Steady-state pharmacokinetics and tissue distribution of anthraquinones of Rhei Rhizoma in rats

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Journal of Ethnopharmacology

j ou rn a l h o m e pa g e:w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m

Steady-state pharmacokinetics and tissue distribution of anthraquinones of Rhei Rhizoma in rats

Chi-ShengShiaâ,Shang-YuanTsaiâ,Jung-ChihLin^b,Ming-LiLi^c,Miau-HwaKo^d,Pei-DawnLeeChaoâ, Yu-ChingHuangâ,Yu-ChiHouâ^,ê^,∗

aSchoolofPharmacy,CollegeofPharmacy,ChinaMedicalUniversity,Taichung,Taiwan

bDepartmentofIntegratedChineseandWesternMedicine,ChungShanMedicalUniversityHospital,Taichung,Taiwan

cCardiovascularDivision,SurgeryDepartment,ChinaMedicalUniversityHospital,Taichung,Taiwan

dDepartmentofAnatomy,CollegeofMedicine,ChinaMedicalUniversity,Taichung,Taiwan

eDepartmentofMedicalResearch,ChinaMedicalUniversityHospital,Taichung,Taiwan

a r t i c l e i n f o

Articlehistory:

Received1March2011

Receivedinrevisedform2August2011 Accepted2August2011

Available online 9 August 2011

Keywords:

Anthraquinone Tissuedistribution Pharmacokinetics Polyphenol

a b s t r a c t

Aimofthestudy:RheiRhizoma,therhizomeofRheumpalmatumL.(RP),isapopularherbinclinical Chinesemedicine.RPisabundantinpolyphenolicanthraquinones,whichhavebeenreportedtoshow variousbeneﬁcialbioactivities.Thisstudyinvestigatedthepharmacokineticsandtissuedistributionof anthraquinonesfollowingseven-doseadministrationofRPdecoctiontorats.

Materialsandmethods:SixSprague-Dawleyratsweregiven2.0g/kgofRPtwicedailyforsevendosesand bloodsampleswerecollectedatdesignatedtimeafterthe7thdose.Anothersixratsweresacriﬁcedat 30minafterthe7thdoseandorgansincludingliver,kidney,lungandbrainwerecollected.Serumand tissuespecimenswereassayedbyHPLCbeforeandafterhydrolysiswith␤-glucuronidaseandsulfatase, respectively.

Results:Pharmacokineticanalysisindicatedthattheanthraquinonesinserummainlypresentedasglu- curonides/sulfatesandcontainedhigherratioofsulfateswhencomparedwithsingle-doseadministration ofRP.Contrarytotheﬁndinginserum,tissueanalysisdiscoveredmainlyfreeformofanthraquinonein mostorgansassayed,suchasaloe-emodinandrheininkidney,liver,lung;emodininliver,lung;trace ofchrysophanolinkidneyandliver.Inallbrains,neitherfreeformsnortheirglucuronides/sulfateshave beendetected.

Conclusions:Theglucuronides/sulfatesofanthraquinoneswerethemajorformsinbloodstream,whereas thefreeformsofmostanthraquinoneswerepredominantinkidneyandliver.

1. Introduction

Anthraquinones are a group of polyphenolicconstituents in RheiRhizoma (the rhizome of Rheum palmatumL., RP), which is commonly usedin clinical Chinese medicineas potent laxa- tive,antibacterial, anti-inﬂammatory, anticancer,antispasmodic andcholereticagents(Shenand Li,1997;Shiaetal.,2011).The anthraquinonesofRPincludealoe-emodin,rhein,emodin,chrysophanoland physcion(chemicalstructuresshown in Fig.1)and theirglycosides(Li,2002).Numerousstudiesreportedthatthese anthraquinones exhibited beneﬁcial bioactivities such as anticancer(Shietal.,2001;Pecereetal.,2003;Kuoetal.,2004;Guo etal.,2007;Huangetal.,2007),antivirus(Shuangsuoetal.,2006),

∗ Correspondingauthorat:SchoolofPharmacy,ChinaMedicalUniversity,91 Hsueh-ShihRd,Taichung,Taiwan.Tel.:+886422053366x5107;

fax:+886422031028.

E-mailaddress:hou5133@gmail.com(Y.-C.Hou).

antioxidation (Caiet al., 2004) and anti-diabetes (Zheng et al., 2008).Despitethesepositiveresults,itremainsunclearwhether thoseactivitiesfoundinvitrocanexistinvivo.

Our previous study documenting the pharmacokinetics of anthraquinones after single dosing of RP has pointed out that the free formsof aloe-emodin,emodin and chrysophanolwere notdetectedinbloodbyHPLC/UVdetection,whereas theirglu- curonides/sulfateswerepredominant.The onlyfreeformfound inthecirculationwasrhein,which alsopresentedmainlyasits glucuronides/sulfates(Shiaetal.,2009).Later,anotherourstudy reportingthetissuedistributionofﬂavonoidsofScutellariaeRadix (root of Scutellariaebaicalensis)disclosed thatthefree forms of baicaleinandwogoninwerethemajorformsinmanyorgans,in contradictiontotheﬁndinginblood(Houetal.,2011).Therefore, inordertoelucidatetherealmolecularformsandconcentrations ofrhubarbanthraquinonesinthecirculationandinvariousorgans atthesteadystate,pharmacokineticandtissuedistributionstud- iesfollowingrepeateddosingofRPdecoctionwereconductedin rats.

doi:10.1016/j.jep.2011.08.003

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O O

HO OH

CH₃

Aloe-emodin Rhein Emodin

Chrysophanol Physcion

O O

HO OH

CH3

H3CO O

O

HO OH

CH₂OH

O O

HO OH

COOH

O O

HO OH

CH3

HO

Fig.1.Chemicalstructuresofanthraquinones.

2. Materialsandmethods 2.1. Chemicals

Aloe-emodin (purity95%),rhein(purity95%),emodin(purity 90%), chrysophanol (purity 98%), physcion (purity 95%), ␤- glucuronidase (type B 1,666,400units/g, from bovine liver), sulfatase(typeH1,14,400units/g,fromHelixpomatia,containing 300,000units/gof␤-glucuronidase)and2-methlylanthraquinone (purity95%)werepurchasedfromSigmaChemicalCo.(St.Louis, MO, U.S.A.). Acetonitrile, methanol and ethyl acetate were LC grade and purchased from Mallinckrodt Baker, Inc. (Phillips- burg, NJ, U.S.A.). l(+)-Ascorbic acid was obtained from RdH Laborchemikalien GmbH & Co. KG (Seelze, Germany). Other reagentswereHPLCgradeoranalyticalreagentgrade.Milli-Qplus water(Millipore,Bedford,MA,U.S.A.)wasusedthroughout this study.

2.2. Instrumentation

The HPLC apparatus included a pump (LC-10AT, Shimadzu, Japan),anUVspectrophotometricdetector(SPD-10AVP,Shimadzu, Japan), a ﬂuorescence detector (RF-10A, Shimadzu, Japan) and chromatopac(C-R6A,Shimadzu,Japan)withanautomaticinjec- tor(SIL-10A,Shimadzu,Japan)andanAlltechApolloC18column (5␮m,4.6mm×150mm,Alltech,Deerﬁeld,IL,USA).

2.3. PreparationofRPdecoction

TherhizomesofRheumpalmatumwaspurchasedfromaChinese drugstoreinTaichung,Taiwan.Theoriginofthecrudedrugwas identifiedmicroscopicallybythecorrespondingauthorDr.Yu-Chi Hou.Water(5l)wasaddedto250gcrudedrugsandheatingon agasstove.Afterboiling,gentleheatingwascontinueduntilthe volumereducedtoabout2.5landthenthemixturewasfiltered whilehot.Thefiltratewasgentlyboileduntilthevolumereduced tolessthan500mlandsufficientwaterwasaddedtomake500ml, whichwasimmediatelydividedintoaliquotsandfrozenat−20^◦C forlateruse.

2.4. QuantitationofRPdecoctionanditsacidhydrolysate

Rhubarbdecoction(3.0ml)wasmixedwith7.0mlofmethanol and centrifuged to remove polysaccharide. Then the properly diluted rhubarb decoction (100␮l) was added with 100␮l of 2-methylanthraquinone solution (50.0␮g/ml in methanol) as internalstandardand20␮lweresubjectedtoHPLCanalysis.The mobilephaseconsistedofacetonitrile(A)–0.1%phosphoricacid (B)andprogramedin agradientmannerasfollows:A/B: 50/50 (0–10min),85/15(15–22min)and50/50(27min).Thedetection wavelengthwassetat250nmandtheﬂowratewas1.0ml/min.

Acid hydrolysis of 1ml of the decoction was conducted by adding1mlof1.2NHClandthehydrolysiswasconductedat80^◦C inawaterbathfor6h.Thehydrolysatewasthenaddedwithsufﬁ- cientmethanoltomake2.0mlbeforeHPLCanalysis.Thecontents of glycosides of aloe-emodin, rhein, emodin, chrysophanoland physcionweredeterminedbysubtractingtheconcentrations of eachaglyconeinthedecoctionfromthoseofcorrespondentagly- coneinthedecoctionhydrolysate.

2.5. Animals

MaleSprague-DawleyratsweresuppliedbyNationalLabora- toryAnimal Center(Taipei, Taiwan) and keptinAnimal Center ofChineseMedicalUniversity.Theanimalstudyadheredto“The Guidebook forthe Careand Useof LaboratoryAnimals(2002)”

(PublishedbytheChineseSocietyfortheAnimalScience,Taiwan, ROC).TheanimalprotocolwasapprovedbyInstitutionalAnimal CareandUseCommittee(IACUC)ofChinaMedicalUniversity.Rats werehousedina12-hlight–darkcycle,constanttemperatureenvi- ronmentpriortostudy.

2.6. Drugadministration,bloodandtissuecollection

Forpharmacokineticstudy,sixratsweighing390–440gwere fastedfor12hbeforedrugadministration.Afterfasting,ratswere administeredorallywith2.0g/kgofRPdecoctiontwicedailyfor7 dosesviagastricgavageinordertoreachsteady-state.Inthe4th daymorning,bloodsamples(0.6ml)werewithdrawnfromrats underanesthesiawithisoﬂuraneat15,30,60,120,240,480,720 and1440minafterthe7thdoseviacardiopuncture.Thebloodsam- pleswerecollectedin microtubesand centrifugedat10,000×g

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for15mintoobtaintheserum,whichwasstoredat−20^◦Cuntil analysis.

For tissue distribution study, six rats weighing 350–450g wereadministeredRPdecoctionwiththesamedosageregimen describedaboveinpharmacokineticstudy.Inthe4thdaymorning, ratsweresacriﬁcedat30minafterthe7thdoseandabloodsam- plewascollectedviacadiopuncture.Followingimmediatesystemic perfusionwithcoldsaline,organsincludingliver,lung,kidneyand brainwereremoved,washedwithcoldsaline, blotteddry with ﬁlterpaperandthenaccuratelyweighed.Thetissueswerehomog- enizedwithcoldsaline(3g/5ml)andthehomogenateswerestored at−30^◦Cuntilanalysis.

2.7. Quantitationofanthraquinonesandtheir glucuronides/sulfatesinserumandtissues

Thedeterminationofanthraquinoneconcentrationsinserum andtissuehomogenatesfollowedapreviousmethodwithminor modiﬁcation(Shiaet al.,2009).For theassayofanthraquinone aglycones, 100␮l of serum or tissue homogenate was mixed with 50␮l of pH 5 acetate buffer and 50␮l of ascorbic acid (100mg/ml). Themixture wasacidiﬁedwith50␮lof 0.1N HCl andpartitionedwith250␮lofethylacetate(containing1␮g/ml of2-methylantraquinoneasinternalstandard).Theethylacetate layerwasevaporatedunderN2 gastodrynessandreconstituted with50␮lofmethanol,then20␮lwassubjectedtoHPLCanalysis.

Forthequantitationofglucuronidesandsulfates,100␮lofserum ortissuewasmixedwith50␮lof␤-glucuronidaseandsulfatase, respectively,inpH5acetatebuffer(1000units/ml)and 50␮lof ascorbicacid(100mg/ml),thenincubatedat37^◦Cfor10min,which hadbeendeterminedbyapreliminarytimestudy.Afterhydroly- sis,theserumortissuehomogenatewasacidiﬁedwith50␮lof 0.1NHClandthelaterprocessesfollowedthosedescribedabove.

Themobilephaseconsistedofacetonitrile(A)and0.1%phosphoricacid(B),andagradientelutionprogramwasrunasfollows:

A/B:50/50(0–10min),85/15(15min),80/20(20min)and50/50 (25min).Forserumassay,thewavelengthofUVdetectorwasset at250nm.Fortissueassay,ﬂuorescencedetectionwasperformed withwavelengthsof435and515nmforexcitationandemission, respectively.Theﬂowratewas1.0ml/min.

For calibrator preparation, 100␮l of serum or tissue homogenate each spiked with various concentrations of anthraquinones was added with 50␮l of pH 5 buffer. The laterprocedurewasthesameasthatdescribedaboveforserum sample.Thecalibrationgraphwasplottedbylinearregressionof thepeakarearatios(anthraquinonestointernalstandard)against concentrationsofanthraquinones.

The systemsuitability wasevaluated through intra-day and inter-dayanalysisofprecisionandaccuracy.Recoverywasassessed bycomparingthepeakareaobtainedfromextractedsamplespiked withstandards tothat obtainedfrom unextractedstandards in extracted sample matrix, which represents100% recovery.The lowerlimitofquantitation(LLOQ)representsthelowestconcentra- tionofanalysisinasamplethatcanbedeterminedwithacceptable precisionand accuracy,whereaslimit ofdetection(LOD) repre- sentsthelowestconcentrationofanalysisinasamplethatcanbe detected(withS/N>3).

2.8. Dataanalysis

Thepeakserum ortissueconcentration(Cmax)wasobtained fromexperimentalobservation.Thepharmacokineticparameters were analyzed by noncompartmentmodel withthe aidof the program WinNonlin (version 1.1 SCI software, Statistical Con- sulting, Inc., Apex, NC, U.S.A.). The area under the serum or tissue concentration–time curve (AUC0–t) was calculated using

Table1

Contents(␮g/ml)ofaloe-emodin,rhein,emodin,chrysophanolandphyscioninRP decoctionbeforeandafteracidhydrolysis.

Constituents Beforehydrolysis Afterhydrolysis

Aloe-emodin 119.5 613.6

Rhein 577.9 910.1

Emodin 114.4 552.6

Chrysophanol 90.5 471.1

Physcion 38.9 199.5

trapezoidal rule to the last point. The concentrations of anthraquinone sulfates in tissue were obtained by subtract- ing theconcentration of anthraquinoneglucuronides from that ofcorrespondent anthraquinoneglucuronides/sulfates.Unpaired Student’st-testwasusedforstatisticalcomparisontakingp<0.05 assigniﬁcant.

3. Results

3.1. QuantitationofanthraquinonesinRPdecoction

ThequantitationofanthraquinonesinRPdecoctionwasper- formed by HPLC analysis before and after acid hydrolysis. The concentrationsofeachanthraquinoneintheRPdecoctionbefore andafteracidhydrolysiswereshowninTable1.Themostabun- dant anthraquinone in RP decoction was rhein and the least wasphyscioneitherbeforeorafteracidhydrolysis.Aloe-emodin, emodin,chrysophanolandphyscionwerepredominantlypresent inglycosideforms,whereasrheinmainlyexistedinfreeform.

3.2. Quantitationofanthraquinonesandtheir glucuronides/sulfatesinserumandtissues

The quantitation of anthraquinones and their glucuronides/sulfates in serum essentially employed a method published previously (Shia et al., 2009). The serum analysis revealedthatrhein freeformwaspresentin allspecimens, and emodinfreeformexistedonlytransientlyintheearlyphaseafter dosing,whereasthefreeformsofaloe-emodin,chrysophanoland physcion were not detected.The glucuronides/sulfates of aloe- emodin,rhein,emodinandchrysophanolpredominantlyexisted inbloodstream,whilenotraceofphyscionglucuronides/sulfates had been detected. The mean serum concentration–time pro- ﬁles of each anthraquinone afterthe 7th dose of RPdecoction in six rats are shown in Fig. 2. All proﬁles revealed a second peak, a clear indication of enterohepatic circulation for these anthraquinones. The pharmacokinetic parameters of rhein free form, glucuronides/sulfates (G/S) and glucuronides (G) of each anthraquinonearelistedinTable2.Thesystemicexposures(area under the concentration–time curve, AUC) of anthraquinones ranked as follows: rhein G/S>rhein>emodin G/S>aloe-emodin G/S>chrysophanolG/S.

Theassaymethodoftissuehomogenateswasestablishedand validated in this study. The calibration ranges of aloe-emodin (0.04–4.0␮g/ml), rhein (0.01–20.0␮g/ml), emodin and chrysophanol(0.01–2.0␮g/ml)invarioustissuehomogenateswerewith goodlinearities.Themethodvalidationindicated thatallcoefﬁ- cientsofvariation inintra-runandinter-runanalysisofvarious tissuehomogenateswerebelow9.9%,andtherelativeerrorswere below 19.2%. For liver homogenate, theLLOQs and LODs were 0.04and0.01␮g/mlforaloe-emodinandchrysophanol,0.08and 0.02␮g/ml for rhein, 0.06 and 0.01␮g/ml for emodin, respectively. For kidney homogenate,the LLOQsand LODswere0.01 and0.006␮g/mlforaloe-emodin,0.06and0.04␮g/mlforrhein, 0.08and0.005␮g/mlforemodin,0.06and0.01␮g/mlforchryso- phanol.Forlunghomogenate,theLLOQsandLODswere0.008and

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Table2

Pharmacokineticparametersofanthraquinonesandtheirglucuronides/sulfates(G/S)andglucuronide(G)after7thdoseofRP(2g/kg)insixrats.

Constituents Metabolites Cmax(nmol/ml) AUC0–720(nmolmin/ml) MRT0–720(min)

Aloe-emodin G/S 2.1±0.3 288.0±63.4 147.5±19.4

G 1.5±0.2 151.7±30.9 105.5±20.1

Rhein Freeform 5.8±0.7 1250.2±167.0 243.1±23.2

G/S 17.1±1.7 3209.4±398.0 236.8±13.1

G 9.3±1.3 1725.1±294.3 215.5±28.2

Emodin G/S 3.7±0.3 717.0±62.8 235.9±15.2

G 2.1±0.3 391.5±70.7 203.6±24.1

Chrysophanol G/S 1.8±0.2 161.6±47.6 93.5±22.4

G 1.4±0.2 117.6±27.6 136.5±44.2

Cmax,themaximumconcentration(nmol/ml).

AUC0–t,theareaunderconcentration–timecurvetothelasttime(nmolmin/ml).

MRT0–t,themeanresidencetime(min).

Dataexpressedasmean±S.E.

0.002␮g/mlforaloe-emodin,0.04and0.01␮g/mlforrhein,0.006 and0.002␮g/mlforemodin,0.004and 0.001␮g/ml forchryso- phanol,respectively.Therecoveriesofanthraquinonesfromliver, kidneyandlungwere68.6–84.2%,78.0–111.1%and76.9–109.4%, respectively,attestedconcentrations.

Tissue analysis had detectedthe free forms of aloe-emodin, rhein,emodinandchrysophanolinmanyorgansasshowninFig.3.

Thetissuecontentsofeachanthraquinonefreeformwererankedas follows:kidney,liver>lungforaloe-emodin;kidney>liver>lung forrhein;liver>lungforemodin;onlytracesofchrysophanolin

kidney andliver. Amongthe assayedorgans, theconcentration ofrheinfreeforminkidneywasthehighest(24␮g/g)andeven muchhigherthanserumconcentration(14␮g/ml). Withregard totheconcentrationsofglucuronides/sulfates,notracesofthem werefoundinliver.OnlytracesofSofaloe-emodin,rhein,emodin werefoundinlung.BothGandSofaloe-emodin,rhein,emodinand chrysophanolwerepresentinkidney,butinmuchlowerconcen- trationsthantheirfreeforms.NeitherphyscionnoritsG/Shadbeen detectedinalltissuesassayed.Inaddition,thefreeformsandthe G/Sofeachanthraquinonewerenotdetectedinallbrains(Fig.4).

Time (min)

0 200 400 600 800

serum concentration(nmol/ml)

0.0 0.5 1.0 1.5 2.0

Chrysophanol G/S Chrysophanol G Time (min)

0 200 400 600 800

0.0 0.5 1.0 1.5 2.0 2.5

Aloe-emodin G/S Aloe-emodin G

Time (min)

0 200 400 600 800

0 2 4 6 8 10 12 14 16 18

Rhein Rhein G/S Rhein G

Time (min) 2

0 00 400 6 00 800

serum concentration (nmol/ml)

0 1 2 3 4 5

Emodin G/S Emodin G

a b

c d

Fig.2.Mean(±S.E.)serumconcentration–timeproﬁlesofanthraquinonesandtheirglucuronides/sulfates(G/S)andglucuronides(G)afterthe7thdoseofRP(2g/kg)insix rats.(a)Aloe-emodin,(b)rhein,(c)emodin,and(d)chrysophanol.

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Organs

serum liver lung kidney brain

Concentration (nmol/mL)

0.0 0.5 1.0 1.5 2.0 2.5

Concentration (nmol/g)

0.0 0.5 1.0 1.5 2.0 Aloe-emodin 2.5

Aloe-emodin G Aloe-emodin S

Organs

0 5 10 15 20 25 30

0 5 10 15 20 25 Rhein 30

Rhein G Rhein S

Organs

0 1 2 3 4 5 6

0 1 2 3 4 5 Emodin 6

Emodin G Emodin S

Organs

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0 0.5 1.0 1.5 2.0 2.5 Chrysophanol 3.0

Chrysophanol G Chrysophanol S

a b

c d

Fig.3. Concentrationsofanthraquinonesandtheirsulfates(S)andglucuronide(G)inserum(nmol/ml)andvarioustissues(nmol/g)afterthe7thdoseofRP(2g/kg)insix rats.(a)Aloe-emodin,(b)rhein,(c)emodin,and(d)chrysophanol.

Fig.4. Tissuedistributionsofanthraquinonesafterthe7thdoseofRPinrats(P,parentform;S,sulfates;G,glucuronides;A,aloe-emodin;R,rhein;E,emodin;andC, chrysophanol).