杜仲叶抗氧化活性成分研究及桃叶珊瑚甙的制备
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摘要
杜仲叶中含有多种具有药理活性的化学成分,如绿原酸、桃叶珊瑚甙、京尼平甙酸、总黄酮等等,其中桃叶珊瑚甙是杜仲叶中含量丰富的环烯醚萜类化合物之一。本文对建立了杜仲叶中抗氧化活性成分的多种分析检测方法,探讨了杜仲叶提取物对体外自由基的清除活性,优化了杜仲叶中桃叶珊瑚甙的分离纯化工艺,为杜仲叶的综合利用和开发提供了可靠的依据。本研究的主要内容如下:
(1)杜仲叶中抗氧化活性成分检测方法的建立
建立了杜仲叶提取物中总黄酮、绿原酸和桃叶珊瑚甙的紫外可见分光光度和薄层色谱检测方法,并运用高效液相色谱技术建立了同时测定杜仲叶中桃叶珊瑚甙、京尼平甙酸和绿原酸的分析方法,并对三种检测方法进行了比较。结果表明高效液相色谱法最为准确可靠。
(2)杜仲叶提取物抗氧化活性研究
运用简便快捷、结果准确的流动注射化学发光法和简单易行的分光光度法对杜仲叶提取物清除羟基自由基(·OH)、超氧阴离子自由基(O2-·)、过氧化氢(H2O2)和DPPH自由基(·DPPH)的效果进行了系统的研究。结果表明杜仲叶粗提物在低浓度下对羟基自由基和超氧阴离子自由基具有很好的清除作用,对过氧化氢和DPPH自由基也有较好的清除能力。为杜仲叶开发成为具有营养保健功能的天然抗氧化剂奠定了理论基础。
(3)桃叶珊瑚甙的分离纯化研究
通过大孔吸附树脂富集后杜仲叶提取物中桃叶珊瑚甙的含量由粗提液中的4.42%上升至22.11%;通过硅胶G柱层析进一步分离纯化,桃叶珊瑚甙的含量提升到52.83%;最后经制备TLC色谱两次纯化,可得到含量为92.02%的桃叶珊瑚甙产品。
(4)双水相体系分离纯化桃叶珊瑚甙新工艺探讨
运用聚乙二醇(PEG)/葡聚糖40000(D40)双水相体系,对杜仲叶提取液中桃叶珊瑚甙的分离纯化工艺进行了初步探讨。结果表明:由质量分数为11%PEG4000和8%D40组成的双水相体系,加入样品溶液8g,在pH值为7,温度60℃时,D40相萃取率达到66.32%,桃叶珊瑚甙纯度由粗提物中的8.750%提高到48.67%。该方法具有操作条件温和、无溶剂残留、耗时少、对桃叶珊瑚甙的单级分离提纯效率高等优点。
There are many kinds of effective components in Eucommia ulmoides leaves, such as chlorogenic acid, aucubin, geniposidic acid and flavonoids etc. Aucubin is one kind of iridoid compounds and it is richly contained in Eucommia ulmoides leaves. In this dissertation, the detection methods of antioxidative components, isolation and purification of aucubin and antioxidative activity of extract from Eucommia ulmoides leaves were investigated and compared. This work is useful to the development of Eucommia ulmoides leaves. The main contents list as fallow:
(1)Establishment of the detection methods of antioxidative components in Eucommia ulmoides leaves
The determination of chlorogenic acid, aucubin and total flavonoids of Eucommia ulmoides leaves by UV spectrophotometry and TLC, and the simultaneous determination of aucubin,geniposide acid and chlorogenic acid by HPLC were established and compared. The results showed that HPLC was exactly and dependable.
(2)Antioxidative activity of the extract of Eucommia ulmoides leaves To provide theoretical evidences for developing novel natural antioxidants, the extract of Eucommia ulmoides leaves were assessed for their antioxidative effects on hydroxyl, superoxide anion, DPPH free radical and hydrogen peroxide by chemiluminescence and UV spectrophotometry. As a result, the extract has the best ability to scavenge hydroxyl, superoxide anion, DPPH free radical and hydrogen peroxide at low density. Therefore, the foundation has been developed for natural antioxidant with nutritional and healthy functions of aucubin from Eucommia ulmoides leaves.
(3)Preparation of aucubin
In this experiment, the aucubin was separated and purified by macroporous absorption resin, silica gel column chromatography and preparative thin layer chromatography from the extract of Eucommia ulmoides leaves. The results demonstrated that the content of aucubin was successively increased from 4.42% to 22.11%, then 52.83% and 92.02% by isolation of macroporous absorption resin, silica gel column chromatography and preparative thin layer chromatography respectively.
(4)Extraction and separation of aucubin from the leave of Eucommia ulmoides by aqueous two-phase system
Extract and separate aucubin from the leaves of Eucommia ulmoides in aqueous two-phase system(ATPS) formed by macromolecular compound PEG4000 and dextran 40000 was investigated in a preliminary stage. The results showed that the best ATPS was composed of 11% PEG4000 and 8% D40, and addition of sample was 8 g at 60℃, pH value was 7.The highest extraction rate could reach to 66.32% after three times.The content of aucubin in the extract from liquid of aqueous two-phase extraction could rised up to 48.67% from 8.750%. This method has the advantages of mild conditions, solvent free residues and less time-consumed, and the obvious efficiency in single stage separation and purification of aucubin.
(1)杜仲叶中抗氧化活性成分检测方法的建立
建立了杜仲叶提取物中总黄酮、绿原酸和桃叶珊瑚甙的紫外可见分光光度和薄层色谱检测方法,并运用高效液相色谱技术建立了同时测定杜仲叶中桃叶珊瑚甙、京尼平甙酸和绿原酸的分析方法,并对三种检测方法进行了比较。结果表明高效液相色谱法最为准确可靠。
(2)杜仲叶提取物抗氧化活性研究
运用简便快捷、结果准确的流动注射化学发光法和简单易行的分光光度法对杜仲叶提取物清除羟基自由基(·OH)、超氧阴离子自由基(O2-·)、过氧化氢(H2O2)和DPPH自由基(·DPPH)的效果进行了系统的研究。结果表明杜仲叶粗提物在低浓度下对羟基自由基和超氧阴离子自由基具有很好的清除作用,对过氧化氢和DPPH自由基也有较好的清除能力。为杜仲叶开发成为具有营养保健功能的天然抗氧化剂奠定了理论基础。
(3)桃叶珊瑚甙的分离纯化研究
通过大孔吸附树脂富集后杜仲叶提取物中桃叶珊瑚甙的含量由粗提液中的4.42%上升至22.11%;通过硅胶G柱层析进一步分离纯化,桃叶珊瑚甙的含量提升到52.83%;最后经制备TLC色谱两次纯化,可得到含量为92.02%的桃叶珊瑚甙产品。
(4)双水相体系分离纯化桃叶珊瑚甙新工艺探讨
运用聚乙二醇(PEG)/葡聚糖40000(D40)双水相体系,对杜仲叶提取液中桃叶珊瑚甙的分离纯化工艺进行了初步探讨。结果表明:由质量分数为11%PEG4000和8%D40组成的双水相体系,加入样品溶液8g,在pH值为7,温度60℃时,D40相萃取率达到66.32%,桃叶珊瑚甙纯度由粗提物中的8.750%提高到48.67%。该方法具有操作条件温和、无溶剂残留、耗时少、对桃叶珊瑚甙的单级分离提纯效率高等优点。
There are many kinds of effective components in Eucommia ulmoides leaves, such as chlorogenic acid, aucubin, geniposidic acid and flavonoids etc. Aucubin is one kind of iridoid compounds and it is richly contained in Eucommia ulmoides leaves. In this dissertation, the detection methods of antioxidative components, isolation and purification of aucubin and antioxidative activity of extract from Eucommia ulmoides leaves were investigated and compared. This work is useful to the development of Eucommia ulmoides leaves. The main contents list as fallow:
(1)Establishment of the detection methods of antioxidative components in Eucommia ulmoides leaves
The determination of chlorogenic acid, aucubin and total flavonoids of Eucommia ulmoides leaves by UV spectrophotometry and TLC, and the simultaneous determination of aucubin,geniposide acid and chlorogenic acid by HPLC were established and compared. The results showed that HPLC was exactly and dependable.
(2)Antioxidative activity of the extract of Eucommia ulmoides leaves To provide theoretical evidences for developing novel natural antioxidants, the extract of Eucommia ulmoides leaves were assessed for their antioxidative effects on hydroxyl, superoxide anion, DPPH free radical and hydrogen peroxide by chemiluminescence and UV spectrophotometry. As a result, the extract has the best ability to scavenge hydroxyl, superoxide anion, DPPH free radical and hydrogen peroxide at low density. Therefore, the foundation has been developed for natural antioxidant with nutritional and healthy functions of aucubin from Eucommia ulmoides leaves.
(3)Preparation of aucubin
In this experiment, the aucubin was separated and purified by macroporous absorption resin, silica gel column chromatography and preparative thin layer chromatography from the extract of Eucommia ulmoides leaves. The results demonstrated that the content of aucubin was successively increased from 4.42% to 22.11%, then 52.83% and 92.02% by isolation of macroporous absorption resin, silica gel column chromatography and preparative thin layer chromatography respectively.
(4)Extraction and separation of aucubin from the leave of Eucommia ulmoides by aqueous two-phase system
Extract and separate aucubin from the leaves of Eucommia ulmoides in aqueous two-phase system(ATPS) formed by macromolecular compound PEG4000 and dextran 40000 was investigated in a preliminary stage. The results showed that the best ATPS was composed of 11% PEG4000 and 8% D40, and addition of sample was 8 g at 60℃, pH value was 7.The highest extraction rate could reach to 66.32% after three times.The content of aucubin in the extract from liquid of aqueous two-phase extraction could rised up to 48.67% from 8.750%. This method has the advantages of mild conditions, solvent free residues and less time-consumed, and the obvious efficiency in single stage separation and purification of aucubin.
引文
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