Chapter 40

Effect of the Antioxidant Supplement

Pyrroloquinoline Quinone Disodium Salt
(BioPQQ) on Cognitive Functions
Yuji Itoh, Kyoko Hine, Hiroshi Miura, Tatsuo Uetake, Masahiko Nakano,
Naohiro Takemura, and Kaoru Sakatani

Abstract Pyrroloquinoline quinone (PQQ) is a quinone compound first identified

in 1979. It has been reported that rats fed a PQQ-supplemented diet showed better
learning ability than controls, suggesting that PQQ may be useful for improving
memory in humans. In the present study, a randomized, placebo-controlled, doubleblinded study to examine the effect of PQQ disodium salt (BioPQQ) on cognitive
functions was conducted with 41 elderly healthy subjects. Subjects were orally
given 20 mg of BioPQQ per day or placebo, for 12 weeks. For cognitive
functions, selective attention by the Stroop and reverse Stroop test, and visualspatial cognitive function by the laptop tablet Touch M, were evaluated. In the
Stroop test, the change of Stroop interference ratios (SIs) for the PQQ group was
significantly smaller than for the placebo group. In the Touch M test, the stratification analyses dividing each group into two groups showed that only in the lower
group of the PQQ group (initial score < 70), did the score significantly increase.
Measurements of physiological parameters indicated no abnormal blood or urinary
adverse events, nor adverse internal or physical examination findings at any point in
the study. The preliminary experiment using near-infrared spectrometry (NIRS)
suggests that cerebral blood flow in the prefrontal cortex was increased by the
Y. Itoh K. Hine H. Miura
Department of Psychology, Keio University, Tokyo, Japan
T. Uetake
CX Medical Japan Co., Inc., Tokyo, Japan
M. Nakano (*)
Niigata Research Laboratory, Mitsubishi Gas Chemical Co., Inc., Niigata, Japan
e-mail: masahiko-nakano@mgc.co.jp
N. Takemura
Department of Electrical and Electronics Engineering, Laboratory of Integrative Biomedical
Engineering, College of Engineering, Nihon University, Tokyo, Japan
K. Sakatani
Department of Electrical and Electronics Engineering, Laboratory of Integrative Biomedical
Engineering, College of Engineering, Nihon University, Tokyo, Japan
Department of Neurological Surgery, School of Medicine, Nihon University, Tokyo, Japan
Springer Science+Business Media, New York 2016
C.E. Elwell et al. (eds.), Oxygen Transport to Tissue XXXVII, Advances in
Experimental Medicine and Biology 876, DOI 10.1007/978-1-4939-3023-4_40

319

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Y. Itoh et al.

administration of PQQ. The results suggest that PQQ can prevent reduction of brain
function in aged persons, especially in attention and working memory.
Keywords PQQ Brain Memory Attention Supplement

1 Introduction
Pyrroloquinoline quinone (PQQ) is a water-soluble quinone compound first identified in 1979 from bacteria as a cofactor for redox enzymes [1, 2]. It is contained in
various everyday foods and beverages such as parsley, green tea or fermented
soybeans [3]. PQQ is also detected in various human organs and tissues [4], and
is especially high in human breast milk [5]. PQQ has anti-oxidative and mitochondrial biogenesis capacities [6]. Reduced PQQ has shown strong anti-oxidant capacity, 7.4x greater than ascorbic acid [7]. It was reported that PQQ inhibited cell death
of cultured neuroblastoma in a dose-dependent manner [8], and increased expression of nerve growth factor (NGF) [9]. In addition, it has been demonstrated that
rats fed a PQQ-supplemented diet showed better learning ability than controls at the
early stage of the Morris water maze test [10]. These results suggest that PQQ is
potentially effective for preventing neurodegeneration caused by oxidative stress,
and may improve memory. However, the effect of PQQ on cognitive function in
humans is unknown. If PQQ can maintain or improve cognitive function in the
aged, PQQ may be useful for active and healthy aging. In the present study, we
examined the effect of PQQ on cognitive function in the aged in a randomized,
double-blinded, placebo-controlled trial. We evaluated cognitive functions
employing the Stroop and reverse Stroop test for selective attention, and the laptop
tablet Touch M for visual-spatial cognitive function. Our hypothesis was that lower
interference ratio in the Stroop test or higher score in the Touch M can be achieved
by the intake of PQQ.

2 Methods
2.1

Test Substance, Study Design and Subjects

The test substance was provided in the form of a two-piece hard capsule containing
10 mg of PQQ disodium salt (BioPQQ) manufactured by Mitsubishi Gas Chemical Co., Inc. (Tokyo, Japan) or placebo. Each capsule was ingested with a cup
of water once a day after breakfast for 12 weeks. The dose of PQQ disodium
salt (20 mg/day) was based on our previous animal study [10], which was far
beyond the estimated daily intake of PQQ from everyday foods and beverages
(0.010.4 mg/day).

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The study was a randomized, placebo-controlled, double-blinded two-group

parallel study, conducted in compliance with the Declaration of Helsinki (2008)
and The Ethical Guideline of Epidemiological Study (issued in 2004 by the
Ministry of Education, Culture, Sports, Science and Technology, and the Ministry
of Health, Labor and Welfare). The study protocol (#1112-05-001MGC03352) was
approved by the ethical committee of Nihon University Itabashi Hospital (Tokyo,
Japan) on 17th April 2012 (Approved #24-1). The 42 volunteers were randomly
divided into two groups, the placebo group (n 21) and the PQQ group (n 21).
All participants underwent internal and physical examinations, peripheral blood
test and urinalysis test at baseline (before intake, 0 weeks) and at 12 weeks.

2.2

Evaluations

The Stroop and reverse Stroop test examine selective attention ability, which are
frequently used to examine individual differences in cognitive ability. Participants
took a group version of the Stroop and reverse Stroop test [11] at 0 and 12 weeks. In
each test, participants were required to choose and check one color name or patch
that corresponded to a sample color name or ink color from five alternatives. Low
Stroop interference ratios (SIs) and reverse Stroop interference ratios (RIs) are
considered to indicate high attention ability and ratios are higher in elderly persons
as a result of reduced attention ability. Previous study showed the range of mean SIs
and RIs of each generation covering 792 years old was from 8.20 to 40.9 and
10.89 to 23.30, respectively [12]. We regard SIs as more important, as RIs might
be affected by sensory sensitivity for color, which is sometimes low in elderly
persons [12]. Statistical analysis methods are shown with the results below.
Touch M is a simple evaluation system for visual-spatial cognitive function
utilizing a laptop tablet with a 12-in. size screen originally developed by Yamato
Sangyo Ltd. (Yokohama, Japan) [13]. The score of Touch M (from 0 to 100 points)
is calculated from the reproduction fidelity of the order of the targets appearance,
which is taken to reflect visual-spatial cognitive function, working memory and
action procedure. Studies to date have shown that the score decreases according to
aging after the 60-year age point has been reached. A score below 70 points can be
regarded to reflect a decline in brain functions [13].

3 Results
3.1

Subjects

One subject in the placebo group dropped out for personal reasons. A total of
41 volunteers completed the study. The placebo group contained 20 subjects

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(7 male, 13 female) with a mean age of 58.4  5.2 (SD) years. The PQQ group
contained 21 subjects (7 male, 14 female) with a mean age of 58.6  5.1 (SD) years.
Measurements of physiological parameters indicated no abnormal blood or
urinary adverse events, nor adverse internal or physical examination findings at
any point in the study.

3.2

Stroop and Reverse Stroop Test

The SIs and RIs were calculated at 0 and 12 weeks for each participant, according to
Watanabe et al [12]. The changes in the SIs were 0.4 for the PQQ group (8.0 for
0 weeks and 8.4 for 12 weeks) and 3.1 for the placebo group (8.9 and 12.0,
respectively). A one factor ANOVA revealed no significant difference between
the groups (F(1,39) 0.72, p > 0.10). Here, F stands for variance ratio likewise
below. One sample t-test did not reject the hypothesis that the average change for
both groups was 0 (t(40) 1.10, p > 0.10). This fact suggests that no effect was
caused by practice of the test. The changes in the RIs were 2.2 for the PQQ group
(9.0 for 0 weeks and 11.2 for 12 weeks) and 2.9 for the placebo group (12.9 and
10.0, respectively). The difference in the changes in RIs between the groups was
calculated as significant (F(1,39) 4.34, p < 0.05). Although one-sample t-tests
revealed that the changes between 0 and 12 weeks were non-significant for both
groups ( p > 0.10), these results seem to show that the placebo, not the test substance, has an effect in improving attention ability. However, the difference
between the two groups might be an artifact that can be attributed to the performances in the 0 weeks test that were slightly higher for the placebo group than the
PQQ group (F(1, 39) 3.34, p < 0.01) by one factor ANOVA. An ANCOVA for
the RIs changes with the group as a factor and the 0 weeks RIs as a covariant
revealed no effects of group, nor the interaction between group and 0 weeks RIs, but
an effect of 0 weeks RIs.
Each of the interference ratios was calculated with performances in two of the
subtests that were susceptible to noise. As the SIs is the main target of the analyses
and discussion, we conducted an additional analysis removing the outliers. Three
participants (one from the PQQ group and two from the placebo group) whose
0 weeks or 12 weeks SIs were more than 2 SD from the mean SIs (for all
participants) were removed. The change for the PQQ group (0.9, 9.0 for
0 weeks and 8.1 for 12 weeks) was significantly smaller (F(1,36) 5.41,
p < 0.05) than for the placebo group (5.2, 8.7 for 0 weeks and 14.1 for 12 weeks)
(Fig. 40.1). A one-sample t-test showed significant change in the SIs for the placebo
group (t(17) 2.41, p < 0.05) whereas no significant change was detected for the
PQQ group (t(19) 0.59, p > 0.10).

Effect of the Antioxidant Supplement Pyrroloquinoline Quinone Disodium Salt. . .

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323

p <0.05

Change of SIs

5.2

4
2
0
0.9

2
4

PQQ

Placebo

(n=20)

(n=18)

Fig. 40.1 Mean changes of the Stroop interference ratios (SIs) from 0 to 12 weeks. Error bars
indicate SEs. p value of paired t-test, comparison between 0 and 12 weeks

Placebo group

PQQ group
100

100

0 weeks

0 weeks

p<0.01

12 weeks

12 weeks

80

Score (point)

Score (point)

80

60

40

20

60

40

20

Total

70

< 70

(n=21)

(n=11)

(n=10)

Total
(n=20)

70

< 70

(n=14)

(n=6)

Fig. 40.2 Mean values of the Touch M score. Error bars indicate SEs. p value of paired t-test,
comparison between 0 and 12 weeks. Stratification analyses were performed, dividing each group
into two groups at the threshold of an initial score of 70 points

3.3

Touch M

Touch M results were analyzed by two-tail paired t-test with setting a significant
level at 5 %. In the PQQ group, the mean score had a tendency to increase from
72.4 at 0 weeks to 77.6 at 12 weeks (t(20) 1.68, p 0.10), while in the placebo
group the score did not significantly differ between 0 weeks (74.8) and 12 weeks
(72.2) (t(19) 0.85, p > 0.10) (Fig. 40.2). Stratification analyses were performed,
dividing each group into two groups at the threshold of an initial score of 70 points.

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In the PQQ group, the mean score of higher group (initial score  70points) did not
differ between 0-weeks (85.4) and 12 weeks (83.2) (t(10) 0.55, p > 0.10); however, that of the lower group (initial score <70 points) significantly increased from
58.1 at 0 weeks to 71.5 at 12 weeks (t(9) 3.88, p < 0.01). In the placebo group, the
mean score of the higher group (initial score  70 points) decreased from 85.4 at
0 weeks to 78.9 at 12 weeks (t(13) 1.59, p < 0.10), and that of the lower group
(initial score < 70 points) did not differ between 0 weeks (50.2) and 12 weeks (56.3)
(t(5) 1.29, p > 0.10).

4 Discussion
The change in SIs in the Stroop test within the PQQ group was significantly lower
than that within the placebo group when the three outliers were omitted, and the
Touch M score significantly increased only in the PQQ group with the lower initial
score of < 70 points after 12 weeks.
It was expected that the SIs and RIs would decrease between 0 weeks and 12 weeks
only for the PQQ group if the 12-week intake of the substance improves participants
cognitive ability. We did not observe such a decrease in either the SIs or the RIs. These
data show no effects of PQQ on cognitive ability. However, it is premature to
conclude that PQQ has no effect on cognitive ability, because the Stroop test measures
only limited aspects of cognitive ability and the indices are not very stable.
There are several reasons to focus on the additional analysis of the SIs with
exclusion of outliers: the SIs seem to be a better index than the RIs because changes
of RIs might be canceled out by changes in color sensitivity, and both SIs and RIs
are susceptible to noise [12], so removal of outliers is desirable. In the additional
analysis, there was a significant difference between changes in the SIs at 0 weeks
and 12 weeks for the PQQ and placebo groups. However, the change for the PQQ
group was not significant, while the SIs increased significantly from 0 weeks to
12 weeks for the placebo group. It seems that unknown factors increased the SIs for
both the PQQ and placebo groups and the improvement of cognitive ability by PQQ
canceled out the increase of the SIs for the PQQ group. Hakoda and Sasaki [11]
observed an increase in SIs by repetition with a similar group version of the Stroop
and reverse Stroop test. Although the reason for the increase was not clear, this
observation might support the discussion above.
PQQ is an anti-oxidant compound. As such compounds often cause improvement of brain functions, a similar effect was expected with PQQ [14]. Moreover,
our preliminary testing on NIRS during a working memory task suggested that the
blood flow detected as cerebral blood oxygenation may influence such improvement. The results of this study suggest that PQQ can prevent a reduction of brain
functions in aged persons, especially in attention and working memory. Further
study on the mechanism involved is needed.

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325

Acknowledgments This research was supported in part by a Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Exploratory
Research 25560356), and grants from Alpha Electron Co., Ltd. (Fukushima, Japan) and Iing
Co., Ltd. (Tokyo, Japan).

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