*3.2. Antioxidant Activity*

The antioxidant activity of amaranth samples of each growth stage was determined as radical scavenging activity (TEAC, PCL-ACL, and DPPH assay) and ability to reduce Fe3<sup>+</sup> to Fe2<sup>+</sup> (FRAP). Additionally, Fe2<sup>+</sup> chelating ability of extracts was determined because phenolic compounds form stable complexes with ferrous ions, and in this way, decrease the extent of free ions to Fenton's reaction in which highly reactive •OH are generated [31]. The results of antioxidant activity are presented in Table 2 and Figures 1 and 2. The significantly higher TEAC was recorded for amaranth in vegetative, early flowering and grain fill stages as opposed to in shooting and budding stages (*p* < 0.05), both when results were expressed on the basis of extract and fresh matter of plant. The FRAP ranged from 469 to 830 μmol Fe<sup>2</sup>+/g extract with the highest value observed for the early vegetative stage and the lowest, again, for the shooting and budding stages of the plant. The FRAP expressed on plant fresh matter basis was less varied between amaranth growth stages (17.4–24.6 μmol Fe2+/g). The PCL-ACL ranged from 422 to 858 μmol TE/g extract and from 16.5 to 23.9 μmol TE/g FM, respectively. For both types of PCL-ACL expression, amaranth in vegetative stages had significantly higher activity compared to plants in subsequent morphological states (*p* < 0.05). In turn, the Fe2<sup>+</sup> chelating ability of extracts ranged from 16.1—19.9% for late vegetative and shooting stages to 37.5% for the medium vegetative stage (Figure 1). The changes of antiradical activity against DPPH• of amaranth extracts with increasing assay content as well as the EC50 values, are presented in Figure 2. The DPPH• scavenging activity expressed as EC50 showed significant differences between certain growth stages of amaranth (*p* < 0.05). The highest antiradical activity was obtained for extracts from plants in the early flowering and grain fill stages with the lowest EC50 value. The highest EC50 values (294–317 μg/mL), which did not differ statistically from each other (*p* ≥ 0.05), were found for late vegetative, shooting, and budding stages.

To the best of our knowledge, the changes of antioxidant activity of amaranth aerial parts during the growth cycle have not been previously demonstrated, although the differences in the antioxidant potential of various morphological parts of the plant were shown [16,19,37]. In general, FRAP, DPPH•, or ABTS•<sup>+</sup> scavenging capacity decreased in this order: Leaves <sup>≥</sup> flowers >> stem <sup>&</sup>gt; seeds. Among some amaranth species, this relationship was demonstrated for *A. caudatus* [19]. It is known that environmental factors, such as light and temperature, also play an important role in amaranth antioxidant metabolism. Khandaker et al. [38] found that the antioxidant activity of leaves of red amaranth (*A. tricolor*) was higher under full sunlight intensity than under dark conditions. Modi [39] studied the effect of growth temperature on yield, nutritional value, and antioxidant activity of the leaves of five *Amaranthus* spp. (*A. hybridus* var. *cruentus*, *A. hypochondriacus*, *A. tricolor*, *A. thunbergii*, and *A. hybridus*) harvested at 20, 40, and 60 days after sowing and found significant differences between growth temperature and stage of development. Authors concluded that for greater nutritional benefit, *Amaranthus* should be grown under warm conditions and that younger leaves are preferable. The maximum antioxidant activity, independent of the temperature and species considered, was found in leaves after 60 days of sowing, corresponding to an intermediate stage between late vegetative and shooting found in our study.

**Figure 1.** Fe2<sup>+</sup> chelating ability of the amaranth extracts. EV, early vegetative; MV, medium vegetative; LV, late vegetative; S, shooting; B, budding; EF, early flowering; GF, grain fill. Different letters above bars indicate significant differences among means (*p* < 0.05).

**Figure 2.** DPPH• scavenging activity of the amaranth extracts. EV, early vegetative; MV, medium vegetative; LV, late vegetative; S, shooting; B, budding; EF, early flowering; GF, grain fill. Different letters above bars indicate significant differences among means (*p* < 0.05).


**Table 2.** Antioxidant activity of the amaranth extract and fresh matter (FM) in different growth stages.

TEAC, Trolox equivalent (TE) antioxidant capacity; FRAP, ferric-reducing antioxidant power; PCL-ACL, photochemiluminescence-antioxidant capacity of lipid-soluble substances. abcd Means with the different lowercase letters in the same column are significantly different (*p* < 0.05).
