Antifungal effect of (R) and (S)-citronellal enantiomers and their predictive mechanism of action on Candida albicans from voriconazole-resistant onychomycoses / Efeito antifúngico dos enantiômeros (R) e (S)-citronelal e seu mecanismo de ação preditivo sobre Candida albicans de onicomicoses resistentes ao voriconazol

Onychomycosis is the most common disease affecting the nail unit and accounts for at least 50% of all nail diseases. In addition, Candida albicans is responsible for approximately 70% of onychomycoses caused by yeasts. This study investigated the antifungal effect of (R) and (S)-citronellal enantiomers, as well as its predictive mechanism of action on C. albicans from voriconazole-resistant onychomycoses. For this purpose, in vitro broth microdilution and molecular docking techniques were applied in a predictive and complementary manner to the mechanisms of action. The main results of this study indicate that C. albicans was resistant to voriconazole and sensitive to the enantiomers (R) and (S)-citronellal at a dose of 256 and 32 µg/mL respectively. In addition, there was an increase in the minimum inhibitory concentration (MIC) of the enantiomers in the presence of sorbitol and ergosterol, indicating that these molecules possibly affect the integrity of the cell wall and cell membrane of C. albicans. Molecular docking with key biosynthesis proteins and maintenance of the fungal cell wall and plasma membrane demonstrated the possibility of (R) and (S)-citronellal interacting with two important enzymes: 1,3-ß-glucan synthase and lanosterol 14α-demethylase. Therefore, the findings of this study indicate that the (R) and (S)-citronellal enantiomers are fungicidal on C. albicans from onychomycoses and probably these substances cause damage to the cell wall and cell membrane of these micro-organisms possibly by interacting with enzymes in the biosynthesis of these fungal structures.

A onicomicose é a doença mais comum que afeta a unidade ungueal e representa pelo menos 50% de todas as doenças ungueais. Além disso, a Candida albicans é responsável por aproximadamente 70% das onicomicoses causadas por leveduras. Nesse estudo, foi investigado o efeito antifúngico dos enantiômeros (R) e (S)-citronelal, bem como seu mecanismo de ação preditivo sobre C. albicans de onicomicoses resistentes ao voriconazol. Para este propósito, foram aplicadas técnicas in vitro de microdiluição em caldo e docking molecular de forma preditiva e complementar para os mecanismos de ação. Os principais resultados deste estudo indicam que C. albicans foi resistente ao voriconazol e sensível aos enantiômeros (R) e (S)-citronelal na dose de 256 e 32 µg/mL respectivamente. Além disso, houve aumento da concentração inibitória mínima (CIM) dos enantiômeros na presença do sorbitol e do ergosterol, indicando que estas moléculas possivelmente afetem a integridade da parede e da membrana celular de C. albicans. O docking molecular com proteínas chave da biossíntese e manutenção da parede celular e da membrana plasmática fúngica, demonstraram a possibilidade do (R) e (S)-citronelal interagir com duas importantes enzimas: 1,3-ß-glucan sintase e lanosterol 14α-demetilase. Portanto, os achados desse estudo indicam que os enantiômeros (R) e (S)-citronelal são fungicidas sobre C. albicans de onicomicoses e provavelmente essas substâncias causem danos a parede e a membrana celular desses microrganismos possivelmente por interagir com as enzimas da biossíntese destas estruturas fúngicas.

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A

Chemical compositions, radical scavenging capacities and antimicrobial activities in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata from Turkey / Composições químicas, capacidades radicais eliminadoras e atividades antimicrobianas em sementes de Satureja hortensis L. e Mentha spicata L. subsp. spicata da Turquia

The present study determined some biological compounds, radical scavenging activity and antimicrobial capacity in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata. Alpha-linolenic acid (C18:3 n3) has been found to be the major polyunsaturated fatty acid of Satureja hortensis L. (66.24 ± 1.24%) and Mentha spicata L. subsp. spicata (48.17 ± 1.01%). Linoleic acid (C18:2 n6) is identified as the second major polyunsaturated fatty acid in the present study and oleic acid (C18:1 n9) is determined as the major monounsaturated fatty acid. Current study showed that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low levels of saturated fatty acids. It has been demonstrated that ergosterol (263.1 ± 2.14 µg/g), stigmasterol (39.07 ± 0.91 µg/g) and beta-sitosterol (14.64 ± 0.49 µg/g) have been found in Mentha spicata L. subsp. spicata, while ergosterol (69.41 ± 1.75 µg/g) and beta-sitosterol (19.81 ± 1.14 µg/g) have been determined in Satureja hortensis L. Also, this study determined that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low lipide-soluble vitamin content. Furthermore, it has been found that Satureja hortensis L. contains naringenin (612.57 ± 2.57 µg/g), morin (86.97 ± 1.12 µg/g), quercetin (22.87 ± 0.75 µg/g), and kaempferol (20.11 ± 0.94 µg/g) while naringenin (135.91 ± 1.91 µg/g), naringin (61.23 ± 2.15 µg/g) and quercetin (47.51 ± 1.17 µg/g) have been detected as major flavonoids in the seeds of Mentha spicata L. subsp. spicata. The results of the present study suggest that methanol extracts of Satureja hortensis L. and Mentha spicata L. subsp. spicata have significant free radical scavenging activity. The present results revealed that Satureja hortensis L. and Mentha spicata L. subsp. spicata showed major activity against gram-positive and gram-negative microorganisms, fungi and yeast.(AU)

O presente estudo determinou alguns compostos biológicos, atividade de eliminação de radicais e capacidade antimicrobiana em sementes de Satureja hortensis L. e Mentha spicata L. subsp. spicata. O ácido alfa-linolênico (C18: 3 n3) foi o principal ácido graxo poliinsaturado de Satureja hortensis L. (66,24 ± 1,24%) e Mentha spicata L. subsp. spicata (48,17 ± 1,01%). O ácido linoléico (C18: 2 n6) é identificado como o segundo principal ácido graxo poliinsaturado no presente estudo e o ácido oleico (C18: 1 n9) é determinado como o principal ácido graxo monoinsaturado. O estudo atual mostrou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixos níveis de ácidos graxos saturados. Foi demonstrado que ergosterol (263,1 ± 2,14 µg/g), estigmasterol (39,07 ± 0,91 µg/g) e beta-sitosterol (14,64 ± 0,49 µg/g) foram encontrados em Mentha spicata L. subsp. spicata, enquanto o ergosterol (69,41 ± 1,75 µg/g) e beta-sitosterol (19,81 ± 1,14 µg/g) também foram determinados em Satureja hortensis L., este estudo determinou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixo teor de vitaminas lipossolúveis. Além disso, verificou-se que S. hortensis L. contém naringenina (612,57 ± 2,57 µg/g), morina (86,97 ± 1,12 µg/g), quercetina (22,87 ± 0,75 µg/g) e kaempferol (20,11 ± 0,94 µg/g) enquanto a naringenina (135,91 ± 1,91 µg/g), a naringina (61,23 ± 2,15 µg/g) e a quercetina (47,51 ± 1,17 µg/g) foram detectadas como flavonóides importantes nas sementes de Mentha spicata L. subsp. spicata. Os resultados do presente estudo sugerem que os extratos metanólicos de S. hortensis L. e Mentha spicata L. subsp. spicata tem significativa atividade de eliminação de radicais livres. Os presentes resultados revelaram que Satureja hortensis L. e Mentha spicata L. subsp. spicata mostrou atividade importante contra microrganismos gram-positivos e gram-negativos, fungos e leveduras.(AU)

Chemical compositions, radical scavenging capacities and antimicrobial activities in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata from Turkey

Abstract The present study determined some biological compounds, radical scavenging activity and antimicrobial capacity in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata. Alpha-linolenic acid (C18:3 n3) has been found to be the major polyunsaturated fatty acid of Satureja hortensis L. (66.24 ± 1.24%) and Mentha spicata L. subsp. spicata (48.17 ± 1.01%). Linoleic acid (C18:2 n6) is identified as the second major polyunsaturated fatty acid in the present study and oleic acid (C18:1 n9) is determined as the major monounsaturated fatty acid. Current study showed that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low levels of saturated fatty acids. It has been demonstrated that ergosterol (263.1 ± 2.14 µg/g), stigmasterol (39.07 ± 0.91 µg/g) and beta-sitosterol (14.64 ± 0.49 µg/g) have been found in Mentha spicata L. subsp. spicata, while ergosterol (69.41 ± 1.75 µg/g) and beta-sitosterol (19.81 ± 1.14 µg/g) have been determined in Satureja hortensis L. Also, this study determined that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low lipide-soluble vitamin content. Furthermore, it has been found that Satureja hortensis L. contains naringenin (612.57 ± 2.57 µg/g), morin (86.97 ± 1.12 µg/g), quercetin (22.87 ± 0.75 µg/g), and kaempferol (20.11 ± 0.94 µg/g) while naringenin (135.91 ± 1.91 µg/g), naringin (61.23 ± 2.15 µg/g) and quercetin (47.51 ± 1.17 µg/g) have been detected as major flavonoids in the seeds of Mentha spicata L. subsp. spicata. The results of the present study suggest that methanol extracts of Satureja hortensis L. and Mentha spicata L. subsp. spicata have significant free radical scavenging activity. The present results revealed that Satureja hortensis L. and Mentha spicata L. subsp. spicata showed major activity against gram-positive and gram-negative microorganisms, fungi and yeast.

Resumo O presente estudo determinou alguns compostos biológicos, atividade de eliminação de radicais e capacidade antimicrobiana em sementes de Satureja hortensis L. e Mentha spicata L. subsp. spicata. O ácido alfa-linolênico (C18: 3 n3) foi o principal ácido graxo poliinsaturado de Satureja hortensis L. (66,24 ± 1,24%) e Mentha spicata L. subsp. spicata (48,17 ± 1,01%). O ácido linoléico (C18: 2 n6) é identificado como o segundo principal ácido graxo poliinsaturado no presente estudo e o ácido oleico (C18: 1 n9) é determinado como o principal ácido graxo monoinsaturado. O estudo atual mostrou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixos níveis de ácidos graxos saturados. Foi demonstrado que ergosterol (263,1 ± 2,14 µg/g), estigmasterol (39,07 ± 0,91 µg/g) e beta-sitosterol (14,64 ± 0,49 µg/g) foram encontrados em Mentha spicata L. subsp. spicata, enquanto o ergosterol (69,41 ± 1,75 µg/g) e beta-sitosterol (19,81 ± 1,14 µg/g) também foram determinados em Satureja hortensis L., este estudo determinou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixo teor de vitaminas lipossolúveis. Além disso, verificou-se que S. hortensis L. contém naringenina (612,57 ± 2,57 µg/g), morina (86,97 ± 1,12 µg/g), quercetina (22,87 ± 0,75 µg/g) e kaempferol (20,11 ± 0,94 µg/g) enquanto a naringenina (135,91 ± 1,91 µg/g), a naringina (61,23 ± 2,15 µg/g) e a quercetina (47,51 ± 1,17 µg/g) foram detectadas como flavonóides importantes nas sementes de Mentha spicata L. subsp. spicata. Os resultados do presente estudo sugerem que os extratos metanólicos de S. hortensis L. e Mentha spicata L. subsp. spicata tem significativa atividade de eliminação de radicais livres. Os presentes resultados revelaram que Satureja hortensis L. e Mentha spicata L. subsp. spicata mostrou atividade importante contra microrganismos gram-positivos e gram-negativos, fungos e leveduras.

Immunomodulation Properties of Solid-State Fermented Laetiporussulphureus Ethanol Extracts in Chicken Peripheral Blood Monocytes In Vitro

Laetiporus sp. is recognized as a fungal species traditionally used for medicinal purposes. This study investigated the in-vitro effects of solid-state fermented Laetiporussulphureus ethanol extracts (LSE) for their immunomodulatory potential. Bioactive levels detected in the LSE on different days throughout the fermentation period revealed that the 12th day was the most efficient, with 7.19 ± 0.66 GAE/g DM crude phenolic content, 2.71 ± 0.03 UAE/g DM crude triterpenoid content, 12.93 ± 0.88 GCE/g DM crude polysaccharides, and 96.44 ± 0.2 mg/g DM ergosterol content. In-vitroLSE tests on chPBMC showed no cytotoxicity within a range of 0.05-1 mg/mL, but LPS-inhibited cell viability was improved, as well as LPS-induced nitric oxide (NO) production and mRNA levels of nuclear factor kappa B (NFB), Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), and interleukin (IL)-1were attenuated Furthermore, the direct application of LSE on chPBMC showed a small but not significant increase in NFB, TLR4, and iNOS mRNA expression compared with the control group. These results indicate the potential of LSE to modulate LPS-triggered inflammation processes involving TLR4 and NFB mediation. However, further experiments are required to determine the specific pathway.(AU)

Immunomodulation Properties of Solid-State Fermented Laetiporussulphureus Ethanol Extracts in Chicken Peripheral Blood Monocytes In Vitro

Laetiporus sp. is recognized as a fungal species traditionally used for medicinal purposes. This study investigated the in-vitro effects of solid-state fermented Laetiporussulphureus ethanol extracts (LSE) for their immunomodulatory potential. Bioactive levels detected in the LSE on different days throughout the fermentation period revealed that the 12th day was the most efficient, with 7.19 ± 0.66 GAE/g DM crude phenolic content, 2.71 ± 0.03 UAE/g DM crude triterpenoid content, 12.93 ± 0.88 GCE/g DM crude polysaccharides, and 96.44 ± 0.2 mg/g DM ergosterol content. In-vitroLSE tests on chPBMC showed no cytotoxicity within a range of 0.05-1 mg/mL, but LPS-inhibited cell viability was improved, as well as LPS-induced nitric oxide (NO) production and mRNA levels of nuclear factor kappa B (NFB), Toll-like receptor 4 (TLR4), inducible nitric oxide synthase (iNOS), and interleukin (IL)-1were attenuated Furthermore, the direct application of LSE on chPBMC showed a small but not significant increase in NFB, TLR4, and iNOS mRNA expression compared with the control group. These results indicate the potential of LSE to modulate LPS-triggered inflammation processes involving TLR4 and NFB mediation. However, further experiments are required to determine the specific pathway.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.(AU)

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Abstract Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.(AU)

Chemical compositions, radical scavenging capacities and antimicrobial activities in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata from Turkey

Abstract The present study determined some biological compounds, radical scavenging activity and antimicrobial capacity in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata. Alpha-linolenic acid (C18:3 n3) has been found to be the major polyunsaturated fatty acid of Satureja hortensis L. (66.24 ± 1.24%) and Mentha spicata L. subsp. spicata (48.17 ± 1.01%). Linoleic acid (C18:2 n6) is identified as the second major polyunsaturated fatty acid in the present study and oleic acid (C18:1 n9) is determined as the major monounsaturated fatty acid. Current study showed that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low levels of saturated fatty acids. It has been demonstrated that ergosterol (263.1 ± 2.14 µg/g), stigmasterol (39.07 ± 0.91 µg/g) and beta-sitosterol (14.64 ± 0.49 µg/g) have been found in Mentha spicata L. subsp. spicata, while ergosterol (69.41 ± 1.75 µg/g) and beta-sitosterol (19.81 ± 1.14 µg/g) have been determined in Satureja hortensis L. Also, this study determined that Satureja hortensis L. and Mentha spicata L. subsp. spicata have low lipide-soluble vitamin content. Furthermore, it has been found that Satureja hortensis L. contains naringenin (612.57 ± 2.57 µg/g), morin (86.97 ± 1.12 µg/g), quercetin (22.87 ± 0.75 µg/g), and kaempferol (20.11 ± 0.94 µg/g) while naringenin (135.91 ± 1.91 µg/g), naringin (61.23 ± 2.15 µg/g) and quercetin (47.51 ± 1.17 µg/g) have been detected as major flavonoids in the seeds of Mentha spicata L. subsp. spicata. The results of the present study suggest that methanol extracts of Satureja hortensis L. and Mentha spicata L. subsp. spicata have significant free radical scavenging activity. The present results revealed that Satureja hortensis L. and Mentha spicata L. subsp. spicata showed major activity against gram-positive and gram-negative microorganisms, fungi and yeast.

Resumo O presente estudo determinou alguns compostos biológicos, atividade de eliminação de radicais e capacidade antimicrobiana em sementes de Satureja hortensis L. e Mentha spicata L. subsp. spicata. O ácido alfa-linolênico (C18: 3 n3) foi o principal ácido graxo poliinsaturado de Satureja hortensis L. (66,24 ± 1,24%) e Mentha spicata L. subsp. spicata (48,17 ± 1,01%). O ácido linoléico (C18: 2 n6) é identificado como o segundo principal ácido graxo poliinsaturado no presente estudo e o ácido oleico (C18: 1 n9) é determinado como o principal ácido graxo monoinsaturado. O estudo atual mostrou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixos níveis de ácidos graxos saturados. Foi demonstrado que ergosterol (263,1 ± 2,14 µg/g), estigmasterol (39,07 ± 0,91 µg/g) e beta-sitosterol (14,64 ± 0,49 µg/g) foram encontrados em Mentha spicata L. subsp. spicata, enquanto o ergosterol (69,41 ± 1,75 µg/g) e beta-sitosterol (19,81 ± 1,14 µg/g) também foram determinados em Satureja hortensis L., este estudo determinou que Satureja hortensis L. e Mentha spicata L. subsp. spicata tem baixo teor de vitaminas lipossolúveis. Além disso, verificou-se que S. hortensis L. contém naringenina (612,57 ± 2,57 µg/g), morina (86,97 ± 1,12 µg/g), quercetina (22,87 ± 0,75 µg/g) e kaempferol (20,11 ± 0,94 µg/g) enquanto a naringenina (135,91 ± 1,91 µg/g), a naringina (61,23 ± 2,15 µg/g) e a quercetina (47,51 ± 1,17 µg/g) foram detectadas como flavonóides importantes nas sementes de Mentha spicata L. subsp. spicata. Os resultados do presente estudo sugerem que os extratos metanólicos de S. hortensis L. e Mentha spicata L. subsp. spicata tem significativa atividade de eliminação de radicais livres. Os presentes resultados revelaram que Satureja hortensis L. e Mentha spicata L. subsp. spicata mostrou atividade importante contra microrganismos gram-positivos e gram-negativos, fungos e leveduras.

Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides (Melanconiaceae) / Fotoesteróis isolados do fungo endofítico Colletotrichum gloeosporioides (Melanconiaceae)

Endophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by 1H and 13C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.

Fungos endofíticos são fungos que colonizam os tecidos internos das plantas. Existem poucos estudos de compostos isolados de fungos endofíticos de plantas da Amazônia. Assim, o objetivo deste estudo foi o isolamento e identificação estrutural de sitosterol (1), estigmasterol (2), sitostenona (3), esqualeno (4), ergosterol (5) e peroxido de ergosterol (6) do fungo Colletotrichum gloeosporioidesisolado como endofítico de Virola michelli, uma planta típica da Amazônia, usada na medicina popular no combate a infecções de pele. Os compostos foram isolados por cromatografia em coluna de sílica e identificados por RMN 1H e 13C e EM. A presença de fitoesteróis em fungos é rara e este é o primeiro relato do isolamento dos fitoesteróides sitosterol, estigmasterol e sitostenona do gêreno Colletotrichum.

Phytosterols isolated from endophytic fungus Colletotrichum gloeosporioides (Melanconiaceae) / Fotoesteróis isolados do fungo endofítico Colletotrichum gloeosporioides (Melanconiaceae)

Endophytic fungi are fungi that colonize internal tissues of plants. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, the aim this study was the isolation and structural identification of sitosterol (1), stigmasterol (2), sitostenone (3), squalene (4), ergosterol (5) and ergosterol peroxide (6) from fungus Colletotrichum gloeosporioidesisolated as endophytic from Virola michelli, a typical Amazon plant, used in folk medicine against skin infection. Compounds were isolated by chromatography column on silica and identified by 1H and 13C NMR and MS. The presence of phytosterols in fungi is rare and this is the first report of the isolation of the phytosterols sitosterol, stigmasterol and sitostenone from the genus Colletotrichum.(AU)

Fungos endofíticos são fungos que colonizam os tecidos internos das plantas. Existem poucos estudos de compostos isolados de fungos endofíticos de plantas da Amazônia. Assim, o objetivo deste estudo foi o isolamento e identificação estrutural de sitosterol (1), estigmasterol (2), sitostenona (3), esqualeno (4), ergosterol (5) e peroxido de ergosterol (6) do fungo Colletotrichum gloeosporioidesisolado como endofítico de Virola michelli, uma planta típica da Amazônia, usada na medicina popular no combate a infecções de pele. Os compostos foram isolados por cromatografia em coluna de sílica e identificados por RMN 1H e 13C e EM. A presença de fitoesteróis em fungos é rara e este é o primeiro relato do isolamento dos fitoesteróides sitosterol, estigmasterol e sitostenona do gêreno Colletotrichum.(AU)

Terpinen-4-ol, tyrosol, and -lapachone as potential antifungals against dimorphic fungi

This study aimed to evaluate the in vitro antifungal activity of terpinen-4-ol, tyrosol, and -lapachone against strains of Coccidioides posadasii in filamentous phase (n = 22) and Histoplasma capsulatum in both filamentous (n = 40) and yeast phases (n = 13), using the broth dilution methods as described by the Clinical and Laboratory Standards Institute, to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these compounds. The mechanisms of action of these compounds were also investigated by analyzing their effect on cell membrane permeability and ergosterol synthesis. The MIC and MFCf these compounds against C. posadasii, mycelial H. capsulatum, and yeast-like H. capsulatum, were in the following ranges: 350-5720 µg/mL, 20-2860 µg/mL, and 40-1420 µg/mL, respectively for terpinen-4-ol; 250-4000 µg/mL, 30-2000 µg/mL, and 10-1000 µg/mL, respectively, for tyrosol; and 0.48-7.8 µg/mL, 0.25-16 µg/mL, and 0.125-4 µg/mL, respectively for -lapachone. These compounds showed a decrease in MIC when the samples were subjected to osmotic stress, suggesting that the compounds acted on the fungal membrane. All the compounds were able to reduce the ergosterol content of the fungal strains. Finally, tyrosol was able to cause a leakage of intracellular molecules.(AU)

The leaf breakdown of Picramnia sellowii (Picramniales: Picramniaceae) as index of anthropic disturbances in tropical streams / Decomposição de folhas de Picramnia sellowii (Picramniales: Picramniaceae) como índice de distúrbio antrópicos em riachos tropicais

There are few studies in tropical regions exploring the use of leaf breakdown process as a functional tool to assess anthropic disturbance on aquatic ecosystems. We assessed the effects of water pollution due to human activities on the leaf breakdown rate of Picramnia sellowii in streams of the southeastern Brazil. The experiment was carried out for 60 days in three reference streams and three streams impaired by organic pollution and absence of riparian vegetation. Three litter bags were incubated in each stream containing 3 ± 0.05 g of P. sellowii leaves. The reference streams presented higher values of dissolved oxygen and lower values of nutrients, turbidity, electrical conductivity, total impermeable area and water temperature. The leaf breakdown rate (k) differed significantly between the reference (k = 0.014 ± 0.003 d–1) and impaired streams (k = 0.005 ± 0.001 d–1). The leaves incubated in the reference streams contained greater fungal biomass (measured as ergosterol concentration) and abundance of invertebrates, as well as greater presence of shredders, with k values being related to the biomass of these organisms. Overall, there were clear differences between the leaf mass loss in the reference and impaired streams. These results reinforce the negative effect of urbanization on leaf breakdown and fungal and shredder biomass.(AU)

Na região tropical são escassos os estudos que exploram o uso do processo de decomposição foliar como ferramenta funcional para avaliar os efeitos das perturbações antrópicas sobre os ecossistemas aquáticos. Nosso objetivo foi avaliar os efeitos dos impactos antrópicos sobre a taxa de decomposição de Picramnia sellowii em riachos no Sudeste do Brasil. O experimento foi realizado durante 60 dias em três riachos referências e três impactados por poluição orgânica e sem cobertura vegetal. Em cada riacho foram incubados três sacos contendo 3 ± 0,05 g de folhas secas de P. sellowii. Os riachos referência apresentaram maiores concentrações de oxigênio dissolvido e menores valores de nutrientes, turbidez, condutividade elétrica, TIA (área total impermeável) e temperatura. O coeficiente de decomposição (k) diferiu entre os riachos referência (k = 0,014 ± 0,003 d–1) e os impactados (k = 0,005 ± 0,001 d–1). Nas folhas incubadas nos riachos referência foi observada maior biomassa de fungos e abundância de invertebrados, assim como a presença de fragmentadores, estando o k relacionado com a biomassa destes organismos. A decomposição se mostrou sensível para avaliar impactos antrópicos nos ecossistemas aquáticos uma vez que foram observadas nítidas diferenças entre a perda de massa foliar nos riachos referências e impactados. Estes resultados reforçam o efeito negativo da urbanização sobre a decomposição e biomassa de fungos e fragmentadores.(AU)

Uso promissor do óleo essencial de Rosmarinus officinalis L. e Origanum majorana L. na esporotricose cutânea experimental por Sporothrix brasiliensis resistente ao itraconazol, e susceptibilidade de isolados aos óleos vegetais

O itraconazol é o fármaco de eleição no tratamento da esporotricose humana e animal, entretanto, o surgimento de Sporothrix brasiliensis com resistência in vitro tem incentivado os estudos com plantas da família Lamiaceae como fontes promissoras de moléculas antifúngicas. Dessa família, os óleos essenciais de alecrim (Rosmarinus officinalis L.) e manjerona (Origanum majorana L.) têm se destacados pela atividade antifúngica e baixa citotoxicidade. Essa tese objetivou avaliar a atividade dos óleos essenciais de plantas da família Lamiaceae em isolados clínicos de Sporothrix spp. e na esporotricose cutânea experimental por S. brasiliensis resistente ao itraconazol. Quarenta ratos Wistar adultos machos foram inoculados via subcutânea no coxim plantar esquerdo (2×105 células/mL) e, dez dias após, receberam tratamento oral (1 mL) por 30 dias nos seguintes grupos experimentais (n=10): controle com solução salina com propilenoglicol 1% (CONT); itraconazol a 10 mg/kg (ITRA); óleos essenciais de alecrim a 90 mg/ml (ALEC) e manjerona a 30 mg/ml (MANJ). Semanalmente, a evolução clínica era acompanhada e a eutanásia realizada para histopatologia e contagem fúngica (retro-isolamento). Ainda, os óleos foram avaliados por cromatografia gasosa com espectrometria de massas, e o mecanismo de ação pelos ensaios de proteção do sorbitol e efeito do ergosterol. Os resultados revelaram que os animais dos grupos ALEC e MANJ apresentaram remissão dos sinais clínicos, com ausência de edema e exsudato no membro inoculado, ao passo que CONT e ITRA mantiveram os sinais da doença. Ainda, a disseminação do S. brasiliensis para os órgãos foi menos observada no fígado, baço, linfonodos, rins e testículos dos grupos ALEC e MANJ em comparação ao CONT e ITRA (P < 0.05), revelando uma proteção sistêmica a partir da infecção subcutânea. Essa observação foi relacionada com a composição química, cujos compostos prevalentes foram terpineol-4 (34,09%) e -terpineno (14,28%) em manjerona, e 1,8-cineol (47,91%) e cânfora (12,47%) em alecrim. Não houve alteração nos valores da concentração inibitória mínima (CIM) quando adicionado o protetor osmótico, demonstrando ausência de atividade à nível de parede celular, ao passo que o uso do ergosterol elevou os valores de CIM, revelando que ambos óleos apresentaram atividade a nível de complexação com o ergosterol. Esse estudo demonstrou a eficácia do uso oral de alecrim e manjerona no tratamento da esporotricose cutânea experimental, sendo promissores como antifúngicos. Palavras-ch

Itraconazole is the drug of choice for the treatment of sporotrichosis, however, the emergence of Sporothrix brasiliensis with in vitro resistance has encouraged studies with plants of the family Lamiaceae as promising sources of antifungal molecules. From this family, essential oils of rosemary (Rosmarinus officinalis L.) and marjoram (Origanum majorana L.) have been highlighted for their antifungal activities and low cytotoxicity. This thesis aimed to evaluate the effectiveness of the essential oils of the plants from Lamiaceae family in clinical isolates of Sporothrix spp. and in the experimental cutaneous sporotrichosis by itraconazole-resistant S. brasiliensis. Forty male adult Wistar rats were inoculated subcutaneously in the left footpad (2×105 cells/mL) and, ten days later, received oral treatment (1 mL) for 30 days in the following experimental groups (n=10): control with solution saline with 1% propylene glycol (CONT); itraconazole at 10 mg/kg (ITRA); essential oils of rosemary at 90 mg/mL (ROSM) and marjoram at 30 mg/mL (MARJ). The clinical follow-up was done weekly, as well as the euthanasia for histopathology and fungal burden (retro-isolation). Furthermore, the oils were evaluated by gas chromatography with mass spectrometry, and the mechanism of action by the assays of sorbitol protection and ergosterol effect. The results showed that the animals from ALEC and MANJ groups presented remission of the clinical signs, with no edema and exudate in the inoculated limb, whereas CONT and ITRA maintained the signs of the disease. Also, the dissemination of S. brasiliensis to the organs was less observed in the liver, spleen, lymph nodes, kidneys and testicles of the ALEC and MANJ groups compared to the CONT and ITRA (P <0.05), revealing systemic protection from the subcutaneous infection. This observation was related to the chemical composition, whose prevalent compounds were terpineol-4 (34.09%) and -terpinene (14.28%) in marjoram, and 1,8-cineole (47.91%) and camphor (12.47%) in rosemary. There was no change in minimum inhibitory concentration (MIC) values when the osmotic protector was added, showing an absence of activity at the cell wall level, whereas the use of ergosterol increased MIC values, revealing that both oils presented activity at the level of complexion with ergosterol. This study demonstrated the effectiveness of the oral use of rosemary and marjoram in the treatment of experimental cutaneous sporotrichosis, being promising as antifungal.

CARACTERIZAÇÃO DAS CÉLULAS-TRONCO MESENQUIMAIS DERIVADAS DE POLPA DENTÁRIA E CORDÃO UMBILICAL DE CÃES

O campo de medicina regenerativa para animais de companhia vem crescendo nos últimos anos. As células-tronco mesenquimais (MSC) podem ser definidas como células-tronco adultas, multipotentes, com capacidade de se diferenciar em vários tipos celulares. A Sociedade Internacional de Terapia Celular (ISCT) estabeleceu critérios mínimos para definir MSC. As MSC podem ser isoladas de diferentes fontes como: polpa dentária e cordão umbilical. A polpa dentária contém uma população de células-tronco com localização perivascular e potencial de diferenciação em múltiplas linhagens semelhante à MSC obtidas a partir de outros tecidos. O fluconazol é um composto antifúngico usado isoladamente ou em combinação com outras drogas, por ter um amplo espectro. Ele exerce sua atividade antifúngica inibindo a síntese de ergosterol fúngico em um dos passos finais de sua biossíntese. Portanto, ele pode ser usado na coleta de dentes e na cultura de células-tronco de polpa dentária. Outra fonte de MSC utilizada é o cordão umbilical, onde sua coleta é menos invasiva, não gera riscos ao paciente e seu material normalmente seria descartado. O objetivo deste estudo foi isolar, caracterizar e avaliar o potencial de proliferação e formação de colônias das células-tronco mesenquimais derivadas de polpa de dente canina (cDPSCs) e do tecido do cordão umbilical de cães (cUCSC). Foram coletadas três amostras de polpa dentária e três de cordão umbilical. As amostras foram dissociadas mecanicamente e isoladas por digestão enzimática usando colagenase tipo II. As células foram expandidas e foi realizada a diferenciação para as linhagens adipogênica, osteogênica e condrogênica. Para diferenciação osteogênica, o meio foi trocado três vezes por semana, em seguida as células foram coradas com Alizarina Red evidenciando os cristais de cálcio. Para diferenciação adipogênica, o meio foi trocado três vezes por semana e então as células foram coradas com Oil Red evidenciando os vacúolos lipídicos. Para a diferenciação condrogênica o meio foi trocado três vezes por semana e foram coradas com azul de toluidina evidenciando os mucopolissacarídeos. Para o ensaio clonogênico foram plaqueadas 300 células/poço em placas de seis poços e cultivadas durante nove dias, em seguida as colônias foram coradas com violeta cristal para serem contadas. Para o ensaio de proliferação, foram plaqueadas 1x104 células/poço em placa de seis poços, então a cada cinco dias foi realizada a dissociação enzimática e as células foram contadas em câmara de Neubauer. As cDPSC e cUCSC apresentaram aderência ao plástico e morfologia fibroblastoide após cultivo de acordo com os critérios mínimos da ISCT. Tanto as cDPSCs quanto as cUCT apresentaram potencial de diferenciação para as três linhagens (osteogênica, adipogênica e condrogênica). As cUCSC apresentaram maior taxa de proliferação (p=0.0414) e número de unidades formadora de colônia (p=0.0002) quando comparada as cDPSC na passagem três. Conclui-se que as cDPSC e cUCSC apresentaram as características de células-tronco mesenquimais e que as cUCSC até a passagem três possuem uma maior taxa de proliferação e maior quantidade de unidades formadoras de colônias que as cDPSC, podendo ser utilizadas na terapia celular.

The field of regenerative medicine for pets has been growing in recent years. Mesenchymal stem cells (MSCs) can be defined as adult, multipotent stem cells with the ability to differentiate into several cell types. The International Society of Cell Therapy (ISCT) has set minimum criteria for defining MSCs. These MSCs can be isolated from different sources such as: dental pulp and umbilical cord. The dental pulp contains a population of stem cells (DPSC) with perivascular location and differentiation potential in multiple lineages similar to MSCs obtained from other tissues. Fluconazole is an antifungal compound used alone or in combination with other drugs because it has a large spectrum. It exerts its antifungal activity by inhibiting ergosterol fungal synthesis in one of the final steps of its biosynthesis. Therefore, it can be used in teeth collection and stem cell culture of dental pulp. Another source of MSCs used is umbilical cord. The collection is less invasive, painful and with lower mortality when compared to collection of the bone marrow. The aim were isolate, characterize and evaluate the potential of proliferation and colonies formation of mesenchymal stem cells derived from dental pulp (cDPSC) and umbilical cord tissue of dogs (cUCSC). Three samples of dental pulp and umbilical cord were collected. The samples were mechanically dissociated and isolated by enzymatic digestion using type II collagenase and cultivate with IMDM and fetal bovine serum. The differentiation was made for adipogenic, osteogenic and chondrogenic lineages. For osteogenic differentiation, the medium was changed three times per week for seven days, then the cells were stained with Alizarin Red evidencing calcium crystals. For adipogenic differentiation, the medium was changed three times per week for 21 days, then the cells were stained with Oil Red evidencing lipid vacuoles. For chondrogenic differentiation the medium was changed three times at week for 21 days, then stained with toluidine blue evidencing mucopolysaccharides. For clonogenic assay, 300 cells/well were plated in 6-well plates and cultured for nine days, then colonies were stained with crystal violet and counted. For proliferation assay, 10,000 cells/well were plated in six-well plate, enzymatic dissociation was performed every five days and cells were counted in a hemocytometer. The cDPSC and cUCSC showed adherence to the plastic and fibroblastroid morphology after culture according to guidelines of the ISCT. In addition, they also shown differentiation potential for the three lineages: osteogenic, adipogenic and chondrogenic. The cUCSC presented higher number of colony forming units when compared to cDPSC at third passage. Regarding proliferation, cUCT presented a higher rate when compared to cDPSCs at third passage. In conclusion, cDPSC and cUCSC showed characteristics of mesenchymal stem cells and cUCSC at third passage had a higher rate of proliferation and more of colony-forming units in comparison to cDPSC, can be used in cell therapy.

Identification of genes expressed by Cryptococcus gattii during iron deprivation

Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.

Toxigenic potential of Fusarium graminearum isolated from maize of northwest Argentina

Twenty six isolates of Fusarium graminearum from grains of maize hybrids harvested in ±west Argentina were grown on autoclaved rice grain to assess their ability to produce type B trichothecenes. Chemical analysis indicated that 38% of isolates were nivalenol (NIV) producers only, 31% were major NIV producers with high DON(deoxynivalenol)/NIV ratios, 8% were major DON producers with minor NIV production, and 23% were DON producers only. Isolates showed a high variability in their toxigenic potential which was not related to fungal biomass. The distribution of the different chemotypes as well as the high and the low trichothecene-producing Fusarium isolates could not be associated to a geographical origin. Our results confirmed for the first time that isolates of Fusarium graminearum from maize of northwest Argentina are able to produce DON and NIV. A substancial contamination with both NIV and DON is likely in maize from northwest Argentina. Their contents should be quantified in regional surveillances for mycotoxin contamination.(AU)

CARACTERIZAÇÃO QUÍMICA E AVALIAÇÃO DE ATIVIDADES BIOLÓGICAS DE EXTRATOS E CONSTITUINTES DE Ourateafieldingiana(Gardner) Engl.

A planta Ouratea fieldingiana (Gardner) Engl., conhecida popularmente como batiputá, é uma espécie arbórea pertencente a família das Ochnaceae. O óleo das sementes do fruto de batiputá é encontrado em larga escala no litoral cearense e é usado empiricamente em processos cicatrizantes e outras patologias. Este estudo descreve a caracterização estrutural dos principais compostos químicos presentes nos extratos de O. fieldingiana e avalia seu potencial biológico, investigando as atividades cicatrizante, antifúngica, antioxidante e anticolinesterásica in vitro, in silico e in vivo. O principal mecanismo de ação do composto antifúngico foi investigado por docking molecular. Os óleos das sementes e os extratos das folhas foram extraídas com etanol em aparelho Soxhlet e por maceração, respectivamente. Ambos os extratos foram submetidos a cromatografia em coluna de sílica gel para isolamento dos constituintes principais e análise por cromatografia liquida de alta eficiência. As estruturas dos compostos foram estabelecidas por espectroscopia de 1H e 13C-NMR e identificadas, por comparação com dados da literatura, como amentoflavona e canferol-3-O-rutinosídeo. As atividades antioxidantes dos extratos foram determinadas pelos métodos de inibição dos radicais livres DPPH e ABTS. Em geral, os extratos com maior atividade antioxidante corresponderam àqueles com maior teor de compostos fenólicos e flavonoides. Os extratos etanólicos e os dois compostos isolados apresentaram atividade antifúngica relevante contra várias cepas de Candida. Os achados in silico revelaram que o composto amentoflavona acoplou à proteína CYP450 em seu sítio ativo devido à baixa estabilização de energia (-9,39 kcal / mol), indica um possível mecanismo de ação pela inibição da biossíntese de ergosterol de fungos Candida. Foi avaliado o efeito antinociceptivo do canferol-3-O-rutinosídeo (Kpf), isolado da planta O. fieldingiana, sobre a nocicepção orofacial e possíveis mecanismos de ação. Kpf não alterou o sistema locomotor do peixe e reduziu significativamente o comportamento nociceptivo orofacial induzido por todos os agentes nocivos em comparação com o grupo controle. O extrato de O. fieldingiana contendo os diversos flavonoides revelou uma excelente atividade cicatrizante em experimento com camundongos, sendo melhor que os flavonoides isolados, portanto o sinergismo dos diversos constituintes contribui para sua maior ação. Foi desenvolvido um produto cicatrizante eficiente à base de extrato das folhas da planta.

The plant Ouratea fieldingiana (Gardner) Engl., popularly known in Brazil as batiputá, is a tree species belonging to the Ochnaceae family. The oil of the seeds of the fruit of batiputá is found in large scale in the coast of Ceará and is used empirically in wound healing processes and other pathologies. This study describes the characterization of main chemical compounds present in extracts of O. fieldingiana and evaluates their biological potential investigating the wound healing, antifungal, antioxidant and anticholinesterase activities in experiments in vitro, in silico and in vivo. The action mechanism of main antifungal compound was investigated by molecular docking. The seeds and leaves were extracted with ethanol in a Soxhlet apparatus and by maceration, respectively. Both extracts were subjected to silica gel column chromatography for isolation of main constituents. The structures of compounds were established by 1H and 13C-NMR spectroscopy and identified by comparison with literature data as amentoflavone and kaempferol 3-O-rutinoside, respectively. The antioxidant activities of the extracts were determined by the DPPH and ABTS free radical inhibition methods. In general, the extracts with the highest antioxidant activity corresponded to those with higher content of phenolic compounds and flavonoids. The ethanol extracts and two isolated compounds, presented relevant antifungal activity against several Candida strains. The in silico findings revealed that the compound amentoflavone coupled with the CYP450 protein in its active site, due to the low energy stabilization (-9.39 kcal/mol), indicating a possible mechanism of action by inhibition of the ergosterol biosynthesis of Candida fungi. Kpf did not alter the fishs locomotor system and significantly reduced the orofacial nociceptive behavior induced by all noxious agents compared to the control group. The extract of O. fieldingiana containing the various flavonoids revealed an excellent healing activity, being better than the isolated flavonoids, so the synergism of the several constituents contributes to its greater action. An efficient wound healing product was developed using the plant leaf extract.

Utilização de extrato hidroetanólico e óleo essencial de Origanum vulgare com associações no tratamento da esporotricose experimental por Sporothrix brasiliensis

A busca por novos fármacos com ação antimicrobiana tem sido extensivamente estudada em especial com extratos vegetais, entretanto, a pesquisa da ação destas frente a fungos ainda é escassa. Dentre as micoses de importância em saúde pública, a esporotricose tem emergido nos últimos anos como uma importante doença zoonótica em grandes centros urbanos, acometendo gatos, cães e humanos. O tratamento desta micose é realizado com a administração diária de antifúngicos, em especial o itraconazol, que é considerado o fármaco de eleição tanto para animais como para humanos. Entretanto, os princípios ativos com ação nos fungos do complexo Sporothrix schenckii, disponíveis comercialmente, são restritos. Neste sentido, verifica-se a necessidade de outras opções terapêuticas para o tratamento da esporotricose. Dessa forma, a atividade in vitro de plantas demonstrada por estudos preliminares com isolados do complexo Sporothrix schenckii impulsionaram a realização do presente estudo visando determinar a atividade in vivo dos extratos vegetais de Origanum vulgare para o tratamento da esporotricose cutânea experimental. Para isso, foram utilizados 60 ratos machos da linhagem Wistar que foram divididos em 6 grupos experimentais, inoculados com S. brasiliensis para o desenvolvimento da esporotricose cutânea e submetidos a 30 dias de tratamento com itraconazol, óleo essencial de O. vulgare, extrato hidroalcoólico de O. vulgare, associação do óleo com itraconazol, associação do extrato com o itraconazol e um grupo controle tratado somente com o veículo. Durante os 30 dias, os animais eram avaliados semanalmente e 2 animais por grupo eram eutanasiados para a coleta de material para analisar o desenvolvimento da doença e a evolução dos diferentes tratamentos. Ao final dos 30 dias, os animais remanescentes de cada grupo foram eutanasiados e, também, tiverem materiais biológicos coletados para as análises. Em todas as necropsias, foram coletadas amostras de fígado, rim, baço, testículo, ponto de inoculação e linfonodo regional, além de amostras de sangue. Como resultados obtidos, somente os animais pertencentes aos grupos ITRA e CONT não apresentação desaparecimento total dos sinais clínicos observados ao final dos 30 dias de tratamento, também foi possível observar que os animais pertencentes aos grupos tratados com o extrato hidroalcoólico de O. vulgare e com o óleo essencial de O. vulgare não apresentaram nenhuma alteração microscópica em rins, fígado e baço durante todo o experimento, indicando assim um possível efeito dessa planta na disseminação da doença. O Sporothrix brasiliensis foi retroisolado do linfonodo regional de todos animais de todos os grupos nas quatro necropsias realizadas. As análises de estresse oxidativo revelaram não existir diferença em seus níveis em nenhum dos grupos avaliados. O estudo avaliando a ação do óleo e do extrato pode determinar que ambos atuam a nível de ergosterol da membrana do fungo.

The search for new drugs with antimicrobial action has been extensively studied 11 especially with vegetal extracts, however, the research of their action against fungi is 12 still scarce. Among the mycoses of public health importance, sporotrichosis has 13 emerged in recent years as an important zoonotic disease in large urban centers, 14 affecting cats, dogs and humans. The treatment of this mycosis is carried out with daily 15 administration of antifungals, especially itraconazole, which is considered the drug of 16 choice for both animals and humans. However, the commercially available active 17 principles with action in the fungi of the Sporothrix schenckii complex are restricted. In 18 this sense, there is a need for other therapeutic options for the treatment of 19 sporotrichosis. Thus, the in vitro activity of plants demonstrated by preliminary studies 20 with isolates of the Sporothrix schenckii complex stimulated the realization of the 21 present study in order to determine the in vivo activity of the vegetal extracts of 22 Origanum vulgare for the treatment of experimental cutaneous sporotrichosis. For this, 23 60 male Wistar rats were divided into 6 experimental groups inoculated with S. 24 brasiliensis for the development of cutaneous sporotrichosis and submitted to 30 days 25 of treatment with itraconazole, O. vulgare essential oil, hydroalcoholic extract of O. 26 vulgare, association of the oil with itraconazole, association of the extract with 27 itraconazole and a control group treated only with the vehicle. During the 30 days, the 28 animals were evaluated weekly and 2 animals per group were euthanized for the 29 collection of material to analyze the development of the disease and the evolution of 30 the different treatments. At the end of the 30 days, the remaining animals from each 31 group were euthanized and also had biological materials collected for the analyzes. At 32 all necropsies, samples of liver, kidney, spleen, testis, inoculation point and regional 33 lymph node were collected, as well as blood samples. As results obtained, only the 34 animals belonging to the ITRA and CONT groups did not show complete 35 disappearance of the clinical signs observed at the end of the 30 days of treatment, it 36 was also possible to observe that the animals belonging to the groups treated with the 37 hydroalcoholic extract of O. vulgare and with the essential oil of O. vulgare did not 38 present any microscopic changes in the kidneys, liver and spleen throughout the 39 experiment, thus indicating a possible effect of this plant on the spread of the disease. 40 Sporothrix brasiliensis was retroisolated from the regional lymph node of all animals of 41 all groups at the four necropsies performed. Analyzes of oxidative stress revealed no 42 difference in their levels in any of the groups evaluated. The study evaluating the action 43 of oil and extract may determine that both act at the ergosterol level of the fungus 44 membrane.