Bin-Seng Low Bee-Hong Ng Wai-Peng Choy Kah-Hay Yuen Kit-Lam Chan

Bioavailability and Pharmacokinetic Studies of Eurycomanone from Eurycoma longifolia

Original Paper

Abstract A validated HPLC analysis of eurycomanone (1), a bioactive quassinoid, in rat plasma following oral and intravenous administration of Eurycoma longifolia Jack extract was developed for pharmacokinetic and bioavailability studies. Relatively high plasma eurycomanone concentrations were detected after an intravenous 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), volume of distribution (Vd) and clearance (CL) were 0.88  0.19 h1, 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 mg/mL and 4.40  0.98 h, respectively. The plasma concentration of the quassinoid after oral administration was much lower than after intravenous application 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 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 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 metabolism. Key words Eurycoma longifolia Jack Simaroubaceae eurycomanone pharmacokinetics bioavailability

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Introduction Eurycoma longifolia Jack, a tall tree belonging to the Simaroubaceae family, is commonly known as Tongkat Ali in Malaysia, Pasak Bumi in Indonesia and Cay ba binh 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 bleeding gums [2]. From its roots, several classes of chemical constituents 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-resistant Gombak A and chloroquine-sensitive D10 strains of Plasmodium falciparum parasites [3]. Besides its antiplasmodial activity, 1 also exhibited potent antipyretic [7] and cytotoxic activities against KB, P388 [12] and MCF-7 [17] tumour cells in vitro. In addition, 1 has been reported as the chemical constituent of E. longifolia with the highest yield [1]. Therefore, any information on the disposition and bioavailability of 1 will be highly important if its pre-clinical bioactivities are to be clinically evaluated. However, to date, no study has been published pertaining

Affiliation School of Pharmaceutical Sciences, University Sains Malaysia, Penang, Malaysia Correspondence Kit-Lam Chan School of Pharmaceutical Sciences University Sains Malaysia 11800 Penang Malaysia Phone: +60-4-657-6836 Fax: +60-4-657-6836 E-mail: klchan@usm.my Received December 9, 2004 Accepted April 6, 2005 Bibliography Planta Med 2005; 71: 803807  Georg Thieme Verlag KG Stuttgart New York DOI 10.1055/s-2005-871259 Published online July 29, 2005 ISSN 0032-0943

to its bioavailability and pharmacokinetics. Thus, the present study is the first report comparing its oral and intravenous bioavailability and pharmacokinetic data, as well as investigating its lipophilicity and stability under different pH conditions. This may affect its absorption in the gastrointestinal tract.

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. Standardized eurycomanone-enriched E. longifolia extract, (F2), containing 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. Blood samples of 0.5 mL were removed from the tail vein of the rats at 0 (pre-dose), 20 min, 40 min, 1, 2, 4, 6 and 8 h after intravenous injection and at 0 (pre-dose), 1, 2, 4, 6, 8, 10, 12 and 16 h after oral administration. They were then transferred into heparinized microcentrifuge 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 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 at 1000 g for 10 min. The clear supernatant layer was collected, and 20 mL were injected for analysis in the HPLC system. Method validation For the assay validation, a stock solution of pure eurycomanone (1) at a concentration of 1000 mg/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 mg/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 withinday accuracy and precision were determined for each concentration based on eight measurements for a single day, whereas the between-day values were obtained over six consecutive days of the validation period. A separate calibration curve using standard substances was calculated on each day of the analysis. The accuracy was expressed as the percentage error whereas the precision was denoted by the coefficient of variation. The recovery values were estimated from the HPLC chromatogram by comparing the peak heights of the plasma samples to the peaks of known concentrations of 1 and to those of the same concentrations prepared directly in aqueous solutions. Data analysis The following pharmacokinetic parameters were estimated from the data obtained for intravenous (i. v.) administration: elimination rate constant, (ke); biological half-life, (t1/2); volume of distribution, (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 concentration values could not be reliably used to estimate the pharmacokinetic 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-

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Original Paper

Material and Methods Materials Eurycomanone (1) was isolated and purified from the roots of Eurycoma longifolia Jack following the protocol described previously [3]. Compound 1 was obtained with a 57.8 % yield. It had 27 a melting point of 252 254 8C and an optical rotation of [a]D : + 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 comparison of its spectroscopic data to literature data reported previously [1], [3], [8]. A voucher specimen of the plant has been deposited 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, Japan), a Gilson 115 UV detector (Wisconsin, US) and a Hitachi D2500 Chromato-integrator (Tokyo, Japan). A Metaphase Crestpak C18 (4.6 mm i.d 250 mm) column was used for chromatographic 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 mg/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 experiments 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 intravenous pharmacokinetic studies. Animals were kept in cages

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

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/keAUC0. The value of AUC0 was determined by adding the area from the time zero to the last sampling time (AUC0t) with the area from the last sampling time to infinity (AUCt). The former was calculated using the trapezoidal 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 mg/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 analysed using the HPLC method as described above, except that the mobile phase was modified to a 4 : 6 mixture of methanol and water. 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 NaH2PO42 H2O (adjusted to pH 4 with glacial acetic acid) and 0.05 M Na2HPO42 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 separating funnels to yield a concentration of 30 mg/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 shaken vigorously 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 aliquot of 2.0 mL was collected from the n-octanol phase. The concentration of 1 in the withdrawn samples was determined using the HPLC method as described previously. Subsequently, the partition 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.

Table 1

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

Within-day Accuracy (% error) Precision (CV, %) 9.7 11.6 6.5 10.9 5.0 7.1 5.3 3.3 Between-day Accuracy (% error) 11.6 9.8 6.1 0.9 3.3 0.6 0.4 0.9 Precision (CV, %) 13.0 12.9 13.5 5.1 10.2 8.8 10.9 8.4

Concentration [mg/mL]

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8

9.5 1.2 10.4 10.0 0.5 0.3 1.1 0.1

Original Paper

Results The between-day, within-day accuracy and precision values of the analytical method for eurycomanone (1) are presented in Table 1. The coefficient of variation value (CV) for precision and the percentage error values for between-day and within-day measurements 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 correlation coefficient of 0.999. A detection limit of 0.02 mg/mL was observed at a signal to noise ratio of 4 : 1, whereas the lower limit of quantification (LLOQ) was 0.10 mg/mL. The chromatograms obtained with the blank plasma, plasma spiked with 10 mg/mL eurycomanone (1) and plasma containing 0.45 mg/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 therefore suitable for preparation of the calibration curve using standard 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 plasma concentration of 1 showed a rapid decline from 5.08  1.99 to

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Fig. 1 HPLC chromatograms showing (a) blank rat plasma with an endogenous peak (E), (b) blank rat plasma spiked with 10 mg/mL of eurycomanone (1) and its retention time (min) and (c) rat plasma 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 of detection = 0.02 mg/mL and limit of quantification = 0.10 mg/mL.

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

Fig. 2 Plasma concentration-time curve of eurycomanone (1) after oral administration of F2 containing 9.8 mg/ kg of 1 and intravenous injection of F2 containing 1.96 mg/ kg of 1. Values are presented as mean  S.E. (n = 5). Limit of detection = 0.02 mg/mL and limit of quantification = 0.10 mg/ mL.

(Table 3). Based on these results, 1 was more soluble in the aqueous 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) in the rat plasma was developed in which its analyte peak at 19 20 min was clearly separated from the plasma endogenous substances. Its peak was discrete and reproducible with a precision (% CV) value for the various standard concentrations (Table 1) 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 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, the volume 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 following 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

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Original Paper

Fig. 3 Plasma concentration-time curve of eurycomanone (1) after an oral administration of F2 containing 9.8 mg/kg of 1. Values are presented as mean  S.E. (n = 5). Limit of detection = 0.02 mg/mL and limit of quantification = 0.10 mg/ mL.

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  0.04 mg/mL after 16 h (Fig. 3). Relatively higher plasma concentrations of 1 were observed after i. v. injection than after oral administration. A comparison of the AUC0 values (Table 2; the values are normalized regarding the dose differences) of 1 given either orally as F2 extract or intravenously revealed 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 numerical values of Cmax and Tmax following oral application were 0.33 0.03 mg/mL and 4.40  0.98 h, respectively. This suggests that the absorption was relatively slow reaching a peak concentration 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 incubation 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 2

Numerical values of pharmacokinetic parameters: AUC0, ke, t1/2, Vd, CL, Cmax and Tmax after intravenous and oral administration of eurycomanone (1)
Oral administration AUC0 [mgh/mL] Cmax [mg/mL] Tmax [h] 3.11  0.35 0.33  0.03 4.40  0.98

Intravenous administration AUC0 [mgh/mL] ke [h1] t1/2 [h] Vd [L/kg] CL [L/h/kg] 5.93  1.24 0.88  0.19 1.00  0.26 0.68  0.30 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 eurycomanone (1) at pH 1, 4 and 7

Eurycomanone calculated from HPLC [mg/m] 2.17 4.07 1.86 3.54 2.90 5.27 Total content of eurycomanone [mg/ml] 10.87 203.54 9.31 176.74 14.48 263.25 0.055 1.26 0.053 1.28 Octanol/ buffer P

Solvent layer

Octanol (pH 1.0) Buffer pH 1.0 Octanol (pH 4.0) Buffer pH 4.0 Octanol (pH 7.0) Buffer pH 7.0

0.053

1.27

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

compound was stable at pH 1, 4 and 7, hence its poor oral bioavailability 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 enterocytes 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 attributable to low stability of 1 under the pH conditions of the gastrointestinal tract. Instead, it may be due to its poor membrane permeability arising from its low P value and/or high first-pass metabolism.

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Acknowledgement The authors wish to thank the Ministry of Science, Technology and Innovation, Malaysia, for a topdown grant from the Intensive Research on Priority Areas (IRPA).

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