A Qualitative and Quantitative Evaluation of Terpenoid and Alkaloid

in Root and Stem of Pasak Bumi (Eurycoma longifolia Jack)

Anisa Rahmalia, Rizkita R. Esyanti, and Iriawati

Plant Science and Biotechnology Scientific Group
School of Life Sciences and Technology,
Bandung Institute of Technology
e-mail: [email protected]; [email protected]

Received 21 December 2010, accepted for publication 28 January 2011

Abstract
Herbal, as health solution, regained its popularity because many studies showed advantages of using it. One of the
most industrially needed herbal materials is the extract of pasak bumi (Eurycoma longifolia Jack), which is popular
for its alleged sexual-enhancing properties. E. longifolia was known to produce secondary metabolites, especially
terpenoid and alkaloid. The presence of major alkaloids and terpenoids in root and stem of E. longifolia was
evaluated quantitatively by analyzing the ethanol extract of root and stem of E. longifolia using GC-MS. Qualitative
analysis was conducted through colorimetric test on both extracts by using Dragendorff and Lieberman-Burchad
reagents. Colorimetric test showed that there was a difference in both terpenoid and alkaloid amounts on ethanol
extract of both root and stem. GC-MS analysis showed that the ethanol extract of E. longifolia from the root
contained 14.631% terpenoid and that from the stem was only 7.781%. The ethanol extract of E. longifolia from the
stem contained 1.785 % of alkaloid, while that from the root was 5.117 % from total compounds. Major alkaloids
found on both organs were 3-Methyl-1-oxo-2,3-dihydro-1H-pyrazolo[4,3-c][1,10] phenanthroline, which showed
higher concentration in root. Major terpenoid in root included 2H-1-Benzopyran-2-one, 3-phenyl- (coumarin
derivate), whilst that in stem was Stigmasterol. These substances have different activities in target cells. Therefore,
the knowledge of active compounds in organ parts of pasak bumi will lead to a more efficient production process.
Keywords : Alkaloid, Eurycoma longifolia, GC-MS, Histochemistry, Terpenoid

Abstrak
Herbal merupakan solusi kesehatan alternatif yang kembali marak digunakan karena manfaatnya yang beragam.
Salah satu bahan herbal yang banyak diperlukan di industri adalah ekstrak pasak bumi (Eurycoma longifolia Jack)
yang terkenal karena kemampuannya meningkatkan aktivitas seksual. E. longifolia dikenal mengandung terpenoid
dan alkaloid. Kehadiran dan kadar alkaloid dan terpenoid utama pada akar dan batang E. longifolia dievaluasi
secara kualitatif dengan analisis GC-MS ekstrak etanol dari akar dan batang, sedangkan secara kuantitatif
dilakukan melalui analisis colorimetrik menggunakan reagen Dragendorff dan Lieberman -Burchard. Hasil uji
colorimetrik menunjukkan adanya perbedaan jumlah alkaloid dan terpenoid baik di ekstrak etanol akar maupun
batang. Hasil analisis GC-MS menunjukan bahwa ekstrak etanol akar mengandung 14,631% terpenoid, sedangkan
batangnya mengandung 7,781% terpenoid. Ekstrak etanol batang E. Longifolia mengandung 1,785% alkaloid,
sementara akarnya mengandung 5,117% alkaloid dari total senyawa. Alkaloid utama yang ditemukan pada kedua
organ adalah senyawa 3-Methyl-1-oxo-2,3-dihydro-1H-pyrazolo[4,3-c][1,10] phenanthroline, dengan konsentrasi
lebih tinggi pada akar. Terpenoid utama yang ditemukan di akar adalah 2H-1-Benzopyran-2-one, 3-phenyl-
(turunan coumarin), sementara pada akar adalah Stigmasterol. Senyawa-senyawa tersebut memiliki aktivitas
berbeda pada setiap sel. Oleh karena itu, pengetahuan mengenai senyawa aktif yang terkandung dalam organ
pasak bumi dapat meningkatkan efisiensi proses industri sesuai dengan tujuan produksinya.
Kata kunci : Alkaloid, Eurycoma longifolia, GC-MS, Histokimia, Terpenoid

1. Introduction as pasak bumi. Pasak bumi is popular for its alleged

sexual-enhancing properties as observed by Ang and
Health is considered as one of the main issues
Cheang (1999). Therefore, in recent years, the
today. Therefore, some people consumed herbal drug
demand for pasak bumi in Indonesia is increasing.
for health maintenance. Herbal drug acts like any
Pasak bumi has several active compounds in a
other drug, either curing or acting as supplement that
group of terpenoid and alkaloid. These compounds
support body system to operate. Such fact leads to
are believed to have potential, such as on sexual
vast development of herbal industry and demanding
activity as suggested by Baker et al. (1999),
the industry to increase quality and quantity of their
anticancer (Wang et al., 2002) and antimalaria (Kuo
material. One of the most industrially needed herbal
et al., 2004). Several researchers, including Bedir et
materials is Eurycoma longifolia, commonly known

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al. (2003), reported that terpenoid could be obtained 3. Results and Discussion
from pasak bumi ethanol extract. Pasak bumi-derived
Qualitative analysis was conducted by using
aphrodisiac compound is commonly obtained from
colorimetric test. The results showed that the color of
its root, but it may also be possible to obtain the
the stem extract was slightly lighter than that of the
compound from other organ parts of pasak bumi as
root extract. Verification of alkaloid presence in the
suggested by Ang and Sim (1997). However, most
ethanol extract of root and stem of pasak bumi
researches focused on pasak bumi root extract, while
(Figure 1) showed that the ethanol extract of both
the stem organ was rarely used. Therefore, the study
root and stem treated with the Dragendorf turned into
of chemical compound in stem is necessary.
orange with similar color intensity. Lieberman-
Identification of terpenoid and alkaloid on
Burchard (terpenoid) test showed deep-brown color
root and stem of pasak bumi can be performed both
on root extract test (top-row of Figure 1A), while the
qualitatively or quantitatively. The presence of
stem extract showed transparent brownish color
alkaloids in pasak bumi can be tested histochemically
(bottom row Figure 1B). Result of colorimetric test
by direct application of Dragendorff and Jefferey
could suggest that the level of terpenoid on root
reagents onto pasak bumi fresh-cut of root and stem.
ethanol extract was higher than that on stem ethanol
However, further analysis is needed to confirm detail
extract, but alkaloid level showed no difference on
content of the extract.
both extracts. In order to reconfirm the colorimetric
Effectivity and efficiency of raw material
test result, root and stem ethanol extracts were
usage are important in herbal industry, that is by only
analyzed by gas-chromatography mass-
utilizing parts of pasak bumi that specifically has
more yet specific alkaloid and terpenoid in it. spectrofotometric (GC-MS).
Therefore, this research was conducted to study the
presence of major terpenoid and alkaloid on pasak
bumi stem and root, both qualitatively and
quantitatively (by GC-MS analysis of ethanol extract
of pasak bumi).
2. Methods
2.1 Extraction
Plant materials used in this experiment were
the root and stem of pasak bumi (Eurycoma
longifolia Jack) collected from West Kalimantan in
Juli 2009. Five hundreds gram of simplisia were
placed on different glass-jars, and submerged in 5 L
of 96% ethanol for 24 hours. The materials were then
filtrated and evaporated by distillation process until Figure 1. Colorimetric tests show the ethanol extract
only 500 mL remained. The filtrates were then of root (A) and stem (B) without treatment, with
analyzed with GC-MS and colorimetric method. Dragendorff treatment and with Lieberman-Burchard
treatment, respectively.
2.2 Qualitative Analysis
GC-MS analysis of pasak bumi root ethanol
Colorimetric analysis of terpenoid was extract showed 52 different compounds. Each peak
conducted by using Lieberman Burchard method has a different retention time and abundance. The
according to Goddel (2010). Positive result of retention time showed the character of the compound.
terpenoid was shown by color shift from extract color Compound with higher polarity could be detected
to brown color. Alkaloid colorimetric-test was earlier than less polar compound, because it has
performed using Dragendorff method as described by different interaction with the immobile phase of Gas-
Goddel (2010). Positive test-result was shown by Chromatography-column used. Moreover, compound
color change, from extract-color to deep orange with lower boiling point was detected earlier on GC-
color. MS than the compound with higher boiling point
2.3 Quantitative Analysis (Schwender, 2009).
The retention time of root extract ranged from
GC-MS analysis of ethanol extracts was then 10 to 30 minutes. The first peak was detected on
conducted by using HP-5MS fused phenyl methyl minute 11,723 read as 2-Furancarboxaldehyde, 5-
silox capillary column. GC-MS analysis resulting in (hydroxymethyl)- a member of terpenoid group,
chromatogram was compared to complete library. while the last peak was on minute 26.601 and read as
Percent of total compound was calculated according 2-Methyl-Z,Z-3,13-octadecadienol, which was also a
to the equation below: member of terpenoid group.
Result of root ethanol extract showed seven
Percent of total = Core area compound ×100%
major compounds of terpenoids and five major
Total core area of all desired compound
 Rahmalia et al., Qualitative and Quantitative Evaluation of Terpenoid and Alkaloid in Root and Stem ……….. 51

compounds of alkaloids (Table 1). The highest of compounds in the stem ethanol extract of pasak
terpenoid content in root ethanol extracts was 2H-1- bumi ranged from 10 to 30 minutes. First peak
Benzopyran-2-one, 3-phenyl-, it comprised about emerged at minute 11.134 read as 2-Cyclohexen-1-
21.44 % among all compounds. The highest alkaloid one,2-methyl-5-(1methylethenyl)- ,(S)-, from
content in root ethanol extract was 3-Methyl-1-oxo- terpenoid group. Last peak was at minute 29.927 read
2,3-dihydro-1H-pyrazolo[4,3c] [1,10] phenan- as 4,22 Stigmastadiene-3-one, which was also a
throline, it made up to 27.83 % of major terpenoid member of terpenoid group.
and alkaloid in the root ethanol extract. The stem ethanol extract of pasak bumi
The highest terpenoid compound in the root consisted of ten major terpenoids and three alkaloids
ethanol extract was 2H-1-Benzopyran-2-one, 3- (Table 2). The highest constituent of terpenoid in
phenyl-, a derivate of coumarin. Coumarin was stem ethanol extracts was stigmasterol, while that of
known as agent to enhance sperm activity, and gave alkaloid was 3-Methyl-1-oxo-2,3-dihydro-1H-
hormonal effects on mammals. It was observed by pyrazolo[4,3-c][1,10]phenanthroline.
Al-Qarawi (2005), that coumarin in Ruta chalepensis Percentage of total compound was shown in
extract could increase sperm density and motility in Table 3.2, it represented volume domination of each
mice epididimys. Another function of coumarin was compound among those 13 compounds only, but not
to inhibit leukemia cell proliferation and coumarin the percentage of total volume domination of the
could regulate the factor on G1-phase of cell cycle on whole total extract. Terpenoid comprised about 7.781
leukemia cell (Wang et al., 2002). % of total chemical constituent detected in
GC-MS analysis of stem ethanol extract of chromatogram GC-MS analysis, while the alkaloid
pasak bumi resulted in 60 peaks. The retention-time group comprised 1.785 % of the total.
Table 1. Alkaloids and terpenoids found in the ethanol extract of the root of pasak bumi
No Compound Total %
1 2-Furancarboxaldehyde, 5-(hydroxymethyl)- 3.04%
2 Vanillin 2.57%
3 1H-2-Benzopyran-1-one, 3,4-dihydro-8-hydroxy-3- 5.79%
Terpenoid methyl-
4 Benzaldehyde, 3-ethoxy- 4.63%
5 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol 5.12%
6 2H-1-Benzopyran-2-one, 3-phenyl- (coumarin 21.44%
derivate)
7 2-Methyl-Z,Z-3,13-octadecadienol 4.64%
1 6-Tert.butyl-2,3-dicyanonaphthalen 3.59%
2 6,7-Dimethoxy-1,4-dihydro-2,3-quinoxalinedione 10.90%
Alkaloid 3 1-Amino-9-fluorenone 2.50%
4 3-Methyl-1-oxo-2,3-dihydro-1H-pyrazolo[4,3- 27.83%
c][1,10]phenanthroline
5 Oxazole, 2,2'-(1,4-phenylene)bis[4-methyl-5-phenyl- 2.31%

Table 2. Alkaloids and terpenoids found in the ethanol extract of the stem of pasak bumi
No Compound Total %
1 2-Cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)-, (S)- 0.94%
2 2,7-Octadien-4-ol, 2-methyl-6-methylene-, (S)- 1.15%
3 Dibutyl phthalate 6.73%
Terpenoid 4 trans-3,4-Dimethoxy-2-ethoxy-.beta.-methylstyrene 1.64%
5 1,2-Benzenedicarboxylic acid, 2-butoxyethyl butyl ester 2.63%
6 Campesterol 4.85%
7 Stigmasterol 50.54%
8 D-Homoandrosta-4,17-dien-3-one,,17a-dihydroxy- 1.76%
9 Stigmasterol, 22,23-dihydro- 7.25%
10 4,22Stigmastadiene-3-one 8.58%
1 9H-Pyrido[3,4-b]indole-1-carboxylic acid, methyl ester 2.37%
Alkaloid 2 6H-Indolo[3,2,1-de][1,5]naphthyridn-6-one 1.79%
3 3-Methyl-1-oxo-2,3-dihydro-1H-pyrazolo[4,3- 9.77%
c][1,10]phenanthroline
 Fifty percent of stem ethanol extract was Ang H.H, Cheang H.S., 1999, Studies on the
stigmasterol. Stigmasterol is a member of anxiolitic activity of Eurycoma Longifolia
phytosterol. Stigmasterol is a derivate of phytosterol, Jack Roots in Mice, Japan.Journal of
and Bouick et al. (1999) suggested that certain Pharmacology, 79, 497.
concentration of some plant sterol could influence the Ang, H.H., Sim, M. K., 1997, Effect of Eurycoma
cellular proliferation of T-lymphosites, a cell longifolia Jack on sexual behavior of male
responsible in immune system for killing pathogens. rats, Archive of Phamcal Research, 20:6,
Stigmasterol in E. longifolia extract had also been 656-658.
tested and proven to be potential against lung cancer Baker, V.A., Hepburn, P.A., Kennedy, S.J., Jones,
(Kuo et al., 2004). P.A., Jea, L.J., Sumpter, J.P., Ashby, J.,
Another application of stigmasterol was 1999, Safety evaluation of phytosterol esters.
studied by Barbosa et al. (1999). It showed that stem Part 1. Assessment of oestrogenicity using a
extract of Mucuna aterrima contained stigmasterol combination of in vivo and in vitro assays,
and sitosterol, which were widely known as Food and Chemical Toxicology. 37:1, 13-2.
nemacidal, and they were used to increase the
mortality level of Meloidogyne incognita, a round Barbosa, C.A., Barcelos, F.F., Demuner, A.J., Santos,
worm nematode. M.A., 1999, Chemical constituent from
Terpenoid was a major compound found in Mucuna aterrima with activitities against
both ethanol extracts of pasak bumi. The root extract Meloidogyne incognita and Heterodera
contained 14.631% terpenoid and the stem extract gycines, NEMATROPICA, 29, 1.
contained only 7.781% terpenoid. This result Bedir,E., Abu-Gazar,H., Ngewedson, J.N., Khan, I.
confirmed previous colorimetric observation by A. Eurycomaoside, 2003, A new
Lieberman-Burchard method that showed greater Quassinoid-type glycoside from roots of
color change in the root extract compared to that in Eurycoma longifolia. Chemical and
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