Indian Journal of Traditional Knowledge
Vol. 12(2), April 2013, pp. 225-230
Hepatoprotective activity of Eurycoma longifolia Jack. roots
Ruqiah ganda putri panjaitan1*, Ekowati handharyani2, Chairul3 & Wasmen manalu4
1
Department of Biology Education, University of Tanjungpura, Pontianak, Indonesia,
2
Department of Pathology, Faculty of Veterinary Medicine IPB, Indonesia,
3
Department of Botany, Biology LIPI, Bogor, Indonesia,
4
Department of Physiology, Faculty of Veterinary Medicine IPB, Indonesia
E-mail: [email protected]
Received 28.09.12, revised 06.01.13
The hepatoprotective activity of Eurycoma longifolia Jack. roots methanol-water fraction on therapeutic dose was
evaluated in carbon tetrachloride (CCl4)-induced rats. Hepatic cells were still normal after three months administration of
methanol-water fraction at therapeutic dose. The administration of methanol-water fraction on therapeutic dose prior to
CCl4-induced or CCl4-induced prior to methanol-water fraction resulted in suppression of ALT and AST. Histopathological
and ultrastructure studies confirmed that methanol-water fraction protected hepatic cells. It is concluded that the methanolwater fraction of pasak bumi roots has a hepatoprotective activity.
Keywords: Eurycoma longifolia Jack., Therapeutic dose, Hepatoprotective
IPC Int. Cl.8: A61K 36/00, A01D 7/54
Hepatic damage is associated with distortion of
metabolic function. Liver disease is still a world
problem, because liver is a regulator of various
important metabolic functions. Liver diseases are
mainly caused by toxic chemicals, excess of
consumption of alcohol, infections, drugs, and
autoimmune disorders1-3.
Carbon tetrachloride (CCl4) is an established
hepatotoxin. It is believed to require activation by
hepatic microsomal mixed function oxidase to
trichloromethyl free radical (*CCl3), and the presence of
oxygen trichloromethylperoxyl radical (*CCl3O2) which
is more reactive. Trichloromethyl free radical binds to
lipids and protein components of the membrane leading
to covalent binding, while trichloromethylperoxyl
radical interacts with polyunsaturated fatty acids and
causes lipid peroxidation4.
Natural products recently attracted attention as
health beneficial foods (physiologically functional
foods) and as source materials for drug development.
Herbal medicines derived from plant extracts are
increasingly being utilized to treat a wide variety of
clinical diseases5. Many traditional remedies employ
herbal drugs for treatment of liver diseases6-7.
——————
*Corresponding author
Eurycoma longifolia Jack. (pasak bumi in
Indonesian language), a medicinal plant belonging
to Simaroubaceae family, is cultivated widely in
some Southeast Asia regions such as Indonesia,
Malaysia, Thailand, and Vietnam. The roots of
this plant contains a series of quassinoids8-15,
canthin-6-one alkaloids, β-carboline alkaloids14,15,
tirucallane-type triterpenes15, squalene derivatives15,16,
and biphenylneolignans15. Some of these constituents
were shown to possess cytotoxic15, antimalarial14,15,17,
, and aphrodiasiac activities19-21, but no information
in literature was found concerning its possible
hepatoprotective. Therefore, to investigate the
in vivo hepatoprotective activity, we assessed the
hepatoprotective activity of E. longifolia Jack.
methanol-water fraction. Prior to conducting the
study, we established the safety level of the fraction
by determining median lethal dose (LD50) and
subchronic toxicity of the fraction to determine the
safety of the fraction.
Methodology
Animals
Sprague-Dawley rats (225 ± 25 gm) and DDY
mouse (30 ± 5 gm) were purchased from the Faculty
of Husbandry, Bogor Agricultural University,
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INDIAN J TRADITIONAL KNOWLEDGE VOL 12, NO. 2 APRIL 2013
Indonesia. Animals were provided with standard
rodent pellet diet, the food and water were allowed
ad libitum.
the last administration, blood was collected allowed to
clot and serum separated. Liver was dissected out for
histopathological study.
Collection of plant material
Hepatoprotective activity of methanol-water fraction of
Eurycoma longifolia Jack. roots
The fresh roots of E. longifolia Jack. were
collected from Betung Karihun National Park and
Gunung Palung Ketapang National Park, West
Kalimantan. The plant specimen was authenticated by
Herbarium Bogoriensis LIPI Bogor, Indonesia
(348/IPH.1.02/If.8/2004).
Preparation of plant extract and partitions
The process first, the air-dried roots (12.5 kg) of
E. longifolia Jack. were made into a coarse powder.
The second, the powdered material was dissolved in
methanol and subjected to maceration process. The
extract was filtrated with evaporated under reduce
pressure and vacuum-dried (2.75%). Finally, 95% of
methanol extract was partitioned with n-hexane,
chloroform, and ethyl acetate. The fractions obtained
were as follows n-hexane fraction (4.34%),
chloroform fraction (28.79%), ethyl acetate fraction
(7.22%), and residue (methanol water fraction)
(53.74%).
Determination of oral median lethal dose (LD50),
hepatoprotective median effective dose (ED50), and sub
chronic toxicity liver of methanol-water fraction of Eurycoma
longifolia Jack. roots
Median lethal dose was determined according to
Weil method 22. This experiment used DDY mouse
(30 ± 5 gm) that were purchased from Faculty of
Husbandry, Bogor Agricultural University, Indonesia.
Animals were divided into four groups based on dose
level to determine LD50 value. The toxic symptoms
and mortality rate were recorded after 24 hrs of per
oral administration of the methanol-water fraction.
Moreover, to determine ED50 hepatoprotective the
animals were divided into four groups based on dose
level. The hepatoprotective activity was recorded at
the ninth day, after seven consecutive days of per oral
administration of the methanol-water fraction and
CCl4 at dose 0.1 ml/kg body weight intraperitoneal
on the eighth day. The hepatoprotective activity of
E. longifolia Jack. compared to silymarin.
Sub chronic toxicity was conducted for three
months. Group I (positive control) animals were
administered aquadest (2 ml/kg body weight, per oral)
and group II (test group) animals were received
methanol-water fraction of E. longifolia Jack roots at
therapeutic dose. Animals were sacrificed 24 hrs after
Rats were divided into three groups that each group
has three rats. Group I (negative control) animals
were administered aquadest (2 ml/kg body weight,
per oral) and group II (positive control) animals were
administered silymarin at dose 25 mg/kg body weight,
per oral. Animals group III were administrated
methanol-water fraction at therapeutic dose per oral.
Aquadest, silymarin, and methanol-water fraction
were administered orally daily for seven consecutive
days, and at the eighth day, the experimental rats were
injected CCl4 at dose 0.1 ml/kg body weight. Animals
were sacrificed 24 hrs after CCl4 injection, blood was
collected allowed to clot and serum separated. Liver
was dissected out for histopathological study.
Currative effect of methanol-water fraction of Eurycoma
longifolia Jack. roots
Rats were divided into three groups that each group
has three rats. Group I (negative control) animals
were administered aquadest (2 ml/kg body weight, per
oral) and group II (positive control) animals were
administered silymarin at dose 25 mg/kg body weight,
per oral. Animals group III were administrated
methanol-water fraction at therapeutic dose per oral.
At the first day, the experimental animals injected
CCl4 at dose 0.1 ml/kg body weight. Then, at the
second until eighth day, aquadest, silymarin, and
methanol-water fraction were administered orally
daily for seven consecutive days. Animals were
sacrificed 24 hrs after the last administration, blood
was collected allowed to clot and serum seperated.
Liver was dissected out for histopathological and
ultrastructure study.
Biochemical estimations
The blood samples from rat heart were collected,
and then the rats were sacrificed by cervical
dislocation. Blood serum was obtained by
centrifugation. The activities of serum alanine
transaminase (ALT) and aspartate transaminase (AST)
were determined using assay kit ST. Reagensia.
Histopathological examination
The rat liver was dissected out and fixed in 10%
formalin, then dehydrated in gradual ethanol, cleared
in xylol and embedded in paraffin23. The paraffin
PANJAITAN et al.: HEPATOPROTECTIVE ACTIVITY OF EURYCOMA LONGIFOLIA JACK. ROOTS
227
sections were prepared and stained with haematoxylin
and eosin, and examined using light microscope.
adminstration of methanol-water
therapeutic dose for three months.
Transmission electron microscope
Hepatoprotective activity of methanol-water fraction of
Eurycoma longifolia Jack. roots
The rat liver was dissected out and fixed in 5%
glutaraldehyde, then dehydrated in gradual ethanol,
and embedded in propylene oxide. The ultrathin
sections were prepared and stained with uranyl acetate
and triple lead, and examined using electron
microscope24.
Statistical analysis
The data were expressed as mean (n=3). Results
were analyzed statistically by one-way ANOVA
followed by Tukey’s multiple comparison using SPSS
11.5 version for Windows. The difference was
considered significant at p<0.05.
Results
Determination of oral median lethal dose (LD50),
hepatoprotective median effective dose (ED50), and subchronic
toxicity liver of methanol-water fraction of Eurycoma
longifolia Jack. roots
The result showed that the oral LD50 value has
more than 15 gm/kg body weight and caterogized
practically non toxic. ED50 value has not more than
the range of lethal dose, and base on the ED50 value
we determine the therapeutic dose. Therapeutic dose
was used for sub chronic toxicity. Compared to
aquadest (ALT enzymes on 0 and 3rd months are
134.70±11.97 U/L and 137.42±29.63 U/L and AST
enzymes on 0 and 3rd months are 437.66±98.94 U/L
and 320.10±72.46 U/L), there were no significant
differences in ALT (0 and 3rd months are
123.39±22.25 U/L and 100.90±24.49 U/L) and
AST (0 and 3rd months are 384.05±122.38 U/L
and 299.52±15.66 U/L) enzymes concentrations.
Histopathological studies confirmed that the hepatic
morphology in male rat was still normal after
fraction
on
Compared with aquadest (ALT 161.70±7.37 U/L
and AST 330.67±42.00), the therapeutic dose
administration of methanol-water fraction prior to
CCl4-induced resulted in suppression of ALT
(91.78±9.63 U/L) and AST (249.50±20.5 U/L)
enzymes as well as silymarin (ALT 105.09±21.62 U/L
and AST 310.25±2.45 U/L) after CCl4-induced.
Histopathological studies confirmed that the
methanol-water fraction gave similar results to
silymarin (Fig.1).
Currative effect of methanol-water fraction of Eurycoma
longifolia Jack. roots
The results demonstrated that compared with
aquadest (ALT 231.57±67.66 U/L and AST
303.87±105.04 U/L), the CCl4-induced prior to
therapeutic dose administration of methanol-water
fraction resulted in suppression of ALT
(136.97±46.19 U/L) and AST (322.80±112.89 U/L)
enzymes as well as silymarin (ALT 143.57±37.00 U/L
and AST 321.33±25 U/L). Histopathological studies
confirm that the methanol-water fraction restored
hepatic cells as well as silymarin (Fig. 2). After
0.1 ml/kg body weight CCl4, compared with
methanol-water fraction on therapeutic dose (Fig. 3 A),
the administration of 2 ml/kg body weight aquadest
(Fig. 3 B) caused serious ultrastructural changes
in the hepatocytes of rats. The prominent changes
were organelle abnormality and focal cytoplasmic
degeneration. Mitochondria abnormalities were
observed, incluiding variations in size and shape, and
reduction of cristae. Then, the rough endoplasmic
reticulum was disarranged and apparently degenerated.
Fig. 1—A-C: Liver sections of aquadest 2 ml/kg body weight, silymarin 25 mg/kg body weight, and methanol-water fraction
at therapeutic dose prior to CCl4 0.1 ml/kg body weight. Fig. 1 A-C showed liver degeneration on difference degree. Bar = 20 µm.
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INDIAN J TRADITIONAL KNOWLEDGE VOL 12, NO. 2 APRIL 2013
Fig. 2—A-C: Liver sections of 0.1 ml/kg body weight CCl4-induced prior to administered 2 ml/kg body weight, silymarin 25 mg/kg body
weight, and methanol-water fraction at therapeutic dose. Fig. 2 A showed necrosis hepatic cells, and Fig. 2 C almost near silymarin group
liver architecture. Bar = 20 µm.
Fig. 3—A-B: Ultrastructure changes in the hepatocytes of
0.1 ml/kg body weight CCl4-induced prior to administered
aquadest 2 ml/kg body weight and methanol-water fraction at
therapeutic dose (A and B). Carbon tetrachloride induced caused
organelle abnormality and focal cytoplasmic degeneration (A).
Normal ultrastructure of hepatocytes in administration of
methanol-water fraction at therapeutic dose (B).
Discussion
The previous study 25showed that the oral
administration of 500 mg/kg body weight methanol
extract and its derived fractions (n-hexane,
chloroform, ethyl acetate, and methanol-water) of
E. longifolia Jack. for seven consecutive days had no
significant effects on liver function, so it means that
their compound relative non toxic to consume.
Furthermore, the investigation to select one fraction
of E. longifolia Jack. (methanol extract and n-hexane,
chloroform, ethyl acetate, and methanol-water
fraction of methanol extract), the administration of
500 mg/kg body weight methanol-water fraction of
E. longifolia Jack. possess hepatoprotective
activity that evidenced by the significant inhibition
in the elevated levels of ALT and AST serum
enzyme activities induced by CCl4. Moreover, its
histopathological study gave similar results to
silymarin26. According to 27-29 it has been showed that
protective agents exert their action against CCl4
induced liver injury by impairment of CCl4-mediated
lipid peroxidation and decreased production of free
radical derivatives.
Based on the value of LD50 and ED50 of methanolwater fraction, 30 Lu (1995) has categorized an extract
with the value of LD50 more than 15 gm/kg body
weight in practically non toxic. Furthermore, the sub
chronic toxicity showed that the administration of
methanol-water fraction at therapeutic dose for three
month have no alteration in histopathological studies.
The investigation of hepatoprotective and currative
effect of methanol-water fraction showed that it has
potensial effect as hepatoprotector.
Alanine aminotransferase (ALT) is a specific
cytosol liver enzyme, and its increase in the blood
serum is specific for changes in the liver function.
Aspartate aminotransferase (AST) is widely
distributed enzyme, which is found in many tissues
and organs, with high activity in the liver. Increased
AST enzyme in serum is a sensitive marker of liver
damage31. Estimating the activities of serum marker
enzymes like ALT and AST can make assessment of
liver function. When liver cell plasma membrane is
damaged, various of enzymes normally located in the
cytosol are released into the blood stream. Their
estimations in the serum are a useful quantitative
marker of the extent and type of hepatocellular
damage. Compound which is decreased ALT and
AST serum enzyme of course increased the activity
of GST, which metabolizes toxic compounds to non
PANJAITAN et al.: HEPATOPROTECTIVE ACTIVITY OF EURYCOMA LONGIFOLIA JACK. ROOTS
toxic ones, means they have an increasing protective
activity of the liver32. Histopathological dan
ultrastructure studies confirm that the administration
of methanol-water fraction at therapeutic dose
preserved the structural integrity of the hepatocellular
membrane and restored hepatic cells in a therapeutic
dose (Figs. 1,2,3).
Actually, the content of methanol-water fraction
that has hepatoprotective activity is not clear, but 15
Kuo et al. (2004) reported that they have isolated
totally 19 quassinoids of four types such as
eurycomalactone, laurycolactone, klaineanone, and
longilactone that were distributed in non polar until
polar fraction of E. longifolia Jack. roots. A range of
studies has demonstrated that triterpenoid can reduce
the amounts of CCl4 metabolite and thus protect the
liver 5,7,33. Therefore, the hepatoprotective activity of
methanol-water fraction may be supported by
bioactive compound. We assume that the methanolwater fraction inhibited lipid peroxidation and
recovered the decrease hepatic GSH level induced by
CCl4 towards normalization. In addition, the
mechanism liver protection of methanol-water fraction
is related by glutathione-mediated detoxification as
well as free radical suppressing activity.
Conclusion
We concluded that methanol-water fraction of
E. longifolia Jack roots has potential activity as
hepatoprotector at therapeutic dose. As long as it is
used based on the therapeutic dose no negative
effect on liver function obtained.
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Aknowledgement
This research supported by research grant from
DIKTI Indonesia, so we would like to express our
thanks to DIKTI.
15
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