Atividade antifúngica e possível mecanismo de ação do óleo essencial de folhas de Ocimum gratissimum (Linn. ) sobre espécies de Candida / Antifungal activity and potential action mechanisms of essential oil of Ocimum gratissimum (Linn. ) leaves against Candida species

RESUMO A espécie Ocimum gratissimum (Linn.), popularmente conhecida como alfavaca-cravo, é muito utilizada na medicina popular. A planta apresenta inúmeros compostos, sendo o eugenol o constituinte majoritário do seu óleo essencial e o provável responsável pela sua atividade antimicrobiana. O objetivo deste estudo foi determinar o potencial antifúngico e o possível mecanismo de ação do óleo essencial extraído das folhas de O. gratissimum L. (OEOg) sobre cepas-padrão de Candida. Para avaliação da atividade antimicrobiana foi determinada a Concentração Inibitória Mínima (CIM), o efeito do tempo de exposição, o efeito modulador na atividade de antifúngicos (ATF) de uso clínico e a ação do OEOg nas fases de crescimento exponencial e estacionário de leveduras do gênero Candida. O mecanismo de ação do OEOg foi verificado por captação do cristal violeta e avaliação da morfologia microbiana pela técnica de microcultivo. Também foi avaliada a toxicidade do OEOg sobre hemácias humanas. O OEOg apresentou boa atividade antifúngica sobre cepas de Candida, sendo capaz de reduzir a taxa de crescimento das cepas de Candida a partir de quatro horas de exposição, além de ter modulado positivamente a atividade do cetoconazol para C. tropicalis ATCC 13803 e reduzir o número de células viáveis em todas as fases de crescimento microbiano. O OEOg foi capaz de promover o aumento discreto da captação do cristal violeta e provocou alterações na micromorfologia das células de Candida spp., sugerindo que seu alvo de ação seja o envoltório celular. Observou-se baixa toxicidade do OEOg sobre hemácias humanas. Os resultados encontrados mostraram que o OEOg possui boa atividade sobre o gênero Candida, com mecanismo de ação mediado possivelmente pela ocorrência de danos no envoltório celular, além de ter sido observada baixa toxicidade, indicando do OEOg é promissor no desenvolvimento e elaboração de um novo fármaco com potencial atividade para o tratamento de doenças fúngicas.

ABSTRACT Ocimum gratissimum (Linn.) is a medicinal plant popularly known as “wild basil” widely used in traditional medicine. The plant has numerous compounds, and eugenol is the major constituent of its essential oil and likely responsible for its antimicrobial activity. The aim of this study was to determine the antifungal activity and the potential mechanism of action of the essential oil extracted from the leaves of O. gratissimum L. (OEOg) against standard strains of Candida. The following experiments were performed: determination of Minimum Inhibitory Concentration (MIC), determination of the effect of exposure time to OEOg; evaluation of the modulating effect of OEOg in antifungal (ATF) activity for clinical use; determination of the effects of the OEOg on different growth phases of Candida spp; determination of crystal violet (CV) uptake and the microculture of yeast technique. OEOg showed good antifungal activity against Candida, being able to reduce microbial growth during 24 hours of contact and also the number of viable cells at all stages of growth. OE positively modulates the activity of ketoconazole for C. tropicalis ATCC 13803. Increased uptake of CV and also the inhibition of Candidavirulence factors were also observed, which indicates the occurrence of damage in the cell envelope. These findings, coupled with the low toxicity of OEOg on human erythrocytes, indicate that “wild basil” is a promising plant for the development of a new drug with a potential activity to treat fungal diseases.

Determinação in vitro do potencial antimicrobiano de preparações caseiras de plantas medicinais utilizadas para o tratamento de doenças infecciosas / In vitro determination of the antimicrobial potential of homemade preparations based on medicinal plants used to treat infectious diseases

The majority of the population in developing countries uses plants or plant preparations in their basic health care. Many plant species used nowadays in folk medicine have been proved to have antimicrobial properties. However, several factors, such as incorrect preparation of the plants, can interfere with the effectiveness of the treatment. The purpose of this study was to assess the use of homemade preparations of medicinal plants in the treatment of infectious diseases, by in vitro determination of their antimicrobial potential. Based on recipes elicited by questionnaires that were previously applied to a participant population, the samples were prepared in a similar manner and analyzed by the agar diffusion method. Members of 41 families, whose children attend a center of education that serves several needy communities in the city of Fortaleza (Ceará, Brazil), were interviewed; of these, 97.6% said they had used herbal therapy as a means to treat infectious diseases. In replies to a total of 39 questionnaires, 97 different homemade preparations of medicinal plants were cited. Out of 45 samples subjected to an in vitro assessment of antimicrobial activity, 25 (55.6%) had some inhibitory effect on the growth of at least one of the microorganisms used. Most of the plants with known antimicrobial properties and cited by respondents showed variations in their in vitro activity, according to the manner in which they were prepared.

Grande parte da população de países em desenvolvimento utiliza plantas ou preparações vegetais nos cuidados básicos à saúde. Muitas das espécies vegetais utilizadas na medicina popular apresentam propriedades antimicrobianas comprovadas; no entanto, diversos fatores, como a metodologia incorreta de preparo, podem interferir na eficácia do tratamento. O presente trabalho teve como objetivo avaliar a utilização de preparações caseiras de plantas medicinais para o tratamento de doenças infecciosas através da determinação in vitro do potencial antimicrobiano. A partir de informações contidas em questionários que foram previamente aplicados à população participante, as amostras foram preparadas de forma semelhante e analisadas através do método de difusão em ágar. Foram entrevistadas 41 famílias, das quais 97,6% afirmaram utilizar as plantas medicinais como opção terapêutica no tratamento de doenças. Em um total de 39 questionários, foram citadas 97 diferentes preparações caseiras de plantas. Das 45 amostras (indicadas para tratar algum sinal ou sintoma que podem estar relacionado a um quadro infeccioso) submetidas à avaliação da atividade antimicrobiana in vitro, 55,6% (25 amostras) apresentaram atividade inibitória sobre o crescimento de pelo menos um dos microrganismos utilizados. A maioria das plantas com propriedade antimicrobiana conhecida e que foram citadas pelos entrevistados apresentaram variações na sua atividade in vitro de acordo com o modo como foram preparadas.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Some snake venom proteins present enzymatic activities, such as L-amino acid oxidase (LAAO). The aim of this paper was to investigate the effect of Bothrops leucurus total venom (BleuTV) and its fraction LAAO (BleuLAAO) on bacteria, yeast, and promastigote forms of Leishmania amazonensis and Leishmania chagasi, and epimastigote forms of Trypanosoma cruzi. BleuTV was isolated with a Protein Pack 5PW® (Waters Corporation, USA), and several fractions were obtained. BleuLAAO was purified to high molecular homogeneity, and its N-terminal amino acid sequence shared a high degree of amino acid conservation with other LAAOs. BleuTV inhibited Staphylococcus aureus growth in a dose-dependent manner, with a minimum inhibitory concentration (MIC) of 25 ìg/mL, which corresponded to its minimum lethal concentration (MLC). BleuTV also inhibited the growth of promastigote forms of L. chagasi and L. amazonensis, with respective IC50 values of 1.94 ìg/mL and 5.49 ìg/mL. Furthermore, it repressed T. cruzi growth with an IC50 of 1.14 ìg/mL. However, BleuLAAO did not inhibit the growth of the microorganisms studied and was not toxic to macrophages. BleuTV had low toxicity against macrophages at the concentrations studied. In conclusion, whole venom from Bothrops leucurus inhibited the growth of some microorganisms, including S. aureus, Leishmania sp., and T. cruzi.

Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens

Dental caries and periodontal disease are associated with oral pathogens. Several plant derivatives have been evaluated with respect to their antimicrobial effects against such pathogenic microorganisms. Lippia sidoides Cham (Verbenaceae), popularly known as "Alecrim-pimenta" is a typical shrub commonly found in the Northeast of Brazil. Many plant species belonging to the genus Lippia yield very fragrant essential oils of potential economic value which are used by the industry for the commercial production of perfumes, creams, lotions, and deodorants. Since the leaves of L. sidoides are also extensively used in popular medicine for the treatment of skin wounds and cuts, the objective of the present study was to evaluate the composition and antimicrobial activity of L. sidoides essential oil. The essential oil was obtained by hydro-distillation and analyzed by GC-MS. Twelve compounds were characterized, having as major constituents thymol (56.7 percent) and carvacrol (16.7 percent). The antimicrobial activity of the oil and the major components was tested against cariogenic bacterial species of the genus Streptococcus as well as Candida albicans using the broth dilution and disk diffusion assays. The essential oil and its major components thymol and carvacrol exhibited potent antimicrobial activity against the organisms tested with minimum inhibitory concentrations ranging from 0.625 to 10.0 mg/mL. The most sensitive microorganisms were C. albicans and Streptococcus mutans. The essential oil of L. sidoides and its major components exert promising antimicrobial effects against oral pathogens and suggest its likely usefulness to combat oral microbial growth.