Antileishmanial activity of a linalool-rich essential oil from Croton cajucara

Abstract

The in vitro leishmanicidal effects of a linalool-rich essential oil from the leaves of Croton cajucara against Leishmania amazonensis were investigated. Morphological changes in L. amazonensis promastigotes treated with 15 ng of essential oil per ml were observed by transmission electron microscopy; leishmanial nuclear and kinetoplast chromatin destruction, followed by cell lysis, was observed within 1 h. Pretreatment of mouse peritoneal macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these macrophages and L. amazonensis, with a concomitant increase by 220% in the level of nitric oxide production by the infected macrophages. Treatment of preinfected macrophages with 15 ng of essential oil per ml reduced by 50% the interaction between these cells and the parasites, which led to a 60% increase in the amount of nitric oxide produced by the preinfected macrophages. These results provide new perspectives on the development of drugs with activities against Leishmania, as linalool-rich essential oil is a strikingly potent leishmanicidal plant extract (50% lethal doses, 8.3 ng/ml for promastigotes and 8.7 ng/ml for amastigotes) which inhibited the growth of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and which presented no cytotoxic effects against mammalian cells.

FIG. 1.

Chemical structure of linalool.

FIG. 2.

Time courses of the viabilities of L. amazonensis promastigotes and mouse peritoneal macrophages in the absence or presence of linalool-rich essential oil (15.0 ng/ml) extracted from C. cajucara. The viabilities of both parasites and macrophages were calculated as described in the text. •, L. amazonensis promastigotes (control); ○, L. amazonensis promastigotes and essential oil; ▪, L. amazonensis amastigotes (control); □, L. amazonensis amastigotes and essential oil; ▾, mouse peritoneal macrophages (control); ▿, mouse peritoneal macrophages and esssential oil

FIG. 3.

Effects of linalool-rich essential oil (15.0 ng/ml) extracted from C. cajucara on promastigote forms of L. amazonensis observed by transmission electron microscopy. (A) Control parasites; (B to E) parasites treated for 5 (B), 10 (C), 15 (D), and 30 (E) min, showing promastigotes with different degrees of damage. Note the disruption of flagellar membranes (arrowheads in panels B and C), the mitochondrial swelling (C and D), and the gross alterations in the organization of the nuclear and kinetoplast chromatins (C and D). After 30 min in the presence of essential oil the parasites were completely destroyed (E). N, nucleus; K, kinetoplast, F, flagellum. Bars, 1 μm.

FIG. 4.

Effects of linalool-rich essential oil extracted from C. cajucara on the L. amazonensis-macrophage interaction. The parasites and/or the mouse peritoneal macrophages were either not treated or treated with 15, 1.5, and 0.2 ng of essential oil per ml 20 min prior to the macrophage-parasite interactions. The adherent cultured macrophages and the free parasites were washed once and resuspended in fresh culture medium. Dead parasites were removed from the medium by centrifugation (1,000 × g, 5 min), and intact living L. amazonensis promastigotes were then added to the macrophage culture plate wells. Association indices were determined by light microscopy, with 600 cells in triplicate coverslips counted after 90 min of interaction. Each bar represents the mean ± standard error of at least three independent experiments, which were performed in triplicate. The association indices from assays performed with macrophages and/or parasites pretreated with essential oil are significantly different from the association indices for control (nontreated) macrophages.

FIG. 5.

Effects of linalool-rich essential oil extracted from C. cajucara on nitric oxide production by mouse peritoneal macrophages (mφ). The parasites and/or the macrophages were either not treated or treated with 15, 1.5, and 0.2 ng of essential oil per ml 20 min prior to the macrophage-parasite interactions. The adherent cultured macrophages and the free parasites were washed once and resuspended in fresh culture medium. Dead parasites were removed from the medium by centrifugation (1,000 × g, 5 min), and intact living L. amazonensis promastigotes were then added to the macrophage culture plate wells. The supernatants from control and L. amazonensis-infected macrophages were recovered, and the nitrite concentration of each system was determined by the Griess reaction, as described in the Materials and Methods section. Each bar represents the mean ± standard error of at least three independent experiments, which were performed in triplicate. The asterisks indicate that the result is significantly different from that for the infected macrophages when neither the macrophages nor the parasites were treated with essential oil.