Food Chemistry 145 (2014) 599–607

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

Phenolic profile and antioxidant activity of Serbian polyfloral honeys

Uroš Gašić a, Silvio Kečkeš b, Dragana Dabić c, Jelena Trifković a, Dušanka Milojković-Opsenica a
Maja Natić a, Živoslav Tešić a,⇑
a
Faculty of Chemistry, University of Belgrade, P.O. Box 51, 11158 Belgrade, Serbia
b
Analysis, Gandijeva 76a, 11070 Belgrade, Serbia
c
Innovative Center, Faculty of Chemistry Ltd., Studentski trg 12-16, 11158 Belgrade, Serbia

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

Article history: A total of 58 polyfloral honey samples from different regions in Serbia were studied to determine their
Received 17 May 2013 phenolic profile, total phenolic content and antioxidant capacity. UHPLC-LTQ OrbiTrap MS made possible
Received in revised form 9 August 2013 the identification of 36 compounds: 24 flavonoids, two abscisic acids, and 10 phenolic acids and their
Accepted 20 August 2013
derivatives. Quantification was done using 14 available standards. Data on phenolics and abscisic acids
Available online 2 September 2013
allowed the discrimination and classification of honeys in accordance to their geographical origin, using
pattern recognition techniques, principal component analysis and partial least squares discriminant anal-
Keywords:
ysis. Samples originated from Vojvodina and Zlatibor region were clearly distinguished from those from
Polyfloral honey
Phenolic profile
the rest of Serbia because of the presence of dicaffeoylquinic acid, ellagic acid, caffeic acid phenethyl
UHPLC-LTQ OrbiTrap MS ester, and chlorogenic acid, among others. A good correlation (r = 0.865) was observed between total phe-
Geographical origin nolic content and radical-scavenging activity. Total phenolic content ranged from 0.03 to 1.39 mg GAE/g
Pattern recognition methods and radical scavenging activity ranged from 1.31% to 25.61%.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Numerous flavonoids (such as apigenin, pinocembrin, pinobanksin,

Generally, honeys are classified as monofloral or polyfloral.
Monofloral honeys are produced by one plant species containing
predominantly its nectar with minor nectar contributions from
other botanical origins. Polyfloral honey has several plant sources,
none of which is predominant. In practical terms it can be consid-
ered as a blend of several monofloral honeys with significant nec-
tar or honeydew contributions from different plants.
Phenolic extracts from different plants have been shown to
have antioxidant as well as antimicrobial activity (Viuda-Martos,
Ruiz-Navajas, Fernandez-Lopez, & Perez-Alvarez, 2008). Similarly,
polyphenols, i.e. flavonoids and phenolic acids, are considered as
one of the important group of components identified in honey hav-
ing antioxidant activity. Available literature indicates that until
now there have been only a few studies to determine the total phe-
nolic content and antioxidant activity of Serbian honeys (Gorjano-
vić et al., 2013; Savatović et al., 2011; Tumbas et al., 2012).
The UHPLC-LTQ OrbiTrap MS technique is reliable for the
unambiguous detection of phenolic acids, their derivatives, and fla-
vonoid aglycones based on their molecular masses and fragmenta-
tion pattern. In our previous investigation (Kečkeš, Gašić, Ćirković
Veličković, Milojković-Opsenica, Natić, & Tešić, 2013), a total of
43 polyphenols were identified in Serbian unifloral honeys.
⇑ Corresponding author. Tel./fax: +381 11 2639 357.
E-mail address: ztesic@chem.bg.ac.rs (Ž. Tešić).
kaempferol, quercetin, galangin, chrysin, and luteolin) and pheno-
lic acids (caffeic, gallic, cinnamic, protocatechuic, p-coumaric, and
chlorogenic acids) were identified in samples. The samples were
classified according to their botanical origin using principal com-
ponent analysis (PCA). PCA resulted in two discriminant clusters
in the set of different monofloral honeys. Similarities between hon-
eys derived from perennial plants were observed, as well as among
honeys derived from annual plants.
Different methods for testing authenticity of geographical ori-
gin of honey exist. Classical pollen analysis is a reference method
used for routine control of honey authenticity, whereas physico-
chemical parameters, such as pH, acidity and electrical conductiv-
ity are the methods of quality control. Determination of sugars,
amino acids, minerals, and flavonoids in combination with chemo-
metric data evaluation are methods used to identify geographical
origin (Bogdanov & Martin, 2002; Primorac et al., 2008; Lazarević,
Andrić, Trifković, Tešić, & Milojković-Opsenica, 2012; Wang & Li,
2011). Mineral and trace elements concentration in different honey
types depend largely on the elemental composition of flowers,
with regard to their geographical origin (Bilandžić et al., 2011). Dif-
ferent sugars (Consonni, Cagliani, & Cogliati, 2013) and amino acids
(Belitz et al., 2009) have been used to detect the geographical ori-
gin of honey. Stable isotopes (Kropf et al., 2010) and Raman spec-
troscopy (Goodacre, Radovic, & Anklam, 2002) are among the most
reliable techniques for the detection of the geographical origin of
honey. Wang et al., 2009 reported a new MALDI-TOF-MS method

0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.08.088
600 U. Gašić et al. / Food Chemistry 145 (2014) 599–607
for determination of the geographical origin of honey by finger-
printing and bar-coding honey proteins in samples produces in
various regions in the USA.
Although propolis-derived polyhenolics are not helpful for the
determination of botanical origin, these compounds can be used
together with other polyphenols as markers of geographical origin
of honey (Ferreres et al., 1992). Tomás-Barberán, Ferreres, García-
Viguera, and Tomás-Lorente (1993) have compared the flavonoid
profiles of different honey samples from various regions in the
world and reported flavonoid profiles characteristic for the pres-
ence of propolis-derived flavonoids in honeys from the Northern
Hemisphere, where poplars are native. Martos, Ferreres, and Tomá-
s-Barberán (2000) have compared the geographical variations in
the flavonoid profiles between Australian and European Eucalyptus
honeys. Australian Eucalyptus honeys contains the propolis-de-
rived flavonoids, such as pinobanksin, pinocembrin, and chrysin,
in very small concentrations and the European Eucalyptus honeys
are relatively rich in these propolis-derived flavonoids. Therefore,
studies of flavonoid profiles of honey could be related to its geo-
graphical origin.
The main goal of this study was to determine characteristic
phenolic profiles by identifying flavonoids and phenolic acids in
Serbian polyfloral honeys. The possibility of verifying the regional
origin based on specific phenolic compounds, using multivariate
statistical methods, was examined. The variables discriminating
honey samples from different regions were identified and success-
ful models for further prediction were developed. Also, total phe-
nolic content (TPC) and the radical-scavenging activity (RSA) of
honey samples were determined, and correlation between these
parameters was evaluated.
2. Experimental
2.1. Chemicals and materials
Acetonitrile and formic acid (both MS grade), methanol (HPLC
grade), sodium carbonate, hydrochloric acid, and Folin–Ciocalteu
reagent were purchased from Merck (Darmstadt, Germany). The
SPE cartridges used for extraction and concentration of samples
were Strata C18–E (500 mg/3 mL) obtained from Phenomenex
(Torrance, CA).
2,2-Diphenyl-1-picrylhydrazyl(DPPH) and flavonoid standards
(rutin (quercetin 3-O-rutinoside), luteolin, quercetin, apigenin,
kaempferol, chrysin, pinocembrin, and galangin) were purchased
from Fluka AG (Buchs, Switzerland), whereas cis, trans-abscisic,
protocatechuic, chlorogenic (3-O-caffeoylquinic), caffeic, p-couma-
ric, and ellagic acids were supplied by Sigma–Aldrich (Steinheim,
Germany).
Ultrapure water (ThermoFisher TKA MicroPure water purifica-
tion system, 0.055 lS/cm) was used to prepare standard solutions
and blanks. Syringe filters (13 mm, PTFE membrane 0.45 lm) were
purchased from Supelco (Bellefonte, PA). Filter paper (Whatman
No. 1) was supplied by Merck (Darmstadt, Germany).
2.2. Honey samples
A total of 58 polyfloral honey samples were collected from five
different regions of Serbia (Fig. Supplementary1) during the 2009
harvesting season were provided by ‘‘The Association of the Bee-
keeping Organizations of Serbia’’ (SPOS) (www.spos.info). The geo-
graphical origin of the samples (Table 1) was specified by the SPOS
based on the information provided by beekeepers. The honeys
were stored at room temperature in the dark before analysis.
2.3. Extraction of phenolics from honey samples
2.3.1. Preparation of sample extracts for UHPLC-LTQ OrbiTrap MS
analysis
The method used for extraction and isolation of phenolics from
the honey was a modification of the method described in the liter-
ature (Michalkiewicz, Biesaga, & Pyrzynska, 2008). Honey samples
(5 g) were mixed with 5 mL of ultrapure water, adjusted to pH 2
with 0.1% HCl and homogenised in an ultrasonic bath for 30 min
at room temperature. The samples were then filtered through filter
paper to remove solid particles. An SPE cartridge was conditioned
by washing with 3 mL of acetonitrile and 9 mL of ultrapure water.
The filtrate was passed through cartridge, which was then washed
with 6 mL of acidified water to remove all sugars and other polar
constituents of honey. The adsorbed compounds were eluted with
acetonitrile (1.5 mL). The extracts were filtered through a 0.45-lm
PTFE membrane filter to be analysed by UHPLC-HESI-MS/MS.
2.3.2. Sample preparation for TPC and RSA
Samples were prepared according to the slightly modified
method proposed by Meda, Lamien, Romito, Millogo, and Naco-
ulma (2005). Each honey sample (5 g) was mixed with 15 mL ultra-
pure water, homogenized in ultrasonic bath for 15 min at room
temperature, transferred to 50 mL volumetric flask, and filled with
ultrapure water. The solution was then filtered through 0.45 lm
PTFE membrane and analyzed for determination of TPC and RSA.
2.4. UHPLC-LTQ OrbiTrap MS
A 1000 mg/L stock solution of a mixture of flavonoids (rutin,
luteolin, quercetin, apigenin, kaempferol, chrysin, pinocembrin,
and galangin), phenolic acids (protocatechuic, chlorogenic, caffeic,
p-coumaric, and ellagic acid) and cis, trans-abscisic acid was pre-
pared in methanol. Dilution of the stock solution with methanol
yielded working solutions at concentrations of 0.025, 0.050,
0.100, 0.250, 0.500, 0.750, and 1.000 mg/L. All stock and working
solutions were stored in the dark at 4 °C and were stable for at least
3 months. Calibration curves were obtained by plotting the peak
areas of the compounds identified relative to the peak area against
the concentration of the standard solution. Calibration curves re-
vealed good linearity, with r2 values exceeding 0.99 (peak areas
vs. concentration).
Chromatographic separations were performed using an ultra-
high-performance liquid chromatography (UHPLC) system consist-
ing of a quaternary Accela 600 pump and Accela autosampler
(ThermoFisher Scientific, Bremen, Germany). The analytical col-
umn used for separations was a Hypersil gold C18 column
(50  2.1 mm, 1.9 lm particle size) from ThermoFisher Scientific.
The mobile phase consisted of (A) water containing 1% formic acid
and (B) acetonitrile. The gradient program was as follows: 0.0–
10.0 min, 5–95% B; 10.0–12.0 min, 95% B; 12.0–12.2 min, 95–5%
B; 12.2–15.0 min, 5% B. The injection volume for all samples was
5 lL, and the flow rate was 300 lL/min.
The UHPLC system was coupled to a linear ion trap-OrbiTrap
hybrid mass spectrometer (LTQ OrbiTrap MS) equipped with
heated electrospray ionisation probe (HESI-II, ThermoFisher Scien-
tific, Bremen, Germany). The mass spectrometer was operated in
negative ion mode. Parameters of the ion source were as follows:
source voltage 5 kV, capillary voltage 40 V, tube lens voltage
80 V, capillary temperature 275 °C, sheath and auxiliary gas flow
(N2) 42 and 11 (arbitrary units). MS spectra were acquired by full
range acquisition covering m/z 100–900. For fragmentation studies,
a data-dependent scan was performed by deploying collision-in-
duced dissociation (CID). The normalised collision energy of the
collision-induced dissociation (CID) cell was set at 35 eV.
Table 1
Contents of phenolics and cis, trans-abscisic acid (mg/kg) in polyfloral honeys produced in different geographical regions of Serbia.

No Geographical region PrA, ChA, CaA, CoA, ElA, Rut, Lut, Que, AbA, Api, Kae, Chr, Pin, Gal, RSA, TPC,
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg % (mg/g)
1 Zlatibor 0.50 0.06 0.49 4.45 0.86 0.07 0.06 0.17 8.31 0.37 0.16 1.31 0.80 0.29 14.76 0.87
2 Zlatibor 0.54 0.10 0.32 3.59 1.04 0.03 0.04 0.21 3.28 0.16 0.13 0.28 0.13 ND 9.04 0.67
3 Zlatibor 0.23 0.11 0.70 2.77 8.48 0.04 0.06 0.22 0.85 0.35 0.28 0.91 1.55 0.31 2.82 0.05
4 Zlatibor 0.57 0.12 0.57 3.93 2.35 0.25 0.06 0.66 1.50 0.41 0.23 3.31 0.63 0.20 12.11 1.04
5 Zlatibor 0.60 0.08 2.33 3.71 4.84 0.12 0.12 0.32 2.40 0.43 0.34 2.13 2.18 0.66 7.59 0.71
6 Zlatibor 0.16 0.10 0.54 7.74 ND ND 0.09 0.17 1.14 0.29 0.19 0.86 0.48 0.22 2.29 0.50
7 Zlatibor 0.18 0.21 0.43 1.43 ND 0.04 0.04 0.18 3.62 0.14 0.10 1.39 0.75 0.22 1.61 0.19
8 Zlatibor 0.49 0.16 0.45 4.50 8.44 0.04 0.06 0.23 2.22 1.36 0.20 0.26 0.37 0.20 3.77 0.22
9 Zlatibor 0.14 0.05 0.47 1.17 1.26 0.03 ND 0.12 2.67 0.15 0.13 0.91 0.60 0.19 1.96 0.37
10 Zlatibor 0.89 0.09 0.36 6.58 1.76 0.04 0.09 0.19 3.11 0.34 0.20 3.53 0.47 0.17 12.22 0.73
11 Zlatibor 0.12 0.06 0.51 1.00 ND ND 0.04 0.12 3.02 0.28 0.14 0.83 0.75 0.26 1.36 0.25
12 Zlatibor 0.65 0.14 0.61 8.05 0.29 0.04 0.24 0.21 0.29 2.04 0.32 0.34 0.51 0.22 9.44 0.57
13 Zlatibor 0.37 0.08 0.33 6.80 ND 0.03 0.11 0.16 ND 0.25 0.17 ND 0.27 0.11 11.86 0.92
14 Zlatibor 0.63 0.06 0.33 3.33 0.34 0.03 0.04 0.22 0.34 0.18 0.21 0.30 0.16 ND 13.03 0.63
15 Zlatibor 0.56 0.27 0.45 6.15 0.62 0.03 0.25 0.19 0.62 3.27 0.29 1.17 0.52 0.30 8.64 0.42
16 Zlatibor 0.62 0.14 0.94 8.45 1.40 0.04 0.08 0.24 2.88 0.60 0.25 0.37 0.66 0.22 18.49 1.16
17 Zlatibor 0.50 0.13 0.48 2.92 1.50 0.22 0.05 0.43 0.92 0.29 0.16 0.30 0.43 0.13 4.56 0.66

U. Gašić et al. / Food Chemistry 145 (2014) 599–607

18 Zlatibor 0.69 0.08 0.38 4.53 1.25 0.05 0.09 0.24 1.25 0.79 0.22 1.69 0.48 0.22 11.18 0.98
19 Zlatibor 0.27 0.14 0.56 4.49 0.50 0.09 0.12 0.34 0.50 0.23 0.27 0.59 1.01 0.46 1.31 0.25
20 Zlatibor 0.84 0.05 0.43 2.35 0.72 0.04 0.07 0.19 0.72 0.42 0.19 1.51 0.61 0.25 15.01 1.19
21 Zlatibor 1.20 0.09 0.46 2.00 ND ND 0.06 0.14 0.46 0.35 0.22 0.33 0.40 0.16 25.61 1.39
22 Zlatibor 0.97 0.18 0.50 1.35 ND 0.06 0.12 0.31 1.44 0.47 0.26 0.81 0.19 0.11 13.29 0.77
23 Zlatibor 0.71 0.07 0.27 0.45 0.28 0.03 0.07 0.16 0.28 0.90 0.18 1.76 0.26 0.10 13.66 1.12
24 South 1.65 0.07 0.72 0.76 3.44 0.04 0.05 0.17 1.17 0.42 0.16 1.83 1.23 0.50 7.88 0.71
25 South 0.13 0.05 0.67 0.58 0.84 0.04 0.05 0.22 1.40 0.49 0.23 1.85 1.56 0.54 3.13 0.51
26 South 0.26 0.10 1.22 0.83 4.69 0.09 0.11 0.23 2.24 0.84 0.37 2.92 4.86 1.38 3.62 0.44
27 South 0.31 0.18 1.99 2.74 2.16 0.63 0.20 0.36 1.72 1.48 0.42 3.36 4.29 1.39 3.06 0.06
28 South 0.39 0.10 0.46 4.83 2.26 0.09 0.22 0.18 0.97 1.05 0.21 1.06 0.92 0.33 10.98 0.64
29 South 0.38 0.05 0.38 1.58 7.64 0.05 0.06 0.20 0.83 0.25 0.21 0.52 0.51 0.21 17.10 0.83
30 South 0.81 0.11 1.06 7.29 4.70 0.15 0.39 0.27 1.27 1.90 0.39 2.74 2.52 1.07 2.38 0.40
31 South 0.18 0.06 1.56 1.38 4.07 0.04 0.07 0.35 1.89 0.34 0.40 1.66 2.34 0.77 1.83 0.43
32 South 0.30 0.11 1.25 8.89 1.03 0.06 0.14 0.39 1.43 0.89 0.38 1.70 3.48 1.14 2.81 0.13
33 South 0.23 0.05 1.37 1.18 2.19 0.04 0.08 0.22 2.19 0.68 0.30 1.34 2.39 0.82 3.69 0.03
34 East 0.37 0.03 2.76 9.97 0.53 0.05 0.12 0.37 4.03 0.71 0.56 4.69 5.76 1.70 2.24 0.39
35 East 0.42 ND 0.59 1.02 4.46 0.20 0.05 0.23 3.60 0.21 0.25 0.97 1.37 0.38 4.45 0.55
36 East 0.23 0.06 0.63 0.79 2.46 0.03 0.05 0.16 2.72 0.44 0.18 1.16 1.55 0.48 1.85 0.08
37 East 0.35 0.07 0.39 2.13 1.24 0.03 0.06 0.13 1.38 0.43 0.17 1.26 0.48 0.17 5.78 0.74
38 East 0.77 0.08 0.44 4.46 3.07 0.07 0.13 0.21 1.33 0.46 0.16 0.70 0.64 0.25 16.23 0.74
39 East 0.86 0.07 0.58 1.79 1.41 0.03 0.12 0.16 1.35 0.33 0.17 0.67 0.55 0.15 4.65 0.21
40 East 0.26 0.05 1.03 4.06 0.83 0.04 0.08 0.21 1.75 0.55 0.31 1.31 1.31 0.73 4.31 0.42
41 East 0.12 0.03 0.23 1.15 1.20 0.06 0.04 0.15 2.83 0.27 0.24 2.22 0.94 0.36 16.44 0.74
42 East 0.49 0.06 0.40 4.42 2.33 0.10 0.11 0.26 0.65 0.44 0.59 0.67 0.61 0.34 25.55 1.36
43 East 0.52 0.03 0.26 2.86 4.91 ND 0.04 0.16 1.37 0.13 0.19 0.34 0.44 0.22 2.86 0.54
44 Vojvodina 0.88 0.08 1.26 1.49 ND 0.17 0.13 0.31 2.21 0.62 0.23 2.40 2.02 0.71 8.86 0.70
45 Vojvodina 0.39 0.32 0.85 2.14 ND 0.21 0.23 0.45 1.42 0.59 0.24 2.61 1.73 0.59 3.01 0.43
46 Vojvodina 0.22 0.67 0.75 4.43 ND 0.42 0.17 0.54 0.65 0.39 0.20 1.40 1.49 0.53 5.29 0.33
47 Vojvodina 0.30 0.41 0.99 2.19 0.69 0.04 0.08 0.39 4.57 ND 0.30 1.40 1.48 0.52 8.78 0.57
48 Vojvodina 0.59 0.67 1.39 1.19 ND 0.27 0.06 0.43 1.12 0.53 0.24 1.46 1.40 0.64 3.78 0.23
49 Vojvodina 0.22 0.39 1.24 7.43 ND 0.12 0.08 0.39 4.65 0.53 0.29 1.08 1.25 0.59 4.23 0.51
50 Vojvodina 0.23 0.51 0.95 4.21 ND 0.11 0.44 0.32 1.08 1.30 0.96 2.29 1.75 0.76 6.07 0.53
51 Vojvodina 0.33 0.11 0.47 0.40 ND 0.18 0.15 0.38 1.16 0.34 0.23 0.86 1.05 0.48 3.85 0.26
52 Vojvodina 0.16 0.10 0.67 5.92 0.99 0.29 0.33 0.33 0.99 0.73 0.68 2.56 1.61 0.59 3.04 0.25
53 Central 0.14 0.08 0.61 1.56 0.72 0.16 0.06 0.22 3.19 0.24 0.17 0.73 0.71 0.23 1.79 0.38
54 Central 0.25 0.05 0.32 1.80 0.84 0.04 0.04 0.17 2.70 0.25 0.17 1.25 1.00 0.34 2.54 0.36

601
(continued on next page)
602 U. Gašić et al. / Food Chemistry 145 (2014) 599–607

ND, not detected; PrA, protocatechuic acid; ChA, chlorogenic acid; CoA, p-coumaric acid; ElA, ellagic acid; Rut, rutin; Lut, luteolin; Que, quercetin; AbA, cis, trans-abscisic acid; Api, apigenin; Kae, kaempferol; Chr, chrysin; Pin,
Flavonoids, phenolic acids and abscisic acid were identified
(mg/g)

and quantified according to their spectral characteristics: mass

0.42
0.76

0.37
0.40
TPC,

spectra, accurate mass, characteristic fragmentation, and charac-

teristic retention time. Xcalibur software (Version 2.1) was used
for instrument control, data acquisition and data analysis. Frag-
RSA,

3.68
7.97
3.43
1.45

mentation mechanism and characteristic fragments were con-

%

firmed using Mass Frontier software (Version 6.0). The

generated MS/MS spectra were processed by ToxID software (Ver-
mg/kg

sion 2.1.1). Internet database of accurate mass spectrometry data,

0.11
0.18
0.77
0.66
Gal,

ChemSpider (www.chemspider.com), was used as a reference li-

brary to identify compounds of interest.
mg/kg

1.77
0.36
2.09
0.30
Pin,

2.5. Determination of the total phenolic content

The TPC was spectrophotometricaly determined with a Folin–

mg/kg

Ciocalteu method reported by Singleton and Rossi (1965), with

1.88
0.23
0.72
1.03
Chr,

some modification. Briefly, 0.3 mL of the sample extracts and

6 mL deionized water were mixed with 0.5 mL of Folin–Ciocalteu
reagent and the solution was incubated 6 min at room tempera-
mg/kg
0.13
0.25
0.37
0.23
Kae,

ture. Next, 3 mL of 20% sodium carbonate were added. After

30 min at 40 °C, absorbance was measured at 765 nm. Gallic acid
was used as standard, concentration 50–250 lg/mL. A mixture of
mg/kg

water and reagent was used as a blank. The results were ex-
0.16

0.46
0.33
0.40
Api,

pressed as mg gallic acid equivalents (GAE) per gram of honey.

2.6. Determination of radical-scavenging activity

mg/kg
AbA,

2.83

2.63
0.71

3.09

The RSA of the extracts of honey samples was evaluated by a

modified method of Li, Wang, Li, Li, and Wang (2008). An aliquot
mg/kg

of 1.0 mL of extract (some extracts were diluted ten times) was

Que,

0.14
0.23
0.21
0.16

mixed with 3 mL of methanol solution of DPPH (71 mM). The

mixture was left for 60 min in the dark (until stable absorption
values were obtained). The reduction of the DPPH radical was
mg/kg

0.09
0.05
0.04
Lut,

ND

measured by monitoring continuously the decrease of absorption

at 515 nm. RSA was calculated as a percentage of DPPH discolor-
ation using the equation:
mg/kg

0.05
0.04
Rut,

ðADPPH  Asample Þ
ND

ND

RSA ð%Þ ¼  100

ADPPH
where ADPPH is the absorbance of methanol solution of DPPHrad-
mg/kg
1.14
2.75
2.63
3.09

pinocembrin; Gal, galangin; TPC, total phenolic content; RSA, radical-scavenging activity.
ElA,

ical, Asample is the absorbance in the presence of honey extract. The

assays were carried out in triplicate and the results were ex-
pressed as mean values.
mg/kg
CoA,

3.88
1.95
1.11
3.44

2.7. Statistical analysis

Principal component analysis (PCA) and partial least squares

mg/kg
CaA,

0.26
0.29
0.86
0.67

discriminant analysis (PLS-DA) were carried out by means of

PLS ToolBox, V.6.2.1, for MATLAB 7.12.0 (R2011a). All data were
group-scaled, i.e. groups of variables were scaled to grand stan-
mg/kg

dard deviation, prior to any multivariate analysis. PCA was carried

ChA,

0.05
0.08
0.03
ND

out as an exploratory data analysis by using a singular value

decomposition algorithm (SVD) and a 0.95 confidence level for
Q and T2 Hotelling limits for outliers. PCA was performed in order
mg/kg
0.17
0.21
0.17
0.10
PrA,

to reduce dimensionality of data space, visualise the structure of

data, identify important variables, and confirm the presence of
outliers. The PLS-DA was performed using the SIMPLS algorithm
Geographical region

without forcing orthogonal conditions to the model in order to

condense Y-block variance into first latent variables. PLS-DA is a
classical PLS regression where the response variable is categorical,
i.e. it indicates the classes or categories of the samples. The aim of
Table 1 (continued)

Central
Central
Central
Central

this supervised technique is to build a mathematical model that

could be used for further classification of unknown samples.
The models were validated using a Venetian blinds validation
No

procedure. The quality of the models was monitored with the fol-
55
56
57
58

lowing parameters: (1) R2cal, the cumulative sum of squares of the

 U. Gašić et al. / Food Chemistry 145 (2014) 599–607
Ys explained by all extracted components, R2CV, the cumulative
fraction of the total variation of the Ys that can be predicted by
all extracted components, and R2pred, the cumulative fraction of
the total variation of the Ys that can be predicted by test compo-
nents (these three values should be as high as possible), and (2)
RMSEC (root mean square errors of calibration), RMSECV (root
mean square errors of cross-validation), and RMSEP (root mean
square errors of prediction); these three values should be as low
as possible, and with the lowest difference between them. Descrip-
tive statistics and Kruskal–Wallis one-way analysis of variance by
ranks test was performed using a demo version of NCSS statistical
software (Hintze, 2001, Number Cruncher Statistical Systems,
Kaysville, UT; www.ncss.com).
Limits of detection (LOD) and limits of quantification (LOQ)
were calculated using standard deviations of the responses (SD)
and the slopes of the calibration curves (S) according to the formu-
las: LOD = 3(SD/S) and LOQ = 10(SD/S). The values of standard
deviations and slopes were obtained from the calibration curves
created in MS Excel. LOD, LOQ, and the recoveries at three concen-
tration levels were determined according to a previously described
method (Kečkeš et al., 2013). These results are given in Supplemen-
tary Data (Table S1).
3. Results and discussion
Generally, there is a growing interest on the effects of natural
antioxidants in food. Different honey types subjected to antioxi-
dant activity tests have demonstrated significant potential, compa-
rable to other foodstuffs (Schramm et al., 2003). Antioxidants
protect cell defence systems against oxidative damage, contribut-
ing to the prevention of certain illnesses, including cardiovascular
diseases, cancer, and diabetes (Viuda-Martos et al., 2008). Antiox-
idant activity of honey partly depends on the phenolics and is clo-
sely related to the floral source of honey. Phenolics’ structural
features and substituents (such as hydroxyl, acyl, methyl, and gly-
cosyl groups) are basic factors influencing the final activity.
3.1. Phenolic profiles of polyfloral honeys
Solid-phase extraction (SPE) combined with ultra-high-perfor-
mance liquid chromatography-OrbiTrap mass spectrometry
(UHPLC-LTQ OrbiTrap MS) was used to analyse the contents of
flavonoids, phenolic acids and abscisic acid in honeys. A total of
36 compounds were identified and 14 of them were quantified
by comparing retention times and MS spectra with available stan-
dards. Selected base peak chromatogram of honey extract from
Vojvodina region is shown in Fig. 1.
In the absence of standards, identification of another 22 peaks
in the chromatograms was based on the search for the [M–H]-
deprotonated molecule and its fragmentation. The exact mass
search and the study of the fragmentation pathways described in
the literature enabled us to obtain as much structural information
as possible. In this way, it was possible to identify two abscisic
acids, different phenolic acids, and many flavonoids. Table 2
Fig. 1. Base peak chromatogram of a honey sample: (1) gallic acid, (2) protocatechuic acid, (4) caffeic acid, (5) p-coumaric acid, (6) ellagic acid, (7) rutin, (8) taxifolin, (9)
dicaffeoylquinic acid, (10) ferulic acid, (11) eriodictyol, (12) trans, trans-abscisic acid, (13) luteolin, (14) quercetin, (15) cis, trans-abscisic acid, (18) apigenin, (20) kaempferol,
(21) pinobanksin, (23) bis-methylated quercetin, (26) chrysin, (30) pinocembrin, (32) galangin, and (34) caffeic acid phenethyl ester (CAPE).
603
summarises the data obtained for all 36 identified compounds.cis,
trans-Abscisic acid was quantified using an available standard
and the other isomer, trans, trans-abscisic acid, was confirmed by
the exact mass search of deprotonated molecule and MS/MS
fragmentation.
A common fragmentation pathway based on the loss of the CO2
group was observed for all phenolic acids identified in samples.
Hydroxybenzoic acids (gallic, protocatechuic, and ellagic acid)
and hydroxycinnamic acids (caffeic, p-coumaric, and ferulic acid),
with a characteristic deprotonated [M–H]molecule and [M–
H44] fragment, were identified. Four caffeic acid derivatives
were detected: chlorogenic acid (identified and quantified using
available standard 3-O-caffeoylquinic acid), dicaffeoylquinic acid,
prenyl caffeate, and caffeic acid phenylethyl ester (identified using
exact mass search of deprotonated molecule [M–H] and its frag-
mentation). All of the caffeic acid derivatives showed negative
product ion at m/z 179 due to the loss of the deprotonated mole-
cule of caffeic acid.
Four subclasses of flavonoids were detected in all honey sam-
ples: flavonols, flavanonols, flavanones, and flavones. In the nega-
tive mode, the loss of groups such as H2O (18 amu), CO
(28 amu), C2H2O (42 amu) and CO2 are common for all flavo-
noids and these characteristic fragments were found (Kečkeš
et al., 2013). Methylated or methoxylated flavonoids were charac-
terised by the loss of CH3 (15 amu) in the negative mode. Also,
complementary fragments from the retro-Diels–Alder (RDA) reac-
tion, which is major fragmentation reaction for flavonoids, were
found in the MS spectra. The fragmentation pattern of flavonoids
is given in detail in our previous paper (Kečkeš et al., 2013).
In most samples flavones luteolin, apigenin, chrysin, and flavo-
nols quercetin, kaempferol, galangin, and glycoside rutin were
identified. As for flavanonols, it was possible to characterise taxif-
olin, pinobanksin, and three derivatives of pinobanksin (pinobank-
sin 5-methylether-3-O-acetate, pinobanksin 5-methylether, and
pinobanksin 3-O-acetate). The fragmentation step observed for
pinobanksin esters was the loss of the acyl group, yielding frag-
ments at 271 and 253 m/z corresponding to [M–acyl] and [M–
acyl–H2O] ions, respectively. Flavanones (eriodictyol, sakuranetin,
and pinostrobin) were identified using exact mass search, and
pinocembrin was quantified using a standard.
The chromatograms of the investigated samples showed similar
profiles. All honey extracts contained somewhat large amount of
flavonoids that are known to originate from propolis, namely chry-
sin, pinocembrin, and galangin (Tomás-Barberán, Martos, Ferreres,
Radovic, & Anklam, 2001). Similar phenolic profiles exist in Slove-
nian honeys (Bertoncelj, Polak, Kropf, Korošec, & Golob, 2011).
The summarised parameters of descriptive statistics for total
intensities obtained from the MS/MS spectra and content of quan-
tified compounds are presented in Table S2 (Supplementary Data)
for each region separately.
Normality of distribution of data was analysed by three tests:
D’Agostino K-squared (skewness normality of residuals), Bonnet-
Seier’s (kurtosis normality of residuals) and Shapiro–Wilk’s (nor-
mal scores correlation) test. A significant deviation from normal
604 U. Gašić et al. / Food Chemistry 145 (2014) 599–607

Table 2
Phenolic profiles of Serbian polyfloral honeys; number of identified compound, target compounds, mean expected retention times, calculated mass, exact mass, mean mass
accuracy (mDa), and MS/MS fragments.

Peak noa Compounds Retention time, min Calculated massb, [M–H]– Exact mass, [M–H]– mDa Mass fragments
d
1 Gallic acid 0.55 169.01473 169.01360 1.13 125
2 Protocatechuic acidc 1.13 153.01972 153.01877 0.95 109
3 Chlorogenic acidc 2.03 353.08782 353.08612 1.70 191, 179, 146
4 Caffeic acidc 2.21 179.03498 179.03427 0.71 135, 161
5 p-Coumaric acidc 2.81 163.04007 163.03949 0.58 119
6 Ellagic acidc 2.91 300.99898 300.99762 1.36 283, 200, 175
7 Rutinc 3.00 609.14610 609.14404 2.06 301
8 Taxifolinb 3.05 303.05101 303.05042 0.59 285, 269, 255, 217
9 Dicaffeoylquinic acidd 3.33 515.11949 515.11725 2.24 191, 179
10 Ferulic acidd 3.41 193.05063 193.05009 0.54 175, 139
11 Eriodictyold 3.53 287.05611 287.05499 1.12 125
12 trans, trans-Abscisic acidd 3.91 263.12889 263.12808 0.81 191, 179
13 Luteolinc 4.15 285.04045 285.03915 1.30 213, 151
14 Quercetinc 4.15 301.03539 301.03400 1.39 151, 179, 121
15 cis, trans-Abscisic acidc 4.20 263.12889 263.12790 0.99 191, 179
16 Sakuranetind 4.36 285.07686 285.07559 1.27 133
17 Rhamnetind 4.36 315.05101 315.04980 1.21 300, 165, 121
18 Apigeninc 4.61 269.04555 269.04437 1.18 149, 151, 173, 183
19 Isorhamnetind 4.67 315.05101 315.04999 1.02 300, 151, 107
20 Kaempferolc 4.70 285.04045 285.03918 1.27 199, 161, 151, 135
21 Pinobanksind 4.71 271.06120 271.06006 1.14 253, 243, 165, 151, 107
22 Methyl quercetind 4.81 315.05101 315.04974 1.27 300, 271, 207, 151
23 Bis-methylated quercetind 5.03 329.06667 329.06589 0.78 315, 165
24 Pinobanksin 5-methylether-3-O-acetated 5.33 327.08699 327.08652 0.47 271, 253
25 Quercetin tetramethyl etherd 5.43 357.09740 357.09537 2.03 300, 207, 179, 161
26 Chrysinc 5.82 253.05063 253.04951 1.12 101, 151, 181, 209, 143
27 Prenyl caffeated 5.83 247.09758 247.09639 1.19 135, 179
28 Pinostrobind 5.87 269.08193 269.08081 1.12 135, 179
29 Pinobanksin 5-methyletherd 5.87 285.07672 285.07532 1.40 271, 165
30 Pinocembrinc 5.92 255.06627 255.06523 1.04 213, 211, 151
31 Acacetind 5.95 283.06120 283.05984 1.36 268, 133, 151, 107
32 Galanginc 5.98 269.04555 269.04443 1.12 151, 183
33 Pinobanksin 3-O-acetated 6.08 313.07120 313.07043 0.77 271, 253
34 Caffeic acid phenethyl ester (CAPE)d 6.12 283.09758 283.09653 1.05 135, 179
35 Methoxychrysind 6.19 283.06120 283.06003 1.17 268, 239, 211
36 Pinobanksin 3-O-propionated 6.66 327.08686 327.08670 0.16 285, 165
a
Peak numbers corresponding to Fig. 1b.
b
Calculated mass of the parent ion using free chemical database, ChemSpider.
c
Confirmed by standard.
d
Confirmed by reference.

distribution for each of the studied variables was observed. There-
fore, for statistical evaluation of differences in the total intensities
of detected compounds, the Kruskal–Wallis test was employed for
each flavonoid, phenolic acid and abscisic acid separately, as vari-
ables, taking the appropriate region as a single factor. Kruskal–
Wallis one-way analysis of variance tests if samples originate from
the same distribution. When the Kruskal–Wallis test led to signif-
icant results, multiple-comparison Z-value test was performed to
identify where the differences occurred or how many differences
actually occurred. Results are presented in Table S2 (Supplemen-
tary Data), with separated regions denoted in parentheses.
Based on Kruskal–Wallis test several compounds, such as chlor-
ogenic acid, ellagic acid, quercetin, dicaffeoylquinic acid and pino-
banksin 5-methylether-3-O-acetate, were identified as parameters
governing a separation of Vojvodina honey samples from those
originating from all other regions. Also, bis-methylated quercetin
and pinobanksin 3-O-propionate differentiate Vojvodina samples
from those from Zlatibor, and Central and East regions. Separation
of Zlatibor honey samples from the rest of Serbia was achieved by
the intensities of ellagic acid, pinocembrin, galangin, eriodictyol,
sacuranetin, pinobanksin, acacetin, caffeic acid phenethyl ester
and methoxychrysin. The rest of the factors are responsible for dif-
ferentiation of only limited numbers of regions, whereas cis, trans-
abscisic acid, gallic acid, pinostrobin and pinobanksin 5-methyle-
ther do not differ among any of the considered regions.
Among the quantified compounds, p-coumaric acid was present
in the highest amount, while the lowest contents were recorded for
luteolin and rutin. Also, median value for chlorogenic acid of honey
samples from Vojvodina was somewhat higher, and for ellagic acid
lower, compared to values from the rest of Serbia (Table S2).
3.2. Geographical origin of polyfloral honey
The identification of geographical origin of honey is a hard task
often requiring the use of several criteria and chemical markers as
well as a combination of several analytical methods with appropri-
ate statistical analyses. The amount of polyphenols in honeys is
closely related not only to the floral variety but also to specific
parameters, such as soil composition and meteorological condi-
tions. Thus, it is possible to relate the phenolic profiles of honeys
of the same botanical source with their geographical origin.
Although polyfloral honeys are produced from nectars (various
meadow flowers and fruit trees) and honeydew, several papers
indicated the possibility of discrimination between samples col-
lected from different regions (Bilandžić et al., 2011; Kropf et al.,
2009).
Multivariate statistical analysis, PCA and PLS-DA were applied
in order to distinguish the honey samples from Serbia according
to their regional origin. Total intensities obtained from the MS/
MS spectra processed through ToxID software served as an input
data matrix.
A PCA resulted in a five-component model which explains
68.99% of total variance. The first principal component, PC1, ac-
counted for 34.76% of the overall data variance, the second one,
 U. Gašić et al. / Food Chemistry 145 (2014) 599–607
Fig. 2. PCA scores plot of Serbian polyfloral honeys of different geographical origin.
Table 3
Statistical performances of the PLS-DA models.
Zlatibor South East Vojvodina Central
region region region
R2cal 0.7139 0.5387 0.4360 0.8440 0.3738
R2CV 0.5371 0.3076 0.1252 0.6781 0.0430
RMSEC 0.2656 0.2318 0.2709 0.1494 0.2332
RMSECV 0.3465 0.2956 0.3786 0.2185 0.3195
PC2, for 11.56% and the third principal component, PC3, for 10.39%.
Mutual projections of factor scores and their loadings for the first
three principal components (PCs) are presented in Fig. 2 and
Fig. S2. Score plot reveals four samples lying outside the Hotteling
T2 ellipse (one sample from Vojvodina, two samples from the South
region and one sample from the Eastern region), suggesting that
they were outliers. They were excluded from further analysis. Tak-
ing into account PC1 score values, two distinctive groups of honeys
belonging to Vojvodina and Zlatibor regions are obtained. Samples
from Zlatibor are firmly clustered, exhibiting small internal vari-
ability. There was some overlapping among honey samples from
the South region and Vojvodina, and Central region and Zlatibor.
The samples from the East region are dissipated in a broader range
of score plots. The loading plot reveals that the most influential
compounds discriminating between honey from Vojvodina and
Zlatibor are methoxychrysin, bis-methylated quercetin, rhamnetin,
sakuranetin, galangin, pinobanksin, pinocembrin, eriodictyol and
caffeic acid.
Honey samples from different geographical areas were mod-
elled simultaneously using PLS-DA. The first model used all of
the 36 identified phenolic compounds to predict the geographical
origin of the samples. According to the variable importance in pro-
jection (VIP) scores, variables that most significantly affected the
PLS-DA calculation were identified. Data from 18 selected parame-
ters were remodelled. The number of the latent variables (LVs) was
selected on the basis of the minimum value of RMSECV, which was
achieved with seven latent variables. Classification and validation
results, expressed as R2cal, R2CV, RMSEC and RMSECV values, for five
new models are presented in Table 3.
Statistical results of PLS-DA models for Zlatibor and Vojvodina
regions were statistically significant, with relatively high values
of R2cal and R2CV, and low difference between RMSEC and RMSECV
values. In contrast, for the three other regions PLS-DA models were
not statistically significant. Hence, data from the phenolic profile of
Serbian polyfloral honeys allow accurate prediction of geographical
origin of honey from Vojvodina and Zlatibor and provides a useful
tool for determination of sample authenticity. The scores plot of
data for these regions also confirmed good predictive ability of
605
PLS-DA modelling (Fig. 3a and b). The contribution of particular
variables with the strongest influence on differentiation according
to geographical origin was analysed using the VIP value (Fig. 3c and
d). The variables with a VIP score higher than one were considered
as the most relevant for explaining certain classes of samples. The
standardised regression coefficient that reveals the significance of
an individual variable in the regression model is shown in Fig. 3e
and f.
The descriptors included in two models are different and a few
mutual parameters are of opposite significance. The compounds
determining the samples originating from Vojvodina are dica-
ffeoylquinic acid, ellagic acid, quercetin, bis-methylated-quercetin,
methoxychrysin, and pinocembrin. Ellagic acid was found in only
two samples originating from Vojvodina; thus the absence of this
compound distinguishes the samples. The compounds that dis-
criminate Zlatibor region from the rest of Serbia are caffeic acid
phenethyl ester, chlorogenic acid, dicaffeoylquinic acid, caffeic
acid, methoxychrysin, isorhamnetin, and bis-methylated querce-
tin. These results are in accordance with the described results of
the Kruskal–Wallis test and PCA.
The compound which gave a highest contribution to the PLS-DA
model proposed for data from Vojvodina is dicaffeoylquinic acid.
This compound was found in all polyfloral honey extracts from
Vojvodina, and in 5 out of 23 samples from Zlatibor. Dicaffeoylqui-
nic acid was previously detected in monofloral Serbian honey sam-
ples originating from plants characteristic for Vojvodina region:
sunflower, basil, and buckwheat (Kečkeš et al., 2013). Dicaffeoyl-
quinic acid appears with the highest positive coefficient in the
model for the data from Vojvodina, contrary to a small negative
coefficient in the model for Zlatibor region. Specific soil composi-
tion, climate, and presence of regional endemic flora could be the
factors that distinguish phenolic profiles of samples from Zlatibor
from those from the rest of Serbia.
In addition to the cross-validation procedure, and in order to
determine the degree of accuracy and sensitivity of PLS-DA models,
the entire data set for Vojvodina and Zlatibor region was divided into
two subsets, calibration set and testing set composed of randomly se-
lected samples. The calibration set consisted of 16 out of 23 samples
from Zlatibor, and 6 out of 9 samples from Vojvodina, while the test-
ing set was composed of the rest of the studied samples. The use of
such subsets of samples resulted in the one LV model including
60.78% variability in the X-variable domain, demonstrating that the
variables are effectively used for building up the model. The ex-
plained calibration variance for the categorical variable was 90.09%
with the following statistical parameters: R2cal = 0.9009,
R2CV = 0.8747, R2pred = 0.5005, RMSEC = 0.1402, RMSECV = 0.1583,
and RMSEP = 0.3623. Good predictive ability of PLS-DA model could
be confirmed by scores plot of data for calibration and test set for
606 U. Gašić et al. / Food Chemistry 145 (2014) 599–607
Fig. 3. PLS-DA, (a and b) scores plots of data for Zlatibor and Vojvodina; (c and d) plots of the variables versus VIP scores in model for Zlatibor and Vojvodina; (e and f) plot of
the coefficients of parameters in model for Zlatibor and Vojvodina, respectively.
Vojvodina and Zlatibor region (Fig. S3, Supplementary Data). The rel-
atively low value of R2pred was, probably, a result of deviation of one
honey sample from Zlatibor from all others (Fig. S3), which could be
also observed on the score plot of the PCA. The mentioned sample
originates from near the border of Zlatibor.
3.3. Antioxidant activity of polyfloral honeys
Antioxidant capacity of honey samples was determined by total
phenolic content and the radical-scavenging activity. Polyfloral
honey samples were characterised with TPC values ranging be-
tween 0.03 and 1.39 mg of gallic acid per g of honey, i.e. 0.05 to
1.39 mg GAE/g for Zlatibor region, 0.03–0.83 mg GAE/g for South
region, 0.08–1.36 mg GAE/g for East region, 0.23–0.70 mg GAE/g
for Vojvodina, and 0.36–0.76 mg GAE/g for Central region. Polyfl-
oral honeys collected from the South, Central, and Vojvodina re-
gions showed similar TPC mean values. The mean value of
0.68 mg GAE/g for honey from the Zlatibor region is somewhat
higher than for other regions. The average content of total pheno-
lics was in good agreement with the values given in the literature
for the polyfloral honeys of surrounding regions (Bertoncelj, Do-
beršek, Jamnik, & Golob, 2007; Dobre, Gâdei, Pătrasßcu, Elisei, & Se-
gal, 2010; Piljac-Žegarac, Stipčević, & Belščak, 2009).
The results of RSA ranged from 1.31% to 25.61%. Some differ-
ences between the mean values of antioxidant activity for samples
of specific regions are noted. The RSA mean value of Zlatibor region
(9.37%) was somewhat higher compared to the East region (8.44%).
Also, RSA mean value for South and Vojvodina regions were similar
(5.65% and 5.21%, respectively), and the lowest value was found for
the Central region (3.48%). The concentration causing 50% inhibi-
tion expressed as micromoles of Trolox equivalents per gram of
honey had a value of 0.58 lmol TE/g. Results expressed as Trolox
equivalents were comparable and consistent to the results found
in the paper published by Gorjanović et al. (2013).
The correlation between the TPC and RSA resulted in a linear
model RSA ¼ 1:55 ð0:79Þ þ 16:01 ð1:24Þ TPC with the following
statistical parameters: r = 0.8646, s = 3.01, t = 12.88 (tcr(56) = 2.00),
F = 165.82, P = 0.0558. Significant correlation between TPC and RSA
indicated that among all active phytochemicals, flavonoids and phe-
nolic acids could be identified as chemicals that account for the anti-
oxidant potential of honey. Our findings are in accordance with
previous reports (Bertoncelj et al., 2007; Piljac-Žegarac et al., 2009).
 U. Gašić et al. / Food Chemistry 145 (2014) 599–607
4. Conclusion
Polyfloral honey samples from different regions in Serbia were
studied to determine their phenolic profile, total phenolic content
and antioxidant capacity. UHPLC-LTQ OrbiTrap MS analysis pro-
vided information about numerous phenolic compounds specific
for the studied polyfloral honeys. The results of this study suggest
that the phenolic profiles varied considerably within samples. Mul-
tivariate statistical analysis, PCA and PLS-DA were performed, in
order to delineate differences between polyfloral honeys according
to their regional origin. The results demonstrated that comprehen-
sive spectral data collected by the accurate mass search in combi-
nation with statistical methods could be successfully applied to
detect the geographical origin of Serbian honeys. Samples obtained
from Vojvodina and Zlatibor region were clearly distinguished
from the rest of Serbia due to the presence of dicaffeoylquinic acid,
ellagic acid, caffeic acid phenethyl ester, and chlorogenic acid.
Among numerous antioxidant constituents, flavonoids and phe-
nolic acids could be identified as components that account for the
total antioxidant activity in samples. Samples collected from the
Zlatibor region showed somewhat higher TPC and RSA mean values
when compared to the other regions. Radical-scavenging activity
correlated well with the total phenolic content.
Acknowledgments
This work was performed within the framework of the research
projects No. 172017 and supported by the Ministry of Education,
Science and Technological Development, Republic of Serbia. The
authors acknowledge the support of the FP7 RegPot project FCUB
ERA GA No. 256716. The EC does not share responsibility for the
content of the article.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2013.
08.088.
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