沙苑子多糖的分离纯化与理化性质研究
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摘要
正文:沙苑子(Astragaius Compianatus R.Br)又名潼蒺藜。主要产地是陕西、河北、东北等地。具有补肝、益肾、明目、固精之功效。主治肝肾虚弱,腰膝酸痛、遗精早泄,小便频数,目暗不明等症。沙苑子用途较广,是开展综合利用的药食兼用植物资源。
为了进一步开发利用沙苑子,本论文对陕西沙苑子的主要营养成分、多糖提取工艺、分离纯化、理化性质和生物活性等方面进行了较为深入的研究,取得了以下试验成果。
沙苑子富含多种氨基酸,微量元素等营养物质,且含量互有不同。沙苑子水溶性部分测得14种氨基酸,其中谷氨基酸含量最高为68%,其次为天冬氨酸14%,并有赖氨酸、苏氨酸、缬氨酸、蛋氨酸、苯丙氨酸、亮氨酸、异亮氨酸等7种人体必需氨基酸,这说明沙苑子具有较高的食用和药用价值。
沙苑子多糖的最佳提取工艺为:热水提取法,温度65℃,加入15倍水,浸提6h,粗多糖最高得率5.06g/kg;超声波辅助提取技术,功率300W,超声时间25min,料液比1:15(W:V),提取得率5.53g/kg,但是超声对多糖结构可能有影响;在传统Sevag法除蛋白的基础上采用Sevag法结合酶法除蛋白,大大提高了除蛋白率。
采用DEAE Sepharose CL-6B离子交换柱和Sephadex G-200凝胶柱层析分离纯化沙苑子多糖,具有较好的分离效果,共得到ACRBⅠ-a、ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ、ACRBⅣ六种级分。其中ACRBⅠ-a量较小不对其进行研究,由SephadexG-200柱层析结果可看出,此分离过程对ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ的分离作用更好。本论文对ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ进行了深入研究。
本试验采用SephadexG-200柱(1.1×100cm)层析法测定多糖的分子量,为了评价该法的精确性,我们将标准分子量葡聚糖T-70和T-40混合后上柱,在前述柱层析条件下获得了很好的分离效果。ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ分子量分别为67kD,75kD,68kD,63kD。
本试验测定多糖的溶解性时,采用单位体积水溶解一定量多糖所用的时间来表示,而没有采用溶解度来表示,这是因为多糖溶液可能形成溶胶而不能准确地评价其溶解性。
沙苑子纯多糖在120℃下进行了10h酸水解,水解完全。气相色谱分析结果表明对于沙苑子多糖,利用硫酸水解、三甲基硅醚化衍生后分析效果较好。
气相色谱,红外光谱和理化特性分析表明ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ均为杂多糖;ACRBⅠ-b和ACRBⅡ-a主要由葡萄糖组成;ACRBⅡ-b主要由鼠李糖、阿拉伯糖、核糖和半乳糖组成;ACRBⅢ主要由甘露糖组成。
Astragaius Compianatus R.Br, which is also named Tong Jili, is mainly distributed in Shaanxi, Hebei, Dongbei district, etc. It has ability to tonifying liver and kidney, improving eyesight, protecting spermatozoon. It also has the function of liver and kidney catheresis, waist and knee soreness, nocturnal emission and premature ejaculation, urination frequency, eyes gloomy etc. Astragaius Compianatus R.Br could be used as medicine, so it has great exploitation value.
In order to ulteriorly exploit resource, the author conducted a series of studyon the nutritional components of Astragaius Compianatus R.Br, mainly researched on Shaanxi Astragaius Compianatus R.Br was conducted and its nutrition ingredients, polysaccharide extraction craft, separation purification, physics and chemistry nature and biological activity were referred. The main results are as follows.
The results showed fruitage and persistent calyx of Astragaius Compianatus R.Br was rich in various amino acids, trace mineral elements and others nutritional components. It contains 14 kind of amino acids, in which the ratio of glutamic acid is 68%, aspartic acid is 14%. It has 7 kinds of human body essential amino acids (lysine, threonine, valine, methionine, phenylalanine, leucine and isoleucine, etc.), which explains that Astragaius Compianatus R.Br has nutritive and pharmaceutical values.
The technique to improve the conditions of extraction of polysaccharides of Astragaius Compianatus R.Br was studied in this paper. The results showed that the optimum conditions of hot water extraction of water-soluble polysaccharides were as follows: the adding water ratio is 1:15, the temperature of extraction was 65℃, the duration of extraction was 6 hours, both the highest extraction ratio of polysaccharides were 96.9g/kg. The optimal conditions of supersonic wave extraction of water-soluble polysaccharides were as follows: the adding water ratio is 1:10 (W:V). The power of supersonic was 200W, the duration of extraction was 15min, the highest extraction ratio of polysaccharides were 118.3 g/kg. The water-soluble polysaccharides were extracted by ratio, may impact the chemical structure of polysaccharides. In addition, Sevag method combined with enzyme method based on traditional Sevag method to eliminate protein was adopted, which could greatly increase removal rate of protein.
Then isolated by DEAE sepharose CL-6B and Sephadex G-200 gel filtration, six glyco conjugates ACRBⅠ- a, ACRBⅠ- b, ACRBⅡ- a, ACRBⅡ- b, ACRBⅢ, ACRBⅣwere at tai-ned,which were tested by gel filtrationon sephadexG-200 and cellulose acetate p-ellicle electrophoresis and the results showed ACRBⅠ- b, ACRBⅡ- a, ACRBⅡ- b, ACRBⅢare homogeneous components.
In order to improve the accuracy, the standard molecular weight glucosan T-70 and T-40 were mixed on the column and it also had a good separation effect. Gas chromatography analysis, infra-red spectra analysis and physical and chemical characters showed that ACRB I -b molecular weight of ACRBⅠ-b is 67kD. ACRBⅡ-a molecular weight of PAVFⅡ-a is 75kD. ACRBⅡ-b molecular weight of which is 68kD.
In determination of polysaccharide solubility, we used the time when polysaccharides dissolve in the unit volume of water but not the usual solubility. This is because the polysaccharide solution possibly forms the sol not to be able to appraise its solubility accurately.
The acid hydrolysis of pure polysaccharides from Astragaius Compianatus R.Br was conducted in 120℃, 10h. The hydrolysis process was complete and they were analyzed by gas chromatography. We found that the sulfuric acid hydrolysis and the trimethyl silicon etherification have good effects.
The gas chromatography analysis, the infrared spectrum and their physicochemical properties indicate that ACRBⅠ-b, ACRBⅡ-a, ACRBⅡ-b, ACRBⅢare the mixed polysaccharide. ACRBⅠ- b and ACRBⅡ- a are mainly composed by the glucose. ACRBⅡ- b is mainly composed by rhamnose, arabinose, ribose and galactose. ACRBⅢis mainly composed by mannose.
为了进一步开发利用沙苑子,本论文对陕西沙苑子的主要营养成分、多糖提取工艺、分离纯化、理化性质和生物活性等方面进行了较为深入的研究,取得了以下试验成果。
沙苑子富含多种氨基酸,微量元素等营养物质,且含量互有不同。沙苑子水溶性部分测得14种氨基酸,其中谷氨基酸含量最高为68%,其次为天冬氨酸14%,并有赖氨酸、苏氨酸、缬氨酸、蛋氨酸、苯丙氨酸、亮氨酸、异亮氨酸等7种人体必需氨基酸,这说明沙苑子具有较高的食用和药用价值。
沙苑子多糖的最佳提取工艺为:热水提取法,温度65℃,加入15倍水,浸提6h,粗多糖最高得率5.06g/kg;超声波辅助提取技术,功率300W,超声时间25min,料液比1:15(W:V),提取得率5.53g/kg,但是超声对多糖结构可能有影响;在传统Sevag法除蛋白的基础上采用Sevag法结合酶法除蛋白,大大提高了除蛋白率。
采用DEAE Sepharose CL-6B离子交换柱和Sephadex G-200凝胶柱层析分离纯化沙苑子多糖,具有较好的分离效果,共得到ACRBⅠ-a、ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ、ACRBⅣ六种级分。其中ACRBⅠ-a量较小不对其进行研究,由SephadexG-200柱层析结果可看出,此分离过程对ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ的分离作用更好。本论文对ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ进行了深入研究。
本试验采用SephadexG-200柱(1.1×100cm)层析法测定多糖的分子量,为了评价该法的精确性,我们将标准分子量葡聚糖T-70和T-40混合后上柱,在前述柱层析条件下获得了很好的分离效果。ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ分子量分别为67kD,75kD,68kD,63kD。
本试验测定多糖的溶解性时,采用单位体积水溶解一定量多糖所用的时间来表示,而没有采用溶解度来表示,这是因为多糖溶液可能形成溶胶而不能准确地评价其溶解性。
沙苑子纯多糖在120℃下进行了10h酸水解,水解完全。气相色谱分析结果表明对于沙苑子多糖,利用硫酸水解、三甲基硅醚化衍生后分析效果较好。
气相色谱,红外光谱和理化特性分析表明ACRBⅠ-b、ACRBⅡ-a、ACRBⅡ-b、ACRBⅢ均为杂多糖;ACRBⅠ-b和ACRBⅡ-a主要由葡萄糖组成;ACRBⅡ-b主要由鼠李糖、阿拉伯糖、核糖和半乳糖组成;ACRBⅢ主要由甘露糖组成。
Astragaius Compianatus R.Br, which is also named Tong Jili, is mainly distributed in Shaanxi, Hebei, Dongbei district, etc. It has ability to tonifying liver and kidney, improving eyesight, protecting spermatozoon. It also has the function of liver and kidney catheresis, waist and knee soreness, nocturnal emission and premature ejaculation, urination frequency, eyes gloomy etc. Astragaius Compianatus R.Br could be used as medicine, so it has great exploitation value.
In order to ulteriorly exploit resource, the author conducted a series of studyon the nutritional components of Astragaius Compianatus R.Br, mainly researched on Shaanxi Astragaius Compianatus R.Br was conducted and its nutrition ingredients, polysaccharide extraction craft, separation purification, physics and chemistry nature and biological activity were referred. The main results are as follows.
The results showed fruitage and persistent calyx of Astragaius Compianatus R.Br was rich in various amino acids, trace mineral elements and others nutritional components. It contains 14 kind of amino acids, in which the ratio of glutamic acid is 68%, aspartic acid is 14%. It has 7 kinds of human body essential amino acids (lysine, threonine, valine, methionine, phenylalanine, leucine and isoleucine, etc.), which explains that Astragaius Compianatus R.Br has nutritive and pharmaceutical values.
The technique to improve the conditions of extraction of polysaccharides of Astragaius Compianatus R.Br was studied in this paper. The results showed that the optimum conditions of hot water extraction of water-soluble polysaccharides were as follows: the adding water ratio is 1:15, the temperature of extraction was 65℃, the duration of extraction was 6 hours, both the highest extraction ratio of polysaccharides were 96.9g/kg. The optimal conditions of supersonic wave extraction of water-soluble polysaccharides were as follows: the adding water ratio is 1:10 (W:V). The power of supersonic was 200W, the duration of extraction was 15min, the highest extraction ratio of polysaccharides were 118.3 g/kg. The water-soluble polysaccharides were extracted by ratio, may impact the chemical structure of polysaccharides. In addition, Sevag method combined with enzyme method based on traditional Sevag method to eliminate protein was adopted, which could greatly increase removal rate of protein.
Then isolated by DEAE sepharose CL-6B and Sephadex G-200 gel filtration, six glyco conjugates ACRBⅠ- a, ACRBⅠ- b, ACRBⅡ- a, ACRBⅡ- b, ACRBⅢ, ACRBⅣwere at tai-ned,which were tested by gel filtrationon sephadexG-200 and cellulose acetate p-ellicle electrophoresis and the results showed ACRBⅠ- b, ACRBⅡ- a, ACRBⅡ- b, ACRBⅢare homogeneous components.
In order to improve the accuracy, the standard molecular weight glucosan T-70 and T-40 were mixed on the column and it also had a good separation effect. Gas chromatography analysis, infra-red spectra analysis and physical and chemical characters showed that ACRB I -b molecular weight of ACRBⅠ-b is 67kD. ACRBⅡ-a molecular weight of PAVFⅡ-a is 75kD. ACRBⅡ-b molecular weight of which is 68kD.
In determination of polysaccharide solubility, we used the time when polysaccharides dissolve in the unit volume of water but not the usual solubility. This is because the polysaccharide solution possibly forms the sol not to be able to appraise its solubility accurately.
The acid hydrolysis of pure polysaccharides from Astragaius Compianatus R.Br was conducted in 120℃, 10h. The hydrolysis process was complete and they were analyzed by gas chromatography. We found that the sulfuric acid hydrolysis and the trimethyl silicon etherification have good effects.
The gas chromatography analysis, the infrared spectrum and their physicochemical properties indicate that ACRBⅠ-b, ACRBⅡ-a, ACRBⅡ-b, ACRBⅢare the mixed polysaccharide. ACRBⅠ- b and ACRBⅡ- a are mainly composed by the glucose. ACRBⅡ- b is mainly composed by rhamnose, arabinose, ribose and galactose. ACRBⅢis mainly composed by mannose.
引文
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