Enhanced cuticular penetration as the mechanism of synergy for the major constituents of thyme essential oil in the cabbage looper, Trichoplusia ni

For the past two decades, the insecticidal activity of botanicals, particularly plant essential oils has been gaining broad research interest. Due to the diverse chemical composition of essential oils, they often exhibit complex interactions among their major constituents, for example, synergistic or antagonistic relationships. Several hypotheses have been proposed for synergistic toxicity, including multiple modes-of-action and inhibition of metabolism, but the mechanism underlying synergy has not been fully elucidated. In the present study, interactions among the four major constituents of thyme (Thymus vulgaris) essential oil against the larvae of the cabbage looper were examined following topical administration. Two statistical models were used to determine the interactions, and several synergistic relationships were identified in natural proportional and equivalent blending ratios, but no antagonistic effects were observed. Among binary mixtures, thymol and p-cymene was synergistic in a topical application assay. GC-MS analyses showed increased recovery of thymol in the mixture both in vivo and in vitro from hemolymph extracts and receiver solutions, respectively, indicating enhanced penetration of thymol through the integument by p-cymene. An injection assay in fifth instar larvae and an in vitro cytotoxicity assay in an ovarian cell line of the cabbage looper showed no internal boosting effect of thymol by p-cymene, suggesting p-cymene facilitates the penetration of thymol, but does not directly contribute to increased toxicity of the mixture. A divided application method confirmed that the increased toxicity of the mixture can be produced only if the two compounds were applied as a mixture, proving p-cymene's role as a penetration enhancer. Contact angle measurement on a waxy surface showed lowered surface tension of thymol in the mixture, suggesting the surface tension can directly influence the penetration and efficacy of a toxicant in pesticide formulation design.