nutrients

Article
Rice Bran Supplement Containing a Functional
Substance, the Novel Peptide Leu-Arg-Ala,
Has Anti-Hypertensive Effects: A Double-Blind,
Randomized, Placebo-Controlled Study
Yutaro Ogawa 1,† , Naohisa Shobako 1,† , Ikuo Fukuhara 2 , Hisao Satoh 3 , Etsuko Kobayashi 1 ,
Takashi Kusakari 1 , Makoto Suwa 1 , Motonobu Matsumoto 1 and Atsushi Ishikado 1, *
1 Health Care R&D, Sunstar Inc., Takatsuki, Osaka 569-1195, Japan; yuutarou.ogawa@jp.sunstar.com (Y.O.);
naohisa.shobako@jp.sunstar.com (N.S.); etsuko.kobayashi@jp.sunstar.com (E.K.);
takashi.kusakari@jp.sunstar.com (T.K.); makoto.suwa@jp.sunstar.com (M.S.);
motonobu.matsumoto@jp.sunstar.com (M.M.)
2 Fukuhara Clinic, Eniwa, Hokkaido 061-1351, Japan; i-feniwa@gray.plala.or.jp
3 New Drug Research Center Inc., Eniwa, Hokkaido 061-1405, Japan; h-satoh@ndrcenter.co.jp
* Correspondence: atsushi.ishikado@jp.sunstar.com; Tel.: +81-72-693-3061
† These two authors contributed equally to this work.




Received: 15 March 2019; Accepted: 26 March 2019; Published: 28 March 2019


Abstract: The anti-hypertensive effect of processed rice bran (PRB) was recently reported, for which
the novel peptide Leu-Arg-Ala (LRA) was identified as the functional substance. The purpose of
this study was to assess the anti-hypertensive effects of a rice bran supplement containing PRB
in individuals with high-normal blood pressure (systolic blood pressure (SBP): 130–139 mmHg
and/or diastolic blood pressure (DBP): 85–89 mmHg) or grade 1 hypertension (SBP: 140–159 mmHg
and/or DBP: 90–99 mmHg). One hundred individuals with high-normal blood pressure or grade 1
hypertension were recruited to participate in this double-blind, randomized, placebo-controlled study.
Participants were randomly allocated to the placebo group (n = 50) or the test group (n = 50). Each
group took four test tablets (43 µg LRA/day) or four placebo tablets daily. The decrease in blood
pressure in the test group compared with the placebo group was the primary outcome. Adverse
events were recorded and hematological/urinary parameters measured to determine the safety of the
supplement, which was the secondary outcome. In total, 87 participants completed the study. The
SBP of the test group at 12 weeks was significantly lower than that of the placebo group (p = 0.0497).
No serious adverse events were observed. Daily consumption of a rice bran supplement containing
PRB can safely improve mildly elevated blood pressure.

Keywords: double-blind randomized placebo-controlled study; anti-hypertensive effect; novel

peptide; rice bran; high-normal blood pressure

1. Introduction
Approximately one billion people worldwide suffer from hypertension, which is thought to be
the main modifiable risk factor for cardiovascular disease [1,2]. Hypertension is also the most common
lifestyle-related disease in Japan, where 34.6% of men and 24.8% of women have a systolic blood
pressure (SBP) exceeding 140 mmHg [3]. Not only grade 1 hypertension (SBP: 140–159 mmHg/diastolic
blood pressure (DBP): 90–99 mmHg), but also high-normal blood pressure (SBP: 130–139 mmHg/DBP:
85–89 mmHg), increase the risk of developing cardiovascular disease [4] (grade 1 hypertension and
high-normal blood pressure are defined in the Japanese Society of Hypertension guidelines for the

Nutrients 2019, 11, 726; doi:10.3390/nu11040726 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 726 2 of 13

management of hypertension [5]). Interventions involving lifestyle modifications, such as dietary

alterations, can prevent hypertension. For individuals with high-normal blood pressure or grade 1
hypertension, blood pressure control based on diet can be important for maintaining their quality
of life. Some substances derived from food, such as peptides and flavonoids, have been reported
to reduce blood pressure in individuals with high-normal blood pressure, grade 1 hypertension, or
both [6–8]. Some of these substances are marketed as ‘foods for specified health use’ (FOSHU) and are
used to reduce the risk of lifestyle-related disease in Japan.
Rice (Oryza sativa) is one of the three major food grains consumed worldwide, and is a staple food
for about half of the world’s population [9]. Rice bran is a by-product of rice polishing, and while its
high protein content and protein efficiency ratio are well known, it is not well utilized in the food
industry, as it is primarily used for rice oil extraction or as feedstuff [10–12]. Some proteins derived from
food materials are used to produce anti-hypertensive peptides. Ile-Pro-Pro (IPP), Val-Pro-Pro (VPP),
and Met-Lys-Pro (MKP) are derived from casein, a protein found in milk, and their anti-hypertensive
effects have been shown in clinical studies in humans [6,13]. Rice bran protein has been identified as a
source of anti-hypertensive peptides, and the anti-hypertensive effect of processed rice bran (PRB) has
been demonstrated in a previous study [14]. A novel anti-hypertensive peptide Leu-Arg-Ala (LRA),
hydrolyzed from vicilin-like seed storage protein At2g28490, was identified as the functional substance
of PRB. Oral administration of a low dose (0.25 mg/kg) of LRA has a potent anti-hypertensive effect in
spontaneously hypertensive rats (SHRs) [14]. It was also reported that LRA has potent vasorelaxant
activity (EC50 = 0.1 µM; the most potent value identified for a grain-derived peptide to date), and that
this is the main mechanism accounting for its anti-hypertensive effect [15].
The purpose of the present study was to determine whether a rice bran supplement containing PRB
can lower blood pressure in humans with high-normal blood pressure or grade 1 hypertension. The
safety of the intervention was also determined as the secondary endpoint. To these ends, a randomized,
double-blind, placebo-controlled clinical trial with an intake period of 12 weeks was conducted.

2. Materials and Methods

The present study was designed and carried out following the CONSORT 2010 statement
guidelines, and a completed copy of the checklist is provided in Table S1.

2.1. Study Design

The study was a 12-week, randomized, double-blind, placebo-controlled clinical trial
(Figures 1 and 2) carried out in accordance with the principles of the Declaration of Helsinki, with
approval from the Institutional Review Board of Sunstar Inc. (17-FH-09), the Miyawaki Orthopedic
Clinic Institutional Review Board (15000093), and the development subcontractors from the New Drug
Research Center Inc. (Hokkaido, Japan), in accord with ethical guidelines for research on humans
(Ministry of Education, Culture, Sports, Science and Technology; Ministry of Health, Labor and Welfare,
Japan). The trial was registered in the UMIN Clinical Trials Registry (UMIN000028762).

2.2. Study Population

Hokkaido residents were recruited to participate in this study through a website from 8 June
2017 to 14 July 2017. Those who wished to participate in this trial were invited to Fukuhara Hospital
(Hokkaido, Japan), where the details of the study and potential risks were thoroughly explained and
written informed consent obtained. Inclusion criteria were age 45–64 years, high-normal blood pressure
(SBP 130–139 mmHg and/or DBP 85–89 mmHg) or grade 1 hypertension (SBP 140–159 mmHg and/or
DBP 90–99 mmHg) during the pre-observation period (at both 6 and 2 weeks prior to administration of
the test supplement or placebo), and a body mass index (BMI) of less than 30.0 kg/m2 . The following
subjects were excluded: women with childbearing potential; pregnant or lactating women; individuals
who had participated in other clinical trials within the 4 weeks preceding this study or who planned
on participating in another study within 4 weeks of the end of this study; those with heart, liver,
Nutrients 2019, 11, 726 3 of 13

kidney, or thyroid disease (or complications thereof); those with a history of cardiovascular disease;
those with diabetes; those with an allergy to brown rice, rice bran, or rice; those who had experienced
adverse reactions to having blood drawn in the past; those who were excessive drinkers (average
30 mL equivalent/day alcohol) or heavy smokers (average 21 cigarettes/day or more); those on an
extremely irregular diet (due to shift work or for other reasons); those on medications that may have
affected this trial, such as anti-hypertensive drugs; those who regularly consumed specific supplements
or foods with functional claims that may have affected this trial and who did not consent to refrain
from consuming them for 1 week before preliminary test 2; those with secondary hypertension;
those with a history of major gastrointestinal surgery such as stomach resection, gastrointestinal
anastomosis, gastrointestinal surgery, or intestinal resection (excluding appendix resection); and those
judged inappropriate for participation in the study based on the participant’s background, physical
examination, and interview by the principal investigator. These exclusion criteria are based on previous
clinical
Nutrients studies
2018, 10, xand
FORthe criteria
PEER REVIEW of the NIPPON DATA 80 study [6,16–20]. 3 of 13
Nutrients 2018, 10, x FOR PEER REVIEW 3 of 13

Figure 1. Flow chart of participant recruitment.

Figure
Figure 1. Flow
1. Flow chart
chart of
of participant
participant recruitment.
recruitment.

Figure 2. Test session schedule. Circles represent when data or samples were measured or collected.
Figure 2. Test session schedule. Circles represent when data or samples were measured or collected.
Figure 2. Test session schedule. Circles represent when data or samples were measured or collected.
2.2. Study Population
2.2. Study Population
Hokkaido residents were recruited to participate in this study through a website from June 8,
2017 to July 14,
Hokkaido 2017. Those
residents who wished
were recruited to participate
to participate in study
in this this trial were a
through invited to from
website Fukuhara
June 8,
Hospital (Hokkaido, Japan), where the details of the study and potential risks were
2017 to July 14, 2017. Those who wished to participate in this trial were invited to Fukuhara thoroughly
explained and written informed consent obtained. Inclusion criteria were age 45–64 years,
Hospital (Hokkaido, Japan), where the details of the study and potential risks were thoroughly
high-normal blood pressure (SBP 130–139 mmHg and/or DBP 85–89 mmHg) or grade 1
explained and written informed consent obtained. Inclusion criteria were age 45–64 years,
hypertension (SBP 140–159 mmHg and/or DBP 90–99 mmHg) during the pre-observation period (at
Nutrients 2019, 11, 726 4 of 13

2.3. Randomization
Participants were randomly assigned to two groups by the block randomization method, stratified
by age and sex and subsequently stratified by condition (SBP or DBP). The randomization was
performed by an allocation coordinator independent from the investigators, and the randomization
code was kept in a sealed envelope to maintain blinding. Thus the investigators, participants, and
other study personnel were blinded to the treatment assignments for the duration of the study.

2.4. Interventions
The trial was conducted in Hokkaido, Japan between 26 July and 9 December 2017. All study
products (the test food and the placebo) were prepared as tablets that were identical in terms of
appearance and flavor. The nutrient composition of the test products is described in Table 1. The rice
bran supplements were prepared by rice bran hydrolyzation [14]. Rice bran was supplied by SUNBRAN
(Yamagata, Japan). The LRA content in the test food was analyzed by liquid chromatography/mass
spectrometry, and LRA was not detected in the placebo tablets. Participants in both groups took
four tablets once a day with water after breakfast. Participants were asked to record the following
information in a daily diary: drugs and FOSHUs that were consumed, schedules (such as when they
awoke, slept, ate meals, and took the tablets), amount of alcohol consumed, and any symptoms that
they noticed. Participants were advised to maintain their usual lifestyle habits, such as physical activity,
throughout the study.

Table 1. Composition of the tablets used in this trial.

Test Placebo
Calories (kcal/day) 4.45 5.42
Protein (g/day) 0.26 0.00
Fat (g/day) 0.05 0.04
Carbohydrate (g/day) 0.75 1.27
Sodium (mg/day) 7.39 0.00
LRA (µg/day) 43.00 0.00

2.5. Outcomes
The primary outcomes of the study were SBP and DBP each week from the date on which the first
dose of tablets was consumed. Safety, a secondary outcome, was evaluated by the number of adverse
events, adverse event occurrence rate, number of side effects, and side effect occurrence rate.

2.6. Procedures
Blood pressure, pulse rate, and body temperature—Blood pressure, pulse rate, and body
temperature were measured seven times in total: at 6 and 2 weeks before starting to take the
tablets, on the day the participants started taking the tablets, and at 4, 8, 10, and 12 weeks after starting
to take the tablets. Blood pressure was measured using an automatic sphygmomanometer (HEM-759P;
Omron, Kyoto, Japan) based on the method described in the Japanese Society of Hypertension
guidelines for the management of hypertension [5], when the subject had been seated at rest (feet
uncrossed, no talking) for at least 10 min after arriving at the hospital. The average value of two
measurements with similar values (difference < 5 mmHg) was recorded as the blood pressure. Before
visiting for blood pressure measurements, participants were asked to do the following: eat dinner
by 21:00 on the day before the test, refrain from alcohol and avoid eating and drinking after 21:00 on
the day before the test (participants were allowed to drink approximately ~200 mL of water up to 2 h
before the measurement was taken), and refrain from smoking until after the measurement was taken
to avoid influence on the outcome values such as blood pressure, blood glucose, and blood lipids.
Nutrients 2019, 11, 726 5 of 13

Weight and height—Body weight was measured a total of six times: at 6 weeks before starting to
take the tablets, on the day that participants began taking the tablets, and at 4, 8, 10, and 12 weeks after
starting to take the tablets. Body height was measured once—at 6 weeks before starting to take the
tablets—and the measured value was used to calculate the BMI.
Laboratory tests—Laboratory tests were carried out five times in total: at 6 weeks before starting
to take the tablets, on the day the participants started to take the tablets, and at 4, 8, and 12 weeks after
starting to take the tablets. Blood (10 mL) and urine (7 mL) were collected.
For blood tests, white blood cell count, erythrocyte count, hemoglobin, hematocrit, platelet count,
total protein, albumin, total bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate
dehydrogenase, alkaline phosphatase isozymes, γ-glutamyltransferase, creatine kinase isoenzyme,
total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides,
fasting blood glucose, hemoglobin A1c (national glycohemoglobin standardization was only performed
for preliminary test 1), uric acid, urea nitrogen, creatinine, sodium, chloride, potassium, and calcium
were measured. Urinalysis was performed to determine specific gravity, pH, ketone bodies, occult blood
reaction, urobilinogen (qualitative), bilirubin (qualitative), sugar (qualitative), protein (qualitative),
sodium, creatinine, potassium, estimated daily urinary sodium excretion, and estimated daily urinary
potassium excretion. Gram creatinine correction was performed for the estimated daily urinary sodium
excretion and estimated daily urinary potassium excretion. These parameters were measured by an
auto-analyzer (BM8040—JEOL, Tokyo, Japan; XN9100—Sysmex, Hyogo, Japan; or US3500—Eiken
Chemical, Tokyo, Japan).

2.7. Statistical Analysis

Sample size was calculated based on the results from a previous study (a test performed to
determine the optimum concentration) in which administration of 43 µg of LRA derived from 1 g of
PRB significantly reduced blood pressure compared with the placebo group (unpublished data).
The parameters analyzed in this study are presented as means and standard deviation (SD). The
efficacy analysis was based on the per-protocol set. The safety analysis was based on a modified
intention-to-treat principle (full analysis set).
To compare the numerical data for the placebo group and the test group at each time point,
an F-test of equality of variances was performed. In the case of equal variance Student’s t-test was
performed, while in the case of unequal variance an Aspin–Welch t-test was performed. For the
semi-quantitative values derived from the urinalysis, a Mann–Whitney test was performed. For all
two-sided tests, the significance level was set at 5% or 1%.
For the efficacy analysis, a repeated-measures analysis of variance was also performed to compare
the blood pressure at week 0 with the blood pressure at weeks 4, 8, 10, and 12 in each group. If a
significant difference was found, the average values were compared by Dunnett’s method.
For the safety assessment, Fisher’s exact test was performed to detect differences between groups
in terms of the number of side effects and number of adverse events. For the laboratory test data and
physical findings, a repeated-measures analysis of variance was performed. If a significant difference
was found, the average values were compared by Dunnett’s method. A Wilcoxon signed-rank test was
performed for the semi-quantitative values obtained from the urinalysis.
A subgroup analysis was planned by dividing the subjects into two groups (high-normal blood
pressure and grade 1 hypertension) according to the blood pressure values recorded when the
participants started taking the tablets.

3. Results

3.1. Participants
In total, 250 participants were recruited and screened. One hundred participants were enrolled
and randomly allocated to the test or placebo group (Figure 1). Four participants (two in the test group
Nutrients 2019, 11, 726 6 of 13

and two in the placebo group) withdrew before further assessment for personal reasons unrelated
to the trial. Nine participants (four in the test group and five in the placebo group) did not fulfill
the inclusion criteria, as their baseline blood pressures were not classified to grade 1 hypertension or
high-normal hypertension. Two participants withdrew for reasons unrelated to the trial. Data from
participants whose nutritional surveys and medication were judged by the principal investigator as
having the potential to interfere with interpretation of the results were excluded before code breaking.
The overall dropout rate was 13% (13 out of 100). The demographic characteristics and baseline
data for each group are shown in Table 2. None of the participants were taking anti-hypertensive
medication during the trial. Sixteen participants took agents such as intestinal drugs during the test
period. As the principal investigator judged that none of these were related to the test food, these
participants were included in the statistical analysis. The estimated salt intake of each group was not
significantly different between both groups during the test period (Table S2).

Table 2. Demographic characteristics and baseline data of the participants.

Placebo Test p
Male/Female 16/27 15/29
Age (years) 54.1 (6.0) 53.8 (5.7) 0.7697
Body weight (kg) 65.1 (9.8) 63.0 (11.3) 0.3641
Body mass index (kg/m2 ) 24.8 (2.7) 24.3 (3.0) 0.4534
SBP (mmHg) 141.9 (8.5) 141.0 (8.5) 0.6242
DBP (mmHg) 88.9 (6.4) 89.4 (7.0) 0.7150
Body temperature (◦ C) 36.5 (0.3) 36.4 (0.3) 0.2501
Pulse rate (beats/min) 69.9 (9.6) 71.0 (9.8) 0.6157
Total cholesterol (mg/dL) 217.3 (32.1) 213.6 (29.4) 0.5722
HDL-C (mg/dL) 65.0 (13.7) 64.9 (16.8) 0.9725
LDL-C (mg/dL) 130.3 (25.8) 126.1 (29.2) 0.4804
Triglycerides (mg/dL) 110.3 (53.4) 113.3 (68.8) 0.8216
Uric acid (mg/dL) 5.4 (1.2) 5.2 (1.2) 0.5373
Fasting blood glucose (mg/dL) 90.8 (8.6) 89.8 (7.2) 0.5697
ALT (GPT) (U/L) 21.1 (9.8) 25.6 (19.1) 0.1742
Each value is expressed as the mean (SD). SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL-C:
high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; ALT: alanine aminotransferase;
GPT: glutamic-pyruvic transaminase.

3.2. Blood Pressure Response

The mean SBP values at each time point in all subjects with high-normal blood pressure and
grade 1 hypertension in the test (n = 43) and placebo (n = 43) groups are shown in Table 3 and Figure 3.
The mean SBP in the test group was significantly lower than that in the placebo group at 12 weeks
(p = 0.0497). There was no difference in effectiveness between male and female participants. In contrast,
the mean DBP did not differ significantly from that of the placebo group at any subsequent time point.

Table 3. Changes in SBP and DBP in all subjects with high-normal blood pressure and
grade 1 hypertension.

Placebo Test
Weeks n Mean (SD) n Mean (SD) ∆BP p
0.3
0 43 140.0 (7.2) 43 140.3 (7.9) 0.8528
(−2.9 to 3.5)
SBP
−4.3
12 42 137.1 (8.9) 41 132.8 (10.7) 0.0497
(−8.6 to −0.006)
0.9
0 43 89.4 (6.1) 43 90.3 (5.6) 0.4985
(−1.7 to 3.4)
DBP
−0.1
12 42 87.0 (5.8) 41 87.0 (8.0) 0.9687
(−3.1 to 3.0)
Each value is expressed as the mean (SD). ∆BP indicates the difference in the averages of the test group and the
placebo group (95% confidence interval). SBP: systolic blood pressure; DBP: diastolic blood pressure.
 -0.1
12 42 87.0 (5.8) 41 87.0 (8.0) 0.9687
(−3.1 to 3.0)
Each value is expressed as the mean (SD). ΔBP indicates the difference in the averages of the test
group and the placebo group (95% confidence interval). SBP: systolic blood pressure; DBP: diastolic
blood pressure.
Nutrients 2019, 11, 726 7 of 13

Nutrients 2018, 10, x FOR PEER REVIEW 8 of 13

*
(−6.0 to 2.8)
(B) Grade 1 HT Placebo Test
Week n Mean (SD) n Mean ΔBP
(SD) p
0.5
0 24 144.0 (7.1) 24 144.5 (7.8) 0.8247
(−3.9 to 4.8)
SBP
−2.8
12 23 139.1 (6.9) 24 136.3 (11.1) 0.2995
(−8.3 to 2.6)
Figure 3. Changes in SBP over 12 weeks in individuals who received the test food 0.2 or the placebo.
0 24 93.4 (4.2) 24 93.7 (4.2) 0.8495
* Significantly different from the placebo group (p < 0.05). Each value is expressed(−2.2 tomean
as the 2.7) ± SD.
DBP
Figure 3. Changes in SBP over 12 weeks in individuals who received the test 0.6 food or the
12 23 89.8 (4.7) 24 90.5 (6.3) 0.6989
Next, stratified analysis was conducted. The results for the participants with
(−2.6high-normal blood
to 3.9) as the
placebo. * Significantly different from the placebo group (p < 0.05). Each value is expressed
pressure
Eachare
mean shown
value
± SD. in Figure
is expressed as 4A
the and
meanTable
(SD).4A. Significant
High-normal BP:reductions
high-normalin blood
SBP were observed
pressure; Grade in the
1 HT:
high-normal blood pressure
grade 1 hypertension. subgroup
ΔBP indicatesatthe
week 8 (p = 0.0014)
difference and week
in the averages of12 = 0.0397)
the(ptest group compared with
and the placebo
the placebo group,
group (95% while the
confidence DBP
interval).
Tabledid4. not differ
Results fromsignificantly
the stratifiedfrom that of the placebo group. In the
analysis
grade 1 hypertension subgroup, no significant difference was observed (Figure 4B and Table 4B).
(A) High-normal BP Placebo Test
Week n Mean (SD) n Mean (SD) Δ BP p
0.1
0 19 135.0 (3.0) 19 135.1 (3.7) 0.9426
(−2.1 to 2.3）
SBP
−6.8
12 19 134.7 (10.5) 17 128.0 (8.2) 0.0397
(−13.2 to −0.3)
1.7
0 19 84.3 (4.2) 19 86.0 (4.0) 0.2205
DBP (−1.0 to 4.4)
12 19 83.6 (5.3) 17 82.0 (7.6) −1.6 0.4631

Figure 4. Changes in SBP over 12 weeks in participants who received the test food or the placebo
(A) high-normal BP; (B) grade 1 hypertension. * p < 0.05, ** p < 0.01, significantly different from the
placebo group. Each value is expressed as the mean ± SD.

Figure 4. Changes in SBP over 12 weeks in participants who received the test food or the placebo
3.3. Safety
(A): high-normal BP; (B): grade 1 hypertension. * p < 0.05, ** p < 0.01, significantly different from the
A total of 17 adverse events were reported throughout the study, with 12 reported by the placebo
placebo group. Each value is expressed as the mean ± SD.
group and 5 by the test group. All reported adverse events are listed in Table S3. The principal
investigator
3.3. Safety judged that none of these adverse events were related to the test food. No significant
differences in body weight and risk factors (BMI, blood lipid, uric acid, blood glucose, and liver
A total
function) of 17the
between adverse events
placebo groupwere reported
and the throughout
test group the study,
were observed at thewith
end 12 reported
of the by the
trial (Table 5).
placebo group and 5 by the test group. All reported adverse events are listed in Table S3.
The values of all of the parameters measured at 0 and 12 weeks to assess the safety of the intervention The
principal
are showninvestigator
in Table S4. judged that none of these adverse events were related to the test food. No
significant differences in body weight and risk factors (BMI, blood lipid, uric acid, blood glucose,
and liver function) between the placebo group and the test group were observed at the end of the
trial (Table 5). The values of all of the parameters measured at 0 and 12 weeks to assess the safety of
the intervention are shown in Table S4.

Table 5. Body weight, risk factors, and representative values of liver function at 12 weeks

Placebo Test p
Nutrients 2019, 11, 726 8 of 13

Table 4. Results from the stratified analysis.

(A) High-normal BP Placebo Test

Week n Mean (SD) n Mean (SD) ∆ BP p
0.1
0 19 135.0 (3.0) 19 135.1 (3.7) 0.9426
SBP (−2.1 to 2.3)
−6.8
12 19 134.7 (10.5) 17 128.0 (8.2) 0.0397
(−13.2 to −0.3)
1.7
0 19 84.3 (4.2) 19 86.0 (4.0) 0.2205
DBP (−1.0 to 4.4)
−1.6
12 19 83.6 (5.3) 17 82.0 (7.6) 0.4631
(−6.0 to 2.8)
(B) Grade 1 HT Placebo Test
Week n Mean (SD) n Mean (SD) ∆BP p
0.5
0 24 144.0 (7.1) 24 144.5 (7.8) 0.8247
SBP (−3.9 to 4.8)
−2.8
12 23 139.1 (6.9) 24 136.3 (11.1) 0.2995
(−8.3 to 2.6)
0.2
0 24 93.4 (4.2) 24 93.7 (4.2) 0.8495
DBP (−2.2 to 2.7)
0.6
12 23 89.8 (4.7) 24 90.5 (6.3) 0.6989
(−2.6 to 3.9)
Each value is expressed as the mean (SD). High-normal BP: high-normal blood pressure; Grade 1 HT:
grade 1 hypertension. ∆BP indicates the difference in the averages of the test group and the placebo group
(95% confidence interval).

Table 5. Body weight, risk factors, and representative values of liver function at 12 weeks.

Placebo Test p
Body weight (kg) 65.3 (9.7) 64.1 (11.8) 0.5882
Body mass index (kg/m2 ) 24.9 (2.8) 24.7 (3.1) 0.7985
Total cholesterol (mg/dL) 211.4 (33.2) 215.5 (31.8) 0.5642
HDL-C (mg/dL) 65.8 (14.5) 65.3 (18.5) 0.9091
LDL-C (mg/dL) 126.1 (25.3) 128.6 (31.9) 0.6868
Triglycerides (mg/dL) 98.0 (58.5) 107.7 (72.0) 0.4969
Uric acid (mg/dL) 5.4 (1.1) 5.2 (1.4) 0.3613
Fasting blood glucose (mg/dL) 90.7 (8.2) 92.2 (9.3) 0.4495
ALT (GPT) (U/L) 23.1 (9.9) 29.0 (29.7) 0.2184
Each value is expressed as the mean (SD). HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density
lipoprotein cholesterol; ALT: alanine aminotransferase; GPT: glutamic-pyruvic transaminase.

4. Discussion
In the present study, it was demonstrated that taking an anti-hypertensive food product for
12 weeks reduced SBP. Hypertension is one of the important risk factors for cardiovascular disease. It is
well established that medical therapies for hypertension can reduce the rate of death from cardiovascular
disease, myocardial infarction, and stroke [21,22]. Thus, lowering blood pressure through the use of a
food-derived material could represent a powerful approach for preventing cardiovascular disease and
maintaining quality of life. Food-derived substances such as polyphenols and peptides have been
shown to have anti-hypertensive effects in human clinical studies [23,24]. For example, digestion of
milk casein containing the hypotensive peptide Met-Lys-Pro (MKP) showed anti-hypertensive activity
in humans with high-normal blood pressure [6]. In this study, hydrolyzed rice bran protein containing
a novel peptide Leu-Arg-Ala (LRA) as a functional substance was evaluated. LRA reduced SBP in SHRs
and its minimal effective dose was 0.25 mg/kg, which is comparable with the pharmaceutical dose [14].
As shown in Figure 3 and Table 3, the test food caused a significant reduction in SBP compared with
the placebo at 12 weeks. Administration of the test food also resulted in a significant reduction in SBP
(−7.5 mmHg, p < 0.05) compared with baseline. Twelve-week interventions with treatments such as
Nutrients 2019, 11, 726 9 of 13

angiotensin II type 1 receptor blockers and DASH (Dietary Approaches to Stop Hypertension) diets
reduced SBP by 5–10 mmHg in patients with grade 1 hypertension [25,26]. The decrease in SBP induced
by the test food is potentially comparable with that induced by these treatments. This clinical study
supports our hypothesis that the rice bran supplements containing LRA are effective anti-hypertensive
agents in humans.
Previous clinical studies have shown that rice bran reduces SBP when combined with other
interventions such as energy restriction or the administration of sesame oil [27,28]. This is the first
study to show that a rice bran-based food material alone can reduce blood pressure without being
combined with another intervention.
The activity of most anti-hypertensive food-derived peptides is based on angiotensin I-converting
enzyme (ACE) inhibition [29–31]. Some peptides have been shown to exert their anti-hypertensive
effects through nitric oxide (NO)-mediated vasorelaxation. NO is an important vasodilator that is
produced from arginine in the endothelial layer and causes relaxation of vascular smooth muscle.
LRA induces vasorelaxation by activating endothelial NO synthase. LRA also induces vasorelaxation
of the mesenteric artery in SHRs in a dose-dependent manner, and its EC50 value was reported
to be 0.1 µM, the most potent value for all known grain-derived anti-hypertensive materials [15].
Chlorogenic acid and peptides derived from chicken collagen hydrate are also reported to improve
NO-mediated vasorelaxation and significantly reduce SBP, but not DBP [32,33]. Physical activities
such as aerobic exercise and isometric exercise also promote NO bioavailability in the endothelial
layer and are likely to reduce SBP more than DBP [34]. Taken together, these studies suggest that
NO-mediated vasorelaxation lowers SBP more effectively than DBP, which also appears to be the case
for the anti-hypertensive effect of rice bran supplement containing the functional substance LRA tested
in this study. Enhanced NO-mediated vasorelaxation improves vascular function, as measured by
flow-mediated dilation. Further studies are needed to investigate whether our food-derived product
also improves vascular function.
In this study, a difference in effectiveness between male and female participants was not observed,
whereas some studies have shown estrogen to activate the NO system and play a key role in such a
difference [35]. LRA is also reported to show a less potent but specific ACE-inhibitory activity, and
other anti-hypertensive pathways might also contribute to the hypotensive activity [14]. Therefore, the
anti-hypertensive effect of LRA might not be affected by sex difference.
Our stratified analysis showed that test food reduced SBP in subjects with high-normal blood
pressure (Figure 4A). Several studies have shown that high-normal blood pressure is a risk factor
for cardiovascular disease. For example, the Framingham study found that there is an elevated risk
of cardiovascular complications in patients with an SBP of ≥120 mmHg [36], while the NIPPON
DATA 80 study reported that the rate of stroke and circulatory disease is higher in subjects with
high-normal blood pressure compared with those with normal blood pressure [37]. In the SPRINT
study, significant reductions in cardiovascular events and all-cause mortality were observed in an
intensive therapy group with an SBP of <120 mmHg in comparison with a standard therapy group
with an SBP of <140 mmHg [38]. As these studies demonstrate, it is important to prevent patients
with high-normal blood pressure from progressing to serious disease, and especially important to
reduce the SBP in these patients. In the present study, SBP was significantly lowered by the test food
even in subjects with high-normal blood pressure, suggesting that the test material may help prevent
the development of cardiovascular disorders. Otherwise a significant difference was not observed
in the grade 1 hypertension subgroup. As shown in Figure 4B, blood pressure was not stable in
this subgroup and the effectiveness was not accurately evaluated. In addition, it has been reported
that the anti-hypertensive effect of a milk casein diet including MKP was shown not in a grade 1
hypertension subgroup but in a high-normal blood pressure subgroup [6]. This result suggests that
food-derived anti-hypertensive peptide might be more effective for persons with high-normal blood
pressure, although further study is needed to determine the anti-hypertensive effect of PRB, including
LRA for patients with grade 1 hypertension.
Nutrients 2019, 11, 726 10 of 13

As shown in Table 5 and Supplementary Tables S3 and S4, the prevalence of adverse events was
low and the rice bran supplement did not affect body weight, blood lipid level, and blood glucose
metabolism. Safety was also demonstrated previously by an excessive consumption test (5 g PRB/day,
5-fold intake over this study) (unpublished data). These results suggest that the rice bran supplement
could be utilized for functional foods such as FOSHU and safely prevent hypertension. In a previous
study, the anti-hypertensive effect of milk casein hydrate for normotensive humans was determined,
whereby no hypotensive effect was observed [39]. In similar manner, the rice bran supplement also
might not lead to excessive blood pressure reduction in normotensive individuals. Further studies are
required to elucidate the effect of anti-hypertensive food materials in normotensive humans.
The strengths of this study are three-fold. First, this is the first report showing that a rice bran
supplement alone can reduce blood pressure without being combined with another intervention.
The dietary intervention in this study appears to be feasible for clinical practice, especially in Asian
countries where rice is a staple food. It was also shown that the anti-hypertensive effect of the rice
bran supplement was independent of any reduction in body weight, decrease in blood lipid level,
or change in glucose metabolism. Second, the SBP was effectively reduced in human subjects with
high-normal blood pressure and grade 1 hypertension in a trial with a relatively small but sufficient
sample size. Third, the safety of the rice bran supplement was demonstrated, as it was associated with
a low prevalence of adverse events. However, this study also has some limitations. The intervention
period lasted only 12 weeks, and only one dose of the rice bran supplement was tested. In addition, the
mechanism underlying the anti-hypertensive effect observed was not investigated. It is also possible
that the test food contained one or more functional substances other than LRA. Finally, only individuals
with high-normal blood pressure and grade 1 hypertension, without other serious disease such as
diabetes, were assessed in this trial; the effectiveness of this intervention should also be tested in
patients with grade 2 and 3 hypertension, other diseases, or individuals with a smoking habit.
To sum up, a 12-week intervention involving LRA derived from PRB effectively reduced the SBP
in individuals with grade 1 hypertension and high-normal blood pressure. Stratified subgroup analysis
showed that this anti-hypertensive effect was statistically significant in the subgroup with high-normal
blood pressure.

5. Conclusions
In conclusion, it has been shown for the first time that 12-week administration of a rice bran
supplement containing the functional peptide LRA significantly reduced the SBP in patients with grade 1
hypertension and high-normal blood pressure in comparison with a placebo group in a double-blind,
randomized, placebo-controlled study. Stratified analysis showed that the rice bran supplement had a
potent anti-hypertensive effect on subjects with high-normal blood pressure, indicating that the rice
bran supplement could be useful for preventing the progression of patients with pre-hypertension to
grade 1 hypertension.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/4/726/s1,

Table S1: CONSORT 2010 checklist of information to include when reporting a randomized trial, Table S2:
Measured urine sodium and creatinine, estimated salt intake of participants, Table S3: All of adverse events
reported throughout the study. Table S4: Values of body weight, BMI, hematological parameters measured in
this study.
Author Contributions: Y.O., N.S., E.K., and A.I. conceived of and designed the experiments. I.F. was the principal
investigator. H.S. performed statistical analysis. N.S. wrote the paper. Y.O., T.K., M.S., M.M., and A.I. critically
edited the drafted manuscript and contributed important intellectual content.
Funding: This study was conducted by New Drug Research Center Inc., a CRO company under financial support
from Sunstar Inc.
Acknowledgments: The authors thank Atsunori Kashiwagi (Kusatsu General Hospital), who was not involved
in conducting this study, for providing helpful advice about writing the manuscript. The authors thank Emily
Crow and Hugh McGonigle, from Edanz Group (www.edanzediting.com/ac) for checking the English grammar of
the manuscript.
Nutrients 2019, 11, 726 11 of 13

Conflicts of Interest: Y.O., N.S., E.K., T.K., M.S., M.M., and A.I. are employed by Sunstar Inc. I.F. declare no
conflicts of interest. H.S. is employed by New Drug Research Center Inc. Test tablets were prepared by Sunstar Inc.

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