姜黄素新剂型：自乳化和亚微乳给药系统的研究
|
摘要
姜黄素(Curcumin, Cur)是从姜科姜黄属植物的根茎中提取的有效成分,具有抗肿瘤、抗炎、抗病毒、抗氧化等多种药理作用。但Cur水溶性差,性质不稳定,体内代谢迅速,生物利用度低,体内难以达到有效浓度,严重制约了其开发成有效的抗癌药。如何改善Cur的各方面缺点,制备生物利用度高、用药量低的Cur制剂已经成为近年来药学工作者亟待解决的课题。
自乳化药物传递系统(self-microemulsifying drug delivery system,SMEDDS)是由药物、油相、非离子型表面活性剂和助表面活性剂组成的热力学稳定的均一液体剂型。它在水相中温和搅拌的情况下,能够形成粒径小于100nm的纳米乳粒,即为口服微乳。它可用作疏水性药物的载体,改善水不溶性药物的口服吸收,提高药物的生物利用度。
静脉注射亚微乳剂(sub-microemulsion)作为一种新型药物传递系统,是脂溶性药物特别是抗癌药物、麻醉药物及抗炎药物的良好载体。将药物制备成亚微乳剂可以解决疏水性药物的注射问题,增加药物稳定性,减少注射给药的刺激性,改善药物的组织分布特性,直接把药物运送到特定器官靶区,增加抗癌药物的作用强度等优点。
因此,本文系统地研究了Cur的理化性质、稳定性,在此基础上研制了姜黄素口服自乳化和静脉用亚微乳给药系统,通过正交设计、单因素考察等方法优化处方和制备工艺,并进行相关质量评价,采用药动学、药效学实验对两种剂型的体内过程、体内外抗肿瘤活性及作用机制进行研究。
第一部分处方前研究
处方前研究是制剂研究的基础,本部分重点考察了Cur的稳定性、溶解度和油水分配系数,为Cur口服SMEDDS和脂肪乳剂的处方设计提供依据。
研究方法:(1)建立HPLC测定制剂中Cur含量的体外分析方法;(2)测定Cur在不同pH介质中、光照及高温条件下的稳定性;(3)测定CurMCT溶液在高温条件下的稳定性;(4)应用正辛醇-水体系测定了Cur的油水分配系数;(5)测定Cur在不同的油相、表面活性剂、助表面活性剂中的溶解度。
结果显示:(1)HPLC法具有灵敏度高、辅料无干扰的特点,适用于Cur SMEDDS和亚微乳剂的质量控制;(2)Cur室温条件下在水、pH4、pH 5.8 PBS中比较稳定,在pH6.8条件下,48内药物降解17%,与pH3、pH7.8的PBS溶液混合后,立即产生白色沉淀;(3) Cur在室内光和4500lux光强下比较稳定,在80℃水浴条件下,24h内药物降解35%,降解半衰期t1/2为92.40 h-1;Cur MCT溶液在高温条件下比较稳定,12h内几乎没有药物降解;(4) Cur的油水分配系数为19204±192(Log P为4.210±0.005),说明其疏水性很强;(5)油相组成对Cur的溶解度有一定的影响,Cur在LCT中的溶解度最低,为1067μg·g-1,在MCT中的溶解度最高,为3332μg·g-1;在自乳化系统的油相中,Cur在肉豆蔻酸异丙酯的溶解度大于油酸乙酯;助表面活性剂中,Cur在乙醇中的溶解度大于丙二醇。
第二部分Cur SMEDDS的处方筛选、工艺研究和质量评价
选择SMEDDS常用辅料,利用正交试验、假三元相图、自乳化效率和粒径评价等方法,确定Cur SMEDDS的处方组成,并对该处方进行体外质量评价。
研究方法:(1)利用正交试验和假三元相图,确定不同辅料的自乳化能力;(2)通过溶解度试验确定Cur SMEDDS的基本辅料组成;(3)以自乳化效率和粒径为评价指标,考察油相比例、乳化剂/辅助乳化剂比例对粒径和自乳化效率的影响,进一步优化处方;(4)通过溶解度试验以及加水稀释后自乳化后溶液的状态,确定药物含量;(5)通过测定乳化后乳剂的pH值、自乳化时间、粒径、形态特征、表面电荷、稳定性和体外释放度对Cur SMEDDS进行体外质量评价。
结果显示:(1)油相中油酸乙酯和肉豆蔻酸异丙酯的自乳化能力优于大豆油;表面活性剂中,聚氧乙烯氢化蓖麻油RH40的自乳化能力优于吐温80和聚氧乙烯蓖麻油;助表面活性剂中乙醇、丙二醇的自乳化能力优于大于聚乙二醇400;(2)处方前研究表明Cur在肉豆蔻酸异丙酯的溶解度大于油酸乙酯在乙醇中的溶解度大于丙二醇,选择溶解度和自乳化能力强的成分作为SMEDDS的辅料;(3)当m(肉豆蔻酸异丙酯):m(聚氧乙烯氢化蓖麻油RH40):m(乙醇)=20:60:20时,系统的自乳化能力适宜,所形成的微乳体系稳定性好,为优选处方;(4)Cur在SMEDDS的溶解度为107.60 mg·g-1,比水中的溶解度(0.4320ug·g-1)增大了24.9万倍,综合乳化后微乳状态,确定载药量为5%。(5) Cur SMEDDS可在4min内乳化完全,乳化后的微乳pH值接近中性,平均粒径为31.98nm,Zeta电位为-19.69mV,以0.25%SDS的纯净水为释放介质,10min内药物可释放完全。8h内微乳溶液含量保持在94%以上,低温和常温状态下SMEDDS稳定。
第三部分Cur亚微乳的处方筛选、工艺研究和质量评价
通过单因素考察优化Cur亚微乳的处方组成和制备工艺,并进行体外质量评价。
研究方法:(1)以离心稳定性参数、粒径大小及粒度分布等为主要评价指标,通过单因素考察筛选Cur亚微乳处方;(2)通过高压均质法制备乳剂,考察了高压均质温度、压力及次数对乳剂性质的影响,研究姜黄素亚微乳的制备工艺;(3)通过考察亚微乳剂灭菌前后的pH、外观、粒径和含量变化,确定灭菌条件;(4)通过测定亚微乳剂的pH值、粘度、粒径、表面电荷、形态特征、产率对Cur亚微乳进行体外评价;(5)通过溶血试验评价姜黄素亚微乳剂的安全性;(6)通过影响因素和长期稳定性试验研究制剂稳定性。
结果显示:(1)姜黄素亚微乳处方为MCT15%,卵磷脂1.8%,pluornic F-68 2%,油酸0.5%,甘油2.25%,加水至100%,载药量为3mg/mL。(2)制备方法是将油相、卵磷脂、稳定剂于80℃溶解,加入处方量的药物,磁力搅拌下溶解或分散,作为油相。将甘油、Pluronic F-68分散于注射用水中作为水相,预热至80℃,将油相缓慢滴加入水相中,超声后(800W,10min)制成初乳。在60℃、900bar条件下进行高压均质,循环11次,充氮气熔封于安瓿中,100℃、45min条件下流通蒸汽灭菌。(3)体外质量评价表明姜黄素亚微乳pH5.83,平均粒径为247nm, Zeta电位为-40.78mV,制剂产率为98.24%,产品粘度为5m Pa·s,制剂体外不产生溶血。(4)稳定性研究结果表明影响,本品长时间贮存应在低温避光条件下。长期稳定性实验表明,本品在6±2℃试验条件下放置3个月,外观、粒径和含量均无显著变化,稳定性良好。
第四部分Cur微乳和亚微乳的体内药物动力学研究
采用高效液相色谱法,以姜黄素的血药浓度测定为依据,研究姜黄素自乳化制剂口服和亚微乳注射后的体内药物动力学过程,为姜黄素自乳化制剂和亚微乳的应用提供参考依据。
研究方法:采用液液萃取法提取血浆样本中的药物,HPLC法测定Cur浓度,应用DAS软件计算药物动力学参数,研究微乳口服和亚微乳静脉注射给药后的体内药动学过程,并与口服Cur混悬剂和静脉注射Cur溶液剂后的药动学过程进行对比。
研究结果:(1)姜黄素微乳和混悬液小鼠灌胃给药后的药动学过程符合单室模型。姜黄素微乳的药动学过程与姜黄素混悬液相比,体内吸收加快4倍,达峰浓度Cmax提高3倍左右,体内半衰期延长8倍左右,AUC明显增大,相对生物利用度为1273.25%。微乳给药后明显提高了姜黄素的吸收量和体内存留时间,提高了药物的生物利用度。(2)姜黄素亚微乳和溶液注射给药后的药动学过程符合二室模型,亚微乳给药后中央室消除速度常数K10变小,为溶液组的1/10,体内消除半衰期t1/2延长2倍左右。从体内滞留时间上看,亚微乳MRT为16.73min,而溶液组2.576min,延长6倍左右。亚微乳的AUC为溶液组的1.3倍,以上结果均表明姜黄素亚微乳制剂消除较慢,达到一定的缓释效果,在一定程度上延长了体内循环时间。
第五部分姜黄素微乳和亚微乳的体内外抗肿瘤作用
将Cur微乳和亚微乳作用于K562细胞,研究两种剂型的体外抗肿瘤效果及作用机制;通过小鼠H22移植瘤模型研究两种剂型的体内抗肿瘤作用。
研究方法:(1)台盼蓝排染法和集落形成法观察Cur微乳和亚微乳对细胞增殖的影响;(2)光学显微镜观察细胞形态变化;(3)AO/EB荧光染色法观察细胞凋亡;(4) Annexin V-FITC/PI双染后流式细胞仪检测细胞凋亡率;(5) Western blot方法观察新剂型对K562与细胞增殖和凋亡有关的信号转导通路中信号分子的影响;(6)应用H22小鼠移植肿瘤模型进行体内实验,观察Cur微乳和亚微乳的抗肿瘤活性。
研究结果:(1)姜黄素微乳和亚微乳均对K562细胞具有一定的抑制作用,并呈量效和时效关系,作用程度与姜黄素DMSO溶液相当或略高;(2) Cur微乳和亚微乳可抑制K562细胞的集落形成;(3)Cur微乳和亚微乳可触发K562细胞凋亡;(4)Cur微乳和亚微乳上调K562细胞Hsp 70蛋白含量,下调P210bcr/abl、Erk1/2、p-erk、p-AKT、NF-КB、PKC、Bcl-2、、Hsp 90蛋白含量;(5)姜黄素微乳口服给药对肝癌H22移植瘤具有明显的抑制作用,50、100、200 mg·kg-1给药剂量的抑瘤率分别为33.70%、47.95%、57.79%,与空白组相比有显著性差异;(6)姜黄素亚微乳腹腔注射给药对肝癌H22移植瘤具有明显的抑制作用,60、120、180mg·kg-1/d给药的抑瘤率分别为42.50%、35.57%、45.55%,与空白组相比有显著性差异(P<0.05或0.01)。
Curcumin (Cur) is a natural poly-phenolic compound isolated from turmeric (Curcuma longa L) which has demonstrated anti-cancer, anti- inflammatory, anti-virus, antioxidant properties. However, due to its water insolubility, instability and low bioavailability in vivo, the application of curcumin as a potential anti-tumor drug has been strongly impeded. To design Cur dosage forms which have high bioavailability and low dose has been highlighted as a major problem.
self-microemulsifying drug delivery system (SMEDDS) is an liquid dosage form which consists of a mixture of drug, oil, surfactant and cosurfactant, and the gentle mixing of these ingredients in aqueous media can generate oil-in-water microemulsion droplets of solubilized drugs with a mean droplet size≤100 nm. It is considered that such SMEDDS may increase drug solubility, improve the absorption of drugs and bioavailability, thereby it has been proven possible to formulate preparations suitable for hydrophobic drugs.
Sub-microemulsion is a novel drug delivery system which has been employed as carriers for lipid soluble drugs especially anti-cancer drugs, anti- inflammatory drugs and anesthetic drugs. This preparation possesses a number of advantages such as increasing stability, improving drug distribution in vivo, target function and administrating of hydrophobic drugs in the way of intravenous injection.
In this article,the physic-chemical properties, stability, solubility of Cur were investigated, and Cur SMEDDS and sub-microemulsion were prepared. The formulation and preparation technology were optimized by orthogonal design and single- factor study, the quality was evaluated, and the pharmacokinetics and pharmacodynamics in vitro and in vivo were investigated.
Part one Pre-formulation study
The stability, solubility and oil/water partition were determined in order to provide some evidence for the design of Cur SMEDDS and sub-microemulsion.
METHODS:(1)The method of high performance liquid chromatography (HPLC) was set up to determine the concentration of Cur.(2)The stability of Cur at different pH phosphate buffered saline (PBS), light and high temperature were determined;(3)The stability of Cur-MCT solution at high temperature was measured;(4)The oil/water partition coefficient of Cur was determined by using octanol-water system;(5)The solubility of Cur was detemined in different ratio soybean oil and medium-chain triacylglycerol (MCT) mixtures, surfactant and cosurfactant were investigated.
Results:(1)HPLC was accurate and suitable for the determination of Cur in SMEDDS and sub-microemulsion;(2)Cur was relatively stable in ethanol-water, ethanol-pH 4 PBS and ethanol-pH 5.8 PBS at room temperature, but it degradated about 17% in pH6.8 PBS within 48h,white precipitation produced immediately when Cur ethanol solution mixed with pH3、pH7.8 PBS solution;(3) Cur ethanol-water solution was relatively stable at in door light and 4500lux light condition; it degraded about 35% at 80℃water bath within 24h,t1/2 was 92.40 h-1, while Cur MCT solution was relatively stable at 80℃;(4) The oil/water partition coefficient of Cur was 19204±192(Log P为4.210±0.005) which demonstrated strongly hydrophobic property;(5)The composition of oil phase had influence on the solubility of Cur. The solubility of Cur in LCT was lowest which was 1067μg·g-1,while it was highest in MCT which was 3332μg·g-1;Among the oil phase of SMEDDS, the solubility of Cur in isopropyl myristate was more than ethyl oleate; Among the cosurfactant, the solubility of Cur in ethanol was more than 1.2-propylene glycol.
Part two Formulation optimization, preparation technology and quality evaluation of Cur SMEDDS
Based on orthogonal experiment, pseudo-ternary phase diagram conduction, self-emulsifying efficacy and particle size determination, the formulation of Cur SMEDDS was studied, then the quality was evaluated.
METHODS:(1) Pseudoternary phase diagrams were constructed and orthogonal design was used to compare the o/w microemulsion forming capacity of different oil/surfactant/co-surfactant;(2) The solubility of curcumin in various oils and cosurfactants was investigated to find suitable ingredients with a good solubilizing capacity;(3) Droplet size and self-emulsifying efficacy were determined to obtain the concentration range of oil, surfactant and cosurfactant for forming stable microemulsion;(4) The drug loading was determined by solubility test and dilution test;(5) To evaluated the quality of the optimized formulation of Cur SMEDDS, pH, self-emulsifying time, particle size, morphologic characteristics, surface charge, stability and release behavior in vitro were determined.
RESULTS:(1)Among the oil phase of SMEDDS, the self-emulsifying capacity of IPM and ethyl oleate were superior to soybean oil; Among the cosurfactant, ethanol and 1.2-propylene glycol were superior to PEG 400; Among the surfactant, Cremophor RH40 was superior to Tween 40 and Cremophor EL; (2) According solubility test, IPM, Cremophor and ethanol were selected as the excipients of Cur SMEDDS; (3) Droplets analysis showed that SMEDDS composed of 20% ethanol, 60% RH 40, 20% IPM would be a stable dosage form for curcumin; (4) The solubility of Cur in SMEDDS was 107.60 mg·g-1,which was 249 thousands times of that of water. But in consideration to the status of micro-emulsion, drug loading was defined to 5%; (5) Cur self-microemulsifying delivery system can emulsified completely within 4min. Mean particle size of the resultant emulsion was 31.98nm, Zeta potential was -19.69mV , and pH approximated to neutral. The dissolution of curcumin self-microemulsifying formulation at 10min was 100% and the content of drug in microemulsion maintained above 94% with 8h. Cur SMEDDS was stable at low temperature (4-8℃) and room temperature.
Part Three Formulation optimization, prepare technology and quality evaluation of Cur sub-microemulsion
The formulation and preparation technology of Cur sub-microemulsion were optimized by single-factor experiment, then the quality was evaluated.
METHODS:(1)Using stability constant, particle size and distribution as indexes, the formulation of Cur sub-microemulsion was optimized by single-factor experiment ;( 2 ) Cur sub-microemulsion was prepared by high pressure homogenization. The temperature, pressure and cycle times during homogenization were investigated to optimized the technology;(3)Sterilization was determined by evaluating the pH、appearance、particle size and content before and after treatment;(4)To evaluated the quality Cur sub-microemulsion, pH, viscosity, particle size, morphologic characteristics、surface charge、yield were evaluated. (5) Haemolysis test was used to evaluated the safety; (6) Stability was investigated by influential factor and long term stability experiment.
RESULTS:(1) The optimized formulation was as follows: MCT15%,egg yolk lecithin 1.8%,pluronic F-68 2%,oleic acid 0.5%,glycerol 2.25%,added water to 100%,drug loading ratio was 3mg/mL. (2) Cur sub- microemulsion was prepared as follows: oil phase (lecithin, MCT, Cur and oleic acid) and water phase (glycerol, pluronic F68 and water) were warmed up to 80℃, the oil phase was dropped into water phase under stirring, the mixture was pre-emulsified using the ultrasonic at 800W for 10min. Final emulsification was carried out by passing the coarse emulsion through a high pressure homogenizer 11 times at a pressure of 900bar, then sealed after adding nitrogen, flowing steam sterilized at 100℃for 45min.(3)Mean particle size of Cur sub-microemulsion was 247nm, Zeta potential was -40.78mV, and pH was 5.83, viscosity was 5m Pa·s, yield was 98.24%,haemolysis test indicated that Cur sub-microemulsion had no hard effect on blood. (4) Cur-microemulsion should be stored in a dark, low temperature area. Stability test indicated that there was no change with appearance, particle size and content after it was stored at 6±2℃for 3 months.
Part Four Pharmacokinetics of Cur SMEDDS and sub-microemulsion
By analysing the concentration of drug in plasma, the pharmacokinetics of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous administration were investigated, which would provide some evidents for anti-cancer efficacy.
METHODS:Cur in plasma were extracted by liquid-liquid extraction, drug concentration was determined by HPLC, pharmacokinetics parameters were obtained by using DAS software, pharmacokinetics behaviors of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous injection were compared with Cur suspension and Cur solution.
RESULTS: (1) Pharmacokinetic behaviors of Cur micro-emulsion and suspension after gavage in mouse were accorded with one-compartment model. Compared with suspension, absorption of micro-emulsion speeded up 4 times, Cmax increased 3 times, T1/2 prolonged 8 times. The developed SMEDDS formulation improved the oral bioavailability of curcumin significantly, and the relative oral bioavailability of SMEDDS compared with curcumin suspension was 1213%. (2) Pharmacokinetic behaviors of Cur sub-microemulsion and solution after intravenous injection were accorded with two-compartment model. K10 of sub-microemulsion decreased which was one tenth of that of solution, t1/2 prolonged 6 times. MRT prolonged 6 times which was 16.73min and 2.576min,respectively. AUC of sub-microemulsion was 1.3 times of that of solution. All the results demonstrated that Cur sub-microemulsion had some sustained released effect.
Part Five The anti-cancer effect of Cur SMEDDS and sub-microemulsion in vivo and in vitro
Anti-tumoral action and relevant mechanism induced by Cur microemulsion and sub-microemulsion in K562 cells were investigated, then the anti-tumor effects of two formulations were evaluated in vivo by mouse H22 transplanted tumor model.
METHODS:(1) Trypan blue exclusive staining and Colony assay were used to observe the inhibitory effects of two formulations on the growth of K562; (2) Morphologic change was observed by light microscope; (3) AO/EB fluorescent staining was to observe the apoptosis; (4) FITC-Annexin-V/PI flow cytometry was used to detect apoptotic rate; (5) Western blotting was used to analyze the impact of Cur formulations on cell proliferation apoptosis-related signaling molecules and partner protein; (6) anti-tumor effects of two formulations were evaluated in vitro by mouse H22 transplanted tumor model.
RESULTS: (1) Cur microemulsion and sub-microemulsion inhibited K562 cells growth with time- and dose-dependent manners and the inhibitory rates were equilibrant or higher than Cur DMSO solution; (2) Two formulations inhibited K562 CFUs; (3) AO/EB fluorescent staining showed that K562 cell apoptosis was induced by Cur formulations; (4) Cur formulations up-regulated Hsp 70 level, down-regulated P210bcr/abl, Erk1/2, p-Erk, p-AKT, NF-КB, PKC, Bcl-2, Hsp 90 level in K562 cells; (5) Significant anti-tumor effect on H22 transplantation tumor was observed after Cur microemulsion oral administration. After treatment at dose of 50、100、200 mg·kg-1, the inhibitory rates were 33.70%、47.95%、57.79% , respectively; (6) After intraperitoneal injection at dose of 60、120、180mg·kg-1/d, the inhibitory rate of Cur sub-microemulsion was 42.50%、35.57%、45.55%,respectively, which showed significant differences compared with control(P<0.05 or 0.01).
自乳化药物传递系统(self-microemulsifying drug delivery system,SMEDDS)是由药物、油相、非离子型表面活性剂和助表面活性剂组成的热力学稳定的均一液体剂型。它在水相中温和搅拌的情况下,能够形成粒径小于100nm的纳米乳粒,即为口服微乳。它可用作疏水性药物的载体,改善水不溶性药物的口服吸收,提高药物的生物利用度。
静脉注射亚微乳剂(sub-microemulsion)作为一种新型药物传递系统,是脂溶性药物特别是抗癌药物、麻醉药物及抗炎药物的良好载体。将药物制备成亚微乳剂可以解决疏水性药物的注射问题,增加药物稳定性,减少注射给药的刺激性,改善药物的组织分布特性,直接把药物运送到特定器官靶区,增加抗癌药物的作用强度等优点。
因此,本文系统地研究了Cur的理化性质、稳定性,在此基础上研制了姜黄素口服自乳化和静脉用亚微乳给药系统,通过正交设计、单因素考察等方法优化处方和制备工艺,并进行相关质量评价,采用药动学、药效学实验对两种剂型的体内过程、体内外抗肿瘤活性及作用机制进行研究。
第一部分处方前研究
处方前研究是制剂研究的基础,本部分重点考察了Cur的稳定性、溶解度和油水分配系数,为Cur口服SMEDDS和脂肪乳剂的处方设计提供依据。
研究方法:(1)建立HPLC测定制剂中Cur含量的体外分析方法;(2)测定Cur在不同pH介质中、光照及高温条件下的稳定性;(3)测定CurMCT溶液在高温条件下的稳定性;(4)应用正辛醇-水体系测定了Cur的油水分配系数;(5)测定Cur在不同的油相、表面活性剂、助表面活性剂中的溶解度。
结果显示:(1)HPLC法具有灵敏度高、辅料无干扰的特点,适用于Cur SMEDDS和亚微乳剂的质量控制;(2)Cur室温条件下在水、pH4、pH 5.8 PBS中比较稳定,在pH6.8条件下,48内药物降解17%,与pH3、pH7.8的PBS溶液混合后,立即产生白色沉淀;(3) Cur在室内光和4500lux光强下比较稳定,在80℃水浴条件下,24h内药物降解35%,降解半衰期t1/2为92.40 h-1;Cur MCT溶液在高温条件下比较稳定,12h内几乎没有药物降解;(4) Cur的油水分配系数为19204±192(Log P为4.210±0.005),说明其疏水性很强;(5)油相组成对Cur的溶解度有一定的影响,Cur在LCT中的溶解度最低,为1067μg·g-1,在MCT中的溶解度最高,为3332μg·g-1;在自乳化系统的油相中,Cur在肉豆蔻酸异丙酯的溶解度大于油酸乙酯;助表面活性剂中,Cur在乙醇中的溶解度大于丙二醇。
第二部分Cur SMEDDS的处方筛选、工艺研究和质量评价
选择SMEDDS常用辅料,利用正交试验、假三元相图、自乳化效率和粒径评价等方法,确定Cur SMEDDS的处方组成,并对该处方进行体外质量评价。
研究方法:(1)利用正交试验和假三元相图,确定不同辅料的自乳化能力;(2)通过溶解度试验确定Cur SMEDDS的基本辅料组成;(3)以自乳化效率和粒径为评价指标,考察油相比例、乳化剂/辅助乳化剂比例对粒径和自乳化效率的影响,进一步优化处方;(4)通过溶解度试验以及加水稀释后自乳化后溶液的状态,确定药物含量;(5)通过测定乳化后乳剂的pH值、自乳化时间、粒径、形态特征、表面电荷、稳定性和体外释放度对Cur SMEDDS进行体外质量评价。
结果显示:(1)油相中油酸乙酯和肉豆蔻酸异丙酯的自乳化能力优于大豆油;表面活性剂中,聚氧乙烯氢化蓖麻油RH40的自乳化能力优于吐温80和聚氧乙烯蓖麻油;助表面活性剂中乙醇、丙二醇的自乳化能力优于大于聚乙二醇400;(2)处方前研究表明Cur在肉豆蔻酸异丙酯的溶解度大于油酸乙酯在乙醇中的溶解度大于丙二醇,选择溶解度和自乳化能力强的成分作为SMEDDS的辅料;(3)当m(肉豆蔻酸异丙酯):m(聚氧乙烯氢化蓖麻油RH40):m(乙醇)=20:60:20时,系统的自乳化能力适宜,所形成的微乳体系稳定性好,为优选处方;(4)Cur在SMEDDS的溶解度为107.60 mg·g-1,比水中的溶解度(0.4320ug·g-1)增大了24.9万倍,综合乳化后微乳状态,确定载药量为5%。(5) Cur SMEDDS可在4min内乳化完全,乳化后的微乳pH值接近中性,平均粒径为31.98nm,Zeta电位为-19.69mV,以0.25%SDS的纯净水为释放介质,10min内药物可释放完全。8h内微乳溶液含量保持在94%以上,低温和常温状态下SMEDDS稳定。
第三部分Cur亚微乳的处方筛选、工艺研究和质量评价
通过单因素考察优化Cur亚微乳的处方组成和制备工艺,并进行体外质量评价。
研究方法:(1)以离心稳定性参数、粒径大小及粒度分布等为主要评价指标,通过单因素考察筛选Cur亚微乳处方;(2)通过高压均质法制备乳剂,考察了高压均质温度、压力及次数对乳剂性质的影响,研究姜黄素亚微乳的制备工艺;(3)通过考察亚微乳剂灭菌前后的pH、外观、粒径和含量变化,确定灭菌条件;(4)通过测定亚微乳剂的pH值、粘度、粒径、表面电荷、形态特征、产率对Cur亚微乳进行体外评价;(5)通过溶血试验评价姜黄素亚微乳剂的安全性;(6)通过影响因素和长期稳定性试验研究制剂稳定性。
结果显示:(1)姜黄素亚微乳处方为MCT15%,卵磷脂1.8%,pluornic F-68 2%,油酸0.5%,甘油2.25%,加水至100%,载药量为3mg/mL。(2)制备方法是将油相、卵磷脂、稳定剂于80℃溶解,加入处方量的药物,磁力搅拌下溶解或分散,作为油相。将甘油、Pluronic F-68分散于注射用水中作为水相,预热至80℃,将油相缓慢滴加入水相中,超声后(800W,10min)制成初乳。在60℃、900bar条件下进行高压均质,循环11次,充氮气熔封于安瓿中,100℃、45min条件下流通蒸汽灭菌。(3)体外质量评价表明姜黄素亚微乳pH5.83,平均粒径为247nm, Zeta电位为-40.78mV,制剂产率为98.24%,产品粘度为5m Pa·s,制剂体外不产生溶血。(4)稳定性研究结果表明影响,本品长时间贮存应在低温避光条件下。长期稳定性实验表明,本品在6±2℃试验条件下放置3个月,外观、粒径和含量均无显著变化,稳定性良好。
第四部分Cur微乳和亚微乳的体内药物动力学研究
采用高效液相色谱法,以姜黄素的血药浓度测定为依据,研究姜黄素自乳化制剂口服和亚微乳注射后的体内药物动力学过程,为姜黄素自乳化制剂和亚微乳的应用提供参考依据。
研究方法:采用液液萃取法提取血浆样本中的药物,HPLC法测定Cur浓度,应用DAS软件计算药物动力学参数,研究微乳口服和亚微乳静脉注射给药后的体内药动学过程,并与口服Cur混悬剂和静脉注射Cur溶液剂后的药动学过程进行对比。
研究结果:(1)姜黄素微乳和混悬液小鼠灌胃给药后的药动学过程符合单室模型。姜黄素微乳的药动学过程与姜黄素混悬液相比,体内吸收加快4倍,达峰浓度Cmax提高3倍左右,体内半衰期延长8倍左右,AUC明显增大,相对生物利用度为1273.25%。微乳给药后明显提高了姜黄素的吸收量和体内存留时间,提高了药物的生物利用度。(2)姜黄素亚微乳和溶液注射给药后的药动学过程符合二室模型,亚微乳给药后中央室消除速度常数K10变小,为溶液组的1/10,体内消除半衰期t1/2延长2倍左右。从体内滞留时间上看,亚微乳MRT为16.73min,而溶液组2.576min,延长6倍左右。亚微乳的AUC为溶液组的1.3倍,以上结果均表明姜黄素亚微乳制剂消除较慢,达到一定的缓释效果,在一定程度上延长了体内循环时间。
第五部分姜黄素微乳和亚微乳的体内外抗肿瘤作用
将Cur微乳和亚微乳作用于K562细胞,研究两种剂型的体外抗肿瘤效果及作用机制;通过小鼠H22移植瘤模型研究两种剂型的体内抗肿瘤作用。
研究方法:(1)台盼蓝排染法和集落形成法观察Cur微乳和亚微乳对细胞增殖的影响;(2)光学显微镜观察细胞形态变化;(3)AO/EB荧光染色法观察细胞凋亡;(4) Annexin V-FITC/PI双染后流式细胞仪检测细胞凋亡率;(5) Western blot方法观察新剂型对K562与细胞增殖和凋亡有关的信号转导通路中信号分子的影响;(6)应用H22小鼠移植肿瘤模型进行体内实验,观察Cur微乳和亚微乳的抗肿瘤活性。
研究结果:(1)姜黄素微乳和亚微乳均对K562细胞具有一定的抑制作用,并呈量效和时效关系,作用程度与姜黄素DMSO溶液相当或略高;(2) Cur微乳和亚微乳可抑制K562细胞的集落形成;(3)Cur微乳和亚微乳可触发K562细胞凋亡;(4)Cur微乳和亚微乳上调K562细胞Hsp 70蛋白含量,下调P210bcr/abl、Erk1/2、p-erk、p-AKT、NF-КB、PKC、Bcl-2、、Hsp 90蛋白含量;(5)姜黄素微乳口服给药对肝癌H22移植瘤具有明显的抑制作用,50、100、200 mg·kg-1给药剂量的抑瘤率分别为33.70%、47.95%、57.79%,与空白组相比有显著性差异;(6)姜黄素亚微乳腹腔注射给药对肝癌H22移植瘤具有明显的抑制作用,60、120、180mg·kg-1/d给药的抑瘤率分别为42.50%、35.57%、45.55%,与空白组相比有显著性差异(P<0.05或0.01)。
Curcumin (Cur) is a natural poly-phenolic compound isolated from turmeric (Curcuma longa L) which has demonstrated anti-cancer, anti- inflammatory, anti-virus, antioxidant properties. However, due to its water insolubility, instability and low bioavailability in vivo, the application of curcumin as a potential anti-tumor drug has been strongly impeded. To design Cur dosage forms which have high bioavailability and low dose has been highlighted as a major problem.
self-microemulsifying drug delivery system (SMEDDS) is an liquid dosage form which consists of a mixture of drug, oil, surfactant and cosurfactant, and the gentle mixing of these ingredients in aqueous media can generate oil-in-water microemulsion droplets of solubilized drugs with a mean droplet size≤100 nm. It is considered that such SMEDDS may increase drug solubility, improve the absorption of drugs and bioavailability, thereby it has been proven possible to formulate preparations suitable for hydrophobic drugs.
Sub-microemulsion is a novel drug delivery system which has been employed as carriers for lipid soluble drugs especially anti-cancer drugs, anti- inflammatory drugs and anesthetic drugs. This preparation possesses a number of advantages such as increasing stability, improving drug distribution in vivo, target function and administrating of hydrophobic drugs in the way of intravenous injection.
In this article,the physic-chemical properties, stability, solubility of Cur were investigated, and Cur SMEDDS and sub-microemulsion were prepared. The formulation and preparation technology were optimized by orthogonal design and single- factor study, the quality was evaluated, and the pharmacokinetics and pharmacodynamics in vitro and in vivo were investigated.
Part one Pre-formulation study
The stability, solubility and oil/water partition were determined in order to provide some evidence for the design of Cur SMEDDS and sub-microemulsion.
METHODS:(1)The method of high performance liquid chromatography (HPLC) was set up to determine the concentration of Cur.(2)The stability of Cur at different pH phosphate buffered saline (PBS), light and high temperature were determined;(3)The stability of Cur-MCT solution at high temperature was measured;(4)The oil/water partition coefficient of Cur was determined by using octanol-water system;(5)The solubility of Cur was detemined in different ratio soybean oil and medium-chain triacylglycerol (MCT) mixtures, surfactant and cosurfactant were investigated.
Results:(1)HPLC was accurate and suitable for the determination of Cur in SMEDDS and sub-microemulsion;(2)Cur was relatively stable in ethanol-water, ethanol-pH 4 PBS and ethanol-pH 5.8 PBS at room temperature, but it degradated about 17% in pH6.8 PBS within 48h,white precipitation produced immediately when Cur ethanol solution mixed with pH3、pH7.8 PBS solution;(3) Cur ethanol-water solution was relatively stable at in door light and 4500lux light condition; it degraded about 35% at 80℃water bath within 24h,t1/2 was 92.40 h-1, while Cur MCT solution was relatively stable at 80℃;(4) The oil/water partition coefficient of Cur was 19204±192(Log P为4.210±0.005) which demonstrated strongly hydrophobic property;(5)The composition of oil phase had influence on the solubility of Cur. The solubility of Cur in LCT was lowest which was 1067μg·g-1,while it was highest in MCT which was 3332μg·g-1;Among the oil phase of SMEDDS, the solubility of Cur in isopropyl myristate was more than ethyl oleate; Among the cosurfactant, the solubility of Cur in ethanol was more than 1.2-propylene glycol.
Part two Formulation optimization, preparation technology and quality evaluation of Cur SMEDDS
Based on orthogonal experiment, pseudo-ternary phase diagram conduction, self-emulsifying efficacy and particle size determination, the formulation of Cur SMEDDS was studied, then the quality was evaluated.
METHODS:(1) Pseudoternary phase diagrams were constructed and orthogonal design was used to compare the o/w microemulsion forming capacity of different oil/surfactant/co-surfactant;(2) The solubility of curcumin in various oils and cosurfactants was investigated to find suitable ingredients with a good solubilizing capacity;(3) Droplet size and self-emulsifying efficacy were determined to obtain the concentration range of oil, surfactant and cosurfactant for forming stable microemulsion;(4) The drug loading was determined by solubility test and dilution test;(5) To evaluated the quality of the optimized formulation of Cur SMEDDS, pH, self-emulsifying time, particle size, morphologic characteristics, surface charge, stability and release behavior in vitro were determined.
RESULTS:(1)Among the oil phase of SMEDDS, the self-emulsifying capacity of IPM and ethyl oleate were superior to soybean oil; Among the cosurfactant, ethanol and 1.2-propylene glycol were superior to PEG 400; Among the surfactant, Cremophor RH40 was superior to Tween 40 and Cremophor EL; (2) According solubility test, IPM, Cremophor and ethanol were selected as the excipients of Cur SMEDDS; (3) Droplets analysis showed that SMEDDS composed of 20% ethanol, 60% RH 40, 20% IPM would be a stable dosage form for curcumin; (4) The solubility of Cur in SMEDDS was 107.60 mg·g-1,which was 249 thousands times of that of water. But in consideration to the status of micro-emulsion, drug loading was defined to 5%; (5) Cur self-microemulsifying delivery system can emulsified completely within 4min. Mean particle size of the resultant emulsion was 31.98nm, Zeta potential was -19.69mV , and pH approximated to neutral. The dissolution of curcumin self-microemulsifying formulation at 10min was 100% and the content of drug in microemulsion maintained above 94% with 8h. Cur SMEDDS was stable at low temperature (4-8℃) and room temperature.
Part Three Formulation optimization, prepare technology and quality evaluation of Cur sub-microemulsion
The formulation and preparation technology of Cur sub-microemulsion were optimized by single-factor experiment, then the quality was evaluated.
METHODS:(1)Using stability constant, particle size and distribution as indexes, the formulation of Cur sub-microemulsion was optimized by single-factor experiment ;( 2 ) Cur sub-microemulsion was prepared by high pressure homogenization. The temperature, pressure and cycle times during homogenization were investigated to optimized the technology;(3)Sterilization was determined by evaluating the pH、appearance、particle size and content before and after treatment;(4)To evaluated the quality Cur sub-microemulsion, pH, viscosity, particle size, morphologic characteristics、surface charge、yield were evaluated. (5) Haemolysis test was used to evaluated the safety; (6) Stability was investigated by influential factor and long term stability experiment.
RESULTS:(1) The optimized formulation was as follows: MCT15%,egg yolk lecithin 1.8%,pluronic F-68 2%,oleic acid 0.5%,glycerol 2.25%,added water to 100%,drug loading ratio was 3mg/mL. (2) Cur sub- microemulsion was prepared as follows: oil phase (lecithin, MCT, Cur and oleic acid) and water phase (glycerol, pluronic F68 and water) were warmed up to 80℃, the oil phase was dropped into water phase under stirring, the mixture was pre-emulsified using the ultrasonic at 800W for 10min. Final emulsification was carried out by passing the coarse emulsion through a high pressure homogenizer 11 times at a pressure of 900bar, then sealed after adding nitrogen, flowing steam sterilized at 100℃for 45min.(3)Mean particle size of Cur sub-microemulsion was 247nm, Zeta potential was -40.78mV, and pH was 5.83, viscosity was 5m Pa·s, yield was 98.24%,haemolysis test indicated that Cur sub-microemulsion had no hard effect on blood. (4) Cur-microemulsion should be stored in a dark, low temperature area. Stability test indicated that there was no change with appearance, particle size and content after it was stored at 6±2℃for 3 months.
Part Four Pharmacokinetics of Cur SMEDDS and sub-microemulsion
By analysing the concentration of drug in plasma, the pharmacokinetics of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous administration were investigated, which would provide some evidents for anti-cancer efficacy.
METHODS:Cur in plasma were extracted by liquid-liquid extraction, drug concentration was determined by HPLC, pharmacokinetics parameters were obtained by using DAS software, pharmacokinetics behaviors of Cur SMEDDS after oral administration and Cur sub-microemulsion after intravenous injection were compared with Cur suspension and Cur solution.
RESULTS: (1) Pharmacokinetic behaviors of Cur micro-emulsion and suspension after gavage in mouse were accorded with one-compartment model. Compared with suspension, absorption of micro-emulsion speeded up 4 times, Cmax increased 3 times, T1/2 prolonged 8 times. The developed SMEDDS formulation improved the oral bioavailability of curcumin significantly, and the relative oral bioavailability of SMEDDS compared with curcumin suspension was 1213%. (2) Pharmacokinetic behaviors of Cur sub-microemulsion and solution after intravenous injection were accorded with two-compartment model. K10 of sub-microemulsion decreased which was one tenth of that of solution, t1/2 prolonged 6 times. MRT prolonged 6 times which was 16.73min and 2.576min,respectively. AUC of sub-microemulsion was 1.3 times of that of solution. All the results demonstrated that Cur sub-microemulsion had some sustained released effect.
Part Five The anti-cancer effect of Cur SMEDDS and sub-microemulsion in vivo and in vitro
Anti-tumoral action and relevant mechanism induced by Cur microemulsion and sub-microemulsion in K562 cells were investigated, then the anti-tumor effects of two formulations were evaluated in vivo by mouse H22 transplanted tumor model.
METHODS:(1) Trypan blue exclusive staining and Colony assay were used to observe the inhibitory effects of two formulations on the growth of K562; (2) Morphologic change was observed by light microscope; (3) AO/EB fluorescent staining was to observe the apoptosis; (4) FITC-Annexin-V/PI flow cytometry was used to detect apoptotic rate; (5) Western blotting was used to analyze the impact of Cur formulations on cell proliferation apoptosis-related signaling molecules and partner protein; (6) anti-tumor effects of two formulations were evaluated in vitro by mouse H22 transplanted tumor model.
RESULTS: (1) Cur microemulsion and sub-microemulsion inhibited K562 cells growth with time- and dose-dependent manners and the inhibitory rates were equilibrant or higher than Cur DMSO solution; (2) Two formulations inhibited K562 CFUs; (3) AO/EB fluorescent staining showed that K562 cell apoptosis was induced by Cur formulations; (4) Cur formulations up-regulated Hsp 70 level, down-regulated P210bcr/abl, Erk1/2, p-Erk, p-AKT, NF-КB, PKC, Bcl-2, Hsp 90 level in K562 cells; (5) Significant anti-tumor effect on H22 transplantation tumor was observed after Cur microemulsion oral administration. After treatment at dose of 50、100、200 mg·kg-1, the inhibitory rates were 33.70%、47.95%、57.79% , respectively; (6) After intraperitoneal injection at dose of 60、120、180mg·kg-1/d, the inhibitory rate of Cur sub-microemulsion was 42.50%、35.57%、45.55%,respectively, which showed significant differences compared with control(P<0.05 or 0.01).
引文
[1]汪海慧,成扬.姜黄素药理作用的研究进展[J].上海中医药大学学报, 2007,21(6):73-74
[2]Han SS,Chung ST,Robertson DA.et a1.Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1,C-myc,Bcl-XL,NF-kappa B and P53[J].Clin Immunol,1999,93(2):152-161.
[3]Ushida J,Sugie S,Kawabata K,et al.Chemopreventive effect of curcumin on N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats[J].Jpn J Cancer Res,2000,91(9):893-898.
[4]许建华,赵蓉,柯丹如,黄自强.姜黄素对人肝癌Bel7402细胞杀伤动力学及周期时相的影响.福建医科大学学报1998;32:236-9
[5]Xu J H, Chen Y Z. Curcumin Inducing Apoptosis and Inhibiting Expression of P210bcr-abl Protein in Human Chronic Myelogenous Leukemia K562 Cells. FASEB J, 2000;14(8):1169
[6] Wang Y J,Pan M H,Cheng A L,et a1.Stability of curcumin in buffer solutions and characterization of its degradation products[J].J Pharm Biomed Arm,1997,15(12):1867-1876.
[7] Ravindranath V,Chandrasekhara N.In Vitro studies on the intestinal absorption of curcumin in rats[J].Toxicol,1981,20(2-3):25l-257.
[8]Shoba G,Joy D,Joseph T,et a1.Influence of pipefine on the pharmacokinetics of curcumin in animals an d human volunteers[J].Planta Medica,1998,64:353-359
[9]仲明远,全山从,胡晋红.姜黄素制剂学研究进展[J].中成药,2007,29(2):255-257
[10]Pouton C W.Formulation of self emulsifying drug delivery systems[J],Adv Drug Deliv Rev,1997,25(1):47-58
[11]Constantinides P P . Lipid microemulsions for improving drug dissolution and oral absorption:Physical and biopharmaceutical aspects[J].Pharm Res,1995.12(11):1561-1572
[12]Gao Zhong Gao, Choi Han Gon, Shin Hee Jong. Physico-chemical characterization and evaluation of a microemulsion system for oral delivery of cyclosporin A [J].Int J Pharm, 1998,161: 75-86
[13]David SS. Pharmaceutical aspects of intravenous fat emulsion [J]. J Hosp Pharm,1974, 32(8&9):149-171
[14]Collins-Gold LC, Lyons RT, Bartholow LC. Parenteral emulsions for drug delivery [J]. Adv Drug Deliv Rew, 1990,5: 189-208
[15]Constantinides PP, Lambert KJ, Tustian AK, et al. Formulation development and antitumor activity of a filter-sterilizable emulsion of pacilitaxel [J]. Pharm Res, 2000, 17(2): 175-182
[16] Brime B, Moreno MA, Frutos G, et al. Amphotericin B in oil-water lecitin-based microemulsion: formulation and toxicity evaluation [J]. J Pharm Sci, 2002, 91(4): 1178-1185
[17] Akkar A, Muller RH. Intraveous itraconazole emulsion produced by SolEmuls technology [J]. Eur J Pharm Biopharm, 2003, 56(1): 29-36
[18] Takahashi T. Emulsion and activated carbon in cancer chemotherapy [J]. J Pharm Sci, 1993, 82(11): 1069-79
[19] Flora KP, Cradock JC, Kelley JA. The hydrolysis of spirohydantion mustard [J]. J Pharm Sci, 1982, 71(11): 1206-11
[20]张继芬,侯世祥,刘惠莲.小鼠血浆中姜黄素的含量测定及药动学研究[J].中国药学杂志,2007, 42 (4): 288-231
[21]徐文,孙进,张婷婷,等.HPLC法测定冬凌草甲素的平衡溶解度和表观油水分配系数[J].沈阳药科大学学报,2007,24(4):220-222
[22]吴义辉,伍玉甜.药物在辛醇-水体系分配系数的应用[J].广东药学,2000,10(4):12—14
[23]周庆辉,平其能.自乳化药物传递系统的应用与前景[J].药学进展,2001,25(3):134-137
[24]李莉,王中彦,莫凤奎.自乳化药物传递系统的研究概况[J].药学专论,2005,14(2):27
[25] Chen, Y., Li, G., Wu, X. G., et al. Self- Microemulsifying Drug Delivery System (SMEDDS) of Vinpocetine: Formulation Development and in vivo Assessment[J]. Biological & Pharmaceutical Bulletin,2008,31:118-125.
[26]Cui, S. M., Zhao, C. S., Chen, D. W., et al. Self-Microemulsifying Drug Delivery Systems (SMEDDS) for Improving In vitro Dissolution and Oral Absorption of Pueraria Lobata Isoflavone[J]. Drug Development and Industrial Pharmacy, 2005, 31:349-356.
[27]Kang, B. K., Lee, J. S., Chon, S. K., Jeong, S. Y., Yuk, S. H., Khang, G., Lee, H. B., & Cho, S. H. Development of self-microemulsifying drug delivery systems (SMEDDS) for oral bioavailability enhancement of simvastatin in beagle dogs[J]. International Journal of Pharmaceutics, 2004,274, 65-73.
[28]Shah NH, Carvajal MT, Patel CI , Infeld MH, Malick AW.Self-emulsifying drug delivery systems (SEDDS) with polyglycolized glycerides for improving in vitro dissolution and oral absorption of lipophilic drugs[J]. Int J Pharm,1994,106(1):15
[29]Pouton, C. W. Effects of inclusion of a model drug on the performance of self-emulsifying formulations [J]. The Journal of Pharmacy and Pharmacology, 1985,37:1-11.
[30]Pouton, C. W. Self-emulsifying drug delivery systems: assessment of the efficiency of emulsification[J]. International Journal ofPharmaceutics, 1985,27, 335–348.
[31] Swenson, E. S., Milisen, W. B., Curatolo, W. Intestinal permeability enhancement: efficacy, acute local toxicity and reversibility[J]. Pharmaceutical Research, 1994,11, 1132–42
[32]胡海燕,徐雄良,龚涛,等.不同溶解性质的药物在微乳中的溶解度和体外释放特征[J].中国药学杂志,2006,31(9):678-682
[33]杨根生,严琴英,王文喜,等.紫杉醇口服自乳化给药系统的处方研究[J].浙江工业大学学报,36 (1):5-11
[34]王思玲,孙长山,陈闯,等.比较Gaussian曲线分布与Nicomp多波型分布分析在纳米分散系粒径测定中的应用[J].沈阳药科大学学报,2003,20(5):321-324
[35]Bok Ki Kang, Jin Soo Lee , Se Kang Chon, et al. Development of self-microemulsifying drug delivery systems (SMEDDS) for oral bioavailability enhancement of simvastatin in beagle dogs[J]. International Journal of Pharmaceutics,2004,274:65-73
[36]崔福德.药剂学(第五版)[M].北京人民卫生出版社,2003,39
[37]汤明辉,平其能.去氢骆驼蓬碱口服乳剂的物理稳定性研究[J].2002,18(1):4-6
[38]郭涛,储晓琴,宋洪涛,等.静脉注射用大蒜油亚微乳的制备[J].中国药学杂志,2005,40(8):602-605
[39]Levy MY,Schutze W,Fuhreret C,et al.Characterization of diazepam submicron emulsion interface: role of loeic acid [J]. J Microencapsulation,1944,11(1):79-92
[40]Tamilvanan S. Oil-in-water lipid emusions: implication for parenteral and ocular delivering systems[J].Progress in Lipid Research,2004,43:489-533
[41]Aslihan A, Rainer H. Muller.Intravenous itraconazole emulsionsproduced by SolEmuls technology [J].European Journal of Pharmaceutics and Biopharmaceutics, 2003, 56 29–36
[42]Junghanns, J. U., Buttle, I., Muller, R. H., et al. SolEmuls? Technology: A way to overcome the drawback of parenteral administration of insoluble drugs[J]. Pharmaceutical Development and Technology, 2007, 12(5): 437-445
[43]田玲玲,唐星,何海冰,等.依托泊苷静脉注射亚微乳的制备及其体内外评价[J].药学学报,2007,42(8):892-897
[44]Tetsuo Yamaguchi. Lipid microspheres as drug carriers: a pharmaceutical point of view [J]. Adv Drug Deliv Rev, 1996, 20: 117-1130
[45]Fang Li, Tao Wang, Hai Bing He, et al.The properties of bufadienolides-loaded nano-emulsion and submicro-emulsion during lyophilization [J]. International Journal of Pharmaceutics 349 (2008) 291–299
[46]张利,侯世祥,王昶光.葫芦素脂肪乳剂成型性影响因素考察[J].中南药学,2004,2(2):74-77
[47]廖颂明.中/长链脂肪乳注射液制备工艺研究[J].广东药学院学报, 2001, 17(3):170-172
[48]王浩,顾吉晋,邓英杰.以泊洛沙姆188为助乳化剂制备双氯芬酸钠亚微乳的研究.中国药房, 2008, 19(25):1965-1968
[49]刘晓峰,张郑瑶,邓旭明,等.纳米TCP/明胶/鹿茸多肽复合材料的生物相容性评价,中国修复重建外科杂志,2009,23(5):598-601
[50]郑少辉,邓意辉.动态光散射技术在静脉注射亚微乳剂粒径评价中的应用,中国药剂学杂志, 2005,3(3): 125-128
[51]孟玉芳,龚明涛,张钧寿.含药静脉注射用脂肪乳剂的研究进展.中国医药工业杂志, 2004, 35( 9): 558-561
[52]温彩霞,许建华,黄秀旺.姜黄素在小鼠体内各脏器的药代动力学研究.中药药理与临床, 2007, 23(4):25-27
[53]赵荣丽,侯世祥,毛声俊,等.川芎嗪脂肪乳在小鼠体内分布的研究.中南药学,2008,6(3): 264-267
[54]Constantinides, P. P. Lipid microemulsion for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharmaceutical research, 1995, 12:1561-1572.
[55]杨春艳,毛莎.西罗莫司口服液服用方法的改进.护理学杂志:外科版,2009,24(9): 82-82
[56] Shah, N. H., Carvajal, M. T., Patel, C. I., Infeld, M. H., & Malick, A. W. Self-emulsifying drug delivery system (SEDDS) with polyglycolysed glycerides for improving in vitro dissolution and oral absorption of lipophilic drugs. International Journal of Pharmaceutics, 1994, 106:15-23.
[57]Chen, Y., Li, G., Wu, X. G., Chen, Z. Y., Hang, J. G., Qin, B., Chen, S., & Wang, R. H. Self-Microemulsifying Drug Delivery System (SMEDDS) of Vinpocetine: Formulation Development and in vivo Assessment. Biological & Pharmaceutical Bulletin, 2008, 31: 118-125.
[58] Trevaskis, N. L., Charman, W. N., & Porter, C. J. Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update. Advanced Drug Delivery Reviews, 2008, 60: 702-716.
[59] Sha, X. Y., Yan, G. J., Wu, Y. J., Li, J. C., & Fang, X. L. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. European Journal of Pharmaceutical Sciences, 2005, 24:477–486.
[60] Woo, J. S., Lee, C. H., Shim, C. K., Hwang, S. C. Enhanced oralbioavailability of paclitaxel by co-administration of the P-glycoprotein inhibitor KR30031. Pharmaceutical research, 2003,20:24–30.
[61]Peng-fei Yue, Hai-long Yuan, Ming Yang, et al. Preparation, characterization, and pharmacokinetic evaluation of puerarin submicron emulsion. Journal of pharmaceutical Science and Technology, 2008,62(1):32-44
[62]田玲玲,唐星,何海冰,等.依托泊苷静脉注射亚微乳的制备及其体内外评价.药学学报, 2007, 42(8): 892-897
[63]魏立,孔佩艳,陈幸华,等.阻抑正常骨髓基质SDF21作用对HL260细胞生物学性质的影响.第三军医大学学报,2004,26(23): 2158-2161
[65]毛咏秋,姜愚,雷松,等.流式细胞术PI、Annexin V/PI和APO2.7法检测细胞凋亡的比较.华西医学,2005,20(3):505-506
[66]吴丽贤,许建华,吴国华,等.姜黄素对K562细胞增殖的影响及其与P210bcr/abl激活的Ras信号途径的关系.中国药理学通报,2003 19(1):33-37
[67]唐印华,王玺,博挽澜,等.三氧化二砷抑制小鼠H22肝癌移植瘤血管生长的实验研究.2008,16(7):1091-1094
[68]杨金霞,王学美,朱伟,等.干、鲜壁虎冻干粉对小鼠H22肝癌体内外抑制作用.世界华人消化杂志2007,15(2):157-160
[69]张金娟,温彩兰,张贵林.艾迪注射液对裸鼠人胃癌移植瘤的抑瘤作用研究.贵阳医学院学报,2009年34(6):662-664
[70]陈蕾.静脉输液脂肪乳剂致静脉炎的预防.中国临床医药研究杂志.2008, 181:52-53
[71] Swenson, E. S., Milisen, W. B., Curatolo, W. Intestinal permeability enhancement: efficacy, acute local toxicity and reversibility. Pharmaceutical Research, 1994, 11:1132–42
[72]胡大裕,李高,陈鹰,等.几种表面活性剂的细胞毒性研究[J].中国药师, 2006,9(4):294-296
[73]李昕,王晖,药物吸收促进剂的毒性研究进展.中国医药工业杂志, 2005,36(1):51-53
[1]汪海慧,成扬.姜黄素药理作用的研究进展[J].上海中医药大学学报,2007,21(6):73-74
[2]Wahlstrom, B, Blennow, G. A study on the fate of curcumin in the rat [J]. Acta Pharmacol. Toxicol. (Copenhagen) 1978, 43 (2), 86–92.
[3]Ravindranath,V.,Chandrasekhara,N. Absorption and tissue distribu- tion of curcumin in rats [J]. Toxicology 1980, 16 (3), 259–65.
[4]Ravindranath,V., Chandrasekhara, N. Metabolism of curcumin-studies with [3H]curcumin [J]. Toxicology 1981, 22 (4), 337-44.
[5]Pan, M. H., Huang,T.M., Lin,J.K. Biotransformation of curcumin through reduction and glucuronidation in mice [J]. Drug Metab. Dispos.1999, 27 (4), 486–94.
[6] Perkins S,Verschoyle R. D., Hill K.,et al. Chemopreventive efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of familial adenomatous polyposis [J]. Cancer Epidemiol Biomarkers Prev,2002,11(6), 535–40.
[7]Marczylo T. H., Verschoyle R. D., Cooke D. N.,et al. Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine [J]. Cancer Chemother Pharmacol.2007,60 (2),171-7.
[8]Ravindranath V., Chandrasekhara N. In vitro studies on the intestinal absorption of curcumin in rats [J]. Toxicology 1981, 20 (2–3), 251–7.
[9]Garcea G., Jones D. J., Singh R. ,et al. Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration [J]. Br. J. Cancer 2004, 90 (5), 1011–5.
[10]Garcea G., Berry D. P., Jones D. J. ,et al. Consumption of the putative chemopreventive agent curcumin by cancer patients: assessment of curcumin levels in the colorectum and their pharmacodynamic consequences [J]. Cancer Epidemiol. Biomarkers. Prev. 2005, 14 (1),120–5.
[11] Holder,G. M., Plummer, J. L., Ryan, A. J. The metabolism and excretion of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene- 3,5-dione) in the rat [J]. Xenobiotica,1978, 8(12),761–8.
[12]Hoehle S.I.,Pfeiffer E.,Solyom A.M.,et al.Metabolism of curcu- minoids in tissue slices and subcellular fractions from rat liver [J]. J. Agric. Food Chem. 2006,54(3),756–64.
[13]Asai, A., Miyazawa, T. Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma [J]. Life Sci. 2000, 67 (23), 2785–93.
[14]Ireson, C., Orr, S., Jones, D. J. ,et al. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production [J]. Cancer Res. 2001, 61 (3), 1058–64.
[15]Sharma, R. A., McLelland, H. R., Hill, K. A. ,et al. Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer [J]. Clin. Cancer Res. 2001, 7 (7), 1894–900.
[16]Shoba, G, Joy, D, Joseph, T,et al. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers [J]. Planta Med 1998, 64 (4), 353–6.
[17]Cruz-Correa, M., Shoskes, D. A., Sanchez, P. ,et al. Combination treatment with curcumin and quercetin of adenomas in familial adenomatous polyposis [J]. Clin. Gastroenterol. Hepatol. 2006, 4 (8), 1035–8.
[18]Verma S. P., Salamone E., Goldin B. Curcumin and genistein,plant natural products, show synergistic inhibitory effects on the growth of human breast cancer MCF-7 cells induced by estrogenic pesticides [J]. Biochem. Biophys. Res. Commun. 1997, 233 (3), 692–6.
[19] Fang J. Y., Hung C. F., Chiu H. C.,et al. Efficacy and irritancy of enhancers on the in-vitro and in-vivo percutaneous absorption of curcumin [J]. J. Pharm. Pharmacol. 2003,55 (5), 593–601.
[20]Karikar C., Maitra A., Bisht S. ,et al. Polymeric nanoparticle- encapsulated curcumin (“nanocurcumin”):a novel strategy for human cancer therapy [J]. J. Nanobiotechnol.2007, 5, 3.
[21]Tiyaboonchai W., Tungpradit W., Plianbangchang P. .Formulation and characterization of curcuminoids loaded solid lipid nanoparticles [J]. Int. J. Pharm. 2007, 337 (1–2), 299–306. (63)
[22]Li, L., Braiteh, F. S., Kurzrock, R. Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis,curcumin: in vitro and in vivo effects on proliferation, apoptosis,signaling, and angiogenesis [J]. Cancer 2005, 104 (6), 1322–31.
[23]Li, L., Ahmed, B., Mehta, K. ,et al. Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis,and angiogenesis in colorectal cancer [J]. Mol. Cancer Ther. 2007,6 (4), 1276–82.
[24]Kunwar, A., Barik, A., Pandey, R. ,et al. Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study [J]. Biochim. Biophys. Acta 2006, 1760 (10), 1513–20.
[25]Ma, Z., Shayeganpour, A., Brocks, D. R. ,et al. High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin [J]. Biomed. Chromatogr. 2007,21 (5), 546–52.
[26]Liu, A., Lou, H., Zhao, L. ,et al. Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin [J].J. Pharm. Biomed. Anal. 2006, 40 (3), 720–7.
[27] Maiti, K., Mukherjee, K., Gantait, A,et al. Curcumin–phospholipid complex: Preparation, therapeutic evaluation and pharmacokinetic study in rats [J]. Int. J. Pharm. 2007,330 (1–2), 155–63.
[28] Mosley, C. A., Liotta, D. C., Snyder, J. P. Highly active anticancer curcumin analogues [J]. AdV. Exp. Med. Biol. 2007, 595:, 77–103.
[29]John, V. D., Kuttan, G., Krishnankutty, K. Anti-tumour studies of metal chelates of synthetic curcuminoids [J]. J. Exp. Clin. Cancer Res. 2002, 21 (2), 219–24.
[30] Sui, Z., Salto, R., Li, J. ,et al. Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes [J]. Bioorg. Med. Chem. 1993, 1 (6), 415–22.
[31]Mishra, S., Narain, U., Mishra, R.,et al. Design, development and synthesis of mixed bioconjugates of piperic acid-glycine, curcumin-glycine/alanine and curcumin-glycine-piperic acid and their antibacterial and antifungal properties [J]. Bioorg. Med. Chem.2005, 13 (5), 1477–86.
[2]Han SS,Chung ST,Robertson DA.et a1.Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1,C-myc,Bcl-XL,NF-kappa B and P53[J].Clin Immunol,1999,93(2):152-161.
[3]Ushida J,Sugie S,Kawabata K,et al.Chemopreventive effect of curcumin on N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats[J].Jpn J Cancer Res,2000,91(9):893-898.
[4]许建华,赵蓉,柯丹如,黄自强.姜黄素对人肝癌Bel7402细胞杀伤动力学及周期时相的影响.福建医科大学学报1998;32:236-9
[5]Xu J H, Chen Y Z. Curcumin Inducing Apoptosis and Inhibiting Expression of P210bcr-abl Protein in Human Chronic Myelogenous Leukemia K562 Cells. FASEB J, 2000;14(8):1169
[6] Wang Y J,Pan M H,Cheng A L,et a1.Stability of curcumin in buffer solutions and characterization of its degradation products[J].J Pharm Biomed Arm,1997,15(12):1867-1876.
[7] Ravindranath V,Chandrasekhara N.In Vitro studies on the intestinal absorption of curcumin in rats[J].Toxicol,1981,20(2-3):25l-257.
[8]Shoba G,Joy D,Joseph T,et a1.Influence of pipefine on the pharmacokinetics of curcumin in animals an d human volunteers[J].Planta Medica,1998,64:353-359
[9]仲明远,全山从,胡晋红.姜黄素制剂学研究进展[J].中成药,2007,29(2):255-257
[10]Pouton C W.Formulation of self emulsifying drug delivery systems[J],Adv Drug Deliv Rev,1997,25(1):47-58
[11]Constantinides P P . Lipid microemulsions for improving drug dissolution and oral absorption:Physical and biopharmaceutical aspects[J].Pharm Res,1995.12(11):1561-1572
[12]Gao Zhong Gao, Choi Han Gon, Shin Hee Jong. Physico-chemical characterization and evaluation of a microemulsion system for oral delivery of cyclosporin A [J].Int J Pharm, 1998,161: 75-86
[13]David SS. Pharmaceutical aspects of intravenous fat emulsion [J]. J Hosp Pharm,1974, 32(8&9):149-171
[14]Collins-Gold LC, Lyons RT, Bartholow LC. Parenteral emulsions for drug delivery [J]. Adv Drug Deliv Rew, 1990,5: 189-208
[15]Constantinides PP, Lambert KJ, Tustian AK, et al. Formulation development and antitumor activity of a filter-sterilizable emulsion of pacilitaxel [J]. Pharm Res, 2000, 17(2): 175-182
[16] Brime B, Moreno MA, Frutos G, et al. Amphotericin B in oil-water lecitin-based microemulsion: formulation and toxicity evaluation [J]. J Pharm Sci, 2002, 91(4): 1178-1185
[17] Akkar A, Muller RH. Intraveous itraconazole emulsion produced by SolEmuls technology [J]. Eur J Pharm Biopharm, 2003, 56(1): 29-36
[18] Takahashi T. Emulsion and activated carbon in cancer chemotherapy [J]. J Pharm Sci, 1993, 82(11): 1069-79
[19] Flora KP, Cradock JC, Kelley JA. The hydrolysis of spirohydantion mustard [J]. J Pharm Sci, 1982, 71(11): 1206-11
[20]张继芬,侯世祥,刘惠莲.小鼠血浆中姜黄素的含量测定及药动学研究[J].中国药学杂志,2007, 42 (4): 288-231
[21]徐文,孙进,张婷婷,等.HPLC法测定冬凌草甲素的平衡溶解度和表观油水分配系数[J].沈阳药科大学学报,2007,24(4):220-222
[22]吴义辉,伍玉甜.药物在辛醇-水体系分配系数的应用[J].广东药学,2000,10(4):12—14
[23]周庆辉,平其能.自乳化药物传递系统的应用与前景[J].药学进展,2001,25(3):134-137
[24]李莉,王中彦,莫凤奎.自乳化药物传递系统的研究概况[J].药学专论,2005,14(2):27
[25] Chen, Y., Li, G., Wu, X. G., et al. Self- Microemulsifying Drug Delivery System (SMEDDS) of Vinpocetine: Formulation Development and in vivo Assessment[J]. Biological & Pharmaceutical Bulletin,2008,31:118-125.
[26]Cui, S. M., Zhao, C. S., Chen, D. W., et al. Self-Microemulsifying Drug Delivery Systems (SMEDDS) for Improving In vitro Dissolution and Oral Absorption of Pueraria Lobata Isoflavone[J]. Drug Development and Industrial Pharmacy, 2005, 31:349-356.
[27]Kang, B. K., Lee, J. S., Chon, S. K., Jeong, S. Y., Yuk, S. H., Khang, G., Lee, H. B., & Cho, S. H. Development of self-microemulsifying drug delivery systems (SMEDDS) for oral bioavailability enhancement of simvastatin in beagle dogs[J]. International Journal of Pharmaceutics, 2004,274, 65-73.
[28]Shah NH, Carvajal MT, Patel CI , Infeld MH, Malick AW.Self-emulsifying drug delivery systems (SEDDS) with polyglycolized glycerides for improving in vitro dissolution and oral absorption of lipophilic drugs[J]. Int J Pharm,1994,106(1):15
[29]Pouton, C. W. Effects of inclusion of a model drug on the performance of self-emulsifying formulations [J]. The Journal of Pharmacy and Pharmacology, 1985,37:1-11.
[30]Pouton, C. W. Self-emulsifying drug delivery systems: assessment of the efficiency of emulsification[J]. International Journal ofPharmaceutics, 1985,27, 335–348.
[31] Swenson, E. S., Milisen, W. B., Curatolo, W. Intestinal permeability enhancement: efficacy, acute local toxicity and reversibility[J]. Pharmaceutical Research, 1994,11, 1132–42
[32]胡海燕,徐雄良,龚涛,等.不同溶解性质的药物在微乳中的溶解度和体外释放特征[J].中国药学杂志,2006,31(9):678-682
[33]杨根生,严琴英,王文喜,等.紫杉醇口服自乳化给药系统的处方研究[J].浙江工业大学学报,36 (1):5-11
[34]王思玲,孙长山,陈闯,等.比较Gaussian曲线分布与Nicomp多波型分布分析在纳米分散系粒径测定中的应用[J].沈阳药科大学学报,2003,20(5):321-324
[35]Bok Ki Kang, Jin Soo Lee , Se Kang Chon, et al. Development of self-microemulsifying drug delivery systems (SMEDDS) for oral bioavailability enhancement of simvastatin in beagle dogs[J]. International Journal of Pharmaceutics,2004,274:65-73
[36]崔福德.药剂学(第五版)[M].北京人民卫生出版社,2003,39
[37]汤明辉,平其能.去氢骆驼蓬碱口服乳剂的物理稳定性研究[J].2002,18(1):4-6
[38]郭涛,储晓琴,宋洪涛,等.静脉注射用大蒜油亚微乳的制备[J].中国药学杂志,2005,40(8):602-605
[39]Levy MY,Schutze W,Fuhreret C,et al.Characterization of diazepam submicron emulsion interface: role of loeic acid [J]. J Microencapsulation,1944,11(1):79-92
[40]Tamilvanan S. Oil-in-water lipid emusions: implication for parenteral and ocular delivering systems[J].Progress in Lipid Research,2004,43:489-533
[41]Aslihan A, Rainer H. Muller.Intravenous itraconazole emulsionsproduced by SolEmuls technology [J].European Journal of Pharmaceutics and Biopharmaceutics, 2003, 56 29–36
[42]Junghanns, J. U., Buttle, I., Muller, R. H., et al. SolEmuls? Technology: A way to overcome the drawback of parenteral administration of insoluble drugs[J]. Pharmaceutical Development and Technology, 2007, 12(5): 437-445
[43]田玲玲,唐星,何海冰,等.依托泊苷静脉注射亚微乳的制备及其体内外评价[J].药学学报,2007,42(8):892-897
[44]Tetsuo Yamaguchi. Lipid microspheres as drug carriers: a pharmaceutical point of view [J]. Adv Drug Deliv Rev, 1996, 20: 117-1130
[45]Fang Li, Tao Wang, Hai Bing He, et al.The properties of bufadienolides-loaded nano-emulsion and submicro-emulsion during lyophilization [J]. International Journal of Pharmaceutics 349 (2008) 291–299
[46]张利,侯世祥,王昶光.葫芦素脂肪乳剂成型性影响因素考察[J].中南药学,2004,2(2):74-77
[47]廖颂明.中/长链脂肪乳注射液制备工艺研究[J].广东药学院学报, 2001, 17(3):170-172
[48]王浩,顾吉晋,邓英杰.以泊洛沙姆188为助乳化剂制备双氯芬酸钠亚微乳的研究.中国药房, 2008, 19(25):1965-1968
[49]刘晓峰,张郑瑶,邓旭明,等.纳米TCP/明胶/鹿茸多肽复合材料的生物相容性评价,中国修复重建外科杂志,2009,23(5):598-601
[50]郑少辉,邓意辉.动态光散射技术在静脉注射亚微乳剂粒径评价中的应用,中国药剂学杂志, 2005,3(3): 125-128
[51]孟玉芳,龚明涛,张钧寿.含药静脉注射用脂肪乳剂的研究进展.中国医药工业杂志, 2004, 35( 9): 558-561
[52]温彩霞,许建华,黄秀旺.姜黄素在小鼠体内各脏器的药代动力学研究.中药药理与临床, 2007, 23(4):25-27
[53]赵荣丽,侯世祥,毛声俊,等.川芎嗪脂肪乳在小鼠体内分布的研究.中南药学,2008,6(3): 264-267
[54]Constantinides, P. P. Lipid microemulsion for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharmaceutical research, 1995, 12:1561-1572.
[55]杨春艳,毛莎.西罗莫司口服液服用方法的改进.护理学杂志:外科版,2009,24(9): 82-82
[56] Shah, N. H., Carvajal, M. T., Patel, C. I., Infeld, M. H., & Malick, A. W. Self-emulsifying drug delivery system (SEDDS) with polyglycolysed glycerides for improving in vitro dissolution and oral absorption of lipophilic drugs. International Journal of Pharmaceutics, 1994, 106:15-23.
[57]Chen, Y., Li, G., Wu, X. G., Chen, Z. Y., Hang, J. G., Qin, B., Chen, S., & Wang, R. H. Self-Microemulsifying Drug Delivery System (SMEDDS) of Vinpocetine: Formulation Development and in vivo Assessment. Biological & Pharmaceutical Bulletin, 2008, 31: 118-125.
[58] Trevaskis, N. L., Charman, W. N., & Porter, C. J. Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update. Advanced Drug Delivery Reviews, 2008, 60: 702-716.
[59] Sha, X. Y., Yan, G. J., Wu, Y. J., Li, J. C., & Fang, X. L. Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells. European Journal of Pharmaceutical Sciences, 2005, 24:477–486.
[60] Woo, J. S., Lee, C. H., Shim, C. K., Hwang, S. C. Enhanced oralbioavailability of paclitaxel by co-administration of the P-glycoprotein inhibitor KR30031. Pharmaceutical research, 2003,20:24–30.
[61]Peng-fei Yue, Hai-long Yuan, Ming Yang, et al. Preparation, characterization, and pharmacokinetic evaluation of puerarin submicron emulsion. Journal of pharmaceutical Science and Technology, 2008,62(1):32-44
[62]田玲玲,唐星,何海冰,等.依托泊苷静脉注射亚微乳的制备及其体内外评价.药学学报, 2007, 42(8): 892-897
[63]魏立,孔佩艳,陈幸华,等.阻抑正常骨髓基质SDF21作用对HL260细胞生物学性质的影响.第三军医大学学报,2004,26(23): 2158-2161
[65]毛咏秋,姜愚,雷松,等.流式细胞术PI、Annexin V/PI和APO2.7法检测细胞凋亡的比较.华西医学,2005,20(3):505-506
[66]吴丽贤,许建华,吴国华,等.姜黄素对K562细胞增殖的影响及其与P210bcr/abl激活的Ras信号途径的关系.中国药理学通报,2003 19(1):33-37
[67]唐印华,王玺,博挽澜,等.三氧化二砷抑制小鼠H22肝癌移植瘤血管生长的实验研究.2008,16(7):1091-1094
[68]杨金霞,王学美,朱伟,等.干、鲜壁虎冻干粉对小鼠H22肝癌体内外抑制作用.世界华人消化杂志2007,15(2):157-160
[69]张金娟,温彩兰,张贵林.艾迪注射液对裸鼠人胃癌移植瘤的抑瘤作用研究.贵阳医学院学报,2009年34(6):662-664
[70]陈蕾.静脉输液脂肪乳剂致静脉炎的预防.中国临床医药研究杂志.2008, 181:52-53
[71] Swenson, E. S., Milisen, W. B., Curatolo, W. Intestinal permeability enhancement: efficacy, acute local toxicity and reversibility. Pharmaceutical Research, 1994, 11:1132–42
[72]胡大裕,李高,陈鹰,等.几种表面活性剂的细胞毒性研究[J].中国药师, 2006,9(4):294-296
[73]李昕,王晖,药物吸收促进剂的毒性研究进展.中国医药工业杂志, 2005,36(1):51-53
[1]汪海慧,成扬.姜黄素药理作用的研究进展[J].上海中医药大学学报,2007,21(6):73-74
[2]Wahlstrom, B, Blennow, G. A study on the fate of curcumin in the rat [J]. Acta Pharmacol. Toxicol. (Copenhagen) 1978, 43 (2), 86–92.
[3]Ravindranath,V.,Chandrasekhara,N. Absorption and tissue distribu- tion of curcumin in rats [J]. Toxicology 1980, 16 (3), 259–65.
[4]Ravindranath,V., Chandrasekhara, N. Metabolism of curcumin-studies with [3H]curcumin [J]. Toxicology 1981, 22 (4), 337-44.
[5]Pan, M. H., Huang,T.M., Lin,J.K. Biotransformation of curcumin through reduction and glucuronidation in mice [J]. Drug Metab. Dispos.1999, 27 (4), 486–94.
[6] Perkins S,Verschoyle R. D., Hill K.,et al. Chemopreventive efficacy and pharmacokinetics of curcumin in the min/+ mouse, a model of familial adenomatous polyposis [J]. Cancer Epidemiol Biomarkers Prev,2002,11(6), 535–40.
[7]Marczylo T. H., Verschoyle R. D., Cooke D. N.,et al. Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine [J]. Cancer Chemother Pharmacol.2007,60 (2),171-7.
[8]Ravindranath V., Chandrasekhara N. In vitro studies on the intestinal absorption of curcumin in rats [J]. Toxicology 1981, 20 (2–3), 251–7.
[9]Garcea G., Jones D. J., Singh R. ,et al. Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration [J]. Br. J. Cancer 2004, 90 (5), 1011–5.
[10]Garcea G., Berry D. P., Jones D. J. ,et al. Consumption of the putative chemopreventive agent curcumin by cancer patients: assessment of curcumin levels in the colorectum and their pharmacodynamic consequences [J]. Cancer Epidemiol. Biomarkers. Prev. 2005, 14 (1),120–5.
[11] Holder,G. M., Plummer, J. L., Ryan, A. J. The metabolism and excretion of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene- 3,5-dione) in the rat [J]. Xenobiotica,1978, 8(12),761–8.
[12]Hoehle S.I.,Pfeiffer E.,Solyom A.M.,et al.Metabolism of curcu- minoids in tissue slices and subcellular fractions from rat liver [J]. J. Agric. Food Chem. 2006,54(3),756–64.
[13]Asai, A., Miyazawa, T. Occurrence of orally administered curcuminoid as glucuronide and glucuronide/sulfate conjugates in rat plasma [J]. Life Sci. 2000, 67 (23), 2785–93.
[14]Ireson, C., Orr, S., Jones, D. J. ,et al. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production [J]. Cancer Res. 2001, 61 (3), 1058–64.
[15]Sharma, R. A., McLelland, H. R., Hill, K. A. ,et al. Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer [J]. Clin. Cancer Res. 2001, 7 (7), 1894–900.
[16]Shoba, G, Joy, D, Joseph, T,et al. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers [J]. Planta Med 1998, 64 (4), 353–6.
[17]Cruz-Correa, M., Shoskes, D. A., Sanchez, P. ,et al. Combination treatment with curcumin and quercetin of adenomas in familial adenomatous polyposis [J]. Clin. Gastroenterol. Hepatol. 2006, 4 (8), 1035–8.
[18]Verma S. P., Salamone E., Goldin B. Curcumin and genistein,plant natural products, show synergistic inhibitory effects on the growth of human breast cancer MCF-7 cells induced by estrogenic pesticides [J]. Biochem. Biophys. Res. Commun. 1997, 233 (3), 692–6.
[19] Fang J. Y., Hung C. F., Chiu H. C.,et al. Efficacy and irritancy of enhancers on the in-vitro and in-vivo percutaneous absorption of curcumin [J]. J. Pharm. Pharmacol. 2003,55 (5), 593–601.
[20]Karikar C., Maitra A., Bisht S. ,et al. Polymeric nanoparticle- encapsulated curcumin (“nanocurcumin”):a novel strategy for human cancer therapy [J]. J. Nanobiotechnol.2007, 5, 3.
[21]Tiyaboonchai W., Tungpradit W., Plianbangchang P. .Formulation and characterization of curcuminoids loaded solid lipid nanoparticles [J]. Int. J. Pharm. 2007, 337 (1–2), 299–306. (63)
[22]Li, L., Braiteh, F. S., Kurzrock, R. Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis,curcumin: in vitro and in vivo effects on proliferation, apoptosis,signaling, and angiogenesis [J]. Cancer 2005, 104 (6), 1322–31.
[23]Li, L., Ahmed, B., Mehta, K. ,et al. Liposomal curcumin with and without oxaliplatin: effects on cell growth, apoptosis,and angiogenesis in colorectal cancer [J]. Mol. Cancer Ther. 2007,6 (4), 1276–82.
[24]Kunwar, A., Barik, A., Pandey, R. ,et al. Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study [J]. Biochim. Biophys. Acta 2006, 1760 (10), 1513–20.
[25]Ma, Z., Shayeganpour, A., Brocks, D. R. ,et al. High-performance liquid chromatography analysis of curcumin in rat plasma: application to pharmacokinetics of polymeric micellar formulation of curcumin [J]. Biomed. Chromatogr. 2007,21 (5), 546–52.
[26]Liu, A., Lou, H., Zhao, L. ,et al. Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin [J].J. Pharm. Biomed. Anal. 2006, 40 (3), 720–7.
[27] Maiti, K., Mukherjee, K., Gantait, A,et al. Curcumin–phospholipid complex: Preparation, therapeutic evaluation and pharmacokinetic study in rats [J]. Int. J. Pharm. 2007,330 (1–2), 155–63.
[28] Mosley, C. A., Liotta, D. C., Snyder, J. P. Highly active anticancer curcumin analogues [J]. AdV. Exp. Med. Biol. 2007, 595:, 77–103.
[29]John, V. D., Kuttan, G., Krishnankutty, K. Anti-tumour studies of metal chelates of synthetic curcuminoids [J]. J. Exp. Clin. Cancer Res. 2002, 21 (2), 219–24.
[30] Sui, Z., Salto, R., Li, J. ,et al. Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes [J]. Bioorg. Med. Chem. 1993, 1 (6), 415–22.
[31]Mishra, S., Narain, U., Mishra, R.,et al. Design, development and synthesis of mixed bioconjugates of piperic acid-glycine, curcumin-glycine/alanine and curcumin-glycine-piperic acid and their antibacterial and antifungal properties [J]. Bioorg. Med. Chem.2005, 13 (5), 1477–86.