Sensors & Transducers Magazine (S&T e-Digest), Vol.52, Issue 2, February 2005, pp.

300 - 309

Sensors & Transducers

ISSN 1726-5479
© 2005 by IFSA
http://www.sensorsportal.com

Potentiometric Fingerprint Profiling of Eurycoma longifolia

Extracts (Tongkat Ali) Using Multichannel Artificial
Lipid-Polymer Membrane Sensor
Ismail ZHARI1*, Ahmad MOHD NOOR2, Oon – Sim CHEW1
and Shafiqual Islam A.K.M.1
1
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Minden Penang, Malaysia
2
Northen Malaysia University College of Engineering, 02600 Arau, Perlis, Malaysia
Tel: 604-6533888 ext 4222, fax: 604-6563443, e-mail: zhari@usm.my

Received: 22 December 2004 / Accepted: 14 February 2005 / Published: 17 February 2005

Abstract: This paper reports a novel application of biomimetic sensor system for phytomedicine
analysis. The in-house fabricated multichannel artificial lipid-polymer membrane sensor consists of
eight non-specific lipid-polymer membranes with partially overlapping selectivity and cross-sensitivity
towards complex liquid extracts from Eurycoma longifolia. The generated multidimensional electrical
potential response pattern due the interaction of the lipid sensing element with the chemical constituent
present in the liquid samples were analyzed qualitatively by potentiometric fingerprint profile and
further processing using chemometric pattern recognition algorithms namely hierarchical cluster
analysis (HCA) and principal component analysis (PCA). The score plot of the PCA of the marker
compounds and various sample treatment (mode of extraction and drying method) indicated the sensor
responded linearly towards increasing concentration. Further quantification of the E. longifolia isolates
and extracts using simple linear regression gives good calibration curve (R2 >0.90) except
eurycomanone (R2 = 0.55). This study extends the areas of sensor application, thus demonstrating the
capability of the developed sensor system as a promising tool for quality control and standardizing
phytomedicinal products.

Keywords: Lipid-polymer membrane, Eurycoma longifolia, Potentiometric fingerprint, Principal

component analysis, Hierarchical cluster analysis.
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1. Introduction
1.1 A Novel Approach of Sensor Technology for Phytomedicine Analysis

Currently, healthcare professionals are facing a paradigm shift in promoting health by introducing
evidence-based traditional herbal medicine. Novel research approaches and methods used to evaluate
the safety, efficacy and quality of herbal medicines are being sought in recent years. Quality control
and standardization of phytomedicine poses many analytical challenges as herbal medicine is a
complex system of mixtures.

Over the century, conventional separation-detection approaches were among the popular techniques
used to solve the analytical problems of multicomponent samples such as food and beverage, wine,
flavor and fragrance, environmental samples and etc. Amongst other, chromatographic techniques
namely thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid
chromatography (HPLC) are commonly used as profiling method for medicinal plant research [1].
However, these methods are time consuming, expensive and depends largely on the polarity of the
mobile phase (TLC and HPLC) and volatilities of the compounds (GC) to be separated.

With the advent of biomimetic sensor technology developed on the basis of mechanism found in
human biological systems which incorporate array of non-specific biomaterial as sensing element [2],
rapid and reliable multicomponent analysis can be accomplished without prior separation. For the first
time, our research group successfully developed a taste-sensing system mainly for quality control and
standardization of herbal medicinal plant [3]. This newly defined area of sensory analysis and
application has gained increasing popularity and acceptance not only among the research scientists but
also the herbal product manufacturers and regulatory organization [4-6].

1.2 Operating Principle of Multichannel Artificial Lipid-Polymer Membrane Sensor

This novel research methodology for phytomedicine analysis are based on the bioelectronic tongue that
mimicks the human gustatory system through the incorporation of biological lipid material as sensing
element [7-8]. Figure 1 illustrates the main component of the sensor system with a simplified diagram
explaining the action of sensor mechanism.

The in-house fabricated sensor consists of eight non-specific sensors coated with different lipid-
polymer membranes with partially overlapping selectivity and cross sensitivity towards the complex
mixture of liquid herbal extract. Firstly, variations in the electrochemical properties of the chemical
substances in solution undergo different mechanism of response involving both electrostatic and
hydrophobic interactions with the biomembrance. Secondly, the characteristic electrical response
generated is acquired using a multi-interface meter. Thirdly, the generated potential electrical response
was visualized using a star-plot graph in order to observe simultaneously the characteristic
potentiometric fingerprint of a particular herbal sample. Lastly, a multidimensional output of the
sensor array was further analyzed using pattern recognition tools namely hierarchical cluster analysis
(HCA) and principal component analysis (PCA). By using these two chemometric data analysis
algorithms, relevant information regarding the physicochemical properties of the analyzed liquid was
extracted from the raw sensor readings thus facilitating the analyst to correlate the observable pattern
with the phytochemistry of the herbs.

In this report, a systematic approach for quality control and standardization of Eurycoma longifolia,
locally known as Tongkat Ali the “Malaysia Ginseng” that possesses immense therapeutic properties.
The in-house fabricated sensor is used as a fingerprinting device for (1) potentiometric characterization
of the E. longifolia marker compounds and extracts, (2) identify the variation of physicochemical
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properties of the E. longifolia extracts by chemometric data analysis and (3) quantification of the E.
longifolia isolated marker compounds and extracts using simple linear regression analysis.

DA
30 60

40
D:T=9:1 OA
20 20
Electrical potential

0
10 -20

0 2 3 OAm -40 DOP

-10
-20 TOMA D:T=5:5

-30
Processing Unit D:T=3:7
DA
OA

OAm
5;5
3;7

9;1
DOP

TOMA

Characteristic
Potentiometric Fingerprint
Electrical potential different
(mV) of eight electrode channels
4
Multi-Interface
Meter
1
10

Chemical substance change the Working Ag/AgCl Reference Class C Class E

membranes electrical potential electrode electrode
Class D
0

Class A Unknown

-10

Class B

-20
Complex mixture of liquid herbal sample -30 -20 -10 0 10 20

Biological Sensing Layer *

For simplicity, only single working Pattern Recognition Model
(Physicochemical Transducer) electrode is shown. (Chemometrics Data Analysis)

Fig. 1. Schematic diagram of the artificial lipid - polymer membrane based sensor system.

2. Experimental
2.1. Artificial Lipid-Polymer Membrane Preparation

Eight kinds of lipid analogs (Table 1) were used for membrane preparation. The lipid materials and the
sensor array arrangement are similar to those reported [9], with an addition of channel No. 8
(DOP:TOMA=9:1). Each lipid was mixed in a small beaker with 200 mg polyvinyl chloride (PVC)
and 0.25 ml dioctyl phenylphosphonate (DOPP) (plasticizer), and dissolved in 10 ml tetrahydrofuran
(THF) [9]. The solution was then poured into the glass casting ring resting on the glass plate. A pad of
filter papers weighted with heavy weight was placed on top of the glass ring and left for a sufficient
time (24 hours) to allow the solvent to evaporate. The lipid polymer membranes thus prepared a
transparent, colorless as a soft film of 200 µm thickness.

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Table 1. Lipid materials used for the membrane preparation

Channel No.
Lipid Material Formula Structure Quantity (µl)
(Charge)
1 (- ve) Decyl alcohol (DA) C10H21OH 100.0
2 ( - ve) Oleic acid (OA) C17H33COOH 100.0
3 ( - ve) Dioctyl phosphate (DOP) (C8H17)2POOH 100.0
4 (neutral) DOP:TOMA=5:5 (D:T=5:5) - 26.7 + 6.7
5 (+ ve) DOP:TOMA=3:7 (D:T=3:7) - 16.0 + 47.2

Trioctyl methyl ammonium

6 (+ ve) (C8H17)3CH3N+Cl- 67.2
chloride (TOMA)

Oleyl amine (OAm)

7 (+ ve) C17H33CH2NH2 12.5 + 12.5
(Decyl alcohol + Oleyl amine)
8 ( - ve) DOP:TOMA=9:1 (D:T=9:1) - 48.2 + 6.7

2.2. Multichannel Sensor Electrode Fabrication

Stable Ag/AgCl coating was prepared by electrolytic method. The silver wires 0.5 mm diameter and
2.5 cm length was thoroughly cleaned with detergent and then washed with multiple rinses of distilled
water. The silver wires were then placed in concentrated HNO3 for one hour, following which were
thoroughly washed with distilled water. Silver chloride was then electrically deposited on the silver
wire. A solution of 0.1 N NaCl titrated to pH 11 to 12 with 6 M NaOH is used. A 5 to 10 mA current
was then passed through the electrolytic cell for 30 minutes with the silver wire acting as positive
electrode and a platinum wire serving as the negative electrode [10].

The lipid membrane was cut into round pieces using borer and hammer. The membranes were fitted on
the bottom of the PVC tube such that the inner part of the cylinder was isolated from the outside, as
shown in the Fig. 2a. A glass tube is inserted through the other end of the PVC tube. The PVC tube
was then filled with 3 M KCl solution. The other end of the glass tube was sealed with a stopper that
held the Ag/AgCl electrode. The ends of the Ag/AgCl wires were inserted inside the KCl solution.

To prevent interference due to different electric charges, eight kinds of lipid membranes were
separated into two groups according to their charges as shown in Fig. 2b. Electrical potential
measurements were taken from two separate beakers.

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Coaxial wire Reference electrodes

Ag/AgCl

3 M KCl
Lipid
membrane Negative charge electrodes
(a) (b)
Positive charge electrodes
Fig. 2. (a) Sensor working electrode and (b) Multichannel electrode arrangement.

2.3. Electrical Potentiometric Measurement

One reference electrode (Orion) was used for each group of electrodes. The electrical potentials
between the measuring electrodes and the reference electrodes were measured using 8-Channel High
Impedance Multi-Interface Meter (Fylde Scientific U.K.). Prior to use, the electrodes were conditioned
in 1mM KCl for one hour for better sensitivity and reproducibility [7] followed by washing with
deionized distilled water. Then, the electrodes were left in the sample for one minute before taking
reading for another minute.

The electrical potential of E. longifolia marker compounds namely 6α-hydroxyeurycomalactone 0.030,

0.016, 0.009, 0.005 and 0.003 mg/ml; 9-methoxycanthin-6-one, eurycomalactone and eurycomanone
of 0.010, 0.006, 0.003, 0.002 and 0.001 mg/ml and finally the spray dried and freeze dried water
extract 0.100%, 0.056%, 0.031%, 0.018% and 0.010%, methanol extract 0.200%, 0.112%, 0.062%,
0.036% and 0.020% and capsule 0.180%, 0.310%, 0.560% and 1.000% were measured from lower to
higher concentration. Each sample was measured three times. The electrodes were washed at least
twice, each time one minute, for each consecutive measurement with distilled water to avoid charge
carry over effect [11].

The electrical potential response of the sample was measured as the difference between the potential of
the sample and 1mM KCl. The subtracted electrical potential data were then treated with suitable
chemometric pattern recognition algorithms namely hierarchical cluster analysis (HCA) and principal
component analysis (PCA) for both qualitative and quantitative analysis of both marker compounds
and herbal extract samples using MS Excel and statistical software package SPSS 11.5 for Windows.

3. Results and Discussion

3.1 Potentiometric Fingerprint of E. longifolia Marker Compounds

The electrical potential pattern of the E. longifolia marker compounds namely 9-methoxy-canthin-6-
one, eurycomalactone, eurycomanone and 6-hydroxyeurycomalactone at different concentration are
shown in Figure 3. By visual pattern recognition, the potentiometric fingerprint of the marker
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compounds seems to have similar response pattern compared with each other. This may be due to the
similar chemical property (all are quassinoids) except for 9-methoxy-canthin-6-one which is an
alkaloid. Generally, the marker compounds have complex chemical structure and are neutral molecule.
Marker compounds bind to the hydrophobic part of the membranes causing a change in the membrane
charge density. Thus with the presence of these molecules, the electrical potential of the membranes
will slightly change. From the magnitude of the potential electrical response (mV), we can concluded
that the positively charge lipid membrane immobilize with TOMA as a transducer gives the highest
response toward the isolated marker compounds.

6-hydroxy-eurycomalactone 9-methoxycanthin-6-one Eurycomalactone Eurycomanone

DA
DA DA DA
20 20
30 20
D:T=9:1 10 OA 10
D:T=9:1 20 OA D:T=9:1 OA D:T=9:1 10 OA
10 0 0 0
0 -10 -10 -10
OAm -10 DOP OAm -20 DOP OAm -20 DOP OAm -20 DOP

TOMA D:T=5:5 TOMA D:T=5:5 TOMA D:T=5:5 TOMA D:T=5:5

D:T=3:7 D:T=3:7 D:T=3:7 D:T=3:7

0.003 mg/ml 0.005 mg/ml 0.001 mg/ml 0.002 mg/ml 0.001 mg/ml 0.002 mg/ml 0.001 mg/ml 0.002 mg/ml
0.009 mg/ml 0.016 mg/ml 0.003 mg/ml 0.006 mg/ml 0.003 mg/ml 0.006 mg/ml 0.003 mg/ml 0.006 mg/ml
0.030 mg/ml 0.010 mg/ml 0.010 mg/ml 0.010 mg/ml

Fig. 3. The potentiometric fingerprint of E. longifolia marker compounds.

3.2 Potentiometric Fingerprint of the E. longifolia herbal extracts

Comparatively, the potentiometric fingerprint of the spray dry and freeze dry water extract are
identical as illustrated in Figure 4. This indicates the fact that the complex chemical constituents from
the water extract are similar irrespective to the drying process. Freeze drying and spray drying are
commonly use in the preparation of phytopharmaceutical into a solid or powder form with sufficient
stability for distribution and storage [12].

On the contrary, significant differences of the potentiometric fingerprint of different solvent extract
namely water and methanol extracts were recorded. This indicated the polarity of the solvent play an
important role in determining the extraction of bioactive fractions of the herbal extract. Water extract
of E. longifolia is reportedly used for its aphrodisiac properties [13] whereas the methanol extract is
indicatedly used for antimalarial and antiplasmodial activity [14].

A closer look at the magnitude of the electrical response (mV) with regards to the analyte
concentration, it is observed that negative charge electrodes DA and OA increases with increasing
concentration whereas positive charge electrodes TOMA and D:T=3:7 decreases with increasing
concentration (see Fig. 4). On the other hand, the herbal extracts has only a little effect on negative
charge electrodes (DOP and D:T=9;1), neutral electrode (D:T=5:5) and positive charge electrode
(OAm). Although some of the electrode channel do not give significant response towards increasing
chemical concentration, it is still not excluded for future data analysis because this redundant sensor
response may be very useful for discriminating similar samples. Besides, the sensors are non-specific
and displaying cross-sensitivity to multiple components in the liquid herbal samples. The overlapping
signal response output may not be a problem with the help of chemometric data analysis.
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Spray dry water extract Freeze dry water extract Methanol extract Tongkat Ali Capsule
DA DA DA
60 DA 20
50
60
10
D:T=9:1
40
OA D:T=9:1 OA D:T=9:1 OA
0
0
40
20 D:T=9:1 20
OA
0
-50 -10
0 -10
-20 0
-20
-20

OAm -40 DOP OAm -40 DOP OAm -150 DOP OAm -30 DOP

TOMA D:T=5:5 TOMA D:T=5:5 TOMA D:T=5:5 TOMA D:T=5:5

D:T=3:7 D:T=3:7 D:T=3:7 D:T=3:7

0.010% 0.018% 0.031% 0.010% 0.018% 0.031% 0.020% 0.036% 0.180% 0.310%
0.056% 0.100% 0.056% 0.100% 0.062% 0.112% 0.560% 1.000%
0.200%

Fig. 4. The potentiometric fingerprint of E. longifolia extracts.

3.3 Potentiometric Fingerprint Mapping by Chemometric Approaches

3.3.1 Hierarchical Cluster Analysis (HCA)

HCA was carried out using the subtracted electrical potential data obtained from the array of the sensor
response in order to identify the similarity and dissimilarity of the physicochemical properties of the E.
longifolia samples treated with different mode of extraction and drying method. By using this
unsupervised pattern recognition algorithms, a group of objects (samples) will be assigned to its
respective classes (cluster) so that similar object are in the same classes [15]. This study employs
Average Linkage (between groups) and square Euclidean distance as a geometric distance measure
between objects. The horizontal scale (0-25) indicates similarity and dissimilarity among the samples.

The dendogram in Figure 5 clearly shows three distinct clusters (A-C) formed at similarity distance of
five. The formation of cluster B validates the results obtained from the potentiometric fingerprint
profile of the E. longifolia water extract treated with different drying method (freeze drying and spray
drying) as discussed earlier. Misclassification of high concentration methanol extract (0.112% and
0.200%) indicated that the particular complex chemical constituents might be identical with the
Tongkat Ali capsule to group under cluster A.

Fig. 5. Dendrogram using Average Linkage method on various samples treatment.

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3.3.2 Principal Component Analysis (PCA)

Figure 6 shows the two dimension score plot of the mulitidimensional sensor array reading by using
PCA, the unsupervised pattern recognition algorithm [15]. The arrow shows the increase of the analyte
concentration. Generally, the concentration increases in a specific direction thus giving a good
indication that the sensors responded linearly towards increasing concentration for both the isolated
marker compounds and extracts.

The study samples having similar electrochemical properties tends to group together in the two
dimensional space. Eurycomalactone, eurycomanone, 9-methoxycanthin-6-one that has less electrical
charge is nearly located with the methanol extract whereas 6α-hydroxyeurycomalactone, which has
more electric charge, is located near the water extracts. Interestingly, water extract freeze dried and
spray dried has almost the same phytochemical composition and physicochemical properties are
projected close to each other and the concentrations also increase in the same direction.

1
PC2

-1

-2

-3
-2 -1 0 1 2 3 4

PC1

Fig. 6. PCA score plot of spray dry water extract Ð, freeze dry water extract U, methanol extract Õ, isolates of
E. longifolia 6α-hydroxyeurycomalactone Ë, 9-methoxycanthin-6-one , eurycomalactone Ã, eurycomanone Ô
and capsule Ñ at different concentrations.

3.4 Quantification of Marker Compounds and Herbal Extracts

Extended application of PCA from the above data to quantify the E. longifolia marker compounds and
extracts are shown in Figure 7 (only three graphs are shown). The contribution rates (variances) of the
data of each sample to PC1 were 95.66%, 81.06%, 97.19%, 78.58%, 65.01%, 73.33%, 74.12% and
73.08% for water extract, water extract freeze dry, methanol extracts, 6a-hydroxyeurycomalactone, 9-
methoxycanthin-6-one, eurycomalactone, eurycomanone and Tongkat Ali capsule respectively.
The R2 (correlation coefficient) values of the regression line were 0.9974, 0.9741 0.9984, 0.9376,
0.9317, 0.9073, 0.5502 and 0.9969 for water extract spray dry, water extract freeze dry, methanol
extracts, 6a-hydroxyeurycomalactone, 9-methoxycanthin-6-one, eurycomalactone, eurycomanone and
Tongkat Ali capsule respectively. Generally, a good calibration curve with R2 > 0.9 except the case of
eurycomanone (R2 = 0.55) indicate that PC1 increase in proportion to the analyte concentration in
logarithmic scale (see Fig. 7). Therefore, the developed sensor could be used for quantification of
unknown herbal samples.
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1.5 1.5
1.5

R2 = 0.9974 R2 = 0.9317 R2 = 0.9969

1.0 1.0
1.0

.5 .5 .5

PC1
PC1

PC1
0.0 0.0 0.0

-.5 -.5 -.5

-1.0 -1.0 -1.0

-1.5 -1.5 -1.5

-2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -.8 -1.2 -1.0 -.8 -.6 -.4 -.2 0.0 .2 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2

Log Concentration Lon Conc Log Concentration

Log Concentration

(a) (b) (c)

Fig. 7. Plot of PC1 vs. Log concentration for (a) spray dried water extract, (b) 9-methoxy-canthin-6-one
and (c) Tongkat Ali capsule.

4. Conclusions
The in-house fabricated multichannel artificial lipid-polymer membrane sensor device could be used
to perform qualitative fingerprint profiling on a wide variety of herbal extract and formulation.
Moreover, the developed sensing system also possesses the capability to quantitate the whole herbal
extract and more importantly determine the level of marker compounds found in a particular herbal
formulation as reported above. With the incorporation of this novel biomimetic sensor technology,
quality control and standardization of herbal medicine is hopefully no longer a time consuming,
complicated and expensive task to be accomplished.

Acknowledgements

Financial support from the Ministry of Science, Technology and Environmental, Malaysia through
IRPA No. 304/Pkim/640041/K105 for this research is acknowledged. The authors also thank Forest
Research Institute Malaysia (FRIM), Malaysia and Prof Chan Kit Lam from School of Pharmaceutical
Sciences, USM for supplying the plant materials and isolated marker compounds.

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