全 文 ：第 29卷 第 5期
2010年 10月
大 豆 科 学
SOYBEANSCIENCE
Vol.29 No.5
Oct. 2010
ExtractionsofIsoflavonesfromLeguminosae:aReview
XIAOYong-ying
(SoutheastUniversityHospital, Nanjing210096, Jiangsu, China)
Abstract:Naturalsourcesofisoflavonesincludesoyandsoyproducts, suchasredclover, kudzu, etc.Therearemainly12
isoflavonesinsoybeans:genistin, daidzin, glycitinandtheirrespectivemalonyl, acetylandagluconeforms.As12 diferent
soyisoflavoneshaveawidevariationinpolarities, developmentofanoptimizedextractionprocedureforallisoflavoneshas
beenachalengingtask.Theextractionsolventsusedinconventionalextractionmethodsincludemethanol, ethanol, acetoni-
trile, acetone, n-butanol, dimethylsulphoxide, Genapol, etc., whicharenotenvironmental-friendly.Andresearchershave
developedvariousextractiontechniques, suchassupercriticalfluidextraction(SFE), ultrasound-assistedextraction(UAE),
pressurized-liquidextraction(PLE), solid-phaseextraction(SPE), andmicrowaveassistedextraction(MAE)toextract
isoflavonesfromthesourcesefficientlyandenvironmental-harmless.Thispaperreviewstheconventionalandnewlydeveloped
extractiontechniquesofisoflavonesfromLeguminosae.
Keywords:Isoflavones;Extractionsolvents;Extractiontechniques
中图分类号:TS214.2 文献标识码:A 文章编号:1000-9841(2010)05-0889-05
豆科植物异黄酮提取技术研究进展
肖永英
(东南大学医院 ,江苏南京 210096)
Received:2010-04-19
Biography:XIAOYong-ying(1973-), female, PharmacistofChinesemedicine, M.Sc(NaturalScience)-PlantPhysiology.
Email:xiao@seu.edu.cn.
摘 要:豆和豆制品 ,红三叶草 , 葛藤中都含有异黄酮 , 包括染料木苷(Genistin), 黄豆苷(Daidzin), 黄豆黄苷(Gly-
citin)以及它们的衍生物如丙二酰(malonyl),乙酰(acetyl), 苷元(aglucone)等。由于不同的组分在原料中的含量
差别很大 , 寻找最优的异黄酮提取法是很困难的。传统的提取法利用甲醇 、乙醇 、乙腈 、丙酮 、n-丁醇 、二甲基亚砜 、
Genapol等有机溶剂提取异黄酮。有机溶剂的大量使用对环境和人体都造成较大损害。近几年来 , 研究人员已发
现几种高效的 、环境友好型的提取法:超临界流体萃取法(SFE), 超声波辅助萃取法(UAE), 高压液体萃取法
(PLE), 固相萃取法(SPE), 微波辅助萃取法(MAE)等。文章综述了从豆科植物中提取异黄酮的溶剂提取法和新
近发展的提取技术。
关键词:异黄酮;溶剂;提取技术
Isoflavonesarenaturalplanthormones[ 1] andnat-
uralantioxidants[ 2-4] .Inthepast20years, therewere
intensiveresearchesfocusingontheisoflavones, dueto
theirpotentialbeneficialefectsonhumanhealth.The
Isoflavonesareheterociclicphenolsthatwithclosesim-
ilaritytoestrogensinstructureandhadadiphenolic
charactersimilartolignanswhichdisplayingbothes-
trogenicandantiestrogenicactivities, influencingsex
hormonemetabolismandtheirbiologicalactivities[ 5] .
Theisoflavoneshavebeenimplicatedinawiderange
ofhealthconditionsincludingmenopause[ 6] , cardio-
vasculardisease[ 7] , osteoporosis[ 8] andcancer[ 5, 9] .
Isoflavonesareveryabundantinsoyandsoyproducts,
suchasredcloverandkudzu, etc.Inrecentyears,
researchersareeagertofindeficientandenvironmen-
talyfriendlyextractiontechniquestoextractisofla-
vonesfromtheirsources.
1 Sourcesofisoflavones
Thereareseveralnaturalsourcesofisoflavones:
soyandsoyproducts, redcloverandkudzu.Thereare
12mainisoflavonesinsoybeanssuchasgenistin, daid-
zin, glycitinandtheirrespectivemalonyl, acetyland
agluconeforms.Theconcentrationsoftheseformsmay
varyinsoyfoodsdependingonthetypeofprocesing
thatused[ 10] .Theevidenceintheliteraturesuggests
thatthebiologicalefectsofsoyisoflavonesdonotde-
pendontheglucosideform[ 11] .Theapparentbioavail-
abilitysequenceofisoflavoneswereglycitein>daidzein
>genistein[ 12] .Thereporthigherabsorptionofaglu-
890 大 豆 科 学 5期
consinfastedratssuggeststhatabsorptionofaglucons
maybefasterinanemptystomach[ 13] .The6-O-malo-
nylformspredominateinrawandunprocessedsoy-
beans, thesemalonylformswildecarboxylatewiththe
timeafterextraction.Ithasbeenreportedthat0.2 ～
0.3 molmalonylformscanconverttotob-glucosides
everyhouratroom temperature[ 14] .Maintenanceof
autosamplertemperaturesat5℃ hasbeenusedtomin-
imizethisphenomenon[ 15] .Thisinstabilityalsoneces-
sitatesinpromptanalysisofextractswithinabout10h
ofextractiontominimizeartifactformation[ 16] .
Insoybeans, theconjugatesofgenistein, daidzein
andglyciteinwerefoundinanapproximateratioof6∶
3∶1, althoughtheisoflavonecontentwasinfluencedby
genetics, cropyear, growthlocationfactors[ 17-18] .The
contentoftheisoflavonesinsoybeanswas0.1 ～ 0.5 g
· 100g-1 soybeans.Itisreportedthatsoybeansprouts
have higher amounts of isoflavones than soy-
beans[ 19-20] , andsoybeansproutcotyledonhasthe
highestamountsofisoflavonesamongthepartsofthe
soybeansprout, includingcotyledon, hypocotylsand
root[ 21] .
Theisoflavoniccompositionofredcloverwasthe
subjectofrecentpublications[ 22-24] .Themaincom-
poundsidentifiedincludeformononetin, biochaninA
aswelastheirglycosidesortheir6“ -O-malonylated
derivatives”.Otherminoraglyconescanalsobepres-
entsuchasglycitein, irilone, orobol, pratensein,
pseudobaptigeininandprunetin.
Theisoflavonecontentofkudzuwasquitecomplex
andseemstohavebeenlessstudiedthanthoseofsoy
andRedClover[ 25-26] .Puerarin(daidzein7-C-β -D-
glucoside), daidzin(daidzein7-O-β -D-glucoside)
anddaidzeinarethemainisoflavonesidentifiedina
methanolicextractofPuerariaradix[ 27] .Isoflavonedi-
glucosidessuchasdaidzin-4-O-glucosideandpuerarin-
4-O--glucoside, alongwithotherunusualisoflavones,
werealsoidentifiedinkudzuextractsbyLC-MS/MSa-
nalysis[ 28] .
2 Extractionsolvents
Since12diferentsoyisoflavoneshaveawidevar-
iationinpolarities, developofanoptimizedextraction
procedureforalisoflavonesisachalengingtask.Dif-
ferentresearchershavedevelopedvarioussolventsand
techniquesforextractionofisoflavonesfromsoybeans.
Theprincipalextractionsolventsaremethanol, etha-
nol, acetonitrile, acetone, n-butanol, dimethylsulph-
oxideandgenapol, etc.
In1980s, 80% methanolwasthepreferredex-
tractionsolvent[ 29] .Murphy[ 14] atemptedtodetermine
theoptimumextractionprotocolforthevariousisofla-
voneformsthatwereknownatthattimebycomparing
methanol, ethanol, acetoneandacetonitrilewithand
withouttheadditionofhydrochloricacid.Farmakalidis
andMurphy[ 30] demonstratedthat80%acetonitrilewas
superiorto80%methanolinextractingtheacetyl-glu-
cosidesfromdefatedsoyflakes.Murphyetal.[ 16]
haveextractedavarietyofsoyfoodswithacidifiedace-
tonitrilewithvaryingamountsofwaterthatwereopti-
mizedforeachfoodtype.GrifithandColison[ 31] have
compared80% methanoland50% acetonitrilewith
andwithoutacidforextractionofsoyproteinfromsoy
supplements.Theseresultsindicatethat50% acetoni-
trileextractionwithoutacidificationisabetermethod.
Murphyetal.[ 32] comparedfourdiferentacidified
solvents53% acetonitrile, 53% methanol, 53% etha-
noland53% acetoneanddemonstratedthatacetoni-
trilewassuperiortoacetone, ethanolandmethanolin
extractingthe12phytoestrogenicsoyisoflavoneforms.
Penalvoetal.[ 33] comparedanotherfourdiferentsam-
plepreparation(extractionandhydrolysis)procedures
withthreediferentsolvents(80%ethanol, 80%acid-
ifiedethanol, and80% methanol)atvarioustempera-
ture(60 ～ 100°C)fordiferenttimeintervals(30 ～
240min)andindicatedthat80% EtOHcontaining1
mol·L-1 HClhadbeterextractionefects.
LinandGiusti[ 34] comparedfrequentlyusedsol-
ventsystemsandtoevaluatetheefectsofpolarityand
acidityontherecoveryofisoflavonesfromsoybeans.
Theyusedsixsolventsystems, whicharethecombina-
tionsofthreepolaritylevels(83% acetonitrile, 80%
methanol, and58% acetonitrile)andtwoaciditylev-
els(nonacidifiedandacidified)intheirstudy.Sol-
ventswithahigherpolarityextractedasignificantly
higheramountoftotalisoflavones.Forindividual
isoflavones, 58% acetonitrile(highestpolarity)ex-
tractedeitherthehighestorequalyamountsthanother
solvents, while83% acetonitrile(lowestpolarity)ex-
tractedeitherthelowestorequalyamountsthanother
solventsexceptfortheaglyconeform.Besides, acidifi-
cationsignificantlyreducedtherecoveryofthemalo-
nylglucosideformandthetotalisoflavones.Amongthe
sixsolventsystemsexamined, 58% acetonitrileaque-
oussolutionwithoutacidwasthebestsystemforex-
tractionofisoflavonesfromsoybeans.
Defatedsoysampleswereextractedwith80%
5期 肖永英:豆科植物异黄酮提取技术研究进展 891
acetonitrile, 80% methanoland80% ethanolbystir-
ringandsonicatingsamples.Theauthorssuggested
thatextractionrepeatlyisessentialfortheoptimumex-
tractionandisoflavoneyieldssignificantlydecreased
withoneextractioncycle[ 35] .Theyfoundsimilar
isoflavonesyieldswithalthreesolvents.
Luthriaetal.[ 36] investigatedtheimpactofex-
tractionsolventsandtechniquesontheassayofisofla-
vonesfromsoybean.Comparisonoffourpreviouslyop-
timizedandcommonlydeployedsolventmixtures(ace-
tonitrile∶water58∶42 (v/v);ethanol∶water70∶30
(v/v);methanol∶water90∶10 (v/v);superheated
pressurizedwater)wascariedoutfortheextractionof
isoflavones.Inaddition, theyalsoexaminedtheex-
tractioneficienciesofthreeadditionalnewsolvent
mixtures(dimethylsulphoxide∶acetonitrile∶water, 5
∶58∶37 (v/v/v);dimethylsulphoxide∶ethanol∶wa-
ter, 5∶70∶25(v/v/v);Genapol∶water5∶95(v/v))
fortheextractionofisoflavonesfromsoybeans.Opti-
mumtotalisoflavonesrecoveriesfromsoybeansamples
wereobtainedwithdimethylsulphoxide∶ethanol∶wa-
ter(5∶75∶25, v/v/v)solventmixtureusingaPLE.
Xuetal.usedn-butanol/watertwo-phasesolvent
systemassolventstoextractisoflavonesfromstemof
Puerarialobata(Wild.)Ohwi.Theresultsshowed
that1∶1(v/v)n-butanol/watertwo-phasesolventsys-
temcannotonlyrealizethecombinationofsolid-liquid
extractionandliquid-liquidpurification, butalsoresult
inthemaximumyieldsofdaidzeinaswelastotal-
isoflavones[ 37] .Furthermore, theyobservedthat1∶1
(v/v)n-butanol/waterresultsinthemaximumyieldof
total-isoflavones[ 38] .
Isoflavonesfrom cotyledonsofsoybeansprouts
wereextractedwithaqueousethanol[ 20] .Themaximum
amountofisoflavoneinaqueousethanolextracts(11.6
mg·g-1 solid)wasobtainedwhenisoflavonesincoty-
ledons(2.18 mg· g-1 solid)wereextractedwith80
～ 90% (v/v)aqueousethanolabove90°Cformore
than100 min.
3 Extractiontechniques
Generaly, isoflavoneshavebeenextractedusing
aqueousmethanol, ethanol, oracetonitrile, acetone
withsimplemixing, soxhlettechniquesforafewto
severalhours.Toseekmoreenvironmentfriendly
methodsandincreasetheproductivityoftheisofla-
vones, newerextractiontechniqueshavebeendevel-
oped.
3.1 Supercriticalfluidextraction(SFE)
Supercriticalfluidextraction(SFE)ofersseveral
advantagesoverconventionalextractionmethodssuch
asincreasedselectivity, expeditiousness, automaticity,
environmentalsafety, dramaticalydecreaseduseofor-
ganicsolvents, higherspeedandbeterreproducibili-
ty[ 39-43] .
Carbondioxide, themostcommonlyusedsuper-
criticalfluid, assolvent, ischemicalyinert, non-tox-
ic, non-flammableandisanacceptedfoodgradesol-
vent.Theseadvantageshaveatractedincreasinginter-
estfromresearchers, especialyfromfood, pharmacy
andenvironmental- engineeringindustries.However,
duetothelimitedsolubilityofpolarorganiccompounds
inSC-CO2 ortotheirinteractionwiththematrix,
quantitativeextractionofthesecompoundswithpure
SC-CO2 isnotpossible.Theadditionofapolarmodifi-
ertoSC-CO2 isthesimplestandthemostefectiveway
toobtainadesiredpolarityofCO2 basedfluids.Modi-
fierscanalsoovercomeinteractionsbetweenanalyte
andmatrix, increasingtheextractioneficiencyofpolar
organiccompounds.Polarmodifiercanbemetha-
nol[ 40, 41, 43] , ethanol[ 42] .
3.2 Ultrasound-assistedextraction(UAE)
Theenhancementofextractioneficiencyoforgan-
iccompoundsbyultrasoundisatributedtothephe-
nomenonofcavitationproducedinthesolventbythe
passageofanultrasonicwave.Cavitationbubblespro-
ducedandcompressedduringtheapplicationofultra-
sound.Theincreaseinthepressureandtemperature
causedbythecompressionleadstothecolapseofthe
bubble.Withthecolapseofbubble, aresultant
“shockwave” passesthroughthesolventandenhances
themixing.Ultrasoundalsoexertsamechanicalefect,
alowinggreaterpenetrationofsolventintothesample
matrix, solidandliquidphaseandcoupledwiththe
enhancedmastransferandsignificantdisruptionof
cels, viacavitationbubblecolapse, increasesthere-
leaseofintracelularproductintothebulkmedium.
TheuseofhighertemperaturesinUAEcanincrease
theeficiencyoftheextractionprocessduetothein-
creasingcontactsurfaceareabyenhancingthenumber
ofcavitationbubblesformed.Theextractioneficiency
ofUAEhasbeeninvestigatedbyseveralresearch-
ers[ 44-47] .
3.3 Pressurized-liquidextraction(PLE)
SincetheuseofPLEbyRostagnoetal.[ 48] toex-
tractisoflavonesfromsoybeans, pressurizedliquidex-
892 大 豆 科 学 5期
tractionwassuccesfulyusedbyseveralresearch-
ers[ 24, 36, 49, 50] .PLEisasamplepreparationtechnique
wheretemperatureandpressureareusedtoaccelerate
extractionofcompoundsfromsolidandsemi-solidsam-
ples.Pressureisusedtoincreasethecontactbetween
theextractingfluidandsample.Temperatureisusedto
breaktheanalyte-matrixbonds.SincePLEisconduc-
tedatelevatedpressuresitalowsliquidextractionat
temperaturesabovetheboilingpointsofthesolventat
atmosphericpressure, therebyimprovestheanalyte
solubilityanditsdesorptionfromthematrix.Moreo-
ver, temperaturecandramaticalymodifytherelative
permitivityoftheextractingfluid, increasingselectivi-
ty[ 51] .Thistechniquealowsasmalerrequiredvolume
oftheextractionsolvent, ashortertheanalysistime
andlesnecessaryhandlingstoproducemoreprecise
results[ 52] .Moreover, PLEofersthepossibilityofper-
formingtheextractionsunderaninertatmosphereand
protectingfromlight, whichrepresentsanatractive
advantagesincemanycompoundsaresensitivetothese
twofactors[ 53] .
3.4 Solid-phaseextraction(SPE)
Solid-phaseextraction(SPE)isasimplesample
preparationtechniquethatcanbeusedtoclean-upex-
tractsaswelasforextracting/concentratingsoyisofla-
vonesfromliquidfoodsandextracts.Mitanietal.[ 54]
proposedanautomatedon-lineintubesolidphasemi-
croextractioncoupledtohighperformanceliquidchro-
matography(HPLC)forthedeterminationofdaidzein
andgenisteininsoyfoods.Recently, severalresearch-
ershaveusedSPEsuccesfuly[ 55-58] .
3.5 Microwaveassistedextraction(MAE)
Microwave-assistedextraction(MAE)isbased
upontheselectiveandrapidlocalizedheatingofmois-
tureinthesamplebymicrowaves.Duetothelocalized
heating, presurebuildsupwithinthecelsofthesam-
pleleadingtoafasttransferofthecompoundsfromthe
celsintotheextractingsolvent, usualytransparentto
microwaves, thendidnotheatedbythem.Additional-
ly, byusingclosedvesselstheextractioncanbeper-
formedatelevatedtemperaturestoacceleratethemass
transformationoftargetcompoundsfromthesample
matrix.Therefore, MAEcanreducethesolventa-
mountand/orenhancetheextractioneficiency.More-
over, MAEcanbeappliedtoseveralsamplessimulta-
neouslyandtheextractiontimecanbealsoreduced
dramaticaly.Recently, MAEhasbeenusedbysever-
alresearchers[ 59-61] .
AcknowledgementsTheauthorwouldliketothank
therefereesandeditorsfortheircarefulyreading, use-
fulsuggestionsandmodificationofEnglish.
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(下转第 896页)
896 大 豆 科 学 5期
单株粒重和百粒重严重降低的结论相一致 。
株高 、底荚高度 、主茎节数和有效分枝数随着
钼浓度的增加而增加 ,当高钼浓度为 0.30 mg·
kg-1时仍未表现出抑制作用 ,表明这些性状能承受
更高的钼浓度。 Adriano[ 10]曾报道 ,大豆营养器官
对高钼的忍耐力非常大 ,当大豆叶片钼含量达到 80
mg·kg-1时 ,其生长仍未出现异常。
由于田间环境比较复杂 ,钼元素也未必完全被
大豆吸收 ,而且病害虫害重叠交叉对记载叶片缺钼
或钼毒害症状造成一定困难。因此 ,对田间初筛出
的典型品种需通过水培进一步验证 ,使低钼和钼毒
害指标更为可信 。同时可以通过水培精确控制各
种营养元素 ,更直观 、准确的评价叶片缺钼或钼毒
害典型症状 。
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