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Journal of Ethnopharmacology 86 (2003) 6973

Antimicrobial activity of propolis samples from


two different regions of Anatolia
Murat Kartal a , Sulhiye Yldz b , Serdar Kaya a ,
Semra Kurucu a, , Glat Topu c
a

Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey


Department of Microbiology, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
.
.
c Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul
University, Istanbul, Turkey
b

Received 4 April 2002; received in revised form 10 January 2003; accepted 23 January 2003

Abstract
Antimicrobial activity of two propolis samples from Kazan and Marmaris regions in Turkey were investigated by the disc diffusion method.
Antimicrobial activity was tested with four different ethanolic extracts (30, 50, 70, and 96% ethanol) of each sample against seven Gram
positive, four Gram negative bacteria and one fungus culture. The activity was found to be mainly due to caffeic acid and its esters. An isomeric
mixture containing 3,3-dimethylallyl caffeate, and isopent-3-enyl caffeate was isolated from Kazan propolis samples.
2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Propolis; Antimicrobial activity; Caffeic acid esters

1. Introduction
Propolis is a complex resinous hive product collected by
bees from certain plant sources in the neighborhood. The
presence of propolis within the hive may also provide an environment not suitable for the growth of bacteria and other
microorganisms. Propolis composition is directly related to
that of bud exudates collected by bees from various trees
(Mochida et al., 1985; Ghisalberti, 1979). Propolis in general
contains a variety of chemical compounds such as polyphenols (flavonoid aglycones, phenolic acids, and their esters,
phenolic aldehydes, alcohols, and ketones), terpenoids,
steroids, amino acids, and inorganic compounds (Dimov
et al., 1991; Volpert and Elstner, 1993; Moreno et al., 2000).
Many biological properties, including antibacterial,
(Mochida et al., 1985; Ghisalberti, 1979; Velikova et al.,
2000; Pepeljnjak et al., 1985), antifungal (Dimov et al.,
1991; Schneidewind et al., 1979; Murad et al., 2002), antiviral (Amoros et al., 1992; Amoros et al., 1994), localanaesthetic (Paintz and Metzner, 1979), anti-inflammatory

Corresponding author. Tel.: +90-312-212-6805x2354;


fax: +90-312-213-1081.
E-mail address: kartal@pharmacy.ankara.edu.tr (S. Kurucu).

(Strehl et al., 1994; Miyataka et al., 1997), antioxidant (Sun


et al., 2000; Isla et al., 2001), hepatoprotective (Gonzales
et al., 1995), immunostimulating (Dimov et al., 1991),
and cytostatic (Frenkel et al., 1993), activities have been
ascribed to propolis. It can be found in the market in different forms such as tablets, capsules, toothpaste, mouthwash
preparations, face creams, ointments, lotions, and solutions
(Burdock, 1998). The medical applications of propolis led
to an increased interest in its chemical composition as well
as its origin (Bankova et al., 1989).
Caffeic acid phenethyl ester (CAPE) has been identified
as one of the major biologically active principles in propolis
with chemopreventive and antitumor properties. Russo et al.
(2002) also reported that CAPE plays an important role in
the antioxidant activity of propolis. Antibacterial activity of
propolis is reported to be due to flavonoids, aromatic acids,
and its esters. The mechanism of this activity is attributed to
a synergism between phenolic and other compounds in the
resin (Burdock, 1998).
The present study investigated the antimicrobial activity
and comparison of ethanolic extracts in two propolis samples
collected in different regions of Turkey. The antimicrobial
activity of a 3,3-dimethylallyl caffeate, and isopent-3-enyl
caffeate mixture which was isolated from Kazan propolis
samples; was also investigated.

0378-8741/03/$ see front matter 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00042-4

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M. Kartal et al. / Journal of Ethnopharmacology 86 (2003) 6973

2. Material and methods


2.1. Propolis origins

repeated and the extracts combined for each sample during


5 days. Consequently, eight different ethanolic extracts were
prepared and kept in the dark at room temperature until use.

Propolis samples were collected from two different localities in Turkey, Kazan-Ankara in April (Central Anatolia)
and Marmaris-Mugla in May (Southwestern Anatolia), representing two different regions in Turkey.
Hand collected propolis were kept desiccated and in the
dark up to their processing. Voucher specimens are deposited
in the Department of Pharmacognosy, Faculty of Pharmacy,
University of Ankara, Turkey.

2.7. Preparation of inoculum

2.2. Chemicals

Antimicrobial activity of two propolis samples were investigated by the disc diffusion method (Bauer et al., 1966).
The antimicrobial screening was performed using MHA, and
MHA with 5% defibrinated sheep blood for bacteria and
SDA for yeast.
All extracts of propolis were weighed under aseptic conditions in sterile volumetric flasks, and dissolved with 70%
sterile ethanol to obtain 0.1 mg/ml extract concentration.
These solutions were impregnated on sterile paper discs of
6 mm diameter (20 l per disc) and discs were let to dry
overnight to remove any residual solvent which might interfere with the determination. The solvent control (ethanol)
did not show any antimicrobial activity. Commercial discs of
Ampicilline (10 g-Oxoid) and Ketoconazole (50 g) were
used as positive control.
Seeded agar plates were prepared and inoculated with
0.1 ml of inoculum. Discs were then placed on the seeded
agar plates. Plates were incubated at 35 C for 1820 h for
bacteria and 48 h for Candida albicans. The zones of growth
inhibition around the discs were measured after 1820 h of
incubation at 35 C for bacteria and 2448 h for Candida
albicans. All determinations were made in duplicate.

Column chromatography was carried out on Merck silica


gel 60 (0.0630.200 mm). Petroleum ether, chloroform, and
methanol were obtained from Merck Chemicals Inc.
2.3. Instruments
1H

and 13 C NMR spectra were determined on a Bruker


AC-200 L in CDCl3 , MS were run on a VG Zabspec
spectrometer.
2.4. Medium
The antimicrobial screening was performed using
Mueller-Hinton Agar, (MHA, Oxoid) and MHA with 5%
defibrinated sheep blood for bacteria and Sabouraud Dextrose Agar (SDA, Oxoid) for yeast.
2.5. Test microorganisms
Gram positive, Gram negative bacteria and yeast like
fungi were used for antimicrobial activity studies. Gram
positive bacteria: Staphylococcus aureus (ATCC 25923),
Staphylococcus epidermidis (ATCC 12228), Enterococcus
faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633),
Corynebacterium diphtheriae (RSKK 632), Streptococcus
pneumoniae, and Streptococcus pyogenes (obtained from
clinical material). Gram negative bacteria: Pseudomonas
aeruginosa (ATCC 27853), Escherichia coli (ATCC
25922), Klebsiella pneumoniae (RSKK 256), Branhamella
catarrhalis (obtained from clinical material). Candida albicans (ATCC 10231) was used for antifungal activity test.
All microorganisms were provided by Department of Microbiology, Faculty of Pharmacy, Ankara University.
2.6. Extraction for antimicrobial studies
Dried and sliced (5 g) propolis samples from two different
regions were macerated in 100 ml ethanol with four different
concentrations (30, 50, 70, and 96%) for 5 days. Each sample
was filtered and concentrated in a rotary evaporator under
reduced pressure at 50 C. This extraction procedure was

All microorganisms were cultured overnight at 37 C in


the trypticase soy broth (Oxoid) and used as inoculum. The
turbidity of the suspensions were adjusted to the McFarland
0.5 turbidity standard.
2.8. Antimicrobial activity test

2.9. Isolation of isomeric mixture


Dried and sliced (500 g) propolis samples from KazanAnkara were macerated with 3 l of methanol for 5 days. The
methanolic extract was filtered and continued to maceration
with 2 l methanol. Methanolic extracts were combined and
concentrated under vacuum to yield a residue (50 g).
The methanolic extract was fractionated by flash column
chromatography (11 cm 40 cm) over silica gel eluting with
500 ml petroleum ether and then 500 ml CHCl3 . The CHCl3
fraction was evaporated in vacuo to yield a residue 30 g. The
CHCl3 extract was fractionated by column chromatography
(3.7 cm 70 cm) on silica gel. First elution was started with
chloroform then polarity increased with methanol. A total of
100 ml fractions were collected and combined according to
TLC results. The fractions: 5158 were separated using column chromatography over silica gel. This fraction yielded a
mixture of two isomer compounds (100 mg). Spectral methods (1 H NMR, 13 C NMR, and MS) were used for the structural determination of isomeric compounds. The structures

M. Kartal et al. / Journal of Ethnopharmacology 86 (2003) 6973

71

of 1 and 2 in the mixture were identified also by comparison of their spectral data with those reported in literature
(Bankova et al., 1989; Hashimoto et al., 1988).

3. Results and discussion


The disc diffusion method was used to determine the inhibition zones of the eight different ethanolic extracts from
two propolis samples. The seven Gram positive, four Gram
negative standard bacterial strains and one fungus have
been used. According to the results in the Table 1; different
ethanolic extracts of two propolis samples showed antibacterial activity against S. aureus, S. epidermidis, and B. subtillis. Ethanolic extracts from Kazan propolis samples are also
effective on C. diphtheriae, B. catarrhalis, and C. albicans.
Antimicrobial activity of the ethanolic extracts against the
S. pyogens, P. aeruginosa, E. coli, K. pneumoniae, and E.
faecalis was not observed. Inhibition zones observed for 70
and 96% ethanolic extracts of Kazan sample were slightly
larger than those observed for 30 and 50% ethanolic extracts.
Consequently, antimicrobial screening clearly indicated
that Kazan samples of propolis had much more powerful antimicrobial activity when compared with Marmaris propolis sample (Table 1). Therefore, Kazan propolis sample was
used as the material for isolation study. On the other hand,
previous studies revealed that compounds responsible for
antimicrobial activity were mainly found to be caffeic acid
esters (Schneidewind et al., 1979). The caffeic acid esters
were isolated by column chromatography, their structures
were elucidated and the antimicrobial activity was tested.
The composition of Kazan propolis seems to be directly related to the bud exudate of Populus species and their hybrids
around Kazan.

Fig. 1. Compounds isolated from Kazan propolis.

The mass spectroscopic findings are indicative of the


presence of a caffeic acid pentenyl ester in the mixture. The
fragmentation by HREIMS of the isomeric mixture showed
the ions, m/z (%) = 248 [M]+ (100), 233 (17), 180 (64),
163 (67), 134 (70), 117 (57). The caffeic acid esters were
characterized by 1 H and 13 C NMR spectroscopy (Table 2).
The spectral results showed that the mixture contained
3,3-dimethylallyl caffeate (1), and isopent-3-enyl caffeate
(2).
The isomeric mixture isolated from Kazan was effective
against S. aureus, S. epidermidis, and B. subtillis. C. diphtheriae, B. catarrhalis, and C. albicans but not effective on
S. pyogens, P. aeruginosa, E. coli, K. pneumoniae, and E.
faecalis. Table 1 shows that isomeric mixture is more active
than standard Ampicilline on S. aureus and S. epidermidis
strains, but less active on B. subtilis and showed almost same
activity with Ketoconazole on C. albicans.
Recent studies showed that CAPE and propolis components are able to permeate the in vitro porcine buccal
mucosa in Franz cells. That is why propolis is suggested to

Table 1
Antimicrobial activity of different ethanolic extracts from Kazan and Marmaris propolis samples (mean values of the diameters of inhibition zones in mm)
Bacteria/fungus

Staphylococcus aureus
Staphylococcus epidermidis
Enterococcus faecalis
Bacillus subtilis
Corynebacterium diphtheriae
Streptococcus pneumoniae
Streptococcus pyogenes
Pseudomonas aeruginosa
Escherichia coli
Klebsiella pneumoniae
Branhamella catarrhalis
Candida albicans

Isolated mixture

Ethanolic extract from Kazan

Ethanolic extract from Marmaris

Positive control

Y1

K1

K2

K3

K4

M1

M2

M3

M4

AMP

KET

14
16

14.5
13

10
14

9
10

9.5
10

8
9

9
10

9
10

8
8

11
12

11
12

8
10

11
12

11
11

10.5

8
8

10

9
9

11.5

8
11

9
9

20
15

15

16

K1, 30% ethanolic extract from Kazan-Ankara propolis; K2, 50% ethanolic extract from Kazan-Ankara propolis; K3, 70% ethanolic extract from
Kazan-Ankara propolis; K4, 96% ethanolic extract from Kazan-Ankara propolis; M1, 30% ethanolic extract from Marmaris-Mugla propolis; M2,
50% ethanolic extract from Marmaris-Mugla propolis; M3, 70% ethanolic extract from Marmaris-Mugla propolis; M4, 96% ethanolic extract from
Marmaris-Mugla propolis; Y1, 3,3-dimethylallyl caffeate and isopent-3-enyl caffeate mixture isolated from Kazan-Ankara propolis samples; AMP,
Ampicilline; KET, Ketoconazole. Sterile paper discs (6 mm) soaked with (20 l per disc) 0.1 mg/ml of each ethanolic extract or isolated mixture.
Commercial discs of Ampicilline (10 g-Oxoid) and Ketoconazole (50 g) were used as positive control. The tests were done in duplicate.

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M. Kartal et al. / Journal of Ethnopharmacology 86 (2003) 6973

Table 2
1 H and 13 C NMR data of compounds 1 and 2 in CDCl
3
Position

1H

NMR (1)

13 C

NMR (1)

, ppm (J)
1
2
3
4
5
6

1
2
3
4
5

7.10 d (J = 1.9 Hz)

6.86
6.98
7.59
6.26
4.71
5.41

d (J = 7.8 Hz)
dd (J = 1.8 and 7.8 Hz)
d (J = 16.0 Hz)
d (J = 16.0 Hz)
d (J = 7.5 Hz, 2H)
dddd (J = 1.2, 7.5, and 8.0 Hz)

1.75 s (3H)
1.78 s (3H)

1H

NMR (2)

13 C

NMR (2)

, ppm (J)
127.3
114.4
143.9
146.5
114.4
122.4
145.2
115.4
112.3
126.6
127.3
25.7
25.7

be used in the stomatological field for its antimicrobial and


anti-inflammatory properties, as well as for its analgesic
qualities (Ceschel et al., 2002). Spectral results revealed
that the mixture composed of two compounds which were
identified as 3,3-dimethylallyl caffeate and isopent-3-enyl
caffeate (Fig. 1). According to the antimicrobial results
of the isomeric mixture, it has been shown that the activity was found to be mainly due to caffeic acid esters
(3,3-dimethylallyl caffeate, and isopent-3-enyl caffeate) in
Kazan propolis samples.

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7.10 d (J = 1.9 Hz)

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