Preparative isolation of highly polar free radical inhibitor from Floccularia luteovirens using hydrophilic interaction chromatography directed by on-line HPLC-DPPH assay.

Wild edible macro fungi Floccularia luteovirens proved to be a valuable source for the identification of novel lead molecules with therapeutic potential. Nevertheless, the chemical constituents of Floccularia luteovirens are rarely reported due to absence of efficient purification methods. In this study, a hydrophilic interaction chromatography directed by on-line HPLC-DPPH assay has been developed and successfully applied for the isolation of free radical inhibitor from the methanolic extract of Floccularia luteovirens. Using a hydrophilic interaction chromatographic column coupled with the HPLC-DPPH assay for screening the potential radical scavengers, the mid-pressure hydrophilic interaction chromatography (HILIC) proved to be more efficient in the pretreatment stage, yielding the fraction rich in free radical scavengers in good yield (5.9% recovery from 130.0 g of fresh F. luteovirens). From highly potent fraction, the target compound was isolated using the Click XION preparative chromatography with 17.2% recovery. The isolated compound was L-(+)-ergothioneine, where the purity (>95%) and antioxidant activity of were confirmed by chromatography and HPLC-DPPH assay, while the structure of this compound was elucidated from HR ESI-MS and NMR data. This method proved to be very efficient for the recognition and isolation of highly polar free radical inhibitors from fungi extracts, and is also applicable for the purification of highly polar compounds from other sources.