1. 234
International Journal of Research in
Pharmacology & Pharmacotherapeutics
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656
IJRPP | Volume 2 | Issue 1 | 2013
Research article
In vivo Antimalarial Activity of Areal Part Extracts of Gardenia lutea and
Sida rhombifolia
*
Baye Akele
University of Gondar, College of Medicine and Health, P.o.Box 196, Gondar, Ethiopia.
ABSTRACT
Malaria is an endemic disease which affects 40% of world’s population and it is distributed widely, mainly due
to the multi-drug resistance developed by Plasmodium falciparum. It remains the leading cause of death among
parasitic diseases. Resistance to all known antimalarial drugs, except for the artemisinin derivatives, has
developed to various degrees in several countries. Hence, there is a huge demand to develop antimalarial drug.
In line with this notion, the hydroalcoholic leaves extracts of Gardenia lutea and Sida rhombifolia were tested
for their in vivo activity against Plasmodium berghei. The extracts showed significant antimalarial activity at
doses of 200, 400 and 600mg/Kg. The plant extracts also exhibited safety profile at tested doses of 500, 1000
and 2000mg/Kg. To conclude, 80% methanol extracts of Gardenia lutea and Sida rhombifolia exhibits
significant antimalarial activity with acceptable margin of safety.
KEY WORDS: Antimalarial, Gardenia lutea, Sida rhombifolia, and safety
INTRODUCTION
Malaria is one of the leading killers of children
under age five, accounting for almost 1 death in 10
worldwide and nearly 1 deaths in 5 in Sub-Saharan
Africa [1, 2, 3]. It is estimated to account for 300
million to 500 million illnesses and nearly 1
million deaths each year. More than 80 percent of
the world’s malaria deaths occur in sub-Saharan
Africa with 90 percent of those deaths in children
under five years of age [4].
Malaria creates significant human morbidity,
suffering and economic loss, being responsible for
70 million to 80 million cases of the global malaria
burden each year and it also places a tremendous
burden on national health systems and individual
families [4,5,6]. The proportion of government
budget allocations to health varies from less than
5% in several countries in Africa, Asia and the
WHO Eastern Mediterranean Region, to well over
_________________________________
* Corresponding author:
E-mail address: wondimakele@yahoo.com
20% in some countries in the Americas [7].
Economists estimate that malaria accounts for
approximately 40 percent of public health
expenditures in Africa and causes an annual loss of
$12billion, or 1.3 percent of the continent’s gross
domestic product [4]. The economic impact of
malaria is disproportionately felt by the poor [8].
According to the World Health Organization
(WHO), malaria is endemic in 91 countries,
predominantly in Africa, Asia and Latin America,
with about 40% of the world’s population at risk
and it is distributed widely, mainly due to the
multi-drug resistance developed by Plasmodium
falciparum. It remains the leading cause of death
due to parasitic diseases with approximately 300
million clinical cases annually resulting in huge
death, primarily in children. Resistance to all
known antimalarial drugs, except for the
artemisinin derivatives, has developed to various
degrees in several countries [9, 10].

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Drug-resistant strains of malaria have accelerated
antimalarial drug research over the last two
decades. There is a consensus that new drugs to
treat malaria are urgently needed. Many approaches
to antimalarial drug discovery are available. While
synthetic pharmaceutical agents continue to
dominate research, attention increasingly has been
directed to natural products. Investigation of plantderived compounds is a valid strategy, and this
approach can benefit from traditional knowledge of
populations from malarious regions. Natural
products afforded two of the most important
currently available drugs to treat malaria
falciparum, quinine and artemisinin. The first one,
a quinoline alkaloid, was isolated from Cinchona
species used for treatment of fevers and/or malaria
by South America Peruvian Indians and has been a
template for the synthesis of chloroquine,
antimalarial drug that was used extensively.
Artemisinin is responsible for the antimalarial
activity of Artemisia annua, a species of millenar
traditional use in China [11].
The success of artemisinin, isolated from Artemisia
annua, and its derivatives for the treatment of
resistant malaria has focused attention on the plants
as a source of antimalarial drugs [11]. The world’s
poorest are the worst affected, and many treat
themselves with traditional herbal medicines.
Ethnobotanical information about antimalarial
plants, used in traditional herbal medicine, is
essential for further evaluation of the efficacy of
plant antimalarial remedies and efforts are now
being directed towards discovery and development
of new chemically diverse antimalarial agents.
Indeed, in malaria endemic areas, plant remedies
are still widely used but mostly without assurance
of their efficacy. Validation of traditionally used
plants to treat malaria is important and requires
clinical trials which must be preceded by
phytochemical and toxicological studies that are
necessary to guarantee efficacy and safety of herbal
preparations [12, 13].
The plant Sida rhombifolia is claimed for its
promising antimalarial activity in Ethiopia, China
[14] and India [15] traditional medicine. While, an
in vitro study previously conducted on extracts
from the fruit pulp of Gardenia lutea has shown a
counter activity against 3D7-chloroquine and
pyrimethamine sensitive and Dd2-chloroquine
resistant and pyrimethamine sensitive Plasmodium
falciparum strains [16]. These plants are claimed
for their antimalarial activity based on either in
vitro activity test or traditional folk knowledge
though there is no remarkable in vivo studies have
been reported so far to strengthen the preclinical
study profile. Therefore, this study aims at
investigating the in vivo antimalarial activity of
extracts from a traditionally used medicinal plants,
Gardenia lutea and Sida rhombifolia.
METHODS
Plant Collection and sample preparation
The leaves Sida rhombifolia were collected in the
outskirt of Woldia town, North Wollo, Ethiopia and
Gardenia lutea was collected outskirt of Adirkay,
Western Tigray, Ethiopia.
The plants were
collected from Decemer, 2011 to January 2012.
Taxonomical identification was carried out at
National Herbarium, Department of Biology,
Faculty of Science, Addis Ababa University. The
plant parts were garbled and dried in the processing
room, and were then powdered and kept at room
temperature in a well-closed and amber colored
bottle until extracted.
Each of the powdered leaves (100g) of Gardenia
lutea and Sida rhombifolia were extracted by cold
maceration with 500 ml of 80 %( v/v) methanol.
Maceration was carried out for 48 hrs with
intermittent shaking with a shaker. The extracts
were then filtered with filter paper (Whatman No 3)
and the marcs were remacerated. This extraction
was repeated four times using 300ml hydroalcohol
at a time. The successive filtrates were collected
and concentrated with Rota Vapour at 400C to
remove alcohol. The remaining water was dried in
an oven at 400C with intermittent stringing and the
dried plant extracts were weighed, packed in a
glass container and kept in a refrigerator for future
use.
In vivo antimalarial activity
In vivo antimalarial activity test of each extract was
performed using a 4-day standard suppressive test
[17]. This is the most widely used preliminary test,
in which the efficacy of a compound is assessed by
comparison of blood parasitemia and mouse
survival time in treated and untreated mice [18].
Plasmodium berghei, chloroquine sensitive
plasmodium strain that is most widely employed in
rodent malaria parasite, was used to infect Swiss
albino mice for a four day suppressive test. The P.
berghei was subsequently maintained in the
laboratory by serial blood passage from mouse to
mouse. For the study, a donor mouse with a rising
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parasitemia of 20% was sacrificed, and its blood
was collected in a slightly heparinized syringe from
the auxiliary vessels. The blood was diluted with
Trisodium Citrate (TC) medium so that each 0.2 ml
contained approximately 107 infected red cells [5].
Each animal received inoculum of about 10 million
parasites per gram body weight, which is expected
to produce a steadily rising infection in mice.
The infection of the recipient mice was initiated by
needle passage of the above mentioned parasite
preparation, from the donor to healthy test animals
via an intraperitoneal route [5]. Therefore, P.
berghei infected red blood cells were
intraperitoneally injected into the mice from the
blood diluted with TC medium so that each 0.2 ml
had approximately 10 6 - 107 infected red cells
(parasite per kg of body weight). Each mouse was
infected with single inoculum of 0.2 ml blood.
On day 0, the test mice were injected with 0.2 ml
7
of 2X10 parasitized erythrocytes, (P. berghei
ANKA strain) intraperitoneally. After 2 hr, the
infected mice were weighed and randomly divided
into four groups of six mice per cage. Three of the
groups were made to receive extract treatments,
while the fourth group received the vehicle
% Paracetaemia =
% Suppression =
(negative control) and the fifth received
chloroquine (the standard antimalarial drug).
Groups 1, 2 and 3, which served as treatment
groups, received the extracts orally at 200mg/Kg,
400mg/kg and 600mg/Kg doses respectively [5].
Group 4, served as a negative control, received the
vehicle (7% Tween 80, 3% ethanol in water. Group
5 received the standard drug chloroquine phosphate
(10 mg/kg) and served as a positive control [19].
On days 1 to 3, animals in the experimental groups
were treated again (with the same dose of the
extract and same route daily) as in the day 0. On
day 4 (i.e. 24 hr after the last dose or 96 hr postinfection), blood smear from all test animals was
prepared using Giemsa stain. Level of parasitemia
was determined microscopically by counting 4
fields of approximately 100 erythrocytes per field.
The difference between the mean value for the
negative control group (taken as 100%) and those
of the experimental groups was calculated and
expressed as a percentage of suppression.
Percentage
parasitaemia
and
percentage
suppression were calculated using the following
formula:
Number of infected RBC
X100
Number of total RBC
Paracetaemia in negative control- Parastemia in treatment group
X100
Paracetaemia in negative control
Untreated control mice typically die in about one
week after infection. For treated mice the survivaltime (in days) was recorded, and the mean survival
time was calculated in comparison with that of the
negative group [20-23].
In vivo acute toxicity test
The extracts were tested for their oral acute toxicity
in mice. Three groups of mice, each group
consisting of six male mice, were used for testing
acute toxicity. The mice in each group were fasted
over night and weighed before test. Test extracts
were dissolved in 70% Tween 80 and 30% ethanol.
This solution was further diluted 10-fold with
sterile distilled water to give a stock solution
containing 7% Tween 80 and 3% ethanol [21].
Mice in groups one and two were given orally
500,1000 and 2000 mg/kg/day of the each extracts
respectively while mice in the control group (group
three) were treated with the vehicle. After
administration of the substance food was withheld
for a further 2 hr period [22].
The mice were closely observed during the first 30
minutes after dosing. They were then observed
periodically during the first 24 hr (with special
attention to the first 4 hr) and once daily thereafter
for a total of 14 days. Attention was given to
toxicity signs including changes in skin, eyes
(blinking), tremors, convulsion, lacrimation,
muscle weakness, sedation, urination, salivation,
diarrhea, lethargy, sleep, coma and also death.
Twenty-four hours later, the % mortality and
weight of mice in each group and for each test
compound at each dose level was recorded [23].
The toxicity study was designed to demonstrate the
approximate safe dose that could be used for
subsequent experiments rather than to provide
complete toxicity data on the compounds.
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Data analysis
Results of the study were expressed as mean ±
standard deviation. Statistical significance for
suppressive test was determined by one-way
ANOVA at 95% confidence limits (p=0.05). Data
on body weight and survival time were analyzed.
All the data were analyzed using Microsoft office
excel 2007.
lutea and Sida rhombifolia extracted by maceration
with 80% methanol and the percentage yields were
12.5% and 10.25% respectively.
In Vivo antimalarial activity test
The 4-day suppressive test, which is a commonly
used and standard test for antimalarial screening
[5], was used in this study. The percentage of
parasitemia and percentage of inhibition of the
plant extracts is depicted in table 1.
RESULT
Extraction
Dried and pulverized leaves of 200g Gardenia
Table 1: Parasitemia suppressive test of hydro alcoholic extracts of leaves extracts of Gardenia lutea and
Sida rhombifolia against P. berghei in mice
Test substance
Dose in mg/Kg
% parasitemia
% inhibition
200
29.4 + 0.31
53.4
400
27.7 + 0.23
56.1
600
26.9 + 0.15
57.3
Vehicle (-)
1ml
63.1+ 0.27
00
Choroquine ( +)
10
00
100
200
27.3 ± 0.19
50.1
400
26.1 ± 0.41
52.3
600
25.2 ± 0.17
53.9
Vehicle (-)
1ml
54.7 ± 0.12
00
Chloroquine (+ )
10
00
100
1.
2.
Gardenia lutea
Sida rhombifolia
P ˂ 0.05 and each value is expressed as mean±SD
In Vivo acute toxicity test
Gross behavioral and physical observation like hair
erection, urination, muscle weakness, sedation and
convulsion, reduction in feeding activity in the test
mice were used as indicators of acute toxicity
effects. The test mice were monitored once daily
for 14 days but no sign of toxicity was observed in
mice treated with Gardenia lutea extract while
minor effects were observed for Sida rhombifolia.
Increased in body weight is observed in mice
treated with hydroalcoholic extract of Sida
rhombifolia. During the first 24hr of the
experimental period no death occurred in any of the
test groups. The result of acute toxicity study is
shown in Table 2.
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Table 2: Data for the acute toxicity studies
Test substances
Dose mg/Kg
Wt. before test
Wt. after test
Gardenia lutea
500
32.5 + .12
32.2 + 0.0
0
1000
29.7 + 1.1
30.6 + 0.37
0
2000
30.4 + .28
30.4 + 0.20
0
500
29.4 + .34
31.1 + 0.26
0
1000
28.2 + .07
29.6 + 0 .11
0
2000
26.1 + .17
29.6 + 0 .39
0
1ml/100g
31.4 + .5
31.53 + 0.07
0
Sida rhombifolia
Control ( -)
% Mortality
Key: Values are M ± SD, P<0.05
DISCUSSION
The percentage yield of Gardenia lutea and Sida
rhomnifolia extracts in this study were 12.5% and
10.25% respectively. Logeswari et.al [24] obtained
a percentage yield of as high as 7.8% while Poojari
et al [25] obtained 18.8% for Sida rhombifolia
extract. Geographical and seasonal variation of the
collected plant materials, method of extraction and
eluting power of solvent could account for this
variation.
Roots and leaves extracts ( 80% methanol) of
Gardenia lutea possessed moderate in vitro activity
against both tested P. falciparum strains in the
range 2.55 - 6.13µg/ml against K1 strain and 1.65
µg/ml against NF54 strain. However, the stem
extract did not show any antimalarial activity [26].
An experiment which was done by Ahmed et al
[16] demonstrated that fruit pulp extract of
Gardenia lutea showed greater than 96.47 %
inhibition of Plasmodium falciparum at a
concentration of 50 µg/ml when activity test was
performed in vitro. Roots extracts of Gardenia
lutea also showed some cytotoxicity towards
cultured cell lines [26]. In addition to its
antimalarial activity, its stem bark 80% methanol
extract of exhibited significant antimicrobial
activity against common pathogens including
Bacillus cereus, Neisseria gonorrhea, Shigella
flexineri and Shigella dysentriea [27].
The tabulated results of this study indicated that
hydro alcoholic extracts of Gardenia lutea
displayed a very good in vivo activity against the P.
berghei malaria parasite. The comparison analysis
indicated that 200 mg/kg hydro alcoholic extract of
Gardenia lutea showed statistically significant
difference on day 4 parasitemia level, compared to
the negative control. The antimalarial activity of
Gardenia lutea dose dependent, i.e., as the dose
increase from 200mg/kg to 600mg/Kg, the
percentage of inhibition creeps from 53.4 to 57.3%.
Hence, this experiment substantiates other in vitro
studies carried out so far Ahmed et al [16], Ali et al
[26] and others. This study also attests the
traditional use of the plant in different parts of the
world including Ethiopia.
The methanol extract of the plant constituents
unsaturated phenols, triterpenes and saponins [16,
27]; however, it is devoid of flavonoids,
cardenolides,
cyanogenic
glycosides
and
anthraquinone [27]. Several investigations have
been published in the field of antiplasmodials of
plant origin related to different bioactive functional
groups classified as: terpenoids, alkaloids,
unsaturated fatty acids, volatile oils and phenolic
compounds including flavonoids and quinines [27].
Hence, the antimalarial activity of Gardenia lutea is
most probably attributed to unsaturated phenols,
triterpenes, and saponins. Isolation and structural
elucidation of these classes of compounds could
lead to the discovery of novel antimalarial agent.
Many in vitro activity tests have been done so far
on the plant Sida rhombifolia which attests its wide
array of activity. Sida rhombifolia exhibits very
good antioxidant effect against in vitro DPPH assay
method for its high phenolic content [28, 29].It has
anti-inflammatory activity via xanthine oxidase
inhibition
[29,
30].
Furthermore,
oral
administration of hydroalcoholic leaves extract
(400 mg/kg) of Sida rhombifolia decreased oedema
induced by carrageenan injection which clearly
demonstrates its anti-inflammatory activity by
another mechanism [31]. The ethanol extract of
dried aerial part of Sida rhombifolia produced
significant (P<0.001) writhing inhibition in acetic
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acid-induced writhing in mice at the oral dose of
250 and 500 mg/kg of body weight comparable to
the standard drug diclofenac sodium at the dose of
25 mg/kg of body weight [32,33]. The crude
ethanolic extract also produced the most prominent
cytotoxic activity against brine shrimp Artemia
salina [33]. Apart from all these experimental
studies, the plant Sida rhombifolia is claimed for its
promising antimalarial activity in china [14],
Ethiopia [5] and India [15] traditional medicine.
As clearly depicted in table 1, hydro alcoholic
extracts of Sida rhombifolia showed a very good
activity against the P. berghei malaria parasite.
Like Gardenia lutea, 200 mg/kg hydro alcoholic
extract of sida rhombifolia showed statistically
significant difference on day 4 parasitemia level,
compared to the negative control and its effect is
dose dependent. The antimalarial activity of Sida
rhombifolia is comparable to Gardenia lutea.
Hence, the experimental result of this study makes
clear the traditional claim and use of Sida
rhombifolia for malaria treatment.
The extract of dried leaf part of Sida rhombifolia
showed the presence of reducing sugar, steroids,
alkaloids, gums, flavonoids and glycosides [14,
24]. The leaf extract also contains ascorbic-acid,
ash,
beta-carotene,
beta-phenethylamine,
carbohydrates, fat, fiber, niacin, protein,
pseudoephedrine, riboflavin, saponin, tannins,
thiamin, triterpenoids and essential metals [14]. In
general, many experimental works confirmed that
diverse chemical classes are present in different
extract of Sida rhombifolia. Hence, rigorous
isolation and activity test should be done to unravel
the general chemical class and the specific
compound responsible for its antimalarial activity.
Considering the toxicity profile of the extracts, the
data indicated that the extract of Gardenia lutea
has not showed significant change between weight
before and after test. However, the effect in change
in body weights of Sida rhombifolia extract is
significant. This increase in body weight may
attribute to increase in appetite of mice, the
presence of several micro nutrients and
immunomodulatory substances, in addition to the
anti-parasitic activity [14, 24, 33]. During the first
24hr of the experimental period no death occurred
in any of the plant extract. This is in line with the
work of Assam et al [33] that no abnormal
symptoms and death of the rats was observed up to
16 g/kg in hydro extract of Sida rhombifolia. The
research finding of Assam et al [33] further
illustrates the significant change of liver enzymes
such as ALT, AST, ALP and CRT at higher dose.
In general, Sida rhombifolia extract can be
classified as non toxic since the limited dose of an
acute toxicity is generally considered to be 5.0 g/kg
[33].Gardenia lutea is also very safe drug at least
up to the maximum dose used in this experiment,
i.e., 2000mg/Kg.
CONCLUSION
The aqueous-methanol (1v:4v) extract of Gardenia
lutea and Sida rhombifolia demonstrated effective
in vivo antimalarial activity. These plants extracts
also exhibited safety profile at a maximum dose of
2000mg/kg. Further research needs to be carried
out to identify the active molecules and evaluate
sub-acute or chronic toxicities.
ACKNOWLEDGMENT
The author would like to thank the Ministry of
Science and Technology, Federal Democratic
Republic of Ethiopian for financing this research
project.
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