Preparative Fractionation of Brazilian Red Propolis Extract Using Step-Gradient Counter-Current Chromatography.

Propolis is a resinous bee product with a very complex composition, which is dependent upon the plant sources that bees visit. Due to the promising antimicrobial activities of red Brazilian propolis, it is paramount to identify the compounds responsible for it, which, in most of the cases, are not commercially available. The aim of this study was to develop a quick and clean preparative-scale methodology for preparing fractions of red propolis directly from a complex crude ethanol extract by combining the extractive capacity of counter-current chromatography (CCC) with preparative HPLC. The CCC method development included step gradient elution for the removal of waxes (which can bind to and block HPLC columns), sample injection in a single solvent to improve stationary phase stability, and a change in the mobile phase flow pattern, resulting in the loading of 2.5 g of the Brazilian red propolis crude extract on a 912.5 mL Midi CCC column. Three compounds were subsequently isolated from the concentrated fractions by preparative HPLC and identified by NMR and high-resolution MS: red pigment, retusapurpurin A; the isoflavan 3(R)-7-O-methylvestitol; and the prenylated benzophenone isomers xanthochymol/isoxanthochymol. These compounds are markers of red propolis that contribute to its therapeutic properties, and the amount isolated allows for further biological activities testing and for their use as chromatographic standards.

Pfaffia paniculata Extract, a Potential Antimicrobial Agent against Candida spp., Pseudomonas aeruginosa, and Streptococcus mutans Biofilms.

The World Health Organization (WHO) has prioritized developing new drugs against specific bacteria and fungi, such as Enterobacteriaceae and Candida spp. While Pfaffia paniculata is commonly called the "cure-everything", its scientifically proven benefits are limited to anti-inflammatory and antioxidant actions. Therefore, this study aims to determine the spectrum of antimicrobial activity of Pfaffia paniculata and assess its cytotoxicity. Thus, broth microdilution test was conducted according to the CLSI M7-A9 and M27-A3 reference methods. After screening, microbial species with minimum inhibitory concentration (MIC) values were selected for biofilm tests. These tests evaluated biomass using the crystal violet (CV) test, metabolic activity using the MTT assay, and structural analysis via Scanning Electron Microscopy (SEM). Cytotoxicity was evaluated in human gingival fibroblasts (FMM-1). There were reductions of 29.4 and 42.7% in CV and MTT assays for Candida spp. biofilm. S. mutans and P. aeruginosa biofilms showed a decrease of 15.7 and 28.6%, respectively. Cell viability tests indicated 55.1, 56.9, and 65.5% of viability after contact with 1.93, 0.96, and 0.48 mg/mL of the extract, respectively. The P. paniculata extract showed antimicrobial action, displayed MIC values, and antibiofilm action on P. aeruginosa, S. mutans, and C. albicans. The cytotoxicity on the FMM-1 cell line was dose-dependent. Therefore, P. paniculata extract holds significant potential for developing new drugs.

Effects of natural antimicrobial compounds propolis and copaiba on periodontal ligament fibroblasts, molecular docking, and in vivo study in Galleria mellonella.

Root canal treatment addresses infectious processes that require control. Occasionally, the radicular pulp is vital and inflamed, presenting a superficial infection. To preserve pulpal remnants, conservative procedures have gained favor, employing anti-inflammatory medications. This study investigated the effects of propolis (PRO), and copaiba oil-resin (COR) associated with hydrocortisone (H) and compared their impact to that of Otosporin® concerning cytotoxic and genotoxic activity, cytokine detection, and toxicity in the Galleria mellonella model. Human periodontal ligament fibroblasts (PDLFs) were exposed to drug concentrations and evaluated by the MTT assay. Associations were tested from concentrations that did not compromise cell density. Genotoxicity was evaluated through micronucleus counting, while cytokines IL-6 and TGF-ß1 were detected in the cell supernatant using ELISA. Molecular docking simulations were conducted, considering the major compounds identified in PRO, COR, and H. Increasing concentrations of PRO and COR were assessed for acute toxicity in Galleria mellonella model. Cellular assays were analyzed using one-way ANOVA followed by Tukey tests, while larval survivals were evaluated using the Log-rank (Mantel-Cox) test (α = 0.05). PRO and COR promoted PDLFs proliferation, even in conjunction with H. No changes in cell metabolism were observed concerning cytokine levels. The tested materials induce the release of AT1R, proliferating the PDFLs through interactions. PRO and COR had low toxicity in larvae, suggesting safety at tested levels. These findings endorse the potential of PRO and COR in endodontics and present promising applications across medical domains, such as preventive strategies in inflammation, shedding light on their potential development into commercially available drugs.

Randomized, double-blind, placebo-controlled clinical trial on the effects of propolis and chlorhexidine mouthrinses on gingivitis / Ensaio clínico duplo-cego randomizado e placebo-controlado dos efeitos de enxaguatórios a base de própolis e clorexidina na saúde gengival, para análise

Objective: The aim of this study was to compare the effects of typified propolis and chlorhexidine mouthrinses on gingival health in a randomized double-blind placebo-controlled clinical trial. Material and Methods: Sixty participants were randomized to 3 mouthrinse study groups: 1) 2% typified propolis (n = 20); 2) 0.12% chlorhexidine (n = 20), and 3) placebo (n = 20). Participants rinsed unsupervised twice a day for 28 days. The Papillary Bleeding Score (PBS) was measured on the mesio-buccal surfaces of all teeth at baseline and 28 days thereafter. Co-variance analysis was employed to compare PBS average values and the number of sites with PBS ≥ 2 among study groups. Sub-group analysis was further applied to participants who were < 40 years-old. Results: The results show efficacy of propolis mouthrinse when comparing before and after treatment protocols significantly for a reduction of mean PBS scores. For younger participants propolis mouthrinse was superior to all groups in reducing mean PBS scores and significant when compared to 0.12% chlorhexidine mouthrinse. Conclusion: The efficacy of 2% typified propolis mouthrinse was demonstrated in reducing the levels of gingival inflammation. These results need to be duplicated by other investigators under similar study protocols.

Objetivos: O propósito deste estudo foi o de comparar os efeitos de enxaguatórios a base de própolis tipificada e clorexidina na saúde gengival, empregando-se um ensaio clínico duplo cego, randomizado, e placebo-controlado. Material e Métodos: Sessenta participantes foram randomizados em 3 grupos de enxaguatórios, a saber: própolis tipificada 2% (n = 20), clorexidina 0,12% (n = 20) e placebo (n = 20). Os participantes bochecharam duas vezes ao dia por 28 dias os respectivos enxaguatórios. Medidas do índice de sangramento papilar (PBS) foram feitas na superfície mésio-bucal de todos os dentes dos participantes no tempo basal e após 28 dias. Análise de co-variância foi empregada permitindo comparações entre os grupos das médias do PBS e do número de sítios ≥ 2. Análise de sub-grupo foi efetuada em participantes com idade < 40 anos. Resultados: Constataram-se os efeitos positivos do enxaguatório de própolis a 2% na reducão da inflamação gingival após uso não supervisionado por 28 dias. Análise no sub-grupo de parcipantes com idade < 40 anos constatou superioridade do enxaguatório de própolis quando comparado com o enxaguatório de clorexidina a 0,12%. Conclusão: Este ensaio clínico demonstrou a eficácia de enxaguatório de própolis tipificada a 2% na redução da inflamação gengival. Estes resultados necessitam ser duplicados por outros investigadores.

Effects of typified propolis on mutans streptococci and lactobacilli: a randomized clinical trial / Efeitos da própolis tipificada nos estreptococcos do grupo mutans e lactobacilos: ensaio clínico randomizado

Objective: The aim of this study was to determine in a randomized, double-blind, placebo-controlled clinical trial the effects of typified propolis and chlorhexidine rinses on salivary levels of mutans streptococci (MS) and lactobacilli (LACT). Methods: One hundred patients were screened for salivary levels of MS >100,000 CFUs/mL of saliva. All patients presented with at least one cavitated decayed surface. Sixty patients met entry criteria. Subjects were adults 18-55 years old. After restoration of cavitated lesions patients were randomized to 3 experimental groups:1) PROP-alcohol-free 2% typified propolis rinse (n= 20); 2) CHX- 0.12% chlorhexidine rinse; 3) PLplacebo mouthrinse. Patients rinsed unsupervised 15 mL of respective rinses twice a day for 1 min for 28 days. Patients were assessed for the salivary levels of MS (Dentocult SM) and LACT (Dentocult LB) at baseline, 7-day, 14-day, and at 28-day visits (experimental effects) and at 45-day visit (residual effects). General linear models were employed to analyze the data. Results: PROP was superior to CHX at 14-day and 28-day visits in suppressing the salivary levels of MS (p < .05). PROP was superior to PL at all visits (p < .01). The residual effects of PROP in suppressing the salivary levels of MS could still be observed at the 45-day visit, where significant differences between PROP and CHX (p < .05), were demonstrated. PROP was significantly superior than CHX in suppressing the levels of salivary LACT at the 28-day visit (p < .05). Conclusion: Typified propolis rinse was effective in suppressing cariogenic infections in caries-active patients when compared to existing and placebo therapies.

Objetivo: O objetivo deste estudo foi determinar, em um estudo randomizado, duplo-cego, placebo-controlado os efeitos da própolis tipificada e clorexidina sobre os níveis salivares de estreptococos do grupo mutans (EM) e lactobacilos (LACT). Métodos: Cem pacientes foram selecionados para níveis salivares de MS > 100.000 UFC/mL de saliva. Todos os pacientes apresentaram pelo menos uma superfície cariada com cavitação. Sessenta pacientes preencheram os critérios de inclusão. Os indivíduos eram adultos com 18-55 anos de idade. Após a restauração das lesões cavitadas, os pacientes foram randomizados para três grupos experimentais: 1) PROP- bochecho livre de álcool de 2% de própolis tipificada (n = 20), 2) CHX- bochecho de clorexidina 0,12%, 3) PL- bochecho placebo. Os participantes bochecharam sem supervisão 15 mL dos enxaguatórios duas vezes por dia, durante 1 minuto, durante 28 dias. Os pacientes foram avaliados para os níveis salivares de MS (Dentocult SM) e LACT (Dentocult LB) na linha de base, e após 7 dias, 14 dias, 28 dias (efeitos experimentais) e 45 dias (efeitos residuais). Modelos lineares foram utilizados para analisar os dados. Resultados: PROP foi superior ao CHX nas visitas de 14 dias e de 28 dias na supressão dos níveis salivares de SM (p < 0,05). PROP foi superior ao PL em todas as visitas (p < 0,01). Os efeitos residuais de PROP na supressão dos níveis salivares de MS ainda foi observado na visita de 45 dias, onde diferenças significativas entre PROP e CHX (p < 0,05) foram demonstradas. PROP foi significativamente superior a CHX na supressão dos níveis salivares de LACT na visita de 28 dias (p < 0,05). Conclusão: O enxaguatório de própolis tipificada foi eficaz na supressão de infecções cariogênicas em pacientes com atividade de cárie quando comparado a terapias existentes e ao placebo

Potential utility of hyperbaric oxygen therapy and propolis in enhancing the leishmanicidal activity of glucantime / A utilidade da terapia de oxigenação hiperbárica e própolis em potencializar a atividade leishmanicida do glucantime

In this study we investigated the efficacy of hyperbaric oxygen (HBO) therapy, alone or combined with the pentavalent antimonial glucantime on Leishmania amazonensis infection. In parallel, the effect of Brazilian red propolis gel (propain) alone or combined with glucantime on L. amazonensis infection was evaluated. The inhibition of the infection in macrophages treated with glucantime in combination with HBO exposition was greater than that of macrophages treated with glucantime alone or HBO alone. The susceptible mouse strain BALB/c infected in the shaved rump with L. amazonensis treated with glucantime and exposed to HBO showed: time points in the course of the disease in which lesions were smaller than those of mice treated with glucantime alone and revascularization of the skin in the lesion site; interferon-gamma (IFN-g) levels were not elevated in lymph node cells from these animals. Propain alone was not efficient against lesions, although less exudative lesions were observed in animals treated with propain alone or combined with glucantime. These results reveal the potential value of HBO and red propolis in combination with glucantime for treating cutaneous leishmaniasis and encourage further studies on the effect of more aggressive HBO, propolis and glucantime therapies on different mouse models of leishmaniasis.

Nesse trabalho foi avaliada a eficácia da terapia da oxigenação hiperbárica (HBO), aplicada em combinação ou não com o tratamento com glucantime, durante a infecção com Leishmania amazonensis. O efeito de gel da própolis vermelha de origem brasileira (propaina) aplicado em combinação ou não com o tratamento com glucantime, também foi avaliado durante infecção com esse parasita. A inibição da infecção de macrófagos tratados com glucantime em combinação com HBO foi maior que a de macrófagos tratados apenas com glucantime ou HBO. A linhagem murina susceptível, BALB/c, infectada no dorso com L. amazonensis, tratada com glucantime e exposta a HBO, mostrou durante o curso da doença, fases em que as lesões eram menores do que a de camundongos apenas tratados com glucantime; observou-se revascularização da pele da lesão e baixa produção de interferon-gama em células de linfonodos desses animais. O tratamento com propaina não foi eficiente na cura das lesões, apesar de lesões menos exsudativas serem observadas em animais tratados com propaina ou propaina combinada ao tratamento com glucantime. Os resultados demonstram que tanto HBO como a própolis vermelha em combinação com glucantime, são promissoras no tratamento da leishmaniose cutânea. Novos estudos devem ser realizados para avaliar tratamentos e outros protocolos em diferentes modelos murinos da leishmaniose.

Evaluation of the in vitro antimicrobial activity of an ethanol extract of Brazilian classified propolis on strains of Staphylococcus aureus

Staphylococcus aureus (S. aureus) is one of the most frequent causes of hospital acquired infections. With the increase in multiple drug resistant strains, natural products such as propolis are a stratagem for new product discovery. The aims of this study were: to determine the in vitro antimicrobial activity of an ethanol extract of propolis; to define the MIC50 and MIC90 (Minimal Inhibitory Concentration - MIC) against 210 strains of S. aureus; to characterize a crude sample of propolis and the respective ethanol extract as to the presence of predetermined chemical markers. The agar dilution method was used to define the MIC and the high performance liquid chromatography (HPLC) method was used to characterize the samples of propolis. MIC results ranged from 710 to 2,850 µg/mL. The MIC50 and MIC90 for the 210 strains as well as the individual analysis of American Type Culture Collection (ATCC) strains of Methicillin-susceptible Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) were both 1,420 µg/mL. Based on the chromatographic analysis of the crude sample and ethanol extracted propolis, it was concluded that propolis was a mixture of the BRP (SP/MG) and BRP (PR) types. The results obtained confirm an antimicrobial activity in relation to the strains of the S. aureus tested.

Effect of green propolis addition to physical mechanical properties of glass ionomer cements

OBJECTIVE: This study investigated the mechanical properties of glass ionomer cements (GICs) combined with propolis as a natural antimicrobial substance. MATERIAL AND METHODS: Typifed green propolis, as an ethanolic extract (EEP) or in the lyophilized form (powder), was incorporated to specimens of Ketac Fil Plus, ChemFlex and Ketac Molar Easymix GICs. For each test, 8 specimens of each material were prepared. For water sorption and solubility tests, specimens were subjected to dehydration, hydration and redehydration cycles until a constant mass was obtained for each step. Measurements were recorded using a digital balance of 10-4 g precision. For the diametral tensile strength test, specimens were tested in a universal test machine at 0.5 mm/min crosshead speed after 24 h storage in deionized water. Data were evaluated by one-way ANOVA and Tukey's tests (p<0.05). RESULTS: The addition of propolis to GIC clearly increased water sorption compared to pure material. Solubility was material-dependent and was not clearly evident. For the diametral tensile strength test, association with propolis altered negatively only Chemfex. CONCLUSION: It may be concluded that incorporation of propolis to GICs alters some properties in a material-dependent condition.

Effects of Brazilian propolis on Leishmania amazonensis

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 æg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

Caracterizaçäo de própolis brasileira por fluorescência de raios-X: evidência de contaminaçäo por chumbo / Characterization of brazilian propolis by X-ray fluorescence: contamination by lead evidency

Foram estudadas amostras de própolis de diferentes origens, tendo sido identificada a presença de vários elementos metálicos. A própolis bruta e seus constituintes foram caracterizadas por análise térmica, prevendo com isto possíveis fraudes que possam ser encontradas neste valioso produto elaborado pelas abelhas.

Extração líquido-líquido de compostos presentes na própolis / Liquid-liquid extraction of propolis compounds

Uma amostra de própolis produzida pela abelha do tipo apis mellifera foi submetida a um processo para extração de alguns compostos farmacologicamente ativos presentes na própolis. Usou-se um extrator líquido-líquido para separação dos compostos, os quais foram identificados por cromatografia líquida de alta eficiência (CLAE). Os compostos analisados foram ácidos fenólicos (p-cumárico e derivados), um flavonóide e um derivado de canferol. O processo de extração permitiu isolar o ácido 3-prenil-4-hidroxicinâmico e o ácido 3,5-diprenil-4-hidroxicinâmico para a fase aquosa ou orgânica, respectivamente.

Análise química, toxicológica e antiulcerogênica preliminar de uma amostra de própolis da região do Paraná / Preliminary chemical, toxicological and anti-ulcerogenic analysis o of sample of propolis from the region of Paraná

O presente trabalho investigou a constituição química e as propriedades toxicológicas e farmacológicas de uma amostra de própolis oriunda da região de Rio Azul (PR), Brasil. As análises químicas foram realizadas por cromatografia líquida de alta resolução acompanhadas do estudo físico-químico da velocidade de oxidação por permanganato de potássio. O ensaio toxicológico consistiu na determinação da toxicidade aguda em dose simples, dose letal mediana (DL50) e Screening Hipocrático em camundongos, pela administração per os da amostra. A análise da atividade antiulcerogênica foi conduzida em protocolo de úlcera induzida pela administração subcutânia de indometacina a ratos albinos. Os resultados das análises químicas indentificaram alguns compostos derivados do ácido e o 3-metoxi-4-hidroxicinamaldeído, bem como outros ainda não indentificados.Do ponto de vista toxicológico a amostra pesquisada não evidenciou efeitos deletérios sobre a função do sistema nervoso central, autônomo e atividade motora, revelando valor de DL50 superior a 5000mg.kg 1. A dose de 1,0 g.kg1 de própolis foi competente em reduzir o índice médio das lesões ulcerativas em 72,06 por cento nas unidades experimentais tratadas com a indometacina, evidenciando significante atividade antiulcerogênica.