Novel Plant-Based Metabolites as Disinfectants against Acanthamoeba castellanii

Abstract

Due to global warming, coupled with global water shortages and the reliance of the public on household water tanks, especially in developing countries, it is anticipated that infections caused by free-living amoebae such as Acanthamoeba will rise. Thus, the development of novel disinfectant(s) which can target pathogenic free-living amoebae effectively is warranted. Herein, we extracted and isolated several plant-based secondary metabolites as novel disinfectants for use against pathogenic Acanthamoeba. The identity of the compounds was confirmed by nuclear magnetic resonance and tested for antiamoebic activities against clinical isolate of A. castellanii, belonging to the T4 genotype. Amoebicidal assays revealed that the compounds tested showed antiamoebic properties. Betulinic acid and betulin exhibited parasite killing of more than 65%. When tested against the cyst stage, betulinic acid, betulin, and vanillic acid inhibited both encystation and excystation processes. Furthermore, the plant-based metabolites significantly inhibited the binding capability of A. castellanii to host cells. Finally, most of the tested compounds displayed minimal cytotoxic activities against human cells and noticeably perturbed amoeba-mediated host cell cytotoxicity. Notably, both alkaloid and betulinic acid showed 20% cytotoxic effects, whereas betulin and lupeol had cytotoxic effects of 24% and 30%, respectively. Overall, our findings indicate that plant-based natural compounds demonstrate anti-Acanthamoebic properties, and they have potential candidates for water disinfectants or contact lens disinfecting solutions, as well as possible therapeutic drugs against Acanthamoeba infections.

Keywords: Acanthamoeba castellanii; betulin; betulinic acid; cytopathogenicity; cytotoxicity; encystation; excystation; plant-based natural compounds.

Figure 1

Compounds and their structures isolated from medicinal plants.

Figure 2

Compounds isolated from medicinal plants presented notable amoebicidal activities against amoeba. Briefly, isolated compounds at a concentration of 100 µg/mL were incubated with 5 × 10⁵ of A. castellanii overnight, at 30 °C. Following this, viable amoebae were calculated using haemocytometer microscopically. The data are expressed as the mean ± standard error. p values were determined using two-sample t-test, two-tailed distribution, (*) is p < 0.05.

Figure 3

Compounds significantly blocked the A. castellanii binding to human cells. Adhesion assays were carried out to examine whether A. castellanii interact with human cells. p values were determined using two-sample t-test, two-tailed distribution, (*) is < 0.05. The data are represented as the mean ± standard error of three independent experiments performed in duplicate.

Figure 4

Effects of isolated compounds on the encystation and excystation of A. castellanii. The results revealed that compounds inhibited both encystment and excystment processes in A. castellanii, compared with the negative control: (a) the encystation process; (b) the excystation effects. The data are presented as the mean ± standard error. p values were calculated using a two-sample t-test with two-tailed distribution; (*) denotes that p < 0.05.

Figure 5

Compounds isolated presented higher cell viability effects against human cell lines. Notably, all compounds showed negligible cytotoxic effects against HeLa cell lines.

Figure 6

The cytopathogenicity of human cells was declined by pretreating A. castellanii with compounds. The results revealed considerable inhibition of amoeba-mediated host cytotoxicity when compared with amoeba (untreated). Data are presented as mean ±  standard error. p values were calculated using a two-sample t-test with two-tailed distribution, and (*) denotes that p < 0.05.