| Objective:Dingzhi pill is a famous formula which consists of four herbs made in accordance with the ratio(3:3:2:2), namely ginseng, polygala, hoelen and Acorus gramineus, initially recorded in the Chinese ancient book Bei-Ji-Qian-Jin-Yao-Fang compiled by Sun Si-Mao. The reasonable formula has been clinically used for the treatment of Alzheimer’s disease up to now. Ginsenosides of the main medicinal material in ginseng acting as “monarch herb” in Dingzhi pills has the low oral bioavailability, hard to be absorbed through the gastrointestinal tract. Acorus tatarinowii Schott, as “adjuvant drug” in Dingzhi pills, has the effects of resolving dampness, having appetite and inducing medicine upward. This paper proposes to explore the promotion of saponin constituents in the intestinal absorption whether depend on the compatibility of Acorus tatarinowii Schott and provide scientific basis for clinical medication.Method:1. To research on the changes of pharmacokinetic parameters about AUC, Cmax,Tmax of ginsenosides Rg1, Re, Rb1 after oral administration of Dingzhi pills with or without Acorus tatarinowii Schott to rats with a view to exploring whether Acorus tatarinowii Schott enhances the bioavailability of ginsenosides.2. To investigate the influence of intestinal absorption on ginsenosides Rg1, Re,Rb1 by the compatibility of Acorus tatarinowii Schott and its volatile oil using in situ single-pass intestinal perfusion model in rats and to clarify the absorption characteristics of ginsenosides in the various intestinal segments by comparing Ka and Papp.3. To investigate the influence of Q, Ka and Papp on ginsenosides Rg1, Re and Rb1 by the compatibility of Acorus tatarinowii Schott and its volatile oil using in vitro everted gut sac model in rats and to explore the mechanism of action of volatile oil ofAcorus tatarinowii Schott promoting the intestinal absorption of saponin constituents whether due to rely on inhibiting the exocytosis effect of P-g P.Results:1. Pharmacokinetic model was established and UHPLC-ESI-MS/MS method was used to detect the changes of pharmacokinetic parameters about AUC, Cmax, Tmax of ginsenosides Rg1,Re, Rb1 after oral administration of Dingzhi pills with or without Acorus tatarinowii Schott to rats. The results showed that AUC0-t, AUC0-∞ and Cmax of ginsenosides Rg1, Re and Rb1 in Dingzhi pills were 1.91, 1.67, 2.03 and 1.93, 1.25,1.92 and 3.81, 4.22, 1.46 times of them in the formula without Acorus tatarinowii Schott, respectively. Tmax was no significant difference between Dingzhi pills with or without Acorus tatarinowii Schott. It shows that Acorus tatarinowii Schott cuold significantly promote the absorption of ginsenosides Rg1, Re and Rb1 and enchance their bioavailability.2. In situ single-pass intestinal perfusion model was established and UHPLCESI-MS/MS method was used to investigate the effect on Ka and Papp of ginsenosides Rg1, Re and Rb1 in the various intestinal segments by the compatibility of Acorus tatarinowii Schott and its volatile oil. The results showed that the Ka and Papp of ginsenosides Rg1, Re and Rb1 in the various intestinal segments of rats after administration of extract Ⅰ, Ⅱ and Ⅲ were(0.022~0.085),(0.024~0.113),(0.144~0.535) h-1 and(0.050~0.191),(0.054~0.258),(0.324~1.221) ×10-3 cm·min-1,respectively, which indicated that the three ginsenosides were difficult to be absorbed in extract Ⅰ and Ⅱ but well absorbed in extract Ⅲ. The Ka of ginsenosides Rg1, Re and Rb1 in the various intestinal segments after administration of extract Ⅲ were(3.97~8.35) times of extractⅠ, which indicated that volatile oil of Acorus tatarinowii Schott was the primary efficacy material for promoting the intestinal absorption of saponin constituents.The Ka of ginsenosides Rg1, Re and Rb1 in duodenum, jejunum, ileum and colon of rats after administration were(0.038~0.518),(0.077~0.535),(0.059~0.353) and(0.022~0.229) h-1, respectively, which indicated that the best intestinal segment of absorption was jejunum.3. In vitro everted gut sac model was established by jejunum, which was the best intestinal segment of absorption in the in situ single-pass intestinal perfusion model.Jejunum tested by glucose kit and LDH kit had definite feasibility and integrity in 120 min.UHPLC-ESI- MS/MS method was used to research the effect on Q, Ka and Papp of ginsenosides Rg1, Re and Rb1 in jejunum by the compatibility of Acorus tatarinowii Schott and its volatile oil. The results showed that the Q-t curve of the three ginsenosides after administration were all showed upward trend in 120 min. The Q of ginsenosides Rg1, Re and Rb1 after administration of extract Ⅲ with or without verapamil were both significantly improved compared with extract Ⅰ and Ⅱ, which indicated that volatile oil of Acorus tatarinowii Schott and verapamil were both promoted the intestinal absorption of saponin constituents. The Ka and Papp of ginsenosides Rg1, Re and Rb1 in jejunum of rats after administration of extract Ⅰ, Ⅱand Ⅲ were(0.377~0.450),(0.523~0.647),(1.338~1.884) ng·min-1·cm-2 and(4.870~8.560),(9.742~12.499),(23.144~26.434) ×10-3 cm·h-1, respectively, which indicated that the three ginsenosides were difficult to be absorbed in extract Ⅰ and Ⅱbut well absorbed in extract Ⅲ. The Ka of ginsenosides Rg1, Re and Rb1 in jejunum of rats after administration of extract Ⅱ and Ⅲ were(1.20~1.47) and(3.09~4.76) times of extractⅠ, respectively, which indicated that volatile oil of Acorus tatarinowii Schott was the primary efficacy material for promoting the intestinal absorption of saponin constituents, the same as in situ single-pass intestinal perfusion model.The Ka and Papp of ginsenosides Rg1, Re and Rb1 in jejunum of rats after administration of extract Ⅱ with low, medium and high dose of volatile oil of Acorus tatarinowii Schott were(1.325~1.876),(1.338~1.884),(1.340~1.887) ng·min-1·cm-2and(23.035~26.177),(23.144~26.434),(23.127~26.467) ×10-3 cm·h-1, respectively,which indicated that volatile oil of Acorus tatarinowii Schott had no significant effect on the ginsenosides absorption by changing the dosage.The Ka and Papp of ginsenosides Rg1, Re and Rb1 in jejunum of rats after administration of extract Ⅲ with verapamil, as one kind of P-g P inhibitors, were(1.347~1.886) ng·min-1·cm-2 and(23.290~26.612) ×10-3 cm·h-1, respectively, which had significantly improved compared with extract Ⅰ and Ⅱ, but had no significant difference compared with extract Ⅲ, which indicated that the mechanism of action of volatile oil of Acorus tatarinowii Schott may be related to inhibiting the the exocytosis effect of P-g P. In order to in-depth study the mechanism of action of Acorus tatarinowii Schott, UPLC-FLR detector was used to evaluate the absorption of Rho-B acting as one kind of P-g P substrates in vitro models of rats. The results showed that the Ka of Rho-B in jejunum of rats after administration of verapamil,volatile oil of Acorus tatarinowii Schott and the mixture of verapamil and volatile oil were 1.34, 1.66 and 1.73 times of the control group, respectively, which indicated that volatile oil of Acorus tatarinowii Schott and verapamil were both promoted the intestinal absorption of Rho-B. The volatile oil group had significantly improved compared with verapamil group, which indicated that volatile oil of Acorus tatarinowii Schott promoting the intestinal absorption was not only related to inhibiting the the exocytosis effect of P-g P, but also related to the other mechanisms of action still under investigation. But the volatile oil group had no significant difference compared with the mixture, may be related to the saturability of P-g P binding site in the intestinal tract.Conclusion:In this paper, the effective saponin constituents in Dingzhi pills could be promoted by the compatibility of Acorus tatarinowii Schott in the intestinal absorption, inducing medicine upward.In the pharmacokinetic model, Acorus tatarinowii Schott cuold significantly promote the AUC0-t, AUC0-∞, Cmax of ginsenosides Rg1, Re and Rb1 and then enchance their bioavailability.In the in situ single-pass intestinal perfusion model, volatile oil of Acorus tatarinowii Schott was the primary efficacy material for promoting the intestinal absorption of saponin constituents and the best intestinal segment of absorption was jejunum by comparing the Ka and Papp of ginsenosides Rg1, Re and Rb1 in the various intestinal segments.In the in vitro everted gut sac model, jejunum, the best intestinal segment of absorption in the in situ single-pass intestinal perfusion model, was used to establish the model. Volatile oil of Acorus tatarinowii Schott was the primary efficacy material for promoting the intestinal absorption of saponin constituents by comparing the Q,Ka and Papp of ginsenosides Rg1, Re and Rb1 in jejunum, the same as in situ single-pass intestinal perfusion model. Volatile oil of Acorus tatarinowii Schott had no significant effect on the ginsenosides absorption by changing the dosage. In order to in-depth study the mechanism of action of Acorus tatarinowii Schott, Rho-B, as one kind of P-g P substrates, was detected by UPLC-FLR. Volatile oil of Acorus tatarinowii Schott and verapamil were both promoted the intestinal absorption of Rho-B after administration. The volatile oil group had significantly improved compared with verapamil group, which indicated that volatile oil of Acorus tatarinowii Schott promoting the intestinal absorption was not only related to inhibiting the the exocytosis effect of P-g P, but also related to the other mechanisms of action still under investigation. But the volatile oil group had no significant difference compared with the mixture, may be related to the saturability of P-g P binding site in the intestinal tract. |
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