Synergistic Effect of Garcinia Mangostana Combined With Chlorhexidine on Acanthamoeba Triangularis Trophozoites and Cysts

Background: Various parts of Garcinia mangostana Linn, including its pericarp have been traditionally used to treat different types of diseases. This study was carried out to determine the anti-Acanthamoeba activity of G. mangostana pericarp extract against Acanthamoeba triangularis. Methods: The G. mangostana ethanolic pericarp extract was screened for the anti-Acanthamoeba activity and determined its minimal inhibition concentrations (MICs) by the microdilution method. Then, the time-kill kinetic assay of the extract was determined. The synergistic effect of G. mangostana extract and chlorhexidine was performed using the checkerboard method. Parasite morphology was detected by scanning electron microscopy (SEM). Results: The MICs of extract were assessed on trophozoites and cysts with 250 and 4000 µg/mL, respectively. More so, at 2×MIC of extract exhibited inhibitory activity against trophozoites and cyst of A. triangularis for up to 7 days. Checkerboard assays showed synergistic activity of extract (500-1000 μg/mL) plus chlorhexidine (3.90-15.62 μg/mL) at a fractional inhibitory concentration index (FICI) of 0.18-0.37. The lowest FICI (0.18) displayed good synergism resulting in up to 16-fold reduction of drug MIC and reducing to 8-fold of extract MIC. The viability of cysts decreased to 12.28±3.03 cells/mL. FICI interpretation equal to 1 is considered an additive effect on Acanthamoeba trophpzoites. The SEM results clearly showed that Acanthamoeba cells treated with a single drug of chlorhexidine and its combination with G. mangostana extract caused cell membrane damage and irregular cell shapes comparing with the control. Conclusion: A good combinatorial relationship displayed by G. mangostana extract and chlorhexidine suggest a more reliable evidenced based therapeutic strategy. Therefore, this approach is promising and could be employed as an alternative treatment method for the management of Acanthamoeba infection.


Introduction
Acanthamoeba is an opportunistic pathogen that is isolated from diverse natural environments [1]. This organism exists in two main forms: the trophozoite, which is the invasive stage; and the cysts, which is the highly resistant stage in a very harsh environment [2]. Acanthamoeba spp. have been divided into three groups (I, II, and III) based on characteristics of cyst such as size and shape. However, Acanthamoeba spp. were then classi ed into 20 different genotypes (T1-T20 Genotype) based on 18S rRNA gene sequencing. Genotype T4 is the most commonly isolated in both clinical and environmental samples, followed by genotype T3 and T5. In addition, the T4 genotype is the most virulent because it has a higher potential of binding to host cells than other genotypes [3].
Acanthamoeba spp. are the causative agents of amebic keratitis (AK) and granulomatous amebic encephalitis (GAE). AK can cause permanent vision loss or blindness [4]. The rate of infectious keratitis is becoming alarming in recent time which is an issue that could be linked to a sudden increase in the population of contact lens wearers [2]. Similarly, Acanthamoeba encysts sprawled deep within the corneal stroma [5], as such, the cyst wall becomes impermeable to the existing drugs and this has reduced further research in the areas of drug formulations and designed for this organism.
Garcinia mangostana Linn is generally known as mangosteen and belongs to the family Clusiaceae. It is a tropical plant, widely grown in Southeast Asia [6]. The pericarps of this fruit have been used for traditional medicine in treating many diseases [7]. The xanthone group, enriched in G. mangostana extract exhibited anti-oxidant properties, anti-bacterial activity, anti-diabetic activity, anti-tumour, antiin ammatory, anti-allergenic, anti-malarial activity, anti-bacteria, cytoprotective and anti-cancer properties [8]. In 2017, Cantos and Lee [9] reported the ovicidal activity of peel and seed extracts from G. mangostana against Ascaris suum. Next year, Canton and Jagunap [10] found that the extract from pericarp had better-inhibited ova fertilization than mebendazole.
Having searched through all available information on this plant, we discovered to our surprise that there is no single report on the anti-amoebic activity of G. mangostana extract against Acanthamoeba spp. Hence, our study sought to investigate the effective concentration of G. mangostana extract for inhibiting Acanthamoeba spp. and demonstrate synergistic effects of extract from G. mangostsana pericarp and chlorhexidine on anti-amoebic activity against A. triangularis.
Materials And Methods

Preparation of plant extracts
Dried mangosteen pericarp powder was extracted with ethanol. 50 g of powdered mangosteen pericarp was soaked in 200 mL of ethanol for 7 days. The extract was ltered through Whatman No. 1 using vacuum and pressure pump. The solution was evaporated to dryness under reduced pressure using a rotary evaporator to obtain a G. mangostana extract. The extract was dissolved in 100% DMSO and stored at -20 °C until use.

Cultivation of A. triangularis
A. triangularis WU19001, a strain from the recreational reservoir at Walailak University, Nakhon Si Thammarat Thailand was used in this study [11]. The parasite was grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose). The trophozoites were observed after 48-72 hours of incubation at room temperature and were cultured in this medium for 1 week in order to obtain mature cysts. Trophozoites cells and cysts were centrifuged at 4000 rpm for 5 minutes and re-suspended in fresh PYG thereafter. The viability was investigated using 0.1% trypan blue assay and adjused to 2 × 10 5 cells/mL. 3. In vitro effect of plant extract against A. triangularis One hundred microliters of trophozoites and cysts suspension (2 × 10 5 cells/mL) were added to 96-well microplates that contained 100 µl of plant extract ( nal concentrations of 4000 µg/mL). Chlorhexidine and 1% DMSO were used as positive and negative controls respectively. Viability was determined using 0.1% trypan blue that was obtained by manual count with inverted microscopy. The relative percentage of parasite viability was de ned as (mean of the treated parasite/mean of the control) × 100. 4. Determination of minimal inhibitory concentration (MIC) of G. mangostana extract against A. triangularis The minimum inhibitory concentration (MIC) for plant extract was determined using microtiter broth dilution method [11]. Plant extract was diluted to give a nal concentration of 4000, 2000, 1000, 500, 250, 125, 62.5 µg/mL into 96-well microplate. Then 100 µl of 2 × 10 5 cells/mL of trophozoites and cysts were inoculated in each well. Chlorhexidine and 1% DMSO were included as positive and negative control, respectively. The plates were incubated at room temperature for 24 hours. The MIC value of the extract was de ned as the lowest concentration that inhibited > 90% of growth, when compared with the negative control.

Time-kill Assay
Time-kill kinetics of the ethanolic extract of G. mangostana was carried out following the procedure as described [12] with modi cation. 100 µL of 2 × 10 5 cells/mL of trophozoites and cysts were inoculated into microcentrifuge tube containing PYG with extract at nal concentration 2 × MIC, MIC, 0.5 × MIC, and 0.25 × MIC and incubated at room temperature for 1 week. The aliquots of A. triangularis were removed and counted at 24 hours intervals using hemocytometer. A. triangularis treated with 1% DMSO was used as a control. 6. Combination of G. mangostana extract with chlorhexidine in targeting trophozoites and cysts of A. triangularis Checkerboard assay method was used to evaluate the interaction between G. mangostana extract and chlorhexidine against A. triangularis. The checkerboard method [13] used was the microdilution assay performed in a 96-well plate with nal volume of 200 µL. G. mangostana extract and chlorhexidine were diluted with PYG to obtain 4 times to its nal concentrations of 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC and MIC. Fifty µl of extract and chlorhexidine were transferred to a microtiter plate containing 100 µL of 2 × 10 5 cells/mL of trophozoites and cysts. The plates were incubated at room temperature for 24 hours. The viability of the extract was de ned as the lowest concentration that inhibited > 90% of growth, when compared with the negative control. The assessment of results was de ned as Fractional Inhibitory Concentration Index (FICI) that was calculated utilizing the following The combination was considered synergistic for ΣFIC ≤ 0.5, additive for 0.5 < ΣFIC ≤ 1, indifferent for 1 < ΣFIC < 4, and antagonistic for ΣFIC ≥ 4, according to EUCAST de nition.

Scanning Electron Microscopic (sem) Study
Acanthamoeba trophozoites and cysts were treated with chlorhexidine and G. mangostana extract. After incubation, cells were collected by centrifugation at 4000 rpm for 5 minutes and re-suspended in phosphate buffer saline (PBS). Cells in DMSO (1%) were used as negative controls. Samples were xed with 2.5% glutaradehyde overnight. The sample was further dehydrated with graded alcohol series (20%, 40%, 60%, 80%, 90%, and 100% ethanol), mounted on aluminum stubs, and allowed to dry using a critical point dryer. The samples were then coated with gold particles and the morphology of Acanthamoeba trophozoites and cysts after treatment was subsequently examined under SEM (SEM-Zeiss, Munich, Germany) at the Center for Scienti c and Technological Equipment, Walailak University, Nakhon Si Thammarat, Thailand.

Statistical Analysis
The experiments were performed in triplicate. All data were recorded and entered using the statistical package software (SPSS Inc. Chicago, IL, USA). The data were expressed as mean ± SD. Statistical analysis was analyzed by the two-tailed unpaired Student's t-test. In all analyzes, P < 0.05 was considered statistically signi cant.

G. mangostana extract exhibited anti-Acanthamoeba activity against A. triangularis
Anti-Acanthamoeba activity was performed to observe the effects of the G. mangostana extract on viability of A. triangularis. The extract showed signi cant reduction in trophozoites viability as compared to the control (P < 0.05) (Fig. 1a). At 250-2000 µg/mL of extract reduced the trophozoites viability to 5.8-8.8%. Furthermore, the viability percentages of cysts decreased to 10.7-13.8% after incubated with 2000-4000 µg/mL of extract (P < 0.05) (Fig. 1b).

Minimum inhibitory concentration (MIC) of G. mangostana extract against A. triangularis
Extract of G. mangostana pericarp was determined for its anti-Acanthamoeba potential by MIC using microtiter dilution broth method. As shown in Table 1, the MICs of mangosteen ethanol extract against A. triangularis trophozoites and cysts were 250 and 4000 µg/mL, respectively. The MIC values of chlorhexidine against trophozoites and cysts were 7.81 and 62.5 µg/mL, respectively. Similarly, population of cysts exhibited an increase number from 2.0 × 10 5 cells/mL to 9.7 × 10 6 cells/mL ( Fig. 2a and b). All concentrations of G. mangostana extract inhibited the growth of Acanthamoeba trophozoites and cysts when compared with the control. At 2 × MIC of the extract displayed strongest inhibitory activity in both forms of Acanthamoeba. After 7 days, the number of viable trophozoites were detected at 2.1 × 10 5 cells/mL in the treatment with 2 × MIC extract (Fig. 2a), while the viable cysts was 1.3 × 10 5 cells/mL (Fig. 2b).
4. Synergistic effects of G. mangostana extract in combination with chlorhexidine against A. triangularis by the checkerboard assay Evaluation of synergistic effects between the extract and drug was determined in this study. Figure. 3 shows the viable trophozoites in combination test. The lowest FIC values (0.5) for each agent reduced viability of trophozoites to 12.72 × 10 5 cells/mL. FICI interpretation equal to 1 is to be considered an additive effect (Table 2). G. mangostana extract showed synergistic (FICI < 0.5) activity with chlorhexidine against A. triangularis cysts. Set of combinations of extract showed FICI ranged from 0.18 to 0.37 ( Table 2). The lowest FICI (0.18) displayed good synergy resulting in up to16-fold reduction of drug MIC and a reduction to 8-fold of extract MIC. The viability of cysts decreased to 12.28 ± 3.03 cells/mL (Fig. 4).

SEM analysis of G. mangostana extract effects on A. triangularis
The alterations due to the action of the extract was con rmed by SEM micrograph are shown in Figs

Discussion
Herb-drug interaction may offer potential health promoting effect by augmenting drug e cacy or diminishing potential side effects. As such, we have presented an effective treatment of Acanthamoeba infections with herb-drug based combination strategy in this study. Also, we have highlighted the potential of anti-Acanthamoeba activity of G. mangostana ethanol extract. It is a natural source that provides active components of G. mangostana also known as the "queen of tropical fruits". The G. mangostana extracts have anti-oxidant, anti-tumoral, anti-allergic, anti-in ammatory, anti-bacterial and anti-viral activities [8]. A few studies have reported the effects of G. mangostana extract on anti-parasitic activity but there was no report on its Acanthamoebic effects.
A. triangularis WU19001 was a selected strain parasite used in this study. A. triaugularis was characterized in group II (type 4) typically a wrinkled ectocyst and an endocyst which could be stellate, polygonal, triangular, or oval [14]. A. triangularis was originally isolated from the human feces documented in France [15]. In 2008, the rst case of keratitis caused by A. triangularis had been reported in Korea [16]. Also, six isolates of A. triangularis had also been reported from cases with contaminated contact lens in southeastern Korea. In support of those previous ndings, our study revealed that G. mangostana extract from pericarp exhibits anti-Acanthamoeba activity against A. triangularis. The MIC values of G. mangostana extract were 250 and 4000 µg/ml against Acanthomoeba trophozoites and cysts, respectively (Table 1). Our nding is in agreement with earlier reported studies that found the compounds of G. mangostana extract with anti-parasitic activities. In literature, xanthones have been identi ed as the main compound of G. mangostana extract. Of this, xanthones have been isolated from pericarp, whole fruit, leaves and bark [3]. α-and γ-mangostin are the most abundant xanthones found in the pericarp of mangosteen fruit. Other xanthones in mangosteen pericarp include β-mangostin, gartanin, 8-deoxygartanin, garcinones A, B, C, D and E, mangostinone, 9-hydroxycalabaxanthone and isomangostin, among others [3,17]. Interestingly, the α-mangostin compound of G. mangostana has been reported with IC 50 value of 17 µM against Plasmodium falciparum [18]. Another study showed that α-mangostin and δ-mangostin exhibited anti-malarial activity against P. falciparum chloroquine-resistant strain with IC 50 value 0.2 and 121.2 µM, respectively [19]. Furthermore, a previous study has reported the use of G. mangostana pericarp in the management of amoebic dysentery [8]. Xanthones block the polymerization process, and accumulation of soluble heme-drug complexes increases the osmotic pressure in the vacuole, causing its lysis on trophozoite stage [20]. The synthetic Caged Garcinia xanthones (CGXs) exhibit anti-malarial activity against P. falciparum with low toxicity. It was reported that CGXs treatment affect malaria parasites relating to morphological changes, signi cant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation [21]. Benzophenone (isoxanthochymol) was isolated from the roots of G. polyantha which showed strong chemosuppressive activity of parasitic growth found in P. falciparum [22]. The benzophenones (guttiferone E, isoxanthochymol, and guttiferone H) isolated from G. xanthochymus exhibited antiplasmodial activity with IC 50 values in the range of 4.71-11.40 µM [23]. The tetraprenylated benzophenone 7-epiclusianone (7-epi) was isolated from the fruits of G. brasiliensis showed effective against Schistosoma mansoni [24]. In addition, bi avanones (GB-1a, GB-1, and GB-2), another active compounds, were isolated from G. kola. These three bi avanones displayed the potent inhibitory activity in vitro against P. falciparum proliferation and also antimalarial potency through oral administration in mice infected with P. berghei without signs of acute toxicity [25]. As kolaviron (KV), a bi avonoid fraction from G. kola seeds that has also been reported to exhibit anti-malarial activities in P. berghei-infected mice [26]. The puri ed bio avonoid of the fruit pericarp of G. brasiliensis, showed signi cant activity as inhibitors of Leishmania's proteases, with mean (± SD) IC 50 values of 15.0 ± 1.3 µg/mL [27]. Interestingly, the effect of xanthones has also been increasingly reported on targeting the bacterial membrane as a result of rapid bactericidal action led to reducing resistance process and mutational possibility [28]. Therefore, xanthones, bi avanones, and/or benzophenones may play a major role on the effect against A. triangularis in this study, however, it further suggests for more comprehensive studies to evaluate the mechanism of action on these compounds against A. triaugularis.
In fact, the eradication of Acanthamoeba infection seems impossible due to highly resistance of the cysts to anti-amoebic drugs. Therefore, we also investigated the effective concentration of G. mangostana extract against A. triangualris trophozoite and cyst stages. Our results interestingly showed that G. mangostana extract can inhibit A. triangularis in a longer incubation period in PYG medium. It's worthy to note that the number of Acanthamoeba trophozoite and cysts were reduced signi cantly (P < 0.05) in the presence of G. mangostana extract (MIC and 2 × MIC nal concentrations). To our observation, the G. mangostana concentrations was found to have the highest growth-inhibitory for trophozoites at day 3 and cysts at day 2 ( Figs. 2a and 2b) though there are few remaining A. triangularis survived due to diminishing the effect of Garcinia extract on the days after. To the best of our knowledge, we have shown the rst potentially report of G. mangostana extract in its capacity to signi cantly inhibit Acanthamoeba cysts in an enrichment medium up to one week. This study have also shown the survival rate of A. triangularis (< 10% in trophozoites and < 15% in cysts) within the same period. Based on these results, it should therefore be better considered a 1-week than 3 days [29] as a general protocol for observation on the growth inhibitory effect of plant extract against A. triangularis. It is also very encouraging to explore the results shown that G. mangostana extract of 2 × MIC remarkably inhibited the growth of both trophozoites and cysts up to 7 days. Our nding is demonstrated similarly to a previous report on the growth inhibition of A. culbertsoni and A. castellanii incubated in the nonpolar extract of Gastrochilus panduratum from day 2 to day 7 [12]. Overall, this study therefore suggests that G. mangostana extract is a promising plant that shows the remarkable effect against A. triangularis infections.
Chlorhexidine is the mainstay drug of choice for Acanthamoeba keratitis and encephalitis due to it is active against both cysts and trophozoites [5]. In this study, a single drug chlorhexidine was therefore used against A. triangularis and successfully exhibited inhibitory activity on trophozoites and cysts with MIC values of 7.81 and 62.5 µg/mL (Table 1), respectively. Our nding is in consistent with an earlier report on chlorhexidine used against A. triangularis exhibited amoebicidal and cysticidal properties at 200 µg/mL (0.02%) [30]. While, a recent study [3] evaluated the e cacy of miltefosine, another orphan drug for the treatment of keratitis and encephalitis, against Acanthamoeba spp. cysts of genotypes T3, T4 and T5. Interestingly, the minimal cysticidal concentration (MCC) of 38.72 mM and 77.44 mM of miltefosine showed the effect against Acanthamoeba genotypes T4/T5 and T3, respectively. This suggests for further study to evaluate the effect of miltefosine against A. triangularis. In a matter of fact, a single drug used for treating infectious diseases includes this parasitic infection not only causes side effects, long-term use, costly, and drug resistance. Therefore, the combination approach is constantly introduced to encounter those pitfalls. Chlorhexidine has frequently been used in combination with aromatic diamidines [5], aminoglycosides, imidazoles and polyene [31], however these chemicals show side effects on keratocytes found in cases of human keratitis [32]. Recently, the nding of novel compounds with anti-Acanthamoeba activity of plant and herbs has been very encouraging to evaluate a source of new molecules with anti-Acanthamoeba effects. At the same time, there is no data on the use of synergistic interaction between plant extract and synthetic drug against Acanthamoeba infection till date. To support this, our study also revealed an additive effect of a combination between G. mangostana extract and chlorhexidine against Acanthamoeba trophozoites (viable trophozoites < 90%) (Fig. 3). An additive effect occurs when substance added together enhances or improves the e cacies but not to the extent of synergic interaction [33]. Our better results were obtained when G. mangostana extract was combined with chlorhexidine to produce a synergistic effect against A. triangularis cysts. The FICI values demonstrated the synergy for concentration of 3.90 to 15.62 µg/mL of chlorhexidine and 500 to 1000 µg/mL of G. mangostana extract as shown the viability of less than 10% (Fig. 4). It is interestingly found that the concentration of chlorhexidine can be 4-16 times lower in the presence of G. managostana extract but presenting the effects against cysts while reducing its toxicity. This nding is in line with a previous study demonstrated a synergistic effect against A. polyphaga from a combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties [34]. From this study, it appears to be a promising combined agents to ght against the infection and especially resistance pattern of Acanthamoeba spp. in the future.
The mode of action considered in this study was con rmed by scanning electron microscopy (SEM) as shown in Figs. 5 and 6. Treated trophozoites showed similar at cells and smooth surface as a result of total destruction of acanthopodia in the presence of G. mangostana extract and chlorhexidine. In combination, morphology of trophozoites were rounded and being observed of the presence of pores on its surface. Chlorhexidine is positively charged and ionic with the negatively charged plasma membrane of the parasite, resulting in membrane structure that gives rise to permeability modulation, ionic leakage, and cytoplasmic disruptions causing cellular damage and cell death [35][36]. The control cysts showed regular morphological characteristics. Overall, A. triangularis cysts were at and morphological deformity (irregular in shape and size) as a result on destruction of the ectocyst walls after treatment given with G. mangostana extract, chlorhexidine and in combination of chlorhexidine/G. mangostana extract.

Conclusion
In line with the data obtained from this study, G. mangostana ethanolic extract from pericarp possesses anti-Acanthamoeba activity against both trophozoites and cysts. Moreover, it is clear that we have presented for the rst time, an anti-Acanthamoeba activity with emphasis on herb-drug combinatorial approach. This study therefore recommends further research in this area as it could provide an alternative treatment method for the management of Acanthamoeba infections.  Inhibitory activity was carried out using trypan blue exclusion assay. 1% DMSO was used as negative control. The relative percentange of viability was de ned as (mean of the treated /mean of the control)×100.  Combination of G. mangostana extract and chlorhexidine for anti-amoebic effects on trophozoites of A.
triangularis. Parasites were grown in PYG medium in the presence of extract alone and in combination with chlorhexidine for 24 hours. Inhibitory activity was carried out using trypan blue exclusion assay. 1% DMSO was used as negative control. The relative percentage of viability was de ned as (mean of the treated /mean of the control)×100.

Figure 4
Combination of G. mangostana extract and chlorhexidine for anti-amoebic effect on cysts of A.
triangularis. Parasites were grown in PYG medium in the presence of extract alone and in combination with chlorhexidine for 24 hours. Inhibitory activity was carried out using trypan blue exclusion assay. 1% DMSO was used as negative control. The relative percentage of viability was de ned as (mean of the treated /mean of the control)×100.