Monomethylsulochrin isolated from biomass extract of Aspergillus sp. against Leishmania amazonensis: In vitro biological evaluation and molecular docking.

Leishmaniasis represents a serious world health problem, with 1 billion people being exposed to infection and a broad spectrum of clinical manifestations with a potentially fatal outcome. Based on the limitations observed in the treatment of leishmaniasis, such as high cost, significant adverse effects, and the potential for drug resistance, the aim of the present study was to evaluate the leishmanicidal activity of the compounds pseurotin A and monomethylsulochrin isolated from the biomass extract of Aspergillus sp. The chromatographic profiles of the extract were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV), and the molecular identification of the pseurotin A and monomethylsulochrin were carried out by electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS) and nuclear magnetic resonance (NMR). Antileishmanial activity was assayed against promastigote and intracellular amastigote of Leishmania amazonensis. As a control, cytotoxicity assays were performed in non-infected BALB/c peritoneal macrophages. Ultrastructural alterations in parasites were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential were determined by flow cytometry. Only monomethylsulochrin inhibited the promastigote growth (IC50 18.04 ± 1.11 µM), with cytotoxicity to peritoneal macrophages (CC50 5.09 91.63 ± 1.28 µM). Activity against intracellular amastigote forms (IC50 5.09 ± 1.06 µM) revealed an increase in antileishmanial activity when compared with promastigotes. In addition to a statistically significant reduction in the evaluated infection parameters, monomethylsulochrin altered the ultrastructure of the promastigote forms with atypical vacuoles, electron-dense corpuscles in the cytoplasm, changes at the mitochondria outer membrane and abnormal disposition around the kinetoplast. It was showed that monomethylsulochrin leads to a decrease in the mitochondrial membrane potential (25.9%, p = 0.0286). Molecular modeling studies revealed that monomethylsulochrin can act as inhibitor of sterol 14-alpha-demethylase (CYP51), a therapeutic target for human trypanosomiasis and leishmaniasis. Assessed for its drug likeness, monomethylsulochrin follows the Lipinski Rule of five and Ghose, Veber, Egan, and Muegge criteria. Furthermore, monomethylsulochrin can be used as a reference in the development of novel and therapeutically useful antileishmanial agents.

Carajurin: a anthocyanidin from Arrabidaea chica as a potential biological marker of antileishmanial activity.

Leishmaniasis is a group of neglected tropical diseases whose treatment with antimonials bears limitations and has changed little in over 80 years. Medicinal plants have been evaluated as a therapeutic alternative for leishmaniasis. Arrabidaea chica is popularly used as a wound healing and antiparasitic agent, especially as leishmanicidal agent. This study examined the leishmanicidal activity of a crude extract (ACCE), an anthocyanidin-rich fraction (ACAF), and three isolated anthocyanidins from A. chica: carajurin, 3'-hydroxy-carajurone, and carajurone. We evaluated the antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis and determined cytotoxicity in BALB/c peritoneal macrophages, as well as nitrite quantification, using the Griess method. Molecular docking was carried out to evaluate interactions of carajurin at the nitric oxide synthase enzyme. All compounds were active against promastigotes after 72 h, with IC50 values of 101.5 ± 0.06 µg/mL for ACCE and 4.976 ± 1.09 µg/mL for ACAF. Anthocyanidins carajurin, 3'-hydroxy-carajurone, and carajurone had IC50 values of 3.66 ± 1.16, 22.70 ± 1.20, and 28.28 ± 0.07 µg/mL, respectively. The cytotoxicity assay after 72 h showed results ranging from 9.640 to 66.74 µg/mL for anthocyanidins. ACAF and carajurin showed selectivity against intracellular amastigote forms (SI> 10), with low cytotoxicity within 24 h, a statistically significant reduction in all infection parameters, and induced nitrite production. Molecular docking studies were developed to understand a possible mechanism of activation of the nitric oxide synthase enzyme, which leads to an increase in the production of nitric oxide observed in the other experiments reported. These results encourage us to suggest carajurin as a biological marker of A. chica.

GC-MS Characterization of Antibacterial, Antioxidant, and Antitrypanosomal Activity of Syzygium aromaticum Essential Oil and Eugenol.

Syzygium aromaticum has a diversity of biological activities due to the chemical compounds found in its plant products such as total phenolic compounds and flavonoids. The present work describes the chemical analysis and antimicrobial, antioxidant, and antitrypanosomal activity of the essential oil of S. aromaticum. Eugenol (53.23%) as the major compound was verified by gas chromatography-mass spectrometry. S. aromaticum essential oil was more effective against S. aureus (MIC 50 µg/mL) than eugenol (MIC 250 µg/mL). Eugenol presented higher antioxidant activity than S. aromaticum essential oil, with an EC50 of 12.66 and 78.98 µg/mL, respectively. S. aromaticum essential oil and eugenol exhibited Trypanosoma cruzi inhibitory activity, with IC50 of 28.68 ± 1.073 and 31.97 ± 1.061 µg/mL against epimastigotes and IC50 of 64.51 ± 1.658 and 45.73 ± 1.252 µg/mL against intracellular amastigotes, respectively. Both compounds presented low cytotoxicity, with S. aromaticum essential oil displaying 15.5-fold greater selectivity for the parasite than the cells. Nitrite levels in T. cruzi-stimulated cells were reduced by essential oil (47.01%; p = 0.002) and eugenol (48.05%; p = 0.003) treatment. The trypanocidal activity of S. aromaticum essential oil showed that it is reasonable to use it in future research in the search for new therapeutic alternatives for trypanosomiasis.

Vernonia brasiliana (L.) Druce induces ultrastructural changes and apoptosis-like death of Leishmania infantum promastigotes.

The present study aimed to evaluate the antileishmanial effect, the mechanisms of action and the association with miltefosine of Vernonia brasiliana essential oil against Leishmania infantum promastigotes. This essential oil was obtained by hydrodistillation and its chemical composition was determined by gas chromatography-mass spectrometry (GC-MS). The antileishmanial activity against L. infantum promastigotes and cytotoxicity on DH82 cells were evaluated by MTT colorimetric assay. Ultrastructural alterations were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential, in the production of reactive oxygen species, and analysis of apoptotic events were determined by flow cytometry. The association between the essential oil and miltefosine was evaluated using the modified isobologram method. The most abundant component of the essential oil was ß-caryophyllene (21.47 %). Anti-Leishmania assays indicated an IC50 of 39.01 ±â€¯1.080 µg/mL for promastigote forms after 72 h of treatment. The cytotoxic concentration for DH82 cells was 63.13 ±â€¯1.211 µg/mL after 24 h of treatment. The effect against L. infantum was proven through the ultrastructural changes caused by the oil, such as kinetoplast and mitochondrial swelling, vesicles in the flagellar pocket, discontinuity of the nuclear membrane, nuclear fragmentation and condensation, and loss of organelles. It was observed that the oil leads to a decrease in the mitochondrial membrane potential (35.10 %, p = 0.0031), increased reactive oxygen species production, and cell death by late apoptosis (17.60 %, p = 0.020). The combination of the essential oil and miltefosine exhibited an antagonistic effect. This study evidences the antileishmanial action of V. brasiliana essential oil against L. infantum promastigotes.

The Influence of Anthocyanidin Profile on Antileishmanial Activity of Arrabidaea chica Morphotypes.

Arrabidaea chica Verlot (crajiru) is a plant used in folk medicine as an astringent, anti-inflammatory, wound healing and to treat fungal and viral diseases such as measles chickenpox and herpes. Arrabidaea chica has several morphotypes recognized but little is known about its chemical variability. In the present study the anthocyanidin profile of A. chica morphotypes collected in two seasons (summer and winter) have been examined and their activity against Leishmania infection compared. High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD-UV) and by tandem mass spectrometry with electrospray ionization (ESI-MS/MS) were used for anthocyanidin separation and identification. Antileishmanial activity was measured against promastigote forms of Leishmania amazonensis. Multivariate analysis, principal component analysis (PCA) and Pearson's correlation were performed to classify morphotypes accordingly to their anthocyanidin profile. The presence of 6,7,3',4'-tetrahydroxy-5-methoxyflavylium (3'-hydroxy-carajurone) (1), carajurone (2), 6,7,3'-trihydroxy-5,4'-dimethoxy-flavylium (3'-hydroxy-carajurin) (3) and carajurin (4), and three unidentified anthocyanidins were detected. Two different groups were recognized: group I containing 3'-hydroxy-carajurone; and group II with high content of carajurin. Among anthocyanidins identified in the extracts, only carajurin showed significant statistical correlation (p = 0.030) with activity against L. amazonensis. Carajurin could thus be considered as a pharmacological marker for the antileishmanial potential of the species.

Aniba rosaeodora (Var. amazonica Ducke) Essential Oil: Chemical Composition, Antibacterial, Antioxidant and Antitrypanosomal Activity.

Aniba rosaeodora is one of the most widely used plants in the perfumery industry, being used as medicinal plant in the Brazilian Amazon. This work aimed to evaluate the chemical composition of A. rosaeodora essential oil and its biological activities. A. rosaeodora essential oil presented linalool (93.60%) as its major compound. The A. rosaeodora essential oil and linalool showed activity against all the bacteria strains tested, standard strains and marine environment bacteria, with the lower minimum inhibitory concentration being observed for S. aureus. An efficient antioxidant activity of A. rosaeodora essential oil and linalool (EC50: 15.46 and 6.78 µg/mL, respectively) was evidenced by the inhibition of the 2,2-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical. The antitrypanosomal activity of A. rosaeodora essential oil and linalool was observed at high concentrations against epimatigote forms (inhibitory concentration for 50% of parasites (IC50): 150.5 ± 1.08 and 198.6 ± 1.12 µg/mL, respectively), and even higher against intracellular amastigotes of T. cruzi (IC50: 911.6 ± 1.15 and 249.6 ± 1.18 µg/mL, respectively). Both A. rosaeodora essential oil and linalool did not exhibit a cytotoxic effect in BALB/c peritoneal macrophages, and both reduced nitrite levels in unstimulated cells revealing a potential effect in NO production. These data revealed the pharmacological potential of A. rosaeodora essential oil and linalool, encouraging further studies.

Vernonia polysphaera Baker: Anti-inflammatory activity in vivo and inhibitory effect in LPS-stimulated RAW 264.7 cells.

Species of the Vernonia genius are widely distributed across the world. In traditional communities, they are commonly used in popular medicine for the treatment of inflammatory diseases. The objective of the present study was to evaluate the anti-inflammatory activity of Vernonia polysphaera Baker hydroalcoholic extract. A λ-carrageenan-induced paw edema and peritonitis model was established in BALB/c mice. The in vitro activity of the extract was measured on LPS-stimulated RAW 264.7 cells. There was no toxic effect on mice or on the cells treated with the extract. Animals treated with V. polysphaera extract demonstrated inhibition of paw edema in comparison with the untreated animals at all the analyzed doses. In peritonitis, treatment with the extract at a dose of 500 mg/kg resulted in a lower total leukocyte count in the peritoneal fluid and blood and lower levels of IL-1ß, IL-6, TNF-α and PGE-2 than the control group. Cells treated with 50 and 100 µg/mL of the extract exhibited lower levels of nitrite and pro-inflammatory cytokine production and lower COX-2, NF-κB expression. The V. polysphaera extract demonstrated an anti-inflammatory effect, interfering with cell migration, reducing pro-inflammatory cytokine levels and COX-2 expression and consequent interference with PGE-2, as well as inhibiting NF-κB transcription.

Endlicheria bracteolata (Meisn.) Essential Oil as a Weapon Against Leishmania amazonensis: In Vitro Assay.

The difficulties encountered and the numerous side effects present in the treatment of cutaneous leishmaniasis have encouraged the research for new compounds that can complement or replace existing treatment. The growing scientific interest in the study of plants, which are already used in folk remedies, has led our group to test Endlicheria bracteolata essential oil against Leishmania amazonensis. Several species of the Lauraceae family, or their compounds, have relevant antiprotozoal activities Therefore, the biological potential on L. amazonensis forms from the essential oil of Endlicheria bracteolata leaves was verified for the first time in that work. The antileishmanial activity was evaluated against promastigotes and intracellular amastigotes, and cytotoxicity were performed with J774.G8, which were incubated with different concentrations of E. bracteolata essential oil. Transmission electron microscopy and flow cytometry were performed with E. bracteolata essential oil IC50. Promastigote forms showed E. bracteolata essential oil IC50 of 7.945 ± 1.285 µg/mL (24 h) and 6.186 ± 1.226 µg/mL (48 h), while for intracellular amastigote forms it was 3.546 ± 1.184 µg/mL (24 h). The CC50 was 15.14 ± 0.090 µg/mL showing that E. bracteolata essential oil is less toxic to macrophages than to parasites. Transmission electron microscopy showed that E. bracteolata essential oil treatment is capable of inducing mitochondrial damage to promastigote and intracellular amastigote forms, while flow cytometry showed ΔÑ°m disruption in treated parasites. These results could bring about new possibilities to develop products based on E. bracteolata essential oil to treat cutaneous leishmaniasis, especially for people who cannot receive the conventional therapy.

Cinnamomum zeylanicum, Origanum vulgare, and Curcuma longa Essential Oils: Chemical Composition, Antimicrobial and Antileishmanial Activity.

The resistance mechanisms of bacteria and protozoans have evidenced the need of discover new compounds with potential pharmaceutical activity against pathogenic microorganisms. Medicinal plants have been for centuries a promising alternative as sources of new drugs. The objective of this work was to evaluate the chemical composition, antimicrobial and antileishmanial activities of Cinnamomum zeylanicum, Origanum vulgare, and Curcuma longa essential oils. Chemical analysis was performed by gas chromatography-mass spectrometry. Antimicrobial activity was performed by disk diffusion and minimum inhibitory concentration (MIC) test. Antileishmanial activity was performed against antipromastigote and intracellular amastigote of Leishmania amazonensis. Cytotoxic and nitrite production were realized in BALB/c peritoneal macrophages. The major compounds of the essential oils were cinnamic aldehyde (46.30%) in C. zeylanicum, cis-p-menth-2-en-1-ol (33.88%) and linalyl acetate (13.90%) in O. vulgare, and turmerone (55.43%) in C. longa. The MIC showed significant antimicrobial activity of C. longa essential oil against S. aureus (83.3 ± 14.43 µg/mL). Antipromastigote activity showed IC50 values >500 µg/mL to C. zeylanicum, 308.4 ± 1.402 µg/mL to O. vulgare, and 405.5 ± 1.119 µg/mL to C. longa essential oil. Activity against intracellular amastigote of L. amazonensis showed IC50 of 63.3 ± 1.369 µg/mL and cytotoxic was not observed, resulting in selectivity index higher than 15.79 to parasite. C. longa essential oil decreased nitrite production in peritoneal macrophages, but not in Leishmania-infected cells. The chemical composition of the three essential oils is directly associated to its potential biological action, as the antimicrobial activity. C. longa presented a potent antileishmanial activity against promastigote and intracellular amastigote of L. amazonensis, although this activity is not linked to nitric oxide, since C. longa essential oil inhibits its production.

Carapa guianensis Aublet (Andiroba) Seed Oil: Chemical Composition and Antileishmanial Activity of Limonoid-Rich Fractions.

Leishmaniasis is a complex of diseases caused by protozoa of the genus Leishmania and affects millions of people around the world. Several species of plants are used by traditional communities for the treatment of this disease, among which is Carapa guianensis Aubl. (Meliaceae), popularly known as andiroba. The objective of the present work was to conduct a chemical study of C. guianensis seed oil and its limonoid-rich fractions, with the aim of identifying its secondary metabolites, particularly the limonoids, in addition to investigating its anti-Leishmania potential. The chemical analyses of the C. guianensis seed oil and fractions were obtained by electrospray ionization mass spectrometry (ESI-MS). The cytotoxic activity was tested against peritoneal macrophages, and antileishmanial activity was evaluated against promastigotes and intracellular amastigotes of Leishmania amazonensis. All the C. guianensis seed oil samples analyzed exhibited the same pattern of fatty acids, while the limonoids 7-deacetoxy-7-hydroxygedunin, deacetyldihydrogedunin, deoxygedunin, andirobin, gedunin, 11ß-hydroxygedunin, 17-glycolyldeoxygedunin, 6α-acetoxygedunin, and 6α,11ß-diacetoxygedunin were identified in the limonoid-rich fractions of the oil. The C. guianensis seed oil did not exhibit antileishmanial activity, and cytotoxicity was higher than 1000 µg/mL. Three limonoid-rich oil fractions demonstrated activity against promastigotes (IC50 of 10.53±0.050, 25.3±0.057, and 56.9±0.043µg/mL) and intracellular amastigotes (IC50 of 27.31±0.091, 78.42±0.086, and 352.2±0.145 µg/mL) of L. amazonensis, as well as cytotoxicity against peritoneal macrophages (CC50 of 78.55±1.406, 139.0±1.523, and 607.7±1.217 µg/mL). The anti-Leishmania activity of the limonoid-rich fractions of C. guianensis can be attributed to the limonoids 11ß-hydroxygedunin and 6α,11ß-diacetoxygedunin detected in the chemical analysis.

In vitro activity of Morinda citrifolia Linn. fruit juice against the axenic amastigote form of Leishmania amazonensis and its hydrogen peroxide induction capacity in BALB/c peritoneal macrophages.

OBJECTIVE: The current treatment of leishmaniasis induces strong side effects and increasing numbers of cases of resistance to reference drugs have been reported. The discovery of the therapeutic properties of active substances in plant extracts represents an interesting field of research into a more efficient treatment against leishmaniasis. Morinda citrifolia, commonly known as noni, has demonstrated promising results as antileishmanial and immunomodulator. Thus, the aim of this work was to evaluate activity against axenic amastigote and hydrogen peroxide induction capacity by M. citrifolia fruit juice. RESULTS: Phytochemical screening identified anthraquinones, flavonoids, alkaloids, terpenoids, steroids, saponins, coumarins, phenolic compounds, tannins, anthocyanidins and chalcones. Noni juice exhibited dose-dependent activity and an IC50 of 240.1 µg/mL for axenic amastigotes. An absence of endotoxins was observed at the concentrations analyzed, while no cytotoxic effects were identified. Noni juice induced hydrogen peroxide production in BALB/c peritoneal macrophages but not in macrophages infected with Leishmania amazonensis. M. citrifolia fruit juice exhibited antileishmanial activity against L. amazonensis axenic amastigotes and activated macrophages by hydrogen peroxide induction, asserting its potential for further research into new forms of leishmaniasis treatment.

Morinda citrifolia Linn. fruit (Noni) juice induces an increase in NO production and death of Leishmania amazonensis amastigotes in peritoneal macrophages from BALB/c.

Leishmaniasis is a complex disease that is considered a serious public health problem. Due to the absence of an effective vaccine and debilitating chemotherapy better therapies are urgently needed. This situation has stimulated the search for alternative treatments such as the use of herbal medicines. Several studies conducted with Morinda citrifolia Linn. have shown various biological activities such as antitumor, immunomodulation and antileishmanial activity, however its mechanisms of action are still unknown. This study aimed to analyze the activity of M. citrifolia fruit juice against Leishmania amazonensis and its action on peritoneal macrophages from BALB/c infected with L. amazonensis. Activity against the promastigote forms showed IC50 at 275.3 µg/mL. Transmission electron microscopy was used to evaluate the ultrastructural alterations in the promastigotes treated with the juice and the results showed cytoplasmic vacuolization, lipid inclusion and increased activity of exocytosis. The juice treatment presented an IC50 at 208.4 µg/mL against intracellular amastigotes and led to an increased nitrite production in infected and non-infected macrophages. When macrophages were pre-treated with iNOS inhibitors, aminoguanidine or 1400W, the intracellular amastigotes increased, demonstrating the important role of NO production in M. citrifolia fruit activity. In conclusion, our results reveal that treatment with M. citrifolia fruit juice can increase NO production in peritoneal macrophages and this ability has an important role in the killing of L. amazonensis intracellular amastigotes.

Ultrastructural Changes and Death of Leishmania infantum Promastigotes Induced by Morinda citrifolia Linn. Fruit (Noni) Juice Treatment.

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV. Morinda citrifolia Linn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity of M. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes in Leishmania infantum promastigotes caused by M. citrifolia fruit juice treatment. M. citrifolia fruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50 values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes of L. infantum treated with M. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed that M. citrifolia fruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles in L. infantum promastigotes treated with M. citrifolia fruit juice. M. citrifolia fruit juice was active against L. infantum in the in vitro model used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis.

Leishmanicidal, cytotoxicity and wound healing potential of Arrabidaea chica Verlot.

BACKGROUND: Leishmaniasis includes a wide complex of diseases that affect humans and other mammals, and can range from a mild cutaneous form to a severe visceral type. The safety of the standard treatment using pentavalent antimony is a concern due to its toxic effects. The search for alternative, effective and less toxic treatments has led to the testing of natural products. The present study aimed to evaluate the cytotoxic, leishmanicidal and healing potential of Arrabidaea chica. METHODS: The crude ethanolic extract, as well as the chloroform, methanol and ethyl acetate fractions of A. chica were prepared and phytochemical analysis was performed. Cytotoxic evaluation was carried out through MTT colorimetric assay, and the 50% cellular cytotoxicity was determined. After that, the effect of the extract and fractions against Leishmania amazonensis promastigotes, at intervals of 24, 48 and 72 h, was analyzed, and 50% inhibitory concentration was determined. The healing effect of the plant was also tested in surgical lesions in Swiss mice skin. RESULTS: Phytochemical screening showed that the crude extracts contained flavonoids, tannins, anthocyanidins and chalcones. The leishmanicidal potential of A. chica produced satisfactory results in concentrations of between 60 and 155.9 µg/mL. Cytotoxic assay revealed a 50% reduction in viable cells at a concentration of 189.9 µg/mL. The healing results indicated that the treated group exhibited more pronounced signs of lesion resolution in the early period, but this pattern did not persist throughout the treatment. CONCLUSIONS: The results of the present study demonstrate that A. chica has cytotoxic and leishmanicidal potential but its healing effect must be better studied.

In Vitro Antileishmanial Activity of Essential Oil of Vanillosmopsis arborea (Asteraceae) Baker.

The search for new immunopharmacological chemical agents to treat various diseases caused by bacteria, fungi, and protozoa, such as leishmaniasis, for example, has led to the exploration of potential products from plant species and their main active ingredients. Antimonial drugs are the current treatment for leishmaniasis. These drugs cause major side effects and frequent discontinuation of treatment. In this study, we evaluated the in vitro leishmanicidal activity of essential oil of Vanillosmopsis arborea (VAEO) and its major compound α -bisabolol against Leishmania amazonensis. The essential oil and α -bisabolol showed activity against promastigotes (IC50 7.35 and 4.95 µ g/mL resp.) and intracellular amastigotes (IC50 12.58 and 10.70 µ g/mL, resp.). Neither product showed any cytotoxicity on treated macrophages. The ultrastructural analysis of promastigotes incubated with VAEO or α -bisabolol at 30 µ g/mL, showed morphological changes with the accumulation of vesicles electrodense lipid inclusions. The results give evidence that both VAEO and α -bisabolol have potential as new therapeutic agents against leishmaniasis.