[Investigation of Cytotoxic and Antileishmanial Activity of Hybrid Silver Nanoparticle Complexes: Potential Drug Candidates against Leishmania Species]. / Hibrit Gümüs Nanoparçacik Komplekslerinin Sitotoksik ve Antilaysmanyal Aktivitesinin Arastirilmasi: Leishmania Türlerine Karsi Potansiyel Ilaç Adaylari.

In recent years, isolation of resistant Leishmania species to drugs in use has made it necessary to search alternative molecules that may be drug candidates. In this study, it was aimed to investigate the cytotoxic and in vitro antileishmanial activity of hybrid silver nanoparticle (AgNP) complexes. In this study, three types of nanoparticles (NPs), oxidized amylose-silver (OA-Ag) NPs, oxidized amylose-curcumin (OA-Cur) NPs and oxidized amylose-curcumin-silver (OA-CurAgNP) nanoparticles were synthesized. The cytotoxic activity of the synthesized nanoparticles was determined against L929 mouse fibroblasts and the in vitro antileishmanial activity was determined against Leishmania tropica, Leishmania infantum and Leishmania donovani isolates by the broth microdilution method. It was observed that the hybrid OA-CurAgNP complex obtained by combining curcumin and silver nanoparticles showed cytotoxic effects against L929 mouse fibroblasts at concentrations of 1074 µg/mL and above. IC50 values expressing the antileishmanial activity of the hybrid OA-CurAgNP complex against L.tropica, L.infantum and L.donovani isolates, were found to vary between 95-121 µg/mL, 202-330 µg/mL and 210-254 µg/mL, respectively. Resistance development has emerged as a major challenge in the treatment of leishmaniasis in recent times. Metallic nanoparticles are considered excellent candidates for medical applications due to their chemical and physical properties, as well as their prolonged circulation in the body. The current drugs used for leishmaniasis treatment are highly toxic, while nanoparticles offer advantages such as low toxicity and easy cellular uptake due to their nanoscale dimensions. The identification of strong efficacy in these particles may contribute scientific evidence for their potential use in leishmaniasis treatment. Therefore, the therapeutical value of OA-CurAgNP complex alone in combination with existing drugs should be examined.

ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

[The Effect of Oleuropein on The Mitochondrial Membrane Potential and Generation of Reactive Oxygen Species on Leishmania tropica Promastigotes]. / Oleuropein'in Leishmania tropica Promastigotlari Üzerinde Mitokondri Membran Potansiyeli ve Reaktif Oksijen Türlerinin Olusumuna Etkisi.

Leishmania parasites, which are reported to be endemic in 98 countries around the world, infect humans as well as wild and domestic carnivores and small mammals, and are transmitted by sand flies (Phlebotomus, dwarf sandflies). It is reported that 350 million people are at risk and two million new cases are seen in the world every year. It has been reported that different drugs (topical paromomycin, oral miltefosine, ketoconazole, rifampin, and zinc) have been tried in studies especially in endemic regions in the treatment of cutaneous leishmaniasis, and response to treatment has been obtained at different rates. Today, the search for alternative treatments continues and many studies have been carried out for this purpose. For centuries, olive leaf extracts have been used to maintain health. Oleuropein has numerous health benefits, including antioxidant, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, antiviral activities, cardio- and neuroprotective, hepatoprotective effects. The aim of this study was to determine and understand the mode of action of oleuropein, the cell death mechanisms caused by oleuropein in L.tropica promastigotes. In this study, the phenolic and flavonoid content of oleuropein was determined by HPLC method. The antioxidant capacity and the amount of oleuropein were determined. Afterwards, morphological and physiological (mitochondrial membrane potential, formation of reactive oxygen species, Annexin V binding) changes triggered by oleuropein in L.tropica promastigotes were investigated using flow cytometry. Our studies revealed that apoptotic properties such as mitochondrial dysfunction, production of reactive oxygen species, flip-flop action of phosphatidylserine could induce cell death in L.tropica promastigotes. It has been observed that oleuropein induced typical apoptotic morphological features in L.tropica promastigotes. Total phenolic content and total flavonoid content values of oleuropein extract were determined as 33 mg/g and 229 mg/g. The radical removal method was used to investigate the antioxidant capacity of methanol extracts against free radicals. Total antioxidant content of oleuropein extract was determined as 87%. In addition, the amount of oleuropein in the oleuropein extract was determined as 21. 1% by HPLC. The oleuropein dose that killed 50% of L.tropica promastigotes, that is the IC50 value, was detected as 46.6 µg/mL after 24 hours. It was observed that the parasites in the control group preserved their typical morphological features with a single nucleus, flagella, kinetoplast and narrow cell body at both 24 and 48 hours. It was observed that as oleuropein concentrations increased, the and kinetoplasts of L.tropica promastigotes could not be distinguished from each other, they moved away from the narrow cell body structure, they lost their flagella and turned into a round form, and they moved away from the typical form of the parasite. The percentage of Annexin V+ apoptotic cells was found to be 2.9 ± 0.4% in the untreated control group, and 38.1 ± 6.9% in the oleuropein-treated group. Polarization in the mitochondrial membrane of healthy promastigotes caused an approximately 1.7-fold change in the direction of depolarization in oleuropein-treated promastigotes. According to these findings, oleuropein triggered mitochondria-related death in L.tropica promastigotes. Moreover, 1.4 ± 0.2 fold increase in reactive oxygen species production was detected in oleuropein-treated promastigotes compared to the untreated control group. Comparisons between groups were made using the independent sample t test method. In conclusion, phenolic compounds of olive leaf extract oleuropein induced apoptotic cell death in L.tropica promastigotes. Our results support that olive products such as oleuropein may have anti-parasitic effects.

GC/MS analysis, anti-leishmanial and relaxant activity of essential oil of Chenopodium ambrosioides (L.) from Malakand region.

Chenopodium ambrosioides is abundantly available in Malakand region. As constituents and concentrations of essential oils vary based on its geographical location, we carried our current study to extract and evaluate its possible relaxant activity in rabbits' jejunum and anti-leishmanial activity against promastigotes of Leishmania tropica. The essential oil was obtained from aerial fresh parts through steam distillation followed by GC/MS analysis. Antispasmodic activity was performed on spontaneous and KCl induced contractions. Curves for calcium concentration response (CCRCs) were prepared with and without different concentrations of essential oils and verapamil - a standard calcium channel blocker as per our reported procedures. GC/MS analysis indicated that the essential oil contains 4-carene (56.59%) and o-cymene (41.46%), the two most abundant compounds previously reported from this species. The LD50 value for acute toxicity is 279.66±2.2mg/kg. The essential oil have significant antileishmanial activity with LC50 of Log10 (1.83±0.0026) ×10-6mg/ml, potent relaxant activity on rabbits' jejunal preparations with respective EC50 = 1.46±0.15mg/ml for spontaneous activity. For KCl (80mM) induced contractions, EC50=0.26±0.02mg/ml. In CCRCs, the oil produced a right shift as exhibited by verapamil. More, its relaxant activity, which is mediated through calcium channel blocking mechanism, proves a rationale for its traditional use in gut spasm.

In vitro Anti-Leishmanial Activity of Essential Oils Extracted from Plants Growing in Northern Cyprus Against Leishmania tropica

Objective: Natural plant products are considered as a source of novel and effective compounds for the treatment of leishmaniasis. In this study, the in vitro activities of essential oils obtained from Origanum dubium (OD), Origanum majorana (OM), Salvia fruticosa (SF) and Laurus nobilis (LN) plants in Northern Cyprus were investigated against Leishmania tropica. Methods: Leishmania tropica strain (MHOM/TR/2012/CBCL-LT) was obtained. RPMI-1640 was added to 96-well plates in 100 µL aliquots, 100 µg/mL essential oil was added to the first well of each row and serial 2-fold dilutions were performed. A promastigote suspension was pipetted into all wells, and the plates were incubated. The promastigotes were enumerated using a haemocytometer. Results: OD essential oil was effective at killing all promastigotes at a minimum inhibitor height (MIC)=0.2 µg/mL and had high activity at the lowest concentrations. Both SF and LN oils had MIC=1.56 µg/mL and LD50=0.78 µg/mL. SF was observed to impair promastigote morphology at the lowest concentrations, while LN did not exert any effect at concentrations <0.2 µg/mL. OM essential oil was found to have a MIC=3.13 µg/mL and a LD50=1.56 µg/mL. Conclusion: All tested essential oils inhibited promastigotes of Leishmania tropica. OD essential oil demonstrated the highest anti-leishmanial activity. Amaç: Bitkilerden elde edilen dogal ürünlerin leishmaniasis tedavisi için yeni ve etkili bilesiklerin üretilmesine öncülük edecegi düsünülmektedir. Çalismamizda, Kuzey Kibris'ta yetisen Origanum dubium (OD), Origanum majorana (OM), Salvia fruticosa (SF) ve Laurus nobilis (LN) bitkilerinden elde edilen uçucu yaglarin Leishmania tropica'ya karsi in vitro etkinlikleri arastirilmistir. Yöntemler: Çalismamizda, Leishmania tropica susu (MHOM/TR/2012/CBCL-LT) kullanildi. Düz tabanli 96'lik plaklarda, tüm kuyucuklara 100 µL RPMI-1640 ve ilk kuyucuklara 100 µg/mL uçucu yaglar eklenerek, seri dilüsyonlari yapildi. Ardindan tüm kuyucuklara Leishmania tropica promastigot süspansiyonundan pipetlendi ve inkübe edildi. Hemositometre yöntemiyle promastigotlarin sayisi incelendi. Bulgular: OD yaginin minimum inhibitör konsantrasyonu (MIK)=0,2 µg/mL'de tüm promastigotlari öldürürken, en düsük konsantrasyonlarda bile etkili oldugu görülmüstür. SF ve LN uçucu yaglarinin ikisinde de MIK=1,56 µg/mL, LD50=0,78 µg/mL olarak saptanmistir. SF'nin en düsük konsantrasyonlarinin bile promastigot morfolojisini bozdugu görülürken, Laurus nobilis'in ise 0,2 µg/mL'den sonraki konsantrasyonlarda etkisini kaybettigi belirlenmistir. OM uçucu yaginin MIK=3,13 µg/mL, LD50=1,56 µg/mL oldugu görülmüstür. Sonuç: Kullanilan tüm uçucu yaglarin Leishmania tropica promastigotlarini inhibe ettigi görülürken, en yüksek anti-leishmanial etkinlik Origanum dubium uçucu yaginda bulunmustur.

Metabolite Biomarkers of Leishmania Antimony Resistance.

Leishmania parasites cause leishmaniasis, one of the most epidemiologically important neglected tropical diseases. Leishmania exhibits a high ability of developing drug resistance, and drug resistance is one of the main threats to public health, as it is associated with increased incidence, mortality, and healthcare costs. The antimonial drug is the main historically implemented drug for leishmaniasis. Nevertheless, even though antimony resistance has been widely documented, the mechanisms involved are not completely understood. In this study, we aimed to identify potential metabolite biomarkers of antimony resistance that could improve leishmaniasis treatment. Here, using L. tropica promastigotes as the biological model, we showed that the level of response to antimony can be potentially predicted using 1H-NMR-based metabolomic profiling. Antimony-resistant parasites exhibited differences in metabolite composition at the intracellular and extracellular levels, suggesting that a metabolic remodeling is required to combat the drug. Simple and time-saving exometabolomic analysis can be efficiently used for the differentiation of sensitive and resistant parasites. Our findings suggest that changes in metabolite composition are associated with an optimized response to the osmotic/oxidative stress and a rearrangement of carbon-energy metabolism. The activation of energy metabolism can be linked to the high energy requirement during the antioxidant stress response. We also found that metabolites such as proline and lactate change linearly with the level of resistance to antimony, showing a close relationship with the parasite's efficiency of drug resistance. A list of potential metabolite biomarkers is described and discussed.

Novel coumarin-isatin hybrids as potent antileishmanial agents: Synthesis, in silico and in vitro evaluations.

Leishmaniasis being one of the six major tropical diseases that affects nearly 0.7-1.3 million people annually, has so far limited and high toxic therapeutic options. Herein, we report the synthesis, in silico, and in vitro evaluations of novel coumarin-incorporated isatin hydrazones (Spf-1 - Spf-10) as highly potent and safe antileishmanial agents. Molecular docking was initially carried out to decipher the binding confirmation of lead molecules towards the active cavity of the target protein (Leishmanolysin gp63) of Leishmania tropica. Among all the docked compounds, only Spf-6, Spf-8, and Spf-10 showed high binding affinities due to a pattern of strong conventional hydrogen bonds and hydrophobic π-interactions. The molecular dynamics simulations showed the stable pattern of such bonding and structure-based confirmation with a time scale of 50 ns towards the top compound (Spf-10) and protein. These analyses affirmed the high stability of the system. Three out of ten compounds evaluated for their antileishmanial activity against Leishmania tropica promastigotes and amastigotes were found to be active at micromolar concentrations (IC50 range 0.1-4.13 µmol/L), and most importantly, they were also found to be highly biocompatible when screened for their toxicity in human erythrocytes.

Bioactive Levan-Type Exopolysaccharide Produced by Pantoea agglomerans ZMR7: Characterization and Optimization for Enhanced Production.

Levan is an industrially important, functional biopolymer with considerable applications in the food and pharmaceutical fields owing to its safety and biocompatibility. Here, levan-type exopolysaccharide produced by Pantoea agglomerans ZMR7 was purified by cold ethanol precipitation and characterized using TLC, FTIR, 1H, and 13C NMR spectroscopy. The maximum production of levan (28.4 g/l) was achieved when sucrose and ammonium chloride were used as carbon and nitrogen sources, respectively, at 35°C and an initial pH of 8.0. Some biomedical applications of levan like antitumor, antiparasitic, and antioxidant activities were investigated in vitro. The results revealed the ability of levan at different concentrations to decrease the viability of rhabdomyosarcoma and breast cancer cells compared with untreated cancer cells. Levan appeared also to have high antiparasitic activity against the promastigote of Leishmania tropica. Furthermore, levan had strong DPPH radical scavenging (antioxidant) activity. These findings suggest that levan produced by P. agglomerans ZMR7 can serve as a natural biopolymer candidate for the pharmaceutical and medical fields.

The leishmanicidal effect of Lucilia sericata larval saliva and hemolymph on in vitro Leishmania tropica.

BACKGROUND: Leishmaniasis is a major parasitic disease worldwide, except in Australia and Antarctica, and it poses a significant public health problem. Due to the absence of safe and effective vaccines and drugs, researchers have begun an extensive search for new drugs. The aim of the current study was to investigate the in vitro leishmanicidal activity of larval saliva and hemolymph of Lucilia sericata on Leishmania tropica. METHODS: The effects of different concentrations of larval products on promastigotes and intracellular amastigotes of L. tropica were investigated using the mouse cell line J774A.1 and peritoneal macrophages as host cells. The 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct observation and counting method were used to assess the inhibitory effects and cell cytotoxicity of the larval products. The effects of larval products on the amastigote form of L. tropica were quantitatively estimated by calculating the rate of macrophage infection, number of amastigotes per infected macrophage cell, parasite load and survival index. RESULTS: The 50% cytotoxicity concentration (CC50) value of both larval saliva and hemolymph was 750 µg/ml, and the 50% inhibitory concentration (IC50) values were 134 µg/ml and 60 µg/ml for larval saliva and larval hemolymph, respectively. The IC50 for Glucantime, used a positive control, was (11.65 µg/ml). Statistically significant differences in viability percentages of promastigotes were observed for different doses of both larval saliva and hemolymph when compared with the negative control (p ≤ 0.0001). Microscopic evaluation of the amastigote forms revealed that treatment with 150 µg/ml larval hemolymph and 450 µg/ml larval saliva significantly decreased the rate of macrophage infection and the number of amastigotes per infected macrophage cell. CONCLUSION: Larval saliva and hemolymph of L. sericata have acceptable leishmanicidal properties against L. tropica.

A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica.

Background: Cutaneous leishmaniasis caused by Leishmania species (L. spp) is one of the most important parasitic diseases in humans. To gain information on the metabolite variations and biochemical pathways between L. spp, we used the comparative metabolome of metacyclic promastigotes in the Iranian isolates of L. major and L. tropica by proton nuclear magnetic resonance (1H-NMR). Methods: L. tropica and L. major were collected from three areas of Iran, namely Gonbad, Mashhad, and Bam, between 2017 and 2018, and were cultured. The metacyclic promastigote of each species was separated, and cell metabolites were extracted. 1H-NMR spectroscopy was applied, and the data were processed using ProMatab in MATLAB (version 7.8.0.347). Multivariate statistical analyses, including the principal component analysis and the orthogonal projections to latent structures discriminant analysis, were performed to identify the discriminative metabolites between the two L. spp. Metabolites with variable influences in projection values of more than one and a P value of less than 0.05 were marked as significant differences. Results: A set of metabolites were detected, and 24 significantly differentially expressed metabolites were found between the metacyclic forms of L. major and L. tropica isolates. The top differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased more in L. tropica than L. major (P<0.005), whereas asparagine, 3-hydroxybutyrate, L-proline, and kynurenine were increased significantly in L. major (P<0.01). The significantly altered metabolites were involved in eight metabolic pathways. Conclusion: Metabolomics, as an invaluable technique, yielded significant metabolites, and their biochemical pathways related to the metacyclic promastigotes of L. major and L. tropica. The findings offer greater insights into parasite biology and how pathogens adapt to their hosts.

Edible mushroom (Flammulina velutipes) as biosource for silver nanoparticles: from synthesis to diverse biomedical and environmental applications.

The current study reports advanced, ecofriendly and biosynthesized silver NPs for diverse biomedical and environmental applications using Flammulina velutipes as biosource. In the study, a simple aqueous extract of F. velutipes was utilized to reduce the AgNO3 into stable elemental silver (Ag0) at a nanometric scale. The NPs had average size of 21.4 nm, spherical morphology, and were highly stable and pure. The characterized nanoparticles were exploited for a broad range of biomedical applications including bacteriocidal, fungicidal, leishmanicidal, in vitro antialzheimer's, antioxidant, anti-diabetic and biocompatibility studies. Our findings showed that F. velutipes mediated AgNPs exhibited high activity against MDR bacterial strains and spore forming fungal strains. All the tested urinary tract infection bacterial isolates, were resistant to non-coated antibiotics but by applying 1% of the synthesized AgNPs, the bactericidal potential of the tested antibiotics enhanced manifolds. The NPs also exhibited dose-dependent cytotoxic potential against Leishmania tropica with significant LC50 of 248 µg ml-1 for promastigote and 251 µg ml-1 for amastigote forms of the parasite. Furthermore, promising antialzheimer and antidiabetic activities were observed as significant inhibition of α-amylase, α-glucosidase, acetylcholinesterase (AChE) and butrylcholineterase (BChE) were noted. Moreover, remarkable biocompatible nature of the particles was found against human red blood cells. The biosynthesized AgNPs as photocatalyst, also resulted in 98.2% degradation of indigo carmine dye within 140 min. Owing to ecofriendly synthesis, biosafe nature and excellent physicochemical properties F. velutipes AgNPs can be exploited as novel candidates for multifaceted biomedical and environmental applications.

Phytofabrication of cobalt oxide nanoparticles from Rhamnus virgata leaves extract and investigation of different bioactivities.

The tunable cobalt oxide nanoparticles (CoONPs) are produced due to the phytochemicals present in Rhamnus virgata (RhV) leaf extract which functions as reducing and stabilization agents. The synthesis of CoONPs was confirmed using different analytical techniques: UV-Vis spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), dynamics light scatterings (DLS), Fourier-transform infrared spectroscopy (FTIR), energy dispersive X-ray, and Raman spectroscopy analyses. Furthermore, multiple biological activities were performed. Significant antifungal and antibacterial potentials have been reported. The in vitro cytotoxic assays of CoONPs revealed strong anticancer activity against human hepatoma HUH-7 (IC50 : 33.25 µg/ml) and hepatocellular carcinoma HepG2 (IC50 : 11.62 µg/ml) cancer cells. Dose-dependent cytotoxicity potency was confirmed against Leishmania tropica (KMH23 ); amastigotes (IC50 : 58.63 µg/ml) and promastigotes (IC50 : 32.64 µg/ml). The biocompatibility assay using red blood cells (RBCs; IC50 : 4,636 µg/ml) has confirmed the bio-safe nature of CoONPs. On the whole, results revealed nontoxic nature of RhV-CoONPs with promising biological potentials.

The potential therapeutic role of PTR1 gene in non-healing anthroponotic cutaneous leishmaniasis due to Leishmania tropica.

BACKGROUND: Drug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non- healing Leishmania tropica isolates and on MA transfected non-healing one to PTR1. METHODS: Two non-healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA-rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme-linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50 ) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages. RESULTS: All three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non-healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non-healing and healing isolates (p < 0.05). CONCLUSION: The results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non-healing cases. These findings have future implications in the prophylactic and therapeutic modality of non- healing Leishmania isolates to drug.

In vitro anti-leishmanial activity of Prunus armeniaca fractions on Leishmania tropica and molecular docking studies.

Prunus armeniaca (L.) is a member of the Rosaceae, subfamily Prunoideae, shows anticancer, antitubercular, antimutagenic, antimicrobial, antioxidant, and cardioprotective activities. Here we fractionated the leaves extract of this highly medicinally important plant for antileishmanial activity. In the current study, the leaves extract was fractionated and characterized using column and thin layer chromatography by n-hexane, ethyl acetate, and methanol solvents. Twelve fractions were isolated and subjected for evaluation of their cytotoxicity and in vitro antileishmanial activity against promastigotes and amastigotes of Leishmania tropica. Among all fractions used, the fraction (F7) exhibited the strongest antileishmanial activity. The bioactive fraction was further characterized by spectroscopy (FTIR, UV-Vis), and GC-MS analysis. The in silico docking was carried out to find the active site of PTR1. All derived fractions exhibited toxicity in the safety range IC50 > 100 µg/ml. The fraction (F7) showed significantly the highest antipromastigotes activity with IC5011.48 ± 0.82 µg/ml and antiamastigotes activity with IC50 21.03 ± 0.98 µg/ml compared with control i.e. 11.60 ± 0.70 and 22.03 ± 1.02 µg/ml respectively. The UV-Vis spectroscopic analysis revealed the presence of six absorption peaks and the FTIR spectrum revealed the presence of alkane, aldehyde, carboxylic acid, thiols, alkynes, and carbonyls compounds The GC-MS chromatogram exhibited the presence of nine compounds: (a) benzeneethanol, alpha, beta dimethyl, (b)carbazic acid, 3-(1 propylbutylidene)-, ethyl ester, (c)1, 2-benzenedicarboxylic acid, diisooctyl ester, (d)benzeneethanamine a-methyl, (e)2aminononadecane, (f)2-heptanamine-5-methyl, (g)cyclobutanol, (h)cyclopropyl carbine, and (i)nitric acid, nonyl ester. Among all compounds, the 1, 2-benzenedicarboxylic acid, diisooctyl ester bound well to the PTR1 receptor. Fraction (F7) showed acceptable results with no cytotoxicity. However, in vivo studies are required in the future.

Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

[Determination of Antimony Resistance Mechanism of Leishmania tropica Causing Cutaneous Leishmaniasis in Turkey]. / Türkiye'de Kutanöz Leysmanyazis Etkeni Leishmania tropica'da Antimon Direnç Mekanizmasinin Belirlenmesi.

World Health Organization reported that approximately one billion people are at risk in endemic areas, one million cases of cutaneous leishmaniasis (CL) and approximately 300,000 cases of visceral leishmaniasis (VL) were reported per year in the last five years. The number of deaths due to VL is reported to be approximately 20,000 per year. Approximately 2500 cases/year have been reported as CL, caused by Leishmania tropica and Leishmania infantum, in Turkey. The significant increase observed in many cities mainly in the provinces of Mediterranean and Aegean regions in cases and foci in recent years, suggests that there may be an increase in this infections in the following years as well. In Turkey, the causative agent of CL is L.tropica and meglumine antimoniate is used in the treatment of CL. We aimed to determine antimony resistance genes specific for L.tropica by comparing the gene and protein expressions of antimony-resistant and non-resistant L.tropica strains. L.tropica isolates obtained from 3 CL patients without antimonate resistance from Aegean, Mediterranean and Southeastern regions of Turkey were provided to transform into 3 resistant isolates against meglumine antimony in the laboratory conditions. Gene expression alterations by microarray method; protein profiles by two-dimensional gel electrophoresis (2D-PAGE) and relevant proteins by MALDI-TOF/TOF MS of these isolates were accomplished and compared. L.tropica isolates from 10 CL patients who did not respond to antimony therapy were analyzed for resistance to antimonial compounds and quantitative real-time polymerase chain reaction was performed to detect the expression of genes responsible for resistance development. Moreover, differences in protein expression levels in isolates with and without antimony resistance were determined by comparing protein profiles and identification of proteins with different expression levels was carried out. Enolase, elongation factor-2, heat shock protein 70, tripanthione reductase, protein kinase C and metallo-peptidase proteins have been shown to play roles in L.tropica isolates developing resistance to antimonial compounds and similar expression changes have also been demonstrated in naturally resistant isolates from patients. In conclusion, it was revealed that L.tropica strains in our country may gain resistance to meglumine antimoniate in a short time. It is foreseen that if the patients living in our country or entering the country are treated inadequately and incompletely, there may be new, resistant leishmaniasis foci that may increase the number of resistant strains and cases rapidly.

[Investigation of Anti-leishmanial Effects of Bee Products (Honey, Propolis) on Leishmania tropica Promastigotes]. / Ari Ürünlerinin (Bal, Propolis) Leishmania tropica Promastigotlari Üzerine Antileysmanyal Etkilerinin Arastirilmasi.

This study was aimed to investigate the anti-leishmanial effects of bee products (honey and propolis) by using the causative agent of cutaneous leishmaniasis Leishmania tropica promastigotes, in in vitro culture. In vitro anti-leishmanial efficacy of honey (pine, flower and chestnut) and propolis used in the study were evaluated using the microdilution method. Honey, which is a bee product, was dissolved with RPMI medium containing fetal calf serum (FCS) and diluted in the same medium, and serial dilutions were prepared in concentrations between 62.5-1000 mg/ml. Propolis, on the other hand, was dissolved with ethyl alcohol and only 2.5 µl was used from all these concentrations since the alcohol content was more than 50% in these concentrations prepared and we thought that this rate would negatively effect the parasite development. Then, RPMI containing FCS was diluted in the medium and serial dilutions were prepared at concentrations between 50-800 µg/ml. To the dilutions prepared, the promastigot suspension was added so that their final concentrations in the wells were 1 x 106 promastigot/ml and then the medium was incubated for 24 and 48 hours in 26°C. After the incubation, promastigotes were determined microscopically for morphology, mobility and live parasite density, and cell viability was determined by MTS method and 50% inhibitor concentrations (IC50) were compared with control groups. Anti-leishmanial activity of propolis (50, 100, 200, 400 and 800 µg/ml) and honey (62.5, 125, 250, 500 and 1000 mg/ml) on promastigotes was evaluated in vitro. In microscopic examinations, pine honey showed anti-leishmanial activity starting from 62.5 mg/ml, flower honey 250 mg/ml, and chestnut honey 125 mg/ml, and pine honey was more effective on promastigotes (p< 0.05), and propolis was effective from 100 µg/ml concentration. It has been determined that very low concentrations of propolis caused changes in the morphological structure of the parasites and were more effective than the other bee products. The prevention of cell proliferation and decreasing of the IC50 values according with the time of pine honey (IC50= 109.28 mg/ml), flower honey (IC50= 248.07 mg/ml), chestnut honey (IC50= 147.65 mg/ml) and propolis (IC50= 82.98 µg/ml) applied on L.tropica promastigot cell culture was determined by MTS method. In this study, it was found that various concentrations of pine, flower, chestnut honey and propolis showed anti-leishmanial activity on L. tropica promastigotes. It has been observed that pine honey is more effective on promastigotes after 48 hours of incubation period, and propolis is more effective in both morphology and cell inhibition of the parasites even at very low concentrations. It is believed that these data can be used as an alternative treatment method against cutaneous leishmaniasis infections and further studies are required.

The potential role of nicotinamide on Leishmania tropica: An assessment of inhibitory effect, cytokines gene expression and arginase profiling.

Leishmaniasis represents a major health concern worldwide which has no effective treatment modality. Nicotinamide (NAm) has been used for a wide range of applications from anticancer to antimicrobial usage. This study aimed to assess the effect of NAm combination on Leishmania tropica Inhibition, as well as on cytokines gene expression and arginase (ARG) activity in L. tropica-infected macrophages in an in vitro model. The leishmanicidal effects of NAm and Glucantime (meglumine antimoniate, MA) alone and in combination (NAm/MA) were evaluated using a colorimetric assay and macrophage model. Additionally, immunomodulatory effects and enzymatic activity were assessed by analyzing Th1 and Th2 cytokines gene expression and ARG level, respectively, in infected macrophages treated with NAm and MA, alone and in combination. Findings indicated that the NAm/MA combination demonstrated greater inhibitory effects on L. tropica promastigotes and amastigotes compared with each drug individually. Docking results proved the affinity of NAm to IFN-γ, which can affirm the increased levels of IFN-γ, IL-12p40 and TNF-α as well as reductions in IL-10 secretion with a dose-response effect, especially in the combination group. The NAm/MA combination also showed a significant reduction in the level of ARG activity at all concentrations used compared to each drug individually. These findings indicate higher effectiveness of NAm plus MA in reducing parasite growth, promoting immune response and inhibiting ARG level. This combination should be considered as a potential therapeutic regimen for treatment of volunteer patients with anthroponotic cutaneous leishmaniasis (ACL) in future control programs.

Molecular Docking, Antioxidant, Anticancer and Antileishmanial Effects of Newly Synthesized Quinoline Derivatives.

BACKGROUND: Due to the pressing need and adverse effects associated with the available anti-cancer agents, an attempt was made to develop the new anti-cancer agents with better activity and lesser adverse effects. OBJECTIVE: Synthetic approaches based on chemical modification of quinoline derivatives have been undertaken with the aim of improving anti-cancer agents' safety profile. METHODS: In the present study, quinoline derivatives 6-hydroxy-2-(4-methoxyphenyl) quinoline-4-carboxylic acid (M1) and 2-(4-chlorophenyl)-6-hydroxyquinoline-4-carboxylic acid (M3) were synthesized by the reaction of aldehyde and pyruvic acid. The complete reaction was indicated by thin-layer chromatography. Newly synthesized M1and M3were tested for in silico and in vitro studies. RESULTS: M1 and M3 were docked against selected targets. Both the test compounds showed good affinity against all targets except the p300\CBP-associated factor target as there was no H-bond formed by M1. IC50 values of M1 and M3 against 1, 1-diphenyl-picrylhydrazyl free radical scavenging activity were 562 and 136.56ng/mL, respectively. In brine shrimp lethality assay, M1 and M3 showed IC50 value of 81.98 and 139.2ng/mL, respectively. IC50 values recorded for M1 and M3 in tumor inhibition activity were 129 and 219µg/mL, respectively. M1 and M3 exhibited concentration-dependent anti-cancer effects against human cell lines of hepatocellular carcinoma (HepG2) and colon cancer (HCT-116). Against HepG2 cells, M1 and M3 exhibited IC50 of 88.6 and 43.62µg/mL, respectively. M1 and M3 utilized against HCT-116 cell lines possessed IC50 values of 62.5 and 15.3µg/mL. M1 and M3 also showed an anti-leishmanial effect with IC50 values of 336.64 and 530.142µg/mL, respectively. CONCLUSION: From the results of pharmacological studies, we conclude that the newly synthesized compound showed enhanced anti-oxidant, anti-cancer and anti-leishmanial profile with good yield.