Optimization of Crude Polysaccharide Extraction and Purification from the Black Outer Layer of the Inonotus obliquus Sterile Conk and Evaluation of the Purified Polysaccharide's In Vitro Antioxidant Activity, Inhibitory Effects on Tumor Cell Viability, and Immunomodulatory Activity.

Inonotus obliquus (I. obliquus) is a medicinal fungus rich in polysaccharides, polyphenols, and triterpenoids. However, studies on purified polysaccharide from the black outer layer of the I. obliquus sterile conk remain limited. In this study, crude polysaccharides were extracted by hot-water extraction, optimized using response surface methodology, and further purified by DEAE-52 ion-exchange chromatography and Sephacryl S-400 HR gel filtration chromatography. The structural characteristics and in vitro biological activities of the purified polysaccharide (IOP) were investigated. Under the optimized conditions, the crude polysaccharide extract yield reached 24.64 ± 0.48%. IOP contained 88.97 ± 1.33% total sugars and 10.91 ± 0.37% uronic acids, with a weight-average molecular weight of 78.0 kDa. Monosaccharide analysis revealed that IOP consisted of glucose, galactose, galacturonic acid, and glucuronic acid, and FT-IR analysis revealed the characteristic functional groups of polysaccharides. IOP exhibited concentration-dependent antioxidant activities, inhibited the cell viability of A549, SW1990, 4T1, and B16F10 cells with low cytotoxicity toward L929 fibroblasts, and stimulated the production of NO, TNF-α, IL-6, and IL-1β in RAW264.7 macrophages. These findings provide preliminary evidence that the purified IOP fraction obtained from the black outer layer of the I. obliquus sterile conk exhibits in vitro antioxidant, inhibitory effects on tumor cell viability, and immunomodulatory activities under the tested experimental conditions.