Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

Abstract

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×106 parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC50 values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC50 values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC50 values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC50 values. For assays with high cell concentrations (2×109 parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC50 values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.

Effects of terpenes on fluidity and lipid extraction in phospholipid membranes

“…Collectively, these results demonstrated that terpenes also cause the formation of two components in a cell membrane and that an increase in the terpene concentration leads to an increase in the population of the most dynamic spin probe component [16].…”

“…Moreover, at their respective IC 50 values, these terpenes caused the lysis of 4 to 9% of cells, and this percentage reached up to approximately 50% at concentrations two to three times higher than the IC 50 values [16]. The IC 50 values of the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole were 0.008, 0.549, 0.678 and 4.697 mM, respectively [16]. This result demonstrated that the in vitro cytotoxicity of nerolidol was similar to that of miltefosine, which is the first effective oral drug approved for the treatment of visceral and cutaneous leishmaniasis [32].…”

“…With its high membrane-water partition coefficient, nerolidol was the terpene that caused the greatest changes in the lipid bilayer, leading to an apparent disintegration at the highest Recently, EPR spectroscopy showed that the terpenes nerolidol, (+)-limonene, α-terpineol and 1,8-cineole dramatically increase the molecular dynamics of the lipid component in the plasma membrane of L. amazonensis promastigotes at terpene concentrations similar to those that inhibit 50% of the parasite growth [16]. Moreover, at their respective IC 50 values, these terpenes caused the lysis of 4 to 9% of cells, and this percentage reached up to approximately 50% at concentrations two to three times higher than the IC 50 values [16]. The IC 50 values of the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole were 0.008, 0.549, 0.678 and 4.697 mM, respectively [16].…”

“…The intercellular lipid matrix of the outermost skin layer, the stratum corneum, which is the major permeability barrier of the skin, becomes more fluid in the presence of the monoterpenes L-menthol and 1,8-cineole [12,13], and several monoterpenes increase the membrane partition coefficients of the small water-soluble spin labels TEMPO and DTBN in stratum corneum membranes [14,15]. Recently, EPR spectroscopy has been used to show that terpenes can cause pronounced increases in the plasma membrane fluidity of Leishmania amazonensis promastigotes at terpene concentrations similar to those that inhibit the growth of the parasite [16]. In addition, the terpenes nerolidol, (+)-limonene, α-terpineol and 1,8-cineole at concentrations that inhibited the growth of Leishmania parasites by 50% caused cell lysis of 4 to 9%, and this percentage rose to approximately 50% for concentrations that were double or triple the IC 50 values [16].…”

“…Recently, EPR spectroscopy has been used to show that terpenes can cause pronounced increases in the plasma membrane fluidity of Leishmania amazonensis promastigotes at terpene concentrations similar to those that inhibit the growth of the parasite [16]. In addition, the terpenes nerolidol, (+)-limonene, α-terpineol and 1,8-cineole at concentrations that inhibited the growth of Leishmania parasites by 50% caused cell lysis of 4 to 9%, and this percentage rose to approximately 50% for concentrations that were double or triple the IC 50 values [16]. The cytotoxicity of seven monoterpenes and the sesqui-terpene nerolidol in cultured fibroblast cells also correlates with the hemolytic potential of these terpenes [17].…”