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Introduction

Eurycoma longifolia Jack, a tall tree belonging to the Simarouba-

ceae family, is commonly known as ªTongkat Aliº in Malaysia,

ªPasak Bumiº in Indonesia and ªCay ba binhº in Vietnam [1].

The roots of this plant are used as traditional medicine for fever,

after birth, for healing of boils, wound ulcer, syphilis and bleed-

ing gums [2]. From its roots, several classes of chemical constitu-

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], squalene derivatives

[23], [24], [25] and biphenylneolignan [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-

tant Gombak A and chloroquine-sensitive D10 strains of

Plasmodium falciparum parasites [3]. Besides its antiplasmodial

activity, 1 also exhibited potent antipyretic [7] and cytotoxic ac-

tivities against KB, P388 [12] and MCF-7 [17] tumour cells in

vitro. In addition, 1 has been reported as the chemical constitu-

ent of E. longifolia with the highest yield [1]. Therefore, any infor-

mation on the disposition and bioavailability of 1 will be highly

important if its pre-clinical bioactivities are to be clinically eval-

uated. However, to date, no study has been published pertaining

Bioavailability and Pharmacokinetic Studies of Eurycomanone from Eurycoma longifolia

Bin-Seng Low

Bee-Hong Ng

Wai-Peng ChoyKah-Hay Yuen

Kit-Lam Chan

AffiliationSchool of Pharmaceutical Sciences, University Sains Malaysia, Penang, Malaysia

CorrespondenceKit-Lam Chan ´ School of Pharmaceutical Sciences ´ University Sains Malaysia ´ 11800 Penang ´ Malaysia ´

Phone: +60-4-657-6836 ´ Fax: +60-4-657-6836 ´ E-mail: [email protected]

Received December 9, 2004 ´ Accepted April 6, 2005

Bibliography Planta Med 2005; 71: 803±807 ´ Georg Thieme Verlag KG Stuttgart ´ New York

DOI 10.1055/s-2005-871259 ´ Published online July 29, 2005

ISSN 0032-0943

Abstract

A validated HPLC analysis of eurycomanone (1), a bioactive quas-

sinoid, in rat plasma following oral and intravenous administra-

tion of Eurycoma longifolia Jack extract was developed for phar-macokinetic and bioavailability studies. Relatively high plasma

eurycomanone concentrations were detected after an intrave-

nous injection of 10 mg/kg extract F2 containing 1.96 mg/kg of

the quassinoid. However, it declined rapidly to zero after 8 h. Its

mean elimination rate constant (ke), biological half-life (t1/2), vol-

ume of distribution (Vd) and clearance (CL) were 0.88 0.19 h±1,

1.00 0.26 h, 0.68 0.30 L/kg and 0.39 0.08 L/h/kg, respectively.

Following oral administration of eurycomanone, its Cmax and Tmax

values were detected as 0.33 0.03 m g/mL and 4.40 0.98 h,

respectively. The plasma concentration of the quassinoid after

oral administration was much lower than after intravenous ap-

plication in spite of the oral dose being 5 times higher. The re-

sults indicate that eurycomanone is poorly bioavailable when

given orally. A comparison of the AUC0®¥ obtained orally to that

obtained after an intravenous administration (normalized for

dose differences) revealed that the absolute bioavailability of

the compound was low with 10.5%. Furthermore, the compoundappeared to be well distributed in the extravascular fluids be-

cause of its relatively high Vd value. The poor oral bioavailability

was not attributed to instability problems because eurycoma-

none has been shown to be stable under different pH conditions.

Thus, its poor oral bioavailability may be due to poor membrane

permeability in view of its low P value and/or high first-pass me-

tabolism.

Key words

Eurycoma longifolia Jack ´ Simaroubaceae ´ eurycomanone ´ phar-

macokinetics ́ bioavailability

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to its bioavailability and pharmacokinetics. Thus, the present

study is the first report comparing its oral and intravenous bio-

availability and pharmacokinetic data, as well as investigating

its lipophilicity and stability under different pH conditions. This

may affect its absorption in the gastrointestinal tract.

Material and Methods

Materials

Eurycomanone (1) was isolated and purified from the roots of

Eurycoma longifolia Jack following the protocol described pre-viously [3]. Compound 1 was obtained with a 57.8% yield. It had

a melting point of 252±2548C and an optical rotation of [a ]D27:

+ 34.28 (c 0.03, pyridine), with an FAB-MS molecular ion peak at

m/z = 408 [M]+ [1], [3]. Its structure was confirmed by compari-

son of its spectroscopic data to literature data reported previous-

ly [1], [3], [8]. A voucher specimen of the plant has been depos-

ited at the Penang Botanical Garden, Malaysia, with Reference

No. 785±117 [1]. HPLC-grade acetonitrile was purchased from

Merck (Darmstadt, Germany).

Instrumentation

The HPLC system consisted of a Jasco PU-980 pump (Tokyo, Ja-pan), a Gilson 115 UV detector (Wisconsin, US) and a Hitachi D-

2500 Chromato-integrator (Tokyo, Japan). A Metaphase Crestpak

C18 (4.6 mm i.d 250 mm) column was used for chromatograph-

ic separation. The analytical 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;

UV wavelength, 238 nm. The calibration curve using standard

substances for determining the concentration of eurycomanone

(1) was y = 1250.3x±43.2, r = 0.999 (y = peak height in mV;

x = 1 in m g/mL).

Animals

Male Sprague-Dawley rats, weighing about 300 g (12 weeks old),

were purchased from the animal house of the Universiti Sains

Malaysia and maintained in a 12 h light-dark cycle at ambient

room temperature. Animals were maintained for one week and

starved overnight with free access to water before the experi-

ments were performed. The animal experiments were conducted

in accordance with the European Agency for the Evaluation of

Medical Product Guidelines (EMEA/CVMP/133/99-Final).

Blood sampling

Five male Sprague-Dawley rats were used in the oral versus in-

travenous pharmacokinetic studies. Animals were kept in cages

with food pellets, and water was given ad libitum. Twelve hours

prior to the study, the food pellets were removed and only water

was given. The animals were fasted throughout the study. Stand-

ardized eurycomanone-enriched E. longifolia extract, (F2), con-

taining 19.6% of 1 dissolved 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

50 mg/kg F2 (equivalent to 9.8 mg/kg of 1). The treated rats were

placed in restraining cages when their blood was collected. Bloodsamples of 0.5 mL were removed from the tailveinof the rats at 0

(pre-dose), 20 min, 40 min, 1, 2, 4, 6 and 8 h after intravenous in-

jection and at 0 (pre-dose), 1, 2, 4, 6, 8, 10, 12 and 16 h after oral

administration. They were then transferred into heparinized mi-

crocentrifuge tubes. The blood samples were centrifuged at

1800 g for 15 min, and the plasma was next separated and kept

frozen until analysis.

Sample preparation

50 m L of the plasma sample were deproteinized by adding 2.5 m L

of 70% perchloric acid. The mixture was mixed for 30 sec on a

vortex andthen centrifuged at 1000 g for 10 min. The clear super-

natant layer was collected, and20 m L were injected for analysis inthe HPLC system.

Method validation

For the assay validation, a stock solution of pure eurycomanone

(1) at a concentration of 1000 m g/mL was prepared by dissolving

the quassinoid in the mobile phase. A standard calibration curve

of 1 at 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 m g/mL was prepared

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-

tion based on eight measurements for a single day, whereas thebetween-day values were obtained over six consecutive days of

the validation period. A separate calibration curve using stand-

ard substances was calculated on each day of the analysis. The

accuracy was expressed as the percentage error whereas the pre-

cision was denoted by the coefficient of variation. The recovery

values were estimated from the HPLC chromatogram by compar-

ing the peak heights of the plasma samples to the peaks of

known concentrations of 1 and to those of the same concentra-

tions prepared directly in aqueous solutions.

Data analysis

The following pharmacokinetic parameters were estimated from

the data obtained for intravenous (i.v.) administration: elimina-

tion rate constant, (ke); biological half-life, (t1/2); volume of dis-

tribution, (Vd), area under plasma concentration-time curve

(AUC0®¥ ) and clearance (CL). For the oral administration data,

only the AUC0®¥ was estimated because the levels obtained

were very low, being near to the limit of detection. Thus, the con-

centration values could not be reliably used to estimate the phar-

macokinetic parameter ke. Peak concentration (Cmax) and time to

reach Cmax (Tmax) following oral administration were obtained

from the actual data. Additionally, the absolute bioavailability of

eurycomanone was estimated from the ratio of the AUC0®¥ of the

oral data over that of the i.v. data. The ke was calculated from the

slope of the plasma concentration versus time curve (after loga-

Low B-S et al. Bioavailability and Pharmacokinetic¼ Planta Med 2005; 71: 803 ±807

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rithmic transformation) whereas t1/2 was calculated using the

relationship, t1/2 = ln 2/ke. As for Vd, it was calculated from the

relationship, V = dose/ke´AUC0®¥ . The value of AUC0®¥ was de-

termined by adding the area from the time zero to the last sam-

pling time (AUC0® t) with the area from the last sampling time to

infinity (AUCt®¥ ). The former was calculated using the trapezoi-

dal methods whereas the latter was estimated by dividing the

last measurable plasma drug concentration by ke. From AUC0®¥ ,

CL was calculated from the relationship, CL = dose/AUC0®¥ [27].

Stability study

The chemical stability of eurycomanone (1) was determined at

pH 1, 4 and 7. The media used were 0.1 M HCl for 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 8C, 1.0 mg of 1 was dissolved in each of the three

reactions at a concentration of 40 m g/mL. Samples of 1.0 mL

were collected from each reaction at intervals of 0, 0.5, 1.0, 2.0,

4.0, 6.0 and 24.0 h. The experiment was conducted in duplicate.

The concentration of 1 in the withdrawn samples was then ana-

lysed using the HPLC method as described above, except that the

mobile phase was modified to a 4:6 mixture of methanol andwater.

Partition coefficient study

The lipophilicity of eurycomanone (1) was determined using the

n-octanol/water partition coefficient (P ). The aqueous phase

used for this study was 0.1 M HCl (pH 1), 0.05 M NaH2PO4´2 H2O

(adjusted to pH 4 with glacial acetic acid) and 0.05 M Na 2HPO4´2

H2O (adjusted to pH 7 with glacial acetic acid). About 10.0 mL of

each phase and 0.3 mg of 1 were added into each of the three se-

parating funnels to yield a concentration of 30 m g/mL. Into each

of the separating funnels, about 10.0 mL of n-octanol were then

added. The mixtures in the three separating funnels were shakenvigorously and left to stand for 2 hours. An aliquot of 0.2 mL was

withdrawn from the aqueous phase (pH 1, 4 and 7), and an ali-

quot of 2.0 mL was collected from the n-octanol phase. The con-

centration of 1 in the withdrawn samples was determined using

the HPLC method as described previously. 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.

Results

The between-day, within-day accuracy and precision values of

the analytical method for eurycomanone (1) are presented in Ta-

ble 1. The coefficient of variation value (CV) for precision and thepercentage 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 standard substances

for determination of plasma eurycomanone (1) was found to be

linear, with an intercept of 43.2, a slope of 1250.3 and a correla-

tion coefficient of 0.999. A detection limit of 0.02 m g/mL was ob-

served at a signal to noise ratio of 4: 1, whereas the lower limit of

quantification (LLOQ) was 0.10 m g/mL.

The chromatograms obtained with the blank plasma, plasma

spiked with 10 m g/mL eurycomanone (1) and plasma containing

0.45 m g/mL eurycomanone (1) 4 h after an oral administration of 50 mg/kg of F2 containing 9.8 mg/kg of 1 are shown in Figs. 1a, b

and c. It can be seen that 1 (retention time of around 19 min)

lacked interference by endogenous compounds and was there-

fore suitable for preparation of the calibration curve using stand-

ard substances.

The curve profiles of the mean concentration of eurycomanone

(1) in the rat plasma versus time after intravenous (10 mg/kg F2

containing 1.96 mg/kg of 1) and oral (50 mg/kg F2 containing

9.8 mg/kg of 1) administrations are compared in Fig. 2. The plas-

ma concentration of 1 showed a rapid decline from 5.08 1.99 to

Fig. 1 HPLC chromatograms showing (a) blank rat plasmawith an endogenous peak (E), (b) blank rat plasma spikedwith 10 m g/mL of eurycomanone (1) and its retention time(min) and (c) rat plasma containing 0.45 m g/mL eurycoma-none (1) and its retention time (min) after 4 h oral adminis-tration 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; limitof detection = 0.02m g/mL and limit of quantification =0.10m g/mL.

Table 1 Within-day and between-day precision and accuracy (n = 6)of the assay method

Concentration [ m m g/mL] Within-day Between-day

Accuracy (% error)

Precision(CV, %)

Accuracy (% error)

Precision(CV, %)

0.1 ± 9.5 9.7 ±11.6 13.0

0.2 ± 1.2 11.6 ± 9.8 12.9

0.4 10.4 6.5 ± 6.1 13.50.8 ±10.0 10.9 ± 0.9 5.1

1.6 ± 0.5 5.0 ± 3.3 10.2

3.2 0.3 7.1 0.6 8.8

6.4 1.1 5.3 ± 0.4 10.9

12.8 ± 0.1 3.3 0.9 8.4


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0 m g/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 0.04 m g/mL after 16 h (Fig. 3). Relatively higherplasma concentrations of 1 were observed after i.v. injection

than after oral administration. A comparison of the AUC0®¥ val-

ues (Table2; the values are normalized regarding the dose differ-

ences) of 1 given either orally as F2 extract or intravenously re-

vealed that the absolute bioavailability of the quassinoid was

only 10.5%. Its mean elimination rate constant, ke, and biological

half-life (t1/2) values obtained from i.v. injection were 0.88 0.19

h-1 and 1.00 0.26 h, respectively. This indicates that 1 has a very

short biological half-life. Its mean volume of distribution, Vd, and

mean clearance, CL, estimated from i.v. data were 0.68 0.30 L/

kg and 0.39 0.08 L/h/kg, respectively. In contrast, its mean nu-

merical values of Cmax and Tmax following oral application were

0.33 0.03 m g/mL and 4.40 0.98 h, respectively. This suggests

that the absorption was relatively slow reaching a peak concen-

tration following 4 hours of dosing (Fig. 3 and Table 2).

The concentrations of 1 kept at 37 8C for either 1/2 hour or 24

hours at pH 1 were analyzed as 96.38% 2.93 S.E. and 96.09 %

4.08 S.E.; at pH 4, its concentrations were 94.90% 1.38 S.E. and

98.54% 0.74 S.E., and at pH 7, they were 98.01% 3.02 S.E and

97.69% 3.92 S.E. respectively. No significant changes in the

compound concentration were observed over a 24-hour incuba-

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

and 7 were determined as ±1.27, ±1.28 and ±1.26 respectively

(Table 3). Based on these results, 1 was more soluble in the aqu-

eous phase than the organic phase. In addition, the P value

showed that its solubility and partition coefficient value were

not influenced by the pH.

Discussion

A rapid direct HPLC assay for measuring eurycomanone (1)inthe

rat plasma was developed in which its analyte peak at 19±20minwas clearly separated from the plasma endogenous substan-

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 sub-

stances has an accuracy (% error) range within the accepted

range of 80±100% for bioanalytical method validation [27]. The

accuracy at its LLOQ was at the low end with 80.6% whereas the

accuracy at higher concentrations increased to 96.2 ± 100.6%.

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, thevolume of distribution (Vd) of 1 was found to be approximately

0.68 0.30 L/kg. In view of the relatively high Vd and its low P

value, the drug may be well distributed in the extravascular

fluids [28]. The low absolute bioavailability (10.5%) of 1 follow-

ing oral administration may be due to pre-systemic metabolism

or first-pass effect (in the gut wall and liver) prior to reaching the

general circulation. In having a high aqueous solubility, 1 may be

absorbed inefficiently by the gastrointestinal tract [29]. The

Fig. 3 Plasma con-centration-timecurve of eurycoma-none (1) after anoral administrationof F2 containing

9.8mg/kg of 1. Val-ues are presented asmean S.E. (n = 5).Limit of detec-tion = 0.02 m g/mLand limit of quantifi-cation = 0.10 m g/mL.

Fig. 2 Plasma con-centration-timecurve of eurycoma-none (1) after oraladministration of F2containing 9.8mg/kg of 1 and intrave-nous injection of F2containing 1.96 mg/kg of 1. Values arepresented as mean

S.E. (n = 5). Limitof detec-tion = 0.02 m g/mLand limit of quantifi-cation = 0.10 m g/mL.

Table 2 Numerical values of pharmacokinetic parameters: AUC0®¥ ,

ke, t1/2, Vd, CL, Cmax and Tmax after intravenous and oral admin-

istration of eurycomanone (1)

Intravenous administration Oral administration

AUC0®¥ [m g´h/mL] 5 .93 1.24 AUC0®¥ [m g´h/mL] 3.11 0.35

ke [h±1] 0.88 0.19 Cmax [m g/mL] 0.33 0.03

t1/2 [h] 1.00 0.26 Tmax [h] 4.40 0.98

Vd [L/kg] 0.68 0.30

CL [L/h/kg] 0.39 0.08

Values are presented as mean S.E. of rats (n = 5).

Table 3 The P values of n-octanol-water partition coefficient for eury-comanone (1) at pH 1, 4 and 7

Solvent layer Eurycomanonecalculated fromHPLC [ m m g/m]

Total content of eurycomanone [ m m g/ml]

Octanol/ buffer

P

Octanol (pH 1.0) 2.17 10.87 0.053 ±1.27

Buffer pH 1.0 4.07 203.54

Octanol (pH 4.0) 1.86 9.31 0.053 ±1.28

Buffer pH 4.0 3.54 176.74

Octanol (pH 7.0) 2.90 14.48 0.055 ±1.26

Buffer pH 7.0 5.27 263.25


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compound was stable at pH 1, 4 and 7, hence its poor oral bio-

availability was unlikely to be caused by its degradation in the

non-favourable pH conditions of the gastrointestinal tract. The

permeability properties of 1 as well as its metabolism in the en-

terocytes and liver are worth of being pursued for development

of better oral bioavailability strategies.

In conclusion, eurycomanone (1) exhibited low bioavailability, a

short biological half-life and appeared to be well distributed in

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, it may be due to its poor mem-

brane permeability arising from its low P value and/or high

first-pass metabolism.

Acknowledgement

The authors wish to thank the Ministry of Science, Technology

and Innovation, Malaysia, for a ªtopdownº grant from the Inten-

sive Research on Priority Areas (IRPA).

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