The Study On Substance Benchmarks Of Classical Famous Prescriptions Guizhi-Shaoyao-Zhimu Decoction

This subject was supported by the National Science and Technology Major Project for’Significant New Drugs Creation’.It comed from the project of’Research and Development of New Drugs of Classical Famous Prescription Guizhi-Shaoyao-Zhimu Decoction Based on Consistency Evaluation’（No.:2018ZX09721004-009）.As one of classical famous prescription,Guizhi-Shaoyao-Zhimu Decoction（GSZD）originates from Synopsis Golden Chamber.It could be expelling wind and removing damness,activating stagnated Yang and replenishing the vital essence and removing heat.It was also an excellent prescription for the treatment of Rheumatoid Arthritis（RA）.The research group took GSZD as the research object,according to the requirements of the task,the substance benchmarks of GSZD of the classical famous prescription was established based on the traditional boiling decoction method and the modern quality control method.According to the previous literature research,the modern clinical medical case excavation,clinical application and"Chinese Pharmacopoeia"to determine the varieties,sources and processing products and functions of GSZD.The herbal medicines in GSZD were cassia twig,radix paeoniae Alba,rizoma anemarrhenae,radix liquiritiae,Chinese ephedra,Saposhnikovia divaricate,bighead atractylodes rhizome,ginger,processing radix Aconiti Praeparata.For the variety sources herbal medicine,the variety of Gancao was Glycyrrhiza uralensis Fisch.,and variety of Mahuang is Ephedra sinica Stapf.According to the original record,refer to the previous research of the research group to form the“classical famous prescriptions”,and determine that the prescription dose is 1 liang equals 3 g,and the single-prescription prescription is 93 g.Refer to the classical famous prescriptions,the fried amount recorded in the original was used as the boiling end point.And cooking control,pots and heating methods were also been studied.Based on the prescription of the decocting time in the decocting specification,the boiling time in accordance with the consensus of the clinical decocting method was established,and the subsequent drying process was examined.Therefore,based on the principle of“comply with the classics and conform to the pharmacopoeia”,a classical famous prescriptions that maintains the“original prescriptions,original dose,and original boiling methods”has been established.To establish the correlation between HPLC fingerprint and free scavenging radicals activity of substance benchmarks of Guizhi-Shaoyao-Zhimu Decoction（GSZD）.It provides a basis for the quality evaluation and the modernization of classical prescriptions.Shimadsu GL-sciences AQ-C18 column（4.6 mm×250 mm,5μm）was used with acetonitrile-0.1%formic acid solution as the mobile phase for gradient elution.The detective wave length was 254 nm;the column temperature was set at32℃;the injection volume was 20μL;and the flow rate was 1.0 mL·min^-1.10batches of substance benchmarks of GSZD were determined.The HPLC fingerprint of GSZD was established by using the Similarity Evaluation System for Chromatographic Fingerprint of TCM（2004 A edition）.The activity of scavenging free radicals was studied by1,1-diphenyl-2-trinitrophenylhydrazine（DPPH）method,and the fingerprint-efficacy relationship was studied by Pearson bivariate correlation analysis.Fingerprints of GSZD were established,26 common peaks were calibrated and similarities ranging from 0.929 to 0.998.Eight of the chromatographic peaks were identified using the reference comparison method:gallic acid,mangiferin,paeoniflorin,glycyrrhizin,asparagus,5-O-methylvisamicin,cinnamic acid,glycyrrhizic acid Ammonium.Among them,14（Paeoniside）,20,12（mangiferin）,13,23（cinnamic acid）shared peak content changes were negatively correlated with free radical scavenging activity.The fingerprint has good precision,repeatability and stability,and the common peak separation degree is good.It can be used for the quality evaluation of GSZD.Ultra-high performance liquid chromatography（UPLC）had a good separation of GSZD.In the exploration and optimization of the chromatographic conditions,the three key chromatographic peaks in the original HPLC chromatogram（No.4 peak gallic acid,No.12 peak mangiferin and No.25 peak ammonium glycyrrhizinate）were used as indicators.The relative retention times of these three peaks were consistent under the optimized UPLC conditions,which ensures the overall similarity of the chromatogram after conversion from HPLC conditions to UPLC conditions.From the positive and negative ion scan modes,a total of 31 major peaks were observed and 28 of them were identified.Six of them were steroidal saponins,and the content was high,which was not observed under previous HPLC-UV conditions.In order to effectively control the quality of the various parts of the substance benchmarks,in accordance with the requirements of medicine compound preparations and their substance benchmarks of ancient classical famous prescription（consultation paper）.Ensuring the quality of the“standard decoction”batch is consistent and traceable by the dry ointment yield,content determination,fingerprint or characteristic map.15batches of"substance basis"were prepared from more than 15 batches of raw materials,and were subjected to decoction,concentration（drying）,etc.,with their average values as the benchmark.The quality profile study of medicinal materials,decoction pieces,“standard decoction”and material benchmarks should be carried out to determine the key quality attributes,to achieve quality control throughout the process,and to ensure that the quality of the material reference batches was basically consistent and traceable.