STUDY OF PREPARATIONS OF BEE POLLEN EXTRACTS , ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY

The aim of this study was to determine the antioxidant activity, phenolic content and antibacterial activity of pollen extracts obtained with different concentrations of ethanol. Each extraction condition (ethanol solutions from 40 to 90%) had a different effect in the phenolic compounds content. Although, the pollen extract obtained at 60, 70 and 80% of ethanol showed relatively higher levels of phenolic compounds (>10 mg/g) and did not present statistical significant difference between the extraction conditions. The amount of total phenolics ranged from 3.6 to 8.1 and 6.6 to 10.9 mg GAE/g for Alagoas state and Parana state pollen, respectively. The higher value for antioxidant activity index was 83.30% for the pollen from Alagoas state and 81.15 % for Parana state pollen. The highest degree of antioxidant activity was found in the extraction at 60% of ethanol solution for Parana state pollen, which also showed the highest concentration of polyphenol compounds. Staphylococcus aureus was inhibited by the ethanolic extract of Alagoas state pollen in all the concentrations of solvent, except the ethanolic extract of pollen at 90%. The extract at 60% of ethanol solution (Parana sample) inhibited Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella sp.


INTRODUCTION
Pollen, as well as other apicultural products, has gained increased attention for its therapeutic properties, as antibacterial (GARCÍA et al., 2001;PROESTOS et al., 2005), antifungicidal (GARCÍA et al., 2001), anti-caryogenic (ALMAS et al., 2001) and immunomodulatory (GEBARA et al., 2002) effects.Other potential applications of pollen include its use in apitherapy and as a functional food in the food industry due to pollen nutritional properties.Bee gathered pollen is considered a valuable special food with varied enhancing effects in health (BOGDANOV, 2004).
The therapeutic action has been attributed to several phenolic compounds with antioxidant activity, present in these products.All bee-derived products such as honey, propolis and pollen have been applied for centuries in traditional medicine as well as in nutritional supplementation (ISLA et al., 2001).
The bioactive properties of apicultural pollen extracts can be increased using a solvent suitable for its extraction, improving the activity of free radicals sequestration (antirust activity) (DI PAOLA-NARANJO et al., 2004).Appropriate extracts of pollen can be used as functional food or alimentary supplement had the amount of phenolic composites and its capacity of sequestered the responsible free radicals for it carcinogenesis and stress oxidative (TANG et al., 2005).Pollen grains have specific characteristics according to the floral species or cultivation methods, but the quality depends on the collection process, cleanness, drying and storage applied by beekeepers with the objective to increase the product shelf-life.
Honey-bee collected pollen can be considered as a potential source of energy for human consumption.Pollen contains nutritional compounds like carbohydrates, proteins, amino acids, lipids, vitamins, minerals and traces of micronutrients (SERRA BONVEHÍ & ESCOLÁ JORDÁ, 1997).In addition, pollen contains significant amounts of polyphenolic substances, mainly flavonoids (ALMEIDA-MURADIAN et al., 2005).These phenolic compounds are essential for the plants physiology due to its contribution in the morphology (form and structure).The polyphenols are involved in plants growth and reproduction; they also supply resistance against pathogens by the action of the phytoalexins and protection against plagues increasing the astringency of the plant as a food, becoming indigestible to predators.The polyphenols constitute one of the most numerous metabolic groups of plants and are integral part of people and animals diet (COOK & SAMMAN, 1996).
Until recently the interest in these compounds has been focused in the harmful effects on health caused by the ability of certain polyphenols to bind and precipitate macromolecules, such as proteins, carbohydrates and digestive enzymes, reducing the digestibility of foods.Nowadays, the interest in phenolic compounds has increased due to the antioxidant and free radical scavenging activities (DORMAN et al., 2003).
Several researchers found out that polyphenols are antioxidants with redox properties which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers (CALDWELL, 2003).The polyphenols also have metal chelation properties (RICE-EVANS et al., 1996).These compounds league metals to it (chelation of metals) and react with free radicals and genotoxic susbtances or carcinogenics (TANG et al., 2005).Epidemiologic studies have shown a correlation between an increased consumption of phenolics antioxidants and a reduced risk of cardiovascular disease and certain types of cancer (COOK & SAMMAN, 1996).
The fact that French people have a low incidence of coronary heart disease, despite having a diet rich in fat and being heavy smokers, is also known to be correlated with wine consumption (KANNER et al., 1994).Despite much interest in the antioxidant activity of red wine, it is uncertain which of the phenols exhibit the greatest antioxidant effect (MINUSSI et al., 2003).These researchers showed a positive correlation between total phenolic and total antioxidant potential, for all wines analyzed.Some authors state the necessity of additional inquiries to evaluate the relative composition of polyphenolic substances in pollen and extracts of pollen, as well as the differences in its specification to evaluate its function and contribution regarding to the antioxidant activities (SERRA BONVEHÍ & ESCOLÁ JORDÁ, 1997).
The aim of this work was to determine the phenolic content, antioxidant and antibacterial activities of pollen relating to the concentration of ethanol used as solvent.Pollen samples were collected in Parana and Alagoas states, in the south and northeastern regions of Brazil, respectively.The purpose of this study was also to demonstrate a possible relationship between phenolic content and antioxidant activity of pollen.

Preparation of pollen ethanolic extracts (PEE)
The pollen samples (2g) were milled, homogenized and extracted individually using 15 mL of ethanol solution as extraction solvent in different concentrations (PEE 40, PEE 50, PEE 60, PEE 70, PEE 80 and PEE 90%) at temperature of 70ºC for 30 min with constant agitation.The supernatant was separated and the solid residue was re-extracted.Then, the ethanol extracts of pollen were combined and stored at 5ºC for further analysis.All samples were extracted in duplicate.

Total phenolic content of pollen ethanolic extracts (PEE)
Total polyphenols content was quantified according to the Folin-Ciocalteau spectrophotometric method using galic acid as reference standard (SINGLETON et al., 1965).In each sample of 0,5 mL, 2 mL (1:10 dilution) of Folin-Ciocalteau s reagent and 2 mL of Na 2 CO 3 (4%w/v) were added, storing this mixture in dark at room temperature for 2 hours.The absorbance of all samples was measured at 740 nm using a UV-VIS spectrophotometer.Results were expressed as milligrams of gallic acid equivalent per gram of dry weight of pollen (mg GAE/g dw) and are presented as the mean of duplicate analyses.

Antioxidant activity of pollen ethanolic extracts (PEE)
The antioxidant activity was determined by the oxidation of -carotene and linoleic acid.-carotene (5 mg) (Sigma Co) was dissolved in 5 mL of chloroform and was pipetted into a flask containing 60 mg of linoleic acid and 200 mg of Tween 40 (polyoxyethylene sorbitan monopalmitate).The chloroform was removed using a rotoevaporator at 40ºC for 5min; afterwards, 50 ml of distilled water was added slowly with vigorous agitation to the residue, to form an emulsion.Aliquots of 1 mL of this emulsion were added to a tube containing 0.5 mL of PEE diluted 1:10, and the absorbance was measured at 470 nm, immediately, against a blank, consisting of the emulsion without -carotene.The tubes were placed in a water bath at 40ºC and absorbance of all the samples at 470 nm were taken at zero time (t=0).Measurement of absorbance was continued until the color of -carotene disappeared in the control reaction (t=180 min) at 60 min intervals.The antioxidant activity of the extract was evaluated in terms of bleaching of -carotene (ALENCAR, 2002).Antioxidant index (AI) was calculated using the following equation: AI = ( -carotene content after 3 h of assay/initial -carotene content) x 100

Antibacterial activity of pollen ethanolic extracts (PEE)
The disk diffusion assay described in detail by Bauer et al. (1966) was used in this study, with some modifications.The stock cultures of bacterials (Staphylococcus aureus ATCC 25.923, Bacillus cereus, Bacillus subtillis ATCC 21.332, Pseudomonas aeruginosa ATCC 15.442 and Klebsiella sp) were grown in nutrient broth (Difco Co) at 26-27°C for 24 h in a shaker.Aliquots of 40 L of pollen ethanolic extract were applied in paper disk and placed in plates containing nutrient agar that was previously inoculated with active cultures of these microorganisms with sterile swabs.Antibacterial activity was assessed by measuring the diameter of the inhibition zone around each disk after 24 hours of inoculation at 37ºC.The control (40 L of ethanol) used in all the plates and extracts analized in duplicate.Gentamicin (40 L) was used as the positive control for bacteria.

Statistical analysis
Data were submitted to the analysis of variance (ANOVA).Tukey test was used to evaluate the significant differences between means (comparison of means) at the level of p<0.05, using the software MSTAT-C (MSTAT-C, 1998).Correlation analyses of antioxidant activity (Y) versus total phenolic content (X) were carried out using the correlation and regression program Statgraphics plus 4.1 and OriginPro 7.0 (OriginLabCorporation).

Total phenolic content of pollen ethanolic extracts (PEE)
Several authors have found phenolic and flavonoid compounds in pollen (PROESTOS et al., 2005).Recent studies of total polyphenols in pollen collected by native bees of the region of Vienna in Austria indicated that the pollen collected by bees generally show characteristic amounts of total polyphenols with some variations due to its botanical origin.
Total phenolic compounds content of pollen extracts was solvent-dependent.The amount of total phenolics varied in different conditions of extraction and ranged from 3.6 to 8.1 and 6.6 to 10.9 mg GAE/g of dry material to Alagoas and Parana pollen, respectively (Figures 1 and 2).The highest total phenolic levels were detected in pollen extracts from Parana samples (Figure 1). Figure 1 shows that each extraction condition had a different effect in the phenolic compounds content.Pollen extracted in ethanol at 60, 70 and 80% of concentration showed relatively higher levels of phenolic compounds (>10 mg/g) and did not present significant difference between the extraction conditions.
Nevertheless, statistical difference was found between the other extraction conditions (40, 50 and 90%).These results showed that the extraction conditions (solvent concentrations) influenced the extraction of phenolic compounds of Parana pollen (Figure 1).Kroyer & Hegedus (2001) achieved 8.2 mg/g of polyphenolics substances in natural bee-collected flower pollen, which could be significantly increased in the extracts (21.4-24.6 mg/g) with the highest content of total polyphenols in the ethanol extract.Comparing Figures 1 and 2, it can be observed that the content of total phenolic compounds of PEE 60, 70 and 80 % from Parana pollen (10.8, 10.9 and 10.6 mg/g) was higher than the one from Alagoas (6.2, 8,1 and 6.3 mg/g,).
The content of total phenolic compounds of Alagoas pollen varied from 3.6 to 8.1 mg g -1 .Pollen extracted in ethanol at 70 % of concentration showed relatively higher levels of phenolic compounds (8.1 mg/g) and have significant difference between the other extraction conditions (Figure 2).The ethanolic extractions performed in Alagoas pollen samples did not have significant differences between the treatment conditions, PEE at 60% and 90%, in the level of 95% according to the Tukey test.
The determination of total phenolic compounds through Folin-Ciocalteau method is important in many food materials.Folin-Ciocalteu reagent, a mixture of phosphotungstic (H 3 PW 12 O 40 ) and phosphomolybdic (H 3 PMo 12 O 40 ) acids, is reduced to blue oxides of tungstene (W 8 O 23 ) and molybdene (Mo 8 O 23 ) during phenol oxidation.Blue coloration is followed at 760 nm and reflects the quantity of polyphenols usually expressed as gallic acid equivalent (GAE) or catechin equivalent.This method was useful and rapid for evaluating pollen samples because bee pollen contains many kinds of phenolics (SERRA BONVEHÍ et al., 2001).

Antioxidant activity of pollen ethanolic extracts (PEE)
The -carotene bleaching method is based on the loss of the yellow colour of -carotene due to its reaction with radicals which are formed by linoleic acid oxidation in an emulsion.In this method, the -carotene undergoes rapid discoloration in the absence of an antioxidant.The linoleic acid free radical formed upon the abstraction of a hydrogen atom from one of its diallylic methylene groups attacks the highly unsaturated -carotene molecules.As a result, -carotene will be oxidized and broken down in part, subsequently the system looses its chromophore and characteristic orange colour, which can be monitored spectrophotometrically (DORMAN et al., 2003).
The absorbances of all samples at 470 nm were taken at zero time and every 60 min until the color of -carotene disappeared in the control reaction.The antioxidation index was calculated as being the reason the tax of bleaching of each group enters on the control tax.
In this work, the extracts from both samples of pollen were found to hinder the extent of -carotene bleaching by neutralizing the linoleate free radical and other free radicals formed in the system.The antioxidant activity through -carotene-linoleate model system of ethanol pollen extracts (40 to 90%) are shown in Figures 3 and 4. The antioxidant ability of the control decreased drastically versus time.All the extraction conditions with regard to the concentration of ethanol, presented some antioxidant activity, therefore they were superior to the control.The highest antioxidant activity index was found in the Alagoas pollen extracted with ethanol at 70%, after 3 hours of treatment (AA= 83.3%).Nevertheless, the highest degree of antioxidant activity was achieved in the PEE at 60% of Parana pollen, with index around 81% (Figures 3 and 4).This treatment condition also has the highest concentration   of polyphenol compounds, although low correlation between the content of total polyphenols and the antioxidant activity of the pollen preparations (Figure 5).The correlation between antioxidant activity (Y) and total phenolic contents (X) of ethanolic extracts of Parana pollen presented a correlation coefficient of R = 0.73 (Y = 63.68 + 1.28 X) and Alagoas pollen showed a correlation coefficient of R = 0.78 (Y = 65.54+ 1.96 X) according to Figure 5. Theses results suggest that the antioxidant activity of Parana pollen and Alagoas pollen are influenced by the phenolic compounds (Figure 5).However other authors found that the antioxidant activity had a higher correlation (71%) with total phenolic content in plant extracts of basil (Ocimum basilicum L) which are used in the traditional medicine, as a culinary herb (JAVANMARDI et al., 2003).

Antibacterial activity of pollen ethanolic extracts (PEE)
Antibacterial activity of all pollen extract analized was according to the disk diffusion assay, and the results are shown in Tables 1 and 2 applied (different ethanol solution concentrations); however Bacillus cereus was inhibited by the pollen of Alagoas in PEE at 50 and 60 % of ethanol.Bacillus subtilis bacteria were inhibited for Parana pollen in PEE at 40%, 50%, 60% and 70% (Table 1) and for Alagoas pollen in PEE at 50%, 70% and 90% (Table 2).In this study, the PEE of Parana pollen at 90% showed the biggest clear zones around each disk (7.0 mm) against Klebsiella sp.Nevertheless, Klebsiella was also inhibited by the pollen from Alagoas in extracts of 60 and 70 %.The Alagoas extract pollen at 70%, contained highest concentrations of phenolic content with antioxidant and antibacterial activity.
Pseudomonas aeruginosa bacteria were inhibited by 80% and 90% and Staphylococcus aureus bacteria were inhibited by extracts of pollen at 50%, 60%, 70% and 80 % of ethanol solution (Table 2).These different patterns of sensitivity are due to different phenolic compounds in pollen (ALMEIDA-MURADIAN et al., 2005).
Gentamicin was the antibiotic used for comparison, showing different ways of action to each bacterium.The control proved that the ethanol used in the extractions did not have inhibiting action.

CONCLUSION
The content of total phenolic compounds in pollen extracts was found to be solvent-dependent.The best extraction conditions, relating to biological properties, were PEE at 70 ethanol solution.The amount of total phenolics varied in different conditions of extraction and ranged from 3.6 to 8.1 and 6.6 to 10.9 mg GAE/g of dry material to Alagoas and Parana pollen, respectively.Although too little antibacterial activity was observed in extract of Parana pollen at 80% and 90% of ethanol, theses extracts showed higher amounts of total phenolic content with antioxidant activity above of 80%.In such a way it is possible that the concentration of phenolic compounds does not determine the antibacterial activity, but the nature of phenolic compounds present in extracts.As different extracts exhibited different antioxidant and antibacterial activities, there may be different kinds of phenolic content in different pollen extract.According to the results obtained, pollen seems to have interesting biological properties, and can be considered as a functional food.Other studies will be necessary to establish quality parameters to standardize the Brazilian product.Due to the great biodiversity of Brazil, more studies are necessary for a better understanding of the functional properties of pollen.

FigureFigure 2
Figure 1 Polyphenolic compounds in pollen of Parana state.
Figure 5 Linear correlation of antioxidant activity versus total phenolic content of 40-90% ethanol concentration of Parana pollen and Alagoas pollen.

a
PEE -Pollen Ethanolic Extract b Antibiotic gentamicin c diameter of the inhibition zone * ethanol

Table 1
Antibacterial activity of Parana state pollen ethanolic extracts (PEE).Antibiotic gentamicin c diameter of the inhibition zone * ethanol a Table 2 Antibacterial activity of Alagoas state pollen ethanolic extracts (PEE).
a PEE Pollen Ethanolic Extract b