Extraction, purification, characterization, and in vitro anticoagulant activity of polysaccharides from Cordyceps militaris

Abstract

To elucidate the anticoagulant effects of Cordyceps militaris polysaccharides and provide a scientific basis for their application in functional foods and biomedicine. Crude polysaccharide (CC-1) was extracted by water extraction and ethanol precipitation, and then purified by papain-mediated deproteinization and dialysis to obtain polysaccharide CC-2. Chemical composition was analyzed using the phenol–sulfuric acid method, the meta-hydroxybiphenyl method, and a BCA protein assay kit. Molecular weight was determined by high-performance gel permeation chromatography (HPGPC). Monosaccharide composition was analyzed by PMP pre-column derivatization HPLC (PMP-HPLC). Structural features were characterized by Fourier transform infrared (FT-IR) and ultraviolet (UV) spectroscopy, and scanning electron microscopy (SEM). In vitro anticoagulant activity was evaluated by measuring activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) levels. The yield of CC-2 was 2.16%. Compared with CC-1, CC-2 showed significantly higher total sugar and uronic acid contents and a decreased protein content. The molecular weight of CC-2 was 5908.612 Da. Its monosaccharide composition consisted primarily of rhamnose, glucuronic acid, glucose, galactose, and xylose in a molar ratio of 5:1:4:11:4. FT-IR indicated the presence of acetyl groups and a furanose ring conformation. SEM revealed a microstructure composed of irregular lumps and spherical particles. In vitro , CC-2 significantly prolonged PT, APTT, and TT ( P < 0.001) by approximately 2.10, 2.46, and 2.79 fold, respectively, and significantly reduced FIB levels ( P < 0.001). Its FIB-lowering effect was superior to that of the positive control, breviscapine. Cordyceps militaris polysaccharides exhibit significant in vitro anticoagulant activity, acting through potential multi-pathway mechanisms and pronounced fibrinogen reduction. These findings support their potential development as functional food ingredients or biomedical agents for the prevention of thrombotic cardiovascular and cerebrovascular diseases.