# Metabolite Profiling of _Christia__vevertilionis_ Leaf Metabolome via Molecular Network Approach
## 3 Results and Discussion
### C-glycosflavone Derivatives
An additional fragment ion observed at \(m/z\) 413 for [(M\(-\)H)-(acyl+H)-OH]\({}^{-}\) further indicated the loss of \(O\)-acyl groups via cleavage from the sugar unit, which specifically refers to the C-6 position of the hexose unit, as shown in Figure 4. Based on these MS/MS fragmentation data, the nodes with precursor ions [M \(-\) H]\({}^{-}\) at \(m/z\) 567.073, 499.085, 517.098, and 473.109 were, therefore, putatively identified as apigenin-6-C-(6\({}^{\circ}\)-O-dihydroxybenzoyl)-glucose (**36**), apigenin-6-C-(6\({}^{\circ}\)-O-croonyl)-glucoside (**37**), apigenin-6-C-(6\({}^{\circ}\)-O-malonyl)-glucoside (**43**), and apigenin-6-C-(6\({}^{\circ}\)-O-acetyl)-glucose (**44**), respectively. The identification of **43** was also further supported by the protonated molecular ion at \(m/z\) 519.114 [M + H]\({}^{+}\), which exhibited MS/MS data at \(m/z\) 415 [(M+H)-(malonyl+H)-OH]\({}^{+}\), 313 [(M+H)-malonyl-120]\({}^{+}\), and 271 [(M+H)-malonylhexose]\({}^{+}\). Through a systematic analysis of the MS/MS fragmentation pattern, the nodes with precursor ions at \(m/z\) 565.155 [M + H]\({}^{+}\) and 563.140 [M \(-\) H]\({}^{-}\) were characterized as a mono-C-glycosflavucing \(O\)-pentoside. A careful analysis of negative ion mode MS/MS fragmentation data for the spectral node with a precursor ion at \(m/z\) 563.140 (Figure S10) revealed that this compound possessed characteristic fragment ions of C-glycosidic linkage at \(m/z\) 443 for [(M\(-\)H)-120]\({}^{-}\) and 473 for [(M\(-\)H)-90]\({}^{-}\), which correspond to the internal cleavage of the sugar moiety. Meanwhile, a direct neutral loss of 132 Da from both the transition ions at \(m/z\) 443 and 473 further suggested the presence of one unit of pentosyl moiety (C\({}_{5}\)H\({}_{8}\)O\({}_{4}\)) bound to the aglycone molecule via an \(O\)-linkage. Additional fragment ions exhibited at \(m/z\) 269 [aglycone]\({}^{-}\) and 282 [aglycone + 12]\({}^{-}\) provided information regarding the aglycone unit (apigenin) and the presence of C-glycosidic linkage in this compound, respectively. Aided by NMR spectral data (detailed in Section 3.2), the linkages of this compound were confirmed to be positioned at C-6 and 4\({}^{\prime}\)-OH of the aglycone unit. Moreover, the characteristic of the pentosyl moiety was also revealed to be similar to that of apiofuranose on the basis of its NMR spectral data. Therefore, this compound was deduced as the new derivative of **35**, named apigenin-6-C-\(\beta\)-glucoside 4\({}^{\prime}\)-O-\(\alpha\)-apiofuranoside (**28**). With a mass difference of 172 Da from that of **28**, nodes with precursor ions at \(m/z\) 737.229 [M + H]\({}^{+}\) and 735.214 [M \(-\) H]\({}^{-}\) were predicted to be a dimalonylated derivative of **28**. A detailed inspection of the negative ion mode MS/MS fragmentation pattern of this compound (**47**) revealed that it has similar characteristic fragment ions as **28** (Figure S11), which supports that it has a similar chemical scaffold as **28**. The proposed  
Figure 4: Proposed mechanism for the formation of fragment ion at \(m/z\) 413 in acylated derivatives of **35**. main fragmentation pathways for both compounds are presented in Figure 5. The fragment ions formed at \(m/z\) 473 for [(M\(-\)H)-90-86-86]\({}^{-}\) and 443 for [(M\(-\)H)-120-86-86]\({}^{-}\) may have resulted from internal cleavages at position 0,\(A\)\" and 0,3\({}^{\prime}\) of the C-glucose moiety, which supports the attachment of each of the malonyl units to the hydroxyl positions of C-4\" and C-6\", respectively. The existence of a fragment ion at \(m/z\) 545 for [(M\(-\)H)-(86+H\({}_{2}\)O)-86]\({}^{-}\) further indicated the specific cleavage of the two malonyl units at C-4\" and C-6\" of the glucoside unit (Figure 6A). In addition, the significant presence of a fragment ion at \(m/z\) 249 [1,3B]\({}^{-}\), generated from cleavage at positions 1 and 3 of ring-C, via the RDA mechanism, provided evidence for the presence of an unsubstituted pentosyl moiety, linked to ring-B of the aglycone (Figure 6B).