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.     

Suppression of postprandial hypertriglyceridemia in rats and mice by oolong tea polymerized polyphenols

Oolong tea-polymerized polyphenols (OTPP) are characterized polyphenols produced from semi-fermented tea (oolong tea). In the present study, we evaluated the suppressive effects of oolong tea extract and OTPP on postprandial hypertriglyceridemia in rats and mice. Lymphatic recovery of triglycerides in rats cannulated in the thoracic duct was delayed by the administration of oolong tea extract at 100 and 200 mg per head, and more effectively than with green tea extract. OTPP delayed lymphatic triglyceride absorption at 20 mg/head, though (-)-epigallocatechin gallate (EGCG) did not do so at the same dose. OTPP also suppressed postprandial hypertriglyceridemia after administration of olive oil in mice. The area under the curve (AUC) of plasma triglycerides was significantly decreased, by 53% and 76%, in the 500 and 1,000 mg/kg OTPP groups respectively, as compared with the control group. These results suggest that OTPP is responsible for the suppression of hypertriglyceridemia by ingestion of oolong tea.     

The anti-biofouling effect of polyphenols against Streptococcus mutans

Biofouling is a process of surface colonization by microorganisms through cell adhesion and production of extracellular polymers (polysaccharides and proteins). It often causes serious problems in the chemical, medical and pharmaceutical industries. Recently, it was demonstrated that some natural phenolic compounds found in plants and vegetables have an antibiofouling effect, reducing formation of biofilm by Gram-negative bacteria. In this study, Streptococcus mutans, a Gram-positive bacterium was investigated for the antibiofouling effect of polyphenols. It was hypothesized that the two enzymes, glucosyltransferase and fructosyltransferase, produced by S. mutans, would be inhibited by the natural phenolic compounds. When these two enzymes were inhibited, less (or no) biofilms were formed. Enzymes were separated from a S. mutans culture medium, and their activities were measured with five different polyphenols using microtiter-plates and high-performance liquid chromatography. The results of minimum inhibitory concentration (MIC) were used to determine the enzyme inhibition effect of polyphenols on biofilm formation without killing the cells. Most of the polyphenols used showed considerable reduction of biofilm formation. Gallic acid and tannic acid showed significant enzyme inhibition effects below their MICs.     

Inhibitory mechanisms of tea polyphenols on the ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway

In this study, we investigated the effect of tea polyphenols, (-)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (-)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.     

High-molecular-weight polyphenols from oolong tea and black tea: purification, some properties, and role in increasing mitochondrial membrane potential

High-molecular-weight polyphenols from oolong and black teas increased mitochondrial membrane potential, as measured by a method using ciliated protozoan Tetrahymena and rhodamine 123. These polyphenols, referred to as mitochondrial activation factors (MAFs), were purified from oolong and black teas by solvent extraction and Toyopearl column chromatography. The number-average molecular weights of the MAFs were 9,000 to 18,000, and the weight-average molecular weights were 15,000 to 25,000. The MAFs increased the mitochondrial membrane potential more than catechins did. Treatment of the MAFs with tannase indicated that they contained galloyl residues. When the MAFs were hydrolyzed with HCl-n-BuOH, cyanidin and delphinidin were detected. The partial structure of the MAFs was analyzed by pyrolysis-gas chromatography-mass spectrometry, and nine compounds were identified. These results suggest that MAFs are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates with intermonomeric linkages of B-ring to B-ring and C-ring to A-ring.     

Potent suppressive effect of green tea polyphenols on tobacco-induced mutagenicity

Antimutagenic activity of green tea (Camellia sinensis) was studied using Salmonella typhimurium strains (TA 102) (Ames test). Aqueous tobacco extract was found to be mutagenic to S. typhimurium TA 102 at concentration of 50 mg/plate. Green tea polyphenols was found to inhibit the mutagenicity of tobacco in a concentration-dependent manner. Concentrations needed for 50% inhibition of mutagen-induced revertant formation was found to be 5 mg/plate. Green tea polyphenols was also found to inhibit the urinary mutagenicity in rats induced by tobacco extract. Moreover green tea polyphenols were found to inhibit in vitro nitrosation reaction produced by reaction sodium nitrite and methyl urea and further inhibition of mutagenicity indicating that green tea has dual action to bring out a reduction in the mutagenic and carcinogenic potential of tobacco.     

Inhibitory effect of water-soluble chitosan on growth of Streptococcus mutans

The present study was undertaken to evaluate the effects of pH and the degree of polymerization of chitosan on the inhibition of growth of Streptococcus mutans. Three types of chitosan, polymer, oligomer and monomer, were used at 4% (W/V) and three different levels of pH: 6.0, 6.5 and 7.4. Bactericidal activity was calculated by the growth ratio. Chitosan oligomer significantly inhibited bacterial growth at a pH value of 6.5. All three types of chitosan strongly inhibited bacterial growth at pH 6.0. Furthermore, nearly complete inhibition was obtained with 2%(W/V) chitosan solution at constant pH 6.5. This study is the first to report that water-soluble chitosan directly suppresses the growth of the typical cariogenic bacterium S. mutans even at pH 6.5, without causing demineralization of the tooth surface.     

Analysis of tea polyphenols

Tea is the most highly consumed beverage in the world, other than water. However, unlike water, tea contains substantial amounts of polyphenols that have unique biological activities and may be responsible for many of the health benefits of tea. As a result, it is essential to be able to measure the various tea-associated polyphenols. Total polyphenol content is currently measured by using methodology based on reducing activity. Several HPLC systems with detectors that, collectively, have wide ranges in sensitivity have been developed for analysis of individual flavonoids in tea and biological samples, and for theaflavins in tea. Catechins also have been measured in plasma by solid phase extraction, addition of a chromophore, and colorimetric quantification. Except for theaflavins in tea, routine and robust methods for the measurement of polyphenol condensation products (dimers and thearubigens) in tea and biological samples have not been developed. Although in vitro and animal studies suggest substantial metabolism of flavonoids in the gastrointestinal tract, only a single HPLC procedure has been assembled for monitoring the metabolic products of quercetin in urine of human subjects.     

Inhibitory effect of green and black tea on tumor growth

The administration of green tea, black tea, or (-)-epigallocatechin gallate inhibited the growth of established nonmalignant and malignant tumors in tumor-bearing mice. In experiments with black tea, we found that its oral administration inhibited DNA synthesis and enhanced apoptosis in both nonmalignant and malignant tumors in tumor-bearing mice.     

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.     

Antimicrobial effect and membrane-active mechanism of tea polyphenols against Serratia marcescens

In this study, we investigated the antimicrobial effect of tea polyphenols (TP) against Serratia marcescens and examined the related mechanism. Morphology changes of S. marcescens were first observed by transmission electron microscopy after treatment with TP, which indicated that the primary inhibition action of TP was to damage the bacterial cell membranes. The permeability of the outer and inner membrane of S. marcescens dramatically increased after TP treatment, which caused severe disruption of cell membrane, followed by the release of small cellular molecules. Furthermore, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis was used to study the difference of membrane protein expression in the control and TP treatment S. marcescens. The results showed that the expression of some metabolism enzymes and chaperones in TP-treated S. marcescens significantly increased compared to the untreated group, which might result in the metabolic disorder of this bacteria. Taken together, our results first demonstrated that TP had a significant growth inhibition effect on S. marcescens through cell membrane damage.     

复方茶多酚含漱液对正畸儿童牙面菌斑中致龋菌的影响

目的观察复方茶多酚含漱液对正畸儿童牙面菌斑中细菌总数和变形链球菌数的影响,以及牙菌斑内原位pH的改变。方法选择42例戴用固定矫治器的正畸儿童,随机分为2组,试验组用复方茶多酚含漱液漱口,对照组用蒸馏水漱口。分别于戴用矫治器前,戴入后1月采集上下颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数,同时测定牙菌斑原位pH。结果对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P0.01),牙菌斑原位pH较戴用前降低(P0.01)。试验组与对照组戴用后1个月相比,试验组细菌总数及变形链球菌数明显少于对照组(P0.01),牙菌斑原位pH高于对照组(P0.01)。结论戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,牙菌斑原位pH较戴用前降低,应用茶多酚含漱液可明显抑制正畸儿童口腔内变形链球菌数,减少龋坏发生。     

茶多酚与锌联合应用对口腔致臭菌产臭水平的影响

目的评价茶多酚与锌联合应用对口腔致臭菌产臭水平的影响。方法选择口腔主要3种致臭菌[牙龈卟啉单胞菌(P.g),中间普氏菌(P.i),具核梭杆菌(F.n)],观察不同比例混合的茶多酚与锌离子溶液在4 h后对3种致臭菌产生挥发性硫化物水平的影响,挥发性硫化物值通过HalimeterTM测得,记录数据,并进行数据的统计分析处理。结果当茶多酚与锌离子的体积比为1.5︰0.5、1.0︰1.0、0.5︰1.5时,抑制P.g产VSCs的能力最优;当茶多酚与锌离子的体积比为1.0︰1.0、0.5︰1.5时,抑制P.i及F.n产VSCs的能力最优。结论茶多酚与锌离子联合应用的最佳体积比可能为1.0︰1.0、0.5︰1.5,此时抑制3种致臭菌产VSCs的效果均较好。     

Chemosystematics of tea trees based on tea leaf polyphenols as phenetic markers

This study examined the polyphenols of tea leaves as chemotaxonomic markers to investigate the phenetic relationship between 89 wild (the small-leaved C. sinensis var. sinensis and large-leaved C. sinensis var. assamica), hybrid, and cultivated tea trees from China and Japan. (?)-Epigallocatechin 3-O-gallate, EGCG (1); (?)-epigallocatechin, EGC (2); (?)-epicatechin 3-O-gallate, ECG (3); (?)-epicatechin, EC (4); (+)-catechin, CA (5); strictinin, STR (6); and gallic acid, GA (7) were used as polyphenolic markers. Of the 13 polyphenol patterns observed, Principal Component Analysis (PCA) indicated that the structure-types of the flavonoid B-rings, such as the pyrogallol-(EGCG (1) and EGC (2)) and catechol-(ECG (3) and EC (4)) types, greatly influenced the classification. Ward’s minimum-variance cluster analysis was used to produce a dendrogram that consisted of three sub-clusters. One sub-cluster (A) was composed of old tea trees ‘Gushu’ cha (C. sinensis var. assamica) and cv ‘Taidi’ cha, suggesting that relatively primitive tea trees contain greater amounts of compounds 3 and 4 and lower amounts of compounds 1 and 2. The other two sub-clusters B and C, made up of Chinese hybrids (sub-cluster B) and Japanese and Taiwanese tea trees (sub-cluster C), had lower contents of 3 and 4 than sub-cluster A. Therefore, PCA and cluster analysis indicated that the greater the amounts of 1 and 2 (and the lower of 3 and 4), the more recent the origin of the tea line. Based on morphological characteristics, geographical information, and the historical information on tea trees, these results show good agreement with the current theory of tea tree origins, and this suggests that the Xishuangbanna district and Puer City are among the original sites of the tea tree species.     

Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols

Forty-three polyphenols from tea leaves were evaluated for their anti-oxidative effect against lipid peroxidation by the ferric thiocyanate method in vitro. Among these, 1,4,6-tri-O-galloyl-beta-D-glucose (hydrolyzable tannin) showed the highest anti-oxidative activity against lipid peroxidation, even stronger than that of 3-tert.-butyl-4-hydroxyanisole (BHA). The assay demonstrates that tea polyphenols, except for desgalloylated dimeric proanthocyanidins that possess a catechin structure in the upper unit and desgalloylated flavan-3-ols, and excepting theaflavin 3,3'-di-O-gallate, had more anti-oxidative activity than that of alpha-tocopherol. The chemical structure-activity relationship shows that the anti-oxidative action advanced with the condensation of two molecules of flavan-3-ols as well as with 3-O-acylation in the flavan skeleton such as that by galloyl, (3'-O-methyl)-galloyl, and p-coumaroyl groups.     

Antimutagenic activity of green tea polyphenols

For centuries green tea has been a widely consumed beverage throughout the world. It is known to contain a number of pharmacologically active compounds. In this study water extracts of green tea (WEGT) and their major constituents, green tea polyphenols (GTP), were examined for antimutagenic activity. WEGT and GTP were found to significantly inhibit the reverse mutation induced by benzo[alpha]pyrene (BP), aflatoxin B1 (AFB1), 2-aminofluorene, and methanol extracts of coal tar pitch in Salmonella typhimurium TA100 and/or TA98 in the presence of a rat-liver microsomal activation system. GTP also inhibited gene forward mutation in V79 cells treated with AFB1 and BP, and also decreased the frequency of sister-chromatid exchanges and chromosomal aberrations in V79 cells treated with AFB1. The addition of GTP during and after nitrosation of methylurea resulted in a dose-dependent inhibition of mutagenicity. Studies to define the mechanism of the antimutagenic activity of GTP suggest that it may affect carcinogen metabolism, DNA adduct formation, the interaction of ultimate carcinogen or the scavenging of free radicals.     

Fragrances in oolong tea that enhance the response of GABAA receptors

We electrophysiologically investigated the effect of some fragrant compounds in oolong tea on the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABAA receptors) which were expressed in Xenopus oocytes. Of the tested fragrances in oolong tea, cis-jasmone, jasmine lactone, linalool oxide and methyl jasmonate significantly potentiated the response. Among these, cis-jasmone and methyl jasmonate potently potentiated the response, having a respective dissociation constant of the compound (Kp) and maximum potentiation (Vm) of 0.49 mM and 322% for cis-jasmone, and 0.84 mM and 450% for methyl jasmonate. Inhalation of 0.1% cis-jasmone or methyl jasmonate significantly increased the sleeping time of mice induced by pentobarbital, suggesting that these fragrant compounds were absorbed by the brain and thereby potentiated the GABAA receptor response. Both of these compounds may therefore have a tranquillizing effect on the brain.     

Effects of oolong tea on metabolism of plasma fat in mice under restraint stress

We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T 1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9 +/- 29.9mg/dl 35min after Intralipid administration in control stressed mice, 478.7 +/- 26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3 +/- 18.4 mg/dl in the stressed group given 1,000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some non-specific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.