| Berberine (Ber) is an isoquinoline alkaloid, used in the clinical treatment of gastroenteritis and bacillary dysentery. Over the last decade, a large number of studies have shown that Ber can significantly reduce symptoms in patients with typeâ…¡diabetes in additional to its effects of blood glucose and liquids regulating, insulin secretion promoting and insulin resistance improving. However Ber showed excessive doses when used for lowering blood sugar. Stuctural modification of Ber and it was discovered that 8-hydroxy dihydroberberine (BerH) was significantly superior to Ber on treating diabetic rat models. And the dose of BerH group was lower than Ber group an order of magnitude. Besides, the results of the absorption of Ber and BerH in intestine which was investigated by in situ perfusion model showed that ther was a significant increase in intestinal absorption after the introduction of hydroxyl to Ber. Caco-2 cell model which has been widely used abroad is simple and reproductive when used as studing drug absorption transport operation method. Therefore, in order to clarify the intestinal absorption mechanism of BerH, this subject was performed in two-pronged approach: one is comparative study of dissolution between Ber and BerH, the other is that Caco-2 cell model was employed to compare the intestinal absorption of Ber and BerH and further understand BerH's absorption mechanism. Caco-2 cell model can be used to study proliferation of Ber and BerH, transfer of energy dependence, three main types of transporters Status and degree of intervention and its absorption. The concentration of Ber and BerH were determined by HPLC the amount of drug membrane transport, membrane transport parameters were calculated and the apparent permeability coefficient Papp and the efflux rate of ER values were calculated according to drug transmembrane transport amount and compared to find differences between the two membrane transportion.The main research was summarized as follows:1 Investigation on the dissolution of Ber and BerH According to Pharmacopoeia, pddle method was selected to study the dissolution of Ber and BerH at 37℃. Then, sampling at different time periods(15,30,45,60,90,120 min), HPLC as an analysis method was used to determine the concentrations of Ber and BerH amd dissolution curve was drawn. The results showed that the dissolution equilibrium time of Ber was 30min at 37℃, while BerH needed 90min. When reached the dissolved equilibrium, the concentration of Ber was 45μmol/L,but BerH 0.35μmol/L. This suggested the solubility of BerH was much less than Ber and BerH was extremely difficult to dissolve in water.2 Time and Concentration-dependent absorption test The absorption of Ber and BerH was investigated by Caco-2 cell monolayer transport experiments. Before the transport experiments, Caco-2 cell monolayers were washed three times with HBSS, then the plates were incubated in the transport medium at 37℃. Effects of different time periods (0.5, 1.0, 1.5, 2.0 h), different concentrations (25,50μmol/L)Ber,BerH through the cell monolayer from the APâ†'BL and BLâ†'AP direction of the concentration of the two compounds retained, AP side of the administration for the uptake and transport experiments, BL side for the outflow experiment, and HPLC as an analysis method was used to determine the concentrations of Ber and BerH. The results showed that BerH's apparent permeability coefficient of the two-direction transport (Papp) were orders of magnitude in 10-5, so we concluded that BerH was one of the good transport compounds; however, Ber's were 10-6, far less than BerH; in the APâ†'BL direction, the Papp of Ber at concentration of 25,50μmol/L were very close, slightly larger than the high concentration of low density, and Papp at anytime had no significant change; however, BerH's Papp in 25,50μmol/L vary greatly, the Papp of 50μmol/L was about three times higher than 50μmol/L group. The experimental results indicated that both Ber and BerH were concentration independent compounds and initially indentified two compounds'transmembrane transportion were non-simple diffusion process, there might be transport carriers involved in, but there are differences.3 Energy-dependent absorption test Caco-2 cell model was used to study the influences of ATP on transmembrane transport and absorption of Ber and BerH. In this part, adding ATP energy failure agent ( sodium azide and deoxyglucose), AP-side delivery, BL side of the sample, each sample was assayed by HPLC. The results showed that the apparent permeability coefficient of the experimental group was significantly different from the control group (P<0.05). Sodium azide and 2 - deoxy-D-glucose inhibited the two-way membrane transport of both Ber and BerH across Caco-2 cell monolayer which suggested that both of the two compounds needed cellular energy consumption when acrossing Caco-2 cell monolayer form the two directions. It was initially proved that ATP binding cassette family of membrane transport proteins were involved in the transmembrane transport of BerH process.4 The study on the effects of P-gp substrate verapamil and organic cation transport system inhibitors cimetidine Caco-2 cell monolayer was employed to study the influences of P-gp substrate verpamil and organic cation tranport system inhibitors cimetidine on the transportion of Ber and BerH. In this section, adding P-gp substrate verpamil and organic cation tranport system inhibitors cimetidine respectively, and administrating in both AP and BL side, the effects of transfer protein inhibitors was investigated.The results showed that the apparent permeability coefficient of the experimental group was significantly different from the control group (P<0.05).Relative to the control group, verapamil could increase the APâ†'BL direction of the apparent permeability coefficient Papp ( P<0.05) of BerH, and reduced BerH's BLâ†'AP direction of the apparent permeability coefficient Papp ( P<0.05); while verapamil only slightly increased the APâ†'BL direction of the apparent permeability coefficient Papp of Ber after 60min, furthermore, the reduction of Ber's BLâ†'AP direction of the apparent permeability coefficient Papp was not obvious, either; After adding cimetidine, the APâ†'BL direction of the apparent permeability coefficient Papp ( P>0.05) of both Ber and BerH was only slightly changed. It meaned that the transport of BerH, as well as Ber, existed P-gp transport efflux transporter protein function acrossing Caco-2 cell monolayer and both were non-dependent organic cation transporter. Generally speaking, this article determined the solubility and dissolution rate of BerH are significantly lower than Ber by a comparative study between the two dissolved in water and dissolution performance, which meaned hydrophilicity did not increase after introducing hydroxyl into Ber's structure and denied the reason of BerH's increasing absorption was the increase of dissolution. On this basis, Caco-2 cell models were used to further develop and compare the transmembrane transport of Ber and BerH. The similarities and differences between them were summarized as follows: both were non-simple diffusion, P-gp efflux involved in but non-dependent organic cation transporter. Besides, the simple diffusion coefficient of the two compounds suggested that the transport of BerH was much better than Ber, which might be the reason for the significant increasion of BerH's intestinal absorption earlier in animal models in vivo. Moreover, related to Ber, BerH was more sensitive to P-gp inhibitors. According to this, the medicinal materials which had the inhibition of P-gp drug pump function could be considered in the process of formulation development, which helped to improve the intestinal absorption of BerH, thereby enhanced its bioavailability. |
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