-
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20Hydrolyzable Tannins: Gallotannins, Ellagitannins, and Ellagic
Acid
Michael Jourdes, Laurent Pouységu, Stéphane Quideau, Fulvio
Mattivi, Pilar Truchado, and Francisco A. Tomás-Barberán
CONTENTS
20.1
Introduction..................................................................................................................................
43620.1.1
OccurrenceinFoodandMedicinalPlants......................................................................
43720.1.2
AntioxidantActivityofHydrolyzableTanninsandEA..................................................
437
20.2
HPLCAnalysis............................................................................................................................
43820.3
UVSpectrophotometryDetection...............................................................................................
43820.4
QuantitativeIndirectAnalysisofETsUsingHPLC-DADafterAcid Hydrolysis.......................
440
20.4.1
Procedure.........................................................................................................................
44020.4.1.1
StandardsandSolvents....................................................................................
44020.4.1.2
SamplingandExtractionofPolyphenols.........................................................
44020.4.1.3
SamplePreparation..........................................................................................
44020.4.1.4
AcidHydrolysis................................................................................................
441
20.4.2
HPLC-DAD-ESI-MSAnalysis.......................................................................................
44120.4.2.1
HPLC-DADAnalysis......................................................................................
44120.4.2.2
HPLC-ESI-MSAnalysis..................................................................................
441
20.4.3
MolarExtinctionCoefficients.........................................................................................
44120.4.3.1
MolarExtinctionCoefficientatMaximalAbsorptioninMethanol................
44120.4.3.2
MolarExtinctionCoefficientat260nminMethanol......................................
44220.4.3.3
MolarExtinctionCoefficientforHPLCAnalysis...........................................
442
20.4.4
UVQuantification...........................................................................................................
44220.4.5
PrincipleandApplicationoftheMethod........................................................................
442
20.4.5.1 PresenceofETsandEACsinRubus
Extracts.................................................
44220.4.5.2 FourProductsAreObtainedfromETsUsing
AcidHydrolysisinMethanol...........................................................................
44220.4.5.3
DetailedCompositionofBlackberries.............................................................
44320.4.5.4
DataInterpretation...........................................................................................
44320.4.5.5
ComputationandInterpretationofmDP.........................................................
445
20.5
HPLC-MS-MSAnalysis..............................................................................................................
44720.6
NMRAnalysis..............................................................................................................................
447
20.6.1
Gallotannins....................................................................................................................
44720.6.1.1
IsolatedGallotannins.......................................................................................
44820.6.1.2
EnzymaticallyandChemicallySynthesizedGallotannins.............................
448
20.6.2
Ellagitannins....................................................................................................................
45020.6.2.1
AbsoluteConfigurationofETAxiallyChiralBiarylGroups.........................
45020.6.2.2
DeterminationofthePositionofETGalloyl-DerivedAcylUnits...................45120.6.2.3
DeterminationoftheAbsoluteConfigurationofthe
AnomericCarbon..............................................................................................451
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436 Handbook of Analysis of Active Compounds in Functional
Foods
20.1 Introduction
Thechemicalstructuresofthehydrolyzabletanninsarebasicallycomposedofacentralsugarcore,typically
a glucose unit, to which gallic acid moieties are
esterified.β-Glucogallin is the
simplestglucosylgallateknownandservesasagalloylunitdonorinthebiosynthesisofthefullygalloylatedβ-d-glucopyranose
(β-PGG), which is itself considered to be the immediate precursor
of the
twosubclassesofhydrolyzabletannins,thatis,gallotanninsandellagitannins(ETs)(Figure20.1)(Gross1999,
2008; Niemetz and Gross 2005; Quideau et al. 2011).
Gallotannins are the result of
furthergalloylationsofβ-PGGandarecharacterizedbythepresenceofoneormoremeta-depsidicdigalloylmoieties,
as exemplified with the hexagalloylglucose
3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucopyranose(1ainFigure20.1).Alternatively,β-PGGcanbesubjectedtointra-andintermolecular
20.6.2.4
AbouttheC-GlucosidicEllagitannins..............................................................45320.6.2.5
AbouttheFlavano-Ellagitannins.....................................................................
454
Acknowledgments...................................................................................................................................455References..............................................................................................................................................
456
CO2H
Gallic acidGallotannis
1a(an example of
hexagalloylglucose)
Meta-depsidebond formation
OH
OHOH
OH
O
O
Digalloylmeta-depside
O
HO
HO
OH
OH
OH
+ 4 G
+ n G
–n [H]
IntramolecularC–C coupling
Ellagitannins
Geraniin
O
O
OO
3
OG
OG
OGOG
S
HO OH
OHOH
OH
HO
HO
HOHO
HO
HO
O OHHDPunit HO
4
4 3 2
64C1
1C4
OOO
O
R
OGO
GO
GO
OG
OGOG
GO
GO
+ UDP-Glc– UDP
HO
HO
OHOHOH
OG
O
O
O
H
OH
OHOH
O
O
ODHHDP
unit
HHDPunit
O HO
HO
O
O
OO
R
O
O
H
HO
HO
HO
β-glucogallin(G donor)
β-PGG
R = OH, tellimagrandin IR = β-OG, tellimagrandin II
FIGURE 20.1
Biosynthesisofhydrolyzabletannins(gallotanninsandellagitannins).
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Hydrolyzable Tannins 437
phenolic coupling processes that create connections between
spatially adjacent galloyl residues
byformingC–CbiarylandC–Odiaryletherbonds.Theso-calledhexahydroxydiphenoyl(HHDP)biarylunit
generated by intramolecular coupling is the structural
characteristic that defines hydrolyzabletannins as ETs. The nature
of the atropisomeric form of these chiral biaryl motifs, such as
the(S)-HHDPunitofthetellimagrandinsorthe(R)-HHDPunitofgeraniin,isdeterminedbythepositionofthegalloylmotifsontheglucopyranosecoreineitherits4C1-orits1C4-conformation.Besides,theHHDPmotifissusceptibletomanyadditionaltransformations,amongwhichitsoxidationleadstothedehydrohexahydroxydiphenoyl
(DHHDP) unit characteristic of the dehydroellagitannin
naturalproducts, suchasgeraniin
(QuideauandFeldman1996;KhanbabaeeandvanRee2001a;Feldman2005;Pouységuet al.2011).
ETsreleaseellagicacid(EA)uponhydrolysis.Thisoccursspontaneouslyinthegastrointestinaltractunderphysiologicalconditions(Larrosaet al.2006).Inaddition,freeEAanditsglycoconjugatedderiva-tiveswithsugarsarealsofoundinmostET-containingplants.
20.1.1 Occurrence in Food and Medicinal Plants
ETsarepresentinsignificantamountsinmanyberries,includingstrawberries,redandblackraspber-ries(Zafrillaet al.2001),blackberries,andnuts,includingwalnuts(Fukudaet al.2003),pistachios,cashewnuts,chestnuts,oakacorns(Cantoset al.2003),andpecans(Villarreal-Lozoyaet al.2007).Theyarealsoabundantinpomegranates(Gilet al.2000),andmuscadinegrapes(Leeet al.2005),andareimportantconstituentsofwood,particularlyoakwood(GlabasniaandHofmann2006).ETscanbe
incorporated into several food products such as wines, and
whiskies, through migration
fromwood tothefoodmatrixduringdifferentagingprocesses.EAhasalsobeenfoundinseveraltypesof
honey and this phytochemical has been proposed as a honey floral
marker for heather
honey(Ferrereset al.1996).FreeEAanddifferentglycosidederivativesarealsopresentinthesefoodprod-ucts,
includingglucosides,rhamnosides,arabinosides,andthecorrespondingacetylesters(Zafrillaet al.2001).
Inapreviousreview,itwasdocumentedthattherewerenoreliablefiguresavailableontheETdietaryburdenbutthatitwouldprobablynotexceed5mg/day(CliffordandScalbert2000).Sincethen,anum-berofstudieshaveshownthat
theETcontentofseveralfoodproductscanbequitehigh.Aglassofpomegranatejuicecanprovideasmuchas300mg,araspberryserving(100gofraspberries)around300mg,astrawberryserving70mg,andfourwalnutssome400mgofETs.Asaresult,theintakeofdietaryETscanbemuchhigherthanpreviouslyestimated(CliffordandScalbert2000),especiallyifsomeoftheseET-richfoodsareregularlyconsumedinthediet.
20.1.2 Antioxidant Activity of Hydrolyzable Tannins and EA
ET-richfoodsgenerallyshowahighfree-radicalscavengingactivityevaluatedin
vitro.Especiallyrele-vantistheantioxidantactivityofpomegranatejuice(Gilet al.2000).ThisstudyshowsthatpomegranatejuicehastwicetheantioxidantactivityofredwineandthatthisisduetotheextractionofETsfromthefruithuskduringjuicemanufacturing(Gilet al.2000).Thisremarkableantioxidantactivityhasbeenthedrivingpowerofresearchonthebiologicalactivityofthesepowerfulantioxidantsfrompomegranate,andisusedbythefoodindustrytomarketpomegranatejuiceproductsassuper-antioxidantfood.ETsarealsoresponsibleforarelevantpartoftheantioxidantactivityobservedinstrawberries(Hannum2004),raspberries(Zafrillaet al.2001),blackberries,walnuts(Blomhoffet al.2006),andpecans(Villarreal-Lozoyaet al.2007).Thisantioxidantactivitycanprobablyberelatedtothebiologicalactivityreportedforthesefoodproducts.
InparalleltothosestudiesoftheantioxidantactivityofET-richfood,clinicalstudieshavealsoshownrelevantbiologicalactivitiesthathavebeenassociatedwiththeseantioxidants,althoughnodirectevi-denceofthebiologicalactivityofthesepolyphenolshasbeendemonstrated.SeveralclinicalstudieshavereportedrelevantbiologicalactivityaftertheintakeofET-richfoods,especiallyregardingtheprotectiveeffectagainstcardiovasculardiseasesandcancer.
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20.2 HPLC Analysis
ETscanbeanalyzedbyhigh-performanceliquidchromatography(HPLC)usingreversed-phasecol-umnswithmethanol,acetonitrile,andwatergradients.Theadditionof1%offormicoraceticacidtothe
water solvent helps increasing the resolution of the chromatograms
through the separation
insharperpeaksavoidingpeaktailing.Ingeneral,complexETmixturesareobservedintheextracts.SomeeffectsoftheETstructureonthechromatographicretentionandelutionorderhavebeenreported(Salminen
et al. 1999; Moilanen and Salminen 2008). In general, the
occurrence of free galloylgroups in the ET molecule increases
the retention times, while the formation of an HHDP in
thehydrolyzabletanninmoleculedecreasestheretentiontime.Theopeningoftheglucopyranosering,asithappensinsomeC-glycosylETs(i.e.,vescalagin,castalagin),alsodecreasestheretentiontime.ETswithacyclicsugar,andwithoutgalloylationinC-1,producetwopeakscorrespondingtotheα-andβ-anomer,
while galloylated ETs at C-1 produce only one chromatographic peak.
This has beenreportedinpunicalaginandpunicalin,
thecharacteristicETsofpomegranate, thatshowtwopeaksfor each
ET corresponding to both α- and β-anomer (Gil et al. 2000).
Acyclic epimers
havinghydroxyl groupsatC-1oftheglucosecanbedistinguishedfromeachothersincetheorientationofthehydroxylgroupcausesvescalagin-typeETs
toelutebefore thecastalagin-typeones (MoilanenandSalminen2008).
EAispresent innature inafreestateor
incombinationwithdifferentsugarsandformingmethylethers.EAhexosides(glucosides),deoxyhexosides(rhamnosides),andpentosides(xylosidesandarabino-sides)aswellasglucuronideshavebeenreportedasPhaseIImetabolitespresentinbiologicalfluids.Inaddition,acetylatedderivativesofEApentosideshavebeenreportedinraspberries(Zafrillaet al.2001).Glucuronides
are the first eluting metabolites, followed by hexosides
(glucosides),
desoxyhexosides(rhamnosides),andpentosides(xylosidesfirstandarabinosides).FreeEAelutesaftertheglycosidesbutearlierthantheacetylpentosides.EAmethylethersandsulfates(thatareoftenfoundinbiologicalfluidsaftertheintakeofETsandEA)elutewithlongerretentiontimesthanfreeEA,andtheretentiontimeincreaseswiththenumberofmethylethersintroducedontheEAmolecule.
The chromatographic behavior of the microbial metabolites of ETs
and EA, known as
urolithins(metabolitesrelatedtoEAinwhichoneofthelactoneringshasbeenremovedbythecolonicmicrobiota),followsasimilartrendasEAderivatives,increasingtheretentiontimewhendecreasingthenumberofhydroxylgroupsontheurolithinnucleus,andwhenincreasingthenumberofmethylethers(González-Barrioet
al.2011).Again, the introductionof aglucuronylconjugationdecreases
the retention
time,whiletheintroductionofasulfateresidueincreasestheretentiontime.Inaddition,thechromatographicpeaks
corresponding to sulfate conjugates are broader, hence decreasing
the chromatographicresolution.
20.3 UV Spectrophotometry Detection
TheUVspectraofthedifferentETs,gallotannins,andEAderivativesareeasilyrecordedintheanaly-sis
of the extracts byHPLC-diode-arraydetection
(DAD)analysis.Theoccurrenceof
freegalloylgroupsintheETmoleculeproducestwoabsorptionmaximainthespectrum,onearound270–280nmand
another around210–220nm.Thehigher thenumberof thegalloyl groupswith
respect to
theHHDPunits,thesteeperisthevalleybetweenthetwomaxima(Salminenet al.1999),andthemaxi-mumforBI(thebandbetween270and290nm)appearsathigherwavelengths.InHHDP-richtannins,thevalleybetweenthetwoabsorptionmaximaevendisappearsfromtheUVspectrum,asisthecaseofbis-HHDP-glucopyranose,andnodefinedmaximumisobservedfor
theabsorptionbandaround270–280nm(Table20.1).
ThechangefromcyclictoacyclicsugarsalsohasasubstantialeffectontheUVspectrum.FreeEAshowsaUVspectrumcharacterizedbytwoabsorptionbandsat365–380and253–255nm,
withacharacteristicshapethatallowsitseasydetectionandidentificationintheUV-DADchromato-grams.
In general, substitution with pentoses, hexoses, and glucuronides
produces shifts of the UV
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Hydrolyzable Tannins 439
maximatoshorterwavelengths.Thiseffectisalsoobservedwhenmethylethersareintroducedonthephenolichydroxyls,andisparticularlyevidentwhenasulfateresidueisintroducedasthehypsochromicshifts
in wavelengths are more marked. The same effects are observed for
the urolithin
derivatives.UrolithinA,themainETandEAmetaboliteinmammals,hasaUVspectrumclosetothatofEA,butitisclearlydistinctive
toallow theunambiguousdifferentiationofbothcompounds
(Figure20.2).ThesameeffectsontheUVspectrumdescribedfortheEAconjugationarevalidforurolithinconjugations(González-Barrioet al.2011).UrolithinswithdifferenthydroxylationpatternsshowcharacteristicUVspectrathatcanbeusedforthestructuraldifferentiationofthemetabolitespresentinbiologicalfluidsusingHPLCcoupledtoUVdetection(DAD).
TABLE 20.1
UVSpectraofEllagitannins.EffectoftheOccurrenceofGalloylorHexahydroxydiphenoylResidues
Compound BI (nm) BII (nm)% of BI Respect BII
Absorbance
Gallicacid 276 219 38
Pentagalloyl-glucopyranose 285 220 42
Trigalloyl-HHDP-glucopyranose 283 215 27
Digalloyl-HHDP-glucopyranose 280 211 23
Galloyl-bis-HHDP-glucopyranose
279 214 19
Bis-HHDP-glucopyranose 270i 205i –
Source: ExtractedfromSalminen,J.P.et al.1999.J. Chromatogr.
A864:283–291.
400350
365
300
λ (nm)
Ellagic acid
Urolithin A
250
253
2000
300
600
mAU
900
1200
400350
356
305
300
λ (nm)
250
246280
200
0
100
200
mAU
300
(a)
(b)
FIGURE 20.2 UVspectraof(a)ellagicacidand(b)urolithinA.
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440 Handbook of Analysis of Active Compounds in Functional
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20.4 Quantitative Indirect Analysis of ETs Using HPLC-DAD
after Acid Hydrolysis
Acidhydrolysisisthemostpracticalandwidelyemployedtechniqueusedtoquantifythehydrolyzabletanninspresentinvegetableextracts.Asanexample,inthecaseofRubus,theuseofbothHPLCanalysisafterhydrolysis(Vrhovseket al.2008)anddirectHPLCanalysis(Gasperottiet al.2010)hasdemon-stratedthattheaveragestructureofETsiswellconservedinthedifferentgenotypes,whiledifferingintermsofabsoluteconcentration.ThismeansthatforroutinequantificationofcomplexETs,themethodusinganalysisafterhydrolysisisstillappropriateandabletoprovideusefulinformation.
However,caremustbetakentochoosetheappropriatesolventforextractionandthewholeessay,sincethesenaturalcompoundsarenoteasytomanage,andeachstep(extraction,hydrolysis,HPLCanalysis,anddataprocessing)shouldbecarefullyconsideredinordertoprovideconsistentdata.Moreover,EAispoorlysolubleandcanprecipitate
ifnotproperlyhandled,escapingdetection
(Törrönen2009).Asaconsequence,theETcontentreportedintheliteratureishighlyvariable,sincedifferentextractionandacidhydrolysisconditionssignificantlyaffecttheyieldofEA(Törrönen2009).EarlierstudiesreliedonthequantificationofreleasedEAanddidnotconsiderotherphenolicsformedduringhydrolysis,whichmayprovidehelpfulinformationonthechemicalstructureofETs.
Forthispurposewedescribehowtoperformandinterpretanoptimizedhydrolyticprocedure(6hwith4MHCl),whichincludesquantificationofallthefourproductsofhydrolysisandprovidesaratio-naleforestimatingthemeandegreeofpolymerization(mDP)ofRubusETs.TheapproachdescribedbelowisessentiallyasreportedbyVrhovseket al.(2006),withsomeminorimprovementsintermsofquantification,
inorder to includeall theproductsofhydrolysis in thecomputationof
themDP.Thecompositionofeightblackberrysamplesisdiscussedasanexampleoftheapplicationofthismethod.Suchanapproachmayalsobeextendedtotheanalysisofhydrolyzable
tanninsfromotherbotanicalsources,providedthatsufficientinformationontheirstructureisavailable(Koponenet al.2007).
20.4.1 Procedure
20.4.1.1 Standards and Solvents
AllchromatographicsolventsshouldbeHPLCgrade:acetonitrile,methanol,diethylether,hexane,for-micacid,aceticacid,andhydrochloricacid.EAstandard(purity≥96%)andmethylgallatestandard(purity≥98%)arebothavailablefromFluka(Steinheim,Germany).Sanguisorbicacid(SA)andmethylsanguisorboate
are not currently available, but their concentration can be
estimated by applying
theavailablemolarextinctioncoefficients(seeSection20.4.3).
20.4.1.2 Sampling and Extraction of Polyphenols
Freshlycollected samplesofblackberries (Rubus fruticosus)
fromeightdifferentcultivarswerepro-duced under standardized
conditions in the experimental fields of the Edmund Mach
Foundation(Vrhovseket al.2008).PolyphenolswereextractedfromfreshlycollectedberriesfollowingthemethodofMattiviet al.(2002)inwhich60goffreshfruitarehomogenizedinamodel847-86Osterizerblenderatspeedonein250mLofacetone/watermixture(70/30v/v)for1min.Priortoextraction,thefruitandextractionsolutionshouldbecooledto4°Ctolimitenzymaticandchemicalreactions.Thecentrifugedextractscanbestoredat–20°Cuntilanalysis,conditionsunderwhichthecompositionremainsstableforafewmonths.
20.4.1.3 Sample Preparation
Analiquot (20mL) of the extract is evaporated to dryness in a
100mL pear-shaped flask by
rotaryevaporationunderreducedpressureat40°C.Thesampleisthenbroughtbackto20mLwithmethanolimmediatelypriortoprocessing,duetothelimitedsolubilityofEAanditsderivatives.
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Hydrolyzable Tannins 441
20.4.1.4 Acid Hydrolysis
A 6 h hydrolysis with 4M HCl at 85°C has been shown to provide
the maximal yield for the
fourhydrolysisproductsofRubusETsandhasalsobeenreportedtobeappropriateforstrawberryextracts(Vrhovseket al.2006;Mertzet al.2007).Inordertocarryoutacidhydrolysisin4MHCl,16.6mLof37%HClare
added to the samplepreparedas aboveand themixture is thendiluted
to50mLwithmethanol.Afterhydrolysis,thesampleisbroughtbacktoitsinitialvolume(50mL)withmethanol.Analiquot(10mL)isthenadjustedtopH2.5with5NNaOHanddilutedto20mLwithmethanol.Finally,an
aliquot (2mL) is filtered with 0.22μm, 13mm polytetrafluoroethylene
(PTFE) syringe-tip
filters(Millipore,Bedford,MA)andtransferredintoLCvialsforHPLCanalysis.
20.4.2 HPLC-DAD-ESI-MS Analysis
20.4.2.1 HPLC-DAD Analysis
HPLCanalysisbeforeandafterhydrolysiswascarriedoutaccordingtoVrhovseket al.(2006)usingaWaters2690HPLCsystemequippedwithWaters996DAD(WatersCorp.,Milford,MA),andEmpowerSoftware(Waters).Separationiscarriedoutusinga250×2.1mmi.d.,5μm,endcappedreversed-phasePurospherStarcolumn(Merck)and4×4mm,5μm,Purospherprecolumn.Thesolventsare:A(1%formicacidinwater)andB(acetonitrile).Thegradientsareasfollows:from0%to5%Bin10min,from5%to30%Bin30min.Thecolumnisthenwashedwith100%ofBfor2minandequilibratedfor5minpriortoeachanalysis.Theflowrateis0.8mL/min,theoventemperaturesetat40°C,andtheinjectionvolumeis10μL.EAand
itsderivativesaredetectedandquantifiedbyUVdetectionat260nm.EA(RT=30.8min)
is quantified following calibration with an EA standard
(concentration range
of10–200mg/L).Methylsanguisorboate(RT=34.6min)andfreeSA(RT=25.1min)arequantifiedfol-lowingcalibrationwiththepurestandardisolatedaccordingtoVrhovsekandco-workers(2006).Methylgallate(RT=21.8min)isquantifiedfollowingcalibrationwiththecorrespondingstandardcompoundwithintheconcentrationrange3–30mg/L.
20.4.2.2 HPLC-ESI-MS Analysis
DetailedcompoundidentificationwascarriedoutusingtheMicromassZQelectrosprayionization-massspectrometry(ESI-MS)system(Micromass,Manchester,UK).Themassspectrometry(MS)detectoroperatedatcapillaryvoltage3000V,extractorvoltage6V,sourcetemperature105°C,desolvationtem-perature200°C,conegasflow(N2)30L/h,anddesolvationgasflow(N2)450L/h.Theoutletof
theHPLCsystemwassplit(9:1)totheESIinterfaceofthemassanalyzer.ESI-massspectrarangingfromm/z100to1500weretakeninnegativemodewithadwelltimeof0.1s.Theconevoltagewassetinscanmodeatthevaluesof20,40,and60V.TypicalionsfortheESI-MSdetectioninnegativemodeare:EA,molecularionatm/z301;SA,molecularionatm/z469andmainfragmentatm/z301;methylsanguisor-boate,molecularionatm/z483andmainfragmentsatm/z315andm/z301;andmethylgallate,molecu-larionatm/z183.
20.4.3 Molar Extinction Coefficients
Caremustbetakeninchoosingtheappropriatestandardandcomparingdataobtainedusingdifferentmethods.Herewesummarizeacomprehensivelistoftheexperimentalvaluesofthemolarextinctioncoefficients
reported in the literature (Vrhovseket al.2006;Gasperotti
et al.2010)andexpressedasM−1cm−1.
20.4.3.1
Molar Extinction Coefficient at Maximal Absorption in Methanol
In methanol, at maximal absorption, EA: ε254nm=40,704,
ε365nm=8066; methyl
sanguisorboate:ε254nm=58,543,ε371nm=13,022;methylgallate:ε274nm=11,818.
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20.4.3.2 Molar Extinction Coefficient at 260 nm in Methanol
In methanol, at 260nm, EA: ε260nm=32,099, sanguiin H-6:
ε260nm=72,070, lambertianin C:ε260nm=104,344.
20.4.3.3 Molar Extinction Coefficient for HPLC Analysis
In the conditions suggested for HPLC analysis with UV detection
(Section 20.4.2), the molarextinctioncoefficient isas
follows:EA:ε260nm=35,822 (solvent:21%ofacetonitrile
in1%formicacidinwater;v/v),methylsanguisorboate:ε260nm=45,114(23.9%ofacetonitrilein1%formicacidinwater;v/v).
IntheslightlydifferentconditionssuggestedforHPLCanalysisbyGasperottiet al.(2010),wheretheseparationisadaptedtoaC18Lunacolumn(solvent:88%ofacetonitrileand12%of1%formicacidinwater;v/v):EA:ε260nm=28,266,sanguiinH-6:ε260nm=63,615,lambertianinC:ε260nm=95,744.
20.4.4 UV Quantification
Toencouragetheapplicationofthismethod,overcomingthelackofamethylsanguisorboatestandard,themolarabsorbivityofpurestandardsofmethylsanguisorboateandEA,measuredattheoptimalwave-length
for UV detection in HPLC analysis, can be exploited. The ratio of
the molar absorbivity
atλ=260nmofmethylsanguisorboatevs.EAis1259.Thisvalueisinagreementwiththepresenceofthreeandtwogalloylunits
inmethylsanguisorboateandEA,respectively,andhasbeenfoundtobeconsistent
with the experimental response of the two compounds in the HPLC
analysis conditionsreportedabove(Vrhovseket al.2006).
20.4.5 Principle and Application of the Method
20.4.5.1 Presence of ETs and EACs in Rubus Extracts
The13structuresofRubusETsthusfardescribed(Gasperottiet al.2010)areinagreementwiththeassumptionthatRubusoligomericETscontainonlythesanguisorboyllinkingestergroup,besidesthewell-knownEAandgallicacidmoieties.AllknownRubusoligomericETsshareacommonstructure,originatinginC–Ooxidativecoupling.Morespecifically,thelinkingunitinRubusETscomesfromthedonationofgalloylhydroxyloxygentoformanetherlinkagetoanHHDPgroup,whichproducestheclassofGOD-typeETs
(Okudaet al. 2009).Blackberrieswere reported to
containonaverage1080mg/kgofETsand200mg/kgofellagicacidconjugates(EACs).LambertianinC(Figure20.3)isthemainETinblackberries,withanaveragelambertianinC/sanguiinH-6ratioof1.7(range0.9–3.4).Itmustbeunderlinedthatbesidesthe13knownETs,Rubusextractscontainatleastfiveotherminorcompounds,whosestructuresarestillunknown(Gasperottiet al.2010).
20.4.5.2
Four Products Are Obtained from ETs Using Acid Hydrolysis in Methanol
ThepresenceofEAandoneortwounidentifiedcompoundswithabsorbancespectraverysimilartothatofEAafteracidhydrolysisofredraspberryandstrawberrysampleshasbeenreportedbysomeauthors(Rommel
and Wrolstad 1993; Mattila and Kumpulainen 2002; Määttä-Riihinen
et al. 2004).
Morerecently,Vrhovseket al.(2006)demonstratedtheformationofmethylgallate,methylsanguisorboate,andaminorunknownEAderivative,named“derivative1,”duringhydrolysis,inadditiontoEA.OnthebasisofUVandMSdataalreadyreported(Vrhovseket al.2006)andfurtherconfirmationbyaccurateMSandMS/MS,thelatterwasshowntobeSA.
TheupdatedschemeofthereactionisshowninFigure20.3.OligomericETs,suchasintheexampleoflambertianinC,donotreleaseonlyellagicacidandmethylgallate.Alsothesanguisorboyllinkingestergroupsarehydrolyzed,yieldingmethylsanguisorboateasthemainproduct,onlyalimitedfraction
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Hydrolyzable Tannins 443
ofsanguisorbicunitescapesesterificationandcanbefoundafterhydrolysisinitsfreeform.Fourcom-poundsarequantifiedbyHPLCafterthehydrolysis.
20.4.5.3 Detailed Composition of Blackberries
Table20.2givesthequantitativecompositionofeightblackberrysamples,analyzedusingtheHPLC-DADmethodofGasperottiet al.(2010).EAandETswerequantifiedusingUVdetectionat260nm.EA
and its conjugates were quantified following calibration with EA
standard. Sanguiin H-6 andlambertianin C were quantified following
calibration with the pure standard, and other ETs
werequantifiedasequivalentsofsanguiinH-6.Foreachknownstructure,Table20.2alsogivesthetheo-reticalnumberofthethreemoieties(ellagic,gallic,andSAs),thatshouldbereleasedfromcompletehydrolysis.
20.4.5.4 Data Interpretation
Undertheseconditions,theamountofEAmeasuredafterhydrolysisnotonlyderivesfromthebreak-downofETs,butalso
includesfreeEAandtheproductof
thehydrolysisofEAglycosides,usuallypresentinRubusextracts(Gasperottiet al.2010).TheinterferenceoffreeEAwiththeassaycanbeavoidedbyusingablankanalysisofthesamplebeforehydrolysis.However,thismakesitnecessarytodoublethenumberofHPLCanalysesandisthereforenotusuallyperformed.OtherEACs,suchasthemethyl-EAglycosides(i.e.,peaks25and26inTable20.2),areexpectedtoreleasethedifferentisomersofmethyl-EA,whichdonotinterferewiththeETestimate,sinceunderthesuggestedconditionstheyelute
as separate peaks (two peaks with molecular ion at m/z 315 in the
case of blackberries) aftermethylsanguisorboate.
OH
OH OH
O
OH
OH OH OH OH
OH
O
OH
OH
OH
OHHO OH
O
OO
OOO
O
O
OH
OH OH OH OH
OH
O
O
OH
OH
O
OH
OH
OH
OHHO OH
O
OOO
OOO
O
O
OH
OH OH OH OH
OH
O
O
OH
OH
O
OH
OH
OH
OHHO OH
O
OOO
OOO
O
O O
OH
OH
O
OO OH
OH
OOH
OH
O
OO OH
OH
O
O
OH
OH
OO
H3C
OH
OH
O
OO OH
OH
O
O
OH
OH
OO
H
OH
OH OH
OOCH3
Lambertianin C(trimer)
Ellagic acidMethyl
sanguisorboateSanguisorbic acid
(minor)
Methyl gallate
FIGURE 20.3
Updatedschemeforhydrolysis,whichaccountsforthepresenceofoligomers.Besidesellagicacidandmethylgallate,thesanguisorboyllinkingestergroupsarereleasedmainlyasmethylsanguisorboate.Alimitedfractionofsanguisorbicunitescapesesterificationandcanbefoundafterhydrolysisinitsfreeform.
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444 H
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TABLE 20.2
QuantificationofEllagitanninsandEllagicAcidConjugatesinBlackberries,Expressedinmg/kg,QuantifiedUsingtheDirectHPLC-DADMethodSuggested
Cultivar 1 2 3 4 5 6 9 10 13 14 15 16 17 18 19Sanguiin
H-2Lambertianin
CSanguiin
H-6 EA 25 26Total ETs
Total EACs
Apache 14.2 10.0 13.6 11.0 10.6 13.3 155.3 26.2 34.9 n.d. n.d.
36.2 163.1 n.d. 9.5 16.1 205.1 152.5 74.1 86.3 68.4 872 229
Blacksatin 19.0 9.1 18.7 19.1 12.3 14.7 213.9 25.3 42.5 n.d. 9.0
16.7 222.4 10.4 12.8 18.0 353.0 324.9 45.6 75.6 n.d. 1342 121
Cacak 13.9 10.2 17.9 n.d. n.d. 14.1 83.4 28.6 n.d. 10.2 n.d.
42.8 95.3 8.9 15.2 n.d. 671.7 256.2 61.9 96.9 53.3 1268 212
Hulltornless 29.6 10.3 27.3 n.d. 10.1 17.5 99.5 20.5 28.7 n.d.
9.3 19.5 97.9 14.5 19.2 20.5 559.2 550.5 103.5 108.7 69.0 1534
281
Kotata 16.8 9.7 24.2 n.d. n.d. 15.4 164.3 n.d. 24.8 n.d. n.d.
n.d. 179.5 n.d. 11.1 n.d. 671.5 257.6 85.0 93.1 90.4 1375 269
LochnessG 17.2 9.0 16.4 13.6 16.3 15.4 149.8 36.2 21.3 8.6 11.2
26.2 149.4 11.9 12.0 10.8 299.7 292.5 50.4 73.4 n.d. 1118 124
Lochtay 15.7 10.6 21.8 n.d. n.d. 12.5 111.8 14.0 23.0 n.d. n.d.
18.1 132.5 9.5 13.3 10.9 756.1 354.4 68.3 102.2 79.2 1504 250
Triplecrown 16.8 11.4 20.9 n.d. n.d. 11.5 81.8 30.0 n.d. 17.9
n.d. 57.1 91.2 9.2 14.8 10.9 615.7 279.4 72.7 115.4 39.3 1269
227
Molecularsize 2 2 3 ? 2 3 ? 2 ? 3 ? ? 3 2 3 1 3 2 0 0 0
n°ofellagicacidunits
2 3 3 2 3 2 3 3 2 3 1 4 3 1 0 0
n°ofgallicacidunits
1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0
n°ofsanguisorbicacidunits
1 1 2 1 2 1 3 2 2 2 1 2 1 0 0 0
n°ofmethyl–ellagicunits
0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1
Source: AdaptedfromGasperotti,M.et al.2010.J. Agric. Food
Chem.58:4602–4616.
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Itshouldbehighlightedthattheabsolutevaluesobtainedwiththetwoindependentmethods,thatis,indirectHPLC-DADmeasurementoftotalETsplusEACsafterhydrolysis,externallycalibratedwiththebuildingunits(EA,methylsanguisorboate,methylgallate,andSA)areonlycomparableintermsoftheorderofmagnitude,butdonotoverlapwiththemorepreciseHPLCdataobtainedseparatelyforETsandEACsusingthedirectquantificationmethod(Gasperottiet al.2010),wheresanguiinH-6andlam-bertianinCwereusedasexternalstandardfortheETs,andEAforEACs.Theindirectmethodwasreportedtohaveamuchlowerrepeatability,withCV%around12%forthetwomajorcompoundsand18%
for theminor compounds (Vrhovsek et al. 2008), and canbe
considered as an acceptable
andcheaperwayofprovidinganestimateofthetotalquantityofEAderivatives.
20.4.5.5 Computation and Interpretation of mDP
The simultaneous quantification of the four main hydrolytic
products of Rubus ETs
providesdirect measurementoftherelativemolarabundanceofthebuildingunitsofETs.Accordingtothemethod
developed by Vrhovsek et al. (2006), and also including the
presence of free SA in
theupdated method,theestimateofthemDPofRubusETscanbetheoreticallyderivedfromthemolarratiobetweenthesanguisorboylunits(methylsanguisorboateplusSA)andtheEAproducedinthereaction,
R[MS+SA]/[EA]. The experimental value is highly reproducible, with
a CV% of around 2%(Vrhovsek et al. 2008). Taking into
consideration the structure of the major known Rubus
ETs(Gasperottiet al.2010),andassumingcompletehydrolysis,thisratioisexpectedtoincreasefrom0forthemonomers(galloyl-bis-HHDP-glucosides)uptoavalueof0.60fortetramericlambertianinD,with
intermediate values for dimeric sanguiin H-6 and trimeric
lambertianin C (Vrhovsek et al.2006). For oligomeric
compounds such as sanguiin H-6 and lambertianin C, which have
beenshown toaccountfor67%(range41–83%)ofETsinblackberries(Gasperottiet al.2010),aswellasfor
the other lambertianin oligomers, this ratio is expected to
increase according to the
equationR[MS+SA]/[EA]=(DP−1)/(DP+1).Inconclusion,thevalueofR[MS+SA]/[EA]canbeobtainedexperimen-tally
from HPLC analysis of the hydrolytic products of raw Rubus extract
and can be used forcomputation of the mDP of Rubus ETs, which can
be derived from the following
equation:mDP=(R[MS+SA]/[EA]+1)/(1–R[MS+SA]/[EA]).
Table20.3givesanexampleofpracticalworkflow.FromtheexperimentalvaluesobtainedfromtheHPLCrunafterhydrolysis,expressedinmg/L,thedatacanbeconvertedintomg/kginordertogivetheconcentrationintheberriesandcanbeconvertedinmmol/L,whichareusedforthecomputationofRandmDP.
TheapplicationofthismethodtotheeightraspberrysamplesinourexamplegivesanaveragemDPofca.1.9
(Table20.3).Thisvalue isslightlyhigher thanreported
inaprevioussurvey(Vrhovseket al.2008),alsoduetotheinclusionofthecontributionoffreeSAintheupdatedformula.Thecorrectionisnotmajorsincethelatterisonaverageca.7timeslessconcentratedthanmethylsanguisorboate(lastcolumninTable20.3).AnmDPvaluecloseto2suggeststhatthesumofoligomerswithDP>2(suchastrimerlambertianinC),orwithalowercontentofEA(suchasthepeaksofdimers1,5,and10,aswellasofthetrimers3,6,17,and19inTable20.2)orwithahigherpresenceofSA(suchasthemonomersanguiinH-2,inadditiontothepeaksofdimer18andtrimer14inTable20.2)roughlybalancethesumofthemonomersandfreeEAintermsofconcentration.SucharesultisinacceptableagreementwiththedetailedHPLCdataofETsandEACsreportedinTable20.2.
ItshouldbekeptinmindthatmDPcomputedaccordingtothismethodisestimated,whichcouldleadtomisleadingvalues
ifdirectlyapplied tohydrolyzable tanninsofadifferentnature
(Koponenet al.2007).However,sufficientdataareavailabletosupportitsapplicationtotheanalysisofETsinbotanicalspeciescharacterizedbythepresenceofGOD-typeETs,asinthecaseofRubus(e.g.,raspberries,black-berries,boysenberries),Sanguisorba,andstrawberrysamples.
Aftercarefulcharacterizationofhydrolysisproducts,itcouldalsoinprinciplebeextendedtoothersourcesofETs,onceenoughstructuralinformationonthechemicalstructureofETsandtheproductsofdegradationisavailable.
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TABLE 20.3
QuantificationofEllagitanninsandEllagicAcidConjugatesinBlackberries,QuantifiedUsingHPLC-DADafterAcidHydrolysis
Met
hylg
alla
te (
mg/
L)
SA (
mg/
L)
EA
(m
g/L
)
Met
hyls
angu
isor
boat
e (m
g/L
)
Met
hylg
alla
te
(mm
ol/L
)
SA (
mm
ol/L
)
EA
(m
mol
/L)
Met
hyls
angu
isor
boat
e (m
mol
/L)
Met
hylg
alla
te (
mg/
kg)
SA (
mg/
kg)
EA
(m
g/kg
)
Met
hyls
angu
isor
boat
e (m
g/kg
)
R =
[M
S +
SA]/
[EA
]
mD
P =
(R
+ 1
)/(1
– R
)
Sum
ET
s m
g/kg
Rat
io M
S/SA
6.5 5.0 138.3 62.7 0.035 0.011 0.457 0.129 27.0 20.7 576.0 261.3
0.306 1.882 901 12.3
12.3 11.3 168.4 69.2 0.067 0.024 0.557 0.143 51.1 47.2 701.9
288.5 0.300 1.856 1130 5.9
16.9 15.8 277.7 125.4 0.092 0.034 0.919 0.259 70.2 65.9 1156.9
522.6 0.318 1.934 1875 7.7
16.6 16.4 287.6 121.1 0.090 0.035 0.952 0.250 69.3 68.5 1198.5
504.5 0.299 1.855 1903 7.2
9.8 15.8 189.9 94.7 0.053 0.034 0.628 0.196 40.9 65.7 791.3
394.6 0.364 2.147 1352 5.8
14.6 10.9 179.1 65.4 0.079 0.023 0.593 0.135 60.7 45.5 746.3
272.5 0.267 1.729 1165 5.8
18.7 15.9 261.0 97.3 0.101 0.034 0.864 0.201 77.7 66.3 1087.7
405.4 0.272 1.746 1697 5.9
20.9 26.7 295.4 135.5 0.113 0.057 0.977 0.280 87.0 111.3 1230.7
564.4 0.344 2.050 2097 4.9
Note:
FromtheexperimentalvaluesobtainedfromtheHPLCrunafterhydrolysisoftheextract,expressedinmg/L,thedatacanbeconvertedintommol/L,whichareusedforthecomputa-tionofRandmDP,andintomg/kginordertogivetheconcentrationintheberries.
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20.5 HPLC-MS-MS Analysis
To obtain information of the molecular masses of ETs and EA
derivatives preliminarily
detectedby HPLC-DAD,HPLC-ESI-MSanalysesofthecrudeextracts,orfractionsobtainedfromthem,canbecarriedout.However,thechromatographicconditionshavetobechangedastheacidcompositionofthemobilephaseforHPLC-MSanalysismustbemilderthanthoseforHPLC-DADanalysis,and0.1–0.4%formicacidshouldbeusedinstead(Salminenet al.1999).MSanalysisinthenegativemodeprovidesmoreinformationontheETandEAconjugatestructuresthanthoseinthepositivemode.
FormostETs, it ispossible toobtainm/zvaluescorresponding
to[M–H]−, [M–2H]2−,or
[2M–H]−dependingonthemassofthecompound.Often,bothmonomericanddimericETsgivethesamem/zvalues,
but the [M–H]− and the [M–2H]2− signals canbe separatedby their
isotopicm/z
values.For[M–H]−,theisotopicsignalsdifferin1m.u.,whileforthe[M–2H]2−,theisotopicdifferencesareonly0.5m.u.
ETfragmentationisproducedbythesequentiallossesofgalloylresidues(m/z152,169,or170)andHHDP(hexahydroxydiphenic)residues(m/z301).ThegalloylunitsattachedtophenolichydroxylsofothergalloylmoleculesaremorecleavableinthenegativeESI-MSthanthegalloylunitsattacheddirectlytotheglucosecore(Salminenet al.1999).Thismeansthatinthefirstcase,lossesof152unitsarefoundintheMSspectrumwhileinthelastones,lossesof169or170unitsareobservedinstead.InthecaseofETscontainingcatechinmoieties,thecharacteristiccleavagereleasesanm/zof289fromtheflavan-3-olresidue.
ForacyclicepimershavinghydroxylgroupsatC–1,
theycanbedistinguishedbythelossofwater[M–H2O]− from the
vescalagin-type ETs, while this is hardly observed from
castalagin-type ETs(MoilanenandSalminen2008).
ForETderivativeswithcatechins,itispossibletoshowtheplaceofsubstitution.Whencatechinisaddedtovescalagin-typeETs,asisthecaseofhippophaeninB,itislinkedatC-1oftheglucoseresidue,asthe[M–H2O]−signalisnotobservedandthe[M–catechin–H]−isobservedinstead.
Inaddition, ithasbeenshownthat thecatechinadditiondidnotoccurat
thecarboxyl(–COOH)group, since the MS data showed both the cleavage
of catechin and carboxyl group[M–catechin–H–COOH]2−.
CharacteristicMSfragmentationofvescalaginare:1103[M–H]−;1085[M–H2O–H]−;1041[M–H2O–COOH]−;529[M–H–COOH]2−;and520[M–H2O–H–COOH]2−.
CharacteristicMSfragmentationofhippophaeninBare:1375[M–H]−;1085[M–catechin–H]−;687[M–2H]2−;665[M–H–COOH]2−;520[M–catechin–H–COOH]2−;and289[catechin–H]−.
OligomericETscanbedetectedandcharacterizedbyLC/ESI-MS,byexaminationof
themultich-argedions,andbylookingattheisotopicions(Karonenet al.2010).
ForEAconjugates, themolecularmasses are easilyobserved in
thenegativemodeof
theHPLC-ESI-MSas[M–H]−pseudomolecularions(m/z463forEA-hexosides;m/z447forEA-rhamnosides,andm/z433forEA-pentosides),andthelossofthesugarresidue(M-162forhexosides,M-146forrhamno-sides,andM-132forpentosides)releasedtheEAmolecule(m/z301).
Inaddition,HPLC-ESI-MSallowsthedetectionofEToligomers,dimersandtrimersbeingquitecom-mon(i.e.,oenotheinAandB,respectively),andevenhexamericandheptamericETshavebeenrecentlyevidenced(Karonenet al.2010).Fortheidentificationoftheseoligomers,theuseofhigh-resolutionMS,andtheanalysisoftheisotopicpatternsareessentialforthedetectionofthepentamers,hexamers,andheptamers(Karonenet al.2010).
20.6 NMR Analysis
20.6.1 Gallotannins
Asfarasthegallotanninsareconcerned,moststudiesconcernthedetectionandidentificationofthesepolygalloylglucosesinvariousnaturalsources(e.g.,thetraditionalChineseherbGalla
chinensis)using
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448 Handbook of Analysis of Active Compounds in Functional
Foods
analyticaltechniques(Mueller-Harvey2001)suchasHPLC-MS(Salminenet al.1999;Tianet al.2009)orMALDI-TOFmassspectrometryprotocols(Xianget al.2007),orrelyingondegradationstudies,forexample,usingtannases(i.e.,galloylesterase)(Mingshuet al.2006)orperformingmildmethanolysisinmethanolicacetatebuffer(pH5.5)(Haslamet al.1961),followedbymassspectrometryanalysisoftheresidues.Severalgallotannins,fromhexa-uptotetradecagalloylglucoses,werethusobservedinsuchinvestigations,butnostructuraldeterminationofthedifferentisomerscouldbeachieved.Incontrast,puregallotannins,whichwereobtainedeitherthroughisolationfromnaturalsourcesorbyenzymaticor
chemical synthesis, could be successfully characterized by nuclear
magnetic resonance (NMR)spectroscopy.
20.6.1.1 Isolated Gallotannins
In theearly1980s,Nishiokaandcoworkersreported the
isolationofseveralpuregallotanninsfromvariousmedicinalplantextractssuchasRhus
semialata(Chinesegall),Quercus infectoria(Turkishgall), Paeoniae
albiflora (syn. P. lactiflora), and Moutan cortex (Nishizawa
et al. 1980a,b,
1982,1983a,b).Eachcomponentcouldbeseparatedfromtheplantextractaccordingtothedegreeofgalloyla-tionusingacombinationofSephadex®LH-20columnchromatographyandnormal-phaseHPLC.Thestructuresoftheresultingindividualgallotanninswerenextdeterminedmainlyby1Hand13CNMRspectroscopy.Thepositionofdigalloyldepsidemotifsontotheglucopyranosecorewasdeterminedbycomparisonofthe13CNMRchemicalshiftsrecordedinacetone-d6ofboththeglucosecarbonatomsand
the carbonyl atoms of the galloyl groups with those of β-PGG.
Typically, a downfield shift
of0.4–0.6ppmisobservedforaglucosecarbonatomlinkedtoadigalloyldepside,whichisinaccordancewiththedifferencemeasuredbetweentheestermethylcarbonsofmethylmeta-digallateandmethylgallate
(δ52.2and51.9ppm,respectively). Inaddition,
thecarbonylcarbonsignalsof theproximaldepsidically
linkedgalloylmoietieswereobservedat ca.0.6ppmupfield.These
13CNMRspectro-scopicanalysesallowedNishiokaandcoworkerstodemonstratethatthedigalloyldepsidemoietyofthehexagalloylglucoseisolatedfromtherootofPaenioae
albiflora(i.e.,P. radix)andMoutan
cortexwasattachedtotheC-6positionoftheglucopyranose(i.e.,1d),butthatofGalla
chinensiswasrandomlydistributedamongtheC-2,C-3,andC-4positions(i.e.,1b,1a,and1c,respectively)(Nishizawaet al.1980a).Similar13CNMRspectraanalysisledtothestructuraldeterminationofsixheptagalloylgluco-ses,
featuringeither twodigalloylgroups (i.e.,2a–e) orone
trigalloylmotif (i.e.,2f),
andevenoneoctagalloylglucosebearingthreedigalloylgroups(i.e.,3)(Figure20.4).Moreover,theinvestigationscarriedoutbyNishiokaandcoworkersalsorevealedthatthepreviouslyreportedmeta-depsidicdigal-loylunits(Fischer1914;Nierensteinet al.1925)areinfactequilibriummixturesofmeta-andpara-depsidically
linked galloyl units resulting from intramolecular
transesterification (Nishizawa et
al.1982).Thisconclusionwassupportedbytheanalysisandcomparisonof13CNMRspectra,runinace-tone-d6,ofpuregallotanninswiththoseofmethylmeta-andpara-digallate,notablybycarefullyexam-iningboththecarbonylandthearomaticregions.
20.6.1.2
Enzymatically and Chemically Synthesized Gallotannins
Withintheframeworkoftheirstudiesonthebiosynthesisofhydrolyzabletannins,Grossandcowork-ers
intensively investigated enzymatic synthesis of gallotannins (Gross
1999, 2008; Niemetz andGross2005).Experimentscarriedoutin
vitrowithcell-freeextractsfromleavesofstaghornsumac(Rhus typhina)
and β-PGG as a standard acceptor substrate led to the isolation of
β-glucogallin-dependentgalloyltransferases(NiemetzandGross2005;Gross2008).Itwasfoundthatnoneoftheseenzymesdisplayedhighsubstratespecificity,butsomeofthempreferentiallyacylatedβ-PGGtogivethe2-,3-,or4-O-depsidicdigalloylatedhexagalloylglucoses1a–c,whileotherspreferentiallycata-lyzed
the galloylation of hexa- and heptagalloylglucoses to furnish, for
example, 3-O-trigalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucopyranose
(2f) and higher galloylated gallotannins (Figure
20.4).Structuraldeterminationof1a–cand2fwasaccomplishedmainlyby1HNMRspectroscopy,andfurthercomparisonwiththematerialsisolatedbytheNishiokagroup.The1HNMRspectrarecorded
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inacetone-d6werecomparedwiththatofβ-PGG,andmeta-depsidicdigalloylmoietiesweretypi-callydetectedfromthediagnosticappearanceofdoubletsforthearomaticprotonsoftheproximalgalloylgroup,insteadoftheinitiallyobservedsetoftwosinglets,astheresultofthedisymmetryintroducedbytheformationofthem-depsidiclink.Inaddition,thesesignalswereshiftedfromthe7.00–7.10ppm
region (singlets) to significantlyhigherδ valuesof7.25–7.55ppm
(Hofmann1996;Gross1999).Incontrast,thearomatichydrogensofnewlyintroduceddistalgalloylresiduesgener-allydisplayedtheexpectedsharpsingletsat7.10ppm(Hofmann1996;Gross1999).Itisworthnot-ingthatnoNMRevidenceofmeta/para-depsidicdigalloylequilibriummixtureswasreportedbytheGrossgroup.
Tothebestofourknowledge,despiteafewchemicalstudiesondepsidemotifscarriedoutintheearly1900s
(Fischer 1914; Nierenstein et al. 1925), no total chemical
synthesis of “complex” (or
“true”)gallotannins,asopposedto“simple”gallotannins,thatis,theirmono-topentagalloylglucoseprecursors,hasbeenreportedthusfar.Thechemicalelaborationofmeta-depsidicdigalloylunitsacylatingaglucosecorehasbeenreportedonlybyRomaniandcoworkersintheirsynthesisofthe2,3-bis-O-digalloylglu-cose
(Arapitsas et al. 2007). They also showed that UV–visible
spectra of compounds featuring
them-depsidicdigalloylmoietydisplayacharacteristicshoulderat300nm(Arapitsaset al.2007).Inordertodeterminetheinfluenceofthegallotannindepsidiclinkonthebiologicalactivitiesofhydrolyzabletannins,Quideauandcoworkersrecentlyengagedeffortsinthetotalsynthesisofthehexagalloylglucose1a,theheptagalloylglucose2f,aswellasthedecagalloylglucose,referredtoas“tannicacid”andwhosecommercialsampleisknownfornotbeingastructurallywell-definedgallotanninbutratheracomplexmixture
of various gallotannin species and derivatives thereof
(Mueller-Harvey 2001; Romani et
al.2006).Fullcharacterizationofthechemicallypuresyntheticgallotannins1a,2f,andtannicacid(Figure20.5),aswellastheirα-anomericanalogs,wasaccomplishedby1Dand2DNMRexperiments(i.e.,1Hand13CNMR,COSYH–H,heteronuclearmultiplequantumcoherence,andheteronuclearmultiplebond
OGG
OGGOG
OGGO
GO O1d6
GGOOG
OGGO
OG
OO 1c1b 4
GGOOG
OGGO
OG
Hexagalloylglucoses
O 1a
3
GOGO
2OG
OG
OG
OGOG
OGGGGO
GGG = trigalloyldepside
OHOH
OHO
OO
OH
H
OO
O
HO
HO
GO O2f
GGO
GG = digalloyldepside
OGOGG
OHOH
OHO
OO
OH
HO
OHOH
OHO
GO
OG
OO 2c2b 4
2 3GGO
OGG
OGG
OGG
OGG
OGG
OGG
OG
OG
OG
OG
G = galloyl
OG
GO
GGO
GGO
GO
GO
GOGO
OG
Heptagalloylglucoses
Octagalloylglucoses
O
O
O
2a
2d 2e
3 3
3
6
3
3
2
6
O
2
6
2
GGOGGO
4
OG
OG
FIGURE 20.4
Selectedexamplesofdepsidicdi-andtrigalloyl-containingglucoses.
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coherence[HMBC])andmassspectrometricanalysis(Sylla2010).Themeta/para-depsidicequilibriumwasconfirmedby1Hand13CNMR,andcouldevenbeevaluatedasaca.2:1ratioby1HNMRspectro-scopicanalysis.
20.6.2 Ellagitannins
TheETsconstitutethesecondsubclassofhydrolyzabletannins,whichistodaycomposedofnearly1000membersthathavebeenidentifiedandfullycharacterizedfollowingtheirisolationfromvariousplants(HaslamandCai1994;QuideauandFeldman1996;Okudaet al.2009;Yoshidaet al.1992,2009).ThechiralityoftheircharacteristicHHDP(i.e.,6,6′-dicarbonyl-2,2′,3,3′,4,4′-hexahydroxybiphenyl)unitsisaconsequenceofthepreventionoffreerotationaroundtheirbiarylaxis,whichisimpededbythepresenceofthephenolichydroxylgroupsandtheglucose-esterifiedcarboxylgroupsortho-positionedrelativelytothatcarbon–carbonbondaxis(QuideauandFeldman;1996;KhanbabaeeandvanRee2001b).Thedeter-minationoftheconfiguration(RorS)ofthisaxialchiralityoratropisomerismisanessentialstepofthestructuralcharacterizationofETs(Figure20.6),togetherwiththedeterminationoftheregiochemistryofallgalloylandgalloyl-derivedunitsontheglucosecoreandthatofthestereochemistryattheanomericpositionofthelatter.
20.6.2.1
Absolute Configuration of ET Axially Chiral Biaryl Groups
Until1982,
theaxialchiralityofETHHDPbiarylunitswasdeterminedbyachemicaldegradationprocedurebeginningwithamethylationstepusingdimethylsulfateandpotassiumcarbonate(Tanakaet al.1986;Yoshidaet al.2000).Thepermethylatedbiarylunitwasthencleavedfromtheglucopyra-nosecorebymethanolysisusingsodiummethoxideinmethanol.Thechiralityofthehexamethoxydi-phenic
acid derivative thus released under its native atropisomeric form
was then determined bycomparison with atropisomerically pure
standards, the main drawback of this procedure being
thedegradationofthesamples.
However,since1982,amoresuitableandnondestructiveprocedurehasbeendevelopedusingcirculardichroismspectroscopytoestablishtheabsolutestereochemistryofHHDPunitslinkedtotheETglu-copyranosecore.Okudaet al.(1982a,b,1984)showedthattheCottoneffectsobservednear220–230and
HO
HO
HO
HO
HO HO
HO
HO
HO
HO
HO
OH
OH OH
OH OH
OH
OH
OH
OH
O O
O
O
O
OO
O
O
O
OO
OO
OO
OO
O O
OO
O
O
OO
H
Tannic acid(a decagalloylglucose)
H
H
HH
FIGURE 20.5 Structureoftannicacid.
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Hydrolyzable Tannins 451
250–260nm were correlated with the absolute configuration of the
HHDP units. An
(S)-configuredHHDPgroupischaracterizedbyapositiveandanegativeCottoneffectat220–230and250–260nm,respectively,whereasan(R)-configuredHHDPgroupexhibitsinsteadanegativeandapositiveeffectatthe
same values, respectively. Similar Cotton effects are also observed
in the case of other
HHDP-derivedgroupssuchastheDHHDP,chebuloyl,andsanguisorboylgroups,aswellasfortheC-glucosidicETnonahydroxyterphenoyl(NHTP)group(Yoshidaet al.2000).Moreover,thischaracteristicCottoneffectisnotinfluencedbythepositionofthebiarylgrouporbythepresenceofgalloylgroupsontheglucopyranosecore,makingthisprocedureapplicabletoanyETs(Okudaet al.1982a,b).Interestingly,ETHHDPunitslinkedtothe2,3-or4,6-positionsofa4C1-glucopyranosecore,ortothe1,6-positionsofa1C4-glucopyranosecore,arepredominantly,andtoalargeextent,(S)-configured,whilethoselinkedtothe2,4-or3,6-positionsofa1C4-glucopyranosecoreareessentiallyalways(R)-configured(QuideauandFeldman1996;KhanbabaeeandvanRee2001b).
20.6.2.2
Determination of the Position of ET Galloyl-Derived Acyl Units
ProtonandcarbonNMRanalyses,togetherwithpartialhydrolysisproceduresundermildconditions,provideuseful
structural informationonETs, such as thenature andnumberof galloyl
andgalloyl-derivedacylgroupsesterifiedtotheglucopyranosecore(e.g.,galloyl,HHDP,DHHDP,sanguisorboylgroups).However,thecrucialstepinthestructuraldeterminationofmonomericoroligomericETistoestablishthepositionofeachacylgroup.Forthispurpose,2Dlong-rangeHMBCexperimentsprovideastraightforwardassignmentbyestablishingtwokeythree-bondcorrelations;onebetweentheestercar-bonylcarbonandanaromaticprotonoftheacylunit,andanotheronebetweenthesameestercarbonylcarbonandtheprotonontheglucopyranosepositionatwhichtheesterbondisconnected,asshownonthepunicalaginstructureinFigure20.7.
20.6.2.3
Determination of the Absolute Configuration of the Anomeric Carbon
ThedeterminationoftheabsoluteconfigurationattheanomericpositionoftheglucopyranosecoreisanotherimportantpointinthestructuralelucidationofETs,especiallysinceinsomecasesbothα-andβ-anomercanbe
insolvent-dependentequilibrium, like in thecaseofpunicalagin
(Figure20.7)
(Luet al.2008).Furthermore,insomedimericETs,forexample,eachglucopyranosemoietycanpossessananomericcenterwithadifferentconfiguration.Forexample,insanguiinH-6,theglucosemoiety“1”displaysaβ-configurationatthatlocus(i.e.,equatorialorientationofthesanguisorboylgroup),whereastheglucose“2”showsanα-configuration(Figure20.8)(Tanakaet al.1985;Gasperottietal.,2010;Koolet al.2010).
OH
OHOO (S)-chebuloyl
OO
HH
HOHOOCOH
OHOH(S)-HHDP
OO
HO HO
HOOH
OHOH(R)-HHDP
OO
HO HO
HO
OHOH
OH
OHOH(S)-sanguisorboyl
OO
O
O
HO HO
HO O
OH
OH
OHOH
OH(S)-DHHDP
O
O
H
OO
O
O
H
O
HO
HOHO
HO
HO
FIGURE 20.6
Structureofthe(R)-HHPD,(S)-HHPD,(S)-chebuloyl,(S)-DHHPD,and(S)-sanguisorboylunits.
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Thestereochemistryattheanomericpositioncanbeeasilydeterminedbymeasuring,onthe1HNMRspectrum,thecouplingconstantbetweentheanomericprotonH-1andtheadjacentprotonH-2.Alargecouplingconstant(JH-1,H-2>5Hz)generallyindicatesaβ-glucosidicconfiguration(i.e.,substituentwithanequatorialorientation),whereasasmallcouplingconstant(JH-1,H-2≈0–5Hz)indicatesanα-glucosidicconfiguration(i.e.,substituentwithanaxialorientation).Thepresenceorabsenceofagalloylorgalloyl-derivedacylgroupat
theanomericpositionhasonlyaminorinfluenceonthevalueof
thisH-1,H-2couplingconstant(Table20.4).Moreover,thechemicalshiftoftheanomericprotonH-1ofanα-anomerusuallypresentsalower-fieldshiftcomparedtothatofthecorrespondingβ-anomer(Hatanoet al.1988).ThesedifferencesarealsoobservedinoligomericellagitanninssuchasthedimersSanguiinH-6andrubusuarinB,andinthetrimerslambertianinCandrubussuarinC(Gaspertottietal.,2010).Incontrast,thechemicalshiftsoftheglucopyranosiccarbonsC-1,C-2,C-3,andC-5ofanα-anomerareup-fieldedcomparedtothoseofitscorrespondingβ-anomer.Forexample,thechemicalshiftsofthecarbonsC-1,C-2,C-3,andC-5oftheα-punicalagin(i.e.,withanaxiallyorientedhydroxylgroupattheanomericposition)areshiftedupfieldby4.2,1.9,2.4,and4.8ppm,respectively,comparedwiththecorrespondingsignalsoftheβ-punicalagin(Tanakaet al.1986).
OH
OH
OHOH
OHO O
OO
O
O
O O
O
O 123
54
6
OO
O
H
H
HH
HH
H
A
B
C
D
F
E
OH
OHOH
HOHO
HOPunicalagin
HO
HO HOHO
HO
HO
FIGURE 20.7 Structureofpunicalagin.
OHHO
HO
HO
HO
HO HO
HO
HO
OHOH
OH
OH
OHOH
OH
OH
OH
OH
Glucose 2
Glucose 1
Sanguiin H-6OO
OO
O
O
OO O
OO
O
OOO
O
OOO
OOO
O
HO
HO
HOHO
HO
HO
HO
HO
HO
FIGURE 20.8 StructureofsanguiinH-6.
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20.6.2.4 About the C-Glucosidic Ellagitannins
TheC-glucosidicETsconstituteanimportantsubclassofETswiththestructuralparticularityofhavingahighlycharacteristicC–ClinkagebetweenthecarbonC-1ofanopen-chainglucosecoreandthecar-bonC-2oftheO-2galloyl-derivedmoietyofa2,3,5-NHTPunit(alsoknownastheflavogalloylgroup),suchasinvescalaginandcastalagin.ThischaracteristicC–Clinkageonanopen-chainglucosecorecanalsooccurwitha2,3-HHDPunit,suchasincasuarininandstachyurin(Figure20.9).VescalaginanditsC-1epimercastalaginwerethefirstmembersofthisETsubclasstobeisolatedfromCastanea(chestnut)andQuercus(oak)speciesin1971(Mayeret al.1967,1969,1971).However,theirstructures,aswellasthoseofstachyurinandcasuarinin(Okudaet al.1981,1982c,1983),werefullydeterminedmuchlaterwhenNishioka’sgrouprevisedtheassignmentoftheconfigurationattheC-1positionofallC-glucosidicETs(Nonakaet al.1990).ThisstructuralrevisionwasbasedontheobservationofspatialcorrelationsbetweentheprotonsH-1andH-3byNuclearOverhauserEffectSpectroscopy(NOESY)inthecorre-spondingspectraofvescalaginandstachyurin.Thesecorrelationsarenotobservedforcastalaginandcasuarinin,
forwhichtheseprotonsH-1andH-3areorientedtowardadifferentsideof
themolecule(Figure20.9).Furthermore,theobservationofsuchdiagnosticcross-peaksonthe2DNOESYspectraof
TABLE 20.4
ChemicalShiftandCouplingConstant(JH-1,H-2)oftheAnomericProtonH-1
α-Anomer β-Anomer
GeminDa 5.31(d,J=4.0Hz) 4.78(d,J=7.5Hz)TellimagrandinIa
5.57(d,J=4.0Hz) 5.13(d,J=8.0Hz)Pedunculagina 5.50(d,J=3.5Hz)
5.09(d,J=8.0Hz)Punicalaginb 5.34(d,J=3.5Hz) 5.01(d,J=8.0Hz)(see
casuarictin)Potentillinc
6.63(d,J=4.0Hz)(seepotentillin)Casuarictinc 6.22(d,J=9.0Hz)
SanguiinH-6d 6.50(d,J=3.5Hz)(glucose2)
6.01(d,J=8.5Hz)(glucose1)
a Hatanoet al.(1988).b Tanakaet al.(1986).c
Okudaet al.(1984).d Koolet al.(2010).
OH
OHHO
HO
HO
HO
HO
HO
HO
HOHO
HO
Castalagin: R1 = H, R2 = OHVescalagin: R1 = OH, R2 = H
Casuarinin: R1 = H, R2 = OHStachyurin: R1 = OH, R2 = H
OHOH
OO
OO
O
O
OO
O
OH 2
3 1456
R2R1
OH
OH
OHHO
HO
HO
HO
HO
HO
HO
HO
O
HOOH
OHOH
OO
OOO
OO
O
OH 2
3 1456
R2R1
OH
FIGURE 20.9 Structures of main monomeric C-glycosidic
ellagitannins vescalagin, castalagin, casuarinin,
andstachyurin.
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vescalaginandstachyurinwasconfirmedby1DNuclearOverhauserEffect(NOE)experiments.TheseNMRexperimentsunambiguouslyestablished
thatbothprotonsH-1andH-3areα-oriented
(R2=H);thustheC-1hydroxylgroupsofvescalaginandstachyurinareβ-oriented(Figure20.9).
However,NOESYandNOEexperimentsarenotabsolutelyrequiredfordeterminingthestereochem-istryattheC-1positionofC-glucosidicETs.Infact,thisstereochemistrycanalsobeeasilyestablishedfromthevalueofthecouplingconstantbetweentheprotonH-1andtheprotonH-2.AsfirstreportedbyNishioka’sgroup,aratherlargeH-1,H-2couplingconstant(JH-1,H-2>4Hz)indicatesanα-orientationoftheC-1
substituent, like in thecaseofcastalaginandcasuarinin,whereasa
small couplingconstant(JH-1,H-2≈
0–2Hz)correspondstoaβ-orientedsubstituent,likeinthecaseofvescalaginandstachyurin(Nonakaet al.1990).
20.6.2.5 About the Flavano-Ellagitannins
TheC-glucosidicETssubclassalsoincludestheflavano-ETs(alsoknownascomplextannins),whicharehybridentitiescomposedofaC-glucosidicETmoietyderived,forexample,fromvescalaginorstachyu-rin,
andaflavanoidmoiety (e.g.,flavan-3-ols,procyanidines,
andanthocyanins). In
theseflavano-EThybrids,thetwomoietiesareconnectedviaC–ClinkagebetweenthecarbonC-1oftheC-glucosidicETandthecarbonC-8orC-6oftheAringoftheflavanoid.Dependingonthenatureofeachmoiety,thesecomplextanninspresentalargediversityofstructuressuchasthecatechin-basedstenophyllaninsA/B(Nonakaet al.1985)andacutissiminsA/B(Ishimaruet al.1987)ortheepicatechin-basedcamelliatan-ninsA,B,andF,andmalabathrinsAandE(Hatanoet al.1991;Okudaet al.2009;Yoshidaet al.2009).Moreover,thesecomplextanninscanbefurthertransformedbyoxidativemeanstogeneratedifferenttypesofderivativessuchasthemongolicainsA/B(Nonakaet al.1988),whichcontainao,m-hydroxy-phenylcyclopentenonemotif
(alsoknownas thedihydrofurangroup).Recently,new
typesofcoloredflavano-ETs,namedanthocyano-ETs,wereobtainedbyhemisynthesis
in aqueousacidicmedia
fromvescalaginandtheanthocyaninoeninoritsmalvidinaglycone(Quideauet al.2005;Jourdeset al.2009;Chassainget al.2010).Allof
theisolatedandcharacterizedflavano-ETstodatepresentaβ-orientedlinkagebetweentheflavanoidunitandtheC-glucosidicETmoiety.Theseβ-configurationsweredeter-mined
by the observation of a small coupling constant between the proton
H-1 and the proton
H-2(JH-1,H-2≈0–2Hz).SuchastereoselectivitywasrecentlyrationalizedthroughmolecularmodelingstudiesbytheQuideaugroup(Quideauet al.2003,2005).
AfterhavingcharacterizedtheC-glucosidicETmoietyusingsomeofthestrategieshighlightedabove,thecrucialelementofthestructuralelucidationofaflavano-ETstructureistoestablishbywhichAringcarbon
(C-8′ or C-6′) the flavanoid unit is connected to the carbon C-1 of
the C-glucosidic ET
unit(Okudaet al.2009;Yoshidaet al.2009).Thisconnectivitycanbeestablishedbytheobservationoftwo-andthree-bondHMBCcorrelationsbetweenprotonH-1andcarbonsC-7′,C-8′,andC-8′ainthecaseofaC-8′/C-1linkage,whereasaC-6′-linkedflavanoidunitwillshowcorrelationsbetweenprotonH-1andcarbonsC-5′,C-6′,andC-7′(Figure20.10)(Jourdeset al.2009;Quideauet al.2003,2005).Moreover,inorder
to establish this connectivity unambiguously, the HMBC data can be
supported by those ofRotational Nuclear Overhauser Effect
Spectroscopy (ROESY) experiments that reveal, for
example,through-spaceconnectivitiesbetweentheprotonH-2′andH-6′oftheB-ringoftheflavanoidunitandtheprotonH-1,H-2,andH-3oftheC-glucosidicETunit.Suchspatialcorrelationsareonlypossibleinthecase
of the C-8′/C-1 linkage between the flavanoid and C-glucosidic ET
units (Figure 20.10).
SuchROESYexperimentswereusedtoconfirmthestructureofthecamelliatanninsAandB(Hatanoet al.1991),aswellasthatofthehemisynthesizedanthocyano-ETs(Chassainget al.2010).
Hatanoet al.(1995)alsousedadifferentstrategytoestablishtheconnectivitybetweenanepicatechinunitandaC-glucosidicETunit(i.e.,5-O-desgalloylstachyurin-derivedunit)inthecaseofthecamellia-tanninsCandEisolatedfromCamellia
japonicaleaves(Figure20.11).Aftermethylationofallphenolichydroxylgroupsusingdimethyl
sulfate andpotassiumcarbonate in
acetone,NOEexperimentswereperformedbysuccessivelyirradiatingtheflavanoidA-ringmethoxygroupsatpositionsO-5′andO-7′.TheseexperimentsrevealedthatforcamelliatanninEinwhichtheepicatechinunitisconnectedbyitsC-8′centertothe5-O-desgalloylstachyurin-derivedunit,theirradiationofbothmethoxygroupsatO-5′andO-7′resultsinNOEsignalswiththeA-ringaromaticprotonH-6′(Hatanoet al.1995).Incontrast,
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Hydrolyzable Tannins 455
inthecaseofcamelliatanninC,onlytheirradiationofthemethoxygroupatO-7′gaveanNOEsignalwithanA-ringaromaticproton(i.e.,H-8′),thusestablishingthattheepicatechinunitislinkedtothecarbonC-1oftheC-glucosidicETunitbyitsA-ringcarbonC-6′(Figure20.11).
ACKNOWLEDGMENTSWearegratefultoUrskaVrhovsek,DomenicoMasuero,andMattiaGasperottifortheircollaborationinthisresearch,andtoLaraGiongoandMarcellaGrisentiforprovidingtheblackberrysamples.ThisworkhasbeensupportedbytheSpanishMICINN(ConsoliderIngenio2010-Fun-C-FoodCSD2007-0063)andFundaciónSenecadelaRegiondeMurcia(grupodeexcelenciaGERM06,04486).PilarTruchadoholdsaPhDgrantfromtheSenecaFoundation(Murcia,Spain).StéphaneQuideaualsowishestothanktheConseil
InterprofessionnelduVindeBordeauxandtheConseilRégionald’Aquitaine(Bordeaux,France)fortheirfinancialsupportofhisteam’sresearchonhydrolyzabletannins.
Camelliatannin C: R = HHexadeca-O-methylcamelliatannin C: R =
Me
Camelliatannin E: R = HHexadeca-O-methylcamelliatannin E: R =
Me
O
ROORRO
O
OROR
OR
OO
OO
OR
OO
ORRO
RO
OR
RO
HO OR
O
OH
OR
RO
HOH
HH
A
C
B
8' 6'
5'
7'
OR
O
ROORRO
O
OROR
OR
OO
OO
OR
OO
ORRO
RO
OR
RO
HO
HOH
H
O
OR
OR
OHOR
ROH
AC
B8'
6' 5'
7'
FIGURE 20.11
StructuresofcamelliatanninsCandE,aswellastheirpolymethylatedderivativeshexadeca-O-methyl-camelliatanninsCandE.
Camelliatannin A 1-Deoxyvescalagin-(1β → 8)-oenin: R =
β-D-glucose
OO
OO
OH
OHOH
O
HO
OO
OHHO
HO
HO
HO
HO
OH
O
OHHO
OH
O
OH
OH
HO
18'
HO
H
H
H2
345
6
H A
C
B
6'
5'
7'
8'a
6"
2"
OO
OO
OH
OHOH
O
HO
OO
OHHO
HO
HO
HO
O
HO
O O
OHHO
HO
HO
HO
OH
O
OR
OH
OMeHO
MeO
12
345
6
H
H
HH
+
8' 6'
5'
7'
8'a
6"
2"H2'
A
C
B
FIGURE 20.10
StructuresofcamelliatanninAand1-deoxyvescalagin-(1β→8)-oenin.
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Acknowledgments20.1 Introduction20.2 HPLC Analysis20.3 UV
Spectrophotometry Detection20.5 HPLC-MS-MS Analysis20.6 NMR
AnalysisREFERENCES学霸图书馆link:学霸图书馆