12 Drug Discoveries & Therapeutics. 2011; 5(1):12-17.

Brief Report DOI: 10.5582/ddt.v5.1.12

Antibacterial activities of Sesbania grandiflora extracts

Pimporn Anantaworasakul1, Srikanjana Klayraung2, Siriporn Okonogi1,*
1
Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand;
2
Faculty of Science, Maejo University, Chiang Mai, Thailand.

ABSTRACT: In this study, Sesbania grandiflora, a compounds to develop better drugs against several
plant in the Leguminosae family, was investigated degenerative diseases like cancer, diabetes, Alzheimer's,
for its antibacterial activities. The agar well diffusion arteriosclerosis as well as diseases caused by pathogenic
assay as well as the agar and broth dilution assays microorganisms (1,2).
were used for determination of antibacterial From previous studies reported by different research
activities. The crude ethanolic extracts obtained from groups, it was demonstrated that many naturally
different parts of this plant exhibited different potent occurring compounds found in plants have been shown
activities. The stem bark has the most potential to to possess biological activities such as antibacterial
yield an extract with the highest antibacterial action. (3-5), antifungal (3,6,7), antiviral (3,8), anti-allergic
The fractionation of the stem bark with different (4), anti-cholinesterase (5), antioxidant (9-11), anti-
solvents indicated that the fractionated extracts inflammatory (4,5,10), antitumor (12,13), anti-
obtained from ethyl acetate or butanol possessed the tyrosinase (14), anti-plasmodial (15), and cytotoxic
most pronounced antibacterial activity. The kinetic effects (10,15).
study of bactericidal activities revealed that the The plant genus Sesbania, family Leguminosae,
butanol fractionated extract of the stem bark was is comprised of about 60 species which are widely
effective against Gram negative bacteria. This study distributed throughout tropical and subtropical regions.
suggests that the stem bark of S. grandiflora contains Most species are annual herbs or shrubs, but a few are
promising antibacterial substances for clinical small trees (16). Many species of Sesbania are used
purposes. for soil improvement as green manners or agroforestry
trees (17). Sesbania grandiflora, syn. Aeschynomene
Keywords: Sesbania grandiflora, antibacterial activity, grandiflora, is a small erect tree that grows rapidly, is
crude extract, pathogenic bacteria, killing kinetics a sparsely branched tree that provides light shade, and
is often grown as an ornamental (18). S. grandiflora is
widely used in Thailand both for food and medicine.
Its leaves and flowers are utilized as food whereas its
1. Introduction stem has been long used as a traditional medicine for
treatment of ulcers in the oral cavity. It was reported
In Thailand and other Asian countries like China, that all parts of S. grandiflora are utilized for medicine
Japan, and India, medicinal plants are widely used by in Southeastern Asia and India including preparations
all sections of the population either directly as folk derived from the roots, bark, gum, leaves, flowers and
remedies or in different indigenous systems of medicine fruits. In the Philippines, the stem bark of this plant
or indirectly in the pharmaceutical preparations of has been used for the treatment of thrush and infantile
modern medicines. The world health organization disorders of the stomach. In Cambodia, the pounded
recently accepted an inventory of more than 20,000 bark is applied to scabies. The juice of the leaves
species of medicinal plants. Thai medicinal plants and is considered anthelmintic and tonic and is used to
their products are used to control diverse diseases and treat worms, biliousness, fever, gout, itchiness, and
disorder symptoms such as cough, fever, bronchitis, leprosy (19). It was also reported that the root of this
itching, pneumonia, ulcers, and diarrhea. Researchers plant could be applied externally as a poultice against
are hence increasingly turning their attention to inflammation. The semisolid mass containing powdered
medicinal plants looking for new bioactive or lead roots of S. grandiflora in an appropriate amount of
water demonstrated a decrease of rheumatic swelling
*Address correspondence to: when applied externally with moderate rubbing to the
Dr. Siriporn Okonogi, Faculty of Pharmacy, Chiang lesion (20). Although S. grandiflora has been used
Mai University, Chiang Mai 50200, Thailand. for treatment in many diseases, most of the biological
e-mail: [email protected] activities have not yet been adequately evaluated.

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 Drug Discoveries & Therapeutics. 2011; 5(1):12-17. 13

The present study deals with the investigation of clinical pathogenic bacterial strains of Bacillus
antibacterial activity of S. grandiflora. Several parts of cereus, Enterobacter cloacae, Enterococcus faecalis,
the whole plant such as leaves, branches, stem barks, Pseudomonas aeruginosa, Serratia marcescens, and
and stem cores were screened for their potency on Staphylococcus epidermidis obtained from the Faculty
growth inhibition of bacteria. The pathogenic organisms of Medicine, Chiang Mai University, Thailand were
were selected for the study on the basis of their clinical used throughout the study. The bacteria were sub-
and pharmaceutical importance as well as their potential cultured individually on fresh Tryptic Soy Agar (TSA)
to cause contamination of food and drugs. The high and incubated overnight at 37C. Each bacterial
potent activity extracts of the most potential parts of strain was then suspended in 0.9% NaCl, and diluted
the plant were further investigated deeply on bacterial to a McFarland turbidity standard No. 0.5 (21). This
killing kinetics. bacterial suspension was used for testing antibacterial
activity.
2. Materials and Methods
2.4. Determination of antibacterial activities by agar
2.1. Plant materials diffusion

S. grandiflora samples were collected in fresh condition The bacterial inhibitory effect of S. grandiflora extracts
from local areas of Northern Thailand. The plant was was carried out by standard agar well diffusion method
identified and deposited in the Herbarium of the Faculty (21). The TSA plate was seeded with the NS suspension
of Pharmacy, Chiang Mai, Thailand. of different bacteria. In each plate, a 6 mm diameter
well was cut out using a sterile cork borer. Using a
2.2. Preparation of plant extracts sterile autopipette, 50 L of the proper diluted extract
was carefully added into the wells. The same volume of
2.2.1. The crude extracts 50% ethanol was used as a negative control. The plates
were incubated overnight at 37C. The antibacterial
Fresh material leaves, branches, stem bark, and stem activity was evaluated by measuring the diameter of
core were collected and washed with distilled water. inhibition zone (DIZ). The experiment was carried out
Samples were cut into small pieces and dried at 50C for in triplicate and the means of diameter of the inhibition
24 h. The dried samples were pulverized into powder, zone were calculated.
and then macerated with 95% ethanol for 48 h 3 at
room temperature. The filtrates from the same part were 2.5. Determination of minimum inhibitory concentration
pooled together and subjected to the rotary evaporator (MIC)
at 45C and under vacuum for solvent removal. The
crude extracts obtained were investigated further for 2.5.1. Agar dilution method
antibacterial activity.
The MIC of fractionated extract of the stem bark
2.2.2. Fractionation of the extracts was determined against the test pathogenic bacteria
by a standard agar dilution method (22). Briefly, to
The dried powder of stem bark was used in this study the tubes containing 9 mL melted Mueller-Hinton
because its crude ethanolic extract showed the highest Agar (MHA) media, serial dilutions of the extract
antibacterial activity. The plant powder was first were added to 10 mL and poured into a sterile plate.
macerated in hexane for 48 h 3 at room temperature. Sample concentrations in the agar plates were in the
The residue after the third filtration was dried at room range of 0.63-20.00 mg/mL. Equivalent amounts of
temperature to ensure hexane was completely removed. sterile distilled water and sterile 50% ethanol were
The dried residue was further macerated respectively used as controls. Ten L of each bacteria suspension
in three solvents as follows; ethyl acetate, butanol and with turbidity equivalent to McFarland No. 0.5 was
methanol, in the same manner as hexane. The filtrates inoculated into each agar dilution plate. The inoculated
of the same solvent were pooled together. The solvent plates were incubated overnight at 37C. The MIC
was removed using a rotary evaporator at 45C under was defined as the lowest concentration of the samples
vacuum. The fractionated extracts obtained were which prevented this change and exhibited complete
subjected to antibacterial activity tests. inhibition of bacterial growth (23).

2.3. Bacterial strains 2.5.2. Broth dilution method

Standard strains of Escherichia coli ATCC 25922, The MIC was also determined by using a broth dilution
Staphylococcus aureus ATCC 25923, Salmonella method (24). Briefly, the extract was first dissolved in
typhi DMST 5784, Shigella sonnei DMST 561 and sterile 50% ethanol. The sample obtained was added

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14 Drug Discoveries & Therapeutics. 2011; 5(1):12-17.

to Mueller-Hinton Broth (MHB). This extract solution limit of quantification. In these few cases, the colonies
was serially diluted two-fold with MHB to obtain were counted and the resulting CFU/mL was used to
the extract concentration in a range of 0.63-10.00 provide an estimate of the number of viable cells. It
mg/mL. Each bacterial suspension obtained from a is noted in the appropriate figure legends when these
McFarland turbidity standard No. 0.5 was inoculated estimates were used. Results of killing kinetic studies
into the broth dilution tube. The tubes were incubated are expressed as the difference between the log CFU/
overnight at 37C. Negative controls were prepared mL at the indicated time point and the log CFU/mL of
with un-inoculated bacterial suspension and the positive the inoculums at time zero. Bactericidal activity was
controls contained inoculated bacterial suspension defined as decreasing the original inoculums after 24 h
without the test samples. After incubation overnight at of incubation (30).
37C, the micro-dilution tubes were checked visually
to detect growth inhibition of the bacteria. The growth 3. Results and Discussion
end points were determined by comparing the amount
of growth in the tubes containing the test samples with The crude extracts of 4 parts of S. grandiflora showed
that in the negative control. Acceptable growth must different yields. The leaf extract gave the highest yield
occur in the positive control. The MIC was defined as of 23.3% (w/w) followed by the stem bark with a yield
the lowest concentration of the samples which is able to of 13.3% (w/w). All of them were first screened for
inhibit any visible bacterial growth (25). their antibacterial activity against E. coli and S. aureus
as representations for Gram negative and Gram positive
2.6. Determination of minimum bactericidal concentration bacteria, respectively. Gentamicin at a concentration of
(MBC) 75 g/mL was used as a positive control. It was found
as shown in Table 1 that the stem bark extract showed
To determine MBC, a method of MIC determination the strongest inhibition against both pathogens with
was slightly modified. Briefly, the samples were taken a DIZ of 9.4 and 13.7 mm for E. coli and S. aureus
from the tube of the MIC assays, where no visible respectively whereas the leaf extracts gave no inhibition
turbidity (growth) was observed, and streaked on zone.
freshly prepared TSA plates. The plates were incubated MIC of the fractionated extracts of the stem bark
overnight at 37C so as to determine the MBC (26). was further examined against ten strains of pathogenic
The MBC was defined as the lowest concentration of bacteria. The results in Table 2 indicated that the
the samples with no bacteria growth on the surface of extract with ethyl acetate possessed the strongest
the medium in the plates (27,28). antibacterial activity followed by the butanol extract.
The MIC of ethyl acetate extract against all tested
2.7. Kinetics of bacteria killing pathogens was lower than other solvents. It was found
that the yield of ethyl acetate extract was 1.75% which
Kinetics of bacteria killing was investigated by a broth was a little lower than that of the methanol extract but
dilution method (29). Briefly, an overnight culture in higher than that of the butanol and hexane extracts.
TSA was adjusted with normal saline (0.9% NaCl) to Considering the antibacterial activity, the ethyl
a McFarland Standard No. 0.5. The adjusted culture acetate and butanol extracts were selected for further
was added to MHB with the test extract at the same investigation.
concentration of MBC and incubated for an appropriate The antibacterial activity in the ethyl acetate
time of 1.5, 3, 4.5, and 6 h, at 37C, to achieve a and the butanol extracts determined by agar dilution
logarithmic growth phase. All cultures contained method were confirmed by using the broth dilution
approximately 1.5 106 CFU/mL at the initiation of
the time course and were maintained at 37C. The
actual initial concentrations were determined by ten- Table 1. Diameter of inhibition zone of growth inhibition of
crude extract (100 mg/mL) from each part of S. grandiflora
fold serial dilution with a normal saline solution of and gentamicin (75 g/mL) against different bacterial
the inoculums and plating the serial dilutions on TSA strains using the agar well diffusion methoda
to calculate the logarithm of colony forming units per Diameter of inhibition zone (mm)b
milliliter extract (log CFU/mL). At 0, 1.5, 3, 4.5, 6, Samples
and 24 h, samples were removed and serially diluted E. coli S. aureus
with normal saline. The log CFU/mL in the culture at Leaves extract NZ c
NZ
each time point was determined by spreading on freshly Branches extract 7.8 0.3 10.7 1.0
prepared TSA plates and incubating overnight at 37C. Stem bark extract 9.4 0.1 13.7 0.6
Stem core extract NZ 8.9 0.2
As a general rule, plates containing greater than 30,
Gentamicin 16.3 0.9 23.1 2.5
but less than 300 colonies were used to quantify viable a
CFU/mL. In some instances, the resulting number of No bacterial growth in the negative control plate.
b
Data are presented as means S.D. (n = 3).
colonies was less than 30 and considered below the c
NZ represents no inhibition zone.

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 Drug Discoveries & Therapeutics. 2011; 5(1):12-17. 15

method. In this experiment, B. cereus, E. faecalis, possessed a similar killing rate against the tested Gram
S. aureus, and S. epidermidis, as representatives for positive bacteria. Both extracts showed killing activity
Gram positive bacteria and S. typhi and S. sonnei, as against E. faecalis within 30 min (Figure 1A). For S.
representatives for Gram negative bacteria were used aureus, more than 90% of cells were killed within 1.5
as test microorganisms. The results shown in Table 3 h (Figure 1B). It was found that the butanol extract had
demonstrate that the MIC values determined by broth a high ability to kill S. typhi and S. sonnei, the tested
dilution method were equal or less than those obtained Gram negative bacteria, faster than the ethyl acetate
by the agar dilution method. Moreover the results extract. The results demonstrated that both extracts
indicated that the MBC values of extracts against could kill S. typhi and S. sonnei within 1.5 h. However,
the Gram positive bacteria were equal to the MIC the butanol extract showed complete inhibiton against
values on corresponding bacteria. Regarding the Gram both strains at 6 h whereas the complete killing time
negative bacteria, it was found that the MBC values
were higher than the MIC values on corresponding
bacteria.
To study the killing kinetics effect of the highest
potential antibacterial activities, the ethyl acetate and
the butanol extracts with a concentration at MBC, were
subjected to test four pathogens including E. faecalis,
S. aureus, S. typhi, and S. sonnei. In this experiment E.
faecalis and S. aureus were used as the model of Gram
positive bacteria whereas S. typhi and S. sonnei were
used as the model of Gram negative microorganisms.
The bactericidal kinetics was examined using time
course experiments measuring the number of surviving
bacteria, Log CFU/mL. It was seen that both extracts

Table 2. The agar dilution method was used to determine

MIC of the extract with different organic solvents from stem
bark of S. grandiflora against different bacterial strainsa
MIC for test samples (mg/mL)
Microorganisms
Hexane Ethyl Butanol Methanol
acetate

Bacillus cereus > 20 2.5 2.5 2.5

Enterobacter cloacae > 20 5 20 > 20
Enterococcus faecalis 20 5 5 10
Escherichia coli > 20 5 20 > 20
Pseudomonas aeruginosa > 20 10 20 20
Salmonella typhi > 20 5 2.5 10
Serratia marcescens > 20 10 20 > 20
Shigella sonnei > 20 5 5 20
Staphylococcus aureus 10 2.5 2.5 5 Figure 1. Killing kinetic curves of Gram positive bacteria,
Staphylococcus epidermidis 20 2.5 2.5 10 E. faecalis (A) and S. aureus (B), with ethyl acetate (EtOAc)
a
and butanol (BuOH) extracts, from stem bark of S.
No bacterial growth in the negative control plate. grandiflora.

Table 3. The broth dilution method was used to determine MIC and MBC of the ethyl acetate and the butanol extracts
from stem bark of S. grandiflora against some different bacterial strainsa

MIC for test samples (mg/mL) MBC for test samples (mg/mL)
Microorganisms
Ethyl acetate Butanol Ethyl acetate Butanol

Bacillus cereus 1.25 1.25 1.25 1.25

Enterococcus faecalis 5 5 5 5
Salmonella typhi 1.25 1.25 2.5 2.5
Shigella sonnei 2.5 2.5 5 5
Staphylococcus aureus 2.5 2.5 2.5 2.5
Staphylococcus epidermidis 2.5 2.5 2.5 2.5
a
No bacterial growth in the negative control plate.

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16 Drug Discoveries & Therapeutics. 2011; 5(1):12-17.

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The authors are grateful the RGJ Grant for financial
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support and the Graduate School, Chiang Mai AC, Barroso JG, Pedro LG, Viljoen AM. Seasonal
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of Medicine, Chiang Mai University, Thailand for the biological activity of solvent extracts of three South
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