The scientific hypothesis of this study was to examine the differences and relations between free phenolic content and antioxidant capacity measured by two methods (DPPH with IC50, and ABTS tests) in three types of pseudocereals (amaranth, buckwheat, and quinoa), and also differences between samples themselves.

Three types of pseudocereals and their products (13 samples), bought from food markets of different origin, were analyzed. There were amaranth, buckwheat, and quinoa. Amaranth (4 samples of Indian, Hungarian, Czech and German origin; grains (AG), flour (AF), wholemeal flour (AWF) and particles (AP)); buckwheat (6 samples of Poland, Czech and Latvia origin; grains (BG1, BG2), flour (BF1, BF2), wholemeal flour (BWF) and groats (BGR)); and quinoa (3 samples of Peruvian and Bolivian origin; different types of grains (white QGW, red QGR, black QGB)).

For the determination of free phenolics content (FP) in the pseudocereals modified spectrophotometric method with Folin-Ciocalteau reagent (Vollmannová et al., 2013) was used.

The extracts of pseudocereal samples were prepared from 1 g of homogenized pseudocereal sample and 25 mL of methanol (80% (v/v); Penta Chemicals, CZ) with stirring in a shaker for 8 h at room temperature. The extract was afterward filtered through a paper filter and used for the analyses of FP.

To extracts (1 mL) with 5 mL of distilled water, Folin- Ciocalteau reagent (2.5 mL; Penta Chemicals, CZ), was added and after agitation, it was left for 3 min in the dark at room temperature. Then sodium carbonate solution (7.5 mL, 20% (w/v); Penta Chemicals, CZ) was added to a mixture and mixed again. The content was then filled up to 50 mL. After 2 h of the extract standing in the dark at room temperature the absorbance of samples was measured at wavelength 765 nm (Libra S6 Biochrom spectrometer, GB) against blank. As a standard a gallic acid was used. FP values were expressed as gallic acid equivalents (GAE), mg.100g^-1 sample. Determinations were made in triplicate.

Antioxidant activity of pseudocereals was assessed as a total antioxidant capacity (TAC). It was evaluated by a modified spectrometric method with DPPH reagent (Vollmannová et al., 2013) and also with ABTS reagent (Škrovánková et al., 2018).

For both determinations there was used the same extraction process for analyzed samples. 1 g of homogenized pseudocereal sample was mixed with 25 mL methanol (80% (v/v); Penta Chemicals, CZ) with stirring in a shaker for 8 h at room temperature. The extract was afterward filtered through a paper filter and used for the analyses.

DPPH method : To pseudocereal extract (0.1 mL) a DPPH (1,1-diphenyl-2-picrylhydrazyl) solution in methanol (3.9 mL; 1 mM; Sigma Aldrich, CZ) was added. The mixture was shaken vigorously on a Vortex mixer in capped glass and left in the dark for 10 min. (room temperature). The absorbance of samples (A) and absorbance of control samples (AC) was measured on the spectrometer (Libra S6 Biochrom, GB) at λ = 515 nm against a blank. The pseudocereal inactivations (I) were calculated from the decrease of absorbance (%) according to relation (1) and the results were then expressed, using the calibration curve of standard (trolox), as trolox equivalents (TE) in mg.100g^-1 sample. Average results were obtained from three parallel determinations.

(1) I = A C − A A C ⋅ 100

IC50 method : For the determination of 50% antioxidant inactivation, to scavenge 50% of DPPH free radicals, the most effective pseudocereal type, buckwheat, was used. For IC50 method there were prepared five diluted methanolic buckwheat extract solutions for each sample, in the range 1 – 10 mg.mL^-1. The reaction mixtures were made the same way as for TAC (DPPH) determination. From the results of inactivation for these extract concentrations the IC50 values were quantified by linear regression.

ABTS method : To 50 μL of pseudocereal extracts 4 mL of the reactive radical mixture composed of ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; Sigma Aldrich, CZ) (12 mL; 3.5 mM) with K₂S₂O₈ (0.06 M; Lukes, CZ) and acetic buffer (pH 4.3), was added. The solution was shaken vigorously on a Vortex mixer and left to react without light exposure for 30 min at room temperature. Pseudocereal samples absorbance (A) and absorbance of control samples (AC) were then measured by a spectrometer (Libra S6 Biochrom, GB) at wavelength 734 nm against a blank. Inactivations (I) were calculated from the decrease of absorbance (%) according to relation (1). Results of TAC (ABTS) were calculated from inactivation using a calibration curve with trolox as a standard. It was expressed as trolox equivalents (TE) in the mg.100g^-1 sample. Average results were obtained from three parallel determinations.

All data were expressed as mean values ± standard deviation (SD), every determination was made in triplicate. Statistical analysis of the results was made by Statistica program, StatSoft version 9.0 (Dell, USA) using parametric test comparing mean values of two independent assortments (Student t-test). Differences at a 95% confidence level (p <0.05) were considered statistically significant. Correlations between the evaluated parameters were obtained using Pearson’s correlation coefficient (r).

Antioxidants that could donate electrons, such as polyphenols, vitamin C (ascorbic acid), and vitamin E (tocopherols), were evaluated by the method with Folin- Ciocalteau reagent.

As Li et al. (2013) found out predominant polyphenols in pseudocereals, such as buckwheat, are free phenolics (FP), accounted for about 94.07% of whole polyphenol content.

The amounts of FP in the pseudocereal samples in our study (Table 1) ranged from 12.4 to 678.1 mg GAE.100g^-1 with the average 208 mg GAE.100g^-1 pseudocereal sample. There were marked differences between individual pseudocereals (p <0.05, Student t-test).

The highest content of FP was analyzed for the buckwheat products; quinoa and amaranth products reached much lower values. These findings are in agreement with Alvarez-Jubete et al. (2010) results for pseudocereal seeds determination. Also Vollmannová et al. (2013) and Gorinstein et al. (2008) detected for pseudocereals this order of polyphenols amount.

The average FP value for buckwheat seeds and products in our research was 357 mg GAE.100g^-1, that is up to 3 times higher than in quinoa (average 141 mg GAE.100g^-1) and up to 10 times than in amaranth (average 34 mg GAE.100g^-1) seeds and their products. The highest amount was analyzed for buckwheat wholemeal flour followed by other types of buckwheat flours.

In Alvarez-Jubete et al. (2010) study polyphenol amount for buckwheat seeds was 323 mg GAE.100g^-1, for amaranth 21.2 and for quinoa it was 71.7 mg GAE.100g^-1. Buckwheat values of our determination were similar also to the contents of Salehi et al. (2018) study that were in the range from 265 to 430 mg of caffeic acid equivalents per 100g.

In a study of Gorinstein et al. (2007), the total phenol content in amaranth and quinoa reached similar values (40.5 – 43 and 91.2 mg GAE.100g^-1, respectively) as in our study, for buckwheat it was much less, 60 mg GAE.100g^-1. Vollmannová et al. (2013), however, found in seeds of selected pseudocereals a higher amount of phenolics than evaluated in our study for buckwheat; quinoa, and amaranth seeds and their products.

To evaluate the antioxidant potential, the antioxidant activity of all selected pseudocereal seeds and their products was measured. Two methods, DPPH and ABTS tests were used. The results of total antioxidant capacity (TAC) are summarized in Table 1.

The TAC values for the DPPH method were in the range from 25 to 473.9 of trolox equivalents per 100 grams of pseudocereal sample with an average 166 mg TE.100g^-1. Results of ABTS test were from 118.2 to 3524.8 mg TE.100g^-1, the average 1280 mg TE.100g^-1. For the results of both methods there were marked differences between pseudocereals (p <0.05, Student t-test), similarly as for free phenols.

From the evaluation of the results it can be seen that the pseudocereal type with higher antioxidant values is buckwheat, followed by quinoa and amaranth. It is the same order as for FP values. These findings are in agreement with the values previously reported by Alvarez- Jubete et al. (2010) for pseudocereal seeds. The studies of Gorinstein et al. (2007), Gorinstein et al. (2008) and Vollmannová et al. (2013) introduced for pseudocereals the same order for antioxidant capacity results.

Buckwheat samples for DPPH and ABTS test in our study reached 2 – 19 and 2 – 30 times higher values than in quinoa samples (average 92 and 860 mg TE.100g^-1, respectively) and 2 – 6 and up to 5 times, respectively, than in amaranth (average 41 and 263 mg TE.100g^-1) seeds and their products. As for particular samples, the best product with the highest antioxidant activity, measured by both methods, was buckwheat wholemeal flour followed by other types of buckwheat flours and buckwheat grain (BG1).

A similar trend of buckwheat samples is shown for IC50 values in Figure 1. IC50 expresses the concentration of buckwheat extracts requisite for 50% inhibition, therefore the highest antioxidant capacity is showed by the lowest IC50 value. IC50 concentrations of buckwheat extracts were in the range 2.391 – 6.520 mg.mL^-1, the average 4.218 mg.mL^-1. Wholemeal flour (BWF) had the lowest IC50, nearly 3 times lower than the sample with the highest IC50 value (buckwheat grain BG2). So wholemeal flour has the highest antioxidant activity what could be seen also from DPPH and ABTS tests.

Alvarez-Jubete et al. (2010) reported analogous order of antioxidant capacity for pseudocereal seeds, determined by DPPH assay, the value of 620 mg TE.100g^-1 for buckwheat, 57.7 for quinoa, and 28.4 mg TE.100g^-1 for amaranth, respectively. In the study of Salehi et al. (2018) TAC results (DPPH test) in buckwheat seed samples varied from 268 to 628 mg TE.100g^-1. Zielińska et al. (2012) determined TAC in buckwheat seeds 215 mg TE.100g^-1.

Despite some variations in exact values of antioxidant potential in our research and other studies, they are comparable, and buckwheat could be reported as the greatest source of polyphenols with the highest antioxidant activity amongst pseudocereals and also some other cereals too (Gorinstein et al., 2007; Gorinstein et al., 2008; Gallardo, Jiménez and García-Conesa, 2006; Zieliński and Kozlowska, 2000).

Pseudocereal samples exhibited similar order of samples for FP and TAC values (DPPH and ABTS assays). The relationships between them are shown by the correlations in Figure 2. They are strongly related to a correlation factors r = 0.9666 and 0.9565, respectively. In the research of Sun and Ho (2005) there was also found a significant correlation (0.96) between polyphenols content and antioxidant activity (DPPH method), in buckwheat extract. In amaranth and quinoa extracts there were reported weak correlations between polyphenols content and antioxidant activity by Nsimba, Kikuzaki and Konishi (2008) study.