款冬花多糖提取纯化工艺研究及结构鉴定
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摘要
本课题对陕南秦巴山区特产药用植物款冬花多糖活性成分的提取、相关动力学、分离纯化、结构鉴定以及体外抗氧化性等方面进行了系统的研究。主要研究成果如下：
(1)对款冬花多糖的水提法、超声提取法两种提取工艺进行了对比研究,并优化了提取工艺参数。水提法优化工艺条件为：提取温度84℃,时间124min,液料比21(mL／g),提取3次,在此优化条件下,多糖提取率为1.862%；超声提取法优化工艺条件为：提取功率90W,温度71℃,时间40min,液料比25(mL／g),提取3次,在此优化工艺条件下,多糖提取率为2.204%。上述数据表明超声提取法具有提取温度低、时间短、提取率较高的优点,但超声法有可能会对多糖结构造成一定的影响。
(2)分别建立了款冬花多糖水提、超声提取的动力学模型,并求得了在水提和超声条件下,款冬花多糖的提取速率常数、相对萃余率、活化能、半衰期以及有效扩散系数等一系列的动力学参数。研究结果可为款冬花多糖的提取工艺条件优化以及工艺设计提供相应的理论依据和参考。
(3)研究了款冬花多糖的脱蛋白、脱色工艺,并对工艺条件进行了优化。
多糖脱蛋白工艺：通过对盐酸法、三氯乙酸法、Sevage法和木瓜蛋白酶结合Sevage法四种方法的比较,木瓜蛋白酶结合Sevage法是一种较为理想的方法,该法优化的工艺条件为：木瓜蛋白酶用量为2％，pH值7.0,酶解时间93min,酶解温度65℃,再结合3次Sevage法脱蛋白,其蛋白脱除率为82.63%,多糖保留率为87.83%。
多糖脱色工艺：通过对活性炭法、双氧水法、反胶束溶液法、大孔树脂法四种脱色方法对比研究,得到了一条大孔树脂吸附法脱除款冬花多糖色素的工艺路线,并求得了款冬花多糖色素的吸附动力学、热力学等相关参数。通过静态、动态吸附和解吸试验表明：XD-5是脱除款冬花多糖色素的理想树脂。色素脱除的优化条件为：多糖的上样浓度为5mg/mL,上样量为2BV,流速为1.5BV／h；在此工艺条件下,款冬花多糖的脱色率为90.04%,多糖保留率为85.38%,蛋白脱除率在前期基础上为91.42%。
(4)脱蛋白、脱色的款冬花多糖(FPI)经DEAE-ecllulose柱和sephadexG-200柱分离,共得到了FPA、FPB、FPC三个多糖分级组分,并对产率较高的FPA、FPB进行了理化性质研究。研究结果表明：FPA为含有糖醛酸的多糖,其多糖含量为93.1%,其中糖醛酸含量为10.7%；分子量为456.6KDal;单糖组成为甘露糖,葡萄糖,半乳糖；单糖的摩尔比为：2.467：1.158：1；键型构成主要为1→2键、1→键、1→6键、1→3等；1H-NMR谱图分析表明表明其组成单糖为α、β构型。
FPB为单一多糖,其含量为99.2%；分子量为5.5KDal:单糖组成为甘露糖、葡萄糖；单糖的摩尔比为：1.798：1；键型构成主要为1→键、1→3和1→3,6键等,1H-NMR谱图分析表明表明其组成单糖为β构型。
(5)抗氧化活性测定结果表明,款冬花多糖具有一定的自由基清除能力,尤其对亚硝基、DPPH自由基具有较好的清除能力,且在一定浓度范围内对二者的清除作用呈现良好的量效关系。
本研究结果为款冬花资源的综合利用提供了一条新途径,对同类研究具有一定的参考和借鉴作用,具有较好的经济、社会价值以及应用前景。
The thesis is aimed to develop herbal resources of Southern Shannxi through researching the dried buds and flowers of Tussilago farfara. This thesis has a systematic study focused on the extraction,related dynamics,isolation,purification, structure characterization and external antioxidant activity of polysaccharide from Tussilago farfara. This paper, mainly achieves the following results:
(1) Tussilago farfara polysaccharide was obtained by using hot water extraction and ultrasonic wave extraction.On the basis of one-factor tests,the method of response surface analysis(RSA) was carried out to determine the optimal condition of the extraction. The using hot water optimum extraction conditions were as follows:84℃,124min,21 (water/dry weight),3 times. The extract rate of polysaccharide is 1.862% based on the above mentioned conditions. The ultrasonic wave extraction optimal condition were:ultrasonic extraction power 90W,71℃,40min,25 (water/dry weight),3 times.Under the optimized condition,the extraction rate of polysaccharide was 2.204%. The result shows that the ultrasonic method is time-saving,lower temperature and higher the extraction rate of polysaccharide,compared with using hot water. However, the polysaccharide structure may be changed by using of ultrasonic wave extraction.
(2) Respectively, the kinetic model in extracting the polysaccharide from Tussilago farfara were establishde by using hot water and ultrasonic wave. And the polyphenol and flavonoids rate constant of extraction, residual rate of extraction, activation energy, half-life, a series of effective diffusion coefficient of the kinetic parameters were obtained. The results provide the theoretical basis and reference for the design and optimization of extraction conditions used in the polysaccharide from Tussilago farfara.
(3) Process of removing protein and decolorization were studied and optimized.
Process of removing protein:By comparing the four methods of protein removal including by hydrochloric acid, trichloroacetic acid, Sevage method and papain-Sevage, papain Sevage method was choosed as the optimal method finally. The optimum conditions were substrate concentration 2% of papain, pH 7.0, temperature 65℃, hydrolysis time 93min, repeat 3 times with Sevags method. The amount of protein removed was 82.63% while polysaccharide remained 87.83% during the deproteinization process.
Process of decolorization:Contrasting four decolorization methods such as hydrogen peroxide oxidation method, activated carbon attaching method, reverse micelle solution method and macroporous rosin adsorption method, it was macroporous rosin adsorption process that had the most effect in decolorizing polysaccharide from the dried buds and flowers of Tussilago farfara. Through static adsorption, desorption, dynamic adsorption, desorption experiments, relative parameters of adsorption kinetics and thermodynamics are acknowledged. The result is shown that XD-5 resin was used to determine the optimum adsorption conditions for this method. The sample was 2BV with the current velocity of 1.5BV/h meanwhile the sample mass concentration was 5.0 mg/mL. The decolorazation rate was 90.04%, and the protein removal was 91.42% in all after disposal while polysaccharide retention rate was 85.38%.
(4) Three FPI that was deproteinized,decolored and dialyzed fractions,FPA,FPB and FPC,were got by DEAE-Cellulose column and sephadex G-200 using different solvent. The physicochemical properties of FPA and FPB were studied.The result was show that FPA was a kind of water soluble polysaccharide. The total sugar content is 93.1%,and the uronic acid is 10.7%.The average molecular weight of FPA is 456.6K Dal.The unit monosaccharide of FPA was identified as mannose,glucose, galactose with a ratio of 2.467∶1.158∶1,the sugar chain of FPA is manily composed of 1→2 linked,1→6 linked,1→linked and 1→3 linked.1H-NMR and IR spectrum of FPA results showed that monosaccharide of FPA wereαandβ-pyranose.
FPB was a kind of water soluble polysaccharide. The total sugar content is 99.2%.The average molecular weight of FPB is 5.5KDal.The unit monosaccharide of FPB was identified as mannose,glucose with a ratio of 1.798∶1,the sugar chain of FPB is manily composed of 1→linked, 1→3 linked and 1→3,6 linked.1H-NMR and IR spectrum of FPB results showed that monosaccharide of FPA were manilyβ-pyranose.
(5) Tussilago farfara polysaccharides had the cleaning capability of superoxide radical,hydroxy radical, nitroso and DPPH.Especially the nitroso and DPPH had satisfied scavenging effect. The cleaning capability of nitroso and DPPH had a dose-effect relationship obviously.
The results of this study provide a new way for the resources of Tussilago farfara of comprehensive utilization and for similar studies certain reference, with better economic, social value and prospects.
(1)对款冬花多糖的水提法、超声提取法两种提取工艺进行了对比研究,并优化了提取工艺参数。水提法优化工艺条件为：提取温度84℃,时间124min,液料比21(mL／g),提取3次,在此优化条件下,多糖提取率为1.862%；超声提取法优化工艺条件为：提取功率90W,温度71℃,时间40min,液料比25(mL／g),提取3次,在此优化工艺条件下,多糖提取率为2.204%。上述数据表明超声提取法具有提取温度低、时间短、提取率较高的优点,但超声法有可能会对多糖结构造成一定的影响。
(2)分别建立了款冬花多糖水提、超声提取的动力学模型,并求得了在水提和超声条件下,款冬花多糖的提取速率常数、相对萃余率、活化能、半衰期以及有效扩散系数等一系列的动力学参数。研究结果可为款冬花多糖的提取工艺条件优化以及工艺设计提供相应的理论依据和参考。
(3)研究了款冬花多糖的脱蛋白、脱色工艺,并对工艺条件进行了优化。
多糖脱蛋白工艺：通过对盐酸法、三氯乙酸法、Sevage法和木瓜蛋白酶结合Sevage法四种方法的比较,木瓜蛋白酶结合Sevage法是一种较为理想的方法,该法优化的工艺条件为：木瓜蛋白酶用量为2％，pH值7.0,酶解时间93min,酶解温度65℃,再结合3次Sevage法脱蛋白,其蛋白脱除率为82.63%,多糖保留率为87.83%。
多糖脱色工艺：通过对活性炭法、双氧水法、反胶束溶液法、大孔树脂法四种脱色方法对比研究,得到了一条大孔树脂吸附法脱除款冬花多糖色素的工艺路线,并求得了款冬花多糖色素的吸附动力学、热力学等相关参数。通过静态、动态吸附和解吸试验表明：XD-5是脱除款冬花多糖色素的理想树脂。色素脱除的优化条件为：多糖的上样浓度为5mg/mL,上样量为2BV,流速为1.5BV／h；在此工艺条件下,款冬花多糖的脱色率为90.04%,多糖保留率为85.38%,蛋白脱除率在前期基础上为91.42%。
(4)脱蛋白、脱色的款冬花多糖(FPI)经DEAE-ecllulose柱和sephadexG-200柱分离,共得到了FPA、FPB、FPC三个多糖分级组分,并对产率较高的FPA、FPB进行了理化性质研究。研究结果表明：FPA为含有糖醛酸的多糖,其多糖含量为93.1%,其中糖醛酸含量为10.7%；分子量为456.6KDal;单糖组成为甘露糖,葡萄糖,半乳糖；单糖的摩尔比为：2.467：1.158：1；键型构成主要为1→2键、1→键、1→6键、1→3等；1H-NMR谱图分析表明表明其组成单糖为α、β构型。
FPB为单一多糖,其含量为99.2%；分子量为5.5KDal:单糖组成为甘露糖、葡萄糖；单糖的摩尔比为：1.798：1；键型构成主要为1→键、1→3和1→3,6键等,1H-NMR谱图分析表明表明其组成单糖为β构型。
(5)抗氧化活性测定结果表明,款冬花多糖具有一定的自由基清除能力,尤其对亚硝基、DPPH自由基具有较好的清除能力,且在一定浓度范围内对二者的清除作用呈现良好的量效关系。
本研究结果为款冬花资源的综合利用提供了一条新途径,对同类研究具有一定的参考和借鉴作用,具有较好的经济、社会价值以及应用前景。
The thesis is aimed to develop herbal resources of Southern Shannxi through researching the dried buds and flowers of Tussilago farfara. This thesis has a systematic study focused on the extraction,related dynamics,isolation,purification, structure characterization and external antioxidant activity of polysaccharide from Tussilago farfara. This paper, mainly achieves the following results:
(1) Tussilago farfara polysaccharide was obtained by using hot water extraction and ultrasonic wave extraction.On the basis of one-factor tests,the method of response surface analysis(RSA) was carried out to determine the optimal condition of the extraction. The using hot water optimum extraction conditions were as follows:84℃,124min,21 (water/dry weight),3 times. The extract rate of polysaccharide is 1.862% based on the above mentioned conditions. The ultrasonic wave extraction optimal condition were:ultrasonic extraction power 90W,71℃,40min,25 (water/dry weight),3 times.Under the optimized condition,the extraction rate of polysaccharide was 2.204%. The result shows that the ultrasonic method is time-saving,lower temperature and higher the extraction rate of polysaccharide,compared with using hot water. However, the polysaccharide structure may be changed by using of ultrasonic wave extraction.
(2) Respectively, the kinetic model in extracting the polysaccharide from Tussilago farfara were establishde by using hot water and ultrasonic wave. And the polyphenol and flavonoids rate constant of extraction, residual rate of extraction, activation energy, half-life, a series of effective diffusion coefficient of the kinetic parameters were obtained. The results provide the theoretical basis and reference for the design and optimization of extraction conditions used in the polysaccharide from Tussilago farfara.
(3) Process of removing protein and decolorization were studied and optimized.
Process of removing protein:By comparing the four methods of protein removal including by hydrochloric acid, trichloroacetic acid, Sevage method and papain-Sevage, papain Sevage method was choosed as the optimal method finally. The optimum conditions were substrate concentration 2% of papain, pH 7.0, temperature 65℃, hydrolysis time 93min, repeat 3 times with Sevags method. The amount of protein removed was 82.63% while polysaccharide remained 87.83% during the deproteinization process.
Process of decolorization:Contrasting four decolorization methods such as hydrogen peroxide oxidation method, activated carbon attaching method, reverse micelle solution method and macroporous rosin adsorption method, it was macroporous rosin adsorption process that had the most effect in decolorizing polysaccharide from the dried buds and flowers of Tussilago farfara. Through static adsorption, desorption, dynamic adsorption, desorption experiments, relative parameters of adsorption kinetics and thermodynamics are acknowledged. The result is shown that XD-5 resin was used to determine the optimum adsorption conditions for this method. The sample was 2BV with the current velocity of 1.5BV/h meanwhile the sample mass concentration was 5.0 mg/mL. The decolorazation rate was 90.04%, and the protein removal was 91.42% in all after disposal while polysaccharide retention rate was 85.38%.
(4) Three FPI that was deproteinized,decolored and dialyzed fractions,FPA,FPB and FPC,were got by DEAE-Cellulose column and sephadex G-200 using different solvent. The physicochemical properties of FPA and FPB were studied.The result was show that FPA was a kind of water soluble polysaccharide. The total sugar content is 93.1%,and the uronic acid is 10.7%.The average molecular weight of FPA is 456.6K Dal.The unit monosaccharide of FPA was identified as mannose,glucose, galactose with a ratio of 2.467∶1.158∶1,the sugar chain of FPA is manily composed of 1→2 linked,1→6 linked,1→linked and 1→3 linked.1H-NMR and IR spectrum of FPA results showed that monosaccharide of FPA wereαandβ-pyranose.
FPB was a kind of water soluble polysaccharide. The total sugar content is 99.2%.The average molecular weight of FPB is 5.5KDal.The unit monosaccharide of FPB was identified as mannose,glucose with a ratio of 1.798∶1,the sugar chain of FPB is manily composed of 1→linked, 1→3 linked and 1→3,6 linked.1H-NMR and IR spectrum of FPB results showed that monosaccharide of FPA were manilyβ-pyranose.
(5) Tussilago farfara polysaccharides had the cleaning capability of superoxide radical,hydroxy radical, nitroso and DPPH.Especially the nitroso and DPPH had satisfied scavenging effect. The cleaning capability of nitroso and DPPH had a dose-effect relationship obviously.
The results of this study provide a new way for the resources of Tussilago farfara of comprehensive utilization and for similar studies certain reference, with better economic, social value and prospects.
引文
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