Enrichment Detection Of Active Compounds In Tamping And Clam Shells And Rapid Screening Of PAMPA

Zhishi is the dried young fruit of Citrus aurantium L.and its cultivars,Citrus sinensis Osbeck.Zhiqiao is the dried immature fruit of Citrus aurantium L.and its cultivars.Chemical composition of Zhishi and Zhiqiao were complex and diverse,including flavonoids,coumarins,alkaloids,volatile,limonoids.Flavonoids and coumarins.are the main chemical constituents of Zhishi and Zhiqiao,mainly including They exhibit diversified bioactivities including anti-oxidant,anti-allergy,anti-inflammatory,anti-tumor,pressure boosting,heart strengthen and gastrointestinal promotion function.The phenolic compounds are in a wide range of solubilities and contents,and other coexisting impurities in the sample increased interference These cause great challenging in the pretreatment and analysis of the samples.At present,traditional Chinese medicine is mainly based on one or a few indicators to screening of extract process.However,the traditional Chinese medicine is a complex system,which play a pharmacological effect mainly through the absorption of the overall compound.Therefore,in the study,the main effective chemical constituents of total flavonoids and coumarins.are of Zhishi and Zhiqiao were purified and enriched by solid phase extraction method and then quantified and compared,with HPLC fingerprint of different species of Aurantii Fructus Immaturus developed.Furthermore,the rapid highthroughput screen of extract process through in vitro absorption for of Zhishi and Zhiqiao were studied by PAMPA model from two levels of monomer and herbal extract.By means of exploring the difference in effective components between Zhishi and Zhiqiao and exploring the mechanism may exist in vitro intestinal absorption we aim at providing an important basis for the quality evaluation of Zhishi and Zhiqiao,and providing a new method for the preparation of extract process of Zhishi and Zhiqiao from the perspective of bioavailability.1.The purification,enrichment and determination of active compounds in Zhishi and Zhiqiao.1.1 Study on the extraction technology of Zhishi and ZhiqiaoTo ensure the efficacy of Zhishi and Zhiqiao,it is important to develop a reasonable extraction process.In order to determine the optimal extraction process of the active ingredients,firstly,the extraction effect of reflux and ultrasonic was compared.Furthermore,factorial design was used to screen.And then extraction process was optimized by orthogonal design method with key factor methanol concentration,solvent multiple and extraction time as investigation factor and the extraction yield of 16 active ingredients including eriocitrin,rutin,naringin,naringin,hesperidin,neohesperidin,naringenin,hesperidin,rhoifolin,sinensetin,nobiletin,5-demethylnobiletin,tangeretin,7-hydroxycoumarin,bergapten auraptene and bergamotin were used as indicators.Consider the content disparity of the composition in the sample,multi-wavelength switching method was used to quantify the content of 16 components.The data were analyzed by multi-index comprehensive evaluation method,and the extraction effect of different extraction techniques was evaluated.The optimum extraction process was as follows,with 125 times 75%methanol as extraction solvent.,ultrasonic extraction for 30 minutes.1.2 Optimization of Solid Phase Extraction Scheme of Zhishi and ZhiqiaoChemical composition in Zhishi and Zhiqiao is complex with many compound type,solubility and content in which were in a big difference.Some components of the content were relatively low,and susceptible to other impurities in the sample interference,affecting its accurate quantitative.Considering solid phase extraction method is simple,set extraction and separation in one time,and have a good purification and enrichment role in the rare components,which was applied to enrichment and purification of Zhishi and Zhiqiao.20 kinds of flavonoids and coumarin from Zhishi and Zhiqiao,were used in the study.Extraction sorbent,breakthrough volume,washing solvent,elution solvent,and elution solvent volume were comprehensively optimized to screen the best purification and enrichment program.The best program was Strata-X as sorbent,the column was preconditioned with 2 mL of methanol followed by 5 mL of water,the breakthrough volumn was 23 mL.After sample loading,the column was sequentially washed with 2mL of 30%methanol and eluted with 10mL of 100%methanol.The results showed that the content of total flavonoids and coumarins in Zhishi and Zhiqiao was increased by 35.55%-68.48%after solid phase extraction.It is suggested that the solid phase extraction method has a good purification and enrichment effect.1.3 Determination and Classification of Zhishi and ZhiqiaoAfter optimization of Solid Phase Extraction Scheme,the developed and validated method was applied to the elucidation of 50 batches of zhishi samples from different species as well as zhishi and zhiqiao at different development stages.Consider the chemical diversity of the components to be tested,the rapid detection and identification of 20 flavonoids and coumarins chemical constituents in Zhishi and Zhiqiao were established by high performance liquid chromatography with multi-wavelength switching method.The method was validated in terms of linearity,accuracy,precision,repeatability,and recovery testing,and its results meet the requirement which can be used as the determination method for the quantification of 20 kind of compounds in Zhishi and Zhiqiao.The results showed that content of different kinds of chemical composition,including dihydroflavone,polymoxylated flavonoids,coumarin in Zhishi were higher than those in Zhiqiao at different development stage.The composition of neohesperidin and naringin were different in the distribution of different kinds of Zhishi.With the help of multivariate statistical analysis software,we realized the classification and identification of different varieties of Zhishi and Zhiqiao at different development stages.In general,the solid phase extraction method has a good application value in the pretreatment of Zhishi and Zhiqiao.1.4 Study on HPLC fingerprint of different species of Aurantii Fructus ImmaturusThis study is to establish an HPLC fingerprint by HPLC-DAD method and simultaneous quantitative analysis of 17 components of 18 batches of Citrus aurantium L.and 10 batches of Citrus sinensis Osbeck.The data calculation was performed with similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine(Version 2004A)together with SIMCA-P 13.0 software to clarify the differential marker between two different species of Aurantii Fructus Immaturus.The results showed that the neohesperidin,naringin,rhoifolin,tangeretin,hesperidin,5-demethylnobiletin,auraptene,have great contribution to the quality control between different species of Aurantii Fructus Immaturus.And these ingredients have significant influence in the clustering analysis of different species of Aurantii Fructus Immaturus.This method has good precision stability and repeatability that could provide basis for quality control and evaluation of Aurantii Fructus Immaturus.2.Rapid Screening of Chemical Constituents of Zhishi and Zhiqiao by PAMPAIn order to investigate the possible absorption of active compounds and the mechanism of intestinal absorption in Zhishi and Zhiqiao,thus providing a new method for the preparation of Zhishi and Zhiqiao.The following studies were conducted.2.1 Establishment and Quality Evaluation of PAMPA ModelThe artificial membrane was established on a 96-well filter plate by using soybean lecithin desolved in dodecane as a membrane material to simulate the small intestinal epithelial membrane.Through the optimization of membrane volume and concentration,the artificial membrane was evaluated in three ways,the appearance observation,fluorescein sodium leak detection and four positive control drug propranolol,carbamazepine,warfarin,furosemide.The results showed that the artificial membrane with volume of 10 ?L and establishment with the concentration of 5%soybean lecithin was reliable and could be used as PAMPA model for rapid screening.2.2 Screening of Monomer Compounds on PAMPA ModelThe PAMPA screening study was carried out on 43 chemical constituents including different structural types of Zhishi and Zhiqiao reported in the literature.With the advantage of HPLC-QqQ-MS/MS method,the rapid analysis of 43 components in 25 min was realized.The effective permeation coefficient value of each monomer compound was calculated under three concentration,high,medium and low.The result showed that the effective permeation coefficient value is an intrinsic property of the compound,which is independent of the concentration.The effective permeability coefficient value of different structural types indicates that the small intestinal absorption of coumarin,polymethoxyflavones,flavanones,aglycones,flavonoid aglycones and limonoids is good and the glycosides are poorly absorbed.2.3 Study on the Screening of the whole Traditional Chinese medicine extract in PAMPA ModelTraditional Chinese medicine play a holistic effect in clinical application,so the use of PAMPA model to establish the overall screening of traditional Chinese medicine is of great significance.In the study,different extracts of Zhishi and Zhiqiao were extracted by different solvent extraction and pretreated with solid phase extraction methods.And then the different extracts of Zhishi and Zhiqiao were used to study on the PAMPA model.The results showed that the coexisting components had a certain effect on the absorption in the whole extract of Chinese herbal medicine,and some of them showed the effect of absorption promotion,such as hesperidin,rhoifolin and diosmin,which may be related to the increase of solubility.The whole extract absorption was further enhanced after purified by solid phase extraction.Conclusion:In this paper,two aspects of work were mainly carried out.Firstly,the solid phase extraction method was used to establish the purification and enrichment scheme of phenolic chemical constituents from Zhishi and Zhiqiao,coupled with the HPLC-DAD multi-wavelength switching quantification method,we successfully realized the discrimination of Zhishi samples from different species as well as Zhishi and Zhiqiao samples in different development stages.The results showed that there were significant differences among the chemical constituents between different varieties of Zhishi,and the concentration of chemical constituencies between Zhishi and Zhiqiao in different development stages was different.During fruit development,the contents of phenolic chemistry ingredients include flavonoids,flavanones,polymethoxyflavones and coumarins displayed a declining trend.The difference in efficacy of Zhishi and Zhiqiao may be related to the different concentration between them.Furthermore,study on rapid screening of monomeric and whole extracts of Zhishi and Zhiqiao by PAMPA in vitro absorption model.The results showed that effective passive permeability values of the compound is the intrinsic property of the compound,which is independent of the concentration,and the strength of the absorption is related to the lipid solubility of the compound.Traditional Chinese medicine as a whole has an interaction between its internal components and affects its absorption.Through the purification process some of the components interfere absorption can be removed,and promote the absorption of compounds.It is suggested that the preparation of traditional Chinese medicine should take the compatibility between the internal compounds and the purification into account.Removing some of the interfering components,so that the Chinese medicine can exert its best absorption within a relatively suitable purity range and improve its bioavailability.This study provides a new approach to the preparation of Chinese medicine from the perspective of bioavailability.