Chemical and bioactive characterization of pot-pollen produced by Melipona and Scaptotrigona stingless bees from Paria Grande, Amazonas State, Venezuela

doi: 10.9755/ejfa.2015-05-245

Chemical and bioactive characterization of pot-pollen produced by Melipona and Scaptotrigona stingless bees from Paria Grande, Amazonas State, Venezuela

INTRODUCTION
Bee pollen -as well as honey and propolis, has been widely studied for Apis mellifera, as recently reviewed (Vit, 1999).In Venezuela the botanical origin (Barth et al., 2011) and the chemical composition of Apis mellifera pollen loads from Los Andes in Cacute (Vit et al., 2008) and Misintá (Vit and Santiago, 2008), Mérida state.Also the antioxidant activity of aqueous, ethanolic and methanolic extracts of yellow, ochre, orange and brown pollen loads was compared (Pérez-Pérez et al., 2012).
The Warime Cooperative of Paria Grande Meliponicultors in the Amazonas state, Venezuela operates since year 2005.These Piaroa or Huottuja meliponicultors −also named stingless bee keepers, harvest honey and pollen produced in cerumen pots by stingless bees (Meliponini) (Pérez and Salas, 2008) and currently is expanded to 120 producers as informed by the president Mr. Alfonso Pérez in 2013.Studies on chemical composition of pot-pollen produced by stingless bees from the genus Melipona, will support the inclusion in sanitary procedures required for controlled marketing, to support this initiative of ancestral knowledge and recovery of native Indian traditions and environmental protection.This pollen is very sour and creamy compared with dried Apis mellifera pollen loads, because Meliponini ferment their pollen inside the nest; possibly it is richer in lactic acid as we found in stingless bee pot-honey (Vit et al., 2011).Menezes et al. (2012) proposed method to collect unfermented Meliponini pollen loads to increase acceptance, because in Brazil consumers prefer unfermented pot-pollen of Scaptotrigona depilis.However, in Venezuela sour taste of pot-pollen is highly appreciated because it reminds lemon, indeed when mixed with water it is named "pollen-lemonade".
Bee pollen is used for its nutritional value in the human diet (Ioirish, 1985;Krell, 1996).It is made up of natural flower pollen mixed with nectar and bee secretions, compacted in the corbiculae (Michener, 1999).Honey and other bee products, such as royal jelly, pollen and propolis may be used as functional foods because of their naturally high antioxidant potential (Viuda-Martos et al., 2008).Silva et al. (2006) studied the composition and free radical scavenging activities of two samples of yellow and brown pollen loads of Melipona subnitida from the Brazilian Northerneast.These authors isolated naringenin, isorhamnetin, and D-mannitol from the Mimosa gemmulata yellow pollen, and β-sitosterol, tricetin, selagin, and 8-methoxiherbacetin from the Fabaceae brown pollen.The EC 50 of ethanolic extracts in yellow and brown pollen was 104.5 ± 0.5, and 106.1 ± 1.3 µg/mL.Later, Silva et al. (2009) evaluated free radical scavenging activity of ethanolic extracts of pollen loads from stingless bee Melipona rufiventris "uruçu amarela", finding a concentration of a scavenger of active oxygen species of 104.1 ± 1.2 µg/mL.In recent studies, Tohamy et al. (2014) investigated the anti-mutagenic, antihistopathologic and antioxidant effects of water extracts of Egyptian bee pollen (WEBP) in induced hepatic, renal, testicular and genotoxicity in male albino mice (Mus muscullus), finding that bee pollen is potent in exerting an ameliorative effect.
In Venezuela and Ecuador, bee pollen is not regulated, only honey norms for Apis mellifera are available from the Venezuelan Commission of Norms (COVENIN 1984 a,b) and the Ecuadorian Technical Norm from the Ecuadorian Institute of Normalization (NTE INEN, 1988).Besides the well known commercial pollen loads of Apis mellifera, there is another type of pollen produced in cerumen pots by stingless bees (Meliponini), this pot-pollen is produced by the Warime Cooperative of Meliponicultors in Paria Grande, and needs to be characterized.The objective of this work is to analyze the proximal content (moisture, ash, proteins and fat), and bioactivity indicators (total antioxidant activity TAA, flavonoid and polyphenol contents) of potpollen collected, processed and stored in cerumen pots by Melipona (Michmelia) sp.aff.eburnea and Scaptotrigona cf.ochrotricha stingless bees with a similar palynological origin in Paria Grande, Amazonas state, Venezuela.

MATERIALS AND METHODS
Pot-pollen samples: Approximately 100 g of pot-pollen produced by two stingless bee species were collected from three nests in Paria Grande, Amazonas state, Venezuela, and were kept frozen at -20°C until analysis.
Proximal analyses: Proximal analyses were done in triplicate following oficial analytical methods (AOAC, 1999).Moisture, ash and fat were done by gravimetric methods; proteins were measured by the microKjeldhal method following sulfuric acid (Merck, Darmstadt, Germany) digestion, ammonia distillation by vapor flow with sodium hydroxide (Sigma Aldrich, USA) and sodium thyosulphate (Merck, Darmstadt, Germany), collection in boric acid (Sigma Aldrich, USA) and titration with hydrochloric acid); carbohidrates were calculated by difference.
Ethanolic homogenate preparations: A weight of 100 ± 10 mg of each pot-pollen type was placed on a glass homogenizer (Thomas No. A3528, USA), and 5 mL of ethanol 95% (v/v) (Riedel de Haën, Europe) were added, and homogenized on an ice bath.Homogenates were centrifuged in a BHG Optima II (USA) centrifuge at 3,000 rpm for 10 min, and supernatants were used for biochemical analysis (Pérez-Pérez et al., 2012).
Total antioxidant activity by the ABTS method: For the method of ethanolic decolorization of ABTS solution (Sigma, Canada), ABTS was dissolved in water to a 7 mM concentration.ABTS radical cation (ABTS +• ) was produced by reacting 7 mM stock solution with potassium persulfate (Merck, Darmstadt, Germany) to a final concentration of 2.45 mM (in water), in the dark at room temperature (RT) for 12-16 h before use (Re et al., 1999) [43].For this pollen analysis, the ABTS +• solution was diluted with 20% ethanol (v/v) up to 0.60-0.70absorbance units at 735 nm, 100 µL of homogenates were diluted in ethanol, and mixed with 7.5 mL of ABTS +• solution diluted in ethanol 20% (v/v).Absorbance values were measured 6 min after mixture.A solution of 8 mM Trolox (Sigma, Steinheim, Germany) was used as antioxidant standard.Trolox was diluted to obtain 1, 2, 4 and 8 µM in 5 mM PBS buffer (pH 7.4).Decolorization percentages at 734 nm after 6 min were calculated and plotted as a function of different Trolox concentrations, and TAA was reported accordingly.TAA value for pollen is equivalent to Trolox concentration that produces the same decolorization percentage.Purified antioxidants like quercetin, melatonin and lipoic acid were used as controls of the antioxidant activity.Results are expressed as µg Trolox equivalents (TEAC) per 100 g honey.
Flavonoid content: Flavonoid content was measured using a modification of the aluminum chloride method (Woisky and Salatino, 1998).The flavonoid concentration was measured with a calibration curve using standard solutions of quercetin (Sigma, Steinheim, Germany) diluted up to 25, 50 and 100 µg/mL in ethanol 80% (v/v).Standard solutions (0.5 mL) were mixed with 1.5 mL 95% (v/v) ethanol, 0.1 mL of aluminum chloride (Fisher Scientific, New Jersey, USA) 10% (w/v), 0.1 mL of potassium acetate (Sigma Aldrich, USA) 1M and 2.8 mL of distilled water.After incubation at ambient temperature for 30 min, absorbance was recorded at 415 nm.In a similar way, 0.5 mL of ethanolic extracts of pollen homogenates reacted with aluminum chloride for flavonoid content determinations.Results are expressed as mg quercetin equivalents (QE) per 100 g honey.
Polyphenol content: Polyphenol content was analyzed by spectrometry at 765 nm using Folin-Ciocalteu (Sigma-Aldrich, St. Louis, USA) reagent (Singleton, 1999).One hundred microliters of pollen homogenates were mixed with 500 µL of Folin-Ciocalteu's reagent diluted 1/10 with water, to which 400 µL of sodium carbonate was added (Sigma, Steinheim, Germany) 7.5% (w/v).Absorbance at 765 nm was recorded after 10 min of reaction at 37°C, against a blank with MQ water instead of ethanolic extract.The polyphenol concentration was estimated with a calibration curve using a solution of 0.1 g/L of gallic acid (Sigma, Steinheim, Germany) as standard (0, 0.25, 0.05 and 0.1 g/L).Results are expressed as mg equivalents of gallic acid (GAE) per 100 g honey.

Statistical analysis
All experiments were done in triplicate.Data were analyzed by ANOVA (P< 0.05), with average comparison through post hoc Scheffé test using SPSS 12.0 software (SPSS, 2004) to compare variations of physicochemical composition and bioactivity in the two pollen types with different entomological origin (Melipona sp. and Scaptotrigona sp.).Media ± SD are given for each parameter.

RESULTS
The pot-pollen produced by two species of Melipona aff.eburnea and Scaptotrigona cf.ochrotricha had two dominant pollen types; one of the Fabaceae Papilionoideae family 46% in the Melipona, and 20% in the Scaptotrigona pot-pollen, and the other of the Malpighiaceae family that was found as less frequent accessory pollen 26% in Melipona, but dominant with 54% in Scaptotrigona pot-pollen.Other pollen types from Apocinaceae, Arecaceae, Bixaceae, Euphorbiaceae, Fabaceae Mimosoideae, Lamiaceae, Malvaceae, Myrtaceae, and Poaceae families were also detected with frequencies lower than 3%.
The chemical composition of pot-pollen produced by Melipona and Scaptotrigona bees from Paria Grande, were done by proximal analysis moisture, ash, fat, protein and carbohydrate contents is given in Table 1.
In Table 2 is presented data on total antioxidant activity (TAA), flavonoid and polyphenol contents of ethanolic homogenates of Melipona and Scaptotrigona pot-pollen, besides the total antioxidant activity of purified antioxidants like quercetin, melatonin and lipoic acid used as controls of the antioxidant activity.

DISCUSSION
Stingless bee origin is estimated in the late Cretaceous (Engel and Michener, 2013), and since then also their interactions with plants (Lunau, 2004).Bees forage plants based on a cost-benefit balance.Besides floral attractiveness to a particular stingless bee species, other factors determine pollen choices and rate of visitation: 1. Presence of competitors, 2. Distance from the nest to the resources, 3. Bee communication strategy, 4. Number of bees in the nest, 5. Type of soil, 6. Temperature, 7. Light (Kajobe, 2007;Corbet et al., 2008).
The dominant pollen types were from the family Fabaceae Papilionoideae (46%) Melipona, and Malpighiaceae (54%) Scaptotrigona pot-pollen.The higher fat content of Scaptotrigona pollen could be explained by the oil glands of Malpighiaceae.Although most genera of Malpighiaceae are generally pollinated by oil collecting bees like the Centridini group (Rego et al., 2006).Novais and Absy (2013) studied pollen pots of Amazonian Tetragonisca angustula in Belterra and Santarém, Pará, Brazil found that Byrsonima, Cecropia, Clidemia hirta, Davilla kunthii, Eriope, Myrcia and Vismia guianensis were the most significant pollen types with frequencies above 70%.Absy et al. (1984) recommend that plants of identified pollen sources should be cultivated near to meliponaries to support stingless beekeeping as potentially sustainable economic activity.Particularly, planting native species of the genera Astrocaryum, Bactris, Byrsonima, Citharexylum, Couma, Euterpe, and Vitex, is adviced to facilitate beekeeping especially in seasons of pollen scarcity; and considering that with the pollination service of the bees, local food production may also increase (Rech and Absy, 2011).These authors reported Scaptotrigona sp.pot-pollen with three secondary pollen types from two species of the family Fabaceae Papilionoideae: Aldina latifolia (24.2%),Swartzia dolichopoda (30.8%), and Vitex cymosa Lamiaceae (34.5%) The botanical origin of the genus Byrsonima (Malpighiaceae) found here as a less frequent source in the Melipona and Scaptotrigona pot-pollen was also reported for Centris (Ribeiro et al., 2008) and Cephalotrigona femorata with a secondary frequency of 33.4 % (Absy et al., 1984).The Malpighiaceae family offers oil from elaiophore calyx glands to pollinators (Pedro, 1994).However, pollination by pollen-collecting bees has probably shifted to remove their calyx glands in a group of the over 950 species of Malpighiaceae in the New World, after an almost obligate relationship of very specialized pollinators that explain the floral conservatism of Neotropical Malpighiaceae (Anderson, 1979).
The TAA of ethanol extracts of pot-pollen from Melipona was lower than that of Scaptotrigona (Table 2).Similarly, lower values of polyphenol contents were also observed in the Melipona pot-pollen, compared to Scaptotrigona potpollen.There is a positive relation between polyphenol content and antioxidant activity (TAA values), but not with the flavonoid content, as previously informed in another research for unifloral honey (Vit et al., 2010).Freire et al. (2012) determined phenolic and flavonoid contents, and antioxidant properties of twenty-five bee pollen harvested during a nine-month period from the Canavieiras municipality (Northeastern Brazil).The total phenolic contents ranged from 41.5 to 213.2 mg GAE/g, and antioxidant activities based on the (ABTS) correlated with the total phenolic contents.Morais et al. (2011) determined phenolic content and antioxidant properties of pollen from five Portuguese Natural-Parks such as Parque Nacional Peneda Gerês (PNPG); Parque Natural do Montesinho (PNM); Parque Natural do Alvão (PNA); Parque Natural da Serra da Estrela (PNSE) and Parque Natural do Douro Internacional (PNDI).The phenolic contents, determined 10.5 and 16.8 mg GAE/g of extract, in bee pollen from PNM and PNDI, respectively.The free radical scavenging measured showed the highest effective extract -PNM with EC 50 2.16, followed by PND with 2.24 mg/mL.Several methods for determining the antioxidant activity in honey have been used, and among them the ABTS assay is referred to by many authors.This assay assesses the total radical scavenging capacity based on the ability of a compound to scavenge the stable ABTS radical cation (ABTS •+ ) (Suarez-Alvarez et al., 2009).Besides to the simplicity of this assay, there is little correlation between the TEAC value and the number of electrons an antioxidant can give away.In spite of this, the ABTS assay is considered an easy and accurate method for use in the antioxidant capacity studies in honey because it allows to determine the radical scavenging ability present in the honey by the hydrogen-donation reaction.On the other side, honey phytochemicals are mainly represented by the extensive class of phenolic compounds, being the major class the flavonoids (flavonols, flavanols and flavones), followed by phenolic acids (benzoic acids, phenylacetic and hydroxycinnamic acids).In pollen samples, the highest quantity of flavonoids exists like glycosides, namely aglycones, being quercetin the majoritarian compound (Bogdanov, 2011).
It has been widely demonstrated that flavonoids are very effective as scavengers of reactive oxygen species (ROS): peroxide, hydroxyl and superoxide radicals, as well as against reactive nitrogen species (RNS) like nitric oxide and peroxyl nitrite (Yen and Lai, 2003).Moreover, flavonoids have high affinity for proteins and other biological macromolecules, such as hormones and nucleic acids, and for divalent ions of metals; and also have high catalytic ability for the electron transfer and act as radical scavengers.Flavonoids are capable as act like antioxidants because have substituents with a high capacity of donate hydrogen/electrons with adequate reduction potentials; and the ability to relocate the resulting radical (Rivas-Gonzalo and García-Alonso, 2002).
It has been described some structural criteria necessary for effectiveness in capturing radicals of flavonoid molecules, through ABTS radical cation assay, among them: (i) the presence of the structure 3',4'-o-dihydroxy on B aromatic ring (catechol), which gives greater stability to the formed radical; (ii) the existence of hydroxyl groups on carbons 5 and 7 of A ring; (iii) the double bond located at position 2,3 in conjunction with the 4-oxo and 3-hydroxy on C ring, responsible for the relocation from the B ring (the peroxyl radicals produced are stabilized by the resonance effect of the aromatic nuclei); and (iv) the appearance of the hydroxyl groups in position 3 and 5 with 4-oxo function in rings A and C, and with 2,3 double bond between positions generate the maximum potential free radical scavenger (Burda and Oleszek, 2001;Heim et al., 2001;Rivas-Gonzalo and García-Alonso, 2002).Depending of its structure, the antioxidant activity of some flavonoids present in pollen samples cannot be determined by ABTS assay because the mechanism involved is not electron/hydrogen transfer, the principal mechanism evaluated in this method.
More recently, Pascoal et al. (2014) evaluated the biological activities of eight commercial bee pollens purchased from the market.Phenolic contents varied from 32.15 to 18.55 GAE/g, flavonoids varied from 10.14 to 3.92 QE/g.All the samples exhibited antimutagenic and antimicrobial activity, being Staphylococcus aureus the most sensitive and Candida glabrata the most resistant of the microorganisms studied.These authors observed positive correlations between polyphenols, flavonoids, antioxidant activity and antimicrobial activity of Apis mellifera bee pollen.These are few evidences about the antioxidant activity of honey, pollen and propolis and its relationship with total polyphenol content, and especially flavonoid concentration.
There is no statistical data available for the bee industry in Venezuela, and this lack of organization is reflected in the low participation in international debates on biodiversity protection of feral bees endangered of extinction (Bouga, 2013), contamination of bee products with genetically modified organisma (GMO) and its residues (Haefeker, 2013), or the effects of pesticides in honeybee and authenticity of honey (Maxwell, 2013), as recently discussed in round tables of the APIMONDIA XXXXIII International Congress of Apiculture held in Kiev, Ucrania from the 29 th September to the 4 th .October 2013.
The fact that pollen regulations still need to be created in Venezuela, offers the advantage to suggest the inclusion of pot-pollen produced by Meliponini bees different from Apis mellifera, like Melipona and Scaptotrigona.Expanded quality criteria (microbiological, contaminants, trace elements, vitamins) recommended in the review for dried Apis mellifera pollen pellets (Campos et al., 2008) could also be adopted for fresh bee pollen; in their draft these authors suggested maximum moistures of 6-8 g/100, maximum ash of 6 g/100, minimum protein of 15 g/100, minimum fat of 1.5 g/100 g, and instead of carbohydrates measured by difference, a minimum of total sugars 40 g/100g.

CONCLUSIONS
In the current research moisture and ash contents measured with gravimetric methods, proteins (micro Kjeldahl), ether extract (Soxhlet), and bioactivity indicators measured by spectrophotometric methods to evaluate total antioxidant activity as free radical scavengers, flavonoid and polyphenol contents were compared in pot-pollen produced by Melipona (Michmelia) sp.aff.eburnea Friese, 1900 and Scaptotrigona cf.ochrotricha (Buysson, 1892) in Paria Grande, Amazonas state, Venezuela.The characterization is useful for a pot-pollen regulation database, and also as a basis for pharmaceutical studies.
The proximal composition and bioactivity of bee pollen produced by two species of stingless bees in Paria Grande, Amazonas state, Venezuela let the following observations: 1. Pot-pollen is a rich source of oligoelements in the diet given the high ash content measured in this study, 2. The pollen spectra varied according to the stingless bee species, 3. Fat content of pollen varied according to the stingless bee species and the plant they visited foraging pollen.
To our knowledge, this is the first report on the biological activities of Venezuelan stingless bee pot-pollen, and there is very small quantity of evidence of antioxidant activity on stingless bee pollen (Silva et al., 2006;Silva et al., 2009).Our data suggest that the ethanol extract of pot-pollen have a potent antioxidant activity, similar or higher than that found for purified antioxidants like quercetin, melatonin and lipoic acid, probably due to its polyphenol content and fermentive process.This is very important to support the use of pollen as a very energetic nutritional supplement since ancient times.