Antibacterial and Cytotoxic Potential of a Brazilian Red Propolis

Objective: To evaluate in vitro the effect of a red propolis ethanolic extract (RPE) in the prevention of growth of a cariogenic biofilm and its cytotoxic potential. Material and Methods: Minimum inhibitory and bactericidal concentrations (MIC and MBC) of RPE against Streptococcus mutans and Lactobacillus casei were determined. The cytotoxic potential of 0.4% RPE in oral fibroblasts was observed after 1, 3 and 5 min of contact. Cellulose membrane disks (13 mm, N=12) were used for biofilm formation (24 h) of S. mutans and L. casei, which were treated (1 min) with 0.4% RPE or 0.12% Chlorhexidine (CHX). The control group of biofilm formation was not submitted to any treatment. Serial dilutions were then made to evaluate microbial viability. Descriptive data analysis and, for microbial viability, Mann Whitney test were performed (p≤0.05). Results: RPE showed similar MIC and MBC (4.46 mg/mL) against S. mutans and, for L. casei, they were 8.92 mg/mL (MIC) and 17.85 mg/mL (MBC). CHX presented MIC and MBC <0.00002 mg/mL for S. mutans and 0.00047 mg/mL for L. casei. After 1, 3 and 5 min, the RPE exhibited, respectively, 69.38%, 43.91% and 40.36% of viable cells. The RPE (6.55) and CHX (6.87) presented similar efficacy to reduce the total number of viable bacteria (p>0.05). Regarding the total number of viable bacteria (Log10 CFU/mL), the RPE (6.55) and CHX (6.87) presented similar efficacy (p>0.05). Conclusion: Red propolis extract showed antibacterial activity against the tested strains, exhibited acceptable cytotoxicity and reduced the colonization of S. mutans and L. casei in a biofilm membrane model.

Potencial terapêutico da tintura de Bauhinia Forficata Link frente à cárie dentária / Therapeutic potential of Bauhinia Forficata Link tincture against dental carie

Avaliou-se o potencial terapêutico frente à cárie dentária da tintura hidroalcoólica (20%) de Bauhinia forficata Link (TBF). O estudo foi dividido em três artigos. O primeiro avaliou a constituição química mineral, pH, sólidos solúveis totais (°Brix) da TBF, suas concentrações mínimas inibitória (CMI) e microbicida (CMM) frente aos Streptococcus mutans (SM), o perfil de crescimento/morte (48h) desse microrganismo na presença da TBF, a citotoxicidade da tintura frente a fibroblastos orais (L929) na concentração máxima de 2,33mg/mL, e a ação antibacteriana da TBF, comparada aos controles (clorexidina 0,12% - CHX, dentre eles), sobre um biofilme de SM formado (48h) em blocos de esmalte bovino (n=48), após 3 dias de tratamento diário (1 min). No segundo artigo, investigou-se a CMI e CMM frente ao SM e Streptococcus sanguinis, Candida albicans, Fusubacterium nucleatum, Lactobacillus casei, Prevotella nigrescens, Bifidobacterium dentium; e a ação antimicrobiana da TBF sobre um biofilme de pool salivar de adultos (1mL, n=3, foram misturados e 20µL semeados para formação do biofilme) em membranas de celulose (n=4, 48h) tratadas em um único momento (membranas submersas por 1 min). A ação antimicrobiana foi quantificada pela contagem de Streptococcus spp. (S) e microrganismos totais (MT). No terceiro artigo, observou-se os conteúdos fenólicos da TBF, perfil de crescimento/morte dos microrganismos testados na presença da tintura, a citotoxicidade da TBF (23,21 e 200 mg/mL) até 5 min de contato com fibroblastos orais e seus efeitos: antimicrobiano frente um biofilme formado de pool salivar de crianças (contagem de S e MT e avaliação da altura do biofilme por microscopia confocal) e sobre a perda mineral do esmalte dentário (n=60; % de perda de microdureza-%PMD) após tratamentos diários (TBF e controles) por 7 dias (50µL/1min). Os dados foram analisados descritivamente e inferencialmente (p<0,05). No primeiro artigo, detectou-se cálcio, fósforo e magnésio na composição da tintura, pH=5,8 e ºBrix=17. A TBF apresentou CIM e CBM de 1.11mg/mL e promoveu redução óptica (0,32 nm) do SM após 48h (p<0,05). A tintura obteve baixos valores de morte celular (fibroblastos orais) (p<0,05) e em 2.32 mg/mL reduziu os níveis de SM do biofilme similar à CHX (p>0,05), sendo efetiva em sua redução (p<0,05). No segundo artigo, as CMI e CMM variaram de 1,31-23,2mg/mL e a TBF reduziu S e MT comparada ao não tratamento do biofilme (p<0.05), porém com ação inferior à CHX (p<0,05). No artigo três, a TBF apresentou grande quantidade de compostos fenólicos e ácidos clorogênicos. Na CMM de cada microrganismo gerou uma redução óptica após 48h; promoveu, na maior CMM (23,21mg/mL) baixa citotoxicidade (3.20% de células mortas em 5 min) e reduziu S e MT comparada ao controle de crescimento (CC; p<0,05), mas com ação inferior à CHX (p<0,05). Além disso, não alterou a altura da biomassa formada sobre os blocos (p>0,05) e permitiu menor %PMD comparada ao CC (p<0,05). Portanto, conclui-se que a TBF é uma substância com potencial terapêutico frente à cárie dentária, por apresentar ação antimicrobiana frente a diferentes biofilmes dentários com uso tópico oral seguro. (AU)

It was evaluated the therapeutic potential against dental caries of a hydroalcoholic tincture (20%) of Bauhinia forficata Link (TBF). The study was divided into three articles. The first one evaluated the mineral chemical composition, pH, total soluble solids (°Brix) of TBF. The TBF minimum inhibitory (MIC) and microbicide (MMC) concentrations against Streptococcus mutans (SM); the growth / death profile of SM (48h) with TBF; and the cytotoxicity of the tincture in oral fibroblasts (L929) at a maximum concentration of 2.33 mg/mL were also evaluated. The antibacterial action of TBF, compared to controls (chlorhexidine 0.12% - CHX, among them), on a SM biofilm formed (48h) on bovine enamel blocks (n=48) after 3 days of daily treatment (1 min) was observed. In the second article, MIC and MMC against SM and Streptococcus sanguinis, Candida albicans, Fusubacterium nucleatum, Lactobacillus casei, Prevotella nigrescens, Bifidobacterium dentium were investigated. The antimicrobial action of TBF was also tested against a salivary pool biofilm of adults (1mL, n=3, were mixed and 20µL seeded for biofilm formation) on cellulose membranes (n=4, 48h) treated in a single moment (membranes submerged during 1 min). The antimicrobial action was quantified by counting Streptococcus spp. (S) and total microorganisms (TM). In the third article, the phenolic contents of the TBF; growth/death profile of the tested microorganisms in the presence of the tincture; and the cytotoxicity (23.21 and 200 mg/mL) of TBF up to 5 min of contact with oral fibroblasts were evaluated. The antimicrobial effect of TBF against a biofilm formed from a salivary pool of children (counting S and TM and evaluation of biofilm height by confocal microscopy) on enamel bovine blocks of teeth and the tincture effect against the dental enamel mineral loss (n=60; % microhardness loss- %MHL) after daily treatments (TBF and controls) of the biofilm until 7 days (50µL/1 min) were observed. Data were analyzed descriptively and inferentially (p<0.05). In the first article, calcium, phosphorus and magnesium were detected. The, pH was 5.8 and ºBrix=17. TBF showed MIC and MMC of 1.11mg/mL and was able to decrease the optics density (0.32 nm) of SM after 48 hours (p<0.05). Low values of cell (oral fibroblasts) death (p<0.05) was obtained in the presence of TBF and it (2.32 mg/mL) reduced SM levels of the biofilm similarly to CHX (p>0.05), being effective in SM reducing (p<0.05). In the second article, the MIC and MMC ranged from 1.31-23.2mg/mL and TBF reduced S and MT compared to non-biofilm treatment (p<0.05), but with an antimicrobial action lower than CHX (p <0.05). In the third article, the TBF presented large amount of phenolic compounds and chlorogenic acids. The TBF at the MMC of each microorganism reached an microbial optical reduction after 48h. Even with the highest TBF MMC (23. 21mg/mL), a low cytotoxicity (3.20% of dead cells at 5 min) was observed. And the tincture (23. 21mg/mL) reduced S and TM compared to growth control (CC, p<0.05), but lower than CHX (p<0.05). In addition, TBF did not change the height of the biomass formed on the blocks (p>0.05) and allowed lower %MHL compared to CC (p<0.05). Therefore, it is concluded that TBF is a substance with therapeutic potential against dental caries, since it presents antimicrobial action against different dental biofilms with safe oral topical use. (AU)

Physical properties and antibacterial activity of herbal tinctures of calendula (Calendula officinalis L) and cashew tree (Anacardium occidentale L)

Objective: To evaluate in vitro the pH, Brix and antibacterial activity of herbal tinctures of calendula (Calendula officinalis L.) and cashew tree (Anacardium occidentale L.) against the following bacterial strains: Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 10557), Streptococcus salivarius (ATCC 9758), Enterococcus faecalis (ATCC 29212) and Eikenella corrodens (ATCC 23834). Material and Method: The strains were reactivated in agar Brain Heart Infusion (BHI) and seeded in blood agar with BHI. Susceptibility tests were performed and the strains were incubated in microaerophilia (37 °C) for 48 h. Chlorhexidine at 0.12% was used as a positive control and distilled water as a negative control. Dilution of the tinctures was carried out from 1:1 (pure form) to 1:64 in alcohol 70%, which is found in their composition, in order to evaluate a possible influence of alcohol on the antibacterial activity of the tinctures. O Bx readings were made by refractometry using an Abbe refractometer and the pH was measured using a digital pH meter. Results: The calendula tincture had pH=5.43 and 18 °Brix and cashew tree tincture had pH=7.2 and 17 °Brix. The cashew tree tincture did not presentantibacterial activity against S. oralis, but it inhibited the growth of all other bacteria up to the 1:64 dilution. The calendula tincture presented antibacterial activity against S. oralis up to 1:16 dilution (6.5 mm halo diameter); against S. mutans and S. salivarius up to 1:32 dilution (7 and 7.5 mm halo diameter, respectively); and against E. faecalis and E. corrodens up to 1:64 dilution. Alcohol 70% did not show any antibacterial activity against the bacterial strains. Conclusion: The tinctures of calendula and cashew tree have soluble solids, have different pH values and present a significant antibacterial activity against several bacterial strains, except for the cashew tree tincture against S. oralis, which was ineffective...

Avaliação da atividade antibacteriana das tinturas de Hamamelis virginiana L. e Stryphnodendron rotundifolium Mart. frente a bactérias bucais / Evaluation of the antibacterial activity of Hamamelis virginiana L. andStryphnodendron rotundifolium Mart. tinctures against oral bacteria

Objetivo: Avaliar, in vitro, a atividade antibacteriana dastinturas de Hamamélis (Hamamelis virginiana L.) eBarbatimão (Stryphnodendron rotundifolium Mart.) sob as bactérias Streptococcus mutans (ATCC 25175),Streptococcus oralis (ATCC 10557) e Streptococcussalivarius (ATCC 9758). Materiais e Metodos: Utilizou-se aclorexidina 0,12% como controle positivo. Água destilada e álcool 70% foram escolhidos como controles negativos.Efetivou-se a diluição das tinturas desde 1:1 (forma pura)até 1:64 (D6) em álcool 70%, o qual está presente na fórmula das tinturas fitoterápicas. O meio de cultura bacteriana utilizado foi o Brain Heart Infusion (BHI), havendo as emeadura em placas de petri com auxílio de swabs.Realizou-se o teste de suscetibilidade e, em seguida, foram incubadas a 37°C, em microaerofilia, por 48 horas. O estudo foi realizado em duplicata e os halos foram mensurados como auxílio de um paquímetro manual. Resultados: O controle positivo apresentou halos de inibição de 16, 15, e 16 mmpara S. mutans, S. oralis e S. salivarius, respectivamente.Nenhuma das tinturas avaliadas, em quaisquer formas,diluídas ou puras, obteve halos de inibição para estas bactérias bucais. A água destilada e o álcool também não promoveram atividade. Conclusão: As tinturas da Hamamélise Barbatimão não apresentam atividade antibacteriana sobreS. mutans, S. oralis e S. salivarius.

Objective: To evaluate the in vitro the antibacterial activity ofthe Hamamelis (Hamamelis virginiana L.) and Barbatimão(Stryphnodendron rotundifolium Mart.) tinctures againstStreptococcus mutans (ATCC 25175), Streptococcus oralis(ATCC 10557) and Streptococcus salivarius (ATCC 9758).Material and Methods: Chlorhexidine (0.12%) was used aspositive control. Distilled water and 70% alcohol were chosenas negative controls. The dilution of the tinctures was carriedout from 1:1 (pure form) to 1:64 (D6) in 70% alcohol, which isfound in the tinctures formula. The culture medium Brain Heart Infusion (BHI) was used, and microorganisms were inoculatedon petri plates using swabs. Plates were incubated at 37 ºC,in microaerophilia, for 48 hours. Tests were performed induplicate and halos were measured using a manual caliper.Results: The positive control showed inhibition halos of 16,15, and 16 mm on S. mutans, S. oralis and S. salivarius,respectively. None of the tinctures evaluated, in any form(diluted or pure), were found to show inhibition halos onthese oral bacteria. Distilled water and 70% alcohol did not exhibit activity. Conclusion: The tinctures of Hamamelis and Barbatimão did not demonstrated antibacterial activity on S.mutans, S. oralis and S. salivarius.