Chemical composition of propolis from Canada, its antiradical activity and plant origin

Natural Product Research, Vol. 20, No. 6, 20 May 2006, 531 536 Chemical composition of propolis from Canada, its antiradical activity and plant origin ROUMEN CHRISTOVy, BORYANA TRUSHEVAz, MILENA POPOVAz, VASSYA BANKOVAz* and MICHEL BERTRANDy yregional Center for Mass Spectrometry, Department of Chemistry, University of Montreal, Quebec, Canada zinstitute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria (Received 15 June 2004; in final form 25 September 2004) The chemical composition of propolis from two regions in Canada was studied: Boreal forest and Pacific coastal forest that lay outside the area of distribution of Aigeiros poplars, the usual propolis source plants. In the sample from Victoria, p-hydroxyacetophenone, benzyl hydroxybenzoate and cinnamic acid were the major components, accompanied by significant amounts of dihydrochalcones, which allowed the identification of its plant source: Populus trichocarpa of Section Tacamahaca. Three dihydrochalcones were new for propolis. The sample from Richmond was characterized by large amounts of p-coumaric and cinnamic acid, typical for poplars of Section Leuce, subsection Trepidae, its plant source was identified as P. tremuloides. Both samples showed good radical scavenging activity against DPPH. Obviously, Northern type propolis is a promising potential source of biologically active substances and deserves further investigation. Keywords: Propolis; Dihydrochalcones; Populus 1. Introduction Propolis (bee glue) is a sticky dark-colored material that honeybees collect from living plants, mix it with wax and use it in the construction and adaptation of their nests. Propolis is also used as an embalming substance to cover hive invaders, which bees have killed but cannot transport out of the hive [1]. It has been used in folk medicine since ancient times and is now known to be a natural remedy with pronounced antibacterial, antifungal, antioxidative, antitumoral, immunomodulatory and other beneficial activities [2,3]. The plant source is of crucial importance for the chemical composition and thus for the biological activity of propolis in a particular region. Bud exudates of poplar trees are the main source of bee glue in the Temperate *Corresponding author. Email: bankova@orgchm.bas.bg Natural Product Research ISSN 1478-6419 print: ISSN 1029-2349 online ß 2006 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410500056918

532 R. Christov et al. Zone [4] and chemical data show a clear preference to Populus species belonging to the Section Aigeiros [5 8]. However, propolis from northern regions, where Aigeiros poplars are absent, has received little attention. In Northern Russia, birch Betula verrucosa and trembling aspen Populus tremula (Sec. Leuce) are documented as propolis plant sources [9], which leads to specific chemical characteristics. In North America, only bee glue from Sydenham, Ontario (Canada), has been analyzed and found to originate from the bud exudates of poplars from Section Aigeiros: P. deltoides Marsh., P. fremontii Wats. or P. maximoviczi Henry [10]. The objective of this work is to study the chemical composition and radical scavenging activity of bee glue from regions in Canada that lay outside the area of distribution of Aigeiros poplars: Boreal forest and Pacific coastal forest regions. The chemical composition was studied by GC-MS and the alcohol extract was silylated because most of its components were not volatile enough for GC without derivatization. 2. Experimental 2.1. Propolis samples The propolis samples were collected in the summer of 2002 at an apiary near Sidney, in the region of Victoria International Airport, Vancouver Island, British Columbia, and at an apiary at St. Claude in the region of Richmond, Quebec. 2.2. Extraction and sample preparation Propolis, grated after cooling, was extracted for 24 h with 70% ethanol at room temperature (1 : 10 w/v). The extract was evaporated to dryness. About 5 mg of the residue was mixed with 75 ml of dry pyridine and 50 ml of bis(trimethylsilyl)trifluoroacetamide (BSTFA), heated at 80 C for 20 min and analyzed by GC-MS. 2.3. GC-MS analysis The GC-MS analysis was performed with a Fisons 8060 gas chromatograph connected with Autospec-Tof magnetic sector MS system (Micromass), equipped with a DB-5MS column, 30 m 0.25 mm, 0.25 um film thickness. GC conditions: splitless injection mode (40s), injector temperature 300 C, temperature program: initial temperature 80 C (1 min hold) and up to 300 C(6 C min 1 ) with 15 min hold. Column interface T 280 C and ionization source T 250 C. Ionization voltage 70 ev. 2.4. Identification of compounds The identification was accomplished using computer searches on commercial libraries. In some cases, when identical spectra had not been found, only the structural type of the corresponding component was proposed based on its mass spectral fragmentation. The components of ethanol extracts of propolis were determined by considering their areas as a percentage of the total ion current. Some components remained unidentified because of the lack of authentic samples and library spectra of the corresponding compounds.

Chemical composition of propolis from Canada 533 2.5. DPPH free radical scavenging activity DPPH free radical scavenging activity was measured according to the procedure described in [11]. In brief, the extracts were dissolved in ethanol (0.21 mg ml 1 ), the solutions analyzed (250 ml) were diluted to 2 ml with ethanol and 1 ml DPPH solution was added (0.02% in ethanol). The resulting solution was thoroughly mixed and absorbance was measured at 517 nm after 30 min. The scavenging activity was determined by comparison of the absorbance with blank (100%), containing only DPPH and solvent. Caffeic acid was used as a positive control. 3. Results and discussion The chemical composition of the ethanol extracts of both samples was investigated by GC-MS after silylation. The results obtained showed the distinct chemical profiles of the two specimens (table 1). In the sample from Victoria, p-hydroxyacetophenone was the major component, accompanied by benzyl hydroxybenzoate, cinnamic acid and significant amounts of dihydrochalcones. We found five dihydrochalcones, three of them for the first time in propolis: 2 0,6 0 -dihydroxy-4 0,4-dimethoxydihydrochalcone (1), 2 0,4 0,6 0,-trihydroxy- 4-methoxydihydrochalcone (2) and 2 0,6 0,4-tryhydroxy-4 0 -methoxydihydrochalcone (3). Dihydrochalcones are considered to be characteristic for bud exudates of poplars of the Section Tacamahaca but not of Section Aigeiros [12,13] and have been found in propolis only rarely and in low concentrations [6]. Obviously, the plant source of this propolis was bud exudate of a poplar of Section Tacamahaca. Two species of this Section are widespread throughout Canada: P. trichocarpa Torr. et Gray and P. balsamifera L., but black cottonwood P. trichocarpa is regarded as the Pacific coastal species of poplar [14]. The major components of P. trichocarpa bud exudates have been found to be p-hydroxyacetophenone, benzyl hydroxybenzoate and cinnamic acid, accompanied by the three dihydrochalcones 1, 2 and 3 [12]. These compounds were important constituents of the sample from the region of Victoria, too, and thus its plant source is definitely P. trichocarpa. The only other chemically proven propolis source from Section Tacamahaca is P. suaveolens in Mongolia [15]. The sample from Richmond region was characterized by large amounts of p-coumaric and cinnamic acids, while acetophenones and dihydrochalcones were completely absent. This sample, as expected, also lacked the typical compounds of Section Aigeiros bud exudates: series of pinobanksin 3-O alkanoates and caffeic acid derivatives [6]. The high concentration of cinnamic and p-coumaric acid and the low concentrations of flavonoids are typical for poplars of Section Leuce, subsection Trepidae, such as P. tremula [5] in European Boreal forests [9]. Its close relative P. tremuloides is the most probable propolis source plant in the Canadian Boreal forest. Our samples showed very good radical scavenging activity, compared to the well-known antioxidant caffeic acid, used as a positive control. These results are in accordance with previous ones published on antioxidative activity of propolis from different geographic origin [11,16,17]. The presence of diverse phenolic compounds, although different in different samples, is a good explanation for this type of activity. For the sample from Richmond, the high concentrations of p-coumaric acid and its esters are responsible for the radical scavenging action. For the sample from

534 R. Christov et al. Table 1. Chemical composition of ethanol extract of propolis samples (% of total ion current). a Compound Victoria Richmond Aromatic acids Benzoic acid 1.6 9.7 Dihydroxycinnamic acid 0.4 0.3 Z-cinnamic acid 0.3 E-cinnamic acid 10.3 9.1 3-Phenyl-3-hydroxypropanoic acid 1.4 Methoxyphenylpropanoic acid 0.6 4-Hydroxybenzoic acid 0.6 Z-p-coumaric acid 0.6 E-p-coumaric acid 3.4 18.8 Ferulic acid 1.0 3.1 Caffeic acid 0.8 Other aromatics Benzyl alcohol 0.1 0.3 4-Hydroxybenzaldehyd 0.3 Hydroquinone 0.6 Cinnamyl alcohol 0.4 0.1 Hydroxyacetophenone 16.8 Fatty acids Oleic acid 0.7 Stearic acid 0.1 Palmitic acid 0.3 Esters Benzyl benzoate 2.4 0.3 Benzyl methoxybenzoate 5.0 Benzyl hydroxybenzoate 5.0 Benzyl dihydroxybenzoate 2.0 Benzyl Z-p-coumarate 0.2 0.3 Benzyl E-p-coumarate 0.8 5.4 Phenethyl p-coumarate 0.6 Benzyl ferulate 0.5 1.5 Benzyl caffeate 0.1 0.3 Phenethyl caffeate 0.1 Cinnamyl caffeate 0.3 Pinostrobin chalcone 0.3 Pinocembrin 0.1 2.4 Pinobanksin 0.2 1.2 Sakuranetin 1.1 Isosakuranetin 0.2 Alpinone 0.1 Pinobanksin 3-O-acetate 1.4 Galangin 0.2 2.0 Dihydrochalcones 2 0,6 0 -Dihydroxy-4 0 methoxydihydrochalcone 1.9 2 0,4 0,6 0 -Trihydroxydihydrochalcone 0.6 2 0,6 0 -Dihydroxy-4 0,4-dimethoxydihydrochalcone b 1.6 2 0,4 0,6 0 -Trihydroxy-4-methoxydihydrochalcone b 1.3 2 0,6 0,4-Trihydroxy-4 0 -methoxydihydrochalcone b 1.0 Others Glycerol 0.5 Hexoses 7.9 26.5 Sesquiterpenes 12.0 0.2 a The ion current generated depends on the characteristics of the compound concerned and is not a true quantification. b New for propolis.

Chemical composition of propolis from Canada 535 Table 2. Radical scavenging activity of Canadian propolis. Sample DPPH radical scavenging activity, % inhibition a Victoria 79 5 Richmomd 65 7 Caffeic acid 58 6 a Mean value of three measurements SD. Victoria, dihydrochalcones might be of special importance in this respect, as they are known to have significant radical scavenging activity against DPPH [18]. This is a difference to propolis originating from poplars of the Section Aigeiros, where the major antioxidant and radical scavenging compounds have been identified as caffeic acid derivatives and flavones [19] (table 2). 4. Conclusions Our samples originate from two climatic and vegetation regions of Canada: the Boreal forest to the northeast of Montreal, and the Pacific coastal forest of British Columbia [20], where poplars of Section Aigeiros are not present. The results obtained demonstrate that honeybees are able to find suitable plant sources of bee glue in the absence of their most preferred source P. nigra L., in the northern regions of North America. These sources were identified as P. trichocarpa (Sec. Tacamahaca) and P. tremuloides (Sec. Leuce). Both samples demonstrated significant radical scavenging activity against DPPH, due to the high concentrations of phenolic compounds: p-coumaric acid and its esters in the Richmond sample; hydroxyacetophenone and dihydrochalcones in the Victoria sample. Obviously, Northern type propolis is a promising potential source of biologically active substances and deserves further investigation. Acknowledgements The authors wish to thank Mr. Jean Porret, Director of Service d action humanitaire et communautaire de l Universie de Montreal for the propolis sample. References [1] E.L. Ghisalberti, Bee World, 60, 59 84 (1978). [2] G.A. Burdock, Food & Chem. Toxicol., 36, 347 363 (1998). [3] A.H. Banksota, Y. Tezuka, S. Kadota, Phytother. Res., 15, 561 571 (2001). [4] V.S. Bankova, S.L. De Castro, M.C. Marcucci, Apidologie, 31, 3 15 (2000). [5] V. Bankova, L. Kuleva, Animal Sci., 2, 94 98 (in Bulgarian) (1989). [6] W. Greenaway, T. Scaysbrook, F.R. Whatley, Bee World, 71, 107 118 (1990). [7] C. Garcia-Viguera, W. Greenaway, F.R. Whatley, Z. Naturforsch., 47c, 634 637 (1992). [8] E. Wollenweber, St. Buchmann, Z. Naturforsch., 52c, 530 535 (1977). [9] S.A. Popravko, Chemical composition of propolis, its origin and standardization, In A Remarkable Hive Product: PROPOLIS, pp. 15 18, Apimondia Publ. House, Bucharest (1978). [10] C. Garcia-Viguera, F. Ferreres, F.A. Tomas-Barberan, Z. Naturforsch., 48c, 731 735 (1993).

536 R. Christov et al. [11] A.H. Banskota, Y. Tezuka, I.K. Adnyana, K. Midorikawa, K. Matsushige, K. Message, A.G.H. Huertas, S. Kadota, J. Ethnopharm., 72, 239 246 (2000). [12] S. English, W. Greenaway, F.R. Whatley, Phytochemistry, 30, 531 533 (1991). [13] W. Greenaway, S. English, E. Wollenweber, R.F. Whatley, J. Chromatogr., 472, 393 400 (1989). [14] T.C. Brayshow, Can. Field-Nat., 79, 91 95 (1965). [15] V. Bankova, A. Dyulgerov, S. Popov, L. Evstatieva, L. Kuleva, O. Pureb, Z. Zamjansan, Apidologie, 23, 79 85 (1992). [16] S. Scheller, T. Wilzcok, S. Imielski, W. Krol, J. Gabrys, J. Shani, Int. J. Rad. Biol., 57, 461 645 (1990). [17] Li-Chang Lu, Yue-Wen Chen, Cheng-Chun Chou, J. Food Drug Anal., 11, 277 282 (2003). [18] G.W. Plumb, S.J. Chambres, N. Lambert, B. Bartolome, R.K. Heaney, S. Wanigatunga, O.J. Aruoma, B. Halliwell, G. Williamson, J. Food Lipids, 3, 171 178 (1996). [19] T. Hamazaka, Sh. Kumazawa, T. Fujimoto, T. Nakayama, Food Sci. Technol. Res., 10, 86 92 (2004). [20] A.C. Carder, J. Range Management, 23, 263 267 (1970).