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Antimicrobial Activity of Nepeta rtanjensis Essential Oil a
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Gordana Stojanović , Niko Radulović , Jelena Lazarević , Dragoljub Miladinović & Dejan Đoković
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Department of Chemistry, Faculty of Natural Sciences and Mathematics, Višegradska 33, 18000, Niš, Serbia, Montenegro b
Department of Medicinal Chemistry, Faculty of Medicine, B. Tasković 81, 18000, Niš, Serbia, Montenegro c
Faculty of Chemistry, Studentski trg 16, 11001, Belgrade, Serbia, Montenegro
Version of record first published: 28 Nov 2011.
To cite this article: Gordana Stojanović, Niko Radulović, Jelena Lazarević, Dragoljub Miladinović & Dejan Đoković (2005): Antimicrobial Activity of Nepeta rtanjensis Essential Oil, Journal of Essential Oil Research, 17:5, 587-589 To link to this article: http://dx.doi.org/10.1080/10412905.2005.9699004
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rtanjensis J. Essent. Oil Res., 17,N. 587-589 (September/October 2005)
Antimicrobial Activity of Nepeta rtanjensis Essential Oil Gordana Stojanovi´c ,* Niko Radulovi´c and Jelena Lazarevi´c Department of Chemistry, Faculty of Natural Sciences and Mathematics, Višegradska 33, 18000 Niš, Serbia and Montenegro
Dragoljub Miladinovi´c Department of Medicinal Chemistry, Faculty of Medicine, B. Taskovi´c 81, 18000 Niš, Serbia and Montenegro
Dejan Đokovi´c
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Faculty of Chemistry, Studentski trg 16, 11001, Belgrade, Serbia and Montenegro Abstract The antimicrobial activity and the composition of the essential oil of aerial parts of Nepeta rtanjensis Diklic´ et Milojevic´ wild growing and cultivated in Serbia were evaluated. The main compound in both samples was characterized as 4aα,7α,7aβ-nepetalactone with 83.6% (wild) and 77.9% (cultivated). α-Copaene, germacrene D and δ-cadinene were present less than 5% in the oils. The antimicrobial activity of the oils was investigated by disk diffusion method. The oil-ethanol solution in three dilutions, tested against five bacteria and a fungus, was effective towards all bacteria but not against Aspergillus niger. The oils showed bacteriostatic activity even in 1:60 dilution. Key Word Index Nepeta rtanjensis, Lamiaceae, antimicrobial activity, essential oil composition, nepetalactone.
Introduction The Serbian endemic plant Nepeta rtanjensis Diklic´ et Milojevic´ belongs to the widespread genus Nepeta L., Lamiaceae (comprising about 250 species, central and southern Europe, the near and middle east, central and southern Asia). Nepeta rtanjensis was found and described in 1974 in the spontaneous flora on only few localities on the Rtanj mountain in the south-east of Serbia (1). The composition of the oil of N. rtanjensis was previously the subject of several studies (2-4). However, no biological assays were performed to the best of our knowledge. Due to the known biological activity of nepetalactone (5-10) that occurs as the main constituent of some Nepeta oils, we decided to investigate the composition and the antimicrobial activity of N. rtanjensis oil. A comparison of the antimicrobial activity and composition of the oils of cultivated and wild growing plants is also presented.
Experimental The plant material was collected in September 2001, the wild growing N. rtanjensis from locations on the Rtanj Mountain, and the cultivated plant from Soko Banja. Voucher specimens are deposited in the Herbarium Moesicum Doljevac (HMD). The air-dried (in the shade and up to constant weight, moisture 8.3%) and ground plant material was submitted to hydrodistillation for 2.5 h in a Clevenger-type apparatus.
The analysis of the oils was achieved by means of GC and GC/MS and the constituents were identified by comparison of their mass spectra to those from the MS library (Adams89, Nist92). Analytical GC: A Varian model 3700 gas chromatograph, equipped with a 60 m x 0.32 mm fused silica capillary column, with a 0.3 μm film thickness of PTE-5 and FID was used for GC measurements. The operating conditions were: temperature program 60°-250°C at 4.3°C/min and an injector and detector temperature of 250°C; carrier gas was H2 (2 mL/min). GC/MS analyses were performed on a Finigan Mat, model 8230, equipped with fused silica 30 m x 0.25 mm, PTE-5 capillary column, with a film thickness 0.25 µm, a carrier gas was H2 (2.5 mL/min) with the same temperature program as for analytical GC. Electronspray ionization was performed at 70 eV. The antimicrobial investigations were performed using the agar diffusion technique (11) against the Gram-negative bacteria Klebsiella pneumoniae ATCC 13076, Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076, Escherichia coli 95, the Gram-positive Staphylococcus aureus ATCC 6538, and the fungus Aspergillus niger ATCC 16404. The bacteria were cultivated on “Antibiotica medium 1” (Difco Laboratories, Detroit, MI) or fungus on Sabouraud Dextose Agar (Unipath). The bacteria were suspended in sterile physiological saline with reference to the value 0.5 of the McFarland scale. The standardized inocula were placed on the surface of the media
*Address for correspondence:
[email protected]
Received: July 2003 Revised: December 2003
1041-2905/05/0005-0587$6.00/0—© 2005 Allured Publishing Corp. Vol. 17, September/October 2005
Accepted: February 2004 Journal of Essential Oil Research/587
Stojanovic´ et al.
Table I. Comparative percentage composition of the oils from wild growing and cultivated Nepeta rtanjensis (compounds are listed in order of elution from the PTE-5 capillary column) Compound
Wild growing plants %
Cultivated plants% Compound
1,8-cineole 0.6 p-cymene 0.5 a-copaene 1.2 4aα,7α,7aβ-nepetalactone 83.6 g-thujaplicin 0.7 germacrene D 3.8
0.8 0.9 5.5 77.9 1.1 1.7
Wild growing plants %
Cultivated plants%
0.3 2.2 1.0 0.9
0.9 1.5 1.5 0.9
94.8
92.7
cis-calamenene δ-cadinene cadina-1,4-diene a-calacorene Total
Table II. Antimicrobial activity of the oils of wild growing and cultivated Nepeta rtanjensis (growth inhibition zones, mm)
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Microorganisms Test samples Dilutions Zones*
K. pneumoniae
P. aeruginosa
S. enteritidis
E. coli
S. aureus
A. niger
1:10 1:30 1:60 1:10 1:30 1:60 1:10 1:30 1:60 1:10 1:30 1:60 1:10 1:30 1:60 1:10 1:30 1:60
Wild growing 12.7 mm** Bacteriostatic 4.8 Bactericidal 4.8
3.0 0.5
3.0 0.0
6.5 3.5
3.5 1.0
3.0 0.0
9.0 4.5
8.0 0.5
8.0 0.0
9.0 5.5
6.0 2.0
6.0 10.5 0.0 5.0
9.0 3.0
6.3 1.0
6.0 0.5
3.5 0.0
3.5 0.0
Cultivated 12.7 mm** Bacteriostatic 5.0 Bactericidal 0.5
3.0 0.0
3.0 10.0 0.0 5.5
6.8 4.0
6.0 0.0
8.0 5.0
6.5 5.0
3.5 0.5
6.0 2.0
5.0 1.5
2.0 1.0
6.0 3.0
5.0 0.5
3.0 0.0
3.0 0.0
0.0 0.0
Antibiotic ampicillin 10 mm** Bacteriostatic 3.5 Bactericidal 1.8
2.0 0.0
2.8 0.0
2.5 0.0
8.0 4.0
2.5 0.0
2.0 0.0
in the case of A. niger the zones are fungicidal and fungistatic; **the diameters of the used discs
*
according to the Kirby-Bauer technique (11). The ethanol solutions of the oils (1:10, 1:30 and 1:60 dilutions) and ethanol were placed on 12.7 mm in diameter sterile empty paper disks (thickness 0.8 mm) “Antibiotica Test Blättchen” (Schleicher and Schuell, Dassel, Germany). The samples were tested at the dose of 50 μL. Three paper disks, imbibed with three different dilutions of the oil, and the standard antibiotic disk (Ampicillin 10 μg, Torlak, Belgrade, 10 mm in diameter) were placed evenly (no closer than 24 mm from center to center) on the surface of the agar plate (100 mm) by using a sterile forceps. The micro-organisms were incubated at 35°C aerobically and after 24 h of incubation the zones of inhibition were measured to the nearest 0.1 millimetre using a “Fisher-Lilly Antibiotic Zone Reader” (Fisher Scientific Co., USA).
Results and Discussion The yield of yellowish highly fragrant oils was 1.5025 g (0.83%, w/w on a dried weight basis) and 0.8234 g (1.03%, w/w) for wild growing and cultivated plants, respectively. Our yields were more than previously found for the same species (2), and also for most other plant species from this genus except for N. phyllochlamys (12). The composition of the oils differed only quantitatively for the wild and cultivated plants. The main component was 4aa,7a,7ab-nepetalactone 83.6% for the wild and 77.9% for 588/Journal of Essential Oil Research
the cultivated plant. This lactone was also the major constituent of the oils of the same species in previous investigations in the following quantities 86.4% (2) and 64% (4). The oil of N. crassifolia (13) contained 92.6% 4aa,7a,7ab-nepetalactone, while the oil of N. racemosa (14) contained in approximately equal amounts the three isomeric nepetalactones, 4aa,7a,7ab(33.6%), 4aa,7a,7ab- (25.6 %) and 4aa,7a,7ab- nepetalactone (24.4%). 4aa,7a,7ab-Nepetalactone was shown to be a feline attractant (8), and a sex pheromone for aphids (5,9) and silver moths (6), but has also been evaluated against populations of subterranean termites (7). Other identified oil constituents are shown in Table I. Previously, p-cymene, g-thujaplicin, germacrene D, cis-calamenene, δ-cadinene, cadina-1,4-diene and a-calacorene were not identified from the oil of N. rtanjensis (2), however, germacrene D was the main component of the oil of N. sorgerae (12). A minor constituent of our oils 1,8-cineole (present in 0.6% for the wild and 0.8% for the cultivated) was the main constituent of N. italica, N. sulfuriflora, N. nuda subsp. nuda and N. nuda ssp. albiflora (12). As it can be seen from Table I, 10 compounds were identified, representing 94.8% and 92.7% of the oil of the wild growing and cultivated plants, respectively. The results of the antimicrobial assay of the N. rtanjensis oil are presented in Table II. The control disk with ethanol showed no activity at all. The activity of the ethanol oil solution in 1:10 dilution (4.6 µL of the pure oil per disk) was greater Vol. 17, September/October 2005
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N. rtanjensis
than the activity of the standard antibiotic. Escherichia coli and S. aureus were especially non-resistant to that dilution with the width of the bactericidal zone 5.5 mm and 5.0 mm for the oil isolated from the wild growing N. rtanjensis. The 1:30 dilution (1.6 µL of the oil per disk) showed approximately equal activity as the standard antibiotic against all the micro-organisms. The oil showed bacteriostatic activity even in 1:60 dilution (0.8 µL of the oil per disk). In the case of the strains of A. niger, the tested samples inhibited the growth of the colonies but no fungicidal activity occurred. However, all the other micro-organisms screened were moderately susceptible to the tested samples. Analyzing Table II it can be seen that microbiological activity of the oil from the wild growing N. rtanjensis showed stronger activity to strains of S. enteritidis, E. coli and S. aureus, but for the rest of the microorganisms the oil obtained from the cultivated plant had stronger activity. The inhibitory effects on the tested micro-organisms could be related to nepetalactone (10), 1,8-cineole (10) and g-thujaplicin (15). The differing activity of the oils of the wild and cultivated plants could possibly be attributed to the varying content of these components in the oils.
3. 4.
5. 6.
7.
8.
9. 10.
11.
12.
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