Study On The Evaluation System Of Bio Availability For Chinese Medicine Orally And Its Application To Pharmaceutics Based On Flos Tonicerae-Fructus Forsythiae Herb Couples

It is difficult for us to evaluatethe system of bio-pharmaceutics for Chinese medicine preparations owning to their multi-components resulted in multi-targets and multi-acitivities. Preparation formulation designation and technologies applications are also difficult. How to establish the quantifiable evaluation system of bio-pharmaceutics and the suitable pharmaceutical technologies are not only the basic scientific problem of Chinese medicine preparations, but also the key to traditional Chinese medicine (TCM) modernization.The establishment of evaluation system of bio-pharmaceutics for Chinese medicine preparations exists some problems below:Firstly, it is difficult to identify the effective ingredients from TCM; Secondly, it is difficult to elucidate the ADME of multi-components in vivo and the mechanism of mutual interaction, which results in the difficulty of evaluation system of bio-pharmaceutics establishment; Thirdly, the complexity of physicochemical properties between active ingredients and associated consistuents leads to difficulties of Chinese medicine preparations formulation designation and technologies application. The problems about how to identify the active components, how to establish the evaluation system of bio-pharmaceutics and how to improve the integrated bioavailability of active ingredients using pharmaceutical technologies need to be further investigated.Therefore,"Flos Lonicerae-Fructus Forsythiae herb couple" was selected as model drug. The creative theories to identify the chemical markers were provided including①"Identify the active components in Flos Lonicerae-Fructus Forsythiae herb couple based on Drug-drug interaction combined with ΣPK-PD",②"Evidence the effective compounds in Flos Lonicerae-Fructus Forsythiae herb couple based on the efficacy enlarged". Besides, the idea about "Study the integrated pharmacokinetics-Integrated AUC based on the anti-viral activity-IC50" was performed to evaluate the bioavailability of "Flos Lonicerae-Fructus Forsythiae herb couple". In addition, absorption enhancers was performed to improve the integrated AUC to enhance the anti-viral effect based on the theory about "Regulate the anti-viral activity of commercially available Flos Lonicerae-Fructus Forsythiae herb couple preparations (Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, Qing-Re-Jie-Du oral liquid) based on Integrated AUC"On the basis of the theory "Study on the ingredients in vitro and in vivo in Flos Lonicerae-Fructus Forsythiae herb couple qualitatively and quantitatively based on UHPLC-LTQ-Orbitrap-MS combined with UPLC-ESI-MS/MS", we found that35components in virto in Flos Lonicerae-Fructus Forsythiae herb couple were identified using UHPLC-LTQ-Orbitrap, including7phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, caffeic acid and quinic acid),5phenylethanoid glycosides (isoforsythoside, forsythoside A, forsythoside B, forsythoside D and forsythoside E),7flavones (rutin, luteolin, astragalin, hyperoside, isoquercitrin, quercetin and luteoloside),2isoflavones (genistin and genistein),9lignans (arctiin, phillyrin, pinoresinol-β-D-glucoside, epipinoresinol-β-D-glucoside, pinoresinol monomethyether-β-D-glucoside, epipinoresinol, pinoresinol, arctigenin and phillygenin),2saponins (dipsacoside B and macranthoidin B),3iridoids (loganin, sweroside and centauroside). Based on the obtained reference standards and literatures, a rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed for the simultaneous determination of forsythoside B, loganin, macranthoidin B, dipsacoside B, rutin, arctiin, phillyrin, pinoresinol-β-D-glucoside,3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, isoquercitrin, hyperoside, astragalin, luteoloside, genistin, arctigenin, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, quercetin, luteolin, genistein, quinic acid, caffeic acid, isoforsythoside and forsythoside A. Besides, we also found that32components in vivo in Flos Lonicerae-Fructus Forsythiae herb couple were identified using UHPLC-LTQ-Orbitrap-MS, including29prototype compounds, containing7phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, caffeic acid and quinic acid),5phenylethanoid glycosides (isoforsythoside, forsythoside A, forsythoside B, forsythoside D and forsythoside E),7flavones (rutin, luteolin, astragalin, hyperoside, isoquercitrin, quercetin and luteoloside),2isoflavones (genistin and genistein),7gnans (phillyrin, pinoresinol-β-D-glucoside, epipinoresinol-β-D-glucoside, epipinoresinol, pinoresinol, arctigenin and phillygenin),1iridoids (loganin). Besides,3metabolites (pinoresinol-o-glucuronide, epipinoresinol-o-glucuronide and phillygenin-o-glucuronide) identified by the Met Works software from Flos Lonicerae-Fructus Forsythiae herb couple were also discovered. On the basis of hypothesis "the constituents absorbed into blood might be the effective ingredients in TCM",29compounds screened from blood could be considered as maker compounds in Flos Lonicerae-Fructus Forsythiae herb couple. But based on the obtained reference standards and literatures,23ingredients in vivo could be treated as marker compounds for bio-pharmaceutics in vivo, including7phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid,3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, caffeic acid and quinic acid),3phenylethanoid glycosides (isoforsythoside, forsythoside A and forsythoside B),7flavones (rutin, luteolin, astragalin, hyperoside, isoquercitrin, quercetin and luteoloside),2isoflavones (genistin and genistein),2lignans (phillyrin, pinoresinol-β-D-glucoside and arctigenin),1iridoids (loganin).On the basis of theory about Identify the chemical markers in Flos Lonicerae-Fructus Forsythiae herb couple based on Drug-drug interaction (DDI) combined with ΣPK-PD", DDI in vitro, DDI in situ and DDI in vivo were simultaneously performed to study the difference after or before compatibility. The result showed that isoforsythoside,3,5-dicaffeoylquinic acid, neochlorogenic acid,3,4-dicaffeoylquinic acid and forsythoside A were the main ingredients resulted in difference between herb couple and single herb. Besides,3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, isoforsythoside, forsythoside B and forsythoside A were identified by^PK-PD combined with PLS analysis, and the ingredients of excellent correlation were3,4-dicaffeoylquinic acid in Flos Lonicerae extracts and isoforsythoside in Fructus Forsythiae extracts.On the basis of theory "Evidence the effective compounds in Flos Lonicerae-Fructus Forsythiae herb couple based on the efficacy enlarged", the observations from in vitro Caco-2cell showed that the absorption of phenylethanoid glycosides in Fructus Forsythiae extract so with that in monomers was mainly restricted by the tight junctions, and influenced by efflux transporters (P-gp and MRP2), and the absorption of phenolic acids in Flos Lonicerae extract as with that in monomers was mainly restricted by the tight junctions, and influenced by efflux transporters (P-gp, MRP2, and BCRP). Besides, it was found that the permeation of phenylethanoid glycosides in Fructus Forsythiae extract in the apical-to-basolateral direction was also high than that in the basolateral-to-apical direction in the presence of middle concentration (some of them had significance) and the efflux ratios of phenylethanoid glycosides such as isoforsythoside and forsythoside B in Fructus Forsythiae extract was higher significantly than that of monomers, which might be attributed to the influence of other ingredients in Fructus Forsythiae extract on the efflux transporters (P-gp and MRP2), though the effect was weak (efflux ratios<1.0) which indicated that the permeability of phenylethanoid glycosides in Fructus Forsythiae extract so with that in monomers was mainly restricted by the tight junctions. In addition, the permeation of phenolic acids in Flos Lonicerae extracts in the apical-to-basolateral direction was also high than that in the basolateral-to-apical direction in the presence of middle concentration (some of them had significance) and the efflux ratios of phenolic acids in Flos Lonicerae extracts was lower significantly than that of monomers, which might be attributed to the influence of other ingredients in Flos Lonicerae extracts on the efflux transporters (P-gpN MRP2and BCRP), and the effect was weak (efflux ratios<1.0) which indicated that the permeability of phenolic acids in Flos Lonicerae extracts so with that in monomers was mainly restricted by the tight junctions. And effect of chito-oligosaccharide (COS) on the absorptions of caffeic acid derivatives in Flos Lonicerae and Fructus Forsythiae were also studied, and found that the intestinal absorptions of caffeic acid derivatives can be enhanced by COS, and treatment with Fructus Forsythiae extract and Flos Lonicerae extract with COS prevented MDCK cell damage upon influenza virus propagation better than that of control. All findings above suggested that caffeic acid derivatives in Flos Lonicerae-Fructus Forsythiae herb couple could be considered as chemical markers to control the antiviral activity ofFlos Lonicerae-Fructus Forsythiae herb couple.On the basis of theory "Study the integrated pharmacokinetics-Integrated AUC based on the antiviral activity-IC50", we found that the order of antiviral activity for caffeic acid derivatives were forsythoside A>forsythoside B>3,4-dicaffeoylquinic acid≈Isoforsythoside>3,5-dicaffeoylquinic acid>neochlorogenic acid>chlorogenic acid>cryptochlorogenic acid, but the bioavailability of isoforsythoside, forsythoside A and forsythoside B were lower largely than that of chlorogenic acid. In order to elucidate the reasonability of the theory about "IC-50, not AUC as weight coefficient index as integrated pharmacokinetics", the integrated AUC and antiviral effect of Flos Lonicerae-Fructus Forsythiae herb couple as control group were performed to compare with that of Flos Lonicerae-Fructus Forsythiae herb couple knocked in forsythoside A as experimental group, the result showed that the antiviral effect in experimental group was better significantly than that in control group, and the integrated A UC cauculated by IC50, not AUC as weight coefficient index was also improved significantly. In addition, we also found that the integrated pharmacokinetics parameters using IC-50as weight coefficient index, such as T1/2、AUC、Cmax and MRT calculated by DAS2.1.1could fully take eight caffeic acid derivatives’pharmacokinetics parameters into account. In short, the results indicated that the weight coefficient by IC50as index had reasonability.On the basis of the theory "Regulate the antiviral activity of commercially available Flos Lonicerae-Fructus Forsythiae herb couple preparations (Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, Qing-Re-Jie-Du oral liquid) based on Integrated AUC", we found that the permeation of caffeic acid derivatives in Flos Lonicerae-Fructus Forsythiae herb couple in the apical-to-basolateral direction was high than that in the basolateral-to-apical direction in the presence of middle concentration (some of them had significance), and the efflux ratios was weak (efflux ratios<1.0) which indicated that the permeability of caffeic acid derivatives in Flos Lonicerae-Fructus Forsythiae herb couple so with that in monomers was mainly restricted by the tight junctions. Besides, the reasonabilities of four preparations (Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, Qing-Re-Jie-Du oral liquid) selected were screened based on the three standards as follows:Firstly,26compounds in vitroreferred above in preparations need to be detected successfully by UPLC-ESI-MS/MS; Secondly, the imprortance of Flos Lonicerae-Fructus Forsythiae herb couple in preparations need to be evaluated by comparison of antiviral activity of Flos Lonicerae-Fructus Forsythiae herb coupleknocked outor knocked inin preparations. Thirdly, the main intestinal absorption barrier of caffeic acid derivatives in preparations was similar to that in Flos Lonicerae-Fructus Forsythiae herb couple. The results found that the commercially available preparations (Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, Qing-Re-Jie-Du oral liquid) could meet the requirements above. In addition, the integrated A UCof caffeic acid derivatives in the four preparations can be enhanced by COS significantly, and treatment with Flos Lonicerae-Fructus Forsythiae herb couple preparations with COS prevented MDCK cell damage upon influenza virus propagation better than that of control, but treatment with the preparations knocked out Flos Lonicerae-Fructus Forsythiae herb couplewith COS prevented MDCK cell damage upon influenza virus propagation non-significantly, compared with that of control, which all indicated that the antiviral activity improvement by COS in Flos Lonicerae-Fructus Forsythiae herb couple preparations resulted from the improvement of the integrated A UC of caffeic acid derivatives.All in all, the theory about "Study on the ingredients in vitro and in vivo in Flos Lonicerae-Fructus Forsythiae herb couple qualitatively and quantitatively based on UHPLC-LTQ-Orbitrap-MS combined with UPLC-ESI-MS/MS" as premise, the theory about "Identify the chemical markers in Flos Lonicerae-Fructus Forsythiae herb couple based on Drug-drug interaction combined withK-PD" and "evidence the effective compounds in Flos Lonicerae-Fructus Forsythiae herb couple based on the efficacy enlarged" as methods, the theory about "Study the integrated pharmacokinetics-Integrated AUC based on the anti-viral activity-IC50" as purpose, and the theory about "Regulate the anti-viral activity of commercially available Flos Lonicerae-Fructus Forsythiae herb couple preparations(Shuang-Huang-Lian oral liquid, Yin-Qiao-Jie-Du tablet, Fufang-Qin-Lan oral liquid, Qing-Re-Jie-Du oral liquid) based on Integrated AUC as application, will all exert exemplary significance for developing bio-pharmaceutics of Chinese medicine preparations.