沙棘叶多糖的提取、功能及其分子修饰的研究
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摘要
本文以沙棘叶多糖为研究对象,系统地研究了其提取、分离纯化以及分子修饰(硫酸酯化、羧甲基化和金属离子螯合作用)的工艺条件。通过对沙棘叶多糖的抗氧化性及分子修饰前后的抑菌功能进行了初步研究,为其在食品、医药及保健品等方面的应用奠定基础。主要研究内容如下：
(1)沙棘叶多糖的提取工艺研究。以沙棘叶多糖得率为指标对水提、微波辅助及超声波辅助三种提取方法进行比较,并对超声波提取法进行了响应面分析优化得到最佳提取工艺条件,即当液固比为52.5：1、超声功率为607.1W、超声时间为42.3min时,此条件下多糖得率的预测值为7.74%,验证值为7.68%。
(2)沙棘叶多糖的最佳脱蛋白法是Sevag法(氯仿：正丁醇=4：1),其去蛋白六次后蛋白质的清除率可达78.96%,此时多糖的损失率仅为33.75%。
(3)沙棘叶多糖经过除蛋白和脱色,除去小分子杂质,为精制的沙棘叶多糖(SJ),经DEAE-52离子交换层析的分离纯化,得到了3个级分,分别为SJ-1、SJ-2和SJ-3。各级分经紫外光谱法和冻融分析可知3个级分均为单一组分,其含量分别为11.64%、77.67%、10.69%。经气相色谱分析,沙棘叶多糖SJ-2级分是由鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖五种单糖组成,其质量分数分别为鼠李糖15.18%、阿拉伯糖13.22%、甘露糖20.38%、葡萄糖33.03%、半乳糖18.19%。
(4)沙棘叶多糖有很好的体外抗氧化能力(羟自由基、超氧阴离子的清除力,总抗氧化活性),当浓度为1200μg·mL-1时,对O2·-的清除率可达100%,比同浓度的抗血坏酸溶液要高18.92%。此外通过急性毒理试验研究证明沙棘叶多糖属于实际无毒级,其LD50>15 OOOmg-kg-1·bw-1, MTD>30 OOOmg·kg-1·bw-1·d-1。
(5)确定了沙棘叶多糖的最佳硫酸酯化工艺：酯化温度60℃,氯磺酸与吡啶的体积比3：1,酯化时间为2.5h,在此条件下的多糖酯化取代度为0.51。红外光谱法检测显示硫酸基与SJ-2形成硫酸酯化合物。
(6)确定了沙棘叶多糖的最佳醚化工艺：异丙醇浓度为80%,氢氧化钠与一氯乙酸的摩尔比2.5：1,作用时间3h,作用温度50℃,在此条件下的多糖羧甲基化取代度为0.73。
(7)确定了沙棘叶多糖的最佳金属离子螯合工艺：多糖与锌离子质量比5：1,锌离子的初始浓度为6mg/mL,螯合时间为8h,此条件下的最佳螯合率为73.89%。
(8)琼脂平板扩散法测定结果表明沙棘叶多糖对大肠杆菌、枯草杆菌、金黄色葡萄球菌均有一定的抑制作用,经化学修饰后的三种多糖衍生物对菌种的抑制效果明显增强,其中金属离子螯合多糖的抑制作用最强,其次是硫酸酯化多糖和羧甲基化多糖。
This paper systematically studied that extraction, separation, purification and molecule modification (sulfated esterification and carboxymethyl ether and metal ion chelate) of polysaccharide from the leave in Hippohae rhamnoides L.. We studied the antioxidant and bacteriostatic functions on the polysaccharide and molecule modified polysaccharide from the leave in Hippohae rhamnoides L., which established a basis for them in foodstuffs, medicine and health supplies, etc. The main contents are as follows:
(1) We made some studies on the extraction process of polysaccharide from the leave in Hippohae rhamnoides L.. In order to study the extract technology of polysaccharide from the leave in Hippohae rhamnoides L., this article used yield as the criterion and compared the follows 3 extraction methods:aqueous extract, microwave-assisted and supersonic-assisted and through response surface analysis on ultrasonic method optimized the best conditions:when the liquid-solid ratio of 52.5:1, ultrasonic power of 607.1 W, ultrasonic time of 42.3min, the polysaccharide yield was maximum with estimated value was 7.74%, and verified value was 7.68%.
(2) The best method for deproteinization of polysaccharides from the leave in Hippohae rhamnoides L. was Sevag, which composed with chloroform and butanol (V/V=4:1). After six times deproteinization of polysaccharides, the clearance ratio of protein was 78.96%, then the loss ratio of polysaccharides was only 33.75%. In addition through acute toxicology test, it proved that the polysaccharide from the leave in Hippohae rhamnoides L. are non-toxic lever in actually and the LD50>15000mg-kg-1-bw-1, MTD>30000mg-kg-1·bw-1-d-1.
(3) Via removing protein, decolor and removing small molecules, the polysaccharides from the leave in Hippohae rhamnoides L. were convert into three ingredients (SJ-1, SJ-2 and SJ-3) by DEAE-cellulose. Analyzed by the tests of ultraviolet spectroscopy and freezing-thawing, we found that the contents are 11.64%,77.67% and 10.69% in each. In further, we used the technology of gas chromatography to test the contents of the polysaccharides (SJ-2), and we foud that it was comprised of 15.18% of Rhamnose,13.22% of Arabinose,20.38% of Mannose,33.03% of Glucose and18.19% of Galactose.
(4) The polysaccharide from the leave in Hippohae rhamnoides L. had certain degree antioxidant capacities in vitro, (Scavenging ability of hydroxyl radical and superoxide anion, Total antioxidant activity). When the concentration of polysaccharide was 1200μg-mL-1, the removal effect towards superoxide anion was of higher than Vc by 18.92%.
(5) The optimum sulfated esterifing technics was determined:the volume ratio of chlorosulfonic acid and pyridine was 3:1, esterifing temperature was 60℃, and esterifing reaction time was 2.5h. Under this condition, the substituted ratio of polysaccharide was 0.51. Infrared spectrometry spectra showed that sulfated compounds may be formed sulfate and SJ-2.
(6) The optimum etherified technics was determined:the concentration of isopropyl alcohol was 80%, the molare ratio of sodium hydroxide and monochloroacetic acid was 2.5:1, etherification time was 3h, and etherification temperature was 50℃. Under this condition, the substituted ratio of carboxymethyl was 0.73.
(7) The optimum chelated technics was determined:the volume ratio of polysaccharide and zinc ion was 5:1, the initial concentration of zinc ion was 6mg/mL, and chelating reaction time was 8h. Under this condition, the chelated volume ratio of polysaccharide was 73.89%.
(8) The results tested by the method of agar diffusion showed that the polysaccharide restrainted the growth of Escherichia coli, Bacillus subtilis and Staphylococcus aureus in some certain and the effects would be enhanced after molecular modification. Results showed that the chelated polysaccharide had the best effects towards the three bacterias and sulfated polysaccharide and etherified polysaccharide in next.
(1)沙棘叶多糖的提取工艺研究。以沙棘叶多糖得率为指标对水提、微波辅助及超声波辅助三种提取方法进行比较,并对超声波提取法进行了响应面分析优化得到最佳提取工艺条件,即当液固比为52.5：1、超声功率为607.1W、超声时间为42.3min时,此条件下多糖得率的预测值为7.74%,验证值为7.68%。
(2)沙棘叶多糖的最佳脱蛋白法是Sevag法(氯仿：正丁醇=4：1),其去蛋白六次后蛋白质的清除率可达78.96%,此时多糖的损失率仅为33.75%。
(3)沙棘叶多糖经过除蛋白和脱色,除去小分子杂质,为精制的沙棘叶多糖(SJ),经DEAE-52离子交换层析的分离纯化,得到了3个级分,分别为SJ-1、SJ-2和SJ-3。各级分经紫外光谱法和冻融分析可知3个级分均为单一组分,其含量分别为11.64%、77.67%、10.69%。经气相色谱分析,沙棘叶多糖SJ-2级分是由鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖五种单糖组成,其质量分数分别为鼠李糖15.18%、阿拉伯糖13.22%、甘露糖20.38%、葡萄糖33.03%、半乳糖18.19%。
(4)沙棘叶多糖有很好的体外抗氧化能力(羟自由基、超氧阴离子的清除力,总抗氧化活性),当浓度为1200μg·mL-1时,对O2·-的清除率可达100%,比同浓度的抗血坏酸溶液要高18.92%。此外通过急性毒理试验研究证明沙棘叶多糖属于实际无毒级,其LD50>15 OOOmg-kg-1·bw-1, MTD>30 OOOmg·kg-1·bw-1·d-1。
(5)确定了沙棘叶多糖的最佳硫酸酯化工艺：酯化温度60℃,氯磺酸与吡啶的体积比3：1,酯化时间为2.5h,在此条件下的多糖酯化取代度为0.51。红外光谱法检测显示硫酸基与SJ-2形成硫酸酯化合物。
(6)确定了沙棘叶多糖的最佳醚化工艺：异丙醇浓度为80%,氢氧化钠与一氯乙酸的摩尔比2.5：1,作用时间3h,作用温度50℃,在此条件下的多糖羧甲基化取代度为0.73。
(7)确定了沙棘叶多糖的最佳金属离子螯合工艺：多糖与锌离子质量比5：1,锌离子的初始浓度为6mg/mL,螯合时间为8h,此条件下的最佳螯合率为73.89%。
(8)琼脂平板扩散法测定结果表明沙棘叶多糖对大肠杆菌、枯草杆菌、金黄色葡萄球菌均有一定的抑制作用,经化学修饰后的三种多糖衍生物对菌种的抑制效果明显增强,其中金属离子螯合多糖的抑制作用最强,其次是硫酸酯化多糖和羧甲基化多糖。
This paper systematically studied that extraction, separation, purification and molecule modification (sulfated esterification and carboxymethyl ether and metal ion chelate) of polysaccharide from the leave in Hippohae rhamnoides L.. We studied the antioxidant and bacteriostatic functions on the polysaccharide and molecule modified polysaccharide from the leave in Hippohae rhamnoides L., which established a basis for them in foodstuffs, medicine and health supplies, etc. The main contents are as follows:
(1) We made some studies on the extraction process of polysaccharide from the leave in Hippohae rhamnoides L.. In order to study the extract technology of polysaccharide from the leave in Hippohae rhamnoides L., this article used yield as the criterion and compared the follows 3 extraction methods:aqueous extract, microwave-assisted and supersonic-assisted and through response surface analysis on ultrasonic method optimized the best conditions:when the liquid-solid ratio of 52.5:1, ultrasonic power of 607.1 W, ultrasonic time of 42.3min, the polysaccharide yield was maximum with estimated value was 7.74%, and verified value was 7.68%.
(2) The best method for deproteinization of polysaccharides from the leave in Hippohae rhamnoides L. was Sevag, which composed with chloroform and butanol (V/V=4:1). After six times deproteinization of polysaccharides, the clearance ratio of protein was 78.96%, then the loss ratio of polysaccharides was only 33.75%. In addition through acute toxicology test, it proved that the polysaccharide from the leave in Hippohae rhamnoides L. are non-toxic lever in actually and the LD50>15000mg-kg-1-bw-1, MTD>30000mg-kg-1·bw-1-d-1.
(3) Via removing protein, decolor and removing small molecules, the polysaccharides from the leave in Hippohae rhamnoides L. were convert into three ingredients (SJ-1, SJ-2 and SJ-3) by DEAE-cellulose. Analyzed by the tests of ultraviolet spectroscopy and freezing-thawing, we found that the contents are 11.64%,77.67% and 10.69% in each. In further, we used the technology of gas chromatography to test the contents of the polysaccharides (SJ-2), and we foud that it was comprised of 15.18% of Rhamnose,13.22% of Arabinose,20.38% of Mannose,33.03% of Glucose and18.19% of Galactose.
(4) The polysaccharide from the leave in Hippohae rhamnoides L. had certain degree antioxidant capacities in vitro, (Scavenging ability of hydroxyl radical and superoxide anion, Total antioxidant activity). When the concentration of polysaccharide was 1200μg-mL-1, the removal effect towards superoxide anion was of higher than Vc by 18.92%.
(5) The optimum sulfated esterifing technics was determined:the volume ratio of chlorosulfonic acid and pyridine was 3:1, esterifing temperature was 60℃, and esterifing reaction time was 2.5h. Under this condition, the substituted ratio of polysaccharide was 0.51. Infrared spectrometry spectra showed that sulfated compounds may be formed sulfate and SJ-2.
(6) The optimum etherified technics was determined:the concentration of isopropyl alcohol was 80%, the molare ratio of sodium hydroxide and monochloroacetic acid was 2.5:1, etherification time was 3h, and etherification temperature was 50℃. Under this condition, the substituted ratio of carboxymethyl was 0.73.
(7) The optimum chelated technics was determined:the volume ratio of polysaccharide and zinc ion was 5:1, the initial concentration of zinc ion was 6mg/mL, and chelating reaction time was 8h. Under this condition, the chelated volume ratio of polysaccharide was 73.89%.
(8) The results tested by the method of agar diffusion showed that the polysaccharide restrainted the growth of Escherichia coli, Bacillus subtilis and Staphylococcus aureus in some certain and the effects would be enhanced after molecular modification. Results showed that the chelated polysaccharide had the best effects towards the three bacterias and sulfated polysaccharide and etherified polysaccharide in next.
引文
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