猴头菌水溶性多糖的分离纯化与结构表征(英)

The isolation and structural characterization of a water-soluble polysaccharide from Hericium erinaceusZHANG An-qiang a FU Li a SUN Pei-long a ZHANG Jing-song b,*PAN Ying-jie ca College of Biological & Environmental Engineering, Zhejiang University of T echnology, Hangzhou, 310014, C hinab Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106,chinac College of Food Science, Shanghai Fisheries University, Shanghai 200090, C hinaAbstract: A novel water-soluble heteropolysaccharide termed HEPF2 was isolated from the fruiting bodies ofHericium erinaceus. HEPF2 had a molecular weight of 1.66⨯104 Da and was composed of fucose, galactose andglucose in the ratio of 1.00:3.69:5.42, as well as a small amount of 3-O-methylrhamnose. Its structural characteristics were further investigated by FT-IR, sugar analysis, methylation analysis, partial acid hydrolysis andNMR spectroscopy. Based on the data obtained, HEPF2 was found to be a heteropolysaccharide composed of(1→4)-linked glucosyl, (1→6)-linked glucosyl and (1→6)-linked galactosyl residue, attached to the O-2 of mainchain with terminal fucosyl residue or a minor of terminal gulcosyl and galactosyl residues. NMR spectra showedthat (1→4)-linked glucosyl wa s β configuration, (1→6)-linked galactosyl, (1→2,6)-linked galactosyl were α configuration and terminal fucosyl residue was also α configuration.Keywords: fungi, Chinese traditional herb, heteropolysaccharide, purification, structure elucidation猴头菌水溶性多糖的分离纯化与结构表征张安强a傅立a孙培龙a 张劲松b,*潘迎捷ca浙江工业大学生物与环境工程学院杭州 310004b上海农业科学院食用菌研究所上海 201106c上海水产大学食品学院上海 200090摘要：于猴头菌子实体中分离得到一种新型的水溶性杂多糖HEPF2，分子量大小为1.66⨯104 Da，该多糖由岩藻糖、半乳糖和葡萄糖以1.00:3.69:5.42比例构成，同时也含有微量的3-O-甲基鼠李糖。

进一步利用傅立叶变换红外光谱法、糖组成分析、甲基化分析、部分酸水解法和核磁共振法等方法进行结构鉴定，检测结果表明，该杂多糖中包含1→4、1→6结合的葡萄糖和1→6结合的半乳糖残基，连接于主链的侧链残基，包括岩藻糖残基、少数的端基葡萄糖和半乳糖残基。

核磁共振法检测结果还表明，1→4结合葡萄糖为β构型，(1→6)结合半乳糖、(1→2,6)结合半乳糖和端基葡萄糖均为α构型。

关键词：大型真菌，传统中草药，杂多糖，纯化，结构分析* C orresponding au thor. E-mail address: zhangjs888@IntroductionHericium erinaceus is a traditional Chinese medicinal fungus, which is distributed throughout China and used to treat gastric ulcers, chronic gastritis and other digestive tract-related diseases. Both the fruiting bodies and mycelia of the fungus have been reported to contain bioactive polysaccharide (Zhou 2000). It has been shown that the polysaccharides from H. erinaceus exhibited various pharmacological activities, for example, enhancement of immune system, antitumor, hypoglycemic and anti-aging properties (Zhou 2000; Nie 2003). The main aim of this work is to find an efficient route to extraction, isolation and fractionation of the polysaccharides present in H. erinaceus and also its structural characterization, as a contribution to better identify correlations between structure and functionality. In this paper, the isolation and structural investigation of HEPF2, a novel neutral polysaccharide purified from the fruiting bodies of H. erinaceus, is described.1 MATERIALS AND METHODS1.1 MaterialsFruiting bodies were purchased from Pan’an in Zhejiang Province, China. DEA E-Sepharose Fast Flow and Sephacryl S-100, S-200, S-300 High Resolution were purchased from Amersham Pharmacia Biotech. Dextrans and the monosaccharides (D-Gal, D-Ara, L-Fuc, L-Rha, D-man, D-Xyl and D-Glc) were from Sigma. All other reagents were of A.R. grade and made in China. HPLC was carried out on a Waters 2695 HPLC system (2695 HPLC Pump, 2414 Refractive Index Detector). GC-MS was carried out using a Thermo Finnigan V oyager GC/MS with Trace 2000 GC, and NMR spectra were determined with a V arian INOV A 500.1.2 Isolation and purificationThe total fruiting bodies of H. erinaceus were first exhaustively extracted with ethanol under reflux for 12 h to remove lipids. After filtration, the residue was air dried and extracted 3 times for 2 h each with boiling water. The combined aqueous filtrate was concentrated into one-tenth of the original volume, and 95% ethanol was added to the aqueous filtrate until the final alcohol concentration reached 30%. Precipitated material was removed by centrifugation (10000 rpm, 10 min, 4 ︒C), and 95% ethanol was again added slowly to 60% final concentration. The precipitate was separated out and lyophilized termed HEPF60. A portion of HEPF60 was dissolved in water and the insoluble residue was removed by centrifugation. The supernatant was applied to a DEA E-Sepharose Fast Flow column (XK 26 ⨯100 cm), eluted first with distilled water and then with 0-2 M gradient of NaCl. The fractions were collected by an auto-collector and compounds were detected by means of the phenol-sulfuric acid assay (Zhang 1999). HEPF60-B was obtained from the 0-2 M gradient NaCl fraction elute, which was further purified by gel permeation chromatography on a column of Sephacryl S-300 High Resolution (XK 26 ⨯ 100 cm). Eluting with water, the main fraction was collected, dialyzed and lyophilized to get a white purified polysaccharide (HEPF2), whose molecular weight range was detected on a linked column SN of TSK PWXL 4000 and 3000 gel filtration columns firstly.1.3 Determination of purity and molecular weightDetermination of the homogeneity and molecular weight of samples was done by HPLC on a linked column of TSK PW XL 4000 and 3000 gel filtration columns, eluting with 0.1 M phosphate buffer solution (PBS) and 0.3 M NaNO3at PH 7.0 with a flow rate of 0.6 mL /min. The column was kept at 30.0 ± 0.1 ︒C. The linear regression was calibrated by dextrans (T-700, 580, 300, 110, 80, 70, 40, 9.3, 4). All samples were prepared as 0.2 % (w/v) solutions, and 10 µL of solution was analyzed in each run.1.4 Monosaccharide composition analysisHEPF2 (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 ︒C for 2 h, and the monosaccharide composition was determined by high-performance anion-exchange chromatography (HPA EC) using a Dionex LC30 equipped with a CarboPacTM PA20 column (3 mm ⨯150 mm). The column was eluted with 2 mM NaOH (0.45 mL/min) and the monosaccharides were monitored using a pulsed amperometric detector (Dionex) (Y ang 2005). 1.5 Methylation analysisV acuum dried polysaccharide (2 mg) was methylated by the Kalyan’s(1992) method. Complete methylation was confirmed by the disappearance of the hydroxyl peak (3200~3700 cm-1) in the IR spectrum. The permethylated product was hydrolyzed by treatment with HCO2H (88%, 0.5 mL), H2O (0.1 mL) and CF3CO2H acid (0.05 mL) at 100 ︒C for 16 h. The partially methylated sugars in the hydrolysate were reacted with NaBH4and acetylated with acetic anhydride, and the resulting mixture of alditol acetates was analyzed by GC-MS.1.6 Partial acid hydrolysis Oligosaccharides were produced by partial acid hydrolysis of the polysaccharide. HEPF2 (100 mg) was hydrolyzed by 0.05 M CF3COOH (50 mL) for 1 h at 100 ︒C. The acid was removed by co-distillation with CH3OH. The hydrolyzate was dissolved in distilled water (0.5 mL), and dialyzed against distilled water for 24 h. The dialysate was purified by gel permeation chromatography on a column of Sephacryl S-100 High Resolution (XK 26 ⨯100 cm) and lyophilized (HEPF2dw1). The nondialysate was purified by gel permeation chromatography on a column of Sephacryl S-300 High Resolution (XK 26 ⨯ 100 cm) and was lyophilized to give a degraded polymer (HEPF2dn1). A portion of HEPF2dn1 was methylated and its glycosyl linkage composition was determined as described above. HEPF2dn1 (30 mg) was further hydrolyzed by 0.1 M CF3COOH (15 mL) for 1 h at 100 ︒C. After partial acid hydrolysis of HEPF2dn1, the hydrolysate was dialyzed. A depolymerized product (HEPF2dn2) was obtained and separated by Sephacryl S-200 High Resolution (XK 26 ⨯100 cm) chromatography. The dialysate was concentrated and lyophilized, designated HEPF2dw2. A portion of HEPF2dn2 was methylated and its glycosyl linkage composition was determined as described above. The glycosyl residue compositions of HEPF2dw1, HEPF2dn1, HEPF2dw2 and HEPF2dn2 were determined by GC as alditol acetates (Albersheim et al.1967) and analyzed by GC-MS using a DB-5 column (30 m ⨯0.25 mm ⨯0.25 µm) at a temperature program as follows: 80 ︒C to 200 ︒C at a rate of 5 ︒C/min, increasing to 215 ︒C at a rate of 2 ︒C/min, and finally to 280 ︒C at a rate of 20 ︒C/min. The injector and detector heater temperatures were both 250 ︒C.1.7 NMR analysisHEPF2 (30 mg) was lyophilized three times in D2O (0.5 mL). The 1H NMR (25 ︒C, 60 ︒C) and 13C NMR (25 ︒C) spectra were determined in 5-mm tubes using a V arian INOV A 500 NMR spectrometer. 1H chemical shifts were referenced to residual HDO at δ4.78 (25 ︒C) as internal standard. 13C chemical shifts were determined in relation to DSS (δ 0.00) calibrated externally.2 Results2.1 Isolation, determination of purity and molecular weight of HEPF2HEPF2 was purified by anion-exchange (Fig.1) and gel filtration chromatography from the fruiting bodies of H. erinaceus. HPLC eluted as a single symmetrical peak, indicating it was a homogeneous polysaccharide (Fig.2). Correlation with the calibration curve of dextran standards, the molecular weight of HEPF2 was 1.66⨯104 Da. Lack of absorption at 280 nm by UV scanning indicated that HEPF2 contained no protein.Fig. 1 The elu tion of HEPF60 isolated from the fruiting b odies of H. erinaceus by DEAE-Sepharose F.F. column chromatography. OD490: The absorbance of the compou nds detected by means of phenol-sulfuric acid assay (Zhang 1999) under 490nm.Fig. 2 High Performance Liquid Chromatog raphy (HPLC) elu tion of HEPF2 isolated from the fruiting b odies of H. erinaceus.2.2 FTIR s pectroscopic characterizationThe infrared spectrum of the polysaccharide was shown in Fig.3. The broadly-stretched intense peak at 3400 cm-1was due to the hydroxyl stretching vibration of the polysaccharide. The band at 2925.5 cm-1 was due to C-H stretching of CH2 groups. The band at 1646.9 cm-1 showed that the occurrence of bound water. The bands approximately in the regain of 3400, 2930 and 1650 cm-1are characteristic of a carbohydrate ring (Li et al.2008). Three absorption peaks between 1010 and 1160 cm-1attributed to the stretching vibrations of pyranose ring. Two stretching peaks at 1161 and 1041 cm-1 in the IR spectrum suggested the presence of C-O bonds (Zhao et al. 2007). No absorption peaks at 1740 cm-1 indicated that there were no uronic acids. 2.3 Sugar and methylation analysisSugar analysis and GC-MS revealed the presence of fucose, galactose, glucose, in the molar ratio of 1.00:3.69:5.42, as well as a s mall amount of 3-O-methylrhamnose.The alditol acetates of the methylated material from the polysaccharide were analyzed by GC-MS using a DB-5 MS fused silica capillary column. The polysaccharide showed that the presence of 1,4,5-tri-O-acetyl-2,3,6-tri-O- methyl-D-glucitol, 1,5,6-tri-O-acetyl-2,3,4-tri-O- methyl-D-glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra -O-methyl-D-glucitol,1,5,6-tri-O-acetyl-2,3,4-tri -O-methyl-D-galactitol,1,2,5,6-tetra-O-acetyl-3, 4-di-O-methyl-D-galactitol,Fig. 3 FT-IR spectrum of the polysaccharide HEPF2 isolated from the fruiting bodies of H. erinaceus.1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-galacti tol,1,5-di-O-acetyl-2,3,4-tri-O-methyl-L-fucitol, 1,5-di-O-acetyl-2,3,4-tri-O-methylrhamnose in a molar ratio of 3.31:2.13:0.25:2.03:1.11:0.21:1.00:0.19(Table 1). These results indicated that (1→4)-linked Glc p, (1→6)-linked Glc p, (1→6)-linked Gal p, (1,2→6)-linked Gal p, nonreducing-end Glc p, nonreducing-end Gal p, nonreducing-end Fuc p were present in the polysaccharide. Besides, it also contained a minor terminal 3-O-methyl rhamnose residue.Table 1. The methylation analysis of HEPF2Methylated sugar Type of linkage MolarratioMajor mass fragment (m/z)2,3,4-Me3-Rha p1-linked3-O-Me-Rha p0.19 43,71,89,101,117,131,145,1612,3,4-Me3-Fuc p1-linked Fuc p 1.00 43,72,89,101,115,117,131,161,175 2,3,4,6-Me4-Glc 1-linked Glc p0.25 43,71,87,101,117,129,145,161,205 2,3,6-Me4-Glc p1,4-linked Glc p 3.31 45,71,87,101,117,129,143,161,203,233 2,3,4-Me3-Glc p1,6-linked Glc p 2.13 43,71,87,101,117,129,161,173,189,233 2,3,4,6-Me4-Gal p1-linked Gal p0.21 43,71,87,101,117,129,145,161,205 2,3,4-Me4-Gal p1,6-linked Gal p 2.03 43,87,99,101,117,129,161,173,189,233 3,4-Me2-Gal p1,2,6-linked Gal p 1.11 43,71,87,99,129,159,173,189,2332.4 Partial acid hydrolysisDue to the complexity of the HEPF2 structure, partial acid hydrolysis experiment was carried out with the polysaccharide. After partialacidhydrolysis of HEPF2, the hydrolysate was dialyzed. The dialysate was purified by gel permeation chromatography on a column of Sephacryl S-100 High Resolution and lyophilized to give HEPF2dw1. The nondialysate was subjected to gel permeation chromatography on a column of Sephacryl S-300 High Resolution and was lyophilized to give a degraded polymer (HEPF2dn1). HEPF2dn1 was further hydrolyzed by 0.1 M CF3COOH for 1 h at 100 C. The nondialysate was subjected to gel permeation chromatography on a column of Sephacryl S-200 High Resolution and was lyophilized to give a degraded polymer (HEPF2dn2). The dialysate was concentrated and lyophilized, termed HEPF2dw2. HPAEC spectrum of HEPF2dw1 and HEPF2dw2 revealed the presence of fucose, 3-O-methyl rhamnose and a few galactose, glucose. This result indicates that the nonreducing-end D-galactopyranosyl, nonreducing-end glucopyranosyl, nonreducing-end fucopyranosyl moieties were destroyed during partial acid hydrolysis. Compositional analysis of the degraded polysaccharide HEPF2dn1 suggested that it was composed of fucose, galactose and glucose in a molar ratio of 0.04:1:2.34 (Table 2). The contents of fucose and 3-O-methyl rhamnose in HEPF2dn1 decreased remarkably compared with those of native polysaccharide. It was in agreement with the results of methylation analysis of HEPF2 that the fucosyl residues and 3-O-methyl rhamnose residues were terminal residues. Compositional analysis of the degraded polysaccharide HEPF2dn2 suggested that it was composed of fucose, galactose and glucose in a molar ratio of 0.07:1:4.27 (Table 2). In comparison with the composition of the native polysaccharide, the proportion of glucose significantly increased, while the content of fucose and galactose decreased remarkably. The methylation analysis of HEPF2dn2 showed the presence of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-glucitol, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-galactito l, 1,2,5,6-tetra-O-acetyl -3,4-di-O-methyl-D-galactitol,1,5-di-O-acetyl-2,3,4-tri-O-methyl-L-fucitol, in a molar ratio of 3.8:1.5:1.00:0.25:0.13 (Table 3). In comparison with the methylation analysis of the native polysaccharide, the proportion of (1→6)-linked galactose and (1→2,6)-linked galactose residues decreased, while the proportion of (1→4)-linked and (1→6)-linked glucosyl residues significantly increased. These results established that the backbone chain of the native polysaccharide was composed of 1,4 and 1,6 linked glucosyl residue and 1,6 and 1,2,6 linked galactosyl residues, with terminal fucosyl residue or a minor of terminal glucosyl and galactosyl residues.Table 2 Glycosyl composition of HEPF2 and its partial acid hydrolysatesGlycosyl residues Molar ratioHEPF2 HEPF2dw1 HEPF2dn1 HEPF2dw2 HEPF2dn2 3-O-methyl-rhamnose 0.28 1.00 0.01 1.00 n.d.a Fucose 1.00 6.17 0.04 6.26 0.07Galactose 3.69 1.25 1.00 3.36 1.00Glucose 5.42 0.74 2.34 0.53 4.27a Note: non detected. HEPF2dw1 was given from partial acid hydrolysis of HEPF2 by gel permeation chromatography S-100. HEPF2dn1 was given from The nondialysate by gel permeation chromatography S-300. HEPF2dn2 was from the nondialysate of further hydrolyzing of HEPF2dn1 by gel permeation chromatography S-200. Concentrated and lyophilized the dialysate, termed HEPF2dw2.2.5 NMR analysisThe 1H (500 MHz, 25 ︒C, 60 ︒C) and 13C (500 MHz, 25 ︒C) NMR experiments were carried out. The 500 MHz 1H NMR spectrum (60 ︒C, Fig. 4) of the native polysaccharide mainly contained signals for five anomeric protons at δ 5.10, 5.06, 5.00, 4.74 and 4.54. One CH3-C group at δ 1.27 (J5, 6 5.6 Hz) corresponded to the chemical shift of H-6 of Fuc. Other sugar protons were in the region of δ3.50~4.41 along with a signal for an O-methyl group at δ3.50. Sugar residues were designated A-E according to the decreasing chemical shifts of the anomeric configuration. Methylation analysis showed the highest proportion of residues was (1→4)-linked Glc p, and in the 1H NMR spectrum the highest proportion was residue E. So residue E was designated as a (1→4)-linked Glc p The large coupling constants J H-2, H-3 and J H-3, H-4(~10 Hz) were observed for E, further indicating that it was a D-glucosyl moiety. The anomeric chemical shift for moiety E at δ 4.54 and appearing as a doublet, indicated that the D-glucose was an β-linked.Fig. 4 500-MHz 1H NMR spectru m of HEPF2 isolated from the fruiting bodies of H. erinaceus at 60 ︒C. A-E were donated by the decreasing chemical shifts of the anomeric configuration.The other signals for anomeric protons were in the region of δ5.00~5.10, indicating 1-linked Fuc p, (1→6)-linked Gal p, (1→2,6)-linked Gal p were α configuration. The 13C NMR spectrum (Fig. 5) of the polysaccharide mainly contained signals for five anomeric carbons at δ106.14、106.07, 105.89, 101.4 and 101.2. Sugar ring carbons linked to oxygen in the region of δ63.12~80.6 and one CH3-C groups (C-6 of Fuc) at δ18.4. In addition, a minor signal at δ57.5 could be assigned to an O-methyl group which, based on GC-MS data, was probably due to 3-O-methyl-rhamnose. The presence of β-D-(1→4)-linked Glc p was proved by the signals at δ80.6. The O-substituted C-6 was shown by the signals at δ 70.05 and δ 69.9, and the signal of the unsubstituted C-6 was at δ64.1and δ 59.1. 4.2.6 ConclusionBased on the data presented above, HEPF2 was composed of fucose, galactose and glucose in a molar ratio of 1.00:3.69:5.42 and a minor of 3-O-methyl-rhamnose. Methylation analysis showed it contained (1→4)-linked glucosyl, (1→6)-linked glucosyl, (1→6)-linked galactosyl, (1→2,6)-linked galactosyl, terminal fucosyl residue, and a minor of terminal 3-O-methyl-rhamnose, terminal glucosyl andgalactosyl residues.Fig. 5 500-MHz 13C NMR spectrum of HEPF2 isolated from the fruiting b odies of H. erinaceus at 25 ︒C. Fuc C-6: This peak was a result of one CH3-C groups (C-6 of Fuc) at δ 18.4.Partial hydrolysis and acetolysis indicated that the polysaccharide was composed of (1→4)-linked glucosyl, (1→6)-linked glucosyl and (1→6)-linked galactosyl residue, attached to the O-2 of main chain with terminal fucosyl residue or a minor of terminal gulcosyl and galactosyl residues. NMR spectra showed that (1→4)-linked glucosyl was β configuration, (1→6)-linked galactosy l, (1→2,6)-linked galactosyl were α configuration and terminal fucosyl residue was also α configuration.3 DISCUSSIONIn the former fungi examined, polysaccharides composed of β-glucans, α-D-manmans, have been reported to be the major components of the cell wall and the intercellular matrix, with the latter found mainly in yeast cell walls and medicinal mushrooms (Vingradov et al.1998). 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