Journal of Apicultural Research

ISSN: 0021-8839 (Print) 2078-6913 (Online) Journal homepage: http://www.tandfonline.com/loi/tjar20

Phytochemical screening, antioxidant and

antibacterial activities of some commercial extract
of propolis

Ticiano Gomes do Nascimento, Adriana dos Santos Silva, Patrícia Beltrão

Lessa Constant, Sâmia Andrícia Souza da Silva, Maria Aline Barros Fidelis
de Moura, Clinston Paulino de Almeida, Valdemir da Costa Silva, Amanda
Barbosa Wanderley, Irinaldo Diniz Basílio Júnior & Pierre Barnabé Escodro

To cite this article: Ticiano Gomes do Nascimento, Adriana dos Santos Silva, Patrícia Beltrão
Lessa Constant, Sâmia Andrícia Souza da Silva, Maria Aline Barros Fidelis de Moura, Clinston
Paulino de Almeida, Valdemir da Costa Silva, Amanda Barbosa Wanderley, Irinaldo Diniz
Basílio Júnior & Pierre Barnabé Escodro (2018): Phytochemical screening, antioxidant and
antibacterial activities of some commercial extract of propolis, Journal of Apicultural Research, DOI:
10.1080/00218839.2017.1412563

To link to this article: https://doi.org/10.1080/00218839.2017.1412563

Published online: 13 Feb 2018.

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Journal of Apicultural Research, 2018
https://doi.org/10.1080/00218839.2017.1412563

ORIGINAL RESEARCH ARTICLE

Phytochemical screening, antioxidant and antibacterial activities of some commercial
extract of propolis
Ticiano Gomes do Nascimentoa* , Adriana dos Santos Silvab , Patrı́cia Beltrão Lessa Constantb , Sâmia Andrı́cia
Souza da Silvaa , Maria Aline Barros Fidelis de Mouraa , Clinston Paulino de Almeidaa , Valdemir da Costa
Silvaa , Amanda Barbosa Wanderleya , Irinaldo Diniz Bası́lio Júniora and Pierre Barnabé Escodroc
a
PostGraduate Program of Pharmaceutical Science, School of Nursing and Pharmacy and PostGraduate Program of Nutrition, College of
Nutrition of the Federal University of Alagoas, Maceió-Alagoas, Brazil; bPostGraduate Program of Food Science and Technology, Department of
Food Science and Technology, Federal University of Sergipe, São Cristóvão-Sergipe, Brazil; cPostGraduate Program of Veterinary Science,
Viçosa-Alagoas, Brazil
(Received 11 November 2016; accepted 11 September 2017)

This study investigated the chemical composition, flavonoids, phenolic compounds, antioxidant activity and antibacterial
activity of commercial propolis extracts produced in the Sergipe and Alagoas States of Brazil as potential bioproducts for
the food and pharmaceutical industries. Four samples were analyzed, three brown propolis extracts and one red propolis
extract, and were characterized through phytochemical screening, chemical, chromatographic profile and antibacterial
activity. Phytochemical analysis detected the presence of triterpenoids and phenolic compound in propolis extracts. Pro-
polis extracts showed total phenolic content between 9 and 15% and total flavonoids >2%. Propolis extracts showed
excellent antioxidant activity with inhibition of the Free radical DPPH˙ between 97 and 60%, which confirm the results
obtained in total phenolics, total flavonoids content and antibacterial activity. The chromatographic profile showed differ-
ences for brown propolis samples and quite different from the red propolis extract, which present flavonoids as isoflavo-
noids, pterocarpans, chalcones and guttiferones. Commercial propolis extract (propolis extract C and propolis extract D)
showed excellent activity for Staphylococcus aureus ATCC 25923 and moderate activity for Pseudomonas aeruginosa ATCC
27853. The chemical characterization of propolis extracts is fundamental in the process of standardization and monitoring
of the chemical composition susceptible to geographic and seasonal variation. These results point to new possibilities of
use as bio-preservative of processed foods as well as in the development of pharmaceuticals and nutraceuticals products
from propolis extract C and D in its formulation actuate on the inhibition of some pathogenic microorganisms strains.

Examen de las actividades fitoquı́mica, antioxidante y antibacteriana de extractos comerciales de propó-

leos

Este estudio investigó la composición quı́mica, los flavonoides, los compuestos fenólicos y las actividades antioxidante y
antibacteriana de extractos comerciales de propóleos producidos en los Estados de Sergipe y Alagoas de Brasil como
bioproductos potenciales para las industrias alimentaria y farmacéutica. Se analizaron cuatro muestras, tres extractos
de propóleos marrón y uno de propóleos rojo, y se caracterizaron con un cribado fitoquı́mico, quı́micamente, por su
perfil cromatográfico y su actividad antibacteriana. El análisis fitoquı́mico detectó la presencia de triterpenoides y com-
puestos fenólicos en los extractos de propóleos. Los extractos de propóleos mostraron un contenido fenólico total de
entre 9% y 15% y un contenido total de flavonoides > 2%. Mostraron una actividad antioxidante excelente con una
inhibición de radicales libres DPPH˙ de entre el 97% y el 60&, lo que confirma los resultados obtenidos sobre los con-
tenidos totales de fenoles y flavonoides y la actividad antibacteriana. El perfil cromatográfico mostró diferencias en las
muestras de propóleos marrones y bastante diferencia con respecto al extracto de propóleos rojo, que presenta tanto
flavonoides como isoflavonoides, pterocarpanos, chalconas y guttiferones. Los extractos comerciales de propóleos (ex-
tractos de propóleos C y D) mostraron una excelente actividad hacia Staphylococcus aureus ATCC 25923 y una activi-
dad moderada hacia Pseudomonas aeruginosa ATCC 27853. La caracterización quı́mica de los extractos de propóleos es
fundamental en el proceso de estandarización y monitorización de la composición quı́mica susceptible a la variación
geográfica y estacional. Estos resultados apuntan a nuevas posibilidades de uso como biopreservativos de alimentos
procesados, ası́ como en el desarrollo de productos farmacéuticos y nutracéuticos de los extractos de propóleos C y
D en su formulación actúan sobre la inhibición de algunas cepas de microorganismos patógenos.
Keywords: Propolis extracts; phytochemical screening; total flavonoid content; total phenolic content; chromato-
graphic profile; antioxidant activity; antibacterial activity

Introduction were the pioneers in the art of beekeeping (Couto &

Since ancient times the honey bee (Apis mellifera) has been Couto, 2006). The main products from bees are honey,
valued for its products; it is assumed that the Egyptians royal jelly, beeswax, pollen and propolis. Propolis is a pro-
duct made by bees from the collection of plants resins

*Corresponding author. Email: ticianogn@yahoo.com.br

© 2018 International Bee Research Association

2 T.G. do Nascimento et al.
and wax which are biotransformed by the bee using sali-
vary secretion resulting in material which is produced for
a defensive function in the hive. Egyptians and Romans
extensively used propolis as anti-putrefactive, cicatrizing
agent and antiseptic properties (Sforcin & Bankova, 2011).
The origin of the resin can determine the quality of
propolis, its biological activity and its medicinal use
(Couto & Couto, 2006). Its color varies from yellow, red,
green, brown and black, according to origin (Daugsch,
Moraes, Fort, & Park, 2008). The variety of botanical
composition which the bees seek the resinous material
for the preparation of propolis, in many cases are plants
considered medicinal, then propolis usually has in its com-
position, bioactive compounds of high biological value,
which are phenolic compounds, flavonoids, xanthones,
propolones, guttiferones and terpenes (Almutairi et al.,
2014; de Mendonça et al., 2015; Do Nascimento et al.,
2016; Kardar et al., 2014; Raghukumar, Vali, Watson,
Fearnley, & Seidel, 2010; Siheri et al., 2014). According to
several researchers (Bankova, 2005; Lu, Chen, & Chou,
2005; Seidel, Peyfoon, Watson, & Fearnley, 2008; Sforcin,
Fernandes, Lopes, Bankova, & Funari, 2000), propolis raw
material and its products can suffer variations on the bio-
logical activity and consequently on the quality due to cli-
mate, seasonal, geographical area, production processes
in hive and extract processing.
In Brazil, several types of propolis have different
chemical compositions, according to the geographical
region in which they are located (Daugsch et al., 2008;
Silva et al., 2008; Trusheva et al., 2006). Brazilian propolis
is classified into several groups due to the great diversity
of flora and is grouped into 13 different groups, according
to the chemical composition and biological activities (Cas-
tro, Cury, & Rosalen, 2007). The range of biological activi-
ties of propolis is higher in tropical areas of the world,
reflecting the plant diversity of these regions (Bankova,
2005) and consequent flora around the hive. Several stud-
ies of propolis have been developed, in particular, related
to its antioxidant, anti-inflammatory, cancer cytotoxicity
and antimicrobial capacity (Bueno-Silva et al., 2013; de
Mendonça et al., 2015; Do Nascimento et al., 2016; Dota,
Consolaro, Svidzinski, & Bruschi, 2011; Jacob, Parolia,
Pau, & Davamani Amalraj, 2015; Nina et al., 2016). Some
investigation (Cabral et al., 2009; Fernandes-Júnior, Lopes,
Colombari, Monteiro, & Vieira, 2006; Portilho, Melo,
Guerra, Batista, & Fernandes, 2013) evaluated the antibac-
terial activity of propolis from different regions of Brazil
and the world achieving very promising results (Inui et al.,
2014; Kalogeropoulos, Konteles, Troullidou, Mourtzinos,
& Karathanos, 2009; López, Schmidt, Eberlin, & Sawaya,
2014; Lu et al., 2005; Santos et al., 2002; Seidel et al.,
2008). Propolis extract has been used as preservatives of
foods products as juices, meat, sausage, fish, seafood and
other (Ali, Kaseem, & Atta-Alla, 2010; Casquete, Castro,
Jácome, & Teixeira, 2016). Casquete et al. (2016) report
the antibacterial activity of propolis against Listeria innocua
as a bio-preservative of traditional sausage during 62 days
of storage at 4 ˚C and thus can be used as natural bio-
preservative of meats and sausages. Vargas-Sanchez et al.
(2014) find a decrease in Listeria monocytogenes population
during 15 days of storage at 2 ˚C. Fatma, Kassem, and
Atta-Alla (2010) has demonstrated the application of pro-
polis extract as antioxidant and preservative of processed
meat products.
This study investigated the chemical composition, fla-
vonoids, phenolic compounds, antioxidant activity and
antibacterial activity of commercial propolis extracts
commercialized at the Sergipe and Alagoas States from
Brazil as potential bioproduct for the food and
pharmaceutical industries.
Materials and methods
The analytical standards biochanin A, chrysin, caffeic
acid, chlorogenic acid, p-coumaric acid, gallic acid,
daidzein, isoliquiritigenin, quercetin, pinobanksin, for-
mononetin, pinocembrin were purchased from
Sigma-adrich (St. Louis, MO, USA) and liquiritigenin was
purchased from extrasynthese (Lyon Nord, France).
Three brown propolis samples were obtained commer-
cially in drugstores and nutraceutical food stores from
the Provinces of Sergipe-Brazil (Sample A), Ceará-Brazil
(Sample B) and Rio de Janeiro-Brazil (Sample C), respec-
tively. A red propolis extract sample from Alagoas
(Sample D) was analyzed under the same conditions and
was taken as our internal control due to great antibac-
terial activity described in the literature. The soluble
solid content and phytochemical screening assays were
determined using the methodology (Matos, 1997). The
chromatographic profile was performed using the
methodology developed in the laboratory for red
propolis extract. The total flavonoids and phenolic com-
pounds follow-up methodology described (Bankova
et al., 2016; Singleton & Rossi, 1965; Woisky & Salatino,
1998). The antioxidant activity was evaluated according
to the methodology described (Alves & Kubota, 2013).
Determination of solid soluble content
A volume of 2 ml of extract was added to the aluminum
plate of an infrared drying balance from Shimadzu
(Tokyo, Japan) programmed in automatic mode to warm
up to a temperature of 120 ˚C until a constant weight
of the sample. The results were determined in triplicate
and expressed as a percentage of soluble solid content
and follow the expression (% solid soluble content =
[(initial mass of the commercial propolis extract − final
mass of the commercial propolis extract)/initial mass of
the commercial propolis extract] × 100).
Phytochemical screening
The phytochemical screening tests were performed
with propolis extract at concentrations from 2.000–
0.250 mg/ml. The methodology used was described in
(Matos, 1997). The samples were assayed using specific
 Antioxidant and antibacterial activity of commercial propolis extract
tests for steroid/triterpenoid, anthocyanin, anthocyani-
din, flavones flavonols, pyrogallic tannin, phebaphenic
tannins, chalcones, flavonols and aurones, leucoantho-
cyanidins, catechins, flavanones, flavanonones, and xan-
thones.
Chromatographic profile
The chromatographic profile of the commercial propolis
extracts were performed using a liquid chromatography
with an ultra performance coupled to diode array detec-
tor (UPLC-DAD) from Shimadzu (Tokyo, Japan). The
UPLC-DAD was composed of the following modules:
high-pressure pump (LC-20ADXR model) degasser
(DGU-20A3R model), Auto-injector (SIL-20AgXR
model) chromatographic oven column, diode array
detector (EPDM-20A model) and fluorescence detector
(RF-20A model), a controller (model CBM-20A) and
Lab solution from Shimadzu software.
The separation of flavonoids and other constituents
occurred using a Phenomenex (Torrance, CA, USA)
reverse phase column (C18, 150 × 4.6 mm; 5 μm), a
mobile phase consisting of solvent A (Milli-Q water) and
solvent B (acetonitrile), pumped at a flowrate of 0.3 ml/
min. The initial gradient of elution consisted of water
(70%) and acetonitrile (30%) (V/V). The column was
eluted with a variation (B) percentage as follows: 0–2 min
30% of B, 2–5 min 36% of B, 5–8 min 46% of B, 8–11 min
52% of B, 11–14 min 52% of B, 14–17 min 57% of B, 17–
20 min 62% of B, 20–24 min 62% of B, 24–28 min 68% of
B, 28–32 min 72% of B, 32–36 min 90% of B, 36–42 min
97% of B, 42–50 min 100% of B, 50–55 min 100% of B,
55–57 min the acetonitrile was reduced to 30% and this
condition was maintained up to 60 min. A theoretical
concentration of 480 μg/ml was prepared, filtered
through a 0.22 μm filter and 2 μl of the sample was
injected into UPLC-DAD (Do Nascimento et al., 2016).
Total flavonoid content
The total flavonoid content was determined using the
spectrophotometric method of reaction with aluminum
chloride (prepared at a concentration of 5% AlCl3) in
methanol. Brown propolis and red propolis extracts
were diluted to concentrations corresponding to 100,
150, 200, 250 and 300 μg/ml in the amber flask (5 ml)
were added 100 μl of 5% AlCl3. The reaction was per-
formed in the dark for 30 min. The absorbances were
determined at 425 nm. The total flavonoid content was
determined using a calibration curve of quercetin in
concentrations seven points. Y = 0.046X − 0.018 where
Y is the absorbance and X is the concentration;
R2 = 0.993). The total flavonoid content was expressed
as equivalents of quercetin in mg per gram of propolis
extract using the dry matter content.
3
Total phenolic content
Folin-Ciocalteu method was used for total phenolic
determination. The absorbance was measured at
760 nm and compared with the standard curve of gallic
acid in seven points of the calibration curve
(Y = 0.153X − 0.086 where Y is the absorbance and X is
the concentration; R2 = 0.995). The total phenolics con-
tent was expressed in mg equivalent of gallic acid per
gram of propolis extract considering the dry matter
content.
Antioxidant activity
The propolis extract was an assessment by the DPPH˙
Free radical scavenging capacity (2,2-diphenyl-1 picrylhy-
drazyl). Propolis extracts were diluted to obtain concen-
trations of 100, 50, 25, 10, 5, 2.5 and 1 μg/ml and 2.0 ml
of DPPH 1 mM solution previously prepared was added
to a 5 ml amber flask, completed the volume with abso-
lute ethanol and then after 30 min, the absorbance was
measured at 518 nm. Absorbance values at 518 nm were
converted into the antioxidant activity percentage (AOA
%) using method previously described (Choi et al., 2002).
Antibacterial activity
The tests for evaluation of antimicrobial activity were
performed according to standard NCCLS (Performance
Standards for Antimicrobial Disk Susceptibility Tests,
2003), with some adjustments. We used the disk
diffusion method using Staphylococcus aureus ATCC
25923 and Pseudomonas aeruginosa ATCC 27853 strains.
The antibiotic Ciprofloxacin (5 μg) and Ampicillin
(10 μg) were used as positive control of P. aeruginosa
and S. aureus respectively. A second disk without the
antibiotic (containing the methanol solvent) was used as
a negative control. In the remaining four disks different
concentrations were used (1000, 690, 490, 250 and
140 μg/ml), and the sample D a differential concentra-
tion (690, 490, 250 and 140 μg/ml). After plating, the
plates were inverted and incubated at 35 ˚C for 24 and
48 h and their inhibition halos measured after this time.
Results
Solid soluble content, total flavonoid content, and
total phenolic content
Commercial propolis extracts showed differences in the
values of solid soluble mass in ethanol. The commercial
extract of propolis A showed a percentage of 6.22%
± 0.61, while the commercial samples of propolis B, C
and D was observed values of 4.32%, ± 0.55, 13.69%
± 0.39 and 12.57% ± 0.65, respectively.
Table 1 shows the percentages of total flavonoids
and total phenolic content found at commercial samples
of propolis extracts. The values showed that the sample
C (propolis from Rio de Janeiro-Brazil) was expressed
4 T.G. do Nascimento et al.

Table 1. Total flavonoids and phenolic compounds of the commercial propolis extracts.

Sample % Total flavonoids (m/m) % Total phenolic compounds (m/m)

a–b, a–c*
A 3.642 ± 0.231 9.388 ± 0.877a–b**
B 6.530 ± 0.399b–c, b–d* 10.268 ± 0.801b–d**
C 7.054 ± 0.538c–d* 9.745 ± 0.428c–d**
D 2.830 ± 0.410a–d* 12.220 ± 0.124a–d**
Notes: The results are mean ± standard deviation of replicates (n = 3). Instruction 03/2001 – Brazilian Agricultural Agency: Low flavonoid content
lower than 1.0% (m/m); Intermediate flavonoid content between 1.0 and 2.0% (m/m) and High flavonoid content >2.0% (m/m) of flavonoid.
*All results were different among themselves (p < 0.05) using Tukey´s test.
**Different from the samples (A), sample (B) and sample (D) (p < 0.05) using Tukey´s test.

Table 2. Antioxidant activity (%) of commercial propolis extracts using DPPH assay.

% DPPH Scavenging capacity

Sample 100 μg/ml 50 μg/ml 25 μg/ml 10 μg/ml
A 96.8 ± 0.010 92.3 ± 0.086 81.2 ± 0.013 70.4 ± 0.402
B 97.2 ± 0.042 95.7 ± 0.087 92.6 ± 0.172 78.0 ± 0.116
C 97.4 ± 0.009 97.1 ± 0.013 95.7 ± 0.026 93.3 ± 0.050
D 97.0 ± 0.010 92.3 ± 0.020 86.6 ± 0.076 79.0 ± 0.165
Notes: The results are mean ± standard deviation of replicates (n = 3). No different from the samples (A), sample (B), sample (C) and sample (D)
(p < 0.05) using Tukey´s test.

as a higher percentage of flavonoids and the sample A
(Sergipe-Brazil) the lowest percentage of the brown
propolis samples.
The red propolis extract showed values of 2.83%.
Significance differences were observed among all sam-
ples studied. However, all samples presented values
>2.0% (mass/mass) and are classified in the group of
high flavonoid concentration according to specific legis-
lation from Agricultural Agency (BRASIL, 2001). The
total phenolic compounds of propolis extracts showed
values between 9.388 and 12.220%. The samples A and
C did not present significance differences between
themselves but sample D presented significance differ-
ences with all brown propolis extract with p-value
<0.05. Therefore, besides flavonoids, other phenolic
compounds are also present in propolis including pheno-
lic lipids studied as anacardic acid and guttiferones that
are present in brown propolis and red propolis,
respectively.
Antioxidant activity
The DPPH radical scavenging activity using commercial
propolis extracts are shown in Table 2. Phenolic com-
pounds present in propolis extracts showed activity
effective in sequestering the DPPH radical, with similar
percentages around 97%. There is no significance differ-
ence among propolis extract using Anova one-way
Tukey´s test with p value < 0.05. The antioxidant activ-
ity showed values similar to those found by (De Melo,
Matsuda, Freitas, Barth, & Almeida-Muradian, 2014). The
researcher studied the antioxidant capacity of propolis
from different regions of the Brazil and average values
of 88% for the Southeast, 89% for propolis of Rio de
Janeiro and 75.7% for the Northeast propolis. Some
papers correlate a probable existence of phenolic com-
pounds present in the ethanolic solution, with the posi-
tive action of antioxidant activity (Silva, Moreira, Melo,
& Lima, 2012). Antioxidant activity of propolis is attribu-
ted to flavonoid components, among which we can
mention quercetin, daidzein, genistein and apigenin
(Kamiya, Nishihara, Hara, & Adachi, 2012). Quercetin
and daidzein were detected in the Brazilian red propolis
of Alagoas (Franchi et al., 2012). Other studies have also
detected the antioxidant activity of the flavonoid
isoliquiritigenin (Oldoni et al., 2011; Ondrias, Stasko,
Hromadová, Suchy, & Nagy, 1997) and pinobanksin
(Blanco-Ayala et al., 2013) and kaempferol and CAPE
(phenethyl caffeate) (Kumazawa, Hamasaka, &
Nakayama, 2004) in propolis samples.
Phytochemical screening and chromatographic
analysis
Commercial extracts of brown propolis (A)–(C) were
similar in phytochemical screening for the presence of
flavones, flavonols and xanthones, and triterpenoids.
The commercial extract of propolis of Alagoas (D)
showed the presence of phlobaphenes tannins, flavones,
flavonols and xanthones and chalcones and aurones,
beside catechins and triterpenoids (de Mendonça et al.,
2015). A high percentage of antioxidant activity demon-
strated by free radical sequestration method DPPH and
high percentage concentration of total phenolics and
total flavonoids corroborate the qualitative results of
phytochemical screening that detected the presence of
several phenolic compounds during the course of this
phytochemical test.
The chromatographic profile of commercial extracts
of brown propolis showed similar between themselves
and totally different from the chromatographic profile of
the commercial extract of red propolis (sample D). The
 Antioxidant and antibacterial activity of commercial propolis extract 5

Figure 1. Chromatographic profile of brown propolis extract at the wavelength of 230 nm and red propolis extract at the wave-
length of 280 nm commercialized on the market of Alagoas and Sergipe (Brazil). (A) Propolis Extract from Sergipe-Brazil, (B) Propo-
lis Extract from Ceará-Brazil, (C) Propolis Extract from Rio de Janeiro-Brazil and (D) Propolis Extract from Alagoas-Brazil.
Notes: Samples (A), (B) and (C) were identified as chromatographic peaks: (a) p-coumaric acid (RT 7.98 UV λ 227, 293 nm); (b) RT
8.91 UV λ 227, 293 nm; (c) RT 10.01 UV λ 227, 288 nm; (d) RT 15.72 UV λ 230, 291 nm; (e) RT 21.03 UV λ 233, 292 nm; (f) RT
22.02 UV λ 228, 290 nm; (g) RT 29.2 UV λ 254, 312 nm; (h) RT 40.0 UV λ 254 nm; (i) RT 42.95 UV λ 256 nm; (j) RT 47.65 UV λ
254 nm and (k) RT 52.95 UV λ 251 nm. Sample (D) were identified the flavonoids: daidzein (1); liquiritigenin (2); pinobanskin (RT
15.78 UV λ 208, 295 nm); quercetin (3); isoliquiritigenin (4); formononetin (5); pinocembrin (6) and biochanin A (7) in UPLC-DAD
profile at the wavelength of 280 nm for Sample D (red propolis extract).

maximum wavelength in UV-PDA detector for the sam-
ples A–C was at 230 nm and eleven (11) main chromato-
graphic peaks were detected (Figure 1). The specific
compounds from brown propolis extract were identified
using main techniques: (1) Injection of the analytical stan-
dards in comparison with the retention time and, (2)
purity peak correlation using UV-PDA spectra library
and, (3) scientific literature search. The chromatographic
peaks showed a UV profile similar to the polyphenols
like cinnamic acids (p-coumaric acid, caffeic acid, ferulic
acids; UV 230, 290 nm), protocateuchuic acids (vanillic
acid, p-hydroxybenzoic acids, syringic acid UV 260,
290 nm), caffeoylquinic acid (chlorogenic acid, cryp-
tochlorogenic acid, neochlorogenic acid), dicaffeoylquinic
acid (cynarine UV 295 and 325 nm) and glyconic querce-
tin derivatives (peltatoside UV 243, 326 nm) (Antolovich,
Prenzler, Robards, & Ryan, 2000; Maróstica Junior et al.,
2008; Mejı́a-Giraldo, Winkler, Gallardo, Sánchez-Zapata,
& Puertas-Mejı́a, 2016). Different phenolic acids and
cinnamates and flavonoids were identified in green
propolis from Minas Gerais and São Paulo as: artepillin
C, dupranin, Baccarin, and, p-coumaric acid, caffeic acid,
ferulic acid, gallic acid and, 3-prenylcinamate allyl, cin-
namyl caffeate, cinnamyl coumarate, dihydrocinnamyl fer-
ulate and, pinobaksin, pinocembrin, quercetin, kampferol,
chrysin and kampferide (Chang, Piló-Veloso, Morais, &
Nascimento, 2008; Hata et al., 2012; Salomão et al.,
2008).
The red propolis (sample D) presented maximum
wavelength in UV-PDA detector at 280 nm (Figure 1).
The identification of different phytochemical classes was
identified in red propolis extract by the technique of
injection of the analytical standard and comparison of
the retention time and purity peak correlation using
UV-PDA spectra library. The presence of polyisopreny-
lated guttiferones (Guttiferone E, xanthochymol, guttif-
erone B), terpenes (alpha-amyrin and beta-amyrin)
pterocarpan (medicarpin) isoflavan (vestitol and
neovestitol), isoflavones (formononetin and biochanin
A), chalcone (isoliquiritigenin), flavonone (naringenin),
dihydro flavonol (pinobanksin), flavonols (kaempferol),
phenolic acids (ferulic acid) flavan (catechin) were identi-
fied in the red propolis extracts as cited in scientific lit-
erature (Bueno-Silva et al., 2013; Daugsch et al., 2008;
6 T.G. do Nascimento et al.

Table 3. Antibacterial activity of propolis extract using disk diffusion technique for Staphylococcus aureus ATCC 25923 and
Pseudomonas aeruginosa ATCC 27853.

Inhibition halo (mm)

Samples Concentration (μg/disk) Staphylococcus aureus Pseudomonas aeruginosa
Propolis extract A 1000 25 10
690 22 8
490 22 <8
250 18 <8
Propolis extract B 1000 19 8
690 19 8
490 17 <8
250 16 <8
Propolis extract C 1000 19 12
690 19 10
490 18 11
250 18 11
Propolis extract D 690 19 12
490 19 11
250 17 10
140 16 11
Note: The results are mean ± standard deviation of replicates (n = 3).

(A) (B)

7A 2B
2A
7B 3B
3A
8A 4B
8B
9A 4A
1A 1B
9B
5B
10A 5A 10B
6B
6A

(C) (D)

2C 3C
2D 3D
7D
7C
4C
4D
8D
8C

1C 5C 1D
9D 5D
9C
6C 6D
10C 10D

Figure 2. Antibacterial activity of commercial extract of propolis using disk-diffusion method. Photos A and B inhibition halos
against Staphylococcus aureus ATCC 25923. Photo (A) 1A ampicillin antibiotic 10 μg. 2A: negative control. Propolis extract A: 3A
(1000 μg/ml); 4A (690 μg/ml); 5A (490 μg/ml); 6A (250 μg/ml). Propolis extract D: 7A (690 μg/ml); 8A (490 μg/ml); 9A (250 μg/ml);
10A (140 μg/ml). Photo (B) 1B ampicillin antibiotic 10 μg. 2B: negative control. Propolis extract B: 3B (1000 μg/ml); 4B (690 μg/ml);
5B (490 μg/ml); 6B (250 μg/ml). Propolis extract C: 7A (1000 μg/ml); 8A (690 μg/ml); 9A (490 μg/ml); 10A (250 μg/ml). Photos C
and D inhibition halos against Pseudomonas aeruginosa ATCC 27853. Photo (C) 1C ciprofloxacin antibiotic 5 μg. 2C: negative con-
trol. Propolis extract A: 3C (1000 μg/ml); 4C (690 μg/ml); 5C (490 μg/ml); 6C (250 μg/ml). Propolis extract D: 7C (690 μg/ml); 8C
(490 μg/ml); 9C (250 μg/ml); 10C (140 μg/ml). Photo (D) 1D ampicillin antibiotic 10 μg. 2D: negative control. Propolis extract B:
3D (1000 μg/ml); 4D (690 μg/ml); 5D (490 μg/ml); 6D (250 μg/ml). Propolis extract C: 7D (1000 μg/ml); 8D (690 μg/ml); 9D
(490 μg/ml); 10D (250 μg/ml).
 Antioxidant and antibacterial activity of commercial propolis extract 7

Inui et al., 2014; de Mendonça et al., 2015; Righi et al.,
2011; Silva et al., 2008; Trusheva et al., 2006).
Antibacterial activity
Quantitative Analysis of the inhibition halos of the
microorganism S. aureus shows the greatest diameter
(19 mm) at concentrations above 1000 μg/ml and
16 mm in the lower concentration (250 μg/ml) for
brown propolis extracts. Red propolis extract presented
the same antibacterial activity in relation brown propolis
extract. The inhibition halos were lower than 10 mm
for microorganism P. aeruginosa proven resistant to
brown propolis A and B.
In qualitative analysis, It was possible to observe the
growth of some bacteria colonies inside the halos in
brown propolis mainly in low concentrations but it was
not observed into red propolis. The results show a
greater sensitivity to Gram-positive microorganisms
(S. aureus ATCC 25923) in comparison with Gram-nega-
tive microorganism tested (P. aeruginosa ATCC 27853).
These results corroborate with found in scientific investi-
gations (Cabral et al., 2009; Fernandes-Júnior et al., 2006;
Portilho et al., 2013), these authors tested extracts of
propolis from different regions of the country, in Gram-
positive and Gram-negative microorganisms, including S.
aureus and P. aeruginosa, noting a higher sensitivity of
Gram-positive microorganisms to extracts (Seidel et al.,
2008; Sforcin et al., 2000) (Figure 2).
The commercial propolis extracts with higher per-
centage of solid soluble mass in ethanol [Propolis
extract C (13.69% ± 0.389) and propolis extract D
(12.57% ± 0.65)] and higher percentage of total phenolic
compounds (see Table 1) also showed a slightly higher
antimicrobial activity against gram-negative bacteria
P. aeruginosa ATCC 27853 (see Table 3), observing
inhibition halos between 12–10 mm. However, the pro-
polis extracts A and propolis extract B presented a
lower percentage of solid soluble mass in ethanol and a
lower percentage of total phenolic compounds and thus
showed lower halos of inhibition (<8 mm). Other com-
pounds different from flavonoids can actuate in the inhi-
bition of gram-positive and gram-negative bacteria
strains (Cushnie & Lamb, 2005; da Silva, de Souza,
Matta, de Andrade, & Vidal, 2006). da Silva et al. (2006)
observed a strong correlation between the antibacterial
activity and total phenolic compounds content but a
weaker correlation for total flavonoids levels.
Discussion
Commercial propolis extracts showed a phytochemical
profile for triterpenoids and phenolic compounds mainly
phenolic acids and flavonoids with high antioxidant activity.
The chromatographic profile showed small differences for
brown propolis samples being brown propolis extract C
with higher levels of phenolic compounds, total flavonoids
and quite different chromatographic profile of the red pro-
polis extract because of the unique characteristics of phe-
nolic compounds and flavonoids such as isoflavonoids,
pterocarpans, chalcones and guttiferones present in your
composition. Commercial propolis extract (propolis
extract C and propolis extract D) showed excellent activ-
ity for S. aureus ATCC 25923 and moderate activity for P.
aeruginosa ATCC 27853. The chemical characterization of
propolis extracts is fundamental in the process of standard-
ization and monitoring of the chemical composition sus-
ceptible to geographic and seasonal variation. These results
point to new possibilities of use as bio-preservative of pro-
cessed foods as well as in the development of pharmaceuti-
cals and nutraceuticals products from propolis extract C
and D in its formulation actuate on the inhibition of some
pathogenic microorganisms strains.
Funding
This work was supported by the CNPq [grant number
446630/2014-4]; Edital Universal/MCT/CNPq [number 014/
2014].
ORCID
Ticiano Gomes do Nascimento http://orcid.org/0000-0002-
3856-8764
Adriana dos Santos Silva http://orcid.org/0000-0002-8613-
8320
Patrı´cia Beltrão Lessa Constant http://orcid.org/0000-0001-
7095-940X
Sâmia Andrı´cia Souza da Silva http://orcid.org/0000-0002-
9878-1719
Maria Aline Barros Fidelis de Moura http://orcid.org/0000-
0002-8068-8946
Clinston Paulino de Almeida http://orcid.org/0000-0002-9519-
309X
Valdemir da Costa Silva http://orcid.org/0000-0002-2069-
2812
Amanda Barbosa Wanderley http://orcid.org/0000-0002-6362-
9397
Irinaldo Diniz Bası´lio Júnior http://orcid.org/0000-0001-9526-
0646
Pierre Barnabé Escodro http://orcid.org/0000-0002-9409-
660X
References
Ali, F.H., Kaseem, G., & Atta-Alla, O.A. (2010). Propolis as a
natural decontaminant and antioxidant in fresh oriental
sausage. Veterinaria Italiana, 46, 167–172.
Almutairi, S., Edrada-Ebel, R.-A., Fearnley, J., Igoli, J.O., Alotaibi,
W., Clements, C.J., … Watson, D.G. (2014). Isolation of
diterpenes and flavonoids from a new type of propolis
from Saudi Arabia. Phytochemistry Letters, 10, 160–163.
doi:10.1016/j.phytol.2014.08.022
Alves, E., & Kubota, E.H. (2013). Conteúdo de fenólicos, flavo-
noides totais e atividade antioxidante de amostras de pró-
polis comerciais. Revista de Ciências Farmacêuticas Básica e
Aplicada, 34(1), 37–41.
Antolovich, M., Prenzler, P., Robards, K., & Ryan, D. (2000).
Sample preparation in the determination of phenolic com-
8 T.G. do Nascimento et al.
pounds in fruits. The Analyst, 125, 989–1009. doi:10.1039/
000080i
Bankova, V. (2005). Chemical diversity of propolis and the
problem of standardization. Journal of Ethnopharmacology,
100(1–2), 114–117. doi:10.1016/j.jep.2005.05.004
Bankova, V., Bertelli, D., Borba, R., Conti, B.J., da Silva Cunha,
I.B., Danert, C., … Zampini, C. (2016). Standard methods
for Apis mellifera propolis research. In V. Dietemann, J.D.
Ellis, & P. Neumann (Eds.), The COLOSS BEEBOOK, Volume
III: Standard methods for Apis mellifera hive product research.
Journal of Apicultural Research. doi:10.1080/
00218839.2016.1222661
Blanco-Ayala, T., Lugo-Huitrón, R., Serrano-López, E.L., Reyes-
Chilpa, R., Rangel-López, E., Pineda, B., … Rorres-Ramos,
M. (2013). Antioxidant properties of xanthones from Calo-
phyllum brasiliense: Prevention of oxidative damage induced
by FeSO4. BMC Complementary Alternative Medicine, 13,
1287. doi:10.1186/1472-6882-13-262
BRASIL. (2001). Instrução Normativa nº 3, de 19 de janeiro de
2001. Aprova os Regulamentos Técnicos de Identidade e
Qualidade de Apitoxina, Cera de Abelha, Geléia Real,
Geléia Real Liofilizada, Pólen Apı́cola, Própolis e Extrato
de Própolis.
Bueno-Silva, B., Alencar, S.M., Koo, H., Ikegaki, M., Silva, G.V.J.,
Napimoga, M.H., & Rosalen, P.L. (2013). Anti-inflammatory
and antimicrobial evaluation of neovestitol and vestitol
Isolated from Brazilian red propolis. Journal of Agricultural
and Food Chemistry, 61(19), 4546–4550. doi: 10.1021/
jf305468f
Cabral, I.S.R., Oldoni, T.L.C., Prado, A., Bezerra, R.M.N., Alen-
car, S.M., Ikegaki, M., & Rosalen, P.L. (2009). Composição
fenólica, atividade antibacteriana e antioxidante da própolis
vermelha brasileira. Quı´mica Nova, 32(6), 1523–1527.
doi:10.1590/S0100-40422009000600031
Castro, M.L., Cury, J.A., & Rosalen, P.L. (2007). Própolis do
sudeste e nordeste do Brasil: Influência da sazonalidade na
atividade antibacteriana e composição fenólica. Quı´mica
Nova, 30(7), 1512–1516.
Chang, R., Piló-Veloso, D., Morais, S.A.L., & Nascimento, E.A.
(2008). Analysis of a Brazilian green propolis from Baccha-
ris dracunculifolia by HPLC-APCI-MS and GC-MS. Revista
Brasileira de Farmacognosia, 18(4), 549–556. doi:10.1590/
S0102-695X2008000400009
Choi, C.W., Kim, S.C., Hwang, S.S., Choi, B.K., Ahn, H.J., Lee,
M.Y., … Kim, S.K. (2002). Antioxidant activity and free rad-
ical scavenging capacity between Korean medicinal plants
and flavonoids by assay guided comparison. Plant Science,
163, 1161–1168. doi:10.1016/S0168-9452(02)00332-1
Couto, R.H.N., & Couto, L.A. (2006). Apicultura: Manejos e pro-
dutos (3rd ed.). Jaboticabal: FUNEP.
Cushnie, T.P.T., & Lamb, A.J. (2005). Antimicrobial activity of
flavonoids. International Journal of Antimicrobial Agents, 26(5),
343–356. doi:10.1016/j.ijantimicag.2005.09.002
da Silva, J.F.M., de Souza, M.C., Matta, S.R., de Andrade, M.R., &
Vidal, F.V.N. (2006). Correlation analysis between phenolic
levels of Brazilian propolis extracts and their antimicrobial
and antioxidant activities. Food Chemistry, 99(3), 431–435.
Daugsch, A., Moraes, C.S., Fort, P., & Park, Y.K. (2008).
Brazilian red propolis—Chemical composition and botani-
cal origin. Evidence-Based Complementary and Alternative
Medicine, 5(4), 435–441. doi:10.1093/ecam/nem057
De Melo, A.A.M., Matsuda, A.H., Freitas, A.S., Barth, O.M.,
Almeida-Muradian, L.B. (2014). Capacidade antioxidante da
própolis. Pesquisa Agropecuária Tropical, 44(3), 341–348.
doi:10.1590/S1983-40632014000300004
de Mendonça, I.C., Porto, I.C.C.M., do Nascimento, T.G., de
Souza, N.S., Oliveira, J.M.S., Arruda, R.E.S., … Barreto, F.S.
(2015). Brazilian red propolis: Phytochemical screening,
antioxidant activity, and effect against cancer cells. BMC
Complementary Alternative Medicine, 15, 467. doi:10.1186/
s12906-015-0888-9
Do Nascimento, T.G., Silva, P.F., Azevedo, L.F., Da Rocha,
L.G., Porto, I.C.C.M., Lima e Moura, T.F.A., … Watson,
D.G. (2016). Polymeric nanoparticles of Brazilian red
propolis extract: Preparation, characterization, antioxidant
and Leishmanicidal activity. Nanoscale Research Letters, 11
(301), 1–16. doi:10.1186/s11671-016-1517-3
Dota, K.F., Consolaro, M.E., Svidzinski, T.I., & Bruschi, M.L.
(2011). Antifungal activity of Brazilian propolis microparti-
cles against yeasts isolated from vulvovaginal candidiasis.
Evidenced-Based Complementary Alternative Medicine, 2011,
201953. doi:10.1093/ecam/neq029
Fatma, H.A., Kassem, G.M., & Atta-Alla, O.A. (2010). Propolis
as a natural decontaminant and antioxidant in fresh orien-
tal sausage. Journal Veterinaria Italiana, 46, 167–172.
Fernandes-Júnior, A., Lopes, M.M.R., Colombari, V., Monteiro,
A.C.M., & Vieira, E.P. (2006). Atividade antimicrobiana de
própolis de Apis melifera obtida três regiões do Brasil. Ciên-
cia Rural, 36(1), 294–297. doi:10.1590/S0103-84782006000
100047
Franchi, Jr., G.C., Moraes, C.S., Toreti, V.C., Daugsch, A.,
Nowill, A.E., & Park, Y.K. (2012). Comparison of effects of
the ethanolic extracts of Brazilian propolis on human
leukemic cells as assessed with the MTT assay. Evidenced-
Based Complementary Alternative Medicine, 2012(6), 918–
956. doi:10.1155/2012/918956
Hata, T., Tazawa, S., Ohta, S., Rhyu, M.-R., Misaka, T., &
Ichihara, K. (2012). Artepillin C, a major ingredient of
Brazilian propolis, induces a pungent taste by activating
TRPA1 channels. PLoS ONE, 7(11), e48072. doi:10.1371/
journal.pone.0048072
Inui, S., Hatano, A., Yoshino, M., Hosoya, T., Shimamura, Y.,
Masuda, S., … Kumazawa, S. (2014). Identification of the phe-
nolic compounds contributing to antibacterial activity in etha-
nol extracts of Brazilian red propolis. Natural Product Research,
28(16), 1293–1296. doi:10.1080/14786419.2014.898146
Jacob, A., Parolia, A., Pau, A., & Davamani Amalraj, F. (2015).
The effects of Malaysian propolis and Brazilian red propolis
on connective tissue fibroblasts in the wound healing pro-
cess. BMC Complementary Alternative Medicine, 15, 363.
doi:10.1186/s12906-015-0814-1
Kalogeropoulos, N., Konteles, S.J., Troullidou, E., Mourtzinos,
I., & Karathanos, V.T. (2009). Chemical composition,
antioxidant activity and antimicrobial properties of propolis
extracts from Greece and Cyprus. Food Chemistry, 116(2),
452–461. doi:10.1016/j.foodchem.2009.02.060
Kamiya, T., Nishihara, H., Hara, H., & Adachi, T. (2012). Etha-
nol extract of Brazilian red propolis induces apoptosis in
human breast cancer MCF-7 cells through endoplasmic
reticulum stress. Journal of Agricultural and Food Chemistry,
60, 11065–11070. doi:10.1155/2014/639856
Kardar, M.N., Zhang, T., Coxon, G.D., Watson, D.G., Fearnley,
J., & Seidel, V. (2014). Characterization of triterpenes and
new phenolic lipids in Cameroonian propolis. Phytochem-
istry, 106, 156–163. doi:10.1016/j.phytochem.2014.07.016
Kumazawa, S., Hamasaka, T., & Nakayama, T. (2004). Antioxi-
dant activity of propolis of various geographic origins. Food
Chemistry, 84(3), 329–339. doi:10.1016/S0308-8146(03)
00216-4
López, B.G.-C., Schmidt, E.M., Eberlin, M.N., & Sawaya,
A.C.H.F. (2014). Phytochemical markers of different types
of red propolis. Food Chemistry, 146(1), 174–180.
doi:10.1016/j.foodchem.2013.09.063
Lu, L.-C., Chen, Y.-W., & Chou, C.-C. (2005). Antibacterial
activity of propolis against Staphylococcus aureus. Interna-
tional Journal of Food Microbiology, 102(2), 213–220.
doi:10.1016/j.ijfoodmicro.2004.12.017
 Antioxidant and antibacterial activity of commercial propolis extract
Maróstica Junior, M.R.M., Daugsch, A., Moraes, C.S., Queiroga,
C.L., Pastore, G.M., & Parki, Y.K. (2008). Comparison of
volatile compounds and polyphenolic compounds in
Brazilian green propolis and its botanical origin Baccharis
dracunculifolia. Ciência e Tecnologia de Alimentos, 28(1), 178–
181. doi:10.1590/S0101-20612008000100026
Matos, F.J.A. (1997). Introduction to experimental phytochemistry
(2nd ed.). Fortaleza: UFC.
Mejı́a-Giraldo, J.C., Winkler, R., Gallardo, C., Sánchez-Zapata,
A.M., & Puertas-Mejı́a, M.A. (2016). Photoprotective
potential of Baccharis antioquensis (Asteraceae) as natural
sunscreen. Photochemistry and Photobiology, 92, 742–752.
NCCLS. (2003). Performance standards for antimicrobial disk sus-
ceptibility tests; approved standard–Eighth edition (NCCLS doc-
ument M2-A8. Wayne, PA: NCCLS. [ISBN 1-56238-485-6].
Nina, N., Lima, B., Feresin, G.E., Giménez, A., Salamanca Capu-
siri, E., & Schmeda-Hirschmann, G. (2016). Antibacterial
and leishmanicidal activity of Bolivian propolis. Letters in
Applied Microbiology, 62(3), 290–296. doi:10.1111/lam.12543
Oldoni, T.L.C., Cabral, I. S. R., d’Arce, M.A.B.R., Rosalen, P.L.,
Ikegaki, M., Nascimento, A.M., & Alencar, S.M. (2011). Iso-
lation and analysis of bioactive isoflavonoids and chalcone
from a new type of Brazilian propolis. Separation and Purifi-
cation Technology, 77(2), 208–213. doi:10.1016/j.sep-
pur.2010.12.007
Ondrias, K., Stasko, A., Hromadová, M., Suchy, V., & Nagy, M.
(1997). Pinobanksin inhibits peroxidation of low density
lipoprotein and it has electron donor properties reducing
alpha-tocopherol radicals. Die Pharmazie, 52, 566–567.
Portilho, D.R., Melo, I.A., Guerra, R.C., Batista, H.L., & Fernan-
des, C.H.C. (2013). Avaliação da Atividade antibacteriana e
antifúngica da própolis produzida na no estado de tocan-
tins. Revista Cientı´fica do ITPAC, 6(2), 1–8.
Raghukumar, R., Vali, L., Watson, D., Fearnley, J., & Seidel, V.
(2010). Antimethicillin-resistant Staphylococcus aureus
(MRSA) activity of ‘pacific propolis’ and isolated prenylfla-
vanones. Phytotherapy Research, 24(8), 1181–1187.
doi:10.1002/ptr.3096
Righi, A.A., Alves, T.R., Negri, G., Marques, L.M., Breyer, H., &
Salatino, A. (2011). Brazilian red propolis: Unreported sub-
stances, antioxidant and antimicrobial activities. Journal of
the Science of Food and Agriculture, 91(13), 2363–2370.
doi:10.1002/jsfa.4468
Casquete, R., Castro, S.M., Jácome, S., & Teixeira, P. (2016).
Antimicrobial activity of ethanolic extract of propolis in
“Alheira”, a fermented meat sausage. Cogent Food & Agricul-
ture, 2, 1125774. doi:10.1080/23311932.2015.1125774
Salomão, K., Pereira, P.R.S., Campos, L.C., Borba, C.M.,
Cabello, P.H., Marcucci, M.C., & de Castro, S.L. (2008).
Brazilian propolis: Correlation between chemical composi-
9
tion and antimicrobial activity. Evidence-Based Complemen-
tary and Alternative Medicine, 5(3), 317–324. doi:10.1093/
ecam/nem058
Santos, F.A., Bastos, E.M.A., Uzeda, M., Carvalho, M.A.R.,
Farias, L.M., Moreira, E.S.A., & Braga, F.C. (2002). Antibac-
terial activity of Brazilian propolis and fractions against oral
anaerobic bacteria. Journal of Ethnopharmacology, 80(1),
1–7. doi:10.1016/S0378-8741(02)00003-X
Seidel, V., Peyfoon, E., Watson, D.G., & Fearnley, J. (2008).
Comparative study of the antibacterial activity of propolis
from different geographical and climatic zones. Phytotherapy
Research, 22(9), 1256–1263. doi:10.1002/ptr.2480
Sforcin, J.M., & Bankova, V. (2011). Propolis: Is there a potential
for the development of new drugs? Journal of Ethnopharma-
cology, 133(2), 253–260. doi:10.1016/j.jep.2010.10.032
Sforcin, J.M., Fernandes, Jr., A., Lopes, C.A.M., Bankova, V., &
Funari, S.R.C. (2000). Seasonal effect on Brazilian propolis
antibacterial activity. Journal of Ethnopharmacology, 73(1–2),
243–249. doi:10.1016/S0378-8741(00)00320-2
Siheri, W., Igoli, J.O., Gray, A.I., Nasciemento, T.G., Zhang, T.,
Fearnley, J., … Watson, D.G. (2014). the isolation of
antiprotozoal compounds from Libyan propolis. Phytother-
apy Research, 28(12), 1756–1760. doi:10.1002/ptr.5194
Silva, B.B., Rosalen, P.L., Cury, J.A., Ikegaki, M., Souza, V.C.,
Esteves, A., & Alencar, S.M. (2008). Chemical composition
and botanical origin of red propolis, a new type of
Brazilian propolis. Evidence-Based Complementary and Alter-
native Medicine, 5(3), 313–316. doi:10.1093/ecam/nem059
Silva, Q.J., Moreira, A.C.C.G., Melo, E.A., Lima, V.L.A.G.
(2012). Compostos fenólicos e atividade antioxidante de
genótipos de ciriguelas (Spondia purpurea L.). Alimentos e
Nutrição, 21(1), 73–80.
Singleton, V.L., & Rossi, J.A. (1965). Colorimetry of total pheno-
lics with phosphomolybdic-phosphotungstic acid reagents.
American Journal of Enology and Viticulture, 16, 144–168.
Trusheva, B., Popova, M., Bankova, V., Simova, S., Marcucci,
M.C., Miorin, P.L., … Tsvetkova, I. (2006). Bioactive
constituents of brazilian red propolis. Evidence-Based
Complementary and Alternative Medicine, 3(2), 249–254.
doi:10.1093/ecam/nel006
Vargas-Sanchez, R., Javier-Saiz, E., Torrescano-Urrutia, G.,
Acedo-Felix, E., Carvajal-Millan, E., Gonzalez-Cordova, A.,
& Sanchez-Escalante, A. (2014). Antioxidant and antimicro-
bial properties of commercial propolis in beef patties. Jour-
nal of Food science, 79, C1409–C1504. doi:10.1111/1750-
3841.12533
Woisky, R.G., & Salatino, A. (1998). Analysis of propolis: Some
parameters and procedures for chemical quality control.
Journal of Apicultural Research, 37(2), 99–105. doi:10.1080/
00218839.1998.11100961