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.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

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

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.

Caracterização de corantes solvatocrômicos como marcadores físico-químicos de medicamentos homeopáticos e de amostras biológicas de sobrenadantes

O presente estudo tem a finalidade de determinar se corantes solvatocrômicos podem ser utilizados como método de escolha de medicamentos homeopáticos para tratamento de sistemas vivos infectados com diferentes agentes infecciosos. Tal método permite analisar possíveis alterações no momento dipolo da água quando em contato com diluições homeopáticas de diferentes origens. Métodos: A pesquisa se desenvolveu a partir de sobrenadantes (SNs) colhidos de macrófagos RAW 264.7 estimulados com bacilos Calmette-Guérin (BCG) (cepa vacinal) e tratados com Silicea terra 6, 30, 200 cH, Zincum metallicum 6, 30, 200 cH e controles; desafiados com Encephalitozoon cuniculi (E. cuniculi) e tratados com Phosphorus 6, 30, 200 cH e controles; ou ainda ativados com LPS (mimetizando infecção por bactérias Gram negativas) e tratados com Aspirina 15 cH e controles. Os SNs descongelados foram diluídos 1:10 em água pura estéril e 1:100 em solução hidro-alcooólica 30%, sendo sucussionados por 100 vezes em braço mecânico automático (Autic®). Os respectivos medicamentos também foram preparados em solução hidro- alcooólica 30% do mesmo lote e sucussionados da mesma forma. Todas as amostras foram codificadas aleatoriamente, em números consecutivos, por um técnico não envolvido com a experimentação. Os códigos só foram quebrados após a análise estatística, de forma que todo o estudo foi realizado em cego. Antes de cada experimento, as amostras foram submetidas a 40 agitações verticais manuais, filtradas em filtro de malha 0.22 micrômetros e adicionadas aos corantes na proporção 1:60. Seis diferentes corantes solvatocrômicos (BDN, NNDMIA, Cumarina, Rodanina, Violeta metileno e Vermelho do Nilo) foram testados, a fim de cobrir todo o espectro de luz visível. A leitura em espectrofotômetro foi feita em cubetas descartáveis e a concentração dos corantes foi definida segundo padrões previamente estabelecidos, utilizando álcool absoluto como solvente. Todas as preparações foram feitas em capela de fluxo laminar e com o mínimo de incidência de luz possível sobre os corantes, até o momento da leitura. As condições ambientais foram controladas diariamente, sendo medidas temperatura, umidade e fluxo magnético. Antes de cada série de medidas, o equipamento foi calibrado com o mesmo álcool absoluto usado na preparação dos corantes e o comprimento de onda correspondente ao pico de absorbância foi registrado. As amostras foram analisadas em triplicata, em três séries experimentais consecutivas (N=9). O perfil de absorbância de cada medicamento foi comparado com a absorbância dos respectivos SNs. O método estatístico usado foi a ANOVA de uma via, seguido de Tukey, sendo os valores de p<0,05 considerados significantes. Testes de normalidade foram feitos por Shapiro- Wilk e eventuais outliers foram removidos após inspeção do QQ plot. Conclusão: Concluiu-se, portanto, que a Rodanina é um bom marcador de solventes submetidos à sucussão, provavelmente em função da atividade oxidativa associada; Cumarina é marcador de Phosphorus 6cH e de sobrenadantes tratados com Aspirina 15 cH e LPS; BDN é marcador de Silicea terra 6 cH, 30 cH e 200 cH, Zincum metallicum 30 cH e 200cH e Aspirina 15cH; Violeta metileno é marcador de sobrenadantes tratados com Silicea terra 30 cH e 200cH, bem como Zincum metallicum 6 cH e 200cH. Os demais corantes não apresentaram interações específicas com as amostras estudadas.

The present study aims to determine whether solvatochromic dyes can be used as a method of choice for homeopathic medicines to treat living systems infected with different infectious agents. This method allows analyzing possible changes in the dipole moment of water when in contact with homeopathic dilutions of different origins. The research was developed from supernatants (SNs) collected from RAW 264.7 macrophages stimulated with bacilli Calmette-Guérin (BCG) (vaccinal strain) and treated with Silicea terra 6, 30, 200 cH, Zincum metallicum 6, 30, 200 cH, and controls. Supernatants challenged with Encephalitozoon cuniculi (E. cuniculi) and treated with Phosphorus 6, 30, and 200 cH and controls, or even from macrophages activated with LPS (mimicking infection by Gram-negative bacteria) treated with Aspirin 15 cH and controls. The thawed SNs were diluted 1:10 in sterile water, then 1:100 in 30% hydro-alcoholic solution and submitted to 100 succussions in a robotic mechanical arm (Autic®). The respective medicines were also prepared in 30% hydro- alcoholic solution from the same batch, being succussed in the same way. All samples were randomly coded in consecutive numbers by a technician not involved in the experiment. The codes were only broken after statistical analysis; thus, the study was performed entirely blind. Before each experiment, the samples were submitted to 40 manual vertical agitations, filtered in a 0.22- micrometer mesh filter, and added to the dyes in a 1:60 ratio. Six different solvatochromic dyes (BDN, NNDMIA, Coumarin, Rhodanine, Methylene Violet, and Nile Red) were tested to cover the whole visible light spectrum. The reading was done in a spectrophotometer in disposable cuvettes, and the concentration of the dyes was defined according to previously established standards, using absolute alcohol as a solvent. All preparations were carried out in a laminar flow hood, with an as little light incidence as possible on the dyes, until the moment of reading. The environmental conditions were controlled daily, being measured temperature, humidity, and magnetic flux. Before each series of measurements, the equipment was calibrated with the same absolute alcohol used in the preparation of the dyes, and the wavelength corresponding to the peak of absorbance was recorded. The samples were analyzed in triplicate, in three consecutive experimental series (N=9). The absorbance profile of each drug was compared with the absorbance of the respective SNs. The statistical method used was one-way ANOVA, followed by Tukey, with p<0.05 being considered significant. The Shapiro-Wilk test checked normality, and eventual outliers were removed after inspection of the QQ plot. Therefore, it was concluded that Rodanine is a good marker of solvents subjected to succussion, probably due to its associated oxidative activity; Coumarin is a marker of Phosphorus 6cH and of supernatants treated with Aspirin 15cH and LPS; BDN is a marker of Silicea terra 6 cH, 30 cH and 200 cH, Zincum metallicum 30 cH and 200cH and Aspirin 15cH; Methylene violet is a marker of supernatants treated with Silicea terra 30 cH and 200cH, as well as Zincum metallicum 6 cH and 200cH. The other dyes did not show specific interactions with the samples studied.

Análise do óleo essencial de Origanum majorana relacionada ao seu potencial tóxico e antineoplásico

O câncer é apontado como um problema de saúde pública de proporções mundiais, 11 uma vez que as taxas de incidência são crescentes, a mortalidade é alta e as 12 opções de tratamento farmacológico disponíveis apresentam efeitos adversos 13 graves e vêm perdendo sua eficácia ao longo do tempo, devido ao desenvolvimento 14 de mecanismos de resistência pelas células neoplásicas. Frente a essa realidade, a 15 indústria farmacêutica tem nas plantas medicinais um amplo painel de moléculas 16 potencialmente eficazes e menos tóxicas a serem estudadas. A tualmente já está 17 cientificamente comprovado qu e os compostos químicos presentes nas plantas 18 medicinais podem proteger as células de eventos carcinogênicos, agindo contra a 19 promoção e a progressão tumorais. Porém, os mesmos princípios ativos que 20 conferem as propriedades terapêuticas a essas plantas, ta mbém podem ser os 21 responsáveis pelos seus efeitos tóxicos e reações adversas. Nesse contexto, o 22 presente trabalho tem como objetivos estudar a citotoxicidade do óleo essencial de 23 Origanum majorana (OEOM) em células espermáticas suínas e hemácias e o seu 24 me canismo de ação intracelular em linhagens celulares não neoplásicas (MDBK 25 Madin Darby Bovine Kidney) e neoplásicas (B16F10 melanoma murino) e 26 determinar a sua composição química. A determinação da toxicidade do OEOM in 27 vitro foi avaliada nas células es permáticas através de parâmetros de cinética (CASA 28 = Computer Assisted Sperm Analisys) e de estrutura (citometria de fluxo), o 29 mecanismo de ação nas células MDBK e B16F10 foi avaliado por citometria de fluxo, 30 e a atividade hemolítica em eritrócitos caninos , pelo teste em meio líquido e em meio 31 sólido com ágar sangue de ovinos. Para a identificação dos compostos químicos do 32 óleo essencial foi utilizado cromatógrafo a gás acoplado a detector de massas 33 (GC/MS QP 2010SE Shimadzu, Japão), equipado com auto injet or AOC 20i. As 34 quantificações foram feitas por área normatizada e as identificações dos compostos 35 pelo espectrômetro de massas, utilizando a biblioteca NIST 8 do GC/MS. O OEOM é 36 composto majoritariamente por terpenos, principalmente por gama terpinen o , cis 37 sabineno hidratado e 4 terpineol. Na avaliação imediata da cinética espermática o 38 OEOM reduziu a motilidade total (MT) e progressiva (MP) e a distância média 39 percorrida (DAP) em todas as concentrações testadas, enquanto que, após 24 horas, 40 as concentraçõe s tóxicas foram de 1 a 4 mg.mL 1 . Na citometria de fluxo, as 41 estruturas afetadas foram as mitocôndrias (PMM) em todas as concentrações e a 42 membrana, que teve a sua fluidez reduzida com 0,25 e 1 mg.mL 1 de OEOM. As 43 concentrações de OEOM capazes de provocar hemólise em ambos os tipos 44 celulares foram 1, 2 e 4 mg.mL 1 , chegando a percentuais de 90 a 100% de hemólise. 45 Nos testes com a linhagem celular MDBK observou se redução de espécies reativas 46 de oxigênio (ERO)e lipoperoxidação (LPO) em todas as concentrações de oxigênio (ERO)e lipoperoxidação (LPO) em todas as concentrações utilizadas de utilizadas de 1 OEOM, nas células MDBK, e também na fase S do ciclo celular. Nas células B16F10, OEOM, nas células MDBK, e também na fase S do ciclo celular. Nas células B16F10, 2 os tratamentos com o OEOM reduziram ERO e as células em fase G2 e os tratamentos com o OEOM reduziram ERO e as células em fase G2 e 3 aumentaram as células na fase S. As concentrações de 0,25 e 0,5 mg.mLaumentaram as células na fase S. As concentrações de 0,25 e 0,5 mg.mL--1 1 4 causaram redução na flcausaram redução na fluidez e aumento do potencial de membrana mitocondrial, uidez e aumento do potencial de membrana mitocondrial, 5 respectivamente na linhagem MDBK, enquanto LPO nas células B16F10 diminuíram respectivamente na linhagem MDBK, enquanto LPO nas células B16F10 diminuíram 6 na concentração de 0,5 mg.mLna concentração de 0,5 mg.mL--11.. Os resultados obtidos nesse estudo apontam para Os resultados obtidos nesse estudo apontam para 7 a elevada toxicidade celular demonstrada, pra elevada toxicidade celular demonstrada, principalmente, nas concentrações entre incipalmente, nas concentrações entre 8 1 e 4 mg.mL1 e 4 mg.mL--11 do OEOM nas células espermáticas suínas e nas hemácias. Quanto do OEOM nas células espermáticas suínas e nas hemácias. Quanto 9 ao mecanismo de ação do OEOM, é possível supor que seu efeito seja citostático ao mecanismo de ação do OEOM, é possível supor que seu efeito seja citostático 10 para as células neoplásicas utilizadas, devido à interferênciapara as células neoplásicas utilizadas, devido à interferência no ciclo celular, sem no ciclo celular, sem 11 exercer efeitos tóxicos na linhagem MDBK, uma vez que os parâmetros relacionados exercer efeitos tóxicos na linhagem MDBK, uma vez que os parâmetros relacionados 12 à membrana plasmática e produção e ERO reduziram. A viabilidade celular não foi à membrana plasmática e produção e ERO reduziram. A viabilidade celular não foi 13 alterada em nenhuma das linhagens utilizadas nos testes. Considerando osalterada em nenhuma das linhagens utilizadas nos testes. Considerando os 14 resultados desse estudo, concluiresultados desse estudo, conclui--se que o OEOM é uma promissora fonte de se que o OEOM é uma promissora fonte de 15 moléculas com potencial terapêutico para o tratamento do câncer, uma vez que é moléculas com potencial terapêutico para o tratamento do câncer, uma vez que é 16 capaz de promover a interrupção da proliferação celular da linhagem neoplásica, capaz de promover a interrupção da proliferação celular da linhagem neoplásica, 17 mesmo em concentrações mesmo em concentrações baixas. A toxicidade apresentada pelo OEOM nas células baixas. A toxicidade apresentada pelo OEOM nas células 18 sanguíneas e espermáticas foi mais evidente nas concentrações mais elevadas e sanguíneas e espermáticas foi mais evidente nas concentrações mais elevadas e 19 não foi observada nas células MDBK.não foi observada nas células MDBK.

Cancer is identified as a public health problem of worldwide proportions, since 12 incidence rates are increasing, mortality is high and the pharmacological treatment 13 options available have serious adverse effects and have been losing their 14 effectiveness over time due to the development of resistance mechanisms by 15 neoplastic cells. Faced with this reality, the pharmaceutical industry has in medicinal 16 plants a wide panel of potentially effective and less toxic molecules to be studied. It is 17 now scientifically proven that chemical compounds present in medicinal plants can 18 protect cells from carcinogenic events, acting against tumor promotion and 19 progression. However, the same active ingredients that give therapeutic properties to 20 these plants, may also be responsible for their toxic effects and adverse reactions. In 21 this context, the present work aims to study the cytotoxicity of the essential oil of 22 Origanum majorana (OEOM) in swine sperm cells and red blood cells and its 23 mechanism of intracellular action in non-neoplastic (MDBK - Madin Darby Bovine 24 Kidney) and neoplastic cell lines ( B16F10 - murine melanoma) and determine its 25 chemical composition. The determination of OEOM toxicity in vitro was evaluated in 26 sperm cells through kinetic parameters (CASA = Computer Assisted Sperm 27 Analyzes) and structure (flow cytometry), the mechanism of action in MDBK and 28 B16F10 cells was evaluated by flow cytometry. , and hemolytic activity in canine 29 erythrocytes, by testing in liquid and solid media with sheep blood agar. For the 30 identification of the chemical compounds of the essential oil, a gas chromatograph 31 coupled to a mass detector (GC / MS-QP 2010SE-Shimadzu, Japan) was used, 32 equipped with an AOC-20i auto injector. The quantifications were made by 33 standardized area and the identification of the compounds by the mass spectrometer, 34 using the NIST 8 library of the GC / MS. OEOM is composed mainly of terpenes, 35 mainly gamma-terpineno, cis-sabinene hydrate and 4-terpineol. In the immediate 36 evaluation of sperm kinetics, OEOM reduced total (MT) and progressive (MP) motility 37 and average distance traveled (DAP) in all concentrations tested, whereas, after 24 38 hours, toxic concentrations were 1 to 4 mg.mL -1. In flow cytometry, the affected 39 structures were the mitochondria (PMM) in all concentrations and the membrane, 40 which had its fluidity reduced with 0.25 and 1 mg.mL-1 of OEOM. The concentrations 41 of OEOM capable of causing hemolysis in both cell types were 1, 2 and 4 mg.mL-1, 42 reaching percentages of 90 to 100% of hemolysis. In tests with the MDBK cell line, a 43 reduction in reactive oxygen species (ROS) and lipoperoxidation (LPO) was 44 observed in all concentrations of OEOM used in MDBK cells, and also in the S phase 45 of the cell cycle. In B16F10 cells, treatments with OEOM reduced ROS and G2 cells 1 and increased cells in S phase. The concentrations of 0.25 and 0.5 mg.mL-1 caused 2 a reduction in fluidity and an increase in membrane potential mitochondrial, 3 respectively in the MDBK lineage, while LPO in B16F10 cells decreased in the 4 concentration of 0.5 mg.mL-1. The results obtained in this study point to the high 5 cellular toxicity demonstrated, mainly, in the concentrations between 1 and 4 mg.mL-1 6 of the OEOM in the swine sperm cells and in the erythrocytes. As for the mechanism 7 of action of the OEOM, it is possible to assume that its effect is cytostatic for the 8 neoplastic cells used, due to the interference in the cell cycle, without exerting toxic 9 effects in the MDBK lineage, since the parameters related to the plasma membrane 10 and production and ERO reduced. Cell viability was not altered in any of the strains 11 used in the tests. Considering the results of this study, it is concluded that OEOM is a 12 promising source of molecules with therapeutic potential for the treatment of cancer, 13 since it is able to promote the interruption of cell proliferation of the neoplastic 14 lineage, even at low concentrations. The toxicity shown by OEOM in blood and 15 sperm cells was more evident at higher concentrations and was not seen in MDBK 16 cells.

Prebiotic inulin: Useful dietary adjuncts to manipulate the livestock gut microflora

In recent years, there has been a growing appreciation on the relevance of gastrointestinal microflora in both ruminants and non-ruminants owing to revelation of their role in several physiological functions including digestion, nutrient utilization, pathogen exclusion, gastrointestinal development, immunity system, gut gene expression and quality of animal products. The ban imposed on the use of antibiotics and hormones in feed has compelled animal researchers in finding an alternative which could overcome the issues of conventional feed additives. Though the concept of prebiotic was evolved keeping in mind the gastrointestinal flora of human beings, presently animal researchers are exploring the efficiency of prebiotic (inulin) for modulating the gut ecosystem of both ruminants and non-ruminants. It was revealed that prebiotic inulin is found to exhibit desirable changes in the gut of non-ruminants like poultry, swine, rabbit etc for augmenting gut health and improvement of product quality. Similarly, in ruminants the prebiotic reduces rumen ammonia nitrogen, methane production, increase microbial protein synthesis and live weight gains in calves. Unlike other feed additives, prebiotic exhibits its effect in multipronged ways for overall increase in the performances of the animals. In coming days, it is expected that prebiotics could be the part of diets in both ruminants and non-ruminants for enabling modulation of gut microflora vis a vis animals productivity in ecological ways.(AU)

Avaliação da atividade antiviral do peptídeo P34 e moléculas da classe da 4-tiazolidinona frente a vírus de importância veterinária

A busca de novas substâncias com atividade antiviral levou este estudo a investigar a ação do peptídeo antimicrobiano P34, produzido pelo Bacillus sp. P34, frente ao herpesvírus bovino tipo 1 (BoHV-1) e de moléculas sintetizadas da 2-picolilamina e 2-aminoetilpiperidina derivadas da classe da 4-tiazolidinona denominadas V19 (2a), V20 (2b), V23 (2c), V28 (2d), V29 (2e), AK11 (1a), AK16 (1d), AK17 (1e), AK18 (1b) e AK20 (1c) frente a diversos vírus que infectam animais. Inicialmente foram determinadas as concentrações não citotóxicas dos diferentes compostos, em diferentes linhagens celulares, através dos métodos MTT e vermelho neutro. A seguir a atividade antiviral foi avaliada mediante um ensaio de inibição do efeito citopático. Em todos os testes, o título viral em cultivo celular foi comparado ao título viral na presença de cada composto para a determinação do percentual de inibição (PI). O P34 demonstrou um PI de 99,94% e alto índice de seletividade (SI de 22,9). Os testes para identificar o modo de ação indicaram que o peptídeo P34 não atua sobre as células MDBK, ou receptores celulares. Em concentrações citotóxicas o P34 obteve efeito virucida e 100% de inibição viral nos ensaios de redução viral e de penetração. Foi evidenciada total inibição na produção das partículas de BoHV-1 quando as células foram tratadas com o P34 posterior a 8 h de infecção. Houve redução significativa (p< 0.01) do título do BoHV-1 na presença do P34 após 8 h de infecção (PI de 90%), chegando a 99,9% de PI após 18 h de tratamento pósinfecção. O P34 inibiu o BoHV-1 tanto no meio extracelular, inibindo a infecção celular, quanto no meio intracelular, após a infecção, impedindo o egresso. Com relação às moléculas derivadas da 4-tiazolidinona a menos tóxica foi a AK 16 (157 µg/mL) nas quatro linhagens de células (RK13, MDCK, CRFK e MDBK) e as mais tóxicas foram as AK11, AK18 e AK20 (19 µg/mL). O PI da molécula AK16 foi de 96,9%, 90,1% e 90,1% contra o EAV, FCV e CPV-2, respectivamente. O composto AK17 mostrou PI de 90,1% contra o EAV, a V20 de 78% contra BVDV, a V28 de 94,4% contra EIV e 78% contra BVDV e a V29 de 94,4% contra CPV-2. As moléculas V20 e V28 foram escolhidas para dar continuidade ao estudo frente ao BVDV. Foram determinados para V20 um SI de 16,8 e de 14,73 para V28. Nos ensaios de inibição da produção de partículas virais, adsorção, competição com receptores celulares, penetração viral e efeito virucida não foram observadas diferenças significativas entre os títulos do BVDV com e sem tratamentos com as moléculas. A associação das moléculas V20 e V28 aumentou de 78 para 82% o PI, não sendo verificada alteração no PI quando as moléculas foram associadas com os antivirais interferon- 2b ou ribavirina. Através da determinação da curva de crescimento do BVDV foi evidenciada atuação no período entre 1 a 5 h pós-infecção com o BVDV, sugerindo ação posterior à entrada viral, mas antes do egresso, provavelmente no período de síntese do RNA e enzimas virais. Os resultados demonstram que o peptídeo P34 e os compostos V20 e V28 derivados da 4tiazolidinona podem ser considerados promissores antivirais contra BoHV-1 e BVDV.

The search for new substances with antiviral activity has led this study to investigate the action of the antimicrobial peptide P34, produced by Bacillus sp. P34 against bovine herpesvirus type 1 (BoHV-1) and of the synthetic molecules 2-picolylamine and 2-aminoethylpiperidine, which are derived from the class of thiazolidin-4-one named V19 (2a), V20 (2b), V23 (2c), V28 (2d), V29 (2e), AK11 (1a), AK16 (1d), AK17 (1e), AK18 (1b) and AK20 (1c) in opposition to several viruses that infect animals. Initially non-cytotoxic concentrations of the different compounds were determined, in different cell lines, through the MTT and neutral red methods. Then, the antiviral activity was evaluated by an assay of the cytopathic effect inhibition. In all tests, the viral titer in cell culture was compared to the viral titer in the presence of each compound to determine the percentage of inhibition (PI). The P34 demonstrated a PI of 99.94% and high selectivity index (SI 22.9). Tests to identify the mode of action indicated that the peptide P34 has no effect on MDBK cells, or cell receptors. In cytotoxic concentrations P34 obtained virucidal effect and 100% inhibition in viral reduction viral and penetration assays. Total inhibition was observed in the production of the BoHV-1 particles when cells were treated with P34 after 8 h of infection. There was a significant reduction (p <0.01) in BoHV-1 titer in the presence of P34 after 8 h of infection (PI 90%), reaching 99.9% PI after 18 h postinfection treatment. P34 inhibited BoHV-1 as extracellularly, inhibiting cell infection, as intracellularly, after infection, preventing its egress. Regarding molecules derived from thiazolidin-4-one the less toxic was AK16 (157 µg/mL) in the four cell lines (RK13, MDCK, CRFK and MDBK), and the most toxics were AK11, AK18 and AK20 (19 µg/mL). The PI of AK16 molecule was 96.9%, 90.1% and 90.1% against EAV, FCV and CPV-2, respectively. The compound AK17 showed PI of 90.1% against EAV, V20 of 78% against BVDV, V28 of 94.4% against EIV and 78% against BVDV and the V29 of 94.4% against CPV-2. V20 and V28 molecules were chosen to continue the study against BVDV. To V20 and V28 molecules, were determined SI of 16.8 and 14.73, respectively. In assays of inhibition of production of viral particles, adsorption, competing with cellular receptors, viral penetration and virucidal effect no significant differences were observed between titers the BVDV with and without treatment with the molecules. The association of V20 and V28 molecules increased the PI from 78 to 82%, and changes in the PI were not verified when the molecules were associated with antiviral interferon- 2b and ribavirin. Through the determination of BVDV growth curve, was observed activity in the period between 1 to 5 h post-infection with BVDV, suggesting further action on viral entry, but probably, before the egress, in the period of RNA synthesis and viral enzymes. The results demonstrate that peptide P34 and compounds V20 and V28 derived from thiazolidin4-one can be considered as promising antiviral against BoHV-1 and BVDV.

Poison as cure: a clinical review of botulinum toxin as an invaluable drug

Botulinum toxin is the most potent toxin known. It is readily absorbed from mucosal surfaces. If dispersed as an aerosol or mixed in the food or water it can lead to a large outbreak of botulism. The disease presents as a symmetric descending paralysis in an afebrile patient. Cranial nerve involvement with diplopia, dysarthria, dysphonia, dysphagia and respiratory paralysis is seen after a variable incubation period. The treatment is mainly supportive. The source of the toxin is Clostridium botulinum, an anaerobic gram-positive spore-forming organism. Some other species of Clostridium like C. butyricum and C. baratii also produce the toxin. The toxin is heat labile and can be inactivated by heating at 100°C for 10 minutes. The toxin acts at the peripheral cholinergic nerve terminals at the neuromuscular junctions, postganglionic parasympathetic ganglia, etc, and affects neurotransmitter release by inhibiting exocytosis. Clinical uses in various medical fields were found for it.(AU)

Poison as cure: a clinical review of botulinum toxin as an invaluable drug

Botulinum toxin is the most potent toxin known. It is readily absorbed from mucosal surfaces. If dispersed as an aerosol or mixed in the food or water it can lead to a large outbreak of botulism. The disease presents as a symmetric descending paralysis in an afebrile patient. Cranial nerve involvement with diplopia, dysarthria, dysphonia, dysphagia and respiratory paralysis is seen after a variable incubation period. The treatment is mainly supportive. The source of the toxin is Clostridium botulinum, an anaerobic gram-positive spore-forming organism. Some other species of Clostridium like C. butyricum and C. baratii also produce the toxin. The toxin is heat labile and can be inactivated by heating at 100°C for 10 minutes. The toxin acts at the peripheral cholinergic nerve terminals at the neuromuscular junctions, postganglionic parasympathetic ganglia, etc, and affects neurotransmitter release by inhibiting exocytosis. Clinical uses in various medical fields were found for it.

An alternative for antibiotic se in poultry: probiotics

Over the past 50 years, there has been increasing amounts of antibiotics used prophylactically and as growth promoters. Today, there is a consumer and governmental outcry to eliminate that practice from poultry and livestock production. Evidence has been accumulated to show that there is a link between risk of zoonotic disease and growth promoting antibiotic usage in livestock and poultry. Therefore, alternatives to the use of growth promoting antibiotics must be found to promote growth or production at or near the genetic potential of the modern day broiler, turkey, and egg producer. The use of probiotics has many potential benefits and include modified host metabolism, immuno-stimulation, anti-inflammatory reactions, exclusion and killing of pathogens in the intestinal tract, reduced bacterial contamination on processed broiler carcasses, enhanced nutrient absorption and performance, and ultimately decreased human health risk. The development of these factors generally can be ascribed to the ability of most probiotic products to balance and maintain the intestinal microflora in poultry species.

An alternative for antibiotic se in poultry: probiotics

Over the past 50 years, there has been increasing amounts of antibiotics used prophylactically and as growth promoters. Today, there is a consumer and governmental outcry to eliminate that practice from poultry and livestock production. Evidence has been accumulated to show that there is a link between risk of zoonotic disease and growth promoting antibiotic usage in livestock and poultry. Therefore, alternatives to the use of growth promoting antibiotics must be found to promote growth or production at or near the genetic potential of the modern day broiler, turkey, and egg producer. The use of probiotics has many potential benefits and include modified host metabolism, immuno-stimulation, anti-inflammatory reactions, exclusion and killing of pathogens in the intestinal tract, reduced bacterial contamination on processed broiler carcasses, enhanced nutrient absorption and performance, and ultimately decreased human health risk. The development of these factors generally can be ascribed to the ability of most probiotic products to balance and maintain the intestinal microflora in poultry species.