Inhibitory effects of Oenothera biennis (evening primrose) seed extract on Streptococcus mutans and S. mutans-induced dental caries in rats.

BACKGROUND: Oenothera biennis (evening primrose) seed extract (OBSE) is known to contain polyphenols, which may possess antioxidant activities. Polyphenols extracted from several plants are reported to exhibit cariostatic activities by inhibiting mutans streptococcus growth and glucosyltransferase activities. The purpose of the present study was to examine the inhibitory effects of OBSE on the development of dental caries, both in vitro and in vivo. METHODS: OBSE was investigated for its inhibitory effects on cellular aggregation, hydrophobicity, sucrose-dependent adherence and insoluble glucan synthesis. Furthermore, biofilm formation was examined in the presence of OBSE, using confocal microscopic imaging. An animal experiment was also performed to examine the in vivo effects. RESULTS: OBSE induced a strong aggregation of Streptococcus mutans MT8148 cells, while cell surface hydrophobicity was decreased by approximately 90% at a concentration of 0.25 mg/ml. The sucrose-dependent adherence of the MT8148 cells was also reduced by addition of OBSE, with a reduction rate of 73% seen at a concentration of 1.00 mg/ml. Additionally, confocal microscopic observations revealed the biofilm development phase to be remarkably changed in the presence of OBSE. Furthermore, insoluble glucan synthesis was significantly reduced when OBSE was present at concentrations greater than 0.03 mg/ml. In an animal experiment, the caries scores in rats given OBSE (0.05 mg/ml in drinking water) were significantly lower than those in rats given water without OBSE. CONCLUSION: Our results indicate that OBSE has inhibitory activity on dental caries.

Remineralization effects of gum arabic on caries-like enamel lesions.

OBJECTIVE: Gum arabic is a natural polysaccharide exudate from Acacia senegal and other related African species of Acacia. Gum arabic is considered to have an ability to enhance remineralization, because of its high concentration of Ca(2+). However, the caries preventive capacity of gum arabic has been scarcely investigated. We evaluated the cariostatic activities of gum arabic using histopathological methods to determine its effects on remineralization. DESIGN: Following incubation in demineralization solution, human third molars were exposed to 10 mg/ml of gum arabic, sodium fluoride at 1000 ppm (NaF), or double distilled water (DW, negative control), then subjected to demineralization-remineralization cycles. Before and after demineralization-remineralization cycles, contact microradiographs of each sample were taken and mineral distribution quantities were calculated. RESULTS: The remineralization ratio of the molars exposed to gum arabic was similar to that of those exposed to NaF, while the ratios of both were significantly greater than that of those exposed to DW. CONCLUSIONS: Gum arabic enhanced the remineralization of caries-like enamel lesions in vitro, suggesting its inhibitory effects towards dental caries.

Phex mutation causes the reduction of npt2b mRNA in teeth.

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.

Antibacterial activity of polyphenol components in oolong tea extract against Streptococcus mutans.

The purpose of the present study was to determine the antibacterial activity of oolong tea extract on oral streptococci, including Streptococcus mutans and Streptococcus sobrinus, and to identify the response to its components. Antibacterial activity was found when the extract was added to S. mutans cells in chemically defined medium but not in complex broth media. Further, pretreatment with bovine serum albumin reduced the antibacterial activity. The extract showed antibacterial activity against all of the oral streptococci examined, with the highest activity against S. mutans MT8148R. This activity was found to originate from a monomeric polyphenol-rich fraction, and it was stronger than that of pure polyphenols. Moreover, some combinations of monomeric polyphenols showed the highest level of antibacterial activity. These results suggest that the antibacterial activity of oolong tea extract is caused by a synergistic effect of monomeric polyphenols, which can easily bind to proteins.

Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148.

An oolong tea polyphenol (OTF6) has been shown to possess a strong anti-glucosyltransferase (GTF) activity and inhibit experimental dental caries in rats infected with mutans streptococci. The effects of OTF6 on the functional domains of GTFs of Streptococcus mutans, an N-terminal catalytic domain (CAT), and a C-terminal glucan-binding domain (GBD), were examined. The maximum velocity of glucan synthesis by recombinant GTFB (rGTFB) and GTFD (rGTFD) became significantly slower in the presence of OTF6, however, Km values remained stable when compared in their absence. These results suggest that OTF6 reduces glucan synthesis by non-competitively inhibiting the GBD of S. mutans GTFB and GTFD. Further, the recombinant proteins of CAT (rCAT) and GBD (rGBD) were expressed using Escherichia coli, and purified by affinity column chromatography. rGBD but not rCAT was found to possess dextran-binding activity, which was shown to be inhibited by OTF6. These results indicate that OTF6, a polymeric polyphenol specific for oolong tea is able to reduce glucan synthesis by inhibiting the GBD of S. mutans GTFB.

Identification of cariostatic substances in the cacao bean husk: their anti-glucosyltransferase and antibacterial activities.

The cacao bean husk has been shown to possess two types of cariostatic substances, one showing anti-glucosyltransferase (GTF) activity and the other antibacterial activity, and to inhibit experimental dental caries in rats infected with mutans streptococci. In the present study, chromatographic purification revealed high-molecular-weight polyphenolic compounds and unsaturated fatty acids as active components. The former, which showed strong anti-GTF activity, were polymeric epicatechins with C-4beta and C-8 intermolecular bonds estimated to be 4636 in molecular weight in an acetylated form. The latter, which showed bactericidal activity against Streptococcus mutans, were determined to be oleic and linoleic acids, and demonstrated a high level of activity at a concentration of 30 microgram/mL. The cariostatic activity of the cacao bean husk is likely caused by these biologically active constituents.

Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments.

Cacao bean husk extract (CBH) was examined for inhibitory effects on the caries-inducing properties of mutans streptococci in vitro and on caries development in specific pathogen-free Sprague-Dawley rats infected with mutans streptococci. CBH reduced the growth rate of almost all oral streptococci examined, which resulted in the reduction of acid production. Furthermore, insoluble glucan synthesis by the glucosyltransferases from Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was significantly inhibited by CBH. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CBH. The administration of CBH in drinking water resulted in significant reductions of caries development and dental plaque accumulation in rats infected with either Strep. sobrinus 6715 or Strep. mutans MT8148R, and the minimum cariostatic concentration was 1.0 mg/ml. These results indicate that CBH possesses powerful anticariogenic potential.

Cariostatic activity of cacao mass extract.

Chocolate is suspected to contain some caries-inhibitory substances. The cariostatic activity of cacao mass extract (CM), the main component of chocolate, was examined in vitro and in experimental animals. CM showed no detectable effects on the cellular growth and acid production of mutans streptococci. On the other hand, the cell-surface hydrophobicity of mutans streptococci was significantly reduced by the presence of CM. Furthermore, insoluble glucan synthesis by the glucosyltransferases from either Streptococcus mutans MT8148R or Strep. sobrinus 6715 was inhibited by CM, but not significantly. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CM. Finally, CM in both a 40% sucrose diet and drinking water resulted in reductions of caries development and plaque accumulation in rats infected with Strep. sobrinus 6715, but not significantly. These results indicate that cacao mass extract possesses some anticariogenic potential, but its anticaries activity is not strong enough to suppress significantly the cariogenic activity of sucrose.

Inhibitory effects of oolong tea extract on caries-inducing properties of mutans streptococci.

The inhibitory effects of oolong tea extract (OTE) on the caries-inducing properties of mutans streptococci were examined in vitro. OTE reduced the rate of acid production by mutans streptococci accompanied with the retardation of growth rate of mutans streptococci, while the action by chromatographically isolated oolong tea polyphenol (OTF6) was weak. On the other hand, both oolong tea products decreased cell surface hydrophobicity of almost all the oral streptococci examined in the present study, and also induced cellular aggregation of Streptococcus mutans, Streptococcus oralis, Streptococcus sanguis or Streptococcus gordonii. In these reactions, OTF6 showed a more pronounced activity than OTE. Furthermore, the oolong tea products inhibited the adherence of mutans streptococci to saliva-coated hydroxyapatite. These results suggest that OTF6 may inhibit bacterial adherence to the tooth surfaces by reducing the hydrophobicity of mutans streptococci, and OTE may inhibit caries-inducing activity of mutans streptococci by reducing the rate of acid production.

Comparison of the cariostatic effects between regimens to administer oolong tea polyphenols in SPF rats.

The cariostatic effect of oolong tea polyphenols administered according to several regimens was examined in specific pathogen-free (SPF) Sprague-Dawley rats given both a diet containing 20% sucrose and infected with S. sobrinus 6715. The crude preparation (OTE) of oolong tea polyphenols showed the most prominent effect on caries reduction in SPF rats when OTE was administered in the drinking water beginning 1 day prior to the inoculation of S. sobrinus 6715, when compared with chromatographically isolated polyphenol fractions (OTF1 and OTF6) of OTE. Reduction in caries development was found even when OTE was given 1 day after inoculation of the organism. OTE was shown to significantly inhibit dental caries in rats at the concentrations of either more than 5 microg/ml in drinking water or more than 10 microg/g in diet. OTF1 and OTF6 also showed significant inhibition of caries induction, with the minimum inhibitory concentration of OTF6 being 50 microg/ml in drinking water and the minimum inhibitory concentration of OTF1 being 100 microg/g in diet. These results indicate that cariostatic activity of OTE was effective even after the establishment of S. sobrinus in the oral cavity and was more effective in drinking water than in diet. Furthermore, OTE may contain some anticaries substances that affect the virulence of S. sobrinus other than glucosyltransferases.

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus.

Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of D-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.

Reduction of dental plaque deposition in humans by oolong tea extract.

The inhibitory effect of oolong tea extract (OTE) containing polymerized polyphenols on plaque deposition was examined in 35 human volunteers. Thirty-five human volunteers, aged 18-29 years, who received extensive oral prophylactic procedures were requested to refrain from all oral hygiene procedures for 4 days, and to rinse their mouth with 0.5 mg/ml OTE solution in 0.2% ethanol before and after every intake of food and before sleeping at night. No restriction regarding meals was given during the test period, except to refrain from teas or coffee. Plaque deposition was evaluated after disclosing the teeth with Erythrocin at the termination of this experiment. The study was repeated 1 week after the first trial, but only 0.2% ethanol without OTE was used for mouthrinsing in the second trial. OTE was found to significantly inhibit plaque deposition in volunteers, although mouthrinsing with OTE solution had no significant effect on the number of mutans streptococci in unstimulated whole saliva.

Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.

Oolong tea polyphenols inhibit experimental dental caries in SPF rats infected with mutans streptococci.

An extract of oolong tea (semifermented tea leaves of Camellia sinensis) and its chromatographically isolated polyphenolic compound was examined for in vitro inhibitory effects on glucosyltransferases (GTases) of mutans streptococci and on caries development in Sprague-Dawley rats infected with mutans streptococci. The samples showed no detectable effect on the growth of mutans streptococci. However, insoluble glucan synthesis from sucrose by the GTases of Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was markedly inhibited, as was sucrose-dependent cell adherence of these mutans streptococci. The administration of the oolong tea extract and the isolated polyphenol compound into diet 2000 and drinking water resulted in significant reductions in caries development and plaque accumulation in the rats infected with mutans streptococci. The active components in the oolong tea extract were presumptively identified as polymeric polyphenols which were specific for oolong tea leaves. These results indicate that the oolong tea polyphenolic compounds could be useful for controlling dental caries.

The occurrence of interglobular dentin in incisors of hypophosphatemic mice fed a high-calcium and high-phosphate diet.

The incisor dentin of hypophosphatemic (Hyp) mice was examined histopathologically to determine whether the multiple occurrences of interglobular dentin would be influenced by the serum phosphate level. Both normal and Hyp mice (12 weeks of age) were divided into two groups. The mice in one group were given a control diet (1.42% Ca, 1.16% P) and the other a high-calcium and high-phosphate diet (2.00% Ca, 3.00% P) for 30 days. Blood was collected from the mice every fifth day for measurement of the calcium and phosphate concentrations in serum. Both ground and decalcified cross-sections were prepared from incisors from the mandible and maxilla for microscopic examination. The levels of serum Ca and P were almost constant in normal mice, regardless of diet. On the other hand, serum P levels in Hyp mice fed the control diet were significantly lower than those in normal mice. The ten days' feeding of the high-Ca/-P diet significantly elevated the serum P level in Hyp mice, and it reached a level similar to that of the normal mice. However, histopathological examination showed no significant changes in incisor dentin of Hyp mice fed the high-Ca/-P diet, and interglobular dentin still occurred. These results suggest that the multiple formations of interglobular dentin, which is the most outstanding feature of X-linked hypophosphatemic vitamin-D-resistant rickets, are not influenced in Hyp mice by the short-time normalization of the serum phosphate level.

Structural deformities of deciduous teeth in patients with hypophosphatemic vitamin D-resistant rickets.

Structural deformities of deciduous teeth from patients with hypophosphatemic vitamin D-resistant rickets (HVDRR) (1 male and 2 female patients) were examined by means of transmitted light microscopy, contact microradiography, and x-ray microanalysis. Freshly extracted teeth were fixed in formalin and subsequently hemisected longitudinally through the midline. One half was prepared for ground sections and the other half for decalcified sections. Neither gross nor microscopic abnormalities were present in enamel of patients with HVDRR. The concentration of calcium and phosphorus and the calcium/phosphorus ratio of the enamel of patients with HVDRR were nearly equal to those of normal teeth, although the degree of radiopacity was less in HVDRR. On the other hand, numerous microscopic abnormalities in the dentin of patients with HVDRR were found, such as interglobular dentin, wide predentin zones, and tubular defects. The concentration of phosphorus in the dentin of a patient with familial HVDRR was extremely low. Furthermore, formation of reparative dentin was observed at the pulp horn of teeth in patients with HVDRR that had been subjected to definite attrition at the corresponding dentin site.

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme.

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.