灯盏花素口服吸收机制及其自微乳剂的研制
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摘要
灯盏花素是从菊科飞蓬属(ErigeronL.)植物短葶飞蓬Erigeron breviscapus (Vant.) Hand.-Mazz.中提取的黄酮类有效成分,其主要成分为灯盏乙素。具有确切的心血管药理活性,但其口服制剂生物利用度低,疗效差。为探明灯盏花素生物利用度低的原因,本文采用caco-2细胞及大鼠在体肠灌流模型对其口服吸收过程的各环节进行考察,考察因素包括时间、温度、介质pH、药物浓度、p-糖蛋白抑制剂、制剂辅料及不同肠段对药物吸收的影响,并在此基础上制备灯盏花素口服自微乳以提高其生物利用度。
以灯盏花素中主要成分灯盏乙素为指标,对灯盏花素在人工胃液、不同pH(pH6.0,6.8 7.4)K-R液、不同肠段内容物及不同肠段肠黏膜组织匀浆液中的稳定性进行了考察,结果显示药物在人工胃液、不同肠段内容物及黏膜组织匀浆液中的稳定性较差,对影响灯盏花素稳定性的影响因素考察结果表明,灯盏花素对胃肠道内酶及介质pH敏感,其在K-R液中的稳定性随介质pH升高而降低。0.1%EDTA-2Na和0.1% Vc均可增强药物在K-R液中的稳定性,而以0.1% Vc对药物稳定性的增强作用最为显著。
通过MTT实验考察了药物及一些辅料对caco-2细胞的毒性作用,结果表明这些辅料对Caco-2细胞的毒性大小顺序为PEG400<普朗尼克F68<多烯酸乙酯<灯盏花素<中链脂肪酸甘油酯<油酸乙酯<吐温80<聚氧乙烯蓖麻油<<油酸。
通过caco-2细胞模型,研究了灯盏花素的口服吸收机制,并考察了吐温80、普朗尼克F68及聚氧乙烯蓖麻油对其跨细胞膜转运的影响。
实验发现,灯盏花素在Caco-2细胞单层上的摄取受温度及药物浓度的影响,Caco-2细胞对灯盏花素的摄取量随着温度的增加而增加,在考察的浓度范围内,灯盏花素细胞摄取量随药物浓度的增加而呈线性增加。pH的变化及p-糖蛋白抑制剂维拉帕米的加入对灯盏花素的细胞摄取无显著影响,F68、Tween80及Cremophor EL的加入可增加灯盏花素的细胞摄取量。Caco-2细胞转运实验表明,药物AP-BL及BL-AP的Papp受温度、F68、Tween80及Cremophor EL的影响,而受pH、浓度、维拉帕米的影响不大。药物在AP→BL及BL→AP的Papp均随温度的升高及三种表面活性剂浓度的升高而增加。灯盏花素细胞摄取与转运实验结果表明灯盏花素吸收方式以被动扩散为主。
在体肠灌流是研究小肠吸收机制的一种重要的方法,我们通过大鼠在体肠灌流研究了灯盏花素的吸收特性。灯盏花素在十二指肠、空肠、回肠和结肠中的吸收系数分别为0.0702h-1,0.0871h-1,0.0663h-1和0.0273h-1,t检验结果表明,灯盏花素在十二指肠、空肠、回肠中的吸收系数无显著性差异,在结肠中的吸收系数与其它肠段比较有显著性差异,各肠段吸收系数大小顺序为空肠>十二指肠>回肠>结肠。
在进一步的研究中,我们分别以油酸乙酯、中链脂肪酸甘油酯和多烯酸乙酯为油相来制备灯盏花素微乳,以磷脂为增溶剂增加灯盏花素在三种油相中的溶解度,并绘制了三种油相的灯盏花素微乳伪三元相图。根据伪三元相图,我们设计具有不同油相以及不同油相与表面活性剂比例的了九个乳剂处方,以Caco-2细胞为模型,对这九个处方促进灯盏花素的吸收效果进行了考察。结果表明九个乳剂处方均在不同程度上增加了灯盏花素的吸收,其促进药物的吸收作用与乳剂中油相的种类及乳滴大小有关。三种油相对乳剂促进灯盏花素吸收的大小顺序为油酸乙酯>多烯酸乙酯>中链脂肪酸甘油酯,乳滴粒径大小与乳剂的促进吸收作用呈负相关,相关系数>0.7。
在以上实验的基础上,制备了灯盏花素自微乳剂,处方由35.1%Tween80.17.5%PEG400、38、6%油酸乙酯、3.9%磷脂、4.7%灯盏花素及0.2%维生素E组成。以上处方制备的灯盏花素自微乳化药液0.5g分散在100ml水中可形成均一透明的微乳,乳滴粒径为43nm。形成的微乳采用葡聚糖凝胶G-50色谱法分离游离药物,高效液相色谱法测定包裹率,结果为(93.2±1.0)%。凝胶柱色谱法回收率为98.07%,凝胶柱色谱法加样回收率为97.9%。高效液相色谱法回收率为99.94%,药物在12～360μg/mL浓度范围内呈良好线性关系。采用透析法测定了灯盏花素微乳中药物在不同介质中的释放度,结果微乳在水中释放较慢且不完全,药物在pH6.8PBS中的释放缓慢、平稳,在pH6.8PBS中的释放速率符合higuchi方程Q=14.19t1/2+0.963(R=0.9934)。
建立了大鼠血浆药物浓度测定方法。分别以灯盏花素混悬液和灯盏花素注射液做为参考,计算灯盏花素自微乳剂的相对生物利用度与绝对生物利用度。绘制了三种制剂不同途径给药后的血药浓度—时间曲线,并用DAS软件计算出了三种制剂的药动学参数,结果表明灯盏花素自微乳的绝对生物利用度为51.8%,灯盏花素混悬液的绝对生物利用度为6.72%,灯盏花素自微乳剂相对于灯盏花素混悬液的生物利用度为770.1%。由此可见,与混悬液相比,灯盏花素自微乳剂的生物利用度提高了6.7倍。
Breviscapine, which is mainly composed of breviscapus (af lavone glucuronide), is a well-known bioactive constituentextracted from the traditional Chinese medicine Erigeron breviscapus (Vant.) Hand.-Mazz. Breviscapine is extensively used to treat ischemic cerebrovascular and cardiovascular diseases in China. However, the oral bioavailability of breviscapine was found to be quite poor in rats. To increase its oral bioavailability, Caco-2eells and in situ perfusion method in rats were applied to study the meehanism of absorption after oral administration of brevescapine, such as time, temperature, pH, concentration, p-glycoprotein inhibitor,excipients and different intestine segments were investigated, and self-microemulsion was prepared to enhance bioavailability of breviscapine.
Stability of breviscapus in artificial gastric juice, K-R fluid at different pH value (pH 6,0.6.8,7.4), contents of different parts of intestine, and homogenates of different parts of intestine mucosa were studied. The results showed that scruetallarin degraded significantly in artificial gastric juice, the contents of intestine and homogenates of intestine mucosa. Analysis on influence factors for stability of breviscapus suggested that breviscapus is sensitive to gastrointestinal Enzymes and pH, stability of breviscapus decreases with pH increasing in K-R fluid.0.1% EDTA-2Na is helpful for improving the stability of scretellarin in K-R fluid but 0.1% Vc can improve its stability significantly.
The cytotoxicity of several excipients on Caco-2 cells was evaluated by the MTT test. It showed that the toxic order of theses excipients to Caco-2 cells was F68< Ethyl polyenoate The absorption mechanism of breviscapus and the effects of Tween 80, F68 and Cremophor ELel-40 on breviscapus across the intestinal membrane was studied by the human colon adenocarcinoma(Caco-2) cell model.
Uptake of breviscapus by Caco-2 cell monolayers was affected by temperature and concentration of breviscapus, but not by the change in the apical pH, and the P-glycoprotein inhibitor (verapamil) showed no pronounced effects. Uptake of breviscapus by Caco-2 cells was increased with temperature elevating, and the uptake was non-saturable and increased linearly with rising of concentration of breviscapus over the range of concentration tested. The permeability coefficient of apical to basolateral and basolateral to apical were affected by temperature, F68, Tween80 and Cremophor EL efficiently, the permeability coefficient of apical to basolateral and basolateral to apical were increased with the increasing of the temperature and the concentration of these there surfactants. However, the pH, concentration, and verapamil had no marked influence on the permeability coefficient of both apical to basolateral and basolateral to apical. These results indicated that the uptake and transport of breviscapus were passive diffusion as the dominating process.
In situ perfusion in rats is one of the most important methods in the research of the drug's absorption. In this study, the absorption activities of breviscapus in the intestine were investigated. The absorption rate constants of breviscapus were 0.0702h-1,0.0871h-1,0.0663h-1 and 0.0273h-1,respectively in duodenum, jejunum, ileum and colon. Student's t test indicated that there was no significant difference amone the Ka of breviscapus in duodenum, jejunum and ileum, and that the Ka of breviscapus in colon was significantly lower than that in other segment. The Ka values of the intestinal segment was jejunum> duodenum>jejunum>ileum>> colon.
In further study, we separately use ethyl oleate, Ethyl polyenoate and medium chain length fatty acid triglyceride as oil phase to prepare breviscapus microemusion. Phospholipid was selected to increase the solubility of breviscapus in these three oil phase. Pseudo-ternary phase diagrams were constructed identifying the efficient micro emulsification region. Nine breviscapus O/W emulsion formulations with different oil pahse, different proportion of oil phase and surfactant were prepared and their effects on the absorption of breviscapus were studied with Caco-2 cells model. The results showed that all of these nine emulsions have absorption enhancement effect and the effect was related to the oil phase and particle size of the emulsions. The absorption enhancement effect order of these three oil phase is medium chain length fatty acid triglyceride Finally, self-microemulsion composed of 35.1% Tween80,17.5%PEG400,38.6% ethyl oleate,3.9% phospolipid,4.7% breviscapus and 0.2%VE was prepared. When 0.5g self-microemusion fluid was dispersed in 100ml water, it showed a transparent system and particle size is 43nm. The microemulsion and free breviscapus were separated by sephadex G-50 chromagography. Entrapment efficiency was detected by HPLC. The drug recovery of the chromatography method was 98.07%, the sample recovery of the chromatography method was 97.9%. The recovery of the determination method was 99.94% and the calibration curve was linear in the range of 12～360μg/mL. The entrapment efficiency of breviscapus microemulsion was (93.2±1.0)%. Cumulative release percent for breviscapus microemulsion in different mediums was investigated by dialysis method, release of the drug from microemulsion in water was extremely slow and uncomplet in 36h. The microemulsion provided a slow and steady drug release in pH6.8 PBS. Release of the drug in pH6.8PBS from microemulsion was in accordance with Higuchi equation: Q=14.19t1/2+0.963 (R=0.9934).
The quantitative analysis method of plasma containing breviscapus has been built. Breviscapus suspension was used as a comparison to study the relative bioavailability and breviscapus injection was used a comparison to study the absolute bioavailability. The mean plasma concentration-time curves were drew and pharmacokinetic parameters were calculated by DAS soft ware. The relative bioavailability of breviscapus self-microemulsion is 770.1% with breviscapus suspension as control. The absolute bioavailability of the self-microemulsion and suspension were 6.72% and 51.8% respectively.
以灯盏花素中主要成分灯盏乙素为指标,对灯盏花素在人工胃液、不同pH(pH6.0,6.8 7.4)K-R液、不同肠段内容物及不同肠段肠黏膜组织匀浆液中的稳定性进行了考察,结果显示药物在人工胃液、不同肠段内容物及黏膜组织匀浆液中的稳定性较差,对影响灯盏花素稳定性的影响因素考察结果表明,灯盏花素对胃肠道内酶及介质pH敏感,其在K-R液中的稳定性随介质pH升高而降低。0.1%EDTA-2Na和0.1% Vc均可增强药物在K-R液中的稳定性,而以0.1% Vc对药物稳定性的增强作用最为显著。
通过MTT实验考察了药物及一些辅料对caco-2细胞的毒性作用,结果表明这些辅料对Caco-2细胞的毒性大小顺序为PEG400<普朗尼克F68<多烯酸乙酯<灯盏花素<中链脂肪酸甘油酯<油酸乙酯<吐温80<聚氧乙烯蓖麻油<<油酸。
通过caco-2细胞模型,研究了灯盏花素的口服吸收机制,并考察了吐温80、普朗尼克F68及聚氧乙烯蓖麻油对其跨细胞膜转运的影响。
实验发现,灯盏花素在Caco-2细胞单层上的摄取受温度及药物浓度的影响,Caco-2细胞对灯盏花素的摄取量随着温度的增加而增加,在考察的浓度范围内,灯盏花素细胞摄取量随药物浓度的增加而呈线性增加。pH的变化及p-糖蛋白抑制剂维拉帕米的加入对灯盏花素的细胞摄取无显著影响,F68、Tween80及Cremophor EL的加入可增加灯盏花素的细胞摄取量。Caco-2细胞转运实验表明,药物AP-BL及BL-AP的Papp受温度、F68、Tween80及Cremophor EL的影响,而受pH、浓度、维拉帕米的影响不大。药物在AP→BL及BL→AP的Papp均随温度的升高及三种表面活性剂浓度的升高而增加。灯盏花素细胞摄取与转运实验结果表明灯盏花素吸收方式以被动扩散为主。
在体肠灌流是研究小肠吸收机制的一种重要的方法,我们通过大鼠在体肠灌流研究了灯盏花素的吸收特性。灯盏花素在十二指肠、空肠、回肠和结肠中的吸收系数分别为0.0702h-1,0.0871h-1,0.0663h-1和0.0273h-1,t检验结果表明,灯盏花素在十二指肠、空肠、回肠中的吸收系数无显著性差异,在结肠中的吸收系数与其它肠段比较有显著性差异,各肠段吸收系数大小顺序为空肠>十二指肠>回肠>结肠。
在进一步的研究中,我们分别以油酸乙酯、中链脂肪酸甘油酯和多烯酸乙酯为油相来制备灯盏花素微乳,以磷脂为增溶剂增加灯盏花素在三种油相中的溶解度,并绘制了三种油相的灯盏花素微乳伪三元相图。根据伪三元相图,我们设计具有不同油相以及不同油相与表面活性剂比例的了九个乳剂处方,以Caco-2细胞为模型,对这九个处方促进灯盏花素的吸收效果进行了考察。结果表明九个乳剂处方均在不同程度上增加了灯盏花素的吸收,其促进药物的吸收作用与乳剂中油相的种类及乳滴大小有关。三种油相对乳剂促进灯盏花素吸收的大小顺序为油酸乙酯>多烯酸乙酯>中链脂肪酸甘油酯,乳滴粒径大小与乳剂的促进吸收作用呈负相关,相关系数>0.7。
在以上实验的基础上,制备了灯盏花素自微乳剂,处方由35.1%Tween80.17.5%PEG400、38、6%油酸乙酯、3.9%磷脂、4.7%灯盏花素及0.2%维生素E组成。以上处方制备的灯盏花素自微乳化药液0.5g分散在100ml水中可形成均一透明的微乳,乳滴粒径为43nm。形成的微乳采用葡聚糖凝胶G-50色谱法分离游离药物,高效液相色谱法测定包裹率,结果为(93.2±1.0)%。凝胶柱色谱法回收率为98.07%,凝胶柱色谱法加样回收率为97.9%。高效液相色谱法回收率为99.94%,药物在12～360μg/mL浓度范围内呈良好线性关系。采用透析法测定了灯盏花素微乳中药物在不同介质中的释放度,结果微乳在水中释放较慢且不完全,药物在pH6.8PBS中的释放缓慢、平稳,在pH6.8PBS中的释放速率符合higuchi方程Q=14.19t1/2+0.963(R=0.9934)。
建立了大鼠血浆药物浓度测定方法。分别以灯盏花素混悬液和灯盏花素注射液做为参考,计算灯盏花素自微乳剂的相对生物利用度与绝对生物利用度。绘制了三种制剂不同途径给药后的血药浓度—时间曲线,并用DAS软件计算出了三种制剂的药动学参数,结果表明灯盏花素自微乳的绝对生物利用度为51.8%,灯盏花素混悬液的绝对生物利用度为6.72%,灯盏花素自微乳剂相对于灯盏花素混悬液的生物利用度为770.1%。由此可见,与混悬液相比,灯盏花素自微乳剂的生物利用度提高了6.7倍。
Breviscapine, which is mainly composed of breviscapus (af lavone glucuronide), is a well-known bioactive constituentextracted from the traditional Chinese medicine Erigeron breviscapus (Vant.) Hand.-Mazz. Breviscapine is extensively used to treat ischemic cerebrovascular and cardiovascular diseases in China. However, the oral bioavailability of breviscapine was found to be quite poor in rats. To increase its oral bioavailability, Caco-2eells and in situ perfusion method in rats were applied to study the meehanism of absorption after oral administration of brevescapine, such as time, temperature, pH, concentration, p-glycoprotein inhibitor,excipients and different intestine segments were investigated, and self-microemulsion was prepared to enhance bioavailability of breviscapine.
Stability of breviscapus in artificial gastric juice, K-R fluid at different pH value (pH 6,0.6.8,7.4), contents of different parts of intestine, and homogenates of different parts of intestine mucosa were studied. The results showed that scruetallarin degraded significantly in artificial gastric juice, the contents of intestine and homogenates of intestine mucosa. Analysis on influence factors for stability of breviscapus suggested that breviscapus is sensitive to gastrointestinal Enzymes and pH, stability of breviscapus decreases with pH increasing in K-R fluid.0.1% EDTA-2Na is helpful for improving the stability of scretellarin in K-R fluid but 0.1% Vc can improve its stability significantly.
The cytotoxicity of several excipients on Caco-2 cells was evaluated by the MTT test. It showed that the toxic order of theses excipients to Caco-2 cells was F68< Ethyl polyenoate
Uptake of breviscapus by Caco-2 cell monolayers was affected by temperature and concentration of breviscapus, but not by the change in the apical pH, and the P-glycoprotein inhibitor (verapamil) showed no pronounced effects. Uptake of breviscapus by Caco-2 cells was increased with temperature elevating, and the uptake was non-saturable and increased linearly with rising of concentration of breviscapus over the range of concentration tested. The permeability coefficient of apical to basolateral and basolateral to apical were affected by temperature, F68, Tween80 and Cremophor EL efficiently, the permeability coefficient of apical to basolateral and basolateral to apical were increased with the increasing of the temperature and the concentration of these there surfactants. However, the pH, concentration, and verapamil had no marked influence on the permeability coefficient of both apical to basolateral and basolateral to apical. These results indicated that the uptake and transport of breviscapus were passive diffusion as the dominating process.
In situ perfusion in rats is one of the most important methods in the research of the drug's absorption. In this study, the absorption activities of breviscapus in the intestine were investigated. The absorption rate constants of breviscapus were 0.0702h-1,0.0871h-1,0.0663h-1 and 0.0273h-1,respectively in duodenum, jejunum, ileum and colon. Student's t test indicated that there was no significant difference amone the Ka of breviscapus in duodenum, jejunum and ileum, and that the Ka of breviscapus in colon was significantly lower than that in other segment. The Ka values of the intestinal segment was jejunum> duodenum>jejunum>ileum>> colon.
In further study, we separately use ethyl oleate, Ethyl polyenoate and medium chain length fatty acid triglyceride as oil phase to prepare breviscapus microemusion. Phospholipid was selected to increase the solubility of breviscapus in these three oil phase. Pseudo-ternary phase diagrams were constructed identifying the efficient micro emulsification region. Nine breviscapus O/W emulsion formulations with different oil pahse, different proportion of oil phase and surfactant were prepared and their effects on the absorption of breviscapus were studied with Caco-2 cells model. The results showed that all of these nine emulsions have absorption enhancement effect and the effect was related to the oil phase and particle size of the emulsions. The absorption enhancement effect order of these three oil phase is medium chain length fatty acid triglyceride
The quantitative analysis method of plasma containing breviscapus has been built. Breviscapus suspension was used as a comparison to study the relative bioavailability and breviscapus injection was used a comparison to study the absolute bioavailability. The mean plasma concentration-time curves were drew and pharmacokinetic parameters were calculated by DAS soft ware. The relative bioavailability of breviscapus self-microemulsion is 770.1% with breviscapus suspension as control. The absolute bioavailability of the self-microemulsion and suspension were 6.72% and 51.8% respectively.
引文
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