| Objiective:This paper is on the basis of existing researches to continue with depth. Previous researches in our laboratory have successfully formulated the novel self-microemulsifying drug delivery systems (SMEDDS) of the main poorly water-soluble active ingredients including Puerarin (PUE), Baicalin (BA) and Berberine Hydrochloride (BH) which extracted from Gegen-Qinlian decoction with the natural emulsifier (NE) instead of50percent of nonionic surfactants (NS). In the present study, the absorption mechanism of PUE, BH and BA was investigated and effects on the absorption with each other were explored on various levels, including transepithelial transport experiments across Caco-2monolayers (in vitro), intestinal perfusion experiments (in situ) and oral bioavailability in rats (in vivo). In addition, the ability of improving absorption of two kinds of SMEDDS (NE-SMEDDS and NS-SMEDDS) to PUE, BH and BA and reducing the toxicity of NS-SMEDDS through replacing50percent of NS were also researched. At the same time, the absorption of BA promoted by phospholipid complex (PC) and PC coupled with SMEDDS were also investigated in this paper.Methods:(1) Constructed Caco-2cell model to study the cell monolayer’s transport characteristics of PUE, BH or BA and its SMEDDSs in vitro. Research contents included following aspects:â‘ The effects of concentration, time, P-glycoprotein inhibitors (verapamil) and its different SMEDDS (NE-SMEDDS and NS-SMEDDS) on the transfer mechanism of single ingredient(PUE, BH and BA);â‘¡the absorption mechanism of single ingredient in the combination of all the three ingredients (PUE+BH+BA) and their SMEDDS and effects on the absorption with each other;â‘¢the absorption mechanism of BA at different ratio of drug to phospholipid (1:2.5,1:3and1:4) of BA-PC and the absorption of BA in a combination of SMEDDS and BA-PC. All above were investigated through the comparison of the apparent permeability coefficients (Papp), cumulative permeate quantity (â–³Q), and drug efflux ratio (ER).(2) In vivo, constructed rat’s intestinal perfusion model to research the absorption of the small intestine of PUE, BH, BA and its SMEDDS.â‘ The study groups included PUE, BH or BA single ingredient and its SMEDDSs,(PUE+BH+BA), and (PUE+BH+BA)-SMEDDS. The main absorption site and characteristics in intestine of PUE, BH, BA were studied by comparing the absorption rate constant (Ka) and apparent permeability coefficients (Peff).â‘¡The effects on the intestine absorption of BA in BA-PC and BA-PC-SMEDDS whose drug to phospholipid were1:2.5and1:4were studied by comparing Ka and Peff, too.(3) Established rat’s mesenteric lymph transport model, to research the correlation of lymphatic transport and blood absorption of (PUE+BH+BA) and its SMEDDSs (NE-SMEDDS and NS-SMEDDS).â‘ The lymph samples were collected every other one hour after oral administration groups and totally collected12hours.â‘¡the blood samples were collected at different time point (0,0.17,0.25,0.5,0.75,1,2,4,6,8,10and12h) through eyes.â‘¢The lymph and blood samples were dissolved again with mobile phase after precipitating protein with Methyl Cyanides, and the concentration of each ingredient in samples was determined by HPLC. Then we compared the difference and correlations between blood and lymph transport results of three ingredients in SMEDDS.(4) Investigated the cytotoxicity of two kinds of SMEDDS (NE-SMEDDS and NS-SMEDDS) by MTT, and research the influence on the membrane integrity of SMEDDS through immunofluorescence and detecting transepithelial electric resistance (TEER).Results:(1) The results of the transport of PUE, BH or BAin Caco-2cell model indicated: it was difficult for PUE, BH and BA to absorb in intestine and all of them belong to passive diffusion. All of them were the potent inhibitors of P-glycoprotein, however, the efflux rate of BH was respectively4.85times and8.78times higher than PUE and BA. PUE, BH and BA interacted on each other:BH and BA were significantly enhanced by PUE, but reduced by each other, while the effect of BA was more significant; PUE was decreased by BH or BA, but the effect of BH was more significant. PUE-SMEDDS and BH-SMEDDS promoted the absorption of PUE and BH, meanwhile, the transport rate of NE-SMEDDS was faster than NS-SMEDDS and the absorption mechanism of PUE and BH were not changed. The lipid solubility and biomembrane transport volume of BA could be increased by PC, but the absorption of BA-PC gradually descended with the increase of drug-phospholipid rate. However, it was helpful to promote the dispersion of BA-PC when made into BA-PC-SMEDDS, which was more obvious with higher drug-phospholipid rate, and the absorption of BA in BA-PC-NS-SMEDDS was totally higher than BA-PC-NE-SMEDDS. Otherwise, the absorption of PUE, BH and BA in (PUE+BH+BA)-SMEDDS was simultaneously increased by SMEDDS.(2) On the basis of Caco-2cell model, the results of in situ single pass perfusion model further indicated:The absorption of PUE or BA in rat’s intestine was a first-order process with the passive diffusion mechanism. The main intestinal absorption site of PUE or BA single ingredient was in colon, while BH in the upper part of small intestine. PUE-SMEDDS, BH-SMEDDS and BA-PC-SMEDDS promoted the absorption of PUE, BH and BA in rat’s each intestinal segment, meanwhile, the main site of PUE, BH or BA signal ingredient absorbed was not changed. The similar influence on BA of BA-PC and BA-PC-SMEDDS also existed in rat in situ single pass perfusion model. And the interact effects on PUE, BH and BA each other in (PUE+BH+BA) were same as the results of Caco-2cell model, too. After PUE, BH and BA combined together, that is (PUE+BH+BA), the main intestinal absorption site of PUE, BH and BA was all in colon. When (PUE+BH+BA) was made into SMEDDS, the absorption of each ingredient was increased simultaneously, and the main absorption site of each one was still all in colon.(3) The results of the lymphatic transport of PUE+BH+BA and its SMEDDSs (NS-SMEDDS and NE-SMEDDS) were as follows:PUE, BH and BA could be absorbed both through blood and lymphatic circulation in different administration groups, while the lymphatic transport amount was poorer than blood absorption. The AUC0â†'12h (blood+lymph) of each ingredient was ranked as PUE>BA>BH(P<0.05), and it was observed that the absorption of BH was the lowest in three ingredients in both blood and lymphatic transportation. The percent rate of AUC0â†'12h(lymph)/AUC0â†'12h(blood+lymph) was ranked as PUE>BH>BA, which meant BA was absorbed mainly through blood circulation in each administration group. SMEDDS promoted the lymphatic transport of PUE, BH and BA significantly, especially to PUE. NS-SMEDDS and NE-SMEDDS enhanced PUE markedly different. PC coupled with SMEDDS increased the lymphatic transport of BA and the effect of PC-NS-SMEDDS was relatively more prominent than PC-NE-SMEDDS.(4)Through MTT, TEER detecting and immunofluorescence investigation on SMEDDSs, which including blank NS-SMEDDS, blank NE-SMEDDS,(PUE+BH+BA)NS-SMEDDS and (PUE+BH+BA)NE-SMEDDS, the results indicated that SMEDDS changed the membrane integrity and fluidity through damaging membrane tight junction and skeleton protein, which caused the increasing membrane permeability and drug transport volume. The role of membrane penetration of NS-SMEDDS containing more tween-80was greater than NE-SMEDDS.Conclusion:The intestine absorption mechanism of PUE, BH and BA was mainly passive diffusion, and all of them were the potent inhibitor of P-glycoprotein. When PUE, BH and BA combined together, interactions generated on the absorption of each other. NS-SMEDDS and NE-SMEDDS simultaneously promoted the absorption of PUE, BH and BA through opening membrane tight junction and decreasing the membrane integrity. In addition, the cytotoxicity of NE-SMEDDS was lower than NS-SMEDDS, while the integral impact of promoting those ingredients’absorption in NE-SMEDDS was poorer than NS-SMEDDS. PC coupled with SMEDDS increased the lymphatic transport of BA efficiently. |
PDF Full Text Request