## 3 Results and Discussion
### NMR and HPLC-MS\({}^{n}\) Analysis of Secondary Metabolites  
HEX, EA, and DCM solvents allowed the extraction of the most abundant lipophilic constituents of the plant material. The respective spectra, reported in Figures 2-5, support the fact that the most abundant compounds in these fractions are lipids. The assignments of the main signals of these lipids are reported in Table 2.  
In the EA fraction, some minor signals could be observed in the aromatic region and suggest the presence of some phenolic derivatives. In particular, two ortho-coupling doublets at \(\delta\) 7.73 and 6.40 indicate the presence of derivatives bearing an ortho-para disubstituted ring, as well as the singlet at \(\delta\) 6.50. The meta-coupling doublets at \(\delta\) 6.13-6.36 could be ascribed to position 3 and positions 6 and 8 of flavonoid derivatives. Moreover, some minor signals at \(\delta\) 7.74, 7.34, and 7.19 indicate the presence of phenolic compounds. \begin{table}
\begin{tabular}{c c} \hline
**Signal Assignment** & **ppm** \\ \hline CH\({}_{3}\) FA: All fatty acids except linoleic acid (-CH\({}_{2}\)-CH\({}_{3}\)) & 0.93 \\ CH\({}_{2}\) FA: Acyl chains [-(CH\({}_{2}\))\({}_{n}\)-] & 1.31–1.35 \\ CH\({}_{2}\) FA1: Acyl chains (-CH\({}_{2}\)-CH\({}_{2}\)-COOH) & 1.63 \\ CH\({}_{2}\) FA2: Mono- and polyunsaturated fatty acids (-CH\({}_{2}\)-CH=CH-) & 2.10 \\ CH\({}_{2}\) COO: Acyl chains in unsaturated fatty acids (-CH\({}_{2}\)-COOH) & 2.35 \\ CH\({}_{2}\) FA CH: (-CH=CH-CH\({}_{2}\)-CH=CH-) & 2.80 \\ TGL1: Triacylglycerglycerols (-CH\({}_{2}\)-OCO-) & 4.23 \\ TGL2: Triacylglycerglycerols (-CH-OCO-) & 5.30 \\ US: unsaturated fatty acids (-CH=CH-) & 5.40 \\ \hline \end{tabular}
\end{table} 
Table 2: Main signals detected in lipophilic fractions attributable to fatty acids. Figure 3: 1H-NMR spectrum of the EA fraction. Figure 2: 1H-NMR spectrum of the HEX fraction. Figure 4: \({}^{1}\)H-NMR spectrum of the DCM fraction. Figure 5: Magnification of the aromatic area in the \({}^{1}\)H-NMR spectrum of the DCM fraction. On the other hand, the spectrum of the BUT fraction (Figure 6) presents enlarged signals suggesting the presence of several polysaccharides. Nevertheless, some resonances can be useful for a screening of the main compounds. Broad signals in the aliphatic part can be ascribed to the non-oxygenated positions of quinic acid moieties, as well as several aromatic signals supporting the presence of phenols. For an exhaustive characterization of phenolic compounds, the chemical composition of the crude methanol extract was explored further by HPLC-MS\({}^{n}\). As shown in Table 3 and Figure 7, the analysis allowed us to identify 23 compounds as polyphenols. These constituents accounted for 7.26 mg/g of dried plant material, and phenolic acids were the most representative (5.83 mg/g). Tricafeoylhexaric acid (1.76 mg/g DW), tetracafeoylhexaric acid (1.41 mg/g DW), 3,5-dicaffeoylquinic acid (1.04 mg/g DW), caffeoyl thiexoside (0.35 mg/g DW), and chlorogenic acid (0.29 mg/g DW) were identified as the most abundant ones. Their MS\({}^{n}\) spectra are shown in Figures S1-S5 of the Supplementary Materials. Among the flavonoids, the most abundant ones were tricin (0.24 mg/g DW) and its 7-glucoside (0.26 mg/g DW), ruin (0.22 mg/g DW), apigenin (0.18 mg/g DW), and patuletin galactoside (0.15 mg/g DW). In a paper dated 1992, [PERSON] et al. reported the identification of 11 flavonoids in the aerial parts of _P. spinosa_ harvested in Egypt. Patuletin 3-O-galactoside and tricin 7-O-glucoside, also detected in our study, were two of these, together with other tricin, patuletin, and quercetin glycosides [13]. More recently, [PERSON] reported the isolation of tricin 7-O-glucoside from the aerial parts of _P. spinosa_ harvested in Algeria, together with tricin, quercetin, patuletin 7-galactopyranoside, and patuletin-3-O-\(\alpha\)-L-rharnmopyranosyl (1-6)-\(\beta\)-D-galactopyranoside [24]. A more exhaustive characterization of phenols from the aerial parts of Algerian _P. spinosa_ has been presented by [PERSON] et al.