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A novel polysaccharide from Malus halliana Koehne with coagulant activity
Carbohydrate Research 485 (2019) 107813
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
A novel polysaccharide from Malus halliana Koehne with coagulant activity a,1
a,1
a
Xiaofeng Zhang , Qi Yu , Huimin Jiang , Changyang Ma Jinmei Wanga,b,∗∗, Wen-Yi Kanga,b,∗ a b c
a,b
c
, Hui Min David Wang ,
T
National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China Kaifeng Key Laboratory of Functional Components in Health Food, Kaifeng, 475004, China Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung City, 402, Taiwan
ARTICLE INFO
ABSTRACT
Keywords: Malus halliana Koehne Polysaccharides Structural characterization Coagulant activity
A novel polysaccharide in Malus halliana Koehne, named MHP-W, was isolated and purified by DEAE-52 cellulose and Sephadex G-100 columns. Structural features were identified by high performance size-exclusion chromatography (HPSEC), fourier transform infrared (FT-IR) spectrometer, gas chromatography (GC) and (1D & 2D) NMR Spectroscopy. Structural characterization showed that the molecular weight of MHP-W was 353 kDa composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2.59: 0.15: 0.23: 0.25: 9.70. The existence of β-glycosidic bond between the sugar units was confirmed by FT-IR and NMR spectroscopy. The effects of MHP-W on active part thrombin time (APTT), protothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) were screened by a cell-based coagulation activity model. MHP-W could significantly shorten TT (p < 0.001) and increase FIB (p < 0.05) as compared with the control group. The results showed that MHP-W promoted bloodclotting through endogenous and exogenous coagulation pathways as well as increasing fibrinogen content, which indicated that MHP-W had procoagulant activities in vitro.
1. Introduction Malus halliana Koehne belongs to the family Rosaceae. M. halliana flowers are tasteless, bitter, plain, through liver-channel according to the theory of Traditional Chinese Medicine [1]. It has been used for treatment of irregular menstruation. Its flowers are used to make sweet and delicious candies in China. We had carried out chemical analysis and pharmacological research on M. halliana. The results showed that many chemical compounds, including flavonoids, terpenoids, sterols, volatile compounds were found in M. halliana. Pharmacological research indicated that M. halliana possessed the capability of protecting the liver, antioxidant, inhibition of α-glycosidase and improving functional constipation [2–6]. Hu and coworkers found that sugar synthesis and metabolism in the Malus halliana roots were affected by Fe deficiency [7]. Currently, there is an increasing interest on polysaccharides. The previous studies indicated that polysaccharides possessed multifarious functions such as immunomodulatory, antioxidation, antitumor, antiviral, anti-stress, radiation resistance and anti-aging [8–13]. The biological activity of the polysaccharide is closely related to its
structure [14]. For example, medicinal mushrooms polysaccharides, which have β-(1 → 3) linkages in the main chain of the glucan and additional β-(1 → 6) branch points, have antitumor and immunomodulating activity [15,16]. The aim of the current study was to isolate and identify polysaccharides from M. halliana. After structural characterization, potential coagulation activities were analyzed by the use of an ex vivo in vitro coagulation assay. 2. Materials and methods 2.1. Plant material Malus halliana Koehne flowers were collected in March 2014 in Kaifeng (Henan, China) and identified by Professor Chang-qin Li. The voucher specimens were deposited at National R & D Center for Edible Fungus Processing Technology, Henan University. 2.1.1. Animals Male rabbit (1.8–2.4 kg) obtained from the Experimental Animal
Corresponding author. National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China. Corresponding author. National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China. E-mail addresses: [email protected] (X. Zhang), [email protected] (Q. Yu), [email protected] (J. Wang), [email protected] (W.-Y. Kang). 1 These authors contributed equally to this work. ∗
∗∗
https://doi.org/10.1016/j.carres.2019.107813 Received 30 April 2019; Received in revised form 24 August 2019; Accepted 8 September 2019 Available online 10 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Elution curve of the crude polysaccharides on DEAE-52 (A), elution curve of MHP-W on Sephadex G-100 column (B) Table 1 Molecular weights of MHP-W Samples
MHP-W
Molecular weight (g/mol) Mn
Mp
Mw
Mz
Mw/Mn
261900
268300
352600
679600
1.360
Mp: the peak molecular weight. Mz: Z average molecular weight. Mw/Mn: the ratio between the molecular weight and the number average molecular weight.
Center of Henan Province (Zhengzhou, Henan, China) was maintained at 25 ± 2 °C and humidity 45–65%, under a 12/12 h light/dark cycle, with free access of water and food in standard cage. 2.1.2. Reagents and instruments DEAE-cellulose-52 was acquired from Whatman (Germany). Sephadex G-100 was obtained from Pharmacia (USA). Standard monosaccharides (L-arabinose, D-xylose, L-rhamnose, D-mannose, Dglucose, D-galactose, D-fucose) were purchased from Dr. Ehrenstorfer GmbH Company (Germany). Pyridine was purchased from Tianjin Damao Chemical Reagent Factory (China). Hydroxylamine hydrochloride and trifluoroacetic acid were from Merck Company (Germany). Yunnan Baiyao injection, 2.775 g/L calcium chloride solution, (Tianjin Pharmaceutical Group Co, Ltd. Xinzheng, 1109051, China). APTT (Lot: 1121911), PT (Lot: 105295), TT (Lot: 121168), FIB (Lot: 132107) assay kits (Shanghai sun biotech Co, Ltd., China) HF6000 Semi-Automated Coagulation Analyzer was purchased from Jinan (China). TGL-16 gR high speed centrifuge was obtained from Shanghai (China). TRACE1310 Gas chromatograph was purchased from Thermo company (USA). CBS-B automatic collector was from Shanghai (China). LL-1500 freeze drier was purchased from Thermo company (USA).
Fig. 2. UV full wavelength scanning of MHP-W
2.2. Extraction and purification of polysaccharides from the Malus halliana The dried flowers of M. halliana (500 g) were extracted with boiling water for three times (1:20 dilution, 3 h for each time). The combined aqueous extracts were concentrated in vacuo and followed by precipitation in 95% ethanol (final concentration 70%) at 4 °C overnight, and then centrifuged at 8000 rpm for 15 min. After centrifugation, the precipitate was dissolved in distilled water and deproteinized 5–10 times by the Sevag method with a mixture of chloroform/1-butanol (4:1 v/v) [17] until protein could not be detected. The protein-free sample was dialysed in distilled water and precipitated with 95% ethanol (final concentration 70%) at 4 °C overnight and centrifuged, the precipitate was redissolved in distilled water and dialyzed for 2 days, lyophilized to
Fig. 3. Infrared spectra of MHP-W
obtain crude polysaccharides. The crude polysaccharide (250 mg) were dissolved in 8 mL of distilled water, filtered through 0.45 μm filters and loaded onto DEAE-52 column (2.5 × 60 cm), sequentially eluted with distilled water and 0.1, 0.2 M NaCl solution at a rate of 0.8 mL/min, collected 6 mL eluent per tube by the automatic collector, and analyzed by the phenol-sulfuric acid method [18]. Three main fractions were collected. The elution curve was drawn with the absorbance as ordinate and the number of 2
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2.3. Structural analysis 2.3.1. Determination of molecular weight The molecular weight of polysaccharide was analyzed by high performance size-exclusion chromatography (HPSEC) according to the People's Republic of China Pharmacopoeia (2015 Edition) in Beijing Center for Physical and Chemical Analysis. 2.3.2. UV spectral analysis The polysaccharide samples were dissolved in distilled water at a concentration of 1 mg/mL and UV spectra were recorded, scanning from 200 nm to 400 nm with UV spectrophotometer. 2.3.3. FT-IR spectral analysis MHP-W (2 mg) were mixed with KBr powder and pressed into a 1 mm pellet for FTIR analysis. FTIR spectra were recorded in the absorbance from 4000 to 400 cm−1. 2.3.4. Determination of monosaccharide compositions MHP-W (10 mg) was hydrolyzed in ampoules with 2 M trifluoroacetic acid (2 mL) for 12 h at 110 °C. After trifluoroacetic acid was removed, the hydrolysates were mixed with 10 mg hydroxylamine hydrochloride and 0.5 mL pyridine and incubated at 90 °C for 30 min. After the reaction completed, the reactants were cooled at room temperature. Acetic anhydride (0.5 mL) was added and incubated at 90 °C for 30 min. The reactants was filtered through 0.22 μm filters and analyzed by gas chromatography fitted with a HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). The initial temperature was 100 °C, increasing to 230 °C at 4 °C ·min−1 for 10 min with N2 as the carrier gas, and flow rate was 2 mL/min, sample volume was 2 μL. The same procedure was used to derivatize the standard monosaccharide (rhamnose, arabinose, xylose, mannose, glucose and galactose). 2.3.5. NMR spectroscopy analysis MHP-W (25 mg) were dissolved with D2O (0.5 mL) at room temperature. 1H and 13C NMR spectra were recorded at 25 °C. HSQC, 1H–1H COSY, HMBC experiments were conducted by the standard Bruker pulse sequence.
Fig. 4. GC chromatogram for monosaccharide composition analysis of MHP-W 1. Rhamnose; 2. Arabinose; 3. Xylose, 4. Mannose; 5. Glucose; and 6. Galactose (A) GC spectrum of monosaccharide reference, (B) GC spectrum showing the monosaccharide compositions of MHP-W
2.3.6. Methylation analysis Eight mg dried MHP-W were dissolved in 0.5 mL DMSO with constant stirring to ensure a complete dissolution. Fifty mg sodium hydroxide was added,the vessel was filled with N2, and the mixture was stirred. Methyl iodide (0.2 mL)was added slowly with ultrasonic treatment for 30 min at 20 °C, the reaction carried out under the protection of nitrogen. The reaction was terminated by adding 1 mL of distilled water. The methylated polysaccharides were extracted with 0.5 mL chloroform. The chloroform extracts were washed by equal volume of water for three times, and passed through sodium sulfate to remove water, and then concentrated to dryness under reducing pressure at 50 °C. The reaction process was repeated until the absorption peak (3400 cm−1) of hydroxyl disappeared entirely in infrared spectrums. The methylated polysaccharides were hydrolyzed with 2 mol/L of acetic acid (0.5 mL) for 2 h at 110 °C, and then reduced by NaBH4 (5 mg) for 2 h at room temperature. The reduced products were further acetylated with acetic anhydride (0.5 mL) at 110 °C for 1 h. The acetylates were dissolved and filtered before GC-MS analysis.
Table 2 GC-MS of alditol acetate derivatives from the methylated product of MHP-W. Methylated sugar
Retention time
Peak area percentage
Molar ratios
Fragments linkage type
3,4-Me2- Araf
20.70
0.33
1.8
2,4-Me2-Gal 2,3,4-Me3-Gal 2,3,4,5-Me4Araf
20.95 24.36 27.38
1.91 5.63 4.78
10.6 31.3 26.5
1,2,5-linked Araf 1,3,6-linked Gal 1,6-linked Gal 1-linked Araf
tubes as abscissa. The fractions eluted by distilled water were concentrated at 48 °C by rotary vacuum evaporator, then dialyzed (Molecular weight cut-off 8000–14000 Da) at 4 °C for 48 h and freeze-dried. The samples (50 mg) were dissolved in 4 mL of distilled water, filtered by 0.22 μm filters and loaded onto Sephadex G-100 (1.5 × 100 cm) and then eluted with distilled water at a flow rate of 0.4 mL/min, the eluent was detected by the phenol-sulfuric acid method [19] and then the eluent was collected and concentrated, dialyzed and freeze-dried. Finally, the polysaccharide was obtained.
2.3.7. Blood coagulation assays MHP-W (1 mg) was dissolved in saline (200 μL) to prepare a concentration of 5 mg/mL solution. The breviscapine (8 mg) was dissolved in saline (600 μL) to obtain a concentration of 13.33 mg/mL solution. Yunnan Baiyao was prepared with the same method. The male rabbit blood was collected from rabbit ear vein containing sodium citrate at 0.109 mol/L tube (citrate/blood: 1/9, v/v) and gently mixed. Then, the blood was centrifuged at 3000 rpm for 15 min at 5 °C to obtain the 3
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Fig. 5. A. 1H NMR spectrum of MHP-W. B. HMBC spectrum of MHP-W of MHP-W
13
C NMR spectrum of MHP-W. C. 1H–1H COSY spectrum of MHP-W. D.1H–13C HSQC spectrum of MHP-W. E. 1H–13C
serum fractions. 25 μL of sample solution was added to the test cup with 100 μL of plasma and 100 μL of APTT reagent pre-warmed at 37 °C. After incubation at 37 °C for 5 min, 100 μL of 0.025 mol/L CaCl2 was added and the solidification time recorded. Finally, the clotting time was recorded. For PT assay, samples (25 μL) were mixed with serum (100 μL) and incubated at 37 °C for 3 min. The PT assay reagent (200 μL), which was pre-hatched for 10 min at 37 °C, was then added and clotting time was recorded. TT and FIB assays were performed according to the manufacture's specifications. For all the clotting assays, saline was used as blank control group. Breviscapine and Yunnan Baiyao were used as the positive control group and the time for clot formation was recorded by Semi-Automated Coagulation Analyzer (Jinan Hanfang Medical Devices Co., Ltd. China).
2.3.7.1. Statistical analysis. All the experimental results were expressed as mean ± standard deviation (SD). The date was statistically analyzed by SPSS19.0 using one-way analysis of variance (ANOVA). 3. Results and discussion 3.1. Extraction and purification The crude polysaccharides from M. Halliana flower were obtained by hot water extraction and alcohol precipitation. The polysaccharides were then further isolated by DEAE-52. Three major fractions eluted with 0, 0.1 and 0.2 M NaCl were collected, concentrated, dialyzed and lyophilized to obtain MP-W, MP-1 and MP-2 (Fig. 1A). Then, the MP-W fraction was further purified by Sephadex G-100 column to obtain
Fig. 5. (continued) 4
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Fig. 5. (continued)
purified polysaccharides MHP-W. The results are shown in Fig. 1B.
results showed that MHP-W did not contain protein and nucleic acid.
3.2. Molecular weight of polysaccharides analysis
3.4. FT-IR spectra analysis
The molecular weight of polysaccharides is just an average distribution of the length of the chain, and different molecular weights are measured in different ways. Even same polysaccharides show a strong variation of the molecular weight (Mw) and the number average molecular weight (Mn). The general dispersion coefficient judges the quality of homogeneous polysaccharide molecules. As shown in Table 1, the average molecular weight of MHP-W was 353 kDa.
The infrared spectroscopy of MHP-W is shown in Fig. 3. There was a larger and wider peak at 3410.29 cm−1, which belonged to the O–H stretching vibration, and there was a small peak in the vicinity of the 2924.09 cm−1, which belonged to the C–H stretching vibration of the alkyl group. 3410.29 cm−1 and 2924.09 cm−1 were the characteristic absorption peaks of carbohydrate. The absorption peak at 1651.07 cm−1 was the stretching vibration of the C]O, and the variable angle stretching vibration of C–H at 1418.71 cm−1. Absorption at 1072.47 cm−1 was the C–O–C stretching vibrations of pyranose ring. The absorption peak near 850 cm−1 was the characteristic absorption peak of α-glucan [20]. The absorption peak near 890 cm−1 was the characteristic absorption of β-glucan [20]. The absorption peak at
3.3. UV spectral analysis The MHP-W was scanned by UV spectra and there were no characteristic absorption peaks detected at 280 nm and 260 nm (Fig. 2). The
Fig. 5. (continued) 5
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Fig. 5. (continued)
897.90 cm-1 showed that there was a β-glycosidic bond [21].
Table 3 1 H and13C NMR chemical shifts of MHP-W recorded in D2O Sugar residue β-1,6-linked-Gal (A) β-1,3,6-Gal (B) α-1,2,5-Araf (C) α-1- Araf (D)
H C H C H C H C
1
2
3
4
5
6
4.47 106.3 4.53 106.07 5.00 110.64 5.24 112.06
3.54 73.63 3.65 72.80 3.85 86.74 4.21 84.21
3.70 75.50 3.74 83.01 4.14 79.31 3.94 79.42
3.97 71.50 4.14 71.40 3.92 85.64 4.13 86.18
3.92 76.64 3.94 79.42 3.88 72.43 3.83 64.19
4.04 72.41 4.03 72.41 3.77
3.5. Determination of monosaccharide composition of polysaccharides by gas chromatography Six monosaccharides were selected as the mixed standard, and the quantity of each monosaccharide standard (m1) was precisely weighed, the peak area (a1) of the monosaccharide was obtained according to the gas chromatogram of the polysaccharide sample. Then the mass of each monosaccharide composition was obtained, and the molar ratio was obtained by (m2) at the molecular weight of each monosaccharide. The results are shown in Fig. 4A and Fig. 4B. MHP-W was composed of arabinose, xylose, mannose, glucose and galactose with a molar ratio of 2.59: 0.15: 0.23: 0.25: 9.70, respectively. 3.6. Methylation analysis of MHP-W The methylation method is an important means to determine the polysaccharide structure, from which the glycosidic linkages of polysaccharides can be judged. The basic principle is that the free hydroxyl groups are completely methylated, and then the methylated polysaccharides are hydrolyzed into partial methylated polysaccharides. The position of the hydroxyl group on the sugar is the position of the polysaccharide glycosyl linkage. Finally, the monosaccharide is hydrolyzed to obtain the derivative for GC-MS analysis. According to the relative retention value and the mass spectrum of each component in the gas chromatogram, the position of the methyl group and the acetyl
Fig. 6. Deducted structure of MHP-W
Table 4 The effect of MHP-W on plasma coagulation parameters Group
Blank Breviscapine Yunnan Baiyao MHP-W
Plasma coagulation parameters APTT (s)
PT (s)
TT (s)
FIB (g/L)
19.70 ± 0.16 25.40 ± 0.18*** 18.18 ± 0.17*** 19.95 ± 0.24
10.33 ± 0.17 11.15 ± 0.24*** 9.32 ± 0.22*** 10.31 ± 0.14
13.63 ± 0.38 16.43 ± 0.34*** 11.38 ± 0.25*** 10.53 ± 0.26***￥￥￥
4.24 ± 0.14 4.93 ± 0.15*** 5.02 ± 0.14*** 4.32 ± 0.16*
Data represent mean ± SD. n = 4; Compared with saline, ***p < 0.001. Compared with Yunnan Baiyao,￥￥￥p < 0.001. 6
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group on the derivative can be judged to confirm the monosaccharide composition and the glycosidic linkage and the position of the oligosaccharide. The results of GC-MS analysis (Table 2) showed that MHP-W contains four linkage forms: 1,2,5-linked Araf, 1,3,6-linked Gal, 1,6-linked Gal and 1-linked Araf.
4.47 ppm) and C-6 of residue of Residue B (δ 72.41 ppm). C-1 of Residue A (δ 106.3 ppm) and H-6 of Residue B (δ 4.03 ppm). The cross peaks between H-1 of Residue B (δ 4.53 ppm) and C-6 of Residue A (δ 72.41 ppm). C-1 of Residue B (δ 106.07 ppm) and H-6 of Residue A (4.04 ppm), which suggested that Residue A was linked to Residue B at the 1, 6-position, and the backbone of MHP-W consisted of consecutively →6)-β-Gal-(1 → 6)-β-Gal-(3–1→. C-3 of Residue B (δ 83.01 ppm) and H-5 of Residue C (δ 3.92 ppm) indicated Residue C was linked to Residue B at the 3-position. The cross peak δ 5.24/86.74 ppm showed the correlation between H-1 of Residue D and C-2 of Residue C, indicating that →α-Araf-(1 → 2)-α-Araf-(5–1→ probably existed in the branch chain. All the data is shown in Fig. 5E. Based on the monosaccharide composition, methylation and GC-MS analysis and NMR spectroscopy, we proposed a structural unit of MHPW as shown in Fig. 6.
3.7. NMR analysis of MHP-W The signals of MHP-W from 1H NMR and 13C NMR spectra were assigned on the basis of monosaccharide composition, glycosidic linkage analysis, chemical shifts and then analyzed. Most of the polysaccharide proton resonance peaks in the 1H NMR spectra were in the range of 3.0–4.5 ppm and several overlap occurred, whereas the chemical shift of the anomeric hydrogen was generally in the range of 4.5–5.5 ppm. The type and number of glycosidic bonds could be determined by anomeric hydrogen. The α-type glycoside heterologous proton was over δ 5.0, while the β-type was generally less than δ 5.0. In Fig. 5A, four anomeric proton signals were found in the 1H NMR spectra. They were designated as A (4.47 ppm), B (4.53 ppm), C (5.08 ppm) and D (5.24 ppm), according to the reference that 4.79 ppm was the symbol of the residual solvent (D2O). Thus, MHP-W contained both α-type and β-type glycosidic bond. The 13C NMR spectra of MHPW are presented in Fig. 5B. The resonance signals between δ 98 ppm and δ 110 ppm in 13C NMR spectra belonged to the anomeric carbon atoms of monosaccharide. Residues A, B, C and D were assigned at 106.3 ppm, 106.07 ppm, 110.64 ppm and 112.06 ppm, respectively. All the 1H and 13C signals were assigned using homonuclear 1H/1H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence spectroscopy (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC). Residue A: the intensive anomeric signals of residue A at 4.47 ppm and 106.3 ppm (Fig. 5D) corresponded to a β-linked residue with relatively high content of MHP-W. This residue was tentatively assigned as β-1, 6-Gal [22,23] compared with the reported data and peak intensity. The proton assignments of residue A were obtained from COSY as shown in Fig. 5C and Table 3. The corresponding 13C NMR chemical shifts of residue A (δ 73.63, 75.49, 71.55, 76.64 and 72.41 ppm for C-2, C-3, C-4, C-5 and C-6, respectively) were revealed by HSQC spectrum. All the 1H NMR and 13C NMR chemical shifts of residue A were consistent with the previous reports [24] and the corresponding intensity was supported by the result of methylation analysis (Table 3), indicating that the residue A was β-1,6-linked-Gal. Residue B: the intensive anomeric signals of residue B at 4.53 ppm and 106.07 ppm corresponded to a β-linked residue. The proton assignments of residue B were obtained from COSY as shown in Table 3. The corresponding 13C NMR chemical shifts of residue A (δ 72.82, 83.01, 71.40, 79.44 and 72.41 ppm for C-2, C-3, C-4, C-5 and C-6, respectively) were revealed by HSQC spectrum. Residue A was tentatively assigned as β-1,3,6-Gal [22] compared with the reported data. Remaining sugar residues: Residues C and D were determined by the same method as above. The similar 1H NMR and 13C NMR chemical shifts between residues C and D suggested that these two spin systems were similar. For most of monosaccharides with α or β form, the chemical shift of C-1 was the range of δ 90–106 ppm. α-Ara is δ 106–110 ppm when it presents as furanoside [25]. The chemical shifts of residues C and D were 110.64 ppm and 112.06 ppm, respectively, indicating that they were α- Araf. When the C-5 of Araf was not substituted, its chemical shift was the range of δ 60–65 ppm [23]. The C-5 of residue C was δ 72.43 ppm, while residue D was δ 64.19 ppm, indicating that residue D was α-1-linked-Araf. Residue C was assigned as α-1,2,5-Araf compared with previous reports. Once the 1H NMR and 13C NMR chemical shifts of all sugar residues were completely assigned, the sequences of these residues were determined by observing inter- and intra-residual connectivities in HMBC spectrum (Fig. 5E). Cross peaks were found between H-1 of residue A (δ
3.8. Coagulation assays in vitro The essence of blood coagulation is the process of transformation of soluble fibrinogenin plasma to insoluble fibrin. The key to this transformation is the occurrence of a series of complex enzymatic reactions that require the involvement of multiple coagulation factors. According to the activation pathway of coagulation factor and the difference of coagulation factors, it can be divided into endogenous coagulation pathway, exogenous coagulation route and coagulation common pathway [26]. According to the results presented in Table 4, MHP-W significantly shortened TT (p < 0.001) as compared to the saline control, and its effect was higher than that of the positive control Yunnan Baiyao (p < 0.001). For APTT and PT no major changes were observed by MHP-W. MHP-W significantly increased FIB (p < 0.05) although to a lesser degree as compared to Yunnan Baiyao and Breviscapine. Results showed that MHP-W promoted blood clotting through endogenous and exogenous coagulation pathways as well as increased fibrinogen content. The results indicated that MHP-W have procoagulant activities in vitro. 4. Conclusions In our study, we prepared a crude polysaccharide from M. halliana flower by water extraction and alcohol precipitation and the resulted polysaccharide was further fractioned into MP-W, MP-1 and MP-2. The MP-W fraction was further purified by Sephadex G-100 column to obtain the purified polysaccharide MHP-W. The average molecular weight of MHP-W was 353 kDa. A further analysis revealed a primary structure of MHP-W to be composed of arabinose, xylose, mannose, glucose and galactose. The majority residues were proven to be 1-linked Araf and 1,6-linked Gal. The coagulation assay showed that MHP-W significantly shortened TT and increase FIB, indicative of a procoagulant activity mainly through endogenous and exogenous coagulation pathways as well as through mechanisms of increased fibrinogen content. Acknowledgments This work was supported by Science and Technology Planning Project of Kaifeng City (1808003), Science and Technology Research Project of Henan Province (182102110473). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.107813. References [1] Chinese Herbalism Editorial Board, SATCM of the People's Republic of China.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
A novel polysaccharide from Malus halliana Koehne with coagulant activity a,1
a,1
a
Xiaofeng Zhang , Qi Yu , Huimin Jiang , Changyang Ma Jinmei Wanga,b,∗∗, Wen-Yi Kanga,b,∗ a b c
a,b
c
, Hui Min David Wang ,
T
National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China Kaifeng Key Laboratory of Functional Components in Health Food, Kaifeng, 475004, China Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung City, 402, Taiwan
ARTICLE INFO
ABSTRACT
Keywords: Malus halliana Koehne Polysaccharides Structural characterization Coagulant activity
A novel polysaccharide in Malus halliana Koehne, named MHP-W, was isolated and purified by DEAE-52 cellulose and Sephadex G-100 columns. Structural features were identified by high performance size-exclusion chromatography (HPSEC), fourier transform infrared (FT-IR) spectrometer, gas chromatography (GC) and (1D & 2D) NMR Spectroscopy. Structural characterization showed that the molecular weight of MHP-W was 353 kDa composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2.59: 0.15: 0.23: 0.25: 9.70. The existence of β-glycosidic bond between the sugar units was confirmed by FT-IR and NMR spectroscopy. The effects of MHP-W on active part thrombin time (APTT), protothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) were screened by a cell-based coagulation activity model. MHP-W could significantly shorten TT (p < 0.001) and increase FIB (p < 0.05) as compared with the control group. The results showed that MHP-W promoted bloodclotting through endogenous and exogenous coagulation pathways as well as increasing fibrinogen content, which indicated that MHP-W had procoagulant activities in vitro.
1. Introduction Malus halliana Koehne belongs to the family Rosaceae. M. halliana flowers are tasteless, bitter, plain, through liver-channel according to the theory of Traditional Chinese Medicine [1]. It has been used for treatment of irregular menstruation. Its flowers are used to make sweet and delicious candies in China. We had carried out chemical analysis and pharmacological research on M. halliana. The results showed that many chemical compounds, including flavonoids, terpenoids, sterols, volatile compounds were found in M. halliana. Pharmacological research indicated that M. halliana possessed the capability of protecting the liver, antioxidant, inhibition of α-glycosidase and improving functional constipation [2–6]. Hu and coworkers found that sugar synthesis and metabolism in the Malus halliana roots were affected by Fe deficiency [7]. Currently, there is an increasing interest on polysaccharides. The previous studies indicated that polysaccharides possessed multifarious functions such as immunomodulatory, antioxidation, antitumor, antiviral, anti-stress, radiation resistance and anti-aging [8–13]. The biological activity of the polysaccharide is closely related to its
structure [14]. For example, medicinal mushrooms polysaccharides, which have β-(1 → 3) linkages in the main chain of the glucan and additional β-(1 → 6) branch points, have antitumor and immunomodulating activity [15,16]. The aim of the current study was to isolate and identify polysaccharides from M. halliana. After structural characterization, potential coagulation activities were analyzed by the use of an ex vivo in vitro coagulation assay. 2. Materials and methods 2.1. Plant material Malus halliana Koehne flowers were collected in March 2014 in Kaifeng (Henan, China) and identified by Professor Chang-qin Li. The voucher specimens were deposited at National R & D Center for Edible Fungus Processing Technology, Henan University. 2.1.1. Animals Male rabbit (1.8–2.4 kg) obtained from the Experimental Animal
Corresponding author. National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China. Corresponding author. National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng, 475004, China. E-mail addresses: [email protected] (X. Zhang), [email protected] (Q. Yu), [email protected] (J. Wang), [email protected] (W.-Y. Kang). 1 These authors contributed equally to this work. ∗
∗∗
https://doi.org/10.1016/j.carres.2019.107813 Received 30 April 2019; Received in revised form 24 August 2019; Accepted 8 September 2019 Available online 10 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Elution curve of the crude polysaccharides on DEAE-52 (A), elution curve of MHP-W on Sephadex G-100 column (B) Table 1 Molecular weights of MHP-W Samples
MHP-W
Molecular weight (g/mol) Mn
Mp
Mw
Mz
Mw/Mn
261900
268300
352600
679600
1.360
Mp: the peak molecular weight. Mz: Z average molecular weight. Mw/Mn: the ratio between the molecular weight and the number average molecular weight.
Center of Henan Province (Zhengzhou, Henan, China) was maintained at 25 ± 2 °C and humidity 45–65%, under a 12/12 h light/dark cycle, with free access of water and food in standard cage. 2.1.2. Reagents and instruments DEAE-cellulose-52 was acquired from Whatman (Germany). Sephadex G-100 was obtained from Pharmacia (USA). Standard monosaccharides (L-arabinose, D-xylose, L-rhamnose, D-mannose, Dglucose, D-galactose, D-fucose) were purchased from Dr. Ehrenstorfer GmbH Company (Germany). Pyridine was purchased from Tianjin Damao Chemical Reagent Factory (China). Hydroxylamine hydrochloride and trifluoroacetic acid were from Merck Company (Germany). Yunnan Baiyao injection, 2.775 g/L calcium chloride solution, (Tianjin Pharmaceutical Group Co, Ltd. Xinzheng, 1109051, China). APTT (Lot: 1121911), PT (Lot: 105295), TT (Lot: 121168), FIB (Lot: 132107) assay kits (Shanghai sun biotech Co, Ltd., China) HF6000 Semi-Automated Coagulation Analyzer was purchased from Jinan (China). TGL-16 gR high speed centrifuge was obtained from Shanghai (China). TRACE1310 Gas chromatograph was purchased from Thermo company (USA). CBS-B automatic collector was from Shanghai (China). LL-1500 freeze drier was purchased from Thermo company (USA).
Fig. 2. UV full wavelength scanning of MHP-W
2.2. Extraction and purification of polysaccharides from the Malus halliana The dried flowers of M. halliana (500 g) were extracted with boiling water for three times (1:20 dilution, 3 h for each time). The combined aqueous extracts were concentrated in vacuo and followed by precipitation in 95% ethanol (final concentration 70%) at 4 °C overnight, and then centrifuged at 8000 rpm for 15 min. After centrifugation, the precipitate was dissolved in distilled water and deproteinized 5–10 times by the Sevag method with a mixture of chloroform/1-butanol (4:1 v/v) [17] until protein could not be detected. The protein-free sample was dialysed in distilled water and precipitated with 95% ethanol (final concentration 70%) at 4 °C overnight and centrifuged, the precipitate was redissolved in distilled water and dialyzed for 2 days, lyophilized to
Fig. 3. Infrared spectra of MHP-W
obtain crude polysaccharides. The crude polysaccharide (250 mg) were dissolved in 8 mL of distilled water, filtered through 0.45 μm filters and loaded onto DEAE-52 column (2.5 × 60 cm), sequentially eluted with distilled water and 0.1, 0.2 M NaCl solution at a rate of 0.8 mL/min, collected 6 mL eluent per tube by the automatic collector, and analyzed by the phenol-sulfuric acid method [18]. Three main fractions were collected. The elution curve was drawn with the absorbance as ordinate and the number of 2
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2.3. Structural analysis 2.3.1. Determination of molecular weight The molecular weight of polysaccharide was analyzed by high performance size-exclusion chromatography (HPSEC) according to the People's Republic of China Pharmacopoeia (2015 Edition) in Beijing Center for Physical and Chemical Analysis. 2.3.2. UV spectral analysis The polysaccharide samples were dissolved in distilled water at a concentration of 1 mg/mL and UV spectra were recorded, scanning from 200 nm to 400 nm with UV spectrophotometer. 2.3.3. FT-IR spectral analysis MHP-W (2 mg) were mixed with KBr powder and pressed into a 1 mm pellet for FTIR analysis. FTIR spectra were recorded in the absorbance from 4000 to 400 cm−1. 2.3.4. Determination of monosaccharide compositions MHP-W (10 mg) was hydrolyzed in ampoules with 2 M trifluoroacetic acid (2 mL) for 12 h at 110 °C. After trifluoroacetic acid was removed, the hydrolysates were mixed with 10 mg hydroxylamine hydrochloride and 0.5 mL pyridine and incubated at 90 °C for 30 min. After the reaction completed, the reactants were cooled at room temperature. Acetic anhydride (0.5 mL) was added and incubated at 90 °C for 30 min. The reactants was filtered through 0.22 μm filters and analyzed by gas chromatography fitted with a HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). The initial temperature was 100 °C, increasing to 230 °C at 4 °C ·min−1 for 10 min with N2 as the carrier gas, and flow rate was 2 mL/min, sample volume was 2 μL. The same procedure was used to derivatize the standard monosaccharide (rhamnose, arabinose, xylose, mannose, glucose and galactose). 2.3.5. NMR spectroscopy analysis MHP-W (25 mg) were dissolved with D2O (0.5 mL) at room temperature. 1H and 13C NMR spectra were recorded at 25 °C. HSQC, 1H–1H COSY, HMBC experiments were conducted by the standard Bruker pulse sequence.
Fig. 4. GC chromatogram for monosaccharide composition analysis of MHP-W 1. Rhamnose; 2. Arabinose; 3. Xylose, 4. Mannose; 5. Glucose; and 6. Galactose (A) GC spectrum of monosaccharide reference, (B) GC spectrum showing the monosaccharide compositions of MHP-W
2.3.6. Methylation analysis Eight mg dried MHP-W were dissolved in 0.5 mL DMSO with constant stirring to ensure a complete dissolution. Fifty mg sodium hydroxide was added,the vessel was filled with N2, and the mixture was stirred. Methyl iodide (0.2 mL)was added slowly with ultrasonic treatment for 30 min at 20 °C, the reaction carried out under the protection of nitrogen. The reaction was terminated by adding 1 mL of distilled water. The methylated polysaccharides were extracted with 0.5 mL chloroform. The chloroform extracts were washed by equal volume of water for three times, and passed through sodium sulfate to remove water, and then concentrated to dryness under reducing pressure at 50 °C. The reaction process was repeated until the absorption peak (3400 cm−1) of hydroxyl disappeared entirely in infrared spectrums. The methylated polysaccharides were hydrolyzed with 2 mol/L of acetic acid (0.5 mL) for 2 h at 110 °C, and then reduced by NaBH4 (5 mg) for 2 h at room temperature. The reduced products were further acetylated with acetic anhydride (0.5 mL) at 110 °C for 1 h. The acetylates were dissolved and filtered before GC-MS analysis.
Table 2 GC-MS of alditol acetate derivatives from the methylated product of MHP-W. Methylated sugar
Retention time
Peak area percentage
Molar ratios
Fragments linkage type
3,4-Me2- Araf
20.70
0.33
1.8
2,4-Me2-Gal 2,3,4-Me3-Gal 2,3,4,5-Me4Araf
20.95 24.36 27.38
1.91 5.63 4.78
10.6 31.3 26.5
1,2,5-linked Araf 1,3,6-linked Gal 1,6-linked Gal 1-linked Araf
tubes as abscissa. The fractions eluted by distilled water were concentrated at 48 °C by rotary vacuum evaporator, then dialyzed (Molecular weight cut-off 8000–14000 Da) at 4 °C for 48 h and freeze-dried. The samples (50 mg) were dissolved in 4 mL of distilled water, filtered by 0.22 μm filters and loaded onto Sephadex G-100 (1.5 × 100 cm) and then eluted with distilled water at a flow rate of 0.4 mL/min, the eluent was detected by the phenol-sulfuric acid method [19] and then the eluent was collected and concentrated, dialyzed and freeze-dried. Finally, the polysaccharide was obtained.
2.3.7. Blood coagulation assays MHP-W (1 mg) was dissolved in saline (200 μL) to prepare a concentration of 5 mg/mL solution. The breviscapine (8 mg) was dissolved in saline (600 μL) to obtain a concentration of 13.33 mg/mL solution. Yunnan Baiyao was prepared with the same method. The male rabbit blood was collected from rabbit ear vein containing sodium citrate at 0.109 mol/L tube (citrate/blood: 1/9, v/v) and gently mixed. Then, the blood was centrifuged at 3000 rpm for 15 min at 5 °C to obtain the 3
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Fig. 5. A. 1H NMR spectrum of MHP-W. B. HMBC spectrum of MHP-W of MHP-W
13
C NMR spectrum of MHP-W. C. 1H–1H COSY spectrum of MHP-W. D.1H–13C HSQC spectrum of MHP-W. E. 1H–13C
serum fractions. 25 μL of sample solution was added to the test cup with 100 μL of plasma and 100 μL of APTT reagent pre-warmed at 37 °C. After incubation at 37 °C for 5 min, 100 μL of 0.025 mol/L CaCl2 was added and the solidification time recorded. Finally, the clotting time was recorded. For PT assay, samples (25 μL) were mixed with serum (100 μL) and incubated at 37 °C for 3 min. The PT assay reagent (200 μL), which was pre-hatched for 10 min at 37 °C, was then added and clotting time was recorded. TT and FIB assays were performed according to the manufacture's specifications. For all the clotting assays, saline was used as blank control group. Breviscapine and Yunnan Baiyao were used as the positive control group and the time for clot formation was recorded by Semi-Automated Coagulation Analyzer (Jinan Hanfang Medical Devices Co., Ltd. China).
2.3.7.1. Statistical analysis. All the experimental results were expressed as mean ± standard deviation (SD). The date was statistically analyzed by SPSS19.0 using one-way analysis of variance (ANOVA). 3. Results and discussion 3.1. Extraction and purification The crude polysaccharides from M. Halliana flower were obtained by hot water extraction and alcohol precipitation. The polysaccharides were then further isolated by DEAE-52. Three major fractions eluted with 0, 0.1 and 0.2 M NaCl were collected, concentrated, dialyzed and lyophilized to obtain MP-W, MP-1 and MP-2 (Fig. 1A). Then, the MP-W fraction was further purified by Sephadex G-100 column to obtain
Fig. 5. (continued) 4
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Fig. 5. (continued)
purified polysaccharides MHP-W. The results are shown in Fig. 1B.
results showed that MHP-W did not contain protein and nucleic acid.
3.2. Molecular weight of polysaccharides analysis
3.4. FT-IR spectra analysis
The molecular weight of polysaccharides is just an average distribution of the length of the chain, and different molecular weights are measured in different ways. Even same polysaccharides show a strong variation of the molecular weight (Mw) and the number average molecular weight (Mn). The general dispersion coefficient judges the quality of homogeneous polysaccharide molecules. As shown in Table 1, the average molecular weight of MHP-W was 353 kDa.
The infrared spectroscopy of MHP-W is shown in Fig. 3. There was a larger and wider peak at 3410.29 cm−1, which belonged to the O–H stretching vibration, and there was a small peak in the vicinity of the 2924.09 cm−1, which belonged to the C–H stretching vibration of the alkyl group. 3410.29 cm−1 and 2924.09 cm−1 were the characteristic absorption peaks of carbohydrate. The absorption peak at 1651.07 cm−1 was the stretching vibration of the C]O, and the variable angle stretching vibration of C–H at 1418.71 cm−1. Absorption at 1072.47 cm−1 was the C–O–C stretching vibrations of pyranose ring. The absorption peak near 850 cm−1 was the characteristic absorption peak of α-glucan [20]. The absorption peak near 890 cm−1 was the characteristic absorption of β-glucan [20]. The absorption peak at
3.3. UV spectral analysis The MHP-W was scanned by UV spectra and there were no characteristic absorption peaks detected at 280 nm and 260 nm (Fig. 2). The
Fig. 5. (continued) 5
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Fig. 5. (continued)
897.90 cm-1 showed that there was a β-glycosidic bond [21].
Table 3 1 H and13C NMR chemical shifts of MHP-W recorded in D2O Sugar residue β-1,6-linked-Gal (A) β-1,3,6-Gal (B) α-1,2,5-Araf (C) α-1- Araf (D)
H C H C H C H C
1
2
3
4
5
6
4.47 106.3 4.53 106.07 5.00 110.64 5.24 112.06
3.54 73.63 3.65 72.80 3.85 86.74 4.21 84.21
3.70 75.50 3.74 83.01 4.14 79.31 3.94 79.42
3.97 71.50 4.14 71.40 3.92 85.64 4.13 86.18
3.92 76.64 3.94 79.42 3.88 72.43 3.83 64.19
4.04 72.41 4.03 72.41 3.77
3.5. Determination of monosaccharide composition of polysaccharides by gas chromatography Six monosaccharides were selected as the mixed standard, and the quantity of each monosaccharide standard (m1) was precisely weighed, the peak area (a1) of the monosaccharide was obtained according to the gas chromatogram of the polysaccharide sample. Then the mass of each monosaccharide composition was obtained, and the molar ratio was obtained by (m2) at the molecular weight of each monosaccharide. The results are shown in Fig. 4A and Fig. 4B. MHP-W was composed of arabinose, xylose, mannose, glucose and galactose with a molar ratio of 2.59: 0.15: 0.23: 0.25: 9.70, respectively. 3.6. Methylation analysis of MHP-W The methylation method is an important means to determine the polysaccharide structure, from which the glycosidic linkages of polysaccharides can be judged. The basic principle is that the free hydroxyl groups are completely methylated, and then the methylated polysaccharides are hydrolyzed into partial methylated polysaccharides. The position of the hydroxyl group on the sugar is the position of the polysaccharide glycosyl linkage. Finally, the monosaccharide is hydrolyzed to obtain the derivative for GC-MS analysis. According to the relative retention value and the mass spectrum of each component in the gas chromatogram, the position of the methyl group and the acetyl
Fig. 6. Deducted structure of MHP-W
Table 4 The effect of MHP-W on plasma coagulation parameters Group
Blank Breviscapine Yunnan Baiyao MHP-W
Plasma coagulation parameters APTT (s)
PT (s)
TT (s)
FIB (g/L)
19.70 ± 0.16 25.40 ± 0.18*** 18.18 ± 0.17*** 19.95 ± 0.24
10.33 ± 0.17 11.15 ± 0.24*** 9.32 ± 0.22*** 10.31 ± 0.14
13.63 ± 0.38 16.43 ± 0.34*** 11.38 ± 0.25*** 10.53 ± 0.26***￥￥￥
4.24 ± 0.14 4.93 ± 0.15*** 5.02 ± 0.14*** 4.32 ± 0.16*
Data represent mean ± SD. n = 4; Compared with saline, ***p < 0.001. Compared with Yunnan Baiyao,￥￥￥p < 0.001. 6
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group on the derivative can be judged to confirm the monosaccharide composition and the glycosidic linkage and the position of the oligosaccharide. The results of GC-MS analysis (Table 2) showed that MHP-W contains four linkage forms: 1,2,5-linked Araf, 1,3,6-linked Gal, 1,6-linked Gal and 1-linked Araf.
4.47 ppm) and C-6 of residue of Residue B (δ 72.41 ppm). C-1 of Residue A (δ 106.3 ppm) and H-6 of Residue B (δ 4.03 ppm). The cross peaks between H-1 of Residue B (δ 4.53 ppm) and C-6 of Residue A (δ 72.41 ppm). C-1 of Residue B (δ 106.07 ppm) and H-6 of Residue A (4.04 ppm), which suggested that Residue A was linked to Residue B at the 1, 6-position, and the backbone of MHP-W consisted of consecutively →6)-β-Gal-(1 → 6)-β-Gal-(3–1→. C-3 of Residue B (δ 83.01 ppm) and H-5 of Residue C (δ 3.92 ppm) indicated Residue C was linked to Residue B at the 3-position. The cross peak δ 5.24/86.74 ppm showed the correlation between H-1 of Residue D and C-2 of Residue C, indicating that →α-Araf-(1 → 2)-α-Araf-(5–1→ probably existed in the branch chain. All the data is shown in Fig. 5E. Based on the monosaccharide composition, methylation and GC-MS analysis and NMR spectroscopy, we proposed a structural unit of MHPW as shown in Fig. 6.
3.7. NMR analysis of MHP-W The signals of MHP-W from 1H NMR and 13C NMR spectra were assigned on the basis of monosaccharide composition, glycosidic linkage analysis, chemical shifts and then analyzed. Most of the polysaccharide proton resonance peaks in the 1H NMR spectra were in the range of 3.0–4.5 ppm and several overlap occurred, whereas the chemical shift of the anomeric hydrogen was generally in the range of 4.5–5.5 ppm. The type and number of glycosidic bonds could be determined by anomeric hydrogen. The α-type glycoside heterologous proton was over δ 5.0, while the β-type was generally less than δ 5.0. In Fig. 5A, four anomeric proton signals were found in the 1H NMR spectra. They were designated as A (4.47 ppm), B (4.53 ppm), C (5.08 ppm) and D (5.24 ppm), according to the reference that 4.79 ppm was the symbol of the residual solvent (D2O). Thus, MHP-W contained both α-type and β-type glycosidic bond. The 13C NMR spectra of MHPW are presented in Fig. 5B. The resonance signals between δ 98 ppm and δ 110 ppm in 13C NMR spectra belonged to the anomeric carbon atoms of monosaccharide. Residues A, B, C and D were assigned at 106.3 ppm, 106.07 ppm, 110.64 ppm and 112.06 ppm, respectively. All the 1H and 13C signals were assigned using homonuclear 1H/1H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence spectroscopy (HSQC), and heteronuclear multiple bond correlation spectroscopy (HMBC). Residue A: the intensive anomeric signals of residue A at 4.47 ppm and 106.3 ppm (Fig. 5D) corresponded to a β-linked residue with relatively high content of MHP-W. This residue was tentatively assigned as β-1, 6-Gal [22,23] compared with the reported data and peak intensity. The proton assignments of residue A were obtained from COSY as shown in Fig. 5C and Table 3. The corresponding 13C NMR chemical shifts of residue A (δ 73.63, 75.49, 71.55, 76.64 and 72.41 ppm for C-2, C-3, C-4, C-5 and C-6, respectively) were revealed by HSQC spectrum. All the 1H NMR and 13C NMR chemical shifts of residue A were consistent with the previous reports [24] and the corresponding intensity was supported by the result of methylation analysis (Table 3), indicating that the residue A was β-1,6-linked-Gal. Residue B: the intensive anomeric signals of residue B at 4.53 ppm and 106.07 ppm corresponded to a β-linked residue. The proton assignments of residue B were obtained from COSY as shown in Table 3. The corresponding 13C NMR chemical shifts of residue A (δ 72.82, 83.01, 71.40, 79.44 and 72.41 ppm for C-2, C-3, C-4, C-5 and C-6, respectively) were revealed by HSQC spectrum. Residue A was tentatively assigned as β-1,3,6-Gal [22] compared with the reported data. Remaining sugar residues: Residues C and D were determined by the same method as above. The similar 1H NMR and 13C NMR chemical shifts between residues C and D suggested that these two spin systems were similar. For most of monosaccharides with α or β form, the chemical shift of C-1 was the range of δ 90–106 ppm. α-Ara is δ 106–110 ppm when it presents as furanoside [25]. The chemical shifts of residues C and D were 110.64 ppm and 112.06 ppm, respectively, indicating that they were α- Araf. When the C-5 of Araf was not substituted, its chemical shift was the range of δ 60–65 ppm [23]. The C-5 of residue C was δ 72.43 ppm, while residue D was δ 64.19 ppm, indicating that residue D was α-1-linked-Araf. Residue C was assigned as α-1,2,5-Araf compared with previous reports. Once the 1H NMR and 13C NMR chemical shifts of all sugar residues were completely assigned, the sequences of these residues were determined by observing inter- and intra-residual connectivities in HMBC spectrum (Fig. 5E). Cross peaks were found between H-1 of residue A (δ
3.8. Coagulation assays in vitro The essence of blood coagulation is the process of transformation of soluble fibrinogenin plasma to insoluble fibrin. The key to this transformation is the occurrence of a series of complex enzymatic reactions that require the involvement of multiple coagulation factors. According to the activation pathway of coagulation factor and the difference of coagulation factors, it can be divided into endogenous coagulation pathway, exogenous coagulation route and coagulation common pathway [26]. According to the results presented in Table 4, MHP-W significantly shortened TT (p < 0.001) as compared to the saline control, and its effect was higher than that of the positive control Yunnan Baiyao (p < 0.001). For APTT and PT no major changes were observed by MHP-W. MHP-W significantly increased FIB (p < 0.05) although to a lesser degree as compared to Yunnan Baiyao and Breviscapine. Results showed that MHP-W promoted blood clotting through endogenous and exogenous coagulation pathways as well as increased fibrinogen content. The results indicated that MHP-W have procoagulant activities in vitro. 4. Conclusions In our study, we prepared a crude polysaccharide from M. halliana flower by water extraction and alcohol precipitation and the resulted polysaccharide was further fractioned into MP-W, MP-1 and MP-2. The MP-W fraction was further purified by Sephadex G-100 column to obtain the purified polysaccharide MHP-W. The average molecular weight of MHP-W was 353 kDa. A further analysis revealed a primary structure of MHP-W to be composed of arabinose, xylose, mannose, glucose and galactose. The majority residues were proven to be 1-linked Araf and 1,6-linked Gal. The coagulation assay showed that MHP-W significantly shortened TT and increase FIB, indicative of a procoagulant activity mainly through endogenous and exogenous coagulation pathways as well as through mechanisms of increased fibrinogen content. Acknowledgments This work was supported by Science and Technology Planning Project of Kaifeng City (1808003), Science and Technology Research Project of Henan Province (182102110473). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.carres.2019.107813. References [1] Chinese Herbalism Editorial Board, SATCM of the People's Republic of China.
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