Food Chemistry 116 (2009) 452461

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Chemical composition, antioxidant activity and antimicrobial properties

of propolis extracts from Greece and Cyprus
Nick Kalogeropoulos a,*, Spyros J. Konteles b, Elena Troullidou a, Ioannis Mourtzinos a, Vaios T. Karathanos a
a
Laboratory of Chemistry Biochemistry Physical Chemistry of Foods, Department of Nutrition-Dietetics, Harokopio University, 70 El. Venizelou Ave.,
Kallithea, 176 71 Athens, Greece
b
Department of Food Technology, Technological Educational Institute of Athens, 12 Ag. Spyridonos St., Egaleo, 122 10 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Chemical composition, antioxidant activity and in vitro antimicrobial activity of twelve propolis ethanolic
Received 17 December 2008 extracts (PEE) from mainland Greece, Greek islands, and east Cyprus were determined. The PEE studied
Received in revised form 23 January 2009 contained signicant amounts of terpenes and/or avonoids, anthraquinones mainly emodin and chry-
Accepted 19 February 2009
sophanol and low amounts of phenolic acids and their esters, presenting differences from typical Euro-
pean propolis, and similarities to East Mediterranean propolis. Simple polyphenols and terpenic acids
content ranged between 11.9373.5 and 7.23286.5 mg/g of PEE, respectively, with anthraquinones rep-
Keywords:
resenting the 1.328.9% of simple polyphenols. Despite differences in composition, the PEE samples
Propolis
Flavonoids
exhibited signicant antioxidant, antibacterial, and antifungal activities, affecting a wider spectrum of
Terpenes microorganisms than the food grade antibacterial nisin, and presenting lower or no activity against sev-
Anthraquinones eral Lactobacillus strains. The presence of signicant amounts of terpenoids seemed to enhance the anti-
Antioxidant activity microbial activity of PEE. The conclusion, given the non-toxic and natural origin of PEE, is that, besides
Antimicrobial activity their potential pharmaceutical and nutraceutical value, propolis balsams from Greece and Cyprus are
Nisin attractive candidates for use as natural antioxidant and microbicidal additives in food systems, especially
those containing lactic acid bacteria.
2009 Elsevier Ltd. All rights reserved.

1. Introduction factors, as well as by the collection season (Ahn et al., 2007; Bank-
ova, De Castro, & Marcucci, 2000; Kujumgiev et al., 1999).
Propolis is a resinous, strongly adhesive natural substance, col- Propolis is considered responsible for the low incidence of bac-
lected by honeybees (Apis mellifera L.) from buds and leaves of teria and moulds within the hive. The action against microorgan-
trees and plants, mixed with pollen as well as enzymes secreted isms is an essential characteristic of propolis, and humans have
by bees (Marcucci, 1995). Bees use it as a general-purpose sealer, used it for centuries for its pharmaceutical properties (Bankova
to smooth out the internal walls of the hive and as protective bar- et al., 2000; Ghisalberti, 1978). Besides its antibacterial, antifungal
rier against intruders (Burdock, 1998). and antiviral properties, propolis presents many other benecial
In general, propolis is composed of 50% resin and vegetable bal- biological activities such as antioxidant, antiinammatory, antitu-
sam, 30% wax, 10% essential and aromatic oils, 5% pollen and 5% mor, hepatoprotective, local anesthetic, immunostimulatory, anti-
various other substances, including organic debris (Burdock, mutagenic, etc. (Banskota, Tezuka, & Kadota, 2001; Burdock,
1998). Wax and organic debris are removed during processing, 1998; Kim, Lee, Aum, & Kim, 2008; Kujumgiev et al., 1999). For
usually by ethanolic extraction, and the propolis tincture (balsam) these reasons propolis has been used as a popular remedy in folk
thus obtained, contains the bulk of propolis bioactive constituents. medicine, in apitherapy, as a constituent of biocosmetics, health
More than 300 compounds, among which polyphenols, terpenoids, foods and in numerous other purposes (Bankova et al., 2000; Bans-
steroids, sugars and amino acids have been detected in raw prop- kota et al., 2001; Ghisalberti, 1978). Although reports of allergic
olis. Their abundance is inuenced by botanical and geographical reactions are not uncommon, propolis is relatively non-toxic, with
a non-observed effect level (NOEL) of 1400 mg/kg body weight/day
in a mouse study (Burdock, 1998).
Abbreviations: BSTFA, bis-(trimethylsilyl)-triuoroacetamide; DPPH, 1,1-diphe- Propolis antioxidant, antibacterial and antifungal properties,
nyl-2-picrylhydrazyl radical; FRAP, ferric reducing antioxidant potential; MIC, combined with the fact that several of its constituents are present
minimum inhibitory concentration; PEE, propolis ethanolic extract; TIC, total ion
current; TMS, trimethylsilyl ether.
in food and/or food additives, and are recognised as Generally
* Corresponding author. Tel.: +30 210 9549 251x367; fax: +30 210 9577 050. Recognised as Safe (GRAS) (Burdock, 1998), make it an attractive
E-mail address: [email protected] (N. Kalogeropoulos). candidate as a natural preservative in new food applications. This

0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.02.060
N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461 453

meets the demand for natural antioxidants and antimicrobials, by the ACA-DC collection (Laboratory of Dairy Research, Agricul-
fuelled by the increasing consumer awareness for natural, mini- tural University of Athens, Greece) while Lactobacillus fermentum
mally processed foods with traditional preservatives absent or at F 12, Lactobacilus casei LC 14, Lactobacillus delbrueckii sub. del-
very low concentrations (Han & Park, 1995; Tosi, R, Ortega, & brueckii LDD-C1, Lactobacillus plantarum LP 101, and Lactobacillus
Cazzoli, 2007). helveticus LH 09 were provided by Laboratory of Microbiology, Har-
Due to geomorphological characteristics, the Greek ora pre- okopio University, Athens, Greece. Lactic acid bacteria working cul-
sents high biodiversity with many endemic plants (Melliou & Chi- tures were maintained on Man Rogosa and Sharpe (MRS, Merck,
nou, 2004), something that is also true for Greek islands and Darmstand, Germany) agar slants while the remaining strains were
Cyprus, as a result of the islands isolation in relation to continental maintained on Tryptone Soy agar slants (TSA, Merck) supple-
lands. This is expected to differentiate the composition of Greek mented with 0.6% yeast extract (Sigma, St. Louis, MO, USA),
and Cypriot propolis from that of typical European ones. (TSYEA). Yeasts were maintained on yeast extract-peptone-dex-
In literature, scarce data can be found about the composition, trose (YPD) agar slants. All slants were stored at 4 C, and sub-cul-
antimicrobial and antioxidant activity of Greek propolis extracts, tured twice per month.
and no data at all about propolis from Cyprus. The chemical com-
position and antimicrobial activity of one propolis extract and of 2.2. Methods
the volatiles from ve propolis obtained from Greek mainland
and one Greek island have been reported by Melliou and Chinou 2.2.1. Samples collection and propolis extract preparation
(2004) and Melliou, Stratis, and Chinou (2007), respectively, while Propolis samples were obtained from several locations of cen-
Velikova et al. (2000) have studied the composition of propolis tral and southern Greece, Aegean Sea islands, and Cyprus (Larnaka,
from Greece, Bulgaria, Turkey and Algeria. SE Cyprus), as indicated in Fig. 1 and Table 1. Samples were col-
In the present study, we are reporting the chemical composition lected during springsummer of 2007; in two cases KAR-I and
including quantitative data for several simple polyphenols and LAR-I samples collected during 2006 were obtained. Voucher
terpenic acids the antioxidant activity, and the antimicrobial specimens are deposited in the Laboratory of Chemistry Bio-
properties of twelve propolis samples collected from 10 localities chemistry Physical Chemistry of Foods, Department of Nutri-
of mainland Greece, Greek islands and Cyprus. Moreover, we com- tion-Dietetics, Harokopio University, Athens, Greece. Crude
pare the inhibitory spectra of propolis samples with that of nisin, a propolis samples were frozen (20 C), grounded in a chilled grin-
GRAS antibacterial peptide used for several years in foods. der and small amounts (10 g) of pulverised crude propolis were ex-
tracted under stirring with a 10-fold volume of 70% ethanol
solution in tightly closed bottles, for 3 days. Extraction was carried
2. Materials and methods out at ambient temperature in the dark. To remove waxes and less
soluble substances, the suspensions were subsequently frozen at
2.1. Materials 20 C for 24 h, then ltered with Whatman No. 1 lter paper.
The freezing-ltration cycle was repeated three times. The nal l-
2.1.1. Reagents and chemicals trates represent the balsam (tincture) of propolis and are referred
Bis-(trimethylsilyl)-triuoroacetamide (BSTFA), analytical grade to as PEE (propolis ethanolic extract). The solutions were evapo-
ethanol, quercetin, 3-(4-hydroxyphenyl)-1-propanol, homovanillic rated to near dryness on a rotary evaporator under reduced pres-
acid, phloretic acid, oleanolic acid, cinnamic acid, Folin-Ciocalteu re- sure at 40 C, and then freeze-dried. The resulting powders were
agent, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbox- dissolved in 80:20 ethanol:water in order to get 5% w/v PEE stock
ylic acid) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) were solutions. The ethanolic extraction yields were determined gravi-
obtained from Aldrich (Steinheim, Germany). Tyrosol, proto- metrically in aliquots of the extracts and found to range from
catechuic acid, sinapic acid, o-coumaric acid, 3,4-dihydroxy-phenyl- 23.9% to 61.2% of raw propolis samples (Table 1).
acetic acid, and caffeic acid, were purchased from Fluka (Steinheim,
Germany); ascorbic acid, 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), 2.2.2. Derivatisation and GCMS analysis of propolis extracts
p-hydroxy-benzoic acid, p-hydroxy-phenylacetic acid, ursolic acid, PEE constituents were determined by GCMS operating either in
vanillin, p-coumaric acid, chlorogenic acid, catechin, syringic acid, SCAN (total ion current, TIC) or selective ion monitoring (SIM) mode
gallic acid, resveratrol, and ferulic acid were obtained from Sigma (Table 2). Samples were derivatised prior to analysis. For this pur-
(Steinheim, Germany); vanillic acid was obtained from Serva (Hei- pose, a proper volume of PEE, containing no more than 1 mg of
delberg, Germany), pinocembrin, kaempferol, chrysin, naringenin, dry extract, was transferred into GC vials. The internal standard
acacetin and apigenine from Extrasynthse (Genay-Sedex, France), was added 50 ll of 3-(4-hydroxyphenyl)-1-propanol solution
myricetin and epicatechin from Fluka Biochemika (Steinheim, Ger- (19.2 lg/ml) the sample was evaporated to dryness under nitro-
many), genistein from Alfa Aesar (Karlsruhe, Germany), and abietic gen, and derivatised by the addition of 250 ll BSTFA at 70 C for
acid from MP Biomedicals LLC (Irvine, CA). Nisin powder (1 g = 20 min. An aliquot (1 ll) of the derivatised sample was injected into
40 106 IU) was purchased from Sigma Chemicals (St. Louis, USA). the gas chromatograph at a split ratio 1:20. An Agilent (Wallborn,
Germany) HP series GC 6890 N coupled with a HP 5973 MS detector,
2.1.2. Bacterial strains and culture preparations split splitless injector and an HP 7683 autosampler were em-
In this study, 18 pathogenic and non-pathogenic bacterial ployed. Mass selective (MS) detector operated under electron im-
strains (target strains) and two pathogenic fungi were used. Shi- pact ionisation (70 eV) and MS scan range was 50800 Da.
gella dysenteriae NCTC 2966, Salmonella typhimurium NCTC Analysis of the samples was achieved using an HP-5 MS capillary
12023, Enterobacter aerogenes NCTC 10006, Yersinia enterocolitica column (5% phenyl 95% methylsiloxane, 30 m 0.25 mm
NCTC 10460, Escherichia coli NCTC 09001, Staphylococcus aureus 250 lm). Carrier gas was helium at a ow rate of 0.7 ml/min, injec-
NCTC 6571 (I), S. aureus ATCC 25923 (II), Staphylococcus epidermidis tor and MS detector transfer line temperatures were set at 220 C
NCTC 11047, Bacillus cereus NCTC 7464 (I), B. cereus ATCC 9139 (II), and 300 C, respectively. To obtain the total ion chromatograms
Listeria monocytogenes NCTC 10357 (I), L. monocytogenes ATCC (GCMS operating in SCAN mode) of derivatised samples, the fol-
7644 (II) and the yeasts Candida tropicalis ATCC 13801 and Candida lowing temperature program was followed: oven initially at
albicans ATCC 10231 were provided by Agrolab S.A. (Agrolab S.A., 100 C, temperature increased at 5 C/min to 310 C, hold 8 min at
Athens, Greece). Lactobacillus bulgaricus ACA-DC 101 was provided 310 C. Under these conditions more than 80 PEE components in
454 N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461

KAR
SKO LES

KAL
MEG TIN
TRI
ARF

Aegean Sea
GREECE
CRE

LAR
CYPRUS

Fig. 1. Propolis sampling sites.

Table 1 2008). Anthraquinones were quantitated by means of the pinocem-

Propolis collection sites and percent yield of propolis ethanolic extraction. brin reference curve.
Code Collection Geographical location Ethanolic extract The derivatised samples were rechromatographed by GCMS
area/year of crude propolis operating in selective ion monitoring (SIM) mode. Identication
(%w/w) of chromatographic peaks was made by comparing the 0.05 Rt
Greece and ratios of target and qualier ions of each compound with those
TRI Trikorfo/2007 Messinia, Southern Peloponnese 27.5 of standards, while quantication was carried out by employing 3-
ARF Arfara/2007 Messinia, Southern Peloponnese 61.2
(4-hydroxyphenyl)-1-propanol as internal standard. Internal stan-
KAL Kalavryta/2007 Achaia, North Peloponnese 49.4
MEG Megalopolis/ Arcadia, Central Peloponnese 53.2 dard quantication was performed based on a series of nine stan-
2007 dard mixtures of the individual polyphenols and terpenic acids
KAR-I Karditsa/2006 Thessaly, Central Greece 40.1 containing the same amount of internal standard as that of sam-
KAR-II Karditsa/2007 Thessaly, Central Greece 58.6 ples. Linearity was obtained for all target compounds detected in
CRE Aloides/2007 Crete island, Southern Aegean Sea 23.9
samples in the range of quantication limit. In this way, 27 simple
TIN Pirgos/2007 Tinos island, Central Aegean Sea 26.8
SKO Glossa/2007 Skopelos island, NW Aegean Sea 27.5 polyphenols and two terpenic acids namely oleanolic and ursolic
LES Moria/2007 Lesvos island, East Aegean Sea 25.3 were quantitatively determined. Retention times, target and
Cyprus qualier ions for the trimethylsilyl ethers (TMS) of the 29 com-
LAR-I Larnaca/2006 East Cyprus 48.2 pounds and the internal standard are given in Table 2.
LAR II Larnaca/2007 East Cyprus 40.4
2.2.3. Determination of total polyphenols
The total polyphenol content of PEE were determined by the Fo-
the form of their TMS ethers were identied using their mass spec- lin-Ciocalteu colourimetric method, adapted to microscale (Ar-
tra and by reference to NIST 98 (NIST MS search v6.1d) and Wiley nous, Makris, & Kefalas, 2002). The results were expressed as mg/
275 (Wiley, New York, NY) mass spectra libraries, as well as by ana- g caffeic acid equivalents (CAE).
lysing pure standards and by reference to literature. In this way, the
qualitative analysis of PEE was achieved, while additionally the 2.2.4. Measurement of free radical scavenging activity (DPPH assay)
avonoids apigenin, pinocembrin, pinobanksin and pinobanksin- The ability of PEE constituents to scavenge the stable free rad-
O-acetate, the anthraquinones 1,8-dihydroxy-3-methylanthraqui- ical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was estimated accord-
none (chrysophanol), 1,3,8-trihydroxy-6-methylanthraquinone ing to the procedure described by Arnous et al. (2002) and the
(emodin), 2,7-dihydroxy-5-methoxy-3-methylanthraquinone, and results were expressed as mmol Trolox equivalents per g of PEE.
1,7-dihydroxy-3-methoxy-6-methylanthraquinone, and the ter-
penic acids isopimaric, abietic and dehydroabietic were quantita- 2.2.5. Measurement of reducing power (FRAP assay)
tively determined. Abietic acid, apigenine and pinocembrin were The reducing ability of PEE was determined by the ferric reduc-
identied and quantied by analysing a series of nine standard solu- ing/antioxidant potential (FRAP) assay. This procedure involves the
tions of the specic compounds. The TMS derivatives of isopimaric reduction of ferric tripyridyltriazine (FeIII-TPTZ) complex to a blue
and dehydroabietic acids were identied by means of the Wiley coloured FeII-TPTZ by samples antioxidants. For the determination,
275 mass spectra library (Wiley, New York, NY) and quantied by the protocol described by Arnous et al. (2002) was followed. Ascor-
the abietic acid reference curve. Pinobanksin, and pinobanksin- bic acid was used as a positive control to construct a reference curve,
O-acetate were identied by the characteristic ions of their TMS and the results were expressed as mmol ascorbic acid per g of PEE.
derivatives (Neacsu et al., 2007) and were quantied by means of
the pinocembrin reference curve. Among the anhtraquinones de- 2.2.6. Antimicrobial action assay
tected, chrysophanol and emodin were identied by the character- The in vitro inhibitory activity of propolis extracts against thir-
istic ions of their TMS derivatives (Zuo, Wang, Lin, Guo, & Deng, teen Gram positive, ve Gram negative bacteria and two pathogenic
N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461 455

Table 2 for 48 h at 37 C under anaerobic conditions (BBL GasPak system,

Simple polyphenols and terpenic acids quantitated by GCMS. Becton Dickinson Microbiology Systems, Cockeysville, MD). The
Compound GCMS run Target ion Qualier ions inhibitory activity of the samples was detected as a clear zone
mode (m/z)a (m/z)a around the wells. Minimum Inhibitory Concentration (MIC) was
Vanillin SIMb 194 209 dened as the lowest concentration of the sample that caused a
Cinnamic acid SIM 205 220 clear (13 mm) zone of inhibition. As a negative control, the etha-
Tyrosol SIM 179 267, 282 nol: water (80:20 v:v) solution was used. All tests were carried out
p-Hydroxybenzoic acid SIM 267 223, 193
p-Hydroxyphenylacetic acid SIM 252 296, 281
in triplicate and the results were averaged.
I.Sc SIM 206 191, 179
Phloretic acid SIM 192 310 2.2.7. Statistical analysis
Vanillic acid SIM 297 267, 312 All the analyses were duplicated unless otherwise specied, and
Homovanillic acid SIM 326 267, 311
o-Coumaric acid SIM 293 308, 147
the results presented are the averages of the obtained values. Data
Protocatechuic acid SIM 193 355, 370 manipulation was performed by means of Microsoft Excel (Micro-
3,4-Dihydroxyphenylacetic acid SIM 384 267, 179 soft Corp., Redmond, WA). Hierarchical cluster analysis was carried
Syringic acid SIM 327 342, 312 out by Statgraphics Plus for Windows 4.0 (Statistical Graphics
p-Coumaric acid SIM 308 293, 219
Corp., Herndon, VA)
Gallic acid SIM 281 458, 443
Ferulic acid SIM 338 323, 308
Caffeic acid SIM 396 219, 381
Sinapic acid SIM 368 353, 338 3. Results and discussion
Resveratrol SIM 444 445, 443
Chrysin SIM 383 384 3.1. PEE chemical composition
Epicatechin SIM 368 355, 474
Naringenin SIM 473 296
Catechin SIM 368 355, 474 3.1.1. Overview
Genistein SIM 473 According to the results of the GCMS analysis, the PEE samples
Kaempferol SIM 559 560 from Greece and Cyprus contained more than 100 compounds,
Chlorogenic acid SIM 345 307, 324 more than 80 of which were identied. Among the identied PEE
Quercetin SIM 647 559, 575
Myricetin SIM 735 647, 575
constituents several compounds with known antioxidant, antiin-
Oleanolic acid SIM 203 320, 482 ammatory and antimicrobial activities, like avonoids, terpenes,
Ursolic acid SIM 203 320, 482 anthraquinones, phenolic acids and their esters were present.
Dehydroabietic acid SCANd The chemical composition of PEE as % of TIC is presented in Table
Abietic acid SCAN
3 and summarised in Table 4, while the quantitative (w/w) results
Isopimaric acid SCAN
Pinocembrin SCAN for individual polyphenols and terpenic acids are given in Table 5.
Pinobanksin SCAN It is generally accepted that propolis from temperate climatic
Pinobanksin-O-acetate SCAN zones, like Europe, North America and the non-tropical regions of
Chrysophanol (1,8-dihydroxy-3- SCAN Asia, originate mainly from the bud exudates of Populus species
methylanthraquinone)
and their hybrids, and are rich in avonoids, phenolic acids and
1,6-Dihydroxy-8-methoxy-3- SCAN
methylanthraquinone their esters (Bankova, Popova, Bogdanov, & Sabatini, 2002; Bank-
Emodin (1,3,8-trihydroxy-6- SCAN ova et al., 2000), while propolis from tropical regions, where no
methylanthraquinone) poplars and birches exist, are rich in prenylated benzophenons,
1,7-Dihydroxy-3-methoxy-6- SCAN
diterpenes and avonoids (Ahn et al., 2007; Bankova, 2005; Bans-
methylanthraquinone
Apigenin SCAN kota et al., 2001). From the analytical results obtained, it is obvious
that, besides differences among individual constituents, the Greek
a
Trimethylsilyl (TMS) ether derivatives of the compounds.
b
and Cypriot propolis samples share characteristics that differenti-
SIM = selective ion monitoring.
c
I.S. = internal standard, 3-(4-hydroxyphenyl)-1-propanol.
ate them from typical European propolis, like the presence of
d
SCAN = total ion current monitoring. anthraquinones and terpenes in signicant amounts, both when
expressed as % of TIC and as mg/g of PEE (Tables 4 and 5), and
the relatively low abundance of phenolic acids and their esters.
fungi was investigated by the agar well diffusion assay. For this pur- By performing hierarchical cluster analysis in the data of Table 4,
pose, PEE stock solutions (5% w/v) were serially twofold diluted after excluding aliphatic acids and alcohols, the propolis samples
with 80:20 ethanol:water and the diluted solutions were used for were divided into two groups: one comprised of samples from cen-
the assay. The sensitivity of the target strains was also tested tral Peloponnese (MEG) and central Greece (KAR-I and KAR-II),
against nisin, a food grade proteinaceous antibiotic, and their anti- which contained low amounts of terpenes and were rich in anthra-
microbial spectra were compared. quinones, avonoids, phenolic acids and their esters, and a second
For the assay, 15 ml of the appropriate agar medium according one comprised of the rest of PEE samples (graph not shown).
to the target strain tested, were added into Petri dishes. The melted
and tempered (at 45 C) agar was previously inoculated with 3.1.2. Terpenes
150 ll of the target cell suspension. The suspensions were pre- The majority of the 12 PEE samples studied contained signi-
pared by diluting overnight cultures of the target strain into saline cant amounts of terpenes, ranging from 1.43% to 41.87% of TIC,
solution to, approximately, 106 cfu/ml using McFarland turbidity while in seven samples, they comprised more than 30% of TIC (Ta-
standards (bioMerieux S.A., Marcy l toile, France). The plates bles 3 and 4). Terpenoids have been also observed in propolis from
were dried for 1 h and then, using a sterile cylinder, wells of Mediterranean areas like Sicily, Turkey and Algeria (Bankova et al.,
7.0 mm diameter were made and lled up with 100 ll of the di- 2002; Velikova et al., 2000), and Greece (Melliou & Chinou, 2004;
luted PEE solutions. In order to obtain comparable results, all sam- Melliou et al., 2007). Based on pollen analysis, Melliou and Chinou
ples were treated under the same conditions in the same plate for (2004) proved that a signicant source of propolis from mainland
each microorganism. The plates were incubated for 24 h at 37 C Greece was Coniferae trees especially Pinus sp. the resin of
and the results were recorded. Lactobacillus strains were incubated which is rich in terpenic acids like abietic, dehydroabietic and
456 N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461

Table 3
Composition of propolis extracts from Greece and Cyprus assessed by GCMS as trimethylsilyl ethers derivatives (% of total ion currenta).
Compoundb Rtc Composition
TRI ARF KAL MEG KAR- KAR- CRE TIN SKO LES LAR- LAR-
I II I II
Alcohols
Ethylene glycol 5.77 0.21 0.41 0.32 0.45 0.39 0.37 0.52 0.24 0.29 0.24 0.43 0.62
Phenethyl alcohol 6.07 0.30 0.69
Glycerol 6.99 0.61 1.43 0.86 0.26 1.41 0.45 6.80 3.21 1.42 1.24 1.62 1.12
Aliphatic acids
Succinic acid 7.69 0.13 0.27 0.16 0.08 0.21
Malic acid (hydroxybutanedioic acid) 11.67 0.10 0.05 0.09 0.34 0.48 0.32 0.26 0.11
Azelaic acid 18.24 0.40
Hexadecanoic acid 22.96 0.28 0.27 0.39 0.28 0.88 0.51 0.34 0.78 0.63 1.02
Oleic acid 26.04 0.65 0.36 0.57 0.43 0.97 0.51 1.00 0.72 1.37 1.10 0.80 1.65
2-Hexenedioic acid 32.96 0.52 0.33 0.41 0.47 0.70 0.98
Aromatic acids
Benzoic acid 6.42 0.57 1.99
3,4-Methylenedioxycinnamic acid 6.73 0.31 0.57 0.49 0.68 0.53 0.58 0.80 0.41 0.47 0.11 0.64 0.93
Cinnamic acid 12.70 0.24 0.76
3,4-Dimethoxycinnamic acid 22.71 0.29 0.28 0.74 1.13 0.63 0.21
Ferulic acid 23.68 0.42 0.63 0.82 0.32 0.09
Caffeic acid 24.84 0.25 0.85 1.56 0.81 0.20 0.47
p-Coumaric acid 28.57 0.54 0.82 2.85 0.48 2.13 0.80 0.83
D-9-Tetrahydrocannabinol acid 30.53 0.20 0.43 0.54 1.75 0.66 0.40 0.29 0.36
Gallic acid 33.93 0.81 1.73 0.63
Phloroglucinic acid 35.47 0.20 0.24 1.44 2.15 1.11 0.53 0.74 0.41 0.94 1.83
Esters
Ethyl benzoate 5.18 0.38
3-Methyl-2-butenyl isoferulate 28.68 0.19 0.34 1.22 0.54
Cinnamyl cinnamate 29.45 0.27 1.49 1.17 0.55 0.06 0.20 1.16
Cinnamic acid ester 31.15 5.21 7.46
Benzyl ferulate 33.48 0.45 0.28 0.20 0.48 1.16 1.79 0.21
Phenylethyl caffeate (CAPE) 35.24 1.18 2.31 1.26 2.05 3.17 1.56 0.70 1.30 0.77 1.26 1.49
Cinnamyl caffeate 38.41 0.24 0.47 0.89 1.17 1.84 1.46 0.42 1.36 1.31
Flavonoids
Pinostrobin (chalcone) 31.25 0.23 1.37 0.43 0.84 0.35 0.72 0.33
Pinocembrin 31.50 5.18 5.83 6.83 11.52 8.96 18.03 4.01 5.24 4.44 0.92 0.66 3.41
Pinobanksin 32.33 0.40 1.08 6.21 3.70 3.38 2.59 1.52 1.39 1.17 0.53
Pinobanksin-3-O-acetate 33.57 2.16 5.35 1.96 5.84 7.61 8.97 0.79 0.48 2.94 4.86 2.77 1.92
Chrysine 34.06 0.79 1.13 0.46 0.92 3.06 4.50 0.51 1.73 0.73 1.35
Galangin 34.57 0.36 0.90 1.09 1.92 1.25 0.39 0.63 0.48 0.23 0.15
Pinobanksin isobutanoate 34.98 0.64 1.04 1.30 1.23 0.73 0.40 0.59 0.24 1.02 0.5
Naringenin 36.26 0.58 1.41 1.18 2.64 2.47 0.84 1.20 0.35 0.41 1.05
Kaempferol 39.19 0.85 0.88 1.63
Apigenine 39.90 0.2 0.28 0.22 1.61 0.28 0.23
Quercetin 39.94 0.21 0.25 0.15 0.30 0.18
Anthraquinones
Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) 34.48 1.88 5.08 6.87 12.55 9.93 4.83 0.31 2.36 0.17 0.10 0.79
1,6-Dihydroxy-8-methoxy-3-methylanthraquinone) 34.69 1.08
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) 35.55 1.57 3.33 6.65 10.30 4.37 6.04 1.49 1.18 0.29
1,7-Dihydroxy-3-methoxy-6-methylanthraquinone 35.67 0.64 1.44 0.51 0.54 0.65 0.32
Ketones
5-Hydroxy-1,7-diphenyl-3-heptanone 29.58 1.78
(3e,5e,7e)-6-Methyl-8-(2,6,6-trimethyl-1-cyclohexenyl)-3,5,7- 30.74 3.79 2.52 0.30 0.40 1.62 2.40 2.73 0.90
octatrien-2-one
Sugars
D-Fructose 18.85 5.78 2.98 0.74 2.17 6.43 6.77 16.99 11.84 6.26 15.65 6.31 11.5
Sorbose 19.10 0.31 0.38 1.22 0.69 0.31 0.79 0.98 0.87
Inositol 19.39 0.34 1.15
D-Xylose 20.57 0.46 4.71
D-Glucose 20.61 1.30 1.20 1.85 2.60 3.37 1.74 0.90 2.14 2.21
D-Glucitol 21.55 0.75 0.47 0.28 4.42
D-Mannose 22.41 1.90 2.02 3.26 3.96 4.82 2.86 1.08 8.27 2.85 4.05
Sucrose 33.69 0.76 0.52 1.51 3.46 1.36 1.41 0.55 19.65 1.24
Terpenes
Thymol 7.80 0.11 0.10 0.36
Menthol 24.67 0.46 0.25 0.29 0.42
Thunbergol 27.04 0.34
Pimaric acid 28.03 2.45 1.42 0.45 0.47 0.60 2.23 3.10 1.33 0.11
Totarol 28.19 4.33 4.49 0.31 0.47 0.99 1.65 2.20 1.52
Phytol 28.23 3.90 2.05 0.61 0.60 2.71 2.57 3.61 2.91 0.31
Dehydroabietic acid 29.03 0.49 0.21 2.85 0.01 0.07 0.04 0.20 0.17 0.27 1.57 1.74 3.05
Abietic acid 29.50 0.12 0.03 32.59 0.06 0.03 0.42 0.52 0.17 0.79 1.00 1.73
Isopimaric acid 32.23 27.99 19.02 5.88 1.00 1.78 2.93 14.96 22.87 28.96 1.50 0.88 1.81
(continued on next page)
N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461 457

Table 3 (continued)
Compoundb Rtc Composition
TRI ARF KAL MEG KAR- KAR- CRE TIN SKO LES LAR-I LAR-
I II II
Coeluting terpenes (trans-caryophyllene, geranyl acetone, caryophyllene 36.90 24.90
based diterpene)
Aristolone 42.56 0.20 1.79 2.03 1.16 0.44 0.28 0.54 0.74
a-Amyrin 43.00 3.58 2.04
Hop-22(29)-en-3-beta-ol 43.11 5.63 2.98 0.21
Urs-12-en-24-oic acid, 3-oxo-, methyl ester 44.30 1.42 0.76 1.35 0.61
Others
D-Glucosamine 17.06 0.26 0.53 0.39
2-Methyl quinoline (quinaldine) 29.11 1.35 0.98 0.94 0.34 0.95 1.32 1.52 0.64
p-Phenylazodiphenylamine 31.01 0.33 0.79 4.77 5.32 2.48 1.78 0.43
7,8-Dimethylbenzo[b]naphtho[2,3-d]thiophene 31.69 0.81 1.04 0.63 0.67 1.21
5,10-Dihydro-1,2,3,4-tetraphenylbenzocyclooctene 32.10 0.32 0.44 2.05 3.03
Indole-2-acetic acid 32.82 0.48 0.74 1.05 0.31 0.44 0.77
Phenanthrene 33.31 1.21 2.68 0.58 2.81 4.04 3.37 0.73 0.74
Scopolin 33.82 0.32 1.03
Thianthrene 34.26 0.31 0.34 0.75 0.24
2-(30 -Hydroxyphenylamino)-5-methyl-4-oxo-3,4-dihydropyrimidine 35.33 0.55 0.35 0.21 0.94
1,2,3-Triphenyl azulene 37.10 0.50 0.57
p-Tert-butyl-phenol 37.65 0.68 0.72 2.81

: not detected.
a
The ion current generated depends on the characteristics of the compound concerned and it is not a true quantitation.
b
Compound names do not include the trimethylsilyl (TMS) substituents.
c
Retention times in min on a HP5 MS column.

Table 4
Main classes of the PEE constituents (% of TICa).

Code Collection site Alcohols Aliphatic acids Phenolic acids Phenolic acid esters Anthraquinones Flavonoids Sugars Terpenes
Greece
TRI Trikorfo 0.82 1.68 1.50 2.33 3.45 10.75 10.05 39.94
ARF Arfara 1.84 1.01 3.62 4.89 8.41 18.64 6.72 30.07
KAL Kalavryta 1.48 1.05 6.65 2.73 14.16 19.38 0.74 41.87
MEG Megalopolis 1.40 0.43 8.92 3.70 24.29 31.17 2.17 1.43
KAR-I Karditsa 1.80 1.31 8.57 8.56 14.30 28.89 13.89 3.27
KAR-II Karditsa 0.82 0.79 4.07 5.90 11.38 37.18 16.79 4.64
CRE Rethymno 7.32 3.44 0.80 0.00 0.54 4.80 28.51 34.17
TIN Tinos 3.45 2.18 1.44 0.76 0.31 7.26 19.01 38.45
SKO Skopelos 1.71 2.67 1.08 2.13 4.50 12.90 9.44 37.40
LES Lesvos 1.48 3.05 2.07 3.29 2.75 10.68 49.35 4.35
Cyprus
LAR-I Larnaca 2.05 1.43 1.58 7.78 0.10 6.03 17.94 34.95
LAR-II Larnaca 1.74 2.88 2.76 8.95 1.08 8.91 20.23 8.25
a
TIC = total ion current. The ion current generated depends on the characteristics of the compound concerned and cannot be considered as a true quantitation.

isopimaric (Joye & Lawrence, 1967). With the exception of samples jor fraction 24.9% of TIC of one Cyprus propolis, namely LAR-I
from Central Peloponnese (MEG) and Central Greece (KAR-I, KAR- (Table 3). High levels of trans-caryophyllene and caryophyllene
II), these terpenic acids were among the major terpenes deter- oxide at levels up to 2.49% and 7.21% of TIC, respectively, were re-
mined in PEE from Greece and Cyprus (Tables 3 and 5). Signicant ported in terpenes-rich propolis from Turkish Mediterranean coast
amounts of resin terpenic acids have also been reported in propolis (Sahinler & Kaftanoglu, 2005).
from Turkish Anatolia (Kartal, Kaya, & Kurucu, 2002). Phytol, at levels from 0.3% to 3.9%, was also observed in the
An interesting nding was the presence of the diterpene totarol, majority of samples (Table 3). Phytol is an acyclic diterpene alco-
which was identied by means of the m/z ions of its TMS deriva- hol, present in all plants as chlorophyll esters. It is a precursor
tive (Cox, Yamamoto, Otto, & Simoneit, 2007). Totarol, which was for vitamins E and K1, and has been proved to possess antimicro-
detected in seven Greek and one Cypriot propolis, comprising bial properties (Inoue et al., 2005).
0.314.3% of TIC (Table 3), is present in southern hemisphere coni- Aristolone was detected in propolis from Greek islands (CRE,
fers, characterizing together with other diterpenes the tropical SKO, TIN, LES), Cyprus (LAR-I, LAR-II) and South Peloponnese
propolis (Cox et al., 2007). Totarol was also detected in Greek prop- (TRI, ARF) (Table 3). Propolis from Tinos (TIN) and Crete (CRE) is-
olis by Melliou and Chinou (2004) and Melliou et al. (2007) the lands contained additionally a-amyrin, one hopenol and one urse-
rst record of totarol in European propolis. Totarol is a known noic acid isomers (Table 3). In Tinos (TIN), thunbergol was detected
antimicrobial agent against Gram positive bacteria (Cowan, in propolis, which was reported for the rst time in Turkish prop-
1999), and totarol isolated from Greek propolis showed a specic olis from Kazan (Kartal et al., 2002).
activity against S. aureus and S. epidermidis, comparable to that of As far as terpenes are concerned, propolis balsams from
standard antibiotics (Melliou & Chinou, 2004). Greece and Cyprus exhibit similarities with propolis from East-
The sesquiterpene trans-caryophyllene together with a caryo- ern Mediterranean, and differences from the typical European
phyllene based diterpene and geranyl acetone constituted the ma- ones.
458 N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461

Table 5
Simple polyphenolsa and terpenic acids quantitated by GCMS in propolis from Greece and Cyprus (mg/g dry ethanolic extract). Each value represents the average of two
determinations. Standard deviations were less than 10%.

Compounds TRI ARF KAL MEG KAR-I KAR-II CRE TIN SKO LES LAR-I LAR-II
Polyphenols
Vanillin 0.03 0.04 0.01 0.04 0.21 0.97 trb ndc 0.05 0.11 nd nd
Cinnamic acid 0.31 0.22 1.68 4.00 0.79 1.67 0.05 0.04 0.15 0.05 0.04 0.10
p-OH benzoic acid 0.08 0.05 0.06 0.06 0.07 0.08 0.10 0.11 0.11 0.19 0.05 0.07
p-OH phenylacetic acid 0.04 nd 0.02 0.02 0.02 nd 0.06 0.06 0.03 0.02 0.03 0.03
Phloretic acid 0.08 0.09 0.03 0.05 0.08 0.08 0.19 0.28 0.07 0.14 0.04 0.17
Vanillic acid 0.05 0.05 0.06 0.07 0.05 0.08 0.05 0.05 0.06 0.05 0.04 0.04
o-Coumaric acid 0.06 0.03 0.04 0.02 nd nd 0.05 0.07 0.05 nd tr 0.05
Protocatechuic acid 0.10 0.07 0.05 0.06 0.06 0.08 0.13 0.07 0.33 0.11 0.10 0.17
Syringic acid 0.03 nd 0.04 0.05 0.03 0.03 0.04 0.04 0.04 0.05 0.03 0.04
p-Coumaric acid 0.44 0.75 0.25 0.31 0.75 2.18 0.07 0.08 0.67 0.40 0.12 0.19
Gallic acid 0.06 0.06 0.04 nd 0.05 0.04 0.04 0.04 0.07 0.10 0.04 0.11
Ferulic acid 0.46 0.71 0.79 1.81 1.07 0.84 0.09 0.11 0.26 0.16 0.10 0.13
Caffeic acid 1.74 3.87 0.22 0.21 3.55 3.29 0.14 0.15 1.14 6.70 0.16 0.31
Chrysin 45.44 87.86 118.5 145.7 96.75 40.38 0.26 1.27 34.77 19.64 0.24 5.94
Epicatechin 0.06 0.10 0.04 0.06 0.04 0.04 0.05 0.05 0.06 0.07 0.05 0.04
Naringenin 0.31 0.59 0.64 0.94 0.44 0.38 0.22 0.13 0.58 0.42 0.07 0.28
Catechin 0.08 0.10 0.07 0.08 0.07 0.07 0.07 0.07 0.08 0.07 0.06 0.07
Genistein 0.05 0.06 nd 0.06 0.05 nd nd nd nd 0.06 0.04 0.07
Kaempferol 0.92 1.74 3.75 3.42 1.73 1.80 0.08 0.21 0.80 2.35 0.08 1.28
Chlorogenic acid nd 0.08 0.05 0.05 0.02 nd 0.12 0.14 0.11 0.13 0.09 0.10
Quercetin 0.20 0.36 nd 0.41 nd nd 0.05 0.06 0.13 0.35 0.05 0.09
Apigenine 9.20 15.85 11.89 13.03 12.49 6.63 nd nd 0.25 2.96 nd nd
Pinocembrin 48.03 33.34 36.90 51.60 43.71 104.8 16.47 38.07 32.45 16.47 3.70 20.26
Pinobanksin 3.68 6.21 33.74 16.52 16.59 16.81 0.32 0.51 11.31 24.24 0.55 1.59
Pinobanksin-3-O-acetate 19.98 30.76 11.09 26.81 37.09 52.18 3.39 0.38 21.82 55.72 4.29 5.67
Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) 16.85 28.12 37.46 53.96 46.89 36.37 nd nd 16.19 2.87 0.10 0.74
1,6-Dihydroxy-8-methoxy-3-methylanthraquinone 3.16 4.99 5.98 8.20 5.83 2.22 nd nd 3.22 0.33 nd nd
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) 10.49 13.77 32.93 36.61 21.04 29.95 0.30 4.70 7.87 20.83 1.80 4.08
1,7-Dihydroxy-3-methoxy-6-methylanthraquinone 1.91 1.40 4.79 9.30 4.03 3.27 nd 0.78 1.72 0.16 tr 1.12
Terpenic acids
Dehydroabietic acid 20.59 5.68 27.03 0.06 1.53 1.13 3.55 5.60 9.56 82.34 17.75 46.97
Abietic acid 4.74 1.05 345.1 1.42 0.96 0.28 7.64 16.70 5.75 49.42 16.57 23.44
Isopimaric acid 259.6 108.8 1.94 4.45 8.66 17.05 61.76 166.3 210.1 18.46 5.50 17.44
Oleanolic acid 1.26 0.91 0.36 0.44 0.53 2.78 0.35 0.37 1.96 11.54 0.46 1.67
Ursolic acid 0.35 0.41 0.44 0.86 0.45 0.49 0.26 0.32 0.54 0.29 0.28 tr

Sum of polyphenols 163.8 231.3 301.2 373.5 293.5 304.3 22.34 47.47 134.4 154.8 11.87 40.63
Sum of terpenic acids 286.5 116.9 374.9 7.23 12.13 21.73 73.56 189.3 227.9 162.1 40.56 89.52
a
Among the single polyphenols determined, traces of tyrosol, homovannilic acid, 3,4-dihydroxy-phenylacetic acid, sinapic acid, resveratrol and myricetin observed in some
samples were not included in the Table.
b
tr = trace (<0.01 mg/g).
c
nd = not detected.

3.1.3. Anthraquinones 3.1.5. Phenolic acids and their esters

A characteristic of the propolis samples studied was the pres- Phenolic acids and esters comprised 3.2117.13% of TIC, the
ence of anthraquinones at levels ranging from 0.54% to 26.34% of higher values being observed in propolis from Cyprus (LAR-I,
TIC, and 0.3108.1 mg/g of PEE (Tables 3 and 5). Among the anthra- LAR-II), central Peloponnese (MEG) and Central Greece (KAR-I,
quinones detected, the known bioactive compounds chrysophanol KAR-II) and the lower in propolis from Crete (CRE) island (Table
and emodin (Tang, Wan, Zhu, Chen, & Huang, 2008) predominated. 4). The phenolic acids observed on a w/w basis ranged from 0.75
Anthraquinones have also been detected in propolis from Egypt to 9.34 mg/g of PEE (Table 5). As these compounds possess signif-
(Abd El Hady & Hegazi, 2002) and Turkey, where chrysophanol at icant antibacterial, antiinammatory, hepatoprotective and antiox-
levels as high as 4.54% and 15.1520.82% of TIC were reported idant activity (Bankova, 2005), their presence in the PEE studied is
(Silici & Kutluca, 2005; Silici, nl, & Vardar-nl, 2007). considered benecial. Especially the presence of caffeic acid phen-
ylethyl ester (CAPE), which has been reported to show antitumour
3.1.4. Flavonoids activity (Bankova, 2005), at levels 0.703.17% of TIC (Table 3).
Propolis from Greece and Cyprus contained avonoids at levels of
4.837.18% of TIC, and 8.8182.6 mg/g of PEE (Tables 4 and 5), with 3.2. Total polyphenols
the higher values observed in propolis from central Peloponnese
(MEG) and central Greece (KAR-I, KAR-II) and the lower in PEE from The total polyphenol content of the propolis studied ranged be-
the islands of Crete (CRE) and Tinos (TIN) (Table 4). Flavonoids are tween 80.2 and 338.5 mg GAE/g PEE (Table 6). These values are
synthesised by plants as a response to environmental stress and within the range of 31.2299 mg GAE/g PEE, reported for propolis
microbial infections, and are known to have antioxidant, antiinam- from several regions of the world (Kumazawa, Hamasaka, & Nakay-
matory and antimicrobial properties (Bankova, 2005; Cowan, 1999; ama, 2004). The higher polyphenol content was observed in PEE
Cushnie & Lamb, 2005; Pietta, 2000). Among the compounds charac- from Central and North Peloponnese (MEG, KAL) and Central
terising the European propolis, the avonoids pinocembrin, pino- Greece (KAR-I, KAR-II). Total polyphenol content correlated very
banksin, pinobanksin-3-O-acetate, chrysin, and galangin were well (p < 0.01) with DPPH antioxidant activity (R = 0.905) and
present in the majority of the propolis studied (Tables 3 and 5). reducing power (R = 0.832). In relation to main classes of PEE con-
N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461 459

Table 6
Total polyphenol content, free radical scavenging capacity on DPPH and reducing power in propolis extracts from Greece and Cyprus. Values are mean SD obtained from
analyses in triplicate.

Code Collection site Total poyphenolsa (mg CAEb/g PEEc) DPPH scavenging capacityd (mmol Trolox/g PEE) Reducing powere (mmol AAEf/g PEE)
TRI Trikorfo 146.2 7.3 0.60 0.04 3.13 0.16
ARF Arfara 184.6 7.4 0.55 0.03 3.09 0.22
KAL Kalavryta 250.6 17.5 0.76 0.05 3.13 0.25
MEG Megalopolis 338.5 13.2 1.11 0.07 3.35 0.27
KAR-I Karditsa 283.5 21.3 1.05 0.04 3.34 0.20
KAR-II Karditsa 322.0 13.1 0.99 0.03 3.24 0.13
CRE Rethymno 80.2 3.2 0.33 0.03 2.14 0.11
TIN Tinos 107.7 5.4 0.65 0.03 2.75 0.08
SKO Skopelos 146.2 10.2 0.62 0.02 2.89 0.14
LES Lesvos 136.3 8.2 0.45 0.02 2.65 0.19
LAR-I Larnaca Cyprus 85.7 5.1 0.46 0.03 2.41 0.10
LAR-II Larnaca Cyprus 100.4 7.2 0.58 0.03 2.63 0.08
a
Total polyphenol content was determined by the Folin-Ciocalteu assay.
b
CAE = caffeic acid equivalent.
c
PEE = propolis ethanolic extract.
d
Free radical scavenging capacity was measured with DPPH (1,2-diphenyl-2-picrylhydrazyl) radical.
e
Reducing power was determined by the ferric reducing antioxidant power (FRAP) assay.
f
AAE = ascorbic acid equivalent.

stituents (Table 4), total polyphenol content correlated very well and the anthraquinones (R = 0.7500.886), while correlating well
(p < 0.01) with phenolic acids (R = 0.848), anthraquinones (R = (p < 0.05) with cinnamic acid, vanillic acid, kaempferol, pinocem-
0.923) and avonoids (R = 0.957), while in relation to individual brin, and ursolic acid.
polyphenols and terpenic acids (Table 5), total polyphenols corre-
lated very well (p < 0.01) with cinnamic acid (R = 0.836), vanillic 3.5. Antimicrobial activity
acid (R = 0.791), ferulic acid (R = 0.918), chrysin (R = 0.818), pino-
cembrin (R = 0.750), kaempferol (R = 0.741), apigenine (R = 0.735), The antimicrobial activity of Greek and Cypriot propolis etha-
all the anthraquinones (R = 0.8070.956), and ursolic acid (R = nolic extracts were tested against eighteen bacterial strains, both
0.760). pathogenic and non-pathogenic, as well as against two pathogenic
fungi and the results are presented in Table 7. The inhibitory spec-
3.3. Antioxidant activity (DPPH) assay tra of PEE were compared with the inhibitory spectrum of nisin, a
known food grade antimicrobial peptide (bacteriocin) produced by
The propolis extracts studied exhibited signicant activity to- Lactococcus lactis subsp. lactis and used as natural preservative in
wards scavenging DPPH radicals, ranging from 0.33 to 1.11 mmol processed cheese, milk, canned foods, pasteurised liquid egg, our
Trolox equivalents/g PEE (Table 6). There was a trend for higher products and elsewhere.
DPPH values in propolis from central Peloponnese (MEG) and cen- The sensitivity of Gram positive bacteria to PEE varied among
tral Greece (KAR-I, KAR-II). the strains tested and the PEE used. S. aureus, S. epidermidis, B. cer-
DPPH assay values correlated very well (p < 0.01) with total eus and L. monocytogenes strains were sensitive against all the PEE
polyphenols as mentioned in Section 3.2, and additionally corre- tested. Among the pathogenic strains the lowest Minimum Inhibi-
lated very well (p < 0.01) with reducing power assay values tory Concentration (MIC) of PEE was observed for the two L. mon-
(R = 0.838). In relation to the main classes of PEE constituents (Ta- ocytogenes strains and both B. cereus strains while the highest MIC
ble 4), DPPH values correlated very well with phenolic acids was observed for the (non-pathogenic) S. epidermidis (Table 7).
(R = 0.838), anthraquinones (R = 0.859) and avonoids (R = 0.905). Although all the PEE samples inhibited all the Gram positive path-
Among the polyphenols determined quantitatively (Table 5) DPPH ogenic bacteria tested, their inhibitory activity against six strains of
values correlated very well (p < 0.01) with cinnamic acid the lactic acid bacteria group was rather occasional (Table 7). The
(R = 0.775), ferulic acid (R = 0.857), pinocembrin (R = 0.714) and PEE samples did not inhibit L. delbrueckii subsp. delbrueckii and L.
the anthraquinones (R = 0.7240.876), while also correlating well plantarum, but they did inhibit L. fermentum and L. helveticus while
(p < 0.05) with vanillic acid, chrysin and the terpene ursolic acid. L. bulgaricus and L. casei were inhibited only by the KAL and LAR-I
(Table 7) samples which exhibited the strongest antibacterial
3.4. Reducing power (FRAP) assay activity. Bozcuk-Erdem and lmez (2004) reported no inhibition
of L. casei RSKK 591 with four Turkish propolis extracts, while three
The PEE reducing power values, expressed as mmol ascorbic Turkish and one Brazilian propolis extracts inhibited L. acidophilus
acid equivalents (AAE), ranged between 2.14 and 3.35 mmol AAE/ ATCC 4356 (Koru et al., 2007). However, it should be pointed out
g PEE (Table 6), with relatively higher values observed in mainland that the MIC of all the PEE tested was lower for pathogenic bacteria
Greece samples (Peloponnese and Central Greece) and lower in like S. aureus, L. monocytogenes and B. cereus than for lactic acid
Greek islands and Cyprus. The reducing power values correlated bacteria (Table 7). This observation suggests that low concentra-
very well (p < 0.01) with total polyphenols and DPPH assay values tions of propolis extracts could be possibly used in fermented
as mentioned in Sections 3.2 and 3.3, respectively. In relation to the products, aiming to selectively inhibit the growth of pathogenic
main classes of PEE constituents (Table 4), the reducing power val- bacteria allowing the survival of starter culture strains like lactic
ues correlated very well (p < 0.01) with phenolic acids (R = 0.711), acid bacteria.
anthraquinones (R = 0.799) and avonoids (R = 0.823), while in Regarding the sensitivity of the Gram negative bacteria tested
relation to individual polyphenols and terpenic acids (Table 5) only the KAL sample from North Peloponnese and LAR-I sample
the reducing power values correlated very well (p < 0.01) with from Cyprus inhibited three out of four Gram negative bacteria
ferulic acid (R = 0.811), chrysin (R = 0.802), apigenin (R = 0.804), that were tested. KAL PEE was very rich in diterpenic acids, like
460 N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461

Table 7
Minimum inhibitory concentrations (MIC)a of PEE and nisin towards selected strains of Gram (+), Gram () bacteria and yeasts.

Target strains Propolis extracts (5% w/v) NISIN

TRI ARF KAL MEG KAR-I KAR-II CRE TIN SKO LES LAR-I LAR-II
MIC (mg/mL) MIC (IU/mL)
Shigella dysenteriae 2.50 2.50
Salmonella typhimurium 2.50 2.50
E. coli O157:H7 5.00 5.00
Y. enterocolitica
E. aerogenes 2.50 2.50
S. aureus I 0.30 0.30 0.15 0.60 0.30 0.30 0.60 0.60 0.30 0.30 0.15 0.60
S. aureus II 0.30 0.30 0.15 0.60 0.30 0.30 0.60 0.60 0.30 0.30 0.15 0.60 2000
S. epidermidis 1.25 1.25 0.30 1.25 0.60 1.25 2.50 2.50 1.25 1.25 0.30 1.25
B. cereus I 0.08 0.04 0.02 0.08 0.08 0.08 0.08 0.08 0.15 0.08 0.02 0.02
B. cereus II 0.08 0.04 0.04 0.08 0.08 0.08 0.08 0.08 0.15 0.08 0.02 0.02
L. monocytogenes I 0.08 0.08 0.08 0.15 0.08 0.08 0.08 0.15 0.15 0.08 0.04 0.30
L. monocytogenes II 0.04 0.08 0.08 0.15 0.08 0.08 0.08 0.15 0.15 0.04 0.04 0.30 2000
L. delbrueckii subsp. delbrueckii 1000
L. bulgaricus 0.60 0.60 250
L. fermentum 2.50 2.50 0.60 2.50 2.50 2.50 2.50 2.50 2.50 2.50 0.60 2.50 250
L. casei 0.30 0.60 125
L. plantarum 500
L. helveticus 0.60 0.60 0.15 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.15 0.60 125
Candida albicans 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
Candida tropocalis 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
a
Values are the average of three measurements.

dehydroabietic, abietic and isopimaric (Tables 3 and 5) known to best agents to protect their hives against bacterial and fungal
contribute to the antibacterial activity of propolis (Bankova et al., infections.
1996; Popova, Silici, Kaftanoglu, & Bankova, 2005), accompanied Concerning the antimicrobial activity of nisin, it did not inhibit
with signicant amounts of avonoids, anthraquinones, phenolic any of the Gram negative bacteria and the fungi tested. Moreover,
acids and esters (Table 4). LAR-I PEE was the only sample that con- some strains of the Gram positive pathogenic bacteria, like L. mon-
tained high amounts of caryophyllene and its derivative, as well as ocytogenes, B. cereus and S. aureus, were not inhibited by nisin but
geranyl acetone and increased concentrations of phenolic acids and they were by all PEE samples applied. On the contrary, all the Lac-
their esters (Tables 3 and 4). Caryophyllene has known antiinam- tobacillus strains were sensitive to nisin (Table 7), a known disad-
matory and antifungal activities (Sabulal et al., 2006). Therefore it vantage for applying nisin in fermented foods as, along with
seems that the presence of signicant amounts of terpenoids spoilage and pathogenic bacteria, desirable starter cultures (e.g.,
combined with other bioactive compounds is responsible for lactic acid bacteria) are also inhibited.
the broad spectrum of microorganisms inhibited by the KAL and Regarding the in vitro antimicrobial spectra of nisin and propolis
LAR-I propolis samples. Melliou et al. (2007) reported that the vol- and based on the range of bacteria tested, it seems that the propolis
atiles of Greek propolis inhibited four different species of Gram inhibitory spectrum is broader and its activity stronger even at
negative bacteria (E. coli, E. cloacae, K. pneumoniae, P. aeruginosa). very low concentrations compared to that of nisin. This should
Ethanolic extract of Bulgarian propolis inhibited 90.9% of the Gram not be surprising since PEE contained a very heterogeneous collec-
negative bacteria tested (Boyanova, Kolarov, Gergova, & Mitova, tion of substances with different and possibly synergistic anti-
2006) while Bankova et al. (1996) found no inhibitory activity of microbial mode of actions while nisin has only one.
Brazilian and Bulgarian propolis extracts against a strain of the
Gram negative bacterium E. coli. Also, Brazilian and Korean propo-
lis extracts inhibited the Gram negative bacterium S. typhimurium 4. Conclusions
ATCC 13311, but failed to inhibit the Gram negative Pseudomonas
aeruginosa ATCC 15523 (Choi et al., 2006). Greek and Cypriot propolis ethanolic extracts were shown to be
In general, with the exception of the terpene-rich KAL and LAR-I very rich in bioactive compounds, possessing antioxidant, antibac-
samples which presented the strongest microbicidal activity, no terial and antifungal activities. Their composition presented differ-
correlation could be established between PEE composition and ences from typical European propolis and similarities with East
their antimicrobial spectrum since similar antimicrobial activities Mediterranean propolis. Despite differences in the chemical com-
were observed among samples with entirely different chemical position of propolis from different geographical locations, the PEE
composition. Although more than 300 constituents have been iden- studied exhibited similar antibacterial and antifungal activities:
tied in propolis samples, biological activity is mainly due to a few they inhibited Gram positive pathogens and fungi, but did not af-
classes of substances such as avonoids, terpenes, phenolic acids fect several lactic acid bacteria, inhibiting in all cases a wider spec-
and their esters, which have been reported to possess antimicrobial trum of microorganisms than the food grade antibiotic nisin. There
activities, and in combination considered to act synergistically is evidence that the biological action of propolis extracts is to some
(Bankova, 2005; Marcucci, 1995). This could offer an explanation extend inuenced by their terpene content.
for the selective and strong antimicrobial activity of propolis Given the non-toxic and natural origin of propolis and the re-
from different regions of Greece and Cyprus, as their ethanolic sults obtained on their antioxidant and antimicrobial action, it is
extracts were very rich in terpenes and aromatic compounds concluded that, besides their potential pharmaceutical use, low
(avonoids, anthraquinones, phenolic acids and esters) the com- concentrations of the propolis balsams studied could be efcient
bined levels of which comprised 23.184.8% of propolis extracts protective agents for use as antioxidant and microbicidal addi-
(Table 4). It also conrms the known ability of bees to collect the tives in food systems, especially in fermented products, aiming
to selectively inhibit the growth of pathogenic bacteria while
N. Kalogeropoulos et al. / Food Chemistry 116 (2009) 452461 461

allowing the survival of starter culture strains like lactic acid Kartal, M., Kaya, S., & Kurucu, S. (2002). GCMS analysis of propolis samples
from two different regions of Turkey. Zeitschrift fr Naturforschung, Teil C, 57,
bacteria.
905909.
Kim, D.-M., Lee, G.-D., Aum, S.-H., & Kim, H.-J. (2008). Preparation of propolis
nanofood and application to human cancer. Biological and Pharmaceutical
References Bulletin, 31, 17041710.
Koru, O., Toksoy, F., Acikel, C. H., Tunca, Y. M., Baysallar, M., Guclu, A. U., et al.
Abd El Hady, F. K., & Hegazi, A. G. Z. (2002). Egyptian propolis: 2. Chemical (2007). In vitro antimicrobial activity of propolis samples from different
composition, antiviral and antimicrobial activities of East Nile Delta propolis. geographical origins against certain oral pathogens. Anaerobe, 13, 140145.
Zeitschrift fr Naturforschung, Teil C, 57, 386394. Kujumgiev, A., Tsvetkova, I., Serkedjieva, Y., Bankova, V., Christov, R., & Popova, S.
Ahn, M. R., Kumazawa, S., Usui, Y., Nakamura, J., Matsuka, M., Zhu, F., et al. (2007). (1999). Antibacterial, antifungal and antiviral activity of propolis from different
Antioxidant activity and constituents of propolis collected in various areas of geographic origins. Journal of Ethnopharmacology, 64, 235240.
China. Food Chemistry, 101, 14001409. Kumazawa, S., Hamasaka, T., & Nakayama, T. (2004). Antioxidant activity of propolis
Arnous, A., Makris, D. P., & Kefalas, P. (2002). Correlation of pigment and avanol of various geographic origins. Food Chemistry, 84, 329339.
content with antioxidant properties in selected aged regional wines from Marcucci, M. C. (1995). Propolis: Chemical composition, biological properties and
Greece. Journal of Food Composition and Analysis, 15, 655665. therapeutic activity. Apidologie, 26, 8399.
Bankova, V. (2005). Recent trends and important developments in propolis Melliou, E., & Chinou, I. (2004). Chemical analysis and antimicrobial activity of
research. e Comprehensive Alternative Medicine, 2, 2932. Greek propolis. Planta Medica, 70, 515519.
Bankova, V., Marcucci, M. C., Simova, S., Nikolova, N., Kujumgiev, A., & Popov, S. Melliou, E., Stratis, E., & Chinou, I. (2007). Volatile constituents of propolis from
(1996). Antibacterial diterpenic acids of Brazilian propolis. Zeitschrift fr various regions of Greece Antimicrobial activity. Food Chemistry, 103, 375380.
Naturforschung, Teil C, 51, 277280. Neacsu, M., Eklund, P. C., Sjholm, R. E., Pietarinen, S. P., Ahotupa, M. O., Holmbom,
Bankova, V. S., De Castro, S. L., & Marcucci, M. C. (2000). Propolis: Recent advances in B. R., et al. (2007). Antioxidant avonoids from knotwood of Jack pine and
chemistry and plant origin. Apidologie, 31, 315. European aspen. Holz Als Roh- und Werkstoff, 65, 16.
Bankova, V., Popova, M., Bogdanov, S., & Sabatini, A. (2002). Chemical composition Pietta, P.-G. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63,
of European propolis: Expected and unexpected results. Zeitschrift fr 10351042.
Naturforschung, Teil C, 57, 530533. Popova, M., Silici, S., Kaftanoglu, O., & Bankova, V. (2005). Antibacterial activity of
Banskota, A. H., Tezuka, Y., & Kadota, S. (2001). Recent progress in pharmacological Turkish propolis and its qualitative and quantitative chemical composition.
research of propolis. Phytotherapy Research, 15, 561571. Phytomedicine, 12, 221228.
Boyanova, L., Kolarov, R., Gergova, G., & Mitova, I. (2006). In vitro activity of Sabulal, B., Dan, M., John, A., Kurup, R., Pradeep, N. S., Valsamma, R. K., et al. (2006).
Bulgarian propolis against 94 clinical isolates of anaerobic bacteria. Anaerobe, Caryophyllene-rich rhizome oil of Zingiber nimmonii from South India:
12, 173177. Chemical characterization and antimicrobial activity. Phytochemistry, 67,
Bozcuk-Erdem, G., & lmez, S. (2004). Inhibitory effect of Bursa Propolis on dental 24692473.
caries formation in rats inoculated with Streptococcus sobrinus. Turkish Journal of Sahinler, N., & Kaftanoglu, J. (2005). Natural product propolis: Chemical
Zoology, 28, 2936. composition. Natural Product Research, 19, 183188.
Burdock, G. A. (1998). Review of the biological properties and toxicity of bee Silici, S., & Kutluca, S. (2005). Chemical composition and antibacterial activity of
propolis (propolis). Food Chemistry and Toxicology, 36, 347363. propolis collected by three different races of honeybees in the same region.
Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Journal of Ethnopharmacology, 99, 6973.
Reviews, 12, 564582. Silici, S., nl, M., & Vardar-nl, G. (2007). Antibacterial activity and
Cox, R. E., Yamamoto, S., Otto, A., & Simoneit, B. R. T. (2007). Oxygenated di- and phytochemical evidence for the plant origin of Turkish propolis from different
tricyclic diterpenoids of southern hemisphere conifers. Biochemical Systematics regions. World Journal of Microbiology and Biotechnology, 23, 17971803.
and Ecology, 35, 342362. Tang, W., Wan, M., Zhu, Z., Chen, G., & Huang, X. (2008). Simultaneous
Cushnie, T. P. T., & Lamb, A. (2005). Antimicrobial activity of avonoids. determination of eight major bioactive compounds in Dachengqi Tang (DT)
Antimicrobial Agents, 26, 343356. by high-performance liquid chromatography. Chinese Medicine, 3, 5. Available
Ghisalberti, E. L. (1978). Propolis: A review. Bee World, 60, 5984. on-line from <http://www.cmjournal.org/content/3/1/5>.
Han, S. K., & Park, H. K. (1995). A study on the preservation of meat products by Tosi, E. A., R, E., Ortega, M. E., & Cazzoli, A. F. (2007). Food preservative based on
natural propolis: Effect of EEP on protein change of meat products. Korean propolis: Bacteriostatic activity of propolis polyphenols and avonoids upon
Journal of Animal Science, 37, 551557. Escherichia coli. Food Chemistry, 104, 10251029.
Joye, N. M., & Lawrence, R. V. (1967). Resin acid composition of pine oleoresins. Velikova, M., Bankova, V., Sorkun, K., Houcine, S., Tsvetkova, I., & Kujumgiev, A.
Journal of Chemical and Engineering Data, 12, 279282. (2000). Propolis from the Mediterranean region: Chemical composition and
Inoue, Y., Hada, T., Shiraishi, A., Hirose, K., Hamashima, H., & Kobayashi, S. (2005). antimicrobial activity. Zeitschrift fur Naturforschung, Teil C, 55, 790793.
Biphasic effects of geranylgeraniol, teprenone, and phytol on the growth Zuo, Y., Wang, C., Lin, Y., Guo, J., & Deng, Y. (2008). Simultaneous determination of
of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 49, anthraquinones in radix Polygoni multiori by capillary gas chromatography
17701774. coupled with ame ionisation and mass spectrometric detection. Journal of
Chromatography A, 1200, 4348.