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Analysis of derivatized and underivatized theanine enantiomers by high-performance liquid chromatography/atmospheric pressure ionization-mass spectrometry.
Rapid Commun Mass Spectrom. 2004;18(3):251-6.
Desai MJ, Armstrong DW.
Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Theanine, a naturally occurring non-proteinic amino acid found in tea leaves, has demonstrated wide-ranging physiological activity, from lowering blood pressure to enhancing the anti-tumor activity of chemotherapeutic drugs. The chiral nature of theanine suggests that enantiospecificity plays a significant role in its various pharmacological functions. Using the Chirobiotic T (teicoplanin) chiral stationary phase, native and derivatized theanine enantiomers were separated and detected via high-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization mass spectrometry (API-MS). With the use of flow rates compatible with each ionization source, native theanine standards achieved excellent sensitivity and detection limits (10 ng/mL) for both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Optimum sensitivity and detection limits for derivatized theanine standards were achieved using ESI-MS. The enantiomeric composition of six commercially available L-theanine samples was evaluated using the high-flow APCI-MS method and confirmed with photodiode array detection. Five of the six products contained significant amounts of D-theanine. Only one product, SunTheanine(R), appeared to contain only the L-theanine enantiomer. Copyright 2004 John Wiley & Sons, Ltd.
PMID: 14755608 [PubMed - in process]
Theanine and glutamate transporter inhibitors enhance the antitumor efficacy of chemotherapeutic agents.
Biochim Biophys Acta. 2003 Dec 5;1653(2):47-59.
Sugiyama T, Sadzuka Y.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, 422-8526 Shizuoka, Japan.
Biochemical modulation has played an important role in the development of cancer chemotherapy. The combined effects of theanine, a specific amino acid in green tea, and glutamate transporter inhibitors on the antitumor activity of doxorubicin (DOX), were investigated and we clarified the biochemical mechanisms of action of these modulators.In M5076 ovarian sarcoma-bearing mice, theanine significantly enhanced the inhibitory effect of DOX on tumor growth and increased the DOX concentration in the tumor, compared to DOX-alone group. Furthermore, the oral administration of theanine or green tea similarly enhanced the antitumor activity of DOX. Moreover, the combination of theanine with DOX suppressed the hepatic metastasis of ovarian sarcoma. In contrast, an increase in DOX concentration was not observed in normal tissues, such as liver and heart. Namely, theanine did not enhance, rather it tended to normalize the increase of lipid peroxide (LPO) levels and reduction of glutathione peroxidase activity as indicators of the DOX-induced side toxicity. On the other hand, in vitro experiments proved that theanine inhibited the efflux of DOX from tumor cells, supporting a theanine-induced increase in the DOX concentration in tumors in vivo. Moreover, theanine significantly inhibited the glutamate uptake by M5076 cells similar to specific inhibitors. Two astrocytic high-affinity glutamate transporters, GLAST and GLT-1, were expressed in M5076 cells. These results suggested that the inhibition of DOX efflux was induced by theanine-mediated inhibition of glutamate transporters. The reduction in the concentration of glutamate in tumor cells caused by theanine induced decreases in the intracellular glutathione (GSH) and GS-DOX conjugate levels. As the expression of MRP5 in M5076 cells was confirmed, it is suggested that the GS-DOX conjugate was transported extracellularly via the MRP5/GS-X pump in M5076 cells and that theanine affected this route. Namely, theanine increases the concentration of DOX in a tumor in vivo through inhibition of the glutamate transporter via the GS-X pump. Similarly, dihydrokainate (DHK) and L-serine-O-sulfate (SOS), specific glutamate transporter inhibitors, indicated the enhancement of the DOX antitumor activity via inhibition of glutamate uptake. Therefore, we revealed the novel mechanism of enhancement of antitumor efficacy of DOX via the inhibition of glutamate transporters. Similarly, theanine enhanced the antitumor activities of other anthracyclines, cisplatin and irinotecan. Consequently, the modulating effect of theanine on the efficacy of antitumor agents is expected to be applicable in clinical cancer chemotherapy.
Publication Types:
PMID: 14643924 [PubMed - indexed for MEDLINE]
Capillary electrophoretic determination of theanine, caffeine, and catechins in fresh tea leaves and oolong tea and their effects on rat neurosphere adhesion and migration.
J Agric Food Chem. 2003 Dec 3;51(25):7495-503.
Chen CN, Liang CM, Lai JR, Tsai YJ, Tsay JS, Lin JK.
Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Section 1, Jen-Ai Road, Taipei, Taiwan 100.
Theanine, caffeine, and catechins in fresh tea leaves and oolong tea were determined by using capillary electrophoresis (CE). CE separated these tea polyphenols from three other tea ingredients, namely, caffeine, theophylline, and theanine, within 8 min. The young leaves (apical bud and the two youngest leaves) were found to be richer in caffeine, (-)-epigallocatechin gallate (EGCg), and (-)-epicatechin gallate (ECg) than old leaves (from 5th to 7th leaves). On the other hand, the old leaves (from 8th to 10th leaves) contained higher levels of theanine, (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). Results from a comparison of fresh young tea and oolong tea compositions indicated oolong tea contained more theanine and catechins than fresh young tea. Furthermore, it was found that the levels of theanine, EGC, and EGCg in young leaves rose markedly with the withering process. Caffeine did not markedly change. However, fully or partially fermented teas (oolong tea or pauchong tea) have a common initial step in the withering process. Fresh tea leaves or oolong tea extract (0.1%, w/v) markedly inhibited neurosphere adhesion, cell migration, and neurite outgrowth in rat neurospheres. Theanine (348 micrograms/mL) and caffeine at high concentration (50 micrograms/mL) did not inhibit neurosphere adhesion or migration activities, but EGCg at 20 micrograms/mL effectively inhibited neurosphere adhesion for 24 h. These results indicated that EGCg might affect neural stem cell survival or differentiation.
PMID: 14640605 [PubMed - indexed for MEDLINE]
Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection.
J Chromatogr A. 2003 Sep 26;1013(1-2):183-9.
Kato M, Gyoten Y, Sakai-Kato K, Toyo'oka T.
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada Shizuoka, Shizuoka 422-8526, Japan.
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 mM phosphate buffer (pH 5.5) containing 0.05 mM 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.
PMID: 14604119 [PubMed - in process]
Antigens in tea-beverage prime human Vgamma 2Vdelta 2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses.
Proc Natl Acad Sci U S A. 2003 May 13;100(10):6009-14. Epub 2003 Apr 28.
Kamath AB, Wang L, Das H, Li L, Reinhold VN, Bukowski JF.
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Human gammadelta T cells mediate innate immunity to microbes via T cell receptor-dependent recognition of unprocessed antigens with conserved molecular patterns. These nonpeptide alkylamine antigens are shared by tumor cells, bacteria, parasites, and fungi but also by edible plant products such as tea, apples, mushrooms, and wine. Here we show that priming of gammadelta T cells with alkylamine antigens in vitro results in a memory response to these antigens. Such priming results also in a nonmemory response to whole bacteria and to lipopolysaccharide, characterized by IL-12-dependent secretion of IFN-gamma by gammadelta T cells and by gammadelta T cell proliferation. Drinking tea, which contains l-theanine, a precursor of the nonpeptide antigen ethylamine, primed peripheral blood gammadelta T cells to mediate a memory response on reexposure to ethylamine and to secrete IFN-gamma in response to bacteria. This unique combination of innate immune response and immunologic memory shows that gammadelta T cells can function as a bridge between innate and acquired immunity. In addition, these data provide an explanation for the health benefits of tea.
PMID: 12719524 [PubMed - indexed for MEDLINE]
[Determination of theanine in tea leaves by HPLC-inhibited chemiluminescence with Co2+ catalyst]
Se Pu. 2002 Nov;20(6):550-3.
Zhou GM, Yang GM, Qi DM, Deng CY, Peng JD.
School of Chemistry and Chemical Engineering, Southwest-China Normal University, Chongqing 400715, China. gmzhou@swnu.edu.cn
A method for the separation and determination of theanine in tea leaves has been developed, based on inhibited chemiluminescence of cobalt-catalyzed luminol-H2O2 reaction by theanine. Separations were performed on a YWG-C18 column using a mobile phase of 0.01 mol.L-1 sodium acetate-acetic acid buffer at pH 5.5 at a flow rate of 0.8 mL.min-1. The optimum concentrations of Co2+, luminol, and H2O2 were 2 micrograms.L-1, 0.25 mmol.L-1, and 0.5 mmol.L-1, respectively. Under the conditions mentioned above, the relative peak area (Y) of inhibited chemiluminescence of theanine was linear with the concentration (X) of theanine in the range of 0.2 g.L-1-5.0 g.L-1 and its linear regression equation was Y = 33,862 x + 1.0605 (r = 0.9983). The average recovery was 97.82% with a RSD of 2.16%.
PMID: 12683006 [PubMed - indexed for MEDLINE]
Inhibition by theanine of binding of [3H]AMPA, [3H]kainate, and [3H]MDL 105,519 to glutamate receptors.
Biosci Biotechnol Biochem. 2002 Dec;66(12):2683-6.
Kakuda T, Nozawa A, Sugimoto A, Niino H.
Central Research Institute, Itoen Ltd., 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-0516, Japan. t-kakuda@itoen.co.jp
In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.
PMID: 12596867 [PubMed - indexed for MEDLINE]
Theanine, gamma-glutamylethylamide, is metabolized by renal phosphate-independent glutaminase.
Biochim Biophys Acta. 2003 Mar 17;1620(1-3):47-53.
Tsuge H, Sano S, Hayakawa T, Kakuda T, Unno T.
Laboratory of Nutritional Biochemistry, Department of Food Science, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Japan. tsuge@cc.gifu-u.ac.jp
The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.
PMID: 12595072 [PubMed - indexed for MEDLINE]
Neuroprotective effects of the green tea components theanine and catechins.
Biol Pharm Bull. 2002 Dec;25(12):1513-8.
Kakuda T.
Central Research Institute, Itoen, Ltd, Shuzuoka, Japan.
The neuroprotective effects of theanine and catechins contained in green tea are discussed. Although the death of cultured rat cortical neurons was induced by the application of glutamic acid, this neuronal death was suppressed with exposure to theanine. The death of hippocampal CA1 pyramidal neurons caused by transient forebrain ischemia in the gerbil was inhibited with the ventricular preadministration of theanine. The neuronal death of the hippocampal CA3 region by kainate was also prevented by the administration of theanine. Theanine has a higher binding capacity for the AMPA/kainate receptors than for NMDA receptors, although the binding capacity in all cases is markedly less than that of glutamic acid. The results of the present study suggest that the mechanism of the neuroprotective effect of theanine is related not only to the glutamate receptor but also to other mechanisms such as the glutamate transporter, although further studies are needed. One of the onset mechanisms for arteriosclerosis, a major factor in ischemic cerebrovascular disease, is probably the oxidative alteration of low-density lipoprotein (LDL) by active oxygen species. The oxidative alterations of LDL were shown to be prevented by tea catechins. Scavenging of *O(2)(-) was also exhibited by tea catechins. The neuroprotective effects of theanine and catechins contained in green tea are a focus of considerable attention, and further studies are warranted.
PMID: 12499631 [PubMed - indexed for MEDLINE]
Direct determination of free amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection.
J Chromatogr A. 2002 Dec 27;982(2):237-44.
Ding Y, Yu H, Mou S.
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China.
Determination of amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection was developed. Amino acids and sugars were separated on an anion-exchange column at a flow-rate of 0.25 ml/min by using ternary gradient elution consisting of deionized water, 0.25 M sodium hydroxide, and 1.0 M sodium acetate. Under optimized conditions, theanine was separated from glutamine and three sugars (glucose, fructose, and sucrose) were eluted earlier than the neutral amino acids to avoid their interference with each other. RSDs of the peak area of analytes were lower than 4.6%. Detection limits for the analytes ranged from 0.12 to 4.9 pmol. The linearities for all analytes were two or three orders of magnitude with the correlation coefficients greater than 0.99. This method was applied to determination of amino acids and sugars in green tea with satisfactory results.
PMID: 12489879 [PubMed - indexed for MEDLINE]
[Glutamate transporter mediated increase of antitumor activity by theanine, an amino acid in green tea]
Yakugaku Zasshi. 2002 Nov;122(11):995-9.
Sadzuka Y, Yamashita Y, Kishimoto S, Fukushima S, Takeuchi Y, Sonobe T.
University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. sadzuka@u-shizuoka-ken.ac.jp
We have confirmed that theanine, a major amino acid in green tea, enhances the antitumor activity of doxorubicin (DOX) without an increase in DOX-induced side effects. We believe that the action of theanine is due to decreases in glutamate uptake via inhibition of the glutamate transporter, intracellular glutathione (GSH) synthesis, GS-DOX conjugate level, and subsequent extracellular transport of GS-DOX by the MRP5/GS-X pump. To increase the clinical usefulness of theanine, we examined its effects on the antitumor activity of cisplatin and irinotecan (CPT-11), which a known to be transported by the efflux system related to MRP. Cisplatin decreased tumor volume in M5076 tumor-bearing mice. Furthermore, the combination of theanine with cisplatin increased the decrease in tumor volume as compared with the cisplatin-alone group. Tumor volume in the CPT-11-alone group did not show a decrease, but the combination of theanine with CPT-11 significantly reduced tumor volume. The concentration of cisplatin in the tumor was significantly increased by combination with theanine, and thus we assume that it correlated with the enhancement on the antitumor activity of theanine. On the other hand, changes in drug concentrations with theanine were not observed in normal tissues, but rather it is indicated that theanine tends to reduce their concentrations. Therefore theanine enhances the antitumor activity not only of DOX but also of cisplatin or CPT-11.
PMID: 12440157 [PubMed - indexed for MEDLINE]
Effects of dietary powdered green tea and theanine on tumor growth and endogenous hyperlipidemia in hepatoma-bearing rats.
Biosci Biotechnol Biochem. 2002 Apr;66(4):711-6.
Zhang G, Miura Y, Yagasaki K.
Department of Applied Biological Science, Tokyo Noko University, Fuchu, Japan.
The effects of dietary powdered green tea (PGT) and theanine on in vivo hepatoma growth and cancerous hyperlipidemia were investigated in rats that had been implanted with a rat ascites hepatoma cell line of AH109A cells. The hepatoma-bearing rats were fed with a 20% casein diet (20C), 20C containing 2% PGT, or 20C containing 0.1% theanine for 14 days. Dietary PGT significantly and time-dependently reduced the solid tumor volume and weight as did dietary theanine. The hepatoma-induced endogenous hyperlipidemia, which was characterized by rises in the serum cholesterol (hypercholesterolemia) and triglyceride (hypertriglyceridemia) levels, was significantly suppressed by PGT and theanine supplementation. Bile acid excretion into the feces was significantly higher in the PGT- and theanine-fed rats than in the control rats. This inhibition of hypercholesterolemia may have resulted from tumor growth suppression as well as increased excretion of steroids from the body. These results suggest that PGT had both anti-proliferative activity toward hepatoma cells and hypolipidemic activity in the hepatoma bearing rats. They also suggest that theanine was, at least in part, responsible for the PGT actions.
PMID: 12036040 [PubMed - indexed for MEDLINE]
Effect of dihydrokainate on the antitumor activity of doxorubicin.
Cancer Lett. 2002 May 28;179(2):157-63.
Sadzuka Y, Yamashita Y, Sugiyama T, Sonobe T.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Japan.
For biochemical modulation, components of green tea have been shown to be useful modulators in combination with doxorubicin (DOX). We have confirmed that theanine enhances the antitumor activity of DOX due to inhibition of DOX efflux from tumor cells. Because theanine is a glutamate analogue, we found that it is associated with a change in the drug transport system on the tumor cell membrane, in particular glutamate transporters. We examined the effect of dihydrokainate (DHK), one of the useful glutamate transporter inhibitors. DHK also inhibits DOX efflux significantly and reduces the glutamate uptake by Ehrlich ascites carcinoma cells. The potential contribution of glutamate transporters not only to glutamate uptake but also to cell membrane export of DOX has been shown. In addition, the combination of DHK with DOX significantly enhances the antitumor activity of DOX, by 1.8-fold (P<0.001). The DOX concentration in tumors significantly increases on combination with DHK and is correlated with the reduced tumor weight. On the other hand, DHK tends to reduce the DOX concentration in normal tissues. We expect that DHK has different actions in tumor and normal tissues because different isoforms of glutamate transporters are expressed in the two tissues. Thus, the results suggest that DHK is a novel and useful modulator for inducing enhancement of antitumor activity.
PMID: 11888670 [PubMed - indexed for MEDLINE]
Enhancement of the activity of doxorubicin by inhibition of glutamate transporter.
Toxicol Lett. 2001 Sep 15;123(2-3):159-67.
Sadzuka Y, Sugiyama T, Suzuki T, Sonobe T.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, 422-8526, Shizuoka, Japan. sadzuka@u-shizuoka-ken.ac.jp
Theanine enhanced doxorubicin (DOX) induced antitumor activity by increasing the concentration of DOX in the tumor through the inhibition of efflux of DOX from tumor cells. As theanine reduced the level of glutamate via suppression of the glutamate transporter in tumor cells, we studied the change in the intracellular concentration of glutathione (GSH) and the correlation with the GSH S-conjugate export (GS-X) pump. The reduction in the concentration of glutamate in tumor cells caused by theanine, induced decreases in the intracellular GSH and GS-DOX levels. The expression of MRP5 in M5076 cells, was confirmed. We concluded that the GS-DOX conjugate was transported extracellularly via the MRP5/GS-X pump in M5076 cells and that theanine affected this route. Namely, theanine increases the concentration of DOX in a tumor in vivo through inhibition of the glutamate transporter via the GS-X pump.
PMID: 11641044 [PubMed - indexed for MEDLINE]
Inhibition of glutamate transporter by theanine enhances the therapeutic efficacy of doxorubicin.
Toxicol Lett. 2001 Apr 30;121(2):89-96.
Sugiyama T, Sadzuka Y, Tanaka K, Sonobe T.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, 422-8526, Shizuoka, Japan.
Theanine, a major amino acid existing in green tea, enhanced the antitumor activity of doxorubicin (DOX) due to inhibition of DOX efflux from tumor cells. In order to clarify the mechanism, we have investigated the contribution of glutamate transporters to the action of theanine, because theanine is a glutamate analogue. In M5076 ovarian sarcoma cells, glutamate transport inhibitors reduced the efflux of DOX, as well as theanine. Incidentally, theanine significantly inhibited the glutamate uptake by M5076 cells in a concentration-dependent manner similar to specific inhibitors. These results suggested that the inhibition of DOX efflux was induced by the inhibition of glutamate transport by theanine. In addition, RT-PCR and Western blot analysis revealed the expression of GLAST and GLT-1, astrocytic high-affinity glutamate transporters, in M5076 cells. Thus, theanine was shown to competitively inhibit the glutamate uptake by acting on these glutamate transporters. This action suggested the contribution of glutamate transporters to the inhibition of DOX efflux by theanine. We revealed the novel mechanism of enhancement of the antitumor efficacy of DOX via the inhibition of glutamate transporters by theanine.
PMID: 11325559 [PubMed - indexed for MEDLINE]
Protective effect of gamma-glutamylethylamide (theanine) on ischemic delayed neuronal death in gerbils.
Neurosci Lett. 2000 Aug 11;289(3):189-92.
Kakuda T, Yanase H, Utsunomiya K, Nozawa A, Unno T, Kataoka K.
Central Research Institute, Itoen, Ltd., Shizuoka, Japan. itn00527@nifty.ne.jp
We examined the protective effect of gamma-glutamylethylamide (theanine) on ischemic delayed neuronal death in field CA1 of the gerbil hippocampus. One microliter of theanine from each three concentrations (50, 125 and 500 microM) was administered through the lateral ventricle 30 min before ischemia. Transient forebrain ischemia was induced by bilateral occlusion of the common carotid arteries for 3 min under careful control of brain temperature at approximately 37 degrees C. Seven days after ischemia, the number of intact CA1 neurons in the hippocampus was assessed. Ischemia-induced neuronal death in hippocampal CA1 region was significantly prevented in a dose-dependent manner in the theanine-pretreated groups. These findings indicate that theanine might be useful clinically for preventing ischemic neuronal damage.
PMID: 10961661 [PubMed - indexed for MEDLINE]
Improvement of idarubicin induced antitumor activity and bone marrow suppression by theanine, a component of tea.
Cancer Lett. 2000 Oct 1;158(2):119-24.
Sadzuka Y, Sugiyama T, Sonobe T.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, 422-8526, Shizuoka, Japan. saduka@ys.7.u-shizoka-ken.ac.jp
We have examined the effect of theanine, a specific amino acid in green tea, on idarubicin (IDA)-induced antitumor activity and toxicity. In combination with theanine, IDA (0.25 mg/kg per day x4 days, a dose that does not show antitumor activity) had significant antitumor activity in P388-bearing mice. The IDA concentration in the tumors in the theanine plus IDA group increased to twice the level in the IDA alone group. Furthermore, the decrease in tumor weight caused by IDA at 1.0 mg/kg per day x4 days (at this dose IDA exhibits antitumor activity) was significantly amplified by theanine. The numbers of leukocyte and bone marrow cells decreased significantly on IDA injection. Theanine significantly reversed these changes. These results suggest that theanine selectively moderates the IDA-induced toxicities. Until recently, the antitumor activity and related toxicities of this chemotherapeutic agent in leukemia could not be distinguished. Theanine increases the IDA-induced antitumor activity and ameliorates the toxicities.
PMID: 10960760 [PubMed - indexed for MEDLINE]
Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography.
J Chromatogr A. 2000 Apr 21;876(1-2):235-42.
Aucamp JP, Hara Y, Apostolides Z.
Department of Biochemistry, University of Pretoria, South Africa.
A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.
PMID: 10823519 [PubMed - indexed for MEDLINE]
Inhibiting effects of theanine on caffeine stimulation evaluated by EEG in the rat.
Biosci Biotechnol Biochem. 2000 Feb;64(2):287-93.
Kakuda T, Nozawa A, Unno T, Okamura N, Okai O.
Central Research Institute, Itoen Ltd., Shizuoka, Japan. ITN00527@nifty.ne.jp
In this study, the inhibiting action of theanine on the excitation by caffeine at the concentration regularly associated with drinking tea was investigated using electroencephalography (EEG) in rats. First, the stimulatory action by caffeine i.v. administration at a level higher than 5 micromol/kg (0.970 mg/kg) b.w. was shown by means of brain wave analysis, and this level was suggested as the minimum dose of caffeine as a stimulant. Next, the stimulatory effects of caffeine were inhibited by an i.v. administration of theanine at a level higher than 5 micromol/kg (0.781 mg/kg) b.w., and the results suggested that theanine has an antagonistic effect on caffeine's stimulatory action at an almost equivalent molar concentration. On the other hand, the excitatory effects were shown in the rat i.v. administered 1 and 2 micromol/kg (0.174 and 0.348 mg/kg) b.w. of theanine alone. These results suggested two effects of theanine, depending on its concentration.
PMID: 10737183 [PubMed - indexed for MEDLINE]
Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance.
Toxicol Lett. 2000 Apr 3;114(1-3):155-62.
Sadzuka Y, Sugiyama T, Sonobe T.
School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Japan. sadzuka@ys7.u-shizuoka-ken.ac.jp
Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.
PMID: 10713480 [PubMed - indexed for MEDLINE]
Metabolism of theanine, gamma-glutamylethylamide, in rats.
J Agric Food Chem. 1999 Apr;47(4):1593-6.
Unno T, Suzuki Y, Kakuda T, Hayakawa T, Tsuge H.
Central Research Institute, Itoen Ltd., Sagara-cho, Haibara-gun, Shizuoka, Japan. ITN00533@nifty.ne.jp
The metabolism of theanine, one of the major amino acid components in tea (Camellia sinensis), was studied in rats. High-performance liquid chromatography (HPLC) with fluorometric detection was used to evaluate the nature of theanine's metabolites in plasma, urine, and tissues. In the urine samples collected after administration of 100, 200, and 400 mg each of theanine, intact theanine, L-glutamic acid, and ethylamine, these compounds were detected in a dose-dependent manner. When 200 mg of theanine was orally administered to rats, the plasma concentrations of theanine and ethylamine reached their highest levels about 0.5 and 2 h after administration, respectively. It seems most likely that the enzymatic hydrolysis of theanine to glutamic acid and ethylamine was accomplished in the kidney. These results indicate that orally administered theanine is absorbed through the intestinal tract and hydrolyzed to glutamic acid and ethylamine in the rat kidney.
PMID: 10564022 [PubMed - indexed for MEDLINE]
Membrane transport and antitumor activity of pirarubicin, and comparison with those of doxorubicin.
Jpn J Cancer Res. 1999 Jul;90(7):775-80.
Sugiyama T, Sadzuka Y, Nagasawa K, Ohnishi N, Yokoyama T, Sonobe T.
Department of Pharmaceutical Engineering, School of Pharmaceutical Sciences, University of Shizuoka.
We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5-fold that of doxorubicin. In terms of the 50% cell growth-inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor-bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3-fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7-fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.
PMID: 10470291 [PubMed - indexed for MEDLINE]
Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity.
Immunity. 1999 Jul;11(1):57-65.
Bukowski JF, Morita CT, Brenner MB.
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. jbukowski@rics.bwh.harvard.edu
Approximately 4% of peripheral blood T cells in humans express a T cell receptor with markedly restricted germline gene segment usage (V gamma 2 V delta 2). Remarkably, these T cells expand 2- to 10-fold (8%-60% of all circulating T cells) during many microbial infections. We show here that these T cells recognize a family of naturally occurring primary alkylamines in a TCR-dependent manner. These antigenic alkylamines are secreted to millimolar concentrations in bacterial supernatants and are found in certain edible plants. Given the large numbers of memory V gamma 2 V delta 2 T cells in adult humans, recognition of alkylamine antigens offers the immune system a response of the magnitude of major superantigens for alpha beta T cells and may bridge the gap between innate and adaptive immunity.
PMID: 10435579 [PubMed - indexed for MEDLINE]
Time-dependent changes of amino acids in the serum, liver, brain and urine of rats administered with theanine.
Biosci Biotechnol Biochem. 1999 Apr;63(4):615-8.
Terashima T, Takido J, Yokogoshi H.
School of Food and Nutritional Sciences, University of Shizuoka, Japan.
Time-dependent changes of theanine (gamma-glutamylethylamide) and other amino acids in various tissues of rats were investigated during the 24 hrs after theanine administration. When theanine (4 g/kg of body weight) was intragastrically administered to rats, the concentrations of theanine in the serum, liver and brain were significantly increased 1 hr after its administration, and thereafter gradually decreased, but reached the maximum level in the brain after 5 hrs. Theanine in these tissues had completely disappeared 24 hrs after its administration. In contrast, the administration of theanine resulted in the concentrations of theanine, urea, ethylamine and glutamic acid in the urine being significantly enhanced. These results suggest that theanine might be degraded via glutamic acid.
PMID: 10361674 [PubMed - indexed for MEDLINE]
Combination of theanine with doxorubicin inhibits hepatic metastasis of M5076 ovarian sarcoma.
Clin Cancer Res. 1999 Feb;5(2):413-6.
Sugiyama T, Sadzuka Y.
School of Pharmaceutical Sciences, University of Shizuoka, Japan.
Theanine is a peculiar amino acid existing in green tea leaves, which was previously indicated to enhance the antitumor activity of doxorubicin. In the present study, the effect of combination of theanine with doxorubicin against hepatic metastasis of M5076 ovarian sarcoma was investigated. The primary tumor was significantly reduced by the combined treatment on M5076 transplanted (s.c.) mice. The liver weight of control mice increased to twice the normal level because of hepatic metastasis of M5076. In contrast, the injection of doxorubicin alone or theanine plus doxorubicin suppressed the increase in liver weight and inhibited hepatic metastasis. Moreover, the liver weights and metastasis scores demonstrated that theanine enhanced the inhibition of hepatic metastasis induced by doxorubicin. Furthermore, in vitro experiments indicated that theanine increased the intracellular concentration of doxorubicin remaining in M5076 cells. This action suggests that theanine leads the enhancement of the suppressive efficacy of doxorubicin on hepatic metastasis in vivo. Therefore, it was proved that theanine increased not only the antitumor activity on primary tumor but also the metastasis-suppressive efficacy of doxorubicin. The effect of theanine on the efficacy of antitumor agents is expected to be applicable in clinical cancer chemotherapy.
PMID: 10037191 [PubMed - indexed for MEDLINE]
Enhancing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma.
Cancer Lett. 1998 Nov 13;133(1):19-26.
Sugiyama T, Sadzuka Y.
School of Pharmaceutical Sciences, University of Shizuoka, Japan. d7210@mail.p.u-shizuoka-ken.ac.jp
We have investigated the combined treatment of components of green tea with adriamycin against M5076 ovarian sarcoma, which exhibits low sensitivity to adriamycin. In M5076 tumor-bearing mice, the injection of adriamycin alone did not inhibit tumor growth, whereas the combination of theanine and adriamycin significantly reduced the tumor weight to 62% of the control level. When combined with theanine, effective antitumor activity of adriamycin was observed without an increase in the dosage. Theanine specifically increased the adriamycin concentration in the tumor by 2.7-fold. In contrast, theanine decreased the adriamycin concentrations in normal tissues. On the other hand, in vitro experiments proved that theanine inhibited the efflux of adriamycin from tumor cells, suggesting a theanine-induced increase in the adriamycin concentration in such tumors in vivo. Furthermore, the oral administration of theanine or green tea similarly enhanced the antitumor activity of adriamycin. In conclusion, the combination of theanine with adriamycin showed antitumor efficacy in spite of the non-effective dose of adriamycin on M5076 ovarian sarcoma. We have found that the modulating action of theanine is useful in clinical cancer chemotherapy.
PMID: 9929156 [PubMed - indexed for MEDLINE]
Theanine-induced reduction of brain serotonin concentration in rats.
Biosci Biotechnol Biochem. 1998 Apr;62(4):816-7.
Yokogoshi H, Mochizuki M, Saitoh K.
Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, University of Shizuoka, Japan. yokogosi@fnsl.u-shizuoka-ken.ac.jp
Following the administration of theanine, the brain tryptophan content significantly increased or tended to increase, but the contents of serotonin and 5-hydroxyindole acetic acid (5HIAA) decreased. The use of inhibitors of serotonin metabolism enable us to speculate that theanine reduced serotonin synthesis and also increased serotonin degradation in the brain.
PMID: 9614715 [PubMed - indexed for MEDLINE]
Modulation of cancer chemotherapy by green tea.
Clin Cancer Res. 1998 Jan;4(1):153-6.
Sadzuka Y, Sugiyama T, Hirota S.
School of Pharmaceutical Sciences, University of Shizuoka, Japan.
Biochemical modulation has played an important role in the development of cancer chemotherapy. We have directed our attention to the intake of common beverages and investigated the effects of green tea and tea components on the antitumor activity of doxorubicin. We carried out the combined treatment of toxorubicin and green tea on Ehrlich ascites carcinoma tumor-bearing mice. The oral administration of green tea enhanced 2.5-fold the inhibitory effects of doxorubicin on tumor growth. The Doxorubicin concentration in the tumor was increased by the combination of green tea with doxorubicin. In contrast, the increase in doxorubicin concentration was not observed in normal tissues after green tea combination. Furthermore, the enhancement of antitumor activity of doxorubicin induced by green tea was observed in M5076 ovarian sarcoma, which has low sensitivity to doxorubicin. These results suggest that drinking green tea can encourage cancer chemotherapy and may improve the quality of life of clinical patients.
PMID: 9516964 [PubMed - indexed for MEDLINE]
Effect of theanine, r-glutamylethylamide, on brain monoamines and striatal dopamine release in conscious rats.
Neurochem Res. 1998 May;23(5):667-73.
Yokogoshi H, Kobayashi M, Mochizuki M, Terashima T.
School of Food and Nutritional Sciences, The University of Shizuoka, Yada, Shizuoka, Japan. yokogosi@fns1.u-shizuoka-ken.ac.jp
Theanine, r-glutamylethylamide, is one of the major components of amino acids in Japanese green tea. Effect of theanine on brain amino acids and monoamines, and the striatal release of dopamine (DA) was investigated. Determination of amino acids in the brain after the intragastric administration of theanine showed that theanine was incorporated into brain through blood-brain barrier via leucine-preferring transport system. The concentrations of norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5HIAA) in the brain regions were unaffected by the theanine administration except in striatum. Theanine administration caused significant increases in serotonin and/or DA concentrations in the brain, especially in striatum, hypothalamus and hippocampus. Direct administration of theanine into brain striatum by microinjection caused a significant increase of DA release in a dose-dependent manner. Microdialysis of brain with calcium-free Ringer buffer attenuated the theanine-induced DA release. Pretreatment with the Ringer buffer containing an antagonist of non-NMDA (N-methyl-D-aspartate) glutamate receptor, MK-801, for 1 hr did not change the significant increase of DA release induced by theanine. However, in the case of pretreatment with AP-5, (+/-)-2-amino-5-phosphonopentanoic acid; antagonist of NMDA glutamate receptor, the theanine-induced DA release from striatum was significantly inhibited. These results suggest that theanine might affect the metabolism and/or the release of some neurotransmitters in the brain, such as DA.
PMID: 9566605 [PubMed - indexed for MEDLINE]
Hypotensive effect of gamma-glutamylmethylamide in spontaneously hypertensive rats.
Life Sci. 1998;62(12):1065-8.
Yokogoshi H, Kobayashi M.
Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, University of Shizuoka, Yada, Japan. yokogosi@fns1.u-shizuoka-ken.ac.jp
The effect of gamma-glutamylmethylamide(GMA), one of the components of green tea extract, on the blood pressure in spontaneously hypertensive rats (SHR) was investigated. The effect of glutamic acid and r-glutamylethylamide (theanine), which is structurally similar to GMA, was also examined. When SHR were injected with glutamic acid (2000mg/kg), the blood pressure was not altered. The same dose of theanine decreased it significantly. GMA administration to SHR reduced the blood pressure significantly, and its degree of hypotensive action was more effective than that by theanine administration.
PMID: 9519808 [PubMed - indexed for MEDLINE]
Influence of green tea and its three major components upon low-density lipoprotein oxidation.
Exp Toxicol Pathol. 1997 Dec;49(5):329-35.
Yokozawa T, Dong E.
Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Japan.
The abilities of green tea extract and its three major components to inhibit lipid peroxidation in low-density lipoprotein (LDL) catalyzed by copper were tested in vitro using malondialdehyde as a parameter of antioxidant activity. The results demonstrated that green tea extract markedly delays peroxidation with a dose-dependent pattern. Of the three components, polyphenols had the strongest action. Similar action was also shown in the theanine-treated group but was weaker than in the former, whereas caffeine had a very limited effect. Based on these data, it is concluded that green tea extract can effectively inhibit peroxidation and that this activity is due largely to the polyphenols it contains. According to the ultraviolet spectra, copper chelation is suggested to be one of the possible mechanisms of LDL antiperoxidation.
PMID: 9455677 [PubMed - indexed for MEDLINE]
Chewing-gum flavor affects measures of global complexity of multichannel EEG.
Neuropsychobiology. 1997;35(1):46-50.
Yagyu T, Wackermann J, Kinoshita T, Hirota T, Kochi K, Kondakor I, Koenig T, Lehmann D.
The KEY Institute for Brain-Mind Research, University Hospital of Psychiatry, Zurich, Switzerland.
Global complexity of spontaneous brain electric activity was studied before and after chewing gum without flavor and with 2 different flavors. One-minute, 19-channel, eyes-closed electroencephalograms (EEG) were recorded from 20 healthy males before and after using 3 types of chewing gum: regular gum containing sugar and aromatic additives, gum containing 200 mg theanine (a constituent of Japanese green tea), and gum base (no sugar, no aromatic additives); each was chewed for 5 min in randomized sequence. Brain electric activity was assessed through Global Omega (Omega)-Complexity and Global Dimensional Complexity (GDC), quantitative measures of complexity of the trajectory of EEG map series in state space; their differences from pre-chewing data were compared across gum-chewing conditions. Friedman Anova (p < 0.043) showed that effects on Omega-Complexity differed significantly between conditions and differences were maximal between gum base and theanine gum. No differences were found using GDC. Global Omega-Complexity appears to be a sensitive measure for subtle, central effects of chewing gum with and without flavor.
Publication Types:
- Clinical Trial
- Randomized Controlled Trial
PMID: 9018023 [PubMed - indexed for MEDLINE]
The effects of theanine, as a novel biochemical modulator, on the antitumor activity of adriamycin.
Cancer Lett. 1996 Aug 2;105(2):203-9.
Sadzuka Y, Sugiyama T, Miyagishima A, Nozawa Y, Hirota S.
School of Pharmaceutical Sciences, University of Shizuoka, Japan.
We studied the effects of theanine, a component of green tea leaves, on the antitumor activity of adriamycin (ADR) from the biochemical modulation view point. In vitro, theanine inhibited the ADR efflux from Ehrlich ascites carcinoma cells and maintained the ADR concentration in tumor cells. Theanine enhanced the inhibitory effect of ADR on tumor growth by 2.1-fold in vivo, and increased 2.9-fold the ADR concentration in the tumor, compared to the ADR alone group. An increase in ADR concentration was not observed in normal tissues, such as the heart and liver. Theanine did not enhance, rather tended to normalize the increase of lipid peroxide level and reduction of glutathione peroxidase activity as indicators of the ADR-induced side toxicity.
PMID: 8697445 [PubMed - indexed for MEDLINE]
Effects of a component of green tea on the proliferation of vascular smooth muscle cells.
Biosci Biotechnol Biochem. 1995 Nov;59(11):2134-6.
Yokozawa T, Oura H, Nakagawa H, Sakanaka S, Kim M.
Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, Japan.
The effects of a component of green tea on the proliferation of smooth muscle cells were measured in terms of [3H]thymidine uptake. When green tea tannin mixture was added to the medium of cultured smooth muscle cells, it suppressed the proliferation of the cells dose-dependently. Similarly to the effects of the green tea tannin mixture, (-)-epigallocatechin 3-O-gallate, its main ingredient, had an inhibitory effect on smooth muscle cell proliferation at a low concentration. (-)-Epicatechin 3-O-gallate was also an effective component. Among four types of gallate-free tannin, (-)-epigallocatechin, (-)-epicatechin, and (+)-catechin showed significant dose-dependent inhibition of smooth muscle cell proliferation. However, caffeine and theanine were found to have no such action.
PMID: 8541655 [PubMed - indexed for MEDLINE]
Reduction effect of theanine on blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats.
Biosci Biotechnol Biochem. 1995 Apr;59(4):615-8.
Yokogoshi H, Kato Y, Sagesaka YM, Takihara-Matsuura T, Kakuda T, Takeuchi N.
School of Food and Nutritional Sciences, University of Shizuoka, Japan.
The effect of theanine, one of the components of green tea, on the blood pressure and brain 5-hydroxyindoles in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) was investigated by intraperitoneally administering theanine. The effect of glutamine, which is structurally similar to theanine, was also examined. When SHR were injected with various amounts of theanine (0, 500, 1000, 1500, and 2000 mg/kg), the change was dose-dependent, and a significant decrease in blood pressure was observed with the high doses (1500 and 2000 mg/kg). A dose of 2000 mg/kg of theanine did not alter the blood pressure of WKY, while the same dose to SHR decreased it significantly. On the other hand, glutamine administration to SHR did not change either the blood pressure or the heart rate. The brain 5-hydroxyindole level was significantly decreased by theanine administration to both WKY and SHR, the decrease being dose-dependent.
PMID: 7539642 [PubMed - indexed for MEDLINE] | 
