*3.3. Phenolic Compound Profile*

The HPLC-DAD analysis showed that the phenolic profile was characterized by 17 compounds (Figure 3). The absorption maxima of UV-Vis spectra of compounds and MRM Q1/Q3 ion pairs used for their identification are listed in Table 3. Compounds **10**, **13**, and **17** were identified as caffeic acid, rutin, and kaempferol-3-*O*-rutinoside, respectively, by comparison with standards. Their presence in the extracts was confirmed by HPLC-MS/MS analysis. This analysis allowed also showing the presence of twelve hydroxycinnamic acids (1–12) and quercetin glucoside (15) in the extracts. Two other compounds (**14** and **16**) were tentatively identified as hydroxycinnamic acid derivatives based on the shape of UV spectra with maxima absorption at 315–329 nm and a shoulder at the shorter wavelength [34]. Most of the identified compounds (caffeic acid, caffeoyl-, coumaroyl- and feruloylglucaric isomers, feruloyl- and caffeoyl- quinic acids, rutin, kaempferol-3-*O*-rutinoside, and quercetin glucoside) were detected previously in leaves, seeds, and other aerial parts of *A. caudatus* [10,18–20]. Coumaroylquinic acids were found in stems of *A. spinosus* [35]. In turn, the free phenolic acids, such as, ferulic, *p*-coumaric, *p*-hydroxybenzoic, vanillic, sinapic, gallic, and protocatechuic acids, as well as betacyanins, were not identified in amaranth in the present study, although, according to literature data, they were determined in leaves, flowers, stalks and seeds of *A. caudatus* [10,11,19,40]. The lack of identification of betacyanins in our samples probably results from the selection of the betacyanin-free *A. caudatus* genotype for experiments.

**Figure 3.** HPLC-DAD chromatogram of the phenolic compounds present in the amaranth extract.


**Table 3.** Chromatographic and spectral data of the phenolic compounds identified in amaranth extracts.

<sup>1</sup> Compound number corresponds to peak number in Figure 3. <sup>2</sup> Retention time (tR) of HPLC-DAD separation. <sup>3</sup> Maximum absorption (λmax) of UV-Vis spectrum in HPLC-DAD analysis. <sup>4</sup> Ion pair of multiple reaction monitoring (MRM) of HPLC-MS/MS analysis.

The contents of individual phenolic compounds (**1**–**16**) in different growth stages of amaranth, expressed both per gram of extract and per gram of fresh matter of plant, are presented in Tables 4 and 5, respectively. Compound **17** (kaempferol-3-*O*-rutinoside) was not quantified because its content in the samples was very low (< 1 μg/g FM). The predominant compound in all samples was rutin (15.0–36.2 mg/g extract; 418–1169 μg/g FM), and its content consisted of approximately 95% of the sum of flavonols. Compounds **11** and **16** were the most abundant hydroxycinnamic acid derivatives. The huge amount of rutin in comparison with other phenolic compounds was in line with literature data on aerial components, especially leaves, of different species of amaranth [19,20,41]. In turn, high content of hydroxycinnamic acid derivatives with very high quantity of caffeic acid derivatives was found by Neugart et al. [18] in *A. cruentus* leaves. These authors reported a 3.3–3.4-fold higher content of caffeic acid derivatives than quercetin glycosides in leaves harvested at the 8-12 leaf stage. In our study, the content of hydroxycinnamic acid derivatives predominated over the quantity of flavonols in the early vegetative stage, but with a lower difference between values (718 vs. 439 μg/g FM). Plant growth stage significantly affected the amount of individual phenolic compounds (Tables 4 and 5). The rutin content (and sum of flavonols) increased for subsequent growth stages of plant, and the highest value was measured in early flowering and grain fill stages at the same level (*p* ≥ 0.05). Interestingly, on the contrary, the sum of hydroxycinnamic acid derivatives was found to be the highest in amaranth of the early vegetative stage, and this value decreased with age of amaranth for values expressed both on the basis of extract and fresh matter of plant. Our observation regarding the changes of rutin content during the amaranth growth cycle confirmed the trends previous reported by Kalinova and Dadakova [42]. These authors determined the rutin contents in leaves, flowers, stems, and seeds of six *Amaranthus* spp. (*A. caudatus*, *A. hypochondriacus*, *A. hybrid*, *A. retroflexus*, *A. cruentus*, and *A. tricolor*) and found that the rutin content in leaves was related to the developmental stage of the crop and that it usually increased with plant aging.



Expressed as caffeic acid equivalents. Expressed as *p*-coumaric acid equivalents. Expressed as ferulic acid equivalents. Expressed as quercetin equivalents. Tr, traces. Meanswith different lowercase letters in the same row are significantly different (*<sup>p</sup>* < 0.05). Compound number corresponds to peak number in Figure 3.

1



Expressed as caffeic acid equivalents. Expressed as *p*-coumaric acid equivalents. Expressed as ferulic acid equivalents. Expressed as quercetin equivalents. Tr, traces. with different lowercase letters in the same row are significantly different (*<sup>p</sup>* < 0.05). Compound number corresponds to peak number in Figure 3.
