Meliponini Geopropolis Extracts Induce ROS Production and Death in Leishmania amazonensis Promastigotes and Axenic Amastigotes In Vitro

Abstract

Leishmania amazonensis, a cause of cutaneous leishmaniasis in Brazil, is a neglected disease with toxic and inconsistently effective treatments. The parasite's survival depends on managing oxidative stress, making redox-regulating enzymes potential therapeutic targets. Geopropolis, a resinous product from native stingless bees, shows promising antiparasitic effects. This study aims to evaluate the anti-L. amazonensis activity of geopropolis produced by Melipona bicolor, M. marginara, M. mondury, and M. quadrifasciata (two samples), targeting enzymes responsible for the parasite's redox balance. Ethanol extracts of geopropolis produced by each bee (BCRL, MRGT, MNDY, MNDA(1), and MNDA(2), respectively) were analyzed for total phenolics and flavonoids. Promastigotes and axenic amastigotes were treated with various extract concentrations, and parasite viability was assessed using the resazurin reduction method. Cytotoxicity was tested on peritoneal macrophages, RAW 264.7, VERO cell lines (MTT assay), and erythrocytes (hemolysis assay). Additionally, mitochondrial dehydrogenase activity, reactive oxygen species (ROS) production, the inhibition of recombinant arginase, and autophagic activity were also evaluated in treated parasites. MRGT showed the highest levels of phenolics (762 mg GAE/g) and flavonoids (345 mg QE/g). MDRY was more effective against promastigote and axenic amastigote forms (IC₅₀ = 168 and 19.7 µg/mL, respectively). MRGT showed lower cytotoxicity against RAW 264.7 and VERO (CC₅₀ = 654 µg/mL and 981 µg/mL, respectively). Erythrocytes exhibited reduced sensitivity to MNDA(2) (HC₅₀ = 710 µg/mL). The activity of dehydrogenases and LiARG was reduced by treating the parasites with the extracts following the induction of ROS and autophagic activity. These results highlight geopropolis extracts as a source of substances with anti-L. amazonensis activity capable of inducing oxidative stress on the parasite.

Keywords: human cutaneous leishmaniasis; leishmanicidal activity; mitochondrial activity; native bee products.

Figure 1

Spectral profiles and chemometric analysis of geopropolis extracts. (a) One-dimensional ¹H NMR spectra of geopropolis extracts; (b) Principal Component Analysis (PCA) plot, showing the grouping of extracts based on their chemical profiles; (c) loadings plot highlighting the spectral regions (0.04 ppm buckets) that contribute most significantly to the differentiation of geopropolis extracts; (d,e) key spectral regions in the lower left (3.351 ppm) and upper right (3.390 ppm) identified as the primary contributors to the extracts’ discrimination. BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata.

Figure 2

The effect of geopropolis extracts on the growth of L. amazonensis promastigotes. (a) Parasites treated with BCLR extract; (b) Parasites treated with MDRY extract; (c) Parasites treated with MNDA(1) extract; (d) Parasites treated with MNDA(2) extract; (e) Parasites treated with MRGT extract; and (f) Parasites treated with SbIII (reference drug). BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata; and SbIII: antimony potassium tartrate trihydrate. The experiments were performed in triplicate, and the results are expressed as mean ± standard error.

Figure 3

The effects of treatment with geopropolis extracts on mitochondrial dehydrogenases in L. amazonensis promastigotes after treatment with the extracts at concentrations corresponding to the IC₅₀ and 2×(IC₅₀). (a) Parasites treated with geopropolis extracts for 4 h; (b) Parasites treated with geopropolis extracts for 12 h; (c) Parasites treated with geopropolis extracts for 24 h; and (d) Parasites treated with geopropolis extracts for 48 h. CNTL: negative control (untreated); BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata. The bars in the graphs represent the mean values derived from two independent experiments, each performed in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test, comparing each treatment group with the control (untreated cultures). Significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

LiARG inhibition activity of geopropolis extracts. Concentration–response bar graph of enzyme inhibition by (a) BCLR; (b) MDRY; (c) MNDA(1); (d) MNDA(2); (e) MRGT; (f) SbIII; and (g) quercetin. (h) Half-maximum enzyme inhibition activities of geopropolis extracts, SbIII, and quercetin. BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata; SbIII: antimony potassium tartrate trihydrate; n.a.: not active. The bars in the graphs and the values in the table represent the mean values ± standard error obtained from three independent experiments, with each experiment being conducted in triplicate. Different letters in (h) indicate significant differences (p < 0.05) between samples through statistical analysis using one-way ANOVA with Tukey’s multiple-comparisons test.

Figure 5

Effect of geopropolis treatment on intracellular ROS production in L. amazonensis. Promastigotes were treated with IC₅₀ or 2×IC₅₀ concentrations of each extract or with AAPH (1 mM) as a control. (a) Parasites treated with geopropolis extracts for 4 h; (b) Parasites treated with geopropolis extracts for 12 h; (c) Parasites treated with geopropolis extracts for 24 h; and (d) Parasites treated with geopropolis extracts for 48 h. CNTL: negative control (untreated); BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata; AAPH: Parasites exposed to 2,2′-azobis(2-methylpropionamidine) dihydrochloride (oxidative stress inducer). The bars in the graphs represent the mean values obtained from two independent experiments, with each experiment conducted in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test, comparing each treatment group with the control (untreated cultures). Significance levels are denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 6

Effect of geopropolis extracts on autophagic activity in L. amazonensis. Promastigotes were treated with IC₅₀ or 2×(IC₅₀) concentrations of each extract. Non-treated parasites were used as controls. (a) Parasites treated with geopropolis extracts for 4 h; (b) Parasites treated with geopropolis extracts for 12 h; (c) Parasites treated with geopropolis extracts for 24 h; and (d) Parasites treated with geopropolis extracts for 48 h. CNTL: negative control (untreated); BCLR: geopropolis extract from Melipona bicolor; MDRY: geopropolis extract from M. mondury; MNDA(1): geopropolis extract from M. quadrifasciata; MNDA(2): geopropolis extract from M. quadrifasciata; MRGT: geopropolis extract from M. marginata. The bars in the graphs represent the mean values obtained from two independent experiments, with each experiment conducted in triplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test, comparing each treatment group with the control (untreated cultures). Significance levels are as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Therefore, no comparison was performed in this case, as the purpose was to assess the relative impact of the treatments compared to the untreated control.

Figure 7

Effects of geopropolis extract treatment on non-infected peritoneal macrophages. (a) BCLR: geopropolis extract from Melipona bicolor; (b) MDRY: geopropolis extract from M. mondury; (c) MNDA(1): geopropolis extract from M. quadrifasciata; (d) MNDA(2): geopropolis extract from M. quadrifasciata; (e) Sb III: antimony potassium tartrate trihydrate. The bars in the graphs represent the mean values obtained from two independent experiments, with each experiment conducted in duplicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test, comparing each treatment group with the control (untreated cultures). Significance levels are as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.