Anticariogenic activity of Nelumbo nucifera leaf extract in oral healthcare.

BACKGROUND: We aimed to evaluate the antimicrobial effect of the Nelumbo nucifera leaf extract. There have been no studies related to dental caries inducing bacteria up to now. OBJECTIVE: This study reviewed the inhibitory effect of glucose transferase (GTase) activation and acid production to confirm the anticariogenic activity of Nelumbo nucifera leaf extract. METHODS: This study used 100 g Nelumbo nucifera leaves cultivated in Yeongcheon-si, Gyeongbuk, after adding 70% methanol tenfold. The leaves were then concentrated (Gotary vacuum evaporator; N-Nseries, EYELA Co., Japan) and were placed under an aspirator (A-3S, EYELA Co., Japan) and a freeze dryer (Ilshin Lab Co., Korea). The anticariogenic effect of Nelumbo nucifera leaves extract was investigated using the growth inhibitory effect, as well as GTase activation. RESULTS: Among the nine kinds of oral-disease-causing bacteria, the Nelumbo nucifera leaf extract most effectively inhibited the growth of Streptococcus anginosus (S. anginosus), but it was difficult to inhibit the growth of Streptococcus oralis (S. oralis). For the anticariogenic effect of Nelumbo nucifera leaf extract, GTase activation was inhibited by at least 50% in all the nine types of bacteria, including Streptococcus mutans (S. mutans). It was shown that Nelumbo nucifera leaf extract had the strongest GTase activation inhibitory effect (85%) in S. anginosus. In addition, Nelumbo nucifera leaf extract showed an acid production inhibitory effect in the nine types of strains by maintaining almost pH 6.2 even after being cultured for 24 hours in the Nelumbo-nucifera-leaf-extract-added culture, while the control culture without Nelumbo nucifera leaf extract showed only about pH 5.0 after 4 hours. CONCLUSIONS: In conclusion, Nelumbo nucifera leaf extract showed the strongest GTase activation inhibitory effect in S. anginosus. Based on this, it was confirmed that Nelumbo nucifera leaf extract showed anticariogenic activity against oral cavity disease microorganisms.

Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

[Effect of Chelerythrine on glucosyltransferase and water-insoluble glucan of Streptococcus mutans].

PURPOSE: To study the effect of Chelerythrine on glucosyltransferase and extra-cellular synthesis of water-insoluble glucan of Streptococcus mutans. METHODS: The Chelerythrine was used as the experimental group with concentrations ranging from 24.4microg/ml to 390.6microg/ml prepared with BHI broth medium with contained 2% glucose, and BHI culture medium was used as the control group. Streptococcus mutans was added to each group, after cultured for 24 hours in the test tubes, centrifugation was followed. The supernatants were divided into two batches. One batch of solutions was used to extract glucosyltransferase, Bradford method and Somogyi method were used to measure the content of total protein and enzyme activity, and the specific activity was calculated. Another batch of solutions was used to measure the content of water-insoluble glucan by anthrone method. The data was statistically analyzed by One-way ANOVA using SPSS13.0 software package. RESULTS: The glucosyltransferase and water-insoluble glucan of Streptococcus mutans decreased gradually with the increase of each concentration of Chelerythrine. There were highly significant differences among total sample groups, and between glucosyltransferase activity or specific activity of each experimental group and control group as well (P<0.01); Except for the group of 24.4microg/ml Chelerythrine, there were highly significant differences of water-insoluble glucan between each experimental group and control group (P<0.01).There was positive correlation between glucosyltransferase activity and water-insoluble glucan content (r=0.883, P<0.01). CONCLUSION: Chelerythrine could inhibit the glucosyltransferase and extra-cellular synthesis of water-insoluble glucan of Streptococcus mutans.

Glycogen debranching enzyme in bovine brain.

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no alpha-glucosidase or alpha-amylase activities and degraded phosphorylase limit dextrin of glycogen with phosphorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an alpha-1,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.