Antioxidant Activities and Phenolic Composition of Extracts
from Greek Oregano, Greek Sage, and Summer Savory
V. EXARCHOU,†N. NENADIS,‡M. TSIMIDOU,*,‡ I. P. GEROTHANASSIS,†
A. TROGANIS,§AND D. BOSKOU‡
Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina,
Ioannina GR-45110, Greece, Laboratory of Food Chemistry and Technology, School of Chemistry,
Aristotle University of Thessaloniki, Thessaloniki GR-54124, Greece, and Department of Biological
Applications and Technologies, University of Ioannina, Ioannina GR-45110, Greece
(Greek sage), and
(summer savory) were examined as potential sources of phenolic antioxidant compounds. The
antioxidant capacities (antiradical activity by DPPH•test, phosphatidylcholine liposome oxidation,
Rancimat test) and total phenol content were determined in the ethanol and acetone extracts of the
dried material obtained from the botanically characterized plants. The ground material was also tested
by the Rancimat test for its effect on the stability of sunflower oil. The data indicated that ground
material and both ethanol and acetone extracts had antioxidant activity. Chromatographic (TLC, RP-
HPLC) and NMR procedures were employed to cross-validate the presence of antioxidants in ethanol
and acetone extracts. The major component of all ethanol extracts was rosmarinic acid as determined
by RP-HPLC and NMR. Chromatography indicated the presence of other phenolic antioxidants, mainly
found in the acetone extracts. The presence of the flavones luteolin and apigenin and the flavonol
quercetin was confirmed in the majority of the extracts by the use of a novel 1H NMR procedure,
which is based on the strongly deshielded OH protons in the region of 12-13 ppm without previous
chromatographic separation. This deshielding may be attributed to the strong intramolecular six-
membered ring hydrogen bond of the OH(5)‚‚‚CO(4) moiety.
KEYWORDS: Phenolic antioxidants; Greek oregano; Greek sage; summer savory
Aromatic plants have been studied as sources of different
classes of natural antioxidants. Some plants, grown wild or
cultivated, have been exploited commercially for many years
(1-4). Among the various medicinal and culinary herbs, some
endemic species are of particular interest for small countries
because they may be used for the production of raw materials
or preparations containing phytochemicals with significant
antioxidant capacities and health benefits.
A number of plants characterized by a high carvacrol content,
known as “oregano” plants (5,6), and Satureja hortensis
(summer savory), a closely related plant (7), have been reported
as sources of rosmarinic acid and other phenols (8,9), but their
compositional data are incomplete and they are not exploited
to the same extent as Rosmarinus officinalis.SalVia fruticosa,
known as Greek sage, that may also be rich in rosmarinic acid
(10,11), has not received much attention in comparison to SalVia
The purpose of this study was to evaluate three perennial
plants of the family Lamiaceae, Oregano Vulgare L. ssp. hirtum
(Link) Ietswaart (Greek oregano) and S. fruticosa (Greek sage),
collected in Greece, and S. hortensis (summer savory), collected
in Lithuania. The plants were examined as potential sources of
phenolic antioxidant compounds within the framework of
research projects for the development of preparations containing
natural antioxidants. Commonly used assays (DPPH radical
scavenging, liposome and Rancimat tests) and chromatographic
(TLC, RP-HPLC) and NMR procedures were employed to
cross-validate the importance of the plant material to provide
extracts containing potent antioxidants.
MATERIALS AND METHODS
Plant Material.Dried material from botanically characterized O.
Vulgare ssp. hirtum and S. fruticosa was a gift of the Botany Department
(Mediterranean Agronomic Institute of Chania, Crete, Greece). Lithua-
nian S. hortensis was collected, characterized, and dried under the
guidance of Professor R. Venskoutonis (Kaunas University, Vilnius,
Lithouania). The summer savory of Bulgarian origin was a commercial
sample. The dried material was ground to pass a 0.4 mm sieve and
* Corresponding author. Tel: ++30310997796. Fax: ++30310997779.
†Section of Organic Chemistry and Biochemistry, Department of
Chemistry, University of Ioannina.
‡Laboratory of Food Chemistry and Technology, School of Chemistry,
Aristotle University of Thessaloniki.
§Department of Biological Applications and Technologies, University
5294 J. Agric. Food Chem. 2002, 50, 5294−5299
10.1021/jf020408a CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/14/2002
extracted with ethanol and acetone in a Soxhlet apparatus for 6 h. The
extracts were purged with nitrogen and kept in a dark and cool place
until analyzed. The percent yield of essential oil was measured in a
Clevenger apparatus. Plant material was collected immediately after
blooming and air-dried at room temperature in the dark.
Standards. Caffeic acid (97%) and rosmarinic acid (97%) were from
Fluka AG (Buchs, Switzerland). Chlorogenic acid (97%), quercetin
(99%), and rutin (90%) were from Riedel de Hae¨n (Seelze, Germany).
Apigenin (95%) and kaempferol were purchased from Sigma-Aldrich
Chemie GmbH (Steinheim, Germany). Luteolin (95%) was from Ro¨th
Solvents and Reagents. All solvents and reagents from various
suppliers were of the highest purity needed for each application. The
Folin-Ciocalteu reagent was from Merck (Darmstadt, Germany). 1,1-
Diphenyl-2-picrylhydrazyl radical (DPPH•), L-R-phosphatidylcholine
(lecithin, ∼40%) from soybean, and copper acetate monohydrate were
purchased from Sigma (St. Louis, MO).
Apparatus. A U-2000 Hitachi spectrophotometer (Tokyo, Japan)
was used for the measurement of absorbance in the UV/vis region.
Induction periods of lipid substrates were measured using a Rancimat
617 apparatus (Metrohm AG, Herisau, Switzerland). A high-perfor-
mance liquid chromatograph consisting of a Thermoquest Spectra
System quaternary pump, model P4000 (Austin, TX), a Rheodyne
Model 9725 (Cotati, CA) injection valve with a 20 µL fixed loop, a
Laballiance column oven, model 505 (State College, PA), and a diode
array Fasma 406 scanning detector (HPLC/DAD) (Rigas Laboratories,
Thessaloniki, Greece) was used. The analysis of phenolic compounds
was performed on a 125 mm ×4 mm i.d., 5 µm, Nucleosil 100-5 C18
column (Macherey-Nagel, Du¨ren, Germany). The data from the diode
array detector were stored and processed with EZChrom chromato-
graphic software (Scientific Software Inc., San Ramon, CA). All NMR
experiments were performed on a Bruker AMX-400 MHz spectrometer
equipped with a z-gradient unit. Spectra were obtained from ethanol
and acetone extracts dissolved in CD3OH and CD3COCD3, respectively.
Chemical shifts and integrals were measured with reference to the
internal standard TMSP-d4or TMS (δ)0.000), of known concentra-
tion, depending on the solvent used. NMR data were processed using
UXNMR (Bruker) software. 2D NMR parameters are described in
previous studies (15).
Determination of Total Phenol Content of Plant Extracts. The
content of total phenols in the plant extracts was measured by the
Folin-Ciocalteu assay (16). Results were expressed as mg of caffeic
acid/100 g of extract. Yields of the extracts were based on weight
Estimation of Antiradical Activity by the DPPH•Test. The
antiradical activity of extracts, containing 50 mg/100 g of total phenols
expressed as caffeic acid, was determined according to ref 17. Results
were expressed as DPPH•% scavenging )a×100/b, where a)
[(absinitial -abst)/absinitial]extract and b)[(absinitial -abst)/absinitia)]caffeic acid.
Caffeic acid solution (500 mg/L of ethanol) was conventionally accepted
to result in 100% reduction of the DPPH radical.
Rancimat Test. Samples of sunflower oil (2.5 g) containing 0.02%
w/w extract or 2% w/w ground material were subjected to oxidation at
120 °C (air flow 20 L/h). Induction periods, IP (h), were recorded
automatically in duplicate. The coefficient of variation (CV) (%) of
the method was 3.3 (n)3). Protection factors (PF) were then calculated
from these values (PF )IPextract/IPcontrol).
Phosphatidylcholine Liposome Oxidation. Lecithin was suspended
in doubly distilled water at a concentration of 8 mg/mL by stirring
with a glass rod and sonication for approximately 5 min. Liposome
formation was obtained through additional sonication with a rod (UP
200S, Dr. Hielscher, GmbH, Berlin, Germany) (2.5 min for 10 mL
aliquots of the liposome sample). Ethanol solutions of the plant extracts
were added into Erlenmeyer flasks at a final concentration of ap-
proximately 50 mg/100 g. Caffeic acid solutions (500 mg/L) were used
for comparison. Liposome aliquots were weighed into the flasks and
diluted with doubly distilled water to a final lecithin concentration of
0.8% w/w. The samples were oxidized by adding cupric acetate (3 µM)
and shaking at 37 °C in the dark. Liposome oxidation was monitored
according to ref 18.
Chromatographic Analyses. Thin-layer chromatography was con-
ducted on analytical silica gel plates (Riedel de Hae¨n, Seelze, Germany).
The presence of specific phenolic compounds was detected by
comparison with standards of caffeic, chlorogenic, and rosmarinic acids,
quercetin, rutin, apigenin, kaempferol, and luteolin. Two different
developing systems were used: (a) CHCl3/CH3COOEt/HCOOH, 5:4:1
(v/v/v) (19), and (b) CH3COOEt/CH3OH/H2Ο, 77:13:10 (v/v/v) (20).
For visualization, plates were sprayed with (a) FeCl3(2% in ethanol)
and (b) AlCl3(1% in ethanol) (21). For the HPLC/DAD analysis of
the selected extracts, 1% v/v formic acid (solvent A) and acetonitrile
(solvent B) were used (22). The elution system was as follows: 0-10
min, 10-13% solvent B; 10-25 min, 13-70% solvent B; 25-29 min,
70% solvent B; 29-30 min, 70-10% solvent B; 30-40 min, 10%
solvent B. The flow rate was 1 mL/min, and the injection volume was
20 µL. Peak characterization was based on spiking with standards and
UV spectra matching. Eight point calibration curves, constructed for
rosmarinic acid and other standards, were used for quantification.
Nuclear Magnetic Resonance Spectroscopy. NMR spectra of the
ethanol and acetone extracts were obtained in CD3OH and CD3COCD3
solutions without prior chromatographic separation. For the differentia-
tion of caffeic from rosmarinic acid, a combination of variable-
temperature 2D NMR techniques was used (15). The methodology was
a combination of variable-temperature two-dimensional 1H-1H double-
quantum-filtered correlation spectroscopy (1H-1H DQF-COSY), 1H-
13C heteronuclear multiple-quantum coherence (1H-13C HMQC), and
1H-13C heteronuclear multiple-bond correlation (1H-13C HMBC)
gradient NMR spectroscopy. For the identification of apigenin, luteolin,
and quercetin, only conventional 1D proton NMR spectroscopy was
used. The suppression of the water resonance was achieved with the
use of the Watergate pulse sequence for gradient (23).
RESULTS AND DISCUSSION
A screening assay of the antioxidant activity of ethanol
extracts from the three plants indicated that all of them have a
high capacity to scavenge free radicals (Table 1). This capacity
coincides with a high total phenol content, but it is not
proportional. Thin-layer chromatography verified the presence
of phenolic compounds; the main component appeared to be
rosmarinic acid while three different bands of flavonoids were
In light of this evidence, new plant material was selected just
after blooming for further examination. A screening of the
antioxidant activity of the dried material using the Rancimat
test indicated that all of the plants were good sources of
antioxidants: PFO.Vulgare, 2.1; PFS.hortensis, 1.3; and PFS.fruticosa, 3.4.
Ethanol and acetone extracts were then prepared. Ethanol was
chosen as an industrial polar nontoxic solvent and acetone as a
less polar solvent, often used to extract active phenolic
constituents from plants (2,24,25). The total phenolic content
Table 1. Essential Oil Content, Extraction Yield, Total Phenol Content
(mg of Caffeic Acid/100 g of Extract), and Antioxidant Activity of
Ethanol Extracts of the Plant Species under Investigation
material)a% yield of
ssp. hirtumd3.7 46.5 9800 30.1 ±0.9
ssp. hirtume6.1 45.5 15100 34.8 ±1.5
S. hortensise,f0.2 66.9 13200 75.2 ±0.6
S. hortensisg2.6 30.3 11600 49.9 ±0.4
S. fruticosae1.1 82.6 5000 33.2 ±1.2
aAs determined in a Clevenger apparatus. bMean value of two measurements.
cMean value of three measurements ±SD (standard deviation). dGrown wild.
eCultivated. fLithuanian origin. gBulgarian origin.
Antioxidants from Greek Oregano and Sage and Summer Savory J. Agric. Food Chem., Vol. 50, No. 19, 2002 5295
of the new extracts was measured, and their antioxidant activity
was examined using three different assays. Extracts were tested
toward their DPPH•scavenging activity. Their efficiency toward
oxidation of bulk oil under accelerated conditions (120 °C) was
evaluated using the Rancimat apparatus. Finally, the activity
of the extracts in a multiphase system was examined using the
phosphatidylcholine liposome assay (18). Results of the DPPH•
and Rancimat tests and total phenol content of the extracts are
presented in Table 2. Results from the phosphatidylcholine
liposome assay are given in Figure 1. The data indicated that
the six extracts had antioxidant activity. Differences were
observed in the activity between the ethanol and acetone
extracts, which cannot be explained by the total phenol content.
This can be attributed to the different composition of the ethanol
and acetone extracts. The active compounds of the essential oils
of O. Vulgare and S. hortensis (mainly carvacrol) may contribute
to the antioxidant activity (26). This effect is not shown in the
Rancimat test because of the volatility of these phenols. The
essential oil of S. fruticosa does not contain carvacrol or thymol
or other antioxidants in the volatile fraction, and thus the
difference in the polarity of the solvent does not affect the
scavenging activity (11). Greek oregano, summer savory, and
Greek sage seem to be good sources of natural antioxidants and
can be further considered for commercial exploitation.
Assignment of the antioxidant activity to specific phenolic
compounds was attempted using chromatographic and NMR
procedures. Thin-layer chromatography indicated the presence
of rosmarinic acid in both ethanol and acetone extracts. Caffeic
acid seemed to be present only in the ethanol extracts of O.
Vulgare ssp. hirtum and S. hortensis. Bands of flavonoids were
also observed near the baseline and the solvent front on the
TLC plates. RP-HPLC was used to characterize further the
phenolics of each extract and to check the levels of caffeic and
rosmarinic acids. The respective HPLC data are shown in Table
3. High levels of rosmarinic acid were found in all extracts
whereas the presence of caffeic acid in trace amounts was
confirmed in the extracts of oregano and summer savory.
Comparing the results of Tables 2 and 3, it can be generally
concluded that the extracts that are rich in rosmarinic acid have
higher radical scavenging activity. This caffeic acid derivative,
common in many plants and very often present in our diet, is a
strong radical scavenger and has been reported to be more
effective in relation to Trolox (27). The presence and levels of
caffeic and rosmarinic acids in the extracts were also examined
by the use of two-dimensional NMR spectroscopy (15) without
previous chromatographic separation. To improve resolution of
strongly overlapped signals, techniques based on the temperature
dependence of proton chemical shifts were applied (15). In
practice, by variable-temperature 2D DQF-COSY spectra at 243
K, the cross-peak connectivity of the H3a and H2a protons of
rosmarinic acid (Figure 2) is differentiated from that of caffeic
acid (Figure 2) and other overlapping cross-peaks. Further
Figure 1. Antioxidant activity of ethanol and acetone extracts (50 mg of caffeic acid/100 g) in lecithin liposomes at 37 °C.
Table 2. Essential Oil Content, Extraction Yield, Total Phenolic
Content (mg of Caffeic Acid/100 g of Extract), and Antioxidant Activity
of Ethanol and Acetone Extracts of the Plants under Investigation
ssp. hirtum 2.9
ethanol 59.2 9700 99.1 1.2
acetone 22.9 17400 54.4 1.4
S. hortensis 0.7
ethanol 31.4 6400 95.8 1.2
acetone 9.9 21500 33.0 1.2
S. fruticosa 0.9
ethanol 61.9 8100 98.5 1.7
acetone 23.8 9900 97.6 2.2
aAs determined in a Clevenger apparatus. bMean value of two measurements;
CV (%) )3, n)5. cPF )protection factor.
Table 3. Caffeic and Rosmarinic Acid Content of Ethanol and Acetone
Extracts As Determined by RP-HPLC at 326 nm and NMR Techniques
(mg/100 g of
(mg/100 g of
plant extract HPLC NMR HPLCaNMRb
ethanol trctr 1271 ±115 904 ±74
acetone nddnd 231 ±15 173 ±12
ethanol tr tr 2137 ±223 2517 ±215
acetone nd nd 249 ±11 267 ±17
ethanol nd nd 1483 ±154 1185 ±83
acetone nd nd 556 ±53 359 ±29
aMean value of three measurements ±SD (standard deviation). bMean value
of four measurements ±SD (standard deviation). ctr )traces. dnd )not detected.
5296 J. Agric. Food Chem., Vol. 50, No. 19, 2002 Exarchou et al.
improvement in resolution and better assignment information
were achieved by the use of two-dimensional 1H-13C hetero-
nuclear techniques. The NMR analysis confirmed the presence
of rosmarinic and caffeic acids and cross-validated the levels
obtained by HPLC (Table 3).
The antioxidant activity of the extracts may also be partly
due to the presence of other phenolic compounds such as
flavonoids. On the HPLC chromatograms some peaks having
the same RRTs with apigenin and luteolin or quercetin (Figure
2) were located. These peaks had UV spectra that practically
matched fully with those of the respective standards. The
literature related to the presence of such flavonoids in the plants
under investigation is generally limited. Apigenin and quercetin
derivatives have been found in oregano plants (28,29). Karakaya
et al. (30) reported the presence of quercetin and luteolin in S.
officinalis infusions. The flavonoid composition of S. fruticosa
and S. hortensis is not known.
The presence of these three flavonoids was investigated using
NMR techniques. The 1H NMR spectra of the three flavonoids
in CD3COCD3and CD3OH solutions indicated a significantly
deshielded signal in the region of 12-13 ppm, as shown in
Figure 3. This resonance may be attributed to the hydroxyl
proton OH(5) of flavonoids which participates in a strong six-
membered ring intramolecular hydrogen bond with CO(4) and,
therefore, is strongly deshielded (31)(Figure 2). As a rule the
1H NMR resonances of the -OH groups appear at room
temperature as broad signals especially in protic solvents, owing
to the mobility of the hydrogen and its fast exchange, on the
NMR time scale, with the protons of the solvent. However, by
decreasing the temperature, the proton exchange rate is reduced
and the -OH peaks are revealed as sharp peaks (32). In acetone
solution the OH(5) resonances of the three flavonoids are clearly
observed as sharp singlets even at room temperature (Figure
3A). In methanol-d3solution a relatively broad -OH(5)
resonance at 12-13 ppm commences to appear at 280 K.
Variable-temperature 1H NMR spectroscopy of apigenin,
luteolin, and quercetin in CD3OH showed that at 240 K the
OH(5) signals appeared as sharp singlets (Figure 3B). The
observation of the OH signal in protic solvent was achieved by
the use of the Watergate pulse sequence for gradient, which
does not eliminate fast-exchanging OH resonances with the
solvent. Since the region of 12-13 ppm in the 1H NMR spectra
of the extracts is not as crowded as the aromatic one, the
identification of flavonoids can be based only on 1D proton
NMR spectroscopy, without the need for two-dimensional
techniques, which are very time-consuming. It is notable that
the OH(5) resonance is more deshielded in the flavones apigenin
and luteolin compared to that of the flavonol quercetin. This
can be attributed to the presence of the OH(3) group in quercetin
(Figure 2) that attenuates the electron density of the C(O)
oxygen and, thus, decreases the strength of the OH(5)‚‚‚OC
Figure 2. Structural formulas of rosmarinic acid (1), caffeic acid (2), and
apigenin (3), luteolin (4), and quercetin (5). Figure 3. Selected region of the 400 MHz 1H NMR spectra of (A) (a)
quercetin, (b) luteolin, and (c) apigenin, in acetone-d6at 300 K, and of
(B) (a) quercetin, (b) luteolin, and (c) apigenin, in methanol-d3at 240 K.
Antioxidants from Greek Oregano and Sage and Summer Savory J. Agric. Food Chem., Vol. 50, No. 19, 2002 5297
intramolecular hydrogen bond. As a result it is possible to
distinguish the flavones apigenin and luteolin from the flavonol
It is important to define the temperature at which the 1H NMR
spectra of the extracts must be carried out in order to avoid
undesirable broadening of the OH peaks. Therefore, detailed
variable-temperature 1H NMR spectra of the extracts in CD3-
COCD3and CD3OH solutions were obtained. With the use of
the proper temperature and by spiking with standards, the two
flavones (luteolin and apigenin) and quercetin were detected in
ethanol and acetone extracts of Greek sage (Figure 4). In the
ethanol extract of Greek oregano and summer savory none of
the three flavonoids was detected by NMR, possibly due to their
low concentration that was below the detection limit. Luteolin
and apigenin were found in their acetone extracts. To the best
of our knowledge this is the first application of the -OH spectral
region for the analysis of real samples.
It can be concluded from the above that the flavonoids
investigated have a rather small contribution to the total
antioxidant activity of the extracts. The quantities of the
flavonoids estimated by HPLC were insignificant, below the
quantitative detection limits for apigenin, quercetin, or luteolin
(0.1 µg/25 µL). In the case of Greek sage, less polar phenolic
compounds that have been identified in other sage species (33)
may be responsible for the high capacity of the acetone extracts
(Figure 5;Table 2). For the rest of the plants the main active
ingredient is rosmarinic acid, an important phytochemical, which
has been found to be a potent active substance against human
immunodeficiency virus type 1 (HIV-1) (34).
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Received for review April 5, 2002. Revised manuscript received June
24, 2002. Accepted June 25, 2002. This research was in part supported
by the Copernicus ERBIC15CT96100 program and in part financed
by the Greek General Secretariat of Research and Technology (EPET
Antioxidants from Greek Oregano and Sage and Summer Savory J. Agric. Food Chem., Vol. 50, No. 19, 2002 5299