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222
Document heading
doi: 10.1016/S2221-6189(13)60131-6
Antimicrobial activity of
Gymnema sylvestre
(Asclepiadaceae)
Beverly C. David
*
, G. Sudarsanam
Department of Botany, S.V. University, Tirupati-517502, A.P., India
ARTICLE INFO ABSTRACT
Article history:
Received 14 March 2013
Received in revised form 19 March 2013
Accepted 25 March 2013
Available online 20 June 2013
Keywords:
Antibacterial
Antifungal
Gymnema sylvestre
*Corresponding author: Mrs. Beverly C David, Research Scholar, Department of
Botany, S.V. University, Tirupati-517502, A.P., India.
E-mail: beverlydavid2011@gmail.com
1. Introduction
Plants have been used for the treatment of various diseases
all over the world before the advent of modern clinical
drugs and are known to contain substances that can be used
for therapeutic purposes or as precursors for the synthesis of
useful drugs
[1]
. Thus over 50% of these modern drugs are of
natural products origin and as such play an important role
in drug development in the pharmaceuti
cal industry
[2]
.
Infectious diseases are the number one cause of death
world-wide, and in tropical countries it accounts for
approximately
50% of deaths. This may be due to poverty
and increasing incidence of multiple drug resistance.
Bacterial resistance to almost all antibacterial agents
has been reported
[3]
. This resistance is largely due to
indiscriminate use of antimicrobial drugs commonly used
in treatment of infectious diseases.
Apart from resistance,
some antibiotics have serious undesirable side effects
which limit their applications, so there is urgent need to
develop new antimicrobial agents that are very effective
with minimal side effects, and represent a potential source
of novel antibiotic prototypes
[4]
.
Gymnema sylvestre
(G. sylvestre) (Asclepiadaceae) is a
large tropical liane native to central and weste
rn India and
can be also found in tropical
Africa and in Australia
[5]
.
G. sylvestre is one of the important anti-diabetic medicinal
plants.
There is a growing demand for G. sylvestre leaves
in pharmaceutical trade.
Gymnemic acid, the active
ingredient of this plant, is extracted from leaves and
used widely as an anti-diabetic
[6]
, anti-sweetner
[7]
and
antihypercholesterolemia
[8]
. It also has stomachic, diuretic
and cough suppre
ssant properties
[9]
. The plant has been
reported to possess antimicrobial
[10]
and ethnoveterinary
medicinal properties
[11]
.
In addition, it possesses hepatoprotective, and anti-
saccharine activities
[12]
. Hence, because of these properties,
G. sylvestre is most important for plant prospecting.
In the
present study, the selection of this plant for evaluation
was based on its traditional usages.
Although very limited
w
ork has been done on the antimicrobial activity of
this endangered medicinal plant, it needs further study
for verification of its activity against disease-causing
microorganisms.
Objective:
To evaluate antimicrobial activities of aqueous, methanol, chloroform and hexane
extract of leaves plant of Gymnema sylvestre
(G. sylvestre).
Methods:
The antimicrobial screening
of the extracts of G. sylvestre against most prevalent microbes like Staphylococcus aureus
(S.
aureus
), Bacillus cereus (B. cereus), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E.
coli
), Candida albicans (C. albicans), Candida tropicalis (C. tropicalis), Candida krusei (C. krusei)
and Candida kefyr
(C. kefyr) by agar well diffusion method, minimum inhibitory concentration,
minimum bactericidal concentration, minimum fungicidal concentration w
ere carried out.
Results:
The aqueous and methanol leaf extract showed significant antibacterial and antifungal
activities against the selected microorganisms when compared to the standard drugs respectively.
Conclusions:
The dried scale leaves of G. sylvestre might represent a new antimicrobial source
with stable, biologically active components that can establish a scientific base for the use in
modern medicine.
Journal of Acute Disease (2013)222-225
Contents lists available at ScienceDirect
Journal of Acute Disease
journal homepage: www.jadweb.org
223
Beverly C. David et al./ Journal of Acute Disease (2013)222-225
2. Materials and methods
2.1. Plant material
The G. sylvestre were leaves collected during June-July of
2012 in and around Vellore, Tamilnadu and authenticated
by
Department of Botany. The voucher specimens were kept
in the
Department of Botany in Voorhees College Vellore,
Tamilnadu, India.
2.2. Extraction procedure
Shade dried leaves (200 g) were coarsely powdered and
subjected to successive solvent extraction by continuous hot
extraction
(soxhlet). The extraction was done with different
solvents in their increasing order of polarity such as
Aqueous, methanol, chloroform and Hexane. Each time the
marc was air dried and later extracted with other solvents.
All the extracts were concentrated by distilling the solvent
in a rotary flash evaporator.
The extracts were preserved in
airtight containers and kept at
4-5 曟 until further use.
2.3. Test organisms
The microorganisms used for the test were Staphylococcus
aureus
(S. aureus), Bacillus cereus (B. cereus), Escherichia coli
(E. coli) and Klebsiella pneumoniae (K. pneumoniae), Candida
albicans
(C. albicans), Candida tropicalis
(
C. tropicalis
)
,
Candida krusei
(
C. krusei
)
and Candida kefyr
(C. kefyr). All the
stock cultures used in the study were obtained from
Institute
of
Microbial Technology (IMTECH), Chandigarh, India.
2.4. Culture media and inoculums preparation
Nutrient agar/Potato Dextrose Agar (Himedia, India.) was
used as the media for the culturing of strains.
Loops full of
all the microbial cultures were inoculated in the nutrient
broth
(NA) at 37 曟 for 72 h.
2.5. Antimicrobial activity study
Antimicrobial activity of the G. sylvestre leaves
extracts viz. aqueous, methanol, chloroform and
Hexane
were determined, using the agar well diffusion assay
method
[13]
. Approximately 20 mL of molten and cooled
media was poured in sterilized petri dishes.
The plates
were left overnight at room temperature to check for any
contamination to appear.
The test organisms were grown in broth for 24 h. A 100
m
L broth culture of each test organism (1伊10
5
cfu/mL) was
used to prepare lawns.
Agar wells of 5 mm diameter were
prepared with the help of a sterilized stainless steel cork
borer.
Five wells were prepared in the agar plates. The wells
were labeled as
A, B, C, D and E.
‘
A’ well was loaded with 10
毺L of aqueous leaves extracts,
‘
B’ well was loaded with 10
毺L of methanol leaves extracts,
‘
C’ well was loaded with 10
毺L of chloroform leaves extracts,
‘
D’ well was loaded with
hexane leaves extracts and
‘
E’ well was loaded with positive
control drugs.
Various bactericides/fungicides (Table 1) were used as
positive controls.
The plates containing the organisms and
leaves extracts were incubated at
37
曟
. The plates were
examined for evidence of zones of inhibition, which appear
as a c
lear area around the wells
[14]
. The diameter of such
zones of inhibition was measured using a meter ruler and the
mean value for each organism was recorded and expressed
in millimeter.
Table 1
Antimicrobial activity of different extracts of leaves of G. sylvestre
against test organisms.
Microorganisms
Zone of inhibition in mm (mg/mL)
AE ME CE HE Reference drug
S. aureus 7 - - 7 18
E. coli - 15 - - 21
B. cereus - 8 - 9 20
K. pneumoniae - - - - 17
C. albicans - 12 - - 19
C. tropicalis - - - - 20
C. kefir 8 - - - 16
C. krusei 10 11 - - 23
AE-Aqueous extract, ME-Methanol extract, CE-Chloroform extract,
HE-Hexane extract.
2.6. Determination of minimum inhibitory concentration
(MIC), minimum bactericidal concentration (MBC) and
minimum fungicidal concentration (MFC)
Antimicrobial activity was measured using a dilution
technique
[15]
. The plant extract (100 mg) was solubilized
in
1 mL of dimethyl sulfoxide (DMSO) and serially two
fold diluted
Nutrient broth (Himedia, India) to obtain a
concentration range of
12.5-100.0 mg/mL.
Nutrient broths containing only DMSO diluted in the
same way, which did not influence microbial growth, were
included as controls.
The test strains were suspended
in sterile physiological
Tris buffer (pH 7.4, 0.05 M),
homogenized and adjusted to an optical density of
0.05 at
530 nm (equivalent to 1伊10
6
CFU/mL).
This suspension was used asthe inoculums for the test
in the agar plates.
Culture suspensions (100 毺L) were
inoculated using a microp
ipette.
The minimal inhibitory concentration (MIC) was defined
224
Beverly C. David et al./ Journal of Acute Disease (2013)222-225
as the minimal concentration of the plant extract which
completely inhibited the visible growth
(turbidity) of the
bacteria in tubes.
The minimal bactericidal concentration (MBC), minimal
fungicidal concentration
(MFC) was defined as the minimal
concentration of the extract which completely inhibited the
visible growth of the test strains on solid media in petri
dishes that were inc
ubated at 37
曟
for 72 h.
3. Results
In the present study the antimicrobial activity of leaf
extracts viz. aqueous, methanol, chloroform and hexane were
evaluated against eight test spp.
(Table 1). In the first stage the
leaf extracts of G. sylvestre, i.e. aqueous, methanol, chloroform
and hexane applied on isolates of each test strains.
Methanol
leaf extract of G. sylvestre showed significant antimicrobial
activity against E. coli, B. cereus,
C. albicans and C. kefyr. The
aqueous leaves extract recorded an intermediate antimicrobial
activity against S. aureus, C. krusei and C. kefyr while on the
other hand the hexane leaf extract revealed antimicrobial effect
on S. aureus, B. cereus but chloroform leaf extract did not show
significant antimicrobial effect on test strains.
The inhibitory
activities of all the four extracts of the leaf reported in the
present study were compared with standard
antimicrobics,
Chlorompenicol, Ketoconazole and Itraconazole.
The results on MIC studies of leaf extract (Methanol), against 8
isolates of test organisms recorded lowest
(50 mg/mL) for E. coli
and C. krusei when compared with that of other
species viz. S.
aureus
(75
mg/mL
)
, B. cereus
(75
mg/mL
)
and K. pneumoniae
(75
mg/mL
)
whereas the MIC for C. albicans,
C. tropicalis and
C. kefyr were recorded as
100 mg/mL. Further the MIC studies of
aqueous leaf extract agains
t test organisms recorded lowest for
C. krusei
(50 mg/mL) when compared with that of other species
viz S. aureus
(75 mg/mL) and C. kefyir (50 mg/mL). The leaf
extract
(hexane) MIC was recorded for B. cereus and S. aureus as
50 mg/mL; 75 mg/mL respectively (Tables 2 & 3).
Table 2
Determination of minimum inhibitory concentration (MIC) for
G. sylvestre.
Microorganisms
MIC (mg/mL)
AE ME CE HE Reference drug
S. aureus 75 75 >100 75 12.5
E. coli >100 50 >100 >100 12.5
B. cereus >100 75 >100 50 12.5
K. pneumoniae >100 75 >100 >100 12.5
C. albicans >100 >100 >100 >100 12.5
C. tropicalis >100 >100 >100 >100 12.5
C. kefir 75 >100 >100 >100 12.5
C. krusei 50 50 >100 >100 12.5
AE-Aqueous extract, ME-Methanol extract, CE-Chloroform extract,
HE-Hexane extract.
Table 3
Determination of minimum bactericidal concentration (MBC) and
minimum fungicidal concentration (MFC) for G. sylvestre.
Microorganisms MBC/MFC (mg/mL)
AE ME CE HE Reference drug
S. aureus 75 75 >100 75 12.5
E. coli >100 50 >100 >100 12.5
B. cereus >100 75 >100 50 12.5
K. pneumoniae >100 75 >100 >100 12.5
C. albicans >100 >100 50 >100 12.5
C. tropicalis >100 >100 >100 >100 12.5
C. kefir 75 >100 >100 >100 12.5
C. krusei 50 50 >100 >100 12.5
4. Discussion
The therapeutic value of medicinal plants lies in
the various chemical constituents’ presents in them.
The bioactivity of plant extracts was attributed to
phytochemical constituents.
For instance, plant rich in
tannins have antibacterial potential due to their basic
character that allows them to react with proteins to
form stable water so
luble compounds thereby killing
the bacteria by directly damaging its cell membrane
[16]
.
Flavonoids are a major group of phenolic compounds
reported for their antiviral
[17]
, antimicrobial
[18]
and
spasmolytic properties
[19]
. Alkaloids isolated from
plant are commonly found to have antimicrobial
properties
[20]
. Extract of the seeds of Vitex agnus-castus
(Family:Lamiaceae) was reported to possess antimicrobial
activity which was as
sociated with its alkaloids, saponins,
tannins, flavonoids, and glycosides contents
[21]
.
The antimicrobial activity of the leaf extracts of G.
sylvestre as recorded in present study might therefore be
attributed to the presence of above phytochemicals
i.e.
flavonoids, terpenoids, amino acids, glycosides, tannins,
amino acids and carbohydrates.
From the above study, it is concluded that the dried
scale leave
s of G. sylvestre might represent a new
antimicrobial source with stable, biologically active
components that can establish a scientific base for the use
in modern medicine.
Further studies are needed to isolate
and characterize the bioactive principles to develop new
antimicrobial drugs from G. sylvestre.
Conflict of interest
The authors declare they have no conflict of interests.
225
Beverly C. David et al./ Journal of Acute Disease (2013)222-225
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