外来入侵植物黄顶菊中活性成分的分离分析、结构鉴定及活性研究
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摘要
外来入侵植物黄顶菊是我国危害最为严重的恶性杂草之一，具有发生量大、繁殖迅速、传播速度快的特点。相比简单的防治方法，资源化利用黄顶菊具有更大优势。为综合防控黄顶菊，开发和利用其巨大的生物资源，需对黄顶菊的活性成分进行深入细致的研究。活性成分的分离纯化是关键。逆流色谱（Counter-current chromatography，CCC）是近几十年发展起来一种连续有效的液液分配色谱技术，它不使用固态支撑体或载体，从而避免了不可逆吸附造成的化学变性及失活等问题，具有样品回收率高，重现性好，溶剂消耗量少，洗脱方式多样等优点。近年来一系列逆流色谱新方法及新仪器的研发，改进了逆流装置，扩展了逆流色谱的应用范围，提高了逆流色谱的分离效率。因此广泛应用于天然产物中有效成分的分离和纯化。本论文主要采用逆流色谱法分离纯化黄顶菊中的活性物质。
本论文系统的研究了外来入侵植物黄顶菊中活性物质的分析方法和分离提纯方法。建立了黄顶菊活性物质的回流粗提工艺；建立了逆流色谱分离纯化黄顶菊中活性物质单体的方法，实现了黄顶菊中活性物质十种单体的分离纯化及结构鉴定；建立了高效液相色谱法定量分析黄顶菊中主要活性物质单体的方法和黄顶菊叶的高效液相色谱指纹图谱；初步评价了从黄顶菊中分离得到的主要黄酮类物质的抗氧化活性。主要研究成果如下：
1.通过单因素和正交实验确定了黄顶菊总黄酮最佳提取工艺：80%乙醇，料液比1:30（w/v），每次回流75min，回流2次。应用最优提取工艺分别提取不同部位，不同生长期，不同地区黄顶菊样品，并测定其总黄酮含量。黄顶菊中黄酮类化合物含量较高的的部位是花部和叶部，含量较高的生长期是开花期，河北省采集的黄顶菊叶中总黄酮含量较其它地区的高。
2.使用逆流色谱和制备液相色谱分离纯化了黄顶菊中四种主要的黄酮苷，并通过ESI-MS，~1H NMR，~(13)C NMR，HSQC和HMBC等方法进行了结构鉴定。对比了高速逆流色谱（HSCCC）与高效逆流色谱（HPCCC）的分离效率，研究了洗脱-推出逆流色谱法（EECCC）分离黄顶菊粗提物的方法。使用GS10AB-A型HSCCC仪，乙酸乙酯-甲醇-水（25:1:25, v/v）体系，一次可分离约400mg黄顶菊粗品，得到3.6mg纯度97%以上的万寿菊素-3-O-葡萄糖苷，4.4mg纯度98%以上的紫云英苷以及4.2mg纯度97%以上的异槲皮苷和6-甲氧基山奈酚-3-O-葡萄糖苷的混合物，采用制备高效液相色谱法进一步将两种物质分离。采用HSCCC二氯甲烷-甲醇-水（5:3:2, v/v）体系，从逆流分离黄酮苷回收样品中分离出高纯度的四种黄酮醇。研究结果表明逆流色谱是一种从黄顶菊中分离活性物质的有效手段。
3.采用逆流色谱两种溶剂系统从黄顶菊中分离纯化了异鼠李素-3-硫酸酯，并鉴定结构。相比传统的多组分有机溶剂-水体系，单组分有机溶剂-盐水体系更适合极性物质异鼠李素-3-硫酸酯的分离。采用正丁醇-0.25%NaCl（1:1，v/v）溶剂体系，一次HSCCC实验可从约400mg未经任何前处理的黄顶菊粗品中分离纯化出2.1mg纯度大于97%的异鼠李素-3-硫酸酯。研究结果表明单组份有机溶剂-盐水逆流色谱体系特别适合制备分离大极性物质异鼠李素-3-硫酸酯。
4.建立了一种简单灵敏的HPLC-DAD同时测定黄顶菊样品中六种主要物质含量的方法，并进行了方法学考察。测定了不同部位，不同生长期，不同地区黄顶菊样品中活性物质的含量，明确了其动态变化规律。首次建立了黄顶菊叶的HPLC指纹图谱，评估和鉴别了十二批来自中国四个不同地区的黄顶菊叶样品，并计算其相似度。将HPLC指纹图谱研究和主成分含量测定结合在一起，可以为黄顶菊的物种鉴别提供化学依据。
5.将黄顶菊中含量最高的黄酮苷紫云英苷研制成国家标准样品，资源化利用黄顶菊。采用连续进样逆流色谱法大量制备分离了黄顶菊中的紫云英苷，并进行了多种手段的结构确证以及HPLC纯度检查。对其进行了均匀性与稳定性考察，均符合标准样品要求，最后通过多家实验室共同定值，确定紫云英苷标准样品纯度定值结果为98.49%。
6.对黄顶菊中分离得到的五种主要黄酮类物质进行初步抗氧化性研究，评价了其清除DPPH自由基，总抗氧化能力T-AOC，清除超氧阴离子自由基和羟自由基的能力。结果表明五种物质均具有一定的自由基清除能力和抗氧化能力，为综合开发利用黄顶菊为天然抗氧化剂提供基础。
Flaveria bidentis (L.) Kuntze was one of the most harmful invasive weedin China. It has very strong reproductive and survival abilities, and spreadsfast. Compared with simple controlling methods, utilization of the weed inuseful purposes might give more benefits. In order to utilize and control F.bidentis, bioactive compounds from F. bidentis should be deeply studied. Themost important point is separation and purification. Counter-currentchromatography (CCC), being a support-free liquid–liquid partitionchromatography, eliminates irreversible adsorption of sample onto the solidsupport, and has been widely used in preparative separation of naturalproducts. CCC is an advanced technique that it needs minimum samplepre-treatment and cleanup procedures, and permits many elution modes. Inaddition, a series of new CCC assays and apparatus extends the applicationfields of CCC and improves the separation efficiency of CCC separation and devices. CCC was mainly used for the separation of bioactive compoundsfrom F. bidentis in this thesis.
In this thesis, multiple separation and analysis methods were studied forthe bioactive compounds from F. bidentis. The optimized extraction method,CCC separation methods of10compounds, HPLC analysis of the maincompounds and fingerprint of F. bidentis leaves were established. In addition,the antioxidant properties of the main flavonoids were estimated. The mainresearch results were as follows:
1. The optimized extraction method of total flavonoids from F. bidentiswas established by single factor and orthogonal design experiments: F.bidentis powder was extracted (refluxed) with80%(v/v) ethanol for75min (2times). The total flavonoids in F. bidentis samples from different origins,different parts of the plant and different growing periods were tested. Resultsshowed that the highest flavonoids contents were flower and leaf, the bloomperiod and the leaf sample from Hebei province.
2. CCC and preparative HPLC (pre-HPLC) were successfully used for theseparation of four main flavonol glycosides from F. bidentis, and the fourcompounds were identified by ESI-MS，~1H NMR，~(13)C NMR，HSQC andHMBC. The separation efficiency of the high-speed counter-currentchromatography (HSCCC) was compared with the high performancecounter-current chromatography (HPCCC). Using GS10AB-A HSCCC bythe two-phase solvent system ethyl acetate-methanol-water (25:1:25, v/v), about400mg of the crude extract was separated, yielding3.6mg ofpatuletin-3-O-glucoside at a purity of over97%;4.4mg of astragalin at apurity of over98%and4.2mg of a mixture of quercetin-3-O-glucoside and6-methoxykaempferol-3-O-glucoside constituting over97%of the fraction.Then the mixture was separated by pre-HPLC. Four pure flavonol wereseparated using dichloromethane-methanol-water (5:3:2, v/v) by HSCCC.
3. The first preparative separation of a flavonoid sulphateisorhamnetin-3-sulphate from F. bidentis by counter-current chromatography(CCC) was presented. Two kinds of solvent systems were used. Aone-component organic/salt-containing system composed of n-butanol-0.25%sodium chloride aqueous solution (1:1, v/v) was used. As a result,2.1mg ofisorhamnetin3-sulphate with a purity of over97%has been isolated from400mg of crude extract without pre-enrichment. Compared with the conventionalorganic/aqueous system, the one-component organic/salt-containing aqueoussystem was more suitable for the separation of isorhamnetin-3-sulphate, andpurer target compound was obtained from the crude extract withoutpre-enrichment using the new solvent system.
4. A simple and sensitive high performance liquid chromatography methodcoupled with photodiode array detection (HPLC-DAD) was developed forsimultaneous determination of6major constituents in F. bidentis. The contentsof the6compounds in F. bidentis samples from different origins, differentparts of the plant and different growing periods were tested. The chemical fingerprint of F. bidentis leaves was established using raw materials of12batches in China. The results indicate that this multi-component determinationmethod in combination with chromatographic fingerprint analysis is suitablefor quantitative analysis and identification of F. bidentis.
5. Astragalin which content was the highest in F. bidentis was developedas a national reference material. A gram scale of astragalin was separated bycontinuous injection HSCCC. It was identified by many methods and thepurity was determined by HPLC. The uniformity and stability were inspected,and results fitted the requirement of reference material. The definite value wasfinally determined by combined value determination, and was98.49%.
6. The antioxidant properties of the main flavonoids were estimated.DPPH scavenging activity, T-AOC, superoxide radicals scaving effect andhydroxyl radicals scaving effect were tested. Results showed that the5flavonoids processed a certain ability of antioxidation.
本论文系统的研究了外来入侵植物黄顶菊中活性物质的分析方法和分离提纯方法。建立了黄顶菊活性物质的回流粗提工艺；建立了逆流色谱分离纯化黄顶菊中活性物质单体的方法，实现了黄顶菊中活性物质十种单体的分离纯化及结构鉴定；建立了高效液相色谱法定量分析黄顶菊中主要活性物质单体的方法和黄顶菊叶的高效液相色谱指纹图谱；初步评价了从黄顶菊中分离得到的主要黄酮类物质的抗氧化活性。主要研究成果如下：
1.通过单因素和正交实验确定了黄顶菊总黄酮最佳提取工艺：80%乙醇，料液比1:30（w/v），每次回流75min，回流2次。应用最优提取工艺分别提取不同部位，不同生长期，不同地区黄顶菊样品，并测定其总黄酮含量。黄顶菊中黄酮类化合物含量较高的的部位是花部和叶部，含量较高的生长期是开花期，河北省采集的黄顶菊叶中总黄酮含量较其它地区的高。
2.使用逆流色谱和制备液相色谱分离纯化了黄顶菊中四种主要的黄酮苷，并通过ESI-MS，~1H NMR，~(13)C NMR，HSQC和HMBC等方法进行了结构鉴定。对比了高速逆流色谱（HSCCC）与高效逆流色谱（HPCCC）的分离效率，研究了洗脱-推出逆流色谱法（EECCC）分离黄顶菊粗提物的方法。使用GS10AB-A型HSCCC仪，乙酸乙酯-甲醇-水（25:1:25, v/v）体系，一次可分离约400mg黄顶菊粗品，得到3.6mg纯度97%以上的万寿菊素-3-O-葡萄糖苷，4.4mg纯度98%以上的紫云英苷以及4.2mg纯度97%以上的异槲皮苷和6-甲氧基山奈酚-3-O-葡萄糖苷的混合物，采用制备高效液相色谱法进一步将两种物质分离。采用HSCCC二氯甲烷-甲醇-水（5:3:2, v/v）体系，从逆流分离黄酮苷回收样品中分离出高纯度的四种黄酮醇。研究结果表明逆流色谱是一种从黄顶菊中分离活性物质的有效手段。
3.采用逆流色谱两种溶剂系统从黄顶菊中分离纯化了异鼠李素-3-硫酸酯，并鉴定结构。相比传统的多组分有机溶剂-水体系，单组分有机溶剂-盐水体系更适合极性物质异鼠李素-3-硫酸酯的分离。采用正丁醇-0.25%NaCl（1:1，v/v）溶剂体系，一次HSCCC实验可从约400mg未经任何前处理的黄顶菊粗品中分离纯化出2.1mg纯度大于97%的异鼠李素-3-硫酸酯。研究结果表明单组份有机溶剂-盐水逆流色谱体系特别适合制备分离大极性物质异鼠李素-3-硫酸酯。
4.建立了一种简单灵敏的HPLC-DAD同时测定黄顶菊样品中六种主要物质含量的方法，并进行了方法学考察。测定了不同部位，不同生长期，不同地区黄顶菊样品中活性物质的含量，明确了其动态变化规律。首次建立了黄顶菊叶的HPLC指纹图谱，评估和鉴别了十二批来自中国四个不同地区的黄顶菊叶样品，并计算其相似度。将HPLC指纹图谱研究和主成分含量测定结合在一起，可以为黄顶菊的物种鉴别提供化学依据。
5.将黄顶菊中含量最高的黄酮苷紫云英苷研制成国家标准样品，资源化利用黄顶菊。采用连续进样逆流色谱法大量制备分离了黄顶菊中的紫云英苷，并进行了多种手段的结构确证以及HPLC纯度检查。对其进行了均匀性与稳定性考察，均符合标准样品要求，最后通过多家实验室共同定值，确定紫云英苷标准样品纯度定值结果为98.49%。
6.对黄顶菊中分离得到的五种主要黄酮类物质进行初步抗氧化性研究，评价了其清除DPPH自由基，总抗氧化能力T-AOC，清除超氧阴离子自由基和羟自由基的能力。结果表明五种物质均具有一定的自由基清除能力和抗氧化能力，为综合开发利用黄顶菊为天然抗氧化剂提供基础。
Flaveria bidentis (L.) Kuntze was one of the most harmful invasive weedin China. It has very strong reproductive and survival abilities, and spreadsfast. Compared with simple controlling methods, utilization of the weed inuseful purposes might give more benefits. In order to utilize and control F.bidentis, bioactive compounds from F. bidentis should be deeply studied. Themost important point is separation and purification. Counter-currentchromatography (CCC), being a support-free liquid–liquid partitionchromatography, eliminates irreversible adsorption of sample onto the solidsupport, and has been widely used in preparative separation of naturalproducts. CCC is an advanced technique that it needs minimum samplepre-treatment and cleanup procedures, and permits many elution modes. Inaddition, a series of new CCC assays and apparatus extends the applicationfields of CCC and improves the separation efficiency of CCC separation and devices. CCC was mainly used for the separation of bioactive compoundsfrom F. bidentis in this thesis.
In this thesis, multiple separation and analysis methods were studied forthe bioactive compounds from F. bidentis. The optimized extraction method,CCC separation methods of10compounds, HPLC analysis of the maincompounds and fingerprint of F. bidentis leaves were established. In addition,the antioxidant properties of the main flavonoids were estimated. The mainresearch results were as follows:
1. The optimized extraction method of total flavonoids from F. bidentiswas established by single factor and orthogonal design experiments: F.bidentis powder was extracted (refluxed) with80%(v/v) ethanol for75min (2times). The total flavonoids in F. bidentis samples from different origins,different parts of the plant and different growing periods were tested. Resultsshowed that the highest flavonoids contents were flower and leaf, the bloomperiod and the leaf sample from Hebei province.
2. CCC and preparative HPLC (pre-HPLC) were successfully used for theseparation of four main flavonol glycosides from F. bidentis, and the fourcompounds were identified by ESI-MS，~1H NMR，~(13)C NMR，HSQC andHMBC. The separation efficiency of the high-speed counter-currentchromatography (HSCCC) was compared with the high performancecounter-current chromatography (HPCCC). Using GS10AB-A HSCCC bythe two-phase solvent system ethyl acetate-methanol-water (25:1:25, v/v), about400mg of the crude extract was separated, yielding3.6mg ofpatuletin-3-O-glucoside at a purity of over97%;4.4mg of astragalin at apurity of over98%and4.2mg of a mixture of quercetin-3-O-glucoside and6-methoxykaempferol-3-O-glucoside constituting over97%of the fraction.Then the mixture was separated by pre-HPLC. Four pure flavonol wereseparated using dichloromethane-methanol-water (5:3:2, v/v) by HSCCC.
3. The first preparative separation of a flavonoid sulphateisorhamnetin-3-sulphate from F. bidentis by counter-current chromatography(CCC) was presented. Two kinds of solvent systems were used. Aone-component organic/salt-containing system composed of n-butanol-0.25%sodium chloride aqueous solution (1:1, v/v) was used. As a result,2.1mg ofisorhamnetin3-sulphate with a purity of over97%has been isolated from400mg of crude extract without pre-enrichment. Compared with the conventionalorganic/aqueous system, the one-component organic/salt-containing aqueoussystem was more suitable for the separation of isorhamnetin-3-sulphate, andpurer target compound was obtained from the crude extract withoutpre-enrichment using the new solvent system.
4. A simple and sensitive high performance liquid chromatography methodcoupled with photodiode array detection (HPLC-DAD) was developed forsimultaneous determination of6major constituents in F. bidentis. The contentsof the6compounds in F. bidentis samples from different origins, differentparts of the plant and different growing periods were tested. The chemical fingerprint of F. bidentis leaves was established using raw materials of12batches in China. The results indicate that this multi-component determinationmethod in combination with chromatographic fingerprint analysis is suitablefor quantitative analysis and identification of F. bidentis.
5. Astragalin which content was the highest in F. bidentis was developedas a national reference material. A gram scale of astragalin was separated bycontinuous injection HSCCC. It was identified by many methods and thepurity was determined by HPLC. The uniformity and stability were inspected,and results fitted the requirement of reference material. The definite value wasfinally determined by combined value determination, and was98.49%.
6. The antioxidant properties of the main flavonoids were estimated.DPPH scavenging activity, T-AOC, superoxide radicals scaving effect andhydroxyl radicals scaving effect were tested. Results showed that the5flavonoids processed a certain ability of antioxidation.
引文
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