Protoberberine alkaloids exhibit significant antitumor and angiogenesis-regulating activities, yet their specific effects on angiogenesis intervention and metabolic regulatory mechanisms remain systematically elucidated. In this study, ultra-high performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q Exactive Orbitrap HRMS) was employed to identify the chemical composition profiles of Coptidis Rhizoma and Corydalis yanhusuo. The anti-angiogenic effects of the major protoberberine-type alkaloid components were evaluated using the quail chorioallantoic membrane (qCAM) model and human umbilical vein endothelial cell (HUVEC) proliferation assays, while vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels were detected via qPCR experiments. Further analysis was conducted using desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) technology to examine the metabolic profile changes induced by the intervention of key anti-angiogenic components. The interaction between these components and critical angiogenesis targets was characterized by integrating network pharmacology with molecular docking technology, followed by a "structure-activity" analysis. A total of 13 alkaloid components were identified in Coptidis Rhizoma, and 17 alkaloid components were identified in Corydalis yanhusuo, with protoberberine-type alkaloids being the predominant type in both. Both the qCAM model and HUVEC experiments demonstrated that oxyberberine exhibited the most significant anti-angiogenic activity. These metabolic abnormalities characterized by suppressed energy and lipid metabolism, may inhibit angiogenesis by affecting cell membrane structure (phosphatidic acid, sphingomyelin, phosphatidylcholine), signal transduction (VEGFR2), oxidative stress, and inflammatory responses. Molecular docking and network pharmacology analyses demonstrated that C-8 oxidation enhances oxyberberine's binding affinity to EGFR and JAK2 through reduced binding energy and increased weak interactions, ultimately conferring superior anti-angiogenic efficacy compared to berberine. These findings systematically elucidate the structure-activity relationship and metabolic basis of protoberberine-type alkaloids in angiogenesis modulation, providing novel insights for anti-angiogenic drug development.