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An efficient procedure for ultrasound-assisted enzymatic extraction of crude polysaccharides from Trichosanthes Fructus (crude TFP) using response surface methodology (RSM) was developed. The Box–Behnken design was applied to optimize the effects of pH (X1), enzyme amount (X2), extraction temperature (X3), and liquid-to-solid ratio (X4) on the extraction. The statistical analysis indicated that the independent variables (X4, X2, and X3), the quadratic coefficients (X12, X22, X32, and X42), and the interaction coefficient (X1X3) had significant impact on the yield of crude TFP. The optimal conditions were determined as follows: pH 4.5, enzyme amount 5000 u/g, extraction temperature 45°C, and liquid-to-solid ratio 30 ml/g. The experimental yield of crude TFP was 6.58%, which was very close to the predicted yield of 6.71%. TFPI was then purified and characterized with Sephadex G-100 column, UV-Vis, GPC, and FT-IR. The average molecular weight of TFPI was calculated to be 1.49 × 105 Da. TFPI exhibited strong reducing power and possessed not only remarkable scavenging activities against ABTS•+ and DPPH radicals, but also high antitumor activities in C4-2, DU145, and PC3 cells. The results suggest that Trichosanthes Fructus and TFPI could be a novel potent natural medicine with antioxidant and antitumor activities.

Proteomics and intestinal flora were used to determine the mechanism of immune modulatory effects of Flammulina velutipes polysaccharide on immunosuppressed mice. The results showed that compared with the model group, F. velutipes polysaccharide could increase thymus and spleen indices and improve thymus tissue structure in mice; IL-2 and IL-4 contents were significantly increased and IL-6 and TNF-α contents were significantly decreased; serum acid phosphatase (ACP), lactate dehydrogenase (LDH) and total antioxidant capacity (T-AOC) activities were increased (P < 0.05); in the liver, superoxide dismutase (SOD) and catalase (CAT) activities were increased (P < 0.001), while malondialdehyde (MDA) content was decreased (P < 0.001). Proteomics discovered that F. velutipes polysaccharides may exert immune modulatory effects by participating in signaling pathways such as immune diseases, transport and catabolism, phagosomes and influenza A, regulating the immune-related proteins Transferrin receptor protein 1 (TFRC) and Radical S-adenosyl methionine domain-containing protein 2 (RSAD2), etc. Gut microbial studies showed that F. velutipes polysaccharides could increase the abundance of intestinal flora and improve the flora structure. Compared to the model group, the content of short-chain fatty acids (SCFAs) and the relative abundance of SCFA-producers Bacteroides and Alloprevotella were increased in the F. velutipes polysaccharide administration group, while Lachnospiraceae_NK4A136_group and f_Lachnospiraceae_Unclassified decreased in relative abundance. Thus, F. velutipes polysaccharide may play an immunomodulatory role by regulating the intestinal environment and improving the balance of flora.

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Two polysaccharides were isolated successfully from Flammulina velutipes and identified as CHFVP-1 (24.44 kDa) and CHFVP-2 (1497 kDa). Based on the results of Fourier transform-infrared spectroscopy (FT-IR), gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), and nuclear magnetic resonance (NMR) spectroscopy regarding the structure of CHFVP-1 and CHFVP-2, CHFVP-1 was constructed with the backbone of→6)-α-D-Galp-(1→ and the branch of Galp by an →3,6)-α-D-Manp-(1→attached with T-β-D-Glcp or t-α-L-Fucp side chains. Meanwhile, the CHFVP-2 was a glucan with the construction of →6)-β-D-Glcp-(1→ and T-β-D-Glcp. Moreover, the coagulant activity in vitro of CHFVP-1 and CHFVP-2 was evaluated, and the results showed that CHFVP-1 exerts procoagulant activity by shortening the activated partial thromboplastin time (APTT) and thrombin time (TT), while CHFVP-2 did not reveal a definite coagulant activity. The finding would benefit the further application of F. velutipes in the field of medicine.