Ethics
This clinical study adhered to the ethical standards of the Declaration of Helsinki of 1964, as modified by subsequent revisions, and to the ethical guidelines for epidemiological research of the Ministry of Education, Culture, Science, and Technology and the Ministry of Health, Labor and Welfare of Japan. The experimental protocol (no. 2016-009) was approved by the Institutional Review Board of Kirin Group Japan Integrated Beverages, Kirin Company, Ltd. This study was registered with the University Hospital Medical Information Network (UMIN) Clinical Trials Registry as UMIN000026566 and was conducted in compliance with the protocol as registered. This clinical trial was conducted from March 2017 to May 2017 in Kirin company ltd, Kanagawa, Japan.
Participants
Healthy adult males and females aged 20 to 65 years old, who had subjective symptoms of eyestrain, were enrolled, and written informed consent was obtained from each individual. The exclusion criteria were as follows: a possible onset of allergy symptoms; treatment for or a history of drug addiction or alcoholism; any history of a serious disease (e.g., heart disease, respiratory disorder, digestive disturbance, endocrine disorder, metabolic disturbance, or food allergy); constant use of pharmaceuticals for a chronic malady; any history of ophthalmic disease; a surgical history related to a digestive organ; donation of more than 200 mL of blood or blood components within a month prior to this study or over 400 mL of blood or blood components within three months prior to this study; constant use of pharmaceuticals, dietary supplements, or functional foods affecting eyestrain; excessive alcohol consumption; and possible pregnancy, pregnancy, or lactation. Also excluded participants were those unable to stop drinking on the day before the experimental day; participants or possible participants in another clinical study; those judged ineligible on the experimental day, prior to the beginning of the study, by the site investigators for any reason.
Target sample size
Based on a preliminary human study, administration of aG-rutin PS 500 mg was expected to reduce HFC1 value by 0.8 with a standard deviation of 1.6 compared to the placebo group. Setting the significance level at 5% by a paired t-test and the power at 0.95, the number of subjects required per group was estimated to be 18. To account for an expected dropout rate of 10%, the number of subjects required per group was estimated to be 20.
Test food
We used aG-rutin PS as an MGR-containing food ingredient which was purchased from Toyo Sugar Refining Co.,Ltd (Tokyo, Japan), Ceolus® FD301 as microcrystalline cellulose which was purchased from Asahi Kasei Corporation (Tokyo, Japan), hard gelatin capsule (capsule size 1) which was purchased from Capsugel Japan company. (Kanagawa, Japan). We prepared active and placebo capsules using dark brown capsuls. Table 1 shows prescription of test capsules. There was no significant difference in the appearance and other properties between the active and placebo capsules. MGR was measured by high-performance liquid chromatography using a previously described method [12]. The active capsule contained 188.5 mg of MGR, and the placebo capsule did not contain MGR.
Study design
The present study was designed as a randomized, double-blind, placebo-controlled, crossover study. The site investigator enrolled the participants. The participants were divided into two groups by stratified randomization using table of random numbers by the assigning controller, and each group was assigned either the active or placebo capsule before the first trial period. Randomization block size was 2. The assigning controller kept the assignment list in a sealed container until the trial was completed. The participants, investigators, and any persons concerned with the study, excluding the assigning controller, remained blinded.
During the test period, the participants were forbidden to take a pharmaceutical for eyestrain, drink alcohol excessively, vigorously exercise, take food containing ingredients affecting eyestrain, participate in another clinical study, and participate in blood donation or sampling.
On the day before the experimental day, the participants were forbidden to have a drink, vigorously exercise, consume food containing food ingredients affecting eyestrain or blood flow, as well as drink and eat after 22:00. The participants were asked to maintain a daily life as usual; keep a diary every day and submit it on the experimental day; contact the site investigator immediately if they felt ill during or after the test period; contact the clinic immediately if they were unable to visit the test room on the experimental day; and keep the test information confidential.
On the experimental day, the participants were forbidden to take food or beverage without water, have VDT such as that on a smartphone or personal computer (PC) working, and smoke.
First, the subjects acclimated to the test room conditions for 20 min. Before the sample intake, we measured the eye status as the pre-intake data (0 min). Then, the participants took the test food (two capsules) with 100 mL of water. After the test food intake, the subjects did work at VDT with PC for 30 min and relaxed for 20 min. Eyestrain was measured after relaxing. We evaluated the primary endpoint; high-frequency component 1 (HFC1) as an objective eyestrain parameter, measured by AA-2 accommodometer (Nidec Corp., Tokyo, Japan) according to the manufacturer’s instructions [13]. We also evaluated the secondary endpoint; subjective symptoms of eyestrain, dry eye, and sleep, stiffness of the neck and shoulder, and waist stiffness using a visual analog scale (VAS) questionnaire [14], with the left side of VAS showing worse feelings and the right side showing better feelings. The VAS score was calculated as a distance from the left (worst) side. The study protocol is accessed in Kirin company.
Statistical analysis
Results were calculated as the mean ± standard deviation using the Ekuseru-Toukei 2010 statistical software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Significance of the differences in HFC1 or VAS between the groups taking the placebo and active compound was estimated using paired t-tests.