Antileishmanial and synergic effects of Rhanterium epapposum essential oil and its main compounds alone and combined with glucantime against Leishmania major infection

Abstract

Cutaneous leishmaniasis (CL) is a widespread disease affecting both humans and animals globally. Currently, common treatments (e.g., glucantime (GC) for CL treatment have many side effects that limit their use. The current experimental study aims to assess the in vitro, in vivo, and potential mechanisms of action of Rhanterium epapposum essential oil (REE) and its main compounds β-Myrcene (MC), camphene (CP), and limonene (LN) alone and in combination against Leishmania major. In vitro effects of REE and its main compounds were evaluated on amastigote forms, infection in macrophages cells stimulation of nitric oxide (NO), and stimulation of the cellular immunity in macrophages. In vivo efficacy of REE and its main constituents was also assessed in mice with CL through evaluating parasite burden, oxidative stress and proinflammatory-related genes. A concentration-dependent reduction in the average number of amastigotes was observed, with statistical significance (p < 0.001); whereas the results revealed synergistic effects when REE, MC and LN were combined with GC. REE and main compounds mainly in combination elicited a dose-dependent elevation in NO production and the expression levels of inducible nitric oxide synthase (iNOS), interferon gamma (IFN-γ), and tumor necrosis factor (TNF-α) genes in macrophages. Notably, mice treated with a combination of REE, MC, and GC showed the complete recovery of CL lesions after 28 days of treatment and resulted in a reduction of tissue malondialdehyde levels and a significant increase (p < 0.001) in the gene expression levels of the antioxidant enzymes. Topical treating CL-infected mice with REE and its main compounds alone particularly in conjunction with GC, significantly increased (p < 0.001) the expression levels of IFN-γ and interleukin (IL-12), while also causing a notable reduction in IL-4 expression. The findings of the current experimental research revealed the high in vitro and in vivo antileishmanial efficacy of REE and its main compounds MC, CP, and LN mainly in combination with GC; which indicated the high synergic effects of these compounds.

Keywords: Herbal medicines; In vitro; In vivo; Leishmanicidal; Leishmanisis.

Graphical abstract

Fig. 1

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on inhibitory rate of Leishmania major amastigotes (A) and infection rate (B) in macrophage cells. ∗p < 0.05, ∗∗p < 0.001; ∗∗∗p < 0.001 compared to normal saline. + p < 0.05, ++ p < 0.01, and +++ p < 0.001 compared to GC. (n = 3).

Fig. 2

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on nitric oxide (NO) production in macrophage cells. ∗p < 0.05, ∗∗p < 0.001; ∗∗∗p < 0.001 compared to normal saline. + p < 0.05 compared to GC. (n = 3).

Fig. 3

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on the expression level of interferon gamma (A) and tumor necrosis factor α (B) in macrophage cells. ∗p < 0.05, ∗∗p < 0.001; ∗∗∗p < 0.001 compared to normal saline. + p < 0.05 and ++ p < 0.01 compared to GC. (n = 3).

Fig. 4

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on lesion size of CL-infected mice. ∗p < 0.05, ∗∗p < 0.001; ∗∗∗p < 0.001 compared to normal saline.

Fig. 5

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on the mean number of amastigotes (A), and the parasite burden (B) in the skin of CL-infected mice. ∗p < 0.05, ∗∗p < 0.001; ∗∗∗p < 0.001 compared to normal saline. + p < 0.05 compared to GC.

Fig. 6

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on the tissue level of A: malondialdehyde (MDA) and the expression level of (B) the antioxidant enzymes of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) genes in the CL-infected mice. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001 compared with control normal saline group. + p < 0.05 compared to GC.

Fig. 7

Effects of Rhanterium epapposum essential oil (REE), β-Myrcene (MC), camphene (CP), limonene (LN) and glucantime (GC) alone and in combination on the pro-inflammatory cytokines genes include IFN-γ, IL-4, and IL-10 genes in the CL-infected mice. ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001 compared with control normal saline group. + p < 0.05 compared to GC.