玳玳果黄酮降脂提取物在体肠吸收过程和转运机制研究

Research on Intestinal Absorption Process and Transfer Mechanism in Vivo of the Flavonoid Lipid-lowering Extract from Daidai Fruit

关键词:  在体单向肠灌流模型  肠吸收特性  玳玳果黄酮降脂提取物  新橙皮苷  柚皮苷  UPLC-MS/MS

Key words:  in situ single-pass intestinal perfusion model  intestinal absorption characteristics  flavonoid lipid-lowering extract from Daidai fruit  neohesperidin  naringin  UPLC-MS/MS

摘要:

目的 探究玳玳果黄酮降脂提取物肠吸收部位、过程及转运机制。方法 采用大鼠在体单向肠灌流模型，以提取物效应组分新橙皮苷及柚皮苷为指标，芦丁为内标，运用UPLC-MS/MS技术，建立玳玳果黄酮降脂提取物肠灌流液定量分析法；考察提取物在各肠段的吸收过程及特性；加入外排转运蛋白P-gp抑制剂维拉帕米（verapamil，VP），评价P-gp对提取物吸收的影响及肠道吸收参数（表观吸收系数P_app、吸收速率常数K_a和药物累计吸收百分比P%值）的变化；并以不同质量浓度提取物探究效应组分的吸收转运过程。结果 玳玳果黄酮降脂提取物在不同肠段吸收量顺序为十二指肠>结肠>空肠≈回肠；与十二指肠比较，其他肠段的新橙皮苷及柚皮苷的肠道吸收参数P_app、K_a及P%均有显著性差异（P<0.05），十二指肠的新橙皮苷和柚皮苷的P%约是其余肠段的5~7倍；加入VP后，效应组分新橙皮苷及柚皮苷的P_app、K_a、P%均有显著提高，且在十二指肠的吸收量明显增加，P%约是VP空白组的4倍；提取物高、中、低浓度组柚皮苷的P_app、K_a无显著变化；中、低浓度组的新橙皮苷P_app、K_a随浓度增加略有上升；中浓度组的新橙皮苷P_app、K_a比高浓度组增大，P_app差异有统计学意义（P<0.05）。结论 玳玳果黄酮降脂提取物的主要吸收部位是十二指肠；其效应组分在肠道的吸收受P-gp影响显著，各组分吸收参数变化趋势相同，不存在竞争性或非竞争性抑制作用；低浓度下表观吸收均属一级动力学过程；柚皮苷的吸收机制可能为被动转运，新橙皮苷的吸收机制可能为载体介导的促进扩散。

Abstract:

OBJECTIVE To study the intestinal absorption site, process and transport mechanism of the flavonoid lipid-lowering extract from Daidai fruit. METHODS Rat in situ single-pass intestinal perfusion model was used, with active parts neohesperidin and naringin as index, rutin as internal standard. Quantitative analysis method of the flavonoid lipid-lowering extract from Daidai fruit intestinal perfusate was established by UPLC-MS/MS. The extract absorption process and characteristics of different intestine segments were observed. P-gp efflux pumps inhibitor verapamil(VP) was added. The effect of P-gp on the absorption of the extract, and the changes in intestinal absorption parameters(apparent absorption coefficient P_app, absorption rate constant K_a, and percentage of accumulative absorption P%) were evaluated. The absorption and transfer process of the active parts were explored using extract samples at different concentration levels. RESULTS The flavonoid lipid-lowering extract from Daidai fruit was absorbed in the following order:duodenum>colon>jejunum≈ileum. Compared with duodenum, the absorption parameters(P_app, K_a, and P%) of neohesperidin and naringin at all the other intestine segments had significant difference(P<0.05). The P% values of neohesperidin and naringin at duodenum was about 5-7 times of those at the other intestine segments. After adding VP, the P_app, K_a, and P% values of the active parts of the extract(neohesperidin and naringin) significantly increased. The amount absorbed at duodenum also showed a significant improvement, with P% value being 4 times of VP blank group. For extracts at all three concentration levels(high, middle, and low), there was no significant changes in the P_app and K_a values of naringin. For extracts at middle and low concentration levels, the P_app and K_a values of neohesperidin increased slowly as the concentration level of the samples increases. For extracts at middle concentration level, the P_app and K_a values of neohesperidin was larger than those for the extracts at high concentration level. The difference in P_app values had statistical significance(P<0.05). CONCLUSION The main absorption site of the flavonoid lipid-lowering extract from Daidai fruit is duodenum. P-gp have significant effects on the intestinal absorption of the active parts of the extract. Both active parts show similar changes for their absorption parameters, which implies that on P-gp efflux pathway, neohesperidin and naringin do not have competitive or non-competitive inhibitory effect on each other. Under low concentration, their apparent absorption at intestine follows the first order kinetics. The absorption mechanism of naringin is probably passive transport, and the neohesperidin is probably facilitated diffusion by vector-mediation.