The initial structural features and in vitro biological study of crude polysaccharides from Calocybe indica (CICP) extracted by hot water followed by ethanol precipitation was investigated. High-performance gel permeation chromatography, HPLC-DAD, UV, IR and NMR spectroscopy, X-ray diffraction, scanning electron microscopy, and Congo red methods were used to determine structural features. The results revealed that CICP is a hetero-polysaccharide with a molecular weight of 9.371 × 104 Da and 2.457 × 103 Da which is composed of xylose, mannose, fucose, rhamnose, arabinose, galactose, and glucose. The antioxidant activity of CICP was evaluated using radical scavenging activity (three methods), reducing ability (three methods), metal chelating activity, and lipid peroxidation inhibition activity (two methods). It was found that the antioxidant capacity is concentration-dependent and EC50 values were found to be 1.99–3.82 mg/mL (radical scavenging activities), 0.78–2.78 mg/mL (reducing ability), 4.11 mg/mL (metal chelating activity), and 0.56–4.18 mg/mL (lipid peroxidation inhibition activity). In vitro anticoagulant assay revealed that CICP could prolong activated partial thromboplastin time (APTT), thrombin time (TT), but not prothrombin time (PT). CICP exhibited antiproliferative activity on HeLa, PC3, HT29, HepG2, and Jurkat cell lines with IC50 (μg/mL) values of 148.40, 143.60,151.00, 168.30, and 156.30, respectively. The above findings suggested that CICP could be considered a natural antioxidant and cancer preventative.

An acidic polysaccharide fraction was obtained from Calocybe indica (CIP3a) after subjecting it to hot water extraction followed by purification through DEAE-cellulose 52 and Sepaharose 6B column chromatography. The CIP3a was further modified using chloroacetic acid to yield carboxymethylated derivatives (CMCIP3a). The modified polysaccharide was characterized using various spectroscopic methods. In addition, further antioxidant, antitumor and anticoagulant activities were also investigated. The polysaccharides CIP3a and CMCIP3a were heterogeneous in nature and composed of various molar percentages of glucose, arabinose and mannose with molecular weights of 1.456 × 103 and 4.023 × 103 Da, respectively. The NMR and FT-IR data demonstrated that the carboxymethylation on the polysaccharide was successful. In comparison to CIP3a polysaccharides, the modified derivatives had lower sugar and protein contents, and higher levels of uronic acid. The in vitro antioxidant activity showed that CMCIP3a with higher molecular weight displayed an elevated ability in scavenging the DPPH radical, ABTS, superoxide, hydroxyl radical, ferric reducing power, cupric reducing power and erythrocyte hemolysis inhibition with an EC50 value of 2.49, 2.66, 4.10, 1.60, 3.48, 1.41 and 2.30 mg/mL, respectively. The MTT assay results revealed that CMCIP3a displayed a dose-dependent inhibition on five cancer cells (HT29, PC3, HeLa, Jurkat and HepG-2) in the range of 10–320 μg/mL. The APTT, PT and TT were significantly extended by CMCIP3a in relation to dosage, indicating that the anticoagulant effect of CIP was both extrinsic and intrinsic, along with a common coagulation pathway. These findings demonstrated that carboxymethylation might effectively improve the biological potential of the derivatives and offer a theoretical framework for the creation of novel natural antioxidants, low-toxicity antitumor and antithrombotic drugs.