ExtractionofPhenolicsfromPlants

AnnE.Hagerman2002

Themethodusedtoextractphenolicsmustbeplannedwithcaresinceallresultsoftheanalysesare
affectedbytheextractionstep.Solventandmethodofhomogenizationaffectbothrecoveryof
extractionandselectivityofextraction.Somesolventsareincompatiblewithsomemethodsofanalyses.
Inparticular,acetoneinhibitsallproteinprecipitationassaysexcepttheradialdiffusionassay.Crude
plantextractsarenotchemicallystable,andcanchangerapidlyduringaging.Phenolicscanbeextracted
fromfresh,frozen,driedorlyophilizedplantsamples.Sampletreatmentdoesaffectphenolic
extractability,soallsamplesshouldbetreatedsimilarly[Hagerman,J.Chem.Ecol.14,453461(1988);
CorkandKrockenberger,J.Chem.Ecol.17,123134(1991);Orians,C.M.J.Chem.Ecol.21,1235(1995);
Lindroth,R.L.;Koss,P.A.J.Chem.Ecol.22,765(1996).

Lyophilizingtissue
Drytissueiseasytohandle,butphenolicsarefrequentlynotstabletosundryingordryinginanoven.
Toconvenientlylyophilize,orfreezedry,planttissue,cutthefreshtissueintosmallpieceswithscissors,
andplaceitinamortarwithliquidnitrogen.Grindwiththepestletoyieldafinepowder,keepingit
frozenwithliquidnitrogenthroughout.PutthesampleinabagmadeofMiracloth,andlyophilize
immediately.(Whensamplesarelyophilized,orfreezedried,waterisremovedbysublimation.Avariety
oftypesoflyophilizerscanbeobtainedfrommakersofrefrigerationorvacuumequipment).

Extractionwithasonicator
Asonicatoruseshighenergysoundwavestoforcesolventtopenetrate"unwettable"material.Ina
sonicatingbath,multiplesamplescaneasilybehandledatonce.Useabout2gdryweightor8gwet
weightoftheplanttissue.Ifusingfreshorfrozentissue,grinditfirstwithliquidnitrogenwithamortar
andpestle.Transferthesampletoa50mLscrewcapplasticcentrifugetubeandrecordthesample
weight.Add20.0mL70%acetone(acetone:water,70:30)andsonicate30minat4C.Centrifuge10min
at2500xg.Removesupernatantandrecordexactvolume.Savesupernatantat4C.Repeatthe
extractionthreemoretimes.Wethenusuallycombinethefirsttwoextractions(about75%ofthetotal
phenolics)andthesecondtwoextractions(about20%ofthetotalphenolics)foranalyses.

Extractionwithahomogenizer
Tortietal.[J.Chem.Ecol.21,117125(1995)]showedthatforsomesamplesphenolicscanbeextracted
efficientlyandreproduciblyusingaPolytrontypehomogenizer.Caremustbetakennottocross
contaminatesampleswiththehomogenizerblade,whichmustbecleanedthoroughlytoremovetraces
ofplantdebris."Stringy"samplesaredifficulttohomogenize,andthevolumeofliquidmustbefairly
largetoaccommodatetheblade.

Copyright 2002 by Ann E. Hagerman. All rights reserved.