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 anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Curcuma longa rhizome extract and Curcumin reduce the adhesion of Acanthamoeba triangularis trophozoites and cysts in polystyrene plastic surface and contact lens.

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 < 0.05). Also, an 80-90% decrease in adhesion of trophozoites and cysts to the plastic surface was detected following the treatment with C. longa extract and Curcumin at 1/2 × MIC, compared to the control. In the contact lens model, approximately 1 log cells/mL of the trophozoites and cysts was reduced when the cells were treated with Curcumin, when compared to the control. Pre-treatment of the plastic surface with Curcumin at 1/2-MIC reduced 60% and 90% of the adhesion of trophozoites and cysts, respectively. The reduction in 1 Log cells/mL of the adhesion of A. triangularis trophozoites was observed when lenses were pre-treated with both the extract and Curcumin. Base on the results obtained from this study, A. triangularis trophozoites treated with C. longa extract and Curcumin have lost strong acanthopodia, thorn-like projection pseudopodia observed by scanning electron microscope. This study also revealed the therapeutic potentials of C. longa extract and Curcumin, as such, have promising anti-adhesive potential that can be used in the management/prevention of A. triangularis adhesion to contact lenses.

PLGA nanoparticles loaded with Gallic acid- a constituent of Leea indica against Acanthamoeba triangularis.

Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Curcuma longa ethanol extract and Curcumin inhibit the growth of Acanthamoeba triangularis trophozoites and cysts isolated from water reservoirs at Walailak University, Thailand.

CURCUMA LONGA: (C. longa) rhizome extract has been traditionally used to treat many infections. Curcumin, a pure compound isolated from the plant, has been documented to possess a wide spectrum of pharmacological effects. The present study aimed to investigate the effects of Thai medicinal plant extracts including C. longa extract and Curcumin on Acanthamoeba triangularis, a causative agent of human Acanthamoeba keratitis. The parasite was isolated from the recreational reservoir at Walailak University, Thailand. The organism was identified as A. triangularis using morphology and 18S rDNA nucleotide sequences. The pathogen was tested for their susceptibility to ethanol extracts of Thai medicinal plants based on eye infection treatment. The ethanol C. longa extract showed the strongest anti-Acanthamoeba activity against both the trophozoites and cysts, followed by Coscinium fenestratum, Coccinia grandis, and Acmella oleracea extracts, respectively. After 24 h, 95% reduction of trophozoite viability was significantly decreased following the treatment with C. longa extract at 125 µg/mL, compared with the control (P < 0.05). The extract at 1,000 µg/mL inhibited 90% viability of Acanthamoeba cyst within 24 h, compared with the control. It was found that the cysts treated with C. longa extract at 500 µg/mL demonstrated abnormal shape after 24 h. The MIC values of C. longa extract and Curcumin against the trophozoites were 125 and 62.5 µg/mL, respectively. While the MICs of the extract and curcumin against the cysts were 500 and 1,000 µg/mL, respectively. The results suggested the potential medicinal benefits of C. longa extract and Curcumin as the alternative treatment of Acanthamoeba infections.

Medicinal Plants with anti-Acanthamoeba Activity: A Systematic Review.

BACKGROUND: Recently, herbal medicine has received much attention in the literature. Several essential oils or plant extracts have been found to have anti-Acanthamoeba properties against trophozoites and cysts of Acanthamoeba spp. OBJECTIVE: The aim of this systematic review is to introduce anti-Acanthamoeba properties of some essential oils or plant extracts; perhaps the results of this research will be used to prevent and treat infectious diseases. METHODS: All published papers in English and Persian databases were systematically searched for some specific keywords to find articles that have influenced plant compounds on Acanthamoeba up to April 2018. Articles related to the subject were selected and studied. RESULTS: A total of 51 articles including 136 experiments (128 in vitro and 8 in vivo) between 1999 and 2018, met our eligibility criteria. Totally, 110 species of plants belonging to 34 families, mainly Lamiaceae (15 plant species) and Asteraceae (13 plant species) were studied against trophozoites and cysts of Acanthamoba in in vitro and in vivo. Most of the plant's species were Citrus (6 species), Allium (5 species), Peucedanum, Piper, Lippia, and Olive (4 species). The most frequently used parts were leaves, aerial parts, flowers, bark, rhizomes, and seeds. CONCLUSIONS: Recent studies have shown that many natural compounds have high anti-parasitic properties and low toxicity. Our research team hopes that the information provided in present systematic review can improve new experimental and clinical trials and herbal combination therapy. Further studies are needed to understand the molecular mechanisms in the anti-amoebic reactions of plant species and ocular toxicity of extracts in animal models.

Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

Efficient therapeutic effect of Nigella sativa aqueous extract and chitosan nanoparticles against experimentally induced Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a devastating, painful corneal infection, which may lead to loss of vision. The development of resistance and failure of the currently used drugs represent a therapeutic predicament. Thus, novel therapies with lethal effects on resistant Acanthamoeba are necessary to combat AK. In the present study, the curative effect of Nigella sativa aqueous extract (N. sativa) and chitosan nanoparticles (nCs) and both agents combined were assessed in experimentally induced AK. All inoculated corneas developed varying grades of AK. The study medications were applied on the 5th day postinoculation and were evaluated by clinical examination of the cornea and cultivation of corneal scraps. On the 10th day posttreatment, a 100% cure of AK was obtained with nCs (100 µg/ml) in grades 1 and 2 of corneal opacity as well as with N. sativa 60 mg/ml-nCs 100 µg/ml in grades 1, 2, and 3 of corneal opacity, highlighting a possible synergistic effect. On the 15th day posttreatment, a 100% cure was reached with N. sativa aqueous extract (60 mg/ml). Moreover, on the 20th day posttreatment, N. sativa (30 mg/ml) provided a cure rate of 87.5%, while nCs (50 µg/ml) as well as N. sativa 30 mg/ml-nCs 50 µg/ml yielded a cure rate of 75%; the lowest percentage of cure (25%) was obtained with chlorhexidine (0.02%), showing a non-significant difference compared to the parasite control. The clinical outcomes were in agreement with the results of corneal scrap cultivation. The results of the present study demonstrate the effectiveness of N. sativa aqueous extract and nCs (singly or combined) when used against AK, and these agents show potential for the development of new, effective, and safe therapeutic alternatives.

Effect of povidone iodine, chlorhexidine digluconate and toyocamycin on amphizoic amoebic strains, infectious agents of Acanthamoeba keratitis - a growing threat to human health worldwide.

INTRODUCTION: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. MATERIAL AND METHODS: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. RESULTS: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. CONCLUSIONS: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.

Influence of Artemisia annua L. on toll-like receptor expression in brain of mice infected with Acanthamoeba sp.

The treatment of acanthamoebiasis is a still a problem. Our previous studies showed that the application of extracts from Artemisia annua L. significantly prolonged the survival of mice infected by Acanthamoeba. This plant has medicinal properties in the treatment of human parasitic diseases. The aim of this study was to evaluate the effects of A. annua on expression of Toll-like receptors (TLRs) 2 and 4 in brain of mice with Acanthamoeba infection. Mice were infected with Acanthamoeba sp. strain Ac309 (KY203908) by intranasal inoculation without and after application of A. annua extract. The administration of extract from A. annua significantly reduced the level of expression of TLR2 and modified the level of expression of TLR4. A. annua extract is a natural substance that is well tolerated in animals and may be considered as a combination therapy in treatment of acanthamoebiasis. Our study suggested that A. annua extract may be used as an alternative therapeutic tool.

Anti-Acanthamoeba activity of Tunisian Thymus capitatus essential oil and organic extracts.

Acanthamoeba species are free-living amoebae widely distributed in the environment and which cause serious human infections. The treatment of Acanthamoeba infections is always very difficult and not constantly effective. More efficient drugs against Acanthamoeba must be developed and medicinal plants can be useful in this case. Our research focused on the examination of the anti-Acanthamoeba activity of the essential oil and the ethanolic-aqueous extract from Thymus capitatus L. The essential oil showed best activity with an IC50 of 2.73 µg/ml. The conducted Bio-guided fractionation of thyme extract result to the identification of two active compounds against the trophozoite stage of Acanthamoeba: thymol and 2,3-dihydroxy-p-cymene. The results have clearly shown that the investigated products may be successfully used against Acanthamoeba infections. These molecules that are found in plants may be an alternative for the development of new drugs.

Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 µg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection.

Evaluation of the effectiveness of tea tree oil in treatment of Acanthamoeba infection.

Eye diseases caused by amoebae from the genus Acanthamoeba are usually chronic and severe, and their treatment is prolonged and not very effective. The difficulties associated with therapy have led to attempts at finding alternative treatment methods. Particularly popular is searching for cures among drugs made of plants. However, no substances with total efficacy in treating Acanthamoeba keratitis have been identified.Results of our semi in vivo studies of tea tree oil simulating eyeball infection demonstrated 100% effectiveness in the case of both trophozoites and cysts of amoebae from the genus Acanthamoeba. The action of tea tree oil indicates that this is the first substance with a potential ability to quickly and effectively remove the amoebae from the eye. Tea tree oil has the ability to penetrate tissues, which allows it to destroy amoebae in both the shallow and deep layers of the cornea. The present research into the use of tea tree oil in the therapy of Acanthamoeba infection is the first study of this type in parasitology. It offers tremendous potential for effective treatment of Acanthamoeba keratitis and other diseases caused by these protozoa.

INVESTIGATION OF AMOEBICIDAL POTENTIAL OF ARACHIS HYPOGAEA L. PERICARP ON CYSTS OF ACANTHAMOEBA ASTRONYXIS T7 GENOTYPE.

Acanthamoeba is an opportunistic pathogen cauAing keratitis and fatal encephalitis. Early diagnosis, followed by intense treatment using a drugs mixture is a necessity for effective therapy. Many natural compounds have proved lethal effects, yet the search for original natural amebicidal agents is still of current concern. This study investigated the acanthamoebicidal effect of A. hypogaea L. pericarp; total ethanol extract and its successive fractions, n-hexane, dichloro-methane, ethyl acetate, and methanol as well as resveratrol. Acanthamoebae were isolated and cultivated on E. coli seeded non-nutrient agar, genotyped, and the in vitro acanthamoebicidal potentials of different concentrations of A. hypogaea L. pericarp; total extract ethanol and its successive fractions and resveratrol compound was investgated on cysts of A. astronyxis T7 genotype. The results showed variable degrees of lethal potentials were obtained by all examined A. hypogaea L. pericarp ethanol extract and its successive fractions, with the highest mean of non-viable cysts on the first and the second days. bf the study by total ethanol extract followed by the methanol fraction . On the third day of the study n-hexane and ethyl acetate gave the highest mean of non-viable cysts. Resveratrol showed the lowest mean of non-viable cysts count all through the study duration. Significantly higher difference was observed between all examined A. hypogaea L. pericarp extract and fractions and chlorohexidine, except for resveratrol compound the difference was found to be noinsignificant.

Effects of Lead Phytochemicals of Radix Scutellariae on Acanthamoeba.

Purpose: Acanthamoeba keratitis (AK), which is associated with noncompliant use of contact lenses, remains difficult to treat due to delayed diagnosis and paucity of therapeutic agents. Although improvements in activity against Acanthamoeba infection have been achieved in disinfecting solutions for soft contact lenses, such modifications have not been extended to those for special rigid gas permeable (RGP) contact lenses, which are increasingly used for myopia control in children. Phytochemicals present in herbs used for traditional Chinese medicine may be effective as therapeutic or preventive agents. The purpose of this study was to investigate amoebicidal properties of lead phytochemicals of Radix scutellariae alone and in combination with multipurpose (disinfecting) solutions (MPS) for RGP lenses. Methods: Viability of Acanthamoeba castellani and A. polyphaga trophozoites was determined following exposure to four phytochemicals: baicalin, baicalein, wogonoside, and oroxylin A and both alone and in combination with four RGP MPS, using a modified stand-alone technique. Results: As individual agents, wogonoside and oroxylin A showed highest activity against A. castellani and A. polyphaga trophozoites, respectively. For both organisms, the combination of baicalein and oroxylin A was superior. Effectiveness of MPS alone did not exceed 0.27 log reduction, but addition of combined baicalein and oroxylin A resulted in 0.92 and 0.64 log reductions of A. castellani and A. polyphaga, respectively. Conclusions: The combination of baicalein and oroxylin A enhanced the activity of MPS for RGP contact lenses against trophozoites of two pathogens, A. castellani, and A. polyphaga, and offers a potential therapeutic and/or preventative agent for AK.

Riboflavin and ultraviolet A as adjuvant treatment against Acanthamoeba cysts.

BACKGROUND: Experimental studies have shown that the standard dose of riboflavin (R) or R + ultraviolet-A (UVA) as solo treatment are not able to exterminate Acanthamoeba cysts or even trophozoites. The purpose of this study is to determine whether the application of R + UVA can enhance the cysticidal effects of cationic antiseptic agents in vitro. METHODS: The log of either polyhexamethylene biguanide or chlorhexidine minimal cysticidal concentration in solutions containing riboflavin (concentrations 0.1, 0.05 and 0.025%) plus either Acanthamoeba castellanii cysts or Acanthamoeba polyphaga cysts was determined and compared in groups treated with UVA 30 mW/cm(2) for 30 min and in control groups (with no exposure to UVA). A permutation test was used to determine the P value associated with treatment. RESULTS: Regardless of the riboflavin concentration and UVA treatment condition, no trophozoites were seen in plates where the cysts were previously exposed to cationic antiseptic agent concentrations ≥200 µg/mL for Acanthamoeba castellanii samples and ≥100 µg/mL for A. polyphaga samples. There was no statistical evidence that R + UVA treatment was associated with minimal cysticidal concentration (P = 0.82). CONCLUSION: R + UVA in doses up to 10 times higher than recommended for corneal crosslinking does not enhance the cysticidal effect of either polyhexamethylene biguanide or chlorhexidine in vitro.

Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 µg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 µg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency.

Abietane diterpenoids from Salvia sclarea transformed roots as growth inhibitors of pathogenic Acanthamoeba spp.

Amoebae from the genus Acanthamoeba are known agents leading to various diseases such as granulomatous amoebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumitis (AP), chronic lung infection, and skin infections. It is known that various synthetic anti-Acanthamoeba substances are ineffective. Therefore, other substances, e.g., natural plant compounds, are the focus of biological investigations regarding anti-parasite activity. In this work, the ability of four abietane diterpenoids (ferruginol, salvipisone, aethiopinone, and 1-oxo-aethiopinone) to inhibit Acanthamoeba growth is reported. All investigated compounds were active against Acanthamoeba growing in vitro. Among them, ferruginol demonstrated the highest activity against Acanthamoeba. This compound inhibited Acanthamoeba growth by about 72% in a 3-day exposure period (IC50 17.45 µM), while aethiopinone and 1-oxo-aethiopinone demonstrated this activity at the level of 55-56%. Salvipisone reduced the growth of Acanthamoeba in vitro culture by 39%. For this compound, the value of IC50 was 701.94 µM after 72 h of exposure.

Activity assessment of Tunisian olive leaf extracts against the trophozoite stage of Acanthamoeba.

The olive tree (Olea europaea, Oleaceae) has historically provided huge economic and nutritional benefits to the Mediterranean basin. In fact, olive leaf extracts have also been used by native people of this area in folk medicine to treat fever and other diseases such as malaria. Recently, several studies have focused on the extraction of high-added-value compounds from olive leaves. However, no previous studies have been developed in order to evaluate the activity of these extracts against Acanthamoeba. In the present work, olive leaf extracts from five different Tunisian varieties of olive trees (Chemlali Tataouine, Zarrazi, Toffehi, Dhokkar, and Limouni) were obtained by using three different solvents, and their activity against the trophozoite stage of Acanthamoeba castellanii Neff was screened. The IC50/96 h (50% parasite growth inhibition) was chosen as the appropriate and comparable data to give as previously described. It could be observed that the amoebicidal activity was dose dependent. Trophozoite growth was inhibited by all the tested extracts with IC50 ranging from 8.234 ± 1.703 µg/ml for the alcoholic mixture of the Dhokkar extract to 33.661 ± 1.398 µg/ml for the methanolic extract of the Toffehi variety. The activity in fact was affected especially by the tested variety and not by the solvent extraction, the Dhokkar variety being the most active one as mentioned above.

Effect of low concentrations of benzalkonium chloride on acanthamoebal survival and its potential impact on empirical therapy of infectious keratitis.

IMPORTANCE: The significant antiacanthamoebal effect of benzalkonium chloride, at or below concentrations used for preservation of common ophthalmic preparations, should be understood both when choosing empiric antibiotic therapy for infectious keratitis and when assessing the persistent rise in Acanthamoeba cases in the United States since 2003. OBJECTIVE: To characterize the antiacanthamoebal efficacy of low concentrations of benzalkonium chloride (BAK) for drug preservation and therapeutic effect against Acanthamoeba. DESIGN: Experimental study with a review of the literature. SETTING: Laboratory. EXPOSURES: A concentration of 10(4) trophozoites of 3 well-characterized clinical strains of Acanthamoeba were exposed at 0.5, 2.0, 3.5, 5.0, and 6.5 hours to BAK (0.001%, 0.002%, and 0.003%), moxifloxacin hydrochloride (0.5%), and moxifloxacin (0.5%) + BAK (0.001% and 0.003%) with hydrogen peroxide (3%) and amoeba saline controls. MAIN OUTCOMES AND MEASURES: Amoeba survival was calculated using the most probable number method recorded as log kill values. The relationship of BAK concentration and exposure time as well as the relative effect of BAK and moxifloxacin on acanthamoebal survival were analyzed. RESULTS: Amoebicidal activity of BAK is both time dependent and concentration dependent in pooled and strain-stratified analyses (P < .001). Moxifloxacin demonstrated no significant independent inhibitory effect or additive effect to BAK efficacy on acanthamoebal survival. The profound antiacanthamoebal effect of BAK, 0.003%, was similar to that of hydrogen peroxide for certain strains. CONCLUSIONS AND RELEVANCE: Low concentrations of BAK, previously demonstrated to concentrate and persist in ocular surface epithelium, exhibit significant antiacanthamoebal activity in vitro at or below concentrations found in commercially available ophthalmic anti-infectives. The unexplained persistence of the Acanthamoeba keratitis outbreak in the United States, clusters abroad, and clinical studies reporting resolution or modification of Acanthamoeba keratitis without specific antiacanthamoebal therapy suggests that other contributing factors should be considered, including changes in the formulations used for empirical therapy of presumed infectious keratitis occurring in the same period.