Beruflich Dokumente
Kultur Dokumente
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
0378-8741/03/$ see front matter 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0378-8741(03)00042-4
70
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.
2.2. 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.
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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).
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
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.
72
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
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
References
Amoros, M., Simoes, C.M.O., Girre, L., Sauvager, F., Cormier, M., 1992.
Synergistic effect of flavones and flavonols against Herpes simplex
virus type 1 in cell culture. Comparison with the antiviral activity of
propolis. Journal of Natural Products 55, 17321740.
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-butyl-2-enyl caffeate. Journal of Natural Products 57,
644647.
Bankova, V.S., Popov, S.S., Marekov, N.L., 1989. Isopentenyl cinnamates from poplar buds and propolis. Phytochemistry 28, 871
873.
Bauer, A.W., Kirby, M.D.K., Sherries, J.C., Truck, M., 1966. Antibiotic
susceptibilities testing by standard single disc diffusion method. American Journal of Clinical Pathology 45, 493496.
Burdock, G.A., 1998. Review of the biological properties and toxicity
of bee propolis (propolis). Food and Chemical Toxicology 36, 347
363.
Ceschel, G.C., Maffei, P., Sforzini, A., Lombardi Borgia, S., Yasin,
A., Ronchi, C., 2002. In vitro permeation through porcine buccal
mucosa of caffeic acid phenetyl ester (CAPE) from a topical mucoadhesive gel containing propolis. Fitoterapia 73 (Suppl. 1), 44
52.
Dimov, V., Ivanovska, N., Manolova, N., Bankova, V., Nikolov, N.,
Popov, S., 1991. Immunomodulatory action of propolis. Influence on
anti-infectious protection and macrophage function. Apidologie 22,
155162.
6.86
6.98
7.60
6.39
4.31
2.40
d (J = 7.8 Hz)
dd (J = 1.8 and 7.8 Hz)
d (J = 16.0 Hz)
d (J = 16.0 Hz)
t (J = 7.5 Hz, 2H)
t (J = 7.5 Hz, 2H)
1.78 s (3H)
4.76 s and 4.81 s (2H)
127.3
114.4
143.9
146.5
114.4
122.4
145.2
115.4
112.3
127.3
118.4
25.7
22.5
Frenkel, K., Wei, H., Bhimani, R., Ye, J., Zadunaisky, J.A., Ferraro, T., Conney, A.H., Grunberger, D., 1993. Inhibition of tumor
promoter-mediated processes in mouse skin and bovine lens by caffeic
acid phenethyl ester. Cancer Research 53, 12551261.
Ghisalberti, E.L., 1979. Propolis: a review. Bee World 60, 5984.
Gonzales, R., Corcho, I., Remirez, D., Rodriguez, S., Ancheta, O., Merino,
N., Gonzales, A., Pascual, C., 1995. Hepatoprotective effects of propolis extract on carbon tetrachloride-induced liver injury in rats. Phytotherapy Research 9, 114117.
Hashimoto, T., Tori, M., Asakawa, Y., Wollenweber, E., 1988. Synthesis
of two allergenic constituents of propolis and poplar bud excretion.
Zeischrift fr Naturforschung 43c, 470472.
Isla, M.I., Moreno, M.I.N., Sampietro, A.R., Vattuone, M.A., 2001. Antioxidant activity of Argentina propolis extracts. Journal of Ethnopharmacology 76, 165170.
Miyataka, H., Nishiki, M., Matsumoto, H., Fujimoto, T., Matsuka, M.,
Satoh, T., 1997. Evaluation of propolis. I. Evaluation of Brazilian
and Chinese propolis by enzymatic and physico-chemical methods.
Biological & Pharmaceutical Bulletin 20, 496501.
Mochida, S., Haga, M., Takino, Y., 1985. Chemical constituents and
antimicrobial activity of Japanese propolis. Apimondia. In: The XXXth
International Apicultural Congress, Nagoya-Japan. pp. 455456.
Moreno, M.I.N., Isla, M.I., Sampietro, A.R., Vattuone, M.A., 2000.
Comparison of the free radical-scavenging activity of propolis from
several regions of Argentina. Journal of Ethnopharmacology 71,
109114.
Murad, J.M., Calvi, S.A., Soares, A.M.V.C., Bankova, V., Sforcin, J.M.,
2002. Effect of propolis from Brazil and Bulgaria on fungucidal activity of macrophages against Paracoccidioides brasiliensis. Journal of
Ethnopharmacology 79, 331334.
Paintz, M., Metzner, J., 1979. Zur lokalanasthetischen wirkung vin propolis
und einigen inhaltsstofen. Pharmazie 34, 839841.
Pepeljnjak, S., Jalsenjak, I., Maysinger, D., 1985. Flavonoid content in
propolis extracts and growth inhibition of Bacillus subtilis. Pharmazie
40, 122123.
Russo, A., Longo, R., Vanella, A., 2002. Antioxidant activity of propolis:
role of caffeic acid phenethyl ester and galangin. Fitoterapia 73 (Suppl.
1), S21S29.
Schneidewind, E.-M., Bge, A., Kala, H., Metzner, J., Zschunke, A.,
1979. Identification of an antimicrobially active constituent isolated
from propolis. Pharmazie 34, 103106.
Strehl, E., Volpert, R., Elstner, E.F., 1994. Biochemical activities of propolis extracts. III. Inhibition of dihydrofolate reductase. Zeischrift fr
Naturforschung 49c, 3943.
73
Velikova, M., Bankova, V., Tsvetkova, I., Kujumgiev, A., Marcucci, M.C.,
2000. Antibacterial ent-kaurene from Brazilian propolis of native stingless bees. Fitoterapia 71, 693696.
Volpert, R., Elstner, E.F., 1993. Biochemical activities of propolis extracts.
II. Photodynamic activities. Zeischrift fr Naturforschung 48c, 858
862.