Production, isolation, and identification of dihydroepiepoformin as an IL-1 receptor antagonistic component in Penicillium patulum.

extracted twice with an equal volume of ethyl acetate. The organic layer after concentration was dissolved in MeOH/waterand passed through a column of Diaion HP-20. The column was eluted with a stepwise water/ MeOHgradient. Active fractions were combined, freezedried, and dry-packed to a silica column. Elution with a CH2Cl2/MeOH gradient resolved the activity into gentisyl alcohol and mixture of EEF and DHEEF. The separation of DHEEFfrom EEF was achieved by using preparative HPLC(Waters Prep 3000) procedures. The mixture was loaded onto a C-18 preparative radial compression cartridge (2.5 x 10cm) via a 5ml loop. The column was then developed isocratically with 15% MeOHin water for 15 minutes. The flow rate was 4.5ml/minute. Since DHEEFdoes not have a UV chromophore, the fractions were collected with a ISCO 200 fraction collector using constant volume mode and peak separator. The fractions near the EEF peak were then analyzed by proton NMR(300 MHz, Bruker AM300). The peak fraction of DHEEFwas estimated to be 90% pure by measuring the NMRsignal intensities.