Major Promastigotes Research Articles

Therapeutic evaluation of activatable melittin for selective treatment of leishmaniasis in vitro and in vivo

Leishmaniasis is a major public health problem worldwide. Because of its high prevalence, the development of an effective treatment is especially important. Drug delivery systems are promising pharmaceutical formulations for improving the therapeutic index of drugs. In this study, activatable melittin (AM) peptide was designed and encapsulated with albumin and liposome for selective targeting of Leishmania infected cells. The effect of AM was determined by measuring its cytotoxicity and pathologic effects using Giemsa and haematoxylin/eosin staining, respectively. The results of this study showed that the toxicity of designed AM is reduced compared to wild-type melittin, in addition to the use of AM-loaded albumin nanoparticle and liposome containing AM and their therapeutic role against cutaneous leishmaniasis caused by L. major. The encapsulation of AM with albumin and liposome as a non-toxic carrier at a concentration of 25 µg/mL can improve and optimise the antileishmanial effects of this drug, so, infected cells treated with AM in albumin nanoparticle and liposome had less Leishmania major promastigotes. These forms of the drug could be a good alternative to the current drugs so performance of further in vivo studies is suggested.

In Vitro Activity of Green Synthesized Silver Nanoparticles via Thymus Vulgaris Extract against Leishmania major.

We aimed to assess the in vitro effects of the green synthesized silver nanoparticles (Ag NPs) via Thymus vulgaris (thyme) against Leishmania major infection. We have prepared T. vulgalis silver nanoparticles (TSNPs) by adding thyme extract to the silver nitrate aqueous solution (0.2 mM), and evaluated their antileishmanial activity. The viability of L. major promastigotes was assessed in the presence of various concentrations of TSNPs by direct counting after 24 h. The MTT assay was used to identify the viability of promastigotes. The same procedures were assessed in uninfected macrophage cells. The apoptotic effects of nanoparticles on L. major promastigotes were determined by flow cytometry assay using annexin staining. To evaluate anti-amastigotes activity of TSNPs, light microscopic observation was used to determine the number of parasites within the macrophages in each well. The effect of TSNPs on promastigotes and amastigotes of L. major was effective and had a reverse relationship with its concentration. TSNPs, inhibited the growth rate of L. major amastigotes and, the IC50 value of these nanoparticles was estimated 3.02 μg/mL (28 μM) after 72h. The results of flow cytometry showed that the toxic effects of TSNPs on promastigotes after 24 hours were statistically significant (P<0.05) and showed 69.51% of apoptosis. TSNPs had an inhibitor effect on promastigote and amastigote forms of L. major in vitro. It might be considered as a candidate for the treatment of this infection.

Gelatin Zymography of Major Proteases in Exosomes of Leishmania major Promastigotes.

Enzymatic digestion of extra cellular matrix proteins by proteinases of Leishmania promastigotes is a complex process. Hence, studies on functional proteomics of these enzymes can help select these enzymes as possible vaccine candidates or selecting candidates for chemotherapy and immunotherapy. Several proteolytic enzymes are involved in virulence of Leishmania spp. These enzymes are mostly serine, cysteine and metalloproteases. We aimed to detect proteases in Leishmania promastigote exosomes. Serine, cysteine and metalloproteases were investigated in exosomes and lysate of L. major promastigote using gelatin zymography. The study was carried out in the Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, in 2021. Zymography findings of metalloproteinases showed transparent bands, including a 63-kDa glycoprotein (GP63). This glycoprotein is a major surface metalloproteinase. In addition, transparent bands belonged to serin proteases and cathepsin were demonstrated in gels associated to Leishmania promastigote lysate and exosomes. Several metalloproteases, serin proteases and cathepsins were shown in promastigote lysate and exosomes of L. major, which could purified and used as fractions for immunodiagnostic.

Antimalarial and Antileishmanial Flavonoids from Calendula officinalis Flowers

Calendula officinalis L. (Asteraceae), commonly known as English or pot marigold, is an herbaceous plant with edible flowers. In this study, UPLC-ESI-MS/MS analysis was used for tentative identification of compounds in marigold flower methanol extract (MFE). In addition, RP-HPLC-DAD analysis was used to quantify the flavonoids hesperidin and rutin in MFE. The antileishmanial potentials of the crude extract and compounds were evaluated against Leishmania major promastigotes and amastigotes. Further, in vivo 4-day antimalarial testing of the extract and compounds was carried out at doses of 25 mg kg−1 per day using mice infected with ANKA strain of Plasmodium berghei, following standard procedure. Molecular docking studies were carried out to assess the binding mode of flavonoids against the vital targets of L. major, including pteridine reductase 1 and farnesyl diphosphate synthase enzymes. The in silico antimalarial potentials of flavonoids were evaluated against wild-type Plasmodium falciparum dihydrofolate reductase-thymidylate synthase and phosphoethanolamine methyltransferase enzymes. Twenty compounds were tentatively identified by UPLC-ESI-MS/MS analysis of MFE, of which, seven flavonoids, six saponins, three phenolic acids, three fatty acids, and a triterpene glycoside were identified. MFE phytochemical analysis revealed that hesperidin content was 36.17 mg g−1 extract, that is, 9.9-fold their content of rutin (3.65 mg g−1 extract). The method was validated to ensure reproducibility of the results. The tested samples exhibited antileishmanial potentials against L. major promastigotes, with IC50 values of 98.62, 118.86, and 104.74 ng µL−1 for hesperidin, rutin, and MFE, respectively. Likewise, hesperidin showed inhibitory potentials against L. major amastigote with an IC50 value of 108.44 ± 11.2 µM, as compared to miltefosine. The mean survival time, parasitemia, and suppression percentages showed similar results for the three samples against ANKA strain of P. berghei. The docking studies showed good binding affinities of rutin and hesperidin with numerous H-bonding and van der Waals interactions. Marigold flowers are nutraceuticals, presenting important sources of bioactive flavonoids with potential against neglected tropical diseases.

Cocrystallization of methyltestosterone with salicylic acid and 2,2′-disulfanediyldibenzoic acid - Effect on solubility of methyltestosterone and growth of leishmania parasite (amastigotes and promastigotes)

Methyltestosterone is a class II anabolic androgenic steroidal drug, structurally resembled to natural testosterone, produced by body. Two cocrystals of methyltestosterone with thiosalicylic acid and salicylic acid were successfully furnished by using solid state grinding method with the aim to study changes in biological activities and solubility. During cocrystallization the coformer thiosalicylic acid dimerize into 2, 2′-disulfanediyldibenzoic acid and furnished co-crystal I. Characterization of cocrystals 1 and II was carried out on the basis of single-crystal X-ray diffraction technique, IR spectroscopy, DSC/TGA analysis. The solubility of prepared cocrystals I and II were found to be significantly improved in different dissolution media i.e. phosphate buffer (pH 6.8, 7.4), 0.1 N HCl and distill water. Methyltestosterone and both cocrystals were found to be non-cytotoxic while tested against BJ human normal fibroblast cell line. Non-cytotoxic cocrystals I and II were further evaluated for anti-leishmanial activity (amastigotes and promastigotes). Both cocrystal I (IC50 = 11.9 µM) and II (IC50 = 27.2 µM) shows potent anti-leishmanial activity against L. major promastigote (extracellular / in blood parasite stage) and amastigote (intracellular / inside macrophage cell) stage in comparison to the standard drugs miltefosine (IC50 = 25.5 ± 0.42 µM) and methyltestosterone (IC50 = 37.42±0.57 µM). Infected macrophage cellular assay together with AFM analysis are carried out to study the effect of synthesized cocrystals on growth of amastigotes stage of L. major. The promising result demonstrates the effect of cocrystallization on solubility and anti-leishmanial potential of anabolic androgenic steroidal drug methyltestosterone for the first time.

Old Dogs with New Tricks: Antiparasitic Potential of Structurally Diverse 5-Nitrofuran and 5-Nitrothiophene Imines and Acyl Hydrazones

Miscellaneous imines and acyl hydrazones were prepared from 5-nitrofuraldehyde and 5-nitrothiophene-2-carboxaldehyde. Their activities against Toxoplasma gondii and Leishmania major parasites were evaluated. Promising antiparasitic effects and selectivities were observed for certain acyl hydrazones and imines. Cobalt(II) and copper(II) complexes conserved the high anti-Toxoplasma activities of 3-hydroxy-2-naphthoic carboxyl hydrazone (2a). In addition, sound activities against L. major promastigotes were observed for various analogs of 2a (2b and 2i) and pyrid-2-ylpyrazole-based imines (3g and 3h). Relatively low toxicities to kidney cells and macrophages indicate promising selectivity profiles for these compounds.

Antileishmanial potentials of azacitidine and along with meglumine antimoniate on Leishmania major: In silico prediction and in vitro analysis.

This study aimed to investigate the in vitro and in silico antileishmanial activity of azacitidine (AZA) on Leishmania major promastigotes and amastigotes. The in silico method was used to evaluate the possibility of the interaction of AZA into the binding pocket of inducible nitric oxide synthase (iNOS), a leading defensive oxidative metabolite. Following that, in vitro anti-promastigote, and anti-amastigote activity of AZA was determined using an MTT assay and a macrophage model, respectively. Cytotoxic effects of AZA and meglumine antimoniate (MA) were also assessed by MTT assay on murine macrophages. All experiments were performed in triplicate. The results showed that AZA interacted with Ser133, Gln134, and Lys13 amino acids of iNOS, and the molecular docking score was obtained at -241.053 kcal/mol. AZA in combination with MA significantly (P<0.001) inhibited the growth rate of nonclinical promastigote (IC50 247.6±7.3 μM) and 8.5-fold higher of clinical intramacrophage amastigote stage (29.8±5.3 μM), compared to the untreated group. A significant upsurge of Th1 subsets and transcription genes and a meaningful decline in Th2 cytokines subclasses at the equivalent concentrations of AZA and MA was observed (P<0.001). The apoptosis effect of AZA along with MA was significantly induced on L. major in a dose-dependent manner (P<0.001). The present study demonstrated that AZA possesses antileishmanial activity in in vitro and in silico models. However, AZA combined with MA was more effective than AZA alone in inhibiting the growth rate of promastigotes and amastigotes of L. major. This study indicates that AZA in combination with MA demonstrated a potent antileishmanial mechanism, promoting immune response and enhancing an immunomodulatory role toward the Th1 pathway. This experimental study is a basic study for applying more knowledge about the mechanisms of AZA along with MA in animal models in the future.

In silico and in vitro potentials of crocin and amphotericin B on Leishmania major: Multiple synergistic mechanisms of actions.

A significant barrier to optimal antileishmanial treatment is low efficacy and the emergence of drug resistance. Multiple approaches were used to monitor and assess crocin (a central component of saffron) mixed with amphotericin B (AmpB) potential in silico and in vitro consequences. The binding behavior of crocin and iNOS was the purpose of molecular docking. The results showed that crocin coupled with AmpB demonstrated a safe combination, extremely antileishmanial, suppressed Leishmania arginase absorption, and increased parasite death. This natural flower component is a robust antioxidant, significantly promoting the expression of the Th1-connected cytokines (IL12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (Elk-1, c-Fos, and STAT-1). In comparison, the expression of the Th2-associated phenotypes (IL-10, IL-4, and TGF-β) was significantly reduced. The leishmanicidal effect of this combination was also mediated through programmed cell death (PCD), as confirmed by the manifestation of phosphatidylserine and cell cycle detention at the sub-GO/G1 phase. In conclusion, crocin with AmpB synergistically exerted in vitro antileishmanial action, generated nitric oxide and reactive oxygen species, modulated Th1, and Th2 phenotypes and transfer factors, enhanced PCD profile and arrested the cell cycle of Leishmania major promastigotes. The main action of crocin and AmpB involved wide-ranging mechanistic insights for conducting other clinical settings as promising drug candidates for cutaneous leishmaniasis. Therefore, this combination could be esteemed as a basis for a potential bioactive component and a logical source for leishmanicidal drug development against CL in future advanced clinical settings.

Anti-leishmanial, immunomodulatory and additive potential effect of Piperine on Leishmania major: The in silico and in vitro study of Piperine and its combination

BackgroundPiperine (Pn), an indole alkaloid compound found in pepper, is an effective compound with anti-leishmanial medications that administered alone or in combination. This study aimed to use Pn for possible biochemical targets and to assess mechanisms of anti-leishmanial action and immunomodulatory roles. MethodsThe ability of Pn to bind to interleukin-12P40 (IL-12P40) and interferon-γ (IFN-γ) was investigated using molecular docking. The leishmanicidal effect of Pn, meglumine antimoniate (Glucantime®; MA), and Pn plus MA was assessed on Leishmania major promastigotes and amastigotes. A real-time PCR was applied to quantify cytokines gene expression in drug-treated murine macrophages. ResultsThe molecular docking findings indicated that Pn could bind to IL-12P40/IFN-γ. In silico analyses showed an affinity of Pn to IL-12P40/IFN-γ, with the MolDock score of −236.91 and −64.87 kcal/mol, respectively. Pn plus MA reduced the proliferation rate of promastigote and amastigote forms of L. major compared to each drug alone (IC50 = 43.22 and 19.41 μg/mL, respectively). Moreover, the combination drug demonstrated no cytotoxicity as the selectivity index (SI) was 14.81. Also, Th1-related cytokines were upregulated, while Th2-related cytokines were downregulated in Pn combination-treated murine macrophages. ConclusionsThe superior effectiveness of combination therapy on L. major warrants further investigations on the clinical potential of this combination in the treatment of leishmaniasis.

Nucleoside-Derived Metallohydrogel Induces Cell Death in Leishmania Parasites.

Self-assembled hydrogels by virtue of their unique 3D network and tunability have extensively been explored for bio-medical applications like tissue engineering, delivery and release of therapeutic agents, etc. Herein, we demonstrate for the first-time nucleoside-based biocompatible hydrogels with a remarkable leishmanicidal effect against both Leishmania major promastigotes and amastigotes and no cytotoxic effect on the macrophage cell line. In this work, a series of biocompatible hydrogels have been synthesized by silver ion-driven self-assembly of natural nucleoside and nucleotide-like cytidine and 5'-GMP. The supramolecular metallogel obtained from the assembly of cytidine and boronic acid is capable of inducing apoptotic-like cell death of protozoan parasite by causing damage to the membrane as well as DNA. These hydrogels could find promising applications in combating cutaneous leishmaniasis by topical treatment.

A New Approach to the Investigation of Therapeutic Efficiency of Novel Carum copticum Nanoparticles against Leishmania major Promastigotes

Current therapies for Leishmaniasis are associated with several side effects as well as drug resistance. Sensitivity and resistance of Leishmania major to Glutamine are referred to as those isolates which are responsive or non-responsive to one or two full courses of treatment by Glucantime systematically and/or intra-lesionally, respectively. In this study, We assessed a new approach to the investigation of the therapeutic efficiency of novel Carum copticum Nanoparticles against Leishmania major Promastigotes. First, the Carum copticum Nanoparticles were synthesized and liposomal Carum copticum was applied as a new therapeutic approach substituted for current therapy. In this experimental study, liposomal Carum copticum was prepared using the thin film hydration method and characterized based on encapsulation efficiency, size, and zeta potential. Carum copticum was successfully loaded into the liposome. The surface charge of the nanoparticle was neutral and the size of the nanoparticle was 176.5 nm. Liposomal Carum copticum beared spherical shape without any agglomeration. Results revealed that liposomal Carum copticum carried a significant effect, compared to the control sample, on parasite growth in both logarithmic and stationary phases. The result of this study signifies that the Carum copticum Nanoparticles induces a better and more tangible effect on the survival of Leishmania major promastigotes.

In vitro Evaluation of the Potent Antileishmanial Activity of Ferula tabasensis Alone or in Combination with Shark Cartilage Extract Against the Standard Iranian Strain of Leishmania major (MRHO/IR/75/ER).

The available drugs for the treatment of leishmaniasis are highly toxic and extremely expensive, with low efficiency; therefore, the development of effective therapeutic compounds is essential. The present study aimed to explore the antileishmanial effects of ethyl acetate extract, methanol extract, and fractions 1-4 (F1-F4) of Ferula tabasensis, alone or in combination with shark cartilage extract (ShCE), on L. major in vitro. In this study, ethyl acetate, methanol, and n-hexane extracts were extracted from the aerial roots of F. tabasensis by the maceration method. The silica gel column chromatography was used to separate n-hexane extracts at varying polarities (F1-F4 fractions). Subsequently, the effects of extracts and fractions against promastigotes were assessed by the parasite counting method microscopic inhibition test and MTT assay. Besides, their effects on the infected macrophage cells and the number of amastigotes were investigated. Cytotoxicity was evaluated in non-infected J774A.1 macrophage cells. Finally, apoptosis induction of promastigotes, including infected and non-infected macrophages, was evaluated. The results indicated the highly potent activity of F. tabasensis extracts and F1-F4 fractions, alone or in combination with ShCE, against L. major promastigotes and amastigotes in a dose-dependent manner (P < 0.05). The F1 fraction and methanol extract showed markedly higher toxicity compared to the other extracts and fractions, with 50% inhibitory concentrations (IC50/72h) of 2.4 ± 0.29 and 2.9 ± 0.55 µg/mL against promastigotes and 1.79 ± 0.27 µg/mL and 1.39 ± 0.27 µg/mL against amastigotes (P < 0.001). Moreover, they had a high selectivity index (SI) due to the low toxicity of macrophages (P < 0.0001). The results of flow cytometry indicated that the percentages of apoptotic promastigote cells in contact with IC50 concentrations of F1 and methanol extract alone after 72 h were 43.83 and 43.93%, as well as 78.4%, and 65.45% for their combination with ShCE, respectively.Also, apoptosis of infected macrophages induced by F1 and methanol extracts was estimated at 68.5% and 83.7%, respectively. In this study, the F1 fraction and methanol extract of F. tabasensis showed potent efficacy against L. major, associated with low toxicity and apoptosis induction. Therefore, they can be promising therapeutic candidates in future animal and even human studies.

Copper-Catalyzed Asymmetric Hydroboration Reaction of Novel Methylene Isoindolinone Compounds through Microwave Irradiation and Their Antileishmanial and Antitoxoplasma Activities.

The aim of this study was devoted into molecular docking calculations to discover the potential antileishmania and antitoxoplasma activities of newly synthesized compounds obtained by applying a practical and simple method under microwave irradiation. All these compounds were tested in vitro for their biological activity against Leishmania major promastigotes, amastigotes, and Toxoplasma gondii tachyzoites. Compounds 2a, 5a, and 5e were the most active against both L. major promastigotes and amastigotes, with IC50 values of less than 0.4 μM mL-1. Compounds 2c, 2e, 2h, and 5d had a strong antitoxoplasma activity of less than 2.1 μM mL-1 against T. gondii. We can conclude that aromatic methyleneisoindolinones are potently active against both L. major and T. gondii. Further studies for mode of action evaluation are recommended. Compounds 5c and 5b are the best drug candidates for antileishmania and antitoxoplasma due to their SI values being over 13. The docking studies of compounds 2a-h and 5a-e against pteridine reductase 1 and T. gondii enoyl acyl carrier protein reductase reveal that compound 5e may be an effective antileishmanial and antitoxoplasma drug discovery initiative.

Development of a novel immunoFET technology-based POC assay for detection of Leishmania donovani and Leishmania major.

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory-based techniques, which are time-consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point-of-care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania-specific antigens were serially diluted in 1× PBS from a concentration of 106 -102 parasites/mL. A specific polyclonal antibody-based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology-based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen-antibody at the gate surface generate an electrical signal that can be measured by semiconductor field-effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 μm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 106 to 102 parasites/mL in 1× PBS. At the concentration of 104 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub-threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno-FET biosensors might allow earlier and faster detection of leishmaniasis.

Anti-leishmanial activity of sanguinarine and nano chitosan is modulated by increased ROS production and upregulated TNF-α and iNOS expression

The current study aimed to evaluate the anti-leishmanial activity of sanguinarine and nano chitosan (CS NPs) on Leishmania major, potentially mediated through gene expression of TNF-α and iNOS. To identify the potential targets for sanguinarine, the energy-minimized structure was docked using AutoDock 4.3 software into the active sites of selected peterdin reductase (PTR1) and arginase (ARG-L), which were modeled using homology modeling programs. The colorimetric MTT assay was used to determine the viability of L. major promastigotes subjected to different concentrations of sanguinarine and CS NPs in a concentration- and time-dependent manner. Reactive oxygen species (ROS) levels were measured in treated promastigotes. TNF-α and iNOS expression was determined in PBMCs infected at 24, 48, and 72 h after treatment with sanguinarine and CS NPs. The sanguinarine - PTR1 and ARG-L interactions and calculated energy of these enzymes indicated an excellent affinity for sanguinarine with both enzymes. The IC50 values of the sanguinarine in combination with 10% CS NPs) were 124.76 and 89.25 μM after 24 and 48 h, respectively. Sanguinarine-CS NPs enhanced the expression of TNF-α and iNOs in L. major-infected PBMCs after 24 h (p < 0.05). Sanguinarine-CS NPs could therefore reflect a promising anti-leishmanial agent. Sanguinarine and CS NPs in combination significantly inhibited the proliferation and viability of L.major promastigotes whilst concurrently regulating the expression of TNF-α and iNOS and increasing ROS levels. To better understand the effect of CS NPs and sanguinarine on leishmaniasis, further studies are required in an experimental murine model.

Natural products derived steroids as potential anti-leishmanial agents; disease prevalence, underlying mechanisms and future perspectives.

Leishmaniasis is a vector-borne infection caused by protozoan parasites from the genus leishmania and is among the most neglected tropical diseases. It is highly prevalent disease, affecting about 350 million population worldwide. Only limited number of anti-leishmanial agents are approved for clinical use till now and they are associated with side effects and have limited efficacy. Subsequently, natural products based discovery of more safe and effective drugs against leishmania is under scientific consideration. Various studies reported the efficacy of natural products against intracellular and extracellular forms of leishmania species. This work is aimed to evaluate current literature focused on the anti-leihmanial efficacy of steroidal moieties from natural products and their mechanism of action. Compounds including steroidal saponins, steroidal alkaloids and phytosterols were found to exhibit considerable anti-leishmanial efficacy. For instance, steroidal saponin, (25R)-spirost-5-en-3b-ol,3-O-α-rhamnopyranosyl-(1→4)-α-rhamnopyranosyl-(1→4)-[a-rhamnopyranosyl-(1→2)]-glucopyranoside isolated from A. paradoxum has completely eradicated Leishmania major promastigotes at 50µgmL-1 dose. Spirostanic saponins isolated from Solanum paniculatum L. were effective against Leishmania amazonensis promastigotes. Turgidosterones isolated from Panicum turgidum exhibited high leishmanicidal potentials against Leishmania donovani promastigotes with IC50 of 4.95-8.03µgmL-1 and even better activity against amastigotes exhibiting an IC50 of 4.50-9.29µgmL-1. Likewise, racemoside-A from Asparagus racemosus was found effective against an antimonial sensitive (AG83) and antimonial resistant (GE1F8R) strains of the L. donovani. Moreover, steroidal alkaloids including hookerianamide-1, hookerianamide-H, hookerianamide-J, hookerianamide-K, dehydrosarsalignone, vagenine-A, sarcovagine-C, holaphylline, saracodine, holamine, 15-α hydroxyholamine, holacurtin, N-desmethyl holacurtine and elasticine has exhibited time and dose-dependent efficacy against various strains of leishmania. β-sitosterol was found active against multiple strains of leishmania. These compounds mainly exhibit their therapeutic efficacy via liberation of ROS, mitochondrial depolarization, morphological and ultra-structural changes, accumulation of lipid droplets, depletion of non-protein thiols and triggering apoptotic pathways. In conclusion, leishmaniasis is a major health problem in many countries. Plants-derived steroids moieties have reveled efficacy against leishmaniasis and is a source of lead compounds. Further detailed molecular studies are warranted for the discovery of more effective and safe anti-leishmanial drugs.

Evaluation of the Antiparasitic and Antifungal Activities of Synthetic Piperlongumine-Type Cinnamide Derivatives: Booster Effect by Halogen Substituents.

A series of synthetic N-acylpyrrolidone and -piperidone derivatives of the natural alkaloid piperlongumine were prepared and tested for their activities against Leishmania major and Toxoplasma gondii parasites. Replacement of one of the aryl meta-methoxy groups by halogens such as chlorine, bromine and iodine led to distinctly increased antiparasitic activities. For instance, the new bromo- and iodo-substituted compounds 3 b/c and 4 b/c showed strong activity against L. major promastigotes (IC50 =4.5-5.8 μM). Their activities against L. major amastigotes were moderate. In addition, the new compounds 3 b, 3 c, and 4 a-c exhibited high activity against T. gondii parasites (IC50 =2.0-3.5 μM) with considerable selectivities when taking their effects on non-malignant Vero cells into account. Notable antitrypanosomal activity against Trypanosoma brucei was also found for 4 b. Antifungal activity against Madurella mycetomatis was observed for compound 4 c at higher doses. Quantitative structure-activity relationship (QSAR) studies were carried out, and docking calculations of test compounds bound to tubulin revealed binding differences between the 2-pyrrolidone and 2-piperidone derivatives. Microtubules-destabilizing effects were observed for 4 b in T. b. brucei cells.

Preparation of Thymol Nanoliposome and Solid Lipid Nanoparticle and Evaluation of their Inhibitory Effects on Leishmania Major Promastigotes

Introduction: Thymol has an antiprotozoal effect. Nanoparticulate systems are useful carriers for both small and large drug molecules, which can protect them from some chemical and biological damages as well as target drug delivery to specific organs or receptors. In this work, the nano-liposomal system and solid lipid nanoparticles loaded by thymol were prepared and the effectiveness of them were evaluated on Leishmania major promastigotes.&#x0D; Methods: Several formulations of nano-liposomes and solid lipid nanoparticles were prepared, and the amount of thymol loading, in-vitro release profile, particle size, and zeta potential were evaluated. Finally, the best formulations were serially diluted and incubated for 24, 48, and 72 hours on Leishmania major promastigotes, which cultured on Novy–MacNeal–Nicolle medium, and the results were analyzed.&#x0D; Results: The highest loading of thymol in nano-liposomes (92%) was seen in the formulations made with phosphatidylcholine (Called L3), and among the solid lipid nanoparticles, the formulation prepared with glycerol monostearate (S1) had the most entrapment efficiency of thymol (87%). These formulations were selected to evaluate the release rate of thymol. The results showed that S1 has a slower release rate than L3; this may be due to the presence of Glycerol monostearate in solid lipid nanoparticles structure. The best formulations, L3 and S1, were chosen for anti-Leishmaniosis assessment; which showed that all three forms of free thymol, nanoliposomes, and solid lipid nanoparticles inhibited Leishmania major. The half maximal inhibitory concentration (IC50) of free thymol, nanoliposome, and solid lipid nanoparticles for a 24-hour incubation are 7.8, 62.5, and 125, respectively, which decrease to 7.8, 7.8, and 15.6 for 48 hours and 7.8, 0.49, and 0 for 72 hours of incubation.&#x0D; Conclusion: Thymol has a significant effect on the inhibition of Leishmania major promastigotes and usage of thymol in the form of liposomes or solid lipid nanoparticles can sustain the drug release and have a lower IC50 during the longer incubation time.

The in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major.

The emerging of drug resistant against Leishmania parasites prompts scientists to seek for novel therapeutic strategies against theses infectious protozoan parasites. Among different strategies, the use of larvae secretions could be suggested as a possible therapy with low side effects. Accordingly, the current study evaluated the in vitro and in vivo effects of Lucilia sericata larval secretions on Leishmania major, the causative agent of cutaneous leishmaniasis (CL). After preparation of L. sericata larval stages (L2 and L3) secretions, the potential effects of secretions were evaluated against L. major promastigotes and amastigotes (in vitro) using MTT assay. The cytotoxicity effects of secretions were also checked on uninfected macrophages. In addition, in vivo experiments were also conducted to investigate the effects of larvae's secretions on the CL lesions induced in the BALB/c mice. Although the increased concentration of larvae secretions exhibited a direct effect on the promastigotes proliferation (viability), contrarily, L2 secretions at a concentration of 96μg/ml represented the highest inhibitory effect on parasite (amastigotes) burden in infected macrophages. Interestingly, L3 secretions > 60μg/ml induced inhibitory effects on amastigotes. The results relevant to the cytotoxicity effects of L2 and L3 secretions on uninfected-macrophages showed a dose dependent correlation. In vivo results were also significant, compared to the positive control group. This study suggested the plausible inhibitory effects of L. sericata larvae's secretions on the L. major amastigotes and CL lesions progression. It seems that the characterization of all effective components/proteins in the larvae secretions and their specific targets in parasite structure or in cell (macrophage) responses could further reveal more details regarding the anti-leishmanial properties of these compounds.

Modulating leishmanial pteridine metabolism machinery via some new coumarin-1,2,3-triazoles: Design, synthesis and computational studies

In accordance with WHO statistics, leishmaniasis is one of the top neglected tropical diseases, affecting around 700 000 to one million people per year. To that end, a new series of coumarin-1,2,3-triazole hybrid compounds was designed and synthesized. All new compounds exerted higher activity than miltefosine against L. major promastigotes and amastigotes. Seven compounds showed single digit micromolar IC50 values whereas three compounds (13c, 14b and 14c) displayed submicromolar potencies. A mechanistic study to elucidate the antifolate-dependent activity of these compounds revealed that folic and folinic acids abrogated their antileishmanial effects. These compounds exhibited high safety margins in normal VERO cells, expressed as high selectivity indices. Docking simulation studies on the folate pathway enzymes pteridine reductase and DHFR-TS imparted strong theoretical support to the observed biological activities. Besides, docking experiments on human DHFR revealed minimal binding interactions thereby highlighting the selectivity of these compounds. Predicted in silico physicochemical and pharmacokinetic parameters were adequate. In view of this, the structural characteristics of these compounds demonstrated their suitability as antileishmanial lead compounds.