Detergent-free parasite transformation and replication assay for drug screening against intracellular Leishmania amastigotes.

Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse effects, variable efficacy, the development of resistant parasites, high cost, parenteral administration and lack of availability in endemic areas. Therefore, active searching for new antileishmanial drugs has been done for years, mainly by academia. Drug screening techniques have been a challenge since the intracellular localization of Leishmania amastigotes implies that the host cell may interfere with the quantification of the parasites and the final estimation of the effect. One of the procedures to avoid host cell interference is based on its detergent-mediated lysis and subsequent transformation of viable amastigotes into promastigotes, their proliferation and eventual quantification as an axenic culture of promastigotes. However, the use of detergent involves additional handling of cultures and variability. In the present work, cultures of intracellular amastigotes were incubated for 72 h at 26 °C after exposure to the test compounds and the transformation and proliferation of parasites took place without need of adding any detergent. The assay demonstrated clear differentiation of negative and positive controls (average Z´ = 0.75) and 50% inhibitory concentrations of compounds tested by this method and by the gold standard enumeration of Giemsa-stained cultures were similar (p = 0.5002) and highly correlated (r = 0.9707). This simplified procedure is less labor intensive, the probability of contamination and the experimental error are reduced, and it is appropriate for the automated high throughput screening of compounds.

Anti-Leishmania activity of the Mayan medicinal plant Thouinia paucidentata Radlk extracts.

Mexico's Yucatan Peninsula is an endemic area of cutaneous leishmaniasis, locally known as the chiclero's ulcer, and Mayan traditional medicine which refers to the use of Thouinia paucidentata Radlk, known as k'an chuunup. Aqueous and organic leaves extracts were evaluated against promastigotes and amastigotes of Leishmania mexicana. Toxicity tests of extracts were performed using Vero and J774A.1 macrophage cell lines. The composition of the most active extracts was analysed by GC-MS. The n-hexane and ethyl acetate extracts showed potent anti-Leishmania activity against the promastigote form, and remarkably, n-hexane extract exhibited potent activity against the amastigote form. Both extracts showed low toxicity on Vero both not on J774A.1 cells. Analysis of both bioactive extracts identified as more abundant compounds, germacrene D-4-ol and thunbergen in n-hexane, and thunbergol in ethyl acetate extracts. Our study presents T. paucidentata as anti-Leishmania phytomedicine supporting its medicinal use and contributes to the understanding of its phytochemical composition.

In vitro and in vivo antileishmanial effects of Nectaroscordum koelzi extract against Leishmania major.

Due to the increasing resistance to common medicinal compounds, the use of medicinal plants has received special attention. Therefore, the current survey was designed to study the antileishmanial effects of Nectaroscordum koelzi Trautv. methanolic extract against Leishmania major. In this study, after preparing the methanolic extract of N. koelzi, its effect on the amastigotes of L. major and triggering the nitric oxide (NO) were measured. Then, the in vivo effect of the methanol extract on cutaneous leishmaniasis in mice was evaluated. The best anti-amastigote effect was for the methanol extract of N. koelzi along with meglumine antimony with 50% inhibitory concentrations value of 17.4 µg/ml (p < 0.001). The 50% cytotoxic concentrations values of methanol extract, meglumine antimoniate, and methanol extract + meglumine antimoniate were 596.3, 784.6, and 296.4 µg/ml, respectively. Macrophages treated with the methanolic extract markedly (p < 0.001) induced the release of nitric oxide. After 28 days of treatment, lesions were completely (p < 0.001) healed in mice treated with the methanolic extract (100 mg/kg) + meglumine antimoniate (25 mg/kg). N. koelzi methanolic extract mainly in combination with meglumine antimoniate showed favorable antileishmanial effects on L. major, concluding that the methanolic extract of N. koelzi can be used for the production of new leishmanicidal agents agaist cutaneous leishmaniasis. Although we revealed that NO trigerring and inhibition of infection in host cells are the antileishmanial mechanism action of N. koelzi methanolic extract, more studies must be performed to clear the mechanisms and its safety.

In vitro Anti-Leishmanial Activities of Methanol Extract of Brucea antidysenterica J.F. Mill Seeds and Its Solvent Fractions.

Introduction: Leishmaniasis is one of the neglected tropical diseases, threatening lives of about 350 million people globally. Brucea antidysenterica seeds are used for the treatment of cutaneous leishmaniasis in the traditional medicine in Ethiopia. Objective: This study aimed to evaluate Brucea antidysenterica seeds' anti-leishmanial activity in vitro. Methods: The crude (80% methanol) extract of Brucea antidysenterica seeds and its fractions were evaluated for their anti-leishmanial activities against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania aethiopica, and for their cytotoxic effects against mammalian cells. The quantitative estimations of total phenolic compounds (TPCs), flavonoids (TFCs) and alkaloids (TACs) were determined, spectrophotometrically. Median inhibitory concentration (IC50) and median cytotoxic concentration (CC50) of the extract and its solvent fractions were calculated using GraphPad Prism 9.1.0 computer software. Data was presented as mean ± standard error of the mean (SEM). Results: The crude extract and its hexane, ethyl acetate and butanol fractions showed anti-leishmanial activities, with IC50 values of 4.14-60.12 µg/mL against promastigotes, and 6.16-40.12 µg/mL against amastigotes of both Leishmania species. They showed moderate cytotoxicity against Vero cell lines and peritoneal mice macrophages, with CC50 values of 100-500 µg/mL, but >1600 µg/mL against red blood cells. Selectivity indices ranged from 7.97 to 30.97. The crude extract, and its ethyl acetate and hexane fractions possessed 54.78-127.72 mg of gallic acid equivalent TPC, 18.30-79.21 mg of quercetin equivalent TFC, and 27.62-97.22 mg of atropine equivalent TAC per gram of extracts. Conclusion: The seeds of the plant possessed anti-leishmanial activities against L. aethiopica and L. donovani that might provide a scientific justification for its use in the treatment of leishmaniasis by traditional healers. Future works are recommended to isolate, purify and identify the possible secondary metabolites attributed to the anti-leishmanial activity.

Chemical composition, antileishmanial, and cytotoxic effects Ferula macrecolea essential oil against Leishmania tropica.

Background: The current study was aimed to evaluate the chemical composition, as well as antileishmanial and cytotoxic effects of the essential oil of Ferula macrecolea and its main compound, terpinolene, against promastigotes and amastigotes of Leishmania tropica. Methods: The chemical composition of the essential oil was analyzed by a gas chromatograph connected to a mass spectrometer (GC/MS). The MTT (3-(4.5-dimethylthiazol-2-yl) - 2.5-diphenyl tetrazolium bromide) assay was used to study the effects of the essential oil and terpinolene against promastigotes while the macrophage model was used for evaluating the effect of F. macrecolea essential oil against amastigotes of L. tropica as well as assessing cytotoxicity. The Griess reaction assay was employed to study the nitric oxide (NO) produced by treating macrophage cells with the essential oil and terpinolene. Furthermore, the effect of the essential oil and terpinolene on plasma membrane permeability and inhibition of infection in macrophages was evaluated. Results: The main compounds were terpinolene (77.72%), n-nonanal (4.47%), and linalool (4.35%), respectively. The 50% inhibitory concentrations (IC50) of the essential oil, terpinolene, and glucantime against promastigotes were 27.6, 11.6, and 32.8 µg/mL, respectively; however, their IC50 values against amastigotes were 42.3, 19.6, and 56.9 µg/mL, respectively. The 50% cytotoxic concentrations of the essential oil, terpinolene, and glucantime were 471.3, 207.3, and 1165.3 µg/mL, respectively. The production of NO in macrophage cells after treatment with the essential oil and terpinolene was increased in a dose-dependent manner (p < 0.001). The results revealed that by increasing the concentration of the essential oil and terpinolene, the permeability of the parasites' plasma membrane was significantly changed (p < 0.001). The pre-incubation of Leishmania parasites with F. macrecolea essential oil and terpinolene significantly declined the rate of cell infection by 74.8% and 79.4%, respectively (p < 0.001). Conclusion: The results of the present study indicated that F. macrecolea essential oil, especially its main compound, i.e., terpinolene, has a potent antiparasitic effect on the promastigote and amastigote stages of L. tropica. Considering the advantages of medicinal plant products over their chemical counterparts, it is suggested that in the continuation of this study, the effect of F. macrecolea essential oil, especially terpinolene, on laboratory animals, and in case of high efficiency, in humans be evaluated.

Stimulation of metacyclogenesis in Leishmania (Mundinia) orientalis for mass production of metacyclic promastigotes.

Leishmania (Mundinia) orientalis is a human pathogen causing leishmaniasis and studies on the properties of metacyclic promastigotes, the parasite's infective stage, are required for a better understanding of its transmission and infection. However, information on cultivation for mass production of L. orientalis metacyclic promastigotes and factors that stimulate their metacyclogenesis is limited. Therefore, the objective of this study was to develop a suitable methodology for generating promastigote cultures containing a high proportion and number of L. orientalis metacyclic promastigotes. Various media, i.e., Schneider's insect medium, Medium 199 and Grace's insect medium, supplemented with various quantities of dithiothreitol, Basal Medium Eagle vitamins, pooled human urine, and fetal bovine serum, were optimized for metacyclogenesis. The results revealed that the optimum culture medium and conditions of those tested were Schneider's insect medium supplemented with 100 µM dithiothreitol, 1% (v/v) Basal Medium Eagle vitamins, 2% (v/v) pooled human urine, and 10% (v/v) fetal bovine serum, pH 5.0 at 26°C. We also demonstrated that L. orientalis metacyclic promastigotes could be purified and enriched by negative selection using peanut lectin. Under these culture conditions, the highest yield of metacyclic promastigotes was obtained with a significantly higher percentage of parasite survival, resistance to complement-mediated lysis, and infection index in THP-1 macrophage cells compared to parasites cultured without media supplements at neutral pH. This is the first report providing a reliable method for mass production of L. orientalis metacyclic promastigotes for in vivo infections and other experimental studies of this emerging parasite in the future.

Screening of medicinal plants unraveled the leishmanicidal credibility of Garcinia cowa; highlighting Norcowanin, a novel anti-leishmanial phytochemical through in-silico study.

Leishmaniasis, one of the most prevalent yet neglected parasitic causes of death, yearns for therapeutic control and treatment. Severely toxic and inefficient modern-day pentavalent antimonials, caters the search for naturally derived drugs, as efficient alternatives for disease treatment. The anti-promastigote activity of ten different plants selected for their ethnomedicinal properties revealed significant leishmanicidal capacity; the most potent being Garcinia cowa methanolic extract with an IC50 value of 21.4 µg/ml. Garcinia cowa, a plant endemic to North-Eastern India that is of the Clusiaceae family, is replete with such medicinal qualities as antimicrobial, antiviral, antiparasitic, and antiproliferative activities. Computational biology with its tools such as molecular docking has opened new horizons aimed at a better understanding of biological systems, complexes, and their interactions, and subsequently drug discovery via in silico techniques. Therefore, an in-silico study was designed to evaluate the binding capability of six phytochemicals- cowanin, cowanol, cowaxanthone, norcowanin, rubraxanthone, and a basic xanthone, found in Garcinia cowa against Pentamidine, a synthetic anti-leishmanial drug. The active sites of three characteristic enzymes belonging to the Leishmania donovani parasite: O-acetylserine sulfhydrylase (OASS), Trypanothione reductase (TryR), and N-Myristoyltransferase (NMT) were chosen as target proteins. Results revealed lower binding energies and higher affinities, of nearly all the phytochemicals with respect to Pentamidine, indicating their leishmanicidal potential. Norcowanin showed the lowest average binding of - 9.8 kcal/mol against all the three enzymes under study.

Study of the in vitro and in vivo antileishmanial activities of nimodipine in susceptible BALB/c mice.

BACKGROUND & OBJECTIVES: Pentavalent antimonials are the standard treatment for cutaneous leishmaniasis (CL), however, treatment failures are frequent. Nimodipine, a calcium channel blocker is known to show promising antiprotozoal effects. Here, we investigated the antileishmanial effect of Nimodipine in both in vitro and in vivo BALB/c mice model of CL. We also compared the in vivo effect with amphotericin B and meglumine antimoniate in the experimental CL mice model. METHODS: Colorimetric alamar blue assay and J774 A.1 mouse macrophage cells were used to determine the effect of nimodipine on promastigotes and amastigotes viability, respectively. Then, the in vivo activity of nimodipine was compared to that of conventional therapies in both the early and established courses of Leishmania major infection in susceptible non-healing BALB/c mice. RESULTS: Nimodipine was highly active against promastigotes and amastigotes of L. major with IC50 values of 49.40 and 15.03 µM, respectively. In the early model, the combination therapy with meglumine antimoniate and nimodipine showed no parasites in the spleen or footpad of animals. The footpad thickness was significantly lower in mice treated with either nimodipine (1 mg/kg or 2.5 mg/kg) or amphotericin B compared to the control group in the established lesions model. However, no complete remission was observed in the footpad lesion of any of the treatment groups (nimodipine, amphotericin B, meglumine antimoniate, and combination therapy). INTERPRETATION & CONCLUSION: The effect of nimodipine was comparable to that of amphotericin B and meglumine antimoniate in early and established CL lesion models. Since nimodipine is more cost-effective than conventional therapies, our results merit further investigation in other animal models and voluntary human subjects.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis.

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

Detection and characterization of an albumin-like protein in Leishmania donovani.

The protozoan parasite, Leishmania donovani, undergoes several molecular adaptations and secretes many effector molecules for host cell manipulation and successful parasitism. The current study identifies an albumin-like secretory protein, expressed in its extracellular promastigote forms. A leishmanial complementary DNA sequence of a partial gene has been cloned, and the encoded peptide (14 kD) is used for the production of polyclonal antibody. This targeted antibody identifies a large native protein (66.421 kD), expressed stage-specifically in promastigotes. Through electron microscopic studies, the native protein is found to be localized in the flagellar pocket and flagella and at the surface of the promastigotes. This native protein is purified with the same customized antibody for future characterization and sequencing. The sequence analysis reveals its homology with the mammalian serum albumin. It is evidenced from in silico studies that this albumin-like protein remains associated with long-chain fatty acids while in vitro studies indicate its close association with membrane cholesterol. Since antibody-mediated blocking compromises the parasite infectivity, these leishmanial albumin-like molecules are hereby proposed to play an instrumental role in the infectivity of L. donovani to peripheral blood monocyte cells. Thus, identification and characterization of an albumin-like protein in L. donovani promastigotes may be interpreted as a molecular adaptation candidate. It may be hypothesized that the parasite mimics the mammalian system for importing fatty acids into the intracellular amastigotes, facilitating its host cell infectivity.

In vitro evaluation of traditionally used Surinamese medicinal plants for their potential anti-leishmanial efficacy.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant-based preparations are extensively used in Surinamese folk medicine for treating leishmaniasis, but often without a scientific rationale. AIM OF THE STUDY: To evaluate 25 Surinamese medicinal plants for their potential efficacy against leishmaniasis. MATERIALS AND METHODS: Concentrated plant extracts were evaluated for their effect on the viability of L. (V.) guyanensis AMC, L. (L.) major NADIM5, and L. (L.) donovani GEDII promastigotes, as well as intracellular amastigotes of L. (L.) donovani BHU814 in infected THP-1 cells. Selectivity was assessed by cytotoxicity against THP-1 cells. RESULTS: The only plant extract that showed potentially meaningful anti-leishmanial activity was that from Solanum lycocarpum that displayed mean IC50 values of about 51, 61, and <16 µg/mL against L. (V) guyanensis, L. (L) major, and L. (L) donovani promastigotes, respectively; about 374 µg/mL against L. (L) donovani amastigotes; and >500 µg/mL against THP-1 cells. The Bryophyllum pinnatum, Inga alba, and Quassia amara extracts displayed moderate to high IC50 values against promastigotes (about 51 to >500 µg/mL) and/or amastigotes (about 224 to >500 µg/mL) but were relatively toxic to THP-1 cells (IC50 values <16 to about 42 µg/mL). The remaining plant extracts exhibited in many cases IC50 values close to, around, or above 500µg/mL against promastigotes, amastigotes, and THP-1 cells. CONCLUSIONS: The S. lycocarpum preparation may be useful against leishmaniasis and may have a good safety index, warranting further investigations into its active constituents and mechanism(s) of action.

Antileishmanial and cytotoxic effects of essential oil and methanolic extract of Myrtus communis L.

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were α-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 µg/ml against promastigotes, respectively. These values were 11.6 and 40.8 µg/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 µg/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves.

AIM: The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study. METHOD: In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope. RESULTS: The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 µgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine. CONCLUSION: This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis.

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves / 中国天然药物

AIM@#The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study.@*METHOD@#In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope.@*RESULTS@#The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 μgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine.@*CONCLUSION@#This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis.

Atividade antiparasitária in vitro e citotóxica de cariofileno e eugenol contra Trypanossoma cruzi e Leishmania brasiliensis / Actividad antiparasitaria in vitro citotóxica de cariofileno y eugenol contra Trypanosoma cruzi y Leishmania brasiliensis / In vitro cytotoxic and antiparasitic activity of caryophyllene and eugenol against Trypanosoma cruzi and Leishmania brasiliensis

Introdução: as doenças negligenciadas persistem por conta de falhas da ciência e acometem principalmente países em desenvolvimento, como exemplos podemos citar a doença de Chagas e a leishmaniose. Objetivo: avaliar o potencial antiparasitário in vitro de um terpenóide componente de óleo essencial, o cariofileno e o eugenol, contra as formas epimastigota e promastigota de T. cruzi e L. brasiliensis, respectivamente, bem como verificar sua citotoxicidade em células de mamíferos. Métodos: para os estudos in vitro de T. cruzi, foi usado o clone B5-CL, estavelmente transfectadas com o gene de Escherichia coli ß-galactosidase (lacZ). Os ensaios de inibição de promastigotas foram realizadas utilizando a estirpe de L. braziliensis, cultivadas a 22 ºC em meio de Schneider de Drosophila suplementado com FBS a 20 %. Para os testes de atividade antiepimastigota, antipromastigota foram utilizados placas de 96 poços com culturas que não tinham atingido a fase estacionária. Os ensaios de citotoxicidade utilizado estirpe de fibroblastos NCTC929 cultivadas em Meio Essencial Mínimo (Sigma). A viabilidade dessas linhagens através da utilização de resazurina como um método colorimétrico. Resultados: as substâncias cariofileno e eugenol foram testadas quanto à atividade antiepimastigota, antipromastigota e quanto à citotoxidade. Foi visto um efeito clinicamente relevante do cariofileno contra os parasitas T. cruzi e L. brasiliensi. Conclusões: os resultados mostram que o cariofileno obteve um melhor resultado quando comparado ao eugenol, sendo capaz de inibir o crescimento dos parasitas testados mostrando uma alternativa contra T. cruzi e L. brasiliensi. Em relação à citotoxidade novos testes deverão ser realizados para futuros testes in vivo.

Introducción: las enfermedades desatendidas persisten debido a los fracasos de la ciencia y afectan principalmente a los países en desarrollo, como ejemplos se pueden citar la enfermedad de Chagas y la leishmaniasis. Objetivo: evaluar el potencial de un componente in vitro terpenoide antiparasitario aceite esencial, el cariofileno y eugenol contra epimastigotes y promastigotes de Trypanosoma cruzi y Leishmania brasiliensis, respectivamente, así como comprobar su citotoxicidad en células de mamífero. Métodos: para los estudios in vitro de Trypanosoma cruzi, se utilizó el clon B5-CL, transfectadas establemente con el gen de la ß galactosidasa de Escherichia coli-(lacZ). Los ensayos de inhibición se realizaron con la cepa promastigotes de Leishmania braziliensis, se cultivó a 22 °C en medio de Schneider de Drosophila suplementado con FBS 20 %. Para la actividad de las pruebas antiepimastigota y antipromastigota, se utilizaron placas de 96 pocillos con cultivos que no habían alcanzado la fase estacionaria. En los ensayos de citotoxicidad se utilizó NCTC929, cepa de fibroblastos cultivados en medio esencial mínimo (Sigma). La viabilidad de estas cepas se evaluó mediante el uso de la resazurina como un método colorimétrico. Resultados: las sustancias cariofileno y eugenol se ensayaron para determinar la actividad antiepimastigota y antipromastigota, respecto a citotoxicidad. Se observó un efecto clínicamente relevante de cariofileno contra los parásitos Trypanosoma cruzi y Leishmania brasiliensis. Conclusiones: los resultados muestran que con el cariofileno se ha obtenido un mejor resultado en comparación con eugenol, siendo capaz de inhibir el crecimiento de parásitos que presentan una alternativa probada contra Trypanosoma cruzi y Leishmania brasiliensis. En cuanto a las pruebas de citotoxicidad, nuevos estudios deben llevarse a cabo para realizar más pruebas in vivo.

Introduction: neglected diseases continue to exist due to failures in science. They mainly affect developing countries. Two examples of neglected diseases are Chagas' disease and leishmaniasis. Objective: evaluate the potential of an in vitro antiparasitic terpenoid essential oil component, caryophyllene and eugenol, against epimastigotes and promastigotes of Trypanosoma cruzi and Leishmania brasiliensis, respectively, and verify their cytotoxicity in mammal cells. Methods: for in vitro studies of Trypanosoma cruzi, the B5-CL clone was used, stably transfected with the beta-galactosidase gene of Escherichia coli-(lacZ). Inhibition assays were performed on the promastigote strain of Leishmania braziliensis, cultured at 22°C in a Schneider's medium of Drosophila supplemented with 20% FBS. For anti-epimastigote and anti-promastigote activity tests, 96-well plates were used with cultures which had not reached their stationary stage. NCTC929 was used in cytotoxicity tests, a strain of fibroblasts cultured in minimum essential medium (Sigma). The viability of these strains was evaluated using resazurine as colorimetric method. Results: the substances caryophyllene and eugenol were assayed to determine their anti-epimastigote and anti-promastigote activity with respect to cytotoxicity. it was found that caryophyllene has a clinically relevant effect against the parasites Trypanosoma cruzi and Leishmania brasiliensis. Conclusions: results show that caryophyllene was more effective than eugenol, inhibiting the growth of parasites and thus constituting a proven alternative against Trypanosoma cruzi and Leishmania brasiliensis. As to cytotoxicity testing, new studies should be conducted in which more in vivo tests are performed.

Inhibitory Activity of Eleven Artemisia Species from Iran against Leishmania Major Parasites.

OBJECTIVES: Annual incidence of cutaneous leishmaniasis is increasingly growing and development of the alternative drugs against it is a major concern. Artemisia genus is a traditional medicinal plant in Iran. The aim of this study was to examine the leishmanicidal activity of various Iranian Artemisia species extracts. MATERIALS AND METHODS: Different extracts were gathered from eleven Iranian Artemisia species. Their leishmanicidal activities against the growth of Leishmania major (L. major) promastigotes were examined as the half maximal inhibitory concentration (IC50) using MTT assay. RESULTS: Obtained results showed that ethanol extracts especially those taken from A. ciniformis, A. santolina and A. kulbadica have the strongest effects. CONCLUSION: Looking for the effective leishmanicidal agents from natural resources in Iran, we found that the ethanol extract of collected Artemisia species had significant effect on in vitro leishmanicidal activity and may be suitable candidates in the treatment of leishmaniasis.