## 3 Results and Discussion  
### Isoflavones Content in Soybeans  
It is widely considered that soybeans are one of the best sources of isoflav and well-digestible protein among legumes [29]. Moreover, [PERSON] et al. [30] reflected that the extraction method employed is of paramount importance to the overall process of isoflavine isolations. Up to the present moment, the following observations have been recorded that in many experiments for the extraction of isoflavines, binary extractants have been used, e.g., aqueous methanol solutions typically 60-80% [31]. In this study, the investigation focuses on isoflaves determined in soybean seeds after their extraction using three solvents, namely ethanol, propanediol, and water, in different proportions. The HPLC-DAD chromatogram (Figure 2) shows the consecutive peaks: (1) daidzin, (2) glycitin, (3) genistin, (4) malonyldaidzin, (5) malonylglycitin, and (6) malonylgenistin. The identity of the analytes tested was confirmed by ultra-performance liquid chromatography-electrospray ionization-time of flight-mass spectrometry (UPLC-ESI-TOF-MS) analysis (Figure S1). As demonstrated in the present study, the daidzin, genistin, malonylenavidin, malonyldaidzin, and malonylglycitin were the predominant isoflaves present in the majority of tested extracts. However, the results of the quantitative analysis (Table 2) suggest that there is considerable variation in the amount of isoflaves present in the samples and depending on the mixture of solvents employed. It was shown that the use of three-component extractants is the most efficient method for all extracted isoflaves. The smallest extraction yield was obtained for 100% ethanol. The daidzin content was 2.5 \(\pm\) 0.5 mg/g DM, while the presence of other isoflaves was not detected. When extracts were prepared using 100% water, the levels of daidzin, genistin, and malonylglycitin were found to be quite low, ranging from 0.039 to 0.070 mg/g DM, but the levels of malonyldaidzin and malonylgenistin exhibited marked values of 0.3945 and 0.438 mg/g DM, respectively. Malonylgenistin and malonyldaidzin reached a slightly significant concentration also in the extract of 100% propanediol (0.2295 and 0.1765 mg/g DM, respectively), but significantly lower results were obtained for daidzin, malonylglycitin, and genistin (0.1005, 0.041, and 0.985 mg/g DM, respectively). Extraction of the target analytes was more efficient in the case of binary mixtures of 50/50% water/ethanol. The levels of malonylgenistin were found to be the highest, ranging from 0.759 to 0.551 mg/g DM. The lowest content in this binary mixture (50/50% water/ethanol) was malonylglycitin, with a range  
Figure 2: The chromatogram of high-performance liquid chromatography with diode array detection (HPLC-DAD) at 254 nm for the extraction mixture of 33/33/33% (\(\
u/\
u/\
u\)). of 0.101 mg/g DM. Quite good results of extraction were obtained in the case of the binary mixture of 50/50% propanediol/water. Levels of malonyldaidzin were observed to be the most abundant, with a range of 1.14 mg/g DM. Other isoflavones also exhibited high concentrations: 1.07, 0.7405, 0.2655, and 0.227 mg/g DM for malonylglycitin, malonylgenistin, and daidzin, respectively. The binary mixture of ethanol/propanediol 50/50% was found to be the least effective of all the mixtures. The obtained contents of isoflavones in soybean seeds are comparable to the results of other researchers [32; 33]. In the study provided by [PERSON] and [PERSON] [32], the characterization of isoflavone profiles was conducted through the utilization of column chromatography and HPLC-DAD-ESI/MS spectrometry analysis. They extracted black soybean with 0.1 N HCl and methanol, and the predominant isoflavone detected was genistin, whose average content was 0.518 mg/g DM, followed by daidzin (0.416 mg/g) and glycitin (0.087 mg/g). [PERSON] et al. [28] observed the highest concentrations of isoflavones in the soybean cultivars Caloria, Safir, and Brovallia, with total values of 3813, 3198, and 4030 mg/g DM, respectively. [PERSON] et al.