Herb-drug interaction may offer potential health promoting effect by augmenting drug efficacy 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-inflammatory, anti-bacterial and anti-viral activities . 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 . A. triangularis was originally isolated from the human feces documented in France . In 2008, the first case of keratitis caused by A. triangularis had been reported in Korea . Also, six isolates of A. triangularis had also been reported from cases with contaminated contact lens in southeastern Korea. In support of those previous findings, 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 finding is in agreement with earlier reported studies that found the compounds of G. mangostana extract with anti-parasitic activities. In literature, xanthones have been identified as the main compound of G. mangostana extract. Of this, xanthones have been isolated from pericarp, whole fruit, leaves and bark . 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 IC50 value of 17 µM against Plasmodium falciparum . Another study showed that α-mangostin and δ-mangostin exhibited anti-malarial activity against P. falciparum chloroquine-resistant strain with IC50 value 0.2 and 121.2 µM, respectively . Furthermore, a previous study has reported the use of G. mangostana pericarp in the management of amoebic dysentery . 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 . 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, significant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation . Benzophenone (isoxanthochymol) was isolated from the roots of G. polyantha which showed strong chemosuppressive activity of parasitic growth found in P. falciparum . The benzophenones (guttiferone E, isoxanthochymol, and guttiferone H) isolated from G. xanthochymus exhibited antiplasmodial activity with IC50 values in the range of 4.71–11.40 µM . The tetraprenylated benzophenone 7-epiclusianone (7-epi) was isolated from the fruits of G. brasiliensis showed effective against Schistosoma mansoni . In addition, biflavanones (GB-1a, GB-1, and GB-2), another active compounds, were isolated from G. kola. These three biflavanones 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 . As kolaviron (KV), a biflavonoid fraction from G. kola seeds that has also been reported to exhibit anti-malarial activities in P. berghei-infected mice . The purified bioflavonoid of the fruit pericarp of G. brasiliensis, showed significant activity as inhibitors of Leishmania's proteases, with mean (± SD) IC50 values of 15.0 ± 1.3 µg/mL . 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 . Therefore, xanthones, biflavanones, 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 significantly (P < 0.05) in the presence of G. mangostana extract (MIC and 2 × MIC final 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 first potentially report of G. mangostana extract in its capacity to significantly 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  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 finding 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 . 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 . 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 finding is in consistent with an earlier report on chlorhexidine used against A. triangularis exhibited amoebicidal and cysticidal properties at 200 µg/mL (0.02%) . While, a recent study  evaluated the efficacy 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 , aminoglycosides, imidazoles and polyene , however these chemicals show side effects on keratocytes found in cases of human keratitis . Recently, the finding 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 efficacies but not to the extent of synergic interaction . 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 finding 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 . From this study, it appears to be a promising combined agents to fight against the infection and especially resistance pattern of Acanthamoeba spp. in the future.
The mode of action considered in this study was confirmed by scanning electron microscopy (SEM) as shown in Figs. 5 and 6. Treated trophozoites showed similar flat 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 flat 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.