Studies On Analysis,Synthesis And Isomerization Of Hypolipidemic Active Compound Piperlonguminine From Natural Productc Piper Longum L

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.