摘要
三子叶植物纲原始花被亚纲委陵菜属植物三叶委陵菜（Protentilla freynianaBornm）或是其变种中华三叶委陵菜为多年生草本植物，分布于华东，华中，西南地区的山坡草地，具有清热解毒之功效，民间以其根茎或全草入药。但遗憾的是迄今为止三叶委陵菜尚未被作为一种中药材为中国药典（2000年）或各地地方标准所收录，亦少见国内外对其化学成分以及生物活性的研究报道。本文研究了微波辅助提取三叶委陵菜的方法和工艺，大孔吸附树脂分离纯化三叶委陵菜中黄酮类化合物的最佳条件，采用高效液相色谱-质谱仪分析了三叶委陵菜中黄酮类化合物的成分和含量，从五个方面测定了三叶委陵菜中黄酮类化合物的抗氧化性和清除自由基的能力。
课题对三叶委陵菜的研究包括四个部分：
(1)研究了三叶委陵菜中黄酮类物质的微波提取工艺，采用微波辅助水蒸气蒸馏法提取三叶委陵菜黄酮，单因素试验考察乙醇浓度、微波功率、提取时间、回流温度、料液比对三叶委陵菜黄酮提取率的影响，确定了微波萃取仪提取三叶委陵菜黄酮的最佳工艺条件为：以75%乙醇作溶剂，在料液比为1：25，温度70℃，功率400W的条件下微波提取5min，效果最好，提取率为4.24%。
(2)以三叶委陵菜黄酮的吸附率、解析率为指标,考察了六种大孔吸附树脂对三叶委陵菜中总黄酮的分离纯化性能，筛选出最佳的大孔吸附树脂,同时采用高效液相色谱法进行分析检测表征了分离纯化的效果；分析了原液浓度、pH和树脂用量以及洗脱剂浓度和pH对吸附解析的影响。实验结果表明,大孔吸附树脂D101对三叶委陵菜总黄酮有很好的吸附和解析性能,并确定了合适的吸附解析条件为:吸附液浓度为2.0mg/mL、pH=6、树脂用量与吸附液量比(g.mL-1)为1:30；平衡浓度达0.36mg/mL时,即树脂已达到吸附饱和,饱和吸附量为49.1mg/g干树脂；洗脱剂乙醇浓度为50%，pH=6。
(3)利用了高效液相色谱-串联质谱分析法来分析并测定三叶委陵菜黄酮中的生物活性物质的成分和含量，最终确定了7种主要成分，它们分别是表儿茶素，二苯乙烯苷，牡荆素，金丝桃苷，芦丁，淫羊藿苷和斛皮苷，同时利用外标法确定了其含量分别为73.02mg/g，14.53mg/g，15.05mg/g，12.51mg/g，160.35mg/g，27.10mg/g和144.52mg/g；同时根据相关文献信息推断出了两种成分，其分别为橙皮苷和槲皮素-3-芸香糖鼠李糖苷。
(4)从总体抗氧化能力、对Fe~(2+)诱发卵黄脂蛋白PUFA过氧化体系的抑制能力、清除羟基自由基能力、清除超氧阴离子自由基能力、清除DPPH自由基能力五个方面评价了四种委陵菜中黄酮类化合物的活性，并与其他的合成抗氧化剂Vc和PG进行了比较。实验结果表明，在一定浓度范围内，四种委陵菜中黄酮类化合物在上述前四个方面的性能有着固定的顺序：三叶（福建）>三叶（河北）>西南（河北）>西南（福建），但在清除DPPH自由基方面稍有不同；与合成抗氧化剂Vc和PG相比其清除DPPH自由基的能力基本相同。
Protentilla freyniana Bornm is perennial herbs, located on the grasslands andmountain slopes of East China, central China and Southwest, with clearing anddetoxifying, and the folk put its rhizome or herb into medicine. But so far theProtentilla freyniana Bornm has not been accepted as a kind of Chinese herbalmedicine for the Chinese Pharmacopoeia (2000) or local standards, and the reports arerare at home and abroad about its chemical composition and biological activity. In thispaper, we study techniques of microwave extraction about flavones from Protentillafreyniana Bornm, the techniques of separation and purification of flavones fromProtentilla Freyniana Bornm, the component and the content of Protentilla freynianaBornm analysed by LC-MS, and the antioxidant activities and free radical scavengingof Protentilla Freyniana Bornm were also evaluated.
The researches about flavones of Protentilla freyniana Bornm in this thesisincluded four aspects:
(1) The microwave extraction of flavones from Protentilla freyniana Bornm wasstudied．The effect of the ethanol concentration, the microwave power, the extractiontime, the refluxing temperature and the ratio of material to liquid on microwaveextraction process of Protentilla freyniana Bornm was researched with single factorexperiment. The optimum conditions were:75%ethanol，material-liquid ratio1：25，70℃e xtracted for5minutes with microwave power400W.The average yield offlavones was4.24%．
(2) To selected the best macroporous adsorption resin, we inspected the separation andpurification of6kinds of macroporous adsorption resin for the flavonoids ofProtentilla freyniana Bornm with the adsorption rate and desorption rate as an index,and detected the purification effect by HPLC analysis; Analyses of filtrationconcentration, pH, resin content and the concentration and pH of ethanol for theinfluence of adsorption and desorption have been studied. Experimental results showthat D101of macroporous adsorption resin has the best adsorption and desorptionperformance and the appropriate adsorption and desorption conditions is: adsorption liquid concentration to2.0mg/mL, pH=6, resin content and adsorption fluid volumeratio (g/mL) for1:30; Balance concentration to0.36mg/mL, namely resin has reachedadsorption saturation, saturated the adsorption quantity for49.1mg/g dry resins;ethanol concentration for50%, pH=6.
(3) The kinds and contents of the flavones of Protentilla freyniana Bornm areidentified by high performance liquid chromatography-Mass Spectrometry (LC-MS).The results show that there are seven substances mainly, and they are Epicatechin,2,3,5,4-tetrahydroxyl diphenylethylene-2-o-glucoside, Vitexin, Hypericin, Rutin,Icariin and Astragaline, meanwhile their contents are73.02mg/g,14.53mg/g,15.05mg/g,12.51mg/g,160.35mg/g,27.10mg/g and144.52mg/g separately.
(4) The antioxidant activities and free radical scavenging of Protentilla freynianaBornm and potentilla fulgens were evaluated based on total antioxidant ability, yolklipoprotein, hydroxyl radical and superoxide anion scav-enging activities, andscavenging DPPH radical. The various antioxidant activities were compared withsynthetic antioxidants such as ascorbic acid (Vc) and propyl gallate(PG).The resultsrevealed that within certain range of concentration, four kinds of Protentilla freynianaBornm compound in the first four aspects have a fixed order: Potentilla freyniana(Fujian)> Potentilla freyniana (Hebei)> potentilla fulgens (Hebei)> potentillafulgens (Fujian), but in the DPPH free radical scavenging a slightly different; andcompared to synthetic antioxidants such as Vc and PG, their activity performance issimilar.
引文
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