Jaime Aparecido Cury

In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens

Arnica and propolis have been used for thousands of years in folk medicine for several purposes. They possess several biological activities such as anti-inflammatory, antifungal, antiviral and tissue regenerative, among others. Although the antibacterial activity of propolis has already been demonstrated, very few studies have been done on bacteria of clinical relevance in dentistry. Also, the antimicrobial activity of Arnica has not been extensively investigated. Therefore the aim here was to evaluate in vitro the antimicrobial activity, inhibition of adherence of mutans streptococci and inhibition of formation of water-insoluble glucan by Arnica and propolis extracts. Arnica montana (10%, w/v) and propolis (10%, w/v) extracts from Minas Gerais State were compared with controls. Fifteen microorganisms were used as follows: Candida albicans--NTCC 3736, F72; Staphylococcus aureus--ATCC 25923; Enterococcus faecalis--ATCC 29212; Streptococcus sobrinus 6715; Strep. sanguis--ATCC 10556; Strep. cricetus--HS-6; Strep. mutans--Ingbritt 1600; Strep. mutans--OMZ 175; Actinomyces naeslundii--ATCC 12104, W 1053; Act. viscosus OMZ 105; Porphyromonas gingivalis; Porph. endodontalis and Prevotella denticola (the last three were clinical isolates). Antimicrobial activity was determined by the agar diffusion method and the zones of growth inhibition were measured. To assess cell adherence to a glass surface, the organisms were grown for 18 h at 37 degrees C in test-tubes at a 30 degree angle. To assay water-insoluble glucan formation, a mixture of crude glucosyltransferase and 0.125 M sucrose was incubated for 18 h at 37 degrees C in test-tubes at a 30 degree angle. Arnica and propolis extracts (20 microl) were added to these tubes to evaluate the % of inhibition of cell adherence and water-insoluble glucan formation. The propolis extract significantly inhibited all the microorganisms tested (p < 0.05), showing the largest inhibitory zone for Actinomyces spp. The Arnica extract did not demonstrate significant antimicrobial activity. Cell adherence and water-insoluble glucan formation were almost completely inhibited by the propolis extract at a final concentration of 400 microg/ml and 500 microg/ml, respectively. The Arnica extract showed slight inhibition of the adherence of the growing cells (19% for Strep. mutans and 15% for Strep. sobrinus) and of water-insoluble glucan formation (29%) at these same concentrations. Thus, the propolis extract showed in vitro antibacterial activity, inhibition of cell adherence and inhibition of water-insoluble glucan formation, while the Arnica extract was only slightly active in those three conditions.

The Role of Sucrose in Cariogenic Dental Biofilm Formation—New Insight

Dental caries is a biofilm-dependent oral disease, and fermentable dietary carbohydrates are the key environmental factors involved in its initiation and development. However, among the carbohydrates, sucrose is considered the most cariogenic, because, in addition to being fermented by oral bacteria, it is a substrate for the synthesis of extracellular (EPS) and intracellular (IPS) polysaccharides. Therefore, while the low pH environment triggers the shift of the resident plaque microflora to a more cariogenic one, EPS promote changes in the composition of the biofilms’ matrix. Furthermore, it has recently been shown that the biofilm formed in the presence of sucrose presents low concentrations of Ca, Pi, and F, which are critical ions involved in de- and remineralization of enamel and dentin in the oral environment. Thus, the aim of this review is to explore the broad role of sucrose in the cariogenicity of biofilms, and to present a new insight into its influence on the pathogenesis of dental caries.

Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity.

ABSTRACT Propolis, a resinous bee product, has been shown to inhibit the growth of oral microorganisms and the activity of bacterium-derived glucosyltransferases (GTFs). Several compounds, mainly polyphenolics, have been identified in this natural product. The present study evaluated the effects of distinct chemical groups found in propolis on the activity of GTF enzymes in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads. Thirty compounds, including flavonoids, cinnamic acid derivatives, and terpenoids, were tested for the ability to inhibit GTFs B, C, and D from Streptococcus mutans and GTF from S. sanguinis (GTF Ss). Flavones and flavonols were potent inhibitors of GTF activity in solution; lesser effects were noted on insolubilized enzymes. Apigenin, a 4′,5,7-trihydroxyflavone, was the most effective inhibitor of GTFs, both in solution (90.5 to 95% inhibition at a concentration of 135 μg/ml) and on the surface of sHA beads (30 to 60% at 135 μg/ml). Antibacterial activity was determined by using MICs, minimum bactericidal concentrations (MBCs), and time-kill studies. Flavanones and some dihydroflavonols, as well as the sesquiterpene tt-farnesol, inhibited the growth of S. mutans and S. sobrinus; tt-farnesol was the most effective antibacterial compound (MICs of 14 to 28 μg/ml and MBCs of 56 to 112 μg/ml). tt-Farnesol (56 to 112 μg/ml) produced a 3-log-fold reduction in the bacterial population after 4 h of incubation. Cinnamic acid derivatives had negligible biological activities. Several of the compounds identified in propolis inhibit GTF activities and bacterial growth. Apigenin is a novel and potent inhibitor of GTF activity, and tt-farnesol was found to be an effective antibacterial agent.

Antimicrobial activity of propolis on oral microorganisms.

Abstract. Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin.

In situ anticariogenic potential of glass ionomer cement

The purpose of this study was to compare the amount of fluoride in plaque formed on glass ionomer cement or composite and to evaluate the effects of fluoride released on growth of cariogenic microflora, fluoride uptake, and secondary caries formation under in situ conditions of a high cariogenic challenge. Ten adult volunteers took part in this crossover study performed in two phases of 28 days. Eighty enamel blocks were randomly restored with glass ionomer cement (Chelon-Fil-Espe) or composite (Silux). During each phase of the study, an acrylic resin appliance, containing four enamel blocks restored with the same material, was constructed for each of the volunteers. During the experimental period, all subjects used fluoride-free dentifrice, refrained from brushing the restored enamel blocks, and immersed the appliances into 20% sucrose solution eight times a day. Fluoride levels, mutans streptococci, and lactobacilli were assessed in dental plaque. Fluoride uptake and microhardness profiles were determined in enamel around the restorations. Statistical analyses indicated a significantly higher level of fluoride (p < 0.05) and a lower level of mutans streptococci plaque formed on glass ionomer cement. Analysis of variance in a split-plot model indicated that in the enamel around the glass ionomer restoration the fluoride uptake was significantly greater (p < 0.025) and the mineral loss significantly lower (p < 0.01). The results show that glass ionomer cement presents a broad anticariogenic effect and may be of value in preventing secondary caries, even under conditions of a high caries risk.

Protective Effect of the Dental Pellicle against Erosive Challenges in situ

The acquired dental pellicle helps prevent erosion, but the protection level is unknown. This in situ study tested whether a two-hour pellicle protects against different erosive challenges by orange juice. Subjects wore palatal appliances loaded with either enamel or dentin specimens. Pellicle was allowed to form, or not (control), on the surfaces of the specimens intra-orally for 2 hrs before the erosive challenges of 0 (control), 10, 20, and 30 minutes’ duration. Specimens were randomly removed from the appliances after each challenge. Percentage of surface microhardness change (%SMC) was determined for the enamel specimens, and that of mineral loss and lesion depth for the dentin specimens. Enamel specimens with the pellicle showed a significantly lower %SMC, only after the 10-minute challenge. No protection was found for dentin. It was concluded that the acquired pellicle reduced dental erosion, but that this effect was limited to the less severe erosive challenge on enamel surfaces.

pH-cycling models to evaluate the effect of low fluoride dentifrice on enamel de- and remineralization

Since the currently available pH-cycling models do not differentiate the anti-caries potential of dentifrices with low fluoride (F) concentration, two models were developed and tested in the present. Bovine enamel blocks were subjected to the models and treated with F solutions containing from 70 to 280 microg F/mL in order to validate them in terms of dose-response effect. The models were also tested by evaluating the dentifrices Colgate Baby (500 microg F/g, as a low fluoride dentifrice), Tandy (1,100 microg F/g, as an active F-dentifrice) and Crest (1,100 microg F/g, as positive control). Enamel mineral loss or gain was assessed by surface and cross-sectional microhardness, and lesion depth was analyzed by polarized light microscopy. The pH-cycling models showed F dose-response effect either reducing enamel demineralization or enhancing remineralization. The low F dentifrice presented anti-caries potential, but it was not equivalent to the dentifrices containing 1,100 microg F/g. These data suggest that the models developed in this study were able to evaluate the anti-caries potential of low F dentifrice either on resistance to demineralization or on enhancement of remineralization.

Relationship among Dental Plaque Composition, Daily Sugar Exposure and Caries in the Primary Dentition

The relationship among daily sugar exposure, dental plaque composition and caries patterns in the primary dentition is not known. Three groups of 20 children (18–48 months old) were studied: (1) caries-free (CF), (2) presenting pit and fissure caries (PFC), and (3) with nursing caries (NC). Dental caries index (dmfs) was assessed and the frequency of daily sugar exposure (TS) and its forms were estimated by questionnaires. Fluoride (F), calcium (Ca), inorganic phosphorus (Pi), insoluble polysaccharide (IP) and mutans streptococci were quantified in dental plaque. The three groups of children were statistically different regarding daily sugar exposure. Concentrations of F, Ca and Pi were lower and the IP and mutans streptococci statistically higher in dental plaque from NC children than those from CF children. PFC children also showed statistically lower F and P levels in their plaque than CF children. Statistically significant correlations were found between IP, TS and dmfs from NC and PFC children. A statistically significant correlation between mutans streptococci in plaque from anterior teeth and dmfs for NC children was also observed. These results suggest that the dietary sugar experience changes the biochemical and microbiological composition of dental plaque, which could partly explain the different caries patterns observed in primary dentition.

In situ relationship between sucrose exposure and the composition of dental plaque

The aim of this study was to analyze the composition of dental plaque according to sucrose exposure. Twelve adult volunteers took part in this crossover study done in four phases of 28 days each. For each phase of the study, an acrylic resin appliance containing four human dental enamel blocks was constructed for each volunteer. A 20% sucrose solution was dripped onto the enamel blocks from 0 to 8 times/day. The volunteers were randomly assigned to the treatments. During the experimental period all the subjects used fluoride-free dentifrice, refrained from brushing the enamel blocks and drank water fluoridated at 0.70 ppm F. After each phase the concentrations of fluoride (F), calcium (Ca), phosphorus (P) and total carbohydrate were determined in dental plaque. Statistical analyses showed that frequent sucrose exposure significantly (p < 0.05) reduced the F, Ca and P concentrations in dental plaque, but increased the alkali-soluble carbohydrate concentration. The results suggest that the cariogenicity of dental plaque formed in the presence of sucrose cannot be attributed only to its higher porosity, but the lower inorganic concentration may also be important.

Chemical Composition and Botanical Origin of Red Propolis, a New Type of Brazilian Propolis

Red propolis is a new type of Brazilian propolis. This material, as well as the secretions of 20 plant species that are often mentioned as its probable botanical source, have been investigated by RP-HPTLC. Phytochemical evidence based on UV-VIS spectra, RP-HPLC and GC-MS, showed Dalbergia ecastophyllum (L.) Taub. to be the main source of red propolis in Alagoas state. The propolis and plant resin showed high relative percentages of the isoflavonoids 3-Hydroxy-8,9-dimethoxypterocarpan and medicarpin. To our knowledge this is the first report of the secretion of a leguminous species being the source of propolis.