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Lactic acid bacteria (LAB) are safe microorganisms that have been used in the processing of fermented food for centuries. The aim of this study was to isolate Lactobacillus from fresh tea leaves and examine the impact of an isolated strain on intestinal barrier integrity. First, the presence of Lactobacillus strains was investigated in fresh tea leaves from Kagoshima, Japan. Strains were isolated by growing on De Man, Rogosa and Sharpe (MRS) agar medium containing sodium carbonate, followed by the identification of one strain by polymerase chain reaction (PCR) and pheS sequence analysis, with the strain identified as Lactiplantibacillus plantarum and named L. plantarum LOC1. Second, the impact of strain LOC1 in its heat-inactivated form on intestinal barrier integrity was investigated. Strain LOC1, but not L. plantarum ATCC 14917T or L. plantarum ATCC 8014, significantly suppressed dextran sulfate sodium (DSS)-induced decreases in transepithelial electrical resistance values of Caco-2:HT29-MTX 100:0 and 90:10 co-cultures. Moreover, in Caco-2:HT29-MTX co-cultures (90:10 and 75:25), levels of occludin mRNA were significantly increased by strain LOC1 compared with untreated co-cultures, and strain LOC1 had higher mRNA levels of MUC2 and MUC4 mucins than L. plantarum ATCC 14917T and L. plantarum YT9. These results indicate that L. plantarum LOC1 may be used as a safe probiotic with beneficial effects on the intestinal barrier, suggesting that fresh tea leaves could be utilized as a safe source for isolating probiotics.

Lactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to “monopolize” inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and

Lactic acid bacteria (LAB) are safe microorganisms that have been used in the processing of fermented food for centuries. The aim of this study was to isolate Lactobacillus from fresh tea leaves and examine the impact of an isolated strain on intestinal barrier integrity. First, the presence of Lactobacillus strains was investigated in fresh tea leaves from Kagoshima, Japan. Strains were isolated by growing on De Man, Rogosa and Sharpe (MRS) agar medium containing sodium carbonate, followed by the identification of one strain by polymerase chain reaction (PCR) and pheS sequence analysis, with the strain identified as Lactiplantibacillus plantarum and named L. plantarum LOC1. Second, the impact of strain LOC1 in its heat-inactivated form on intestinal barrier integrity was investigated. Strain LOC1, but not L. plantarum ATCC 14917T or L. plantarum ATCC 8014, significantly suppressed dextran sulfate sodium (DSS)-induced decreases in transepithelial electrical resistance values of Caco-2:HT29-MTX 100:0 and 90:10 co-cultures. Moreover, in Caco-2:HT29-MTX co-cultures (90:10 and 75:25), levels of occludin mRNA were significantly increased by strain LOC1 compared with untreated co-cultures, and strain LOC1 had higher mRNA levels of MUC2 and MUC4 mucins than L. plantarum ATCC 14917T and L. plantarum YT9. These results indicate that L. plantarum LOC1 may be used as a safe probiotic with beneficial effects on the intestinal barrier, suggesting that fresh tea leaves could be utilized as a safe source for isolating probiotics.

Allergies are a global issue. However, medical intervention for allergy treatment is limited. Recent studies have focused on allergy prevention with food components. Both the lactic acid bacteria Enterococcus faecalis IC-1 (IC-1) and the flavonoid luteolin have been shown to have an anti-allergic effect. Following the concept of diet diversity and synergistic effects, the purpose of this study was to explore the anti-allergic activity of a combination of IC-1 and luteolin. A combination of IC-1 and luteolin had anti-allergic effects with the passive cutaneous anaphylaxis reaction and strongly inhibited mast cell degranulation in a Caco-2/RBL-2H3 cells co-culture system. Oral administration with a combination of IC-1 and luteolin significantly decreased blood IgE content in ovalbumin (OVA)-induced allergic mice and alleviated symptoms of an allergy attack. These might have been done through correcting Th2 shifting with the allergy state since a combination of IC-1 and luteolin increased the IFN-γ content in blood. Furthermore, in vitro differentiation of the Th2 subset confirmed that a combination of IC-1 and luteolin inhibited naive T cell from differentiating into a Th2 subset through inhibition of Th2 transcription factor GATA-3 expression. The combination also promoted the gene expression level of IFN-γ in mice bone marrow-derived dendritic cells. These results suggested a synergistic anti-allergic effect using a combination of IC-1 and luteolin both in reducing IgE content and in inhibiting mast cell degranulation during the allergy state.

The main cause of arteriosclerosis is atherosclerosis in the aorta. Atherosclerosis is recognized as a chronic inflammatory condition that begins with the dysfunction or activation of arterial endothelium. Low-density lipoprotein (LDL) and especially its oxidized form play a key role in endothelial dysfunction and atherogenesis. Recent studies showed that senescent cells are involved in the development and progression of atherosclerosis, and eliminating senescent cells suppresses the senescence-associated secretory phenotype. We previously reported that molecular hydrogen-rich water (HW) has antioxidant and anti-inflammatory effects in numerous diseases. Here, we used LDL receptor-deficient mice fed a high-fat diet (HFD) for 13 weeks as a model for atherosclerosis and evaluated the effects of continuous administration of HW. The numbers of endothelial cells in the atheroma expressing the senescence factors p16INK4a and p21 decreased in HFD-fed mice given HW compared with HFD-fed mice given control water. Furthermore, macrophage infiltration and Tnfα expression in the atheroma were also suppressed. These results suggest that vascular aging can be suppressed by HW.

Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.

The mechanism of sediment formation during the storage of green tea beverage was investigated. Green tea extract was separated by Diaion HP-20 column chromatography, and a sediment-formation test was performed. Results showed that at least one compound of the substance causing flock sediment was contained in each of the HP-20 nonadsorbed and adsorbed fractions. From the following fractionations and structure analyses, the substance in the HP-20 adsorbed fraction was determined to be 1-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-beta-D-glucose (strictinin), which is one of the ellagitannins. Strictinin was hydrolyzed to ellagic acid by heat-sterilization processes such as retort sterilization or the ultra-high temperature processing used during the manufacturing of tea beverages. Ellagic acid combined with proteins in the HP-20 nonadsorbed fraction to form an irreversible sediment of green tea beverage; ellagic acid and proteins were confirmed to be present in that sediment. The HP-20 adsorbed fraction contained little strictinin and formed hardly any sediment, suggesting that control of the strictinin content is significant in avoiding sediment formation during the manufacturing process of tea beverages.

We prospected the antidotes from natural oriental drugs for botulinum neurotoxin type A. The crude extracts of Galla Rhois and Geranii Herba showed a potent antidote activity for botulinum neurotoxin type A. The antidote titer of the crude extracts of black tea was higher than those of Galla Rhois and Geranii Herba. No antidote activity was detected in the crude extracts of the followings: Gambir, Rosae Multiflorae Fructus, Scutellariae Radix, Coptidis Rhizoma, Sophorae Flos, Puerariae Radix, Houttuyniae Herba, Swertiae Herba, Galla Halepensis. Although both Galla Rhois and Galla Halepensis are gall-apples and contain tannin as the main component, Galla Rhois showed the potent antidote activity while Galla Halepensis did not.

Banaba [Lagerstroemia speciosa (L.) Pers.] has been used as a folk medicine for diabetes in the Philippines. Using bioassay-guided separation, valoneaic acid dilactone (1) was isolated from the leaves as a potent alpha-amylase inhibitor. A simple and efficient method for the quantitative determination of valoneaic acid and its derivatives in Banaba extract was established. Valoneaic acid exists as the structural part of the polyphenols, which like flosin A, reginin A, and lagerstroemin, are characteristic constituents of Banaba. These derivatives were hydrolyzed to valoneaic acid by HCl and extracted with 2-butanone. This extract was subjected to HPLC analysis, and the contents of valoneaic acid determined as the whole valoneaic acid contents. Using this method, the whole valoneaic acid contents were measured in eight Banaba leaf decoctions. The alpha-amylase-inhibiting activities of the decoctions were dependent on the whole valoneaic acid contents. In addition, a strong linear correlation was observed between the whole valoneaic acid contents and total polyphenol contents. This analytical procedure is applicable to the chemical evaluation of Banaba.

紅茶から抽出したテアルビジン分画の,生ハム抽出液に添加したボツリヌス神経毒素に対する効果を確認した.テアルビジン分画は,ボツリヌス神経毒素が神経終末細胞表面へ結合する前に混合することにより濃度依存的にに毒素の作用を阻止した.B型毒素の経口毒性を調べるため,毒素と生ハム由来の上清液との混合試料をマウスに経口投与した.本毒素は濃度依存的にマウスに運動麻痺作用を発現した.さらにテアルビジン分画の抗毒素能を調べるたあ,B型毒素,テアルビジン分画と生ハム上清液との混合試料をマウスに経口投与した.テアルビジン分画はマウスの運動麻痺を濃度依存的に阻止した.テアルビジン分画の食品への応用性を調べるため,テアルビジン分画に浸漬した生ハム試料を用いて官能試験を実施した.生ハムは熱湯抽出したテアルビジン分画に浸漬すると食味の悪化を呈した.しかしテアルビジン分画からタンニンなどの夾雑物を除去することにより,その抗毒素能は高まるとともに食味の悪化を防げると予想した.

フィリピンで糖尿病に有効であるとされているバナバについてXODに対する阻害効果に関する評価を行った.(1) バナバ葉熱水抽出物は緑茶,ルイボス茶,杜仲茶と比較して強いXOD阻害効果を示した.(2) バナバ葉熱水抽出物を合成吸着樹脂であるダイアイオンHP-20に供し,そのメタノール溶出フラクションを回収したところ,XOD阻害活性が上昇した.(3) HPLCを用いた分析より,バナバ葉成分としてエラグ酸の存在を認め(乾燥重量として3.1%),エラグ酸がバナバ葉熱水抽出物のXOD阻害効果に関与している可能性があることを推察した.

The hypoglycemic effects of Lagerstroemia speciosa L., known by the Tagalog name of banaba in the Phillipines, were studied using hereditary diabetic mice (Type II, KK-AY/Ta Jcl). The mice were fed a test diet containing 5% of the hot-water extract (HWE) from banaba leaves, 3% of the water eluent of the partial fraction unadsorbed onto HP-20 resin of HWE (HPWE), and 2% of the methanol eluent of the partial fraction adsorbed onto HP-20 resin of it (HPME) for a feeding period of 5 weeks. The elevation of blood plasma glucose level in non-insulin dependent diabetic mice fed the cellulose as control (CEL) diet were almost entirely suppressed by addition of either HWE or HPME in place of cellulose in the CEL diet. Water intakes were inclined to increase gradually in the group fed either CEL or HPWE, but lower in the mice fed either HWE or HPME than in the animals given either CEL or HPME. The level of serum insulin and the amount of urinary excreted glucose were also lowered in mice fed HWE. Plasma total cholesterol level was also lowered in mice fed the either HWE or HPME. It is suggested that HWE, especially HPME, obtained from banaba leaves have beneficial effects on control of the level of plasma glucose in non-insulin dependent diabetes mellitus.

The suppression of cytochrome P450 1A1 (CYP1A1) expression was examined in mouse hepatoma Hepa-1c1c7 cells treated with serum prepared from (−)-epigallocatechin-3-gallate- and green tea extract-administered rats. Catechins were found in the rat plasma after the administration. In Hepa-1c1c7 cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 expression was suppressed by treatment with the rat serum. It is concluded that catechins can possibly modulate CYP1A1 expression.

In order to develop naturally occurring antioxidants from edible plants, the antioxidative effect of hot water extracts of Lagerstroemia speciosa leaves, known by the Tagalog name of banaba in the Phillipines, was studied. The content of tannin in banaba extract was 36.8% in dry weight. Banaba extract showed strong antioxidative activity in a linoleic acid autoxidation system. Banaba extract was found to have a potent radical scavenging action on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide radicals (O(2-) ) generated by a hypoxanthine (HPX)/xanthine oxidase (XOD) system. In vitro lipid peroxidation of rat liver homogenate induced by tert-butyl hydroperoxide (BHP) was inhibited by the addition of banaba extract in a dose-dependent manner. From these results, banaba extract was demonstrated to be useful as an antioxidant or free radical scavenger to protect biological systems against oxidative stress.

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.

Dioxins cause various adverse effects through binding to an aryl hydrocarbon receptor (AhR) and transformation of the receptor. In this study, we investigated whether black tea extract suppresses AhR transformation. Dried black tea leaves were extracted with 75% ethanol, and the extract was pretreated to the rat liver cytosol fraction 10 min prior to addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transformed AhR was detected by electrophoretic gel mobility shift assay. Black tea extract suppressed AhR transformation in a dose-dependent manner, and the IC50 value against 1 nM TCDD-induced AhR transformation was 8.9 microg/ml. The result suggests that intake of black tea has a potential to suppress the AhR transformation, leading protection from dioxin toxicity.

Environmental contaminants such as dioxins enter the body mainly through diet and cause various toxicities through transformation of the aryl hydrocarbon receptor (AhR). We previously reported that certain natural flavonoids at the dietary level suppress the AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we identified lutein and chlorophyll a and b from green tea leaves as the novel antagonists for AhR. These active compounds suppressed AhR transformation dose-dependently with the 50% inhibitory concentration (IC(50)) values against 0.1 nM TCDD-induced AhR transformation at 3.2, 5.0, and 5.9 microM, respectively. (-)-Epigallocatechin gallate, which is the most abundant flavonoid in green tea leaves, also showed stronger suppressive effects than did other major tea components, with the IC(50) value of 1.7 microM. Thus, these pigments of green tea leaves have the potential to protect from dioxin toxicity through the suppression of AhR transformation.

Banaba [Lagerstroemia speciosa (L.) Pers.] has been used as a folk medicine for diabetes in the Philippines. Using bioassay-guided separation, valoneaic acid dilactone (1) was isolated from the leaves as a potent alpha-amylase inhibitor. A simple and efficient method for the quantitative determination of valoneaic acid and its derivatives in Banaba extract was established. Valoneaic acid exists as the structural part of the polyphenols, which like flosin A, reginin A, and lagerstroemin, are characteristic constituents of Banaba. These derivatives were hydrolyzed to valoneaic acid by HCl and extracted with 2-butanone. This extract was subjected to HPLC analysis, and the contents of valoneaic acid determined as the whole valoneaic acid contents. Using this method, the whole valoneaic acid contents were measured in eight Banaba leaf decoctions. The alpha-amylase-inhibiting activities of the decoctions were dependent on the whole valoneaic acid contents. In addition, a strong linear correlation was observed between the whole valoneaic acid contents and total polyphenol contents. This analytical procedure is applicable to the chemical evaluation of Banaba.

Both growth and theanine accumulation in tea callus cultures were improved by the combination of 4 mg/liter benzyladenine and 2mg/liter indol-3-butyric acid, but were strongly inhibited by the addition of 2,4-dichlorophenoxyacetic acid. The optimum initial concentration of carbon source was 30g/liter sucrose. Upon the addition of more than 30 g/liter of sucrose, the callus fresh weight was increased, but the theanine formation was not improved.

The botulinum neurotoxin produced by Clostridium botulinum exhibits the strongest neurotoxicity, and causes botulism in mammals. We have found an inactivator for clostridial neurotoxins in black tea extract (thearubigin fraction) as a natural foodstuff. In this study, we have isolated and identified the inactivators. The activity against the neuromuscular blocking action of botulinus neurotoxin type A was examined in mouse phrenic nerve diaphram preparation. The purification procedure of the inactivators was as follows. Tea was extracted with aqueous acetone, and then filtrated and lyophilized. It was also extracted with n-hexane, chloroform, ethyl acetate, n-butylalchol and water, so the activity of the antidote was recognized to be in the n-butylalchol layer (named the thearubigin fraction). A two-step reversed phase HPLC was developed for the thearubigin fraction. Three flavonoids were found to have the major activity. The structural elucidation of the compounds by means of NMR spectrascopy revealed, kaempfenol-3-O-[glc-(6-1)-rha-(3-1)-glc];keampfetrin, kaempferol-3-O-[glc-(6-1)-rha];nicotiflorin and quercetin glycoside.

The effects of a green tea (Camellia sinensis) extract on ethanol metabolism in ICR male mice were studied. A crude green tea extract (GTE) and the tea components as (-)-epigallocatechin gallate (EGCg), (-)-epigallocatechin (EGC), and caffeine were administered before the tests. One hour later, the mice were orally given 2g/kg body weight (b.w.) of ethanol (20% ethanol w/v). The results show that the levels in the blood and liver of ethanol and acetaldehyde were lower, and that the levels of acetate and acetone were higher than in the controls orally given 500 mg/kg b.w. of GTE. After the administration of 75 mg/kg b.w. and 225 mg/kg b.w. of EGCg, the acetate and acetone concentrations in the blood and liver were lower than in the controls. The mice given caffeine at the same dose as that in GTE showed almost the same effects as the group treated with GTE. This suggests that EGCg and caffeine, the principal components of GTE, both had an effect on ethanol metabolism.

In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 μM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.