Terpinen-4-ol: estudo do efeito sinérgico/aditivo, adesão em co-cultura e alteração dos fatores de virulência sobre Candida spp
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Data
2016-03-22
Autores
Tonon, Caroline Coradi [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O gênero Candida pode ser encontrado em até 50% dos indivíduos saudáveis não causando danos aparentes, porém, sob condições predisponentes como doenças sistêmicas ou condições fisiológicas, pode tornar-se patogênico causando inflamação e destruição tecidual. Candida spp. na forma de biofilmes são importantes no desenvolvimento de infecções, pois estão associados a altos níveis de resistência a agentes antimicrobianos. Terapias alternativas com extratos naturais abrem novas perspectivas para prevenção e controle das doenças bucais na busca de efeitos terapêuticos favoráveis. O Terpinen-4-ol é um monoterpeno que atua na indução da perda da membrana e apresenta amplo espectro de atividade antimicrobiana e atividade anti-inflamatória. O objetivo deste estudo foi avaliar o efeito antifúngico, sinérgico/aditivo, inibição da adesão em células orais e alteração dos fatores de virulência do Terpinen-4-ol sobre Candida albicans e Candida tropicalis. Assim, foi realizada a identificação da Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM) do Terpinen-4-ol sobre cepas padrão de C. albicans (ATCC 90028) e C. tropicalis (ATCC4563), empregando-se o método de microdiluição em caldo. Biofilmes mono e dual-espécies foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (UFC/mL). O efeito do Terpinen-4-ol na adesão de C.albicans e C. tropicalis foi realizado em co-cultura com células orais NOK Si como também os fatores de virulência (proteases, fosfolipases e hemolisina) foram avaliados in vitro. A CIM para Terpinen-4-ol foi 2,31 mg/ml para ambas espécies e a CFM 0,002 mg/ml e 0,004 mg/ml respectivamente para Candida albicans e Candida tropicalis. Quando testados em combinação houve efeito aditivo e sinergismo (T-4-ol 1,06 mg/ml e Nis 0,00012 mg/ml) para Candida albicans. Em biofilme, os resultados apresentaram aditismo para ambas as espécies. O Terpinen-4-ol demonstrou uma redução na adesão de Candida tropicalis em células NOK Si, já a Nistatina teve maior efeito tanto para Candida albicans quanto para Candida tropicalis. Quanto à atividade enzimática, Terpinen-4-ol e Nistatina não demonstraram ação na produção de proteinase, fosfolipase e hemolisina quando comparados ao grupo controle (sem tratamento). A combinação de Terpinen-4-ol e Nistatina apresentou efeito antifúngico potencializado sobre as duas espécies de Candida avaliadas, mostrando efeito sinérgico/modulador em culturas planctônicas e biofilmes. Terpinen-4-ol reduz a adesão de C. tropicalis e a Nistatina reduz a adesão de ambas as espécies em células orais imortalizadas (NOK Si). As concentrações sub-inibitorias (CIM50) de Terpinen-4-ol e Nistatina utilizadas não apresentaram atividade na produção de proteinase, fosfolipase e hemolisina.
The genus Candida can be found in up to 50% of healthy individuals without causing apparent damage, however, under predisposing conditions such as systemic diseases or physiological conditions, can become pathogenic causing inflammation and tissue destruction. Candida spp. in the form of biofilms are important in the development of infections because they are associated with high levels of resistance to antimicrobial agents. Alternative therapies with natural extracts open new perspectives for prevention and control of oral diseases in search of favorable therapeutic effects. The Terpinen-4-ol is a monoterpene engaged in membrane loss of induction and presents broad spectrum of antimicrobial and anti-inflammatory activity. The aim of this study was to evaluate the antifungal effect, synergistic / additive inhibition of adhesion on oral cells and modification of virulence factors of Terpinen-4-ol of Candida albicans and Candida tropicalis. Thus, the identification of Minimum Inhibitory Concentration was performed (MIC) and Minimum Fungicidal Concentration (MFC) of Terpinen-4-ol on standard strains C. albicans (ATCC 90028) and C. tropicalis (ATCC4563), using the method microdilution broth. Biofilms mono and dual-species were prepared using the microtiter plate static model and quantitated by colony forming units (CFU / mL). The effect of Terpinen-4-ol in the adhesion on C. tropicalis and C. albicans was carried out in co-culture with oral cells NOK Si as well as virulence factors (proteases, phospholipases and hemolysin) were evaluated in vitro. The MIC for Terpinen-4-ol was 2.31 mg / ml for both species and CFM 0.002 mg / ml and 0.004 mg / ml respectively for Candida albicans and Candida tropicalis. When tested in combination there was synergism and additive effect (T-4-ol 1.06 mg / ml and 0.00012 mg Nis / ml) for Candida albicans. In the biofilm, the results presented aditismo for both species. The Terpinen-4-ol was a reduction in adhesion of Candida tropicalis on cells NOK Si, Nystatin already had most effective for Candida albicans and for Candida tropicalis. For enzymatic activity, Terpinen-4-ol and Nystatin showed no action on the production of proteinase, phospholipase and hemolysin when compared to the control group (no treatment). The combination of Terpinen-4-ol and Nystatin presented antifungal effect potentiated on the two Candida species evaluated, showing synergistic / modulatory effect on planktonic and biofilm cultures. Terpinen-4-ol reduces the adhesion of C. tropicalis and Nystatin reduces the adhesion of both species in oral immortalized cells (NOK Si). The sub-inhibitory concentrations (MIC50) of Terpinen-4-ol and Nystatin used did not show activity in the production of proteinase, phospholipase and hemolysin.
The genus Candida can be found in up to 50% of healthy individuals without causing apparent damage, however, under predisposing conditions such as systemic diseases or physiological conditions, can become pathogenic causing inflammation and tissue destruction. Candida spp. in the form of biofilms are important in the development of infections because they are associated with high levels of resistance to antimicrobial agents. Alternative therapies with natural extracts open new perspectives for prevention and control of oral diseases in search of favorable therapeutic effects. The Terpinen-4-ol is a monoterpene engaged in membrane loss of induction and presents broad spectrum of antimicrobial and anti-inflammatory activity. The aim of this study was to evaluate the antifungal effect, synergistic / additive inhibition of adhesion on oral cells and modification of virulence factors of Terpinen-4-ol of Candida albicans and Candida tropicalis. Thus, the identification of Minimum Inhibitory Concentration was performed (MIC) and Minimum Fungicidal Concentration (MFC) of Terpinen-4-ol on standard strains C. albicans (ATCC 90028) and C. tropicalis (ATCC4563), using the method microdilution broth. Biofilms mono and dual-species were prepared using the microtiter plate static model and quantitated by colony forming units (CFU / mL). The effect of Terpinen-4-ol in the adhesion on C. tropicalis and C. albicans was carried out in co-culture with oral cells NOK Si as well as virulence factors (proteases, phospholipases and hemolysin) were evaluated in vitro. The MIC for Terpinen-4-ol was 2.31 mg / ml for both species and CFM 0.002 mg / ml and 0.004 mg / ml respectively for Candida albicans and Candida tropicalis. When tested in combination there was synergism and additive effect (T-4-ol 1.06 mg / ml and 0.00012 mg Nis / ml) for Candida albicans. In the biofilm, the results presented aditismo for both species. The Terpinen-4-ol was a reduction in adhesion of Candida tropicalis on cells NOK Si, Nystatin already had most effective for Candida albicans and for Candida tropicalis. For enzymatic activity, Terpinen-4-ol and Nystatin showed no action on the production of proteinase, phospholipase and hemolysin when compared to the control group (no treatment). The combination of Terpinen-4-ol and Nystatin presented antifungal effect potentiated on the two Candida species evaluated, showing synergistic / modulatory effect on planktonic and biofilm cultures. Terpinen-4-ol reduces the adhesion of C. tropicalis and Nystatin reduces the adhesion of both species in oral immortalized cells (NOK Si). The sub-inhibitory concentrations (MIC50) of Terpinen-4-ol and Nystatin used did not show activity in the production of proteinase, phospholipase and hemolysin.
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Palavras-chave
Candida albicans, Candida tropicalis, Biofilmes, Biofilm