Participants
Eighteen healthy volunteers (9 males, 9 females; ages 23.4 ± 1.85 years) participated in 3 experimental trials at the same time of day with an interval of 7 days between trials. All participants were requested to avoid eating or drinking anything but water for three hours before the start of each trial. 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. All subjects provided informed consent after receiving an explanation of the experimental protocol.
Procedure
Figure 1 shows the experimental procedure. All participants were required to complete sessions on a total of 3 study days with 1-week intervals between experiment days; total session time per day was about 1.5 hours. Experimental sessions were held from 09:00 to 10:30 in July. The room temperature was 25.8°C. On the day of the experiment, when participants entered the room, they were seated and asked to rest for ten minutes. During the resting time, participants rinsed their mouths with a cup of water. After resting, the first subjective assessment and the first saliva collection were completed to obtain baseline data. Participants then drank one of the beverage samples, followed by the second subjective assessment. They then began the 31-minute mental stress load task session (2 Uchida-Kraepelin (U-K) tests with a 1-minute rest). The stress load task was followed by a measurement session that included the second saliva collection and third subjective assessment. At about 35 minutes after sample ingestion, participants performed another 31-minute mental stress load task session. Finally, a third saliva collection and fourth subjective assessment were completed.
Treatment
A cross-over, single blind, randomized design was used in this study. Three separate trials were performed in which the participants ingested one of three beverage samples (water as a control and two kinds of green tea: Sagara and shaded white tea) each day. Sagara and shaded white tea were made from the Japanese Yabukita tea plant by a different process. Shaded white tea was specifically grown in perfectly blocked sunlight before plucking. Tea samples were prepared using each of their ordinary brewing method for serving these different kinds of green tea [13]. Sagara was extracted with 900 ml of 90°C hot water for 30 seconds from 20 g of Sagara tealeaf; shaded white tea was extracted with 900 ml of 70°C hot water for 2 minutes from 30 g of the tealeaf. After extraction, tealeaves were removed and 250 ml of the tea sample was cooled to 25°C and then poured into a paper cup for subjects to drink. The warm water used as the control sample was prepared by cooling hot water to 25°C.
Levels of amino acid components in the two tea samples were measured in sulfosalicylic acid deproteinized samples by Ion Exchange Chromatography using a JLC-500/V AminoTac™ Amino Acid Analyzer (JEOL Ltd., Tokyo, Japan) [14]. L-theanine (Suntheanine, Taiyo Kagaku Co., Ltd, Mie, Japan) and the amino acids mixture standard solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were used as reference amino acid standards. The concentrations of caffeine and catechins in the two tea samples were measured with the Acquity UPLC System (Waters Corp., Milford, MA, US), using Acquity BEH Shield RP18 column with 2.1 × 100 mm inner diameter and 1.7 μm particle size [15].
Stress load task
The U-K test was used as the stress load task. The test, a questionnaire modified from Kraepelin’s arithmetic test and developed by Uchida [16], is widely used as a mental stressor [17–19]. In this study, participants were given a pre-printed paper containing 15 lines of random, single-digit, horizontally aligned numbers, and asked to perform calculations as quickly and accurately as possible for 15 minutes. After a one-minute rest period, the test was repeated, so that one stress-load session lasted about thirty-one minutes. Two sessions were performed in each trial. The average number of answers and percentage of correct answers for each test were used as indices of task performance.
Subjective assessment
The POMS and VAS, used for subjective ratings on mood state, were completed before ingestion (baseline data), immediately after ingestion, and again after the two mental task sessions were completed.
We used a short version of POMS to assess distinct affective mood states. The POMS is a popular tool used widely among psychologists and scientists from many fields. Six identifiable mood or affective states were scored from the POMS: Tension-Anxiety, Depression-Dejection, Anger-Hostility, Vigor-Activity, Fatigue-Inertia, and Confusion-Bewilderment. Total Mood Disturbance (TMD) was then calculated from these six scores. A higher TMD score indicated a more negative affective state; that is, positive changes in mood were reflected by negative changes to TMD scores. All of the scores were used for analysis in this study.
Participants’ feelings of pressure, drowsiness, stress, relaxation, fatigue, reassurance and tension were assessed using VAS. A continuous, 10-cm VAS rating scale was used, with the 0 end point representing ‘do not feel’ and the 10 end point indicating ‘strongly feel’. Subjects were asked to make a mark on the line that represented their mood at the time. Immediately after ingestion, two more scales were also included in the VAS asking about preference and familiar experience with the drink samples.
Saliva collection and CgA measurements
Saliva was collected in Salivette tubes (Sarstedt, Nümbrecht, Germany) and centrifuged at 3,000 rpm. The supernatant was transferred into Eppendorf tubes and frozen at -80°C for later measurement. Concentration of CgA in the saliva samples was subsequently determined by ELISA (Yanaihara Institute Inc., Shizuoka, Japan). At the same time, total protein was measured with the dye-binding assay of Bradford (Bio-Rad Protein Assay, Hercules, CA, US). Measured values of CgA were divided by the protein concentration and used for further analysis.
Statistical analysis
Data were analyzed using IBM SPSS Statistics version 19 (Chicago, IL, USA). All data are expressed as the mean ± standard error, and P < 0.05 was considered significant.
Time effects
Across the four time points (before intake, immediately after intake, after U-K test two, and after U-K test four), the effect of time in POMS scores were analyzed by Friedman tests and Wilcoxon signed rank tests with Bonferroni correction.
Kruskal-Wallis tests and Mann-Whitney U-tests with Bonferroni correction were conducted to detect differences of CgA concentrations among the four time points.
Sample effects
Repeated measure one-way analysis of variance (ANOVA) and Bonferroni post hoc tests were performed to detect differences of task performance among the three sample conditions (warm water, Sagara and shaded white tea).
Changes of VAS score regarding mood state and changes of POMS score from baseline were analyzed by Kruskal-Wallis tests.
VAS scores immediately after intakes asking about preference and familiar experience with the drink samples and the concentrations of CgA were analyzed by Friedman tests and Wilcoxon signed rank tests with Bonferroni correction.