Effect of Hydrodistillation Time on the Composition and Bioactivities ofMelicope pteleifolia Leaf Essential Oils

Introduction: Melicope pteleifolia leaf essential oils (EOs) are traditionally used for inflamed/infected skin, yet the impact of hydrodistillation time on volatile profiles - especially β-ocimenes - and in-vitro bioactivities has not been systematically compared. This study aimed to compare 3, 4, and 6 h distillation (EO3/EO4/EO6) and clarify how time - dependent composition changes relate to anti-inflammatory (NO inhibition) and antibacterial effects. Methods: EOs extracted at 3, 4, and 6 hours were characterized by GC - MS, FT-IR, and UV - Vis spectroscopy. Anti-inflammatory activity was assessed by NO inhibition in LPS-stimulated RAW 264.7 macrophages. Antibacterial effects against Staphylococcus aureus and other pathogens were evaluated via agar well diffusion. All tests were run in triplicate. Results: EO3 was monoterpene-rich (73.29%), dominated by (E)-β-ocimene 50.82% and (Z)-βocimene 12.22% (total 63.04%), whereas EO4/EO6 shifted to sesquiterpenes (78.64% and 83.95%). EO3 showed the strongest NO inhibition (IC50 = 4.03 µg/mL); EO4 and EO6 were less active (ANOVA, p < 0.05). EO3 also produced a 32.3 mm inhibition zone vs S. aureus at 50 µg/mL, while activity against Gram-negative strains was weaker Discussion: Shorter distillation enriched β-ocimene-rich monoterpenes and coincided with stronger NO-inhibitory and anti-S. aureus responses. Chemometric trends (PCA/HCA/PLS-R) were exploratory, given n = 3 oils and support a correlative composition - activity link rather than causality. Differences between Gram-positive and Gram-negative bacteria align with expected permeability/barrier effects. Conclusion: Hydrodistillation time modulates M. pteleifolia EO composition and in-vitro effects; 3 h favored β-ocimene-rich profiles and stronger activities. Findings are in-vitro and correlative; in-vivo/mechanistic studies are warranted for topical applications.