# Structural Identification and Coagulation Effect of _Flammulina velutipes_ Polysaccharides
## 3 Results and Discussion
### NMR Analysis of CHFVP-1 and CHFVP-2
Regarding the complete \({}^{1}\)H and \({}^{13}\)C chemical shifts of residue B as well as previous work [35], residue B was \(\rightarrow\)6)-\(\alpha\)-D-Gal\(p\)-(1\(\rightarrow\). The heterotopic hydrogen and carbon signals of H-1 and C-1 were \(\xi\) 5.11/105.25. According to the literature, residue C was inferred to be an \(\alpha\)-configuration residue. Other signals from H-2 to H-6a were identified from the results of COSY spectrum (Figure 5c), including H-2 (\(\xi\) 3.91), H-3 (\(\xi\) 3.99), H-4 (\(\xi\) 4.07), H-5 (\(\xi\) 4.22), and H-6a (\(\xi\) 4.00). In addition, the matching \({}^{13}\)C chemical shifts were C-2 (\(\xi\) 69.75), C-3 (\(\xi\) 80.37), C-4 (\(\xi\) 71.21), C-5 (\(\xi\) 71.61), and C-6 (\(\xi\) 74.25) in the HSQC spectrum (Figure 5d). The chemical shifts of all \({}^{1}\)H and \({}^{13}\)C of residue C were basically consistent with the literature [29], which indicated that residue C was \(\rightarrow\)3,6)-\(\alpha\)-D-Man\(p\)-(1\(\rightarrow\). Based on the terminal \({}^{13}\)C and \({}^{1}\)H shift at 55.06/104.44, the \(\alpha\)-configuration of the D residue was inferred. In the HSQC spectrum (Figure 5d), the cross-peak at 81.23/18.525 indicated the H6/C6 of T-\(\alpha\)-L-Fuc\(p\). The remaining hydrogen chemical shifts of D come from the COSY (Figure 5c) spectrum, and the \({}^{13}\)C chemical shifts associated with it were determined in the HSQC spectrum (Figure 5d). Compared with the literature, the \({}^{1}\)H and \({}^{13}\)C chemical shifts for residue D were assigned [26]. The NMR chemical shifts and significant connectivity are summarized in Tables 4 and 5. Due to the large molecular weight and viscosity of CHFVP-1 and the wide distribution of branched chains, the NMR data of the sample were incomplete, and only partial sugar \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Glycosyl Residue**} & \multirow{2}{*}{**Sugar Linkage**} & \multicolumn{6}{c}{**Chemical Shift \& H/C (ppm)**} \\ \cline{3-8}  & & **H1/C1** & **H2/C2** & **H3/C3b** & **H4/C4** & **H5/C5** & **H6a/C6** \\ \hline A & H & 4.53 & 3.35 & 3.49 & 3.66 & 3.86 & 3.77/3.90 \\ T-\(\beta\)-D-Glc\(p\) & C & 106.29 & 75.97 & 78.78/72.3 & 72.83 & 71.08 & 63.832 \\ B & H & 5.00 & 3.89 & 4.21 & 4.07 & 3.79 & 3.91/3.70 \\ \(\rightarrow\)6)-\(\alpha\)-D-Gal\(p\)-(1\(\rightarrow\) & C & 100.79 & 72.43 & 71.56 & 71.21 & 76.21 & 69.45 \\ C & H & 5.11 & 3.91 & 3.99 & 4.07 & 4.22 & 4.00 \\ \(\rightarrow\)3,6)-\(\alpha\)-D-Man\(p\)-(1\(\rightarrow\) & C & 105.25 & 69.75 & 80.37 & 71.21 & 71.61 & 74.25 \\ D & H & 5.06 & 3.86 & 3.91 & 4.074 & 4.17 & 1.23 \\ T-\(\alpha\)-L-Fuc\(p\) & C & 104.44 & 71.35 & 71.56 & 71.21 & 71.77 & 18.52 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: \({}^{1}\)H and \({}^{13}\)C NMR chemical shifts of CHFVP-1 fraction recorded in D\({}_{2}\)O at 291 K.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Sugar Residue**} & \multirow{2}{*}{**Sugar Linkage**} & \multicolumn{6}{c}{**H-1/C-1 (ppm) Connectivities**} \\ \cline{3-6}  & & \(\delta_{\mathbf{H}}\)/\(\delta_{\mathbf{C}}\) & \(\delta_{\mathbf{H}}\)/\(\delta_{\mathbf{C}}\) & **Residue** & **Atom** \\ \hline A & T-\(\beta\)-D-Glc\(p\) & 4.53 & 71.77 & D & C-5 \\  & & 106.29 & 3.35 & A & H-2 \\ B & \(\rightarrow\)6)-\(\alpha\)-D-Gal\(p\)-(1\(\rightarrow\) & 5.001 & 71.77 & D & C-5 \\  & & 100.79 & & & \\ C & \(\rightarrow\)3,6)-\(\alpha\)-D-Man\(p\)-(1\(\rightarrow\) & 5.11 & 74.25 & C & C-6 \\  & & 76.21 & B & C-5 \\  & & 80.37 & C & C-3 \\  & & 1 residue fragments were inferred. According to the correlation between the peaks of \({}^{13}\)C and \({}^{1}\)H from the HMBC spectrum (Figure 5e), the glycosides of the CHFVP-1 residues were linked as follows: AC-1/AH-2; AH-1/DC5; BH-1/DC-5; CC-1/AH-6; CH-1/BC-5/CC-6/3; DH-1/B/C/DC-4; DC-1/AH-2.