Polyphenolic profile and antioxidant and antibacterial activities of monofloral honeys produced by Meliponini in the Brazilian semiarid region

    Polyphenolic profile and antioxidant and antibacterial activities of monofloral honeys produced by Meliponini in the Brazilian semiarid region Jana´ına Maria de Sousa, Evandro de Souza, Gilmardes Marques, Bruno ˆ Meireles, Angela Cordeiro, Beatriz Gull´on, Maria Manuela Pintado, Marciane Magnani PII: DOI: Reference:

S0963-9969(16)30088-6 doi: 10.1016/j.foodres.2016.03.012 FRIN 6204

To appear in:

Food Research International

Received date: Revised date: Accepted date:

23 September 2015 25 February 2016 6 March 2016

Please cite this article as: de Sousa, J.M., de Souza, E., Marques, G., Meireles, ˆ Gull´ B., Cordeiro, A., on, B., Pintado, M.M. & Magnani, M., Polyphenolic profile and antioxidant and antibacterial activities of monofloral honeys produced by Meliponini in the Brazilian semiarid region, Food Research International (2016), doi: 10.1016/j.foodres.2016.03.012

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Polyphenolics profile, antioxidant and antibacterial activity of monofloral honeys produced

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by Meliponini in the Brazilian semiarid region

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Running Title: Bioactivity of Meliponini honeys

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Janaína Maria de Sousa a, Evandro de Souza b, Gilmardes Marques a, Bruno Meireles c, Ângela

a

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Cordeiro c, Beatriz Gullón d, Maria Manuela Pintado d, Marciane Magnani a*

Laboratory of Microbial Process in Foods, Department of Food Engineering, Center of

Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal

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Technology, Federal University of Paraíba, Campus I, 58051-900, João Pessoa, Paraíba, Brazil

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Laboratory of Fuel, Federal University of Paraíba, Campus I, 58051-900, João Pessoa, Paraíba,

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Escola Superior de Biotecnologia, Universidade Católica do Porto, Porto, Portugal

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University of Paraíba, Campus I, 58051-900, João Pessoa, Paraíba, Brazil

* Corresponding author: Laboratory of Microbial Processes in Foods, Department of Food Engineering, Technology Center, Federal University of Paraíba, Campus I, 58051-900, João Pessoa, Brazil e-mail: [email protected]; Phone number: + 55 83 3216 7576; Fax number: + 55 83 3216 735

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Abstract This study assessed the polyphenolic profile, the antioxidant and antibacterial activities of

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monofloral honeys produced by Meliponini in Brazilian semiarid region. Honeys from Ziziphus

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joazeiro Mart. (juazeiro) and Croton heliotropiifolius Kunth (velame branco) showed the highest

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total phenolic contents (TPC) and the greatest antioxidant activity in assays with DPPH and ABTS•+ radicals. Honeys from Mimosa quadrivalvis L. (malícia) presented the strongest anti-peroxyl

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activity in ORAC assay. Juazeiro honeys showed the highest quantities of trans-cinnamic, pcoumaric, ellagic and ferulic acids, as well as of cathecin, rutin, hesperetin and chrysin when

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compared to the other honeys produced by the same bee species. Malícia honeys showed the greatest quantities of myricetin, quercetin and kaempferol among the studied honeys. Honeys with

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highest TPCs presented the highest antimicrobial activity. The results showed the impact of the

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of Meliponini honeys.

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floral source on the polyphenolic profile as well as on the antioxidant and antimicrobial properties

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Key-words: Meliponini, honey, radical scavenger, antimicrobial effect

1. Introduction

Honeys are members of a class of natural substances that have recently attracted attention for having high therapeutic interest. In the long human tradition, honey has been used not only as a nutrient but also as a medicine. Despite the relevant importance of polyphenolic compounds, which are recognized as the major constituents and responsible for the health-promoting properties of honey (Meda Lamien, Romito, Millogo & Nacoulma, 2005; Habib, Meqbali, Kamal, Souka & Ibrahim, 2014), their identification and quantification are of great interest for understanding their contributions to the overall bioactivity of honey (Manzanares, García, Galdón, Rodríguez &

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Romero, 2014). In honeys, polyphenolic compounds, comprising phenolic acids and flavonoids, are also considered potential markers of botanical origin (Alvarez-Suarez et al., 2012).

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Antioxidant activity, or simply the antioxidant capacity of honeys, is the ability and potential

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of these products to reduce oxidative reactions within food systems and human health (Frankel,

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Robinson, & Berenbaum 1998). The antioxidants that naturally occur in honey contribute to its antioxidant capacity (Silva et al., 2013b). Furthermore, the phenolic profiles of honeys and consequently their antioxidant capacity depend on the floral sources visited by the bee (Meda et al.,

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2005; Habib et al., 2014).

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Apart from their antioxidant activity, honeys also possess broad antimicrobial activity that is capable to inhibit the growth of many foodborne pathogens and bacteria of clinical importance (Vorlova, Karpiskova, Chabiniokova, Kalabova, & Brazdova, 2005). The antimicrobial properties

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of honeys were first attributed to their high sugar concentration and the presence of peroxide compounds (primarily hydrogen peroxide) (Weston, Brocklebank & Lu, 2000). However,

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polyphenols have been strongly related to the antimicrobial properties of monofloral honeys (Rodriguez, Mendoza, Iturriga & Castaño-Tostado, 2012). Moreover, studies have found that

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honeys from specific floral sources present stronger antibacterial activity than other types of honey (Silici, Sagdic, & Ekici, 2010; Hussain et al., 2015). The predominance of particular botanical species in honey is influenced by geographical, seasonal and environmental factors (Andrade, Ferreres & Amaral, 1997). The phenolic profile of honeys is variable depending primarily on their floral source but also on their entomological origin (Kirnpal-Kaur, Tan, Boukraa & Gan 2011; Belitz, Grosch & Schieberle 2009). Thus, honeys that are produced by distinct bee species or that are collected from different locations possess different active compounds, and they consequently exhibit differences in their biological properties (Kirnpaul-Kaur et al., 2011). Monofloral honeys are of interest because they have specific phenolic profiles related to the honey-producing plant (Habib et al., 2014; Cavazza, Corradini, Musci, & Salvadeo, 2013; Silva et

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al., 2013b). This characteristic is particularly relevant in the Brazilian semi-arid region, where an unique biodiversity of stingless bees (Meliponini) and native flora is found. The occurrence of only

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two seasons during the year (dry and rainy) with availability of specific major botanical sources

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favors the production of different monofloral honeys with particular characteristics in this region

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(Silva et al., 2013b; Sousa et al., 2015).

Only a few studies on the bioactivity of monofloral honeys produced by the Meliponini are available, although it is well-known that these products possess differences, including their phenolic

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profile, when compared with honeys produced by bees belonging to Apis genera (Silva et al.,

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2013b; Sousa et al. 2013). Among the Meliponini species, Melipona subnitida Ducke (jandaíra) and M. scutellaris Latrelle (uruçu) typically visit botanical species that are available only during the rainy or dry season in the semiarid region of Brazil, thus producing different types of honeys during

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the year (Sousa et al., 2013). Considering these aspects, the aims of the present study were to determine for the first time a) the antioxidant activity of rare monofloral honeys produced by

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distinct stingless bee species in the Brazilian semi-arid region by using distinct methods; b) the total phenolics and flavonoids as well as the individual phenolic profile of the studied monofloral

strains.

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honeys; and c) the antibacterial activity of the whole honeys against different pathogenic bacterial

2. Materials and methods 2.1. Honey samples The experimental design included four different monofloral honeys produced by two different stingless bee species in the semiarid region of Brazilian northeastern (Fig. 1) collected in three different occasions (4 x 2 x 3). Each of the 24 samples analyzed was composed by a mixture of honeys collected in four different beekeeper’s meliponaries specific for each stingless bee species. The honey samples from Ziziphus joazeiro Mart. (juazeiro) and Mimosa quadrivalvis L.

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(malícia) produced by the stingless bees M. subnitida Ducke (jandaíra) and M. scutellaris Latrelle (uruçu) were collected during the 2012 dry season, while the honey samples from Mimosa arenosa

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willd Poir (jurema branca) and Croton heliotropiifolius Kunth (velame branco) produced by the

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both bee species (jandaíra and uruçu) were collected during the 2013 rainy season. The samples

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were stored in sterilized amber glass containers, shipped to the laboratory and maintained at 6 - 8 ºC in the dark until analysis (Sousa et al., 2015). When the analysis was delayed for more than a month after sampling, the honeys were frozen at -18 ºC. To ensure the botanical source, the monofloral

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honeys were submitted to melissopalynological analysis (Louveaux, Maurizio & Vorwohl, 1978).

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Briefly, a total of 10 g of each honey sample was diluted in 20 mL of distilled water and centrifuged at 4000 rpm for 20 min. The sediment was dried at 40 ºC and mounted in microscope slides with Entellan Rapid (Merck, 1.07961.0500). The honeydew elements and pollen grains (n = 500) were

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counted and identified in 20 distinct optical areas (Nikon Optiphot II microscope; 400 x and 1000x). The pollen grains were compared to reference images of Pollen and Apicultural Plants of

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Laboratory of Entomology, ESALQ, São Paulo, Brazil (ESALQ, 2009). All honey samples included in the study contained more than 85% pollen grains of the same botanical origin, being

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characterized as monofloral honey samples (Table 1).

2.2. Test strains

The strains Listeria monocytogenes 3375, Staphylococcus aureus 18N, Escherichia coli CINF1, Salmonella spp. CINF2, and Pseudomonas aeruginosa CINF3 that were isolated from food samples were obtained from the Collection of Innovation Center and Business Support of Porto (CINATE, Porto, Portugal). The strains were stored in cryovials with glycerol at 15% (v/v) and maintained at -80 ºC before use. The inoculum of the each bacteria strain used in the antimicrobial testing were obtained after preparing the suspensions in sterile saline solution (0.85% NaCl w/v) from overnight cultures grown in Mueller-Hinton (MH) agar (Biokar Diagnostics, Beauvais,

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France) at 37 °C. Each strain was grown in MH broth (Biokar Diagnostics, Beauvais, France) at 37 °C for 18 – 20 h (late exponential growth phase), harvested by centrifugation (4500 g, 15 min, 4

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°C), washed twice in sterile saline solution and re-suspended in MH broth to obtain standard cell

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suspensions at which the OD reading at 660 nm (OD660) was 0.1, which provided viable cell counts

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of approximately 8 log CFU/mL when they were pour-plated onto MH agar (McMahon et al., 2008).

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2.3. Total phenolic contents (TPC)

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The concentration of total phenolics was measured by using Folin-Ciocalteu phenol reagent (Habib et al., 2014). Briefly, each honey (1 g) was diluted in ultrapure water (H2O) (10 mL) and filtered through grade 1 Whatman® qualitative filter paper. An aliquot of 0.5 mL (filtrate) was

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mixed with 2.5 mL of 0.2 N Folin-Ciocalteu reagent (Sigma Aldrich, Germany) for 5 min, and then 2 mL of 7.5% sodium carbonate (Na2CO3) (Labosi, Paris, France) was added to the solution. After

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the reaction mixture incubated at room temperature for 2 h, its absorbance was measured and compared with a methanol blank at 760 nm by using a spectrophotometer (UV1240 Shimadzu). A

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sugar analogue (an artificial honey whose composition reflects the approximate sugar composition of honey) was used to check possible interference of the main sugar components present in the honeys (Piljac-Žegara, Stipčević, & Belščak, 2009). (Piljac-Žegara, Stipčević & Belščak, 2009). The analogue was prepared considering an average of sugar and moisture previously detected in assayed honeys by dissolving 1.7 g of sucrose, 50.5 g of fructose and 37.5 g of glucose in 24 mL of deionized water and assayed similarly (Sousa et al., 2015). Gallic acid (Sigma Aldrich®, Germany) (0 - 100 mg/mL) was used as a standard to produce the calibration curve. The TPC were expressed as mg of gallic acid equivalents (GAE) /100 g honey.

2.4. Total flavonoid contents

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The total flavonoid contents were measured by using a colorimetric assay (Meda et al., 2005). A 5 mL aliquot of honey solution (1:10 g/mL ddH2O) was mixed with 5 mL of a methanolic

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solution (2 g/100 mL) of aluminum trichloride (AlCl3) (Labosi, Paris, France). Absorption readings

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at 415 nm (UV1240 Shimadzu) were measured after 10 min and compared with a blank sample

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consisting of a 5 mL honey solution along with 5 mL methanol without AlCl3. The total flavonoid content was determined using a standard curve with quercetin (Sigma Aldrich, Germany) (0 - 50 mg/L) as the standard. The mean of three readings was used and expressed in mg of quercetin

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equivalents (QE)/100 g of honey.

2.5. Antioxidant assays

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2.5.1 DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical-scavenging assay The scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (Sigma-

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Aldrich®, Germany) and the antioxidant activity were determined according to a procedure described elsewhere (Meda et al., 2005). The honey samples were dissolved in methanol (100

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mg/mL), and 0.75 mL of each sample was mixed with 1.5 mL of DPPH (Fluka Chemie, Switzerland) in methanol (0.05 mg/mL), with methanol serving as the blank sample. The mixtures were left for 15 min at room temperature, and the absorbance was measured at 517 nm. Quercetin (0 - 25 µg/mL) and ascorbic acid (Labosi, Paris, France) (0 - 50 µg/mL) were used as positive controls. The measurements were performed in triplicate. The index for DPPH radical scavenging activity (RSA), which was expressed as the percent inhibition, was calculated as follows: RSA [ % ] = [(absorbance control – absorbance sample) / absorbance control x 100] (eq. 1.)

2.5.2. ABTS•+ free radical-scavenging assay

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ABTS•+ [2,2`- azino-bis (3-thylbenzothiazoline-6-sulfonic acid)] is a cation radical that was obtained through the reaction of a 2 mM phosphate-buffered stock solution of 2,2´-azinobis (3-

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ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma-Aldrich) with potassium

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persulfate (POCh). The mixture was left to stand for 24 h at room temperature. Prior to analysis, the

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ABTS•+ solution was diluted with a 75 µM phosphate buffer solution at pH 7.0 (PBS) to produce a solution with an absorbance (ƛ ) of 0.20 ± 0.03 at ƛ = 734 nm. Fifty microliters of methanolichoney solution (1:10) was mixed with 5 mL of the ABTS•+ cation radical solution, and after 6 min,

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the absorbance was measured at a wavelength of 734 nm using a spectrophotometer (UV1240

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Shimadzu). The control test was made with methanol instead of the methanolic honey solution. The measurements were performed in triplicate. The antioxidant activity was determined using standard curves for Trolox (Sigma-Aldrich, Germany; 0.00 for 2000 µmol Trolox/mL; Y = -0.0002x +

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0.6934; R = 0.991), and the results were expressed in μmol TE/100 g (Lianda et al., 2012).

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2.5.3. Oxygen radical absorbance capacity (ORAC) assay An ORAC assay was performed by using a procedure described elsewhere (Gorjanovic´ et

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al., 2013). All reagents were prepared in PBS, and Trolox (6.25 - 200 µM) was used as a standard. The honey solutions (1:10) were suitably diluted (10 - 60 v/v) in PBS. To each well of a 96-well microplate, 150 µL of fluorescein (0.08 µM) and 25 µL of honey solution were added. The plate was incubated for 10 min at 37 ºC and then 25 mL of 150 mM AAPH [L (+)-ascorbic acid, epicatechin gallate, 2,2’-azobis (2-methylpropionamidine) dihydrochloride] were added to each well to start the reaction. The plate was shaken automatically for 3 s, and the fluorescence was measured every 2 min for 120 min at 37 ºC at emission and excitation wavelengths of 485 and 530 nm, respectively, by using a microplate fluorescence reader (SynergyTM Multi-Detection Microplate Reader; Bio-Tek1 Instruments, Inc., USA). The ORAC values were expressed as µmol of Trolox equivalent per gram of honey (µmol TE/100 g of honey).

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2.6. Extraction of phenolic compounds from honey The different honey samples (50 g each) were mixed (1:5) with acidified water (which was

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acidified to pH 2.0 with 1 M HCl) until completely fluid and then filtered through cotton to remove

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the solid particles. The filtrate was then passed through a column (25 x 2 cm) of Amberlite XAD-2

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resin (pore size = 9 nm, particle size = 0.3-1.2 mm). Sugars and polar compounds were eluted with acidified water (350 mL) (fraction 1); the column was washed with 300 mL of neutral water (fraction 2), and phenolic compounds were further eluted with methanol (600 mL). The methanol

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extracts were concentrated to 10 mL under a vacuum at 40 ºC in a rotary evaporator (Alvarez-

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Suarez et al., 2010)

2.7. Chromatographic analysis and identification of phenolics

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HPLC analyses were performed by using a Shimadzu Prominence LG2OAT HPLC equipped with a photodiode array detector (SPDM2O) and a reversed-phase column (Shimpack

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CLCODS, 4.6 mm x 250 mm x 5 mm). For the benzoic and cinnamic acid derivatives, the mobile phase consisted of a mixture of 2% aqueous acetic acid in water (A) and acetonitrile: methanol (2:1)

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(B) at a flow rate of 1 mL/min. A gradient elution was used, starting with 20% B up to 15 min, 30% B at 20 min, 40% B at 30 min and isocratic at 40% B up to 45 min. The flavonoids were separated by using a mobile phase consisting of 1% aqueous acetic acid (A) and methanol (B) at a flow rate of 1 mL/min. The mobile phase was delivered by using the following solvent gradient: 0–3 min 40% B, 5-15 min 45% B, 17-25 min 50% B, 27-35 min 55% B and 35-40 min 70% B. The injection volume was 10 µL. The identification of phenolic compounds was based on the retention times, the UV-spectra and a chromatographic comparison (co-injection) with authentic markers from Sigma Aldrich® (Silva et al., 2013a).

2.8. Antibacterial assays

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The antibacterial activities of the honey samples were measured by determining the minimum inhibitory concentration (MIC) using a microdilution assay (CLSI, 2012; Boorn, Khor,

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Sweetman, Tan, Heard, & Hammer, 2009). The honey samples were prepared at concentrations

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ranging from 1 - 20 mL/100 mL in Muller Hinton Broth (MHB) and filtration-sterilized through

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qualitative 0.22 µm filter paper (Orange Scientific, Braine-l’Alleud, Belgium). Approximately 50 μL of each of the tested honey concentrations was dispensed into each well of a 96-well microplate containing 100 µL of MH broth. Subsequently, 50 μL of bacterial

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suspension (approximately 6 log CFU/mL) was added to each well. The microplate was loosely

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wrapped with cling wrap to prevent evaporation. Each plate included controls without honey. The system was incubated at 37 °C for 24 h. The MIC values were assessed by visual readings and

2.9. Statistical analysis

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confirmed as the lowest concentrations capable of inhibiting visible bacterial growth.

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All assays were performed in three independent experiments in triplicate, and the results are expressed as the averages ± standard deviations (SD) of the assays. For the phenolic and flavonoid

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contents as well as for antioxidant and antimicrobial assays, statistical analyses were performed to determine the significant differences (p ≤ 0.05) by using a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey's test. Pearson’s linear correlation coefficients (r) between selected parameters were also calculated in bivariate linear correlations. For MIC determination assays, the results are expressed as modal values because the MIC values were the same in all repetitions. Sigma Stat 3.5 computer software (Jandel Scientific Software, San Jose, California) was used for the statistical analysis of the data.

3. Results and Discussion

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3.1. Total phenolic (TPC) and flavonoid contents (TFC) The TPC in the assessed honey samples varied from 31.5 ± 2.1 to 126.6 ± 2.7 mg GAE/100 g

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with differences (p ≤ 0.05) among samples from distinct floral sources (Table 2). The honeys from

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juazeiro produced by both studied bee species presented the highest (p ≤ 0.05) TPC (JJ: 126.6 ± 2.7

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and JU: 61.1 ± 3.7 mg GAE/100 g), and the lowest (p ≤ 0.05) TPC values were observed in honeys from malícia (31.5 ± 2.1 and 34.2 ± 1.5 mg GAE/100 g). The average TPC for all analyzed honeys was 56.32 ± 28.5 mg GAE/100 g.

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A previous study reported the highest TPC (120 mg GAE/100 g) in honey produced by

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jandaíra from Mimosa caesalpiniifloia Benth (Silva et al., 2013b), a botanical species that is typical of the Brazilian semiarid region. The highest TPC (240 and 148.29 mg GAE/100 g) was also observed for heterofloral honeys produced by Meliponini in Paraguay and Argentina (Vit,

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Gutiérrez, Rodríguez-Malaver, Aguilera, Fernández-Díaz, & Tricio, 2009). The TFC in the studied honeys varied from 4.2 ± 0.6 to 1.9 ± 0.1 mg QE/100 g and differed

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(p ≤ 0.05) between honeys produced by the same bee species using different botanical sources (Table 2). Honeys from juazeiro and malícia showed the highest (p ≤ 0.05) TFC (approximately 4.2

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± 0.2 mg QE/100 g), and honeys from velame branco presented the lowest TFC (p ≤ 0.05) (JVB: 2.4 ± 0.1 mg QE/100 g and UVB: 1.9 ± 0.1 mg QE/100 g). The average TFC for all the analyzed honeys was 2.97 ± 0.9 mg QE/100 g. The TFC of the honeys analyzed in our study was higher than those reported for honeys produced by Apis mellifera from Citrus spp. (0.17 mg QE/100 g) and Robinia pseudoacacia L. (acácia) (1.75 mg QE/100 g) (Lianda, Sant’Ana, Echevarria, & Castro, 2012) and for honeys from Tilia platyphyllos (lime) (0.95 mg QE/100 g) (Can, Yildiz, Sahin, Turumtay, Silici, & Kolayli, 2015). The influence of the floral source on the TPC and TFC of honeys from the same geographic region was already reported (Alvarez-Suarez et al. 2010; Silva et al., 2013a; Silva et al., 2013b). Researchers have stated that the environmental and climatic conditions where the plants grow

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define their metabolism and directly impact the nectar composition that is offered to the bee for honey production (Al-Mamary, Al-Meeri & Al-Habori, 2002; Perna, Simonetti & Gambacorta,

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2013; Flores et al., 2015). In particular, regions characterized by a hot and humid climate with very

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high exposure to sunlight (as in northeastern Brazil) are known to exert a marked influence on the

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polyphenolic content of plants (Tenore, Ritieni, Campiglia & Novellino 2012). Sun-exposed plants (such as juazeiro) can contain much more total phenolics than the same varieties or other when

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3.2. DPPH-free radical-scavenging assay

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grown in the shade (Tenore et al., 2012).

DPPH is a stable, nitrogen centered radical, and it has been widely used to test the free radical-scavenging ability of various substances or compounds (Habib et al., 2014). The reduction

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capability of DPPH was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. The scavenging activity of DPPH radicals (RSA%) varied from 11.2 ± 1.3% to

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46.9 ± 1.9%. The honeys from juazeiro and velame branco that were produced by jandaíra species showed the greatest (p ≤ 0.05) RSA%. According to the Pearson’s correlation, these results were

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influenced by the TPC (r = 0.8662; p ≤ 0.01) and by the antioxidant activity of the DPPH radicals in mgs of QEAC/100 g (r = 0.9976) and DPPH mg in AEAC/100 g (r = 0.9525) (Table 3). Previous studies on honeys produced by A. mellifera showed a positive correlation between the TPC and RSA% (Escuredo, Miguez, Fernandez-Gonzalez, & Seijo, 2013; Meda et al., 2005). However, the RSA% of a sample cannot be predicted only on the basis of its TPC and TFC, as observed in the present study (Table 2). In honeys, the antioxidant capacity could be a result of the combined activity of a wide range of compounds found therein, including phenolics, peptides, organic acids, enzymes, minerals, Maillard reaction products and possibly other minor components (Escuredo et al., 2013; Sant'Ana et al., 2012; Gheldof Wang & Engeseth, 2002).

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3.3. ABTS•+ free radical-scavenging assay The ABTS method measures the ability of compounds to capture the 2,2'-azinobis (3-

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ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) radical, which can be generated in chemical and

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enzymatic reactions (Kuskoski, Asuero, Troncoso, Filho, & Fett, 2005). There was a significant (p

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≤ 0.05) variation in the scavenger activity of ABTS•+ free radicals among the tested honey samples. Similar to the findings observed in the DPPH assays, the honeys from juazeiro and velame branco showed the highest (p ≤ 0.05) ABTS•+-scavenging activities (JJ: 81.6 ± 4.5; UJ: 46.5 ± 5.9 and

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JVB: 35.3 ± 1.9; UVB: 39.6 ± 1.0 μmol TE/100 g). The lowest (p ≤ 0.05) scavenging activity for the ABTS•+ radical (JA: 24.8 ± 0.9 and UA: 23.2 ± 2.9 μmol TE/100 g) was observed for malícia

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honeys (p ≤ 0.05). A previous study detected stronger and weaker ABTS•+-scavenging activities for honeys from Fagopyrum esculentum (buckwheat) (66.35 μmol TE/100 g) and Robinia

Fortuna & Witczak, 2011).

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pseudoacacia (acácia) (31.61 μmol TE/100 g), respectively (Socha, Juszczak, Pietrzyk, Gałkowska,

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The honeys tested here presented higher inhibition potentials against the ABTS•+ radical (up to four-fold) than against DPPH (Table 2). These results could be related to the specific action

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monitored in the ABTS•+ assay, which indicate the antioxidant activity of compounds with both hydrophilic and lipophilic characteristics (Meda et al., 2005; Kuskoski et al., 2005). In general, the antioxidant properties of different honey samples depend on the structures of their phenolic compounds, as a consequence of the ability of these compounds to donate the hydrogen ion or electrons for the free radicals (Gašic´ et al., 2014). The positive correlations (Table 3) between the TPC and the ABTS•+ radical inhibition capacity (r = 0.9853, p ≤ 0.01) and between the ABTS•+ radical inhibition capability and the TFC (r = 0.8228, p ≤ 0.05) indicate that phenolics and flavonoids are the primary factors responsible for the antioxidant properties of the studied honeys. Consequently, these results reinforce the influence of the botanical source on honey antioxidant properties (Piljac-Žegarac, Stipčević & Belščak, 2009; Lianda et al. 2012).

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3.4. ORAC

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The ORAC method consists of measuring the decrease in fluorescence of a protein that

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results from the loss of its conformation when it suffers oxidative damage caused by a source of

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peroxyl radicals (ROO•) (Zulueta, Esteve, & Frigola, (2009). The ORAC radical scavenging effect of the honey samples varied from 8.9 ± 0.1 to 54.3 ± 0.3 μmol TE/100 g GAE/100 g, with differences (p ≤ 0.05) among honeys from distinct bee species and floral sources. For this assay,

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honeys from malícia showed antioxidant potential that was up to 4-fold higher than the other tested

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honeys (Table 2). Significant correlations (p ≤ 0.01) were found between the ORAC and RSA (r = 0.9363), PCT (r = 0.9667), FCT (r = 0.8957), DPPH (r = 0.9347) and ABTS (r = 0.9851) (Table 3). The potential advantages (and differences) of the ORAC assay compared with the other

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methods include the use of peroxyl radicals as reactants, with a redox potential and mechanism of reaction (i.e., hydrogen atom vs. electron transfer) similar to those of physiological oxidants, and

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the use of a physiological pH. Thus, the antioxidants react with an overall charge and protonation state which are similar to those found in the human body (Bisby, Brooke & Navaratnam, 2008;

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Gorjanovic de et al., 2013). In general, compounds with more hydroxyl substitutions possess stronger anti-peroxyl radical activities. Particularly in flavonoids, the di-OH substitution at C-3' and C-4' plays an important role in the peroxyl radical-absorbing activity (Cao, Sofic, & Prior, 1997). The structure-bioactivity relation could explain the highest antioxidant properties presented by malícia honeys in ORAC assay, considering that these honeys showed the lowest TPC among those produced by the same bee species. Malícia honeys presented the highest quantities of the flavonoids kaempferol, quercetin and myricetin (Table 4), which possess hydroxyl group substituents on C-3' and C-4'. When similar total phenolic contents occur, they do not necessarily correspond to the same antioxidant responses. The response of different phenolic compounds in

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Folin-Ciocalteu assay also depends on their particular chemical structures (Atoui, Mansouri, Boskou, & Kefalas, 2005).

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When considering the activity results of the DPPH, ABTS•+ and ORAC assays, discordance

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among the antioxidant potentials was also observed for monofloral honeys from acacia, lime and

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singing bean produced by A. mellifera (Meda et al., 2005; Alvarez-Suarez et al., 2010; Socha et al., 2011). This variation is attributed to the profile of phenolic compounds in each honey. Different chemical structures possess distinct capabilities to capture the assayed radicals, particularly with

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respect to the number and position of their hydroxyl groups (Atoui et al., 2005). At the present time,

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no single available assay for testing the antioxidant capacity provides all the desired information. An evaluation of the overall antioxidant capacity may require multiple assays to generate an “antioxidant profile” encompassing reactivity towards both aqueous (DPPH and ABTS) and

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lipid/organic radicals (ORAC) directly via radical quenching and radical-reducing mechanisms (DPPH; ABTS; FRAP and ORAC) and indirectly via metal complexing (FRAP) (Alvarez-Suarez et

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3.5 Phenolic profile

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al., 2012).

The results obtained for the polyphenolic composition of honey samples are presented in Table 4. The identity of most of the phenolic compounds in the analyzed honey samples were positively confirmed by comparing them with standards. However, the other compounds present in samples with similar phenolic spectra and chromatographic behavior could not be identified because of the lack of availability of standard compounds. Flavonoids and benzoic and cinnamic acids are the most common phenolic compounds found in honeys produced by A. mellifera (Alvarez-Suarez et al., 2012; Sabatier Amiot, Tacchini & Aubert, 1992). In the Meliponini, the flavonoids reported as prevalent are naringenin, quercetin and isorhamnetin along with gallic, vanillic, 3,4-dihydroxybenzoic and cumaric acid (Silva et al., 2013). However, to the best of our

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knowledge, only a few studies are available on the profile of phenolic compounds of Meliponini honeys from heterofloral sources (Vit et al., 2009; Silva et al., 2013a; Silva et al., 2013b), and no

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studies assessing these characteristics in Meliponini honeys from monofloral sources are available.

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The presence of the phenolic compounds in the tested monofloral honeys was determined

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using fourteen reference compounds generally detected in honey samples. The HPLC-DAD analyses of the studied honeys revealed the influence of the floral source on their polyphenolic profile. Generally, the HPLC chromatograms of the phenolic acids and flavonoids indicated that the

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honeys from the same floral source had similar phenolic profiles, with the exception of honey from

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juazeiro. All phenolic acids, particularly p-coumaric, ellagic, and 3,4-hydroxybenzoic acid and the flavonoids rutin, catechin, chrysin and naringenin were detected in higher amounts in juazeiro honeys in comparison with the other honeys produced by the same bee species (Fig. 2), suggesting a

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particular profile for juazeiro honeys. An early study of honeys produced in arid regions with climatic conditions similar to those found in northeastern Brazil showed a high quantity of rutin in

(Habib et al., 2014).

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honeys from Z. spina-christi, suggesting that it is a marker in honeys from the Ziziphus species

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Observing the Table 4, in malícia honeys, the phenolic acid detected in higher quantities was 3,4-hydroxybenzoic acid, along with the flavonoids quercetin, miricetin and kaempferol. Interestingly, a previous study on the pollen loads of jandaira honey that contained 98.9% Mimosa gemmulata L. pollen indicated the presence of naringenin. This finding suggested that naringenin may be specific to honeys from the Mimosa species (Silva et al., 2006). In the present study, two honeys from the same genera, namely malícia (Mimosa quadrivalvis L.) and jurema branca (Mimosa arenosa L.), showed variations in their naringenin contents in honeys that were produced by both bee species. Additionally, naringenin was present in all the honey samples, suggesting that it is not a marker of Mimosa honeys, as previously proposed.

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3.6. Antimicrobial activity The antimicrobial activity of polyphenolic compounds (phenolic acids and flavonoids) is

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well known. We have chosen to assess the antimicrobial properties of the entire honey (when

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diluted in growth media) because total honey may be more beneficial than its isolated constituents.

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The individual bioactive components in honeys can exhibit changes in their antibacterial effects in the presence of other compounds, establishing a synergistic effect (Silici, et al., 2010). The target bacterial strains exhibited different sensitivities for the different tested monofloral

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honey samples, as measured by the determination of MIC. Honey from juazeiro and velame branco, which were produced by both bee species, displayed the lowest MICs (honeys from juazeiro: <7.5 –

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10 mL/100 mL; honeys from velame branco: 10 – 20 mL/100 mL). The highest MICs were displayed by honeys from malícia (> 20 g/100 mL against all test strains) (Table 5). In general, a

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ranking of the floral source of honeys in terms of antimicrobial activity is ordered as follows: juazeiro > velame branco > jurema branca > malícia. The samples that showed the strongest

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antimicrobial activities also presented the highest TPC. It is known that antibacterial activity of honeys depends on many factors. Some studies have

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reported antimicrobial activity in monofloral or multifloral honeys produced by A. mellifera (Elbanna, et al., 2014) or Meliponini (Silva et al., 2013a) and different methods have been used to determine this activity. Some researchers state that the H2O2 produced by the action of glucose oxidase or the non-peroxide activity is the primary aspect related to the antimicrobial activity of honeys (Elbanna et al., 2014). The effects of the non-peroxide activity as part of the antimicrobial properties of honeys are more pronounced in ripe honeys (relative to that of the present study), in which only a small amount of peroxide (which is not sufficient to inhibit bacterial growth) is found (Bizerra, Silva & Hayashi, 2012). The antibacterial activity of honeys has consistently been found to be related to the polyphenols found therein (Silici, et al., 2010; Brudzynski, Abubaker, St-Martin & Castle, 2011). The non-peroxide compounds could mostly be related to the antibacterial effects

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presented by the studied honey samples, because the samples with the highest phenolic contents presented the smallest MICs against the tested bacterial strains. Moreover, the presence of higher

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amounts of flavonoids such as rutin, cathecin and chrysin in honeys from juazeiro could also

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contribute to its stronger antimicrobial activity. Flavonoids are capable to interfere with the

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structural and functional properties of bacterial membranes, which in turn become increasingly permeable to protons, ultimately leading to the loss of cell integrity with leakage of cytoplasmic content (Kirnpal-Kaur et al., 2011).

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Considering the detected MICs, the sensitivity of the tested bacteria was observed to have

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the following order: Salmonella spp. > S. aureus > E. coli/P. aeruginosa > L. monocytogenes. These results are in accordance with the literature, which reports that different bacteria differ in their sensitivity to honeys and that Salmonella spp. was the most sensitive followed by S. aureus, L.

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monocytogenes and E. coli (Rodriguez et al., 2012), and S. aureus is more sensitive to honey than P. aeruginosa (Alvarez-Suarez et al., 2010). These results are noteworthy because Salmonella spp.

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is a gram-negative bacteria, and it has commonly presented higher resistance to antimicrobials when compared with gram-positive bacteria (e.g., S. aureus and L. monocytogenes) (Mazzarrino et al.,

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2015). Overall, the whole Meliponini honeys were capable to inhibit bacteria growth, and further studies could thus be performed to assess the contribution of their individual compounds to establish their inhibitory effects and their possible modes of action in bacterial cells.

4. Conclusions The honeys from juazeiro and velame branco that were produced by jandaíra and uruçu Meliponini bees showed the highest TPC and the highest antioxidant (when measured as a scavenger of DPPH and ABTS•+ radicals) and antibacterial (when measured by MIC determination) activities. Otherwise, honeys from malícia that were produced by both bee species studied showed the highest potential to capture oxygen radicals and the greatest quantities of

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quercetin, myricetin and kaempferol. This study describe for the first time relevant quantities of some phenolic acids and flavonoids in rare juazeiro and malicia honeys produced by Meliponini and

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characterize Meliponini honeys bioactivities related to floral source.

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Acknowledgments

The authors would like to thank to Coordination of Improvement of Higher Education Personnel (CAPES, Brazil) for the Science Without Borders scholarship awarded to the first author

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(Sousa, J.M.B.) and the National Council for Scientific and Technological Development (CNPq -

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Brazil) and the Foundation for Science and Technology (FCT - Portugal) by financial support. Moreover, the authors would like to thank the beekeepers for providing the honey samples.

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Figure Legend

Agreste region, state of Paraíba.

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Norte;

Seridó region, state of Rio Grande do

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honey samples and their respective geographic coordinates.

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Figure 1. Map of the semiarid region of northeastern Brazil showing the distribution of study

Figure 2. Chromatogram of the Ziziphus joazeiro L. (juazeiro) honey produced by jandaíra

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(Melipona subnitida); honey sample JJ; peaks identified 1: 2.4-dihydroxybenzoic acid; 2: 3.4-

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hydroxybenzoic acid; 3: ferulic acid; 4: ellagic acid,; 5: trans-cinnamic acid; 6: p-coumaric acid; 7: syringic acid; 8 myricetin; 9: quercetin; 10: catechin; 11: rutin; 12: kaempferol; 13: hesperetin; 14:

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naringenin; and 15: chrysin

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Figure 1.

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Figure 2.

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Table 1. Melissopalynological analysis of monofloral honeys produced by Meliponini species (jandaíra and uruçu) in the semiarid region of northeastern Brazil. Honey samples

Z.

M.

M.

joazeiro

quadrivalvis arenosa Kunth

Mart.

L.

willd Poir

Rhamnaceae

Z. joazeiro Mart.

PP

nd

nd

(88.7%)

Fabaceae

Spondias mombin

OP

nd

L.

(2.7%)

M. quadrivalvis L.

nd

PP (87.4%)

Mimosa tenuiflora

nd

OP (2.9%)

Fabaceae

M. arenosa willd

nd

nd

Poir IP

juliflora (Sw) DC

(6.1%)

Anadenanthera

nd

nd

nd

nd

nd

nd

nd

PP

nd

AP (5.4%)

(86.7%) OP (2.3%) nd

(88.4%)

nd

nd

nd

nd

nd

OP (1.8%)

nd

OP (2.9%) PP

nd

nd

(87.5%) nd

PII

nd

nd

nd

IIP

nd

nd

(5.6%)

PP (88.2%)

nd

nd

nd

PP (85.1%)

nd

OP

nd

nd

nd

nd

nd

PII (3.1%)

OP

nd

IP

IIP (4.0%)

nd

Croton campestris

Centratherum

nd

nd

Marsypianthes

nd

nd

(6.7%)

(1.3%) nd

nd

(2.7%) nd

punctatum Cass

Lamiaceae

nd

nd

nd

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Compositae

nd

Poir

nd

Croton

St. Hil

Kunth

nd

C. heliotropiifolius

PII (6.9%)

sonderianus Muell Euphorbiaceae

L.

willd

C.

nd

Kunth Euphorbiaceae

heliotropiifolius

Mart.

PP

M.

D

Prosopis

macrocarpa Benth. Euphorbiaceae

PP

joazeiro quadrivalvis arenosa

(89.5%)

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Fabaceae

nd

M.

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Fabaceae

nd

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Fabaceae

nd

nd

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Rhamnaceae

C.heliotropiifolius Z.

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Botanical origin

Family

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Botanical

Uruçu (M. scutellaris Latrelle)

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Jandaíra (M. Subnida Duke)

IP

(6.0%)

nd

nd

nd

nd

nd

nd

nd

nd

IP

nd

(7.4%) nd

nd

nd

chamaedrys

(4.66%)

Kuntze Turneraceae

Turnera subulata

nd

nd

nd

PII (4.3%)

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

IIP (3.8%)

OP

OP (2.8%)

OP

OP (2.6%)

OP

OP

OP

OP (1.7%)

(2.7)

(2.0%)

(1.9%)

100

100

100

L. Chrysobalanaceae

Licania rigida Benth Other

(2.5%) Total pollen (%)

100

(1.8%) 100

100

100

100

Data represent analyses of 500 pollen grains (n = 500) counted in twenty distinct optical areas in three different samples; standard deviation of means was ≤ 0.5. PP: predominant pollen (> 45%); AP: Accessory pollen (16-45%); IP: Isolated pollen (3-15%); OP: Occasional pollen (<3%); nd: not detected

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Table 2. Total phenolic and flavonoid contents and antioxidant activity (DPPH, ABTS and ORAC) of monofloral honeys produced by Meliponini species (jandaíra and uruçu) in the semiarid region of

Parameters

T

northeastern Brazil Meliponini/monofloral honeys

Juazeiro

Malícia

Velame

Jurema

Juazeiro

(JJ)

(JA)

branco

branca

(JVB)

(JJB)

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2.7

GAE/100 g)

0.6

DPPH (mg

23.6

AEAC/100 g)

1.0

DPPH (mg

10.8

QEAC/100 g)

0.5

ORAC (μmol

22.1

TE/100 g)

0.4

ABTS (μmol

81.6

TE/100 g)

4.5 46.9

±

4.1

±

±

±

11.0

4.6

±

±

33.7 2.7

Aa

±

24.8 0.9

Aa

±1.9

23.3 1.4

±

±

19.9

9.0

Ab

0.8 Ba

±

9.6

±

35.3

Bb

± 1.9

Cc

±

±

±

Cc

±

0.8 4.4

40.1

Ab

± 3.2

±

Cc

14.9

±

ACb

±

0.3 26.3

4.4

Aa

±

22.7

18.9

±

1.6 6.2

±

±

0.9 22.6

±

46.5

Ba

±

±

29.5 3.4

±

(UJB)

3.8

4.0

±

4.5

1.6

±

±

±

±

11.2

6.0

8.9

±

Dc

±

±

11.7 BCb ±

4.6

Bca

±

27.5

11.7Bc

±

0.5 Aa

±

1.0

0.5

±

0.1 Cd

39.6

±

2.1Bc

0.2 Ba

0.1

Cb

±

0.5 Bcb

0.4

54.3 ±0.3 23.2

4.3

37.5Cb 2.1

Ac

1.7 Db

Aa

1.3

1.9

ABa

0.1 Dc

±2.9 Ba

(UVB)

1.5

0.3 Cb

branca

54.0

Ab

Jurema

branco

34.2 ±

0.7 Ba

5.9 Cc

Cb

0.4 Ba

1.0 Abc

0.0

1.6

±

0.2

10.7

±

61.1

Ba

Malícia Velame (UA)

3.7

Ac

0.4

Cd

0.6

Cc

2.4 0.1

Ab

± 1.8 Cc

0.4 Cb

2.6

Bc

0.5

Cb

0.6 Cc

0.7 Aa

48.1

± 3.7 Aa

0.4 Aa

57.5

TE

4.2

.1 Aa

CE P

TFC (mg

± 31.54 ±2

Ab

NU

GAE/100 g)

Cd

(UJ)

MA

126.6

Aa

D

TPC (mg

RSA (%)

uruçu (M. scutellaris Latrelle)

IP

jandaíra (M. subnitida Duke)

26.7Cb

±

1.8 Bab

±

24.6BCab ± 0.4

Mean values of three analyses of three different samples ± standard deviations. TP C= total phenolic content; TFC=

AC

total flavonoids content; AEAC= ascorbic acid equivalent antioxidant content; QEAC= quercetin equivalent antioxidant content; RSA= radical scavenging activity. Different capital letters (A, B, C) in the same row indicate significant differences (p ≤ 0.05) between honeys from the same botanical source and honeys produced by distinct bee species according to the Tukey’s test. Different lowercase letters (a, b, c) in the same row indicate significant differences (p ≤ 0.05) between honeys from different floral sources and those produced by the same bee species according to the Tukey’s test.

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31

Table 3. Correlation matrix of antioxidant activity (ORAC, ABTS and DPPH), total phenolic and flavonoid contents and the RSA (%) of monofloral honeys produced by Meliponini species

FCT

RSA

(mg

(mg

(%)

GAE/100 g)

QEAC/100

ORAC

g) PCT (mg

1

AEAC/100 g)

g)

0.9377**

0.8957**

0.8228*

0.9178**

0.9403**

0.9363**

0.8917**

0.9976**

0.9525**

1

0.9851**

0.9347**

0.8941**

1

0.8930**

0.8671**

1

0.9705**

D

NU

MA

1

TE CE P AC

DPPH (mg

g)

1

00 g)

g)

AEAC/100

0.8313 **

(μmol/TE/1

QEAC/100

QEAC/100

0.8663**

ORAC

DPPH (mg

(mg

0.9853**

RSA (%)

00 g)

(mg

0.9667**

g)

(μmol/TE/1

DPPH

0.8662**

QEAC/100

ABTS•+

DPPH

0.7700*

GAE/100 g) FCT (mg

ABTS•+

IP

PCT

SC R

Variables

T

(jandaíra and uruçu) in the semiarid region of northeastern Brazil.

*significant at (p ≤ 0.05); **significant at (p ≤ 0.01)

1

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32

Table 4. Phenolic acids and flavonoids of monofloral honeys produced by Meliponini species

T

(jandaíra and uruçu) in the semiarid region of northeastern Brazil.

1.1 ± 0.0 4.2 ± 0.1

Catechin

14.9 ± 0.4 86.1 ± 0.2 4.9 ± 0.2

Kaempferol

AC

Quercetin

Rutin

0.9 ± 0.0

SC R

0.5 ± 0.0

3.8 ± 0.1 2.1 ± 0.1

1.4 ± 0.1

0.1 ± 0.0

0.2 ± 0.0

1.0 ± 0.0

1.9 ± 0.3

4.4 ± 0.3

MA

1.2 ± 0.2

10.0 ± 0.5 3.7 ± 0.1

5.0 ± 0.1

4.9 ± 0.1

25.7

22.3 ± 0.2 11.1 ± 0.1 24.3 ± 0.5 10.8 ± 0.2 32.7 ± 0.1 11.6 ± 0.2 138.3

NU

0.3 ± 0.1

2.4 ± 0.1

10.9

29.5

21.1 ± 0.4 23.3 ± 0.4 0.7 ± 0.1

4.0 ± 0.1

4.2 ± 0.2

1.8 ± 0.1

2.7 ± 0.1

1.7 ± 0.0

4.4 ± 0.1

1.5 ± 0.0

0.3 ± 0.0

5.3 ± 0.2

7.1 ± 0.1

3.2 ± 0.1

6.1 ± 0.5

3.8 ± 0.3

2.8 ± 0.1

10.5 ± 0.4 55.5 ± 0.5 4.7 ± 0.1

CE P

trans-cinnamic acid

Total contents (µg/1 g) Flavonoids Myricetin

3.6 ± 0.1 2.0 ± 0.1 9.8 ± 0.3 4.0 ± 0.2 2.5 ± 0.1 2.2 ± 0.1 27.1

1.0 ± 0.0

D

Ellagic acid

Syringic acid

24.0 ± 0.2 18.0 ± 0.1 41.0 ± 0.1 13.0 ± 0.2 45.0 ± 0.4 12.0 ± 0.2 183

TE

2,4dihydroxybenzoic acid 3,4-hydroxybenzoic acid Ferulic acid

p-coumaric acid

Jurema Branca

IP

Phenolic acids

Meliponini/monofloral honeys jandaíra (M. subnitida Duke) uruçu (M. scutellaris Latrelle) Juazeiro Malícia Velame Jurema Juazeiro Malícia Velame Branco Branca Branco 16.9 ± 25.5 ± 17.2 ± 30.0 ± 0.8 1.3 ± 0.2 3.5 ± 0.2 0.1 0.2 1.3 ± 0.2 0.1

Hesperetin

7.7 ± 0.2

0.3 ± 0.3

0.5 ± 0.0

0.3 ± 0.0

4.1 ± 0.1

Naringenin

8.7 ± 0.1

0.5 ± 0.0

13.0 ± 0.1 140.6

6.5 ± 0.2 0.3 ± 0.0

9.1 ± 0.1

Chrysin

0.3 ± 0.0 0.7 ± 0.0

0.5 ± 0.0

10.9 ± 0.2 101.1

1.0 ± 0.0 0.7 ± 0.0 1.5 ± 0.1 0.3 ± 0.0 1.4 ± 0.1 5.3 ± 0.2 27.4

18.0 ± 0.1 17.3 ± 0.2 1.2 ± 0.1 6.0 ± 0.1 5.8 ± 0.3 0.3 ± 0.0 0.2 ± 0.0 0.9 ± 0.1 49.7

0.4 ± 0.0 1.5 ± 0.2 1.1 ± 0.1 0.2 ± 0.0 0.9 ± 0.1 4.5 ± 0.2 9.92

3.9 ± 0.1 3.2 ± 0.2 0.6 ± 0.0 6.7 ± 0.2 3.6 ± 0.1 0.7 ± 0. 0.6 ± 0.0 0.5 ± 0.0

Total Contents 57.8 19.1 19.3 19.8 (µg/1 g) Mean values of three analyses of three different samples (µg/1 g of honey) ± standard deviations

3.0 ± 0.5

5.5 ± 0.1 2.0 ± 0.1 0.1 ± 0.0 3.5 ± 0.2 2.6 ± 0.1 0.3 ± 0.0 5.2 ± 0.0 0.7 ± 0.0 19.9

ACCEPTED MANUSCRIPT

33

T

Table 5. Minimum inhibitory concentration (MIC; mL/100 mL) of monofloral honeys produced by

IP

Meliponini (jandaíra and uruçu) in the semiarid region of northeastern Brazil against pathogenic

P. aeruginosa CINF3

JA

> 20

> 20

JVB

20

20

UJ UA

> 20

> 20

20

20

> 20

Salmonella sp. CINF2

L. monocytogenes 3375

7.5

20

> 20

> 20

20

20

20

20

> 20 > 20

> 20

10

10

20

> 20

> 20

> 20

MA

JJB

10

S. aureus 18N 7.5

NU

Target bacterial Honey strains samples * E. coli CINF1 JJ 10

SC R

bacterial strains.

> 20

UVB

20

AC

CE P

TE

D

20 20 10 20 UJB > 20 > 20 > 20 > 20 20 JJ: honeys from juazeiro produced by jandaíra bees; JA: honeys from malícia produced by jandaíra bees; JVB: honeys from velame branco produced by jandaíra bees; JJB: honeys from jurema branca produced by jandaíra bees; UJ: honeys from juazeiro produced by uruçu bees; UA: honeys from malícia produced by uruçu bees; UVB: honeys from velame branco produced by uruçu bees; and UJB: honeys from jurema branca produced by uruçu bee.

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Graphical abstract

34

ACCEPTED MANUSCRIPT

IP

T

Highlights

SC R

 Ziziphus joazeiro honeys presented the highest quantities of phenolic acids;  M. quadrivalvis honeys presented the strongest anti-peroxyl radical activity;

AC

CE P

TE

D

MA

NU

 Z. joazeiro and C. heliotropiifolius honeys showed high antibacterial activities

35