Evaluation of the biocidal activity of origanum vulgare l. essential oil in nanoemulsion

Biocides are substances or mixtures that destroy or neutralise harmful agents through non-mechanical mechanisms (1). Among the natural alternatives, essential oils (EOs), such as Origanum vulgare L. (OEO), have received attention due to their antimicrobial properties for their antimicrobial properties. They can replace or complement synthetic antimicrobials, but their application is limited (2). To overcome these limitations, these volatile oils have been incorporated into various nano-sized colloidal carriers (3) . Different studies have reported that nanoemulsions (NEs) to deliver essential oils (EOs) improve their water solubility and facilitate their use in antimicrobial formulations, considering that the nanometre size of the droplets may enhance the interaction with microbial cells (4). This study evaluated the antifungal activity of lipid-based NEs containing OEO and its primary component, carvacrol (CAR), against Candida albicans. The NEs were prepared using EO and soy phosphatidylcholine (soy-PC) as the main surfactant. The amount of soy-PC was carefully optimized to produce homogeneous NEs with a stable interfacial film capable of ensuring long-term dispersion stability. Co-surfactants (phenylacetic acid (PAA), benzyl alcohol (BA) and benzalkonium chloride (BK) were included in the formulation to modify the surface characteristics of the NEs and impart negative or positive charges. CAR was also encapsulated in NEs and compared with OEO dispersed in Tween 80 aqueous solution. GC/MS and HS-GC/MS analyses evaluated the formulations' chemical composition and evaporation behaviour. Antimicrobial efficacy was tested on three vaginal C. albicans isolates using broth microdilution assays to determine the MIC and MFC. Literature suggests that positively charged nanodroplets may enhance fungal uptake due to electrostatic interactions with negatively charged membranes. However, our research found no significant advantage for positively charged NEs over negatively charged ones regarding antifungal activity. Both charged-NEs exhibited comparable inhibition, suggesting that surface charge is not a critical factor for activity in this system. Soy-PC and charged non- phospholipid surfactants effectively produce stable oil-in-water NEs via a low-energy emulsification method. Nanoencapsulated OEO and CAR are more effective against fungi than non-formulated OEO, even when negatively charged NEs are used. These formulations are a promising alternative as green biocides. References and Citations 1. Regolamento - 528/2012 - EN - EUR-Lex. [cited 2025 Feb 12]. Available from: https://eur- lex.europa.eu/eli/reg/2012/528/oj/eng. 2. Li L, Li X, McClements DJ, Jin Z, Ji H, Qiu C. Recent progress in the source, extraction, activity mechanism and encapsulation of bioactive essential oils. Crit Rev Food Sci Nutr. 2024 Dec 25:1-19. doi: 10.1080/10408398.2024.2439040 3.  Jugreet BS, Suroowan S, Rengasamy RRK, Mahomoodally MF. Chemistry, bioactivities, mode of action and industrial applications of essential oils. Trends Food Sci Technol. 2020;101:89–105. doi: 10.1016/j.tifs.2020.04.025. 4. Paolicelli P., Petralito P., Trilli J., Di Muzio L., Garzoli S., Casadei M.A.; Encapsulation of Essential Oils within Lipid-Based Formulations for Enhanced Antimicrobial Activity, Terpenes Medicinal Chemistry Lessons From Nature (2023) 2: 94https://doi.org/10.2174/9789815123647123020006 Bentham Science Publisher Online ISSN 2972-340X