This study aimed to establish an efficient extraction process for polysaccharides from yellow-feathered chicken intestine (YFCI-P), to analyze the structural characteristics of its key components, and to evaluate their in vitro anticoagulant and antithrombotic activities, thereby providing a theoretical basis for the high-value utilization of chicken intestinal by-products.

Single-factor experiments combined with Response Surface Methodology (RSM) were used to optimize the enzymatic hydrolysis extraction conditions of YFCI-P; The anticoagulant potency of the enzymatic hydrolysate was determined by the sheep plasma method. The polysaccharide components YFCI-P1 and YFCI-P2 were obtained by separation and purification through gel filtration column chromatography. Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) levels were assessed to evaluate the anticoagulant activity of the polysaccharides, alongside an assessment of in vitro thrombolytic activity. The molecular weight of YFCI-P1 was determined by High-Performance Gel Permeation Chromatography (GPC/HPLC). Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) techniques were applied to analyze its functional group composition and glycosidic linkage patterns.

The optimum enzymatic hydrolysis conditions were determined as follows: temperature 55 ℃, enzyme-to-substrate ratio 0.9%, time 3 h, sodium chloride concentration 3%, solid-to-liquid ratio 1:1 (g·mL^-1), and pH 8.5. Under these conditions, the anticoagulant potency of the enzymatic hydrolysate reached 5.13 U·mL^-1. The predicted value from the response surface model showed good agreement with the experimental value. Two polysaccharide fractions, including YFCI-P1 and YFCI-P2, were obtained through separation and purification by gel filtration chromatography. In vitro anticoagulant activity studies demonstrated that at a concentration of 1000 μg·mL^-1, YFCI-P significantly prolonged APTT to 485.7 s; YFCI-P1 significantly prolonged PT to 600 s; and YFCI-P2 significantly prolonged TT to 367 s, suggesting their anticoagulant effects via the intrinsic and extrinsic pathways, extrinsic and common pathways, and intrinsic and common pathways, respectively. Molecular characterization revealed that YFCI-P1 had a weight-average molecular weight (Mw) of 10.2 kDa and contained uronic acid, acetyl amino, and sulfate groups. Its backbone consisted of repeating disaccharide units composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) linked alternately by β-1, 4 and β-1, 3 glycosidic bonds, exhibiting the characteristic features of glycosaminoglycans. Furthermore, YFCI-P, YFCI-P1, and YFCI-P2 promoted tissue plasminogen activator (tPA) generation and demonstrated in vitro thrombolytic activity, with clot dissolution rates of 42.38%, 36.91%, and 19.74% at 1000 μg·mL^-1, respectively.

An efficient and stable extraction process for YFCI-P was successfully established, and the optimized parameter combination demonstrated promising potential for industrial application. YFCI-P1 was identified as a polysaccharide exhibiting structural features characteristic of glycosaminoglycans. Moreover, YFCI-P, YFCI-P1, and YFCI-P2 all exhibited significant in vitro anticoagulant and thrombolytic activities.