Determination of orcinol glucoside by LC-MS in Curculigo orchioides and its application to a pharmacokinetic study

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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

Determination of orcinol glucoside by LC-MS in Curculigo orchioides and its application to a pharmacokinetic study

Tong- tong Lv

^a,∗,1

, Cheng-guo Ju

^a,∗,1

, Bo-nan Liu

^a

, Xiao-hang Ren

^a

, Guo-shun Shan

^a

, Tian-zhu Jia

^a,b

aSchoolofPharmacy,LiaoningUniversityofTraditionalChineseMedicine,Dalian,China

bChineseMateriaMedicaProcessingEngineeringCenterofLiaoningProvince,Dalian,China

a r t i c l e i n f o

Articlehistory:

Received15April2019 Accepted15August2019 Availableonline30October2019

Keywords:

Excretion

Plasmaconcentration Tissuedistribution Wine-processingtechnology

a b s t r a c t

Thisstudywasdesignedtoexplorethepharmacokineticregularityoftheplasmaconcentration,tissue distribution and excretionof orcinol glucosidefrom aqueous extracts of raw and processed Cur- culigoorchioides Gaertn.,Hypoxidaceae. Theexperimentﬁrstusedanultrahigh-performanceliquid chromatography-tandemmassspectrometryapproachwithmultiplereactionmonitoringandaposi- tivemodetoseparateorcinolglucosidefromnaringintoobtaintheplasmaconcentrationcurves,bar graphoftissuedistributionandexcretioncurves.Theseresultsmightbebeneﬁcialforreasonableclinical applicationofC.orchioidesandforfurtherdevelopmentofitswineandsalt-processingmechanism.

Introduction

ThedriedrhizomeofCurculigoorchioidesGaertn.,Hypoxidaceae, isatraditionalChineseherbalmedicinethathasbeenusedforcen- turiesinthetreatmentofdecliningstrength(Wangetal.,2017), jaundice,asthmaetc.(Wangetal.,2019).Curculigoorchioideshas variousprocessedproducts,whichhavealonghistoryofmedical usein China. Wine-processing and salt-processing are special- izedtechnologyinvolvingsautéingChinesericewineandsaline solutioninanappliedprocessingtechnology.Wine-processedC.

orchioides(WXM)ispartofChinesePharmacopoeia.Salt-processed C.orchioides(SXM)isaninnovativetechnologydesigned byour research group, guided by the theories of traditional Chinese medicineof“Ru YanZouShen”and“XiangFanWeiZhi”.These theorieshavethousandsofhistoryinclinicalusesinChina(Tao etal.,2017;Chenetal.,2018;Hejazietal.,2018).Furthermore, wine-processingand salt-processingcan reduce or remove the side-effectsofC.orchioides,whilealsochangingitspropertiesand strengtheningitsefﬁcacy.

On thebasis of prophasepharmacodynamics studies byour group, the increasing absorption of C. orchioides in vivo was mostlikelyduetoprocessing.ThemajoractiveingredientsofC.

orchioidesarephenolicglycosidesandmainlyincludecurculigo-

∗ Correspondingauthor.

E-mails:Alcucucu@126.com(T.t.Lv),jcg@lnutcm.edu.cn(C.Ju).

1TongtongLvandChengguoJucontributedequallytothiswork.

side,orcinol glucoside (1), curculigineA, anacardosideetc.;the mostcommoningredientisorcinolglucoside,anactiveconstituent thatisacommontraditionalChinesemedicinewithdiversebene- ﬁcialbiologicalandpharmacologicalactivities.Constituentorcinol glucoside(1)hashighsafety marginsand novelpharmacother- apeutic applications in cancer treatment that include diverse beneﬁcialbiologicalandpharmacologicalfunctions(Nahaketal., 2018), as wellas antioxidant, antidepressant, adaptogenic, and neuroprotective activities, among others (Ge et al., 2014); in addition,1maybeapharmacodynamicsubstancethatenhances theimmuneactivityofRAW264.7cells,accordingtoourresearch results.

Ultimately,orcinolglucoside(1)ischosen astheanalytefor thisexperiment,inwhichwestudyitsabsorption,distributionand excretioninrats.Themethodinvolvesthesupportoftimepharma- cologyandaneffective,rapidandsensitiveUPLC-MS/MSapproach combinedwithmultiplereactionsmonitoringbyoptimizingsepa- rationandfindingthebestdaughterionfordeterminationoforcinol glucoside(1).Thisstudyreports,forthefirsttime,thepharmacoki- neticparametersoftheoralbioavailability,tissuedistributionand excretionoforcinolglucosideand comparestheinfluenceofW

https://doi.org/10.1016/j.bjp.2019.08.005

creativecommons.org/licenses/by-nc-nd/4.0/).

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andSwithrawC.orchioides(RXM),whichcancontributetofurther clinicalapplicationsofC.orchioides.

Materialandmethod

Chemicalsandreagents

Orcinolglucoside (1)and naringin (2)werepurchased from theMustCompany(Sichuan,China).Chineserice wineand salt werepurchased fromZhejiangBrandTowerShaoxingWineCo., Ltd.(Zhejiang, China)and DalianSalt Industry Co.,Ltd. (Dalian, China),respectively. Waterfor UPLCanalysiswaspuriﬁed with a Milli-Qacademic water puriﬁcation system (Millipore, USA).

UPLC-MS grade methanol (Merck KGaA, Darmstadt, Germany), acetonitrile (Merck KGaA, Darmstadt, Germany), spectroscopy gradeformicacid(MerckKGaA,Darmstadt,Germany)andanal- ysisgrademethanol (KermolChemical Reagent,Tianjin,China).

C samples were collected from Kangmei Pharmaceutical Co., Ltd. in Chinaand authenticatedby Professor Yanjun Zhaifrom theDepartment of Identiﬁcation of Chinese Medicine,Liaoning University of Traditional Chinese Medicine (Liaoning, China) (180200431).RXM,WXMandSXMwerepreparedinaccordance withthePharmacopoeia,i.e.,themethodofstir-fryingwithwine and stir-frying with salt-water(Chinese Pharmacopoeia, 2015).

Theaboveprocedurewascompletedundertheguidanceofthe ProcessingofChineseMateriaMedica.

Standardsolutionpreparation

Standardstocksolutionsof1and2werepreparedinmethanol ata concentration of630 and 3.128␮g/ml. Thestock solutions weredilutedwithmethanoltoprepareworkingstandard solutions.Plasmacalibrationstandardswerepreparedbyspikingblank plasmawithanappropriateamountofstandardsolutionsand2 workingsolutiontoensureﬁnalseriesconcentrations.Qualitycon- trol(QC)samplesatthreelevelswerepreparedusingthesame procedureatlow,middleandhighconcentrations.2stocksolutions ofurineandtissueswerepreparedinthesamewayasplasma.All thesolutionswerestoredat4^◦C.

Equipmentandoperatingconditions

Quantitativeanalysisof1wasachievedontheWatersAcquity UPLC H-Class Xevo TQD triple quadrupole mass spectrometer system(WatersCorporation,Milford,MA,USA)equippedwithelec- trosprayionizationoperatedinthepositive ionizationmodeby multiplereactionmonitoring.Dataacquisitionandanalysiswere accomplishedwithMasslynxsoftware.Chromatographicsepara- tionwasoptimizedonaWatersACQUITYUPLCBEHC18column (1.8␮m)at30^◦C,alongwithamobilephaseﬂowrateat0.3ml/min.

(A)acetonitrileand(B)0.1%formicacidinwaterconstitutedthe mobilephase,andtheﬁnalelutionconditionwasappliedasfol- lows:0–4min:3%A;4–5min:13%A;5–7min:21%A;7–9min:25%

A;9–12min:28%A.Theinjectionvolumewas1␮l.Theautosampler wasmaintainedat8^◦C.

TheanalysiswasperformedonaWatersH-classUPLC-xevo TQD.TheESI sourceofthemass spectrometerwasoperatedin thepositivemode.Detectionwasperformedbymultiplereaction monitoringatm/z287.13→125.02(CE:8V,CV:22V)for1,m/z 581.32→273.12(CE:20V,CV:32V)for2astheinternalstandard.

Sourcetemperatureanddesolvationtemperaturewere150^◦Cand 350^◦C.Thedesolvationgasﬂowwas700l/h.Thecapillaryvoltage andconevoltageweresetto3.0kvand25v,respectively.MS/MS spectraof1and2areshowninFig.1inthesupportinginformation.

Biologicalsampletreatment

Ratplasma(100␮l)wasspikedwith40␮lof2ina1.5-mlEppen- dorftube.Themixturewasvortexedfor15s.Aconventionalprotein precipitationmethodwasappliedby360␮lmethanol.Then,the mixture wasvortexed for 3minand centrifuged at12,800 × g forcefor 5min,followingtransferof supernatanttoanEP tube andevaporationtodrynessunderagentlenitrogenstream.Finally, theresiduewasreconstitutedin150␮lacetonitrilewatersolution (50:50),mixedfor30sonavortex-mixerandthencentrifugedat 12,800gforcefor5min.Then,1␮lofsupernatantwasinjectedfor analysis.Raturine(100␮l)samplewaspretreatedfollowingasimi- larproceduretothatofplasma.Tissues(0.5g)werehomogenizedin normalsalineataproportionof1:8(w/v),andtissuehomogenate wascentrifugedat12,800gforcefor10minat4^◦C.Then,tissue supernatant(100␮l)wasprocessedusingasimilarapproachas plasmasamples.Deﬁningtheintestineastheblanktissueforthe follow-upstudy,blanktissuesupernatantwasprocessedusingthe sameprocedureasthetissuessamples.

Bioanalyticalmethodvalidation(FDA,2013) Selectivity

Theselectivityofthismethodwastestedandensuredbyanal- ysesofblanksamplesoftheplasma,urineandtissuesupernatant obtainedfromsixrats,showninFig.2andFig.3.Therewasno interference,andtheselectivityofthemethodwasensuredatthe lowerlimitofquantiﬁcation.

Accuracy,precision,recoveryandmatrixeffect

Theaccuracyoftheanalyticalmethodwasmeasuredusingsam- plescontainingknownamountsoftheanalyte,andthemeanvalue waswithin15%.Theprecisionoftheanalyticalmethodwasmea- suredusingsixdeterminationsperconcentration,anditdidnot exceed15%of thecoefﬁcientof variationateach concentration level,asshowninTable1(Supportinginformation).Recoveryper- tainstotheextractionefﬁciencyoftheanalyticalmethodwithin thelimitsofvariability.Recoveryof1didnotneedtobe100%,but theextentofrecoveryof1andoftheinternalstandardwascon- sistent,precise,andreproducible.Inthisstudy,therecoveryand matrixeffectexperimentswereperformedbycomparingtheana- lyticalresultsforextractedsamplesatthreeconcentrations(low, medium,andhigh)withunextractedstandardsthatrepresent100%

recoveryinTable2(Supportinginformation).

Calibrationcurve

The calibration curve for 1 showed good linearity over the concentrationranges.Theresultswereﬁttedtolinearregression analysisusing1/x²astheweightingfactor.Thecoefﬁcientsofdeter- mination(r²)weregreaterthan0.99inallvalidationbatchesin Table3(Supportinginformation).

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Stability

Stabilitytestingwasperformedbyevaluatingthestability of 1duringsamplecollectionandhandling,afterlong-term(frozen attheintendedstoragetemperature)andshort-term(benchtop, roomtemperature)storage,andafterthreefreezeandthawcycles andtheanalyticalprocessinTable4(Supportinginformation).

Biologicalsamplepreparation

Plasmasamplepreparation:Bloodsampleswerecollectedfrom theocularveinof18ratsclassiﬁedrandomlyintothreegroups:

RXM,WXMandSXM.Bloodsampleswerecontainedinpolypropy- lene tubes withheparin as an anti- coagulant at 0.0833, 0.25, 0.4167,0.6667,1,1.5,2,3.5, 5.5,8.5,12 and24hafterintragas- tricadministrationofwaterdecoctionofR,WandSatadoseof 20g/kg.Thevolumeofbloodateachtimepointwas0.5ml.The sampleswerecentrifugedat3733g ×forcefor 15minandthe supernatantplasmawasgatheredandstoredat−80^◦Cuntilbeing injectedintotheUPLC-MSsystemforanalysis.Blankplasmasam- pleswereacquiredatpredose.

Urinesamplepreparation:Fortheexcretionstudy,1wasnot discoveredinallfeces.Theurinewascollectedfrom18ratskept inmetaboliccagesandclassiﬁedrandomlyintothreegroups of R,WandS;thecollectionsweretakenattimeintervalsof0–3, 3–6,6–9,9–12,12–24,24–36,36–48and48–72hafterintragastric administrationofwaterdecoctionofRXM,WXMandSXMatadose of20g/kgandstoredat−80^◦Cuntilanalysis.

Tissuedistributionstudy:Thetissues,includingkidney,gonad, spleen,lung,stomach,intestine,liver,heartandbrain,werecol- lectedafterexecution at0.1667, 0.6667,1.5, 0.5455,5.5hafter intragastricadministrationofawaterdecoctionofRXM,WXMand SXMatadoseof20g/kgfrom90ratsclassiﬁedrandomlyintothree groupsandateachtimepointfor6rats.Thetissuessampleswere storedat−80^◦Cuntiltreated.

Resultsanddiscussion

Theroutinedoseof C forhumanconsumption, accordingto theChinesePharmacopoeia(2015),was3–10g/day.Thedosein ratswascalculatedas0.9g/kg.Wedetermined20timesthedose.

Thecontentof 1 inR,W and Swasquantitativelyanalyzed as 456.8,430.7and432.1␮g/mlinaqueousextractofRXM,WXMand SXMusingourpreviousmethod.Ensuringthedifferencebetween groupsandreducingtheimpactofthelossoftheextractionpro- cessofthedrugontheexperiment, thedosewasappropriately ampliﬁedbasedontheprescribeddoseinthePharmacopoeia.The speciﬁcgastric concentrationwas20g/kg, and thevolumewas 1ml/100g.

Themostfundamentaladvantageofnoncompartmentalmod- elingistheuseoffewerandlessrestrictiveassumptionsthanin thecompartmentalmethods.Theassumptionsofthenoncompart- mentalmethodsareoftenmorereadilysubjecttoexperimental verification;thesemethodseliminatetheproblemresultingfrom theinabilitytofitalldatasetsinastudytothesamecompartmen- talmodel andtheresultingdifficultyinmakingcomparisonsof parameterestimates(WilliamR.Gillespie,1991).Therefore,anon- compartmentalmethodwaschoseninthisstudy,andthemean concentrationtimecurvesof1fittedanoncompartmentalanalysis model(Fig.1).Inaddition,thepharmacokineticparametersafter oraladministrationofwaterdecoctionofRXM,WXMandSXMwere calculatedandareshowninTable1.

Orcinolglucoside(1)ofRXMwasquicklyabsorbedtoachieve maximumplasma concentration2hafteradministration, while 1 of WXM and SXM were absorbed to achieve the maximum plasma concentration more rapidly. The area under the curve

Fig.1.Plasmaconcentrationcurvesoforcinolglucosidefromdifferentprocessing products.

Fig.2.Excretionstabilityoforcinolglucosidefromdifferentprocessingproductsin raturine.

(AUC0-∞)for1wasfoundtobe99.73±14.325␮g/l*hinWXM, 71.458±1.378␮g/l*hinRXMand75.646±7.216␮g/l*hinSXM, which suggests the same efﬁcacy due to thereabsorption and enterohepaticcirculation.(Fengetal.,2018;Zhang etal.,2018;

Zhouetal.,2018;Taoetal.,2018).Moreover1wasnotfoundin ratfeces butwasfoundin rat urine,along witha 100% degra- dationafter48h(Fig.2).FromtheFig.2,we couldseethatthe accumulativeexcretionratesof1inWXMandSXMwerealways lower than RXM, where 1 in RXM had 62.24% excretion rate, while50.02%inWXMand47.05%inSXM.Thepharmacokinetic parameters of 1 in tissue matrices are summarized in Fig. 3.

Considerablelevelsweredetectedinallstudiedtissues.Compared withplasmaexposure,higherlevelsof1weredetectedintissues includingthekidney,stomachandintestine, whilelowerlevels of1weredetectedinliver,gonadandlungtissues(Zhangetal., 2013;Xuetal.,2017;Wangetal.,2018).And,hardlyany1was detectedinspleen,heartand braintissues,whichsuggeststhat 1cannotcrosstheblood-brainbarrier.AsshowninFig.3,after intragastricadministration,1rapidlydistributedtovarioustissues, includingthekidney, stomach,intestine, liver,gonad and lung.

TheWXMandSXMexhibitedasimilarpatterntotheRXM,which probablyexplainstheefﬁcacyandlegitimacyofwine-processing andsaline-processing.InalloftheRXM,WXMandSXMgroups, the highest concentrationof 1 in thetissues showed this ten-

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Table1

Pharmacokineticparametersoforcinolglucosidefromdifferentprocessingproductsinratplasma.

Pharmacokineticparameters RXM WXM SXM

AUC(0-t)(mg/l*h) 71.443±1.376 99.591±14.473 75.48±7.112

AUC(0-∞)(mg/l*h) 71.458±1.378 99.730±14.325 75.646±7.216

MRT(0-∞)(h) 1.892±0.056 1.913±0.285 2.130±0.191

Tmax(h) 2.000±0.000 1.417±0.204 0.973±0.304

T1/2(h) 0.864±0.227 0.819±0.211 0.821±0.117

Vz/F(l/kg) 347.554±87.013 242.17±79.243 313.654±39.196

CLz/F(L/h/kg) 279.972±5.430 204.003±29.054 266.380±25.062

Cmax(mg/l) 31.089±3.596 40.786±6.611 26.381±2.638

Eachvaluerepresentsmean±SD(n=6);AUC(0-t),areaundertheplasmaconcentration-timefromzerotothelastquantiﬁabletime-point;AUC(0-∞)areaundertheplasma concentration-timecurvefromzerotoinﬁnity;MRT(0-∞),meanresidencetimezero;Cmax,maximumplasmaconcentration;Tmax,timetomaximumconcentration;t1/2, eliminationhalf-life;

Fig.3.Pharmacokineticproﬁleoforcinolglucosidefromdifferentprocessingproductsinthetissues.

dency: RXM (intestine>kidney>stomach>liver>gonad=lung);

WXM (intestine>kidney>stomach>liver>lung>gonad); SXM (intestine>stomach>kidney>liver>gonad=lung).

Inaddition,thesigniﬁcantincreaseinAUC0-tandCmaxvalues suggested thatwine-processing couldincrease thebioavailabil- ity.ComparedwithRXMandWXM,thepharmacokineticcurveof SXMhasnobimodalphenomenon,whichmayexplaintheeffect ofsalt-processingonC.Thereweresigniﬁcantdifferencesinthe threegroupsbasedontheparallelsamplingpoints.1fromSXM andWXMreachedCmaxmorerapidlythanR.Thisoutcomecould beexplainedbyintestinalabsorptionof1inthepriorCaco-2exper- iment.Wine-processingcouldenhanceintestinalabsorptionof1.

At0.67hand1.5h,theconcentrationof1washighestinthespleen comparedwithotherorgans,whichsuggeststhekidneyandintes- tinemayserveasthereceptorof1.Urineexcretionisthemajor excretionrouteoforal 1fromwater decoction.We canfurther examineitsmetabolitesinfutureexperiments.Theseresultsmight showthatprocessingproductswereexcretedmorerapidlyand noresorption and explain the differencein absorptionof 1 in ratplasma.Althoughthecontentof1inWXMwashigherthan RXMandreversedinSXM,thewine-processingandsalt-processing mightplayacrucialroleinenhancingtheabsorptionandbioavail- abilityofthe1,whichmightbeduetotheprocessingtechniqueof traditionalChinesemedicineusedforthousandsofyears.

Conclusion

Furthermore,alldatawereanalyzedusingMasslynx4.1,DAS2.1 andOrigin7.Inthisstudy,anaccurateandpreciseH-ClassXEVO TQDapproachbasedonmethanolprecipitationandtheprocess- ingtechniquewasestablishedandvalidatedforthesimultaneous determinationof1inratplasma,urineandvarioustissuesandfor anapplicationtoacomparativepharmacokineticstudyofaque- ousextractsofrawand processedC.Thestudyinvestigatedrat plasma,tissueandurineexcretionandexploredtheregularabsorp- tion,tissue distributionand metabolicpathway of1 in vivoby usingaqueousextractsofrawandprocessedC,whichcouldsave resourcesandpermitcomparisonsbetweenrawandprocessedC.

Conﬂictofinterest

Theauthorsdeclaredthattheyhavenoconﬂictsofinterestto thiswork.Wedeclarethatwedonothaveanycommercialorasso- ciativeinterestthatrepresentsaconﬂictofinterestinconnection withtheworksubmitted.

Authors’contributions

TLandCJwrotethemanuscript.TLandGScarriedoutthedata analyses.TLandCJdesignedthestudy.Theremainingauthorscon-

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tributedtorefiningtheideas,carryingoutadditionalanalysesand finalizingthepaper.Alltheauthorshavereadthefinalmanuscript andapprovedsubmission.

Ethicaldisclosures

Protectionofhumanandanimalsubjects.

Theauthorsdeclarethattheproceduresfollowedwereinaccor- dancewiththeregulationsoftherelevantclinicalresearchethics committeeandwiththoseoftheCodeofEthicsoftheWorldMed- icalAssociation(DeclarationofHelsinki).

Conﬁdentialityofdata.

Theauthorsdeclarethatnopatientdataappearinthisarticle.

Righttoprivacyandinformedconsent.

Theauthorsdeclarethatnopatientdataappearinthisarticle.

Acknowledgments

The work was supported by Natural Science Foundation of LiaoningProvince(20180550241).

AppendixA. Supplementarydata

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.

1016/j.bjp.2019.08.005.

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