In Vitro Evaluation of the Angiogenic Potential of Plantago Major Extract in Enhancing the Wound-Healing Process

<i>Plantago major</i> extracts have demonstrated considerable efficacy in promoting wound healing. However, there is limited research evaluating the angiogenic potential of <i>P. major</i> extract using Matrigel-based assays alongside gene expression analysis of key angiogenic markers such as vascular endothelial growth factor A and vascular endothelial growth factor receptor 2. This study evaluated the angiogenic effects of <i>P. major</i> extracts obtained by several extraction techniques: ultrasound-assisted extraction for leaves and both ultrasound-assisted extraction and maceration for non-leaf components using <i>in vitro</i> human umbilical vein endothelial cells. Aucubin content was analyzed using an HPTLC-densitometer, revealing the highest aucubin content in the non-leaves extract obtained from ultrasound-assisted extraction (16.75%). Furthermore, an <i>in vitro</i> experiment with human umbilical vein endothelial cells was conducted to assess <i>P. major</i> 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 extraction 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 A and vascular endothelial growth factor receptor 2 showed no significant upregulation in the leaves extract obtained from ultrasound-assisted extraction-treated group, and low replication numbers limited some assays. These findings suggest that <i>P. major</i> leaf extract may promote angiogenesis through mechanisms beyond vascular endothelial growth factor signaling, but further studies with higher statistical power and broader mechanistic approaches are warranted.