## 3 Results and Discussion
### Quantification of Compounds by HPLC  
The HPLC method (or other chromatographic techniques) has been frequently used for the determination of the profile of chemical constituents of propolis, since it allows its quantification and separation; however, its high cost makes its application as a routine activity not recommended [80; 81]. The intrinsic complexity of propolis was demonstrated in the large number of chromatographic peaks obtained from the HPLC method; however, it was noted that the peaks found showed good resolution between them, with a high likelihood of identifying new compounds (Figure 2). Several chemical classes, such as isoflavoids, prenylated benzophenones, terpenes and flavonoids, have been found in previously researched Brazilian red propolis samples [38; 82]. However, the marked presence of isoflavoids in its composition makes the red propolis from the state of Alagoas in Brazil an atypical case of chemical composition [83]. Concerning the identification and quantification of the chemical species of interest in the samples (red propolis extracts) (Table 2), only formononetin was identified in all the obtained extracts, and kaempfer was found and quantified in eight of the ten samples studied here. It was not possible to quantify the kaempfer in the control sample (E:UT) and in the E:25\(-\)30 sample given that this compound was present at a concentration below the quantification limit (result confirmed by repeating the analysis in triplicate). The values of these two components (formononetin and kaempfer) in the obtained extracts are shown in Figure 3. Therefore, formonetin was the most abundant chemical species found in the extracts, and its profile was confirmed by the HPLC method [84,85]. Formononetin concentrations ranged from 4.54 \(\pm\) 0.01 to 8.36 \(\pm\) 0.25 mg:g\({}^{-1}\) (samples E:UT and E:50\(-\)20, respectively), with significant differences between the extracts and the control. Being a biomarker particular to Brazilian red propolis [86], formononetin was present in significant amounts in all extracts, independent of extraction conditions. The kaempfer concentration ranged from 0.53 \(\pm\) 0.07 to 1.04 \(\pm\) 0.02 mg:g\({}^{-1}\) (samples E:75\(-\)30 and E:50\(-\)20, respectively), with significant differences (\(p<0.05\)) between the extracts in which the compound was found. Among the extracts studied, sample E:50\(-\)20 showed the  
Figure 3: Quantification of (**a**) formononetin and (**b**) kaempfer contents with chromatographic method (HPLC) in the red propolis extracts obtained under different conditions. Values presenting the same letter in the same analysis do not show significant differences (\(p>0.05\)) in [PERSON]’s test at 95% confidence. Samples E:UT and E:25\(-\)30 presented values lower than the quantification limit for kaempfer. Average of analyses obtained from triplicates (\(n=3\)). Figure 2: Chromatographic profile of the red propolis extract, kaempfer (10) and formononetin (11) (sample E:50\(-\)20). better concentrations of the considered compounds, with significant differences (\(p<0.05\)) when compared with the control, thus showing that the application of an ultrasound pretreatment at a temperature of 50 \({}^{\circ}\)C is efficient at increasing commonentin extraction by approximately 60% and allows for significant kaempfer extraction. The efficiency of the pretreatment with ultrasound technology for the improved yield of these compounds can also be confirmed through a comparative analysis of the control (E:UT) and the extracts obtained at the same temperature and over different exposure durations (E:25\(-\)10, E:25\(-\)20 and E:25\(-\)30), with significant differences (\(p<0.05\)) between the extracts and the control. For the extracts obtained at 75 \({}^{\circ}\)C, regardless of the duration of exposure to the ultrasound, there were no significant differences in the formononetin yield.