POLYPHENOLS AND ANTIOXIDANT ACTIVITY IN PSEUDOCEREALS AND THEIR PRODUCTS

Pseudocereals are important as gluten-free crops that could be utilized as functional foods. They contain proteins with high biological value and also bioactive compounds such as phenolic compounds, flavonoids, vitamins, and minerals that can possess positive health effects on the body. Three types of pseudocereals (amaranth, buckwheat, and quinoa) were evaluated for polyphenols and antioxidant activity. Spectrophotometric methods were used for the determination of free phenols amount with Folin-Ciocalteu reagent, and total antioxidant capacity (TAC) with DPPH and ABTS reagents. Free phenols, the predominant part of polyphenols, were in pseudocereals in the range from 12.4 to 678.1 mg GAE.100g-1. The highest content of FP was found in buckwheat products (146.8 – 678.1 mg GAE.100g-1); quinoa and amaranth products reached much lower values (up to 226.1 mg GAE.100g-1). Antioxidant activity was in an agreement with the FP amounts order, the highest TAC values were again for buckwheat products (167.3 – 473.9 and 876.9 – 3524.8 mg TE.100g-1), followed by quinoa (78.2 – 100.6 and 738.9 – 984.5 mg TE.100g-1) and amaranth ones (25.0 – 69.7 and 118.2 – 431.4 mg TE.100g-1). A high positive correlation between FP amount and TAC values was evaluated for analyzed pseudocereals. The highest content of free phenols and the best antioxidant potential showed buckwheat wholemeal flour, so buckwheat could be characterized as a great source of free phenols with high antioxidant activity.


INTRODUCTION
Pseudocereals are important gluten-free crops where belong especially amaranth, buckwheat, and quinoa. Their great nutrient properties and also suitability for the preparation of gluten-free foodstuffs (Alvarez-Jubete et al., 2010) predestinate them for the utilization as functional foods. They are known to have good nutritional value, specifically because of proteins with high biological value. It is due to the presence of essential amino acids (especially lysine and tryptophan) in a higher content (Kocková and Valík, 2011). Due to their starch content they are also sources of energy. They contain also natural antioxidants, high levels of flavonoids (e.g. rutin, hyperoside, vitexin, isovitexin, orientin, isoorientin, catechin and epicatechin gallate in buckwheat), vitamins and minerals (Salehi et al., 2018;Tomotake et al., 2007;Kiprovski et al., 2015).
Amaranth (Amaranthus spp.) is a rich source of proteins, with well-balanced amino acid composition and good bioavailability. It has higher lysine content than other cereal grains (López et al., 2019; Tovar-Pérez, Lugo-Radillo and Aguilera-Aguirre, 2019). Amaranth is known also due to some potential health benefits (decreasing plasma cholesterol levels, reducing blood glucose levels and anemia) that have been conducted in experimental animal models (Caselato-Sousa and Amaya-Farfán, 2012). Its seeds contain a good amount of polyphenols such as flavonoids with quite high antioxidant activity (Vollmannová et al., 2013). To the important phenolics, there belong caffeic acid, ferulic acid and p-hydroxybenzoic acid (Klimczak, Małecka and Pachołek, 2002).
One of the most important pseudocereal sources for functional foods is common buckwheat (Fagopyrum esculentum Moench). To the functional substances in buckwheat belong flavonoids, phytosterols, fagopyrins, fagopyritols, phenolic compounds, resistant starch, dietary fiber, lignans, vitamins, minerals and antioxidants (Ahmed et al., 2014). Middling and bran buckwheat flours could be used to develop functional foods due to phenolic compounds presence. Phenolics are present there in the free and bound form. They are concentrated mainly in the outer layer (hull and bran) as the hull is removed before the milling of the buckwheat (Martín-García et al., 2019). The study of Li et al. (2013) showed that rather than buckwheat flours, hulls and brans are a better source of antioxidants. The health-promoting properties of buckwheat are expressed due to the content of antioxidants such as phenolic acids, rutin (quercetin-3-rutinoside), and fagopyrin, and specific proteins (Ölschläger et al., 2008;Sytar et al., 2016). To the other health benefits belong, similarly as for amaranth, plasma cholesterol level reduction, antidiabetic properties, and also antiinflammatory effect and improvement of hypertension conditions (Giménez-Bastida and Zieliński, 2015).
Quinoa (Chenopodium quinoa Willd.) is a plant of the Chenopodiaceae family. It is also a gluten-free crop that is suitable for coeliac patients because it contains very little or no prolamin (Jancurová, Minarovičová and Dandár, 2009). It is exceptionally high in lysine that is not overly abundant in the vegetable kingdom. Quinoa seeds contain also phytohormones that have a good impact on human nutrition (Vega-Gálvez et al., 2010).
Processing of crops (procedures, extraction methods, used temperature, type of present compounds) can modify the polyphenol content of foods in several ways This study aimed to assess differences in antioxidants of pseudocereals, concretely amaranth, buckwheat and quinoa, by comparison of free phenols content and total antioxidant capacity.

Scientific hypothesis
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.

Determination of Free Phenolic Content
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.

Determination of Antioxidant Activity
Antioxidant activity of pseudocereals was assessed as a total antioxidant capacity (TAC). It was evaluated by a modified spectrometric method with DPPH reagent 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) 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.

Statistical analysis
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).

Content of phenolics
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 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  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.

Antioxidant activity
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 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

CONCLUSION
Pseudocereals contain bioactive compounds such as phenolic compounds, flavonoids that can possess positive health effects on the body.
Amaranth, buckwheat, and quinoa were evaluated by spectrophotometric methods for the determination of free phenols amount and total antioxidant capacity. Free phenols in pseudocereals were in the range from 12.4 to 678.1 mg GAE.100g -1 . The highest contents of FP were found in buckwheat products; quinoa and amaranth products reached much lower values (up to 226.1 mg GAE.100g -1 ). Evaluated antioxidant activity, the highest TAC values were determined again for buckwheat products (up to 473.9 (DPPH test) and 3524.8 (ABTS test) mg TE.100g -1 ), followed by quinoa (up to 100.6 and 984.5 mg TE.100g -1 , respectively) and amaranth ones (up to 69.7 and 431.4 mg TE.100g -1 , respectively). Antioxidant capacity values by two evaluation methods (DPPH, ABTS) are in agreement with polyphenols content order. The highest content of free phenols, and also the best antioxidant potential, showed buckwheat wholemeal flour. Our study is generally in agreement with the findings of previously reported researches focused on pseudocereals. Buckwheat therefore could be characterized as a great source of free phenols with high antioxidant activity and thus could be used as seed for production of high nutritional quality products, especially for people who do not could eat cereal products due to the gluten presence. Buckwheat seeds could be also added to other cereal products to heighten their nutritional quality. Toledo, F., Katrich, E., Trakhtenberg, S. 2007