Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 72 No. 4 pp.

757ñ767, 2015 ISSN 0001-6837

Polish Pharmaceutical Society

CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF SOME

MEDICINAL PLANTS FROM LAMIACEAE FAMILY
MARIOLA KOZ£OWSKA1*, AGNIESZKA E. LAUDY2, JAROS£AW PRZYBY£3,
MA£GORZATA ZIARNO4 and EWA MAJEWSKA1

1
Department of Chemistry, 3Department of Vegetable and Medicinal Plants, Warsaw University of Life
Sciences (WULS-SGGW), Nowoursynowska 159C, 02-776 Warszawa, Poland
2
Department of Pharmaceutical Microbiology, Warsaw Medical University,
Oczki 3, 02-007 Warszawa, Poland
4
Department of Biotechnology, Microbiology and Food Evaluation, Warsaw University of Life Sciences
(WULS-SGGW), Nowoursynowska 166, 02-787 Warszawa, Poland

Abstract: Chemical composition and antibacterial activity of aqueous (ethanolic and methanolic) extracts from
herbs often used in Polish cuisine and traditional herbal medicine including thyme (Thymus vulgaris L.), rose-
mary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), peppermint (Mentha piperita L.) and sage
(Salvia officinalis L.) were compared. The aqueous ethanolic extracts contained slightly higher levels of phe-
nolics compared to the aqueous methanolic extracts. In turn, GC-MS analysis showed that the aqueous
methanolic extracts of thyme, rosemary and sage contained several additional compounds such as eugenol or
ledol. The present studies also indicated that the bacterial species applied in the experiment exhibited different
sensitivities towards tested extracts. Staphylococcus aureus strains were found to be the most sensitive bacteria
to aqueous (ethanolic and methanolic) rosemary and sage extracts and aqueous methanolic thyme extract.
Klebsiella pneumoniae ATCC 13883 and Proteus vulgaris NCTC 4635 were more susceptible to the aqueous
methanolic thyme extract. However, Listeria monocytogenes 1043S was the most sensitive to the aqueous
ethanolic rosemary extract. Gram-positive bacteria were generally more sensitive to the tested extracts than
Gram-negative ones.

Keywords: spice extracts, total phenolics, GC-MS analysis, antibacterial activity

Herbs, especially these belonging to the of climate, soil composition, the type of solvent used
Lamiaceae family are popular aromatic plants grow- in the extraction process, and also on the volume of
ing in many regions of the world. Some of them are inoculum used and culture medium or the type of
extensively used to enhance the flavor and aroma of strains within the same species of bacteria (5, 6).
foods, and to improve the overall quality of the Some microorganisms has also become resistant to
product (1). They are also the basic source of phyto- the present used antibiotics. The Gram-positive bac-
chemical compounds which have a beneficial effect terium Staphylococcus aureus is still responsible for
on health or play an active role in amelioration of post-operative wound infections, toxic shock syn-
diseases. Many studies showed that herbs from the drome, osteomyelitis and the Gram-negative bac-
Lamiaceae family have a potent antioxidant and terium Escherichia coli causes urinary tract infec-
antibacterial activities, mostly due to the quantity tion (7). Therefore, one of the routine approaches to
and quality of phenolic compounds present in them the research of biologically active substances is the
(2, 3). Among these, eugenol, carvacrol and thymol systematic screening of microorganisms and plants
which are the major components of essential oils, that are sources of many useful therapeutic agents
are primarily responsible for their bactericidal/bac- (8). The main purpose of this study was to verify and
teriostatic properties (4). It was also observed, that compare the chemical composition and characterize
the antimicrobial effect of plant extracts varies from the antibacterial activities of selected herbs extracts
one herb to another in different regions of the world. against Gram-positive and Gram-negative bacteria.
This may be due to many factors such as: the effect It is worth emphasizing, that herbs extracts were

* Corresponding author: e-mail: mariola_kozlowska@sggw.pl; phone: +48 22 593 7614, fax: +48 22 593 76 35

757
758 MARIOLA KOZ£OWSKA et al.

obtained by using methanol (70%) as the solvent adding 5 mL of 20% sodium carbonate in distilled
usually utilized for polyphenol extraction, and water. The mixture was kept at room temperature
ethanol (70%) which is an environmentally friendly in the dark for 1 h and then the absorbance was
solvent and safe for human consumption. Because measured at 765 nm. Total phenolics were
the plant material may contain some varying expressed as mg gallic acid equivalents (GAE)
amounts of bacteria or protozoa, the extracts were per gram of extract. All measurements were per-
prepared by procedure involving the use of organic formed in triplicate.
solvents and elevated temperature.
HPLC analysis
EXPERIMENTAL Herbs extracts were dissolved in ethanol (70%)
and methanol (70%), respectively, and filtered with
Chemicals Supelco Iso-Discô Syringe Tip Filter Unit, PTFE
Reagents for GC-MS and HPLC analysis (ace- membrane, diameter 25 mm, pore size 0.20 µm and
tone, methanol, acetonitrile, phosphoric acid) were subjected to HPLC. The analyses were performed
obtained from Merck (Germany). Standards for using a Shimadzu chromatograph equipped with
HPLC were obtained from ChromaDex. Methanol, autosampler SIL-20, photodiode array detector
ethanol, toluene, dimethyl sulfoxide (DMSO), sodi- SPD-M10A VP DAD and Class VP 7.3 chromatog-
um carbonate and Folin-Ciocalteu reagent were raphy software. A modern C-18 reversed-phase col-
purchased from POCH (Gliwice, Poland) and umn with core-shell technology (Phenomenex
Sigma-Aldrich Chemicals (PoznaÒ, Poland), KinetexÆ 2.6 µm, C18, 100A, 100 ◊ 4.60 mm i.d.)
respectively. All solvents and chemicals were of was used as solid phase. The chromatographic sepa-
analytical grade. ration was carried out using deionized water as
mobile phase, adjusted to pH 3 with phosphoric acid
Plant material and extraction as solvent A and acetonitrile adjusted to pH 3 with
The aerial part of thyme (Thymus vulgaris L.) phosphoric acid as solvent B programmed in gradi-
and rosemary (Rosmarinus officinalis L.), oregano ent. The following conditions were applied: flow
(Origanum vulgare L.), peppermint (Mentha piperi- rate 1.3 mL/min, oven temperature 32OC, total time
ta L.) and sage (Salvia officinalis L.) leaves were of analysis 10 min, injection volume 1 µL. UV-spec-
collected at the beginning of flowering time from tra were recorded between 190 and 450 nm. Peak
the garden-plot near ZamoúÊ, in July 2013. They identification was confirmed by comparison of
were dried at room temperature and then extracted retention time and spectral data with adequate
with ethanol (70%) and methanol (70%), respective- parameters of standards used. Hesperetin-7-gluco-
ly, according to the method described by Koz≥owska side, hesperetin-7-rutinoside and hesperetin were
et al. (9). Briefly, 10 g of each dried plant material detected at 285 nm, caffeic acid at 300 nm, ros-
was mixed with 250 mL of aqueous ethanol and marinic acid at 330 nm, apigenin-7-glucoside, api-
aqueous methanol and heated on water bath for 10 h genin-7-rutinoside and apigenin at 336 nm, luteolin-
at 45OC. Next, the filtration was carried to separate 7-glucoside, luteolin-7-rutinoside and luteolin at
the plant residue and then the solvent was removed 347 nm. The content of the determined compounds
to dryness in a rotary evaporator at 40OC. Obtained was calculated in mg of each compound per gram of
plant extracts were stored frozen until further use dry weight (DW) of extract.
(-26OC). The yields of the herbs extracts were as fol- Characteristic parameters of HPLC analysis
lows: rosemary (aqueous ethanolic ñ E) ñ 21.2%, (calibration equation, linear range, LOD, LOQ, cor-
rosemary (aqueous methanolic ñ M) ñ 19.4%, relation coefficient) are listed in Table 2.
oregano (E) ñ 26.6%, oregano (M) ñ 30.6%, thyme Commercially available standards were separately
(E) ñ 25.3%, thyme (M) ñ 31.2%, sage (E) ñ 18.4%, dissolved in methanol according to the
sage (M) ñ 24.3%, peppermint (E) ñ 12.3%, pepper- ChromaDexís Tech Tip 0003. The stock solutions
mint (M) ñ 19.2%, were injected (0.5, 1.0, 2.0, 5.0 and 10 µL) on a col-
umn in six replicates (n = 6) using SIL-20AC HT to
Total phenolic content generate a five-point calibration curve for the each
Total amount of phenolic compounds was standard compounds separately, using LC Solution
measured in the herbs extracts with a standard Version 1.21 SP1 chromatography software.
Folin-Ciocalteu reagent (10) which was added Standard curve parameters, LOD and LOQ were
(0.5 mL) to 1 mL of each spice extract solution calculated with statistical service e-stat (http://www.
dilluted with water. The color was developed by chem.uw.edu.pl/stat/e-stat/).
 Chemical composition and antibacterial acrivity of some... 759

GC-MS analysis matographic paper) were dripped with tested herbs

Extracts were analyzed by GC-MS using an extract solutions to load 2 mg of a given extracts per
Agilent 7890A gas chromatograph coupled with an disc, and were placed on the agar plates uniformly
Agilent 5975C mass spectrometer. An HP-5MS cap- inoculated with the test microorganisms. The paper
illary column (30 m ◊ 250 µm ◊ 0.25 µm film thick- discs with 70% ethanol and 70% methanol were
ness) was used for gas chromatographic separation. used as negative control. Nitrofurantoin was used as
The GC oven temperature was maintained at 40OC a positive control (300 µg/disc). The diameter of the
for 5 min, then programmed at 10OC/min to 300OC clear zone surrounding the disc after 18 h incubation
for 10 min. The injection volume was 1 µL with a at 35 ± 2.5OC was the measure of antimicrobial
split ratio of 1 : 5. Helium was used as carrier gas at activity of a given extracts. For MIC (minimum
a flow rate 1 mL/min. The mass spectrometer was inhibitory concentration) determination, the solu-
operated in electron impact mode with ionization tions of the tested spice extracts were prepared in
electron energy of 70 eV. Acquired data were dimethyl sulfoxide (DMSO), and were added to liq-
processed with MSD Chemstation E. 02.00.493 uid solution of the agar medium to form two-fold
software. All compounds were identified from their serial dilutions covering the range from 31.3 to 2000
mass spectra, by comparison of their retention times mg/L. The plate of agar medium with DMSO was
(RT) with those of standard compounds and with the used as a control of the bacterial growth in the pres-
spectrum of the known components stored in the ence of the solvent, the so-called negative control.
National Institute Standard and Technology (NIST) Next, solidified agar plates were inoculated using 2
library. µL aliquotos. The final inoculum of all studied
organisms was 104 colony forming units (CFU)/mL,
Antibacterial activity except the final inoculum of E. faecalis ATCC
Test microorganisms 29212 which was 105 CFU/mL.
Extracts were individually tested against a
panel of microorganisms including Gram-positive Statistical analysis
bacteria: Staphylococcus aureus ATCC 6538P, S. Data were analyzed using analysis of variance
aureus NCTC 4163, S. aureus ATCC 25923, S. epi- (ANOVA) with post-hoc Tukeyís HSD at the confi-
dermidis ATCC 12228, Enterococcus faecalis dence level α = 0.05 (Statgraphics Centurion XV
ATCC 29212, E. hirae ATCC 10541, Bacillus sub- software, Statpoint Inc., Warrenton, Virginia, USA).
tilis ATCC 6633, Geobacillus stearothermophilis
ATCC 7953 and Gram-negative bacteria: RESULTS AND DISCUSSION
Escherichia coli ATCC 25922, E. coli NCTC 8196,
Klebsiella pneumoniae ATCC 13883, K. pneumoni- Total phenolics content, HPLC and GC/MS
ae ATCC 700603, Proteus vulgaris NCTC 4635, P. analysis
vulgaris ATCC 13315, P. mirabilis ATCC 12453, The total phenolics content in herbs extracts
Listeria monocytogenes 1043S, Pseudomonas was analyzed by the Folin-Ciocalteu method and
aeruginosa ATCC 27853, P. aeruginosa NCTC varied from 137.60 to 228.30 mg gallic acid per
6749, Stenotrophomonas maltophilia ATCC 13637, gram of extract (Table 1). The highest phenolic con-
Bordetella bronchiseptica ATCC 4617. The tent was found in aqueous ethanolic extract of R.
microorganisms were obtained from the own collec- officinalis and aqueous methanolic extract of S.
tion of the Department of Pharmaceutical officinalis but the lowest in aqueous methanolic
Microbiology, Medical University of Warsaw extract of O. vulgare. Generally, the aqueous
(Warszawa, Poland). ethanolic extracts of plant spices extracts had the
slightly higher total phenolic content compared to
Antimicrobial screening the aqueous methanolic extracts with the exception
Antibacterial activity of herbs extracts was of extracts from S. officinalis and M. piperita. The
examined by the disc-diffusion method and the MIC values obtained in present study were slightly high-
method under standard conditions using Mueller- er for rosemary extracts, and lower for thyme and
Hinton II agar medium (Beckton Dickinson) accord- peppermint extracts, compared to those reported by
ing to the guidelines established by the CLSI (11, Gramza-Michalowska et al. (13). Gallego et al. (14)
12). For the disc-diffusion assay, the solutions of reported a value of 219 mg GAE/g of lyophilized
tested herbs extracts were prepared in methanol powder after ethanol extraction of rosemary leaves
(70%) or ethanol (70%), respectively. Sterile filter and 334 mg GAE/g of lyophilized powder for thyme
paper discs (9 mm diameter, Whatman No. 3 chro- leaves. These results were higher for thyme extract
 760

Table 1. Contents of determined phenolic compounds (mg/g DW of extract) and total phenolics (mg GAE/g of extract) of the aqueous ethanolic (E) and aqueous methanolic (M) herbs extracts.

Herbs extracts
Compounds R. officinalis O. vulgare T. vulgaris S. officinalis M. piperita
E M E M E M E M E M
a,b a,b a a b,c b,c c c a,b a,b
Caffeic acid 1.45 ± 0.07 1.43 ± 0.02 1.43 ± 0.02 1.42 ± 0.07 1.46 ± 0.69 1.46 ± 0.06 1.46 ± 0.19 1.51 ± 0.07 1.46 ± 0.02 1.41 ± 0.01
d f a b c h e g c a,b
Rosmarinic acid 50.43 ± 0.41 60.89 ± 0.12 25.02 ± 1.85 28.42 ± 0.97 35.33 ± 1.29 103.29 ± 1.14 56.62 ± 1.35 70.66 ± 1.47 34.52 ± 0.91 27.85 ± 0.69
a b c d
Luteolin - - - - - - 0.86 ± 0.02 0.48 ± 0.02 4.80 ± 0.15 2.88 ± 0.18
Luteolin- a,b c d e f g d a
- - 10.08 ± 0.89 13.53 ± 0.23 16.11 ± 0.36 38.30 ± 0.57 41.29 ± 0.23 49.65 ± 0.15 16.46 ± 0.41 8.58 ± 0.87
7-O-glucoside
Luteolin- a b
- - - - - - - - 36.58 ± 0.81 19.42 ± 1.08
7-O-rutinoside
a a b b,c
Apigenin - - - - - - 7.64 ± 0.21 7.93 ± 0.19 7.49 ± 0.37 6.29 ± 0.05
Apigenin- a a,b c c
- - - - - - 9.49 ± 0.29 11.68 ± 0.45 3.98 ± 0.06 3.47 ± 0.06
7-O-glucoside
Apigenin- a a,b c c,d
- - - - - - 10.21 ± 0.51 13.06 ± 0.62 4.08 ± 0.13 3.68 ± 0.04
7-O-rutinoside
a a,b
Hesperetin - - - - - - - - 15.14 ± 0.13 14.00 ± 0.04
MARIOLA KOZ£OWSKA et al.

Hesperetin- a b
- - - - - - - - 36.81 ± 1.03 27.73 ± 0.15
7-O-glucoside
Hesperetin- a b
- - - - - - - - 16.78 ± 0.65 12.93 ± 0.03
7-O-rutinoside
f c d,e a e d b e,f c b,c
Total phenols 228.30 ± 5.30 184.40 ± 3.40 204.60 ± 4.40 137.60 ± 3.20 217.60 ± 5.70 200.40 ± 5.20 175.20 ± 6.30 225.90 ± 8.90 184.70 ± 2.90 179.90 ± 2.30
Values represent the mean ± standard deviation; Means with different lowercase letters in the same row denote a significant differences at the level α = 0.05; ñ = not detected.
 Chemical composition and antibacterial acrivity of some... 761

than those found in the present study. However, GC-MS analysis showed that among the
MarcinË·k et al. (15) indicated significantly higher detected compounds in the aqueous ethanolic and
amount of total phenols in methanol extract from aqueous methanolic extracts of thyme, oregano
oregano in comparison with thyme and sage. These and peppermint 2,3-dihydro-3,5-dihydroxy-6-
differences may be mainly attributed to diversities methyl-4(H)-pyran-4-one (DDMP) was found.
of the analytical methods, extraction methods of This compound is not a typical constituent of alco-
spices and herbs and their geographic origin. hol extracts of herbs and spices from the
Among selected phenolics, identified in both herbs Lamiaceae family, but its presence in both leaf and
extracts by HPLC method (Table 1), rosmarinic acid flower extracts of Lantana camara might be the
was the most predominant phenolic compound. The reason for their larvicidal activity (20). Other
highest content of rosmarinic acid was observed in important compounds identified in extracts (Table
aqueous methanolic extract of T. vulgaris and S. 3) from thyme, oregano and rosemary were thy-
officinalis, while the extracts from oregano and pep- mol, carvacrol, camphor, borneol and α-terpineol.
permint showed the lowest values. Its content in They are also major components of essential oils
examined Lamiaceous species was comparable to obtained from spices and herbs exhibiting antibac-
the results of other researchers (16, 17). Another terial activity (21). Moreover, the aqueous ethano-
phenolic acid that was identified in all spices lic extract of oregano contained n-heptanoic acid,
extracts was caffeic acid (18). Its concentration was 3-methoxy-2,4,6-trimethylphenol and 2-methyl-4-
similar in both, the aqueous ethanolic and aqueous (4-methylcyclohexyl)butanoic acid. However, in
methanolic extracts. In addition to phenolic acids, M. piperita extract, palmitic acid, α-linolenic acid
extracts from M. piperita contained the following and phytol were present. Phytol was also the char-
flavones and flavanones: hesperetin, hesperetin-7- acteristic constituent of alcohol extract of
glucoside, hesperetin-7-rutinoside, apigenin, api- Plectranthus amboinicus (Lour) belonging to the
genin-7-glucoside, apigenin-7-rutinoside, luteolin, mint family (Lamiaceae) (22). It is a diterpene
luteolin-7-glucoside, luteolin-7-rutinoside. However, which showed antibacterial activity against
luteolin, apigenin, apigenin-7-glucoside and api- Staphylococcus aureus by causing damage to cell
genin-7-rutinoside were present in S. officinalis membrane, leading to a leakage of potassium ions
extracts. These biologically active ingredients from from bacterial cells (20). Generally, GC-MS
hydrophilic extracts of thyme, sage, oregano were analysis showed that in comparison to aqueous
previously described by other researchers (16, 19). ethanolic extracts, the aqueous methanolic
Of special interest are flavones, which were evaluat- extracts, except extract from peppermint, con-
ed as antioxidants using the linoleic acid oxidation tained several additional compounds such as
system and effectively protected biological systems eugenol, 2,1,3-benzothiadiazole or ledol. Ledol, β-
against various oxidative stresses by inhibition of cubebene and the sesquiterpenes are also the major
superoxidase anion production in the xanthine/xan- compounds in the essential oil composition of
thine oxidase system. Judean sage (Salvia judaica Boiss.) (23).

Table 2. Characteristic parameters of the HPLC analysis.

Standard Calibration equation R2 (n = 6) Linear range (mg/mL) LOD LOQ

(mg/mL) (mg/mL)
Caffeic acid y = 3E-07x + 0.2899 0.9997 0.4992 - 9.9840 0.3069 0.5115
Rosmarinic acid y = 3E-06x + 0.4653 0.9998 1.1210 - 22.418 0.5843 0.9739
Luteolin y = 1E-05x + 0.2504 0.9999 2.2700 - 45.400 0.7101 1.1835
Luteolin-7-O-glucoside y = 1E-05x + 0.2285 0.9999 2.3850 - 47.700 0.6425 1.0709
Luteolin-7-O-rutinoside y = 1E-05x + 0.2285 0.9999 2.3850 - 47.700 0.6425 1.0709
Apigenin y = 3E-06x + 0.4001 0.9998 0.9955 - 19.910 0.5136 0.8560
Apigenin-7-O-glucoside y = 1E-05x + 0.5126 0.9999 2.4425 - 48.850 0.6344 1.0574
Apigenin-7-O-rutinoside y = 1E-05x + 0.5126 0.9999 2.4425 - 48.850 0.6344 1.0574
Hesperetin y = 2E-06x + 0.4712 0.9997 0.9450 - 18.900 0.5764 0.9607
Hesperetin-7-O-glucoside y = 2E-06x + 0.4712 0.9997 0.9450 - 18.900 0.5764 0.9607
Hesperetin-7-O-rutinoside y = 2E-06x + 0.4712 0.9997 0.9450 - 18.900 0.5764 0.9607a
Table 3. Compounds identified in aqueous ethanolic (E) and aqueous methanolic (M) herbs extracts by GC-MS.
762

[%]
Molecular
Compounds RT MW R. officinalis O. vulgare T. vulgaris S. officinalis M. piperita
formula
E M E M E M E M E M
1 4-Hydroxy-4-methyl-2-pentanone 7.161 116 C6H12O12 72.80 54.58 32.18 28.87 73.69 19.72 90.27 47.69 88.04 81.06
2 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-
pyran-4-one 13.449 144 C6H8O4 -b - 2.96 4.22 2.57 4.43 - - 2.11 2.36
3 Camphor 13.507 152 C10H16O 1.35 2.03 - - - - - - - -
4 Borneol 13.849 154 C10H18O 2.31 2.38 - - - - - - - -
5 α-Terpineol 14.223 154 C10H18O 1.92 2.25 - - - - - - - -
6 5-(Hydroxymethyl)-2-
furancarboxaldehyde 14.770 126 C6H6O3 - - - 2.22 - - - - - -
7 Thymol 15.667 150 C10H14O - - 24.36 16.29 23.74 3.71 - - - -
8 Indole 15.768 117 C8H7N - - 1.33 0.82 - - - - - -
9 2-Methyl-4-(4-methylcyclohexyl)
butanoic acid 16.001 198 C12H22O2 - - 4.96 - - - - - - -
10 2,6-Dimethoxyphenol 16.542 154 C8H10O3 - - - - - 2.74 - - - -
11 2,1,3-Benzothiadiazole 17.696 136 C6H4N2S - 5.48 - 1.89 - 4.46 - - - -
12 o-Methoxy-α,α-dimethylbenzyl alcohol 17.753 166 C10H14O2 - - - - - 7.85 - - - -
13 Ledol 19.723 222 C15H26O - - - - - - - 5.62 - -
14 Propyl-propanedioic acid 19.856 146 C6H10O4 - 14.45 - - - - - - - -
MARIOLA KOZ£OWSKA et al.

15 Butyraldehyde semicarbazone 19.859 129 C5H11N3O 4.63 - - - - 5.89 - - - -

16 n-Heptanoic acid 19.938 130 C7H14O2 - - 17.06 - - - - - - -
19 Nitro-L-arginine 19.967 219 C6H13N5O4 - - - 15.93 - - - - - -
20 Palmitic acid 23.423 256 C16H32O2 - - 1.29 - - 0.60 - 1.30 2.24 3.14
21 1-Naphthalenepropanol,
α-ethenyldecahydro α-5,5,8a
tetramethyl-2-methylene-, 24.584 290 C20H34O - - - - - - 6.53 22.71 - -
[1S-[1α(S*),4aβ,8a. α ]]-
22 Phytol 24.911 296 C20H40O - - - - - - - - 1.64 3.38
23 α-Linolenic acid 25.143 278 C18H30O2 - - 1.29 - - 1.49 - 2.09 2.69 4.92
 Chemical composition and antibacterial acrivity of some... 763

Antibacterial activity
M. piperita
M

-
-
-

-
-
-
-
-
The antibacterial activities of the herbs
extracts from the tested samples in terms of
E
the minimum inhibitory concentrations (MIC)
-
-
-

-
-
-
-
-
and the diameters of inhibition zones (IZ) are
S. officinalis

1.75

3.90
reported in Table 5 and Table 4, respectively.
M

-
-

-
-
-
-
The solvents used in this studies, 70%
methanol and 70% ethanol in the disc-diffu-
sion assay as well as DMSO in the MIC deter-
E

-
-
-

-
-
-
-
-
mination method, did not inhibit growth of
tested bacterial strains. According to the
3.58

1.36

5.74
results, aqueous methanolic and aqueous
M

-
T. vulgaris

ethanolic extract of R. officinalis showed the

[%]

broader spectrum activity against all tested

Gram-positive bacteria (IZ, 12-19 mm and
E

-
-
-

-
-
-
-
-
MIC, 0.125-0.5 mg/mL) and against four
Gram-negative bacteria (IZ, 11-17 mm and
MIC, 0.25-0.5 mg/mL). Among Gram-posi-
2.01
M

-
-
-

-
-

-
-

tive bacteria, E. faecalis and E. hirae were the

O. vulgare

most sensitive bacteria to the aqueous ethano-

lic rosemary extract but among Gram-negative
8.47
E

-
-
-

-
-
-
-

bacteria, E. coli ATCC 25922, K. pneumoniae

ATCC 700603, P. vulgaris NCTC 4635, P.
aeruginosa ATCC 27853 and S. maltophilia
were more susceptible to aqueous methanolic
3.14

4.93

3.57
M

-
-
-
-
-
R. officinalis

extract. On the other hand, the aqueous

methanolic extract of T. vulgaris appeared
more active against microorganisms studied
3.60

than the aqueous ethanolic extract. It is inter-

E

-
-

-
-
-
-
-

esting to note that aqueous methanolic extract

from T. vulgaris was the only effective extract
C19H14N2O
Molecular

C9H7IN20
C22H30O3

C15H22N6
C10H12O2
C10H14O2
C10H14O2
C10H14O2
C10H14O
formula

against K. pneumoniae with MIC value of 0.5

mg/mL. The distinct antibacterial activity of
both S. officinalis extracts was observed
against S. aureus strains, S. epidermidis and B.
MW

150
342

286

286

286
164
166
166
166

bronchiseptica with a MIC value of 0.5

RT ñ Retention time (min); MW ñ Molecular weight; ñ = not detected.

mg/mL and B. subtilis, and G. stearother-

mophilis with MIC value of 0.25 mg/mL.
27.463
26.236

27.624

28.261

28.264
28.919
29.859
29.860
29.876

Also, both M. piperita extracts were active

RT

against G. stearothermophilis (MIC, 0.25

mg/mL) but the aqueous ethanolic extract was
Di(6,7,8,9-tetrahydro-5H-[1,2,4]triazolo

more effective against P. aeruginosa NCTC

2-(1,1-Dimethylethyl)-1,4-benzenediol
6-Iodo-2-methylquinazolin-4(3H)-one

6749. However, the extracts from oregano

4-Methoxy-2,3,6-trimethyl-phenol
3-Methoxy-2,4,6-trimethyl-phenol
4,6-Bis(1,1í-dimethylethyl)-2í,5í-

showed moderate antibacterial activity against

3-(4-Methoxyphenyl)benzo[f]
dimethoxy-1,1í-biphenyl-2-ol

all tested bacteria. The growth of P. aerugi-

[4,3-a]azepin-3-yl)methane

nosa, an opportunistic pathogen, responsible

Compounds

in healthcare institutions, for serious and often

fatal nosocomial infections and an indicator of
environmental contamination, was inhibited
quinazoline

by both T. vulgaris extracts and aqueous

Carvacrol

Eugenol

ethanolic extract from M. piperita. In turn, S.

Table 3. Cont.

maltophilia - a β-lactam antibiotics resistant

rods that is closely related to the Pseudomonas
species, was the most sensitive microorganism
24

25
26
27

28

29
30
31
32
 764

Table 4. Antimicrobial activity of the aqueous ethanolic (E) and aqueous methanolic (M) herbs extracts.

Herbs extracts/Zones of inhibition (IZ) (mm)

Microorganism O. vulgare T. vulgaris R. officinalis M. piperita S. officinalis Nitrofurantoin*
E M E M E M E M E M
Gram-positive bacteria
S. aureus ATCC 6538P 11 11 16 15 19 18 15 12 16 15 24
S. aureus NCTC 4163 - - 11 14 16 17 10 - 12 15 23
S. aureus ATCC 25923 - - 13 14 17 17 11 11 12 13 22
S. epidermidis ATCC 12228 11 11 13 15 19 19 12 12 15 15 29
E. faecalis ATCC 29219 - - - - 12 12 - - - - 22
E. hirae ATCC 10541 - - - - 12 12 - - - - 19
B. subtilis ATCC 6633 11 11 13 13 16 16 13 10 14 13 28
G. stearothermophilis ATCC 7953 11 11 13 13 16 16 13 11 14 13 27
Gram-negative bacteria
E. coli ATCC 25922 - - - - - - - - - - 24
E. coli NCTC 8196 - - - - - - - - - - 24
K. pneumoniae ATCC 13883 - - 12 10 10 10 11 - - 10 23
K. pneumoniae ATCC 700603 - - - - - - - - - - 12
P. vulgaris NCTC 4635 - - 12 12 16 17 - - - 11 17
MARIOLA KOZ£OWSKA et al.

P. vulgaris ATCC 13315 - - 12 12 13 13 - - 10 11 -

P. mirabilis ATCC 12453 - - 12 12 13 13 - - - 10 10
L. monocytogenes 1043 S - - - - 14 14 - - 12 12 23
P. aeruginosa ATCC 27853 - - - - - - - - - - -
P. aeruginosa NCTC 6749 - - 11 11 - - 11 10 - - -
S. maltophilia ATCC 13637 - - 11 11 - 11 12 10 - - -
B. bronchiseptica ATCC 4617 12 13 16 17 13 15 15 13 10 14 -
*References compound, 300 µg per disc (Mast Diagnostics, Merseyside, UK); ñ = not detected.
Table 5. Antimicrobial activity of the aqueous ethanolic (E) and aqueous methanolic (M) herbs extracts.

Herbs extracts/MIC (mg/mL)

Nitrofurantoin*
Microorganism O. vulgare T. vulgaris R. officinalis M. piperita S. officinalis
(µg/mL)
E M E M E M E M E M
Gram-positive bacteria
S. aureus ATCC 6538P 2c 2c 1b 0.5a 0.5a 0.5a 2c 2c 0.5a 0.5a 25
c c b b a a c c a
S. aureus NCTC 4163 2 2 1 1 0.5 0.5 2 2 0.5 1b 25
c c b a a a c c b b
S. aureus ATCC 25923 2 2 1 0.5 0.5 0.5 2 2 1 1 25
S. epidermidis ATCC 12228 1c 1c 1c 0.5b 0.25a 0.25a 1c 1c 0.5b 0.5b 12.5
E. faecalis ATCC 29219 >2d >2d >2d >2d 0.25a 1b >2d >2d 2c 2c 12.5
d d d d a b d d c
E. hirae ATCC 10541 >2 >2 >2 >2 0.25 1 >2 >2 2 2c 25
d d c c a a d d b b
B. subtilis ATCC 6633 2 2 0.5 0.5 0.125 0.125 2 2 0.25 0.25 12.5
G. stearothermophilis ATCC 7953 2d 2d 0.5c 0.5c 0.125a 0.125a 0.25b 0.25b 0.25b 0.25b 12.5
Gram-negative bacteria
E. coli ATCC 25922 >2c >2c 2b 1a 2b 1a >2c >2c 2b 2b 6.25
d d b b a a b b b a
E. coli NCTC 8196 >2 >2 >2 >2 2 2 >2 >2 >2 2 12.5
K. pneumoniae ATCC 13883 2c 2c 1b 0.5a 1b 1b 1b 1b 1b 1b 25
c c c c b a c c c b
K. pneumoniae ATCC 700603 >2 >2 >2 >2 2 1 >2 >2 >2 2 400
P. vulgaris NCTC 4635 2c 2c 1b 0.5a 1b 0.5a 2c 2c 1b 1b 100
b b a a a a b b a a
P. vulgaris ATCC 13315 2 2 1 1 1 1 2 2 1 1 >400
P. mirabilis ATCC 12453 2b 2b 1a 1a 1a 1a 2b 2b 1a 1a >400
Chemical composition and antibacterial acrivity of some...

e e d c a b e e c c
L. monocytogenes 1043 S >2 >2 2 1 0.25 0.5 >2 >2 1 1 50
P. aeruginosa ATCC 27853 >2c >2c 2b 1a 2b 1a 2b 2b 2b 2b >400
P. aeruginosa NCTC 6749 2c 2c 0.5a 0.5a 1b 1b 0.5a 1b 1b 1b >400
c c b a b a c c b
S. maltophilia ATCC 13637 2 2 1 0.5 1 0.5 2 2 1 1b >400
b b a a a a b b a a
B. bronchiseptica ATCC 4617 1 1 0.5 0.5 0.5 0.5 1 1 0.5 0.5 >400
*References compound, 300 µg per disc (Mast Diagnostics, Merseyside, UK); Means with different lowercase letters in the same row denote a significant differences at the level α = 0.05.
765
766 MARIOLA KOZ£OWSKA et al.

to the aqueous methanolic extracts from thyme and cantly higher amount of rosmarinic acid that is the
rosemary. However, the growth of L. monocyto- predominant nonvolatile polyphenol is present in the
genes which is one of the most virulent foodborne aqueous methanolic plant extracts. The aqueous
pathogen, was reduced by the aqueous ethanolic and methanolic plant extracts also contained several
aqueous methanolic rosemary extracts (MIC, 0.25 additional compounds that are important con-
and 0.5 mg/mL, respectively). In this paper, the stituents of plant essential oils exhibiting antibacter-
aqueous methanolic extract of T. vulgaris inhibited ial activity. The presence of these compounds may
the growth of S. aureus strains similarly to Al-Bayati have an influence on greater antibacterial activity of
research (24). Its antibacterial activity could be thyme aqueous methanolic extract as compared to
associated to the presence of phenolic compounds the aqueous ethanolic extract. The antibacterial
such as carvacrol and thymol, which are the most activity of the remaining both types of extracts did
active constituents of many spices, especially essen- not show statistically significant differences.
tial oils. Moreover, the occurrence of different kind Therefore, the aqueous ethanolic plant extracts
of chemical compounds in plant extracts can pro- could also be used as natural antibacterial agents
mote their synergistic effect and results in a greater instead of aqueous methanolic extracts.
antimicrobial activity. Shan et al. (25) also found
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