molecules
ArticleUtilization of Flavonoid Compounds from Bark andWood. III. Application in Health Foods
Sosuke Ogawa 1, * ID , Yosuke Matsuo 2 ID , Takashi Tanaka 2 ID and Yoshikazu Yazaki 3
1
2

3

*

Mimozax Co., Ltd., 4291-1, Miyauchi, Hatsukaichi-shi, Hiroshima 738-0034, Japan
Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University,
1-14 Bunkyo-Machi, Nagasaki 852-8521, Japan; y-matsuo@nagasaki-u.ac.jp (Y.M.);
t-tanaka@nagasaki-u.ac.jp (T.T.)
Department of Chemical Engineering, Monash University, Clayton, VIC 3800, Australia;
yoshi.yazaki@monash.edu
Correspondence: ogawa@mimozax.co.jp; Tel.: +81-829-30-6100

Academic Editor: Tsukasa Iwashina
Received: 25 June 2018; Accepted: 22 July 2018; Published: 26 July 2018


Abstract: Dietary supplements ACAPOLIA® and ACAPOLIA PLUS have been sold in Japan under
the classification “Foods in General” for a number of years. In April 2015, the classification of“Foods with Function Claims” was introduced in Japan to make more products available to the
public that were clearly labeled with functional claims based on scientific evidence. In order toobtain recognition of ACAPOLIA PLUS under this new classification, the following informationneeded to be established. The safety of the bark extract of Acacia mearnsii was shown from thehistory of the long-term safe consumption of the extract as a health supplement, together with several
additional clinical safety tests. Robinetinidol-(4α,8)-catechin was detected by high performanceliquid chromatography (HPLC) in the supplement and was suitable for use as the basis of thequantitative analysis. In clinical tests, the amount of change in the plasma glucose concentration in
the initial 60 min after rice consumption by a test group who had been given the Acadia extract wassignificantly lower than the glucose concentration in the group that was given a placebo. The blood
glucose incremental areas under the curve (IAUC) in the first 60 min after rice consumption werealso significantly lower in the Acacia group. The functional mechanisms were explained in termsof the inhibition of the absorption of glucose in the small intestine and the reduction in the activityof the digestive enzymes caused by proanthocyanidins derived from A. mearnsii bark. As a result,
ACAPOLIA PLUS was accepted as a “Food with Function Claims” in August 2016. ACAPOLIA
PLUS is now sold under this new classification. The growth of a typical intestinal bacterium isinhibited by an extract containing flavonoid compounds from A. mearnsii bark; thus, one of the future
directions of study must be a comprehensive investigation of the effect that flavonoid compounds,
proanthocyanidins, have on intestinal bacteria.
Keywords: Acacia mearnsii bark; wattle tannin; proanthocyanidins; functional substance; health foods;
foods with function claims

1. Introduction
Due to the strong antioxidant activity of flavonoid compounds, bark extracts from the Frenchmaritime pine (Pinus pinaster, synonym P. maritima) and radiata pine (Pinus radiata) have beencommercialized as nutritional supplements. These commercial products are Pycnogenol andEnzogenol, respectively. The background and development of Pycnogenol and the basic difference inthe preparation processes between Pycnogenol and Enzogenol have been described [1]. Due to thediscovery that the superoxide scavenging activity (SOSA) value of wattle tannin is approximately ten
Molecules 2018, 23, 1860; doi:10.3390/molecules23081860

www.mdpi.com/journal/molecules

Molecules 2018, 23, x
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1860

2 of 9times that of the extracts of the pine bark supplements, chemical, biochemical, and clinical studies ontimes thatof theextractsof thepine
barktannin
supplements,
andsupplement
clinical studies
wattletanninwereconducted[1].
Wattlehas been chemical,
developedbiochemical,
as a nutritional
andonwattletanninwereconducted[1].
Wattletanninhas
beendeveloped
asanutritional
supplement®
marketed as ACAPOLIA in Japan since 2007. The details of these studies have beenreported® in Japan since 2007. The details of these studies
and marketed
ACAPOLIA
haveadded
been vitamins
reportedpreviously[1]. asA further
supplement,
ACAPOLIA PLUS, which is ACAPOLIA® with® with added vitaminspreviously[1].
A
further
supplement,
ACAPOLIA
PLUS,
which
is
ACAPOLIA
has been sold since 2012 under the category of “Foods in General”, as shown in Figure 1.
has beensold since
2012definition
under the ofcategory“FoodsGeneral”,
shownininGeneral”
Figure 1.and “Foods
In Japan,the legal“food”ofhasbeen individedintoas“FoodsJapan,
the legal
definitionof “food”
been individedintocannot“FoodsGeneral”
with In
HealthClaims”.
A productclassified
as hasa “Foods
General”
beinlabeledwithandany“Foods
health
with
HealthClaims”.
Aproductclassified
asa“Foodsin
General”
cannotbelabeledwithany healthbenefits. A product accepted under the category of “Foods with Health Claims” can be labeledwithbenefits. A
product
acceptedunderfunction
the categoryof “Foods
with HealthofClaims”
can be aslabeledwithspecifiedhealthclaimsor nutrient
claims.
The advertisement
foods classed“Foodsin
specifiedhealthclaimsornutrient
function
claims.
The
advertisementoffoodsclassedas“Foodsin
General” is strictly controlled. The classification “Foods in General” is the base level and cannot be
General”
isstrictly
controlled.
The
classification“Foodsin
General”
is
the
base
level
and
cannotbedescribed further. The next category, “Foods with Health Claims”, was divided, until recently, into
described
TheHealthnext category,“Foodswith
HealthClaims”,
was divided,
recently,
into“Foodsforfurther.
SpecifiedUses” and“Foodswith
Nutrient
Function
Claims”.until
In orderto make“FoodsforSpecifiedHealthUses”
and“Foodswith
Nutrient
Function
Claims”.
In
order
to
make
more
more products that were clearly labeled with functional claims based on scientific evidence available
products
that wereclearly
withconsumers
functionaltoclaims
scientificchoices,
evidencetoto
the generalpublic,andlabeled
to enable
makebased
moreoninformedanavailable
additional
the generalpublic,andFunction
to enable
consumers
to make more
informed
an inadditional
category“Foodswith
Claims”
was introducedin April
2015,choices,
as shown
Figure 1 category[2,3].
“Foods with Function Claims” was introduced in April 2015, as shown in Figure 1 [2,3].

Figure 1. Categories of food products in Japan in April 2015.
Figure 1. Categories of food products in Japan in April 2015.

The acceptanceacceptance ofof ACAPOLIA
ACAPOLIA PLUS
PLUS under
under thethe category
category “Foods“Foods with
with Function
Function Claims”
Claims” offers
offers aa
Thecommercial advantage.
advantage. As
As aa requirement,
requirement, thethe following
following scientificscientific tests
tests were
were conductedconducted andand accepted
acceptedcommercial
bytheSecretary-GeneraloftheConsumer
Affairs
AgencyinJapan.
by the Secretary-General of the Consumer Affairs Agency in Japan.
1. TheThe supplement
supplement safety
safety has
has toto be
be substantiated.
substantiated.
1.
2.
An
analyticalmethodforthequalitativeand quantitative
quantitative determination
determination ofof thethe functional
2. An analytical method for the qualitative andfunctional
substanceor
substancesmustbeestablished.
substance or substances must be established.
3. The
The effectivenesseffectiveness ofof thethe supplement
supplement must
must be3.
be substantiated.
substantiated.
4.
The
functional
mechanisms
must
be
fully
explained.
4. The functional mechanisms must be fully explained.
This paper
paper briefly
briefly describes
the processThis
describes theprocess whereby
whereby thethe healthhealth food
food supplement
supplement containing
containing
proanthocyanidins(flavonoids)
derivedfrom
Acaciamearnsii
bark
came
to
be
acceptedinthe“Foodsproanthocyanidins (flavonoids) derived from Acacia mearnsii bark came to be accepted in the “Foods
with
Function
Claims”
categoryforthecontrolofblood
glucoseinhumans.
with Function Claims” category for the control of blood glucose in humans.
2. Results
2. Results
2.1. ACAPOLIA PLUS Safety
2.1. ACAPOLIA PLUS Safety
In order to substantiate the ACAPOLIA PLUS safety requirements, a history of consumption of
In orderto substantiate
the ACAPOLIA
PLUS
requirements,
a historyof consumptionofA. mearnsii
bark
extract by humans
and a safety
testsafety
on functional
substances(whichin this case wasA.
mearnsii
bark
extractbyhumansandasafetytest
onfunctional
substances(which
in
this
case
was
proanthocyanidins-derived A. mearnsii bark) were assessed as described below.
proanthocyanidins-derived
A. mearnsii bark) were assessed as described below.
As was previously indicated, ACAPOLIA® has been on the market in Japan for more than tenAs
waspreviously
indicated,
ACAPOLIA® has been on the market in Japan for more than ten
years, and ACAPOLIA PLUS for a shorter period. Both supplements are manufactured by Mimozax
years, and ACAPOLIA PLUS for a shorter period. Both supplements are manufactured by Mimozax
Co., Ltd. (Hiroshima, Japan). The tablets of both ACAPOLIA® and ACAPOLIA PLUS contain a daily

Molecules 2018, 23, 1860

3 of 9Co., Ltd. (Hiroshima, Japan). The tablets of both ACAPOLIA® and ACAPOLIA PLUS contain a daily
dose of 250 mg of the extract. In the time that these flavonoid-containing supplements have been on
the market, they have been consumed by more than 100,000 people. There have been no instancesof serious adverse events reported by consumers to Mimozax Co., Ltd (Hiroshima, Japan). during
this time. Therefore, it was concluded that a daily dose of 250 mg of the extract could be taken safely.
However, additional specific safety tests were undertaken as described below.
Using healthy male adult subjects, four safety tests of tablets which contain proanthocyanidins
derived from A. mearnsii bark as the functional substance were undertaken, see Table 1, and reported
by Kataoka et al. [4]. Two of the tests were single-dose tests and the other two were 4-week repeateddose tests. The results of both the single-dose safety study and the 4-week repeated dose safety study
showed no safety concerns for daily doses of up to 1000 mg of the extract. These results supported thesafety of the product.
Table 1. Clinical test conditions of four different tests.
Test No.

Clinical Test Conditions

Study I

Twenty subjects were divided to 4 groups of 5 subjects and took study diets of 2, 4, 6 and8 tablets (each 250, 500, 750 and 1000 mg as the A. mearnsii bark extract) during a single trial.

Study II

Five subjects took study diets of 12 tablets (1500 mg as the A. mearnsii bark extract) during asingle trial.

Study III

Twenty-two subjects were divided into 2 groups and each group took a study diet of either
2 tablets (a daily total of 250 mg of the A. mearnsii bark extract) or 4 tablets (a daily total of
500 mg of the A. mearnsii bark extract) over a 4-week trial period.

Study IV

Twenty-five subjects were divided into 2 groups and each group took a study diet of either
6 tablets (a daily total of 750 mg of the A. mearnsii bark extract) or 8 tablets (a daily total of1000 mg as the A. mearnsii bark extract) over a 4-week trial period.

1*

2*

1*: single-dose test, 2*: 4-week repeated dose safety test.

These results were considered sufficient for the supplement safety requirement. However, if theSecretary-General of the Consumer Affairs Agency in Japan decided that the history of consumption
by humans based on actual intake data was not satisfactory, further safety tests would be needed toshow that the supplements were safe up to a level of five times the recommended daily intake. In orderto satisfy the guideline and confirm the safety of the functional substance, additional overdose studies
were undertaken in which a total of 29 healthy adult subjects were involved. The trials and results
were reported by Ogawa et al. [5] in 2017. The subjects were randomized to an Acacia group (whoeach received 1875 mg of A. mearnsii bark extract per day) or a Placebo group (who received zeroextract). The trial lasted for four weeks in a double-blind, parallel study. The results indicated that adaily intake of 1875 mg of an Acacia dietary supplement was safe for healthy adults.
2.2. Qualitative and Quantitative Analyses of ACAPOLIA PLUS
In circumstances where the functional substance of a supplement is a natural product, it isnecessary to identify the origin of the functional substance and the chemical equivalent of the functional
substance. The A. mearnsii extract used as the raw material for ACAPOLIA PLUS has been produced asa food-grade substance in South Africa for a number of years; methods of qualitative and quantitative
analyses of the functional substance of the supplement have been researched and confirmed as follows:
2.2.1. Qualitative Analyses
Robinetinidol-(4α,8)-catechin, shown in Figure 2, was reported as being a constituent of A. mearnsii
and, to date, has not been isolated from other plants [6–10]. Therefore, whether A. mearnsii extract
is included in any preparation may be determined by establishing if robinetinidol-(4α,8)-catechin ispresent. The qualitative analysis for proanthocyanidins was performed by HPLC, as shown in Figure 3.
The detailed method of qualitative analysis is described below.

Molecules2018,2018,23,
23,x xFOR
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Molecules

4 4ofof9 9extractisisincluded
includedininany
anypreparation
preparationmay
maybebedetermined
determinedby
byestablishingestablishingififrobinetinidol-(4α,8)robinetinidol-(4α,8)extract
Molecules 2018, 23, 1860
4 of 9catechinispresent.
The
qualitative
analysisforproanthocyanidins
wasperformed
byHPLC,
HPLC,
catechin is present. The qualitative analysis for proanthocyanidins was performed byasas
shownininFigure
Figure3.3.The
Thedetailed
detailedmethod
methodofofqualitative
qualitativeanalysis
analysisisisdescribed
describedbelow.
below.
shown
One
tabletcontainingA.
mearnsii
extractwaspowderedandextracted
with60%
aqueousethanol
One tablet
mearnsii
extract
waswaspowderedand extractedwith 60%
aqueousethanoltabletcontaining
containingA.A.
mearnsii
extract
powderedand extracted
with
60% aqueous
(2.0
mL).
After
filtration(membranefilter,
0.45
μL),
10
μL
of
filtratewas
analyzed
by
HPLC.
(2.0
mL).(2.0
After
filtration(membranefilter, 0.45μL),0.45
10 µL),
μL of10filtrate
analyzedby HPLC.
ethanolmL).
After filtration(membranefilter,µL of wasfiltrate
was analyzed
byRobinetinidol-(4α,8)-catechin(preparedfromA.
mearnsii
extract
[10])
was
also
analyzedasastandard
Robinetinidol-(4α,8)-catechin(preparedfromA.
mearnsii
extract
[10])
was
also
analyzedasastandard
HPLC. Robinetinidol-(4α,8)-catechin (prepared from A. mearnsii extract [10]) was also analyzed as a(t
23.1min).
min).
Analytical
HPLCwaswas
performed
usinga aCosmosil
Cosmosil
5C-ARII(NacalaiTesque,
Tesque,Tesque,
Kyoto,(t
R R23.1
Analytical
HPLC
performed
using
5C
1818
-ARII
Kyoto,
standard(tR 23.1
min). Analytical
HPLC
was performedusing
a Cosmosil
5C(Nacalai
18 -ARII (Nacalai
Japan)column
columncolumn(250× ×4.6
4.6mm,i.d.)withwithai.d.)agradientgradient
4%–30%(39min)andand
30%–75%(15min)min)
Japan)(250mm,
ofof4%–30%min)
Kyoto,
Japan)(250
×i.d.)
4.6mm,
with elution
aelution
gradient
elutionof(394–30%(3930%–75%min)and(1530–75%
◦
CH
3
CN
in
50
mM
H
3
PO
4
at
35
°C
(flow
rate,
0.8
mL/min;detection,
Jasco
photodiodearray
detectorCH
CN in
503 CNmMinH50
3PO
4 atH
353 PO°C4(flow
0.8 mL/min;detection,detection,
Jasco photodiodearray detector(15 3min)CHmM
at 35 rate,
C (flow
rate, 0.8 mL/min;
Jasco photodiodearray
MD-2010).
MD-2010).
detector
MD-2010).
OH
OH
OH
OH
HO
HO

OO

OH
OH
OH
OH

HO
HO

OH
OH

OO

OH
OH
OH
OH

OH
OH

robinetinidol-(4α,8)-catechin (1)robinetinidol-(4α,8)-catechin(1)
Figure2. Structureofofrobinetinidol-(4α,8)-catechin.
robinetinidol-(4α,8)-catechin.
FigureFigure 2.
2. Structure
Structure of robinetinidol-(4α,8)-catechin.

FigureHPLC
profilesof of(a)
ACAPOLIA
PLUS(1 tablet/10mL) and(b)androbinetinidol-(4α,8)-catechin.
Figure3.
HPLC
profilesof(a)(a)ACAPOLIA
ACAPOLIA
PLUS
tablet/10mL)
and(b)(b)robinetinidol-(4α,8)robinetinidol-(4α,8)Figure
3.3.
HPLC
profiles
PLUS(1(1tablet/10mL)
Detection
wavelength:
230
nm;
injected
volume:
10
µL
for
each
sample.
catechin.Detection
Detectionwavelength:
wavelength:230
230nm;
nm;injected
injectedvolume:
volume:1010μL
μLfor
foreacheachsample.
sample.
catechin.

2.2.2.Quantitative
QuantitativeAnalyses
Analyses
2.2.2.
Quantitative
Analyses
2.2.2.
Themethod
methodfor
forthethequantitative
quantitativeanalysis
analysisof
proanthocyanidinswas
wasdeveloped
developedbased
basedon
onthe
the
The
methodfor
analysis
ofofproanthocyanidins
wasdeveloped
based
on
the
The
the
quantitative
proanthocyanidins
Folin-Ciocalteu
method.
The
Folin-Ciocalteu
methodisthemajormethodusedforthequantification
Folin-Ciocalteu
method.
The
Folin-Ciocalteu
methodisthemajormethodusedforthequantification
of
Folin-Ciocalteu method. The Folin-Ciocalteu method is the major method used for the quantification
ofphenols
phenolsandpolyphenols[11].
However,otherantioxidant
compounds,
suchascorbic
acid,
also
phenols
andandpolyphenols[11].[11].
However,otherantioxidant
compounds,
suchsuchasascorbic
acid, acid,
also
yield
of
polyphenols
However,otherantioxidant
compounds,
asasascorbic
also
yield
coloredproducts
when
thismethod
is
used
[12].
It
is
essential
toremove
them
in
order
coloredproducts
when
thismethod
is
used
[12].
It
is
essential
toremove
them
in
order
to
accurately
yield colored products when this method is used [12]. It is essential to remove them in order toto
accuratelythe
determine
theproanthocyanidins
amountofofproanthocyanidins
proanthocyanidins
thetablets.
tablets.
Therefore,
afterthethepowdereddetermineamounttheofin the tablets.
after
the powderedtablets
wereaccurately
determineamountininTherefore,
theTherefore,
after
powderedtabletswerewere
suspended
inwaterwater
andtreatedtreatedwith
withultrasound,thesolution
solutionwas
wasfiltered.
filtered.
The
filtratesuspendedinsuspended
water andin
treatedwithultrasound,thesolution was
filtered.
The
filtrate
wasappliedtablets
andultrasound,theThe
filtrateappliedC
cartridge,
andthethenon-adsorbed
non-adsorbed(including
constituents(including
ascorbicacid)were
were
towas
aC
the non-adsorbed
constituents
ascorbicacid)ascorbic
werewashedwith
18 cartridge,
wasappliedtotoa aCand
1818cartridge,
andconstituents(includingacid)
washedwithwater.No
Noproanthocyanidins
proanthocyanidins
weredetectedinthe
thewatereluate.
eluate.The
Theincluding
adsorbed
water.
Nowithproanthocyanidins
were detected in thewater
eluate.inThe
adsorbed
constituentswashedwater.
weredetectedwater
adsorbedproanthocyanidins were eluted with methanol, and this solution was used for the quantitative

Molecules 2018, 23, 1860

5 of 9estimation of the proanthocyanidins with the use of the Folin-Ciocalteu reagent. (+)-Catechin wasused as a positive control, and the proanthocyanidins content was evaluated as a catechin equivalent.
(+)-Catechin is commercially available and has a structure similar to those of the proanthocyanidins
contained in A. mearnsii [10]. Therefore, accurate quantitative values reflecting the proanthocyanidins
content can be obtained.
In order to increase the accuracy of the value of the proanthocyanidins content, theproanthocyanidins fraction prepared from A. mearnsii extract could be used as a positive control.
However, the A. mearnsii extract contains phenolic compounds other than proanthocyanidins,such as monomer catechins [10]. Therefore, removing these from the quantitative values affords
a more accurate estimate of the proanthocyanidins content. As a result of the quantitative analysis,
six tablets of ACAPOLIA PLUS contain 120 mg (catechin-equivalent) or 163 mg (A. mearnsii
proanthocyanidins-equivalent) of proanthocyanidins. A detailed method of the quantitative analysis
is described below.
Tablets containing A. mearnsii extract were powdered using a mortar and pestle. Upon grinding,
100 mg of this powder was suspended in water using a 10 mL measuring flask, and treated withultrasound for 3 min. The suspension was filtered through a membrane filter (0.45 µm), and the filtrate(1.0 mL) was applied to water-substituted Sep-Pak Plus C18 (Short, 360 mg/0.7 mL, Waters, Milford,
MA, USA). The non-adsorbed constituents were eluted with water (2.0 mL), after which the adsorbed
constituents containing the proanthocyanidins were eluted with 3.0 mL methanol. The methanol-eluted
solution was diluted to 5 mL in a measuring flask, and water (1400 µL) was added to the diluted
solution (600 µL). The resulting solution was diluted to concentrations of 50% and 25% with 30%
aqueous methanol, and these solutions were subjected to quantitative analysis. The prepared sample
solution or a standard solution (30% aqueous solution of (+)-catechin (Tokyo Chemical Industry
Co., Tokyo, Japan) (50.0, 25.0, 12.5, 6.25 µg/1.0 mL)) (100 µL) was applied to a 96 well microplate.
After which, 25 µL of Folin-Ciocalteu’s phenol reagent (Kanto Chemical Co., Tokyo, Japan) was added
to each well and left to stand for 3 min. After standing, 25 µL of 10% aqueous sodium carbonate
solution was added to each well and allowed to stand for 1 h with light shielding at room temperature
to allow the color to develop. After one hour, the UV/vis absorbance was measured at 690 nm using
an Emax precision microplate reader (Molecular Devices, San Jose, CA, USA).
2.3. Effectiveness Clinical Trial with ACAPOLIA PLUS on Suppressing Postprandial Blood Glucose Elevation
The scientific evidence for the proposed functional claims must be explained by one of thefollowing methods. The first is a clinical trial with a finished product, and the second is a systematic
literature review on a finished product or functional substance. In this case, a clinical trial withthe finished product, ACAPOLIA PLUS, was conducted and reported by Takeda et al. [13] in 2016.
Subjects in this study were as follows; (1) non-diabetic men and women aged between 40 and 60 yearsof age; and (2) possessed a fasting plasma glucose (FPG) level of between 110–125 mg/dL or an
impaired glucose tolerance (120 min glucose level in a 75 g oral glucose tolerance test (OGTT), between
140–199 mg/dL). A total of 13 subjects were finally selected for this study. The changes in the blood
glucose levels were monitored over a period of 120 min after taking ACAPOLIA PLUS or a placebo
before consuming 200 g of cooked rice. The results showed that the amount of change in the plasma
glucose concentration in the initial 60 min after rice consumption in the Acacia group (the group given
ACAPOLIA PLUS) was statistically lower than the group that was given a placebo, see Figure 4A.
The blood glucose incremental postprandial areas under the curve (IAUC), shown in Figure 4B, in theinitial 60 min after the consumption of rice were also significantly lower in the Acacia group. This study
was conducted with relatively few participants and therefore should be repeated with larger numbers ofparticipants. However, from the limited study, it was concluded that the ACAPOLIA PLUS containing
proanthocyanidins derived from A. mearnsii bark did reduce the absorption of carbohydrates andlimited the rise of blood glucose in humans.

Molecules 2018, 23, x FOR PEER REVIEW
Molecules 2018, 23, 1860

6 of 96 of 9Figure 4. The amount of change in the plasma glucose concentration (A) and incremental area under the
Figureof4.blood
The amountchangein(B)theofplasma
glucoseindividuals
concentration(A) and incremental
area
under
curve
glucoseof(0–60
min)
non-diabetic
administeredcooked rice.
Each
bar
the
curve
of
blood
glucose
(0–60
min)
(B)
of
non-diabetic
individuals
administeredcookedrice.
Eachrepresents the mean for 13 non-diabetic individuals ± standard deviation.  Extract of Acacia bark,
barPlacebo
represents
the mean for
13 non-diabetic
individuals
standard deviation.
□ Extract of Acacia bark,* Significantly
different
from placebop < 0.05±(Students’t-test)■ Placebo * Significantly different from placebo p < 0.05 (Students’ t-test)

2.4. Functional Mechanisms
2.4. Functional Mechanisms
The functional mechanisms for the reduction in the carbohydrate absorption and the limit of
The functional
mechanisms
forafterthe reduction
the carbohydrate
absorption
andthe limit inof thethethe blood
glucose risein humans
taking theinextractof A. mearnsii
bark wereexplainedblood
glucoseriseinhumansafter
takingtheextractofA.
mearnsii
bark
were
explained
inthefollowing terms of two potential mechanisms. Namely, the inhibition of the absorption of glucose infollowingterms ofby
twomechanisms.
Namely,intheinhibition
the absorptionof theglucoseinthesmall intestine
thepotential
bark extract
and the reduction
thedigestiveof
enzymeactivity by
extract.
the smallintestine
by
the
bark
extract
andthereductioninthedigestive
enzymeactivitybythe
Ikarashi et al. [14] revealed that the extract significantly inhibited the increase in plasma
extract.
glucose concentration after maltose, sucrose, or glucose loading in vivo. This inhibitory effect may
Ikarashi et al.
extract
significantly
inhibited the increasein plasma
glucosebe attributable
to [14]the revealed
inhibitionthatof theglucoseuptakevia a sodium-dependent
glucosetransporter
concentration
after
maltose,sucrose,or
glucoseloading
in
vivo.
This
inhibitory
effectmay(SGLT) and glucose transporter (GLUT) in the small intestine. It has been reported that there beisattributable
totheinhibitionofglucoseuptakeviaasodium-dependent
glucosetransporter(SGLT)
inhibition of digestive enzymes such as lipase [10,14], α-amylase [10,15], and glucosidase [14] by
andextract
glucosefromtransporter(GLUT)
the small
It has beenthat there isfrom
inhibitionoftheA. mearnsii
bark.inKusano
et intestine.
al. [10] revealed
that reported
proanthocyanidins
the bark
digestive
enzymessuchaslipase[10,14],α-amylase[10,15],andglucosidase
[14]
by
the
extract
fromof A. mearnsii exhibited strong α-amylase inhibition activity, comparable to the results seen with
A. mearnsii
bark.
Kusanoet al.than[10] thoserevealed
proanthocyanidins
bark ofleaf
A. extracts.
mearnsiiblack
tea andmuch
strongerseenthatwithgreen tea, oolongfromtea, theor guava
exhibitedstrongα-amylase
inhibition
activity,
comparable
totheresults
seen
with
black
tea and
The proanthocyanidins exhibited strong lipase inhibition activity. The most active proanthocyanidins
much stronger
than thoseseen with green
tea, oolong
tea,andor shown
guavatoleaf
extracts.
The
fraction
was characterizedby spectroscopic
and chemical
methods
containtetramericproanthocyanidins
exhibitedstronglipaseinhibition
activity.
The
mostactiveproanthocyanidins
to octameric compounds mainly composed of robinetinidol units. Ikarashi et al. [14] revealed thatfractionwasby inhibited
spectroscopic
and chemical
methods
andshown
to containtheextract
ofcharacterized
A. mearnsii bark
the activityof lipase,maltase,andsucrase.
Matsuotetramericet al. [15]tooctamericcompounds
mainlycomposedofrobinetinidol
units.
Ikarashi
et
al.
[14]
revealedthereported that the hot-water bark extracts were carefully fractionated into six fractions usingthat
DiaionextractofA.
mearnsiibark
inhibitedtheactivityoflipase,maltase,andsucrase.
Matsuoetal.
[15]HP20 column chromatography with monitoring using thin-layer chromatography (TLC) employing
reported
that thehot-water
barkTheextracts
were(2–6)
carefully
fractionatedintocompounds
six fractionsand
using
Diaiontwo
different
solvent
systems.
fractionscontained
phenolic
exhibited
HP20 column
chromatography
with monitoring
using thin-layer
chromatography(TLC)
employing
α-amylase
inhibitory
activity. Contrastingly,
the fraction
1 which contained
sugars andpolyols
did nottwo
different
solvent
systems.
The
fractions(2–6)
contained
phenolic
compounds
andexhibited
αexhibit any α-amylase inhibitory activity. Spectroscopic results clearly indicated that fractions withamylaseinhibitoryactivity
activity.
Contrastingly,
the fraction 1 oligomers
which contained
sugars and polyols
notstronginhibitory
contained
proanthocyanidins
with catechol-type
B-ringsdid
ratherexhibitany
α-amylase
inhibitory
activity.
Spectroscopic
results
clearly
indicatedthatfractionswiththan pyrogallol-type B-rings. HPLC analysis of the pyrolysis products showed peaks for pyrocatechol
strongonly
inhibitory
activity
proanthocyanidins
oligomers
B-rings rather
were
observedin the contained
mixtures obtained
from the fractions
withwithhighcatechol-typeinhibitory activities.
than Inpyrogallol-type
HPLC analysis
the pyrolysis
products showed
peaks for pyrocatechola clinical test,B-rings.
the ACAPOLIA
PLUSofreduced
the absorptionof carbohydrates
and limited
were
only
observed
inthemixturesobtainedfromthefractionswithhighinhibitory
the rise of blood glucose in humans. Inhibition of the increase in the plasma glucoseactivities.
concentration
In a clinical test, the
ACAPOLIA
PLUS
reducedthe absorptionof carbohydrates
and limited
theby proanthocyanidins
derivedfrom A.
mearnsii
in vivo,and α-amylase
and glucosidase
inhibition
rise of bloodin humans. derived
Inhibitionfromof thethe plasma
concentrationbyactivities
by glucose
proanthocyanidins
A. increase
mearnsiiinbark
in vitro glucose
were consideredas theproanthocyanidins
derivedfromA.
mearnsii
in
vivo,
and
α-amylase
andglucosidase
inhibition
functional mechanisms.

Molecules 2018, 23, 1860

7 of 9

3. Discussion
3.1. ACAPOLIA PLUS Containing Flavonoid Compounds as a “Food with Function Claims”
The supplement safety has been confirmed, an accurate analytical method for the qualitativeand quantitative determination of the functional substance has been developed, the supplement
effectiveness has been confirmed, and the functional mechanisms explained all conditions required
for the reclassification of ACAPOLIA PLUS. These data were submitted to the Secretary-General of
the Consumer Affairs Agency for classification under “Foods with Function Claims” [2] and were
accepted in August 2016. Sales of ACAPOLIA PLUS have commenced with a new label detailing thefollowing functions. ACAPOLIA PLUS contains 163 mg of proanthocyanidins derived from Acacia
bark as the functional substance in each daily dose, and the supplement has functions that reduce theabsorption of carbohydrates and limit the postprandial rise of blood glucose in humans for a person
who has a high blood glucose level or an easily increased blood glucose level.
3.2. Further Research of Biological Activities Based on a Microbiome Changed by Flavonoids
The effectiveness of clinical trials for the product containing proanthocyanidins of A. mearnsii
bark in suppressing postprandial blood sugar elevation and the mechanism by which this occurs
have been described. The bark extract inhibited the absorption of glucose in the small intestineand reduced the activity of the digestive enzymes in the digestive tract. It is believed that thebark extract functions in the digestive tract without being absorbed in the body. The question to
be asked is, does the bark extract produce another effect in the small and large intestine or not?
The answer to this question may lead to additional applications in health foods as “Food with Function
Claims”. In addition, upon consideration of the other possibilities for the use of proanthocyanidinsof A. mearnsii bark in dietary supplements, Ogawa et al. [16] reviewed the biological activities in aprevious paper in 2018. According to that review, as the growth of a typical intestinal bacterium such as
Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Serratia marcescens was inhibited by the extractfrom A. mearnsii bark, further study must investigate the effect that proanthocyanidins had on intestinalbacteria. Several papers reported the relationships between flavonoids and microbiome as follows.
Goto et al. [17] reported that the effects of tea catechins on fecal contents and metabolites of elderly
people. Tea catechins (300 mg), which were divided into three doses per day, were a meal supplement
each day for six weeks. The tea catechins extract contain 62% catechins, including (−)-epicatechin(6.5%), (−)-epigallocatechin (18.5%), (−)-epicatechin gallate (7.0%), and (−)-epigallocatechin gallate(EGCG) (30.5%). In the same clinical test, Hara [18] reported that the amounts of Lactobacillus andBifidobacteria referred to as “beneficial bacteria” increased, and the quantities of Clostridium, Bacteroides,
and E. coli referred to as “bad bacteria” decreased while tea catechin was taken. In another cinical test,
Okubo et al. [19], investigated the effects of tea polyphenol intake (0.4 g/volunteer, three times per day,for four weeks) on the fecal microflora, the bacterial metabolites, and pH using eight healthy humanvolunteers. Counts of Clostridium spp. significantly decreased during the tea polyphenol intake, whichcontains (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin gallate,(−)-gallocatechin gallate, and (−)-epigallocatechin gallate. Unno et al. [20] also reported that EGCG
inhibited the growth of Clostridium groups in vivo.
Research has indicated that decreasing the level of the intestinal bacteria, Clostridium spp., bytreatment with EGCG affects the biological activity of the host animals. Ikarashi et al. [21] revealed
that the intake of a high-dose of green tea polyphenols results in a liver-specific decrease in the
drug-metabolizing enzyme cytochrome P450 3A (CYP3A) expression level during an in vivo test.
Ikarashi et al. [22] evaluated the liver-specific decrease in the CYP3A expression level observed after
a high-dose intake of EGCG. Owing to the fact that EGCG, which is not absorbed in the intestine,causes a decrease in the level of lithocholic acid (LCA)-producing bacteria of Clostridium cluster XIVain the colon, the level of LCA in the liver decreases, resulting in a decrease in the nuclear translocation

Molecules 2018, 23, 1860

8 of 9of pregnane X receptor (PXR), which in turn leads to the observed decrease in the expression levelof CYP3A.
As not all the effects of proanthocyanidins derived from A. mearnsii bark on the intestinalbacteria of host animals have been studied, biological activities focused on the connection between theintestinal bacteria and the bark material must be determined. In the future, the relationships between
proanthocyanidins derived from A. mearnsii bark and intestinal bacteria will likely be discovered,at which time the biological activities for host animals will be fully disclosed.
4. Conclusions
This paper briefly describes the process whereby the health food supplement ACAPOLIA PLUS,
containing proanthocyanidins (flavonoids) derived from A. mearnsii as the functional substance,
became approved under the “Foods with Function Claims” legislation for the control of blood glucose
in humans. The following were revealed. The safety of the bark extract of A. mearnsii was establishedfrom the history of the safe consumption of the extract when taken as a health supplement andseveral additional clinical safety tests. Robinetinidol-(4α,8)-catechin in the supplement was detectedby HPLC, and quantitative analyses detected 120 mg (catechin-equivalent) or 163 mg (A. mearnsii
proanthocyanidins-equivalent) of proanthocyanidins in six tablets of the supplement. In the clinical test,
the amount of change in the plasma glucose concentration in the initial 60 min after rice consumption
in the Acacia group decreased significantly compared to the reduction found in the Placebo group.
The blood glucose IAUC in the initial 60 min was significantly lower in the Acacia group. The functional
mechanisms for the reduction in the absorption of carbohydrates and the limiting of the blood glucose
rise in humans after taking the extract were explained in terms of the inhibition of the absorption of
glucose in the small intestine and the reduction in the digestive enzymes activity by proanthocyanidins
derived from A. mearnsii bark.
A potential further direction for the proanthocyanidins (flavonoids) in other supplements withnew functional claims was discussed. As it is known that the growth of a typical intestinal bacterium isinhibited by the extract from A. mearnsii bark, and the possibilities for the effective use of the flavonoid
compounds, proanthocyanidins, on the control of intestinal bacteria were briefly discussed.
Author Contributions: S.O. and Y.Y. conceived and designed the experiments, analyzed the data and wrote thepaper; Y.M. and T.T. conceived and designed the experiments and analyzed the data of Section 2.2 and wrote
this section.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Frank Lawson of the Department of Chemical Engineering,
Monash University for his invaluable discussions and critical reading of this manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
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