Japanese Green Tea Research Articles

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.

Comparative Performance of Hens Fed Diets Containing Korean, Japanese and Chinese Green Tea

This experiment was carried out to investigate the effects of Korean, Japanese and Chinese green tea on laying performance and egg quality in hens. A total of 168 Tetran Brown hens aged 40 weeks were assigned to 7 treatments in a completely randomized design. Each treatment had 4 replicates accommodating 6 layers per replication. The seven dietary treatments were: 1) control diet with no green tea added, 2) diet containing 1.0% Korean green tea (1.0% KGT), 3) diet containing 2.0% Korean green tea (2.0% KGT), 4) diet containing 1.0% Japanese green tea (1.0% JGT), 5) diet containing 2.0% Japanese green tea (2.0% JGT), 6) diet containing 1.0% Chinese green tea (1.0% CGT), and 7) diet containing 2.0% Chinese green tea (2.0% CGT). Egg production rate of the layers fed diets containing 1.0 or 2.0% green tea powders were significantly increased compared to that of the control (p 0.05). Green tea feeding to layers tended to reduce the overall cholesterol content of egg yolk. Particularly, 1.0 or 2.0% CGT significantly depressed the total cholesterol content of egg yolk (p<0.05). In conclusion, incorporation of 1.0 or 2.0% Korean, Japanese and Chinese green tea into layer diets regardless of origin had favorable effects on laying performance and egg quality profiles. Among the three green tea sources, the Chinese green tea powder had the highest reducing effect on cholesterol content in egg yolk. (Key Words : Green Tea, Hen, Egg Production, Feed Intake, Egg Shell Thickness, Yolk Cholesterol)

Molecular and Sensory Studies on the Umami Taste of Japanese Green Tea

Aimed at defining the key drivers for the quality-determining umami taste of a high-grade powdered green tea, called mat-cha, a bioactivity-guided fractionation using solvent extraction, solvent precipitation, preparative chromatographic separations, and human psychophysical experiments was applied on freshly prepared mat-cha. Liquid chromatography-tandem mass spectrometry and one-/two-dimensional nuclear magnetic resonance studies on isolated fractions led to the identification of l-theanine, succinic acid, 3,4,5-trihydroxybenzoic acid (gallic acid), and (1R,2R,3R,5S)-5-carboxy-2,3,5-trihydroxycyclohexyl-3,4,5-trihydroxybenzoate (theogallin) as umami-enhancing compounds in the green tea beverage, and it can be shown by sensory studies that these compounds are able to raise the umami intensity of sodium l-glutamate proportionally.

照射緑茶の電子スピン共鳴法による検知

With electron spin resonance (ESR) spectroscopy, we revealed various radicals in commercially available Japanese green tea before and after γ-irradiation. The representative ESR spectrum of green tea is composed of sextet centered at g=2.0, a singlet at the same g value and a singlet at g=4.0. The first signal is attributable to a signal with hyperfine interaction of the Mn2+. The second signal is due to an organic free radical. The third signal may originate from the Fe3+. Upon irradiation, a pair of signals was newly appeared at the symmetric positions on both sides of the organic free radical signal. According to the theory of radical production in irradiated solid, a linear relationship is expected between the radiation dose and the radical yield. We determined the unreported irradiation dose by the linear extrapolation.

Japanese Green Tea Export and International Marketing

Japanese Green Tea Export and International Marketing

Protective effect of Japanese green tea against cognitive impairment in the elderly, a two-years follow-up observation

target selective, glial neuroinflammatory responses that are linked to hippocampal neuronal dysfunction, neuronal loss, and spatial learning deficits [2,3,4], and provided a proof of concept that targeting the neuroinflammatory cycle could attenuate disease progression related endpoints. Objective: To take these exploratory basic science results toward development of safe and effective, AD-relevant lead compounds with oral bioavailability, by chemical diversification of the privileged pyridazine core fragment. Methods: Compounds were synthesized and taken through our hierarchical biological screening process involving a) cell-based screening for concentration-dependent and selective inhibition of glia inflammation responses, and b) in vivo assays using mouse models of A -induced neuroinflammation, synaptic dysfunction and neuronal death, in order to discover compounds with in vivo anti-neuroinflammatory activity. An oral toxicological screen was also incorporated into the discovery process [4] by using a multi-day, escalating dose standard protocol. Results and Conclusions: We report here a proof of concept that our approach can produce safe and effective compounds that are orally bioavailable in an animal model of AD-related disease progression. These compounds suppress production of neuroinflammatory cytokines and other biomarkers indicative of A -induced neuroinflammation, and protect against neuronal dysfunction in the hippocampus. These first generation experimental therapeutics are amenable to further medicinal chemistry refinement in future drug development. Current research is focused on medicinal chemistry refinement and safety evaluations as a foundation for phase I clinical investigations. [1] J Med Chem 45: 563-566. [2] Neurobiol. Aging 25: 1283-1292. [3] J Mol Neurosci 24: 115-122. [4] Curr Alzheimer’s Res, in press. (Support: ISOA, Alzheimer’s Association, PhRMA, and NIH grants AG13939, AG21184, NS47586; AG00260, NS46942.

Influence of Manufacturing Conditions and Crop Season on the Formation of 4-Mercapto-4-methyl-2-pentanone in Japanese Green Tea (Sen-cha)

4-Mercapto-4-methyl-2-pentanone is one of the most strongly contributing odorants in the volatile fraction of a Japanese green tea (sen-cha) infusion, and on the basis of the results of an aroma extract dilution analysis, the contribution of this compound to the flavor of the sen-cha infusion varied according to the degree of heating of the tea leaves during the roasting process. The concentration of this odorant in the sen-cha infusion, as with other roasty odorants, increased with the increasing roasting temperature. However, the slope of the increase curve differed with the odor compound, and even if roasting was done at a low temperature, at which the other roasty odorants hardly increased, 4-mercapto-4-methyl-2-pentanone still increased and reached a maximum at 112 degrees C. On the other hand, the amount of 4-mercapto-4-methyl-2-pentanone in sen-cha was a maximum in the first crop, then decreasing in the order of the second and third crops. These results suggested that the amount of 4-mercapto-4-methyl-2-pentanone was closely involved with the quality of sen-cha and that the concentration was dependent on the roasting conditions for the green tea leaves, which might be accompanied by an enzymatic reaction.

Composition of pyrolyzate from Japanese green tea

Green tea is a beverage that is produced by hot-water extraction of the leaves of tea (Camellia sinensis L.). As the amount of green tea consumption has increased, the importance of using the residue has grown. To address this problem, chemical compositions of two pyrolyzates obtained with an electric kiln, L and G, which were obtained from the tea leaf product and its hot-water extraction residue, respectively, were examined. Both pyrolyzates were alkaline, in contrast to the acidic pHs that are typical of pyrolyzates from wood materials. The major constituents were nitrogen-containing compounds, which accounted for up to about 70% (based on gas chromatography-mass spectrometry peak area). Caffeine was the main compound in both samples, although it might not be a pyrolysis product but actually sublimates from the samples and then condenses back into the pyrolyzates. Other nitrogen-containing compounds were formed mainly from proteins and amino acids. Acids and phenols, which are typical components in pyrolyzates obtained from wood materials and contribute to acidic pH, were rare in both samples. Other neutral compounds are known as compounds in pyrolyzates from wood materials. The pyrolyzates obtained in this experiment were novel and may have new uses.

Paradoxical Effects of Green Tea (Camellia Sinensis) and Antioxidant Vitamins in Diabetic Rats

We tested the hypothesis that green tea prevents diabetes-related tissue dysfunctions attributable to oxidation. Diabetic rats were treated daily with tap water, vitamins C and E, or fresh Japanese green tea extract. After 12 months, body weights were decreased, whereas glycated lysine in aorta, tendon, and plasma were increased by diabetes (P < 0.001) but unaffected by treatment. Erythrocyte glutathione and plasma hydroperoxides were improved by the vitamins (P < 0.05) and green tea (P < 0.001). Retinal superoxide production, acellular capillaries, and pericyte ghosts were increased by diabetes (P < 0.001) and improved by green tea and the vitamins (P variable). Lens crystallin fluorescence at 370/440 nm was ameliorated by green tea (P < 0.05) but not the vitamins. Marginal effects on nephropathy parameters were noted. However, suppressed renal mitochondrial NADH-linked ADP-dependent and dinitrophenol-dependent respiration and complex III activity were improved by green tea (P variable). Green tea also suppressed the methylglyoxal hydroimidazolone immunostaining of a 28-kDa mitochondrial protein. Surprising, glycoxidation in tendon, aorta, and plasma was either worsened or not significantly improved by the vitamins and green tea. Glucosepane cross-links were increased by diabetes (P < 0.001), and green tea worsened total cross-linking. In conclusion, green tea and antioxidant vitamins improved several diabetes-related cellular dysfunctions but worsened matrix glycoxidation in selected tissues, suggesting that antioxidant treatment tilts the balance from oxidative to carbonyl stress in the extracellular compartment.

Cultivar Identification and Analysis of the Blended Ratio of Green Tea Production on the Market Using DNA Markers

Japanese green teas are produced by blending different materials and there are cases of discrepancy between the contents and the label of the product. Therefore, a technique for identifying tea cultivars among product contents is strongly required. A simple CTAB (Cetyl trimethyl ammonium bromide) extraction method with a short time pre-incubation yielded DNA is suitable for cultivar identification by CAPS (cleaved amplified polymorphic sequence) analysis. The extracted DNA was analyzed with seven CAPS markers to identify cultivars.

Identification of Volatile Compounds which Enhance Odor Notes in Japanese Green Tea using the OASIS (Original Aroma Simultaneously Input to the Sniffing port) method

We evaluated the volatile compounds of Japanese green tea using GC/O with the original aroma simultaneously input to the sniffing port (OASIS) method in order to determine the relationship between the volatile compounds and the green tea aroma. Application of OASIS to the volatile fractions of Japanese green tea infusions revealed 11 odor-active peaks. Those of cis-1,5-Octadien-3-one, 4-mercapto-4-methyl-2-pentanone, 3-methylnonane-2,4-dione, geraniol, decanal and cis-3-hexenol emphasized the green note of the tea. 2-Acetylpyrrole, 2-ethyl-3,5-dimethylpyrazine and 2-acetyl-3,5-dimethylpyrazine emphasized the roast note. Indole strengthened the overall green tea odor. β-Ionone emphasized the sweet note in the green tea. Of these odorants, 2-acetyl-3,5-dimethylpyrazine was identified for the first time as a volatile compound in tea.

Separation of Flavonoids by Semi-Micro High-Performance Liquid Chromatography with Electrochemical Detection

Abstract A highly sensitive and selective method was developed for the determination of 15 flavonoids by high-performance liquid chromatography with electrochemical detection (HPLC–ECD) using a microbore column. The 15 flavonoids were divided into two groups according to their hydrophobicity and were resolved by two isocratic systems, namely, methanol–water (1:1 and 3:7, v/v) containing 0.5% phosphoric acid. The retention factor (k) of each flavonoid linearly correlated with the logarithmic partition of coefficients between 1-octanol–water (log P) values. The detection limits (S/N = 3) of the flavonoids tested were in the range of 2–25 fmol; that is 600 times more sensitive than conventional HPLC with UV detection. The relative standard deviations (RSD, n = 5) were less than 5% at 1 pmol for the flavonoids tested. By the present method, only 100 μL of bottled Japanese green tea were enough to determine the flavonoid contents.

PCR‐based amplicon length polymorphisms (ALPs) at microsatellite loci and indels from non‐coding DNA regions of cloned genes as a means of authenticating commercial Japanese green teas

AbstractWe have developed PCR‐based DNA markers with the objective of authenticating, and discouraging the fraudulent commercialization of, the forty‐four most cultivated Japanese green teas. For this purpose we have generated amplicon length polymorphisms (ALPs) by targeting microsatellite loci using nucleotide information from other species and indels located in the non‐coding regions of phenylalanine ammonia‐lyase (PAL) and dihydroflavonol 4‐reductase (DFR) genes in tea. One useful indel was detected in the unique intron of PAL and three others in introns 2, 3 and 5 of the DFR gene. All four nuclear microsatellite loci were polymorphic showing three to four alleles per locus, while only one out of the four chloroplast loci was variable with two distinct haplotypes. PCR primers were designed to amplify small but variable PCR fragments in such a way that they were applicable to low amounts of highly degraded DNA. The markers devised here were sufficient to differentiate all the teas, although a combination of several loci was necessary for most of them. This single‐step PCR‐based methodology is more effective than the PCR‐RFLP technique commonly used for identifying food materials and will be useful for the certification of commercial Japanese green teas. Copyright © 2004 Society of Chemical Industry

Modulation of β-lactam resistance in Staphylococcus aureus by catechins and gallates

Aqueous extracts of Japanese green tea ( Camellia sinensis) are able to reverse β-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). We have attributed the capacity to reverse oxacillin resistance in the homogeneous PBP2a producer BB568 and in EMRSA-16 to (−)-epicatechin gallate (ECG) and (−)-catechin gallate (CG). Minimum inhibitory concentration (MIC) values for oxacillin were reduced from 256 and 512 to 1–4 mg/l, respectively, in the presence of these polyphenols. In addition, (−)-epigallocatechin gallate (EGCG) had a moderate capacity to modulate oxacillin activity against S. aureus BB568, but none against EMRSA-16. ECG, CG and EGCG increased the sensitivity of EMRSA-15 to oxacillin. The gallate moiety was essential for the oxacillin-modulating activity of ECG, as both (−)-epicatechin and (−)-epicatechin-3-cyclohexylcarboxylate were unable to reverse resistance to oxacillin. Gallic acid and three alkyl gallates (methyl gallate, propyl gallate, and octyl gallate) did not modulate β-lactam resistance in MRSA. Octyl gallate exhibited direct antibacterial activity against S. aureus BB568 (16 mg/l). Modulation of β-lactam resistance by ECG significantly enhanced the activities of flucloxacillin and the carbapenem antibiotics imipenem and meropenem against 40 MRSA isolates, with MIC 90 values for the antibiotics reduced to the susceptibility breakpoint or below. Consequently, EGCG, CG and, particularly, ECG warrant further investigation as agents to combat β-lactam resistance in S. aureus.

日本と欧米におけるアレルギー制御の試み

. Pollinosis by Japanese Cedar in spring has become national phenomenon. Steroid treatment in atopic dermatitis and bronchial asthma is essential, but causes side effects when used improperly. Sophisticated research in allergy will going to develop a new therapy using drugs such as humanized anti-IL5. anti-IgE or DNA-based immunotherapy. Clinical Trials of anti-IL5 remain controversial but the trials using anti-IgE or DNA-based immunotherapy are promising. Our projects involve the traditional and affordable resources such as simple preventive tips and Japanese green tea, and are going to extend clinical trials after preliminary experiments. Comparative reviews of allergy control between Japan and Western countries are introduced from the aspects of culture, food, climate and history.

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 m M phosphate buffer (pH 5.5) containing 0.05 m M sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.

Rooibos tea: equilibrium and extraction kinetics of aspalathin

A mobile phase for the extraction of aspalathin was developed and used to study the kinetics of extraction of the compound from Rooibos tea. The rate of infusion of aspalathin into water at 80 °C was measured for Rooibos tea, using a leaf of size 1.00–1.40 mm. The first order rate constant ( k obs=23.4×10 −4 s −1) was determined from the rate of increase of aspalathin concentration in a tea infusion with time and the result interpreted using a steady state model. The results were compared with those of rose-hip, black and green teas. The first order rate constant for extraction of aspalathin was found to be comparable with that of l-ascorbic acid in rose-hip tea. However, the value was about five times smaller than that of flavanols from Japanese green tea and 3–10 times smaller than the extraction of caffeine from black Assam Bukial and green Chun Mee teas. The pH of the liquor at equilibrium was found to be 4.6. This value is comparable to that of both black and rose-hip tea but 20% smaller than that of green Chun Mee tea.

Measuring Astringency of Beverages Using a Quartz-Crystal Microbalance

Two methods for measuring astringency were developed using a lipid-coated and gelatin-immobilized quartz-crystal microbalance (QCM). It was found that the level of adsorption of astringent tannins onto the lipid membrane significantly increased in the presence of peptides, while NaCl, tartaric acid, quinine-sulfite, sucrose, and glutamic acid did not show the same level of adsorption. The lipid-coated QCM specifically responded to astringent tannins in the presence of peptides, and the gelatin-immobilized QCM specifically responded to polyphenols. The responses of these sensors to red wines, Japanese green teas, and beers successfully coincided with the astringent intensities in the sensory evaluation.

Inhibitory effects of green tea catechins on protein tyrosine phosphatase in Prevotella intermedia.

Members of the Prevotella intermedia group possess protein tyrosine phosphatase (PTPase). The purpose of this study was to investigate the effects of catechin derivatives from Japanese green tea on the activity of PTPase in P. intermedia and related organisms. Multilocus enzyme electrophoresis of alkaline phosphatase derived from P. intermedia, Prevotella nigrescens, Prevotella pallens and Porphyromonas gingivalis revealed a species-specific migration pattern. Among the tea catechin derivatives tested, (-)-epigallocatechin gallate (EGCg), similar to orthovanadate, a specific inhibitor for PTPase, was effective in inhibiting the PTPase activity in P. intermedia at 0.5 microm, and related species at 5 microm. The results suggested that the inhibitory effect observed is due to the presence of galloyl moiety in the structure. In contrast, neither the green tea catechins nor orthovanadate inhibited the phosphatase activity in P. gingivalis, suggesting that this organism possessed a different family of alkaline phosphatase.

Identification of processed Japanese green tea based on polymorphisms generated by STS-RFLP analysis.

In Japan, tea is generally sold blended, though 90% of the total production is clonal. Due to the increasingly strict consumer need and taste, however, more and more Japanese green teas are being sold under their particular cultivar name. Moreover, tea made from Yabukita, a much appreciated cultivar originally developed in Japan, has recently been produced and imported from a neighboring country. This paper describes a simple and inexpensive methodology capable of identifying fresh and processed Japanese green teas to discourage its fraudulent commercialization. The study was based on 46 main tea cultivars, and polymorphism detected through STS-RFLP analysis of the coding and noncoding DNA regions of three genes, namely phenylalanine ammonia-lyase, chalcone synthase, and dihydroflavonol 4-reductase, for which nucleotide information was available. All 46 tea cultivars analyzed could be easily distinguished using a combination of codominant DNA markers. Yabukita displayed a unique profile when PAL intron was digested with DdeI, thus allowing its rapid authentication at low cost.