| ObjectiveUnprocessed and processed of traditional Chinese medicine used in different situation mainly refers to the different effects of the same medicinal material after processing.Ziziphi Spinosae Semen is a kind of traditional Chinese medicine commonly used as"raw,ripe and different treatment".The clinical application is mostly raw and fried products.The raw Ziziphi Spinosae Semen is flat and cleanser.After frying,it is easy to crush and decoct out,which can enhance its function of treating deficiency and annoyance and sleepleness,nourishing the heart and collecting sweat,and thus achieve the purpose of"effective ripening".The effect,function and internal quality of Ziziphi Spinosae Semen changed in different degrees during the process of ripening,so its quality control standard also changed to some extent.The content determination indexes of spinosin,and jujuboside A in Chinese Pharmacopoeia could not reflect the quality difference of integrity and specificity of Ziziphi Spinosae Semen before and after processing.Liu cx member of team,according to the theory of traditional Chinese medicine formulae compatibility principle,preparation and clinical application features,put forward the traditional Chinese medicine(including traditional Chinese medicine,medicine yinpian,medicinal broth,extracts,preparation,etc.)itself exists or processing and preparation process of closely related to the medicinal properties,efficacy of the concept of"quality of traditional Chinese medicine(TCM)markers"So this topic first by central composite design-response surface method optimization craft Ziziphi Spinosae Semen benevolence making,combination with the determination of the index component content,fingerprint,UPLC-Q-TOF/MS,the methods of multivariate statistical analysis from the perspective of"quantity"and"qualitative"before and after analysis and comparison of Ziziphi Spinosae Semen benevolence making chemical composition change,finally through the simulation,Fried Ziziphi Spinosae Semen benevolence and diazepam saturated competition experiments in vitro,molecular docking test before and after the screening of Ziziphi Spinosae Semen benevolence making give play to the role of sedative hypnotic quality markers,in order to establish quality Ziziphi Spinosae Semen benevolence"born cooked treat"marker specificity and integrity of the quality standards to provide the reference.Methods1 Central Combinative Design(CCD)-Response Surface Methodologies(RSM)to optimize the frying process of Ziziphi Spinosae SemenThis topic according to the central composite design principle of the response surface method,set up time to examine factors,making the temperature and making adenosine,magnoflorine,spinosin,6’’’-feruloylspinosin、jujuboside A and betulinic acid content of 6 kinds of ingredients comprehensive and extract rate scores selected as the optimized indexes,optimizing the Ziziphi Spinosae benevolence best making process.According to the optimized technology,the contents of 6 index components of 15batches of jujube kernel from 3 producing areas before and after processing were analyzed and compared,which provided stable and feasible technological conditions for the subsequent screening of quality markers of"raw,ripe and different treatment"of Ziziphi Spinosae semen.2 Fingerprint and component difference of Ziziphi Spinosae semen before and afterAccording to the process parameters in the first chapter,UPLC method was used to establish the fingerprint of raw and fried Ziziphi Spinosae semen from 15 batches of3 producing areas on the market,and the chemical composition changes of Ziziphi Spinosae semen before and after processing were initially clarified by combining similarity analysis,principal component analysis and cluster analysis.Furthermore,the chemical composition changes of Ziziphi Spinosae semen before and after frying were studied by UPLC-Q-TOF-MS,and multivariate statistical analysis was carried out by UNIFIL and Masslynx V4.1 software to find the significant difference of the chemical composition of Ziziphi Spinosae semen before and after frying.3 Receptor-ligand binding molecular docking technology was used to screen sedative and hypnotic active components of Ziziphi Spinosae Semen before and after fryingAccording to the principle of the receptor and the ligand binding,build,Fried extract of ziziphi spinosae semen(zs)and diazepam in the rats had GABA(gamma-aminobutyric acid)receptor in vitro experimental model of saturated competition,compare different concentration,Fried extract of semen ziziphi spinosae semen(zs)inhibition rate of diazepam,using HPLC and HPLC-MS screening,Fried Ziziphi Spinosae Semen can compete with diazepam replacement GABA receptors play a role of sedative hypnotic,the active ingredient,in combination with the active ingredient of the ionic strength,its combination with GABA receptor rate calculation,using molecular docking to authenticate the combination rate size.Results1 Central Combinative Design(CCD)-Response Surface Methodologies(RSM)to optimize the frying process of Ziziphi Spinosae Semen.Response surface methodology(RSM)was used to investigate the extraction rate and the contents of six components,including adenosine,magnoflorine,spinosin,6’’’-feruylspinosin,jujuboside A and betulinic acid.The optimal frying process of Ziziphi Spinosae Semen was obtained by using comprehensive weight scoring method.The frying temperature was 125℃and the frying time was 6.5min.The average comprehensive score of the three validation tests was 92.3075,and the relative error with the predicted value of 91.8706 was 0.48%.2 Study on fingerprint and component difference of Ziziphi Spinosae Semen before and afterAccording to the results of similarity evaluation software,determine the raw and fried Ziziphi Spinosae Semen fingerprint peaks of 15,respectively,each batch of similarity of similar samples,and the stability of retention time,difference is small,but there was some difference of peak area of each chromatographic peak,clustering analysis and the analysis of PCA and OPLS-DA,according to the results of raw Ziziphi Spinosae Semen and Fried products obviously divided into two categories,namely raw Ziziphi Spinosae Semen clustering for category,Fried Ziziphi Spinosae Semen for a class,suggests Ziziphi Spinosae Semen benevolence has certain differences before and after processing.The results of UPLC-Q-TOF/MS and multivariate statistical analysis showed that 33 common components,including flavonoids,saponins and alkaloids,were identified in raw and fried Ziziphi Spinosae Semen under positive and negative ion detection modes.The analysis results of Progenesis QI software showed that there were 16 different components in Ziziphi Spinosae Semen before and after processing,among which the contents of 6components,including adenosine,spinosin,6’’’-feruylspinosin,jujuboside A and betulinic acid,increased,while the contents of 10 components,such as magnoflorine decreased.These changes may be the material basis of"effective ripening"of Ziziphi Spinosae Semen after processing.3 In vitro saturation competition experiment and molecular docking to screen sedative and hypnotic active ingredients before and after frying Ziziphi Spinosae SemenBoth raw and fried Ziziphi Spinosae Semen extracts could competitively bind to GABA receptors in rat brain with diazepam,and the competitive binding effect of fried Ziziphi Spinosae Semen was stronger than that of raw Ziziphi Spinosae Semen,and there was a certain dose-effect correlation.HPLC results showed that characteristic peaks appeared at 6.26±0.02min,7.56±0.02min,16.56±0.02min in raw and fried Ziziphi Spinosae Semen group,diazepam group+brain tissue group,raw and fried Ziziphi Spinosae Semen+brain tissue group+diazepam group,respectively.LC-MS analysis identified the three components as mass charge ratio M/Z 608.1741,M/Z784.2215 and M/Z 455.3631,respectively.Combined with the literature reports and the rule of mass spectrometry,the three compounds were identified as spinosin,6’’’-feruylspinosin,and betulinic acid,respectively.According to the peak surface of mass spectrometry,the order of binding rate of each component was 6’’’-feruylspinosin>betulinic acid>spinosin,and the fried Ziziphi Spinosae Semen was higher than the raw Ziziphi Spinosae Semen.The binding rate of the three compounds to GABBA receptor was verified by molecular docking,and the order was 6’’’-feruylspinosin(-9.6)>betulinic acid(-9.2)>spinosin(-8.9),which was consistent with the results of HPLC-MS analysis.That is to say,these three components may be the quality markers of"raw,ripe and different treatment"of Ziziphi Spinosae Semen.ConclusionThis topic from the index component content before and after the fingerprint determination,making comparison,chemical composition change,saturated competition model in vitro of four angles,systematically illustrates the Ziziphi Spinosae Semen benevolence after making material base,give play to the role of efficiency for clinical reasonable choice of processed products of ziziphi spinosae semen,and establish and improve the quality of integrity,specificity standard for reference. |
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