| 题名 | 石榴中主要化学成分的HPLC-MS分析 |
| 作者 | 王爱红 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 尤进茂 |
| 关键词 | 石榴 高效液相色谱质谱联用 脂肪酸 三萜酸 多糖 植物激素 |
| 学位专业 | 中药学 |
| 中文摘要 | 石榴（Punica grunatum L.）为石榴科石榴属落叶灌木或小乔木，在中国各地均有分布，其种质资源丰富，根据用途不同可分为食用型和观赏型石榴。食用型石榴果实味道酸甜可口，是备受青眯的水果之一。近期研究证实其花、皮、籽和果汁等均表现出多种生物活性，如抗氧化、预防和治疗肿瘤、抑菌、抗动脉粥样硬化和抗炎等，尤其是石榴花提取液对糖尿病的高效预防和治疗作用。为进一步阐述石榴药理活性成分，促进石榴生物学活性和营养价值的研究，本试验对石榴的不同组织花、皮、籽和果汁中的组成成分进行了系统分析，同时还对石榴花多糖的体外抗氧化活性进行了研究。 主要内容如下： （1）合成了灵敏度高、专属性强的荧光衍生试剂2-[2-(对甲苯磺酸酯)乙基]吡唑并[3,4-b]-7-甲基喹啉（MPB），建立了基于柱前MPB标记HPLC-FLD-MS技术的25种脂肪酸分析方法，利用该方法对石榴各组织中游离脂肪酸（Free fatty acid, FFA）进行定性定量分析。结果表明，石榴所有部位中脂肪酸成分相似，石榴皮和汁中含量相对较少。 （2）选择2-( 7H-二苯并[ a，g]咔唑) 乙基对甲苯磺酸酯（DBCETS）为柱前荧光标记试剂，结合HPLC/FLD-APCI/MS技术，建立灵敏度高、分离度好、专属性强的三萜酸分析方法。6种三萜酸标记物在35 min内即可实现基线完全分离，检测线可达ng/mL。将该方法应用于石榴花、皮和籽中三萜酸的组成及含量测定，发现各组织中三萜酸成分相似但含量有显著差异，石榴花中三萜酸含量明显高于石榴籽和皮。 （3）采用响应面RSM法优化石榴花多糖的微波提取参数，在预测的最佳条件下提取率可达10.2%；建立了基于柱前1 -萘基-3-甲基-5-吡唑啉酮（NMP）标记的HPLC-DAD方法，测定石榴花多糖的单糖组成；同时建立体外抗氧化体系，采用1,1-苯基-2-苦肼基自由基（DPPH）自由基和羟基自由基的清除试验，研究石榴花多糖的抗氧化活性。 （4）植物激素为植物受激时产生的微量信号分子，此次试验用微量草酸刺激石榴花，在不同时间段采摘样品，利用建立的基于苯并[b]吖啶酮-5-乙基对甲苯磺酸酯（BAETS）标记的HPLC-FLD- MS分析方法，对上述样品中植物激素进行定性定量分析，发现经草酸刺激后石榴花中植物激素的含量和种类发生明显变化。 |
| 英文摘要 | Pomegranate (Punica grunatum L.) is a deciduous shrub or small tree, which distributes in many areas of China, and the germplasm resources are abundant. According to different uses, it can be divided into edible and ornamental pomegranate. Edible pomegranate is one of the most favorite fruit for its sweet and sour taste. Recently, studies have confirmed that different pomegranate parts exhibited a variety of biological activities, such as antioxidant, prevention and treatment of cancer, antibacterial, anti-atherosclerotic and anti-inflammatory, the pomegranate flower exhibited activities on prevention and treatment of diabetes. In order to illustrate the pharmacologically active ingredients in pomegranate and promote the further research on biological activities and nutritional value of pomegranate, a systematic analysis of chemical compositions in different pomegranate parts including flower, peel, seed and juice was carried out in our study. The antioxidant activity of pomegranate flower polysaccharide was also evaluated by in vitro experiments. The major contents are as follows: (1) A novel pre-column fluorescence labeling reagent has been first developed for highly sensitive and selective determination of fatty acids by HPLC with fluorescence detection and online mass spectrometric identification. The developed method was employed to analyze free fatty acids composition in pomegranate sample. The result showed that all of pomegranate organs have the similar free fatty acid compositions. (2) A sensitive and selective method using 2-(7H-dibenzo[a,g]carbazol-7-yl)ethyl-4-methyl benzenesulfonate (DBCETS) as labeling reagent for the analysis of 6 triterpene acids by HPLC with fluorescence detection and on-line mass spectrometric identification was developed. The completed separation of 6 triterpenic acids was achieved within 35 min with the low detection limits of 1.16-1.42 ng/mL. The proposed method was successfully applied to the analysis of triterpene acids composition in different pomegranate organs. It was found that the profile of triterpene acids was similar but the content was obviously different in all of pomegranate organs, higher value of triterpene acids was existed in flower. (3) The response surface methodology was used to optimize the microwave extraction parameters for pomegranate flower polysaccharide (PFP) extraction. Under the predicted conditions, the extraction yield was up to 10.2%. Meanwhile, a pre-column derivatization method using1-(2-Naphthyl)-3-methyl-5-pyrazolone (NMP) as saccharide labeling reagents followed by HPLC-DAD has been developed for the saccharide composition analysis of PFP. Then, DPPH radical activity assay and hydroxyl radical scavenging activity assay were used to evaluate the anti-oxidation activity of PFP, the results suggested that PFP have strong radical scavenging abilities on DPPH and hydroxyl radical. (4) Plant hormone is one kind of trace signaling molecules produced when plant was stimulated. In this study, A sensitive and selective method using 2-(5-benzoacridine) ethyl-p-toluenesulfonate (BAETS) as labeling reagent followed by HPLC-FLD-MS analysis has been developed for the determitation of 8 plant hormones in pomegranate flower samples induced with oxalic acid. It was found that formic acid can make pomegranate flower produce new kinds of plant hormones as well as the change of relative content. |
| 公开日期 | 2014-09-04 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.nwipb.ac.cn/handle/363003/4033]  |
| 专题 | 西北高原生物研究所_中国科学院西北高原生物研究所 |
| 推荐引用方式 GB/T 7714 | 王爱红. 石榴中主要化学成分的HPLC-MS分析[D]. 北京. 中国科学院研究生院. 2014. |
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