Research Paper
Effects of Methanol Extract of Wedelia chinensis
Osbeck (Asteraceae) Leaves against Pathogenic Bacteria
with Emphasise on Bacillus cereus
I. DARAH, S. H. LIM* AND K. NITHIANANTHAM
Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang,
Malaysia
Darah, et al.: Methanol Extract of Wedelia chinensis Leaves Against Bacteria
The antibacterial activity of the methanol extract of Wedelia chinensis leave was studied and tested against three
pathogenic Gram positive bacteria (Bacillus cereus, B. subtilis and Stapylococcus aureus) and three pathogenic Gram
negative bacteria (Escherichia coli, Proteus rettgeri and Pseudomonas aeruginosa) by the disk diffusion assay and
broth dilution methods. The extract exhibited favourable antibacterial activity against the bacterial cells but was
more potent against Gram positive bacteria with the minimum inhibition concentration of 3.12 to 6.25 mg/ml
compared to the Gram negative bacteria which had minimum inhibition concentration values of 25 mg/ml. The
time-kill study suggested that the extract possessed bactericidal properties at higher concentrations and eradicated
the growth of bacterial cells. The major abnormalities occurred to the bacterial cells after exposed to the extract
were complete alterations in their morphology and collapsed of the cells beyond repair. The methanol extract of
W. chinensis may be an effective antibacterial agent to treat bacterial infections.
Key words: Antimicrobial activity, minimum inhibition concentration, pathogenic bacterial, Wedelia chinensis
Various therapeutic benefits available in plants are
becoming of interest amongst researchers to search
for alternatives for combating the rising prevalence
of global antimicrobial resistance problems. Moreover,
concerns on the safety of some chemical in drugs
have prompted an increased interest in natural
additives.
Furthermore, the spread of drug resistant pathogens is
one of the most serious threats to successful treatment
of microbial diseases[1]. Bacteria for example have
shown a remarkable ability to endure and adapt
to their environment including the development
of different mechanisms of resistance to most old
and new antibacterial agents[2]. Bacterial adaptation
to antibiotics has been very successful, and over
the years, the increase in antibiotic resistance has
generated a considerable worldwide public health
problem[3]. In addition, it was found that the synthetic
antibiotics not only costs, but also have caused some
side effects in the treatment of infectious disease[4].
Thus, scientists are forced to search new antimicrobial
*Address for correspondence
E-mail: limshehhong77@gmail.com
September - October 2013
substances from various sources and there is a need
to develop alternative antimicrobial drugs for the
treatment of infectious diseases from medicinal
plants[5].
W. chinensis is a traditionally used medicinal herb in
Ayurveda, Siddha and Unani system of medicines[6].
Traditionally, the fruits, leaves and stem are used in
child birth and in the treatment of bites and stings,
fever and infection. The leaves are used in the
treatment of kidney dysfunction, cold, wounds and
amenorrhea[7]. The leaves are also used for dyeing
hair and for promoting their growth besides been
used in treating elephantiasis, toothache, headache and
cancer[8,9]. The tonic of the leaves is used in cough
and cephalagia. Decoction of the plant is used in
menorrhagia and skin diseases[10]. The plant has also
found its use in inflammations, helmintic diseases
and liver disorders[11]. The plant has been used as
astringent, bitter, acrid, antiinflammatory, cardiotonic,
treatment of wounds, seminal weakness and viral
hepatitis [12‑14]. The plant is scientifically reported
to possess antioxidant property which indicates its
usefulness in reducing anxiety and stress in emotional
conditions[15].
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Since W. chinensis exhibited various medicinal
properties, this study further evaluated the
antimicrobial activity of the crude methanol extract
from these leaves against some bacteria species. Its
effect on the selected bacterial growth and structure
degeneration were studied and evaluated.
MATERIALS AND METHODS
Methanol (Fisher Scientific, United Kingdom),
chloramphenicol (Sigma‑Aldrich, St. Louis, USA),
nutrient agar and nutrient broth (Merck, Germany),
filter paper No. 1 (Whatman plc, Kent, UK),
6 mm antibiotic disk (GF A, Whatman, Kent, UK),
Shaker (Infors HT Ecotron, Switzerland), rotary
evaporator (Eyela, China), UV spectrophotometer
Genesys 10 uv (Spectronic Unicam, USA), scanning
electron microscope Leica Cambridge S‑360 (Leica
Cambridge, UK) and transmission electron
microscope Philips CM12 (Philips, Eindhoven,
Netherland) were used in our study.
Collection, processing and extraction of plant
sample:
The fresh sample of W. chinensis leaves was collected
around the Penang Island, Malaysia. The leaves
were rinsed thoroughly under running tap water
and the clean samples were then dried in an oven
at 45° for 4‑7 days until they were completely
dried before grinding them into powder form. The
dried and finely ground (0.5 mm) of sample was
extracted with methanol by using the modified
method of Darah and Annie‑Clara[16]. Approximately,
40 g of dried powdered plant sample was soaked in
400 ml of methanol at room temperature (30±2°)
for 3 consecutive days with frequent agitation.
The mixture was filtered using a muslin cloth and
followed by Whatman No. 1 ilter paper. The iltrate
was then concentrated in a rotary evaporator under
reduced pressure until oily paste formed and kept at
cool dry place until further used.
Microorganisms and cultural maintenance:
Six pathogenic bacterial species which consisted
of three Gram positive (B. cereus, B. subtilis
and S. aureus) and three Gram negative (E. coli,
P. rettgeri and P. aeruginosa) bacteria were
obtained from the Industrial Biotechnology Research
Laboratory (IBRL) Culture Collection, School of
Biological Sciences, Universiti Sains Malaysia were
used throughout the study. The bacterial cultures were
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maintained on nutrient agar slants at 37° for 24 h.
All the cultures were kept at 4° until further used.
Subculturing was done at every 4 weeks to maintain
their viability.
Antibacterial activity:
The antibacterial activity of the extract against the
test bacteria were determined following the method
described by Tong et al.[17] with slight modiications.
Test bacteria were cultured on nutrient agar plates
and incubated at 37° for 24 h. Bacterial suspensions
were prepared by inoculating one loopful of a pure
colony into 5.0 ml of sterile distilled water. Suficient
inoculums were added until the turbidity equal to 0.5
McFarland standards which approximately equivalent
to 1.5×105 cells/ml.
One milliliter of the suspension was added into
15.0 ml of sterilized molten nutrient agar aseptically.
The mixtures were mixed well by swirling the
plates left and right and then they were left on the
bench to solidify. The commercial antibiotic disk
GF A with 6.0 mm diameter was used to screen
the antibacterial activity. Each of the sterile disks
was then impregnated with 20 µl of the extracts of
100.0 mg/ml of extract stock. Chloramphenicol at
the concentration of 30 µg/ml was used as a positive
control and methanol was used as a negative control.
All the impregnated disks were air dried before
placing them on the agar surface. The plates were
incubated at 37° for 24 h and the antibacterial activity
was determined by measuring the diameter of the
inhibition zones formed around the disks.
Determination of minimum inhibitory concentrations:
The determination of minimum inhibitory
concentration (MIC) was carried out by the liquid
dilution method[16]. The complete protocol of the MIC
test is found in the M7‑T2 publication of the National
Committee for Clinical Laboratory Standards [18].
Briefly, different extract preparations were subjected
to a serial dilution using sterile nutrient broth medium
as a diluents to give inal crude extract concentrations
between 1.275 and 200 mg/ml. The tubes were
inoculated with the bacterial suspension (20 µl/ml
broth), homogenized, and incubated at 37° for 24 h.
The lowest dilution of the extract that retained its
inhibitory effect resulting in no growth (absence of
turbidity) of a microorganism was recorded as the
MIC value of the extract. The bacterial growth was
indicated by the turbidity. Each test was performed
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in triplicate and repeated twice. A control experiment
was run in parallel to study the impact of the solvent
(without plant component) on growth of the test
bacteria.
Time‑kill study of B. cereus:
Bacterial suspension of B. cereus was prepared as
described previously. Methanol (1 ml) extract stock
was added into conical flasks containing 23.0 ml
of sterilized nutrient broth and 1.0 ml of inoculum.
The inal concentrations of the extracts in the flasks
were at 1.56 mg/ml (half MIC), 3.13 mg/ml (MIC)
and 6.25 mg/ml (2MIC). Control with 1.0 ml
of methanol was used n this experiment [19] . The
experiments were conducted in triplicate and all the
flasks were incubated in a shaker (Infors HT Ecotron,
Switzerland) incubator at 37° with agitation of
150 rpm. One milliliter of the mixture from each flask
was withdrawn at every 4 h intervals starting from
0 h until 48 h of cultivation and the bacterial cell
growth was monitored by measuring optical density
at 540 nm.
Scanning and transmission electron microscope
observations:
The B. cereus suspension was prepared as described
previously. To each sample, 1.0 ml of the 24 h old
bacterial suspension was inoculated in a 50.0 ml
conical flask containing 30.0 ml of sterilized nutrient
broth and incubated in a shaker at 37°, 150 rpm for
18 h. The bacterial suspension was then added to the
extract stock solution (the inal concentration in each
flask was at the MIC value) and incubated at the
required incubation time (12, 24 and 36 h). As for a
negative control, a conical flask containing bacterial
suspension was added with 1.0 ml of methanol.
The sample preparation for Scanning (SEM) and
transmission (TEM) electron microscopy were
done following the method describes by Marez[20]
and Yogalatha et al. [19] respectively. The prepared
samples were then viewed under a scanning (Leica
Cambridge, S‑360, United Kingdom) and
transmission (Philips CM12, Eindhoven, Netherland)
electron microscopes.
RESULTS AND DISCUSSION
The present study has shown that methanol extract
of W. chinensis leaves has promising antibacterial
activity, and this is probably the reason for its wide
use as traditional medicine. Table 1 shows that all
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the six test bacteria exhibited zone of inhibition,
which were smaller when compared to zones of
inhibition produced by the commercial antibiotic,
chloramphenicol (30 µg/ml). The results also showed
that Gram positive bacteria, B. cereus, B. subtilis and
S. aureus produced bigger zone of inhibition of 14.0,
12.0 and 15.0 mm, respectively compared to Gram
negative bacteria E. coli, P. rettgeri and P. aeruginosa
which produced 7.0, 8.0 and 7.0 mm of inhibition
zones, respectively.
Gram positive bacteria, B. cereus, B. subtilis and
S. aureus exhibited lower MIC values of 3.12,
6.25 and 6.25 mg/ml, respectively compared to
the Gram negative bacteria, E. coli, P. rettgeri
and P. aeruginosa which exhibited MIC values of
25 mg/ml extract (Table 1). The results indicated that
Gram positive bacteria were more susceptible to the
extract compared to Gram negative bacteria.
The activity of the plant against both Gram positive
and Gram negative bacteria can be indicative of the
presence of broad spectrum antibiotic compounds
or simply general metabolic toxins in the plant.
Generally, Gram negative bacteria are more
resistant than Gram positive bacteria[21‑24]. The same
characteristics were observed in other antimicrobial
studies of plant extract against pathogenic
bacteria[22,23].
Time‑kill studies were performed over a period
of 48 h with the B. cereus cells being exposed
to MIC (3.13 mg/ml), 1/2MIC (1.65 mg/ml) and
2MIC (6.25 mg/ml) values of the extract and the
results are shown in ig. 1. At 1/2MIC (1.56 mg/ml)
the extract demonstrated a drastic drop in OD after
TABLE 1: ANTIMICROBIAL ACTIVITY
Microorganism
Gram-positive bacteria
Bacillus cereus
Bacillus subtilis
Staphylococcus
aureus
Gram-negative
bacteria
Escherichia coli
Proteus rettgeri
Pseudomonas
aeruginosa
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Diameter zone of
Minimum
inhibition (mm)
inhibitory
Methanol Chloramphenicol concentration
(mg/ml)
extract
14.0±0.3 20.00±0.3
12.0±0.4 21.00±0.2
15.0±0.3 21.00±0.3
3.13
6.25
6.25
7.0±0.3
8.0±0.5
7.0±0.2
25.00
25.00
25.00
22.00±0.2
20.00±0.3
20.00±0.4
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16 h, which leads to the stationary phase of the
bacterial growth compared to the control. At the
values of MIC (3.13 mg/ml) and 2MIC (6.25 mg/ml),
the extract produced cell eradication after 12 h. Based
on the results obtained from the time‑kill studies,
it was obviously seen the potency of the methanol
extract of W. chinensis leaves as antibacterial agents
against pathogenic bacteria.
Fig. 2 represents the morphological changes of the
nontreated and treated B. cereus. Fig. 2a shows
the SEM micrographs of bacterial cells without the
methanol extract treatment. The igure revealed the
normal rod shape cell structure without any shrinkage
or cavity formation as the surface was smooth and
regular. Fig. 2b shows the morphology of the cell
after 12 h of treatment with the extract. The bacterial
cells started to show multiple defects with many of
cells exhibited crumpled or shrunken cell surface.
Fig. 2c revealed more formation of crumpled cells
and some the cells formed cavities. After 36 h of
exposure (ig. 2d), the bacterial cells were seemed to
be totally deformed and many collapsed cells were
seen. At this stage, the cells had lost their original
rod shape as compared to the control cells in ig. 2a.
These conditions can be seen clearly in ig. 3 where
TEM studies were conducted in the extract treated
cells. Fig. 3a shows the untreated cells, with typical
rod shaped cells of Bacillus sp. Fig. 3b shows the
cells after 12 h exposure to the extract. The cells
showed some alteration in the internal structures
with shrunken cytoplasm and the cells seems to start
Fig. 1: Effects on the growth of Bacillus cereus at different extract
concentration.
Effects of Wedelia chinensis leaves methanol extract on the growth of
Bacillus cereus at different concentration of the extract. The control
suspension ( ), 1/2 MIC ( ), MIC ( ), 2MIC (
).
536
losing its rod shaped structure. The worst condition
occurred on the cells after 24 h of exposure to
the extract (fig. 3c) and finally the cells leakage
occurred (fig. 3d) where the internal structures of
cells including cytoplasm and organelles were found
outside the cells. There were some holes formed on
the cell wall which reflect of the cell leakages. The
sequences exhibited in ig. 3 acted as proves of what
happening to the cells in ig. 2, which caused by the
effect of W. chinensis leaves methanol extract.
Hyde et al. [25], suggested that the morphological
changes of the antibiotic‑treated bacteria occur when
the antimicrobial agent attacked the cell membrane.
In this case, the bioactive compound of the methanol
extract of W. chinensis leaves that locked on the
cell surface structure had permeabilized the bacterial
membranes. Any disruption in cell wall integrity
will have a great influence in bacterial growth. This
prediction was coincided well with the findings
of Sasidharan et al.[26], who reported the methanol
extract of macroalgae Gracilaria changii exerted
its inhibitory effect on the cell wall of the bacterial
cells which led to the complete damage of the
cells. Various studies were reported to investigate
the mechanism of actions involved in bacterial
killing process. Among them are the interactions of
antibacterial compound with the cell membrane[27]. As
shown by the SEM and TEM micrographs where the
cells became crumpled and exhibited the formation of
holes, these damages may indicate the lost of cellular
materials and organelles from the cell cytoplasm[28].
a
b
c
d
Fig. 2: SEM micrographs.
SEM micrographs of the Bacillus cereus cells after treatment with
the methanol extract of Wedelia chinensis. (a) control at 0 h (×5000),
(b) at 12 h (×6000), (c) at 24 h (×6000) and (d) at 36 h of treatment
times (×6000).
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a
c
b
d
Fig. 3: TEM micrographs.
TEM micrographs of the Bacillus cereus cells after treatment with
the methanol extract of Wedelia chinensis. (a) control at 0 h, (b) at 12
h, (c) at 24 h and (d) at 36 h of treatment times.
These unstable and altered cells were collapsed
beyond repair and inally led to cell death.
The ability of the plant bioactive compounds to
cause disintegration of bacterial colonies, probably
results from their interference with the bacterial cell
wall[29]. Majority of plant extracts have been reported
to be more active against Gram‑positive bacteria than
the Gram‑negative bacterial strains[30]. Theoretically,
the Gram‑negative bacteria bear an extra outer
membrane (OM) which includes the asymmetric
distribution of the lipids with phospholipids and
lipopolysaccharide (LPS) located in the inner and
outer leaflets, respectively can act as additional barrier
which hinders the movement of foreign substance into
the cell[31]. This characteristic is absent in the Gram‑
positive bacteria and the cell wall of Gram‑positive
bacteria contains lipotheichoic acids (LTA) that
represent unique and essential structural components
to the cells and should be good drug targets to the
bioactive compounds of W. chinensis.
The results obtained from this study proved that
W. chinensis leaves methanol extract can be used to
treat bacterial infections topically as it acted directly
to the target bacteria cell wall. In order to assure the
right time of using the extract against the bacteria, we
conducted a study on the effect of addition the extract
to the bacterial growth proile. Fig. 4 shows that the
control cells grew well and achieved its logarithmic
proile at 8 h of cultivation time and then it entered
the initial stationary phase at 12 h of cultivation time,
followed by a stationary phase from 20 h onward.
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Fig. 4: Effects on the growth of Bacillus cereus at MIC value.
Effect of addition the methanol extract of Wedelia chinensis leaves
(at MIC value of 3.13 mg/ml) on the growth proile of Bacillus cereus.
Arrows indicated the time (h) of extract addition to the bacterial
culture. Control (
), 8 h ( ), 16 h ( ), 24 h (
).
Therefore, the additions of the extract were done at 8,
16 and 24 h of cultivation time in response to those
phases. The results revealed that the growth of the
treated cells decreased once the extract was added
without having any exception on the time of addition.
This condition is good in the sense that the extract
can be used at any time of the bacterial growth phase
to treat its infection.
Generally, the methanol extract was more active
than other extracts [32]. This may be attributed to
the presence of soluble phenolic and polyphenolic
compounds. Even though there were reports that
methanol extract demonstrated inhibitory effects
to B. subtilis, P. aeruginosa and S. aureus but not
E. coli[33], yet recent research activities on antibacterial
activities of crude extracts have implicated the
methanol extract for being more active than the
other solvents extracts [34,35] . In this study three
Gram positive (B. cereus, B. subtilis and S. aureus)
and three Gram negative (E. coli, P. rettgeri and
P. aeruginosa) were used as test microorganisms.
All these are pathogenic bacteria that are known to
cause several diseases and infections in humans and
animals. For instance, S. aureus and P. aeruginosa are
most common pathogens causing serious infections
while E. coli is an opportunistic pathogen at the site
of cut wound.
The MIC values of the extract against all the test
bacteria were determined using broth dilution
methods and the results showed that MIC values
for Gram‑positive bacteria were between 3.12 to
6.25 mg/ml which were more susceptible than
Gram‑negative bacteria which exhibited the MIC
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537
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values of 25.00 mg/ml. The broader spectrum of
activities could be due to the synergic effects of
the various components in the W. chinensis leaves
extract. The exact antibacterial mechanism of the
extract is not known, but it can be attributed to
the presence of the major phytochemicals such as
flavonoids, terbinoids and tannins that were detected
in the studies that were reported previously by several
researchers[6,36,37].
Based on these results, it can be concluded that
W. chinensis leaves methanol extracts have a great
potential as antibacterial agent to treat infectious
diseases caused by a range of pathogenic bacteria.
The study provides support for the use of these
plants in the management of infectious diseases.
The findings can form the basis for further studies
to prepare and optimise preparation of the herbal
extract to further evaluate them against a wide range
of bacterial strains.
12.
13.
14.
15.
16.
17.
18.
19.
20.
ACKNOWLEDGEMENTS
21.
This work was supported by a research grant provided by
Universiti Sains Malaysia.
22.
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September - October 2013
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Indian Journal of Pharmaceutical Sciences
Accepted 28 June 2013
Revised 17 June 2013
Received 19 February 2013
Indian J Pharm Sci 2013;75(5):533-539
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