Characterization and biological evaluation of selected Mediterranean propolis samples. Is it a new type?

Accepted Manuscript Characterization and biological evaluation of selected Mediterranean propolis samples. Is it a new type? Dr Konstantia Graikou, Milena Popova, Olga Gortzi, Vassya Bankova, Prof. Dr Ioanna Chinou PII:

S0023-6438(15)30119-5

DOI:

10.1016/j.lwt.2015.08.025

Reference:

YFSTL 4896

To appear in:

LWT - Food Science and Technology

Received Date: 28 April 2015 Revised Date:

24 July 2015

Accepted Date: 7 August 2015

Please cite this article as: Graikou, K., Popova, M., Gortzi, O., Bankova, V., Chinou, I., Characterization and biological evaluation of selected Mediterranean propolis samples. Is it a new type?, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.08.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization and biological evaluation of selected Mediterranean propolis

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samples. Is it a new type?

3 Konstantia Graikoua*, Milena Popovaa,b, Olga Gortzic, Vassya Bankovab, Ioanna

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Chinoua*

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a

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Pharmacy,

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[email protected])

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Division of Pharmacognosy & Chemistry of Natural Products, Department of of

Athens,

Greece

([email protected];

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University

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b

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of Sciences, Sofia, Bulgaria ([email protected]; [email protected])

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c

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Larissa, Temponera street, Karditsa, Greece ([email protected])

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Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy

Department of Food Technologies, Technological Educational Institution (T.E.I.) of

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14 *Prof. Dr Ioanna B. Chinou

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University of Athens, Department of Pharmacy,

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Division of Pharmacognosy & Chemistry of Natural Products

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Panepistimiopolis-Zografou, 157 71 Athens, Greece

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e-mail: [email protected]

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Tel: +30-210-7274595

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Fax: +30-210-7274115

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* Dr Konstantia Graikou

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University of Athens, Department of Pharmacy,

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Division of Pharmacognosy & Chemistry of Natural Products

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Panepistimiopolis-Zografou, 157 71 Athens, Greece

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e-mail: [email protected]

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Tel: +30-210-7274283

29 Dr Milena Popova was in the University of Athens as a postdoctoral scholar from

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Bulgarian Academy of Sciences.

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ACCEPTED MANUSCRIPT Abstract

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Chemical composition, antioxidant and antimicrobial activity of thirty two (32)

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selected Mediterranean propolis extracts from mainland Greece, Greek islands,

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Cyprus, Croatia, and Algeria were determined. Chemo-geographical patterns within

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Mediterranean propolis were further analyzed by chemometrics.

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Knowledge of propolis composition, which depends on the geographical and climatic

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origin and its biological properties from different geographic regions samples, is

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extremely valuable with respect to the problem of propolis standardization.

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This work concluded that the Greek propolis samples share characteristics that

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differentiate them from typical European propolis, like the presence of diterpenes in

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significant amounts and the relatively low quantity of phenolic acid esters and besides

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their potential pharmaceutical and nutraceutical value, they are also attractive

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candidates for use as natural antioxidant and microbicidal additives in food systems.

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Keywords: Mediterranean propolis; Chemical composition; Antioxidant activity;

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Antimicrobial activity.

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Chemical compounds studied in this article

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Isocupressic acid (PubChem CID: 6438138); Pimaric acid (PubChem CID: 10116);

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Agathadiol (PubChem CID: 5316778); Totarol (PubChem CID: 326995); Communic

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acid (PubChem CID: 12303809); Pinocembrin (PubChem CID: 68071); Pinobanksin

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(PubChem CID: 73202); Pinobanksin-3-O-acetate (PubChem CID: 148556); Chrysin

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(PubChem CID: 5281607); Galangin (PubChem CID: 5281616).

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Abbreviations: BSTFA, bis-(trimethylsilyl)-trifluoroacetamide; MIC, minimum

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inhibitory concentration; P.F., induction period with antioxidant/induction period

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without antioxidant; TMS, trimethylsilyl ether; CNS, central nervous system.

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Introduction

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Propolis is a natural resinous substance, collected by honeybees (Apis mellifera L.)

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from buds and exudates of plants, mixed with pollen as well as enzymes secreted by

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bees. It has been considered to be used in the beehive to smooth out the internal walls

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of the hive and as a protective barrier against their enemies (Bankova, 2005; Toreti,

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Sato, Pastore & Park, 2013). Since the 1960’s, numerous studies have revealed the

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variability of propolis’ composition depending on the plant source (Bankova, De

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Castro & Marcucci, 2000; Salatino, Fernandes-Silva, Righi & Salatino, 2011).

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Because of the very complex chemical composition, GC-MS became the most often

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used method in the 1980’s for rapid chemical characterization of propolis samples of

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different geographic and plant origin. More than 300 compounds (polyphenols,

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terpenoids, steroids, sugars, amino acids and others) have been detected in raw

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propolis. Their abundance is influenced by botanical and geographical factors, as well

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as by the collection season (Ahn et al., 2007; Bankova et al., 2000; Kujumgiev et al.,

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1999). The different types of propolis as they are referred in bibliography (Sforcin &

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Bankova, 2011) are: i) Poplar type (Populus spp. which is originate mainly from

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Europe, non-tropic regions of Asia, New Zealand and North America), ii) Birch type

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(Betula verrucosa which is derived from Russia), iii) Green type (Baccharis spp.

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characteristic of Brazil) iv) Red type (Dalbergia spp. which is located in Brazil,

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Mexico and Cuba), v) Clusia type (from Clusia spp. from Cuba and Venezuela) and

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vi) Pacific type (Macaranga tanarius which is originate from Indonesia, Taiwan and

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Okinawa Prefecture) and vii) the most recent, Mediterranean type (plants mainly from

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Cupressaceae family which is located in Greece, Sicily and Malta).

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Propolis is considered responsible for the low incidence of bacteria and moulds within

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the bee-hive. It is well known, that it possesses antibacterial, antifungal and antiviral

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hepatoprotective, cytotoxic, immunostimulating, etc (Bankova et al., 2000). These activities

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recently have been scientifically proved (Banskota, Tezuka & Kadota, 2001; Sforcin &

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Bankova, 2011) and they are recorded due to its chemical complexicity. Moreover,

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propolis is extensively used in food and beverages to improve health and prevent

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diseases, as a constituent of biocosmetics and in numerous other purposes (Bankova et

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al., 2000; Banskota et al., 2001).

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Due to geomorphological characteristics, the Greek flora presents high biodiversity

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with many endemic plants, which is expected to differentiate the composition of

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Greek propolis from that of typical European ones (Melliou & Chinou, 2004; Popova,

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Chinou, Marekov & Bankova, 2009; Popova, Graikou, Chinou & Bankova, 2010).

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In the present study, we are reporting the chemical composition, the antioxidant

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activity and the antimicrobial properties of thirty two propolis samples collected from

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the Mediterranean (mainland Greece, Greek islands, Cyprus, Croatia and Algeria)

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compared with a typical European sample from Bulgaria. Moreover, we compare their

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chemical profile in order to define the type of Mediterranean propolis, as well as to

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find out the most active samples in accordance to the assayed activities and their

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potential uses as natural antioxidants and antimicrobial additives in food systems.

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2. Materials and methods

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2.1.Propolis samples

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Propolis samples were obtained from several locations of central, southern and

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northern Greece, Aegean Sea islands, and Cyprus as well as from two different

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regions of Croatia and from Algeria, as indicated in Table 1. Samples were collected

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during spring–summer of 2008-2011. Voucher specimens are deposited in the

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Division of Pharmacognosy & Chemistry of Natural Products, Department of

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Pharmacy, University of Athens, Greece. Crude propolis samples were frozen

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(−20 °C) and grounded in a chilled grinder.

114 2.2. Extraction and sample derivatization

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Small amounts (10 g) of pulverised crude propolis were extracted with a 10-fold

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volume of 70% ethanol solution extensively for 24 hr at room temperature. The

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solutions were evaporated to near dryness on a rotary evaporator under reduced

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pressure at 40 °C and then freeze-dried.

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About 5 mg of the residue were mixed with 50 µL of dry pyridine and 75 µL of

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bis(trimethylsilyl)trifluoracetamide (BSTFA) and heated at 80 ºC for 20 min. The

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isolated compounds used as reference compounds were subjected to the same

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procedure for derivatization as about 1 mg of the pure compound was mixed with 10

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µL of dry pyridine and 15 µL of BSTFA. The derivatized ethanolic extracts and pure

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compounds as TMS derivatives were analyzed by GC-MS.

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2.3.GC-MS analysis

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The GC-MS analysis (Popova et al., 2010) was performed with a Hewlett Packard

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Gas Chromatograph 5890 Series II Plus linked to Hewlett Packard 5972 mass

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spectrometer system equipped with a 30 m long, 0.25 mm id and 0.5 µm film

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thickness HP5-MS capillary column. The temperature was programmed from 100 to

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300 ºC at a rate of 5 ºC/min. Helium was used as a carrier gas, flow rate 0.7 mL/min.

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Split ratio 1:20, injector temperature 280 ºC, ionization voltage 70 eV. The

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identification of the compounds was performed using NIST98 and Wiley mass

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spectral databases and comparison of mass spectra and retention time of reference

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compounds as well as by using literature.

137 2.4.Rancimat method

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2.4.1. Preparation of oil

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Sunflower oil (Sol, Elais S.A., Athens, Greece) was purified from trace metals and

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other prooxidants via adsorption chromatography to yield purified sunflower oil

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triacylglycerol fractions according to the method described by Fuster, Lampi, Hopia,

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& Kamal-Eldin (1998). A glass column (40 x 2.5 cm i.d.) (wrapped with aluminum

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foil to prevent light-induced oxidations during the purification process), plugged with

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glass wool, was packed with 250 g of alumina (activated at 100°C for 8 h and then at

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200°C for 12 h) suspended in n-hexane, capped with sea sand, and conditioned by

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prewashing with 200 ml of n-hexane. The oil (100 ml) was dissolved in an equal

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volume of hexane and passed through the column, which was then washed with 200

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ml of n-hexane. The hexane (total volume 300 ml) was evaporated using a rotary

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evaporator and the triacylglycerols were collected in an aluminum foil-wrapped flask.

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2.4.2. Rancimat method

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The method used, was a modification of the method reported by Lalas & Tsaknis

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(2002). About three grams of purified sunflower oil and added propolis extract (in a

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concentration of about 70ppm) were accurately weighed into the reaction vessel of the

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Rancimat 743 (Metrhom LTD, Herisau, CH 9101, Switzerland) along with another

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vessel with a sample of purified sunflower oil (control). One ml of absolute ethanol

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was added in order to dissolve the extract and mixed well. The reaction vessels were

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placed in the apparatus. The conditions were set at 60oC and 7 L/h. The protection

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factor (P.F.) was calculated as P.F. = (induction period with antioxidant)/(induction

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period without antioxidant). A protection factor greater than that of 1 indicates

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inhibition of the lipid oxidation. The higher the value, the better the antioxidant

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activity (Lalas & Dourtoglou, 2003).

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All assays were carried out in triplicates. The data were analyzed using ANOVA test,

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and results were expressed as the mean (RSD% by PASW Statistics 18).

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168 2.5. Antimicrobial bioassay

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In vitro antibacterial studies, firstly, were carried out by the disc diffusion method by

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measuring the zone of inhibitions against the two Gram-positive bacteria:

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Staphylococcus aureus (ATCC 25923), S. epidermidis (ATCC 12228), the four Gram-

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negative bacteria: Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC

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13047), Klebsiella pneumoniae (ATCC 13883) and Pseudomonas aeruginosa (ATCC

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227853), the pathogen fungi Candida albicans (ATCC 10231), C. tropicalis (ATCC

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13801) and C. glabrata (ATCC 28838) as well as against the oral pathogens

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Streptococcus mutans and S. viridans, both sensitive strains, clinically isolated. The

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tested extracts were dissolved in ethanol. For each experiment control disc with pure

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solvent was used as blind control. All the paper discs had a diameter of 6 mm and

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were deposited on the surface of the seeded trypticase soya-agar Petri dishes. The

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plates were inoculated with the tested organisms to give a final cell concentration of

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107cell/mL and were incubated for 48h at 370C. The fungi were grown on Sabouraud’s

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agar at 250C for 48 h.

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ACCEPTED MANUSCRIPT The MIC values for the most active extract were determined using the dilution method

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in 96-well plates. Stock solutions of the tested extract were prepared at 10 mg/ml. Serial

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dilutions of the stock solutions in broth medium (100 µl of Müller-Hinton broth or on

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Sabouraud broth for the fungi) were prepared in a microtiter plate (96 wells). Then 1 µl of

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the microbial suspension (the inoculums, in sterile distilled water) was added to each

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well. For each strain, the growth conditions and the sterility of the medium were checked

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and the plates were incubated as referred above. MICs were determined as the lowest

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concentrations

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concentrations 4-88 µg/ml) was used in order to control the sensitivity of the tested

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bacteria, while 5-flucytocine and intraconazole (at concentrations 0.5, 25 µg/ml) as

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controls against the tested fungi (Sanofi, Diagnostics Pasteur at concentrations of 30, 15

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and 10 µg/ml) and finally the alkaloid sanguinarine (at concentration 80 µg/ml) in order

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to control the oral pathogens. For each experiment, any pure solvent used was also

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applied as blind control. The experiments in all cases were repeated three times and

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the results were expressed as average values.

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3. Results and discussion

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3.1. Chemical characterization

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The collection sites for thirty three propolis samples from the Mediterranean (25 from

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Greece, 5 from Cyprus, 1 from Algeria and 2 from Croatia and 1 from Bulgaria) are

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shown in Table 1.

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According to the results of the GC–MS analysis, the propolis extracts from the

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Mediterranean contain about 150 compounds. The main chemical classes of

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compounds identified are listed in Table 2.

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applied PCA to analyze the large amount of analytical data in Table 2. The obtained

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two-dimensional plot (Fig.1) covers 94% of the total variation and clearly separates

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the samples into three groups. The largest group consists of 15 samples rich in

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diterpenes, originating from Southern Greece (Peloponnese, Crete, Athens and South

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Aegean Islands). Some of these samples contained no flavonoids at all, while in others

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there were low amounts of flavonoids. A second group contains 10 samples, coming

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mainly from Central and Northern Greece, together with the Algerian and the two

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Croatian samples. The samples of the second group are characterized by relatively

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high amounts of flavonoids and low diterpene content. The samples of the smallest

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third group (5 samples) are characterized by the predominance of sugars and sugar

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derivatives and show no geographic homogeneity.

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This grouping, clearly connected to their geographic origin, and location of their

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collection, reflects the availability of different plant sources used by bees for propolis

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production. In our case, pollen analysis of studied propolis samples (personal data)

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showed that approx 90% of the rich in diterepens propolis samples were coming from

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Coniferae trees and especially from Pinus sp.

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Populus sp., which has been considered as the major source of the so-called European

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propolis was not identified, not even in traces, revealing the special character of the

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Mediterranean propolis samples (Melliou & Chinou, 2004).

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The major compounds in the diterpene-rich samples were isocupressic acid, pimaric,

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imbricatoloic acid, agathadiol, totarol, 13-epi-torulosal, communic acid, 13-epi-

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cupressic acid, abietic acid and ferruginolon. Similar diterpene content has been also

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observed as the main chemical category in samples from Mediterranean areas like

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Malta, Sicily, Turkey and Algeria (Popova et al., 2011; Bankova, Popova, Bogdanov

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different regions of Greece (Melliou & Chinou, 2004; Popova et al., 2009; Popova et

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al., 2010) in previous work from our team. This specific terpenic profile has recently

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leaded to the identification of its main plant source of Mediterranean propolis: the

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resin cypress trees Cupressus sempervirens (Popova et al., 2012).

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The flavonoids in the studied samples were the same as in the well-known poplar type

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European propolis (Bankova et al., 2002): pinocembrin, pinobanksin, pinobanksin-3-

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O-acetate, chrysin, and galangin were the most abundant compounds. Their plant

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source is the bud exudate of trees of the genus Popupus, mainly P. nigra. The samples

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of the “flavonoids-rich” group originate from regions where P. nigra grows. The low

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concentration of flavonoids in samples from the “diterpenic” group could be

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explained by the role of poplar buds as minor propolis source in the respective areas

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of collection, where poplars are not widespread.

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It is interesting to note that we were did not identify anthraquinone constituents in any

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of the studied propolis samples, even in traces amount, which is in accordance to the

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chemical profile of the main plant source of Mediterranean propolis, contrary to

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Kalogeropulos et al. (2009), where the presence of high amounts of antraquinones in

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several Greek propolis samples was reported.

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3.2. Antioxidant activity

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Many authors (von Pongracz, Hoffmann-la Roche & Basel, 1984; Lalas, Gortzi,

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Tsaknis & Sflomos, 2007) have earlier proved that the antioxidant activity of some

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biological compounds (like α-tocoperol) is highly affected by the temperature of

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determination. These compounds appear to work well at or near the temperature of the

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human body. The temperature of 60oC (during the determination of the induction

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that the higher the temperature was, the lower the antioxidant activity appeared

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(results not shown). Especially, at a temperature over 75οC all samples appeared to

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promote sunflower oils oxidation.

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The protection by various propolis ethanol extracts on purified sunflower oil was

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studied (Table 3). Seventeen of the tested samples showed antioxidant activity

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(P.F.>1). One of the extracts (Cyp2) showed no action on the oxidation of sunflower

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oil (P.F.=1), as fifteen extracts proved to posses prooxidant activity (P.F.<1).

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The antioxidant activity (Guo et al., 2011) of propolis extracts can possibly be

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attributed to the polyphenol content as indicated by the significantly positive

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correlation. Moreover, the correlation coefficient between reducing power and

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antioxidant activity was also statistically significant suggesting that the main

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constituents of EP contribute to the antioxidant activity are total polyphenol. Previous

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studies showed that there was a strong positive correlation between antioxidant

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activities and total phenol, and the content of flavonoids largely influenced the

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antioxidant activity of propolis extracts.

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Therefore, many of the studied Greek propolis extracts proved to be rich sources of

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natural antioxidants and can possibly be exploited as protective agents against various

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free radical related degenerative diseases. It is well-known that antioxidants prevent

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free radical induced tissue damage by preventing the formation of radicals,

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scavenging them, or by promoting their decomposition. The beneficial role of free

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radicals in normal physiological states like ageing and common health problems (such

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as infertility, cancer or systemic diseases of the cardiovascular system e.g.

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atherosclerosis, and disease of other systems as CNS, immune, endocrine anomalies)

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are well evaluated (Pramod, Singh & Singh, 2013).

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The ethanolic extracts of the studied propolis samples were also evaluated for their

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antimicrobial activity against six Gram-negative and -positive bacterial strains, three

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human-pathogenic fungi and two oral pathogens. The results of these tests showed

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interesting and promising antimicrobial activity (Table 4) confirming the traditional

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reputation of propolis as an antimicrobial agent (Banskota et al., 2001).

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It is noteworthy that some of our samples with the highest percentage of diterpenes

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like Cret2, Andr and Alex (80.4%, 73.3%, and 81.9% respectively) exhibited the

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highest antimicrobial activity against all tested microorganisms, while they showed a

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specific strong activity against Gram-positive bacteria (S. aureus, S. epidermidis, S.

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mutans).

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Chemometric approaches (cluster analysis, PCA) to the antibacterial data failed to

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produce clearly distinct sample groups, so no unambiguous correlations could be

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found between chemical composition and antimicrobial activity, as well as between

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geographic origins. Expectedly, samples with high concentration of sugars and sugar

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derivatives were of lowest activity.

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The finding demonstrates that the chemical differences in propolis samples don’t

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necessarily result in drastic differences in their antimicrobial activity.

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4. Conclusion

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Mediterranean propolis could be confirmed as a new type of propolis consisting

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mainly from diterpenes and produced based to Conifer trees among Cupressaceae and

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Pinaceae which are widespread in the Mediterranean area. Recently, such potential

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type of so-called Mediterranean propolis has been referred and used in many different

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of the Mediterranean African countries (Zhang et al., 2014) as well as Mediterranean

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European countries (Melliou & Chinou 2004; Kalogeropoulos, Konteles, Troullidou,

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Mourtzinos, & Karathanos, 2009; Popova et al., 2009; Popova et al., 2010; Popova et

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al., 2011; Popova et al., 2012)

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As far as it concerns the isolation of diterpenes, it should be noted that it is generally

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considered as a characteristic of tropical propolis and especially Brazilian one

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(Bankova et al., 1996). Although later, as previously referred, diterpenes have been

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isolated and structurally determined for the first time from propolis of European

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origin, from Greece and Sicily (Italy) (Melliou & Chinou 2004, Bankova et al. 2002).

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Diterpenes have also been occasionally identified from propolis of Mediterranean

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origin (Turkey, Algeria). In all these cases, the plant origin of the studied propolis

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samples have not been determined and the presence of diterpenes has been just

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considered as unexpected (Bankova et al. 2002).

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In conclusion, the Mediterranean propolis group consists mainly of samples rich in

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diterpenes, which are comparable with Brazilian propolis samples, showing a

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particular diterpenic chemical profile (Bankova et al., 1996). These so-called

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Mediterranean propolis group has a completely different chemical profile compared

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with that of poplar European propolis (Bankova et al., 2002), as well as with that of

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propolis samples from New Zealand, China, Chile (Midorikawa et al., 2001) and from

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different countries of Africa (Papachroni et al., 2015).

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Although more than 300 constituents have been identified in propolis samples,

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biological activity is mainly due to a few classes of substances such as flavonoids,

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terpenes, phenolic acids and their esters, which have been reported to possess

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antimicrobial activities, and in combination considered to act synergistically

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antimicrobial activity of propolis from different regions of Greece and Cyprus, as

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their ethanolic extracts were very rich in terpenes and aromatic compounds

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(flavonoids, phenolic acids and esters). It also confirms the known ability of bees to

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collect the best agents to protect their hives against bacteria. In conclusion,

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Mediterranean propolis need further research, as all propolis types, in order to

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overcome the problem of standardization, which is an obstacle to wider use of

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propolis in the food as well as pharmaceutical industry.

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341 Acknowledgements

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The authors wish to thank Dr S. Karabournioti (Director of the Chemical and

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Analytical Laboratory of “Attiki” Bee-Culturing Company) for the pollen analyses of

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the propolis samples.

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346 References

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Ahn, M. R., Kumazawa, S., Usui, Y., Nakamura, J., Matsuka, M., Zhu, F., et al.

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(2007). Antioxidant activity and constituents of propolis collected in various

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areas of China. Food Chemistry, 101, 1383-1392.

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Bankova V., Marcucci, M. C., Simova S., Nikolova N., Kujumgiev A. & Popov S.

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(1996). Antibacterial Diterpenic Acids from Brazilian Propolis. Zeitschrift für

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Naturforschung, Teil C, 51, 277-280.

Bankova, V. S., De Castro, S. L., & Marcucci, M. C. (2000). Propolis: recent advances in chemistry and plant origin. Apidologie, 31, 3-15.

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Bankova, V., Popova, M., Bogdanov, S., & Sabatini, A. G. (2002). Chemical

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composition of European propolis: expected and unexpected results. Zeitschrift

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different geographic origins. Journal of Ethnopharmacology, 64, 235–240.

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Lalas, S., & Dourtoglou, V. (2003). Use of rosemary extract in preventing oxidation

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400

diterpene compounds in mediterranean propolis from Greece. Journal of

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Agricultural & Food Chemistry, 58, 3167-3176.

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(2011). The specific chemical profile of Mediterranean propolis from Malta.

404

Food Chemistry, 126 (3), 1431-1435. Popova, M., Trusheva, B., Cutajar, S., Antonova, D., Mifsud, D., Farrugia, C., &

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407

of Mediterranean type propolis. Natural Product Communications, 7, 569-570.

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human health and disease. International Journal of Current Research and

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Propolis research and the chemistry of plant products. Natural Products

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development of new drugs?. Journal of Ethnopharmacology, 133, 253-260.

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Toreti, V. C., Sato, H. H., Pastore, G. M. & Park, Y. K. (2013). Recent progress of

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propolis for its biological and chemical compositions and its botanical origin.

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Evidence-Based Complementary and Alternative Medicine, Article ID 697390.

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(2014). Chromatographic analysis with different detectors in the chemical

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characterisation and dereplication of African propolis. Talanta, 120, 181-190.

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ACCEPTED MANUSCRIPT Table 1. Propolis collection areas Country code Mikri Mantinia, Kalamata Messenia, Southern Peloponnese GR Verga, Kalamata Messenia, Southern Peloponnese GR Meligalas Messenia, Southern Peloponnese GR Mani Laconia, Southern Peloponnese GR Taygetos Central Peloponnese GR Arcadia Arcadia, Central Peloponnese GR Epidaurus Argolis, East Peloponnese GR Chania Chania, Western Crete GR Sfakia Chania, Western Crete GR Sisses Rethymno, Central Crete GR Rethymno Rethymno, Central Crete GR Zaros Heraklion, Central Crete GR Pallini Athens, Attica GR Andros Cyclades Islands, South Aegean GR Kos Dodecanese Islands, South Aegean GR Euboea Central Greece, South Aegean GR Kephalonia Ionian Islands, West Greece GR Karpenissi Evrytania, Central Greece GR Karditsa Thessaly, Central Greece GR Preveza Epirus, Northwest Greece GR Chalkidiki Central Macedonia, North Greece GR Alexandroupoli Thrace, North Greece GR Didymoticho West Thrace, North Greece GR Evros East Macedonia, North Greece GR Pafos South West Cyprus CY Nicosia Central Cyprus CY Limassol South Cyprus CY Limassol South Cyprus CY Famagusta North East Cyprus CY Lokrum Island Island of Dubrovnik, Croatia HR Trogir Split-Dalmatia County, Croatia HR Algeria North Africa DZ Dobrich, Yambol Eastern Bulgaria BG Geographical location

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Collection area

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Sample code Pel1 Pel2 Pel3 Pel4 Pel5 Pel6 Pel7 Cret1 Cret2 Cret3 Cret4 Cret5 Ath Andr Kos Eub Kefal Karp Kard Prev Chal Alex Did Evros Cyp1 Cyp2 Cyp3 Cyp4 Cyp5 Cro1 Cro2 Alg Bul

ACCEPTED MANUSCRIPT Table 2. Main chemical groups identified in propolis (percent TIC, TMS derivatives) Flavonoids

Sugars

63.7 44.9 55.1 46.0 71.7 69.4 53.8 50.4 80.4 41.8 22.9 57.5 71.8 73.3 71.3 7.4 14.6 4.7 10.2 16.4 5.7 81.9 6.3 1.8 0.7 3.5 18.5 8.9 32.6 4.8 8.6 12.4 -

10.5 21.1 1.0 8.7 2.7 4.7 1.4 3.5 0.9 2.4 35.2 39.6 3.4 33.5 46.7 29.5 1.6 32.2 42.6 38.5 17.1 0.7 15.5 0.7 35.5 35.2 48.6 40.7

10.0 13.3 11.9 12.1 2.17 20.5 12.9 5.2 49.0 42.0 25.0 17.7 12.9 5.6 12.8 4.0 32.2 6.6 2.0 15.3 0.4 6.1 11.3 9.2 45.7 49.8 20.9 2.1 2.6 5.0 2.0 10.8

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Diterpenes

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Aromatic Acid Esters 2.1 4.5 1.5 1.5 0.6 0.7 0.9 5.4 1.1 0.4 2.9 6.9 3.2 17.1 13.6 28.9 19.2 18.1 5.6 26.3 16.5 1.1 2.3 11.3 17.0 15.2 16.2 13.8 15.9

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Pel1 Pel2 Pel3 Pel4 Pel5 Pel6 Pel7 Cret1 Cret2 Cret3 Cret4 Cret5 Ath Andr Kos Eub Kefal Karp Kard Prev Chal Alex Did Evros Cyp1 Cyp2 Cyp3 Cyp4 Cyp5 Cro1 Cro2 Alg Bul

AC C

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Aliphatic Aromatic Acids Acids <0.1 2.4 1.4 3.5 1.3 0.7 2.8 0.2 0.9 2.2 2.3 0.1 7.3 0.1 1.9 0.1 2.5 6.5 4.5 0.3 1.1 0.2 3.1 0.2 6.2 3.20 4.8 <0.1 7.0 7.4 1.0 1.1 4.9 4.4 6.8 <0.1 <0.1 1.9 5.4 1.5 3.6 0.5 0.7 5.1 0.1 10.7 0.7 2.0 0.9 17.9 0.3 2.5 4.5 2.5 4.0 1.5 1.1 7.4 7.8

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ACCEPTED MANUSCRIPT Table 3. Protection Factor of various propolis extracts on purified sunflower oil. Protection Factor 1.23 (0.03) 0.57(0.02) 0.83 (0.11) 0.85 (0.02) 1.04 (0.06) 0.90 (0.09) 0.64 (0.03) 1.00 (0.02) 1.04 (0.01) 0.47(0.01) 1.04 (0.02) 1.13 (0.04) 0.88 (0.04) 0.96 (0.03) 1.46 (0.09) 1.21(0.02) 1.11(0.03)

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Sample code Kard Prev Chal Alex Did Evros Cyp1 Cyp2 Cyp3 Cyp4 Cyp5 Cro1 Cro2 Alg Bul BHT α-tocopherol

SC

Protection Factor 1.06 (0.09) 1.14 (0.04) 0.95 (0.02) 1.28 (0.09) 0.81 (0.01) 1.11 (0.01) 1.53 (0.07) 1.57 (0.11) 1.09 (0.07) 0.94 (0.01) 1.03 (0.01) 1.57 (0.08) 0.74 (0.02) 0.96 (0.02) 0.95 (0.01) 1.72 (0.10) 0.99 (0.01) 1.10 (0.05)

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Sample code Pel1 Pel2 Pel3 Pel4 Pel5 Pel6 Pel7 Cret1 Cret2 Cret3 Cret4 Cret5 Ath Andr Kos Eub Kefal Karp *

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Values are means of triplicate determinations and standard deviation (SD) is given in parenthesis.

ACCEPTED MANUSCRIPT Table 4. Antimicrobial activities (zones of inhibition/ and MIC mg/ml, n=3) of the

S. epidermidis

P. aeruginosa

E. cloacae

K. pneumoniae

E. coli

S. mutans

S. viridans

C. albicans

C. tropicalis

C. glabrata

Pel1 Pel2 Pel3 Pel4 Pel5 Pel6 Pel7 Cret1 Cret2 Cret3 Cret4 Cret5 Ath Andr Kos Eub Kefal Karp Kard Prev Chal Alex Did Evros Cyp1 Cyp2 Cyp3 Cyp4 Cyp5 Cro1 Cro2 Alg Bul Netilmicin Intraconazole 5-flucytocine sanguinarine

16/0.90 17/0.80 15/0.97 16/1.15 16/1.00 15/1.28 22/ 0.15 22/ 0.10 24/ 0.12 22/ 0.17 15/0.87 22/0.15 23/ 0.09 23/ 0.10 17/0.90 16/1.20 15/1.16 12/1.10 15/0.77 12/0.98 17/0.90 23/ 0.05 16/0.70 15/0.88 17/0.65 11/1.00 15/0.90 12/0.97 16/0.72 15/0.82 17/0.75 16/0.80 NT 21/ 0.004 NT NT NT

18/0.49 18/0.50 16/0.88 20/0.23 14/1.30 13/1.44 24/0.08 22/ 0.12 25/ 0.05 23/ 0.10 14/0.94 23/0.10 25/ 0.04 24/ 0.08 15/1.15 12/1.50 14/1.12 12/0.99 15/0.95 12/1.00 16/0.88 22/ 0.15 15/1.00 15/0.97 18/0.50 12/0.95 14/0.87 12/0.99 16/0.75 16/0.77 18/0.54 15/0.96 NT 25/ 0.004 NT NT NT

17/0.67 13/1.45 12/1.90 18/0.68 14/1.20 14/1.27 20/0.34 15/ 1.00 22/ 0.24 20/ 0.30 13/1.12 16/1.10 21/ 0.27 20/ 0.24 15/1.14 16/1.10 15/1.26 10/1.98 12/1.80 13/1.10 15/1.00 15/0.98 12/1.87 12/1.94 13/1.55 10/1.94 13/1.35 11/1.90 15/1.00 11/1.84 12/1.90 12/1.75 NT 20/ 0.088 NT NT NT

17/0.47 13/1.27 13/1.85 18/0.25 15/1.12 14/135 17/0.39 16/ 0.55 18/ 0.30 17/ 0.42 13/1.32 17/1.08 17/ 0.46 17/ 0.40 16/1.05 16/1.30 14/1.38 11/1.75 11/1.95 12/1.34 15/1.39 15/1.28 11/1.90 12/1.81 10/1.90 12/1.22 12/1.25 11/1.20 15/1.02 12/1.80 13/1.87 12/1.78 NT 23/ 0.008 NT NT NT

17/0.50 13/1.64 12/1.89 18/0.38 14/1.34 13/156 18/0.45 17/ 0.60 17/ 0.44 16/ 0.48 13/1.15 17/0.65 16/ 0.68 18/ 0.32 15/1.12 14/1.66 13/1.80 10/1.95 13/1.58 10/1.92 14/1.60 16/ 0.77 13/1.64 12/1.79 11/1.62 10/1.94 12/0.88 10/1.87 16/0.93 11/1.77 12/1.72 15/0.98 NT 22/ 0.008 NT NT NT

14/1.10 14/0.96 13/1.10 13/1.56 15/1.09 15/1.15 17/0.35 18/ 0.37 18/ 0.40 17/ 0.32 12/1.45 19/0.60 18/ 0.47 16/ 0.69 16/0.96 14/1.25 13/1.15 11/1.88 14/1.45 10/1.87 14/1.35 17/0.58 13/1.58 15/0.85 12/1.58 11/1.76 13/1.00 10/1.98 15/0.74 15/0.88 14/0.96 16/0.74 NT 24/ 0.010 NT NT NT

18/0.50 17/0.70 16/0.88 17/0.61 15/1.00 14/1.22 22/0.09 19/ 0.75 20/ 0.17 22/ 0.15 14/0.79 18/0.55 21/ 0.15 18/ 0.22 16/ 0.78 17/0.50 15/1.14 12/1.15 15/0.88 12/1.00 17/0.64 20/0.32 15/0.90 16/0.93 20/0.18 12/0.90 15/0.67 13/0.80 17/0.55 15/0.90 16/0.78 16/0.69 28/ 0.015 24/ NT NT NT 0.015

16/0.96 17/0.67 17/0.72 16/1.70 17/0.50 16/0.70 22/0.10 15/ 0.80 20/ 0.18 21/ 0.14 14/0.85 17/0.22 20/ 0.20 19/ 0.15 18/ 0.30 18/0.45 16/1.33 12/1.10 15/1.00 11/1.12 18/0.40 19/ 0.35 15/1.06 16/0.88 19/0.36 13/0.98 15/0.59 12/0.92 17/0.48 16/0.85 17/0.65 17/0.65 28/ 0.015 25/ NT NT NT 0.015

13/0.98 11/1.25 10/1.50 15/0.93 12/1.50 11/1.80 13/1.00 18/ 0.75 14/ 0.94 13/ 0.90 12/1.12 15/1.05 13/ 0.98 12/ 1.37 11/ 1.75 12/1.30 11/1.82 9/2.05 9/2.00 9/2.20 11/1.43 17/ 0.88 9/2.05 10/1.67 11/1.25 9/2.00 11/1.13 9/2,23 13/1.10 10/1.40 11/1.32 10/1.38 NT NT 20 0.01 NT

15/0.72 12/1.13 12/1.40 15/0.65 13/1.54 12/1.67 14/0.45 19/0.28 16/0.47 15/0.55 13/1.50 18/0.25 15/0.52 13/1.49 12/1.59 13/1.54 13/1.60 10/1.95 10/1.87 9/2.12 13/1.50 18/0.50 10/1.90 11/1.52 15/0.72 11/1.91 12/1.24 10/1.87 14/0.70 12/1.55 12/1.50 11/1.47 NT NT 22 0.001 NT

15/0.70 13/1.00 13/1.28 17/0.28 13/1.45 12/1.60 17/0.38 19/ 0.25 17/ 0.35 15/ 0.58 13/1.62 18/0.26 16/ 0.44 13/ 1.55 13/ 1.32 14/1.65 13/1.68 10/2.10 11/1.90 10/1.98 13/1.72 18/ 0.52 10/1.98 12/1.47 16/0.80 10/1.94 12/1.28 10/1.90 14/0.82 13/1.68 15/0.76 12/1.54 NT NT 23 0.0001 NT

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Tested compounds

S. aureus

studied propolis ethanol extracts

ACCEPTED MANUSCRIPT Figure 1. A PCA two-dimensional plot showing separation of propolis extracts into three clusters according to their chemical analysis. Projection of the cases on the factor-plane ( 1 x 2) Cases with sum of cosine square >= 0,00

30 3220 17 30 19 31 23

24 2516 21

29

1

2 4

0

-20 18

-30

26

11

10

27

-40 -50 -60 -80

3 7 12

28

-10

8

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Factor 2: 21,73%

10

22 65 9 15 14 13

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20

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40

-60

-40

-20

0

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Factor 1: 72,88%

20

40

60

80

Active

ACCEPTED MANUSCRIPT Highlights •

Chemical composition of thirty two selected Mediterranean propolis samples

•

Greek propolis samples share characteristics that differentiate them from European propolis Confirmation of a new type diterpene rich propolis proposed to be called

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•

Mediterranean type

Propolis samples through chemometrics were separated into three groups

•

Strong antimicrobial activity of propolis from different regions of Greece

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ACCEPTED MANUSCRIPT

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Supplementary file Characterization and biological evaluation of selected Mediterranean propolis samples. Is it a new type?

a

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Konstantia Graikoua*, Milena Popovaa,b, Olga Gortzic, Vassya Bankovab, Ioanna Chinoua* Division of Pharmacognosy & Chemistry of Natural Products, Department of Pharmacy, University of Athens, Greece

b

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([email protected]; [email protected])

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria ([email protected];

[email protected])

([email protected])

Table of Contents

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Department of Food Technologies, Technological Educational Institution (T.E.I.) of Larissa, Temponera street, Karditsa, Greece

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Table 1: Composition of propolis extracts from Mediterranean area Table2: Composition of propolis extracts from Mediterranean area (Samples from Peloponnese and Crete) Table 3: Composition of propolis extracts from Mediterranean area (samples from Cyprus)

ACCEPTED MANUSCRIPT

Table 1: Composition of propolis extracts from Mediterranean area

8.83 10.51 10.58 12.39 13.52 15.42 17.13 18.5522.06 17.93 18.51 19.11 22.07 23.11 23.44 23.89 24.00 24.04 24.38

hylitol vanillic acid ribonic acid p-coumaric acid manoyl oxide sugar dimethoxycinnamic acid hexadecanoic acid ethyl ester hexadecanoic acid labda-8(17),12,13-trien

Kefal

<0.1 0.1 0.2 2.3 0.1

0.1

1.0

0.4

11.4

11.8

0.1

0.2 0.1 0.2 0.6

0.1

0.4 0.3

12.0 1.3

0.2

0.5 0.1

0.3 0.2

2.3

Prev

0.4 0.3 <0.1

0.5

3.5

20.8

0.5

Chal

Alex

Did

<0.1

0.3

0.1 0.1

<0.1

Kard

0.1

0.7

0.3 0.3

Karp

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17.7

Eub

0.3 1.4 0.6

SC

sugars

Kos

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8.59

Аndros

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3.66 6.26 6.64 7.30 8.09

Ath

4.8

1.7

Evr

Cro1

<0.1

Cro2

Alg

<0.1

<0.1

<0.1

<0.1

0.8

0.2 0.1

0.3 0,8 0.6

0.4

0.4

Bul

<0.1 0.1 1.0 1.0 0.2 0.1 0.4 0.1

0.3 0.4

0.2

3.6 0.8 0.1

0.6

12.6

0.4

6.1

0.1

0.1

10.1

2.3

2.1

2.0

8.6 0.1

0.1

EP

3.47

Compound 2-hydroxypropanoic acid glycolic acid phenylethyl alcohol benzoic acid glycerol butanedioic acid dihydroxypropanoic acid butendioic acid hydrocinnamic acid hydroquinone mallic acid cinnamic acid p-hydroxybenzoic acid pentandioic acid

AC C

RT

1.2 8.3

0.2 1.5 2.3 0.6

1.0

0.1

<0.1

1.3

1.2 0.9

0.5 0.1 0.3 0.6

<0.1

0.4

0.5

0.6

<0.1 0.1 0.2

0.1

1.2

0.1 0.1

2.2 0.6

0.5 0.9

0.5

0.4

0.5 1.2

1.8

2.6

0.3

ACCEPTED MANUSCRIPT

27.10 27.21 27.38 27.54 27.64 27.70 27.86 27.95 28.31 28.53 28.75 28.85 28.98 29.24 29.30 29.32 29.44

<0.1

0.7

0.3 0.1

1.1 <0.1 1.8

1.9 2.3 0.3

<0.1 0.5 <0.1

RI PT

1.6

0.3

0.9

1.1

2.1

2.3 0.5

1.5 0.1

1.1 0.1

SC

27.07

0.5

0.1

0.1

0.5

4.5

0.2

0.3

0.3

0.6

1.8

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26.94

0.2

0.1

0.6 <0.1 0.4

0.1

1.7

0.5

2.3 0.3

0.1

0.3

0.8

1.3

0.1

0.4

0.2

0.1

0.1

0.8

<0.1

0.7

0.3

0.5 0.1

2.7 1.9

2.6

3.1

0.3

1.6

0.7 0.2 0.1

2.6

4.3

4.4

0.9

2.1

0.3

0.2

0.8

1.2

TE D

26.69

0.1 0.2 0.1

EP

26.27

ferullic acid 13-epi-manool caffeic acid pentenyl p-coumarate 2-methyl-2-butenylferulate 3-methyl-2-butenylferulate 3-methyl-3-butenyl caffeate octadecadienoic acid octadecanoic acid ethyl ester octadecenoic acid communal pentenyl caffeate octadecanoic acid sempervirol mixture * ferruginol 14,15-dinor-13-oxo8(17)-labden-19-oic acid copalol 3-methyl-3-butenylferulate 18-hydroxyabieta8,11,13-triene 2-methyl-2-butenylferulate communic acid totarol totarol and 13-epi-torulosal

AC C

24.80 25.78 25.95 26.26

5.7 6.5

0.4 0.3

<0.1

0.7 0.2

0.8

2.7

0.8

1.3

0.6 0.5 4.1

<0.1 6.3

1.6 0.2

0.9

0.1

1.2

0.9 0.5

0.3

0.1

1.6

0.6

8.8

0.8 0.1 <0.1

0.9

0.7

10.4 5.9

0.5

1.5 2.6

1.8 1.6

0.6 0.4

0.8 9.1 5.9

1.1 0.7

0.3 2.5

0.3

2.3

1.7

5.0

0.8

ACCEPTED MANUSCRIPT

30.45 30.69 30.71 30.95 31.34 31.48 31.49 31.69 32.02 32.04 32.23 32.34 32.42 32.54 32.65

0.2

1.8

3.2

1.8

2.7 1.7

10.4

2.7

<0.1

0.7 0.1

3.9 2.01

2.7

0.1

0.5

0.9

3.0

0.2

1.4

2.1

4.2 1.7

5.9 4.8 1.3

0.8

10.4

0.3

1.1

RI PT

0.1

1.3

3.7

1.6

1.2

1.7

SC

(30.0 – 30.15) 30.24 30.25

<0.1

0.4

M AN U

30.10

0.1

1.1 3.1

0.3

4.6

0.4

0.7

0.6 0.3

0.3

0.3

<0.1

0.1

2.2

2.6

1.2

1.9

2.2

1.9

1.0

0.1

1.5

0.1

0.1

3.2

13.2

1.2

8.0

2.4

0.1

0.7

0.9

1.8

1.1

<0.1

0.9

1.7

1.2

1.0

1.1

0.4

10.9

3.8

0.5

2.5

1.6

2.8

1.9

2.6

1.5

3.1

0.7

0.4

0.4

3.5

1.1

0.6

0.3

<0.1

0.6

0.3

5.1

0.1

0.8

TE D

29.96

3.4

0.2

EP

29.85

isopimaric acid isopentyl caffeate neoabietic acid 3-methyl-3butenylcaffeate 2-methyl-2butenylferulate 3-methyl-2butenylferulate pimaric acid and imbricataloic acid 13-epi-torulosоl dehydroabietic acid 2-methyl-2butenylcaffeate 3-methyl-2butenylcaffeate abietic acid 13-epi-cupressic acid dihydroxyabieta-8-1113-triene ferruginolon benzyl ferulate trihydroxymonomethoxy chalcone m=502 isoagatholal pinostrobin chalcone benzylcaffeate 2-hydroxyferruginol pinostrobin pinocembrin chalcone methyl ester of 3,4-

AC C

29.48 29.51 29.75

1.7

0.7

0.4 0.5

1.4 0.9

4.7

0.6

0.4

2.0

1.4

1.4 3.5

0.5 1.4

4.1 1.4 0.1

5.5 0.9

8.3

2.4

1.3

0.5

5.0

0.1 3.9

0.4 4.0 2.0

2.2

2.1

0.1 4.2

2.3

1.6

ACCEPTED MANUSCRIPT

35.30 35.45 35.88 36.01 36.32 36.63 36.84 36.94 37.03

0.4

4.3

3.2

0.2 0.3 0.5 1.8 5.5 0.5

1.4

1.9

0.5

10.0

0.5

0.6

0.4

5.4 10.2

27.3

0.7

4.8 0.9

5.1 0.6 0.6 0.9 0.2

0.5 1.0

0.5 3.1 0.4

0.7

1.4

2.9

2.8

0.7

4.8

10.4

0.3

1.2

0.3

0.2

3.2

0.3 0.4 1.0 1.6 3.1 0.1

32.1

7.9 1.5

5.1

4.3 2.4 0.3

0.4 0.5 0.8 1.1 3.0 0.4

7.7

<0.1 0.8

0.6 13.0 4.4 0.8

8.7

4.4

0.6

0.4

1.7 2.1 6.8

0.8

0.2

4.5 0.4

1.2

0.9 1.1 0.8

0.7

1.5

1.2 1.4 2.4

3.7

0.8

3.2 4.2 5.6 0.7 1.9

1.0 1.6 4.0

4.8

3.6

2.1

0.8

3.0

4.1

2.0

1.5

1.1

2.9

7.6

9.0

4.8

0.8

3.5

5.1

3.4

5.2

4.8

5.1

0.5

1.1 5.5 5.0

1.8

1.1

<0.1

10.0

0.5

0.5

0.4

1.3

2.7

6.7 5.7 0.4

M AN U

22.9

5.9

SC

0.8

1.5

RI PT

2.0

TE D

34.93 34.94

6.4

EP

34.65

16.8

AC C

(32.732.8) 32.86 32.94 33.04 33.19 33.39 33.58 33.70 33.77 33.79 33.9 34.01 34.11 34.29 34.38

dimethoxymandelic acid agathadiol and imbricatoloic acid pinocembrin totarolon pinobanksin chalcone sugar sakuranetin isocupressic acid pinobanksin phenethyl caffeate sugar tectochrysin 6/7-hydroxyferruginol junicedric acid acetylisocupressic acid sugar dihydroxymonomethoxy flavanone m=430 pinobanksin 3-O-acetate sugar 13(14)-dehydro junicedric acid benzyl caffeate chrysin galangin pinobanksin 3-butanoate phenylethylcaffeate dihydroxymonomethoxy flavone m=428 tetracosanoic acid kaempferol dimethylether

8.5 3.2

0.9

1.6

1.7

3.0 3.3 4.1 0.4 1.8

2.5

1.4

4.8 0.5 2.3 0.5 3.9

0.5

0.2 3.5

3.5 5.4 6.6 1.1 2.4

2.3 4.9 3.8 1.0 2.3

1.6 0.7 2.0 0.5 1.1

2.6 8.4 6.7 0.8 2.4

2.9 1.3 12.4 1.1 2.6

1.0

1.4

0.1

1.1

0.8

1.4

0.1

0.7

ACCEPTED MANUSCRIPT

39.34 39.85 40.54 40.83 42.543.7 43.84 44.30 44.1845.80 47.30 47.5647.93

1.8

0.9

0.7

0.9

1.2

0.1 0.4

1.3

0.6

0.4

<0.1 0.8

0.3 0.9

0.7

0.8

1.6

1.0

0.3

0.3 0.3

0.2

5.0

0.2

0.4 0.2 0.9

0.3

0.9

0.5

0.8

1.0

triterpene

7.2

triterpene keton secocycloartane triterpenic alcohol amyrine type triterpenic acid

0.1

triterpenic ketoacid

2.5

RI PT

0.3

SC

37.76 38.46 39.09

1.5

1.2

M AN U

37.62

coniferyl benzoate cinnamyl p-coumarate alpinon chalcone pinobanksin-3pentanoate naringenin tetrahydroxyflavanone cinnamyl ferulate 18-succinyloxyabietadiene cinnamyl caffeate kaempferol quercetin dimethylether

1.7

0.7

1.3

3.2 1.0

0.9

2.4

0.5

0.8

0.1 1.1

0.3

2.3

0.2

14.9

<0.1 <0.1

0.7

0.4

TE D

37.16 37.19 37.34

AC C

EP

* mixture of labda-(17),13e-dien-19-carboxy-15-yl oleate and labda-8(17),13e-dien-19-carboxy-15-yl palmitate

0.1

1.0

0.3 0.7 2.8

ACCEPTED MANUSCRIPT

Table 2: Composition of propolis extracts from Mediterranean area (Samples from Peloponnese and Crete)

25.07 25.78 25.95 26.94

Pel 1

Pel2

Pel3

<0.1

0.4

0.2

sugars

10.0

Pel5

Pel6

0.5 13.3

0.1

8.8

TE D

10.0

M AN U

0.1 <0.1

0.1

<0.1 <0.1 0.3

0.4 0.9

0.2 0.9 0.3

Cret2

Cret3

0.7 0.4

1.7

0.1

4.1

0.1

0.1 <0.1

1.3

0.2

0.9

1.2

0.2

<0.1

0.1 0.1 0.1

Cret4

0.2

0.1

Cret 5 0.1 0.1 0.1 3.0 0.2 0.1 0.1 0.3

<0.1 0.3 15.7

10.6

3.0

39.1

33.7

0.1

13.4 0.3 0.3

0.1 0.2

<0.1 0.2

0.3

1.8

0.4

0.2

1.0

0.3 0.2

2.2 0.5

0.2 0.2

<0.1 0.3 0.1

<0.1 0.5 <0.1

0.5

0.2 0.7

0.3

1.3

1.3 0.3

<0.1

1.0

1.5

<0.1

0.4 0.3

Cret1 0.1 0.1

0.3

0.7

EP

sugar

Pel7 <0.1

SC

0.7

hylitol ribonic acid gluconic acid gallic acid manoyl oxide

dimethoxycinnamic acid hexadecanoic acid labda-8(17),12,13-trien manool ferullic acid 13-epi-manool caffeic acid 3-methyl-3-butenyl caffeate

Pel4 <0.1 <0.1

RI PT

Compound 2-hydroxypropanoic acid glycolic acid benzoic acid glycerol butanedioic acid dihydroxypropanoic acid hydroxyhexanoic acid mallic acid butandioic acid cinnamic acid pentandioic acid

AC C

RT 3.47 3.66 6.64 7.30 8.09 8.59 11.30 12.39 13.38 13.52 17.13 18.55 22.06 17.93 19.11 21.20 22.66 23.11 23.44 23.80 23.89 24.04 24.38

<0.1

1.1 0.3

0.6 0.1

ACCEPTED MANUSCRIPT

29.24 29.32 – 29.44 29.48 29.51 29.70 29.75 29.85 29.90 (30.0 – 30.1) 30.24 30.25 30.45 30.69 30.71

0.51

0.39 <0.1

0.6

1.0

0.3 0.1 0.1

1.9

0.8

2.1

1.5

6.1

1.1

0.5

2.9

2.1

0.6

0.4

0.1

7.0

2.5

1.6

3.6

2.3

4.3

1.8

3.4

4.9

0.3

0.4

1.6

3.7

6.4

0.3 0.2

3.2

0.4

<0.1 1.5 0.5 0.1 0.7 0.4 0.5

0.1

SC

2.1

0.3 3.6 0.2 0.7 0.5 0.2 0.2

0.1 0.4

2.0

RI PT

0.9 0.6

M AN U

28.85

1.9

0.5

2.5

4.6

3.3

7.0

1.2

1.1 0.45

0.6

4.3

3.4

4.0

6.9

1.9 <0.1

1.8

6.1

3.3

1.4

2.0

0.9

1.1

5.7

4.9

4.4 2.3

3.5

3.1

5.0

6.1

3.6 <0.1 2.3

2.3

0.3

6.7

0.7

1.4

10.8 3.8

0.2 2.2 0.2 0.4 0.4 0.2 0.2 2.1

<0.1

1.4

0.7

<0.1 3.5 0.4

14.6

TE D

28.53

1.3

EP

28.31

octadecadienoic acid octadecenoic acid communal octadecanoic acid sempervirol mixture * ferruginol 14,15-dinor-13-oxo8(17)-labden-19-oic acid copalol 18-hydroxyabieta8,11,13-triene communic acid totarol totarol and 13-epi-torulosal isopimaric acid isopentyl caffeate pimaric acid neoabietic acid 3-methyl-3butenylcaffeate imbricataloic acid pimaric acid and imbricataloic acid 13-epi-torulosоl dehydroabietic acid 2-methyl-2butenylcaffeate 3-methyl-2butenylcaffeate abietic acid

AC C

27.07 27.21 27.38 27.64 27.70 27.86 27.95

0.1

0.4

0.2

0.1

20.4

9.5

8.8

4.7

3.1

4.3

9.9

10.8

0.5 0.5

5.8 3.1

4.7

0.6 0.2

1.0

1.0

10.6

10.6 0.2 0.3

0.2

0.4

0.3 0.8

0.1

5.8

2.7

1.4

0.7

0.5

0.6

ACCEPTED MANUSCRIPT

33.02 33.19 33.58 33.64 33.7933.96 34.01 34.11 34.29 34.38 34.65 34.93 34.94 35.30 35.45 35.88

6.0 4.8

1.6 0.6

2.1

1.3 4.2

3.4

3.1 0.2 1.1

6.7

4.3 4.7

1.8

0.1

SC

3.3 1.3

0.3 0.3

1.1 0.4

RI PT

3.6 0.3

2.5

2.2 <0.1

1.3 0.9

1.2

0.4 <0.1

1.5 0.5

6.2 0.1

1.1

1.6

3.3 0.2

2.3

4.1

2.7

2.6

3.3

4.1

4.5

2.7

<0.1 0.5

0.9

2.7 3.6

8.3

0.1 0.3

0.8

0.6 9.2

1.0 16.3

29.1

2.6 13.9

13.0

0.2 22.0

0.2

0.5

0.8

4.9

8.6

0.2

0.5 2.9 1.6 0.2

0.6 1.5 2.8 2.8

1.2

1.4

0.2 2.0 1.7

0.8

0.2 0.6 0.4 1.2

1.4

0.7 1.0

0.7

1,63

5.5 0.3

1.1

18.2

6.8

sugar 6/7-hydroxyferruginol junicedric acid acetylisocupressic acid sugar dihydroxymonomethoxy flavanone m=430 pinobanksin 3-o-acetate sugar benzyl ferrulate 13(14)dehydrojunicedric acid benzyl caffeate chrysin

1.1 0.5

M AN U

(32.732.8) 32.86 32.94

0.6 <0.1

14.4

16.1

TE D

32.02 32.04 32.34 32.54

0.7 <0.1

0.4

3.2 1.5

1.2

<0.1 2.0

0.5

0.7

0.3 0.7 0.2 0.6

1.0

0.6

EP

31.69

13-epi-cupressic acid ferruginolon trihydroxymonomethoxy chalcone m=502 isoagatholal pinostrobin chalcone 2-hydroxyferruginol pinocembrin chalcone agathadiol agathadiol and imbricatoloic acid pinocembrin totarolon podocarp-8,11,13-triene3-one sugar isocupressic acid tectochrysin

AC C

30.95 31.48

6.2

6.7

1.5 2.0

1.2

1.6

1.3

0.4 1.0

0.4

0.7

0.6

1.5

<0.1

0.3 2.6

0.1 1.1

0.7

0.6

0.1 0.6

ACCEPTED MANUSCRIPT

37.62 39.09 39.34 39.78 39.85 42.52 44.30 44.1845.80 47.30

0.6

1.9

<0.1 0.6

1.4 0.7

0.7 <0.1 0.3

0.7 1.8 0.16 0.4

1.3

<0.1

0.6

0.8 0.3

0.8

0.2

0.1

0.7

0.3

0.1

RI PT

1.7

SC

36.94 37.19 37.34

0.6

1.3

0.5

0.1

0.5

6.2

3.2

3.6

1.2

0.5

9.7

0.5

0.4

0.2

0.4

0.4

0.6 0.3

3.8

0.4

0.3

1.1

* mixture of labda-(17),13e-dien-19-carboxy-15-yl oleate and labda-8(17),13e-dien-19-carboxy-15-yl palmitate

EP

2.5

0.3

0.5

AC C

0.1

0.2

M AN U

36.84

galangin pinobanksin 3-butanoate phenylethylcaffeate dihydroxymonomethoxy flavon m=428 tetracosanoic acid cinnamyl p-coumarate alpinon chalcone pinobanksin-3pentanoate cinnamyl ferulate 18-succinyloxyabietadiene 18-succinyloxyabieta8,11,13-triene cinnamyl caffeate triterpene secocycloartane triterpenic alcohol amyrine type triterpenic acid

TE D

36.01 36.32 36.63

0.3

ACCEPTED MANUSCRIPT

Table 3: Composition of propolis extracts from Mediterranean area (samples from Cyprus)

30.00 – 30.15 30.25

Cyp2

Cyp3 <0.1

Cyp4

<0.1

<0.1 <0.1

<0.1

0.1 0.1

0.2 0.3

0.1 0.2

0.9

3.3

<0.1 0.4 3.6

1.3 5.5

TE D

0.7 0.1 0.2

49.1

0.2

0.5

0.7

3.8 <0.1 <0.1

0.2

0.6

0.2

1.9

<0.1 20.0

<0.1 2.1

0.4 <0.1 0.58 <0.1 1.5 7.8 <0.1

1.6 <0.1 0.2 1.4 14.1 15.3 0.8 0.8 1.8 3.3

M AN U

<0.1 29.6

9.2 0.1

SC

0.7

6.9

Cyp5

RI PT

Cyp1

EP

Compound 2-hydroxypropanoic acid glycerol butanedioic acid hydrocinnamic acid hydroquinone mallic acid cinnamic acid sugars vanillic acid ribonic acid p-coumaric acid manoyl oxide sugar dimethoxycinnamic acid hexadecanoic acid ferullic acid caffeic acid octadecadienoic acid octadecenoic acid pentenyl caffeate octadecanoic acid mixture * communic acid totarol isopimaric acid neoabietic acid pimaric acid and imbricataloic acid dehydroabietic acid

AC C

RT 3.47 7.30 8.09 10.51 10.58 12.39 13.52 18.55-22.06 18.51 19.11 22.07 23.11 23.44 23.89 24.04 24.80 25.95 27.07 27.21 27.54 27.64 27.86 29.24 29.30 29.48 29.75

<0.1

<0.1

0.4

2.0

3.4

<0.1

ACCEPTED MANUSCRIPT

3.7 <0.1 1.6

1.5

1.7 <0.1

RI PT

3.6

2.9

5.4 1.3

SC

0.1 2.0

5.3

3.0 0.6 5.5

1.3 0.7 7.6

0.9 4.8

M AN U

4.8

3.4 1.3

0.8 0.1

1.9 10.0

2.0

0.2

TE D

32.86 33.04 33.19 33.58 33.70 33.77 33.79 34.29 34.93 35.30 35.45 35.88 36.01 36.32 36.63 37.03 37.11 37.34 37.62 37.76 39.09 40.83 42.5-43.7

<0.1 0.2

14.6 1.3

1.0 1.9

0.7

1.0 10.0

1.5 9.9

EP

32.7-32.8

2-methyl-2-butenylcaffeate abietic acid 13-epi-cupressic acid dihydroxyabieta-8-11-13-triene isoagatholal pinostrobin chalcone pinocembrin chalcone agathadiol and imbricatoloic acid pinocembrin pinobanksin chalcone sugar isocupressic acid pinobanksin phenethyl caffeate sugar acetylisocupressic acid pinobanksin 3-O-acetate 13(14)-dehydro junicedric acid benzyl caffeate chrysin galangin pinobanksin 3-butanoate phenylethylcaffeate kaempferol dimethylether kaempferol methylether alpinon chalcone pinobanksin-3-pentanoate naringenin cinnamyl ferulate quercetin dimethylether triterpene

0.7

<0.1

1.0

0.4

11.8 6.5

AC C

30.45 30.71 30.95 31.34 32.02 32.04 32.54

0.4 10.0 1.5 1.8

2.0

0.5 <0.1 <0.1

1.9

<0.1

ACCEPTED MANUSCRIPT

44.18-45.80

triterpenic alcohol amyrine type

1.2

AC C

EP

TE D

M AN U

SC

RI PT

* mixture of labda-(17),13e-dien-19-carboxy-15-yl oleate and labda-8(17),13e-dien-19-carboxy-15-yl palmitate