防风多糖的提纯、结构分析及生物活性研究
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摘要
多糖是广泛存在于植物、动物、微生物组织中的一类天然高分子化合物,是维持生命正常运转的基本物质之一,具有抗氧化、抗衰老、调节免疫等多种生理活性,且无毒副作用,已被广泛应用于医药和保健食品领域。防风为伞形科多年生草本植物,是国家重点保护的野生药材之一。防风的药用部位主要是未抽花植株的干燥的根,具有解热、镇静、镇痛、抗炎、抗肿瘤、调节免疫等多种生理功能,临床上被用来治疗伤风感冒、急性结膜炎、高血压、肥胖症、中风、痔疮等症,但其临床应用多以古代文献记载为依据,对其具体的活性成分和作用机制尚缺乏系统深入的研究。防风多糖是防风主要的活性成分之一,决定着其多项生理功能,因此对其进行深入系统的研究将能为防风资源的进一步开发利用提供必需的理论依据。本论文对防风多糖的提取、分离纯化、分子修饰、结构分析、生物活性等方面进行了比较系统的研究,主要实验结果如下:
1.采用传统热水浸提法、超声强化提取法、微波辅助提取法三种不同方法提取防风多糖,优化出传统热水浸提法的最佳提取工艺为:提取温度75℃、固液比1:25、提取时间3h、提取次数3次:超声强化提取法的最佳提取工艺为:超声功率1000W、提取时间25min、固液比1:25;微波辅助提取法的最佳提取工艺为:微波功率560W、固液比1:30、提取时间10min。与传统热水浸提法相比,超声强化提取法和微波辅助提取法均可以提高防风多糖的提取率,其中以微波辅助提取法的效果更为明显,在最佳提取工艺条件下,微波辅助提取法的多糖提取率为7.639%,远高于传统热水浸提法的4.913%,而提取时间也由3h缩短到10min,大大节约了时间、降低了成本,因此微波辅助提取法是一种快速、高效的提取防风多糖的好方法。
2.粗多糖经过脱色、除蛋白和柱层析分离纯化,得到两种防风多糖SPS-Ⅰ、SPS-Ⅱ,纯度鉴定结果表明SPS-Ⅰ、SPS-Ⅱ为均一的多糖。苯酚-硫酸法和硫酸-咔唑法分别测得SPS-Ⅰ、SPS-Ⅱ中的多糖和糖醛酸含量分别为93.1790%、71.0493%和6.4525%、29.1153%,高效液相凝胶色谱(HPGPC)法测得SPS-Ⅰ、SPS-Ⅱ的分子量分别为32700 Da和68100Da。利用气相色谱测得,SPS-Ⅰ中各种单糖组成的摩尔比为:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=0.80:9.34:1.42:3.98:7.40 SPS-Ⅱ中各种单糖组成的摩尔比则为:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=2.34:5.49:1.22:2.40:9.04。部分酸水解、高碘酸氧化和Smith降解实验结果表明,SPS-Ⅰ的主链是由Gal、Ara和Rha构成,Glu、Gal、Ara和Man构成侧链,而SPS-Ⅱ中,Gal、Ara、Glu和Rha除了构成主链组成外,还有一部分参与了侧链的构成。SPS-Ⅰ、SPS-Ⅱ的相邻糖残基间的连接方式以1→2、1→6、1→3等键型为主,少数以1→4键型连接,糖苷键以α-构型为主,而且表现出吡喃糖的特征吸收。
3.防风多糖(SPS)经三氧化硫-吡啶法硫酸酯化得到硫酸酯化防风多糖(S-SPS),得率为143.4513%,硫酸钡比浊法测得S-SPS的硫酸基含量为19.3853%,取代度DS为0.412。GC分析结果表明,SPS和S-SPS的单糖组成基本相同,均由鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖组成,只是各种单糖之间的比例略有差别。
4.抗氧化实验结果表明,防风多糖具有一定的清除自由基、抗脂质过氧化的能力,对于不同的自由基,效果不尽相同,其中对·OH和DPPH·具有较强的清除作用,在SPS、SPS-Ⅰ、SPS-Ⅱ和S-SPS四种防风多糖中,以S-SPS和SPS-Ⅱ的效果较好,在8mg/mL时,二者对·OH的清除率均在90%以上。抑菌实验结果表明,防风多糖具有一定的抑菌性,并且对所抑菌的种类具有选择性,对青霉、毛霉和大肠杆菌无抑制作用,而对枯草芽孢杆菌的抑制效果较好。在几种防风多糖中,以S-SPS的效果最好,其对枯草芽孢杆菌的最小抑菌浓度(MIC)为1mg/mL,远低于SPS的最小抑菌浓度(4mg/mL)。总之,防风多糖经硫酸酯化后,其水溶性和抗氧化活性明显提高,抑菌性能也有所增强,这可以为防风多糖的开发利用提供借鉴与参考。
Polysaccharide,a kind of natural polymers in tissue of plant,animal and microbe,is one of basic materials to maintain normal operation of the lives,and has many physiological functions such as anti-oxidation,anti-decrepitude,immune regulation and so on while poses no poisonous effect to the body,so it has been applied to clinical service and sold in health product market.Saposhnikovia devaricata(Turcz.) Schischk,one of most critical wild Chinese medicinal materials,belongs to Umbelliferous,perennial and herbaceous plant.Its dry root with no florescence could be treated cold, acute conjunctivitis,high blood pressure,adiposity,apoplexy and hemorrhoid for the physiological functions of anti-febrile,balsamic,antalgic,anti-agnail,anti-tumour,immune regulation and so on.As for most of application in clinic,there is a lack of systemic and in-depth research on specific active ingredient and functionary mechanism,only according to recordation of ancient document.As one of main active ingredients of Saposhnikovia devaricata,polysaccharide is involved in its many physiological functions,so these research on Saposhnikovia devaricata polysaccharides will provide necessary theory for farther exploiture of Saposhnikovia devaricata.In the present paper,we studied the polysaccharides from the roots of Saposhnikovia devaricata,including process of extraction, isolation,purification,molecular modification,structure analysis and biological activity.The primary results were as follows:
1.Polysaccharides from Saposhnikovia divaricata(Turez.) Schischk were obtained through traditional hot water extraction,ultrasonic-intensified extraction and microwave-assisted extraction to optimize the best extraction process in view of yield of polysaccharide by single factor test and orthogonal experiment.The results showed that the optimal conditions of traditional hot water extraction were temperature 75℃,extraction time three hours once,solid-liquid ratio 1:25 and extracting three times,the optimal conditions of ultrasonic-intensified extraction were ultrasonic power of 1000 W,extraction time of 30 min and solid-liquid ratio of 1:25,the optimal conditions of microwave-assisted extraction were microwave power of 560 W,extraction time of 10 min and solid-liquid ratio of 1:30.Compared to traditional hot water extraction,both of ultrasonic-intensified extraction and microwave-assisted extraction could increase the yield of polysaccharide,effect of microwave-assisted extraction was more significant,under the optimal conditions,its yield of polysaccharide increased from 4.913%to 7.639%,while extraction time shortened from 3h to 10min. Therefore,microwave-assisted extraction was one more quick and efficient method to extract Saposhnikovia devaricata polysaccharides with fewer time and lower cost.
2.Two kinds of homogeneous Saposhnikovia devaricata polysaccharides SPS-Ⅰand SPS-Ⅱwere attained from the crude polysaccharide through de-coloring by active carbon adsorption method, de-proteinizating by Sevag method,purifying by DEAE-52 cellulose coltmm chromatography and Sephadex G-200 gel filtration chromatography.In SPS-Ⅰand SPS-Ⅱ,the contents of potysaccharides and uronic acids,determined by phenol-sulfuric acid method and sulfuric acid carbazole method, were 93.1790%,71.0493%and 6.4525%,29.1153%,respectively.The molecular weight of SPS-Ⅰand SPS-Ⅱwere 32700 and 68100Da by the determination of high performance gel permeation chromatography(HPGPC).Gas chromatography(GC) analysis showed that SPS-Ⅰwas composed of rhamnose,arabinose,mannose,glucose,galactose in molar ratios of 0.80:9.34:1.42:3.98:7.40 and SPS-Ⅱwas also composed of rhamnose,arabinose,mannose,glucose,galactose in molar ratios of 2.34:5.49:1.22:2.40:9.04.After the methods of partial hydrolysis with acid,periodate oxidation and Smith degradation analysis,the results indicated that the main chain of SPS-Ⅰwas made up of galactose,arabinose and rhamnose,and the side chain of SPS-Ⅰwas composed of glucose,galactose, arabinose and mannose,while in SPS-Ⅱ,galactose,arabinose,glucose and rhamnose formed not only the main chain but also the side chain.In SPS-Ⅰand SPS-Ⅱ,the main linking of were 1→2,1→6 and 1→3 linkage,the minority was 1→4 linkage,the glycosidic conformation was mostlyα-linked glycosidic bond and characteristic of pyranose.
3.Sulfated Saphoshnikovia devaricata polysaccharide(S-SPS) was gamed from Saphoshnikovia devaricata polysaccharide(SPS) by the method of sulfur trioxide pyridine complex with the yield of 143.4513%,the conten of SO_4~(2-) was 19.3853%and the degree of substituent(DS) was 0.412.Gas chromatography(GC) analysis results showed that both of SPS and S-SPS were composed of rhamnose,arabinose,mannose,glucose and galactose,only proportion of each monosaccharide has a little difference.
4.The results of antioxidative experiments revealed that Saposhnikovia devaricata polysaccharides had some functions of scavenging free radicals and inhibiting lipid peroxidation and scavenging effects on DPPH.and·OH were especially significant.S-SPS and SPS-Ⅱhave better effect among the four polysaccharides of SPS,SPS-Ⅰ,SPS-Ⅱ,S-SPS and the scavenging rate of·OH could exceed 90%when the experimental concentration was 8mg/mL.The results of antibacterial experiments showed that Saposhnikovia devaricata polysaccharides inhibited selectively growth of some microbes and had better inhibitory effect on Bacillus subtilis,while had no visible inhibitory effects on Escherichia coli,Penicillum chrysogenum and Mucor mucedo.Among different Saposhnikovia devaricata polysaccharides,S-SPS had best antibacterial effect and its minimal inhibitory concentration(MIC) to Bacillus subtilis was 1mg/mL,greatly lower than that of SPS(4mg/mL).In conclusion,the sulfation of SPS was a considerable molecular modification method for improving soluble property in water and enhancing antioxidative activities and antibacterial ability, providing some reference for farther exploiture and using of the resource of Saposhnikovia devaricata.
1.采用传统热水浸提法、超声强化提取法、微波辅助提取法三种不同方法提取防风多糖,优化出传统热水浸提法的最佳提取工艺为:提取温度75℃、固液比1:25、提取时间3h、提取次数3次:超声强化提取法的最佳提取工艺为:超声功率1000W、提取时间25min、固液比1:25;微波辅助提取法的最佳提取工艺为:微波功率560W、固液比1:30、提取时间10min。与传统热水浸提法相比,超声强化提取法和微波辅助提取法均可以提高防风多糖的提取率,其中以微波辅助提取法的效果更为明显,在最佳提取工艺条件下,微波辅助提取法的多糖提取率为7.639%,远高于传统热水浸提法的4.913%,而提取时间也由3h缩短到10min,大大节约了时间、降低了成本,因此微波辅助提取法是一种快速、高效的提取防风多糖的好方法。
2.粗多糖经过脱色、除蛋白和柱层析分离纯化,得到两种防风多糖SPS-Ⅰ、SPS-Ⅱ,纯度鉴定结果表明SPS-Ⅰ、SPS-Ⅱ为均一的多糖。苯酚-硫酸法和硫酸-咔唑法分别测得SPS-Ⅰ、SPS-Ⅱ中的多糖和糖醛酸含量分别为93.1790%、71.0493%和6.4525%、29.1153%,高效液相凝胶色谱(HPGPC)法测得SPS-Ⅰ、SPS-Ⅱ的分子量分别为32700 Da和68100Da。利用气相色谱测得,SPS-Ⅰ中各种单糖组成的摩尔比为:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=0.80:9.34:1.42:3.98:7.40 SPS-Ⅱ中各种单糖组成的摩尔比则为:鼠李糖:阿拉伯糖:甘露糖:葡萄糖:半乳糖=2.34:5.49:1.22:2.40:9.04。部分酸水解、高碘酸氧化和Smith降解实验结果表明,SPS-Ⅰ的主链是由Gal、Ara和Rha构成,Glu、Gal、Ara和Man构成侧链,而SPS-Ⅱ中,Gal、Ara、Glu和Rha除了构成主链组成外,还有一部分参与了侧链的构成。SPS-Ⅰ、SPS-Ⅱ的相邻糖残基间的连接方式以1→2、1→6、1→3等键型为主,少数以1→4键型连接,糖苷键以α-构型为主,而且表现出吡喃糖的特征吸收。
3.防风多糖(SPS)经三氧化硫-吡啶法硫酸酯化得到硫酸酯化防风多糖(S-SPS),得率为143.4513%,硫酸钡比浊法测得S-SPS的硫酸基含量为19.3853%,取代度DS为0.412。GC分析结果表明,SPS和S-SPS的单糖组成基本相同,均由鼠李糖、阿拉伯糖、甘露糖、葡萄糖和半乳糖组成,只是各种单糖之间的比例略有差别。
4.抗氧化实验结果表明,防风多糖具有一定的清除自由基、抗脂质过氧化的能力,对于不同的自由基,效果不尽相同,其中对·OH和DPPH·具有较强的清除作用,在SPS、SPS-Ⅰ、SPS-Ⅱ和S-SPS四种防风多糖中,以S-SPS和SPS-Ⅱ的效果较好,在8mg/mL时,二者对·OH的清除率均在90%以上。抑菌实验结果表明,防风多糖具有一定的抑菌性,并且对所抑菌的种类具有选择性,对青霉、毛霉和大肠杆菌无抑制作用,而对枯草芽孢杆菌的抑制效果较好。在几种防风多糖中,以S-SPS的效果最好,其对枯草芽孢杆菌的最小抑菌浓度(MIC)为1mg/mL,远低于SPS的最小抑菌浓度(4mg/mL)。总之,防风多糖经硫酸酯化后,其水溶性和抗氧化活性明显提高,抑菌性能也有所增强,这可以为防风多糖的开发利用提供借鉴与参考。
Polysaccharide,a kind of natural polymers in tissue of plant,animal and microbe,is one of basic materials to maintain normal operation of the lives,and has many physiological functions such as anti-oxidation,anti-decrepitude,immune regulation and so on while poses no poisonous effect to the body,so it has been applied to clinical service and sold in health product market.Saposhnikovia devaricata(Turcz.) Schischk,one of most critical wild Chinese medicinal materials,belongs to Umbelliferous,perennial and herbaceous plant.Its dry root with no florescence could be treated cold, acute conjunctivitis,high blood pressure,adiposity,apoplexy and hemorrhoid for the physiological functions of anti-febrile,balsamic,antalgic,anti-agnail,anti-tumour,immune regulation and so on.As for most of application in clinic,there is a lack of systemic and in-depth research on specific active ingredient and functionary mechanism,only according to recordation of ancient document.As one of main active ingredients of Saposhnikovia devaricata,polysaccharide is involved in its many physiological functions,so these research on Saposhnikovia devaricata polysaccharides will provide necessary theory for farther exploiture of Saposhnikovia devaricata.In the present paper,we studied the polysaccharides from the roots of Saposhnikovia devaricata,including process of extraction, isolation,purification,molecular modification,structure analysis and biological activity.The primary results were as follows:
1.Polysaccharides from Saposhnikovia divaricata(Turez.) Schischk were obtained through traditional hot water extraction,ultrasonic-intensified extraction and microwave-assisted extraction to optimize the best extraction process in view of yield of polysaccharide by single factor test and orthogonal experiment.The results showed that the optimal conditions of traditional hot water extraction were temperature 75℃,extraction time three hours once,solid-liquid ratio 1:25 and extracting three times,the optimal conditions of ultrasonic-intensified extraction were ultrasonic power of 1000 W,extraction time of 30 min and solid-liquid ratio of 1:25,the optimal conditions of microwave-assisted extraction were microwave power of 560 W,extraction time of 10 min and solid-liquid ratio of 1:30.Compared to traditional hot water extraction,both of ultrasonic-intensified extraction and microwave-assisted extraction could increase the yield of polysaccharide,effect of microwave-assisted extraction was more significant,under the optimal conditions,its yield of polysaccharide increased from 4.913%to 7.639%,while extraction time shortened from 3h to 10min. Therefore,microwave-assisted extraction was one more quick and efficient method to extract Saposhnikovia devaricata polysaccharides with fewer time and lower cost.
2.Two kinds of homogeneous Saposhnikovia devaricata polysaccharides SPS-Ⅰand SPS-Ⅱwere attained from the crude polysaccharide through de-coloring by active carbon adsorption method, de-proteinizating by Sevag method,purifying by DEAE-52 cellulose coltmm chromatography and Sephadex G-200 gel filtration chromatography.In SPS-Ⅰand SPS-Ⅱ,the contents of potysaccharides and uronic acids,determined by phenol-sulfuric acid method and sulfuric acid carbazole method, were 93.1790%,71.0493%and 6.4525%,29.1153%,respectively.The molecular weight of SPS-Ⅰand SPS-Ⅱwere 32700 and 68100Da by the determination of high performance gel permeation chromatography(HPGPC).Gas chromatography(GC) analysis showed that SPS-Ⅰwas composed of rhamnose,arabinose,mannose,glucose,galactose in molar ratios of 0.80:9.34:1.42:3.98:7.40 and SPS-Ⅱwas also composed of rhamnose,arabinose,mannose,glucose,galactose in molar ratios of 2.34:5.49:1.22:2.40:9.04.After the methods of partial hydrolysis with acid,periodate oxidation and Smith degradation analysis,the results indicated that the main chain of SPS-Ⅰwas made up of galactose,arabinose and rhamnose,and the side chain of SPS-Ⅰwas composed of glucose,galactose, arabinose and mannose,while in SPS-Ⅱ,galactose,arabinose,glucose and rhamnose formed not only the main chain but also the side chain.In SPS-Ⅰand SPS-Ⅱ,the main linking of were 1→2,1→6 and 1→3 linkage,the minority was 1→4 linkage,the glycosidic conformation was mostlyα-linked glycosidic bond and characteristic of pyranose.
3.Sulfated Saphoshnikovia devaricata polysaccharide(S-SPS) was gamed from Saphoshnikovia devaricata polysaccharide(SPS) by the method of sulfur trioxide pyridine complex with the yield of 143.4513%,the conten of SO_4~(2-) was 19.3853%and the degree of substituent(DS) was 0.412.Gas chromatography(GC) analysis results showed that both of SPS and S-SPS were composed of rhamnose,arabinose,mannose,glucose and galactose,only proportion of each monosaccharide has a little difference.
4.The results of antioxidative experiments revealed that Saposhnikovia devaricata polysaccharides had some functions of scavenging free radicals and inhibiting lipid peroxidation and scavenging effects on DPPH.and·OH were especially significant.S-SPS and SPS-Ⅱhave better effect among the four polysaccharides of SPS,SPS-Ⅰ,SPS-Ⅱ,S-SPS and the scavenging rate of·OH could exceed 90%when the experimental concentration was 8mg/mL.The results of antibacterial experiments showed that Saposhnikovia devaricata polysaccharides inhibited selectively growth of some microbes and had better inhibitory effect on Bacillus subtilis,while had no visible inhibitory effects on Escherichia coli,Penicillum chrysogenum and Mucor mucedo.Among different Saposhnikovia devaricata polysaccharides,S-SPS had best antibacterial effect and its minimal inhibitory concentration(MIC) to Bacillus subtilis was 1mg/mL,greatly lower than that of SPS(4mg/mL).In conclusion,the sulfation of SPS was a considerable molecular modification method for improving soluble property in water and enhancing antioxidative activities and antibacterial ability, providing some reference for farther exploiture and using of the resource of Saposhnikovia devaricata.
引文
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