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REVISTA DE BIOLOGIA E CIÊNCIAS DA TERRA ISSN 1519-5228
Volume 16 - Número 2 - 2º Semestre 2016
ATIVIDADE ANTIOXIDANTE E COMPOSTOS FENÓLICOS DE PRÓPOLIS
VERMELHA DO ESTADO DE SERGIPE, BRASIL.
Yzila Liziane Farias Maia de Araújo1
; Lucyana Santos de Mendonça1
; Nayjara Carvalho Gualberto2
;
Sara Cuadros Orellana3
; Juliana Cordeiro Cardoso4
; Sona Jain5
; Edilson Divino de Araújo1
; Narendra Narain2
RESUMO
A própolis é uma resina natural extraída das plantas e modificada pelas abelhas. Este estudo teve
como objetivo avaliar a atividade antioxidante da própolis vermelha brasileira por DPPH,
peroxidação lipídica e métodos de quimiluminescência, juntamente com a análise de seu conteúdo
fenólico por calorimetria e cromatografia líquida ultrarrápida. Todas as amostras de própolis
analisadas neste estudo mostraram atividade antioxidante por DPPH ∙ teste que foi confirmado por
peroxidação lipídica e quimiluminescência métodos mais sensíveis. A peroxidação lipídica mostrou
inibição na gama de 48,08 % a 93,37 %, valores melhores do que os citados em trabalhos
anteriores. Os extratos com maior atividade antioxidante também mostram o melhor conteúdo de
fenólicos totais, no entanto, não apresentaram melhores níveis de flavonoides. Presença de
flavonoides (catequina, epicatequina e formononetina) foi confirmado em todas as amostras
analisadas.
Palavras-chave: Resina, Peroxidação lipídica, Quimiluminescência, Flavonoides.
ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENT OF THE RED PROPOLIS
FROM THE STATE OF SERGIPE, BRAZIL.
ABSTRACT
Propolis is a natural resin extracted from the plants and modified by the honeybees. This study
aimed to evaluate the antioxidant activity of Brazilian red propolis by DPPH, lipid peroxidation and
chemiluminescence methods, together with the analysis of its phenolic content by calorimetry and
ultra fast liquid chromatography. All the propolis samples analyzed in this study showed
antioxidant activity by DPPH∙ test which was confirmed by more sensitive lipid peroxidation and
chemiluminescence methods. Lipid peroxidation showed inhibition in the range of 48.08% to
93.37%, values better than those cited in previous report. The extracts with the highest antioxidant
activity also showed the best total phenolic content, however, did not show better levels of
flavonoids. Presence of flavonoids (catechin, epicatechin and formononetin) was confirmed in all
the analyzed samples.
Keywords: Bee glue, Lipid peroxidation, Chemiluminescence, Flavonoids.
73
1. Introduction
Propolis is a complex mixture of resinous
substances collected by the bees from various
plant parts (LOTTI et al., 2010). The resin
collected by the bees contains a variety of
chemical compounds such as flavonoid
aglycones, phenolic acids and their esters,
phenolic aldehydes, alcohols and ketones,
sesquiterpene, quinones, coumarins, steroids,
amino acids and inorganic compounds
(PIERINI et al., 2013) which have diverse
biological properties, including the anti-
inflammatory, antimicrobial, antioxidant,
antitumor, antiulcer and anti-HIV activities
(HUANG et al., 2014).
Brazilian propolis was divided into 12
types by Park et al., (2000). In 2007, a new type
of propolis called the red propolis was found
along the sea and rivers of the Northeastern
Brazil and was classified as propolis belonging
to group 13 (DAUGSCH et al., 2007). Red
propolis of Sergipe has been reported to have
antitumoral and immunomodulatory properties
(FROZZA et al., 2013) and wound healing
activities (ALBUQUERQUE JUNIOR et al.,
2009).
Studies with the Brazilian red propolis
have shown a great potential for this product
including its use in the prevention and treatment
of cardiovascular diseases such as
atherosclerosis (ALBUQUERQUE JUNIOR et
al., 2009; IIO et al., 2012).
Several methods can be utilized for the
extraction of natural chemical compounds from
propolis. One such method includes the use of
ultrasound. The use of ultrasound in the
extraction processes has drawn increasing
attention due to its higher efficiency and
reduced extraction time, compared to
conventional extraction methods, such as in
Soxhlet and maceration (LUQUE-GARCÍA;
CASTRO, 2003). Using this method, the
antioxidant activity and chemical composition
of the red propolis samples collected from the
Northeast Brazil was determined, which is
detailed in this manuscript.
2. Materials and Methods
2.1. Propolis
The red propolis samples were collected
from two different apiaries located in the lower
Sao Francisco region, Sergipe, Brazil. Five
samples were collected from the apiary CJ (S
10°26'04'', W 36°30'34'') and four samples from
the apiary CP (S 10°28'25'', W 36°26'12”). The
samples were stored at -20°C.
2.2. Chemicals
All the commercially available chemicals
such as 1,1-diphenyl-2-picrylhydrazylradical
(DPPH), thiobarbituric acid (TBA), luminol and
xanthine, xanthine oxidase (XOD) were
purchased from Sigma-Aldrich
2.3. Preparation of hydroalcoholicic
extracts (HAE) and Yied determination
1g of propolis sample was extracted with
12.5 ml of 70% (v/v) ethanol using ultrasound
for 60 min and then the mixture was centrifuged
and filtered to obtain its hydroalcoholic extract
(HAE). The dry weight was obtained from
respective hydroalcoholic extracts and used for
yield determination. All the tests were carried
out using 5% HAE.
2.4. Total polyphenol and flavonoid
contents
The total polyphenols were determined
using the Folin-Ciocalteau method (LEE et al.,
2003; FUNARI; FERRO, 2006), and expressed
as gallic acid equivalents in mg phenol/g of
propolis.
The flavonoid content was determined
using the colorimetric method of aluminum,
using 1.0 mL of the 1:10 diluted sample, 3.0 mL
95% ethanol, 0.5 ml of 10% aluminum nitrate,
0.2 mL potassium acetate and 5.6 mL of
distilled water. The blank was prepared with 1.0
mL diluted sample, 3.0 mL 95% ethanol, 0.2
mL of 1 M potassium acetate and 5.8 mL of
distilled water. After 20 minutes of incubation
at RT, the absorbance was measured at 415 nm.
Results were expressed as mg of flavonoids / g
of propolis using quercetin as a reference.
2.5. Ultra Fast Liquid Chromatography –
UFLC
Chromatographic analysis was carried out
using ultra fast liquid chromatography – UFLC
(Shimadzu Co.), equipped with a quat pump, a
degasser, a diode array detector (DAD) and a
reversed phase column (XP-ODS 50 x 3 mm;
particle size, 2.2 micrometers).
The binary mobile phase was composed of
water (solvent A) and methanol (solvent B)
according to the method described by Alencar et
al., (2007) and Cabral et al., (2009) with
modifications. The HAE was dissolved in
methanol (50 mg /mL) and filtered with a 0.45
μm filter (Millipore). Aliquots of 2 μL of 5% of
HAE (w/v) were injected into the UFLC system.
UFLC was performed using a linear gradient
elution as follows: 40% B increased to 60% B
after 22.5 min, a hold at 90% B for 37.3- 42.3
min, followed by a decrease to 30% B after 42.3
min with a solvent flow rate of 0.4 mL/min. The
following authentic standards of flavonoids and
phenolic acids were used: formononetin,
quercetin, 3-hydroxy-7-methoxyflavone,
catechin, epicatechin and propyl gallate. All the
tests were performed in triplicates using three
samples from each apiary
2.6. DPPH radical scavenging activity
The effect of DPPH radical was evaluated
by the method described by Brand-Williams et
al., 1995 and Tominaga et al., 2005 with
adaptations. The assay mixture contained 1.0
mL of 100mM sodium acetate buffer, 50 μL of
HAE, 1.0 ml of 95% ethanol, and 500 μL of 200
mM DPPH solution. Absorbance was recorded
at 517 nm after 10 min of incubation at room
temperature, in the dark. Blank containing the
same amount of ethanol and DPPH solution was
used as the negative control. The percentage
inhibition was plotted against phenol content
and IC50 (concentration of total phenol able to
scavenge 50% of DPPH free radical) was
determined.
2.7. Lipid peroxidation assay
Reaction mixture was prepared by adding
10 µL of each sample to 1 mL of a reaction
medium containing 130 mM KCl, and 10 mM
TrisHCl pH7.4, and mitochondria was added to
yield a final concentration of 1 mg of protein.
Later 50 µL ferrous ammonium sulfate and
2mM sodium citrate were added and the
samples were incubated at 37°C for 30 minutes.
Mitochondria was isolated from male Wistar
rats livers by differential centrifugation as
described by Pedersen et al., 1978, and the
mitochondrial protein content was determined
by the biuret reaction (CAIN; SKILLETER,
1987). For the determination of TBA-reactive
compound, 1 mL of 1% thiobarbituric acid
(TBA), 0.1 mL of 10M NaOH and 0.5 mL of
H3PO4 were added, followed by incubation in a
water bath for 20 minutes at 80°C. The TBA-
reactive compounds were extracted with 2 mL
of n-butanol and the samples were then
centrifuged at 9800 g for 10 min. The readings
were taken at 535 nm, using a Hitachi U2001
spectrophotometer. The experiments were
performed in triplicates together with blank
(without mitochondria), positive control
(without sample) and negative control (without
iron). The IC50 values were determined using
the GraphPad Prism ®.
2.8. Xanthine/luminol/XOD system
Samples of red propolis extract were
diluted in hydro alcoholic solution to a
concentration of 5 mg/mL and subsequent
dilutions were prepared in 0.1 M glycine buffer
pH 9.4. To verify free radical scavenging
activity of the propolis extract, a solution
containing 400 μL of 1 mM EDTA and 0.1 M
glycine pH 9.4 were added to a test tube
together with 150 μL of 6 mM xanthine, 10 μL
of test sample and 10 μL of 0.6 mM luminol
solution. The reaction was initiated with the
addition of 100 μL of freshly prepared cold 20
mU/mL xanthine oxidase solution. The
scavenging activity was determined after an
incubation of 5 minutes at 25 °C in an
Autolumat LB 953 luminometer. The results
were expressed according to the concentration
of propolis extract in the reaction medium
(MARQUELE-OLIVEIRA et al., 2005). The
IC50 values were determined using GraphPad
Prism® software.
2.9. Statistics
All the measurements were performed in
triplicates. The average was determined with
Assistat 7.7 Beta (registre INPI 0004051-2), and
used for the analysis of variance (ANOVA) and
Tukey test for means comparison, when
necessary.
3. Results and Discussion
The hydroalcoholic extracts of all the
propolis samples presented yield ranging from
9.45% to 74.62% (w/w) with the exception of
two samples that showed lower yield than the
value determined by Ministry of Agriculture
(less than 11%), as shown in Table 1 (BRASIL, 2001).
Table 1. Yield, flavonoid content (mg quercetina/g própolis), total phenols (mg fenol/g propolis), DPPH∙
and lipid
peroxidation of the hydroalcoholic extracts of red propolis. Same letters in the same column represent statistically
identical values for the Tukey test (p<0,05)
Funari et al., (2007) obtained a yield of
38.34% (w/w) using cold ethanol extracts of
propolis from the state of São Paulo, while
work done by Longhine et al., (2007) using
ethanol extracts of propolis from Paraná using
turboextractor showed an yield of 3.68% to
14.90% (w / w). Alencar et al., (2007) acquired
a yield of 58.20% (w / w) with ethanol extracts
of propolis using water bath at 70 ° C from the
state of Alagoas.
The propolis has different characteristics
according to their geographical location, and its
chemical composition is related to the climate,
the genetic characteristics of bees, the
methodology for conducting the tests and the
time of year that propolis was produced
(LASKAR et al.,2010; MIGUEL et al., 2010).
However, the results of this study suggest that
the forging activity of the honey bees can differ
between the hives of the same apiary This
suggests that the genetic characteristics of the
queen and foraging habits of the workers may
be related to this issue, facts also commented
by Silva e Azevedo, (2006). Propolis samples
from different boxes of the same apairy differed
in their yield, confirming the fact that the choice
of the plant to collect the resin varies between
the bees of the same apiary. This variable is
therefore important in determining the final
yield of propolis samples. Yield can also be
related to the type and volume of extracting
solvent, the method and the extraction time
(GRIGORIS et al., 2005).
3.1. Total polyphenol, flavonoid, and free
radical scavenging activity of DPPH ▪
The amount of total phenols in the
propolis samples analyzed in this study ranged
from 1.35% to 2.27% exceeding the minimum
limit of 0.50%. 70% of propolis samples
analyzed met the minimum value of 0.25%
(w/w) of flavonoid (Table 1) required by the
Ministry of Agriculture (BRASIL, 2001).
The content of phenolic compounds
ranged from 13.52 to 22.78 mg phenol/g
propolis, values comparable to those cited in the
literature (CASTRO et al., 2007; DAUGSCH et
al., 2008). The values expressed in mg phenol
equivalent to gallic acid/g propolis acid can be
seen in Table 1.
Despite the importance of classifying the
content of polyphenols and flavonoids in
propolis samples, it is important to note that in
natural extracts, which are a mixture of different
polyphenols and flavonoids, antioxidant activity
can not be directly related to these chemical
compounds, as two different polyphenols can
interact with the Folin-Ciocalteau forming
products with similar absorbance, but with very
different antioxidant activity (MARQUELE-
OLIVEIRA et al., 2008).
The flavonoid content (mg propolis/g
quercetin) of propolis samples analyzed in this
study presented values well below the values
reported in the literature with other types of
Brazilian propolis. The colorimetric method
utilized in this study can also be an interfering
agent for the evaluation of flavonoids in these
samples. Other analytical methods may exhibit
Extracts
Yield
(%m/m)
Flavonoids
(mg/g)
Total phenol
(mg/g)
DPPH▪
IC50 (μg/mL)
Lipid
peroxidation (%)
CP 03 74.6 ± 0.00 g
0.00 ± 0.00 d
13.52 ± 0.28d
324.3 ± 17.8 e
60.77 ± 0.43 e
CP 14 11.7 ± 0.00 g
0.03 ± 0.01 c
22.70 ± 0.17a
127.2 ± 7.90 c,f
91.74 ± 0.37 a
CP 18 11.8 ± 0.00 d
0.04 ± 0.01 b,c
21.91 ± 0.28a
133.3 ± 0.70 f
69.53 ± 2.55 d
CP 19 20.4 ± 0.00 b
0.03 ± 0.01 c
21.54 ± 0.46b
194.3 ± 9.00 d
92.63 ± 0.51 a
CP 29 15.5 ± 0.00 cd
0.00 ± 0.00 d
22.78 ± 0.28a
138.4 ± 0.00 f
93.37 ± 0.75 a
CJ 04 9.4 ± 0.00 a
0.04 ± 0.02 b,c
17.70 ± 0.40c
405.3 ± 20.0 a
48.08 ± 0.68 g
CJ 06 9.3 ± 0.00 f
0.07 ± 0.02 a
21.06 ± 0.44b
225.3 ± 4.60 b
83.33 ± 0.77 b
CJ 09 18.6 ± 0.00 f
0.00 ± 0.00 d
20.92 ± 0.54b
101.6 ± 1.30 c
75.37 ± 0.34 c
CJ 10 24.3 ± 0.00 c
0.05 ± 0.01 b
20.39 ± 0.57b
218.4 ± 5.20 b,d
91.15 ± 0.43 a
different results than those observed in this
study.
Samples that showed the best IC50 values
in DPPH test were CJ 09 (101.6 mg/mL), CP 14
(127.2 mg/mL), CP 18 (133.6 mg/mL) and CP
29 (138.4 mg/mL), and these samples also
showed higher levels of total phenols 4.12, 3.93,
2.50 and 3.60% respectively. However, these
samples did not show better levels of
flavonoids, and the sample CJ 09 that showed
the best antioxidant activity showed no
flavonoid content by colorimetric method that
was used here (Table 1). It is well established
that phenolic compounds, such as the ones
present in propolis, work as antioxidants by
breaking the chain reaction of lipids (TOREL et
al., 1986), inhibiting chemiluminescence
reactions (GEORGETTI et al., 2003),
scavenging reactive oxygen species (BORS et
al., 1990), and so forth. Nevertheless, no
correlation between phenolic and flavonoid
contents and antioxidant activity has been
confirmed. Reports published till date suggests
that the antioxidant properties arise from
complex mechanisms or synergistic interactions
between compounds (RAMANAUSKIENE et
al., 2007; MARQUELE-OLIVEIRA et al.,
2008).
Red propolis extract analyzed in this study
showed similar DPPH results to those obtained
by Pinheiro (2009) and Frozza et al., (2013)
with red propolis from the same geographic
region, with IC50 of 294μg/mL and IC50 of
270.13μg/mL, respectively.
3.2. Lipid peroxidation assay and
Xanthine/luminol/XOD system
According to Halliwell e Gutteridge
(1990), the detection and measurement of lipid
peroxidation is evidence most cited in the
literature that demonstrates the involvement of
free radical reactions in toxicology and human
diseases. Use of mitochondria as the source of
lipids may mimic human physiological
conditions. From the results of DPPH test,
concentration of 0.25 mg/mL which showed the
best antioxidant activity was chosen to for lipid
peroxidation and chemiluminescence test.
The red propolis extracts showed
inhibitory properties for lipid peroxidation by
preventing the formation of reactive species
against 2-thiobarbitric acid (TBA). The extracts
showed an inhibition of 48.08% to 93.37% at a
concentration of IC50 0.95 µg/mL, which were
better than those cited in the literature.
Argentina propolis extracts showed inhibition
from 20 to 80% (ISLA et al., 2001). According
to Jasprica et al., (2007), a decrease in the
concentration of malondialdehyde (an indicator
of lipid peroxidation) by 23.2% was observed in
volunteer men patients in Croatia after treatment
with propolis, demonstrating its antioxidant
action. Marquele-Oliveira et al., (2005) found in
their study IC50 values equivalente to 16µg/mL
and 12µg/mL for fluid and glycolic extract of
brown propolis.
As for chemiluminescence technique,
Dodeigene et al., (2000) argued that within the
techniques of antioxidant activity, this is an
advantageous method because of its high
sensitivity and speed. When compounds like
propolis are added to a chemiluminescent
solution, the light emission is reduced,
indicating antioxidant activity by removing any
radical generated in this system. The
chemiluminescence activity of the propolis
samples was analysed using one extract from
each apiary (CP29 and CJ10). Samples that
showed the best lipid peroxidation inhibition
were chosen for this assay. Luminol was used as
a detector in the chemiluminiscence test, where
it is oxidized by the superoxide anion.
The inhibition of the luminiscence by the
reduction of the superoxide was measured and
the extracts of red propolis from Sergipe were
shown to possess inhibiting activity for the
enzyme XOD, revealing IC50 of 0.096 µL/ml
(approximately 96 µg/ml) for sample CP29 and
IC50 of 0.089 µL/ml (approximately 89 µg/ml)
for sample CJ10 and inhibiting concentration
ranging between 15.66 to 89.80% as shown in
Figure1. As can bee seem in Figure 1, no
significant difference was found between
samples CP29 and CJ10. Pascual et al., (1994)
found similar values of IC50 ranging from 50 to
95 µg/mL in propolis samples from Cuba by the
method of xanthine-xanthine oxidase.
Figure 1. Inhibition of light emission from
xanthine/luminol/XOD luminescent reactions with
luminol found for different concentrationsof: (■) CP 29
and (♦) CJ 10. Results are mean±SD of three experiments
run in parallel.
3.3. Ultra Fast Liquid Chromatography –
UFLC
The hydroalcoholic extracts of propolis
showed a complex composition, with several
peaks at different retention times as analyzed by
UFLC-DAD. The chromatographic profiles of
the hydroalcoholic extracts of red propolis
indicated the presence of approximately 20
compounds with totally distinct profiles.
Presence of flavonoids such as catechin,
epicatechin and formononetin was confirmed as
shown in Figure 2.
Figure 2. Chromatograms of red propolis samples from two different regions of the river São Francisco
basin with three samples from apiary Cajuipe (CJ) and three from capivara (CP). 1- Catechin, 2-
Epicatechin and 3- Formononetin.
And the amount of catechin was
quantified to 0.26 to 0.88 mg/ml extract of
propolis. Catechins belong to a group of
polyphenols that have high antioxidant activity
and presents possible mechanisms of action
against cancer (HO et al., 1992; RODGERS et
al., 1998; PINHEIRO et al., 2014). The
flavonoid that appeared with the highest
intensity in the red propolis samples is
formononetin which is a chemical marker
present in the samples of red propolis (JAIN et
al., 2014; MENDONÇA et al., 2015). The
presence of formononetin in propolis shows
estrogenic and antifungal activity. According to
Moraes (2009), it has been shown that when
mammals consume this isoflavin it is
metabolized into daidzein, which is one of
isoflavins aglycones present in soy, and is used
in the prevention and treatment of menopausal
symptoms, and treatment of prostate cancer and
breast cancer.
4. Conclusion
Propolis extracts analyzed in this study
showed yield and total phenols comparable to
other similar reports with red propolis. IC50
values for lipid peroxidation were infact better
than previous reports suggesting higher
potential of the propolis samples from the state
of Sergipe. Presence of flavonoids such
catechin, epicatechin and formononetin known
for their medicinal properties was confirmed by
UFLC and it was possible to quantify the
catechin content.
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The binary mobile phase was composed of
water (solvent A) and methanol (solvent B)
according to the method described by Alencar et
al., (2007) and Cabral et al., (2009) with
modifications. The HAE was dissolved in
methanol (50 mg /mL) and filtered with a 0.45
μm filter (Millipore). Aliquots of 2 μL of 5% of
HAE (w/v) were injected into the UFLC system.
UFLC was performed using a linear gradient
elution as follows: 40% B increased to 60% B
after 22.5 min, a hold at 90% B for 37.3- 42.3
min, followed by a decrease to 30% B after 42.3
min with a solvent flow rate of 0.4 mL/min. The
following authentic standards of flavonoids and
phenolic acids were used: formononetin,
quercetin, 3-hydroxy-7-methoxyflavone,
catechin, epicatechin and propyl gallate. All the
tests were performed in triplicates using three
samples from each apiary
2.6. DPPH radical scavenging activity
The effect of DPPH radical was evaluated
by the method described by Brand-Williams et
al., 1995 and Tominaga et al., 2005 with
adaptations. The assay mixture contained 1.0
mL of 100mM sodium acetate buffer, 50 μL of
HAE, 1.0 ml of 95% ethanol, and 500 μL of 200
mM DPPH solution. Absorbance was recorded
at 517 nm after 10 min of incubation at room
temperature, in the dark. Blank containing the
same amount of ethanol and DPPH solution was
used as the negative control. The percentage
inhibition was plotted against phenol content
and IC50 (concentration of total phenol able to
scavenge 50% of DPPH free radical) was
determined.
2.7. Lipid peroxidation assay
Reaction mixture was prepared by adding
10 µL of each sample to 1 mL of a reaction
medium containing 130 mM KCl, and 10 mM
TrisHCl pH7.4, and mitochondria was added to
yield a final concentration of 1 mg of protein.
Later 50 µL ferrous ammonium sulfate and
2mM sodium citrate were added and the
samples were incubated at 37°C for 30 minutes.
Mitochondria was isolated from male Wistar
rats livers by differential centrifugation as
described by Pedersen et al., 1978, and the
mitochondrial protein content was determined
by the biuret reaction (CAIN; SKILLETER,
1987). For the determination of TBA-reactive
compound, 1 mL of 1% thiobarbituric acid
(TBA), 0.1 mL of 10M NaOH and 0.5 mL of
H3PO4 were added, followed by incubation in a
water bath for 20 minutes at 80°C. The TBA-
reactive compounds were extracted with 2 mL
of n-butanol and the samples were then
centrifuged at 9800 g for 10 min. The readings
were taken at 535 nm, using a Hitachi U2001
spectrophotometer. The experiments were
performed in triplicates together with blank
(without mitochondria), positive control
(without sample) and negative control (without
iron). The IC50 values were determined using
the GraphPad Prism ®.
2.8. Xanthine/luminol/XOD system
Samples of red propolis extract were
diluted in hydro alcoholic solution to a
concentration of 5 mg/mL and subsequent
dilutions were prepared in 0.1 M glycine buffer
pH 9.4. To verify free radical scavenging
activity of the propolis extract, a solution
containing 400 μL of 1 mM EDTA and 0.1 M
glycine pH 9.4 were added to a test tube
together with 150 μL of 6 mM xanthine, 10 μL
of test sample and 10 μL of 0.6 mM luminol
solution. The reaction was initiated with the
addition of 100 μL of freshly prepared cold 20
mU/mL xanthine oxidase solution. The
scavenging activity was determined after an
incubation of 5 minutes at 25 °C in an
Autolumat LB 953 luminometer. The results
were expressed according to the concentration
of propolis extract in the reaction medium
(MARQUELE-OLIVEIRA et al., 2005). The
IC50 values were determined using GraphPad
Prism® software.
2.9. Statistics
All the measurements were performed in
triplicates. The average was determined with
Assistat 7.7 Beta (registre INPI 0004051-2), and
used for the analysis of variance (ANOVA) and
Tukey test for means comparison, when
necessary.
3. Results and Discussion
The hydroalcoholic extracts of all the
propolis samples presented yield ranging from
9.45% to 74.62% (w/w) with the exception of
two samples that showed lower yield than the
value determined by Ministry of Agriculture
Fonseca, M. J. V. Assessment of the
antioxidant activities of Brazilian extracts of
propolis alone and in topical pharmaceutical
formulations. Journal of Pharmaceutical and
Biomedical Analysis, v.39, p.455-462, 2005.
Marquele-Oliveira, F., Fonseca, Y.M.,
Georgetti, S. R., Vicentini, F. T. M. C.,
Bronzati, V., Fonseca, M. J. V. Evaluation of
the antioxidant activity as an additional
parameter to attain the functional quality of
natural extracts. Latin American Journal of
Pharmacy, v.27, n.3, p. 325–332, 2008.
Mendonça, L.S., Mendonça, F.M.R., Maia-
Araújo, Y.L.F., Araújo, E.D., Ramalho, S.A.,
Narain, N., Padilha, F.F., Jain, S., Orellana,
S.C., Cardoso, J.C. (Accepted) Chemical
markers and antifungal activity of red propolis
from Sergipe, Brazil. Food Science and
Technology (Campinas).
Miguel, M.G., Nunes, S., Dandlen, S. A.,
Cavaco, A.M. and Antunes, M.D. Phenols and
antioxidant activity of hydro-alcoholic extracts
of propolis from Algarve, South of Portugal.
Food and Chemical Toxicology, v.48, p.3418–
3423, 2010.
Moraes, C.S. Isolamento e identificação de
formononetina da própolis de João Pessoa-PB,
estudo de sua sazonalidade e avaliação de suas
atividades biológicas. 2009. Tese de doutorado,
Universidade Estadual de Campinas. Faculdade
de Engenharia de Alimentos. São Paulo, SP,
Brasil.
Park, Y.K., Ikegaki, M., Alencar, S.M.
Classification of Brazilian propolis by both
physicochemical methods and biological
activity. Mensagem Doce, v.58, p.2-7. 2000.
Pascual C., Gonzales R., Torricella R. G.
Scavenging action of propolis extract against
oxygen radicals. Journal Ethnopharmacology,
v.4, p.9-13, 1994.
Pedersen P.L., Greenawalt J.W., Reynafarje B.,
Hullihen J., Decker G.L., Soper J.W.
Bustamente E. Preparation and characterization
of mitochondria and submitochondrial particles
of rat liver and liver-derived
tissues. Methods in Cell Biology, v..20, p.411–
481, 1978.
Pierini, G. D., Granero, A.M., Di Nezio, M.S.,
Centurión, M.E., Zon, M.A., Fernández, H.
Development of an electroanalytical method for
the determination of lead in Argentina raw
propolis based on bismuth electrodes.
Microchemical Journal, v.106, p.102–106,
2013.
Pinheiro, M.S. Avaliação da atividade
antimicrobiana e citoprotetora gástrica dos
extratos de mangaba, caju e própolis vermelha.
2009. Dissertação de mestrado, Universidade
Tiradentes – UNIT. Aracaju, SE, Brasil, 2009.
Pinheiro, K. S., Ribeiro,D.R, Alves, A.V.F.,
Pereira-Filho, R.N., Oliveira, C.R., Lima, S.O.
Modulatory activity of brazilian red propolis on
chemically induced dermal carcinogenesis.
Acta Cirúrgica Brasileira, v.29, n.2, p.111-
117, 2014.
Ramanauskiene, K., Kalveniene, Z.,
Kasparaviciene, G., Briedisis, V. Assay of
correlation between antioxidant activity of
propolis extracts and their chemical
components. European Journal of
Pharmaceutical Sciences, v.32, p. S45, 2007.
Rodgers, E.H., Grant, M.H. The effect of the
flavonoids, quercetin, myricetin and
epicathechin on the growth and enzyme
activities of MCF7 human breast cancer cells.
Chemical Biologic Interactions, v.116, n.1-2,
p.213-228, 1998.
Silva, F.A.S.E., Azevedo, C.A. A New Version
of The Assistat-Statistical Assistance Software.
In: World Congress on Computers in
Agriculture, 4, Orlando-FLUSA: Anais.
Orlando: American Society of Agricultural
Engineers, p.393-396, 2006.
Tominaga, Y., Kobayachi, Y., Goto,
T., Kasemura, K., Nomura, M., Yasshi, Y. DPPH
Radical–scavenging effect of
several phenylpropanoid compounds and their
glycoside derivatives. Journal of the
Pharmaceutical Society of Japan, v. 125, n.4,
p.371–375, 2005.
Torel, J., Cillard, J., and Cillard, P. Antioxidant
activity of flavonoids and reactivity with peroxy
radical. Phytochemistry, v.25, n.2, p. 383–385,
1986.
______________________________________
1. Departamento de Biologia,
Universidade Federal de Sergipe, São Cristóvão,
Sergipe – Brasil (ylmaia@yahoo.com.br –
Maia-Araújo, Y.L.F),
(lucyana_biologia@yahoo.com.br – Mendonça,
L.S.) e (edaraujo@yahoo.com.br – Araújo,
E.D.);
2. Departamento de Engenharia de
Alimentos, Universidade Federal de Sergipe,
São Cristóvão, Sergipe – Brasil
(nayjaracarvalho@hotmail.com – Gualberto,
N.C.) e (narendra.narain@gmail.com – Narain,
N.);
3. Fundação Oswaldo Cruz, Centro de
Pesquisa René Rachou, Belo Horizonte, Minas
Gerais, Brasil (sara_cuadros@yahoo.com –
Cuadros-Orellana, S.);
4. Instituto de Tecnologia e Pesquisa
(ITP), Aracaju, Sergipe – Brasil
(juaracaju@yahoo.com.br – Cardoso, J.C.);
5. Departamento de Morfologia,
Universidade Federal de Sergipe, São Cristóvão,
Sergipe – Brasil (sonajain24@yahoo.com –
Jain, S.) *.
Corresponding author*
Sona Jain, Av. Marechal Rondon, S/N, Bairro
Rosa Elze. Universidade Federal de Sergipe,
Departamento de Morfologia, São Cristóvão –
SE. CEP 49100-000. e-mail:
sonajain24@yahoo.com
82

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Artigo bioterra v16_n2_10

  • 1. REVISTA DE BIOLOGIA E CIÊNCIAS DA TERRA ISSN 1519-5228 Volume 16 - Número 2 - 2º Semestre 2016 ATIVIDADE ANTIOXIDANTE E COMPOSTOS FENÓLICOS DE PRÓPOLIS VERMELHA DO ESTADO DE SERGIPE, BRASIL. Yzila Liziane Farias Maia de Araújo1 ; Lucyana Santos de Mendonça1 ; Nayjara Carvalho Gualberto2 ; Sara Cuadros Orellana3 ; Juliana Cordeiro Cardoso4 ; Sona Jain5 ; Edilson Divino de Araújo1 ; Narendra Narain2 RESUMO A própolis é uma resina natural extraída das plantas e modificada pelas abelhas. Este estudo teve como objetivo avaliar a atividade antioxidante da própolis vermelha brasileira por DPPH, peroxidação lipídica e métodos de quimiluminescência, juntamente com a análise de seu conteúdo fenólico por calorimetria e cromatografia líquida ultrarrápida. Todas as amostras de própolis analisadas neste estudo mostraram atividade antioxidante por DPPH ∙ teste que foi confirmado por peroxidação lipídica e quimiluminescência métodos mais sensíveis. A peroxidação lipídica mostrou inibição na gama de 48,08 % a 93,37 %, valores melhores do que os citados em trabalhos anteriores. Os extratos com maior atividade antioxidante também mostram o melhor conteúdo de fenólicos totais, no entanto, não apresentaram melhores níveis de flavonoides. Presença de flavonoides (catequina, epicatequina e formononetina) foi confirmado em todas as amostras analisadas. Palavras-chave: Resina, Peroxidação lipídica, Quimiluminescência, Flavonoides. ANTIOXIDANT ACTIVITY AND PHENOLIC CONTENT OF THE RED PROPOLIS FROM THE STATE OF SERGIPE, BRAZIL. ABSTRACT Propolis is a natural resin extracted from the plants and modified by the honeybees. This study aimed to evaluate the antioxidant activity of Brazilian red propolis by DPPH, lipid peroxidation and chemiluminescence methods, together with the analysis of its phenolic content by calorimetry and ultra fast liquid chromatography. All the propolis samples analyzed in this study showed antioxidant activity by DPPH∙ test which was confirmed by more sensitive lipid peroxidation and chemiluminescence methods. Lipid peroxidation showed inhibition in the range of 48.08% to 93.37%, values better than those cited in previous report. The extracts with the highest antioxidant activity also showed the best total phenolic content, however, did not show better levels of flavonoids. Presence of flavonoids (catechin, epicatechin and formononetin) was confirmed in all the analyzed samples. Keywords: Bee glue, Lipid peroxidation, Chemiluminescence, Flavonoids. 73
  • 2. 1. Introduction Propolis is a complex mixture of resinous substances collected by the bees from various plant parts (LOTTI et al., 2010). The resin collected by the bees contains a variety of chemical compounds such as flavonoid aglycones, phenolic acids and their esters, phenolic aldehydes, alcohols and ketones, sesquiterpene, quinones, coumarins, steroids, amino acids and inorganic compounds (PIERINI et al., 2013) which have diverse biological properties, including the anti- inflammatory, antimicrobial, antioxidant, antitumor, antiulcer and anti-HIV activities (HUANG et al., 2014). Brazilian propolis was divided into 12 types by Park et al., (2000). In 2007, a new type of propolis called the red propolis was found along the sea and rivers of the Northeastern Brazil and was classified as propolis belonging to group 13 (DAUGSCH et al., 2007). Red propolis of Sergipe has been reported to have antitumoral and immunomodulatory properties (FROZZA et al., 2013) and wound healing activities (ALBUQUERQUE JUNIOR et al., 2009). Studies with the Brazilian red propolis have shown a great potential for this product including its use in the prevention and treatment of cardiovascular diseases such as atherosclerosis (ALBUQUERQUE JUNIOR et al., 2009; IIO et al., 2012). Several methods can be utilized for the extraction of natural chemical compounds from propolis. One such method includes the use of ultrasound. The use of ultrasound in the extraction processes has drawn increasing attention due to its higher efficiency and reduced extraction time, compared to conventional extraction methods, such as in Soxhlet and maceration (LUQUE-GARCÍA; CASTRO, 2003). Using this method, the antioxidant activity and chemical composition of the red propolis samples collected from the Northeast Brazil was determined, which is detailed in this manuscript. 2. Materials and Methods 2.1. Propolis The red propolis samples were collected from two different apiaries located in the lower Sao Francisco region, Sergipe, Brazil. Five samples were collected from the apiary CJ (S 10°26'04'', W 36°30'34'') and four samples from the apiary CP (S 10°28'25'', W 36°26'12”). The samples were stored at -20°C. 2.2. Chemicals All the commercially available chemicals such as 1,1-diphenyl-2-picrylhydrazylradical (DPPH), thiobarbituric acid (TBA), luminol and xanthine, xanthine oxidase (XOD) were purchased from Sigma-Aldrich 2.3. Preparation of hydroalcoholicic extracts (HAE) and Yied determination 1g of propolis sample was extracted with 12.5 ml of 70% (v/v) ethanol using ultrasound for 60 min and then the mixture was centrifuged and filtered to obtain its hydroalcoholic extract (HAE). The dry weight was obtained from respective hydroalcoholic extracts and used for yield determination. All the tests were carried out using 5% HAE. 2.4. Total polyphenol and flavonoid contents The total polyphenols were determined using the Folin-Ciocalteau method (LEE et al., 2003; FUNARI; FERRO, 2006), and expressed as gallic acid equivalents in mg phenol/g of propolis. The flavonoid content was determined using the colorimetric method of aluminum, using 1.0 mL of the 1:10 diluted sample, 3.0 mL 95% ethanol, 0.5 ml of 10% aluminum nitrate, 0.2 mL potassium acetate and 5.6 mL of distilled water. The blank was prepared with 1.0 mL diluted sample, 3.0 mL 95% ethanol, 0.2 mL of 1 M potassium acetate and 5.8 mL of distilled water. After 20 minutes of incubation at RT, the absorbance was measured at 415 nm. Results were expressed as mg of flavonoids / g of propolis using quercetin as a reference. 2.5. Ultra Fast Liquid Chromatography – UFLC Chromatographic analysis was carried out using ultra fast liquid chromatography – UFLC (Shimadzu Co.), equipped with a quat pump, a degasser, a diode array detector (DAD) and a reversed phase column (XP-ODS 50 x 3 mm; particle size, 2.2 micrometers).
  • 3. The binary mobile phase was composed of water (solvent A) and methanol (solvent B) according to the method described by Alencar et al., (2007) and Cabral et al., (2009) with modifications. The HAE was dissolved in methanol (50 mg /mL) and filtered with a 0.45 μm filter (Millipore). Aliquots of 2 μL of 5% of HAE (w/v) were injected into the UFLC system. UFLC was performed using a linear gradient elution as follows: 40% B increased to 60% B after 22.5 min, a hold at 90% B for 37.3- 42.3 min, followed by a decrease to 30% B after 42.3 min with a solvent flow rate of 0.4 mL/min. The following authentic standards of flavonoids and phenolic acids were used: formononetin, quercetin, 3-hydroxy-7-methoxyflavone, catechin, epicatechin and propyl gallate. All the tests were performed in triplicates using three samples from each apiary 2.6. DPPH radical scavenging activity The effect of DPPH radical was evaluated by the method described by Brand-Williams et al., 1995 and Tominaga et al., 2005 with adaptations. The assay mixture contained 1.0 mL of 100mM sodium acetate buffer, 50 μL of HAE, 1.0 ml of 95% ethanol, and 500 μL of 200 mM DPPH solution. Absorbance was recorded at 517 nm after 10 min of incubation at room temperature, in the dark. Blank containing the same amount of ethanol and DPPH solution was used as the negative control. The percentage inhibition was plotted against phenol content and IC50 (concentration of total phenol able to scavenge 50% of DPPH free radical) was determined. 2.7. Lipid peroxidation assay Reaction mixture was prepared by adding 10 µL of each sample to 1 mL of a reaction medium containing 130 mM KCl, and 10 mM TrisHCl pH7.4, and mitochondria was added to yield a final concentration of 1 mg of protein. Later 50 µL ferrous ammonium sulfate and 2mM sodium citrate were added and the samples were incubated at 37°C for 30 minutes. Mitochondria was isolated from male Wistar rats livers by differential centrifugation as described by Pedersen et al., 1978, and the mitochondrial protein content was determined by the biuret reaction (CAIN; SKILLETER, 1987). For the determination of TBA-reactive compound, 1 mL of 1% thiobarbituric acid (TBA), 0.1 mL of 10M NaOH and 0.5 mL of H3PO4 were added, followed by incubation in a water bath for 20 minutes at 80°C. The TBA- reactive compounds were extracted with 2 mL of n-butanol and the samples were then centrifuged at 9800 g for 10 min. The readings were taken at 535 nm, using a Hitachi U2001 spectrophotometer. The experiments were performed in triplicates together with blank (without mitochondria), positive control (without sample) and negative control (without iron). The IC50 values were determined using the GraphPad Prism ®. 2.8. Xanthine/luminol/XOD system Samples of red propolis extract were diluted in hydro alcoholic solution to a concentration of 5 mg/mL and subsequent dilutions were prepared in 0.1 M glycine buffer pH 9.4. To verify free radical scavenging activity of the propolis extract, a solution containing 400 μL of 1 mM EDTA and 0.1 M glycine pH 9.4 were added to a test tube together with 150 μL of 6 mM xanthine, 10 μL of test sample and 10 μL of 0.6 mM luminol solution. The reaction was initiated with the addition of 100 μL of freshly prepared cold 20 mU/mL xanthine oxidase solution. The scavenging activity was determined after an incubation of 5 minutes at 25 °C in an Autolumat LB 953 luminometer. The results were expressed according to the concentration of propolis extract in the reaction medium (MARQUELE-OLIVEIRA et al., 2005). The IC50 values were determined using GraphPad Prism® software. 2.9. Statistics All the measurements were performed in triplicates. The average was determined with Assistat 7.7 Beta (registre INPI 0004051-2), and used for the analysis of variance (ANOVA) and Tukey test for means comparison, when necessary. 3. Results and Discussion The hydroalcoholic extracts of all the propolis samples presented yield ranging from 9.45% to 74.62% (w/w) with the exception of two samples that showed lower yield than the value determined by Ministry of Agriculture
  • 4. (less than 11%), as shown in Table 1 (BRASIL, 2001). Table 1. Yield, flavonoid content (mg quercetina/g própolis), total phenols (mg fenol/g propolis), DPPH∙ and lipid peroxidation of the hydroalcoholic extracts of red propolis. Same letters in the same column represent statistically identical values for the Tukey test (p<0,05) Funari et al., (2007) obtained a yield of 38.34% (w/w) using cold ethanol extracts of propolis from the state of São Paulo, while work done by Longhine et al., (2007) using ethanol extracts of propolis from Paraná using turboextractor showed an yield of 3.68% to 14.90% (w / w). Alencar et al., (2007) acquired a yield of 58.20% (w / w) with ethanol extracts of propolis using water bath at 70 ° C from the state of Alagoas. The propolis has different characteristics according to their geographical location, and its chemical composition is related to the climate, the genetic characteristics of bees, the methodology for conducting the tests and the time of year that propolis was produced (LASKAR et al.,2010; MIGUEL et al., 2010). However, the results of this study suggest that the forging activity of the honey bees can differ between the hives of the same apiary This suggests that the genetic characteristics of the queen and foraging habits of the workers may be related to this issue, facts also commented by Silva e Azevedo, (2006). Propolis samples from different boxes of the same apairy differed in their yield, confirming the fact that the choice of the plant to collect the resin varies between the bees of the same apiary. This variable is therefore important in determining the final yield of propolis samples. Yield can also be related to the type and volume of extracting solvent, the method and the extraction time (GRIGORIS et al., 2005). 3.1. Total polyphenol, flavonoid, and free radical scavenging activity of DPPH ▪ The amount of total phenols in the propolis samples analyzed in this study ranged from 1.35% to 2.27% exceeding the minimum limit of 0.50%. 70% of propolis samples analyzed met the minimum value of 0.25% (w/w) of flavonoid (Table 1) required by the Ministry of Agriculture (BRASIL, 2001). The content of phenolic compounds ranged from 13.52 to 22.78 mg phenol/g propolis, values comparable to those cited in the literature (CASTRO et al., 2007; DAUGSCH et al., 2008). The values expressed in mg phenol equivalent to gallic acid/g propolis acid can be seen in Table 1. Despite the importance of classifying the content of polyphenols and flavonoids in propolis samples, it is important to note that in natural extracts, which are a mixture of different polyphenols and flavonoids, antioxidant activity can not be directly related to these chemical compounds, as two different polyphenols can interact with the Folin-Ciocalteau forming products with similar absorbance, but with very different antioxidant activity (MARQUELE- OLIVEIRA et al., 2008). The flavonoid content (mg propolis/g quercetin) of propolis samples analyzed in this study presented values well below the values reported in the literature with other types of Brazilian propolis. The colorimetric method utilized in this study can also be an interfering agent for the evaluation of flavonoids in these samples. Other analytical methods may exhibit Extracts Yield (%m/m) Flavonoids (mg/g) Total phenol (mg/g) DPPH▪ IC50 (μg/mL) Lipid peroxidation (%) CP 03 74.6 ± 0.00 g 0.00 ± 0.00 d 13.52 ± 0.28d 324.3 ± 17.8 e 60.77 ± 0.43 e CP 14 11.7 ± 0.00 g 0.03 ± 0.01 c 22.70 ± 0.17a 127.2 ± 7.90 c,f 91.74 ± 0.37 a CP 18 11.8 ± 0.00 d 0.04 ± 0.01 b,c 21.91 ± 0.28a 133.3 ± 0.70 f 69.53 ± 2.55 d CP 19 20.4 ± 0.00 b 0.03 ± 0.01 c 21.54 ± 0.46b 194.3 ± 9.00 d 92.63 ± 0.51 a CP 29 15.5 ± 0.00 cd 0.00 ± 0.00 d 22.78 ± 0.28a 138.4 ± 0.00 f 93.37 ± 0.75 a CJ 04 9.4 ± 0.00 a 0.04 ± 0.02 b,c 17.70 ± 0.40c 405.3 ± 20.0 a 48.08 ± 0.68 g CJ 06 9.3 ± 0.00 f 0.07 ± 0.02 a 21.06 ± 0.44b 225.3 ± 4.60 b 83.33 ± 0.77 b CJ 09 18.6 ± 0.00 f 0.00 ± 0.00 d 20.92 ± 0.54b 101.6 ± 1.30 c 75.37 ± 0.34 c CJ 10 24.3 ± 0.00 c 0.05 ± 0.01 b 20.39 ± 0.57b 218.4 ± 5.20 b,d 91.15 ± 0.43 a
  • 5. different results than those observed in this study. Samples that showed the best IC50 values in DPPH test were CJ 09 (101.6 mg/mL), CP 14 (127.2 mg/mL), CP 18 (133.6 mg/mL) and CP 29 (138.4 mg/mL), and these samples also showed higher levels of total phenols 4.12, 3.93, 2.50 and 3.60% respectively. However, these samples did not show better levels of flavonoids, and the sample CJ 09 that showed the best antioxidant activity showed no flavonoid content by colorimetric method that was used here (Table 1). It is well established that phenolic compounds, such as the ones present in propolis, work as antioxidants by breaking the chain reaction of lipids (TOREL et al., 1986), inhibiting chemiluminescence reactions (GEORGETTI et al., 2003), scavenging reactive oxygen species (BORS et al., 1990), and so forth. Nevertheless, no correlation between phenolic and flavonoid contents and antioxidant activity has been confirmed. Reports published till date suggests that the antioxidant properties arise from complex mechanisms or synergistic interactions between compounds (RAMANAUSKIENE et al., 2007; MARQUELE-OLIVEIRA et al., 2008). Red propolis extract analyzed in this study showed similar DPPH results to those obtained by Pinheiro (2009) and Frozza et al., (2013) with red propolis from the same geographic region, with IC50 of 294μg/mL and IC50 of 270.13μg/mL, respectively. 3.2. Lipid peroxidation assay and Xanthine/luminol/XOD system According to Halliwell e Gutteridge (1990), the detection and measurement of lipid peroxidation is evidence most cited in the literature that demonstrates the involvement of free radical reactions in toxicology and human diseases. Use of mitochondria as the source of lipids may mimic human physiological conditions. From the results of DPPH test, concentration of 0.25 mg/mL which showed the best antioxidant activity was chosen to for lipid peroxidation and chemiluminescence test. The red propolis extracts showed inhibitory properties for lipid peroxidation by preventing the formation of reactive species against 2-thiobarbitric acid (TBA). The extracts showed an inhibition of 48.08% to 93.37% at a concentration of IC50 0.95 µg/mL, which were better than those cited in the literature. Argentina propolis extracts showed inhibition from 20 to 80% (ISLA et al., 2001). According to Jasprica et al., (2007), a decrease in the concentration of malondialdehyde (an indicator of lipid peroxidation) by 23.2% was observed in volunteer men patients in Croatia after treatment with propolis, demonstrating its antioxidant action. Marquele-Oliveira et al., (2005) found in their study IC50 values equivalente to 16µg/mL and 12µg/mL for fluid and glycolic extract of brown propolis. As for chemiluminescence technique, Dodeigene et al., (2000) argued that within the techniques of antioxidant activity, this is an advantageous method because of its high sensitivity and speed. When compounds like propolis are added to a chemiluminescent solution, the light emission is reduced, indicating antioxidant activity by removing any radical generated in this system. The chemiluminescence activity of the propolis samples was analysed using one extract from each apiary (CP29 and CJ10). Samples that showed the best lipid peroxidation inhibition were chosen for this assay. Luminol was used as a detector in the chemiluminiscence test, where it is oxidized by the superoxide anion. The inhibition of the luminiscence by the reduction of the superoxide was measured and the extracts of red propolis from Sergipe were shown to possess inhibiting activity for the enzyme XOD, revealing IC50 of 0.096 µL/ml (approximately 96 µg/ml) for sample CP29 and IC50 of 0.089 µL/ml (approximately 89 µg/ml) for sample CJ10 and inhibiting concentration ranging between 15.66 to 89.80% as shown in Figure1. As can bee seem in Figure 1, no significant difference was found between samples CP29 and CJ10. Pascual et al., (1994) found similar values of IC50 ranging from 50 to 95 µg/mL in propolis samples from Cuba by the method of xanthine-xanthine oxidase.
  • 6. Figure 1. Inhibition of light emission from xanthine/luminol/XOD luminescent reactions with luminol found for different concentrationsof: (■) CP 29 and (♦) CJ 10. Results are mean±SD of three experiments run in parallel. 3.3. Ultra Fast Liquid Chromatography – UFLC The hydroalcoholic extracts of propolis showed a complex composition, with several peaks at different retention times as analyzed by UFLC-DAD. The chromatographic profiles of the hydroalcoholic extracts of red propolis indicated the presence of approximately 20 compounds with totally distinct profiles. Presence of flavonoids such as catechin, epicatechin and formononetin was confirmed as shown in Figure 2. Figure 2. Chromatograms of red propolis samples from two different regions of the river São Francisco basin with three samples from apiary Cajuipe (CJ) and three from capivara (CP). 1- Catechin, 2- Epicatechin and 3- Formononetin.
  • 7. And the amount of catechin was quantified to 0.26 to 0.88 mg/ml extract of propolis. Catechins belong to a group of polyphenols that have high antioxidant activity and presents possible mechanisms of action against cancer (HO et al., 1992; RODGERS et al., 1998; PINHEIRO et al., 2014). The flavonoid that appeared with the highest intensity in the red propolis samples is formononetin which is a chemical marker present in the samples of red propolis (JAIN et al., 2014; MENDONÇA et al., 2015). The presence of formononetin in propolis shows estrogenic and antifungal activity. According to Moraes (2009), it has been shown that when mammals consume this isoflavin it is metabolized into daidzein, which is one of isoflavins aglycones present in soy, and is used in the prevention and treatment of menopausal symptoms, and treatment of prostate cancer and breast cancer. 4. Conclusion Propolis extracts analyzed in this study showed yield and total phenols comparable to other similar reports with red propolis. IC50 values for lipid peroxidation were infact better than previous reports suggesting higher potential of the propolis samples from the state of Sergipe. Presence of flavonoids such catechin, epicatechin and formononetin known for their medicinal properties was confirmed by UFLC and it was possible to quantify the catechin content. REFERENCES Albuquerque Junior, R. L. C., Barreto, A. L. S., Pires, J.A., Reis, F. P., Lima, S. O., Ribeiro, M.A.G., Cardoso, J. C. Effect of bovine type-I collagen-based films containing red propolis on dermal wound healing in rodent model. International Journal of Morphology, v. 27, n.4, p. 1105-1110, 2009. Alencar, S.M., Oldoni, T.L.C., Castro, M.L., Cabral, I.S.R., Costa-Neto, C.M., Cury, J.A., Rosalen, P.L., Ikegakid, M. Chemical composition and biological activity of a new type of Brazilian propolis: red propolis. Journal of Ethnopharmacology, v. 113, n. 2, p. 278- 283, 2007. Bors, W., Heller, W., Michel, C., Saran, M. Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods in Enzymology, v.186, p.343-355, 1990. Brand-Williams, W., Cuvelier, M.E., Berset, C. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology, v. 28, p. 25–30, 1995. Brasil. Ministério da Agricultura, Pecuária e do Abastecimento. (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 Apícola, Própolis e Extrato de Própolis, conforme consta dos Anexos desta Instrução Normativa. Diário Oficial da União, Seção 1, Página 18. Cabral, I.S.R., Oldoni, T.L.C., Prado, A., Bezerra, R. M. N., Alencar, S. M. Composição fenólica, atividade antibacteriana e antioxidante da própolis vermelha brasileira. Química Nova, v. XY, p.1-5, 2009. Cain, K, Skilleter, D.N. Preparation and use of mitochondria in toxicological research. In Snell, K.; Mullock , B. (Eds.), Biochemical Toxicology, IRL Press, Oxford. p. 217–254, 1987. Castro, M.L., Cury, J.A., Rosalen, P.L., Alencar, S.M., Ikegaki, M., Duarte, S., Koo, H. Própolis do Sudeste e Nordeste do Brasil: influencia da sazonalidade na atividade antibacteriana e composição fenólica. Química Nova, v. 30, n.7, p. 1512-1516, 2007. Daugsch, A., Moraes, C.S., Fort, P., Park, Y.K. Brazilian Red Propolis - Chemical Composition and Botanical Origin. Evidence-Based Complementary and Alternative Medicine, v.5, n.4, p.435-441, 2007. Daugsch, A., Moraes, C.S., Fort, P., Park, Y.K. Brazilian red propolis - chemical composition and botanical origin. Evidence-Based Complementary and Alternative Medicine, v. 5, p. 435–441, 2008. Dodeigene, L., Thumus, R., Lejeune. Chemiluminescence as a diagnostic tool. A review. Talanta, v.51, p.415–439, 2000.
  • 8. The binary mobile phase was composed of water (solvent A) and methanol (solvent B) according to the method described by Alencar et al., (2007) and Cabral et al., (2009) with modifications. The HAE was dissolved in methanol (50 mg /mL) and filtered with a 0.45 μm filter (Millipore). Aliquots of 2 μL of 5% of HAE (w/v) were injected into the UFLC system. UFLC was performed using a linear gradient elution as follows: 40% B increased to 60% B after 22.5 min, a hold at 90% B for 37.3- 42.3 min, followed by a decrease to 30% B after 42.3 min with a solvent flow rate of 0.4 mL/min. The following authentic standards of flavonoids and phenolic acids were used: formononetin, quercetin, 3-hydroxy-7-methoxyflavone, catechin, epicatechin and propyl gallate. All the tests were performed in triplicates using three samples from each apiary 2.6. DPPH radical scavenging activity The effect of DPPH radical was evaluated by the method described by Brand-Williams et al., 1995 and Tominaga et al., 2005 with adaptations. The assay mixture contained 1.0 mL of 100mM sodium acetate buffer, 50 μL of HAE, 1.0 ml of 95% ethanol, and 500 μL of 200 mM DPPH solution. Absorbance was recorded at 517 nm after 10 min of incubation at room temperature, in the dark. Blank containing the same amount of ethanol and DPPH solution was used as the negative control. The percentage inhibition was plotted against phenol content and IC50 (concentration of total phenol able to scavenge 50% of DPPH free radical) was determined. 2.7. Lipid peroxidation assay Reaction mixture was prepared by adding 10 µL of each sample to 1 mL of a reaction medium containing 130 mM KCl, and 10 mM TrisHCl pH7.4, and mitochondria was added to yield a final concentration of 1 mg of protein. Later 50 µL ferrous ammonium sulfate and 2mM sodium citrate were added and the samples were incubated at 37°C for 30 minutes. Mitochondria was isolated from male Wistar rats livers by differential centrifugation as described by Pedersen et al., 1978, and the mitochondrial protein content was determined by the biuret reaction (CAIN; SKILLETER, 1987). For the determination of TBA-reactive compound, 1 mL of 1% thiobarbituric acid (TBA), 0.1 mL of 10M NaOH and 0.5 mL of H3PO4 were added, followed by incubation in a water bath for 20 minutes at 80°C. The TBA- reactive compounds were extracted with 2 mL of n-butanol and the samples were then centrifuged at 9800 g for 10 min. The readings were taken at 535 nm, using a Hitachi U2001 spectrophotometer. The experiments were performed in triplicates together with blank (without mitochondria), positive control (without sample) and negative control (without iron). The IC50 values were determined using the GraphPad Prism ®. 2.8. Xanthine/luminol/XOD system Samples of red propolis extract were diluted in hydro alcoholic solution to a concentration of 5 mg/mL and subsequent dilutions were prepared in 0.1 M glycine buffer pH 9.4. To verify free radical scavenging activity of the propolis extract, a solution containing 400 μL of 1 mM EDTA and 0.1 M glycine pH 9.4 were added to a test tube together with 150 μL of 6 mM xanthine, 10 μL of test sample and 10 μL of 0.6 mM luminol solution. The reaction was initiated with the addition of 100 μL of freshly prepared cold 20 mU/mL xanthine oxidase solution. The scavenging activity was determined after an incubation of 5 minutes at 25 °C in an Autolumat LB 953 luminometer. The results were expressed according to the concentration of propolis extract in the reaction medium (MARQUELE-OLIVEIRA et al., 2005). The IC50 values were determined using GraphPad Prism® software. 2.9. Statistics All the measurements were performed in triplicates. The average was determined with Assistat 7.7 Beta (registre INPI 0004051-2), and used for the analysis of variance (ANOVA) and Tukey test for means comparison, when necessary. 3. Results and Discussion The hydroalcoholic extracts of all the propolis samples presented yield ranging from 9.45% to 74.62% (w/w) with the exception of two samples that showed lower yield than the value determined by Ministry of Agriculture
  • 9. Fonseca, M. J. V. Assessment of the antioxidant activities of Brazilian extracts of propolis alone and in topical pharmaceutical formulations. Journal of Pharmaceutical and Biomedical Analysis, v.39, p.455-462, 2005. Marquele-Oliveira, F., Fonseca, Y.M., Georgetti, S. R., Vicentini, F. T. M. C., Bronzati, V., Fonseca, M. J. V. Evaluation of the antioxidant activity as an additional parameter to attain the functional quality of natural extracts. Latin American Journal of Pharmacy, v.27, n.3, p. 325–332, 2008. Mendonça, L.S., Mendonça, F.M.R., Maia- Araújo, Y.L.F., Araújo, E.D., Ramalho, S.A., Narain, N., Padilha, F.F., Jain, S., Orellana, S.C., Cardoso, J.C. (Accepted) Chemical markers and antifungal activity of red propolis from Sergipe, Brazil. Food Science and Technology (Campinas). Miguel, M.G., Nunes, S., Dandlen, S. A., Cavaco, A.M. and Antunes, M.D. Phenols and antioxidant activity of hydro-alcoholic extracts of propolis from Algarve, South of Portugal. Food and Chemical Toxicology, v.48, p.3418– 3423, 2010. Moraes, C.S. Isolamento e identificação de formononetina da própolis de João Pessoa-PB, estudo de sua sazonalidade e avaliação de suas atividades biológicas. 2009. Tese de doutorado, Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos. São Paulo, SP, Brasil. Park, Y.K., Ikegaki, M., Alencar, S.M. Classification of Brazilian propolis by both physicochemical methods and biological activity. Mensagem Doce, v.58, p.2-7. 2000. Pascual C., Gonzales R., Torricella R. G. Scavenging action of propolis extract against oxygen radicals. Journal Ethnopharmacology, v.4, p.9-13, 1994. Pedersen P.L., Greenawalt J.W., Reynafarje B., Hullihen J., Decker G.L., Soper J.W. Bustamente E. Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues. Methods in Cell Biology, v..20, p.411– 481, 1978. Pierini, G. D., Granero, A.M., Di Nezio, M.S., Centurión, M.E., Zon, M.A., Fernández, H. Development of an electroanalytical method for the determination of lead in Argentina raw propolis based on bismuth electrodes. Microchemical Journal, v.106, p.102–106, 2013. Pinheiro, M.S. Avaliação da atividade antimicrobiana e citoprotetora gástrica dos extratos de mangaba, caju e própolis vermelha. 2009. Dissertação de mestrado, Universidade Tiradentes – UNIT. Aracaju, SE, Brasil, 2009. Pinheiro, K. S., Ribeiro,D.R, Alves, A.V.F., Pereira-Filho, R.N., Oliveira, C.R., Lima, S.O. Modulatory activity of brazilian red propolis on chemically induced dermal carcinogenesis. Acta Cirúrgica Brasileira, v.29, n.2, p.111- 117, 2014. Ramanauskiene, K., Kalveniene, Z., Kasparaviciene, G., Briedisis, V. Assay of correlation between antioxidant activity of propolis extracts and their chemical components. European Journal of Pharmaceutical Sciences, v.32, p. S45, 2007. Rodgers, E.H., Grant, M.H. The effect of the flavonoids, quercetin, myricetin and epicathechin on the growth and enzyme activities of MCF7 human breast cancer cells. Chemical Biologic Interactions, v.116, n.1-2, p.213-228, 1998. Silva, F.A.S.E., Azevedo, C.A. A New Version of The Assistat-Statistical Assistance Software. In: World Congress on Computers in Agriculture, 4, Orlando-FLUSA: Anais. Orlando: American Society of Agricultural Engineers, p.393-396, 2006. Tominaga, Y., Kobayachi, Y., Goto, T., Kasemura, K., Nomura, M., Yasshi, Y. DPPH Radical–scavenging effect of several phenylpropanoid compounds and their glycoside derivatives. Journal of the Pharmaceutical Society of Japan, v. 125, n.4, p.371–375, 2005. Torel, J., Cillard, J., and Cillard, P. Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry, v.25, n.2, p. 383–385, 1986.
  • 10. ______________________________________ 1. Departamento de Biologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe – Brasil (ylmaia@yahoo.com.br – Maia-Araújo, Y.L.F), (lucyana_biologia@yahoo.com.br – Mendonça, L.S.) e (edaraujo@yahoo.com.br – Araújo, E.D.); 2. Departamento de Engenharia de Alimentos, Universidade Federal de Sergipe, São Cristóvão, Sergipe – Brasil (nayjaracarvalho@hotmail.com – Gualberto, N.C.) e (narendra.narain@gmail.com – Narain, N.); 3. Fundação Oswaldo Cruz, Centro de Pesquisa René Rachou, Belo Horizonte, Minas Gerais, Brasil (sara_cuadros@yahoo.com – Cuadros-Orellana, S.); 4. Instituto de Tecnologia e Pesquisa (ITP), Aracaju, Sergipe – Brasil (juaracaju@yahoo.com.br – Cardoso, J.C.); 5. Departamento de Morfologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe – Brasil (sonajain24@yahoo.com – Jain, S.) *. Corresponding author* Sona Jain, Av. Marechal Rondon, S/N, Bairro Rosa Elze. Universidade Federal de Sergipe, Departamento de Morfologia, São Cristóvão – SE. CEP 49100-000. e-mail: sonajain24@yahoo.com 82