{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:29:46Z","timestamp":1792805386505,"version":"4.5.0"},"reference-count":34,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T00:00:00Z","timestamp":1774310400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Higher Education Commission","award":["NRPU 17549"],"award-info":[{"award-number":["NRPU 17549"]}]},{"name":"Open Access Program from the American University of Sharjah","id":[{"id":"https:\/\/ror.org\/001g2fj96","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cannabis sativa L. leaves (CSL) are a rich in bioactive compounds and known for their medicinal and recreational uses. In this study, a natural hydrophobic deep eutectic solvent (HDES) system composed of menthol and thymol (1:1) was employed for the efficient extraction of bioactive compounds from CSL. Extraction of bioactives was optimized at various conditions involving DES\/ethanol ratio, temperature, and extraction time, as well as shaking speed through statistical models including response surface methodology (RSM) and artificial neural network (ANN). The maximum bioactive yield, equal to 70% (w\/w) of powdered CSL, was achieved at optimized values of 5.5 mL DES, 4.5 mL ethanol, and 225 rpm shaking speed at 55 \u00b0C for 107.5 min. It was observed that slightly adjusting the shaking speed and temperatures customized the nature of bioactives with more antioxidant, antidiabetic, and antimicrobial properties. The extracts of CSL produced while applying natural HDES were found to be non-toxic during hemolytic assay. Overall, HDES when mixed with ethanol in 55:45 ratio produced CSL extracts with an ample level of phenolics (133.75 mg GAE\/g) and flavonoids (120.05 mg QE\/g). GC-MS analysis of CSL extracts produced by HDES revealed the presence of multiple bioactives like tetrahydrocannabivarin, cannabidiol, cannabinol, cannabidivarol, dl-menthol, levomenthol, and 4-hydroxy-3-methylacetophenone. Based on these findings, it can be concluded that HDES in combination with ethanol may work as an efficient extraction solvent to recover CSL bioactives without compromising their antioxidant features and safety for use in food and pharmaceutical applications.<\/jats:p>","DOI":"10.3390\/ijms27072933","type":"journal-article","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T12:58:59Z","timestamp":1774357139000},"page":"2933","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Natural Hydrophobic Deep Eutectic Solvent-Based Enhanced Extraction of Bioactive Compounds from Cannabis sativa L. Leaf for Pharmaceutical Applications"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-0045-9765","authenticated-orcid":false,"given":"Serwat","family":"Naz","sequence":"first","affiliation":[{"name":"Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9818-6832","authenticated-orcid":false,"given":"Sumia","family":"Akram","sequence":"additional","affiliation":[{"name":"Division of Science and Technology, University of Education Lahore, Lahore 54770, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6644-9079","authenticated-orcid":false,"given":"Rabia","family":"Naeem","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6892-079X","authenticated-orcid":false,"given":"Haroon","family":"Iftikhar","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7826-0556","authenticated-orcid":false,"given":"Rizwan","family":"Ashraf","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Agriculture, Faisalabad 38000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noor Ul Ain","family":"Khalid","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9100-6365","authenticated-orcid":false,"given":"Muhammad","family":"Shahid","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Agriculture, Faisalabad 38000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Imad A.","family":"Abu-Yousef","sequence":"additional","affiliation":[{"name":"Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, Sharjah P.O. Box 26666, United Arab Emirates"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0240-1057","authenticated-orcid":false,"given":"Amin F.","family":"Majdalawieh","sequence":"additional","affiliation":[{"name":"Department of Biology, Chemistry and Environmental Sciences, College of Arts and Sciences, American University of Sharjah, Sharjah P.O. Box 26666, United Arab Emirates"},{"name":"Advanced Biosciences and Bioengineering Research Center, American University of Sharjah, Sharjah P.O. Box 26666, United Arab Emirates"},{"name":"Bioinformatics and Computational Biology Research Group, American University of Sharjah, Sharjah P.O. Box 26666, United Arab Emirates"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4013-038X","authenticated-orcid":false,"given":"Muhammad","family":"Mushtaq","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Government College University Lahore, Lahore 54000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1016\/j.rser.2012.10.019","article-title":"Potential of bioenergy production from industrial hemp (Cannabis sativa): Pakistan perspective","volume":"18","author":"Rehman","year":"2013","journal-title":"Renew. Sustain. Energy Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Andre, C.M., Hausman, J.-F., and Guerriero, G. (2016). Cannabis sativa: The plant of the thousand and one molecules. Front. Plant Sci., 7.","DOI":"10.3389\/fpls.2016.00019"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.metabol.2018.09.005","article-title":"The epidemiology of obesity","volume":"92","author":"Chooi","year":"2019","journal-title":"Metabolism"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Valizadehderakhshan, M., Shahbazi, A., Kazem-Rostami, M., Todd, M.S., Bhowmik, A., and Wang, L. (2021). Extraction of Cannabinoids from Cannabis sativa L. (Hemp)\u2014Review. Agriculture, 11.","DOI":"10.3390\/agriculture11050384"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Kinghorn, A.D., Falk, H., Gibbons, S., and Kobayashi, J. (2017). Phytochemistry of Cannabis sativa L.. Phytocannabinoids: Unraveling the Complex Chemistry and Pharmacology of Cannabis sativa, Springer International Publishing.","DOI":"10.1007\/978-3-319-45541-9"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mauriello, A., Giudice, C.D., Vecchio, G.E., Correra, A., Maratea, A.C., Grieco, M., Amata, A., Quagliariello, V., Maurea, N., and Proietti, R. (2025). Takotsubo Syndrome and Oxidative Stress: Physiopathological Linkage and Future Perspectives. Antioxidants, 14.","DOI":"10.3390\/antiox14050522"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1177\/2047487319881021","article-title":"The global epidemics of diabetes in the 21st century: Current situation and perspectives","volume":"26","author":"Standl","year":"2019","journal-title":"Eur. J. Prev. Cardiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s13679-024-00602-y","article-title":"Long-Term Efficacy and Safety of Nutritional and Pharmacological Strategies for Obesity","volume":"14","author":"Barrea","year":"2025","journal-title":"Curr. Obes. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"127202","DOI":"10.1016\/j.molliq.2025.127202","article-title":"Natural deep eutectic solvents (NADES) for sustainable extraction of bioactive compounds from medicinal plants: Recent advances, challenges, and future directions","volume":"425","author":"Huang","year":"2025","journal-title":"J. Mol. Liq."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"110957","DOI":"10.1016\/j.molliq.2019.110957","article-title":"Green extraction of cannabidiol from industrial hemp (Cannabis sativa L.) using deep eutectic solvents coupled with further enrichment and recovery by macroporous resin","volume":"287","author":"Cai","year":"2019","journal-title":"J. Mol. Liq."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8015","DOI":"10.1021\/acssuschemeng.1c03762","article-title":"The Power of the United Nations Sustainable Development Goals in Sustainable Chemistry and Engineering Research","volume":"9","author":"Anastas","year":"2021","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1016\/j.tplants.2020.05.005","article-title":"Phytocannabinoids: Origins and biosynthesis","volume":"25","year":"2020","journal-title":"Trends Plant Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"115895","DOI":"10.1016\/j.trac.2020.115895","article-title":"Recent advances in scaling-up of non-conventional extraction techniques: Learning from successes and failures","volume":"127","author":"Belwal","year":"2020","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"116336","DOI":"10.1016\/j.jece.2025.116336","article-title":"Hydrophobic deep eutectic solvents as green solvents for phenolic compounds extraction from olive mill wastewater","volume":"13","author":"Illoussamen","year":"2025","journal-title":"J. Environ. Chem. Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"143065","DOI":"10.1016\/j.foodchem.2025.143065","article-title":"Ultrasound-assisted ternary temperature-responsive deep eutectic solvents for extraction of carotenoids from tomato samples","volume":"473","author":"Shen","year":"2025","journal-title":"Food Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"104324","DOI":"10.1016\/j.tifs.2023.104324","article-title":"Application of deep eutectic solvents in the extraction of anthocyanins: Stability, bioavailability, and antioxidant property","volume":"144","author":"Foroutani","year":"2024","journal-title":"Trends Food Sci. Technol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1016\/j.matpr.2019.11.133","article-title":"Assessment of maceration, ultrasonic and microwave assisted extraction for total phenolic content, total flavonoid content and kaempferol yield from Cassia alata via microstructures analysis","volume":"19","author":"Ling","year":"2019","journal-title":"Mater. Today Proc."},{"key":"ref_18","first-page":"17","article-title":"Effect of extraction solvent on total polyphenols content and antioxidant activity of Cannabis sativa L.","volume":"38","author":"Koren","year":"2018","journal-title":"Lek. Sirovine"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6961","DOI":"10.15666\/aeer\/1703_69616979","article-title":"Phytochemical screening, total phenolic and flavonoids contents and antioxidant activities of Citrullus colocynthis L. and Cannabis sativa L.","volume":"17","author":"Ahmed","year":"2019","journal-title":"Appl. Ecol. Environ. Res"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"C\u00e1sedas, G., Moliner, C., Maggi, F., Mazzara, E., and L\u00f3pez, V. (2022). Evaluation of two different Cannabis sativa L. extracts as antioxidant and neuroprotective agents. Front. Pharmacol., 13.","DOI":"10.3389\/fphar.2022.1009868"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Haddou, S., Elrherabi, A., Loukili, E.H., Abdnim, R., Hbika, A., Bouhrim, M., Al Kamaly, O., Saleh, A., Shahat, A.A., and Bnouham, M. (2024). Chemical Analysis of the Antihyperglycemic, and Pancreatic \u03b1-Amylase, Lipase, and Intestinal \u03b1-Glucosidase Inhibitory Activities of Cannabis sativa L. Seed Extracts. Molecules, 29.","DOI":"10.3390\/molecules29010093"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"120145","DOI":"10.1016\/j.molliq.2022.120145","article-title":"Dynamics of type V menthol-thymol deep eutectic solvents: Do they reveal non-ideality?","volume":"365","author":"Morineau","year":"2022","journal-title":"J. Mol. Liq."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mushtaq, M., and Akram, S. (2020). Ionic Liquid for the Extraction of Plant Phenolics. Nanotechnology-Based Industrial Applications of Ionic Liquids, Springer.","DOI":"10.1007\/978-3-030-44995-7_5"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4501261","DOI":"10.1155\/2020\/4501261","article-title":"Optimization of extraction conditions of phytochemical compounds and anti-gout activity of Euphorbia hirta L. (Ara Tanah) using response surface methodology and liquid chromatography-mass spectrometry (LC-MS) analysis","volume":"2020","author":"Abdullah","year":"2020","journal-title":"Evid.-Based Complement. Altern. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"101420","DOI":"10.1016\/j.fochx.2024.101420","article-title":"Modeling and optimization of microwave-assisted extraction of total phenolics content from mango (Mangifera indica) peel using response surface methodology (RSM) and artificial neural networks (ANN)","volume":"22","author":"Quintana","year":"2024","journal-title":"Food Chem. X"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Basri, M., Rahman, R.N.Z.R.A., Ebrahimpour, A., Salleh, A.B., Gunawan, E.R., and Rahman, M.B.A. (2007). Comparison of estimation capabilities of response surface methodology (RSM) with artificial neural network (ANN) in lipase-catalyzed synthesis of palm-based wax ester. BMC Biotechnol., 7.","DOI":"10.1186\/1472-6750-7-53"},{"key":"ref_27","first-page":"I1.1.1","article-title":"Determination of total phenolics","volume":"6","author":"Waterhouse","year":"2002","journal-title":"Curr. Protoc. Food Anal. Chem."},{"key":"ref_28","first-page":"75","article-title":"Total phenolic, total flavonoid contents and antioxidant potential of the wild edible mushroom Clitocybe odora","volume":"27","author":"Sevindik","year":"2024","journal-title":"Tarim Ve Doga Derg."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1069","DOI":"10.1007\/s11696-023-03144-0","article-title":"Chemical fingerprinting of Nepeta L. species and antioxidant analysis of their leaves extract","volume":"78","author":"Rasheed","year":"2024","journal-title":"Chem. Pap."},{"key":"ref_30","first-page":"124","article-title":"Wild mushrooms: A potential source of nutritional and antioxidant attributes with acceptable toxicity","volume":"22","author":"Sharif","year":"2017","journal-title":"Prev. Nutr. Food Sci."},{"key":"ref_31","first-page":"240","article-title":"In vitro exposure of whole blood to a cannabinoid mixture impairs the quality of red blood cells and platelets","volume":"21","author":"Lampron","year":"2023","journal-title":"Blood Transfus"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"15593258211050491","DOI":"10.1177\/15593258211050491","article-title":"Biofilm Inhibition and Antibacterial Potential of Different Varieties of Garlic (Allium sativum) Against Sinusitis Isolates","volume":"19","author":"Shahid","year":"2021","journal-title":"Dose-Response"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Casillo, A., D\u2019Angelo, C., Imbimbo, P., Monti, D.M., Parrilli, E., Lanzetta, R., Gomez d\u2019Ayala, G., Mallardo, S., Corsaro, M.M., and Duraccio, D. (2023). Aqueous Extracts from Hemp Seeds as a New Weapon against Staphylococcus epidermidis Biofilms. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242216026"},{"key":"ref_34","first-page":"100243","article-title":"Total phenolic content, antioxidant activity, GC-MS characterization, and antimicrobial activity of leaf and flower extracts of Porophyllum ruderale","volume":"7","author":"Dzigbor","year":"2025","journal-title":"Pharmacol. Res.-Nat. Prod."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2933\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T05:24:39Z","timestamp":1774502679000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/7\/2933"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,24]]},"references-count":34,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ijms27072933"],"URL":"https:\/\/doi.org\/10.3390\/ijms27072933","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,24]]}}}