桦褐孔菌化学成分及药理活性研究
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摘要
桦褐孔菌的学名为Inonotus obliquus (Pers.:Fr.) Pilát(以下简称IO),在俄罗斯又被俗称为Chaga。该菌外表黑色,常生于白桦、银桦、榆树和赤杨等活立木的树皮下。IO主要分布于北半球北纬45~50°的地区,如俄罗斯西西伯利亚、波兰、芬兰和中国的黑龙江、长白山等地。从十六世纪开始,IO就被俄罗斯、波兰和大多数波罗的海国家用来治疗胃癌、心血管疾病和糖尿病等。虽然IO的用量在逐年增加,但其化学组成和药理作用还有待进一步研究。
本研究对IO的化学成分进行了综合分析。先通过分级萃取的方法将IO按石油醚、丙酮、甲醇和水的顺序依次进行萃取。通过气相色谱质谱联用技术对IO石油醚萃取物(IOPE)和丙酮萃取物(IOA)的化学成分进行了分析。其中,IOPE中共检出25个主要组分,占总成分的95.6%,其中含量最高的是甾体化合物,共6个,占总量91.6%。在IOA的气相色谱质谱分析中,鉴定出58个主要组分,占总成分的86.60%。分析结果表明,IOA中含量最高仍是甾体化合物,共14个,占总量77.1%。
通过单因素实验及正交实验对IO多糖萃取过程工艺条件进行优化,确定了多糖水萃取的最佳工艺参数。由实验确定的最佳萃取条件为:萃取时间为240 min,萃取温度为90 oC,液料比为14:1,萃取次数为7次。通过用水和稀碱溶液萃取、醇沉、脱蛋白、DEAE柱分离和Sephadex G-100柱纯化等工艺,从IO中分离得到8种多糖。
对纯多糖IOPS-2结构进行了分析。凝胶层析法测得IOPS-2的相对分子量为2.76×104。由气相色谱分析、高碘酸氧化-Smith降解、甲基化和部分酸水解等方法分析可知IOPS-2为葡甘露聚糖,主要由端基葡萄糖,1,3-连接的D-吡喃葡萄糖,1,3-连接的D-吡喃甘露糖,1,4,6-连接的D-吡喃甘露糖及1,4,6-连接的D-吡喃葡萄糖构成,其摩尔数比为1.0:2.5:8.3:3.2:0.5。
本研究对IOPE、IOA、IOM和IOW的药理活性进行了研究。体内实验证明,IO萃取物具有抑制肿瘤的生长的作用,其抑瘤率随萃取溶剂极性的增大而增大。体外实验结果表明,IOPE和IOA对肿瘤的生长有一定的细胞毒性,能在体外抑制肿瘤的生长,而水萃取物基本不具有细胞毒性。此外,IO各萃取物给药组小鼠血清对K562肿瘤细胞有较大的细胞毒性,且细胞毒作用随着萃取溶剂极性的增大而增大。
实验研究了IO各萃取物免疫作用,结果表明,IO各萃取物具有提高荷瘤小鼠脾指数和胸腺指数的作用,并且有助于促进淋巴细胞的增殖和增强NK、LAK细胞的活性。
The mushroom Inonotus obliquus (Fr.) Pilát (IO),which name is Chaga in Russian, is a black parasitic fungus that grows on living trunks of the mature birch, elm, alder or beech trees. It is mainly found at latitudes of 45?N–50?N, such as Western Siberia, Poland, Finland、Changbai Mountain and Heilong River in China. Traditionally, it has been used for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes since the 16th century in Russia, Poland and most of the Baltic countries. However, its chemical components and pharmacological actions have not been well documented, in spite of its increasing usage.
A comprehensive analytical study on the chemical constituents of IO was performed. The stepwise extractions of IO were performed using petroleum ether, acetone, methanol and water orderly. The main chemical constituents in the extract with petroleum ethe(rIOPE) and extract with acetone(IOA) from IO were analyzed by GC-MS. 25 chemical constituents (relative content: 95.6%) of IOPE were identified by using GC-MS. The primary component were 6 steroids (relative content: 91.6%). 58 chemical constituents (relative content: 86.6%) of IOA were identified by using GC-MS. The primary components were 14 steroids (relative content: 77.1%).
Several factors that affecting the extraction process of polysaccharide were studied through single factor and orthogonal experiments. The optimal technological conditions of polysaccharide extraction from IO were decided: the extraction time being 240 minutes; the temperature being 90 oC; the ratio of solid material to water being 1:14; the times of extraction being 7. 4 purified polysaccharides and 4 purified acid polysaccharides were prepared with process as the following: extracting, ethanol precipitating, Sevag method deproteinizing, DEAE-cellulose chromatographic fractionating, sephadex chromatographic purifying.
The structure of Polysaccharide IOPS-2 was analyzed. Molecular weight of IOPS-2 estimated to be 2.76×104 detected by gel chromatography. IOPS-2 has been investigated by GC analysis, periodate oxidation, Smith degradation, methylation and partial acid hydrolysis analysis. The results showed that it is a glucomannan, composed of terminal Glcp, (1→3)-linked Glcp, (1→3)-linked Manp, (1→4,6)-linked Manp, (1→4,6)-linked Glcp in the molar ratios of 1.0: 2.5: 8.3: 3.2: 0.5.
The pharmacological activities of extracts from IO (IOPE, IOA, IOM and IOW ) were studied. In vivo, the test showed that the extracts of IO have inhibition effects on tumor, the inhibition ratio increased with the polar of solvent. In vitro test, the result show that IOPE and IOA have cell toxicity on tumor cell, at the same time the IOW have no cell toxicity. In vitro test, the results also show that IO extracts containing serums have cytotoxic activity on K562 cell. The higher polarity the extract solvents have, the higher activity the serums have.
To study the immunoregulatory effects of IO extracts, the results show that the extracts can increase the spleen index and thymus index of tumor-bearing mice, induce the proliferation of spleen lymphocytes, and increase the activities of NK and LAK cell.
本研究对IO的化学成分进行了综合分析。先通过分级萃取的方法将IO按石油醚、丙酮、甲醇和水的顺序依次进行萃取。通过气相色谱质谱联用技术对IO石油醚萃取物(IOPE)和丙酮萃取物(IOA)的化学成分进行了分析。其中,IOPE中共检出25个主要组分,占总成分的95.6%,其中含量最高的是甾体化合物,共6个,占总量91.6%。在IOA的气相色谱质谱分析中,鉴定出58个主要组分,占总成分的86.60%。分析结果表明,IOA中含量最高仍是甾体化合物,共14个,占总量77.1%。
通过单因素实验及正交实验对IO多糖萃取过程工艺条件进行优化,确定了多糖水萃取的最佳工艺参数。由实验确定的最佳萃取条件为:萃取时间为240 min,萃取温度为90 oC,液料比为14:1,萃取次数为7次。通过用水和稀碱溶液萃取、醇沉、脱蛋白、DEAE柱分离和Sephadex G-100柱纯化等工艺,从IO中分离得到8种多糖。
对纯多糖IOPS-2结构进行了分析。凝胶层析法测得IOPS-2的相对分子量为2.76×104。由气相色谱分析、高碘酸氧化-Smith降解、甲基化和部分酸水解等方法分析可知IOPS-2为葡甘露聚糖,主要由端基葡萄糖,1,3-连接的D-吡喃葡萄糖,1,3-连接的D-吡喃甘露糖,1,4,6-连接的D-吡喃甘露糖及1,4,6-连接的D-吡喃葡萄糖构成,其摩尔数比为1.0:2.5:8.3:3.2:0.5。
本研究对IOPE、IOA、IOM和IOW的药理活性进行了研究。体内实验证明,IO萃取物具有抑制肿瘤的生长的作用,其抑瘤率随萃取溶剂极性的增大而增大。体外实验结果表明,IOPE和IOA对肿瘤的生长有一定的细胞毒性,能在体外抑制肿瘤的生长,而水萃取物基本不具有细胞毒性。此外,IO各萃取物给药组小鼠血清对K562肿瘤细胞有较大的细胞毒性,且细胞毒作用随着萃取溶剂极性的增大而增大。
实验研究了IO各萃取物免疫作用,结果表明,IO各萃取物具有提高荷瘤小鼠脾指数和胸腺指数的作用,并且有助于促进淋巴细胞的增殖和增强NK、LAK细胞的活性。
The mushroom Inonotus obliquus (Fr.) Pilát (IO),which name is Chaga in Russian, is a black parasitic fungus that grows on living trunks of the mature birch, elm, alder or beech trees. It is mainly found at latitudes of 45?N–50?N, such as Western Siberia, Poland, Finland、Changbai Mountain and Heilong River in China. Traditionally, it has been used for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes since the 16th century in Russia, Poland and most of the Baltic countries. However, its chemical components and pharmacological actions have not been well documented, in spite of its increasing usage.
A comprehensive analytical study on the chemical constituents of IO was performed. The stepwise extractions of IO were performed using petroleum ether, acetone, methanol and water orderly. The main chemical constituents in the extract with petroleum ethe(rIOPE) and extract with acetone(IOA) from IO were analyzed by GC-MS. 25 chemical constituents (relative content: 95.6%) of IOPE were identified by using GC-MS. The primary component were 6 steroids (relative content: 91.6%). 58 chemical constituents (relative content: 86.6%) of IOA were identified by using GC-MS. The primary components were 14 steroids (relative content: 77.1%).
Several factors that affecting the extraction process of polysaccharide were studied through single factor and orthogonal experiments. The optimal technological conditions of polysaccharide extraction from IO were decided: the extraction time being 240 minutes; the temperature being 90 oC; the ratio of solid material to water being 1:14; the times of extraction being 7. 4 purified polysaccharides and 4 purified acid polysaccharides were prepared with process as the following: extracting, ethanol precipitating, Sevag method deproteinizing, DEAE-cellulose chromatographic fractionating, sephadex chromatographic purifying.
The structure of Polysaccharide IOPS-2 was analyzed. Molecular weight of IOPS-2 estimated to be 2.76×104 detected by gel chromatography. IOPS-2 has been investigated by GC analysis, periodate oxidation, Smith degradation, methylation and partial acid hydrolysis analysis. The results showed that it is a glucomannan, composed of terminal Glcp, (1→3)-linked Glcp, (1→3)-linked Manp, (1→4,6)-linked Manp, (1→4,6)-linked Glcp in the molar ratios of 1.0: 2.5: 8.3: 3.2: 0.5.
The pharmacological activities of extracts from IO (IOPE, IOA, IOM and IOW ) were studied. In vivo, the test showed that the extracts of IO have inhibition effects on tumor, the inhibition ratio increased with the polar of solvent. In vitro test, the result show that IOPE and IOA have cell toxicity on tumor cell, at the same time the IOW have no cell toxicity. In vitro test, the results also show that IO extracts containing serums have cytotoxic activity on K562 cell. The higher polarity the extract solvents have, the higher activity the serums have.
To study the immunoregulatory effects of IO extracts, the results show that the extracts can increase the spleen index and thymus index of tumor-bearing mice, induce the proliferation of spleen lymphocytes, and increase the activities of NK and LAK cell.
引文
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