nutrients
ArticleA 12-Week-Long Intake of Bilberry Extract (Vaccinium
myrtillus L.) Improved Objective Findings of Ciliary
Muscle Contraction of the Eye: A Randomized,
Double-Blind, Placebo-Controlled, Parallel-Group
Comparison Trial
Marie Kosehira 1, *, Naomichi Machida 1, * and Nobuyoshi Kitaichi 2
1
2*

Research Department, Omnica Co., Ltd, 4-21-7 Itabashi, Itabashi Ward, Tokyo 173-0004, Japan
Department of Ophthalmology, Health Sciences University of Hokkaido, Sapporo 002-8072, Japan;
nobukita@hoku-iryo-u.ac.jp
Correspondence: kosehira@omnica.co.jp (M.K.); machida@omnica.co.jp (N.M.);
Tel.: +81-3-5944-4201 (M.K. & N.M.); Fax: +81-3-5944-4211(M.K. & N.M.)

Received: 27 January 2020; Accepted: 22 February 2020; Published: 25 February 2020


Abstract: A 12-week-long randomized, double-blind, placebo-controlled, parallel-group comparison
trial was conducted to determine the effects of long-term standardized bilberry extract (SBE) intake on
tonic accommodation of ciliary muscle caused by visual display terminal (VDT) tasks. This study wascompliant with the accordance with CONSORT 2010 statement. A total of 109 healthy adult men andwomen aged 20–60 years were recruited and randomized into SBE and placebo groups. The subjects
in the SBE and placebo groups were administered 240 mg of SBE and placebo, respectively, once dailyfor 12 weeks. Tests were performed before and after VDT tasks at week 0, 4, 8, and 12; high-frequency
component (HFC)-1 value was the evaluation outcome. Results showed that post-load HFC-1 values
at weeks 8 and 12 were significantly improved in the SBE group than in the placebo group (p = 0.014
and 0.017, respectively). This study shows that oral consumption of 240 mg SBE extract for 12 weeks
relieves the tonic accommodation of the ciliary muscle caused by VDT tasks and near-vision tasks.
Keywords: bilberry; asthenopia; clinical trial; accommodation; refraction; accommodative
microfluctuation

1. Introduction
Owing to the increased distribution of visual display terminal (VDT) devices, our eyes areconstantly being overworked [1,2]. According to the survey conducted by the Ministry of Health,Labour and Welfare, the proportion of workers performing VDT tasks is increasing; as per the 2008
survey, approximately 50% of all workers were found to be engaged in VDT tasks for ≥4 h/day. Of theworkers experiencing physical fatigue and VDT work-related symptoms, 91.6% experience “ocularfatigue and ocular pain,” indicating a high load of VDT tasks on the eyes [1]. Recently, the use of
VDT devices, such as smartphones and tablets when used and placed close to the eyes, is rapidly
spreading among people. Individual smartphone ownership has quadrupled from 14.6% in 2011 to
56.8% in 2016, and ≥90% smartphone users are 40 years old or younger [3]. Because individuals use
VDT devices, such as personal computers and smartphones, close to their eyes for long periods of time,the eye muscles become fatigued and experience tension, making focus adjustment difficult. Further,because more and more young people are starting to experience symptoms similar to presbyopia, it isextremely important to prevent and reduce ocular fatigue to improve the quality of life of VDT users.

Nutrients 2020, 12, 600; doi:10.3390/nu12030600

www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 600

2 of 9Bilberry (Vaccinium myrtillus L.) contains 15 types of low-molecular anthocyanin glycosides
made up of a combination of five types of aglycones (cyanidin, delphinidin, malvidin, peonidin,
and petunidin) and three types of monosaccharides (glucose, galactose, and arabinose). These 15
V. myrtillus anthocyanins (VMAs) serve as the main components of bilberry. Bilberry extract obtained
via the separation and refinement of these 15 VMAs has been used as a component of functional foods
wherein the “tertiary” function of the food [4] is intentionally regulated instead of just consuming thebilberry. Different extraction methods yield bilberry extracts with different overall compositions; even
if the bilberry extracts share the same VMAs, their benefits and safety could vary [5,6]. In the present
paper, we refer to bilberry extract that has had reproducibility of effect confirmed in several tests [7–10]
as standardized bilberry extract (SBE).
We confirmed that SBE consumption suppresses or relieves convergence, miosis, and declined
accommodation function caused by VDT tasks [7–10]. Furthermore, we established intervention
conditions for the tonic accommodation of the ciliary muscle caused by VDT tasks and verified
the minimum effective dose of SBE [8] and minimum period for efficacy expression [9]. This
12-week randomized, double-blind, placebo-controlled, parallel-group comparison trial examining
SBE consumption aimed to determine the effects and safety of long-term SBE consumption on the tonic
accommodation of ciliary muscles caused by VDT tasks.
This is the first trial for the efficacy of Bilberry extract in accordance with the CONSORT 2010
statement (Figure S1, Table S1).
2. Methods and Subjects
2.1. Trial Design and Subjects
To evaluate the effect of SBE consumption on ocular fatigue symptoms, we conducted a randomized,
double-blind, placebo-controlled, parallel group human clinical trial. The trial strictly adhered to tenetsof the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving
Human Subjects, and the trial plan was approved by the Huma R&D ethical review board (approvaldate 15 January 2016). This study was registered at https://www.umin.ac.jp/ctr/index.htm (UMINregistration number: UMIN000020866). All subjects were provided with a sufficient explanation before
participation in the trial, and written informed consent was obtained. The ocular fatigue test was
performed by the Medicine Evaluation Research Center, whereas the safety test was performed by the
K Medical Office TOC Building Clinic.
A total of 109 healthy adult men and women aged 20–60 years responded to the Human R&D
public request for trial participation. Subjects whose jobs involved VDT tasks and those who had
subjective ocular fatigue symptoms were selected. Interviews, body measurements, blood tests,
urine tests, and ocular fatigue tests were conducted during the pretrial screening, and preliminary
questionnaires were administered. Subjects who met any of the following criteria were excluded fromthe trial:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Serious diseases of the digestive system, liver, pancreas, heart, or kidney, or a history of sucha diseasecurrently under treatment with pharmaceuticals
ocular diseases other than ametropia (hyperopia, myopia, or astigmatism)
existing drug and food allergies
history of, or under treatment for, drug dependence and substance abusenightshift or irregular work hours
history of refractive surgeries; LASIK (laser-assisted in situ keratomileusis) or others
users of reading glasses
current consumption of BE, pharmaceuticals, or supplements that may improve ocular fatigue
participated in another clinical trial within the last 1 monthNutrients 2020, 12, 600

11.
12.
13.

3 of 9pregnant, breastfeeding, or planning for pregnancy during the trial period
blood sampling of ≥ 200 mL within the last 1 month, anddeemed illegible by the trial supervisor or trial attending physician

During the pretrial screening, the subjects’ dominant eyes were determined using the hole-in-thecard method. The subjects were randomized into SBE and placebo groups by staff who were not
involved in the intervention trial; sex and age were balanced between the groups. The staff blinded thesubjects by masking the identification of tested foods with symbols, and the tested foods were securely
managed by the same staff.
According to our previous trials on SBE consumption [8], high-frequency component (HFC) of
accommodative microfluctuation was measured. We calculated the power spectrum of HFC based on
measured values using fast Fourier transformation [11]. The significance level was set at <5%. Under
this condition, the number of subjects required to set the statistical power at 0.8 or 0.9 was 88 or 117,
respectively. The goal case number was chosen as 120 such that the statistical power would be ≥0.8,
even if the subjects dropped out during the trial period.
2.2. Test Foods
The SBE group was administered a hard capsule containing 240 mg SBE (Omnica Co., Ltd,Tokyo, Japan), which was the same SBE as used in previous reports [7–10]. The placebo group was
administered a control capsule without SBE. Table 1 shows the composition of the tested foods. The
concentration of VMA in SBE was 36.89% when determined using HPLC with cyanidin 3-glucosidechloride as the anthocyanin reference and 35.82% when determined using a quantitative methodthat employs the absolute purity of the reference material. The true value of cyanidin 3-glucosidechloride in SBE measured by converting the reference material of absolute purity was 3.83% and thetrue concentration of a total of 15 VMAs was 29.00%. Approximately 68.0 mg of caramel color wasadded to the placebo food to differentiate it from the SBE food.
Table 1. Composition of standardized bilberry extract (SBE) and placebo foods (mg/day).
SBE Food

Placebo Food

SBE *

240.0

0

Dextrin

27.2

51.0

Starch

45.6

193.8

Calcium stearate

22.1

22.1

Silicon dioxide (fine)

5.1

5.1

Caramel color

068* Functional components: total anthocyanins of 36.89%.

2.3. Consumption Methods and Schedule
The subjects consumed SBE or placebo food once daily for 12 weeks without changing their daily
routine. The SBE and placebo foods were to be consumed on an empty stomach before breakfast
or lunch.
Figure 1 shows the flow of tests performed before and after the VDT tasks at week 0, 4, 8, and 12.
Each subject wore an eye mask upon arrival at the trial venue, rested for 10 min, and then had their
HFC of accommodative microfluctuation measured before VDT loading. Measurements were made in
the order of the dominant eye followed by the nondominant eye.

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4 of4 9of 9

FigureFigure 1.
1. Test
Test schedule.
schedule.
Figure1. Testschedule.
Figure1. Testschedule.

BeforeBefore the load test, thethe subjects
subjects performed
performed VDTVDT tasks forfor 4040 minmin inin aa room
room with
with anan illuminanceilluminance
of
500
±
50
lux.
The
load
task
was
as
follows.
A
stringwasattached
toasmartphone(Appleof Before
±
lux.
The
load
task
was
as follows.
A
stringattachedawith
smartphone(Apple Inc.
Inc.
the
load
test,
the
subjects
performed
VDT
tasks
for
40
min
atoroomwith
an illuminance
Before
the50
load
test,
the
subjects
performed
VDT
tasks
for was40 minin ainroom
an illuminance
iPhone
5),
which
was
loopedoverthesubjects’
necks
such
that
the
screenandthesubjects’eyes
were
iPhone
5),
which
was
loopedoverthesubjects’
necks
such
that
the
screenandthesubjects’eyes
wereof 50050 lux.
load
as follows.
A string
attacheda smartphone(Appleof 500± 50± lux.
TheTheloadtasktask
waswasas follows.
A string
waswasattached
to atosmartphone(AppleInc.Inc.
30
cm 5),
apart.
Thewassubjects
playedagame
game
inthisthisposition
such
that
they
all hada similar
VDT
load.
30
apart.
The
subjects
playedainposition
such
that
they
all
had
a
similar
VDT
load.
The
iPhonewhichloopedoverthesubjects’
necks
such
that
the
screenandthesubjects’eyes
were
iPhone 5), which was looped over the subjects’ necks such that the screen and the subjects’ eyes were
The
game
was
a
simpleoneofcollecting
fallingdebris:Tetris®.
As
thepost-load
test,
HFC
was
game
was
a
simpleoneofcollecting
fallingdebris:Tetris®.
As
thepost-load
test,
HFC
was
measured
30 cm
apart.
subjects
playeda gamein thispositionsuch
they
all hada similar
VDT
load.
30 cm
apart.
TheThesubjects
playeda gamein thispositionsuchthatthatthey
all hada similar
VDT
load.
measured
1the
min
after
theoneend
the VDT task.
1 min
after
ofone
VDT
game
aend
simpleofoftask.
collecting
fallingdebris:Tetris®.
post-load
HFC
TheThegame
waswasa simpleofcollecting
fallingdebris:Tetris®.
As As
thethepost-load
test,test,
HFC
waswasmeasured
1 min
after
of theVDT
task.
measured
1 min
after
the theendendof theVDT
task.
2.4. Test
Test Items
2.4.
items
The
ocular tests
performed
at week
0-thefirstfirst
daydayof intervention-(0w),The
tests(HFC(HFCmeasurements)
measurements)were
were
performed
at week
0-theof intervention2.4.2.4.
TestTest
Items
week
4
(4w),
week
8
(8w),
and
week
12
(12w).
The
clinicaldiary
wascheckeddaily(consumption
(0w),
week
4 tests(4w),(HFC
weekmeasurements)
8 (8w), and wereweek performed
12 (12w). at
The
clinicaldiarydaywasdaily
ocularweek
0-theof checkedinterventionTheTheoculartests(HFC measurements)
were performed
at week
0-thefirstfirstday of interventionof the tested food,healthcondition,
adverseevents,etc.). events,
The safety
test
was
performed
during the(consumptionofthetestedfood,healthcondition,
adverseetc.).
The
safetytest
wasperformed
(0w),
week
4 (4w),week
8 (8w),week(12w).
clinicaldiarychecked
daily
(0w),
week
4 (4w),week
8 (8w),
andandweek
12 12(12w).
TheTheclinicaldiary
waswascheckeddaily
screening
and atweekand
12 at(Table2).12 (Table 2).
duringthe(SCR)
screening
(SCR)
week
(consumptionofthetestedfood,healthcondition,
adverseevents,etc.).
The
safetytest
wasperformed(consumption of the tested food, health condition, adverse events, etc.). The safety test was performed
duringscreening(SCR)
at week
12 (Table
duringthethescreening(SCR)
andandat week
12 (Table
2). 2).
Table 2. Tested
Tested foods and test schedule.
Table

Table
2.Screening
Testedfoods
testschedule.
Examination
periodweek
weeks
8 weeks 12 weeks
12 weeksTable
2. Testedfoods
andandtestschedule.
Examination
periodScreening
00week44weeks
8 weeks
Ophthalmic
examination（HFC）●•●
Ophthalmic
examination
(HFC)
•●
•●
•●
Examination
periodScreening
0 week
4 weeks
8 weeks
12 •weeks
Examination
periodScreening
0 week
4 weeks
8 weeks
12 weeks
Anthropometric●●
Ophthalmic
examination（HFC）
Ophthalmic
examination（HFC）
● ● •
● ●
● ●
● ●
● •●
AnthropometricDoctor's questions●●
Anthropometric●
Anthropometric●● •●questions
Blood Doctor’s
test / Urinary
test
●•
●
Doctor's
questions
Doctor's
questions
● ●
● ●
Diary
Blood
test/Urinary
test
•
•
Blood
/ UrinaryBloodtesttest/ Urinary
testtest
● ●
● ●
Test
points:
●,
Diary
entry
period:DiaryDiary
Diary

2.4.1. Ocular Fatigue Test

points:Diary
entry
period: .
TestTestpoints:●, Diary
entry
period:Testpoints:•,●,Diary
entry
period:2.4.1.
Ocularfatigue
test
Using
the
ocularaccommodation function analysis software AA-2 (NIDEK, Nagoya, Japan)
2.4.1.
OcularFatigueTest
2.4.1.
OcularFatigueTestinstalledintheAutorefractor/keratometer
ARK-560A(NIDEK),
theAA-2
HFC wasexamined
accordingtoUsingaccommodation
functionanalysis
software(NIDEK,
Nagoya,Japan)
Using
the theocularaccommodation
functionanalysis
software
AA-2(NIDEK,
Nagoya,Japan)
Using
theocular
ocularaccommodation
functionanalysis
software
AA-2(NIDEK,
Nagoya,Japan)
the reportby Kajita
et al. [12]. In brief, the ARK-560A
accommodation
response
waveform
was measured
8 timesinstalled
ininthe(NIDEK),
accordinginstalledin theAutorefractor/keratometer
ARK-560A(NIDEK),
thetheHFC
waswasexamined
accordingto totoinstalled
theAutorefractor/keratometer
Autorefractor/keratometer
ARK-560A(NIDEK),
theHFC
HFC
wasexaminedexamined
accordingbychanging
thepositionofthetargetatthe0.5
diopterinterval
from
far
to
near
positions
betweenthereportbyKajita
et
al.
[12].
In
brief,
theaccommodation
response
waveformwasmeasured8times
the reportby Kajita
et al.et[12].
In brief,
the accommodation
response
waveformwasmeasured
8 timesthe reportby Kajita
al. [12].
In brief,
the accommodation
response
waveformwasmeasured
8 times by+0.5
and −3.0D
based
on of
thetheminimum
calculated
for fromeach measurement.
Examineeswerebychanging
positiontargetatrefraction
the0.5
diopterinterval
far
topositions
near
positions
betweenby changing
thepositionoftargetatthe
the0.5diopter
diopterintervalfromto nearpositions
betweenchanging
thethepositionof thetargetat
0.5interval
fromfarfarto nearbetween+0.5 and
instructed
to maintainclear
gaze at thetarget atcalculated
each step.for
Frequency
spectra, obtained
through
fast
and
−3.0D
based
onatheminimum
refractioneachmeasurement.
Examinees
were+0.5+0.5
and
−3.0D
based
on minimumtheminimum
refraction
calculated
for measurement.
eachmeasurement.
Examinees
were
−3.0D
based
on
the
refraction
calculated
for eachExaminees
were instructed
Fouriertransformoftheobtained
waveforms,
wereconvertedtologarithm,
and
this
was
integrated
instructed
to maintaina cleargaze
at theat eachstep.
Frequency
spectra,obtainedthrough
fast
instructed
to maintaingaze
attargetthetargetat eachstep.
Frequency
spectra,obtainedthroughto maintaina cleara clear
gaze
at theattargeteach
step.
Frequency
spectra,obtainedthroughfastfast
Fourierbythefrequencyof
1.0–2.3
Hztoobtain
HFC.
The
fractionfromtheminimum
refraction
to−0.75D
Fouriertransformoftheobtained
waveforms,
wereconverted
to logarithm,
and
this
was
integrated
Fouriertransformof theobtained
waveforms,
wereconverted
logarithm,
and
this
was
integrated
transformof theobtained
waveforms,
wereconverted
to tologarithm,
and
this
was
integrated
by thewasdefinedas theHFC-1 Hz
range,and themean
HFCin thefrom
HFC-1
range
was definedas tothe−0.75D
HFC-1
by
the
frequencyof
1.0–2.3
toobtain
HFC.
The
fractiontheminimum
refraction
by thefrequencyof
1.0–2.3
Hztoobtain
HFC.
The
fractionfromtheminimum
refraction
to−0.75D
frequency of 1.0–2.3 Hz to obtain HFC. The fraction from the minimum refraction to −0.75D was definedvalue.
When
human
eyes
are fixedupona stationary
target,subjectively,
refraction
to bedefinedas theHFC-1
range,
mean
HFC
in theHFC-1
range
definedas seemstheHFC-1
waswasdefinedas theHFC-1
range,
andandthe themean
HFCin theHFC-1
range
waswasdefinedas theHFC-1
stationary;
however,
in reality,
the uponciliarya muscle
repeatedly
contracts andrelaxes,seems
leadingvalue.
When
human
eyes
fixed
stationary
target,subjectively,
refraction
to to
be a
value.
When
human
eyes
are arefixed
upon a stationary
target,subjectively,
refraction
seems to besinusoidal
fluctuationinrefraction.
Thisfluctuationiscalledaccommodative
microfluctuation
andisstationary;
however,
in reality,
ciliary
musclerepeatedly
contracts
relaxes,leading
stationary;
however,
in reality,
the theciliary
musclerepeatedly
contracts
andandrelaxes,leading
to ato asinusoidal
fluctuationin refraction.
fluctuation
is calledaccommodative
microfluctuation
sinusoidal
fluctuationin refraction.
ThisThis
fluctuation
is calledaccommodative
microfluctuation
andandis isNutrients 2020, 12, 600

5 of 9as the HFC-1 range, and the mean HFC in the HFC-1 range was defined as the HFC-1 value. Whenhuman eyes are fixed upon a stationary target, subjectively, refraction seems to be stationary; however,in reality, the ciliary muscle repeatedly contracts and relaxes, leading to a sinusoidal fluctuationin refraction. This fluctuation is called accommodative microfluctuation and is divided into low
frequency components (gradual fluctuations of <0.6 Hz) and high-frequency components (relatively
fast fluctuations of 1.0–2.3 Hz). The low frequency components are generated by accommodation that
adjusts the point of focus, whereas the high-frequency components originate from fluctuations in therefractive power of the lens, reflecting the activity of the ciliary muscle [11,13,14].
2.4.2. Safety Evaluation
For safety evaluation, blood tests, urine tests, body measurements, and interviews were conducted
before and after the start of the trial. All undesirable or unintentional signs, symptoms, and illnesses
that occurred during the consumption of the tested foods in this trial were considered as adverse
events, regardless of the causal relationship with the tested food.
2.5. Statistical Processing
Analysis of covariance (ANCOVA) was used to compare the HFC-1 values between the groups.
Values < 10% were considered a significant trend, and those < 5% were considered a significantdifferencein two-tailed tests.
Data were analyzed using SAS 9.3 (SAS Institute, Inc, Cary, NC, USA) and Microsoft Excel 2013
(Microsoft Corp.).
3. Results
3.1. Subject BackgroundThe subjects were recruited between January 18 and February 29, 2016, and the trial was conductedfrom April 11 to July 21, 2016. Of the 206 subjects who underwent pretrial primary screening using
blood and urine tests as well as a questionnaire, 175 were selected to proceed to the secondary screening.
Of these 175 subjects, 14 people were declined from the study for personal reasons and 161 underwent
HFC-1 measurement as the secondary screening; from these 161 subjects, 120 were determined to beeligible based on their results.
Of the 120 eligible subjects, 38 were men and 82 were women; the mean age of the subjects was
35.16 ± 7.38 years. Before the trial started, 11 subjects withdrew due to personal reasons and theremaining 109 subjects (55 in the SBE group and 54 in the placebo group) completed the prescribedschedule and trial. Table 3 presents the background information of the 109 subjects. For the HFC-1
analysis, 97 out of 109 were able to complete the trial according to the instructions provided by thetester. The age distribution of the subjects was 10 subjects in their 20s, 18 subjects in their 30s, and20 subjects in their 40s in the placebo group and 10 subjects in their 20s, 14 subjects in their 30s, and25 subjects in their 40s in the SBE group. Therefore, the age distribution of both groups is similar
and there was no difference in lifestyle. Figure 2 presents the flow from enrollment of the subjects toanalysis of the data.
Table 3. Subjects’ background information.
Variables

Placebo

SBE

Gender (male/female) (n)Age (years)
Height (cm)Body weight (kg)Body mass index (kg/m2)

54 (16/38)
35.46 ± 6.96
163.72 ± 8.03
56.78 ± 8.71
21.06 ± 2.3

55 (18/37)
36.18 ± 7.14
163.11 ± 8.71
57.77 ± 10.65
21.58 ± 3.13

Nutrients 2020, 12, 600

6 of 9Nutrients 2020, 12, 600

6 of 9Figure 2. Flow chart of the study (n = total subjects).
Figure2. Flow chart of the study (n = total subjects).

3.2.Impact
Impacton
onOcularOcularFatigue
FatigueBased
Basedon
onHFC-1
HFC-1Values
Values
3.2.
We
preloadand post-load
HFC-1 valuesof the SBE
and placebo
at week
0, 4,the8,Asforconfirmed
the difference
betweenthe before-loadand post-load
HFC-1
values foods(∆HFC-1),
when
and 12. atWhenmadeversus
the post-loadat week
0 (SBE
placebo,52.21
versus50.72)
variation
weekwe0 (SBE
placebo, HFC-1
1.135 versus
0.123)was versus
covariant,
the SBE
group
presented
covariant, post-load
HFC-1
in the group
SBE groupcomparedwith thoseinsignificantly
lower values
thanvalues
the placebo(Tablesignificantly
4 and Figureimproved3B) at week
4 (SBE versus
placebo,theplacebogroup(Table4
andFigure
3)
at
week
8
(SBE
versusplacebo,50.64
versus
51.13,
p=0.014)
0.478 versus 1.296, p = 0.018) and week 12 (SBE versus placebo, 0.053 versus 0.347, p = 0.049).
and week 12 (SBE versus placebo, 50.65 versus 50.67, p = 0.017).

0w
4w
8w

0w
4w
8w

12w 12w

Before VDT load
BeforePost VDTVDT loadload
Post VDT load
Before VDT load
Before VDT load
Post VDT load
Post VDT load
BeforeVDT
VDTloadload
Before
Post
VDT
load
Post VDT load
BeforeBeforeVDT
VDTloadload
Post
PostVDT
VDTloadload

Table 4. HFC-1 values.
Table 4. HFC-1 values.
Placebo
SBE
PlaceboSBE
p Value(n =48)(n = 49)
(n
=
48)
(n
p Value
Mean
±
SD
Mean
± = 49) SD
Mean
±
SD
Mean
±
SD
50.60
±
4.13
51.08
±
3.84
4.13 52.2151.08 ±± 4.163.84
50.7250.60 ± ± 4.28
50.72
±
4.28
52.21
±
4.16
49.51
±
4.13
50.90
±
5.04
49.51
±
4.13
50.90
±
5.04
50.81
±
4.92
51.37
±
4.29
0.225
50.81
±
4.92
51.37
±
4.29
0.225
50.9450.94 ± ± 4.93
4.93 50.0850.08 ±± 4.684.68
51.1351.13 ± ± 5.19
5.19 50.6450.64 ±± 4.704.70 * 0.014* 0.014
4.33 50.5950.59 ±± 4.204.20
50.3250.32 ± ± 4.33
5.13 50.6550.65 ±± 4.034.03 * 0.017* 0.017
50.6750.67 ± ± 5.13

Valuesshown
the means± standard
deviations;
BeforeVDT andloadPost
HFC-1
and HFC-1.
Post VDT
load
Valuesare areshown
as theasmeans± standard
deviations;
Before VDTload HFC-1
VDT load
Statisticalsignificance:* p < 0.05significance:
versus placebo
visualdisplayterminal.
HFC-1. Statistical* pgroup.
< 0.05VDT:
versusplacebogroup.
VDT: visual display terminal.

Nutrients 2020, 12, 600
Nutrients 2020, 12, 600

7 of 97 of 9FigureFigure3.3.Value
Valuechanges
changesatatweek
week0,0,4,4,8,8,and
and12.
12.Changes
Changesin
inthe
theHFC-1
HFC-1values.
values. Statistical
Statistical significance:
significance:* *pp<<0.05
0.05versus
versusplacebo
placebogroup.
group.

3.3. Safety
3.3. Safety
The incidences of adverse events during the trial period were surveyed based on the subjects’
The incidences of adverse events during the trial period were surveyed based on the subjects’ (n(n = 109) journals and interviews by physicians. Multiple occurrences of the same adverse event in the= 109) journals and interviews by physicians. Multiple occurrences of the same adverse event in thesame subject were considered as one case (Table 5). All adverse events that occurred were determinedsame subject were considered as one case (Table 5). All adverse events that occurred were determined
to be unrelated to the tested foods by the physicians. In addition, the physicians determined that there
to be unrelated to the tested foods by the physicians. In addition, the physicians determined thatwere no changes in the general blood, blood biochemistry, and urine tests associated with the tested
there were no changes in the general blood, blood biochemistry, and urine tests associated with thefood consumption.
tested food consumption.
Table 5. Adverse events in the standardized bilberry extract (SBE) and placebo groups.
Table 5. Adverse events in the standardized bilberry extract (SBE) and placebo groups.
Adverseevents
EventsAdverseCommoncold
cold
CommonHeadaches
Headaches
Digestive
symptoms(stomachache(stomachacheorordiarrhea)
diarrhea)
Digestive
symptoms
Menstrual
pain
Menstrual pain
Other
Other

Placebo (n= 54) (n = SBE
Placebo54) (n = 55)SBE (n = 55)
12
11
6
3
17

12
11
6
3
17

10
18
6
4
24

10
18
6
4
24

4.4. Discussion
Discussion
InInthethepresent
presentstudy,
study,suppression
suppressionofofand
andreliefreliefininocularocularfatigue,
fatigue,withwithciliary
ciliarymuscle
musclecontraction
contraction
causedload
asas
thetheindex,
waswasconfirmed.
ThisThisis onetheoflarge-scalerandomized,
double-blind,causedby
byVDT
VDT
load
index,
confirmed.
is of
one
the large-scalerandomized,
doubleplacebo-controlled
clinicalclinical
trials concerning
ocular fatigue
that havein recentblind, placebo-controlled
trials concerning
ocular fatigue
thatbeen
haveconductedbeen conductedin years.
recentThe
subjects
that werethat
includedin the present
trial
were healthy
individuals
ocular fatigue.
Theyears.
The subjects
were includedin thepresent
trial were
healthy withindividuals
with ocularsubjects
in
the
SBE
group
consumed
240
mg
of
SBE
once
daily
for
12
weeks.
fatigue. The subjects in the SBE group consumed 240 mg of SBE once daily for 12 weeks.
With
ofof
each
subject
as asa reference,
wewe
moved
the thetargetfromfrom
the farthe
Withthetheminimum
minimumrefraction
refraction
eachsubjecta reference,
moved
targetthetofartonear
in eightin
steps
andsteps
measuredfrequencytheof HFCof accommodative
the positions
near positions
eight
and themeasuredfrequencyof HFC ofmicrofluctuation.
accommodative
These
values wereThese
digitizedby the“Accommodative
measurement
softwareAA-2” asmicrofluctuation.
valueswere
digitized by thefunction“Accommodative
functionmeasurement
HFC[15]. AA-2”
HFC is as
generated
viaHFCvibrationof the ciliarymuscle andincreases
excessive
load [16,17].
software
HFC [15].
is generated
via vibrationof theciliary withmuscleand increases
withIna trial thatexamined
simple questionnaire
HFC values,
excessive
load[16,17]. theIn asubjective
trial thatsymptoms
examined via
theasubjective
symptoms andvia ameasured
simple questionnaire
comparedwith subjects
who did
not experiencemuch ocularfatigue,
who routinely
experienced
and measured
HFC values,
comparedwith subjects
who did
not those
experiencemuch ocularfatigue,ocularfatigue
had a greater
increasein HFC
following
VDT increasetasks. Ininaddition,
the subjects
who
those who
routinely
experienced
ocularfatigue
had a greater
HFC following
VDT tasks.
responded
that
VDT
tasks
eitherfatiguedorextremely
fatiguedtheireyes
demonstratedagreater
In addition, the subjects who responded that VDT tasks either fatigued or extremely fatigued theireyes demonstrated a greater increase in HFC compared with subjects who only experienced mild or
little ocular fatigue [18]. As such, HFC is sufficiently and specifically correlated with subjective ocularNutrients 2020, 12, 600

8 of 9increase in HFC compared with subjects who only experienced mild or little ocular fatigue [18]. As such,
HFC is sufficiently and specifically correlated with subjective ocular fatigue and is widely used toevaluate ocular fatigue [12,19,20]. In addition, we used HFC to confirm the effects of SBE consumption
on ocular fatigue [8,9]. In the present trial, we defined the range from the minimum refraction to−0.75 D as the HFC-1 range and used the mean HFC in the HFC-1 range fraction to evaluate ocularfatigue. To eliminate the impact of pre-consumption values in each group, we performed intergroupcomparison using ANCOVA.
Due to the continuous consumption of SBE, compared with the placebo group, the post-VDT load
HFC-1 values showed a significant improvement at weeks 8 and 12, suggesting that SBE suppresses
the tonic accommodation of ciliary muscles in response to VDT load. The observable effects fromweeks 8 to 12 of SBE consumption indicate that 12 week long-term consumption of SBE would notattenuate its efficacy.
Anthocyanin in bilberry extract is a highly soluble antioxidant with a flavylium ion structure [21].
It is found in the ocular tissues [22] and suppresses various inflammatory factors in the body [23].
Because it is possible that anthocyanins migrate and are distributed from the blood to the ciliarymuscle, they can suppress and relieve the tension in the ciliary muscle, thereby potentially reducing
ocular fatigue.
In conclusion, the present trial demonstrated that 12-week oral consumption of 240 mg of
SBE ameliorates and relieves the tonic accommodation of ciliary muscles caused by VDT tasks andnear-vision tasks.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/3/600/s1,
Figure S1: CONSORT 2010 Flow Diagram, Table S1: CONSORT 2010 checklist of information to include whenreporting a randomised trial.
Author Contributions: Conceptualization, M.K.; Methodology, M.K. and N.K.; Formal analysis, M.K.; Data
curation, M.K. and N.M.; Project administration, M.K.; Writing—Original Draft Preparation, M.K. and N.M.;
Writing–Review & Editing, M.K. and N.K. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by funding from Omnica Co., Ltd., Japan.
Acknowledgments: We are grateful to all staff members of the Medicine Evaluation Research Center and K
Medical Office TOC Building Clinic for their dedicated cooperation. We also wish to thank members of the New
Drug Research Center Inc. for performing the statistical analyses. Finally, we would like to thank all members of
Omnica Co., Ltd. for their invaluable support of this research.
Conflicts of Interest: M.K. and N.M. are employees of Omnica Co., Ltd., Japan.

Abbreviations
HFC
SBE
VDT
VMA

high-frequency component
standardized bilberry extract
visual display terminals
Vaccinium myrtillus anthocyanin

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