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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.

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.

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.

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.

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.

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.

Citrus essential oils, including lemon essential oil, have long been used widely in aromatherapy and alternative medicine. This study was designed to assess the effects of flavour components in lemon essential oil on physical and psychological stress. In this evaluation, acute cold stress and communication box techniques were used to apply stress after intraperitoneal administration of essential oil components such as limonene, γ-terpinene and citral. Serum corticosterone and monoamines in brain tissues were then determined. In the present study, it found the presence of perillic acid, a limonene metabolite, at concentrations of 1.5–2.5 μg/mL in serum and 0.4–0.6 μg/g in brain tissue collected 3 h after administration. The research also showed that the lemon components R-limonene, citral and γ-terpinene inhibited elevation of serum corticosterone levels and cerebral monoamine levels. S-limonene, a stereoisomer of R-limonene, seemed to have stronger effect than other monoterpenes and inhibited brain monoamines elevation on psychological stress. These findings suggest that limonenes, and particularly S-limonene, have a potent stress-alleviating effect, and the possibility that different stereoisomers of limonene have different levels of activity in their effect on stress responses. These results suggest a possibility that ingestion of lemon essential oil containing components such as limonene and citral alleviates both physical and psychological stress. Copyright © 2007 John Wiley & Sons, Ltd.

Interaction between tea catechins, such as epicatechin gallate (ECg) and epigallocatechin gallate (EGCg), and isotropic bicelle model lipid membranes was investigated by solution NMR techniques. (1)H NMR measurements provided signals from the B-ring and the galloyl moiety in ECg and EGCg that were obviously shifted, and whose proton T1 relaxation times were shortened upon interaction of the catechins with the bicelles. These results indicate that the B-ring and the galloyl moiety play an important role in this interaction. Nuclear Overhauser effect spectrometry experiments demonstrated that the B-ring and the galloyl moiety are located near the gamma-H in the phospholipid trimethylammonium group. On the basis of these findings, we propose that ECg and EGCg interact with the surface of lipid membranes via the choline moiety.

Propolis is a resinous substance collected by honeybees from various plant sources. The composition of propolis depends on time, vegetation, and the area of collection. This study examined the antioxidant activity of propolis from various areas of China: Heilongjiang, Neimongol, Hebei, Shandong, Shanxi, Gansu, Henan, Hubei, Sichuan, Hunan, Yunnan and Hainan. Ethanol extracts of propolis (EEP) were prepared and evaluated for their antioxidant activities by β-carotene bleaching, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging, and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assays. Furthermore, the major constituents in EEP were identified by high-performance liquid chromatography (HPLC) analysis with a photodiode array (PDA) and mass spectrometric (MS) detection, and each component was quantitatively analyzed. All propolis samples except that from Yunnan had relatively strong antioxidant activity accompanied by high total polyphenol contents. Propolis with strong antioxidant activity contained large amounts of antioxidative compounds, such as caffeic acid, ferulic acid and caffeic acid phenethyl ester. On the other hand, propolis from Yunnan and Hainan had compounds not present in propolis from other areas.

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. The composition of propolis depends on time, vegetation, and the area of collection. This study examined the antioxidant activity of propolis from various areas of Korea: Chilgok, Cheongju, Geochang, Muju, Pocheon, and Sangju. Ethanol extracts of propolis (EEP) were prepared and evaluated for their antioxidant activity by beta-carotene bleaching, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assays. Furthermore, the major constituents in EEP were identified by high-performance liquid chromatography analysis with a photodiode array and mass spectrometric detection, and each component was quantitatively analyzed. EEP from Cheongju and Muju had relatively strong antioxidant activity accompanied by high total polyphenol contents. Propolis from Cheongju contained large amounts of antioxidative compounds, such as caffeic acid, kaempferol, and phenethyl caffeate. On the other hand, propolis from Pocheon had compounds not seen in propolis from other areas.

(+)-Catechin derivatives with different alkyl chain lengths were synthesized from (+)-catechin and various straight chain alkylaldehydes in the presence of methyl mercaptan, and their antibacterial activities against Gram-positive bacteria were evaluated. The antibacterial activity increased markedly with elongation of the alkyl chain lengths of the derivatives and reached a maximum at a chain of four to seven carbons. Subsequently, interaction of the (+)-catechin derivatives with a model membrane using liposome was investigated. The derivatives with a chain of three carbons or more were found to have very strong affinity for the membrane. The injury action of the derivatives against the membrane was examined with liposome in which calcein was enclosed as a fluorescent indicator. The leakage was observed in the derivatives with chain lengths of four carbons or more. Particularly the derivatives with chains longer than five carbons are considered to destroy the liposome membrane judging from the degree of the fluorescent leakage. These results implied that the lipophilicity and disrupting ability of the (+)-catechin derivatives to the liposome membrane participate in their antibacterial activity.

Propolis is a resinous substance collected by honeybees from various plant sources. The antioxidant activities of propolis of various geographic origins, i.e., Argentina, Australia, Brazil, Bulgaria, Chile, China (Hebei, Hubei, and Zhejiang), Hungary, New Zealand, South Africa, Thailand, Ukraine, Uruguay, United States, and Uzbekistan were compared. Ethanol extracts of propolis (EEP) were prepared and evaluated for antioxidant activities of EEP samples by the β-carotene bleaching and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay systems. Major constituents were identified in EEP by HPLC analysis with photo-diode array (PDA) and mass spectrometric (MS) detection, and quantitatively analyzed. EEP from Argentina, Australia, China, Hungary and New Zealand had relatively strong antioxidant activities, and were also correlated with the total polyphenol and flavonoid contents. Propolis with strong antioxidant activity contained antioxidative compounds such as kaempferol and phenethyl caffeate.

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.

Quercetin and its glucosides exist in plant foods and are recovered in human plasma as glucuronide and sulfate conjugates. Quercetin and its conjugates show antioxidant activity in model experiments. It remains obscure, however, whether these conjugates retain these biological functions in vivo. We investigated the interaction of quercetin conjugates with lipid bilayers using liposome systems. Less quercetin conjugate was incorporated into liposomes than quercetin aglycone. We also studied the vascular permeability of quercetin-3-glucuronide using cell culture inserts. Incubation of human aortic endothelial cells (HAECs) with IL-1alpha resulted in increased permeability of quercetin-3-glucuronide. Furthermore, quercetin-3-glucuronide showed no suppressive effect on TNF-alpha-induced cell expression of intercellular adhesion molecule-1 (ICAM-1) on HAECs. These observations suggest that circulating conjugates of quercetin pass through the endothelium to reach vascular smooth muscle cells and exert their biological effects in the blood vessels during inflammation followed by deconjugation of the conjugates.

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.

Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.

Two antioxidative compounds were isolated from the oil of sesame seeds roasted at 230°C for 15 min and identified as sesaminols (isomers) by MS and NMR analysis. Although sesaminols are present in refined unroasted sesame seed oil, we have demonstrated their presence also in the oil from the roasted sesame seed without refining for the first time. Furthermore, the amounts of sesaminols in sesame seeds were increased by roasting. Sesamol and melanoidin-like browning compounds have been believed to contribute to the antioxidant activity of roasted sesame seed oil, but the present study revealed that sesaminols also play an important role in the antioxidant activity of roasted sesame seed oil.

Three parameters, i.e., the proportion of the amount incorporated into the liposomes, the partition coefficient in a system of n-octanol/phosphate buffered saline, and the retention times by HPLC, were measured to determine the lipophilicity of isoflavonoids. The presence of a hydroxyl group at 5-position of the A-ring and a methoxyl group at 4'-position of the B-ring in the isoflavonoid structure increased the three parameters. The localization of isoflavonoids in lipid bilayers was investigated by a liposome system with fluorescent probes. The location of the isoflavonoid depended on its structure. The cytotoxicity of isoflavonoids was investigated by a colony-formation assay with Chinese hamster lung fibroblast V79 cells. The structure-activity relationship of the cytotoxic activity partly reflected those of the three parameters. This suggests that the biological activities of isoflavonoids in vitro could be attributable to their affinity for lipid components in the cases where the estrogen receptors have no role.

Eighteen flavonoids including two new compounds, four aromatic carboxylic acids, and eleven phenolic acid esters including one new compound were isolated and identified from the ethyl acetate soluble fraction of the 70% ethanol extract of Uruguayan propolis. The new compounds were elucidated as pinobanksin 3-(2-methyl)butyrate (1; recently reported in Usia, T.; Banskota, A. H.; Tezuka, Y.; Midorikawa, K.; Matsushige, K.; Kadota, S. J. Nat. Prod. 2002, 65, 673-676) pinobanksin 3-isobutyrate (2), and 2-methyl-2-butenyl ferulate (24). The constituents isolated from Uruguayan propolis in this study were similar to those of propolis of European and Chinese origin. Thus, it is suggested that the Uruguayan propolis has a plant origin similar to those of propolis from Europe and China.

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. 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.

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.

We established a simple method to quantify hydrogen peroxide (H2O2) generated during the aerobic heating process of (+)-catechin solution. Using this method, we found that non-enzymatic H2O2 formation from catechin depended on pH, temperature, incubation time, and the presence of O2 in the solution. The formation of oxidized products of (+)-catechin, which was estimated by measuring the absorbance of the solution at 430 nm, also depended on these factors. The H2O2 formation was inhibited by various kinds of superoxide dismutase (SOD) with almost the same dose dependency. Although the oxidation of (+)-catechin was enhanced by superoxide (O2−), neither catalase nor H2O2 had any effects on the oxidation. These results suggest that O2−, rather than H2O2, participated in the autoxidation of (+)-catechin, which was coupled with the H2O2 formation.

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.

Active oxygen species, e. g., hydrogen peroxide (H2O2), superoxide anion radical (O2-), hydroxyl radical (·OH) and lipid hydroperoxides, have been implicated as mediators of oxidative cell injuries in a variety of clinical conditions. Some metabolites of carcinogenic compounds, cigarette smoke and glucosone, an intermediate of the Maillard reaction, were found to produce H2O2 in their autoxidation processes, and to induce both cytotoxicity and DNA single-strand breaks in mammalian cells. Inhibition of these cell injuries by catalase indicated that H2O2 was the critical species. In contrast to the above prooxidant effects, antioxidant effects of food ingredients on H2O2-induced cytotoxicity were also investigated. Polyphenols, e. g., caffeic acid ester and flavonoids, were effective for suppression of H2O2-induced cytotoxicity on mammalian cells. A study of the structure-activity relationship revealed that the affinity of polyphenols for cell membranes and the presence of an ortho-dihydroxy moiety in their structure were essential for their protective action.

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.

The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

Antioxidative activity of pea bean (Phaseolus vulgaris L.) extract was evaluated by using a linoleic acid system, and the methanol extract exhibited strong antioxidative activity as measured by the thiocyanate method. The crude methanol extract was partitioned between then-butanol phase (BP) and the water phase (WP). Then, the antioxidative activity of the BP and the WP was determined by using a linoleic acid system. The WP showed strong antioxidative activity, while BP showed only weak activity as measured by the thiocyanate method. Next, the synergistic antioxidative action of WP with α-tocopherol was examined by using linoleic acid and liposome systems. The WP had a synergistic effect with α-tocopherol in both the food model and liposome systems. For purification and isolation of the antioxidative substances of the pea bean, preparative high-performance liquid chromatography was carried out with an octadecylsilyl column. Five fractions were collected, and antioxidative activity was determined in a linoleic acid system. Although fraction 1 had strong activity by the thiocyanate method, the purification of this active fraction was difficult; therefore, the partly characterized active fraction was investigated. The contents of total phenolics and sugars were 0.31±0.01 mg/g of fraction 1 and 406.1±0.1 mg/g, respectively. The ninhydrin chromogenic reaction was positive, and the ultraviolet absorption spectral λ max value in distilled water was 264.0 nm, indicating that the water-soluble antioxidative components from pea bean may be a new type of antioxidant. Isolation and identification are currently being investigated.

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.

Lipid peroxidation of liposome made of egg lecithin was induced by glucosone (D-arabino-hexos-2-ulose), a secondary product of Maillard reaction or glycation of protein. Lipid peroxidation was assessed with measurement of TBARS (thiobarbituric acid reacting substances), POV (peroxide value), and HPLC measurement of MDA (malondialde-hyde). EDTA and DTPA (diethylenetriamine-pentaacetic acid) in-hibited the lipid peroxidation assessed by each method described above, indicating involvement of metal ions. The observed reduction of Fe3+ to Fe2+ by glucosone might be a critical step of the lipid peroxidation. Our findings suggest a possible role of lipid peroxidation of low-density lipo-proteins (LDL) induced by glucosone in atherosis caused by diabetes mellitus.

Glucosone (D-arabino-hexos-2-ulose), a typical enediol product formed both in the Maillard reaction and gamma-radiolysis of sugars, decreased survival of Chinese hamster lung V79 cells, which were incubated under MEM for 4 h. Inhibition of the decrease in cell survival by catalase and SOD suggests the role of active oxygen species, namely H2O2 and O2-, in the biological effects of glucosone. H2O2 was formed in the medium during oxidative degradation of glucosone. Inhibition of the formation of H2O2 by SOD indicates that the formation of H2O2 and the consequent decrease of the cell survival was enhanced by O2-. These results suggest that the mechanisms of the effects of glucosone on the mammalian cells in the absence of Cu2+ are different from those in the presence of Cu2+.