EEG-Based Neurophysiological Assessment of Pharmacopoeial-Quality Essential Oils and Development of Stable Micro-Emulsion Systems for Aromatherapy Applications

Background/Objectives: Despite the widespread use of essential oils in aromatherapy, objective evidence supporting their neurophysiological effects remains limited. This study evaluated the effects of selected pharmacopoeial-quality essential oils (lavender, rosemary, lemon, and peppermint as well as a blend of these oils) on electroencephalographic (EEG) activity following inhalation and developed and characterized microemulsion formulations guided by exploratory EEG response patterns, with preliminary in vitro cytotoxicity assessment. Methods: EEG recordings were obtained from 28 healthy volunteers following inhalation of the essential oils. Neurophysiological responses were evaluated using exploratory paired comparisons between each oil and its corresponding grape-seed oil control, together with repeated-measures ANOVA of normalized EEG responses. Selected oils were formulated as microemulsions (M1-M3), which were characterized by their physicochemical properties. Cytotoxicity was assessed in HaCaT human keratinocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Exploratory paired comparisons suggested localized EEG changes, particularly in the alpha-1 frequency band. However, these effects did not remain statistically significant after correction for multiple comparisons, and repeated-measures ANOVA of normalized EEG responses did not demonstrate significant overall differences among oil conditions, although the alpha-1 band exhibited the most distinct response pattern. Microemulsions exhibited nanoscale droplet sizes and favorable physicochemical stability. The IC50 values of M1 and M2 were greater than 2000 µg/mL, whereas M3 had an IC50 of approximately 1220 µg/mL. Conclusions: Pharmacopoeial-quality essential oils produced exploratory, region-specific EEG response patterns, with the alpha-1 band showing the most consistent response profile. The developed microemulsion formulations demonstrated suitable physicochemical stability and generally low cytotoxicity, supporting their further evaluation as aromatherapy product prototypes.