Chemical composition, botanical evaluation and screening of radical scavenging activity of collected pollen by the stingless bees Melipona rufiventris (Uruçu-amarela)

CHEMICAL SCIENCES

Chemical composition, botanical evaluation and screening of radical scavenging activity of collected pollen by the stingless bees Melipona rufiventris (Uruçu-amarela)

Tania M.S. Silva^I; Celso A. Camara^I; Antonio C.S. Lins^II; Maria de Fátima Agra^II; Eva M.S. Silva^III; Igor T. Reis^III; Breno M. Freitas^III

^IDepartamento de Química, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, 52171-900 Recife, PE, Brasil

IILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, Caixa Postal 5009, 58051-970, João Pessoa, PB, Brasil

IIIDepartamento de Zootecnia, CCA, Universidade Federal do Ceará, Caixa Postal 12168, 60021-970 Fortaleza, CE, Brasil

^{Correspondence to}

ABSTRACT

Stingless bees in Brazil are indigenous and found all over the country. Bee pollen is used for its nutritional value in the human diet. It is made up of natural flower pollen mixed with nectar and bee secretions. In order to evaluate the chemical composition, free radical scavenging activity, and botanical origin, sample of pollen loads from stingless bee, Melipona rufiventris (Uruçu amarela) was studied. The EtOAc extract of pollen of Melipona rufiventris yielded the following compounds: p-hydroxycinnamic acid, dihydroquercetin, isorhamnetin, isorhamnetin3-O-(6"-O-E-p-coumaroyl)-β-D-glucopyranoside, luteolin, and quercetin. This is the first report of the isolation of isorhamnetin3-O-(6"O-E-p-coumaroyl)β-D-glucopyranoside from pollen. The free radicalscavenging activities of different solvent extracts of pollen were determined using DPPH assay. This activity decreases in the order: EtOAc>EtOH>Hexane extract. It appears that the EtOAc extract of the pollen is a good scavenger of active oxygen species. The botanical evaluation of pollen loads showed the composition by two pollen types, with the dominant type (97.3%) being Scopariadulcis (L.) (Scrophulariaceae) and the minor one Senna obtusifolia (L.) Irwin & Barneby (Fabaceae). This suggests a specific foraging behavior in Melipona rufiventris bees, even in an environment with such a rich botanical diversity as the Northeastern Brazil.

Key words: antioxidant activity, bee pollen, flavonoids, Melipona rufiventris, Uruçu amarela.

RESUMO

As abelhas sem ferrão são espécies indígenas encontradas em todo o Brasil. Seu pólen é utilizado devido ao seu valor nutricional na dieta humana. É produzido a partir de pólen floral misturado com néctar e líquidos secretados pelas abelhas. Visando avaliar a composição química, a atividade sequestradora de radicais livres e a origem botânica foi estudado o pólencoletado pela abelha sem ferrão Melipona rufiventris (Uruçu-amarela). Do extrato acetato de etila foram isolados os compostos: ácido phidroxicinâmico, dihidroquercetina, isoramnetina, 3O(6"OEpcoumaroil)βDglicopiranosideoisoramnetina, luteolina e quercetina. Esta é a primeira vez que a 3O(6"OEpcoumaroil)βDglicopiranosideoisoramnetina é isolada de pólen apícola. A atividade sequestradora de radicais livres de vários extratos com solventes diferentes foi determinada pelo teste com DPPH (difenilpicrilhidrazida). A atividade mostrou a ordem decrescente para os extratos AcOEt>EtOH>Hexano. O extrato AcOEt apresenta melhor atividade sequestradora de radicais. A avaliação botânica palinológicamostrou que o pólen era composto de dois tipos, um majoritário(97.3%) proveniente de Scoparia dulcis (L.) (Scrophulariaceae) e outro minoritário proveniente de Senna obtusifolia (L.) Irwin& Barneby (Fabaceae). Estes resultados sugerem o comportamento de forragem bastante específico exibido pela abelha Melipona rufiventris, mesmo em um ambiente tão rico em diversidade vegetal como o Nordeste do Brasil.

Palavras-chave: atividade antioxidante, pólen de abelha, flavonóides, Melipona rufiventris, Uruçu amarela.

INTRODUCTION

The Meliponin is a bee group of more than 300 species encountered around the world. They are characterized as having social and bearing an atrophied and nonfunctional sting, which justifies their popular name of stingless bees (Roubik 1989). Stingless bees are found in tropical and subtropical regions of South and Central America, Africa, Southeastern Asia and Australia. In Brazil, they are indigenous and present all over the country, although species differ from region to region (Nogueira-Neto 1997, LimaVerde and Freitas 2002).

Besides their importance as major pollinators of most wild plants and some cultivated species, honey and bee pollen of meliponins are also source of food, and medicines (geopropolis) and income to rural populations. There is a strong culture of bee keeping and using their products in Brazil and other parts of the world where these bees are native (Freitas 1999).

One of their products, bee pollen, is used for its nutritional value in the human diet. It is made up of natural flower pollen mixed with nectar and bee secretions, and is rich in sugars, proteins, lipids, vitaminsand flavonoids (3-5% dry weight) (TomasLorente et al.1992). Although bee pollen found in the market is harvested from honey bee (Apis mellifera) colonies, manyother social bee species also collect and use pollen, suchas the stingless bees (Nogueira-Neto 1997).

During ancient times, people throughout the world commonly used pollen, for the goodness and medicinal properties. Some of the reasons why ancient people used beepollen are the same for its use today. To date, no scientific evidence has been cited to disprove the claimed properties of beepollen (Campos et al. 1997). In recent years, the physiological functionality of natural food shas received much attention, due to increasing interestin human health. Among natural products, honey bee derived apicultural products such as pollen and propolishave been applied for centuries in traditional medicine aswell as in food diets and supplementary nutrition (Kroyerand Hegedus 2002). Much work has been conducted onthe chemistry and properties of propolis and pollen. Hundreds of chemical compounds have been identified from them. In general, compared to many standard humanfoods, pollens are rich in protein, low in fat and possessa wealth of minerals and vitamins (Campos et al. 1996).

Reactive oxygen species (ROS) are produced in allmammalian cells as the result of normal cellular metabolism and due to the activation of oxidantproducing enzymes in response to exogenous stimulus. The balance between ROS production and antioxidant defenses determines the degree of oxidative stress. Generation of ROS has been associated with cell signaling, stress responses, cell proliferation, aging, and cancer development. The ability of ROS to induce cellular damage and to cause cell death opens the possibility to utilize this property in the treatment of cancer through a free radical mediated mechanism (Villamor et al. 2004). It has been reported that both pollen and propolis extracts and their respective isolated compounds have free radical scavenging activity (Campos et al. 2003, Kumazawaet al. 2004).

The major aim of the present study is to determine the chemical composition and the antiradical activity of pollen loads collected and processed by stingless bee Melipona rufiventris. In a previous work the chemical composition of the closely related solitary bee species Melipona subnitida Ducke was studied (Silva et al.2006). Both species, among others, are end angered byextinction, mostly because their native environments are being destructed, and is well documented the sharp foraging range of these species (Gathmann and Tscharntke 2002).

MATERIALS AND METHODS

SAMPLE

The pollen loads of Melipona rufiventris known as uruçu-amarela was collected directly from pollen pots of four Melipona rufiventris colonies kept in Pacoti (4º13^" 40^"" S, 38º55^" 17^"" W, 830 m above sea level) Stateof Ceará, Brazil, in 2003.

EXTRACTION AND SEPARATION

Pollen loads of Melipona rufiventris (67.6 g) were extracted with EtOH in an ultrasound bath and the extractfiltered and concentrated under reduced pressure. The crude residue was dissolved in MeOH:H2O (7:3) and fractioned with nhexane and then with EtOAc. These solvents were evaporated to dryness. The EtOAc fraction (6.1 g) was subsequently submitted to column chromato graphy using Sephadex^® LH-20 and MeOH aselution system. The collected flavonoid fractions were analyzed by commercial TLC aluminum sheets (Mercksilica gel 60 F254), with the spot visualization donewith spraying diphenylboryloxyethylamine (NP) solution in MeOH and observing under UV light (366 nm).The EtOAc extract of the pollen of Melipona rufiventris yielded the phenolics, 1 (1.1 mg), 2 (10.0 mg), 3 (28.0 mg), 4 (15.0 mg), 5 (5.0 mg), and 6 (10.0 mg).

FREE RADICAL-SCAVENGER ACTIVITY

The free radical-scavenger activity was determined bythe 1,1-diphenyl-2- picrylhydrazyl (DPPH) assay, as described previously (Silva et al. 2006). The antiradicalactivity of the EtOH extract, hexane and EtOAc fractionswere evaluated using a dilutions series (with the use of ethanol as solvent in all cases), in order to obtain a larg espectrum of sample concentrations. This involved the mixing of 2 mL of DPPH solution (100µM in ethanol)with an appropriate amount of extract or compound, followed by homogenization. After 30 min, quantify ofthe remaining DPPH radicals was recorded from absorption at λ 517 nm. Three analytical replicates (n = 3)were carried out on each extract for antiradical activity determinations. Measurements were averaged, and the results are given as mean ± standard deviation (S.D.). Antiradical efficiency was established using regression analysis at a 95% significance level (P < 0.05). Results are presented in EC₅₀ values, which represent the concentration of sample required to scavenge 50% of the dpphradicals available (table I). the reference standard were ascorbic acid (EC₅₀ = 2.48±0.03µg/ml) and butylatedhydroxyl toluene bht (4.47 ± 0.08µg/ml).

BOTANICAL ORIGIN OF THE POLLEN LOADS

Samples of pollen loads (2.0 g) were hydrated in waterin order to free pollen grains from the loads mass. After being completely homogenized, they were washed, centrifuged and mounted in glycerin jelly according tothe method of Maurizio and Louveaux (1965). Five slides with glycerin jelly were mounted from each sample. Pollen grains were counted (500 per slide, at least)to establish the identity and frequency of the pollentypes. Pollen types were identified by comparison with slides from the palynotheca of the Bee Laboratory (Universidade Federal do Ceará, Brazil).

Pollen types were classified according to their frequency in the sample into dominant (> 45%), accessory (45% < x > 3%) and isolated (< 3%) (Maurizio and louveaux 1965).

RESULTS AND DISCUSSION

IDENTIFICATION OF THE COMPOUNDS

The NMR spectra were used to identify the structuresof the phenolics isolated from pollen loads of Melipona rufiventris as p-hydroxycinnamic acid (1), dihydroquercetin (2), isorhamnetin (3), isorhamnetin3-O-(6"O-E-pcoumaroyl)-β-D-glucopyranoside (4), luteolin (5),and quercetin (6), Figure 1. Their spectral data, particularly ¹³C NMR, were in close agreement with literature values, for dyhydroquercetin, isorhamnetin, luteolin and quercetin (Agrawal 1989, Markham and Chari1982), and for isorhamnetin3-O-(6"O-E-p-coumaroyl)-β-D-glucopyranoside (4) (Romussi et al. 1988). Table I showed the ¹H and ¹³NMR data for compound 4.

Flavonoids are widely recognized as reliable chemotaxonomic markers in plants in general (Emerenciano et al. 2001, Harborne and Turner 1984). Recently, this evidence was obtained for pollen flavonoids (Campos et al. 1997) and has been recognized previouslyas potentially useful taxonomic markers (Meurer 1988, TomasLorente 1992). In addition, pollen flavonoids are arguably quality factors in terms of their phytotherapeutic value as antioxidants and radical scavengers (Pietta2000, Cook and Samman 1996), and their involvement as essential components in pollen germination and pollentube grown (Ylstra et al. 1992).

With the exception of the acylated flavonoid, isorhamnetin 3-O-(6"O-E-p-coumaroyl) βDglucopyranoside isolated from pollen loads collected by Melipona rufiventris we isolate and report only flavonoid aglycone. This profile of flavonoid distribution has bee nshown also in other stingless beepollens as Melipona subnitida (Silva et al. 2006), and Scaptotrigona bipunctata (Lins et al. 2003).

Dihydroquercetin (Strohl and Seikel 1965), isorhamnetin (TomasLorente et al. 1992), and luteolin (Campos et al. 2002), have already been isolated from pollen. This is however the first report of isorhamnetin-3-O(6"O-E-p-coumaroyl) -β-D-glucopyranoside (4) in bee pollen.

Antiradical activity of EtOH extract, and hexane and EtOAc fractions of bee pollen according to the DPPH radicalscavenging method.

The free radical scavenging activities of differentsolvent extracts of pollen loads of Melipona rufiventris were determined using the DPPH assay. The results, expressed as EC₅₀ values, were calculated by regression analysis as present in Table I (the correlation coefficients of the regression curves are also present in the Table). The EtOAc fraction showed potent free radicalscavenging activity on the DPPH radical compared to EtOH extract. The hexane extract was inactive (EC₅₀>500µg/mL).

The polyphenolics present in the EtOAc extract could be responsible for the activity. Quercetin, luteolin (Nessa et al. 2004), isorhamnetin and dihydroquercetin (Edenharder and Grunhage 2003) are already tested forthe antiradicalar activity.

The complete assignment for compound (4) is showed in Table II and the corresponding chemical structure is shown in Figure 1.

BOTANICAL ORIGIN

Bee pollen studied here, collected directly from hives, consisted of a mixture of pollen types of different species of plants. The pollen loads was composed by only two pollen types, where the dominant (97.3%) type was Scoparia dulcis (L.) (Scrophulariaceae) and the isolated one Senna obtusifolia (L.) Irwin & Barneby (Fabaceae) (2.7%).

Mixture of different pollen types in one pollen bee is not common in social bees. Apis mellifera, a polylectic species, is known for its high fidelity to individual sources of pollen (Roubik 1989). Stingless bees are less generalist than A. mellifera in their foraging behavior (Nogueira-Neto 1997). Not surprisingly, only two pollen types were present in the pollen loads studied, suggesting that Senna obtusifolia contributed with little pollen and Scoparia dulcis was the major source of pollen to Melipona rufiventris in the observed period, even though the colonies were placed in an environment with a rich and varied flora, as in Brazilian SemiArid Northeast. This fact corroborates the foraging specificity to Melipona rufiventris.

CONCLUSION

In pollen loads of Melipona rufiventris the EtOAc mainly comprised of p-hydroxycinnamic acid, dihydroquercetin, isorhamnetin, isorhamnetin isorhamnetin-3-O-(6"O-E-p-coumaroyl)-β-D-glucopyranoside, luteolin, and quercetin. To the best of our knowledge, this is the first report of the isolation of isorhamnetin isorhamnetin3O(6"OEpcoumaroyl) βDglucopyranoside from pollen. The botanical evaluation of bee pollen showed that it was composed by two pollen types, with the dominant type (97.3%) being Scoparia dulcis and the isolated one, Senna obtusifolia. This result suggests a specific foraging behavior in Melipona rufiventris bees, even in an environment as rich in diversity as the Northeastern Brazilian flora. The free radical scavenging effectiveness of the extracts showed that EtOAc extract was the most active. This is the first study of pollen loads from stingless bee Melipona rufiventris, a native species of Northeastern Brazil. The results of these trials will behelpful for the commercial production of the stingless beepollen for pharmaceutical or nutritional use. Other bioactivity determinations are now being carried out inorder to give us more information about the potentialityof these pollens. It suggests that the extracts of the pollen are good scavengers of active oxygen species. This property of pollen seems to be important in the prevention of various diseases such as cancer, cardiovascular diseases, and diabetes, among others.

ACKNOWLEDGMENTS

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto do Milênio do SemiÁrido (IMSEAR), Rede Nordestina de Biotecnologia (RENORBIO), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Programa Institucional de Bolsas de Iniciação Científica (PIBIC). The authors also thank Prof. J. Bhattacharyya (Laboratório de Tecnologia Farmacêutica UFPB) for useful help in the preparation of the manuscript.

Manuscript received on January 16, 2008; accepted for publication on December 8, 2008; presented by FERNANDO GALEMBECK

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Publication Dates

• Publication in this collection
  26 May 2009
• Date of issue
  June 2009

History

• Accepted
  08 Dec 2008
• Received
  16 Jan 2008