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, Chairul

3 & Wasmen manalu

4

1Department of Biology Education, University of Tanjungpura, Pontianak, Indonesia, 2Department of Pathology, Faculty of Veterinary Medicine IPB, Indonesia,

3Department of Botany, Biology LIPI, Bogor, Indonesia, 4Department of Physiology, Faculty of Veterinary Medicine IPB, Indonesia

E-mail: ruqiah.gpp@gmail.com

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 methanol-

water 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

.

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, ——————

*Corresponding author

INDIAN J TRADITIONAL KNOWLEDGE VOL 12, NO. 2 APRIL 2013

226

Indonesia. Animals were provided with standard

rodent pellet diet, the food and water were allowed

ad libitum.

Collection of plant material

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

the last administration, blood was collected allowed to

clot and serum separated. Liver was dissected out for

histopathological study.

Hepatoprotective activity 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.

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.

Transmission electron microscope

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

adminstration of methanol-water fraction on

therapeutic dose for three months.

Hepatoprotective activity of methanol-water fraction of

Eurycoma longifolia Jack. roots

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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Discussion

The previous study 25

showed 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 methanol-

water 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

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).

PANJAITAN et al.: HEPATOPROTECTIVE ACTIVITY OF EURYCOMA LONGIFOLIA JACK. ROOTS

229

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 methanol-

water 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.

Aknowledgement

This research supported by research grant from

DIKTI Indonesia, so we would like to express our

thanks to DIKTI.

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