Study Of Promotion Absorption Methods Of Effective Components Of Fufang Danshen Preparation

Compound danshen tablet, a famous traditional medicine recipe containing Radix Salvia miltiorrhiza (Danshen in Chinese), Radix Panax notoginseng (Sanqi) and borneol (Bingpian), is widely used for improving coronary and cerebral circulation. This study is mainly focused on the lipophilic and hydrophilic components of danshen, P. Notoginseng saponins, which have been considered to be reponsible for the clinical efficacy. Among them, salvianolic acid B (SAB), tanshinone IIA (TS), ginsenoside Rb1 (Rb1), ginsenoside Rgi (Rg1)and notoginsenoside R1 (R1) are major active ingredients in the fraction, are selected as phytochemical markers for the quality control of our study. In this study, the pharmacokinetics of marker compounds after oral administration of single herb extract and different combinations of constitutional herbs in FDP were investigated, and the potential herb-herb interactions among the ingredients in the multi-herb medicine were eplored. Multiple models including in situ perfusion model in rats, Caco-2 monolayer cell model and kinetic research in vivo were employed to systemically investigate the effect to promote absorption and action mechanism of absorption enhancers, on the basis of the physical and chemical characteristics of effective components and factors that affect on theirs absorption.The TLC and HPLC methods were developed for the qualitation and quantitation assay of TS, SAB, R1, Rb1 and Rg1. The technical condition for extraction and purification of lipophilic and hydrophilic components of danshen was screened by single factor and orthogonal desigin experiment, using the content and the transfer rate of TS and SAB as index also companying the physical and chemical properties and stability of the efficient components. The tanshinone extract was obtained by supercritical carbon dioxide fluid extraction (SFE-CO2) from S. miltiorrhiza with the extraction yield of TS up to 93.4%, a content of 42.1%. Then, the residue of S. miltiorrhiza was extracted with 50% acidic ethanol (pH=2) using percolation technique to obtain SAB. The corresponding yield was 93.4% with a content of 10.6%.The percolate was concentrated and further purified on a column of D101 macroporous resin. The SAB content of the obtained phenolic acids extract was 58.4% with a transfer rate of 79.7%.A rapid, sensitive and specific UPLC-ESI-MS/MS method was developed and validated for simultaneous determination of SAB, TS, Rb1, Rg1 and R1 in rat plasma after oral administration of compound danshen tables and different combinations of its constituent herbs. The results indicated that the herb-herb interactions could be accounting for the different pharmacokinetic behaviors of active constituents administered in combination versus in single-herb extracts, however, which were not significant in most cases (p>0.05). The co-occurring components performed independent action in vivo, without competitive inhibition of the absorption, distribution, metabolism and excretion. Borneol, as potential absorption enhancer, lead to different degree of increased adsorption amount of saponins. The AUC0-72h of Rg1, Rb1 and R1 is 64.0%,31.0% and 7.7% respectively. The absorption of Rg1 was significant enhanced (p<0.05). The enhanced absorption effect of borneol is related to the molecular structure and physical chemical properties.On the basis of the physical and chemical characteristics of effective components and factors that affect theirs absorption, kinetic research in vivo were employed to systemically investigate the absorption enhancing effect of different absorption enhancers, including D-α-tocopherol polyethylene glycol 1000 succinate (TPGS), polyethylene glycol caprylic acid capric acid glycerides (Labarsol), poloxamer 188 (F68), sodium caprate (SC) and sodium desoxycholate (SDC). The difference of the enhancing effect of each absorption enhancer was much larger, and which showed a dose-dependent behavior. The promoting effects on TS was in the order of TS was TPGS> Labrasol> F68≈SDC> SC. Compared with the control group, the Cmax and AUC0-72h of TS increased by 2.5 and 2.6-fold,1.5 and 1.4-fold,1.8 and 1.3-fold,, respectively. The role of various absorption enhancers for TS was directly related to the maximum additive concentration and inhibition degree of P-gp on the intestinal epithelial cells. The promotion degree of SC for SAB, R1, Rg1 and Rb1 was most significant. With the addition of 4.0% SC, the Cmax and AUC0-72h of SAB, R1, Rg1 and Rb1 increased by 4.0 and 1.7-fold,5.6 and 2.0-fold,3.6 and 1.5-fold,1.7 and 2.0-fold compared with the control group, respectively. The significant absorption enhancement of SC for BCSIII drugs showed non-specificity and dose-dependent. Therefore SC was chosen for the enhancer of BCSIII drugs.In our study, in situ perfusion model in rat and Caco-2 monolayer cell model were selected to verify the enhancing effect of absorption enhancer, explore the action mechanism and mucosal toxicity. Tween-80 was added to avoid the effect of drug solubility and dissolution rate on the absorption of TS. Hence, it can be used directly to evaluate the influence of the enhencers on the drug permeation. The role of various absorption enhancers for TS was directly related to the inhibition degree of P-gp on the intestinal epithelial cells. Yet instinct HLB and CMC values of the enhancers were responsible for its effect intensity. Combined with the TEER values of Caco-2 cell monolayer as well as the Papp of AP-BL when co-administrated with absorption enhancers, the membrane permeability of SAB, R1, Rg1 and Rb1 were enhanced by regulation of tight junctions between epithelial cells. The regulations of tight junctions were mainly through phospholipase C-dependent pathway, Ca2+-E-cadherin pathway and tyrosine kinase-phosphatase pathways to change the tight junctions, so that it can increase the membrane permeability of the drug.