银杏酮酯口服自微乳化给药系统的研究
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摘要
自微乳化药物传递系统(SMEDDS)是由药物、油相、乳化剂和助乳化剂组成的均一、透明的溶液,在环境温度和温和搅拌的条件下,在水相中自发乳化形成粒径在10～100nm的微乳。其特点是粒径小、微乳液澄清透明、药物增溶量大、制剂稳定,药物存在于细小的油滴中,能快速分布于整个胃肠道,减少了由于药物与胃肠壁的直接接触而引起的刺激;药物在油/水两相之间分配,依靠细小油滴的巨大比表面积大大提高了水难溶性药物的溶出,提高了药物的生物利用度,同时可以避免水不稳定药物的水解及药物对胃肠道的不良刺激。
SMEDDS处方由油相、乳化剂、助乳化剂与药物等组成。质量评价指标主要有乳剂粒径、自乳化效率、伪三元相图、相平衡、乳滴的极性、乳滴电荷、药物体外释放速率、体内药动学与生物利用度等。SMEDDS的制备主要通过微乳的伪三元相图完成。伪三元相图的制备方法有加水滴定法、加油滴定法、加乳化剂滴定法及交替加入法等。制备相图时,通常考察在乳化剂和助乳化剂的不同Km(Km为乳化剂和助乳化剂的质量比)下微乳的形成情况,以乳化剂和助乳化剂为伪三元相图的1个顶点,油相和水相为另外2个顶点,根据各组分在临界点时所占总量的百分比来确定该点在相图中的位置,将每个临界点连成曲线即得该组分在一定Km值下的伪三元相图,通过伪三元相图找出微乳化区域最大的组分。
SMEDDS所选择的活性成分多为脂溶性或水难溶性的药物,能改善药物的口服吸收,增加其生物利用度。相比与传统提高生物利用度的方法,SMEDDS具有更加突出的优点:(1)对难溶性或脂溶性药物具有较高的增溶能力;(2)较高的物理化学稳定性;(3)能填充到硬胶囊或软胶囊,制备工艺简单,容易放大生产。因此,本课题选择SMEDDS运载银杏酮酯进行研究。
银杏酮酯(GBE50)是从银杏叶中提取、精制而成的经国家食品药品监督管理局批准正式生产的二类中药新药和生产批文(国药准字Z20000049)的银杏原料药,其为棕黄色至黄棕色的粉术;有特殊香气,味苦。它主要含有黄酮醇苷、萜类内酯等化学成分,其中银杏总黄酮≥44%,黄酮醇苷≥24%、游离黄酮≥20%;萜类内酯≥6%;银杏酸≤5ppm。
GBE50主要含有黄酮和内酯类两大成分,药理作用主要在血液系统方面、心血管方面、脑血管方面等有明显的保护作用。GBE50临床上主要用于治疗心脑血管疾病,如冠心病、心绞痛、心肌梗塞、脑栓塞、脑血管痉挛等。
GBE50中的银杏萜类内酯难溶于水,黄酮中苷元类水溶性也差,影响内酯和黄酮类的溶解吸收,普通制剂口服吸收差,生物利用度低,且制剂苦感较重,影响制剂的临床使用和推广。为克服GBE50现有剂型的不足,提高生物利用度以提高临床效果,结合自微乳化给药系统,本论文研究GBE50-SMEDDS,由于SMEDDS的特点,增大了比表面积,药物释放快,可以增加GBE50的吸收速度和生物利用度,起到速效和高效的作用。
为了系统研究GBE50-SMEDDS,本文通过测定GBE50在不同溶剂中的平衡溶解度与伪三元相图对组分进行了筛选,最终选定,乳化剂为聚氧乙烯蓖麻油Cremophor EL、助乳化剂为丙二醇1,2-Propanediol、油相为肉豆蔻酸异丙酯IPM,即Cremophor EL/丙二醇/IPM作为自微乳化的基本配方,制备了GBE50自微乳化制剂并对其性质(外观,粒径分布,自微乳化效率与微乳电镜下状态)进行了研究,最终GBE50-SMEDDS处方组成为GBE50 0.12g、丙二醇0.3g、Cremophor EL0.5g、IPM0.25g、Vit E0.03g。油相IPM含量为20.8%,乳化剂Cremophor EL的含量为41.7%,GBE50载药量为10%。GBE50-SMEDDS与水、0.1M HCl或6.8PBS接触后,轻微搅拌下很快在3min后均能白化形成均匀稳定的微乳,粒径为20～50nm。微乳在透射电镜观察为均匀球状液滴。
选用了两种介质水与0.1M HCl,分别以GBE50-SMEDDS为固体制剂与微粒制剂进行了溶出度与释放度的测定。GBE50-SMEDDS的溶出与释放在两种介质中差异均不大。体外溶出测定中,GBE50-SMEDDS胶囊与GBE50粉末胶囊的溶出在前10分钟相差不大,但10分钟后,溶出差异渐大,45分钟后GBE50-SMEDDS胶囊的溶出可达80%以上,而GBE50粉末胶囊仅为40%;总体液平衡反向透析法中,GBE50-SMEDDS体外释放快,15分钟能达到约30%,45分钟能有70%,90分钟后趋于平衡,最后能达到90%。溶出度与释放度结果均表明,GBE50-SMEDDS的体外溶出度高,能有效提高GBE50的溶出与释放。
GBE50-SMEDDS质量标准研究中,采用铝基硅胶薄层板法,以正已烷-乙酸乙酯(4:6)为展开剂,对GBE50中银杏内酯A与B进行了鉴别。采用反相高效液相色谱法对自微乳化组合物中总黄酮醇苷的含量进行了测定,以甲醇-0.5%磷酸(54:46)为流动相;流速为1ml/min;检测波长为368nm,以槲皮素、山柰素与异鼠李素为对照品,槲皮素回归方程为A=110973C-160122,r=0.999 5,线性范围0.163～1.141μg,平均回收率为99.25%,RSD为0.44%;山奈素回归方程为A=97764C-182398,r=0.999 5,线性范围0.2196～1.5372μg,平均回收率为99.20%,RSD为0.43%;异鼠李素回归方程为A=92938C-36124,r=0.999 8,线性范围0.0552～0.3864μg,平均回收率为99.31%,RSD为0.25%。结果表明,GBE50-SMEDDS含有银杏内酯A与B;总黄酮醇苷含量不低于2.4%。该方法简便快速,结果准确可靠,可作为该制剂的质量标准。
以GBE50-SMEDDS外观性状、自微乳化效率(时间)、微乳粒径与总黄酮醇苷的含量为指标,考察了GBE50-SMEDDS的初步稳定性研究。低温试验中,GBE50-SMEDDS于4℃冰箱冷藏放置5天与10天后,其外观性状、自微乳化时间、形成微乳的粒径及药物含量,与0天结果比较,没有明显变化,表明GBE50-SMEDDS低温条件下稳定。高温试验中,GBE50-SMEDDS于60℃高温放置10天后,其外观性状虽无明显变化,但自微乳化时间、形成微乳的粒径及药物含量,与0天结果比较,有明显变化,表明GBE50-SMEDDS高温条件下不稳定。冷热循环试验(4℃与37℃)中,GBE50-SMEDDS放置5天与10天后仍为澄清、透明褐色溶液,并无药物晶体析出,同时自微乳化时间、微乳外观与粒径、药物含量无明显改变,表明GBE50-SMEDDS对温度变化的耐受力良好。强光照射试验(4500Lx±500Lx)中,GBE50-SMEDDS经强光照射10天后,颜色有变化,不澄清透明,药物含量明显降低,乳化时间延长,所得乳剂粒径相应增大,表明GBE50-SMEDDS对光照敏感,需避光保存。加速试验中,GBE50-SMEDDS在温度(40±2)℃、相对湿度(75±5)%条件下放置3个月后,其外观性状、自乳化时间、形成微乳的粒径及含量,与0月结果比较,没有明显变化,表明GBE50-SMEDDS加速条件下稳定性良好。留样试验中,GBE50-SMEDDS在室温留样条件下放置6个月,其各项考察指标基本无变化,表明GBE50-SMEDDS稳定性良好。
研究GBE50-SMEDDS加介质自微乳化后微乳的稳定性,GBE50-SMEDDS经0.1M HCl稀释到200 ml后,8h内药物吸光值无明显变化,微乳剂外观与粒径并未发生明显改变,微乳在8h内稳定。
初步稳定性研究结果表明,GBE50-SMEDDS在密封包装与常温贮存下,稳定性高,其外观性状、自微乳化时间、微乳粒径与药物含量均无明显变化。自微乳化制剂加介质后经自微乳化形成的微乳液在8h内稳定,从而保证GBE50在被胃肠道吸收之前以溶解状态的微乳形式存在而不析出,以确保其口服生物利用度。
GBE50-SMEDDS大鼠体内药物代谢动力学研究中,以桑色素为内标,槲皮素、山奈素、异鼠李素为对照品,建立了银杏黄酮的血浆样品分析方法。血药浓度-时间曲线和药物动力学参数显示,GBE50-SMEDDS的Cmax比参比对照颗粒剂提高了0.3 mg/L,Tmax提前了2.7 h,消除速率减小,消除半衰期延长了7.4 h,分布速率增大,分布半衰期提前了2.2h,AUC_(0-∞)提高了13.8 mg.h/L,AUC_(0-∞)是参比对照颗粒剂的152.47%,提高了52.47%。GBE50-SMEDDS新制剂比现有市场剂型杏林颗粒剂生物利用度高,为临床疗效的提高提供了实验基础。
GBE50-SMEDDS小鼠急性毒性实验结果表明,最大剂量组毒副反应明显,反应迟钝,眼睛呆滞,眼角有分泌物,腹部胀满,大便便溏,甚至死亡。毒副反应是由制剂中辅料油相、乳化剂与助乳化剂引起。半数致死量LD50=0.33ml(0.36g)/10g,LD50的95%的可信限0.30～0.37ml(0.33～0.41g)/10g。提示GBE50-SMEDDS小鼠急性口服给药有毒副作用,LD50是正常成人常用剂量(1.2g/60Kg)的1800倍,可见,GBE50-SMEDDS虽有毒副作用,但在临床上按说明使用,是安全的。
GBE50-SMEDDS的动物药效实验结果表明,在GBE50-SMEDDS对急性血瘀模型大鼠血液流变学影响实验中,与正常对照组比较,血瘀模型组的全血黏度比(低切、中切、高切值)与血浆黏度比都有升高(P值分别为0.000,0.000,0.000,0.014),表明造模成功。与血瘀模型组比较,给药阳性对照组能降低全血黏度比(低切、中切、高切值),(P值分别为0.007,0.01,0.004);给药GBE50-SMEDDS高剂量组能降低全血黏度比(低切、中切、高切值)与血浆黏度比(P值分别为0.000,0.000,0.001,0.001);给药GBE50-SMEDDS中剂量组能降低全血黏度比(低切、中切、高切值)与血浆黏度比(P值分别为0.000,0.001,0.002,0.016);低剂量组结果差异虽无显著性意义,但有降低趋势,表明GBE50-SMEDDS高、中、低剂量组有量效关系。各组的红细胞压积结果差异虽无显著性意义(原因可能与试验误差、检测方法及样本量较少等有关),但均有作用趋势,并呈量效关系,说明GBE50-SMEDDS有改善急性血瘀模型大鼠血液流变学的作用,并呈剂量依赖性。
在GBE50-SMEDDS对异丙肾上腺素(ISO)致大鼠心肌缺血的保护作用实验中,与正常对照组比较,缺血模型组大鼠注射ISO后很快出现心肌缺血改变,表现为心电图ST段抬高(ST段30与35 min的P均为0.001),表明造模成功。与缺血模型组比较,给药阳性对照组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.038,0.007);给药GBE50-SMEDDS高剂量组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.005,0.001);给药GBE50-SMEDDS中剂量组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.023,0.007);低剂量组结果差异虽无显著性意义,但有降低趋势,表明GBE50-SMEDDS高、中、低剂量组有量效关系。各组的T波结果差异虽无显著性意义(原因可能与试验误差、检测方法及样本量较少等有关),但均有作用趋势,并呈量效关系。说明GBE50-SMEDDS有改善大鼠心肌缺血损伤引起心电图变化的作用,并呈剂量依赖性。
对大鼠心肌缺血损伤引起心肌三酶异常的影响中,与正常对照组比较,缺血模型组的CK、LDH与AST释放均增多(P均为0.000),表明造模成功。与缺血模型组比较,给药阳性对照组能降低CK、LDH与AST释放(P分别为0.000,0.000,0.002);给药GBE50-SMEDDS高剂量组能降低CK、LDH与AST释放(P均为0.000);给药GBE50-SMEDDS中剂量组均能降低CK、LDH与AST释放(P均为0.000);给药GBE50-SMEDDS低剂量组均能降低CK、LDH释放(P分别为0.005,0.001);GBE50-SMEDDS高、中、低剂量组有量效关系,说明GBE50-SMEDDS有改善大鼠心肌缺血损伤引起心肌三酶异常的作用,并呈剂量依赖性。
在动物药效实验中,GBE50-SMEDDS能改善急性血瘀模型大鼠的血液流变异常,对异丙肾上腺素致大鼠心肌缺血有保护作用,并呈剂量依赖性。等效剂量的GBE50-SMEDDS比对照组现有市场剂型杏林颗粒剂作用趋势好,说明GBE50-SMEDDS能有效提高GBE50的药效作用,为临床疗效的提高提供了动物药理实验基础。
本文结合SMEDDS的特点与GBE50作用效果及现有剂型的不足,从处方筛选、微乳制备、工艺优化、体外评价、质量标准、初步稳定性、体内药动学与药效试验等方面对GBE50-SMEDDS进行了研究,研究结果表明,GBE50-SMEDDS处方组成稳定、质量可控、体外释放快、生物利用度有提高、药效作用增强,值得进一步深入研究。
Self-Microemulsifying Drug Delivery Systems (SMEDDS) compose of drug, oil phase, emulsifier and assist emulsifier, it is homogeneous, transparent solution.SMEDDS can spontaneously emulsifying to form microemulsion with particle size 10～100nm when mixing with water phase, ambient temperature and moderate mixing conditions.Its characteristics are small size, transparent microemulsion clarification, a large quantity of drug solubilization, formulation stability and drug exists in the small oil droplets, the rapid distribution of the entire gastrointestinal tract, reduced stimulation by drugs and the gastrointestinal wall in direct contact; drug at oil/water distribution between the two-phase, small droplets depend on the enormous surface area greatly improve the water insoluble drug dissolution, increased bioavailability of drugs at the same time to avoid water unstable drugs hydrolysis and drugs on the gastrointestinal tract adverse stimulus.
SMEDDS prescription compose of the oil phase, emulsifier , assist emulsifier and drug and so on. The main indicators of quality assessment have microemulsion particle size, self-emulsifying rate, pseudo-ternary phase diagram, phase equilibrium, the polarity of microemulsion drops, microemulsion droplet charge, drug release rate in vitro, in vivo pharmacokinetics and bioavailability, etc. Preparation of SMEDDS mainly use microemulsion pseudo-ternary phase diagram to finish. Pseudo-ternary phase diagram of the preparation methods have add aqueous titration, add oil titration, add emulsifier titration and alternately add these components. Preparation of phase diagram, it is usually inspected at emulsifier and assist emulsifier at the different Km (Km is the ratio of emulsifier and the assist emulsifier) under the formation of microemulsions , emulsifier and assist emulsifier for the pseudo-ternary phase diagram of a vertex, oil phase and water phase for another two vertices, according to each component at the critical point when the percentage of the total percentage to determine the point in the phase diagram of the location of the critical point of each curve that is connected. The component must have at Km value of pseudo-ternary phase diagram, through the pseudo-ternary phase diagram to identify the biggest component microemulsifying regional.
The active ingredients for SMEDDS are selected from the many fat-soluble or water insoluble drugs, SMEDDS can improve oral drug absorption, increase its bioavailability. Compared with the traditional method of bioavailability, the micro-emulsion drug delivery system has a more prominent advantages: (1) have a high solubilization capacity for the insoluble or fat-soluble drugs; (2) higher physical and chemical stability; (3) filling the hard capsule or soft capsule, preparation simple, easy to enlarge the production. Therefore, the topic select self-microemulsifying drug delivery system carrying GBE50 for research.
GBE50 are from the Ginkgo biloba leaf extract, refined formed by the State Food and Drug Administration approved the production of second-class medicine and production approval of new drugs of Ginkgo biloba raw material, it is brown to yellow-brown powder; have a special aroma, bitter taste. It contains flavonoid glycosides, terpene lactones, such as chemical composition, total flavonoids of Ginkgo≥44%, flavone glycosides≥24%, free flavonoids≥20%; terpene lactones≥6%; ginkgolic acid≤5ppm.
GBE50 compose of the main two ingredients: flavonoids and ginkgolide, there is a clear protective effect on pharmacological effects mainly in the blood system, cardiovascular, cerebrovascular and so on. GBE50 for the treatment of major clinical cardiovascular and cerebrovascular diseases such as coronary heart disease, angina, myocardial infarction, cerebral embolism, cerebral vasospasm.
Ginkgolide of GBE50 is difficult to dissolve in water, flavonoid aglycone medium category of water-soluble is bad, the impact of ginkgolide and flavonoids dissolved absorption, oral absorption of ordinary formulations is bad, low bioavailability, and the ordinary formulations is bitter, effecting the clinical use and promotion. To overcome the shortcomings of existing GBE50 formulations to improve bioavailability and clinical effects, combined with self-microemulsifying drug delivery system, this thesis research GBE50-SMEDDS, because of SMEDDS characteristics, increasing the surface area, drug releasing quickly, can increase GBE50 absorption rate and bioavailability, played the role of quick and efficient.
System study for GBE50-SMEDDS, the use of Equilibrium Solubility and Pseudo-ternary phase diagram of the components were screened. Finally selected Cremophor EL/propylene glycol/IPM as a self-microemulsifying basic formula, we prepared GBE50-SMEDDS and study their properties (appearance, self-microemulsifying time, particle size distribution) and in vitro release carried out a mission, eventually consisting of prescription GBE50 0.12g, Propanediol 0.3g, Cremophor EL0.5g, IPM 0.25g, Vit E 0.03g; GBE50-SMEDDS with water, O.IMHCI or 6.8PBS after contact under slight agitation is quickly form a stable microemulsion uniformity in 3 minutes, particle size is 20～50nm. Determination of in vitro dissolution after 45 minutes of dissolution SMEDDS capsule up to more than 80%, while only 40% powder capsules; Bulk-equilibrium reverse dialysis bag technique in reverse, GBE50-SMEDDS quick in vitro release, 15 minutes to achieve about 30%, 45 minutes to have 70%, 90 minutes after the balance, and finally reach 90%.
Using TLC with silica gel thin aluminum plate , Ginkgolides A and B have been identified. Using RP-HPLC of self-microemulsifying composition of total flavonol glycoside contents, measuring method is simple, rapid, accurate and results are reliable, quercetin average recovery was 99.25%, RSD 0.44%; kaempferol average recovery was 99.20%, RSD 0.43%; isorhamnetin average recovery was 99.31%, RSD 0.25%. The results show that, GBE50-SMEDDS containing ginkgolide A and B; total flavonol glycoside content is not less than 2.4%.
Preliminary stability study, results showed that GBE50-SMEDDS has high stability while in a sealed packaging and storage under ambient temperature, the appearance of formulation, self-microemulsifying time, particle size and drug content determination was no significant change . Microemulsion with adding water is stable in 8h, thus ensuring GBE50 absorption in the gastrointestinal tract prior to the status of the microemulsion form of dissolved without precipitation to ensure that its oral bioavailability.
GBE50-SMEDDS rat pharmacokinetics study, use morin as internal standard, quercetin, kaempferol and isorhamnetin as the reference substance, establish the ginkgo flavone analysis method of plasma samples. Plasma concentration-time curves and pharmacokinetic parameters showed that, Cmax of GBE50-SMEDDS increased 0.3 mg/Lthan the reference group, Tmax earlier 2.7 h, the elimination of rate decreases, the elimination half-life extend 7.4 h, the distribution rate increase , the distribution of half-life ahead 2.2h, AUC increased 13.8 mg.h/L, relative bioavailability is the reference of 152.47%, increased 52.47%. GBE50-SMEDDS new formulations has higher bioavailability than market existing agents Xinglin, providing an experimental basis for clinical efficacy improvement.
The results of GBE50-SMEDDS acute toxicity in mice show that the maximum group have obvious toxicity, such as unresponsive, eyes dull, eyes have a discharge, or even death. Toxicity is caused by the agents in the oil phase materials, emulsifier and assist emulsifier.Half lethal dose LD50 = 0.33ml (0.36g)/10g, LD50 of 95% CI is 0.30～0.37ml (0.33～0.41g)/10g. GBE50-SMEDDS has acute toxic side effects of oral administration, LD50 is 1,800 times as the dose commonly used in normal adults (1.2g/60Kg). We can see, GBE50-SMEDDS although has toxic side effects, but it is safe while in clinical use according to instructions.
The animal efficacy results of GBE50-SMEDDS show that in the acute blood stasis model rats hemorrheology experiment, compared with normal control group, blood stasis model group of the whole blood viscosity ratio (low-cut, medium-cut and high-cut values) and plasma viscosity are higher (P=0.000,0.000,0.000,0.014), showing that the model is successful. Compared with blood stasis model group, the positive control group can reduce whole blood viscosity (low-cut, medium-cut and high-cut values), (P=0.007, 0.01, 0.004); GBE50-SMEDDS high dose group can reduce the viscosity of whole blood (low-cut, medium-cut and high-cut values) and plasma viscosity (P=0.000, 0.000, 0.001,0.001); GBE50-SMEDDS medium dose group can reduce whole blood viscosity ( low-cut, medium-cut and high-cut values) and plasma viscosity (P=0.000, 0.001, 0.002,0.016); difference between the results of low-dose group were not significant, but have lower trend, showing GBE50-SMEDDS high, medium , low-dose group, have dose-effect relationship. The group differences in hematocnt results were not significant (due to pilot error and less sample size), but both the role of trends and dose-effect relationship, showed that an improvement in GBE50-SMEDDS acute blood stasis model and the role of hemorheology in rats and dose-dependent manner.
GBE50-SMEDDS on isoproterenol-induced myocardial ischemia experiment, compared with normal control group, ischemic model group rats injected ISO myocardial ischemia soon after the change in electrocardiogram showed ST-segment elevation ( ST-30 and 35 min of P=0.001), indicating that it is a successful model. Compared with ischemic model group, the positive control group may be against ISO-induced myocardial ischemia in ECG changes, manifested as decreased ST segment (ST-30 and 35 min of P=0.038,0.007); GBE50-SMEDDS high-dose group could induced myocardial ischemia in ECG changes, manifested as decreased ST segment (ST-30 and 35 min of P=0.005,0.001); GBE50-SMEDDS medium dose group induce myocardial ischemia-induced changes in the electrocardiogram showed ST segment declined (ST-30 and 35 min of P=0.023,0.007); difference between the results of low-dose group were not significant, but has lower trend, showing GBE50-SMEDD high, medium and low-dose group, have dose-effect relationship. The results of T-wave in each group have no significant differences in statistical (due to pilot error and less sample size), but both have the role of trends and dose-effect relationship. GBE50-SMEDDS has an improvement in myocardial ischemia caused by changes in the role of ECG and has dose-dependent manner.
Compared with normal control group, ischemic model group of CK, LDH and AST release increased (P=0.000), indicating that model is successful. Compared with ischemic model group, the positive control group could reduce CK, LDH and AST release (P=0.000,0.000,0.002); GBE50-SMEDDS high dose group reduced CK, LDH and AST release (P=0.000); GBE50-SMEDDS medium dose group were lower in CK, LDH and AST release (P=0.000); GBE50-SMEDDS low-dose group could reduce the CK, LDH release (P=0.005, 0.001); GBE50-SMEDDS high, medium and low-dose group, have dose-effect relationship. GBE50-SMEDDS improve myocardial ischemic injury in rats, and dose-dependent manner.
Efficacy in animal experiments, GBE50-SMEDDS can improve the acute blood stasis model rats abnormality, and has a protective effect on isoproterenol-induced myocardial ischemia, and dose-dependent manner. Equivalent doses of GBE50-SMEDDS has a better effect than the control group existing market Xinglin granules , GBE50-SMEDDS efficiently improve the efficacy of GBE50, provide the basis of animal in order to improve the clinical efficacy of pharmacological experiments.
SMEDDS处方由油相、乳化剂、助乳化剂与药物等组成。质量评价指标主要有乳剂粒径、自乳化效率、伪三元相图、相平衡、乳滴的极性、乳滴电荷、药物体外释放速率、体内药动学与生物利用度等。SMEDDS的制备主要通过微乳的伪三元相图完成。伪三元相图的制备方法有加水滴定法、加油滴定法、加乳化剂滴定法及交替加入法等。制备相图时,通常考察在乳化剂和助乳化剂的不同Km(Km为乳化剂和助乳化剂的质量比)下微乳的形成情况,以乳化剂和助乳化剂为伪三元相图的1个顶点,油相和水相为另外2个顶点,根据各组分在临界点时所占总量的百分比来确定该点在相图中的位置,将每个临界点连成曲线即得该组分在一定Km值下的伪三元相图,通过伪三元相图找出微乳化区域最大的组分。
SMEDDS所选择的活性成分多为脂溶性或水难溶性的药物,能改善药物的口服吸收,增加其生物利用度。相比与传统提高生物利用度的方法,SMEDDS具有更加突出的优点:(1)对难溶性或脂溶性药物具有较高的增溶能力;(2)较高的物理化学稳定性;(3)能填充到硬胶囊或软胶囊,制备工艺简单,容易放大生产。因此,本课题选择SMEDDS运载银杏酮酯进行研究。
银杏酮酯(GBE50)是从银杏叶中提取、精制而成的经国家食品药品监督管理局批准正式生产的二类中药新药和生产批文(国药准字Z20000049)的银杏原料药,其为棕黄色至黄棕色的粉术;有特殊香气,味苦。它主要含有黄酮醇苷、萜类内酯等化学成分,其中银杏总黄酮≥44%,黄酮醇苷≥24%、游离黄酮≥20%;萜类内酯≥6%;银杏酸≤5ppm。
GBE50主要含有黄酮和内酯类两大成分,药理作用主要在血液系统方面、心血管方面、脑血管方面等有明显的保护作用。GBE50临床上主要用于治疗心脑血管疾病,如冠心病、心绞痛、心肌梗塞、脑栓塞、脑血管痉挛等。
GBE50中的银杏萜类内酯难溶于水,黄酮中苷元类水溶性也差,影响内酯和黄酮类的溶解吸收,普通制剂口服吸收差,生物利用度低,且制剂苦感较重,影响制剂的临床使用和推广。为克服GBE50现有剂型的不足,提高生物利用度以提高临床效果,结合自微乳化给药系统,本论文研究GBE50-SMEDDS,由于SMEDDS的特点,增大了比表面积,药物释放快,可以增加GBE50的吸收速度和生物利用度,起到速效和高效的作用。
为了系统研究GBE50-SMEDDS,本文通过测定GBE50在不同溶剂中的平衡溶解度与伪三元相图对组分进行了筛选,最终选定,乳化剂为聚氧乙烯蓖麻油Cremophor EL、助乳化剂为丙二醇1,2-Propanediol、油相为肉豆蔻酸异丙酯IPM,即Cremophor EL/丙二醇/IPM作为自微乳化的基本配方,制备了GBE50自微乳化制剂并对其性质(外观,粒径分布,自微乳化效率与微乳电镜下状态)进行了研究,最终GBE50-SMEDDS处方组成为GBE50 0.12g、丙二醇0.3g、Cremophor EL0.5g、IPM0.25g、Vit E0.03g。油相IPM含量为20.8%,乳化剂Cremophor EL的含量为41.7%,GBE50载药量为10%。GBE50-SMEDDS与水、0.1M HCl或6.8PBS接触后,轻微搅拌下很快在3min后均能白化形成均匀稳定的微乳,粒径为20～50nm。微乳在透射电镜观察为均匀球状液滴。
选用了两种介质水与0.1M HCl,分别以GBE50-SMEDDS为固体制剂与微粒制剂进行了溶出度与释放度的测定。GBE50-SMEDDS的溶出与释放在两种介质中差异均不大。体外溶出测定中,GBE50-SMEDDS胶囊与GBE50粉末胶囊的溶出在前10分钟相差不大,但10分钟后,溶出差异渐大,45分钟后GBE50-SMEDDS胶囊的溶出可达80%以上,而GBE50粉末胶囊仅为40%;总体液平衡反向透析法中,GBE50-SMEDDS体外释放快,15分钟能达到约30%,45分钟能有70%,90分钟后趋于平衡,最后能达到90%。溶出度与释放度结果均表明,GBE50-SMEDDS的体外溶出度高,能有效提高GBE50的溶出与释放。
GBE50-SMEDDS质量标准研究中,采用铝基硅胶薄层板法,以正已烷-乙酸乙酯(4:6)为展开剂,对GBE50中银杏内酯A与B进行了鉴别。采用反相高效液相色谱法对自微乳化组合物中总黄酮醇苷的含量进行了测定,以甲醇-0.5%磷酸(54:46)为流动相;流速为1ml/min;检测波长为368nm,以槲皮素、山柰素与异鼠李素为对照品,槲皮素回归方程为A=110973C-160122,r=0.999 5,线性范围0.163～1.141μg,平均回收率为99.25%,RSD为0.44%;山奈素回归方程为A=97764C-182398,r=0.999 5,线性范围0.2196～1.5372μg,平均回收率为99.20%,RSD为0.43%;异鼠李素回归方程为A=92938C-36124,r=0.999 8,线性范围0.0552～0.3864μg,平均回收率为99.31%,RSD为0.25%。结果表明,GBE50-SMEDDS含有银杏内酯A与B;总黄酮醇苷含量不低于2.4%。该方法简便快速,结果准确可靠,可作为该制剂的质量标准。
以GBE50-SMEDDS外观性状、自微乳化效率(时间)、微乳粒径与总黄酮醇苷的含量为指标,考察了GBE50-SMEDDS的初步稳定性研究。低温试验中,GBE50-SMEDDS于4℃冰箱冷藏放置5天与10天后,其外观性状、自微乳化时间、形成微乳的粒径及药物含量,与0天结果比较,没有明显变化,表明GBE50-SMEDDS低温条件下稳定。高温试验中,GBE50-SMEDDS于60℃高温放置10天后,其外观性状虽无明显变化,但自微乳化时间、形成微乳的粒径及药物含量,与0天结果比较,有明显变化,表明GBE50-SMEDDS高温条件下不稳定。冷热循环试验(4℃与37℃)中,GBE50-SMEDDS放置5天与10天后仍为澄清、透明褐色溶液,并无药物晶体析出,同时自微乳化时间、微乳外观与粒径、药物含量无明显改变,表明GBE50-SMEDDS对温度变化的耐受力良好。强光照射试验(4500Lx±500Lx)中,GBE50-SMEDDS经强光照射10天后,颜色有变化,不澄清透明,药物含量明显降低,乳化时间延长,所得乳剂粒径相应增大,表明GBE50-SMEDDS对光照敏感,需避光保存。加速试验中,GBE50-SMEDDS在温度(40±2)℃、相对湿度(75±5)%条件下放置3个月后,其外观性状、自乳化时间、形成微乳的粒径及含量,与0月结果比较,没有明显变化,表明GBE50-SMEDDS加速条件下稳定性良好。留样试验中,GBE50-SMEDDS在室温留样条件下放置6个月,其各项考察指标基本无变化,表明GBE50-SMEDDS稳定性良好。
研究GBE50-SMEDDS加介质自微乳化后微乳的稳定性,GBE50-SMEDDS经0.1M HCl稀释到200 ml后,8h内药物吸光值无明显变化,微乳剂外观与粒径并未发生明显改变,微乳在8h内稳定。
初步稳定性研究结果表明,GBE50-SMEDDS在密封包装与常温贮存下,稳定性高,其外观性状、自微乳化时间、微乳粒径与药物含量均无明显变化。自微乳化制剂加介质后经自微乳化形成的微乳液在8h内稳定,从而保证GBE50在被胃肠道吸收之前以溶解状态的微乳形式存在而不析出,以确保其口服生物利用度。
GBE50-SMEDDS大鼠体内药物代谢动力学研究中,以桑色素为内标,槲皮素、山奈素、异鼠李素为对照品,建立了银杏黄酮的血浆样品分析方法。血药浓度-时间曲线和药物动力学参数显示,GBE50-SMEDDS的Cmax比参比对照颗粒剂提高了0.3 mg/L,Tmax提前了2.7 h,消除速率减小,消除半衰期延长了7.4 h,分布速率增大,分布半衰期提前了2.2h,AUC_(0-∞)提高了13.8 mg.h/L,AUC_(0-∞)是参比对照颗粒剂的152.47%,提高了52.47%。GBE50-SMEDDS新制剂比现有市场剂型杏林颗粒剂生物利用度高,为临床疗效的提高提供了实验基础。
GBE50-SMEDDS小鼠急性毒性实验结果表明,最大剂量组毒副反应明显,反应迟钝,眼睛呆滞,眼角有分泌物,腹部胀满,大便便溏,甚至死亡。毒副反应是由制剂中辅料油相、乳化剂与助乳化剂引起。半数致死量LD50=0.33ml(0.36g)/10g,LD50的95%的可信限0.30～0.37ml(0.33～0.41g)/10g。提示GBE50-SMEDDS小鼠急性口服给药有毒副作用,LD50是正常成人常用剂量(1.2g/60Kg)的1800倍,可见,GBE50-SMEDDS虽有毒副作用,但在临床上按说明使用,是安全的。
GBE50-SMEDDS的动物药效实验结果表明,在GBE50-SMEDDS对急性血瘀模型大鼠血液流变学影响实验中,与正常对照组比较,血瘀模型组的全血黏度比(低切、中切、高切值)与血浆黏度比都有升高(P值分别为0.000,0.000,0.000,0.014),表明造模成功。与血瘀模型组比较,给药阳性对照组能降低全血黏度比(低切、中切、高切值),(P值分别为0.007,0.01,0.004);给药GBE50-SMEDDS高剂量组能降低全血黏度比(低切、中切、高切值)与血浆黏度比(P值分别为0.000,0.000,0.001,0.001);给药GBE50-SMEDDS中剂量组能降低全血黏度比(低切、中切、高切值)与血浆黏度比(P值分别为0.000,0.001,0.002,0.016);低剂量组结果差异虽无显著性意义,但有降低趋势,表明GBE50-SMEDDS高、中、低剂量组有量效关系。各组的红细胞压积结果差异虽无显著性意义(原因可能与试验误差、检测方法及样本量较少等有关),但均有作用趋势,并呈量效关系,说明GBE50-SMEDDS有改善急性血瘀模型大鼠血液流变学的作用,并呈剂量依赖性。
在GBE50-SMEDDS对异丙肾上腺素(ISO)致大鼠心肌缺血的保护作用实验中,与正常对照组比较,缺血模型组大鼠注射ISO后很快出现心肌缺血改变,表现为心电图ST段抬高(ST段30与35 min的P均为0.001),表明造模成功。与缺血模型组比较,给药阳性对照组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.038,0.007);给药GBE50-SMEDDS高剂量组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.005,0.001);给药GBE50-SMEDDS中剂量组可对抗ISO所致的心肌缺血的心电图改变,表现为ST段有下降(ST段30与35 min的P分别为0.023,0.007);低剂量组结果差异虽无显著性意义,但有降低趋势,表明GBE50-SMEDDS高、中、低剂量组有量效关系。各组的T波结果差异虽无显著性意义(原因可能与试验误差、检测方法及样本量较少等有关),但均有作用趋势,并呈量效关系。说明GBE50-SMEDDS有改善大鼠心肌缺血损伤引起心电图变化的作用,并呈剂量依赖性。
对大鼠心肌缺血损伤引起心肌三酶异常的影响中,与正常对照组比较,缺血模型组的CK、LDH与AST释放均增多(P均为0.000),表明造模成功。与缺血模型组比较,给药阳性对照组能降低CK、LDH与AST释放(P分别为0.000,0.000,0.002);给药GBE50-SMEDDS高剂量组能降低CK、LDH与AST释放(P均为0.000);给药GBE50-SMEDDS中剂量组均能降低CK、LDH与AST释放(P均为0.000);给药GBE50-SMEDDS低剂量组均能降低CK、LDH释放(P分别为0.005,0.001);GBE50-SMEDDS高、中、低剂量组有量效关系,说明GBE50-SMEDDS有改善大鼠心肌缺血损伤引起心肌三酶异常的作用,并呈剂量依赖性。
在动物药效实验中,GBE50-SMEDDS能改善急性血瘀模型大鼠的血液流变异常,对异丙肾上腺素致大鼠心肌缺血有保护作用,并呈剂量依赖性。等效剂量的GBE50-SMEDDS比对照组现有市场剂型杏林颗粒剂作用趋势好,说明GBE50-SMEDDS能有效提高GBE50的药效作用,为临床疗效的提高提供了动物药理实验基础。
本文结合SMEDDS的特点与GBE50作用效果及现有剂型的不足,从处方筛选、微乳制备、工艺优化、体外评价、质量标准、初步稳定性、体内药动学与药效试验等方面对GBE50-SMEDDS进行了研究,研究结果表明,GBE50-SMEDDS处方组成稳定、质量可控、体外释放快、生物利用度有提高、药效作用增强,值得进一步深入研究。
Self-Microemulsifying Drug Delivery Systems (SMEDDS) compose of drug, oil phase, emulsifier and assist emulsifier, it is homogeneous, transparent solution.SMEDDS can spontaneously emulsifying to form microemulsion with particle size 10～100nm when mixing with water phase, ambient temperature and moderate mixing conditions.Its characteristics are small size, transparent microemulsion clarification, a large quantity of drug solubilization, formulation stability and drug exists in the small oil droplets, the rapid distribution of the entire gastrointestinal tract, reduced stimulation by drugs and the gastrointestinal wall in direct contact; drug at oil/water distribution between the two-phase, small droplets depend on the enormous surface area greatly improve the water insoluble drug dissolution, increased bioavailability of drugs at the same time to avoid water unstable drugs hydrolysis and drugs on the gastrointestinal tract adverse stimulus.
SMEDDS prescription compose of the oil phase, emulsifier , assist emulsifier and drug and so on. The main indicators of quality assessment have microemulsion particle size, self-emulsifying rate, pseudo-ternary phase diagram, phase equilibrium, the polarity of microemulsion drops, microemulsion droplet charge, drug release rate in vitro, in vivo pharmacokinetics and bioavailability, etc. Preparation of SMEDDS mainly use microemulsion pseudo-ternary phase diagram to finish. Pseudo-ternary phase diagram of the preparation methods have add aqueous titration, add oil titration, add emulsifier titration and alternately add these components. Preparation of phase diagram, it is usually inspected at emulsifier and assist emulsifier at the different Km (Km is the ratio of emulsifier and the assist emulsifier) under the formation of microemulsions , emulsifier and assist emulsifier for the pseudo-ternary phase diagram of a vertex, oil phase and water phase for another two vertices, according to each component at the critical point when the percentage of the total percentage to determine the point in the phase diagram of the location of the critical point of each curve that is connected. The component must have at Km value of pseudo-ternary phase diagram, through the pseudo-ternary phase diagram to identify the biggest component microemulsifying regional.
The active ingredients for SMEDDS are selected from the many fat-soluble or water insoluble drugs, SMEDDS can improve oral drug absorption, increase its bioavailability. Compared with the traditional method of bioavailability, the micro-emulsion drug delivery system has a more prominent advantages: (1) have a high solubilization capacity for the insoluble or fat-soluble drugs; (2) higher physical and chemical stability; (3) filling the hard capsule or soft capsule, preparation simple, easy to enlarge the production. Therefore, the topic select self-microemulsifying drug delivery system carrying GBE50 for research.
GBE50 are from the Ginkgo biloba leaf extract, refined formed by the State Food and Drug Administration approved the production of second-class medicine and production approval of new drugs of Ginkgo biloba raw material, it is brown to yellow-brown powder; have a special aroma, bitter taste. It contains flavonoid glycosides, terpene lactones, such as chemical composition, total flavonoids of Ginkgo≥44%, flavone glycosides≥24%, free flavonoids≥20%; terpene lactones≥6%; ginkgolic acid≤5ppm.
GBE50 compose of the main two ingredients: flavonoids and ginkgolide, there is a clear protective effect on pharmacological effects mainly in the blood system, cardiovascular, cerebrovascular and so on. GBE50 for the treatment of major clinical cardiovascular and cerebrovascular diseases such as coronary heart disease, angina, myocardial infarction, cerebral embolism, cerebral vasospasm.
Ginkgolide of GBE50 is difficult to dissolve in water, flavonoid aglycone medium category of water-soluble is bad, the impact of ginkgolide and flavonoids dissolved absorption, oral absorption of ordinary formulations is bad, low bioavailability, and the ordinary formulations is bitter, effecting the clinical use and promotion. To overcome the shortcomings of existing GBE50 formulations to improve bioavailability and clinical effects, combined with self-microemulsifying drug delivery system, this thesis research GBE50-SMEDDS, because of SMEDDS characteristics, increasing the surface area, drug releasing quickly, can increase GBE50 absorption rate and bioavailability, played the role of quick and efficient.
System study for GBE50-SMEDDS, the use of Equilibrium Solubility and Pseudo-ternary phase diagram of the components were screened. Finally selected Cremophor EL/propylene glycol/IPM as a self-microemulsifying basic formula, we prepared GBE50-SMEDDS and study their properties (appearance, self-microemulsifying time, particle size distribution) and in vitro release carried out a mission, eventually consisting of prescription GBE50 0.12g, Propanediol 0.3g, Cremophor EL0.5g, IPM 0.25g, Vit E 0.03g; GBE50-SMEDDS with water, O.IMHCI or 6.8PBS after contact under slight agitation is quickly form a stable microemulsion uniformity in 3 minutes, particle size is 20～50nm. Determination of in vitro dissolution after 45 minutes of dissolution SMEDDS capsule up to more than 80%, while only 40% powder capsules; Bulk-equilibrium reverse dialysis bag technique in reverse, GBE50-SMEDDS quick in vitro release, 15 minutes to achieve about 30%, 45 minutes to have 70%, 90 minutes after the balance, and finally reach 90%.
Using TLC with silica gel thin aluminum plate , Ginkgolides A and B have been identified. Using RP-HPLC of self-microemulsifying composition of total flavonol glycoside contents, measuring method is simple, rapid, accurate and results are reliable, quercetin average recovery was 99.25%, RSD 0.44%; kaempferol average recovery was 99.20%, RSD 0.43%; isorhamnetin average recovery was 99.31%, RSD 0.25%. The results show that, GBE50-SMEDDS containing ginkgolide A and B; total flavonol glycoside content is not less than 2.4%.
Preliminary stability study, results showed that GBE50-SMEDDS has high stability while in a sealed packaging and storage under ambient temperature, the appearance of formulation, self-microemulsifying time, particle size and drug content determination was no significant change . Microemulsion with adding water is stable in 8h, thus ensuring GBE50 absorption in the gastrointestinal tract prior to the status of the microemulsion form of dissolved without precipitation to ensure that its oral bioavailability.
GBE50-SMEDDS rat pharmacokinetics study, use morin as internal standard, quercetin, kaempferol and isorhamnetin as the reference substance, establish the ginkgo flavone analysis method of plasma samples. Plasma concentration-time curves and pharmacokinetic parameters showed that, Cmax of GBE50-SMEDDS increased 0.3 mg/Lthan the reference group, Tmax earlier 2.7 h, the elimination of rate decreases, the elimination half-life extend 7.4 h, the distribution rate increase , the distribution of half-life ahead 2.2h, AUC increased 13.8 mg.h/L, relative bioavailability is the reference of 152.47%, increased 52.47%. GBE50-SMEDDS new formulations has higher bioavailability than market existing agents Xinglin, providing an experimental basis for clinical efficacy improvement.
The results of GBE50-SMEDDS acute toxicity in mice show that the maximum group have obvious toxicity, such as unresponsive, eyes dull, eyes have a discharge, or even death. Toxicity is caused by the agents in the oil phase materials, emulsifier and assist emulsifier.Half lethal dose LD50 = 0.33ml (0.36g)/10g, LD50 of 95% CI is 0.30～0.37ml (0.33～0.41g)/10g. GBE50-SMEDDS has acute toxic side effects of oral administration, LD50 is 1,800 times as the dose commonly used in normal adults (1.2g/60Kg). We can see, GBE50-SMEDDS although has toxic side effects, but it is safe while in clinical use according to instructions.
The animal efficacy results of GBE50-SMEDDS show that in the acute blood stasis model rats hemorrheology experiment, compared with normal control group, blood stasis model group of the whole blood viscosity ratio (low-cut, medium-cut and high-cut values) and plasma viscosity are higher (P=0.000,0.000,0.000,0.014), showing that the model is successful. Compared with blood stasis model group, the positive control group can reduce whole blood viscosity (low-cut, medium-cut and high-cut values), (P=0.007, 0.01, 0.004); GBE50-SMEDDS high dose group can reduce the viscosity of whole blood (low-cut, medium-cut and high-cut values) and plasma viscosity (P=0.000, 0.000, 0.001,0.001); GBE50-SMEDDS medium dose group can reduce whole blood viscosity ( low-cut, medium-cut and high-cut values) and plasma viscosity (P=0.000, 0.001, 0.002,0.016); difference between the results of low-dose group were not significant, but have lower trend, showing GBE50-SMEDDS high, medium , low-dose group, have dose-effect relationship. The group differences in hematocnt results were not significant (due to pilot error and less sample size), but both the role of trends and dose-effect relationship, showed that an improvement in GBE50-SMEDDS acute blood stasis model and the role of hemorheology in rats and dose-dependent manner.
GBE50-SMEDDS on isoproterenol-induced myocardial ischemia experiment, compared with normal control group, ischemic model group rats injected ISO myocardial ischemia soon after the change in electrocardiogram showed ST-segment elevation ( ST-30 and 35 min of P=0.001), indicating that it is a successful model. Compared with ischemic model group, the positive control group may be against ISO-induced myocardial ischemia in ECG changes, manifested as decreased ST segment (ST-30 and 35 min of P=0.038,0.007); GBE50-SMEDDS high-dose group could induced myocardial ischemia in ECG changes, manifested as decreased ST segment (ST-30 and 35 min of P=0.005,0.001); GBE50-SMEDDS medium dose group induce myocardial ischemia-induced changes in the electrocardiogram showed ST segment declined (ST-30 and 35 min of P=0.023,0.007); difference between the results of low-dose group were not significant, but has lower trend, showing GBE50-SMEDD high, medium and low-dose group, have dose-effect relationship. The results of T-wave in each group have no significant differences in statistical (due to pilot error and less sample size), but both have the role of trends and dose-effect relationship. GBE50-SMEDDS has an improvement in myocardial ischemia caused by changes in the role of ECG and has dose-dependent manner.
Compared with normal control group, ischemic model group of CK, LDH and AST release increased (P=0.000), indicating that model is successful. Compared with ischemic model group, the positive control group could reduce CK, LDH and AST release (P=0.000,0.000,0.002); GBE50-SMEDDS high dose group reduced CK, LDH and AST release (P=0.000); GBE50-SMEDDS medium dose group were lower in CK, LDH and AST release (P=0.000); GBE50-SMEDDS low-dose group could reduce the CK, LDH release (P=0.005, 0.001); GBE50-SMEDDS high, medium and low-dose group, have dose-effect relationship. GBE50-SMEDDS improve myocardial ischemic injury in rats, and dose-dependent manner.
Efficacy in animal experiments, GBE50-SMEDDS can improve the acute blood stasis model rats abnormality, and has a protective effect on isoproterenol-induced myocardial ischemia, and dose-dependent manner. Equivalent doses of GBE50-SMEDDS has a better effect than the control group existing market Xinglin granules , GBE50-SMEDDS efficiently improve the efficacy of GBE50, provide the basis of animal in order to improve the clinical efficacy of pharmacological experiments.
引文
[1]Leuner C,Dressman J.Improving drug solubility for oral delivery using solid dispersions.Eur J Pharm Biopharm,2000,50:47
[2]Lawrence MJ,Rees GD.Microemulsion-based media as novel drug delivery systems.Adv Drug Deliv Rev,2000,45:89
[3]Tarr B D,Yalkowsky S H.Enhanced intestinal absorption of cyclosporine in rats through the reduction droplet size[J].Pharm Res,1989,6(1):40-43
[4]Holmberg I Aksnes L,Berlin T et al.Biopharm Drug Disposition,1990,11:15.
[5]Swenson ES,Milisen WB,Curatolo W.Intestinal permeability enhancement efficacy,acute local toxicity and reversibility[J].Pharm Res,1994,11(8):1132
[6]Gershanik T,Benita S.Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs[J].Eur Pharma Biopharmac.2000,50(1):179-188
[7]李国栋,范伟,居红卫,等.自乳化释药系统的研究进展[J].国外医药·合成药、生化药、制剂分册,2001,22(5):304-305
[8]张瑛,张钧寿.盐酸维拉帕米自乳化缓释片的研制及释药行为的探讨[J].中国药科大学学报,2002,33(3):208-210
[9]Pouton C W.Formulation of self-emulsifying drug delivery systems [J]Advanced Drug Delivery Reviews,1997,25(4):47-58
[10]Craig D Q M,Lievens H S R,Pitt K G,et al.An investigation into the physico-chemical properties of self-emulsifying systems using low frequency dielectric spectroscopy,surface tension measurements and particle size analysis[J].Int J Pharm,1993,96(7):147-155
[11]Pouton C W.Effects of the inclusion of a model drug on the performance of self emulsifying formulation[J].J Pharm Pharmacol,1985,37(1):1-11
[12]Florence A T.The oral absorption of micro-and nano-particulates:neither exceptional nor unusual[J].Pharm Res,1997,14(3):259-266
[13]Constantinides P P.Lipid microemulsions for improving drug dissolution and oral absorption:physical and biopharmaceutical aspects[J].Pharm Res,1995, 12(11):1561-1572
[14] Craig D Q M, Barker S A, Banning D,et al. An investigation into mechanisms of self-emulsification using particle sizeanalysis and low frequency dielectric spectroscopy[J].Int J Pharm, 1995, 114(1): 103-110
[15] Khoo S M, Humberstone A J, Porter C J H,et al. Formulation design and bioavailability assessment of lipidic self-emulsifying formulations of halofantrine [J].Int J Pharm, 1998,167: 155-164
[16] Kommuru T R, Gurley B, Khan M A,et al. Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment [J].Int J Pharm, 2001, 212(1): 233-246
[17] Pouton C W. Formulation of Self -emulsifying drug delivery system[J]. Advanced Drug Delivery Reviews,1997,25(4):47-58
[18] Gershanik T, Benzeno S, Beniton S. Interaction of Self-emulsifying lipid drug delivery system with the everted rat intestinal macosa as a function of droplet size and surface charge[J]. Pharm Res,1998,15(6):863-869
[19] Gursoy R N, Benita S. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs [J].Biomed Pharmacother,2004, 58(3): 173-182
[20] Gershanik T, Benita S. Positively-charged self-emulsifying oil formulation for improving oral bioavailability of progesterone[J]. Pharm Dev Technol,1996, 1(2): 147
[21] Shah NH, Carvajal MT, Patel CI, et al. Self-emulsifying drug delivery systems (SEDDS) with polyglycolzed glycerides for improving in vitro dissolution and oral absorption of lipophilic durgs. Int J Pharm, 1994,106(1): 15
[22] Barry RZ, Eggenton J.J Physiol,1972;227(1):201
[23] Gershanik T, Haltner E, Lehr CM,et al. Charge-dependent interaction of self-emulsifying oil formulation(SEOF) with Caco-2cells monolayers: binding, effects on barrier function and cytotoxicity[J].Int J Pharm, 2000,211(1-2):29
[24] Gershanik T, Benita S.Pharm Dev Technol, 1996,1(2): 147
[25] Gershanik T. Benita S. Eur J Pharm Bio,2000;50(2):179
[26]朱盛山.药物新剂型[M].第1版.北京:化学工业出版社,2003:115
[27]Bok Ki Kang,Jin Soo Lee,Se Kang Chon.Development of self-micro-emulsifying drug delivery systems(SMEDDS) for oral biolavailability enhancement of simvastatin in beagle dogs[J].International Journal of Pharmaceutics,2004,274:65-73
[28]Lawrence MJ,Rees GD.Microemulsion-based media as novel drug delivery systems[J].Advanced Drug Delivery Reviews,2000,45(1):89-121
[29]Itoh K,Tozuka Y,Oguchi T,et al.Improvement of physicochemical properties of N-4472 part Ⅰ:formulation design by using self-microemulsifying system.Int J Pharm,2002,238:153-160
[30]陈宗淇,戴闽光.胶体化学[M].北京:高等教育出版社,1985:342-345
[31]Schechter RS.Microemulsions and Releated Systems[M].New York:Mareel Dek-ker,1998:1-200
[32]Craig DQM,Barker SA,Banning D,et al.An investigation into mechanisms of self-emulsification using partical size analysis and low frequency dielectric spectroscopy[J].Int J Pharm,1995,114(1):103-113
[33]徐贵霞,王玉丽,全东琴,等.自乳化释药系统的体外评价.解放军药学学报,9(3):195
[34]Kommuru TR,Gurley B,Khan MA,et al.Self-emulsifying drug delivery systems SEDDS) of coenzymeQ10 formulation,development and bioavailability assessment.Int J Pharmac,2001,212(1):233
[35]张正全,陆彬.微乳给药系统研究概况.中国医药工业杂志,2001;32(3):139
[36]王晓黎,蒋雪涛.微乳在药剂学上的应用.解放军药学学报,2000;16(2):88
[37]Panayiotis P.LiPid microemulsion for improving drug dissoluteion and oral absorption:physical and biopharmaceutical aspects.Pharm Res,1995;12(11):1561
[38]沈熊,吴伟.自乳化和自微乳化释药系统.复旦学报(医学版).2003mar,30(2):180
[39]Shui-Mei Khoo,Andrew J.H et al.Formulation design and bioavailability ssessment of lipidic self-emulsifying formulations of halofantrine.Int J Pharm,998;155
[40]崔升淼,赵春顺,何仲贵.葛根黄酮自微乳化给药系统的体内外评价.中药材,2007(30):684-687
[41]冯少华,肖小河,高文远,等.提高难溶性药物生物利用度方法研究概述.解放军药学学报,21,(5):372-374
[42]沙先宜.9-硝基喜树碱小肠吸收机理及其自微乳化给药系统的研究.复旦大学博士学位论文.
[43]中国药典[S].一部,2005:220,281
[44]日本特许公报.昭46-91(1971)
[45]松本武,松本明子,日本特许公报,平2-31649(1990)
[46]SchwabeWillamar DEPatent 2117429(1972)
[47]日本特许公报,平1-199989(1989)
[48]日本特许公报,平1-258626(1989)
[49]松本武,日本特许公报,昭62-292794(1987)
[50]Natsumoto Takeshi,SelTsuyosi,EP Patent237066(1987)
[51]Kwak,Wie Jong,EP Patent 0402925A_2(1990)
[52]谢培山.银杏叶标准提取物EGb761及银杏叶制剂的质量评价.中国中药杂志,1999,24(2):116-118
[53]王成章,郁青,谭卫红.银杏叶黄酮甙的纯化研究.林产化工通讯.1997,31(4):7-9
[54]钟郁青.银杏叶中双黄酮成分的提取分离及其HPLC法测定.药学学报,1995,30(9):694-697
[55]郑卫平,楼凤昌.银杏内酯的研究概况.药学进展,1999,23(2):82-87
[56]楼凤昌,凌娅,唐于平,等.银杏萜内酯的分离、纯化和结构鉴定.中国天然药物,2004,2(1):11-15
[57]臧丽,李英华.银杏酚酸的研究概况.中国医药工业杂志,2000,31(7):331-334
[58]肖崇厚.中药化学[M].上海:上海科学技术出版社,1997:274
[59]王成章,沈兆邦,陈祥.银杏叶聚戊烯醇化学研究[J].林产化学与工业,1992,12(4):279-285
[60]张永红,王敬勉,廖德胜,等.银杏叶挥发性化学成分的研究[J].天然产物研究与开发,2001,13(2):43-45
[61]黄桂宽,曾麒燕.银杏多糖的化学研究[J].中草药,1997,28(8):459-461
[62]李兆龙,胡季强,罗跃明.银杏叶的开发利用.1995:12-14
[63]郭炎荣.银杏叶提取物的制备及质量控制研究进展.海峡药学,2001,13(2):1-3
[64]李梅,杨正鸿,王坚毅.紫外分光光度法测定复方银杏胶囊中银杏叶总黄酮含量.中国医院药学杂志,1998,18(2):85
[65]张晓琦,陈颍怡.高效液相色谱法测定银杏叶提取物中槲皮素的含量.色谱,1994,12(4):302
[66]Hasler A,Sticher O,Meier B.Identification and determination of he flavonoids from ginkgo biloba by high-performance liquid chro-matography.J Chromatography,1992,605:41
[67]Pietta P G,Mauri P L,Manera E.Animproved HPLC determina-tion of Flavonoids in medicinal plant extracts.J Chromatography,1989,27(9):509
[68]李吉来,于留荣,曾宇珠.薄层扫描法测定银杏叶中总黄酮醇甙的含量.中国中药杂志,1996,21(2):106
[69]Pietta P G,Mauri P L,Rave A.Appllication of micellar electrokinetics capillary chromatography to the determination of flavonoid drags.J Chromatography,1991,549:367
[70]Marc P Maillard,Jean-Lucwolfender,Kurt Hostettmann.Use of iquid chromatography-thermospray mass spectrometry in phytochemical analysis of crude plant extracts,J Chromatography,1993,647:147
[71]姚渭溪.银杏叶中活性成分的提取工艺测定及其进展,中草药,1995,26(3):157
[72]Komoday,Nakamura H,Uchida M.High-performance liquid chromatographic analysis of ginkgolides by using a differential refractormeter,Iyo Kizai Keenkyusho Hokoku,1988,22:83
[73]Wagner H,Bladt S,Hartmann U,et al.Ginkgo biloba-Dcand HPLC analyse von ginkgo-extrakten und ginkgo-extrakte enthaltenden phytopraparaten.Deut Apoth Ztg,1989,129:2421
[74]Van Beek T A,Scheeren H A,Ranto T,et al.Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and phytophyarmaceuticals,J Chromatogr,1991,543:375
[75]Chauret N,Carrier J,Mancini M.Gas chromatography-mas spectrometric analysis of ginkgolides produced by ginkgo bilobacell culture,J Chromatography,1991,588:281
[76]颜玉贞,谢培山,钱浩良.银杏叶提取物及制剂萜类内酯薄层色谱荧光扫描定量测定研究.中国中药杂志,1997,22(3):159
[77]Van Beek T A,Van Veldhuizen H,Lelyveld G P,et al.Quantitation of bilobalide and ginkgolides A,B,C and J by means of nuclear magnetic resonance spectroscopy.Phytochemical Analysis,1993,4:261
[78]Steinke B,Bernd M,Wagner H.Biological standardization of gink-go extracts,Planta Medica,1993,59:155
[79]]Hasler A.色谱法与生物法测定收获季节银杏叶中总黄酮和萜类内酯含量,国外医药-植物药分册,1994,9(6):267
[80]杨光.银杏叶制剂质量与功效的重新评价.首都医药,2003,8(20):22-24
[81]杨光.再论科研开发银杏资源的重要意义.北京中医药,2008,27(6):463-466
[82]国家药品标准WS3-227(Z-028)-2002(Z):40-44
[83]魏云等.银杏酮酯对实验性心肌缺血和血液流变性的影响.中药新药与临床药理,2004,15(6):390-392
[84]闵暘等.银杏酮酯抗心肌梗塞药效学研究.中成药,1998,20(10):32-35
[85]王淑仙,崔艳英,沈连忠.银杏酮酯对动物急性脑缺血保护作用.中成药,1998,20(10):28-32
[86]冯必钧,魏云.银杏酮酯对大鼠局灶性脑缺血及家犬脑血管阻力脑血流量的影响.中华新医药杂志,2004,1(1):7-10
[87]李菲,芮耀诚,陈维洲.银杏叶提取物对溶血磷脂酰胆碱引起的脑微血管损伤的保护作用,中国新药与临床杂志,1998,17(3):149-151
[88]孙莉莎,徐江平,杨雪梅.银杏酮酯对大鼠缺血再灌注损伤的保护作用.中华现代中西医药杂志,2003,1(8):32-34
[89]郑明祺,龚旭龄.银杏黄酮和内酯的药理与临床.中国药房,1997,8(2):85
[90]王庆蓉,朱万仁.醋酸泼尼松水中溶解度及正辛醇/水分配系数的测定[J].玉林师范学院学报(自然科学版),2007,(03):39-41
[91]张婷婷,徐文,胡生亮,等.水飞蓟宾在不同介质中平衡溶解度和表观油水分配系数的测定[J].中国药学杂志,2006,(10):1569-1571
[92]陶涛,赵雁,陈庆华.石杉碱甲的解离常数、表观溶解度和表观油/水分配系数的测定.中国医药工业杂志,2005,36(8):487-489
[93]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.沈阳药科大学学报,2007,(4):220-222
[94]王雷,田青平,谢茵.两性霉素B平衡溶解度和表观油水分配系数的测定.山西医科大学学报,2007,38(12):1082-1084
[95]潘国梁,贾晓斌,魏惠华,等.药用微乳伪三元相图的几种制备方法比较研究.中国药房.2006,17(1):21-23
[96]吕娟丽,刘振华,SE坚成,等.9-硝基喜树碱口服自微乳化系统的制备与体外评价.中国新药杂志,2008,17(10):861-865
[97]崔升淼,赵春顺,何仲贵.葛根黄酮自微乳化软胶囊的制备和溶出度的考察.中成药,2007,29(7):993-996
[98]周晓堂,王晶,王颖,等.吡罗昔康自微乳化药物传递系统的处方筛选与体外评价.药学学报,2008,43(4):415-420
[99]纪海英,张钧寿.尼群地平胶囊自乳化释药系统处方优化研究.中国药科大学学报,2004,35(3):211-214
[100]国家药典委员会编.中华人民共和国药典(二部,2005年版).北京:化学工业出版社,2005:附录Ⅹ C,ⅩⅨ C
[101]张莉,夏运岳.用电子表格Excel计算药物溶出度Weibull分布参数.药学进展,2002,26(1):48-50
[102]杨怡静,平其能,宋梅.固体分散体提高银杏叶片溶出度的研究.中国新药杂志,2004,13(6):521-524
[103]Trotta M.Affluence of phase transformation on indomethacin release from microemulsions[J].J Control Release,1999,60:399-405
[104]Washington C.Drug release from microdisperse systems:a critical review[J].Int J Pharm,1990,58:1-12
[105]Levy MY,Benita S.Drug release from submicronized o/w emulsion:a new in vitro kinetic evaluation model[J].Int J.Pharm,1990,66:29-37
[106]杨光.银杏叶制剂质量与功效的重新评价.首都医药,2003,8(20):22-24
[107]徐任生,等编著.天然产物化学(第二版).科学出版社,2004,9
[108]李小燕,潘洪平.高效液相色谱法测定复方银杏叶片中银杏总黄酮的含量.时珍国医国药,2007,18(7):1599-1600
[109]陈香爱,袁志芳,张兰桐.HPLC法测定银杏叶粉针中总黄醇醇苷和萜类内酯.中草药,2007,38(9):1333-1335
[110]孙丽丽,周沫.HPLC测定银杏叶片中总黄酮醇苷和萜类内酯的含量.华西药学杂志,2007,22(2):201-203
[111]毕殿洲等.药剂学(第四版).人民卫生出版社,227
[112]]梁文权.生物药剂学与药物动力学(第2版).人民卫生出版社,155
[113]谢燕,张钧寿.银杏叶提取物制剂研究概况,中草药,2005,36(8):1267-1269
[114]Zhang YM.Bioavailability of isoflavone in soybeans[J].Foreign Med Sci,2001,28(2):105
[115]Wang QW,Mei QB,Zhou SY,et al.Study on the bioavailability of genistein capsule in SD rats[J].Pharm J Chin PLA,2003,19(3):189-191
[116]Jeff S,Lori C,Marion K,et al.Intestinal up take and biliary excretion of the isoflavone genistein in rats[J].J Nutr,1997,127(7):1260-1268
[117]方子季,许自明,朱秀美,等.高效液相色谱内标法测定银杏叶制品中的总黄酮 含量,解放军药学学报,2002,18(1):51-53
[118]汪凤梅,吴美珍,周莉,等.血浆中银杏黄酮的高效液相色谱测定方法学研究,中国中药杂志,2003,28(3):279-281
[119]汪凤梅,周莉,姚彤炜,等.高效液相色谱法测定口服银杏制剂后兔血中槲皮素和山萘酚浓度.中国医院药学杂志,2005,25(1):10-12
[120]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2006,7
[121]陈奇.中药药效研究思路与方法[M].北京:人民卫生出版社,2005:3,14,42-44,67
[122]白荣,吴晓敏,向国全,等.癫宁片的急性毒性试验研究,中国药业,2008,17(15):9-10
[123]张喜平,程琪辉,裘怡.大黄素胶囊小鼠急性毒性试验.医学研究杂志,2007,36(6):47-50
[124]潘洪平.银杏叶制剂药理作用和临床应用研究进展[J].中国中药杂志,2005,30(2):93
[125]杨卫平,詹亚梅,邱德文,等.人参汤对实验性气虚血瘀证心肌缺血大鼠的血液流变学的影响[J].四川中医,2005,23(8):26
[126]潘志,段富津,魏雄辉.冠心康胶囊对实验性急性心肌缺血大鼠心电检测与血液流变学的影响.辽宁中医杂志,2007,34(12):1804-1805
[127]胡京,杨旻,王燕.冠心康宁合剂抗心肌缺血的实验研究.数理医药学杂志.2006,19(4):415-416
[128]孙伟夫,李晓峰,李彩燕,等.心复康抗心肌缺血的实验研究[J].中国中医药科技,2001,8(5):298.
[129]韦健全,郑子敏,潘勇,等.芒果苷对异丙肾上腺素诱发小鼠心肌缺血的保护作用.中药药理与临床,2008,24(2):16-18
[2]Lawrence MJ,Rees GD.Microemulsion-based media as novel drug delivery systems.Adv Drug Deliv Rev,2000,45:89
[3]Tarr B D,Yalkowsky S H.Enhanced intestinal absorption of cyclosporine in rats through the reduction droplet size[J].Pharm Res,1989,6(1):40-43
[4]Holmberg I Aksnes L,Berlin T et al.Biopharm Drug Disposition,1990,11:15.
[5]Swenson ES,Milisen WB,Curatolo W.Intestinal permeability enhancement efficacy,acute local toxicity and reversibility[J].Pharm Res,1994,11(8):1132
[6]Gershanik T,Benita S.Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs[J].Eur Pharma Biopharmac.2000,50(1):179-188
[7]李国栋,范伟,居红卫,等.自乳化释药系统的研究进展[J].国外医药·合成药、生化药、制剂分册,2001,22(5):304-305
[8]张瑛,张钧寿.盐酸维拉帕米自乳化缓释片的研制及释药行为的探讨[J].中国药科大学学报,2002,33(3):208-210
[9]Pouton C W.Formulation of self-emulsifying drug delivery systems [J]Advanced Drug Delivery Reviews,1997,25(4):47-58
[10]Craig D Q M,Lievens H S R,Pitt K G,et al.An investigation into the physico-chemical properties of self-emulsifying systems using low frequency dielectric spectroscopy,surface tension measurements and particle size analysis[J].Int J Pharm,1993,96(7):147-155
[11]Pouton C W.Effects of the inclusion of a model drug on the performance of self emulsifying formulation[J].J Pharm Pharmacol,1985,37(1):1-11
[12]Florence A T.The oral absorption of micro-and nano-particulates:neither exceptional nor unusual[J].Pharm Res,1997,14(3):259-266
[13]Constantinides P P.Lipid microemulsions for improving drug dissolution and oral absorption:physical and biopharmaceutical aspects[J].Pharm Res,1995, 12(11):1561-1572
[14] Craig D Q M, Barker S A, Banning D,et al. An investigation into mechanisms of self-emulsification using particle sizeanalysis and low frequency dielectric spectroscopy[J].Int J Pharm, 1995, 114(1): 103-110
[15] Khoo S M, Humberstone A J, Porter C J H,et al. Formulation design and bioavailability assessment of lipidic self-emulsifying formulations of halofantrine [J].Int J Pharm, 1998,167: 155-164
[16] Kommuru T R, Gurley B, Khan M A,et al. Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment [J].Int J Pharm, 2001, 212(1): 233-246
[17] Pouton C W. Formulation of Self -emulsifying drug delivery system[J]. Advanced Drug Delivery Reviews,1997,25(4):47-58
[18] Gershanik T, Benzeno S, Beniton S. Interaction of Self-emulsifying lipid drug delivery system with the everted rat intestinal macosa as a function of droplet size and surface charge[J]. Pharm Res,1998,15(6):863-869
[19] Gursoy R N, Benita S. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs [J].Biomed Pharmacother,2004, 58(3): 173-182
[20] Gershanik T, Benita S. Positively-charged self-emulsifying oil formulation for improving oral bioavailability of progesterone[J]. Pharm Dev Technol,1996, 1(2): 147
[21] Shah NH, Carvajal MT, Patel CI, et al. Self-emulsifying drug delivery systems (SEDDS) with polyglycolzed glycerides for improving in vitro dissolution and oral absorption of lipophilic durgs. Int J Pharm, 1994,106(1): 15
[22] Barry RZ, Eggenton J.J Physiol,1972;227(1):201
[23] Gershanik T, Haltner E, Lehr CM,et al. Charge-dependent interaction of self-emulsifying oil formulation(SEOF) with Caco-2cells monolayers: binding, effects on barrier function and cytotoxicity[J].Int J Pharm, 2000,211(1-2):29
[24] Gershanik T, Benita S.Pharm Dev Technol, 1996,1(2): 147
[25] Gershanik T. Benita S. Eur J Pharm Bio,2000;50(2):179
[26]朱盛山.药物新剂型[M].第1版.北京:化学工业出版社,2003:115
[27]Bok Ki Kang,Jin Soo Lee,Se Kang Chon.Development of self-micro-emulsifying drug delivery systems(SMEDDS) for oral biolavailability enhancement of simvastatin in beagle dogs[J].International Journal of Pharmaceutics,2004,274:65-73
[28]Lawrence MJ,Rees GD.Microemulsion-based media as novel drug delivery systems[J].Advanced Drug Delivery Reviews,2000,45(1):89-121
[29]Itoh K,Tozuka Y,Oguchi T,et al.Improvement of physicochemical properties of N-4472 part Ⅰ:formulation design by using self-microemulsifying system.Int J Pharm,2002,238:153-160
[30]陈宗淇,戴闽光.胶体化学[M].北京:高等教育出版社,1985:342-345
[31]Schechter RS.Microemulsions and Releated Systems[M].New York:Mareel Dek-ker,1998:1-200
[32]Craig DQM,Barker SA,Banning D,et al.An investigation into mechanisms of self-emulsification using partical size analysis and low frequency dielectric spectroscopy[J].Int J Pharm,1995,114(1):103-113
[33]徐贵霞,王玉丽,全东琴,等.自乳化释药系统的体外评价.解放军药学学报,9(3):195
[34]Kommuru TR,Gurley B,Khan MA,et al.Self-emulsifying drug delivery systems SEDDS) of coenzymeQ10 formulation,development and bioavailability assessment.Int J Pharmac,2001,212(1):233
[35]张正全,陆彬.微乳给药系统研究概况.中国医药工业杂志,2001;32(3):139
[36]王晓黎,蒋雪涛.微乳在药剂学上的应用.解放军药学学报,2000;16(2):88
[37]Panayiotis P.LiPid microemulsion for improving drug dissoluteion and oral absorption:physical and biopharmaceutical aspects.Pharm Res,1995;12(11):1561
[38]沈熊,吴伟.自乳化和自微乳化释药系统.复旦学报(医学版).2003mar,30(2):180
[39]Shui-Mei Khoo,Andrew J.H et al.Formulation design and bioavailability ssessment of lipidic self-emulsifying formulations of halofantrine.Int J Pharm,998;155
[40]崔升淼,赵春顺,何仲贵.葛根黄酮自微乳化给药系统的体内外评价.中药材,2007(30):684-687
[41]冯少华,肖小河,高文远,等.提高难溶性药物生物利用度方法研究概述.解放军药学学报,21,(5):372-374
[42]沙先宜.9-硝基喜树碱小肠吸收机理及其自微乳化给药系统的研究.复旦大学博士学位论文.
[43]中国药典[S].一部,2005:220,281
[44]日本特许公报.昭46-91(1971)
[45]松本武,松本明子,日本特许公报,平2-31649(1990)
[46]SchwabeWillamar DEPatent 2117429(1972)
[47]日本特许公报,平1-199989(1989)
[48]日本特许公报,平1-258626(1989)
[49]松本武,日本特许公报,昭62-292794(1987)
[50]Natsumoto Takeshi,SelTsuyosi,EP Patent237066(1987)
[51]Kwak,Wie Jong,EP Patent 0402925A_2(1990)
[52]谢培山.银杏叶标准提取物EGb761及银杏叶制剂的质量评价.中国中药杂志,1999,24(2):116-118
[53]王成章,郁青,谭卫红.银杏叶黄酮甙的纯化研究.林产化工通讯.1997,31(4):7-9
[54]钟郁青.银杏叶中双黄酮成分的提取分离及其HPLC法测定.药学学报,1995,30(9):694-697
[55]郑卫平,楼凤昌.银杏内酯的研究概况.药学进展,1999,23(2):82-87
[56]楼凤昌,凌娅,唐于平,等.银杏萜内酯的分离、纯化和结构鉴定.中国天然药物,2004,2(1):11-15
[57]臧丽,李英华.银杏酚酸的研究概况.中国医药工业杂志,2000,31(7):331-334
[58]肖崇厚.中药化学[M].上海:上海科学技术出版社,1997:274
[59]王成章,沈兆邦,陈祥.银杏叶聚戊烯醇化学研究[J].林产化学与工业,1992,12(4):279-285
[60]张永红,王敬勉,廖德胜,等.银杏叶挥发性化学成分的研究[J].天然产物研究与开发,2001,13(2):43-45
[61]黄桂宽,曾麒燕.银杏多糖的化学研究[J].中草药,1997,28(8):459-461
[62]李兆龙,胡季强,罗跃明.银杏叶的开发利用.1995:12-14
[63]郭炎荣.银杏叶提取物的制备及质量控制研究进展.海峡药学,2001,13(2):1-3
[64]李梅,杨正鸿,王坚毅.紫外分光光度法测定复方银杏胶囊中银杏叶总黄酮含量.中国医院药学杂志,1998,18(2):85
[65]张晓琦,陈颍怡.高效液相色谱法测定银杏叶提取物中槲皮素的含量.色谱,1994,12(4):302
[66]Hasler A,Sticher O,Meier B.Identification and determination of he flavonoids from ginkgo biloba by high-performance liquid chro-matography.J Chromatography,1992,605:41
[67]Pietta P G,Mauri P L,Manera E.Animproved HPLC determina-tion of Flavonoids in medicinal plant extracts.J Chromatography,1989,27(9):509
[68]李吉来,于留荣,曾宇珠.薄层扫描法测定银杏叶中总黄酮醇甙的含量.中国中药杂志,1996,21(2):106
[69]Pietta P G,Mauri P L,Rave A.Appllication of micellar electrokinetics capillary chromatography to the determination of flavonoid drags.J Chromatography,1991,549:367
[70]Marc P Maillard,Jean-Lucwolfender,Kurt Hostettmann.Use of iquid chromatography-thermospray mass spectrometry in phytochemical analysis of crude plant extracts,J Chromatography,1993,647:147
[71]姚渭溪.银杏叶中活性成分的提取工艺测定及其进展,中草药,1995,26(3):157
[72]Komoday,Nakamura H,Uchida M.High-performance liquid chromatographic analysis of ginkgolides by using a differential refractormeter,Iyo Kizai Keenkyusho Hokoku,1988,22:83
[73]Wagner H,Bladt S,Hartmann U,et al.Ginkgo biloba-Dcand HPLC analyse von ginkgo-extrakten und ginkgo-extrakte enthaltenden phytopraparaten.Deut Apoth Ztg,1989,129:2421
[74]Van Beek T A,Scheeren H A,Ranto T,et al.Determination of ginkgolides and bilobalide in Ginkgo biloba leaves and phytophyarmaceuticals,J Chromatogr,1991,543:375
[75]Chauret N,Carrier J,Mancini M.Gas chromatography-mas spectrometric analysis of ginkgolides produced by ginkgo bilobacell culture,J Chromatography,1991,588:281
[76]颜玉贞,谢培山,钱浩良.银杏叶提取物及制剂萜类内酯薄层色谱荧光扫描定量测定研究.中国中药杂志,1997,22(3):159
[77]Van Beek T A,Van Veldhuizen H,Lelyveld G P,et al.Quantitation of bilobalide and ginkgolides A,B,C and J by means of nuclear magnetic resonance spectroscopy.Phytochemical Analysis,1993,4:261
[78]Steinke B,Bernd M,Wagner H.Biological standardization of gink-go extracts,Planta Medica,1993,59:155
[79]]Hasler A.色谱法与生物法测定收获季节银杏叶中总黄酮和萜类内酯含量,国外医药-植物药分册,1994,9(6):267
[80]杨光.银杏叶制剂质量与功效的重新评价.首都医药,2003,8(20):22-24
[81]杨光.再论科研开发银杏资源的重要意义.北京中医药,2008,27(6):463-466
[82]国家药品标准WS3-227(Z-028)-2002(Z):40-44
[83]魏云等.银杏酮酯对实验性心肌缺血和血液流变性的影响.中药新药与临床药理,2004,15(6):390-392
[84]闵暘等.银杏酮酯抗心肌梗塞药效学研究.中成药,1998,20(10):32-35
[85]王淑仙,崔艳英,沈连忠.银杏酮酯对动物急性脑缺血保护作用.中成药,1998,20(10):28-32
[86]冯必钧,魏云.银杏酮酯对大鼠局灶性脑缺血及家犬脑血管阻力脑血流量的影响.中华新医药杂志,2004,1(1):7-10
[87]李菲,芮耀诚,陈维洲.银杏叶提取物对溶血磷脂酰胆碱引起的脑微血管损伤的保护作用,中国新药与临床杂志,1998,17(3):149-151
[88]孙莉莎,徐江平,杨雪梅.银杏酮酯对大鼠缺血再灌注损伤的保护作用.中华现代中西医药杂志,2003,1(8):32-34
[89]郑明祺,龚旭龄.银杏黄酮和内酯的药理与临床.中国药房,1997,8(2):85
[90]王庆蓉,朱万仁.醋酸泼尼松水中溶解度及正辛醇/水分配系数的测定[J].玉林师范学院学报(自然科学版),2007,(03):39-41
[91]张婷婷,徐文,胡生亮,等.水飞蓟宾在不同介质中平衡溶解度和表观油水分配系数的测定[J].中国药学杂志,2006,(10):1569-1571
[92]陶涛,赵雁,陈庆华.石杉碱甲的解离常数、表观溶解度和表观油/水分配系数的测定.中国医药工业杂志,2005,36(8):487-489
[93]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.沈阳药科大学学报,2007,(4):220-222
[94]王雷,田青平,谢茵.两性霉素B平衡溶解度和表观油水分配系数的测定.山西医科大学学报,2007,38(12):1082-1084
[95]潘国梁,贾晓斌,魏惠华,等.药用微乳伪三元相图的几种制备方法比较研究.中国药房.2006,17(1):21-23
[96]吕娟丽,刘振华,SE坚成,等.9-硝基喜树碱口服自微乳化系统的制备与体外评价.中国新药杂志,2008,17(10):861-865
[97]崔升淼,赵春顺,何仲贵.葛根黄酮自微乳化软胶囊的制备和溶出度的考察.中成药,2007,29(7):993-996
[98]周晓堂,王晶,王颖,等.吡罗昔康自微乳化药物传递系统的处方筛选与体外评价.药学学报,2008,43(4):415-420
[99]纪海英,张钧寿.尼群地平胶囊自乳化释药系统处方优化研究.中国药科大学学报,2004,35(3):211-214
[100]国家药典委员会编.中华人民共和国药典(二部,2005年版).北京:化学工业出版社,2005:附录Ⅹ C,ⅩⅨ C
[101]张莉,夏运岳.用电子表格Excel计算药物溶出度Weibull分布参数.药学进展,2002,26(1):48-50
[102]杨怡静,平其能,宋梅.固体分散体提高银杏叶片溶出度的研究.中国新药杂志,2004,13(6):521-524
[103]Trotta M.Affluence of phase transformation on indomethacin release from microemulsions[J].J Control Release,1999,60:399-405
[104]Washington C.Drug release from microdisperse systems:a critical review[J].Int J Pharm,1990,58:1-12
[105]Levy MY,Benita S.Drug release from submicronized o/w emulsion:a new in vitro kinetic evaluation model[J].Int J.Pharm,1990,66:29-37
[106]杨光.银杏叶制剂质量与功效的重新评价.首都医药,2003,8(20):22-24
[107]徐任生,等编著.天然产物化学(第二版).科学出版社,2004,9
[108]李小燕,潘洪平.高效液相色谱法测定复方银杏叶片中银杏总黄酮的含量.时珍国医国药,2007,18(7):1599-1600
[109]陈香爱,袁志芳,张兰桐.HPLC法测定银杏叶粉针中总黄醇醇苷和萜类内酯.中草药,2007,38(9):1333-1335
[110]孙丽丽,周沫.HPLC测定银杏叶片中总黄酮醇苷和萜类内酯的含量.华西药学杂志,2007,22(2):201-203
[111]毕殿洲等.药剂学(第四版).人民卫生出版社,227
[112]]梁文权.生物药剂学与药物动力学(第2版).人民卫生出版社,155
[113]谢燕,张钧寿.银杏叶提取物制剂研究概况,中草药,2005,36(8):1267-1269
[114]Zhang YM.Bioavailability of isoflavone in soybeans[J].Foreign Med Sci,2001,28(2):105
[115]Wang QW,Mei QB,Zhou SY,et al.Study on the bioavailability of genistein capsule in SD rats[J].Pharm J Chin PLA,2003,19(3):189-191
[116]Jeff S,Lori C,Marion K,et al.Intestinal up take and biliary excretion of the isoflavone genistein in rats[J].J Nutr,1997,127(7):1260-1268
[117]方子季,许自明,朱秀美,等.高效液相色谱内标法测定银杏叶制品中的总黄酮 含量,解放军药学学报,2002,18(1):51-53
[118]汪凤梅,吴美珍,周莉,等.血浆中银杏黄酮的高效液相色谱测定方法学研究,中国中药杂志,2003,28(3):279-281
[119]汪凤梅,周莉,姚彤炜,等.高效液相色谱法测定口服银杏制剂后兔血中槲皮素和山萘酚浓度.中国医院药学杂志,2005,25(1):10-12
[120]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2006,7
[121]陈奇.中药药效研究思路与方法[M].北京:人民卫生出版社,2005:3,14,42-44,67
[122]白荣,吴晓敏,向国全,等.癫宁片的急性毒性试验研究,中国药业,2008,17(15):9-10
[123]张喜平,程琪辉,裘怡.大黄素胶囊小鼠急性毒性试验.医学研究杂志,2007,36(6):47-50
[124]潘洪平.银杏叶制剂药理作用和临床应用研究进展[J].中国中药杂志,2005,30(2):93
[125]杨卫平,詹亚梅,邱德文,等.人参汤对实验性气虚血瘀证心肌缺血大鼠的血液流变学的影响[J].四川中医,2005,23(8):26
[126]潘志,段富津,魏雄辉.冠心康胶囊对实验性急性心肌缺血大鼠心电检测与血液流变学的影响.辽宁中医杂志,2007,34(12):1804-1805
[127]胡京,杨旻,王燕.冠心康宁合剂抗心肌缺血的实验研究.数理医药学杂志.2006,19(4):415-416
[128]孙伟夫,李晓峰,李彩燕,等.心复康抗心肌缺血的实验研究[J].中国中医药科技,2001,8(5):298.
[129]韦健全,郑子敏,潘勇,等.芒果苷对异丙肾上腺素诱发小鼠心肌缺血的保护作用.中药药理与临床,2008,24(2):16-18