Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine.

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 µM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

Daily oral intake of theanine prevents the decline of 5-bromo-2'-deoxyuridine incorporation in hippocampal dentate gyrus with concomitant alleviation of behavioral abnormalities in adult mice with severe traumatic stress.

Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2'-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress.

Neuroprotective effects of theanine and its preventive effects on cognitive dysfunction.

Theanine (γ-glutamylethylamide) characteristically present in tea leaves (Camellia sinensis). It has a similar chemical structure to glutamate, which is a neurotransmitter related to memory. Theanine passes through the blood-brain barrier and has been shown to have a cerebroprotective effect and a preventive effect on neuronal cell death after transient cerebral ischemia. The neuroprotective effect is partly due to the antagonistic action of theanine on glutamate receptor subtype AMPA and kainate receptors, but the affinity is very low. Theanine also acted on glutamine (Gln) transporter strongly and inhibited the incorporation of extracellular Gln into neurons, which in turn suppressed the conversion of Gln to glutamate by glutaminase, a reaction required for condensation into synaptic vesicles to form a neurotransmitter pool responsible for subsequent exocytotic release upon stimuli. In an investigation of elderly persons with normal or slight cognitive dysfunction, volunteers who ingested powdered green tea containing a high theanine concentration (equivalent to 47.5mgday(-1) of theanine) showed significantly lower decline in cognitive function compared with that of the placebo group. This result suggested that theanine might have improved a slight cognitive dysfunction in elderly persons.

Theanine, an ingredient of green tea, inhibits [3H]glutamine transport in neurons and astroglia in rat brain.

We have previously shown that theanine (=gamma-glutamylethylamide), an ingredient of green tea, has a protective effect against ischemic neuronal death in the hippocampal CA1 region of the gerbil brain without affecting ligand binding to ionotropic receptor subtypes of the neurotransmitter glutamate structurally related to theanine. The neurotransmitter pool of glutamate is thought to be fueled by the entry of the other structural analog glutamine (Gln) and subsequent cleavage by glutaminase. Although theanine did not inhibit [3H]glutamate accumulation, [3H]theanine was actively accumulated in a temperature-dependent and saturable manner in rat brain synaptosomal fractions. The accumulation of [3H]theanine was markedly inhibited by Gln in a concentration-dependent manner, whereas [3H]Gln accumulation was inhibited by theanine vice versa. Both [3H]theanine and [3H]Gln accumulations were decreased after the replacement of sodium chloride with choline chloride, along with similarly high distribution profiles in telencephalic structures. A similar equilibrium was observed within 30 min at 30 degrees C for the accumulations of both [3H]theanine and [3H]Gln in cultured rat neocortical astroglia as well as neurons, whereas theanine inhibited [3H]Gln accumulation in a concentration-dependent manner at 0.1-10 mM. Furthermore, sustained exposure to 10 mM theanine led to a significant decrease in the level of extracellular glutamate released from cultured neurons. These results suggest that the green tea ingredient theanine would be an inhibitor of different transporters capable of transporting Gln across plasma membranes toward the modulation of the glutamate/Gln cycle required for the neurotransmitter pool of glutamate in neurons.

Endothelium-dependent vasorelaxation effect of rutin-free tartary buckwheat extract in isolated rat thoracic aorta.

The aim of this study was to point out the potential of tartary buckwheat on vascular functions. A nonabsorbed fraction of hot-water extract of tartary buckwheat on a SP70 column (TBSP-T), which was free from rutin, was used for this aim. In a contractile experiment using Sprague-Dawley rat thoracic aorta rings contracted by 1.0 microM phenylephrine (PE) or 50 mM KCl, TBSP-T evoked a significant vasorelaxation [EC50 (mg/ml): PE; 2.2; KCl, 1.9]. By a further fractionation of TBSP-T by liquid-liquid partitioning into basic, neutral and acidic fractions, a marked enhancement of vasorelaxation effect was observed only for acidic fraction (EC50, 0.25 mg/ml). The action of acidic fraction was significantly attenuated in endothelium-denuded aortic rings and in the presence of nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 microM). The fraction also enhanced the cyclic guanosine monophosphate (cGMP) production in aortic rings contracted with PE [cGMP (pmol/mg protein): PE, 7.2+/-2.3; PE+Acidic fraction, 35+/-8]. These results indicate that acidic fraction could mediate NO/cGMP pathways, thereby exerting endothelium-dependent vasorelaxation action. In conclusion, tartary buckwheat was proven to regulate vascular tones and have latent acidic candidates except for rutin.

Effects of (-) -epigallocatechin gallate in liver of an animal model of combined (physical and mental) fatigue.

OBJECTIVE: Fatigue can be classified as physical and mental depending on the cause. However, in our daily lives, combined fatigue, which is the combination of physical and mental fatigue, is most often experienced. In this study, the effects of (-)-epigallocatechin gallate (EGCg) on combined fatigue were assessed. METHODS: To produce an animal model of combined fatigue, rats were kept in a cage filled with water to a height of 1.5 cm for 5 d. To evaluate the extent of fatigue, the rats swam with a load of steel rings that weighed approximately 8% of their body weight and were attached to their tails. RESULTS: Fatigued rats treated with EGCg (50 or 100 mg/kg intraperitoneally [not for 25 mg/kg]) for 5 d could swim longer than fatigued animals given saline. Although levels of thiobarbituric acid-reactive substances in the plasma, brain, and skeletal muscle were not different between control and fatigued rats, thiobarbituric acid-reactive substance levels were higher in livers of fatigued animals than in livers of control animals. Oral intake of EGCg (50 or 100 mg/kg) for 5 d significantly decreased thiobarbituric acid-reactive substance levels in livers of fatigued animals. CONCLUSION: These results suggest that EGCg (50 or 100 mg/kg) is effective for attenuating fatigue. EGCg given orally appears to have an antioxidant effect on the oxidatively damaged liver of fatigued animals.

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro.

We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2'-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.

Theanine, gamma-glutamylethylamide, is metabolized by renal phosphate-independent glutaminase.

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.

Analysis of tea catechins in human plasma by high-performance liquid chromatography with solid-phase extraction.

Accurate monitoring of tea catechins in biological samples might provide a means of better evaluation of their benefits. The aim of the present study was to develop a rapid method for extracting tea catechins from human plasma samples with a solid-phase extraction technique and to subsequently measure their concentrations using an HPLC system. A human plasma sample spiked with known concentrations of the analyte standards was passed through a Waters Oasis HLB cartridge. After repeated washing, tea catechins were eluted with 70% dimethylformamide containing 0.1% phosphoric acid, and the resulting eluate was injected into an HPLC system. Analytes were separated on a reverse-phase C18 column using an isocratic mobile phase and detected electrochemically. The coefficient of variation for inter- and intraday reproducibility was less than 5.0% and 6.4%, respectively. Linearity was established for the concentration range of 0.01-1.0 microM. The method was successfully applied to measure tea catechin concentrations in the plasma of two healthy subjects who received a single ingestion of a green tea beverage. The proposed method enables the rapid and accurate quantitation of plasma tea catechins and might prove useful for the evaluation of beneficial health effects of tea consumption.

Urinary excretion of 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a ring-fission metabolite of (-)-epicatechin, in rats and its in vitro antioxidant activity.

There is great interest in the nutritional potential of (-)-epicatechin, a common polyphenolic constituent of many foods and beverages, because of its potent antioxidant capacity. To better evaluate the biological role of (-)-epicatechin, we studied the urinary excretion of 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a ring-fission metabolite of (-)-epicatechin by intestinal microflora, in rats as well as its antioxidant activity in vitro. The method for measuring the urinary levels of (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone was based on the enzymatic hydrolysis of beta-glucuronidase and sulfatase, and was subsequently determined by HPLC coupled to an electrochemical detector. Following administration of (-)-epicatechin at doses of 0, 20, 40, and 80 mumol per rat, (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone were excreted into the urine within 24 h in a dose-dependent manner. Urinary 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone was mostly in the conjugated form, with a higher ratio of conjugation than (-)-epicatechin. We assessed the relative antioxidant potentials for scavenging radicals in the aqueous phase as expressed in the Trolox equivalent antioxidant capacity (TEAC). The results demonstrated that the degradation of (-)-epicatechin into 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone attenuated the antioxidant ability of the former. However, 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone showed stronger antioxidant activity than l-ascorbic acid. These results led us to suppose that 5-(3',4'-dihydroxyphenyl)-gamma-valerolactone, a microbial metabolite of (-)-epicatechin, circulating in the body may also at least be biologically active in terms of contributing to its combined antioxidant effect.