【作者】 李素云；

【导师】 郭长江； 张振清；

【作者基本信息】 中国人民解放军军事医学科学院 ， 营养与食品卫生学， 2010， 博士

【摘要】 在参考有关文献的基础上,本研究构建了体外人类肠吸收模型Caco-2细胞模型,对槲皮素及其主要糖苷在Caco-2细胞模型上的吸收、代谢过程以及相关机制进行了研究,为进一步深入了解槲皮素及其主要糖苷在体内的吸收、代谢过程以及生物利用度等提供实验依据。本研究主要完成了下面六个方面的工作,主要结果如下:1、建立了Caco-2单层细胞吸收及外排模型,优化了相关的实验条件。同时,应用P-gp特异性抑制剂CysA和MRP2特异抑制剂MK571,对受试物与P-gp和MRP2的相互作用进行了考察,结果与预期实验一致,证明该模型可靠。2、摸索了细胞孵育的相关实验条件,建立了样品处理方法和LC/MS检测方法。通过模拟细胞孵育环境,确定实验中细胞孵育时间不超过2.5h;受试物负载量浓度低于100?g/ml时,细胞抑制率均小于5%,对细胞的活性状态没有影响,能够满足实验要求。对含有槲皮素及其糖苷以及甲基化代谢物的PBS溶液,经过一定比例稀释后,可以直接采用LC/MS法测定,方法灵敏度高,线性良好,可以满足本研究的需要。3、考察了槲皮素及其糖苷在Caco-2单层细胞模型上吸收特征。槲皮素在Caco-2细胞单层上的跨膜双向转运,不同浓度各时间点均能在透过面检测到槲皮素,且存在浓度、时间依赖性动态变化,B→A的透过量高于A→B,大约是A→B的2.2倍;加载侧槲皮素的减少也存在浓度、时间依赖性;以两侧槲皮素量的变化做标准计算表观渗透系数(Papp),负载面得到的Papp(A→B)和Papp(B→A)分别是透过面的24倍和8倍左右,负载面槲皮素减少和透过面检测到槲皮素之间相差10倍以上,90%以上的槲皮素在跨膜转运过程中消失,提示槲皮素在跨膜转运过程中可能发生了广泛的代谢和转化。槲皮苷和异槲皮苷在Caco-2单层细胞模型上也存在着双向跨膜转运,并存在浓度、时间依赖性的动态变化,A→B的透过量高于B→A,Papp(A→B)/Papp(B→A)比值,槲皮苷1.38,异槲皮苷2.24,按透过面的测定量计算,150min时槲皮苷的双向透过率分别为5.1%和2.9%,异槲皮苷的双向透过率为5.3%和2.7%。这一结果表明槲皮苷和异槲皮苷均能直接透过Caco-2单层细胞,提示在Caco-2细胞表面可能存在直接摄取槲皮苷和异槲皮苷的转运蛋白,同时也存在跨膜转运过程中的代谢和转化。4、探讨了SGLT1和GLUT2对槲皮素及其糖苷在Caco-2单层细胞模型上吸收中的作用。不同浓度葡萄糖对槲皮素的跨膜转运均有明显抑制作用,但没有表现出浓度依赖性,说明槲皮素和葡萄糖可能通过竞争性结合转运蛋白而非共转运的方式转运,且是以苷元的形式而不是以与葡萄糖结合成糖苷的形式与转运蛋白结合,非浓度依赖性则间接提示了转运载体的转运可能具有饱和作用;根皮苷对不同浓度槲皮素的跨膜转运的影响与葡萄糖相似,提示了SGLT1在槲皮素跨膜吸收中的作用;根皮素的影响与根皮苷不同,其显著增加了槲皮素A→B和B→A的双向透过率。根皮苷和根皮素对槲皮苷的跨膜转运有明显的抑制作用,提示SGLT1和GLUT2可能同时参与了槲皮苷的跨膜转运作用;根皮苷和根皮素对异槲皮苷的作用不同,120min内根皮素没有表现出明显的抑制作用,150min时明显上调了异槲皮苷的透过率,而根皮苷对异槲皮苷的作用与之相反,120min内抑制作用显著,150min时抑制作用消失,提示不同的糖基配体对槲皮素糖苷在Caco-2单层细胞上的吸收和/或代谢会有一定程度的影响。5、研究了解了P-gp和MRP2对槲皮素及其糖苷在Caco-2单层细胞模型上吸收的作用。P-gp抑制剂CysA和MRP2抑制剂MK571可显著提高对侧面槲皮素浓度,提示槲皮素是P-gp和MRP2的底物。CysA和MK571对槲皮苷和异槲皮苷均没有表现出明显的抑制作用,提示二者可能不是P-gp和MRP2的底物。6、分析了槲皮素及其糖苷在Caco-2细胞模型上的代谢产物。在孵育30-150min时间内,槲皮素孵育组的两侧孵育液中均检测到了槲皮素的甲基化产物-异鼠李亭和柽柳黄素,其中异鼠李亭含量约是柽柳黄素的1.5-2.0倍,二者总计含量不超过透过面测到槲皮素的10%和负载面减少量的1%,提示在跨膜转运过程中槲皮素的甲基化作用只是胞内代谢的中间环节,应该仍然存在其他的和进一步的代谢转化;两侧含量的动态变化均具有时间依赖性,90-120min时达峰值,随后呈下降趋势;在槲皮素的双向转运中,A→B的A面异鼠李亭含量明显高于B侧,B→A转运中,30min时B侧异鼠李亭高于A侧,60min以后各时间点,A面含量均高于B侧面;柽柳黄素在A、B两面的分布特征与异鼠李亭相似,提示甲基槲皮素可能是Caco-2细胞顶侧面外排蛋白的底物,且在基底侧也存在胞外的槲皮素甲基化反应。对槲皮素及其糖苷的代谢产物进一步分析后发现,除了异鼠李亭和柽柳黄素外,其他可能的代谢产物尚有槲皮素一硫酸酯(m/z 383→301,153)、槲皮素三硫酸酯(m/z 543→153)、槲皮素葡萄糖醛酸(m/z 479→153)、槲皮素葡糖醛酸化硫酸酯(m/z 559→153)、甲基槲皮素硫酸酯(m/z 397→301,153)和甲基槲皮素葡萄糖醛酸(m/z 493→301,153)。槲皮苷和异槲皮苷孵育后除了检测到微量的甲基化代谢产物外,均未检测到其它代谢产物。综上所述,槲皮素及其糖苷均可以原型形式直接透过Caco-2细胞单层,并在跨膜吸收过程中发生广泛的代谢转化,但槲皮素糖苷的透过和代谢速度较槲皮素苷元缓慢;在细胞中的代谢产物主要为甲基化、硫酸化和葡萄糖醛酸的一级代谢产物。SGLT1是槲皮素及其两个糖苷在肠道吸收中的转运蛋白之一,同时槲皮苷也通过GLUT2转运吸收,GLUT2在槲皮素和异槲皮苷的跨膜吸收过程中的作用仍有待进一步研究。槲皮素是P-gp和MRP2的底物,存在外排现象,而槲皮苷和异槲皮苷的跨膜吸收不受P-gp和MRP2外排作用的影响。更多还原

【Abstract】 A model for the simulation of human intestine epithelia was established from Caco-2 cells based on the procedures reported in literature. The absorption and metabolism of quercetin and its glycosides were studied on this model, in order to provide information on absorption and metabolism of quercetin and its glycosides in vivo and related mechanisms.Six parts were included in this study. The major results were summarized as followings.1. The model of Caco-2 cells was established and culture conditions were optimized. The specific inhibitors for P-gp and MRP2 were applied to validate the model. The results showed that the model was reliable.2. The adequate optimal incubation conditions and LC/MS technique were developed. The incubation time should be less than 2.5h and the concentration of tested chemicals lower than 100ug/ml. The quercetin, quercetin glycosides and methylated metabolites were analyzed using the LC/MS procedure developed in this experiment. The results indicated this LC/MS procedure was sensitive, linear in certain range of concentration.3. The absorption of quercetin and quercetin glycosides was investigated in Caco-2 cell monolayer. The quercetin was transported across the monolayer bilaterally, in concentration and time dependent manners. The transport from basolateral to apical was more than from apical to basolateral. The Papp(A→B) and Papp(B→A) on the loading side were 24 and 8 times higher than the receiver side, respectively, indicating that quercetin underwent extensive transformation after absorption. Quercitrin and isoquercitrin were also transported across the monolayer bilaterally, in concentration and time dependent manners. Possible metabolism also occurred to them during the transportation across the Caco-2 cell monolayer.4. The roles of SGLT1 and GLUT2 in the absorption of quercetin and quercetin glycosides were investigated on Caco-2 cell monolayer. The glucose inhibited the transportation of quercetin across the monolayer, which was in a manner independent of the concentration of glucose. It was, therefore, suggested that quercetin and glucose competed for the same transporters on the monolayer during the absorption processes. Phloridzin and phloretin exhibited an effect similar to glucose on quercetin absorption, indicating that both SGLT1 and GLUT2 were involved in the processes of quercetin absorption. Phloridzin and phloretin acted differently on the absorption of quercitrin and isoquercitrin, which implied that the difference in sugar moiety affected the process of absorption on Caco-2 cell monolayer.5. The roles of P-gp and MRP2 in the absorption of quercetin and quercetin glycosides were also investigated on Caco-2 cell monolayer. The addition of specific inhibitors for P-gp and MRP2 decreased the transportation of quercetin across Caco-2 cell monolayer. However, no effect was observed for quercitrin and isoquercitrin. It was demonstrated that quercetin was the substrate for P-gp and MRP2, whereas quercitrin and isoquercitrin were not.6. The metabolism of quercetin and quercetin glycosides were analyzed on Caco-2 cell monolayer. Two methylated metabolites, isorhamnetin and tamarixetin were detected in the loading and receiver sides, which accounted for 10% of quercetin loaded, suggesting the existence of other metabolic pathways for quercetin. More metabolites were further identified, such as quercetin mono-sulphate, quercetin tri-sulphate, quercetin glucuronide, quercetin glucuronide sulphate, methylquercetin sulphate and methylquercetin glucuronide.In conclusion, quercetin and quercetin glycosides were transported intact across the Caco-2 cell monolayer and experienced an extensive metabolism. The quercetin glycosides appeared relatively slower than quercetin in the process of absorption and metabolism. The methylated, sulphated and glucuronidated metabolites were identified, respectively. The SGLT1 participated in the process of absorption of quercetin and quercetin glycosides, while the GLUT2 was involved in the process of absorption of quercitrin. Quercetin was the substrate for P-gp and MRP2. However, the absorption of quercitrin and isoquercitrin was not affected by P-gp and MRP2.更多还原