Antioxidant and Anti-inflammatory Activities of Glc-Man-Gal Heteroglycans from Paecilomyces hepiali Cs-4 Mycelium by Synergistic Autoclaving-Ultrasonication Extraction

Cordyceps sinensis polysaccharides are valued for their immunomodulatory and antioxidant properties, but the conventional hot water extraction typically achieves yields of only 3%-11%. In addition, the relationship between the extraction technique, polysaccharide structure, and function in Cs-4 ( Paecilomyces hepiali ) mycelium remains poorly understood. This study is the first to apply an autoclaving‑ultrasonic extraction (AUE) method to Cs-4 ( Paecilomyces hepiali ) mycelium, achieving a markedly improved crude polysaccharide yield of 37.35% while preserving high‑molecular‑weight, bioactive polysaccharide fractions. This work provides a foundation for structure–function studies and AUE application of Cs‑4 polysaccharides. In comparison with conventional hot-water extraction (HWE), single-step autoclaving extraction (AE), and ultrasonic-assisted extraction (UE), AUE yielded the highest crude PS with the highest sugar content, while retaining the high-molecular-weight and similar composition of monosaccharides as UE, Glc-Man-Gal heteroglycans. The AUE extract was then precipitated and fractionated with different ethanol precipitation concentrations from 40-80% (v/v) into five PS fractions, PS40, PS50, PS70, and PS80. PS40 and PS50 were rich in glucose with the highest molecular weight (3.98 × 10 6 and 6.81 × 10 5 kDa), while PS70 and PS80 were rich in both mannose and glucose with relatively low molecular weights (4.62 × 10 3 and 8.05 × 10 3 kDa), respectively. All the PS fractions significantly suppressed the lipopolysaccharide (LPS)-stimulated reactive oxygen species (ROS) and NO levels in RAW264.7 macrophages, with PS70 and PS80 demonstrating the most significant effects. In addition, all PS fractions attenuated macrophage proliferation, while PS80 notably suppressed macrophage phagocytic activity. PS40 significantly suppressed IL-1β expression and attenuated the diurnal oscillatory amplitude of both IL-1β and TNF-α expressions, while PS50 uniquely restored the disrupted diurnal rhythmicity of MCP-1 expression in LPS-stimulated macrophages. Based on these findings, the AUE technique, which synergistically integrates AE and UE, emerged as the most efficient method for the extraction of Cs-4 PS, having potential antioxidative and anti-inflammatory properties.