| Objective:Intestinal absorption is an important prerequisite for the efficacy of oral drugs.As an effective tool for predicting intestinal absorption of oral drugs,biopharmaceutics classification system(BCS)has been used to judge whether bioavailability limits may occur when the drug is absorbed orally by studying the core indicators affecting the druggability such as water solubility and intestinal permeability.BCS has been widely used in guiding new drug development and formulation design,predicting the in vitro and in vivo correlation and waiver of bioequivalence studies for drugs.The biopharmaceutics classification system of Chinese materia medica(CMMBCS)developed on the basis of BCS theory is based on absorption and combined with the clinical efficacy of traditional Chinese medicine(TCM).CMMBCS is a scientific framework for classifying traditional Chinese medicines by exploring the essential characteristics of Chinese herbal medicinal ingredients absorption according to excavate the solubility and permeability results of a single compound and its changing laws under multi-compound environment.The research on CMMBCS should be finally implemented to the holistic research level of traditional Chinese medicine compounds,but the characteristics of multi-compound and multi-target for TCM determine that its overall absorption could not be explained by the simple addition of solubility and permeability parameters of contained compounds.In view of the purpose of studying the intestinal absorption for oral drugs is to associate it with the efficacy.Taking Gegen Qinlian tablet as research object and guided by its anti-inflammatory effect,this thesis not only studied the solubility and permeability of representative compounds with anti-inflammatory activity,but also corrected and integrated the solubility and permeability data of each compound by considering the mass weight and efficacy weight in order to obtain the biopharmaceutical parameters which could represent the overall absorption characteristics of Gegen Qinlian tablet.This thesis made a preliminary exploration of the overall CMMCBS research of traditional Chinese medicine compound which will contribute to the selection of quality control compounds and the improvement of traditional Chinese medicine preparation process,as well as supplemented and improved the theoretical framework of CMMBCS and provided data support and research basis for its further development.Methods:The database of Chinese medicine ingredients and molecular docking technology,combined with literature statistics and pharmacopoeia records,were used to preliminarily select representative compounds with anti-inflammatory activity in Gegen Qinlian tablets under consideration of biological activity and content.In order to obtain the efficacy weight of each compound,lipopolysaccharide(LPS)was used to induce RAW264.7 cells to establish an in vitro inflammatory model,and TNF-α and IL-6 were used as targets to investigate the anti-inflammatory effects of representative compounds in Gegen Qinlian tablets.A method for the determination of representative compounds in Gegen Qinlian tablets was established to obtain the mass weight of each compound.The combination of mass weight and efficacy weight was used to integrate the solubility in different media and effective permeability coefficient determined by the typical shake flask method and in situ single pass intestinal perfusion model respectively.Results:(1)The results of molecular docking showed that the compounds with strong binding activity to TNF-a and IL-6 in the four herbs of Gegen Qinlian tablets had a high proportion in total compounds,ranging from 54.55%to 78.04%.It is indicated that Gegen Qinlian tablets have strong anti-inflammatory activity,and its anti-inflammatory mechanism is associated with two targets of TNF-α and IL-6.Combined with literature search and pharmacopoeia records,taking into account both biological activity and content,the twelve compounds(puerarin,daidzin,baicalin,daidzein,wogonoside,baicalein,wogonin,glycyrrhizic acid,coptisine,epiberberine,berberine and palmatine)were identified as representative compounds in the CMMBCS study of Gegen Qinlian tablets.In vitro cell experiments confirmed that twelve compounds inhibited TNF-a and IL-6 secreted by LPS-induced RAW264.7 cells in varying degrees,and the efficacy weights were 0.04,0.12,0.10,0.13,0.05,0.10,0.04,0.04,0.07,0.10,0.10,0.11,respectively.The mixture of twelve compounds had no significant difference compared with the overall pharmacodynamic effect of the extract of Gegen Qinlian tablets,indicating that the selection of representative compounds has certain rationality.(2)Twelve representative compounds could be detected in Gegen Qinlian tablets from different manufacturers and batches,but the proportion of each compound was different and the content had great disparity between different compounds.In order to facilitate the simultaneous detection and accurate quantification of solubility and permeability for multicompound in the overall environment of Gegen Qinlian tablets,the tablet from Jilin Wantong pharmaceutical industry with the batch number of 20170501,which had relatively high integral content of main compounds,was finally selected for demonstration.And the mass weights of twelve compounds were 0.33,0.06,0.19,0.03,0.04,0.06,0.02,0.02,0.02,0.02,0.17,0.04.(3)The solubility results analyzed by the angle cosine method indicated that the dissolving tendency of puerarin,daidzin,daidzein,baicalin,wogonoside and wogonin in Gegen Qinlian tablets was similar,shown as the solubility in the pH4.0 buffer solution was lowest and solubility gradually increased with the increase of pH value in the pH4.0-pH7.4 buffer solution;the dissolving tendency of baicalein and coptisine hydrochloride,epiberberine,berberine hydrochloride and palmatine hydrochloride was similar,shown as the solubility in the pH1.0 buffer solution was highest;the solubility of glycyrrhizic acid is pH-dependent,and its solubility increased with the increase of pH value.It was found that the dissolving tendency of twelve compounds under single compound environment and the whole environment of Gegen Qinlian tablets changed greatly,indicating that the Chinese herbal compound formula environment has a great influence on the solubility of single compound,but its specific mechanism needs further exploration.The dose number(D0)of each compound calculated by the solubility data showed all to be larger than 1,and the D0 value of Gegen Qinlian tablet calculated by combination of mass weight and efficacy weight was 6.34,belonged to low solubility.(4)By analyzing the difference of the absorption parameters of twelve compounds in Gegen Qinlian tablet at high,medium and low drug concentrations,it was known that the absorption fraction of each compound was low,between 3%and 20%;the absorption mechanism of each compound was mainly passive diffusion,in which the absorption of daidzin,baicalein,and wogonoside may also be accompanied by an active transport process.Except that the effective permeability coefficient(Peff)of daidzin,baicalein and wogonin was higher than 5×10-5cm·s-1,the other compounds were less than 5×10-5 cm·s-1.The Penrvalue of Gegen Qinlian tablet was calculated to be 3.826×10-5cm·s-1,belonged to low permeability.The maximum absorbed dose(MAD)of the twelve compounds and the whole Gegen Qinlian tablet were calculated respectively,and the results showed that puerarin,daidzin,baicalin,wogonoside,berberine hydrochloride had higher MAD values with better absorption;but the overall MAD value of Gegen Qinlian tablet was 68.49mg,which was lower than the clinical dose,and the absorption percentage(F)was only 38.41%.Conclusion:The anti-inflammatory mechanism of the Gegen Qinlian tablet may be related to inhibition of TNF-α and IL-6 release.From the absorption point of view,the better absorption components such as puerarin,daidzin,baicalin,wogonoside and berberine hydrochloride could be used as important indicators for quality control of Gegen Qinlian tablets.Based on anti-inflammatory activity,the Gegen Qinlian tablet could be classified as CMMBCS class IV with poor absorption,both the solubility and permeability were the main limiting factors for its absorption.It may be necessary to increase the solubility or permeability of this tablet to improve its absorption,so as to improve its clinical efficacy. |
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