龙须菜多糖硫酸基含量与抗流感病毒活性关系的研究
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摘要
本文研究了龙须菜多糖抗流感病毒活性及其作用机制以及多糖硫酸基含量与抗病毒活性之间的关系。同时优选多糖的提取分离条件和选择适宜的脱硫及硫酸化方法。主要研究结果如下:
用正交实验优选龙须菜多糖的提取条件,所得最适提取条件为:温度100℃,时间5h,料水比1:100;在该条件下提取多糖得率为样品干重的22.97%;经纯化后的龙须菜精多糖(PPGL)的糖含量为94.48%,蛋白质含量为0.95%,其硫酸基含量为13.25%;用DEAE C-52分离PPGL可得到GL-1、GL-2和GL-3三个明显洗脱峰,表明PPGL至少含有3种级分;GL-1、GL-2和GL-3三种级分经紫外分析,它们分别在260nm与280nm波长处均无吸收峰,说明样品中已基本除去蛋白质、核酸等杂质,为较高纯度的组分;GL-1、GL-2和GL-3分别过Sephadex G -100凝胶柱层析所得的洗脱曲线均为单一对称峰,表明其为均一纯品。经测定GL-1、GL-2和GL-3的分子量及硫酸基含量分别为5.1kD、52kD、67kD与0.00%、12.63%、22.61%。
以HCl-甲醇法、稀HCl法和溶剂法对PPGL进行脱硫,所得脱硫多糖的SO42-含量分别为1.75%、0.00%和2.25%;由取代度计算公式得三种脱硫方法的DS分别为0.09、0.00和0.12。经比较认为,在三种脱硫方法中HCl-甲醇法最适合PPGL的脱硫处理。
采用Wolfrom改良法对HCl-甲醇法制得的脱硫多糖进行硫酸化处理,用以制备硫酸化多糖。以SO42-含量和DS的最大值为指标选择硫酸化条件,所得最佳硫酸化条件是:氯磺酸与吡啶体积比为1:4、温度65℃、时间2h,在此条件下制备的硫酸化多糖其SO42-含量为17.53%,DS为2.01。
抗病毒实验结果显示,PPGL对流感病毒H1-364的抑制效果要比对流感病毒H3-616的抑制效果好;选定以H1-364作为病毒株,用PPGL、脱硫多糖、不同硫酸基含量的硫酸化多糖(S1、S2、S3)和纯多糖(GL-1、GL-2和GL-3)八个样品分别对感染H1-364的MDCK细胞进行抗病毒实验,结果表明:PPGL的抗病毒效果最好,其最大病毒抑制率为88.82%;多糖抗病毒能力在一定范围内随多糖中硫酸根含量的增加而增大,当多糖SO42-含量和DS分别在13%和1.10附近时,其抗H1-364病毒的能力最强,而硫酸根含量过高或过低对H1-364病毒的抑制作用都有所下降。
对PPGL抗病毒机理初步研究的结果表明,PPGL对H1-364无直接灭活作用,不影响病毒的释放,可通过干扰病毒对宿主细胞的吸附,抑制病毒的复制实现其抗流感病毒H1-364的作用。
本研究首次探讨龙须菜多糖抗病毒活性,揭示了多糖硫酸基含量与抗病毒活性的关系。研究结果对于丰富龙须菜生理活性的研究内容具有重要的理论意义和学术价值,对合理开发利用龙须菜多糖有重要的指导意义。
In this paper the relationship between SO42- content of polysaccharide from Gracilaria lemaneiformis(PGL) and anti-influenza virus activities was studied, its initial antiviral mechanism was also approached. Extraction conditions of polysaccharide were optimized and suitable desulfuration and sulfation methods were selected at the same time. The main study results were as follows:
Extraction conditions of polysaccharide from Gracilaria lemaneiformis were obtained by the L933orthogonal design, while temperature of 100℃, time of 5 hours, dry alage to water ratio of 1:100 was the optimum, the ratio of polysaccharide to dry alage was 22.97% in this condition; Purified PGL’s(PPGL) polysaccharide content was 94.48%, protein content was 0.95%, SO42- content was 13.25%; Three fractions called GL-1,GL-2 and GL-3 were obtained by DEAE-celluose column chromatograph, it showed that PPGL was composed of three components at lease; Results of ultraviolet analysis and elution from Sephadex G-100 of GL-1, GL-2 and GL-3 showed that three of them were purified well. The average molecular weight and SO42- content of each fraction were estimated at 5.1kD, 52kD, 67kD and 0.00%, 12.63%, 22.61%.
Using hydrochloric acid- methanol, dilute hydrochloric acid and dissolvent methods to deal with PPGL to get desthio-PGL, results showed that SO42- contents of three methods were 1.75%, 0.00% and 2.25%; And the degree of substitution(DS) were 0.09, 0.00 and 0.12 separately. In general, hydrochloric acid- methanol method was the best of the three.
Using reforming Wolfrom method to deal with desthio-PGL from hydrochloric acid- methanol method to obtain sulfated PGL. Taking SO42- content and DS as index, the sulfation condition the volume ratio of chlorosulfonic acid to pyridine of 1:4, temperature of 65℃, time of 4 hours was the optimum; The SO42- content and DS of sulfated PGL was 17.53% and 2.01 separately in this condition.
Antiviral results showed that the inhibiting effect of PPGL to influenza virus H1-364 was better than to influenza virus H3-616; Putting PPGL, desthio-PGL, different SO42- content of sufation samples (S1, S2, S3) and purified ploysacchrides (GL-1, GL-2 and GL-3) on MDCK which was affected by H1-364, results demonstrated that PPGL’s inhibiting effect was the best and it’s max virus inhibition ratio was 88.82%; Polysacchride samples’antiviral ability was better when SO42- content was higher within limits; While SO42- content and DS were near 13% and 1.10, the polysacchride samples got the best antiviral ability, while SO42- content was higher or lower than 13%, the antviral ability reduced.
Antiviral mechanism initial approach showed that PPGL had no direct effect on H1-364, it could not effect virus to release, but it could interfere H1-364 to adsorb on host cell and it also had effect on virus replication.
Study on antiviral activities of polysaccharide from Gracilaria lemaneiformis was firstly approached in this paper, it revealed the relationship between SO42- content and antiviral activities. Study results had significant theory and academic value to physiological activities study of Gracilaria lemaneiformis, they also provided significant instructions for exploitating polysaccharide from Gracilaria lemaneiformis reasonably.
用正交实验优选龙须菜多糖的提取条件,所得最适提取条件为:温度100℃,时间5h,料水比1:100;在该条件下提取多糖得率为样品干重的22.97%;经纯化后的龙须菜精多糖(PPGL)的糖含量为94.48%,蛋白质含量为0.95%,其硫酸基含量为13.25%;用DEAE C-52分离PPGL可得到GL-1、GL-2和GL-3三个明显洗脱峰,表明PPGL至少含有3种级分;GL-1、GL-2和GL-3三种级分经紫外分析,它们分别在260nm与280nm波长处均无吸收峰,说明样品中已基本除去蛋白质、核酸等杂质,为较高纯度的组分;GL-1、GL-2和GL-3分别过Sephadex G -100凝胶柱层析所得的洗脱曲线均为单一对称峰,表明其为均一纯品。经测定GL-1、GL-2和GL-3的分子量及硫酸基含量分别为5.1kD、52kD、67kD与0.00%、12.63%、22.61%。
以HCl-甲醇法、稀HCl法和溶剂法对PPGL进行脱硫,所得脱硫多糖的SO42-含量分别为1.75%、0.00%和2.25%;由取代度计算公式得三种脱硫方法的DS分别为0.09、0.00和0.12。经比较认为,在三种脱硫方法中HCl-甲醇法最适合PPGL的脱硫处理。
采用Wolfrom改良法对HCl-甲醇法制得的脱硫多糖进行硫酸化处理,用以制备硫酸化多糖。以SO42-含量和DS的最大值为指标选择硫酸化条件,所得最佳硫酸化条件是:氯磺酸与吡啶体积比为1:4、温度65℃、时间2h,在此条件下制备的硫酸化多糖其SO42-含量为17.53%,DS为2.01。
抗病毒实验结果显示,PPGL对流感病毒H1-364的抑制效果要比对流感病毒H3-616的抑制效果好;选定以H1-364作为病毒株,用PPGL、脱硫多糖、不同硫酸基含量的硫酸化多糖(S1、S2、S3)和纯多糖(GL-1、GL-2和GL-3)八个样品分别对感染H1-364的MDCK细胞进行抗病毒实验,结果表明:PPGL的抗病毒效果最好,其最大病毒抑制率为88.82%;多糖抗病毒能力在一定范围内随多糖中硫酸根含量的增加而增大,当多糖SO42-含量和DS分别在13%和1.10附近时,其抗H1-364病毒的能力最强,而硫酸根含量过高或过低对H1-364病毒的抑制作用都有所下降。
对PPGL抗病毒机理初步研究的结果表明,PPGL对H1-364无直接灭活作用,不影响病毒的释放,可通过干扰病毒对宿主细胞的吸附,抑制病毒的复制实现其抗流感病毒H1-364的作用。
本研究首次探讨龙须菜多糖抗病毒活性,揭示了多糖硫酸基含量与抗病毒活性的关系。研究结果对于丰富龙须菜生理活性的研究内容具有重要的理论意义和学术价值,对合理开发利用龙须菜多糖有重要的指导意义。
In this paper the relationship between SO42- content of polysaccharide from Gracilaria lemaneiformis(PGL) and anti-influenza virus activities was studied, its initial antiviral mechanism was also approached. Extraction conditions of polysaccharide were optimized and suitable desulfuration and sulfation methods were selected at the same time. The main study results were as follows:
Extraction conditions of polysaccharide from Gracilaria lemaneiformis were obtained by the L933orthogonal design, while temperature of 100℃, time of 5 hours, dry alage to water ratio of 1:100 was the optimum, the ratio of polysaccharide to dry alage was 22.97% in this condition; Purified PGL’s(PPGL) polysaccharide content was 94.48%, protein content was 0.95%, SO42- content was 13.25%; Three fractions called GL-1,GL-2 and GL-3 were obtained by DEAE-celluose column chromatograph, it showed that PPGL was composed of three components at lease; Results of ultraviolet analysis and elution from Sephadex G-100 of GL-1, GL-2 and GL-3 showed that three of them were purified well. The average molecular weight and SO42- content of each fraction were estimated at 5.1kD, 52kD, 67kD and 0.00%, 12.63%, 22.61%.
Using hydrochloric acid- methanol, dilute hydrochloric acid and dissolvent methods to deal with PPGL to get desthio-PGL, results showed that SO42- contents of three methods were 1.75%, 0.00% and 2.25%; And the degree of substitution(DS) were 0.09, 0.00 and 0.12 separately. In general, hydrochloric acid- methanol method was the best of the three.
Using reforming Wolfrom method to deal with desthio-PGL from hydrochloric acid- methanol method to obtain sulfated PGL. Taking SO42- content and DS as index, the sulfation condition the volume ratio of chlorosulfonic acid to pyridine of 1:4, temperature of 65℃, time of 4 hours was the optimum; The SO42- content and DS of sulfated PGL was 17.53% and 2.01 separately in this condition.
Antiviral results showed that the inhibiting effect of PPGL to influenza virus H1-364 was better than to influenza virus H3-616; Putting PPGL, desthio-PGL, different SO42- content of sufation samples (S1, S2, S3) and purified ploysacchrides (GL-1, GL-2 and GL-3) on MDCK which was affected by H1-364, results demonstrated that PPGL’s inhibiting effect was the best and it’s max virus inhibition ratio was 88.82%; Polysacchride samples’antiviral ability was better when SO42- content was higher within limits; While SO42- content and DS were near 13% and 1.10, the polysacchride samples got the best antiviral ability, while SO42- content was higher or lower than 13%, the antviral ability reduced.
Antiviral mechanism initial approach showed that PPGL had no direct effect on H1-364, it could not effect virus to release, but it could interfere H1-364 to adsorb on host cell and it also had effect on virus replication.
Study on antiviral activities of polysaccharide from Gracilaria lemaneiformis was firstly approached in this paper, it revealed the relationship between SO42- content and antiviral activities. Study results had significant theory and academic value to physiological activities study of Gracilaria lemaneiformis, they also provided significant instructions for exploitating polysaccharide from Gracilaria lemaneiformis reasonably.
引文
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