Investigation of Effects of Terpene Skin Penetration Enhancers on Stability and Biological Activity of Lysozyme

The transport of proteins through skin can be facilitated potentially by using terpenes as chemical enhancers. However, we do not know about the effects of these enhancers on the stability and biological activity of proteins which is crucial for the development of safe and efficient formulations. Therefore, this project investigated the effects of terpene-based skin penetration enhancers which are reported as nontoxic to the skin (e.g., limonene, p-cymene, geraniol, farnesol, eugenol, menthol, terpineol, carveol, carvone, fenchone, and verbenone), on the conformational stability and biological activity of a model protein lysozyme. Terpene (5% v/v) was added to lysozyme solution and kept for 24 h (the time normally a transdermal patch remains) for investigating conformational stability profiles and biological activity. Fourier transform infrared spectrophotometer was used to analyze different secondary structures, e.g., α-helix, β-sheet, β-turn, and random coil. Conformational changes were also monitored by differential scanning calorimeter by determining midpoint transition temperature (Tm) and calorimetric enthalpy (ΔH). Biological activity of lysozyme was determined by measuring decrease in A₄₅₀ when it was added to a suspension of Micrococcus lysodeikticus. The results of this study indicate that terpenes 9, 10, and 11 (carvone, l-fenchone, and l-verbenone) decreased conformational stability and biological activity of lysozyme significantly (p < 0.05) less than other terpenes used in this study. It is concluded that smaller terpenes containing ketones with low lipophilicity (log K_ow ∼2.00) would be optimal for preserving conformational stability and biological activity of lysozyme in a transdermal formulation containing terpene as permeation enhancer.Key words: conformational stability, lysozyme, penetration enhancers, protein, terpene