18, 31000 Osijek, Croatia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2717-0555","authenticated-orcid":false,"given":"Zoran","family":"Maksimovi\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jovana","family":"Drlja\u010da Lero","sequence":"additional","affiliation":[{"name":"Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8904-6993","authenticated-orcid":false,"given":"Aleksandra","family":"Gavari\u0107","sequence":"additional","affiliation":[{"name":"Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2704","DOI":"10.1016\/j.arabjc.2015.05.017","article-title":"Influence of harvesting time on composition of the essential oil of Thymus capitatus (L.) Hoffmanns. & Link. growing wild in northern Sicily and its activity on microorganisms affecting historical art crafts","volume":"12","author":"Casiglia","year":"2019","journal-title":"Arab. J. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1688","DOI":"10.1002\/ptr.6109","article-title":"Thymol, thyme, and other plant sources: Health and potential uses","volume":"32","author":"Salehi","year":"2018","journal-title":"Phytother. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1080\/J157v04n01_07","article-title":"Thyme (Thymus vulgaris L.), thymol","volume":"4","author":"Basch","year":"2004","journal-title":"J. Herb. Pharmacother."},{"key":"ref_4","first-page":"1934578X1000501034","article-title":"Essential oil polymorphism of wild growing Hungarian thyme (Thymus pannonicus) populations in the Carpathian Basin","volume":"5","year":"2010","journal-title":"Nat. Prod. Commun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"149","DOI":"10.2478\/s11535-008-0013-x","article-title":"Chemical composition and antimicrobial activity of Thymus pannonicus All.(Lamiaceae) essential oil","volume":"3","year":"2008","journal-title":"Open Life Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"560","DOI":"10.1007\/s13197-017-2965-1","article-title":"Potential use of deodorised water extracts: Polyphenol-rich extract of Thymus pannonicus All. as a chemopreventive agent","volume":"55","author":"Drobac","year":"2018","journal-title":"J. Food Sci. Technol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129525","DOI":"10.1016\/j.chemosphere.2020.129525","article-title":"Supercritical carbon dioxide extraction of plant phytochemicals for biological and environmental applications\u2014A review","volume":"271","author":"Arumugham","year":"2021","journal-title":"Chemosphere"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.cep.2012.02.003","article-title":"Effect of CO2 pre-treatment on scCO2 extraction of natural material","volume":"56","author":"Meyer","year":"2012","journal-title":"Chem. Eng. Process. Process Intensif."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/j.supflu.2012.12.022","article-title":"Influence of pretreatment on supercritical CO2 extraction from Nannochloropsis oculata","volume":"79","author":"Crampon","year":"2013","journal-title":"J. Supercrit. Fluids"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Vladi\u0107, J., Duarte, A.R.C., Radman, S., Simi\u0107, S., and Jerkovi\u0107, I. (2021). Enzymatic and microwave pretreatments and supercritical CO2 extraction for improving extraction efficiency and quality of Origanum vulgare L. spp. hirtum extracts. Plants, 11.","DOI":"10.3390\/plants11010054"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Afonso, A.F., Pereira, O.R., and Cardoso, S.M. (2020). Health-promoting effects of Thymus phenolic-rich extracts: Antioxidant, anti-inflammatory and antitumoral properties. Antioxidants, 9.","DOI":"10.3390\/antiox9090814"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/j.supflu.2007.09.024","article-title":"Pressure effect in supercritical CO2 extraction of plant seeds","volume":"44","author":"Machmudah","year":"2008","journal-title":"J. Supercrit. Fluids"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"112072","DOI":"10.1016\/j.indcrop.2019.112072","article-title":"Production of thymol rich extracts from ajwain (Carum copticum L.) and thyme (Thymus vulgaris L.) using supercritical CO2","volume":"145","author":"Morsy","year":"2020","journal-title":"Ind. Crops Prod."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1016\/j.cherd.2020.09.032","article-title":"Supercritical carbon dioxide extraction of essential oil from tangerine peel: Experimental optimization and kinetics modelling","volume":"164","author":"Xiong","year":"2020","journal-title":"Chem. Eng. Res. Des."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.tibtech.2011.06.014","article-title":"Enzyme-assisted extraction of bioactives from plants","volume":"30","author":"Puri","year":"2012","journal-title":"Trends Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Krakowska-Sieprawska, A., Kie\u0142basa, A., Rafi\u0144ska, K., Ligor, M., and Buszewski, B. (2022). Modern methods of pre-treatment of plant material for the extraction of bioactive compounds. Molecules, 27.","DOI":"10.3390\/molecules27030730"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1111\/j.1749-6632.2009.05255.x","article-title":"Enzyme technology for precision functional food ingredient processes","volume":"1190","author":"Meyer","year":"2010","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.tifs.2005.12.004","article-title":"Recent advances in extraction of nutraceuticals from plants","volume":"17","author":"Wang","year":"2006","journal-title":"Trends Food Sci. Technol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1016\/j.ifset.2008.05.004","article-title":"Effect of pretreatment with microwaves on mechanical extraction yield and quality of vegetable oil from Chilean hazelnuts (Gevuina avellana Mol)","volume":"9","author":"Uquiche","year":"2008","journal-title":"Innov. Food Sci. Emerg. Technol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.supflu.2015.08.006","article-title":"Microwave pretreatment of Moringa oleifera seed: Effect on oil obtained by pilot-scale supercritical carbon dioxide extraction and Soxhlet apparatus","volume":"107","author":"Decorti","year":"2016","journal-title":"J. Supercrit. Fluids"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1111\/j.1472-8206.2009.00738.x","article-title":"Citral inhibits cell proliferation and induces apoptosis and cell cycle arrest in MCF-7 cells","volume":"23","author":"Chaouki","year":"2009","journal-title":"Fundam. Clin. Pharmacol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1016\/j.biopha.2017.10.038","article-title":"Antiproliferative and apoptosis inducing effects of citral via p53 and ROS-induced mitochondrial-mediated apoptosis in human colorectal HCT116 and HT29 cell lines","volume":"96","author":"Sheikh","year":"2017","journal-title":"Biomed. Pharmacother."},{"key":"ref_23","first-page":"107","article-title":"Antidiabetic Potential and Metabolite Profile of Leaf and Stem Extract of Castanopsis tungurrut (Blume) A. DC","volume":"1","author":"Nabila","year":"2024","journal-title":"J. Pharm. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1515\/znc-2007-7-808","article-title":"Screening of antibacterial activities of twenty-one oxygenated monoterpenes","volume":"62","author":"Kotan","year":"2007","journal-title":"Z. F\u00fcr Naturforschung C"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1590\/S0102-695X2007000400002","article-title":"Chemical composition and antitumor activity of different wild varieties of Moroccan thyme","volume":"17","author":"Jaafari","year":"2007","journal-title":"Rev. Bras. Farmacogn."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3200","DOI":"10.3390\/molecules15053200","article-title":"Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells","volume":"15","author":"Zu","year":"2010","journal-title":"Molecules"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e080","DOI":"10.3989\/gya.0955142","article-title":"Essential oils from Egyptian aromatic plants as antioxidant and novel anticancer agents in human cancer cell lines","volume":"66","author":"Ramadan","year":"2015","journal-title":"Grasas Y Aceites"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.tifs.2019.01.020","article-title":"Thymus spp. plants-Food applications and phytopharmacy properties","volume":"85","author":"Salehi","year":"2019","journal-title":"Trends Food Sci. Technol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1002\/1097-0215(20001015)88:2<287::AID-IJC22>3.0.CO;2-U","article-title":"Overcoming cisplatin resistance in vitro by a free and liposome-encapsulated bile acid derivative: BAMET-R2","volume":"88","author":"Briz","year":"2000","journal-title":"Int. J. Cancer"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1515\/znc-2020-0070","article-title":"Essential oil chemical composition, antimicrobial, anticancer, and antioxidant effects of Thymus convolutus Klokov in Turkey","volume":"76","author":"Kockar","year":"2021","journal-title":"Z. F\u00fcr Naturforschung C"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1007\/BF02535593","article-title":"Geraniol interferes with membrane functions in strains of Candida and Saccharomyces","volume":"23","author":"Bard","year":"1988","journal-title":"Lipids"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1128\/mr.59.2.201-222.1995","article-title":"Mechanisms of membrane toxicity of hydrocarbons","volume":"59","author":"Poolman","year":"1995","journal-title":"Microbiol. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1289\/ehp.99107805","article-title":"p53 induction as a genotoxic test for twenty-five chemicals undergoing in vivo carcinogenicity testing","volume":"107","author":"Yang","year":"1999","journal-title":"Environ. Health Perspect."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1080\/01480545.2019.1585445","article-title":"Citral presents cytotoxic and genotoxic effects in human cultured cells","volume":"43","author":"Souza","year":"2020","journal-title":"Drug Chem. Toxicol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.mrgentox.2006.02.005","article-title":"Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS","volume":"606","author":"Bakkali","year":"2006","journal-title":"Mutat. Res.\/Genet. Toxicol. Environ. Mutagen."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1016\/j.foodchem.2013.03.090","article-title":"Cytotoxic impact of phenolics from Lamiaceae species on human breast cancer cells","volume":"141","author":"Berdowska","year":"2013","journal-title":"Food Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1447","DOI":"10.1111\/cns.13889","article-title":"Diazepam diminishes temozolomide efficacy in the treatment of U87 glioblastoma cell line","volume":"28","year":"2022","journal-title":"CNS Neurosci. Ther."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/0022-1759(83)90303-4","article-title":"Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays","volume":"65","author":"Mosmann","year":"1983","journal-title":"J. Immunol. Methods"}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/13\/24\/3457\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:51:07Z","timestamp":1760115067000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/13\/24\/3457"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,10]]},"references-count":38,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["plants13243457"],"URL":"https:\/\/doi.org\/10.3390\/plants13243457","relation":{},"ISSN":["2223-7747"],"issn-type":[{"type":"electronic","value":"2223-7747"}],"subject":[],"published":{"date-parts":[[2024,12,10]]}}}