Agrimonia eupatoria L. (AE) has a rich tradition of use in wound healing improvement across various cultures worldwide. In previous studies, we revealed that Agrimonia eupatoria L. water extract (AE) possesses a rich polyphenolic composition, displaying remarkable antioxidant properties. Our investigations also demonstrated that lipophosphonoxin (LPPO) exhibited antibacterial efficacy in vitro while preserving the proliferation and differentiation of fibroblasts and keratinocytes. Building upon our prior findings, in this study, we intended to examine whether a combination of AE and LPPO could enhance skin wound healing while retaining antibacterial attributes. The antibacterial activity of AE/LPPO against Staphylococcus aureus was evaluated, alongside its effects on fibroblast-to-myofibroblast transition, the formation of extracellular matrix (ECM), and endothelial cells and keratinocyte proliferation/phenotype. We also investigated AE/LPPO's impact on TGF-β1 and VEGF-A signaling in keratinocytes/fibroblasts and endothelial cells, respectively. Additionally, wound healing progression in rats was examined through macroscopic observation and histological analysis. Our results indicate that AE/LPPO promotes myofibroblast-like phenotypic changes and augments ECM deposition. Clinically relevant, the AE/LPPO did not disrupt TGF-β1 and VEGF-A signaling and accelerated wound closure in rats. Notably, while AE and LPPO individually exhibited antibacterial activity, their combination did not lead to synergism, rather decreasing antibacterial activity, warranting further examination. These findings underscore substantial wound healing improvement facilitated by AE/LPPO, requiring further exploration in animal models closer to human physiology.

2nd Department of Surgery Louis Pasteur University Hospital Pavol Jozef Šafárik University 040 11 Košice Slovakia

Department of Biomedical Research East Slovak Institute of Cardiovascular Diseases 040 11 Košice Slovakia

Department of Chemical Theory of Drugs Faculty of Pharmacy Comenius University 832 32 Bratislava Slovakia

Department of Infectology and Travel Medicine Louis Pasteur University Hospital Pavol Jozef Šafárik University 040 11 Košice Slovakia

Department of Microbiology Faculty of Medicine and Dentistry Palacký University 779 00 Olomouc Czech Republic

Department of Natural Drugs Faculty of Pharmacy Masaryk University 601 77 Brno Czech Republic

Department of Pathology Louis Pasteur University Hospital Pavol Jozef Šafárik University 040 11 Košice Slovakia

Department of Pharmacognosy and Botany Faculty of Pharmacy Comenius University 832 32 Bratislava Slovakia

Department of Pharmacology Faculty of Medicine Pavol Jozef Šafárik University 04001 Košice Slovakia

Department of Surgery AGEL Hospital Košice Šaca Pavol Jozef Šafárik University 04001 Košice Slovakia

Institute of Neurobiology Biomedical Research Center of the Slovak Academy of Sciences 04001 Košice Slovakia

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences v v i 160 00 Prague Czech Republic

Prague Burn Center 3rd Faculty of Medicine University Hospital Královske Vinohrady Charles University 100 00 Prague Czech Republic

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