Phytochemical characterization and antiangiogenic effect of <i>Salvadora persica</i> using chorioallantoic membrane (CAM) assay: An <i>in vitro</i>, <i>in vivo</i>, and <i>in silico</i> investigations

Hepatocellular carcinoma (HCC) has a high prevalence and mortality worldwide. Plant Salvadora persica (SP) possesses therapeutically essential phytochemicals. The study evaluated in vitro cytotoxic, antiproliferative, and antiangiogenic effects of SP aqueous (SP-AQ) and ethanolic (SP-E) extracts and in vivo their antiangiogenic effects using the chorioallantoic membrane (CAM) assay. Phytochemicals of SP active extract were identified and studied in silico against the angiogenic protein, vascular endothelial growth factor(VEGF)-A, to determine their interactions. In vitro, in HepG2 cells, the MTT assay (cytotoxicity), immunocytochemistry assay (VEGF-A), RT-qPCR assay (proliferative genes MKI67, PCNA), and in vivo, the CAM assay in fertilized chicken eggs for 3D quantification of blood vessels were performed. Identification of active SP-E extract phytomolecules by gas chromatography- mass spectrometry (GC-MS), and their molecular docking study against VEGF-A by AuToDOCK Tools were performed. In an in vitro study, SP-E extract (IC50 = 46.7 µg/mL) significantly decreased cell viability compared to SP-AQ extract (IC50 = 65.8 µg/mL) and considerably reduced VEGF-A level in HepG2 cells. In vivo study, SP-E extract significantly reduced the diameter of primary, secondary, and tertiary blood vessels, height, and 3D surface roughness parameters of blood vessels compared to SP-AQ extract. The GC-MS analysis revealed (3-methoxyphenyl)acetonitrile and bis(2-ethylhexyl)isophthalate as major phytomolecules in SP-E extract, whereas (3-methoxyphenyl)acetonitrile and naphtho(2,1,8,7-klmn)xanthene exhibited higher docking score against VEGF-A. SP-E extract of SP is a potent cytotoxic, antiproliferative, and antiangiogenic candidate compared to the SP-AQ extract.