| Caco-2 cell model has been applied abroad widely for nearly ten years, as a kind of in vitro model studying intestinal absorption of drugs. It is much more simple, faster, more efficient and more reproducible than the whole animal experiments, and the process of in vitro drug absorption has a good correlation with absorptive process in small intestine after oral administration. So as a quick screening tool, the Caco-2 cell model is used extensively in screening and study of absorptive mechanism during the pre-developing drug period.Mixed system of Chinese medicine is a traditional form of administration. This study aimed to understand its impact on the oral absorpsion of active ingredients, and to explore the potential interaction between active ingredients of Chinese medicine during the process of intestinal absorption. First of all, we established the Caco-2 model. Then we studied absorption of three diester-type alkaloids by this model, which were aconitine, mesaconitine, hypaconitine. They each were in single compounds, mixtures and aconite extract respectively. The bi-directional transportion processes (from apical side to basolateral side and the opposite direction) were researched, and the former was absorptive process, while the latter was excretive process. The administrative concentrations of three alkaloids in single compounds, mixed compounds, and aconite extract were corresponsive on the whole. Their concentrations were 18μmol/L, 70μmol/L and 100μmol/L for aconitine, mesaconitine and hypaconitine respectively. Besides, We also inspected the absorption of three alkaloids in extract with a low concentration, which were 9μmol/L, 35μmol/L and 50μmol/L for aconitine, mesaconitine and hypaconitine respectively. The concentrations of every compound in samples were determined by High Performance Liquid Chromatography (HPLC).The result was that the apparent permeability coefficient (Papp) values of aconitine with the concentration at 18μmol/L from A side to B side in single compound, mixed compounds, and aconite extracts with high and low concentrations were (3.01±0.72)×10-6 cm·s-1, (12.36±1.52)×10-6 cm·s-1, (17.00±0.85)×10-6 cm·s-1 and (13.96±1.24)×10-6 cm·s-1 respectively; While its Papp values from B side to A side in the four samples mentioned above were (38.73±1.87)×10-6 cm·s-1, (26.75±1.39)×10-6 cm·s-1, (17.38±2.11)×10-6 cm·s-1 and (19.13±1.74)×10-6 cm·s-1 respectively; So the efflux ratios Papp(B→A)/ Papp(A→B) of aconitine were 12.89, 2.16, 1.02 and 1.37 respectively. Similarly, for mesaconitine, the Papp(A→B) values in single compound, mixed compounds, and aconite extracts with high and low concentrations were (3.90±0.92)×10-6 cm·s-1, (9.59±1.32)×10-6 cm·s-1, (16.07±0.99)×10-6 cm·s-1, (14.63±1.10)×10-6 cm·s-1 respectively;While its Papp(B→A) values in the four samples were (32.91±1.39)×10-6 cm·s-1, (25.66±1.68)×10-6 cm·s-1, (20.10±2.13)×10-6 cm·s-1, (18.90±0.96)×10-6 cm·s-1 respectively; So the efflux ratios of mesaconitine were 8.44, 2.68, 1.25 and 1.29 respectively. For hypaconitine, the Papp(A→B) values in single compound, mixed compounds, and aconite extracts with high and low concentrations were (15.21±0.90)×10-6 cm·s-1, (18.03±1.45)×10-6 cm·s-1, (20.45±2.01)×10-6 cm·s-1, (21.98±1.30)×10-6 cm·s-1 respectively; While its Papp(B→A) values in the four samples were (26.81±1.20)×10-6 cm·s-1, (29.04±2.30)×10-6 cm·s-1, (20.51±1.44)×10-6 cm·s-1, (21.16±1.63)×10-6 cm·s-1 respectively ; So the efflux ratios of hypaconitine were 1.76, 1.61, 1.00 and 0.96 respectively.The results showed that the Papp values of three diester-type alkaloids are more than 1×10-6 cm·s-1, and the concentration of extract had no influence on the bi-directional transportion. In addition, each alkaloid in single compounds had much smaller absorptive speed from apical side to basolateral side than that in mixed compounds and extract, and the absorptive speed in extract was much bigger than that in mixed compounds. Their differences of excretive speeds from basolateral side to apical side were the opposite. The results showed that three diester-type alkaloids with similar chemical structures promoted absorptive transportion, and inhibited excretive transportion each other, which was more obvious in aconite extract. As a result, the absorptive quantities of each alkaloid had been enhanced apparently.In order to clarify the transportive mechanism of three alkaloids and the mechanism of their interaction in the process of intestinal absorption, we also studied P-glycoprotein -mediated (P-gp-mediated) effect on their excretive transportion and their own inhibitory actions on P-gp-mediated efflux. Verapamil was used as the inhibitor of membrane transporter P-gp and digoxin as its substrate. P-gp inhibitors were added in both apical side and basolateral side, and the initial concentration of digoxin and three alkaloids were 35μmol/L respectively. The results showed that Papp(B→A) values of digoxin when adding aconitine, mesaconitine, hypaconitine and verapamil or not were (7.24±1.46)×10-6 cm·s-1, (10.17±1.35)×10-6 cm·s-1, (6.21±1.30)×10-6 cm·s-1, (3.80±0.99)×10-6 cm·s-1, (15.85±1.12)×10-6 cm·s-1 respectively; The Papp(B→A) values of aconitine while adding verapamil or not were (23.44±1.90)×10-6 cm·s-1 and (18.68±2.17)×10-6 cm·s-1 respectively, and their intracellular accumulation rates were (0.42±0.09)‰and (1.54±0.20)‰respectively; Similarly, the Papp(B→A) values of mesaconitine while adding verapamil or not were (14.43±1.36)×10-6 cm·s-1 and (24.94±2.21)×10-6 cm·s-1 respectively, and their intracellular accumulation rates were (0.15±0.01)‰and (0.56±0.02)‰respectively; The Papp(B→A) values of hypaconitine while adding verapamil or not were (26.86±1.84)×10-6 cm·s-1 and (29.12±2.22)×10-6 cm·s-1 respectively.The experimental results above showed that three diester-type alkaloids inhibited the excretive transportion of digoxin from basolateral side to apical side significantly, and the effect of hypaconitine was the strongest, although their inhibitory actions were inferior to that of verapamil. At the same concentration, the Papp(B→A) value of mesaconitine and hypaconitine decreased while adding verapamil, but that of aconitine increased slightly. However, cellular accumulation rates of both aconitine and mesaconitine were reduced significantly. This result prompted that three diester-type alkaloids may not only be P-gp substrates but also its inhibitors, so they enhanced absorptive transportion and inhibited excretive transportion each other when co-administration. As a result, they showed synergies during the course of absorption. This effect showed more stronger in such a more complex system as extract of Chinese medicine, which elucidated additional components also played an important role on absorption of three alkaloid, thus their bioavailability were improved enormously. Meanwhile, in the clinical treatment, it also hinted us that toxicity may be increased due to synergitic absorption of multi-components in toxic traditional medicine. |
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