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Indian Journal of Experimental Biology
Vol. 47, March 2009, pp. 218-221
Antimicrobial activity of endemic Digitalis
lamarckii Ivan from Turkey
Mehlika Benli1*, Nazife Yiğit2, Fatmagü Geven1, Kerim Güney3,
& Ümit Bingöl1
1Ankara University, Faculty of Science,
Department of Biology, 06100, Ankara, Turkey
2Kirikkale University, Faculty of Science and Arts,
Department of Biology, Kirikkale, Turkey
3Kastamonu University, Faculty of Forestry, Kastamonu, Turkey
Received 25 April 2008
Antimicrobial activity of the methanolic extracts of leaves
and flowers of D. lamarckii Ivan, (Scophulariaceae), an endemic
plant species of Turkey, was tested on ten bacterial and four yeast
strains. Effective antibacterial activity was observed in four
bacterial strains. Minimum inhibitory concentration (MIC) was
calculated by use of liquid culture tests and in all the four
effective bacterial strains, the MIC was found to be ≥199.5
mg/ml. The minimum bactericidal concentration (MBC) of B.
subtilis, S. aureus, and L. monocytogenes was calculated to be
≥199.5 mg/ml, and MBC value for Shigella was calculated as
≥399 mg/ml.
Keywords: Antibacterial, Antimicrobial, Digitalis lamarckii
In Turkey, as well as in the entire world, many
medicinal plants are found and are used in curing
illnesses.
Turkey has a rich flora and is noted for its existing
plant diversity. Anatolia is not only the origin and
diversification centre of many genera and sections but
also has ecological and phytogeographical diversity.
As a consequence, many plant species are highly
endemic in Anatolia1,2.
Digitalis species is distributed in Europe, Western
Asia and the Mediterranean region. Today, around 40
species belonging to different genera of
Scophulariaceae family are known. The endemism
rate of the species is around 50%3. The hazard
category of Digitalis lamarckii species is stated to be
of least concern and low risk4.
The leaves of Digitalis species are used for medical
treatment purposes as they have cardiac, diuretic,
stimulant and tonic characteristics. Digitalis, which
was first used by William Withering in 1785, was
declared as cardiotonic in 1860 and in 1869, the
glycoside called digitalin was isolated from the leaves
of Digitalis purpurea by Natville. In the same year,
Schiemiedeberg named this glycoside “digitoxin”.
These glycosides have hazardous effects for animals
and humans but are used in cardiac drugs as they
reduce pulse rate and slow down blood circulation5,6 .
Digitalis is a general term for steroidal drugs
prepared from the seeds and dried leaves of the genus
Digitalis, which are used as a cardiac stimulant. More
than a quarter of a century has elapsed since the first
demonstration of the presence of digitalis-like
compounds (DLC) in mammalian tissues at the
cellular and molecular levels,. A large number of
scientific reports that have appeared since then
unequivocally support the notion that these
compounds function as hormones in mammals; they
are synthesized and released from the adrenal gland
and by interacting with their receptor, Na+, K+,
ATPase, they affect numerous cellular functions7.
Many cardiologic and floristic studies have been
conducted in Digitalis species8,9. However, there were
no studies on antimicrobial property of this plant.
In this study the extract obtained from the leaves
and flowers of D. lamarckii plant has been tested on
some bacterial and yeast strains and its antimicrobial
activity was evaluated.
Collection of plants ⎯ From the A4, Kastamonu:
Azdavay; Karyatağı Mountain, Yanık Plateau and
Iran-Turan, KG 1493 region, D. lamarckii plants were
collected and dried in the shade. D. lamarckii was
identified at Ankara University Faculty of Science
Herbarium (ANK Herbarium), and its leaves and
flowers were used in tests. The plant samples are
being preserved at Ankara University Faculty of
Science Herbarium (ANK Herbarium).
Preparation of extracts ⎯ For the determination of
antimicrobial activity, 3 g of ground plant parts were
soaked in 30 ml of methanol, and for the minimal
inhibitory concentration (MIC) tests, 10 g of ground
plants were extracted with 100 ml of methanol. It was
heated until the mixture boiled and then cooled, kept
overnight at room temperature. The extracts were
filtered and dried. The dry weight of the remaining
residue was calculated. The dried material was again
_
___________________
*Correspondent author
Telphone: +90 3122126720;
Fax: +90 3122232395
E- mail: mehlikabenli_76@hotmail.com
NOTES
219
re-dissolved in methanol and diluted with deionized
water. After the prepared extract solution was
sterilized at 121°C for 15 min, it was put in sterile
tubes and centrifuged at 5000 rpm for 3 min. The
supernatant of extract was used.
Test microorganisms ⎯ Fresh cultures of the
microorganisms were grown in nutrient broth
(acumedia). The density of microorganisms was
adjusted to Mc Farland 0.5 standard. In the tests, a total
of 14 microorganisms namely; Enterococcus gallinarum
CDC-NJ-4, Enterococcus faecalis ATCC 29212,
Bacillus subtilis RSHI, Escherichia coli RSHI, Shigella
RSHI, Escherichia coli ATCC 25922, Streptococcus
pyogenes ATCC 19615, Staphylococcus aureus ATCC
29213, Listeria monocytogenes ATCC 7644,
Pseudomonas aeruginosa ATCC27853, Saccharomyces
cerevisiae (Pakmaya), Candida albicans 845981, C.
albicans 900628 and C. crusei ATCC 6258 were used.
Antibiotic discs ⎯ The discs amikacin (30 µg/ml;
Eczacibasi), vancomycin (30 µg/ml; Mayne),
penicillin (10 U/ml; I.E.Ulagay), gentamicin (10
µg/disc; I.E.Ulagay), rifampicin (5 µg/ml; Aventis),
tetracycline (30 µg/ml; SIGMA), ampicillin (10
µg/ml; SELVA), chloramphenicol (30 µg/ml;
SIGMA) and erythromycin (15 µg/ml; SIGMA) were
used as positive control. In the MIC tests, gentamicin
(Genta-120 mg; I.E.Ulagay) was used as the standard
antibiotic. As negative control 1 ml of methanol, 5 ml
of deionized water mixture was used.
Determination of antimicrobial activity
⎯
Fresh
cultures of the microorganisms (100 µl) were
inoculated on Muller Hinton Agar (Merck). Agar was
allowed to dry for 15- 20 min in an incubator and on
each plate, three drops of extract was added each of
which was 20 µl. The plates were then incubated at
37ºC for 24 hr and the diameters of inhibition zones
were measured and evaluated. The assays that were
found to be effective were repeated three times. The
positive and negative tests were performed under
same conditions10.
Determination of minimum inhibitory concentration
(MIC)
⎯
On the sensitive bacterial strains, two-fold
liquid dilution tests were made by using Mueller
Hinton Broth (Merck). For each strain, two series of 10
tubes were used; while in the first series the plant
extract was tested, in the second series standard
antibiotic was tested. Whether bacterial growth
occurred or not was determined by observing the
turbidity of the cultures. The tube in which no growth
occurred was evaluated as the minimum inhibitory
concentration (MIC) and then the minimum effective
dose of the extract was calculated10.
Determination of the minimum bactericidal
concentration (MBC)
⎯
The contents of the MIC
tubes having no-growth were spread on Mueller
Hinton Agar plates for colony counting. MBC was
calculated by the determination of whether the
activity of the extract was bacteriostatic or
bactericidal according to the state of growth. If there
was no growth, the extract was identified as
bactericidal10.
Many plants are commonly used as curative and as
antiseptic for skin mucosa lesions and infections of
other systems11.
The leaves and flowers obtained from D. lamarckii
were extracted together and their antimicrobial effect
was tested on 10 bacterial and 4 yeast strains; and on
4 bacteria, effective inhibition zones were observed. It
was noted that D. lamarckii extracts had no effect on
the yeast strains tested (Table 1).
B. subtilis, a bacterium that was susceptible to the
extract, exhibited an inhibition zone of 20 mm and it
was found to be susceptible to all but one of the
standard antibiotics that were tested. The plant extract
exhibited an inhibition zone of 25 mm against S.
aureus; and it was found to be susceptible to all but
two of the antibiotics that were tested. L.
monocytogenes exhibited an inhibition zone of 27 mm
and it was found to be susceptible to five of the
antibiotics while it was resistant against four. Shigella
exhibited the smallest diameter of inhibition zone of
16 mm and showed one of the most effective results
that the plant extract yielded. This particular Shigella
strain was resistant against many antibiotics and
susceptible to only two of the antibiotics. Hence it
was a significant finding, as it was susceptible to D.
lamarckii extract. When the susceptibility of bacterial
strains to antibiotics and the plant extract were
compared, it was observed that the plant extract had
antibacterial activity no less than the antibiotics.
Main stock content concentration of the plant
extract used in MIC tests conducted by use of liquid
dilution method was calculated as 399 mg/ml. The
dose at which the plant extract was effective at a
minimum level (MIC) was calculated. The minimum
inhibitory concentration values of the incubation after
18, 24, 48 and 72 hr were all the same. At the 18th hr,
no turbidity was observed in the tube that was diluted
at a rate of 1/2 and no change occurred in the results
as the incubation time increased. Even though the
INDIAN J EXP BIOL, MARCH 2009
220
bacterial strains were different, the minimum
inhibitory concentration values were the same. In
other words, the MIC value for all the four bacterial
strains was calculated as ≥ 199.5 mg/ml. These results
were quite interesting. Excluding Shigella, the
susceptibilities of the bacterial strains to gentamicin
were the same as their susceptibilities to the extract
(Table 2). Besides, no difference can be observed as
regards to the effect of the extract depending on time.
No turbidity was observed in the same MIC tube after
18, 24, 48 and 72 hr. The Gram negative bacterium
Shigella and Gram positive organism S. aureus, L.
monocytogenes and B. subtilis were observed to have
the same minimum inhibitory concentrations. The
determination of the MIC tubes is performed visually
by looking at the turbidity in the tubes. Considering
the possibility of a visual mistake, MIC results and
MBC results were compared.
With the inoculations made on the agar plates from
MIC tubes, MBC values were assessed and bacterial
growth was not observed when inoculated on agar
plates. When the MBC values were studied, such
values of B. subtilis, S. aureus and L. monocytogenes
were found to be ≥199.5 mg/ml in parallel to MIC
values; and with respect to Shigella, the MIC value
was found to be ≥199.5 mg/ml and the MBC value
was found to be ≥399 mg/ml (Table 2). No bacterial
colony was observed on the plates in the MBC tests.
This result indicates that D. lamarckii plant extract
had bactericidal effects.
Increase in number of resistant bacterial types and
strains against classical chemotherapeutics and
antibiotics (including Penicillin) reflect the necessity
to search for newer antimicrobics. Discovery of plants
with potentiable antimicrobial property has opened up
a new horizon to search for newer antimicrobics in
this field. Plants that have antibacterial activity inhibit
bacteria through mechanisms that are different from
the antibiotics that are currently in use. For this
reason, they are able to take the resistant bacteria
under control12,13. Scientists have identified many
medicinal plants and the effects of many of these
herbal drugs have been scientifically proven14,15.
Consequently, it may be concluded that D.
lamarckii plant extract has antibacterial activity. The
extract is found to be bactericidal in nature. Effective
antimicrobial property obtained by the determination
of the active compound from the plant can account for
new resources to develop newer chemotherapeutics.
Even at a trace level, the presence of antibacterial
agents in the plant will allow development of new
Table 1⎯Comparison of antimicrobial activity of leaf and flower
extract of D. lamarckii and nine standard antibiotics
Diameter of inhibition zone (mm)
Plant
Standard antibiotics
Microorganisms
Digitalis lamarckii
(leaves & flowers)
Amikacin
Vancomycin
Penicillin
Gentamicin
Rifocin
Tetracycline
Ampicilin
Chloramphenicol
Erythnomycin
Enterococcus
gallinarum CDC-
NJ-4
- - 12 - 15 13 - - - 11
Enterococcus
f
aecalis ATCC
29212
- 16 12 - 16 14 - - - 11
Bacillus subtilis
RSHI 20 24 19 22 25 23 12 - 13 24
Escherichia coli
RSHI - 18 - - 18 - - - - -
Shigella RSHI 16 20 - - 19 - - - - -
Escherichia coli
ATCC 25922 - 16 - - 17 - - - - -
Streptococcus
p
yogenes ATCC
19615
- 13 12 - 16 15 - - - 12
Staphylococcus
aureus ATCC
29213
25 17 15 19 17 27 12 - - 18
Listeria
monocytogenes
ATCC 7644
27 25 16 - 27 39 - - - 19
Pseudomonas
aeruginosa
ATCC27853
- 17 - - 15 - - - - -
Saccharomyces
cerevisiae
(Pakmaya)
- - 16 - - 17 10 8 - 11
Candida albicans
845981
- 17 12 - 19 15 - - - 12
Candida crusei
ATCC 6258
- 14 11 - 17 17 - - - 11
Candida albicans
900628
- 17 11 - 16 15 - - - 11
-, resistant
Table 2 ⎯ MIC, MBC values of the D. lamarckii leaf and flowers
extract (mg/ml) and level of sensitivity of gentamicin (µg/ml) on
susceptible bacterial strains
Bacteria Plant
MIC
(mg/ml )
Extract
MBC
(mg/ml)
Gentamicin
sensitivity
(µg/ml)
B. subtilis ≥ 199.5 ≥ 199.5 ≥ 1.875
Shigella ≥ 199.5 ≥ 399 ≥ 3.75
S. aureus ≥ 199.5 ≥ 199.5 < 1.875
L. monocytogenes ≥ 199.5 ≥ 199.5 < 1.875
NOTES
221
biologically originated compounds as a result of
obtaining that active agent from the plant through
different methods and purification processes.
This study was financially supported by a grand
from the Technical and Research Council of Turkey
(TUBITAK, TBAG- HD/ 107(105T542)).
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