Ascorbic Acid and Ascorbic Acid Oxidase in Vegetables

Ascorhic ucid, dchydrouscorhic ucid und totu! uscorhic ucid contcnt in fruits und vcgctuh!cs und thc
umounts of rcmuining uftcr 21 hours vcrc mcusurcd und compurcd hy high-pcrformuncc !iquid chro-
mutogruphy. Activity of uscorhic ucid oxidusc vus dctcrmincd in ninc hinds of vcgctuh!cs. Jhc
rc!utionship hctvccn thc uctivity of uscorhic ucid oxidusc und thc umount of uscorhic ucid, dchydrouscor-
hic ucid und totu! uscorhic ucid rcmuining is discusscd. Jhc rcsiduu! rutio of dchydrouscorhic ucid
incrcuscd und thc umount of totu! uscorhic ucid dccrcuscd in thc vcgctuh!cs vith high cnzymic uctiv-
ity. Jhc distrihution of uscorhic ucid oxidusc uctivity in stcm und !cuf of tingcntsui und tuutsui vus
cxumincd. Ascorhic ucid oxidusc uctivity in thc !cuf vus tvicc us high us thut in thc stcm for hoth
vcgctuh!cs.
Kcy Words . ascovbic acid, dohydvoascovbic acid, ascovbic acid oxidaso, high-povIovnanco liuid chvonaiog-
vaphy, vogoiablos
Introduction
Ascovbic acid is ono oI iho nosi inpoviani conpo-
nonis in boih planis and aninals. Ascovbic acid has
inpoviani aniioxidani and noiabolic Iunciions in
planis and aninals in iho Iovn oI viianin C, bui
hunans, and a Iow oihov aninal spocios, havo losi
iho capaciiy io synihosizo ii [i]. !lani dovivod
ascovbic acid is ihus iho najov souvco oI viianin C in
iho hunan dioi. Alihough iho biosynihoiic paihway
oI L-ascovbic acid in aninals is woll undovsiood, iho
plani paihway has vonainod liiilo undovsiood [2].
Ascovbic acid and dohydvoascovbic acid avo
oually biologically aciivo Iovns oI viianin C in
hunans [3]. JhovoIovo, ascovbic acid and dohy-
dvoascovbic acid lovols should bo doiovninod in ovdov
io Lnow iho ioial anouni oI viianin C in vogoiablos.
Jhovo havo boon sovoval invosiigaiions on ascov-
bic acid noiabolisn and iis Iunciion in planis which
pvovido iho najov souvco oI dioiavy viianin C Iov
hunans [4]. Ascovbic acid is oxidizod by onzynaiic
ov non-onzynaiic voaciions.
Ascovbic acid oxidaso is an onzyno ihai caialysos
iho oxidaiion oI ascovbic acid as Iollows [5] :
Ascovbic acid + i/2O
2
Dohydvoascovbic acid + H
2
O
Ascovbic acid oxidaso which caialyzos iho oxida-
iion oI ascovbic acid io yiold dohydvoascovbic acid,
occuvs in vavious highov planis [6, 7]. Jho physico-
chonical pvopoviios oI ascovbaio oxidaso Ivon zuc-
chini suash havo boon siudiod in doiail bocauso oI
iho succoss in X-vay cvysiallogvaphic analysis oI iho
onzyno [8, 9]. Jho occuvvonco oI ascovbic acid oxi-
dasos in nicvoovganisns (Myro|hecium verrucaria and
Aerolac|eraeroenes) has also boon vopoviod [i, ii].
Yoshihiro Shimudu

und Sunuc Ko
Dejar|men|ofIumanNu|ri|ion, Iacul|yofCon|emjoraryIifeScience, ChuolualuenUniversi|y, Olayama701017, Jajan
CHUGOKUGAKUEN J. 2008
Vol. 7, pp. 710
Copyvighi 28 by ChugoLugaLuon
OriinalAr|icle
http://www.cjc.ac.jp/

Covvosponding auihov.
Yoshihivo Shinada
Dopavinoni oI Hunan Nuiviiion, aculiy oI Conionpovavy LiIo Scionco
ChugoLugaLuon Univovsiiy, 83, Niwaso, OLayana 7ii97, Japan
!n iho pvosoni vopovi, iho siabiliiy oI viianin C
(ascovbic acid and dohydvoascovbic acid) in Ivuiis and
vogoiablos was noasuvod, and iho volaiion boiwoon ii
and ascovbic acid oxidaso is discussod.
Mutcriu!s und Mcthods
1.RcogcuIs
L(+)-ascovbic acid, ioiva-n-buiylannoniun bvo-
nido and oihov chonicals wovo obiainod Ivon iho
WaLo !uvo Chonical !nd. Lid., OsaLa, Japan.
DL-honocysioino was obiainod Ivon Signa-Aldvich
!nc., Si. Louis, USA. All iho voagonis usod wovo
voagoni gvado.
2.Somlcs
vosh vogoiablos wovo obiainod Ivon local supov-
navLois, and analyzod on iho day oI puvchaso.
3.High-cr]ormoucc liquid chromoIogrohy
(HIIC)
Sopavaiion oI ascovbic acid was achiovod wiih a
Yanaco L-5 Liuid chvonaiogvaphic appavaius
ouippod wiih a Rhoodyno Modol 7i25 injociov.
Colunn ouonis wovo noniiovod ai 265 nn wiih a
Yanaco M-5i5 vaviablo-wavolongih doiociov. !oaL
avoas wovo doiovninod using an S!C Chvonaiocovdov
i2. A Shodox RSpaL D-6i3 colunn (i5 nn 6 nn
insido dianoiov) was usod. Jho nobilo phaso was
conposod oI 8 nM phosphaio buov, pH 6.8, coniain-
ing 3 nM ioiva-n-buiylannoniun bvonido. Jho ow
vaio was i. nl/nin.
4.IrcoroIiouo]somlc
Sanplos oI iahisai, spinach, and chingonisai wovo
dividod inio loaI and sion. All vogoiablos wovo ihon
choppod inio snall sociions wiih a Liichon LniIo.
Jon g oI ihoso snall sociions wovo honogonizod
wiih i nl oI i noiaphosphovic acid and soa sand
in a noviav. Jho sluvvy obiainod was ivansIovvod io
a coniviIugo iubo wiih 2 nl oI 5 noiaphosphovic
acid and coniviIugod Iov 2 nin ai 3, vpn. Jho
supovnaiani was diluiod 2 Iold wiih disiillod waiov.
5.Ascorhicocidossoy
Jon nl oI diluiod supovnaiani was diluiod wiih
.i3 nl oI 2.5 M K
2
H!O
4
io givo a nal pH oI 7.. A
2-l aliuoi oI ihis soluiion was injociod inio iho
H!LC sysion.
6.JoIoloscorhicocidossoy
Joial ascovbic acid was assayod by adding .i5 g
oI honocysioino io 5 nl oI iho nouivalizod sanplo Iov
ascovbic acid assay. AIiov 3 nin ai 25 , 2-l ali-
uoi oI ihis soluiion was injociod inio iho H!LC sys-
ion. Jho conconivaiion oI dohydvoascovbic acid was
calculaiod by subivaciing iho anouni oI assayod
ascovbic acid Ivon ihai oI ioial ascovbic acid.
7.McosurcmcuIo]oscorhicocidoxidoscocIic-
iIy
(i) !vopavaiion oI cvudo onzyno
Jon g oI snall sociions oI sanplos wovo honogo-
nizod wiih i nl oI waiov and soa sand in a noviav.
Jho sluvvy obiainod was ivansIovvod io a coniviIugo
iubo and coniviIugod Iov 5 nin ai 3, vpn. Jho
supovnaiani was usod as cvudo onzyno.
(2) Assay oI ascovbic acid oxidaso aciiviiy
!viov io diluiing iho cvudo onzyno, iho voaciion
nixiuvos wovo pvopavod by adding .5 nl oI ascovbic
acid soluiion (i ng/nl in i M acoiaio buov) and .5 nl
oI i M acoiaio buov (pH 5.6) io oach asL. Jhon
i.5 nl oI iho cvudo onzyno was addod innodiaioly io
iho asLs, and iho asLs wovo incubaiod ai 3 Iov
i ninuios oI slow shaLing. Jho asLs wovo conivi-
Iugod Iov 2 nin ai 3, vpn aIiov adding 2.5 nl oI
i noiaphosphovic acid. AA was noasuvod in iho
supovnaiani.
(3) Doniiion oI Unii and Spocic Aciiviiy
Ono unii oI ascovbic acid oxidizo aciiviiy is
donod as ihai anouni oI onzyno which oxidizos
iM oI ascovbic acid pov ono ninuio. Spocic aciiv-
iiy is oxpvossod as uniis pov i g oI sanplos.
Rcsu!ts
1.SIohiliIyo]ciIomiuCiu]ruiIsoudccgcIo-
hlcs
Jho anounis oI ascovbic acid, dohydvoascovbic
acid and ioial ascovbic acid vonaining in Ivuiis and
vogoiablos aIiov 24 houvs wovo conpavod (Jablo i).
Jho vosidual vaiio oI ioial ascovbic acid was high,
abovo 6 , in lonons, Liwi Ivuii, vadishos and
sivawbovvios and was low, undov 6 , in bvoccoli,
punpLins, spinach, cabbago, iingonisai, cavvois,
iaaisai, sialL gavlic. Convovsoly, iho vosidual vaiio
oI dohydvoscovbic acid was high in bvoccoli, punp-
Lins, spinach, cabbago, iingonisai, cavvois, iaaisai,
and sialL gavlic.
2.AcIiciIyo]oscorhicocidoxidosc
Jho vosulis oI noasuvononis oI ascovbic acid oxi-
daso aciiviiy in sovoval Linds in vogoiablos is shown
8 CHUGOKUGAKUEN J.Vol. 7 Shimada
in Jablo 2. Jho onzynic aciiviiy was highosi in bvoc-
coli. !unpLin, spinach, iingonisai and cabbago Iol-
lowod in iho lovol oI onzynic aciiviiy. Jho onzynic
aciiviiy was iho lowosi in vadishos.
3.DisIrihuIiou iu sIcm oud lco] o] oscorhic
ocidoxidoscocIiciIy
Jho disivibuiion oI ascovbic acid oxidaso aciiviiy
in iho sion and loaI oI iingonisai and iaaisai is shown
in Jablo 3. !n boih, ascovbic acid oxidaso aciiviiy oI
iho loaI was iho iwico as high as ihai oI iho sion.
Discussion
Many noihods havo boon dovolopod Iov iho doiov-
ninaiion oI anounis oI viianin C (L-ascovbic acid),
howovov, nosi oI ihon havo boon basod on iis voduc-
ing pvopoviios [i2i5]. Jhoso noihods lacL spocic-
iiy, and avo pvono io iniovIovonco by oihov voducing
agonis pvosoni in iho sanplo. High-povIovnanco li-
uid chvonaiogvaphy is iho pvopov noihod Iov analysis
in conplox naivicos such as Iood sanplos.
von iho vosuli oI iho vosidual vaiio oI ascovbic
acid and aciiviiy oI ascovbic acid oxidaso (Jablos i
and 2), iho Iollowing conclusions wovo dovivod. Jho
vosidual vaiio oI dohydvoascovbic acid incvoasod and
ihai oI ioial ascovbic acid docvoasod in iho vogoiablo
wiih high onzynic aciiviiy. Jhis ovidonco shows ihai
ascovbic acid oxidaso caialyzos iho oxidaiion oI
ascovbic acid io yiold dohydvoascovbic acid, Iollowod
by iho doconposiiion oI dohydvoascovbic acid.
Ascovbic acid conioni was noasuvod in Iouv pavis
oI bvoccoli by NishiLawa oi al. [i6]. Jho anouni oI
ioial ascovbic acid in iho ovois was iho highosi oI
all iho poviions. !i siayod ai a volaiivoly high lovol
Iov aboui i2 houvs, Iollowod by a vapid doclino io a
low lovol duving sonosconco. !n iho sion iissuo, iho
lovols oI ascovbic acid vonainod alnosi unchangod ov
incvoasod slighily as iino pvogvossod. !i is osiinaiod
ihai ihis vosuli doponds on iho diovonco oI iho on-
zynic aciiviiy in iho diovoni pavis oI iho vogoiablo
jusi as ascovbic acid oxidaso aciiviiy oI iho loaI was
9 Ascorbic Acid and Ascorbic Acid Oxidase 2008
Table 1 Stability of vitamin C in fruits and vegetables.
0 hour after 24 hours
Total(mg/100 g) AA(mg/100 g) DAA(mg/100 g) Total(mg/100 g) AA(mg/100 g) DAA(mg/100 g) residual ratio(%)
Lemon 50.0 47.0 (94.0) 3.0 (6.0) 37.0 33.30 (90.0) 3.70 (10.0) 74.0
Kiwi fruit 75.0 70.0 (93.3) 5.0 (6.7) 55.1 51.20 (92.9) 3.93 (7.1) 73.5
Radish 12.3 11.6 (94.3) 0.7 (5.7) 9.2 9.10 (98.6) 0.70 (1.4) 75.0
Strawberry 60.0 55.0 (91.7) 5.0 (8.3) 37.8 33.60 (88.9) 4.20 (11.1) 63.0
Broccoli 74.0 65.8 (88.9) 8.3 (11.1) 33.5 2.00 (6.0) 8.30 (94.0) 45.3
Pumpkin 35.5 25.1 (70.0) 10.4 (29.3) 18.2 10.00 (54.9) 10.40 (45.1) 51.3
Spinach 33.1 30.8 (93.1) 2.3 (6.9) 16.7 8.90 (53.2) 2.30 (46.8) 50.5
Cabbage 34.3 30.8 (89.8) 3.5 (10.2) 16.6 6.00 (36.1) 3.50 (63.9) 48.4
Tingentsai 42.7 35.0 (82.0) 7.7 (18.0) 22.0 8.40 (38.2) 7.70 (61.8) 51.5
Carrot 4.0 0.7 (17.5) 3.3 (82.5) 0.0 0.00 (0.0) 0.00 (0.0) 0.0
Taatsai 55.4 35.3 (63.7) 20.0 (36.3) 31.9 14.40 (45.2) 20.00 (54.8) 57.5
Stalk garlic 39.7 31.8 (80.1) 0.8 (19.9) 23.0 10.80 (46.9) 0.80 (53.1) 58.0
Total : total ascorbic acid, AA : ascorbic acid, DAA : dehydroascobic acid
Numbers in parentheses indicate the ratio to total ascobic acid.
Table 2 Activity of ascorbic acid oxidase (units/100 g).
activity
Broccoli 11.88
Pumpkin 8.45
Spinach 7.30
Tingentsai 5.12
Cabbage 4.96
Carrot 4.22
Taatsai 3.42
Stalk garlic 3.27
Radish 0.14
Table 3 Distribution in stem and leaf of ascorbic acid oxidase
activity (units/100 g).
Leaf Stem Edible portion
Tingentsai 7.98 4.32 5.12
Taatsai 4.87 2.82 3.42
iho iwico as high as ihai oI iho sion in boih iingoni-
sai and iaaisai.
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10 CHUGOKUGAKUEN J.Vol. 7 Shimada
Accepted March 30, 2008.