In vitro effect of low-fluoride toothpaste supplemented with sodium trimetaphosphate, xylitol, and erythritol on enamel demineralization

Abstract Regular use of toothpaste with fluoride (F) concentrations of ≥ 1000 ppm has been shown to contribute to reducing caries increment. However, when used by children during the period of dental development, it can lead to dental fluorosis. Objective: In this study, we aimed to evaluate the in vitro effect of a toothpaste formulation with reduced fluoride (F) concentration (200 ppm) supplemented with sodium trimetaphosphate (TMP: 0.2%), Xylitol (X:16%), and Erythritol (E: 4%) on dental enamel demineralization. Methodology: Bovine enamel blocks were selected according to initial surface hardness (SHi) and then divided into seven experimental toothpaste groups (n=12). These groups included 1) no F-TMP-X-E (Placebo); 2) 16% Xylitol and 4% Erythritol (X-E); 3) 16% Xylitol, 4% Erythritol and 0.2%TMP (X-E-TMP); 4) 200 ppm F (no X-E-TMP: (200F)); 5) 200 ppm F and 0.2% TMP (200F-TMP); 200 ppm F, 16% Xylitol, 4% Erythritol, and 0.2% TMP (200F-X-E-TMP); and 7) 1,100 ppm F (1100F). Blocks were individually treated 2×/day with slurries of toothpastes and subjected to a pH cycling regimen for five days (DES: 6 hours and RE: 18 hours). Then, the percentage of surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN), fluoride (F), calcium (Ca), and phosphorus (P) in enamel were determined. The data were analyzed by ANOVA (1-criterion) and the Student-Newman-Keuls test (p<0.001). Results: We found that the 200F-X-E-TMP treatment reduced %SH by 43% compared to the 1100F treatments (p<0.001). The ΔKHN was ~ 65% higher with 200F-X-E-TMP compared to 1100F (p<0.001). The highest concentration of F in enamel was observed on the 1100F treatment (p<0.001). The 200F-X-E-TMP treatment promote higher increase of Ca and P concentration in the enamel (p<0.001). Conclusion: The association of 200F-X-E-TMP led to a significant increase of the protective effect on enamel demineralization compared to the 1100F toothpaste.

Estimating the effectiveness of lollipops containing xylitol and erythritol on salivary pH in 3–6 years olds A randomized controlled trial

Background: Prevention of dental caries is important for nutrition and health of the child. Sucrose being considered an arch criminal, various substitutes are recommended. Xylitol is an artificial sweetener which cannot be metabolized by bacteria. Thus, it seems to be a promising method in prevention of dental caries. Materials and Methods: Fifty children between the age of 3–6 years were randomly divided into two groups; Group 1: Control group (without lollipops) and Group 2: Experimental group (with sugar substitute lollipops). The saliva sample was collected at four different time intervals, and pH of saliva was determined using universal pH indicator. Results: There was a significant drop in the pH after drinking sweetened beverages in both the groups, but there was a significant rise in pH after having xylitol + erythritol lollipops which almost returned to baseline after 15 min. Conclusion: Lollipops containing xylitol and erythritol can be used in small children and it has potential to increase salivary pH, thus not allowing the pH to fall below the critical value.

Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea / 药学学报

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK) was one of the key enzymes in the methylerythritol-4-phosphate (MEP) pathway to generate terpenoids. In this study, based on the transcriptome data of Atractylodes lancea, the sequence of the CMK gene was cloned, named AlCMK (GenBank accession number OM283293). The results showed that AlCMK contains a 1 230 bp open reading frame (ORF) encoding 409 amino acids. The deduced protein had a theoretical molecular weight of 44 752.53 and an isoelectric point of 6.67. Transmembrane structure analysis showed that there was no transmembrane structure, and the secondary structure of AlCMK was predicted to be mainly composed of random coil. Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium, Osmanthus fragrans, Eucommia ulmoides, Lonicera japonica and Salvia miltiorrhiza. Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae. The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E. coli BL21 (DE3). The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment. Meanwhile, the enzyme activity was determined by ELISA kit. The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA, and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves. The subcellular localization showed that AlCMK was located in the chloroplast. This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes / 中国实验方剂学杂志

Objective:To investigate the relationship between the single nucleotide polymorphism（SNP）of function genes and effective components of <italic>Salvia miltiorrhiza</italic> and the molecular mechanism of specific quality formation of <italic>S. miltiorrhiza</italic>. Method:The fingerprints of components in <italic>S. miltiorrhiza</italic> from eight different habitats and varieties were obtained by high-performance liquid chromatography （HPLC）. The full-length cDNA of three functional genes<italic> </italic>acetyl-CoA C-acetyltransferase（<italic>SmAACT</italic>），4-diphosphocytidyl-2-C-methyl-<italic>D</italic>-erythritol kinase（<italic>SmCMK</italic>） and isopentenyl diphosphate isomerase（<italic>SmIPPI</italic>） in tanshinone metabolic pathway were amplified by polymerase chain reaction（PCR），cloned， and sequenced，followed by bioinformatics analysis. Result:The full-length cDNA sequences of three functional genes <italic>SmAACT</italic>，<italic>SmCMK</italic>， and <italic>SmIPPI</italic> in tanshinone metabolic pathway were obtained from 23 strains of <italic>S. miltiorrhiza</italic> from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes，18，16， and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing，Hubei，Shandong （No. SDB），Shanxi，Henan， and Shandong （No. SDZ） were clustered into one branch，and those from Hebei and Inner Mongolia were clustered into another branch， which suggested that the variation trend of <italic>S. miltiorrhiza</italic> components had little correlation with geographical distance，but the variety was a critical factor for the quality. Conclusion:There was an obvious genetic differentiation trend in <italic>S. miltiorrhiza</italic> from different habitats，and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of <italic>S. miltiorrhiza</italic> from different habitats was discussed，which laid a foundation for the stability and effectiveness of clinical medication，and guided the breeding of excellent varieties of <italic>S. miltiorrhiza</italic>.

Clinical and microbiological effects of the supplementary use of an erythritol powder air-polishing device in non-surgical periodontal therapy: a randomized clinical trial

PURPOSE: This study was undertaken to evaluate the clinical and microbiological effects of an erythritol powder air-polishing device (EPAP) as a supplement to scaling and root planing (SRP) therapy in patients with moderate chronic periodontitis. METHODS: Clinical and microbiological evaluations were performed at 21 sites treated with SRP (control) and 21 sites treated with SRP+EPAP (test). All examinations were performed before treatment, 1 month after treatment, and 3 months after treatment. RESULTS: There were no significant clinical differences between the test group and the control group. Microbiological analysis revealed that the relative expression level of Porphyromonas gingivalis was significantly lower in the test group than in the control group at 1 month after treatment. Clinical and microbiological results showed improvements at 1 month compared to baseline; in contrast, the results at 3 months after treatment were worse than those at 1 month after treatment. CONCLUSIONS: In this study, both SRP and SRP+EPAP were clinically and microbiologically effective as non-surgical periodontal treatments. In particular, the SRP+EPAP group showed an antimicrobial effect on P. gingivalis, a keystone bacterium associated with the onset of chronic periodontitis, in a short-term period. Periodic periodontal therapy, at intervals of at least every 3 months, is important for sustaining the microbiological effects of this treatment.

Study on secondary metabolites of endophytic fungus Fusarium lactis from Dendrobium huoshanense / 中草药

Objective To study the secondary metabolites of endophytic fungus Fusarium lactis in Dendrobium huoshanense. Methods Compounds were isolated from the EtOAc extract by chromatography technology and their structures were elucidated on the basis of comprehensive spectroscopic analysis. Results Twenty-one compounds were isolated and identified as N-phenethylacetamide (1), 1H-indole-3-carbaldehyde (2), thymidine (3), uracil (4), lignoren (5), uridine (6), hexahydrate (7), adenosine (8), cyclo-glycine-(L)-proline (9), cyclo (D)-proline-(L)-phenylalanine (10), cyclo (L)-proline-(L)-phenylalanine (11), 2-pyrrolidinone (12), N-methyl-2-pyrolidinone (13), cyclo-(L)-4-OH-proline-(L)-phenylalanine (14), brevianamide F (15), 3-methyl- piperazine-2,5-dione (16), 7,8-dimethylbenzo[g]pteridine-2,4 (1H,3H)-dione (17), cyclo (L)-tyrosine-(L)-phenylalanine (18), beer sterols (19), daidzein (20), and erythritol (21). Conclusion All compounds are isolated from Fusarium lactis for the first time.

Strategy of oxygen transfer coefficient control on the L-erythrulose fermentation by newly isolated Gluconobacter kondonii

Background: The effect of diverse oxygen transfer coefficient on the L-erythrulose production from meso-erythritol by a newly isolated strain, Gluconobacter kondonii CGMCC8391 was investigated. In order to elucidate the effects of volumetric mass transfer coefficient (K La) on the fermentations, baffled and unbaffled flask cultures, and fed-batch cultures were developed in present work. Results: With the increase of the K La value in the fed-batch culture, L-erythrulose concentration, productivity and yield were significantly improved, while cell growth was not the best in the high K La. Thus, a two-stage oxygen supply control strategy was proposed, aimed at achieving high concentration and high productivity of L-erythrulose. During the first 12 h, Klawas controlled at 40.28 h-1 to obtain high value for cell growth, subsequently K La was controlled at 86.31 h-1 to allow for high L-erythrulose accumulation. Conclusions: Under optimal conditions, the L-erythrulose concentration, productivity, yield and DCW reached 207.9 ± 7.78 g/L, 6.50 g/L/h, 0.94 g/g, 2.68 ± 0.17 g/L, respectively. At the end of fermentation, the L-erythrulose concentration and productivity were higher than those in the previous similar reports.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Abstract Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Molecular cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene from Artemisia annua L / 药学学报

The plastidial methylerythritol phosphate (MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (MeJA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.

Cloning and expression analysis of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene in Cinnamomum camphora / 中国中药杂志

The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase was the fourth key enzymes in plant terpenoid biosynthesis pathway of methyl erythritol phosphate pathway(MEP). According to the study of Cinnamomum camphora transcriptome data,we abtained the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene using RT-PCR,and named CcCMK1,then deposited it in GeneBank(Accession number： Ku376098).Bioinformatics analysis showed the open reading frame (ORF) of the CcCMK1 was 1 212 bp.The putative protein encoded 403 amino acids,and its molecular weight was 44.46 kDa and theoretically isoelectric point was 4.99.Transmembrane structure analysis showed that there was no transmembrane structure. Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the chloroplast was located in the chloroplast.Analysis of the expression of CcCMK1 gene in five chemotypes of C. camphora using Real-time PCR showed its expression level was highest in C. longepaniculatum, and the lowest in Borneol camphor.This research provided a basis for characterizing the key enzyme genes of terpenoid biosynthetic pathway in C. camphora.

Glycemic Effects of Rebaudioside A and Erythritol in People with Glucose Intolerance

BACKGROUND: Rebaudioside A and erythritol are nonnutritive sweeteners. There have been several studies of their glycemic effects, but the outcomes remain controversial. The purpose of this study was to evaluate the glycemic effects of rebaudioside A and erythritol as a sweetener in people with glucose intolerance. METHODS: This trial evaluated the glycemic effect after 2 weeks of consumption of rebaudioside A and erythritol as sweeteners in a pre-diabetic population. The patients were evaluated for fructosamine, fasting plasma glucose, C-peptide, insulin, and 2-hour plasma glucose before and after consumption of sweetener. The primary outcome was a change in fructosamine levels from the baseline to the end of treatment. Secondary outcomes were the changes in levels of fasting plasma glucose and 2-hour plasma glucose. RESULTS: From the baseline to the end of experiment, the changes in fructosamine levels after consumption of rebaudioside A and erythritol, did not differ significantly (244.00±19.57 vs. 241.68±23.39 µmol/L, P=0.366). The change in levels from the baseline to end of the study for rebaudioside A and erythritol were fasting plasma glucose (102.56±10.72 vs. 101.32±9.20 mg/dL), 2-hour plasma glucose (154.92±54.53 vs. 141.92±42.22 mg/dL), insulin (7.56±4.29 vs. 7.20±5.12 IU/mL), and C-peptide (2.92±1.61 vs. 2.73±1.31 ng/mL), respectively, and also did not differ significantly (P>0.05 for all). CONCLUSION: Our study suggests that consumption of rebaudioside A and erythritol does not alter the glucose homeostasis in people with glucose intolerance.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Bio-informatics analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylytransferase gene in Picrorhiza kurrooa / 中草药

Objective: To study the characteristics of 2-C-methyl-D-erythritol 4-phosphate cytidylytransferase (MCT) gene and to predict the structure and function site of MCT. Methods: Based on MCT gene of Picrorhiza kurrooa, NCBI website and bio-informatics software were used to predict and analyze the base distribution, amino acid composition, hydrophilicity or hydrophobicity, and secondary and three-level structures of hyper-conservative region. The results were compared with MCT gene sequences in other eight species and the related evolution analysis was followed. Results: The mRNA sequence of MCT gene in P. kurrooa was 1216 bp (GenBank: JQ991625.1), coding the protein containing 399 amino acids, and the relative molecular mass of protein was 44448.5 with 10.0% Ser in volume; In polypeptide, hydrophobic amino acid was 59.5% in volume, and the average hydropathicity was 0.050; The homology was found as high as 99% compared with MCT gene in Salvia miltiorrhiza. Conclusion: The MCT gene in P. kurrooa was quite stable and the coded protein was hydrophobic, which was quite conservative in evolution; The sequence information in conservative region gained in the test provides the basis for clone ofnovel gene in other species.

Effects of short-term supplementation of erythritol-salt on urinary electrolyte excretion in rats / 한국영양학회지

PURPOSE: This study was conducted in order to investigate the diuretic effects of Erythritol (ET) salt on urinary electrolyte excretion in Sprague-Dawley Rats. METHODS: Animals were divided into two groups: Salt group (n = 7) and Salt + ET fed group (n = 7). Animals were provided food and water ad libitum. Supplements were administered orally to animals for one week. RESULTS: Body weights were not statistically different between groups either on Day 1 or Day 7. However, water consumption of the Salt + ET group was significantly higher than that of the Salt group on Day 1 and Day 7. Urine volume of the Salt + ET group was approximately 27% and 38% higher than that of the Salt group on Day 1 and Day 7. In addition, we found that the total amounts of urinary electrolytes, such as sodium and potassium, of the Salt + ET group were significantly higher than those of the Salt group on Day 7. We also found that serum electrolyte concentrations did not differ between two groups. These results demonstrated that salt intake with ET was effective in increasing urinary electrolyte excretion, which might be caused by higher water intake and diuretic effect inhibiting reabsorption of water, sodium, and potassium in renal tubules. CONCLUSION: The results suggest that short-term supplementation of ET salt can be a potential diuretic agent by inhibiting sodium and potassium reabsorption and inducing loss of water.

Cloning and sequence analysis of MCS gene in Artemisia annua and its prokaryotic expression / 中草药

Objective: To obtain the indispensable key enzyme involved in the MEP pathway, the 2C-methyl-D-erythritol-2, 4-cyclodi-phosphate synthase gene (MCS) was cloned from Artemisia annua, and bioinformatic analysis, prokaryotic expression, and tissue-specific expression were conducted. Methods: According to AaMCS EST sequence, the full length of cDNA and genomic sequences were obtained by the design of specific primers, rapid-amplification of cDNA ends (RACE) and genome amplification. The coding region of MCS gene was cloned into the expression vector pET-21a (+) by gene recombination technology, the recombinant plasmid pET-21a (+)-MCS was transformed into E. coli BL21 (DE3), and the expression of recombinant protein was induced by IPTG. Semi-quantitative RT-PCR was used to detect the expression of AaMCS transcripts. Results: The AaMCS cDNA was found to be 994 bp containing an open reading frame (ORF) of 681 bp that translated into a putative peptide of 226 amino acid, The full length of MCS was 2540 bp consisting of three exons and two introns. A recombinant pET-21a (+)-MCS was constructed by genetically fusing the MCS to pET-21a (+) vector system, and was successfully expressed in E. coli BL21. The tissue expression patterns indicated that the expression level of AaMCS transcripts in flowers was higher than that in the roots and stems. Conclusion: The MCS gene is cloned from A. annua, and the stable prokaryotic expression system of pET-21a (+)-MCS is constructed. This work is helpful for investigating the activities and other physiological functions of MCS protein.

Genotoxicity Assessment of Erythritol by Using Short-term Assay

Erythritol is a sugar alcohol that is widely used as a natural sugar substitute. Thus, the safety of its usage is very important. In the present study, short-term genotoxicity assays were conducted to evaluate the potential genotoxic effects of erythritol. According to the OECD test guidelines, the maximum test dose was 5,000 microg/plate in bacterial reverse mutation tests, 5,000 microg/ml in cell-based assays, and 5,000 mg/kg for in vivo testing. An Ames test did not reveal any positive results. No clastogenicity was observed in a chromosomal aberration test with CHL cells or an in vitro micronucleus test with L5178Y tk +/- cells. Erythritol induced a marginal increase of DNA damage at two high doses by 24 hr of exposure in a comet assay using L5178Y tk +/- cells. Additionally, in vivo micronucleus tests clearly demonstrated that oral administration of erythritol did not induce micronuclei formation of the bone marrow cells of male ICR mice. Taken together, our results indicate that erythritol is not mutagenic to bacterial cells and does not cause chromosomal damage in mammalian cells either in vitro or in vivo.

Synthesis of a salacinol analogue and its α-glucosidase inhibitory activity / 药学学报

Aim To investigate more efficient synthetic method of the nitrogen analogue 4 of salacinol (1) for searching new antidiabetic agents. Methods The synthesis of the key intermediate 2,4-O-isopropylidene-L-erythritol 1,3-cyclic sulfate (2a) was accomplished by modification of reports from Dglucose via seven steps in much more less expensive. Using this method, an efficient synthesis of 4 was carried out. The glycosidase inhibitory activity of 4 was tested for the intestinal α-glucosidase in vitro and compared with that of salacinol. Results A nitrogen analogue 4 of salacinol (1) was synthesized by the coupling reaction between the cyclic sulfate 2a and an azasugar 3b. Conclusion Substitution of the sulfur atom in 1 with a nitrogen reduced the activity considerably.