effect on chemical composition and biological a
Post on 06-Jul-2018
214 Views
Preview:
TRANSCRIPT
-
8/17/2019 Effect on Chemical Composition and Biological A
1/7
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: velaz@guayacan.uson.mx (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:velaz@guayacan.uson.mxhttp://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:velaz@guayacan.uson.mxhttp://dx.doi.org/10.1016/j.foodchem.2011.08.086
-
8/17/2019 Effect on Chemical Composition and Biological A
2/7
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
-
8/17/2019 Effect on Chemical Composition and Biological A
3/7
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
-
8/17/2019 Effect on Chemical Composition and Biological A
4/7
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).
648 D. Valencia et al./ Food Chemistry 131 (2012) 645–651
-
8/17/2019 Effect on Chemical Composition and Biological A
5/7
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.
D. Valencia et al. / Food Chemistry 131 (2012) 645–651 649
-
8/17/2019 Effect on Chemical Composition and Biological A
6/7
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.
650 D. Valencia et al./ Food Chemistry 131 (2012) 645–651
-
8/17/2019 Effect on Chemical Composition and Biological A
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).
References
Ahn, M. R., Kumazawa, S., Hamasaka, T., Bang, K. S., & Nakayama, T. (2004).
Antioxidant activity and constituents of propolis collected in various areas of
Korea. Journal of Agricultural and Food Chemistry, 52, 7286–7292.Amoros, M., Lurton, E., Boustie, J., Girre, L., Sauvager, F., & Cormier, M. (1994).
Comparison of the anti-herpes simplex virus activities of propolis and 3-
methyl-but-2-enyl caffeate. Journal of Natural Products, 57 , 644–647.Bankova, V. (2005). Recent trends and important developments in propolis
research. Evidence-Based Complementary and Alternative Medicine, 2, 29–32.Bankova, V. S., de Castro, S. L., & Marcucci, M. C. (2000). Propolis: recent advances in
chemistry and plant origin. Apidologie, 31, 3–15.Banskota, A. H., Tezuka, Y., & Kadota, S. (2001). Recent progress in pharmacological
research of propolis. Phytotherapy Research, 15, 561–571.Blois, M. S. (1958). Antioxidant Determinations by the Use of a Stable Free Radical.
Nature, 181, 1199–1200.Bonvehi, J. S., & Coll, F. V. (1994). Phenolic composition of propolis from China and
from South America. Z Naturforsch, 49c , 712–718.Bufalo, M. C., Candeias, J. M. G., & Sforcin, J. M. (2009). In vitro Cytotoxic Effect of
Brazilian Green Propolis on Human Laryngeal Epidermoid Carcinoma (HEp-2)
Cells. Evidence-Based Complementary and Alternative Medicine, 6 , 483–487.Chen, C. N., Weng, M. S., Wu, C. L., & Lin, J. K. (2004). Comparison of Radical
Scavenging Activity, Cytotoxic Effects and Apoptosis Induction in Human
Melanoma Cells by Taiwanese Propolis from Different Sources. Evidence-BasedComplementary and Alternative Medicine, 1, 175–185.
Chen, Y. W., Wu, S. W., Ho, K. K., Lin, S. B., Huang, C. Y., & Chen, C. N. (2008).
Characterisation of Taiwanese propolis collected from different locations andseasons. Journal of the Science of Food and Agriculture, 88 , 412–419.
Arzneibuch, Das. Deutsche. (1986). Flavanone. Kommentar, 3, 2226.Garciaviguera, C., Ferreres, F., & Tomasbarberan, F. A. (1993). Study of canadian
propolis by gc-ms and hplc. Zeitschrift Fur Naturforschung C-a Journal of Biosciences, 48, 731–735.
Ghisalberti, E. L. (1979). Propolis: a review. Bee World, 60, 59–84.Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E. M., Efros, L., Caldwell, M., et al.
(1988). Preferential cyto-toxicity on tumor-cells by caffeic acid phenethyl ester
isolated from propolis. Experientia, 44, 230–232.Hernandez, J., Goycoolea, F. M.,Quintero, J., Acosta, A., Castaneda, M.,Dominguez, Z.,
et al. (2007). Sonoran propolis: Chemical composition and antiproliferative
activity on cancer cell lines. Planta Medica, 73, 1469–1474.Isla, M. I., Zampini, I. C., Ordonez, R. M., Cuello, S., Juarez, B. C., Sayago, J. E., et al.
(2009). Effect of Seasonal Variations and Collection Form on Antioxidant
Activity of Propolis from San Juan, Argentina. Journal of Medicinal Food, 12,1334–1342.
Jorge, R., Furtado, N., Sousa, J. P. B., da Silva, A. A., Gregorio, L. E., Martins, C. H. G.,
et al. (2008). Brazilian Propolis: Seasonal Variation of the Prenylated p-Coumaric Acids and Antimicrobial Activity. Pharmaceutical Biology, 46 , 889–893.Li, F., Awale, S., Zhang, H. Y., Tezuka, Y., Esumi, H., & Kadota, S. (2009). Chemical
Constituents of Propolis from Myanmar and Their Preferential Cytotoxicity
against a Human Pancreatic Cancer Cell Line. Journal of Natural Products, 72,1283–1287.
Lima, B., Tapia, A., Luna, L., Fabani, M. P., Schmeda-Hirschmann, G., Podio, N. S., et al.
(2009). Main Flavonoids, DPPH Activity, and Metal Content Allow
Determination of the Geographical Origin of Propolis from the Province of San
Juan (Argentina). Journal of Agricultural and Food Chemistry, 57 , 2691–2698.Lotti, C., Fernandez, M. C., Piccinelli, A. L., Cuesta-Rubio, O., Hernandez, I. M., &
Rastrelli, L. (2010). Chemical Constituents of Red Mexican Propolis. Journal of Agricultural and Food Chemistry, 58, 2209–2213.
Majiene, D., Trumbeckaite, S., Pavilonis, A., Savickas, A., & Martirosyan, D. M. (2007).
Antifungal and Antibacterial Activity of Propolis. Current Nutrition & FoodScience, 3, 304–308.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival -application to proliferation and cyto-toxicity assays. Journal of ImmunologicalMethods, 65, 55–63.
Munstedt, K., & Kalder, M. (2009). Contact allergy to propolis in beekeepers.
Allergologia Et Immunopathologia, 37 , 298–301.Nagy, M.,& Grancai, D. (1996). Colorimetricdetermination of flavanones in propolis.
Pharmazie, 51, 100–101.Paulino, N., Dantas, A. P., Bankova, V., Longhi, D. T., Scremin, A., De Castro, S. L., et al.
(2003). Bulgarian propolis induces analgesic and anti-inflammatory effects in
mice and inhibits in vitro contraction of airway smooth muscle. Journal of Pharmaceutical Sciences, 92, 307–313.
Piccinelli, A. L., Fernandez, M. C., Cuesta-Rubio, O., Hernandez, I. M., De Simone, F., &
Rastrelli, L. (2005). Isoflavonoids isolated from Cuban propolis. Journal of Agricultural and Food Chemistry, 53, 9010–9016.
Popova, M., Bankova, V., Butovska, D., Petkov, V., Nikolova-Damyanova, B., Sabatini,
A. G., et al. (2004). Validated methods for the quantification of biologically
active constituents of poplar-type propolis. Phytochemical Analysis, 15, 235–240.Popova, M., Silici, S., Kaftanoglu, O., & Bankova, V. (2005). Antibacterial activity of
Turkish propolis and its qualitative and quantitative chemical composition.
Phytomedicine, 12, 221–228.Salatino, A., Teixeira, E. W., Negri, G., & Message, D. (2005). Origin and chemical
variation of Brazilian propolis. Evidence-Based Complementary and AlternativeMedicine, 2, 33–38.
Senedese, J. M., Rodrigues, A. R., Furtado, M. A., Faustino, V. D., Berretta, A. A.,
Marchetti, J. M.,& Tavares, D. C. (2008). Assessmentof the Mutagenic Activity of
Extracts of Brazilian Propolis in Topical Pharmaceutical Formulations on
Mammalian Cells In Vitro and In Vivo. Evid Based Complement Alternat Med.Sforcin, J. M., Fernandes, A., Lopes, C. A. M., Bankova, V., & Funari, S. R. C. (2000).
Seasonal effect on Brazilian propolis antibacterial activity. Journal of Ethnopharmacology, 73, 243–249.
Sforcin, J. M., Fernandes Junior, A., Lopes, C. A. M., Funari, S. R. C., & Bankova, V.
(2001). Seasonal effect of Brazilian propolis on Candida albicans and Candida
tropicalis. Journal of Venomous Animals and Toxins, 7 , 1–7.Sforcin, J. M., Kaneno, R., & Funari, S. R. C. (2002). Absence of seasonal effect on the
immunomodulatory action of Brazilian propolis on natural killer activity.
Journal of Venomous Animals and Toxins, 8.Simoes-Ambrosio, L. M. C., Gregorio, L. E., Sousa, J. P. B., Figueiredo-Rinhel, A. S. G.,
Azzolini, A., Bastos, J. K., et al. (2010). The role of seasonality on the inhibitoryeffect of Brazilian green propolis on the oxidative metabolism of neutrophils.
Fitoterapia, 81, 1102–1108.Simone-Finstrom, M., & Spivak, M. (2010). Propolis and bee health: the natural
history and significance of resin use by honey bees. Apidologie, 41, 295–311.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. American Journal of Enologyand Viticulture, 16 , 144–158.
Teixeira, E. W., Message, D., Negri, G., Salatino, A., & Stringheta, P. C. (2010).
Seasonal Variation, Chemical Composition and Antioxidant activity of Brazilian
Propolis Samples. Evidence-Based Complementary and Alternative Medicine, 7 ,307–315.
Usia, T., Banskota, A. H., Tezuka, Y., Midorikawa, K., Matsushige, K., & Kadota, S.
(2002). Constituents of chinese propolis and their antiproliferative activities.
Journal of Natural Products, 65, 673–676.Velazquez, C., Navarro, M., Acosta, A., Angulo, A., Dominguez, Z., Robles, R., et al.
(2007). Antibacterial and free-radical scavenging activities of Sonoran propolis.
Journal of Applied Microbiology, 103, 1747–1756.
Walgrave, S. E., Warshaw, E. M., & Glesne, L. A. (2005). Allergic contact dermatitisfrom propolis. Dermatitis, 16 , 209–215.
Woisky, R. G., & Salatino, A. (1998). Analysis of propolis: some parameters and
procedures for chemical quality control. Journal of apicultural research, 37 (2),99–105.
D. Valencia et al. / Food Chemistry 131 (2012) 645–651 651
top related