Comparative antioxidant activity of pomegranate leaf extract and derived metabolites in single and co-cultured cell...

The maintenance of cellular redox homeostasis with endogenous and exogenous antioxidants is crucial for regulating physiological functions. Plant extracts contain compounds that provide exogenous antioxidants, enhancing cellular protection against oxidative stress. This work aims to investigate the antioxidant potential of the ethanolic total extract (TE) and some derivatives (i.e., ellagic acid/EA, luteolin/LU) of the leaves of Punica granatum L. (pomegranate). Human dermal fibroblast (HDF) and human umbilical vein endothelial (HUVEC) cells were considered both as a single cell line culture and as a co-culture. TE and the single compounds were tested as primary or secondary antioxidants using hydrogen peroxide as a pro-oxidant, assessing cell viability, reactive oxygen species generation, and mRNA expression of antioxidant genes. Briefly, LU and EA showed a significant activity as primary antioxidants in H₂O₂-exposed HDF and HUVEC, respectively. TE showed a significant activity as a secondary antioxidant only in H₂O₂-exposed HDF-HUVEC co-culture, where any activity was observed with LU and EA treatment. Analysis of the mRNA expression of CAT, GPX1, NFE2L2, NOQ1 and SOD1 on HDF-HUVEC co-culture revealed TE ability to induce a strong increase in the expression of GPX1 and NOQ1, not observed on the single cell lines, where TE was mainly able to induce a moderate increase in the expression of SOD1. The results show different activity of the extract/compounds across cell lines and conditions. The co-cultured cell assay confirms the potential of pomegranate leaf extract in preventing oxidative stress-induced cell damage and modulating antioxidant gene expression.