|
Tropical Journal of Pharmaceutical Research
Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
ISSN: 1596-5996 EISSN: 1596-9827
Vol. 8, Num. 4, 2009, pp. 371-375
|
Tropical Journal of Pharmaceutical Research, Vol. 8, No. 4, Aug, 2009, pp. 371-375
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
*Corresponding author: E-mail: gorkemtazeler@yahoo.com
Received: 20 February 2009
Revised
accepted: 25 April 2009
Code Number: pr09048
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 µg/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 µg/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
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 tumors and cancerous ulcers1. Whole plant or
leaves of this species are used in phytotherapy and said to possess sedative,
antispasmodic, diuretic and emmenagogue activities when used as a tea2.
Some Stachys species are used as a tonic and for stomach ailments in
Anatolia3.
Stachys
pseudopinardii R. Bhattacharjee &
Hub.Mor. (Lamiaceae) is endemic to Turkey4. 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 -20°C 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 employed5. Sterile 6 mm disc filter paper disc (Schleicher & Schul, No. 2668, Dassel, Germany) were impregnated with
50 µL of the plant extract. The bacterial cultures were inoculated on Nutrient
Broth (Oxoid) and incubated for 24 h at 37±0.1 °C, while the yeast cultures were inoculated on Malt Extract Broth (Oxoid) and incubated for 48 h
at 28.0±0.1 °C. Adequate amounts of Mueller Hilton Agar (Oxoid) were dispensed
into sterile plates and allowed to solidify under aseptic conditions. The
counts of bacterial and yeast cultures were adjusted to yield 107
108 mL-1 and 105 106 mL-1,
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 37±0.1 °Cfor 24 h while yeast plates were incubated at 28±0.1 °C for 48 h. After incubation, all plates were observed for
zones of growth inhibition and the diameter of these zones was measured in millimetres.
All tests were performed under sterile conditions in duplicate and repeated
three times. Penicillin (10 µg/disc), tobramycin discs (10 µg/disc), ampicillin
(20 µg/disc), nystatin (30 µg/disc), clotrimazole (30 µg/disc) and ketoconazole
(20 µg/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 and Porter, with some modifications6. 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 µg/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 µg/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
µg/mL, respectively, against B. cereus, followed by D. hansenii
and K. fragilis, with MIC/MBC of 32/32 and 64/>128 µg/mL,
respectively. The extracts showed weak antimicrobial avtivity against the other
test microorganisms with MIC/MBC ranging from 1000/1000 to 250/500 µg/mL. These
values were well below those of the standards.
DISCUSSION
Ethanol
was observed as the best solvent for extracting antimicrobial substances from
some plants in a previous study7. 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. anumurensis, S. cydnia, S. aleurites and S. pinardii - was reported. The methanol extracts of Stachys species were effective
only against bacteria8-9. In another study, the ethanol extract of S.
byzantina was found not to be effective against C albicans strains10.
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 Saeedi et al11. 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 bacteria and two C.
albicans strains12. In another work, the essential oils of eight Stachys species (S. alopecuros, S. scardia, S. cretica subsp. cretica, S. germanica subsp. heidrichii, S.
recta, S. euboica and S. menthifolia were tested for their
antimicrobial activity13. 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.
Table 1:
Antimicrobial activity of the ethanol extract of S. pseudopinardii
Microorganism
|
Diameter of zone of inhibition (mm)a
|
Extract
(µg/mL)
|
Standard
|
P
|
AMP
|
TOB
|
NYS
|
KETO
|
CLT
|
Bacillus
subtilis
|
11.0
|
14.0
|
12.0
|
24.0
|
Nt
|
Nt
|
Nt
|
Bacillus
cereus
|
25.0
|
13.0
|
16.0
|
18.0
|
Nt
|
Nt
|
Nt
|
Escherichia
coli
|
6.0
|
16.0
|
14.0
|
10.0
|
Nt
|
Nt
|
Nt
|
Stapylococcus
aureus
|
13.0
|
23.0
|
16.0
|
8.0
|
Nt
|
Nt
|
Nt
|
Pseudomonas
aeruginosa
|
11.0
|
8.0
|
10.0
|
12.0
|
Nt
|
Nt
|
Nt
|
Proteus
vulgaris
|
14.0
|
10.0
|
16.0
|
13.0
|
Nt
|
Nt
|
Nt
|
Salmonella
typhimurium
|
10.0
|
13.0
|
13.0
|
10.0
|
Nt
|
Nt
|
Nt
|
Candida
albicans
|
15.0
|
Nt
|
Nt
|
Nt
|
20.0
|
21.0
|
15.0
|
Debaryomyces
hansenii
|
17.0
|
Nt
|
Nt
|
Nt
|
16.0
|
14.0
|
20.0
|
Kluyveromyces
fragilis
|
16.0
|
Nt
|
Nt
|
Nt
|
18.0
|
16.0
|
18.0
|
Rhodotorula
rubra
|
6.0
|
Nt
|
Nt
|
Nt
|
18.0
|
22.0
|
16.0
|
aZone
of inhibition, including the diameter of the filter disc (6.0 mm); mean value of three independent experiments; Nt = not tested; P = penicillin (10 µg/disc);
TOB = tobramycin discs (10 µg/disc); AMP = ampicillin (20 µg/disc); NYS =
nystatin discs (30 µg/disc); KETO = ketoconazole (20 µg/disc); CLO =
clotrimazole (30 µg/disc).
Table 2:
Minimum inhibitory concentration (MIC) of the ethanol extract of S.
pseudopinardii
Microorganism
|
MIC (MBC or MFC)
|
Extract (µg/mL)
|
Standard
|
ST
|
AMP
|
NYS
|
Bacillus
subtilis
|
500 (>1000)
|
0.5 (0.5)
|
0.5 (2.0)
|
Nt
|
Bacillus
cereus
|
16 (32)
|
4.0 (4.0)
|
8.0 (8.0)
|
Nt
|
Escherichia
coli
|
1000 (1000)
|
4.0 (4.0)
|
64 (128)
|
Nt
|
Stapylococcus
aureus
|
250(500)
|
2.0 (4.0)
|
<0.25 (0.35)
|
Nt
|
Pseudomonas
aeruginosa
|
1000 (1000)
|
1.0 (1.0)
|
16 (32)
|
Nt
|
Proteus
vulgaris
|
250 (500)
|
8.0 (8.0)
|
0.5 (0.5)
|
Nt
|
Salmonella
typhimurium
|
1000 (1000)
|
16 (32)
|
1.0 (4.0)
|
Nt
|
Candida
albicans
|
250 (500)
|
Nt
|
Nt
|
8.0 (16)
|
Debaryomyces
hansenii
|
32 (32)
|
Nt
|
Nt
|
16 (32)
|
Kluyveromyces
fragilis
|
64 (>128)
|
Nt
|
Nt
|
16 (16)
|
Rhodotorula
rubra
|
1000 (1000)
|
Nt
|
Nt
|
16 (16)
|
Nt = not tested; ST = streptomycin; AMP = mpicillin;
NYS = nystatin
Phytochemical analyses of Stachys
species have confirmed the occurrence of diterpenes, phenyl ethanoid
glycosides, flavanoids and saponines14. Flavonoids may be
responsible for their antibacterial activity11. 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.
REFERENCES
- Skaltsa HD,
Lazari DM, Chinou IB, Loukis AE. Composition and
antibacterial activity of the essential oils of Stachys
candida and S.
chrysantha from Southern Greece. Planta Med 1999; 65: 255256.
- Miller LG,
Murray WJ. Herbal Medicinals. A Clinicians Guide. Pharmaceutical Products
Press: Binghampton, NY, 1998; p. 223.
- Baytop T. Therapy
with Medicinal Plants in Turkey-Past and Present (in Turkish), 2nd ed. Nobel
Tip Basimevi: Istanbul, 1999; p. 193.
- Davis PH. Flora of Turkey and the East Aegean Islands, Vol. 7, Edinburgh University Press: Edinburgh, 1982; p. 238.
- Collins CM, Lyne PM.
Microbiological Methods. Butterworths and Co. Ltd., London, 1987; p. 316.
-
Zgoda JR, Porter JR. A convenient microdilution method for screening natural
products against bacteria and fungi. Pharm Biol 2001; 39: 221-225.
-
Jonathan SG,
Fasidi IO. Antimicrobial activities of two Nigerian edible macrofungi,
Lycoperdon pusilum and L. giganteum. Afr J Biomed Res 2003; 6: 85-90.
- Dulger B, Gonuz A.
Antimicrobial activity of some endemic Verbascum, Salvia and Stachys. Pharm Biol
2004; 42: 301-304.
- Dulger B, Ugurlu E, Aki C,
Suerdem TB, Camdeviren A, Tazeler G. Evolution of antimicrobial activity of
some endemic Verbascum, Sideritis and Stachys species from Turkey. Pharm Biol
2005; 43: 270-274.
- Duarte MCT, Figueira GM,
Sartoratto A, Rheder VGL, Delarmelina C. Anti-Candida activity of Brazilian
medicinal plants. J Ethnopharmacol 2005; 97(2): 305-311.
-
Saeedi M, Morteza-Semnani K,
Mahdavi MR, Rahimi F. Antimicrobial studies on Extracts of Four Species of
Stachys. Indian J Pharm Sci 2008; 70(3): 403-406.
- Petrovic S, Ristic M,
Milenkovic M, Kukic J, Antic-Stankovic J, Niketic M. Composition and
antimicrobial activity of essential oil of Stachys plumosa Griseb. Flavour Frag
J 2005; 21(2): 250-252.
- Skaltsa HD, Demetzos C,
Lazari D, Sokovic M. Essential oil analyses and antimicrobial activity of eight
Stachys species from Greece. Phytochemistry 2003; 64(3): 743-752.
- Khanavi M, Sharifzadeh M,
Hadjiakhoondi A, Shafiee A. Phytochemical investigation and anti-inflammatory
activity of aerial parts of Stachys byzantina C. Koch. J Ethnopharmocol 2005; 97:
463-468.
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
|