# Metabolite Profiling of _Christia__vevertilionis_ Leaf Metabolome via Molecular Network Approach
## 3 Results and Discussion
### Structural Elucidation of Isolated Compounds  
From the results of the derepplication process via MN, compounds **28** and **47** (previously described in Section 3.1.2.) were targeted for isolation in order to validate their predicted structures. The selection of these two constituents was mainly based on practical considerations, specifically due to their well-resolved chromatograms and their presence as the major constituents in the methanolic leaf extract of _C. vespertilionis_ (Figure 5b). The ethyl acetate fraction containing these two compounds was subjected to semipreparative reversed-phase HPLC (RP-HPLC). As illustrated in Figure 8, the UV spectra of these compounds exhibited similar UV absorption maxima at 271 and 326 nm. These values were typical for flavonoids with a flavone skeleton, reported to show two maximum absorptions in the range of 250-290 and 310-350 nm [22]. Compound **28** was obtained as a light yellow amorphous solid. Its molecular formula was established as C\({}_{26}\)H\({}_{28}\)O\({}_{14}\) based on the deprotonated molecular ion peak observed at \(m/z\) 563.140 in negative ion mode ESI-MS/MS spectrum (Figure S10). Its \({}^{1}\)H-NMR spectrum (Figures S21 and S22) showed characteristics of a flavonoid diglycoside. An apigenin (4\({}^{\prime}\),5,7-trihydroxyflavone) aglycone was deduced for the compound based on the aromatic proton signals exhibited in the downfield range of \(\delta_{\rm H}\) 6.44 to 7.93. The two proton signals at \(\delta_{\rm H}\) 7.93 (d, \(J\) = 8.9 Hz, H-2\({}^{\prime}\) and H-6\({}^{\prime}\)) and \(\delta_{\rm H}\) 7.20 (d, \(J\) = 8.9 Hz, H-3\({}^{\prime}\) and H-5\({}^{\prime}\)), corresponding to the protons on ring-B, were for the ortho-coupled systems between H-2\({}^{\prime}\) and H-3\({}^{\prime}\) and H-5\({}^{\prime}\) and H-6\({}^{\prime}\). The assignment of these protons was further confirmed by correlations observed in the TOCSY spectra (Figure S23). Moreover, the two sets of one proton singlet, resonating at \(\delta_{\rm H}\) 6.62 (H-3) and \(\delta_{\rm H}\) 6.44 (H-8), further indicated the unsubstituted C-3 and C-8 positions. The occurrence of two units of sugar moiety attached to the aglycone was deducible from the presence of twoomeric protons at \(\delta_{\rm H}\) 4.91 (1H, d, H-1\({}^{\prime}\)) and \(\delta_{\rm H}\) 5.69 (1H, d, H-1). The large \(J\) value (9.9 Hz) for the anomeric proton at \(\delta_{\rm H}\) 4.91 and the small \(J\) value (3.0 Hz) for the anomeric proton at \(\delta_{\rm H}\) 5.69 indicated \(\beta\)- and \(\alpha\)-configurations for the sugar units, respectively. Complete assignment of the remaining resonance of the aglycone unit and the specific site for the linkage to both sugar units were achieved with the aid of HSQC and HMBC spectral data (Figures S24 and S25). The attachment of one sugar unit at position C-6 was confirmed from the HMBC correlations observed between the anomeric proton at \(\delta_{\rm H}\) 4.91 (H-1\({}^{\prime}\)) to C-6 (\(\delta_{\rm C}\) 108.43, \({}^{\prime}\)J), C-5 (\(\delta_{\rm C}\) 160.62, \({}^{\prime}\)J), and C-7 (\(\delta_{\rm C}\) 167.48, \({}^{\prime}\)J). For the other sugar unit, HMBC correlation observed between the anomeric proton at \(\delta_{\rm H}\) 5.69 (H-1\({}^{\prime\prime}\)) to C-4\({}^{\prime}\) (\(\delta_{\rm C}\) 160.17) indicated that the sugar unit was \(O\)-linked to C-4\({}^{\prime}\) on the aglycone. Careful analyses of the  
Figure 8: Semipreparative HPLC-PDA chromatogram of ethyl acetate fraction of _Christia vespertilionis_ methanolic leaf extract. proton and carbon resonances arising from both sugar units, with the combination of HSQC and HMBC correlations, revealed the presence of \(\beta\)-glucose and \(\alpha\)-apiofuranose as the sugar moieties (Figures S24-S26). The assignment of all protons of the two sugar moieties was also confirmed based on TOCSY spectral data (Figures S27 and S28).