Dulger & Aki

Tropical Journal of Pharmaceutical Research, August 2009; 8 (4): 371-375

Pharmacotherapy Group,
Faculty of Pharmacy, University of Benin,
Benin City, 300001 Nigeria.

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Available online at http://www.tjpr.org

Research Article

Antimicrobial Activity of the Leaves of Endemic

Stachys pseudopinardii in Turkey
G Dulger* and C Aki
Department of Biology, Faculty of Science and Arts, Canakkale Onsekiz Mart University, 17100 Canakkale, Turkey

Abstract
Purpose: The ethanol extract of the leaves of Stachys pseudopinardii R. Bhattacharjee and Hub.Mor.
(Lamiaceae) were investigated for their antimicrobial activities.
Methods: The antimicrobial activity of the leaf extract of the plant was tested against Bacillus subtilis
ATCC 6633, Bacillus cereus ATCC 7064, Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC
10538, Proteus vulgaris ATCC 6899, Salmonella typhimurium CCM 5445 and Pseudomonas aeruginosa
ATCC 27853, as well as Candida albicans ATCC 10239, Debaryomyces hansenii DSM 70238,
Kluyveromyces fragilis ATCC 8608 and Rhodotorula rubra DSM 70403, by disc diffusion and
microdilution methods. Selected antibacterial agents (penicillin, tobramycin and ampicillin) and
antifungal agents (nystatin, clotrimazole and ketoconazole) antibiotics were used as positive reference
standards in the tests.
Results: The extracts showed strong antibacterial activity against Bacillus cereus ATCC 7064, with an
inhibition zone of 25.0 mm, and minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) of 16 and 32 g/mL, respectively. Debaryomyces hansenii DSM 70238 was among
the most susceptible of the yeast cultures, with an inhibition zone of 17.0 mm and MIC and minimum
fungicidal concentration (MFC) of 32 and 32 g/mL, respectively. The extract exhibited moderate activity
against the other test microorganisms.
Conclusion: The results demonstrate that the ethanol extract of the leaves of Stachys pseudopinardii
has significant antimicrobial activity and suggest that it may be useful in the treatment of infections.
Key words: Stachys pseudopinardii, ethanol extract, antimicrobial activity, MIC, MBC, MFC

Received: 20 February 2009

Revised accepted: 25 April 2009

*Corresponding author: E-mail: [email protected]

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Dulger & Aki

INTRODUCTION
Medicinal plants have been known for their
healing or disease-curing qualities for
centuries. Stachys species have been
reported in folk medicine to treat genital
tumors, sclerosis of the spleen, inflammatory
1
tumors and cancerous ulcers . Whole plant or
leaves of this species are used in
phytotherapy and said to possess sedative,
antispasmodic, diuretic and emmenagogue
2
activities when used as a tea . Some Stachys
species are used as a tonic and for stomach
3
ailments in Anatolia .
Stachys pseudopinardii R. Bhattacharjee &
Hub.Mor. (Lamiaceae) is endemic to
4
Turkey . A bibliographical survey showed that
there are no reports on the antimicrobial
activity of this plant. Therefore, the aim of this
work was to evaluate the antimicrobial activity
of Stachys pseudopinardii which grows wild
in Turkey.

MATERIALS AND METHODS

Plant material
The plant material was collected from Icel,
Turkey in July and August, 2008. A voucher
specimen (voucher number GD56) of the
plant was deposited in the Biology
Department of Canakkale Onsekiz Mart
University following identification by Ersin
Karabacak of the same Department.
Preparation of extracts
The leaves of the plant were dried in an oven
at 40 C for 12 h and powdered. Each dry
powdered plant material (20 g) was extracted
with 150 mL of 95% ethanol (Merck,
Darmstadt, Germany) for 24 h using a
Soxhlet extractor. The extract was filtered
with Whatman filter paper no.1, and the
filtrate was evaporated under vacuum in a
rotary evaporator at 55 C. The extract yield
obtained was 12.4%. The dry extract, which
was sticky and black, was stored in labeled
sterile screw-capped bottles at -20C pending

use. Prior to testing, 2 g was dissolved in 0.4

L of dimethyl sulfoxide (DMSO) (5 mg/mL).
Test microorganisms
In vitro antimicrobial studies were carried out
on seven bacterial strains (Bacillus subtilis
ATCC 6633, Bacillus cereus ATCC 7064,
Staphylococcus aureus ATCC 6538P,
Escherichia coli ATCC 10538, Proteus
vulgaris ATCC 6899, Salmonella typhimurium
CCM 5445 and Pseudomonas aeruginosa
ATCC 27853) and four yeast strains
(Candida
albicans
ATCC
10239,
Debaryomyces
hansenii
DSM
70238,
Kluyveromyces fragilis ATCC 8608 and
Rhodotorula rubra DSM 70403). They were
all obtained from the Microbiology Research
Laboratory,
Department
of
Biology,
Canakkale Onsekiz Mart University, Turkey.
Disc diffusion method
The paper disc diffusion method was
5
employed . Sterile 6 mm disc filter paper disc
(Schleicher & Schul, No. 2668, Dassel,
Germany) were impregnated with 50 L of
the plant extract. The bacterial cultures were
inoculated on Nutrient Broth (Oxoid) and
incubated for 24 h at 370.1 C, while the
yeast cultures were inoculated on Malt
Extract Broth (Oxoid) and incubated for 48 h
at 28.00.1 C. Adequate amounts of Mueller
Hilton Agar (Oxoid) were dispensed into
sterile plates and allowed to solidify under
aseptic conditions. The counts of bacterial
7
and yeast cultures were adjusted to yield 10
8
-1
5
6
-1
10 mL and 10 10 mL , respectively,
using the standard McFarland counting
method. The test microorganisms (0.1 mL)
were inoculated with a sterile swab on the
surface of appropriate solid medium in plates.
The agar plates inoculated with the test
microorganisms were incubated for 1 h
before placing the extract impregnated paper
disc on the plates. The bacterial plates were
incubated at 370.1 C for 24 h while yeast
plates were incubated at 280.1 C for 48 h.
After incubation, all plates were observed for
zones of growth inhibition and the diameter of
Trop J Pharm Res, August 2009; 8 (4): 372

Dulger & Aki

these zones was measured in millimetres. All

tests were performed under sterile conditions
in duplicate and repeated three times.
Penicillin (10 g/disc), tobramycin discs (10
g/disc), ampicillin (20 g/disc), nystatin (30
g/disc), clotrimazole (30 g/disc) and
ketoconazole (20 g/disc) discs were used as
positive controls.
Microdilution method
Determination of the minimum inhibitory
concentration (MIC) was carried out
according to the method described by Zgoda
6
and Porter, with some modifications . A
dilution series of the extract, ranging from 10
to 0.5 mg/mL, were prepared and then
transferred to the broth in 96well microtitre
plates. The final concentrations were in the
range 1000 to 50 g/mL in the medium.
Before inoculation of the test organisms, the
bacterial and yeast strains were adjusted to
0.5 McFarland and diluted 1:1000 in Mueller
Hinton Broth (Oxoid) and Malt Extract Broth
(Oxoid), respectively. The plates were
incubated at 35 C for 18 24 h for bacteria
and 30 C for 48 h for the yeast cultures. All
the tests were performed in broth and
repeated twice. While the MIC values of the
extracts were defined as the lowest
concentration that showed no growth,
minimum bactericidal concentration (MBC)
and minimum fungicidal concentration (MFC)
were determined by plating samples from
clear wells onto Mueller Hinton Agar and Malt
Extract Agar, respectively. MBC and MFC
were defined as the lowest concentration
yielding negative subculture.
Ampicillin and streptomycin were used as the
standard antibacterial agents, while nystatin
was used as the standard antifungal agent.
Their dilutions ranged from 128.0 to 0.25
g/mL concentrations in microtitre plates.

RESULTS
The antimicrobial activities of Stachys
pseudopinardii extracts against the test
microorganisms examined in this study were

qualitatively and quantitatively assessed by

inhibition zone, MIC, MBC and MFC. The
results are shown in Tables 1 and 2. The
extract of S. pseudopinardii exhibited strong
antimicrobial effects against the test
microorganisms, with inhibition zones ranging
from 6 to 25 mm. Notably, B. subtilis was
more susceptible to the extract (inhibition
zone: 25.0 mm) compared to the standard
antibacterials, ampicillin and tobramycin, and
penicillin whose inhibition zones ranged from
13 18 mm. Similarly, the extract showed
higher antibacterial activity against S. aureus,
P. aeruginosa and Proteus vulgaris than
some of the standard antibiotics. The
antifungal effect of the extract against C.
albicans and K. fragilis was equivalent to
those of the standard antifungal agents,
nystatin and ketoconazole, respectively. D.
hansenii was more susceptible to the extract
than the standard antifungals, except
clotrimazole.
In the microdilution test, the lowest MICs and
MBCs of the extract were 16 and 32 g/mL,
respectively, against B. cereus, followed by
D. hansenii and K. fragilis, with MIC/MBC of
32/32 and 64/>128 g/mL, respectively. The
extracts showed weak antimicrobial avtivity
against the other test microorganisms with
MIC/MBC ranging from 1000/1000 to 250/500
g/mL. These values were well below those
of the standards.

DISCUSSION
Ethanol was observed as the best solvent for
extracting antimicrobial substances from
7
some plants in a previous study . It is likely
that the concentration of extract used in the
test may correlate with the activity of its
chemical components.
To the best of our knowledge, there are no
reports of the antimicrobial activity of Stachys
pseudopinardii. Furthermore, investigations
of antimicrobial activity of the other Stachys
species are few. In previous studies, the
antimicrobial activity of some endemic
Stachys species - S. sivasica, S.
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Dulger & Aki

Table 1: Antimicrobial activity of the ethanol extract of S. pseudopinardii

a

Diameter of zone of inhibition (mm)

Microorganism

Extract
(g/mL)

Bacillus subtilis
Bacillus cereus
Escherichia coli
Stapylococcus aureus
Pseudomonas
aeruginosa
Proteus vulgaris
Salmonella typhimurium
Candida albicans
Debaryomyces hansenii
Kluyveromyces fragilis
Rhodotorula rubra

11.0
25.0
6.0
13.0
11.0

P
14.0
13.0
16.0
23.0
8.0

AMP
12.0
16.0
14.0
16.0
10.0

TOB
24.0
18.0
10.0
8.0
12.0

NYS
Nt
Nt
Nt
Nt
Nt

KETO
Nt
Nt
Nt
Nt
Nt

CLT
Nt
Nt
Nt
Nt
Nt

14.0
10.0
15.0
17.0
16.0
6.0

10.0
13.0
Nt
Nt
Nt
Nt

16.0
13.0
Nt
Nt
Nt
Nt

13.0
10.0
Nt
Nt
Nt
Nt

Nt
Nt
20.0
16.0
18.0
18.0

Nt
Nt
21.0
14.0
16.0
22.0

Nt
Nt
15.0
20.0
18.0
16.0

Standard

Zone of inhibition, including the diameter of the filter disc (6.0 mm); mean value of three independent
experiments; Nt = not tested; P = penicillin (10 g/disc); TOB = tobramycin discs (10 g/disc); AMP =
ampicillin (20 g/disc); NYS = nystatin discs (30 g/disc); KETO = ketoconazole (20 g/disc); CLO =
clotrimazole (30 g/disc).

Table 2: Minimum inhibitory concentration (MIC) of the ethanol extract of S. pseudopinardii

MIC (MBC or MFC)
Microorganism
Bacillus subtilis
Bacillus cereus
Escherichia coli
Stapylococcus aureus
Pseudomonas aeruginosa
Proteus vulgaris
Salmonella typhimurium
Candida albicans
Debaryomyces hansenii
Kluyveromyces fragilis
Rhodotorula rubra

Extract
(g/mL)
500 (>1000)
16 (32)
1000 (1000)
250(500)
1000 (1000)
250 (500)
1000 (1000)
250 (500)
32 (32)
64 (>128)
1000 (1000)

Standard
ST
0.5 (0.5)
4.0 (4.0)
4.0 (4.0)
2.0 (4.0)
1.0 (1.0)
8.0 (8.0)
16 (32)
Nt
Nt
Nt
Nt

AMP
0.5 (2.0)
8.0 (8.0)
64 (128)
<0.25 (0.35)
16 (32)
0.5 (0.5)
1.0 (4.0)
Nt
Nt
Nt
Nt

NYS
Nt
Nt
Nt
Nt
Nt
Nt
Nt
8.0 (16)
16 (32)
16 (16)
16 (16)

Nt = not tested; ST = streptomycin; AMP = mpicillin; NYS = nystatin

anumurensis, S. cydnia, S. aleurites and S.

pinardii - was reported. The methanol
extracts of Stachys species were effective
8-9
only against bacteria . In another study, the
ethanol extract of S. byzantina was found not
10
to be effective against C albicans strains .
However, the essential oil of this plant
showed
anti-Candida
activity.
The
antimicrobial activity of the methanol extracts
of Stachys byzantina, S. inflata, S.

lavandulifolia and S. laxa were studied

against some bacteria and C. albicans by
11
Saeedi et al . The extracts were more active
against Gram-positive bacteria. The extracts,
however, did not show any antifungal activity.
In contrast, the essential oil of S. plumosa
exhibited antimicrobial activity against
12
bacteria and two C. albicans strains . In
another work, the essential oils of eight
Stachys species (S. alopecuros, S. scardia,
Trop J Pharm Res, August 2009; 8 (4): 374

Dulger & Aki

S. cretica subsp. cretica, S. germanica subsp.

heidrichii, S. recta, S. euboica and S.
menthifolia were tested for their antimicrobial
13
activity . The essential oil of S. scardia was
shown to be the most active against both
bacteria and fungi. As can be seen from
these literature data, the essential oils of
Stachys species have antifungal activity
against the yeast cultures, especially C.
albicans, but the antifungal activity was not
observed for the leaf extracts. Notably, in this
study, the extract of S. pseudopinardii
demonstrated antimicrobial activity against
both bacteria and yeast cultures. The
difference between our results and those of
other workers may be due to several factors,
for example, the intra-specific variability in the
production of secondary metabolites. In
addition, there may be differences in the
extraction protocols used to recover the
active metabolites as well as differences in
the assay methods.

REFERENCES
1.

2.
3.
4.
5.
6.

7.

8.

Phytochemical analyses of Stachys species

have
confirmed
the
occurrence
of
diterpenes, phenyl ethanoid glycosides,
14
flavanoids and saponines . Flavonoids may
be responsible for their antibacterial
11
activity . The results indicate that S.
pseudopinardii possessed significant activity
against both bacteria and yeast cultures.
This activity may be indicative of the
presence of metabolic toxins or the
compounds stated above. Therefore, this
plant extract should be analyzed further, as it
might contain a yet unknown compound that
is effective against pathogens.

CONCLUSION
This preliminary evaluation indicated that the
ethanol leaf extract of Stachys pseudopinardii
has significant activity against the test
bacterial and fungal strains used. Further
studies are necessary to identify the main
active constituents.

9.

10.

11.

12.

13.

14.

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