Integration of bioassay-guided high-performance thin-layer chromatography with spectroscopy for assessing phytochemical profile of anti-inflammatory compounds from Helichrysum italicum extracts.

This study investigated the antioxidant and anti-inflammatory properties of Helichrysum italicum flower and leaf extracts using High-Performance Thin-Layer Chromatography coupled with Effect-Directed Analysis (HPTLC-EDA), employing microchemical derivatization and in situ bioassays. The goal was to enhance antioxidant and anti-inflammatory activity through spontaneous fermentation. Although fermentation increased the flavonoid content and enhanced antioxidant activity of the flower extracts, it reduced the total phenolic content and did not improve their anti-inflammatory properties. While fermented ethyl acetate and ethanol leaf extracts showed increased anti-inflammatory activity compared to their non-fermented extracts, it was still lower than that of the non-fermented flower extracts. Notably, the ethyl acetate flower extract showed strong COX-1 inhibition (IC₅₀ = 42.42 µg/band), with a much lower IC50 than salicylic acid (IC50 = 557.58 µg/band) under these assay conditions. Spectroscopic analysis (FTIR, NMR, LC-MS) of bioactive zones isolated via preparative TLC identified triterpenoid acids and santinols, rather than flavonoids, as the primary contributors to anti-inflammatory activity.