Untargeted Metabolomics Profiling and Global Semi ... · For untargeted metabolomics...

Shu Y et al. Untargeted Metabolomics Profiling and … Planta Med Int Open 2020; 7: e45–e57

Original Paper Thieme

Untargeted Metabolomics Profiling and Global Semi-quantitation of a Prescription Chinese Herbal Medicine Formula Yinqiaosan Using UPLC-QTOF-MS with a Single Exogenous Reference Internal Standard

AuthorsYachun Shu1, 2 , Chandana Mannem2, Gang Xu2, Shashank Gorityala2 , Xiao Liu2, 3, Yan Xu2, 4

Affiliations1 AffiliatedHospitalofNanjingUniversityofChinese

Medicine,Nanjing,P.R.China2 DepartmentofChemistry,ClevelandStateUniversity,

Cleveland,OH,USA3 CollegeofPharmacy,NanjingUniversityofChinese

Medicine,Nanjing,P.R.China4 CaseComprehensiveCancerCenter,CaseWestern

ReserveUniversity,Cleveland,OH,USA

Key wordsYinqiaosan,Chineseherbalmedicineformula,untargetedmetabolomicsprofiling,globalsemiquantitativeanalysis,multivariatedataanalysis,UPLC-QTOF-MS

received 03.09.2019 revised 26.02.2020 accepted 10.03.2020

BibliographyDOI https://doi.org/10.1055/a-1141-0119Publishedonline:2020PlantaMedIntOpen2020;7:e45–e57©GeorgThiemeVerlagKGStuttgart·NewYork ISSN2509-9264

CorrespondenceYachunShuAffiliatedHospitalofNanjingUniversityofChineseMedicine155HanzhongRoad210029NanjingCityJiangsuProvinceP.R.China Tel.:+8625-86617141,Fax:+8625-86618555 [email protected]

YanXuDepartmentofChemistry,ClevelandStateUniversity2121EuclidAve.,SI333ClevelandOhio44115-2214USA Tel.:+12166873991,Fax:+12166879298 [email protected]

Supporting Informationforthisarticleisavailableonlineat:http://www.thieme-connect.de/products.

AbstractYinqiaosanisaclassicChineseherbalmedicineformulathathasbeenusedtotreatvariousbacterialandviralinfectionsbyChi-nesemedicinedoctorsforovertwocenturies.Inthiswork,wedevelopedacomprehensivequalitativeandquantitativemeth-odforidentification,quantitation,andqualityassessmentofchemicalconstituentsofYinqiaosanformulainfourdifferentpreparationforms(i.e.,decoction,granule,pill,andtablet),whichemployedultra-performanceliquidchromatographyquadrupoletime-of-flightmassspectrometrywithasingleexogenousreferenceinternalstandardforuntargetedme-tabolomicsprofilingandglobalsemiquantitativeanalysis.Theuseofasingleexogenousreferenceinternalstandardpermit-tednotonlyqualitativeandquantitativeanalysesofmultipleherbalcomponentsinasingleinstrumentrun,butalsocross-comparisonofchemicalcontentsinbetweenallfourYinqia-osanpreparationforms.Theacquiredmasschromatogramswereanalyzed,quantitated,andcomparedusingmultivariatedataanalysisforsimilaritiesanddifferencesofchemicalcon-stituentsinfourYinqiaosanpreparationforms.Forthefirsttime,wewereabletoidentifyover100chemicalconstituentsfromeachpreparationformusingtheavailabledatabase.Amongthe49commonlyidentifiedcompoundsinthe4Yin-qiaosanpreparationforms,16havebeenreportedtohavepharmacologicalactivities,whichmaybeusedinanetworkpharmacologystudyofYinqiaosanforexploringtheunderlyingmechanismoftheherbalformula.

e45

Published online: 2020-04-21

https://orcid.org/0000-0002-2661-5762

https://orcid.org/0000-0002-2514-6589

mailto:[email protected]

mailto:[email protected]


Original Papers Thieme

AbbrevIAtIonS

CHM ChineseherbalmedicineCV coefficientofvariationEBM evidence-basedmedicineESI electrosprayionizationIS internalstandardPCA principalcomponentanalysisPLS-DA partialleastsquaresdiscriminantanalysisQC qualitycontrolQ-marker qualitymarkerRCT randomizedcontrolledtrialSIM selectedionmonitoringTCM traditionalChinesemedicineTIC totalioncurrentUPLC-QTOF-MS ultra-performanceliquidchromatogra-

phy-quadrupoletime-of-flightmassspectrometry

IntroductionCHMformulasarethekeycomponentsofTCMinterventions,whichhavemanypreparationformsincludingdecoctions,granules,pills,tablets,capsules,powders,medicatedteas,medicatedwines,etc.[1].DespitethewidespreaduseofCHMformulasinAsiaandtheirgrowinguseintheWest,thefundamentalissuehinderingCHMin-tegrationintotheWesternmainstreamhealthcaresystemisthatrigorousscientificevidenceofCHMefficacyandsafetyobtainedthroughRCTsorsystematicreviewsofRCTshasbeenlimitedfromEBMperspectives[2].Thisismainlyattributedtotwomajorde-fectsintheRCTstodate:(1)improperdifferentiationorreportingofaTCMpatternorsyndromesincepatterndifferentiationiscru-cialinTCMforprescribingCHMtherapy,and(2)thelackofacom-prehensivequalityassessmentandcontrolofCHMformulassincethechemicalconstituentsinmedicinalplantsvarywithgeograph-icoriginandcultivar,partsinuse,timeofharvest,ecologicalenvi-ronmentandpotentialcontaminationwithheavymetals,pesti-cides,ormycotoxins,processinganddetoxificationmethodaswellasvariousformsofpreparations[1].

Themethodscurrentlyadoptedbytheinternationalpharma-copeialmonographs[3–5]foridentificationandqualityassessmentofmedicinalplantsandCHMformulasemphasizethequalitativeandquantitativedeterminationofagroupofsubjectivelyselectedQ-markerconstituentsthatmayormaynothaveatherapeuticef-fect.Thesemethodscanʼtdistinguishwhetheramarkerconstitu-entisendogenousorfalsifiedbecausenoinformationonqualita-tiveandquantitativechemicalprofilesofmedicinalplantsorCHMformulasisobtained.Therefore,comprehensivequalitativeandquantitativechemicalanalysismethodsaremuchmoredesirable,notonlyforphytochemicalprofilingandqualityassessmentofme-dicinalplantsandCHMformulasbutalsofordetectionofcounter-feits.UntargetedmetabolomicsprofilingusingLC-QTOF-MShasbeensuccessfullyappliedtofingerprintingherbalmedicines[6–8].Ifthistechniquecombineswiththeuseofasingleexogenousref-erenceISforsemiquantitativeanalysisandPCAforpatternrecog-

nition,itwouldprovideamoreadequatemethodforidentificationofchemicalconstituentsandqualityassessmentofmedicinalplantsandCHMformulas.Thisworkdemonstratessuchanat-tempt.

YinqiaosanisaclassicCHMformuladevelopedbytheQingDy-nastyfamousChinesedoctorWuJutong(1758–1836).Itiscom-posedofnineherbsincludingJinyinhua(Flos lonicerae),Lianqiao(Fructus forsythiae),Bohe(Herba menthae),Jingjie(Herba schizone-petae),Niubangzhi(Fructus arctii),Jiegeng(Radix platycodonis),Dandouchi(Semen Sojae preparatum),Gancao(Radix glycyrrhizae),andDanzhuye(Herba lophatheri)[9].BasedonCHMclassification,Yinqiaosanhascoolenergy,anacridtaste,andpropertiesofreleas-ingexterior,clearingheatandremovingtoxicityfromhumanbod-ies[10].IthasbeenusedbyTCMdoctorsforovertwocenturiesastreatmentforvariousbacterialandviralinfectionssuchasinfluen-za[11],hand,foot,andmouthdisease[12],pharyngitis[13],pneu-monia[14],acutetonsillitis[15],measles[16],andmumps[17]. ArecentrandomizedclinicaltrialindicatedthatYinqiaosanplusan-otherCHMformula,Maxingshigan,wasaseffectiveasoseltamivir(alsoknownasTamiflu)forthetreatmentofH1N1influenzaAvirusinfections[18].

Yinqiaosanwasoriginallypreparedintheformofadecoction.Duetotheeaseofuse,carry,andstorage,Yinqiaosanhasbeenpre-paredinvariousformsoverthecourseofhistory,suchasYinqia-osanpeifangkeli(granule),Yinqiaojieduwan(pill),andYinqiao-jiedupian(tablet),whicharebeingusedinterchangeablyinclinicalinterventions.Currently,therearevariousformsofYinqiaosanpro-ducedbynumerousChinesemanufacturers[19].Thelackofcom-prehensivequalitativeandquantitativechemicalanalysismethodsforchemicalprofilingandquantitationnotonlyimpedecompletequalityassessmentofvariousYinqiaosanpreparationforms,butalsoconfusetheassessmentofoutcomesofclinicalstudies[2,20].Therefore,wehavedevelopedacomprehensivequalitativeandquantitativemethodthatcombinesuntargetedmetabolomicspro-filingandglobalsemiquantitativeanalysiswiththeuseofasingleexogenousIS,aswellaspatternrecognitionthroughPCAforiden-tification,quantitation,andqualityassessmentofYinqiaosaninfourpreparationforms(decoction,granule,pill,andtablet).

ResultsandDiscussionOneofthekeytechnicaladvancesofthemethoddevelopedistheimplementationofasingleexogenousreferenceIS(etoposide-d3),whichservesmultiplerolesincludingselectionofretentiontimeandmassshiftwindowsforUPLC-QTOF-MSanalysis,assessementandcorrectionofasamplematrixeffect,peaknormalizationformultivariatedataanalysis,andglobalsemiquantitativeanalysisofmulticomponentsineachYinqiaosanpreparationform,aswellascross-comparisonofchemicalcontentsinbetweenvariousYinqia-osanpreparationforms.

Ourexperimentaldata(notshown)indicatedthattherewerenochromatographicormassspectrometricinterferencestotheISintheblanksolutionsandsamplesofthefourYinqiaosanprepara-tionforms.ThematrixeffectofeachYinqiaosanpreparationformonthemassspectrometricdetectionoftheISexpressedasmatrixfactorwascalculatedbythemeanpeakareaoftheISspikedinsam-plesofeachYinqiaosanpreparationformoverthatoftheISspiked

e46


intheblanksolutions.ThematrixfactorsoffourYinqiaosanprep-arationformsrangedfrom0.87–1.07(▶table 1),indicatingtherewasnosignificantsignalsuppressionorenhancementoftheISinanyofthesamplematrices.

Foruntargetedmetabolomicsprofiling,constituentidentifica-tion,andglobalsemiquantitationofchemicalconstituentsinYin-qiaosanformula,triplicatesampleswerepreparedforeachYinqia-osanpreparationformandthesolutionblank.Atotalof15Yinqia-osansampleswereanalyzedusingtheUPLC-QTOF-MSmethod

developed.Thechromatographicandspectra(MSandMS/MS)datawereacquiredbybothpositiveandnegativeESImodes.Therep-resentativeTICchromatogramsareshownin▶Fig. 1.ChemicalconstituentsofeachYinqiaosanpreparationformwereextractedaftersubtractingTICchromatogramsofthesolutionblanksfromthoseofYinqiaosansamplesolutions.

Thereareseveralnonvolatile,water-solubleQ-markersfromthenineherbsofYinqiaosan[3].Toillustratethistargetedfeatureofthemethoddeveloped,theseQ-markersweretargetedandex-

▶table 1 MatrixeffectsofYinqiaosansamplesonmassspectrometricdetectionoftheISa.

Sample matrix (n = 3) eSI mode PAISb in extracted sample matrix ± SDc PAIS in solution ± SD MFIS

d ± SD

Decoction + (4.75±0.06)×105 (5.4±0.2)×105 0.88±0.03

− (2.34±0.02)×106 (2.68±0.03)×106 0.87±0.01

Granule + (5.4±0.2)×105 (5.4±0.2)×105 1.00±0.05

− (2.52±0.03)×106 (2.68±0.03)×106 0.94±0.02

Tablet + (5.8±0.2)×105 (5.4±0.2)×105 1.07±0.05

− (2.37±0.04)×106 (2.68±0.03)×106 0.88±0.02

Pill + (4.83±0.04)×105 (5.4±0.2)×105 0.89±0.03

− (2.43±0.01)×106 (2.68±0.03)×106 0.91±0.01

aIS=1.69µM,bAIS=meanpeakareaofthespikedIS,cSD=standarddeviation,dMFIS=(PAISintheextractedsamplematrix)/(PAISinthesolution).

Coun

ts

Coun

ts

–TIC Blank

–TIC Decoction

–TIC Granules

–TIC Tablet

–TIC Pill

Retention Time (min)

f

g

h

i

j

x106

3210

x106

3

2

1

0

5 10 15 20 25 30 35 40 45 50 55

5 10 15 20 25 30 35 40 45 50 55

x106

3

2

1

05 10 15 20 25 30 35 40 45 50 55

x106

3

2

1

05 10 15 20 25 30 35 40 45 50 55

x106

3

2

1

05 10 15 20 25 30 35 40 45 50 55

x106+TIC Blank

+TIC Decoction

+TIC Granules

Retention Time (min)

+TIC Tablet

+TIC Pill

3

a

b

c

d

e

2

1

5 10 15 20 25 30 35 40 45 50 55

x106

3

2

1

0

x106

3

2

1

0

x106

3

2

1

0

x106

3

2

1

0

5 10 15 20 25 30 35 40 45 50 55

5 10 15 20 25 30 35 40 45 50 55

5 10 15 20 25 30 35 40 45 50 55

5 10 15 20 25 30 35 40 45 50 55

▶Fig. 1 RepresentativeTICchromatogramsofthesolutionblankandsamplesofvariousYinqiaosanpreparationformsbybothpositiveionizationmode(a–e)andnegativeionizationmode(f–j).

e47



tracted.TherepresentativeSIMchromatogramsofsixQ-markers(i.e.,chlorogenicacid,luteolin7-glucoside,forsythiaside,liquiritin,forsythin,andarctiin)areprovidedin▶Fig. 2.

Duetotheshortageofstandardreferencematerialandthecostconcern,determinationofmulticomponentsforqualityassessmentofCHMformulaiscurrentlynotrequiredbytheChineseregulato-ryagency.InthecaseofYinqiaosanproducts,onlyoneofthe Q-markers(i.e.,arctiin,chlorogenicacid,orforsythin)isregulatedbytheChinesePharmacopoeia[3].SinceYinqiaosanproductscon-tainmultiplebioactivecomponents,thecurrentmethodsforsin-gle-componentanalysiscanneitherassessthequalityofYinqia-osanproductsobjectivelynordetectcounterfeitseffectively.ThemethoddevelopedinthisworkprovidesamuchbettertechnicalsolutionforqualityassessmentofYinqiaosanproducts,whichnotonlyprofilesaproductbutalsoquantitatesmultipleQ-markerssi-multaneously.

IdentificationofchemicalconstituentsineachYinqiaosanprep-arationformwasaccomplishedbyfollowingtheproceduredetailed

in“Dataprocessing,constituentidentification,andstatisticalanal-ysis”inthe“MaterialsandMethods”section.Thedataobtainedwerethensubjectedtomolecularfeatureextractionsusingpossi-bleionadducts,isotopepatterns,andchargestatesaswellasapresetretentiontimeandmasswindow.Itisworthnotingthatthepost-processingfilterofthedataprocessingsoftwarewassetat3outof3toensureeachmolecularfeatureextractedpresentedinallthreemasschromatogramsofthetriplicatemeasurementsforeachYinqiaosanpreparationform.Thetotalnumberofconstitu-entsidentifiedwithchemicalnamesandthenumberofconstitu-entsidentifiedwithmolecularformulasineachpreparationformaresummarizedin▶table 2.Thenumberofchemicalconstituentsfoundindecoction,granule,pill,andtabletformsofYinqiaosanwere302,434,427,and388,respectively.Amongthe4prepara-tionforms,therewere104commonconstituents(▶Fig. 3),49identifiedwithbothchemicalnamesandformulas(▶table 3)and55identifiedonlywithchemicalformulas(▶table 1S,SupportingInformation).TheMS/MSspectraofthe49chemicalconstituents

▶Fig. 2 RepresentativeSIMchromatogramsofsixmarkerconstituentsofYinqiaosanalongwiththeISataconcentrationof1.69μM.

x103 1.801

1

1

1

1

1

1

1

Chlorogenic acid +ESI

+ESI

+ESI

+ESI

+ESI

+ESI

+ESI

–ESI

Luteolin 7-glucoside

Forsythiaside

Liquiritin

Forsythin

14.36

17.36

22.94

25.99

28.99

28.95

30.00

Arctiin

Etoposide d3 (IS)

Etoposide d3 (IS)

x104

x103

x104

x104

x104

x104

x104

6

4

2

0

6

4

4

3

2

2.52

1.5

0.50

5 10 15 20 25 30 35 40 45 50 55

1

1

0

2

0

6

1.51.25

10.75

0.50.25

8

2.5

Coun

ts

Retention time (min)

21.5

10.5

0

6420

0

4

2

0

e48


commonlyidentifiedinallfourYinqiaosanpreparationformsareprovidedin(▶Fig. 1S,SupportingInformation).

SinceeachYinqiaosanpreparationformexertsasimilarthera-peuticeffectandisusedtotreatthesameillnesses,itisreasonabletothinkthatpharmacologicallyactiveconstituentsareamongthe49commonlyidentifiedchemicalconstituentsinallfourprepara-tionforms,whereastheuniquechemicalconstituentsineachprep-arationform(▶tables 2S– 9S,SupportingInformation)maycomefromtheherbsusedbythemanufacturers,wherechemicalcon-stituentsmayvarywithgeographicoriginandcultivar,timeofhar-vestandtheecologicalenvironmentoftheherbs,potentialagri-culturalpollutionsofpesticidesandherbicidesorindustrialcross-contaminationwithotherpharmaceuticalsaswellassidereactionsandby-productsassociatedwiththeuniquemanufacturingcondi-tions(e.g.,temperature,pressure,time,solvent,additives,etc.)ofeachYinqiaosanpreparationform.Thisstudyshowsthatproductprofilingisanindispensablepartofqualityassessment,andtheun-targetedmetabolomicsapproachnotonlyallowsustoprofilebutalsofingerprintanherbalproductintermsofQ-markers,originandcultivars,potentialcontaminations,andsignatureconstituentsas-sociatedwithaparticularpreparationormanufacturingprocess.

MultivariateanalysisoftheacquiredMSdata(i.e.,exactmasstoretentiontimepairandnormalizedpeakarea)wasfirstcarriedoutwithanunsupervisedPCAscoreplottoassessthesimilaritiesofchemicalconstituentsamongthefourYinqiaosanpreparationformsandthereproducibilityofreplicatesamplesofeachprepa-rationformbytheUPLC-QTOF-MSmethod,thenvisualizedbyasu-pervisedPLS-DAscoreplottoestablishrecognitionpatternsofthefourYinqiaosanpreparationforms.

AsshowninthePCAscoreplot(▶Fig. 4a),thedifferencesinchemicalconstituentsamongthefourYinqiaosanpreparationformswereapparent.Thefirsttwoprincipalcomponentsencom-passed72.3%ofthetotalvariance,andthetightgroupingoftrip-licatemeasurementsonthesamplesofeachpreparationformin-dicatedthattheUPLC-QTOF-MSmethoddevelopedhadexcellentreproducibility.ThePLS-DAscoreplot(▶Fig. 4b)wasinagreementwiththePCAscoreplot,andshoweddistinctivepatternsamongthefourYinqiaosanpreparationforms.Hence,thesepatternsmaybeusedforproductdifferentiationandrecognition.

Ahigh-throughputUPLC-QTOF-MSmethodforglobalsemi-quantitativeanalysisofchemicalconstituentsinCHMformulawasdemonstratedinthiswork.Asillustratedbythecontentsof49commonlyidentifiedchemicalconstituentsinfourYinqiaosanpreparationforms(▶table 4),theuseofasingleexogenousrefer-enceIS(etoposide-d3)inthemethodenabledustoperformnotonlyglobalsemiquantitativeanalysisofmulticomponentsineachYinqiaosanpreparationformbutalsocross-comparisonamongthefourpreparationforms.Therefore,themethoddevelopedprovidesanefficientandeconomicalsolutionforbothherbalcontentanal-ysisandproductcomparison.

ThereproducibilityofglobalsemiquantitativeanalysisbytheUPLC-QTOF-MSmethodwasinvestigatedbytriplicatemeasure-mentsofsampleofeachYinqiaosanpreparationform,andthepre-cisionofthesemiquantitativemeasurementswascalculatedintermsofCVofthechemicalcontents.Itwasshown(▶table 4)that91.8–98.0%ofthe49chemicalconstituentsofthe4preparationformshadCVvaluesthatfellwithintherecommendedvalues(CV≤30%)[21],whichindicatedthereliabilityoftheUPLC-QTOF-MSmethod.

Thepharmacologicalactivitiesofthe49commonlyidentifiedchemicalconstituentsinall4Yinqiaosanpreparationformswerein-vestigatedthroughtextmininganddatabasesearching.Sixteenoftheforty-ninechemicalconstituentswerefoundtohavevarious

▶table 2 SummaryofchemicalconstituentsfoundinfourYinqiaosanpreparationforms.

Yinqiaosan Constituents Common constituents

Forms eSI mode Identified Unidentified total eSI mode Identified Unidentified total

Decoction+ 158 37

302

+ 46 21

104

− 21 86

Granule+ 79 177

434− 36 142

Tablet+ 91 163

388

- 3 34− 24 110

Pill + 67 186427

− 38 136

Pill (P)

Granules (G) Tablet (T)

G

P 226

155

13

27

104

3

24245

1

7

162

5

196

35

D

T

Decoction (D)

▶Fig. 3 VenndiagramofthechemicalconstituentsfoundineachpreparationformofYinqiaosan.

e49


Original Papers Thieme▶

tabl

e 3

Common

chemicalcon

stitu

entsdetectedandidentifi

edinallfourYinqiaosanpreparationform

s.

no.

Form

ula

nam

etr

(min

)o

bser

ved

mas

sD

atab

ase

mas

sPr

ecur

sor

ion,

m/z

1C 1

6H18O9

Chlorogenicacid

*

1.80

354.09

6135

4.09

5135

5.10

26[M

+H]+

2C 1

1H12N

2O2

D-Tryptop

han

3.01

204.09

1420

4.08

9120

5.09

86[M

+H]+

3C 3

4H34O18

Isorientin4'-O

-glucoside2''-O-p-hydroxybenzoagte

4.88

730.17

6473

0.17

7773

1.18

41[M

+H]+

4C 2

4H24O

11Echioidinin2'-(6''-acetylglucoside)

6.36

488.13

3248

8.13

3748

9.13

93[M

+H]+

5C 1

9H25N5O

6Asn-Trp-Thr

8.11

419.18

2041

9.18

1742

0.18

95[M

+H]+

6C 2

0H26O9

BruceineD

8.40

410.15

7341

0.15

8341

1.16

44[M

+H]+

7C 1

7H18N

4O5

202-79

18.49

358.12

8635

8.12

8138

1.11

77[M

+Na]

+ ;3

59.138

2[M

+H]+

8C 2

1H28O8

Vernofl

exuo

side

8.49

408.17

8540

8.17

9340

9.18

59[M

+H]+

9C 2

1H24O

12Ca

techin-4-ol3-O

-BeTa-D-galactopyrano

side

9.59

468.12

7446

8.12

7946

9.13

45[M

+H]+

10C 2

8H34O

12Ca

ohuo

sideD

9.79

562.20

5656

2.20

6656

3.21

30[M

+H]+

11C 1

1H14O5

Genipin

11.14

226.08

5322

6.08

5522

7.09

25[M

+H]+

12C 2

0H18N

4O6

Phe-His-O

H11

.14

410.12

1441

0.12

2641

1.12

87[M

+H]+

13C 1

3H22N

4O8S

2Cysteineglutathion

edisulfide

11.19

426.08

8542

6.08

7121

4.05

06[M

+2H]2+

14C 2

0H24N

4O6

Asp-Pro-Trp

11.69

416.16

8141

6.16

7741

7.17

54[M

+H]+

15C 1

8H26O7

Prop

ofolglucuronide

12.04

354.16

7635

4.16

7235

5.17

44[M

+H]+

16C 2

3H20O9

Pong

amosideA

12.25

440.11

1144

0.11

1244

1.11

85[M

+H]+

17C 1

5H12O

4Liqu

iritig

enin

13.37

256.07

4825

6.07

5225

7.08

20[M

+H]+

18C 2

1H21ClN

2O8

Dem

eclocycline

13.43

464.09

7746

4.09

9048

7.08

72[M

+Na]

+ ;4

65.105

2[M

+H]+

19C 3

2H31N

3O5

KT572

013

.78

537.22

3653

7.22

5053

8.23

08[M

+H]+

20C 2

2H22O7

Artoindo

nesianinR

14.13

398.13

7139

8.13

6939

9.14

37[M

+H]+

21C 2

1H20O

11Luteolin7-glucoside

*

14.42

448.10

8744

8.10

3844

9.10

98[M

+H]+

22C 2

2H24O7

Dihydroanhydrop

odorhizol

15.60

400.15

2040

0.15

3140

1.15

94[M

+H]+

23C 2

5H23ClN

2O7

TyrM

e-Phe4Cl-O

H17

.02

498.11

8849

8.12

0149

9.12

58[M

+H]+

24C 2

9H36O15

Forsythiaside *

17

.32

624.20

3562

4.20

5464

7.19

94[M

+Na]

+

25C 2

8H30O

11IkarisosideD

17.98

542.18

1154

2.18

0954

3.18

75[M

+H]+

26C 2

9H32O

12Am

orph

igeninO-glucoside

19.41

572.19

0257

2.19

0157

3.19

77[M

+H]+

27C 2

3H28O

11Albiflo

rin20

.08

480.16

3448

0.16

4048

1.17

06[M

+H]+

28C 2

1H19ClN

2O8

Oxazepamglucuronide

20.73

462.08

3146

2.08

2546

3.09

03[M

+H]+

29C 3

4H30N

2O9

Atalanine

21.18

610.19

2461

0.19

3161

1.19

97[M

+H]+

30C 2

1H22O9

Liqu

iritin

*

22.87

418.12

9341

8.12

8241

9.13

66[M

+H]+

31C 2

8H50N

2O19P 2

N,N'-D

iacetylchitobiosyldiph

osph

odolicho

l23

.52

780.24

8678

0.25

0278

1.25

77[M

+H]+

32C 3

2H42O15

3,4,7-Trihydroxy-5-m

etho

xy-8-prenylflavan4-O

-(beta-D-xylop

yranosyl-

(1-6)-beta-D-glucopyrano

side)

24.24

666.25

0866

6.25

0866

7.25

81[M

+H]+

33C 2

7H34O

11Forsythin

* 25

.88

534.20

8653

4.21

0155

7.20

15[M

+Na]

+

34C 2

3H36O8

3''-H

ydroxypravastatin

26.01

440.24

0544

0.24

1844

1.24

77[M

+H]+

35C 2

1H25ClN

2O3

Cetirizine

27.06

388.15

4838

8.15

4741

1.14

41[M

+Na]

+

36C 2

7H34O

11Arctiin

*

29.02

534.21

0753

4.21

0155

7.20

17[M

+Na]

+

e50


pharmacologicalactivities(▶table 5),rangingfromantibacterial,antiviral,anti-inflammatory,andantifungaltoantioxidant,anti-cancer,andtreatmentofallergicrhinitis[22–52].Thesepharma-cologicalactivecompoundsmayservenotonlyasleadcompoundsinnewdrugdevelopment,butalsoasbaitfortheretrievalofpro-teintargetsinanetworkpharmacologystudy[53,54].ThelattermayhelpustounderstandtheunderlyingactionmechanismofYinqiaosanformula.

Inconclusion,aUPLC-QTOF-MSmethodwiththeuseofasingleexogenousreferenceIShasbeendevelopedforuntargetedmetab-olomicsprofilingandglobalsemiquantitationoftheprescriptionChineseherbalmedicineformulaYinqiaosan.Thechemicalprofilesof4Yinqiaosanpreparationforms(i.e.,decoction,granule,pill,and▶

tabl

e 3

Common

chemicalcon

stitu

entsdetectedandidentifi

edinallfourYinqiaosanpreparationform

s.

37C 3

1H42O15

Erub

erinC

33.29

654.25

0665

4.25

1065

5.25

77[M

+H]+

38C 2

0H30O5

19(R)-Hydroxy-PGA2

34.33

350.20

9335

0.20

9135

1.21

67[M

+H]+

39C 2

0H32O5

Tuberonicacid

35.72

352.22

4735

2.22

4535

3.23

22[M

+H]+

40C 1

5H19NO

Pron

etalol

38.36

229.14

8322

9.14

7523

0.15

57[M

+H]+

41C 1

8H14O

3OvaliteninA

39.93

278.09

3527

8.09

3227

9.10

06[M

+H]+

42C 1

9H26O

6S2-Metho

xyestradiol-1

7β3-sulfate

40.02

382.14

4338

2.14

4140

5.13

36[M

+Na]

+ ;3

83.151

2[M

+H]+

43C 2

3H28O

417

-Phenyltrin

orprostagland

inA2

47.86

368.20

1136

8.20

0536

9.20

85[M

+H]+

44C 2

4H40O

63α

,6α,7β,12α

-Tetrahydroxy-5α

-cho

lan-24

-oicacid

48.05

424.28

2442

4.28

3642

5.28

98[M

+H]+

45C 2

4H41NO

4Ca

ssaidine

48.75

407.30

4440

7.30

3440

8.31

16[M

+H]+

46C 2

4H38O

43α

,12α

-Dihydroxy-5β-chol-6-en-24

-oicacid

51.34

390.27

8739

0.27

8941

3.26

76[M

+Na]

+ ;3

91.286

1[M

+H]+

47C 3

6H64N8O

17Glycopeptide

7.86

880.44

1788

0.44

1687

9.43

33[M

-H]-

48C 3

1H39NO

10S

Labrifo

rmin

16.28

617.23

3261

7.22

7861

6.22

59[M

-H]-

49C 4

9H76O

20LanatosideC

21.15

984.49

0698

4.49

3549

1.23

83[M

-2H

]2- ;983

.485

1[M

-H]-

* Markercon

stitu

ent.

Con

tinued

▶Fig. 4 Visualizationofmultivariatedataanalysis.aThePCAscoreplot,andbthePLS-DAscoreplotoffourYinqiaosanpreparationforms.

10

5

5

0

– 5

– 10

0

– 5

– 20 – 10

DecoctionGranulesPillTablet

DecoctionGranulesPillTablet

0

Decoction

Decoction

Pill

Tablet

Granules

Granules

Tablet

Pill

PC1 (54.8 %)

a

b

PLS1 (54.8 %)

PC2

(17.

5%

)PL

S2 (1

6.6

%)

10 20

– 20 – 10 0 10 20

e51


Original Papers Thieme▶

tabl

e 4

Semiquantitativeanalysisof49common

lyidentifi

edcom

poundsin4Yinqiaosanpreparationform

s(n=3).

no.

Form

ula

nam

eD

± S

D (µ

g/g)

Cv (

%)

G ±

SD (µ

g/g)

Cv (

%)

t ± SD

(µg/

g)Cv

( %

)P

± SD

(µg/

g)Cv

( %

)

1C 1

6H18O9

Chlorogenicacid

*

2.6±0.4

1314

.5±0.3

23.9±0.2

41.4±0.1

9

2C 1

1H12N

2O2

D-Tryptop

han

18±2

1261

±2

43.4±0.1

41.8±0.2

13

3C 3

4H34O18

Isorientin4'-O

-glucosid

e2''-O

-p-hydroxybenzoagte

22±5

2243

±10

2485

±17

2042

±2

5

4C 2

4H24O

11Echioidinin2'-(6''-acetylglucoside)

13±5

3916

±1

45.0±0.2

32.7±0.1

5

5C 1

9H25N5O

6Asn-Trp-Thr

3.9±0.8

2022

±3

1415

±3

205±2

43

6C 2

0H26O9

BruceineD

7±2

2742

±16

3824

±3

147.5±0.5

6

7C 1

7H18N

4O5

202-79

113

±2

1611

3±16

1414

±2

137.5±0.5

7

8C 2

1H28O8

Vernofl

exuo

side

6±2

3049

±7

1321

±1

610

±1

9

9C 2

1H24O

12Ca

techin-4-ol3-O

-BeTa-D-galactopyrano

side

4.1±0.5

1230

.6±0.7

230

±1

53.3±0.2

5

10C 2

8H34O

12Ca

ohuo

sideD

2.8±0.7

2416

±3

205±1

284±1

35

11C 1

1H14O5

Genipin

12.5±0.5

412

2±1

111

±3

2818

±3

15

12C 2

0H18N

4O6

Phe-His-O

H72

±2

360

1±10

210

5±24

2313

8±50

38

13C 1

3H22N

4O8S

2Cysteineglutathion

edisulfide

18.3±0.2

110

9±12

1119

±4

2115

±2

12

14C 2

0H24N

4O6

Asp-Pro-Trp

3.2±0.6

1727

.5±0.7

26.8±0.7

113.3±0.4

13

15C 1

8H26O7

Prop

ofolglucuronide

13±2

1310

5±44

4253

±10

1917

±2

9

16C 2

3H20O9

Pong

amosideA

34±3

1029

2±20

711

9±3

269

±8

12

17C 1

5H12O

4Liqu

iritig

enin

11±1

1188

±3

417

±2

1224

±2

6

18C 2

1H21ClN

2O8

Dem

eclocycline

4.1±0.5

1330

.2±0.8

322

±2

113.5±0.4

12

19C 3

2H31N

3O5

KT572

026

±3

1213

2±5

417

.0±0.8

523

±3

11

20C 2

2H22O7

Artoindo

nesianinR

4.7±0.9

1914

±3

184.1±0.4

94.7±0.4

9

21C 2

1H20O

11Luteolin7-glucoside

*

9±1

1265

±3

512

.2±0.2

22.1±0.2

8

22C 2

2H24O7

Dihydroanhydrop

odorhizol

4.0±0.4

922

.2±0.2

18.4±0.2

34.6±0.4

10

23C 2

5H23ClN

2O7

TyrM

e-Phe4Cl-O

H48

±11

2323

8±61

2627

5±13

512

1±19

16

24C 2

9H36O15

Forsythiaside *

7±2

2319

6±38

2033

6±23

793

±7

7

25C 2

8H30O

11IkarisosideD

27±3

1145

4±15

366

6±26

414

5±14

10

26C 2

9H32O

12Am

orph

igeninO-glucoside

12±2

1488

±2

238

±3

714

.0±0.7

5

27C 2

3H28O

11Albiflo

rin4±1

2940

±6

144±1

242.7±0.7

26

28C 2

1H19ClN

2O8

Oxazepamglucuronide

5±1

2519

±6

327.5±0.4

63.8±0.5

12

29C 3

4H30N

2O9

Atalanine

5.9±0.9

1539

.0±0.6

216

.5±0.7

44.9±0.2

4

30C 2

1H22O9

Liqu

iritin

*

12±3

2619

7±10

530

±3

117.6±0.4

5

31C 2

8H50N

2O19P 2

N,N'-D

iacetylchitobiosyldiph

osph

odolicho

l22

±1

672

±2

29.2±0.3

43.2±0.4

12

32C 3

2H42O15

3,4,7-Trihydroxy-5-m

ethoxy-8-prenylflavan4-O-(beta-

D-xylopyranosyl-(1-6)-beta-D-glucopyranosid

e)3.7±0.6

1515

.9±0.5

31.81

±0.08

44.3±0.4

9

33C 2

7H34O

11Forsythin

* 1.4±0.1

1095

±3

313

8±7

513

±1

11

34C 2

3H36O8

3''-H

ydroxypravastatin

3.7±0.6

175.9±0.7

124.9±0.3

61.4±0.2

12

35C 2

1H25ClN

2O3

Cetirizine

2.03

±0.09

47.0±0.9

1228

±3

1110

.0±0.3

3

36C 2

7H34O

11Arctiin

*

2.1±0.3

1431

8±9

351

±5

116.8±0.6

8

37C 3

1H42O15

Erub

erinC

2.9±0.3

1091

±10

1112

.6±0.3

23.9±0.3

7

e52


tablet)wereobtained,andthe49commonchemicalconstituentsand16pharmacologicalactivecompoundswereidentified.Simul-taneoussemiquantitativeanalysisofthemulticomponentsineachYinqiaosanpreparationformwascarriedoutalongwithuntarget-edmetabolomicsprofiling,andcross-comparisonofthechemicalcontentsinbetweenfourpreparationformswasaccomplished.PCAandPLS-DAanalysesshowedthattheUPLC-QTOF-MSmethoddevelopedwasreproducibleandthechemicalconstituentsfoundinthefourYinqiaosanpreparationformsdisplayeduniquepatternsforproductdifferentiationandrecognition.Themethoddevelopedisusefulfortheidentification,quantitation,andcross-comparisonofchemicalconstituentsnotonlyinYinqiaosanproducts,butalsoinotherCHMformulas.

MaterialsandMethods

Chemicals and Chinese herbal medicine formulasAmmoniumhydroxideandformicacidwerepurchasedfromSig-ma-Aldrich.OptimaLC/MSgradeacetonitrileandmethanol,andHPLCgradewaterwerefromFisherScientific.Deionizedwaterwasobtainedfromanin-houseBarnsteadNanopureewaterpurifica-tionsystem(ThermoScientific)witharesistivitymeterreadingof18.2MΩ-cm.Etoposide-d3(purity,97.8%)waspurchasedfromTorontoResearchChemicalsandusedasthesingleexogenousref-erenceinternalstandardinthiswork.

TheYinqiaosandecoctionwaspreparedbyJiangsuProvincialHospitalofTraditionalChineseMedicine(Nanjing,Jiangsu,China)withninekindsofprocessedCHMs(Jinyinhua,BatchNo.150320;Lianqiao,BatchNo.150718;Bohe,BatchNo.150701;Jingjie,BatchNo.150313;Niubangzhi,BatchNo.150601;Jiegeng,BatchNo.150601;Dandouchi,BatchNo.150401;Gancao,BatchNo.150701;andDanzhuye,BatchNo.150302).Yinqiaosan peifangkeli(granules)weremanufacturedbyJiangyinTianjiangPharmaceuticalCo.(Ji-angyin,Jiangsu,PRC)asindividualpackagesforeachCHM[i.e.,Jiny-inhua(BatchNo.1503112),Lianqiao(BatchNo.1501092),Bohe(BatchNo.1412140),Jingjie(BatchNo.1501094),Niubangzhi(BatchNo.1411156),Jiegeng(BatchNo.1503133),Dandouchi(BatchNo.1410023),Gancao(BatchNo.1502100),andDanzhuye(BatchNo.1501021)].Yinqiaojieduwan(pills)(BatchNo.14033352)wasproducedbyBeijingTongrentang(FengtaiDistrict,Beijing,PRC).Yinqiaojiedupian(tablets)(BatchNo.140695)wasmadebytheYunnanTengyaoPharmaceuticalCo.(Tengyue,Yunnan,PRC).

Preparation of the internal standard solutionThestocksolutionofISwaspreparedbydissolving1.00mgetopo-side-d3powderin1.00mLofmethanoltoaconcentrationof1.00mg/mL.TheworkingsolutionoftheISwaspreparedbya1/10di-lutionofthestocksolutioninmethanoltoaconcentrationof100μg/mL(or169μM).

Preparation of various forms of Yinqiaosan sample solutionsYinqiaosandecoctionsamplesolutionTheYinqiaosandecoctionwaspreparedusingthefollowingproce-dure:First,sevenofthenineprocessedCHMs,Jinyinhua(FlosLoni-cerae,9.00g),Lianqiao(FructusForsythiae,9.00g),Niubangzhi▶

tabl

e 4

Semiquantitativeanalysisof49common

lyidentifi

edcom

poundsin4Yinqiaosanpreparationform

s(n=3).

38C 2

0H30O5

19(R)-Hydroxy-PGA2

8±1

1217

±1

616

.1±0.8

56.2±0.8

13

39C 2

0H32O5

Tuberonicacid

5.6±0.4

721

±3

1618

±3

146±1

17

40C 1

5H19NO

Pron

etalol

3.9±0.6

1523

±2

810

±1

129.4±0.7

7

41C 1

8H14O

3OvaliteninA

1.3±0.3

217±2

282.0±0.5

242.3±0.2

9

42C 1

9H26O

6S2-Metho

xyestradiol-1

7β3-sulfate

4.3±0.6

1426

.4±0.4

22.98

±0.02

18±1

19

43C 2

3H28O

417

-Phenyltrin

orprostagland

inA2

1.4±0.2

127.4±0.7

106.3±0.3

53.5±0.4

11

44C 2

4H40O

63α

,6α,7β,12α

-Tetrahydroxy-5α

-cho

lan-24

-oicacid

1.46

±0.01

111

±1

107.7±0.6

84±2

41

45C 2

4H41NO

4Ca

ssaidine

0.54

±0.06

125.0±0.2

48.0±0.6

72.4±0.4

17

46C 2

4H38O

43α

,12α

-Dihydroxy-5β-chol-6-en-24

-oicacid

4±1

3112

±5

437±2

223.7±0.8

21

47C 3

6H64N8O

17Glycopeptide

12±1

1069

±7

109.4±0.2

38.6±0.1

2

48C 3

1H39NO

10S

Labrifo

rmin

28.7±0.8

318

2±13

712

±4

3313

±1

11

49C 4

9H76O

20LanatosideC

8±2

2428

4±57

2017

±5

2983

±10

13

* Markercon

stitu

ents.D

=decoctio

n,G=granu

le,T=tablet;P=pill.

Con

tinued

e53



(FructusArctii,9.00g),Jiegeng(RadixPlatycodonis,6.00g),Dan-douchi(SemenSojaePreparatum,5.00g),Gancao(RadixGlycyr-rhizae,5.00g),andDanzhuye(HerbaLophatheri,4.00g)werefirstsoakedin696mLofdeionizedwaterinsideaclaypotfor30min;next,thepotwasheatedoveragasrangewithahighflame.Oncethewaterinsidethepotstartedtoboil,thegasflamewasadjustedtolowandthepotcontinuedtobeheatedforanother10min.Atthispoint,theremainingtwoprocessedCHMs,Bohe(HerbaMen-thae,6.00g)andJingjie(HerbaSchizonepetae,5.00g),wereadded,andboilingwascontinuedforanother5min.Attheendofheating,thehotdecoction(<500mL)waspouredintoa500-mLglassbeakerandcooledtoroomtemperature.Thecooleddecoc-tionwastransferredintoa500-mLvolumetricflask,andthebeak-erwasrinsedthreetimeswithdeionizedwater.Therinsesolutionwasthencombinedwiththedecoctionintheflask,andadditionaldeionizedwaterwasaddedtothe500-mLmarkoftheflask.Thesolutionwasvortexedandallowedtosettleonabenchtopfor30min;then,3.00mLofthesupernatantwerepipettedandtrans-ferredintoaborosilicateglasstube(16×100mm;FisherScientif-ic)followedbytheadditionof6.90mLofmethanoland0.100mLoftheISworkingsolution(169μM).Themixturewasvortexedfor30susingaMaxiMixIIVortexMixer(ThermoScientific).Next,1.00mLofthesolutionwastransferredintoa1.5-mLmicrocentrifugetube(VWR),andcentrifugedat18000×gfor10minusingaSor-vallST40Rcentrifuge(ThermoFisherScientific).Aftercentrifuga-tion,500µLofthesupernatantwastransferredintoan8-mmclearglassscrewthreadautosamplervialfromThermoFisherScientificandsubjectedtoUPLC-QTOF-MSanalysis.

Yinqiaosangranule,pill,andtabletsamplesolutionsFirst,1/12.5ofthedailymaximumdoseofeachpreparationformofYinqiaosanwasweighedoutprecisely,andeachsamplewas

transferredintoa50.0-mLcentrifugetube(Corning).Then,deion-izedwaterwasaddedtothe40.0-mLmark.Eachsamplewassoakedindeionizedwaterfor90min,followedbyvortexmixingfor3min,thenplacedinaThermoFisherScientificFS-28ultrasonicbathfor30min.Aftersettlingonabenchtopfor30min,3.00mLofthesu-pernatantwerepipettedandtransferredintoaborosilicateglasstube(16×100mm),andthen6.90mLofmethanoland0.100mLoftheISworkingsolution(169μM)wereadded.Themixturewasvortexedfor30s.Next,1.00mLofthesolutionwastransferredintoa1.5-mLmicrocentrifugetubeandcentrifugedat18000×gfor10min.Aftercentrifugation,500µLofthesupernatantweretrans-ferredintoan8-mmclearglassscrewthreadautosamplervialandsubjectedtoUPLC-QTOF-MSanalysis.

Preparation of QC sampleTheQCsample(800µL)couldbepreparedbymixing200µLofeachofthefourYinqiaosansamplesolutions(see“Preparationofvari-ousformsofYinqiaosansamplesolutions”),andusedwitheachbatchanalysisbymonitoringtheselectivityandreproducibilityofthemethodoverthecourseofanalysisonthe49commonlyiden-tifiedcompounds.

UPLC-QTOF-MS systemTheUPLC-QTOF-MSsystemusedinthisworkconsistedofanAgi-lent1290InfinityUPLCsystemcoupledwithanAgilent6540QTOFmassspectrometer.TheUPLCsystemincludedasolventreservoir,adegasser,aG4220Abinarypump,aG1330Bthermostat,aG4226Aautosampler,aG1316Cthermostattedcolumncompart-ment,andaG4212Adiodearraydetector.ThemassspectrometerwasequippedwithanAgilentjetstreamESIprobe.

LiquidchromatographicseparationwasachievedusinggradientelutiononaWatersACQUITYUPLCBEHC18(2.1mmi.d.×100mm,

▶table 5 PharmacologicalactiveconstituentsfoundinallfourYinqiaosanpreparationforms.

no. name PubChem CID CAS reported pharmacological activity

1 Chlorogenicacid * 1794427 327-97-9 antibacterial,antiviral[26–27]

2 Luteolin7-glucoside * 5280637 5373-11-5 antibacterial,antifungal,antioxidant,anti-inflammatory[28,29],antidiabetic[30]

3 Forsythiaside * 5281773 79916-77-1 antibacterial,antiviral,anti-inflammatory[31–32]

4 Forsythin * 101712 487-41-2 antioxidant,anti-inflammatory[33]

5 Liquiritin * 503737 551-15-5 antioxidant[34],antioxidative,anticancer,andneuroprotective[35]

6 Arctiin * 100528 20362-31-6 antiviral,anti-inflammatory[36–39]

7 D-Tryptophan 9060 153-94-6 antibacterial[40]

8 BruceineD 441788 21499-66-1 anticancer:inhibitshepatocellularcarcinomagrowth[41]

9 202-791 122114 101342-80-7 acalciumchannelagonist[42]

10 Genipin 442424 6902-77-8 anticancer:suppressescolorectalcancercells[43–45]

11 Liquiritigenin 114829 578-86-9 restoresosteoblastdamage[46],attenuatescardiacinjury[47],preventspalmitate-inducedbeta-cellapoptosis[48]

12 Demeclocycline 54680690 127-33-3 forthetreatmentofhyponatremiasecondarytoSIADH[49]

13 KT5720 3844 108068-98-0 reversesmultidrugresistance,achemosensitizer[50–51]

14 Albiflorin 51346141 39011-90-0 amelioratesobesity[52]

15 Cetirizine 2678 83881-51-0 forthetreatmentofallergicrhinitis[53–54]

16 LanatosideC 656630 17575-22-3 anticancer:inducesapoptosisinhumangastriccancercells[55]andhumanhepatocellularcarcinomacells[56]

* Markerconstituents.

e54


1.7µm,130Å)columnwithaninlineVHPfilter(0.5µm,stainlesssteel)fromUpchurchScientific.ThemobilephaseforthepositiveESI-QTOF-MSconsistedofA)0.1%formicacidaqueoussolution(v/v)andB)acetonitrile.ThemobilephaseforthenegativeESI-QTOF-MSconsistedofA)0.05%ammoniumhydroxideaqueoussolution(v/v)andB)acetonitrile.Thegradientelutionprofilewasasfollows:0–4min,5%B;4–7min,5–10%B;7–20min,10–15%B;20–30min,15–22%B;30–35min,22–35%B;35–40min,35–50%B;40–45min,50–70%B;45–50min,70–90%B;50–52min,5%B;52–60min,5%B.Theflowratewas0.4mL/min.Thecolumntemperaturewasat60℃.Thesampleinjectionvolumewas5.00µL.Priortosampleanalyses,thecolumnwasequilibratedwiththemobilephaseforatleast30minataflowrateof0.4mL/min.

TheQTOF-MSwasoperatedinbothpositiveandnegativeESImodes.Thechromatographicandspectradata(.d)wereacquiredforeachESImodeusingAgilentMassHunterDataAcquisitionsoft-ware(VersionB.05.01).Theoperationparametersofthemassspectrometerwereasfollows:dryinggas(N2)temperature,350°C;dryinggasflowrate,10.0L/min;nebulizergas(N2)pressure,35psi;sheathgasflowrate,11.0L/min;sheathgas(N2)temperature,325°C;capillaryvoltage,4000V;nozzlevoltage,500V;fragmen-torvoltage,100V;skimmervoltage,65V;octopoleradio-frequen-cyvoltage,750V;collisionenergy,10,20,and40eV.ThedatawereacquiredbyautoMS/MSmodewithanextendeddynamicrange (2GHz).TheMSscanrangewas50–1000m/zatascanrate5spectra/s.TheMS/MSscanrangewas50–1000m/zatascanrateof3spectra/swithanisolationwidthatnarrow(~1.3m/z).Tomaintainthemassaccuracy,themassspectrometerwascalibrat-edandtunedbeforeanalysis,andinternalreferencemassesfromthereferencemasssolutionwereusedforreal-timemasscorrec-tionatm/z121.0508and922.0098forthepositiveionmode,andm/z112.9885and1033.9881forthenegativeionmodethrough-outtheacquisitionprocess.Thereferencemasssolutionwaspre-paredusinganAgilentAPI-TOFreferencemasssolutionkit(PartNo:G1969-85001).

Method validationThespecificityandreproducibilityoftheUPLC-QTOF-MSmethodwereassessedbyreplicatemeasurementsofsamplesoffourYin-qiaosanpreparationforms.PCAscoreplotswereconstructedusingtheacquiredMSdata,andtheCVswerecalculatedontheconcen-trationsofreplicatemeasurementsof49commonlyidentifiedcompoundsthroughglobalsemiquantitativeanalysis.

Data processing, constituent identification, and statistical analysisThedatafiles(.d)ofreplicatemeasurementsforfourYinqiaosanpreparationformsacquiredbyAgilentMassHunterDataAcquisi-tionsoftwareatthesameionpolarity(i.e.,eitherpositiveornega-tiveESI)werefirstassessedusingAgilentMassHunterQualitativeAnalysissoftware(Version:B.06.00)forthesignal/noiselevel,andretentiontimeandmassshiftswithrespecttothespikedIS,whichwerethenprocessedusingAgilentProfindersoftware(VersionB.06.00)forbatchrecursiveanalysis.Thedatafilesweregroupedintofourpreparationformsandsubjectedtomolecularfeatureex-tractionbyselectingapeakheightthresholdof1000counts,pos-

sibleionadducts,isotopemodelofcommonorganicmolecules,chargestatesuptotwo,aretentiontimewindowof0.10%+0.60min,amasswindowof20.00ppm+2.00mDa(foralignmentoftheISinallrunswiththesamepolarity),andapost-processingfil-terat3outof3replicatemeasurementsforeachYinqiaosanprep-arationform.

Eachmolecularfeatureextracteddatafile(.cef)byProfindersoftwarealongwiththecorrespondingdatafile(.d)byMassHunt-erDataAcquisitionsoftwarewasimportedintoAgilentMassHunt-erQualitativeAnalysissoftware.Using“FindbyFormula”,MS/MSdataalongwithitsMSandretentiontimedatawereextracted.Thenewdatafiles(.cef)inthesameionpolarityofreplicatemeasure-mentsofeachYinqiaosanpreparationformwerethenimportedintoAgilentMassProfilerProfessionalsoftware(Version:B.13.1.1)foranMETLINAMdatabasesearchandmolecularformulagenera-tion.Theselectionofelementsandlimitsformolecularformulagenerationwereasfollows:C(3–156),H(0–180),O(0–40),N(0–20),S(0–14),Cl(0–12),F(0–12),Br(0–10),P(0–9),andSi(0–15)[55].Thetopfiveconstituentswiththehighestscoreswereanno-tated,andcross-checkedwiththeTraditionalChineseMedicineIn-tegratedDatabase[56]andTraditionalChineseMedicineSystemsPharmacologyDatabaseandAnalysisPlatform[57]forfinalnameassignments.Thecombineddatafile(.cef)ofreplicatemeasure-mentsofeachYinqiaosanpreparationformwiththenamesofchemicalconstituentswassavedforglobalsemiquantitativeanal-ysis.

Formultivariatedataanalysis,themolecularfeatureextracteddatafiles(.csv)bytheProfindersoftwarewereimportedintoMe-taboAnalyst4.0[58]intermsofmass,retentiontime,andpeakarea.Themasstoleranceandtheretentiontimetoleranceweresetat0.025and30s,respectively.Samplenormalizationwasper-formedbytheISreferencefeatures(i.e.,mass,retentiontime,andpeakarea),andthedatawerelogtransformedandautoscaled.PCAandPLS-DAwereperformedusingtheISfornormalization.PCAandPLS-DAscoreplotswereconstructedforsimilaritycomparisonofthechemicalconstituentsandreproducibilityassessmentoftheUPLC-QTOF-MSmethodaswellaspatternrecognitionamongthefourYinqiaosanpreparationforms.

Global semiquantitative analysisThedatafiles(.d)withthesameionpolarityofreplicatemeasure-mentsofeachYinqiaosanpreparationformbytheMassHunterDataAcquisitionsoftwareandtheircorrespondingcombineddatafile(.cef)withthenamesofchemicalconstituentsbytheMassProfilerProfessionalsoftwarewereimportedintoAgilentMassHunterQuantitativeAnalysissoftware(Version:B.06.00).Inthemethodsetuptask,theretentiontimewindowwassetat0.6min,etopo-side-d3ammoniumadductwaschosenastheISandflagged,otheridentifiedchemicalconstituentswerechosenastargetsrelativetotheIS,andproperionpolaritywaschosen.Aftervalidatingthemethodsetup,globalsemiquantitativeanalysiswasperformedbasedonthepeakarearatioofeachindividualtargettotheIS,andthequantitationdatawereexportedasexcelfileforreporting.

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Supporting InformationTheMS/MSspectraofthe49chemicalconstituentscommonlyidentifiedinallfourYinqiaosanpreparationforms(▶Fig. 1S),thecommonchemicalconstituentsdetectedbutunidentifiedinallfourYinqiaosanpreparationforms(▶table 1S),andtheuniquecon-stituentsfoundindecoction,granule,pill,andtabletformsofYin-qiaosan(▶tables 2S– 9S)areavailableasSupportingInformation.

AcknowledgementsYachunShuwouldliketoacknowledgethefundingsupportoftheChinaNaturalScienceFoundation(81503216)andJiangsuProv-ince(LGY2016017).

ConflictofInterest

Theauthorsdeclarethattheyhavenoconflictofinterest.

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