# Metabolite Profiling of _Christia__vevertilionis_ Leaf Metabolome via Molecular Network Approach
## 3 Results and Discussion
### C-glycosflavone Derivatives
The predicted structure for compound **47** as nigerin-6-\(C\)-\(C\)-\(C\)-[(4\",6\"-\(O\)-dimalonyl)-glucoside] 4\({}^{\prime}\)-\(O\)-\(a\)-apiofuranoside was further verified with the aid of NMR spectral data (detailed in Section 3.2). A node with precursor ion [M + H]\({}^{+}\) at \(m/z\) 595.166, a mass difference of 162 Da from that of **35**, exhibited characteristic fragment ions at \(m/z\) 433 for [(M+H)-162]\({}^{+}\), 337 for [(M+H)-120-120-120]\({}^{+}\), and 367 for [(M+H)-120-90-H\({}_{2}\)O]\({}^{+}\). The two consecutive losses of 120 Da for the \(m/z\) 337 ion were suspected to have originated from the simultaneous cleavage of two sugar units, providing evidence for the presence of two hexose units in this molecule. Thus, the molecule was assigned as the di-C-glycosflavone, apigenin-6,8-di-C-glucose (also known as vicenin 2) (**30**) [24]. Figure 5: Proposed fragmentation pathways for the major product ions observed in the MS/MS spectra of compound **28** and its dimalonylated derivative (**47**), recorded in negative ion mode. Flavone-\(C\),\(O\)-diglycoside  
Compound **31**, with a deprotonated molecular ion at \(m/z\) 593.151 [M \(-\) H]\({}^{-}\), was assigned as apigenin-6-\(C\)-glucosyl-2\"-\(O\)-glucoside, alternatively known as isovitexin-2\"-O-glucoside, based on fragment ions observed at \(m/z\) 473 for [(M\(-\)H)-120]\({}^{-}\), 413 for [(M\(-\)H)-180]\({}^{-}\), and 293 for [(aglycone+41)-18]\({}^{-}\), which indicate the characteristic of flavone \(O\)-glycosyl-C-glucoside (1\(\rightarrow\)2 interglycosidic linkage) [25]. Flavonol-3-O-glycosides  
In Clusters E (positive ion mode), and Q (negative ion mode), shown in Figure 3, automated GNPS spectral library matching allowed the annotation of several flavonoid-3-O-glycosides. The nodes with precursor ions at \(m/z\) 611.161 [M \(+\) H]\({}^{+}\) and 609.146 [M \(-\) H]\({}^{-}\) were annotated as quercetin-3-O-rutinoside (rutin) (**34**), while the node with a precursor ion at \(m/z\) 463.088 [M \(-\) H]\({}^{-}\) was annotated as quercetin-3-O-glucose or isoquerctrin (**39**), as previously reported in _C. vespertilionis_ [4]. In negative ion mode, these compounds were characterized based on the presence of deprotonated main adjycone product ion [Yo]  
Figure 6: Proposed mechanism for the formation of fragment ions at (**A**) \(m/z\) 545 and (**B**) \(m/z\) 249 in **47**. and also the highly abundant radical aglycone [Y\({}_{\rm o}\)-H]\({}^{-\bullet}\). These are typical fragmentation patterns observed for the flavonoid-3-O-glycosides [26], which result from the neutral losses of saccharide or disaccharide moieties [(M\(-\)H)-gly)]\({}^{-}\) via homolytic and heterolytic cleavages of the glycosidic bond, respectively. Using this information and in comparison with literature data, the remaining compound with precursor ions at \(m/z\) 595.166 [M + H]\({}^{+}\) and 593.151 [M - H]\({}^{-}\) was deduced to be kaempfer-3-O-rutinoside (**41**). In negative ion mode, the existence of fragment ions at \(m/z\) 285 (the main aglycone product ion via elimination of rutinose moiety), 255, 227, and 151 further supported kaempfer as the aglycone moiety [27].