Bis vanillate ester with antioxidant activity and selective modulation of the NO/iNOS pathway in LPS-activated macrophages

Abstract Vanillic acid (VA), a phenolic acid categorized as a polyphenol found in plants and foods, is known for its antioxidant, anti-inflammatory, and other therapeutic properties. The phenolic functional group is well known as a critical structural characteristic for antioxidant action via free radical scavenging and the aromatic system's stability of the produced radical. To enhance VA’s antioxidant activity, a bis vanillate ester (compound 1 ) was designed and synthesized. The bis vanillate ester showed high antioxidant activity in the DPPH free radical scavenging assay, however it had lower ABTS radical scavenging activity. The compound demonstrated higher anti-lipid peroxidation action than VA. The correlation between antioxidant and anti-inflammatory activity urges further investigation into its anti-inflammatory properties in LPS-induced RAW264.7 macrophages. The bis vanillate ester significantly reduced nitric oxide production and iNOS expression without significantly altering the secretion of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, indicating a selective effect on the NO/iNOS pathway under the experimental conditions. Transcriptome profiling showed upregulation of ribosome biogenesis genes ( Nol6, Nop58, Wdr75, Nmd3, and Wdr36 ) and downregulation of cellular stress to hypoxia and lysosome genes ( Atp6v0d2, Hyal1, Lgmn, Psap, and Litaf ). These findings demonstrate that bis vanillate ester selectively modulates the NO/iNOS pathway in LPS-stimulated macrophages while having minimal effects on the measured pro-inflammatory cytokines. Transcriptomic analysis further identified compound-associated changes in genes related to ribosome biogenesis and lysosomal pathways, providing preliminary insights into the cellular responses associated with compound 1 treatment.