Application of micro-TLC to the total antioxidant potential (TAP) measurementby Bronisław K. Głód, Paweł M. Wantusiak, Paweł Piszcz, Elwira Lewczuk, Paweł K. Zarzycki

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Accepted Manuscript

Application of micro-TLC to the total antioxidant potential (TAP) measurement

Paweł K. Zarzycki

PII: S0308-8146(14)01620-3


Reference: FOCH 16585

To appear in: Food Chemistry

Received Date: 12 February 2013

Revised Date: 2 October 2014

Accepted Date: 10 October 2014 micro-TLC to the total antioxidant potential (TAP) measurement, Food Chemistry (2014), doi: 10.1016/j.foodchem.2014.10.058

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Bronisław K. Głód, Paweł M. Wantusiak, Paweł Piszcz, Elwira Lewczuk,

Please cite this article as: Głód, B.K., Wantusiak, P.M., Piszcz, P., Lewczuk, E., Zarzycki, P.K., Application of 1

Application of micro-TLC to the total antioxidant potential (TAP) 2 measurement 3 4

Bronisław K. Głód a,*, Paweł M. Wantusiak a, Paweł Piszcz a, Elwira Lewczuk a, 5

Paweł K. Zarzycki b 6 7 aDepartment of Analytical Chemistry, Institute of Chemistry, Faculty of Science, Siedlce 8

University of Natural Sciences and Humanities, 3 Maja 54, 08-110 Siedlce, Poland 9 bSection of Toxicology and Bioanalytics, Faculty of Civil Engineering, Environmental and 10

Geodetic Sciences, Koszalin University of Technology, Śniadeckich 2, 75-453 Koszalin, 11

Poland 12 13 14 15 * Corresponding author. Tel.: +48 25 6431040; fax: +48 25 6431130. 16 e-mail address: (B.K. Głód) 17 2


DPPH• is commonly applied for estimation of antioxidant capacity of single and complex 19 biological samples, and changes color from purple to yellow during reduction to DPPH-H. 20

Importantly, for some samples, for example colored foods, such reaction cannot be used 21 because of interference from pigments. Therefore, the number of reported quantitative 22 protocols involving off- or on-line sample reaction with DPPH• are based on chromatographic 23 separation of target components. In typical planar chromatographic assay, developed plates 24 are sprayed with DPPH• solution for antioxidant screening. Such approach enables simple 25 visualization of separated spots exhibiting antioxidant activities, but unfortunately, such 26 procedure may also give the misleading signal for colored spots. In the present 27 communication we examined a new approach for measuring antioxidant capacity using 28 quantitative analysis of DPPH• and DPPH-H molecules after reaction with the sample, and 29 then separated from the interfering compounds by micro-thin-layer chromatography. 30

Particularly, the antioxidant capacities of colored food samples (such as herbs and meads) 31 were determined and the results compared with those obtained using the classical photometric 32 assay. The main advantages of the new micro-TLC assay are (i) low cost, (ii) multiple 33 measurements, (iii) short analysis time, (iv) simplification of sample preparation and (v) 34 effective separation of DPPH• signal from interfering compounds. 35 36 37

KEYWORDS: total antioxidant potential, micro-TLC, DPPH•, mead, herbs 38 39 40 41 42 3 1. INTRODUCTION 43

The interest in food natural antioxidants and their positive impact on the human body is 44 constantly increasing. This is because people attach more and more importance to what is 45 consumed and how food quality affects their bodies and health. Natural antioxidants are 46 present in large quantities in fruits, vegetables, red wine, green tea (including white), honey 47 and meads (Tosun & Ustun, 2003; Benzie & Szeto, 1999; Campanella, Bonanni, Favero & 48

Tomassetti, 2003; Głód, Piszcz, Czajka & Zarzycki, 2012; Głód, Piszcz, Czajka & Zarzycki, 49 2011). They have an important role in protecting living organisms from the harmful effects of 50 free radicals. The main task of the antioxidant is defense of aerobic organisms from the 51 harmful effects of free radicals and/or reactive oxygen species and support endogenous 52 antioxidants, and may prevent premature aging of the organism by antioxidants. Today's 53 society lives in a chronic oxidative stress caused by poor eating, habits, addictions (mainly 54 tobacco and alcohol abuse) and increasing environmental pollution. 55

A variety of fresh and processed food products consumed daily contain high levels of 56 antioxidants. For example, there is increasing interest of antioxidant fingerprinting of green 57 tea and red wine (Lambert & Elias, 2010; Porgali & Büyüktuncel, 2012). The traditional 58

Polish meads and herbal meads are characterized by antioxidant properties and health 59 benefits. However, the influence of herbs on the antioxidant properties of honeys is still 60 unknown (Wantusiak, Piszcz, Skwarek & Głód, 2011). 61

Strong antioxidant properties of herbs are assigned mainly due to plant dyes, 62 particularly polyphenols including chlorophylls, flavonoids, carotenoids, betainines and 63 antioxidant vitamins (Wojdyło, Oszmiański & Czemerys, 2007). Mead is an alcoholic 64 beverage with low alcohol content (9-18 %), obtained by the fermentation of wort (honey 65 diluted with water) (Sroka & Tuszyński, 2007; Wantusiak, Piszcz, Skwarek & Głód, 2011). 66

Honey wort may also contain additional ingredients such as hops (hop mead), spices (ginger, 67 4 cloves, cinnamon) and herbs or fruits (Juszczak, Socha, Różnowski, Fortuna & Nalepka, 68 2009; Socha, Juszczak, Pietrzyk & Fortuna, 2009). These additives affect the smell and color 69 of the mead. They may also change their antioxidant properties. 70

Photometric measurement of total antioxidant potential (TAP) can be performed using 71 purple alcoholic solution of DPPH• (2,2-diphenyl-1-picrylhydrazyl) acting as the oxidant. The 72 sample (containing antioxidants) changes color by reducing DPPH• to yellow DPPH-H (2,2-73 diphenyl-1-picrylhydrazine). Absorbance of the resulting solution is measured 74 photometrically at λmax = 517 nm (Sharma & Bhat, 2009; Locatelli, Gindro, Travaglia, 75