No 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 g/ml) and on the surface of sHA beads (30 to 60% at 135 g/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 g/ml and MBCs of 56 to 112 g/ml). tt-Farnesol (56 to 112 g/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.

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.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).

Since in vitro and animal studies suggest that the combination of starch with sucrose may be more cariogenic than sucrose alone, the study assessed in situ the effects of this association applied in vitro on the acidogenicity, biochemical and microbiological composition of dental biofilm, as well as on enamel demineralization. During two phases of 14 d each, fifteen volunteers wore palatal appliances containing blocks of human deciduous enamel, which were extra-orally submitted to four groups of treatments: water (negative control, T1); 2 % starch (T2); 10 % sucrose (T3); and 2 % starch þ 10 % sucrose (T4). The solutions were dripped onto the blocks eight times per day. The biofilm formed on the blocks was analysed with regard to amylase activity, acidogenicity, and biochemical and microbiological composition. Demineralization was determined on enamel by cross-sectional microhardness. The greatest mineral loss was observed for the association starch þ sucrose (P,0·05). Also, this association resulted in the highest lactobacillus count in the biofilm formed (P,0·05). In conclusion, the findings suggest that a small amount of added starch increases the cariogenic potential of sucrose. Among dietary carbohydrates, starch has been pointed out as noncariogenic or slightly cariogenic when used as the sole source of carbohydrate in the diet. This observation has been supported by experiments on dental biofilm acidogenicity (Stephan, 1940;Imfeld, 1977;Lingström et al. 1989), experimental studies with animals (König & Grenby, 1965;Green & Hartles, 1967;Hefti & Schmid, 1979;Bowen et al. 1980), controlled studies in man (Gustaffson et al. 1954), epidemiological data (Marthaler & Froesch, 1967;Fisher, 1968;Newbrun et al. 1980) and in situ experiments (Lingström et al. 1994), which demonstrated that starch is less cariogenic than sucrose.However, while in primitive diets starch was consumed as the main energy source, in contemporary ones it is consumed simultaneously or interspersed with sucrose (Lingström et al. 2000). This combination, which is consumed by both adults and children, may influence dental biofilm composition and consequently dental caries. Thus, a greater prevalence of caries lesions was found in children who consume milk supplemented with a combination of cereal and sucrose (Mattos-Graner et al. 1998). Such observation in human subjects is supported by the results of experimental caries studies in animals (Firestone et al. 1982;Mundorff-Shrestha et al. 1994), suggesting that starch would enhance the cariogenic potential of sucrose.The explanation for the greater cariogenicity of the association of dietary starch with sucrose may reside in the dental biofilm formed. It is well known that the biofilm formed in the presence of sucrose is more cariogenic due to its high concentration of extracellular insoluble polysaccharides (IP), which alter the matrix of the biofilm, making it more porous (Dibdin & Shellis, 1988). These polysaccharides are produced from sucrose by bacterial enzymes named glucosyltransferases....

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch → sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.

Since in vitro pH-cycling models are widely used to study dental caries, they should allow evaluations of fluoride effect on early stages of caries development. Therefore, acid etching on enamel surface must be avoided, enabling surface microhardness (SMH) analysis. In the present study, the pH-cycling model originally described by Featherstone et al.9 (1986) was modified to preserve the enamel surface and to produce early carious lesions that could be evaluated using SMH and cross-sectional microhardness (CSMH) measurements. In order to validate this modified model, a dose-response evaluation with fluoride was made. Human enamel blocks with known SMH were submitted to such regimen with the following treatments: distilled deionized water (DDW; control) and solutions containing 70, 140 and 280 ppm F. Data from %SMH change and deltaZ (mineral loss) showed a statistically significant negative correlation between F concentration in treatment solutions and mineral loss. In conclusion, the modified pH-cycling model allowed the evaluation of changes on the outermost enamel layer during caries development, and a dose-response effect of fluoride reducing enamel demineralization was observed.

Since the effect of iron (Fe) on the cariogenicity of sucrose in humans is unexplored, this study assessed in situ the effect of Fe co-crystallized with sucrose (Fe-sucrose) topically applied in vitro on the acidogenicity, biochemical and microbiological composition of the dental biofilm formed in vivo and on the demineralization of the enamel. During two phases of 14 days each, 16 volunteers wore palatal appliances containing blocks of human enamel, which were submitted to four groups of separate treatments: (1) water; (2) 20% sucrose; (3) 20% (w/v) sucrose plus 18 µg Fe/ml, and (4) 20% (w/v) sucrose plus 70 µg Fe/ml. The solutions were dripped onto the blocks 8 times per day. The biofilms formed on the blocks were analyzed with respect to acidogenicity, biochemical and microbiological composition. Mineral loss was determined on enamel by surface and cross-sectional microhardness. Lower demineralization was found in the blocks subjected to Fe-sucrose (70 µg Fe/ml) than in those treated with sucrose (p < 0.05). This concentration of Fe also reduced significantly the populations of mutans streptococci in the biofilm formed on the blocks. In conclusion, our data suggest that Fe may reduce in situ the cariogenic potential of sucrose and the effect seems to be related to the reduction in the populations of mutans streptococci in the dental biofilm formed.