三叶青地上部分抗炎提取物效应组分群不同生理状态在体肠吸收动力学及转运机制研究

Intestinal absorption kinetics and transport mechanisms in healthy and inflammatory pathological model rats after oral administration of the extract of Tetrastigma hemsleyanum Diels et Gilg’s aerial part

目的:考察三叶青地上部分抗炎提取物(THAA)在不同生理状态大鼠体内的吸收特性和转运机制。方法:采用大鼠在体肠单向灌流模型,建立同时测定THAA效应组分群绿原酸,荭草苷,异荭草苷,异荭草苷-2′-O-鼠李糖苷,牡荆苷,异牡荆苷,牡荆苷-2″-O-鼠李糖苷7个组分含量的液-质联用(UPLC-MS/MS)法,考察比较THAA效应组分群在不同生理状态大鼠不同肠段的累积吸收量,探讨其最佳吸收部位及转运机制,同时选择吸收较好的肠段,考察P-gp外排转运蛋白对THAA肠吸收的影响。结果:正常生理状态,荭草苷、异荭草苷、异荭草苷-2′-O-鼠李糖苷、牡荆苷、异牡荆苷、牡荆苷-2″-O-鼠李糖苷6个组分在各肠段吸收顺序为十二指肠>回肠>空肠>结肠,其中异荭草苷-2′-O-鼠李糖苷、牡荆苷-2″-O-鼠李糖苷在十二指肠及回肠吸收更好(Papp均大于或等于0.2×10-4cm·s-1);绿原酸各肠段吸收顺序为回肠≥十二指肠>空肠>结肠,不过吸收均相对较差(Papp均小于0.2×10-4cm·s-1);同一肠段中各组分的吸收均无显著性差异(P>0.05)。炎症病理状态与正常组比较,THAA效应组分群在各肠段的吸收均有增加;其中荭草苷、异荭草苷、异荭草苷-2′-O-鼠李糖苷、牡荆苷-2″-O-鼠李糖苷在十二指肠及结肠的吸收显著增加;绿原酸、牡荆苷、异牡荆苷在各肠段的吸收略有提高;效应组分群在各肠段的吸收顺序与正常组无显著性差异(P>0.05)。荭草苷等6个组分肠道吸收受外排转运蛋白P-gp影响显著,炎症病理状态下影响更为明显;绿原酸可能不是P-gp的底物,但在炎症病理状态时,可使其吸收参数Papp、Ka、P%增大,且差异有统计学意义(P<0.05)。结论:小肠中上段是THAA效应组分群的主要吸收部位;荭草苷等6个主要特征效应组分可能是P-gp的底物,其肠吸收存在饱和性抑制现象,在体内的吸收机制可能不仅为单纯的被动吸收,还存在主动转运和易化扩散等吸收特征;其中绿原酸在肠道中的吸收可能为促进扩散式。炎症病理状态下,各组分所表现的肠吸收动力学特性及机制与正常生理状态相似,不过肠吸收Papp、Ka、P%均有一定增加,提示炎症病理状态可能因机体内的菌群发生一定变化、因应激促进效应组分群吸收增加而使转运过程产生变化。

OBJECTIVE To explore the absorption characteristics and the transport mechanism of extract Tetrastigma hemsleyanum Diels et Gilg’s aerial part(THAA)in rats with different physiological states.METHODS Using a single-pass intestinal perfusion model of rats, UPLC-MS/MS method was employed for simultaneous content determination of seven effective components of THAA extract, i.e.chlorogenic acid, orientin, isoorientin, isoorientin-2’-O-rhamnoside, vitexin, isovitexin and vitexin-2″-O-rhamnoside.The cumulative absorption amounts of THAA extract at different intestinal segments of rats in different physiological states were compared.Optimal absorption site and transport mechanism were examined.The intestinal segments with relatively better absorption rate were selected for determining the effect of P-gp efflux pumps on intestinal absorption and internal transportation of THAA.RESULTS In normal rats, the order of absorption rates of orientin, isoorientin, vitexin, isovitexin, isoorientin-2’-O-rhamnoside and vitexin-2″-O-rhamnoside was duodenum>ileum>jejunum>colon.Among them, isoorientin-2’-O-rhamnoside and vitexin-2″-O-rhamnoside were better absorbed in duodenum and ileum(Papp ≥0.2×10-4cm·s-1).The order of absorption rates of chlorogenic acid in different intestinal segments was ileum≥duodenum>jejunum>colon.However, absorption was relatively poor(Papp all<0.2×10-4cm·s-1).No significant difference existed in absorption of each component in the same intestinal segment(P>0.05).In inflammatory pathology model, the absorption rates of THAA anti-inflammatory extract were better in all intestinal segments as compared to normal rats.Among them, the absorption rates of orientin, isoorientin, isoorientin-2’-O-rhamnoside and vitexin-2″-O-rhamnoside increased markedly in duodenum and colon.The absorption rates of chlorogenic acid, vitexin and isovitexin rose slightly in all segments.The order of absorption rates of effective component groups in different intestinal segments was not significantly different from that of normal group(P>0.05).The intestinal absorption rates for six components including orientin were significantly affected by P-gp, efflux pumps.The effext was more significant in the physiological state of inflammation.Chlorogenic acid may not be a substrate of P-gp.However, in the pathological state of inflammation, its absorption parameters of Papp,Ka,and P% increased and were statistically significant(P<0.05).CONCLUSION The upper and middle segments of small intestine are the major absorption sites of THAA extract.Under pathological state of inflammation, the absorption of pharmacodynamic components increases due to stress.The six components including orientin may be substrates of P-gp.The absorption mechanism of chlorogenic acid in intestine may be diffusion-promoting.The absorption of the other six components has showed saturation inhibition phenomenon, suggesting that its in vivo absorption mechanism may not be a pure passive absorption process, but also has absorption characteristics such as active transport and facilitated diffusion.Under the pathological state of inflammation, the characteristics and the mechanism of intestinal absorption kinetics of each component are similar to those of normal state.However, Papp,Ka,and P% values of intestinal absorption somewhat increase.Thus flora changes in inflammation state may lead to a higher absorption of drugs and a change of transfer process.