Promastigote Forms Research Articles

EFFECTS OF NYCTANTHES ARBOR-TRITIS (HARSINGAR) PLANT EXTRACTS ON PROMASTIGOTE FORM OF LEISHMANIA DONOVANI IN IN-VITRO CONDITION

Leishmaniasis is a group of disease caused by the members of kinetoplast protozoa of the genus leishmania comprising a group of unicellular organisms which are intracellular parasites in macrophage and other phagocytic cells of the reticuloendothelial system. Visceral leishmaniasis (VL) or Kala-azar a vector-borne parasitic disease caused by protozoan parasite (Chang, KP.; et.al, 1985). In India the traditional methods of treatment are based on the principles of ayurveda and naturopathy. Considering the aforesaid facts this project has been selected to search the plants for the treatment of Kala-azar. The plant products or plant extracts will certainly be cheaper than the allopathic drugs. Therefore, aim is to search a plant which is locally available and if not available, it can be purchased from the local market to provide cheaper and without side effect treatment to Kala-azar (Mishra P. 2008; W.H.O. Tropical diseases 2008. Effects of Nyctanthes arbor-tritis plant extracts on promastigote form of Leishmania donovani in in-vitro condition. Phytochemical screening of the plant extract showed the presence of alkaloid, avonoid, xed oil and fats, saponin, tannin and phenolic compounds. The selected plant obtained from Gaya, Bihar region & their ethanolic extracts will be prepared in the laboratory by Soxhlet apparatus in Ethanol solvent to use during experiment. The effects of plant extracts on the parasite will be studied by providing variable doses to the parasite. The plant extracts at varied concentration will be added into media containing the parasite in the ratio between extract & media determined during experimentation in the laboratory. After incubation at different time intervals of 24, 48 and 72 hours the effect of extracts on the parasite will be studied. The sample of parasite (promastigote of L. donovani) used for experiment (in-vitro) will be routinely culture in laboratory at 25± 2° with in different culture media after collecting parasites (sample) from the blood and bone marrow of patients of Kala-azar in the hospitals or research institutes. The plant extracts exhibit that with the increasing duration of time the efcacy of the plant extracts also increases signicantly in NA taken into the consideration

6-Chloro-3-nitro-2-[(phenylsulfonyl)methyl]imidazo[1,2-b]pyridazine

As part of our ongoing scaffold-hopping work on an antikinetoplastid 3-nitroimidazo[1,2-a]pyridine scaffold, we explored 3-nitroimidazo[1,2-b]pyridazine as a potential new antikinetoplastid series. Using conditions previously described by our lab, we obtained 6-chloro-3-nitro-2-[(phenylsulfonyl)methyl]imidazo[1,2-b]pyridazine with 54% yield. In vitro activity of this compound was evaluated against both the promastigote form of Leishmania donovani, the axenic amastigote form of Leishmania infantum and the trypomastigote blood stream form of Trypanosomabrucei brucei, and its influence on cell viability was assessed on the HepG2 cell line. However, despite good activity against the trypomastigote blood stream form of T. b. brucei (EC50 = 0.38 µM), it showed poor solubility in both HepG2 (CC50 > 7.8 µM) and L. infantum axenic amastigotes (EC50 > 1.6 µM) culture media, associated with a loss of activity against the promastigote form of L. infantum (EC50 > 15.6 µM).

Antioxidant, antibacterial and antileishmanial potential of Micromeria nervosa extracts and molecular mechanism of action of the bioactive compound.

This study aimed to determine the antibacterial and antileishmanial potential of Micromeria nervosa extracts. The identification of the antileishmanial compound and the study of its molecular mechanism of action have also been undertaken. Ethanol extract showed high polyphenol content and diethyl ether extract exhibited high DPPH scavenging and low beta-carotene bleaching activity (IC50=13.04±0.99µg/mL and 200.18±3.32µg/mL, respectively). However, diethyl ether extract displayed high antibacterial activity against Gram-positive strains including methicillin-resistant Staphylococcus aureus (MIC=31.25µg mL-1), Staphylococcus aureus ATCC6538 (MIC=62.5µg mL-1) and Listeria monocytogenes ATCC 19115 (MIC=125µg mL-1) as well as high antileishmanial activity against the promastigote forms of L. infantum and L. major (IC50=11.45µg mL-1 and IC50=14.53µg mL-1, respectively). The active compound was purified using bioassay-guided fractionation and thin layer chromatography, and identified as ursolic acid using high performance liquid chromatography coupled with a photodiode array and mass spectrometry. The purified compound was strongly inhibitory against the promastigote and amastigote forms of L. infantum and L. major (IC50=5.87µg mL-1 and 6.95mL-1, versus 9.56µg mL-1 and 10. 68µg mL-1, respectively) without overt cytotoxicity against Raw 264.7 macrophage cells (SI=13.53 and 11.43, respectively). The commercial compound (ursolic acid) showed similar activity against amastigotes and promastigotes forms of L. infantum and L. major. Moreover, its molecular mode of action against leishmaniasis seems to involve the expression of the ODC and SPS genes involved in thiol pathway. Extracts of Micromeria nervosa can be considered as a potential alternative to antimicrobial and antileishmanial drugs.

Imidazolium salts as an alternative for anti-Leishmania drugs: Oxidative and immunomodulatory activities.

In this study we explored the previously established leishmanicidal activity of a complementary set of 24 imidazolium salts (IS), 1-hexadecylimidazole (C16Im) and 1-hexadecylpyridinium chloride (C16PyrCl) against Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) infantum chagasi. Promastigotes of L. amazonensis and L. infantum chagasi were incubated with 0.1 to 100 μM of the compounds and eight of them demonstrated leishmanicidal activity after 48 h - C10MImMeS (IC50 L. amazonensis = 11.6), C16MImPF6(IC50 L. amazonensis = 6.9), C16MImBr (IC50 L. amazonensis = 6), C16M2ImCl (IC50 L. amazonensis = 4.1), C16M4ImCl (IC50 L. amazonensis = 1.8), (C10)2MImCl (IC50 L. amazonensis = 1.9), C16Im (IC50 L. amazonensis = 14.6), and C16PyrCl (IC50 L. amazonensis = 4).The effect of IS on reactive oxygen species production, mitochondrial membrane potential, membrane integrity and morphological alterations of promastigotes was determined, as well as on L. amazonensis-infected macrophages. Their cytotoxicity against macrophages and human erythrocytes was also evaluated. The IS C10MImMeS, C16MImPF6, C16MImBr, C16M2ImCl, C16M4ImCl and (C10)2MImCl, and the compounds C16Im and C16PyrCl killed and inhibited the growth of promastigote forms of L. amazonensis and L. infantum chagasi in a concentration-dependent manner, contributing to a better understanding of the structure-activity relationship of IS against Leishmania. These IS induced ROS production, mitochondrial dysfunction, membrane disruption and morphological alterations in infective forms of L. amazonensis and killed intracellular amastigote forms in very low concentrations (IC50 amastigotes ≤ 0.3), being potential drug candidates against L. amazonensis.

Selene-Ethylenelacticamides and N-Aryl-Propanamides as Broad-Spectrum Leishmanicidal Agents.

The World Health Organization classifies Leishmania as one of the 17 “neglected diseases” that burden tropical and sub-tropical climate regions with over half a million diagnosed cases each year. Despite this, currently available anti-leishmania drugs have high toxicity and the potential to be made obsolete by parasite drug resistance. We chose to analyze organoselenides for leishmanicidal potential given the reduced toxicity inherent to selenium and the displayed biological activity of organoselenides against Leishmania. Thus, the biological activities of 77 selenoesters and their N-aryl-propanamide derivatives were predicted using robust in silico models of Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania (Viannia) braziliensis. The models identified 28 compounds with >60% probability of demonstrating leishmanicidal activity against L. infantum, and likewise, 26 for L. amazonesis, 25 for L. braziliensis, and 23 for L. major. The in silico prediction of ADMET properties suggests high rates of oral absorption and good bioavailability for these compounds. In the in silico toxicity evaluation, only seven compounds showed signs of toxicity in up to one or two parameters. The methodology was corroborated with the ensuing experimental validation, which evaluated the inhibition of the Promastigote form of the Leishmania species under study. The activity of the molecules was determined by the IC50 value (µM); IC50 values < 20 µM indicated better inhibition profiles. Sixteen compounds were synthesized and tested for their activity. Eight molecules presented IC50 values < 20 µM for at least one of the Leishmania species under study, with compound NC34 presenting the strongest parasite inhibition profile. Furthermore, the methodology used was effective, as many of the compounds with the highest probability of activity were confirmed by the in vitro tests performed.

In vitro effects of lapachol and β-lapachone against Leishmania amazonensis.

Leishmaniasis is a neglected disease that affects millions of people worldwide, and special attention should be given to treatment because the available drugs have limitations, which can lead to low therapeutic adherence and parasitic resistance. This study evaluated the activity of the bioactive naphthoquinones, lapachol and β-lapachone, against Leishmania amazonensis. The cell alterations were evaluated in vitro on promastigote and amastigote forms. The lethal dose (LD50) at 24, 48, and 72 h on the promastigote's forms using lapachol was 75.60, 72.82, and 58.85 μg/mL and for β-lapachone was 0.65, 1.24, and 0.71 μg/mL, respectively. The naphthoquinones significantly inhibited the survival rate of L. amazonensis amastigotes at 83.11, 57.59, and 34.95% for lapachol (82.28, 41.14, and 20.57 µg/mL), and 78.49, 83.25, and 80.22% for β-lapachone (3.26, 1.63, and 0.815 µg/mL). The compounds on the promastigote's forms led to the loss of mitochondrial membrane potential, induced changes in the integrity of the membrane, caused damage to cells suggestive of the apoptotic process, and showed inhibition of tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. The results showed that these naphthoquinones are promising candidates for research on new drugs with anti-Leishmania activity derived from natural products.

Antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, on Leishmania (Leishmania) amazonensis.

Leishmaniasis, a neglected disease caused by the parasite Leishmania, is treated with drugs associated with high toxicity and limited efficacy, in addition to constant reports of the emergence of resistant parasites. In this context, snake serums emerge as good candidates since they are natural sources with the potential to yield novel drugs. We aimed to show the antileishmanial effects of γCdcPLI, a phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum, against Leishmania (Leishmania) amazonensis. Promastigotes forms were exposed to γCdcPLI, and we assessed the parasite viability and cell cycle, as well as invasion and proliferation assays. Despite the low cytotoxicity effect on macrophages, our data indicate that γCdcPLI has a direct effect on parasites promoting an arrest in the G1 phase and reduction in the G2/M phase at the highest dose tested. Moreover, this PLA2 inhibitor reduced the parasite infectivity when promastigotes were pre-treated. Also, we demonstrated that the γCdcPLI treatment modulated the host cell environment impairing early and late steps of the parasitism. γCdcPLI is an interesting tool for the discovery of new essential targets on the parasite, as well as an alternative compound to improve the effectiveness of the leishmaniasis treatment.

Chemical composition, in vitro larvicidal and antileishmanial activities of the essential oil from Citrus reticulata Blanco fruit peel.

Numerous studies have investigated the chemical composition and biological activities of essential oils from different Citrus species fruit peel, leaves and flowers. This paper aims to investigate the chemical composition, larvicidal and antileishmanial activities of essential oil from Citrus reticulata fruit peel (CR-EO). CR-EO was obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was analyzed by GC-MS and GC-FID. Limonene (85.7%), ɣ-terpinene (6.7%) and myrcene (2.1%) were identified as its major components. CR-EO showed high activity against promastigote forms of Leishmania amazonensis (IC50 = 8.23 µg/mL). CR-EO also exhibited high larvicidal activity against third instar Aedes aegypti larvae at a lethal concentration (LC50 = 58.35 µg/mL) and 100% mortality at 150 µg/mL. This study suggests, for the first time, the potential use of CR-EO against this important mosquito-borne viral disease caused by the genus Aedes.

Leishmanicidal activity of the venoms of the Scorpions Brotheas amazonicus and Tityus metuendus.

Leishmaniasis is a vector-transmitted zoonosis caused by different species of the genus Leishmania, with a wide clinical spectrum. It is a public health problem aggravated by a series of limitations regarding treatment. In the search for new therapeutic alternatives, scorpion venoms are a source of multifunctional molecules that act against the natural resistance of pathogens. This work evaluated the antileishmanial potential of Brotheas amazonicus and Tityus metuendus venoms against the promastigote forms of Leishmania amazonensis e Leishmania guyanensis. The venoms of B. amazonicus and T. metuendus were evaluated for their constituents using Fourier Transform Infrared (FTIR). Growth inhibition and death of promastigotes were evaluated in the presence of diferente crude venom concentrations (100 µg/mL, 50 µg/mL, 10 µg/mL, 1 µg/mL) after one hour of incubation at 25 °C. The FTIR spectra of both venoms exhibited bands in approximate regions, revealing that both exhibit similar functional groups. Crude venom from both scorpion species showed similar or superior leishmanicidal effects to the standart drug N-methylglucamine antimoniate. At the highest concentration of 100 µg/mL, cultures of L. guyanensis treated with the venom of B. amazonicus showed the highest mortality percentages, above 28%, while T. metuendus venom showed the highest activity against L. amazonensis, with mortality above 7%. This preliminar study demonstrates that B. amazonicus and T. metuendus venoms can be important tools in the search for new drugs Against leishmaniasis. Next step involves evaluating the activity against the amastigote forms and purifying the venom proteins in order to identify the best anti-leishmania candidates.

Green Synthesis, Characterization and Antibacterial and Leishmanicidal Activities of Silver Nanoparticles Obtained from Aqueous Extract of Eucalyptus grandis

This work describes a green synthesis, the characterization, and biological evaluation of silver nanoparticles (AgNPs). The AgNPs suspension was synthesized using aqueous leaf extract of Eucalyptus grandis, which presented a characteristic band at 407 nm in the UV-Vis spectrum. The AgNPs presented a spherical shape and size of 9.7 ± 0.3 nm. The nanoparticles were stable over a month, indicating that E. grandis’ extract is suitable for their preparation and stabilization. The X-ray analysis showed that the crystallinity of AgNPs corresponded to the centered face phase of silver. The antibacterial and leishmanicidal activities of AgNPs were evaluated. The AgNPs presented antibacterial activity on the Gram-negative bacteria Escherichia coli at 53.9 µg mL–1. The leishmanicidal activity evaluation against promastigote forms of Leishmania infantum, Leishmania amazonensis, and Leishmania braziliensis showed that the biological response is dependent on the volume of AgNP suspension. It was demonstrated that L. infantum was more sensitive to the nanoparticle’s treatment than L. amazonensis and L. braziliensis. The treatment of L. infantum promastigotes with 150 µL of AgNP suspension reduced parasite growth by 67.9%, a result which was similar to the treatment with 1 (66.7%) or 2 µL (70.6%) of amphotericin B used as a positive control.

Synthesis of 4-(4-chlorophenyl)thiazole compounds: in silico and in vitro evaluations as leishmanicidal and trypanocidal agents.

Neglected tropical diseases are a diverse group of communicable pathologies that mainly prevail in tropical and subtropical regions. Thus, the objective of this work was to evaluate the biological potential of eight 4-(4-chlorophenyl)thiazole compounds. Tests were carried out in silico to evaluate the pharmacokinetic properties, the antioxidant, cytotoxic activities in animal cells and antiparasitic activities were evaluated against the different forms of Leishmania amazonensis and Trypanosoma cruzi in vitro. The in silico study showed that the evaluated compounds showed good oral availability. In a preliminary in vitro study, the compounds showed moderate to low antioxidant activity. Cytotoxicity assays show that the compounds showed moderate to low toxicity. In relation to leishmanicidal activity, the compounds presented IC50 values that ranged from 19.86 to 200 µM for the promastigote form, while for the amastigote forms, IC50 ranged from 101 to more than 200 µM. The compounds showed better results against the forms of T. cruzi with IC50 ranging from 1.67 to 100 µM for the trypomastigote form and 1.96 to values greater than 200 µM for the amastigote form. This study showed that thiazole compounds can be used as future antiparasitic agents.

Antileishmanial and cytotoxic activity of dillapiole n-butyl ether

ABSTRACT Among the neglected diseases, American cutaneous leishmaniasis (ACL) still remains highly endemic in some tropical regions. The currently available drugs for treatment are highly toxic, prompting the search for new therapeutic options. The aim of this study was to evaluate the toxic potential of dillapiole n-butyl ether (DBE) against Leishmania amazonensis and L. guyanensis, as well as its toxicity on human peripheral blood mononuclear cells (PBMCs) in vitro. For cell cytotoxicity, concentrations of DBE that ranged from 7.8 to 500 µM were used for 48 and 72 h. For the evaluation of the antileishmanial activity, DBE was tested at concentrations of 0.28 to 18 µM for 24, 48, and 72 h. A value of 36 µM was used for the amastigote assay. The selectivity index (SI) was determined by dividing the CC50/IC50 (macrophages/promastigotes). DBE exhibited a CC50 of 203.9 ± 0.5 µM in 72 h. DBE inhibited promastigote forms with an IC50 of 3.0 µM for both Leishmania species for 72 h. The standard, Pentacarinat®, showed an IC50 of 2.9 µM and 0.3 µM, respectively. The SI of DBE for both species was 67.9 for 72 h. DBE inhibited intracellular forms of L. amazonensis by 65.5% after 48 h. In molecular modeling, DLpOl-F showed two hydrogen bonds (SER418 and 421). DBE demonstrated promising in vitro antileishmanial potential.

A Study of the inhibitory effect of Terpinen-4-ol on Amastigote Forms of Leishmania tropica within Macrophages of Mouse in vitro

It was recorded that Terpinen-4-ol has an anti-parasitic properties, so it will be noteworthy to intensify the studies about this compound.&#x0D; This study aims to test the effectiveness of terpinen-4-ol on amastigote forms of Leishmania parasite in macrophages.&#x0D; This effect was studied by adding increasing concentrations of Terpinen-4-ol to culture wells containing mouse macrophages that were previously incubated with the promastigote forms of the parasites for 24 hours .Then, they were incubated for another 24 hours with increasing concentrations of Terpinen-4-ol. After, Parasites were enumerated into macrophages in wells either treated with Terpinen-4-ol or in control wells.&#x0D; Treatment with Terpinen-4-ol at concentrations (0.01%, 0.02%, 0.05%, 0.1%) (v/v) decreased the viability of the amastigote forms inside macrophages (24.02%, 32.74%, 66.72%, 100%) respectively, compared to control wells (water only). &#x0D; This study demonstrated an activity of Terpinen-4-ol against amastigote forms of Leishmania tropica in vitro with minimal inhibitory concentration (MIC50) value was 0.0416% (v/v).

First evidence of a serine arginine protein kinase (SRPK) in leishmania braziliensis and its potential as therapeutic target

Leishmaniasis is a parasitic disease found in tropical and subtropical regions around the world, caused by parasites of the genus Leishmania. The disease is a public health concern and presents clinical manifestations that can cause death, disability, and mutilation. The parasite has promastigote (vector) and amastigote (vertebrate host) forms and kinase enzymes are involved in this differentiation process. In the present investigation, we show, for the first time, evidence of a serine/arginine protein kinase in Leshmania braziliensis (LbSRPK). Our results show that amastigotes express more LbSRPK than promastigotes. Analogues of SRPIN340 (a known inhibitor of SRPK) were evaluated for their leishmanicidal activity and two of them, namely SRVIC22 and SRVIC32 showed important leishmanicidal activity in vitro. SRVIC22 and SRVIC32 were able to reduce the infection rate in macrophages and the number of intracellular amastigotes by 55 and 60%, respectively. Bioinformatics analysis revealed the existence of two different amino acid residues in the active site of LbSRPK compared to their human homologue (Tyr/Leu-and Ser/Tyr), which could explain the absence of leishmanicidal activity of SRPIN340 on infected macrophages. In order to enhance leishmanicidal activity of the analogues, optimizations were proposed in the structures of the ligands, suggesting strong interactions with the catalytic site of LbSRPK. Although the evidence on the action of inhibitors upon LbSRPK is only indirect, our studies not only reveal, for the first time, evidence of a SRPK in Leishmania, but also shed light on a new therapeutic target for drug development.

In vitro Investigation of Rutin-Loaded PLGA Nanoparticles on Leishmania infantum Promastigotes

Leishmaniasis is a group of illnesses occasioned Leishmania (L.) parasites transmitted by the bites of infected female Phlebotominae class flies and it is endemic in 102 countries. It is seen worldwide, particularly in developing countries. In the present study, the antileishmanial efficacy of free rutin and nanoparticles formed by encapsulating flavonoid rutin in a polymer nanoparticle system on Leishmania infantum promastigotes were contrasted. The efficacy of rutin-loaded PLGA nanoparticles (RT)NPs on the proliferation of promastigote form of L. infantum parasites was examined for the first time by counting the in vitro antileishmanial activities of (RT)NPs using the MTT assay and counting on the thoma slide. It has been observed that (RT)NPs significant affect the proliferation of parasites at concentrations of 1000, 750, and 500 μg/ml at 72nd and 96th hours. The viability% value decreased 10-fold at 1000 μg/ml concentration of (RT)NPs. While the IC50 value of promastigote form of L. infantum parasites was 29.2 ± 4.5 μg/ml in the specimens treated with RT at varied concentrations, the IC50 value of promastigote form of L. infantum parasites was found to be 23.0 ± 2.7 µg/ml in the specimens treated with (RT)NPs. It was observed that the absorbance measurements of (RT)NPs were lower compared to RT at concentrations of 1000, 750, and 500 μg/ml at 48th hour.

In vitro anti-Leishmania activity of new isomeric cobalt(II)complexes and in silico insights: Mitochondria impairment and apoptosis-like cell death of the parasite

The synthesis, physico-chemical characterization and in vitro antiproliferative activity against the promastigote form of Leishmania amazonensis of two new cobalt(II) coordination compounds (i.e. [Co(HL1)Cl2]0.4,2H2O (1) and [Co(HL2)(Cl)(CH3OH)](ClO4).2H2O (2)) are reported, where HL1 = 4-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one and HL2 = 7-{3-[bis(pyridin-2-ylmethyl)amino]-2-hydroxypropoxy}-2H-chromen-2-one. X-ray diffraction studies were performed for complex (2) and the structure of complex (1) was built through Density Functional Theory (DFT) calculations. Complex (1) presented no cytotoxicity to LLC-MK2, but complex (2) was toxic. IC50 against promastigotes of L. amazonensis for complex (1) were 4.90 (24 h), 3.50 (48 h) and 3. 80 μmol L−1 (72 h), and for complex (2) were 2.09, 4.20 and 2.80 μmol L−1, respectively. Due to the high toxicity presented by complex (2) against LLC-MK2 host cells, mechanistic studies, to shed light on the probable mode of leishmanicidal activity, were carried out only for the non-cytotoxic complex. Complex (1) was able to elevate mitochondrial membrane potential of the parasites after treatment. Transmission electron microscopy revealed typical apoptotic condensation of chromatin, altered kinetoplast and mitochondria structures, suggesting that apoptosis-like cell death of the protozoa is probably mediated by an apoptotic mechanism associated with mitochondrial dysfunction (intrinsic pathway). Molecular docking studies with complex (1) upon protein tyrosine phosphatase (LmPRL-1) suggests a plausible positive complex anchoring mainly by hydrophobic and hydrogen bond forces close to the enzyme's catalytic site. These promising results for complex 1 will prompt future investigations against amastigote form of L. amazonensis.

In vitro and in vivo study on antiprotozoal activity of calcium oxide (CaO) and magnesium oxide (MgO) nanoparticles on promastigote and amastigote forms of Leishmania major

Background: Currently, anti-leishmanial drugs have been developed. However, the available compounds have several side effects such as drug resistance and toxicity that cause some limitation for use. The development of nanoparticles (NPs) use in biological research and the proven effectiveness of CaONPs and MgONPs on bacteria and fungi, along with the lack of information about its antileishmanial effects, have motivated this study. CaO and MgONPs possess considerable antibacterial effects because of their alkalinity and active oxygen species. This study has taken into account the impacts of these two NPs on the L. major in vitro and in vivo. Methods: To evaluate the antileishmanial activity of NPs, the cytotoxic effect of CaONPs, MgONPs, and MgOCaONPs against L. major amastigotes, promastigotes, as well as macrophages, was evaluated using counting or MTT assay. The possible apoptosis of L. major by CaONPs, MgONPs, and MgOCaONPs was evaluated via flow cytometry assay. For in vivo study, BALB/c mice were allocated to five groups and the lesions of infected mice with L. major promastigotes were treated with a 200 μg/mL concentration CaONPs, MgONPs, and MgOCaONPs, then the mice underwent a 4-week follow-up to examine the wound diameter and survival rates. Results: The XRD-pattern related to CaONPs and MgONPs indicating the cubic phase and Rocksalt cubic structures. According the effects of nanoparticle on promastigotes the IC50 values of CaONPs, MgONPs, and MgOCaONPs within 72 h were 7.9 ug/mL, 10.3 ug/mL, and 8.0 ug/mL respectively. CaONPs, MgONPs, and MgOCaONPs induced apoptosis in about 7.8%, 53.57%, and 12.8% of promastigotes. All mice presented lesions. MgONPs was the most effective in reducing the size of the lesions. Conclusion: According to the results of the present research, MgONPs and CaONPs showed good in vitro and in vivo effects on L. major promastigotes and intracellular amastigotes especially MgONPs, and also it seems that MgONPs are applicable in Leishmania infection treatment due to their potential antileishmanial effects.

Eugenia gracillima essential oil has pharmaceutical applications in pain and inflammation without toxic effects in mice

Ethnopharmacological relevanceEugenia gracillima is widely used by the population in the manufacture of pulps and jellies, with popular reports of its use in the treatment of infections in the urinary system, respiratory and dermatological problems. A previous study reports that EO from E. gracillima leaves proved to be a promising antioxidant agent in combating the promastigote forms of protozoa. Despite this, this species has been little studied due to its pharmacological properties. Study objectiveIn this study, an essential oil extracted (EO) from Eugenia gracillima leaves was evaluated for its acute toxicity and anti-inflammatory, antinociceptive and behavioral effects in mice. MethodsThe EO was obtained by hydrodistillation, and the composition analysis was performed by gas chromatography coupled to mass spectrometry. Acute toxicity assessment was performed with observation of hematological parameters and histopathological evaluation, as well as tests to investigate antinociceptive, anti-inflammatory activities and behavioral effects. ResultsChromatographic analysis showed D-germacrene (16.10%), γ-muurolene-g (15.60%) and bicyclogermacrene (8.53%) as the majority of compounds. In the toxicity evaluation, no death or physiological changes were observed in mice treated with a single oral dose of up to 5000 mg/kg, and it did not lyse erythrocytes in vitro. The hematological parameters evaluated were not changed after treatment; however, 5,000 mg/kg promoted an increase in transaminase levels. In the histopathological evaluation, only the animals that received the dose of 5000 mg/kg showed discrete leukocyte infiltration around the centrilobular vein in the liver. Antinociceptive activity was detected through tests of acetic acid-induced writhing, formalin, and tail flick, promoted in part by the opioid receptor pathway. In the evaluation of anti-inflammatory activity, a reduction in inflammation was observed in the paw edema test and a decrease in the migration of leukocytes and neutrophils in the peritonitis test. The open field and elevated plus maze tests showed that EO did not affect the animals' motor functions or exploratory activity. ConclusionIt was concluded that the essential oil of E. gracillima has potential for the development of pharmaceutical formulations with analgesic and anti-inflammatory actions in non-toxic concentrations.

New antileishmanial quinoline linked isatin derivatives targeting DHFR-TS and PTR1: Design, synthesis, and molecular modeling studies

In a search for new drug candidates for one of the neglected tropical diseases, leishmaniasis, twenty quinoline-isatin hybrids were synthesized and tested for their in vitro antileishmanial activity against Leishmaniamajor strain. All the synthesized compounds showed promising in vitro activity against the promastigote form in a low micromolar range (IC50 = 0.5084–5.9486 μM) superior to the reference miltefosine (IC50 = 7.8976 μM). All the target compounds were then tested against the intracellular amastigote form and showed promising inhibition effects (IC50 = 0.60442–8.2948 μM versus 8.08 μM for miltefosine). Compounds 4e, 4b and 4f were shown to possess the highest antileishmanial activity against both promastigote and amastigote forms. The most active compounds were proven to exhibit their significant antileishmanial effects through antifolate mechanism, targeting DHFR-TS and PTR1. To evaluate the safety profile of the most active derivatives 4e, 4b and 4f, the in vitro cytotoxicity test was carried out and displayed higher selectivity indices than the reference miltefosine. Molecular docking within putative target protein PTR1 confirmed the high potentiality of the most active compounds 4e, 4b and 4f to block the catalytic activity of Lm-PTR1.

Design, Synthesis, and Antiprotozoal Evaluation of New Promising 2,9-Bis[(substituted-aminomethyl)]-4,7-phenyl-1,10-phenanthroline Derivatives, a Potential Alternative Scaffold to Drug Efflux.

A series of novel 2,9-bis[(substituted-aminomethyl)]-4,7-phenyl-1,10-phenanthroline derivatives was designed, synthesized, and evaluated in vitro against three protozoan parasites (Plasmodium falciparum, Leishmania donovani and Trypanosoma brucei brucei). Pharmacological results showed antiprotozoal activity with IC50 values in the sub and μM range. In addition, the in vitro cytotoxicity of these original molecules was assessed with human HepG2 cells. The substituted diphenylphenanthroline 1l was identified as the most potent antimalarial derivative with a ratio of cytotoxic to antiparasitic activities of 505.7 against the P. falciparum CQ-resistant strain W2. Against the promastigote forms of L. donovani, the phenanthrolines 1h, 1j, 1n and 1o were the most active with IC50 from 2.52 to 4.50 μM. The phenanthroline derivative 1o was also identified as the most potent trypanosomal candidate with a selectivity index (SI) of 91 on T. brucei brucei strain. FRET melting and native mass spectrometry experiments evidenced that the nitrogen heterocyclic derivatives bind the telomeric G-quadruplexes of P. falciparum and Trypanosoma. Moreover, as the telomeres of the parasites P. falciparum and Trypanosoma could be considered to be possible targets of this kind of nitrogen heterocyclic derivatives, their potential ability to stabilize the parasitic telomeric G-quadruplexes have been determined through the FRET melting assay and by native mass spectrometry.