【作者】 潘为高；

【导师】 刘世贵； 高平；

【作者基本信息】 四川大学 ， 遗传学， 2007， 博士

【摘要】 本文系统地研究了藏波罗花的几种提取物体内外抗氧化、抗衰老活性，分离纯化出了两种高效活性物质，并进行了活性物质的结构鉴定和解析；通过对活性物质的药理研究，初步探索了藏波罗花抗氧化、抗衰老的机理。本研究揭示了藏波罗花抗氧化抗衰老的物质基础，有利于藏波罗花药用价值的深度开发和利用。藏波罗花的五种提取物分别为：根的干粉用80％乙醇提取，浓缩干燥得IYS1，得率36.8％；80％乙醇提取过后的残渣，再用水提取，浓缩干燥得IYS2，得率16.9％；根的干粉直接用水提取，浓缩干燥得IYS3，得率49.7％；将IYS1用80％乙醇配制成0.5mg／ml浓度溶液，加适量的乙酸乙酯，发生沉淀，上层浓缩干燥得IYS4，得率7.73％，下层浓缩干燥得IYS5，得率29.0％。利用三个离体指标测定五种提取物的体外抗氧化活性，发现体外活性强弱顺序依次为IYS4＞IYS1＞IYS3＞IYS5＞IYS2。选用活性最强的IYS4作为分离纯化的样品，经正相硅胶柱层析、反相C18柱层析、反相半制备型高效液相，首次从藏波罗花中分离到两个具有优良抗氧化活性的化合物A和B。通过低分辨ESI-MS得出化合物A和B的分子量均为624，进一步通过高分辨HRESI-MS得出它们的分子式均为C₂₉H₃₆O₁₅。通过1D-NMR(¹H NMR，¹³C NMR，DEPT)和2D-NMR（¹H-¹H COSY，HSQC，HMBC，NOESY和TOCSY）解析两个化合物的结构；并进行了化合物A和B水解单糖硅烷化衍生物的GC-MS鉴定，得出化合物A和B的甲基五碳糖均为L-鼠李糖，六碳糖均为D-葡萄糖。化合物A的结构最后鉴定为β-D-Glucopyranoside，2-（3，4-dihydroxyphenyl）ethyl3-0-（6-deoxy-α-L-mannopyranosyl）-，4-[（2E）-3-（3，4-dihydroxyphenyl）-2-propenoate]（9CI）．。化合物B的结构最后鉴定为：β-D-Glucopyranoside，2-（3，4-dihydroxyphenyl）ethyl-3-0-（6-deoxy-α-L-mannopyranosyl）-，6-[（2E）-3-（3，4-dihydroxyphenyl）-2-propenoate]（9CI）．。通过与CA库中所有具有C₂₉H₃₆O₁₅这一分子式，并且结构相似的化合物进行对比，化合物B为一个新的化合物。化合物A和B是两个同分异构体，具有完全相同的分子式C₂₉H₃₆O₁₅。它们的¹H NMR，¹³C NMR，DEPT，¹H-¹H COSY，HSQC，HMBC，NOESY和TOCSY谱也极其相似。化合物A和B的UV／PDA谱基本相同，均在198nm和330nm处具有强吸收。在相同色谱条件下，化合物B要比A的保留时间长。化合物A和B分别均含有多个酚羟基，这可能是它们具有抗氧化活性的结构根源。化合物A首次报道存在于藏波罗花根中（43.1mg／g），是一个具有多种生物活性、用途广泛的化合物。分别利用纯品A和B作外标，采用分析型HPLC测定五种提取物中的这两种化合物的含量，并由IYS1中的含量推断藏波罗花干根中化合物A和B的含量。结果如下：化合物A含量的高低次序为IYS4（380.6mg／g）＞IYS1（113.6mg／g）＞IYS3（40.4mg／g）＞IYS5（28.7mg／g）＞IYS2（7.5mg／g）；化合物B含量的高低次序为IYS4（76.1mg／g）＞IYS1（18.9mg／g）＞IYS3（5.1mg／g）＞IYS5（3.2mg／g）＞IYS2（0.7mg／g）；化合物A+B总含量的高低次序为IYS4（456.7mg／g）＞IYS1（132.5mg／g）＞IYS3（45.5mg／g）＞IYS5（31.9mg／g）＞IYS2（8.2mg／g）。藏波罗花干根中，化合物A和B分别为43.1mg／g和7.1mg／g，可见A的含量远高于化合物B的含量。利用Folin-Ciocalteu试剂法，测定五种提取物中多酚含量，结果如下：多酚含量的高低次序为IYS4（154.85±2.73mg／g）＞IYS1（47.94±0.76 mg／g）＞IYS3（26.18±0.76mg／g）＞IYS5（20.88±0.76 mg／g）＞IYS2（12.21±1.00mg／g）。化合物A和B的多酚含量分别为388.68±0.56mg／g和353.24±3.55mg／g。五种提取物中多酚含量的高低顺序与化合物A含量高低顺序、化合物B含量高低顺序、化合物A和B的总含量高低顺序完全一致，表明多酚含量与化合物A、B或A和B的总含量成绝对正相关关系。采用L16₄₅正交表进行正交工艺设计，研究藏波罗花的最佳提取工艺条件。结果：从得率×多酚极差来看，溶剂用量对得率×多酚影响最大（672.354），其他几个因素影响其次。以得率×多酚作为实验结果进行分析，从效应曲线图可见，最佳工艺条件为：温度60℃，时间为6小时，溶剂浓度为50—65％乙醇，溶剂用量为9—12倍（W／V），搅拌速度400rpm。采用超氧阴离子(O₂^·-)清除实验、羟自由基HFR（^·OH）的清除实验和小鼠肝组织过氧化脂质（LPO）抑制实验进行五种提取物和化合物A和B的体外抗氧化活性测定。超氧阴离子(O₂^·-)清除活性：化合物A（49.3％）＞化合物B（45.4％）＞Vc（34.5％）＞IYS4（16.0％）＞IYS1（5.9％）＞IYS3（1.8％）＞IYS2（0.9％）＞IYS5（0.6％）。羟自由基HFR（^·OH）清除活性：苯甲酸（345min）＞化合物A（330min）=化合物B（330min）＞IYS4（300min）＞IYS1（120min）=Vc（120min）＞IYS3（90min）＞IYS5（60min）＞IYS2（60min）＞空白（15min）。小鼠肝组织过氧化脂质（LPO）抑制活性：化合物A（-97.2％）=化合物B（-97.1％）≈IYS4（-97.4％）≈IYS1（-96.1％）＞IYS3（-92.2％）≈IYS5（-90.6％）＞BHT（-57.6％）≈IYS2（-53.8％）＞Vc（206.3％）。提取物中多酚含量、化合物A和B总含量越高，超氧阴离子(O₂^·-)和羟自由基HFR（^·OH）的清除能力越强，反之，清除能力越弱；二者完全成正相关关系。Vc的超氧阴离子(O₂^·-)清除能力、羟自由基HFR（^·OH）的清除能力要比化合物A或B差。醇提物（IYS1）或醇提物的极性较弱部分（IYS4）要比亲水性的提取物（IYS2、IYS3、IYS5）清除超氧阴离子(O₂^·-)、羟自由基HFR（^·OH）的能力强。Vc有强烈的促LPO生成的作用。只要微量的化合物A或B，就能强烈保护肝组织免受氧化作用，这是其它药物（例如BHT、Vc）所无法比拟的。藏波罗花醇提物抗氧化性能优于水提物，醇提物能明显保护肝组织免受氧化作用。化合物A和B高浓度（1.0000 mg／ml）和低浓度（0.0625 mg／ml）对小鼠肝组织过氧化脂质（LPO）抑制率较低；而中浓度（0.2500 mg／ml）抑制率最高。IYS2、IYS3、IYS5的多酚含量和化合物A和B总含量低，对雌雄果蝇寿命延长作用不明显；IYS1和IYS4多酚含量和化合物A和B总含量高，对雌雄果蝇寿命具有非常明显的延长作用。表明多酚含量及化合物A和B的总含量与果蝇延寿率成正相关。有效成分（多酚含量、化合物A和B总含量）达到一定剂量后，继续增加剂量，并不会使延寿率继续增加，有效成分的延寿作用会出现饱和状态。适当剂量的IYS4具有提高SOD活性、CAT活性、抑制MDA生成的作用，剂量过高反而效果下降。GSH-Px活性随IYS4给药剂量的提高而明显提高。Vc无提高GSH-Px活性的作用并会促进MDA在体内的生成。果蝇体内抗氧化抗衰老活性也与藏波罗花提取物中化合物A和B的总含量直接成正相关关系（或者说化合物A和B具有果蝇体内抗氧化活性）。安全性实验结果显示，藏波罗花80％乙醇提取物IYS1对雌雄小鼠无经口急性毒性；小鼠骨髓嗜多染红细胞微核实验和小鼠精子畸形实验结果均为阴性，未见其有遗传毒性。更多还原

【Abstract】 Antioxidative and anti-aging activities in vivo and in vitro of the extracts of the Tibet-herb Incarvillea younghusbandii Sprague were studied systematically here. Two compounds with antioxidative and anti-aging activities were isolated and identificated. Our studies not only demonstrated the practical application value of Incarvillea younghusbandii Sprague but. also showed some theoretical values to the future research.The five extracts from the roots of Incarvillea younghusbandii Sprague were as follows: IYS1 （extract by ethanol, yield 36.8%）, IYS2 （residue extract by water, yield 16.9%）, IYS3 （extract by water directly, yield 49.7%）, IYS4 （supernatant by ethanol from IYS1, yield 7.73%）, IYS5 （sediment by ethanol from IYS1, yield 29.0%）.Evaluate by O’₂^-, "OH scavenging assay and LPO inhibitory assray, the activities of the five extracts were as follows: IYS4＞IYS1＞IYS3＞IYS5＞IYS2. Compound A and B with antioxidative and antiaging effect were first isolated from the roots of Incarvillea younghusbandii Sprague through ethyl acetate precipitation, silica gel column chromatography, reverse-phase C18 column chromatography and reverse-phase semi-preparative HPLC. Compound A and B had the same molecular weight （624） and the same molecular formula (C₂₉H₃₆O₁₅) which were determined by ESI-MS and high resolution ESI-MS, respectively. Compoung A and B were hydrolyzed and their silylated monosaccharide derivatives were analyzed by GC-MS. Compound A and B consisted a L-rhamnose and a D-glucose. Their structures were identified by 1D-, 2D-NMR and GC-MS as: A,β-D-Glucopyranoside,2-（3,4-dihydroxyphenyl）ethyl-3-O-（6-deoxy-a-L-mannopyran osyl）-,4-[（2E）-3-（3,4-dihydroxyphenyl）-2-propenoate]（9CI）; B,β-D-Glucopyranoside,2-（3,4-dihydroxyphenyl）ethyl-3-O-（6-deoxy-a-L-mannopyran osyl）-,6-[（2E）-3-（3,4-dihydroxyphenyl）-2-propenoate]（9CI）. Compound A （CAS registry number, 61276-17-3）, a famous compound with various bioactivities and widely commercial use had been reported in 768 literatures （see Chemical Abstracts, USA）, but its occurrence in the roots of IYS with a high yield of 43.1mg/g of dry roots was reported here for the first time. Compound B （7.1mg/g in the dry roots） was a novel compound and its structure and bioactivity were reported here for the first time. The UV/PDA spectra of compound A was the same as that of compound B with the maximum absorption at 198nm and 330nm. At the same chromatography conditions, retention time of compoung B was longer than that of compoung A.Compound A and B were used as external standards to quantificated of A and B in IYS1, IYS2, IYS3, IYS4 and IYS5 extracts by analysis HPLC. The level were as follows: A, IYS4 （380.6mg/g）＞IYS1 （113.6mg/g）＞IYS3 （40.4mg/g）＞IYS5 （28.7mg/g）＞IYS2 （7.5mg/g） ; B, IYS4 （76.1mg/g）＞IYS1 （18.9mg/g）＞IYS3 （5.tmg/g）＞IYS5 （3.2mg/g）＞IYS2 （0.7mg/g） ; A+B,IYS4 （456.7mg/g）＞IYS1 （132.5mg/g）＞IYS3 （45.5mg/g）＞IYS5 （31.9mg/g）＞IYS2 （8.2mg/g） .The concentration of compound A （43.1mg/g） in the dry roots of IncarviUea younghusbandii Sprague was far high than that of compound B （7.1rag/g）.Result of total phenolic assay were as follows: IYS4 （154.85±2.73rag/g）＞IYS1 （47.94±0.76 mg/g）＞IYS3 （26.18±0.76mg/g）＞IYS5 （20.88±0.76mg/g）＞IYS2 （12.21±1.00 mg/g） . The level of phenolic content in the five extracts were absolutely positive correlation to the level of compound A, or B or A+B. The level of phenolic content of compound A and B were 388.68±0.56 and 353.24±3.55mg/g, respectively.Solvent volume was major factor （range, 672.354） and others were minor factor in yield×phenolic content. See from effect-curve diagram, the best conditions for yield×phenolic content were as follow: extracted at a temperature of 60℃, extracted for 6h, solvent consisted was 50-65% Alcohol in water, solvent volume used for extracting was 9-12 time, stirring speed was 400rpm.In vitro activities, the higher werethe level of phenolic content and the compound A+B, the higher were the scavenging activity of extracts to superoxide radical anion（O’₂^-） and hydroxyl radicals （OH）. Positive correlations existed between the level of phenolic content, or the compound A+B and superoxide radical anion（O’₂^-）, hydroxyl radicals （’OH） scavenging activity. Both superoxide radical anion（O’₂^-） and hydroxyl radicals （’OH） scavenging activity of Vc were inferiort to those of compound A or B. Vc had not inhibited but raised LPO level in vitro. At a low concentration of 0.0625 mg/mg, compound A and B could intensity protected the mice hepatic tissue from oxidation, while others （such as BHT, Vc） had not this property. The protections of compound A and B at the concentration of 0.2500 mg/ml （medium level） were better than those of at the concentration of 1.0000mg/ml （high level） and 0.0625 mg/ml （low level）.With a low level of phenolic content or compound A+B, the prolong lifespan effects in Drosophila melanogaster of IYS2、IYS3、IYS5 were not significant. With a high level of phenolic content or compound A+B, the prolong lifespan effects in Drosophila melanogaster of IYS1、IYS4 were absolutely significant. So positive correlations existed between the level of phenolic content or compound A+B and the prolong lifespan effects in Drosophila melanogaster. However, higher level of phenolic content or compound A+B could not increase the lifespan of Drosophila melanogaster any more. An appropriate dose of IYS4 could increase SOD and CAT activities and inhibit MDA level in Drosophila melanogaster, while a high dose could not. At any dose, Vc could not increase GSH-Px activity or inhibit MDA level in Drosophila melanogaster. At an appropriate dose, the positive correlation between the level of the polyphenol or compound A+B and the life-span extends percent of fruit fly were significant.The oral acute toxicity in mice was actually not toxic. The mouse micronucleus test for IYS1 was negative and the sperm abnormality frequencies were not significantly affected by IYS1.更多还原