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Antimicrobial and Antioxidant Properties of Rosemary and Sage (Rosmarinus

officinalis L. and Salvia officinalis L., Lamiaceae) Essential Oils

Article in Journal of Agricultural and Food Chemistry · October 2007

DOI: 10.1021/jf0715323 · Source: PubMed

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 J. Agric. Food Chem. 2007, 55, 7879–7885 7879

Antimicrobial and Antioxidant Properties of

Rosemary and Sage (Rosmarinus officinalis L. and
Salvia officinalis L., Lamiaceae) Essential Oils
BILJANA BOZIN,*,† NEDA MIMICA-DUKIC,‡ ISIDORA SAMOJLIK,§ AND
EMILIJA JOVIN‡
Faculty of Medicine, Department of Pharmacy, University of Novi Sad, Hajduk Veljkova 3,
21000 Novi Sad, Serbia, Faculty of Sciences, Deprtment of Chemistry, University of Novi Sad, Trg
D. Obradovica 3, Novi Sad, Serbia, and Faculty of Medicine, Department of Pharmacology and
Toxicology, University of Novi Sad, Hajduk Veljkova 3, Novi Sad, Serbia

The essential oils of rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis L.) were analyzed
by means of gas chromatography–mass spectrometry and assayed for their antimicrobial and
antioxidant activities. Antimicrobial activity was tested against 13 bacterial strains and 6 fungi, including
Candida albicans and 5 dermatomycetes. The most important antibacterial activity of both essential
oils was expressed on Escherichia coli, Salmonella typhi, S. enteritidis, and Shigella sonei. A significant
rate of antifungal activity, especially of essential oil of rosemary, was also exhibited. Antioxidant activity
was evaluated as a free radical scavenging capacity (RSC), together with the effect on lipid
peroxidation (LP). RSC was assessed by measuring the scavenging activity of essential oils on 2,2-
diphenyl-1-picrylhydrazil (DPPH) and hydroxyl radicals. Effects on LP were evaluated following the
activities of essential oils in Fe2+/ascorbate and Fe2+/H2O2 systems of induction. Investigated essential
oils reduced the DPPH radical formation (IC50 ) 3.82 µg/mL for rosemary and 1.78 µg/mL for sage)
in a dose-dependent manner. Strong inhibition of LP in both systems of induction was especially
observed for the essential oil of rosemary.

KEYWORDS: Rosmarinus officinalis; Salvia officinalis; essential oil; chemical composition; antimicrobials;
antioxidants

INTRODUCTION organoleptic properties. Also, they have great potential in the

emerging nutritious industry, because these materials are often
The oxidation of lipids in food, cosmetic, and pharmaceutical
considered as food and medicines, as well, and are used in
products, together with the growth of undesirable microorgan-
prevention and curative treatments throughout the world (4).
isms results in the development of spoilage, off-flavor, rancidity,
Their use in phytotherapy is mostly related to different activities
and deterioration, rendering such products unacceptable for
of secondary biomolecules, especially essential oils, which are
human consumption. It has been estimated that each year as
antimicrobial, spasmolytic, carminative, hepatoprotective, an-
many as 30% of people in industrialized countries suffer from
tiviral, anticarcinogenic, etc. (5, 6). Besides, many essential oils
a foodborne disease. Thus, in the year 2000, at least two million
and isolated compounds were recently qualified as very strong
people died from diarrheal disease worldwide (1). Furthermore,
natural antioxidants (7–10) and proposed as potential substitutes
overproduction of free radicals in organisms and lipid peroxi-
for synthetic antioxidants.
dation in cell membranes has been implicated in various
pathophysiological dissorders, including cardiovascular diseases, Thus, many aromatic plants are today considered as the
mutagenesis, diabetes, ischemia-reperfusion injury, coronary most important sources for the extraction of compounds with
atherosclerosis, Alzheimer’s disease, and cancerogenesis, as well strong antioxidant activity. Rosemary (Rosmarinus officinalis
as the aging process (2, 3). L., Lamiaceae) and sage (SalVia officinalis L., Lamiaceae)
Since ancient times, aromatic herbs and spices have been are two spices widely used in folk medicine, cosmetics,
added to different types of food to improve the flavor and phytopharmacy, and the flavoring of food products (6).
Furthermore, they are defined as very powerful aromatic
plants. Many studies have been focused on various biological
* To whom correspondence should be addressed. Tel/Fax: +39121
422 760. E-mail: [email protected].
activities of the secondary metabolites in these two
†
Department of Pharmacy. spices (5, 7, 11–13). It was found that they possess the best
‡
Deprtment of Chemistry. antioxidant activity among the wide range of herbs and spices
§
Department of Pharmacology and Toxicology. tested (14–17). Furthermore, rosemary is the only spice
10.1021/jf0715323 CCC: $37.00  2007 American Chemical Society
Published on Web 08/21/2007
7880 J. Agric. Food Chem., Vol. 55, No. 19, 2007 Bozin et al.

commercially available for use as an antioxidant in Europe (Dalynn Biological Inc.; Calgary, Canada). The inocula were stored at
and the United States, marketed in an oil soluble form, in +4 °C for further investigations. Dilutions of the inocula were cultured
dry powder, and also in water dispersible or water miscible on solid Malt agar (MA) (Torlak) to verify the absence of contamination
formulations. Phenolic compounds such as carnosol, car- and to check the viability of the inoculum. The minimal inhibitory
concentration (MIC) determination was performed by a serial dilution
nosoic acid, rosmanol, rosmadial, epirosmanol, rosmadiphe-
technique using 96-well microtiter plates. Investigated samples were
nol, rosmarinic acid, etc. (14, 16–18) are considered to have dissolved in Malt medium broth (MB) agar (Torlak) with fungal
antioxidant ability. However, data regarding this activity of inoculum to achieve concentrations of 1.0–5.0 µL/mL. The microplates
essential oils of rosemary and sage, are not abundant and were incubated for 72 h at 28 °C. The lowest concentrations without
the methods for determination are different (11, 15). On the visible growth at the binocular microscope were defined as the
contrary, the antimicrobial activity of the rosemary and sage concentrations that completely inhibited the fungal growth (MIC). The
essential oils is well documented (12, 15, 19–22). But, these minimal fungicidal concentrations (MFC) were determined by a serial
investigations are not so often performed with a precisely subcultivation of 2 µL into microtiter plates containing 100 µL of MB
defined chemical composition of the essential oil in question. per well and further incubation for 72 h at 28 °C. The lowest
Furthermore, new examinations of antimicrobial activity on concentration with no visible growth was defined as the MFC, indicating
99.50% killing of the original inoculum. Bifonazole (in dilution of 1
a wider spectrum of microorganisms, including some new
g/100 mL of ethanol) was used as a synthetic antimycotic for a positive
multiresistant strains of bacteria and fungi, could help the control.
pharmaceutical industry in synthesis or semisynthesis of new EValuation of Antibacterial ActiVity. A collection of 13 test organ-
antibiotics (23). isms, 5 Gram-positive and 8 Gram-negative bacterial strains, was used.
In this study, the antioxidant activity and antimicrobial effects Eight microorganisms of the American type of Culture Collection
of essential oils of rosemary and sage (Rosmarinus officinalis (ATCC) and five microorganisms of the Institute of Public Health,
L. and SalVia officinalis L., Lamiaceae) against some pathogen Faculty of Medicine, University of Novi Sad, Serbia, were isolated
strains of bacteria and fungi are reported. The chemical directly from the patients (IPH), including four multiresistant strains
characterization of the investigated essential oils was performed (IPH-MR) were included in those groups. The source of the bacterial
strains is shown in Table 3. All test organisms were stored at +4 °C
by gas chromatography–mass spectrometry (GC-MS).
on Mueller-Hinton (MH) agar (Torlak) slants, subcultured every two
weeks, and checked for purity.
MATERIALS AND METHODS
For the evaluation of the antibacterial activities of the essential oils,
Plant Material. Leaves of cultivated plants of rosemary (Rosmarinus the hole–plate agar diffusion method was used as described before (9).
officinalis L., Lamiaceae) were collected in July of 2005 in the The bacterial strains were grown on MH slants overnight at 37 °C and
Vojvodina province, Republic of Serbia. Leaves of wild-growing sage checked for purity. After incubation, the bacterial cells were washed
(SalVia officinalis L., Lamiaceae) were collected also in July of 2005 from the surface of agar and suspended in sterile 0.1 M phosphate buffer
in the Sićevo gorge, in south Serbia. Voucher specimens of collected (PB) containing 167 mM NaCl (167 mM NaCl–PB; pH ) 7.4). A
plants (rosemary no. 1980 and sage no. 1979) were confirmed and density of the bacterial suspensions was determined by the McFarland
deposited at the Herbarium of the Department of Biology and Ecology nefelometer (Dalynn Biological Inc.). The samples contained about 2
(BUNS), Faculty of Natural Sciences, University of Novi Sad. × 107 colony-forming units (CFU)/mL. Spreading of 0.1 mL of
Essential Oil Isolation. Air-dried leaves of rosemary and sage were bacterial suspension seeded the surfaces of MH agar plates. The holes
submitted to hydrodistillation according to ref 24, using n-hexane as a 5 mm in diameter on the agar surface were punched, and 15 µL of the
collecting solvent. The solvent was removed under vacuum, and the tested essential oils (50% and 20% solutions in n-hexane) was put in
quantities of the essential oils were determined gravimetrically. them. The plates were incubated overnight at 37 °C, and the diameter
Essential Oil Analysis. Qualitative and quantitative analysis of the of the resulting zone of inhibition was measured. The evaluation of
investigated essential oils was carried out using a Hewlett-Packard the antibacterial activities of the essential oils was carried out in five
5973–6890 GC-MS system, operating in electron ionization (EI) mode repetitions. Penicillin (500 and 1000 µg/mL) was used as a positive
at 70 eV, equipped with a split-splitless injector (200 °C) and a flame antibiotic control. The effect of the solvent (n-hexane) on the microbial
ionization detector (250 °C). Helium was used as carrier gas (1 mL/ growth was also analysed.
min), and the capillary columns used were HP 5MS (30 m × 0.25 Antioxidant Activity. Antioxidant properties of the rosemary and
mm; film thickness 0.25 µm). The temperature programs were 60–280 sage essential oils were evaluated as both free radical scavenging
°C at a rate of 3 °C/min and 60–260 °C at a rate of 3 °C/min, capacity (RSC) and protective effect on the lipid peroxidation (LP).
respectively; the split ratio was 1:10. Coelution and MS analysis based Free Radical ScaVenging Capacity (RSC). RSC was evaluated
the identification of individual compounds on comparison of their measuring the scavenging activity of the examined essential oils on
relative retention times with those of authentic samples (Carl Roth the DPPH and OH radicals.
GmbH, Karlsruhe, Germany). For the other components, mostly The DPPH assay was performed as described before (8). Samples
sesquiterpenes and aliphatic compounds, for which authentic substances containing the amount of 3 mL of methanol solution of the essential
were not available, the identification was performed by matching their oil at different concentrations (from 0.25 to 12.5 µg/mL) were mixed
retention indices and mass spectra with NIST/NBS, Wiley libraries with 1 mL of 90 µM DPPH• solution (Sigma Co., St. Louis, MO). The
spectra, and literature data (25). absorbance of the resulting solutions was recorded spectrophotometri-
Antimicrobial Activity. EValuation of Antifungal ActiVity. A cally (Beckman DU-65) at 515 nm after 1 h at room temperature, against
collection of five test organisms of dermatomyceta and Candida the blank (with the same chemicals, except for the sample). The same
albicans, shown in Table 2 was used for the bioassay. Micromycetes procedure was repeated with tert-butylated hydroxytoluene (BHT;
were isolated directly from patients at the Centre for Preventive Fluka, AG, Buchs, Switzerland) as a positive control. For each sample
Medicine, Military Medical Academy (MMA), Belgrade, Serbia, and four replicates were recorded. The percentage of RSC was calculated
maintained on Sabourand agar (SBA) (Torlak, Belgrade, Serbia). using the following equation:
Cultures were stored at +4 °C and subcultured once a month.
In order to investigate the antifungal activity of essential oils, the RSC(%) ) 100 × (Ablank - Asample ⁄ Ablank)
microdilution technique was performed as described before (8). The
fungal spores were washed from the surface of agar plates with sterile The IC50 value, which represented the concentrations of the essential
0.85% saline water containing 0.1% Tween 80 (v/v) (Torlak). The spore oil that caused 50% inhibition of LP, was determined by linear
suspension was adjusted with sterile saline to a concentration ap- regression analysis from the obtained RSC values.
proximately 1.0 × 105 in a final volume of 100 µL per well. A density The scavenging capacity of the essential oils for hydroxyl radicals
of the fungal suspensions was determined by McFarland nefelometer was evaluated by measuring the degradation of 2-deoxy-D-ribose (Fluka
Activities of Rosemary and Sage Essential Oils J. Agric. Food Chem., Vol. 55, No. 19, 2007 7881

AG) with OH radicals, generated in a Fenton reaction. The degradation I(%) ) (A0 – A1) ⁄ A0 × 100
products are the thiobarbituric acid-reactive substances (TBARS), which
could be determined spectrophotometrically at 532 nm (9). All solutions where A0 was the absorbance of the control reaction (full reaction,
and reagents were freshly prepared by dissolution in 0.05 M without the test compound) and A1 was the absorbance in the presence
KH2PO4–K2HPO4 phosphate buffer. In a test tube, 10 µL of pure of the inhibitor.
essential oil (2.13 µg/mL), 50% or 20% solution (1.065 and 0.425 µg/
mL) in n-hexane with 0.125 mL of H2O2, 0.125 mL of FeSO4, and
0.125 mL of 2-deoxy-D-ribose were mixed and filled up with 0.05 M RESULTS AND DISCUSSION
PB, pH ) 7.4, to a volume of 3 mL. After an incubation period of 1 h
at 37 °C, the extent of deoxyribose degradation was measured by the
The content of essential oils (v/w in dry matter) was very
2-thiobarbituric acid (TBA) (Sigma) reaction. A 1.5 mL amount of similar in rosemary (1.18%) and sage (1.34%). The percentage
TBA reagent (10.4 mL of 10% HClO4, 3 g of TBA, and 120 g of 20% composition of the essential oils is presented in Table 1. The
trichloroacetic acid) and 0.2 mL of 0.1 M EDTA (Sigma) were added total number of chemical constituents identified in essential oils
to the reaction mixture, and the tubes were heated at 100 °C for 20 was for rosemary 38 and for sage 31, representing 98.4% and
min. After the mixture was cooled, the absorbance was measured against 93.2% of the total oil content, respectively.
a blank (containing buffer solution only) at 532 nm. Control with The oil distillated from the Rosmarinus officinalis was found
n-hexane instead of sample was also analyzed. to be composed of approximately equal amounts of the
The absorbance at the end of the experiment was used to calculate oxygenated monoterpenes (46.9%) and monoterpene hydrocar-
the inhibition rate of deoxyribose degradation by the essential oil: bons (46.7%). In the oil obtained from the SalVia officinalis,
the oxygenated monoterpenes were found to be the major class
I(%) ) 100 × (Ablank - Asample ⁄ Ablank)
of substances (57.8%), followed by the oxygenated hydrocar-
Four replicates were recorded for each sample. BHT 0.5 M (220.4 µg/ bons (19.5%). Hydrocarbons limonene (21.7%) and R-pinene
mL) was used as a positive control. (13.5%) and oxygenated monoterpenes camphor (21.6%) and
Rapid Screening for ScaVenging Compounds of Essential Oils. For Z-linalool oxide (10.8%) were identified as the main constituents
fast screening of essential oil compounds on RSC, the dot-blot test on in the rosemary essential oil. Besides, in a considerable amount,
thin-layer chromatography (TLC) silica gel F254 aluminium plates monoterpenes borneol (6.2%), camphene (3.9%), sabinene
(Merck, Darmstadt, Germany) stained with the free radical DPPH• was (2.0%), 1,8-cineole (2.1%), and R-terpineol (1.9%) were also
used (8). An appropriate amount of pure essential oil (5 µL) was placed detected (Table 1). In the essential oil of sage, three compounds
on a silica gel plate and eluted with benzene:ethyl acetate (95:5). After were dominant: R-thujone (19.9%), camphor (18.9%), and
drying, the plates were sprayed with a 0.4 mM solution of DPPH• in viridiflorol (17.5%). They were followed by borneol (5.4%),
methanol, using a Desaga Spray Gun. Sprayed plates gave a purple
1,8-cineole (4.2%), β-thujone (3.8%), and bornyl acetate (3.3%).
background with yellow spots at the location of those compounds that
possessed high RSC. Essential oil compounds responsible for scaveng- Earlier data pertaining to rosemary essential oil point out the
ing activity were identified comparing the DPPH-TLC chromatogram persistence of two chemotypes, Morocco/Tunisian (containing
with the control treated with vanillin–sulphuric acid spray reagent. 38–55% of 1,8-cineole) and Spanish, characterized by a high
Determination of Lipid Peroxidation (LP). The extent of LP was amount of two monoterpene hydrocarbons R-pinene (18–26%)
determined by measuring the color of adduct produced in the reaction and camphene (8–12%) and 1,8-cineole (16–25%) (6). The
between TBA and malondialdehyde (MDA), as an oxidation product investigated essential oil, obtained from the plant material
in the peroxidation of membrane lipids, by the TBA assay (26), with cultivated in the Pannonian plane, has a specific chemical
small modifications (9). For this investigation, two systems of induction, composition and could not be categorized in one of the two
Fe2+/ascorbate and Fe2+/H2O2, were used. In both systems, the control previously described chemotypes. On the contrary, the composi-
with n-hexane instead of the sample was also analyzed and it expressed
tion of the sage essential oil is in accordance with the earlier
no activity.
published data (6, 27). The specificity of this essential oil was
The commercial preparation of liposomes “PRO-LIPO S” (Lucas-
a high amount of viridiflorol, a sesquiterpene alcohol that has
Meyer, Hamburg, Germany) pH ) 5–7 was used as a model-system
of biological membranes. The liposomes, 225–250 nm in diameter, were not been reported yet as one of the major compounds in sage
obtained by dissolving the commercial preparation in demineralized oil.
water (1:10), in an ultrasonic bath. Three concentrations of essential Antimicrobial Activity. Antimicrobial activity along with
oils were prepared for the experiment: pure essential oil (2.13 µg/mL) the antioxidant effectiveness of essential oils is one of the most
and 50% and 20% solution in n-hexane (1.065 and 0.425 µg/mL). examined features, important for both food preservation and
In the Fe2+/ascorbate induced LP, 60 µL of suspension of liposomes control of human and animal diseases of microbial origin.
was incubated with 20 µL of 0.01 M FeSO4, 20 µL of 0.01 M ascorbic Numerous reports suggest strong antibacterial and antifungal
acid and 10 µL of essential oil samples in 2.89 mL of 0.05 M activities of a wide range of essential oils, especially those
KH2PO4–K2HPO4 buffer, pH ) 7.4 (3 mL final solution). belonging to the Lamiaceae family (8–10, 12, 15, 19–23, 28).
The reaction mixture in Fe2+/H2O2 induced LP contained 30 µL of However, in order to get more relevant data about the influence
a suspension of liposomes, 0.125 mL of 9 mM FeSO4, 0.125 mL of of some essential oil compounds on the activity cited, further
0.88 M H2O2, and 10 µL of tested essential oil samples in 2.71 mL of examinations are necessary.
0.05 M KH2PO4–K2HPO4 buffer, pH ) 7.4 (3 mL final solution). The antifungal activity of the tested essential oils against the
Samples were incubated at 37 °C for 1 h. LP was terminated using five examined dermatomycetes and Candida albicans is pre-
the reaction with 1.5 mL of TBA reagent and 0.2 mL of 0.1 M EDTA, sented in Table 2. In general, both examined essential oils
heating at 100 °C for 20 min. After the cooling of solutions and
exhibited notable fungistatic and fungicidal activity. Comparing
precipitation of proteins by centrifugation (4000 rpm for 10 min), the
content of the MDA (TBARS) was determined by measuring the to the bifonazole, the essential oil of rosemary showed lower
absorbance of adduct at 532 nm. Both analyses were compared with MIC especially against C. albicans, Trichophyton tonsurans,
the commercial synthetic antioxidant BHT (0.5 M stock solution, and T. rubrum indicating its significant antifungal effect. Strong
concentration 220.4 µg/mL) as a positive control. All reactions were antifungal activity against C. albicans is in accordance with
carried out in triplicate. The control with n-hexane was also analyzed. earlier published data (20, 22), but no data about the antifungal
The percentage of LP inhibition was calculated by the following effect of this oil on Trichophyton and Microsporum species have
equation: been reported yet.
7882 J. Agric. Food Chem., Vol. 55, No. 19, 2007 Bozin et al.

Table 1. Chemical Composition of the Essential Oil of Rosmarinus officinalis and Salvia officinalis

percentage
a
pick no. compounds RI Rosmarinus officinalis Salvia officinalis identification methodb
Monoterpene Hydrocarbons 44.7 3.0
1 R-pinene 939 13.5 0.5 RT GC MS
2 camphene 951 3.9 0.7 RT* MS
3 sabinene 972 2.0 RT GC MS
5 β-pinene 978 1.1 0.8 RT GC MS
6 E-isolimonene 982 0.3 RT* MS
8 allo-ocimene 1130 0.1 RT* MS
9 limonene 1032 21.7 1.0 RT GC MS
10 β-phellandrene 1034 0.9 RT* MS
12 γ-terpinene 1061 0.6 RT GC MS
13 R-terpinolene 1088 0.6 RT* MS
Aromatic Monoterpene Hydrocarbons 2.0 0.3
7 o-cymene 1020 2.0 0.3
Oxygenated Monoterpenes 46.9 57.8
11 1,8-cineole 1035 2.1 4.2 RT GC MS
14 Z-linalool oxide 1065 10.8 RT* MS
15 Z-sabinene hydrate 1095 0.1 RT* MS
16 linalool 1101 1.1 0.6 RT GC MS
17 R-thujone 1104 19.9 RT GC MS
18 endo-fenchol 1111 0.2 RT* MS
19 β-thujone 1114 0.2 3.8 RT GC MS
20 camphor 1146 21.6 18.9 RT GC MS
21 camphene hydrate 1148 0.1 RT* MS
22 menthone 1156 0.4 RT GC MS
23 borneol 1167 6.2 5.4 RT GC MS
24 neomenthol 1170 0.7 RT* MS
25 terpinen-4-ol 1176 0.7 0.8 RT GC MS
26 R-terpineol 1188 1.9 RT GC MS
27 myrtenol 1196 0.2 RT* MS
28 pulegone 1235 0.1 RT GC MS
29 bornyl acetate 1288 1.4 3.3 RT GC MS
Aromatic Oxygenated Monoterpenes 0.4
30 carvacrol 1298 0.3 RT GC MS
32 methyl eugenol 1401 0.1 RT* MS
Sesquiterpene Hydrocarbons 2.2 10.1
31 R-jlangene 1372 0.1 RT* MS
33 longifolene 1403 0.8 RT* MS
34 E-caryophyllene 1419 1.0 1.8 RT GC MS
35 R-humulene 1452 0.9 2.6 RT GC MS
36 allo-aromadendrene 1462 0.7 RT* MS
37 R-muurolene 1498 0.1 RT* MS
39 Z-R-bisabolene 1504 0.9 RT* MS
40 β-bisabolene 1508 0.1 0.8 RT* MS
41 E,E-R-farnesene 1509 2.5 RT* MS
Oxygenated Sesquiterpenes 0.3 19.5
42 ledol 1563 0.6 RT* MS
43 caryophyllnol 1568 0.2 0.4 RT* MS
44 spathulenol 1576 0.2 RT* MS
45 caryophyllene oxide 1584 0.1 0.7 RT* MS
46 viridiflorol 1593 17.5 RT* MS
51 sclareol 2220 0.1 RT* MS
Aliphatic Compounds 1.9 2.5
4 3-cctenol 976 1.3 RT* MS
38 pentadecane 1501 0.4 RT* MS
47 heptadecane 1700 0.1 RT* MS
48 octadecane 1799 0.2 RT* MS
49 nonadecane 1900 0.3 RT* MS
50 eicosane 2001 0.1 2.0 RT* MS
Total of Identified Compounds 98.4 93.2

a
Retention indices relative to C9–C24 n-alkanes on the HP 5MS column. b (RT) comparison with pure standard retention time; (GC) gas chromatographic coelution with
pure standard; (MS) mass spectrometry; (RT*) comparison of the relative retention time with those obtained from the NIST/NBS, Wiley libraries spectra, and those reported
by Adams (25).
The antibacterial activity of tested essential oils of rosemary tested strains of E. coli, including the multiresistant one, showed
and sage against five strains of Gram-positive and eight strains high sensitivity to the essential oils of rosemary and sage, which
of Gram-negative bacteria is shown in Table 3. In general, tested is of a particular interest. Furthermore, both essential oils
Gram-positive strains of bacteria tested seemed to be more exhibited important activity against Shigella sonei and Salmo-
sensitive to the examined essential oils. These results are in nella typhi.
accordance with the earlier published literature data (12, 15, The essential oil of rosemary exhibited antibacterial and
20–23). However, this study also recorded a notable susceptibil- antifungal activity as expected although in several cases that
ity of the examined Gram-negative pathogenic bacteria. All effect was stronger than in earlier published data (12, 15, 20–22)
Activities of Rosemary and Sage Essential Oils J. Agric. Food Chem., Vol. 55, No. 19, 2007 7883

Table 2. Antifungal Activity of Essential Oilsa of Rosmarinus officinalis, Salvia officinalis, and Bifonazole (as a Positive Control)

Rosmarinus officinalis Salvia officinalis bifonazole

source organism MIC MFC MIC MFC MIC MFC
MMA Candida albicans 30.2 ( 0.3 60.2 ( 0.3 200.0 ( 0.0 250.0 ( 0.0 20.0 ( 0.0 25.2 ( 0.3
MMA Trichophyton mentagrophytes 15.3 ( 0.6 30.0 ( 0.0 60.0 ( 1.0 100.0 ( 0.0 9.7 ( 0.6 9.7 ( 0.6
MMA Trichophyton tonsurans 15.2 ( 0.3 30.0 ( 0.0 60.0 ( 1.0 100.3 ( 0.6 10.0 ( 0.0 10.0 ( 0.0
MMA Trichophyton rubrum 15.0 ( 0.0 30.2 ( 0.3 60.0 ( 1.0 100.3 ( 0.6 10.3 ( 0.6 10.3 ( 0.6
MMA Epidermophyton floccosum 30.0 ( 0.0 30.0 ( 0.0 100.0 ( 0.0 200.2 ( 0.3 10.0 ( 0.0 10.0 ( 0.0
MMA Microsporum canis 30.2 ( 0.3 30.2 ( 0.3 100.2 ( 0.3 200.2 ( 0.3 15.0 ( 0.0 20.0 ( 0.0

a
Concentrations shown in the table were expressed in microliters. If they are expressed in real concentrations of active substances (µg), the expressed values would
be 10 times higher.

Table 3. Antibacterial Activitya of Essential Oilsb of Rosmarinus officinalis, Salvia officinalis, and Penicillin (as a Positive Control)

Rosmarinus officinalis Salvia officinalis penicillin (µg/mL)

source organism 20% 50% 20% 50% 500 1000
ATCC 27853 Pseudomonas aeruginosa 0.0 0.0 0.0 0.0 0.0 0.0
IPH-MR Pseudomonas aeruginosa 0.0 0.0 0.0 0.0 0.0 0.0
ATCC 35218 Escherichia coli 19.8 ( 0.45 23.0 ( 0.71 20.2 ( 0.45 20.4 ( 0.55 0.0 0.0
ATCC 25922 Escherichia coli 22.0 ( 0.71 25.0 ( 0.71 23.8 ( 1.09 24.6 ( 0.55 12.6 ( 0.89 13.4 ( 0.55
IPH-MR Escherichia coli 18.6 ( 0.89 18.2 ( 0.45 13.8 ( 0.84 14.2 ( 0.84 0.0 0.0
IPH-MR Salmonella typhi 18.0 ( 0.00 16.2 ( 0.45 40.2 ( 0.45 50.0 ( 0.00 0.0 0.0
IPH Salmonella enteritidis 26.6 ( 2.07 29.0 ( 0.71 16.6 ( 2.07 10.0 ( 0.71 22.0 ( 1.22 26.2 ( 0.83
IPH-MR Shigella sonei 19.4 ( 0.89 19.6 ( 0.89 21.2 ( 0.83 13.2 ( 0.84 0.0 0.0
ATCC 10240 Micrococcus flavus 30.0 ( 0.00 23.6 ( 2.07 56.0 ( 0.71 60.6 ( 0.89 40.0 ( 0.55 40.0 ( 0.89
ATCC 9341 Sarcina lutea 16.2 ( 1.48 43.2 ( 1.92 21.0 ( 0.71 21.2 ( 0.84 0.0 0.0
ATCC 6538 Staphylococcus aureus 17.2 ( 1.09 29.0 ( 0.71 24.0 ( 1.00 20.0 ( 0.71 48.0 ( 0.00 50.0 ( 0.00
ATCC 12228 Staphylococcus epidermidis 18.2 ( 1.48 20.2 ( 1.48 26.0 ( 1.41 49.2 ( 0.84 14.2 ( 0.44 16.8 ( 0.83
ATCC 10707 Bacillus subtilis 20.2 ( 0.45 21.4 ( 0.89 41.4 ( 1.67 60.2 ( 0.45 38.2 ( 0.44 38.0 ( 0.70

a
The inhibition zone is measured in millimeters, including the hole of 5 mm in diameter. The values shown represent the average of five determinations ( standard
deviations. b All essential oils were diluted in n-hexane.

Table 4. Neutralization of DPPH• by Rosmarinus officinalis and Salvia officinalis Essential Oils and BHT (as a Positive Control) in the DPPH Assay (in
Percentage)

concentrations (µL/mL)
source 0.25 0.50 1.20 2.50 3.12 4.80 6.25 7.20 9.60 12.00 12.50 IC50
Rosmarinus officinalis 13.89 19.44 36.11 47.56 58.33 64.42 66.67 80.55 88.89 92.24 3.82
Salvia officinalis 11.76 23.53 38.23 70.59 87.54 98.99 >100 >100 >100 >100 >100 1.78
BHT 4.62 11.56 23.12 30.11 44.71 55.22 65.07 67.51 70.12 74.22 5.37

possibly due to the specific chemical profile of the essential essential oils possessed stronger antioxidant effects than BHT.
oil, in which limonene and camphor are the main compounds, These findings are in correlation with the earlier published data
followed with high amounts of R-pinene, Z-linalool oxide, and on the notable antiradical activity of both essential oils (11).
borneol (Table 1). The comparison of control TLC analysis with the results of GC-
Antioxidant Activity. The antioxidant potential of plant MS (Table 1) and TLC-DPPH methods accomplished the
products and pure compounds can be evaluated using numerous identification of the constituents most responsible for RSC. For
assays. The first step in these examinations is the screening of the neutralization of DPPH radicals, the most responsible
the potential activity by different in vitro tests. Each of those is compounds were the oxygenated monoterpenes (R- and β-thu-
based on one feature of the antioxidant activity, such as the jone, bornyl acetate, camphor, and menthone) and the mixture
ability of scavenging free radicals, the inhibition of lipid of mono- and sesquiterpene hydrocarbons (Table 5). Although
peroxidation, the chelating of transition metal ions (TMI), etc. found in a small amount in the essential oil of rosemary,
However, in order to get relevant data, a single method for carvacrol (Table 1) exhibited notable scavenging activity, too.
testing antioxidant activities of plant products is not recom- These findings confirm the earlier obtained data on the anti-
mended due to their complex composition (29). Therefore, the oxidant activities of the selected components of essential
antioxidant activity of the tested essential oils of rosemary and oils (7–10). A higher RSC was gained by the sage essential oil
sage has been evaluated in a series of in vitro tests. (IC50 ) 1.78 µL/mL) compared to that expressed by the essential
In the DPPH assay, the ability of essential oils of interest to oil of rosemary (IC50 ) 3.82 µL/mL). This could be explained
act as donors of hydrogen atoms or electrons in transformation partially by the presence of a higher amount of compounds
of DPPH• into its reduced form DPPH–-H was investigated. responsible for this activity, such as bornyl acetate, camphor,
Both examined essential oils were able to reduce the stable, and sesquiterpene hydrocarbons recorded in the sage essential
purple-colored radical DPPH into yellow-colored DPPH–H, oil (Table 1).
reaching 50% of reduction with IC50 values of 3.82 for rosemary Furthermore, in Figure 1, the hydroxyl RSC of the examined
and 1.78 µL/mL for sage (Table 4). Comparison of the DPPH essential oils measured by the deoxyribose assay is shown.
scavenging activity of the investigated essential oils and those Protective effects of the essential oils on 2-deoxy-D-ribose were
expressed by BHT (5.37 µL/mL) showed that both examined assessed through their ability to remove hydroxyl radicals
7884 J. Agric. Food Chem., Vol. 55, No. 19, 2007 Bozin et al.

Table 5. DPPH Scavenging Active Compounds Identified by the TLC

Dot-Blot Technique

source of essential oil compound Rf values

Rosmarinus officinalis carvacrol 0.65
menthone 0.67
bornyl acetate 0.69
camphor 0.80
mixture of mono- and 0.97
sesquiterpene hydrocarbons
Salvia officinalis R- and β-thujone 0.64–0.68
bornyl acetate 0.69 Figure 2. Inhibition of lipid peroxidation (LP) in Fe2+/ascorbate system
camphor 0.80 of induction by the essential oils of Rosmarinus officinalis, Salvia officinalis,
mixture of mono- and 0.98
and BHT (as a positive control; the essential oils and BHT were diluted
sesquiterpene hydrocarbons
in n-hexane (the solvent expressed no antioxidant activity)) in the TBA
assay. The numbers and labels on the right show concentrations expressed
in microliters per milliliter.

Figure 1. Inhibition of degradation of 2-deoxyribose by essential oils (all

examined essential oils were diluted in n-hexane (the solvent expressed
Figure 3. Inhibition of lipid peroxidation (LP) in Fe2+/H2O2 system of
no scavenging activity)) of Rosmarinus officinalis, Salvia officinalis, and
induction by the essential oils of Rosmarinus officinalis, Salvia officinalis,
BHT (as a positive control) in the deoxyribose assay. The numbers and
and BHT (as a positive control; the essential oils and BHT were diluted
labels on the right show concentrations expressed in microliters per
in n-hexane (the solvent expressed no antioxidant activity)) in the TBA
milliliter.
assay. The numbers and labels on the right show concentrations expressed
in microliters per milliliter.
formed in Fenton reaction from the test solution and to prevent
its degradation. However, this feature of the antioxidant activity
free radical scavengers and LP inhibitors. However, from the
of investigated essential oils has not been investigated yet.
obtained results, it is obvious that the assayed essential oils,
Generally, both essential oils inhibited the degradation of
together with the compounds present in them, have different
deoxyribose more than BHT (18.71%), used as a positive
modes of action. Thus, the essential oil of sage exhibited
control. Higher activity was shown by the essential oil of
stronger RSC against DPPH radical, which was found to be in
rosemary (in a range from 17.65 to 32.35% inhibition),
correlation to the content of some oxygenated monoterpene
compared to that exhibited by the sage essential oil (5.56–22.22%).
compounds and sesquiterpene hydrocarbons. The essential oil
The protective effects of the rosemary and sage essential oils of rosemary expressed a better effect against hydroxyl radical
on LP have been evaluated using two systems of induction, and in the processes of the peroxidation of lipids. Furthermore,
Fe2+/ascorbate and Fe2+/H2O2. The inhibition of LP was the study confirmed the earlier published data about significant
determined by measuring the formation of MDA, using lipo- antibacterial and antifungal activity of the investigated essential
somes as an oxidizable substrate. oils against a wide spectrum of bacteria and fungi. The use of
In Figure 2, results of antioxidant activities of the examined investigated essential oils and spices could be useful not only
essential oils and BHT as a positive control in the Fe2+/ascorbate in food and cosmetics production but also as important
system of induction are presented. Both essential oils expressed functional food in the prevention and treatment of various human
stronger antioxidant capacity than BHT (37.04%). In particular, diseases.
the essential oil of rosemary exhibited very high activity (from
56.29 to 75.79% inhibition). In LP induced by the Fenton
LITERATURE CITED
reaction (Fe2+/H2O2), both examined essential oils, together with
BHT, exhibited very similar antioxidant activities (ranging from (1) WHO. Food safety and food borne illness. In World health
33.33 to 66.67%) (Figure 3). Essential oil obtained from Organization Fact sheet; World Health Organization: Geneva,
rosemary exhibited a little bit of a stronger effect on the LP Switzerland, 2002; pp 237.
(33.33–58.33%) than sage essential oil (33.33–40%). In both (2) Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and
systems of induction, the essential oil of rosemary reached 50% medicine; Clarendon Press: Oxford, UK, 1989; pp 23–30.
of LP inhibition and also showed dose-dependent activity. These (3) Smith, M. A.; Perry, G.; Pryor, W. A. Causes and consequences
of oxidative stress in Alzheimer’s disease (1,2). Free Radical Biol.
findings are in accordance with earlier published data about
Med. 2002, 32 (11), 1049.
antioxidant activities of these two essential oils (15). (4) Diplock, A. T.; Charleux, J. L.; Crozier-Willi, G.; Kok, F. J.; Rice-
In conclusion, the results of the antioxidant effects of the Evans, C.; Roberfroid, M.; Stahl, W.; Vina-Ribes, J. Functional
investigated essential oils, obtained with different methods of food science and defence against reactive oxidative species. Brit.
assessment, point out very strong protective activities, both as J. Nutr. 1998, 80, S77–S112.
Activities of Rosemary and Sage Essential Oils J. Agric. Food Chem., Vol. 55, No. 19, 2007 7885

(5) Buchbauer, G.; Jirovetz, L. Aromatherapy-Use of Fragrances and Mediated Oxidative Stress in Caco-2 Cells. J. Agric. Food Chem.
Essential Oils as Medicaments. FlaVour Fragrance J. 1994, 9, 2007, 55, 1193–1199.
217–222. (19) Lis-Balchin, M.; Deans, S. G. Bioactivity of selected plant essential
(6) Bruneton, J. Pharmacognosy, Phytochemistry, Medicinal Plants, oils against Listeria monocytogenes. J. Appl. Bacteriol. 1997, 82,
2nd ed.; Intercept Ltd.: London, 1999; pp 539–540. 759–762.
(7) Ruberto, G.; Baratta, M. T. Antioxidant activity of selected (20) Hammer, K. A.; Carson, C. F.; Riley, T. V. Antimicrobial activity
essential oil components in two lipid model systems. Food Chem. of essential oils and other plant extracts. J. Appl. Microbiol. 1999,
2000, 68, 167–174. 86, 985–990.
(8) Mimica-Dukic, N.; Bozin, B.; Sokovic, M.; Mihajlovic, B.; (21) Managena, T.; Muyima, N. O. Comparative evaluation of the
Matavulj, M. Antimicrobial and Antioxidant Activities of Three antimicrobial activities of essential oils of Artemisia afra, Pteronia
Mentha Species Essential Oils. Planta Med. 2003, 69, 413–419. incana and Rosmarinus officinalis on selected bacteria and yeast
(9) Mimica-Dukic, N.; Bozin, B.; Sokovic, M.; Simin, N. Antimi- strains. Lett. Appl. Microbiol. 1999, 28, 291–296.
crobial and Antioxidant Activities of Melissa officinalis L. (22) Yesil Celiktas, O.; Hames Kocabas, E. E.; Bedir, E.; Vardar Sukan,
(Lamiaceae) Essential Oil. J. Agric. Food Chem. 2004, 52, 2485– F.; Ozek, T.; Baser, K. H. C. Antimicrobial activities of methanol
2489. extracts and essential oils of Rosmarinus officinalis, depending
(10) Bozin, B.; Mimica-Dukic, N.; Simin, N.; Anackov, G. Charac- on location and seasonal variations. Food Chem. 2007, 100 (2),
terization of the Volatile Composition of Essential Oils of Some
553–559.
Lamiaceae Species and the Antimicrobial and Antioxidant Activi-
(23) Cowan, M. M. Plant Products as Antimicrobial Agents. Clin.
ties of the Entire Oils. J. Agric. Food Chem. 2006, 54, 1822–
Microbiol. ReV. 1999, 12 (4), 564–582.
1828.
(24) European Pharmacopeia, 4th ed.; Council of Europe: Strasbourg
(11) Lee, S. E.; Lee, H. S.; Ahn, Y. J. Scavenging Effect of Plant-
Cedex, France, 2002; Vol. 2.8.12, pp 183–184.
Derived Materials on Free Radicals and Active Oxygen Species.
Agric. Chem. Biotechnol. 1999, 42 (1), 40–44. (25) Adams, R. P. Identification of Essential Oil Components by Gas
(12) Daferera, D. J.; Ziogas, B. N.; Polissiou, M. G. GC-MS analysis Chromatography/Mass Spectroscopy; Allured Publishing Corp.:
of essential oils from some greek aromatic plants and their Carol Stream, Illinois, 1995.
fungitoxicity on Penicillium digitatum. J. Agric. Food Chem. 2000, (26) Afanasev, I. B.; Doroszhko, A. I.; Brodski, A. V.; Kostyuk, V. A.;
48, 2576–2581. Potapovitch, A. I. Chelating and free radical scavenging mech-
(13) Kosar, M.; Dorman, H. J. D.; Hiltunen, R. Effect of an acid anism of inhibitory action of rutin and quercetin in lipid
treatment on the phytochemical and antioxidant characteristics of peroxidation. Biochem. Pharmacol. 1989, 38 (11), 1763–1768.
extracts from selected Lamiaceae species. Food Chem. 2005, 91, (27) Couladis, M.; Tzakou, O.; Mimica-Dukic, N.; Jancic, R.; Stojan-
525–533. ovic, D. Essential oil of SalVia officinalis L. from Serbia and
(14) Madsen, H. L.; Bertelsen, G. Spices as antioxidants. Trends Food Montenegro. FlaV. Fragr. J. 2002, 17, 199–126.
Sci. Technol. 1995, 6, 271–277. (28) Morris, J. A.; Khettry, A.; Seitz, E. W. Antimicrobial activity of
(15) Baratta, M. T.; Dorman, H. J. D.; Deans, S. G.; Biondi, D. M.; aroma chemicals and essential oils. J. Am. Oil Chem. Soc. 1979,
Ruberto, G. Chemical Composition, Antimicrobial and Antioxi- 56, 598–619.
dative Activity of Laurel, Sage, Rosemary, Oregano and Coriander (29) Nuutila, A. M.; Puupponen-Pimia, R.; Aarni, M.; Oksman-
Essential Oils. J. Ess. Oil. Res. 1998, 10, 618–627. Caldentey, K. M. Comparision of antioxidant activities of onion
(16) Bicchi, C.; Binello, A.; Rubiolo, P. Determination of Phenolic and garlic extracts by inhibition of lipid peroxidation and radical
Diterpene Antioxidants in Rosemary (Rosmarinus officinalis L.) scavenging activity. Food Chem. 2003, 81, 485–493.
with Different Methods of Extraction and Analysis. Phytochem.
Anal. 2000, 11, 236–242. Received for review May 25, 2007. Revised manuscript received July
(17) Yanishlieva, N. V.; Marinova, E.; Pokorny, J. Natural antioxidants 18, 2007. Accepted July 21, 2007. The Ministry of Sciences and
from herbs and spices. Eur. J. Lipid Sci. Technol. 2006, 108, 776– Environmental Protection, Republic of Serbia, No. 142036, supported
793. this research work.
(18) Wijeratne, S. K.; Cuppett, S. L. Potential of Rosemary (Rosmari-
nus officinalis L.) Diterpenes in Preventing Lipid Hydroperoxide- JF0715323

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