## 3 Results
### Impact of the Solvent and Extraction Method on the Recovery of Phytochemicals  
To investigate the effect of different extraction methods, i.e., homogenizer-assisted extraction (HAE), maceration (MAC), Soxhlet (SOX), and infusion; and different solvents, i.e., ethyl acetate (EA), methanol (MetOH), ethanol (EtOH), and water (H\({}_{2}\)O) on the untargeted metabolomic profile of _R. angustifolius_ extracts; both unsupervised hierarchical cluster analysis (HCA) and supervised orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used. The HCA is reported in Figure 1; as can be observed, three clear clusters were detected. The first cluster was characterized by HAE and MAC extraction methods, using water as the extraction solvent. This cluster suggests a similar extraction capacity among HAE and MAC methods, showing an evident down accumulation of certain metabolites compared to the median intensity of other samples. The second cluster was characterized by the solvent EA, shown as a clear discriminating factor. The third cluster was divided in turn by two different sub-clusters, i.e., the first was characterized by ethanolic extracts clustering together despite the different extraction methods, and the second was driven by MetOH discriminating extraction solvents, showing no differences in the use of different extraction methods. Interestingly, the variable temperature resulted in a critical factor in the recovery of bioactive compounds; indeed, the infusion method showed a phytochemical profile like that of the methanolic extracts (Figure 1). Considering that each solvent was able to promote the selective solubilization of different types of chemical compounds based on their polarity index (H\({}_{2}\)O > EtOH > MetOH > EA), it was interesting to investigate and compare the impact of different extraction methods  
Figure 1: Unsupervised hierarchical cluster analysis of bioactive compounds considering three methods of extraction: homogenizer-assisted extraction (HAE), maceration (MAC), Soxhlet (SOX), and four solvents: ethyl acetate (EA), methanol (MetOH), ethanol (EtOH), and water (H\({}_{2}\)O) of metabolites profile of _R. angustifolius_. using the same solvent. For this purpose, three supervised OPLS-DA multivariate models were created, one for each technique investigated (Figure S1; (A) HAE, (B) MAC, and (C) SOX), aimed to group and discriminate different solvents employed in accordance with the corresponding phytochemical profiles. The three OPLS-DA models built were characterized by excellent accuracy parameters with goodness-of-fit values (R2Y) equal to 0.986, 0.987, and 0.986, and goodness-of-prediction values (Q2Y) equal to 0.886, 0.891, and 0.865, as HAE, MAC, and SOX, respectively. Additionally, the models were cross-validated and inspected for outliers (Figures S2 and S3). For each OPLS-DA model, we selected the variable's importance in projection (VIP) markers used to highlight the BCs most affected by the different extraction solvents. A comprehensive list of VIP markers is reported in Supplementary Materials, divided into classes and sub-classes, including their VIP score (>1.2), cross-validated standard error, and log2 Fold-Change values obtained by pairwise comparison against samples extracted with water. Interestingly, 58 discriminant compounds were detected for the HAE method, mainly characterized by flavonoids, reporting 33 compounds compared to SOX and MAC, i.e., 19 and 12 flavonoids, respectively (Figure 2A). The main flavonoid biomarkers were represented by anthocyanins (cyanidin 3-O-(6''-malonyl-glucoside), vitisin A, cyanidin), flavones (apigenin 7-O-glucose, luteolin 7-O-malonyl-glucose), flavonoids (quercetin 3-O-xylosyl-rutinoside, kaempfer 3-O-glucose), and isoflavonoids (genistin), owing to the best VIP score ranging from 1.20 to 1.42 (Table S2). According to Log2 Fold-Change (LogFC) analysis, these VIP markers were better extracted in MetOH and EtOH solvents than the aqueous solvent, showing 35.39 and 15.81 sum LogFC values, respectively.