Evaluation of Antibacterial and Antidiarrhoeal Activities of <i>Feronia limonia</i> Leaf Extract

doi:10.4236/ajps.2013.411270

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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.

Email: *[email protected]

Received August 19th, 2013; revised September 20th, 2013; accepted October 15th, 2013

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

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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

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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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