SYNERGISTIC ACTIVITIES OF TWO PROPOLIS WITH AMPHOTERICIN B AGAINST SOME AZOLE-RESISTANT CANDIDA STRAINS. PART I

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1 SYNERGISTIC ACTIVITIES OF TWO PROPOLIS WITH AMPHOTERICIN B AGAINST SOME AZOLE-RESISTANT CANDIDA STRAINS. PART I DURAN NIZAMI 1, MUZ MUSTAFA 2, DURAN GULAY GULBOL 3, OZER BURCIN 1, ONLEN YUSUF 4 1 Mustafa Kemal University, Medical Faculty, Department of Microbiology & Clinical Microbiology, Hatay-Turkey, nizamduran@hotmail.com, burcinozer@yahoo.com 2 Mustafa Kemal University, Veterinary Faculty, Department of ParasitologyHatay-Turkey, mmuz@mku.edu.tr 3 Mustafa Kemal University, Health College, Department of Medical Biology&Genetics, Hatay- Turkey, gulaygulbol@gmail.com 4 Mustafa Kemal University, Medical Faculty, Department of Infectious Diseases, Hatay-Turkey, yonlen@mku.edu.tr Propolis is a bee product which has many pharmacological properties and also used in traditional medicine. The objective of this work is to analyze and identify the components in propolis samples collected from the South-Eastern Mediterranean region of Anatolia (Hatay province) and South Marmara region (Bursa province) during The components of both propolis samples were identified by GS-MS analysis. The results of chemical composition show that aromatic acids, aromatic acid esters, flavanols, cinnamic acid esters have been found in two different propolis samples. Biologically active chemical constituents of propolis are highly variable, depending on the plant species, geographic area, and on the season of collection. The antifungal, antibacterial and antiviral properties of the studied samples were microbiologically analyzed for candida strains and the results are presented in the second part of this paper. Keywords: Bursa propolis, Hatay propolis, components identification. INTRODUCTION Recently, new drug researches are focused on natural products. Among these kinds of natural products, one of the most interesting natural product is propolis. Propolis is a bee product which has many pharmacological properties and also used in traditional medicine. Various pharmacological properties of propolis have been reported such as antifungal, antibacterial and antiviral properties. Some natural products like propolis are especially candidates to be new antimicrobial substances in these works. In previous studies, propolis has been reported to be effective against yeast-like fungi (Aksoy et al., 2007; Duran et al., 2008; Onlen et al., 2007a; Duran et al., 2006; Oksuz et al., 2005; Allahverdiyev, 2004; Onlen et al., 2007b). The pharmacological properties of propolis are main attributed to the presence of various components (Banskota, Tezuka and Kadota, 1978). Biologically active chemical constituents of propolis are highly variable, depending on the plant species, geographic area, and on the season of collection (Ghisalberti, 1978). The fungi, especially yeasts belonging to the genus Candida are potentially pathogenic agents for human. Candida species especially C.albicans is found in the normal flora of mouth and gastrointestinal tract of human. Candida strains are opportunistic pathogenic fungi in humans which can cause either septicaemic or mucosal infections (Odds, 1994). These kinds of fungal infections are an important cause of morbidity and mortality hospitalized patients (Beck-Sague and Jarvis, 1993; Edmond et al., 1999; Pittet et al., 1997; Safdar and Armstrong, 2001). 211

2 In this study, we aimed to investigate the existance of synergistic activities of Hatay and Bursa propolis with Amphotericin B against some azole-resistant Candida strains. MATERIALS AND METHODS Sample Collection Propolis samples were collected from the South-Eastern Mediterranean region of Anatolia (Hatay province) and South Marmara region (Bursa province) during Hand-collected propolis samples were kept in a dry and dark place and stored at 4 C until their processing. Each of the propolis samples were sliced into small pieces after cooling at -40 C and then grated. The samples were dissolved in 96% ethanol (1:10, w/v) at 25 C for 14 days in clean dark-brown glass bottles. Airtight bottles shaked up for 3 minutes in every 6 hours for a period of 15 days. After extraction period, the supernatant was filtered twice with Whatman No.4 and No.1 filter papers. The ethyl alcohol extract was then evaporated to dryness under vacuum. Five micrograms of residue were mixed with 75 µl of dry pyridine and 50 µl bis (trimethylsilyl) trifluoroacetamide, heated and supernatant was analyzed by GC-MS (Sorkun, Suer, and Salih, 2001). RESULTS AND DISCUSSION The chemical composition of Bursa and Hatay propolis samples are given in Table 1. Substances Table 1. The percentage composition of identified compounds in Bursa and Hatay propolis samples Bursa Propolis Hatay Propolis Alcohols 3-Methyl-3-buten-1-ol Aromatic Alcohols Phenylethyl Alcohol (E)-11-Hexadecen-1-ol Propen-1-ol Naphthalene-methanol Tetradecy-11-yn-1-ol Olean -12-en-3-ol Benzenemethanol Aromatic Acids 5-phenyl-4-pentenoic acid Benzoic acid Benzenepropanoic acid phenyl-2-propenoic acid Decanoic acid Octadecenoic acid Octadecanoic acid Aromatic Acid Esters Benzene acetic acid, 4-hydroxy methoxymethyl ester Octadecanoic acid-methyl ester

3 Substances Bursa Propolis Hatay Propolis 1,2-benzenedicarboxylic acid, bis (8-methyl nonyl) ester 1,2-benzenedicarboxylic acid, bis (8-methyl propyl) ester 1,2-Benzenedicarboxylic acid, butyl methylonyl ester Benzyl cinnamate ,2-Benzenedicarboxylic acid, diisodecyl ester Aldehydes Benzaldehyde Straight-Chain Acids Tetradecanoic acid Heptadecanoic acid n-hexadecanoic acid Straight-Chain Acids Esters Tetradecanoic acid, ethyl ester Hexadecanoic acid, ethyl ester Heptadecanoic acid, 15-methyl-ethyl ester Flavanols 4H-1-Benzopyran-4-one,5-hydroxy methoxy-2-phenyl 4H-1-Benzopyran-4-one, 2,3-dihydro-5, dihydroxy-2-phenyl 4H-1-Benzopyran-4-one,3,5,7-trihydroxy phenyl Chrysin ,7-dihydroxy-6-methoxy-3( methoxyphenyl) 5-hydroxy-6,7-dimethoxy-3( methoxyphenyl) Hydrocarbons Cyclotetradecane Heptadecane Heptadecane Nonadecene Tricosene Delta-Cadinene Bicyclo(4.4.0) dec-1-ene (Z9(E)-Heptadecane Aromatic Esters 2-Propen-1-one Fatty Acid Ester Ethyl Oleate 2.45 Cinnamic Acid Esters Cinnamyl cinnamate Hydroxy-4-methoxycinnamic acid 2.26 Ketones 2(5H)-Furanone,5,5-diphenyl Phenyl-2-tipyl-acenapthenone (2-Vinyl phenyl)ethanone 0.07 Totarolone

4 Substances Bursa Propolis Hatay Propolis Hinokione Heptadecanoate The variability of constituents of propolis in two samples showed that they were collected from different plants depending on the geographic location. The pharmacological properties of propolis are principally attributed to the presence of various components (Banskota, A.H., Tezuka, Y. and Kadota, 1978). Biologically active chemical constituents of propolis are highly variable, depending on the plant species, geographic area, and on the season of collection (Ghisalberti, 1978). In the literature, the antimicrobial activity of propolis was reported to associate with the presence of flavonoids and derivatives of caffeic acid (Salomao et al., 2004; Prytzyk et al., 2003). It was reported that aromatic acids, aromatic acid esters, flavanols, cinnamic acid esters have been found in two different propolis samples. CONCLUSIONS Propolis may be an important prospect in the treatment of azole-resistant yeast. Although the effects of these two kinds of propolis against the Candida spp. in vitro are promising, further investigation such as microbiological, pharmacological and clinical trials are required. REFERENCES Aksoy, A., Duran, N., Toroglu, S. et al. (2007), Short-term effect of mastic gum on salivary concentrations of cariogenic bacteria in orthodontic patients, Angle Orthod, 77, Allahverdiyev, A., Duran, N., Ozguven, M. et al. (2004), Antiviral activity of the volatile oils of Melissa officinalis L. against Herpes simplex virus type-2, Phytomed, 11, Banskota, A.H., Tezuka, Y. and Kadota, S. (1978), Recent progress in pharmacological research of propolis, Phytother Research, 15, Beck-Sague, C. and Jarvis, W.R. (1993), Secular trends in the epidemiology of nosocomial fungal infections in the United States, , Journal of Infectious Diseases, 167, Duran, G., Duran, N., Culha, G., et al. (2008), In vitro antileishmanial activity of Adana propolis samples on Leishmania tropica: A preliminary study, Parasitol Research, 102, Duran, N., Koc, A., Oksuz, H. et al. (2006), The protective role of topical propolis on experimental keratitis via nitric oxide levels in rabbits, Mollecularand Cellular Biochemistry, 281, Edmond, M.B., Wallace, S.E., McClish, D.K. et al. (1999), Nosocomial bloodstream infections in United States hospitals: a three-year analysis, Cliical Infectious Diseases, 29, Ghisalberti, E.L. (1978), Propolis: a review, Bee World, 60, Odds, F.C. (1994), Pathogenesis of Candida infections. Review., Journal of the American Academy of Dermatology, 31, 2-5. Oksuz, H., Duran, N., Tamer, C. et al. (2005), Effect of propolis in the treatment of experimental Staphylococcus aureus keratitis in rabbits, Ophthalmic Research, 37, Onlen, Y., Duran, N., Atik, E. Et al. (2007a), Antibacterial activity of propolis against MRSA and synergism with topical mupirocin, Journal of Alternative and Complementary Medicine, 13, Onlen, Y., Tamer, C., Oksuz, H. et al. (2007b), Comparative trial of different anti-bacterial combinations with propolis and ciprofloxacin on Pseudomonas keratitis in rabbits, Microbiological Research, 162, Pittet, D., Li, N., Woolson, R.F. et al. (1997) Microbiological factors influencing the outcome of nosocomial bloodstream infections: a 6-year validated, population-based model, Clinical Infectious Diseases, 24, Prytzyk, E., Dantas, A.P., Salomao, K. et al. (2003), Flavonoids and trypanocidal activity of Bulgarian propolis, Journal Ethnopharmacol, 88,

5 Safdar, A., Armstrong, A. (2001) Infectious morbidity in critically ill patients with cancer, Critical Care Clinics,17, Salomao, K., Dantas, A.P., Borba, C.M. et al. (2004), Chemical composition and microbicidal activity of extracts from Brazilian and Bulgarian propolis, Lett Applications Microbioogy, 38, Sorkun, K., Suer, B. and Salih, B. (2001), Determination of chemical composition of Turkish propolis, Z Naturforschung C, 56,

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