A Randomized, Placebo-Controlled Study on the Safety and Efﬁcacy of Daily Ingestion of Green Tea (Camellia Sinensis L.) Cv

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Article A Randomized, Placebo-Controlled Study on the Safety and Efﬁcacy of Daily Ingestion of Green Tea (Camellia sinensis L.) cv. “Yabukita” and “Sunrouge” on Eyestrain and Blood Pressure in Healthy Adults

Mari Maeda-Yamamoto 1,*, Mie Nishimura 2 ID , Nobuyoshi Kitaichi 3, Atsushi Nesumi 4, Manami Monobe 5, Sachiko Nomura 5, Yukihiro Horie 3, Hirofumi Tachibana 6 and Jun Nishihira 2 1 Agri-Food Business Innovation Center, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8517, Japan 2 Department of Medical Management and Informatics, Hokkaido Information University, Ebetsu, Hokkaido 069-8585, Japan; [email protected] (M.N.); [email protected] (J.N.) 3 Department of Ophthalmology, Health Sciences University of Hokkaido, Ishikari-gun, Hokkaido 002-8072, Japan; [email protected] (N.K.); [email protected] (Y.H.) 4 Institute of Fruit and Tea Science, NARO, Makurazaki, Kagoshima 898-0087, Japan; [email protected] 5 Institute of Fruit and Tea Science, NARO, Shimada, Shizuoka 428-8501, Japan; [email protected] (M.M.); [email protected] (S.N.) 6 Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Fukuoka 812-8581, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-29-838-8800

Received: 25 March 2018; Accepted: 3 May 2018; Published: 6 May 2018

Abstract: The green tea (Camellia sinensis L.) cultivar “Sunrouge” contains anthocyanins, catechins and flavonols. To determine whether ingesting green tea containing anthocyanins improves visual function and blood pressure (BP) in healthy adults, a randomized, double-blind, placebo-controlled study was performed. A total of 120 healthy subjects, aged between 20 and 60 years and with a systolic BP (SBP) value of ≤125 and <155 and a diastolic BP (DBP) value <95, or a DBP of ≤75 mmHg and <95 mmHg and a SBP <155 mmHg, were randomly assigned to one of three groups. For 12 weeks, the placebo group received barley extract without catechin; another group received “Sunrouge” extract containing 11.2 mg anthocyanin and 323.6 mg epigallocatechin-3-O-gallate (EGCG); and a third group received “Yabukita” extract containing 322.2 mg EGCG. Home BP, accommodation ability, visual analog scale questionnaires for eyestrain, and metabolic-associated markers were analyzed at weeks 0, 4, 8, and 12 of the intake period. The ingestion of “Sunrouge” tea significantly improved accommodation ability and eyestrain in subjects younger than 45 years and in subjects who operated visual display terminals every day. It also elevated BP. “Yabukita” tea ingestion significantly increased serum adiponectin levels. No adverse effects were observed. We conclude that long-term intake of “Sunrouge” tea containing anthocyanins and flavonols might improve visual function.

Keywords: green tea cv. “Sunrouge” or “Yabukita”; anthocyanins; ﬂavonols; eyestrain reducing effect; blood pressure elevating effect; adiponectin level increasing effect

1. Introduction In recent years, fatigue and eyestrain associated with the use of visual display terminals (VDT) have become increasingly prevalent among younger individuals. In Japan, the penetration ratios of personal computers and smartphones in households reached 76.8% and 72.0%, respectively, in 2015 [1],

Nutrients 2018, 10, 569; doi:10.3390/nu10050569 www.mdpi.com/journal/nutrients Nutrients 2018, 10, x FOR PEER REVIEW 2 of 13

In recent years, fatigue and eyestrain associated with the use of visual display terminals (VDT) have become increasingly prevalent among younger individuals. In Japan, the penetration ratios of Nutrientspersonal2018 computers, 10, 569 and smartphones in households reached 76.8% and 72.0%, respectively, in2 2015 of 13 [1], while the ownership rates of mobile phones reached 128.0% in 2016 [2]. Longer VDT operation whiletimes theresult ownership in increased rates stress of mobile and fatigue phones in reached different 128.0% body inregions, 2016 [2 such]. Longer as tired VDT eyes operation and eye times pain result(91.6%), in increasedstiffness in stress the andshoulders fatigue and in different neck (70.4%), body regions,and lower such back as tired pain eyes (26.6%) and eye[3]. painEyestrain (91.6%), is stiffnessincreasing in due the shouldersto long hours and neckof VDT (70.4%), operation and lowerand its back consequences, pain (26.6%) such [3]. as Eyestrain sleep deprivation is increasing or duemental to longstress. hours It has of been VDT reported operation that and approximately its consequences, 90% such of the as Japanese sleep deprivation population or has mental eyestrain, stress. Itmaking has been it one reported of the factors that approximately that lowers their 90% quality of the Japaneseof life (QOL) population [3]. Research has eyestrain, and drug making development it one offor the eyestrain factors are that scarce; lowers functional their quality foods oflife are(QOL) expected [3]. to Research contribute and to drug the treatment development of eyestrain. for eyestrain are scarce;Green functional tea (Camellia foods sinensis are expected L.) is consumed to contribute worldwide, to the treatment with large of eyestrain.quantities being consumed in JapanGreen and tea China, (Camellia where sinensis it hasL.) been is consumedused for medicinal worldwide, purposes with large for centuries. quantities Green being tea consumed contains inmany Japan functional and China, ingredients where such it has as beencatechins, used caffeine, for medicinal flavonols, purposes vitamins, for and centuries. dietary fiber Green. It has tea containsbeen reported many to functional have various ingredients bioregulatory such asfunctions, catechins, such caffeine, as anti- flavonols,carcinogenic vitamins, and anti and-metastatic dietary fiber.[4], anti It has-obesity been reported[5], anti-diabetes to have various [6], anti bioregulatory-hypercholesterolemic functions, such[7,8], as antioxidative anti-carcinogenic [9], anti and- anti-metastaticcardiovascular [[10],4], anti-obesity anti-allergic [5 [11],], anti-diabetes and anti-dry [6],-eye anti-hypercholesterolemic activities [12], and it is known [7,8], antioxidative to improve the [9], anti-cardiovascularintestinal flora. The[ 10tea], cultivars anti-allergic (cv.) [11 “Yabukita”], and anti-dry-eye and “Sunrouge” activities [13], [12], used and itas is test known samples to improve in this thestudy, intestinal contain flora. catechins, The tea cultivars including (cv.) epigallocatechin “Yabukita” and-3- “Sunrouge”O-gallate (EGCG), [13], used which as test are samples believed in this to study,prevent contain lifestyle catechins,-related includingdiseases. epigallocatechin-3-In addition, “Sunrouge”O-gallate tea (EGCG), is rich in which antioxidative are believed anthocyanins to prevent lifestyle-related[14–16]. It has beendiseases. reported In addition, that tea “Sunrouge” catechins havetea is richan anti in antioxidative-hypertensive anthocyanins effect [17,18], [14 while–16]. Itanthocyanins has been reported are well that known tea catechins to reduc havee eyestrain an anti-hypertensive [19,20]. “Sunrouge” effect [17 water,18], while extract anthocyanins suppressed areacetylcholinesterase well known to reduce activity eyestrain in human [19,20 ]. neuroblastoma “Sunrouge” water SK-N extract-SH cells suppressed [15]. Neostigmine acetylcholinesterase methyl activitysulfate, which in human is an neuroblastomaacetylcholinesterase SK-N-SH inhibitor, cells is [ 15used]. Neostigmineas an eye-focusing methyl medicine sulfate, [21]. which is an acetylcholinesteraseTherefore, in this inhibitor, study, to is clarify used as the an effects eye-focusing of ingesting medicine “Yab [21ukita”]. green tea and “Sunrouge” greenTherefore, tea on eyestrain in this study, and to hypertension, clarify the effects we conducted of ingesting a “Yabukita” 12-week, randomized, green tea and double “Sunrouge”-blind, greenplacebo tea-controlled on eyestrain trial in and Japanese hypertension, adults (aged we conducted 20–60 years) a 12-week,with slightly randomized, elevated blood double-blind, pressure placebo-controlled(BP). trial in Japanese adults (aged 20–60 years) with slightly elevated blood pressure (BP).

2. Materials and Methods

2.1. Study Design This was a double-blind,double-blind, randomized, placebo-controlledplacebo-controlled study. TheThe studystudy protocolprotocol isis shownshown in Figure1 . 1. The The subjects subjects were were recruited recruited by the by Hokkaido the Hokkaido Information Information University University and were and fully were informed fully regardinginformed regarding the content the and content methods and methods of this study. of this Writtenstudy. Written informed informed consents consents were obtainedwere obtained from subjectsfrom subjects before before their enrollment.their enrollment. The screeningThe screening for the for ﬁrst the first volunteers volunteers started started in July in July 2015 2015 and and the studythe study was was completed completed in January in January 2016. 2016.

Figure 1. Schematic representation of the study/protocol. Nutrients 2018, 10, 569 3 of 13

2.2. Subjects We recruited a total of 258 healthy volunteers (aged 21–55 years), of which 120 subjects (50 men, 70 women) were eligible. Key eligibility and exclusion criteria are shown in Table S1. The 120 eligible subjects were randomly assigned to the “Yabukita” group, “Sunrouge” group, or placebo group stratified by sex, age, hospital-measured SBP, and eye strain severity score. Assignments were computer-generated using stratified block randomization at a third-party data center. Doctors, nurses, clinical research coordinators, and statistical analyzers had no knowledge of the assignment information during this trial period. This information was only disclosed after the laboratory and analytical data were fixed and the method of statistical analysis was finalized.

2.3. Test Samples Three types of test drinks were manufactured industrially by Nikken Foods Co. Ltd. (Tokyo, Japan). Green tea (cv. “Yabukita” and “Sunrouge”) was extracted in hot water and the tea extract was concentrated and spray-dried to prepare a composite granulated powder with cyclodextrin. Barley tea extract powder that included no catechins and caffeine was used as the placebo. Subjects drank the tea, obtained by diluting the extract powder at the time of drinking, after each meal. The extracts were analyzed for ﬂavonoids, caffeine, and anthocyanin content by high-performance liquid chromatography using UV-VIS detection [15,22]. The analytical test results are summarized in Table1.

Table 1. Functional components in test teas (mg/day).

“Yabukita” Green Tea “Sunrouge” Green Tea Placebo Barley Tea Anthocyanins 0.0 11.2 * 0.0 EGCG 2 322.2 323.6 0.0 EGC 3 413.1 85.7 0.0 EGCG3”Me 4 0.0 12.3 0.0 Flavonols 54.8 5 40.4 6 0.0 Caffeine 202.5 184.8 0.0 * delphinidin glucosides, 7.9 mg; cyanidin glucosides, 3.3mg; 2 EGCG, epigallocatechin-3-O-gallate; 3 EGC, epigallocatechin; 4 EGCG3”Me, epigallocatchin-3-O-(3-O-methyl)-gallate; 5 myricetin glucosides, 11.7 mg; quercetin glucosides, 32.2 mg; kaempferol glucosides, 10.9 mg; 6 myricetin glucosides, 20.1 mg; quercetin glucosides, 13.4 mg; kaempferol glucosides, 6.9 mg.

2.4. Study Outcomes The primary outcomes measured were systolic blood pressure (SBP), diastolic blood pressure (DBP), accommodation (focusing ability of the eye) after stress or after rest, and the difference between accommodation ability after rest and after stress. Secondary outcomes measured were answers to a visual analog scale (VAS) questionnaire for eyestrain symptoms, intraocular pressure, peripheral blood ﬂow, oxidized low density lipoprotein (Ox-LDL), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), 8-hydroxy-20-deoxyguanosine (8-OHdG), high sensitivity-C-reactive protein (hs-CRP), adiponectin, asymmetric dimethylarginine (ADMA), total homocysteine, and low density lipoprotein cholesterol (LDL-C).

2.5. Measurement of Blood Pressure The subjects performed twice-daily home BP checks by themselves on the upper part of the arm opposite to the dominant hand, using an automated sphygmomanometer. BP measurement was carried out after urination and before breakfast within one hour after getting up in the morning, and after urination and before going to bed in the evening. Measurements were taken three times, with subjects in the sitting position, with ﬁve minutes of rest between measurements. The average of two Nutrients 2018, 10, 569 4 of 13 stable measurement values was assumed to be the day’s measurement. BP values were determined before intake and at 4, 8, and 12 weeks after intake and were calculated as the averages of the BP measurements executed for 5 days prior to the recorded date of measurement.

2.6. Measurement of Accommodation after VDT Work Load To evaluate the effects of tea on eye focusing ability, we measured accommodation using an accommodate (D’ACOMO, WOC, Kyoto, Japan). Brieﬂy, we asked subjects to press the stop button when the cross mark was clearly visible (their far point). Then, subjects were again asked to press the stop button when the cross mark became blurry (their near point). Each subjects’ amplitude of accommodation was measured by the distance between their near point and their far point; the resulting distance (cm) was converted into diopters (D). An increase in the value of D was considered to be an improvement in accommodation. We asked the subjects to play the game “Tetris” using a tablet-type device for 20 min to induce eyestrain (stress period) and then to wear a blindfold during a 10 min break (break period). Accommodation of both of their eyes was monitored twice—once after the stress period and once after the break period. In addition, we asked them to ingest the test food right before the visual load of VDT work. We analyzed three kinds of accommodation values for each eye: accommodation measured after the stress period, the break period, and the difference between the stressing and the break time.

2.7. Measurement of Eyestrain Symptoms We examined changes in eyestrain symptoms before and after (4, 8, 12 weeks) the intake using a visual analog scale (VAS) test. Our test was labeled “I feel extreme eyestrain” (0) and “I have no eyestrain” (100) on the extreme ends of a 100 mm line. The VAS questionnaire for eyestrain symptoms also included labels for “tired eyes,” “blurred vision,” “weight of eyelid,” “an ache behind the eyes,” “red eyes,” “bleary eyes,” “dryness of eyes,” “stiff shoulder,” “lower back pain,” “moodiness,” and “heaviness of the head.”

2.8. Adverse Events Subjects were instructed to record any subjective symptoms regarding body conditions in their diaries, which were submitted to the responsible physician at the beginning of the run-in period; at weeks 0, 4, 8, and 12 of the intake period; and at the end of the withdrawal period.

2.9. Ethics Committee This study protocol was approved by the Ethics Committee of the Hokkaido Information University (Ebetsu, Hokkaido, Japan) in accordance with the principles of the Helsinki Declaration (approval date 22 June 2015; approval number: 2015-08). This study was registered at www.umin.ac. jp/ctr/index.htm (registration number: UMIN000018469, date of registration 29 July 2015).

2.10. Statistical Analysis The statistical analysis was performed using the per protocol analysis set. Values are expressed as means ± standard deviations (SD). Differences between intervention groups were evaluated by one-way ANOVA and the Tukey post-hoc tests. For each group, differences between pre- and post-intake were evaluated using paired t-tests. All statistical analyses were performed with SPSS ver. 19 (IBM Japan, Tokyo, Japan). A p-value of <0.05 was considered signiﬁcant, while a p-value of <0.10 was deﬁned as a marginal difference. Nutrients 2018, 10, 569 5 of 13

3. Results and Discussion Nutrients 2018, 10, x FOR PEER REVIEW 5 of 13 3.1. Number of Subjects and Intake Rate of Test Teas Thirty-eight subjects in the “Yabukita” group, 39 in the “Sunrouge” group, and 37 in the placebo Thirty-eightgroup completed subjects the final in the visit “Yabukita” evaluation group,, and the 39 data in theof these “Sunrouge” subjects were group, used and for 37 the in final the placebo groupanalys completedis. The average the final intake visit rates evaluation, for the experimental and the period dataof were these 98.0 subjects± 3.6%, 97.7 were ± 3.6%, used and for99.4 the final ± 1.4%, as calculated using the intake check sheets of the “Yabukita” group, “Sunrouge” group, and analysis. The average intake rates for the experimental period were 98.0 ± 3.6%, 97.7 ± 3.6%, and placebo group, respectively. The flow diagram for this study is shown in Figure 2. The food survey 99.4 ± using1.4%, theas food calculated frequency using questionnaire the intake showed check no sheets change of in thethe diet “Yabukita” during the group, test period “Sunrouge” in either group, and placebogroup group,(Table S2). respectively. The flow diagram for this study is shown in Figure2. The food survey using the food frequency questionnaire showed no change in the diet during the test period in either group (Table S2).

Enrollment Assessed for eligibility (n=258)

Excluded （n=138) Not meeting inclusion criteria (n=138)

Randomized （n=120)

Allocation "Yabukita green tea" "Sunrouge green tea" "placebo (barley tea)" Allocated to Allocated to Allocated to intervantion intervention (n=40, intervention（n=40, （n=40, 16/24)

Follow-up Did not receive allocated Did not receive allocated Did not receive allocated intervention (n=0) intervention (n=1) intervention (n=1)

Discontinued Discontinued Discontinued intervention intervention intervention （n=2) (n=0) (n=2)

ITT (Intension-to- Continued Continued Continued treat) analysis intervention intervention intervention (Full Analysis Set) (n=38, 15/23) (n=39, 16/23)(n=37, (n=37, 15/22)

FigureFigure 2. Flowchart 2. Flowchart of of the the studystudy ( (nn == total total participants participants,, male/female) male/female)..

3.2. Physical3.2. Physical Characteristics Characteristics The mean age, height, body weight, body mass index (BMI), and body fat ratio for each group Theare mean presented age, in height, Table 2. body These weight, data did body not significantly mass index differ (BMI), among and the body three fat groups, ratio forconfirming each group are presentedthe appropriate in Table2. allocation These data of sub didjects not to signiﬁcantlythe three groups. differ among the three groups, conﬁrming the appropriate allocation of subjects to the three groups. Table 2. Baseline characteristics of subjects taking the test teas “Yabukita”, “Sunrouge”, or the placebo Table 2.barleyBaseline tea. characteristics of subjects taking the test teas “Yabukita”, “Sunrouge”, or the placebo barley tea.Baseline Characteristics “Yabukita” “Sunrouge” Placebo Gender(male/female) (n) 38 (15/23) 39 (16/23) 37 (15/22) Age (years)Baseline Characteristics “Yabukita”49.8 ± 6.7 “Sunrouge”49.5 ± 6.2 Placebo48.5 ± 7.5 Body weight (kg) 64.3 ± 9.7 61.4 ± 11.7 62.1 ± 10.0 Gender(male/female) (n) 38 (15/23) 39 (16/23) 37 (15/22) Body mass index (kg/m2) 23.9 ± 3.3 23.3 ± 3.4 23.3 ± 2.4 Age (years) 49.8 ± 6.7 49.5 ± 6.2 48.5 ± 7.5 Body fat ratio (%) 28.9 ± 6.9 27.9 ± 6.3 28.7 ± 6.7 Body weight (kg) 64.3 ± 9.7 61.4 ± 11.7 62.1 ± 10.0 Home SBP at waking up (mmHg) 126.8 ± 11.9 126.6 ± 14.2 124.6 ± 10.1 Body mass index (kg/m2) 23.9 ± 3.3 23.3 ± 3.4 23.3 ± 2.4 HomeBody DBP fat ratioat waking (%) up (mmHg) 28.9 ± 6.980.5 ± 9.8 27.9 ±81.86.3 ± 9.8 * 28.777.3± 6.7± 7.4 Home SBP at bedtime (mmHg) 123.7 ± 11.9 123.3 ± 12.9 123.7 ± 11.0

Nutrients 2018, 10, 569 6 of 13

Table 2. Cont.

Nutrients 2018Baseline, 10, x FOR Characteristics PEER REVIEW “Yabukita” “Sunrouge” Placebo 6 of 13 Home SBP at waking up (mmHg) 126.8 ± 11.9 126.6 ± 14.2 124.6 ± 10.1 Home DBPHome at bedtime DBP at waking(mmHg) up (mmHg) 80.5 ± 9.875.3 ± 10.0 81.8 ± 9.8 *77.1 ± 8.5 77.3 ± 7.4 74.8 ± 7.0 Pulse (bpm)Home SBP at bedtime (mmHg) 123.7 ± 11.974.6 ± 9.9 123.3 ± 12.975.8 ± 11.1 123.7 ± 11.074.4 ± 10.7 Home DBP at bedtime (mmHg) 75.3 ± 10.0 77.1 ± 8.5 74.8 ± 7.0 Values are shown as the means ± standard deviations. n = number of subjects; SBP, systolic blood Pulse (bpm) 74.6 ± 9.9 75.8 ± 11.1 74.4 ± 10.7 pressure; DBP, diastolic blood pressure; One-way ANOVA and the Tukey post-hoc test vs. placebo; Values are shown as the means ± standard deviations. n = number of subjects; SBP, systolic blood pressure; DBP, paireddiastolic t- bloodtest vs pressure;. 0 week One-way was performed ANOVA and to theanalyze Tukey the post-hoc values test. Statistical vs. placebo; significan paired t-testce: vs.* p 0< week 0.05 wasvs. placeboperformed group. to analyze the values. Statistical signiﬁcance: * p < 0.05 vs. placebo group.

3.3. Primary O Outcomesutcomes

3.3.1. Accommodation Ability (AA) The intraocularintraocular pressurespressuresof of the the right right and and left left eye eye gradually gradually increased increased in allin all test test groups groups (Table (Table S3). SThe3). The intention intention to treat to treat (ITT) (ITT) analysis analysis did did not not show show an an improvement improvement of of AA AA inin thethe green tea tea groups. groups. We performedperformed a stratifiedstratified analysis for age, because eye AA could not be measured accurately in t thehe subjects with presbyopiapresbyopia.. Changes Changes in in the the AA AA of of the the left left eye after rest in non-presbyopicnon-presbyopic subjects younger than 45 years at weeks 4 and 8 significantlysignificantly improved in the “Sunrouge” group ( n = 8) 8) compared to the placebo group ( n = 10) (Figure 33A).A). ChangesChanges inin thethe AAAA ofof thethe leftleft eyeeye afterafter restrest andand after stress in non non-presbyopic-presbyopic subjects younger than 45 years at week 8 significantly significantly improved in the “Sunrouge”“Sunrouge” groupgroup comparedcompared toto thethe placeboplacebo groupgroup (Figure(Figure3 B).3B).

Figure 3. The changes in acc accommodationommodation ability ability from from week 0 to weeks 4, 8 and 12:12: ( A), changes in accommodation abilityability ofof the the left left eye eye after after a rest a rest (under (under 45 years); 45 years); (B) changes (B) changes in accommodation in accommodation ability abilityof the leftof the eye—the left eye difference—the difference between between the stressing the stressing and the break and the time break (under time 45 years);(under (45C) years); changes (C in) changesaccommodation in accommodation ability of the ability left eye of the after left a resteye after (visual a rest display (visual terminals display (VDT)terminals operation). (VDT) operation Statistical). Statisticalsigniﬁcance: significan * p < 0.05,ce: * ** pp << 0.05, 0.01. ** p < 0.01.

Changes in the left eye’s AA after rest in non-presbyopic subjects younger than 45 years of age Changes in the left eye’s AA after rest in non-presbyopic subjects younger than 45 years of age at at week 8 (p = 0.031) significantly improved in the “Sunrouge” group compared to the “Yabukita” week 8 (p = 0.031) significantly improved in the “Sunrouge” group compared to the “Yabukita” group group (Figure 3A). Changes in the difference between AA after rest and AA after stress of the left eye (Figure3A). Changes in the difference between AA after rest and AA after stress of the left eye in in non-presbyopic subjects younger than 45 at weeks 8 and 12 significantly improved in the non-presbyopic subjects younger than 45 at weeks 8 and 12 significantly improved in the “Sunrouge” “Sunrouge” group compared to the “Yabukita” group (Figure 3B). group compared to the “Yabukita” group (Figure3B). At week 8, changes in the AA of the left eye after rest in subjects operating VDT every day At week 8, changes in the AA of the left eye after rest in subjects operating VDT every day significantly improved in the “Sunrouge” group compared to the “Yabukita” group (Figure 3C). significantly improved in the “Sunrouge” group compared to the “Yabukita” group (Figure3C). In a stratified analysis of AA, significant improvement of left eye AA was observed in subjects In a stratified analysis of AA, significant improvement of left eye AA was observed in subjects younger than 45 years and in subjects operating VDT every day. A possible explanation for these younger than 45 years and in subjects operating VDT every day. A possible explanation for these results is the small difference between the near and far point, leading to an inaccurate evaluation of results is the small difference between the near and far point, leading to an inaccurate evaluation of the AA. As for AA improvement being observed only for the left eye, it is thought that this is related the AA. As for AA improvement being observed only for the left eye, it is thought that this is related to eye dominance, and needs further examination. Moreover, it is necessary to measure the AA before to eye dominance, and needs further examination. Moreover, it is necessary to measure the AA before rest and consider this parameter to evaluate the improvement of AA. Improvement of accommodation ability by the “Sunrouge” group was also sufficient compared to other functional foods known to improve eyestrain; thus, we consider “Sunrouge” as a sufficiently valuable tea to improve eyestrain.

3.3.2. Home Blood Pressure

Nutrients 2018, 10, 569 7 of 13 rest and consider this parameter to evaluate the improvement of AA. Improvement of accommodation ability by the “Sunrouge” group was also sufﬁcient compared to other functional foods known to improve eyestrain; thus, we consider “Sunrouge” as a sufﬁciently valuable tea to improve eyestrain.

3.3.2. Home Blood Pressure Morning SBP values at weeks 8 and 12, morning DBP values at weeks 0, 4, 8, and 12, and evening DBP values at weeks 4, 8, and 12 were signiﬁcantly higher in the “Sunrouge” group than in the placebo group, but remained within the normal range for the Japanese population (SBP <140 and DBP <85), as shown in Table3.

Table 3. Changes in home blood pressure after drinking “Sunrouge”, “Yabukita”, or barley infusion tea.

Interventions Week 0 Week 4 Week 8 Week 12 Home SBP at waking up “Yabukita” 126.8 ± 11.9 127.3 ± 11.9 127.3 ± 13.8 129.3 ± 14.4 (mmHg) “Sunrouge” 126.6 ± 14.2 128.8 ± 13.5 # 131.5 ± 13.4 *## 132.9 ± 14.3 *## Placebo 124.6 ± 10.1 125.2 ± 9.4 125.6 ± 9.2 126.7 ± 10.5 Home DBP at waking up “Yabukita” 80.5 ± 9.8 80.3 ± 8.7 80.8 ± 9.7 81.6 ± 1.6 (mmHg) “Sunrouge” 81.8 ± 9.8 * 83.0 ± 8.7 * 85.1 ± 8.8 **## 85.5 ± 1.5 **## Placebo 77.3 ± 7.4 78.4 ± 7.0 78.1 ± 7.7 78.9 ± 1.4 Home SBP at bedtime “Yabukita” 123.7 ± 11.9 126.8 ± 13.5 125.9 ± 15.6 126.3 ± 14.7 (mmHg) “Sunrouge” 123.3 ± 12.9 128.8 ± 13.8 ## 129.9 ± 13.7 ## 129.8 ± 12.7 ## Placebo 123.7 ± 11.0 123.8 ± 13.8 124.7 ± 10.5 124.5± 12.6 Home DBP at bedtime “Yabukita” 75.3 ± 10.0 77.7± 10.3 78.4 ± 10.9 78.8 ± 11.1 (mmHg) “Sunrouge” 77.1 ± 8.5 81.0 ± 8.9 *## 81.9 ± 8.5 **## 82.4 ± 8.2 **## Placebo 74.8 ± 7.0 76.3 ± 7.3 76.1 ± 8.7 76.2 ± 9.0 Values are shown as the means ± standard deviations; SBP, systolic blood pressure; DBP, diastolic blood pressure; One-way ANOVA and the Tukey post-hoc test vs. placebo, paired t-test vs. 0 week were performed to analyze the values. Statistical signiﬁcance: * p < 0.05, ** p < 0.01 vs. placebo group, # p < 0.05, ## p < 0.01 vs. 0 week.

The evening SBP change at weeks 4, 8, and 12, and DBP change at weeks 8 and 12 were significantly higher in the “Sunrouge” group than in the placebo group, as shown in Figure4C,D. The morning SBP change at week 12 was higher in the “Sunrouge” group than in the placebo group, though the difference was not statistically significant (p = 0.052) (Figure4A). Moreover, the morning DBP and morning SBP changes at week 8 were significantly higher in the “Sunrouge” group than in the “Yabukita” group (Figure4A,B). “Sunrouge” strongly raised blood pressure, though within the normal range for the Japanese population; thus, we consider this result clinically relevant. In this study, long-term ingestion of “Sunrouge” containing anthocyanins raised blood pressure, suggesting that “Sunrouge” may be usable as a hypertensive agent. Several studies have reported that consecutive intake of 64 mg of anthocyanins by patients after cardiac infarction lowered BP, due to anthocyanins’ ameliorating effect on hypertension [23,24]. We hypothesized that BP regulation by anthocyanins is related to its antioxidative effect or improvement of the peripheral blood flow. However, the definite improvement of antioxidative markers or peripheral blood flow was not observed with “Sunrouge” ingestion in this study (Table S3); therefore, further investigation is needed to elucidate the relationship between “Sunrouge” intake and BP. Moreover, it is necessary to investigate the effect of “Sunrouge” on BP in subjects who are sensitive to cold temperature. Nutrients 2018, 10, 569 8 of 13

Figure 4. The changes in home-measured blood pressure in the morning and evening from week 0 to weeks 4, 8, and 12: (A) SBP change at waking up; (B) DBP change at waking up; (C) SBP change at bedtime; (D) DBP change at bedtime. Statistical signiﬁcance: * p < 0.05, ** p < 0.01.

3.4. Secondary Outcomes Secondary outcomes were data on intraocular pressure, peripheral blood ﬂow, Ox-LDL, TBARS, SOD, 8-OHdG, hs-CRP, adiponectin, ADMA, total homocysteine, and LDL-C. Among oxidative stress markers, at week 4, TBARS and SOD levels in the “Yabukita” group were signiﬁcantly higher than in the placebo group, while Ox-LDL and urinary 8-OHdG did not differ between the groups, as shown in Table4 and Table S3.

Table 4. Secondary outcomes after drinking “Sunrouge”, “Yabukita”, or barley infusion.

Interventions Week 0 Week 4 Week 8 Week 12 Oxidation marker TBARS (mM) “Yabukita” 13.7 ± 3.5 16.2 ± 5.4 *# 9.9 ± 3.3 ## 12.8 ± 3.8 “Sunrouge” 13.0 ± 3.3 13.9 ± 5.3 9.1 ± 3.5 ## 12,0 ± 4.0 Placebo 13.8 ± 3.7 14.2 ± 3.9 8.5 ± 3.0 ## 11.9 ± 3.5 # SOD (U/L) “Yabukita” 3.2 ± 1.8 2.7 ± 1.8 *# 2.9 ± 1.9 *## 2.7 ± 2.2 ## “Sunrouge” 3.0 ± 1.6 2.4 ± 1.6 ## 2.9 ± 2.2 2.6 ± 2.1 ## Placebo 2.6 ± 0.8 2.0 ± 0.8 ## 2.3 ± 1.2 # 2.1 ± 1.3 ## Values are shown as the means ± standard deviations; TBARS, 2-thiobarbituric acid reactive substances, SOD, superoxide dismutase. Statistical signiﬁcance: * p < 0.05, ** p < 0.01 vs. placebo group, # p < 0.05, ## p < 0.01 vs. 0 week.

3.5. Eyestrain Symptoms We compared the changes listed in the VAS questionnaire for eyestrain symptoms—“tired eyes,” “blurred vision,” “weight of eyelid,” “an ache behind the eyes,” “red eyes,” “bleary eyes,” “dryness of eyes,” “stiff shoulder,” “lower back pain,” “moodiness,” and “heaviness of the head”—from baseline for each week in all test groups. We observed no significant differences among the three groups; therefore, a stratified analysis was conducted for the presence or absence of daily VDT operation and age. Nutrients 2018, 10, x FOR PEER REVIEW 9 of 13 Nutrients 2018, 10, 569 9 of 13 from baseline for each week in all test groups. We observed no significant differences among the three groups; therefore, a stratified analysis was conducted for the presence or absence of daily VDT Asoperation shown and in Figureage. 5, in subjects who performed VDT operations (PC, smartphone, tablet) everyday (placeboAs shown group:in Figure 18 5, people, in subjects “Yabukita” who performed group: VDT 19, operations “Sunrouge” (PC, group: smartphone, 18), changes tablet) in eyestraineveryday at week (placebo 4 (Figure group5A),: 18 and people, changes “Yabukita” in lower group back: 19, pain “Sunrouge” at week 8 group(Figure: 18),5B) changes significantly in eyestrain at week 4 (Figure 5A), and changes in lower back pain at week 8 (Figure 5B) significantly improved in the “Sunrouge” group compared to the placebo group. Moreover, for subjects younger improved in the “Sunrouge” group compared to the placebo group. Moreover, for subjects younger than 45than years 45 years (placebo (placebo group: group 10: people,10 people, “Yabukita” “Yabukita” group: group: 8, “Sunrouge” “Sunrouge” group group:: 8), 8),the the“Sunrouge” “Sunrouge” groupgroup showed showed significant significant improvement improvement in in the the top-heavy top-heavy feelingfeeling at at week week 12 12 compared compared to those to those in the in the placeboplacebo group group (Figure (Figure5C). 5C Subjects). Subjects younger younger than than 45 45 years years inin thethe “Yabukita” group group experienced experienced less less ache behindache behind the eyes the eyes at week at week 12 compared12 compared to to those those in in thethe placeboplacebo group group,, though though the the difference difference was was not statisticallynot statistically significant significant (p = (p 0.052) = 0.052) (Figure (Figure5D). 5D).

Placebo (n=18) Placebo (n=18) Yabukita (n=18) Yabukita (n=18) A50 B *S vs P (p=0.047) Sunrouge (n=19) 50 Sunrouge (n=19) 40 40 *S vs P (p=0.027) 30 30 20 20 10

10 0 VAS ( mm) ( VAS VAS ( mm) ( VAS -10 0 -20 -10 -30 -20 -40 0 4 8 12 0 4 8 12 weeks weeks Placebo (n=10) C Yabukita (n=8) D Placebo (n=10) **S vs P (p=0.008) Yabukita (n=8) Sunrouge (n=8) 80 Sunrouge (n=8) 50 Y vs P (p=0.052) 60 30 40 10

VAS ( mm) ( VAS 20

-10 VAS ( mm) ( VAS 0 -30

-50 -20

-70 -40 0 4 8 12 0 4 8 12 weeks weeks

FigureFigure 5. The 5. changes The changes in eyestrain in eyestrain symptoms symptoms from from weeks weeks 00 to weeks 4, 4, 8 8and and 12: 12: (A ()A changes) changes in eye in eye strainstrain (subjects (subject whos who do VDT do VDT operations operation everyday);s everyday); ( B(B))changes changes in in lower lower back back pain pain (subject (subjectss who who do do VDT operationsVDT operation everyday);s everyday); (C) ( changesC) changes in in top-heavy top-heavy feelingfeeling (under 45 45 years); years); (D ()D changes) changes in ache in ache behind the eyes (under 45 years). Statistical significance: * p < 0.05, ** p < 0.01 vs. placebo group. behind the eyes (under 45 years). Statistical significance: * p < 0.05, ** p < 0.01 vs. placebo group. The biological mechanism of the eye strain reduction effect of anthocyanins is thought to be the Theeffect biological of antioxidants mechanism [25] and of the rhodopsin eye strain regene reductionration [26] effect. The of above anthocyanins results suggest is thought that the to be the effecteyestrain of antioxidants-reducing effect [25 ]of and “Sunrouge” rhodopsin was regeneration higher in subjects [26]. performing The above many results VDT suggest operations. that the eyestrain-reducingBecause changes effect in the of top “Sunrouge”-heavy feeling was significantly higher in improved subjects in performing the “Sunrouge” many group VDT in subjects operations. Because changes in the top-heavy feeling significantly improved in the “Sunrouge” group in subjects younger than 45 years, the eyestrain-reducing effect of Sunrouge may be higher in subjects who do not have presbyopia. A similar result has been reported for blackcurrant anthocyanin. Nakanishi et al. reported that after the intake of 50 mg of blackcurrant anthocyanin concentrate (delphinidin-3-rutinoside; 4.6%, delphinidin-3-glucoside; 1.4%, cyanidin-3-rutinoside; 2.8%, cyanidin-3-glucoside; 0.4%), significant Nutrients 2018, 10, x FOR PEER REVIEW 10 of 13

younger than 45 years, the eyestrain-reducing effect of Sunrouge may be higher in subjects who do Nutrientsnot2018 have, 10, presbyopia. 569 10 of 13 A similar result has been reported for blackcurrant anthocyanin. Nakanishi et al. reported that after the intake of 50 mg of blackcurrant anthocyanin concentrate (delphinidin-3-rutinoside; 4.6%, improvementdelphinidin was-3 seen-glucoside; in eyestrain 1.4%, symptomscyanidin-3-rutinoside; (eye and lower 2.8%, back), cyanidin assessed-3-glucoside; using a0.4%), questionnaire significant [ 19]. Lee etimprovement al. reported thatwas seen the administrationin eyestrain symptoms of anthocyanoside (eye and lower oligomerback), assessed (85 mg) using seemed a questionnaire to improve subjective[19]. symptoms Lee et al. reported and objective that the contrast administration sensitivity of inanthocyanosi subjects withde oligomer myopia and(85 mg) asthenopia seemed [to20 ]. Inimprove the future, subjective a Randomized symptoms Controlledand objective Trial contrast (RCT) sensitivity needs to in be subjects carried with out onmyopia the effect and of “Sunrouge”asthenopia green [20]. tea on the reduction of eyestrain in non-presbyopic subjects with a workload involving increasedIn the future, VDT a Randomized operation. No Controlled blood flow Trial improvement (RCT) needs to was be carried associated out on with the “Sunrouge” effect of intake,“Sunrouge” as assessed green by peripheral tea on the reduction blood flow of eyestrain measurement, in non-presbyopic but these results subjects do with not a workload exclude the possibilityinvolving that consecutiveincreased VDT intake operation. of “Sunrouge” No blood flow tea, whichimprovement rich in was anthocyanins, associated with may “Sunrouge” have positive intake, as assessed by peripheral blood flow measurement, but these results do not exclude the effects on blood flow. Therefore, further examination is needed on blood flow improvement by possibility that consecutive intake of “Sunrouge” tea, which rich in anthocyanins, may have positive “Sunrouge”effectstea. on blood flow. Therefore, further examination is needed on blood flow improvement by “Sunrouge” tea. 3.6. Endothelial Markers Changes3.6. Endothelial in serum Markers homocysteine were significantly higher in the “Yabukita” and “Sunrouge” groups at weekChanges 12 than in serum in the homocysteine placebo group, were as significantly shown in Figure higher6A. in The the changes“Yabukita” in serumand “Sunrouge” adiponectin levels weregroup significantlys at week 12 higherthan in in the the placebo “Yabukita” group, group as shown at weeks in Figure 8 and 6A. 12 thanThe in changes the placebo in serum group (Figureadiponectin6B). It has beenlevels reportedwere significantly that dietary higher supplementation in the “Yabukita” withgroup 0.25% at weeks EGCG 8 and elevated 12 than levelsin the of circulatingplacebo adiponectin group (Figure in non-obese 6B). It has type-2 been reported diabetic that Goto-Kakizaki dietary supplementation rats [27]. Moreover, with 0.25% there EGCG was a significantelevated increase levels in ofplasma circulating adiponectin adiponectin levels in and non plasma-obese LDL type- particle2 diabetic size Goto in subjects-Kakizaki with rats coronary [27]. Moreover, there was a significant increase in plasma adiponectin levels and plasma LDL particle size artery disease after one-month intake of oolong tea containing 245.6 mg of polyphenols [28]. Green tea in subjects with coronary artery disease after one-month intake of oolong tea containing 245.6 mg of catechinpolyphenol administrations [28]. Green to rats tea with catechin high-fat, administration diet-induced to obesity rats with increased high-fat the, diet levels-induced of peroxisome obesity proliferator-activatedincreased the levels receptor of peroxisome (PPAR)γ proliferatorin subcutaneous-activated white receptor adipose (PPAR)γ tissue in and subcutaneous decreased white those of PPARγadiposein visceral tissue white and decreased adipose tissuethose of [29 PPARγ]. in visceral white adipose tissue [29].

Placebo Placebo **Y vs P (p=0.002) A 1.2 Yabukita B 3.0 Yabukita **Y vs P (p=0.008) Sunrouge 2.5 Sunrouge

0.8 *Y vs P

mol/l)

g/ml) μ μ 2.0 (p=0.027) 0.4 1.5 *S vs P (p=0.033) 0.0 1.0

Adiponectin Adiponectin ( 0.5 -0.4 Homocysteine ( Homocysteine 0.0 -0.8 0 4 8 12 0 weeks 12 weeks Figure 6. The changes in homocysteine and adiponectin levels from weeks 0 to weeks 4, 8, and 12: (A) Figure 6. The changes in homocysteine and adiponectin levels from weeks 0 to weeks 4, 8, and 12: change in homocysteine; (B) change in adiponectin. Statistical significance: * p < 0.05, ** p < 0.01 vs. (A) change in homocysteine; (B) change in adiponectin. Statistical significance: * p < 0.05, ** p < 0.01 vs. placebo group. placebo group. In the present study, the changes in serum adiponectin significantly increased in the group Inadministered the present “Yabukita” study, thecontaining changes 322 mg in of serum EGCG. adiponectin Although “Sunrouge” significantly green tea increased contained in as the groupmuch administered EGCG and “Yabukita”caffeine as “Yabukita” containing green 322 tea, mg the ofchange EGCG. in serum Although adiponectin “Sunrouge” did not increase green tea containedupon as the much intake EGCG of “Sunrouge” and caffeine green as tea. “Yabukita” “Sunrouge” green green tea, tea thecontains change epigallocathin in serum- adiponectin3-O-(3-O- methyl)-gallate, one-fifth the epigallocatechin, twice the myricetin, two-fifths of the quercetin and a did not increase upon the intake of “Sunrouge” green tea. “Sunrouge” green tea contains seven-tenths of the kaempferol of “Yabukita” green tea (Table 1). We hypothesize that this difference epigallocathin-3-O-(3-O-methyl)-gallate, one-fifth the epigallocatechin, twice the myricetin, two-fifths is due to a difference in the composition ratios of anthocyanins, catechins, or flavonols, rather than to of the quercetindifferences and in the a seven-tenths EGCG content of between the kaempferol “Sunrouge” of “Yabukita” green tea and green “Yabukita” tea (Table green1). tea. We hypothesizeHowever, that this difference is due to a difference in the composition ratios of anthocyanins, catechins, or

flavonols, rather than to differences in the EGCG content between “Sunrouge” green tea and “Yabukita” green tea. However, it is not clear if caffeine was driving any of these effects, because caffeine was absent from the placebo group. A subgroup analysis was performed between the two subject groups with and without a daily habit of tea drinking (n = 12 and 13 for daily tea drinkers in the placebo and green tea groups, respectively; n = 25 for non-daily tea drinkers in both the placebo group and green tea Nutrients 2018, 10, 569 11 of 13 groups). The change in HDL cholesterol levels from week 0 to week 12 in the “Yabukita” tea group (5.28 ± 9.11 mg/dL) was significantly higher than that in the placebo group (−1.00 ± 6.81 mg/dL) (p = 0.008). This result suggests that the effect of “Yabukita” tea on lipid metabolism is greater in subjects without a daily habit of tea drinking. In this study, “Yabukita” tea did not affect BP, antioxidant actions, endothelial markers, or peripheral blood flow. However, serum adiponectin and HDL-C content were significantly higher in members of the “Yabukita” group who did not have a daily habit of tea drinking. It will be necessary to evaluate the efficacy of “Yabukita” tea in improving lipid metabolism or against body fat accumulation in subjects with a slightly increased cholesterol level or BMI in the future.

3.7. Safety and Hospitalization We evaluated complete blood cell counts (CBCs), liver and renal function, and BP after the ingestion of tea. As shown in Table S2, minimal changes were observed in CBC parameters (white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (Hb) level, hematocrit (Ht), and platelet (Plt) count). Changes were also minimal for liver function (aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH)), renal function (urea nitrogen (BUN), creatinine (CRE), and uric acid (UA)), albumin (Alb), total protein (TP), unsaturated iron binding capacity (UIBC), and total iron binding capacity(TIBC). One subject who ingested the placebo tea exhibited an adverse event related to liver function, and recovered from this symptom within a week. One subject who ingested the “Yabukita” tea was admitted to the hospital because of an acute virus infection. An additional subject who ingested the “Yabukita” tea was admitted to the hospital with venous thrombosis; since the onset of the disease occurred immediately after the start of the study, this subject had likely not ingested the test meal. The relationship between homocysteine and thrombus formation was higher in “Yabukita” group; however, the values of all subjects were in the normal range. Accordingly, the principal investigator judged that there were no serious adverse events related to the study intervention. These results suggest that the intake of “Sunrouge” or “Yabukita” tea containing 324 mg of EGCG, 203 mg of caffeine and 11 mg of anthocyanins for 12 weeks is safe. “Sunrouge” and “Yabukita” teas have large amounts of catechins including 300 mg of EGCG, and it has been reported that a large amount of EGCG may cause hepatic damage [30], but 704mg EGCG per day might be considered safe, based on observed human adverse event data [31]. However, we should carefully watch our intake of catechins.

4. Conclusions Consecutive ingestion of the “Sunrouge” tea extract significantly improved eye AA in subjects younger than 45 years and in subjects operating VDT every day. It also elevated BP. Moreover, in subjects who operated VDT everyday, the “Sunrouge” group noted significant improvements in eyestrain at week 4, lower back pain at week 8, and top-heavy feelings at week 12 when compared to the placebo group. This result shows the possibility that “Sunrouge” is usable as an eyestrain-reducing agent or a hypertensive agent. “Yabukita” tea extract ingestion significantly increased serum adiponectin levels. No adverse effects were observed. These results suggest that the eyestrain-reducing effect of “Sunrouge” is higher in subjects younger than 45 years or in subjects operating VDT. The eyestrain-reducing effect of “Sunrouge” may therefore be higher in subjects who do not have presbyopia. In the future, we need to carry out an RCT on the eyestrain-reducing effect of “Sunrouge” tea for non-presbyopic subjects with a workload involving increased VDT operation. We hypothesize that the difference in efficacy is due to a difference in anthocyanins, catechins, or flavonols composition ratios rather than a difference in EGCG content between “Sunrouge” green tea and “Yabukita” green tea.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/5/569/s1, Table S1. Key eligibility and exclusion criteria, Table S2. Changes in the anthrometric values and biochemical Nutrients 2018, 10, 569 12 of 13

parameters after drinking “Sunrouge”, “Yabukita”, or barley tea, Table S3. Secondary outcomes after drinking “Sunrouge”, “Yabukita”, or barley tea. Author Contributions: J.N., M.M.-Y., M.N., H.T., and N.K. designed the research; J.N. conducted the research; M.M.-Y., and A.N. performed the preparation of test teas; M.M., and S.N. performed the chemical analysis of test teas; N.K., and Y.H. measured the accommodation ability; M.N. performed the statistical analysis. All authors read and approved the final version of the manuscript. Acknowledgments: The authors would like to thank Kaori Ema of the NARO Institute of Fruit and Tea Science for her valuable assistance in the research and data analysis. This study was supported by a grant-in-aid “A scheme of Revitalizing Agriculture and Fisheries in Disaster Area through Deploying Highly Advanced Technology” from of the Ministry of Agriculture, Forestry and Fisheries of Japan. Conflicts of Interest: The authors declare no conflict of interest to disclose for this study.

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