Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

Preparation and evaluation of a niosomal delivery system containing G. mangostana extract and study of its anti-Acanthamoeba activity.

Garcinia mangostana extract (GME) has severe pharmacokinetic deficiencies and is made up of a variety of bioactive components. GME has proven its anti-Acanthamoeba effectiveness. In this investigation, a GME-loaded niosome was developed to increase its potential therapeutic efficacy. A GME-loaded niosome was prepared by encapsulation in a mixture of span60, cholesterol, and chloroform by the thin film hydration method. The vesicle size, zeta potential, percentage of entrapment efficiency, and stability of GME-loaded niosomes were investigated. The values for GME-loaded niosome size and zeta potential were 404.23 ± 4.59 and -32.03 ± 0.95, respectively. The delivery system enhanced the anti-Acanthamoeba activity, which possessed MIC values of 0.25-4 mg mL-1. In addition, the niosomal formulation decreased the toxicity of GME by 16 times. GME-loaded niosome must be stored at 4 °C, as the quantity of remaining GME encapsulated is greater at this temperature than at room temperature. SEM revealed the damage to the cell membrane caused by trophozoites and cysts, which led to dead cells. In light of the above, it was found that GME-loaded niosomes had better anti-Acanthamoeba activity. The study suggested that GME-loaded niosomes could be used as an alternative to Acanthamoeba's therapeutic effects.

Tea tree oil, a vibrant source of neuroprotection via neuroinflammation inhibition: a critical insight into repurposing Melaleuca alternifolia by unfolding its characteristics. / èŒ¶æ ‘æ²¹æŠ‘åˆ¶ç¥žç»ç‚Žç—‡å¹¶æä¾›ç¥žç»ä¿æŠ¤­­æŽ¢ç´¢äº’花千层的潜力.

Over the past few decades, complementary and alternative treatments have become increasingly popular worldwide. The purported therapeutic characteristics of natural products have come under increased scrutiny both in vitro and in vivo as part of efforts to legitimize their usage. One such product is tea tree oil (TTO), a volatile essential oil primarily obtained from the native Australian plant, Melaleuca alternifolia, which has diverse traditional and industrial applications such as topical preparations for the treatment of skin infections. Its anti-inflammatory-linked immunomodulatory actions have also been reported. This systematic review focuses on the anti-inflammatory effects of TTO and its main components that have shown strong immunomodulatory potential. An extensive literature search was performed electronically for data curation on worldwide accepted scientific databases, such as Web of Science, Google Scholar, PubMed, ScienceDirect, Scopus, and esteemed publishers such as Elsevier, Springer, Frontiers, and Taylor & Francis. Considering that the majority of pharmacological studies were conducted on crude oils only, the extracted data were critically analyzed to gain further insight into the prospects of TTO being used as a neuroprotective agent by drug formulation or dietary supplement. In addition, the active constituents contributing to the activity of TTO have not been well justified, and the core mechanisms need to be unveiled especially for anti-inflammatory and immunomodulatory effects leading to neuroprotection. Therefore, this review attempts to correlate the anti-inflammatory and immunomodulatory activity of TTO with its neuroprotective mechanisms.

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

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.

Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation.

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.

Drug Repurposing of Quisinostat to Discover Novel Plasmodium falciparum HDAC1 Inhibitors with Enhanced Triple-Stage Antimalarial Activity and Improved Safety.

Our previous work found that the clinical histone deacetylase (HDAC) inhibitor quisinostat exhibited a significant antimalarial effect but with severe toxicity. In this work, 35 novel derivatives were designed and synthesized based on quisinostat as the lead compound, and their in vitro antimalarial activities and cytotoxicities were systematically evaluated. Among them, JX35 showed potent inhibition against both wild-type and multidrug-resistant parasite strains and displayed a significant in vivo killing effect against all life cycles of parasites, including the blood stage, liver stage, and gametocyte stage, indicating its potential for the simultaneous treatment, chemoprevention, and blockage of malaria transmission. Compared with quisinostat, JX35 exhibited stronger antimalarial efficacy, more adequate safety, and good pharmacokinetic properties. Additionally, mechanistic studies via molecular docking studies, induced PfHDAC1/2 knockdown assays, and PfHDAC1 enzyme inhibition assays jointly indicated that the antimalarial target of JX35 was PfHDAC1. In summary, we discovered the promising candidate PfHDAC1 inhibitor JX35, which showed stronger triple-stage antimalarial effects and lower toxicity than quisinostat.

Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects.

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 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 provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.

Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes.

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.

Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level.

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.

Discovery of fast-acting dual-stage antimalarial agents by profiling pyridylvinylquinoline chemical space via copper catalyzed azide-alkyne cycloadditions.

To identity fast-acting, multistage antimalarial agents, a series of pyridylvinylquinoline-triazole analogues have been synthesized via CuAAC. Most of the compounds display significant inhibitory effect on the drug-resistant malarial Dd2 strain at low submicromolar concentrations. Among the tested analogues, compound 60 is the most potent molecule with an EC50 value of 0.04 ± 0.01 µM. Our current study indicates that compound 60 is a fast-acting antimalarial compound and it demonstrates stage specific action at the trophozoite phase in the P. falciparum asexual life cycle. In addition, compound 60 is active against both early and late stage P. falciparum gametocytes. From a mechanistic perspective, compound 60 shows good activity as an inhibitor of ß-hematin formation. Collectively, our findings suggest that fast-acting agent 60 targets dual life stages of the malarial parasites and warrant further investigation of pyridylvinylquinoline hybrids as new antimalarials.

Resistance to cellular autophagy by Mycobacterium tuberculosis Beijing strains.

Autophagy represents a key pathway in innate immune defense to restrict Mycobacterium tuberculosis growth inside host macrophages. Induction of autophagy has been shown to promote mycobacterial phagosome acidification and acquisition of lysosomal hydrolases, resulting in the elimination of intracellular M. tuberculosis reference strains such as H37Rv. The notorious Beijing genotype has been previously shown to be hyper-virulent and associated with increased survival in host cells and a high mortality rate in animal models, but the underlying mechanism that renders this family to have such advantages remains unclear. We hypothesize that autophagic control against M. tuberculosis Beijing strains may be altered. Here, we discovered that the Beijing strains can resist autophagic killing by host cells compared with that of the reference strain H37Rv and a strain belonging to the East African Indian genotype. Moreover, we have determined a possible underlying mechanism and found that the greater ability to evade autophagic elimination possessed by the Beijing strains stems from their higher capacity to inhibit autophagolysosome biogenesis upon autophagy induction. In summary, a previously unrecognized ability of the M. tuberculosis Beijing strains to evade host autophagy was identified, which may have important implications for tuberculosis treatment, especially in regions prevalent by the Beijing genotype.

In vitro antifungal activities of longan (Dimocarpus longan Lour.) seed extract.

Longan, Dimocarpus longan Lour., contains polyphenolic compounds which exhibit several pharmacological properties. This study aims to evaluate antifungal activities of longan fruit extract in comparison to its active compounds. The results showed that longan seed exhibited antifungal activity against the opportunistic yeasts (Candida species and Cryptococcus neoformans). In contrast, longan pulp and whole fruit did not demonstrate any inhibitory effects. Ellagic acid showed the most potent antifungal activity followed by corilagin and gallic acid, respectively. Ellagic acid inhibited Candida parapsilosis and C. neoformans more effectively than Candida krusei and also some Candida albicans clinical strains. Baidam cultivar possessed higher antifungal activity (MIC=500-4000 µg/ml) as it contained higher contents of ellagic acid and gallic acid than Edor (MIC=1000-8000 µg/ml). For antibacterial activity, only corilagin and gallic acid possessed weak to moderate inhibitory effects against Staphylococcus aureus and Streptococcus mutans, respectively. Longan seed was then applied in the oral care products. Longan effervescent granule (5% extract) significantly reduced adhesion of C. albicans to acrylic strips. Mouthwash containing 0.5% extract exhibited good antifungal activity compared to a commercial product. These findings indicated that longan seed extract and its polyphenolic compounds can be used as an antifungal agent in oral care products for the treatment of opportunistic yeast infection.