女贞子中裂环环烯醚萜苷类成分的研究

【作者】 李阳；

【导师】 孙文基；

【作者基本信息】 西北大学 ， 中药学， 2007， 硕士

【摘要】 女贞子为木犀科（Oleaceae）植物女贞（Ligustrum lucidum Ait．）的干燥成熟果实，始载于《神农本草经》，列为上品，是我国传统的扶正固本中药。味甘、苦，性凉，具有滋补肝肾，明目乌发的功效。临床主要用于治疗眩晕耳鸣，腰膝酸软，目暗不明，须发早白等症。文献研究表明女贞子中主要含有脂溶性极强的三萜类成分，以齐墩果酸为其代表。长期以来，国内外对于齐墩果酸，从化学到药理都进行了深入系统的研究，并建立了多种定量分析方法。然而女贞子按照祖国医学中药的传统用药方法，是作为水煎液服用，因而其中的水溶性活性成份不容忽视。20世纪90年代后的国内外研究表明，女贞子中除低极性的齐墩果酸外，还有含量较高的环烯醚萜苷类水溶性成分。环烯醚萜苷具有保肝利胆、抗肝纤维化、抗菌消炎、抗肿瘤、降血脂、降血糖、降低血液粘度、增强机体免疫调节等多种药理活性，能够反映女贞子药材的药理作用，日益引起广泛重视。本文以女贞子药材作为研究对象，采取溶剂萃取法、硅胶柱层析，特别是制备高效液相色谱分离方法，从中分离到5种化合物，并运用波谱(UV、IR、¹³CNMR、¹HNMR)手段，结合理化常数，鉴定了它们的结构，分别是齐墩果酸、熊果酸、女贞苷、女贞苷G13及D-葡萄糖，其中2种是裂环环烯醚萜苷类成分（女贞苷及女贞苷G13）。为了更进一步研究女贞子中的裂环环烯醚萜苷类成分，本文采取HPLC法，双波长检测、梯度洗脱，同时考察了全国不同产地、不同采集时间女贞子样品中女贞苷及女贞苷G13两种成分的含量。结果表明，分离到的女贞苷和女贞苷G13系女贞子中水溶性裂环环烯醚萜苷类化合物的两种主要成分；不同地区女贞子药材中上述两种成分的含量差别较大，最高分别可达1.089％和0.478％，最低含量则分别为0.026％和0.013％，可能与不同品种来源及加工炮制方法等因素有关；并且发现，在女贞子果实及女贞叶中，女贞苷和女贞苷G13的含量变化趋势一致，均为先增后减，在11月初至中旬左右达到最大值，这与女贞子药材的传统采收季节一致。本实验同时研究了女贞苷和女贞苷G13在区带毛细管电泳中的迁移行为，建立了毛细管区带电泳法快速分离分析以上两种成分的方法。通过各种条件的优化，发现背景缓冲液的pH值、硼砂浓度、分离电压对分离均具有重要意义：pH值极大的影响着各物质在毛细管中的迁移行为；高浓度缓冲液可以获得较好的分离度，但保留时间变长，而且导致通过毛细管的电流增加，从而增大焦耳热，严重影响峰形；分离电压是毛细管电泳的一项重要参数，实验证明，增加组分的迁移速度是减少谱带展宽、提高分辨效率的重要途径，但高电场强度也会导致毛细管的电流增加而增大焦耳热。论文中并且对女贞苷和女贞苷G13的抗氧化活性和抗菌活性进行了初步研究。通过抗氧化活性实验中的数据比较，发现2种成分均有显著的抗氧化活性，剂量效应之间呈正相关，其中女贞苷G13清除DPPH自由基的作用强于女贞苷，而由于两者的结构差异仅在于女贞苷G13比女贞苷多了一个裂环环烯醚苷结构，由此说明糖苷类结构对清除自由基具有积极作用，这对于深入研究女贞子的抗氧化机理具有十分重要的意义。因此，本文以女贞子药材中提取分离得到女贞苷及女贞苷G13作为女贞子药材中有代表性的水溶性指标成分，测定其含量，为2010版中国药典女贞子含量测定项的修订开展了铺垫工作。更多还原

【Abstract】 Fructus Ligustri lucidi （Chinese name Nvzhenzi） is from the dried fruits of Ligustrum lucidum Ait., family Oleaceae, which is a kind of traditional Chinese medicine, sweet or bitter in flavor, cold in nature. It was firstly recorded in the earliest Chinese pharmaceutical monograph Shen Nong’s Herbal （about AD200） and ranked in the first grade. It has the effect of nourishing liver and invigorating the kidney, and promoting the eyesight and blackening the hair. Use for dizziness and tinnitus, soreness and weakness of waist and knee, manifestations of blurred vision and weakness of eyesight, premature whitening of hair.In many related reports, liposoluble triterpenoid was considered as the predominant ingredient in Fructus Ligustri lucidi. Oleanolic acid, regarded as the most important active substance has been investigated deeply and systematically in both its chemistry and pharmacological research. Meanwhile many quantitative analysis methods for it have been set up. However according to Chinese traditional custom, Fructus Ligustri lucidi should be boiled with water before drug regimen. Consequently the water-soluble portion of Fructus Ligustri lucidi could not be ignored.After 20 century the world research indicate that besides the low-polar constituent oleanolic acid, Fructus Ligustri lucidi also contains many water-solubility components, such as high-contents iridoid glucosides. Iridoid glucosides have many biological functions like protect liver and gallbladder, anti-hepatic fibrosis, antibiosis and dephlogisticate, subdue blood-fat, cut down blood-sugar, degrade viscosity, reinforce immunological regulation, etc. We need to give more attention to the iridoid glucosides, which could also reflect the internal quality of Fructus Ligustri lucidi.Here, we study the extraction and separation of the constituents in FructusLigustri lucidi. By using solvent extraction, column chromatography on silica gel ,especially preparative HPLC, we got five chemical compounds, and the structures ofthem were authenticated by means of spectroscopy (UV, IR, ¹³C NMR, ¹H NMR). They are oleanolic acid, ursolic acid, nuezhenoside, G13 and D-glucose. Two of them were secoiridoid glucosides （nuezhenoside and G13）.In order to get further messages of the secoiridoid glucosides in Fructus Ligustri lucidi, we determined two of them （nuezhenoside and G13） in samples from different places and acquisition times by HPLC using double wavelength and gradient elution. From the experiment we found that nuezhenoside and G13 were the two major secoiridoid glucosides in the water-soluble portion of Fructus Ligustri lucidi. And the results of content determination （nuezhenoside and G13） of samples from different places varied greatly. For example, the highest contents for nuezhenoside and G13 were 1.089 and 0.478%, while the lowest contents were 0.026 and 0.013%, respectively. This might be caused by different species sources and different preparation methods, etc. The tendency of the content variation in the fruits was coincident with in the leaves. This experiment led up to the fact that from the beginning to the middle of November the content of nuezhenoside and G13 reached the maximum.Meanwhile, By studying the migration behavior of the above two secoiridoid glucosides on capillary zone electrophoresis （CZE）. Subsequently a quick, accurate capillary electrophoresis method for quantitative analysis of nuezhenoside and G13 was developed. In our experiment, we found that the power of hydrogen, concentration of borax and the separation voltage were three main factors. The power of hydrogen influences the migratory behavior of different materials greatly. Higher concentration of borax can get better resolution. But it probably led up to the fact that retention time will be prolonged and the peak shape will be critically influenced. Sometimes higher separation voltage is the same.Here we also have done some preliminary researches about the antioxidant and antibacterial activities of nuezhenoside and G13. The results revealed that these ingredients showed significant antioxidant activities. A positive correlation existed between total content and antioxidant activity. G13 had shown higher antioxidant activity than nuezhenoside. It implied that the structure of secoiridoid glucoside was positive to antioxidant activity. Otherwise, the results could promote the deep research of the antioxidant mechanism in Fructus Ligustri Lucidi.So, we regarded nuezhenoside and G13 as the typical water-soluble ingredients in Fructus Ligustri Lucidi and establish the quality control system. All the works are prepared for the revision of Chinese Pharmacopeia in 2010.更多还原