Seven pimarane type-diterpenes re-isolated from Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); ent-8(14),15-pimaradien-3b-ol; ent-15-pimarene-8b,19-diol; ent-8(14),15-pimaradien-3b-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 μg∙mL-1. Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.

Recebido em 3/1/07; aceito em 25/9/07; publicado na web em 2/4/08 COMPARATIVE STUDY OF SCREENING TECHNIQUES FOR ANTIBACTERIAL ACTIVITY EVALUATION OF PLANT CRUDE EXTRACTS AND PURE COMPOUNDS. In this work, the effectiveness of four screening techniques (three techniques of the diffusion method and one microdilution broth method) were compared. Evaluated were the ethanolic and dichloromethanic extracts of Miconia rubiginosa (Melastomataceae) against six standard bacteria (ATCC). The results showed statistical disagreement among the three diffusion techniques. Among the diffusion techniques, the well technique displayed the best result. However the microdilution broth method demonstrated to be the most adequate method to evaluate the antibacterial activity of plant crude extracts and pure compounds when compared to the other methodologies.Keywords: antimicrobial activity; screening methods; plant extracts. INTRODUÇÃOAtualmente existem diversas técnicas de screening para definir se o extrato de uma determinada planta possui atividade antimicrobiana, desde as mais simples, que podem ser realizadas rotineiramente, até as mais sofisticadas, que muitas vezes se tornam indisponíveis em alguns laboratórios. Apesar disso, há poucos estudos que relatam qual o melhor método de screening a ser utilizado de acordo com o tipo de extrato a ser testado, mesmo no que se refere às técni-cas mais simples. Por vezes, relatos de resultados em diferentes artigos publicados com uma mesma planta são apresentados de maneira distinta em relação à atividade antimicrobiana, às vezes até mesmo discrepantes, mesmo naqueles em que foram utilizadas as mesmas condições de experimento (ex: solvente utilizado para extração, temperatura, tempo de incubação e microrganismos indicadores), embora as técnicas ou modificações empregadas nos méto-dos de screening para avaliar a atividade antimicrobiana por diferentes autores não tenham sido as mesmas. 1 Os dois métodos mais comumente utilizados para o screening de extratos de plantas com potencial antibacteriano são o de difusão em ágar e de diluição em caldo. O método de difusão em ágar pode ser realizado através das técnicas do disco, do poço ou template. 2-4 Diversas modificações já foram realizadas nas metodologias de screening a fim de se obter resultados mais confiáveis, uma vez que alguns fatores, tais como composição do meio de cultura, microrganismos testados, método de extração, pH e solubilidade das amostras no meio de cultura, podem alterar os resultados, sendo difícil, portanto, padronizar um procedimento. 1 Quando se utiliza o método de difusão vários fatores podem se tornar fontes de erros, tais como composição do meio de cultura, preparação incorreta do meio de cultura, espessura do meio de cultura, densidade do inóculo incorreta, uso de swab com excesso de caldo para inoculação das placas, temperatura e tempo de incubação inadequados, interações entre o antimicrobiano e o meio de cultura, utilização errada da atmosfera de CO 2 quando necessária, leitura prematura, erro na medida das zonas ...

In this work, we used the Minimum Inhibitory Concentration (MIC) technique to evaluate the antibacterial potential of the apitoxin produced by Apis mellifera bees against the causative agents of tooth decay. Apitoxin was assayed in natura and in the commercially available form. The antibacterial actions of the main components of this apitoxin, phospholipase A 2 , and melittin were also assessed, alone and in combination. The following bacteria were tested: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, Lactobacillus casei, and Enterococcus faecalis. The MIC results obtained for the commercially available apitoxin and for the apitoxin in natura were close and lay between 20 and 40µg / mL, which indicated good antibacterial activity. Melittin was the most active component in apitoxin; it displayed very promising MIC values, from 4 to 40µg / mL. Phospholipase A 2 presented MIC values higher than 400µg / mL. Association of mellitin with phospholipase A 2 yielded MIC values ranging between 6 and 80µg / mL. Considering that tooth decay affects people's health, apitoxin and its component melittin have potential application against oral pathogens.

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Triterpene acids (ursolic, oleanoic, gypsogenic, and sumaresinolic acids) isolated from Miconia species, along with a mixture of ursolic and oleanolic acids and a mixture of maslinic and 2-α-hydroxyursolic acids, as well as ursolic acid derivatives were evaluated against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) during the evaluation of the antibacterial activity. All the isolated compounds, mixtures, and semi-synthetic derivatives displayed activity against all the tested bacteria, showing that they are promising antiplaque and anticaries agents. Ursolic and oleanolic acids displayed the most intense antibacterial effect, with MIC values ranging from 30 μg/mL to 80 μg/mL. The MIC values of ursolic acid derivatives, as well as those obtained for the mixture of ursolic and oleanolic acids showed that these compounds do not have higher antibacterial activity when compared with the activity observed with either ursolic acid or oleanolic acid alone. With regard to the structure-activity relationship of triterpene acids and derivatives, it is suggested that both hydroxy and carboxy groups present in the triterpenes are important for their antibacterial activity against oral pathogens.