| Objective:Erzhi pill is a classical tradition oral preparation,consisting of two traditional Chinese medicines,Ligustri Lucidi Fructus and Ecliptae Herba,has the functions of invigorating liver and kidney,nourishing Yin and hemostasis,used to treat menopause syndrome,seborrheic alopecia,vitiligo,subacute thyroiditis,nephrotic syndrome and other diseases.Biopharmaceutics is a process to study the absorption,distribution,metabolism and excretion of drugs and their dosage forms in vivo,bioavailability is the key study index of absorption,good bioavailability is one of the important conditions for drug development,improve drug bioavailability by change dosage forms is an important content for the investigation of biopharmaceutics,both influence each other,complement each other.One of the purpose for formulation design is to change the drug's permeability,which affects the absorption rate and bioavailability.It is a major factor that determines whether the drug has development potential and application prospect.Among the various permeability research models,PAMPA model has the advantages of high-throughput,low cost,convenient detection means and high flexibility.It is more suitable for the permeability study of Chinese traditional medicine with multiple components,and gets the advantages of simple operation,high repeatability and accuracy.In this study,we first tested several chemical substances in Erzhi Pills of separate batches,and used chemometric models for quality analysis.Second,the in vitro permeability PAMPA model was established and optimized,to screen and test permeable components in Erzhi Pills.Finally,we discussed the effect mechanism of the traditional water honey pill preparation technology for its oral bioavailability,and to provide some ideas for the compound preparation study about traditional Chinese medicine.Methods:Using UPLC method to detect the multi-components in the powder samples with multiple batches Erzhi Pill,combined with PLS-DA,HCA and other models in stoichiometry to summarize the content detection results,and select index components.PAMPA was established with egg lecithin and dodecane as film components,and the membrane integrity was investigated with fluorescein sodium leak detection,we optimize the key factors in the model,such as the content of cosolvent and incubation time,and use bilaterally positive drugs to verifie the model permeable.Based on PAMPA model,we screen the permeable components in monomer and Erzhi Pill preparation,and investigate the mechanism of preparation accessories and compatible components on the permeability.Combining the biopharmaceutical classification method to explore the effect of Erzhiwan traditional water honey pill preparation technology on oral bioavailability,further develop the in vitro evaluation method of traditional Chinese medicine preparation.Results:In the quality evaluation study of Erzhi Pills,we found that there were some otherness in the different components of different regions,the results showed that the content of specnuezhenide in each batch was higher than that of 4.0 mg·g-1in China Pharmacopeia,but the content fluctuated greatly among batches.HCA analysis identified compatible samples with similar climate and rainfall as one category.PLS-DA model R2X=0.966,Q2=0.415,permutation test the R2=0.261,Q2=0.587,the model will be anhui,henan,hunan,jiangsu,sichuan five kinds of producing area of Ligustri Lucidi Fructus compatibility sample separation,analyzing the composition of get VIP value is greater than 1 have nuezhenidic acid,specnuezhenide,nuezhenoside G13,oleoside-11-methyl ester,echinacoside,illustrate the five components of Erzhi Pills had a greater influence on the quality of research,can be used as Erzhi Pills index ingredients.Establish the PAMPA model and inspect its integrity,the results show that the apparent permeability coefficient of fluorescein sodium in 4h is 0.056×10-6,which is far lower than the standard line of 0.6×10-6,indicating that the artificial membrane constructed in this experiment is complete and without leakage.The content of the cosolvent DMSO was determined to be 5%,and the incubation time of the drug was 16 h.The PAMPA model was tested bilaterally by using the high permeation drug propranolol and the low permeation drug furosemide as positive control drugs.The results showed that propranolol lg Pe=-3.58,belonging to the high permeation range,and furosemide lg Pe=-7.51,belonging to the low permeation range,the results of bilateral positive drug penetration attribute division is accurate.Using the established PAMPA model to study the permeability of Erzhi Pills,it was found that tyrosol,salidroside,nuezhenidic acid,oleoside-11-methyl ester,echinacoside,isoquercitrin,isochlorogenic acid A,verbascoside,specnuezhenide,wedelolactone and nuezhenoside G13 have 11 kinds of monomer composition,and only two kinds can pass through,which are tyrosol and specnuezhenide.There are three permeable components in Erzhi Pills preparation,namely,tyrosol,specnuezhenide and nuezhenoside G13.As for the composition of tyrosol,the monomer lg Pe=-5.08 is a permeable medium component,and the average lg Pe=-4.47 in pharmaceutical formulations is a moderate permeability component.The composition of specnuezhenide,the monomer lg Pe=-6.80,and the average lg Pe=-6.41in pharmaceutical formulations,all was low-permeability components.Nuezhenoside G13 the monomer was not permeable,and the average lg Pe=-6.41 in pharmaceutical formulations,which was a low-permeability component.In this experiment,we used the PAMPA model to further investigate the influence of Erzhi Pills water-honey pills preparation technology such as auxiliary materials and the prescription formulation on the components permeability.In the study of excipients,four kinds of honey were investigated,chaste honey,acacia honey,medlar honey and jujube honey The degree of honey refining is divided into three grades:tender,medium and old honey.In the research of prescription formulation,the lyophilized Ecliptae Herba was extracted,and combined with the Ligustri Lucidi Fructus powder.Both groups used the honey-free powder group as a control,detect the permeability coefficient,content of 3 permeable components and p H change.The results showed that the components of tyrosol lg Pe were-4.52±0.20,-2.83,-4.32 in the honey addition,extraction from Ecliptae Herba and non-honey powder group,respectively,the components of specnuezhenide lg Pe were-6.9±0.13,-4.96,-7.33,and the nuezhenoside G13 lg Pe were-7.10±0.09,-6.46,-7.54.The p H of the solution was6.05,5.30 and 6.06.The content of tyrosol in the solution was 4.19±0.15?g·m L-1,2.06?g·m L-1,4.27?g·m L-1,specnuezhenide was 284.29±4.28?g·m L-1,296.54?g·m L-1,265.00?g·m L-1,nuezhenoside G13 was 74.51±1.56?g·m L-1,82.93?g·m L-1,68.87?g·m L-1.Through further investigation,it was found that the chemical composition changes of Ecliptae Herba after extraction compared with the powder,which provided scientific basis for the Ecliptae Herba water extraction.Based on the equilibrium solubility and permeability coefficient to study BCS classification research.In monomer,market pills,WM-water decoction,the BCS of tyrosol is classified as?,?and?,the nuezhenide is classified as?,?and?,and the nuezhenide G13 is classified as?.The constituents classification results in preparations are better than monomer,indicating that the quality control can improve the permeability coefficient and change the BCS classification.After the Ecliptae Herba decoction with water,the components of nuezhenide was changed from BCS?to?,indicating that Ecliptae Herba extraction is the key preparation process factor to change the permeability coefficient.Conclusion:The quality of 25 batches of Erzhi Pills were in accordance with the provisions of Chinese pharmacopoeia,but the content of components fluctuated greatly among the samples,indicating that the quality of traditional Chinese medicine was different in different areas.HCA clustering analysis showed that the climatic type and rainfall in the region of Ligustri Lucidi Fructus had great influence on the clustering results.Therefore,in the quality evaluation of Erzhi Pills,the place of origin,climate type and rainfall of Ligustri Lucidi Fructus have certain evaluation value.The PLS-DA model can effectively identify Ligustri Lucidi Fructus from 5 producing areas.The components with VIP value greater than1 are all chemical components in Ligustri Lucidi Fructus,indicating that the quality fluctuation of Erzhi Pills between different batches are related to Ligustri Lucidi Fructus.The permeability of the PAMPA model was verified by using fluorescein sodium,propranolol and furosemide.The results showed that the parallel artificial membrane established in this experiment was complete without leakage and had good permeability,the PAMPA model was successfully established.Based on the PAMPA model,only two of the components?specnuezhenide and nuezhenoside G13?of tyrosol,specnuezhenide,nuezhenoside G13 are consistent with the objectively selected index components of stoichiometry,indicating bioavailability research plays an important role in the quality evaluation of traditional Chinese medicine.It was found that the honey of different types and different refining degree had no obvious effect on the composition permeability,liquid p H and ingredient content.After extraction,the chemical composition was changed,therefore,lower solution p H value,higher composition permeability,the difference in the permeability of the three components in Erzhi Pills between the monomer and the preparation may be related to the extraction of Ecliptae Herba.Represented by the ingredients tyrosol,specnuezhenide,nuezhenoside G13,based on the BCS classification system,it was found that permeability is the influencing factor of Erzhi Pills oral bioavailability,and in the preparation process of Erzhi Pills,Ecliptae Herba extraction process can improve the composition permeability,further improve the Erzhi Pills oral bioavailability. |
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