Int. Res J Pharm. App Sci., 2013; 3(3):13-17
ISSN: 2277-4149
International Research Journal of Pharmaceutical and Applied
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Int. Res J Pharm. App Sci., 2013; 3(3):13-17
Research Article
THE ACUTE TOXICITY OF METHANOLIC EXTRACT OF EURYCOMA LONGIFOLIA JACK
ROOTS AND HISTOPATHOLOGIC CHANGES OF RAT VITAL ORGANS
1
Rumaisa Dhifa Mawaddah, 2Firdalena Meutia, 3Sofia, 4Hanifah Yusuf
Faculty of Medicine University of Syiah Kuala Darussalam Banda Aceh 23111
2,4
Departement of Pharmacology Faculty of Medicine University of Syiah Kuala Banda Aceh 23111
3
Departement of Biochemistry Faculty of Medicine University of Syiah Kuala Banda Aceh 23111
. (Received: 20 May 2013; Accepted: 23 June, 2013; Published: 30 June, 2013)
Corresponding Author’s email: hans_yusuf1104@yahoo.com
1
Abstract: The acute toxicity of methanolic extract of Eurycoma longifolia Jack roots have been studied on rats. The study was
based on increasing used of E. longifolia Jack roots for maintaining health and treatment by the people. This experimental study
used “completely randomized design” and 30 rats were divided in 5 groups (n = 6 each group) for treated and control group. The
rats were given tested extract with a single dose 0; 25; 75; 225; 625 mg/kg BW/day orally. The observed parameters were the fifty
percent of lethal dose, its influences on toxicological profile, the development of body weight and mortality. The microscopic
observation of vital organ (kidney, stomach, and liver) have been done on fifteenth day. The results of acute toxicity test after
giving the single dose extract to males and females rats showed that the fifty percent of lethal dose (LD50 ) is 7498.94 mg/kg BW,
orally. The increasing of body weight was shown on dose 75 mg/ml and 225 mg/kg BW. Hematoxylin-eosin stain was used to
evaluate the histopathologic changes of rat vital organs (kidney, liver , heart, stomatch) and showed the apparent change on dose
675 mg//kg BW like necrosis. The symptom of toxicity were shown on dose 225 and 675 mg//kg BW such as depression,
shallow breath, convulsion, coma and then died.
Keywords: Acute toxicity, LD50, Eurycoma longifolia Jack..
INTRODUCTION
The increasing of drug prices and the limited
purchasing power of people, making traditional medicine as
an alternative for the purpose of maintaining their own
health and treatment1. Although the use of traditional
medicine is widespread and entrenched, but its use has not
been supported by scientific study as well as modern drugs.
The truth efficacy and safety of a drug, including traditional
medicines have been proven through a scientific study and
the test is performed to prove the safety of a drug, known as
toxicity tests. Toxicity test not only for modern drugs but
also important for traditional medicine, especially for
traditional medicine most commonly consumed by the
people.
One of the most popular traditional medicine is
Eurycoma longifolia Jack, known as “tongkat ali“ or
“pasak bumi” plant in Indonesia. Eurycoma longifolia
Jack is the most widely used traditionally to treat a variety
of diseases such as malaria, inflammation, aphrodisiac,
cancer and as a tonic drug2. In Malaysia, the roots of this
plant is best known as an aphrodisiac to address male sexual
dysfunction3,4.
The roots of this plant contain compounds such as
quassinoids, xanthinon alkaloids, β carboline alkaloids,
triterpene trycullane, squalene and biphenolneolignane
derivatives5. The main compounds are a class of quasinoid
with skleton structure composed of C18, C19, and C20.
Extraction is performed on the root of E.longifolia Jack
Rumaisa DM et al., 2013
standardized contains quassinoid with three main types
namely, eurycomanone, 13 β, 21 α-dihydroeurycomanone
and 13 21-epoxyeurycomanone6 have reported that
quassinoid include eurycomanone as antimalarial,
euryomanol and 13 - (β -epoxy) eurycomanone efficacious
as antiulcer.
The scientific studies of the roots, contain
quassinoid derivatives that active against Plasmodium
falciparum in vitro6. The other studies have suggested that
the roots of E. longifolia Jack which grown in Thailand
contain quasinoid that have antimalarial activity and
cytotoxic effects7. Its antiplasmodial activity was tested in
vitro and eurycomanone is the most potent active compound
and have cytotoxic effects against nasopharyngeal epidermal
cells carcinoma8.
Beside that, the extract can raise blood levels of
testosterone9. Based on pharmacological studies obtained
information that compounds in this plants can inhibit cancer
cell growth, whereas quasinoid compound also serves as a
potential anti-leukemia and anti-HIV10.
The chemical compounds contained within the
E. longifolia, Jack has attracted the attention of many
researchers due to a fairly broad biological activity 9 .
Traditional medicinal products containing the roots of
this plant attracted many people in the world, not only
because it can cure malaria, cancer, aphrodisiac, but
this natural ingredient is also the most widely used as
13
Int. Res J Pharm. App Sci., 2013; 3(3):13-17
an ingredient in the drug-tonic10 . Its use as a tonic is
very popular not only consumed by men but also used
by women 11 .
In addition to in vivo studies in mice infected
Plasmodium berghei, were obtained ED50 values between
11.2 mg / kgBW12. But in the study of acute toxicity tests
of ethanolic extract orally in mice, LD 50 values has
obtained 2.6 g / kg BW, and the symptoms of toxicity
were found such as depression, shallow breathing and
convulsions, in that study 95% of mice died at a dose of
0.43 g / kg13 .
Acute toxicity test of methanolic extract of E.
longifolia Jack roots through oral administration in rats
has not been done. The widespread use of traditional
medicines containing E.longifolia Jack roots in the
community, it is felt necessary by the researchers to
assess the acute toxicity on rats that can be known the
safety limits. It is hoped that the results of this study
can be useful as an initial information to the public to
prevent side effects or toxic effects resulting from the
use of traditional medicines containing the roots of E.
longifolia uncontrolled 14 .
Based on the things mentioned above, this study
was conducted to determine the median lethal dose (LD50)
of methanolic extract of E. longifolia Jack roots on rats, the
symptoms of toxicity, the profile change of vital organs
microscopically after oral administration 15.
MATERIALS AND METHODS
Materials:
The roots of E. longofolia Jack were collected from
4 years old plants that grown in Study Forest Park, Faculty
of Forestry University of Lambung Mangkurat, Banjar Baru,
South Kalimantan. The root have been deposited and
identified by a specialist (Prof Dr.Wahyono,SU) from
Departement of Biology Pharmacy, Faculty of Pharmacy
University of Gadjah Mada. A voucher speciment No
BF/123/Ident/Det/III/2010. In this study the methanolic
extract of E. longifolia Jack roots is used as test drug that
made by standard method.
Preparation of White Rats (Rattus norvegicus)
Experimental animals (rats) were acclimatized for 1
week before the study is done in a way kept in cages
measuring 50 x 30 x 50 cm with each cage containing 6
individuals (male and female, five groups, n=6). Cages
placed in a room kept clean, with a 12-hour cycle of light
and 12 hours of dark light. Prior to testing, animal were
weighed and then fasted for 12 hours but the water is still
The result of maceration process of E. longifolia
Jack powdered roots yielded 50 gram of dark brown extract.
The number of animals death were 3 of tested group with
dose 225 mg/kg BW and 6 with dose 675 mg/kg BW and
the observation performed for 24 hours after oral
administration of the extract. Animal mortality data were
used to calculate lethal 50 dose (LD50) using the method of
Rumaisa DM et al., 2013
ISSN: 2277-4149
given, then taken to a laboratory for adaptation to the
environment. Food animal given back 6 hours after
administration of the test drug16. The study protocol was
appoved by Animal Ethics Comittee, Faculty of Medicine
University of Syiah Kuala
Acute Toxicity Test Procedure and Parameters
The serial test dose given only once, on day 1 after
acclimatization. Serial test dose of the extract administered
orally (25 mg/ml, 75 mg/ml, 225 mg/ml, 675 mg/ml) using a
metal sonde to experimental animals (1 ml/200g BW). As a
negative control is used distilled water.
The observation of toxicity symptoms in the form
of behavioral changes conducted 0.5 - 1, 1.5 – 2, 3-6 hours
after administration of the test dose. Development of the
body weight of rats was measured three (3) times during 1
week, that is on day 1; 3; 5 and day 7 after administration of
the test drug and the number of deaths was calculated for 24
hours on day 1.
Subsequently the animals were still alive and
observations continued 2 times a day for 14 days. On day 15
the autopsy was performed by microscopic examination of
the kidney, liver, heart and stomach. The development of
body weight and the profile changes of vital organ were
measured and compared with the control group.
Furthermore the number of the rats death in the treatment
group used to calculate lethal dose 50 (LD50) by the method
of Weil CS (1952).
Histological Assessment
Rats were sacrificed by cervical dislocation and
subsequently kidney, liver, heart and stomach collected from
each rat and fixed with 10 % buffered formalin. Paraffin
blocks were prepared and sections of 5μm were cut on a
microtome and stained with hematoxylin and eosin. The
tissue sections were examined and compared with negative
controls.
Data analysis
All data were collected and processed statistically,
the lethal dose 50 (LD50) was calculated by the method Weil
CS (1952). The body weight development between
treatment groups were examined using Analysis of Variance
(ANOVA) at the level of significance 0.05 and the results
were expressed as mean ± SD. The symptoms of toxicity or
changes in animal behavior qualitatively assessed by
Ngatidjan guide (2006).
RESULT AND DISCUSSION
Weil CS manually and obtained LD50 value 7498.94 mg/kg
BW.
The LD50 is defined as "a single dose of a
compound that is statistically expected to kill 50% of test
animals". Other researchers obtained LD50 value of the
methanolic extract in mice of 6.180 mg / kg13, whereas the
LD50 value of the ethanolic extract in mice at 2600 mg / kg
orally.
14
Int. Res J Pharm. App Sci., 2013; 3(3):13-17
ISSN: 2277-4149
The difference in the LD50 value of the extract can
be affected by various factors, such as the concentrations of
compounds present in the extract, species, age, weight, sex
of animal used, nutritional, environmental temperature,
humidity, and air circulation. Besides that the LD50 value is
also influenced by health factors, animal stomach contents,
route of administration and dosage form drug testing and
how the implementation of the acute toxicity test.
weight in animals due to treatment is also influenced by
various factors such as: nutrition, environmental
temperature, the potential toxicity of the compound
contained in the extract.
Symptoms of toxicity animals were observed
before and after administration of single dose and the
behavioral observation covers locomotoric activity, central
nervous and autonomic nervous system, defication and
urination (Thompson, 1985). Oral single dose administration
of the extract didn’t affect the behavior of male and female
rats compared to controls group during the intensive
observation in 0,5 -1 hour. However, after 1.5 - 2 hour
administering a single dose of the extract showed an
increase in locomotor activity behavior and aggressiveness.
Sensitivity to pain slightly increased in group dose 225 and
675 when compared with the control group. Reflexes and
awareness increased with increase in dose while the
respiratory rate showed more increased in those groups.
The observations on the development of animal
body weight (rats) were performed for 7 days after
administration of a single dose of the extract. Weighing
were performed before treatment on day 1; 3; 5 and 7 after
oral administration of the extract shown in Table 1. The
result showed that on day 1 there was no significant
difference between all treatment groups. The same analysis
conducted on the development of body weight on day 3, the
results showed that there was no significant difference
between the control groups vs. group 25 mg /kg BW, as well
as between group 75 mg / kg BW vs group 225 mg /kg BW.
However, a significant difference (*) was seen between the
control groups vs 75 and 225 mg/kg BW. On day 5 there is a
significant difference (*) between all treatment groups,
except between group 75 and 225 mg/kg BW. Whereas on
day 7 there are significant differences (*) between all dose
groups, except group 75 and 225 mg/kg BW.
On behavioral observations after 3-6 hours of the
extract in group 225 to group 675 showed significant
differences in behavior compared to the control group. The
acute toxicity test results after administration of the extract
with single dose 675 mg /kg BW orally seen a decrease in
locomotor activity, aggressiveness, reflexes, awareness and
tremor when compared to the control group. While
symptoms of toxicity begin to occur in toxic doses 225 - 675
mg / kg BW in the form of depression, shallow breathing,
convulsions, coma and death.
The weight development in 675 mg/kg BW group
can be observed only on day 1, because all experimental
animals in this group died within 24 hours after being
treated with the extract. The decrease or increase in body
Table 1. The Development of Rats Body Weight on Acute Toxicity of The Extract Eurycoma longifolia Jack Roots
Rats Body Weight
Day 3**
Day 5**
Level doses (mg/kg
BW)
Day 1*
0
158,95 ± 5,21
134,52 ± 5,49
169,38 ± 7,75
155,47 ± 6,03
25
159,28 ± 7,16
133,15 ± 8,03
151,33 ± 9,88
162,50 ± 9,84
75
225
177,78 ± 12,49
181,22 ± 7,18
153,73 ± 11,01
152,40 ± 2,16
191,32 ± 14,07
187,20 ± 9,01
191,23 ± 14,68
199,20 ± 14,50
675
185,87 ± 10,30
-
-
-
Day 7**
* before given the extract of E.longifolia Jack roots
** after given the extract of E.longifolia Jack roots
Rumaisa DM et al., 2013
15
Int. Res J Pharm. App Sci., 2013; 3(3):13-17
ISSN: 2277-4149
A
B
C
D
E
F
G
H
Histologic specimens of rats tissues were collected
after 16 days of treatment. Tissue samples were stained with
hematoxylin and eosin. The histological pictures were taken
at following magnifications at 400x (A: Kidney; C: Liver; E:
Heart; G: Stomatch of control grroup) and (B: Kidney; D:
Liver; F: Heart; H: Stomatch of treated grroup in dose 675
mg/kg BW).
2.
3.
4.
Conclusions
The lethal 50 dose (LD50) of methanolic extract of
Eurycoma longifolia Jack roots using the method of Weil
CS manually were obtained LD50 value 7498.94 mg/kg BW,
orally. The administration of a single dose of the extract
with doses up to 225 mg / kg BW affect on the development
of animal body weight (rats) during the 7 days of
observation. The extract in oral administration on rats affect
behavior primarily locomotor activity, aggressiveness,
reflexes, awareness, sensitivity to pain and respiratory
system. While symptoms of toxicity begin to occur in toxic
doses 225 - 675 mg / kg BW in the form of depression,
shallow breathing, convulsions, coma and died.
5.
6.
7.
Acknowlegment
The authors are grateful to Prof. Dr. Mustofa, M.Kes
from Departement of Pharmacology and Toxicology for his
assistance. Authors would like to thank Ria from
Departement of Biology Faculty of Mathematic and Natural
Science University of Syiah Kuala Banda Aceh Indonesia
for her assistance.
8.
9.
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1. CHROMATOGRAPHIC SEPARATION FOR CILAZAPRIL AND RELATED IMPURITIES DETERMINATION
M Sarat And C Rambabu
Abstract
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2. RP­HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF OMEPRAZOLE AND CINITAPRIDE IN TABLETS
N.V.M.S.Bhagavanji
Abstract
3.
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Pages (7­12)
THE ACUTE TOXICITY OF METHANOLIC EXTRACT OF EURYCOMA LONGIFOLIA JACK ROOTS AND HISTOPATHOLOGIC CHANGES OF RAT VITAL
ORGANS
Rumaisa Dhifa Mawaddah, Firdalena Meutia, Sofia, Hanifah Yusuf
Abstract
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4. A REVIEW: ANIMAL MODELS USED IN THE SCREENING OF ANTIEPILEPTIC DRUGS
Kasthuri.S*, Karthigadevi.K, Manjulakshmi.P, S. Kavimani
Abstract
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5. VARIABLES EFFECTING DRUG ENTRAPMENT EFFICIENCY OF MICROSPHERES: A REVIEW
Kaur Dupinder*, Saini Seema
Abstract
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6. NEW DRUG DEVELOPMENT: A REVIEW
Dr. Anil Kumar M.H., Mr. Varun Gopinath. Abstract
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No of Download=1678
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7. DETERMINATION OF SELENIUM CONCENTRATION IN DIFFERENT SPECIES OF RICE CONSUMED IN BANDUNG INDONESIA
Holis A. Holik*, Herlianda Bianti, Mutakin, Rizky Abdulah
Abstract
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8. WITHANIA COAGULANS AND PSIDIUM GUAJAVA­ AN OVERVIEW
Roshni Barad*, Purvi Soni, Miss Siddhi Upadhyay, Dr. Umesh Upadhyay Abstract
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