（17）
Food Preservation Science VOL．24 No．11998〔Article〕
17
AscorbicAcidandβ−CaroteneAffecttheChlorophyllDegradationinStored
Spinach（5i）inacia olemcea L．）Leaves
YAMAUCHINaoki＊and
Alley
E．WATADA＊＊
＊／−∴∴・・ト∴・一二・・・ト・・∴∴●／∴．・㍉∴t二・日ト∴‥．し，．．′∴・− ∴ ト‥・∴‥∵ ∴ 十・．
；巨＊／／・一一∴′一一′・∴−LJ・り′‥こ／二、・・．．・・・．／∴．・∴‥∴卜∴・■′・／、・．．〝．ト．・・‥、ト−．・一∫・／、′．・∴
Sgγ血ち と侶84βg独〃沼gノ朋か20705−2350 ひS．A
Effects of antioxidants，L−aSCOrbic acid（AsA）and PMCarOtene，On Chlorophyll（Chl）
degradationbyaperoxidaseMhydrogenperoxidesystemweredetermined，andtherelationshipof
theChldegradationtotheantioxidantcontentsinstoredspinach（5i）inacia oleYtZCeaL．）leaves
treatedwithethylenewasstudied・TheantioxidantsreducedChldegradationbyinhibitingthe
peroxidase．hydrogenperoxidereactionsystem．AsAandβqcarotenecontentsinspinachleaves
decreasedduringstorageat25℃，thedecreasebeinggreaterwithAsAthanwithp−CarOtene．
DegradationofChlandAsAwashastenedwhenthespinachleavesweretreatedwithlOppm
ethylene，andthedegradationofAsApreceded that ofChl．Thus，AsAwasmoreeffectivethan
β￣CarOteneinprotectingthe Chl，buttheeffect waslostwhen AsA content dropped belowa
thresholdlevel．
（ReceivedJun．27，1997）
Yellowing ofleafy vegetables occurs with
duringstorage7）・Thus，the peroxidase plays an
degradationofchlorophyll（Chl）．Thedegradation
importantroleinChldegradationandappearsto
OfChlisknOwntobecatalyzedbyvariousspecific
be affectedby ethylene treatment．
enzymesl卜4）
Chl degradation alsois influenced by the
Peroxidaseis one of the enzymes which are
SurrOunding componentswithin the organelle8）I9）
involvedinChldegradation4）・5）・Inparsleyleaves，
The components such as L−aSCOrbic acid（AsA）
Chl was degraded by the peroxidasein the
andβてarOtenelocalizedin the chloroplasts can
peroxidase￣hydrogen
peroxide
systemcontaining
p】ay a protective role as a antioxidant8），9）．The
apigenin，a major flavonein theleaves4）．In this
quantityorthecontentofantioxidantsrequiredin
degradativereaction，132−hydroxychlorophylladid
thetissueforprotecting the Chlisunknown．
not accumulateasanintermediate5）andthefinal
Theobjectivesofthisstudyweretodetermine①
product was a colorlesslow molecular weight
theeffects ofantioxidants，AsA andノ㌢carotene，
COmpOund・The peroxidase−hydrogen peroxide
On
Chldegradation
by
the
peroxidase岬hydrogen
SyStemalsoregulatedChldegradationinspinach
perxide systemin stored spinachleaves，②the
leaves，andtheperoxidaseactivityincreasedwith
effects of antioxidants on a phenolic compound，
yellowlng Ofleaves6）・Peroxidase requires a
Whichis required for the peroxidase−hydrogen
amajorflavonoidintheflavedotissue，andwas
peroxidesystem，and③therelationshipbetween
the antioxidant contents and the timing of the
ChangesinChlcontentinspinachleavesheldinair
found to decrease with ethylene degreenlng
Or ethylene atmosphere．
flavonoid
for
oxidation
to
degrade
Chl’in
the
flavedoofSatsumamandarinfruit7）・Hesperidinis
18
FoodPreservationScienceVOL・24 No．11998 （18）
MateriaJs and Methods
（Figt2），thereactionwasstoppedbytheaddition
OflO％ metaphosphoric acid．One mE of the
l．Plant materiaI
reactionsolutionobtainedwasthenaddedtolmE
Fresh‘Hybrid612’spinachleaves（＄t）inacia
Of2，6−dichlorophenolindophenol（60／Jg／mE）．
Ole7ueaL・）Werestoredinacontainerhavinga
COntinuous stream of humidified air with or
Degradation ofAsA was determinedby reading
the decrease at520nm．
WithoutlOppmethyleneat25℃．Subsampleswere
・remOVed daily andleaf bladeswithoutmidribs
Chls were extracted from spinachleaves and
PurifiedpartiallybythemethodofYosHIURA and
Were uSed for the analysis．
IRIYAMAl隼
2・Analyses of ch．0rOPhyH，L−aSCOrbic acid and
β−Ca「Otene
Results and Discussion
Chl was extracted by 80％ acetone and
l・EffectsofL−aSCOrbicacidandβ−CarOteneOn
determined by the photometric analysis as
the chlorophylldegradation by the peroxidase−
describedbyARNONlO）．
hydrogen peroxide system
AsA was extracted by using O．1M citric acid
Chlandp−COumaricacidwererapidlydagraded
COntainlnglFeMEDTAandseparatedonaHPLC
in the antioxidant free peroxidase−hydrogen
（Whatman PartisilPAC column，4．6×100mm）
PerOXidesystem（Fig．1）．AdditionoflmMAsA
usinglmMKH2PO4（pH3．0）asthemobilephase
tothesysteminhibiteddegradationforabout2min
ataflowrateofl．OmEpermin．Concentrationwas
before both Chl and p NCOumaric acid were
determined on absorbance at 245 nm with a
degradedataratesimi1artothatofthecontrol．A
Perkin−Elmer LCu95spectrophotometerll）．
β￣CarOteneWaSeXtraCtedfromleavesthatwere
5mMAsAcompletelyinhibited Chldegradation
duringthe5minreactiontime．MostofthelmM
frozeninliquidnitrogenandstoredatM80℃．The
AsAaddedtotheperoxidase−hydrogenperoxide
hexane extract was dried under nitrogen and
dissolvedinasmallquantityofchloroform，Which
SyStemWaSOXidizedrapidlyanddepletedwithin2
min of the reaction time．
WaS analyzed by HPLC．A Vydac201TP54C18
㌻carotenealsohadaninhibitoryeffectonChl
COlumn（4・6×250mm）wasusedfortheseparation
degradationintheperoxidasesystem（Fig．2）．In
Of carotenoidswith methyl alcohol：chloroform
（90：10）asthemobilephase．Theflowratewas
measuremeht of the effect ofβ，CarOtene On9−
l．Om恩permin andthe absorbance was monitored
reactionmixturewithβ−CarOteneatthebeginning
at450nm with an HPlO40A photodiode array
detector12），
OfthereactiollWaShigherascomparedwiththat
3．Chlorophylldegradation reaction
COumaric acid oxidation，the absorbance of the
withoutβてarOtene（control），becauseβrcarotene
has the absorbance at310nm・The pattern of
To determine the effect ofantioxidants on Chl
inhibitionofβ−CarOteneWaSdifferentfromthatof
degradation，partiallypurifiedChlsinethanoIwere
AsA・Withβqcarotene，amarkeddegradationof
incubated at25℃in Triton X−100，P−COumaric
Chl was notedinitially，but the extent of Chl
acid，Whichis almost ubiquitousin plants13），
degradationwasnotasgreatasthatofthecontrol．
hydrogen peroxide，perOXidase（Sigma TypeII，
Aftertheinitialdrop，therateofdecreaseinChl
horseradish），phosphatebuffer（pH6．0）andwith
withβ−C云rotenewassimilart。that。fc。ntr。1．0。t
Or Without antioxidants，aS Shownin Fig．1and
Fig．2．
Of accordancewith theinhibition of AsA，β−
Degradation of Chl，針−COumaric acid orβ−
effectonpTCOumaricacidoxidation，aSisapparent
CarOtene WaS determined spectrophotometrically
in Fig・2・Bothβ−CarOtene andp−COumaric acid
byreadingtheabsorbanceat668，3100r450nm，
WeredegradedwiththeadvanceofChldegradation．
respectively．In measurement of AsA oxidation
AsAand針CarOteneareknowntoplayaroleas
CarOtene，however，did not show aninhibitory
（19）
〔Article〕Chl・DegradationAffectedbyAntioxidants 19
0．7
Ch−orophyll＊l
0．9
Chloroph州＊1
︵巨の霊︶ の2月LOSqく
盲
〇〇
諾0．5
q）
2
何
．工】
○
∽
⊥】
く 0．3
0
0．6
夕べ0＝maricacid＊2
クーCOumaricacid＊2
︵声占蒜︶ むOu遥LOS毒
β−CarOtOn。＊2
0 1 2 3 4 5
1 2 3
Reaction time（min）
Fig・l Degradation of chlorophyllin a petoxidase−
hydrogenperoxidesystemwithorwithoutascorbicacid
4 5
Reaction time（min）
Fig・2 Degradation of chlorophyllin a peroxidase−
hydrogenperoxide systemwith orwithoutβ−CarOtene
＊1Reactionmixture二chlorophylls（a−30〃g，bLllpg）in
＊1ReactionmiXture：chlorophylls（aL30pg，b−11JLg）in
ethanol（finalconc・−4％）＋0・04％TritonX−100十0．04〟M
ethanol（finalconc∴4％）＋0・08％TritonXlOO＋0．04FEM
PNCOumaric acid＋1mM or5mM sodium ascorbate十
P−COumaricacid＋10FLMp−CarOtene＋0．012％hydrogen
0・012％hydrogen peroxide ＋ horseradish peroxidase
PerOXide＋horseradishperoxidase（SigmaTypeII，20／‘
（SigmaTypeII，20FLg）＋76mMphosphatebuffer（pH
6．0），2．5mgin a totalvolume
VOlume
＊2 Reactionmixture：4％ethanol＋0．04％TritonX−100＋
g）＋76mMphosphatebuffer（pH6．0），2．5mEinatotal
＊2 Reactionmixture：4％ethanol＋0．08％TritonX−100＋
0・04／（M p−COumaric acid＋1mM sodium ascorbate＋
0・04ノ∠Mp−COumaricacid＋101‘M針CarOtene＋0．012％
0・012％hydrogen peroxide＋horseradish peroxidase
hydrogen peroxide十horseradish peroxidase（Sigma
（SigmaTypeII，20FLg）十76mMphosphatebuffer（pH6．
TypeII，20pg）＋76mMphosphatebuffer（pH6．0），2．5m且
0），2．5m且in a totalvolume
in a total volume
20 FoodPreservationScienceVOL24 No．11998 （20）
an antioxidantin plants B）・9）and this was
about65％ofAsAwasdepletedinspinachleaves
demonstrated in this studyin inhibiting Chl
heldin air and about90％inethylene treated
degradation．However，themechanismofinhibition
leaves：Thelevelinaironday4wassimilartothat
byAsAandβ−CarOtenearedifferent．AsAinhibits
inethylene，Whichwasverylow．
Chldegradation bythe reduction of oxidizedp−
PuCarOtene COntent Ofleaves held in air or
COumaric acid，Whereas βq carotene has an
ethlenedecreasedataboutthesamerateandabout
inhibitory effect on Chl degradation sinceβ−
30％oftheinitialamountwaslostbyday4（Fig．
CarOteneitselfisdegradedbyoxidizedp−COumaric
acid．
3B）．
2・Chlorophyll，L−aSCOrbic acid and P−CarOtene
maintainingtheintegrityofChl，aSnOtedbytheir
COntentSin stored splnaChleaves
Both AsA andβPcarotene were effectivein
protective rolein the peroxidase M hydrogen
Chlacontentofleavesheldinairshowedalmost
peroxide system．The protective role waslost
nochangebyday2anddecreased30％byday4
When AsA was depleted as noted bothwith the
（Tablel）．Ethylenetreatmenthastenedtherateof
isolatedChlintheperoxidase−hydrogenperoxide
Chldegradationandthecontentdecreased60％by
SyStemandinthewholesplnaChtissuesystem．In
day4．
the wholespinach tissue system，Chlcontent did
AsAcontentdecreasedrapidlyinleavesheldin
notdecrease untilday2，Whereas AsA showed a
aitorethylene atmosphere（Fig．3A）．Byday2，
decreasein dayl．Thisimplies that a sufficient
Tablel Changesin chlorophyll a content of
SplnaChleaves during storage at25℃
Daysin storage
Atmosphere o 2
amount of AsA was available up to dayl to
PrOteCttheChl，butafterdayl，theleveldropped
belowthethresholdlevelforprotection・Ethylene
Ethylene 125．1±5．3＊（100＊＊）100．8±4，0（81）47．7±2．6（38）
hastened the decrease of both Chl and AsA at
Air 125．1±5．3（100）124．7±9．4（100）88．9±4．1（71）
aboutthesametime，Whichwasafterdayl．Itis
＊mg／100gFW，Means±SE ＊＊％
unknownfromthesefindingsifethyleneinitially
hadaneffectonAsA，Whichsubsequentlywould
0 0
P．6和 0云﹂00S＜−﹂
affect Ch1，0r Whether the effect occurred
Simultaneously onboth AsA and Chl．
ThedechleinAsAmayalsobeduetothedeclinein
theregenerationofitself・Monodehydroascorbate
︵茫gOL＼ぎ︶
reductase′ Which
occurs
ubiquitouslyin
the
Chloroplast，mitochondria，microsomeandcytosol，
WaS related to the major reducing system for
Ouの；完nTq
regenerating AsAin the cucumber fruit15）
Dehydroascorbate
reductase，localizedin
chloro山
plast，WaS alsoinvolvedin regeneration of AsA
in splnaChleaveslG）・The activity of these
reducingenzymesmaybeaffectedbyethylene，but
furtherstudiesareneededtoconfirmthistheory．
We greatfully acknowiedge Mr．Willard
Douglas，Beltsvi11eAgriculturalResearchCenter，
Daysin storage
Fig・3 L−AscQrbic acid（A）andβてCarOtFne（B）
COntentSinsplnaChleavesstoredinalrOralrwithlO
ppm ethylene at25℃
The vertical barS rePreSent SE．
for his excellent technicalassistance．
Literature Cited
l）AMIR−SHAPIRA，D．，GoLDSHUMIDT，E．E．and
、．ご1■、
〔Article〕
Ch・lDegradationAffectedbyAntioxidants
21
ALTMAN・A・：Proc・∧厄tl・Acad・Sciリ84，1901
貯蔵ホウレンソウのクロロフィル分解に及ぼすアスコ
（1987）
2）MARTINOIA，E・，DALLING，M・J・andMATILE，P．
∴乙丹肋儲噸勧由り07，269（1982）
3）KATO，M・andSHIMIZU，S・：PhlntCellPhysiol”
26，1291（1985）
4）YAMAUCHI，N・andMINAMIDE，T・：JJ履）an．
510C．助〟．Sc・ブリ54，265（1985）
ルビン酸およびβ一カロチンの影響について
山内直樹＊・AlleyE．Watada＊＊
＊山口大学農学部 ■
（〒753 山口市吉田167ト1）
＊＊米国農務省ベルツヴィル農業研究センター
5）YAMAUCHI，N・andWATADA，A・E・：JJ4）an．
（メリーランド州ベルツヴィル）
Soc・肋γ才．Sc㍑63，439（1994）
6）YAMAUCHI，N・andWATADA，A．E∴JAmer．
SDC一理フ7ィ．Scg．，116，58（1991）
7）YAMAUCHI・N・・ⅩIA，Ⅹ・andHASHINAGA，FJJ
ノ郎α犯・Soc・肋γ才．Sc7．，66，283（1997）
8）pELL，E・J・and STEFFEN，K．L．（eds）：Active
Oxygen／0Xidative Stress and Plant Metab。1ism
（AmericanSocietyofPlantPhysiologists），P．131
貯蔵したホウレンソウ（勤乃αCα油化Cgα上．）のベ
ルオキンダーゼによるクロロフィル（Cbl）分解に及ぼ
す還元型アスコルビン酸（AsA）およびβ−カロテンの
影響について検討した0ホウレンソウの貯蔵は25℃下，
加湿した10ppmエチレンおよび空気を通気することに
よって行い，貯蔵中のChl，AsAおよび針カロテン含
9）LARSON，R・A・：勒tochemist7y，27，969（1988）
量を測定した0また標品のベルオキシターゼを使用し，
ベルオキシダーゼー過酸化水素系によるChl分解に及ぼ
10）ARNON・D・I・：PlantPjv）Siol．，24，1（1949）
すAsAと針カロテンの影響についても調べた。
（1991）
11）WATADA，A・E・‥HortSci．，19，334（1982）
12）KHACHIK・F・：J Agric・Fbod Chem．，34，603
（1986）
13）HARBORNE，J，B．＝ Phytochemical
反応系に添加されるとChl分解は抑制され，これら抗酸
化剤はベルオキンダーゼー過酸化水素系によるChl分解
M。th。ds
（ChapmanandHall，London），P．41（1973）
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∧毎ごねオCαCZd＆且犯即弼gリ24，612（1979）
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22，867（1981）
AsAと針カロテンがベルオキシダーゼー過酸化水素
を効果的に抑制することが示された。ホウレンソウの貯
蔵に伴うAsA含量はChl含量の低下に伴い減少し，その
減少程度は針カロテン含量の減少に比べ顕著であった。
エチレン処理はAsAおよびChlの減少を促進し，AsA
の減少はChlの低下に先行して生じた。
以上の結果から，AsAおよび針カロテンはChl分解
に関与するベルオキンダーゼー過酸化水素系に対して抑
制作用を持ち，ホウレンソウの貯蔵に伴うChl分解につ
いては，特にAsAの一定レベル以下の減少が分解要因
になっているものと推察した。
（平成9年6月27日受理）