蒙古黄芪中多糖、皂甙及活性蛋白的提取分离
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摘要
中草药活性成分的提取分离技术是目前的研究热点之一。为了高效利用黄芪资源,探索黄芪中新的活性成分,本文研究了蒙古黄芪中多糖、皂甙和活性蛋白的提取分离方法。得到的主要结果如下:
黄芪多糖提取的试验结果表明:以80%的乙醇脱脂后,水提取2次,第一次提取60 min,第二次提取70 min可提取出大部分的多糖;本文首次应用纤维素酶辅助提取黄芪多糖,取得了很好的效果,在多糖含量变化不大的情况下,多糖的得率提高率达20%以上。纤维素酶的最佳处理条件为:加酶量60 U/g;酶处理时间90 min;处理温度50℃。分级醇沉和分步醇沉的试验结果表明醇沉浓度为80%比较合理。采用一系列不同截留分子量的中空纤维膜对黄芪水提物进行了较系统的超滤分离,结果表明黄芪多糖的分子量分布较宽,在各分子量阶段的分布是很不均匀的,高分子量和低分子量的糖分布较多,选用截留分子量50 kDa的中空纤维膜可以截留大部分的多糖。酶法脱除黄芪多糖中蛋白质的结果表明:所选五种蛋白酶均能脱除黄芪多糖中的蛋白质,且明显优于Sevag法,将中性蛋白酶1直接应用于未经醇沉的提取浓缩液,加酶量30 U/g生药,在50℃下处理5 h可以去除约68%的蛋白,其多糖含量和得率与对照相比变化不大。对鸡生长发育及免疫活性的试验结果表明去除蛋白后的精制多糖的增重效果和免疫活性得到加强。
采用水提醇沉法、醇提法和纤维素酶辅助水提醇沉法进行了黄芪皂甙的提取试验结果表明:醇提法稍好于水提醇沉法,经纤维素酶处理后,醇沉上清液中的黄芪皂甙浓度提高率达8%以上。对黄芪皂甙分离效果的试验表明:AB-8大孔吸附树脂对黄芪皂甙的分离效果优于正丁醇萃取法:AB-8树脂柱层析分离黄芪皂甙的效果受树脂柱长度、洗脱用乙醇浓度和提取用乙醇浓度的影响较大,柱长55 cm、洗脱用乙醇浓度80%以及提取用乙醇浓度60%为较好参数。
首次研究了黄芪中一种活性蛋白的分离、纯化和鉴定。通过硫酸铵分级沉淀及离子交换柱层析,得到了一种纯的蛋白质,简称Astramonin 1。该蛋白是一种糖蛋白,总糖含量为19.6%。在天然状态下以同源二聚体的形式存在,分子量为61.1 kDa,单一亚基的分子量约为29.6 kDa。该蛋白对一些病原真菌具有明显的抗真菌活性,特别是对灰葡萄孢菌有很强的抑制能力。N端15个氨基酸残基为ESGINLQGDATLANN,同源性分析表明,Astramonin 1是一种新蛋白。该蛋白能凝集兔和人的血红细胞,特别对不同血型的人血表现出明显的特异性,表明Astramonin 1是一种凝集素。糖抑制性实验表明该凝集素属半乳糖结合型的凝集素。凝集活性的最适pH稳定范围很宽,在pH 4.5～7.5范围内,其凝集活性没有变化。Astramonin 1凝集活性耐受温度达65℃,是一种热稳定性较好的凝集素。
The extraction and separation of active components from Chinese medicinal herbs at high efficiency is one of the key research topics today. This study was carried out to broaden the use of Aslralagus mongolicus as well as to discover its new effective components. Major conclusions are as follow:1. Extraction and separation of Astragalus polysaccharide (APS)Dynamic analysis on the extraction process of Astragalus polysaccharide (APS) showed that double aqueous extraction at 60 min and 70 min was able to extract most of the APS from Astralagus mongolicus Bge. Effect of cellulase treatment on the APS extraction was also investigated in this study. Although content of the APS remained unchanged, yield of the APS increased for more than 20% after the cellulase treatment. Optimal condition for APS extraction was as follow: cellulose dosage 60U/g, incubation time 90 min and temperature 50 ℃. An ethanol precipitation concentration at 80% was found to be reasonable after studying the graded ethanol precipitation and stepwise ethanol precipitation. A series of hollow fiber ultrafiltration membrane was used to separate water-soluble extract systematically and membrane with cut-off of 50 kDa was found to be suitable for the separation of APS from other components. This report also describes the enzymatic removal of protein from the APS and this method was more superior than the Sevag's method. According to the immunologic test, the APS was an effective immunostimulating component. Its immunostimulating activity was strong and was better for live-wight growth.2. Extraction and separation of Astragalus sapnonin (AS)Various methods for the extraction and isolation of Astragalus sapnonin (AS), such as water-extraction, ethanol extraction and cellulase pretreatment, were carried out in this study. Results showed that ethanol extraction was slightly better than water-extration. Concentration of AS in ethanol precipitation supernatant was increased by enzyme pretreatment and showed an increment of more than 8%. Resin absorbing method significantly better than extraction refined by n-butyl alcohol. The rate and yield of AS were high when it was extracted by chromatography using AB-8 macroporous adsorption resin at 60% ethanol, resin column of 55 cm and elution concentration of 80%.3. Extraction, Purification and identification of bioactive proteinA novel bioactive protein (Astramonin 1) was isolated from the roots of Astragalus monghollcus using a combination of ammonium sulfate fractionation and ion exchange chromatographies. Astramonin 1 was a dimeric protein (61.1 kDa) and was composed of two identical subunits each with a
molecular mass of 29.6 kDa. The protein was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified protein hemagglutinated both rabbit and human erythrocytes, but with a preference for blood types O (native) and AB (trypsin-treated). Among the various carbohydrates tested, the protein was best inhibited by D-galactose and its derivatives with pronounced preference for lactose, Astramonin 1 may therefore be regarded as a galactose-specificic lectin. N-terminal amino acid sequence of Astramonin 1 was determined as ESGINLQGDATLANN. The optimal pH range for hemagglutinating activity was between pH 4.5 and pH 7.5, and the hemagglutinating activity was stable up to 65℃. Besides, it exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp. and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.
黄芪多糖提取的试验结果表明:以80%的乙醇脱脂后,水提取2次,第一次提取60 min,第二次提取70 min可提取出大部分的多糖;本文首次应用纤维素酶辅助提取黄芪多糖,取得了很好的效果,在多糖含量变化不大的情况下,多糖的得率提高率达20%以上。纤维素酶的最佳处理条件为:加酶量60 U/g;酶处理时间90 min;处理温度50℃。分级醇沉和分步醇沉的试验结果表明醇沉浓度为80%比较合理。采用一系列不同截留分子量的中空纤维膜对黄芪水提物进行了较系统的超滤分离,结果表明黄芪多糖的分子量分布较宽,在各分子量阶段的分布是很不均匀的,高分子量和低分子量的糖分布较多,选用截留分子量50 kDa的中空纤维膜可以截留大部分的多糖。酶法脱除黄芪多糖中蛋白质的结果表明:所选五种蛋白酶均能脱除黄芪多糖中的蛋白质,且明显优于Sevag法,将中性蛋白酶1直接应用于未经醇沉的提取浓缩液,加酶量30 U/g生药,在50℃下处理5 h可以去除约68%的蛋白,其多糖含量和得率与对照相比变化不大。对鸡生长发育及免疫活性的试验结果表明去除蛋白后的精制多糖的增重效果和免疫活性得到加强。
采用水提醇沉法、醇提法和纤维素酶辅助水提醇沉法进行了黄芪皂甙的提取试验结果表明:醇提法稍好于水提醇沉法,经纤维素酶处理后,醇沉上清液中的黄芪皂甙浓度提高率达8%以上。对黄芪皂甙分离效果的试验表明:AB-8大孔吸附树脂对黄芪皂甙的分离效果优于正丁醇萃取法:AB-8树脂柱层析分离黄芪皂甙的效果受树脂柱长度、洗脱用乙醇浓度和提取用乙醇浓度的影响较大,柱长55 cm、洗脱用乙醇浓度80%以及提取用乙醇浓度60%为较好参数。
首次研究了黄芪中一种活性蛋白的分离、纯化和鉴定。通过硫酸铵分级沉淀及离子交换柱层析,得到了一种纯的蛋白质,简称Astramonin 1。该蛋白是一种糖蛋白,总糖含量为19.6%。在天然状态下以同源二聚体的形式存在,分子量为61.1 kDa,单一亚基的分子量约为29.6 kDa。该蛋白对一些病原真菌具有明显的抗真菌活性,特别是对灰葡萄孢菌有很强的抑制能力。N端15个氨基酸残基为ESGINLQGDATLANN,同源性分析表明,Astramonin 1是一种新蛋白。该蛋白能凝集兔和人的血红细胞,特别对不同血型的人血表现出明显的特异性,表明Astramonin 1是一种凝集素。糖抑制性实验表明该凝集素属半乳糖结合型的凝集素。凝集活性的最适pH稳定范围很宽,在pH 4.5～7.5范围内,其凝集活性没有变化。Astramonin 1凝集活性耐受温度达65℃,是一种热稳定性较好的凝集素。
The extraction and separation of active components from Chinese medicinal herbs at high efficiency is one of the key research topics today. This study was carried out to broaden the use of Aslralagus mongolicus as well as to discover its new effective components. Major conclusions are as follow:1. Extraction and separation of Astragalus polysaccharide (APS)Dynamic analysis on the extraction process of Astragalus polysaccharide (APS) showed that double aqueous extraction at 60 min and 70 min was able to extract most of the APS from Astralagus mongolicus Bge. Effect of cellulase treatment on the APS extraction was also investigated in this study. Although content of the APS remained unchanged, yield of the APS increased for more than 20% after the cellulase treatment. Optimal condition for APS extraction was as follow: cellulose dosage 60U/g, incubation time 90 min and temperature 50 ℃. An ethanol precipitation concentration at 80% was found to be reasonable after studying the graded ethanol precipitation and stepwise ethanol precipitation. A series of hollow fiber ultrafiltration membrane was used to separate water-soluble extract systematically and membrane with cut-off of 50 kDa was found to be suitable for the separation of APS from other components. This report also describes the enzymatic removal of protein from the APS and this method was more superior than the Sevag's method. According to the immunologic test, the APS was an effective immunostimulating component. Its immunostimulating activity was strong and was better for live-wight growth.2. Extraction and separation of Astragalus sapnonin (AS)Various methods for the extraction and isolation of Astragalus sapnonin (AS), such as water-extraction, ethanol extraction and cellulase pretreatment, were carried out in this study. Results showed that ethanol extraction was slightly better than water-extration. Concentration of AS in ethanol precipitation supernatant was increased by enzyme pretreatment and showed an increment of more than 8%. Resin absorbing method significantly better than extraction refined by n-butyl alcohol. The rate and yield of AS were high when it was extracted by chromatography using AB-8 macroporous adsorption resin at 60% ethanol, resin column of 55 cm and elution concentration of 80%.3. Extraction, Purification and identification of bioactive proteinA novel bioactive protein (Astramonin 1) was isolated from the roots of Astragalus monghollcus using a combination of ammonium sulfate fractionation and ion exchange chromatographies. Astramonin 1 was a dimeric protein (61.1 kDa) and was composed of two identical subunits each with a
molecular mass of 29.6 kDa. The protein was a glycoprotein with a neutral carbohydrate content of 19.6%. The purified protein hemagglutinated both rabbit and human erythrocytes, but with a preference for blood types O (native) and AB (trypsin-treated). Among the various carbohydrates tested, the protein was best inhibited by D-galactose and its derivatives with pronounced preference for lactose, Astramonin 1 may therefore be regarded as a galactose-specificic lectin. N-terminal amino acid sequence of Astramonin 1 was determined as ESGINLQGDATLANN. The optimal pH range for hemagglutinating activity was between pH 4.5 and pH 7.5, and the hemagglutinating activity was stable up to 65℃. Besides, it exerted antifungal activity against Botrytis cincerea, Fusarium oxysporum, Colletorichum sp. and Drechslera turcia but not against Rhizoctonia solani and Mycosphaerella arachidicola.
引文
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