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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Evaluation of bioactive compounds potential and antioxidant activity of

brown, green and red propolis from Brazilian northeast region
Julianna Karla Santana Andradea, Marina Denadaia,⁎, Christean Santos de Oliveiraa,
Maria Lucia Nunesb, Narendra Naraina
a
Laboratory of Flavor and Chromatographic Analysis, PROCTA, Federal University of Sergipe, 49100-000 São Cristóvão, SE, Brazil
b
Department of Food Technology, Federal University of Ceara, CEP 60020-180 Fortaleza, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the present study was to determine the contents of bioactive compounds present in brown, green and
Propolis red species of propolis cultivated in the Brazilian northeast states of Alagoas and Sergipe. The contents of
Bioactive compounds phenolic compounds, flavonoids and antioxidant activity (DPPH, ABTS+, FRAP, ORAC) were determined.
Flavonoids Identification and quantification of phenolic and flavonoid compounds were performed by using UHPLC-QqQ-
Antioxidant activity
MS/MS system. The results revealed high contents of total phenolics and flavonoids. Among the three species,
UHPLC-QqQ-MS/MS
the antioxidant potential had higher capacity in the red propolis. The presence of some of bioactive compounds
viz. acacetin, artepellin C, eriodictyol, gallic acid, isorhamnetin, protocatechuic acid, vanillin and vanillic acid in
Brazilian red propolis is reported for the first time in this work. Positive correlation between total phenolics
versus the FRAP and ORAC methods was established which led to conclusion that antioxidant activity of propolis
is mainly due to its phenolic compounds.

1. Introduction The chemical composition of propolis varies according to the geo-

Propolis is a resinous substance, dark in color and it is collected by
honeybees, mainly from leaf and flower buds, stems and cracks in the bark
of many species of trees. This material is transported to the hive and mixed
with beeswax, producing a strongly adhesive substance, which for cen-
turies has been used worldwide in traditional medicine (Daugsch, Moraes,
Fort, & Park, 2008; Pellati, Orlandini, Pinetti, & Benvenuti, 2011).
In South America, Brazil is well known for its green propolis, produced
by Apis mellifera, which collect resins mainly from a native shrub Baccharis
dracunculifolia (López, Schmidt, Eberlin, & Sawaya, 2014). However, due
to the large Brazilian biodiversity, there are 13 types of propolis, which
include less common brown and red propolis and these are classified based
on the place of production where these are found (Sawaya et al., 2004).
Since propolis is a natural product characterizing for several biological and
pharmacological properties, it has attracted interest of researchers in the
last decades. The therapeutic properties of propolis are well known in
popular medicine, due its antiseptic, antitumoral (Franchi et al., 2012;
Frozza et al., 2017), antiinflammatory (Bufalo et al., 2013; Franchi et al.,
2012), immunomodulatory (Bufalo et al., 2013), antioxidant (Franchi
et al., 2012), antibacterial, antimicrobial activities (Bufalo et al., 2013;
Franchi et al., 2012; Sforcin & Bankova, 2011; Szliszka et al., 2013; Viuda-
Martos, Ruiz-Navajas, Fernandez-Lopez, & Perez-Alvarez, 2008).
graphic region, climate, environmental conditions and collecting season
(López et al., 2014; Sawaya, Barbosa da Silva Cunha, & Marcucci,
2011). Although phenolic compounds are the most abundant in pro-
polis, yet > 300 compounds have been identified in different species,
such as phenolic acids, flavonoids including flavones, flavanones, fla-
vonols and chalcones, terpenes, aromatic aldehydes, alcohols, fatty
acids, stilbenes, steroids, amino acids, lignans and sugars (Akyol et al.,
2013; da Silva Frozza et al., 2013; Righi et al., 2011).
Raw propolis cannot be used as feedstock and it must be purified.
This process should remove the waxy material and preserve the fraction
of polyphenols, which are considered to contribute most to the curative
effects than the other constituents of propolis. The most popular tech-
nique for the production of propolis extracts is ethanol extraction due to
the fact that active substances of propolis are more easily soluble in
etanol (Pietta, Gardana, & Pietta, 2002). According to other authors,
extraction using the hydroalcoholic solvent (ethanol) have been de-
scribed as the most suitable means for the extraction of biologically
active phenolic components from propolis materials (Cottica et al.,
2011; Frozza et al., 2017; López et al., 2014; Pellati et al., 2011). Sun,
Wu, Wang, and Zhang (2015) stated in his study that ethanol/water
solvents have a significant effect on the phenolic composition and an-
tioxidant properties of the propolis extracts.

⁎
Corresponding author.
E-mail address: marrydenadai@gmail.com (M. Denadai).

http://dx.doi.org/10.1016/j.foodres.2017.08.066
Received 11 July 2017; Received in revised form 29 August 2017; Accepted 30 August 2017
0963-9969/ © 2017 Published by Elsevier Ltd.

Please cite this article as: Andrade, J.K.S., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.08.066
J.K.S. Andrade et al.
The compounds daidzein, biochanin A (López et al., 2014; Silva
et al., 2015), pinocembrin and quercetin (Daugsch et al., 2008; de
Mendonça et al., 2015) are biomarkers of Brazilian red propolis. Besides
these compounds, the red propolis is known to possess chemical sub-
stances which were not found in other varieties of propolis, such as
vestitol and neovestitol, C-glycoside, liquiritigenin, isoliquiritigenin,
formononetin, and medicarpin (Bueno-Silva et al., 2013; da Silva
Frozza et al., 2013; López et al., 2014; Piccinelli et al., 2011). However,
the principal constituents of Brazilian green propolis are caffeic acid, p-
coumaric acid, ferulic acid, naringenin, kaempferol, isorhamnetin, sa-
kurametin, pinocembrin, kaempferide and artepellin C (Szliszka et al.,
2013).
In Brazil, green and red propolis species are the most studied
(Frozza et al., 2017; Hatano et al., 2012; Silva et al., 2015), although
there are some reports in literature on other species, like brown pro-
polis (Bittencourt et al., 2015). However, there are no reports on the
presence and comparative data on phenolic compounds and antioxidant
activity among the various varieties of Brazilian propolis. Thus the in-
vestigation on the chemical composition of different species and their
comparative evaluation becomes important.
In recent years, the use of Ultra-high Performance Liquid
Chromatographic system coupled with Tandem Mass Spectrometry
(UHPLC-QqQ-MS/MS) is becoming the most commonly employed
method for determination of phenolic compounds, due to the system's
high sensitivity, selectivity and high-throughput capability. Thus the
objective of this study was to use UHPLC-QqQ-MS/MS system to identify
and quantify phenolic compounds and to evaluate their antioxidant ac-
tivity in brown, red and green propolis species grown in the states of
Alagoas and Sergipe, pertaining to the Northeast region in Brazil.
2. Materials and methods
2.1. Analytical standards and reagents
All the organic solvents employed were of HPLC grade. Water used
for the mobile phase was purified through a Milli-Q system (Millipore,
São Paulo, Brazil; Direct-Q® 3UV). The solvents acetonitrile and formic
acid used were of HPLC grade 98% of purity obtained from Sigma
Aldrich and Fluka Analytica (St Louis, MO, USA). Apigenin (C15H10O5),
acacetin (C16H12O5), artepellin C (C19H24O3), biochanin A (C16H12O5),
cinnamic acid (C9H8O2), α-cyano-4-hydroxycinnamic acid (C10H7O3N),
caffeic acid (C9H8O4), ferulic acid (C10H10O4), caffeic acid phenyl ester
(CAPE) (C17H16O4), (+)-catechin (C15H14O6), chrysin (C15H10O4),
epicatechin (C15H14O6), eriodictyol (C15H12O6), ethyl gallate
(C9H10O5), gallic acid (C7H6O5), isorhamnetin (C16H12O7), kaempferide
(C16H12O6), kaempferol (C15H10O6), luteolin (C15H10O6), narigenin
(C15H12O5), p-coumaric acid (C9H8O3), protocatechuic acid (C7H6O4),
pinocembrin (C15H12O4), quercetin-3-glucoside (C21H20O12), chrolo-
genic acid (C16H18O9), rutin (C27H30O16), vanillin (C8H8O3) and va-
nillic acid (C8H8O4) were purchased from Sigma-Aldrich (Saint Louis,
MO, USA). The reagents and standards: etanol; aluminum chloride;
sodium carbonate, potassium phosphate buffer, sodium citrate, ferrous
sulfate; Folin-Ciocalteu phenol reagent; fluorescein; 6-hydroxy-2,5,7,8-
tetramethylchromo-2-carboxylic acid (Trolox); 2,2-diphenyl-1-pi-
crylhydrazyl radical (DPPH%); 2,2′-azino-bis (3-ethylbenzthiazoline) 6-
sulfonic acid (ABTS+); 2,2-Azobis (2-Amidino-Propane) dichloride
(AAPH); and FRAP reagent were obtained from Sigma Aldrich and
Fluka Analytica (St Louis, MO, USA).
2.2. Propolis samples
Brown, red and green raw propolis samples were collected from
apiaries located at Marechal Deodoro, Alagoas state, Brazil, at co-
ordinates 9° 45′34.454″ S, 35° 50′24.986″ W in January 2016 while the
raw red propolis was collected from apiary, located in Brejo Grande,
Sergipe, Brazil, at coordinates 10° 25′28″ S, 36° 27′44″ W, in July 2016.
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The samples of brown, green and red propolis were collected for a
period of 7 days in the afternoon time at about 16 h. Three batches of
propolis sample weighing about 50 g each were collected to carry out
the analysis. Raw propolis were ground using a mill (IKA, Brazil, A11
basic) and the powder was later stored in glass containers which were
maintained at 0 °C.
2.3. Preparation of propolis extracts
Raw propolis sample (2 g) was extracted with 15 mL of ethanol:-
water (70:30, v/v) in an ultrasound bath apparatus (3L Alpha Plus) at
35 °C for 60 min. The extracts were centrifuged at 400g for 10 min,
using a centrifuge (Eppendorf, 5810R). The supernatants were col-
lected, dried at 35 °C in a drying oven, and resuspended in 2 mL of
ethanol:water (70:30, v/v). Finally, samples were filtered through
0.2 μm cellulose filters (Millipore, Bedford, MA, USA) and injected in
the UHPLC-QqQ-MS/MS system.
2.4. Determination of total phenolics content
The total content of phenolic compounds was determined by using
Folin-Ciocalteu phenol reagent (Shetty, Curtis, Levin, Witkowsky, & Ang,
1995). One milliliter of the ethanolic extract was transferred to test tubes
and 1 mL of 95% ethanol solution, 5 mL of distilled water and 0.5 mL of
Folin-Ciocalteu reagent (1N) were added, followed by homogenization in
vórtex. Later, 1 mL of sodium carbonate solution 5% (w/v) was added. The
test tubes were kept in dark for 60 min, and then homogenized in vórtex.
The absorbance was measured at a wavelength of 725 nm against a blank
consisting of 95% ethanol solution, using a spectrophotometer (Molecular
Devices, Sunnyvale, CA, USA; SpectraMax M2). For the quantification of
these extracts, a calibration curve was constructed from the analysis of
different concentrations of gallic acid varying from 0.0008–0.1 mg/mL, and
its data on absorbances based on calibration equation: y = 10.571x
+ 0.0111 (r2 = 0.9958). Results were expressed in terms of milligrams of
gallic acid equivalent (GAE) per g of fresh sample weight.
2.5. Determination of total flavonoids contents
The total flavonoids content was determined according to the
method proposed by Meda, Lamien, Romito, Millogo, and Nacoulma
(2005). Five hundred microliters of extract were transferred to test tube
and 0.5 mL of a 20 mg·mL− 1 methanolic solution of aluminum chloride
(ALCL3) was added. Samples were homogenized on a vortex and left in
the dark for 30 min. The spectrophotometer (Molecular Devices, Sun-
nyvale, CA, USA; SpectraMax M2) was set at the wavelength of 415 nm
and absorbance reading measured. The calibration curve was con-
structed from different concentrations of quercetin varying from
0.0008–0.1 mg/mL, and its data on absorbances based on calibration
equation: y = 21.874x − 0.0047 (r2 = 0.9998). The results were ex-
pressed in terms of milligrams of quercetin per g of propolis.
2.6. Determination of antioxidant activity
2.6.1. DPPH assay
The antioxidant capacity was determined by the DPPH radical (2,2-
diphenyl-1-picrylhydrazyl) scavenging method recommended by Kwon,
Vattem, and Shetty (2006) with slight modifications. A 250 μL aliquot
of extract was mixed with 1.25 mL of DPPH. After 5 min, the absor-
bance was read at 517 nm using a spectrophotometer (Molecular De-
vices, Sunnyvale, CA, USA; SpectraMax M2). The readings were com-
pared with the controls, containing 95% ethanol instead of extract. The
percentage inhibition was calculated by Eq. (1). Different concentra-
tions of Trolox varying from 0 to 0.0014 mmol Trolox/mL were used to
construct the calibration curve based on calibration equation:
y = − 421.1x + 0.7193 (r2 = 0.9958), and expressed the results in
terms of μmol Trolox per g of propolis.
J.K.S. Andrade et al.
% Inhibition =
Abscontrol − Absextract
× 100
Abscontrol (1)
where: Abscontrol = absorbance of the control sample and Ab-
sextract = absorbance of the extract. Result was expressed as percentage
inhibition.
2.6.2. ABTS+ assay
Free radical ABTS+ was captured according to the methodology
proposed by Nenadis, Wang, Tsimidou, and Zhang (2004), with slight
modifications. An aliquot of 30 μL of extract was transferred to test
tubes with 3.0 mL of the ABTS+ radical and homogenized in vortex.
After 6 min, the absorbance value was read in the spectrophotometer
(Molecular Devices, Sunnyvale, CA, USA; SpectraMax M2) at 734 nm.
Different concentrations of Trolox varying from 0 to 0.0018 mmol
Trolox/mL were used to construct the calibration curve based on cali-
bration equation: y = − 247.07x + 0.6706 (r2 = 0.996). Results were
expressed in terms of μmol Trolox per g of propolis.
2.6.3. FRAP assay
The antioxidant activity through the iron reduction method (FRAP)
was determined according to the methodology proposed by Thaipong,
Boonprakob, Crosby, Cisneros-Zevallos, and Hawkins Byrne (2006). For
the analysis, an extract aliquot of 90 μL was transferred into test tubes,
with 270 μL of distilled water, mixed with 2.7 mL of the FRAP reagent,
homogenized on a vortex and kept at 37 °C in a water bath. The ab-
sorbance value was read in a spectrophotometer (Molecular Devices,
Sunnyvale, CA, USA; SpectraMax M2) at 595 nm after 30 min. A cali-
bration curve, ranging from 0 to 0.00064 mmol Trolox/mL, was con-
structed based on calibration equation: y = 1740.5x + 0.1223
(r2 = 0.998). The results were expressed in terms of μmol Trolox per g
of propolis.
2.6.4. ORAC assay
The absorption capacity of the oxygen radical (ORAC) was analyzed
according to the methodology proposed by Albarici (2009), with slight
modifications. 1.50 mL of fluorescein working solution (63 mmol·L− 1)
and 0.75 mL of sample were added, and tubes were maintained at 37 °C
for 15 min. Later, 0.75 mL of AAPH working solution was added. The
experiment was performed in a 96-well microplate and 350 μL of the
sample was added. The antioxidant activity was monitored at every 5 min
by measuring fluorescence for 1000 min in a spectrophotometer (Mole-
cular Devices, USA; SpectraMax M2) using an excitation filter of 485 nm
and emission of 520 nm. Different concentrations of Trolox varying from
5.0–90.0 μmol Trolox/mL were used to construct the calibration curve
based on equation: y = 0.4339x + 0.5079 (r2 = 0.9984). The results
were expressed in terms of μmol Trolox per g of propolis.
2.7. Identification and quantification of phenolic and flavonoid compounds
using UHPLC-QqQ-MS/MS system
Identification and quantification of phenolic and flavonoid com-
pounds present in propolis was performed using an Agilent UHPLC-MS/
MS system of the Triple Quadrupole type (Agilent 6490 Triple Quad LC-
MS/MS), with electrospray ionization in positive mode, except for
caffeic acid phenethyl ester (CAPE) which was in negative mode, in
SRM (Selected Reaction Monitoring) experiments. The column used was
Ascents Express F5 (150 × 2.1 mm, 2.7 μm particle; Sigma Aldrich)
operating at a flow rate of 0.2 ml·min− 1 and a temperature of 40 °C.
The mobile phases used were ultrapure water + 0.1% formic acid
(Phase A) and acetonitrile + 0.1% formic acid (Phase B). The injection
volume was 2 μL. The gradient elution was as follows: 0.0–1.0 min,
15% B; 7.0–9.0 min, 25% B; 13.0–16.0 min, 30%; 17.0 min, 35%;
21.0–23.0 min, 40% B; 25.0 min, 45% B; 28.0 min, 50% B; 30.0 min,
15% B.
The parameters of the mass spectrometer were: gas temperature was
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200 °C, gas flow: 12 L·min− 1, nebulizer: 20 psi, sheat gas temp: 400 °C,
sheat gas flow: 11 L·min− 1, capillary voltage: 3500 V, nozzle: 500 V,
dwell time: 9.8, acceleration cell voltage: 5 V.
The standard calibration curve was prepared using eight data
points, covering the concentration ranges (20–1000 ng·mL− 1). All
analyses were performed in triplicate.
2.8. Statistical analysis
The test results were evaluated by the method of analysis of var-
iance (ANOVA) with comparison of means by Tukey test at 95% con-
fidence level with the help of SAS version 9.0 software. The correlation
between results (Total phenolics, flavonoids, DPPH, ABTS*, FRAP and
ORAC) was determined from the Pearson correlation coefficient, which
quantifies the linear association between two quantitative variables.
This correlation was determined using Statistica 7.0 software.
3. Results and discussion
3.1. Total phenolic and flavonoid contents
Phenolic and flavonoid compounds are the main components re-
sponsible for the functional property of propolis. Table 1 shows sig-
nificant difference by Turkey's test (p < 0.05) for total phenolic and
flavonoids composition among brown, green and red varieties. Red
propolis had 91.3 mg GAE·g− 1 of total phenolics, which was the highest
among all varieties. Regarding flavonoids content, green propolis
showed the highest amount (59.45 mg quercetin·g− 1 of propolis) when
compared with red and brown.
Some studies with Brazilian propolis have reported the phenolic
concentrations ranging from 48 to 87 mg GAE·g− 1 for propolis of states
of Paraná (Cottica et al., 2011) and 49–100 mg GAE·g− 1 for the etha-
nolic extracts of green propolis from the state of São Paulo
(Mello & Hubinger, 2012), although the results vary depending on the
collecting region and species. Bittencourt et al. (2015) reported with
respect to the content of total phenolics the value of 48.24 ± 1.09 mg
GAE·g− 1 of dry weight for ethanolic extract of Brazilian brown propolis
and 185.52 ± 1.09 mg GAE·g− 1 of dry weight for ethanolic extract of
Brazilian green propolis.
Regarding the flavonoid content, the red propolis (31.48 mg of
quercetin·g− 1 of propolis) exhibited a similar result was found by Righi
et al. (2011) for red propolis obtained from Maceio, state of Alagoas, in
northeastern Brazil (32.9 mg of quercetin·g− 1 of propolis). According
to these authors, flavonoid contents of Brazilian red propolis were
found to vary from 27 and 43 mg·g− 1. Due to the geographical origin of
the samples, there is diversity in polyphenol content of propolis, which
affects the biological activity and pharmacological effects among the
species (Falcao et al., 2010; Gómez-Caravaca, Gómez-Romero, Arráez-
Román, Segura-Carretero, & Fernández-Gutiérrez, 2006). Cottica et al.
(2011) obtained values of total flavonoids between 10 and 26 mg
quercetin·g− 1 of Brazilian propolis extract from Paraná. Hatano et al.
(2012), obtained the flavonoid content of 51.9 ± 2.4 mg·g− 1 of
ethanolic extract of Brazilian green propolis from the state of Minas
Gerais. Wang et al. (2016) obtained values of 53.0 ± 0.22 mg
Table 1
Total phenolic compounds and flavonoids contents (mean ± standard deviation; n = 9)
in brown, green and red propolis.
Propolis Total phenolic compounds (mg Total flavonoids content (mg
GAE·g− 1 of propolis) quercetin·g− 1 of propolis)
Brown 55.74 ± 0.48c 30.89 ± 0.20c
Green 90.55 ± 1.52b 59.45 ± 0.82a
Red 91.32 ± 0.49a 31.48 ± 0.56b
⁎
Means followed by same lower-case letters in the same column do not differ statistically
from each other at the 5% significance level (p < 0.05).
J.K.S. Andrade et al.
quercetin·g− 1 of ethanol extract from Brazilian propolis of Olimpia of
states São Paulo.
It is observed that the green and brown Brazilian propolis of the
state of Alagoas and the Brazilian red propolis of the state of Sergipe,
obtained different concentrations of phenolics and flavonoids among
themselves and when compared to the propolis of the varieties (brown,
green and red) from other Brazilian states. This can be explained due to
the fact that the chemical composition of propolis can vary significantly
depending on the different geographic regions, climatic conditions, the
vegetation growing around hives, as well as the time of collection and
these differences can influence the biological activity (López et al.,
2014; Piccinelli et al., 2011; Sawaya et al., 2011).
3.2. Identification and determination of phenolic and flavonoids compounds
by UHPLC-QqQ-MS/MS
Some phenolic compounds cannot be separated with good efficiency
in a single LC-MS system. Tandem mass spectrometric technique is a
powerful tool to provide better information and the characteristic
spectra profile of the target compounds. The SRM mode of UHPLC-QqQ-
MS/MS is a highly sensitive technique used to determine phenolic
compounds based on the monitoring of specified molecular ion and
their respective ion transitions, which avoid interference of overlapping
LC peaks (Sherwood et al., 2009; Wu et al., 2016).
For quantification of target compounds, a calibration curve was
prepared by analyzing at different concentrations (20–1000 ng/mL).
Regression analysis produced r2 values above 0.99 and maximum de-
tection limit (MDL) ranged from 0.02 to 1.12 ng·mL− 1 (Table 2).
In order to obtain maximum sensitivity for identification of all
compounds, two SRM transitions were optimized for each compound.
The first transitions were used for quantification, and the second ones
for confirmatory purposes. The data on precursor and product ions,
with associated collision energy for fragmentation and the retention
times are presented in Table 2. The target compounds were identified in
accordance with the SRM chromatogram of the two selected transitions,
and their corresponding retention times. The chromatographic separa-
tion of the 28 target compounds was optimized in order to get the best
resolution, efficiency and selectivity, particularly for enantiomeric se-
paration of isomers, in case of (+)-catechin and epicatechin, which
exhibit the same molar mass and the same patterns of fragmentation,
consequently resulting in identical product ions. After testing several
conventional reverse phase stationary phases, the Ascentis Express F5
column provided a proper resolution of these isomeric catechins.
The quantification of individual compounds was calculated using
the peak areas of the identified compounds relative to the peak areas of
the corresponding analytical standard. The data on contents of flavo-
noids and phenolic acids found in propolis samples are presented in
Table 3. The chromatographic profile of the three propolis species is
show in Fig. 1.
The results indicate that the 3 propolis species (brown, green and red)
had similar bioactive profile composition, but these were quantitatively
different. The flavonoids artepellin C, kaempferide, kaempferol, pinocem-
brim, and the phenolic acids such as p-coumaric, chrologenic and caffeic
acids were the most abundant compounds found in brown and green pro-
polis. However, kaempferide was the majoritary compound (6.04 mg·g− 1)
in green propolis followed by p-coumaric acid (5.34 mg·g− 1), artepellin C
(4.80 mg·g− 1), chrologenic acid (2.81 mg·g− 1), kaempferol (1.48 mg·g− 1)
and caffeic acid (1.06 mg·g− 1). Brown propolis contained higher quantity
(3.72 mg·g− 1) of artepellin C followed by kaempferide (3.48 mg·g− 1), p-
coumaric acid (2.64 mg·g− 1), chrologenic acid (1.76 mg·g− 1) and pino-
cembrim (1.66 mg·g− 1). Artepellin C was identified in all 3 propolis species
analyzed in this work. As expected, artepellin C concentration was higher in
green propolis, due to its characteristic composition in this specie (Hata
et al., 2012; Ikeda et al., 2011). Major compounds found in red propolis
were luteolin (1.75 mg·g− 1), naringenin (0.96 mg·g− 1), kaempferol
(0.59 mg·g− 1), pinocembrin (0.41 mg·g− 1) and biochanin A (0.39 mg·g− 1).
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These compounds presented the higher concentration in red propolis sam-
ples, when compared with the other species.
Acacetin, artepellin C, eriodictyol, gallic acid, isorhamnetin, pro-
tocatechuic acid, vanillin and vanillic acid were identified in red pro-
polis analyzed in this work. Fig. 2 illustrates the SRM chromatograms of
the quantification transitions of these phenolic compounds in red pro-
polis extract. The presence of these compounds in Brazilian red propolis
is reported for the first time in this work.
The distinct chemical composition of red propolis plays an im-
portant role in the treatment of human diseases, such as dental caries,
candidiasis, cancer, skin wound and oxidative stress-related conditions
(Freires, de Alencar, & Rosalen, 2016). Biochanin A and daidzein, which
are well known biomarkers of Brazilian red propolis, were also found in
the samples. Daidzein has significant antiinflammatory potential, and
previous studies demonstrated that administration of this compound
significantly reduces the number of neutrophils, release antiin-
flammatory cytokines, and other benefits as well (Franchin et al.,
2017). Additionally, the presence of a 5-hydroxil group in the chemical
structure of biochanin A appears to enhance inhibitory activity of some
enzymatic systems, including testosterone 5α-reductase, which con-
verts testosterone to dihydrotestosterone, as reported by Bae et al.
(2012). The inhibition of this enzyme represents an important phar-
macological approach against androgen dependent diseases, such
prostate hyperplasia.
In general, the results obtained in this study exhibit more quantities of
polyphenolic compounds in ethanolic extracts from green propolis, when
compared with the red and brown extracts. These results are in agreement
with total phenolic and flavonoid results, which demonstrate that green
propolis exhibited the highest amount of these class of compounds. In an
earlier study reported by (Barbaric et al., 2011), the chemical composition
of ethanolic extracts from propolis was found to have lower levels of the
phenolic compounds viz. ferulic acid (0.0358 to 0.3022 mg·g− 1 of pro-
polis), p-coumaric acid (0.00441 to 0.1562 mg·g− 1 of propolis), pino-
cembrin (0.2487 to 7.6651 mg·g− 1 of propolis), apigenin (0.0019 to
0.0189 mg·g− 1 of propolis) and kaempferol (0.0697 to 0.2931 mg·g− 1 of
propolis). When determining the phenolic compounds in propolis from
China, Sun et al. (2015) found low quantities of p-coumaric acid
(1.40 mg·g− 1), cinnamic acid (0.10 mg·g− 1), kaempferol (0.59 mg·g− 1)
and quercetin (0.10 mg·g− 1). Vargas-Sánchez et al. (2014), in a previous
study with Mexican propolis, found similar results for kaempferol
(1.45 mg·g− 1) when comparing with the green propolis of the present
work. In the same study, pinocembrin (1.65 mg·g− 1) and luteolin
(0.87 mg·g− 1) also exhibited similar results for brown and red propolis,
respectively.
Daidzein, formononetine and biochanin A were identified by Silva
et al. (2015) in red propolis from Brazilian northeast region. These
compounds are biomarkers of chemical composition for red propolis
species (Franchi et al., 2012; López et al., 2014; Piccinelli et al., 2011).
Frozza et al. (2017) found low quantities of biochanin A (0.27 mg·g− 1)
in Brazilian red propolis from the state of Alagoas. Other phenolic
compounds, such as isoliquiritigenina, pinocembrina and quercetin
were also identified in red propolis samples (Daugsch et al., 2008; de
Mendonça et al., 2015).
Among the several target polyphenolic compounds found in the
different propolis species (brown, green and red) from Brazil, artepillin
C, kaempferide, p-coumaric acid, luteolin, chrologenic acid, kaemp-
ferol, caffeic acid and pinocembrin were in majority. These compounds
have been reported in the literature as beneficial for human health.
Some authors have attributed antibacterial, antitumoral and antiin-
flammatory activities for artepillin C (Hata et al., 2012; Ikeda et al.,
2011; Paulino et al., 2008) and p-coumaric acid (Punithavathi, Prince,
Kumar, & Selvakumari, 2011), including other therapeutic properties.
Chrologenic acid has demonstrated good antioxidant activity in in vitro
studies (Farooqui & Farooqui, 2012), preventing the DNA oxidation
resulting in the prevention of many chronical diseases. Caffeic acid,
luteolin and kaempferol have been studied by many authors (Bufalo
J.K.S. Andrade et al. Food Research International xxx (xxxx) xxx–xxx

Table 2
Analytical MS/MS parameters retention time, linearity parameters, correlation coefficients and detection limits for phenolic and flavonoid compounds.

Compound Molecular ESI polarity Precursor ion Fragments Collision Retention Calibration equationa Correlation MDL
formula m/z energy (eV) time (min) coefficient (r2) (ng·mL− 1)

Acacetin C16H12O5 + 285 242b 25 24.2 y = 6587 + 69212x 0.998 0.022

270
Apigenin C15H10O5 + 271 153b 25 17.7 y = 11116 + 2146x − 0.4294x2 0.999 0.077
119
Artepellin C C19H24O3 + 301 245b 15 29.3 y = 12979 + 1680x + 0.7351x 2
0.995 0.017
189
Caffeic acid C9H8O4 + 181 135b 25 4.4 y = 433.1 − 558.4 0.998 0.400
163 15
Biochanin A C16H12O5 + 285 253b 25 24.5 y = 8429.9 + 5661.8x − 1.45x2 0.997 0.130
152
Caffeic acid phenethyl C17H16O4 − 283 161b 25 23.3 y = 121.8 − 565.1x 0.999 0.150
ester (CAPE) 179 15
Chrysin C15H10O4 + 255 153b 25 25.6 y = 3297 + 29158x 0.998 1.29
209
(+)-Catechin C15H14O6 + 291 139b 15 3.1 y = 1460 − 2538x 0.998 0.116
123
Epicatechin C15H14O6 + 291 139b 15 3.6 y = 1965 − 28235x 0.997 2.39
123
Daidzein C15H10O4 + 255 199b 25 11.8 y = 164591 + 8489x − 2,96x 2
0.997 0.229
137
Ethyl gallate C9H10O5 + 199 127b 15 5.6 y = 976.8 − 6209x 0.998 0.103
153
Eriodictyol C15H12O6 + 289 153b 30 13.1 y = 2707 − 15399x 0.998 1.61
145 25
Chrologenic acid C16H18O9 + 355 163b 15 3.16 y = 10531 − 57618x 0.996 0.149
145 25
Gallic acid C7H6O5 + 171 109b 15 2.17 y = 171 − 2135x 0.995 16.5
125
Cinnamic acid C9H8O2 + 149 103b 25 12.5 y = 1139 − 10095x 0.998 0.749
131 15
α-Cyano-4- C10H7O3N + 190 172b 15 8.48 y = 242 + 1725x 0.997 9.33
hydroxycinnamic 144
acid
Isorhamnetin C16H12O7 + 317 302b 25 18.6 y = − 6352 + 1254x + 0.18x2 0.995 2.03
229 35
Luteolin C15H10O6 + 287 153b 35 15.0 y = 2600 + 50848x 0.998 1.51
135 35
Kaempferide C16H12O6 + 301 258b 35 24.3 y = 1296 − 6160x 0.997 0.013
286 25
Kaempferol C15H10O6 + 287 153b 35 17.6 y = 1193 − 6547x 0.997 1.80
121 25
Narigenin C15H12O5 + 273 153b 35 16.0 y = 3146 − 7237x 0.998 0.040
119
p-Coumaric acid C9H8O3 + 165 147b 15 6.51 y = 971.3 − 6661x 0.997 1.12
91 35
Protocatechuic acid C7H6O4 + 155 65b 15 2.80 y = 414.6 − 2985x 0.997 0.270
137 35
Pinocembrin C15H12O4 + 257 153b 15 23.1 y = − 68899 + 4477x 0.998 0.070
131 25
Ferulic acid C10H10O4 + 195 177b 15 7.47 y = 2811 − 28884x 0.997 0.192
134 20
Quercetin-3-glucoside C21H20O12 + 465 303b 15 7.24 y = 1541 − 7238x 0.998 0.926
85 35
Rutin C27H30O16 + 611 303b 35 6.46 y = 840.7 − 3036x 0.997 0.066
465 15
Vanillin C8H8O3 + 153 93b 15 6.51 y = 9927 − 72786x 0.998 0.054
65 35
Vanillic acid C8H8O4 + 169 93b 15 4.57 y = 20015 − 3631x + 0.4320x2 0.996 0.221
65 35

MDL - Method Detection Limits.

a
Y is the value of the peak area. X is the concentration of the standard compound.
b
Quantitation transition.

et al., 2013; Chen et al., 2008; Filomeni et al., 2012) against different
cancer cell lines and these compounds, inhibit significantly the pro-
liferation of tumoral cells.
The main characteristic of chemical composition of propolis from
tropical locals, particularly in Brazil, is the presence of non-typical
flavonoids, such kaempferide, and prenylated phenylpropanoids
(Falcao et al., 2010; Gómez-Caravaca et al., 2006). These observations
are in agreement with our findings, which exhibited that kaempferide
was the major compound found in green and brown propolis species.
The phenolic contents in propolis may vary depending on the origin of

5
J.K.S. Andrade et al. Food Research International xxx (xxxx) xxx–xxx

Table 3 Bărnuţiu (2011) observed lower DPPH values ranging from

Phenolic and flavonoid compounds contents (mg·g− 1) in brown, green and red propolis. 0.29 ± 0.10 to 1.24 ± 0.01 mmol Trolox·g− 1 propolis of Transylva-
nian propolis extracts. Kalogeropoulos, Konteles, Troullidou,
Compounds Content (mg·g− 1 of propolis)
Mourtzinos, and Karathanos (2009) found DPPH values for propolis
Brown Green Red from Greece ranging from 0.33 to 1.11 mmol Trolox·g− 1 propolis.
Vargas-Sánchez et al. (2014), when analyzing raw propolis from
Acacetin 0.08 ± 0.011 0.11 ± 0.01 0.08 ± 0.00
Mexico, found a percentage inhibition of the DPPH radical of 64.8%,
α-Cyano-4-hydroxycinnamic acid < LQ < LQ < LQ
Apigenin 0.03 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 which is lower than found in this work.
Artepellin C 3.72 ± 0.00 4.80 ± 0.03 0.01 ± 0.00 The results of ORAC exhibited higher antioxidant responses for
Biochanin A 0.01 ± 0.00 0.01 ± 0.00 0.39 ± 0.02 green and red propolis (6734 and 6665 μmol Trolox·g− 1 propolis, re-
Caffeic acid 0.59 ± 0.06 1.06 ± 0.04 < LQ spectively) when compared with brown propolis (5343 μmol
Caffeic acid phenethyl ester 0.09 ± 0.01 0.29 ± 0.02 < LQ
Trolox·g− 1 propolis). However, no significant difference (p < 0.05)
(CAPE)
± Catechin 0.01 ± 0.00 0.01 ± 0.00 < LQ was observed by Tukey test. The results of ORAC tests on propolis, and
Chlorogenic acid 1.76 ± 0.03 2.81 ± 0.01 0.01 ± 0.00 the antioxidant responses may vary due the differences in species and
Chrysin 0.14 ± 0.04 0.07 ± 0.01 < LQ their geographic localization (Hatano et al., 2012; Silva et al., 2011;
Cinnamic acid 0.10 ± 0.01 0.10 ± 0.00 < LQ
Sun et al., 2015).
Daidzein 0.001 ± 0.00 < LQ 0.12 ± 0.00
Epicatechin 0.01 ± 0.00 0.01 ± 0.00 < LQ
Eriodictyol 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 3.4. Pearson correlation coefficients
Gallic acid 0.04 ± 0.00 0.04 ± 0.00 0.01 ± 0.00
Isorhamnetin 0.40 ± 0.03 0.42 ± 0.01 0.01 ± 0.00 The mechanism used to measure the antioxidant capacity is dif-
Kaempferide 3.48 ± 0.03 6.04 ± 0.08 < LQ
ferent in each method, which may cause slight differences between the
Kaempferol 0.61 ± 0.19 1.48 ± 0.00 0.59 ± 0.00
Luteolin 0.04 ± 0.02 0.01 ± 0.00 1.75 ± 0.01 results. Thus, all methods were compared using the Pearson correlation
Narigenin 0.15 ± 0.03 0.25 ± 0.03 0.96 ± 0.03 coefficient (r) between the variables (Total Phenolic contents,
p-Coumaric acid 2.64 ± 0.22 5.34 ± 0.28 0.01 ± 0.00 Flavonoids, DPPH, ABTS*, FRAP and ORAC), which quantifies the
Pinocembrin 1.66 ± 0.15 0.93 ± 0.08 0.41 ± 0.02
linear association between two quantitative variables. This correlation
Protocatechuic acid 0.18 ± 0.01 0.11 ± 0.00 0.05 ± 0.00
Quercetin-3-glucoside 0.24 ± 0.06 0.45 ± 0.06 < LQ
data are presented in Table 5.
Rutin 0.13 ± 0.02 0.16 ± 0.02 0.05 ± 0.00 A very strong positive correlation was observed between total
Trans-isoferulic acid 0.06 ± 0.00 0.15 ± 0.02 < LQ phenolics and the FRAP and ORAC methods (r = 0.9888 and
Vanillin 0.04 ± 0.01 0.02 ± 0.00 0.01 ± 0.00 r = 0.9980, respectively), which suggests the relation of the phenolic
Vanillic acid 0.02 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
compounds with the antioxidant activity. As for the DPPH method,
⁎
LQ: limit of quantification. there was a strong positive correlation (r = 0.9492) with the FRAP
method and a very strong and positive correlation between the DPPH
and ABTS* methods (r = 0.9744). The antioxidant activity evaluated
by the ABTS method showed a strong positive correlation (r = 0.8542)
the samples, and its differences may affect the biological activity and with the FRAP assay. The ORAC method presented a very strong cor-
pharmacological effects. relation (r = 0.9774) with the iron reduction potential (FRAP).
The correlation observed between the parameters shows statistically
that the flavonoids are not responsible for the antioxidant activity, since
3.3. Antioxidant activity the Pearson correlation coefficient was 0.0526 and − 0.1730 for the
DPPH and ABTS* assays, respectively and of 0.3643 and 0.5529 for the
The Table 4 presents the results of DPPH, ABTS+, FRAP and ORAC FRAP and ORAC assays, respectively. This fact suggests that the anti-
activities of different species of propolis. All the extracts possessed oxidant activity of brown, green and red Brazilian propolis is related to
potent and diverse antioxidant activity with significant variations other phenolic compounds, and not directly related to the class of fla-
(p < 0.05). Red propolis exhibited the higher antioxidant potential in vonoids.
DPPH, ABTS+ and FRAP methods, followed by green and brown spe- According to Cottica et al. (2011), a negative correlation (− 0.9082)
cies. was observed between DPPH and total flavonoids content in a Brazilian
The antioxidant activity of three propolis when compared with the propolis collected in Maringá in the state of Paraná. In addition, the
results of propolis reported in other studies, show wide difference in authors also showed that the FRAP value may not be related to the
their antioxidant potential. FRAP antioxidant assay exhibited the values flavonoid content, since the correlation coefficient was only 0.4915.
ranging from 471 to 633 mmol Trolox·g− 1propolis. Turan et al. (2015) This fact corroborated with the results presented in this study. Similar
reported FRAP value of 311.0 ± 2.5 mg Trolox·g− 1 in extracts of results were also reported by Can, Yildiz, Şahin, Asadov, and Kolayli
propolis cultivated in Turkey, which was lower than the result herein (2015), showing a very strong and positive correlation (0.98) between
obtained. the FRAP values and the total phenolic compounds content in the
With regard to DPPH assay, inhibition percentage varied from 86 to propolis samples, which suggests that the total antioxidant activity of
90.7%. Red propolis exhibited the higher inhibition (90.72%) of DPPH the propolis is due to their phenolic compounds.
radical, followed by the green (88.53%) and brown (86.06%) propolis.
Skaba et al. (2013) found low values of DPPH (1230.07 ± 135.55 4. Conclusions
μmol Trolox·g− 1) and ABTS%+ (1223.06 ± 137.40 μmol Trolox·g− 1)
in ethanolic extract of Brazilian green propolis from the state of Minas Ethanolic extracts of Brazilian propolis from different species
Gerais, Brazil. In other study, Bonvehí and Gutiérrez (2011) obtained (brown, green and red) presented high flavonoid and phenolic com-
values of ABTS%+ between 560 and 1430 μmol Trolox·g− 1 in ethanolic pounds contents. In addition, the results obtained by DPPH, ABTS,
extract of propolis from Spain. Mihai, Mărghitaş, Dezmirean, and FRAP and ORAC assays revealed high antioxidant activity, indicating

6
J.K.S. Andrade et al.
Fig. 1. Total ion chromatogram (TIC) of ethanolic extracts of Brazilian propolis (brown, green and red) in the SRM mode. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
that propolis is a promising source of biologically active polyphenols.
The UHPLC-QqQ-MS/MS system provided good selectivity and effi-
ciency, in the determination of 28 phenolic compounds with good
precision and accuracy. Comparing the 3 different propolis species
analyzed, brown and green propolis displayed higher amounts of
bioactive compounds than the red species, which reveals the differences
in the chemical composition of the different species.
7
Food Research International xxx (xxxx) xxx–xxx
The results on total phenolic compounds and flavonoids in all 3 propolis
species (brown, green and red) obtained from the Alagoas and Sergipe
states of Brazilian northeastern region were correlated with their anti-
oxidant activities (DPPH, ABTS, FRAP and ORAC). A very strong positive
correlation between total phenolics and the FRAP and ORAC methods was
established which led to conclusion that antioxidant activity of propolis is
mainly due to its phenolic components composition.
J.K.S. Andrade et al. Food Research International xxx (xxxx) xxx–xxx

Fig. 2. SRM chromatograms of ethanolic extract of red propolis according to quantification transitions of acacetin, artepellin C, gallic acid, eriodictyol, isorhamnetin, protocatechuic acid,
vanillin and vanillic acid.

Table 4
Antioxidant activity (mean ± standard deviation; n = 9) of brown, green and red propolis.

Propolis Antioxidant activity (μmol Trolox·g− 1 propolis)

DPPH ABTS+ FRAP ORAC

Brown 4431.00 ± 45.11bB 1868.45 ± 131.39cC 471.51 ± 37.79bD 5343.84 ± 114.94bA

Green 4554.35 ± 80.20aB 2214.96 ± 20.61bC 604.20 ± 20.72aD 6734.87 ± 124.56aA
Red 4663.80 ± 68.01aB 2913.55 ± 95.26aC 633.18 ± 40.20aD 6665.75 ± 58.78aA

⁎
Means followed by same lower-case letters in the same column do not differ statistically from each other at the 5% significance level (p < 0.05).
⁎⁎
Means followed by same capital letters in the same row do not differ statistically from each other at the 5% significance level (p < 0.05).

Abbreviations
Table 5
Pearson correlation coefficients (r) of total phenolic compounds, flavonoids and anti-
oxidant activity (DPPH, ABTS*, FRAP and ORAC).
ABTS%+ 2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)
ABTS radical cation scavenging activity
Parameters Total Flavonoids DPPH ABTS⁎ FRAP ORAC AAPH 2,2-Azobis (2-Amidino-Propane) dichloride
phenolic DPPH radical scavenging activity
Total phenolic 1 0.4994 0.8914 0.7669 0.9888⁎ 0.9980⁎⁎
DPPH% 2,2-diphenyl-1-picryhydrazyl radical
Flavonoids 1 0.0526 −0.1730 0.3643 0.5529 FRAP ferric reducing antioxidant power
DPPH 1 0.9744⁎ 0.9492 0.8612 GAE gallic acid equivalent
ABTS 1 0.8542⁎ 0.7250 Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
FRAP 1 0.9774⁎
ALCL3 methanolic solution of aluminum chloride
ORAC 1
ORAC oxygen radical absorbance capacity
⁎
Significant at 5% probability (p < 0.05).
⁎⁎
Significant at 1% probability (p < 0.01).

8
J.K.S. Andrade et al.
Acknowledgment
Special thanks are also extended to Apicola Almar ltda, Maceio,
Alagoas, Brazil for supply of the green and brown propolis and to Dr.
Juliana Cordeiro Cardoso of the University Tiradentes, Aracaju, Brazil
for supplying the red propolis samples.
Funding sources
All authors gratefully acknowledge financial support received from
CNPq, Brazil vide research project ‘Instituto Nacional de Ciência e
Tecnologia de Frutos Tropicais’ (Project 465335/2014-4) in developing
this work. Authors (Julianna Andrade, Marina Denadai) acknowledge
and thank CAPES (Ministry of Education, Brazil) for their fellowships.
Notes
The authors declare that there is no competing financial interest.
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