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Fulltext Potential anti-Acanthamoeba and anti-adhesion activities of Annona muricata and Combretum trifoliatum extracts and their synergistic effects in combination with chlorhexidine against Acanthamoeba triangularis trophozoites and cysts

Mitsuwan W, Sin C, Keo S, Sangkanu S, de Lourdes Pereira M, Jimoh TO, et al.

Heliyon, 2021 May;7(5):e06976.
PMID: 34027178 DOI: 10.1016/j.heliyon.2021.e06976

Plants with medicinal properties have been used in the treatment of several infectious diseases, including Acanthamoeba infections. The medicinal properties of Cambodian plant extracts; Annona muricata and Combretum trifoliatum were investigated against Acanthamoeba triangularis. A total of 39 plant extracts were evaluated and, as a result, 22 extracts showed positive anti-Acanthamoeba activity. Of the 22 extracts, 9 and 4 extracts showed anti-Acanthamoeba activity against trophozoites and cysts of A. triangularis, respectively. The minimum inhibitory concentration of A. muricata and C. trifoliatum extracts against trophozoites and cysts was 500 and 1,000 μg/mL, respectively. The combination of A. muricata at 1/4×MIC with chlorhexidine at 1/8×MIC demonstrated a synergistic effect against trophozoites, but partial synergy against cysts. A 40% reduction in trophozoites and 60% of cysts adhered to the plastic surface treated with both extracts at 1/2×MIC were noted comparing to the control (P < 0.05). Furthermore, a reduction of 80% and 90% of trophozoites adhered to the surface was observed after pre-treatment with A. muricata and C. trifoliatum extracts, respectively. A 90% of cysts adhered to the surface was decreased with pre-treatment of A. muricata at 1/2×MIC (P < 0.05). A 75% of trophozoites and cysts from Acanthamoeba adhered to the surface were removed after treatment with both extracts at 4×MIC (P < 0.05). In the model of contact lens, 1 log cells/mL of trophozoites and cysts was significantly decreased post-treatment with both extracts compared to the control. Trophozoites showed strong loss of acanthopodia and thorn-like projection pseudopodia, while cysts demonstrated retraction and folded appearance treated with both extracts when observed by SEM, which suggests the potential benefits of the medicinal plants A. muricata and C. trifoliatum as an option treatment against Acanthamoeba infections.
Fulltext Curcuma longa rhizome extract and Curcumin reduce the adhesion of Acanthamoeba triangularis trophozoites and cysts in polystyrene plastic surface and contact lens

Mitsuwan W, Sangkanu S, Romyasamit C, Kaewjai C, Jimoh TO, de Lourdes Pereira M, et al.

Int J Parasitol Drugs Drug Resist, 2020 12;14:218-229.
PMID: 33238231 DOI: 10.1016/j.ijpddr.2020.11.001

Curcuma longa and Curcumin have been documented to have a wide spectrum of pharmacological effects, including anti-Acanthamoeba activity. Hence, this study sought to explore the anti-adhesion activity of C. longa extract and Curcumin against Acanthamoeba triangularis trophozoites and cysts in plastic and contact lenses. Our results showed that C. longa extract and Curcumin significantly inhibited the adhesion of A. triangularis trophozoites and cysts to the plastic surface, as investigated by the crystal violet assay (P
Fulltext Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level

Boonhok R, Sangkanu S, Norouzi R, Siyadatpanah A, Mirzaei F, Mitsuwan W, et al.

Parasitology, 2021 Aug;148(9):1074-1082.
PMID: 33966667 DOI: 10.1017/S0031182021000718

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.
Fulltext Anti-Acanthamoeba synergistic effect of chlorhexidine and Garcinia mangostana extract or α-mangostin against Acanthamoeba triangularis trophozoite and cyst forms

Sangkanu S, Mitsuwan W, Mahabusarakam W, Jimoh TO, Wilairatana P, Girol AP, et al.

Sci Rep, 2021 Apr 13;11(1):8053.
PMID: 33850179 DOI: 10.1038/s41598-021-87381-x

Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is effective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the effectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4-1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the first time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future.
Fulltext Comparative efficacy of Knema retusa extract delivery via PEG-b-PCL, niosome, and their combination against Acanthamoeba triangularis genotype T4: characterization, inhibition, anti-adhesion, and cytotoxic activity

Chimplee S, Mitsuwan W, Zulkifli M, Eawsakul K, Ongtanasup T, Sangkanu S, et al.

PeerJ, 2024;12:e18452.
PMID: 39559326 DOI: 10.7717/peerj.18452

BACKGROUND: Acanthamoeba spp. is a waterborne, opportunistic protozoan that can cause amebic keratitis and granulomatous amebic encephalitis. Knema retusa is a native tree in Malaysia, and its extracts possess a broad range of biological activities. Niosomes are non-ionic surfactant-based vesicle formations and suggest a future targeted drug delivery system. Copolymer micelle (poly(ethylene glycol)-block-poly(ɛ-caprolactone); PEG-b-PCL) is also a key constituent of niosome and supports high stability and drug efficacy. To establish Knema retusa extract (KRe) loading in diverse nanocarriers via niosome, PEG-b-PCL micelle, and their combination and to study the effect of all types of nanoparticles (NPs) on Acanthamoeba viability, adherent ability, elimination of adherence, and cytotoxicity.
METHODS: In this study, we characterized niosomes, PEG-b-PCL, and their combination loaded with KRe and tested the effect of these NPs on Acanthamoeba triangularis stages. KRe-loaded PEG-b-PCL, KRe-loaded niosome, and KRe-loaded PEG-b-PCL plus niosome were synthesized and characterized regarding particle size and charge, yield, encapsulation efficiency (EE), and drug loading content (DLC). The effect of these KRe-loaded NPs on trophozoite and cystic forms of A. triangularis was assessed through assays of minimal inhibitory concentration (MIC), using trypan blue exclusion to determine the viability. The effect of KRe-loaded NPs was also determined on A. triangularis trophozoite for 24-72 h. Additionally, the anti-adhesion activity of the KRe-loaded niosome on trophozoites was also performed on a 96-well plate. Cytotoxicity activity of KRe-loaded NPs was assessed on VERO and HaCaT cells using MTT assay.
RESULTS: KRe-loaded niosome demonstrated a higher yielded (87.93 ± 6.03%) at 286 nm UV-Vis detection and exhibited a larger size (199.3 ± 29.98 nm) and DLC (19.63 ± 1.84%) compared to KRe-loaded PEG-b-PCL (45.2 ± 10.07 nm and 2.15 ± 0.25%). The EE (%) of KRe-loaded niosome was 63.67 ± 4.04, which was significantly lower than that of the combination of PEG-b-PCL and niosome (79.67 ± 2.08). However, the particle charge of these NPs was similar (-28.2 ± 3.68 mV and -28.5 ± 4.88, respectively). Additionally, KRe-loaded niosome and KRe-loaded PEG-b-PCL plus niosome exhibited a lower MIC at 24 h (0.25 mg/mL), inhibiting 90-100% of Acanthamoeba trophozoites which lasted 72 h. KRe-loaded niosome affected adherence by around 40-60% at 0.125-0.25 mg/mL and removed Acanthamoeba adhesion on the surface by about 90% at 0.5 mg/mL. Cell viability of VERO and HaCaT cells treated with 0.125 mg/mL of KRe-loaded niosome and KRe-loaded PEG-b-PCL plus niosome exceeded 80%.
CONCLUSION: Indeed, niosome and niosome plus PEG-b-PCL were suitable nanocarrier-loaded KRe, and they had a greater nanoparticle property to test with high activities against A. triangularis on the reduction of adherence ability and demonstration of its low toxicity to VERO and HaCaT cells.
Phytochemical, anti-Acanthamoeba, and anti-adhesion properties of Garcinia mangostana flower as preventive contact lens solution

Sangkanu S, Mitsuwan W, Mahboob T, Mahabusarakam W, Chewchanwuttiwong S, Siphakdi P, et al.

Acta Trop, 2022 Feb;226:106266.
PMID: 34890540 DOI: 10.1016/j.actatropica.2021.106266

Acanthamoeba keratitis infection extends due to the growing number of contact lens users. Indigenous plants including Garcinia mangostana play a vital role in human health and well being. Many species of this plant have been reported with myriads of potent medicinal properties. However, the aims of this study were, for the first time, to isolate compounds from the flower of G. mangostana and to test their anti-Acanthamoeba and anti-adhesion activity against Acanthamoeba triangularis. Powdered flowers of G. mangostana were extracted and chromatographed on a silica gel column. The structures of the compounds were established with the aid of 1H NMR. More so, the anti-Acanthamoeba and anti-adhesion properties were tested on a 96-well polystyrene microtiter plate and soft contact lenses. Scanning electron microscope (SEM) was used to determine the features of A. triangularis on contact lenses. Eight pure compounds were obtained, namely 9-hydroxycalabaxanthone, tovophillin A, garcinone E, garcinone B, α-mangostin, gartinin, 8-deoxygartinin and γ-mangostin. The extract and pure compounds exhibited anti-Acanthamoeba activity with MIC values in the range of 0.25-1 mg/mL. In addition, the extract and α-mangostin displayed significant activity against the adhesion of A. triangularis trophozoites both in polystyrene plate and in contact lenses at 0.5 × MIC (0.25 mg/mL). Furthermore, α-mangostin has the potential to remove A. triangularis adhesion in contact lenses similar to a commercial multipurpose solution (MPS). SEM study confirmed that crude extract and α-mangostin are effective as solutions for contact lenses, which removed A. triangularis trophozoites within 24 h. Alpha-mangostin was non-toxic to Vero cells at a concentration below 39 μM in 24 h. Crude extract of G. mangostana flower and its α-mangostin serve as candidate compounds in the treatment of Acanthamoeba infection or as lens care solution, since they can be used as a source of natural products against Acanthamoeba and virulence factor associated with the adhesion of A. triangularis.
Fulltext Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens

Chuprom J, Sangkanu S, Mitsuwan W, Boonhok R, Mahabusarakam W, Singh LR, et al.

PeerJ, 2022;10:e14468.
PMID: 36523474 DOI: 10.7717/peerj.14468

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.
Fulltext Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation

Boonhok R, Sangkanu S, Phumjan S, Jongboonjua R, Sangnopparat N, Kwankaew P, et al.

PeerJ, 2022;10:e13657.
PMID: 35811814 DOI: 10.7717/peerj.13657

BACKGROUND: Curcumin is an active compound derived from turmeric, Curcuma longa, and is known for its benefits to human health. The amoebicidal activity of curcumin against Acanthamoeba triangularis was recently discovered. However, a physiological change of intracellular pathways related to A. triangularis encystation mechanism, including autophagy in the surviving amoeba after curcumin treatment, has never been reported. This study aims to investigate the effect of curcumin on the survival of A. triangularis under nutrient starvation and nutrient-rich condition, as well as to evaluate the A. triangularis encystation and a physiological change of Acanthamoeba autophagy at the mRNA level.
METHODS: In this study, A. triangularis amoebas were treated with a sublethal dose of curcumin under nutrient starvation and nutrient-rich condition and the surviving amoebas was investigated. Cysts formation and vacuolization were examined by microscopy and transcriptional expression of autophagy-related genes and other encystation-related genes were evaluated by real-time PCR.
RESULTS: A. triangularis cysts were formed under nutrient starvation. However, in the presence of the autophagy inhibitor, 3-methyladenine (3-MA), the percentage of cysts was significantly reduced. Interestingly, in the presence of curcumin, most of the parasites remained in the trophozoite stage in both the starvation and nutrient-rich condition. In vacuolization analysis, the percentage of amoebas with enlarged vacuole was increased upon starvation. However, the percentage was significantly declined in the presence of curcumin and 3-MA. Molecular analysis of A. triangularis autophagy-related (ATG) genes showed that the mRNA expression of the ATG genes, ATG3, ATG8b, ATG12, ATG16, under the starvation with curcumin was at a basal level along the treatment. The results were similar to those of the curcumin-treated amoebas under a nutrient-rich condition, except AcATG16 which increased later. On the other hand, mRNA expression of encystation-related genes, cellulose synthase and serine proteinase, remained unchanged during the first 18 h, but significantly increased at 24 h post treatment.
CONCLUSION: Curcumin inhibits cyst formation in surviving trophozoites, which may result from its effect on mRNA expression of key Acanthamoeba ATG-related genes. However, further investigation into the mechanism of curcumin in A. triangularis trophozoites arrest and its association with autophagy or other encystation-related pathways is needed to support the future use of curcumin.
Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects

Sama-Ae I, Sangkanu S, Siyadatpanah A, Norouzi R, Chuprom J, Mitsuwan W, et al.

F1000Res, 2022;11:1274.
PMID: 36936052 DOI: 10.12688/f1000research.126227.1

Background : Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti- Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of the agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results : The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions : The results obtained provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.
Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis

Chuprom J, Kidsin K, Sangkanu S, Nissapatorn V, Wiart C, de Lourdes Pereira M, et al.

Vet Res Commun, 2023 Jun;47(2):523-538.
PMID: 36260188 DOI: 10.1007/s11259-022-09999-0

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.
Fulltext Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes

Boonhok R, Sangkanu S, Chuprom J, Srisuphanunt M, Norouzi R, Siyadatpanah A, et al.

Pathogens, 2021 Jul 04;10(7).
PMID: 34357992 DOI: 10.3390/pathogens10070842

Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.

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