2576 J. Agric. Food Chem.

2000, 48, 2576−2581

GC-MS Analysis of Essential Oils from Some Greek Aromatic Plants

and Their Fungitoxicity on Penicillium digitatum
Dimitra J. Daferera,† Basil N. Ziogas,‡ and Moschos G. Polissiou*,†
Laboratory of General Chemistry and Laboratory of Phytopathology, Agricultural University of Athens,
Iera Odos 75, 118 55 Athens, Greece

The isolated essential oils from seven air-dried plant species were analyzed by gas chromatography-
mass spectrometry (GC-MS). Thymus vulgaris (thyme), Origanum vulgare (oregano), and Origanum
dictamus (dictamus) essential oils were found to be rich in phenolic compounds representing 65.8,
71.1, and 78.0% of the total oil, respectively. Origanum majorana (marjoram) oil was constituted of
hydrocarbons (42.1%), alcohols (24.3%), and phenols (14.2%). The essential oil from Lavandula
angustifolia Mill. (lavender) was characterized by the presence of alcohols (58.8%) and esters (32.7%).
Ethers predominated in Rosmarinus officinalis (rosemary) and Salvia fruticosa (sage) essential oils,
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constituting 88.9 and 78.0%, respectively. The radial growth, conidial germination, and production
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of Penicillium digitatum were inhibited completely by oregano, thyme, dictamus, and marjoram
essential oils at relatively low concentrations (250-400 µg/mL). Lavender, rosemary, and sage
essential oils presented less inhibitory effect on the radial growth and conidial germination of P.
digitatum. Conidial production of P. digitatum was not affected by the above oils at concentrations
up to 1000 µg/mL. Apart from oregano oil, all essential oils were more effective in the inhibition of
conidial germination than of radial growth. The monoterpene components, which participate in
essential oils in different compositions, seem to have more than an additive effect in fungal inhibition.

Keywords: GC-MS analysis; essential oils; fungitoxicity; antimicrobial; Penicillium digitatum

INTRODUCTION quantitative) analysis is indispensable for the evalua-

tion of the biological activity of the essential oil.
Aromatic plants produce volatile C10 and C15 terpenes
that are derived from the isoprene unit. These sub- The antimicrobial activity of essential oils against
stances, which are known as essential oils, can be important human pathogenic microorganisms has been
isolated from various parts of plants by steam distilla- examined in detail (Farag et al., 1989; Paster et al.,
tion or other modified methods. A wide variety of 1990; Adam et al., 1998; Smith-Palmer et al., 1998;
terpene hydrocarbons, cyclic or noncyclic, and their Hammer et al., 1999; Marino et al., 1999; Cosentino et
oxygenated isoprenoid compounds are present in es- al., 1999). The inhibitory effects of the main essential
sential oils as mixtures. oil components or the total oil on microorganisms that
The chemical composition of a plant essential oil cause food spoilage have been also studied (Thompson,
depends on a number of parameters, such as the 1989; Ismaiel and Pierson, 1990; Mahmoud, 1994;
enviromental conditions, the season that the aromatic Basilico and Basilico, 1999). Recently, interest in the
plants have been collected, the dehydration procedure, application of essential oils to control plant pathogens
the storage conditions under which the collected plants has increased (Gorris et al., 1994; Thanassoulopoulos
were kept until their essential oil extraction, the applied and Laidou, 1997; Reddy et al., 1998; Arras et al., 1993,
method for the isolation of the essential oil, and the 1995).
analysis conditions (column, programmed temperature), Penicillium digitatum is one of the most common
which are used for the identification of the compounds postharvest pathogens, causing green mold rots in
(Hawthorne et al., 1993; Kokkini et al., 1997; Tarantilis Citrus species. A number of fungicides such as benz-
and Polissiou, 1997; Russo et al., 1998). imidazoles, aromatic hydrocarbons, and sterol biosyn-
Pharmacology, pharmaceutical botany, medical and thesis inhibitors are in use as postharvest treatments
clinical microbiology, phytopathology, and food preser- to control the pathogen. A serious problem in the
vation are some fields in which the essential oils can effective use of these chemicals is the development of
be applied. The antimicrobial activity of essential oils resistance by P. digitatum. The application of higher
and their pure compounds from plant species of the concentrations of chemicals to control the resistant
Lamiaceae family has been reported by several research- strains, if possible, increases the risk of high levels of
ers, although few of them have studied the chemical toxic residues in the products. In that case, the problem
composition of the applied essential oils. A gas chroma- is particularly serious because fruits are often consumed
tography-mass spectrometry (GC-MS) (qualitative and in a relatively short time after harvest.
Our objectives in the present work were, first, to
* Corresponding author (telephone ++ 30 1 529 42 41; fax determine the chemical composition of the essential oils
++ 30 1 529 4265; e--mail [email protected]). from Thymus vulgaris (thyme), Origanum vulgare
† Laboratory of General Chemistry. (oregano), Origanum dictamus (dictamus), Origanum
‡ Laboratory of Phytopathology.
majorana (marjoram), Lavandula angustifolia Mill.
10.1021/jf990835x CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/20/2000
Essential Oil of Greek Aromatic Plants J. Agric. Food Chem., Vol. 48, No. 6, 2000 2577

(lavender), Rosmarinus officinalis (rosemary), and Salvia with a conidial suspension and incubated for 7 days in an
fruticosa (sage) by GC-MS analysis and, second, to incubation cabinet at 25 °C. The total mycelial mass that was
evaluate the efficacy of the above oils and their pure produced in each dish was transferred to a flask with 50 mL
major components on the radial growth, conidial ger- of sterile water. The flasks were agitated vigorously, and the
concentration of conidia in the resulting spore suspension was
mination and production of P. digitatum.
evaluated by counting with a hemocytometer and converted
to spores per square centimeter of plate culture (Ziogas and
EXPERIMENTAL PROCEDURES Girgis, 1993).
Materials. All of the dried aromatic plants, apart from
marjoram, were collected from Crete (Greece) and stored at RESULTS AND DISCUSSION
room temperature in darkness. Marjoram was collected from Oil Composition. The compositions of essential oils
Attiki (Greece), air-dried, and stored under the same condi-
tions.
from T. vulgaris (thyme), O. vulgare (oregano), O.
Pure commercial essential oil components were purchased dictamus (dictamus), S. fruticosa (sage), O. majorana
from the Sigma-Aldrich Co. (marjoram), L. angustifolia Mill. (lavender), and R.
The test organism, P. digitatum, was provided by the officinalis (rosemary) were determined by comparing the
Laboratory of Phytopathology of the Agricultural University relative retention times and the mass spectra of oil
of Athens (AUA). components with those of authentic samples and mass
Methods. Isolation of the Essential Oils. The essential oils spectra from data library. Most of the essential oils were
were isolated according to the Lickens-Nickerson method, characterized by the dominant presence of one or two
using a microsteam distillation-extraction apparatus for substances. Only in marjoram essential oil have a large
organic solvents lighter than water. number of substances been found.
The apparatus consisted of a main body, a coldfinger, a 100 T. vulgaris, O. vulgare, and O. dictamus are a group
mL water flask, and a 5 mL solvent flask. Four milliliters of
of plants having their essential oils characterized by the
the extracting solvent (diethyl ether) was heated in the solvent
flask and condensed on the coldfinger above the place where predominant presence of thymol (Table 1).
the water condensed. The sample (10 g) to be analyzed was Thyme essential oil was characterized by the presence
heated in the water flask with water. The vapor, which also of γ-terpinene (4.3%), p-cymene (23.5%), carvacrol (2.2%),
contained the volatile organic compounds, condensed on the and thymol (63.6%), which composed 93.6% of the total
coldfinger. Both phases returned via the connecting tubes into oil. In oregano essential oil the most abundant com-
their flasks, and the procedure was started again. Refluxing pounds were also γ-terpinene (12.7%), p-cymene (9.9%),
was continued for ∼2 h. In that way, all of the aroma carvacrol (7.8%), and thymol (63.3%), which participated
constituents concentrated in the extracting solvent. Inert gas in the mixture at 93.7%. Along with thymol (78%),
(N2) was introduced into the main body of the apparatus to p-cymene (10.1%) and γ-terpinene (7.9%) constituted
avoid the oxidation of molecules during the procedure. All
diethyl ether extracts were stored at 4 °C until their analysis
96% of dictamus oil. Oregano oil also has been charac-
by GC-MS or their usage in bioassays. terized as a thymol chemotype by Russel et al. (1998).
Analysis Conditions. Apart from marjoram oil, the analysis In contrast to our results, carvacrol instead of thymol
of all essential oils was performed using a Hewlett-Packard was determined as the main compound in oregano and
5890 II GC, equipped with a HP-5 capillary column (30 m, dictamus oils by other researchers (Sivropoulou et al.,
0.25 mm i.d., 0.25 µm film thickness) and a mass spectrometer 1996; Baser et al., 1993). In some other cases the
5971 A as detector (method A). The carrier gas was helium, percentages of carvacrol and thymol in the total oil were
at a flow rate of 1 mL/min. Column temperature was initially almost equal (Adam et al., 1998; Russo et al., 1998).
60 °C for 5 min, then gradually increased to 160 °C at 4 °C/ The analysis of the marjoram oil gave a large number
min, and finally increased to 240 °C at 15 °C/min. For GC-MS
of constituents. Among them were detected 3-thujene
detection an electron ionization system was used with an
ionization energy of 70 eV. The extracts were diluted 1:100 (2.8%), β-myrcene (3.8%), 2-carene (7.8%), 2-ethyl-m-
(v/v) with diethyl ether, and 1.0 µL of the diluted samples was xylene (5.2%), 3-carene (10.4%), terpinen-4-ol (7.8%),
injected automatically in splitless mode. Injector and detector sabinene hydrate (6.0%), R-terpineol (4.2%), and thymol
temperatures were set at 250 and 280 °C, respectively. (14%). Two chemotypes of O. majorana were found in
Marjoram essential oil analysis was performed using a Fison the literature, the cis-sabinene hydrate/tepinen-4-ol
8000 GC, equipped with a CP-Sil 8 (30 m, 0.32 mm i.d.) chemotype and the carvacrol/thymol chemotype (Ko-
capillary column and a mass spectrometer 800 as detector maitis et al., 1992; Bellomaria et al., 1993; Baser et al.,
(method B). Column temperature was initially 60 °C for 5 min, 1993). High amounts of carvacrol (78.3-79.5%) and
then gradually increased to 240 °C at 4 °C/min, and kept there thymol (11.55%) have been reported by Baser et al.
for 5 min. In this case, 1.0 µL of the diluted sample was
injected manually in splitless mode.
(1993) and Pino et al. (1997) for O. majorana from
Measurement of Fungitoxicity. A stock of pure essential oils Turkey and Cuba, respectively. Generally, the Orig-
of the aromatic plants was prepared to be used in all bioassays. anum species is characterized by the presence of two
The diethyl ether extracts of each plant were combined, and major biochemically related groups of compounds (Sk-
the solvent was evaporated by a flow of nitrogen gas at room oula et al., 1999). The first group includes the aromatic
temperature. monoterpenes such as p-cymene, thymol, carvacrol,
Inhibition of mycelial growth of P. digitatum was deter- their precursor γ-terpinene, and their derivatives. The
mined by daily measuring of the radial growth on PDA plates second group includes the thujanes, such as sabinene,
containing the respective essential oil at a range of concentra- sabinene hydrate, and their derivatives. According to
tions, for 10 days at 25 °C (Ziogas and Girgis, 1993). Plates Skoula et al. (1999) there is a clear division into sabinyl-
were inoculated with 2 mm disks from PDA on which conidia
rich and carvacrol-rich plants with no intermediates
had been allowed to germinate.
For spore germination assays, conidia were plated on PDA
observed.
medium with and without the essential oil. The proportion of From our results, the specified sample from Greek O.
conidia capable of producing germ tubes was counted after 12 majorana included also a high amount of thymol (14%),
h of incubation at 25 °C. and this is reported for the first time about Greek
To determine conidial production in the absence and pres- marjoram. It seems that this sample of O. majorana
ence of the examined essential oil, PDA plates were inoculated plants is characterized by an intermediate situation due
2578 J. Agric. Food Chem., Vol. 48, No. 6, 2000 Daferera et al.

Table 1. Quantitative Composition of the Essential Oils from Thyme (T), Oregano (O), Dictamus (D), and Marjoram (M)
composition (%)
tR-T,O,Da (min) tR-Mb(min) certainty of
by method A by method B component identification T O D M
hydrocarbons
5.67 4.87 3-thujene **c -e 0.3 0.3 2.8
5.07 3,6,6-trimethyl-2-norpinene ** - - - 1.9
5.79 R-pinene ***d 0.3 0.6 0.3 -
7.71 β-myrcene *** - - - 3.8
7.84 β-pinene ** - 0.5 0.5 -
8.07 2-carene ** - - - 7.8
8.32 2-ethyl m-xylene ** - - - 5.2
8.50 m-mentha-6,8-diene ** - - - 4.2
9.01 R-terpinene *** 1.0 1.0 0.9 -
9.15 p-cymene *** 23.5 9.9 10.1 -
9.72 3-carene ** - - - 10.4
10.74 γ-terpinene *** 4.3 12.7 7.9 -
12.14 sabinene ** - - - 1.3
26.88 23.14 β-caryophyllene *** 1.3 0.5 0.4 2.6
31.77 26.15 β-bisabolene ** - 0.4 - 2.1
alcohols
11.32 sabinene hydrate ** - - - 6.0
12.49 11.45 linalool *** - 0.6 0.4 3.8
14.85 13.87 borneol *** 1.4 0.5 - 2.5
15.52 14.44 terpinen-4-ol ** 0.6 0.3 0.3 7.8
14.92 R-terpineol *** - - - 4.2
phenols
20.87 18.84 carvacrol *** 2.2 7.8 - 0.2
21.58 19.60 thymol *** 63.6 63.3 78.0 14.0
esters
17.49 linalyl acetate ** - - - 3.4
others 1.8 1.6 0.9 16

total 100 100 100 100

a tR-T,O,D is the retention time for the compounds of T, O, and D, according to method A of GC-MS analysis. b tR-M is the retention time
for the compounds of M, according to method B of GC-MS analysis. c **, tentative identification from mass spectra data. d ***, positive
identification from mass spectrum and retention time which agree with authentic compound. e Not determined.

to the environmental conditions and the agricultural Table 2. Quantitative Composition of the Essential Oil
treatments (irrigation) which developed. The essential from Lavender (L), Rosemary (R), and Sage (S)
oils’ chemical composition varies and depends of locality, tR-L,R,Sa
composition %
the climatic conditions, and the season that the plants (min) by certainty of
were collected (Kokkini et al., 1997; Russo et al., 1998). method A component identification L R S
The chemical composition of rosemary and sage hydrocarbons
essential oils was characterized by the predominant 5.79 R-pinene ***b -d 2.7 2.3
presence of 1,8-cineole, which composed 88.9 and 78% 5.92 ocimene **c - 0.7 -
of the total oils, respectively (Table 2). Except for 1,8- 7.84 β-pinene ** - - 3.7
cineole, R-pinene (2.7%), p-cymene (0.7%), ocimene 8.11 β-myrcene *** - - 1.4
9.15 p-cymene *** - 0.7 -
(0.7%), borneol (1.5%), R-terpineol (1.3%), and camphor
23.44 2-carene ** - - 0.3
(2.4%) were detected in rosemary essential oil. The 26.88 β-caryophyllene *** 0.3 - 1.0
monoterpenes R-pinene (2.3%), β-pinene (3.7%), β-caryo- 28.77 R-caryophyllene ** - - 0.2
phyllene (1.0%), β-myrcene (1.4%), linalool (0.7%), R-ter-
alcohols
pineol (2.7%), thujone (4.2%), and camphor (1.2%) also 12.49 linalool *** 44.5 - 0.7
participated in the case of sage oil, except for 1,8-cineole. 14.85 borneol *** 3.9 1.5 -
The same terpenes have been detected in the Greek sage 15.19 myrcenol ** - - 1.0
essential oil by Sivropoulou et al. (1997). In the case of 15.52 terpinen-4-ol ** 6.9 - 0.4
lavender, linalool (44.5%), linalyl acetate (32.7%), and 15.85 R-terpineol *** 3.5 1.3 2.7
1,8-cineole (4.8%) were dominant in the mixture, rep- ethers and ketones
resenting 82% of the total oil (Table 2). The same 9.46 1,8-cineole *** 4.8 88.9 78.0
terpenes were also found to be the main components of 12.73 thujone *** - - 4.2
lavender oil by Adam et al. (1998). 13.76 camphor *** - 2.4 1.2
Effect of Essential Oils and Their Main Compo- esters
nents on Radial Growth of P. digitatum. Pure 18.95 borneol acetate ** - - 0.2
commercial thymol, the basic compound of thyme, 19.01 linalyl acetate ** 32.7 - -
others 3.4 1.8 2.7
oregano, dictamus, and marjoram oils, and carvacrol,
which differs from thymol only with regard to the total 100 100 100
position of the hydroxyl group at the aromatic ring, were a tR-L,R,S is the retention time for the compounds of L, R, and S,
tested independently for their effectiveness in the according to method A of GC-MS analysis. b ***, positive identi-
inhibition of the radial growth of P. digitatum. A dose- fication from mass spectrum and retention time which agree with
dependent inhibition of mycelial growth was observed authentic compound. c **, tentative identification from mass spec-
in both cases. The ED50 value, the concentration causing tra data. d Not determined.
Essential Oil of Greek Aromatic Plants J. Agric. Food Chem., Vol. 48, No. 6, 2000 2579

Figure 3. Effect of lavender, rosemary, and sage essential

Figure 1. Effect of carvacrol, thymol, and R-terpineol on oils on radial growth of P. digitatum.
radial growth of P. digitatum.

Figure 4. Inhibition of conidial germination in P. digitatum

Figure 2. Effect of oregano, thyme, dictamus, and marjoram by oregano, thyme, dictamus, and marjoram essential oils.
essential oils on radial growth of P. digitatum.

50% inhibition of radial growth, and the minimal

inhibitory concentration (MIC) were approximately 47
and 160 µg/mL, respectively, for carvacrol and 79 and
200 µg/mL, respectively, for thymol (Figure 1). A
comparable MIC value (125 µg/mL) was also found for
carvacrol fungitoxic action by Caccioni and Guizzardi
(1994).
Pure commercial cymene, cineole, linalool, β-myrcene,
R-terpineol, and R-pinene were also tested for their
fungitoxicity to the radial growth of P. digitatum at
concentrations up to 400 µg/mL. Only R-terpineol was
effective, reducing by 46% the radial growth of P. Figure 5. Inhibition of conidial germination in P. digitatum
digitatum at a concentration of 400 µg/mL (Figure 1). by lavender, rosemary, and sage essential oils.
A dose-dependent inhibition of P. digitatum mycelial
growth was also caused by thyme, oregano, dictamus, fectively the conidial germination of P. digitatum (Fig-
and marjoram essential oils (Figure 2). Mycelial growth ure 4). A complete inhibition of germination was ob-
was totally inhibited by these oils at 300, 250, 300, and served at the concentration of ∼250 µg/mL. A similar
400 µg/mL, respectively. fungitoxitity on spore germination was obtained with
Lavender, rosemary, and sage essential oils were pure carvacrol. The MIC of carvacrol on spore germina-
slightly fungitoxic on the mycelial growth of P. digi- tion of P. digitatum was also determined at the level of
tatum (Figure 3). The radial growth was inhibited 29.5, 250 µg/mL by Caccioni and Guizzardi (1994).
24.0, and 9.0% at 1000 µg/mL by lavender, rosemary, Conidial germination was also found to be more
and sage essential oils, respectively. sensitive than the radial growth to lavender, rosemary,
Carvacrol and thymol seem to have more than an and sage essential oils (Figure 5). Thus, at 1000 µg/mL
additive effect on fungal inhibition. This conclusion is of the above oils conidial germination was inhibited 2.0,
drawn by the fungitoxicity of oregano, thyme, and 2.7, and 5.0 times more than the radial growth. Conidial
dictamus essential oils in relation to their composition production was also found to be sensitive to thyme,
in thymol and carvacrol. Thus, oregano oil [carvacrol oregano, dictamus, and marjoram essential oils. A
(7.8%) plus thymol (63.3%), total ) 71%) and thyme oil complete inhibition of conidial production was observed
[carvacrol (2.2%) plus thymol (63.6%), total ) 66%) were at the concentration of 250 µg/mL of thyme, oregano,
more fungitoxic to P. digitatum than dictamus oil and dictamus essential oils. At the same concentration,
[thymol (78%)] at 250 µg/mL oil concentration (Figure marjoram essential oil inhibited the conidial production
2). by 84% (Figure 6). Lavender, rosemary, and sage
Effect of Essential Oils on Conidial Germination essential oils were found to be nonfungitoxic in conidial
and Production of P. digitatum. The oregano, thyme, production of P. digitatum at concentrations up to 1000
dictamus, and marjoram essential oils inhibited ef- µg/mL.
2580 J. Agric. Food Chem., Vol. 48, No. 6, 2000 Daferera et al.

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