【作者】 曾晓会；

【导师】 王培训；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 高脂血症是人体脂质代谢失常导致血浆内脂质浓度超过正常范围的病症,是引发动脉粥样硬化、冠心病及高血压的主要危险因素。近年来,高脂血症的发病率呈不断上升的趋势,多数降脂药仅有短期疗效,长期使用则会出现明显的毒副反应。辨证论治、因人、因时、因地制宜的个体化治疗体系是中医的诊治特点,中药通过多环节、多层次、多靶点的整合调节作用,符合人体多样性的特点。故从中药、天然药物中寻找安全有效的调脂药物是目前发展的趋势。姜黄素所具有的降血脂、抗突变、抗氧化、清除自由基等药理作用,使其成为在治疗高脂血症方面较有发展前途的药物。但是,姜黄素本身存在的一些问题,例如脂溶性高、水中溶解度小、稳定性差、在肝脏和肠道内易分解转化等,导致其口服血药浓度低、生物利用度差、用药量大,从而大大限制了临床推广使用和新药的开发。针对这些问题,本课题将姜黄素与已知的肝、肠内葡萄糖醛酸转移酶抑制剂-胡椒碱合用,通过抑制肝肠内姜黄素的葡萄糖醛酸化,从而减少姜黄素的代谢;并采用微囊化技术,以增加其稳定性和水溶性,促进其吸收,减少服用次数,维持持久药效,以提高生物利用度。本课题的主要研究内容如下:研究目的:本课题以期寻找姜黄素与胡椒碱最佳组方比例;探讨通过微囊化工艺改进后,姜黄素在动物体内的药代动力学过程,以及加入胡椒碱后对姜黄素药代动力学过程的影响;考察姜黄素和胡椒碱组方-复方姜黄微囊的调脂效应,并与单独应用姜黄素相比,加入胡椒碱后对姜黄素的调脂作用的影响。以期解决一直围绕姜黄素水溶性差、稳定性不好、易分解代谢、药理活性低等问题,最大的发挥姜黄素的调脂效应,为姜黄素和胡椒碱组方提供佐证,并为将复方姜黄微囊开发为安全、有效调脂新药打下基础。研究方法:①通过传代培养复制Caco-2细胞模型;采用“门静脉二步灌流分离法”进行正常和高脂血症模型大鼠肝脏原位灌流,然后进行悬浮培养建立原代肝细胞模型。进行细胞活性评定后,开展药物细胞毒性实验,计算最大无毒浓度,由此确定药效实验最大给药剂量。然后以与姜黄素肝肠内代谢密切相关的β-GD为观察指标,观察姜黄素与胡椒碱不同比例降低β-GD活性的差异。通过尾静脉注射Triton WR-1339 250mg/kg复制急性小鼠高脂血症模型,考察姜黄素与胡椒碱不同比例的调脂效应,以期确定姜黄素与胡椒碱组方的最佳比例。②通过灌胃给予不同剂量的姜黄素微囊制剂,用HPLC测定血浆和胆汁中姜黄素的含量,用DAS2.1软件计算结果,分析姜黄素微囊在大鼠体内的药动学过程。并考察姜黄素与胡椒碱为9:1、36:1和144:1时组方-复方姜黄微囊的大鼠体内药动学过程,以及评价胡椒碱对姜黄素药代动力学过程的影响。③将动物分为空白对照组、模型对照组、阳性药物组、复方姜黄微囊高剂量组、复方姜黄微囊低剂量组、姜黄素高剂量组和姜黄素低剂量组。通过尾静脉注射TritonWR-1339复制急性小鼠高脂血症模型,和灌服高脂乳剂复制慢性食饵性大鼠高脂血症模型,检测动物血清、肝脏组织、粪便和胆汁中脂质、脂肪代谢酶、胆汁酸、载脂蛋白等成分的含量,对姜黄素和胡椒碱组方的调脂效应进行评价。同时与单独应用姜黄素进行比较,探讨胡椒碱对姜黄素调脂效应的影响。研究结果:①Caco-2细胞在3-5天内处于对数增殖期,故选择3-5天时的细胞进行药效研究。药物细胞毒性实验研究结果显示,胡椒碱TC₅₀=1.74mmol/L,TC₁=0.084 mmol/L,姜黄素TC₅₀=10.7mmol/L,TC₁=0.25mmol/L,TC₀=0.266 mmol/L,故姜黄素和胡椒碱分别以0.266mmol/L和0.084 mmol/L为药理实验研究的最大给药浓度。②与细胞对照组相比,在1h和3h时,姜黄素与胡椒碱组方在8:1-33:1之间均有显著降低肝细胞内β-GD活性的作用;姜黄素与胡椒碱组方在8:1-65:1之间均有显著降低Caco-2细胞内β-GD活性的作用。③与模型组比较,姜黄素与胡椒碱配比C组、配比D组和配比E组（10.4:1-41.6:1）均明显降低小鼠血清TC含量（P＜0.05-0.01）;配比D组和配比E组（20.8:1-41.6:1）也明显降低小鼠血清TG含量（P＜0.01）。单独应用姜黄素有明显降低TC含量的作用（P＜0.05）;单独应用胡椒碱没有显著性降低TC和TG水平的作用（P＞0.05）。其中配比E组（41.6:1）对TC和TG的抑制率为最高。通过聚类分析发现配比D组（20.8:1）和配比E组抑制TC和TG含量的百分率属同一类,即抑制效应无显著性差异。故在相同抑制效应情况下,应选择药物剂量小即配比D组作为药效实验的最佳配比剂量。④确定了分别用乙酸乙酯和酸分解法萃取血浆和胆汁中姜黄素的方法;精密度、稳定性和加样回收率等方面的研究结果提示所建立的方法符合生物样品检测的要求。⑤姜黄素微囊口服制剂在大鼠体内的代谢过程出现双峰现象。姜黄素高、中、低剂量的Cmax分别为1.16、0.55、2.66 mg/L;Tmax分别为1.5、1.1和0.78h;AUC分别为3.79、2.60和5.92。由此可见,姜黄素的药动学符合非线性过程,低剂量AUC和Cmax明显比高、中剂量高。与姜黄素脂质体和混悬液相比,本室制备的姜黄素微囊制剂低剂量以58.6mg/kg灌胃给药,将姜黄素的峰浓度提高到2.66mg/L,分别是姜黄素混悬液和脂质体的5.78和5.02倍;姜黄素微囊的AUC提高到5.92 mg·h/L,分别为姜黄素混悬液和脂质体的7.6倍和8.4倍,说明姜黄素微囊制剂明显提高了血药浓度和生物利用度。⑥对姜黄素和胡椒碱不同比例组成的复方进行了药动学研究,胡椒碱为姜黄素1/9、1/36、1/144和单独应用姜黄素时,Cmax分别为3.45、4.14、2.43、2.23 mg/L:Tmax分别为2、3、1.05、0.75h;AUC分别为13.47、16.49、8.7、8.07。由此可见,加入胡椒碱后,Cmax和AUC明显升高,Tmax延长,尤其以姜黄素:胡椒碱=36:1的Cmax和AUC升高最明显。其Cmax为4.14mg/L,分别是姜黄素混悬液和姜黄素脂质体的9倍和7.8倍;AUC为16.49 mg·h/L,分别是姜黄素混悬液和姜黄素脂质体的21倍和23.6倍。⑦姜黄素单独应用对血清、肝脏和粪便中脂质（TC、TG）含量均有降低作用,揭示姜黄素具有一定的降脂效应;但对血清和肝脏中脂质（HDL-C和LDL-C）含量无显著性影响;对胆汁和粪便中胆汁酸、脂肪代谢酶和载脂蛋白也无显著性影响。⑧姜黄素与胡椒碱以20:1组成的复方姜黄微囊有明显降低血清、肝脏中脂质（TC、TG和LDL-C）和升高HDL-C含量的作用;还有促进胆汁和粪便中TC、TG和胆汁酸分泌和排泄的作用;增强血液载脂蛋白（apoAI、apoB及apoAI/apoB）活性的作用;增强血液和肝脏脂质代谢酶（LPL、HL、LCAT）的活性;此外,还有增强抗脂质过氧化酶（SOD和GSH-PX）的活性等作用。⑨与单独应用姜黄素相比,复方姜黄素微囊在降低血液和肝脏中脂质含量;促进胆汁和粪便中TC、TG、胆汁酸排泄:增强载脂蛋白、脂质代谢酶和抗脂质过氧化酶的活性等方面有显著性差异。研究结论:①通过离体细胞实验的研究,并结合以高脂血症小鼠为模型开展的姜黄素与胡椒碱配比筛选结果,认为20:1是姜黄素与胡椒碱的最佳配比。②建立了稳定的血浆和胆汁中姜黄素的HPLC检测方法。姜黄素微囊口服制剂在大鼠体内的药代动力学符合非线性过程。在血浆中的姜黄素药时曲线呈双峰现象,可能存在肝肠循环;也可能是因为姜黄素快速分布到其他组织,再次吸收入血导致第二个峰的出现,具体原因尚需进一步研究证实。低剂量的AUC和Cmax明显比高、中剂量高,可能是因为低剂量最接近临床拟用量和动物药效剂量,所以更易吸收入血。③与姜黄素脂质体和混悬液相比,本室制备的姜黄素微囊制剂明显提高了血药浓度和生物利用度。与单独应用姜黄素相比,加入胡椒碱后姜黄素的血药浓度和生物利用度明显提高,提示胡椒碱作为肝肠内葡萄糖醛酸苷酶结合抑制剂,可能通过抑制肝肠内姜黄素的葡萄糖醛酸化,提高了姜黄素的生物利用度。通过开展本研究,可望为其新制剂的设计和临床合理用药提供参考。④复方姜黄微囊对高脂血症小鼠模型和慢性食饵性高脂血症大鼠模型均有显著调脂作用;与单独应用姜黄素相比,复方姜黄素微囊的综合调脂效应明显强于单独应用姜黄素。⑤复方姜黄微囊调脂作用可能与以下几点有关:（1）抑制肠道外源性脂质吸收,增加粪便中脂质（胆固醇和胆汁酸）含量,促进脂质排泄;（2）增强血液载脂蛋白（apoAI、apoB及apoAI/apoB）和脂肪酶（LPL、HL、LCAT）活性,促进血脂转化,加速血液TC和TG的代谢及脂质肝脏转运;（3）增强肝脏脂质代谢酶活性,加速肝脏对TC、TG代谢,增加胆汁分泌及胆汁中TC含量;（4）通过增强抗脂质过氧化能力（SOD和GSH-PX）,抑制脂质过氧化;（5）通过降低血液与肝脏脂质含量,抑制肝脏脂肪样病变。通过本课题的研究,为姜黄素和胡椒碱组方提供了实验依据,并验证了复方姜黄微囊有明确的调脂效应,提示复方姜黄微囊可能是一种有发展前景的有效调脂新药,以期为降脂中药新药的研发提供参考,探索出一套适合于中药新药研究的新思路和方法。更多还原

【Abstract】 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.更多还原