【作者】 黎霜；

【导师】 黄可龙；

【作者基本信息】 中南大学 ， 制药工程， 2011， 博士

【摘要】 深绿卷柏,药用卷柏属植物,由于良好的治疗效果,已引起许多领域的广泛关注。本文运用多种谱学技术对深绿卷柏中的双黄酮类化合物进行了一系列具有原创性的研究工作并取得以下很有意义的研究成果。一、在单因素实验基础上,利用响应面分析法优化深绿卷柏总黄酮超声辅助提取的工艺参数,得到最佳工艺条件为：提取温度65℃,乙醇浓度70%,提取时间50min。在此条件下,提取的总黄酮含量为4.4mg/g,提取率为7.7%。以IC50值(半数抑制浓度)为次参考指标,得出了不同提取条件下提取得到的深绿卷柏粗提物抗氧化活性数据。在最优工艺条件下,IC5o值范围在20.22-46.64mg/mL之间。二、以天然药物化学为基础,运用化学及物理分离分析方法对深绿卷柏全草的乙醇提取物进行分离、纯化,得到穗花杉双黄酮和橡胶树双黄酮2个双黄酮单体化合物以及正十八烷酸、β-谷甾醇、豆甾醇等3个单体化合物。通过核磁共振、质谱等光谱学技术确定了这些单体的化学结构。三、运用GC-MS、HPLC-MS等现代的色谱技术,分析深绿卷柏中的各化学成分。从深绿卷柏中分析鉴定了59种化学成分。其中包括n-octacosane、9-octyl-hexacosane、3-phenyl-2-butanol等42种挥发性成分以及17种双黄酮化合物。穗花杉双黄酮、橡胶树双黄酮为深绿卷柏主要成分。采用反向HPLC法建立了同时测定穗花杉双黄酮和橡胶树双黄酮的检测方法。回收率大于95%。结果表明：所建立的检测方法适用于评价深绿卷柏药材的品质。采用HPLC法建立了深绿卷柏药材的指纹图谱检测方法,确定活性组分的分布及活性化合物的位置。依据此分离体系,考察了广西五个不同产地,15个样品的液相色谱图,得到深绿卷柏的参照液相指纹图谱。运用中药指纹图谱相似度计算软件对这些色谱峰进行评价,表明色谱图间的相关系数大于0.9。色谱图中有14个共有峰,可以确认为深绿卷柏的特征指纹峰。四、采用负离子检测模式下的电喷雾多级串联质谱(ESI-MS)方法对穗花杉双黄酮和罗伯斯特双黄酮的质谱裂解行为进行了研究,讨论了C3’-C8"与C3’-C6"连接的双黄酮化合物结构特征与质谱裂解行为之间的异同。对卷柏属植物中其他不同连接方式的双黄酮化合物的裂解规律进行总结,得到规律性的认识：(1)双黄酮化合物在相同的质谱条件下可以在C环发生0,4健断裂,生成m/z=375的碎片离子。对于C-O-C连接的双黄酮化合物,C-O健易发生均衡碎裂,质谱图中产生较强的mm/z=271的碎片离子峰。这些离子峰为双黄酮化合物的特征离子峰。(2)空间结构对双黄酮的裂解途径有重要影响。与C3’-C8"相比C3’-C6"和C2’-C8"连接的双黄酮更易于在离子碎片中脱去水分子出现环化的过程,生成丰度响应值较高的碎片离子m/z=519、m/z=309以及m/z=401；C3’-C3"’连接模式则是在离子碎片中形成内酯而出现环化的过程,产生m/z=371的碎片。C-O-C连接的双黄酮化合物中,C-O健出现非均衡碎裂产生较强m/z=285的碎片离子峰。此离子碎片中存在邻位酚羟基,更易于进一步碎裂而产生m/z为133、199、257等碎片离子。此规律可有效用于深绿卷柏中双黄酮化合物的定性鉴别,共鉴定出的17个双黄酮化合物。五、选取深绿卷柏中有代表性的芹菜素、穗花杉双黄酮、橡胶树双黄酮三个化合物分别与牛血清白蛋白作用。系统探索了在不同温度及浓度的条件下它们之间的作用机理,指出：(1)芹菜素与穗花杉双黄酮的猝灭率与浓度都遵循Stern-Volmer方程以及双对数曲线方程。但是,芹菜素对牛血清白蛋白的荧光猝灭是静态猝灭,穗花杉双黄酮对牛血清白蛋白的荧光猝灭是动态猝灭。(2)药物分子的结构对其与血清的结合有重要影响。A：穗花杉双黄酮与牛血清白蛋白之间的结合力比芹菜素强3-20倍,比橡胶树双黄酮强8-10倍。说明在黄酮与血清的结合过程中,羟基扮演了非常重要的作用。芹菜素与牛血清白蛋白氨基酸残基的结合距离最小,橡胶树双黄酮最大。说明在与牛血清作用的过程中,结合距离不仅与结合力有关,还与分子的体积有关。B：在本实验条件的药物浓度下,芹菜素对牛血清白蛋白二级结构的影响大于穗花杉双黄酮。这可能与芹菜素有着更小的分子尺寸有关。更多还原

【Abstract】 Selaginella doederleinii Hieron (SD), belongs to the family Selaginellaceae. It has been used as a traditional Chinese medicine and has attracted great interest among researchers because of good pharmaceutical, in recent years. A series of original creative investigations about biflavonoids of SD have been accomplished and some significant outcomes obtained through the by multi-spectroscopic method.1. Applied the central composite design combined with response surface methodology to optimize the parameter of ultrasonic-assisted extraction the total flavonoids from SD. The results indicate that the maximum extract of total flavonoids was 4.4 mg/g by using 70% ethanol as solvent, extract 50 min at 65℃. Antioxidant activity of extracts, determined by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, IC50 ranged from 20.22 to 46.64 mg/ml of raw SD. The optimization condition for antioxidant activity was similar to extract of total flavonoids. Yield increase of the total flavonoids depends on the cooperative effect of three selected parameters in the process of ultrasonic-assisted extract. Antioxidant activity more reckon on the ethanol concentration. Moreover, it was also found that there was significant correlation between the bioactivities and content of total flavonoids.2. Basing on natural medicine chemistry, utilizing physical and chemical methods, two bi-flavonoids, amentoflavone (AM) and Heveaflavone (HE) were purified from the ethanol extraction of SD. Meanwhile, the stearic acid,β-sitosterol and stigmasterol were also isolated. The structures of them were identified by NMR, ESI-MS spectroscopies.3. Direct identification of constituent of SD by gas chromatography with mass spectrometry (GC-MS) and high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSn) technique was established. There were 59 kinds chemical compounds have been analyzed. Including 42 components of essential oil, such as n-octacosane,9-octyl-hexacosane,3-phenyl-2-butanol. There were 17 components of bi-flavonoids, which linkage by different C-C and C-O-C. AM and HE were the principal ingredient of SD. A reversed-phase high performance liquid chromatography (RP-HPLC) procedure was developed for simultaneous determination of AM and HE compounds. The variation in precision and reproducibility in peak area was<1.6 and <1.8%, respectively. The correlation coefficients, r, of calibration curves of the 2 compounds were>0.99. The spike recovery of the method was> 95%. That indicated the method could be used to quantify the quality of SD. A fingerprint profile method was developed using a HPLC method which can identify the distributions of major active components. According to the separation system of optimality conditions,15 samples from 5 regions of GuangXi province were analyzed. A standardization fingerprint profile of SD can be obtained by similarity calculation for traditional Chinese medicines fingerprints software. The correlation coefficients, r, of 15 peaks of active compounds were>0.9 by the software calculation. There are 14 peaks were the owned by all samples, which can be identified as the characteristic of SD.4. Amentoflavone and robustaflavone were investigated by negative ion electrospray ionization tandem mass spectrometry (ESI-MS). The relationship between their structural features(C3’-C8" or C3’-C6")and the corresponding characteristic ions fragmentation behavior was discussed. The conclusions were obtained about the fragmentation behavior of bi-flavonoid, which linkage by different position.(1) Bi-flavonoid, belongs to the family Selaginellaceae, have the same fragmentation pathways. Under the condition of mass spactrum, the bond of C-ring broken at 0,4 positions, and than gave the principal fragment ion at m/z=375 by lossing of neutral species. For the bi-flavonoid of C-O-C linkage, there was a fragmentation pathway that the bond of C-O broken by balanced method, and than would bring to the principal fragment ion at m/z=271. All of these ions may be the characteristic ions.(2) It was very important that the stereostructure of bi-flavonoid effects on the fragmentation pathway. Compare to the C3’-C8", bi-flavonoid of C3’-C6" and C2’-C8", dehydrating will be easy to produce cyclization process, and than gave the principal fragment ion at m/z=519, m/z=309 or m/z=401. For C3’-C3"’, may produce lactones, and than gave the principal fragment ion at m/z=371. As far as the bi-flavonoid of C-O-C linkage, the fragmentation ions m/z=285 has stranger abundance value because the bond of C-O broken by unbalanced method. There was p-phenolic group on the fragmentation ions, which will be easy broken. It may bring the fragment ion at m/z=133,199 or 257. The rules may be used to distinguish the bi-flavones of SD. There were 17 constituents of bi-flavones were quality analyzed.5. Apigenin, Amentoflavone and Heveaflavone were selected to investigate the binding to bovine serum albumin. Systemic study the interaction mechanisms under different temperature and concentration. The conclusion is that:(1) It was found that two different quenching mode, static mechanism for apigenin, and the dynamic mechanism for amentoflavone, although they conformed Stern-Volmer equation.(2) The relationship between molecule structure and the BSA binding to flavonoids.A:It was found that the affinity of amentoflavone binding to BSA increases 3-20 times than apigenin, increasing 8-10 times than heveaflavone. Results show that the hydroxyl groups on the melcules might play a major role in binding flavonoids to BSA. The binding distance between apigenin and BSA was smallest. The binding distance between heveaflavone and BSA was largest. It indicated that the binding distance not only was affected by affinity, but also the molecule size.B:under the experiments conditions, the effect of AP on the submain conformational of BSA was stronger than AM, which may be affected by the molecule size.更多还原