【作者】 娜日苏；

【导师】 博格日勒图；

【作者基本信息】 内蒙古大学 ， 植物学， 2013， 博士

【摘要】 我们课题组多年来对降血脂蒙药--荜茇进行了深入的研究,首次发现了天然化合物荜茇宁(代号GBN)有显著的降血脂活性,且毒性极低,并能够防止早期动脉粥样硬化(AS)的发生。此研究成果获得了2项国家发明专利,入选国家重大新药创制专项的候选药课题。本文首先分析了十余种不同产地的荜茇和胡椒当中主要的两种生物碱成分胡椒碱和荜茇宁的含量,发现日本和越南产的荜茇中荜茇宁的含量较高,分别是1.93mg/g和1.82mg/g；印度和印度尼西亚产的含量为0.8mg/g；中国海南产含量最低,为0.42mg/g。而胡椒中的荜茇宁的含量更低,黑胡椒中荜茇宁的平均含量仅为0.15mg/g。由上述结果可知,从天然产物中提取荜茇宁成本极高。继而又进行了荜茇宁的化学合成,确定了最佳合成工艺并进行了中试放大实验,合成了3个批次的GBN样品。将合成的3批样品作为降血脂候选化合物进行了系统的质量控制研究。根据该化合物的合成路线及结构特点,依照《中国药典》2010版(二部)、《化学药物研究技术指导原则》和《一类化学药质量要求》,进行了性状、理化性质、鉴别(TLC、UV、IR、HPLC)、杂质检查、含量测定以及稳定性的研究,有关物质检查和含量测定方法采用高效液相色谱法,十八烷基硅烷键合硅胶填充剂色谱柱,乙腈-水(50：50)为流动相,检测波长为338nm。建立的方法具有良好的专属性,最小检出量为1.5ng,含量测定方法在0.2-1.0μg范围内线性关系良好(r=0.99994)。日内和日间精密度平均标准偏差(RSD)分别为0.38%和0.89%。首次发现GBN溶液对光极不稳定,在4500Lux的光照箱中照射15分钟后主成分峰被降解,产生另外3种降解物,经LC-MS分析,证实与GBN共属同分异构体。为得到各异构体化合物,将GBN配成一定浓度的溶液,放入光照箱(4500Lux)照射,诱导其产生异构体,采用半制备高效液相色谱,通过梯度洗脱的方法,分离并收集得到了GBN及其另外2种异构体的化合物。分离得到的化合物按保留时间先后分别命名为GBN-P1,GBN-P2和GBN-P3;采用多种波谱技术,包括UV、MS、MR(1H-MR、13C-MR、DEPT-13C-MR、1H-1HCOSY、HSQC、HMBC)等,确证了GBN-P1,GBN-P2和GBN-P3的结构分别为N-异丁基-5-(3,4-亚甲双氧苯基)-2E,4E-戊二烯酰胺(白色晶体)；N-异丁基-5-(3,4-亚甲双氧苯基)-2Z,4E-戊二烯酰胺(白色晶体)；N-异丁基-5-(3,4-亚甲双氧苯基)-2E,4Z-戊二烯酰胺(灰白色晶体)。为研究GBN的异构体对生物活性是否有影响,用光照诱导产生50%异构体的混合物(GBNiso-mix)与纯GBN对比进行了动物降血脂药效实验,采用大鼠高脂血症(HLP)模型,研究异构体对GBN降血脂活性的影响,结果表明Iso-mix高剂量组和低剂量组均可显著降低HLP大鼠血清总胆固醇(TC)、甘油三酯(TG),并呈剂量依赖性,与GBN的活性没有显著差异。用MTT法通过HepG2细胞对GBN3种异构体进行了细胞毒性的比较,以辛伐他汀和胡椒碱做为阳性对照,结果表明3种异构体对HepG2细胞的毒性都很低,GBN-P2毒性显著低于GBN-P2和GBN-P3,均显著低于辛伐他汀和胡椒碱。GBN-P1, GBN-P2和GBN-P3的LC50分别是0.407mM,0.587mM,0.360mM;胡椒碱和辛伐他汀分别为0.192mM,0.102mM。体外考察了GBN-P1, GBN-P2和GBN-P3对HepG2细胞脂质代谢相关蛋白,低密度脂蛋白受体(LDLR)基因表达的影响,用10μM浓度的GBN-P1,GBN-P2, GBN-P3以及阳性对照药辛伐他汀处理HepG2细胞,以配药溶剂处理细胞作为空白对照。三种异构体均能不同程度上调HepG2细胞LDLR基因的表达,相互间没有显著差异。采用脂多糖(LPS)和γ干扰素(interferon-γ,IFN-γ)联合刺激小鼠巨噬细胞系RAW264.7建立体外炎症模型,考察GBN及其异构体对过量产生的NO的抑制作用,并以辛伐他汀做阳性对照。结果表明,GBN-P1,GBN-P2,GBN-P3均能显著抑制NO的产生,并呈剂量依赖关系。本研究首次分离得到了GBN的另外2种同分异构体,并通过动物实验和细胞实验进行体内、体外毒性和活性的比较,明确了GBN顺反异构体之间毒性及降血脂活性没有显著差异,为GBN的成药性评价提供有价值的理论依据和实验数据。以化学一类创新药的要求建立了降血脂候选药GBN的一套科学、合理的质量控制方法,从而有效地检验了该药用化合物的质量,为建立临床前研究的质量标准提供了充分的资料,对民族医药的发展具有重要的意义。更多还原

【Abstract】 The natural compound Piperlonguminine (GBN), separated from Piper Longum L. has been found to have a remarkable Hypolipidemic activity and low toxicity and it could prevent early Atherosclerosis. This research achievement has obtained two national invention patents and it was elected as a candidate of the "State Project for Essential Drug Research and Development".A simple and convenient method was established for simultaneous quantitative determination of piperine and piperlonguminine in more than ten types of dried fruits of Piper longum and allied plants of different origins. A sample of P. retrofractum from Ishigaki, Japan and a sample of P. longum from Vietnam, showed high contents of piperlonguminine at1.93mg/g and1.82mg/g, respectively. The content of piperlonguminine from the samples collected in India and Indonesia showed the normal level at0.8mg/g and the lowest content level of0.42mg/g was showed from the sample collected in Hainan China. It was found that the average content of Piperlonguminine (0.15mg/g) was significantly lower than pipeline (40mg/g) in P. nigrum. Based on above results, it would be very expensive to extract piperlonguminine from natural products. Therefore we had developed a method to chemically synthesize piperlonguminine, determined the synthesis process and made pilot scale experiments of GBN synthesis.We had conducted a systematic quality control study for GBN as a new synthetic drug candidate. It was found for the first time that GBN in solution tends to transform to three other cis-trans isomers. It was discussed in detail in this article for the method of separating the GBN cis-trans isomers and whether their isomers have impacts on GBN’s biological activity and toxicity, since a proper clarification for this question is very crucial for future drug research development.Using the optimal synthetic route, we had synthesized three batches of samples, with lot numbers20101027,20101220and20110117. We had conducted the researches for their characteristics, physical and chemical properties, identifying methods (TLC method, UV, IR, HPLC, etc.), impurities inspecting methods, method to determine content and studied their stability based on their synthesizing process and structural characteristic referring to "Chinese Pharmacopoeia",2010Edition (2),"Chemical Pharmaceutical Research technical guidelines", and "The quality requirement of chemical drug."Utilized high-performance liquid chromatography (HPLC)(octadecyl silane bonded silica filled column, acetonitrile-water (50:50) as mobile phase, detection wavelength on338nm), we confirmed the method to inspect related substance and determine the content. The method has good specificity, with the lowest detection limit of1.5ng (S/N=3). The established method of determination showed good linear relationship (r=0.99994) in the range of0.2-1.0μg, the inter-day RSD were0.38%and the intra-day RSD were1.16%.The solution GBN is very unstable under light. A15minutes exposure under4500Lux light box gave four degraded components, as confirmed by HPLC and LC-MS analysis, which confirmed that the four components are isomers.In order to obtain pure isomers, we treated GBN under light (4500Lux) for radiation to induce isomerization. The4isomers were separated using semi-preparative high performance liquid chromatography and3of isomers were assembled through gradient elution method. The obtained isomers were named GBN-P1, GBN-P2, GBN-P3, according to their retention time on HPLC. Using a variety of spectroscopic techniques, including UV、IRMS、 NMR(1H-NMR、13C-NMR、DEPT-13C-NMR、1HCOSY、HSQC、HMBC) and so on, we confirmed the structure of3isomers. They are N-isobutyl-5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienamide (white crystal); N-isobutyl-5-(3,4-methylenedioxyphenyl)-2E,4Z-pentadienamide (white crystal) and N-isobutyl-5-(3,4-methylenedioxyphenyl)-2Z,4E-pentadienamide (gray crystal) respectively. The fourth isomer is N-isobutyl-5-(3,4-methylenedioxyphenyl)-2Z,4Z-pentadienamide.Considering that it would consume large amount of organic solvents to produce sufficient amount of isomeric compounds by HPLC for animal experiments, we had conducted the study on the lipid-lowering activity using50%GBN and isomer mixture which was induced by light. Three pure isomers were used on the cell cytotoxicity comparison study and in vitro biological activity comparison study.To investigate the influence of the isomerization on the Cholesterol-lowering activity of GBN, we used50%light induced GBNiso-mix and pure GBN on the HLP model. The results showed that GBNiso-mix high and low dose group could both significantly reduce Total Cholesterol (TC) and Triglycerides (TG), although it showed dose-dependency. There was no significant difference with GBN at high and low dose group.Using simvastatin and pipeline as positive control groups, we compared the cell cytotoxicity of3GBN isomers using MTT method on HepG2cells. The results showed that the3isomers have no significant cell cytotoxicity on HepG2cells, and all of the three isomers showed significantly lower toxicity than simvastatin and piperine. The LC50of GBN-P1, GBN-P2and GBN-P3were0.407mM,0.587mM and0.360mM, respectively. The LC50of Piperine and simvastatin were0.192mM and0.102mM, respectively.We had conducted the study on the impact of GBN-P1, GBN-P2and GBN-P3to the gene expression of lipid metabolism related genes such as LDLR in HepG2cells. We treated HepG2cells with GBN-P1, GBN-P2, GBN-P3at the concentration of lOuM. The results showed that all three isomers could increase LDLR gene expression on HepG2cells inordinately, without any significant difference. Moreover, we investigated the impact of GBN isomers on LPS/IFN-y induced nitric oxide (NO) production in RAW264.7Macrophages. Our results indicated that tree isomers significantly suppressed LPS/IFN-y induced NO production in a dose-dependent manner.During this research, two isomers of GBN were obtained for the first time, and the comparison of their in vivo and in vitro toxicity and activities were researched via animal and cell experiments. It has been confirmed that there was no significant difference in the toxicity and cholesterol-lowering activity between GBN cis trans isomers. It provided valuable theoretical basis and experimental data for the evaluation of the drug ability of GBN.In this thesis, a set of scientific and reasonable method of quality control was established for GBN according to the requirement of a First Class Innovative Chemical Hypolipidemic candidate drug. Thus, the quality of the medicinal compound was effectively tested and it provided the sufficient information and reference for the establishment of quality standards for pre-clinical studies, offered reliable experimental data and theoretical basis to the application of this kind of Hypolipidemic drug, which is beneficial to human beings.更多还原