Antiamoebic activity of plant-based natural products and their conjugated silver nanoparticles against Acanthamoeba castellanii (ATCC 50492)

Abstract

Acanthamoeba spp. are the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis (GAE). The current options to treat Acanthamoeba infections have limited success. Silver nanoparticles show antimicrobial effects and enhance the efficacy of their payload at the specific biological targets. Natural folk plants have been widely used for treating diseases as the phytochemicals from several plants have been shown to exhibit amoebicidal effects. Herein, we used natural products of plant or commercial sources including quercetin (QT), kolavenic acid (PGEA) isolated from plant extracts of Polyalthia longifolia var pendula and crude plant methanolic extract of Caesalpinia pulcherrima (CPFLM) as antiacanthamoebic agents. Furthermore, these plant-based materials were conjugated with silver nanoparticles (AgNPs) to determine the effects of the natural compounds and their nanoconjugates against a clinical isolate of A. castellanii from a keratitis patient (ATCC 50492) belonging to the T4 genotype. The compounds were conjugated with AgNPs and characterized by using ultraviolet visible spectrophotometry and atomic force microscopy. Quercetin coated silver nanoparticles (QT-AgNPs) showed characteristic surface plasmon resonance band at 443 nm and the average size distribution was found to be around 45 nm. The natural compounds alone and their nanoconjugates were tested for the viability of amoebae, encystation and excystation activity against A. castellanii. The natural compounds showed significant growth inhibition of A. castellanii while QT-AgNPs specifically exhibited enhanced antiamoebic effects as well as interrupted the encystation and excystation activity of the amoebae. Interestingly, these compounds and nanoconjugates did not exhibit in vitro cytotoxic effects against human cells. Plant-based compounds and extracts could be an interesting strategy in development of alternative therapeutics against Acanthamoeba infections.

Keywords: Acanthamoeba; Antiamoebic; Caesalpinia pulcherrima; Polyalthia longifolia; Quercetin; Silver nanoparticles.

Fig. 1

Chemical structure of quercetin a and b Kolavenic acid (PGEA)

Fig. 2

a UV–Vis spectra of quercetin conjugated silver nanoparticles (QT-AgNPs) showing characteristic surface plasmon resonance band at 443 nm confirming the formation of QT-AgNPs. b AFM topographic image recorded on an AFM instrument

Fig. 3

Amoebicidal effects of QT, PGEA, CPFLM and the nanoconjugates of the natural compounds above mentioned against A. castellanii. 5 × 10⁵A. castellanii was incubated with 5 and 10 μM (or mg/mL) of the natural compounds and the nanoconjugates for 24 h at 30 °C then the viability of A. castellanii was determined by staining with 0.1% Trypan Blue. The natural products QT, PGEA and CPFLM showed significant inhibition of A. castellanii growth when compared to the negative control (amoeba alone) (*P < 0.05, using two sample T-test, two-tailed distribution). Nanoparticles conjugation enhanced the antimaoebic activity of natural products as compared to AgNPs and compounds alone (*P < 0.05, using two sample T-test, two-tailed distribution). #shows significance as compared to compound and AgNPs alone

Fig. 4

Representative effects of naturals compounds and nanoconjugates on inhibiting the viability of A. castellanii. a Amoeba alone; b amoeba + solvent control; c amoeba + chlorhexidine 100 μM (positive control); d amoeba + QT 10 μM; e amoeba + QT-AgNPs 10 μM; f amoeba + PGEA 10 μM; g amoeba + PGEA-AgNPs 10 μM; h amoeba + CPFLM 10 mg/mL; i amoeba + CPFLM-AgNPs 10 mg/mL; j amoeba + AgNPs alone 10 μM

Fig. 5

Effects of QT, PGEA, CPFLM and the nanoconjugates of the natural compounds above mentioned on the encystation of A. castellanii. 5 × 10⁵ amoebae were incubated with encystation media (E.M, including 10% glucose and 5 μM MgCl₂) and respective 5 and 10 μM of compounds or nanoconjugates for 72 h at 30 °C. Then the cysts were counted after adding 0.05% of Sodium dodecyl sulfate (SDS). 10 μM of QT-AgNPs showed significantly enhanced encystation activity as compared to QT and AgNPs alone (#P < 0.05, using two sample T-test, two-tailed distribution). PGEA and CPFLM alone showed significant inhibition of A. castellanii encystation when compared to the negative control (amoeba + E.M) (*P < 0.05, using two sample T-test, two-tailed distribution)

Fig. 6

Effects of QT, PGEA, CPFLM, and the nanoconjugates of natural compounds mentioned above on the excystation inhibition of A. castellanii cysts. 1 × 10⁵ cysts were incubated with 5 and 10 μM (or mg/mL) of natural compounds or nanoconjugates for 72 h at 30 °C then the viability of A. castellanii trophozoites were determined by staining with 0.1% Trypan blue. The nanoconjugates of quercetin (10 μM) showed significantly increased results as compared to AgNPs and QT alone (#P < 0.05, using two samples T-test, two-tailed distribution). PGEA and CPFLM alone showed significant inhibition of A. castellanii excystation when compared to the negative control (cysts alone) (*P < 0.05, using two samples T-test, two-tailed distribution)

Fig. 7

QT, PGEA, CPFLM and their silver nanoconjugates showed limited cytotoxicity to HaCaT cells. Briefly, 10 μM of test samples were added to HaCaT cells and incubated for 24 h at 37 °C in a 5% CO₂ incubator. The compounds and nanoparticles showed less than 30% cytotoxicity at 10 μM