【作者】 张建业；

【导师】 刘宏民；

【作者基本信息】 郑州大学 ， 有机化学， 2005， 博士

【摘要】 本文分两部分内容,一部分是重要民间草药马鞭草科植物马鞭草的化学成分研究,另一部分是重要的中药材红花的指纹图谱研究。 马鞭草中的马鞭草苷(又称山茱萸苷)、戟叶马鞭草苷和毛蕊花苷是重要的免疫调节化合物,其中马鞭草苷是我国重要传统中成药六味地黄丸中山茱萸的重要活性成分,但在山茱萸中的含量低,而在马鞭草中有较高含量。本文在研究马鞭草化学成分的基础上对上述三个主要活性成分的含量测定、提取分离条件和结构改造等方面进行了研究。发现了马鞭草苷的自动氧化现象,研究了影响自动氧化的因素,并对其机理进行了分析。在此基础上研究了不同产地马鞭草的HPLC指纹图谱,并扩展研究了菊科植物红花的指纹图谱,建立了红花中药材、红花黄色素和红花黄色素氯化钠注射液的HPLC指纹图谱。 一、对马鞭草的化学成分进行了研究,共从马鞭草全草的乙醇提取物中分离出七个化合物,通过理化方法和光谱分析确证它们分别是7α,22β-二羟基谷甾醇、熊果酸、3α,24-二羟基齐墩果酸、胡萝卜苷、马鞭草苷、戟叶马鞭草苷和毛蕊花苷。LC-MS联用法初步确定了马鞭草苷母液中的化合物为3,4-二氢马鞭草苷。 二、研究并建立了戟叶马鞭草苷和马鞭草苷(流动相为水-乙腈(90:10),流速为1mL/min)、毛蕊花苷(流动相:水-乙腈-乙酸(80:10:10),流速为1mL/min)的HPLC含量分析方法及马鞭草提取液的SPE(固相萃取)处理方法,方法学(系统适用性)研究表明所建立的方法准确、快速、灵敏。 通过研究确定了上述三种化合物的提取条件为:80%乙醇与干燥马鞭草以75:10(体积(mL):重量(g))的比例在65℃提取12h,连续提取3次;并证明了该提取条件可应用于中试设备。完善了大量提取制备戟叶马鞭草苷、马鞭草苷和毛蕊花苷三种化合物的方法,并对它们进行了大量的制备,为三者衍生物的合成及构效关系研究打下了坚实的基础。 三、发现并确定了马鞭草提取液中马鞭草苷的自动氧化现象,初步研究表明毛蕊花苷对自动氧化有较大影响,进一步研究表明温度和毛蕊花苷的浓度对马鞭草苷的自动氧化均有影响。初步分析了自动氧化现象的机理。这对马鞭草苷的提取、制备和保更多还原

【Abstract】 Two parts of researching work were included in the dissertation. The first part was the study of chemical constituents of Verbena officinalis L. (Verbenaceae), which was an important folk herb medicine. And the second was the study of fingerprint analysis of Flos Carthami (dried flower petals of Carthamus tinctorius L. (Compositae)).Verbenalin (also named cornin), hastatoside and verbascoside were the important immunoregulational compounds of Verbena officinalis L.. And verbenalin was a prominent constituent of Cornus officinalis, which was one species of Liuwei Dihuang Wan, which was a well-known Chinese traditional patent medicine and widely used. But the content of verbenalin in Cornus officinalis was lower than in Verbena officinalis L.. The methods of HPLC quantitative analysis, extraction and isolation of three main bio-active constituents above-mentioned have been established based on the chemical constituents investigating of Verbena officinalis L.. The structural modification of three were designed and prepared, too. The autoxidation of verbenalin in the extraction solution of Verbena officinalis L. was discovered and studied, its possible mechanism was described. The HPLC fingerprint analysis of Verbena officinalis L. was carried out. Furthermore, fingerprint analysis of Flos Carthami, safflower yellow pigments, safflower yellow pigments and sodium chloride injection were also studied by HPLC.1. The chemical constituents of Verbena officinalis L. have been investigated. Seven compounds have been isolated from the alcohol extract of the whole grass and they were determined as 7a,22P-dihydroxysitosterol, ursolic acid, 3<x,24-dihydroxyoleanoic acid, β-sitosterol-3-O-β-D-glucoside, verbenalin, hastatoside and verbascoside on the basis of spectral data. 3,4-dihydroverbenalin in the extract was determinded by liquid chromatography-tandem mass spectrometry.2. HPLC quantitative analysis methods of hastatoside, verbenalin (mobile phase was H2O-CH3CN (90:10, v/v) and flow rate was 1 mL/min) and verbascoside (mobile phase was H2O-CH3CN-CH3COOH (90:10:10, v/v) and flow rate was 1 mL/min) were studied and established. The SPE (solid phase extraction) pretreatment method of the extract of Verbena officinalis L. was confirmed, too. Methodology (system suitable) studies proved that all the methods were accurate, rapid and sensitive.The extraction condition of the three compounds, that a 10-g dried grass was extracted with 75 ml 80% ethanol in a water bath (temperature was 65 ℃) for 12 h and extracted three times, was studied and determined. The method was proved to be suitable for middle-scale extraction. The extraction and preparation method of verbenalin, hastatoside andverbascoside was consummated. Three compounds were prepared in large scale and it has become possible for the investigation of their structure-activity relationship (SAR).3. The autoxidation of verbenalin in the extract of Verbena officinalis L. was discovered and studied. Verbascoside was proved to be an important effect factor to the autoxidation by primarily studies. It was found that the autoxidation velocity was affected by the temperature and the concentration of verbascoside by further studies. The possible mechanism of the autoxidation was described. It would be important for the extraction, preparation and storage of verbenalin.4. The contents of verbenalin, hastatoside and verbascoside in the grasses of Verbena officinalis L. from different sources were compared and showed great variant. The HPLC fingerprints of their extract displayed variant and correlative.HPLC method of fingerprint analysis of Flos carthami, of which the mobile phase was methanol- acetonitrile-water-phosphoric acid (25:0.7:75:0.285, v/v) and the flow rate was 0.85 mL/min, was established firstly. A set of orthogonal tests was carried out to determine the method of preparing the extract of Flos Carthami, which was a 1.0-g sample of the dried flower powder (particle diameters were about 400 um) was extracted with 100 ml water in a water bath (temperature range was from 65 °C to 75 °C) for 20 min.A standardized procedure, including preparing and analyzing samples, was established by a set of experiments. Then the characteristic HPLC fingerprints of Flos Carthami, safflower yellow pigments, safflower yellow pigments and sodium chloride injection were established by analyzing 10 batches of samples of each with the standardized procedure. The parameters of three sets of fingerprints were listed as following. For Flos Carthami, I, serial numbers of "common peaks" (relative retention times) were: 1 (0.339), 2 (0.405), 3 (0.461), 4 (0.559), 5 (0.657), 6 (0.896), S (1.000), 8 (1.291), 9 (1.557), 10(2.370), 11(3.358), 12 (4.031), 13 (4.434), 14 (5.941), 15 (10.419); II, the relative peak areas of PG1-5 and peak 15 should be controlled as: PG1-5: S: 15 = (0.259 - 0.431): 1.000: (0.437 - 0.655). For safflower yellow pigments, serial numbers of common peaks (relative retention times) were: 1’ (0.336), 2’ (0.380), 3’ (0.651), S (1.000), 5’ (1.312), 6’ (1.554), 7’ (2.390), 8’ (2.748). For safflower yellow pigments and sodium chloride injection, serial numbers of common peaks (relative retention times) were: 1" (0.345), 2" (0.479), 3" (0.659), S (1.000), 5" (1.331), 6" (1.554), 7" (2.375), 8" (2.731).In succession, the fingerprint of Flos carthami was used to identify the raw herbs from different sources in China. It was indicated that the fingers showed variant. The correlation of fingerprints of Flos carthami, safflower yellow pigments, safflower yellow pigments and sodium chloride injection was discussed. The viewpoints towards the establishing offingerprint of Traditional Chinese Medicine were put forward.5. The structural modification of verbenalin, hastatoside and verbascoside were designed and carried out. The structures of 13 derivatives were determined by the spectral data, and 9 of them were new compounds. The vitro immunoactivity of verbascoside and some of its derivatives have been detected. That of the other compounds were in testing. It was discovered that shift of the methoxyl of the substrate was occurred when the substrate was acetylated in base condition. The possible mechanism was discussed.o OAcThe method of selective cleavage of acetyl with dibutyltin oxide was used to cleavage of acetyl of nonaacetylverbascoside. The products of partly or whole cleavage of acetyl were prepared.In conclusion, The extract and isolation method of three main bio-active chemical compounds, verbenalin, hastatoside and verbascoside, of Verbena officinalis L. has been studied. Three of them were isolated and prepared in large scale. Their derivatives were designed and prepared. The structures of 13 compounds of the derivatives were determined base on the spectral data, 9 of them were new compounds. It was discovered that shift of the methoxyl of the substrate was occurred in the acetylation reaction. The autoxidation of verbenalin in the extraction solution of Verbena officinalis L. was discovered and studied. Its possible mechanism was discussed. The contents of verbenalin, hastatoside and verbascoside of Verbena officinalis L. from different sources and their HPLC fingerprints were compared and showed variant and correlative. A standardized procedure of fingerprint analysis of Flos Carthami, safflower yellow pigments, safflower yellow pigments and sodium chloride injection were established. The correlation of three sets of fingerprints was also studied. Flos carthami from different sources in China was compared by the fingerprint analysis.更多还原