Theanine

Contents

What is theanine

What is theanine

L-Theanine, also known as L-gamma-glutamylethylamide or N-gamma-ethyl-L-glutamine, is a member of the class of compounds known as glutamine and derivatives ¹⁾. These compounds contain glutamine or a derivative thereof resulting from a reaction of glutamine at the amino group or the carboxy group, or from the replacement of any hydrogen of glycine by a heteroatom. L-Theanine is slightly soluble (in water) and a moderately acidic compound (based on its pKa). L-Theanine can be found in saliva. L-theanine, 2-Amino-4-(ethylcarbamoyl) butyric acid, an amino acid found in green tea (Camellia sinensis), is sold in the United States as a dietary supplement to reduce stress and improve cognition and mood ²⁾. Tea is known to be a rich source of flavonoid antioxidants and tea also contains L-theanine that may modulate aspects of brain function in humans ³⁾. Theanine (γ-glutamylethylamide) is the major amino acid in tea and is thought to be a flavorous constituent of tea leaves. Theanine constitutes between 1 and 2% of the dry weight of tea3 which corresponds to 25-60mg per 200ml serving. Within tea the predominant form of theanine is the L isomer. The analysis of 17 tea samples carried out by Ekborg-Ott et al., revealed that the average theanine content of black teas was 1.40%, as compared to 1.42% in green teas, and 1.16% in oolong teas. The widest range of theanine content (0.60–1.72%) was detected in oolong samples. One of the black tea samples contained the highest amount of theanine (2.38%) ⁴⁾. L-theanine has been reported to produce anxiolytic effects in humans ⁵⁾, including increasing alpha wave activity, which is associated with relaxed alertness ⁶⁾.

Figure 1. L-theanine

Although the mechanism of action for the pharmacological effects of L-theanine remain to be established, L-theanine increases CNS levels of GABA, dopamine, and serotonin, as well as inhibits glutamate receptors ⁷⁾. Moreover, studies indicate that L-theanine increases brain-derived neurotrophic factor (BDNF) levels ⁸⁾, ⁹⁾ and L-theanine has also been show to have antagonistic effects on N-methyl d-asparate (NMDA) receptors ¹⁰⁾. Thus, the observations that BDNF levels can be mediated by glutamate actions at NMDA receptors ¹¹⁾ suggest that the antagonistic properties of L-theanine on NMDA receptors may contribute to L-theanine’s behavioral effects. Glutamate is released during opioid withdrawal ¹²⁾ and, thus, L-theanine’s antagonistic effects on NMDA receptors ¹³⁾ may account for its ability to reduce opioid withdrawal signs in morphine-dependent rhesus monkeys. Additionally, given the anxiolytic properties of serotonin and GABA ¹⁴⁾, L-theanine may produce its anxiolytic-like actions via its effects on these neurotransmitters.

The regulatory status of theanine varies by country. In Japan, L-theanine has been approved for use in all foods, including herb teas, soft drinks, and desserts. Restrictions apply to infant foods. In the United States, the Food and Drug Administration (FDA) considers theanine to be generally recognized as safe (GRAS) and allows its sale as a dietary supplement. The German Federal Institute for Risk Assessment, an agency of their Federal Ministry of Food and Agriculture, objects to the addition of L-theanine to beverages. The European Food Safety Authority EFSA advised negatively on health claims related to L-theanine and cognitive function, alleviation of psychological stress, maintenance of normal sleep, and reduction of menstrual discomfort ¹⁵⁾. Therefore, health claims for L-theanine are prohibited in the European Union ¹⁶⁾. L-Theanine is found in mushrooms and is a constituent of tea (Camellia sinensis) ¹⁷⁾ and in the edible bay boletes mushroom Boletus badius ¹⁸⁾. L-Theanine has been shown to exhibit neuroprotectant and neuroprotective functions in laboratory test tube study ¹⁹⁾. The research found that cells treated with L- theanine showed decreased production of nitric oxide resulting from the down-regulated protein levels of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). These results indicate that the inhibition of the N-methyl d-asparate (NMDA) subtype of glutamate receptors and its related pathways is the crucial point of the neuroprotective effect of L- theanine in the cell model ²⁰⁾.

L- theanine is thought to cross the blood-brain barrier to exert its effects directly on the brain within 30 minutes ²¹⁾. L- theanine’s brain effects were first reported in humans following a randomized, placebo-controlled, crossover study comparing placebo, 50 and 200mg L-theanine. The study was originally published in Japanese ²²⁾ and later reviewed in English ²³⁾. Relative to placebo, the higher dose of L-theanine increased power in the alpha frequency band (8-13Hz) of the electroencephalogram (EEG) across parietal and occipital sites after approximately 40 minutes, which indicates that L- theanine relaxes the mind without inducing drowsiness. This effect has only been established at higher doses than that typically found in a cup of black tea (~20mg).

However, L-theanine was effective only in participants classified as highly anxious using a manifest anxiety scale. A more recent study has since confirmed that L-theanine is most effective in individuals who generally have high levels of anxiety ²⁴⁾.

In the study ²⁵⁾ on the effect of L-theanine on the resting brain activity in young healthy human volunteers demonstrated that L-theanine significantly modulates the resting state of brain activity. L-theanine enhanced the power in the alpha-1 frequency band (8-10Hz), replicating previous reports ²⁶⁾. In that experiment, the power in the alpha band increased linearly with time and this linear increase was significantly enhanced by L-theanine. This increase in alpha-band activity supports a role for L-theanine in achieving a relaxed but alert mental state via a direct influence on the central nervous system.

Furthermore, activity in the alpha-band is also a key component in focused attentional processes ²⁷⁾. Directed deployment of alpha appears to be critical to the ability to suppress distracting visual information during highly demanding tasks. Recently, L-theanine has also been shown to enhance this so-called alpha attention effect also ²⁸⁾. In a follow-up study, other researchers tested whether an analogous alpha-mediated attention effect seen in visuospatial attention tasks is also affected by L-theanine ingestion. L-Theanine, at a dosage of 250 mg, was not found to increase the differential effect of attention. However, in a replication of the previous intersensory attention study ²⁹⁾, overall tonic alpha was greatly reduced on L-theanine ³⁰⁾.

L-theanine and caffeine

In recent years, several potential health benefits of drinking tea (Camellia sinensis) have come to light through systematic study of the effects of its constituent compounds ³¹⁾. Although anecdotal evidence abounds, the psychological and neurophysiological effects of tea have received relatively little experimental investigation and thus remain unclear. Popular claims have centered on generalized state changes such as the reduction of stress and induction of relaxed wakefulness. Psychopharmacological studies have indeed demonstrated mood effects that support these claims and have further shown that tea affects elements of cognition ³²⁾. Although caffeine (1,3,7-trimethylxanthine) is by far the constituent most studied, with findings of increased alertness and speeded reaction time predominant ³³⁾, there exists evidence that caffeine alone cannot fully account for the positive effects of tea drinking. Tea has been shown to raise skin temperature to a higher level ³⁴⁾, to increase critical flicker fusion threshold ³⁵⁾ and to reduce physiological stress responses and increase relaxation ratings ³⁶⁾ when compared with coffee or other control beverages matched for caffeine level.

L-Theanine (γ-glutamylethylamide), a unique amino acid present almost exclusively in the tea plant, has recently received research interest in the neuroscience community with findings of neuroprotective effects ³⁷⁾ and mood effects indexed both by subjective self-reports ³⁸⁾ and via psychological and physiological responses to stress ³⁹⁾. Using electroencephalographic (EEG) recordings in humans, Kobayashi et al. ⁴⁰⁾ and Juneja et al. ⁴¹⁾ reported that activity within the alpha frequency band (8–14 Hz) increased in reaction to L-theanine ingestion when measured during a state of rest. This was of interest to the attention community, as the alpha rhythm has long been known to be sensitive to overall attentional states (i.e., intensity aspects such as arousal) ⁴²⁾ and, further, is involved in the biasing of selective attention ⁴³⁾. In intersensory attention tasks, where the relevant modality is cued ∼1 s before a compound audiovisual target stimulus, parieto-occipital alpha power in the intervening period is increased for attend-visual trials relative to attend-auditory trials ⁴⁴⁾. In Gomez-Ramirez et al. ⁴⁵⁾, this differential effect of cue information on anticipatory alpha amplitude was found to be larger on ingestion of 250 mg of L-theanine relative to placebo. In addition, tonic (background) alpha amplitude was relatively decreased for L-theanine, in apparent contradiction to the findings of Juneja et al. ⁴⁶⁾. In a follow-up study, other researchers tested whether an analogous alpha-mediated attention effect seen in visuospatial attention tasks is also affected by L-theanine ingestion. L-Theanine, at a dosage of 250 mg, was not found to increase the differential effect of attention. However, in a replication of the previous intersensory attention study ⁴⁷⁾, overall tonic alpha was greatly reduced on L-theanine ⁴⁸⁾.

Table 1. Theanine and caffeine content of teas

[Source ]

Human intervention studies on black tea

Three of the human intervention studies provided (described in the two publications/reports) investigated the effects of black tea characterized by its content of tea solids, caffeine and l‐theanine on measures of attention ⁵⁰⁾.

Two randomized, double‐blind, placebo‐controlled, cross‐over, two‐period, intervention studies are reported in one publication ⁵¹⁾.

The first study included 26 healthy volunteers (mean age 30.7 ± 11.2 years; 20 females) who were regular caffeine consumers. The authors stated that the sample size was loosely based on previous observational tea studies. Participants abstained from caffeinated foods for 15 hours before visiting the laboratory. After completing baseline attention tests, participants consumed one 200 mL beverage, followed by a second 200 mL beverage 50 min after the first. Beverages were either black tea (providing 520 mg of tea solids, 50 mg of caffeine and 23 mg of l‐ theanine per serving; cumulative amount of 1,040 mg tea solids, 100 mg caffeine and 46 mg l‐theanine) or placebo (i.e. coloured and tea‐flavoured water). The black tea was prepared by pouring 235 mL of boiled, de‐ionised water onto a PG Tips tea bag. The tea was passively infused for 60 seconds after which the teabag was removed and allowed to drip over the cup for 3 seconds. The 200 mL of the infusion was then poured and served in a fresh cup. The placebo was prepared by adding 10 mg caramel colour, 10 μL red food colour, 7 mg tea flavor, 150 mg oak tannin and 150 mg grapeseed tannin powders to 200 mL boiled, de‐ionized water. The authors noted that the placebo tea flavors and colors have no known effects on cognitive performance. Interventions were allocated using a Latin square design such that the order of the beverages was counterbalanced across participants, with visits separated by 6–14 days.

Attention performance was measured at baseline and after each serving using two standardized tasks: the Attention Switching Task and the Intersensory Attention Task.

The Attention Switching Task measured ability to shift attention between different stimuli displayed on a screen. Participants had to respond to even numbers when the font color was purple and vowels when the font color was red. The font color (and therefore the task) was switched every three trials in a predictable manner. The Intersensory Attention Task measured ability to attend to stimuli presented in the visual and auditory modalities. On each trial, an auditory cue instructed participants to attend to either the auditory or visual modality. This was followed by the presentation of stimuli in either the auditory modality or the visual modality (unisensory), or both modalities (intersensory), and participants had to report whether the stimuli were the same or different.

The proportion of correct responses and reaction times on the attention tests were analysed using a 2 × 2 mixed model analysis of covariance (ANCOVA) with a repeated‐measures covariance structure. Subjects were modelled as a random effect, and treatment (black tea or placebo) and session were used as within‐subject factors. Baseline scores and treatment order were included as covariates.

On the Attention Switching Task, participants were significantly more accurate after consuming black tea compared to placebo, but reaction times did not differ significantly. The increased accuracy with no increase in reaction time can be interpreted as an improvement in attention. On the Intersensory Attention Task, participants were more accurate in the black tea condition on both the auditory and visual intersensory subtasks, and had faster reaction times on the visual intersensory subtask, but not on the auditory‐attention subtask.

In the second study with a similar design, 32 healthy volunteers (mean age 30.3 ± 10.1 years; 15 females) who were regular caffeine consumers and had abstained from caffeinated foods for 15 hours, consumed three 200 mL servings of a beverage administered at 40‐min intervals over the course of 80 min. Each serving contained either black tea (cumulative amount of 1,140 mg tea solids, 90 mg caffeine and 36 mg l‐theanine) or placebo. Methods for the allocation of interventions and washout periods were as in study one. Sample size was reported to be based on the results of study one.

The tea was prepared by pouring 190 mL of boiled, de‐ionized water onto a Lipton Yellow Label tea bag. The tea was passively infused for 90 seconds, after which the tea bag was removed and allowed to drip over the cup for 3 seconds. A quantity of 10 mL of water at room temperature was added. The placebo was prepared by matching color and flavor with the tested back tea as in study one.

Participants were assessed on the Attention Switching and Intersensory Attention Tasks at baseline and after each serving. Results were analyzed as in study one. Accuracy on the Attention Switching Task was significantly greater after black tea compared to placebo, but there were no significant differences in reaction time. Performance on the Intersensory Attention Tasks showed no significant effects of black tea compared to placebo.

These two studies with a similar design showed a consistent effect of black tea on attention when attention performance was assessed using the Attention Switching Task. The effects of black tea on attention when using Intersensory Attention Tasks were inconsistent between the studies, with the first study showing a significant effect of black tea on attention and the second study showing no effect compared to placebo. These two studies taken together support an effect of black tea on attention, and that the effect is observed for 2–3 servings of black tea consumed over 50–80 min providing a cumulative dose of tea solids, caffeine and l‐theanine of about 1 g, 90–100 mg and 36–46 mg, respectively.

Giesbrecht et al. ⁵²⁾ reported on a randomized, four‐period, cross‐over, placebo‐controlled, double‐blind study of the effects of black tea consumption on attention. A total of 40 healthy regular caffeine consumers (mean age 26.1 ± 8.5 years; 20 females) were recruited. Participants were instructed to refrain from food and beverages containing caffeine or l‐theanine in the 24 h prior to testing.

At each test session, participants consumed two servings of a beverage (200 mL each) separated by an interval of 75 min. The beverages were:

regular strength tea (cumulative amount of 1,224 mg tea solids, 100 mg caffeine and 38 mg l‐theanine);
1.5 strength tea (cumulative amount of 1,704 mg tea solids, 150 mg caffeine and 57 mg l‐theanine);
double strength tea (cumulative amount of 2,126 mg tea solids, 200 mg caffeine and 76 mg l‐theanine);
placebo (deionised water with food grade colours and flavours, 0 mg caffeine and 0 mg l‐theanine).

Each participant received all four beverages (each beverage on a different testing day, four testing days in total per participant) in a balanced order achieved with a Williams square design for 40 participants. Testing days were separated by 7 ± 2 days and the study duration for each participant was approximately 5 weeks.

The primary outcome was accuracy on the Attention Switching Task. A power analysis based on the results by De Bruin et al. ⁵³⁾ estimated that a sample of 32 participants was necessary to detect a difference in accuracy of 1.6% with a power of 0.90 and an alpha of 0.05. Allowing for a dropout rate of 20% and considering the study design, 40 subjects were recruited for the study. The Attention Switching Task was performed according to the protocol reported by De Bruin et al. ⁵⁴⁾.

The secondary outcome was performance on the Attention Network Test (ANT), which measures three different components of attention (Alerting, Orienting and Executive Control) within a single task. On each trial, participants are required to indicate the direction of a central arrow which is surrounded by flanker arrows. Each trial is preceded by one of four possible cue conditions.

The attention tasks were administered at baseline, 40 min after the first serving of the beverage, and 35 min after the second serving.

Intention‐to‐treat analyses were conducted on all participants who received at least one test beverage. Per protocol (PP) analyses excluded participants on medical grounds, non‐compliance with the protocol or on the basis of extreme results in cognitive tasks. No participants were excluded from data analyses because of missing data. No formal imputation was performed in the event of excluded or missing data. There were no drop outs and no missed visits.

The primary response was based on a contrast comparing the interventions corresponding to 0 cups and 2 cups of tea.

Intention‐to‐treat analyses on the Attention Switching Task showed that regular strength black tea significantly reduced reaction time, but had no effect on accuracy as compared to placebo. The reduction in reaction time with no reduction in accuracy can be interpreted as an improvement in attention. Compared to placebo, 1.5 strength black tea did not significantly reduce reaction time, but did improve accuracy. Double strength black tea compared to placebo significantly reduced reaction time and increased accuracy.

In dose–response intention‐to‐treat analyses, a significant linear dose–response effect for accuracy (increased) and reaction time (decreased) was observed across beverages with increasing doses of tea solids, caffeine and l‐theanine. Similar results were obtained in per protocol analyses.

Intention‐to‐treat analyses on the Attention Network Test (ANT) showed that all three black tea beverages increased accuracy compared to placebo, but did not differ significantly from each other. There was a significant linear dose–response effect. All three doses of black tea significantly reduced reaction times compared to placebo and there was a significant linear dose–response effect. Similar results were obtained in per protocol analyses.

This study shows an effect of black tea on attention, and that the effect is observed for two servings of black tea consumed over 75 minutes providing a cumulative dose of tea solids, caffeine and l‐theanine of 1,224 mg, 100 mg and 38 mg, respectively.

Mechanism of action

The effect depends on the concerted action of two substances, caffeine and l‐theanine, both of which are present in black tea. Caffeine has a structure that is similar to adenosine, which is a central nervous system neuromodulator. When adenosine binds to its receptors, neural activity slows down. Caffeine binds to the same receptors, and when this occurs it prevents adenosine from binding and thus slowing neural activity. Some of these receptors occur in the striatum, which explains the ability of caffeine to enhance motor activity, as well as general neural activity ⁵⁵⁾. Caffeine also affects the dopaminergic system ⁵⁶⁾, which is involved in the control of attention. The effects of caffeine on the central nervous system and consequent influence on the control of attention are well established ⁵⁷⁾.

With regard to the possible effects of l‐theanine on the central nervous system, l‐theanine can cross the blood–brain barrier ⁵⁸⁾ and it was hypothesized that l‐theanine may compete with endogenous amino acids for transport into the brain, and may modulate the activity of neurotransmitters involved in attention processes, including glutamate, glycine and gamma‐aminobutyric acid (GABA) ⁵⁹⁾. Such modulation could occur by l‐theanine binding to a variety of receptors and blocking signal transduction between neurons ⁶⁰⁾. L-Theanine may indirectly affect neurotransmitter concentrations by interfering with the availability of their precursors, and thus affect cognition. The proposed mechanism of action for l‐theanine is speculative, and that no evidence was provided on the effects of l‐theanine on attention.

The claim related to caffeine and increased attention has already been evaluated with a positive outcome ⁶¹⁾. The evidence provided by consensus opinions/reports and by the majority of the studies submitted for the scientific substantiation of the claim showed good consensus on the role of caffeine in increasing attention, measured by a range of psychometric tasks, in healthy individuals of both sexes, at doses of at least 75 mg. Caffeine has a plasma half‐life of about 4 hours with range of about 2–8 hours, and that the kinetics of caffeine are linear up to very high (~ 500 mg) doses ⁶²⁾. In this context, the cumulative doses of caffeine consumed over a period of 90 min are likely to exert similar effects on attention that the same dose of caffeine consumed on a single occasion.

Among the human studies assessing the effects of caffeine and l‐theanine on attention in a different product matrix than black tea, and they could provide evidence that caffeine and l‐theanine in combination have a greater effect on attention than caffeine alone to support a claim on black tea rather than on caffeine.

Among the studies provided which investigated the effects of caffeine and/or l‐theanine on attention but did not use black tea as the intervention, two investigated the effects of caffeine and l‐theanine in combination but not the effects of caffeine alone ⁶³⁾, ⁶⁴⁾ and two did not report on between‐group statistical comparisons for the caffeine plus l‐theanine vs the caffeine interventions ⁶⁵⁾, ⁶⁶⁾. No conclusions can be drawn from these studies with respect to the effects of the combination of caffeine and l‐theanine vs caffeine alone on attention.

Five additional human intervention studies addressed the effects of caffeine plus l‐theanine vs caffeine alone on attention ⁶⁷⁾, ⁶⁸⁾, ⁶⁹⁾, ⁷⁰⁾, ⁷¹⁾.

In a randomized, four‐period, cross‐over, placebo‐controlled study already provided with the previous application, Rao and Nobre ⁷²⁾ investigated the effects of caffeine and l‐theanine consumption on attention. A total of 20 healthy volunteers (aged between 18 and 35 years, 10 females) were recruited. The study beverages (tea base) contained, per cup, 49 mg caffeine and 49 mg l‐theanine (CT‐high), 49 mg caffeine and 23 mg l‐theanine (CT‐low), 49 mg caffeine and no l‐theanine (C), and 5.27 mg of l‐theanine and no caffeine (placebo). The Panel notes that the nature of the tea base was not specified.

Participants were asked to refrain from drinking beverages containing caffeine, including coffee, tea, cola or other soft drinks, chocolate (including any chocolate products) on the day of the experiment prior to arrival at the laboratory. Three cups of the test beverages were ingested over 80 min, each administration being separated by 40‐min intervals. Each session lasted approximately 3 h. The respective cumulative doses for the study beverages were 147 mg caffeine and 147 mg l‐theanine (CT‐high), 147 mg caffeine and 69 mg l‐theanine (CT‐low), 147 mg caffeine with no l‐theanine (C), and 16 mg l‐theanine and no caffeine (placebo). The order of experimental conditions was counterbalanced between participants.

Attention performance was measured with five standardised tasks: Flanker Interference Task (FLA), Attentional Blink Task (AB), Cued Spatial Orienting Task (CUED), Visual Oddball Task and Auditory Oddball Task. The series of five tasks were performed successively in a block, with a total of three blocks per session. The first block was administered 40 min after consuming the first drink, the second block 40 min after consuming the second drink, and the third block 50 min after consuming the third drink.

In the Flanker Interference Task (FLA), participants were significantly more accurate in the CT‐high (49 mg caffeine and 49 mg l‐theanine), CT‐low (49 mg caffeine and 23 mg l‐theanine) and C (49 mg caffeine and no l‐theanine) conditions compared to placebo, but the three caffeine‐containing conditions did not differ from each other. There were no significant differences involving reaction time. There were no significant differences between conditions in performance on the Attentional Blink Task (AB) task. In the Cued Spatial Orienting Task (CUED), there was a significant main effect of condition, but individual comparisons between the three caffeinated conditions and placebo were not reported. There were no significant differences for reaction time. In the Visual Oddball Task, participants were more accurate in the C (49 mg caffeine and no l‐theanine) and CT‐high (49 mg caffeine and 49 mg l‐theanine) conditions compared to placebo, but performance in the CT‐low (49 mg caffeine and 23 mg l‐theanine) and placebo conditions did not differ significantly, and there were no significant differences involving reaction time. In the Auditory Oddball Task, accuracy was significantly higher in all the caffeinated conditions compared to placebo, but comparisons between the caffeine plus l‐theanine and caffeine alone conditions were not reported. There were no significant differences for reaction time.

This study does not show an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on attention.

Dodd et al. ⁷³⁾ carried‐out a randomized, four‐period, cross‐over, placebo‐controlled, double‐blind study with 24 healthy participants (mean age 21.8 years) consuming two capsules providing a cumulative dose of (a) 75 mg caffeine, (b) 50 mg l‐theanine, (c) 75 mg caffeine and 50 mg l‐theanine and (d) placebo. Participants attended four study visits separated by at least 48 h. Attention tests administered at each study visit included Serial 3 subtractions, Serial 7 subtractions, RVIP, Choice Reaction Time and Stroop Colour‐Word Test. Attention performance was examined with a mixed model and significant effects or interactions were further explored with Bonferroni‐corrected pairwise comparisons. There were no significant differences between the caffeine and caffeine plus l‐theanine treatments on any attention measure.

This study does not show an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on attention.

Foxe et al. ⁷⁴⁾ carried‐out a randomized, four‐period, cross‐over, placebo‐controlled, double‐blind study with 21 healthy participants (mean age 26 years) consuming one 200 mL serving containing (a) 50 mg caffeine, (b) 100 mg l‐theanine, (c) 50 mg caffeine and 100 mg l‐theanine, and (d) placebo. Participants attended four study visits separated by an average of 4 days. The order of treatment across days was randomised and counterbalanced across participants The Sustained Attention to Response Inhibition Task (SART) was administered at each study visit. Sustained Attention to Response Inhibition Task (SART) omission errors, commission errors and reaction times were examined with Generalized Linear Mixed Models in which treatment order, caffeine, l‐theanine and the interaction of Caffeine x l‐Theanine were fitted as fixed effects, and subject was included as a random effect. There were no significant differences between the caffeine and caffeine plus l‐theanine treatments on any attention measure.

This study does not show an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on attention.

Owen et al. ⁷⁵⁾ carried‐out a randomized, three‐period, cross‐over, placebo‐controlled, double‐blind study with 27 healthy participants (mean age 28.3 years) consuming one 250 mL serving containing (a) 50 mg caffeine, (b) 100 mg l‐theanine and (c) 50 mg caffeine and 100 mg l‐theanine. Participants attended three study visits separated by at least 7 days. Attention at each study visit was measured by RVIP and Attention Switching tests administered twice at 60 min and 90 min post‐ingestion. Attention scores were examined with a mixed model analysis of variance (ANOVA). There were no significant treatment effects for the RVIP test, but there was a significant treatment x time interaction for number of correct responses on the Attention Switching Test. At 60 min post‐ingestion, the number of correct responses was significantly higher in the caffeine and l‐theanine intervention compared to the caffeine intervention, but at 90 min post‐ingestion the number of correct responses was significantly higher in the caffeine intervention compared to the caffeine and l‐theanine intervention.

This study does not show an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on attention.

Kahathuduwa et al. ⁷⁶⁾ carried‐out a randomized, five‐period, cross‐over, placebo‐controlled, open‐label study with 20 healthy male volunteers (mean age 21.9 years) consuming one 150 mL serving of (a) caffeine (160 mg), (b) l‐theanine (200 mg), (c) caffeine (160 mg) and l‐theanine (200 mg) combined (all in water), (d) black tea and (e) placebo (water). The caffeine and l‐theanine content of the black tea dose was not reported. Each subject was given five treatments in five consecutive days.

Simple visual reaction time (SVRT) and recognition visual reaction time (RVRT) tasks were administered at baseline and again from 30 min until 1 h post‐consumption. The RVRT task was a type of go/no go task in which participants were required to respond to only one of the two stimuli. Reaction time was measured in both tasks, but accuracy was measured only in the recognition visual reaction time (RVRT) task. An auditory odd‐ball task was also administered during which auditory event‐related potentials (ERPs) were recorded. The event‐related potential (ERP) is an electrophysiological measure of cortical activity in which the N2 negative wave occurring 200 ms after stimulus onset is related to covert (internal) orienting of attention towards relevant information, and increased selective attention is identified by a greater amplitude in the N2 component. Because the baseline correction for averaged waveforms was not possible owing to technical limitations, the N2‐P300 peak to peak amplitude was obtained by measuring the amplitude difference between the N2 and the P300 peaks.

Recognition visual reaction time (RVRT) reaction time and accuracy data were examined in a time x treatment two‐way within‐subject ANOVA model. The Panel notes that no statistical comparisons between the l‐theanine and caffeine plus l‐theanine treatments were reported for either RVRT reaction time or accuracy. A one‐way ANOVA on post‐dose N2‐P300 amplitudes showed a significant treatment effect and N2‐P300 amplitudes were significantly greater in the caffeine plus l‐theanine treatment compared to the caffeine treatment.

This study shows an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on an electrophysiological measure of attention.

The four studies ⁷⁷⁾, ⁷⁸⁾, ⁷⁹⁾, ⁸⁰⁾ did not show an effect of the combination of caffeine and l‐theanine above the effect of caffeine alone on measures of attention performance, and that one study ⁸¹⁾ reported improved performance on one electrophysiological measure of attention with the caffeine plus l‐theanine intervention compared to caffeine alone. No study showed an effect on behavioural measures of attention performance.

These studies do not show an effect of caffeine and l‐theanine on attention above the effect of caffeine alone, and that the mechanism of action for l‐theanine on attention is speculative. It is well established that caffeine increases attention in healthy adult individuals of both sexes at doses of at least 75 mg ⁸²⁾ and that cumulative doses of caffeine consumed over a period of 90 minutes are likely to exert similar effects on attention as the same dose of caffeine consumed on a single occasion owing to the pharmacokinetics of caffeine ⁸³⁾. Therefore, the effect of black tea on attention observed in the three human intervention studies, two studies by De Bruin et al. ⁸⁴⁾; Giesbrecht et al., 2012 ⁸⁵⁾ can be explained by its caffeine content. Therefore, owing to its caffeine content, black tea improves attention. In order to obtain the claimed effect, 2–3 servings of black tea providing at least 75 mg of caffeine in total should be consumed within 90 min.

Interestingly, caffeine induces panic attacks and anxiety at high doses ⁸⁶⁾ and also reduced sleep efficiency ⁸⁷⁾, an effect that was partially blocked by L-theanine ⁸⁸⁾. L-theanine may counteract the stimulatory effect of caffeine. In rats, theanine administered intravenously after caffeine dosing, and at approximately the same dose, blunted the stimulant effect of caffeine seen on electroencephalographic recordings. When given by itself in a smaller dose (20-40% of the original dose), theanine administration resulted in excitatory effects, suggesting a dual activity of theanine, depending on the dose ⁸⁹⁾.

L-theanine benefits

L-theanine anxiety

In clinical studies, L-theanine increased alpha wave activity, which is associated with decreases in stress and anxiety ⁹⁰⁾. L-theanine had anxiolytic effects in comparison with placebo during a relaxed state, but lacked these effects during an experimentally induced anxiety state ⁹¹⁾ suggesting that it can decrease baseline anxiety, but does not affect anticipatory anxiety. However, in an acute stress task L-Theanine reduced heart rate and salivary immunoglobulin A responses as compared to a placebo ⁹²⁾. In another clinical study L-theanine increased calmness but decreased alertness ⁹³⁾. Co-administered with antipsychotic treatments, L-theanine reduced anxiety symptoms in schizophrenia and schizoaffective disorder patients ⁹⁴⁾, an effect that was significantly associated with increases in serum levels of brain-derived neurotrophic factor (BDNF) ⁹⁵⁾.

Owen et al. ⁹⁶⁾ conducted a study to compare 50 mg caffeine, with and without 100 mg L-theanine, on cognition and mood in healthy volunteers. The effects of these treatments on word recognition, rapid visual information processing, critical flicker fusion threshold, attention switching and mood were compared to placebo in 27 participants. Performance was measured at baseline and again 60 min and 90 min after each treatment (separated by a 7-day washout). Caffeine improved subjective alertness at 60 min and accuracy on the attention-switching task at 90 min. The L-theanine and caffeine combination improved both speed and accuracy of performance of the attention-switching task at 60 min, and reduced susceptibility to distracting information in the memory task at both 60 min and 90 min. These results replicate previous evidence which suggests that L-theanine and caffeine in combination are beneficial for improving performance on cognitively demanding tasks.

Cancer

For cancer, L-theanine in conjunction with chemotherapy the dose is speculative, as no human studies have been performed. L-theanine has been studied extensively in test tubes and lab animals for its effects on tumor cells and the sensitivity of those cells to chemotherapeutic agents. It appears theanine competitively inhibits glutamate transport into tumor cells, which causes decreased intracellular glutathione (GSH) levels. Theanine also inhibits the efflux of chemotherapeutic agents, such as doxorubicin, idarubicin, cisplatin, and irinotecan, causing them to accumulate in tumor cells. Theanine also protects normal cells from damage by these drugs via antioxidant activity, specifically by maintaining cellular glutathione levels ⁹⁷⁾.

L-theanine, given along with doxorubicin, reduced the size of ovarian tumors and decreased metastases to the liver as well ⁹⁸⁾. In another study, theanine almost doubled the effect of doxorubicin in Erlich ascites carcinoma, while increasing the drugʼs concentration in tumor cells threefold ⁹⁹⁾. It appears theanine exerts an additive effect along with chemotherapy by reducing transport of glutamic acid into the cell, decreasing GSH levels in the cell, and increasing the concentration of the drug in tumor cells. Theanine also protects normal cells from damage by chemotherapeutic drugs ¹⁰⁰⁾, ¹⁰¹⁾.

Theanine increases the antitumor effect of doxorubicin (DOX) with decreasing adverse reaction. Glutathione S-transferase activity did not change in the theanine-alone group whereas increased in the theanine and doxorubicin (DOX)-combined group. These results suggested that theanine combination increased the conjugate with DOX and glutathione, promoted the efflux of glutathione S-transferase-DOX conjugates from the liver, and decreased DOX concentration in the liver ¹⁰²⁾. Similarly, theanine enhanced the antitumor activities of other anthracyclines, cisplatin and irinotecan ¹⁰³⁾. Consequently, the modulating effect of theanine on the efficacy of antitumor agents is expected to be applicable in clinical cancer chemotherapy.

Alcoholic liver injury

Li et al. ¹⁰⁴⁾ evaluated the protective effects of l-theanine on ethanol-induced liver injury in vitro and in vivo. The results revealed that l-theanine significantly protected hepatocytes against ethanol-induced cell cytotoxicity which displayed by decrease of viability and increase of LDH and AST. Furthermore, the experiments of DAPI staining, pro-caspase3 level and PARP cleavage determination indicated that l-theanine inhibited ethanol-induced L02 cell apoptosis. Mechanically, l-theanine inhibited loss of mitochondrial membrane potential and prevented cytochrome c release from mitochondria in ethanol-treated L02 cells. l-Theanine also prevented ethanol-triggered ROS and MDA generation in L02 cells. l-Theanine restored the antioxidant capability of hepatocytes including glutathione content and SOD (Superoxide Dismutase) activity which were reduced by ethanol. In vivo experiments showed that l-theanine significantly inhibited ethanol-stimulated the increase of ALT (Alanine Transaminase), AST (Aspartate Transaminase), TG (Triglyceride) and MDA (3,4-methylenedioxyamphetamine) in mice. Histopathological examination demonstrated that l-theanine pretreated to mice apparently diminished ethanol-induced fat droplets. In accordance with the in vitro study, l-theanine significantly inhibited ethanol-induced reduction of mouse antioxidant capability which included the activities of SOD, CAT and GR, and level of glutathione. These results indicated that l-theanine prevented ethanol-induced liver injury through enhancing hepatocyte antioxidant abilities ¹⁰⁵⁾.

Cardiovascular effects

A dose-dependent hypotensive effect was demonstrated in spontaneously hypertensive rats, but not in normotensive ones. The effect may have been related to reduction in central levels of dopamine and serotonin ¹⁰⁶⁾. Whether humans will experience similar results has yet to be determined; however, theanine might find a place in antihypertensive treatment regimens. In healthy volunteers, synthetic theanine 200 mg muted the increase in heart rate response to an acute stress test, while theanine has been observed to antagonize the hypertensive effect of caffeine. L-theanine exerts a weak (compared with green tea) antioxidant action as measured by the inhibition of LDL oxidation ¹⁰⁷⁾.

CNS effects

Although the pharmacological effects of theanine are uncertain, several researchers have proposed a number of mechanisms by which it may act on the CNS. These include the inhibition of glutamate receptors, increasing the concentration of gamma-aminobutyric acid (GABA), increasing dopamine and serotonin in specific brain regions, and neuroprotective blockage of multiple glutamate receptor subtypes in the hippocampus, suggesting a potential role in Parkinson disease ¹⁰⁸⁾.

L-theanine is able to cross the blood-brain barrier, and most research has focused on its relaxing effect. Studies have evaluated the effect of theanine alone or in combination with caffeine in concentrations naturally found in tea, and use self-reporting measures of stress and electroencephalographic recordings of brain activity. However, study data are inconsistent in methodology and outcome measures, making comparisons difficult.

An effect of wakeful relaxation of theanine alone is apparent in most published data. The effect is weak in comparison with benzodiazepines, and may differ with consumption of theanine in the relaxed state versus an already anxious state. Some studies suggest a positive contribution to cognitive performance ¹⁰⁹⁾.

Immune system functioning

A limited number of studies conducted primarily by 2 groups of researchers suggest L-theanine may enhance the action of immune system components.

Results of in vitro studies, a pilot study, and a small clinical trial suggest that the enhancement of gamma delta T lymphocytes may play a role in the observed decrease in cold and influenza symptoms. A combination preparation was used in the clinical trial ¹¹⁰⁾.

Other researchers have evaluated the combined effect of L-cystine and L-theanine on the immunologic response to vaccinations in the elderly and to exercise in athletes, as well as in rats, and suggest an enhanced immunologic response ¹¹¹⁾.

L-theanine dosage

Data supporting a clinical role for theanine are weak. Studies reporting an anxiolytic effect used single doses of theanine 200 to 250 mg ¹¹²⁾, ¹¹³⁾. No dosage of L-theanine is suggested for enhanced immune system functioning.

L-theanine crosses the blood-brain barrier, with effects evident within 30 minutes and measurable up to 5 hours after administration ¹¹⁴⁾.

Theanine side effects

Few adverse reactions have been reported. Adverse reactions recorded in human pharmacokinetic studies using tea extracts include headache, dizziness, and GI symptoms. Clinical trials used small numbers of participants and reported poorly on adverse events. One study among elderly participants recorded a higher number of reported headaches among those receiving 4 doses of theanine 250 mg ¹¹⁵⁾.

L-theanine is generally well tolerated, and has a median lethal dose (LD ₅₀ ) of greater than 5,000 mg/kg in rats. It is not mutagenic or carcinogenic in animals or bacteria ¹¹⁶⁾.

Information regarding safety and efficacy in pregnancy and lactation is lacking ¹¹⁷⁾.

A toxicological study in rats showed no effect on behavior, morbidity, mortality, body weight, hematology, or urinalysis. An increased incidence of renal tubule adenomas in a small number of female rats given high dosages (400 mg/kg body weight per day) was attributed to genetic predisposition ¹¹⁸⁾.

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