A Study On Supramolecular "Imprinting Template" Characteristics And Metabolic Laws Of Lonicerae Japonicae Flos And Lonicerae Flos Supramolecular Guest Molecule

Lonicerae japonicae flos（Jinyinhua,JYH,in Chinese）and lonicerae flos（Shanyinhua,SYH,in Chinese）are one of the most commonly used traditional Chinese Medicine,which have the functions of clearing away heat,toxic material and dispersing wind heat.In 2005,Pharmacopoeia Commission had JYH divided into JYH and SYH,triggering“double flower struggle”.The key point of"double battle"is the debate of the two drugs as the same kind of traditional Chinese Medicine.The core problem is how to establishtheresearchtechniquetocorrectlyevaluatetheclinical equivalence of different pieces.Therefore,if we can clarify the equivalent heterogeneity mechanism of JYH and SYH,we will provide a scientific basis for breakdown problem of JYH and SYH.Objective:Based on the theory of supramolecular"imprinting template",using JYH and SYH as a model drug and SD rats as the experimental animal,using liquid chromatography-mass spectrometry and GC-MS detection technologyasdetectiontechnologys.theywereusedtostudy supramolecular"imprinting template"characteristics and metabolic laws of JYH and SYH supramolecular objects.This will lay the foundation for the study of the equivalent heterogeneity between JYH and SYH.Methods:1.Chemical composition database of JYH and SYH was established by organizing literature and database that were published at domestic and foreign and related to JYH and SYH.The main contents of the database included Chinese and English name,molecular weight,chemical structure,commonmassspectrometryfragmentions,molecular connectivity index（MCI）,attribution and so on.2.The water extract ingredients and volatile oil of JYH and SYH were identified by LC-MS and GC-MS,and the average MCI of the different components was calculated.3.Based on the theory of supramolecular"imprinting template",the JYH,SYH and its preparations were subjected to cross-blotting test.The fingerprints were analyzed by high performance liquid chromatography to explore the action law of JYH and SYH.4.The chemical compositions of water extract in JYH and SYH were distributedin the blood,urine and feces of rat by LC-MS detection.Results:1.The average MCI of various chemical constituents in JYH water extract was 9.06 for organic acids,10.93 for flavonoids,9.74 for iridoid,and 29.11 for saponins,and the average MCI of various chemical constituents in SYH water extract were organic acids 8.70,flavonoids 10.52,iridoid terpenes 9.38,saponins 30.56.The overall mean for the MCI of water extract ingredients in JYH and SYH were compared.The results showed F=1.26×10^-4<F_{0.05（1,6）}=8.813,then P>0.05.There was no significant difference in the mean value of MCI for water extract ingredients in JYH and SYH.The MCI of various components of JYH and SYH was analyzed by wilcoxon rank sum test.The result showed P equal 0.713 which was more than the 0.05.The results showed that there was no significant difference in the MCI of ingredientsbetween the JYH and SYH.2.MCI and retention time of water extract components in JYH and SYH are curvecorrelation.ThecorrelationequationofJYHwas Y=0.177X²-2.028X+11.886（R²=0.678）.The correlation equation of SYH was Y=0.281X²-3.577X+17.023（R²=0.750）.Y represented the MCI and X represented the retention time.By fitting a curve,in water extract components of JYH,when t _R>5.72min,MCI was positively correlated with t _R..In water extract components of SYH,when t _R>6.36min,MCI was positively correlated with t _R.As reported in the literature,MCI was related to solubility.3.Analysis result of volatile oil in JYH and SYH:JYH volatile oil containing the aldehydes、ketones、ethers,the acids,ester were more than SYH in terms of species,and the hydrocarbons and alcohols of the SYH were more than JYH in terms of species.The average MCI for each component were calculated.The average MCI of hydrocarbons,alcohols,aldehydes,ketones,ethers,acids and esters in JYH are 8.99,5.11,5.21,7.77,7.95;The average MCI of hydrocarbons,alcohols,aldehydes,ketones,ethers,acids and esters in SYH were 4.99,5.50,4.18,8.04,7.69.The overall mean for the MCI of volatile oil components in JYH and SYH were compared.The results showed F=0.7210<F_{0.05（1,8）}=7.5709,then P>0.05,There was no significant difference in the mean value of molecular connectivity index.The paired t test was used to analyze the MCI of various volatile oil components in JYH and SYH.The statistical parameters were t=1.146<t_{0.05（n=4）}=2.776,P=0.316>0.05.The results showed that there was no significant difference in the molecular connectivity index between the volatile oil of JYH and SYH.4.Crossed blotting results of JYH and SYH:The adsorption constants of SYH dregs adsorpting JYH extract,JYH biloba extract and JYH silver thorny detoxification extract respectively were 0.620,0.585 and 0.359,and the adsorption constants of JYH dregs adsorpting SYH extract,SYH biloba extract and SYH silver thorny detoxification extract respectively were0.698、0.676 and 0.229.Pairwise T test for both adsorption constants,statistic t=-0.182,freedom degree df=2,bilateral P=0.873>0.05.The results showed that the adsorption constants of JYH and SYH with each other had no significant difference.5.The law of metabolism of JYH and SYH in rats:The time distribution rule of chemical components of the two herbs in the plasma,urine and feces was similar.The distribution rule of the chemical constituents of JYH and SYHintheplasma,urineandfeceswasanalyzedbyusingthe supramolecular"imprinting template"characterization parameters—MCI.The results showed that the average MCI of the organic acids were 10.85and 10.85respectively,and the iridoid terpenes were 8.12 and 8.23respectively in JYH and SYH plasma,which indicated that there were similarity supramolecular"imprinting template"in JYH and SYH plasma;In the feces,JYH and SYH were identified the same type of prototype components,which were organic acids and saponins.Organic acids average MCI of JYH and SYH were 10.56 and 10.03 respectively,and saponins were29.11 and 30.84 respectively.These data suggest that the excreted prototype component of the JYH and SYH by the fecal excretionhas a similar supramolecular"imprinting template".In the urine,there was a big differencethe composition species between JYH and SYH,of which the averageMCIwerenotcomparable.Afteranalysisthechemical constituents distribution in the blood,urine and feces of rats in JYH rats,the results showed that 3,4-di-caffeoylquinic acid,3,5-di-caffeoylquinic acid and 4,5-di-caffeic acid were detected in the plasma,urine and feces,and chlorogenic acid and its isomers can only be detected in urine or feces.The iridoid components（secologanoside,sweroside and secoxyloganin）were only detectable in plasma,and the saponinsare（macranthoidin A and dipsacoside B）can only be found in the feces;Compared with JYH group,the distribution rule of organic acids and iridoid terpenes were similar in JYH and SYH.The main differences in the metabolism of JYH and SYH are shown in the following two aspects:1.Hyperoside and luteoloside can be detected in the urine of SYH group;2.Macranthoidin B and dipsacoside B can be detected in the urine,feces and plasma of the SYH group.Conclusion:There are significant differences in the water extract and volatile oil between JYH and SYH,but the both have the same or similar“imprinting template”;The cross-blotting experiment in vitro indicate that JYH is the same or similar to the chemical composition"imprinting template"of SYH;Organic acids,iridoid ethers and saponins are the main blood component of JYH and SYH,which are very close to the average MCI of organic acids and iridoid terpenes.These show that the two herbs into the blood components have similar supramolecular"imprinting template".Based on the above conclusions,it is shown that it is feasible to clarify the equivalent heterogeneity between JYH and SYH with the action law of supramolecular"imprinting template",and it laid the foundation for heterogeneous equivalent research.