# Metabolite Profiling of _Christia__vevertilionis_ Leaf Metabolome via Molecular Network Approach
## 3 Results and Discussion
#### 3.1.2 Flavonoids  
A total of 20 compounds were identified as flavonoids, which included mono- and dihydroxyflavones, C-glycosflavone derivatives, flavonol 3-O-glycos, and flavone-C,O-diglycosides. These compounds were mapped into 4 distinct clusters (B, C, D, and E) based on positive ion mode MS/MS and 3 distinct clusters (O, P, and Q) and one \"self-loop\" node based on negative ion mode MS/MS, according to their substituent groups and types of glycosidic linkages (Figure 3). Monohydrooxyflavones  
Interpretation of the MS/MS spectral data for a node in Cluster B (Figure 3) with a precursor ion [M \(+\) H]\({}^{+}\) at \(m/z\) 239.128 led to the tentative identification of a monohydroxyflavone that could be assigned as 5 or 7-hydroxyflavone (**18**). The MS/MS fragmentation of **18** produced a prominent fragment ion at \(m/z\) 137 [1\({}^{,}\)3A]\({}^{+}\), generated from the cleavage of ring-C of the flavone at positions 1 and 3, via the retro-Diels-Alder (RDA) mechanism, thus verifying that the hydroxyl group was attached to ring-A. Other fragment ions exhibited at \(m/z\) 221 for [(M\(+\)H)-H\({}_{2}\)O]\({}^{+}\), 109 for [1\({}^{,}\)3A-CO]\({}^{+}\), and 93 for [O\({}^{,}\)A]\({}^{+}\) further support this suggested structure. For the node with a precursor ion [M \(+\) H]\({}^{+}\) at \(m/z\) 401.181, the compound differed from **18** by 162 Da, indicating that a sugar moiety (hexose) is present within its parent structure. This compound (**19**) showed similar fragment ions as **18**, i.e., at \(m/z\) 239, 221, 137, 109, and 93 (Figure S6), suggesting that the aglycone moiety could also be a 5 or 7-hydroxyflavone. A base peak ion observed at \(m/z\) 239 for [(M\(+\)H)-162]\({}^{+}\), due to the direct loss of sugar moiety, suggested an O-glycosylation of the phenolic hydroxyl group of **19**. Since 5-OH substituent on a flavonoid structure has very low reactivity towards glycosylation [21], compound **19** was thus characterized as 7-hydroxyflavone glucoside. Meanwhile, the remaining node with a precursor ion at \(m/z\) 418.207 was identified as the ammonium adduct ion, [M \(+\) NH\({}_{4}\)]\({}^{+}\), of **19** on the basis of similar fragment ions observed at \(m/z\) 239, 221, 137, 109, and 93. Dihydroxyflavones  
Analysis of the MS/MS fragmentation pattern for the nodes in Clusters C and O (Figure 3) suggested that the compounds were the 5,7-dihydroxyflavone (also known as chrysin (**20**)) and its acylated derivatives (**21** and **25**). The node with a precursor ion at \(m/z\) 255.123 [M \(+\) H]\({}^{+}\) (**20**) exhibited fragment ions at \(m/z\) 237 for [(M\(+\)H)-H\({}_{2}\)O]\({}^{+}\), 209 for [(M\(+\)H)-H\({}_{2}\)O-CO]\({}^{+}\), and 153 for [(M\(+\)H)-(\({}^{1,}\)3B cleavage)]\({}^{+}\), with the latter fragment ion having higher relative abundance. This fragmentation pattern was found similar to that of previously reported data for chrysin [22]. Moreover, based on its MS/MS spectrum, obtained in this present study, the additional fragment ion exhibited at \(m/z\) 177 for [(M\(+\)H)-C\({}_{6}\)H\({}_{5}\)-H]\({}^{+}\), which is attributed to a direct loss of ring-B via homolytic cleavage, and the migration of the hydrogen atom at C-3 (hydrogen atom abstraction) to the resulting ring-B further confirmed that the dihydroxyl substitution was both on ring-A (Figure S7). The presence of **20** was also supported by the MS/MS data in negative ion mode with the precursor ion [M - H]\({}^{-}\) at \(m/z\) 253.108, which yielded fragment ions at \(m/z\) 175 for [(M - H)-C\({}_{6}\)H\({}_{5}\)-H]\({}^{-}\) and 151 for [(M - H)-(1,3 B cleavage)]\({}^{-}\).