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Anti-stress effects of drinking green tea with lowered caffeine and enriched theanine, epigallocatechin and arginine on psychosocial stress induced adrenal hypertrophy in mice.

ABSTRACT

Background: Theanine, an amino acid in tea, has significant anti-stress effects on animals and humans. However, the anti-stress effects of drinking green tea have not yet been elucidated.

Hypothesis/purpose: The present study aimed to explore anti-stress effects of green tea and roles of tea components in a mouse model of psychosocial stress.

Study design: We examined anti-stress effects of three types of green teas, theanine-rich "Gyokuro", standard "Sencha", and Sencha with lowered caffeine (low-caffeine green tea). Furthermore, the roles of tea components such as caffeine, catechins, and other amino acids in anti-stress effects were examined.

Methods: To prepare low-caffeine green tea, plucked new tea leaves were treated with a hot-water spray. Mice were psychosocial^ stressed from a conflict among male mice under confrontational housing. Mice consumed each tea that was eluted with room temperature water ad libitum. As a marker for the stress response, adrenal hypertrophy was compared with mice that ingested water.

Results: Caffeine was significantly lowered by spraying hot-water on tea leaves. While epigallocatechin gallate (EGCC) is the main catechin in tea leaves, epigallocatechin (EGC) was mainly infused into water at room temperature. Adrenal hypertrophy was significantly suppressed in mice that ingested theanine-rich and low-caffeine green tea that were eluted with water at room temperature. Caffeine and EGCG suppressed the anti-stress effects of theanine while EGC and arginine (Arg) retained these effects.

Conclusion: These results suggest that drinking green tea exhibits anti-stress effects, where theanine, EGC and Arg cooperatively abolish the counter-effect of caffeine and EGCG on psychosocial stress induced adrenal hypertrophy in mice.

Keywords:

Anti-stress effect

Arginine

Caffeine

Epigallocatechin

Green tea

Theanine

Introduction

Theanine (L-theanine), the major amino acid and a sweet umami component of green tea (Camellia sinensis (L.) Kuntze), has significant anti-stress effects on animals and humans (Kimura et al., 2007; Unno et al., 2011, 2013a, 2013b). In those studies, purified theanine had been used. However, whether simply drinking green tea exerts similar anti-stress effects is not known. We have shown that the anti-stress effect of theanine was blocked by caffeine and catechins, two other main components of tea (Unno et al., 2013a). Each tea component also provides a distinct taste such as umami (theanine and other amino acids), bitterness (caffeine), and astringency (catechins), which all together determine the overall taste of a specific type of green tea. It is therefore important to know the relationship between the biological function and taste of green tea based on the balance of theanine, caffeine, catechins and other amino acids.

The relative amounts of these tea components vary depending on cultivation conditions such as the harvest season of tea leaves, sunshine conditions, breeds of tea plant, and geography. All these factors affect the taste and grade of specific green teas. Gyokuro, a high-grade green tea, is prepared by placing in the shade for about three weeks before being harvested. This process increases both theanine and caffeine in tea leaves (Sasaoka et al., 1964; Konishi et al., 1972). Gyokuro is thus umami-rich, but as a consequence, very expensive. To fully infuse theanine, an umami component, from Gyokuro, it is further necessary to steep in low-temperature water (40-60[degrees]C), whereas at a high temperature (~90[degrees]C), catechins and caffeine are preferentially infused (Shimotoku et al., 1982; Monobe et al., 2010).

Sencha, a more commonly consumed "popular" green tea, is grown under full sun, which results in reduced theanine and increased catechins compared to Gyokuro. Furthermore, a grade of Sencha green tea is generally distinguished by the amount of amino acids, although a standard value has not been defined. In this experiment, a higher grade Sencha was used to prepare Sencha with lowered caffeine content (low-caffeine Sencha). A middle grade Sencha was used as the standard. In this study, we propose a practical way of maximizing the anti-stress effect of Sencha green tea as a daily consumable drink by enriching theanine and lowering caffeine and catechin through the temperature-sensitive kinetics of water elution of each tea component. The anti-stress effect of these three types of green tea was examined.

In the present study, the anti-stress effect of drinking green tea was evaluated using our unique mouse model of psychosocial stress evoked by confrontational housing. We previously demonstrated that adrenal hypertrophy is a suitable marker for the stress response (Unno et al., 2013 a). That is, two male mice were housed in the same cage separated with a partition to establish a territorial imperative. Then, the partition was removed and mice were co-housed confrontationally. As a marker for the stress response, changes in the adrenal gland were studied and compared with group-housed control mice. Significant adrenal hypertrophy, which was observed in mice under confrontational housing, had developed in 24 h and persisted for at least 1 week. The cell size in the zona fasciculata of the adrenal gland, from which glucocorticoid is mainly secreted, increased in mice under confrontational housing, which was accompanied by a flat diurnal rhythm of corticosterone and ACTH in blood. In addition, consumption of paroxetin, an antidepressant drug, significantly suppressed adrenal hypertrophy of mice under confrontational housing. Therefore, adrenal hypertrophy is suitable for evaluating the anti-stress effect.

The present study examined whether the ingestion of green tea prevents and relieves psychosocial stress. Furthermore, individual and combined effects of each tea component were examined and the best balance among theanine, caffeine, catechins and other amino acids was explored.

Materials and methods

Preparation of low-caffeine Sencha green tea

Plucked new tea leaves were treated with a hot-water spray at 95[degrees]C as described previously (Maeda-Yamamoto et al., 2007). To compare the reduction of caffeine, tea leaves were treated with heated water spray for 180 s and 280 s. After centrifugal dehydration, green tea was prepared through a standard manufacturing process namely rolling and drying. We termed this low-caffeine Sencha green tea (Fig. 1). Tea components in both dried tea leaves and eluates were measured by HPLC.

Measurement of tea components by HPLC

The chemical composition of dried tea leaves are generally measured to compare the grade and taste of green teas. However, in this study, we focused on the biological functions of green tea components in an elution steeped at room temperature. The temperature was selected to reduce the elution of catechins and caffeine (Shimotoku et al., 1982; Monobe et al., 2010). Therefore, tea components in both dried tea leaves as described next and eluates that are described in Section (Green tea infusion at room temperature and measurement of tea components by HPLC) were measured by HPLC.

The amount of caffeine, catechins and amino acids in tea leaves was measured according to a method described in Horie et al. (2002). In brief, powdered tea leaves (25 mg) were shaken in 5 ml of an acetonitrile-water solution (1:1 v/v) at 180 rpm for 1 h. Catechins and caffeine in the filtrate were measured by HPLC (SCL-10Avp, Shimadzu, Japan; Develosil packed column ODS-HG-5, 150 x 4.6 mm, Nomura Chemical Co. Ltd., Japan). Gradient elution from the column was performed at 40[degrees]C at a flow rate of 1.0 ml/min using a two-gradient elution system with mobile phases A (acetonitrile/water/phosphoric acid (10:400:1, v/v/v)) and B (mobile phase A/methanol (1000:500, v/v)). Catechins and caffeine were measured at 280 nm.

Next, free amino acids in tea leaves were measured using another elution condition. In brief, 10 mg of powdered tea leaves was extracted with 5 ml of water after adding polyvinylpolypyrrolidone (10 mg) to remove polyphenols. Alanine (Ala), arginine (Arg), aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gin), serine (Ser) and theanine, major amino acids in the tea leaves, were measured by HPLC as described above using homoserine as an internal standard (Goto et al., 1993). Elution from the column was performed at 40[degrees]C at a flow rate of 1.0 ml/min using the mobile phase of acetonitrile and 5 mM citric acid buffer (pH 6.0). These amino acids were detected at an excitation wavelength of 340 nm and at 450 nm of emission wavelength (RF-535 UV detector, Shimadzu, Japan).

The HPLC profiles of standard catechins and caffeine, and those in Sencha were shown in Fig. SI a and b, respectively. The HPLC profiles of standard amino acids and those in Sencha were shown in Fig. Sic and d, respectively. The relative standard deviation (RSD%) of precision and repeatability were < 5.0%. The recoveries of catechins, caffeine, and free amino acids were 99 [+ or -] 4%, 98 [+ or -] 4%, and 98 [+ or -] 3%, respectively.

Green tea infusion at room temperature and measurement of tea components by HPLC

Three types of drinking green tea were prepared for mouse studies: theanine-rich Gyokuro, low-caffeine Sencha green tea, and standard Sencha green tea. Green tea leaves (3g) were added to 11 of tap water at room temperature and filtered after stirring for 6 min (Fig. 2). Amino acids and non-gallate catechins such as (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) are infused easily into water at room temperature. On the other hand, caffeine and gallate catechins such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are hardly soluble in room temperature water and are retained in tea leaves (Shimotoku et al., 1982; Monobe et al., 2010). Each infusion was used to measure the tea components by HPLC and for animal studies.

Catechins and caffeine in the infusions were measured by HPLC as described in Section (Measurement of tea components by HPLC). Free amino acids in the infusions were also measured by HPLC as described in Section (Measurement of tea components by HPLC).

Animals, housing conditions for stress experiment

Male ddY mice (Sic: ddY, 4 weeks old) were purchased from Japan SLC Co. Ltd (Shizuoka, Japan) and kept in a conventional condition in a temperature- and humidity-controlled environment with a 12-12 h light-dark cycle (light period, 08:00-20:00 h; temperature, 23 [+ or -] 1[degrees]C; relative humidity, 55 [+ or -] 5%). Mice were fed with a normal diet (CE-2; Clea Co. Ltd. Tokyo, Japan) and water ad libitum. All experimental protocols were approved by the University of Shizuoka Laboratory Animal Care Advisory Committee (approval no. 136068 and 166197) and were in accordance with the guidelines of the US National Institutes of Health for the care and use of laboratory animals.

Mice at 4 weeks of age were housed in a group of six in a cage for 5 days for adaptation. The mice were then divided into two groups: confrontationally-housed and group-housed. For confrontational housing, a standard polycarbonate cage was divided into two identical subunits by a stainless-steel partition as previously described (Unno et al, 2013a). In brief, two mice were housed in a partitioned cage for 1 week (single housing): then the partition was removed to expose the mice to confrontational stress for 24 h (confrontational housing). Each cage was placed in a Styrofoam box in order to avoid social contact between cages.

Ingestion of green tea in mice

The effects of theanine-rich Gyokuro, low-caffeine Sencha, and standard Sencha were examined in eight groups of mice (6 mice/group, total n = 48). Mice consumed each tea ad libitum for 8 days (single housing for 7 days and confrontational housing for 1 day) (Fig. 2). Mice of group housing also consumed each tea ad libitum for 8 days. The drinking volume of each tea was measured. At the end of the experimental period, mice were sacrificed and adrenal glands were weighed. RSD of precision of weighting of the adrenals within groups (confrontation and group housing) was <10.0%.

Ingestion of individual and combined tea components in mice

The effects of individual and combined tea components such as theanine, caffeine, catechins and other amino acids, were examined in 34 groups of mice (4 mice/group, total n = 136). Tea components used were as follows: L-theanine (Suntheanine; Taiyo Kagaku Co. Ltd., Yokkaichi, Japan), EGCG (Sunphenon EGCg, Taiyo Kagaku Co. Ltd.), EGC (Sunphenon EGC, Taiyo Kagaku Co. Ltd.), ECG (Tokyo Kasei Co. Ltd., Tokyo, Japan), Arg, Glu, and Gin (Wako Pure Chemical Co. Ltd., Osaka, Japan). Mice consumed individual or combined tea components in tap water ad libitum for 8 days as described above. The drinking volume of each tea component was measured. At the end of the experimental period, mice were sacrificed and adrenal glands were weighed.

Statistical analysis

Data are expressed as mean [+ or -] SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Bonferroni's post hoc test for multiple comparisons. ANOVA was assessed using a statistical analysis program (StatPlus, Analyst-Soft Inc., online version). Differences were considered to be significant at p < 0.05.

Results

Preparation of low-caffeine Sencha green tea

First, we optimized a method to lower the level of caffeine in Sencha tea leaves, while retaining theanine and other components, by using a hot-water treatment (Maeda-Yamamoto et al, 2007). Spraying a heated-water shower at 95[degrees]C preferentially eluted caffeine from plucked new tea shoots over 90s (Maeda-Yamamoto et al, 2007). We treated Sencha tea leaves with a hot-water shower for 180 or 280s (Fig. 1). As shown in Table 1, caffeine decreased to 1/4-1/5 of the level of non-treated tea leaves, whereas the amount of catechins such as EGCG and EGC did not change significantly. EGCG was the most abundant followed by EGC. Theanine decreased slightly to 93% and 83% after hot-water treatment for 180s and 280s, respectively. The amount of other amino acids did not change. Taken together, treatment with hot water for 180s is sufficient to significantly lower caffeine without altering the other tea components. We used Sencha leaves treated with a hot-water spray for 180s to create "low-caffeine Sencha" to measure the tea components and for animal studies.

Green tea infusions at room temperature

To reduce the elution of caffeine and gallate catechins, each type of tea leaves (3g) was steeped in water (11) at room temperature by stirring for 6 min (Fig. 2). The amounts of caffeine, catechins and theanine in theanine-rich Gyokuro, low-caffeine Sencha and standard Sencha green tea were compared (Table 2). The reduction of caffeine in low-caffeine Sencha was significant compared to Gyokuro (> 170-fold) and standard tea (> 70-fold) (Table 2). The most common catechin was non-gallate catechins such as EGC and EC, while EGCG was almost absent in each tea elution. The total amount of catechins was highest in the standard tea. Although the amount of theanine in the infusion from low-caffeine Sencha is half of that from Gyokuro, it is 2.5-fold higher than that from standard Sencha (Table 2). Other amino acids such as Glu and Arg were slightly higher in low-caffeine green tea than in other teas (Table 2). Collectively, low-caffeine Sencha steeped in water at room temperature is characterized by a negligible amount of caffeine, significantly reduced gallate catechins and enriched theanine and other amino acids.

Anti-stress effects of green tea on a mouse model of psychosocial stress

The anti-stress effects of drinking low-caffeine Sencha, theanine-rich Gyokuro, and standard Sencha were tested on a mouse model of psychosocial stress evoked by confrontational housing. Adrenal hypertrophy, a typical stress response in living organisms, was used as a marker for assessing the degree of stress (Unno et al., 2011, 2013a).

Each type of green tea was administered to mice as drinking water for 8 days. The adrenal weights of group and confrontational housing mice that consumed [H.sub.2]O or each green tea were measured (Fig. 3a). The changes of adrenal weight from group-housing mice to confrontational-housing ones were compared (Fig. 3b). The adrenal hypertrophy was significantly lower in mice that consumed low-caffeine and theanine-rich green teas than those that consumed water (F (3, 20) = 9.560; p = 4.0 x [10.sup.-4]; [[eta].sup.2] = 0.35; one-way ANOVA). To examine why significant suppression of adrenal hypertrophy was not observed in mice of the standard green tea group, ingestion of each tea component was compared among the three green tea groups of mice under confrontational housing. The drinking volume of the theanine-rich group was significantly higher than that of the standard and low-caffeine green tea groups (F (2, 15) =17.661; p = 1.1 x [10.sup.-4]; [[eta].sup.2] = 0.49; one-way ANOVA; Table 3). The ingestion of caffeine, a diuretic, was significantly higher in the theanine-rich green tea than in other groups (F (2, 15)= 573.58; p = 6.8 x [l0.sup.-15]; [[eta].sup.2] = 0.98; one-way ANOVA), suggesting an increase in the volume of theanine-rich green tea that had been drunk. The ingestion of EGCG was also significantly higher in the theanine-rich group than in the other groups (F (2, 15)= 121.74; p = 5.3 x [l0.sup.-10]; [[eta].sup.2] = 0.89; one-way ANOVA). The ingestion of theanine and Arg was significantly higher in low-caffeine and theanine-rich groups than in the standard green tea group (theanine. F (2, 15) = 454.93; p = 3.8 x [10.sup.-14]; [[eta].sup.2] = 0.97; Arg, F (2, 15) = 80.50; p = 9.5 x [10.sup.-9]; = 0.84; one-way ANOVA, Table 3). The effects of these tea components were then examined in detail.

Anti-stress effects of tea components on a mouse model of psychosocial stress

Individual and combined effects of each tea component on adrenal hypertrophy were examined in 34 groups of mice. The volumes that had been consumed were not significantly different among these groups. Mean volume was 10.00 [+ or -] 1.21 ml/mouse/day. Mean body weight, which was 31.28 [+ or -] 1.07 g, was not different among these groups.

First, the relationship between theanine and caffeine was assessed. Adrenal hypertrophy in mice under confrontational housing was significantly suppressed by drinking theanine (0.32 and 3.2 mg/kg/day), but not in mice that ingested caffeine (0.32 and 3.2 mg/kg/day) (Fig. 4a). While adrenal hypertrophy was significantly suppressed in mice that ingested theanine (3.2 mg/kg/day) with caffeine (0.32 mg/kg/day), the suppression was abolished in mice that ingested theanine with high doses of caffeine (F (8, 26) = 6.567; p = 1.1 x [10.sup.-4]; [[eta].sup.2] =0.45; one-way ANOVA). The dose-dependent suppression of caffeine against theanine suggests the importance of a lower amount of caffeine in green tea for the anti-stress effect of theanine.

EGC, which is 10 (w/w) times higher than theanine, did not suppress the effect of theanine, and low dose of EGC (3.2 mg/kg/day) showed an anti-stress effect (F (6, 20) = 6.853; p = 4.6 x [10.sup.-4]; [[eta].sub.2] = 0.45; one-way ANOVA; Fig. 4b). Unlike EGC, the gallate catechins EGCG and ECG strongly abolished the effect of theanine (F (6, 20)= 1.219; p = 0.34; [[eta].sup.2] = 0.07; one-way ANOVA; Fig. 4c). Adrenal hypertrophy was significantly suppressed in mice that ingested Arg but not Glu and Gin (F (5, 20)= 11.972; p = 1.9 x [10.sup.-5]; [[eta].sup.2] =0.56; one-way ANOVA; Fig. 4d).

The combined effects of theanine, caffeine, EGC, EGCG and Arg showed that Arg significantly enhanced the anti-stress effect of theanine in the presence of caffeine or EGCG on psychosocial stress induced adrenal hypertrophy in mice. On the other hand, EGC suppressed the effect of EGCG, resulting in an enhanced anti-stress effect of theanine (F (10, 33) = 4.433; p = 5.5 x [l0.sup.-4]; [[eta].sup.2] = 0.33; one-way ANOVA; Fig. 4e).

Discussion

Ann-stress effect of theanine, and counteracting effects of caffeine and catechins on theanine

Adrenal hypertrophy, a typical stress response in living organisms, was significantly suppressed in stressed mice that ingested theanine (0.32 mg/kg). This was much lower than the concentration of theanine in standard Sencha green tea (5.0 mg/kg), suggesting that other tea components suppress the effect of theanine. Although caffeine has been reported to antagonize theanine (Kakuda et al., 2000), we found that EGCG remarkably suppressed the anti-stress effect of theanine. Whereas the oral bioavailability of catechins and the distribution of EGCG in the brain is very low (Nakagawa et al., 1997; Suganuma et al., 1998), the suppression of EGCG against theanine suggests that EGCG was incorporated into the brain and abolished the effect of theanine.

On the other hand, EGC had an anti-stress effect at a low concentration (3.2 mg/kg), and suppressed the effect of EGCG (Fig. 4e). The relationship among theanine, EGCG and EGC suggests that EGC antagonizes EGCG, whereas EGCG antagonizes theanine. Since the blood-brain permeability of EGCG was significantly lowered by the same molar amount of EGC using a model of the blood-brain barrier (unpublished data), intestinal and blood-brain permeability of EGCG may be competitively suppressed by EGC. A lowered incorporation of EGCG into the brain results in lowered suppression against theanine. However, EGC was needed at more than three (w/w) times higher level (or 5 times higher molar ratio) than EGCG to compete inhibition, suggesting that EGC is a weak inhibitor of EGCG.

If so, then why did EGC have no effect on theanine? Catechins are excreted by efflux transporters expressed on the cell membrane (Kadowaki et al, 2008) and the activity of efflux transporters are suppressed by the presence of a gallate moiety (Annaba et al., 2010; Farabegoli 2010). Efflux of EGC from the brain may reduce suppression against theanine.

Whereas green tea is usually steeped in hot water and EGCG is infused as the main catechin, non-gallate catechins such as EGC and EC are mainly eluted into water at room temperature. EGCG is abundant in tea leaves; therefore, regulation of catechin composition by steep temperature is important for determining the anti-stress effect of green tea.

The amount of caffeine was higher in Gyokuro than in standard Sencha. However, the relative amount of caffeine and gallate catechin against theanine was higher in standard Sencha than in Gyokuro. These results suggest that a balance among theanine, caffeine and catechins is important for anti-stress effect of green tea. A reduction of caffeine resulted in a relative increase of theanine in Sencha. In addition, low-caffeine Sencha is useful for people such as infants, pregnant women and elderly people who do not want to take a high dose of caffeine. If drinking green tea has similar benefits for humans as mice, it is expected to reduce stress in many people by switching to the daily ingestion of green tea from other beverages.

Anti-stress effects of amino acids in green tea

Theanine incorporated into brain is reported to reduce the release of glutamate from pre-synapse to the synaptic cleft by acting as a Gin transporter and inhibiting the incorporation of extracellular Gin into neurons, which suppresses the conversion of Gin to Glu by glutaminase (Kakuda et al., 2008; Kakuda 2011). Glu can be decarboxylated into [gamma]-amino butyric acid (GABA). In the hippocampus of mice that ingested theanine (6 mg/kg) in drinking water for 2 weeks, the levels of Glu and pyroglutamic acid were significantly reduced, and conversely GABA increased (Inoue et al., 2016), indicating that theanine modulates GABA production from Glu. Glu is the main excitatory neurotransmitter while GABA is the main inhibitory neurotransmitter in the brain. Changes in Glu and GABA metabolism may play important roles in the control of neuronal excitability. Since GABA in tea and foods is not able to cross the blood-brain barrier and as such must be synthesized within neurons (Martin and Rimvall, 1993), modulation of GABA production by theanine would be important in the brain.

Whereas suitable synaptic excitation is important, excessive excitation damages nerve cells and triggers neurodegenerative diseases (Mehta et al., 2013), suggesting that the suppression of excessive Glu is important. On the other hand, excessive synaptic inhibition may be attributed to Down syndrome, and EGCG suppresses over-expression of the GABA pathway in Down syndrome mouse models (Souchet et al., 2015). This data supports the notion that theanine and EGCG modulate GABA production antagonistically.

Arg is considered to be an important regulator in the central nervous system through nitric oxide synthesis (Virarkar et al., 2013) and has vital functions in physiological stress and anxiety (Gulati and Ray, 2014). Although the amount of Arg is lower than that of theanine (Tables 1 and 2), Arg has a rather higher anti-stress effect than theanine (Fig. 4). High contents of both theanine and Arg are important for the anti-stress effects of green tea, which is characteristic of high-grade green teas (Mukai et al., 1992; Miyauchi et al., 2014). These results indicate that high-grade green tea has an anti-stress effect. Theanine is the major amino acid in all tea cultivars. However, the difference in Arg content among cultivars was larger than the other amino acids, and cultivars native to Japan had more Arg than Assam hybrid cultivars (Ikeda et al., 1993). A quality assurance study that considers the functions of green tea is necessary.

Conclusions

Ingestion of green tea, such as low-caffeine Sencha and theanine-rich Gyokuro that were eluted with water at room temperature, exhibited an anti-stress effect on mice under stressful conditions. In these infusions, the amount of theanine, EGC and Arg were relatively enriched and caffeine and EGCG were lowered. The data of individual and combined effects of each tea component indicates that the ingestion of theanine and Arg, the main amino acids in green tea, suppressed adrenal hypertrophy in mice, while caffeine and gallate catechins such as EGCG and ECG abolished the anti-stress effects of theanine. In addition, non-gallate EGC retained the anti-stress effect of theanine by suppressing EGCG.

Since Gyokuro exhibited anti-stress effects in mice, ingestion of high-grade green tea such as Gyokuro is highly expected to have an anti-stress effect in humans. However, because the content of caffeine is high in Gyokuro, low-caffeine Sencha is a valuable green tea to suppress stress in people who do not want to take a high dose of caffeine.

ARTICLE INFO

Article history:

Received 4 January 2016

Revised 13 July 2016

Accepted 19 July 2016

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

This research study was supported by a Grant-in-Aid for Scientific Research (KAKENHI 23617014 and 15K00828) and a grant for specially promoted research of the University of Shizuoka. We thank Dr. Hara at University of Chicago for her valuable discussion.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.07.006.

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Keiko Unno (a,c), *, Ayane Hara (a), Aimi Nakagawa (a), Kazuaki Iguchi (a), Megumi Ohshio (b), Akio Morita (b), Yoriyuki Nakamura (c)

(a) Department of Neurophysiology. School of Pharmaceutical Sciences. University of Shizuoka. Shizuoka. Japan

(b) Department of Functional Plant Physiology. Faculty of Agriculture. Shizuoka University. Shizuoka. Japan

(c) Tea Science Center. Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka. Shizuoka. Japan

Abbreviations: Ala. alanine; Arg. arginine; Asn, asparagine; Asp. aspartic acid; (+) C. (+)-catechin; EC, (-)-epicatechin; ECC, (-)-epicatechin gallate; EGC, (-)epigallocatechin; EGCG, (-)-epigallocatechin gallate; GABA, [gamma]-amino butyric acid; Glu, glutamate; Gin, glutamine; Ser, serine.

* Correspondence author at: University of Shizuoka, School of Pharmaceutical Sciences, Department of Neurophysiology, 52-1 Yada. Suruga-ku, Shizuoka 4228526. Japan. Fax: +81 54 264 5909.

E-mail address: unno[R]u-shizuoka-ken.ac.jp (K. Unno).

http://dx.doi.Org/10.1016/j.phymed.2016.07.006

Table 1
Amount of caffeine, catechins and amino acids in tea leaves
that were sprayed with hot-water shower.

Treatment       Caffeine (mg/g)
time (s)

0             21.0 [+ or -] 0.42
180         * 4.99 [+ or -] 0.22
280         * 4.03 [+ or -] 0.12

Treatment   Catechins (mg/g)
time (s)
                EGCC             ECC             ECC

0               42.6            24.9            8.57
            [+ or -] 0.57   [+ or -] 0.64   [+ or -] 0.06
180             37.1            22.9            7.47
            [+ or -] 0.64   [+ or -] 0.57   [+ or -] 0.18
280             47.8            26.8             8.9
            [+ or -] 4.95   [+ or -] 3.04   [+ or -] 0.78

Treatment   Catechins (mg/g)
time (s)
                 EC              CG

0               8.09            0.19
            [+ or -] 0.47   [+ or -] 0.02
180             6.67            0.15
            [+ or -] 0.03   [+ or -] 0.04
280             8.36            0.11
            [+ or -] 0.87   [+ or -] 0.01

Treatment   Catechins (mg/g)
time (s)
                (+)C            Total

0               0.96            85.3
             [+ or -] 0.07  [+ or -] 1.77
180             0.84            76.1
             [+ or -] 0.07  [+ or -] 1.70
280             0.96            92.9
             [+ or -] 0.03  [+ or -] 9.55

Time (s)    Free amino acids (mg/g)

            Theanine   Glu    Arg    Asp    Gln

0             7.91     1.53   3.96   0.80   1.28
180           7.36     2.34   4.71   2.04   1.21
280           6.58     1.97   4.58   1.17   0.84

Time (s)    Free amino acids (mg/g)

               Ser      Ala   Asn    GABA   Total

0             0.42     0.19   0.06   0.07   16.2
180           0.62     0.28   0.31   0.19   19.1
280           0.72     0.34   0.08   0.23   16.5

The amount of each tea component in dried tea leaves was
measured by HPLC.

(* p < 0.05)

EGCC. (-)-epigallocatechin gallate: ECC. (-)-
epigallocatechin; ECC. (-)-epicatechin gallate; EC. (-)-
epicatechin; CC. (-)-catechin gallate; (+)C, (+)-catechin;
Clu. glutamic acid; Arg. arginine; Asp. aspartic acid; Gin.
glutamine; Ser. serine; Ala, alanine; Asn. asparagine; CABA.
[gamma]-butyric acid.

Table 2
Amount of caffeine, catechins and amino acids in the eluate
from each green tea.

Green tea        Caffeine (mg/l)

Low-caffeine      0.07
Theanine-rich    12.1
Standard          5.3

Green tea        Catechins (mg/l)

                 EGCC   ECC   ECG   EC     CG    (+)C   Total

Low-caffeine     0.2    8.0   0.1    7.3   0     0.6    16.1
Theanine-rich    0.3    5.3   7.2   10.4   0.5   1.9    25.5
Standard         0.2    7.6   8.1   14.6   0     1.3    31.7

Green tea        Free amino acids (mg/l)

                 Theanine   Glu    Arg    Asp   Gin

Low-caffeine     50         12.0   13.4   8.0   10.7
Theanine-rich    102         5.8    8.8   3.6    5.2
Standard         18          4.4    6.6   2.9    3.9

Green tea        Free amino acids (mg/l)

                 Ser   Ala   Asn   CABA   Total

Low-caffeine     3.2   1.6   0.7   1.1    101
Theanine-rich    3.5   1.3   0.8   0      131
Standard         1.3   0.5   0.2   0.4    39

Tea leaves (3 g) from each green tea were added to 11 of
water at room temperature, stirred for 6 min, then filtered.
The amount of each tea component in the eluate was measured
by HPLC.

Table 3
Ingestion of green tea and main tea components consumed by
mice.

Green tea           Low-caffeine            Theanine-rich

Body weight (g)     32.58 [+ or -] 2.46     33.96 [+ or -] 1.19
Ingestion volume    9.40 [+ or -] 0.95      * 12.86 [+ or -] 1.20
  (ml/day/mouse)
Caffeine (mg/kg)    * 0.02 [+ or -] 0.002   * 4.6 [+ or -] 0.4
EGCG (mg/kg)        0.06 [+ or -] 0.005     * 0.11 [+ or -] 0.009
EGC (mg/kg)         23 [+ or -] 0.5         2.0 [+ or -] 0.2
Theanine (mg/kg)    * 14.5 [+ or -] 13      * 38.6 [+ or -] 3.1
Arg (mg/kg)         * 3.9 [+ or -] 0.4      * 3.3 [+ or -] 0.3

Green tea           Standard

Body weight (g)     33.62 [+ or -] 2.45
Ingestion volume    9.40 [+ or -] 1.31
  (ml/day/mouse)
Caffeine (mg/kg)    1.5 [+ or -] 0.2
EGCG (mg/kg)        0.06 [+ or -] 0.007
EGC (mg/kg)         2.1 [+ or -] 0.5
Theanine (mg/kg)    5.0 [+ or -] 0.6
Arg (mg/kg)         1.8 [+ or -] 0.2

The value of each component of low-caffeine and theanine-
rich green teas was compared with that of the standard green
tea group (mean [+ or -] SD, n = 6. * p < 0.05).


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Author:Unno, Keiko; Hara, Ayane; Nakagawa, Aimi; Iguchi, Kazuaki; Ohshio, Megumi; Morita, Akio; Nakamura, Y
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
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Date:Nov 15, 2016
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