酸枣仁抗氧化活性物质的提取、分离及其活性的研究
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摘要
酸枣仁是我国常见的中药材之一,其有效成分具有较高的抗氧化活性,可以开发成为具有抗氧化作用的药物或保健品。本文系统研究酸枣仁提取物有效成分的抗氧化活性,不但以活性追踪法找到酸枣仁中抗氧化活性最好的组分并进行谱图鉴定,还进一步研究各活性组分抗氧化活性的协同作用,为以后以酸枣仁为原料生产抗氧化、抗衰老功能性食品药品提供参考与依据。
本实验研究了不同方法分离得到的酸枣仁油的抗氧化活性,得出酸枣仁油中含有大量不饱和脂肪酸,这一含量决定了其抗氧化活性的强弱。实验证明,超临界CO2萃取法与微波辅助萃取法得到的酸枣仁油的抗氧化活性远远优于索氏萃取法得到的酸枣仁油,并且与人工合成抗氧化剂BHT活性相当。
建立了酸枣仁中抗氧化活性物质的提取工艺路线。通过单因素法、正交实验法、响应面设计法,以清除DPPH自由基的能力、清除羟自由基的能力、总抗氧化能力、总还原能力为检测指标,最终确定最优提取路线。该提取条件是：石油醚脱脂处理后的酸枣仁粉末(40目)在59.1%的乙醇溶液中(1：10)进行回流提取4.5小时(76.5℃)提取一次,收集提取液,减压浓缩,得粗品。该条件下得到的酸枣仁粗品抗氧化活性最好。
确定了有机溶剂萃取后乙酸乙酯相为活性最好的部分,并研究了聚酰胺树脂分离纯化酸枣仁乙酸乙酯萃取相的条件。实验证明,分离条件为上样量5mg；洗脱体系为0-27min内,30%甲醇等度洗脱；27-37min内,30%-80%甲醇线性梯度洗脱；37-75min内,80%甲醇等度洗脱；流速5mL/min时,分离出分离物B为抗氧化活性最好的部分。
研究了C18填料中低压制备分离聚酰胺分离物B的条件。实验证明,分离条件为洗脱体系为0-60min内由甲醇：水=40：60线性变化为甲醇：水=50：50；流速为5mL/min时,分离出分离物B2为酸枣仁中抗氧化活性最好的组分。
通过质谱(MS)和核磁共振(NMR)鉴定了分离出的活性物质的分子量和结构,鉴定出聚酰胺分离物A为酸李碱,C18填料分离物B1为斯皮诺素,C18填料分离物B2为6'''阿魏酰斯皮诺素。
研究了提取分离纯化过程中不同操作单元所得物质的抗氧化活性比较,发现随着各操作单元的逐渐增加分离物质的抗氧化活性也逐步增加,最后单品6'''阿魏酰斯皮诺素活性最好。
研究了酸枣仁提取物：酸李碱、斯皮诺素与6'''阿魏酰斯皮诺素抗氧化活性之间的相互关系。实验发现不同配比混合后,酸李碱、斯皮诺素、6'''阿魏酰斯皮诺素在清除DPPH自由基的反应中呈现拮抗作用,在总抗氧化能力的反应中为拮抗作用,而在金属离子螯合能力的反应中为协同作用的相互关系。
Ziziphus jujube Mill. var. spinosa is a common Chinese medicinal plant, the effective compounds of Ziziphus jujube which have high antioxidant activity can be developed into natural antioxidants or health products. This dissertation studied the antioxidant activity of effective compounds of Ziziphus jujube systematically. The compounds whose antioxidant activities are highest have been found by bioassay-guided method and identified. We also study the synergy of the isolated compounds.
The antioxidant activity of Ziziphus jujube oils isolated by different methods has been studied. The oils contain plenty of unsaturated fatty acid, and the antioxidant activities of oils isolated by supercritical CO2 extraction and microwave assisted extraction are as well as that of synthetic antioxidant BHT, but much higher than that of the oil isolated by Soxhlet extraction.
The condition for extracting of antioxidant compounds of Ziziphus jujube has been established. After study by single factor experiment, orthogonal experimental and response surface design, the optimum conditions are determined as follows:adding the powder of Ziziphus jujube defatted by petroleum ether into 10 times ethanol by volume radio of 59.1%, circulation refluxing one time at the temperature of 76.5℃for 4.5 hours, collecting the extract and concentrating by reduced pressure to obtain the crude product whose antioxidant activity is highest.
Ethyl acetate phase has been determined to be the part whose antioxidant activity is highest, and the condition for isolating of antioxidant compounds by polyamide resins has been established. The best condition is:feeding concen-tration is 5mg/mL, the flow rate of elution is 5mL/min. A gradient is formed by varying the proportion of water (A) and MeOH (B). The elution system was:0-27 min, isocratic 30% B; 27-37 min, linear gradient 30-80% B; 37-75 min, isocratic 80% B, and the fraction B has the highest antioxidant activity.
The condition for isolating of antioxidant compounds by low-pressure preparation C18 column chromatography has been optimized. The best condition is:linear elution solution is MeOH/H2O=4:6, v/v for 60 minutes, the flow rate of elution is 5mL/min, and the fraction B2 has highest antioxidant activity.
The molecular weights and structures of the active fraction A, B1, and B2 are analyzed by the mass spectrometry (MS) and nuclear magnetic resonance (NMR), and the result is that A is zizyphusine, B1 is spi- nosin, and B2 is 6"'-feruloylspinosin.
The antioxidant activities of different products from operating units of extraction, separation, and purification processes, and different compounds of Ziziphus jujube have been tested.6"'-feruloylspinosin is proved to have highest antioxidant activity.
The interaction among zizyphusine, spinosin, and 6"'-feruloylspinosin has been studied. The results have shown the interactions among zizyphusine, spinosin and 6"'-feruloylspinosin were synergistic and additive effects by using metal ion chelating method, and antagonistic effects by both T-AOC and DPPH methods.
本实验研究了不同方法分离得到的酸枣仁油的抗氧化活性,得出酸枣仁油中含有大量不饱和脂肪酸,这一含量决定了其抗氧化活性的强弱。实验证明,超临界CO2萃取法与微波辅助萃取法得到的酸枣仁油的抗氧化活性远远优于索氏萃取法得到的酸枣仁油,并且与人工合成抗氧化剂BHT活性相当。
建立了酸枣仁中抗氧化活性物质的提取工艺路线。通过单因素法、正交实验法、响应面设计法,以清除DPPH自由基的能力、清除羟自由基的能力、总抗氧化能力、总还原能力为检测指标,最终确定最优提取路线。该提取条件是：石油醚脱脂处理后的酸枣仁粉末(40目)在59.1%的乙醇溶液中(1：10)进行回流提取4.5小时(76.5℃)提取一次,收集提取液,减压浓缩,得粗品。该条件下得到的酸枣仁粗品抗氧化活性最好。
确定了有机溶剂萃取后乙酸乙酯相为活性最好的部分,并研究了聚酰胺树脂分离纯化酸枣仁乙酸乙酯萃取相的条件。实验证明,分离条件为上样量5mg；洗脱体系为0-27min内,30%甲醇等度洗脱；27-37min内,30%-80%甲醇线性梯度洗脱；37-75min内,80%甲醇等度洗脱；流速5mL/min时,分离出分离物B为抗氧化活性最好的部分。
研究了C18填料中低压制备分离聚酰胺分离物B的条件。实验证明,分离条件为洗脱体系为0-60min内由甲醇：水=40：60线性变化为甲醇：水=50：50；流速为5mL/min时,分离出分离物B2为酸枣仁中抗氧化活性最好的组分。
通过质谱(MS)和核磁共振(NMR)鉴定了分离出的活性物质的分子量和结构,鉴定出聚酰胺分离物A为酸李碱,C18填料分离物B1为斯皮诺素,C18填料分离物B2为6'''阿魏酰斯皮诺素。
研究了提取分离纯化过程中不同操作单元所得物质的抗氧化活性比较,发现随着各操作单元的逐渐增加分离物质的抗氧化活性也逐步增加,最后单品6'''阿魏酰斯皮诺素活性最好。
研究了酸枣仁提取物：酸李碱、斯皮诺素与6'''阿魏酰斯皮诺素抗氧化活性之间的相互关系。实验发现不同配比混合后,酸李碱、斯皮诺素、6'''阿魏酰斯皮诺素在清除DPPH自由基的反应中呈现拮抗作用,在总抗氧化能力的反应中为拮抗作用,而在金属离子螯合能力的反应中为协同作用的相互关系。
Ziziphus jujube Mill. var. spinosa is a common Chinese medicinal plant, the effective compounds of Ziziphus jujube which have high antioxidant activity can be developed into natural antioxidants or health products. This dissertation studied the antioxidant activity of effective compounds of Ziziphus jujube systematically. The compounds whose antioxidant activities are highest have been found by bioassay-guided method and identified. We also study the synergy of the isolated compounds.
The antioxidant activity of Ziziphus jujube oils isolated by different methods has been studied. The oils contain plenty of unsaturated fatty acid, and the antioxidant activities of oils isolated by supercritical CO2 extraction and microwave assisted extraction are as well as that of synthetic antioxidant BHT, but much higher than that of the oil isolated by Soxhlet extraction.
The condition for extracting of antioxidant compounds of Ziziphus jujube has been established. After study by single factor experiment, orthogonal experimental and response surface design, the optimum conditions are determined as follows:adding the powder of Ziziphus jujube defatted by petroleum ether into 10 times ethanol by volume radio of 59.1%, circulation refluxing one time at the temperature of 76.5℃for 4.5 hours, collecting the extract and concentrating by reduced pressure to obtain the crude product whose antioxidant activity is highest.
Ethyl acetate phase has been determined to be the part whose antioxidant activity is highest, and the condition for isolating of antioxidant compounds by polyamide resins has been established. The best condition is:feeding concen-tration is 5mg/mL, the flow rate of elution is 5mL/min. A gradient is formed by varying the proportion of water (A) and MeOH (B). The elution system was:0-27 min, isocratic 30% B; 27-37 min, linear gradient 30-80% B; 37-75 min, isocratic 80% B, and the fraction B has the highest antioxidant activity.
The condition for isolating of antioxidant compounds by low-pressure preparation C18 column chromatography has been optimized. The best condition is:linear elution solution is MeOH/H2O=4:6, v/v for 60 minutes, the flow rate of elution is 5mL/min, and the fraction B2 has highest antioxidant activity.
The molecular weights and structures of the active fraction A, B1, and B2 are analyzed by the mass spectrometry (MS) and nuclear magnetic resonance (NMR), and the result is that A is zizyphusine, B1 is spi- nosin, and B2 is 6"'-feruloylspinosin.
The antioxidant activities of different products from operating units of extraction, separation, and purification processes, and different compounds of Ziziphus jujube have been tested.6"'-feruloylspinosin is proved to have highest antioxidant activity.
The interaction among zizyphusine, spinosin, and 6"'-feruloylspinosin has been studied. The results have shown the interactions among zizyphusine, spinosin and 6"'-feruloylspinosin were synergistic and additive effects by using metal ion chelating method, and antagonistic effects by both T-AOC and DPPH methods.
引文
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[2]陈晖.野生酸枣的利用及人工栽培技术[J].中国农村小康科技,2006,4：34-35
[3]姚煜.酸枣仁提取工艺及成分研究[D].北京：首都师范大学,2005
[4]Sun Yanfang, Liang Zongsuo, Shan Changjuan, etc. Comprehensive evaluation of natural antioxidants and antioxidant potentials in Ziziphus jujube Mill. var. spinosa (Bunge) Hu ex H. F. Chou fruits based on geographical origin by TOPSIS method[J]. Food Chemistry,2011,124:1612-1619
[5]史琪荣,周耘,周萍等.中药酸枣仁的研究概况[J].药学实践杂志,2004,22(2)：94-98
[6]郭胜明,范晓雯,何健伟.酸枣仁总黄酮的中枢抑制作用[J].中药材,1998,21(11)：578-579
[7]Jiang Jianguo, Huang Xiaojuan, Chen Jian, etc. Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from Semen Ziziphus jujube [J]. Natural Product Research,2007,21(4):310-320
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