Journal of ApiProduct and ApiMedical Science 3 (2): 67 - 74 (2011) © IBRA 2011 DOI 10.3896/IBRA.4.03.2.01

ORIGINAL RESEARCH ARTICLE

A palynological analysis of Brazilian propolis samples

Alex da Silva de Freitas1*, Ortrud Monika Barth1,2, Érika de Oliveira Sales1, Adriana Hitomi Matsuda3 and Ligia Bicudo de Almeida-Muradian3

1Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Botânica, Laboratório de Palinologia, Rio de Janeiro, RJ, Brasil. 2 Fundação Oswaldo Cruz, Instituto Oswaldo Cruz. Avenida Brasil 4365, 21040-900 - Rio de Janeiro, RJ, Brasil. 3 Universidade de São Paulo, Faculdade de Ciências Farmacêuticas. Cidade Universitária, São Paulo, SP, Brasil.

Received 09 December 2010, accepted subject to revision 26 January 2011, accepted for publication 14 February 2011.

*Corresponding author: Email: [email protected]

Summary

Twenty eight propolis samples, collected in different apiaries located in the Northeast, Southeast and South Brazilian regions were examined for content. In order to facilitate analysis, wax and resin was removed with ethanol, followed by acetolysis and ultrasound treatment. The target number of pollen grains was 300 or more per sample. The dominant pollen types detected in the Northeast samples were verrucosa ( state) and Borreria ( state), followed by Acacia (Rio Grande do Norte state). In the Southeast samples, Minas Gerais state were dominated by Asteraceae followed by Eucalyptus, Cecropia and Solanaceae. In the São Paulo and Rio de Janeiro state samples, the dominant pollen types were Asteraceae and Eucalyptus. In the South region samples, Asteraceae (Paraná state) and Eucalyptus (Rio Grande do Sul state) were dominant. Pollen grains of nectariferous were also common and dominant, and pollen of anemophilous taxa were also frequent. Analysis of the pollen types found in propolis samples from the Brazilian regions demonstrates that samples from the Northeast could be distinguished from those from the South and Southeast.

Keywords: Propolis, pollen, phytogeography,

types. The country is currently the world’s third largest producer. The Introduction colour of Brazilian propolis ranges from yellow, dark greenish to Propolis is characterized by a mixture of resin exudates originating brown, depending upon the source of vegetation and the local from several plant sources, including waxes, essential oils extracted environmental factors of production. Some samples are fibrous and from woody plants and flowers. Five percent of its weight comprises hard, while others are elastic and malleable, having complex pollen grains as a contaminant (Ghisalberti 1979, Warakomska & chemical composition (Teixeira et al. 2003, Salatino et al. 2005, Maciejewicz 1992, Barth et al. 1999). Bees use propolis for several Marcucci et al. 2001). purposes: to seal the hives in order to prevent mechanical damage, The growing variety and availability of apiculture in the to act as a thermoregulatory agent and to avoid exposure to air Brazilian market derivatives encourages research into the various currents and to prevent colony infestation (Manrique & Soares 2002, propolis types, in order to control the quality of this product and Teixeira et al. 2003). potentially increase apiculture business prospects in Brazil, as well as Throughout history propolis has been used in medicine in the international market (Luz et al. 2007). Scientific knowledge and its biochemical compounds have been studied. Its medicinal about the geographical and botanical origin of propolis needs to be properties are increased with the addition of glandular secretion expanded (Teixeira et al. 2003). from the bee head. The biological and antiseptic properties of Few studies focus on the palynological analysis of propolis propolis have been widely described in pharmacological studies in Brazil. The first study was performed by D'Albore (1979), who (Marcucci 1995, Kujumgiev et al. 1999, Banskota et al. 2001). analysed several propolis samples including three from Brazil. Brazilian vegetation provides a large range of propolis Additional studies were undertaken by Barth (1998), Barth et al. 68 Freitas, Barth, Sales, Matsuda, Almeida-Muradian

(1999), Bastos (2001), Barth & Luz (2009), Freitas et al. (2010), Table 1. Characterisation of Brazilian propolis samples. with further studies of geopropolis samples of stingless bees Region Origin/ Trichomes Organic Pollen (Meliponinae) by Barth (2006) and Barth & Luz (2003). Municipality matter grains The present study aimed to analyse and characterize BA (Irecê) ++++ +++ +++ pollen grains in propolis samples obtained from several Brazilian North- east RN (João + +++ ++ states in order to define the phytogeographic regions from which the Câmara) samples were obtained. MG1 (Barbacena) ++++ - ++

MG2 (Itabira) ++ - ++ MG3 (Lavras) ++++ - + Materials and methods MG4 (Patrocínio) ++++ - + MG5 (Juiz de ++++ - + Twenty eight propolis samples produced in different Brazilian states, Fora) covering the Northeast, Southeast and South regions were analysed. SP1 (Salesópolis) ++++ - + The apiaries used in the present study, represent different locations SP2 (Barra do ++++ - + of propolis collection, with João Câmara (RN), Içara (SC) and Pelotas Chapéu) (RS) closer to the sea than the other samples, which were obtained SP3 (São Carlos) ++++ - ++ from inland apiaries. South- SP4 (Cajamar) ++++ - ++ Palynological processing of the samples followed the east RJ1 (Itaboraí) ++++ - + standard methodology (Barth, 1998; Barth et al. 1999), using 0,5g RJ2 (Rio Bonito) ++ + +++ of scraped propolis and extraction overnight with ethanol. Next, the RJ3 (Santa ++ - +++ Isabel) sediment was treated with KOH, ultrasound and sieved to eliminate RJ4 (Japeri) +++ - +++ large fragments. In this stage, two microscope slides were mounted RJ5 (Barra do +++ - +++ with sediment obtained after centrifugation for observation of plant Piraí) RJ6(Santa +++ - ++ trichomes and other organic residues that may be destroyed in Isabel) sequence. Then acetolysis (Erdtman, 1952) was applied, and two RJ7 (Visconde de + - +++ Mauá) additional microscope slides were prepared using glycerin jelly, one RJ8 (Petrópolis) ++ - +++ stained with basic fuchsin and the other without stain. RJ9 (Itaboraí) +++ - +++ Approximately 300 pollen grains in each sample were PR1 (São José da ++++ - ++ counted and the definition of pollen classes presented by Zander Boa Vista) (Louveaux et al. 1978) was used for qualitative and quantitative PR2 (São Mateus ++++ - + analyses. Samples were observed using light and polarized light do Sul) microscopy. Pollen type identification followed the principles used by PR3 (Wenceslau ++++ - + Brás) Barth (1989) and Roubik & Moreno (1991). South PR4 (Ivaí) ++++ - + SC1 (Içara) ++ ++ +++ SC2 (Anitapolis) +++ - ++ Results RS1 (Pelotas) ++++ + + RS2 (Taquara) ++ ++ + The majority of pre-acetolysis samples contained abundant (-) = without elements; (+) = few; (++) = medium: (+++) = trichomes, scarce organic matter and variable amounts of pollen frequent structured elements. grains (Table 1). Organic matter was largely absent in samples from Brazilian states: BA = Bahia, RN = Rio Grande do Norte, GG = Minas the South region. Gerais, SP= São Paulo, RJ = Rio de Janeiro, PR = Paraná, SC = Santa In seven post-acetolysis samples, the number of pollen Catarina, RS = Rio Grande do Sul. grains present in two slides did not exceed 300 (Table 2 and 3). Except for one, the four remaining samples of Minas Gerais state Eighteen samples of the Southeast region were analysed (MG) showed a low content of pollen grains. (Table 3; Fig 1). The dominant pollen types were of Asteraceae The dominant pollen types occurring in the Northeast (comprising several genera with pollen grains without cristae) in the region samples were of Mimosa verrucosa from Bahia state (BA), three states, and of Eucalyptus in São Paulo (SP) and Rio de Janeiro and Borreria densiflora from Rio Grande do Norte state (RN), this (RJ) states. Accessory pollen types (A) comprised these two pollen last sample contained Acacia as the accessory pollen type (Table 2; types and in addition Cecropia, Mimosa scabrella, Salvia, Poaceae Fig. 1). and Solanaceae pollen types. Important isolated pollen types (I) are frequent. Pollen in Brazilian propolis 69

Table 2. Frequencies of pollen types of propolis samples from three distinct regions in Brazil.

Northeast South Pollen Types States States BA RN PR1 PR2 PR3 PR4 SC1 SC2 RS1 RS2 Amaranthaceae Alternanthera PI Anacardiaceae PI PI Tapirira PI Arecaceae PI PI PI PI PI

PA PA PD PD PA PA PA PA Asteraceae PI 17.9% 33.5% 46.7% 71.8% 22.5% 18.3% 29.5% 15.9%

Cyperaceae PI Euphorbiaceae PI PI Melastomataceae/ Combretaceae PI PI Mimosaceae Mimosa scabrella PI PD Mimosa verrucosa 60.6% PI PI PA Acacia 22.0% Myrtaceae PA PA PA PA PA PA PD PI Eucalyptus 38.4% 21.7% 32,1% 42.1% 34.0% 27.2% 50.3%

Myrcia PI Rubiaceae Borreria verticillata PI PD Borreria densiflora 63.9%

Psycotria PI PI Sapindaceae Paullinia PI Solanaceae PI PA Solanum PI PI 15.6% Theaceae PI Ulmaceae Celtis PI Number of pollen grains counted 305 36 307 313 321 323 300 306 353 352

PD = dominant pollen (> 45%), PA = accessory pollen (15% to 45%), PI = isolated pollen (3% to15%). Brazilian states: BA = Bahia, PR = Paraná, RN = Rio Grande do Norte, RS = Rio Grande do Sul, SC = Santa Catarina 70 Freitas, Barth, Sales, Matsuda, Almeida-Muradian

RJ9 PI PA 33.1% PI PA 27.5% PI PI PI 323

RJ8 PI PI PD 68.6 % PI 347

RJ7 PI PA 24.0% PI PI PI PA 18.4% PI 331

RJ6 PD 56.9% PA 21.1% PI PI 246

RJ5 PA 23.8% PA 18.2% PI 302

RJ4 PA 42.7% PI PA 24.4% PI PI PI 307

RJ3 PD 52.3% PA 16.5% PI PI PI 340

RJ2 PA 29.0% PA 16.5% PI PI PI PI PI PI 327

RJ1 PI PI PI PI PA 18.3 % PI PI PI 180

Brazilian states: MG = Minas Gerais, RJ = Rio de Janeiro, SP = São Paulo São = SP Janeiro, de Rio = RJ Gerais, Minas = MG states: Brazilian

Southest States

SP4 PI PD 82.9 % 316

SP3 PI PD 78.3 % 304

(3% to15%).

in Brazil. in

SP2 PI PD 52.5 % PI PI PI 324

regions

isolated pollen isolated

SP1 PA 17.3 % PD 58.3 % PI 317

MG5 PI PA 22.3 % PI PA 25.5 % PA 20.4 % 314

MG4 PI PI PI PI 181

MG3 PD 53.6 % PI PA 17.5 % PI 252

MG2 PA 24.0 % PI PI PI PI PI PA 22.8 % 158

MG1 PI PA 39.0 % PI 64

of pollen types of propolis samples from three distinct three from samples propolis of types pollen of

Frequencies

3.

Pollen Types Anacardiaceae Tapirira Schinus Arecaceae Asteraceae Bignoniaceae Tabebuia Arrabidea Boraginaceae Cordia Brassicaceae Brassica Caesalpiniaceae Lamiaceae Salvia Melastomataceae/ Combretaceae Mimosaceae scabrella Mimosa verrucosa Mimosa Myrtaceae Eucalyptus Myrcia Poaceae Rubiaceae Borreria Rutaceae Citrus Sapindaceae Paullinia Solanaceae Ulmaceae Celtis Urticaceae Cecropia of Number grains pollen counted

Table PI = 45%), to (15% pollen = accessory (> PA 45%), pollen = dominant PD Pollen in Brazilian propolis 71

Fig. 1 Pollen grains and pollen types of propolis samples: 1-2: Acacia (Mimosaceae). 3-4: Borreria densiflora (Rubiaceae). 5-6: Myrcia (Myrtaceae). 7-9: Mimosa verrucosa (Mimosaceae). 10-12: Eucalyptus (Myrtaceae). 13-14: Solanaceae. 15-16: Eupatorium (Asteraceae). 17-18: Bidens (Asteraceae). 19-20: Cocos (Arecaceae). 21: Cecropia (Cecropiaceae). 72 Freitas, Barth, Sales, Matsuda, Almeida-Muradian

The South Brazilian region was represented by eight were identified as an accessory pollen type in a sample from Rio samples of propolis, four from the Paraná state (PR) and two from Grande do Sul (RS) state. Barth et al. (1999) also observed accessory each of the Santa Catarina (SC) and Rio Grande do Sul (RS) states. Eucalyptus pollen grains in one propolis sample of Rio Grande do Sul Asteraceae and Eucalyptus pollen types were dominant, and they (RS) state. also frequently occured as secondary / accessory pollen types (Table Several species of Eucalyptus are cultivated in Brazil, 2). One sample of Rio Grande do Sul (RS) state contained Solanum mainly in the South and Southeast regions. Their pollen morphology as an accessory pollen type. Important isolated pollen types were is so similar that species characterization is not possible, as in the diverse (Fig. 1). present paper. Montenegro et al. (2001) observed that Eucalyptus globulus in Chile was one of the preferred sources of propolis by bees, according to the significant and sustained presence of pollen Discussion grains in samples. From chemical evidence, the species Eucalyptus citridora was an indicator of a possible source of propolis in Brazil The propolis samples analysed in the present study were (Lopes et al. 2003). characterized by pollen assemblages, providing evidence for Asteraceae plants occur in several types of vegetation, as different types of plants and vegetation. In the Brazilian Northeast pioneers and mainly in areas with open vegetation as pasturelands, region, pollen grains of Mimosa verrrucosa (Bahia state) and supplying bees with pollen and nectar. Pollen grains of the Borreria densiflora were the most frequent, followed by Acacia (Rio Asteraceae family were detected in all propolis samples analysed, but Grande do Norte state) (Table 2). Barth (1989) analysed honey a detailed knowledge of the vegetation for genus or species samples of the Northeast region, where Mimosa verrucosa was identification is needed. Baccharis dracunculifolia DC. (Bastos et al. suggested as one of the most characteristic pollen types in the Bahia 2000, Bastos 2001) is the main species supplying bees with resin (BA) state. However, the two samples analysed in the present study from leafs, and resulting in green propolis production of significant showed no detectable Eucalyptus pollen grains, unlike the report by commercial value. Barth & Luz (2009) of a propolis sample from Bahia. Pollen types of low frequency are useful to characterize the Pollen grains of Asteraceae and Eucalyptus occurred in all phytogeographical region surrounding the apiaries. With this, and of the samples of the three states in the Southeast region, as including a large number of samples by location, a higher resolution dominant, accessory or isolated pollen types (Table 3). Cecropia and of the composition of vegetation can be obtained, contributing to a Solanaceae pollen types also were present as accessory pollen in better interpretation of sources used by bees for propolis, honey and samples of Minas Gerais (MG) state, and Mimosa scabrella, Salvia bee pollen production. These important pollen types identified in the and Poaceae pollen types in samples of Rio de Janeiro (RJ) state. present propolis samples showed that the majority of plant diversity Bastos et al. (2000) and Bastos (2001) observed a high occurred in the Southeast region of Brazil. frequency of Baccharis dracunculifolia DC. (Asteraceae) in samples A quantitative comparison of some propolis and honey of green propolis from Minas Gerais state, besides pollen grains of pollen spectra showed large variations. The propolis samples Eucalyptus. These were observed also in propolis samples by Barth analysed in the present paper comprised 33 pollen types, including (1998) and Barth et al. (1999), who suggested that Eucalyptus and four dominant nectariferous plant taxa, and in addition 29 taxa with a Eupatorium (Asteraceae) were well represented in propolis samples frequency higher than 3%, 19 of them from nectariferous plants. of the Southeast and South Brazilian regions. In another study (Freitas et al. 2010), pollen analysis of 24 Freitas et al. (2010), analyzing brownish propolis samples propolis samples obtained in the Atlantic coast region next to Rio de obtained in three locations of the Atlantic side of the state of Rio de Janeiro city was presented. Ten pollen types, with frequency higher Janeiro, verified Eucalyptus and Mimosa verrucosa pollen types both than 3% were identified. Eucalyptus was dominant in nine samples, as dominant and accessory pollen. Cecropia and Mimosa scabrella Mimosa caesalpiniaefolia in one sample only, and four nectariferous pollen types occurred both also as accessory and important isolated plants were recognized in addition. Honey samples of Morro Azul, Rio pollen grains. Anacardiaceae, Arecaceae (mainly Cocos nucifera), de Janeiro (RJ) state (Luz et al. 2007), showed 26 pollen types, Asteraceae, Citrus and Poaceae pollen grains were frequent. Barth seven of which were dominant but only five came from nectariferous (1998) and Barth et al. (1999) observed the presence of Eucalyptus plants, and 19 taxa more were identified, 14 of them from as dominant pollen in propolis samples from Rio de Janeiro (RJ) and nectariferous plants. Minas Gerais (MG) states, and Barth & Luz (2003) in geopropolis Honeys from Ceará state (Sodré et al. 2007) contained 20 samples from São Paulo (SP) state. pollen types, including five dominant nectariferous plant taxa, 15 In propolis samples of the South region, Eucalyptus and taxa with frequency higher than 3%, 13 of them from nectariferous Asteraceae pollen grains were detected mainly at dominant and plants. In honeys from the Piauí state (Sodré et al. 2007), between accessory levels (Table 2). Solanaceae pollen grains, in addition, the 25 pollen types identified, four of five proceeded from Pollen in Brazilian propolis 73

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