金耳子实体多糖的分离纯化、结构鉴定、分子修饰和生物活性的研究
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摘要
金耳(Tremella aurantialba Bandoni et Zang),俗称脑耳、金木耳,是我国一种稀有的珍贵食用菌和药用菌,金耳胶质细腻,清润可口,其滋补营养价值,优于银耳、黑木耳等其他胶质菌,是“三耳”(金耳、银耳、黑木耳)中的佼佼者,具有化痰、止咳、定喘、调气、平肝阳之功效,据文献报道,金耳多糖主要具有增强免疫、降血糖、降血脂、抗肿瘤、抗血栓等作用。目前关于金耳的研究主要集中于对金耳的人工驯化、营养成分和生物活性的研究,多糖作为金耳的主要生理活性成分之一,也受到了国内外越来越多的关注,但由于过去多糖技术和分析手段的落后,金耳多糖的化学研究主要集中于对其基本的理化性质、单糖组成和连接位点的分析,多糖的药理活性研究也主要是关于粗多糖的研究,而对均一多糖的结构分析和生物活性还很少涉及。为了系统地研究金耳子实体多糖的化学结构和生物学特性,本研究对金耳子实体多糖进行了提取、分离纯化、结构鉴定、分子修饰和生物活性研究,为合理开发和利用金耳资源奠定扎实可靠的理论基础。
1金耳子实体多糖分离纯化和理化性质的研究
金耳子实体经乙醇脱脂和热水抽提得粗提物TAP、TAP经超滤分级得三组分TAPA、TAPB和TAPC,总得率为3.84%,其中TAPA最高,为3.67%,TAPA的多糖含量也比较高,为62.2%。TAPA经阴离子交换后得到TAPA1-TAPA5五个组分,其中TAPA1的得率在五个组分中最高为31.6%；TAPA1经Sephacryl S-500凝胶层析并经检测后得到三个均一多糖：TAPA11、TAPA21和TAPA51.三个均一多糖均为白色絮状样品,均易溶于水,温度升高溶解度升高；在常温和加温条件下均不溶于二甲亚砜。
2金耳子实体多糖的结构鉴定
通过单糖组成分析、甲基化分析、红外谱和NMR谱等技术,研究了金耳子实体的2种均一多糖TAPA11和TAPA21的化学结构。
2.1 TAPA11的结构鉴定
均一多糖TAPA11的分子量为1.35×106,单糖组成主要由甘露糖、木糖、葡萄糖醛酸组成,其摩尔比约为5：4：1,并含有少量葡萄糖、半乳糖醛酸,是一种酸性多糖；甲基化结果表明,甘露糖主要以1,3.、1,2,3-、1,2,3,4-、1,4-和1-连接；木糖主要以1,2-、1,3-、1,4-和1-连接；葡萄醛酸以1,4-连接。由NMR谱进一步分析结果表明,得出TAPA11的四个结构片段为：
因此,TAPA11可能的结构为：
2.2 TAPA21的结构鉴定
均一多糖TAPA21的相对分子质量为7.6×105,单糖组成和摩尔比为Man:Xyl:GluA=3:3:1,并含有少量Glc和GalA,是一含葡萄糖醛酸的酸性多糖,结合甲基化结果和NMR图谱分析结果,表明TAPA21的一级结构主要是由以下三个片段组成：
片段Ⅰ组成主链,是由α-(1→3)糖苷键连接的甘露聚糖组成的,Ⅱ和Ⅲ组成侧链,分别连接于主链Ⅰ的2位和4位。TAPA21的一级结构的重复单元可能如下所示：
3均一多糖TAPA11的分子修饰的研究
为了进一步提高均一多糖TAPA11的生物学活性及研究多糖的构效关系,对TAPA11进行了衍生化。采用氯磺酸-吡啶法制备了硫酸酯化多糖TAPA11-s,得率为65.25%,利用BaSO4浊度法测得硫酸基含量为2.88 %,取代度为0.05；并对TAPA11-s的红外光谱和核磁共振波谱进行了初步分析,结果均证实了与计算结果相吻合的低取代度；均一多糖溶解于DMSO中以吡啶、乙酸酐为酯化试剂制备了TAPA11的乙酰化多糖衍生物TAPA11-ac,得率分别为56%,乙酰基含量为5.81%,取代度为0.23；TAPA11经脱乙酰基作用制得了TAPA11的脱乙酰基衍生物TAPA11-deac,得率为75%,经测定乙酰基含量和取代度均为0,表明了通过乙酰基作用乙酰基已完全被脱去。
4金耳子实体生物活性的研究
分别选择了小鼠(BALB/C57)脾淋巴细胞、小鼠巨噬细胞RAW264.7、来源于大鼠肾上腺嗜铬细胞未成熟神经元的细胞系PC12,以脾淋巴细胞的增殖活性、NO的释放量和H202诱导的PC12细胞损伤修复活性三个指标考察了金耳子实体多糖及其衍生物的生物活性。
4.1金耳多糖及其衍生物对刺激小鼠脾淋巴细胞的增殖作用作用研究
采用不同溶剂对金耳子实体依次提取所得组分四组分TA冷水提(TAP1)、TA热水提(TAP)、TA草酸铵提(TAP2)、TA碱提(TAP3)对淋巴细胞均有一定的增殖作用,TAP2的活性最低,其他三组分的活性相当,其中TAP1、TAP的刺激增殖的作用呈现明显的浓度依赖性关系；四个组分在试验浓度下的活性均低阳性对照的活性。综合考虑四个组分的提取得率和提取工艺简化程度,选择了TAP做进一步的分离纯化的研究材料。粗多糖TAP经超滤后所得三组分中,TAPA和TAPB相比原样品TAP活性有所提高,TAPA活性提高显著,且呈一定的剂量依赖性,TAPA在高浓度时刺激淋巴细胞增殖的活性接近于阳性对照的活性。TAPA经阴离子交换层析后所得各组分中,组分TAPA1刺激淋巴细胞增殖的活性随浓度升高而活性增加,呈现浓度依赖性。TAPA1经凝胶层析分离得到的三种均一组分TAPA11、TAPA21和TAPA51中,TAPA11的体外免疫刺激活性最好,高于TAPA在相同浓度下的增殖率。三种衍生物TAPA11-s、TAPA11-ac和TAPA11-deac中,TAPA11-s免疫活性最好,在高浓度下刺激淋巴细胞增殖的活性高于阳性对照,说明TAPA11经硫酸酯化可以提高其生物活性。
4.2金耳均一多糖TAPA11及其衍生物对小鼠巨噬细胞株RAW264.7生成NO的影响
TAPA11对RAW264.7产生NO的量在10μg／ml时就出现明显增加,且与样品浓度呈剂量-效应依赖关系,50μg／ml时释放NO的量与阳性对照LPS(1μg／ml)相当;三种衍生物中,乙酰化衍生物TAPA11-ac作用稍明显,50μg／ml的NO的释放量达到了214%,但低于阳性对照LPS(1μg／ml)的水平(258.9%),其次是硫酸化衍生物TAPA11-s,50μg／ml的NO释放量为185.7%,脱乙酰基衍生物TAPA11-deac最差(50μg／ml的NO释放量仅为158.7%),结果表明多糖对巨噬细胞RAW264.7产生NO的影响作用有可能不仅与多糖上的取代基有关系,还可能与取代基的含量(或取代度)有关。
4.3金耳子实体多糖及其衍生物对经H2O2诱导的PC12氧化损伤的修复活性的研究
研究了总提取物TAP、超滤后各组分、阴离子层析交换所得各组分、凝胶层析所得的均一多糖及衍生物对经H2O2损伤的PC12的修复作用。结果表明,样品TAP、TAPA、TAPB、TAPC、TAPA1-TAPA5、TAPA11对经H2O2损伤的PC12细胞的均具有一定的修复作用；乙酰化样品TAPA11-ac的修复作用在500mg／mL时的修复率157.63%,与阳性对照NGF的修复率(166.8%)相当,可见乙酰化修饰后的样品与原样品TAPA11相比较可以明显提高经H202损伤的PC12细胞的修复活性；硫酸化样品TAPA11-s和脱乙酰基样品TAPA11-deac的修复作用明显低于原样品TAPA11和阳性对照NGF。
Tremella aurantialba Bandoni et Zang. (known as Jin'er in China), is a kind of edible and medicinal basidiomycetous fungus belonging to the family Tremellaceae. When cooked, it has tender flesh with good appearance, a delicate flavor and pleasant fragrance. It is reported that T. aurantialba exhibits a range of pharmacological properties such as enhancement of the immune system, anti-diabetic, anti-hyperlipidemic, and anti-tumor activity, and anti-thrombotic effects, etc. At present, researches on T. aurantialba have been developed, but most of them were focused on the planting method, nutritional element and biological activities. Although some researches were done on pharmacology, they were mostly on the basis of crude extracts. As an important extract, polysaccharide had attracted more and more attention in past few years. A large part of papers dealed with monosaccharide composition and few ones focused on polysaccharide structure elucidation in past decades due to analysis and technique lag. There was no more detail and useful structural data of polysaccharide provided for development of polysaccharide medicines and functional foods, as well as for expounding biological mechanism. To further develop and utilize the fugus T. aurantialba resourses, isolation, purification, structural elucidation were studied herein, combination with chemical modification and pharmacological tests.
1 Isolation, purification and physic-chemical characterization of polysaccharides from T. aurantialba fruiting bodies
The fruiting bodies of T. aurantialba were defatted by 95% ethanol, followed by extraction with hot water and prepared crude material TAP. Ultrafiltration and a series of chromatographic technologies was applied to isolate TAP, affording three crude polysaccharide fractions(TAPA, TAPB and TAPC) and five semi-purified polysaccharide fractions (TAPA1-TAPA5)and three homogeneous polysaccharide fractions TAPA11, TAPA21 and TAPA51. TAPA11, TAPA21 and TAPA51 were white powder samples, and they are soluble in water, but not in DMSO.
2 Studied on structures of three homogeneous polysaccharides from T. aurantialba fruiting bodies
The structures of TAPA11 and TAPA21 were investigated by composition analysis, methylation analysis, FT-IR, NMR spectroscopic methods (1D NMR:1H NMR,13C NMR, 2D NMR:COSY, TOCSY, HMQC, HMBC and NOESY spectra).
2.1 Primary structure of polysaccharide TAPA11
TAPA11, with a molecular mass of 1.35×106 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.5:4:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted, 1,3-disubstituted and 1,4-disubstituted xylopyranose residues, and 1-substituted, 1,3-disubstituted,1,2,3-trisubstituted and 1,2,3,4-tetrasubstituted mannopyranose residues, and 1,4-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included four sequences as follows:
So, the predicted repeating primary structure of polysaccharide TAPA11was as follows:
2.2 Primary structure of polysaccharide TAPA21
TAPA21, with a molecular mass of 7.6×105 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.3:3:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted and 1,3-disubstituted xylopyranose residues, and 1,3-disubstituted,1,2,3- and 1,3,4-trisubstituted mannopyranose residues, and 1-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included three sequences as follows:
So, the predicted repeating primary structure of polysaccharide TAPA21 was as follows:
3 Chemical modification of homopolysaccharide TAPA11
To further enhance the biological activity and be easy to investigate the relationship between structure and functions, chemical modification for fraction TAPA11, including sulfation, acetylation and deacetylation, were carried out, and derivatives TAPAll-s, TAPA11-ac and TAPA11-deac were prepared, respectively. TAPA11-s, was prepared by the chlorosulfonic acid-pyridine method. The total yield of the sulfated product was approximately 65%, and the content of sulfur was determined to be～2.88% by BaSO4 method, so the D.S. value was calculated to be 0.05. TAPA11-ac was prepared using Pyridine and Acetic anhydride as esterifying agent. The total yield of product was approximately 56%, and the content of acetyl was determined to be 5.81% and the D.S. value was calculated to be 0.23. As for TAPAll-deac, the total yield of product was approximately 75%, and the content of acetyl and the D.S. value were determined to Zero, repectively, indicating that the deacetylation was finished completely from the TAPA11.
4 Pharmacological properties for polysaccharide fractions
All polysaccharide fractions were tested in vitro by BALB/C57, macrophages RAW264.7 and PC 12 cell line in our research.
4.1 Proliferation of mice spleen lymphocytes in vitro
T. aurantialba fruiting bodies were extracted by four different solvent one by one and prepeared four different extracts (TAP1, TAP, TAP2 and TAP3). Four samples were assayed by proliferation of mice spleen lymphocytes in vitro, and the result showed that four fractions had property to promote proliferation of mouse spleen lymphocytes. TAP1, TAP and TAP3 showed a better activity compared with TAP2, and at the same time samples TAP land TAP had the property in dose-dependent manner. So TPA was selected to further be isolated and investigated. Immunobiological activity assay showed that three fractions of crude polysaccharides (TAPA, TAPB and TAPC), obtained by ultrafiltration (UF) according to different molecular weight, could significantly increase mouse splenocytes (MSLs) proliferation in vitro at variouse testing concentrations. Compared with TAP, TAPA showed markedly increased activity, does-dependently, at the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA (6μg/mL). The results indicated that UF can play some effect to isolate some fractions which have a better property to stimulate the proliferation of MSLs. Five fractions TAPA 1-TAPA5 were prepared from TAPA after anion-exchange chromatography. Five fractions TAPA 1-TAPA5 markedly increased proliferation rates at various testing concentrations (50,200,500μg/mL) compared with the blank reference. At the concentration of 500μg/mL, samples TAPA1 and TAPA5, almost showed similar MSLs stimulation potency to PHA which was served as a positive reference. Among three samples TAPA11、TAPA21、TAPA51, obtained from TAPA1 after Gel-chromatography, TAPA11 had the best MSLs stimulation potency. At 500μg/mL, TAPA11 had higher the proliferation rate of MSLs (429%) compared with its native fraction TAPA (its proliferation rate was 389%). Among three derivatives, TAPA11-s showed the highest immunobiological activity at various concentrations compared with their original fraction TAPA11, in apparent dose-dependent manner. At the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA, which indicated sulfation of polysaccharide TAPA 11 was effective.
4.2 Effect of samples on nitric oxide production by macrophages RAW264.7
Exposure of RAW264.7 cells to increasing concentrations (10-50μg/mL) of TAPA11 for 24 h resulted in significant increases in NO production (based on nitrite accumulation) compared with untreated controls. Cells treated with 50μg/mL TAPA11 produced the same NO compared with LPS, which was served as a positive control. Among three derivatives, TAPA11-ac was the best one in comparison with the others samples TAPA11-s and TAPA 11-deac.The data indicated that effect of samples on nitric oxide production by macrophages RAW264.7 might be related to not only degree of acetyl group but also different groups.
4.3 Activity of oxidative injury Rehabilitation of PC12 induced with H2O2
All samples were applied to investigate rehabilitation rate of PC 12 cell after treatment with H2O2, and the results indicated that samples TAP、TAPA、TAPB、TAPC、TAPA1-TAPA5、TAPA11 had properties to rehabilitate PC12 cell. APA11-ac exhibited a strong bioactivity to rehabilitate PC 12 cell, at the concentration of 500μg/mL, the rehabilitation rate was 157.63%, which was very close to that of positive control NGF and its rehabilitation rate was 166.8%. The data also revealed that acetylation could improve rehabilitation rate of polysaccharide to PC 12 after treatment with H2O2. However, two samples TAPA11-s and TAPA11-deac exhibited a lower bioactivity to rehabilitate PC12 cell in comparison with the native polysaccharide TAPA11 and NGF.
1金耳子实体多糖分离纯化和理化性质的研究
金耳子实体经乙醇脱脂和热水抽提得粗提物TAP、TAP经超滤分级得三组分TAPA、TAPB和TAPC,总得率为3.84%,其中TAPA最高,为3.67%,TAPA的多糖含量也比较高,为62.2%。TAPA经阴离子交换后得到TAPA1-TAPA5五个组分,其中TAPA1的得率在五个组分中最高为31.6%；TAPA1经Sephacryl S-500凝胶层析并经检测后得到三个均一多糖：TAPA11、TAPA21和TAPA51.三个均一多糖均为白色絮状样品,均易溶于水,温度升高溶解度升高；在常温和加温条件下均不溶于二甲亚砜。
2金耳子实体多糖的结构鉴定
通过单糖组成分析、甲基化分析、红外谱和NMR谱等技术,研究了金耳子实体的2种均一多糖TAPA11和TAPA21的化学结构。
2.1 TAPA11的结构鉴定
均一多糖TAPA11的分子量为1.35×106,单糖组成主要由甘露糖、木糖、葡萄糖醛酸组成,其摩尔比约为5：4：1,并含有少量葡萄糖、半乳糖醛酸,是一种酸性多糖；甲基化结果表明,甘露糖主要以1,3.、1,2,3-、1,2,3,4-、1,4-和1-连接；木糖主要以1,2-、1,3-、1,4-和1-连接；葡萄醛酸以1,4-连接。由NMR谱进一步分析结果表明,得出TAPA11的四个结构片段为：
因此,TAPA11可能的结构为：
2.2 TAPA21的结构鉴定
均一多糖TAPA21的相对分子质量为7.6×105,单糖组成和摩尔比为Man:Xyl:GluA=3:3:1,并含有少量Glc和GalA,是一含葡萄糖醛酸的酸性多糖,结合甲基化结果和NMR图谱分析结果,表明TAPA21的一级结构主要是由以下三个片段组成：
片段Ⅰ组成主链,是由α-(1→3)糖苷键连接的甘露聚糖组成的,Ⅱ和Ⅲ组成侧链,分别连接于主链Ⅰ的2位和4位。TAPA21的一级结构的重复单元可能如下所示：
3均一多糖TAPA11的分子修饰的研究
为了进一步提高均一多糖TAPA11的生物学活性及研究多糖的构效关系,对TAPA11进行了衍生化。采用氯磺酸-吡啶法制备了硫酸酯化多糖TAPA11-s,得率为65.25%,利用BaSO4浊度法测得硫酸基含量为2.88 %,取代度为0.05；并对TAPA11-s的红外光谱和核磁共振波谱进行了初步分析,结果均证实了与计算结果相吻合的低取代度；均一多糖溶解于DMSO中以吡啶、乙酸酐为酯化试剂制备了TAPA11的乙酰化多糖衍生物TAPA11-ac,得率分别为56%,乙酰基含量为5.81%,取代度为0.23；TAPA11经脱乙酰基作用制得了TAPA11的脱乙酰基衍生物TAPA11-deac,得率为75%,经测定乙酰基含量和取代度均为0,表明了通过乙酰基作用乙酰基已完全被脱去。
4金耳子实体生物活性的研究
分别选择了小鼠(BALB/C57)脾淋巴细胞、小鼠巨噬细胞RAW264.7、来源于大鼠肾上腺嗜铬细胞未成熟神经元的细胞系PC12,以脾淋巴细胞的增殖活性、NO的释放量和H202诱导的PC12细胞损伤修复活性三个指标考察了金耳子实体多糖及其衍生物的生物活性。
4.1金耳多糖及其衍生物对刺激小鼠脾淋巴细胞的增殖作用作用研究
采用不同溶剂对金耳子实体依次提取所得组分四组分TA冷水提(TAP1)、TA热水提(TAP)、TA草酸铵提(TAP2)、TA碱提(TAP3)对淋巴细胞均有一定的增殖作用,TAP2的活性最低,其他三组分的活性相当,其中TAP1、TAP的刺激增殖的作用呈现明显的浓度依赖性关系；四个组分在试验浓度下的活性均低阳性对照的活性。综合考虑四个组分的提取得率和提取工艺简化程度,选择了TAP做进一步的分离纯化的研究材料。粗多糖TAP经超滤后所得三组分中,TAPA和TAPB相比原样品TAP活性有所提高,TAPA活性提高显著,且呈一定的剂量依赖性,TAPA在高浓度时刺激淋巴细胞增殖的活性接近于阳性对照的活性。TAPA经阴离子交换层析后所得各组分中,组分TAPA1刺激淋巴细胞增殖的活性随浓度升高而活性增加,呈现浓度依赖性。TAPA1经凝胶层析分离得到的三种均一组分TAPA11、TAPA21和TAPA51中,TAPA11的体外免疫刺激活性最好,高于TAPA在相同浓度下的增殖率。三种衍生物TAPA11-s、TAPA11-ac和TAPA11-deac中,TAPA11-s免疫活性最好,在高浓度下刺激淋巴细胞增殖的活性高于阳性对照,说明TAPA11经硫酸酯化可以提高其生物活性。
4.2金耳均一多糖TAPA11及其衍生物对小鼠巨噬细胞株RAW264.7生成NO的影响
TAPA11对RAW264.7产生NO的量在10μg／ml时就出现明显增加,且与样品浓度呈剂量-效应依赖关系,50μg／ml时释放NO的量与阳性对照LPS(1μg／ml)相当;三种衍生物中,乙酰化衍生物TAPA11-ac作用稍明显,50μg／ml的NO的释放量达到了214%,但低于阳性对照LPS(1μg／ml)的水平(258.9%),其次是硫酸化衍生物TAPA11-s,50μg／ml的NO释放量为185.7%,脱乙酰基衍生物TAPA11-deac最差(50μg／ml的NO释放量仅为158.7%),结果表明多糖对巨噬细胞RAW264.7产生NO的影响作用有可能不仅与多糖上的取代基有关系,还可能与取代基的含量(或取代度)有关。
4.3金耳子实体多糖及其衍生物对经H2O2诱导的PC12氧化损伤的修复活性的研究
研究了总提取物TAP、超滤后各组分、阴离子层析交换所得各组分、凝胶层析所得的均一多糖及衍生物对经H2O2损伤的PC12的修复作用。结果表明,样品TAP、TAPA、TAPB、TAPC、TAPA1-TAPA5、TAPA11对经H2O2损伤的PC12细胞的均具有一定的修复作用；乙酰化样品TAPA11-ac的修复作用在500mg／mL时的修复率157.63%,与阳性对照NGF的修复率(166.8%)相当,可见乙酰化修饰后的样品与原样品TAPA11相比较可以明显提高经H202损伤的PC12细胞的修复活性；硫酸化样品TAPA11-s和脱乙酰基样品TAPA11-deac的修复作用明显低于原样品TAPA11和阳性对照NGF。
Tremella aurantialba Bandoni et Zang. (known as Jin'er in China), is a kind of edible and medicinal basidiomycetous fungus belonging to the family Tremellaceae. When cooked, it has tender flesh with good appearance, a delicate flavor and pleasant fragrance. It is reported that T. aurantialba exhibits a range of pharmacological properties such as enhancement of the immune system, anti-diabetic, anti-hyperlipidemic, and anti-tumor activity, and anti-thrombotic effects, etc. At present, researches on T. aurantialba have been developed, but most of them were focused on the planting method, nutritional element and biological activities. Although some researches were done on pharmacology, they were mostly on the basis of crude extracts. As an important extract, polysaccharide had attracted more and more attention in past few years. A large part of papers dealed with monosaccharide composition and few ones focused on polysaccharide structure elucidation in past decades due to analysis and technique lag. There was no more detail and useful structural data of polysaccharide provided for development of polysaccharide medicines and functional foods, as well as for expounding biological mechanism. To further develop and utilize the fugus T. aurantialba resourses, isolation, purification, structural elucidation were studied herein, combination with chemical modification and pharmacological tests.
1 Isolation, purification and physic-chemical characterization of polysaccharides from T. aurantialba fruiting bodies
The fruiting bodies of T. aurantialba were defatted by 95% ethanol, followed by extraction with hot water and prepared crude material TAP. Ultrafiltration and a series of chromatographic technologies was applied to isolate TAP, affording three crude polysaccharide fractions(TAPA, TAPB and TAPC) and five semi-purified polysaccharide fractions (TAPA1-TAPA5)and three homogeneous polysaccharide fractions TAPA11, TAPA21 and TAPA51. TAPA11, TAPA21 and TAPA51 were white powder samples, and they are soluble in water, but not in DMSO.
2 Studied on structures of three homogeneous polysaccharides from T. aurantialba fruiting bodies
The structures of TAPA11 and TAPA21 were investigated by composition analysis, methylation analysis, FT-IR, NMR spectroscopic methods (1D NMR:1H NMR,13C NMR, 2D NMR:COSY, TOCSY, HMQC, HMBC and NOESY spectra).
2.1 Primary structure of polysaccharide TAPA11
TAPA11, with a molecular mass of 1.35×106 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.5:4:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted, 1,3-disubstituted and 1,4-disubstituted xylopyranose residues, and 1-substituted, 1,3-disubstituted,1,2,3-trisubstituted and 1,2,3,4-tetrasubstituted mannopyranose residues, and 1,4-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included four sequences as follows:
So, the predicted repeating primary structure of polysaccharide TAPA11was as follows:
2.2 Primary structure of polysaccharide TAPA21
TAPA21, with a molecular mass of 7.6×105 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.3:3:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted and 1,3-disubstituted xylopyranose residues, and 1,3-disubstituted,1,2,3- and 1,3,4-trisubstituted mannopyranose residues, and 1-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included three sequences as follows:
So, the predicted repeating primary structure of polysaccharide TAPA21 was as follows:
3 Chemical modification of homopolysaccharide TAPA11
To further enhance the biological activity and be easy to investigate the relationship between structure and functions, chemical modification for fraction TAPA11, including sulfation, acetylation and deacetylation, were carried out, and derivatives TAPAll-s, TAPA11-ac and TAPA11-deac were prepared, respectively. TAPA11-s, was prepared by the chlorosulfonic acid-pyridine method. The total yield of the sulfated product was approximately 65%, and the content of sulfur was determined to be～2.88% by BaSO4 method, so the D.S. value was calculated to be 0.05. TAPA11-ac was prepared using Pyridine and Acetic anhydride as esterifying agent. The total yield of product was approximately 56%, and the content of acetyl was determined to be 5.81% and the D.S. value was calculated to be 0.23. As for TAPAll-deac, the total yield of product was approximately 75%, and the content of acetyl and the D.S. value were determined to Zero, repectively, indicating that the deacetylation was finished completely from the TAPA11.
4 Pharmacological properties for polysaccharide fractions
All polysaccharide fractions were tested in vitro by BALB/C57, macrophages RAW264.7 and PC 12 cell line in our research.
4.1 Proliferation of mice spleen lymphocytes in vitro
T. aurantialba fruiting bodies were extracted by four different solvent one by one and prepeared four different extracts (TAP1, TAP, TAP2 and TAP3). Four samples were assayed by proliferation of mice spleen lymphocytes in vitro, and the result showed that four fractions had property to promote proliferation of mouse spleen lymphocytes. TAP1, TAP and TAP3 showed a better activity compared with TAP2, and at the same time samples TAP land TAP had the property in dose-dependent manner. So TPA was selected to further be isolated and investigated. Immunobiological activity assay showed that three fractions of crude polysaccharides (TAPA, TAPB and TAPC), obtained by ultrafiltration (UF) according to different molecular weight, could significantly increase mouse splenocytes (MSLs) proliferation in vitro at variouse testing concentrations. Compared with TAP, TAPA showed markedly increased activity, does-dependently, at the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA (6μg/mL). The results indicated that UF can play some effect to isolate some fractions which have a better property to stimulate the proliferation of MSLs. Five fractions TAPA 1-TAPA5 were prepared from TAPA after anion-exchange chromatography. Five fractions TAPA 1-TAPA5 markedly increased proliferation rates at various testing concentrations (50,200,500μg/mL) compared with the blank reference. At the concentration of 500μg/mL, samples TAPA1 and TAPA5, almost showed similar MSLs stimulation potency to PHA which was served as a positive reference. Among three samples TAPA11、TAPA21、TAPA51, obtained from TAPA1 after Gel-chromatography, TAPA11 had the best MSLs stimulation potency. At 500μg/mL, TAPA11 had higher the proliferation rate of MSLs (429%) compared with its native fraction TAPA (its proliferation rate was 389%). Among three derivatives, TAPA11-s showed the highest immunobiological activity at various concentrations compared with their original fraction TAPA11, in apparent dose-dependent manner. At the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA, which indicated sulfation of polysaccharide TAPA 11 was effective.
4.2 Effect of samples on nitric oxide production by macrophages RAW264.7
Exposure of RAW264.7 cells to increasing concentrations (10-50μg/mL) of TAPA11 for 24 h resulted in significant increases in NO production (based on nitrite accumulation) compared with untreated controls. Cells treated with 50μg/mL TAPA11 produced the same NO compared with LPS, which was served as a positive control. Among three derivatives, TAPA11-ac was the best one in comparison with the others samples TAPA11-s and TAPA 11-deac.The data indicated that effect of samples on nitric oxide production by macrophages RAW264.7 might be related to not only degree of acetyl group but also different groups.
4.3 Activity of oxidative injury Rehabilitation of PC12 induced with H2O2
All samples were applied to investigate rehabilitation rate of PC 12 cell after treatment with H2O2, and the results indicated that samples TAP、TAPA、TAPB、TAPC、TAPA1-TAPA5、TAPA11 had properties to rehabilitate PC12 cell. APA11-ac exhibited a strong bioactivity to rehabilitate PC 12 cell, at the concentration of 500μg/mL, the rehabilitation rate was 157.63%, which was very close to that of positive control NGF and its rehabilitation rate was 166.8%. The data also revealed that acetylation could improve rehabilitation rate of polysaccharide to PC 12 after treatment with H2O2. However, two samples TAPA11-s and TAPA11-deac exhibited a lower bioactivity to rehabilitate PC12 cell in comparison with the native polysaccharide TAPA11 and NGF.
引文
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