**1. Introduction**

Development of products which positively affect the consumer health is an important aspect followed by the food industry. This assignment may be realized when process of the industry is concentrated on the natural antioxidants. Antioxidants present in food can protect against lipid and protein autoxidation. It is very important to quantify the antioxidant properties of different types of food. In addition, a special attention is devoted to the processing methods to maintain the beneficial antioxidant properties of food [1].

Presently, wheat flour is widely used in bread making; however, other types of flour are also used. Rye and spelt types of flour are preferred due to the content of micro- and macronutrients and fibre [2, 3]. Recently, the potential usage of buckwheat flour as a functional component in food has been demonstrated [4]. Buckwheat (*Fagopyrum esculentum* Moench), commonly, cultivated in Russia and China, is added to the other cereal grains because of likenesses in usage [5]. Buckwheat is a rich source of nutrients (lysine, vitamins B, carbohydrates) [6] and antioxidants such as vitamin E, glutathione, phytic acid [7], phenolic acids and flavonoids mainly rutin, with anti-inflammatory, anti-carcinogenic and anti-glycation properties [4, 8, 9]. Buckwheat polyphenols can function as antioxidants in one or more possible ways: as reducing agents, as compounds that scavenge free radicals, as chelating agents of metals that catalyse oxidation reactions and thus limiting their ability to initiate free radical chain reactions or by inhibiting oxidative enzymes such lipoxygenases [10–13]. However, processing conditions may considerably affect biological activity of polyphenols. There are many studies regarding negative effects of thermal processing on the phenolic compounds including flavonoids. The type of heat transfer and processing conditions are the major factors responsible for the observed decrease in the flavonoid content in food [7, 14–16]. Having all these evidences, buckwheat mill products seem to be an attractive ingredient in the bakery industry [17–20]. The recent evidences have shown that the intake of bread with addition of buckwheat flour in the recipe resulted in a positive increase of antioxidant potential in humans [17]. This finding, due to the quality properties of buckwheat bread as described by Lin et al. [21], can make it favourable for developing a healthy diet. Recently, a number of food products containing buckwheat has been investigated such as buckwheat-enhanced ginger nutty cakes [18], buckwheat enriched wheat bread [17, 20, 21] and buckwheat cakes [18, 19, 22]. Therefore, due to the nutritional value and beneficial effects on human health, buckwheat and partially buckwheat-based products form a pool of potential functional food [4, 10].

For the overall characterization of a new food product, more often the antioxidative capacity is used. There is a variety of analytical methods to assess the antioxidant capacity of food. However, there is no single standard method that would be used to determine the antioxidant capacity of the complex matrix and give consistent, unquestionable results in confrontation with other analytical methods. Therefore, it is advisable to use more than one method. A part of analytical methods is based on scavenging of non-natural free radicals, other deal with lipid peroxidation chemical markers. These methods need a little preparation, small amounts of reagents and are quick [13].

Many antioxidant capacity approaches have been suggested to assess antioxidant properties of food products and to clarify their relationships with antioxidants. Among them, ABTS, DPPH, reducing power assay and metal chelating activity are used for the assessment of antioxidant capacity of food [1, 23–25]. The ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6 sulfonic acid) is a spectrophotometric method based on inhibition of green colour in the presence of an antioxidant. DPPH (2,2-diphenyl-1-picrylhydrazyl) is based on the assumption that antioxidants are the hydrogen donors. This spectrophotometric method uses the DPPH radical, which changes from purple to yellow in the attendance of antioxidant compounds. However, ABTS is soluble in water and in alcoholic solutions, but DPPH is soluble only in organic solvents. The photochemiluminescence (PCL) method is based on the scavenging activity against the superoxide anion radical. The chelation of Fe(II) ions may cause significant antioxidative effects by delaying metal-catalysed oxidation [26, 27]. The reducing power assay involves the formation of coloured complexes, in the presence of antioxidants, with potassium ferricyanide, trichloroacetic acid and ferric chloride. The increase of absorbance of the reaction mixture is related to the reducing power of the samples. Currently, a mixture of the methods should be used to evaluate the antioxidant capacity of food *in vitro* to cover all aspects of antioxidant effectiveness [1, 12, 25]. Therefore, the aim of this study was to characterize the dark wheat bread with exogenous buckwheat addition as a source of antioxidative activity for humans.
