Abstract The potential of H. virginiana L. was evaluated against Candida spp. (C. albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, and C. tropicalis) and bacteria (Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus mutans). Effect on murine macrophages (RAW 264.7) was also evaluated with respect to cytotoxicity and production of cytokines (IL-1β and TNF-α) and nitric oxide (NO). The most effective concentrations of the extract were determined by microdilution broth. These concentrations were analyzed on biofilms, after 5 min or 24 h exposure. Cytotoxicity was performed by MTT assay and quantification of cytokines and NO by ELISA and Griess reagent, respectively. The extract acted against the planktonic forms and provided significant reductions of all the microbial biofilms; besides, showed no cytotoxic effect, except at 100 mg/mL, after 24 h exposure. There was cytokine production; however, a modulatory effect was observed in groups exposed to lipopolysaccharide (LPS) from E. coli. NO production was similar or higher than the control group. Thus, H. virginiana L. extract showed antimicrobial and antibiofilm effects; absence of cytotoxicity for RAW 264.7; anti-inflammatory action; and potential to fight infections through the NO production. H L spp C (C albicans dubliniensis glabrata guilliermondii krusei tropicalis Acinetobacter baumannii coli faecalis pneumoniae aureus mutans. mutans . mutans) 264.7 2647 264 7 IL1β ILβ IL 1β β TNFα TNF α TNF-α NO. (NO) broth biofilms 2 exposure reagent respectively besides 10 mgmL mg mL mg/mL however LPS (LPS E group Thus effects antiinflammatory anti inflammatory action 264. 26 (NO 1

Abstract Candida spp. are naturally opportunistic and can promote infections. These yeasts can form biofilm, after penetration and adhesion to the biotic or abiotic surfaces. Preexisting diseases, treatments with drugs and radiation therapy, medical procedures, and parafunctional habits favor the installation of a fungal infection. Increased resistance to the available antifungals has become a concern. Therefore, alternative methods to control them have been evaluated, including the use of plant substances. In this study, the antibiofilm effect of R. officinalis L. extract was analyzed on C. albicans, C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis. A phytochemical analysis of the extract was performed. Biofilms were formed for 48 h and exposed to the different concentrations of the extract (50, 100, and 200 mg/mL) for 5 min or 24 h. The effect of the plant extract was compared to the antifungal nystatin. Rosmarinus officinalis L. extract was constituted of phenols and flavonoids, highlighting the presence of chlorogenic acid derivatives in its composition. Biofilm reductions were observed after exposure to the plant extract for both periods. The plant extract provided a reduction similar to the antifungal. Thus, R. officinalis L. extract showed antibiofilm effect on Candida spp. comparable to the nystatin.

Adjuvants play an important role in vaccine formulations by increasing their immunogenicity. In this study, the phenolic compound-rich J fraction (JFR) of a Brazilian green propolis methanolic extract stimulated cellular and humoral immune responses when co-administered with an inactivated vaccine against swine herpesvirus type 1 (SuHV-1). When compared to control vaccines that used aluminium hydroxide as an adjuvant, the use of 10 mg/dose of JFR significantly increased (p < 0.05) neutralizing antibody titres against SuHV-1, as well as the percentage of protected animals following SuHV-1 challenge (p < 0.01). Furthermore, addition of phenolic compounds potentiated the performance of the control vaccine, leading to increased cellular and humoral immune responses and enhanced protection of animals after SuHV-1 challenge (p < 0.05). Prenylated compounds such as Artepillin C that are found in large quantities in JFR are likely to be the substances that are responsible for the adjuvant activity.

Sistemas microemulsionados (MES) oferecem inúmeras vantagens como liberadores de fármacos já que favorecem a absorção do princípio ativo e são, em muitos casos, mais eficientes do que outros métodos de liberação de substâncias bioativas. Neste trabalho, um MES foi obtido visando uma formulação para uso tópico, contendo extrato etanólico de própolis (PEE). Neste estudo, Diagramas de fase pseudoternários foram utilizados para caracterização das regiões de microemulsão, bem como para definição da região de Winsor IV do sistema PEE-MES (Tween 80 como tensoativo e álcool etílico como co-tensoativo, em baixo percentual). Os resultados obtidos indicaram que o melhor sistema microemulsionado contém C/S (cotensoativo/tensoativo) na razão 1,0, fornecendo uma microemulsão do tipo O/A com ampla região de Winsor IV. A formulação PEE-MES poderá vir a ser utilizada comercialmente para uso tópico no combate a inflamações de boca e garganta. Considerando o amplo uso de própolis em formulações convencionais, onde sua solubilidade representa um dos maiores problemas, o sistema PEE-MES disponibiliza este produto natural para aplicações farmacológicas, com boa solubilidade em um sistema microemulsionado do tipo O/A. Adicionalmente, o efeito bactericida do sistema PEE-MES foi comprovado e uma nova metodologia de extração de própolis é apresentada.

Microemulsion systems (MES) offer advantages as drug delivery systems, among them favour drug absorption, being in most case more efficient than other methods in delivering of drug. In this work a new MES was obtained in order to be applied as a pressurized aerosol formulation containing bee propolis ethanolic extract (PEE). For that, pseudoternary phase diagrams were used to characterize the microemulsions boundaries and also to define the Winsor IV microemulsion region of the PEE-MES system containing Tween 80 as surfactant and the cosurfactant ethyl alcohol in small percentage. The obtained results indicated that the best MES was composed by Tween 80 and ethyl alcohol with C/S (cosurfactant/surfactant) ratio equal to 1.0, since it provided a large boundaries in the obtained O/W microemulsion region. This PEE-MES formulation, in which bee propolis consisting as oil phase, is herein designed for topical uses (PEE-MES spraying) in order to treat mouth and throat inflammatory infections. Considering the very large uses of bee propolis in conventional vehicles, MES type of delivery system has to be compatible with achieving the highest drug aim loadings, determined substantially by the specific MES application (drug solubilization in water systems) improving in this case, propolis farmacological aplications. Additionally, PEE-MES antibacterial effect was evidenced and the microemulsion system PEE-MES was also used as newest chemical approach for extraction of bee propolis material from resinous hive.

Durante a extração de própolis foram variados alguns fatores para determinar como eles afetam o rendimento e o teor de fenóis totais. Seis amostras diferentes da região sudeste do Brasil foram testadas. Os resultados indicam que os maiores rendimentos de extratos de própolis obtidos por maceração, que também apresentam o maior número de componentes, foram aqueles usando 70% (v/v) ou maior proporção de etanol no solvente. A extração em Soxhlet resultou em um maior rendimento. Não foram observadas diferenças entre extratos de macerações com ou sem luz. Um aumento no rendimento da extração foi observado entre os extratos obtidos por maceração durante 10 e 30 dias, porém o teor de fenóis não variou significativamente nesses extratos. O teor de fenóis totais de todos os extratos variou entre 6,41 e 15,24% mas nenhuma correlação direta foi encontrada com qualquer um dos fatores testados.

During the extraction of propolis several factors were varied in order to determine how they affected the yield and phenolic composition of the obtained extracts. Six samples of green propolis from the Southeastern region of Brazil were tested. The results indicate that the highest yield of propolis extracts obtained by maceration, which also had the greatest number of components, were those using 70% (v/v) ethanol or more as a solvent. The Soxhlet extraction procedure resulted in even higher yields. No differences were observed between extracts macerated in the presence or absence of light. An increase in yield was observed between extracts macerated for 10 and 30 days although the phenolic content did not vary significantly. The total phenolic content of all extracts varied from 6.41 to 15.24 % but no direct correlation could be found between any of the factors tested.

Própolis brasileira, proveniente do estado de São Paulo, foi submetida à extração usando vários solventes, resultando em extratos com diferentes composições. Estes extratos foram submetidos à Cromatografia em Camada Delgada (CCD). Análise bioautográfica das placas de CCD permitiu identificar as frações com atividade antimicrobiana, que foram então analisadas por Cromatografia Líquida de Alta Eficiência (CLAE). Ensaios in vitro freqüentemente utilizados para avaliar a atividade de própolis frente a bactérias Gram-positivas foram comparados para determinar qual renderia os resultados mais consistentes. A atividade bactericida destes extratos foi analisada por Diluição Seriada em Tubos e por testes de Difusão em Agar. O método de Diluição em Tubos permitiu obter os resultados mais consistentes e a Concentração Bactericida Mínima dos extratos variou entre 2,5 e 20,0 mg/mL, para as espécies de bactérias Gram-positivas testadas. Os resultados do método de Difusão em Agar foram diretamente proporcionais à hidrossolubilidade dos extratos, e não avaliaram a atividade bactericida corretamente. A atividade bactericida desta amostra resultou da combinação de vários componentes, identificados por CLAE, que foram extraídos preferencialmente usando etanol 50% como solvente.

Brazilian propolis from São Paulo state was submitted to extraction using several solvents, resulting in extracts with different composition. These extracts were submitted to Thin Layer Chromatography (TLC). Bioauthographic analysis of the TLC plates identified fractions with inhibitory activity, which were then analysed by High Performance Liquid Chromatography (HPLC). In vitro assays, commonly used to evaluate the activity of propolis against Gram-positive bacteria, were compared to determine which rendered the most consistent results. The bactericidal activity of these extracts were analysed by Serial Dilution in Tubes and Agar Plate Diffusion. Serial Dilution in Tubes obtained the most consistent results, with the Minimal Bactericidal Concentration of the extracts ranging between 2.5 and 20.0 mg/mL, for the species of Gram-positive bacteria tested. The results of the Agar Plate Diffusion were directly proportional to the hydro-solubility of the extracts and did not evaluate their bactericidal activity correctly. The bactericidal activity of this sample of propolis was due to the combined effect of several components that were identified by HPLC and were best extracted using 50% ethanol as a solvent.