## 3 Results and Discussion
### Ion Identity Molecular Network Investigation of P. mauritianum Ethyl Acetate Extract
Their structures suggested by Sirius were similar to the level 2 annotation structures of neighbor nodes. Therefore, they were attributed the level 2 annotation. Finally, compounds that were isolated and fully characterized by NMR during our study were pinpointed as level 1 annotations. Table S1, which summarises the information for level 1 and 2 annotations (73 features), can be found in the supplementary information. The annotation results provided by Sirius combined with the GNPS spectral databases matches, and NMR structural elucidation of purified compounds were investigated in the context of the IIMN. This approach allowed the expansion of the annotation coverage and identified triterpenoids and flavonoids in their free, methylated, or glycosylated forms as the main classes of compounds of the _P. mauritianum_ ethyl acetate extract. Figure 3: IIMN zoomed view showing three clusters containing flavonoids. For readability purposes, the maximum number of edges is limited to five. (Edges with the lowest cosine are hidden). Structures displayed in blue were isolated and characterized by NMR (level 1 annotation, three displayed out of three). Structures in black are consensus results given by GNPS and Sirius annotation tools (level 2 annotation, square-shaped features, 11 displayed out of 14) or a Sirius match (level 3 annotation). The biggest cluster in terms of the number of nodes (Figure 2, cluster 1) harbours 79 features, 70 of which were labelled as triterpenoids, meaning the plant extract is rich in compounds from this class. The ion identity molecular network approach and a manual curation of the network allowed the identification of the different adducts generated during the ionisation process (mainly [M-XH\({}_{2}\)O+H]\({}^{+}\), [M+NH\({}_{4}\)]\({}^{+}\), and [M-CO+H]\({}^{+}\)) gathered in cluster 1. Finally, up to 21 different triterpenes (features with at least a level 2 annotation) were annotated in the extract, whereas only 4 were previously reported in _P. mauritianum_[3; 5]. These later were also all identified in the present work: asiatic acid (ID 43), corrosion acid (ID 44), unsolic acid (ID 56), and oleanic acid (ID 59) (Figure 2, Table S1). In addition, several lupane triterpenes, which is a class never reported in the plant, such as lupeone (ID 41) or betunolic acid (ID 36), were identified for the first in _P. mauritianum_ to our knowledge. Free and glycosylated flavonoids were reported in previous plant investigations. For instance, kaempfer and quercetin, both free or linked to an unspecified sugar, were previously detected by HR-MS/MS analyses [4; 6]. In the present cluster, one feature was identified as quercetin (ID 11, Table S1), one as kaempfer (ID 8), and three features as glycosylated kaempfer (ID 25, 27, and 28). Sugar of compound 13 was identified later by NMR as glucose. Methoxylated flavonoids have never been isolated or reported from _P. mauritianum_. A cluster (Figure 3, bottom left, dark blue) contains 10 features identified as polymethoxylated flavonoids. For instance, feature ID 21 was identified as gardenin B. Coelution of PMFs during the purification process made it difficult to obtain pure compounds, but NMR analysis of several fractions collected during the purification process confirmed the presence of several PMFs in the extract. Finally, a cluster (Figure 3, bottom right, light green) contains seven features identified as chalcones, a class of flavonoids not reported in _P. mauritianum_ yet. For instance, feature ID 14 was annotated as flavokawin b, a methylated chalcone. Chalcones are largely distributed in plants and are often reported for their biological activity [14]. Therefore, a chalcone was later isolated to confirm the cluster annotations and evaluate its activity.