Pollen analysis of Brazilian propolis

This article was downloaded by: [179.107.44.135] On: 26 April 2015, At: 05:58 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Grana Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/sgra20 Pollen analysis of Brazilian propolis Ortrud Monika Barth a b a Departamento de Virologia , Instituto Oswaldo Cruz FIOCRUZ , Avenida Brasil 4365, Rio de Janeiro, 21045–900 b Departamento de Botânica, Instituto de Biologia , Universidade Federal do Rio de Janeiro, CCS , Bloco A, Ilha do Fundão, Rio de Janeiro, 21949–900, Brazil Published online: 03 Sep 2009. To cite this article: Ortrud Monika Barth (1998) Pollen analysis of Brazilian propolis, Grana, 37:2, 97-101, DOI: 10.1080/00173139809362650 To link to this article: http://dx.doi.org/10.1080/00173139809362650 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. 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Downloaded by [179.107.44.135] at 05:58 26 April 2015 Eleven samples of Brazilian propolis were analysed for their pollen content: seven from the state of Rio de Janeiro, three from Minas Gerais and one from Rio Grande do Sul. A standard methodology for the analysis of Brazilian samples of propolis in terms of their pollen content is proposed. The preparation of a sample of the sediment just prior to acetolysis is useful in order to examine plant hairs (trichomes) and other plant structures not resistant this procedure. The most important pollen types detected were Eupatorium-type, Cecropia-type and Eucalyptus-type, each one certainly comprising more than one plant species with similar morphology. Mimosa caesalpiniaefolia type dominated in a sample collected next to the city of Rio de Janeiro. It was possible to characterize a sample coming from the state of Rio Grande do Sul relative to the samples obtained from the two other states. The influence of the large city of Rio de Janeiro could be recognized in the pollen spectra of propolis collected in its vicinity. Pollen of plant species characteristic of the southeast Atlantic forest could be determined in some samples. The importance of pollen grains from anemophilous plant species is emphasized. O. M. Barth, Departamento de Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, 21045-900 Rio de Janeiro, and Departamento de Botânica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, CCS, Bloco A, Ilha do Fundão, 21949-900 Rio de Janeiro, Brazil. (Manuscript accepted 15 May 1998) When bee keepers prepare propolis from plant exudates, resins and bee waxes in order to seal up gaps in the beehives, they unintentionally introduce pollen grains. These pollen grains come from the flowers visited by the bees for nectar and pollen and also from wind pollinated plants. Because of this, identification of the plant species whose pollen occurs in propolis samples, allows a characterization of the vegetation surrounding the beehive and frequently also the geographical region from which the propolis was collected (D'Albore 1979, Warakomska & Maciejewicz 1992). The richness of the plant species used by the bees to prepare propolis is an indicator of good quality and good medicinal properties. Propolis has long been used in local medicine and its biochemical composition has been analysed (Banková et al. 1983). Its anti-bacterial activity is well known (Grange & Davey 1990). Anti-viral experiments have also been carried out to demonstrate its use in containing the spread of viruses (König & Dustmann 1988). Single or compound hairs from plants (trichomes), especally leaves, are commonly an additional component of propolis. Some of these indicate the plant species from which they were collected by their morphological characteristics (D'Albore 1979, Theobald et al. 1979). Pollen can occur in propolis firstly through sticking to plant resins in the case of wind pollinated flowers, secondly by being introduced from stored pollen loads inside the hives, or thirdly by being transported on the hairs of bees which have been in contact with pollen in the field. A general study characterizing the phytogeographical regions of several countries and continents producing propolis was carried out by D'Albore (1979). Three of the samples of propolis analysed by him came from Brazil. His © 1998 Scandinavian University Press. ISSN 0017-3134 results showed that, with the exception of a few samples, it may be possible to determine the phytogeographical origin of the propolis by pollen analysis. The southeast region of Brazil comprises the Atlantic forest, one of the richest forests, in terms of the number of plant species and species diversity, in the world (Hueck 1966). Propolis collected there is considered to be of the best quality. The Atlantic forest formation stretches from the state of Rio Gande do Sul, southern Brazil, north along the Brazilian Atlantic coast as far as Rio Grande do Norte, and extends about 4000 km in a north-south direction. A variety of phytogeographical regions occur and each has a characteristic plant assemblage. Propolis collected from this area varies in composition and quality. Pollen analysis of propolis has not previously been carried out in Brazil. The data obtained from other countries provide only a reference for initiating the present studies. The only data available are that of D'Albore (1979) who analysed 56 samples of propolis from different countries and continents, three of them from Brazil. There is little melissopalynological data from Brazil, the first being the thesis published by Santos (1961). More recently, Barth (1989) described and illustrated the pollen morphology of the most important Brazilian honey plants and presented a list of published papers on this topic. The present study aims to analyse and characterize propolis samples from the Atlantic forest region in order to recognise the phytogeographic regions from which the samples were collected. The data obtained may be compared with that obtained from areas other than the Atlantic forest. MATERIALS AND METHODS Eleven samples of propolis collected during the last years were used to establish a method for routine prepartion. They were obtained Grana 37 (1998) Downloaded by [179.107.44.135] at 05:58 26 April 2015 98 Ο. Μ. Barth from bee hives from the southeastern region of Brazil, from the states of Rio de Janeiro and Minas Gérais (Table I). The samples were either very hard or elastic; in the latter case the propolis could not be scraped, but was cut into very small pieces for dissolving. All the samples were kept in the cold at — 20°C. Several experiments were made to extract resins and waxes from the samples. According to Warakomska & Maciejewicz (1992), only 55-73.9% of the initial weight of a propolis sample remains when only ethanol extraction is used; 17.6-31.6% when this is followed by benzol and acetone extraction and 8.5-13.2% after a final filtration. We confirmed these data on the Brazilian propolis samples. One problem is that a large amount of organic matter remains in the final sediment. The pollen cannot be easily detected and identified using these methods. We decided to apply the acetolysis method (Erdtman 1952) to our samples in order to reduce the organic component of the sediment after chemical extraction, as in the procedure described by D'Albore (1979). We made a first extraction using only ethanol (overnight or several days) in contrast to the ethanol-chloroformaceton (1:1:1) mixture used by D'Albore (1979) and obtained the same results. It is important to use 10% KOH, followed by an ultrasonic treatment before acetolysis on the first sediment obtained. We also prepared a slide of the sediment before the acetolysis stage, in order to obtain information about plant hairs (trichomes) and other organic elements that do not survive this treatment. The protocol we propose is as follows: - weigh 0.5 g of the scraped propolis - extract with 15 ml ethanol overnight or for some days, shaking the vial at intervals - divide the suspension into two centrifuge tubes and centrifuge - resuspend the sediment with 13 ml of ethanol in each tube - centrifuge and decant - add 12 ml of 10% KOH and boil for 2 min in a waterbath - put the tubes in an ultrasonic agitator for 5 min - centrifuge and decant - add 13 ml of distilled water - transfer the sediment to another centrifuge tube, through a 0.3 mm mesh sieve in order to remove large organic particles - centrifuge, decant and prepare a slide of the sediment with glycerine-jelly (to observe any organic material such as plant hairs, etc.) - add 5 ml of glacial acetic acid and leave to stand overnight - centrifuge and decant - add the acetolysis mixture (9 volumes of acetic anhydride and 1 volume of concentrated sulphuric acid) and leave for 3 min at 80°C in a waterbath - centrifuge and decant - wash the sediment with distilled water Table I. Samples of propolis analysed. Reference number PI P2 P3 P4 P5 P6 P7 P8 P9 P10 Pli Grana 37 (1998) State of origin Beekeeper Rio de Janeiro (Mage) Minas Gérais (Viçosa) Rio de Janeiro (Rio Bonito) Minas Gérais Minas Gérais (Divino do Carangola) Rio de Janeiro (Mendes) Rio de Janeiro (city) Rio de Janeiro (city) Rio de Janeiro (city) Rio Grande do Sul Rio de Janeiro (Barra do Pirai) Vedolin, 1995 Alexandre, 1996 Cândido, 1996 Elias, 1996 ?, 1996 - add a mixture of glycerine and water 1:1 and leave for at least 30 min - centrifuge and decant, prepare the final slides, one with unstained glycerine-jelly and another with basic fuchsin-stained glycerinejelly - store the remainder of the sediment in glycerol. Except for sample P2, which was poor in pollen grains, the counts totalled between 150 and 250 pollen per microscope slide prepared, sometimes more. RESULTS A great variety of pollen types were detected in the propolis samples (Figs. ΙΑ-D). In order to have some basis for comparison, the pollen grains were counted, although it was realised that such counts do not represent a quantitative evaluation of propolis plant origin. Six samples (P1-P6) were prepared and counted twice, in order to observe variations in pollen type frequencies between different parts of the same propolis sample. All these data are presented in Table II. In accordance with melissopalynological criteria (Louveaux et al. 1970), we have used the following designations of pollen frequency: PD for dominant (more than 45%), PA for accessory (15-45%), and PI for isolated but important to characterize the phytogeographical origin of the sample (3-15%). The last group, PO, for isolated and ocasionally observed pollen grains (less than 3%), comprises a great number of less important plant species, sometimes represented by only a single grain, is not considered in Table II. The total number of pollen counted for each sample varies; when no more important pollen types (PI) were encountered, the counting was stopped. The range of pollen types in some samples was so high, however, that the saturation curve was not achieved. In three of the propolis samples examined, pollen of a single plant species occured at percentage as high as those of PD; these were Eucalyptus (sample PI), Eupatorium-type (sample P3) and Mimosa caesalpiniaefolia-typs (sample P7). Several pollen types occur as PA, some of them belong to wind pollinated species as in sample P6. A variety of organic elements were detected in the sediments in addition to pollen. As described in "Methods", one microscope slide was prepared before acetolysis. In this plant hairs or trichomes were frequently observed. In all the propolis samples studied, only two types of unbranched trichomes were observed: a single cell long hair (like Fig. 5.2.h of Boronia, Rutaceae, in Theobald et al. 1979) and a multicellular unbranched hair (like Fig. 5.2.S of Inga, Mimosoideae, in Theobald et al. 1979: Fig. 3). Brownish coloured spores and fungal filaments were frequently noticed (Fig. IE). DISCUSSION Américo, 1996 Pereira, 1996 Pereira, 1996 Pereira, 1996 Domanski, 1996 Arnaldo, 1996 When the propolis extraction was made exclusively with a mixture of organic solvents (ethanol, benzol, acetón) less sediment was obtained than by ethanol extraction alone. D'Albore (1979) used a mixture of ethanol, chloroform and acetone, followed by KOH, ethanol and acetolysis. For clear visibility of the pollen, good preparations were obtained by Pollen analysis of Brazilian propolis 99 5 / <Î3 .7.2 rff m f Of Downloaded by [179.107.44.135] at 05:58 26 April 2015 B f-m •Ο) · χ ο,· Fi^. /. Α-D. Pollen from acetolysed propolis samples. (A) Sample P8: Anacardiaceae (1), Arecaceae (2), Asteraceae (3), Solanaceae (4), Eucalyptus sp. (5); scale bar = 50 μΐη. (Β) Sample P7: Asteraceae ( 1 ), Combretaceae (2), Eucalyptus sp. (3), Persea americana (4), Triumfetta sp. (5); scale bar = 50 μιη. (C) Sample P8: Arecaceae (1), Asteraceae (2), Bombacaceae (3), Solanaceae (4); scale bar = 50 μΐη. (D) Sample ΡΙΟ: Hyptis sp. (1), hyphae and spores of fungi (2); scale bar=l(^m. (E) Sample P6: propolis extract before acetolysis; one long single cell plant hair and several pluricellular plant hairs (2) (200x); scale bar = 50 μπι. a simple extraction method with pure ethanol, followed by KOH, ultrasonic and acetolysis treatment. By this method the organic material was dissolved and the pollen concentration increased. Pollen slides prepared with unstained glycerine-jelly show the typical colour of the oxidised exines of empty grains. The use of stained glycerine-jelly helps in the detection of small-sized pollen against the background of residual organic matter and in differentiating them from fungal spores. In order to test the method, a second sample from the six propolis specimens was processed in the same way. The results obtained were similar. A standartization of this pollen preparation method for propolis samples would allow a comparison of the results obtained by different authors and in different regions. The data presented in Table II show that the most common pollen types belong to the Compositae: Eupatorium-type, Moraceae: Cecropia-type and Myrtaceae: Eucalyptus-type. These types generally represent more than one species, with very similar pollen and it was dificult or impossible to distinguish them. With respect to their phytogeographical origin, only Cecropia-type pollen was not found in the sample from Rio Grande do Sul (P10). Myrcia-, Palmae-, Coffea-, Triumfetta- and SWarøm-pollen types appear with a regular frequency (PA). Mimosa caesalpiniaefolia-typc pollen was found as dominant (PD) in a Grana il (1998) 100 Ο. Μ. Barth Table II. Most frequent pollen types in the samples of propolis analysed. »Pollen types described and illustrated in Barth (1989). Pl-Pl 1 =samples of propolis (Table I). PD=dominant (>45%), PA=accessory (15-45%), PI = isolated (<15%) pollen grains. Samples of propolis Downloaded by [179.107.44.135] at 05:58 26 April 2015 Pollen types* Anacardiaceae Mangifera* Bignoniaceae Tabebuia Bombacaceae Bombax Combretaceae/ Melastomataceae* Compositae Ambrosia Elephaniopus* Eupatorium* Gochnatia Montanoa* Vemonia* Flacourtiaceae Casearia Gramineae Paspalum* Labiatae Hyptis* Leg. Mimosaceae Mimosa caesalpiniaefolia* Leg. Caes. Dehnix Leg. Pap. Moraceae Brosimum Cecropia* Myrtaceae Eucalyptus* Myrda* Palmae* Piperaceae Piper Polygonaceae Antigonon leptopus Rubiaceae Borreria verlicillata* Coffea Rutaceae Zanthoxylum* Sapindaceae Serjania Solanaceae Solarium* Tiliaceae Triumfetta* Ulmaceae Celtis* not identified PI P2 P3 P4 P5 P6 1st 2nd sample sample 1st 2nd sample sample 1st 2nd sample sample 1st 2nd sample sample 1st 2nd sample sample 1st 2nd sample sample P8 P9 P10 Pll PI PI PI PI PI PI PI PI PA PA PI/PA PD PI/PA PA PA PA PA PI PI PA PI PA PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PD PI PI PI PI PD PI PD PA PI PI PA PI PA PA PI PI PA PA PA PI PA PA PA PI PA PI PI PA PA PI PI PI PA PA PI PI PA PA PI PI PI/PA PI PA PI PI PI PI PA PI/PA PI PI PI PI sample from the vicinity of Rio de Janeiro. Shrubs of this species flower in February and are a valuable source for bee keepers. Grana 37 (1998) P7 The majority of the pollen types represented by only a few pollen grains (PI) could belong to samples from the state of Rio de Janeiro (RJ) and Minas Gérais (MG). Labiatae: Pollen analysis of Brazilian propolis Downloaded by [179.107.44.135] at 05:58 26 April 2015 Hyptis-type and the Solanum-type were found only in samples from RJ, while Rubiaceae: Coffea-type occurred only from MG. The pollen spectra of the samples from RJ and MG do not show significant differences with respect to the less frequent pollen types (PI). At present samples of propolis coming from these states cannot be characterized on the basis of pollen analysis. One sample from RJ (P7) looked different and contained no Compositae pollen; another two samples of RJ (P8, PI 1) showed typical elements of the southeast Atlantic forest. In conclusion, it was possible to identify such plant associations as forests, bushes and savannas from where the Brazilian propolis samples analysed here were collected. D'Albore (1979) emphasized the necessity of applying acetolysis to the propolis sediment obtained after extraction. We agree with him when seeing the large quantity of organic matter that remained in our final preparations without acetolysis treatment. Warakomska and Maciejewicz (1992) do not use acetolysis in order to preserve plant trichomes and secretory discs. We did not find any morphological types of plant hair in our samples that were of taxonomic value. At present we prefer to use acetolysis but prepare an extra slide before the treatment, in order to screen the samples. In D'Albore's data (1979) from three Brazilian propolis samples, all the pollen types identified occur at less than 20% of total pollen counted and no dominant pollen type was detected; which indicates a high floristic diversity. (His percentage classes are different from the standard, defined by Louveaux et al. 1970). The three samples he examined probably originated in the south of Brazil as Ilex is well represented, and so are Rosaceae {Pinus- and Prunus-poüen types) Proteaceae (Roupala) and Mimosa scabrella. The anaemophilous pollen types of our samples form an important proportion of the total pollen. Cecropia is the most frequent wind pollinated pollen type and appears at more than 20% of the total in samples P4, P5, P6, P8 and PI 1. However, pollen of this genus is absent in samples obtained from hives located near the city of Rio de Janeiro (P7 and P9) and from Rio Grande do Sul (P10). Other anaemophilous pollen types found are Ambrosia (Compositae), Brosimum (Moraceae), Piper (Piperaceae) and 101 Celtis (Ulmaceae), which occasionally reach values exceeding 15%. These data show that anaemophilous pollen types are an important constituent of Brazilian propolis. The low quantity of such pollen recorded by D'Albore (1979) may be explained by the geographical origin of the samples, probably from the south of Brazil. This is in agreement with the data obtained for our single sample from the state of Rio Grande do Sul. ACKNOWLEDGEMENTS I am grateful to Ronaldo L. Justo and Vânia M. L. Dutra for technical assistance and to the beekeepers listed in Table I who provided the propolis samples. Financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) is acknowledged. REFERENCES Bankova, V. S., Popov, S. S. & Marekov, N. L. 1983. A study on flavonoids of propolis. - Journal of Natural Products 46:471-474. Barth, O. M. 1989. O Pólem no Mel Brasileiro. - Gráfica Luxor, Rio de Janeiro. D'Albore, G. R. 1979. L'origine géographique de la propolis. Apidologie 10: 241-267. Erdtman, G. 1952. Pollen Morphology and Plant Taxonomy. Angiosperms. - Chron. Bot., Waltham MA. Grange, J. M. & Davey, R. W., 1990. Antibacterial properties of propolis (bee glue). - Journal of the Royal Society of Medicine 83: 159-160. Hueck, K. 1966. Die Wälder Südamerikas. - G. Fischer, Stuttgart. Koenig, B. & Dustmann, J. H. 1988. Baumharze, Bienen und antivirale Chemotherapie. - Naturwissenschaftliche Rundschau 41: 43-53. Louveaux, J., Maurizio, A. & Vorwohl, G. 1970. Methodik der Melissopalynologie. - Apidologie 1: 193-209. Santos, C. F. de O. 1961. Morfologia e Valor Taxonômico do Pólen das Principals Plantas Apícolas. - Thesis. Esc. Sup. Agricult Luiz de Queiroz, Univ.Sao Paulo, Piracicaba SP. Theobald, W. L., Krahulik, J. L. & Rollins, R. C. 1979. Trichome Description and Classification. - In: Anatomy of the Dicotyledons. Vol. I. (ed. C. R. Metcalf & L. Chalk), pp. 40-53. - Clarendon Press, Oxford. Warakomska, Z. & Maciejewicz, W. 1992. Microscopic analysis of propolis from Polish regions. - Apidologie 23: 277-283. Grana 37 (1998) View publication stats