Study On The Absorption And Metabolism Of Forsythoside A In Shuang-huang-lian Oral Liquid

Oral administration is the popular way of drug application (a mainly used way in drug's clinical application).The ADME of the active ingredients in traditional Chinese medicine (TCM) is the key point for TCM research; Furthermore, the absorption and metabolism of the active ingredients is not only the key factor to influence the bioavailability, but the key factor to affect the TCM action. In order to establish the basic methods for the biopharmaceutical research of TCM and to scientifically illustrate the composition theory of TCM, it is profitable to reveal the absorption and metabolism mechanism of active ingredients. Besides, the comprehension of the factors that influence absorption, metabolism, clarification of the roles, effect extent of transports and emzyme as well as the investigation of the interactions of various active ingredients are also important in the process of absorption and metabolism.Studying the ADME and the interactions of TCM, especially the active ingredients, by means of biopharmaceuticals and pharmacokinetics not only provides foundation and valuable information for the formulation screening, technique modifying, quality evaluating in addition to rational drug application, but illustrates scientificity of compounds.Shuang-Huang-Lian (SHL), a traditional Chinese formula containing Lonicerae japonicae flos (LJF), Scutellariae radix (SR) and Forsythiae fructus (FF), is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. Forsythoside A (FTA) is one of the main active ingredients in Forsythiae fructus, a key herb in SHL. In the present study, in situ gastrointestinal circulation model, in vivo pharmacokinetic model and in vitro metabolism model in Sprague-Dawley rat liver microsomes were studied to investigate the absorption and metabolism of FTA in SHL. The reasons that bioavailability of FTA in SHL was higher than that in herbs were demonstrated from absorption and metabolism perspective.In pharmacokinetics study, Male Sprague-Dawley rats were randomly assigned into four groups (A-D). The animals were fasted for 12 h, but were given access to water prior to the oral administration of the extracts prepared above with a dose of 10riig-kg-1 based on the content of FTA. After dosing for 0,10,20,30,40,55,70,100,160,250,600,1440min, blood was collected from the pre-intubated catheter and put into tubes with heparin and ascorbic acid (2μg) at predetermined time points. Subsequently, plasma was prepared by centrifugation at 5000 rpm for 7 min and stored at-80℃for LC-MS analysis. Pharmacokinetic parameters were estimated by plasma concentration versus time profiles using Pharmacokinetics Program DAS 1.0. The results showed that Cmax and AUCoâ†'1440 of FTA all increased and T1/2 prolonged in SHL, FF+ LJF and FF+SR compared with FF, which indicated that compatibility of herbs in SHL changed the ADME of FTA obviously resulting in the bioavailability of FTA improvement in SHL,The results from in situ gastro perfusion model showed that calculated A of FTA was less than 10%, and had no difference from that obtained after perfusing FTA with different concentrations in stomach, which indicated that the absorption of FTA was passive diffusion in stomach. The observations from in situ gastro perfusion model showed that A/%(h-1) of FTA in FF+LSF, FF+SR and SHL had no differences compared with that in FF, and were less than 10%.The results from in situ intestinal circulation model showed that the residue of FTA with different concentrations had little significant difference from that obtained after perfusing via duodenum, jejunum, ileum and colon, which indicated that the absorption of FTA was passive diffusion and had no difference in different segments of rat intestine. The residue of FTA increased to 466.160 and 463.429μg respectively when Cyclosporine (4μg·mL-1) or Midazolam (50μmol·L-1) was added to the circulation fluid, which showed significant difference compared to the control group (P<0.05). Moreover, the residue of FTA showed a tendency of increase with the increase of Cyclosporine or Midazolam. When Digoxin(50μmol·L-1) or EDTA(10μg·mL-1) was added to the circulation fluid, the residue of FTA decreased to 325.110 and 369.888μg respectively, which showed significant difference compared to the control group (P<0.05). Beside, the residue of FTA showed a tendency of reduction with the increase of Digoxin or EDTA. However, there is no significant change in the absorption of FTA when the different concentrations of Mannitol were added to the circulation fluid. The results above indicated that the absorption of FTA was mainly passive diffusion and involved paracellular route meanwhile. In addition, the substrates of P-gp or CYP3A4 had dose-dependent effects on the absorption of FTA. The observations from in situ intestinal circulation model showed that A/%(h-1) of FTA in FF+LSF, FF+SR and SHL were all reduced greatly compared with that in FF. Intestinal absorption of FTA with chlorogenic acid in Lonicerae japonicae flos and baicalin, baicalein in Scutellariae radix of different concentrations in situ approach illustrated that the calculated A of forsythosideA in FF showed a reduction trend with the content of chlorogenic acid in Lonicerae japonicae flos and baicalin, baicalein in Scutellariae radix increasing.The observations from Sprague-Dawley iat liver microsomes in presence ofβ-NADPH or UDPGA showed that FTA maybe the substrates of CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3, UGT1A1 and UGT1A9 respectively; Chlorogenic acid maybe the substrates of CYP3A4, CYP2C9, CYP1A2, CYP2C19, UGT1A6, UGT1A3 and UGT1A1; Baicalin maybe the substrates of CYP3A4, CYP2C19, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; Baicalein maybe the substrates of CYP3A4, CYP2E1 and UGT1A6. It was also found that the residue of FTA in SHL, FF+LJF and FF+SR were greatly increased compared with that in FF in Sprague-Dawley rat liver microsomes in presence ofβ-NADPH or UDPGA, which indicated that the metabolism of FTA in SHL may be influenced by chlorogenic acid in LJF acting on the CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3 and UGT1A1; baicalin in SR acting on the CYP3A4, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; baicalein acting on the CYP3A4 and UGT1A6 respectively.It is rarely reported to explore the absorption and metabolism mechanism of active ingredients and their interaction by multi-angle methods and multi-biopharmaceutical models. There is little literature about the preparation design of TCM under the guidance of biopharmaceutical principles. The reasons that pharmacokinetics of FTA in SHL higher than that in herb extracts from absorption and metabolism perspective were little to be reported. This study prepared for SHL pharmaceuticals secondary development and provided new approaches for the in vivo research of TCM, which enriched the content of biopharmaceutics and pharmacokinetics.