Plantago major extracts have demonstrated considerable efficacy in promoting wound healing. However, there is limited research evaluating the angiogenic potential of P. major extract using Matrigel-based assays alongside gene expression analysis of key angiogenic markers such as vascular endothelial growth factor A VEGFA and vascular endothelial growth factor receptor 2VEGFR2
.This study evaluated the angiogenic effects of P. major extracts obtained by several extraction techniques: ultrasound-assisted extraction (UAE) for leaves (LU) and both ultrasound-assisted extractionUAE and maceration for non-leaf components (NLC and NLU) using in vitro human umbilical vein endothelialHUVEC cells. Aucubin content was analyzed using an HPTLC-dDensitometer, revealing the highest aucubin content in the non-leaves extract obtained from ultrasound-assisted extraction NLU extract (16.75%). Furthermore, an in vitro experiment with human umbilical vein endothelial cellsHUVEC was conducted to assess P. major extract’s effect on cell viability, migration, and the formation of capillary-like structures (tube formation). All extracts maintained cell viability above 80% at concentrations below 250 µg/mL. The leaves extract obtained from ultrasound-assisted extractionLU extract at 31.25 µg/mL showed the greatest wound closure (80.29%) and the highest branching length (2756.41 cm). However, gene expression of vascular endothelial growth factor AVEGFA and vascular endothelial growth factor receptor 2VEGFR2 showed no
significant upregulation in the leaves extract obtained from ultrasound-assisted extractionLU43 treated group, and low replication numbers limited some assays. These findings suggest that P. major leaf extract may promote angiogenesis through mechanisms beyond vascular endothelial
growth factor VEGF signaling, but further studies with higher statistical power and broader mechanistic approaches are warranted
