**2. Results and Discussions**

Both the extraction and the instrumental method used were optimized within the present study. Three different extraction procedures are tested and compared: tinctures, microwave-assisted (MAE), and ultrasound assisted extraction (UAE). The details regarding the extraction optimisation are presented in the Supplementary materials along with details on the optimisation of the electrospray ionization parameters and mobile phase selection. According to the experimental results (Supplementary Figure S1), the UAE method was subjected for further method validation.

#### *2.1. Identification of Phenolic Compounds in Alfalfa and Red Clover Sprouts*

Identification and quantification of polyphenols in plant material are of great interest as they make a significant contribution to its total bioactivity. A specific non-target UHPLC-Q-Orbitrap HRMS method for rapid identification of the samples components was developed, optimized, and validated. A total of 59 polyphenolic compounds were simultaneously identified including nine phenolic acids, 22 isoflavones and glucoside derivatives, 11 flavone, six flavanone, and nine flavonols. Among them, 30 major compounds were unambiguously quantified by comparing with reference standards. The retention time, compound name, formula, *m*/*z* values of adduct ions, and MS/MS fragment ions in negative ESI mode, mass error, and accurate molecular mass are shown in Table 1.

For the compounds without available references, the structures were presumed based on high-accuracy analysis of deprotonated precursors and fragment ions of specific components. The chemical elemental composition for each peak was assigned within a mass error of 2 ppm. Based on literature [4,12,13,17,18,20,25], a self-built chemical database of known polyphenolic compounds in seeds and sprouts was achieved. A total of 29 compounds were identified in the analysed extracts (Table 2, Supplementary Figure S2).

The fragmentation pattern of polyphenols in negative electrospray ionization has been extensively studied [26–31]. The retro-Diels–Alder (rDA) reaction, loss of a methyl radical, and the mechanism of eliminating CO, CH2CO, and CO2 at ring C are followed by successive specific fragmentations that were previously described. MS <sup>2</sup> data obtained in the present study was consistent of literature sources [31–33].

A number of 13 isoflavones and two isoflavones glycosides were identified and listed in Table 2. In the MS-MS spectra of the methoxylated isoflavones, the loss of the [M-H-CH3] - radical anion, loss of a hydrogen atom in these radical anions, and the neutral losses of CO and CO2 were commonly observed [31].

Four isomeric peaks displayed the same [M – H]<sup>−</sup> at *m*/*z* 283.06 (C16H12O5) in the extracted ion chromatogram of extracts of alfalfa and red clover spouts (Figure 1) and were assigned as biochanin A, prunetin, calycosin, and glycitein after the comparison with the fragmentation pattern in the mentioned databases based on the presence of some key fragments (Table 3, Supplementary Figure S3). In both alfalfa and red clover sprouts corresponding to the germination days 3 to 5, a fifth peak at *m*/*z* 283.06 was displayed at 16.58 min. (mass error 1.3) and it was assigned to 5,7-dihydroxy-2- -methoxyflavone based on the pattern fragmentation.

Glycitein, which is available as a reference standard, was identified by comparing with the retention time and fragmentation pattern of the reference solution. The fragment ion *m*/*z* 147.00 corresponding to [M-H-CH3-CO-B-ring]<sup>−</sup> is a characteristic fragment ion of glycitein, which can be used to differentiate glycitein from its isomers and was identified only in glycitein spectra. The rest of the compounds were deduced based on the presence of diagnostic fragment ions. The loss of CH3· (*m*/*z* 268) followed by loss of a hydrogen atom (*m*/*z* 267) was characteristic of all four compounds. The loss of CO· moiety from demethoxylated precursors generated the ion *m*/*z* 240 and the loss of CO2 generated fragment *m*/*z* 224. Loss of a hydrogen atom was observed in the fragments *m*/*z* 240 and *m*/*z* 224, which both gave peaks at *m*/*z* 239 and *m*/*z* 223. Both *m*/*z* 240 and *m*/*z* 239 ions further lose CO·and CO2 to produce *m*/*z* 212 and *m*/*z* 211 ions, respectively, in biochanin and glycitein. In an attempt to differentiate biochanin, prunetin and isoprunetin Fra ´nski (2018) [31] showed that biochanin and prunetin cannot be differentiated by MS/MS experiments in which fragmentation occurs in a collision chamber. In the previously mentioned study, by recording full scan mass spectra at high cone voltages, differences in ions' abundance were obtained. We tried to overlap that information on our data but the differences were not consistent and were not repeatable in real vegetal samples.

**Table 1.** The 30 compounds identified from alfalfa and red clover sprouts by UHPLC-Q-Exactive with structures confirmed by comparison with reference standards.




**Figure 1.** Extracted chromatograms for [M <sup>−</sup> H]<sup>−</sup> 286.06, full MS <sup>2</sup> (50–330 *m*/*z*). The isomeric peaks were assigned to (**1**) glycitein, (**2**) calycosin, (**3**) prunetin, and (**4**) biochanin A.

**Table 3.** Key ions in HRMS-MS spectra (*m*/*z* with relative abundances (%) in parenthesis) for the identification of biochanin A and isomers.


Among possible differences between those four compounds, the mass spectral decompositions in the MS2 mode concerning the retrocyclization cleavages are of special interest. The ions of type [M-H-CH3-CO-CO2-CO]−(*m*/*z* 168) characteristic for glycitein, [M-H-CO-B-ring]−(*m*/*z* 167.03) characteristic for prunetin and glycitein and [M-H-CH3-CO-B-ring]<sup>−</sup>(*m*/*z* 147) were used in tentative compound identification (Table 3). Ions [A-ring fragment]−(*m*/*z* 135.00) and [B-ring fragment]−(*m*/*z* 132.02) were characteristic for biochanin and were not detected in the rest of the isomers.

The peak at the retention time of 18.22 displayed at *m*/*z* 267.03 (C15H8O5) was deduced as coumestrol based on the presence of diagnostic fragment ions *m*/*z* 266.03 produced by loss of H and *m*/*z* 239.03, 223.04 and 211.01 (mzCloudeTM). Biachi (2016) suggested the fragmentation pattern for coumestrol [34]. The loss of CO· moiety from the precursor ion generated the ion *m*/*z* 239.03 and the loss of CO2, the ion *m*/*z* 223. The *m*/*z* 239.03 ion further lose CO·and CO2 to produce *m*/*z* 211.04 and *m*/*z* 167.10 ions, respectively. All fragments were identified in the alfalfa extracts samples.

At 12.46-min and 22.02-min hexose glycoside of coumestrol and, respectively, biochanin A were identified. The loss of sugar moieties was observed, producing a fragment ion [M-H-Glc]− corresponding to the aglycone form. For apigenin glucoside, two isomers were observed and assigned as vitexin (apigenin8-*C*-glucoside) and apigetrin (apigenin-7-glucoside). The fragmentation of each isomer was predicted using MS Fragmenter software. Due to their structural differences, fragment *m*/*z* 340.05 [M-C3H8O3] − was specific only for apigetrin (Figure 2).

**Figure 2.** Proposed pathway for fragment *m*/*z* 340.05, specific for apigetrin.

Peaks observed at the retention time 15.30 and 18.1921 minutes displayed the same exact mass *m*/*z* 256.0735 and was assigned as liquiritigenin and isoliquiritigenin (C15H12O4) in reference to the mass spectral database and the previously reported study [33,34]. The diagnostic ions were *m*/*z* 135.00, 119.04, which were consistent with typical [1,3A <sup>−</sup> H]<sup>−</sup> and [1,3B <sup>−</sup> H]<sup>−</sup> fragments [33].

Three isomeric peaks with the same [M − H]<sup>−</sup> ion at *m*/*z* 299.06 (C16H12O6) displayed at 15.24, 18.20, and 19.70 min in the extracted ion chromatogram of alfalfa samples and were assigned to chryosoeriol, tectorigenin, and pratensein based on literature [12,15] and fragmentation pattern (Supplementary Figure S4). The fragments at m/z 284.03 formed by demethoxylation and m/z 256.06 generated by the future loss of CO were characteristic for all three compounds (Table 4). Fragment ions at *m*/*z* 267.03 [M-H-HO-CH3] <sup>−</sup> and 255.06 [M-H-CO2] − are also detected in the MS2 spectrum of tectorigenin. The minor fragment ion at *m*/*z* 135 [M-H-Aring]− correspond to the cleavage of the B ring pathway. The results were consistent with previous studies [35,36]. The formation of an ion at *m*/*z* 151.04 comprising the ring A of the flavone skeleton was also c haracteristic for the mass spectral decomposition in tectorigenin.

The extracted chromatogram for *m*/*z* 285.04 [M − H]<sup>−</sup> in the red clover spout extracts revealed two peaks at 11.74 min 17.06 min. The last one was identified as kaempferol according to the retention time and fragmentation pattern of the reference standard. According to the fragment interpretation using MS Fragmenter software and the published data, the first peak was identified as baptigenin. In the MS/MS, spectrum were detected signals at *m*/*z* 269.04 corresponding to the [M -OH]− fragment, *m*/*z* 240.04 as [M-H-CO2-H]<sup>−</sup>, *m*/*z* 136.01 as [M-B-ring-C2H]<sup>−</sup>, and 109.02 corresponding to the [B ring]−.

Peck at [M − H]<sup>−</sup> *m*/*z* 297.04 (RT 17.21) was identified in the red clover sprouts samples as irilone, which is in agreement with other studies [25,37]. The diagnostic ions were: *m*/*z* 269.04 corresponding to [M-H-CO]−, *m*/*z* 252.04 [M-H-CO2-H]−, *m*/*z* 178.99 [M-H-B ring-CO]−, *m*/*z* 133.02 [M-C8H5O4] −. Isoflavone pseudobaptigenin [M − H]<sup>−</sup> *m*/*z* 281.04 (RT 21.29) was identified in both alfalfa and red clover sprout samples based on the diagnostic ions *m*/*z* 285.05 [M-H-CO]−, *m*/*z* 251.03 [M-H-CO-H2] −, and *m*/*z* 135.00 [M-C9H7O2] <sup>−</sup>. Azelaic acid, [M − H]<sup>−</sup> at *m*/*z* 187.09 (RT 15.11), which is naturally

occurring in wheat, rye, barley, oat seeds, and sorghum, was identified in all analysed samples based on the diagnostic ions *m*/*z* 171.10 [M-OH]−, *m*/*z* 125.09 [M-CH3O4] <sup>−</sup>, and *m*/*z* 123.08 [M-CH4O3] −, according to the MS Fragmenter Software.

The other compounds listed in Table 2 were identified according to their molecular mass, formula, MS/MS fragments, and related literature by using the same approach. Regarding those compounds, distribution in the analysed samples and the variation during germination is shown in Supplementary Figure S5.

**Table 4.** Key ions in HRMS-MS spectra (*m*/*z* with relative abundances (%) in parenthesis) for identifying tectorigenin A and isomers.


The isoflavone aglycones biochanin A, pseudobaptigenin, calycosin, prunetin, and pratensein previously reported in the red clover aerial part [16,18,19] were detected in both red clover and alfalfa seeds and sprouts. Comestrol, tricin, vitexin (apigenin 8-C-glucoside), and tectorigenin were found only in alfalfa sprout samples, while afrormosin, baptigenin, and irilone were detected only in red clover samples. Afrormosin and irilone appear due to various modifications of the isoflavones A-ring and have been reported in aerial parts of plant form *Fabaceae* family [25].

Although biochanin A, formononetin, genistein, daidzein, and their glycosides are commonly determined in red clover as phytoestrogens of interest [18,38,39], other compounds identified in the present study that may contribute to the significant estrogenic activity of red clover include medicarpin, liquiritigenin, and isoliquiritigenin [19]. All of these compounds were found both in alfalfa and red clover sprouts.

Chrysoeriol and its glycoside, previously reported in the aerial part of alfalfa [20,38], were identified in both species of spouts. Medicarpin and chrysoeriol show significant antiangiogenic and cytotoxic activity [40] suggesting an interesting potential in cancer therapy and justify further studies.

Coumestrol was identified only in alfalfa sprouts. However, alfalfa sprout was recognised as a major source of coumestrol [19]. Tricin was detected in all samples of alfalfa seeds and sprouts, which is consistent with other studies reporting tricin identification in the alfalfa aerial part [12]. Luteolin-7-*O*-glucoside, kaempferol-3-*O*-glucoside, kaempferol-3-*O*-rutinoside previously reported in red clover, alfalfa, and mung bean sprouts [6] were identified in both species in different germination stages.

The compounds identified in sprouts' samples exhibit a wide range of biological effects, including antioxidant, antimicrobial, phytoestrogenic effects, and anticarcinogenic activity [13,15,19,40]. Our study findings confirm that both alfalfa and red clover sprouts are important sources of isoflavones besides soy and soy-derived products. Furthermore, according to the literature, while widely used soy is a source of poorly absorbed isoflavones glycosides, red clover and alfalfa contains easily-absorbed free aglycones forms [5].

### *2.2. Quantification Result*

The developed UPLC-HRMS/MS method was applied for the routine determination of 30 compounds in the extracts of seeds and sprouts of alfalfa and red clover identified in Table 1a. The method was validated according to the section "Quantitative Method validation." Validation parameters are provided in Supplementary Table S1. The result of the quantification of the target compounds in alfalfa and red clover seeds and sprouts were shown in Tables 5 and 6. The analysis was conducted in duplicate. The obtained data were expressed as mean values ± standard deviations.

Among the quantified compounds, ononin, naringin, and epicatechin levels were below quantification limits. The results of the quantitative analysis are consistent with other studies [6,18,39]. Germination is a process known to be accompanied by a spectrum of significant changes in metabolites. Phenolic compounds are already present in the earliest plant stages and have crucial functions in plants' evolution and adaptation. While the total polyphenols' content varies, there are some kinetic transformations of the individual components that are directed towards reducing or increasing the activity over time to support specific metabolic pathways [25].

The quantitative results showed a high content in catechin, hesperetin, and quercetin in red clover seeds. The content in catechin sharply decreased after the first day of germination to a value almost six-fold lower and it continued to be reduced over time, up 20-fold on the fifth day of red clover germination. Quercetin concentration was four times lower on the fifth day of germination when compared to red clover seeds. The concentration of hesperetin in the red clover seeds linearly decrease 3.47-fold from the first to the fifth day of germination. Rather, rutin concentration increased during germination, reaching the maximum concentration on the fifth day of germination.

Respecting the alfalfa seeds and sprouts, lower variation of compounds' concentrations was registered for the sprouts in the second, third, and fourth day of germination. However, clear differences were observed between the seeds and spouts. For example, the considerably higher concentrations of kaempferol, quercetin, syringic acid, and hyperoside were measured in the seeds. Hesperitin dynamics during germination was reversed when compared to red clover, registering a marked increase in the third day of germination, which is followed by a slight decrease.

The dynamics of ferulic acid and *p*-coumaric acid concentration during germinations was similar in alfalfa to the one in red clover, with higher concentrations of *p*-coumaric acid and lower concentrations of ferulic acid in alfalfa than in red clover during germination. *p*-coumaric acid can be converted into caffeic acid via hydroxylase activity during plant germination [25]. According our results, *p*-coumaric acid showed a similar decrease during germination for both species. In alfalfa sprouts, almost double the amount of *p*-coumaric acid was measured when compared to red clover. However, caffeic acid was detected only in red clover samples, which suggests different metabolic pathways. The highest concentrations of caffeic acid were measured in red clover seeds and in sprouts on the third day of germination.

The profile of the isoflavones with estrogenic activity varied greatly with the phenological stage along the germination. In the red clover sprouts, aglycones daidzein and genistein concentrations increased from the first to the fourth day of germination, with a maximum on the third day, while, on the fifth day, the concentration was considerably lower. The concentration of formononetin in red clover increased during germination 12-fold up to day 4 when compared to the seeds. Glycitein was found in the highest concentration on the third day of germination.

Formonetin and glycitein concentrations in alfalfa sprouts on the third germination day were comparable with red clover sprouts in the same germination stage, while the concentrations in genistein, genistin, daidzein, and daidzin were considerably lower. However, other compounds that manifest an estrogenic effect include myricetin and apigenin [41]. Apigenin was found only in alfalfa sprouts with an increasing concentration up to three times higher in sprouts on the third day when compared to seeds. Apigenin was previously reported in alfalfa aerial parts [20,42]. Myricetin concentration in the alfalfa sprouts on the third germination day was about 10-fold higher than in red clover sprouts.


**Table 5.** The results of the quantitative analysis for alfalfa sprouts in μg per g of dried weight vegetal material (alfalfa sprouts samples coded as: ALF – alfalfa, day 1-first day of germination, day 2-second day of germination, day 3-third day of germination, day 4-fourth day of germination, and day 5-fith day of germination).

\* NF – not found

**Table 6.** The results of the quantitative analysis for red clover sprouts in μg per g of dried weight (DW) vegetal material (red clover sprouts samples coded as: RCV - red clover, day 1-first day of germination, day 2-second day of germination, day 3-third day of germination, day 4-fourth day of germination, day 5-fith day of germination).


<sup>\*</sup> NF – not found

In contrast to the red clover sprouts, which contain genistein as the major isoflavons, in alfalfa sprouts, the most abundant isoflavone was formononetin, which is consistent with other studies [37]. Genistein content in alfalfa and red clover spouts, reaching the maximum level on the third day of germination (607.2 μg/g for red clover and 105.8 μg/g for alfalfa), might be considered high, even compared to soybean in which genistein was reported as the major isoflavone, ranging from 84 μg/g to 583 μg/g DW along the reproductive stages [42]. According to the quantitative analysis, the sum of the isoflavones with estrogenic activity (daidzein, genistein, glycosides, apigenin, formononetin, myriceitin, and glycitein) was 906.1 μg/g DW for red clover and 587.5 μg/g DW for alfalfa sprouts on the third day of germination. Due to the different compounds' bioavailability [43], consideration of their biological activity could be speculative.

While rutin concentrations in all red clover samples were significantly higher than in alfala, comparable concentration of naringenin, syringic acid, ellagic acid, isorhamnetin, and pinostrobin were measured in both species during germination. For isorhamentin, the same nonlinear variation was observed for both species with the maximum concentration reached on the fourth day of germination. Isorhamnetin 3-*O*-glucoside was also identified in both species. Isorhamnetin has been previously detected, but not quantified in alfalfa aerial parts [38].

Abscisic acid, which is a plant hormone that has important roles in seed development and maturation [44], was present in relatively small amounts in both red clover and alfalfa in seeds and during germination.

In conclusion, consider the quantitative results for both plants species. Sprouts on the third day of germination could be considered as valuable sources of bioactive polyphenols with a potential health impact. The important health promoting potential of polyphenols resides in various biological activities, including antioxidant, estrogenic, anti-carcinogenic, and vasodilatory [16,25,37]. Different studies described the interactions of phenolic derivatives with intracellular receptors and signaling pathways to induce adaptive responses and regulation of apoptotic genes and mitochondrial function [3,15]. They suggest that *Fabaceae* sprout consumptions may also reduce the risk of osteoporosis, help alleviate menopausal symptoms, and prevent cardiovascular disease, hypertension, and hormone-dependent tumours [5,41].

### *2.3. Multivariate Data Analysis*

Unsupervised classification by PCA and HCA were used in order to show the grouping of the investigated samples. PCA explained 59.68% of the total variation using principal components with a higher contribution brought by PC1 (33.26%) when compared to PC2 (26.42%), (Figure 3). Along PC1, the germinated alfalfa and red clover seeds from the initial seeds can be discriminated. Along the PC2 axis, two clusters including the firs cluster including alfalfa seeds and germinated alfalfa seeds located on the left side and cluster of two grouping the germinated red clover seeds located on the right side are observed. Non-germinated red clover seeds are clearly discriminated from the other seeds. PCA analysis revealed the correlations among the polyphenols' composition of different germinated seeds. Our results showed that ellagic and *p*-coumaric acids, kaempferol, and myricetin represent polyphenol markers of alfalfa seeds, while chlorogenic acid, chrysin, apigenin, and pinocembrin are representative for germinated alfalfa seeds. Red clover seeds were characterised by catechin, naringenin, hyperoside, and syringic acid, while gallic and ferulic acids, quercetin, hesperitin, and abscisic acid characterise the red clover seeds on the first and second day of germination and caffeic acid, pinostrobin, glycitein, rutin, isorhamnetin, genistein, formonetin, daidzein, and genistin are representative of the germinated red clover seeds on day 3, day 4, and day 5.

The Ward's hierarchical clustering method with Euclidean distances as measures of dissimilarity based on the phenolic compounds' profile was applied. The dendrogram shows the clustering of the samples in four separate groups (Figure 4). At a dissimilarity level of 1200000, class 1 refers to the initial alfalfa seeds (ALF seeds) (C1), while class 2 (C2) include the germinated alfalfa seeds in the five days of germination and red clover seeds on the first day of germination. Class 3 (C3) refers to the

initial red clover seeds (RCV seeds) and class 4 (C4) correspond to the germinated red clover seeds, which reveal a polyphenolic composition similar to the initial alfalfa seeds.

**Figure 3.** Bi-plot of the principal components PC1 and PC2 resulted from the PCA analysis with normalized Quatrimax rotation data of the polyphenols' concentrations quantified and the samples (alfalfa and red clover sprouts samples coded as: RCV — red clover, ALF — alfalfa, s — seeds, day 1-first day of germination, day 2-second day of germination, day 3-third day of germination, day 4-fourth day of germination, and day 5-fifth day of germination).

**Figure 4.** Dendrogram of the 12 objects (alfalfa and red clover sprout samples) represented by phenolic compounds' profile obtained by Ward's hierarchical clustering method (hierarchical cluster analysis).

The second PCA-analysis (Figure 5) was performed for the results of the qualitative analysis: identified polyphenols obtained from the screening HRMS/MS <sup>2</sup> and indicated with a present / absent decision.

PCA analysis revealed the correlations among the polyphenols' composition of different germinated seeds. Thereby, coumestrol, tricin, vitexin, tectorigenin, and sissotrin represent polyphenol markers of alfalfa seeds, while isoliquiritigenin, apigetrin, irisolidone kaempferol-*O*-glucoside, medicarpin, irilone, alfalone, and afrormosin are representative for germinated alfalfa seeds. Azaleic acid, isorhamnetin, luteolin-7-glucoside, liquitrigenin, pratensein, pseudobaptigenin, chrysoeriol, chrysoeriol-7-glucoside, and kaempferol-3-rutinoside were characteristic for alfalfa in the five days of germination, while biochanin A, calycosin, prunetin, and baptigenin are characteristic for red clover.

**Figure 5.** Bi-plot of the principal components PC1 and PC2 resulting from the PCA analysis with normalized Quatrimax rotation data of the tentatively identified polyphenols and the samples.

### **3. Materials and Methods**

#### *3.1. Reagents*

The reference standards of 30 compounds (bold in Table 1) were purchased from Sigma–Aldrich (Aquator, Iasi, Romania). Organic solvents' methanol and ethylic alcohol, HPLC grade, were purchased from Merck Romania. Formic acid (98%) was ultrapure water (LC-MS grade) and was purchased from Merck (Merck Romania, Bucharest, Romania). For the calibration of the mass spectrometer, the PierceTM LTQ Velos electrospray ionization (ESI) positive and negative ion calibration solutions (Thermo Fisher Scientific) were used.
