【作者】 韩旻；

【导师】 方晓玲；

【作者基本信息】 复旦大学 ， 药剂学， 2006， 博士

【摘要】 三七总皂苷（Panax notoginseng saponins，PNS）是中药三七的主要成分，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PNS生物利用度低的原因，本文采用Caco-2细胞及动物等体内外模型对其口服吸收过程中的各个环节进行考察，并制备W/O微乳制剂以提高其口服生物利用度。分别考察了PNS在人工胃液、Hanks缓冲液、不同肠段内容物以及肠粘膜匀浆内的稳定性。结果表明PNS中的人参皂苷Rb₁（ginsenoside Rb₁，Rb₁）和人参皂苷Rg₁（ginsenoside Rg₁，Rg₁）单体在人工胃液内易被破坏，而在近中性环境（pH5.5、6.0、6.8、7.4的Hanks缓冲液）内基本保持稳定。Rb₁和Rg₁的单体及其混合物PNS在大肠内容物中易降解，尤以Rb₁降解较为明显；二者在小肠内容物及肠粘膜中则相对稳定。以Caco-2细胞为模型，模拟小肠上皮细胞，考察Rb₁和Rg₁的摄取及转运机制。实验发现，Rb₁和Rg₁的单体及其混合物PNS在Caco-2细胞层的摄取受温度的影响，而pH的变化及其他化合物（环孢菌素）的加入对二者摄取均无显著性影响，同时在实验考查浓度范围内，细胞内Rb₁（或Rg₁）的摄取量随着Rb₁（或Rg₁）单体或混合物PNS的浓度的增加而呈线性增加，Rb₁（或Rg₁）单体与总皂苷中的Rb₁（或Rg₁）在Caco-2细胞中的吸收特性无明显差异。Rg₁的细胞摄取量（1.07±0.16μg/mg protein）（C₀=1mg/ml）相对Rb₁（0.77±0.03μg/mg protein）（C₀=1mg/ml）较高（P＜0.05）。Caco-2细胞转运实验表明，Rb₁和Rg₁单体的转运透过系数(P_app)（Apical-Basolateral）分别为（5.90±1.02）×10^-8 cm/s和（2.59±0.17）×10^-7 cm/s，二者转运都不受环孢菌素影响。通过大鼠不同途径给药后血药浓度参数的比较，考察药物口服吸收过程中各部位对生物利用度的影响。结果表明PNS溶液灌胃（po）（1500 mg/kg）、十二指肠（id）（900 mg/kg）及门静脉（pv）（50 mg/kg）给药后测得Rb₁绝对生物利用度分别为0.71％、2.71％和65.77％；Rg₁绝对生物利用度分别为3.29％、3.41％和50.56％。口服吸收实验结果表明，PNS（包括Rb₁和Rg₁）的肠道吸收机制为单纯被动扩散，吸收过程不受细胞膜内P-gp等外排蛋白的作用，PNS中其他成分对Rb₁或Rg₁的吸收特性无明显影响。胃液的酸性环境、大肠菌丛产生的酶及肝的首过作用均对其口服吸收产生影响，而肠道粘膜的透过性低是其口服吸收差的主要影响因素。为深入了解PNS的体内吸收及药动学行为，分别考察了Rb₁、Rg₁的胆汁排泄及血浆蛋白结合率，并建立了胆汁和血浆样品的分析方法。静脉给药（PNS50mg/kg）后10h，Rg₁、Rb₁分别有（61.48±18.30）％和（3.94±1.49）％由胆汁排泄进入肠腔内，灌胃给药（PNS 1500mg/kg）后12h，Rg₁、Rb₁分别有（0.91±0.51）％，（0.055±0.02）％由胆汁排泄进入肠腔内。在PNS溶液20-200μg/ml浓度范围内，Rb₁、Rg₁的血浆蛋白结合率分别为（80.11～89.69）％和（6.56～12.74）％。结果表明，Rb₁的血浆蛋白结合率较高，胆汁排泄较低；Rg₁的血浆蛋白结合率较低，而胆汁排泄较高。为提高PNS的口服吸收，设计并制备了W/O微乳制剂。选择大豆磷脂/乙醇（SP/EtOH）为微乳的混合表面活性剂，并考察了不同K_m值和油相组成，以及药物加入前后对微乳形成及理化性质的影响。当处方组成为：SP/EtOH（K_m=1/1）、Labrafil M 1944CS、PNS水溶液（400mg/ml），各自质量百分比分别为30％、45％和25％时，微乳外观澄清透明，粒径为17.8±1.7nm，表面活性剂含量较低，而载药量较高。以SP/EtOH（K_m=1/1）[或（SQ/O）/EtOH（K_m=1/1）]和PNS水溶液（400mg/ml）与不同油相分别制备11个W/O微乳处方，于大鼠十二指肠内给药研究其体内药动学性质，实验结果表明大多数微乳处方可明显提高药物的生物利用度。W/O微乳促进药物吸收的作用除与制剂中所含的表面活性剂有关外，不同种类油相的选用也会产生一定影响，实验中发现，对于Rb₁、Rg₁采用不同油相制备的微乳其吸收促进作用大小都有IMWITOR 312≈IPM＞IPP＞2EHP，表明随着脂肪酸碳链的增加，长链(C＞C₁₄)脂肪酸酯的吸收促进作用较中链脂肪酸酯(C₈＜C＜C₁₄)有所降低。研究不同处方微乳稀释20倍后的外翻肠囊吸收，结果表明大多数微乳处方稀释后可不同程度的促进Rb₁、Rg₁的肠吸收，其中，处方3无论对于Rb₁还是Rg₁，都具有非常明显的吸收促进作用。实验中制备大单室脂质体（200～300nm）做为生物膜模型，考察了W/O微乳对脂质体膜流动性的影响。结果表明大多数微乳处方可不同程度的提高脂质体膜流动性，提示微乳促进药物吸收的作用可能与其提高生物膜流动性有关。首次采用PAMPA技术研究了不同微乳处方对药物膜转运的促进作用，计算有效渗透系数P_e，并将结果与大鼠体内肠吸收实验结果作相关性分析，探讨PAMPA分析技术作为制剂体外质量评价的可行性，并分析其影响因素。结果发现，在同等给药条件下，Rb₁的P_e值总体等于或小于Rg₁，这与细胞实验的结果相吻合。另一方面，稀释后微乳（D-ME）中药物的P_e大多高于PNS对照溶液，表明微乳中的组分可以提高药物的膜渗透转运能力，但P_e以及外翻肠囊实验与大鼠体内肠吸收实验结果间的相关性都比较差，原因可能是由于W/O微乳稀释后，体系中的脂质成分分层析出、粘附于肠粘膜表面或PAMPA膜上，从而对实验结果产生了一定的影响而导致的，另外PAMPA自身所缺乏的细胞旁通路转运机制，也可能是制约其全面反映药物吸收性质的一个原因。与此同时，实验发现稀释前微乳（ME）的PAMPA分析结果与大鼠体内肠吸收实验结果间具有相对较好的直线相关性，虽然W/O微乳在体内是以D-ME的形式发挥作用，但ME中药物膜转运能力的差异也可以在一定程度上反映微乳中组分对药物吸收的影响，该实验也进一步表明，除可应用于原料药物的膜转运研究外，在了解药物一定的相关背景知识，并在供试液体系相对单一的条件下，PAMPA可以尝试引入制剂处方研究的某些领域中。更多还原

【Abstract】 As main ingredients of notoginseng, Panax notoginseng saponins （PNS） proved in recent years to possess pharmacological action in cardiovascular system. But bioavailability for both Rg₁ and Rb₁, which are main ingredients of PNS, is very low after oral administration. In present study, Caco-2 cells and rat models were applied to study the mechanism of absorption after oral administration of PNS, and W/O microemulsion were prepared to enhance bioavailability of PNS.Stability of of both Rb₁ and Rg₁ in PNS in artificial gastric juice, HBSS at different pH value （pH 5.5, 6.0, 6.8, 7.4）, contents of different parts of intestine, and homogenates of intestine mucosa were studied. It showed that Rb₁ （and Rg₁） proved to be readily eliminated in stomach, but stable in relatively neutral circumstance. Both Rb₁ and Rg₁ in PNS, especially for Rb₁, degraded significantly in the contents of large intestine. However, both of them kept mainly intact in the contents of small intestine and homogenates of intestinal mucosa.Uptake of both Rb₁ and Rg₁ by Caco-2 cell monolayers was inhibited in low temperature, but not by cyclosporine, and the change in the apical pH showed no pronounced effects. Uptake and transport were non-saturable and increased linearly with rising of concentration of Rb₁ （and Rg₁） over the range of concentration tested, which indicated a passive transport. There was no significant difference between monomer （Rb₁ and Rg₁） and mixture （PNS） for absorption characteristic. Uptake amount of Rg₁（1.07±0.16μg/mg protein） （Co=1mg/ml） in Caco-2 cells was a little higher than that of Rb₁ （0.77±0.03μg/mg protein） （Co=lmg/ml）. Meanwhile, apparent permeability coefficient of （5.90±1.02）×10^-8 cm/s（C₀=1mg/ml） for Rb₁ and （2.59±0.17）×l0^-7cm/s （Co=1mg/ml） for Rg₁ from apical compartment to basolateral compartment predicted an incompletely absorption. Transport of both Rb₁ and Rg₁ were not influenced by cyclosporine.The serum concentration-time profiles of Rb₁ afer tail venous （iv）, portal venous （pv）, intraduodenal （id） and peroral （po） administration to rats were compared to evaluate the the role of stomach, intestine and liver involved in absorption process. The pharmacokinetic behavior of Rb₁ （and Rg₁） after different routes of administration to rats showed that the absolute bioavailability after po （PNS 1500mg/kg）, id （PNS 900mg/kg）, and pv （PNS 50mg/kg） administration is 0.71%, 2.71% and 65.77% respectively for Rb₁and 3.29%, 3.41% and 50.56% respectivelyfor Rg₁.So, it can be concluded that transport across Caco-2 cell monolayers for PNS （include Rb₁ and Rg₁） is a simple passive diffusion process. No efflux transporters in Caco-2 cells and other components in PNS showed effects on it. The elimination in stomach, large intestine and liver contributed to the low bioavailability of PNS, but the low membrane permeability might be a more important factor dominating the extent of absorption.This thesis studies Plasma protein binding and bile excretion of both Rg₁ and Rb₁ for further analysis of PNS. （61.48±18.30）% dose of Rg₁, and （3.94±1.49）% dose of Rb₁ were respectively excreted into bile 10 hours after iv administration （PNS 50mg/ml）, and （0.91±0.51%）% dose of Rg₁ and （0.055±0.02）% dose of Rb, were excreted into bile 12 hours after po administration （PNS 1500mg/ml）. Ranging from 20 to 200μg/ml in PNS concentration, the plasma protein binding degrees of Rg₁ and Rb₁ are respectively 6.56-12.74% and 80.11-89.69%.In further study, SP/EtOH was selected as surfactant/co-surfactant for the preparation of W/O microemulsion to enhance the bioavailability of PNS. Studies are mainly focused on the influence of different K_m and oil, and addition of drug （PNS） on the formation and physico-chemical properties of W/O microemulison. When W/O microemulision is composed of 30% SP/EtOH（K_m-1/1）, 45%Labrafil M 1944CS and 25%PNS solution （400mg/ml）, it shows a transparent system and particle size is 17.8±1.7nm, the content of surfactant is fairly low while that of PNS is relatively high. With the surfactant/co-surfactant of SP/EtOH （K_m=1/1） [or （SQ/O） /EtOH （K_m=1/1） ] and 11 PNS W/O microemulison prepared in different kinds of oils, pharmacokinetic behavior of W/O microemulison was experimented in the rats after intraduodenal administration. The results show that most of microemulison enhances the absorption of Rg₁ and Rb₁ significantly, and the mechanism of the enhanced absorption may be attributed to the effect of surfactant/co-surfactant. Meanwhile, the composition of the oil plays a role that cannot be neglected here and the enhancement of PNS absorption varies with the microemulsion containing different kinds of oil, as follows: BvIWITOR 312^IPM>IPP>2EHP. Accompanying with the rising carbon chain of fatty acid in oil, the rate of absorption enhancement by the long chain glyceride (C> C₁₄) is lower than that by the medium chain glyceride (C₈<C<C₁₄).The experiments are also carried out for the analysis of the microemulsion absorption in everted intestine sac, and it is proved by the results that most ofmicroemulison can enhance the absorption of Rb₁ and Rg₁ after diluted with Kreb’s solution. Among them, microemulision 3 can significantly enhance the absorption of both Rb₁ and Rg₁.Liposome was used as a model to imitate biological membrane, and then W/O microemulison was found to enhance the membrane fluidity of liposome to different extents. It suggests that the effect of W/O microemulison on the absorption enhancement may be related with its enhancement in the membrane fluidity.PAMPA analysis was first applied in the present study of P_erformulation to explore the effects of different W/O microemulison on the absorption of PNS. The value of P_e and the correlation between P_e and Fr, P_e and R were studied to evaluate the feasibility of PAMPA being an in vitro model and the pharmaceutical dosage.The results show that P_e of Diluted-Microemulsion （D-ME） is mostly higher than that of PNS solution （control）, which suggests the components of microemulision can facilitate the membrane P_ermeability of drug. While in the coordinate conditions, P_e of Rg₁ is mainly lower than that of Rb₁, which accords with the results of Caco-2 cell exP_eriments. But for D-ME, neither P_e nor R shows linearity correlation with Fr and this may be attributed to the phase transformation resulted from the separation, precipitation and flocculation of the system after the dilution of the Microemulsion. In some exP_eriments, lipids separated from the system even adhere to PAMPA membrane or intestine mucous membrane, which may also influence the results of the exP_eriment. The linearity correlation between PAMPA and Fr acquired in Microemulison （ME） analysis further indicates that the PAMPA analysis can be brought into not only the investigation of membrane transport of crude drug, but also conditioned preformulation research.更多还原