Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil.

Human tegumentary leishmaniasis (HTL) is a serious tropical disease caused by Leishmania amazonensis. Developing new leishmanicidal agents can help overcome current treatment challenges, such as drug resistance and toxicity. Essential oils are a source of lipophilic substances with diverse therapeutic properties. This study aimed to determine the anti-L. amazonensis activity, cytotoxicity, and chemical profile of Allium sativum essential oil (ASEO). The effect of ASEO on parasite and mammalian cells viability was evaluated using resazurin and MTT assays, respectively. The oil's effect against intracellular amastigotes was also determined. Transmission electron microscopy was used to assess the ultrastructural changes induced by ASEO. In addition, the chemical constituents of ASEO were identified by gas chromatography-mass spectrometry (GC-MS). The cytotoxic potential was evaluated in vitro and in silico. The oil displayed IC50 of 1.76, 3.46, and 3.77 µg/mL against promastigotes, axenic, and intracellular amastigotes, respectively. Photomicrographs of treated parasites showed plasma membrane disruption, increased lipid bodies, and autophagic-like structures. ASEO chemical profiling revealed 1,2,4,6-tetrathiepane (24.84%) and diallyl disulfide (16.75%) as major components. Computational pharmacokinetics and toxicological analysis of ASEO's major components demonstrated good oral bioavailability and better toxicological endpoints than the reference drugs. Altogether, the results suggest that ASEO could be an alternative drug candidate against HTL.

Discriminant Analysis of Brazilian Stingless Bee Honey Reveals an Iron-Based Biogeographical Origin.

Stingless bee honey (SBH) is gaining attention due to its nutritional, sensorial, and medicinal characteristics. This study focuses on the combination of physicochemical properties, antioxidant capacity, mineral profile, and mass spectrometry-based fingerprints, using a chemometric approach to differentiate SBH (n = 18) from three different Brazilian biogeographical zones (Caatinga, Cerrado, and Atlantic Forest). The physicochemical properties of SBH varied, resulting in a wide range of water activity, moisture, total soluble solids, pH, and total and free acidity. The Caatinga honey showed the highest and the lowest contents of phenolics and flavonoids, respectively. The antioxidant free-radical scavenging assays demonstrated that the Brazilian SBH has a high antioxidant potential. The mineral profile of honey samples from the Atlantic Forest revealed higher contents of Ca and Fe while the Cerrado and Caatinga honey showed the highest P contents. Partial Least-Squares Discriminant Analysis (PLS-DA) analysis separated the samples into three groups based on the biogeographical zones of harvest. The main separation factors between groups were the m/z 326 ion and the Fe content. Univariate analysis confirmed that Fe content is important for SBH discrimination. The present results indicate that the origin of SBH can be determined on the basis of mineral profile, especially Fe content.

Antileishmanial Screening, Cytotoxicity, and Chemical Composition of Essential Oils: A Special Focus on Piper callosum Essential Oil.

Leishmania amazonensis is the etiological agent of tegumentary leishmaniasis, a disease characterized by the emergence of cutaneous and mucocutaneous ulcerated lesions that can evolve into severe destruction of skin tissue. Treatment of the disease is often accompanied by high toxicity and variable efficacy. Essential oils stand out for having diverse pharmacological properties. Here, we screened a panel of fourteen essential oils for their anti-L. amazonensis activity, cytotoxicity, and chemical profile. Lippia sidoides (LSEO) and Piper callosum (PCEO) oils displayed the best anti-promastigote and anti-amastigote activities with IC50 of 31 and 21 µg/ml, respectively. PCEO was the safest oil with a desirable selectivity index >10. In addition, PCEO showed no cytotoxicity against the VERO line and erythrocytes. PCEO-treated amastigotes displayed mitochondrial membrane depolarization and high levels of intracellular ROS. Safrole (54.72 %) was the main component of PCEO. The results described here highlight the use of essential oils to combat tegumentary leishmaniasis.

Leishmania infantum arginase: biochemical characterization and inhibition by naturally occurring phenolic substances.

Inhibition of Leishmania arginase leads to a decrease in parasite growth and infectivity and thus represents an attractive therapeutic strategy. We evaluated the inhibitory potential of selected naturally occurring phenolic substances on Leishmania infantum arginase (ARGLi) and investigated their antileishmanial activity in vivo. ARGLi exhibited a Vmax of 0.28 ± 0.016 mM/min and a Km of 5.1 ± 1.1 mM for L-arginine. The phenylpropanoids rosmarinic acid and caffeic acid (100 µM) showed percentages of inhibition of 71.48 ± 0.85% and 56.98 ± 5.51%, respectively. Moreover, rosmarinic acid and caffeic acid displayed the greatest effects against L. infantum with IC50 values of 57.3 ± 2.65 and 60.8 ± 11 µM for promastigotes, and 7.9 ± 1.7 and 21.9 ± 5.0 µM for intracellular amastigotes, respectively. Only caffeic acid significantly increased nitric oxide production by infected macrophages. Altogether, our results broaden the current spectrum of known arginase inhibitors and revealed promising drug candidates for the therapy of visceral leishmaniasis.

Development of Nanoemulsions to Enhance the Antileishmanial Activity of Copaifera paupera Oleoresins.

Based on the ethnopharmacological evidences about the antileishmanial activity of Copaifera spp. oleoresins, the effects of crude extracts and fractions of oleoresin of two specimens from Copaifera paupera were evaluated on Leishmania amazonensis and Leishmania infantum strains. The oleoresin rich in α-copaene (38.8%) exhibited the best activity against L. amazonensis (IC50 = 62.5 µg/mL) and against L. infantum (IC50 = 65.9 µg/mL). The sesquiterpene α-copaene isolated was tested alone and exhibited high antileishmanial activity in vitro with IC50 values for L. amazonensis and L. infantum of 17.2 and 11.4 µg/mL, respectively. In order to increase antileishmanial activity, nanoemulsions containing copaiba oleoresin and α-copaene were developed and assayed against L. amazonensis and L. infantum promastigotes. The nanoemulsion containing α-copaene (NANOCOPAEN) showed the best activity against both species, with IC50 of 2.5 and 2.2 µg/mL, respectively. This is the first report about the antileishmanial activity of α-copaene.

Cytotoxicity and anti-Leishmania amazonensis activity of Citrus sinensis leaf extracts.

CONTEXT: Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease characterized by lesional polymorphism and the commitment of skin surface. Previous reports demonstrated that the Citrus genus possess antimicrobial activity. OBJECTIVE: This study evaluated the anti-L. amazonensis activity of Citrus sinensis (L.) Osbeck (Rutaceae) extracts. MATERIALS AND METHODS: Citrus sinensis dried leaves were subjected to maceration with hexane (CH), ethyl acetate (CEA), dichloromethane/ethanol (CD/Et - 1:1) or ethanol/water (CEt/W - 7:3). Leishmania amazonensis promastigotes were treated with C. sinensis extracts (1-525 µg/mL) for 120 h at 27 °C. Ultrastructure alterations of treated parasites were evaluated by transmission electron microscopy. Cytotoxicity of the extracts was assessed on RAW 264.7 and J774.G8 macrophages after 48-h treatment at 37 °C using the tetrazolium assay. In addition, Leishmania-infected macrophages were treated with CH and CD/Et (10-80 µg/mL). RESULTS: CH, CD/Et and CEA displayed antileishmanial activity with 50% inhibitory activity (IC50) of 25.91 ± 4.87, 54.23 ± 3.78 and 62.74 ± 5.04 µg/mL, respectively. Parasites treated with CD/Et (131.2 µg/mL) presented severe alterations including mitochondrial swelling, lipid body formation and intense cytoplasmic vacuolization. CH and CD/Et demonstrated cytotoxic effects similar to that of amphotericin B in the anti-amastigote assays (SI of 2.16, 1.98 and 1.35, respectively). Triterpene amyrins were the main substances in CH and CD/Et extracts. In addition, 80 µg/mL of CD/Et reduced the number of intracellular amastigotes and the percentage of infected macrophages in 63% and 36%, respectively. CONCLUSION: The results presented here highlight C. sinensis as a promising source of antileishmanial agents.

Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis.

The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 µg/mL against C. neoformans and 152 µg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 µg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents.

Natural Products: Insights into Leishmaniasis Inflammatory Response.

Leishmaniasis is a vector-borne disease that affects several populations worldwide, against which there are no vaccines available and the chemotherapy is highly toxic. Depending on the species causing the infection, the disease is characterized by commitment of tissues, including the skin, mucous membranes, and internal organs. Despite the relevance of host inflammatory mediators on parasite burden control, Leishmania and host immune cells interaction may generate an exacerbated proinflammatory response that plays an important role in the development of leishmaniasis clinical manifestations. Plant-derived natural products have been recognized as bioactive agents with several properties, including anti-protozoal and anti-inflammatory activities. The present review focuses on the antileishmanial activity of plant-derived natural products that are able to modulate the inflammatory response in vitro and in vivo. The capability of crude extracts and some isolated substances in promoting an anti-inflammatory response during Leishmania infection may be used as part of an effective strategy to fight the disease.

Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania spp.

Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite's ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 µg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents.

In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi.

BACKGROUND: Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells. METHODS: Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil. RESULTS: The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 µg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%. CONCLUSION: These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds.

Leishmanicidal activity of polyphenolic-rich extract from husk fiber of Cocos nucifera Linn. (Palmae).

The available therapy for leishmaniasis, which affects 2 million people per annum, still causes serious side effects. The polyphenolic-rich extract from the husk fiber of Cocos nucifera Linn. (Palmae) presents antibacterial and antiviral activities, also inhibiting lymphocyte proliferation, as shown by our group in previous works. In the present study, the in vitro leishmanicidal effects of C. nucifera on Leishmania amazonensis were evaluated. The minimal inhibitory concentration of the polyphenolic-rich extract from C. nucifera to completely abrogate parasite growth was 10 microg/ml. Pretreatment of peritoneal mouse macrophages with 10 microg/ml of C. nucifera polyphenolic-rich extract reduced approximately 44% the association index between these macrophages and L. amazonensis promastigotes, with a concomitant increase of 182% in nitric oxide production by the infected macrophage in comparison to nontreated macrophages. These results provide new perspectives on drug development against leishmaniasis, since the extract of C. nucifera at 10 microg/ml is a strikingly potent leishmanicidal substance which inhibited the growth of both promastigote and amastigote developmental stages of L. amazonensis after 60 min, presenting no in vivo allergenic reactions or in vitro cytotoxic effects in mammalian systems.