Free-living amoebae (FLA) are protozoa that live autonomously in diverse environments, such as soil and fresh water, and feed on bacteria, fungi, and algae. However, Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba species are pathogenic and can infect humans and cause life-threatening diseases [1]. Although human infections by these FLA are rare, they can cause rapidly progressive and severe central nervous system (CNS) infections that are almost fatal [1].
Primary amebic meningoencephalitis (PAM) is a brain infection caused by Neglaria fowleri. The amoeba enters the brain via the olfactory neuroepithelium and induces an acute hemorrhagic-necrotizing meningoencephalitis accomplished with fulminating inflammation [2]. The rapid progression of PAM can cause death (mortality rate >97%) within 1 or 2 weeks after the initial exposure [2]. Acanthamoeba species cause diseases such as acanthamoeba keratitis and granulomatous amebic encephalitis (GAE) in humans [3]; GAE also has a high lethality of up to 90%, particularly in immunocompromised humans [3]. Such high fatalities of PAM and GAE are mainly attributed to the lack of a reliable and rapid diagnosis method and effective therapeutic drugs. The currently recommended drug treatment regimens either alone or in combination for PAM and GAE have typically been used for treating bacterial and fungal infections [2] but provide no guarantee for successful treatment, and undesirable side effects of these drugs are also a concern. Therefore, the development of novel effective drugs for PAM and GAE is urgently required.
Natural compounds from plants have been used to manage or treat diverse diseases throughout human history [4]. Recently, large screening approaches to find natural compounds with antiamoebic or amoebicidal activity against N. fowleri and Acanthamoeba species from diverse natural resources have been performed [5–9]. These studies demonstrated the amoebicidal or antiamoebic effects of natural compounds or plant extracts against N. fowleri and Acanthamoeba species, suggesting these compounds have potential applications as therapeutic candidates or supplemental compounds for PAM and GAE.
Flavonoids, natural compounds isolated from plants and plant-derived products, have gained attention as attractive alternative drugs or leads for protozoan parasites such as Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, and Trypanosoma cruzi [10–13]. Several flavonoids have recently been proposed as potential candidates for anti-FLA drug development based on these studies [14]. However, their antiamoebic activity for FLA has not yet been experimentally determined. Herein, we evaluated the antiamoebic activity of selected flavonoids against trophozoites of N. fowleri and Acanthamoeba species.
Based on a literature review [10,14], a selection of 18 flavonoids was made according to their potential antiprotozoan activities on other protozoan parasites and included (−)-epicatechin, (−)-epigallocatechin, (±)-catechin hydrate, demethoxycurcumin, luteolin, resveratrol, silybin (A+B) mixture, rosmarinic acid, (−)-trans-caryophyllene, costunolide, kaempferol, apigenin, afzelin, quercetin, genistein, berberine, chelerythrine chloride, and (+)-pinostrobin. Azithromycin dihydrate and miltefosine were included as control drugs. All flavonoids and chemicals were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO; Sigma) or distilled water at 100 mM. The purity of all flavonoids was >95%.
Neglaria fowleri (Carter NF69 strain, ATCC 30215) trophozoites were cultured and maintained in Nelson’s medium containing 5% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco) at 37°C. A. castellanii (ATCC-30868) and A. polyphaga (ATCC-30461) were axenically cultured and maintained in peptone-yeast-glucose medium supplemented with 1% penicillin/streptomycin at 25°C.
C6 rat glial cells (ATCC CCL-107) were cultured in Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in a humidified 5% CO2 incubator.
Neglaria fowleri trophozoites (5×104 cells/well) were seeded on 96-well microplates (Thermo Fisher Scientific, Waltham, MA, USA) in Nelson’s medium and incubated at 37°C overnight. A. castellanii and A. polyphaga trophozoites (5×104 cells/well) were seeded on 96-well microplates (Thermo Fisher Scientific) in peptone-yeast-glucose medium and incubated at 25°C overnight. Different concentrations (0–400 μM) of each flavonoid were used to treat N. fowleri and Acanthamoeba trophozoites followed by incubation at 37°C or 25°C for 48 h, respectively. Azithromycin and miltefosine were included as control drugs with the same procedures. Morphological changes of the amoebae were observed microscopically every 12 h. The viability of the amoebae was accessed with a colorimetric method using the CellTiter-Blue Cell viability assay (Promega, Madison, WI, USA). All experiments were performed in triplicate with 3 replicates. Data are presented as mean±standard deviation (SD) of 3 independent experiments. The inhibitory concentration 50 (IC50) of each compound was calculated using GraphPad Prism 9.1.0 software (GraphPad Software, San Diego, CA, USA). Amoebae treated with 0.1% DMSO were used as negative controls, representing 100% cell viability.
The potential cytotoxicity of each flavonoid against C6 glial cells was analyzed. Cells were seeded in 96-well microplates (Thermo Fisher Scientific; 2×104 cells/well) and incubated overnight until 80% confluence. Serially diluted flavonoids (0–400 μM) or control drugs were used to treat cells as described above. Morphological alterations of the cells were observed via microscopic examination. Cell viability was determined using the CellTiter-Blue Cell viability assay (Promega). All experiments were performed in triplicate with 3 replicates. Data are given as mean±SD of 3 independent assays. The cytotoxicity concentration 50 (CC50) of each flavonoid was calculated using GraphPad Prism 9.1.0 software (GraphPad Software). The sensitivity index (SI) was determined by the ratio between CC50 and IC50. Cells treated with 0.1% DMSO, which was confirmed not to induce the morphological change of the cells under microscopic examination, were used as controls with 100% cell viability.
In vitro anti-N. fowleri activities of 18 flavonoids were evaluated by incubating the amoeba with various concentrations of each flavonoid. Six of the 18 flavonoids showed anti-N. fowleri activity with IC50 values <50 μM (Table 1). Demethoxycurcumin, luteolin, and kaempferol had low IC50 values <30 μM and high SI values >7 and was more effective than miltefosine, a reference drug. Resveratrol and silybin (A+B) also showed low IC50 against N. fowleri but displayed partial effects on C6 glial cells, producing lower SI values of 4.68 and 4.13, respectively. Chelerythrine had the most robust antiamoebic activity against N. fowleri (IC50=11.83±1.15 μM) but showed potent cytotoxicity against C6 glial cells, producing low CC50 and SI values. The other 12 flavonoids showed weak anti-N. fowleri activity (IC50 >65 μM) and low SI values (<4).
The in vitro anti-Acanthamoeba activity of the flavonoids was analyzed. The IC50 values of the flavonoids slightly differed by Acanthamoeba species, and A. polyphaga was generally more sensitive than A. castellanii. Among the 18 flavonoids, 10 showed antiamoebic activity against A. castellanii and A. polyphaga (Table 1). (−)-Epicatechin, (−)-epigallocatechin, demethoxycurcumin, apigenin, resveratrol, rosmarinic acid, silybin (A+B), costunolide, and kaempferol had low IC50 <50 μM for both Acanthamoeba species. In particular, (−)-epicatechin, (−)-epigallocatechin, demethoxycurcumin, rosmarinic acid, and kaempferol displayed high SI values >8. Apigenin, resveratrol, silybin (A+B), costunolide, and (−)-trans-caryophyllene also showed potential antiamoebic activity against Acanthamoeba but also partially affected C6 glial cells, producing relatively low CC50 and SI values. Luteolin was effective only for A. polyphaga. The other seven flavonoids, (±)-catechin, afzelin, quercetin, genistein, berberine, chelerythrine, and (+)-pinostrobin, did not exhibit potential or selective antiamoebic activity against either Acanthamoeba species.
The 18 flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. However, demethoxycurcumin and kaempferol showed promising in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species with low IC50 values (<40 μM) and high SI values (>8). The overlapping antiamoebic activities of demethoxycurcumin and kaempferol against both N. fowleri and Acanthamoeba species suggest these flavonoids could be candidates for drug development for both PAM and GAE. Resveratrol and silybin (A+B) also showed reasonable IC50 and SI values against both N. fowleri and Acanthamoeba species. The antiamoebic activities of kaempferol, demethoxycurcumin, and resveratrol have been reported previously. Kaempferol was shown to block the proliferation and invasion of E. histolytica trophozoites in vitro through the degradation of the cytoskeleton [15] and to induce programmed cell death in N. fowleri [16]. Demethoxycurcumin and resveratrol were toxic to A. castellanii trophozoites and inhibited the binding of the trophozoites to host cells [17]. These studies support the hypothesis that these flavonoids are attractive candidates for anti-FLA drug development. However, further investigations are necessary to understand the underlying antiamoebic mechanisms of these flavonoids at the molecular level and evaluate their biosafety.
The anti-Acanthamoeba activities of (−)-epicatechin, (−)-epigallocatechin, apigenin, and rosmarinic acid have been partially reported [7,10,18]. Consistent with these studies, we confirmed the anti-Acanthamoeba activities of these 4 flavonoids herein, although they did not show significative anti-N. fowleri activities. Interestingly, these flavonoids had different IC50 values against A. castellanii and A. polyphaga, suggesting further investigation is required on these inconsistent antiamoebic activities against different Acanthamoeba species and underlying molecular mechanisms of these flavonoids for anti-Acanthamoeba activities.
Apigenin and luteolin reportedly show potential cytotoxicity against murine macrophages J774A.1 but did induce programmed cell death in A. castellanii [7]. However, the 2 flavones did not show significative cytotoxicity to C6 glial cells in our study, suggesting further evaluation is needed of these flavones as potential anti-Acanthamoeba drug candidates. The antiamoebic activity of luteolin is also a novel finding obtained in this study. Luteolin showed promising IC50 values (<30 μM) and high SI values (>11) for N. fowleri and A. polyphaga, implying substantial and selective antiamoebic activity without toxicity to animal cells but was not effective against A. castellanii. Further studies are therefore required to investigate the molecular mechanism of the antiamoebic activity of luteolin and evaluate its effectiveness as a drug candidate.
A recent in silico analysis demonstrated that chelerythrine and berberine are attractive drug candidates for PAM [19], and the strong antiamoebic activity of chelerythrine against N. fowleri was confirmed in this study to support the in silico results. However, chelerythrine also showed strong toxicity to C6 glial cells, producing very low CC50 and SI values. Considering the high antiamoebic activity of the alkaloid for the N. fowleri, studies using derivatives of chelerythrine should be performed to reduce the toxicity to human cells. Berberine did not affect either N. fowleri or Acanthamoeba species.
In addition to the 18 flavonoids evaluated in this study, flavonoids such as anemonin, conessine, curcumin, isotrilobine, mangostin, and quassin have been proposed as potential candidates with anti-FLA activities because of their known antiamoebic activities for E. histolytica [14]. Further analyses of the anti-FLA activities of these flavonoids would be interesting in drug repurposing.
In conclusion, we experimentally confirmed that 11 flavonoids possess antiamoebic activities against N. fowleri and Acanthamoeba species. These flavonoids exhibited different antiamoebic profiles against the amoeba species and can be applied as alternative or supplement therapeutic agents to treat PAM and GAE. Further studies to determine the molecular mechanisms of the antiamoebic activities of these flavonoids and in vivo studies to evaluate their clinical significances are necessary. Significant variances in drug susceptibility among N. fowleri strains and isolates have also been reported [20], suggesting that additional studies to evaluate the antiamoebic activity of flavonoids for different genotypes or clinical isolates of the amoeba are also required. Moreover, studies to evaluate the antiamoebic activity of flavonoids, alone or in combination with other drugs, are recommended to support the design or formulation of effective treatment regimens.
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