# Structural Identification and Coagulation Effect of _Flammulina velutipes_ Polysaccharides
## 3 Results and Discussion
### NMR Analysis of CHFVP-1 and CHFVP-2  
The NMR spectra of CHFVP-1 are shown in Figure 5, which were the basis for inferring monosaccharide, glycosidic linkages, and chemical shifts. In Figure 5a,b, most of the proton and carbon chemical shifts of CHFVP-1 come from the regions ranging from \(\delta\) 3.0 to 5.50 and \(\delta\) 60 to 110, respectively, which were the typical chemical shifts of the polysaccharide [24]. Furthermore, four anomeric proton and carbon chemical shifts at \(\delta\) 4.53/106.29, \(\delta\) 5.001/100.79, \(\delta\) 5.11/105.25, and \(\delta\) 5.06/104.44 were identified in the \({}^{1}\)H NMR, \({}^{13}\)C NMR, and HSQC in Figure 5 and indicated as A, B, C, and D in CHFVP-1. According to the composition of monosaccharides, methylation results, and literature comparison, the carbon signals at \(\delta\) 18.52 in the \({}^{13}\)C NMR and the proton shift at \(\delta\) 1.23 in the \({}^{1}\)H NMR indicated the existence of terminal-fucose (T-fuc) in CHFVP-1 [25; 26]. The peak at \(\delta\) 105.25 indicated the presence of \({}^{13}\)C of \(\alpha\)-1,3,6-linked D-mannopyrananose units [27; 28; 29]. The peak at \(\delta\) 100.79 meant the existence of C1 of the \(\alpha\)-D-galactose [30]. The signal peak appearing at \(\delta\) 64.0 suggested the presence of the C-6 galactose in the polysaccharide sugar chain structure [31]. The peaks in the region of \(\delta\) 63.83-80.37 indicated the typical shift of carbohydrate. The glycosidic linkage of CHFVP-1 determined by its \({}^{1}\)H NMR spectra in the region of \(\delta\) 4.53-5.11 revealed that there were both \(\alpha\)- and \(\beta\)-configurations in it [32]. According to the anomeric chemical shift at \(\delta\) 4.53/106.29 and literature comparison, the \(\beta\)-configuration of residue A was determined [33]. From the COSY spectrum (Figure 5c), the chemical shifts of H2-H6 can be deduced to \(\delta\) 3.47, \(\delta\) 3.72, \(\delta\) 3.96, \(\delta\) 4.36, and \(\delta\) 3.76, respectively. The \({}^{13}\)C chemical shifts of residue A were obtained from the HSQC spectrum by the chemical shift of \({}^{1}\)H (Figure 5d), and the results are summarized in Table 4. Furthermore, according to the related literature and the chemical shifts of all \({}^{1}\)H and \({}^{13}\)C of residue A [25; 34], residue A should be terminal-\(\beta\)-D-Glc\(p\). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Retention Time (min)** & **PMAA** & **Linkages Types** & **Percentage (\%)** \\ \hline
20.26 & 2,3,4,6-Me\({}_{4}\)-Glc & T-Glc & 28.23 \\
22.33 & 2,4-Me\({}_{2}\)-Man & 1,3,6-Linked-Man & 10.18 \\
23.13 & 2,3,4-Me\({}_{2}\)-Glc & 1,6-Linked-Glc & 19.00 \\
24.14 & 2,3,4-Me\({}_{3}\)-Gal & 1,6-Linked-Gal & 33.25 \\
26.07 & 3,4-Me\({}_{2}\)-Man & 1,2,6-Linked-Man & 9.24 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: GC-MS date for methylation analysis of CHFVP-1. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Retention Time (min)** & **PMAA** & **Linkages Types** & **Percentage (\%)** \\ \hline
20.28 & 2,3,4,6-Me\({}_{4}\)-Glc & T-Glc & 67.01 \\
23.15 & 2,3,4-Me\({}_{3}\)-Glc & 1,6-Linked-Glc & 32.99 \\ \hline \hline \end{tabular} Note: PMAA is the sugar alcohol acetate derivatives.  
\end{table} 
Table 3: GC-MS date for methylation analysis of CHFVP-2. Figure 5: \({}^{1}\)H spectrum (a); \({}^{13}\)C spectrum (b); COSY spectrum (c); HSQC spectrum (d); HMBC spectrum (e). According to the anomeric chemical shift at \(\xi\) 5.00/100.79 and literature comparison, the \(\alpha\)-configuration of residue B was determined. The other chemical shifts of H-2, H-3, H-4, H-5, H-6 and H-6a at \(\xi\) 3.89, \(\xi\) 4.21, \(\xi\) 4.07, \(\xi\) 3.79, \(\xi\) 3.91 and \(\xi\) 3.70 were found from the cross-peaks in the COSY spectra (Figure 5c), and the corresponding carbon signals could be attributed to the correlations of C-H signals in the HSQC spectrum (Figure 5d), which were \(\xi\) 72.43, \(\xi\) 71.56, \(\xi\) 71.21, \(\xi\) 76.21, and \(\xi\) 69.45 for C-2, C-3, C-4, C-5, and C-6, respectively.