The experiment conducted in this study was approved by the research ethics committee of the University of Shizuoka, and was carried out in accordance with the Declaration of Helsinki.
Sixteen healthy volunteers (students, eight men, eight women; ages, 22.8±2.1 years) participated in the experiment individually at similar times of the day at an interval of 7 days. The data from two women were excluded from the analyses because they were absent on at least 2 experiment days owing to temporary illness. All participants were requested to avoid eating or drinking, except for water, from 3 h before the start of each trial.
A cross-over, randomized, placebo-controlled design was used in this study. In total, three separate trials were performed, in which the participants orally took either L-theanine (200 mg, Taiyo Kagaku Co., Tokyo, Japan) + placebo, caffeine (100 mg, Shiratori Pharmaceutical Co., Chiba, Japan) + placebo, or placebo only on each day. Dextrin (Nisshin Pharma Inc., Tokyo, Japan) was used as the placebo. All sample capsules were taken with 250 mL warm water at about 25°C. Treatments were allocated using a Latin square design such that the order of treatments was counterbalanced across participants.
Yokogoshi et al.
] reported that L-theanine increased by 1 h at the latest in the serum, the liver, and the brain after administration, and thereafter decreased sharply in the serum and liver
]. Van der Pijl et al.
] reported that L-theanine plasma concentration reached the peak between 32 and 50 min after oral ingestion, and its half-life ranged from 58 min to 74 min in humans
]. Terashima et al.
]. also reported that L-theanine could influence the secretion and function of neurotransmitters in the central nervous system even at 30 min after oral administration
]. On the other hand, caffeine absorption from the gastrointestinal tract is rapid and reaches 99% in about 45 min after ingestion, while peak plasma caffeine concentration is reached between 15 min and 120 min, and half-life ranges from 2.5 h to 4.5 h after oral ingestion in humans
]. To allow a peak of both L-theanine and caffeine appears during the stress load period, sample treatment was decided to be taken at 36 min before the end of the mental tasks session (DT and AT as defined below), followed by subjective assessment which was performed from 38 min to 43 min, physiological measurement from 44 min to 45 min, and physical stress task session (CPT) from 45 min to 49 min after the sample treatment.
Stress load task
After each sample was taken, an auditory oddball target detection task (DT) lasting for 5 min each and an arithmetic mental task (AT) lasting for 10 min each were both imposed twice as the psychological stress load. In the DT, participants were required to click the left button of a computer mouse as quickly as possible to target stimuli (a single tone of 2,000 Hz lasting for 0.1 s) that occur infrequently and irregularly within a series of standard stimuli (a single tone of 1,000 Hz lasting for 0.1 s). The AT required participants to add two numbers (each from 1 to 9) that were being displayed on a PC monitor and to enter the answer through the keyboard quickly and accurately. The number and accuracy of the answers to the second AT, which was taken from 26 min to 36 min after each sample intake, were used for data analysis.
A cold pressor test (CPT) was taken to establish physical acute stress
]. Participants were asked to immerse their right hand, past the level of the wrist, for 1 min in a bucket filled with slushy ice water (1.5±0.3C) and then to place the hand on the table nearby with a towel underneath the hand.
The Profile of Mood States (POMS) and the visual analogue scales (VAS) for subjective ratings on mood state were also completed before the intake as a basic control and after all of the mental tasks were finished.
The short version of POMS was used to assess distinct affective mood states. POMS is a popular tool that is widely used among psychologists and scientists in many fields. Six identifiable mood or affective states can be measured and were used for analysis in this study: Tension-Anxiety (T-A), Depression-Dejection (D), Anger-Hostility (A-H), Vigor-Activity (V), Fatigue-Inertia (F), and Confusion-Bewilderment (C).
VAS comprised five scales including feelings of fatigue, relaxation, arousal, pressure, and tension. At the end of each trial, the subjects used the scales to rate their painful feelings about accomplishing the CPT and their feelings of annoyance about DT and AT.
Arterial pressure in each participant’s left thumb was recorded continuously by Finometer Pro (FMS, Finapres Measurement Systems, Arnhem, the Netherlands). Simultaneously, skin temperature of the back of the left hand was recorded using a BioAmplifier (Polymate AP1132, TEAC, Tokyo, Japan). The sampling rate was 200 Hz. As baseline data, both the blood pressure and skin temperature were measured for 1 min before the intake. Measurement after mental tasks (AMT) was also made for 1 min at 44 min after the intake of each sample, followed by measurement for 4 min after CPT was started.
Baseline data were calculated by averaging the 1 min data before each intake. Differences in blood pressure and skin temperature from the baseline were calculated using the mean value of every 10-s epoch for the above measurements after intake. The first 10-s epoch of the AMT was described as AMT1, and the second, third, fourth, fifth, and sixth 10-s epochs were described as AMT2, AMT3, AMT4, AMT5, and AMT6, respectively. Similarly, CPT1 to CPT6 for the CPT epochs, and RP1 to RP18 for epochs during the 3-min recovering period after the 1 min CPT were named respectively and used for the analysis.
shows the experimental procedure. Each participant was required to attend a total of 3 study days, which were conducted 7 days apart, to ensure a sufficient washout between conditions. Prior to the start of the experiment, all participants were given the opportunity to familiarize themselves with all of the stress load tasks. The experiments took place in a quiet room. The room temperature was 26.4±1.1°C, and the humidity was 51.5±6.8%. On each experiment day, each participant entered the room and rested for 15 min. During the resting time, a skin-surface temperature probe was attached, and POMS and VAS were completed. After the rest, a 1-min physiological measurement session to obtain baseline data took place, followed by sample treatment. After the oral administration, mental tasks were performed: DT (5 min), rest (2 min), AT (10 min), and rest (2 min); the cycle was then repeated. Then, POMS and VAS and another 1-min measurement were completed again to obtain data after the mental tasks. CPT for 1 min was then started. At the same time, measurement was recorded for 4 min (1 min for CPT, 3 min for RP after CPT). At last, VAS about feelings of DT, AT, and CPT was completed.
Data were analyzed using IBM SPSS Statistics version 19. Prior to the primary statistical analysis, separate, one-way, repeated measures ANOVAs of the baseline data were conducted to ascertain any chance baseline differences across study days prior to the treatments.
L-theanine reduced blood pressure in spontaneously hypertensive rats but not in rats with normal blood pressure
]. Thus it is considerable that L-theanine might act in different ways between people in whom stress increases whose blood pressure in different ways. With this in mind, we divided the participants into two groups after the experiment according to their changes in systolic blood pressure after the mental tasks in the placebo intake condition. The half of participants who showed greater than average changes in blood pressure were sorted into a high-response group and the other half into a low-response group.
Differences in blood pressure and skin temperature from the basic control were calculated and used for a repeated-measures ANOVA with group (high-response group and low- response group), treatment (L-theanine, caffeine, and placebo), and epoch (six epochs for AMT, CPT and 18 epochs for CPT). Repeated-measures ANOVA with group and treatment was also applied to the task performance data. A Tukey’s honestly significant difference (HSD) post hoc test was applied to data groups with significant main effect (P <0.05). Differences in POMS and VAS scores were analyzed using the nonparametric Friedman test to detect differences in treatments. The Wilcoxon signed rank test was further carried out to evaluate the changes among treatments.