7 research outputs found
A Novel Function for Kojic Acid, a Secondary Metabolite from <i>Aspergillus</i> Fungi, as Antileishmanial Agent
<div><p>Kojic acid (KA) is a fungal metabolite used as a topical treatment skin-whitening cosmetic agent for melasma in humans; however its potential as an anti-leishmanial agent is unknown. Chemotherapy is one of the most effective treatments for Leishmaniasis. However, the drugs available are expensive, invasive, require long-term treatment and have severe side effects. Thus, the development of new effective leishmanicidal agents is a necessity. In this study we investigated the anti-leishmanial effect of KA on <i>L. amazonensis</i>, following <i>in vitro</i> and <i>in vivo</i> infections. KA (50 μg/mL) was found to decrease the growth by 62% (IC<sub>50</sub> 34 μg/mL) and 79% (IC<sub>50</sub> 27.84 μg/mL) of promastigotes and amastigotes <i>in vitro</i>, respectively. Ultrastructural analysis of KA-treated amastigotes showed the presence of vesicles bodies into the flagellar pocket, and an intense intracellular vacuolization and swelling of the mitochondrion. During the <i>in vitro</i> interaction of parasites and the host cell, KA reverses the superoxide anions (O<sub>2</sub><sup>-</sup>) inhibitory mechanism promoted by parasite. In addition, 4 weeks after KA-topical formulation treatment of infected animals, a healing process was observed with a high production of collagen fibers and a decrease in parasite burden. Thus, these results demonstrated the great potential of KA as an anti-leishmanial compound.</p></div
Ultrastructural effects of KA on intracellular amastigotes of <i>Leishmania (L.) amazonensis</i>.
<p>(<b>A</b>) General view of untreated infected macrophages showing a typical morphology. Note parasites (<b>stars</b>) within the parasitophorous vacuoles. (<b>B</b>) General view of infected macrophages treated for 1 h and cultivated for 24 h, showing large parasitophorous vacuole. Note reduced number of amastigotes, parasite and flagellar fragments (<b>arrowheads</b>). (<b>C</b>) Infected and treated macrophages presented vacuoles with damaged parasites (<b>stars</b>) or without amastigote forms. (<b>D</b>) Higher magnification of (<b>C</b>); parasites inside PV with alterations in the flagellar membrane (<b>thin arrows</b>) and vesicles inside the flagellar pocket (<b>asterisks</b>). (<b>E</b>) Intracellular amastigotes with membrane profiles in the flagellar pocket (<b>thin arrows</b>) and in the parasite cytoplasm (<b>arrows</b>); intense formation of lipid-like bodies (<b>asterisks</b>) in the cytoplasm of amastigotes forms. (<b>F</b>) Intracellular parasites presented concentric membrane (<b>arrow</b>), kinetoplast swelling (<b>arrow heads</b>) and lipid-like bodies (<b>asterisks</b>). <i>N</i>, nucleus; <i>FP</i>, flagellar pocket; <i>K</i>, kinetoplast; <i>F</i>, flagellum; <i>M</i>, mitochondria; <i>PV</i> parasitophorous vacuole. Bars: (<b>A–C</b>) 5 μm; (<b>D–F</b>) 2 μm.</p
Effect of KA topical formulation on experimental infection with <i>L. amazonensis</i>.
<p>(<b>A</b>) Development of lesions in <i>L. (L.) amazonensis</i>-infected animals treated with KA. The treatment started after 5 weeks post-infection and continued for 4 weeks. Data represent the average measurements of 5 animals for each group. (<b>B</b>) Parasite load in the lesion sites. Amastigotes were quantified after interruption of treatment and the mean number of parasites evaluated in 10 fields reported; (**p<0.001). Results are expressed as the mean number of cells evaluated in 10 fields; (*p<0.05; ***p<0.0001).</p
Superoxide radicals (O<sub>2</sub><sup>−</sup>) detection in the infected macrophages treated with 50 μg/mL KA for 1 h.
<p>(<b>A</b>) Macrophages without infection and untreated. (<b>B</b>) Positive control, interaction with Zymozan (<b>arrows</b>). (<b>C</b>) Macrophage infected with <i>L. (L.) amazonensis</i> (<b>arrows</b>) showed superoxide production inhibition. (<b>D</b>) Macrophages infected with <i>L. amazonensis</i> and treated with KA reverted the inhibitory effect. Bars: 10 μm. (<b>E</b>) Number of infected macrophages that presented formazan deposits. MO+L.a.: macrophages infected with <i>L. amazonensis</i>; MO+L.a.+KA: macrophages infected with <i>L. amazonensis</i> and treated with 50 μg/mL KA; O<sub>2</sub><sup>+</sup>: infected macrophages with formazan deposits; O<sub>2</sub>: infected macrophages without formazan deposites. (***p<0.001).</p
Ultrastructural analysis of skin lesions from infected animals untreated and treated with KA-topical formulation.
<p>Transmission electron microscopy of control (<b>A</b>) and KA-treated animals (<b>B</b>). Note the presence of amastigotes in the parasitophorous vacuoles and few collagen fibers in the control group and absence of intracellular amastigotes with organized fibers in the treated group. Scanning electron microscopy of untreated lesion showed the presence of a large number of amastigotes (<b>arrows</b>) inside the parasitophorous vacuoles (<b>C</b>) and lesion from treated animals showed an intense production of fibrous material suggestive of collagen fibers (<b>D-arrowheads</b>). <i>P</i>, parasite; <i>HCN</i>, host cell nuclei; <i>PV</i>, parasitophorous vacuole. Bars: (<b>A–B</b>) 3 μm, (<b>C–D</b>) 20 μm.</p
KA activity against <i>L. (L.) amazonensis in vitro</i>.
<p>(<b>A</b>) Growth curve of <i>L. (L.) amazonensis</i> promastigotes treated with different KA concentrations (B) Growth curve of <i>L. (L.) amazonensis</i> promastigotes treated with glucantime® (GLU). Results are from three experiments performed in triplicate. *p<0.05; ***p<0.001; compared with control (CTL). (<b>C</b>) Effect of KA on intracellular amastigote survival of <i>L. (L.) amazonensis</i>. Macrophages infected and treated with KA and GLU (50 μg/mL) (***p<0.001 compared with CTL).</p
Skin lesion section of infected animals, untreated and treated with KA topical formulation for one month.
<p>Histopathologycal analysis of lesion site of untreated animal (<b>A–B</b>) showed tissue damage (<b>A</b>) and numerous amastigote forms (<b>arrows</b>) dispersed for all tissue (<b>B</b>); Vehicle group (<b>C–D</b>) showed an intense vacuolization for all tissue (<b>C</b>) and a higher number of amastigotes (<b>arrows</b>) inside the parasitophorous vacuoles (<b>D</b>); KA-treated animals (<b>E–F</b>) presented reduced number of vacuoles in the macrophages and reduced number of amastigotes (<b>E–F</b>); H&E stain. Bars: (<b>A, C, E</b>) 20 μm; (<b>B, D, F</b>) 10 μm.</p