Theanine is an L-glutamate analogue and a non-protein amino acid that is particular to the tea plant (Camellia sinensis
(L.) Kuntze) [1
]. The amount of theanine, which is the most abundant amino acid in green tea leaves, depends on nitrogen supply absorbed from the roots [2
]. Matcha is a fine-powdered green tea that is prepared from tea leaves protected from sunlight. When tea leaves are protected from direct sunlight, their amino acid content, especially theanine, remains high because the hydration of theanine used in the biosynthesis of catechin is lowered [3
]. To make matcha, cultivation under shade for about three weeks is necessary before harvest [5
]. Given their protection from sunlight, catechin content is lower in matcha than in other popular green teas prepared from leaves grown in sunlight [3
]. In addition, matcha has a high content of caffeine because the buds and young leaves of Camellia
plants contain more caffeine than mature leaves [7
]. The balance of these components, such as theanine, caffeine, and catechin, determines the quality of the green tea. A higher content of amino acids indicates a higher level of “umami” ingredients. Therefore, matcha is essentially the best-grade green tea, rich in theanine and caffeine, but with a low content of catechin compared with popular green tea.
Chronic psychosocial stress is associated with the development of depression, mood disorders, and various other stress-related diseases [8
]. Addressing stress-induced alterations with dietary supplements is a potential therapeutic strategy for a healthy life. Green tea is the most popular drink in Asian countries and its consumption and that of theanine has revealed health benefits and medicinal potential for several ailments [11
]. Theanine exhibits an excellent stress-reducing effect on mice and humans [12
]. However, the effect of theanine is antagonized by caffeine and epigallocatechin gallate (EGCG), which are two major components of green tea [14
]. In contrast, the stress-reducing effect of theanine is enhanced by arginine (Arg), which is the second most abundant amino acid in Japanese green tea [17
]. Glutamate (Glu), the third most abundant amino acid in green tea, has no effect on stress in mice [17
]. To enhance the stress-reducing effect of green tea, low-caffeine green tea with reduced caffeine content can be prepared from tea leaves by irrigating them with hot water at 95 °C for three minutes. Published data showed that a significant stress-reducing effect of low-caffeine green tea was observed in participants in their 20 s, 40 s–50 s, and 80 s–90 s relative to barley tea or standard green tea [18
Matcha is expected to have a stress-reducing effect due to its high theanine content, although this has not been scientifically proven. Previous studies described above suggested that differences in the quantities and ratios of green tea components affect the efficiency of its stress-reducing action. Therefore, the stress-reducing effect of matcha, which contains catechins, caffeine, and amino acids whose contents were measured, was evaluated in an animal (mouse) experiment. Mice were stressed using territorial conflict between male mice [14
]. The stress-reducing effect of matcha was evaluated as the suppression of adrenal hypertrophy in stressed mice because adrenal glands are sensitive to stress [14
]. The relationship between suppressed adrenal hypertrophy and quantities or ratios of matcha tea components was examined. Based on these data, test-matcha with contents expected to have a stress-reducing effect, as well as placebo-matcha with contents expected to have no stress-reducing effect, were selected for a clinical trial. Participants, who were selected for a double-blind randomized controlled trial, consumed matcha (3 g) suspended in 500 mL water daily. They were fifth year college students of the University of Shizuoka, School of Pharmaceutical Sciences, Japan, who were assigned to a pharmacy practice outside the university, such as a hospital or a pharmacy. Commitment to a new environment provides a stressful condition for young students. Since anxiety is a reaction to stress, to assess the anxiety of participants, the state-trait anxiety inventory (STAI) test was administered before and on the eighth day of pharmacy practice. In addition, to assess the physiological stress response, the activity of salivary α-amylase activity (sAA), an oral cavity enzyme, was measured as a stress marker of sympathetic excitement [15
]. This enzyme rapidly increases in response to physiological and psychosocial stress [21
]. We examined whether test-matcha was able to reduce participants’ stress. Finally, based on the quantity and ratio of components of each matcha, 76 matcha samples sold in Japan and 67 samples sold abroad were evaluated to determine whether mental function such as anxiety and stress could be expected when humans consume these forms of matcha.
Based on the quantities and ratios of matcha components, the stress-reducing effect of matcha was evaluated by mice experiments using seven kinds of matcha. These matcha samples were selected based on differences in theanine content. Since the amount of theanine in the tea leaves depends on the amount of nitrogen supply in the soil, one matcha with low theanine content was sourced from organic cultivation. The matcha sample containing high amounts of EGCG might be prepared from leaves that were not fully protected from the sunlight before harvest. Additionally, a sample with low amounts of all components was a matcha product containing additives.
Since theanine and Arg reduce stress and caffeine and EGCG antagonizes the action of theanine [16
], we examined how these levels affect adrenal hypertrophy in stressed mice. In mice, adrenal hypertrophy was suppressed when the amount of theanine ingested was 0.32 mg/kg or more. Since the effect of theanine is cancelled by caffeine and EGCG, a molar ratio of caffeine and EGCG to theanine and Arg must be less than 3.6. Although EGC has a stress-reducing effect, EGC needed to be more than 10 times higher than EGCG [17
]. As EGC was not higher than EGCG in matcha, the effect of EGC was not considered. Matcha essentially contains a high amount of caffeine, but if theanine is sufficiently high, the anti-theanine action of caffeine is counteracted. Catechin in matcha is less than in general green teas, but its increase is accompanied by a decrease in theanine. Therefore, an increase in catechin in matcha potentially increases the reduction of the stress-reducing effect of matcha.
Using test-matcha, which meets these conditions, we conducted a study in humans. Whereas the distribution of sAAm, a marker of physiological stress, was similar in the test and placebo groups before matcha was consumed, anxiety (STAI) and physiological stress (sAAm) decreased when test-matcha was consumed. The significant decrease in sAAm at university due to the intake of test-matcha may have contributed to the decrease in anxiety before pharmacy practice. Although caffeine consumption is reportedly linked to anxiety sensitivity [25
], the caffeine content in test- and placebo-matcha were not different (Table 2
). Additionally, the amount of caffeine ingested by participants was about 40 mg/day, which is about one-quarter cup of coffee, so drinking it every day would not cause a problem. Rather, in the case of matcha, the counteracting effects of caffeine and EGCG against theanine and Arg are considered to be important.
Whereas EGCG has been reported to have anxiolytic effects [27
], the antagonistic effect of EGCG on theanine may be due to an increased excitatory synaptic connection by EGCG [28
]. Glutamate is the main excitatory neurotransmitter and EGCG reportedly facilitates the release of glutamate [29
]. Although central glutamatergic activity is crucial to cognitive function, excessive release of glutamate causes excessive excitation. The level of GABA, the main inhibitory neurotransmitter in the brain, is increased by theanine ingestion [30
]. These data suggest that different ratios of EGCG/theanine cause a different balance of excitatory and inhibitory neurotransmitters such as glutamate and GABA.
Since the active component of matcha could be applied to evaluate the stress-reducing effect of matcha in humans, we then tried to assess the stress-reducing effect of matcha marketed in Japan and overseas. When people ingested three grams of these matcha per day, we examined whether stress-reducing effects would be expected. The results showed that, about 42% of matcha sold in Japan was expected to suppress stress versus only one of the matcha marketed overseas, because the amounts of theanine and Arg were low, and the amounts of caffeine and EGCG were high. Matcha is essentially rich in theanine and low in catechin, but not all marketed matcha satisfied this condition. This indicates that greater attention is needed to evaluate the mental function of matcha. Although the number of people drinking green tea as a health beverage is increasing in many countries, low-grade green teas with low amino acid contents are also sold as “matcha” [32
]. Many matcha marketed overseas had low amino acids contents (Figure 5
, Table S2
). The counteracting effect of caffeine and EGCG on theanine is hardly considered. This may cause confusing results. For example, the effect of matcha tea on mood and cognitive performance has been examined [33
]. The mood state measured by a Profile of Mood State (POMS) was not significantly changed by the ingestion of four grams of matcha tea. The matcha tea that those authors used contained 67 mg theanine, 280 mg EGCG, and 136 mg caffeine. Theanine content was slightly less than 17 mg/g. Although the content of Arg was not reported, the molar ratio of caffeine and EGCG against theanine was higher than two. This suggests that the result of POMS might have been different if those authors had used another matcha sample with a lower content of caffeine and EGCG. EGCG has been reported to have various beneficial functions [34
]. However, when expecting mentally positive functions of matcha in anxiety, stress, and mood, the quantities of theanine and Arg must be high, and the ratios of caffeine and EGCG against theanine must be low.