The Influence Of Piperine On Metabolism And Effect Of Regulating Lipid Metabolism Of Curcumin

Hyperlipidemia,a disease of disorder of lipid metabolism in human,leads to the fact that plasma lipid concentrations exceed the normal range.Therefore,It is the major factor that causes atherosclerosis,coronary heart disease and high blood pressure.However,in recent years,the incidence of hyperlipidemia has been rising increasingly,and most of the lipid lowering drugs have only short-term efficacy,while Long-term use will result in toxicity.Therefore,to find safe and effective lipid lowering drugs from the traditional Chinese and natural medicine is the present trend for the treatment of the Hyperlipidemia. On one hand,the traditional Chinese medical diagnosis and treatment is characterized by its dialectic,which means,more specifically,an individual treatment basing on the patient,the time of the illness as well as the place where the treatment is implemented.On the other hand,the traditional Chinese medicine could integrate all its regulatory function through multi-links,multi-levels,multi-targets,which is in accordance with the diversity in human body.Curcumin has lots of pharmacological functions,such as curing hypolipidemic, anti-mutagenic,anti-oxidation,scavenging free radicals,which renders it to become more promising than other drugs in the treatment of hyperlipidemia.However,curcumin itself has a number of problems,such as high-fat-soluble,small water solubility,poor stability, easy decomposition and transformation in the liver and intestine,resulting in low serum concentration,poor bioavailability and large doses,which greatly limit its clinical use and the development of new drugs.To solve these problems,the dissertation combines curcumin with piperine,the glucuronosyltransferase inhibitors of liver and intestinal.By inhibiting the glucuronic acid of curcumin,the metabolism of curcumin will be reduced; and the use of micro - capsule technology will increase its stability and water-soluble, promote its absorption,reduce the frequency of use,and maintain a lasting effect;all the above will enhance its bioavailability.The main research topics of the dissertation are as follows: Objective:The dissertation firstly tries to explore the changes of the pharmacokinetics process of curcumin in rats after the technical improvement through the microcapsules as well as the effect after adding the piperine.Basing on the above experiment,the author manages to find the best set of square ratio of curcumin with piperine;Then,the author also studies the blood lipid regulating effects of Fufang Jianghuang microcystis,a Chinese medicine whose main ingredient is curcumin and piperine and compares the blood lipid regulating effects after the adding of piperine.Finally,The dissertation tries to solve those disadvantages of Curcumin,such an small water solubility,poor stability,easy decomposition and transformation and the low pharmacological activity,so as to realize the full play of the lipid effects of curcumin,to provide support for curcumin and piperine as a group,and to lay foundation for developing Fufang Jianghuang microcystis into a safe and effective new drug.Methods:①To orally give curcumin microcapsules to the rats;to determine the content of curcumin in plasma and bile by HPLC;to process the data by software DAS2.1;to analyze the pharmacokinetics results of curcumin microcystin in rats.Furthermore,when the ratio ofpiperine and curcumin is 1:9,1:36 or 1:144,to inspect the pharmacokinetics process and evaluate of piperine on the pharmacokinetics of curcumin.②To cultivate and copy Caco-2 cell model;to apply "two-step portal vein perfusion separation" into normal and hyperlipidemic rat model of liver perfusion in situ,in suspension culture and then to proceed the establishment of primary liver cell model;to Assess cell activity then to carry out experiment of the cytotoxic drug;to calculate the maximum non-toxic concentrations and to determine the maximum dose effect for the experiments.And then to takeβ-GD as the observation indicators to study the reducing difference ofβ-GD activity;Finally,to combine the experimental results of the rats with pharmacokinetic results to determine the effect of best ratio for curcumin and piperine group.③To divide animals into blank control group,model control group,positive medicine group,Fufang Jianghuang Microcystis high-dose group,Fufang Jianghuang Microcystis low-dose group,high doses of curcumin and curcumin low-dose group;through the tail vein injection of Triton WR-1339 to copy the model of acute hyperlipidemia in rats,and to feed them with high fat emulsion then to copy rats with chronic hyperlipidemia prey model;Then to exam the content of Lipid,fat-metabolizing enzymes,bile acids,and apolipoprotein in animal serum,liver,feces and bile;to evaluate the effect of the Fufang Jianghuang Microcyst;at the same time,to discuss the influence of piperine on the effect of curcumin,then with curcumin alone for comparison.Results:①Having identified the methods of extracting curcumin with ethyl acetate and the acid decomposition method in plasma and bile respectively;the results of precision,stability, recovery and processing suggest that the method established in line with the requirements of testing biological samples.②Bimodal phenomenon of metabolic process has appealed in Microcystis oral formulations of curcumin in rats.When the curcumin is at the level of high,medium and low,the doses of Cmax were 1.16,0.55,2.66 mg / L respectively,the Tmax were 1.5,1.1 and 0.78h respectively,and the AUC y were 3.79,2.60 and 5.92 respectively.This proves that the pharmacokinetics of curcumin is in accordance with the non-linear process,that' s to say,the AUC and Cmax of low-dose,is obviously much higher than that of the high-dose and the middle dose.Compared to the curcumin liposome and suspension,the preparation of the curcumin microcapsules whose peak concentration has been increased to 2.66mg/L when the dose is 58.6mg/kg,which is 5.78 and 5.02 times of liposome and suspension respectivily;the AUC of curcumin microcapsules has been increased to 5.92 mg.h/L,which is 7.6 times and 8.4 times of curcumin liposome and suspension respectively.All the above findings have proved that the curcumin microcapsules have significantly increased the plasma concentration and bioavailability.③Research on the pharmacokinetics of different ratios of the curcumin and piperine group has been implemented:When piperine for curcumin is 1/9,1/36 or 1/144 and a separate application curcumin,Cmax were 3.45,4.14,2.43,2.23 mg/L;Tmax were 2,3,1.05,0.75 h;AUC were 13.47,16.49,8.7,8.07.It can be seen that after adding piperine, Cmax and AUC have been increased greatly and the Tmax has also been extended, especially when the proportion of curcumin and piperine is 1 to 36,Cmax and AUC has been increased with the greatest scale.Cmax is the 4.14mg/L,which is 9 times and 7.8 times of curcumin liposomes and suspension respectively;AUC is 16.49 mg.h/L,which is 21 times and 23.6 times of curcumin liposome and curcumin suspension respectively.④Caco-2 cells proliferate at their maximum speed during the period of 3-5 days,so the efficacy studies of the cell has been carried out within this period Experimental study on cytotoxic drugs has showed that when the piperine TC50 is 0.497mg/mL,TC1 is 0.024mg/mL,curcumin TC50 is 3.9247mg/mL,TC1 is 0.098mg/mL,then the curcumin and piperine is 0.098mg/mL and 0.024mg/mL respectively,which is the greatest concentration of drug delivery. ⑤Compared to The control group,in 1h and 3h,when the group of piperine and curcumin is in 1:8～1:33,they could significantly reduce theβ-GD activity of in liver cells; when piperine and curcumin is in 1:8～1:65,they have the function of significantly reducingβ-GD activity in Caco-2 cells.In vivo study,it has been found that when piperine and curcumin group is 1:20,they could greatly decrease the fβ-GD activity in liver and intestinal tissue,while no significant impact has been found on fβ-GD activity in liver and intestinal tissue by using curcumin only.Therefore piperine may play the role of inhibitingβ-GD activity.⑥Curcumin alone could reduce lipid(TC,TG) content in serum,liver and fecal,which reveals curcumin has a lipid-lowering effect.However,curcumin has no significant impact on lipids(HDL-c and LDL-c) content of serum and liver;nor does it have effect on bile acids,fat-metabolizing enzymes and apolipoprotein of bile and fecal.⑦Fufang Jianghuang Microcystis(Piperine and curcumin in turmeric 1:20)has the function of significantly lowering serum and liver lipid(TC,TG and LDL-c) and increasing HDL-c content in the role;It could promote bile and faeces in TC,TG and bile acid secretion and excretion of the role;In addition to these,it could also enhance blood apolipoproteins(apoAI,apoB and apoAI / apoB) activity,strengthen the blood and liver lipid metabolism enzymes(LPL,HL,LCAT) activity,as well as enhance the anti-lipid peroxidase(SOD and GSH-PX) activity.⑧Compared with the separate application of curcumin,Fufang Jianghuang Microcystis has obvious differences in the following aspects:the reduction of blood and liver lipid content;the promotion of bile and feces of TC,TG;bile acid excretion,the enhancement of apolipoprotein,lipid metabolizing enzymes and anti-lipid oxidase activity.Conclusion:①A stable method to exam the curcumin in plasma and bile by HPLC has been established.②The pharmacokinetics of curcumin microcystis in the rat is consistent with the non-linear process.Curcumin in the plasma concentration-time curve is bimodal.It may be caused by enterohepatic circulation,or by the rapid distribution to other organizations, that's to say the second absorption of the blood leads to the emergence of the second peak. AUC and Cmax of low-dose are significantly higher than that of the high-dose and the medium-dose,which could be explained by the fact that the low-dose is mostly near to that of the clinical efficacy dose and animal consumption,so it is more easily absorbed into the blood. ③Compared with the preparations of curcumin liposome and suspension,the curcumin microcapsules has markedly enhanced the plasma concentration and bioavailability.Compared with the separate application of curcumin,curcumin plasma concentration and bioavailability has markedly been improved after adding the piperine, which suggests that piperine,as the glucuronosyltransferase inhibitor in the liver and intestine,may increase the bioavailability of curcumin.This research is expected to provide rational reference for the design of its new agents and clinical rational drug use.④Through in vitro cells experiments and animal experiments in vivo study and the combination of hyperlipidemia in mice as a model to carry out the ratio of curcumin and piperine screening results with the pharmacokinetic studies,the dissertation has found out that 1:20 is best ratio of the piperine with the curcumin.⑤Fufang Jianghuang microcapsules has a significant hypolipidemic role in mouse model of hyperlipidemia and chronic predator rat model of hyperlipidemia;compared with the separate application of curcumin,Fufang Jianghuang microcapsules is stronger than curcumin alone in the function of regulating the lipid comprehensively.⑥Fufang Jianghuang Microcystis regulation of blood lipids may be related with the following points:(1)Inhibition of intestinal exogenous lipid absorption,increase of fecal lipid(cholesterol and bile acid) levels,and promotion of lipid excretion;(2)Enhancement of the blood apolipoprotein(apoAI,apoB and apoAI / apoB) and lipase(LPL,HL,LCAT) activity,the promotion of lipid transformation,acceleration of the blood TC and TG in liver lipid metabolism and transport;(3)Enhancement of the activity of the liver lipid metabolism and speeding up TC,TG metabolism,and increase of the bile secretion and bile TC content;(4)By enhancing the capacity of anti-lipid peroxidation(SOD and GSH-PX), to inhibit lipid peroxidation;(5)By lowering the blood and liver lipid content,to resist the fatty liver lesions.The study is aimed to provide an experimental basis for the group of curcumin and piperine,and to verify that the compound has a clear effect of regulation of blood lipids, and to suggest that the compound may be a safe and effective new drugs of regulation of blood lipids.The study is aIso expected to provide reference for developing new Chinese medicine for lowering the lipid and to explore a new set of methodology suitable for the development of the new Chinese traditional medicine.