American Journal of Plant Sciences, 2013, 4, 2181-2185
Published Online November 2013 (http://www.scirp.org/journal/ajps)
http://dx.doi.org/10.4236/ajps.2013.411270
Open Access AJPS
2181
Evaluation of Antibacterial and Antidiarrhoeal Activities
of Feronia limonia Leaf Extract
Mohammad Abdul Motalib Momin1, Mizanur Rahaman Khan2, Johir Rayhan1, Afrina Afrose1,
Sohel Rana1, Anjuman Ara Begum1*
1Department of Pharmacy, Jahangirnagar University, Savar, Bangladesh; 2Department of Pharmacy, University of Science and
Technology Chittagong, Chittagong, Bangladesh.
Received August 19th, 2013; revised September 20th, 2013; accepted October 15th, 2013
Copyright © 2013 Mohammad Abdul Motalib Momin et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
ABSTRACT
The present study was carried out to investigate possible antibacterial and antidiarrhoeal activities of ethanol extract of
Feronia limonia leaves. Phytochemical analysis of the crude extract was performed to detect presence of different kinds
of phytoconstituents. The antibacterial activity was investigated against four Gram positive and four Gram negative
bacteria by using disc diffusion method. The plant extract showed moderate antibacterial activity against Gram positive
bacteria namely Staphylococcus saprophyticus and Staphylococcus pyogenes and all tested Gram negative bacteria
namely Escherichia coli, Shigella boydii, Shigella dysentery and Shigella flexneri in dose dependant manner. The re-
sults of castor oil-induced diarrhoeal study showed that Feronia limonia extract significantly reduced the severity &
frequency of diarrhoea in mice at a higher dose of 500 mg/kg compared with the standard drug loperamide (25 mg/kg).
The present study clearly supports the medicinal value of this plant. The overall results indicate the possibility of pres-
ence of some active principles in the plant extract possessing antibacterial and antidiarrhoeal actions.
Keywords: Phytochemical Screening; Antimicrobial; Antidiarrhoeal; Feronia limonia
1. Introduction
Antibiotic resistance developed by bacteria has become a
vital issue all over the world. A good number of antibi-
otics are found to be inactive in recent years largely due
to resistance development through the inappropriate and
injudicious uses of commercial antimicrobial drugs com-
monly employed in the treatment of infectious diseases
[1]. So to combat the problem of microbial resistance and
for substitution with effective ones the developments of
new antibacterial agents are necessary. Diarrhoea is an-
other important health problem around the world, re-
sponsible for more than 5 million deaths annually [2,3].
The investigation of the antimicrobial and antidiarrhoeal
properties of plants has brought attention to the opportu-
nity of producing a safe, economical and easily available
source that could replace the synthetic antimicrobial and
antidiarrhoeal compounds [4,5].
Feronia limonia is a deciduous, slow-growing, erect
tree belonging to family Rutaceae and subfamily Auran-
tioideae [6]. It is distributed in deciduous and arid land-
scapes of several countries in South Asia [7]. Feronia
limonia as a whole, or its parts such as unriped fruit,
riped fruit, root, bark, trunk gum and leaves have a broad
spectrum of traditionally established therapeutic proper-
ties including antimicrobial and antidiarrhoeal effects
[8,9]. Literature survey revealed that leaf extract of the
plant has antioxidant [10], hepatoprotective [10], lar-
vicidal [11], antitumor [12], antidiabetic [13], and CNS
depressant potentials [14]. In the present study, assess-
ment of antibacterial and antidiarrhoeal activities of the
leaves of F. limonia was conducted to find out medici-
nal properties and to justify their traditional use for ail-
ments of diseases.
2. Materials and Methods
2.1. Collection and Preparation of the Plant
Material
The plant material was collected from Khulna district,
*Corresponding author.

Evaluation of Antibacterial and Antidiarrhoeal Activities of Feronia limonia Leaf Extract 2182
Bangladesh and identified by the taxonomist of Bangla-
desh National Herbarium, Dhaka (Accession No. DACB-
34397). The voucher specimens of the plants have been
deposited in the herbarium for future reference. The
leaves of collected plant were sun-dried for one week.
The plant parts were then converted into a coarse powder
with a suitable grinder. The powder was stored in an air-
tight container and kept in a cool, dark and dry place
until analysis commenced.
2.2. Preparation of Plant Extract
About 150 gm of powered material was taken in a clean,
flat bottomed glass container and soaked in 600 ml of
95% ethanol. The container with its contents was sealed
and kept for a period of 14 days accompanying occa-
sional shaking and stirring [15-17]. The whole mixture
then underwent a coarse filtration by a piece of clean,
white cotton material and also using Whatman filter pa-
per. By using a rotary evaporator (Bibby RE200, Sterilin
Ltd., UK) the resultant filtrate was concentrated to pow-
der form through complete evaporation of the extraction
solvent. The filtrate was then air dried at room tempera-
ture to evaporate the extra ethanol. It rendered concen-
trate of reddish color which was designated as crude ex-
tract of ethanol and stored in a refrigerator until further
investigation.
2.3. Experimental Animal
Young Swiss-albino mice (average weight 20 - 25 gm)
were purchased from the Animal Research Branch of the
International Center for Diarrhoeal Disease and Research,
Bangladesh (ICDDRB) for assessing biological activity.
The animals were kept in standard environmental condi-
tions for one week for adaptation after their purchase and
fed ICDDRB formulated rodent food and water. All the
experiments were conducted on an isolated and noiseless
condition.
2.4. Phytochemical Screening
The preliminary phytochemical screening with various
qualitative chemical tests was performed to detect the
presence of various classes of phytoconstituents in 10%
(w/v) solution of the plant extract. Phytoconstituents like
saponins, tannins, alkaloids, steroids, flavonoids, gly-
cosides were identified by characteristic color changes
using different reagents following standard procedures
[18,19].
2.5. Antimicrobial Test
The antimicrobial assay was performed by disc diffusion
method [20-22]. Eight pathogenic bacteria (collected
from the International Centre for Diarrhoeal Disease and
Research, Bangladesh) were inoculated on 16 ml previ-
ously sterilized nutrient agar media, mixed thoroughly
and transferred immediately to the sterile Petri dish in an
aseptic condition using a sterile loop. Prepared plant ex-
tracts (250 µg/disc and 500 µg/disc) and standard kana-
mycin solutions (30 µg/disc) were applied to the corre-
sponding Petri dish. The plates were incubated for 24
hours at 37˚C. After proper incubation, clear zone of in-
hibition around the point of application of sample solu-
tion were measured and expressed in millimeter (mm).
2.6. Antidiarrhoeal Test
The experiment was conducted by previously reported
castor-oil diarrhea model [23]. The mice were screened
initially by giving 0.5 mL of castor oil and only those
showing diarrhoea were selected for the experiment. The
test animals fastened overnight were randomly allocated
to four groups consisting of six mice in each group. The
animals of group I (control) received vehicles only (dis-
tilled water containing 0.1% Tween-80). Group-II (posi-
tive control) received standard antimotility drug lopera-
mide (50 mg/kg body weight) as oral suspension. The
group-III and group-IV (test groups) were treated with
suspension of leaves extract of Feronia limonia at the
oral dose of 250 mg/kg-body weight and 500 mg/kg-
body weight. After one hour treatment with distilled wa-
ter, standard drug or plant extract, each animal was given
0.5 mL of castor oil by oral route. Individual animals of
each group were then placed in separate cages having
adsorbent paper beneath and examined for the presence
of diarrhoea every hour in five hours study after the cas-
tor oil administration. Number of stools or any fluid ma-
terial that stained the adsorbent paper were counted at
each successive hour during the 4-hour period and were
noted for each mouse. The latent period of each mouse
were also counted. At the beginning of each hour new
papers were placed for the old ones.
2.7. Statistical Analysis
Results were expressed as mean ± standard error of mean
(SEM). Statistical analysis for animal experiment was
carried out using one-way ANOVA followed by Dun-
net’s multiple comparisons. The results obtained were
compared with the control group. P values < 0.05 were
considered to be statistically significant.
3. Results and Discussion
3.1. Phytochemical Screening
The crude extract was subjected for chemical group tests
to identify various types of important chemical constitu-
ents. Phytochemical studies showed that alkaloids, ster-
oids, tannins and flavonoids are present in the ethanolic
extract of Feronia limonia (Table 1). However, glyco-
Open Access AJPS

Evaluation of Antibacterial and Antidiarrhoeal Activities of Feronia limonia Leaf Extract
Open Access AJPS
2183
Enterococcus facealis and Streptococcus agalactiae. An-
timicrobial activity of the plant against both Gram posi-
tive and Gram negative bacteria may be due to the pres-
ence of broad spectrum antibiotic compounds [24,25] or
the previously reported compounds like essential oil, rich
in methyl chavicol [26].
side, gum, carbohydrate and saponin were absent in
ethanol extract of the plant.
3.2. Antimicrobial Test
Antibacterial activity of the ethanol extract of Feronia
limonia leaves (250 μg/disc and 500 μg/disc) was evalu-
ated by determining zones of inhibition (mm) against
four Gram positive and four Gram negative bacteria and
compared with the standard antibiotic kanamycin (30
μg/disc) (Table 2). For both gram positive and gram
negative bacteria kanamycin demonstrated almost similar
actions which justified its use as standard in this study.
The study showed the plant extract possessed moderate
dose-dependent antibacterial activity against Gram posi-
tive Staphylococcus saprophyticus and Staphylococcus
pyogenes and all tested Gram negative bacteria. However,
the plant extract was ineffective against Gram positive
3.3. Antidiarrhoeal Test
Castor oil (0.5 mL, p.o.) induced diarrhoea promptly
within one hour in the animals and produced a
considerable amount of stool. The time for diarrhoeal
induction in mice was prolonged by administration of
ethanol extract of leaves of F. limonia at the doses of 250
and 500 mg/kg (Table 3). The plant extract significantly
reduced the total number of faeces as well as of dia-
rrhoeic faeces at a higher dose of 500 mg/kg body
weight.
Table 1. Phytochemical constituents of Feronia limonia leaves.
Extract Alkaloid Glycoside Steroid Gums Carbohydrate Tannins Flavonoids Saponins
Ethanol + − + − − + + −
Table 2. In-vitro antimicrobial activity of ethanolic extract of F. limonia.
Diameter of zone of inhibition (mm)
Bacterial strains Kanamycin (30 μg/disc) Ethanol extract (250 μg/disc) Ethanol extract (500 μg/disc)
Gram positive (+)
Staphylococcus saprophyticus 21 5 7
Enterococcus facealis 20 0 0
Staphylococcus pyogenes 24 6 7
Streptococcus agalactiae 14 0 0
Gram negative (−)
Escherichia coli 24 5 8
Shigella boydii 22 7 9
Shigella dysenteriae 16 7 9
Shigella flexneri 24 7 9
Table 3. Effect of F. limonia on castor oil-induced diarrhoea in mice.
Treatment Latent period (hr) Total number of faeces in 4 hr Mean of defaecation in 4 hour.
Control 0.64 ± 0.27 46 9.2
Loperamide (50 mg/Kg) 2.57 ± 0.06 19 3.8
Extract (250 mg/kg) 0.80 ± 0.21 41 8.2*
Extract (500 mg/kg) 0.96 ± 0.17 32 6.4*
Values are expressed as mean ± SEM (n = 6), *P < 0.01 vs control.
It is well evident that castor oil produces diarrhoea due
to its most active component recinoleic acid which
causes irritation and inflammation of the intestinal mu-
cosa, leading to release of prostaglandins, which results
in stimulation of secretion [27]. Since the ethanol extract
of F. limonia successfully inhibited the castor oil-in-
duced diarrhoea, the extract might have exerted its
antidiarrhoeal action via antisecretory mechanism which
was also evident from the reduction of total number of
wet faeces in the test groups of the experiment.

Evaluation of Antibacterial and Antidiarrhoeal Activities of Feronia limonia Leaf Extract 2184
Furthermore, the standard chemical tests performed in
this study showed that the leaves of the plant species
contain tannins, steroids and flavonoids. Tannins have
been reported in several studies to have antidiarrhoeal
effect [28,29]. In fact, tannins denature proteins and form
protein tannate, which makes the intestinal mucosa more
resistant and reduces intestinal secretion [30]. The
antidiarrhoeal activity of flavonoids has been ascribed to
their ability to inhibit intestinal motility and hydro-elec-
trolytic secretion [31,32]. Hence, the antidiarrhoeal ac-
tivity of the plant may be due to its content of tannins
and/or flavonoids. In addition, the antidiarrhoeal activity
of the plant may be associated with its antimicrobial ef-
fect.
4. Conclusion
The results of the present study indicate that the ethanol
extracts of F. limonia leaves possess significant antibac-
terial and antidiarrhoeal potentials in dose dependant
manner. The present data justify the traditional uses of
this plant for the treatment of various diseases. However,
further studies are required for isolation and purification
of the active principles of the plant responsible for these
effects and to better understand the mechanism of such
actions. As the leaves extracts possess tannin and flavon-
oids, which indicates the presence of antioxidant capacity,
we have further plan to conduct study to investigate an-
tioxidant property.
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