Article
HTTP://DX.DOI.ORG/10.5504/BBEQ.2013.0034
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BIOLOGICALLY ACTIVE COMPOUNDS WITH ANTITUMOR ACTIVITY IN
PROPOLIS EXTRACTS FROM DIFFERENT GEOGRAPHIC REGIONS
Anton Slavov1, Atanas Trifonov1, Lyudmil Peychev2, Stela Dimitrova2, Stela Peycheva2, Velitchka Gotcheva1, Angel Angelov1
University of Food Technologies, Faculty of Technology, Plovdiv, Bulgaria
2
Medical University of Plovdiv, Faculty of Pharmacy, Plovdiv, Bulgaria
Correspondence to: Anton Slavov
E-mail: [email protected]
1
ABSTRACT
Seven propolis samples (S1–S7) from different regions of Bulgaria and eight commercial products of propolis extracts (C8–
C15) from various European countries were studied. Concentrations of five antitumor compounds: CAPE, quercetin, rutin,
p-coumaric acid, and ferulic acid, were determined and a comparison between the propolis extracts from different regions
was made. The results showed that among samples S1–S7, two were distinctive with their high concentrations of CAPE and
quercetin: S1 (Asenovgrad region) – 9843.6 μg/mL and 2575.6 μg/mL, respectively, and S7 (Smolyan region) – 10270.2 μg/mL
and 2575.6 μg/mL, respectively. Sample S4 (Plovdiv region) showed significant amounts of p-coumaric acid (1638.2 μg/mL)
and ferulic acid (1186.5 μg/mL). Sample C11 from the commercial propolis extract products (Bulgaria) was distinctive with its
high concentrations of CAPE (5802.3 μg/mL), p-coumaric acid (2955.2 μg/mL) and ferulic acid (1081.2 μg/mL). Samples C15
(Italy) and C8 (Austria) also contained significant amounts of CAPE, quercetin, p-coumaric acid, and ferulic acid. The overall
results showed high content variability of the five biologically active substances analyzed. Nevertheless, each sample contained
significant amounts of at least four of the biologically active compounds tested, which confirms that propolis remains a valuable
source of antitumor compounds.
Biotechnol. & Biotechnol. Eq. 2013, 27(4), 4010-4013
Keywords: propolis, HPLC, p-coumaric acid, ferulic acid,
caffeic acid phenethyl ester, quercetin, rutin
Introduction
Propolis is a resinous substance collected by honeybees
from botanical sources such as tree buds, sap flows, etc.,
and processed by enzymes (β-glucosidases) produced by
honeybee’s glands (2). Propolis is used by the bees for
sealing unwanted open gaps in the hive and as a construction
material reinforcing the structural stability of the hive. Apart
from these functions, propolis plays an important role as an
antimicrobial agent protecting the bees and the hives from
various contaminants (4).
Recent analyses show that propolis contains bioflavonoids,
terpenoids, various aromatic acids, their esters and salts. It also
contains small amounts of vitamins A, D, E, C, B1, B2, B6, H,
folic acid, nicotinic acid, macro- and micro-elements such as
Ca, Mg, Fe, Zn, Co, Mn, Sr, and essential oils (9, 22).
Propolis has been widely used in home medicine since
ancient times. Nowadays, its biological activities and
beneficial properties are extensively studied and propolis is
known to exhibit antioxidant, antimicrobial, hepatoprotective,
anti-inflammatory and antitumor activity, as well as
immunostimulating properties (6, 10, 12, 14, 20, 22, 25). It
is interesting to note that, despite the differences in origin and
composition, propolis products show very similar biological
activity (6).
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The antitumor effect of propolis is due to its ability to
stimulate macrophage activity and the production of antibodies
destroying tumors and suppressing cancerogenesis. Therefore,
it is used to treat skin cancer, lung cancer, throat and brain
tumors, etc. (6, 7). The antitumor effect is mainly attributed to
several compounds found in propolis: caffeic acid phenethyl
ester (CAPE), artepillin C, ferulic acid and p-coumaric acid
(17, 22, 29). CAPE is amongst the main biologically active
components in propolis. Huang et al. (13) showed in their in
vitro experiments the ability of CAPE to suppress the growth
of human tumor cells. Other researchers found that CAPE is a
potent specific inhibitor of NF-kappa B activation, which may
provide the molecular basis for its multiple immunomodulatory
and anti-inflammatory activities (23). CAPE was also found to
have significant cytotoxycity on oral submucosal fibroblast,
neck metastasis of gingival carcinoma, and tongue squamous
cell carcinoma cells and the results suggest that CAPE-like
compounds could be used as potential chemotherapy agents
against oral cancer (21). P-coumaric acid is a hydroxy
derivative of cinnamic acid. There are three isomers of
cinnamic acid which differ in the position of the hydroxyl group
of the phenyl ring, and p-coumaric acid is the most abundant
isomer in nature. It is found in different edible plants such as
peanuts, tomatoes, carrots, etc. Ferulic acid is another hydroxy
derivative of cinnamic acid and differs from p-coumaric acid
by one methoxy group at C6. Artepillin C – 3-[4-hydroxy-3,5di(3-methyl-2-butenyl)phenyl]-2(E)-propenoic acid, is a low
molecular weight phenolic compound isolated from Brazilian
Biotechnol. & Biotechnol. Eq. 27/2013/4
propolis with antimicrobial, antitumor, apoptosis-inducing,
immunomodulatory and antioxidant properties (24).
Quercetin and rutin are widely distributed flavonoids with
a pronounced antioxidant and antitumor effect, which are also
important representatives of the biologically active substances
in propolis. Quercetin is present in many plants such as edible
berries (11), green tea, capers, lovage, apples, onion, etc. (8),
and rutin is a glycoside of quercetin and the disaccharide
rutinose (α-L-rhamnopyranosyl-(1→6))-β-D-glucopyranose)
found in various fruits such as orange, grapefruit, lemon,
cranberries, mulberry, buckwheat, Japanese pagodatree,
eucalyptus, etc. (8, 18).
The color and chemical composition of propolis vary
widely between the different geographical regions depending
on the botanical source(s), the bee species and the season (27).
This variability makes the comparisons between the biological
activity of propolis products and their standardization
extremely difficult (2, 3, 28).
The aim of the present study was to explore and compare
the chemical composition of propolis extracts from different
geographic regions in terms of several antitumor compounds:
CAPE, p-coumaric acid, ferulic acid, quercetin, and rutin.
on a laboratory shaker for 1 h and filtered. The obtained extracts
were stored in dark plastic bottles at 4 ºC until analyzed.
Materials and Methods
Results and Discussion
Materials
Seven propolis samples from small honeybee farms located in
different regions of Bulgaria and eight commercial products
of propolis extracts from various European countries were
studied (Table 1).
Standards used were HPLC-grade rutin hydrate, p-coumaric
acid, ferulic acid, quercetin, and caffeic acid phenethyl ester
(Sigma–Aldrich Chemie, Germany). All solvents used were
HPLC-grade and obtained from local distributors.
Methods
Preparation of the propolis extract
Each natural propolis sample (100 g) was ground and mixed
with 450 mL of ethanol and 110 mL of distilled water. The
mixture was left for 48 h at ambient temperature, then placed
HPLC analyses
Propolis extracts were analyzed in a high-performance
liquid chromatography system (Thermo Fisher Scientific,
USA). Separation was performed on a reverse-phase column
(Nucleodur 250 mm × 4.6 mm, C-18, 5 μm particles,
Macherey–Nagel, Germany), using a gradient method of water
(with pH = 4, adjusted with 85 % HPLC-grade o-phosphoric
acid) and acetonitrile (AcCN) mobile phase with a flow rate
of 0.9 mL/min. The gradient of the solvents was as follows:
starting with H2O:AcCN = 9:1; 16 min – H2O:AcCN = 6:4;
19 min – H2O:AcCN = 1:9; 21 min – H2O:AcCN = 1:9;
24 min – H2O:AcCN = 9:1; runtime of analysis – 30 min. The
temperature of the column was set at 30 ºC. Spectrophotometric
detection was performed at λ = 335 nm with a photodiode
detector (Surveyor PDA Plus Detector, deuterium and tungsten
halogen lamps, Thermo Fisher Scientific).
Analyses of the samples were performed in sets of 3
repetitions with suitable dilution allowing clear separation of
the investigated propolis extracts components. All results were
expressed as mean ± SD.
The most widely distributed bee species in Europe is Apis
mellifera L., collecting propolis mainly from poplar trees
(Populus sp.). Poplar propolis contains flavonoid aglycones,
hydroxy derivatives of cinnamic acid (p-coumaric acid, ferulic
acid) and their esters (1). This suggests that these compounds
are expected to be present in all tested samples, since they
originate from European countries.
The results from the analysis showed that from the five
investigated biologically active substances only p-coumaric acid
was present in all the investigated samples. Many other studies
have demonstrated presence of p-coumaric acid in propolis from
different parts of the world. Kosalec et al. (19) found p-coumaric
acid in propolis from continental and Adriatic regions of Croatia.
Kalogeropoulos et al. (16) reported its presence in propolis
samples from Greece, and Bankova et al. (5) reported p-coumaric
acid in propolis extracts from Bulgaria and Mongolia. The
TABLE 1
Geographical origin of the analyzed propolis samples
Sample No.
S1
S2
S3
S4
S5
S6
S7
Origin – Propolis samples
Bulgaria, Asenovgrad region
Bulgaria, Veliko Tarnovo region
Bulgaria, Bulgarevo, Varna region
Bulgaria, Voivodinovo, Plovdiv region
Bulgaria, Chirpan region
Bulgaria, Kyilevcha, Shumen region
Bulgaria, Yagodina, Smolyan region
Biotechnol. & Biotechnol. Eq. 27/2013/4
Sample No.
C8
C9
C10
C11
C12
C13
C14
C15
Origin – commercial products
Austria
Italy
France
Bulgaria
Hungary
Germany
Bulgaria
Italy
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concentration which we measured showed high variability of
p-coumaric acid in the fifteen analyzed samples (Fig. 1). The
highest observed concentration was 2955.2 μg/mL in commercial
sample C11, which was almost twice higher than that in product
C8 (1241 μg/ml). Much lower amounts of p-coumaric acid were
detected in products C14 and C15: 596.4 μg/mL and 645.1 μg/mL,
respectively. The same wide variety of p-coumaric acid content
was observed for the Bulgarian propolis samples. The highest
amount was found in sample S4 (1638.2 μg/mL), followed by
sample S1 (1008.0 μg/mL). For the rest of the samples (S6, S5,
S3, S2, and S7) the concentration of p-coumaric acid varied from
140.7 μg/mL to 827.3 μg/mL.
Fig. 1. Concentration of p-coumaric acid in propolis extracts from Bulgaria
and commercial propolis products from other EU countries.
Amongst the five biologically active compounds analyzed,
CAPE was found in highest concentrations, reaching an
order higher than the rest of the compounds in some samples
(Fig. 3). Generally, CAPE was found in significant amounts in
all samples, except for propolis extracts S5 and S6, where it
was not detected. A high CAPE content was also reported by
Kalogeropoulos et al. (16) for Greek propolis samples. Based
on data from other authors as well, it is important to note
that, in contrast to European countries, most tropical propolis
samples do not contain even traces of CAPE (2, 3).
Fig. 3. Concentration of CAPE in propolis extracts from Bulgaria and
commercial propolis products from other EU countries.
Ferulic acid was also present in all analyzed propolis
extracts, except for sample S6 (Fig. 2). Similarly to p-coumaric
acid, ferulic acid has been found in propolis from different
geographic regions in Europe. It was reported in propolis from
Greece (16), continental and Adriatic regions of Croatia (19),
and Bulgaria (5).
Fig. 4. Concentration of quercetin in propolis extracts from Bulgaria and
commercial propolis products from other EU countries.
Fig. 2. Concentration of ferulic acid in propolis extracts from Bulgaria and
commercial propolis products from other EU countries.
The variability of ferulic acid content in the samples was
similar to that of p-coumaric acid. The highest observed
concentration was 1186.5 μg/mL in propolis extract S4,
followed by commercial sample C11 (1081.2 μg/mL). It
is interesting to note that in these two samples the highest
concentrations of p-coumaric acid were also detected. This
could possibly be explained by the fact that, since both ferulic
and p-coumaric acids are derivatives of cinnamic acid, they
are probably both synthesized and accumulated in propolis by
a similar mechanism. Also, the concentration ratios of the two
phenolic acids was found to be similar in all propolis extracts,
except for commercial product C11, where the concentration
of p-coumaric acid is almost three times higher than that of
ferulic acid.
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Maximum values for CAPE content were observed in
Bulgarian propolis extracts: 10270.2 μg/mL in sample S7,
followed by 9843.6 μg/mL and 7159.9 μg/mL for samples S1
and S2, respectively. From the commercial products, sample C11
was shown to have the highest amount of CAPE: 5802.3 μg/mL.
Significant amounts were also found in samples C15, C8 and
C14, varying from 5039.2 μg/mL to 4207.3 μg/mL.
Quercetin and its glycoside rutin are widely distributed
in nature and have been found in propolis samples from
different geographical regions (16, 26, 30). In our experiments
quercetin was found in all samples except for S6 (Fig. 4), with
maximum values, interestingly, observed for those samples
which showed highest CAPE content as well: S1 and S7 with
2575.6 μg/mL and 1616.2 μg/mL quercetin, respectively. From
the commercial propolis samples the highest concentration
(1254.4 μg/mL) was observed in sample C15.
Although rutin was continuously detected in propolis from
different geographical regions from Brazil to China (15, 30),
it was surprisingly found in only half of our analyzed samples
(Fig. 5), with better results for commercial products (5 out 8
samples) than for propolis extracts obtained in the laboratory (2
Biotechnol. & Biotechnol. Eq. 27/2013/4
out of 7 samples). In general, for all rutin-containing samples
the amounts found were quite low, with the highest value of
711 μg/mL determined in sample C12. In Bulgarian propolis
extracts the highest concentration of rutin was found in sample
S4: 488.7 μg/mL.
Acknowledgements
This paper was published with the support of project
BG051PO001/3.3-05-0001 “Science and Business” funded by
Operational programme “Human Resources Development” of
the European Social Fund.
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Fig. 5. Concentration of rutin in propolis extracts from Bulgaria and
commercial propolis products from other EU countries.
Conclusions
The study performed showed that among the seven farmderived samples (S1–S7) from different locations in Bulgaria,
two were distinctive with their high concentrations of CAPE
and quercetin: S1 (Asenovgrad region) and S7 (Smolyan
region), and sample S4 (Plovdiv region) showed significant
amounts of the derivatives of cinnamic acid – p-coumaric acid
and ferulic acid, while samples S5 (Chirpan region) and S6
(Shumen region) were found to have very low concentrations
or lack of the analyzed compounds. These results suggest
that even within the small territory of Bulgaria, the chemical
composition of propolis from different regions varies
significantly. The general observation was that the propolis
samples from the mid-south regions were distinctively richer
in the analyzed biologically active compounds compared to
those from regions located further north and east.
The commercial propolis extract products also showed high
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C11 (Bulgaria) was distinctive with its high concentrations of
CAPE, p-coumaric acid and ferulic acid. Samples C15 (Italy)
and C8 (Austria) also contained significant amounts of CAPE,
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The overall analysis showed high content variability of
the five biologically active substances analyzed in propolis
extracts from honeybee farms and in commercial propolis
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geographical origin (climate conditions, plants available in the
various regions), seasonal differences, method for propolis
extract preparation, etc. Nevertheless, each sample contained
at least four of the biologically active compounds tested, which
confirms that propolis remains a valuable source of antitumor
compounds regardless of its geographical origin. However,
the variability in the chemical composition and the levels of
biologically active compounds in every product makes propolis
extract standardization a highly challenging task.
Biotechnol. & Biotechnol. Eq. 27/2013/4
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