Achillea clypeolata: extraction, phenolic profile, cytotoxic and antioxidant activities, and pro-angiogenic potential in human keratinocytes

Species of the genus Achillea L. (Asteraceae) are recognized as valuable sources of natural antioxidants and are widely used for their diverse pharmacological properties. This study aimed to examine the phenolic composition of the methanolic extract of the aerial parts of yarrow, Achillea clypeolata (ACE), as well as its cytotoxicity, antioxidant effects, and the potential molecular mechanisms involved in angiogenesis, which plays a crucial role in wound healing. The extract's composition was analyzed using the HPLC method. Total polyphenol content (TPC) of 116.69±8.18 mg GAE (gallic acid equivalent)/g dry extract was determined viathe Folin–Ciocalteu assay. Antioxidant activity was assessed spectrophotometrically, showing 38.68±2.29 μmol Trolox equivalents pergram of dry extract based on the ABTS radical inhibition and an IC₅₀ (concentration required to neutralize 50% of free radicals) value of 685.6±9.8 μg/mL for DPPH• radical scavenging. Cytotoxicity was evaluated in HaCaT human keratinocyte cells over a concentration range of 1 to 200 μg/mL, identifying concentrations below 50 μg/mL as non-cytotoxic. The production of intracellular reactive oxygen species (ROS) in HaCaT cells exposed to the extract with or without hydrogen peroxide treatment was measured using the H2DCFDA assay. The results demonstrated that ACE is rich in flavonoids and phenolic acids with significant in vitro antioxidant potential. It also effectively reduced ROS levels in HaCaT cells. The impact of ACE on pro-angiogenic factors (VEGFA, HiF1α, and MIF) was evaluated at the protein level using cell-based ELISA, and it was shown that ACE at 25 μg/mL significantly enhanced the expression of all examined factors. These findings suggest that ACE is a promising bioactive compound with potential uses in the pharmaceutical and cosmetic industries, as well as in the creation of scaffolds that encourage angiogenesis and wound healing.