In vitro controlling of selected human diarrhea causing bacteria by clove extracts (Syzygium aromaticum L.)

Abstract

Antibacterial activity of clove extracts (Syzygium aromaticum L.) was proven against five diarrhea causing bacteria. This was further confirmed when compared with commonly used three commercial antibiotics (ciprofloxacin, tetracycline and erythromycin) as a positive control. Significant differences (P<0.0001) were observed in the effect of the antimicrobial agents (clove extracts and antibiotics), and in the sensitivities of the bacterial species (P<0.0001) to the antimicrobial agents. Clove extracts had significant (P<0.001) activity with the acetone extract demonstrating highest activity followed by antibiotics and other extracts against tested bacteria. The zone of inhibition of clove extracts was ranged from 7.33 to 12.00 mm whereas in antibiotics, it was 0.00 to 11.67 mm. Of all the bacteria, Salmonella typhimurium was the most susceptible against all of the extracts as well as concentrations of clove, while low MIC (180 mgml-1) and MBC (680 mgml-1) of the extracts were observed against Shigella dysenteriae. Consequently, clove has a significant antidiarrheal activity and it could be used as an effective antibacterial agent, alternative to the use of antibiotics.

Full text

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Int. J. Biomol. & Biomed.
RESEARCH PAPER OPEN ACCESS
In vitro
controlling of selected human diarrhea causing bacteria
by clove extracts (
Syzygium aromaticum
L.)
M Mostafizur Rahman1, M Atikur Rahman2, M Soriful Islam3, M Firoz Alam1*
1Biotechnology and Microbiology Laboratory, Department of Botany, Rajshahi University, Rajshahi
6205, Bangladesh
2Forage Biotechnology Laboratory, Department of Animal Bioscience, Division of Applied Life Science,
Gyeongsang National University, Jinju 600-701, South Korea
3Department of Molecular Pathology and Innovative Therapies, Anatomy and Cell Biology, Polytechnic
University of Marche, Via Tronto 10/A, 60020 Ancona,
Italy
Received: 20 June 2011
Revised: 22 July 2011
Accepted: 22 July 2011
Key words: Diarrhea, Syzygium aromaticum, antibiotics, antibacterial activity.
Abstract
Antibacterial activity of clove extracts (Syzygium aromaticum L.) was proven against five diarrhea causing bacteria.
This was further confirmed when compared with commonly used three commercial antibiotics (ciprofloxacin,
tetracycline and erythromycin) as a positive control. Significant differences (P<0.0001) were observed in the effect of
the antimicrobial agents (clove extracts and antibiotics), and in the sensitivities of the bacterial species (P<0.0001) to
the antimicrobial agents. Clove extracts had significant (P<0.001) activity with the acetone extract demonstrating
highest activity followed by antibiotics and other extracts against tested bacteria. The zone of inhibition of clove
extracts was ranged from 7.33 to 12.00 mm whereas in antibiotics, it was 0.00 to 11.67 mm. Of all the bacteria,
Salmonella typhimurium was the most susceptible against all of the extracts as well as concentrations of clove, while
low MIC (180 mgml-1) and MBC (680 mgml-1) of the extracts were observed against Shigella dysenteriae.
Consequently, clove has a significant antidiarrheal activity and it could be used as an effective antibacterial agent,
alternative to the use of antibiotics.
*Corresponding Author: M Firoz Alam  falambiotech@gmail.com
International Journal of Biomolecules and Biomedicine (IJBB)
ISSN: 2221-1063 (Print) 2222-503X (Online)
Vol. 1, No.2, p. 17-26, 2011
http://www.innspub.net

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Int. J. Biomol. & Biomed.
Introduction
In developing countries diarrhea is the most common
causes of morbidity and mortality (Amstrong and
Cohen, 1999) and it caused several million of deaths in
the world annually (Field, 2003). Many bacteria, virus
and protozoa have been isolated from diarrhea
patients, especially Salmonella typhimurium,
Escherichia coli, Shigella dysenteriae, Proteus
mirabilis (Prescott et al., 2005; Eja et al., 2007),
Yersinia enterocolitica (Okwori et al., 2007), Vibrio
cholera (Zuckerman et al., 2007), Campylobacter
jejuni, Clostridium difficile (Prescott et al., 2005) etc.
bacteria are responsible for acute and chronic diarrhea.
Antibiotics are the essential part for combating
harmful bacterial infections in vivo (Kaushik and
Goyel, 2008), but repeated and improper uses of
antibiotics resulting drug-resistant bacteria. To
overcome this problem an alternative therapy is very
much needed and researchers are looking for
developing alternative strategies (Sivam et al., 1997).
The World Health Organization (WHO) has included a
programme for the control of diarrhea, which involves
the use of traditional herbal medicine (Snyder and
Merson, 1982). Various herbs and spices have been
recognized by their medicinal value used as an
alternative antimicrobial agent to antibiotics, and
several plants have been reported to be used in treating
and managing diarrhea diseases (Agunu et al., 2005).
Cloves (Syzygium aromaticum L.) are the aromatic
dried flower buds, and several studies have
demonstrated on potent antibacterial effects of clove
(Cai and Wu, 1996; Bae et al., 1998; Li et al., 2005; Fu
et al., 2007). The present study was undertaken for in
vitro controlling of human diarrhea causing bacteria
by clove extracts using agar disc diffusion assay.
Materials and methods
Collection of plants and Antibiotics
Locally available flower buds of Syzygium aromaticum
L. (Common name: Clove, Family: Myrtaceae) and
three commercial antibiotics namely ciprofloxacin,
tetracycline and erythromycin (Beximco
Pharmaceuticals Ltd., Bangladesh) were used during
antibacterial study.
Collection of bacterial isolates
Five bacteria with the accession number Salmonella
typhimurium BMLRU1021, Escherichia coli
BMLRU1023, Shigella dysenteriae BMLRU1025,
Proteus mirabilis BMLRU1027 and Yersinia
enterocolitica BMLRU1029 were used in this study.
These bacterial strains were isolated and identified
from stool and urine samples (diarrhea associated)
according to Holt et al. (1994) using their respective
standard strain (collected from ICDDRB, Dhaka,
Bangladesh) in Biotechnology and Microbiology
laboratory, Department of Botany, University of
Rajshahi, Bangladesh.
Preparation of crude extracts
Collected clove flower buds were dried for 3 days in
oven under 60oC then crushed into fine powder using
mortar, pestle and electric blender (Nokia, Osaka-
Japan). Ten-gram dried powder of clove was dipped
into 100ml of different organic solvents (methanol,
ethanol and acetone) separately into a conical flask
followed by air tight with rubber corks, and left for 2
days on orbital shaking (IKA Labortechnik KS 250
Basic Orbital Shaker, Staufen, Germany). The well
refined solution was filtrated through Teton cloth and
Whatman No. 1 filter paper in a beaker followed by
evaporation of solvent using water bath (4 holes
analogue, Thermostatic water bath, China) until
formation of semisolid extract. Semi solid extracts
were dissolved into respective solvent and preserved in
airtight screw cap tube at 4°C for further use.
Preparation of antibiotics
Antibiotics solution was prepared as described by
Ekwenye and Elegalam (2005). The commercial
antibiotics ciprofloxacin (500 mg), tetracycline (500
mg) and erythromycin (250 mg) were crushed
manually using mortar and pestle. Ciprofloxacin and
tetracycline were dissolved in 10 ml de-ionized distill

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water separately, and erythromycin was dissolved in 10
ml ethanol (95%). The solutions were preserved at 4°C
until further use.
Antibacterial assay
In vitro antibacterial activity of clove extracts as well as
antibiotics were tested against five studied bacteria
using agar disc diffusion method (Parekh and Chanda,
2007). Under aseptic conditions, sterilized Whatman
no. 1 filter paper discs (6 mm in diameter) were
impregnated with 10 μl of different solvent extracts
(200, 400, and 600 mgml-1) as well as antibiotics
(0.02, 0.04 and 0.06 mgml-1) followed by air-drying
and placed on seeded nutrient agar plates. 30 µl of
bacterial suspension (108 cfu ml-1) was used for
preparing seeded nutrient agar plates. Negative
controls were prepared using respective solvents. The
Petri-plates were incubated at 37°C for 24h. After
incubation, antibacterial activity was determined by
measuring the zone of inhibition in millimeter scale
against the studied bacteria. Each assay was carried
out in triplicate.
Determination of minimum inhibitory concentration
(MIC) and minimum bactericidal concentration
(MBC)
MIC and MBC of plant extracts were determined
according to Doughari et al. (2007). For MIC
determination, 0.5 ml of varying concentrations of the
extracts (150, 180, 200, 220, 250, 280, 300, 320, 350,
380, 400, 420, 450, 480, 500, 520, 550, 580, 600, 620
and 650 mgml-1) were added with nutrient broth (2 ml)
in test tubes, then a loop-full of the test bacteria (108
cfu ml-1) was introduced. A tube containing nutrient
broth was seeded only with the test bacteria, as
described above, to serve as control. The culture tubes
were incubated at 37oC for 24 h. After incubation, the
tubes were examined for microbial growth by
observing for turbidity.
To determine the MBC, for each set of test tubes in the
MIC determination, a loopful of broth was collected
from those tubes that did not show any growth and
inoculated onto sterile nutrient agar by streaking. All
the plates were then incubated at 37oC for 24 h. After
incubation the concentration at which no visible
growth was seen, noted as MBC.
Statistical analysis
Statistical analysis (ANOVA) was performed using
software SPSS (version 10.0; SPSS Inc., Chicago IL,
USA) and MSTAT (version 2.10; Russell, D. Freed,
Michigan State University, USA) and expressed as
mean ± SEM. Least Significant Difference (LSD) test
was used to speculate further if there was a significant
difference within three clove extracts, three antibiotics,
various concentrations, studied bacteria and
interaction effect between them. P values 0.05 were
considered as significant.
Results
The results reveal that studied three concentrations of
clove extract as well as antibiotics have effective
activity against all tested bacteria (Table-1). In clove
extracts, the zone of inhibition was ranged from 0.00
to 12.00 mm. For 200 mgml-1, it was ranged from 0.00
to 7.83 mm, 0.00 to 9.67 mm for 400 mgml-1, and 7.33
to 12.00 mm for 600 mgml-1. In three type of extracts,
acetone gave the best results (10.00 to 12.00 mm)
followed by methanol (7.50 to 9.83 mm) and ethanol
(7.33 to 9.33 mm) at highest concentration (600 mgml-
1). In case of antibiotics, the zone of inhibition was
ranged from 0.00 to 11.67 mm. For 0.02 mgml-1 it was
ranged from 0.00 to 8.67 mm, 0.00 to 9.67 mm for
0.04 mgml-1, and 0.00 to 11.67 mm for 0.06 mgml-1. In
three type of antibiotics, ciprofloxacin gave best results
(10.5 to 11.67 mm) followed by tetracycline (0.00 to
11.00 mm), while erythromycin did not show any
activity. Statistical results of antibacterial activity of
three clove extracts showed significant differences
(P<0.0001) in efficacy among the bacterial strains (S),
concentrations (C) of clove extracts (E), type of extracts
as well as their interaction cases- S×C, S×E, C×E and
S×C×E (Table 2).

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Table 1. Antibacterial activities of three solvents extract of S. aromaticum L. and three antibiotics.
Concentrations
(mgml-1)
Zone of inhibition (mm)
S. typhimurium
E.
coli
S.
dysenteriae
P.
mirabilis
Y. enterocolitica
Clove extracts
200
ET
0.00
0.00
0.00
0.00
0.00
ME
0.00
0.00
0.00
0.00
0.00
AC
7.33 ± 0.33
7.33 ± 0.33
7.83 ± 0.441
0.00
7.50 ± 0.29
400
ET
7.00 ± 0.00
0.00
0.00
7.67 ± 0.33
0.00
ME
8.33 ± 0.33
0.00
0.00
0.00
7.50 ± 0.50
AC
9.33 ± 0.33
9.33 ± 0.33
9.67 ± 0.33
8.17 ± 0.44
9.33 ± 0.33
600
ET
9.33 ± 0.33
7.50 ± 0.29
7.33 ± 0.33
9.33 ± 0.33
8.00 ± 0.58
ME
9.83 ± 0.33
7.67 ± 0.66
7.50 ± 0.29
7.50 ± 0.29
9.33 ± 0.33
AC
11.17 ± 0.17
12.00 ± 0.58
11.17 ± 0.44
10.00 ± 0.58
11.33 ± 0.33
Antibiotics
0.02
CP
8.67 ± 0.33
7.33 ± 0.33
7.33 ± 0.33
7.33 ± 0.33
7.17 ± 0.33
TC
0.00
0.00
0.00
0.00
0.00
ER
0.00
0.00
0.00
0.00
0.00
0.04
CP
9.67 ± 0.33
9.00 ± 0.58
9.50 ± 0.29
8.33 ± 0.33
9.33 ± 0.33
TC
0.00
8.67 ± 0.33
0.00
0.00
0.00
ER
0.00
0.00
0.00
0.00
0.00
0.06
CP
11.00 ± 0.58
11.67 ± 0.33
11.33 ± 0.33
10.50 ± 0.29
10.67± 0.33
TC
0.00
11.00 ± 0.58
0.00
0.00
0.00
ER
0.00
0.00
0.00
0.00
0.00
ET = Ethanol extract; ME = Methanol extract; AC = Acetone extract; CP = Ciprofloxacin; TC = Tetracycline; ER = Erythromycin. *Data were
representing mean zone of inhibition (mm) ± SEM of three replicates.
According to the LSD test results (Table 3), means of
strain, no significant differences were observed among
E. coli, S. dysenteriae and P. mirabilis. But S.
typhimurium and Y. enterocolitica were significantly
different from others and between themselves. Highest
mean value was found against S. typhimurium (6.926)
and decreasing order of sensitivity of selected species
of bacteria against three extracts was Y. enterocolitica
(5.889) > E. coli (4.87) > S. dysenteriae (4.833) > P.
mirabilis (4.741). In case of mean values of
concentration, increasing the concentration level for
extracts had a significant (P<0.05) inhibitory effect on
all test bacteria. The inhibition area that found is larger
as concentration of extracts is increased and highest
for 600 mgml-1 (9.267) followed by 400 mgml-1 (5.089)
and 200 mgml-1 (2.00). The mean value of extracts
shown acetone (8.767) was significantly different from
methanol (3.844) and ethanol (3.744), while no
differences ware found between methanol and ethanol.
Table 2. Statistical results (ANOVA) of antibacterial
activity of three clove extract.
Source of
variation
Degree
of
Freedom
Sum of
squares
Mean
Square
F Value
Prob.
Replication
2
0.104
0.052
0.1718
-
Strains (S)
4
96.94
24.23
81.79
0.00
Concentrations
(C)
2
1196.99
598.50
2019.93
0.00
S×C
8
117.67
14.71
49.64
0.00
Extracts (E)
2
741.92
370.96
1251.98
0.00
S×E
8
204.70
25.59
86.36
0.00
C×E
4
71.06
17.77
59.96
0.00
S×C×E
16
173.70
10.88
36.70
0.00
Error
88
26.563
0.30
-
-
Total
134
2629.94
-
-
-
Like clove extracts, antibacterial activity of three
antibiotics showed significant differences (P<0.0001)
among the bacterial strains (S), concentrations (C) of
antibiotics, type of antibiotics (A) as well as for
interaction items- S×C, S×A, C×A and S×C×A (Table-
4). Mean separation (Table-5) for antibacterial activity
of antibiotics shows that the mean of strain E. coli
(5.296) in the top and significantly different from

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others. Rest of strains shows no differences among
themselves. Here also concentrations of antibiotic were
different from each other. All antibiotics were
significantly different from each other and highest for
ciprofloxacin (9.267) followed by tetracycline (1.311)
and erythromycin (0.00).
Table 3. Analysis of mean data of the antibacterial
activity of three clove extracts.
Variables
Growth inhibition diameter (mm)
Strains
S. typhimurium
6.926 A
E. coli
4.87 C
S. dysenteriae
4.833 C
P. mirabilis
4.741 C
Y. enterocolitica
5.889 B
LSD
0.5148
Concentrations
200 mgml-1
2.00 C
400 mgml-1
5.089 B
600 mgml-1
9.267 A
LSD
0.3988
Extracts
Ethanol
3.744 B
Methanol
3.844 B
Acetone
8.767 A
LSD
0.3988
Means followed by different letter(s) down the column are
significantly different at P<0.05. Data values are means of
three replicates.
Table 4. Statistical analysis of antibacterial activity of
three antibiotics.
Source of
variation
Degree
of
Freedom
Sum of
squares
Mean
Square
F Value
Prob.
Replication
2
0.181
0.091
0.5577
-
Strains (S)
4
107.57
26.89
166.91
0.00
Concentrations
(C)
2
80.12
40.06
248.63
0.00
S×C
8
63.81
7.98
49.51
0.00
Antibiotics (A)
2
2263.22
1131.61
7023.77
0.00
S×A
8
207.28
25.91
160.81
0.00
C×A
4
48.83
12.21
75.77
0.00
S×C×A
16
102.36
6.40
39.71
0.00
Error
88
14.319
0.16
-
-
Total
134
2887.66
-
-
-
The MIC and MBC results of clove extracts are
presented in Table 6. The results reveal that MIC
values were ranged from 180 (S. dysenteriae) to 620
mgml-1 (E. coli and S. dysenteriae). For ethanol
extract, it was ranged from 400 (P. mirabilis) to 620
mgml-1 (E. coli and S. dysenteriae), 400 (S.
typhimurium) to 600 mgml-1 (E. coli, S. dysenteriae
and P. mirabilis) for methanol, and 180 (S.
dysenteriae) to 400 mgml-1 (P. mirabilis) for acetone.
Table 5. Analysis of mean data of the antibacterial
activity of three antibiotics.
Variables
Growth inhibition diameter (mm)
Strains
S. typhimurium
3.259 B
E. coli
5.296 A
S. dysenteria
3.13 B
P. mirabilis
2.907 B
Y. enterocolitica
3.037 B
LSD
0.3782
Concentrations
0.02 mgml-1
2.533 C
0.04 mgml-1
3.633 B
0.06 mgml-1
4.431 A
LSD
0.293
Antibiotics
Ciprofloxacin
9.267 A
Tetracycline
1.311 B
Erythromycin
0.00 0 C
LSD
0.293
Means followed by different letter(s) down the column are
significantly different at P<0.05. Data values are means of three
replicates.
In three types of extract, acetone extract gave lowest
MIC value (180 mgml-1) against S. dysenteriae
followed by methanol and ethanol (400 mgml-1). In
case of MBC values, it was ranged from 220 to 680
mgml-1 under the same strain (S. dysenteriae). For
ethanol extract, it was ranged from 450 (S.
typhimurium and P. mirabilis) to 680 mgml-1 (S.
dysenteriae), 450 (S. typhimurium) to 650 mgml-1 (E.
coli, S. dysenteriae and P. mirabilis) for methanol and
220 (S. dysenteriae) to 450 mgml-1 (P. mirabilis) for
acetone. In three types of extracts, acetone extract gave
lowest MBC value (220 mgml-1) against S. dysenteriae
followed by methanol and ethanol (450 mgml-1).
Discussion
Although, the primary purpose of spices is to impart
flavor and piquancy to food, the medicinal,
antimicrobial and antioxidant properties of spices have
also been exploited (Souza et al., 2005). Cloves are
antimutagenic (Miyazawa and Hisama, 2003), anti-

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inflammatory (Kim et al., 1998), antioxidant (Chaieb et
al., 2007a), antiulcerogenic (Bae et al., 1998; Li et al.,
2005), antithrombotic (Srivastava and Malhotra, 1991)
and antiparasitic (Yang et al., 2003). On the basis of
this information, antibacterial activities of clove
extracts were evaluated for their antidiarrheal
properties.
Table 6. Minimum inhibitory concentration (MIC)
and Minimum bactericidal concentration (MBC) of
three solvent extracts of S. aromaticum L. against
studied bacterial strains.
Extracts
Concentrations (mgml-1)
S.
typhimurium
E.
coli
S.
dysenteriae
P.
mirabilis
Y.
enterocolitica
MIC
ET
420
620
620
400
580
ME
400
600
600
600
420
AC
200
200
180
400
200
MBC
ET
450
650
680
450
620
ME
450
650
650
650
480
AC
250
250
220
450
250
ET = Ethanol extract; ME = Methanol extract; AC = Acetone extract
The antimicrobial activity has been attributed to the
presence of some active constituents in the extracts.
Clove contains a high eugenol (70-90%) content (de
Guzman and Siemonsma, 1999) which is an
antibacterial compound having wide spectra of
antimicrobial effects against enterobacteria (Burt and
Reinders, 2003; Nanasombat and Lohasupthawee,
2005; Chaieb et al., 2007b). The results of this study
exemplifies that clove extracts have potential source of
antidiarrheal properties because extracts and their
concentrations have significant influence on the
growth of diarrhea causing bacteria and also it has
superior antibacterial activity than antibiotics. Several
investigators conducted related investigation and
recommend clove extracts as a source of antibacterial
agent (Nascimento et al., 2000; Saeed and Tariq,
2008). It has also been reported that, clove oil potently
inhibited the growth of different Gram negative
bacteria (Saeed and Tariq, 2008; Lopez et al., 2005).
Clove extracts had a high activity against E. coli (12.00
mm) and previous studies have documented that E.
coli are known to be multi-drug resistant bacteria
(Saeed et al., 2007; Singh et al., 2002). The results
were also in accordance with those reported by many
investigators (Mandee et al., 2003; Smith-Palmer et
al., 2001; Dorman and Deans, 2000; Hammer et al.,
1999; De et al., 1999). All the tested bacteria, which
were resistant to erythromycin and tetracycline, but
significantly inhabited by clove extracts. Salman et al.
(2008) also found comparable results. In this
experiment, extracts showed different degrees of
growth inhibition depending upon the bacterial
strains. These variations were found because strains
are genetically different from each other, and this is
probably due to the differences in chemical
composition and structure of the cell wall of both types
of microorganisms (Kaushik and Goyel, 2008),
microbial growth, exposure of micro- organisms to
plant oil, the solubility of oil or oil components and the
use and quantity of an emulsifier (Bansod and Rai,
2008). Increasing of the concentrations level of
extracts had a significant (P<0.05) inhibitory effect on
all studied bacteria. Similarly Tylor et al. (2001)
reported that active compounds may be present in
insufficient quantities in the crude extracts to show
activity with the dose levels employed and lack of
activity can thus only be proven by using large dose
(Farnsworth, 1993). Extracts prepared in acetone
extract gave better activity than that of other extracts,
and it could be better solubility of active components
in acetone. It has been reported that different
phytoconstituents have different degrees of solubility
in different types of solvents depending on their
polarity (El-mahmood and Ameh, 2007). This
observation clearly indicates that the polarity of
antimicrobial compounds make them more readily
extracted by acetone solvent, and using organic solvent
does not negatively affect their bioactivity against
bacterial species (Kaushik and Goyel, 2008). Of all the
bacteria, S. typhimurium was the most susceptible
against all of the extracts and concentrations of clove
while P. mirabilis was the most resistance bacteria. In

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Int. J. Biomol. & Biomed.
this study the low MIC and MBC values observed for S.
dysenteriae is a good indication of high efficacy against
this bacteria and high MIC and MBC values are
indication of low activity (Doughari et al., 2007). In all
cases, three clove extracts consistently displayed
superior potency when compared with antibiotics,
while extracts are a mixture of various plant
constituents and antibiotic is a refined and purified
product (El-Mahmood and Doughari, 2008).
Comparing among the three extracts with positive
control, acetone extract was found most effective for
antibacterial activity and the degree of antibacterial
property of three extracts can be put in the following
order: acetone > methanol > ethanol.
The results of this study revealed that although crude
extracts of clove are not purified but their activity was
very effective against all tested bacteria, and these
extracts could be used as an effective antimicrobial
agent, alternative to the use of antibiotics.
Acknowledgement
The authors would like to thank the Department of
Pathology, Rajshahi Medical College Hospital,
Rajshahi, Bangladesh, for collecting samples.
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