Optimization of deep eutectic solvents-based ultrasonic-assisted three-phase partitioning extraction of polysaccharides from Pleurotus tuber-regium and its anti-ulcerative colitis activity

Based on the various anti-inflammatory and antioxidant bioactivities of Pleurotus tuber-regium and its polysaccharides (PTPs), they represent promising candidates for complementary or alternative therapeutic strategies against Ulcerative Colitis (UC), but conventional extraction methods are often inefficient or environmentally unfriendly. This study presents a novel integrated deep eutectic solvents (DESs)-based ultrasonic-assisted three-phase partitioning (TPP) extraction technique of PTPs to solve the dual challenges of inefficient traditional extraction methods and the unexplored therapeutic potential of PTPs against UC. First, it establishes an optimized and sustainable extraction process, enhancing the yield and potential bioavailability of PTPs. Single-factor assays followed by Box-Behnken Design (BBD) were employed to optimize key parameters: DES-2 (choline chloride:1,6-hexanediol = 1:2, molar ratio), temperature 40℃, material-liquid ratio 1:33 mg/mL, ammonium sulphate mass fraction 15%, upper and lower phase (ammonium sulphate: DES-2) volume ratio 1:2, time 40 min, power 320 W. Structural characterization utilized UV spectrophotometer (UV-Vis) and Fourier transform infrared (FT-IR) spectrometer, high-performance gel permeation chromatography (HPGPC), and scanning electron microscopy (SEM). Monosaccharide composition was analyzed via PMP derivatization and HPLC. Second, it provides the first comprehensive preclinical evidence, using dextran sulfate sodium (DSS)-induced Drosophila colitis model, to systematically validate the anti-UC efficacy of the optimized PTPs extract. The findings bridge a critical gap, offering both an advanced extraction method for efficient utilization of PTPs and a pharmacological foundation for developing PTPs as a promising nutraceutical or botanical drug candidate for inflammatory bowel disease management.