Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens

Chemicals used

Proteose peptone, Page’s saline, and yeast extract were procured from HiMedia Laboratories (Mumbai, India). Sodium citrate dihydrate (C₆H₅Na₃O₇.2H₂O), disodium phosphate (NaHPO₄), sodium chloride (NaCl), calcium chloride (CaCl₂), glucose, and phenylmethylsulphonyl fluoride (PMSF) were from Sigma Chemical Co. (St. Louis, MO, USA). Potassium dihydrogen phosphate (KH₂PO₄) and magnesium sulfate heptahydrate (MgSO₄.7H₂O) were procured from Labscan (Bangkok, Thailand). Trypan blue (0.4%) was obtained from Gibco BRL (Grand Island, NY, USA). All chemicals and medium components used were of analytical grade.

Preparation of mangostin and its derivatives

The extraction and isolation procedures of mangostin were previously described by Mahabusarakam et al. (2009). Briefly, 4 kg of air-dried powdered pericarps of G. mangostana (Mahabusarakam et al., 2009) were soaked in 2.0 L of dichloromethane (CH₂Cl₂) for 48 h. The extraction was repeated twice, and the combined solution was filtered through Whatman No. 1 filter paper (GE Healthcare Life Science, Buckinghamshire HP7 9NA, United Kingdom) using a vacuum and pressure pump. The collected filtrate was evaporated to dryness under a vacuum at 45 °C using a rotary evaporator (Hei-VAP Advantage HL/G3; Heidolph, Schwabach, Germany). The yellow precipitate was collected. The solid was re-dissolved in dichloromethane and partitioned with 20% (w/v) sodium carbonate solution. The organic layer was separated and washed with water several times until the water layer was no longer basic. The dried organic layer was concentrated, and mangostin was obtained as a yellow crystalline solid.

To prepare epoxide derivative, a mixture of mangostin (5 g, 12 mmol) in dimethylformamide (DMF) (20 mL) and sodium hybrid (2 g) in DMF (10 mL) was stirred at room temperature for 30 min, followed by the addition of 2.5 mL 1-chloro-2,3-epoxypapane. The reaction mixture was then refluxed for 6 h and worked up. The residue was purified by silica gel chromatography and eluted with light petroleum/dichloromethane (4:5) to obtain 1,3-Dihydroxy-6-(2,3-epoxypropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one (C1) (1.5 g, 26% yield) (Mahabusarakam et al., 2006).

The preparation of nitrile derivatives was previously described by Mahabusarakam et al. (2009). A solution of mangostin (2 g, 4.8 mmol) in DMF (10 mL) and sodium hydride (2 g) in DMF (5 mL) was stirred at room temperature for 30 min, followed by addition of 4-chlorobutyronitrile (1.5 mL, 15 mmol). The reaction mixture was then refluxed for 5 h, poured into iced water, neutralized with 3 M hydrochloric acid (HCl), and extracted with dichloromethane. Later, the dichloromethane layer was washed with water and dried over sodium sulfate. The solvent was removed, and the residue was purified by silica gel chromatography. The sample was eluted with the mixed solvent of hexane/dichloromethane (1:8) to give yellow needles of 1-Hydroxy-3,6-di(4-cyanopropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one (C2) (1 g, 43% yield) and 1,3-Dihydroxy-6-(4-cyanopropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one (C3) (0.25 g, 9% yield).

Strain and growth conditions

Acanthamoeba triangularis WU19001 genotype T4 (MW647650) used in this study was previously isolated from the recreational reservoir of Walailak University, Nakhon Si Thammarat, Thailand, by Mitsuwan et al. (2020). The strain was seeded in T-25 tissue culture flasks (SPL Life Science Co., Ltd., Gyeonggi-do, South Korea) with 10 mL of peptone–yeast extract–glucose (PYG) medium containing (g/L) proteose peptone 20, yeast extract 2.0 and glucose 18, C₆H₅Na₃O₇.2H₂O 1.0, CaCl₂ 0.059, MgSO₄.7H₂O 0.98, sodium phosphate dibasic heptahydrate (Na₂HPO₄.7H₂O) 0.355, KH₂PO₄ 0.34, ammonium iron (II) sulfate hexahydrate [Fe(NH₄)₂(SO₄)₂.6H₂O] 0.02 in 1,000 mL distilled water pH 7.0. After incubation in a dark room at ambient temperature for 72 h, A. triangularis trophozoites were observed.

For A. triangularis cyst preparation, the trophozoites grown in PYG medium for 72 h were centrifuged at 4,000 rpm for 5 min in a Sorvall ST 40R centrifuge (Thermo Scientific, Waltham, MA, USA) and then washed twice with Neff’s encystment medium pH 8.9 (Neff et al., 1964) contained (g/L) KCl 7.455, ammediol (2-amino-2-methyl-1,3-propanediol) 2.44, NaHCO₃ 0.084, MgSO₄.7H₂O 1.968, CaCl₂.2H₂O 0.0588. The trophozoites were transferred into T-25 tissue culture flasks containing 10 mL of an encystment medium, and encystation was induced in a dark room at ambient temperature for 7 days. In order to obtain only mature cysts, the cells were collected and then centrifuged at 4,000 rpm for 5 min and treated with sodium dodecyl sulfate (SDS) solution at 0.5% (w/v) final concentration for 20 min to solubilize trophozoites and immature cysts while mature cysts were resistant to SDS solution (Moon et al., 2014). Later, the mature cysts were harvested by centrifugation at 4,000 rpm for 5 min and washed twice with 0.85% (w/v) NaCl solution. The obtained cysts were re-suspended in Neff’s encystment medium.

The viability of A. triangularis trophozoites and cysts was determined by trypan blue exclusion staining. A. triangularis suspension was mixed with the same volume of 0.2% trypan blue and incubated at ambient temperature for 3 min (Sangkanu et al., 2022). The viable (unstained) and dead (stained dark blue) amoebae were enumerated using a hemocytometer (Tiefe Depth Profondeur, Marienfeld, Germany) under an inverted microscope (Nikon ECLIPSE TE2000-S, Tokyo, Japan).

Screening of anti-Acanthamoeba activity of mangostin derivatives

Each of the compounds was screened for anti-Acanthamoeba activity against A. triangularis trophozoites and cysts. Briefly, each of these compounds was dissolved in 100% dimethyl sulfoxide (DMSO) to a 100 mg/mL final concentration and stored at −20 °C until use. The stock solution of each compound was diluted with the cultivation medium to give the final concentration of 2 mg/mL, and the 50 μL of aliquots were added to a 96-well plate (SPL Life Sciences, Seoul, South Korea). A. triangularis trophozoites and cysts were prepared as described above, and an aliquot of 50 μL of cell suspension prepared at 2 $\times$ 10⁵ cells/mL was inoculated into each well of the plate, followed by incubation in a dark room at ambient temperature for 24 h. The final concentration of DMSO was 1% (v/v), and medium containing 1% (v/v) DMSO was used as a negative control, while a final concentration of 0.016 and 0.064 mg/mL chlorhexidine (CHX) solution was used as a positive control for trophozoite and cyst, respectively. The trypan blue exclusion staining was used to determine the number of viable cells present in samples as described above.

Determination of minimal inhibitory concentration (MIC) of mangostin derivatives

The microtiter broth dilution method performed using a 96-well plate was used to determine the MIC value of semi-synthesized compounds against A. triangularis trophozoites and cysts according to the method of Mitsuwan et al. (2020). In this assay, each of the compounds was diluted to give a final concentration of 0.016–1024 mg/mL. The cell suspension was prepared as described above, after which 50 μL of an aliquot of the cell suspension (2 × ${10}^5$ cells/mL) was seeded into each well and incubated at ambient temperature under darkness for 24 h. A. triangularis trophozoites and cysts exposed to 1% (v/v) DMSO and CHX solutions (a final concentration of 0.008–0.128 mg/mL) were used as a negative and positive control, respectively. The plates were incubated in a dark room at ambient temperature for 24 h. The viability was calculated according to the following equation:

$\mathrm{\%}\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=(\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{p}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{e}/\mathrm{N}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})\times 100$

MIC is defined as the lowest concentration of the tested compound that inhibits visible growth of at least 90% of A. triangularis (90% of amoeba died).

Determination of inhibitory concentration 50 (IC₅₀)

IC₅₀ of C1 and CHX against Acanthamoeba trophozoites and cysts was further determined. The protocol was modified from Kolören et al. (2019). In brief, the assay was performed in a 96-well plate. C1 and CHX were prepared with two-fold serial dilution (a final concentration of 0.015–1 mg/mL). Then, 50 µL of 2 × 10⁵ cells/mL of trophozoites and cysts were added to each well and incubated in a dark room at ambient temperature for 24 h. The untreated parasite [medium containing 1% (v/v) DMSO] was included as a negative control. After incubation for 24 h, trypan blue staining was used to determine the number of viable trophozoites and cysts by counting on a hemocytometer. The results are given as percent inhibition compared to control cells (considered as 100%). The IC₅₀ was calculated using the GraphPad Prism, version 7 for Windows (GraphPad Software, San Diego, CA, USA). This experiment was performed in triplicate.

Scanning electron microscopy (SEM) analysis

A. triangularis trophozoites and cysts treated with C1 were carried out to observe any morphological changes using SEM (Zeiss, Munich, Germany) at the Center for Scientific and Technological Equipment, Walailak University, Nakhon Si Thammarat, Thailand (Mitsuwan et al., 2020). The trophozoites were treated with 0.064 mg/mL C1 (1/2×MIC) and CHX 0.008 mg/mL (1/2×MIC). A. triangularis cysts were also treated with CHX (0.032 mg/mL) and C1 (0.032 mg/mL) at 1/2×MIC of CHX. After incubation in a dark room at ambient temperature for 24 h, cells were collected by centrifugation at 4,000 rpm for 5 min and washed three times in 0.01 M phosphate-buffered saline (PBS) solution pH 7.4. Cells were then smeared very thinly onto a sterile cover glass and air-dried. The dried cover glass was placed in a 24-well plate and fixed with 2.5% glutaraldehyde solution overnight at ambient temperature. The samples were then washed three times with PBS solution and dehydrated in a graded series of ethanol solutions from low- to high-density (20%, 40%, 60%, 80%, 90%, and 100%) for 30 min at each step, further mounted on aluminum stubs, and allowed to dry using a critical point dryer. The morphology of A. trianguaris trophozoites and cysts was subsequently analyzed under SEM.

Fluorescence microscopy by acridine orange and propidium iodide staining

A. triangularis cell death after exposure to C1 was identified by AO/PI (acridine orange/propidium iodide) double-staining assay according to the modified method of Kusrini et al. (2020). For the staining, A. triangularis trophozoites were treated with CHX (0.008 mg/mL) and C1 (0.064 mg/mL) at 1/2×MIC in 1.5 mL microcentrifuge tubes, followed by mixing. After incubation for 24 h, samples were centrifuged at 4,000 rpm for 5 min, and the supernatant was discarded and the pellet was washed twice with 0.01 M PBS solution pH 7.4. The pellets were re-centrifuged at 4,000 rpm for 5 min and re-suspended in 100 μL AO/PI staining solution, which was prepared by adding 200 μL of AO (1 mg/mL) and 200 μL PI (1 mg/mL) (Sigma Chemical Co., St. Louis, MO, USA) in 600 μL 0.01 M PBS solution pH 7.4. The cell suspension was then incubated in a dark room at ambient temperature for 10 min since both dyes are light-sensitive and placed onto a slide, carefully covered with a coverslip (Momčilović et al., 2019). The cells were visualized using a fluorescent microscope (Leica TCS SP5 laser scanning confocal microscope; Leica microsystem, Inc., Wetzlar, Germany). Live cells turned into green (AO), whereas the dead appeared red (PI).

Calcofluor white staining

A. triangularis cysts were incubated in the absence or presence of C1 at 1/2×MIC (0.032 mg/mL) for 24 h. The cysts were harvested by centrifugation at 4,000 rpm for 5 min and washed twice with 0.01 M PBS solution pH 7.4. A. triangularis cysts were re-suspended in 2.5% calcofluor white (CFW) staining solution (Sigma Chemical Co., St. Louis, MO, USA) and incubated in a dark room at ambient temperature for 120 min and collected by centrifugation at 4,000 rpm for 5 min. Later, it was washed twice with 0.01 M PBS solution pH 7.4 to remove excess CFW stain and re-suspended in the same buffer solution. Finally, 20 μL cyst suspensions were placed onto a slide, carefully covered with a coverslip, and examined under Olympus, BX-53 fluorescent microscope (Olympus, Tokyo, Japan) with the excitation of 405 nm and emission band pass 420–480 nm.

Cytotoxicity assay

The in vitro cytotoxicity of C1 and CHX was evaluated against the Vero cell (Elabscience, Wuhan, Hubei, China) according to the method of Eawsakul et al. (2017). Vero cells were maintained in Dulbecco’s Modifed Eagle’s (DMEM) medium (Merck KGaA, Darmstadt, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic cocktail containing penicillin G of 100 units/mL and streptomycin of 100 μg/mL. The culture was incubated at 37 °C in a humidified 5% (v/v) CO₂ 95% air atmosphere. The medium was changed every 2 days until 90% confluence was achieved. The cells were then enzymatically detached from the T-flask by adding trypsin- ethylenediaminetetraacetic acid (EDTA) solution (0.25% trypsin, 1 mM EDTA), and subsequently incubated at 37 °C in a humidified incubator (Binder, Tuttlingen, Germany) containing 95% room air/5% CO₂. After incubation, 100 μL of the suspensions were seeded in 96-well plates at a density of 1 × 10⁵ cells/well, and then 100 μL of C1 and CHX at different concentrations was added. Positive and negative controls were prepared by adding the same amount of 1% (v/v) Triton X-100 and 0.01 M PBS solution pH 7.4, respectively. After incubation for 24 h, the cell viability of treated Vero cells was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The medium was removed and cells were incubated with fresh medium containing 0.5 mg/mL of MTT for 4 h. The formed formazan crystal was dissolved by adding 100 mL of DMSO. The absorbance was measured using a microplate reader (Metertech M965; Metertech, Taipei, Taiwan) at 570 nm. The cell viability was calculated using the following equation. Based on ISO 10993-5 standard, the percentage of cell viability above 80% are non-cytotoxic; within 80–60% weak; 60–40% moderate, and below 40% strong cytotoxic, respectively (International Organization for Standardization ISO 10993-5, 2009).

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})=(\mathrm{A}\mathrm{B}\mathrm{t}/\mathrm{A}\mathrm{B}\mathrm{u})\times 100$$

ABt and ABu are the absorbance values of treated and untreated cells, respectively.

Effect of mangostin derivative on A. triangularis encystation

The effect of C1 on A. triangularis encystation was assessed in 96-well plates according to the modified method of Fakae et al. (2020). In brief, C1 was diluted with Neff’s encystment medium to give a final concentration of 1/2×MIC–1/32×MIC. Then, 50 μL of each dilution was added to each well, and an aliquot of 50 μL containing approximately 6 × 10⁵ trophozoites/mL was seeded into each well of the 96-well plate. The trophozoites in the presence of 10 mM phenylmethylsulfonyl fluoride (PMSF) were used as a positive control, whereas the trophozoites exposed to an encystment medium containing 1% (v/v) DMSO served as a negative control. The plates were incubated in a dark room at ambient temperature for 72 h. The viability of A. triangularis cells was quantified by trypan blue exclusion staining using a hemocytometer. To solubilize all trophozoites, a final concentration of 0.5% (w/v) SDS solution was added and incubated at ambient temperature for 30 min with mild shaking, and cysts were enumerated using a hemocytometer. The inhibition effect of mangostin derivative on encystation was calculated according to the following equation:

$$\begin{array}{cc}\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=& 100-(\mathrm{P}\mathrm{o}\mathrm{s}\mathrm{t}\text{-}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}/\\ & \mathrm{P}\mathrm{r}\mathrm{e}\text{-}\mathrm{d}\mathrm{i}\mathrm{g}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r})\times 100\end{array}$$

Effect of mangostin derivative on excystation activity of A. triangularis

The excystation assay was performed to determine the effect of C1 on excystation activity of A. triangularis. The protocol for this assay was obtained from Anwar et al. (2019) with a slight modification. C1 was diluted with a PYG growth medium to give a final concentration of 1×MIC–1/128×MIC. An aliquot of 50 μL of each dilution was added to each well, and cell suspension containing 6 × 10⁵ cysts/mL was prepared as described above and followed by adding 50 μL of this into each well. The cysts in the presence of 10 mM PMSF were served as a negative control, while the cysts without C1 exposure served as a positive control. After incubation in a dark room at ambient temperature for 7 days (Shing, Balen & Debnath, 2021), a final concentration of 0.5% (w/v) SDS solution was added to solubilize all trophozoites and incubated at ambient temperature for 30 min with mild shaking, and cysts were enumerated using a hemocytometer. The excystation rate was calculated according to the following equation:

$$\mathrm{E}\mathrm{x}\mathrm{c}\mathrm{y}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\mathrm{\%})=(\mathrm{N}\mathrm{o}.\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}-\mathrm{N}\mathrm{o}.\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{y}\mathrm{s}\mathrm{t}/\mathrm{N}\mathrm{o}.\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s})\times 100$$

The results are representatives of three experiments presented as mean ± standard error.

Effect of mangostin derivative on the adhesion of A. triangularis to the surface of contact lenses (CL)

The potential of C1 in removing A. triangularis trophozoites and cysts adherent to contact lens (CL) was evaluated according to a previous study (Mitsuwan et al., 2020) with slight modification. In this assay, the commercial CL-1 was removed from the original package and placed in a 24-well plate. Each lens was rinsed twice with 0.01 M PBS solution pH 7.4. A. triangularis trophozoites and cysts were prepared as described above, and an aliquot of 500 μL cell suspension containing 1 × 10⁶ cells/mL was dropped on the CL in a 24-well plate and incubated in a dark room at ambient temperature for 24 h. C1 and CHX at a final concentration of 1/2×MIC (500 μL) were added to each well and incubated at ambient temperature under darkness for 24 h. Two brands of multi-purpose disinfecting (MPD) solutions, including Renu^®freshTM (Bausch & Lomb, Rochester, NY, USA) (MPD-1) and Duna NO RUB (Alcon Laboratories, INC, Fort Worth, TX, USA) (MPD-2) were used as positive controls. Contact lenses (CL) were gently washed with 0.01 M PBS solution pH 7.4 and transferred to a microcentrifuge tube. The samples were then placed in an ice bath for 10 min and shaken in a vortex for 5 min. The total number of cells in the microcentrifuge tube was counted under an inverted microscope.

Statistical analysis

All experiments were carried out in triplicate, and the mean values were registered. The data were compared by one-way ANOVA and the Tukey’s t-test using the statistical package software (SPSS version 26.0, Chicago, IL, USA). Data were expressed as mean ± SD. In all analyzes, p < 0.05 was considered statistically significant.

Effect of mangostin derivatives against A. triangularis

The anti-Acanthamoeba activity of the mangostin derivatives at a final concentration of 1.0 mg/mL against A. triangularis WU19001 trophozoites and cysts was screened through the microtiter broth dilution method. Three semi-synthetic mangostin derivatives were obtained: 1,3-Dihydroxy-6-(2,3-epoxypropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one (C1, epoxide derivative), 1-Hydroxy-3,6-di(4-cyanopropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H xanthen-9-one (C2, nitrile derivative) and 1,3-Dihydroxy-6-(4-cyanopropoxy)-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9-one (C3, nitrile derivative), and their chemical structures were shown in Fig. 1. These tested compounds exhibited inhibitory activities at 1 mg/mL and were further evaluated for their MICs. Among these, C1 showed greater anti-Acanthamoeba activity at 0.128 and 0.064 mg/mL on trophozoites and cysts, respectively (Table 1). Moreover, C1 exhibited anti-Acanthamoeba cysts activity similar as chlorhexidine (CHX). Regarding to mangostin nitrile derivatives included C2 and C3 on A. triangularis trophozoites and cysts, both exhibited much higher MIC values in vitro against trophozoites (0.512 mg/mL) and cyst (>1 mg/mL) compared to those of CHX. C1 and CHX also were shown in demonstrating the inhibitory activity against Acanthamoeba trophozoites with IC₅₀ of 0.056 and 0.014 mg/mL, respectively (Fig. 2). In addition, C1 and CHX exhibited the inhibitory effect against cysts with IC₅₀ values of 0.035 and 0.038 mg/mL, respectively (Fig. 2). In this regard, C1 was selected for further study.

Effect of mangostin derivative on morphological changes of A. triangularis

The morphological changes of the A. triangularis trophozoites upon exposure to C1 at its 1/2×MIC value (0.064 mg/mL) were observed by scanning electron microscopy (SEM). The A. triangularis trophozoites exhibited spine-like structures on their surface known as acanthopodia. Based on the SEM images, shortening and loss of acanthopodia on the amoeba membrane were observed after treated trophozoites with C1 (0.064 mg/mL) (Fig. 3A) and CHX (Fig. 3D) for 24 h. Moreover, trophozoites treated with CHX at 1/2×MIC value (0.008 mg/mL) showed stronger loss of acanthopodia compared to that of C1. On the contrary, untreated cells exhibited numerous acanthopodia on the amoebic surface (Fig. 3G). The most remarkable changes on the amoeba morphology after treated with C1 and CHX were the reduction of cell size, development of abnormal shapes, blebbing on the plasma, pore formation in the cell membrane, and membrane damage (Fig. 3). A large number of pores were detected in treated trophozoites with CHX when compared to those of C1.

SEM images displayed morphological changes in A. triangularis cysts treated with C1 (1/2×MIC value = 0.032 mg/mL). In this condition, cysts became shrunk and showed thick ridges on the wrinkled surface with the observation of flat and irregular shapes (Figs. 4A–4C). The normal morphological characteristics such as triangular shape, smooth surface, and appearance of thin ridges on the wrinkled surface were observed in the untreated cysts (Figs. 4G–4I). The cyst size of treated ones with CHX at 1/2×MIC value (0.032 mg/mL) was smaller compared with treated cysts with C1 (Figs. 4A and 4D). In addition, the result also showed that the cysts treated with CHX had rougher surfaces compared to those treated with C1.

Determination of cell death by fluorescence microscopy-acridine orange/propidium iodide (AOPI) staining

AO/PI double staining is a technique used to differentiate between viable and non-viable cells. In untreated trophozoites, the cells were observed the green cytoplasm and bright green fluorescent of an intact nucleus stained by AO, indicating healthy and viable Acanthamoeba cells (Fig. 5C). In addition, a prominent vacuole was seen in untreated Acanthamoeba trophozoite. Meanwhile, A. triangularis cells treated with C1 and CHX were observed as orange to red granules in cells, indicating non-viable Acanthamoeba cells (Figs. 5A and 5B).

Detection of cysticidal activity by calcofluor white staining

The cysticidal activity of C1 on A. triangularis cysts was evaluated by calcofluor white (CFW) staining assay. This fluorescence stain is known to bind to the cellulose deposition in the cell wall layer of Acanthamoeba cysts (El-Sayed & Hikal, 2015). The cell walls of Acanthamoeba cyst consisting of two layers identified as ectocyst and endocyst were observed in untreated cysts (Figs. 6A, 6B and 6E). Furthermore, our results showed that C1 and CHX had cysticidal activities and caused cyst death based on the loss of CFW staining (Figs. 6C and 6D).

In vitro cell cytotoxicity

The cytotoxicity of C1 and CHX towards Vero cells was investigated using the MTT assay. The IC₅₀ values of C1 for Acanthamoeba trophozoites and cysts were 0.056 and 0.035 mg/mL, respectively. According to ISO 10993-5, at the cell viability exceeds 80%, compound is considered non-cytotoxic, while at cell viability range between 60–80%, as weakly cytotoxic. In this study, the non-cytotoxic dose of C1 was 0.04 mg/mL and the weakly cytotoxic concentration of C1 was 0.08 mg/mL (Fig. 7). Therefore, the results confirmed that C1 had no cytotoxic effect on Vero cells at inhibitory concentrations. The IC₅₀ values of CHX for Acanthamoeba trophozoites and cysts were 0.014 and 0.038 mg/mL, respectively and this obtained data indicated that CHX had strong cytotoxic effect on Vero cells.

Effect of mangostin derivative as an inhibitor on A. triangularis encystation

Encystation of Acanthamoeba leads to cyst formation from vegetative trophozoites. This is essential for parasite survival under harsh conditions. The inhibitory effect of C1 on A. triangularis encystation was evaluated. Based on the results obtained, C1 displayed a high inhibitory effect on A. triangularis encystation at 1/2×MIC (0.064 mg/mL) to 1×32 MIC (0.008 mg/mL) with encystation inhibition of 6.0–9.0% (Fig. 8). Of this, the best encystation inhibition was observed at 1/16×MIC (0.004 mg/mL) (Fig. 8). Meanwhile, 10 mM PMSF was used as a positive control and suppressed up to 90% of trophozoites from forming into cysts (Fig. 8). C1 gave a better inhibitory effect against A. triangularis encystation compared to control group (without C1) (Fig. 8).

Effect of mangostin derivative on the excystation activity of A. triangularis

Excystation assay was conducted to determine the effect of C1 on the ability of A. triangularis to re-emerge from the cyst stage to trophozoites. Cysts treated with PMSF (10 mM) were used as a negative control, while the cysts without C1 exposure were used as a positive control. According to the results, the treatment of amoebic cysts with C1 significantly succeeded in triggering excystation (p < 0.05) at 1/128×MIC, as compared to the control groups (10 mM PMSF and without adding C1) (Fig. 9) with excystation rate of 89.47%. In addition, treatment of A. triangularis cysts with C1 at 1/2–1/8×MICs exhibited moderate efficiency for excystation activity with excystation rate of 23% to 28% (Fig. 9). In our study, the incomplete excystment of A. triangularis in PYG medium at an optimal 10 day period with the excystation rate of 100% was found (Fig. 9).

The ability of mangostin derivative on the removal of adhesive A. triangularis trophozoites and cysts on contact lenses (CL)

The ability of C1 for the removal of adhesive A. triangularis trophozoites and cysts on contact lenses (CL) was investigated. Two commercial multi-purpose disinfecting (MPD-1 and -2) solutions and CHX were used as positive controls, while the CL without C1 exposure was considered as a negative control. Our result showed that C1 had the greatest capability to remove adherent A. triangularis trophozoites on CL compared to a negative control (Fig. 10A). In addition, low numbers of trophozoites and cysts adherent on CL were observed in the presence of C1, CHX, and both MPD-1 and-2 solutions (Figs. 10A and 10B). Therefore, C1 is a good candidate for CL care solution application.