Colour , antioxidant capacity , phenolic and flavonoid content of honey from the Humid Chaco Region , Argentina Color , capacidad antioxidante , contenido de fenoles y flavonoides en mieles de la Región del Chaco Húmedo , Argentina

Resumen. Nuestro objetivo fue correlacionar el color con los componentes fenólicos totales, flavonoides totales y capacidad antioxidante de mieles de la región del Chaco Húmedo. Con este propósito, se seleccionaron 19 muestras representativas durante el período 2009-2012. El análisis del polen mostró una predominancia de especies nativas provenientes de diferentes paisajes como bosques y humedales. Del número total de muestras, once fueron mixtas o multiflorales y ocho muestras estuvieron dominadas por diferentes especies de leñosas nativas nectaríferas. El color de la miel, de acuerdo a la escala Pfund, varió desde ámbar extra claro a ámbar oscuro. El contenido fenólico total varió desde 40,18 a 118,82 mg GAE/100 g de miel. El contenido de flavonoides varió entre 6,94 y 67,76 mg QE/100 g de miel, y la capacidad antioxidante, evaluada por el método del radical DPPH, entre 17,36% y 56,53%. Las mieles color ámbar extra claro y ámbar claro tuvieron menores niveles de compuestos fenólicos totales, flavonoides totales, y capacidad antioxidante que las mieles color ámbar y oscuras. La correlación observada entre color y flavonoides (r=0,78) fue más alta que aquella entre color y fenoles totales (r=0,53). Se observó una relación directa entre el contenido de fenoles y la capacidad antioxidante (r=0,91). Las mieles estudiadas, comparadas con las mieles de otras regiones, fueron ricas en flavonoides y mostraron una notable capacidad antioxidante. Es importante tener en cuenta estas características para la salud humana.


INTRODUCTION
Honey produced by Apis mellifera L. bees exhibits a characteristic colour, aroma and texture according to the plant nectar from which it derives and to the specific environmental conditions.Colour is a sensory attribute of particular importance in the typification of honey and together with consistency influences consumer preference (Aubert & Gonnet, 1983).Honey colour is the first trait taken into account when grading honey for commercial purposes, and therefore it reflects on the price of bulk honey (Gallez, 2007).Raw honey colour depends on the botanical and geographical origin of the product (Terrab et al., 2003a,b).Some authors reported a relationship between honey colour and mineral composition (Balbarrey et al., 2012;González Miret et al., 2005).
From the chemical point of view honey, is a concentrated solution of reducing sugars and lesser amounts of other carbohydrates, proteins, amino acids, organic acids, enzymes, vitamins, and minerals.It has also been found to contain pigments such as carotenoids, volatile components, and polyphenols (White & Doner, 1980;Crane, 1990).Phenolic compounds are directly involved in the colour of honey, mainly through flavonoids (Amiot et al., 1989).Plant nectar is a source of phenolic compounds.The type and concentration of phenolic compounds are the main determinants of the bioactive properties of honey (Mărghitaş et al., 2009).These compounds are considered to reduce the risk of oxidative damage in cells and numerous studies show their role as antioxidants (Aljadi & Kamaruddin, 2004;Baltrusăityté et al., 2007;Bertoncelj et al., 2007).Therefore, honey phenolic compounds confer to this product possible protective effects against various diseases such as heart disease, cancer, atherosclerosis, infection, and inflammation (Martinez-Florez et al., 2002;Jaganathan & Mandal, 2009).
There are very few studies characterizing the honey from the Humid Chaco region in terms of its physico-chemical attributes (Ciappini et al., 2009;Cabrera et al., 2011;Salgado & Maidana, 2014).This region shows a great diversity of native species of melliferous interest and a low volume of honey production (Cabrera et al., 2013).
The aim of the present study was to establish the relationships among colour, antioxidant capacity, and phenolic and flavonoid content for contributing to the characterization of honeys from the native flora of the Humid Chaco region.

Physico-chemical analysis
Colour.The honey samples were liquefied and centrifuged for 5 min at 3000 rpm to eliminate bubbles.Six measurements for each sample were performed on a Pfund colour grader (Koehler Instrument Company Inc.New York) and a corrected average obtained.The honey colour was expressed in mm Pfund and named in accordance with the standard nomenclature.
Determination of total phenolic content.Total phenolic content was determined according to the method described by Marghitaş et al. (2009).Each honey sample was dissolved in water to obtain a 10% solution.Five hundred microlitres of the aqueous solution were mixed with 2.5 mL of Folin-Ciocalteu reagent (0.2 N), and after 5 minutes two ml of a solution of Na 2 CO 3 (75 g/L) were added.All samples were kept at room temperature for 2 h, and their absorbance was measured at 760 nm in a Metrolab RC 325 spectrophotometer.The amount of total phenolic compounds was determined by a calibration curve, using dilutions of a stock solution of gallic acid (0.25 mg/mL) in methanol (70%).The linearity obtained was R 2 =0.993.The results were expressed in mg gallic acid equivalents (GAE)/100 g honey, as the mean of three replications.
Determination of total flavonoid content.The total flavonoid content of honey samples was determined by the method described by Marghitaş et al. (2009).One millilitre of a honey aqueous solution (0.6 mg/mL) was mixed with 0.3 mL of NaNO 2 solution (5%), and after 5 minutes 0.3 mL of AlCl 3 solution (10%) were added.After 6 min, the samples were neutralized with 2 mL of NaOH (1 M) and the absorbance was read at 510 nm in a Metrolab RC 325 spectrophotometer.Different concentrations of quercetin in ethanol (80%) were used for the calibration curve.The linearity obtained was R 2 = 0.9981.The results were expressed in mg quercetin equivalents (QE)/100 g honey, as the mean of three replications.
Antioxidant capacity.The scavenging activity of honey for the radical 2.2-diphenyl-1-picrylhydrazyl (DPPH radical) was used to evaluate the antioxidant capacity of samples, according to the method of Meda et al. (2005) with some modifications.DPPH solutions have an intense violet colour and show a strong absorption band at 517 nm.The method is based on a reduction of colour solution in presence of antioxidant compounds; thus, the less the absorbance at 517 nm, the greater the antioxidant capacity of the sample.The honey samples were diluted with distilled water (25 mg/mL), and 0.75 mL of honey solution was mixed with 1.5 mL of DPPH methanolic solution (0.02 mg/ mL).In parallel, a control solution with 0.75 mL of methanol and 1.5 mL of DPPH was prepared.To eliminate the interference of colour of the honey solution, a blank with 0.75 mL of the honey solution and 1.5 mL of methanol was measured.The mixtures were shaken vigorously and kept in the dark for 15 minutes at room temperature.Their absorbances were measured at 517 nm in a Metrolab RC 325 spectrophotometer.The antioxidant capacity of honey samples was calculated as: where A Control is the absorbance of the control (containing all reagents except the honey solution), A Sample is the absorbance of the honey solution with DPPH, and A Blanc sample is the absorbance of the honey solution with methanol.

Statistical analysis.
The obtained data were subjected to an analysis of variance (ANOVA), measurement comparison, and regression analysis.The comparison between two sample groups was performed using the Student's t-test.Multivariate techniques such as the Principal Component Analysis (PCA) using the 2014 version of the InfoStat software were also carried out in order to summarize the information.Bivariate linear analysis was carried out and correlation coefficients (r) are shown.1).The pollinic description presented in the table refers only to the main native and naturalized species identified in each honey sample.

Pollen
Colour.The average colour of the honey samples was 93.32 ± 30.62 mm Pfund and ranged from extra light amber to dark amber.The lightest sample scored 40.67 mm Pfund and the darkest one was the only one that surpassed 140 mm (Table 1).In the particular case of the darkest honey, in order to get a score to correlate with the other parameters, the scale was extended and the value obtained was 150.50 mm (sample 983).Six samples were identified as light (extra light amber-ELA and light amber-LA), seven as amber (A), and six as dark (D).
Total phenolic content, total flavonoid content and antioxidant capacity.Phenolic compounds or polyphenols constitute one of the most numerous and widely distributed groups of substances in the plant kingdom.Notably, flavonoids and simple phenolic derivatives were the most common polyphenols.
Table 2 shows the results of total phenols, antioxidant capacity and total flavonoids of the analysed honey.Total phenolic content, total flavonoids content, and antioxidant capacity of the honey samples from the Humid Chaco region, grouped by colour are shown in Figure 2. The first group involved ELA and LA ranges.This group showed 40.18 and 47.31 mg GAE/100g honey as minimum and maximum values for phenolic compounds respectively, whereas flavonoids ranged from 6.94 to14.39 mg QE/100g and antioxidant capacity from 17.36 to 27.09%.Within the group named A, the range of phenolic compounds was 46.35-118.82mg GAE/100g, that of flavonoids 9.38-37.47mg QE/100g and that of antioxidant capacity 24.76-60.95%.The third group, named D showed the following ranges: phenolic compounds 52.06-79.06mg GAE/100g, flavonoids 15.95-67.76mg QE/100g and antioxidant capacity 23.43-56.53%.In all three variables, values were significantly higher (t ≤ 0.05) for the honeys above 85 mm Pfund (group ELA-LA) in comparison to honeys below 85 mm Pfund (groups A and D).
Correlations.Plots of the first two components clearly indicated that darker honeys had higher flavonoid content than lighter honeys, and that antioxidant capacity was strictly related to total phenolic content (Fig. 3).Bivariate linear analysis showed a high association between colour and flavonoid content (r=0.78) and between antioxidant capacity and phenolic content (r=0.91).Colour was less correlated with total phenols (r=0.53) and with antioxidant capacity (r=0.51)than with flavonoids.The correlation coefficients between flavonoid content and antioxidant capacity (r=0.45;P<0.01) and that between flavonoid and total phenolic (r=0.48;P<0.01) were lower than those mentioned above.The darkest sample, atypically high in flavonoid content, reduced both coefficients.
Table 1.Colour and pollen types of honey samples from the Humid Chaco region.Percentage of pollen type present in the sample is shown in brackets.

Fig. 1 .
Fig. 1.Geographical location of the sampling area.The dots on the map show the apiaries from where the honey samples were taken.

Table 2 .
Mean, Range and Standard Deviation (SD) for the Total phenolics, Antioxidant capacity and Total flavonoids.Medias, rango, desvío estandar (DS) de Fenoles totales, Capacidad antioxidante y Flavonoides totales.Colour and antioxidant capacity of honey from Chaco