儿茶素氧化产物的分离纯化及生物活性分析
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摘要
儿茶素氧化产物的生物活性日益受到人们的重视和关注,国内外学者都对其进行了许多研究。本实验采用柱色谱与制备型液相色谱对儿茶素氧化产物进行了分离分析,并对分离纯化得到的物质进行了结构鉴定和生物活性的分析。结果表明:
用柱层析分离由碱性氧化反应生成的儿茶素氧化产物。分别采用了MCI GEL CHP20P柱、浓度从10%到70%依次增加的甲醇水溶液,和Sephadex LH-20柱、浓度从20%到60%依次增加的丙酮水溶液,进行梯度洗脱,每次增加梯度为10%,对儿茶素氧化聚合物进行分离。洗脱体系可以将儿茶素氧化聚合物中极性有差别的物质分离开,该两种体系均能有效地将儿茶素与氧化产物分开,甚至能将极性相近的氧化聚合物之间进行分离。此部分实验收集到M6、S7和S10主要含有儿茶素低聚合物的组分。
对柱层析分离中分离得到的M6、S7和S10组分,采用OBD(C18)柱和乙腈-水洗脱体系进行了制备实验。经进一步分离,分别得到M6-2、S7-1、S7-2、S10-1和S10-2。经MS分析和1HNMR和13CNMR分析,参考相关文献,确定M6-2为一种黄酮甙类化合物Kaempferol-3-O-β-D-glucoside,S7-1为EGCG氧化后两分子以C2’-C2’方式连接聚合而成的EGCG二聚体,S7-2为GCG,S10-1为epitheaflagallin-3-O-gallate,S10-2为theaflavin-3-gallate。
对儿茶素氧化产物混合物(P)及分离组分S7的总抗氧化能力(T-AOC)、清除OH·、O2-·能力进行了分析研究,结果表明氧化产物混合物与S7都具有较高的生物活性。差异显著性比较结果显示,P的总抗氧化能力显著高于98%TP和EGCG,清除羟自由基的能力显著高于EGCG、98%TP和80%TFs,清除O2-·能力显著高于80%TFs;S7的总抗氧化能力显著高于EGCG,清除羟自由基的能力显著高于EGCG和98%TP,均与80%TFs相当,而清除O2-·能力显著高于80%TFs。
People pay more attention to the biological activity of oxidation products of catechins increasingly. Scholars home and abroad have done many researches on them. In this paper, column chromatography and preparative HPLC were uesed to separate oxidation products of catechins, the structures of fractions are identified, and the bioability was analyzed. The results of study show:
Column chromatography was used to separate oxidation products of catechins produced in alkalescence condition. Tea oxidation products were fractionated by MCI GEL CHP20P column with 10% step-wise elution of H2O containing increasing proportions of MeOH from 10% to 70% and Sephadex LH-20 column with 10% step-wise elution of H2O containing increasing proportions of acetone from 20% to 60%. Oxides with different polaritis were seperated. Thesse two eluting system can separate catechins and oxides, and different oxides with close polarities availably. M6, S7 and S10 were collected in this part of experiment, which contained oligomers of catechins.
M6, S7 and S10 were applied to a column of OBD(C18) with H2O containing acetonitrile. After purification, M6-2, S7-1, S7-2, S10-1 and S10-2 were get. According to the results of MS and NMR analysis and refer to literatures, the structures of these substance s were determined, M6-2 was Kaempferol-3-O-β-D-glucoside, and S7-1 was dipolymer of EGCG linked at the B-rings through a C-C bond, S7-2 were GCG, S10-1 were epitheaflagallin-3-O-gallate, S10-2 were theaflavin-3-gallate.
The bioability of oxides P and fraction S7 on total antioxidation(T-AOC) and radicals scavenging of OH·, O2-·were tested. The results showed the bioactivity of oxide and S7 were active. Significant difference analysis showed that, the T-AOC of P was significantly higher than that of 98%TP and EGCG, the ability of scavenging OH·of it was significantly higher than that of EGCG, 98%TP and 80%TFs, and the ability of scavenging O2-·of it was significantly higher than that of 80%TFs. The T-AOC of S7 was significantly higher than that of EGCG, the ability of scavenging OH·of it was significantly higher than that of EGCG and 98%TP, it was equal to 80%TFs. And the ability of scavenging O2-·of S7 was significantly higher than that of 80%TFs.
用柱层析分离由碱性氧化反应生成的儿茶素氧化产物。分别采用了MCI GEL CHP20P柱、浓度从10%到70%依次增加的甲醇水溶液,和Sephadex LH-20柱、浓度从20%到60%依次增加的丙酮水溶液,进行梯度洗脱,每次增加梯度为10%,对儿茶素氧化聚合物进行分离。洗脱体系可以将儿茶素氧化聚合物中极性有差别的物质分离开,该两种体系均能有效地将儿茶素与氧化产物分开,甚至能将极性相近的氧化聚合物之间进行分离。此部分实验收集到M6、S7和S10主要含有儿茶素低聚合物的组分。
对柱层析分离中分离得到的M6、S7和S10组分,采用OBD(C18)柱和乙腈-水洗脱体系进行了制备实验。经进一步分离,分别得到M6-2、S7-1、S7-2、S10-1和S10-2。经MS分析和1HNMR和13CNMR分析,参考相关文献,确定M6-2为一种黄酮甙类化合物Kaempferol-3-O-β-D-glucoside,S7-1为EGCG氧化后两分子以C2’-C2’方式连接聚合而成的EGCG二聚体,S7-2为GCG,S10-1为epitheaflagallin-3-O-gallate,S10-2为theaflavin-3-gallate。
对儿茶素氧化产物混合物(P)及分离组分S7的总抗氧化能力(T-AOC)、清除OH·、O2-·能力进行了分析研究,结果表明氧化产物混合物与S7都具有较高的生物活性。差异显著性比较结果显示,P的总抗氧化能力显著高于98%TP和EGCG,清除羟自由基的能力显著高于EGCG、98%TP和80%TFs,清除O2-·能力显著高于80%TFs;S7的总抗氧化能力显著高于EGCG,清除羟自由基的能力显著高于EGCG和98%TP,均与80%TFs相当,而清除O2-·能力显著高于80%TFs。
People pay more attention to the biological activity of oxidation products of catechins increasingly. Scholars home and abroad have done many researches on them. In this paper, column chromatography and preparative HPLC were uesed to separate oxidation products of catechins, the structures of fractions are identified, and the bioability was analyzed. The results of study show:
Column chromatography was used to separate oxidation products of catechins produced in alkalescence condition. Tea oxidation products were fractionated by MCI GEL CHP20P column with 10% step-wise elution of H2O containing increasing proportions of MeOH from 10% to 70% and Sephadex LH-20 column with 10% step-wise elution of H2O containing increasing proportions of acetone from 20% to 60%. Oxides with different polaritis were seperated. Thesse two eluting system can separate catechins and oxides, and different oxides with close polarities availably. M6, S7 and S10 were collected in this part of experiment, which contained oligomers of catechins.
M6, S7 and S10 were applied to a column of OBD(C18) with H2O containing acetonitrile. After purification, M6-2, S7-1, S7-2, S10-1 and S10-2 were get. According to the results of MS and NMR analysis and refer to literatures, the structures of these substance s were determined, M6-2 was Kaempferol-3-O-β-D-glucoside, and S7-1 was dipolymer of EGCG linked at the B-rings through a C-C bond, S7-2 were GCG, S10-1 were epitheaflagallin-3-O-gallate, S10-2 were theaflavin-3-gallate.
The bioability of oxides P and fraction S7 on total antioxidation(T-AOC) and radicals scavenging of OH·, O2-·were tested. The results showed the bioactivity of oxide and S7 were active. Significant difference analysis showed that, the T-AOC of P was significantly higher than that of 98%TP and EGCG, the ability of scavenging OH·of it was significantly higher than that of EGCG, 98%TP and 80%TFs, and the ability of scavenging O2-·of it was significantly higher than that of 80%TFs. The T-AOC of S7 was significantly higher than that of EGCG, the ability of scavenging OH·of it was significantly higher than that of EGCG and 98%TP, it was equal to 80%TFs. And the ability of scavenging O2-·of S7 was significantly higher than that of 80%TFs.
引文
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