Structural Identification and Coagulation Effect of Flammulina Velutipes Polysaccharides

: 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 trans-form-infrared spectroscopy (FT-IR), gas chromatography (GC), gas chromatography–mass spec-trometry (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-Gal p -(1 → and the branch of Gal p by an → 3,6)- α -D-Man p -(1 → attached with T- β -D-Glc p or t- α -L-Fuc p side chains. Meanwhile, the CHFVP-2 was a glucan with the construction of → 6)-β-D-Glc p -(1 → and T-β-D-Glc p . 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 def-inite coagulant activity. The finding would benefit the further application of F. velutipes in the field of medicine. this study, the crude polysaccharides, CHFVP-1 and CHFVP-2, were extracted, isolated, and purified from F. velutipes . Then, the relative molecular weight, physical and chemical properties, monosaccharide composition, residue segments, linkages, and the possible primary structure of the two F. velutipes polysaccharides were analyzed with corresponding methods. Meanwhile, the coagulation activity of the purified F. velutipes polysaccharides was studied in vitro. Funding Resources,


Introduction
Flammulina velutipes, one of the edible mushrooms of Flammulina, is popular in Asia, North America, and Europe, and its production and consumption have increased rapidly due to its rich nutritional values and industrialized cultivation [1]. According to the finding of many researchers, polysaccharides, well-known key functional compounds in F. velutipes fruit bodies, have a variety of bioactivities, such as immunomodulatory [2], antitumor [3], antioxidant [4], anti-proliferation [5], anti-Hepatitis B Virus(HBV) activity [6], hepatocyte protection [7], renoprotective effects [8], and so on.
In our previous study, we found that several polysaccharides have coagulation or anticoagulation activities [9][10][11][12]. Up to date, there is no research on the coagulation effect of the F. velutipes polysaccharides. In this study, the crude polysaccharides, CHFVP-1 and CHFVP-2, were extracted, isolated, and purified from F. velutipes. Then, the relative molecular weight, physical and chemical properties, monosaccharide composition, residue segments, linkages, and the possible primary structure of the two F. velutipes polysaccharides were analyzed with corresponding methods. Meanwhile, the coagulation activity of the purified F. velutipes polysaccharides was studied in vitro.

Materials
F. velutipes sample were dried, ground, and collected from Henan Lonfon Industrial Corporation Limited (Puyang, China) in 2016.

Extraction and Purification of Polysaccharides from F. Velutipes
The polysaccharides of F. velutipes were extracted and purified by the modified method of Zhang [11] and Wang [12]. During the method, lipids, pigments, and small molecular compounds were all washed out from F. velutipes firstly. Then, the residue was extracted with distilled water three times. The supernatant was filtered, combined, and concentrated. After that, the crude polysaccharides were precipitated by adding ethanol and washed successively with anhydrous ethanol, acetone, and petroleum ether. Then, Sevag reagent and dialysis bags were used to deproteinize the crude polysaccharides and remove some salts. Finally, crude F. velutipes polysaccharides (FVP) were obtained after the freeze-dried processing.
First, we dissolved the crude FVP in distilled water and separated it into fractions by a Diethylaminoethyl cellulose-52 column (DEAE-52 cellulose column). Then, the fractions were eluted by solvent gradient chromatography with 0, 0.05, 0.1, 0.2, and 0.5 M NaCl (1.3 mL/min). The phenol-sulfuric acid method was used to monitor the factions of crude FVP, and some completely separated fractions were collected. After dialysis, concentration, and purification with a Sephadex G-100 column, two main fractions, CHFVP-1 and CHFVP-2, were obtained.

Infrared Spectrum Analysis
The infrared spectrum of CHFVP-1 and CHFVP-2 was measured by a Fourier-transform infrared (FT-IR) spectrometer with the KBr pellet method in the range of 4000-400 cm −1 .

Molecular Weight Determination Analysis
The relative molecular weight of CHFVP-1 and CHFVP-2 were commissioned for Beijing Physical and Chemical Analysis and Testing Center and determined according to the 2015 edition of the Pharmacopoeia of the People's Republic of China.

Monosaccharide Composition Analysis
Gas chromatography (GC) in reference [12], a popular method for monosaccharide composition, was adopted in this paper. Briefly, hydrolyze sample with trifluoroacetic acid (TFA) and get the hydrolysates. Mix it with hydroxylamine hydrochloride, inositol hexyl acetate, and pyridine, add acetic anhydride, and bathe the mixture in hot water for 40 min. After that, dissolve it in chloroform. Filter the reactants and analyze it by gas chromatography fitted with a Thermo TG-Waxms capillary column (30 m × 0.32 mm × 0.5 μm). The analysis situation of GC was the same as Wang's research [12]. The related monosaccharide (glucose, arabinose, xylose, ribose, fucose, mannose, fructose, galactose, and rhamnose) was derived with the above method.

Methylation Analysis
In this part, the analysis of CHFVP-1 and CHFVP-2 was consistent with the method of Ciucanu et al. [14]. Briefly, dissolve the sample in the dimethyl sulfoxide (DMSO); then, add NaOH dry powder and CH3I to methylate it. During this step, the absorption (3200-3700 cm −1 ) on infrared spectroscopy was used to judge the success of methylation. The sample after methylation was hydrolyzed at 100 °C by adding Trifluoroacetic Acid (TFA). After cooling, NaOH solution and NaBH4 were added to the sample for reaction, and next, acetic acid was used to neutralize the sample. Then, we added pyridine and acetic anhydride for acetylation before analysis with the GC-Mass Spectrophotometer (MS) system with a J&W DB-5 ms capillary column (30 m × 250 μm × 0.25 μm). The GC situation was that the temperature increased from 110 to 190 °C at the rate of 5 °C/min, to 210 °C at 3 °C/min and finally to 230 °C at 5 °C/min.

Nuclear Magnetic Resonance (NMR) Spectroscopy
Dissolve 15 mg of CHFVP-1 and CHFVP-2 in 0.5 mL of D2O (the internal standard was Tetramethylsilane, TMS), respectively and load them into the NMR tube. NMR experiments were recorded on a Bruker-AV-500 spectrometer at 25 °C. The measured spectra included 1 H, 13 C, H/H correlation spectroscopy (COSY), heteronuclear single quantum coherence spectroscopy (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC).

Coagulation Activities In Vitro
CHFVP-1 and CHFVP-2 were separately dissolved in saline, and 5 mg/mL of sample solutions were obtained. Then, we mixed them together for the next step. Breviscapine were dissolved in saline to get 13.3 mg/mL solution. The preparation method of Yunnan Baiyao was similar to Breviscapine [15]. Rabbits were used as experimental animals, and then PT, APTT, and TT detection kits were used for determination. Sodium citrate in saline was taken into an eppendorf (EP) tube, rabbit ear venous blood was added, and plasma was prepared after being centrifuged at 3000 g/min for 15 min (TGL-16gR, Shanghai Anting Scientific Instrument Co., Shanghai, China). CHFVP-1 and CHFVP-2 were transferred to plasma, and APTT was added. After preheating and incubating it for 5 min, steel balls and CaCl2 solution were added successively and pre-warmed at 37 °C. Finally, the experimental results were recorded. For PT analysis, CHFVP-1 and CHFVP-2 and serum were incubated with the coagulation and minimum steel ball cell at 37 °C for 3 min. After that, PT reagent was added at 37 °C. TT analysis was determined according to the instruction. During the determination, according to many researchers, breviscapine and Yunnan Baiyao were taken as anti-coagulated [16] and pro-coagulated [17] reagents, respectively, while the normal saline was taken in the blank control group. Record the thrombus time with the RAC-030 automatic coagulation analyzer (Shenzhen Leidu Life Science Co., Ltd., Shenzhen 518067, China). The software SPSS 22.0 was used for data analysis.

Preparation of Polysaccharides from F. Velutipes
After the extraction of polysaccharides from F. velutipes, the crude polysaccharides were obtained, and the yield was about 1.33% (on dry; g/g). After the DEAE-52 cellulose column isolation, FVP was separated into five components (Figure 1a). In this study, FVP-1 and FVP-2 were selected for the next step of purification and structural study. Then, the purified yields of FVP-1 and FVP-2 were 8.4% and 36.2% respectively and pu-rified with the Sephadex G-100 column to obtain the pure subfraction CHFVP-1 ( Figure  1b) and CHFVP-2 ( Figure 1c) with a yield of 88.4% and 78.9% (by dry weight).

Spectroscopic Characteristics
The sample of FVP, CHFVP-1, and CHFVP-2 were scanned through ultraviolet spectrum, and the characterization of their absorption spectrum is shown in Figure 2. It could be found that there was no ultraviolet absorption at 260 and 280 nm, which means that FVP, CHFVP-1, and CHFVP-2 did not contain free protein and nucleic acid.
The infrared spectra of CHFVP-1 and CHFVP-2 at the range of 400-4000 cm −1 were scanned and are shown in Figure 3. The two fractions revealed the hydroxyl group (-OH) for the broad and strong absorption peak at around 3400 cm −1 [18]. The peaks at 2935 and 2923 cm −1 indicated the C, H and aliphatic C-H bonds, respectively [19]. Stretching peaks at 1642 and 1653 cm −1 corresponded to free carboxyl groups [20]. The broad absorption bands with intense intensities around 1420-1300 cm −1 could be owing to stretching vibrations of the C-H bond [21]. The absorption band at 1000-1200 cm −1 suggested that the two polysaccharides contained pyranose monomers in their structures [22]. The absorption bands at 882 and 888 cm −1 indicated the β-pyranoside linkage in CHFVP-1 and CHFVP-2 [23].

Molecular Weight and Monosaccharides Composition of CHFVP-1 and CHFVP-2
The average molecular masses of CHFVP-1 and CHFVP-2 were 24.44 and 1497 kDa, respectively ( Table 1). The GC result of standard monosaccharides is shown in Figure 4a. According to the comparison between Figure 4a  1.024 Note: The number average molecular weight (Mn, g/mol) is the statistical average molecular weight of all polymeric chains in the polysaccharides; the weight average molecular weight (Mw, g/mol) is the statistical average of all molecular weights in the polysaccharides; Mp (g/mol) is defined as the molecular weight of the highest peak; Mz (g/mol) is defined as Z-average molecular mass, which is calculated according to the precipitation equilibrium method to determine the chain length; the ratio of Mw/Mn is called the polydispersity coefficient, which is used to describe the molecular mass distribution of polysaccharides.

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 δ 3.0 to 5.50 and δ 60 to 110, respectively, which were the typical chemical shifts of the polysaccharide [24]. Furthermore, four anomeric proton and carbon chemical shifts at δ 4.53/106.29, δ 5.001/100.79, δ 5.11/105.25, and δ 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 δ 18.52 in the 13 C NMR and the proton shift at δ 1.23 in the 1 H NMR indicated the existence of terminal-fucose (T-fuc) in CHFVP-1 [25,26]. The peak at δ 105.25 indicated the presence of 13 C of α-1,3,6-linked D-mannopyranose units [27][28][29]. The peak at δ 100.79 meant the existence of C1 of the α-D-galactose [30]. The signal peak appearing at δ 64.0 suggested the presence of the C-6 galactose in the polysaccharide sugar chain structure [31]. The peaks in the region of δ 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 δ 4.53-5.11 revealed that there were both α-and β-configurations in it [32]. According to the anomeric chemical shift at δ 4.53/106.29 and literature comparison, the β-configuration of residue A was determined [33]. From the COSY spectrum ( Figure  5c), the chemical shifts of H2-H6 can be deduced to δ 3.47, δ 3.72, δ 3.96, δ 4.36, and δ 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-β-D-Glcp.
Based on the terminal 13 C and 1 H shift at δ5.06/104.44, the α-configuration of the D residue was inferred. In the HSQC spectrum (Figure 5d), the cross-peak at δ1.23/18.525 indicated the H6/C6 of T-α-L-Fucp. 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 Table 4 and Table 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 residue fragments were inferred. According to the correlation between the peaks of 13  CHFVP-2 was analyzed by the same way as that used for CHFVP-1. In Figure 6, anomeric proton and carbon signals at δ 4.53/105.88 were determined in the 1 H and 13 C NMR (Table 6 and Table 7) and HSQC spectra in Figure 6 and denoted as A and B in CHFVP-2 [34]. All the 1 H and 13 C signals were assigned by 1 H/ 1 H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), as shown in Figure 6, indicating that residue A was T-β-D-Glcp and B was →6)-β-D-Glcp-(1→.
The structure of CHFVP-2 was proposed based on the above analysis in the same way used for CHFVP-1, and the possible partial structure of CHFVP-2 information was shown as follows:

Effects of CHFVP-1 and CHFVP-2 on Parameter Coagulation Parameters In Vitro
Four kinds of coagulation kits were used to detect the coagulation activity of polysaccharide components, including APTT, PT, and TT in vitro.