Study On The Synthesis And Lipid - Regulating Effect Of Piperine Derivatives

Piperine, derived from Pepper and Piper longum, is an important active alkaloids. As a common medication, Piperine has antioxidant, anti-inflammatory, antineoplastic, anticonvulsion as well as immune adjustment properties. In recent years, many papers have reported the Hypolipidemic and Anti-atherosclerosis Effect of Piperine, but Piperine has the obviously toxicity, which is the major hurdles in the exploitation of clinical application. There were some reports have explored that the derivatives of Piperine had significant hypolipidemic effect and low toxicity. Piperlonguminine is a chemical compounds of natural products fromPiper longum. It is a Piperine derivatives with Piperine as raw material. There were some reports have explored that the derivatives of Piperine had significant hypolipidemic effect and low toxicity. The result of Piperine metabolic product research was showed that the piperonyl of carbon and oxygen five rings was open loops in the Piperine metabolites of human body and It was generated metabolin which had a phenolic hydroxyl structure. In order to examine the phenolic hydroxyl structure of metabolite of lipid effect and toxicity, we synthesized three kinds of compounds GBOT, GBOX and GBNT with Piperine as raw material, authenticated their structures, investigated the hypolipidemic effect and Anti-oxidation effect, and proved the mechanism of hypolipidemic effect, and investigated the interaction with Bovine serum albumin (BSA), to explicit this piperine derivate’s lipid-lowering activities. In this dissertation, including chemical, medical, biological and pharmacological pharmacokinetics study methods has been used, and the main contents consists of the folling five aspects.Firstly, optimized middle test and prepared three piperine derivatives employing piperine as the raw material. After that, HPLC, IR, UV, NMR and MS methods were utilized to determine their structures and purities for further study. The result showed that three derivatives of piperine including GBOT、GBOX、GBNT were synthesized successfully with the semi-synthesis method. Among them, GBNT was found to be a new compound. Besides, all of them can definitely meet the needs of later researches with the purities higher than 98%.Secondly, the cytotoxicity of GBO, GBN, GBOT, GBOX, GBNT and simvastatin on the human liver cancer cell HepG2 was compared by using the MTT method. The result of cytotoxicity test showed that the IC50 of the GBO, GBOT, GBOX, GBN and GBNT and simvastatin was 0.160 mmol·L-1、0.895 mmol·L-1、4.002 mmol·L-1、7.184 mmol·L-1、9.529 mmol·L-1 and 0.520 mmol·L-1 respectively, and the cytotoxicity of GBOT、GBOX、GBNT was lower than simvastatin and piperine.Thirdly, the influence of the three derivatives of Piperine was investigated on the dyslipidemia (HLP) rat model, and the lipid and autioxidant enzyme activities in serum and hepatic tissue were detected, and the pathomorphism of the hepatic tissue was observed. The animal experiment results showed that GBOT and GBNT were able to decrease the content of TC、TG、LDL-C and the AI value significantly in the serum of rat, to enhance the content of HDL-C and to decrease the content of TC、TG in the liver of HLP rat in a dose-dependent; the high-dose group of GBOT whose the effects on decreasing the TG of serum and hepatic and increasing the content of serum HDL-C is better than simvastatin, with statistical significance. Besides that, by observation the pathological morphological changes of the hepatic tissue using the light microscopy and electron microscopy (sem), we can find that GBOT and GBNT were able to significantly decrease the lipid accumulation in the HLP rats liver and to improve the severity of hepatic steatosis and lipid metabolism disorders in HLP rats. GBOX played a significant role in the regulation of TC and TG, but had no effect on LDL-C and HDL-C, and the lipid regulating effect was similar to GBO.It indicated that lipid-lowering activities of the piperine derivate was enhanced by drawing into phenolic hydroxyl structure. GBOT and GBNT were able to significantly enhance the activity of SOD、CAT and GSH-Px in HLP rats liver and to decrease the conent of MDA, thus to enhance the antioxidant ability of engine body. The result showed that GBOT, GBNT in the body reduced the degree of lipid peroxidation, and hence promoted lipid-lowering effect by adjusting the activity of antioxidant enzyme.Fourthly, by RT-PCR technology determinated the influence of mRNA impression of Fat metabolism related genes of GBOT、GBNT for HLP rat of liver, include LCAT, CYP7A1, LDLR and HMG-CoAR. We proved the influence of LCAT、LDLR、CYP7A1 expression of the piperine derivate by the Western blotting. The result showed that the GBOT、GBNT were verified to facilitate the expression of mRNA of LDLR、LCAT、CYP7A1 in HLP rats, but It didin’t influence for the mRNA expression of G CoA-mRNA. In additions, we also proved the influence of GBOT、GBNT on the expression of protein of LCAT、LDLR、 CYP7A1. The GBOT、GBNT were regulated dynamic equilibrium of lipid metabolism on molecule level, exerted lipid-lowering effect.Fifth, we investigated the interaction of GBO, GBOT, GBOX, GBN and GBNT with bovine serum albumin (BSA) by using fluorescence spectra and ultraviolet spectroscopy. The results proved that the five kinds of drugs can stronger quench fluorescence of BSA. GBO and BSA could be formed not hair fluorescent compounds. It was quenched the fluorescence By static quenching method. The GBOT, GBOX, GBN and GBNT were quenched the endogenous fluorescent of BSA by dynamic quenching method. Five kinds of compounds with the combination of the distance between tryptophan residues on the BSA r were less than 7 nm, showed that they could be in the form of the energy transfer with BSA. Hydrophobic force is the main force between GBO, GBOT, GBOX and BSA, while the main forces between GBN and BSA were hydrogen bonding and van der Waals force. Besides, the electrostatic force is the main driving force between GBNT and BSA,and the processes is spontaneous. The comparison of association constant of various drugs and BSA:K(GBO)> K(GBOT)≈K(GBOX)>K(GBN)≈K(GBNT), The comparison of binding-site of various drugs and BSA:n(GBO)>n(GBOT)>n(GBOX)>n(GBN)≈n(GBNT), The results of binding constant and binding site showed that the binding force betwenn GBO and BSA is the strongest which might be the one reason for the certain toxicity of GBO. The binding force between GBOT, GBN, GBNT and BSA is moderate. They not only could be storaged and transported to the target organs in the form of temporary binding with plasma albumin, but also could maintain the plasma concentration in a certain level, which might be the one reason for that the efficacy of GBOT, GBN and GBNT is superior to GBO. This study would provide the basis of pharmacokinetic for development of GBOT and GBNT.The new compound, GBNT, was first synthesised in this dissertation. GBOT and GBNT have significant regulatory function in lipid metabolic disorders of rat and lower cell toxicity are the satisfactory drugs to adjustable the blood-fat. The lipid management mechanism of GBOT and GBNT might be associated with the increasing of mRNA expressions of LDLR, LCAT and CYP7A1, the enhancing of activity of SOD、CAT、GSH-Px, and the improving of antioxidant ability of engine body, which was completely different from the HMG CoA reductase inhibitor mechanism of simvastatin. From the angle of pharmacokinetics, GBOT and GBNT have considerable possibility to be developed clinical medication for that the serum albumin binding of GBOT and GBNT was moderated. The results obtained from our study would not only provide experimental basis for the further research, but also have an active meaning for the development of new drug.