ency of the activity could be performed for the determination of IC50 values. For this, aliquot C of each microfraction was recovered and used to make a fixed-concentration solution in dimethyl sulfoxide to perform a concentration-response analysis and determine IC50 values for anti-angiogenic activity for compounds a to d. Genistein and licoisoflavone A displayed similar levels of potency, with IC50 values of 24.2 mM and 16.7 mM, respectively. MedChemExpress AGI-6780 sophoraisoflavone A and rhynchoviscin were less potent but still clearly anti-angiogenic, with IC50 values of 50.7 mM and 41.3 mM, respectively. All four Microscale Natural Product Discovery in Zebrafish compounds phenocopied the anti-angiogenic effects of the R. viscosa extract in this assay. Since the crude extract also exhibited anti-inflammatory activity, compounds a to d were also assessed using the LPSenhanced leukocyte migration assay in zebrafish larvae. Moderate but significant inhibition of leukocyte migration was observed for genistein and sophoraisoflavone A at 12.5 and 25 mM. Intriguingly, no significant anti-inflammatory activity was observed for licoisoflavone A or rhynchoviscin, indicating some structure-dependent activity differences between these related compounds. Genistein, an isoflavone synthesized by Fabaceae species and usually derived from soybeans, inhibits the tyrosine kinases EGFR, pp60v2src, and pp110gag2fes at pharmacological doses, with negligible effects against serine/ threonine kinases 20347963 such as protein kinase A, protein kinase C, and phosphodiesterase. With regard to its role in inflammation, genistein inhibits LPS-induced nitrite production by cultured macrophages and protects against LPS-induced necrosis by reducing nitric oxide release via the downregulation of inducible nitric oxide synthase. Genistein also inhibits leukocyteendothelium interaction, thereby modulating vascular inflammation, and reduces reactive oxygen species by attenuating the expression of ROS-producing enzymes. Regarding its role in angiogenesis, genistein as well as other isoflavones are known to inhibit mammalian endothelial cell proliferation and migration in vitro. In vivo, genistein has been found to inhibit angiogenesis in mouse models of melanoma and breast cancer and to inhibit retinal neovascularization, as well as to downregulate vascular endothelial growth factor and hypoxia-inducible factor expression, in a mouse model of oxygen-induced retinopathy. To date, no anti-angiogenic or anti-inflammatory activity has previously been reported licoisoflavone A and sophoraisoflavone A. In the initial screen, the inhibition of angiogenesis was dependent on the original amount of each constituent in the extract. The qNMR results enable the correlation of compound amounts with bioactivity. For genistein, the analysis revealed the anti-angiogenic activity of each microfraction to correlate well with its calculated amount and thus the bioactivity profile in the 23713790 initial screen had a direct quantitative link with this compound. For compounds b, c and d, similar activities were observed in the primary screen for microfractions containing these pure compounds, and these results were consistent with the subsequent IC50 analysis for each molecule indicating the ability of this in vivo approach to identify microgram-level quantities of NPs possessing only moderate levels of bioactivity. 
De Novo Identification of the Novel Compound b During the first phase of dereplication and microfractionation compou
