The Substance Benchmarks Research And Development Of Classical Formula ShaoYao-GanCao Decoction

Shaoyao-Gancao decoction(SYGCD)is composed of paeony root and licorice in the 1:1 ratio,originated from the treatise on febrile diseases compiled by Zhongjing Zhang.Its efficacy mainly focuses on harmonizing the liver and spleen,reliefing chronic pain.It is used for the treatment of chronic atrophic gastritis,facial spasm,gastrocnemius or muscle spasm,cough asthma and anaphylactoid purpura,etc.As one of the classical traditional Chinese medicine(TCM)formula in 100 prescriptions released by the government,it is anticipated to provide a certain basis for the research of paeony and licorice-containing TCM prescriptions.However,the existed researches mainly focused on the clinical usage and the pharmaceutical activity of some major components such as paeoniflorin,limited by the insufficient chemical investigation of SYGCD.Moreover,differentiation and recognition of the TCM formulas prepared from white paeony root(WPR)and red paeony root(RPR)remains a hurdle.Aiming at the problems existing in the classic prescription SYGCD and the key technical difficulties of the research and development of the classic prescription pointed out in the application guidelines of the classic recipe,we carried out researches on literature retrival,preparation process of substance benchmarks,quality control methods and in-depth investigation of chemical compounds of SYGCD.1.Literature investigation to clarify prescription informationBased on the databases,such as CNKI,and bibliographic retrial,we draw the following conclusion.The SYGCD originated from treatise on febrile diseases written by Zhongjing Zhang,is composed of paeony root and licorice in 1:1 ratio with each of 12 grams,and commonly used to treat spasmodic pain and podalgia in clinic.The original plant of licorice is Glycyrrhiza uralensis Fisch.,which was processed with honey before use.2.The preparation technology research of SYGCDMultiple batches of collected medicinal materials from original and main producing area were screened by detecting macroscopical characters,thin layer authentication,content and fingerprint according to Chinese Pharmacopoeia and Hong Kong pharmacopoeia standard.The herbal drug material with high quality were processed to corresponding decoction pieces and used for the preparation technology research of SYGCD.The primary parameters such as water volume,immersion time,decocting time,extraction times,condensed temperature and drying method were investigated and the optimal preparation technology was determined by comparing the extraction yield,fingerprint and the content of index component.The optimized preparation process lists as the following:48 g of white paeony root(WPR)and 48 g of Glycyrrhizae Radix et Rhizomes(GRR)were added to casserole with 960 mL ultrapure-water.After immersion for 30 minutes,the mixtures were decocted under boiling water for 1 h and then filtered.Moreover,768 mL of ultrapure-water was added and heated for another 0.5 h after boiling,then filtered and the filtrates combined.The filtrate was concentrated to a density of 1.05～1.10 g/mL under 60 centigrade and lyophilized.3.The establishment of quality control method of SYGCDThe thin layer chromatography method and fingerprint method could reflect the chemical information of each single herbal drug material,and quantitative determination method by high performance liquid of chromatography for the quality control of SYGCD were established.The high-performance layer chromatography method(HPTLC)was successfully developed by investigating mobile phase and chromogenic reagent,and the methodology was futher verified.The optimal fingerprint method of SYGCD was determined by optimizing the extraction conditions including extraction solvent,extraction method and extraction time and chromatographic condition such as mobile phase composition,propotion of organic phase.Five reference peaks were determined:albiflorin,paeoniflorin,Liquiritigenin-7-O-D-apiosyl-4’-O-Dglucoside,glycyrrhizin and glycyrrhizic acid and methodological verification of specificity,precision,reproducibility and stability were carried out.Finally,a method was established to determine the content of paeoniflorin,glycyrrhizin and glycyrrhizic acid after optimizing extraction and chromatographic conditions,and methodological validation results showed satisfactory specificity,linearity,accuracy,precision,repeatability,durability and stability.4.Chemical profiling of SYGCD based on UPLC/LTQ-Orbitrap MSIn negative mode,the substance benchmarks and single herbal drug material samples were analyzed by UPLC/LTQ-Orbitrap full scan method.Except for comparison with reference standards to infer the pathway of representative compounds,an in-house library of SYGCD recording all the chemical components isolated from paeony root and licorice was built by Progenesis SDF studio.As a result,a total of 161 compounds were identified or tentatively characterized in SYGCD,including 84 triterpenoid saponins,10 monoterpenoid glycosides,42 flavonoids and 25 other compounds.The research laid a foundation of further study on the chemical composition of SYGCD and elucidating its pharmacodynamic substances.5.Authentication of paeony in SYGCD based on the non-targeted metabolomicsTotally 20 batches of home-made SYGCD prepared form WPR,RPR and GRR were systematically analyzed by UPLC-Q-TOF/MS and a metabonomic method combining multi-step chemometric analysis was used to screen out the chemical markers to differentiate the authentic SYGCD(prepared from WPR)from false SYGCD(prepared from RPR).As shown in PC A plot,two types of SYGCD were successfully distinguished by monitoring the screened five chemical markers(marker l:m/z 461.13,marker 2:m/z 631.17;maker 3 m/z 631.17,maker 4:m/z 503.18 and paeoniflorin),the marker 2 was deduced to be galloyl-albiflorin.Besides,taking practicability and accessibility into consideration,the UPLC/QDa-SIM method was established for routine analysis of 20 batches of home-made SYGCD,the results showed that the strategy was proved to be effective in authentication of paeony root in SYGCD.By the above mentioned research,we can reach the following conclusions:Firstly,bibliography retrieval was carried out to confirm historical evolution and other information of the prescription,and to provide a theoretical basis for the research and development of the classic famous SYGCD formula;Secondly,the important parameters affecting the modern preparation process of SYGCD were investigated,which provided some reference for the research on the classic recipe technology of other prescriptions;Thirdly,the quality control method of SYGCD was established from three aspects including thin layer identification,characteristic fingerprint and content determination,which provided an effective means for enhancing the quality control of SYGCD;Fourthly,the multi-component chemical information of the authentic SYGCD was systematically characterized,which provided a scientific basis for clarifying its pharmacodynamic substance basis.Finally,the non-targeted metabolomics approach was applied to compare the differences among 20 batches of home-made samples by UPLC/Q-TOF,and 4 chemical markers were screened out by multiple chemometrics methods.Moreover,intuitive screening of the chemical markers was accomplished on single quadrupole MS detector QDa using selected ion monitoring(SIM)method to authenticate samples efficiently.The developed strategy will help control the quality control of the paeony root in TCM prescriptions,and hopefully could be applied to other Chinese herbal medicine materials in TCM prescriptions.