Analytical possibilities for the relative estimation of the antioxidative capacity of honey varieties harvested in different regions of Serbia J. Serb. Chem. Soc. 81 (5) 567–574 (2016) UDC 638.16+543:615.279–188(497.11) JSCS–4868 Short communication 567 SHORT COMMUNICATION Analytical possibilities for the relative estimation of the antioxidative capacity of honey varieties harvested in different regions of Serbia UROŠ M. GAŠIĆ1#, DALIBOR M. STANKOVIĆ2, DRAGANA Č. DABIĆ2#, DUŠANKA M. MILOJKOVIĆ-OPSENICA1#, MAJA M. NATIĆ1#, ŽIVOSLAV Lj. TEŠIĆ1# and JELENA J. MUTIĆ1* 1Faculty of Chemistry, University of Belgrade, P. O. Box 51, 11158 Belgrade, Serbia and 2Innovative Center, Faculty of Chemistry, Studentski trg 12–16, 11158 Belgrade, Serbia (Received 13 March, revised 15 December, accepted 25 December 2015) Abstract: Two different approaches, spectroscopic and electrochemical, were applied for the rough determination of the antioxidative capacity of honey samples. Honey samples of diverse botanical origin were collected in different geographical regions of Serbia. The total phenolic content (TPC) was deter- mined by the Folin–Ciocalteu method. Cyclic voltammograms on a glassy car- bon electrode in KCl supporting electrolyte were used to check the electrode sensitivity to the presence of honey. In order to calculate the Trolox equivalent antioxidant capacity (TEAC) of the studied honey samples, cyclic voltammo- grams were recorded for the Trolox standard. The results were expressed as µmol of Trolox equivalents per kg of sample (µmol TE kg-1). Good correl- ations were observed between the cyclic voltammetry data and the TPC deter- mined by the Folin–Ciocalteu method and the radical scavenging activity (RSA) determined using the DPPH·(1,1-diphenyl-2-picrylhydrazyl) radical test. Cyclic voltammetry appears to be a highly attractive alternative method for a rapid estimation of the antioxidative capacity of honeys. It was found that polyfloral honey samples had the highest, whereas acacia honey showed the lowest values of TPC. Keywords: antioxidant activity; Folin–Ciocalteu method; radical scavenging activity. INTRODUCTION Generally, there is a growing interest on the efficiency of natural antioxi- dants in food. Polyphenols, i.e., flavonoids and phenolic acids, are considered as * Corresponding author. E-mail: jmutic@chem.bg.ac.rs # Serbian Chemical Society member. doi: 10.2298/JSC150313009G 568 GAŠIĆ et al. one of the important groups of components for antioxidant activity identified in honey. The antioxidant activity of honey is closely related to the floral source of the honey. Generally, honeys are classified as monofloral (produced from one plant species) and polyfloral (several plant sources). Different honey types were subjected to antioxidant activity tests and they demonstrated significant potential, comparable to those of the other foodstuff.1 On the other hand, research on the antioxidant capacity of honey samples originating from Serbia is scarce. Avail- able literature indicates that there have only been a few reports to date on the determination of both total phenolic content and antioxidant activity of Serbian honeys by spectroscopic methods2–4 and an electrochemical (polarographic) method.5 Cyclic voltammetry was shown to be sensitive, convenient, rapid and low-cost approach in the quality evaluation of food products beneficial for human health.6,7 Studies on the evaluation of the antioxidant activity of different food products using electrochemical methods were reported.8–11 Due to growing interest in the quality of Serbian honey samples and continuing research on these nutritionally important products, the antioxidant potentials of honey samples of diverse botanical origin are communicated in this paper. The samples were obtained and selected directly by the beekeepers from different parts of the territory of Serbia. Two different approaches, spectroscopic and electrochemical, were applied to check rapidly the antioxidative capacity of the selected honey samples. The total phenolic content was determined by the well-established spec- trophotometric technique using the Folin–Ciocalteu method. The radical scav- enging activity of the honey samples was determined using the 2,2-diphenyl-1- -picrylhydrazyl radical (DPPH•). Cyclic voltammetry was used to check the elec- trochemical response of glassy carbon in the presence of the honey samples. To inspect the applicability of cyclic voltammetry for the rather rapid and prelim- inary determinations, the electrochemically determined results were correlated with the results obtained by spectrophotometric methods already established in the literature for these purposes. EXPERIMENTAL Chemicals and materials Methanol (HPLC grade), sodium carbonate, potassium chloride, hydrochloric acid, Folin–Ciocalteu reagent, and filter paper (Whatman No. 1) were purchased from Merck (Darmstadt, Germany). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Sigma–Aldrich (Steinheim, Germany). 2,2-Diphenyl-1-picrylhyd- razyl·(DPPH•) was purchased from Fluka (Buch, Switzerland). Ultrapure water (Thermo- Fisher TKA MicroPure water purification system, 0.055 µS cm-1) was used to prepare the standard solutions, blanks, and artificial honey analogue (30 % glucose, 40 % fructose, 10 % sucrose and 20 % water). An analogue of honey was made to check whether the main sugars in honey could interfere with the proposed electrochemical assay. Sugar standards (glucose, fructose and sucrose) were purchased from Tokyo Chemical Industry (TCI, Europe, Belgium). DETERMINATION OF ANTIOXIDATIVE CAPACITY OF HONEYS 569 Syringe filters (13 mm, PTFE membrane, 0.45 µm) were purchased from Supelco (Bellefonte, PA, USA). Ethanol (96 vol. %) was from J. T. Baker (Deventer, The Netherlands). Honey samples A total of 27 honey samples collected from different regions of Serbia (Fig. S-1 of the Supplementary material to this Communication) during the 2009 harvesting season were selected and provided by the Association of the Beekeeping Organizations of Serbia (SPOS, www.spos.info). The botanical origins of the samples were specified by the SPOS based on the information provided by beekeepers and sensory characteristics, and confirmed by phys- icochemical analyses and chemometrics.12,13 The honey samples were from: acacia (Robinia pseudoacacia), sunflower (Helianthus annuus), lime (Tilia cordata), giant goldenrod (Soli- dago virgaurea), basil (Ocimum basilicum), oilseed rape (Brassica napus), buckwheat (Fago- pyrum esculentum) and polyfloral meadow honey. The honeys were stored at room tempe- rature in the dark before analysis. Determination of total phenolic content and radical scavenging activity Samples were prepared according to the slightly modified method proposed by Meda et al.14 Each honey sample (5 g) was mixed with 15 mL ultrapure water, homogenized in ultrasonic bath for 15 min at room temperature, transferred to a 50 mL volumetric flask, and filled to the mark with ultrapure water. The solution was then filtered through 0.45 µm PTFE membrane and subjected to the determination of the total phenolic content (TPC) and radical scavenging activity (RSA). The TPC was spectrophotometrically determined by the Folin– –Ciocalteu method reported by Singleton and Rossi,15 with some modifications. Briefly, 0.3 mL of the sample solution and 6 mL deionized water were mixed with 0.5 mL of Folin– Ciocalteu reagent and solution was incubated for 6 min at room temperature. Then, 3 mL of 20 % sodium carbonate solution was added and after keeping the sample at 40 °C for 30 min, the absorbance was measured at 765 nm. Gallic acid was used as the standard, and the calibration curve of gallic acid was prepared in the concentration range between 50 and 250 mg L-1. A mixture of water and Folin–Ciocalteu reagent was used as the blank. The results are expressed as mg gallic acid equivalent (GAE) per kg of honey. The RSA of the honey samples was evaluated by a modified method of Li et al.16 An aliquot of 1.0 mL of sample solution was mixed with 3 mL of a methanolic solution of 1,1- diphenyl-2-picrylhydrazyl (DPPH, 71 mM). The mixture was left for 60 min in the dark (until stable absorption values were obtained). After that, the reduction of the DPPH• absorbance was measured by monitoring continuously the decrease in absorption at 515 nm. The RSA was calculated as a percentage of DPPH• discoloration using the equation: DPPH sample DPPH (%) 100 A A RSA A − = where ADPPH is the absorbance of a methanolic solution of the DPPH •, Asample is the absor- bance in the presence of a honey extract. The assays were performed in triplicate and the results are expressed as mean values. For cyclic voltammetry, honey samples, 1 g of each, were mixed with 20 mL 0.1 M KCl, homogenized in an ultrasonic bath for 10 min at room temperature, then filtered through 0.45 µm PTFE membranes and used for cyclic voltammetry measurements. Trolox was used as the standard. In order to achieve better similarity with the honey matrix, the Trolox standard was prepared and recorded in a solution of artificial honey. The solution of artificial honey was prepared in the same manner as were the honey samples (1 g of artificial honey in 20 mL of 570 GAŠIĆ et al. supporting electrolyte). The scan was taken in the potential range between –200 mV and 800 mV at a scan rate 100 mV s-1. Cyclic voltammograms were recorded for Trolox standard in the concentration range 10 to 100 µmol L-1 In this concentration range, a linear relationship between the response and concentration was obtained. Parameter Q was determined as the area under the oxidation voltammetric peak for the Trolox solutions. The obtained calibration curve, Q = f(concentration of Trolox) was linear with the correlation coefficient R2 = 0.998 and was used to calculate Trolox equivalent antioxidant capacity (TEAC) from the Q para- meter of the studied honeys. The results are expressed as µmol of Trolox equivalents per kg of sample (µmol TE kg-1). Instrumentation A UV/Vis spectrophotometer (GBC UV/Visible Cintra 6) was used for the absorbance measurements and spectra recording, using optical cuvettes of 1 cm optical path. Cyclic voltammograms were recorded on a CHI760B instrument (CH Instruments, Austin, TX, USA). The cell was equipped with a GC electrode, 3 mm in diameter (model CHI104), an auxiliary platinum electrode of a larger area (model CHI221, a cell top including a Pt wire counter electrode) and an Ag/AgCl reference electrode (model CHI111; all poten- tials in the paper are referred to Ag/AgCl). All measurements were performed at ambient temperature. Prior to each run, the surface of the glassy carbon electrode was freshly polished with 1.0, 0.3 and 0.05 μm alumina powder, rinsed with redistilled water and degreased in ethanol in an ultrasonic bath. Statistical analysis Data of all measurements were obtained in triplicate and are expressed as the mean values. Statistical analyses were performed by the NCSS software package.17 RESULTS AND DISCUSSION The results on the total phenolic content (TPC) and of the radical scavenging activiry (RSA) of the honey samples are presented in Table I, together with the Q parameter derived from cyclic voltammograms (CV) as Trolox equivalents. Different values for the CV charge correspond to oxidation of low-formal potential antioxidants and could be an indicator of the antioxidant potential of a sample.18 The highest values were registered for the polyfloral honey samples from South–West Serbia, whereas the lowest CV charge values were generally obtained for the acacia honey samples, as well as for honey from basil harvested in North Serbia. All honey samples were characterized with TPC values ranging between 127.76 mg (acacia – H1) to 887.18 mg (polyfloral – H22) of gallic acid per kg of honey, Table I. The average content of total phenolics was in a good agreement with the values given in the literature for honeys of the surrounding regions.19–21 Generally, polyfloral honey samples had the highest values of TPC, while acacia samples showed the lowest values. Such findings are consistent with literature data. Here, just as an example, we cite a paper published by Bertoncelj et al.22 who reported higher TPC values of polyfloral honeys in comparison to mono- floral honeys (lime and sunflower) is cited. As is visible from Table I, the results of RSA ranged from 1.86 % (acacia honey – H4) to 23.20 % (polyfloral honey – DETERMINATION OF ANTIOXIDATIVE CAPACITY OF HONEYS 571 H22). Among all monofloral honey samples, buckwheat was found to have the highest TPC and RSA values. This was also found in a study of different mono- floral honeys when buckwheat honey was reported to have the highest anti- oxidant activity.23 In general, the results indicated that samples from the Zlatibor region (H5, H20, H22, H23, H25 and H26) were characterized with high TCP and RSA values. It was observed that polyfloral honey samples originating from this region showed different physico-chemical properties than those from the rest of Serbia.24 TABLE I. Total phenolic content (TPC), radical scavenging activity (RSA), CV charge derived from cyclic voltammograms and Q parameter derived from CV of monofloral and polyfloral honey samples Sample Botanical origin TPC mg GAE kg-1 RSA % Q µC Q µmol TE kg-1 H1 Acacia 127.76 2.18 0.041 9.01 H2 279.20 2.93 0.074 16.26 H3 328.22 3.34 0.080 17.58 H4 281.35 1.86 0.084 18.46 H5 368.69 2.51 0.110 24.18 H6 Sunflower 362.42 5.04 0.112 24.62 H7 465.16 9.65 0.168 36.92 H8 246.99 5.95 0.080 17.58 H9 451.19 10.64 0.124 27.25 H10 Lime 320.84 4.04 0.100 21.98 H11 373.49 4.95 0.123 27.03 H12 474.19 6.43 0.090 19.78 H13 483.03 10.84 0.170 37.36 H14 Giant goldenrod 467.11 6.69 0.110 24.18 H15 414.99 5.72 0.120 26.37 H16 Basil 379.63 8.76 0.070 15.39 H17 395.46 4.33 0.130 28.57 H18 Oilseed rape 513.64 13.51 0.180 39.56 H19 372.47 9.45 0.110 24.18 H20 Buckwheat 668.58 14.44 0.210 46.15 H21 Polyfloral 496.40 5.82 0.125 27.47 H22 887.18 23.20 0.300 65.93 H23 782.16 13.81 0.280 61.54 H24 540.63 7.74 0.170 37.36 H25 432.29 5.89 0.140 30.77 H26 631.85 11.86 0.230 50.55 H27 688.81 18.67 0.220 48.35 The data from Table I show that TPC, RSA and Q are in acceptable agree- ment and bring similar information about the relative antioxidant capacity of the investigated honey samples. The correlation matrix for these variables, presented in Table II, shows large positive correlations between all the values. The Q para- 572 GAŠIĆ et al. meter derived from the CV charge was strongly correlated with the TPC, with a correlation coefficient of 0.946. The level of significance for each correlation was p < 0.000001. Such statistically significant correlations clearly indicate that CV is a potentially applicable fast and informative experimental tool for the relative estimation of the antioxidant capacities of honeys. TABLE II. Correlation coefficients between TPC, RSA and Q TPC RSA Q TPC 1 RSA 0.879 1 Q 0.946 0.859 1 CONCLUSION Serbian honey samples from different botanical origins and geographical regions were studied for their relative antioxidant capacity. Two methods, spec- troscopic and electrochemical, were used for this purpose. Polyfloral honey samples had the highest values of TPC, while acacia samples showed the lowest values. Cyclic voltammetry was shown to be a highly attractive alternative method for the rapid relative checking of the antioxidant capacity of honeys. The linear dependence between this method and the commonly used Folin–Ciocalteu method was high with r = 0.946. The TPC values were compared with the anti- oxidant activities of the honey samples and a good correlation was obtained. Such a simple electrochemical technique could be considered as a valuable method for quality control, not only of honey but also for plant-derived food pro- ducts in general. SUPPLEMENTARY MATERIAL A map of the geographical regions of Serbia from where the 27 honey samples under study were collected is available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. This work was performed within the framework of the research projects Nos. 172017 and 172030, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. DETERMINATION OF ANTIOXIDATIVE CAPACITY OF HONEYS 573 И З В О Д AНАЛИТИЧКЕ МОГУЋНОСТИ ЗА РЕЛАТИВНУ ПРОЦЕНУ АНТИОКСИДАТИВНОГ КАПАЦИТЕТА РАЗЛИЧИТИХ СОРТИ МЕДА ПРИКУПЉЕНИХ ИЗ РАЗЛИЧИТИХ РЕГИОНА СРБИЈЕ УРОШ М. ГАШИЋ1, ДАЛИБОР М. СТАНКОВИЋ2, ДРАГАНА Ч. ДАБИЋ2, ДУШАНКА М. МИЛОЈКОВИЋ-ОПСЕНИЦА1, МАЈА М. НАТИЋ1, ЖИВОСЛАВ Љ. ТЕШИЋ1 и ЈЕЛЕНА Ј. МУТИЋ1 1Хемијски факултет Универзитета у Београду, Студентски трг 12–16, п. пр. 158, 11000 Београд и 2Иновациони центар Хемијског факултета Универзитета у Београду, Студентски трг 12–16, п. пр. 158, 11000 Београд Помоћу два различита приступа, спектроскопског и електрохемијског, тестиране су могућности релативне процене антиоксидативног капацитета узорака меда. Узорци меда различитог ботаничког порекла прикупљени су из различитих географских реги- она Србије. Садржај укупних фенола (TPC) одређен је применом Folin–Ciocalteu методе. Циклична волтаметрија (CV) са електродом од стакластог угљеника коришћена је као индикатор антиоксидативног потенцијала кроз промену волтаметријског наелектрисања у присуству узорака меда. Ради израчунавања Тролокс еквивалентa антиоксидативног капацитета (TEAC) испитиваног меда, снимани су и циклични волтамограми са Тролокс стандардом. Резултати су изражени као μmol Тролокс еквивалента по kg узорка (μmol TE kg-1). Показано је да су резултати цикличне волтаметрије у доброј корелацији са резултатима који се добијају применом Folin–Ciocalteu реагенса, као и са антиокси- дативним потенцијалом (RSA) који је одређен употребом 1,1-дифенил-2-пикрилхид- разил (DPPH•) радикалa. Резултати указују на то да је циклична волтаметрија ефикасна метода и да може бити алтернативна метода за брзу релативну процену антиоксида- тивног капацитета меда. Нађено је да највеће вредности TPC имају полифлорни узорци меда, док најмање вредности показује багремов мед. (Примљено 13. марта, ревидирано 15. децембра, прихваћено 25. децембра 2015) REFERENCES 1. D. D. Schramm, M. Karim, H. R. Schrader, R. R. Holt, M. Cardetti, C. L. Keen, J. Agr. Food Chem. 51 (2003) 1732 2. U. Gašić, S. Kečkeš, D. Dabić, J. Trifković, D. Milojković-Opsenica, M. Natić, Ž. Tešić, Food Chem. 145 (2014) 599 3. V. T. Tumbas, J. J. Vulić, J. M. Čanadanović-Brunet, S. M. Đilas, G. S. Ćetković, S. Stajčić, D. I. Štajner, B. M. Popović, Acta periodica technol. 43 (2012) 293 4. S. M. Savatović, D. J. Dimitrijević, S. M. Đilas, J. M. Čanadanović-Brunet, G. S. Ćetković, V. T. Tumbas, D. I. Štajner, Acta periodica technol. 42 (2011) 145 5. S. Ž. Gorjanović, J. M. Alvarez-Suarez, M. M. Novaković, F. T. Pastor, L. Pezo, M. Battino, D. Ž. Sužnjević, J. Food Compos. Anal. 30 (2013) 13 6. V. Roginsky, E. A. Lissi, Food Chem. 92 (2005) 235 7. S. Chevion, M. A. Roberts, M. 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Soc. 78 (2013) 1875. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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