Seasonal Variation in Essential Oil Composition and Bioactivity of Three Ocimum Species from Nepal

The plants from the <i>Ocimum</i> genus, belonging to the Labiatae family, serve as important bioresources of essential oils (EOs) rich in biologically active secondary metabolites, widely used in medicine, food, and cosmetics. This study explored the volatile composition, enantiomeric distribution, and in vitro biological activities of EOs from three <i>Ocimum</i> species native to Nepal: <i>O. tenuiflorum</i> L., <i>O. basilicum</i> L., and <i>O. americanum</i> L. EOs were extracted via hydro-distillation and analyzed using gas chromatography-mass spectrometry (GC-MS) for chemical profiling and chiral GC-MS for enantiomeric composition. Hierarchical cluster analysis was performed for major chemotypes. Antioxidant activity was assessed using DPPH and ABTS assays. Antimicrobial efficacy was evaluated using the microbroth dilution method, and cytotoxicity was tested on NIH-3T3 (normal) and MCF-7 (breast cancer) cell lines via the Cell Counting Kit-8 assay. EO yield was highest in <i>O. tenuiflorum</i> (1.67 ± 0.13%) during autumn and lowest in <i>O. americanum</i> (0.35 ± 0.02%) during winter among all <i>Ocimum</i> spp. The major compounds identified in <i>O. tenuiflorum</i> were eugenol (32.15-34.95%), <i>trans</i>-β-elemene (29.08-32.85%), and β-caryophyllene (19.85-21.64%). In <i>O. americanum,</i> the major constituents included camphor (51.33-65.88%), linalool (9.72-9.91%), germacrene D (7.75-1.83%), and β-caryophyllene (6.35-3.97%). For <i>O. basicilum,</i> EO was mainly composed of methyl chavicol (62.16-64.42%) and linalool (26.92-27.05%). The oxygenated monoterpenes were a dominant class of terpenes in the EOs except for <i>O. tenuiflorum</i> (sesquiterpene hydrocarbon). A hierarchical cluster analysis based on the compositions of EOs revealed at least three different chemotypes in <i>Ocimum</i> species. Chiral GC-MS analysis revealed β-caryophyllene and germacrene D as enantiomerically pure, with linalool consistently dominant in its levorotatory form. <i>O. tenuiflorum</i> exhibited the strongest antimicrobial activity, particularly against <i>Candida albicans</i>, and showed notable anticancer activity against MCF-7 cells (IC₅₀ = 23.43 µg/mL), with lower toxicity to normal cells. It also demonstrated the highest antioxidant activity (DPPH IC₅₀ = 69.23 ± 0.10 µg/mL; ABTS IC₅₀ = 9.05 ± 0.24 µg/mL). The EOs from <i>Ocimum</i> species possess significant antioxidant, antimicrobial, and cytotoxic properties, especially <i>O. tenuiflorum</i>. These findings support their potential application as natural agents in medicine, food, and cosmetics, warranting further validation.