Rev. bras. farmacogn. vol.27 número5

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RevistaBrasileiradeFarmacognosia27(2017)576–579

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

Original

Article

Identification

of

phenolic

compounds

in

Myricaria

bracteata

leaves

by

high-performance

liquid

chromatography

with

a

diode

array

detector

and

liquid

chromatography

with

tandem

mass

spectrometry

Alexander

A. Chernonosov

a,∗

,

Evgeniya

A. Karpova

b

,

Elena

M. Lyakh

b a_Institute_of_Chemical_Biology_and_Fundamental_Medicine,_Siberian_Branch_of_Russian_Academy_of_Sciences,_Novosibirsk,_Russia b_Central_Siberian_Botanical_Garden,_Siberian_Branch_of_Russian_Academy_of_Sciences,_Novosibirsk,_Russia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15November2016 Accepted4July2017 Availableonline2August2017

Keywords: HPLC-DAD LC-MS/MS Flavonol Flavon Ellagicacid

a

b

s

t

r

a

c

t

MyricariabracteataRoyle,Tamaricaceae,isaspecieswithawidegeographicrangethatencompasses East-ernEurope,WesternandCentralSiberia,CentralAsia,andtheHimalayas.Thisplantisusedintraditional folkmedicineinRussia(Siberia)andinChinatypicallyasananalgesicandforthetreatmentofsome infec-tionsandcertaintypesofintoxication.Theaimofthisstudywastoidentifyphenolicconstituentsofthe leavesofM.bracteatafromtwoconsiderablydistantpopulations.Chromatographicprofilesoftheleaves ofM.bracteatawereanalyzedforthefirsttime.Seventeencompounds,mainlymethylethersofquercetin (isorhamnetin,rhamnazin),kaempferol(kaempferide,rhamnocitrin),andellagicacidaswellasquercetin, quercetin3-glucoside,kaempferol,luteolin,chrysoeriol,citricacid,gallicacid,methylgallate,ethyl gal-late,andferulicacidwereidentifiedinhydrolyzedaqueousethanolextractsoftheleaves.Flavonols andellagicacidwerethemajorcompoundsinbothsamples.Isorhamnetinwasthemainflavonoid con-stituent.KaempferideandrhamnazinwerealsoabundantintheflavonoidcomplexoftheleavesofM. bracteatafromtheAltai.ThisstudyshowsthatM.bracteataleavesareasourceofflavonoidswithpossible biologicalactivities.

Introduction

MyricariaDesv.,Tamaricaceae,isagenusofherbswitha

poten-tialfor applicationstoconventional medicine.Myricaria species areusedinfolkmedicineinRussia(Siberianregions)andinmany Asiancountries(morecommonlyinChina,Mongolia,andIndia) forthetreatmentofsomeinfections,certaintypesofintoxication, liverdiseases,scalds,andarthritis(Semenova,1993;Gewali,2008; Kletteretal.,2008;Kirbagetal.,2009;Singh,2012).Somespecies werereportedtohavehighantimicrobialandacetylcholinesterase inhibitoryactivitiesandthuscanbeconsiderednaturalsourcesof antibioticsanddrugsforthetreatmentofneurologicaldisorders suchasAlzheimer’sdisease(Mukherjeeetal.,2007).

High antimicrobial activity of the volatiles of intact

Myri-cariabracteata plantsagainst Staphylococcusepidermidis aswell

asapronouncedantifungalactivityagainstCandidaalbicanswere alsorevealed(LyakhandTsybulya,2009).Crudeethanolextract from the above-ground part of M. bracteata and its fractions

∗ Correspondingauthor.

E-mail:alexander.chernonosov@niboch.nsc.ru(A.A.Chernonosov).

havebeenshowntohaveastrongantimicrobialactivityagainst

Staphylococcusaureus,Enterococcusfaecalis,Micrococcusluteus,and

Pseudomonasaeruginosa(Gonchigetal.,2008).

Theliteraturelacksdetailedinformationonregularconstituents

of M.bracteata, theirgeographic variability, and thekey active

ingredients.Theaimofthisstudywastoidentifythephenolic con-stituentsoftheleavesofM.bracteatafromtwoconsiderablydistant populations.

Materialsandmethods

All chemicals were mass spectrometry (MS) and analytical grade. Chemical reference standards of gallic and ferulic acids

were purchased from Serva (Heidelberg, Germany); quercetin,

kaempferol,rhamnetin, isorhamnetin,luteolin,citric andellagic acidswerepurchasedfromSigma(St.Louis,MO,USA).

ThesamplesofMyricariabracteataRoyle,Tamaricaceae,leaves werecollectedinthefruitingperiodof2011.SampleAwas col-lectedat1800mabovethesealevel(a.s.l.)onapebblefloodplain oftheBolshoiYalomanRiver,1kmfromBolshoiYalomanvillage, OngudayskyRegion,theAltaiRepublic.SampleBwascollectedat 3100ma.s.l.onapebbleandsandyfloodplainoftheTokuz-Bulak

http://dx.doi.org/10.1016/j.bjp.2017.07.001

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A.A.Chernonosovetal./RevistaBrasileiradeFarmacognosia27(2017)576–579 577

River(thelefttributaryoftheGuntRiver),1.5kmfromthe Kuy-gantukoyvillage,ShugnonRegion,theRepublicofTajikistan.The collectedMyricariasamplesweredriedandgroundintopowder usingahouseholdmill.

Apreciselyweighedsampleofair-driedplantmaterial(0.3g) wasexhaustivelyextractedwithanethanol:watermixture(70:30, v/v) in a water bath at 60–70◦_C. _The _aqueous_ethanol _extract washydrolyzedwith2NHClfor2hinaboilingwaterbath.The hydrolysatewaspurifiedbymeansofaC16Diapackcartridgeand redissolvedinethanol.

MassspectrometricanalysiswascarriedoutattheCoreFacility ofMassSpectrometricAnalysis(ICBFMSBRAS).AnAgilent1200 liquidchromatographysystem,withanAgilent6410QQQmass spectrometer(AgilentTechnologies,USA)equippedwithan elec-trosprayionization(ESI)source,servedasanLC-MS/MS system. Thechromatographicseparationwascarriedoutat25◦_C_on_a Zor-baxEclipseXBD-C18Column(4.6mm×150mm,5␮mi.d.)withan

EclipseXBD-C18guardcolumn(4.6mm×12.5mm,5␮mi.d.).

Forthegradientelution,10mM NH4COOHinwater (pH4.0,

adjustedwithformicacid),i.e.,solventA,and10mMNH4COOH(pH

4.0adjustedwithformicacid)inacetonitrile–water95:5(v/v),i.e., solventB,wereused.Therunwasstartedwith0.6mlmin−1_flow

rateatasolventA:solventBratioof100:0(v/v)followedbyalinear gradientto50:50(v/v)forthefirst5min,thento48:52(v/v)from min5tomin10,thento0:100(v/v)frommin10tomin20;and returnedto35:65(v/v)frommin20tomin35,thento100:0(v/v) frommin35tomin40.Thesampleinjectionvolumewas30␮l.All

dataacquisitionandpeakintegrationtaskswereperformedinthe MassHuntersoftware(version1.3)fromAgilentTechnologies.

Atthe firststep of theanalysis, thesamples wereanalyzed in full-scannegativemodefrom 100to1500Da. The multiple-reaction monitoring (MRM) transitions for possible molecular structures that have been proposed for a detected ion

(Sup-plementary data A) were based on the RIKEN MSn spectral

database for phytochemicals (http://spectra.psc.riken.jp), Mass-Bank (http://www.massbank.jp) and the Human Metabolome Database(HMDB)(http://www.hmdb.ca).

The HPLC system for absolute quantification of phenolic

consistedofanAgilent1200withadiodearraydetector(DAD) andtheChemStation(AgilentTechnologies,USA)softwarefordata processing. Thechromatographic separation wascarried out at 25◦_C _on_a_Zorbax_SB-C18_Column_(4.6_mm_×₁₅₀_mm,₅_␮_m_i.d.) withanAgilentGuardColumnHardwareKit(p.n.820888-901).

The mobile phase consisted of MeOH (solvent A) and 0.1%

orthophosphoricacidinwater(solventB).Therunwasstartedat 1mlmin−1_rate_with_a_solvent_A–solvent_B_mixture_at_50:50_(v/v)

followedbyalineargradientto52:48(v/v)forthefirst15min,then to100:0(v/v)frommin15tomin17.Itwasreturnedto50:50(v/v) frommin17tomin20.Thesampleinjectionvolumewas10␮l.

Thequantificationof phenoliccompoundswasconductedas previouslyreported(KarpovaandKhramova,2014)(see Supple-mentarydataB).Allthedatawerereportedasmean±standard deviation(SD)ofthreereplicates.Theresultswerecomparedby Student’sttest.Thedataanalysiswasperformedusingthe Sta-tistica7.0software(StatsoftInc.,Tulsa,OK,USA),anddifferences betweenthemeanswereconsideredstatisticallysignificantatthe 5%level(p<0.05).

Resultsanddiscussion

Seventeenconstituents,mainlyflavonoids,wereidentifiedin thehydrolyzedextractsofeachgeographicsampleoftheleavesof

M.bracteata.Thecompositionoftheleaveswasidentical(Fig.1).

Atthebeginningoftheanalysis,threephenolicacidswere iden-tifiedinM.bracteataleafextract(Table1).Citric(1),gallic(2),ellagic

T

otal ion intenc

ity

M. bracteata (A) from Bolshoi Yaloman, the Altai Republic

M. bracteata (B) from Kuygantukoy, the Republic of Tajikistan Time, min

Time, min

T

otal ion intenc

ity 160000 160000 180000 140000 140000 120000 120000 100000 100000 80000 80000 60000 60000 40000 40000 20000 20000 0 0 -2 -2 0 0 2 2 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 20 20 22 22 24 24 26 26 28 28 30 30 32 32 5000 4000 4000 6000 8000 10000 3000 2000 2000 1000 0 0 4 4 6 6 8 8 10 10 12 12 14 14 16 16 18 18 400 300 200 100 0

7 8 9 10 11 12 1 2 2 7 7 8 8 9 9 10 10 11 11 3 3 4 4 5 5 6 6 13 13 15 15 16 16, 12 12 14 14 17 17

A

B

Fig.1.LC-MS/MSbasepeakchromatogramsofMSinnegativeionmodeforthe hydrolyzedaqueousethanolextractsoftheleavesofMyricariabracteatafromthe Altai(A)andfromtheTajikistan(B).

(7),andferulic(9)acidsweredirectlyidentifiedbycomparisonof theirretention timewiththat ofstandardsandwereconfirmed intheMS/MSspectrum.Citricacidisbeingsuggestedasa con-stituentofM.bracteataforthefirsttimeandcharacterizesthisplant asalkali-tolerant(Nawwaretal.,2013).

Ethylgallate(8)wastentativelyidentifiedaccordingto[M−H]− atm/z197andMS/MSfragmentionsatm/z124and169,wherethe lattercorrespondstogallicacid.Compounds3and4with[M−H]− atm/z183andanMS/MSfragmentionatm/z124(Rendekovaetal., 2015)weretentativelyidentified as methylgallateisomers. An additionalfragmentionatm/z140wasdetectedfor3(Rendekova etal.,2015);however,thisfragmentionwasnotrevealedfor4, probablybecauseoflowquantity.Methylgallateandother deriva-tivesofhydroxybenzoicacidshavebeenreportedtobefrequent constituentsofM.bracteata(Zhangetal.,2011;Zhaoetal.,2005).

A total of ten flavonoid derivatives were identified in M.

bracteataleaves.Quercetin(11),luteolin(10),kaempferol(12)and

isorhamnetin(13)wereidentifiedbycomparisonofretentiontime andtheMS/MSspectrawiththoseofauthenticstandards.

Compound14wastentativelyidentifiedasrhamnocitrinbyits molecularion[M−H]−_at_m/z_299.2_and_fragment_ions_at_m/z_271, 256,and227,whichreflectdecarbonylation,i.e.,[M-H-CO]−_,_the lossofamethylradicalwithsubsequentdoubledecarbonylation,

i.e., [M-H-CH3-CO-H]− and [M-H-CH3-CO-CO-H]−, respectively.

Compound15withdeprotonatedion[M−H]−_at_m/z₃₂₉_and_the fragmentionsm/z314[M-H-CH3]−,m/z299[M-H-2CH3]−,andm/z

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578 A.A.Chernonosovetal./RevistaBrasileiradeFarmacognosia27(2017)576–579

Table1

PhytochemicalconstituentsidentifiedinMyricariabracteataleavesofplantsfromtheAltai(A)andTajikistan(B)andsomephenoliccompoundsconcentrations.

No. TR(min) [M−H]−_,_m/z _Fragment_ions,_m/z _Identified_compounds _{Concentration}_in_leaves,

mg/gofdryweight

A B

1 3.7 191 111,87,67,57 Citricacida

2 6.8 169 125,97,79,69 Gallicacida _1.69_±_0.19b _1.71_±_0.06

3 8.4 183 140,124 MethylgallateI

4 9.3 183 124 MethylgallateII

5 9.7 317 179,151,137,107 Myricetina

6 10 463 301 Quercetinhexoside/Ellagicacidhexoside

7 10.6 301 284,257,229,185 Ellagicacida _5.72

±0.22 4.29±0.15

8 11.1 197 169,124 Ethylgallate

9 11.9 193 178,149,134 Ferulicacida _0.15_±_0.02b _0.11_±_0.03

10 18.2 285 151,133,121,107 Luteolina _0.00_±_0.00 _0.03_±_0.01

11 18.3 301 245,229,179,151,

121,107

Quercetina _0.25_±_0.02 _1.64_±_0.08

12 21.2 285 255,227,211,187,

159,143,117,108, 93

Kaempferola _0.42

±0.06b _0.37

±0.06

13 21.8 315 300,283,271,255,

243,227,164,151, 148,136,107,83, 63

Isorhamnetina _2.25_±_0.06 _11.73_±_0.19

14 24.6 299.2 271,256,255,227,

211,199,151

Rhamnocitrin

15 24.7 329.2 314,299,271,227 Rhamnazin

16 29.3 299 284,255,227,164,

163,132,107,83, 63

Kaempferide

17 29.3 299 226,211,200,199,

183,158,151,133, 107

Chrysoeriol

a_Identified_by_comparison_with_a_standard. b _{Insignificant}_differences_between_samples₍_p_>_0.05).

Kaempferide(16)andchrysoeriol(17)weretentatively identi-fiedbytheirmolecularion[M−H]−_at_m/z₂₉₉_and_{fragmentation} patternscontainingspecificionsatm/z63,83,163,and164orm/z

151,158,and183,respectively.

Compound 5 was tentatively identified as myricetin with a molecularion[M−H]− _at_m/z₃₁₇_and_typical_MS/MS_fragments atm/z179and151thatcorrespondedtoretrocyclizationonthe A–Cring(1,2_A−₎_and_the_consecutive_loss_of_CO₍1,2_A−_-CO), respec-tively.MyricetinhasnotbeendetectedinTamaricaceaebefore,and thelackofthataglyconisregardedasacharacteristicofthefamily (Harborne,1975).ItwasdetectedinthesamplesbyMSinsmall amount.Itsabsenceinotherpreviouslystudiedsamplesof

Myri-cariawasmostlikelyduetothepeculiaritiesofthemethodsor

samplepreparation.

Compound6, withmolecularion [M−H]− _at_m/z ₄₆₃ _and _a fragmention at m/z 301, could beproposed as one of ellagic-acidorquercetinhexosidestructure.Indirectconfirmationofthe quercetinderivativecouldbethefactthatquercetin3-glucoside wasfoundinthemajorityofMyricariaspeciesearlier(Iwashina, 2013).

Concentrationsofsomephenolic compoundsin leavesofM.

bracteataareshown in Table1. Significantdifferencesbetween

thegeographic locations(p<0.05)wererevealedin ellagicacid, quercetin,luteolin,andisorhamnetin.Mostsignificantly,the sam-plesdifferedinconcentrationsofquercetinandisorhamnetin.

Isorhamnetinwasdominantin theleavesofplantsfromthe Tajikistan.Kaempferideandrhamnazinalsomadeasignificant con-tributiontothetotalphenoliccontentintheleavesofplantsfrom theAltai(Fig.1).Ellagicacidwasfoundtobethesecondmajor phenolicconstituentofleavesofM.bracteata.ResultsofbothHPLC andLC-MS/MSanalysisshowedhigherconcentrationofellagicacid insampleAcomparedtosampleB.Citricacid,phenolicacids,and

theirderivatives,luteolin,kaempferol,andrhamnocitrinwerethe minorconstituentsinbothsamples.

TheseresultsareinagreementwiththefindingsofChumbalov etal.(1975)whostudiedsamplesofM.bracteatafromKazakhstan, andincontrasttoWangetal.(2008),reportedflavonesasthemajor activeingredientsofM.bracteata.Inotherstudies,therearenodata ontheabundanceofsuchcompounds.

Abroadgeographicdistributionofthespeciesissuggestiveof quantitativeorqualitative differencesinthechemical composi-tionoftheplantsowingtodifferentenvironmentalcircumstances (Keinänen etal., 1999).Theresultsobtainedin this study indi-catethatthesamplesdiffermainlyinflavonolaccumulation.The chiefdifferencebetweenthesamplesisthelowerconcentrationof isorhamnetinandthehigherlevelsofrhamnazinandkaempferide, othermethylatedmetabolitesofquercetinandkaempferol,in sam-pleA.

There are no reports in the literature about the

quan-tification and variation of phenolic compounds in species M.

bracteata.Our resultssuggest thatit is important todetermine

the chromatographic profile of this species to reveal possible chemotypes.

Theprogress intheresearch onchemicalconstituents ofM.

bracteatafromdifferentgeographicalareasformsthebasisfor

iden-tificationofpharmacologicalleadsinmedicinalplantmaterialsand forsettingthestandardsofqualitycontrol.

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A.A.Chernonosovetal./RevistaBrasileiradeFarmacognosia27(2017)576–579 579

Authors’contributions

AC conducted the HPLC-MS/MS analysis and wrote the

manuscript.EKcontributedtotheHPLCexperimentsandwrote themanuscript.ELcollectedandidentifiedthesamples.Allauthors readandapprovedthefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisworkwassupportedpartiallybyaRussianState-funded project(VI.62.2.2,0309-2016-0007).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.07.001.

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