| Objective:The biopharmaceutics classification system(BCS)is a scientific method to classify drugs based on their water solubility and intestinal permeability.The BCS attributes of drugs can predict their pharmacokinetic properties in vivo and establish the correlation between in vitro and in vivo.And using this as a basis to guide early drug design,new drug development and generic drug consistency evaluation,has become an important tool in each stage of chemical drug basic research and applied drug development.However,unlike the single-component environment of chemical medicine,traditional Chinese medicine(TCM)has the characteristics of multiple components,and the interaction between the components is likely to affect the water solubility and intestinal permeability of the active ingredients,thereby affecting the efficacy of the medicine.Therefore,referring to the theoretical basis of BCS and establishing the biopharmaceutics classification system of Chinese materia medica(CMMBCS)with absorption as the core,it can accurately evaluate the water solubility and intestinal permeability of the active ingredients and the influence mechanism in the compound environment.All of those are helpful to actually guide the formulation design and improvement of proprietary Chinese medicine preparations,simplify the evaluation of the consistency of the efficacy of new Chinese medicines and generic drugs,and improve the development and utilization of preparations.At present,there are still two research bottlenecks in the research of CMMBCS:One is that TCM has the characteristics of multiple ingredients and multiple pharmacological effects,how to accurately screen the active ingredients that can represent the efficacy of the compound prescription;the second is that how to find the change law and the mechanism of the CMMBCS properties of the active ingredient under the several-compound environment.In view of the fact that clinical efficacy is the only indicator for evaluating the effectiveness of TCM,this study starts from the efficacy of TCM compound prescriptions,and establishes a "screeningverification" combined active ingredient research model based on the efficacy of the compound prescriptions,in order to obtain the active ingredients.Then continue to carry out the CMMBCS research,preliminary analysis and discussion of the active ingredients’ solubility and permeability changes and influencing factors in different environments.In order to follow the research ideas from simple to complex,this study initially selected Qishen Yiqi dripping pills(QSYQ)with clear clinical efficacy as the research carrierMethods:Firstly,collected the blood components of QSYQ through literature and built a "component target-disease protein" network based on network pharmacology in order to obtain the key targets and characteristic pathways of the compound’s anti-myocardial ischemia effect,and use this as a basis to obtain potential active ingredients against myocardial ischemia.Secondly,established quantitative detection and analysis methods for the potential active ingredients of QS YQ to obtain content relationship between ingredients in the compound.Established H9C2 cardiomyocyte oxygen-glucose deprivation model(OGD).Taking cell viability as the active index,as well as comprehensively considering potential active ingredients’ structural characteristics,the frequency of literature research,content and pharmacopoeia records to screen out representative active ingredients that can represent the compound.Of course,it cannot be ignored to use cell viability,LDH,CK as the efficacy indicators to comprehensively verify whether the mixed medicinal solution is representative of the compound efficacy.Measured the solubility and effective permeability coefficient of representative active ingredients of QSYQ in different environments and different media by the classic shake flask method and the single-pass intestine perfusionin(SPIP).Analyzed their solubility and permeability changes in different environments,and discussed the main influencing factors of the changes.Results:(1)A total of 18 blood components from QSYQ were collected through literature,and 407 component targets and 672 disease proteins were collected through databases such as TCMSP,Swiss target prediction and OMIM.Using String database and cytoscape software to construct a "component target-disease protein" PPI network,and then 82 characteristic targets were obtained by the screening conditions of Degree,Betweenness,and Closeness.The results of PPI network indicated that QSYQ could regulate various biological functions to protect cardiomyocytes,including nitric oxide biosynthesis,apoptosis,inflammation,and angiogenesis.Additional,ALB,TNF,TP53,AKT1 and VEGFA were the key targets.Using the David database to re-enrich the KEGG pathway of single herbs,it was found that the hypoxic stress pathway HIF-1 signaling pathway was the common signaling pathway of Salviae miltiorrhizae Radix et Rhizoma.Astragali radix,and Notoginseng Radix et Rhizoma.which can be quickly activated under hypoxia to protect cardiomyocytes.Finally,based on the key targets of the"component target-disease protein" PPI network and the regulation targets of the characteristic pathway HIF-1 signaling pathway,it was preliminarily confirmed that the potential active ingredients of QSYQ were astragaloside Ⅳ.calycosin,formononetin,apterocarpan.salvianic acid A.rosmarinic acid,protocatechualdehyde.protocatechuic acid,cryptotanshinone.tanshinone ⅡA.notoginsenoside R1,ginsenoside Rg1,ginsenoside Rbl,trans-nerolidol.(2)Quantitative detection and analysis methods for the 12 potential active ingredients of QSYQ were established to detcect the content of 11 batches of QSYQ from different batches of Tasly Group(apterocarpan and cryptotanshinone were not detcected).The experimental results showed that astragaloside Ⅳ,salvianic acid A.ginsenoside Rg1,ginsenoside Rb1 and trans-nerolidol were high-content ingredients(x>1 mg),while protocatechualdehyde.formononetin,tanshinone ⅡA were micro-ingredients(x<0.05 mg).The content relationship was as follows.salvianic acid A:protocatechuic acid:protocatechualdehyde:rosmarinic acid:calycosin:formononetin:notoginsenoside R1:ginsenoside Rg1:ginsenoside Rbl:astragalosideⅣ:tanshinone ⅡA:trans-nerolidol=18:0.15:3:1:0.25:0.1:0.8:0.9:1.5:1.2:0.03:8.(3)The H9C2 cardiomyocyte OGD model was successfully established under the conditions of 5%CO2,94.2%N2,0.8%O2 through hypoxic fluid and a three-gas incubator.Based on the cell activity results of each potential active ingredients,as well as comprehensively considering thier structural characteristics,the frequency of literature research,content and pharmacopoeia records,salvianic acid A,protocatechualdehyde.rosmarinic acid.formononetin,ginsenoside Rg1.astragaloside Ⅳ,tanshinone ⅡA and trans-nerolidol were finally screened out as the representative active ingredients to represent the whole compound.The mixed culture medium and the QSYQ culture medium of low.medium and high concentration were prepared,and the cell viability,LDH and CK were used as indicators to verify the representativeness of mixed culture medium on the overall efficacy of QSYQ.The efficacy results showed that the mixed culture medium and the QSYQ culture medium at the same concentration had similar efficacy,indicating that the above eight ingredients were representative of the overall efficacy of QSYQ and could be used as representative active ingredients for subsequent the biopharmaceutics classification system of Chinese materia medica research.(4)The dissolving tendency analyzed and compared by the angle cosine method under singlecomponent environment and several-compound environment indicated that the dissolving tendency of salvianic acid A,protocatechualdehyde,rosmarinic acid,formononetin,ginsenoside Rg1,astragaloside Ⅳ,tanshinone ⅡA and trans-nerolidol performed better similarity under several-compound environment.In addition,the results showed that formononetin,ginsenoside Rg1,astragaloside Ⅳ,tanshinone ⅡA and trans-nerolidol under compound environment had higher solubility.Among them,the solubility properties of formononetin and trans-nerolidol changed from low solubility to high solubility,and the saponins of notoginseng exist in the compound environment may be the main factor for this change.(5)The SPIP method was used to obtain the absorption parameters of 8 representative active ingredients under different drug concentrations at low,medium,and high.The results of permeability experiments showed that there was no significant difference in the absorption parameters Peff and Ka of protocatechualdehyde,formononetin,ginsenoside Rg1 and astragaloside Ⅳ at different concentrations.There is a positive correlation between absorption and concentration,indicating that their absorption mechanism is passive diffusion.The experimental results also showed that there was a non-linear negative correlation between the absorption parameters,absorption amount and concentration of salvianic acid A and rosmarinic acid,indicating that their absorption has its own concentration-inhibitory effect.The absorption parameters of tanshinone ⅡA and trans-nerolidol were lower at the high concentration than its at low,indicating that the absorption of the ingredients in the intestine was saturated,and the absorption mechanism of them was passive diffusion,also there may be carrier-mediated facilitated diffusion or active transshipment.This study also compared the absorption parameters of 8 representative active ingredients at high concentrations under singlecomponent environment and compound environment.It was found that there was no significant difference in the absorption parameters of salvianic acid A,formononetin and astragaloside Ⅳ,while the absorption parameters of rosmarinic acid,ginsenoside Rg1,and trans-nerolidol under compound environment had been significantly improved.Trans-nerolidol was the main factor that improved the permeability of rosmarinic acid and ginsenoside Rg1,and its penetration enhancement effect might be non-specific.Conclutions:(1)This study was pharmacodynamic-oriented and integrated a variety of research methods,such as network pharmacology,liquid quantitative analysis,and glucose and oxygen deprivation pharmacological model,to establish a "screening-verification" combined active ingredient research model.It was preliminarily determined that the active ingredients of QSYQ againsted myocardial ischemia were salvianic acid A,protocatechualdehyde,rosmarinic acid,formononetin,ginsenoside Rg1,astragaloside Ⅳ,tanshinone ⅡA and trans-nerolidol.(2)The "multi-level difference comparison method" was used to carry out the CMMBCS study of the active ingredients of QSYQ,it was found that Salvianic acid A and rosmarinic acid belonged to CMMBCS Ⅲ,while others belonged to CMMBCS Ⅰ.(3)The notoginsenosides in the compound could increase the solubility of formononetin,astragaloside Ⅳ,tanshinone ⅡA and trans-nerolidol,while the excipients ccould only increase the solubility of formononetin.Trans-nerolidol could improve the permeability of rosmarinic acid and ginsenoside Rg1,while ginsenoside Rg1 decreased the permeability of rosmarinic acid and tanshinone ⅡA. |
PDF Full Text Request