In Vitro Evaluation of The Angiogenic Potential of Plantago major Extract in Enhancing the Wound Healing Process

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 VEGFA and VEGFR2. This study evaluated the angiogenic effects of P. major extracts obtained by several extraction techniques: ultrasound-assisted extraction (UAE) for leaves (LU) and both UAE and maceration for non-leaf components (NLC and NLU) using in vitro HUVEC cells. Aucubin content was analyzed using HPTLC-Densitometer, revealing the highest aucubin content in the NLU extract (16.75%). Furthermore, an in vitro experiment with HUVEC 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 LU extract at 31.25 µg/mL showed the greatest wound closure (80.29%) and the highest brancing length (2756.41 cm). However, gene expression of VEGFA and VEGFR2 showed no significant upregulation in the LU-treated group, and low replication numbers limited some assays. These findings suggest that P. major leaf extract may promote angiogenesis through mechanisms beyond VEGF signaling, but further studies with higher statistical power and broader mechanistic approaches are warranted.