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Articles
Manjakani Research
The potential of aqueous and acetone extracts of galls of
Quercus infectoria as antibacterial agents
OBJECTIVE:
To evaluate the antibacterial potential of aqueous and acetone
extracts of galls of Quercus infectoria by determination of
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) values.
MATERIALS
AND METHODS: The extracts from the galls of
Q. infectoria at 10 mg/ml were screened against three Gram-positive
bacteria ( Staphylococcus aureus ATCC 25923, Staphylococcus
epidermidis and Bacillus subtilis ) and three Gram-negative
bacteria ( Escherichia coli NCTC 12079 serotype O157:H7, Salmonella
typhimurium NCTC 74 and Pseudomonas aeruginosa ATCC 27853).
The MIC of the extracts were then determined using the twofold
serial microdilution technique at a concentration ranging
from 5 mg/ml to 0.0024 mg/ml. The MBC values were finally
obtained from the MIC microtiter wells which showed no turbidity
after 24 hrs of incubation by subculturing method.
RESULTS:
Out of the six bacterial species tested, S. aureus was the
most susceptible. On the other hand, the extracts showed weak
inhibitory effect against S. epidermidis, B. subtilis , S.
typhimurium and P. aeruginosa while there was no inhibition
zone observed for E. coli O157. The MIC values of the extracts
ranged from 0.0781 mg/ml to 1.25 mg/ml whereas the MBC values
ranged from 0.3125 mg/ml to 2.50 mg/ml. The MBC values of
aqueous extract against S. aureus and S. typhimurium were
higher than their MIC values. The MBC value of acetone extract
against S. aureus was also higher than its MIC value. Interestingly,
however, the MIC and MBC values of acetone extract against
S. typhimurium were the same (1.25 mg/ml).
CONCLUSION:
The aqueous and acetone extracts displayed similarities in
their antimicrobial activity on the bacterial species and
as such, the galls of Quercus infectoria are potentially good
source of antimicrobial agents.
Introduction
Quercus infectoria
Olivier (Fagaceae) is a small tree native of Greece , Asia
Minor and Iran . The galls arise on young branches of this
tree as a result of attack by the gall-wasp Adleria gallae-tinctoria
.[1] The galls are locally known as manjakani
in Malaysia, and are used in combination with other herbs
as drinking remedy by women after childbirth to restore the
elasticity of the uterine wall.[2] Majuphal, as it is widely
known in Indian traditional medicine have been used as dental
powder and in the treatment of toothache and gingivitis.[3],[4]
The galls of Q. infectoria have also been pharmacologically
documented to possess astringent, antidiabetic,[5] antitremorine,
local anaesthetic,[6] antiviral,[7] antibacterial,[8] antifungal,[9]
larvicidal[10] and anti-inflammatory[11] activities. The main
constituents found in the galls of Q. infectoria are tannin
(50-70%) and small amount of free gallic acid and ellagic
acid.[12],[13],[14]
As a result of indiscrimate
use of antimicrobial drugs in the treatment of infectious
diseases, microorganisms have developed resistance to many
antibiotics.[15] There is a need to develop alternative antimicrobial
drugs. One approach is to screen local medicinal plants which
represent a rich source of novel antimicrobial agents. The
present study was carried out to investigate the antibacterial
properties of the galls of Q. infectoria extracted by two
solvents of different polarity.
Materials
and Methods
Plant
materials
The galls of Q. infectoria
used in this study were obtained from the local market and
were identified based on its physical characteristics. The
galls were crushed to small pieces using pestle and mortar
and powdered in an electric grinder.
Preparation
of acetone extract
The acetone extract
was prepared by immersing 100g of the dried material in 500
ml acetone for 24 h at room temperature. The mixture was then
filtered and the process was repeated using the remaining
residue with 300 ml acetone. The two filtrates were added
and concentrated under reduced pressure using a rotary evaporator.
The resulting pellet was finally pounded to dryness under
hot air-dryer to produce a powdery crude acetone extract.
Aqueous
extraction
In the preparation
of aqueous extract, the powdered material was dissolved in
distilled water for 24 hr at 45[degrees] C and centrifuged
at 3000 rpm at 4[degrees] C. The supernatant was then filtered
and the whole process repeated using the remaining residue
with 300 ml distilled water. The filtrates were combined and
freeze-dried at -50[degrees] C under vacuum for 12 h to produce
a fine crystal-like crude aqueous extract. The extracts were
stored in air-tight jars at 4[degrees] C until further use.
Preparation
of extract solution
The extracts were dissolved
in sterile distilled water to a final concentration of 10
mg/ml for disc diffusion assay and a 5 mg/ml concentration
for broth microdilution technique. The choice of dose concentration
was based on our previous finding[16] in which a crude antifungal
extract isolated from sea cucumber species was capable of
inhibiting the growth of filamentous fungi at 10 mg/ml. All
the extracts were sterilized by passing through a 0.45 [micro]m
membrane filter.
Microorganisms
The bacterial strains
used in this study were Staphylococcus aureus ATCC 25923,
Pseudomonas aeruginosa ATCC 27853, Escherichia coli NCTC 12079
(serotype O157:H7), Salmonella typhimurium NCTC 74 and local
clinical isolates of Staphylococcus epidermidis and Bacillus
subtilis. All the bacterial strains were grown and maintained
on nutrient agar slants. The inoculum size of each test strain
was 108 bacteria/ml for disc diffusion assay which was standardized
by adjusting the optical density of the bacterial suspension
to a turbidity corresponding to spectrophotometric absorbance
= 0.08 (OD620 = 0.08) at 620 nm.
Screening
for antibacterial activity
The disc diffusion
method[17] was used to evaluate the antibacterial activity.
Mueller Hinton agar was prepared in the plates as the media
for the test microorganisms. Sterile filter paper discs (Whatman
No. 1, 6 mm) were impregnated with 100 [micro]l of each of
the extracts (10 mg/ml) to give a final concentration of 1
mg/disc and left to dry under the laminar flow cabinet overnight.
The bacterial inoculum was spread evenly onto the surface
of the Mueller Hinton agar plates using a sterile cotton bud
before the extract discs were positioned on the inoculated
agar surface. Each extract was assayed in triplicate. Sterile
distilled water served as negative control. Gentamicin (10
[micro]g/disc) was used as standard to confirm that all the
microorganisms tested were inhibited by the antibiotic. All
the plates were incubated for 24 hr at 37[degrees] C. The
antibacterial activity was interpreted from the size of the
diameter of zone inhibition measured to the nearest millimeter
(mm) as observed from the clear zones surrounding the discs.
Determination
of MIC and MBC values
The minimum inhibitory
concentration (MIC) of the extracts was determined for S.
aureus and S. typhimurium using the twofold serial microdilution
method with saline at a final concentration ranging from 5
mg/ml to 0.0024 mg/ml. The tested extracts were added to sterile
Mueller Hinton broth into microtiter plates before the diluted
bacterial suspension (final inoculum of 105 bacteria/ml) were
added. Each extract was assayed in triplicate. The bacterial
suspensions were used as positive control and extracts in
broth were used as negative control. The MIC values were taken
as the lowest concentration of the extracts in the wells of
the microtiter plate that showed no turbidity after 24 hours
of incubation at 37[degrees] C. The turbidity of the wells
in the microtiter plate were interpreted as visible growth
of the microorganisms. The minimum bactericidal concentration
(MBC) was determined by subculture of the well showing no
apparent growth in a sterile agar plate. The least concentration
showing no visible growth on agar subculture was taken as
MBC value.
Results
Determination
of MIC and MBC
The MIC values of the
aqueous and acetone extracts from the galls of Q. infectoria
against S. aureus and S. typhimurium are shown in [Table:1].
The MIC values of the aqueous and acetone extracts were the
same (0.0781 mg/ml) against S. aureus , whereas the MIC values
of aqueous and acetone extracts against S. typhimurium were
0.6250 mg/ml and 1.25 mg/ml, respectively. The lower MIC values
of both the extracts against S. aureus in comparison to S.
typhimurium suggests that S. aureus showed greater sensitivity
towards the extracts of the Q. infectoria galls.
[Table:2] shows the
result of MBC of the aqueous and acetone extracts from the
galls of Q. infectoria against S. aureus and S. typhimurium
. The MBC value of the aqueous extract against S. aureus was
higher (0.3125 mg/ml) compared to its MIC value of 0.0781
mg/ml. This was also observed for S. typhimurium in which
the MBC values of the aqueous extract was also higher (2.5
mg/ml) compared to its MIC value ie. 0.6250 mg/ml. As for
the acetone extract against S. aureus , although its MBC value
was also higher (0.1563 mg/ml) than its MIC value of 0.0781
mg/ml, it is interesting to note that the MIC and MBC values
of the acetone extracts against S. typhimurium were the same
(1.250 mg/ml). This means that the acetone extracts of the
galls of Q. infectoria , may be considered bactericidal for
S. typhimurium. As for S. aureus , both the aqueous and acetone
extracts were bacteriostatic agents. The aqueous extracts,
however, displayed consistent bacteriostatic activity against
both bacterial species.
Discussion
Our study showed that
the extracts from the galls inhibited the Gram-positive bacteria
better than Gram-negative. Generally, plant extracts are usually
more active against Gram-positive bacteria than Gram-negative
bacteria.[18] Our findings were also supported by other researchers
who reported that the crude powder of the galls of Q. infectoria
was found to be active against S. aureus and B. subtilis while
both the methanol and aqueous extracts were active against
S. epidermidis .[8] In the present study, S. aureus , S. epidermidis
and B. subtilis were also inhibited by the acetone extract.
In addition to these bacterial strains, P. aeruginosa was
also found to be susceptible to both the extracts tested.
This is in accordance with a study[19[ that one of the most
susceptible bacteria to the effect of the ethanol extract
from the galls of Q. infectoria was P. aeruginosa.
The range of MIC values
for both S. aureus and S. typhimurium correlated well with
the results obtained using the disc diffusion method. The
MIC values for both extracts against S. aureus are lower when
compared with Gram-negative bacteria. This shows that the
Gram-positive bacteria is more susceptible to the effect of
the extracts from the galls of Q. infectoria with respect
to its Gram-negative counterpart.
The MBC values were
higher than the MIC values of the extracts against both the
bacteria tested except for acetone extract from the galls
of Q. infectoria . The MIC and MBC values of acetone extract
were the same against S. typhimurium while the MBC values
of both the extracts were 2-4 times greater than their MIC
values for S. aureus . This suggests that the bioactive compound
in the extracts of the galls of Q. infectoria , was bacteriostatic
against S. aureus rather than bactericidal as reported previously.[20]
It is well known that
tannin is a phenolic compound which is soluble in water, alcohol
and acetone, and gives precipitates with protein.[21] The
similarity in the antimicrobial activity of both the aqueous
and acetone extracts suggest that these extracts may have
high total tannin content. The antimicrobial activity seemed
to depend on the contents of tannin in the plant extracts.[22]
High amounts of tannin
present in the galls of Q. infectoria [12],[13],[14] implied
that tannin may be the active compound which may be responsible
for the antibacterial activity in this study. Tannin in plant
extracts was found to possess antibacterial activity.[23]
In conclusion, the
extracts of the galls of Q. infectoria have high potential
as antibacterial agent. This finding provides an insight into
the usage of the galls of Q. infectoria in traditional treatment
of wounds or burns associated with bacterial infections.
Byline: Dayang. Basri, S. Fan
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