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The detection sensitivity of immunostick colorimetric assay has been increased by using a bio-nanocapsule as a scaffold for oriented immobilization of immunoglobulin Gs. This immunostick produced ∼82-folds stronger coloration in the detection of food allergens and reduced detection time by a factor of 5.

Black tea extracts (BTEs) from four different production areas showed a higher aggregation strength for phosphatidylcholine-based liposomes containing cholesterol used as a viral membrane model. Furthermore, the anti-influenza A virus (IAV) activity of each BTE in vitro demonstrated that although Sri Lanka, Kenya, and Assam had higher anti-IAV activities, Darjeeling had a lower anti-IAV activity, showing a correlation between each BTE and the liposome aggregation strength. Moreover, the antiviral activity strength of BTEs was consistent with the antioxidant activity strength of BTEs, suggesting that the component(s) in black tea that exhibits antioxidant activity would also be the component(s) that accounts for its antiviral activity. Thus, our results propose that BTEs exert their antiviral effects by binding not only hemagglutinin and neuraminidase but also viral membranes directly, especially “cholesterol-rich lipid rafts” and affect the membrane structure, causing the virus to aggregate, thereby inhibiting infection of the host cells.

We report a novel scaffold for clustering and oriented immobilization of human IgG1 Fc-fused lectins on biosensors without chemical modifications. This approach uses a bio-nanocapsule (BNC) displaying a tandem form of IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). Incorporating ZZ-BNC effectively increased both the sensitivity and sugar chain-binding capacity compared with the condition without ZZ-BNC.

Black tea is a highly popular beverage, and its pigments, polymerized catechins such as theaflavins (TFs), are attracting attention due to their beneficial health effects. In this study, to test the inhibitory activities of TFs on the intestinal absorption of cholesterol, we investigated their effects on phosphatidylcholine (PC) vesicles in the absence or presence of a bile salt. (−)-Epicatechin gallate, (−)-epigallocatechin gallate, and TFs formed insoluble complexes with PC vesicles. Galloylated TFs such as TF2A, TF2B, and TF3 precipitated far more than other polyphenols. The subsequent addition of taurocholate redispersed the polyphenol-PC complexes, except that a large amount of TF2A remained insoluble. After incubation with taurocholate-PC micelles, TF2A elevated the turbidity of the micelle solution, providing red sediments. The TF2A-specific effect was dependent on the PC concentration. These results suggest that TF2A interacts with PC and aggregates in a specific manner different from catechins and other TFs.

The orientation and density of biosensing molecules on sensor chip should be precisely controlled to improve sensitivity and ligand-binding capacity. We previously developed a ~30-nm bio-nanocapsule (ZZ-BNC), consisting of the hepatitis B virus envelope L protein fused with the tandem form of protein A-derived IgG Fc-binding Z domain (ZZ-L protein). This is used as a robust nanoparticle scaffold to enhance the sensitivity and ligand-binding capacity of IgGs and Fc-fused sensing molecules (Fc-fused receptors). However, due to their rigid particle structure, the surface density of ZZ-L proteins could not be optimized for biosensor functions, and useless ZZ-L proteins become stuck between ZZ-BNC and the sensor chip. Here, we have developed a planar lipid membrane embedded with ZZ-L micelles (ZZ-L membrane), which could modify the surface of any biosensor chip with a controlled density of ZZ-L proteins. Compared with ZZ-BNC, the sensitivity and ligand-binding capacity of IgGs were enhanced about 10-fold with the ZZ-L membrane. Furthermore, the immobilized IgGs could capture their respective antigens almost stoichiometrically, indicating that ZZ-L membrane is the most ideal scaffold for Fc-fused sensing molecules in terms of both clustering and oriented immobilization.

The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.

Several physical and psychological stresses frequently become triggers for gastrointestinal disorders such as ulcer. In this study, we tried to identify serum proteins as potential biomarkers for the evaluation of stress-induced gastric ulcer. By proteomic analysis using rats with gastric ulcer induced by water immersion and restraint (WIR) stress as an animal model, we found quantitative changes in several serum proteins, including creatine kinase muscle M chain (CK-M) and apolipoprotein A-IV (ApoA4) in the stressed rats. On western blotting and enzyme-linked immunosorbent assay (ELISA), we confirmed that serum CK-M was remarkably increased by WIR stress. However, ApoA4 appeared to be decreased by fasting, but not WIR stress, which is usually applied prior to WIR stress. The findings suggest that these two serum proteins might be useful as biomarkers, CK-M for stress-induced gastric ulcer and ApoA4 for starvation.

Catechins are the major polyphenols in green tea leaves. Recent studies have suggested that the catechins form complexes with HSA for transport in human blood, and their binding affinity for albumin is believed to modulate their bioavailability. In this study, the binding affinities of catechins and their analogs were evaluated and the relationship between the chemical structure of each catechin and its binding property were investigated. Comparing these catechins by HPLC analysis with the HSA column, we showed that galloylated catechins have higher binding affinities with HSA than non-galloylated catechins. In addition, pyrogallol-type catechins have a high affinity compared to catechol-type catechins. Furthermore, the binding affinity of the catechin with 2,3-trans structure was higher than those of the catechin with 2,3-cis structure. The importance of the hydroxyl group on the galloyl group and B-ring was confirmed using methylated catechins. These results indicate that the most important structural element contributing to HSA binding of tea catechins is the galloyl group, followed by the number of hydroxyl groups on the B-ring and the galloyl group or the configuration at C-2. Our findings provide fundamental information on the relationship between the chemical structure of tea catechins and its biological activity.

An analysis of hydrogen peroxide in an aqueous extract of cigarette smoke, which contains many redox-active compounds, requires a method with high selectivity. An aqueous extract of the particulate phase of cigarette smoke was analyzed by HPLC with an electrochemical detector (ECD). Samples were prepared by collecting the particulate phase of the cigarette smoke on a glass fiber filter and extracting it with a phosphate buffer. The obtained solution was purified by using a Waters Oasis MCX cation-exchange cartridge, and then analyzed by an HPLC-ECD system with a Shodex KS-801 mixed-mode resin column. Pre-injecting hydrogen peroxide at a high concentration into the HPLC instrument stabilized the analytical results. The recovery of hydrogen peroxide by using an extract of the particulate phase of the cigarette smoke was more than 80%. An increase in the amount of hydrogen peroxide was observed during extraction with the phosphate buffer at higher pH values. In contrast, extraction with phosphoric acid did not increase the amount of hydrogen peroxide during extraction.

(-)-Epigallocatechin gallate (EGCg) is the major component of green tea and is known to show strong biological activity, although it can be easily oxidized under physiological conditions. In this study, we indicate that EGCg is stable in human serum and that human serum albumin (HSA) stabilizes EGCg under aerobic condition. Although EGCg is usually decomposed within 1 h in aqueous solution at neutral pH, EGCg in serum and phosphate buffer (pH 7.4) containing HSA was stable over 1 h, even at neutral and slightly alkaline pH. Under these conditions, EGCg binds to HSA non-covalently. The sulfhydryl group acts as an antioxidant for EGCg oxidation. Incubation of EGCg with HSA is accompanied by the oxidation of a free sulfhydryl group in HSA. These results suggest that the antioxidant property and the binding capacity of HSA contribute to the stabilization of EGCg in human serum.

The biological activity of polyphenols in vitro reflects their affinity for cell membranes and the amount of cellular incorporation. The interaction of polyphenols with phosphatidylcholine was investigated by HPLC with an immobilized artificial membrane (IAM) column. The IAM partition coefficients (K(IAM)) of the polyphenols, calculated by retention times, correlated well with the amounts of polyphenols incorporation into the liposomes.

Polyphenols, a large group of natural antioxidants, are a versatile group of phytochemicals beneficial for disease prevention. In this study, we comprehensively analyzed polyphenols, catechins, procyanidins, simple polyphenols, anthocyanins and flavonoids, in fruits consumed in Japan by high performance liquid chromatography with photo-diode array and mass spectrometric detection to complete the database of food components.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

Propolis is a resinous substance collected by honeybees from various plant sources. We examined the antioxidant activity of Japanese propolis from various areas of Japan: Hokkaido, Akita (Minamiakita and Kazuno), Fukushima (Aizuwakamatsu and Futaba), Gifu, Nagano, Tokyo, Kanagawa, Shizuoka, Okayama, Tottori, Fukuoka and Okinawa. We prepared ethanol extracts of propolis (EEP), and evaluated the antioxidant activity of EEP samples by the β-carotene bleaching and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay systems. Further, we identified the major constituents in EEP by HPLC analysis by a photodiode array (PDA) and mass spectrometric (MS) detection, and quantitatively analyzed each component. EEP from Akita (Minamiakita) and Okinawa had relatively strong antioxidant activity, and was correlated with total polyphenol contents. Propolis from Akita (Minamiakita) contained large amounts of the antioxidative compounds, caffeic acid and phenethyl caffeate. Propolis from Okinawa appeared to have antioxidants not seen in propolis from other areas.

We purified several hundred mgs of four major theaflavins (theaflavin, theaflavin-3-O-gallate, theaflavin-3′-O-gallate, and theaflavin-3,3′-O-digallate). Among the 25 hTAS2Rs expressed in HEK293T cells, hTAS2R39 and hTAS2R14 were activated by theaflavins. Both hTAS2R39 and hTAS2R14 responded to theaflavin-3′-O-gallate. In addition, hTAS2R39 was activated by theaflavin and theaflavin-3,3′-O-gallate, but not by theaflavin-3-O-gallate. In contrast, hTAS2R14 responded to theaflavin-3-O-gallate.

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Tea catechins are classified into cis-type and trans-type from the configuration of the two hydrogens at the 2 and 3 positions on the C-ring. The amount of trans-type catechins incorporated into liposomes was less than that of the respective cis-type catechins. Furthermore, the order of the partition coefficients of catechins in an n-octanol/PBS system is the same as that of the amount incorporated into liposomes. These results indicate that in addition to the number of hydroxyl groups on the B-ring and the presence of the galloyl moiety, the stereochemical structure of the C-ring also governs the hydrophobicity and the affinity for lipid bilayers. Trans-type catechins with the galloyl moiety were located on the surface of the lipid bilayer, as well as cis-type catechins with the galloyl moiety, and perturbed the membrane structure. These different stereochemical structures should influence the affinity for lipid bilayers, the alteration of membrane structures, and the difference in the order of the biological activities.

Interaction of tea catechins with lipid bilayers was investigated with liposome systems, which enabled us to separate liposomes from the external medium by centrifugation. We found that epicatechin gallate had the highest affinity for lipid bilayers, followed by epigallocatechin gallate, epicatechin, and epigallocatechin. Epicatechin gallate and epigallocatechin gallate in the surface of lipid bilayer perturbed the membrane structure.

The new chalcone, 4′-methoxy-bavachromanol (1), was isolated from propolis collected on Jeju, a southern island of Korea together with two known compounds, laserpitin (2) and isolaserpitin (3). The structure of each compound was determined by spectral methods, including mass spectrometry and 2D NMR. The IC50 value of 1 against soybean lipoxygenase was 14.6μM.

We measured ubiquinone (CoQ0) and hydrogen peroxide (H2O2) formed in the process of oxidation of ubiquinol (CoQ0H2). We found that copper-zinc superoxide dismutase and manganese superoxide dismutase inhibited both the CoQ0 formation and the H2O2 formation only in the presence of chelators such as DTPA (diethylenetriaminepentaacetic acid). The amount of H2O2 was almost equal to that of CoQ0, indicating that the H2O2 formation was coupled with the CoQ0 formation. The lack of inhibitory effects of the corresponding heat-inactivated superoxide dismutase (SOD) confirmed that the inhibition by the original SOD was due to its enzymatic activity. We propose that CoQ0H2 oxidation occurs as a chain reaction with superoxide as the chain carrier and that SOD inhibits this reaction by lowering the superoxide concentration.

Antioxidant effects of the phenolic acids, caffeic acid, p-coumaric acid, and ferulic acid, were investigated by monitoring O2 consumption in the linoleic acid micelles in the presence of H2O2 and metmyoglobin. O2 consumption usually consists of two phases: In the first phase, O2 is consumed slowly until the concentration of linoleic acid hydroperoxide reaches a certain value; then in the second phase O2 is consumed rapidly. At pH 7.4, the phenolic acids prolonged the period of the first phase and showed no effect on the second phase. At pH 3.4, the phenolic acids decreased the oxygen consumption rate in the second phase. These results suggest that the main antioxidant effects of the phenolic acids should be ascribed to reduction of ferrylmyoglobin in the first phase at pH 7.4 and inhibition of the chain reaction occurring inside of the micelles in the second phase at pH 3.4.

Human serum albumin (HSA) contributes to the stabilization of (-)-epigallocatechin gallate (EGCg) in serum. We characterize in the present study the mechanisms for preventing EGCg oxidation by HSA. EGCg was stable in human serum or buffers with HSA, but (-)-epigallocatechin (EGC) was unstable. We show by comparing EGCg and EGC in a neutral buffer that EGCg had a higher binding affinity than EGC. This indicates that the galloyl moiety participated in the interaction of EGCg with HSA and that this interaction was of critical importance in preventing EGCg oxidation. The binding affinity of EGCg for HSA and protein carbonyl formation in HSA were enhanced in an alkaline buffer. These results suggest the reversible covalent modification of EGCg via Schiff-base formation, and that the immobilization of EGCg to HSA, through the formation of a stable complex, prevented the polymerization and decomposition of EGCg in human serum.

BACKGROUND: Lectins are proteins that bind specifically to the carbohydrate moiety of glyco-conjugates. Japanese mistletoe lectin given intragastrically affected cytokine gene expression in the mouse intestine. This study examines the actions of Lens culinaris agglutinin (LCA) on the gene expression of gluconeogenic enzymes in the intestine. RESULTS: The results of quantitative real-time reverse transcription–polymerase chain reaction indicated that LCA caused an up-regulation of the gene expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). This change was correlated with an increase in the expression of two transcription factors, HNF1α and HNF4α. Experiments using human colonic cancer Caco-2 cells demonstrated that LCA up-regulated the gene expression of G6Pase and PEPCK whereas insulin had the opposite effect. In addition, the observed up-regulation of HNF4α gene expression in the duodenum raises the possibility that the lectin promotes the colorectal cancer. CONCLUSION: Lentil beans should be cooked well to avoid unfavourable effects of LCA. Copyright © 2011 Society of Chemical Industry

In this study, we investigated the relationship between the stability of catechins and their electrophilic reactivity with proteins. The stability of catechins was evaluated by HPLC analysis. Catechol-type catechins were stable in a neutral buffer, but pyrogallol-type catechins, such as (-)-epigallocatechin gallate (EGCg), were unstable. The electrophilic reactivity of catechins with thiol groups in a model peptide and a protein was confirmed by both mass spectrometry and electrophoresis/blotting with redox-cycling staining. In a comparison of several catechins, pyrogallol-type catechins had higher reactivity with protein thiols than catechol-type catechins. The instability and reactivity of EGCg were enhanced in an alkaline pH buffer. The reactivity of EGCg was reduced by antioxidants due to their ability to prevent EGCg autoxidation. These results indicate that the instability against oxidation of catechins is profoundly related to their electrophilic reactivity. Consequently, the difference in these properties of tea catechins can contribute to the magnitude of their biological activities.

Pyrroloquinoline quinone (PQQ), a putative essential nutrient, is a redox modulator in cell and animal models. Here we characterized PQQ-induced protein oxidative modifications in a model peptide and protein, and we propose that the mechanism of protein modification by PQQ is redox cycling-mediated oxidation. PQQ may contribute to the regulation of intracellular protein functions through its prooxidant action.

Propolis possesses various physiological activities. In this study, we examined the antiangiogenic and antioxidant activities of various components from propolis: acacetin, apigenin, artepillin C, caffeic acid phenethyl ester, chrysin, p-coumaric acid, galangin, kaempferol, pinocembrin, and quercetin. The effects of these components were tested on in vitro models of angiogenesis, tube formation and growth of human umbilical vein endothelial cells (HUVECs). Furthermore, these components were evaluated for their antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferric reducing/antioxidant power (FRAP) assays. Two propolis components, caffeic acid phenethyl ester, and quercetin, possessed strong inhibitory effects on tube formation and on endothelial cell proliferation and, coincidentally, showed strong antioxidant activity. Artepillin C, galangin, and kaempferol also possessed strong antiangiogenic and antioxidant activities to a slightly less degree. In contrast, acacetin, apigenin, and pinocembrin possessed a considerable degree of antiangiogenic activities, although they showed very low antioxidant activities. From these results, we propose that components from propolis such as artepillin C, caffeic acid phenethyl ester, galangin, kaempferol, and quercetin might represent a new class of dietary-derived antioxidative compounds with antiangiogenic activities. These propolis components may have the potential to be developed into pharmaceutical drugs for the treatment of angiogenesis-dependent human diseases such as tumors.

The quartz-crystal microbalance (QCM) technique was applied to investigate the interaction of tea catechins with lipid bilayers. The association constants obtained from the frequency changes of QCM revealed that (−)epicatechin gallate and (−)epigallocatechin gallate interacted with 1,2-dimyristoyl-sn-glycero-3-phosphocholine ca. 1000 times more strongly than (−)epicatechin and (−)epigallocatechin. The results exhibited good correlation with the strength of biological activity.

The mechanisms for tight-junction (TJ) stabilization by beta-lactoglobulin (beta-Lg) were studied. Treatment of Caco-2-SF cells with inhibitors for some enzymes in the intracellular signal transduction pathways and a cytoskeleton-disturbing agent (cytochalasin D) reduced the TJ-stabilizing activity of beta-Lg. So beta-Lg is suggested to modulate the cytoskeletal structure through the activation of phospholipase C and protein kinase C, resulting in the TJ stabilization.

We developed a method to measure the amounts of antioxidative polyphenols and ubiquinones incorporated into the liquid bilayers of liposomes to estimate their affinities for cell membranes. Results were expressed in terms of an "affinity factor", calculated by division of the amount of compound incorporated by the amount added to the liposomal solution. The results reflected dose-dependence of the biological activities of the compound found in earlier in vitro experiments with mammalian and bacterial cells.

Cytotoxicity and DNA single-strand breaks caused by H2O2 were assessed by a colony formation assay and a DNA precipitation assay, respectively, with Chinese hamster V79 cells. In both assays, caffeic acid ethyl ester showed protective effects. The structure-activity relationship showed that the o-dihydroxy structure of caffeic acid ethyl ester was essential for the protective effects.

The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state (31)P and (2)H NMR. The (31)P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The (2)H NMR spectrum of [4-(2)H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase. These (31)P and (2)H NMR observations provide direct experimental evidence that the EGCg molecule interacts with the lipid bilayers.

The new prenylflavonoid, isonymphaeol-B (1), together with three known compounds, nymphaeol-A (2), nymphaeol-B (3), and nymphaeol-C (4), were isolated from propolis collected in Okinawa, the southern-most prefecture of Japan. The structure of each compound was determined by spectral methods, including mass spectrometry and 2D NMR. Each compound had 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging activity.

Green tea contains a high concentration of such catechins as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.

The cytotoxicity of hydrogen peroxide (H2O2) toward Chinese hamster V79 cells was assessed with a colony-formation assay. Decylubiquinone, a coenzyme Q homolog, suppressed H2O2-induced cytotoxicity. Almost all the incorporated decylubiquinone was detected as decylubiquinol after incubating the cells for 4h. These results suggest that decylubiquinol reduced in the cells acted as an antioxidant against H2O2-induced oxidative cell injury.

We investigated the lipid peroxidation in linoleic acid micells caused by H2O2 in the presence of metmyoglobin by monitoring the oxygen consumption. O2 consumption usually consisted of two phases. In the first phase, it occurred slowly and linearly until the concentration of linoleic acid hydroperoxide reached a certain value, rapid consumption, presumably by a chain reaction, then followed in the second phase. No effects of diethylenetriaminepentaacetic acid (DTPA) on the induction period (the period during the first phase) and the maximum oxygen consumption rate (MOCR) in the second phase indicate that free ferric ions liberated from myoglobin had no role in any phases during the lipid peroxidation. The differing dose effects of ascorbic acid, alpha-tocopherol, and sodium nitrite on the induction period and MOCR reflect their respective antioxidative mechanisms during lipid peroxidation.

The tight junction (TJ) of the human intestinal Caco-2-SF monolayer was shown not to have been stably formed. TJ was stabilized by incubating the cell monolayer with beta-lactoglobulin (beta-Lg) and bovine serum albumin (BSA). Thus, the Caco-2-SF cell-culture system will provide a useful model for studying the factors which stabilize TJ and for investigating the mechanism of TJ regulation.