Separation of Lower Polarity Components From <i>Ganoderma lucidum</i> Spores by a Combined Approach of Ultra‐High Pressure Extraction and pH‐Zone‐Refining Counter‐Current Chromatography

An integrated strategy combining ultra-high pressure-assisted extraction and pH-zone-refining counter-current chromatography was developed for the efficient extraction and separation of the lower polar constituents from Ganoderma lucidum spores. The ultra-high pressure extraction parameters were optimized using response surface methodology, achieving a high extraction yield of 1.739% under mild conditions (liquid-to-solid ratio of 21 mL/g, pressure of 268 MPa, time of 7 min), demonstrating higher efficiency and avoidance of thermal degradation compared to conventional techniques. For separation, pH-zone-refining counter-current chromatography was employed for the crude ultra-high pressure extraction using n-hexane/ethyl acetate/methanol/water (20:3:4:1, v/v, 10 mM trifluoroacetic acid in the upper phase and 10 mM aqueous ammonia in the lower phase), yielding Compounds 1-3, Fraction I, and the eluted Fraction II. Subsequently, for Fractions I and II, pH-zone-refining counter-current chromatography was further carried out using n-hexane/ethyl acetate/methanol/water (6:5:4:2, v/v, 10 mM trifluoroacetic acid in the upper phase and 10 mM aqueous ammonia in the lower phase) and n-hexane/ethyl acetate/methanol/water (60:5:40:4, v/v, 10 mM trifluoroacetic acid in the upper phase and 10 mM aqueous ammonia in the lower phase) as the solvent systems, yielding Compounds 4-6 and 7-10, respectively. All these obtained 10 compounds were classified into seven fatty acids, one lipid, one terpenoid, and one phthalate ester, which were all characteristic of low polarity, especially for the fatty acids with chain lengths ranging from C₁₁ to C₂₆. This integrated ultra-high pressure extraction and pH-zone-refining counter-current chromatography approach markedly outperforms conventional techniques by drastically shortening the extraction time and enabling a highly effective, large-scale separation. Consequently, the ultra-high pressure extraction and pH-zone-refining counter-current chromatography platform represents a robust and promising methodology for the systematic investigation of hydrophobic constituents from natural resources.