蒙药荜茇降血脂有效成分荜茇宁的含量分析、合成及其异构体研究
|
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。
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.
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。
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.
引文
[1]赵水平,临床血脂学[M],长沙,湖南科学技术出版社,1997
[2]刘瑞杰,牛好敏,王景富等,高脂血症和相关疾病[M],北京,科学技术文献出版社,1999。
[3]王浩,生物化学[M],北京,人民卫生出版社,2002
[4]中华心血管病杂志编委会血脂异常防止对策专题组,血脂异常防治建议[J],中华心血管病杂志,1997,25,169-175
[5]赵冬,中国人群的血脂流行病学研究[J],临床荟萃,2006,21(5)259-261
[6]卫生部心血管病防治中心,《中国成人血脂异常防治指南》[M],2007,中华医学会
[7]Lusis AJ. Atherosclerosis, Nature,2000,407:233-241
[8]Brown MS, Goldstein JL. Human mutations affecting the low density lipoprotein pathway.Am J Clin Nutr.1977;30:975-978
[9]Todd J,Farmer JA. Optimal low-density lipoprotein levels:evidence from epidemiology and clinical trials. Curr Atheroscler Rep.2006;8:157-162
[10]Horton JD,Goldstein JL,Brown MS. SPEBPs:activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest.2002; 109:1125-1131
[11]Todd J,Farmer JA. Optimal low-density lipoprotein levels:evidence from epidemiology and clinical trials. Curr Atheroscler Rep.2006;8:157-162
[12]Rutishauser J. The role of statins in clinical medicine LDL cholesterol lowering and beyond. Swiss Med Wkly.2009; 203:1-7
[13]杨诗杰,苏汝好,项岚等,降血脂药的应用现状及研究进展[J],医药论坛杂志,2006,27(1)：90-92
[14]鄢琳,曹立亚,雷建军等,9种调血脂药物有效性及安全性评价[J],中国循证医学杂志,2005,5(1)：8-21
[15]王海勇,降血脂药物的研究进展,国外医学药学分册,2004,31(3)160-165
[16]程志清,中医药防治高脂血症[M],北京,人民卫生出版社,2002
[17]潘琴,降血脂药物临床应用的不良反应概述,strait pharmaceutical Journal,2013,25 (5) 293-295
[18]中国科学院中国植物志编辑委员会,中国植物志(第一卷)[M],北京：科学出版社,2004：147
[19]傅立国,陈潭清,郎楷永等,中国高等植物(第三卷)[M].青岛：青岛出版社,2003：325-326
[20]唐·苏敬撰.尚志钧辑较.新修本草(辑复本)[M]合肥：安徽科学出版社,1981：369.
[21]国家药典委员会编.中华人民共和国药典[M],北京：化学工业出版社,2005：163.
[22]Atal C K,Banga S S. Ind. J. Pharm,1962,24:105
[23]Chatterj ee A, Dutta C P. Alkaloids of Piper longum Linn. I. Structure and synthesis of piperlongumine and piperlonguminine[J]. Tetrahedron,1967,23(4):1769-1781
[24]Tabuneng W,Bando H,Amiya T, Studies on the Constituents of the Crude Drug "Piperis Longi Fructus" on the Alkaloids of Fruits of Piper Longum L. [J]. Chem. Pharm. Bull,1983,31(10):3562
[25]Badu D D,Ayim J S K,Dabra T T, et al. △αβ-Dyhydro piperlonguminine, A New Amide from Piper Guineense[J]. Phytochemistry,1976,15:822
[26]Okogum JI, Ekong D E U J. Chem. Soc,1974, Perkin I:2195
[27]Nakatani N,Inatani R, Isobutyl Amides from Pepper (Piper nigrum L.) [J]. Agri. Biol Chem, 1981,45(6):1473
[28]Tackie A N, Badu D D, Ayim J S K, N-isobutyloctadeca-Trans-2-Trans-4-Dienamide:A New Constituent of Piper Guineense[J]. Phytochemical Reports,1975,14:1888
[29]Raina M L, Dhar K L, Atal C K, Occurrence of N-isobutyl Eicosa-Trans-2-Trans-4-Dienamide in Piper nigrum[J]. Planta Medica,1976,30:198
[30]Gupta O P, Gupta S C, Dhar K L,et al, A New Amide from Piper Officinarum[J]. Phytochemistry,1977,16:1436
[31]包照日格图,吴恩,荜茇油非皂化物对小鼠高脂血症的影响[J],中草药,1992,23(4)：197
[32]包照日格图,吴恩,荜茇油非皂化物对小鼠胆固醇代谢的影响[J],中国药理学通报,1991,7(6)：463
[33]斯庆图娜拉,查干布拉格,荜茇研究[J].中国民族医药杂志,2006,3(17)
[34]Min K R, Kim K S, Ro J S, et al.Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells[J]. Planta medica,2004,70(12):1115.
[35]Qi, Hong-Shunl, Liu, Pei2, Inhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloid beta and APP in SK-N-SH Cells, CHINESE JOURNAL OF PHYSIOLOGY,2009,52 (3) 160-168
[36]Rahman T, Rahmatullah M, Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2010,20 (2) 537-540
[37]Silva DR (Silva, Daniel Rodrigues)1, Endo EH (Endo, Eliana Harue)1, Chemical Composition and Antimicrobial Properties of Piper ovatumVahl, MOLECULES,2009,14 (3) 1171-1182
[38]Bezerra DP (Bezerra, Daniel P.)1, Pessoa C (Pessoa, Claudia) 1, In vivo growth inhibition of sarcoma 180 by piperlonguminine, an alkaloid amide from the Piper species, JOURNAL OF APPLIED TOXICOLOGY,2008,28 (5) 599-607
[39]Rodrigues Silva, D; Baroni, S; Svidzinski, A E; Bersani-Amado, C A; Cortez, D A G, Anti-inflammatory activity of the extract, fractions and amides from the leaves of Piper ovatum Vahl (Piperaceae). J Ethnopharmacol,2008,116(3) 569-573
[40]包兰兰,金桩,博格日勒图,胡椒碱降血脂作用的实验研究[J],中国民族医药杂志,2004,1：22.
[41]Piyachaturawat P, Glinsukon T, Toskulkao C. Acute and subacute toxicity of piperine in mice, rats and hamsters[J].Toxicol Lett,1983,16(3-4):351-359
[42]Daware M B, Mujumdar A M, Ghaskadbi S. Reproductive toxicity of piperine in Swiss albino mice[J]. Planta Med,2000,66(3):231-236.
[43]Jin,Z; Borjihan, G; et al, Antihyperlipidemic Compounds from the Fruit of Piper longum L.,Phytotherapy research,2009,23(8)1194-1196
[44]麻春杰,博格日勒图等,荜茇宁降血脂和急性毒性实验研究[J],中华中医药杂志,2008,23(4)321-324
[45]麻春杰,博格日勒图等,荜茇宁对家兔实验性动脉粥样硬化的影响[J],中国中药杂志,2008,33(4)436-440
[46]麻春杰,博格日勒图等,荜茇宁对动脉粥样硬化家兔抗氧化酶活性的影响[J],中国药科大学学报,2008,39(1)68-71
[47]麻春杰,博格日勒图,荜茇宁对高脂血症大鼠血脂代谢及其相关基因表达的影响[J],中草药,2008,39(7)1039-1043
[48]《化学药物质量控制分析方法验证技术指导原则》2005.3指导原则编号[H]GPH5-1
[49]《化学药物质量标准建立的规范化过程技术指导原则》2005.3指导原则编号[H]GPH1-1
50、 [50]周怡,化学药品标准中杂质控制及其限度的建立,,药物生物技术[J],2010,17(2)：181-184
51、 [51]王平,药品质量控制与质量标准,中国职业药师[J],2008,5(7)38-39
52、[52]马磊,化学药物质量标准建立的一般过程,中国新药杂志[J],2009,18(14)1297-1299
[53]马玉楠,冯毅,美国FDA关于上市前化学药药学研究的技术要求,中国新药杂志[J],2005,14(7)805-80
[54]胡胜,袁继容,朱进,等.选择反应法制备高纯油酸[J].应用化工,2005,34(12)：748-751.
[55]解振兵,卢义和,宫素芝,等.混合脂肪酸选择性加氢制高纯度油酸[J].日用化学品科学,2006,29(1)：19-21.
[56]赵国志,刘喜亮,刘智锋,等.反式脂肪酸危害与控制[J].粮食与油脂,2007(1)：7-13.
[57]Nobuyuki Kozukue,Mal-Sun Park, Kinetics of Light-induced Cis-Trans Isomerization of Four Piperines and Their Levels in Ground Black peppers as Determined by HPLC and LC/MS,J.Agric.Food Chem.,Vol.55,No.17,2007
[59]Lee EB, Shin KH and Woo WS, Pharmacological study on piperine. Arch Pharmacol 1984,7:127-132
[60]Kanaki N, Dave M, Padh H and Rajani M, A rapid method for isolation of piperine from the fruits of piper nigrum linn. J Nat Med,2008,62:281-283
[61]Rahman T and Rahmatullah M Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases. Bioorg Med Chem Lett 2010,20:537-540
[62]Bezerra DP, Pessoa C,Moraes MO, Alencar NMN, Mesquita RO, Lima MW, Alves APNN, Pessoa ODL, Chaves JH, Silveira ER and Costa-Lotufo LV, In vivo growth inhibition of sarcoma
180 by piperlonguminine, an alkaloid amide from the Piper species. J Appl Tosicol,2008, 28(5):599-607
[63]Kim KS, Kim JA, Eom SY, Lee SH, Min KR and Kim Y, Inhibitory effect of piperlonguminine on melanin production in melanoma B16 cell line by downregulation of tyrosinase expression. Pigment Cell Res.2005.19:90-98
[64]Lin Z, Liao Y, Venkatasamy R, Hider RC and Soumyanath A Amides from Piper nigrum L. with dissimilar effects on melanocyte proliferation in vitro. J Pharm Pharmacol (2007)59:259-536
[65]Jin Z, Borjihan G, Zhao RG, Sun Z, Hammond GB and Uryu T (2009) Antihyperlipidemic compounds from the fruit of Piper longum L. Phytother Res 23(8):1194-1196
[66]Ma CJ, Alatangaole, Narisu, Zhaorigetau, Zhou HJ and Borjihan G (2008) Effects of piperlonguminine on genes expression of LOX-1 and VCAM-1 in the aortic of atherosclerosis rabbits. Chinese Pharm J 43(19):1449-1553
[67]Ma CJ, Ling X and Borjihan G (2011) Study on the blood lipid-lowering and anti-atherosclerosis function of piper longum L.and its effective Ingredients. World Science Technology/Modernization of Traditional Chinese Medicine and Materia Medica 13(1):133-135
[68]Roger A. Olsen and Gary O.Spessard,A Short, Stereoselective Synthesis of Piperine and Related Pepper-Derived Alkaloids,J.Agric.Food Chem.1981,29,942-944.
[69]Lee SH, Kim DH, Kim JA, Jahng Y, A simple synthesis of piperlonguminine, HETEROCYCLIC COMMUNICATIONS,2005,11 (5) 407-410
[70]张震,陈海峰,化学药品新药研究中的有关物质定性研究,中国新药杂志,2008,17(19)1721-1723
[71]领小,博格日勒图,娜日苏,荧光分析法测定荜茇有效成分荜茇宁,分析实验室,2009,28(6)21-23
[72]中国药典2010版二部,国家药典委员会,中国医药科技出版社
[73]化学药物质量控制分析方法验证技术指导原则,2005.3指导原则编号[H]GPH5-1
[74]化学药物质量标准建立的规范化过程技术指导原则,2005.3指导原则编号[H]GPH1-1
[75]张慧玲,浅谈药品注册中HPLC法测定原料药有关物质的问题,药物分析杂志,2007,27(6)947-948
[76]Nobuyuki Kozukue,Mal-Sun Park, Kinetics of Light-induced Cis-Trans Isomerization of Four Pipelines and Their Levels in Ground Black peppers as Determined by HPLC and LC/MS,J.Agric.Food Chem., Vol.55,No.17,2007
[77]许宏琪,盛龙生,安登魁,用色谱联用技术分析桂腈和溴代苏合香烯顺反异构体,色谱,1995,13(4)272-273
[78]游长江,烯烃光敏顺反异构化反应,感光科学与光化学,2003,3(2)：147-155.
[79]张震,陈海峰,化学药品新药研究中的有关物质定性研究,中国新药杂志,2008,17(19)1721-1723
[80]徐国华,殷国栋等,两个1,4-二酮衍生物的顺反异构体的确定及其1H,13C核磁共振信号归属,华中师范大学学报(自然科学版),2007 41(4)549-551
[81]彭惠琦,核磁振法测定化学位移及自旋耦合常数,实验室研究与探索,1998,6.,36-39
[82]谭英,利用NMR技术对紫苏草顺反异构体的研究[D],湖南师范大学,2003,04
[83]李锋,王敏.顺反异构化过程及其机理[J].化学试剂,2005,27(1)：22-22.
[84]Jin,Z; Borjihan, G; et al, Antihyperlipidemic Compounds from the Fruit of Piper longum L.,Phytotherapy research,2009,23(8)1194-1196
[85]许宏琪,盛龙生,安登魁,用色谱联用技术分析桂腈和溴代苏合香烯顺反异构体,色谱,1995,13(4)272-273
[86]王乃兴,核磁共振谱学在有机化学中的应用[M],化学工业出版社,2006年6月第一版
[87]百度文库,细胞培养基本技术,中国科技大学生命科学学院
[89]Grundy S M, Statin trial and goals of cholesterol lowering therapy, [J], Circulationl,1998,97 (15) 1436-9
[90]Dietschy J M, Dietary fatty acids and the regulation of plasma low dinsity lipoprotein cholesterol concentration [J], J Nutr,1998,128,444-8
[91]麻春杰,荜茇有效成分荜茇宁的调制作用及其机制研究[D],内蒙古大学博士论文,2008
[92]胡苗苗,GB-A对脂代谢紊乱及早期动脉粥样硬化的防治作用及其机制研究[D],上海：中国科学院上海药物研究所,2009.10
[93]王亚利,范春雷,窦晓兵,绞股蓝总皂苷对肝细胞低密度脂蛋白受体表达的影响,中国药理学通报,2010,26(1)：138-139
[94]庄晓,陈志,杜心,吴雅茗,阿魏酸钠对HepG2细胞低密度脂蛋白受体mRNA,中国临床保健杂志,2006,9(2)116-117
[95]Kuhlencordt PJ, Hotten S, Schodel J, Rutzel S, Hu K, Widder J, et al.Atheroprotective effects of neuronal nitric oxide synthase in apolipoprotein E knockout mice[J]. Arterioscler Thromb Vase Biol,2006,26:1539-1544.
[96]Barbato JE, Tzeng E. Nitric oxide and arterialdisease[J]. J Vase Surg,2004,40:187-193.
[97]王忠振,李芳,炎症与动脉粥样硬化及冠状动脉疾病的关系,中国微生态学杂志2012年9月第24卷第9期,862-865]
[98]Peter RH, Inflammatory altertions in the myocantiol microcirculation, J Mol Cell Cardio,1998,30(12):2555-2559
[99]Andrew PJ,Mayer B. Enzymatic function of nitric oxide synthases, Cardiovasc Res, 1999,43:521-531
[100]Barmaes PJ,et al.Time course and cellular localization of lipopolysaccharide induced inducible nitric oxide synthsase messenger RNA expression in the ratinvivo. Crit Care Med, 1997,25:512-518]
[101]Okamoto I, Abe M,Shibata K,et al. Evaluation the role of inducible nitric oxide synthaseusing an overland selective inducible nitric oxide synthase inhibitor in septicling injury produced by Cecalligation and puncture,AmJRespir Crit Care Med,2000,162:716-722
[102]何志旭,周同甫,廖清奎,徐学聚,罗眷华,李敛伯,王树,李丰益,炎性细胞因子及脂多糖诱导产生的一氧化氮对人血管内皮细胞的损伤作用,中国病理生理杂志,2001,17(9)：893—89
[103]麻春杰,阿拉坦高勒,娜日苏等,荜茇宁对动脉粥样硬化家兔主动LOX-1和VCAM-1基因表达的影响,中国药学杂志,2008,43(19)1449-1452。
[104]麻春杰,博格日勒图等,荜茇宁对家兔实验性动脉粥样硬化的影响,中国中药杂志,2008,(4)436-440。
[105]Son, Dong Ju; Kim, Soo Yeon; Han, Seong Su; Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,427 (2) 349-354
[2]刘瑞杰,牛好敏,王景富等,高脂血症和相关疾病[M],北京,科学技术文献出版社,1999。
[3]王浩,生物化学[M],北京,人民卫生出版社,2002
[4]中华心血管病杂志编委会血脂异常防止对策专题组,血脂异常防治建议[J],中华心血管病杂志,1997,25,169-175
[5]赵冬,中国人群的血脂流行病学研究[J],临床荟萃,2006,21(5)259-261
[6]卫生部心血管病防治中心,《中国成人血脂异常防治指南》[M],2007,中华医学会
[7]Lusis AJ. Atherosclerosis, Nature,2000,407:233-241
[8]Brown MS, Goldstein JL. Human mutations affecting the low density lipoprotein pathway.Am J Clin Nutr.1977;30:975-978
[9]Todd J,Farmer JA. Optimal low-density lipoprotein levels:evidence from epidemiology and clinical trials. Curr Atheroscler Rep.2006;8:157-162
[10]Horton JD,Goldstein JL,Brown MS. SPEBPs:activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest.2002; 109:1125-1131
[11]Todd J,Farmer JA. Optimal low-density lipoprotein levels:evidence from epidemiology and clinical trials. Curr Atheroscler Rep.2006;8:157-162
[12]Rutishauser J. The role of statins in clinical medicine LDL cholesterol lowering and beyond. Swiss Med Wkly.2009; 203:1-7
[13]杨诗杰,苏汝好,项岚等,降血脂药的应用现状及研究进展[J],医药论坛杂志,2006,27(1)：90-92
[14]鄢琳,曹立亚,雷建军等,9种调血脂药物有效性及安全性评价[J],中国循证医学杂志,2005,5(1)：8-21
[15]王海勇,降血脂药物的研究进展,国外医学药学分册,2004,31(3)160-165
[16]程志清,中医药防治高脂血症[M],北京,人民卫生出版社,2002
[17]潘琴,降血脂药物临床应用的不良反应概述,strait pharmaceutical Journal,2013,25 (5) 293-295
[18]中国科学院中国植物志编辑委员会,中国植物志(第一卷)[M],北京：科学出版社,2004：147
[19]傅立国,陈潭清,郎楷永等,中国高等植物(第三卷)[M].青岛：青岛出版社,2003：325-326
[20]唐·苏敬撰.尚志钧辑较.新修本草(辑复本)[M]合肥：安徽科学出版社,1981：369.
[21]国家药典委员会编.中华人民共和国药典[M],北京：化学工业出版社,2005：163.
[22]Atal C K,Banga S S. Ind. J. Pharm,1962,24:105
[23]Chatterj ee A, Dutta C P. Alkaloids of Piper longum Linn. I. Structure and synthesis of piperlongumine and piperlonguminine[J]. Tetrahedron,1967,23(4):1769-1781
[24]Tabuneng W,Bando H,Amiya T, Studies on the Constituents of the Crude Drug "Piperis Longi Fructus" on the Alkaloids of Fruits of Piper Longum L. [J]. Chem. Pharm. Bull,1983,31(10):3562
[25]Badu D D,Ayim J S K,Dabra T T, et al. △αβ-Dyhydro piperlonguminine, A New Amide from Piper Guineense[J]. Phytochemistry,1976,15:822
[26]Okogum JI, Ekong D E U J. Chem. Soc,1974, Perkin I:2195
[27]Nakatani N,Inatani R, Isobutyl Amides from Pepper (Piper nigrum L.) [J]. Agri. Biol Chem, 1981,45(6):1473
[28]Tackie A N, Badu D D, Ayim J S K, N-isobutyloctadeca-Trans-2-Trans-4-Dienamide:A New Constituent of Piper Guineense[J]. Phytochemical Reports,1975,14:1888
[29]Raina M L, Dhar K L, Atal C K, Occurrence of N-isobutyl Eicosa-Trans-2-Trans-4-Dienamide in Piper nigrum[J]. Planta Medica,1976,30:198
[30]Gupta O P, Gupta S C, Dhar K L,et al, A New Amide from Piper Officinarum[J]. Phytochemistry,1977,16:1436
[31]包照日格图,吴恩,荜茇油非皂化物对小鼠高脂血症的影响[J],中草药,1992,23(4)：197
[32]包照日格图,吴恩,荜茇油非皂化物对小鼠胆固醇代谢的影响[J],中国药理学通报,1991,7(6)：463
[33]斯庆图娜拉,查干布拉格,荜茇研究[J].中国民族医药杂志,2006,3(17)
[34]Min K R, Kim K S, Ro J S, et al.Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells[J]. Planta medica,2004,70(12):1115.
[35]Qi, Hong-Shunl, Liu, Pei2, Inhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloid beta and APP in SK-N-SH Cells, CHINESE JOURNAL OF PHYSIOLOGY,2009,52 (3) 160-168
[36]Rahman T, Rahmatullah M, Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 2010,20 (2) 537-540
[37]Silva DR (Silva, Daniel Rodrigues)1, Endo EH (Endo, Eliana Harue)1, Chemical Composition and Antimicrobial Properties of Piper ovatumVahl, MOLECULES,2009,14 (3) 1171-1182
[38]Bezerra DP (Bezerra, Daniel P.)1, Pessoa C (Pessoa, Claudia) 1, In vivo growth inhibition of sarcoma 180 by piperlonguminine, an alkaloid amide from the Piper species, JOURNAL OF APPLIED TOXICOLOGY,2008,28 (5) 599-607
[39]Rodrigues Silva, D; Baroni, S; Svidzinski, A E; Bersani-Amado, C A; Cortez, D A G, Anti-inflammatory activity of the extract, fractions and amides from the leaves of Piper ovatum Vahl (Piperaceae). J Ethnopharmacol,2008,116(3) 569-573
[40]包兰兰,金桩,博格日勒图,胡椒碱降血脂作用的实验研究[J],中国民族医药杂志,2004,1：22.
[41]Piyachaturawat P, Glinsukon T, Toskulkao C. Acute and subacute toxicity of piperine in mice, rats and hamsters[J].Toxicol Lett,1983,16(3-4):351-359
[42]Daware M B, Mujumdar A M, Ghaskadbi S. Reproductive toxicity of piperine in Swiss albino mice[J]. Planta Med,2000,66(3):231-236.
[43]Jin,Z; Borjihan, G; et al, Antihyperlipidemic Compounds from the Fruit of Piper longum L.,Phytotherapy research,2009,23(8)1194-1196
[44]麻春杰,博格日勒图等,荜茇宁降血脂和急性毒性实验研究[J],中华中医药杂志,2008,23(4)321-324
[45]麻春杰,博格日勒图等,荜茇宁对家兔实验性动脉粥样硬化的影响[J],中国中药杂志,2008,33(4)436-440
[46]麻春杰,博格日勒图等,荜茇宁对动脉粥样硬化家兔抗氧化酶活性的影响[J],中国药科大学学报,2008,39(1)68-71
[47]麻春杰,博格日勒图,荜茇宁对高脂血症大鼠血脂代谢及其相关基因表达的影响[J],中草药,2008,39(7)1039-1043
[48]《化学药物质量控制分析方法验证技术指导原则》2005.3指导原则编号[H]GPH5-1
[49]《化学药物质量标准建立的规范化过程技术指导原则》2005.3指导原则编号[H]GPH1-1
50、 [50]周怡,化学药品标准中杂质控制及其限度的建立,,药物生物技术[J],2010,17(2)：181-184
51、 [51]王平,药品质量控制与质量标准,中国职业药师[J],2008,5(7)38-39
52、[52]马磊,化学药物质量标准建立的一般过程,中国新药杂志[J],2009,18(14)1297-1299
[53]马玉楠,冯毅,美国FDA关于上市前化学药药学研究的技术要求,中国新药杂志[J],2005,14(7)805-80
[54]胡胜,袁继容,朱进,等.选择反应法制备高纯油酸[J].应用化工,2005,34(12)：748-751.
[55]解振兵,卢义和,宫素芝,等.混合脂肪酸选择性加氢制高纯度油酸[J].日用化学品科学,2006,29(1)：19-21.
[56]赵国志,刘喜亮,刘智锋,等.反式脂肪酸危害与控制[J].粮食与油脂,2007(1)：7-13.
[57]Nobuyuki Kozukue,Mal-Sun Park, Kinetics of Light-induced Cis-Trans Isomerization of Four Piperines and Their Levels in Ground Black peppers as Determined by HPLC and LC/MS,J.Agric.Food Chem.,Vol.55,No.17,2007
[59]Lee EB, Shin KH and Woo WS, Pharmacological study on piperine. Arch Pharmacol 1984,7:127-132
[60]Kanaki N, Dave M, Padh H and Rajani M, A rapid method for isolation of piperine from the fruits of piper nigrum linn. J Nat Med,2008,62:281-283
[61]Rahman T and Rahmatullah M Proposed structural basis of interaction of piperine and related compounds with monoamine oxidases. Bioorg Med Chem Lett 2010,20:537-540
[62]Bezerra DP, Pessoa C,Moraes MO, Alencar NMN, Mesquita RO, Lima MW, Alves APNN, Pessoa ODL, Chaves JH, Silveira ER and Costa-Lotufo LV, In vivo growth inhibition of sarcoma
180 by piperlonguminine, an alkaloid amide from the Piper species. J Appl Tosicol,2008, 28(5):599-607
[63]Kim KS, Kim JA, Eom SY, Lee SH, Min KR and Kim Y, Inhibitory effect of piperlonguminine on melanin production in melanoma B16 cell line by downregulation of tyrosinase expression. Pigment Cell Res.2005.19:90-98
[64]Lin Z, Liao Y, Venkatasamy R, Hider RC and Soumyanath A Amides from Piper nigrum L. with dissimilar effects on melanocyte proliferation in vitro. J Pharm Pharmacol (2007)59:259-536
[65]Jin Z, Borjihan G, Zhao RG, Sun Z, Hammond GB and Uryu T (2009) Antihyperlipidemic compounds from the fruit of Piper longum L. Phytother Res 23(8):1194-1196
[66]Ma CJ, Alatangaole, Narisu, Zhaorigetau, Zhou HJ and Borjihan G (2008) Effects of piperlonguminine on genes expression of LOX-1 and VCAM-1 in the aortic of atherosclerosis rabbits. Chinese Pharm J 43(19):1449-1553
[67]Ma CJ, Ling X and Borjihan G (2011) Study on the blood lipid-lowering and anti-atherosclerosis function of piper longum L.and its effective Ingredients. World Science Technology/Modernization of Traditional Chinese Medicine and Materia Medica 13(1):133-135
[68]Roger A. Olsen and Gary O.Spessard,A Short, Stereoselective Synthesis of Piperine and Related Pepper-Derived Alkaloids,J.Agric.Food Chem.1981,29,942-944.
[69]Lee SH, Kim DH, Kim JA, Jahng Y, A simple synthesis of piperlonguminine, HETEROCYCLIC COMMUNICATIONS,2005,11 (5) 407-410
[70]张震,陈海峰,化学药品新药研究中的有关物质定性研究,中国新药杂志,2008,17(19)1721-1723
[71]领小,博格日勒图,娜日苏,荧光分析法测定荜茇有效成分荜茇宁,分析实验室,2009,28(6)21-23
[72]中国药典2010版二部,国家药典委员会,中国医药科技出版社
[73]化学药物质量控制分析方法验证技术指导原则,2005.3指导原则编号[H]GPH5-1
[74]化学药物质量标准建立的规范化过程技术指导原则,2005.3指导原则编号[H]GPH1-1
[75]张慧玲,浅谈药品注册中HPLC法测定原料药有关物质的问题,药物分析杂志,2007,27(6)947-948
[76]Nobuyuki Kozukue,Mal-Sun Park, Kinetics of Light-induced Cis-Trans Isomerization of Four Pipelines and Their Levels in Ground Black peppers as Determined by HPLC and LC/MS,J.Agric.Food Chem., Vol.55,No.17,2007
[77]许宏琪,盛龙生,安登魁,用色谱联用技术分析桂腈和溴代苏合香烯顺反异构体,色谱,1995,13(4)272-273
[78]游长江,烯烃光敏顺反异构化反应,感光科学与光化学,2003,3(2)：147-155.
[79]张震,陈海峰,化学药品新药研究中的有关物质定性研究,中国新药杂志,2008,17(19)1721-1723
[80]徐国华,殷国栋等,两个1,4-二酮衍生物的顺反异构体的确定及其1H,13C核磁共振信号归属,华中师范大学学报(自然科学版),2007 41(4)549-551
[81]彭惠琦,核磁振法测定化学位移及自旋耦合常数,实验室研究与探索,1998,6.,36-39
[82]谭英,利用NMR技术对紫苏草顺反异构体的研究[D],湖南师范大学,2003,04
[83]李锋,王敏.顺反异构化过程及其机理[J].化学试剂,2005,27(1)：22-22.
[84]Jin,Z; Borjihan, G; et al, Antihyperlipidemic Compounds from the Fruit of Piper longum L.,Phytotherapy research,2009,23(8)1194-1196
[85]许宏琪,盛龙生,安登魁,用色谱联用技术分析桂腈和溴代苏合香烯顺反异构体,色谱,1995,13(4)272-273
[86]王乃兴,核磁共振谱学在有机化学中的应用[M],化学工业出版社,2006年6月第一版
[87]百度文库,细胞培养基本技术,中国科技大学生命科学学院
[89]Grundy S M, Statin trial and goals of cholesterol lowering therapy, [J], Circulationl,1998,97 (15) 1436-9
[90]Dietschy J M, Dietary fatty acids and the regulation of plasma low dinsity lipoprotein cholesterol concentration [J], J Nutr,1998,128,444-8
[91]麻春杰,荜茇有效成分荜茇宁的调制作用及其机制研究[D],内蒙古大学博士论文,2008
[92]胡苗苗,GB-A对脂代谢紊乱及早期动脉粥样硬化的防治作用及其机制研究[D],上海：中国科学院上海药物研究所,2009.10
[93]王亚利,范春雷,窦晓兵,绞股蓝总皂苷对肝细胞低密度脂蛋白受体表达的影响,中国药理学通报,2010,26(1)：138-139
[94]庄晓,陈志,杜心,吴雅茗,阿魏酸钠对HepG2细胞低密度脂蛋白受体mRNA,中国临床保健杂志,2006,9(2)116-117
[95]Kuhlencordt PJ, Hotten S, Schodel J, Rutzel S, Hu K, Widder J, et al.Atheroprotective effects of neuronal nitric oxide synthase in apolipoprotein E knockout mice[J]. Arterioscler Thromb Vase Biol,2006,26:1539-1544.
[96]Barbato JE, Tzeng E. Nitric oxide and arterialdisease[J]. J Vase Surg,2004,40:187-193.
[97]王忠振,李芳,炎症与动脉粥样硬化及冠状动脉疾病的关系,中国微生态学杂志2012年9月第24卷第9期,862-865]
[98]Peter RH, Inflammatory altertions in the myocantiol microcirculation, J Mol Cell Cardio,1998,30(12):2555-2559
[99]Andrew PJ,Mayer B. Enzymatic function of nitric oxide synthases, Cardiovasc Res, 1999,43:521-531
[100]Barmaes PJ,et al.Time course and cellular localization of lipopolysaccharide induced inducible nitric oxide synthsase messenger RNA expression in the ratinvivo. Crit Care Med, 1997,25:512-518]
[101]Okamoto I, Abe M,Shibata K,et al. Evaluation the role of inducible nitric oxide synthaseusing an overland selective inducible nitric oxide synthase inhibitor in septicling injury produced by Cecalligation and puncture,AmJRespir Crit Care Med,2000,162:716-722
[102]何志旭,周同甫,廖清奎,徐学聚,罗眷华,李敛伯,王树,李丰益,炎性细胞因子及脂多糖诱导产生的一氧化氮对人血管内皮细胞的损伤作用,中国病理生理杂志,2001,17(9)：893—89
[103]麻春杰,阿拉坦高勒,娜日苏等,荜茇宁对动脉粥样硬化家兔主动LOX-1和VCAM-1基因表达的影响,中国药学杂志,2008,43(19)1449-1452。
[104]麻春杰,博格日勒图等,荜茇宁对家兔实验性动脉粥样硬化的影响,中国中药杂志,2008,(4)436-440。
[105]Son, Dong Ju; Kim, Soo Yeon; Han, Seong Su; Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,427 (2) 349-354