Two-step dual-layer SPE method to separate antibacterial and antioxidant mushroom compounds

We developed a two-step dual-layer solid-phase extraction (SPE) method to separate and concentrate minor bioactive compounds of golden oyster mushroom (Pleurotus citrinopileatus) methanol extract. The mushroom extract is rich in fatty acids, including the highly abundant (20–35 %) and antibacterial linoleic acid. To eliminate the masking effect of linoleic acid in the bioassay, its removal was achieved by homemade dual-layer SPE that consisted of a lower C₁₈ phase and an upper silica gel phase with the dried-on extract. In the first step the elution was performed with water (aqueous eluate), and then the dried C₁₈ and silica gel phases were eluted separately with methanol (C₁₈ methanol eluate and silica gel methanol eluate, respectively). Thin-layer chromatography (TLC)-effect-directed analysis demonstrated that the aqueous eluate contained sugars and antioxidants (TLC-DPPH); fatty acids were present in the silica gel methanol eluate, in the C₁₈ methanol eluate the biologically active minor secondary metabolites were concentrated (TLC–Bacillus subtilis assay) beside 2 % remained linoleic acid according to HPLC-UV measurement. As a result, the antioxidant and antibacterial metabolites divided, and the low-abundant antibacterial compounds could be detected. Based on this study, the substance found at R_F 0.25 by TLC-DPPH played the most significant role in generating the antioxidant effect of the aqueous eluate, equivalent to 5.07 ± 0.09 µg of gallic acid. Moreover, each SPE fraction and raw extract were tested for antibacterial effect using a microplate assay with a non-pathogenic Gram-positive Bacillus subtilis. The P. citrinopileatus extract (MIC-value – 50 µg/mL) and C₁₈ methanol eluate (MIC-value - 25 µg/mL) inhibited the growth of B. subtilis while the other eluates did not.