竹荪多糖的提取分离、结构分析和生物活性研究
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摘要
本文以竹荪(Dictyophora indusiata)子实体为原料,对其多糖提取工艺、分离纯化、结构鉴定等进行了深入的研究,并对其水溶性多糖抗氧化活性,酸性竹荪多糖和碱性竹荪多糖增强免疫力功效进行了较为系统的研究,研究结果表明:
以福建古田、建阳、顺昌、浙江江山、贵州贵阳和云南红河地区的6种竹荪子实体为原料,测定了其粗蛋白、粗脂肪、粗纤维、粗多糖、灰分、矿质元素和氨基酸含量。6个供试地区的竹荪子实体均富含蛋白,多糖与粗纤维,且脂肪含量低,但多糖含量差异明显,来自福建建阳的样品粗多糖含量达到30.68%,来自贵州的样品多糖含量仅为18.68%;此外,竹荪子实体富含Ca、Mg、Fe和Zn等人体必需的矿质元素,其中来自浙江和贵州的样品中含量最为丰富;通过氨基酸分析,竹荪蛋白中的限制性氨基酸为赖氨酸,不同产地的竹荪样品存在较大差异。
应用响应面分析法优化了竹荪多糖水浸提工艺。研究了提取时间、提取温度、料液比、pH对多糖得率的影响。在此基础上以提取温度、料液比、pH值为考察因素,以多糖得率为指标,采用响应面实验方法,确定了竹荪多糖提取的最优工艺条件为提取温度95℃、pH=2.0、料液比1:33。在此条件下,其多糖得率达到11.717%,实际测得多糖得率为l1.698%。选取DPPH法和ABTS法评价对上述条件提取出的多糖的清除自由基的能力,DPPH法中,竹荪多糖的EC50为0.150mg/mL(Vc的EC50为0.011 mg/mL),ABTS法中,竹荪多糖的EC50为0.909 mg/mL(Vc的EC50为0.209 mg/mL),竹荪多糖具有较好的抗氧化活性。
从竹荪(Dictyophora indusiata)子实体提取水溶性多糖,经Sephadex G75和DEAE-Sepharose FF柱层析纯化得到长裙竹荪多糖Dip-1和Dip-2,采用GPC方法分别测定该多糖的分子量为2100990和19755;分别测定了其单糖组和糖苷键连接分别为:Dip-1的单糖组成为Fru 1.30%,Glc 85.34%, Man 13.36%,糖苷键组成为:非还原端Man /非还原端Glc /→1)-Glc-(6→/→1)-Man-(3,6→= 1:9.3:5.6:1;Dip-2的单糖组成为Xyl 20.6%, Fru 7.7%, Gal 17.7%, Glc 13.1%,Man 40.9%,糖苷键摩尔百分比:→1)-Xyl-(5→/→1)-Gal-(3→/→1)-Gal-(6→/→1)-Glc-(6→/非还原端Glc /→1)-Man-(2→= 18.7:2.4:13.7:1:10.8:37.2。并对水提多糖进行抗氧化动物实验和ORAC方法进行抗氧化实验研究,结果表明水提多糖具有很好的抗氧化作用。
竹荪(Dictyophora indusiata)子实体经水溶液提取后,滤渣采用碱提,1M HCl溶液提取,滤液真空浓缩后得到竹荪粗多糖Di-1;滤渣再用1% NaOH溶液提取,滤液真空浓缩后得到竹荪粗多糖Di-2;用Sevag法除去蛋白,乙醇分级沉淀,分别用30%、80%和95%乙醇沉淀,对80%乙醇沉淀的竹荪多糖Di-1和Di-2分别测定该多糖的单糖组成,糖苷键连接,Di-1的单糖组成为Rha 2.6%; Fru 13.6%; Gal 1.5% ; Glc 64.0% ; Man 18.3%,糖苷键摩尔百分比:→1)-Glc-(6→64.0/→2)-Man-(1→18.3 /→2)-Fru-(1→13.6 =4.7:1.3:1。Di-2的单糖组成为Rha 1.56%; Xyl 3.55% ; Fru 11.13%; Gal 1.45%; Glc 74.36 %; Man 7.95%,糖苷键摩尔百分比:糖苷键摩尔百分比:→1)-Glc-(6→69.8/非还原端Glc 4.6/→2)-Fru-(1→11.1 = 15.2:1:2.4。并对竹荪多糖Di-1和Di-2分别进行免疫动物实验,结果表明:Di-1和Di-2均有不同程度的增强免疫的作用。
The present study focused on the extraction and structural analysis of polysaccharides in the fruiting body of Dictyophora indusiata and their bioactivities. The antioxidant activities about water-soluble polysaccharides were investigated in vitro and in vivo. The immune activities of acid- and alkali-soluble polysaccharides were investigated in vitro and in vivo. The main contents and results are as follows:
The fruiting bodies of Dictyophora indusiata from 6 different regions, including Gutian, Jianyang and Shunchang in Fujian province, Jiangshan in Zhejiang province, Guiyang in Guizhou province, and Honghe in Yunnan province, were investigated. The contents of crude proteins, crude fat, crude fiber, crude polysaccharides, ash, mineral elements and amino acids were determined. It’s showed that the fruiting bodies from different regions all enriched crude proteins, crude polysaccharides and crude fiber with low crude fat, although, there was a marked difference in crude polysaccharides. The content of crude polysaccharides from Jiangyang in Fujian procince was 30.68% when the sample of Guizhou was about 18.68%. The fruiting bodies of Dictyophora indusiata were rich in essential mineral elements such as Ca , Mg, Fe and Zn. The most abundant samples were the samples from Zhejiang and Guizhou. By amino acid analysis, the limited amino acid of Dictyophora indusiata was lysine and it’s changing with the origins.
Aiming at optimization of the extraction of polysaccharides in the fruiting body of Dictyophora indusiata response surface methodology was adopted. The efects of extraction temperature,material-solvent ratio and extraction pH on the yield of polysaccharides were measured by single factor test and response surface method using the extraction rate as index.The optimal conditions were determined as follows:extraction temperature 95℃,material-solvent ratio 1:33 and extraction pH 2.0.Under the optimal conditions,the extraction rate of polysaccharides from Dictyophora indusiata was 11.71%.And the actual extraction rate of the polysaccharides was detected as 11.698%.High in vitro antioxidant activities of polysaccharides from Dictyophora indusiata were observed in antioxidant systems.Its DPPH and ABTS scavenging capabilities were both in dose-dependent manner, with EC50 values being of 0.150 mg/mL and 0.909 mg/mL, respectively.
The water-soluble crude Dictyophora indusiata polysaccharides (DIP)were extracted from the fruiting body of Dictyophora indusiata. They were fractionated by Sephadex G-75 and DEAE-Sepharose fast flow column chromatography to obtain DIPs I and II. The structural features of DIPs I and II were investigated. The results indicated that DIP I was composed of Fru, Glc and Man with molecular weight of 2100.99 kDa , while DIP II comprised Xyl, Fru, Gal, Glc and Man with molecular weight of 18.16 kDa. The glycosidic linkage of DIP I was composed of terminal Man: terminal Glc:→1)-Glc-(6→:→1)-Man-(3,6→with the ratio of 1.0:9.3:5.6:1.0, while DIP II was composed of→1)-Xyl-(5→:→1)-Gal-(3→:→1)-Gal-(6→:→1)-Glc-(6→:terminal Glc :→1)-Man-(2→with the ratio of 10.8: 1.0: 37.2: 18.7: 2.4: 13.7. DIP exhibited the higher antioxidant activities in vivo and vitro assay systems and could be explored as a novel potential antioxidant.
The acid- and alkali-soluble polysaccharides (DIPs I and II)were extracted from the fruiting body of Dictyophora indusiata in this work. DIP I comprised of Glc, Fru and Man, whereas DIP II was of Glc and Fru. Glc was the dominant monosaccharide in both DIPs with relatively molar percentage of > 60%, which constructed the backbone of polysaccharide chain. The analysis of glycosidic linkage indicated that Glc was occurred as→1)-Glc(-6→in DIP II. The immunological assays showed that both DIPs I and II had a noticeable effect on hemolytic antibody level in the tested dosage range.
以福建古田、建阳、顺昌、浙江江山、贵州贵阳和云南红河地区的6种竹荪子实体为原料,测定了其粗蛋白、粗脂肪、粗纤维、粗多糖、灰分、矿质元素和氨基酸含量。6个供试地区的竹荪子实体均富含蛋白,多糖与粗纤维,且脂肪含量低,但多糖含量差异明显,来自福建建阳的样品粗多糖含量达到30.68%,来自贵州的样品多糖含量仅为18.68%;此外,竹荪子实体富含Ca、Mg、Fe和Zn等人体必需的矿质元素,其中来自浙江和贵州的样品中含量最为丰富;通过氨基酸分析,竹荪蛋白中的限制性氨基酸为赖氨酸,不同产地的竹荪样品存在较大差异。
应用响应面分析法优化了竹荪多糖水浸提工艺。研究了提取时间、提取温度、料液比、pH对多糖得率的影响。在此基础上以提取温度、料液比、pH值为考察因素,以多糖得率为指标,采用响应面实验方法,确定了竹荪多糖提取的最优工艺条件为提取温度95℃、pH=2.0、料液比1:33。在此条件下,其多糖得率达到11.717%,实际测得多糖得率为l1.698%。选取DPPH法和ABTS法评价对上述条件提取出的多糖的清除自由基的能力,DPPH法中,竹荪多糖的EC50为0.150mg/mL(Vc的EC50为0.011 mg/mL),ABTS法中,竹荪多糖的EC50为0.909 mg/mL(Vc的EC50为0.209 mg/mL),竹荪多糖具有较好的抗氧化活性。
从竹荪(Dictyophora indusiata)子实体提取水溶性多糖,经Sephadex G75和DEAE-Sepharose FF柱层析纯化得到长裙竹荪多糖Dip-1和Dip-2,采用GPC方法分别测定该多糖的分子量为2100990和19755;分别测定了其单糖组和糖苷键连接分别为:Dip-1的单糖组成为Fru 1.30%,Glc 85.34%, Man 13.36%,糖苷键组成为:非还原端Man /非还原端Glc /→1)-Glc-(6→/→1)-Man-(3,6→= 1:9.3:5.6:1;Dip-2的单糖组成为Xyl 20.6%, Fru 7.7%, Gal 17.7%, Glc 13.1%,Man 40.9%,糖苷键摩尔百分比:→1)-Xyl-(5→/→1)-Gal-(3→/→1)-Gal-(6→/→1)-Glc-(6→/非还原端Glc /→1)-Man-(2→= 18.7:2.4:13.7:1:10.8:37.2。并对水提多糖进行抗氧化动物实验和ORAC方法进行抗氧化实验研究,结果表明水提多糖具有很好的抗氧化作用。
竹荪(Dictyophora indusiata)子实体经水溶液提取后,滤渣采用碱提,1M HCl溶液提取,滤液真空浓缩后得到竹荪粗多糖Di-1;滤渣再用1% NaOH溶液提取,滤液真空浓缩后得到竹荪粗多糖Di-2;用Sevag法除去蛋白,乙醇分级沉淀,分别用30%、80%和95%乙醇沉淀,对80%乙醇沉淀的竹荪多糖Di-1和Di-2分别测定该多糖的单糖组成,糖苷键连接,Di-1的单糖组成为Rha 2.6%; Fru 13.6%; Gal 1.5% ; Glc 64.0% ; Man 18.3%,糖苷键摩尔百分比:→1)-Glc-(6→64.0/→2)-Man-(1→18.3 /→2)-Fru-(1→13.6 =4.7:1.3:1。Di-2的单糖组成为Rha 1.56%; Xyl 3.55% ; Fru 11.13%; Gal 1.45%; Glc 74.36 %; Man 7.95%,糖苷键摩尔百分比:糖苷键摩尔百分比:→1)-Glc-(6→69.8/非还原端Glc 4.6/→2)-Fru-(1→11.1 = 15.2:1:2.4。并对竹荪多糖Di-1和Di-2分别进行免疫动物实验,结果表明:Di-1和Di-2均有不同程度的增强免疫的作用。
The present study focused on the extraction and structural analysis of polysaccharides in the fruiting body of Dictyophora indusiata and their bioactivities. The antioxidant activities about water-soluble polysaccharides were investigated in vitro and in vivo. The immune activities of acid- and alkali-soluble polysaccharides were investigated in vitro and in vivo. The main contents and results are as follows:
The fruiting bodies of Dictyophora indusiata from 6 different regions, including Gutian, Jianyang and Shunchang in Fujian province, Jiangshan in Zhejiang province, Guiyang in Guizhou province, and Honghe in Yunnan province, were investigated. The contents of crude proteins, crude fat, crude fiber, crude polysaccharides, ash, mineral elements and amino acids were determined. It’s showed that the fruiting bodies from different regions all enriched crude proteins, crude polysaccharides and crude fiber with low crude fat, although, there was a marked difference in crude polysaccharides. The content of crude polysaccharides from Jiangyang in Fujian procince was 30.68% when the sample of Guizhou was about 18.68%. The fruiting bodies of Dictyophora indusiata were rich in essential mineral elements such as Ca , Mg, Fe and Zn. The most abundant samples were the samples from Zhejiang and Guizhou. By amino acid analysis, the limited amino acid of Dictyophora indusiata was lysine and it’s changing with the origins.
Aiming at optimization of the extraction of polysaccharides in the fruiting body of Dictyophora indusiata response surface methodology was adopted. The efects of extraction temperature,material-solvent ratio and extraction pH on the yield of polysaccharides were measured by single factor test and response surface method using the extraction rate as index.The optimal conditions were determined as follows:extraction temperature 95℃,material-solvent ratio 1:33 and extraction pH 2.0.Under the optimal conditions,the extraction rate of polysaccharides from Dictyophora indusiata was 11.71%.And the actual extraction rate of the polysaccharides was detected as 11.698%.High in vitro antioxidant activities of polysaccharides from Dictyophora indusiata were observed in antioxidant systems.Its DPPH and ABTS scavenging capabilities were both in dose-dependent manner, with EC50 values being of 0.150 mg/mL and 0.909 mg/mL, respectively.
The water-soluble crude Dictyophora indusiata polysaccharides (DIP)were extracted from the fruiting body of Dictyophora indusiata. They were fractionated by Sephadex G-75 and DEAE-Sepharose fast flow column chromatography to obtain DIPs I and II. The structural features of DIPs I and II were investigated. The results indicated that DIP I was composed of Fru, Glc and Man with molecular weight of 2100.99 kDa , while DIP II comprised Xyl, Fru, Gal, Glc and Man with molecular weight of 18.16 kDa. The glycosidic linkage of DIP I was composed of terminal Man: terminal Glc:→1)-Glc-(6→:→1)-Man-(3,6→with the ratio of 1.0:9.3:5.6:1.0, while DIP II was composed of→1)-Xyl-(5→:→1)-Gal-(3→:→1)-Gal-(6→:→1)-Glc-(6→:terminal Glc :→1)-Man-(2→with the ratio of 10.8: 1.0: 37.2: 18.7: 2.4: 13.7. DIP exhibited the higher antioxidant activities in vivo and vitro assay systems and could be explored as a novel potential antioxidant.
The acid- and alkali-soluble polysaccharides (DIPs I and II)were extracted from the fruiting body of Dictyophora indusiata in this work. DIP I comprised of Glc, Fru and Man, whereas DIP II was of Glc and Fru. Glc was the dominant monosaccharide in both DIPs with relatively molar percentage of > 60%, which constructed the backbone of polysaccharide chain. The analysis of glycosidic linkage indicated that Glc was occurred as→1)-Glc(-6→in DIP II. The immunological assays showed that both DIPs I and II had a noticeable effect on hemolytic antibody level in the tested dosage range.
引文
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