4.4. Extraction and Purification of HEA

The mycelial powder was defatted using supercritical extraction carbon dioxide (SCO₂) under 5000 psi at 60 °C for 1 h, then subjected to solvent extraction with boiling water (1:10 w/v) for 2 h, filtered through 0.22 µm microporous membrane to obtain the aqueous extract which was evaporated under vacuum to one tenth original volume (designated as CCM–1). The filtrate was concentrated under vacuum on a rotary evaporator and freeze-dried (CCM–2). The obtained sample (CCM–2) was then subjected to column separation using the Sephadex^® LH–20 column (id × ℓ = 1.5 × 30 cm) and eluted with double distilled water at a flow rate 1 mL/min. The eluent was collected, each fraction from 3 min, until a total of 40 fractions. The OD at 260 nm was used to detect the absorbance of each tube. The fractions from fractions 33–35 were pooled and subjected to further HPLC-ESI-(+)-MS/MS analysis to identify the purified HEA, and then lyophilized (1.07 ± 0.04 mg/g dry weight). The obtained fraction was used for biochemical analyses of HK–2 cells.