Natural deep eutectic solvents (NADES) made mainly with abundant primary metabolites are being increasingly applied in green chemistry. The advantages of NADES as green solvents have led to their use in novel green products for the food, cosmetics and pharma markets.
Trang 1j ou rn a l h om ep a ge :w w w e l s e v i e r c o m / l o c a t e / c h r o m a
method
Xiaojie Liua, Samantha Ahlgrena,b, Henrie A.A.J Korthoutc, Luis F Salomé-Abarcaa,
Lina M Bayonaa, Robert Verpoortea, Young Hae Choia,d,∗
a Natural Products Laboratory, Institute of Biology, Leiden University, 2333 BE Leiden, The Netherlands
b Division of Pharmacognosy, Faculty of Pharmacy, Uppsala University, 751 05 Uppsala, Sweden
c Fytagoras BV, 2333 BE Leiden, The Netherlands
d College of Pharmacy, Kyung Hee University, 02447 Seoul, Republic of Korea
a r t i c l e i n f o
Article history:
Received 16 October 2017
Received in revised form 3 December 2017
Accepted 4 December 2017
Available online 6 December 2017
Keywords:
Natural deep eutectic solvents
Chemical profiling
Ginkgo
Ginseng
High-performance thin-layer
chromatography
a b s t r a c t
increasinglyappliedingreenchemistry.TheadvantagesofNADESasgreensolventshaveledtotheir useinnovelgreenproductsforthefood,cosmeticsandpharmamarkets.However,oneofthemain diffi-cultiesencounteredinthedevelopmentofnovelproductsandtheirqualitycontrolarisesfromtheirlow vapourpressureandhighviscosity.Thesefeaturescreatetheneedforthedevelopmentofnewanalytical methodssuitedtothistypeofsample.Inthisstudy,suchamethodwasdevelopedandappliedtoanalyse theefficiencyofadiversesetofNADESfortheextractionofcompoundsofinterestfromtwomodel plants,GinkgobilobaandPanaxginseng.Themethoduseshigh-performancethin-layerchromatography (HPTLC)coupledwithmultivariatedataanalysis(MVDA).Itwassuccessfullyappliedtothe compara-tivequali-andquantitativeanalysisofverychemicallydiversemetabolites(e.g.,phenolics,terpenoids, phenolicacidsandsaponins)thatarepresentintheextractsobtainedfromtheplantsusingsixdifferent
molarratios;malicacid-cholinechloride(1:1),malicacid-glucose(1:1),cholinechloride-glucose(5:2), malicacid-proline(1:1),glucose-fructose-sucrose(1:1:1)andglycerol-proline-sucrose(9:4:1).Ofthese mixtures,malicacid-cholinechloride(1:1)andglycerol-proline-sucrose(1:1:1)forG.bilobaleaves,and malicacid-cholinechloride(1:1)andmalicacid-glucose(1:1)forP.ginsengleavesandstemsshowed thehighestyieldsofthetargetcompounds.Interestingly,noneoftheNADESextractedginkgolicacids
asmuchastheconventionalorganicsolvents.Asthesecompoundsareconsideredtobetoxic,thefact thattheseNADESproducevirtuallyginkgolicacid-freeextractsisextremelyuseful.Theeffectofadding differentvolumesofwatertothemostefficientNADESwasalsoevaluatedandtheresultsrevealedthat thereisagreatinfluenceexertedbythewatercontent,withmaximumyieldsofginkgolides,phenolics andginsenosidesbeingobtainedwithapproximately20%water(w/w)
©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Natural products (NPs) are undoubtedly the most plentiful
sourceofnewbioactivecompoundsandplay animportantrole
in our daily lives, being used for their medicinal, nutritional
andcosmeticproperties,andinindustrialapplications.However,
∗ Corresponding author at: Natural Products Laboratory, Leiden University, 2333
BE Leiden, The Netherlands and College of Pharmacy, Kyung Hee University, 02447
Seoul, Republic of Korea.
E-mail address: y.choi@chem.leidenuniv.nl (Y.H Choi).
extractionfromtheirnaturalsourcesisgenerallyacomplex pro-cess, consisting of several steps that involve the use of large volumesoforganicsolvents.Unfortunately,mostofthesesolvents arebannedintheuseofproductsforhumanconsumptiondueto theirtoxicityorareextremelyrestricted.Ingeneral,thesesolvents arealsohighlyvolatile,posingashazardstotheenvironment.For thesereasons,thechoiceofsuitablesolventsisverylimited[1] Thegreen extractionof NPscanbeachievedby using inno-vative extraction techniques and/or sustainable alternatives to conventional solvents Great improvements have been accom-plished with the use of non-conventional techniques such as ultrasound-assisted extraction, microwave-assisted extraction, https://doi.org/10.1016/j.chroma.2017.12.009
0021-9673/© 2017 The Authors Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
Trang 2thesetechniquesevenallowforsolvent-freeextraction,including
microwavehydro-diffusionandgravity,enzyme-assisted
extrac-tion[3]andthermaldesorptionsystems.However,forlarge-scale
extraction, theuse ofsolvents ispractically unavoidable hence
it is necessary tofind alternatives tothe conventional organic
solvents and/or mineralacids Amongst the green options that
have appeared sofar, themost popularare super- or
subcriti-calfluidsandionicliquids.Supercriticalfluidextraction(SFE),in
particular withsupercritical carbon dioxide (SC-CO2), hasbeen
usedextensivelyforyearstoextractmanynaturalbioactive
com-poundsoreliminatetoxiccompoundsfromavarietyofmaterials
[4–6].However,despiteitsenvironmentalbenefits,theefficiency
of CO2 for the extraction of polar/hydrophilic compounds and
macromoleculesislimitedandmanybioactiveornutritional
com-pounds,suchasphenolicglycosidesoralkaloids,arenotextractable
due to their poor solubility in CO2 Other alternatives in the
green list include conventional solvents such as water, certain
agro-solvents(e.g.,ethanolandglycerol)andsurfactantaqueous
solutions[7,8]
Anothertypeofnovelalternativesolventsthathavegained
con-siderableattentionwithinthepastfewyearsareionicliquids(ILs)
ILsaresaltsthatconsist ofcertaincombinationsof cationsand
anionswithmelting pointsthatarebelow100◦C.Theseliquids
possessmanyattractivephysicochemicalpropertiessuchaslow
volatility,someelectrolyticconductivityandtunableviscosityand
miscibility,makingthempromisingsubstitutesfororganicsolvents
in numerous processes like biocatalytic processes, extractions,
catalysis, and electrochemistry [9–11] However, their toxicity,
poorbiodegradabilityandthehighcostofsynthesisingtheirmajor
componentscausemajorhindrancefortheirwidespread
applica-tionasextractionsolvents
Anotheroption,deepeutecticsolvents(DES),aremixturesof
specific organiccompounds (not necessarily ionic)that have a
muchlowermelting pointthaneitheroftheindividual
compo-nentsand,similarly toILs,areliquidsat ambienttemperatures
[12].DESexhibitsimilarphysicochemicalpropertiesascommonly
usedILs(e.g.,highdensityandviscosity)buthavetheadvantages
ofbeingsignificantlycheaper,easiertoprepareandlessimpactful
ontheenvironment.Inadditiontothis,theycanbetailoredtobe
target-specific.Theseuniquepropertiesallowforawiderangeof
applicationsofDESinfieldssuchasextraction,catalysis,materials
chemistry,organicsynthesis,metalprocessing,and
electrochem-istry[13,14],aswellasintheextractionofDNAandasaMediafor
enzymaticreactions[15,16]
AnumberofobstaclesremainintheapplicationofDES,
particu-larlyinthosemadewithsyntheticcomponentsinwhichtoxicity
is anissue Searchingfor alternatives,Choi and hisco-workers
haveexploredmorethan100combinationsofDESusingonly
com-monmetabolitesthat areabundantinplants [17] Theynamed
thesecombinations;‘naturaldeepeutecticsolvents’(NADES)[17]
Theseliquidsarebio-basedDES which arecomposedof twoor
morecompoundsthataregenerallyfunctionalprimary
metabo-lites,i.e.,organicacids,sugars,(poly)alcohols,aminesandamino
acids[17,18].Apartfromsharingthefavorablecharacteristicsof
ILsandDESasdescribedpreviously,NADEShaveadditional
advan-tagesofbeingcomposedofnaturally-occurringcompounds,being
muchmoresustainableandposingpracticallynoenvironmental
hazards.ThishighlightsthegreatpotentialofNADESforits
applica-tioninvariousareas,namelyintheNPfieldasanextractionsolvent
Currently,themoststudiedapplicationofNADESassolventsfor
NPsistheextractionofdifferentphenoliccompoundsfromplant
materialsuchasCarthamustinctoriusflowers[19],Sophorajaponica
flowers[20],Cajanuscajanleaves[21],Catharanthusroseus
flow-ers[22],grapepeel[23],andanthocyaninsfromwine lees[24]
Theyhave also beenappliedfor the extractionof ginsenosides
fromPanaxginseng[25]andfoodcontaminants[26].Recently,the firstcommercialNADESplantextractswerelaunchedascosmetic ingredientsandinthecomingyears anumberofnovel NADES-basedproductscanbeexpectedinthefood,cosmeticandpharma markets
Given the increasing amount of NADES applications, the methodologicalproblemsrelatedtotheanalysisofNADESextracts mustbeaddressed.Theseproblemsarerelatedmainlytothe neg-ligiblevolatilityofNADESthatmakestherecoveryofcompounds fromNADESextractsortheeliminationofinterferingNADES com-ponents very difficult Sofar,most analysesof NADES extracts havebeenfocusedonphenolicsthatposefewmethodological chal-lengesgiventheirhighly-absorbingchromophoresthatallowdirect UV-spectrophotometricanalysiswithfewtonosampleclean-up procedures.Differentchromatographicmethodshavebeenusedto identifyandquantifyindividualcomponents,includingthe deter-minationoftheanthocyanincontentinflowerpetalextractsfrom
C.roseus[19]andflavonoidsinflowerextractsfromS.japonicavia HPLC-DAD,usinganti-solventstrategiesforrecovery[20] Along-sidechromophorecompounds,volatileshavebeenrelativelyeasily analysedbyGCfollowing simpleliquid-liquidextractionfroma cholinechlorideand4-chlorophenol(1:2)NADES[27].However, noneofthesemethodscanbeappliedtothefullrangeofvery chem-icallydiversecompoundsthatarepresentinNADESextractsfrom biologicalmaterials
Withinthepastfivedecades,thinlayerchromatography(TLC) hasadvancedgreatlyfromasimpleplanarchromatographic tech-niqueformajorqualitativeprofilingintoamulti-targetquantitative analytical tool withmany applicationsin thefieldof medicinal plants The main disadvantages of TLC, suchas its low resolu-tionandpoorquantitativeperformance,havebeenconsiderably improved by the optimisation of the conventional TLC set-up Nowadays, the basic steps of TLC-based methods, i.e., sample application,chromatographicdevelopmentanddetection,canbe automatedandcomputer-controlled.Otherundesirable character-istics,suchaslow efficiency,havebeenimprovedwiththeuse
of high-performancethin-layerchromatography (HPTLC)plates, whichusestationaryphaseswithsmallerparticlesizes(5–6M) and high-resolutionsorbentswithimprovedsilica particlesand chemically-modifiedphases.Thesemodernsystemsfeaturehigh reproducibility in retention, detection and quantitative analy-sis For further statistical evaluation, electronic images of the chromatogramscanbegeneratedandusedformultivariatedata analysis.Asfortheidentificationofcompounds,notableprogress hasbeenmadeinthedirectonlinecouplingofHPTLCwithmass spectrometry(MS)[28–31]whichintroducesnewopportunities fortheapplicationofplanarchromatographyinmetabolomics Combiningalloftheseimprovementswiththeadvantagesof multi-sampleanalysisinasinglerunandtheoptionforselective detectionofawiderangeofchemicallydiversecompoundsmakes HPTLCausefultoolforthequalitativeandquantitativeanalysisof NADESextractsfromnaturalproducts.Furthermore,the applica-tionofmultivariateimageanalysistoHPTLCchromatogramsallows fortheextractionofincalculablymoreinformationthanthatmade availablebysimplevisualinspection[31]
Theaimofthecurrentstudywastodevelopasuitableanalytical protocolfortheHPTLCanalysisofNADESplantextractsandthen
toimplementittoinvestigatetheapplicationofdiverseNADES (includingavariationinwatercontent)withtheextractionofthree chemicallydifferentgroupsofactivecompoundsinGinkgobiloba (i.e.,ginkgolides,phenolicsandginkgolicacids)[33]andof ginseno-sidesfromPanaxginseng[34–37].Theinfluenceofadjustmentsto thewatercontentontheextractionabilityandefficacyofthemost efficientNADESwasalsostudiedduetoitsknownimpactontheir physicochemicalproperties
Trang 32 Materials and methods
2.1 Plantmaterial
GinkgobilobaleaveswerecollectedinSeoul,RepublicofKorea
in2012.Panaxginsengleavesandstemswerekindlyprovidedby
Fytagoras(Leiden,theNetherlands).Samplesusedforthisstudy
wereidentifiedbyoneoftheauthors,Dr.Y.H.Choiandavoucher
samplewasdepositedintheNaturalProductsLaboratory,Institute
ofBiology,LeidenUniversity.Thedryplantmaterialwaspowdered
inablenderwithliquidnitrogen
2.2 Chemicalsandreagents
Bilobalide,ginkgolides(A,BandC),ginkgolicacids(C13:0,C15:1
and C17:1) and ginsenosides (Rb1, Rb2, Rb3, Re, Rg1,Rg2 and
Rg3)were purchased fromBiopurify Phytochemicals (Chengdu,
China).Rutin,chlorogenicacidandquercetinwerepurchasedfrom
Sigma(St.Louis,MO, USA).Methanol,ethanol, chloroform,
ace-tone,ethylacetateandtolueneofanalyticalgradewerepurchased
fromSigma.Aceticanhydride, aceticacidand formicacidwere
obtainedfromSigma.Milli-Qwaterwasused.Polyethylene
gly-col400was purchased from Alfa Aesar(Kandel, Germany) All
ofthe NADEScomponents,i.e., cholinechloride (≥98.0%),
glyc-erol(≥99.5%),L-proline(≥99.0%),d-fructose(≥99.0%),d-glucose
(≥99.5%),malicacid(≥99.0%),andsucrose(≥99.5%)wereobtained
fromSigma.Sodiumacetateand2-aminoethyldiphenylborinate
werepurchasedfromSigma.Solidphaseextraction(SPE)cartridges
(OASISHLB3cc)werepurchasedfromWaters(Milford,MA,USA)
Silicagel60F254HPTLCplateswerepurchasedfromMerck
(Darm-stadt,Germany)
2.3 NADESpreparation
TheNADESemployedinthisstudywerecombinationsoffixed
molarratiosasfollows;malicacid-cholinechloride(1:1,N1),malic
acid-glucose(1:1,N2),choline chloride-glucose(5:2,N3), malic
acid-proline(1:1, N4), glucose-fructose-sucrose(1:1:1, N5) and
glycerol-proline-sucrose(9:4:1,N6)(TableS1).ThefirstfiveNADES
werepreparedbystirringmixturesoftheircomponentsat50◦C
until a clearliquid wasformed [19], whereas
glycerol-proline-sucrose(9:4:1)waspreparedusingthefreeze-dryingmethod[25]
AllofthepreparedNADESweremixedwith10%(w/w)water.The
twomostefficientNADESfor theextractionofeachplantwere
foundtobemalicacid-cholinechloride(1:1)and
glycerol-proline-sucrose(9:4:1)forG.biloba,andmalicacid-cholinechloride(1:1)
and malic acid-glucose (1:1)for P.ginseng.These NADES were
selectedtofurtherstudytheeffectofaddingvaryingamountsof
water(0%,10%,20%,30%and40%,w/w)ontheirextraction
prop-erties
2.4 PreparationofreferencecompoundsolutionsforHPTLC
analysis
Allreferencesolutionswerepreparedseparatelyinmethanol
in the following concentrations: bilobalide and ginkgolides B
and C (250g/mL); ginkgolide A and C13:0, C15:1 and C17:1
ginkgolicacids(125g/mL); rutin(1.8g/mL);chlorogenicacid
andquercetin(4g/mL);ginsenosidesRb1,Rb2,Rb3,Re,Rg1,Rg2,
andRg3(30g/mL)
2.5 PreparationofextractsandsamplesolutionsforHPTLC
analysis
Powdered plant material (200mg) was mixed with 4mL
methanolorNADESina15-mLcentrifugationtube.After
vortex-ingfor1min,themixturewasplacedintoawaterbathat40◦Cfor
1handthenultrasonicatedatroomtemperaturefor30min.The mixturewasthencentrifugedat13,000rpmfor20min.Analiquot
of1mLofthesupernatantwasusedfortheHPTLCanalysis follow-ingpre-treatment.Allextractswerepreparedbytriplicate.Extracts preparedwithmethanolwereusedtocomparetheyieldofthe diverseNADESextracts
2.6 RecoveryofsamplesfromNADES TheremovalofNADESfromthemixturewasconductedwith solid-phaseextraction(SPE)usingHLBcartridges[25].Briefly,a cartridgewasplacedinavacuummanifoldandequilibratedwith
5mLofethanol,followedby5mLofwater.Afterloadingtheextract solution(1mL),thecartridgewassubsequentlyrinsedwith6mL
ofwatertwiceandthenelutedwith6mLofethanol.Theethanol eluatewasdriedandre-dissolvedin1mLofmethanolforHPTLC analysis
2.7 GeneralHPTLCanalysis Referenceandsamplesolutions(100L)werespottedby trip-licateonHPTLC Si60 F254,20×10cm (Merck) plates as7mm bands,underastreamofnitrogen,usingtheCAMAG Automatic TLCsampler(ATS4)(CAMAG,Muttenz,Switzerland)witha100L Hamiltonsyringe.TheCAMAGauto-samplesystemiscontrolledby WINCATSsoftware.Allplateswerepreparedsimilarly,spottingthe methanolextractsinthefirstthreelanes,thereferencesolutions
inthenextthreeandthentheNADESextracts
2.8 AnalysisofNADESextractswith10%water(w/w) ThelayoutofthetracksontheHPTLCplatesvariedineachcase For theginkgolidesinG biloba,a total of 13trackswereused, includingtriplicatesofthemethanolextract,fourreferencesand sixdifferentNADESextracts.Bandswereappliedatadistanceof
10mmfromthebottomoftheplateand16mmfromtheleftand therightedges.Forthedeterminationofphenolicsandginkgolic acidsinG.biloba,thenumberoftracksperplatewas12,i.e., tripli-catesofmethanolextract,threereferencesandsixdifferentNADES extracts.Bandswereappliedatadistanceof10mmfromthe bot-tomoftheplateand20mmfromtheleftandtherightedges.For ginsenosidesinP.ginsengleavesandstems,thenumberoftracks perplatewas17,i.e.,duplicatesofmethanolextractofleavesand stemsrespectively,onereferenceandsixdifferentNADESextracts
ofleavesandstemsrespectively.Bandswereappliedatadistance
of10mmfromthebottomoftheplateand18mmfromtheleftand therightedges
2.9 TheuseofNADESwithdifferentwatercontents TheextractsobtainedfromG.bilobamaterialwiththemost effi-cientNADES(N1andN6,seeabove)withdifferentaddedwater contents werethen compared witheach other and againstthe methanolextracts.Inthecaseofginkgolides,thisresultedin17 tracksperplatewith16usedforthephenolicsandginkgolicacids, i.e.,triplicatesofthemethanolextract,threereferences,andthe twoNADESwithfivedifferentwatercontents.Bandswereapplied
atadistanceof10mmfromthebottomoftheplateand16mmfrom theleftandtherightedges.WiththeNADESextractionof ginseno-sidesfromP.ginsengleavesandstems,theeffectofaddedwater contentswasstudiedusingthemostefficientNADESinthiscase, i.e.,N1andN2.Therewere14tracksoneachplateincluding trip-licatesofthemethanolextract,onereference,andtheN1andN2 extractswithfivedifferentwatercontents.Bandswereappliedata distanceof10mmfromthebottomoftheplateand18mmfromthe
Trang 4werethosedescribed forG.bilobaandP.ginsenginthe
applica-tionnotesofCAMAGlaboratory(F-16A,F-16B,F-16C)[38]andthe
ChinesePharmacopeia[39],respectively(TableS2).Derivatisation
reagentswereappliedwithanauto-sprayinginstrument
(Deriva-tizer,CAMAG)
2.10 DeterminationofginkgolidesinGinkgobilobaleaves
Priortosamplespotting,theplateswereimmersedinan
ethano-licsolutionofsodiumacetate(8gNaOAcin200mLof80%aqueous
ethanol) for 2s, allowed todry in the hood for 5min atroom
temperatureandthenactivatedat90◦C for30minasindicated
intheapplicationnotesofCAMAG laboratory(F-16A)[38]
Vol-umesof25Lofmethanolextractsandreferencesolutions,and
80Lof NADESextractsolutions werespotted ontotheHPTLC
plateasdescribed.Theplatewasdevelopedinasaturatedchamber
withamobilephase oftoluene-ethylacetate-acetone-methanol
(20:10:10:1.2, v/v/v/v) After drying the plates to remove the
mobilephase,theywereevenlysprayedwithaceticanhydrideand
heatedat180◦Cfor10min.Densitometricscanningwasperformed
atUV366nm.BilobalideandginkgolidesA,BandCwereusedas
referencecompounds
2.11 DeterminationofphenolicsinGinkgobilobaleaves
Avolumeof25Lofeachreferencesolution,methanolextract,
andNADESextractswasappliedontotheHPTLCsilicaplateand
developedwithamobilephaseconsistingofethylacetate-acetic
acid-formicacid-water(100:11:11:27,v/v/v/v)inachamber
sat-uratedfor20minbeforeuse,accordingtotheapplicationnoteof
CAMAGlaboratory(F-16B)[38].For thederivatisation,theplate
washeatedat100◦C for3minonaTLCPlateHeater(CAMAG),
thendipped firstin NaturalProducts reagent(1%2-aminoethyl
diphenylborinateinmethanol),driedwithcoldair,andthen
sub-sequentlydippedinPEGreagent(5%polyethyleneglycol400in
dichloromethane).DensitometricscanningwasperformedatUV
366nmafterderivatisation.Rutin,chlorogenicacidandquercetin
wereusedasreferencecompounds
2.12 DeterminationofginkgolicacidsinGinkgobilobaleaves
Avolumeof25Leachofreferencesolutionsofginkgolicacids
(C13:0,C15:1 andC17:1),themethanolextract andtheNADES
extracts was spotted separately onto the plate and developed
withamobilephaseconsistingoftoluene-ethylacetate-aceticacid
(8:2:0.2,v/v/v)inasaturatedchamberasindicatedintheF-16-C
oftheapplicationnotesofCAMAGlaboratory[38].Theplatewas
driedwithastreamofcoldairandscannedatUV366nm
2.13 DeterminationofginsenosidesinPanaxginsengleavesand
stems
The plate was developed with chloroform-ethyl
acetate-methanol-water(15:40:22:10)inachambersaturatedfor20min
[39].Ginsenosidesweredetectedbysprayingtheplatewithfreshly
preparedanisaldehyde-sulfuricacidreagentandheatingonaplate
heaterat105◦Cfor5min.Imageswerecapturedunderwhitelight
afterderivatisation.GinsenosidesRb1,Rb2,Rb3,Re,Rg1,Rg2and
Rg3wereusedasstandards
2.14 Imageprocessingandmultivariateanalysis
TheHPTLCchromatogramswereprocessedusingrTLCsoftware
[32]whichconvertstheHPTLCimagesintoanumericaldatamatrix
whichisthenintegrated.Thisprocessgenerateddigitaldataof50
sequential0.02binsoverthefullretentionfactors(Rf)rangefor eachtrack.AnRfof0.02wasselectedasthebinsizebecauseit rep-resentstheindividualbandinasinglebinbutavoidstheRfdrift whichmayresultfrombatch-to-batchvariationfactors.Toreduce thevariationsbetweenthereplicatesthatwereperformedat differ-enttimes,theintensityrecordedforeachRfvaluewasnormalised withrespecttothemethanolextract(referenceextractsample)in eachplate.These0.02Rfbinsweresubjectedtomultivariatedata analysis.Principalcomponentanalysis(PCA)andorthogonal par-tialleastsquare(OPLS)wereperformedwiththeSIMCA-Psoftware (v.14.1,Umetrics,Umeå,Sweden).Theunitvariance(UV)scaling methodwasusedbothforPCAanalysisandOPLSmodelling
3 Results and discussion
ThisstudywasperformedusingsixtypesofNADEStoextract fourgroupsofchemicallydiversebioactivecompounds(phenolics, terpenoidsandphenolicacidsfromG.bilobaleaves,and ginseno-sidesfrom P.ginsengleavesand stems) TheNADES have been grouped into fivetypical types according totheir components, i.e., NADES composedof acids and bases (N1), acids and sug-ars(N2),bases andsugars(N3),aminoacidand acids(N4),and sugarmixtures(N5).InadditiontothesetypicalNADES, glycerol-proline-sucrose(9:4:1,N6)wasselectedbecauseinpreviouswork performedbyJeongandherco-workers,itwasreportedthat gin-senosideswerewell-extractedfromP.ginsengrootsbytheNADES [25].ToreducethehighviscosityoftheNADESasanextraction sol-vent,10%ofwater(w/w)wasaddedtoeachNADES,assuggested
inapreviouslypublishedpaper[18] SimilarlytoconventionalsyntheticDESorILs,NADESextracts have virtually zero vapourpressure which means that the sol-ventcannotberemovedbyevaporationasis generallythecase whenorganicsolventsareusedforextraction.Theremovalofthe extractionsolventisgenerallynecessarywhenanalysinganextract, concentratingthelow-levelcompoundsoravoidingitsinterference withtheanalysis.InthecaseofNADES,twoapproacheshavebeen proposed,namelyliquid-liquidpartitioning[27]andsolidphase extraction(SPE)withdiversesorbents[20,25].However,neither methodcompletelyremovedalloftheNADES.Liquid-liquid parti-tioningisonlypossiblewithnon-polarsolventssuchasn-hexane, dichloromethaneorchloroformbecausemostNADEScomponents aresolubleinpolarormid-polarsolvents,makingitdifficultto sep-aratethemfromtheextractedpolarsecondarymetaboliteswith similarpolarities.Ontheotherhand,SPEhasprovedtobequite effi-cientinpurifyingsecondarymetabolites,thoughNADESresidues maystillcauseproblemsinvariousanalyticalmethods
So,thereisaclearneedforefficientmethodsthatcanbeapplied
tothevastrangeofchemicallydiversecompoundsfoundinNADES extracts.ThinlayerchromatographyhasalongtraditioninNP anal-ysisand,inthepastdecade,hasevolvedintoahighlyimproved techniqueknownasHPTLC[40].Thistechniquemeetsallofthe mentionedrequirementstoagreatextent.Inparticular,the pos-sibilityofthesimultaneousanalysisofseveralsamplesonasingle plateandthepossibilityofapreparativeworkbymassand/orNMR spectroscopyconstituteagreat advantageoverother chromato-graphicmethods[41,42]
TodevelopHPTLCprotocolsfortheanalysisofNADESextracts,
we usedtwo well-knownmedicinalplants asmodels,G biloba andP.ginseng.Theplantmaterialwasextractedwithsixdifferent typesofNADES.TheseextractswerethenanalysedbyHPTLCfor thepresenceoffourdifferenttypesofcompounds;phenolics, ter-penelactonesandalkylphenolsinG.bilobaleaves,andtriterpene saponinsinP.ginsengleavesandstems
TheNADESextractswerefirstanalyseddirectly,withoutany pre-purificationsteps,toevaluatetheinterferenceoftheNADESin
Trang 5Fig 1.High-performance thin-layer chromatographs (HPTLC) of methanol extracts and six natural deep eutectic solvent (NADES) extracts with 10% (w/w) water for ginkgolides (a), ginkgo phenolics (b), and ginkgolic acids (c) of Ginkgo biloba leaves, and ginsenosides of Panax ginseng stems (d) and leaves (e) Ginkgolides were ana-lysed after derivatisation with acetic anhydride at 366 nm using plates that were impregnated with an ethanolic solution of sodium acetate (see details in M&M) Ginkgo phenolics were detected after derivatisation with the natural products reagent (NPR) and PEG 4000 at 366 nm Ginsenosides were visualised under white light after treatment with the anisaldehyde-sulfuric acid reagent The detailed HPTLC conditions were provided in the experimental section M: methanol extract, N1-N6: NADES extracts N1: malic acid-choline chloride (1:1), N2: malic acid- glucose (1:1), N3: choline chloride-glucose (5:2), N4: malic acid-proline (1:1, molar ratio), N5: glucose-fructose-sucrose (1:1:1), N6: glycerol-proline-sucrose (9:4:1) Reference compounds: 1: bilobalide, 2: ginkgolide A, 3: ginkgolide B, 4: ginkgolide C, 5: quercetin, 6: chlorogenic acid, 7: rutin, 8: ginkgolic acids (C13:0, C15:1 and C17:1), 9: ginsenoside Rg3, 10: ginsenoside Rg2, 11: ginsenoside Rg1, 12: ginsenoside Re, 13: ginsenoside Rb3, 14: ginsenoside Rb2, 15: ginsenoside Rb1.
theHPTLCmethodsforthefourdifferentgroupsofNPs.The
pres-enceofNADEScausedseveretailingofspotsinallofthesystems
Itwasclearlynecessarytoperformsomesampleclean-up
proce-dures,henceSPEwasselectedasthemethodofchoicefor this
Becauseoftheirabilitytobindawiderangeofsecondary
metabo-lites,includingglycosides,OasisHLBcartridgesweretestedforthe
purificationofginsenosidesfromtheNADESextractscomposedof
glycerol,prolineandsucrose(9:4:1)[25].TheNADESextractwas
introducedontothecartridgeandtheNADEScomponentswere
removedbyaninitialelutionwithwater,afterwhich the
com-poundsofinterest wereeluted withethanol Thequalityofthe
HPTLCseparationimprovedastailingcompletelydisappeared
TheNADESextractsof G.bilobaleavesandP.ginsengleaves
andstemswerealltreatedinthesamewayandthenanalysedby
HPTLC.Fig.1showsthefourgroupsofmetabolitesthateachcan
bewellvisualisedinthedifferentHPTLCchromatograms,clearly
showingitspowertodetectawiderange ofchemicallydiverse
groupsofmetabolites[43].TheP.ginsengsaponinswithout
UV-chromophoreswereabletobevisualisedat254nmand366nm
aftertreatmentwiththeanisaldehyde-sulfuricacidderivatisation
reagent(Fig.1d,e)
VisualexaminationoftheHPTLCchromatogramsshowedthat
the extractionefficiency of all NADES employed in this study,
exceptfortheall-sugarNADESN5,wassimilartothatofmethanol forginkgolidesandphenolicsfromG.bilobaandforginsenosides
inP.ginsengleavesandstems(Fig.1).Ginkgolicacidswerenot sig-nificantlyextractedbyanyoftheNADES.Ingeneral,plantaliphatic phenolslikeginkgolicacidshaveverylowpolarity,whichmakes themdifficulttobedissolvedinpolarsolvents.MostNADESare categorisedaspolarsolventsandcouldnotadequatelyextractthe non-polarginkgolicacids
Inthecasesofginkgolidesandginsenosides,theNADESextracts showedfewerbandsthanthemethanolextracts,butallofthemain compounds(bilobalide,3ginkgolides,and7ginsenodides)inthe NADESextractswerestillpresentinsimilarconcentrationstothat
ofthemethanolextractswithanexceptionforthesugarmixture (N5)(Fig.1a,d,e).InthecaseofG.bilobaphenolics,NADESextracts displayedmorebands thanthemethanolextracts,forexample, withinthe0.35–0.38Rfrange(Fig.1b).Themoststrikingfeature, however,islowextractionyieldoftheginkgolicacidsinalltested NADESrevealingclearlydifferentextractionprofilesfortheNADES andmethanol(Fig.1c).Ginkgolicacidsareconsideredtobetoxic and thepresenceof thesecompoundsis unwantedin G.biloba extractsthatareusedforhumanconsumption,sotheextractionof theleaveswithNADEScouldresultinhighqualityGinkgo prepa-rationswithverylowginkgolicacidcontent
Trang 6Fig 2.Score plot of principal component analysis (PCA) of natural deep eutectic solvent (NADES) extracts and methanol extracts of ginkgolides (a) and ginkgo phenolics (b) in Ginkgo biloba leaves, and score plots of orthogonal partial least square discriminant analysis of ginkgolides (c) and ginkgo phenolics (d) M: methanol extract 1–6: NADES extracts 1: malic acid-choline chloride (1:1, molar ratio), 2: NADES of malic acid- glucose (1:1), 3: choline chloride-glucose (5:2), 4: malic acid-proline (1:1), 5: glucose-fructose-sucrose (1:1:1), 6: glycerol-proline-sucrose (9:4:1).
Theresultsobtainedinthisstudyhighlightoncemorethegreat
potentialofNADESasagreenalternativesolventfortheextraction
ofphenolics.ThishighextractionpowerofNADESforphenolics
mayberelatedtoH bondinginteractionsbetweenthefunctional
groupsofthecomponents(e.g.,hydroxylandcarboxylgroups)and
thehydroxylgroupsinphenolics.Wehavereportedthe
observa-tionofH bondinginteractionsbetweenquercetinandNADESin
previousstudies[44]
Chromatographicprofilesprovidebasicinformationabout
spe-cificgroupsofcompoundsbut,mostimportantly,cancharacterise
thechemicalcompositionofasampleinaholisticway.Infact,that
istheparadigmofmetabolomics;aimingattheunbiased
analy-sisofallofthemetaboliteswithinanorganism.Inordertofully
takeadvantageofalloftheinformationprovidedbythisprofiling
method,itisnecessarytousebiometricmethodssuchas
multivari-atedataanalysis(MVDA)andmultivariateimageanalysistobeable
toidentifythesimilaritiesanddifferencesbetweenthemeasured
profilesandthencombinethesewithotherobservations,including
themetabolicchangestriggeredbydiseasesorrelatedtoresistance againstherbivores
Allchromatographicprofilingmethodsrequirethe normalisa-tionandalignmentofsignalspriortoMVDA.Forthenormalisation, threecontrolsamples(inthisstudyamethanolextractwasused) werespottedalongsidetheothersamplesoneachplate.The inten-sityateachRfvalueofthesampleswerenormalisedtoamethanol extractinordertominimisethevariationofreplicateson differ-entplates.ThisnormalisationimprovedthequalityoftheMVDA dataquality (Fig.S1) Therecently-developedopen-source soft-ware,rTLC,wasusedforalignmentwhich offersa standardised procedureforimageprocessingandthevisualisationtoolsthatare requiredtocompareHPTLCfingerprintsviadifferentpattern recog-nition andpredictiontechniques[32].Theprocesseddata were furtheranalysedbyPCAandOPLS
ThePCAdataofginkgolidesandginkgophenolicsinG.biloba leavesisshowninFig.2.Nofurtheranalysisoftheginkgolicacids
inNADESextractsofG.bilobaleaveswasperformedduetotheirlow
Trang 7Fig 3. Score plots of principal component analysis (PCA) of data corresponding to ginsenosides in Panax ginseng leaves (a) and stems (b) obtained from HPTLC chromatograms using PC1 and PC2 comparing various natural deep eutectic solvents (NADES) extracts and methanol extracts Orthogonal partial least square-discriminant analysis (OPLS-DA) score plots of ginsenosides in P ginseng leaves (c) and stems (d) The numbering of the extracts is the same as in Fig 2
yieldasshowninFig.1c.InthePCAscoreplotofG.bilobasamples,
NADESextractswereclearlydistinctfromthemethanolextractbut
therewasnosignificantdifferenceamongsttheNADESsolvents
Ifany,inthecaseofphenolics(Fig.2b),malicacid-choline
chlo-ride(N1)andglycerol-proline-sucrose(N6)appearedtobecloserto
themethanolextractsthantheotherNADESextracts.Asupervised
MVDA,OPLS-DA,wasemployedtoobtainamoredetailed
compar-isonbetweenboththemethanolandNADESextracts,includingall
ofthesixtestedNADES.Thisshowedaclearseparationbetween
methanoland NADESextracts(Fig.2d).For theidentificationof
thecontributingmetabolites,anSplotwasusedanditrevealed
thatmethanolextractedhigheramountsofmostmetabolites.All
ofthetestedginkgolides,bilobalide(Rf 0.447),ginkgolidesA(Rf
0.361),B(Rf0.266)andC(Rf0.114)wereextractedlessefficiently
withNADES(Fig.S2a).TheHPTLCanalysisofphenolicsinG.biloba
leavesalsoacknowledgedthatmethanolwasmoreefficientthan
NADES,forexample,forchlorogenicacid(Rf0.52),rutin(Rf0.352)
andquercetin(Rf0.99)(Fig.S2b)
InthecaseofP.ginsengleavesandstems,thereweredifferences amongsttheextractionprofilesobtainedwithmethanol,butalso amongsttheNADESextracts(Fig.3a,b).Thefirstclusterconsisted
ofthemethanolextracts,thesecondofmalicacid-cholinechloride (N1)andmalicacid-glucose(N2),andthethirdgroupedtogether thethreeremainingNADES,cholinechloride-glucose(N3),malic acid-proline(N4) and glycerol-proline-sucrose (N6), implying a similarityintheirchemicalprofiles.Extractsmadewith glucose-fructose-sucrose(N5)formedafourthcluster(Fig.3aandb).To comparetheNADESandmethanolextracts,OPLS-DAwasapplied
totheP.ginsengsamples(Fig.3candd)andtheresultsshowed thatallofthesevenanalysedginsenosidesweremoreefficiently extractedfrombothP.ginsengleavesandstemswithNADES(Fig S3)
Apartfromtheirchemicalcomposition,anotherfactorthathas
agreatinfluenceonthephysicochemicalpropertiesofNADESis theirwatercontent[45].Oneofthepositiveeffectsofincreasing thewatercontentisadecreaseintheirviscosity;oneofthefeatures
Trang 8Fig 4.Orthogonal partial least square-discriminant analysis (OPLS-DA) score plots of different natural deep eutectic solvents (NADES) extracts (a), OPLS score plots of various water contents (0–40%, w/w) (b) and shared-and-unique-structures (SUS)-plots (c) of ginkgolides and ginkgo phenolics in Ginkgo biloba leaves, and ginsenosides in Panax ginseng leaves and stems SUS plots correlate the two OPLS-DA models with the X-axis of NADES composition and OPLS of water contents as Y-axis The numbering of the extracts in Fig 4 a and b is the same as in Fig 2 The numbering of the identified compounds is the same as in Fig 1
thathinderstheiruseasextractionsolvents.Toevaluatetheeffect
ofthewaterratio,differentamountsofwaterwasadded(0–40%,
w/w)tothetwoNADESthathadthehighestextractionyieldsfor
eachplantinthefirstexperiment.Theextractswereanalysedusing
HPTLC-MVDA
ThecontentofginkgolidesandphenolicsinG.bilobaextracts
preparedwithmalicacid-cholinechloride(1:1)(N1)and
glycerol-proline-sucrose (9:4:1) (N6) with varying water ratios were compared.ThedataobtainedfromtheHPLTCchromatograms (X-data)werecombinedwiththewatercontent(Y-data)toevaluate theeffectofthewaterpercentage(w/w)onthetestedNADES.Two differentOPLSmodelswiththewatercontentorcompositionof NADESasY-dataweresetup(Fig.4a,b).Inthescoreplotofthe OPLSmodeling,thetwoselectedNADESextractswereclearly
Trang 9apartfromthedifferentchemicalcompositionsofN1andN6,the
watercontentalsohada largeeffectontheextractionprofiles
ThesetwoOPLSmodels(NADEScompositionofN1andN6,and
watercontent)wereintegratedbyashared-and-unique-structures
(SUS)-plot,in whichdiagonally-alignedmetabolitesareofequal
importanceand sharedbythetwomodels,andthemain factor
influencing extractionyieldof each metabolite canbededuced
(Fig.4c) IntheSUS-plot,theeffectof individualfactors(X-axis
forNADESchemicalcompositionsandY-axisforwatercontent)
wereeasilydistinguishedfor eachmetabolite Asobserved,the
twometabolitesthataremostaffectedbytheNADEScomposition
changeswereginkgolideBandchlorogenicacid,thoughvery
dif-ferently,i.e.,glycerol-proline-sucrose(N6)extractedthehighest
amountofginkgolideBbuttheleastamountofchlorogenicacid
ThewatercontentgreatlyinfluencedNADESextractionyieldsas
seenontheY-axisintheSUS-plot(Fig.4c).Thiswasparticularly
noticeableinthecaseofrutin
Panaxginsengleavesand stemswereextractedwiththetwo
mostefficientNADES,malicacid-cholinechloride(N1)andmalic
acid-glucose(N2),withvaryingvolumesofaddedwaterandthen
analyzsedbyOPLSandSUS-plot,similarlytotheG.bilobasamples
Interestingly,asseeninFig.4,theyieldofthesevenginsenosides
wereinfluencedinadifferentway,eventhoughtheirstructural
differencesareminimal.ThehighestyieldofginsenosidesRg3and
Rg2inbothP.ginsengleavesandstemswereobtainedwithmalic
acid-glucose(N2)whilstmalicacid-cholinechloride(N1)yielded
themostginsenosideRb1fromP.ginsengstems(Fig.4c).Allseven
ginsenosidesofP.ginsengleavesandsixginsenosidesofP.ginseng
stems(exceptforginsenosideRb1)werebestextractedwithNADES
withthehighestaddedwatercontent(Fig.4c)
4 Conclusion
Todevelop a reliableanalytical method for NADESextracts,
aHPTLC-based methodwasemployed.Thismethodwastested
ontwo well-known medicinalplants and proved tobe ableto
deliverreproducible chemicalprofilesfromtheNADESextracts
Theresultsverifiedthattheyieldofbioactivecompoundsobtained
withsixdifferenttypesofNADESissimilartothatofmethanol
in all cases with only one exception The application of
mul-tivariate analysis revealed, however, some clear differences in
extractionselectivityamongstthedifferentNADES.Theaddition
ofwatertotheNADEShadalargeeffectontheefficiencyoftheir
extractionfor theselectedcompounds,increasingtheiryield in
general.Maximum amounts of ginkgolides, phenolics and
gin-senosideswereobtainedwithanadditionofapproximately20%
water to theNADES It is worth noting that themost striking
differencebetween the methanol and NADES extracts wasthe
significant lack in ginkgolic acids in the NADES extracts This
is very promising for further studies, since it suggests
poten-tialfor obtainingpracticallyginkgolic acid-freepreparationsfor
pharmaceuticaluse
Allof the resultsobtainedin this studyshow NADES tobe
promisingextractionsolvents.Adeeperknowledgeofthe
theo-reticalbasisfortheextractionmechanismoftheNADESandtheir
interactionwithsoluteswouldgreatlyfacilitatethedevelopmentof
futureapplications.Asidefromthis,theHPTLC-analyticalmethod
describedherewillbeausefultoolforthisprocess
Acknowledgments
WethankMrDickBruynzeelinBCONInstrument(Sint
Anna-land,theNetherlands)fortechnicalsupportforHPTLCexperiments
andDrEricaG.Wilsonforallvaluablescientificdiscussion
Appendix A Supplementary data
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion, atdoi:https://doi.org/10.1016/j.chroma.2017 12.009
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