Adrian Wiater

INSOLUBLE GLUCANS SYNTHESIZED BY CARIOGENIC STREPTOCOCCI : A STRUCTURAL STUDY

Of the three cariogenic streptococci grown in four various culture media, the strain Streptococcus mutans 20381 was found to produce large amounts of extracellular glucosyltransferase and water‐insoluble, adhesive exopolysaccharide when grown in batch culture on brain‐heart infusion broth. Methylation and nuclear magnetic resonance analyses revealed that the insoluble polymers synthesized by the crude glucosyltransferase preparations were mixed‐linkage (1 → 3), (1 → 6)‐α‐D‐glucans (so‐called mutans) with a greater proportion of 1,3 to 1,6 linkages and major branch points of 3,6‐linked glucose. The percentage content of different types of linkages in glucans varied widely and depended on the strain of cariogenic bacteria used to produce glucosyltransferase, and on the kind of medium utilized to cultivate mutans streptococci. The potential application of insoluble glucan produced by mutans streptococci is discussed.

Variation in total polyphenolics contents of aerial parts of Potentilla species and their anticariogenic activity.

The aerial parts of selected Potentilla species (P. anserina, P. argentea, P. erecta, P. fruticosa, P. grandiflora, P. nepalensis, P. norvegica, P. pensylvanica, P. crantzii and P. thuringiaca) were investigated in order to determine their contents of polyphenolic compounds. The results showed that P. fruticosa has relatively high concentrations of tannins (167.3 ± 2.0 mg/g dw), proanthocyanidins (4.6 ± 0.2 mg/g dw) and phenolic acids (16.4 ± 0.8 mg/g dw), as well as flavonoids (7.0 ± 1.1 mg/g dw), calculated as quercetin. Furthermore, we investigated the in vitro inhibitory effects of aqueous extracts from these species against cariogenic Streptococcus spp. strains. It was found that the tested samples moderately inhibit the growth of oral streptococci. However, all the preparations exhibited inhibitory effects on water-insoluble α-(1→3)-, α-(1→6)-linked glucan (mutan) and artificial dental plaque formation. The extract from P. fruticosa showed the highest anti-biofilm activities, with minimum mutan and biofilm inhibition concentrations of 6.25–25 and 50–100 µg/mL, respectively. The results indicate that the studied Potentilla species could be a potential plant material for extracting biologically active compounds, and could become a useful supplement for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products.

Biological study on carboxymethylated (1→3)-α-D-glucans from fruiting bodies of Ganoderma lucidum.

Water-insoluble, alkali-soluble polysaccharides (ASPs) were isolated from three fruiting bodies of the macromycete fungus Ganoderma lucidum. The structure of ASPs was determined using composition analysis, methylation analysis, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The analysis of the biological activity of the carboxymethylated (CM) (1→3)-α-D-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects against a tumor cell line (human cervical carcinoma HeLa), and two normal human cell lines (colon myofibroblasts CCD-18Co and epithelial cells CCD 841 CoTr). The chemical and spectroscopic investigations indicated that the ASPs from G. lucidum were (1→3)-α-D-glucans. After carboxymethylation (1→3)-α-D-glucans were tested in the range of 25-250 μg/mL concentrations. All the tested CM-(1→3)-α-D-glucans decreased the cellular metabolism of tumor and normal cells after 24h of incubation. The CM-(1→3)-α-D-glucans had no toxic effects on cervical carcinoma cells but reduced the viability of normal cells. The cytotoxic activity of the CM-(1→3)-α-D-glucans was concentration- and cell-type-dependent with normal cells more sensitive to their action than tumor cells. Generally, the CM-(1→3)-α-D-glucans tested did not have a free radical scavenging effect. It was concluded that the carboxymethylated derivatives of (1→3)-α-D-glucans isolated from the G. lucidum fruiting bodies are biologically active and after further detailed studies may be regarded as a dietary or therapeutic supplements.

(1 → 3)-α-d-Glucan hydrolases in dental biofilm prevention and control: A review

Dental plaque is a highly diverse biofilm, which has an important function in maintenance of oral and systemic health but in some conditions becomes a cause of oral diseases. In addition to mechanical plaque removal, current methods of dental plaque control involve the use of chemical agents against biofilm pathogens, which however, given the complexity of the oral microbiome, is not sufficiently effective. Hence, there is a need for development of new anti-biofilm approaches. Polysaccharides, especially (1→3),(1→6)-α-D-glucans, which are key structural and functional constituents of the biofilm matrix, seem to be a good target for future therapeutic strategies. In this review, we have focused on (1→3)-α-glucanases, which can limit the cariogenic properties of the dental plaque extracellular polysaccharides. These enzymes are not widely known and have not been exhaustively described in literature.

In vitro anticariogenic effects of aerial parts of Potentilla recta and its phytochemical profile

This study, for the first time, investigated the in vitro inhibitory effects of Potentilla recta extracts and subfractions obtained with solvents of different polarity (aqueous, 50% ethanol, diethyl ether, ethyl acetate and n‐butanol) against cariogenic Streptococcus spp. strains. It was found that the tested samples inhibited the growth of oral streptococci. Furthermore, all five P. recta preparations exhibited an inhibitory effect on water‐insoluble α‐(1→3)‐,α‐(1→6)‐linked glucan (mutan) and artificial dental plaque formation. The ethyl acetate fraction showed the highest antibiofilm activities especially against S. sobrinus GCM 20381, with minimum mutan and biofilm inhibition concentrations of 6.25 and 25 µg/mL, respectively. The phytochemical profile of active constituents in the investigated samples was analysed. The high polyphenolics (total phenol, phenolic acids, tannins, proantocyanidins, flavonoids) content were found. The ethyl acetate fraction showed the highest concentration of total polyphenol content which may correlate with the high cariogenic activity of this subfraction. The results demonstrate that P. recta extracts and subfractions could become useful supplements for pharmaceutical products as new anticariogenic agents in a wide range of oral care products. Further studies are necessary to clarify the precise bioactive constituents of P. recta responsible for the anticariogenic properties. Copyright

Chemical characterization of a water insoluble (1 → 3)-α-d-glucan from an alkaline extract of Aspergillus wentii

The chemical structure of a water insoluble α-glucan isolated from the cell wall of Aspergillus wentii was described on the basis of total acid hydrolysis, methylation analysis, and 1D and 2D NMR studies (TOCSY, DQF-COSY, NOESY and HSQC) as well as other instrumental techniques. It was established that the analyzed preparation contained a linear polymer composed almost exclusively of (1→3)-linked α-d-glucose, with a molecular mass of about 850kDa. The polymer was divided into subunits separated by a short spacers of (1→4)-linked α-d-glucoses. Each subunit contained about 200 glucose residues.

Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans

Basic cultural parameters affecting mutanase production by Trichoderma harzianum F-340 in shaken flasks and aerated fermenter cultures have been standardized. The best medium for enzyme production was Mandels medium A with initial pH 5.3, supplemented with 0.3% mutan and 0.05% peptone and inoculated with 20% of the 72-h mycelium as inoculum. It was shown that mycelial mass, used in the culture medium as a sole carbon source, induced mutanase synthesis and could be utilized as an inexpensive and easily available substitute for bacterial mutan. Application of optimized medium and cultural conditions enabled us to obtain a high mutanase yield (0.6-0.7 U/mL, 2.0-2.5 U/mg protein) in a short period of time (3-5 days), which was much higher than the best reported in literature. The enzyme in crude state was stable in the pH range of 4.5-6.0, and at temperatures of up to 40oC; its maximum activity was recorded at 45oC and at pH 5.5. The mutanase preparation obtained from the T. harzianum fungus was relatively stable under storage conditions, and showed a high hydrolytic potential in reaction with a mixed-linkage (a-1,3, a-1,6) water-insoluble mutan of streptococcal origin (hydrolysis yield reached a value of 69% in 24 h). Steady-state measurement of the enzymic reaction products during the hydrolysis revealed that mutanase exhibited an exo type of action on mutan. Thin-layer chromatographic analysis showed that glucose was the primary final product of mutan hydrolysis with mutanase. The potential application of mutanase in dentistry is discussed.

Purification and characterization of an extracellular mutanase from Trichoderma harzianum

Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0.33 U ml−1, 1.85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS-PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7.11, and the carbohydrate content in the purified enzyme was 4.42%. The pH and temperature optima were 5.5 and 40°C, respectively. The enzyme remained stable over a pH range of 4.5-6.0 and up to 35° for 1 h. The values of Km and Vmax under standard assay conditions were 1.2 × 10−3 g ml−1 and 8.48 × 10−2 U mg−1, respectively.

Aqueous Extracts of Selected Potentilla Species Modulate Biological Activity of Human Normal Colon Cells

Potentilla L. (Rosaceae) species have been used in traditional and in folk medicine for many years. This study characterized the activity of extracts from aerial parts of selected Potentilla species: P. argentea, P. anserina, P. grandiflora and P. erecta as well as one species of closely related to the genus Potentilla, Drymocallis rupestris (syn. P. rupestris). The biological activities were analyzed using MTT, NR and DPPH assays on CCD 841 CoTr and CCD-18Co cells. Moreover, cell morphology and cytoskeletal actin F-filaments organization and IL-6 and IL-10 levels by ELISA were analyzed after 24 h of incubation. Potentilla extracts at dose levels between 25 and 250 µg/mL were analyzed. For ELISA, 15 µg/mL and 30 μg/mL were chosen. When mitochondrial succinyl dehydrogenase activity was tested (MTT assay) only extract obtained from P. erecta at lower concentrations (up to 125 µg/mL) suppressed metabolism of myofibroblasts, while epithelial cells mitochondrial enzyme activity increased after incubation with all extracts. In Neutral Red (NR) method cellular membrane disturbance of both cell cultures was found after D. rupestris and P. grandiflora addition. Moreover, strong influence on epithelial cells was also found for P. anserina. All extracts showed similar, concentration-dependent free radical scavenging (DPPH) effect. Potentilla extracts, especially at lower concentration, decreased IL-6 production in myofibroblasts but the level of the cytokine was found to be stable in epithelial cells. IL-10 analysis revealed that P. argentea, D. rupestris, P. erecta extracts decrease cytokine level in myofibroblasts, while only when higher concentration were applied, decreased cytokine level produced by epithelial cells was found. F-actin filaments staining revealed that Potentilla extracts significantly influence on cellular cytoskeleton organization. Potentilla extracts influence on cells of human colon wall lining modulating the main features of them (viability, cytokine production). Moreover, as a free radical reducing agents, may be successfully used in the prevention of colon disorders.

In Vitro Anticariogenic Effects of Drymocallis rupestris Extracts and Their Quality Evaluation by HPLC-DAD-MS3 Analysis

In this study, for the first time, we investigated in vitro inhibitory effects of Drymocallis rupestris extracts and their subfractions obtained with solvents of different polarity (aqueous, 50% ethanolic, diethyl ether, ethyl acetate and n-butanolic) against bacterial viability and caries virulence factors of Streptococcus spp. strains. The diethyl ether subfraction (PRU2) showed bacteriostatic and bactericidal activity against mutans streptococci, with minimum inhibitory concentrations (MICs) in the range of 0.75–1.5 mg/mL and minimum bactericidal concentrations (MBCs) in the range of 1.5–3 mg/mL. Furthermore, PRU2 inhibited biofilm formation by Streptococci in a dose-dependent manner. It was also found that all five D. rupestris preparations exhibited diverse inhibitory effects on de novo synthesis of water-insoluble and water-soluble α-d-glucans by glucosyltransferases of the mutans group streptococci. The phytochemical profile of investigated samples was determined by spectrophotometric and chromatographic (HPLC-DAD-MS3) methods. The high polyphenol (total phenol, phenolic acids, tannins, proantocyanidins, and flavonoids) contents were found which correlated with anticariogenic activity of the analyzed samples. The results demonstrate that D. rupestris extracts and their subfractions could become useful supplements for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products. Further studies are necessary to clarify which phytoconstituents of D. rupestris are responsible for anticaries properties.