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Avalidated HPLC analysis of eurycomanone(1),abioactivequas- sinoid, in rat plasma followingoral and intravenous administration of Eurycomalongifolia Jack extract was developed for

pharmacokinetic and bioavailability studies. Relatively high plasma eurycomanoneconcentrations were detected after an intrave- nousinjection of 10mg/kg extract F2c ontaining1.96mg/kgof the quassinoid. However, it declined rapidly to zero after 8 h. Its mean eliminationrate constant (ke), biological half-life (t1/2), volume of distribution (Vd) and clearance (CL) were

0.880.19h1, 1.000.26h,0.680.30L/kgand0.390.08L/h/kg, respectively. Following oraladministration of eurycomanone, itsCmaxandTmax values were detected

as0.330.03mg/mLand4.400.98h, respectively. The plasma concentration of the quassinoidafter oral administration was much lower than after intravenous application in spite of the oraldose being 5 times higher. The re-

sults indicate that eurycomanone is poorly bioavailable when givenorally .A comparison of theAUC0 obtained orallyto that obtained after an intravenous administration ( normalized for dose

differences) revealed that the absolute bioavailability of the compound was lowwith10.5%.Furthermore, the compound appeared to be well distributed in the extravascular fluids

because of its relatively high Vd value. The poor oral bioavailability was not attributed to

instability problems because eurycomanone has been shown to be stable under different pHconditions. Thus, its poor oral bioavailability may be due to poor membrane permeability in viewof it slow Pvalue and/or high first-pass metabolism.

Keywords

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Introduction

EurycomalongifoliaJack, atall tree belonging to the Simarouba- ceae family,is commonly knownas a Tongkat A lioin Malaysia, a Pasak Bumio in Indonesia and aCay ba binho in Vietnam [1].

The roots of this plant are used as traditional medicine forfever, after birth,for healing of

boils,wound ulcer,syphilisandbleed- inggums[2].From its roots, several classes of chemicalconstitu- ents consisting of quassinoids[3],[4],[5],[6],[7],[8],[9],[10], [11], [12], [13], [14], [15],[16], [17], alkaloids [18], [19], [20], [21], [22], tirucallane-type triterpenes [9], squalenederivatives [23],[24],[25]and biphenyl neolignan[26]have been isolated

and characterized. Among the quassinoids studied from this plant [16], [17], [18], eurycomanone(1), has shown antimalarial activity against the Thailand strain(K1)[8],chloroquine-resis-

tantGombakAandchloroquine-sensitiveD10strainsof Plasmodium falciparum parasites [3].Besides its antiplasmodial activity,1alsoexhibitedpotentantipyretic[7]andcytotoxicac-tivitiesagainstKB,P388[12]andMCF-7[17]tumourcellsin

vitro.Inaddition,1hasbeenreportedasthechemicalconstitu-entofE.longifoliawiththehighestyield[1].Therefore,anyinfor- mation on the disposition and

bioavailability of 1 will be highly important if its pre-clinical bioactivities are to be clinicallyeval- uated.However,todate,nostudyhasbeenpublishedpertaining

to its bioavailability and pharmacokinetics.Thus,thepresent study is the first report comparing itsoral and intravenousbio- availability and pharmacokinetic data, as well as investigating itslipophilicity and stability under different pH conditions.This may affect its absorption in thegastrointestinal tract.

Material and Methods


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Materials Eurycoman one(1) was isolated and purified from the roots of Eurycomalongifolia Jackfollowing the protocol described previously[3]. Compound 1 was obtained with a 57.8% yield . It

had a melting point of 252254 VC and an optical rotation of [a]D27: + 34.2V (c 0.03, pyridine),

with an FAB-MS molecular ion peak at m/z=408[M]+[1],[3]. Its structure was confirmed by

comparison of its spectroscopic data to literature data reported previously[1],[3],[8].A voucherspecimen of the plant has been deposited at the PenangBotanicalGarden,Malaysia,withReference

No. 785 117 [1]. HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany).

Instrumentation

The HPLC system consisted of a JascoPU-980pump(Tokyo,Ja- pan), a Gilson 115 UV detector(Wisconsin, US) and a Hitachi D- 2500 Chromato-integrator (Tokyo, Japan). A MetaphaseCrestpak C18(4.6mmi.d250mm) column was used for chromatograph- ic separation. Theanalytical conditions were as follows:mobile phase, acetonitrile:distilled deionized water = 1 : 9[the water was prepared from distilled water deionized through a Maxima ultra pure water

purifier (Elga, England)]; flow-rate, 1.0 mL/min; UVwave length,238nm. The calibration curveusing standard substances for determining the concentration of eurycomanone (1) was y =1250.3x43.2, r = 0.999 (y = peak height in mV; x = 1 in mg/mL).

Animals

Male Sprague-Dawley rats, weighing about 300 g (12 weeks old), were purchased from the

animal house of the UniversitiSains Malaysia and maintained in a12h light-dark cycle at ambientroom temperature . Animals were maintained for one weekand starved overnight with free accessto water before the experiments were performed. The animal experiments were conducted inaccordanceith theEuropeanAgencyfor the Evaluation of Medical Product Guidelines(EMEA/CVMP/133/99-Final).

Blood sampling

Five male Sprague-Dawley rats were used in the oralversus intravenous pharmacokineticstudies. Animals were kept in cages

With food pellets ,and water was given ad libitum. Twelve hours prior to the study, the foodpellets were removed and only water was given. The animals were fasted throughout the study.Stand- ardized eurycomanone-enriched E. longifolia extract, (F2), containing 19.6% of 1dissolved in normal saline was injected into the tail vein of the rats at a dose of 10.0 mg/kg F2(equivalent to 1.96 mg/kg of 1). After two weeks of washout period, all the rats were then

administered F2 orally via a feeding needle at a dose of 50mg/kgF2(equivalent to9.8mg/kgof1).Thet reated rats were placed in restraining cages when their blood was collected. Blood samplesof 0.5mLwere removed from the tail vein of the rat sat0 (pre-dose),20min,40min,1,2,4,6and8h

after intravenous injection and at0(pre-dose),1,2,4,6,8,10,12and16h after oral

administration.They were then transferred into hepar in izedmi- crocentrifuge tubes. The bloodsamples were centrifuged at 1800g for15min,and the plasma was next separated and kept frozenuntil analysis.

Sample preparation

50 mL of the plasma sample were deproteinized by adding 2.5 mL of 70% perchloric acid. The

mixture was mixed for 30 sec on a vortex and then centrifuged t1000g for10min . The clearsuper- natant layer was collected,and20mLwere injected for analysisin the HPLCsystem.


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Method validation

Fortheassayvalidation,astocksolutionofpureeurycomanone

(1)ataconcentrationof1000mg/mLwaspreparedbydissolvingthequassinoidinthemobilephase.Astandardcalibrationcurve

of1at0.1,0.2,0.4,0.8,1.6,3.2,6.4and12.8mg/mLwasprepared by serial dilution of the stock solution

with pooled blank human plasma prior to the determination of recovery, within-day and between-day precision and accuracy of the method. The within- day accuracy and precision were

determined for each concentra- tionbasedoneightmeasurementsforasingleday,whereasthebetween-day values were obtained over six consecutive days of the validation period. A separate

calibration curve using stand- ardsubstanceswascalculatedoneachdayoftheanalysis.Theaccuracywasexpressedasthepercentageerrorwhereasthepre- cision was denoted by the coefficientof variation. The recovery valueswereestimatedfromtheHPLCchromatogrambycompar- ing the

peak heights of the plasma samples to the peaks ofknownconcentrationsof1andtothoseofthesameconcentra- tionsprepareddirectlyinaqueoussolutions.

Data analysis

Thefollowingpharmacokineticparameterswereestimatedfrom the data obtained for intravenous (i.v.) administration: elimination rate constant, (ke); biological half-life, (t1/2); volume of dis-

tribution,(Vd),areaunderplasmaconcentration-timecurve

(AUC0)andclearance(CL).Fortheoraladministrationdata,

onlytheAUC0wasestimatedbecausethelevelsobtained

wereverylow,beingneartothelimitofdetection.Thus,thecon-centrationvaluescouldnotbereliablyusedtoestimatethephar-macokineticparameterke.Peakconcentration(Cmax)andtimeto reach Cmax (Tmax) following oral

administration were obtained from the actual data. Additionally, the absolute bioavailability ofeurycomanonewasestimatedfromtheratiooftheAUC 0ofthe

oraldataoverthatofthei.v.data.Thekewascalculatedfromthe slope of the plasma concentration

versus time curve (after loga-

rithmictransformation)whereast 1/2wascalculatedusingthe

relationship,t1/2=ln2/ke.AsforVd,itwascalculatedfromthe

relationship,V=dose/keAUC0.ThevalueofAUC0wasde- termined by adding the area from thetime zero to the last sampling time (AUC0t) with the area from the last sampling time to

infinity(AUCt).Theformerwascalculatedusingthetrapezoi- dal methods whereas the latter was

estimated by dividing the last measurable plasma drug concentration by ke. From AUC0,

CLwascalculatedfromtherelationship,CL=dose/AUC 0[27].

Stability study The chemical stability of eurycomanone (1) was determined at pH 1, 4 and7. Themedia used were0.1 M HClfor pH 1, 0.05 M NaH2PO42 H2O adjusted to pH 4 with glacial acetic

acid and 0.05 M Na2HPO42 H2O adjusted to pH 7 with glacial acetic acid. After approximately

half an hour for allowing equilibration of the medium to 37 VC, 1.0 mg of 1 was dissolved in

each of the three reactions at a concentration of 40 mg/mL. Samples of 1.0 mLwerecollectedfromeachreactionatintervalsof0,0.5,1.0,2.0, 4.0, 6.0 and 24.0 h. The experiment wasconducted in duplicate. The concentration of 1 in the withdrawn samples was then ana- lysed

using theHPLC method as described above,exceptthatthe mobile phase wasmodifiedtoa4:6mixture of methanol and water.


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Partition coefficient study The lipophilic ityofeurycomanone(1) was determined using the n-octanol/water partition coefficient(P).The aqueous phase used for this study

was0.1MHCl(pH1),0.05MNaH2PO4H2O (adjusted to pH4 with glacial aceticacid)and0.05MNa2HPO4 H2O (adjusted to pH 7 with glacial acetic acid). About 10.0 mL ofeach phase and 0.3mgof1were added into each of the three separating funnelsto yieldaconcentration of30mg/mL.Into each of the separating funnels , about10.0mL of n-octanol werethen added. The mixtures in the three separating funnels were shaken vigorously and left to standfor 2hours. Analiquot of0.2mL was withdrawn from the aqueous phase (pH1,4and7), and anali-

quotof2.0mLwascollectedfromthen-octanolphase.Thecon- centration of 1 in the withdrawnsamples was determined using theHPLC method as described reviously. Subsequently, the par-

tition coefficient, P value, of 1 was determined from the ratio of the its concentration in the n-octanol to that in the aqueous phase for each pH studied.

The between-day, within-day accuracy and precision values of the analytical method for

eurycomanone(1)are presented inTa- ble1.The coefficient of variation value(CV)for precision andthe percentage error values for between-day and within-day meas- urements of 1 were all less

than 14%. Its mean recovery was 92.6 % 6.1 S.E. The calibration curve using standardsubstances for determination of plasma eurycomanone(1) was found tobe linear,with an intercept

of43.2, a slope of1250.3 and a correla- tion coefficient of0.999. A detection limitof0.02mg/mLwas observe data signal to no iseratio of4:1,whereas the lower limit ofquantification (LLOQ) was 0.10 mg/mL.

Results

The chromatograms obtained with the blank plasma,plasma spiked with10mg/mLeurycomanone(1) and plasma containing 0.45 mg/mL eurycomanone (1) 4 h after an oral

administration of 50mg/kgofF2containing9.8mg/kgof1areshowninFigs.1a,bandc.Itcanbeseenthat1(retentiontimeofaround19min) lacked nterference by endogenouscompounds and wasthere- fore suitable for preparation of the calibration curve using stand-ardsubstances.

The curve profiles of the mean concentration of eurycomanone (1) in the rat plasma versus timeafter intravenous (10 mg/kg F2 containing 1.96mg/kgof 1) andoral(50mg/kg F2 containing 9.8mg/kg of 1) administrations are compared in Fig. 2. The plas-

maconcentrationof1showedarapiddeclinefrom5.08 1.99to

0 mg/mL after 8 h when given i. v. Following oral application, it showed a gradual rise followed

by a gradual decline from 0.05 0.05 to 0.06 0.04mg/mL after 16h (Fig.3). Relatively higherplasma concentrations of 1were observed after i.v.injection than after oral administration. Acomparison of the AUC0 values (Table 2; the values are normalized regarding the dose differ-

ences) of 1 given either orally as F2 extract or intravenously revealed that the absolutebioavailability of the quassinoid was only10.5%.Its mean elimination rate constant,ke,and

biological half-life(t1/2)values obtained fromi.v.injectionwere0.880.19 h-1and1.000.26h,respectively.This indicates that1has avery short biological half-life. Its meanvolume of distribution, Vd, and mea nclearance,CL, estimatedfrom i.v. data were 0.680.30L/kgand0.390.08L/h/kg,respectively.Incontrast,itsmeannu-mericalvaluesofCmaxandTmaxfollowingoralapplicationwere

0.330.03mg/mLand4.400.98h,respectively.Thissuggests that the absorption was relativelyslow reaching a peak concen- trationfollowing4hoursofdosing(Fig.3andTable2).


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The concentrations of 1 kept at 37 VC for either 1/2 hour or 24

hoursatpH1wereanalyzedas96.38% 2.93S.E.and96.09%4.08 S.E.; at pH 4, its concentrationswere 94.90 % 1.38 S.E. and 98.54%0.74S.E.,andatpH7,theywere98.01% 3.02S.Eand97.69% 3.92 S.E. respectively. No significant changes in thecompoundconcentrationwereobservedovera24-hourincuba- tion period in media adjusted to three

different pH values. The P values of the n-octanol-water partition coefficient for 1 at pH 1, 4

and7weredeterminedas1.27,1.28and1.26respectively.

Fig. 1 HPLC chromatograms showing (a) blank rat plasma with an endogenous peak (E), (b)blank rat plasma spiked with10mg/mLofeurycomanone(1)anditsretentiontime (min) and (c) ratplasma containing 0.45 mg/mL eurycomanone (1) and its retention time (min) after 4 h oral

administration of 50 mg/kg F2 containing 9.8 mg/kg of 1. Y-axis, peak height (mV); X-axis,chart speed = 2.5 mm/min; limit ofdetection=0.02mg/mLandlimitofquantification= 0.10 mg/mL.

Discussion ArapiddirectHPLCassayformeasuringeurycomanone(1)in therat plasma wasdeveloped in which its analyte peak at 1920min was clearly separated rom the plasmaendogenoussubstan-ces. Its peak was discrete and reproducible with a precision (% CV) value for

the various standard concentrations (Table1) within the limits of less than 20% deviation for the

lower limit of quantification (LLOQ) and 15% deviation of standards other than the LLOQ [27].The calibration curve of the standard substances has an accuracy (% error) range within theaccepted rangeof80100%forbioanalyticalmethodvalidation[27].TheaccuracyatitsLLOQwasatthelowendwith80.6%whereasthe

accuracyathigherconcentrationsincreasedto96.2100.6%.

Following intravenous administration of 10.0 mg/kg of F2 extract (equivalent to 1.96 mg/kg of 1)

into the tail vein of the rats, the volumeofdistribution(Vd)of1wasfoundtobeapproximatelyvalue,

the drug may be well distributed in the extravascular fluids [28]. The low absolute bioavailability(10.5 %) of 1 following oral administration may be due to pre-systemic metabolism orfirst-

passeffect(inthegutwallandliver)priortoreachingthe general circulation. In having a high aqueous

solubility, 1 may be

absorbedinefficientlybythegastrointestinaltract[29].ThecompoundwasstableatpH1,4and7,henceitspoororalbio- availability was unlikely to be caused by its degradation in the non-favourable pHconditions of the gastrointestinal tract. The permeability properties of 1 as well as its metabolismin the en- terocytesandliverareworthofbeingpursuedfordevelopment of better oral bioavailability

strategies.

Inconclusion,eurycomanone(1)exhibitedlowbioavailability,a shortbiologicalhalf-lifeandappearedtobewelldistributedin the extravascular fluids. Its poor oral bioavailability was not

at- tributable to low stability of 1 under the pH conditions of the gastrointestinal tract. Instead, itmay be due to its poor membrane permeability arising from its low P value and/or high first-

pass metabolism.

Acknowledgement

The authors wish to thank the Ministry of Science, Technology

andInnovation,Malaysia,foraatopdownograntfromtheInten- sive Research on Priority Areas(IRPA).


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jurnal1 terapan