桦褐孔菌多糖结构及生物活性研究
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摘要
桦褐孔菌Inonotus obliquus ( Pers . : Fr . ) Pilat属担子菌亚门、层菌纲、非褐菌目、多孔菌科、褐卧孔菌属,在我国主要分布于黑龙江和吉林省的长白山地区。据文献报道桦褐孔菌多糖具有抗肿瘤、免疫调节、抗氧化和降血糖等生理功能。但目前的研究主要集中在粗多糖的药理活性上,对桦褐孔菌多糖的分离纯化和结构研究报道较少。为进一步探讨桦褐孔菌多糖活性与结构之间的关系,本文对桦褐孔菌多糖的提取、分离纯化、理化性质、结构和生物活性等方面进行了较为系统的研究,得到如下结论:
1.桦褐孔菌子实体多糖的提取工艺研究通过单因素分析和正交试验,确定了水溶多糖与碱溶多糖的最佳提取工艺。水溶多糖的适宜提取条件为料液比1:20(w:v)、提取温度90℃、提取时间1 h,多糖得率为0.992%。提取液浓缩后醇沉的条件为提取液浓缩4倍,加3倍乙醇醇析时,多糖最大沉淀率为97.9%。
碱溶多糖的适宜提取条件为料液比为1:20(w:v),NaOH浓度为0.5mol/L,提取时间为1 h,乙醇浓度为85%,在此条件下桦褐孔菌碱溶性多糖的得率为0.659%。
2.桦褐孔菌子实体多糖的分离纯化研究
应用热水煮提和乙醇沉淀的方法,获得了桦褐孔菌多糖IOP,收率为2.67%。IOP的总糖含量为37.2%,蛋白含量为17.8%。经气相色谱分析,IOP中含有鼠李糖(7.59%)、葡萄糖(50.87%)、木糖(10.63%)、半乳糖(14.96%)和甘露糖(15.95%);应用稀碱溶液煮提和乙醇沉淀的方法,获得了桦褐孔菌碱溶多糖AIOP,收率为2.14%。AIOP的总糖含量为31.4%,蛋白含量为16.3%。经GC分析得知,AIOP含有鼠李糖(8.90%)、甘露糖(5.21%)、半乳糖(13.58%)、葡萄糖(62.54%)、木糖(6.71%)和葡萄糖醛酸(3.06%);
建立了DEAE-纤维素和分子筛层析相结合的分级方法,对去蛋白和脱色后的桦褐孔菌多糖进行比较系统的分级,获得了七种多糖级分,其中:两种中性糖级分IOPN-I和IOPN-II;三种酸性糖级分IOPS-I-A、IOPS-I-B和IOPS-II-A;两种碱溶多糖AIOP-I和AIOP-II,其分子量分布均一,大小在3.3×10~3到5.9×10~3之间。
3.桦褐孔菌子实体多糖的结构研究
采用部分酸水解、高碘酸氧化、Smith降解、甲基化和二维核磁共振等方法,对桦褐孔菌多糖中收率和纯度较高的级分IOPN-I进行了结构研究。结果表明IOPN-I主要由β构型糖苷键构成,也有少量a构型糖苷键;其主链部分主要由(1→3)-β-D-Man、(1→3,6)-β-D-Glc、(1→6)-3-O-Me-β-D-Gal、(1→3)-a-D-Glc和(1→6)-β-D-Glc构成;分支点糖残基是(1→3,6)-β-D-Glc;(1→)-β-D-Glc形成主链的非还原末端;Glc构成支链或主链边缘的末端残基;主链核心区每8个己糖残基有1个非还原末端或1个1→6糖苷键。
4.桦褐孔菌子实体多糖的生物活性研究
通过多糖对小鼠免疫系统影响的实验表明,桦褐孔菌多糖能促进B淋巴细胞的增殖;增强巨噬细胞的吞噬功能;提高巨噬细胞分泌TNF-α的能力;增加NO的生成量;提高了荷瘤小鼠的免疫功能。
体内抗肿瘤实验表明,桦褐孔菌多糖在抑制S180肿瘤细胞生长的同时,增加了荷瘤小鼠的相对脾重、相对胸腺重以及小鼠血清中TNF-α的含量。但体外实验证明多糖对S180、Hela、BGC-830及HepG2细胞并没有直接的杀伤作用,说明桦褐孔菌多糖的抗肿瘤作用是通过增强机体的免疫功能来实现的,其抗肿瘤效果由高到低依次为IOPN>IOPS>AIOP。
Inonotus obliquus (Pers.:Fr.) Pilat is well known as one of the most popular medicinal species for its therapeutic effect, which is a white rot fungus belonging to the family Hymenochaetacea. It is mainly found at Changbai Mountain and Heilong River in China.The polysaccharide distilled from I. obliquus has the functions of immunomodulation, anti-tumor, antioxidant and anti-inflammation and has no poisonous effect. But the studies on polysaccharides from Inonotus obliquus have focused on crude polysaccharide, there is still a lack of understanding of all of the Inonotus obliquus polysaccharides. So far there is not any information published about chemical structures on the homgerous component from the polysaccharide.Therefore, The present paper was concerned with the isolation, chemical characterization, evaluation of the immunomodulative and antitumor activities of a polysaccharide from the fruiting body of Inonotus obliquus. The investigation results are as follows:
1. Development of extract technique for polysaccharide from the fruiting body of Inonotus obliquus.
Through single-factor experiments of orthognal experiments, it was found that best yield could be obtained. Polysaccharides of Inonotus obliquus were extracted by hot water and its residue extracted by alkali. The optimal extracting conditions of water soluble polysaccharides from Inonotus obliquus are as follows:the adding ratio of water is 1:20,the temperature of extraction is 90℃, the time of extraction is 1 hours,The extracting ratio of water soluble polysaccharides from Inonotus obliquus were 0.992%.Extracted solution was concentrated, then precipitated after incubation for 4 with 3volumes ethanol.
The optimal extracting conditions of alkali extractional polysaccharide from Inonotus obliquus are as follows:the adding ratio of alkali is 1:20, the concentration of NaOH is 0.5 moL/L, the time of extraction is 1 hour, the concentration of ethanol is 85%.The extracting ratio of alkali soluble polysaccharides from Inonotus obliquus were 0.659%.
2. Extraction and purification of polysaccharides from IOP and AIOP.
The water-soluble polysaccharides were extracted from Inonotus obliquus with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as IOP, was obtained with a yield of 2.67% (w/w). IOP contained 37.2% total carbohydrates and more than 17.8% protein. Sugar composition analysis by GC chromatography indicated that IOP consisted of Rhamnose (7.59%),Glucose (50.87%), Xylose (10.63%),Galactose (14.96%) and Mannose (15.95%).
The alkali-soluble polysaccharides were extracted from its residue with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as AIOP, was obtained with a yield of 2.14 (w/w). IOP contained 31.4% total carbohydrates and more than 16.3% protein. Sugar composition analysis by GC chromatography indicated that AIOP consisted of Rhamnose (8.9%),Mannose (5.21%), Galactose (13.58%), Glucose (62.54%), Xylose (6.71%), and Glucturonic acid (3.06%). IOP was separated on a preparative DEAE-Cellulose chromatographic fractionating, an unbound fraction (IOPN) by water elution and two bound fraction (IOPS-I、IOPS- II) by 1.0 M NaCl elution. Then, IOPN、IOPS-I、IOPS- II and AIOP were separated on Sephades G-100 column into seven fractions IOPN-I、IOPN-II、IOPS-I-A、IOPS-I-B、IOPS-II-A.、AIOP-I and AIOP-II respectively. These seven fractions were homogenous polysaccharides, theirs molecular weights approximately ranging from 3.3×10~3 to 5.9×10~3.
3. Structural analysis of homogeneous component from IOPN-I.
The structural features of IOPN-I were elucidated using partial acid hydrolys, methylation, periodate oxidation, Smith degradationand and 2D NMR analysis. The results show that the IOPN-I mainly formβglycosidic bond configuration, but also a small amount of a glycosidic bond configuration. IOPN-I had a backbone composed of (1→3)-linked-β-D-Mannopyranos -yl, (1→3,6)-linked-β-D-glucopyranosyl, (1→6)-linked-3-O-Me-β-D-galactopyranosyl, (1→3)-linked-α-D-glucopyranosyl, (1→6)-linked-β-D-glucopyranosyl residues in the ratio of 1:1:2:2:1, and terminated with a single non-reducing terminal (1→)-β-D-glucopyranosyl residues at the C-3 position of (1→3,6)-linked-β-D-glucopyranosyl, along the main chain.
4. Study of bioactivity of the polysaccharides from the fruiting body of Inonotus obliquus.
The immunological activity of Inonotus obliquus polysaccharides were studied in mice. IOPN、IOPS and AIOP stimulated the B spleen lymphocyte proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. Pharmacological studies revealed IOPN、IOPS and AIOP could inhibit the growth of Sarcoma 180 tumor remarkably in vivo. Polysaccharides from Inonotus obliquus increased in the relative spleen and thymus weight, expression of tumor necrosis factor-alpha (TNF-α) in serum. However, IOPN、IOPS and AIOP at the concentration up to 300μg/ml did not affect the growth of S180、Hela、BGC-830 and HepG2 tumor cells in vitro. The neutral Inonotus obliquus polysaccharide IOPN had best immunomodulative activities in the acidic polysaccharide IOPS and the alkali-soluble polysaccharides AIOP. These results indicated that the tumor inhibitory activity of the three fractions may be related to immunomodulation.
1.桦褐孔菌子实体多糖的提取工艺研究通过单因素分析和正交试验,确定了水溶多糖与碱溶多糖的最佳提取工艺。水溶多糖的适宜提取条件为料液比1:20(w:v)、提取温度90℃、提取时间1 h,多糖得率为0.992%。提取液浓缩后醇沉的条件为提取液浓缩4倍,加3倍乙醇醇析时,多糖最大沉淀率为97.9%。
碱溶多糖的适宜提取条件为料液比为1:20(w:v),NaOH浓度为0.5mol/L,提取时间为1 h,乙醇浓度为85%,在此条件下桦褐孔菌碱溶性多糖的得率为0.659%。
2.桦褐孔菌子实体多糖的分离纯化研究
应用热水煮提和乙醇沉淀的方法,获得了桦褐孔菌多糖IOP,收率为2.67%。IOP的总糖含量为37.2%,蛋白含量为17.8%。经气相色谱分析,IOP中含有鼠李糖(7.59%)、葡萄糖(50.87%)、木糖(10.63%)、半乳糖(14.96%)和甘露糖(15.95%);应用稀碱溶液煮提和乙醇沉淀的方法,获得了桦褐孔菌碱溶多糖AIOP,收率为2.14%。AIOP的总糖含量为31.4%,蛋白含量为16.3%。经GC分析得知,AIOP含有鼠李糖(8.90%)、甘露糖(5.21%)、半乳糖(13.58%)、葡萄糖(62.54%)、木糖(6.71%)和葡萄糖醛酸(3.06%);
建立了DEAE-纤维素和分子筛层析相结合的分级方法,对去蛋白和脱色后的桦褐孔菌多糖进行比较系统的分级,获得了七种多糖级分,其中:两种中性糖级分IOPN-I和IOPN-II;三种酸性糖级分IOPS-I-A、IOPS-I-B和IOPS-II-A;两种碱溶多糖AIOP-I和AIOP-II,其分子量分布均一,大小在3.3×10~3到5.9×10~3之间。
3.桦褐孔菌子实体多糖的结构研究
采用部分酸水解、高碘酸氧化、Smith降解、甲基化和二维核磁共振等方法,对桦褐孔菌多糖中收率和纯度较高的级分IOPN-I进行了结构研究。结果表明IOPN-I主要由β构型糖苷键构成,也有少量a构型糖苷键;其主链部分主要由(1→3)-β-D-Man、(1→3,6)-β-D-Glc、(1→6)-3-O-Me-β-D-Gal、(1→3)-a-D-Glc和(1→6)-β-D-Glc构成;分支点糖残基是(1→3,6)-β-D-Glc;(1→)-β-D-Glc形成主链的非还原末端;Glc构成支链或主链边缘的末端残基;主链核心区每8个己糖残基有1个非还原末端或1个1→6糖苷键。
4.桦褐孔菌子实体多糖的生物活性研究
通过多糖对小鼠免疫系统影响的实验表明,桦褐孔菌多糖能促进B淋巴细胞的增殖;增强巨噬细胞的吞噬功能;提高巨噬细胞分泌TNF-α的能力;增加NO的生成量;提高了荷瘤小鼠的免疫功能。
体内抗肿瘤实验表明,桦褐孔菌多糖在抑制S180肿瘤细胞生长的同时,增加了荷瘤小鼠的相对脾重、相对胸腺重以及小鼠血清中TNF-α的含量。但体外实验证明多糖对S180、Hela、BGC-830及HepG2细胞并没有直接的杀伤作用,说明桦褐孔菌多糖的抗肿瘤作用是通过增强机体的免疫功能来实现的,其抗肿瘤效果由高到低依次为IOPN>IOPS>AIOP。
Inonotus obliquus (Pers.:Fr.) Pilat is well known as one of the most popular medicinal species for its therapeutic effect, which is a white rot fungus belonging to the family Hymenochaetacea. It is mainly found at Changbai Mountain and Heilong River in China.The polysaccharide distilled from I. obliquus has the functions of immunomodulation, anti-tumor, antioxidant and anti-inflammation and has no poisonous effect. But the studies on polysaccharides from Inonotus obliquus have focused on crude polysaccharide, there is still a lack of understanding of all of the Inonotus obliquus polysaccharides. So far there is not any information published about chemical structures on the homgerous component from the polysaccharide.Therefore, The present paper was concerned with the isolation, chemical characterization, evaluation of the immunomodulative and antitumor activities of a polysaccharide from the fruiting body of Inonotus obliquus. The investigation results are as follows:
1. Development of extract technique for polysaccharide from the fruiting body of Inonotus obliquus.
Through single-factor experiments of orthognal experiments, it was found that best yield could be obtained. Polysaccharides of Inonotus obliquus were extracted by hot water and its residue extracted by alkali. The optimal extracting conditions of water soluble polysaccharides from Inonotus obliquus are as follows:the adding ratio of water is 1:20,the temperature of extraction is 90℃, the time of extraction is 1 hours,The extracting ratio of water soluble polysaccharides from Inonotus obliquus were 0.992%.Extracted solution was concentrated, then precipitated after incubation for 4 with 3volumes ethanol.
The optimal extracting conditions of alkali extractional polysaccharide from Inonotus obliquus are as follows:the adding ratio of alkali is 1:20, the concentration of NaOH is 0.5 moL/L, the time of extraction is 1 hour, the concentration of ethanol is 85%.The extracting ratio of alkali soluble polysaccharides from Inonotus obliquus were 0.659%.
2. Extraction and purification of polysaccharides from IOP and AIOP.
The water-soluble polysaccharides were extracted from Inonotus obliquus with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as IOP, was obtained with a yield of 2.67% (w/w). IOP contained 37.2% total carbohydrates and more than 17.8% protein. Sugar composition analysis by GC chromatography indicated that IOP consisted of Rhamnose (7.59%),Glucose (50.87%), Xylose (10.63%),Galactose (14.96%) and Mannose (15.95%).
The alkali-soluble polysaccharides were extracted from its residue with hot water, precipitated by ethanol and deproteinated using the Sevag method. The polysaccharide mixture, referred to as AIOP, was obtained with a yield of 2.14 (w/w). IOP contained 31.4% total carbohydrates and more than 16.3% protein. Sugar composition analysis by GC chromatography indicated that AIOP consisted of Rhamnose (8.9%),Mannose (5.21%), Galactose (13.58%), Glucose (62.54%), Xylose (6.71%), and Glucturonic acid (3.06%). IOP was separated on a preparative DEAE-Cellulose chromatographic fractionating, an unbound fraction (IOPN) by water elution and two bound fraction (IOPS-I、IOPS- II) by 1.0 M NaCl elution. Then, IOPN、IOPS-I、IOPS- II and AIOP were separated on Sephades G-100 column into seven fractions IOPN-I、IOPN-II、IOPS-I-A、IOPS-I-B、IOPS-II-A.、AIOP-I and AIOP-II respectively. These seven fractions were homogenous polysaccharides, theirs molecular weights approximately ranging from 3.3×10~3 to 5.9×10~3.
3. Structural analysis of homogeneous component from IOPN-I.
The structural features of IOPN-I were elucidated using partial acid hydrolys, methylation, periodate oxidation, Smith degradationand and 2D NMR analysis. The results show that the IOPN-I mainly formβglycosidic bond configuration, but also a small amount of a glycosidic bond configuration. IOPN-I had a backbone composed of (1→3)-linked-β-D-Mannopyranos -yl, (1→3,6)-linked-β-D-glucopyranosyl, (1→6)-linked-3-O-Me-β-D-galactopyranosyl, (1→3)-linked-α-D-glucopyranosyl, (1→6)-linked-β-D-glucopyranosyl residues in the ratio of 1:1:2:2:1, and terminated with a single non-reducing terminal (1→)-β-D-glucopyranosyl residues at the C-3 position of (1→3,6)-linked-β-D-glucopyranosyl, along the main chain.
4. Study of bioactivity of the polysaccharides from the fruiting body of Inonotus obliquus.
The immunological activity of Inonotus obliquus polysaccharides were studied in mice. IOPN、IOPS and AIOP stimulated the B spleen lymphocyte proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. Pharmacological studies revealed IOPN、IOPS and AIOP could inhibit the growth of Sarcoma 180 tumor remarkably in vivo. Polysaccharides from Inonotus obliquus increased in the relative spleen and thymus weight, expression of tumor necrosis factor-alpha (TNF-α) in serum. However, IOPN、IOPS and AIOP at the concentration up to 300μg/ml did not affect the growth of S180、Hela、BGC-830 and HepG2 tumor cells in vitro. The neutral Inonotus obliquus polysaccharide IOPN had best immunomodulative activities in the acidic polysaccharide IOPS and the alkali-soluble polysaccharides AIOP. These results indicated that the tumor inhibitory activity of the three fractions may be related to immunomodulation.
引文
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