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54, 82-88, 2008
J Nutr SciVltamii]ol,

Chestnut Astringent Skin Extract, an ev-Amylase
Carbohydrate Absorption in Rats andInhibitor,
HumansRetards

Takahiro TsuJITixi,
Takeshi TAKixKuiand Tsuneo Suzui([2iBioscience,
2GenkiEhime Ui]iversity,
Shitsukawa,lbon,Ehinie 791-0295, fapan
Jntegrated
eftnterfor
Scienc'ffs,
Healthcare,
3-6-S
ljdabashi,
Chiyoda-ku,Tbkyo I02-8508, Ripan
Pla:･aMedicalCenter.tbr

CReceivedJune21,2007)of carbohydrate-hydrolyzing
enzymcInhibitors
play an important role to conIn thispapez we investigatedthe effcct of an ethanol
troLpostprandialblood glucose Ievels,
astringent
skinextract flrom chestnut(CAS)on alphu-amyEase. Chestnut astringent skin
We also investicxtract strongly inhibited human and porcine pancreatic a]pha-amylase.
absorption
in
rats
and
humans,Oraladmtnthe
effectofCAS
extractoncarbohydrate
gatedsignificantly
istrationof CAS extract to normal
rats fed corn starch (2glkg body weight),
suppressed
the increase of blood glucoselevelsafter starch loadtngin a dose-dependent
20 and 40% suppressionofmanner,
The effective doseof CAS extract required to achieve
body weight, respccwas
estimated
to be 40 and i55 mglkg
the rise in bloodglucose levcl
tivelyChestnutastringent skin extract also suppressed the rise in plasma insulin leve[andIn the type 2 dtabetic
rat model,
CAS extract
rattyacid level,
the fallin plasma non-csterified
the rise in bloodglucose ]evelaftcr starch loadingin a dose-depensignificantly suppressed
the rise in plasma glucose
Chestnut astrtngent skin extract also suppressed
dent manner,mannerin humans, The amountof CASlevelafter boiledrice loadingin a duse-depcndent
in the rise in plasma glucose tevelwas
extract
requiredto achieve
11 and 23`)6suppression
3OO and 600 mgfperson, respectively These results suggcst that CAS extract retards absorptton of carbohydrate
and reduces post-prandial hyperglycemia,
oral carbohydrate
inhibitor,bloodglucose,
Keu l4a)rds chestnut astringent sktn. amylase
tolerance test

Summary

Tormo et al. reported
that a-amylase
inhibitorfrom
The prevalence of diabetesis increasing worldwide{5).
reducedinhoth
non-diabetic
andinmodernlifestyles
2}.
Mostdiabetic
whtte
beans
due to changes
glycemia(1,
andreducedtheintake
of
food
and
type
2
diabeteswhichiscloseiy
diabetic
animalspatients suffer fromwater
DiMagnoetal.
reportedthat
ana-amylase
4).
Diet
and
exercisetherapyarerelatedto obesity (3,
(6),
might
bc usefu1 fortreatment offor preventing and treatingtype 2 diabetes. inhibitorfrom wheat
key factors
type2
dtabetes
mellitus
healthyblood
levelsisofMatntcnance ofpartic(7-9).
glucoseIn the scrceningof inhibitorsof carbohydrate-hydroularimportance and is greatly affected by dietaryTheselyzing enzyme
fTom plant, we discovercd
CAS inhibitedsuchas starchesand glycogen,polysaccharides
ex-amylase,The
chestnutisa deciduousttmber treeof
arebrokendown
bydigesttve
enzymespolysaccharides
a key enzyme
in polysaccharide the genus Castanea, in the beech i'amilyNative chestsuch as cr-amylase,of thenuts
are distributedin the temperate regions
a-Amylasecatalyzes
thefirststep in thedigestion,
fruitsthat
a-1,4-gluNorthern
Hemisphere.
They
bear
burrlikehydrolyzing
thedtgestionof polysaccharides,
nuts,
The
nuts
are
imporcontainedtnthecontain
two
or
three
edtbleTheamylasesarecoside linkages.
andtransform
tant as foodand are exported in largequantittes,Theyof mammals
saliva and pancreatic fluidof East Asia such
as
in
the
alimentaryarecultivated over large areatntooligosaccharides
polysaccharides
Korea
and
We
tnvestigatedtheacttvityisimportantin
China,
potentialofa-amylase
Japan.
canal, Control
inhibitorbecause
andto regulatedietaryuse of CAS as an a-amylaseorder to inhibitexcess energy supply
ofchestnuts gcnerindustrial
CAS-freelevels.
pea
processing
bloodglucoseasignificant
of
CAS
as
a
chestnut wastc
fromvarioussourccs
atesof
cr-amylase
derivedquantity
Inhibitors
In
this
-ve
reportthat
CAS
extract ts a
effectshave
been
investimaterial.
beenreportedand
theirpaper,
have
retardabsorpinhibttorof cr-amylase
and mayofsix
selectedstrongAIietal.
reported
that
extracts
gated.
byperofcarbohydrates
andreducea-amylase
activitytion
post-prandial
strongly
inhibited
Malaysianplants
gtycemia in rats and humans.
E-mail:tsujita@m,chime-u.ac.jp

Abbreviations:'AUC,area underastringent
sktn; ICso,
concentrationof itsactivity; NEIA, non-esteritlcd

the curve; CAS, chestnutof inhibitor to inhibit5O`V,

MA[[]ERIALS AND METHOD
Materials, a-Amylases

human pancreasand humanfattyacid,from
salLvaporctnewere

pancreas,

obtained

from82
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CAS Retards CarbohydrateAbsorption

Solubtestarch was(Tokyo,Japan).
froniWako Purc Chemical Industries,
Ltd,

Sigma-AldrichJapanrcceived

only

obtainedples werecollected

83

Dried chestnut<Osaka,Japan).

astringent(CAS.) heparinizedsyringc

skin

CAS werc obtained from Chuon Co,
Ltd,(Matsuyama,
Japan),
I'reparationofCAS extract. One hundred grams ofthe driedCAS powder were added to 2L of 50{M) vfv
aqueousacetonitrile, followedby stirring
at 37'C for
12 h. The mixture
was filtcred
and the li]trate
wasconcentrated
and lyophilized,
to gcnerate CAS extract powder,designatedCAS-exl.A large-scale
extract
was
prepowder and raw

pared from rau, CAS to mirr]ic industriat-scale
production. One thousand kilogramsof raw CAS was added
to
2,OOO kg of 75% aqueous et,hanol and subjectedi to thesameCASprocedure describedabove. The large-scale
extract
CAS-ex2.
powder was dcsignated

Assay methods.

activitywasdetermined
power of released oltgosacchartde
from soluble starch by the tnethod of MillerC1O)
withthe following
minor modifications. The assay sys-

by measuring

cr-Amylase

the reducing

tem comprised
the tb[[owing components
in a totalvolumeof 1 mL:
100mM sodium phosphate, pH 6.8,
17 mM NaCl, 5 mg soluble starch, 100 tLL of inhibitor
solution,and
10 ILLof enzyme solutton, After incubation at 370C fbr 30min, the reaction was
stoppedbyadditionof O.1 mL

2 N NaOH

and

O.1 mLcolor

reagentacid, 106"mol(4.4ptmol of 3,5-dinitrosalicylic
tetrahydrate andpotassium sodium(+)-tartrate

oi'

40

of NaOII},lbllowedby a 5-min incubution, at
1OOthC and subsequent
Asiomeasurement,
Polyphenols (total
phenolics) in the CAS extract were
determined by thc method of Folin-Denis
12),
(11,
Oivl carbohydrate
tolerance testin }'ats. The experimcnta]
animalby the Animal
protocol was approved
Study Committee of Ehtme LJniversityMalc WistarKing
rats, weighing
190-230g
were
starvcd
overnightCl5h),and dividedinto two groups, The test group
received
2 ml, of corn starch suspension(2glkg body
wetght) containing
1 mL of CAS extract powder solution whilc the controt group recetvcd
corn
starchsusblood
pension and 1 mL of water, Aftcr administration,
samples were co]lected from the tailvein or arteryatregular interva]s,
BIood glucose was measured
usingablood glucose test met,er, GLUCOCARD
tnc.,(Ar]gray
Kyoto. Japan).
After a ]-wk tntervat, the testand control groups were switched and the experiment
was
sLmol

repcated.

Type II diabetic modelrats
obtained from
(GK!jcl)
CLEA Japan,
Inc, {lbkyo,
weighing
31O-360 g
Japan),
were starved
overnight(15h),and subjected to the carbohydratetolerance testas described above,P]asma insulin and freefattyacids were assayed inrats subjected to the carbohydrate
follows,Male Wistar King rats, weighing

normalas

starved(]5h)andtolerance test
190-230 g
dividedinto threeovernight
The
test
groups,group received

2 mL

pension (2glkg body weighOcontaining

wereor corn

starch

1 mL

of

sus-

CAS
Con-

powder so]ution (3OOmglkg body weight),
trol group 1 received
corn
starch suspension(2gfkgbody weight)
and
1 mL water and control
group 2extractwater.

After administration,
blood samfrom the in['erior
vena cava using a
at regularintervals,
andeentri-fuged immediately at, 2,OOO Xg for5 min, Plasma glucose, insulin and non-esterified fatty
acids (NEM) were
determinedusing the GlucoseC-TTtest 1"lako(Wako
Pure Chemical Industries,Ltd,),
thc Morinaga insutinassay
kit ()vlorinaga
Institutcof BiologicalScienceTnc,,
Yokohama, Japan),and the NEEA C-testWako CWako
Pure Chcmical Industries,I,td,),
respectively
Oiul carbohydrate
toJeiwncetestin humaJtsulijects. The
human study protocol was approved by the EthicsCommitteeof Ktmura Hospita] and was carried out in Genki
Plaza Medtcal CenterforHealthcare(Tokyo,
inJapan),
accordancewith thc principlesof the Helsinki
Declaration as revised in 2000, Allsubjects gaveinformed consent,
EIex/en healthy Japanesevolunteers Cmale:feage

male=8:3;

fasting blood

30-59y (averageage

glucose level <126mgfdL:

46.6± 9.3y;
HbAlc

Studieswere done on
three separate mornings
at lcast
3 d apart,, The firstday
subjects
ingested 2OO g of boiledrice and 2OO mL of water and venous
bloodsamples were takcn fordetermination of plasma glucose levelbefore,and 30, 60, 90 and
120 min aftcr eating, Tbe second day subiects ingested
200 g or boi[edrice and 200 mL of water containing
300 m.ff of CAS extract. On Lhe third day they ingested
200 g of botled rice and 200 mL of water contatning
600 mg of CAS extract, after which b[oodsamples were
Laken, The bloodsamples were cent:ifinged immediately
at 2,OOO Xg ibr 5 min
and plasma glucose
was deter<6.5%)

werestarvedovernight.

mined.

Statisticalanalgsis,
Resultsarc expressed as the± SE, The statisticalsignificancc
ofdiffbrences
withandwtthout
CAS extract was assessed(contro])using the pairedStudent's
t-test,mean

RESULT.S

CAS cxtract powder <CAS-ex
IL)isa bittcr-tastingdarkbrown powdez Ikvas found to strongty inhlbitpig pancreatic a-amylase
activtty
A concentraLionof L3,1 ILgfmL
wasdetermined to result in 5O{rainhibition,Becausc
CAS extract was lound to inhibitmammalian
cr-amvlase,we examined
whether the CAS extract could exert
an inhibitoryeffect on blood glucose level
increase afiersLarchloadingin rats that had fasted15 h. Blood glucose Ievels
increased from a baselineof 52,7± ],83 mgl
dL at O min to a peak of 113.6 ± 3,86 mgfdL (increased
bloodglucose value 60.9 ± 3,9mgldL} at 60 min after
starchadministration(2glkg body weight), The rise inbloodglucose was suppressedin a dose-dependcnt
nianner when
CAS extract {10,25, 50, 100 and 300 mglkg
body weight) was given with staich (Fig.
IA).The area
the curve CAUCo.isomin)
for CAS extract administrationwas also found to decreasewith the increase in
CAS extract administration
in a dose-dependcntmanner comparedto administrationof starch
alone
(Fig.
IB), Figure IC shows `Z] ot' A(]C changc
tbr CAS extract
admintstration
relativeto administrationof starchalone.
Based on the observcd
retattonshipbetween
under

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TsujvrA T et at,

84
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ASP,Fff61･i'\4

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Cont

Time(min)

10

25

SO

100

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300

CASextract(mgtKgbodl'weighO

100
200
300
CASextract{mgfKgbodl'weight)

on increase d blood gLucoseconcentrations
the area under the curve (AUC}
CCAS-exl)
CA),
in
norma
1rats,A:
Rats
fasted
for
15
h,
and
CAS
extract(10(i),25 (A),SO CI),1OO {D)
(B),
(C)
were
a dministered.
As a control, rats were givcnonly
and 3OO (e)mgfkg
bodyweight}
starch (2g/kg body weighO
B: AUCo.isnmin ts the area under the curve of the incrernental blood glucose ]eve[up to
starch C2gfkg body wetght)(O),
CAS extract against muCn isf)min forthe cont]'ohvas uscd to estimate 2O and180 min, C: The ratio (CX,)
of AUC[Hsomin for

Fig, 1. Effectsof CAS extract
andvaLuesof AUC

ratio

and409S suppression

Resu]tsarc expressed as meansvalues.

± SE. n=8.

'p<O,O5,"p<O,Ol

A

120

and

'*"p<e,OOI

vs. controL

1.0:sVZC

fiabeg=E18=9.a1:'xn:ac:100ggO.8;L:t
**

so:8=

**ls

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40"aveeo.4Eg:

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Time(min)

lso

±

O
o

3o

6o

go 12oTime(min}lso lsoinsulin(B),and NEEA (C)concentrations
tn norCCAS-exl) on increasedp}asma glucose (A),andstarchCAS cxtract (3Oemgll(gbody weight)(2glkg body wcight) z-rere adminis2, ]). The
tered (e),As a control, rats were given onLy starch (2g/kg bodyweighr) (contro]
L C)･)or on]y water(control"p<O.Ol± SE, nt=8.
and ""'p<O.OOI
vs. controLl.
results are cxprcssed
as means

Fig. 2.
mal

Effcctsol' CAS extract

rats, Rats fastedfor 15 h, and'p<O,05,of
CAS extract
andreduction
ratio
of
the
effective
dose
of
CAS
extract
required
AUCo.isomTn,of blood glucose
to achieve 20 and 40% suppression
wasestimated
to
be
40
and
155 mgfkg
bodylevelriseIC),weight, respectively (Fig,
For the assay of plasma insulin ttnd NEEA on carbowere collectedhydratetolerance test, blood samples
inferior
vena
cava
of
normalrats
2).
from the(Fig.
Plasma glucose levelswere tbund to increase from abaseline value of 47.3 ± O,84 mgfdL at O min to a peakof 149.5 ± 6,5 mgfdL
plasma glucose value(increasedof
102,2 ± 6.4m.uldL) at 60min after administration
Plasmastarchbodyweight)2A),(Fig,
glucose(2g!kgthe amountlcvclsdecreasedslightly
only

A similar

after

pattern was

administration

obscrvedof waterin the plasma insu-

value
of
Iinlevelswhich
also increascd from a baseline
O,432 ± O.Ol3 ngfmL
at O min to a peak of 3.07± O.52
2B),
ngfmL
at 60 min after starch administration(Fig.
Plusma insulin leveLsalso decreased slightly aftcr

of water oply When a CAS extract (300
body ut,eight) was gtven with starch, the rise inplasma glucose (peak106.7 ± 6.8mgldL, increased
insulin (pcak55.2± 6,7mgfdL, at 60min) and
2,04± O.61nglmL at 90min) levelswere suppressedsignificantly, While plasma NEEA leveiswere high upon
at O min),they decreased
fasting(O.68± O,023 mEqfL± O.029
administratjon
significantiy after starch
{O,48
mEq!L
at
40min)
2C),
PIasmaNEEA
levels(Fig.
remained
at a high level±
O,Ol6mEqlL
at
180
(O,69
min)
after admintstrationof water only The drop in
plasma NEEA levelsby administration of starch were
suppressedsjgnificantly
by administratjon of starchwith
CAS extract. The arca under the curve (AUCo.isoof p]asma glucose rise andmin)plasma insulin lcvel
decreasedsignificantly w･ith administration of CAS
extract:
45% suppression of plasma glucose rise andadministration
mgfkg

27[rasuppression of plasma insultnincrease,Conversei}lincreased significantly
AI/]Co.
isomin of plasma NEIYtlevel

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CAS RctardsCarbohydrateAbsorptton

8512%) with(aboutadministrationof CAS extract (Table centrationof polyphenols, about
20%, Heavy metal
1),
content
and bacterial
contents
werevery
Iow. TheIn diabeticrats (GKIjcl),
blood gLucose Ievels extract was found to inhibit cr-amylase
activity
in aincreased from a baselineof 96 ± 8.e8 mgfdL
at O mindose-dependent
manner
witha relatively low 50% inhito a peak of 406 ± 1 9,4 mgfdL
at 60 minaftcr adminisbitionvalue (ICso).
IIuman pancreatic amy]ase
wastration of starch (2gfkg body weighO. The rise of thestrongly
inhibited,The ICsovalue fbrhuman pancreatic
blood glucose Ievels
weresuppressedsignificantly in aamylasewasabout two ttmes lower than that for pig
dose-dependentmannerwhen
CAS extract was given2),Based on the carbohypancreatic amylase {Table
withstarch.
Peak increased blood glucoselevelsweredrate tolerance testin normal rats, 33% suppressionof
3IO ± 18,1. 256 ± 14,7 and 197 ± 14,2mgfdL at 60
the blood glucose Ievel
rise wasobservedwithadminismin for CAS extract
administeredat dosesof O, IOO
trationof thc CAS extract (CAS-ex2}
at 1OO mgfkg
bodyand 300mglkg
body weight,
rcspectively (Fig.
3A),
weight(datanot shown),
AUCo-isominfbr CAS extract administration also dePrior to performing the test on human subjects, thecreased with the increase in CAS extract administration
CAS extract powdcr was assayed forsubchronic
toxicity
in a dose-dependentmannercomparedto administrausing
SD rats (6males and 6 females).
CAS extract powtion of starch aEone <Fig,
3B): 22 and 46% suppressionder (l,OOOmgor 2,OOOmglkg body weightfd) was
lbr CAS extract administration of 100 and 300 mgfkg
administered
to rats via a stomach tube fbr28 d.Dur-body weight, rcspectivety
The largc-sca]eextractionwasdone to mimic industrialproductionforpotentialhuman application.
Ethaused
as an extraction
solvent
instead of acetonitrite,
becausethe toxicityof ethanol islowerthan thatof acetonitrile, A typicalaqueous
ethanolic
CAS cxtract
is
shown
in
Table
2.
It
contains{CAS-ex2)
a high con-'1'ab]e2, Characterizationof

nol wasTabLe 1, Effects of CAS extract CCAS-exl)
on inereased
p[asma gLucose,p]asma insuLin and plasma NERNA[JCo-so tntni in normal rats,2
NEEA

InsuLin(mgXmintdL) (ngXminimL)(mEqXmintL)

Controll 13,OOO ± 2,OOO
369 ± 6.03
94,0 ± 1.70
CAScxtract 7,940 ± 1,550*"* 269 ± 9.77*'* ]05 ±2.97"-550 ± r,700"'59,5 ± 2.24"' ]24 ± 1.44*"
Control2

AmyLase inhibttion
Humanpancreas
HumansH]tvaPercinepancreas
Polypheno}content

isthe area

under

expressed

as

means

vs. controL

1.

the curveup

to 18Oandare'"'p<O.OO](ICso)2
7,5 ± ].2 ptglmL9.4± 1.3 nglmL18.2± 3,2 ptglmL20.7± O.53%
3.3± O.14%
7.4± O.64%
<2
ppm
<10
ppm
<1,Ooolg<100 fg

Arseniccontent
Lead content

Livcbacteria
I,ivefungi(]olibacillusgroup

SE. n=8.

"p<O.O]min±

resultsareexpressed

as

means

±

SE of

six

CAS

extracts.2The

in Fig. 2,
2Resutts

extractva]uc

Ash content

iThe
LAUCo.]seniin

typteal CAS

Parameter

Miater eontent

GlucoseGroupa(CAS-ex2),i

ICsovalue ts dcEinedas the concentrationof inhibitorto inhibit5O% of itsactivity under
the assayed conditiOI]S,

mn,R

400

×

50a!EE.B=Ruafi40anev.fiS

300wx8:-hmg*

30fiU`p<20zassec･v
2oe-=;g:g

*ss

looe
10gsx:ou:H

st

o
O

30

60

90

120

150

180

ControllOOmgiKg300mglKg

Timc(min)Fig. 3. EffecLsof CAS extract ((]AS-ex1)
on lncrease
dblood glucose concentrations(A)and AUCh.ise.i. (B)in diabeticrats
Rats fastedfor15 h, and CAS extract (lOOmgfkg(GKfjcl),body weight (A),300 mg/kg body weight(e)and starch (2gf
kg body weighO ut,ere administercd,
As a control, rats were givenonly starch (2g/kg body wetght) (controL
The results(O).
are expressed

as means± SE, n=8,

'p<O,05,"'p<O.O]and**'p<O.OOI

vs, controL

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TSUJIzAT et al,86th-,::.

80

72YLF Big6og:tX

6.og'1,*,.s:･i

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o

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6o
yo
Time(min)12oContrul

300mg

600mg

Fig,4. Effectsof CAS extract (CAS-ex2)
en increased plasma g]ucoscconcentrations(A)uT]d AIJq)=L2o,min(B>in heatthy
arter ingestion of 2OO g of boiledrice with CAS extract (3OOmglperson
volunteers.
I]lasmaglucose levels were measured
resultsareexpressedus
w･ere gLven only boiLedrice (O).
As a contro], voLunteers(e)and 600mslperson (A)).
meuns± SE, n=1l.
vs, controL']'hc'p<O.05

inhibitor<s)is not athat the cr-amylase
derjvative,The inhibitory activity u,asacetonitrile,a solventcumBody weight
extractable in 50% aqueous
gain, food intake,water intake,urinalysis,
toextractThe
inhibitory
hematology, bloodibiochemistry,
and
mon]y
used
polypbenols,
gross patho[ogyactivity
wtis also
extractablc
in ttqueous
acetone andorgan
weights
cxhibitedi
no differenccs
of toxico]ogicat
not,aqucouscthanol,butnet in 1OO% acctonitrile, acetone,
significancebetwcen control and t]'eatedrats (dataaboutshown).
these results indicated a no-obscrvedethano} or hexane. The CAS extract contained
20`M, polyphenolic material
adverse-etTect
Ievelof CAS extract to be over 2,OOO mg/(Table2), Furthcrmore,
when
CAS extract powder was re-eNtracted with aque!cgbody weightfd,
The effect of CAS extract/ taken wtthousetbanol,
Lhe content of polyphenolicniaterial
boiledrice (200g) on plasma glucose leveljn healthy
in
Fig,
4,
Followtng
increasedabout
3-fold (about609{,)
and the amylase
volunteersts
shownJapanesc
water
inhibitory actjvtty also increasedabout 2,6-{btd (dataadministrationof boi]ed rice with(contr()l),
not show･n). These resuit$ suggest that an active compoplasma glucoselevelsincreasedfrom a baselineoi'
at Ominto a peal< of 166,8 ±nent(s) of the CAS extract may be a polyphenol, Kandra
100,3± 5,Ol mgfdL
et al, have reportcd
that tannic acid, a polypheno],
11,67 mgldL at 6O min. Thc rise in p]asma glucose wasnon-competitivc
type inhibitionof awhen
CASexbibjted mixed
suppressedtn a dose-dependentmannercompared
the jnhibttory
withthehoiledrice.
Peaklevelsol'amylaseacttvity
MJe
extract was
(15).
given
66,5 ± 12.1, 60,5±
activity of tannic acid with that of thc CAS extract andtncreased plasma glucosc were
found that the former had an ICio toward porctnc pan] 1,2,and 52.3± 1l,7mgfdL at 60 min fbrCAS extractof greatcr than 1 mg!mL,
whtch
was
respecttveLy
dosesof O, 300, and 600 mgfperson,(Fig, creatic cu-amylase
over
times
htgher
than
that
oE'
the
CAS
extractforadministration50{Table4A).Starchabsorption
]2o
.,i.)
CAUCe
2). Theretbre, we speculaLethat the majo: active comof CAS cxtract also decreasedwith the increase in dosein a dose-dependent mannercomparcd
to ingestion of
ponents of the CAS extract may not includetannic acid,from the CAS
Purificationof the active components
rice alonc
4B). Suppressionby 11 and 2396 was(Fig,
at doses of 300
extract
could leadto dcvelopmenlof an antihyperglyceadmtnisteredobserv･ed bv CAS extracts
mic
drug.
and
600 mgfperson.
respectively
acttvity
Thc CAS extract also inhibitedcr-glucosidasc
DISCUSSIONof the rat small intestine,
Howevcr,the ICsuvalues of theinhibi- CAS extract (CAS-ex2)
were 50 to 100 times highcr
In this paper, wc repurt a potent cv-amyluse
640 and 940 tLgfmL towards
have been iso- than that fora-amylasc:inhibitors
tor(s)in CAS. Many a-amylase
respectiveiy
The extract
mattase and sucrase activity
latcdfrom plants and microorganisms(13,14).a-Atnyactivity
withan ICsealso inhibited
rat pancreatic lipase
tase tnhibitors are roughly dividedinto two classes,
vatucof
510tLgfmL.
I'roteaselnhibitory
activity toWe
and
non-proteinaceous,presumeprotejnaceous
trypstn andi chymotrypsin
wasvery weal<, ICio
wards
that the cr-amytasc
inhibitor(s)in the CAS extract isnot
over
2 mglmL,
These results suggest thc
values wcreproteinaceous, becausethe inhibitory activ･ity was heat
CAS extract spectfically and strongly inhibitcd cy-amyactivity
wasmt)jnstable:over
9001oof the inhibttory
notlase activitv,
tained fo]lowinga 1 h tncubation at 100"C (dataL,vhen cAs extract was given with starch to thc norshown). The inhibitoryactivity was slight[y extracted jn
insulin,and thc
malrat, the rise in plasma g]ucoseandnotshown),
Therefbrcwe
weter
or in hot water (dataing the sLudy,
effectsontheclinicat

trcatment induced

signs,

survlval

ornonoticeab]eophthalmo]ogy,

also presume

carbohvdrate'l'hus,NII-Electronic
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CAS Retards CarbohvdrateAbsorption

87

faltin plasma NEEA levels
weresignificantly
suppressed
4)
YbonKH,1,eeJH,KimJVXLChojH,ChoiYH,KoSH,Zim2. Table 1),Onc explanation
met R Son HY, 2006. Eptdemtc obesity and type2 diabeforthe suppression(Ftg.
of
tes in Asia. I,ancet
the drop in plasma NEFA Ievclis as fo11ows.PIasma
368:l681-1688.
)H)
Ali
H,
Houghton
PJ,
Soumyanath A. 2006, Atpha-amyNEEA levelsare affected by adipose
tissue tipolysis,
laseinhibitory activity of some ]L･Ialaystan
which
is regulatedpLantsused toby hormones such as insuEinandtreat diabetes;withrcference
to Phyllanthus
parttcular
catecholamines,
and by dtetaryintal<eof nutrition suchamrus,J Ethnopharmacol107: 449-455,
as glucose (16-18).
One of the important metabolic
6)
Tormo MA, Gjl-ExojoI,Romero-de-'1leiada
A, Camptllo
actionsof insulin isthe inhibitionof lipolysts
in adipose
20()6, White bean amy]use
inhtbitoradministeredJE,
tissue (19,20>, Insulin secretion t'rorr]
isletB-cells
isorally reduces
rats. Br JNutr
gJycemia in type 2 diabetic
stimulated by a rise in blood glucose ]evel
There(2l).
96: 539-544.
lore,after eating, bloodglucose levels
increase stimulat7) Layer R Zinsmeister AR, DiMagno RR 1986, Effectof
ing insulin secretion, which in turn causes a decrcase
in plasma NEIA levels
due to the antitipolytic action of
insulin,VL]hcn CAS extract was given wtth food,thedecreasing intraluminaL amylase
activityonstarchdigestionand postprandialgastrointestinal functioninhuman. Gastroente}'ology
91: 4]-48.
g) Boivin M, FLourieB, Rizza RA, Go VL, DtMagno EP
normalrise in bloodglucoselevelwassuppressed,
and1988. Gastrointestinal
and metaboLic
effects of amytase
insulin secretion mras aEso supprcssed,
ZVherefore,llpolyinhibition Lndiabetics.
Gastroenterology
94: 387-394.
sis in adipose
tissue was not suppresscd
andplasma9) r,ankisch M, Laycr R Rizza RA, DiMagno ER 1998.
NEEA levelsremained high,Acute postprandial gastrointcstinal and metabolic
a-Arnylasetnhibttors
have been used in humaneffects of wheat
amylaseinhibitor<1,wtI)
in normaL,
hcalthto inhibitexcess energy supply, to control blood
obese and diabetic
humans. llancreas17: 1 76-181.
glucose levels,and to prevent or treat obesity and diabeIO) MilLcrC;L, 1Y59. Use of dinltrosa]icy]tc
acid reagent
fortes,A significant body of rescarch has lcdto the discovdeterminationor reducing sugar. Anal Chutn 31: 426ery of many
a-amylase
inhibitors,Severalhave been428.
reportedin various11) FolinO, Denis W 1915, A colorimetrlc method forthe
23),
plants, especially grains(22,determinatjonof phenols (andphenol derivatives)
Iegumes (24,25) and root vegetables <26,in27).Tn theurine.
Biol
Chem
ZZ:
305-308.
J
study
we
discoveredthat a CAS extract was aprescnt12} AppcL IID,Govenor Hi),D'Ascenzo M, Siska E, Schultz
stronginhibitorof a-amylasc
and could attenuate
theIC.2OO1, Limitattonsof FoLin assays ef foIiarphcnoltcs
rapjd
increase in bloodglucose foIIowing
consumptionin ecological studics, J Chem Ecol 27: 76]-778,
of a carbohydrate-containing
meal
by delayingor block- 1.3) SvemssonB,FukudaK,NieLsenPK,BonsagerBC.2004,
ing absorptionof carbohydrates.
Chestnutshave been
l'roteinaceousa-amylase
inhibitors. Biochirn Biophys
eaten
since
ancient
timesand CAS has also been eaten
Acta 1696: l45-IS6.
as an associatedmaterial,
As an examplc,
14) Franco OI,,Rigden DJ,MeLo FR, Grossi-de-Sa ME 2002.
ni" isa processed food, madeby boilingthe CAS-associPIant cr-amylase
inhibitorsandthcir interaction withated
materialwithsugar.
eAS is generated in Iarge
insect a-amylase.
gtructure,funetionand potcntialtbr
crop protcction,
EurJ Biochem 269: 397-412,
quantitiesas a waste product during CAS-free pea pro151 Kandra I,,Fyemant G, ZajSczA, Batta G. 2004, Inhibicessing of chestnuts, Therefore, the use of CAS extract
tory effects ef tannin on human sa[ivary cr-amylase,
as a sat'e and inexpensive functional
tbod or therapeutic
Biochem BiophysRes C(mrmun 319: 1265-1271,
agent
adds potential
rralue to chestnut
processing.
16) OkudaH, Morimeto C,TsujitaT,1994. Roleof endogeIn conclusion, thjs investigationsuggcsts that CAS
nous]ipiddropletsin lipotysjs
in rat adipocytes. J Lipid''Shibukawa-

extract might
cr-amylase,

exert

and

an

anti-diabetic

suppressing

effectcarbohydrateby Lnhibiting
absorption

t'rom the intestine, thereby reducing
post-prandial
increase in blood glucose, Theretbre,
CAS extract may
be usefu1 as a potentia] additive to foodsand beverages
to inhjbitcarbohydrate
adserption.
Acki]ouiledgrnents

This study was supported
by ResearchGrants flrom
Rohto Pharmttceutical Co, I,td,(Osaka,Japan)
andChuon Co.Ltd. (AtTatsuyama,
Japan).
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