Food Chemistry 131 (2012) 645651

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

Seasonal effect on chemical composition and biological activities

of Sonoran propolis
Dora Valencia a, Efrain Alday b, Ramon Robles-Zepeda b, Adriana Garibay-Escobar b, Juan C. Galvez-Ruiz b,
Magali Salas-Reyes c, Manuel Jimnez-Estrada d, Enrique Velazquez-Contreras a, Javier Hernandez c,
Carlos Velazquez b,
a

Department of Polymers and Materials (DIPM), University of Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora 83000, Mexico
Department of Chemistry-Biology, University of Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora 83000, Mexico
Unidad de Servicios de Apoyo en Resolucin Analtica, Universidad Veracruzana, Apdo. Postal 575, Xalapa, Ver., Mexico
d
Laboratorio de Productos Naturales, Instituto de Qumica, Universidad Nacional Autnoma de Mxico, Ciudad Universitaria, D.F., 04510, Mexico
b
c

a r t i c l e

i n f o

Article history:
Received 19 March 2011
Received in revised form 2 July 2011
Accepted 30 August 2011
Available online 19 September 2011
Keywords:
Sonoran propolis
Seasonal effect
Antiproliferative activity
Chemical composition

a b s t r a c t
Propolis is widely used as a folk medicine and as a constituent of health foods in many parts of the world.
The main purpose of this study was to evaluate the seasonal effect on the chemical composition and biological activities (antiproliferative and antioxidant activities) of Sonoran propolis (Mexico). Propolis was
collected during all four seasons of the year and chemical composition, antiproliferative, and free-radical
scavenging activities of collected propolis were evaluated by HPLC, MTT, and DPPH assays, respectively.
The relative abundance of the main chemical constituents of propolis was similar in all propolis samples
analysed. In contrast, signicant differences were observed in their antiproliferative activity in the B-cell
lymphoma cancer cell line M12.C3.F6. Propolis collected in the spring showed the highest inhibitory
effect on the growth of cancer cells. All propolis samples had weak free-radical scavenging activity.
Our results indicate that season had a signicant effect on the antiproliferative properties of Sonoran
propolis.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Propolis is a resinous material that is collected by honeybees
from buds, leaves, bark, and exudates of several trees and plants;
(Hernandez et al., 2007; Lotti et al., 2010). This natural product has
a long history of use in traditional medicine dating back at least to
300 BC (Ghisalberti, 1979). Propolis possesses a broad spectrum of
biological activities including anti-cancer (Bufalo, Candeias, &
Sforcin, 2009; Hernandez et al., 2007; Li et al., 2009), antioxidant
(Ahn, Kumazawa, Hamasaka, Bang, & Nakayama, 2004; Lima et al.,
2009; Velazquez et al., 2007), fungicidal (Majiene, Trumbeckaite,
Pavilonis, Savickas, & Martirosyan, 2007; Sforcin, Fernandes Junior,
Lopes, Funari, & Bankova, 2001), antibacterial (Jorge et al., 2008;
Popova, Silici, Kaftanoglu, & Bankova, 2005; Sforcin, Fernandes,
Lopes, Bankova, & Funari, 2000; Velazquez et al., 2007), antiviral
(Amoros et al., 1994), and anti-inammatory (Paulino et al., 2003)
properties among others. As a result of this wide range of biological
activities, propolis is extensively used in the food industry (beverages, health foods, and nutritional supplements), cosmetology,

Corresponding author. Tel./fax: +52 662 259 21 63.

E-mail address: velaz@guayacan.uson.mx (C. Velazquez).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.08.086

alternative medicine products (toothpaste, soap, syrup, and candy)

as well as in home remedies. Unfortunately, propolis can also cause
some serious side effects such as allergic reactions in some individuals (Munstedt & Kalder, 2009; Walgrave, Warshaw, & Glesne,
2005). These observations emphasise the need to extend our knowledge about the chemical and biological characterisation (standardisation) of propolis, which would aid the appropriate use of this
natural product in human health.
Currently, more than 300 compounds, such as phenolic acid,
terpenes, cinnamic acid, caffeic acid, several esters, and avonoids
have been identied as constituents of propolis from different
geographic origins (Paulino et al., 2003; Salatino, Teixeira, Negri, &
Message, 2005; Senedese et al., 2008). The chemical composition
of propolis is qualitatively and quantitatively variable, depending
on the vegetation at the site from which it was collected and the
time of collection (Ahn et al., 2004; Jorge et al., 2008; Lotti et al.,
2010; Piccinelli et al., 2005). The main constituents of propolis in
Europe, China, and North America are avonoids and phenolic acid
esters (Bankova, de Castro, & Marcucci, 2000; Chen, Weng, Wu, &
Lin, 2004; Lotti et al.,2010). Among the main compound classes
found in Brazilian propolis are diterpenes, lignans, prenylated derivatives of p-coumaric acid, sesquiterpenes, and of acetophenones
(Bankova, 2005; Piccinelli et al., 2005). Previously, we identied
and quantied the main chemical constituents of Sonoran propolis,

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D. Valencia et al. / Food Chemistry 131 (2012) 645651

which possess strong antiproliferative activity against cancer cell

lines (Hernandez et al., 2007). Additionally, Sonoran propolis
showed potent antibacterial activity (Velazquez et al., 2007).
A limited number of studies have been conducted to evaluate the
seasonal effect on the chemical composition and biological activities
of propolis. Sforcin et al. found no signicant differences related to
the seasonal effect on the antimicrobial and immunoregulatory
properties of propolis (Sforcin, Kaneno, & Funari, 2002; Sforcin
et al., 2000; Sforcin et al., 2001). On the other hand, Brazilian propolis
collected monthly over a period of one year, had considerable variation with respect to antioxidant activity and total phenolic content
(Teixeira, Message, Negri, Salatino, & Stringheta, 2010). This observation is in agreement with work from the laboratories of Isla and
of Chen who reported that antioxidant activity of propolis depends
on the collection month (Chen et al., 2008; Isla et al., 2009). Additionally, previous reports have shown that seasonality does not signicant change the chemical composition of propolis, but it can
inuence the quantitative chemical prole of propolis (SimoesAmbrosio et al., 2010). In the present study, we evaluated the effect
of seasonality on the chemical composition and biological activity of
propolis collected from a semiarid region of Sonora, Mexico.
2. Materials and methods
2.1. Chemicals
Aluminium chloride anhydrous, citric acid anhydrous, colchicine
(P95%), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,6-di-tert-butyl4-methylphenol (BHT), 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), doxorubicin hydrochloride (P98%), FolinCiocalteu reagent, formic acid,
methanol, ethanol, potassium hydroxide, and sodium carbonate
were purchased from Sigma Chemicals (St. Louis, MO, USA). The following authentic standards of avonoids: chrysin, galangin, acacetin, naringenin, hesperetin, and pinocembrin were purchased from
INDOFINE Chemical Co., Inc., USA. 5-Fluorouracil (P99%) was purchased from Fluka, BioChemika. Caffeic acid phenethyl ester (CAPE)
was synthesized based on the procedure of Grunberger et al. (1988).
2.2. Propolis and methanolic extracts from propolis
Raw propolis was collected during each season of the year,
spring (from March 21, 2008 to June 21, 2008), summer (from June
22, 2008 to September 22, 2008), fall (from September 23, 2008 to
December 21, 2008), and winter (December 22, 2008 to March 20,
2009). The hives were located in the area known as El Coyote, located in Ures, Sonora, Mexico (N 29270 18100 , W 110230 39800 ).
Propolis samples used in this study were collected from 12 hives
each season. Previously, we reported that propolis from this region
possessed strong antiproliferative activity (Hernandez et al., 2007).
Propolis samples (5 g) were cut into small pieces and extracted
three times (at room temperature) with methanol (30 ml) for several days (usually 34 days) with occasional stirring (23 times per
day). Then, the extracts were ltered through Whatman grade No.
4 lter paper and concentrated under reduced pressure in a Yamato RE300 Rotary Evaporator. The methanolic extracts were
washed three times with hexane to remove waxes. The wax-free
methanolic extracts were stored in the dark at 20 C until analysis (Hernandez et al., 2007; Velazquez et al., 2007).
2.3. HPLC analysis
Analysis of propolis constituents was performed on a Varian
ProStar 210 (Walnut Creek, USA) equipped with a LiChrospher 5
RP-18 column (150  4.6 mm, 100 A). The column was eluted

using a water-formic acid/methanol gradient at a ow rate of

1 ml/min. The mobile phase consisted of 5% formic acid in water
(A) and methanol (B). The gradient program was 30% B (0
15 min), 40% B (1520 min), 45% B (2030 min), 60% B (30
50 min), 80% B (5065 min), and 100% B (6571 min). The elution
of the compounds was monitored at 280 nm and 340 nm. The
assignment of peaks was performed using the authentic standards:
pinocembrin, pinobanksin 3-acetate, CAPE, chrysin, galangin, and
acacetin (Hernandez et al., 2007). The peak areas of each chromatogram were measured and the relative abundance of each peak was
calculated (peak area divided by total area of all peaks recorded in
the chromatogram).
2.4. Cell lines
The cancerous cell line, M12.C3.F6 (murine B-cell lymphoma)
was provided by Dr. Emil R. Unanue (Department of Pathology
and Immunology, Washington University in St. Louis, MO, USA).
We used this cancer cell line due to its high sensitivity to anti-cancer drugs and propolis extracts (Hernandez et al., 2007). The cell
line NCTC clone L-929 (normal subcutaneous connective tissue)
was purchased from the American Type Culture Collection (ATCC;
Rockville, MD, USA).
2.5. Antiproliferative assays (cell viability assay)
To evaluate the effect of propolis extract on cell lines, cell proliferation was determined using the MTT assay (Mosmann, 1983)
with some modications (Hernandez et al., 2007). Briey, cells
(1  104 per well, 50 ll) were placed in each well of a 96-well
plate. After 24 h incubation at 37 C in an atmosphere of 5% CO2
to allow cell attachment, aliquots (50 ll) of medium containing
different concentrations of propolis extracts were added and the
cell cultures were incubated for 48 h. Preliminary experiments,
established that use of dimethyl sulfoxide (DMSO) concentrations
ranging from 0.062.0% in the cell cultures caused no cell damage.
Previously, propolis extracts were dissolved in DMSO and subsequently diluted in culture medium. We used the cytotoxic drugs
5-uorouracil, doxorubicin, colchicine, and CAPE (a constituent of
Ures propolis) as positive controls in the antiproliferative assays.
In the last 4 h of the cell culture, 10 ll of a MTT solution (5 mg/
ml) were added to each well. The cell viability was assessed by
the ability of metabolically active cells to reduce tetrazolium salt
to coloured formazan compounds. The formed formazan crystals
were dissolved with acidic isopropyl alcohol. The absorbance of
the samples was measured with an ELISA plate reader (Multiskan
EX, ThermoLabSystem), using a test wavelength of 570 nm and reference wavelength of 650 nm. The antiproliferative activity of
propolis extracts was reported as IC50 values (IC50 was dened as
the concentration of propolis extract required to inhibit cell proliferation by 50%).
2.6. Free-radical scavenging activity (DPPH assay)
Radical scavenging activity was measured by DPPH assay
according to the procedure described by Usia et al. (Blois, 1958;
Usia et al., 2002) with slight modications. Briey, propolis samples dissolved in ethanol (600 ll) were mixed with an equal volume of DPPH solution (300 lM). The resulting solution was
thoroughly mixed by vortex. After 30 min of incubation in the dark
(at room temperature), absorbance was measured at 517 nm in a
spectrophotometer (Aquamate Plus UVVis, Thermo Scientic).
The DPPH radical scavenging activity was determined by comparing the decrease in absorbance of the sample with that of the blank
containing only absolute ethanol. The propolis samples were evaluated at different concentrations (12.5, 25, 50, and 100 lg/ml).

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D. Valencia et al. / Food Chemistry 131 (2012) 645651

Vitamin C (70 lM), CAPE (35 lM), and BHT (140 lM) were used as
antioxidant standards. The free-radical scavenging activity (DPPH
assay) results were expressed as a percentage decrease in the
absorbance with respect to the control values.
2.7. Total phenolic content
Total phenolic content of propolis extracts was determined as
described by Popova et al. (2004, 2005) with slight modications
(Popova et al., 2004; Singleton & Rossi, 1965; Woisky & Salatino,
1998). Forty microlitres of propolis extract were diluted with
300 ll of distilled water and 80 ll of FolinCiocalteu reagent and
120 ll of a 20% sodium carbonate solution were added. The reaction mixture was brought to 1 ml with distilled water and incubated in the dark (at room temperature) during 2 h. The
absorbance was measured at 760 nm in a spectrophotometer
(Aquamate Plus UVVis, Thermo Scientic). The total phenolic content was estimated using a mixture of pinocembrin and galangin
(2:1 w/w) standard.
2.8. Flavone and avonol content
The total avone and avonol content of propolis samples was
estimated by the colorimetric method based on aluminium chloride complex formation as described by Popova et al. (Bonvehi &
Coll, 1994; Popova et al., 2004). Forty microlitres of propolis extract were diluted with 400 ll of methanol and 20 ll of 5% AlCl3
(w/v) were added. The volume was brought to 1 ml with methanol.
The mixture was incubated for 30 min (at room temperature). The
absorbance was read at 425 nm in a spectrophotometer (Aquamate
Plus UVVis, Thermo Scientic). The results were expressed as milligrams per gram of propolis of rutin equivalents.
2.9. Flavanone and dihydroavonol content
The avanone and dihydroavonol contents of propolis samples
were quantied by using a published spectrophotometric method
with some modications (Arzneibuch, 1986; Nagy & Grancai,
1996; Popova et al., 2004). Briey, 40 ll propolis extract were
mixed with 80 ll of 2,4-dinitrophenylhydrazine (DNP) solution
[50 mg DNP in 100 ll 96% sulphuric acid (v/v), diluted to 5 ml with
methanol] and heated at 50 C for 50 min. After cooling (at room
temperature), the mixture was diluted to 400 ll with 10% KOH in
methanol (w/v) and 20 ll of the resulting solution were diluted
to 1 ml with methanol. The absorbance was measured at 486 nm
in a spectrophotometer (Aquamate Plus UVVis, Thermo Scientic). The results were expressed as milligrams of pinocembrin
equivalents per gram of propolis.
2.10. Statistical analysis
Data were analysed using analysis of variance with TurkeyKramer and Duncans multiple comparison tests [Number Cruncher
Statistical Software (NCSS) 2000].

3. Results
3.1. Organoleptic and physical characteristics of collected propolis
Sonoran propolis was collected throughout all four seasons of
the year, from March 21, 2008 to March 20, 2009. Previously, we
have shown that propolis from this region has strong antiproliferative activity on cancer cell lines (Hernandez et al., 2007). Different
amounts of propolis were collected during each season of the year.
The highest amount of collected propolis was obtained during
summer [summer (245 g) > fall (60 g) > spring (45 g) > winter
(8 g)]. The organoleptic (colour and consistency) and physical characteristics of collected propolis varied widely among propolis samples (Table 1).
3.2. Effect of season on the relative abundance of the main chemical
constituents of Sonoran propolis
In a previous study, we identied and quantied the most abundant constituents of Sonoran propolis (Hernandez et al., 2007). In
order to compare the chemical composition of Sonoran propolis
collected during different seasons, methanolic extracts from propolis were analysed by HPLC (Fig. 1). The HPLC chromatographic
proles of all propolis samples looked very similar. The main peaks
in the chromatograms were identied by using standard samples
and by comparing elution patterns to those obtained previously
(Hernandez et al., 2007). We identied six major propolis constituents (pinocembrin, pinobanksin 3-acetate, chrysin CAPE, acacetin,
and galangin) that have been reported in Sonoran propolis (Hernandez et al., 2007). The relative abundance of the main HPLC
peaks of each chromatogram was determined (Table 2). The most
abundant constituents of propolis samples were pinocembrin,
pinobanksin 3-acetate, and chrysin. The relative abundance of
the identied and unidentied compounds was similar in the four
propolis samples analysed. Overall, the season did not have a signicant effect on the relative abundance of the main chemical constituents of propolis samples analysed.
3.3. Season has a signicant effect on the antiproliferative activity of
Sonoran propolis
To evaluate the growth inhibitory activity of Sonoran propolis
samples on cancer cells, we performed antiproliferative assays
using propolis extracts. All propolis samples had an antiproliferative activity on the cancer cell line M12.C3.F6, with spring propolis
showing the highest inhibitory effect on the growth of cancer cells.
The order of antiproliferative activity of propolis extracts on
M12.C3.F6 cells was: spring (IC50 11.6 4.6 lg/ml) > winter (IC50
26.6 11.5 lg/ml) > summer (IC50 49.7 1.4 lg/ml)  fall (IC50
54.5 2.5 lg/ml). All propolis samples showed a low antiproliferative activity on the murine normal cell line L-929 (IC50 > 50 lg/ml)
(Table 3). CAPE (a Sonoran propolis constituent) and the cytotoxic
drugs 5-uorouracil and doxorubicin had potent antiproliferative
activity (IC50 0.61.9 lM) on M12.C3.F6 cells. Cell cultures incubated with DMSO (0.060.5%) did not show any evidence of cell
damage.

Table 1
Organoleptic and physical characteristics of collected propolis.
Season

Propolis collected (g)

Physical aspects

Colour

Consistency

Spring
Summer
Fall
Winter

45
245
60
8

Earthy
Flakes
Earthy
Splinter-like appearance

Browngreen (ochre)
Browngreen (ochre)
Brownyellow
Light browngreen

Sticky
Non-sticky
Sticky
Very sticky

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D. Valencia et al. / Food Chemistry 131 (2012) 645651

Fig. 1. HPLC chromatograms of Sonoran propolis samples (recorded at 340 nm).

3.4. Effect of season on the free-radical scavenging activity of Sonoran

propolis
We investigated the free-radical scavenging activity (FRS) of
propolis samples, using the DPPH assay. Vitamin C, BHT, and CAPE
were used as antioxidant standards. All propolis samples had a

weak FRS activity (spring 16.2% 0.4, summer 17.0% 0.2, fall
19.7% 0.6, and winter 18.5% 0.2, at 100 lg/ml) as compared with
those of antioxidant standards (BHT 51.8% 2.1 at 140 lM; CAPE
65.0% 0.2 at 35 lM; vitamin C 94.6% 0.1 at 70 lM) (Fig. 2). There
were signicant differences (p < 0.05) in the FRS activity of propolis
samples only at the highest concentration tested (100 lg/ml).

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D. Valencia et al. / Food Chemistry 131 (2012) 645651

Table 2
Relative abundance (%) of the main constituents of Sonoran Propolis.
Peak No.

Retention time

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

13.4(S), 13.3(F), 13.5(w),13.1(SP)

22.6(S), 23.9(F), 23.8(w), 23.8(SP)
23.2(S), 24.3(F), 24.2(w), 24.1(SP)
23.7(S), 24.9(F), 24.8(w), 24.8(SP)
(-)(S), 27.6(F), 27.4(w), (-)(SP)
(-)(S), 28.7(F), 28.5(w), (-)(SP)
27.9(S), 30.3(F), 30.0(w), 29.9(SP)
29.2(S), 31.7(F), 31.4(w), 31.2(SP)
31.2(S), 33.9(F), 33.5(w), 33.4(SP)
33.6(S), 36.4(F), 36.0(w), 35.8(SP)
35.7(S), 38.3(F), 37.9(w), 37.8(SP)
37.7(S), 40.3(F), 40.0(w), 39.8(SP)
(-)(S), 42.1(F), 41.8(w), (-)(SP)
41.5(S), 43.8(F), 43.5(w), 43.4(SP)
43.2(S), 45.4(F), 45.1(w), 45.0(SP)
44.2(S), 46.2(F), 45.9(w), 45.9(SP)
45.1(S), 47.2(F), 46.9(w), 46.8(SP)
50.0(S), 51.7(F), 51.4(w), 51.4(SP)
52.4(S), 53.2(F), 53.1(w), 53.1(SP)
53.6(S), 54.0 (F), 53.9(w), 53.9(SP)
54.5(S), 54.7(F), 54.7(w), 54.7(SP)
56.1(S), 56.3(F), 56.3(w), 56.3(SP)
Unassigned peaks

(Pinocembrin)
(Pinobanksin 3-acetate)
(CAPE)
(Chrysin)
(Galangin)
(Acacetin)

Relative abundance (%)

Summer

Fall

Winter

Spring

0.9
1.5
0.8
1.1
(-)
(-)
3.7
1.8
3.5
7.5
21.0
14.3
(-)
3.3
8.2
7.3
6.8
0.7
1.9
1.4
1.6
1.3
11.4

0.9
0.2
0.3
0.8
0.8
0.7
4.9
0.8
2.7
7.1
20.3
12.2
1.7
2.4
10.6
10.2
8.9
0.1
3.5
2.5
2.4
2.1
3.9

1.0
0.3
0.6
0.8
0.7
0.5
3.2
1.3
3.2
5.5
23.1
12.8
1.9
3.7
7.9
11.9
7.2
0.6
2.8
3.3
1.6
1.9
4.2

0.9
1.2
0.88
1.1
(-)
(-)
3.0
2.3
3.7
6.4
22.6
13.6
(-)
4.3
6.6
8.6
6.0
1.6
2.2
2.1
1.4
1.2
10.32

(-)

Not detected.
Summer.
(F)
Fall.
(W)
Winter.
(SP)
Spring.
(S)

Table 3
Antiproliferative activity (IC50)a of Sonoran propolis.
Propolis extract or compound

Spring
Winter
Summer
Fall
CAPEb
5-Fluorouracilb
Colchicineb
Doxorubicin hydrochloride

Cell lines
M12.C3.F6

L-929

11.6 4.6
26.6 11.5
49.7 1.4
54.5 2.5
1.6 0.5
1.9 0.6
ND
0.6 0.01

56.7 13.2
50.7 7.6
51.0 3.0
54.6 5.1
>100
>100
>100
ND

ND: not determined.

a
IC50 values of propolis extracts (lg/ml) or compounds (lM) are representative
of at least three independent experiments. All values represent mean of triplicate
determinations SD.
b
CAPE, 5-uorouracil, colchicine, and doxorubicin hydrochloride were used as
positive controls in the antiproliferative assays.

3.5. Total avonoid and phenolic content in Sonoran propolis

The biological activities of Sonoran propolis could be due to the
presence of diverse phytochemicals including polyphenolics,
mainly avonoids (Banskota, Tezuka, & Kadota, 2001). We used
several spectrophotometric methods for the quantication of the
three main groups of bioactive substances (avones and avonols,
avanones and dihydroavonols, and total phenolics) (Popova
et al., 2004) in Sonoran propolis (Table 4). The fall and summer
propolis extracts showed higher phenolic and avonoid contents
than the winter and spring propolis.
4. Discussion
In this study, we analysed the effect of seasonality on chemical
composition and biological activities (antiproliferative and antioxidant) of Sonoran propolis collected over a 12-month period. The

relative abundance (HPLC analysis) of the main constituents of

propolis was similar in all propolis samples analysed. However,
signicant differences were observed in the antiproliferative activity of the propolis extracts tested.
The largest amount of collected propolis was obtained during
summer. It is thought that the greatest resin collection by bees occurs in late summer through autumn, when the honey ow is reduced (Simone-Finstrom & Spivak, 2010). It has been proposed
that the greater propolis collection (in late summer and early autumn) is due to seasonal changes in the behaviour of the foragers,
and is not the result of changes of climatic conditions or the need
to prepare the hive for winter (Ghisalberti, 1979; Simone-Finstrom
& Spivak, 2010). Additionally, the propolis collection also depends
on the availability of the resin sources found in the hive area.
The season did not have a signicant effect on the relative abundance of the major chemical constituents of Sonoran propolis. The
propolis constituents pinocembrin, pinobanksin 3-acetate, chrysin,
CAPE, acacetin, and galangin were present in all four propolis samples collected. In addition, the HPLC chromatograms obtained from
the propolis samples analysed in this study (20082009) were almost identical to a HPLC chromatogram obtained from a propolis
sample collected in the same area in 20032004 (Hernandez
et al., 2007). These ndings suggest that the chemical constitution
of Ures propolis is very stable and also indicate that the main
botanical source of Sonoran propolis is available during all four
seasons of the year. The presence, in high concentration, of the
avonoids pinocembrin, pinobanksin 3-acetate, and chrysin in
the propolis samples (Table 2) suggests that its main botanical
source is Populus fremonti, which is a perennial tree widely distributed in the propolis collection area (Bankova, 2005; Bankova et al.,
2000; Garciaviguera, Ferreres, & Tomasbarberan, 1993; Hernandez
et al., 2007). All propolis extracts had very similar qualitative HPLC
chromatographic proles. Only slight differences in the relative
abundance of some compounds were observed (Table 2). This
observation is in agreement with previous reports showing that

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D. Valencia et al. / Food Chemistry 131 (2012) 645651

100

75

75

Free-Radical Scavenging activity (%)

100

*
*

50

25

50

25

0.0 12.5 25.0 50.0 100.0 BHT CAPE VIT C

0.0 12.5 25.0 50.0 100.0 BHT CAPE VIT C

Fall propolis ( g/mL)

Summer propolis ( g/mL)

100

100

*
75

75

*
*

50

25

*
0

50

25

0.0 12.5 25.0 50.0 100.0 BHT CAPE VIT C

0.0 12.5 25.0 50.0 100.0 BHT CAPE VIT C

Spring propolis ( g/mL)

Winter propolis ( g/mL)

Fig. 2. DPPH free-radical scavenging activity of propolis extracts. Different concentrations of propolis extract (0100 lg/ml) were used in the DPPH assays. Vitamin C
(70 lM), BHT (140 lM), and CAPE (35 lM) were used as antioxidant standards. The results shown are representative of at least three independent experiments. All values
represent mean of triplicate determinations SD. Signicant differences (p < 0.05) from control are marked with asterisk.

Table 4
Total avonoid and phenolic content in Sonoran propolis (mg/g).

Flavanones and dihydroavonolsa

Flavones and avonolsb
Total phenolicsc

Summer

Fall

Winter

Spring

305.9 5.8
185.9 3.2
601.8 8.2

246.2 9.2
133.0 2.2
629.6 9.1

228.0 3.9
104.5 3.3
532.3 6.7

237.1 7.4
96.8 0.3
427.9 9.9

The results shown are representative of at least three independent experiments. All values represent mean of triplicate determinations SD.
a
Expressed as pinocembrin equivalent.
b
Expressed as rutin equivalent.
c
Expressed as pinocembrin/galangin equivalent.

seasonality does not signicantly change the chemical composition

of propolis (qualitative chromatographic prole), but it can inuence the quantitative chemical prole of propolis (Simoes-Ambrosio et al., 2010).
Signicant differences were found in the abilities of the propolis
samples collected during different seasons to inhibit the growth of
cancer cells. Despite the similarities in the chemical compositions
of the propolis samples (based on HPLC analysis), evident differences in their antiproliferative activities were found. Probably,
those differences could be due to small quantitative variations in
the propolis constituents, which could inuence the biological
activity of propolis. Another possible explanation for those observations could be that unidentied compounds with potent antiproliferative effect are present in different abundance in the propolis
samples tested. Elucidation of the chemical basis for the seasonal
effect on the antiproliferative activity of Sonoran propolis will require additional studies.
Additionally, we found signicant differences (p < 0.05, at the
highest propolis concentration tested (100 lg/ml)) in the antioxidant activity of the propolis extracts. This observation is in agreement with previous reports that showed an effect of the
seasonality on antioxidant activity of propolis (Chen et al., 2008; Isla

et al., 2009; Teixeira et al., 2010). This difference in the antioxidant

activity reinforces the idea that slight modications in the chemical
composition of propolis can greatly inuence its biological activity.
The presence of phenolic compounds contributes signicantly to
the antioxidant activity. We found a correlation between the total
phenolic content of propolis and its antioxidant activity. Fall propolis had the highest phenolic content and the highest FRS activity
and, conversely, spring propolis showed the lowest phenolic content and the lowest antioxidant activity. On the other hand, there
was no correlation between the antiproliferative activity of propolis
and its antioxidant activity or its total avonoid and phenolic content. From a previous study from an our group, we know that the
propolis constituents CAPE and galagin possess potent antiproliferative activity on cancer cells, and those compounds also have significant FRS activity (Velazquez et al., 2007). Further studies are
needed to advance our understanding about the differences in biological activity observed and to relate the chemical composition of
Sonoran propolis to its antiproliferative and antioxidant properties.
Knowledge of the seasonal effects on the chemical and biological
properties of propolis will contribute to the characterisation and
standardisation of this natural product and it could be important
for practical application of propolis in the pharmaceutical and food

D. Valencia et al. / Food Chemistry 131 (2012) 645651

industries. In conclusion, we found that season had no signicant

effects on the relative abundance of the main chemical constituents
of Sonoran propolis. However, there were marked differences in the
antiproliferative activities of this natural product. To our knowledge, this is the rst study that described the seasonal effects on
chemical composition and antiproliferative properties of propolis
collected from a semiarid region of the American continent.
Acknowledgements
We thank professional beekeeper Gilberto Valenzuela for all
facilities provided for propolis collection. We are grateful to Karla
Martinez Robinson for technical support of this research. This work
was partially supported by a Grant from National Council for Science and Technology of Mexico (CONACYT, 83462).
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