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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.

Aims Although periarticular injection plays an important role in multimodal pain management following total hip arthroplasty (THA), there is no consensus on the optimal composition of the injection. In particular, it is not clear whether the addition of a corticosteroid improves the pain relief achieved nor whether it is associated with more complications than are observed without corticosteroid. The aim of this study was to quantify the safety and effectiveness of cortocosteroid use in periarticular injection during THA. Methods We conducted a prospective, two-arm, parallel-group, randomized controlled trial involving patients scheduled for unilateral THA. A total of 187 patients were randomly assigned to receive periarticular injection containing either a corticosteroid (CS group) or without corticosteroid (no-CS group). Other perioperative interventions were identical for all patients. The primary outcome was postoperative pain at rest during the initial 24 hours after surgery. Pain score was recorded every three hours until 24 hours using a 100 mm visual analogue scale (VAS). The primary outcome was assessed based on the area under the curve (AUC). Results The CS group had a significantly lower AUC postoperatively at 0 to 24 hours compared to the no-CS group (AUC of VAS score at rest 550 ± 362 vs 392 ± 320, respectively; mean difference 158 mm; 95% confidence interval (CI) 58 to 257; p = 0.0021). In point-by-point evaluation, the CS group had significantly lower VAS scores at 12, 15, 18, 21, 24, and 48 hours. There were no significant differences in complication rates, including surgical site infection, between the two groups. Conclusion The addition of corticosteroid to periarticular injections reduces postoperative pain without increasing complication rate following THA. Cite this article: Bone Joint J 2020;102-B(10):1297–1302.

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.

The oriented immobilization of sensing molecules (e.g., IgGs, receptors, lectins, and DNA aptamers) on sensor chips is particularly important for maximizing the sensitivity and target-binding capacity of biosensors.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys ≅ GSH NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added.

In this study, we found that hydrogen peroxide was generated during the separate incubation of four catechins, (-)-epicatechin, (-)-epigallocatechin, and their galloylated forms, in the absence or presence of mushroom tyrosinase (EC 1.14.18.1). In particular, autooxidation of (-)-epigallocatechin and enzymatic oxidation of (-)-epicatechin gallate contributed to the increase of hydrogen peroxide. We confirmed the hydrogen peroxide-induced inactivation of tyrosinase and found that hydrogen peroxide was also generated during the tyrosinase-catalyzed synthesis of theaflavins by selectively combining two types of catechins, diphenol- and pyrogallol-types. Elimination of hydrogen peroxide by co-incubation with bovine catalase (EC 1.11.1.6) increased the products in synthetic reactions for TF1, TF2A, TF2B, and TF3 by 15%, 16%, 45%, and 18%, respectively. Based on the prospective mechanism of the tyrosinase-catalyzed synthesis of theaflavins, the yield of TF2B increased by elevating the initial ratio of (-)-epigallocatechin/(-)-epicatechin gallate in the reaction media. Furthermore, under such conditions, there were clear ameliorative effects of catalase on the tyrosinase-catalyzed synthesis of TF2B, reaching 3.1-fold increase of the product compared to the reaction without catalase. These results support a novel strategy to use both tyrosinase and catalase for the efficient synthesis of theaflavins from catechins.

Green tea is well known for its various physiological effects, but there are few studies relating the tastes of tea catechins to their biological activities. We have focused on the interaction of tea catechins with phospholipids to clarify the relationship. The galloyl-type catechins, epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) usually show beneficial properties including chemopreventive, antimutagenic and antioxidant actions more potently than the nongalloyl-type catechins, epicatechin (EC) and epigallocatechin (EGC). The difference in their chemical structures is correlated with their affinity for phospholipid membranes and incorporated amounts into liposomes. This indicates that the interaction of the galloyl-type catechins with phospholipids is always stronger than the nongalloyl-type catechins. Molecular-level insights into how tea catechins interact with lipid membranes were acquired using NMR spectroscopy. Based on solution and solid-state NMR studies with isotropic bicelles and liposomes as models of phospholipid membranes, it is proposed that ECg and EGCg interact with the surface of lipid membranes via the choline moiety of the phospholipids. Since the galloyl-type catechins have been reported to be more bitter and astringent than the nongalloyl-type catechins, both of their tastes and biological activities could be ascribed to the interaction.

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.

This study kinetically characterized the mechanism of the enzymatic synthesis of theaflavins (TFs) from catechins by mushroom tyrosinase (EC 1.14.18.1). In reactions containing one of four catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and their galloylated forms (ECg and EGCg), they were oxidized by tyrosinase with apparent KM values of 3.78, 5.55, 0.80, and 3.05 mM, respectively, and with different consumption rates, of which EC was more than four times higher than those of the others. In reactions with binary combinations of catechins with tyrosinase, the synthesis of TF1 from EC and EGC occurred most efficiently, while the yields of mono- and di-galloylated TFs, TF2A, TF2B, and TF3, were low. Time-dependent changes in concentrations of the reactants suggested that the enzymatic oxidation of catechins and the subsequent non-enzymatic coupling redox reaction between the quinone derived from EC or ECg and the intact pyrogallol-type catechin (EGC or EGCg) proceeded concurrently. The latter reaction induced the rapid decrease of EGC and EGCg and it was remarkable for EGCg. So the efficiency of condensation of a pair of quinones from catechol- and pyrogallol-type catechins is restricted, critically influencing the yield of TFs. Using green tea extracts as mixtures of the four substrate catechins, tyrosinase produced TF1 most abundantly. Furthermore, a remarkable enhancement of production of TF2A and TF2B as well as TF1 was observed, when the initial concentration of EGCg was low. These results suggest that the catechin composition has an impact on yields of 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.

Background Polyphenols in tea are considered beneficial to human health. However, many such claims of their bioactivity still require in vitro and in vivo evidence. Results Using Drosophila melanogaster as a model multicellular organism, we assess the fat accumulation-suppressing effects of theaflavin (TF), a tea polyphenol; epitheaflagallin (ETG), which has an unknown function; and epigallocatechin gallate (EGCg), a prominent component of green tea. Dietary TF reduced the malondialdehyde accumulation related to a high-fat diet in adult flies. Other physiological and genetic responses induced by the high-fat diet, such as lipid accumulation in the fat body and expression of lipid metabolism-related genes, were ameliorated by the addition of TF, ETG, and EGCg, in some cases approaching respective levels without high-fat diet exposure. Continuous ingestion of the three polyphenols resulted in a shortened lifespan. Conclusion We provide evidence in Drosophila that tea polyphenols have a fat accumulation-suppressing effect that has received recent attention. We also suggest that tea polyphenols can provide different desirable biological activities depending on their composition and the presence or absence of other chemical components.

Background Periarticular analgesic injection (PAI) is being used more commonly for pain relief after orthopaedic surgeries. However, there is conflicting evidence regarding the effectiveness of PAI for post-THA pain relief. Questions/purposes In a double-blind, randomized, controlled trial among patients undergoing same-day bilateral THA, with each patient serving as his or her own control, we asked: (1) Did the pain score as measured on a 100-mm VAS differ between the hips that received PAI versus placebo? (2) Were there differences in complications between the treatment and control hips in these patients? Methods Over a 1-year period at one center, 45 patients underwent same-day bilateral THA; three were excluded for prespecified reasons, and two declined participation in this randomized, controlled trial, leaving 40 patients (80 THAs) in the study. Patients randomly received PAI in one hip and placebo in the contralateral hip; patients, surgeons, and nurses were blinded in terms of which hip received the PAI and which hip received a placebo saline injection. The PAI solution included ropivacaine, morphine hydrochloride hydrate, methylprednisolone, ketoprofen, and epinephrine. The primary outcome was the VAS for pain at rest 24 hours after THA, measured using a 100-mm horizontal VAS. The VAS score was compared between two groups and assessed to reach the reported threshold values for the minimum clinically important difference (MCID) of 20 mm for the postoperative VAS score. No patients were lost to followup, and there were no missing data for the primary outcome. Complications that occurred during the trial were recorded prospectively with emphasis on infection, wound complications, nerve palsy and allergic reactions to the injections. Results There were no clinically important differences between hips treated with the PAI and those treated with the placebo injection at any point. The hips that received PAI had less pain than those receiving placebo 24 hours after THA (16 ± 17 mm versus 22 ± 20 mm; mean difference, 6 mm; 95% confidence interval [CI], 2-9 mm; p = 0.006), but this effect size was below the MCID of 20 mm and thus is unlikely to be clinically important. The hips that received PAI also had better VAS scores in the recovery room (38 ± 29 mm versus 52 ± 33 mm; mean difference 14 mm; 95% CI, 5-23 mm; p = 0.004) and 3 hours after THA than placebo controls (28 ± 22 mm versus 37 ± 24 mm; mean difference 9 mm; 95% CI, 2-16 mm; p = 0.010). Neither of these differences exceeded the MCID and likewise were unlikely to be clinically important. No complications, including surgical site infections, were observed in either group. Conclusions Periarticular analgesic injection for pain control after THA did not result in a clinically important reduction in pain at any point examined. Given the expense associated with this PAI mixture and the lack of effectiveness outside this timeframe, we cannot recommend its use. Other mixtures or concentrations of drugs may be helpful in short-stay admissions for THA, but this will require further research. Level of evidence Level I, therapeutic study.

This study aimed to develop a novel method with tea extracts and its components, to reduce the risk of foodborne illnesses caused by the bacterial toxin staphylococcal enterotoxin A (SEA). The potential effect of tea extracts, theaflavins, and epitheaflagallin on staphylococcal growth was studied. A broth microdilution method was used to determine the minimum inhibitory concentration of these samples against an SEA-producing strain, Staphylococcus aureus C-29. The following assays were performed to evaluate various effects on concentrations of no effect on staphylococcal growth. The interactions of theaflavin-rich green tea extracts (TGE), theaflavins, and epitheaflagallin to cultured S. aureus C-29 were determined using Western blot analysis. As a result, all samples suppressed the binding affinity of the anti-SEA antibody to SEA. Since these samples could react directly with SEA, we examined whether they could bind to SEA. Our results demonstrated that binding of the anti-SEA antibody to 4 theaflavins-treated SEA was inhibited in a dose-dependent manner. On the other hand, the production of SEA was significantly decreased by treatment with TGE and epitheaflagallin. Based on the finding that TGE and epitheaflagallin inhibit the production of SEA, we further examined the relative expression levels of sea toxin-encoding genes after treatment with TGE and epitheaflagallin with real-time RT-PCR. TGE and epitheaflagallin significantly supressed the gene transcription of SEA in S. aureus C-29. We then tested whether the samples block the biological activity of SEA in murine spleen cells. TGE, theaflavins, and epitheaflagallin became inactivated the biological activity of SEA. These results suggest that edible and safe compounds in tea can be used to inactivate both pathogens and toxins.

Numerous studies suggest that the ingestion of polyphenols can potentially confer haemodynamic benefits. The association between different polyphenolic chemical structures and their physiologic effects on systemic haemodynamic variables and microcirculation are less well known. This study investigated the effects of a single 10 mg/kg body weight dose of different polyphenolic compounds on haemodynamic and microcirculatory parameters. Key results from laser Doppler flowmetry showed that flavan-3-ols, theaflavins, and quercetin significantly increased cremaster artery blood flow. Flavan 3-ols, theaflavins, (−)-epicatchin, daidzein, and cyanidin significantly elevated mean blood pressure, and flavan-3-ols significantly increased the heart rate. In contrast, hesperidin, epigallocatechin gallate, trans-resveratrol, gnetin C or curcumin did not result in any detectable changes. Our results suggest that polyphenols can contribute variably to alterations in the haemodynamic and microcirculatory status in rats depending on their characteristic chemical structures.

We have established a novel method to evaluate the redox properties of tea polyphenols by HPLC-coulometric-array analysis. We plotted the quantity of electricity (µC) on the vertical axis and the electric potential (mV), adjusted with the associated palladium reference electrode, on the horizontal axis to provide "quantity versus potential (QP) plot". The patterns of the plots correspond to the derivative of a hydrodynamic voltammogram or a current-voltage curve, with the electric potentials of the peaks in the QP plot corresponding to the half-wave potentials in the current-voltage curve. We confirmed that catechins and theaflavins are oxidized depending on the electric potentials of their partial structures, and found that all compounds showing a peak at 0 mV in the QP plots produce hydrogen peroxide (H2O2) during the autoxidation process.

The mechanisms underlying the effect of epigallocatechin gallate (EGCG) on the micellar solubility of cholesterol were examined. EGCG eliminated both cholesterol and phosphatidylcholine (PC) from bile salt micelles in a dose-dependent manner in vitro. When the bile salt micelles contained a phospholipid other than PC, neither cholesterol nor the phospholipid was eliminated following the addition of EGCG. When vesicles comprised of various phospholipids were prepared and, EGCG was added to the vesicles, EGCG effectively and exclusively eliminated only PC. An intermolecular nuclear Overhauser effect (NOE) was observed between PC and EGCG in bile salt micelles with EGCG added, but not between cholesterol and EGCG, by using a NOE-correlated spectroscopy nuclear magnetic resonance method. The results of binding analyses using surface plasmon resonance (SPR) showed that EGCG did not bind to cholesterol. These observations strongly suggest that EGCG decreases the micellar solubility of cholesterol via specific interaction with PC.

In this study, we investigated the memory task performance and the central nervous activity after smelling two kinds of pan-fired Japanese green tea to examine the differences of their physical and psychological effects. Twenty eight subjects (14 male and 14 female) participated in this study. We used Koushun and Kouju for test samples, which were made by different manufacturing processes. After smelling each odor sample, a memory task and an arithmetic task were used to test mental stress. Electroencephalogram (EEG) was recorded before and after smelling the test samples, and EEG activity was estimated for 4 frequency bands (alpha 1, alpha 2, beta 1 and beta 2). The profiles of mood states (POMS) and the visual analog scale (VAS) after mental stress task were completed for subjective assessments. The results showed that the odor of Kouju may induce a positive emotion such as a more familiar and a less hate feeling. It may also affect the EEG band power of beta 1 at right frontal region and improve memory task performance as well.

The present study was performed to investigate the immune-modulating activities of extracts from green soybean (Glycine max) in a 2,4-toluene diisocyanate (TDI)-inducing guinea pig rhinitis model and a human trial study for allergic rhinitis. Hot water extracts of green soybean were chosen for animal experimentation on the basis of their ability to regulate the production of B cell-activating factor of the TNF family and a proliferation-inducing ligand in mouse spleen cells. Green soybean extracts significantly decreased the levels of ovalubumin (OVA)-specific IgE in mice and significantly suppressed the TDI-induced nasal mucosa secretion. An open-label human pilot study was performed on 16 subjects, using Japanese cedar pollinosis. The symptom scores for Japanese cedar pollinosis were better in the long-term green soybean extracts intake group than in the withdrawal short-term intake group. Green soybean extracts had great potential as an orally active immune modulator for the treatment of various allergic diseases.

Islet amyloid polypeptide (IAPP) is a major component of pancreatic amyloid deposits associated with type 2 diabetes. Polyphenols contained in plant foods have been found to inhibit amyloid fibril formation of proteins and/or peptides. However, the inhibition mechanism is not clear for a variety of systems. Here the inhibition mechanism of green tea polyphenols, catechins, on amyloid fibril formation of the IAPP fragment (IAPP22-27), which is of sufficient length for formation of β-sheet-containing amyloid fibrils, was investigated by means of kinetic analysis. A quartz crystal microbalance (QCM) determined that the association constants of gallate-type catechins [epicatechin 3-gallate (ECg) and epigallocatechin 3-gallate] for binding to IAPP22-27 immobilized on the gold plate in QCM were 1 order of magnitude larger than those of the free IAPP22-27 peptide, and also those of epicatechin and epigallocatechin. Kinetic analysis using a two-step autocatalytic reaction mechanism revealed that ECg significantly reduced the rate constants of the first nucleation step of amyloid fibril formation, while the rate of autocatalytic growth was less retarded. (1)H nuclear magnetic resonance studies clarified that a IAPP22-27/ECg complex clearly forms as viewed from the (1)H chemical shift changes and line broadening. Our study suggests that tea catechins specifically inhibit the early stages of amyloid fibril formation to form amyloid nuclei by interacting with the unstructured peptide and that this inhibition mechanism is of great therapeutic value because stabilization of the native state could delay the pathogenesis of amyloid diseases and also the toxicity of the small oligomer (protofibril) is reported to be greater than that of the mature fibril.

Background. Although active ankle movement plays a predominant role in mechanical thromboprophylaxis following total hip arthroplasty (THA), the most effective frequency of movement remains unclear.Materials and Methods.In 29 consecutive patients undergoing THA, the velocity of blood flow in the profunda femoris was measured after various frequencies of ankle movement two days after THA using a pulse wave Doppler ultrasound system. To test the interobserver reliabilities for the velocity measured with Doppler ultrasound system, the intraclass correlation coefficient was calculated based on the measurement in 10 limbs of healthy volunteers.Results.At 0, 1, and 2 minutes after ankle movement, the velocity after movement at 60 contractions per minute was significantly faster than that after movement at 40 or 80 contractions per minute (p=0.0007, repeated-measures analysis of variance). The intraclass correlation coefficient score in two investigators was 0.849 (95% confidence interval, 0.428 to 0.962).Conclusions.Active ankle movement at 60 contractions per minute is recommended in patients receiving THA to obtain optimal venous blood flow.

Many biological activities of green tea have been attributed to a major constituent, (minus;)-epigallocatechin gallate (EGCG). We previously reported that EGCG and an EGCG-free fraction derived from green tea modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, in the mouse liver. EGCG is also known to affect the gene expression of enzymes related to lipid metabolism. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we prepared an EGCG-free water-soluble fraction (GT-W), and examined its effects on the hepatic gene expression of lipogenic enzymes in mice. The results of quantitative real-time PCR assays indicated that the dietary administration of GT-W for 4 weeks reduced the hepatic gene expression of lipogenic enzymes: fatty acid synthase, hydroxymethylglutaryl coenzyme A reductase, and acetyl-coenzyme A carboxylase alpha. Also, the gene expression of sterol regulatory element-binding transcription factor (Srebf)1 and/or Srebf2 was reduced, suggesting that the reduction of Srebfs contributed to the down-regulation of the lipogenic enzymes, since these transcription factors bind the promoter region to enhance their expression. The plasma levels of triglycerides and cholesterol were reduced with statistical significance in the group given a diet containing GT-W. These results suggest that in addition to EGCG, green tea contains some component(s) which may help to prevent arteriosclerosis and obesity.

(−)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.

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.

We recently reported that propolis suppresses tumor-induced angiogenesis through tube formation inhibition and apoptosis induction in endothelial cells. However, molecular mechanisms underlying such angiogenesis suppression by propolis have not been fully elucidated. The aim of this study was to investigate the effects of ethanol extract of Brazilian propolis (EEBP) on two major survival signals, extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt, and to elucidate whether changes in these signals were actually involved in antiangiogenic effects of the propolis. Detection by western blotting revealed that EEBP suppressed phosphorylation of ERK1/2, but not that of Akt. Pharmacological inhibition by U0126 demonstrated that ERK1/2 inactivation alone was enough to inhibit tube formation and induce apoptosis. It was also shown that EEBP and U0126 similarly induced activation of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) and lamin A/C, all of which are molecular markers of apoptosis. These results indicate that inhibition of survival signal ERK1/2, and subsequent induction of apoptosis, is a critical mechanism of angiogenesis suppression by EEBP.

Immunoglobulin E (IgE) plays an important role in allergic diseases. In this study, we found that am ethanol extract of leaves of the camphor tree Cinnamomum camphora (C. camphora )r educed the amount of IgE secreted by human myeloma U266 cells. When the methanol extract was fractionated by extraction with organic solvents, the ethyl acetate fraction showed the highest activity. The fraction was further separated into several subfractions by preparative TLC. We identified the component of one of the active subfractions as dimethylmatairesinol. Authentic dimethylmatairesinol exhibited similar activity. Thus, the extract of C. camphora and its components including dimethylmatairesinol have potential as an anti-allergic agent.

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.

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.

Alpha-lipoic acid (LA) acts as a direct regulator of intracellular redox status through the formation of mixed disulphides. However, as this is reversible, the evidence for the mixed disulphides has not been obtained. This study established a method for the detection and characterization of mixed disulphides by mass spectrometry (MS) and was the first to provide direct evidence for their formation. When cysteine methyl ester was incubated with LA in the presence of iodeacetamide (IAA), a mixed disulphide with mono carbamidomethylation was observed. MS/MS analysis indicated that the LA forms a mixed disulphide with the cysteinyl sulphydryls, while the other sulphydryl group is the carbamidomethylated. The same results were obtained from the incubation of sulfphydryl peptides such as glutathione with LA in the presence of IAA. These results may provide further biological evidence that LA is a potential modifier of intracellular sulphydryls through mixed disulfide formation.

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.

Catechins are the principle polyphenolic compounds in green tea; the four major compounds identified are epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg). Tea catechins tend to attach externally to their targets, such as viral envelopes, cell membranes, or the surface of low-density lipoproteins. In order to further our understanding of the molecular mobility of these compounds in cells, we examined the interaction of tea catechins with lipid membranes using solid-state NMR techniques. Our previous work indicated that the EGCg molecule is incorporated into lipid bilayers in a unique orientation. However, the detailed configuration, orientation, and dynamics of EGCg in lipid bilayers have not been well-characterized. Here, we investigated the orientation and dynamics of EGCg incorporated into multi-lamellar vesicles (MLVs) and bicelles using solid-state NMR spectroscopy.