## 4 Materials and Methods
### NMR Analysis  
For the untargeted NMR analysis, two types of extraction protocols were performed on the dried hop leaves: the Bligh-Dyer extraction and a methanol extraction.  
The first one is a useful extraction method for the isolation of most polar metabolites, obtaining a clean extract free from traces of organic compounds. This extraction was performed according to previous literature articles [48; 49; 50]. In brief, 0.100 g of the dried hop leaves were sequentially added with 0.8 mL of Millipore grade water and 3 mL of methanol/chloroform (2:1% \(v/v\)). The extraction was performed through a sonication step at room temperature for 10 min. Then, the extract was sequentially added with 1 mL of chloroform and 1 mL of Millipore grade water to obtain the biphasic system methanol/chloroform/water (2:2:1.8% \(v/v/v\)). Finally, the upper hydroalcoholic phase was collected, and the residue pellet was extracted again. The obtained extract was dried under N\({}_{2}\) flow and prepared for the NMR analysis. The dried hydroalcoholic phases were solubilized in a solution of 20 mM phosphate buffer/D\({}_{2}\)O containing 0.4 mM 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSPA) and EDTA-d16, and 0.7 mL was transferred in a 5 mm NMR tube.  
Regarding the methanolic extraction, the used protocol is an already established method for hops bitter acids and xanthohumol extraction [30]. In detail, 0.100 g of dried leaves were extracted with 1 mL of MeOD with 10 min of sonication at room temperature. The TSPA internal standard was added to achieve a 1 mM concentration in each extract and directly analyzed by NMR.  
Both the extraction methods were performed in triplicate and analyzed with a JNM-ECZ 600R (JEOL Ltd., Tokyo, Japan) spectrometer operating at the proton frequency of 600.17 MHz equipped with an autosampler.  
The parameters used for the hydroalcoholic fraction analysis were the following: 90\({}^{\circ}\) pulse angle of 9.26 \(\upmu\)s, 128 transients, 4 pre-scans, 65 K data points, and a presaturation pulse sequence to suppress the water signal. Regarding the methanol extract, a double suppression at 4.83 ppm and 3.28 ppm was performed. The other experimental parameters were a 90\({}^{\circ}\) pulse angle of 9.0 \(\upmu\)s, 128 transients, 4 pre-scans, and 65 K data points.  
2D NMR experiments, namely \({}^{1}\)H-\({}^{1}\)H TOCSY and \({}^{1}\)H-\({}^{13}\)C HMBC for the hydroalcoholic extract and \({}^{1}\)H-\({}^{13}\)C HSQC for the methanolic one were performed using previously reported experimental parameters [51]. All the NMR spectra were processed using the JEOL Delta v5.3.1. software (JEOL Ltd., Tokyo, Japan). The 1H NMR spectra were Fourier transformed, manually phased, and automatically base corrected. Both types of spectra were referenced to the internal standard TSPA set at 0.00 ppm.  
The metabolites identification was based on literature NMR data on other plant matrices and hop parts [18; 52; 53], 2D experiments, and standard additions. Signals of the identified metabolites and the internal standard in the hydroalcoholic and methanolic extracts were manually integrated for all the samples. In order to realize an absolute quantification, the area of each signal was referred to as the area of the TSP methyl group, the internal standard with a known concentration of 0.4 mM, taking into account the number of protons (Supplementary Material). The final quantitative results were expressed as mg/100 g of DW \(\pm\) SD.