车前子多糖的分离纯化及其功能性质研究
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摘要
车前子具有药蔬兼用、医食同源之性，被我国卫生部列为可用于保健食品的物品。车前子多糖是车前子中主要的有效成分，位于种皮外表细胞壁层。车前子多糖具有缓泻、降血糖、降血脂、抗炎及免疫活性等多种生物活性，并作为一种亲水性胶体被广泛用于食品、药品行业。欧美等国对车前子多糖的结构、生物活性及其应用研究得相当深入，而我国对其研究甚少。本文以吉安产的大粒车前子多糖为研究对象，在系统研究了车前子多糖的提取、分离纯化、初级结构的基础上，对其抗氧化活性及功能性质进行了初步探索。现将主要研究结论概括如下：
1．建立了以木糖为标准，采用苯酚．硫酸法进行车前子多糖含量测定的方法。对两种测定方法和7种的标准对照物分别进行了比较分析，发现苯酚．硫酸法更适合测定车前子中多糖的含量，以木糖为标准得出的结果更能准确反映真实的多糖含量值。对苯酚．硫酸法测定车前子中多糖含量的测定条件优化为：2 mL样液先加3％苯酚溶液0.5 mL，混匀后加浓硫酸4 mL，迅速摇匀后，室温放置35min，在480 nm处测定其吸光度。平均回收率为98.9％，RSD=1.79％(n=6)。此方法是一种测定车前子多糖含量的理想方法，也可为其他戊糖含量高的多糖的测定提供参考。
2．对车前子多糖制备工艺进行了优化研究，得出的最佳提取条件为：温度100℃，料液比1∶35，水浴时间6 h，提取1次，提取率可达8.5％。最佳沉淀条件为：乙醇浓度60％，提取液浓缩比为2∶1，低温静置6 h。该工艺经济、简约、可行性强，为车前子多糖制备的工业化生产提供理论参考依据。
3．比较Sevage法、酶法-Sevage法联用脱蛋白的效果，发现采用酶法-Sevage法联用在保留较高蛋白脱除率的同时，能获得较高的多糖得率，其组成成份也更为单一。采用Sephacryl-400葡聚糖凝胶层析柱与大分子纯化系统AKTA Purifier100联用对车前子精制多糖进行纯化，得到一个多糖组分，经HPLC鉴定其为一较纯的组分，该组分为糖与蛋白的复合物，命名为SPG(Semen Plantaginis Glycoprotein)。HPGPC法测得SPG重均分子量Mw为1898584道尔顿。
4．对车前子多糖SPG理化性质进行分析，发现SPG为棕色粉末，可溶于水，含有2.92％蛋白质和14.58％糖醛酸。通过GC分析，SPG由木糖、阿拉伯糖、半乳糖、核糖、葡萄糖、甘露糖6种单糖组成，木糖、阿拉伯糖是SPG的主要组成单糖。通过紫外、红外光谱的特征吸收峰，鉴定SPG为酸性糖蛋白，且结构中存在β-型吡喃糖环结构。
5．通过体外抗氧化实验(DPPH·自由基、O_2~-·自由基、·OH自由基和卵黄脂蛋白PUFA过氧化体系)，分别研究了车前子多糖提取物SPWE、车前子粗多糖CSPP和车前子多糖纯品SPG的抗氧化活性。结果表明，SPWE、CSPP、SPG均具有一定的抗氧化活性。一定浓度范围内，随着浓度增加，抗氧化活性逐渐增强。在4种体外抗氧化体系中，SPG的活性较高，在卵黄脂蛋白PUFA过氧化体系中的作用最显著，2 mg／mL的作用效果与同浓度的Vc接近。
6．通过流变仪、物性测定仪、DSC和TGA／SDTA对车前子多糖的流变、凝胶特性和热稳定性进行分析。结果表明，车前子多糖溶液为假塑性流体，表观黏度随浓度的增加逐渐增加；温度升高可导致溶液黏度降低：在pH5～11时较稳定；对NaCl具有良好的兼容性，低浓度CaCl_2对车前子多糖溶液有显著的增稠效应，其增稠效果与离子浓度呈正相关。车前子多糖形成的凝胶为热可逆性凝胶，溶解温度、浓度、pH、盐对凝胶强度具有较大的影响；DSC分析无明显的焓变，推测其无明显的熔化点和凝固点；TGA／SDTA分析发现车前子多糖热稳定性好，预示其在食品、医药工业等行业具有广泛的应用前景。
Semen plantaginis has the dual usages as medicine and as food. It is an approved raw material for healthy food by the Minister of Public Health of China. The polysaccharides which are located in the outer seed-coat (husk) are one of the main active components of Semen plantaginis. The polysaccharides in Semen Plantaginis have many biological activities including laxation effect, hypoglycemic effect, hypolipidemic effect, anti-inflammatory and immunological activity. The polysaccharides in Semen Plantaginis as a hydrophilic colloid were widely used in food and pharmaceutical industries. Many previous researches were focused on the structure, bioactivity and application of the polysaccharides in Semen Plantaginis in foreign countries especially Europe and America, However, few studies were found in China. Therefore, in our paper, the polysaccharides extracted from the seeds of Ji'an Plantago asiatica L were studied. On the basis of systematic analysis on the extraction, isolation, purification and primary structure of this polysaccharides, its antioxidant activity and functional properties were studied preliminarily. The main results in the paper were concluded as follows:
1. A suitable method for the determination of the contents of polysaccharides in Semen Plantaginis by the phenol-H_2SO_4 colorimetry was established, and xylose was used as a standard. The two colorimetry methods and seven standards were compared and analyzed, respectively. The results showed that the colored compounds of xylose and Semen Plantaginis polysaccharides by phenol-H_2SO_4 colorimetry were more stable and had the similar maximum absorption wavelength. It means that the determination value of the content of polysaccharides in Semen Plantaginis by the phenol-H_2SO_4 colorimetry method with xylose as a standard was more near to the true content. The optimum conditions and operating steps are as follows. Firstly, 3 % phenol solution 0.5 mL is added into the sample solution 2 mL. Then sulfuric acid 4 mL is added in. After shaking up rapidly and sufficiently, the mixture is stayed for 35 min at room temperature. Finally the absorbency at 480 nm is determined. The content of polysaccharides in Semen Plantaginis was evaluated as 9.02 % by the optimization method. The average recovery rate was 98.9 % with 1.79 % of RSD (n=6). This method is suitable for the determination of polysaccharides in Semen Plantaginis. It can also be regarded as the reference for the determination of polysaccharides with high pentoses content in other plants.
2. The preparation process of polysaccharides in Semen Plantaginis was studied. The extraction conditions that influenced the extraction yield of polysaccharides were optimized by orthogonal design, the optimum extraction conditions were: 6 h at 100℃, the ration of material to solvent is l:35(w/v), giving the yield of polysaccharides at 8.5%. The optimum precipitation conditions were determined by single factor test, the best precipitation conditions were as follows: the concentration ratio 2, 60% alcohol precipitating for 6 h at low temperature. The optimum conditions are economic, simplicity and feasible, providing a theoretical reference for the industrialization of the preparation of polysaccharides in Semen Plantaginis.
3. The two methods of Sevage method and Enzyme combined with Sevage method were used to remove proteins from the crude polysaccharides in Semen Plantaginis. By comparing the effects, it was found that by Enzyme combined with Sevage method, both the protein removal rate and polysaccharide yield were higher, and the purity of Semen Plantaginis polysaccharides after deproteinated was higher, too. The Sephacryl-400 gel column chromatography in series with the AKTA purifier 100 system was used to purify the polysaccharides in Semen Plantaginis. A main component, named SPG {Semen Plantaginis Glycoprotein), was obtained which was found to be a homogeneous glycoprotein component by HPLC. The molecular weight (Mw) of SPG was 1898584 Da determined by HPGPC.
4. The physicochemical properties of SPG were studied. The results showed that SPG was brown powder and dissolvable in water. SPG contained protein of 2.92% and uronic acid of 14.58%. Arabinose, xylose, mannose, ribose, glucose and galactose were found to be the compositional monosaccharides in SPG by GC. Xylose and arabinose are the two main compositional monosaccharides. By UV spectrum and IR spectrum, SPG was identified as an acid glycoprotein, containing theβ- pyranoid ring.
5. The antioxidant effects of different Semen Plantaginis Polysaccharides (Semen Plantaginis Water Extract, SPWE; Crude Semen Plantaginis Polysaccharides, CSPP; Semen Plantaginis Glycoprotein, SPG) were studied in vitro by different testing systems , which included DPPH radical, superoxide anion radical, hydroxyl radical and the lipid peroxidation of yolk liporeotein system. The results showed that SPWE, CSPP, SPG all had certain antioxidant activities. The correlativity of their effects and the doses are positive, in a certain concentration range. The SPG showed high antioxidant activities in those four antioxidant systems, especially in the lipid peroxidation of yolk liporeotein system. SPG showed comparable antioxidant effects with Vc at the same concentration of 2 mg / mL.
6. The rheological property, gelation property and thermally stability of polysaccharides in Semen Plantaginis were studied by rheometer, TA-XT2i instrument, DSC and TGA/SDTA analysis respectively. It was found that the polysaccharides solution had pseudoplasticity. Apparent viscosity increased with the increase of concentration; the increasing of temperature can reduce the viscosity of the polysaccharides; the viscosity is not affected by the pH 5 toll; It has better compatibility with NaCl, Ca~(2+) obviously improve its viscosity, and the ability is correlated to the concentration of Ca~(2+) ions. The polysaccharides gel was the thermal reversible gel. Dissolution temperature, concentration, pH, salt all have obvious effects on the gel strength. The results of DSC showed no obvious enthalpic transitions for the gel, which indicated no obvious melting point (T_(melt)) and gel point (T_(gel)) for the gel. The results of TGA/SDTA analysis showed the polysaccharides had good thermal stability. All the above results showed that the polysaccharides in Semen Plantaginis had wide application potential in the food, pharmaceutical industries and so on.
1．建立了以木糖为标准，采用苯酚．硫酸法进行车前子多糖含量测定的方法。对两种测定方法和7种的标准对照物分别进行了比较分析，发现苯酚．硫酸法更适合测定车前子中多糖的含量，以木糖为标准得出的结果更能准确反映真实的多糖含量值。对苯酚．硫酸法测定车前子中多糖含量的测定条件优化为：2 mL样液先加3％苯酚溶液0.5 mL，混匀后加浓硫酸4 mL，迅速摇匀后，室温放置35min，在480 nm处测定其吸光度。平均回收率为98.9％，RSD=1.79％(n=6)。此方法是一种测定车前子多糖含量的理想方法，也可为其他戊糖含量高的多糖的测定提供参考。
2．对车前子多糖制备工艺进行了优化研究，得出的最佳提取条件为：温度100℃，料液比1∶35，水浴时间6 h，提取1次，提取率可达8.5％。最佳沉淀条件为：乙醇浓度60％，提取液浓缩比为2∶1，低温静置6 h。该工艺经济、简约、可行性强，为车前子多糖制备的工业化生产提供理论参考依据。
3．比较Sevage法、酶法-Sevage法联用脱蛋白的效果，发现采用酶法-Sevage法联用在保留较高蛋白脱除率的同时，能获得较高的多糖得率，其组成成份也更为单一。采用Sephacryl-400葡聚糖凝胶层析柱与大分子纯化系统AKTA Purifier100联用对车前子精制多糖进行纯化，得到一个多糖组分，经HPLC鉴定其为一较纯的组分，该组分为糖与蛋白的复合物，命名为SPG(Semen Plantaginis Glycoprotein)。HPGPC法测得SPG重均分子量Mw为1898584道尔顿。
4．对车前子多糖SPG理化性质进行分析，发现SPG为棕色粉末，可溶于水，含有2.92％蛋白质和14.58％糖醛酸。通过GC分析，SPG由木糖、阿拉伯糖、半乳糖、核糖、葡萄糖、甘露糖6种单糖组成，木糖、阿拉伯糖是SPG的主要组成单糖。通过紫外、红外光谱的特征吸收峰，鉴定SPG为酸性糖蛋白，且结构中存在β-型吡喃糖环结构。
5．通过体外抗氧化实验(DPPH·自由基、O_2~-·自由基、·OH自由基和卵黄脂蛋白PUFA过氧化体系)，分别研究了车前子多糖提取物SPWE、车前子粗多糖CSPP和车前子多糖纯品SPG的抗氧化活性。结果表明，SPWE、CSPP、SPG均具有一定的抗氧化活性。一定浓度范围内，随着浓度增加，抗氧化活性逐渐增强。在4种体外抗氧化体系中，SPG的活性较高，在卵黄脂蛋白PUFA过氧化体系中的作用最显著，2 mg／mL的作用效果与同浓度的Vc接近。
6．通过流变仪、物性测定仪、DSC和TGA／SDTA对车前子多糖的流变、凝胶特性和热稳定性进行分析。结果表明，车前子多糖溶液为假塑性流体，表观黏度随浓度的增加逐渐增加；温度升高可导致溶液黏度降低：在pH5～11时较稳定；对NaCl具有良好的兼容性，低浓度CaCl_2对车前子多糖溶液有显著的增稠效应，其增稠效果与离子浓度呈正相关。车前子多糖形成的凝胶为热可逆性凝胶，溶解温度、浓度、pH、盐对凝胶强度具有较大的影响；DSC分析无明显的焓变，推测其无明显的熔化点和凝固点；TGA／SDTA分析发现车前子多糖热稳定性好，预示其在食品、医药工业等行业具有广泛的应用前景。
Semen plantaginis has the dual usages as medicine and as food. It is an approved raw material for healthy food by the Minister of Public Health of China. The polysaccharides which are located in the outer seed-coat (husk) are one of the main active components of Semen plantaginis. The polysaccharides in Semen Plantaginis have many biological activities including laxation effect, hypoglycemic effect, hypolipidemic effect, anti-inflammatory and immunological activity. The polysaccharides in Semen Plantaginis as a hydrophilic colloid were widely used in food and pharmaceutical industries. Many previous researches were focused on the structure, bioactivity and application of the polysaccharides in Semen Plantaginis in foreign countries especially Europe and America, However, few studies were found in China. Therefore, in our paper, the polysaccharides extracted from the seeds of Ji'an Plantago asiatica L were studied. On the basis of systematic analysis on the extraction, isolation, purification and primary structure of this polysaccharides, its antioxidant activity and functional properties were studied preliminarily. The main results in the paper were concluded as follows:
1. A suitable method for the determination of the contents of polysaccharides in Semen Plantaginis by the phenol-H_2SO_4 colorimetry was established, and xylose was used as a standard. The two colorimetry methods and seven standards were compared and analyzed, respectively. The results showed that the colored compounds of xylose and Semen Plantaginis polysaccharides by phenol-H_2SO_4 colorimetry were more stable and had the similar maximum absorption wavelength. It means that the determination value of the content of polysaccharides in Semen Plantaginis by the phenol-H_2SO_4 colorimetry method with xylose as a standard was more near to the true content. The optimum conditions and operating steps are as follows. Firstly, 3 % phenol solution 0.5 mL is added into the sample solution 2 mL. Then sulfuric acid 4 mL is added in. After shaking up rapidly and sufficiently, the mixture is stayed for 35 min at room temperature. Finally the absorbency at 480 nm is determined. The content of polysaccharides in Semen Plantaginis was evaluated as 9.02 % by the optimization method. The average recovery rate was 98.9 % with 1.79 % of RSD (n=6). This method is suitable for the determination of polysaccharides in Semen Plantaginis. It can also be regarded as the reference for the determination of polysaccharides with high pentoses content in other plants.
2. The preparation process of polysaccharides in Semen Plantaginis was studied. The extraction conditions that influenced the extraction yield of polysaccharides were optimized by orthogonal design, the optimum extraction conditions were: 6 h at 100℃, the ration of material to solvent is l:35(w/v), giving the yield of polysaccharides at 8.5%. The optimum precipitation conditions were determined by single factor test, the best precipitation conditions were as follows: the concentration ratio 2, 60% alcohol precipitating for 6 h at low temperature. The optimum conditions are economic, simplicity and feasible, providing a theoretical reference for the industrialization of the preparation of polysaccharides in Semen Plantaginis.
3. The two methods of Sevage method and Enzyme combined with Sevage method were used to remove proteins from the crude polysaccharides in Semen Plantaginis. By comparing the effects, it was found that by Enzyme combined with Sevage method, both the protein removal rate and polysaccharide yield were higher, and the purity of Semen Plantaginis polysaccharides after deproteinated was higher, too. The Sephacryl-400 gel column chromatography in series with the AKTA purifier 100 system was used to purify the polysaccharides in Semen Plantaginis. A main component, named SPG {Semen Plantaginis Glycoprotein), was obtained which was found to be a homogeneous glycoprotein component by HPLC. The molecular weight (Mw) of SPG was 1898584 Da determined by HPGPC.
4. The physicochemical properties of SPG were studied. The results showed that SPG was brown powder and dissolvable in water. SPG contained protein of 2.92% and uronic acid of 14.58%. Arabinose, xylose, mannose, ribose, glucose and galactose were found to be the compositional monosaccharides in SPG by GC. Xylose and arabinose are the two main compositional monosaccharides. By UV spectrum and IR spectrum, SPG was identified as an acid glycoprotein, containing theβ- pyranoid ring.
5. The antioxidant effects of different Semen Plantaginis Polysaccharides (Semen Plantaginis Water Extract, SPWE; Crude Semen Plantaginis Polysaccharides, CSPP; Semen Plantaginis Glycoprotein, SPG) were studied in vitro by different testing systems , which included DPPH radical, superoxide anion radical, hydroxyl radical and the lipid peroxidation of yolk liporeotein system. The results showed that SPWE, CSPP, SPG all had certain antioxidant activities. The correlativity of their effects and the doses are positive, in a certain concentration range. The SPG showed high antioxidant activities in those four antioxidant systems, especially in the lipid peroxidation of yolk liporeotein system. SPG showed comparable antioxidant effects with Vc at the same concentration of 2 mg / mL.
6. The rheological property, gelation property and thermally stability of polysaccharides in Semen Plantaginis were studied by rheometer, TA-XT2i instrument, DSC and TGA/SDTA analysis respectively. It was found that the polysaccharides solution had pseudoplasticity. Apparent viscosity increased with the increase of concentration; the increasing of temperature can reduce the viscosity of the polysaccharides; the viscosity is not affected by the pH 5 toll; It has better compatibility with NaCl, Ca~(2+) obviously improve its viscosity, and the ability is correlated to the concentration of Ca~(2+) ions. The polysaccharides gel was the thermal reversible gel. Dissolution temperature, concentration, pH, salt all have obvious effects on the gel strength. The results of DSC showed no obvious enthalpic transitions for the gel, which indicated no obvious melting point (T_(melt)) and gel point (T_(gel)) for the gel. The results of TGA/SDTA analysis showed the polysaccharides had good thermal stability. All the above results showed that the polysaccharides in Semen Plantaginis had wide application potential in the food, pharmaceutical industries and so on.
引文
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