Atividade antifúngica e modo de ação do óleo essencial de Coriandrum sativum L. (coentro) sobre Candida spp. e alvos moleculares afetados na expressão do genoma humano

Orientadores: Pedro Luiz Rosalen, Marta Cristina Teixeira DuarteDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Introdução: A candidíase oral é uma infecção fúngica oportunista da cavidade oral, cujas taxas de prevalência e incidência vêm aumentando significativamente em todo o mundo. Assim, novas estratégias orientadas para gerir esta doença têm sido propostas, dentre as quais está o uso de óleos essenciais (OE) com propriedades antifúngicas. Evidências indicam que o OE de Coriandrum sativum L. (coentro) é um forte agente antifúngico contra Candida e, portanto, investigações devem dar continuidade ao conhecimento gerado. Objetivo: Este estudo buscou avaliar a atividade antifúngica e modo de ação do OE de C. sativum sobre Candida spp., e determinar os alvos moleculares afetados na expressão global do genoma humano. Material e Métodos: C. sativum foi obtido a partir do Banco de Germoplasmas do Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (Universidade Estadual de Campinas, SP, Brasil) cujo OE e fração ativa tiveram o perfil fitoquímico determinado por cromatografia gasosa acoplada a espectrometria de massa. Posteriormente, foram realizados testes com cinco cepas de referência de Candida: Determinação da Concentração Inibitória e Fungicida Mínima (CIM/CFM); modo de ação antifúngica (ensaio do sorbitol e ergosterol); Microscopia Eletrônica de Varredura (MEV) de biofilmes de Candida e testes de inibição de aderência em biofilme. Utilizou-se nistatina, anfotericina B ou caspofungina como controles positivos, além de controles negativos. Também, foi testado o efeito do OE sobre a atividade proteolítica de C. albicans. Por fim, um ensaio de farmacogenômica identificou quais alvos moleculares no genoma humano foram afetados pelo OE e fração ativa de C. sativum. Os testes foram realizados em triplicata de experimentos independentes e os dados foram tratados estatisticamente (ANOVA, pós-teste de Tukey, ?=0,05). Os dados da análise farmacogenômica foram processados nas plataformas GeneGo MetaCore® e David Bioinformatics Resources. Resultados: O perfil fitoquímico EO indicou monoterpenos (37,9%) e sesquiterpenos (62,1%) como compostos principais. Os valores de CIM/CFM para o OE variaram de 15,6 a 62,5 µg/mL. Quanto ao modo de ação, o OE de C. sativum parece se ligar ao ergosterol da membrana celular fúngica, aumentando a permeabilidade iônica e causando morte celular; entretanto, o OE não atua sobre vias de biossíntese da parede celular. Estes achados confirmam as alterações na integridade da morfologia do biofilme verificadas nas análises por MEV. Além disso, o OE apresentou atividade antiaderente em biofilme em baixas concentrações (15,6-62,5 µg/mL) contra as cepas testadas, bem como atividade contra proteases produzidas por C. albicans, sendo estatisticamente significante na CIM (p<0,05). Finalmente, o OE e sua fração ativa apresentaram baixa citotoxicidade em células humanas com CI30 de 359,8 e 366,7 µg/mL, respectivamente. As principais vias afetadas estão relacionadas com quimiocinas e MAP-quinase (apoptose, proliferação) bem como proteínas de adesão. Conclusões: O OE das folhas de C. sativum tem forte atividade antifúngica e antiaderente sobre Candida spp. e atividade anti-proteolítica sobre C. albicans, e atua aumentando a permeabilidade iônica da membrana celular, provavelmente devido ao efeito sinérgico de mono e sesquiterpenos. Análise farmacogenômica indicou baixa citotoxicidade do OE e sua fração ativa com alvos moleculares específicos afetados no genoma humano, o que incentiva o desenvolvimento de novas pesquisas pré-clínicas toxicológicas e clínicas nesta áreaAbstract: Introduction: Oral candidiasis is a common opportunistic fungal infection of the oral cavity with increasingly significant worldwide prevalence and incidence rates. Accordingly, novel specific-targeted strategies to manage this ailment have been proposed, among which is the use of essential oils (EO) with antifungal properties. Coriandrum sativum L. (coriander) EO has proven antifungal activity against Candida species and thus deserves further investigation. Objective: This study aimed to evaluate the antifungal activity and mode of action of the EO from Coriandrum sativum L. leaves on Candida spp., and to determine the molecular targets affected in the whole-genome expression in human cells. Material and Methods: C. sativum was obtained from the Germoplasm Bank of the Research Center for Chemistry, Biology and Agriculture (University of Campinas, SP, Brazil) whose EO and active fraction had their phytochemical profile determined by Gas Chromatography coupled to Mass Spectrometry. Then, we carried out the following tests with five reference strains of Candida spp. (CBS): Minimum Inhibitory and Fungicidal Concentration (MIC/MFC); antifungal mode of action (sorbitol and ergosterol assays); Scanning Electron Microscopy (SEM) analysis of Candida biofilm and tests of inhibition of biofilm adherence. We used nystatin, amphotericin B or caspofungin as positive controls, in addition to negative controls. Also, we tested the effect of C. sativum EO on the proteolytic activity of C. albicans. Next, a pharmacogenomic assay identified which molecular targets in the human genome were affected by C. sativum EO and its active fraction. Tests were performed in triplicate of independent experiments and data were statistically treated (ANOVA, Tukey¿s post-test, ?=0.05). Pharmacogenomic data were processed on GeneGo MetaCore® and DAVID Bioinformatics Resources. Results: The EO phytochemical profile indicated monoterpenes (37.9 %) and sesquiterpenes (62.1 %) as major compounds. The MIC/MFC values for the EO ranged from 15.6 to 62.5 µg/ml. With regard to the mode of action, C. sativum EO may bind to membrane ergosterol, increasing ionic permeability and causing membrane damage to cell death, but it does not act on cell wall biosynthesis-related pathways. This mode of action confirms the changes in the integrity of the biofilm morphology as verified in the analyses by SEM. The EO showed anti-adherent activity at low concentrations (31.2 ¿ 62.5 µg/ml) against all strains tested, as well as activity against proteases produced by C. albicans, with statistical significance at MIC (P < 0.05). Finally, the EO and its active fraction had low cytotoxicity on human cells with IC30 of 359.8 and 366.7 µg/ml, respectively, affecting the pathways of chemokines and MAP-kinase (apoptosis, proliferation), as well as adhesion proteins. Conclusions: The EO from C. sativum leaves has strong antifungal and anti-adherent activity against Candida spp., as well as anti-proteolytic activity against C. albicans, and acts by increasing cell membrane ionic permeability, probably due to the synergistic effect of mono- and sesquiterpenes. Pharmacogenomic analyses revealed low cytotoxicity with specific targets affected in the human genome, which encourages further pre-clinical toxicological screening and clinical research in this fieldMestradoFarmacologia, Anestesiologia e TerapeuticaMestre em Odontologi

The role of Streptococcus mutans Cnm in intracellular invasion, adhesion to human cardiac tissues, and cariogenicity in vivo

Orientador: Pedro Luiz RosalenTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Um dos principais agentes etiológicos da cárie dentária, Streptococcus mutans, dispõe de um sofisticado repertório de mecanismos de sobrevivência e virulência, pelos quais pode causar doenças orais e extra-orais. A capacidade de S. mutans interagir com componentes de matriz extracelular tecidual, tais como colágeno, permite a colonização de diferentes tecidos do hospedeiro. Previamente, demonstramos que a proteína ligante de colágeno Cnm (~120 kDa) promove adesão ávida de S. mutans à dentina radicular ex vivo e aumenta cárie coronária in vivo. Todavia, a influência de Cnm sobre o desenvolvimento de cárie radicular, onde o colágeno é abundante, permanece desconhecida. No Capítulo I, utilizamos um modelo de ratos dessalivados sob alto desafio cariogênico para investigar o papel de Cnm no desenvolvimento de cárie radicular. Ratos infectados com a cepa OMZ175?cnm (knockout) apresentaram menos cárie coronária com envolvimento inicial de dentina quando comparados aos infectados com a cepa selvagem OMZ175 e a complementada OMZ175Acnm::pcnm (p<0,05) após 5 semanas. No entanto, não foram observadas diferenças significativas quanto ao desenvolvimento de cárie radicular. Interessantemente, os ratos infectados com OMZ175 ou OMZ175?cnm::pcnm apresentaram maior exposição radicular (até cerca de 18,5% mais elevada, P <0,05) do que aqueles infectados com OMZ175?cnm ou UA159 (cepa naturalmente negativa para Cnm). Em síntese, este capítulo descreve a contribuição de Cnm no desenvolvimento da cárie, particularmente nos estágios iniciais de lesão dentinária, e destaca seu potencial impacto no desenvolvimento de doenças periodontais e destruição óssea alveolar, a ser investigado em estudos futuros. Há também evidências de que Cnm desempenha um papel importante, e ainda subestimado, no desenvolvimento de doenças cardiovasculares. Contudo, ainda não se conhecem os mecanismos pelos quais patógenos orais Cnm+ colonizam e persistem em tecidos cardíacos humanos. No Capítulo II, utilizamos um sistema de expressão heteróloga em Lactococcus lactis NZ9800 para investigar o papel de Cnm na invasão intracelular, adesão a tecidos cardíacos ex vivo e desenvolvimento de endocardite infecciosa (EI) in vivo. Semelhante a S. mutans, a expressão de Cnm em L. lactis mediou ligação ao colágeno e laminina, invasão de células endoteliais da artéria coronária humana e aumentou a virulência em larvas de Galleria mellonella (p<0,05). Utilizando um modelo de colonização da valva aórtica humana ex vivo, demonstramos que as cepas Cnm+ de S. mutans ou L. lactis colonizaram tecidos da valva aórtica de 10 a 100 vezes mais que as cepas Cnm- (p<0,001). Finalmente, um ensaio de EI em coelhos revelou que a cepa Cnm+ de L. lactis foi recuperada de tecidos cardíacos com uma frequência significativamente mais elevada (~ 70%, p=0,0001) em relação à cepa Cnm-. Em síntese, este capítulo descreve que a presença de Cnm é suficiente para conferir virulência a um organismo não patogênico e confirma a utilidade do sistema heterólogo de L. lactis para caracterização adicional de fatores de virulência bacterianos. Coletivamente, os nossos resultados fornecem evidência de que a ligação a componentes da matriz extracelular tecidual mediada por Cnm é um atributo de virulência importante de S. mutans para o desenvolvimento de doenças orais e sistêmicasAbstract: One of the major etiological pathogens of dental caries, Streptococcus mutans, utilizes a sophisticated repertoire of survival and virulence mechanisms, thereby potentially causing oral and extra-oral diseases. The ability of S. mutans to interact with tissue extracellular matrix components, such as collagen, enables colonization of different host tissues. Previously, we demonstrated that the 120-kDa surface collagen-binding protein Cnm promotes stringent adhesion of S. mutans to root dentin ex vivo and increases coronal caries in vivo. Nevertheless, the impact of Cnm on root caries development, where collagen is abundant, remains unknown. In Chapter I, we used a desalivated rat model under a highly cariogenic challenge to investigate whether Cnm contributes to root caries development. Rats infected with the cnm-knockout strain OMZ175?cnm showed lower levels of slight dentinal smooth-surface caries when compared to the wild type OMZ175 and complemented strain OMZ175?cnm::pcnm (P<0.05) after 5 weeks. Nevertheless, no significant differences were observed for root caries development. Surprisingly, rats infected with OMZ175 or OMZ175?cnm::pcnm showed increased root surface exposure (up to ~18.5% higher, P<0.05) than those infected with OMZ175?cnm or UA159 (naturally defective for Cnm). In summary, this chapter describes the contribution of Cnm to caries development, particularly in early stages of lesion severity, and highlights its potential impact on periodontal diseases and alveolar bone destruction. Cnm has also been reported to play an important and underestimated role in the onset of cardiovascular diseases. Yet, the mechanisms employed by Cnm+ oral pathogen to colonize and persist in human heart tissues are still poorly understood. In Chapter II, we used a heterologous expression system in Lactococcus lactis NZ9800 to investigate the role of Cnm in intracellular invasion, adhesion to cardiac tissues ex vivo and development of infective endocarditis (IE) in vivo. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in Galleria mellonella larvae (P<0.05). Using an ex vivo human heart tissue colonization model, we showed that Cnm+ strains of either S. mutans or L. lactis outcompete their Cnm- counterparts by 10-100 fold for tissue colonization (P<0.001). Finally, a rabbit IE assay showed that the L. lactis Cnm+ strain was recovered from heart tissues at significantly higher frequencies (~70%, p=0.0001) than the L. lactis Cnm- strain. In summary, this chapter describes that Cnm is enough to confer virulence to an otherwise non-pathogenic organism and confirms the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors. Collectively, our results provide evidence that binding to extracellular matrix components mediated by Cnm is an important virulence attribute of S. mutans to cause oral and systemic diseasesDoutoradoFarmacologia, Anestesiologia e TerapeuticaDoutor em Odontologia2013/25080-7FAPES

Antibacterial activity of essential oils and their isolated constituents against cariogenic bacteria: a systematic review

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORDental caries remains the most prevalent and costly oral infectious disease worldwide. Several methods have been employed to prevent this biofilm-dependent disease, including the use of essential oils (EOs). In this systematic review, we discuss the antibacterial activity of EOs and their isolated constituents in view of a potential applicability in novel dental formulations. Seven databases were systematically searched for clinical trials, in situ, in vivo and in vitro studies addressing the topic published up to date. Most of the knowledge in the literature is based on in vitro studies assessing the effects of EOs on caries-related streptococci (mainly Streptococcus mutans) and lactobacilli, and on a limited number of clinical trials. The most promising species with antibacterial potential against cariogenic bacteria are: Achillea ligustica, Baccharis dracunculifolia, Croton cajucara, Cryptomeria japonica, Coriandrum sativum, Eugenia caryophyllata, Lippia sidoides, Ocimum americanum, and Rosmarinus officinalis. In some cases, the major phytochemical compounds determine the biological properties of EOs. Menthol and eugenol were considered outstanding compounds demonstrating an antibacterial potential. Only L. sidoides mouthwash (1%) has shown clinical antimicrobial effects against oral pathogens thus far. This review suggests avenues for further non-clinical and clinical studies with the most promising EOs and their isolated constituents bioprospected worldwide.Dental caries remains the most prevalent and costly oral infectious disease worldwide. Several methods have been employed to prevent this biofilm-dependent disease, including the use of essential oils (EOs). In this systematic review, we discuss the antibacterial activity of EOs and their isolated constituents in view of a potential applicability in novel dental formulations. Seven databases were systematically searched for clinical trials, in situ, in vivo and in vitro studies addressing the topic published up to date. Most of the knowledge in the literature is based on in vitro studies assessing the effects of EOs on caries-related streptococci (mainly Streptococcus mutans) and lactobacilli, and on a limited number of clinical trials. The most promising species with antibacterial potential against cariogenic bacteria are: Achillea ligustica, Baccharis dracunculifolia, Croton cajucara, Cryptomeria japonica, Coriandrum sativum, Eugenia caryophyllata, Lippia sidoides, Ocimum americanum, and Rosmarinus officinalis. In some cases, the major phytochemical compounds determine the biological properties of EOs. Menthol and eugenol were considered outstanding compounds demonstrating an antibacterial potential. Only L. sidoides mouthwash (1%) has shown clinical antimicrobial effects against oral pathogens thus far. This review suggests avenues for further non-clinical and clinical studies with the most promising EOs and their isolated constituents bioprospected worldwide20473297358FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIOR2008/55492-7; 2011/14757-0; 2011/15984-0; 2013/25080-7308644/2011-523038005263/2012-9

Atividade antifúngica de Cimentos de Ionômero de Vidro puros e associados à Cinnamomum zeylanicum

Considerando a importância da atividade antimicrobiana dos materiais restauradores odontológicos, objetivou-se verificar a atividade antifúngica in vitro de Cimentos de Ionômero de Vidro (CIV) puros, associados a nistatina e ao óleo essencial de Cinnamomum zeylanicum (canela) sobre Candida albicans (ATCC 289065). Foram avaliados Vitro Fil®, Maxxion R® e Vitro Cem®. A inibição do crescimento fúngico foi dada pela medição do diâmetro dos halos de inibição nos testes de difusão em meio de cultura sólido. Os CIV foram manipulados de acordo com as orientações dos fabricantes e inseridos em poços, confeccionados no meio de cultura, com 6 mm de diâmetro com o auxílio do sistema Centrix (DFL®). Quando preparadas as associações, as substâncias sintética e natural foram adicionadas ao Cimento de Ionômero de Vidro no momento da manipulação. As placas de Petri foram armazenadas em estufa a 37ºC, sob condições ideais de CO2, durante 48h e posteriormente foi feita a leitura dos resultados com auxílio de um paquímetro. O estudo foi realizado em triplicata e os dados foram analisados a partir do teste de Friedman, seguido de pós-teste de Dunns. Frente a cepa de Candida albicans, houve inibição do crescimento fúngico na maioria das associações, exceto quando o Vitro Fil® foi associado a emulsão do óleo essencial da canela (Cinnamomum zeylanicum).

The Effect of Essential Oils and Bioactive Fractions on Streptococcus mutans

The essential oils (EO) and bioactive fractions (BF) from Aloysia gratissima, Baccharis dracunculifolia, Coriandrum sativum, Cyperus articulatus, and Lippia sidoides were proven to have strong antimicrobial activity on planktonic microorganisms; however, little is known about their effects on the morphology or viability of oral biofilms. Previously, we determined the EO/fractions with the best antimicrobial activity against Streptococcus mutans and Candida spp. In this report, we used a confocal analysis to investigate the effect of these EO and BF on the morphology of S. mutans biofilms (thickness, biovolume, and architecture) and on the metabolic viability of C. albicans biofilms. The analysis of intact treated S. mutans biofilms showed no statistical difference for thickness in all groups compared to the control. However, a significant reduction in the biovolume of extracellular polysaccharides and bacteria was observed for A. gratissima and L. sidoides groups, indicating that these BF disrupt biofilm integrity and may have created porosity in the biofilm. This phenomenon could potentially result in a weakened structure and affect biofilm dynamics. Finally, C. sativum EO drastically affected C. albicans viability when compared to the control. These results highlight the promising antimicrobial activity of these plant species and support future translational research on the treatment of dental caries and oral candidiasis

A pharmacological perspective on the use of brazilian red propolis and its isolated compounds against human diseases

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOPropolis is a complex resinous mixture collected by bees, with high medicinal, historical and economic value. The nutraceutical and pharmacological benefits of propolis have been extensively explored in several fields of medicine as an important resource for prevention and treatment of oral and systemic diseases. A relatively new type of propolis, named red propolis (in Brazil, Brazilian Red Propolis - BRP), has been arousing attention for the promising pharmacological properties of some of its isolated compounds (vestitol, neovestitol, quercetin, medicarpin, formononetin, etc). Due to a distinct chemical composition, BRP and its isolated compounds (mainly isoflavones) affect a wide range of biological targets and could have an impact against numerous diseases as an antimicrobial, anti-inflammatory and immunomodulatory, antioxidant and antiproliferative agent. In this review, we comprehensively address the main aspects related to BRP bioprospection, chemistry and therapeutic potential. Further information is provided on mechanisms of action discovered thus far as well as clinical use in humans and regulatory aspects. As of now, BRP and its isolated molecules remain a fascinating topic for further research and application in biomedical areas and dentistry.Propolis is a complex resinous mixture collected by bees, with high medicinal, historical and economic value. The nutraceutical and pharmacological benefits of propolis have been extensively explored in several fields of medicine as an important resource for prevention and treatment of oral and systemic diseases. A relatively new type of propolis, named red propolis (in Brazil, Brazilian Red Propolis - BRP), has been arousing attention for the promising pharmacological properties of some of its isolated compounds (vestitol, neovestitol, quercetin, medicarpin, formononetin, etc). Due to a distinct chemical composition, BRP and its isolated compounds (mainly isoflavones) affect a wide range of biological targets and could have an impact against numerous diseases as an antimicrobial, anti-inflammatory and immunomodulatory, antioxidant and antiproliferative agent. In this review, we comprehensively address the main aspects related to BRP bioprospection, chemistry and therapeutic potential. Further information is provided on mechanisms of action discovered thus far as well as clinical use in humans and regulatory aspects. As of now, BRP and its isolated molecules remain a fascinating topic for further research and application in biomedical areas and dentistry110267279FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2011/15984-0; 2013/25080-7; 2008/58492-8308644/2011-

Alternative animal and non-animal models for drug discovery and development: bonus or burden?

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOMammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting.Mammalian models have served as a basis for R&D over the past decades. Nevertheless, these models are expensive, laborious, may yield results that cannot always be translated into the human in vivo situation and, more recently, have reverberated great social and ethical dilemmas. Hence, the prospect of changes in the global scientific scenario and the Three Rs principle (Reduction, Replacement and Refinement) have encouraged the development of alternative methods to the use of mammals. Despite the efforts, suitable alternative tests are not available in all areas of biomedical research, as regulatory acceptance requires time, prior validation and robust financial and scientific investment. In this perspective, we aim to shed light on the concepts, challenges and perspectives for implementation of innovative alternative animal and non-animal methods in scientific research. The applicability and meaningfulness of invertebrate animal models, in silico analysis and reverse pharmacology are discussed, among other aspects of relevance in today's scenario. Overall, the use of alternative models, including Artemia salina (brine shrimp), Caenorhabditis elegans (roundworm), Danio rerio (zebra fish), Drosophila melanogaster (fruit fly), Galleria mellonella (greater waxmoth) and in silico modelling, increased 909% from 1990 to 2015, as compared to 154% of conventional mammals in the same period. Thus, technological and scientific advancements in the fields of toxicology and drug development seem to have diminished the need for mammalian models. Today, however, mammals still remain critically indispensable to provide - in most cases -reliable data subsidizing and validating translation into the clinical setting344681686FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2011/15984-0; 2013/25080-7310522/2015-

Revista de Ciências Médicas e Biológicas

Abstract Introduction: The combination of a natural product and a synthetic antifungal may lead to a lower dose of each individual agent and consequent reduction of adverse effects and greater pharmacological synergism. Objective: This study investigated the antifungal activity of the essential oil (EO) from Cinnamomum zeylanicum Blume alone and combined with nystatin on Candida albicans growth and micromorphology. Methodology: We determined the Minimum Inhibitory Concentration (MIC), Fractional Inhibitory Concentration Index (FIC) and the effect of the EO alone and combined with nystatin on the growth kinetics and production of virulence-related structures by the yeasts, such as pseudohyphae and chlamydoconidia. Results: When tested alone, the EO from C. zeylanicum and nystatin showed MIC of 312.5 μg/ml and 64 μg/ml, respectively. When combined, MIC values decreased to 39 μg/ml and 32 μg/ ml for the EO and nystatin, respectively. The value of the Fractional Concentration Index (FIC) was 0.6024, indicating additivity. It could be observed that at all concentrations the products tested alone and in combination were able to reduce the number of CFU/ mL, when compared to the control group (p<0.0001) from 30 min. In addition, both the products alone and combined inhibited production of pseudohyphae and chlamydoconidia compared to the control. Conclusion: The combination between the essential oil from C. zeylanicum and nystatin potentiated the inhibitory effect on C. albicans growth. Furthermore, it reduced the production of pathogenicity-related morphological structures such as pseudohyphae and chlamydoconidia. Key-words: Cinnamomum zeylanicum. Nystatin. Essential Oil. Natural Product. Drug synergism. Candidiasis.Introdução: A combinação entre produtos naturais e antifúngicos sintéticos pode acarretar em uma menor dose individual de cada agente e consequente diminuição dos efeitos adversos e maior sinergismo farmacológico. Objetivo: O presente estudo investigou a atividade antifúngica do óleo essencial (OE) de Cinnamomum zeylanicum Blume sozinho e combinado com nistatina sobre o crescimento e micromorfologia de Candida albicans. Metodologia: Determinou-se a Concentração Inibitória Mínima (CIM), índice de Concentração Inibitória Fracional (FIC) e o efeito do OE sozinho e combinado com nistatina sobre a cinética de crescimento e produção de estruturas de virulência relacionadas tais como pseudohifas e clamidoconídios. Resultados: Quando testados isoladamente, o OE de C. zeylanicum e nistatina apresentaram CIM de 312,5 μg/mL e 64 μg/mL, respectivamente. Quando combinados, os valores de CIM referente ao OE e nistatina diminuíram, respectivamente, para 39 μg/mL e 32 μg/mL. O valor do índice de concentração fracional (FIC) foi de 0,6024, indicando aditividade. Pôde-se observar que, em todas as concentrações, os produtos testados isoladamente e em combinação reduziram o número de UFC/mL a partir de 30 minutos quando comparados ao grupo controle (p<0,0001). Além disso, ambos os produtos sozinhos e combinados inibiram a formação de pseudohifas e clamidoconídios em comparação ao grupo controle. Conclusão: A combinação entre o óleo essencial de C. zeylanicum e nistatina (antifúngico padrão) potencializou o efeito inibitório sobre o crescimento de C. albicans, além disso, reduziu a formação de estruturas morfológicas relacionadas à patogenicidade, tais como pseudohifas e clamidoconídios.Salvado

Combined effect of Cinnamomum zeylanicum blume essential oil and nystatin on Candida albicans growth and micromorphology

Introduction: The combination of a natural product and a synthetic antifungal may lead to a lower dose of each individual agent and consequent reduction of adverse effects and greater pharmacological synergism. Objective: This study investigated the antifungalactivity of the essential oil (EO) from Cinnamomum zeylanicum Blume alone and combined with nystatin on Candida albicans growth and micromorphology. Methodology: We determined the Minimum Inhibitory Concentration (MIC), Fractional Inhibitory Concentration Index (FIC) and the effect of the EO alone and combined with nystatin on the growth kinetics and production of virulence-related structures by the yeasts, such as pseudohyphae and chlamydoconidia. Results: When tested alone, the EO from C. zeylanicum andnystatin showed MIC of 312.5 μg/ml and 64 μg/ml, respectively. When combined, MIC values decreased to 39 μg/ml and 32 μg/ml for the EO and nystatin, respectively. The value of the Fractional Concentration Index (FIC) was 0.6024, indicating additivity. It could be observed that at all concentrations the products tested alone and in combination were able to reduce the number of CFU/mL, when compared to the control group (