# Amaranth Seed Polyphenol, Fatty Acid and Amino Acid Profile
## 3 Results and Discussion
### Surface Response 3D Plots  
The surface response methodology is a statistical method used to depict the relationship between two parameters and the experimental data [26]. In our study, the Box-Benhken design was used for the optimization of the TPC and TFC extraction from amaranth seed flour using four parameters (ultra-turnax speed, solid-to-liquid ratio, methanol concentration and time). In Figures 2 and 3, the evolution of the TPC and TFC as a function of the parameters studied are presented. In the case of the TPC, a positive combined effect of a high RSL and high ultra-turnax speed was observed, and a high speed corelated with a high amount of matrices influenced the extraction of phenolic compounds positively. Regarding the combined effect of the methanol concentration and ultra-turnax speed, it can be observed that a high ultra-turnax speed and a high methanol concentration generated a high TPC concentration. Furthermore, at a low methanol concentration, there was a maximum TPC, and one possible explanation for this may be the complex chemical composition of amaranth that includes other polyphenols more soluble in high water solvent concentrations. The combined effect of methanol and RSP showed a maximum point at a high methanol concentration and high RSL, respectively. In the case of time combined with the ultra-turnax speed and RSL, a direct correlation between the TPC concentration and time/RSL can be observed. The combination of the methanol concentration and time shows that the time did not influence the evolution significantly. In the case of the TFC, a positive combined effect of a high RSL and high ultra-turnax speed was observed, and a high speed corelated with high amount of matrices influenced the extraction of phenolic compounds positively. Regarding the combined effect of the methanol concentration and ultra-turnax speed, it can be observed that a high TFC concentration was achieved at the maximum speed and concentration, and at a low methanol concentration, there was a maximum. One possible explanation may be that because of the complex chemical composition of the amaranth, there can be other flavones more soluble in high water solvent concentrations. The combination of the methanol concentration and time shows that the time did not influence the evolution significantly. The combined effect of methanol and RSP showed a maximum point at a high methanol concentration and high RSL, respectively. In the case of time, it can be observed that its combination with the ultra-turnax speed and RSL had a direct correlation between the TPC concentration and time/RSL. Figure 2: 3D evolution of TPC according to the Box–Benhken design. \(-1\) the low value of the parameter, +1 the highest value of the parameter according to data from Table 1.  
acid, rosmarinic acid, myricetin, luteolin and kaempfer), while 4 were under the limit of detection (gallic acid, protocatechuic acid, 4-hydroxybenzoic acid and quercetin). From the phenolic acids determined, vanillic acid was the most abundant, followed by rosmarinic acid, chlorogenic acid, \(p\)-coumaric acid and caffeic acid. The most abundant flavonoid was kaempfer, followed by myricetin and luteolin. [PERSON] et al. determined three polyphenols in amaranth seed--rutin (10.1 mg/g), nictoiflorin (7.2 mg/g) and isoquercetin (0.5 mg/g)--and regarding phenolic acids, they quantified 4-hydroxybenzoic acid (2.2 mg/g), syringic acid (0.8 mg/g) and vanillic acid (1.8 mg/g) [10]. The polyphenols in amaranth seed present high antioxidative activity and anti-inflammatory capacity as well as miscellaneous effects like anti-fibrotic, anti-bacterial, anti-hypertensive, anti-atherogenic and anti-proliferative effects [27]. Kaempfer, the major phenolic observed in our study, was reported to have a nephprotective effect, preventing DOX-induced cardiac damage, hippocampal damage and neural and lung ischemia injury as well as an antioxidative effect [28; 29]. Myricetin, the second-largest phenolic observed, possesses antimicrobial activity and has a positive effect on microbiota [30].