【作者】 甘瑾；

【导师】 冯颖；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2013， 博士

【摘要】 玛咖(Lepidium meyenii Walp.)是生长在秘鲁海拔4000～4500米的安第斯山脉的一种十字花科植物，具有提高生育能力、抗疲劳、改善性功能、抗氧化、抑制前列腺增生、缓解更年期综合症、增强免疫力等多种功效，因此受到世界各国的广泛关注。研究者认为玛咖的多种保健功能与其含有的生物碱、芥子油苷、甾醇、多酚等次生代谢物质有密切关系，而抗氧化活性也是产生这些功效的基础，因此玛咖抗氧化功能及活性物质基础的研究对玛咖其他生物功能机制的揭示、功能产品的开发具有重要的意义。本论文以云南种植的紫、黄、白3种色型的玛咖为原料，对生物碱、芥子油苷、甾醇、多酚等次生代谢物质的组成及含量进行分析比较，建立和确定了玛咖中次生代谢物质的分析检测方法；通过体外抗氧化活性的检测分析，对抗氧化成分提取及活性进行研究；采用生物活性追踪的方法，筛选出活性较强部分；利用硅胶柱层析的方法将活性较强部位进一步分离为不同的组分，通过各组分次生代谢物质含量和体外抗氧化活性的相关性研究揭示玛咖抗氧化功效的物质基础。并利用对氧化损伤细胞的保护作用进一步验证抗氧化活性组分的抗氧化能力。主要研究结果如下：1、玛咖次生代谢物质组成与含量分析（1）总生物碱：通过对玛咖总生物碱含量比色法与滴定法测定结果的比较，确定酸性染料比色法是测定玛咖总生物碱含量较理想的方法，确定了比色测定的方法与条件：测定波长为414nm，溴麝香草酚蓝显色剂的ph值为7.0，用量为5.0mL。比色法的测定结果显示紫色、黄色、白色玛咖的总生物碱含量分别为4.4078、2.2241、2.9193mg/g，3种颜色的差异达到了极显著水平（P＜0.01），其中紫色玛咖的总生物碱含量最大，与相关文献测定的来源于秘鲁的玛咖干粉的总生物碱含量相近。（2）芥子油苷：采用LC-ESI/MS对玛咖中芥子油苷组成进行的分析和研究表明，3种颜色玛咖中含有相同组分的芥子油苷，即苄基芥子油苷（Benzyl glucosinolate）和甲氧基芐基芥子油苷（Methoxybenzyl glucosinolate），组分中的苄基芥子油苷含量较高，甲氧基芐基芥子油苷含量相对较低，结果与相关文献报道的秘鲁产原料一致。紫、黄、白玛咖鲜样中的芥子油苷总量分别是50.14、46.35、84.57μmol/g，存在显著差异(P＜0.01)，其中白色最高。干燥样品的含量显著低于鲜样（P＜0.01），而不同的干燥方法之间也存在显著差异（P＜0.01），表明不同处理条件对玛咖芥子油苷的水解具有较大影响。（3）甾醇：采用GC-MS对玛咖的甾醇类化合物组成及含量进行分析，研究表明3种色型玛咖中含有相同组分的甾醇类化合物，即β-谷甾醇（β-Sitosterol）和菜油甾醇（Campesterol）。组分中的β-谷甾醇含量较高，菜油甾醇含量相对较低。白、紫、黄玛咖中的甾醇总量分别是36.60、32.38、27.97mg/100g，存在显著差异（P＜0.01），其中白色含量最高。结果显示甾醇的主要组成与秘鲁产玛咖相同。（4）多酚：采用Folin-酚测定法对玛咖中的总多酚含量进行分析，结果表明云南种植的紫、黄、白3种颜色玛咖的多酚含量分别为5.08、4.05、3.57mg/g，3种颜色之间存在显著差异（P＜0.01），其中紫色玛咖含量最高。2、玛咖抗氧化活性成分提取及活性部位研究采用超氧阴离子自由基的清除能力、羟自由基清除能力、还原抗氧化能力及抑制脂质氧化能力等体外抗氧化评价方法，对甲醇和不同浓度乙醇的玛咖提取物的抗氧化活性进行分析，确定了70%乙醇溶液为玛咖抗氧化活性成分最佳提取溶剂，试验结果也表明了云南种植的紫、黄、白3种颜色玛咖具有明显的体外抗氧化活性，抗氧化活性大小顺序为紫色＞黄色＞白色。活性追踪方法显示黄色和紫色玛咖抗氧化活性较强部位为氯仿极性部位。采用H202诱导损伤细胞保护作用的评价方法对黄色和紫色玛咖氯仿部分及氯仿部分的柱分离组分的抗氧化活性进行验证，结果显示柱分离组分对氧化损伤细胞有明显的保护作用，进一步验证了玛咖的抗氧化活性。3、玛咖抗氧化活性物质基础的研究利用相关性分析及多元线性回归的统计方法研究玛咖柱分离组分抗氧化活性与次生代谢物的相关性，研究结果显示，黄色玛咖中多酚含量和生物碱含量与还原抗氧化能力、羟自由基清除能力及脂质氧化抑制率都具有极显著的线性相关（P＜0.01），并且显示抗氧化功效是多酚和生物碱协同作用的结果；紫色玛咖中多酚含量和生物碱含量与还原抗氧化能力呈显著的线性相关（P＜0.01），研究结果揭示了玛咖的主要次生代谢物中多酚和生物碱是玛咖抗氧化能力的主要物质基础。更多还原

【Abstract】 Maca （Lepidium meyeni） is a plant of the Brassicaceae family growing originally at4000～4500meters altitude in the Andean region of Peru．Maca has attracted attention frommany countries around the world, as it has multiple biological functions such asfertility-enhancing, anti-fatigue, improving sexual function, antioxidant, inhibition prostatehyperplasia, reducing menopause syndrome and increasing immunity function. Researchersrecognized these functions were correlative with maca secondary metabolites includingalkaloids,polyphenol, glucosinolates and sterol, and several functions were also attributable toantioxidant. Therefore studies on antioxidant and active substance basis are important forrevealing reactive mechanism and development of functional products.In this study, HPLC, LC-ESI/MS and colorimetry have been applied to analyzecompositions and contents of alkaloids,polyphenol, glucosinolates and sterol in three colortypes of maca cultivated in Yunnan, meanwhile the methods analyzing secondary metaboliteshave been established. In addition, the crude extract solvent was confirmed by antioxidantactivity evaluation in vitro. Then the crude extracts of maca were separated into severalfractions of different polarities based on the bioassay guided method. The fractions whichshowed best antioxidative activity were selected to separate into six constituents by silica gelcolumn chromatography. The antioxidative substance basis was revealed by relativity analysisbetween antioxidative activity and the content of secondary metabolites of six constituents.Further, the antioxidative capacities of constituents were confirmed by resisting injury toH₂O₂-induced damage cells. The main results are as follow:1. Analysis of secondary metabolites compositions and contents in macaThe acidic dye colorimetry is more appropriate for the determination of total alkaloids inmaca cultivated in Yunnan, The conditions of method are: determination wave414nm,dibromothymol-sulfonphthalein dye pH7.0, dye volume5.0mL. The contents of total alkaloids in purple, yellow and white maca were4.4078,2.2241and2.9193mg/g, respectively, thedifference between3color types maca is significant （P＜0.01）. The content of total alkaloidsin purple maca is the highest. which is close to the content of total alkaloids in maca grown inPeru.HPLC and LC-ESI/MS were applied to analyze glucosinolate compositions and contentsof3color types maca cultivated in Yunnan. Three color types of maca contain the samecompositions of glucosinolate, which are benzyl glucosinolate and methoxybenzylglucosinolate.The content of benzyl glucosinolate is higher than that of methoxybenzylglucosinolate.The result was consistent with that of sample cultivated in Peru. The totalglucosinolate contents in purple, yellow and white fresh samples were50.14,46.35, and84.57μmol/g, respectively, with a significant difference （P＜0.01）. Among them, the content ofglucosinolate in white sample is highest. The contents of dried samples were much lower thanthat in fresh samples. The contents of glucosinolate in sample processed by two dry techniquewere different significantly（P＜0.01） too, which showed that effect on glucosinolatehydrolyzed of processing condition was obvious.The compositions and contents of sterols in three color types of maca cultivated in Yunnanwere analyzed by GC-MS．The result showed that all the three types of maca containedβ-sitosterol and campesterol． The content of β-sitosterol was higher than that ofcampesterol．The total sterol contents in white，purple and yellow samples were36.60，32.38，27.97mg/100g，respectively．Among them，the total sterol content in white sample was thehighest．The difference in contents was significant among the three types of sample（P＜0.01）．The composition was consistent with that of sample cultivated in Peru.The contents of total polyphenol in three color types of macacultivated in Yunnan wereanalyzed by Folin-Ciocalteus method. Result indicated that the total polyphenol contents inpurple, yellow and white were35.08,4.05,3.57mg/g，respectively, with a significantdifference （P＜0.01）, the content of polyphneol in purple maca is highest.2. Study on extraction of antioxidative components and the strongest antioxidativefraction The antioxidative activities of components extracted by methanol and ethanol withvarious concentration from maca were determined by evaluating ferric reducing antioxidantpower, scavenging capacity of superoxide anion and hydroxyl free radical, inhibition of lipidoxidation. Result showed70%ethanol was appropriate extract solvent for anitoxidativecomponents in maca, The results indicated that ethanol extracts of3color types of macacultivated in Yunnan all had antioxidative activities, among them, the activity of purple macawas highest,and the activity of yellow maca was higher than that of white maca. It was foundthat the chloroform fractions in purple and yellow maca had the strongest antioxidativecapacities by using the bioassay guided method. The antioxidative activities of chloroformfractions and activity constituents separated from chloroform fractions were also confirmed byresisting injury to H₂O₂-induced damage cells, the protective effects of constituents separatedfrom chloroform fractions by silica gel column chromatography were visible in this test.3. Study on antioxidant substance basis of macaThe relativities were studied between antioxidative activity and the content of secondarymetabolites of constituent separated from chloroform fractions by correlation and linearregression statistic analysis.Result indicated the contents of polyphenol and alkaloids hadsignificant linear correlation with reducing antioxidative capacity, scavenging capacity ofhydroxyl free radical and inhibition of lipid oxidation （P＜0.01） in yellow maca. The resultalso showed that antioxidant capacities resulted from synergic action of polyphenol andalkaloids. In purple maca the contents of polyphenol and alkaloids had significant linearcorrelation with reducingl antioxidative capacity （P＜0.01）. The research reveals thatpolyphenol and alkaloids are the most important substance basis for antioxidation. of maca.更多还原