Determination of isoquinoline alkaloids in extracts of Chelidonium majus L. using emulsion liquid membrane extraction and GC/MS

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www.elsevier.es/rmi

Medicina

e

Investigación

ORIGINAL

ARTICLE

Determination

of

isoquinoline

alkaloids

in

extracts

of

Chelidonium

majus

L. using

emulsion

liquid

membrane

extraction

and

GC/MS

Z. Guo

a

_,

_H.

_Su

a

_,

_R.

_Cai

a

_,

_X.

_Ma

b_,_∗

a_Research_Center_of_Physics_and_Chemistry_Analysis,_Key_Laboratory_of_Analysis_Science_and_Technology,

CollegeofChemistryandEnvironmentalScience,Baoding,China

b_College_of_Traditional_Chinese_Medicine,_Hebei_University,_Baoding,_China

Received25September2015;accepted6October2015 Availableonline16March2016

KEYWORDS

Chelidonium

majusL.;

Isoquinoline alkaloids; Emulsionliquid membrane extraction; GC/MS

Abstract AlkaloidscontainedinChelidoniummajusL.(C.majus)wereinvestigatedbasedon emulsionliquidmembraneextractionfollowedbyGC/MS.Theoil-in-wateremulsionwas pre-paredusingpolybutadiene-styrenerubberkerosenesolutionasemulsifieranditismorestable thanthatoftheoil-in-wateremulsionpreparedwithSpan80.Thekeyparametersaffecting alkaloidsmembranemasstransferratewereinvestigatedandoptimizedusingprotopineas ref-erencesubstance.Theoptimalemulsionliquidmembraneextractionwasperformedthrough dispersing theoil-in-water emulsion inaqueoussample extracts solutionat room tempera-ture(20◦C)for4minwithaceticacidascarrier,whichgavemuchhigherextractionefficiency insignificantlythanconventionalliquid---liquidextractionmethod.Completeseparationofnine alkaloidswasachievedwithinonly4min.Acomparisonwasmadeofalkaloidsfromthesame C.majussampleintwomodels:onewastherawandotherhadundergoneanacidbathasthe processedsample.Theresultshowedthatthealkaloidcontentintherawsampleismorethan thatoftheprocessedsample.ItmeansthatthecontentofalkaloidcontaininginC.majuswill bedecreasewhentherawC.majushasbeenprocessed.

PALABRASCLAVE

Chelidoniummajus

L.;

Alcaloidesde isoquinolina;

DeterminacióndelosalcaloidesdeisoquinolinaenextractosdeChelidonium majusL.utilizandoemulsiónextraccióndemembranalíquidayGC/MS

Resumen Unmétodosimplebasadoenemulsiónporextraccióndemembranalíquidaseguido porGC/MSfuedesarrolladoparalacuantiﬁcacióndealcaloidesenChelidoniummajusL.(C. majus).Laemulsióndeaceiteenaguasepreparóutilizandocauchodepolibutadieno-estireno.

∗_{Corresponding}_author._Tel.:₊₈₆₀₃₁₂_5079385;_fax:₊₈₆₀₃₁₂_5079525.

E-mailaddress:[email protected](X.Ma). http://dx.doi.org/10.1016/j.mei.2016.01.004

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Emulsiónpor extracciónde membranalíquida; GC/MS

Lasolución dequerosenocomo emulsionantefuemásestablequela emulsiónde aceiteen aguapreparadaconSpan80.Seinvestigaronlosparámetrosclavequeafectanalavelocidad detransferenciadelosalcaloidesatravésdelamembrana,yseoptimizóelusodeprotopina comosustanciadereferencia.Serealizólaóptimaemulsiónporextraccióndemembranalíquida atravésdeladispersióndelaemulsióndeaceiteenaguaenextractosdesolucióndemuestras acuosasatemperaturaambiente(20◦C)durante4min conácido acéticocomo portador,lo que otorgóunamayor eficaciaala extracción queel método deextracción líquido-líquido convencional.Laseparacióncompletade9alcaloidesselogróentansolo4min.Serealizóuna comparacióndelos2métodosdeextraccióndelosalcaloidesdelamismamuestradeC.majus: unofueencrudoyotrosesometióaunbañodeácidocomolamuestraprocesada.Elresultado mostróqueelcontenido dealcaloidesenlamuestracrudaesmayorqueelquecontiene la muestraprocesada.EstosignificaqueelcontenidodealcaloideenC.majusserámenorcuando lamuestraencrudohayasidoprocesada.

Introduction

Chronicinﬂammationsareknowntoberesponsibleforthe pathogenesis of many chronic diseases including asthma, rheumatoid arthritis, septic shock, multiple sclerosis, Parkinson’s,Alzheimer’sdiseasesandotherallergicdiseases suchascoloncancer.1---3_Antibiotics_have_little_effect_on_the

chronic inflammations. Oneof the alternatives is toseek novelanti-inflammatoryagentsinherbaldrugplants. Dur-inga screening ontofindcandidates, Chelidoniummajus

L.(C. majus) has exhibited considerable inhibitory activ-ityin itsmethanolextract. Withbeinganti-inﬂammatory, anti-microbial,antiviral, antispasmodicactivities, antifun-galandfungistaticeffects,theC.majusextractshavebeen reportedtocontrolseveraldiseases includingdermatoses, liverandgastrointestinaldisorders,gastriculcer,ﬁghtfever andtreatingtraumaticinjuriesandvarioustypesof carbun-cle,stoppingbleeding,relievingpain,alleviatingswelling4---8

andparasiticinfectionsinhumanandanimals.9---11

C.majus or Greatercelandinebelongstothe Papaver-aceae family, also named as Shanhuanglian, niujinhua, babujin,duanchangcaoandxionghuangcaoinChinese,isa herbaceous perennial plant throughout the world includ-ing Asia, Europe, Northwest Africa and North America.12

Themain chemicalcomponentsofC.majus arealkaloids, ﬂavonoids,andphenolicacids,13_in_which_isoquinoline

alka-loids are considered as mainly pharmacological effective component14,15_and_in_general,_have_been_as_indices_for

esti-mationofqualityofC.majus.AlthoughtheC.majusisused astraditionalfolkmedicine for thetreatment of diseases withlonghistory, it is alsooneof potentially hepatotoxic herbs when being used at relatively high concentration, leadinganherbinducedliverinjury.16,17,7

Thechemicalscreeningandcharacterizationofextracts isconductedbyliquidchromatography(LC),gas chromatog-raphy (GC) or capillary electrophoresis (CE) techniques coupled with mass spectrometer (MS) or other different detectors, providing a great deal of preliminary informa-tion about the content and nature of constituents.12,18---21

Gaschromatographycoupledtomassspectrometry(GC/MS) is one of the most powerful tools for natural products analysis.22---24 _With _the _equipped _capillary _column, _GC/MS

hasadvantagesofhighseparationefﬁciency,lowdetection limitandidentiﬁcationeasy.25---27

Generally, the isolation of bioactive compounds from bio-matrix requires a preliminary fractionation, andthus, a cleanup pretreatment of herbal drug plants is needed priortoGC/MSanalysis.Inrecent years,theemulsion liq-uidmembrane extraction(ELME)hasbeen proven tobea powerfulsample extractiontechnique,28,29 _which _can_give

achannel fromthefeed phasetothetripphasefor weak alkalineorganiccompound,andthus,alkaloidsinherbscan beenrichedfromitsextracts.Separationofalkaloidsfrom traditional Chinese medicines (TCMs) using ELME, as was done in the current study, providesa discriminating ben-eﬁt with respect to attractant analysis. It is known that at least some of thealkaloids willbe transferredtostrip phase.The useof ELMEis believedtoallow for preferen-tialconcentrationofattractivecompoundswhileminimizing collectionoforganicimpurities.30,31 _However,_ELME_suffers

fromtheemulsioninstability, whichlimitsitsapplications at a broader scale. To overcome the emulsioninstability, the alternative wasadded some polymeric compounds to the organic phase in preparation of emulsion, which was considered as a good way for improvement of emulsion stability.31

Our previous works have been the focus of attention for developing newanalytical methodologyfor the use of GC/MScombiningasamplepretreatmenttodeterminethe alkaloidsin plants.32---39 _In _the_present _work, _a_simple _and

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Experimentscomparingethanolextractsfromthecrude andprocessedC.majushavebeen brieﬂydiscussed inthe literature. This method of pretreatment was reported to provide more material than extraction of alkaloids from TCMs.Thepurposeofthismanuscriptistoreportthe identi-tiesofalkaloidsthatarecontainedincrudeandprocessedC. majusandthenanalyzedusingthemethoddescribedbyusin apreviouspaper.32_The_results_reported_herein_provide_the

foundationforthenextmanuscriptinthisseries,inwhich acomparisonismade ofalkaloidscontentfromthe crude andprocessedsameTCMssampleforwhichalkaloidshasa markedlydifferentcontent.

Experimental

Materialsandchemicals

C. majus L. (dried) was purchased from herbal medicine market (Anguo, Hebei, China) and identiﬁed with their voucher numbers. The protopine reference standard was purchased fromShanghai Yuanye Bio-TechnologyCo., Ltd. (Shanghai, China). Matrine was purchased from Chinese controlofpharmaceuticalandbiologicalproducts(Beijing, China). Acetone of HPLC grade was supplied by Beijing RUIZHX Technology Company (Beijing, China). The sol-ventsandother commercial-gradereagentswereobtained from local distributors. Kerosene (boiling point range of 200---240◦C) and butadiene styrene rubber (BR9000) were obtainedfrommanufacturers(Yanshan,Beijing).Thewater usedintheexperimentswasdoublydistilledwaterprepared inourlaboratory.

Samplestreatment

The sample of C. majus was divided two group, one wasundergone a processing drugand other was not.The processing drugwas performed with beingsoaked raw C. majusinHClsolution(2mol/L)for1handdriedintheair. Bothsampleswereextractedwithethanolaqueoussolution (60%)inSoxhlet extractoruntilthesample extracted solu-tion colorless. After ethanol was recovered, two samples extractssolutions(50mL)wereobtained,markedE1forraw

C.majussampleandE2forprocessedC.majussample.

Emulsionextractionofalkaloids

The preparation procedural details of the emulsion employed to extract alkaloids were described in the reference.32 _A _volume _of ₇_mL _0.1_M _H

2SO4 was added to

13mL the membrane phase(kerosene and rubber emulsi-ﬁer)and0.4mLcarrier(36%ofaceticacid)ina25mLbeaker andthemixturewasvortexed(WX500CY,Weiyu,Shanghai, China)at10,000rpmforseveralmin,beforeobtainingabout 20mLfreshwaterinoil(W/O)emulsion.Thefeedphasewas preparedthroughdilutingthesamplesolutionE1orE2with water2-foldandadjustingpH12withNaOHsolution(9.8%), respectively. The fresh emulsion(10mL) was dispersedin thefeedphase(40mL)formingawater-oil-water(W/O/W) emulsionfortheextractionofalkaloidsundermagneticstir (150rpm).

Afteremulsionextraction, theemulsionwasseparated anddemulsified.Theresultingdemulsificationproductstrip phasesolutionwasbasified using 9.8%aqueous NaOHand thenwereextractedwithmethylenedichloride(3×3mL). The organic solvent in extracts was evaporated under N2

blowandthedriedextractsweredissolvedin10mLof ace-tone of HPLC grade in glass vials. The ﬁnal products for GC/MSanalysiswerelimpid.

Liquid---liquidextraction(LLE)

SamplessolutionE1orE2(approximately20mL)wasmixed with 20mL of hydrochloric acid solution (2mol/L). After reﬂuxedunder heating for 1h, the mixture was defatted using methylene dichloride (3× 15mL). Next, the aque-ous layer was collected and basiﬁed to pH 12 using 9.8% aqueousNaOHsolutionandthenwasextractedusing methy-lenedichloride(3×20mL).Theorganicsolventinextracts was evaporated under N2 blow and the dried extracts

were dissolved in 10mL of acetone of HPLC grade in glass vials. The ﬁnal products for GC/MS analysis were limpid.

GC/MSapparatusandmeasurementconditions

Chromatographic separation was performed using an Agi-lent6890NGCsystemequippedwithafusedsilicacapillary column(DB-5MSUI,length:30m,i.d.:0.32mm,ﬁlm thick-nesses:0.5␮m).The carriergas wasHelium ataconstant ﬂow-rateof1min−1_._The_column_temperature_program_was

50◦C,heldfor1min,thenincreasedat40◦Cmin−1_to₂₀₀◦_C,

and was further increased at rate 4◦Cmin−1 _to ₂₈₀◦_C,

ﬁnally,held for10min.The injections(1␮Leach)wereof thesplit mode(10:1) withtheinjector andinterface line temperature set at 260 and 280◦C, respectively. The GC wascoupledtoa5975CMS.Themeasurementwasoperated in positiveelectron impact(EI) ionization mode at 70eV; EMvoltagewas1423V.Thetemperatureofionsourceand quadrupolewas230◦C and150◦C, respectively. The mass spectrawereobtainedinafullscanmode(33---550amu)at 0.25s/scanscanrate.

Calibration was performed using the standard stock solution (STD) and one working solution made from the STDwithitsdilution. The STDof protopine wasdissolved 10mg of protopine in the mixtureof acetone (HPLC) and methylenedichloride (8:2, v:v). Reference standard solu-tion(200mg/L)ofmatrinewaspreparedinwatercontaining 40mgofmatrineina200mLofvolumetricﬂask.

Aseriesofworkingstandardsolutionswerepreparedby dilutionof the STDof protopine withacetone (HPLC).All thesolutionswerestoredat−20◦Cuntiluse.Priorto injec-tion,thesolutions wereeach added 50␮L ofthe matrine referencestandard.

Thecalibrationcurvewasmadebyplottingtheratioof peaksareaoftheanalyteprotopinetomatrineinfunctionof theinjectedmassoftheanalyte.Thecorrelationcoefﬁcient (R2₎_of_the_calibration_curve_was_between_0.99713.

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where ywasthe concentration ofsolution and xwasthe ratioofpeaksareaoftheanalyteprotopinetomatrine.

Results

and

discussion

Effectofemulsiﬁcationtime

Stability of a prepared W/O emulsion is one of the mostimportantfactorsimpactingtheextractionefficiency. Breakageofemulsionnullifiestheseparation efficiencyby transferringbackoftheseparatedsolutetothefeedphase. OurrecentworkhasemphasizedastableW/Oemulsioncan bepreparedusingarubberemulsifierinsteadofSpan80.40

Themostimportantfactorsgoverning theemulsion stabil-itywerestudied.Thesefactorsinvolveemulsificationspeed andtime,stripphasepH,emulsifierconcentrationand vol-umeratioofthestripaqueousphasetoorganicphaseetc. Extendingemulsificationtime,theW/Oemulsionprepared withrubberemulsifierisobviousconsistence,whichaffect thepermeationprocess.

Experiments wereconducted in thelight of ‘‘Emulsion extractionofalkaloids’’sectiondescribedwith emulsiﬁca-tiontimefrom2to4min.ContentofprotopinepergramC. majus(CPC)extractedbythismethodwasregardedasthe assessmentforassessingtheeffectofemulsiﬁcationtimeon masstransferrate.

Quantiﬁcationemployedexternalstandardmethodusing matrineasreferencesubstancewithdifferentconcentration ofprotopine.Thestandardcurveofprotopinewasachieved by measuring the ratio of two peaks areas as a function ofthe concentrationof protopine solution, whichshowed a linear relationship with a good correlation coefﬁcient in theprotopine concentrationrange from2 to25␮g/mL (y=0.12841+104244x, R2₌_0.9971). _Each _experiment _was

performed at least triplicates and the mean values were calculated.

Theeffectofemulsificationtimeshows thattheCPCis increasedwithemulsificationtimeincreasedfrom2to3min anddecreased for further increasein emulsifyingtime to 4min(Fig.1).Forinsufficientemulsificationtime(<3min), theeffectof coalescence wasgreatbecausethe droplets havealargesize,whichleadstotheircoalescence.In con-trast,for alongemulsification time,thebreakageplaysa rolebecauseofhigh internalshearingconducive toavery highnumberofsmalldropletsbyvolumeunit.Thisincreases collision frequency between small droplets conducting to emulsion breakage. Therefore, an emulsification time of 3minwastakenasthe optimumfor furtherstudiesinthe followingexperiments.

FromFig.1,whatevertheemulsiﬁcationtimewasused, an abnormal CPC decrease with increase of extraction time beyond 4min was observed. This phenomenon was also observed in our previous similar work extracting dl

-anabasine from Alangium platanifolium root.32 _The _CPC

decrementcausedbyextendingextractingtimeisnotclear yet.Fortunatelythedecrementisnotafatalfaultbecause themosttargetcompoundscanbetransferredwithin4min. Intheotherward,a4minofextractingtimeislongenough for an emulsion extraction and the abnormal mass trans-portcanbeavoidaslongaslimitingtheextractingtimein 4min.

Figure1 Effectsofemulsificationtimeontheextraction pro-topine.Experimentalconditions---emulsions:volumeratiosof rubberemulsifiertokerosenetointernalstripphase(0.1M sul-furicacidaqueoussolution)are1:12:7;emulsificationspeedis 10,000rpm;C.majusalcoholextractsaqueoussolution(pH12) isfeedphase;10mLoftheemulsionisdispersed40mLthefeed phasefor4minat150rmpofmagnetstirringspeed.

EffectsofpHofthestripphaseonmasstransfer rate

The stripaqueoussolutionpHeffectofmasstransferrate was studied becauseincreasing acidity of the strip phase shouldnotalwayscontributetoanalytemasstransferrate accordingtothepresentliteratures.14,41,42_For_this_purpose

0.05,0.1and0.5Msulfuricacidaqueoussolutionwere pre-paredasthestripphaseandC.majusextractsasthefeed phase.TheCPCatthreedifferentsulfuricacid concentra-tionswasshowninFig.2,which showedthattheorderof transferprotopineamountatdifferentsulfuricacid concen-trationswas 0.05<0.5<0.1M, indicating that thesolution ofincreasingacidityofstripphaseforenhancingthemass transfer rate would be not always available. The reason shouldreferstabilityoftheemulsion,becausethestronger acidsolutionasthestripphasewouldleadtotheW/O/W emulsioninstability.

Selectionofcarrier

Accordingtothemechanismofemulsionextraction,41_with

additionoforganicacidascarrierinthemembranephase should be the beneﬁtof alkaloid mass transfer rate.The reactions of dissociated alkaloidanion A+ _and_the _organic

acidcarrierCHintwointerfacesofoilandwaterare

A++CH ↔ AC+ H+ (I)

AC+ SO42−→ A2SO4+2C− (II)

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Figure 2 Effectsof concentration ofsulfuric acidaqueous solution inthestrip phaseonthe ELMEprotopine efficiency. Experimentalconditions--- emulsions:volumeratiosofrubber emulsifiertokerosenetointernalstripphaseare1:12:7; emulsi-ficationspeedis10,000rpm;C.majusalcoholextractsaqueous solution (pH12) isfeedphase; 10mLofthe emulsionis dis-persed40mLthe feedphasefor 4min at150rmpofmagnet stirringspeed.

extractsasthefeedphaseandtheCPCalsoasthe assess-ment.

ExperimentscomparingtheCPCextractedwithoutadded carrierinthemembranephase,asshowninFig.3,theCPC wasincreasedasaceticacidaddedanddecreased,however, whenmetacresolandoctanoicacidascarrieraddedinthe membranephase.ThedecreaseofCPCwasagreedwiththe decreaseof the usedcarrierpolarity.Increase in theCPC corresponds to increased diffusion of solute carrier com-plexacrossthemembrane.Amongallthreecarriers,acetic acidhas thestrongest polarity.Therefore,it canincrease the protopine’s concentration at the interface, providing aCPCincrease.However,thecarrierslikemetacresoland octanoicacidaretheweakerpolarcompoundsthanacetic acidandhenceaninhibitionofionexchangeofsolutecarrier

Figure3 EffectsofdifferentcarriersontheELMEprotopine efﬁciency. Experimental conditions --- 0.4mL of carrier was added to 13mL of the organic phase as membrane phase; volumeratiosofthemembranephasetostripphase(0.1M sul-furicacidaqueoussolution)is13.4:7;emulsiﬁcationspeedis 10,000rpm;C.majusalcoholextractsaqueoussolution(pH12) isfeedphase;10mLoftheemulsionisdispersed40mLthefeed phasefor4minat150rmpofmagnetstirringspeed.

complexacrosstheinterfacesofoilandwaterprovidingthe CPCdecrease.Therefore,itappearsthatthereisalimitto thecarrierpolaritythatwillgivesigniﬁcantincreaseinthe extractionrate,probablyasa resultof theequilibriumof theionexchangereaction.

Extractionofalkaloids

The above ELM individual optimization was employed for theextractionofalkaloidsfromC.majusextracts.Atotal ion chromatogram (TIC) of C. majus alkaloids extracted byemulsion, acquired in EI mode, is presented in Fig.4. Thepeaknumbersrepresenttheelutionorderofidentiﬁed peaks.Analysisusingthemethodsemployedhereresulted inchromatogramsthatcontainedasmanyas36discernible

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Table1 AlkaloidspresentorsuspectedpresentfromextractionofC.majususingemulsionliquidmembrane.

Peakno. Alkaloids Molecularformulaand

matchingdegree

Retentiontime (min)

a_Content

(␮g/g)

1 Stylopine C19H17NO498 24.514 265

2 Dihydroberberine C20H21NO495 24.67 9.1

3 Chelidonine C20H19NO599 25.038 _b

191

4 Protopine C20H19NO599 25.051

5 Dihydrosanguinarine C20H15NO4--- 27.671 156

6 Dihydrochelerythrine C21H19NO474 28.267 25.6

7 Fumariline C20H17NO530 28.498 11

8 6-Acetonyldihydrochelerythrine C24H23NO553 32.316 5

9 6-Acetonyldihydroavicine C23H19NO5--- 32.483 22.7

---,ManualinterpretationwasusedaccordingtoaspeciﬁcEIfragmentationpatternanditsmolecularmass.

a_{Qualiﬁcation}_of_the_alkaloids_in_C._majus_is_performed_using_protopine_as_reference_compound_through_calculation_of_their_relative

ratioofchromatogrampeakareatoprotopinepeak.

b _The_peaks_observed_in_the_{chromatograms}_were_not_separated.

peaks.Of these,9 alkaloidsidentiﬁcations weremade by matchingsamplemassspectrawiththoseoftheNBSlibrary invarying degreesof certainty.They arelabeledby num-beronthechromatogram.As seen inFig.4,thealkaloids of peaks were not wholly eluted prior to 20min and 3# peak should be correspond to both alkaloids chelidonine andprotopine, whichwerenotdistinguishedthroughtheir chromatogrambehaviorbuttheycanbedistinguishedfrom theirmassspectra.ThepeaknumbersinFig.4correspond tothoselistedinTable1.

Itreportedthatover20differentC.majusalkaloidswere identiﬁedandmainalkaloidsarebenzylisoquinolines.14_But

hereonly9sortofthealkaloidswereseparatedand identi-ﬁed.ThereasonshouldbefromthedriedandagedC.majus

sample.

In order to illustrate the efﬁciency of the method employed here, same the sample was extracted by liquid---liquidextraction (LLE) methodfor comparingtheir extractionefﬁciency.TheC.majussamplewasdividedinto twogroups, one group was the crude and the other was immersedin2Mof hydrochloricacidsolutionfor1hprior toextraction,callingtheformerrawsampleandthelater processedsample.

Protopine was also selected as the reference analyte forassessing theextractionefﬁciencybecauseitwas sec-ondmainalkaloidcontentelutedinchromatogram(Fig.4).

Table2 showsthe separation efﬁciencyachievedbyELME andLLE.HorizontalcomparisonfromTable2,itisobserved thattheCPCextractedbyELMEisabout1.4timetothatby LLE,indicatingthemethoddescribedismoreefﬁciencythan

Table 2 The content of protopine per gram C. majus extractedbyELMorLLE.

SampleofC.majus Extractionmethod

ELM LLE

Raw 191.0 134.6

Processed 141.0 99.2

Experimentalconditionsarethesameasdescribedincaptionof

Fig.3.

LLEinextractionofalkaloids.Columncomparison,theCPC extracted fromthe rawsample is alsoabout 1.4 timesto thatfromtheprocessedsample,indicatingthesome alka-loidcontentlostasC.majusundergoaprocessofacidbath. The alkaloid contentlost can beevaluatedasan alkaloid dissolvedinacidbathprocess.Inotherward,apartof alka-loidscontentinC.majuscanberemovedthroughacidbath sample.

Conclusion

An available andreliable methodbasedonELMcombined withGC/MS wasdeveloped for theextractionof alkaloids fromC.majus.Theoperationalconditionshereforan excel-lent stability of the W/O emulsion prepared with rubber emulsifierwasemulsificationtime3minat10,000rpm stir-ring speed. The organic phase waskerosene with rubber emulsifier (1:15.25,v:v), aceticacid (36%)ascarrier.The stripphasewas0.1Mofsulfuricacidaqueoussolutionand thevolumeratioofstripphasetoorganicphasewas3/7.

Atthe optimumexperimentalconditions, theemulsion extraction timeshouldbelimited within4min. Totalof 9 alkaloidswere separatedandidentiﬁed fromthedried C. majususingthepresentmethod.Ofthem,themostcontent of alkaloid was stylopine and second content was cheli-donine and protopine. The content of alkaloids would be partlyconsumedasC.majussamplewasunderwentanacid bathprocess.IncomparisonwithcurrentlyLLEmethod,the contentof protopine pergram C. majusextracted by the ELME is 1.4 time to that by LLE and hence the present methodoffersan effectivewayforanalysisofalkaloidsin

C.majus.

Funding

Noﬁnancialsupportwasprovided.

Conﬂict

of

interest

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