effect on chemical composition and biological a

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Seasonal effect on chemical composition and biological activitiesof 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 Jiménez-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, Mexicob Department of Chemistry-Biology, University of Sonora, Blvd. Luis Encinas y Rosales s/n, Hermosillo, Sonora 83000, Mexicoc Unidad de Servicios de Apoyo en Resolución Analítica, Universidad Veracruzana, Apdo. Postal 575, Xalapa, Ver., Mexicod Laboratorio de Productos Naturales, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, D.F., 04510, Mexico

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 bio-

logical 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, significant 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 significant 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-inflammatory (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 (bever-

ages, health foods, and nutritional supplements), cosmetology,

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 individ-

uals (Munstedt & Kalder, 2009; Walgrave, Warshaw, & Glesne,

2005). These observationsemphasise the needto extend our knowl-

edge about the chemical and biological characterisation (standardi-

sation) 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 flavonoids

have been identified 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 flavonoids 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 deriv-

atives of p-coumaric acid, sesquiterpenes, and of acetophenones

(Bankova, 2005; Piccinelli et al., 2005). Previously, we identified

and quantified the main chemical constituents of Sonoran propolis,

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2011.08.086

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

E-mail address: [email protected] (C. Velazquez).

Food Chemistry 131 (2012) 645–651

Contents lists available at SciVerse ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m

http://dx.doi.org/10.1016/j.foodchem.2011.08.086mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.08.086http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchemhttp://www.elsevier.com/locate/foodchemhttp://www.sciencedirect.com/science/journal/03088146http://dx.doi.org/10.1016/j.foodchem.2011.08.086mailto:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.08.086


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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 conductedto evaluate the

seasonaleffecton the chemicalcomposition andbiologicalactivities

of propolis. Sforcin et al. found no significant differences related to

the seasonal effect on the antimicrobial and immunoregulatory

properties of propolis (Sforcin, Kaneno, & Funari, 2002; Sforcin

et al., 2000;Sforcinet al., 2001). Onthe other hand, Brazilian propolis

collectedmonthlyover a period of one year, had considerablevaria-

tion with respect to antioxidant activity and total phenolic content

(Teixeira, Message, Negri, Salatino, & Stringheta, 2010). This obser-

vation 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). Addi-

tionally, previous reports have shown that seasonality does not sig-

nificant change the chemical composition of propolis, but it can

influence the quantitative chemical profile of propolis (Simoes-

Ambrosio et al., 2010). In the present study, we evaluated the effect

of seasonality on the chemicalcomposition andbiological activity of

propolis collected from a semiarid region of Sonora, Mexico.

2. Materials and methods

2.1. Chemicals

Aluminium chlorideanhydrous, citric acidanhydrous, colchicine

(P95%), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,6-di-tert -butyl-

4-methylphenol (BHT), 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyl-

tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), doxorubi-

cin hydrochloride (P98%), Folin–Ciocalteu reagent, formic acid,

methanol, ethanol, potassium hydroxide, and sodium carbonate

were purchased from SigmaChemicals (St. Louis, MO, USA). The fol-

lowing authentic standards of flavonoids: chrysin, galangin, acace-

tin, naringenin, hesperetin, and pinocembrin were purchased from

INDOFINE Chemical Co., Inc., USA. 5-Fluorouracil (P

99%) was pur-chased fromFluka, BioChemika. Caffeic acid phenethyl ester (CAPE)

wassynthesized 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’’, lo-

cated in Ures, Sonora, Mexico (N 2927018100, W 11023039800).

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 sev-

eral days (usually 3–4 days) with occasional stirring (2–3 times per

day). Then, the extracts were filtered through Whatman grade No.

4 filter paper and concentrated under reduced pressure in a Yam-

ato 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 analy-

sis (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 5RP-18 column (150 4.6 mm, 100 A). The column was eluted

using a water-formic acid/methanol gradient at a flow 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 (15–20 min), 45% B (20–30 min), 60% B (30–

50 min), 80% B (50–65 min), and 100% B (65–71 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 chromato-

gram 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-can-

cer 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 pro-

liferation was determined using the MTT assay (Mosmann, 1983)

with some modifications (Hernandez et al., 2007). Briefly, 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% CO2to 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 0.06–2.0% in the cell cultures caused no cell damage.

Previously, propolis extracts were dissolved in DMSO and subse-quently diluted in culture medium. We used the cytotoxic drugs

5-fluorouracil, 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 ref-

erence wavelength of 650 nm. The antiproliferative activity of

propolis extracts was reported as IC50 values (IC50 was defined as

the concentration of propolis extract required to inhibit cell prolif-

eration 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 modifications. Briefly, propolis sam-

ples dissolved in ethanol (600 ll) were mixed with an equal vol-

ume 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 UV–Vis, Thermo Scientific).

The DPPH radical scavenging activity was determined by compar-

ing the decrease in absorbance of the sample with that of the blank

containing only absolute ethanol. The propolis samples were eval-uated at different concentrations (12.5, 25, 50, and 100 lg/ml).

646 D. Valencia et al./ Food Chemistry 131 (2012) 645–651


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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 asdescribed by Popova et al. (2004, 2005) with slight modifications

(Popova et al., 2004; Singleton & Rossi, 1965; Woisky & Salatino,

1998). Forty microlitres of propolis extract were diluted with

300ll of distilled water and 80 ll of Folin–Ciocalteu reagent and

120ll of a 20% sodium carbonate solution were added. The reac-

tion mixture was brought to 1 ml with distilled water and incu-

bated in the dark (at room temperature) during 2 h. The

absorbance was measured at 760 nm in a spectrophotometer

(Aquamate Plus UV–Vis, Thermo Scientific). The total phenolic con-

tent was estimated using a mixture of pinocembrin and galangin

(2:1 w/w) standard.

2.8. Flavone and flavonol content

The total flavone and flavonol content of propolis samples was

estimated by the colorimetric method based on aluminium chlo-

ride complex formation as described by Popova et al. (Bonvehi &

Coll, 1994; Popova et al., 2004). Forty microlitres of propolis ex-

tract 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 UV–Vis, Thermo Scientific). The results were expressed as mil-

ligrams per gram of propolis of rutin equivalents.

2.9. Flavanone and dihydroflavonol content

The flavanone and dihydroflavonol contents of propolis samples

were quantified by using a published spectrophotometric method

with some modifications (Arzneibuch, 1986; Nagy & Grancai,

1996; Popova et al., 2004). Briefly, 40ll 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 UV–Vis, Thermo Scien-

tific). 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 Turkey–Kra-

mer and Duncan’s 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 antiprolifer-

ative activity on cancer cell lines (Hernandez et al., 2007). Differentamounts of propolis were collected during each season of the year.

The highest amount of collected propolis was obtained during

summer [summer (245g) > fall (60 g) > spring (45 g) > winter

(8 g)]. The organoleptic (colour and consistency) and physical char-

acteristics of collected propolis varied widely among propolis sam-

ples (Table 1).

3.2. Effect of season on the relative abundance of the main chemical

constituents of Sonoran propolis

In a previous study, we identified and quantified the most abun-

dant 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 prop-

olis were analysed by HPLC (Fig. 1). The HPLC chromatographic

profiles of all propolis samples looked very similar. The main peaks

in the chromatograms were identified by using standard samples

and by comparing elution patterns to those obtained previously

(Hernandez et al., 2007). We identified six major propolis constit-

uents (pinocembrin, pinobanksin 3-acetate, chrysin CAPE, acacetin,

and galangin) that have been reported in Sonoran propolis (Her-

nandez 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 identified and unidentified compounds was similar in the four

propolis samples analysed. Overall, the season did not have a sig-

nificant effect on the relative abundance of the main chemical con-

stituents of propolis samples analysed.

3.3. Season has a significant 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 antiprolifera-

tive 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 (IC5026.6 ± 11.5lg/ml) > summer (IC50 49.7 ± 1.4 lg/ml) fall (IC5054.5 ± 2.5 lg/ml). All propolis samples showed a low antiprolifera-

tive activity on the murine normal cell line L-929 (IC50 > 50lg/ml)(Table 3). CAPE (a Sonoran propolis constituent) and the cytotoxic

drugs 5-fluorouracil and doxorubicin had potent antiproliferative

activity (IC50 0.6–1.9 lM) on M12.C3.F6 cells. Cell cultures incu-

bated with DMSO (0.06–0.5%) 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 45 Earthy Brown–green (ochre) Sticky

Summer 245 Flakes Brown–green (ochre) Non-sticky

Fall 60 Earthy Brown–yellow Sticky

Winter 8 Splinter-like appearance Light brown–green Very sticky

D. Valencia et al. / Food Chemistry 131 (2012) 645–651 647


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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 CAPEwere 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 winter18.5% ± 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 at35 lM; vitaminC 94.6% ± 0.1 at70 lM) (Fig. 2). There

were significant differences ( p < 0.05) in the FRS activity of propolissamples only at the highest concentration tested (100 lg/ml).

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



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3.5. Total flavonoid and phenolic content in Sonoran propolis

The biological activities of Sonoran propolis could be due to the

presence of diverse phytochemicals including polyphenolics,

mainly flavonoids (Banskota, Tezuka, & Kadota, 2001). We used

several spectrophotometric methods for the quantification of the

three main groups of bioactive substances (flavones and flavonols,

flavanones and dihydroflavonols, and total phenolics) (Popova

et al., 2004) in Sonoran propolis (Table 4). The fall and summer

propolis extracts showed higher phenolic and flavonoid 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 antiox-idant) 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,

significant differences were observed in the antiproliferative activ-

ity 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 oc-curs in late summer through autumn, when the honey flow is re-

duced (Simone-Finstrom & Spivak, 2010). It has been proposed

that the greater propolis collection (in late summer and early au-

tumn) 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 significant effect on the relative abun-

dance 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 sam-

ples collected. In addition, the HPLC chromatograms obtained from

the propolis samples analysed in this study (2008–2009) were al-most identical to a HPLC chromatogram obtained from a propolis

sample collected in the same area in 2003–2004 (Hernandez

et al., 2007). These findings 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

flavonoids 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 distrib-

uted 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 profiles. Only slight differences in the relative

abundance of some compounds were observed (Table 2). Thisobservation is in agreement with previous reports showing that

Table 2

Relative abundance (%) of the main constituents of Sonoran Propolis.

Peak No. Retention time Relative abundance (%)

Summer Fall Winter Spring

1 13.4(S), 13.3(F), 13.5(w),13.1(SP) 0.9 0.9 1.0 0.9

2 22.6(S), 23.9(F), 23.8(w), 23.8(SP) 1.5 0.2 0.3 1.2

3 23.2(S), 24.3(F), 24.2(w), 24.1(SP) 0.8 0.3 0.6 0.88

4 23.7(S), 24.9(F), 24.8(w), 24.8(SP) 1.1 0.8 0.8 1.1

5 (-)(S), 27.6(F), 27.4(w), (-)(SP) (-) 0.8 0.7 (-)

6 (-)(S), 28.7(F), 28.5(w), (-)(SP) (-) 0.7 0.5 (-)

7 27.9(S), 30.3(F), 30.0(w), 29.9(SP) 3.7 4.9 3.2 3.0

8 29.2(S), 31.7(F), 31.4(w), 31.2(SP) 1.8 0.8 1.3 2.3

9 31.2(S), 33.9(F), 33.5(w), 33.4(SP) 3.5 2.7 3.2 3.7

10 33.6(S), 36.4(F), 36.0(w), 35.8(SP) 7.5 7.1 5.5 6.4

11 (Pinocembrin) 35.7(S), 38.3(F), 37.9(w), 37.8(SP) 21.0 20.3 23.1 22.6

12 (Pinobanksin 3-acetate) 37.7(S), 40.3(F), 40.0(w), 39.8(SP) 14.3 12.2 12.8 13.6

13 (-)(S), 42.1(F), 41.8(w), (-)(SP) (-) 1.7 1.9 (-)

14 (CAPE) 41.5(S), 43.8(F), 43.5(w), 43.4(SP) 3.3 2.4 3.7 4.3

15 (Chrysin) 43.2(S), 45.4(F), 45.1(w), 45.0(SP) 8.2 10.6 7.9 6.6

16 (Galangin) 44.2(S), 46.2(F), 45.9(w), 45.9(SP) 7.3 10.2 11.9 8.6

17 (Acacetin) 45.1(S), 47.2(F), 46.9(w), 46.8(SP) 6.8 8.9 7.2 6.0

18 50.0(S), 51.7(F), 51.4(w), 51.4(SP) 0.7 0.1 0.6 1.6

19 52.4(S), 53.2(F), 53.1(w), 53.1(SP) 1.9 3.5 2.8 2.2

20 53.6(S), 54.0 (F), 53.9(w), 53.9(SP) 1.4 2.5 3.3 2.1

21 54.5(S), 54.7(F), 54.7(w), 54.7(SP) 1.6 2.4 1.6 1.4

22 56.1(S), 56.3(F), 56.3(w), 56.3(SP) 1.3 2.1 1.9 1.223 Unassigned peaks 11.4 3.9 4.2 10.32

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

Table 3

Antiproliferative activity (IC50)a of Sonoran propolis.

Propolis extract or compound Cell lines

M12.C3.F6 L-929

Spring 11.6 ± 4.6 56.7 ± 13.2

Winter 26.6 ± 11.5 50.7 ± 7.6Summer 49.7 ± 1.4 51.0 ± 3.0

Fall 54.5 ± 2.5 54.6 ± 5.1

CAPEb 1.6 ± 0.5 >100

5-Fluorouracilb 1.9 ± 0.6 >100

Colchicineb ND >100

Doxorubicin hydrochloride 0.6 ± 0.01 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-fluorouracil, colchicine, and doxorubicin hydrochloride were used as

positive controls in the antiproliferative assays.



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seasonality does not significantly change the chemical composition

of propolis (qualitative chromatographic profile), but it can influ-

ence the quantitative chemical profile of propolis (Simoes-Ambro-

sio et al., 2010).

Significant 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 compositionsof the propolis samples (based on HPLC analysis), evident differ-

ences in their antiproliferative activities were found. Probably,

those differences could be due to small quantitative variations in

the propolis constituents, which could influence the biological

activity of propolis. Another possible explanationfor those observa-

tions could be that unidentified compounds with potent antiprolif-

erative 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 re-

quire additional studies.

Additionally, we found significant differences ( p < 0.05, at the

highest propolis concentration tested (100 lg/ml)) in the antioxi-

dant activity of the propolis extracts. This observation is in agree-

ment with previous reports that showed an effect of theseasonality 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 modifications in the chemical

composition of propolis can greatly influence its biological activity.

The presence of phenolic compounds contributes significantly to

the antioxidant activity. We found a correlation between the total

phenolic content of propolis and its antioxidant activity. Fall prop-

olis had the highest phenolic content and the highest FRS activityand, conversely, spring propolis showed the lowest phenolic con-

tent and the lowest antioxidant activity. On the other hand, there

wasno correlation between the antiproliferative activity of propolis

and its antioxidant activity or its total flavonoid and phenolic con-

tent. From a previous study from an our group, we know that the

propolis constituents CAPE and galagin possess potent antiprolifer-

ative activity on cancercells, andthose compounds also have signif-

icant FRS activity (Velazquez et al., 2007). Further studies are

needed to advance our understanding about the differences in bio-

logical 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 importantfor practical application of propolis in the pharmaceutical and food

0.0 12.5 25.0 50.0 100.0

0

25

50

75

100

BHT CAPE VIT C

Summer propolis ( g/mL)

* **

*

*

*

*

0.0 512. 25.0 50.0 100.0

0

25

50

75

100

Fall propolis ( g/mL)

BHT CAPE VIT C

* **

*

**

*

0.0 12.5 25.0 50.0 100.0

0

25

50

75

100

BHT CAPE VIT C

Winter propolis ( g/mL)

* ** *

*

*

*

0.0 12.5 25.0 50.0 100.0

0

25

50

75

100

BHT CAPE VIT C

Spring propolis ( g/mL)

* * **

**

*

F r e e - R a d i c a l S c a v e n g i n g a c t i v i t y ( % )

Fig. 2. DPPH free-radical scavenging activity of propolis extracts. Different concentrations of propolis extract (0–100 lg/ml) were used in the DPPH assays. Vitamin C

(70lM), 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. Significant differences ( p < 0.05) from control are marked with asterisk.

Table 4

Total flavonoid and phenolic content in Sonoran propolis (mg/g).

Summer Fall Winter Spring

Flavanones and dihydroflavonolsa 305.9 ± 5.8 246.2 ± 9.2 228.0 ± 3.9 237.1 ± 7.4

Flavones and flavonolsb 185.9 ± 3.2 133.0 ± 2.2 104.5 ± 3.3 96.8 ± 0.3Total phenolicsc 601.8 ± 8.2 629.6 ± 9.1 532.3 ± 6.7 427.9 ± 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.



7/7

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

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 knowl-

edge, this is the first 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 Sci-

ence and Technology of Mexico (CONACYT, 83462).

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effect on chemical composition and biological a

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