HPLC-PDA and LC-HRMS/MS-based quantitative and qualitative analysis of Glycosmis pentaphylla alkaloids, and investigation of their anticancer potential through network pharmacology.

Glycosmis pentaphylla (Rutaceae) is a medicinal plant of ethnobotanical importance, noted for its diverse alkaloid content and therapeutic potential. This study integrates analytical chemistry and network pharmacology to investigate its major alkaloids. A robust high-performance liquid chromatography method with photodiode array detection (HPLC-PDA) was developed and validated according to ICH Q2(R2) guidelines for the simultaneous quantification of eight key alkaloids (1-8) of G. pentaphylla. Quantitative analysis indicated that des-N-methylacronycine (3) was present at 17.24 mg/g in the root methanolic extract and 3.46 mg/g in the stem methanolic extract. Noracronycine (7) was present at 3.6 mg/g in the root hexane extract and 5.89 mg/g in the stem hexane extract. In the fruit extracts, kokusaginine (2) was the most abundant compound in the methanolic extract (36.23 mg/g), while 3-O-methoxyglycocitrine II (8) was the most abundant in the hexane extract (19.25 mg/g). To ensure specificity and unambiguous identification, qualitative characterization of the crude extract was performed using liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS). All eight alkaloids exhibited a unique fragmentation pattern consistent with radical cation chemistry. Protein-protein interaction (PPI) analysis identified 24 hub genes. Gene Ontology (GO) and KEGG pathway enrichment analyses indicated significant involvement of these hub genes in PI3K-Akt signalling, proteoglycan-mediated oncogenesis, and intracellular signal transduction. A compound-target-pathway network further highlighted 5-hydroxynoracronycine (5) as a potential anticancer lead compound. This study integrates analytical chemistry and systems pharmacology to enable quality assessment and mechanistic insights into the anticancer potential of G. pentaphylla alkaloids.