Biomedical behaviors of CuO/γ-alumina/chitosan nanocomposites: Scolicidal and apoptotic effects on hydatid cysts protoscolices.

Hydatid cysts caused by Echinococcus granulosus are a serious health problem that requires effective treatment. This study aimed to evaluate the scolicidal and apoptotic effects of copper oxide (CuO) and gamma alumina (γ-Al2O3) with or without chitosan (Chit), using Rosmarinus officinalis extract and chemical methods on protoscolices (PSCs) in vitro. The nanomaterials (NMs) were characterized by FTIR, EDS, DLS, XRD, FESEM, PDI, and zeta potential (ZP). Scolicidal and apoptotic effects of NMs were tested against PSCs at different concentrations and exposure times. The CuO NPs showed the highest scolicidal effect (33.26 %) among all NMs at 1.6 mg/mL and 60 min, followed by phytosynthesized CuO/γ-Al2O3 NC (23.41 %). The chitosan-modified CuO/γ-Al2O3 NC and the chemically synthesized CuO/γ-Al2O3 NC had less effect. The CuO NPs and the phytosynthesized CuO/γ-Al2O3 NC also significantly increased the expression of the caspase-3 gene in the PSCs at 0.4 mg/mL, indicating the induction of apoptosis. In conclusion, this study suggests that the phytosynthesized CuO/γ-Al2O3 NC and the CuO NPs could be potential candidates for treating echinococcosis by killing the PSCs through apoptosis. Further studies are needed to verify the in vivo efficacy and toxicity of these NMs and to optimize their delivery and targeting systems.

Scolicidal and apoptotic effects of phyto- and chemically synthesized silver/boehmite nanocomposites on Echinococcus granulosus protoscoleces.

Cystic hydatid disease (CHD) is a zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus). This study aimed to synthesize silver nanoparticles (Ag NPs), silver boehmite nanocomposite (Ag/Bhm NC), and silver boehmite nanocomposite modified with chitosan (Ag/Bhm/Chit NC) using Rosmarinus officinalis (R. officinalis) extract and chemical method, and to evaluate their scolicidal and apoptotic effects on protoscoleces (PSCs) in vitro. The nanomaterials (NMs) were characterized by XRD, FTIR, FESEM, EDS, DLS, PDI, and zeta potential (ZP). The NMs were tested against PSCs at different concentrations (0.2-1.6 mg/mL) and exposure times (10-60 min). The size of Ag NPs, phytosynthesized Ag/Bhm NC, Ag/Bhm/Chit NC, and chemically synthesized Ag/Bhm NC were 25.55, 43, 72.3, and 60.8 nm, respectively. Ag NPs and phytosynthesized Ag/Bhm NC showed the highest scolicidal effect, with 65.34 % and 51.60 % mortality rate at 1.6 mg/mL and 60 min, respectively. Caspase-3 mRNA expression was higher in PSCs treated with Ag NPs and Ag/Bhm NC than in control groups (P < 0.05). Phytosynthesized Ag/Bhm NC had stronger scolicidal and apoptotic effect than chemically synthesized Ag/Bhm NC. Ag/Bhm/Chit NC had a weaker scolicidal effect but higher gene expression than Ag/Bhm NC. In conclusion, this study demonstrates the potential of phytosynthesized Ag NPs and Ag/Bhm NC as effective scolicidal and apoptotic agents against PSCs of hydatid cysts, which may be useful for the treatment of this disease.

Antioxidant Potential of Ethiopian Medicinal Plants and Their Phytochemicals: A Review of Pharmacological Evaluation.

Background: Free radicals are very reactive molecules produced during oxidation events that in turn initiate a chain reaction resulting in cellular damage. Many degenerative diseases in humans, including cancer and central nervous system damage, are caused by free radicals. Scientific evidence indicates that active compounds from natural products can protect cells from free radical damage. As a result, the aim of this review is to provide evidence of the use of diverse Ethiopian medicinal plants with antioxidant properties that have been scientifically validated in order to draw attention and foster further investigations in this area. Methods: The keywords antioxidant, radical scavenging activities, reactive oxygen species, natural product, Ethiopian Medicinal plants, and 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging assay (DPPH) were used to identify relevant data in the major electronic scientific databases, including Google Scholar, ScienceDirect, PubMed, Medline, and Science domain. All articles with descriptions that were accessed until November 2022 were included in the search strategy. Results: A total of 54 plant species from 33 families were identified, along with 46 compounds isolated. More scientific studies have been conducted on plant species from the Brassicaceae (19%), Asphodelaceae (12%), and Asteraceae (12%) families. The most used solvent and extraction method for plant samples are methanol (68%) and maceration (88%). The most examined plant parts were the leaves (42%). Plant extracts (56%) as well as isolated compounds (61%) exhibited significant antioxidant potential. The most effective plant extracts from Ethiopian flora were Bersama abyssinica, Solanecio gigas, Echinops kebericho, Verbascum sinaiticum, Apium leptophyllum, and Crinum abyssinicum. The best oxidative phytochemicals were Rutin (7), Flavan-3-ol-7-O-glucoside (8), Myricitrin (13), Myricetin-3-O-arabinopyranoside (14), 7-O-Methylaloeresin A (15), 3-Hydroxyisoagatholactone (17), ß-Sitosterol-3-O-ß-D-glucoside (22), Microdontin A/B (24), and Caffeic acid (39). Conclusion: Many crude extracts and compounds exhibited significant antioxidant activity, making them excellent candidates for the development of novel drugs. However, there is a paucity of research into the mechanisms of action as well as clinical evidence supporting some of these isolated compounds. To fully authenticate and then commercialize, further investigation and systematic analysis of these antioxidant-rich species are required.

In Vivo Toxicity of Oral Administrated Nano-SiO2: Can Food Additives Increase Apoptosis?

Nano-silicon dioxide (nano-SiO2) has a great deal of application in food packaging, as antibacterial food additives, and in drug delivery systems but this nanoparticle, despite its wide range of utilizations, can generate destructive effects on organs such as the liver, kidney, and lungs. This study is aimed at investigating the toxicological effects of nano-SiO2 through apoptotic factors. For this purpose, 40 female rats in 4 groups (n = 10) received 300, 600, and 900 mg/kg/day of nano-SiO2 at 20-30 nm size orally for 20 days. Relative expression of Caspase3, Bcl-2, and BAX genes in kidney and liver was evaluated in real time-PCR. The results indicated the overexpression of BAX and Caspase3 genes in the liver and kidney in groups receiving 300 and 900 mg/kg/day of nano-SiO2. Bcl-2 gene was up-regulated in the liver and kidney at 600 mg/kg/day compared to the control group. Overexpression of the Bcl-2 gene in the kidney in 300 and 900 mg/kg/day recipient groups was observed (P ≤ 0.05). Histopathological examination demonstrated 600 mg/kg/day hyperemia in the kidney and lungs. In addition, at 900 mg/kg/day were distinguished scattered necrosis and hyperemia in the liver. The rate of epithelialization in the lungs increased. The nano-SiO2 at 300 and 900 mg/kg/day can induce more cytotoxicity in the liver and lung after oral exposure. However, cytotoxicity of nano-SiO2 at 600 mg/kg/day in the kidney and lung was noticed. Hence, the using of nano-SiO2 as an additive and food packaging should be more considered due to their deleterious effects.

Green synthesis of nano-liposomes containing Bunium persicum and Trachyspermum ammi essential oils against Trichomonasvaginalis.

BACKGROUND: Trichomonas vaginalis, a parasitic flagellated protozoan, is one of the main non-viral sexually transmitted diseases worldwide. Treatment options for trichomoniasis are limited to nitroimidazole compounds. However, resistance to these drugs has been reported, which requires the development of new anti-Trichomonas agents that confer suitable efficacy and less toxicity. METHODS: In the present work, we assessed the effectiveness of the liposomal system containing essential oils of Bunium persicum and Trachyspermum ammi against T. vaginalis in vitro. The chemical composition of B. persicum and T. ammi were analyzed using gas chromatography-mass spectrometry (GC-MS). Liposomal vesicles were prepared with phosphatidylcholine) 70%) and cholesterol)30%) using the thin-film method. The essential oils of B. persicum and T. ammi were loaded into the liposomes using the inactive loading method. Liposomal vesicles were made for two plants separately. Their physicochemical features were tested using Zeta-Sizer, AFM and SEM. The anti-Trichomonas activity was determined after 12 and 24 h of parasite cultures in TYI-S-33 medium. RESULTS: After 12 and 24 h of administration, the IC50 of the B. persicum essential oil nano-liposomes induced 14.41 µg/mL and 45.19 µg/mL, respectively. The IC50 of T. ammi essential oil nano-liposomes induced 8.08 µg/mL and 25.81 µg/mL, respectively. CONCLUSIONS: These data suggested that nano-liposomes of the essential oils of B. persicum and T. ammi may be a promising alternative to current treatments for Trichomonas infection.

Hepatoprotective activity of andrographolide possibly through antioxidative defense mechanism in Sprague-Dawley rats.

The aims of this study to assess the efficiency of AGL against acetaminophen (APAP)-induced hepatic toxicity that was generated by mitochondrial oxidative stress and glutathione depletion. Free radical scavenging potentiality was analyzed by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide, nitric oxide, and hydroxyl radical scavenging assays. APAP-induced liver toxicity was formed at a dose level of 640 mg/kg mg/kg BW each, p.o. for 14 days for all experimental rats except the vehicle control group. AGL (5 and 10 mg/kg) were treated orally with negative control and negative control silymarin (50 mg/kg) group. To assess the protective effect, we looked at the levels of serum biochemical markers, liver histoarchitecture, and hepatic antioxidant enzyme activity. AGL showed in vitro anti-oxidant potentialities by scavenging radicals in the respective assays. As evidenced by serum biochemical indicators and relative liver weight, AGL co-administration substantially reduced toxicant-induced hepatic damage. APAP-intoxication increased the malondialdehyde (MDA) level and declined in cellular endogenous antioxidant enzymes such as reduced catalase, superoxide dismutase, and glutathione, where, AGL treatment amended their level. In the same way, histopathological evaluation further verified that AGL protected the hepatocyte from APAP-induced damage. As AGL scavenges toxic free radicals, thereby protects mitochondria and other organelles from reactive oxygen and nitrogen species-mediated stress and its eventual consequence necrosis. Therefore, we propose the hepatoprotective activity of AGL through its antioxidant mechanism.

In vitro efficacy of albendazole-loaded ß-cyclodextrin against protoscoleces of Echinococcus granulosus sensu stricto.

BACKGROUND: Cystic echinococcosis (CE), a widespread helminthic disease caused by the larval stage of the dog tapeworm Echinococcus granulosus represents a public health concern in humans. Albendazole (ABZ) is the first-line treatment for CE; however therapeutic failure of ABZ against CE occurs because of size and location of formed cysts as well its low aqueous solubility and consequently its erratic bioavailability in plasma. Serious adverse effects have also been observed following the long-term use of ABZ in vivo. METHODS: We evaluated the apoptotic effects of ABZ-loaded ß-cyclodextrin (ABZ-ß-CD) against protoscoleces (PSCs) versus ABZ alone. After 15 h of exposure, Caspase-3 enzymatic activity was determined by fluorometric assay in PSCs treated with ABZ and ABZ-ß-CD groups. To assess the treatment efficacy of ABZ-ß-CD against PSCs, mRNA expression of Arginase (EgArg) and Thioredoxin peroxidase (EgTPx) were quantified by Real-time PCR. RESULTS: A significant scolicidal activity of ABZ was observed only at a concentration of 800 µg/mL (100% PSCs mortality rate after 4 days of exposure), while the 200 and 400 µg/mL ABZ reached 100% PSCs mortality rate after 9 sequential days. The 400 µg/mL ABZ-ß-CD had 100% scolicidal rate after 5 days of exposure. Morphological alterations using scanning electron microscopy in treated PSCs revealed that 400 µg/mL ABZ-ß-CD induced higher Caspase-3 activity than their controls, indicating a more potent apoptotic outcome on the PSCs. Also, we showed that the 400 µg/mL ABZ-ß-CD can down-regulate the mRNA expression of EgArg and EgTPx, indicating more potent interference with growth and antioxidant properties of PSCs. CONCLUSIONS: In the present study, a significant scolicidal rate, apoptosis intensity and treatment efficacy was observed in PSCs treated with 400 µg/mL ABZ-ß-CD compared to ABZ alone. This provides new insights into the use of nanostructured ß-CD carriers with ABZ as a promising candidate to improve the treatment of CE in in vivo models.

Potentiation of the Activity of Antibiotics against ATCC and MDR Bacterial Strains with (+)-α-Pinene and (-)-Borneol.

The increasing rates of antimicrobial resistance have demanded the development of new drugs as conventional antibiotics have become significantly less effective. Evidence has identified a variety of phytocompounds with the potential to be used in the combat of infections caused by multidrug-resistant (MDR) bacteria. Considering the verification that terpenes are promising antibacterial compounds, the present research aimed to evaluate the antibacterial and antibiotic-modulating activity of (+)-α-pinene and (-)-borneol against MDR bacterial strains. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of the compounds and antibiotics and further evaluate the intrinsic and associated antibiotic activity. These analyses revealed that (+)-α-pinene showed significant antibacterial activity only against E. coli (MIC = 512 µg.mL-1), while no significant inhibition of S. aureus and P. aeruginosa growth was observed (MIC ≥ 1024 µg mL-1). However, when combined with antibiotics, this compound induced a significant improvement in the activity of conventional antibiotics, as observed for ciprofloxacin, amikacin, and gentamicin against Staphylococcus aureus, as well as for amikacin and gentamicin against Escherichia coli, and amikacin against Pseudomonas aeruginosa. On the other hand, (-)-borneol was found to inhibit the growth of E. coli and enhance the antibiotic activity of ciprofloxacin and gentamicin against S. aureus. The present findings indicate that (+)-α-pinene and (-)-borneol are phytocompounds with the potential to be used in the combat of antibacterial resistance.

Evaluation of the In Vitro Antiparasitic Effect of the Essential Oil of Cymbopogon winterianus and Its Chemical Composition Analysis.

Cymbopogon winterianus, known as "citronella grass", is an important aromatic and medicinal tropical herbaceous plant. The essential oil of C. winterianus (EOCw) is popularly used to play an important role in improving human health due to its potential as a bioactive component. The present study aimed to identify the components of the essential oil of C. winterianus and verify its leishmanicidal and trypanocidal potential, as well as the cytotoxicity in mammalian cells, in vitro. The EOCw had geraniol (42.13%), citronellal (17.31%), and citronellol (16.91%) as major constituents. The essential oil only exhibited significant cytotoxicity in mammalian fibroblasts at concentrations greater than 250 µg/mL, while regarding antipromastigote and antiepimastigote activities, they presented values considered clinically relevant, since both had LC50 < 62.5 µg/mL. It can be concluded that this is a pioneer study on the potential of the essential oil of C. winterianus and its use against the parasites T. cruzi and L. brasiliensis, and its importance is also based on this fact. Additionally, according to the results, C. winterianus was effective in presenting values of clinical relevance and low toxicity and, therefore, an indicator of popular use.

Ferulic acid derivatives inhibiting Staphylococcus aureus tetK and MsrA efflux pumps.

Background: Bacterial resistance to multiple drugs has recently emerged as a serious health problem. Concomitantly, the characterization of new substances with potential antimicrobial activity has been less frequent in the drug development industry. The overexpression of genes encoding efflux pumps that expel antimicrobial drugs from the intracellular environment, lowering these to subinhibitory concentrations, are among the resistance mechanisms predisposing microorganisms to high drug resistance. Staphylococcus aureus is a bacterium found in the normal microbiota of the skin and mucous membranes, and is an opportunistic microorganism capable of causing infections with high rates of morbidity and mortality. TetK is an efflux pump characterized by its ability to provide bacterial resistance to antibiotics from the tetracycline class. This study aimed to evaluate the inhibitory effect of ferulic acid and four of its esterified derivatives against resistant Staphylococcus aureus strains. Method: Ferulic acid derivatives were obtained by esterification and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis. The minimum inhibitory concentrations (MIC) of ferulic acid and its esterified derivatives, ethidium bromide, and antibiotics were obtained using the microdilution test, while the efflux pump inhibition test was conducted by examining reduction in the MICs. Results: Propylferulate was seen to reduce the minimum inhibitory concentration (MIC) of both the control substance ethidium bromide and the tested antibiotic, indicating that this compound is promising for the use of efflux pump inhibition of IS-58 strains. Conclusions: This study provides strong evidence that the molecular basis for this activity is potentially due to the MsrA and TetK efflux pumps. However, further investigations are necessary to prove this hypothesis and elucidate the potentiating mechanism of the modulatory effect.

Microbial resistance: The role of efflux pump superfamilies and their respective substrates.

The microorganism resistance to antibiotics has become one of the most worrying issues for science due to the difficulties related to clinical treatment and the rapid spread of diseases. Efflux pumps are classified into six groups of carrier proteins that are part of the different types of mechanisms that contribute to resistance in microorganisms, allowing their survival. The present study aimed to carry out a bibliographic review on the superfamilies of carriers in order to understand their compositions, expressions, substrates, and role in intrinsic resistance. At first, a search for manuscripts was carried out in the databases Medline, Pubmed, ScienceDirect, and Scielo, using as descriptors: efflux pump, expression, pump inhibitors and efflux superfamily. For article selection, two criteria were taken into account: for inclusion, those published between 2000 and 2020, including textbooks, and for exclusion, duplicates and academic collections. In this research, 139,615 published articles were obtained, with 312 selected articles and 7 book chapters that best met the aim. From the comprehensive analysis, it was possible to consider that the chromosomes and genetic elements can contain genes encoding efflux pumps and are responsible for multidrug resistance. Even though this is a well-explored topic in the scientific community, understanding the behavior of antibiotics as substrates that increase the expression of pump-encoding genes has challenged medicine. This review study succinctly summarizes the most relevant features of these systems, as well as their contribution to multidrug resistance.

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.

Phytochemical, anti-Acanthamoeba, and anti-adhesion properties of Garcinia mangostana flower as preventive contact lens solution.

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.

In vitro scolicidal effect of Calendula officinalis, Artemisia dracunculus, Artemisia absinthium, and Ferula assafoetida extracts against hydatid cyst protoscolices.

Echinococcus granulosus is the etiologic agent of cystic echinococcosis. Numerous research studies have been conducted on natural scolicidal agents to inactivate protoscolices during surgery. This study was undertaken to compare the in vitro scolicidal effects of hydroalcoholic extracts of Calendula officinalis, Artemisia dracunculus, Artemisia absinthium and Ferula assafoetida. The scolicidal activities of the extracts were tested at different concentrations following incubation periods of 10, 30 and 60 min. The chemical composition of the hydroalcoholic extracts were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The major chemical components of C. officinalis, A. dracunculus, A. absinthium and F. assafoetida were identified as n-Docosane (14.17%), 2H-1-benzopyran-2-one, 7-methoxy (54.96%), n-Docosane (9.72%) and 2-methoxy-3-methyl-butyric acid, methyl ester (13.9%), respectively. The results showed that the hydroalcoholic extracts of A. absinthium and F. assafoetida at a concentration of 250 mg/ml resulted in killing 100% of the protoscolices at 60 minutes, while the concentration of 250 mg/ml of hydroalcoholic extract of C. officinalis and A. dracunculus resulted in killing 42.33% and 65.67%, respectively. The findings of the present study showed that A. absinthium and F. assafoetida have potent scolicidal effects. However, additional in vivo studies are required to confirm the efficacy of these plant-derived extracts against hydatid cyst for their clinical use.

In vitro evaluation of albendazole nanocrystals against Echinococcus granulosus protoscolices.

Echinococcus granulosus is a zoonotic parasite causing hydatidosis in humans and animals. This study has been done in order to investigate the effect of albendazole nanocrystals on the viability of E. granulosus protoscolices. The average size and hydrodynamic diameter of albendazole nanocrystals were 976±218 and 1334±502 nm, respectively. Fertile hydatid cysts were isolated from the liver of slaughtered sheep. The isolated cysts were further identified using morphological and molecular techniques. The nucleotide sequence analysis indicated that the genotype of the protoscolices was E. granulosus sensu stricto with 100% similarity. The parasites were examined precisely for susceptibility to albendazole nanocrystals. The results revealed that albendazole nanocrystals are effective in removing protoscolices. It was observed that 1 µg/ml albendazole nanocrystals and albendazole completely inhibited the viability of the protoscolices within 17 and 23 days, respectively. The results suggested that albendazole nanocrystals can be used as an alternative effective treatment for E. granulosus infection.

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.

Scolicidal and Apoptotic Activities of 5-hydroxy-1, 4-naphthoquinone as a Potent Agent against Echinococcus granulosus Protoscoleces.

Cystic hydatid disease (CHD) is a zoonotic disease with different clinical stages caused by the larval stage of the cestode Echinococcus granulosus. It is important to highlight as a public health problem in various regions of the world. In the current study, the efficacy and apoptotic activity of the liposomal system containing juglone (5-hydroxy-1,4-naphthoquinone) were assessed against protoscoleces (PSCs) in vitro. To this aim, firstly, liposomal vesicles were prepared by the thin-film method. Their physico-chemical features were assessed using Zeta-Sizer and Scanning Electron Microscope (SEM). Subsequently, various concentrations (50, 100, 200, 400, and 800 µg/mL) of juglone nanoliposomes at different exposure times (15, 30, 60, and 120 min) were used against PSCs. Results showed that juglone nanoliposomes at all tested concentrations induced scolicidal effect, however, 800 µg/mL and 400 µg/mL of juglone nanoliposomes could reach 100% mortality in 60 and 120 min, respectively. Additionally, we found that caspase-3 mRNA expression was higher in PSCs treated with juglone nanoliposomes compared to control groups (p < 0.001). Therefore, juglone nanoliposomes are suggested to have a more potent apoptotic effect on PSCs. Generally, optimized doses of juglone nanoliposomes could display significant scolicidal effects. Moreover, further in vivo studies are required to evaluate the efficacy of this nanoliposome.

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.

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.

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.

Anti-Acanthamoeba synergistic effect of chlorhexidine and Garcinia mangostana extract or α-mangostin against Acanthamoeba triangularis trophozoite and cyst forms.

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.