Anti-acanthamoebic activity of methanolic extract of Piper sarmentosum leaves

Aims@#Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii.@*Methodology and results@#Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba.@*Conclusion, significance and impact of study@#This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.

Repurposing Auranofin and Evaluation of a New Gold(I) Compound for the Search of Treatment of Human and Cattle Parasitic Diseases: From Protozoa to Helminth Infections.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Anti-Acanthamoeba effect of potassium isostearate for use as a multipurpose solution.

Acanthamoeba is one of the organisms that cause corneal infection. In this study, attention was focused on potassium isostearate (iso-C18K, a branched chain fatty acid salt) for use in a multipurpose solution (MPS) against Acanthamoeba. An anti-amoebic test against Acanthamoeba castellanii ATCC 30010 (trophozoites type) was conducted. As a result, a growth reduction effect of 4 log units (99.99% suppression) was observed after incubation with 150 mM (5.0 w/v%) iso-C18K for 10 minutes. Furthermore, after the amoeba suspension was mixed with iso-C18K, disruption of cell membranes were observed, and the minimum amoebacidal concentration (MAC) at that time was 9.6 mM (0.31 w/v%). To evaluate the effectiveness as an MPS, assessment by verification tests was conducted using contact lenses. Reducing the concentration of iso-C18K caused a decrease in the number of viable cells, which was confirmed at a MAC of 1.2 mM (0.039 w/v%).

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Philippine tsaang gubat (Ehretia microphylla Lam) and ampalaya (Momordica charantia L.) leaf extracts lack amoebicidal activity in vitro

Background@#Amoebiasis is a global health problem affecting poor regions in the world. Few drugs such as metronidazole are available to treat this disease; unfortunately, it is associated with several serious side effects. Tsaang gubat and ampalaya have been used by traditional healers from different cultures to treat dysentery.@*Objective@#The aim of this research was to provide evidence to validate the use of tsaang gubat and ampalaya leaf extracts for dysentery by determining their anti-amoebic activity.@*Methods@#The tsaang gubat and ampalaya leaves were sourced from the University of the Philippines at Los Baños and processed into a lyophilized aqueous extract. Anti-amoebic activity was determined in an in vitro assay using Entamoeba histolytica HK-9 strain against 10 dose levels (18-10,000 μg/mL). The amoeba and leaf extracts were incubated for 24, 48, and 72 hours. The trophozoites were stained with Trypan blue and dispensed into chambers of a Neubauer hemocytometer. The live trophozoites (unstained) were counted under a binocular microscope. The MIC and IC50 were determined. Metronidazole and DMSO served as positive and negative controls, respectively.@*Results@#Tsaang gubat and ampalaya leaves failed to show anti-amoebic activity and even had increased growth of amoeba at all dose levels. The IC50 of tsaang gubat and ampalaya leaf extracts were >500 μg/mL at 24, 48, and 72 hours. Metronidazole was able to eradicate the amoeba parasite at 24 and 72 hours, while exposure to DMSO did not result in inhibition nor death of the parasite.@*Conclusion@#Tsaang gubat and ampalaya aqueous leaf extracts did not exhibit any anti-amoeba activity.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.

Role of fatty acid salts as anti Acanthamoeba agents for disinfecting contact lens.

　Acanthamoeba is found in seawater, fresh water, and soil and is an opportunistic pathogen that causes a potentially blinding corneal infection known as Acanthamoeba keratitis. The anti-amoeba activity of 9 fatty acid salts (potassium butyrate (C4K), caproate (C6K), caprylate (C8K), caprate (C10K), laurate (C12K), myristate (C14K), oleate (C18:1K), linoleate (C18:2K), and linolenate (C18:3K)) was tested on Acanthamoeba castellanii ATCC 30010 (trophozoites and cysts). Fatty acid salts (350 mM and pH 10.5) were prepared by mixing fatty acids with the appropriate amount of KOH. C8K, C10K, and C12K showed growth reduction of 4 log-units (99.99% suppression) in A. castellanii upon 180 min incubation at 175 mM, whereas the pH-adjusted control solution showed no effect. After the amoeba suspension was mixed with C10K or C12K, cell membrane destruction was observed. The minimum inhibitory concentration of C10K and C12K was also determined to be 2.7 mM. Confirmation tests were conducted using contact lenses to evaluate the effectiveness of C10K and C12K as multi-purpose solutions. Experiments using increasing concentrations showed reduced numbers of living cells in C10K (5.5 mM, 10.9 mM) and in C12K (5.5 mM, 10.9 mM). These results demonstrate the inhibitory activity of C10K and C12K against A. castellanii and indicate their potential as anti-amoeba agents.

A case of amebiasis with negative serologic markers that caused intra-abdominal abscess.

A 23-year-old Japanese woman presented with abdominal distention following fever, diarrhea, and abdominal pain during a stay in Taiwan. Serology for the detection of amebic-antibodies and stool microscopic examination were both negative. A computed tomography scan showed a 13 cm diameter abscess spreading from the lower abdominal wall to the pelvic retroperitoneal space. Needle aspiration of the abscess was done under computed tomography guidance, and microscopy of the aspirated fluid revealed trophozoites of Entamoeba. The patient was diagnosed as amebiasis with negative serologic markers that caused intra-abdominal abscess. Intravenous metronidazole treatment for two weeks did not result in any improvement of the abscess. After irrigation and drainage of the abscess, her symptoms resolved. This case report highlights that amebiasis should be considered when indicated by patient history, including travelers returning from endemic areas, and that further evaluation is necessary for diagnosis, even if the serology and stool test are negative.