## 3 Discussion  
In the present work, for the first time, a multi-methodological approach was applied to study the metabolome of dried Chinook hop leaves. The obtained results highlighted the presence of a high number of volatile and semi-volatile metabolites. Overall, up to now, a small number of previous studies have focused on the chemical characterization of hop leaves, with a very small fraction of these involving the volatile content of untreated plant material. In fact, most of the previous articles mainly concerned the determination of \(\alpha\)- and \(\beta\)-acids of hop infloresences [20; 21; 22; 23; 24]. Furthermore, to the best of our knowledge, this is the first work reporting the fatty acid content of hop leaves beyond the investigated cultivar. Our data showed that \(\alpha\)-humulene followed by \(\beta\)-caryophyllene and \(\alpha\)-copaene were the most representative terpene compounds. In our previous study [25], carried out on fresh leaves, we found that \(\beta\)-caryophyllene, \(\alpha\)-humulene, and \(\delta\)-cadinene, in descending order, were the most abundant. This suggests that drying, in general, may influence the volatile profile of this matrix. In addition, as reported by [PERSON] et al. [13], the drying method can induce variations in the content of flavones, thiols, phenols, and pigments in hop leaves. Likewise, genetic and environmental factors greatly affect the content of phytochemical compounds [26]. The detected emission of several terpene compounds from the leaves was in line with what was previously reported by [PERSON] et al. [16], where borneol, \(\gamma\)-cadinene, estragole, \(\alpha\)-copaene, \(\beta\)-farnesene, \(\beta\)-caryophyllene, \(\gamma\)-muroulene, and \(\alpha\)-humulene were emitted by leaf samples of five Lithuanian hop varieties. These compounds were characteristic of the volatile composition of both leaf and cone hops, albeit with different trends [16]. Thanks to PTR-ToF-MS analyses, in addition to some terpenes, which represent about 2% of the total VOC emission, a range of low-molecular-weight compounds such as aldehydes, ketones, alcohols, and sulfur compounds was also found. Among them, aldehydes such as acetaldehyde (C\({}_{2}\)H\({}_{5}\)O\({}^{+}\)) and propanal (C\({}_{3}\)H\({}_{7}\)O\({}^{+}\)) were the most abundant identified compounds, whereas alcohols, ketones, and sulfur showed the lowest emission. For example, the emission amount of CH\({}_{5}\)S\({}^{+}\) (methanethiol) accounted for 0.2% of the total VOC emission (Table 2). The data obtained by the GC-MS and NMR analyses on the derivatized methanolic extract and the hydroalcoholic extract, respectively, were aligned as both techniques highlighted the presence of compounds belonging to other chemical classes, such as sugars which made up the main fraction and the organic acids. Moreover, taken individually, the GC-MS technique has allowed the identification of a series of alcohols, albeit with a low relative percentage; on the other hand, the NMR technique has made it possible to identify a large number of amino acids. In general, IH-NMR was widely used to characterize bitter acids and xanthohumol in hop cones [18; 27; 28], but no NMR study is available on hop leaves. On the other hand, only one study reports the polar metabolite content in hop cones by NMR. [29]. Aparagine, glucose, and fructose have been reported to be the major amino acids and sugars in hop cones [18]. Instead, in our study, glutamate, asparagine, and sucrose were the main ones in hop leaves. Regarding bitter acids, they were not detected in hop leaves due to their low concentration [30; 31]. The obtained results from our investigation highlighted that hop leaves can be a natural alternative source of a large number of bioactive metabolites whose biological effects are well known. In fact, the identified different phytochemical classes play a key role in the prevention of various diseases.