Rev. bras. farmacogn. vol.27 número6

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

UPLC/Q-TOF-MS

profiling

of

phenolics

from

Canarium

pimela

leaves

and

its

vasorelaxant

and

antioxidant

activities

Juan

Wu

a

_,

_Xiao’ai

_Fang

a

_,

_Yan

_Yuan

a

_,

_Yanfen

_Dong

b

_,

_Yanling

_Liang

b

_,

_Qingchun

_Xie

a,∗

_,

_Junfeng

_Ban

a

_,

Yanzhong

Chen

a

_,

_Zhufen

_Lv

a,∗

a_Guangdong_Provincial_Key_Laboratory_of_Advanced_Drug_Delivery_Systems,_Guangong_{Pharmaceutical}_University,_Guangzhou_510006,_China

b_College_of_Basic_Medicine,_Guangong_{Pharmaceutical}_University,_Guangzhou_510006,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15September2017 Accepted26October2017 Availableonline15November2017

Keywords:

Phenolic UPLC/Q-TOF-MS Vasorelaxant Antioxidant Biologicalactivity HPLC-DAD

a

b

s

t

r

a

c

t

CanariumpimelaK.D.Koenig,Burseraceae,havealonghistoryofuseintheChinesetraditionalmedicine treatmentofvariousailmentsincludinghypertension,andourresearchteamhasreportedthe anti-hypertensiveactivityanddelineatedthemechanisminvolvedintheaction.Thefollowingresearchaimsto evaluatethevasorelaxantandantioxidantactivitiesofethanolextractfromC.pimelaleavesandtoanalyze itschemicalcompositionbyultra-performanceliquidchromatography-quadrupoletime-of-flightmass spectrometry(UPLC/Q-TOF-MS)thatmaycorrelatewiththeirpharmacologicalactivities.Theresults showedthatpre-incubationofaorticringswiththeextract(0.3,1,3,10,30and100mg/l)significantly inhibitedthecontractileresponseoftheringstonorepinephrine-inducedcontraction(p<0.01orp<0.05). Crudeethanolextractandrefinedethanolextractshowedahighestinhibitoryeffectagainst 2,2dipheyl-2-picrylhydrazylhydratescavengingactivity(IC50ofcrudeethanolextract=15.42±0.14␮g/mlandIC50 ofrefinedethanolextract=5.72±0.31␮g/ml)and2,2′_-azinobis_{(3-ethyl-benzothiazoline-6-sulphonic} acidammoniumsalt)(ABTS(IC50ofcrudeethanolextract=3.24±0.18␮g/mlandIC50ofrefinedethanol extract=1.88±0.07␮g/ml)scavengingactivity,whichwasconsiderablyhigherthanthatreportedfor butylatedhydroxytolueneandlowerofthatmeasuredforascorbicacid.Moreover,itschemical compo-sitionwasanalyzedbyUPLC/Q-TOF-MS.Sixteencompoundsincludingnineflavonoids,fourtannins,two phenolicacidsandonedianthronewereidentifiedforthefirsttimeasconstituentsofthisspecies.Andof this,sixmajorphenoliccomponentsweresimultaneousquantitativeanalysisbyHPLC-UV,chlorogenic acidisthemajorcompoundsinC.pimelaleaves.Theseresultsindicatethatthephenolic-richextractof

C.pimelaleavesisapromisingnaturalpharmaceuticalforcombatinghypertensionandoxidativestress. ©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

In2012and2013intheUnitedStates(Lindsley,2015),themost dispensedprescriptionswereanti-hypertensivedrugsinall dis-eases(698million).From2003to2013,thedeathrateattributable tohighbloodpressureincreased8.2%,andtheactualnumberof deathsrose34.7%(Mozaffarianetal.,2016), andthatthis prob-lemmayincreaseinthecomingdecade.However,thepathogenesis andpathophysiologyofhypertensionisacomplexheterogeneous disorder,ofwhichisapositiveanddirectcorrelationwithother cardiovascularandmetabolicabnormalities.Oxidativestressesor inefficientantioxidant defensesas one ofthe factorsappear to

∗ _{Corresponding}_authors.

E-mails:xieqchun@163.com(Q.Xie),luzhufen@126.com(Z.Lv).

beinvolvedinthedevelopmentandprogressionofhypertension (Wilcox,2005;Elksetal.,2009).

In recent years, traditional and natural medicine have been extensively used to prevent and heal various cardiovascular problemsincludinghypertension(HoandJie,2007)andthe anti-hypertensiveactivity of plantextract has beencorrelated with its antioxidantproperties (Wilcox, 2005; Elks et al., 2009) and flavonoid contents (Hugel et al., 2016). As antioxidants, these compoundsmaypreventtheprogressiveimpairmentofvascular endothelialcells due tooxidativestress toimproveendothelial function.Inaddition,thereisgrowingevidencethatflavonoid-rich dietshavebeenlinkedtoimprovementsinendothelialfunctionand bloodpressure(Hugeletal.,2016).

CanariumpimelaK.D.Koenig(CPL)belongtothefamily Burs-eraceae, locallycalledas“Wulan”and “Chineseblack olive”or “MuWeizi”isthecharacteristiccashcropofGuangdongProvince (China)(Huang and Wang,2009)because ofits medicinalfood

https://doi.org/10.1016/j.bjp.2017.10.005

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homology(JiangsuNew MedicalCollege,1986).It isindigenous tothesoutheastareaofChinaandhasbeenintroducedtoother Asian tropicaland semi-tropicalregions. Thisplant, whichwas madeintodecoction,wasusedinthefolkloremedicinetotreat anti-bacterium, anti-virus,anti-inflammation and detoxification in China(Ding,1999).In recent years,thestudy ofCPLmainly concentratedoncardiovascular.TheCPLleavesextracthas anti-hypertensiveactivity(Lietal.,1997).Itsextractcanhelptoimprove myocardialbloodperfusionandprotectthemyocardium.The liq-uidextractsfromCPLleaveshavea quickeffectofthenegative inotropicactiononisolatedfrogheartsandanti-positiveinotropic actionofCaCl2,andtheactioncanbeblockedbyatropine(Liang etal.,2006).TheextractsfromleavesofCPLhaveabi-directional effectonheartfunction.Aftertransientinhibition,myocardial con-tractilitywouldtransformtointensificationwiththedescending heartrate(Cenetal.,2012).Theuseofisolatedratheartperfusion LangendorffmodelshowedthatCPLleavesliquidextractionmaybe beneficialtoreducecardiacoxygenconsumption,maintainheart function,playaroleinprotectingthemyocardium(Cenetal.,2013, 2014).Thewaterextractsoffruit,leaves,andbarkofCPLall low-eredtheratsbloodpressurequickly,andtheeffectofextractsof fruitandleavesinloweringbloodpressurewasstrongerthanthatof thebarkextracts(Dongetal.,2006),anditwasfoundthattherewas norelationshipbetweenthedepressurizationeffectofCPLextract andthebasalbloodinstressedhypertensionrats(Dongetal.,2007). Intheisolatedratthoracicaorticannulusperfusiontest,foundthat theleaves, fruit,and barkallhavethevasodilatationeffect,and theleaveswerethemostobvious(Liangetal.,2011).Theleaves extractscanrelaxratthoracicringswithoutendothelium,which mechanismis associatedwithactivating␤-receptor,openingof Ca2+_channel_by_the_receptor_dependent_pathway_as_well_as inhibi-tionofCa2+_influx_in_the_vascular_smooth_muscle_cell._The_extracts alsoincreaseaortaringstensioninpre-contractionwithKClbut arenotassociatedwith␣receptor(Dongetal.,2008).Alltheabove studiesarepointedoutthebenefitsofthisplantineffectof hyper-tensionactivities.However,onlythevolatileoilwasreported(Li etal.,2015),thechemicalcompositionofphenolicsofthisplantthat maycorrelatewithitspharmacologicalactivitiesisstillaproblem. In this context, and following our interest on the study of thechemicalcompositionandvalorizationofCPLleaves,theaim of thisstudy is toevaluatethepotentialof CPLas a cash crop forGuangdongProvinceinChina,determiningtheirvasorelaxant activepartsbythoracicaortaringsofrat,theirantioxidant prop-erties determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and 2,2′_-azinobis (3-ethyl-benzothiazoline-6-sulphonic acid ammonium salt) (ABTS) radical scavenging, analyzingtheirethanol:waterextractsbyUPLC/Q-TOF-MS, and alsoquantitativeanalyzedsixphenolic compounds(chlorogenic acid,vitexin,isovitexin,hyperin,rutin,quercetin)byHPLC-UV.

Materialsandmethods

Chemicals

MethanolwasofHPLCgradeandpurchasedfromLabscience, Inc. (Nevada, USA). Distilled water was supplied by Watson (Guangdong,China).Aceticacidandphosphoric acidof analyti-calgradewerepurchasedfromZhiyuanChemicals Ltd.(Tianjin, China).Rutin andquercetin(BatchNo. 100080-201409and No. 100081-201408)wereobtainedfromtheNationalInstitutesfor Food and Drug Control (Beijing, China). Other reference stan-dards (purity ≥98%) including chlorogenic acid (No. 150511), vitexin(No. 151102),isovitexin (No.151204) and hyperin(No. 150615)werepurchasedfromChengduPurechem-standardCo., Ltd.(Sichuan,China).Norepinephrine(No.20150414)were

pur-chasedfromGrandPharmaceutical(China)Co.,Ltd.Acetylcholine chloride(No.60-31-1)andDPPH(No.C10088334)werepurchased from Shanghai MacklinBiochemical Co., Ltd. (Shanghai,China). Isoprenalinehydrochloride(No.20150301)waspurchased from ShanghaiHarvestPharmaceuticalCo.,Ltd.(Shanghai,China).ABTS (No.F1719012)waspurchasedfromAladdinIndustrialCorporation (Shanghai,China).

Preparationofplantextract

ThefreshleavesofCanariumpimelaK.D.Koenig,Burseraceae, werecollectedduringthemid-August2015fromtheherbal gar-denofZhongshancity,Guangdongprovince,China.Theplantwas taxonomicallyidentifiedbyProfessorJizhuLiuandavoucher speci-men(No.20150825)wasdepositedatSchoolofTraditionalChinese Medicine,GuangdongPharmaceuticalUniversity.

Dried and powdered leaf(20g) of CPLwas transferredto a round-bottomflaskandwasextractedwithethanol:water(50:50, v/v) witha solid–liquidratio 1:50 (m/v).The flaskwasheated (70±1◦_C) _in _an _electric _jacket _under _reflux _for ₈₀_min. _The suspensions were filteredand theextract wasvacuum-filtered, concentratedinarotaryvacuumevaporatorat50–60◦_C₍_Jin_et_al., 2010)andfreeze-driedtoyieldthecrudeethanolextraction(CEE) (4.19g). CEEwasthenpurified byAB-8resinand elutedas fol-lows:water2-bedvolume(BV),30%2BV,50%3BVand95%ethanol 2BV.Whentheeluentwasclarified,beginningtocollectiontillthe end.Sugarswereelutedfirstandtheotherswerecombinedand driedasabovetoaffordabrownphenolicextract–refinedethanol extraction(REE)(2.68g).

EvaluationofCEEandREEonvasculartensioninrats

Adult male and female Sprague Dawley (SD)rats (weighing 280–320g) obtainedfromMedical LaboratoryAnimal Centerof GuangdongProvince(No.SCXK( )2013-0002)werehousedunder conventionallaboratoryconditionsthroughouttheperiodof exper-imentation. Animalexperimentswerecarried outfollowing the guide ofExperimental Animal Ethics Committeeof GuangDong PharmaceuticalUniversity(No.gdpu2016500)forthecareanduse ofLaboratoryanimals.Theywerefedwithstandard pelletsand wateradlibitumandmaintainedat22◦_C_temperature,_85%_relative humidity,anda12hdayandnightcycle.

Thepreparationofisolatedratthoracicaorticringswasbased ontheproceduresdescribedbyQinetal.(2014).Contractionof aortaringswasinducedwithnorepinephrine (NE,0.1mM),and inducedtherelaxationwithacetylcholine (Ach,1␮M). The ten-sionwasmeasuredusingforce–displacementtransducer(Beijing, China)coupledwithaMedLabsbiologicalsignalacquisitionsystem (Nanjing,China).

Relaxation,ameasureofinhibitionofcontractioninaorticring pre-contractedwithphenylephrinewasmeasuredinpercentage andcalculatedasEq.(1):

% Relaxation=Tc−Tt

Tc ∗100 (1)

whereTcstandsforchangeintensionaftercontractionwith norepi-nephrine,whileTtstandsforchangeintensionafteraddingextract. Valueswereexpressedasmean±standarddeviation(SD).

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EvaluationofantioxidanteffectsofCEEandREE

DPPHradicalscavengingassay

TheantioxidantactivityoftheextractofCPLleavesevaluated by2,2dipheyl-2-picrylhydrazylhydrate(DPPH)radicalscavenging. Theextractwasdissolvedinethanoltopreparestocksolutionsover theconcentrationrange0.005–0.1mg/ml,whichwasmixedwith 2mlofDPPHsolution.Then,themixturewasincubatedatroom temperaturefor30mininthedark.Theabsorbanceofthe solu-tionwasmeasuredat517nm.Theantioxidantactivitywasalso expressedinmgofascorbicacidequivalents/gofextract(mgAAE/g ofextract).ThescavengingratewascalculatedusingEq.(2):

Radicalscavenging rate (%)=

1−A_sample A_control

×100 (2)

ABTSradicalcationdecolourizationassay

TheantioxidantactivityoftheextractofCPLleaveswasalso evaluated by ABTS radical cation decolourization assay, which were carried out as reported by ˇSliumpait ˙e et al. (2013) and witha slightly modified.Stock solution(7mM)of 2,2′_-azinobis (3-ethyl-benzothiazoline-6-sulphonicacidammoniumsalt)(ABTS) waspreparedby dissolvingit in 2.45mMpotassium persulfate solutionandstoring themixturein thedarkat room tempera-turefor15–16hbeforeuse.TheABTS•+_stock_solution_was_diluted withphosphatebuffer(PBS,10mM,pH7.4)beforemeasurements toobtaintheabsorptionof0.800±0.030at734nm.Theextractwas dissolvedinPBSanddilutedtoaseriesofconcentrations(0.05,0.10, 0.20,0.23,0.26,0.30mg/ml).A4mlofABTS•+_solution_was_mixed with40␮loftheextractsolutioninthedark,theabsorptionwas readafter10min.Allmeasurementswereperformedintriplicate. AnditsabilitytoscavengeABTS•+_is_compared_with_ascorbic_acid.

Determinationoftotalphenolicsandflavonoidscontents

Thetotalphenoliccontentsoftheextractsweredeterminedby Folin–CiocalteumethodbasedontheproceduresdescribedbySiger etal.(2008).Gallicacidwasusedtocomputethestandard calibra-tioncurve(2,4,6,8,10␮g/ml).Alloftheresultswereexpressedas mgofgallicacidequivalentspergramextract(mgGAE/gofextract). Theanalyseswerecarriedoutintriplicateandtheaveragevalue wascalculatedineachcase.

TotalflavonoidswerecarriedoutasreportedbyIbrahimetal. (2015).Rutinwasusedasstandardandtheequivalents(w/w)were determinedfromastandardconcentrationcurve(0.05,0.10,0.20, 0.30,0.40,0.50mg/ml).Determinationswerecarriedoutin trip-licates;resultswererepresentedasthemean values±standard deviationsandexpressedasmgrutinequivalentspergramextracts (mgRE/gofextract).

UPLC-Q-TOF/MSanalysis

Theleafpowderwasdissolvedinaqueousmethanol(50%(v/v)) inanultrasonic bath(Shumei KQ-300DAultrasonic instrument, Kunshan,China)for30minandthemixturewascentrifugedthrice with9055.8×g for 10min.The clearsupernatant wasused for UPLC-Q-TOF/MSanalysis.

AcquityUPLCsystem(WatersCo.,Massachusetts,USA)coupled withMicro-massQ-TOFmicro(WatersCo.,Massachusetts,USA). UPLCwasusedasaseparationmeansequippedwithabinary sol-ventdeliverysystem,anauto-sampler,andanAcquityUPLCTM_BEH C18column(4.6×150mm,5␮m,WatersCo.,USA).Agradient elu-tionwasachievedusingtwomobilephases:0.1%(v/v)aqueous aceticacid(solventA)andmethanol(B)ataflowrateof0.21ml/min withthefollowinggradientconditions:0min,22%B;3.6min,29% B;4.0min,33%B;12.6min,35%B;15.6min,70%B;17.6min,70%

CEE REE

80

70

60

50

40

30

20

10

0

Chlorogenic acid Vitexin

Isovitexin hyperin Rutin Quercetin

Content(mg/g of e

xtr

act)

Fig.1.ThedifferencesinthecontentsofthesixcomponentsinCEEandREEwere determinedbyHPLC-UV.

B;22min,22%B.Thesampleinjectionvolumewas5␮landthe columntemperaturewasmaintainedat30◦_C.

MassspectrometricanalyseswereperformedonMicro-mass Q-TOFMicroSystemequippedwithanelectrosprayionization(ESI) sourceinnegativeion mode.Theoptimized parametersforthe mass spectrometric analysiswere as follows: capillary voltage, 3000V;sampleconevoltage,30V;extractionconevoltage,2.0V; desolvationgasflowrate,600l/h;desolvationgastemp,300◦_C; collisionenergies,30–35eV.Allmassdataacquisitionwere per-formedonMassLynxsoftware(Version4.1,Waters,Cicero,USA) usinganindependentreferencesprayviaaLockSprayTM_interface toensureaccuracy,lockingthe[M−H]− _of_leucine_enkephalin_at m/z554.2615andtheQ-TOFacquisitionratewassetas0.1sand theinter-scandelayas0.02(Wuetal.,2015).

Quantification

QuantitativeanalyseswereperformedonanAgilent1260 Infin-ity series equipped with a UV detector. The chromatographic separationwascarriedoutonanAgilentZORBAXSB-C18column (250mm×4.6mm,5␮m,AgilentCo.,USA)withthemobilephase consistingof0.5%(v/v)aqueousphosphoricacidandmethanol(B). Thegradientelutionprogramwasasfollows:0min,23%B;8min, 32%B;30min,35%B;50min,70%B;55min,23%B.Theflowrate wasat1ml/min,theUVdetectionwavelengthwassetat360nm, theinjectionvolumewas20␮landthecolumntemperaturewas maintainedat30◦_C.

CalibrationcurveswereobtainedbyHPLCinjectionof chloro-genicacid, vitexin,isovitexin,hyperin,rutin,quercetinstandard solutionsin50%(v/v)aqueousmethanolwiththeconcentration of571.87,25.70,34.96,25.06,55.14and9.91␮g/ml,respectively. Thedatarelevantforobtainingthecalibrationcurvesareshown inTable1.Quantificationofindividualcompounds(Table2,Fig.1) wasobtainedusingthecalibrationdataofthecommercialstandard. Compoundsconcentrationswerecalculatedintriplicateandthe meanvaluecalculatedineachcase.

Statisticalanalysis

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Table1

Regressionequation,correlationcoefficients,linearityranges,LODandLOQforsixanalytes.

Compounds Linearityranges(mg/l) Calibrationcurvea ₍_r2₎ _LODb_(mg/l) _LOQc_(mg/l)

Chlorogenicacid 57–570 y=0.2402x+0.3208 0.9998 0.13 0.48 Vitexin 2.5–25 y=0.3108x− 0.1698 0.9996 0.52 1.89 Isovitexin 3.5–35 y=0.6288x− 0.3924 0.9994 0.44 1.12 Hyperin 2.5–25 y=0.642x − 0.3703 0.9995 0.41 1.02 Rutin 5.5–55 y=0.5521x−0.3689 0.9995 0.97 2.53 Quercetin 1–10 y=0.9747x−0.4955 0.9978 0.21 0.65

a_y₌_peak_area,_x₌_{concentration}_in_mg/l_of_extract. b_LOD,_limit_of_detection.

c _LOQ,_limit_of_{quantification.}

Table2

HPLCquantificationofsixcomponentsidentifiedinCPLleavesextracts.

150612 150820 150930

Means±SD(mg/g) RSD(%) Means±SD(mg/g) RSD(%) Means±SD(mg/g) RSD(%)

Chlorogenicacid 13.39±0.09 0.70 6.71±0.09 1.39 13.42±0.20 1.51

Vitexin 1.63±0.03 1.80 0.56±0.01 1.43 1.05±0.01 1.06

Isovitexin 0.93±0.01 0.66 0.47±0.01 0.92 0.86±0.02 1.76

Hyperin 1.04±0.01 0.86 0.51±0.01 1.35 0.88±0.11 0.89

Rutin 2.04±0.04 1.88 0.85±0.02 1.91 1.62±0.02 1.00

Quercetin 0.35±0.01 1.49 0.16±0.01 1.37 0.37±0.01 0.65

Total(mg/gofleaf) 19.38±2.28 9.26±0.025 18.65±0.06

Allvaluesareexpressedasmean±standarddeviation(n=3).

Resultsanddiscussion

Totalphenolsandflavonoidscontents

TotalphenolsandflavonoidscontentsofCEEandREEofC.pimela

leavesweredeterminedinthisstudy(Table3).Thevalueofthe totalphenoliccontentofCEEandREEwere259.1±55(mgGAE/g ofextract)and 415.6±2.37(mgGAE/g ofextract),respectively. AndthatofthetotalflavonoidsofCEEandREEwere373.9±6.56 (mgRE/gofextract)and824.1±5.86(mgRE/gofextract), respec-tively.Theresultrevealedthatthetotalphenolic(approximately 1.6-fold)andflavonoids(approximately2.2-fold)contentofREE wereincreasedcomparedwithCEE,andthedifferencesbetween CEEandREEweresignificant(p<0.01orp<0.001).Thatwaslikely duetotheproteins,carbohydratesorothersmallpolarhydrophilic moleculesremoval.

EffectofCEEandREEonvasculartensioninrats

Having a relaxingeffect onblood vessels is themost direct effecttoantihypertension. Studieshave reportedthat theeffect ofrelaxingtheaorticringshowedanon-endothelium-dependent vasodilation by Dong et al. (2008). So the relaxation effect of extracts on endothelium-intact aortic rings pre-contracted with 0.1mM NE was evaluated at the addition of cumulative concentrations (0.3, 1.0, 3.0, 10.0, 30.0 and 100mg/ml) in the presentstudy.

The result revealed that CEE (Emax=92.34±9.78%) and REE (Emax=100.18±8.08%)ofCPLleavesextractsboth havea relax-ingeffectonthoracicaortaringspre-contractedwithNEcompared to the normal control group (saline group), and showing a concentration-dependentrelaxeffects(Fig.2).Isoproterenol(ISO), a␤receptoragonist,whichcanexpansionofperipheryblood ves-selasapositivecontrolgroup.ThevasorelaxanteffectofCEEgroup (10,30and100mg/ml),REEgroup(3,10,30and100mg/ml)and ISOgroup(3,10,30and100mg/ml)showsalargesignificance com-paredtothenormalcontrolgroup(p<0.05orp<0.01)(Table4).In addition,bothCEEgroupandREEgrouphaveasimilareffecttoISO group(EC50=0.73±0.00064mg/l)(Fig.2).Atthesame concentra-tion,thevasorelaxanteffectofREEgroup(EC50=5.2±0.002mg/l)

150

100

50

0

-50

-3 -2

IgCa_(g/L)

CEE REE Control

Relaxtion(%)

-1 0

Fig.2. EffectsofCEEandREEofCPLleafonvasorelaxantactivityinrataorticrings. Allvaluesareexpressedasmean±standarddeviation(n=10).a_IgC,_cumulative

concentration.

wasstrongerthanCEEgroup(EC50=10±0.007mg/l).Basedonthe resultsofthetotalphenolicandflavonoidscontentoftheextracts, itisspeculatedthattheactivecompoundsofvasodilationmaybe relatedtoitsphenolicandflavonoidscomponents.

Antioxidantactivity

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Table3

Extractionyields,totalphenoliccontentandflavonoidscontentofCEEandREEofCPLleafextracts.

Exstracts Extractionyield(%) Totalphenoliccontent(mgGAEa_/g_of_extract) _Flavonoids_content_(mg_REd_/g_of_extract)

CEEb _20.95 _259.1_±_5.5 _373.9_±_6.56

REEc _13.4 _415.6_±_2.37** _824.1_±_5.86

ComparewiththetotalphenoliccontentofCEE,**p<0.01. ComparewiththeflavonoidscontentofCEE,_p_<_0.001.

Valuesoftotalphenoliccontentareexpressedasmean±standarddeviation(n=3).

a_GAE,_gallic_acid_equivalents. b _CEE,_crude_ethanol_extract. c _REE,_refined_ethanol_extract. d _RE,_rutin_equivalents.

Table4

VasorelaxationeffectsofCEEandREEonrataorticrings.

Number Blankgroup Isoproterenolgroup(ISO) CPLleafextractgroup

Relaxation(%) FinalconcentrationlgC(g/l) Relaxation(%) CEE REE

FinalconcentrationlgC(g/l) Relaxation(%)

1 0.63±1.47 −5.5 0.9±2.03 −3.5 2.16±2.81 2.98±3.51

2 3.05±2.33 −5.0 8.64±7.45 −3.0 4.68±5.61 5.89±5.65

3 6.81±3.70 −4.5 18.76±9.1** −2.5 14.25±14.69 44.00±13.54**

4 10.57±6.16 −4.0 34.72±16.7** −2.0 65.77±19.49** 91.25±11.91**

5 13.84±6.42 −3.5 50.62±14.18** −1.5 90.38±11.9** 100.18±8.08**

6 19.23±10.38 −3.0 54.16±11.05** −1.0 92.34±9.78** 95.97±7.88**

Comparewithblankgroups,**p<0.01;comparedwithCFE,_p_<_0.05,_p_<_0.01. Allvaluesareexpressedasmean±standarddeviation(n=10).

Table5

Antioxidantactivity.

IC50ofDPPH •

(␮g/ml)

IC50ofABTS •₊

(␮g/ml)

mgAAEa_/g_of

extracts Ascorbicacid 4.23± 0.23 1.78± 0.01 – BHT 20.87± 0.40 – – CEE 15.42± 0.14 3.24± 0.18 274.39± 2.54 REE 5.72±0.31*** 1.88±0.07 _740.52_±_40.65

ComparewithCEE,***p<0.001,_p_<_0.001.

Allvaluesareexpressedasmean± standarddeviation(n=3).

a_AAE,_ascorbic_acid_equivalents.

observed that the ascorbic acid (IC50=4.23±0.23␮g/ml) was onlyslightly more effective DPPH• scavenger compared toREE (IC50=5.72±0.31␮g/mlofextracts),andREEwasmoreeffective DPPH• scavenger compared to CEE(IC50=15.42±0.14␮g/ml of extracts)andBHT(IC50=20.87±0.40␮g/ml),inaddition,the dif-ferencesweresignificant(p<0.001).

ABTS•+ _{decolorization} _assay _gave _quite _similar _results com-paredtothoseobtainedbytheDPPH• assay(Table5).Statistical analysisprovedthat thereweresignificantdifferencesbetween CEE(IC50=3.24±0.18␮g/ml)andREE(IC50=1.88±0.07␮g/ml)in ABTSradicalcationdecolourizationassay(p<0.001),andthe ascor-bicacid(IC50=1.78±0.01␮g/ml)wasonlyslightlymoreeffective scavengercomparedtoREE.

Theantioxidantactivityresultsrevealed thatleavesextracts haveanantioxidantactivityconsiderablyhigherthanthatreported for BHTand lower of thatmeasured for ascorbicacid, and the antioxidantactivityofREEisbetterthanCEE.Sotheextractsmaybe couldprotectthebloodvesselsfromthedetrimentaleffects pro-ducedbyoxidativestressandplayaroleinrelaxingbloodvessels andloweringbloodpressure(Hugeletal.,2016).

AnalysisphenoliccompoundsintheleafextractofCPL

Theidentificationofthecomponentswascarriedoutby HPLC-UV,UPLC-MS/MS.Atotal16compoundshavebeenidentifiedand tentativeidentifiedbycomparingtheirspectraldatawithreference compounds,whenavailable,orcorroboratedwiththeliterature.

100

%

0

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

Relativ

e ab

undance

1

3 4 5 6

7 8

9 10

11 13 14 ₁₅

16 17

12 2

Fig.3.BasepeakchromatogramsdatadetectedinCanariumpimelaL.leaves.

Thebase-peakchromatographyof themethanolextractfromC. pimelaleaveswasdisplayedinFig.3.UPLC-MSdataforthe iden-tifiedcompounds,namelytheirretentiontime,themolecularion [M−H]−_and_the_main_product_ions_obtained_by_UPLC-MS/MS_were providedinTable6.Identifiedphenoliccompoundsbelongedto variousclassesincludingnineflavonoids,fourtannins,two pheno-licacidsandonedianthrone.

Twophenolicacidscompounds(1and2)wereidentified accord-ingtothefragmentation.Peak1wasidentifiedasthequinicacid inthenegative mode,revealing thediagnosticprecursorion of m/z191at 0.800min andproduct ionspectra withm/z 85 and 93,consistingofdatafromtheliterature.Peak2wasassignedto chlorogenicacid(3-O-caffeoylquinicacid)duetothepresenceof characteristicproductionsatm/z191[quinicacid-H]−_,₁₇₉_[caffeic acid-H]−_and₁₆₁_[caffeic_acid-H

2O-H]−.Itwasfurtherconfirmed byreferenceaswell.

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Table6

PhytochemicalconstituentsidentifiedinCanariumpimelaleavesextracts.

Peakno. Rt(min) [M−H]−

(m/z) Calculated(m/z) Formula Fragmentions(m/z) Identifiedcompounds Ref/Std.

1 0.800 191.0576 191.0556 C7H12O6 85,93,127 Quinicacid aRef.

2 1.787 353.8078 353.1036 C16H18O9 191,179,135 Chlorogenicacid bStd.

3 2.635 633.0703 633.0728 C27H22O18 463,301 Corilagin aRef.

4 2.839 401.1617 401.1423 C25H22O5 269,161,101 Unkonwn

-5 3.112 505.1626 505.1506 C30H18O8 343,293,169,151 Protohypericin aRef.

6 3.672 451.1029 451.1501 C24H20O9 341,217,177 CinchonainI aRef.

7 5.420 431.0978 431.1065 C21H20O10 311,283 Vitexin bStd.

8 6.148 431.0987 431.0992 C21H20O10 341,311,283 Isovitexin bStd.

9 6.494 463.0876 463.1071 C21H20O12 300 Hyperoside bStd.

10 6.721 609.1458 609.1792 C27H20O16 300,179,151 Rutin bStd.

11 7.403 433.0771 433.0820 C20H18O11 300,271,255 Quercetin-3-O-arabinoside aRef.

12 10.132 461.0773 461.0720 C21H18O12 315,300 4′-O-methyellagicacid-3-O-␣-l-rhamnopyranoside a_Ref.

13 10.635 451.1029 451.1019 C24H20O9 341,217,189 CinchonainIisomer aRef.

14 12.685 475.0904 475.0877 C22H20O12 460,328,313 3,3′-Di-O-methylellagicacid-4-O-rhamnopyranoside aRef.

15 14.503 301.0331 301.0292 C15H10O7 179,151,121 Quercetin bStd.

16 15.764 343.1539 343.1545 C18H16O7 328,191,163 3,4,3′-Tri-O-methylellagicacid aRef.

17 16.705 537.7937 537.1107 C30H18O10 417,375 Amentoflavone aRef. a_Ref.:_Identified_by_comparison_with_references.

b_Std.:_Identified_by_comparison_with_a_standard.

hexoside, ellagicacidand gallic acidrespectively (Zhang et al., 2016).Peak3exhibitedtheprecursorion[M−H]−_at_m/z₆₃₃_and ionfragmentswere463[M-galloyl-H2O-H]−and301[ellagic acid-H]−_,_coincident_with_a_gallic_group_and_hexosyl_residue,_indicating thatthiswasprobablyanHHDP-hexosyl-gallate,thuspeak3was identifiedascorilagin(Daetal.,2010).Themolecularionofpeak 12was[M−H]−_,_m/z_461._It_yielded_the_major_fragment_at_m/z₃₁₅ withalossofarhamnoseresidue(146u),andafurtherlossofa methylradical(15u)presentanothercharacterizedfragmentat m/z300,whichwasproposedas4′_-O-methyl_ellagic_acid-3-O-_␣_-_l -rhamnopyranosewiththereference(Lietal.,2013).Peak14shared thesimilarfragmentationregularitieswithpeak12,whichisthe lossofrhamnoseresidueandmethyl,withtheexceptionthatpeak 14losttwomethylradicals(15u)duringtheprocess.Thus,peak 14wastentativelyidentifiedas3,3′_-di-O-methyl_ellagic acid-4-O-rhamnopyranose(Siratetal.,2010).Peak16wasassignedtobe methylellagicacidduetoitssamefragmentpatternwithreference identifiedas3,4,3′_{-tri-O-methyl}_ellagic_acid_with_the_molecular_ion atm/z343anddiagnosticfragmentsatm/z328,191and163(Gao etal.,2014).

Furthermore,nineflavonoids(6,7,8,9,10,11,13,15and17) wereidentifiedintheCPLleavesextract.Peak6and13were ten-tativelyproposedascinchonainIandcinchonainIisomer(Zhang etal.,2016)basedontheirsamemolecularion[M−H]−_at_m/z₄₅₁ (C24H20O9)andsimilarmajorfragmentionsat341[M-C6H6O2-H]− and217[M-2C6H6O2-CH2-H]−,whichrevealedthepresenceoftwo dehydrobenzeneringsandtwomethylene.Peak7and8exhibited thesameprecursorion[M−H]−_at_m/z₄₃₁_in_the_negative_mode ofESI/MSspectra.Twomainfragmentionspresentedatm/z341 ([M−H−90]−_),_and₃₁₁_([M

−H−120]−_),_which_confirmed_that_these compoundsaremono-C-glycosideflavonoids.Ithasbeen demon-stratedthataslightdifferencetodistinguishthepositionofthe sugarresidueistoobservethepeakofthe[M−H−18]−_fragment ion.Whenthisispresent,itmeansthesugarsubstituentlieson the6positionasincompound8(isovitexin).Onthecontrary,its absencerevealsthatthesugarislocatedinposition8ascompound (7vitexin)(Gattusoetal.,2006).Thesefragmentationsare consis-tentwiththedatafromliteratureandonlinedatabases(Daetal., 2010).Bothtwocompoundswerefurtherconfirmedbythe authen-ticstandardsaswell.Peak9,peak10,peak11,peak15andpeak 17presentedthesamefragmentionatm/z301withtheformula C15H9O7,whichiscorrespondingtotheaglyconflavonolquercetin. Peak9, peak 10 andpeak 15 werecharacterized ashyper side (quercetin-3-␤-d-galactoside),rutin(quercetin-3-␤-d-rutinoside)

andquercetin,andconfirmedbytheauthenticstandardsand

lit-1

2

0 10 20 30 40 50 60 70

3 4 6

6

t / min

A

B C

5

5 4 3 1

Fig.4.HPLCchromatogramofmixedreferencessolution(A),samplesolution(B), andblanksolution(C)at360nm.(1)Chlorogenicacid,(2)vixetin,(3)isovitexin,(4) hyperin,(5)rutin,(6)quercetin.

erature(Hsuetal.,2017;Zhouetal.,2014)accordingtotheMS andMS/MSdata.Peak11wasconsideredtobequercetin-3-O-␣-l

-arabinofuranoside,yieldtheprecursorion[M−H]−_at_m/z₄₃₃_and majorfragmentionatm/z300([M−H−133]−_),_a_typical_loss_of arab-inoseresidue,whichisconsistentwithfragmentionsreportedin theliterature(Lietal.,2009)andMassbankdatabase.Peak17with themolecularion[M−H]−_at_m/z₅₃₇_and_major_MS2_fragments_at m/z417and375,wasassignedtoamentoflavoneduetoitssame fragmentpatternwithreference(Zhouetal.,2014).

Peak5presentamolecularion[M−H]−_at_m/z₅₀₅_(C

30H18O8), andMS2_fragments_at_m/z_343,_293,₁₆₉_and₁₅₁_were_tentatively identifiedasprotohypericinaccordingtoreference(Holscheretal., 2009).

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acid(69.1%,72.5%,and72.0%)possessedthehighestshareamong thesixcomponentsfromtheextractinthreebatches.Andthetotal amountsofsixcompoundsofCEEandREE(Fig.1)identifiedby HPLCfollowsatrendsimilartothatoftotalphenoliccontentand flavonoidscontent,whichwasfurthervalidatedthatpolyphenols maybeactiveingredients.

Inthecurrentstudy,16compoundsoftheextractwere char-acterized by using UPLC/Q-TOF-MS technique, and all the 16 compoundsarereferencedforthefirsttimeasconstituentsofCPL leaves.UPLC/Q-TOF-MSanalysisrevealedthataqueousmethanol extractcontainsacomplexmixtureofphenoliccompounds includ-ingphenolic acids and flavonoids, of which chlorogenicacid is themajorcompoundsin C.pimelaleaves. Additionally, mostof thesewerereportedtohavevasorelaxantandantioxidanteffects.It wasreportedthatchlorogenicacidcanreduceoxidativestressand improvesNObioavailability,leadingtotheattenuationof endothe-lialdysfunctionandhypertension(Suzukietal.,2006).Quercetin was reported to possess anti- hypertensive action (Marunaka etal.,2017).Isovitexinandvitexinwerereportedtohavea relax-ationeffectonagonist-inducedvascularcontractionregardlessof endothelialfunction(Je etal.,2014).Rutincan improvekidney andheartstructureandfunction(bloodpressure,leftventricular stiffness,vascular responses,echocardiog., histol.)(Diwan etal., 2017).However,furtherseparationandpurificationofbioactive compoundsintheleafextracts,aswellastheelaborationoftheir molecularmechanismsarewarrantedinthenextstudy.

Conclusion

Inthiswork,wehaveevaluatedthevasorelaxantand antiox-idant activity of the ethanolic of CPL leaves, determined the phenolic as active ingredients, and analyzed the compounds by UPLC-MS. Sixteen compounds were identified or tenta-tive identified including two phenolic acids, quinic acid and chlorogenic acid, four tannins, corilagin, 4′_-O-methyl _ellagic acid-3-O-␣-l-rhamnopyranose,3,3′-di-O-methylellagic

acid-4-O-rhamnopyranose and 3,4,3′_{-tri-O-methyl} _ellagic _acid, _and _nine flavonoids, cinchonain I, cinchonain I isomer, vitexin, isovi-texin, hyperin, rutin, quercetin-3-O-arabinoside, quercetin and amentoflavone,andananthrone,protohypericin.Allthe compo-nentsweredeterminedforthefirsttime asconstituentsof this species.Furthermore,sixofthesecompounds,including chloro-genicacid,vitexin,isovitexin,hyperin,rutin andquercetinwere simultaneouslyanalyzedbyHPLC-UVforthefirsttime.The cur-rentresultsindicatethatthephenolic-richextractofCPLleaves isapromisingnaturalpharmaceuticalforcombatinghypertension andoxidativedamages.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthepublication of patientdata.

Right to privacy and informed consent. The authors have

obtainedthewritteninformedconsentofthepatientsorsubjects mentionedinthearticle.Thecorrespondingauthorisinpossession ofthisdocument.

Authors’contributions

YFDandYLLcollectedandidentifiedthesamples.JWandYY performedtheexperimentsinterpretedtheresults,andJWdrafted themanuscript.XAFconductedtheUPLC/Q-TOF-MSanalysisand designedthestudy.QCXandYZCsupervisedthelaboratorywork. ZFLandJFBinterpretedtheresultsandcontributedtocritical read-ingofthemanuscript.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Theauthorsgratefullyacknowledgethetechnicalhelpprovided by Professor Rui in Central Laboratory, Guangdong Pharma-ceutical University. This research was financiallysupported by theGuangdongScienceand TechnologyDepartment (GrantNo. 2013B020311020) and Guangdong R&D Team for Formulation Innovation(GrantNo.2015KCXTD026).

References

Cen,B.H.,Jiang,W.N.,Liu,P.F.,Lin,J.C.,Liang,Y.L.,Dong,Y.F.,2012.Experimental studyontheeffectoftheliquidextractsofleavesofCanariumpimelaKoening onthecardiachemodynamic.J.Med.TheoryPract.25,747–749.

Cen,B.H.,Jiang,W.N.,Liu,P.F.,Lin,J.C.,Dong,Y.F.,Liang,Y.L.,2013.Experimental studyontheeffectoftheliquidextractsofCanariumpimelaLeenhontherats’

exvivocardiacperfusion.ChinaPract.Med.8,4–5.

Cen,B.H.,Jiang,W.N.,Liu,P.F.,Lin,J.C.,Liang,Y.L.,Dong,Y.F.,Lv,Z.F.,2014.Theimpact ofthediverseextractsofCanariumpimelaLeenhleavesonisolatedheartofthe rat.Mod.Med.J.China01,1–4.

Ding,B.P.,1999.PharmacologyofQingguopillsonrelievingcough.ChinaTrad. PatentMed.21,27–28.

Dong,Y.F.,Liang,Y.L.,Luo,J.P.,Luo,Y.,2006.Experimentontheeffectofdifferent partsofCanariumpimelaKoeningonnormalrats’bloodpressureandheartrates. Int.Med.HealthGuidanceNews17,8–9.

Dong,Y.F.,Liang,Y.L.,Wang,J.H.,Luo,Y.,2007.Depressurizationeffectofextractsof leaveofCanariumpimelaKoeninginstress-inducedhypertensiverats.Tianjin Med.J.35,587–589.

Dong,Y.F.,Liang,Y.L.,Luo,Y.,Li,Y.L.,2008.TheeffectofCanariumpimela Koen-ingleavesonratthoracicaortasmoothmusclecells.GuangdongMed.J.29, 1780–1783.

Da,S.C.,Trevisan,M.T.,Rios,J.B.,Erben,G.,Haubner,R.,Pfundstein,B.,Owen,R.W., 2010.Secondaryplantsubstancesinvariousextractsoftheleaves,fruits,stem, andbarkofCaraipadensifoliaMart.FoodChem.Toxicol.48,1597–1606.

Diwan,V.,Brown,L.,Gobe,G.C.,2017.Theflavonoidrutinimproveskidneyand heartstructureandfunctioninanadenine-inducedratmodelofchronickidney disease.J.Funct.Foods33,85–93.

Elks,C.M.,Mariappan,N.,Haque,M.,Guggilam,A.,Majid,D.S.,Francis,J.,2009.

ChronicNF-{kappa}Bblockadereducescytosolicandmitochondrialoxidative stressandattenuatesrenalinjuryandhypertensioninSHR.Am.J.Physiol.Renal Physiol.296,F298–F305.

Elks,C.M.,Reed,S.D.,Mariappan,N.,Shukitt-Hale,B.,Joseph,J.A.,Ingram,D.K., Fran-cis,J.,2011.Ablueberry-enricheddietattenuatesnephropathyinaratmodelof hypertensionviareductioninoxidativestress.PLoSONE6,e24028.

Gao,X.,Wu,J.,Zou,W.,Dai,Y.,2014.Twoellagicacidsisolatedfromrootsof San-guisorbaofficinalisL.promotehematopoieticprogenitorcellproliferationand megakaryocytedifferentiation.Molecules19,5448–5458.

Gattuso,G.,Caristi,C.,Gargiulli,C.,Bellocco,E., Toscano,G.,Leuzzi, U.,2006.

Flavonoidglycosidesinbergamotjuice(CitrusbergamiaRisso).J.Agric.Food Chem.54,3929–3935.

Ho,J.W.,Jie,M.,2007.Pharmacologicalactivityofcardiovascularagentsfromherbal medicine.Cardiov.Hematolog.AgentsMed.Chem.5,273–277.

Holscher,D.,Shroff,R.,Knop,K.,Gottschaldt,M.,Crecelius,A.,Schneider,B.,Heckel, D.G.,Schubert,U.S.,Svatos,A.,2009.Matrix-freeUV-laserdesorption/ionization (LDI)massspectrometricimagingatthesingle-celllevel:distributionof sec-ondarymetabolitesofArabidopsisthalianaandHypericumspecies.PlantJ.60, 907–918.

Huang,H.Y.,Wang,Z.M.,2009.Theproductivesituationandcountermeasuresof

CanariumpimelaLeethinPuningcity.ChinaFruitNews26,30.

Hsu,B.Y.,Lin,S.W.,Inbaraj,B.S.,Chen,B.H.,2017.Simultaneousdeterminationof phenolicacidsandflavonoidsinChenopodiumformosanumKoidz.(djulis)by HPLC-DAD-ESI-MS/MS.J.Pharm.Biomed.Anal.132,109–116.

(8)

Ibrahim,R.M.,El-Halawany,A.M.,Saleh,D.O.,ElNaggar,E.M.B.,El-Shabrawy,A.E.O., El-Hawary,S.S.,2015.HPLC-DAD-MS/MSprofilingofphenolicsfromSecurigera securidacaflowersanditsanti-hyperglycemicandanti-hyperlipidemic activi-ties.Rev.Bras.Farmacogn.25,134–141.

JiangsuNewMedicalCollege,1986.TheDictionaryofChineseMedicinalPlant, Shanghai.ShanghaiSci.Technol.Press,pp.467–472.

Jin,S.N.,Wen,J.F.,Kim,H.Y.,Kang,D.G.,Lee,H.S.,Cho,K.W.,2010.Vascular relax-ationbyethanolextractofXanthocerassorbifoliaviaAkt-andSOCE-eNOS-cGMP pathways.J.Ethnopharmacol.132,240–245.

Je,H.G.,Hong,S.M.,Je,H.D.,Sohn,U.D.,Choi,Y.S.,Seo,S.Y.,Min,Y.S.,Chung,S.J.,Shin, Y.K.,Lee,T.J.,Park,E.S.,Jeong,J.H.,2014.Theinhibitoryeffectofvitexinonthe agonist-inducedregulationofvascularcontractility.Pharmazie69,224–228.

Li,J.,Dong,Y.F.,Liang,Y.L.,Tang,H.Q.,1997.Studyontheantihypertensiveeffectof aqueousextractofCanariumpimelaKoeningleaf.Inform.Trad.Chin.Med.6,39.

Liang,Y.L.,Dong,Y.F.,Luo,Y.,Luo,J.P.,2006.Experimentontheeffectoftheaqueous solublepartofCanariumpimelaKoeningoftheleavesextractsonbloodpressure andheartratesofrats.ActaAcad.Med.Jiangxi46,42–45.

Li,C.,Du,H.,Wang,L.,Shu,Q.,Zheng,Y.,Xu,Y.,Zhang,J.,Zhang,J.,Yang,R.,Ge, Y.,2009.Flavonoidcompositionandantioxidantactivityoftreepeony(Paeonia

sectionmoutan)yellowflowers.J.Agric.FoodChem.57,8496–8503.

Liang,Y.L.,Luo,Y.,Li,Y.L.,Dong,Y.F.,2011.EffectoffruitofCanariumpimelaKoening onvasculartensioninrats.Chin.J.Gerontol.31,3099–3100.

Li,L.,Zhao,Y.,Liu,W.,Feng,F.,Xie,N.,2013.HPLCwithquadrupoleTOF-MSand chemometricsanalysisforthecharacterizationofFoliumTurpiniaefrom differ-entregions.J.Sep.Sci.36,2552–2561.

Lindsley,C.W.,2015.2013Trendsandstatisticsforprescriptionmedicationsin theUnitedStates:CNShighestrankedandrecordnumberofprescriptions dis-pensed.ACSChem.Neurosci.6,356–357.

Li,Z.F.,Li,K.,Li,F.Y.,Chen,X.,Zhao,H.H.,Du,Y.Y.,Li,Q.P.,2015.GC–MSanalysis andantioxidantactivityofvolatileoilfromleavesofCanariumpimelaLeenh.in Rongxiancounty.J.SouthernAgric.46,317–321.

Mozaffarian,D.,Benjamin,E.J.,Go,A.S.,etal.,2016.Executivesummary:heart diseaseandstrokestatistics-2016update:areportfromtheAmericanHeart Association.Circulation133,447–454.

Marunaka,Y.,Marunaka,R.,Sun,H.,Yamamoto,T.,Kanamura,N.,Inui,T.,Taruno, A.,2017.Actionsofquercetin,apolyphenol,onbloodpressure.Molecules22,

http://dx.doi.org/10.3390/molecules22020209,209/1–209/12.

Newman,D.J.,Cragg,G.M.,2007.Naturalproductsassourcesofnewdrugsoverthe last25years.J.Nat.Prod.70,461–477.

Qin,X.,Hou,X.,Zhang,M.,Liang,T.,Zhi,J.,Han,L.,Li,Q.,2014.Relaxationofrataorta byfarrerolcorrelateswithpotencytoreduceintracellularcalciumofVSMCs.Int. J.Mol.Sci.15,6641–6656.

Suzuki,A.,Yamamoto,N.,Jokura,H.,Yamamoto,M.,Fujii,A.,Tokimitsu,I.,Saito, I.,2006.Chlorogenicacidattenuateshypertensionandimprovesendothelial functioninspontaneouslyhypertensiverats.J.Hypertens.24,1065–1073.

Siger,A.,Nogala-Kalucka,M.,Lampart-Szczapa,E.,2008.Thecontentand antioxi-dantactivityofphenoliccompoundsincold-pressedplantoils.J.FoodLipids15, 137–149.

Sirat,H.M.,Rezali,M.F.,Ujang,Z.,2010.Isolationandidentificationofradical scav-engingandtyrosinaseinhibitionofpolyphenolsfromTibouchinasemidecandra

L.J.Agric.FoodChem.58,10404–10409.

ˇSliumpait ˙e,I.,Venskutonis,P.R.,Murkovic,M.,Pukalskas,A.,2013.Antioxidant propertiesandpolyphenolicscompositionofcommonhedgehyssop(Gratiola officinalisL.).J.Funct.Foods5,1927–1937.

Wilcox,C.S.,2005.Oxidativestressandnitricoxidedeficiencyinthekidney:a criticallinktohypertension?Am.J.Physiol.Regul.Integr.Comp.Physiol.289, R913–R935.

Wu,X.,Li,Y.,Wang,Q.,Li,W.,Feng,Y.,2015.Effectsofberberineandpomegranate seedoilonplasmaphospholipidmetabolitesassociatedwithrisksoftype2 diabetesmellitusbyU-HPLC/Q-TOF-MS.J.Chromatogr.B1007,110–120.

Zhang,L.,Tu,Z.,Xie,X.,Lu,Y.,Wang,Z.,Wang,H.,Sha,X.,2016.Antihyperglycemic, antioxidantactivitiesoftwoAcerpalmatumcultivars,andidentificationof phenolicsprofilebyUPLC-QTOF-MS/MS:newnaturalsourcesoffunctional con-stituents.Ind.Crop.Prod.89,522–532.

Zhou,D.,Jin,Y.,Yao,F.,Duan,Z.,Wang,Q.,Liu,J.,2014.ValidatedLC–MS/MSmethod forthesimultaneousdeterminationofhyperosideand2′′_{-O-galloylhyperin}_in