Phenolic Acids, Hydrolyzable Tannins, and Antioxidant Activity of

Article pubs.acs.org/JAFC

Phenolic Acids, Hydrolyzable Tannins, and Antioxidant Activity of Geopropolis from the Stingless Bee Melipona fasciculata Smith Richard Pereira Dutra,†,‡ Bruno Vinicius de Barros Abreu,‡ Mayara Soares Cunha,‡ Marisa Cristina Aranha Batista,‡ Luce Maria Brandaõ Torres,§ Flavia Raquel Fernandes Nascimento,† Maria Nilce Sousa Ribeiro,*,‡ and Rosane Nassar Meireles Guerra† †

Laboratório de Imunofisiologia and ‡Laboratório de Farmacognosia, Universidade Federal do Maranhão, Av. dos Portugueses 1966, 65085-580 São Luı ́s, Maranhão, Brazil § Núcleo de Pesquisa em Fisiologia e Bioquı ́mica, Instituto de Botânica de São Paulo, Av. Miguel Estéfano 3687, Á gua Funda, 04301-012 São Paulo, São Paulo, Brazil S Supporting Information *

ABSTRACT: Geopropolis is a mixture of plant resins, waxes, and soil produced by the stingless bee Melipona fasciculata Smith. This paper describes the antioxidant activity and chemical composition of geopropolis produced by M. fasciculata. The total phenolic content determined with the Folin−Ciocalteu reagent was highest in the ethyl acetate fraction and hydroalcoholic extract. Antioxidant activity was assayed by the in vitro DPPH, ABTS, and FRAP assays. The hydroalcoholic extract and fractions of geopropolis, except for the hexane fraction, exhibited antioxidant activity against DPPH, ABTS, and FRAP. The phenolic compounds were identified by HPLC-DAD-MS on the basis of the evaluation of their UV−vis absorption maxima (λmax) and mass spectral analysis. Eleven compounds belonging to the classes of phenolic acids and hydrolyzable tannins (gallotannins and ellagitannins) were tentatively identified. These compounds are responsible for the antioxidant activity and high phenolic content of geopropolis produced by M. fasciculata. KEYWORDS: geopropolis, Melipona fasciculata, phenolic compounds, hydrolyzable tannins, antioxidant activity, HPLC-DAD-ESI-MS

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INTRODUCTION The products of stingless bees, such as geopropolis, are important sources of bioactive compounds. Geopropolis is produced by some stingless bee species of the genus Melipona from a mixture of vegetable resins, saliva secretions, wax, and soil. Bees store large deposits of this material inside their hives, where it is used to close small cracks, to prevent the entry of air, and as an antimicrobial agent.1 Geopropolis produced by Melipona scutellaris collected in the state of Bahia, northeastern Brazil, has been shown to exhibit antimicrobial activity against Staphylococcus aureus and Streptococcus mutans, as well as antioxidant, anti-inflammatory, antinociceptive, and antiproliferative properties.2,3 Benzophenones have been identified as the major compounds of this geopropolis, but no flavonoids were detected.3 Such compounds were detected in Melipona compressipes.4 In recent years, studies investigating the geopropolis produced by Melipona fasciculata Smith in different regions of the Brazilian state of Maranhao have demonstrated high concentrations of polyphenols, flavonoids, triterpenoids, and saponins.5−7 This geopropolis exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans and Streptococcus mutans biofilms, as well as immunomodulatory properties.8,9 Phenolic compounds exhibit a wide variety of biological properties such as anticancer, antiallergic, antiatherogenic, antihepatotoxic, anti-inflammatory, antimicrobial, antithrombotic, cardioprotective, anti-HIV replication, vasodilatory, and antioxidant activities. The beneficial effects of phenolic © 2014 American Chemical Society

compounds have been attributed to their antioxidant activity.10,11 In a study on Apis mellifera, the polyphenolic composition of propolis was correlated with its antioxidant properties.12 Phenolic compounds have been the focus of interest because of their antioxidant and chemopreventive properties. Particularly, polyphenols seem to be responsible for human health benefits.13 Reactive oxygen species and reactive nitrogen species, including free radicals such as superoxide radical anion, hydroxyl radicals, singlet oxygen, hydrogen peroxide, and nitric oxide, are continuously produced in human cells. Reactive oxygen species and free radical-induced reactions have been associated with degenerative or pathological events such as aging, cancer, heart dysfunction, Alzheimer’s disease, rheumatoid arthritis, hemorrhagic shock, cardiovascular disorders, cystic fibrosis, metabolic disorders, neurodegenerative diseases, gastric ulcerogenesis, and AIDS.14,15 The objective of the present study was to determine the antioxidant activity and chemical composition of geopropolis produced by Melipona fasciculata. Received: Revised: Accepted: Published: 2549

November 6, 2013 February 26, 2014 February 27, 2014 February 27, 2014 dx.doi.org/10.1021/jf404875v | J. Agric. Food Chem. 2014, 62, 2549−2557

Journal of Agricultural and Food Chemistry

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as the samples. The percent inhibition was calculated using the formula

MATERIALS AND METHODS

Reagents and Standards. Ethanol, methanol, hexane, chloroform, ethyl acetate, formic acid, hydrochloric acid, acetic acid, sodium carbonate, sodium acetate trihydrate, and anhydrous sodium sulfate were purchased from Merck (Darmstadt, Germany). All chemicals used in the study were of analytical or HPLC grade. Folin−Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), potassium persulfate, iron(III) chloride hexahydrate, iron(II) sulfate heptahydrate, gallic acid, and ellagic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvents were filtered using a Phenomenex solvent filtration apparatus (Torrance, CA, USA). The water used in this experiment was purified in a Millipore Milli-Q apparatus (Purelab UHQ-PS, Elga). Material. Geopropolis produced by Melipona fasciculata Smith (Figure 1) was collected in November 2008 from the internal parts of

DPPH scavenging activity (%) = 100 − [(A sample − A blank ) × 100/Acontrol ] where Asample = absorbance of the sample after 30 min of reaction, Ablank = absorbance of the blank, and Acontrol = absorbance of the control. The percentage of scavenging activity was plotted against the sample concentration to obtain the IC50, defined as the concentration of sample necessary to cause 50% inhibition. All experiments were done in triplicate. ABTS•+ Assay. Antioxidant activity was evaluated by using the ABTS•+ method (2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) as described by Re et al.17 with modifications. For formation of the ABTS radical, 7 mM ABTS•+ solution was mixed with 2.45 mM potassium persulfate solution, and the mixture was stored in the dark for 16 h. The radical was diluted with ethanol PA to an absorbance of 0.700 ± 0.020 at 734 nm. Three different dilutions of the samples (1− 20.0 μg/mL) were then added to 3.0 mL of ABTS radical in the dark. Absorbance was read in a Lambda 35 UV−vis spectrophotometer (Perkin-Elmer, Inc.) after 6 min of the reaction using ethanol as a blank. Standards of gallic acid, ellagic acid, and Trolox were treated under the same conditions as the samples. The percent inhibition was calculated using the formula

Figure 1. Geopropolis of Melipona fasciculata Smith collected in Maranhao state, Brazil.

ABTS•+ scavenging activity (%) = 100 − [(A sample − A blank ) × 100/Acontrol ]

a beehive located in the municipality of Fernando Falcao, Alto Mearim and Grajau microregion (6°7′30″ S, 44°52′30″ W) (Brazilian savannah), southeastern state of Maranhao, Brazil. Extraction and Fractionation of Crude Geopropolis Extract. The in natura geopropolis was triturated using a knife mill, and the geopropolis powder (500 g) obtained was extracted by maceration with 1000 mL of 70% ethanol/water (70:30, v/v) for 48 h at a solid to solvent ratio of 1:2 (w/v). The extract was filtered through Whatman no. 1 filter paper (Whatman, Durham, UK) in a Buchner funnel and concentrated to a small volume at 40 °C in a rotary evaporator under vacuum, obtaining the hydroalcoholic extract of geopropolis (HEG). The geopropolis extract (HEG, 20 g) was dissolved in 100 mL of methanol/water (1:1, v/v) by stirring, and the solution was subjected to fractionation by liquid−liquid partition chromatography using hexane, chloroform, and ethyl acetate. The solutions were filtered (anhydrous Na2SO4) and concentrated to a small volume at 40 °C in a rotary evaporator under vacuum, obtaining the hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EAF), and hydroalcoholic fraction (HAF). Determination of Total Phenolic Content (TPC). The TPC of all samples was determined with the Folin−Ciocalteu reagent and 20% sodium carbonate. The reaction mixture was kept in the dark for 2 h at room temperature, and absorbance was then measured at 760 nm in a Lambda 35 UV−vis spectrophotometer (Perkin-Elmer, Inc., Waltham, MA, USA).5,7 TPC was calculated from the calibration curve constructed with standard solutions of gallic acid (1.0−30.0 μg/mL) and is expressed as gallic acid equivalent (%). The analyses were carried using three aliquots of each sample, measured in triplicate, and the average value was calculated for each sample. Determination of Antioxidant Activity. DPPH Radical Scavenging Activity. The antioxidant activity of the sample of geopropolis was evaluated by using the DPPH free radical scavenging assay as described by Brand-Williams et al.16 with modifications. The samples were diluted in methanol at different concentrations (1.0− 100.0 μg/mL) and added to a methanol solution of DPPH (40.0 μg/ mL). After 30 min of reaction at room temperature in the dark, the absorbance of each solution was read at 517 nm in a Lambda 35 UV− vis spectrophotometer (Perkin-Elmer, Inc.). Methanol was used as the control, and DPPH solution was used as the blank. Standards of gallic acid, ellagic acid, and Trolox were treated under the same conditions

where Asample = absorbance of the sample after 6 min of reaction, Ablank = absorbance of the blank, and Acontrol = absorbance of the control. The percentage of scavenging activity was plotted against the sample concentration to obtain the IC50, defined as the concentration of sample necessary to cause 50% inhibition. All experiments were done in triplicate. Ferric Reducing Antioxidant Power Assay (FRAP). The method described by Benzie and Strain,18 with some modifications, was used to determine the antioxidant activity based on iron reduction using the FRAP assay. FRAP measures the ferric-reducing ability of a sample in acid medium (pH 3.6), forming an intense blue color as the ferric tripyridyltriazine (Fe3+−TPTZ) complex is reduced to the ferrous (Fe2+) form. FRAP reagent was prepared immediately before analysis by mixing 25 mL of acetate buffer (300 mM, pH 3.6), 2.5 mL of TPTZ solution (10 mM TPTZ in 40 mM HCl), and 2.5 mL of FeCl3·6H2O (20 mM) in aqueous solution. Different concentrations of 100 μL of the samples (1−100.0 μg/mL) were added to 300 μL of distilled water and 3.0 mL of FRAP reagent, and the mixtures were incubated in a water bath at 37 °C for 30 min. The absorbance of the reaction mixture was read at 593 nm in a Lambda 35 UV−vis spectrophotometer (Perkin-Elmer, Inc.) using FRAP solution as a blank. The calibration curve was constructed using different concentrations of FeSO4·7H2O (0−2000 μM) (r2 = 0.9987), and the results are expressed as millimoles of Fe2+ per gram of sample. Standards of gallic acid, ellagic acid, and Trolox were treated under the same conditions as the samples. HPLC/UV−Vis Analysis. HPLC analysis was carried out in a Thermo Finnigan Surveyor Autosampler liquid chromatograph (San Jose, CA, USA) equipped with an injector with a 25 μL loop and a UV detector. A Hypersil BDS C-18 column (250 × 4.6 mm, 5 μm; Thermo Electron Corp., Waltham, MA, USA), protected by a C-18 precolumn (4 × 3 mm, 5 μm, Gemini; Phenomenex), was used. The compounds of the geopropolis extract and fractions were separated at room temperature using a gradient elution program at a flow rate of 1.0 mL/min. The mobile phases consisted of Milli-Q water containing 0.1% formic acid (A) and methanol (B). The following linear gradient was applied: 0−1 min, 5% B; 1−60 min, 5−30% B; 60−90 min, 30− 100% B. The column was reequilibrated for 10 min before the next run. The injection volume into the HPLC system was 25 μL, and UV− 2550

dx.doi.org/10.1021/jf404875v | J. Agric. Food Chem. 2014, 62, 2549−2557


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Table 1. Total Phenolic Content and Antioxidant Activity of the Extract and Fractions of Geopropolisa sample HEG HF CF EAF HAF gallic acid ellagic acid Trolox

TPC (%) 47.78 6.32 20.53 57.90 36.0

± ± ± ± ±

0.04 0.02 0.01 0.02 0.04

DPPH IC50 (μg/mL) a b c d e

5.24 nd 17.86 3.75 8.81 1.83 1.20 5.11

ABTS IC50 (μg/mL)

± 0.12 a ± ± ± ± ± ±

0.05 0.10 0.15 0.03 0.01 0.04

2.56 nd 16.29 1.44 4.41 0.73 1.20 2.36

b c d e f a

FRAP (mmol Fe2+/g)

± 0.17 a ± ± ± ± ± ±

0.02 0.03 0.04 0.04 0.08 0.04

14.70 nd 2.52 17.87 15.06 25.87 51.32 9.09

b c d e c a

± 0.43 a ± ± ± ± ± ±

0.01 0.28 0.98 0.28 1.98 0.10

b c a d e f

Values represent the mean of triplicate measurements ± standard deviation. Different letters in the same column indicate a significant difference (Tukey test, p < 0.05). TPC, total phenolic content; HEG, hydroalcoholic extract of geopropolis; HF, hexane fraction; CF, chloroform fraction; EAF, ethyl acetate fraction; HAF, hydroalcoholic fraction; nd, not detected. a

vis detection was performed at 254 nm. Before injection into the HPLC system, each extract was dissolved in the same solvent used for extraction (HPLC grade) to obtain a final concentration of about 5 mg/mL and then filtered through a 0.22 μm nylon syringe filter obtained from Allcrom (São Paulo, SP, Brazil). HPLC-DAD-ESI-MS Analysis. The HEG and EAF fraction were analyzed with an HPLC system (LC-10AD, Shimadzu) equipped with a photodiode array detector, which was coupled to an Esquire 3000 Plus ion-trap mass spectrometer (Bruker Daltonics, Bremen, Germany) using electrospray ionization (ESI). The conditions for dilution of the samples and the mobile phase composition were the same as described above. The ionization conditions were adjusted as follows: electrospray voltage of the ion source of 40 V, capillary voltage of 4.0 kV, and capillary temperature of 320 °C. Ultrahigh-purity helium (He) was used as the collision gas and high-purity nitrogen (N2) as the nebulizing gas. Nebulization was aided with a coaxial nitrogen sheath gas provided at a pressure of 27 psi. Desolvation was facilitated using a counter current nitrogen flow set at a rate of 7.0 L/ min. Analyses were carried out using full-scan mass spectra in the negative ionization mode and data-dependent MS2 scans from m/z 100 to 3000. The compounds were identified on the basis of the molecular ion mass, fragmentation, UV−visible spectra, or co-injection with standards. Statistical Analysis. All analyses were performed in triplicate. The results are expressed as the mean ± standard deviation and were analyzed using the GraphPad Prism 5.0 program. One-way analysis of variance (ANOVA) and Tukey’s multiple-comparisons test were used to determine significant differences between means. A level of significance of p < 0.05 was adopted. Pearson’s correlation test was used to evaluate the correlation between TPC (%), DPPH free radicalscavenging activity (IC50), ABTS free radical-scavenging activity (IC50), and ferric-reducing ability (mmol Fe2+/g sample).

highest activity among the samples analyzed by the DPPH radical-scaveninging assay, with an IC50 value of 3.75 μg/mL. The HEG extract also exhibited antioxidant activity, with an IC50 value of 5.24 μg/mL. HAF and CF provided IC50 values of 8.81 and 17.86 μg/mL, respectively. The phenolic compounds gallic and ellagic acids, which are strong antioxidants, exhibited the highest DPPH-scavenging activity. The highest antioxidant activity was observed for EAF, which contained the highest levels of total phenols and presented the lowest IC50 value and its radical-scavenging activity exceeded that of Trolox (Table 1). In the ABTS assay, the EAF, which contained the highest levels of phenolic compounds, exhibited the lowest IC50 value (1.44 μg/mL), similar to that of ellagic acid, followed by HEG (IC50 = 2.56 μg/mL), which presented antioxidant activity similar to that of Trolox. HAF and CF also exhibited antioxidant activity, with IC50 values of 4.41 and 16.29 μg/ mL, respectively. In the FRAP assay, EAF, HEG, and HAF presented the best ferric-reducing abilities (17.87, 14.70, and 15.06 mmol Fe2+/g, respectively) which were superior to the antioxidant activity of Trolox. No antioxidant activity was observed for HF in any of the antioxidant assays (Table 1). The differences in antioxidant activity between the same samples demonstrated by the different assays, even when the same standard compound was used, can be explained by the reaction mechanisms of the methods. Correlation between Antioxidant Activity and Total Phenolics Content. The correlations between the results of the DPPH, ABTS, and FRAP methods and TPC are shown in Table 2. A negative correlation was observed for DPPH−TPC (−0.9649), ABTS−TPC (−0.9154), DPPH−FRAP (−0.9670), and ABTS−FRAP (−0.9891), with a low IC50 value in the DPPH and ABTS assays being correlated with a high TPC and a high FRAP value and vice versa. The correlation between TPC and FRAP was positive (0.9034) as was that between DPPH and ABTS (0.9866), indicating that a high reducing

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RESULTS AND DISCUSSION Extraction Yields, Total Phenolic Content, and Antioxidant Activity. The HEG yielded 8.80% (44 g) of a black, bitter-tasting, and resin-smelling substance. Fractionation of the HEG yielded the following fractions: HF (0.22 g), CF (2.30 g), EAF (7.44 g), and HAF (9.30 g). TPC and antioxidant activity of the fractions and extract are shown in Table 1. EAF (57.89%) and HEG (47.78%) had the highest total phenolic content, whereas total phenolic content ranged from 6.32 to 36.0% in the remaining fractions. Abreu et al.,5 analyzing geopropolis from Melipona fasciculata collected in the savannah of Maranhao state, found a TPC ranging from 14.14 to 67.46%. In contrast, Cunha et al.7 reported total polyphenol levels ranging from 7.36 to 36.95% in hydroalcoholic extracts of geopropolis collected in the Western Lowland of Maranhao. In the analysis of DPPH and ABTS, a lower IC50 value indicates higher antioxidant activity. The EAF exhibited the

Table 2. Pearson Correlation Coefficient between the DPPH, FRAP, and ABTS Methods and Total Phenolic Contenta TPC DPPH ABTS

DPPH

ABTS

FRAP

−0.9649

−0.9154 0.9866

0.9034 −0.9670 −0.9891

a

TPC, total phenolic content; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; FRAP, ferric reducing antioxidant power.

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Figure 2. HLPC chromatograms of phenolic compounds detected at 254 nm in the hydroalcoholic extract (HEG) and ethyl acetate fraction (EAF) of geopropolis. Peak numbers correspond to the compounds shown in Table 3.

ability in the FRAP assay is related to the presence of phenolic compounds in the extracts.

The strongest free radical-scavenging activity and reducing ability were observed for the EAF fraction, which presented the 2552



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yl-glucose (HHDP-glucose). The chemical structures of these compounds are shown in Figure 4. Table 3 summarizes the tentatively identified phenolic compounds, their retention time, maximum UV−vis absorbance (λmax), molecular weight, molecular ion [M − H]−, and main product ions obtained by HPLC-MS/MS for the 11 fragmentation peaks of HEG and EAF. The compounds were identified by comparing their fragmentation profiles with reference compounds run under the same experimental conditions. When no standards were available, the compounds were identified on the basis of literature data. The characterization of phenolic compounds is important because these compounds are associated with a variety of health benefits. HPLC with diode array and mass spectrometry detection has proven to be useful for the characterization of individual phenolic compounds, particularly gallotannins and ellagitannins. The results showed losses of 152 and 170 Da from the ion molecule, indicating the presence of galloyl groups, and a loss of 302 Da from the ion molecule, which is characteristic of HHDP groups. Loss of the carboxyl group (44 Da) and water (18 Da) from the ion molecule indicates the presence of a trigalloyl group and C-glucosidic ellagitannins, respectively.29,30 The presence of ellagitannins was confirmed by the data indicating spontaneous lactonization of HHDP forming ellagic acid, whereas the partial lactonization of HHDP yields a product ion at m/z 319, and subsequent decarboxylation yields an ion at m/z 275 [M − H − 44]−. Furthermore, tannins with molecular masses different by 2 units were observed, which are due to differences in one HHDP group or two galloyl groups.31 Phenolic Acids. Compounds 1 and 11 were identified as gallic acid and ellagic acid, respectively, by comparing their retention times, UV−vis spectra, fragmentation pathways observed in MS/MS spectra, and [M − H]− ions at m/z 169 and 301 with literature data. These peaks were confirmed using an authentic standard.32,33 Galloyl Glucose Derivatives and Ellagitannins. Compound 2 was tentatively identified as HHDP-galloylglucose (corilagin). Fragmentation of the molecular ion at m/z 633 [M − H]− produced ion fragments at m/z 481 (HHDP-glucose), due to the loss of the galloyl group, and at m/z 301, corresponding to the HHDP unit after lactonization to ellagic acid.33 Compound 3 showed an [M − H]− ion at m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and was tentatively identified as HHDP-glucose after the loss of one unit of glucose.34 Ellagitannins with [M − H]− ions at m/z 783 and 785 were at m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and were tentatively identified in the samples analyzed. Compound 4, a parental [M − H]− ion at m/z 783 is present, producing ion fragments at m/z 765 (M − 18, loss of water), m/z 481, which correspond to deprotonated HHDPglucose (M − 302 Da, loss of HHDP), and m/z 301, which corresponds to ellagic acid. These fragments were tentatively identified as di-HHDP-glucose (pedunculagin/casuariin).28,33 Compound 5 showed an [M − H]− ion at m/z 635 and fragments at m/z 483 [M − H − 152]−, m/z 465 [M − H − 170], and m/z 313 [M − H − 152 − 152 − 18] −, indicating the loss of a galloyl group, the loss of gallic acid, and the loss of another galloyl group, water along with cross-ring fragmentation of glucose, and at m/z 301, corresponding to the HHDP unit after lactonization to ellagic acid. This compound was at

highest TPC. These results suggest that total phenols, particularly hydrolyzable tannins, present in geopropolis were mainly responsible for the antioxidant activity and reducing ability of geopropolis. Other authors also reported important antioxidant activity of geopropolis obtained from the stingless bees Melipona interrupta and Melipona seminigra19 and from Melipona subnitida,20 which was correlated with the content of phenolic compounds. TPC has been shown to be closely correlated with antioxidant activity.21 Polyphenols have been identified as the most abundant and effective antioxidants in samples of Brazilian propolis produced by Apis mellifera.22−24 In addition, Moreira et al.25 demonstrated that propolis extracts with the highest concentrations of total polyphenols also exhibit the highest antioxidant activity. In the study of Sawaya et al.,26 propolis produced by Scaptotrigona species, a stingless bee found in the Brazilian states of Maranhao and Sao Paulo, suggested that TPC and antioxidant activity are affected by the chemical composition of propolis, season of collection, and geographic origin.26 Identification of Phenolic Compounds. Because EAF and HEG had higher TPC and were more effective against the DPPH radical, the chemical composition of these samples was analyzed by HPLC/UV−vis and HPLC-DAD-MS/MS. The chromatogram obtained by HPLC/UV−vis (254 nm) for HEG and EAF (Figure 2) revealed similar chemical compositions, with various peaks corresponding to phenolic acids, gallotannins, and ellagitannins on the basis of the analysis of the three-dimensional DAD-HPLC chromatogram of EAF, which established the absorption maxima at 254−363 nm (Figure 3; Figure S1 in the Supporting Information). Gallic acid

Figure 3. Three-dimensional DAD-HPLC chromatogram of the ethyl acetate fraction of geopropolis. (See also the Supporting Information.)

derivatives exhibit an absorption maximum at 254 nm, whereas ellagitannins show UV spectra similar to that of ellagic acid, with two absorption maxima at λmax 254 and 365 nm. In contrast, gallotannins have only one absorption maximum at 275 nm due to the presence of galloyl and hexahydroxydiphenoyl groups.27,28 The compounds 1−11 were identified by HPLC-MS/MS as phenolic acids and hydrolyzable tannins (gallotannins and ellagitannins) as indicated by the presence of molecular ions corresponding to gallic acid, ellagic acid, trigallic acid, hexahydroxydiphenic acid (HHDP), and hexahydroxydipheno2553



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Figure 4. Chemical structures of phenolic compounds detected in geopropolis of Melipona fasciculata Smith.

Table 3. Tentative Identification of Phenolic Compounds in Melipona fasciculata Geopropolis by HPLC-DAD-ESI-MS/MSa compd 1 2 3 4 5 6 7 8 9 10 11

RT (min) 11.23 19.26 25.60 27.16 30.53 31.98 35.62 39.25 45.08 51.39 69.88

tentative identification b

gallic acid HHDP-galloylglucosec HHDP-glucosec di-HHDP-glucosec trigalloyl glucosec HHDP-digalloylglucose isomerc valoneic acid dilactonec trisgalloyl-HHDP-glucose isomerc di-HHDP-galloylglucosec trigalloyl-HHDP-glucosec ellagic acidb,c

HPLC-DAD λmax (nm)

MW

[M − H]− (m/z)

272 262 267 265 277 274 254, 370 263 271 277 254, 363

170 634 482 784 636 786 470 952 936 938 302

169 633 481 783 635 785 469 951 935 937 301

MS/MS fragments (m/z) 125 481, 301, 764, 483, 783, 425, 907, 933, 785, 301

301, 275, 481, 465, 709, 301 783, 633, 767,

275 169 301, 275 313, 301 633, 463, 301, 275 301 451, 301, 275 635, 465, 301, 275

RT, retention time; λmax, UV−vis absorption maxima; MW, molecular weight; [M − H]− molecular ion; HHDP, hexahydroxydiphenic acid. bCoinjection of the authentic standard. cCompounds identified for the first time in geopropolis. a

m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and was tentatively identified as trigalloylglucose.28,33,34 Compound 6 with m/z 785 [M − H]− was at m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and was tentatively identified as an HHDP-digalloylglucose isomer (tellimagrandin I) because it contained ions at m/z 633 (loss of a galloyl group), m/z 483 (loss of HHDP), and m/z 313 after the loss of one unit of HHDP, one galloyl group, and water.

These two compounds differ from one another by 2 atomic mass units, suggesting that compound 4 could be formed by binding of two adjacent galloyl groups in HHDP-digalloylglucose by means of intramolecular oxidation.30,33,34 Compound 7 produced [M − H]− ions at m/z 469 and two main fragments at m/z 425 and 301 due to the loss of CO2 from the deprotonated molecule and ellagic acid fragment, suggesting the presence of valoneic acid dilactone.30,35 2554



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Compound 8 showed an [M − H]− ion at m/z 951, which fragmented to m/z 907, 783, and 301. On the basis of the fragmentation patterns, the peak was at m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and was tentatively identified as the trisgalloyl-HHDP-glucose isomer. UV spectral data and the fragment ion at m/z 783 [M − H − 168] indicate the presence of di-HHDP-glucose. The fragment corresponds to the presence of an additional galloyl residue, but only with a C−C bond to one of the HHDP molecules. The fragment ion at m/z 907 ([M − H]− − CO2) agrees with the presence of a free unesterified carboxyl group, and ion m/z 301 corresponds to 1 unit of HHDP after the lactonization process to ellagic acid.28,29,33,36 Compound 9 showed an [M − H]− ion at m/z 935, which fragmented to m/z 633 (−302 Da, loss of HHDP), m/z 481 (loss of a galloyl group), and m/z 301, and subsequent decarboxylation yields an ion at m/z 275. This compound was at m/z 481 [M − H]−, followed by an ion fragment m/z 301 (ellagic acid), and was tentatively identified as di-HHDPgalloylglucose (potentillin/casuarictin).30,32,33 Compound 10 was tentatively identified as trigalloyl-HHDPglucose (tellimagrandin II) on the basis of an [M − H]− ion at m/z 937, followed by ions m/z 785 and 767 (loss of water and a galloyl group, respectively), in addition to the ion fragments m/z 635 (minus one HHDP group), m/z 465 (loss of HHDP and gallic acid groups), and m/z 301, which corresponds to one unit of HHDP after the lactonization process to ellagic acid, followed by decarboxylation producing ion m/z 275. This compound differs from tellimagrandin I by the presence of an additional galloyl unit in the anomeric center of the glucopyranosyl core. Tellimagrandin II is produced by enzymatic transformation of pentagalloylglucose.30,33 Bankova et al.37 demonstrated the presence of gallic acid in geopropolis produced by Melipona compressipes collected in the state of Piaui, Brazil. Gallic acid is one of the main compounds found in propolis samples from stingless bees collected in the Brazilian states of Pernambuco, Parana, and Sao Paulo.38 However, there are no studies in the literature reporting the presence of ellagic acid and hydrolyzable tannins in samples of geopropolis or propolis produced by stinging or stingless bees. Ellagitannins were the main compounds identified in the HEG and in EAF. These phenolic compounds are esters of HHDP with a polyol, usually glucose, have a high molecular weight, and are soluble in water.39 Ellagic acid is a phenolic compound that can occur in free form, glycosylated, or in bound form like ellagitannins. This compound is found in strawberries (Fragaria ananassa D.), raspberries (Rubus idaeus L.), pomegranates (Punica granatum L.), blackberries (Morus nigra L.), and eucalyptus (Eucalyptus globules L.). Ellagic acid and its derivatives (ellagitannin) have been shown to have antiviral, antioxidant, anticarcinogenic, anti-inflammatory, antifibrotic, and chemopreventive properties,11 as well as antileishmanial activity.40 Antioxidants have attracted much interest because of their protective effect against free radical damage, which is the cause of many diseases, including cancer. In the present study, HEG and its EAF exhibited the highest antioxidant activity. This finding is probably due to the high content of polyphenols, such as gallic acid, ellagic acid, and hydrolyzable tannins. A similar correlation between antioxidant activity and phenolic content has been demonstrated in other studies.21 Zahin et al., 41 studying the antioxidant activity of pomegranate (Punica granatum) peel extracts by DPPH free

radical scavenging, identified gallic acid, ellagic acid, HHDPglucose, corilagin, and pedunculagin by HPLC-MS. All of these compounds were detected for the first time in stingless bee products in the present study. The strong correlation observed between antioxidant activity measurements and ellagitannins indicates that high molecular weight polyphenols were the main contributors of antioxidant activity. This can be attributed to the structure of ellagitannins, which is characterized by the presence of several ortho-hydroxy functions that exhibit a greater ability to donate a hydrogen atom and support the unpaired electron when compared to low molecular weight polyphenols. The antioxidant efficiency of ellagitannins and ellagic acid is directly correlated with their degree of hydroxylation and decreases in the presence of a sugar moiety.11 The present results provide further evidence that geopropolis is a rich source of bioactive compounds with potential health benefits, including antioxidant activity, which is related to the presence of phenolic compounds such as phenolic acids, gallotannins, and ellagitannins. Identification of the chemical composition and antioxidant substances is important for the understanding of the biological activity of geopropolis and for the standardization of extracts.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.N.S.R.) E-mail: [email protected]. Phone: (+55) 98 32728592. Funding

This work was supported by Conselho Nacional de Desenvolvimento Cientı ́fico e Tecnológico (CNPq) and Fundaçaõ de Amparo a Pesquisa e ao Desenvolvimento Cientı ́fico e Tecnológico do Maranhão (FAPEMA). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Ozimar Cavalcante Araújo for providing the geopropolis samples. ABBREVIATIONS USED HEG, hydroalcoholic extract of geopropolis; HF, hexane fraction; CF, chloroform fraction; EAF, ethyl acetate fraction; HAF, hydroalcoholic fraction; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; FRAP, ferric reducing antioxidant power; HHDP, hexahydroxydiphenic acid; HPLC, highperformance liquid chromatography; ESI, electrospray ionization; MS, mass spectrometry; DAD, diode array detection; MS/ MS, tandem mass spectrometry; TPC, total phenolic content

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REFERENCES

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Phenolic Acids, Hydrolyzable Tannins, and Antioxidant Activity of

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