Study On The Potential Quality Markers Of Dingkun Dan

Rationales: Dingkun Dan(DKD),a classical traditional Chinese medicine(TCM),it was founded in the fourth year of Qianlong in the Qing Dynasty and named by Qianlong according to legend.Some scholars believed that DKD is made up of the "Bujing Decoction" in the Zhulin Women's Syndrome,and some scholars believed that it came from the “Xusijiangsheng Dan” in the Women's Daquan Recipe,but both of them had similar utilities,and the clinical observations in modern research have also proved that it can be used to invigorate Qi and blood,regulating menses and relieving nervous depression.DKD is currently used to improve dysmenorrhea,irregular menstruation,polycystic ovary syndrome,perimenopausal syndrome(menopausal syndrome),endometriosis and other gynecological diseases,and it can also be used for infertility,repairing endometrial and relieving the pain while postpartum or post-abortion,and prevention of postoperative complications.DKD is consists of Ginseng Radix ex Rhizoma Rubra(GRRR),Cervi Cornu Pantotrichum(CCP),Croci Stigma(CS),Notoginseng Radix Et Rhizoma(NRR),Paeoniae Radix Alba(PRA),Rehmanniae Radix Praeparata(RRP),Angelicae Sinensis Radix(ASR),Atractylodis Macrocephalae Rhizoma(AMR),Lycii Fructus(Ly F),Scutellariae Radix(SR),Cyperi Rhizoma(Cy R),Chuanxiong Rhizoma,(Ch R),Asini Corii Colla(ACC),Corydalis Rhizoma(Co R)and other herbs.The complicated composition of formula brought difficulties to the study of its quality,And the quality control standards contained in the the Pharmacopeia of the People's Republic of China is lack of controllability.In the Chinese Pharmacopoeia,the testing items include microscopic identification(AMR,Ly F,and Glycyrrhizae Radix et Rhizoma),thin layer identification(ASR,Ch R,Ly F,PRA,GRRR,NRR herbs,and paeoniflorin,ginsenoside Rb1,ginsenoside Re,ginsenoside Rg1,notoginsenoside R1 reference compounds),and the lowest contents of ginsenoside Rg1 in each DKD pills [1].These standards can only represent the quality of some of the herbs,and show the limitations in the overall quality control of the formula.Therefore,it is necessary to conduct in-depth research on its quality to better control the quality of DKD.Objective: To establish a suitable detection method for obtaining DKD fingerprints,and find stable,bioactive components on DKD as its quality markers.To improve DKD's quality control standards and provide scientific basis for its internal quality control and ensure that the quality of the formula is stable.Methods: 1.The UPLC-DAD technique was used to detect the methanol extractation of DKD.The detection time was 45 min,the wavelength was 280 nm,and the mobile phase was 0.1% formic acid-water and 0.1% formic acid-methanol.1H-NMR technique was used to detect the two-phase extractation of DKD(the methanol-water phase and chloroform phase),and the magnetic resonance of methanol-water phase extractation was determined by noesyppr 1D.Sequence,locked field with deuterated methanol,internal standard was TSP.The chloroform phase extractation was determined by zg30 sequence locked field with deuterated chloroform,and the internal standard was TMS;2? UPLC-MS technology was used to characterize components of DKD,and the obtained MS data were identified by comparing with herbs,reference compounds,database,and literature,and fitting the accurate molecular weight.In order to clarify the compositions of the formula;3 ?The network pharmacology technology was used to analyze the identified components of DKD.Analyzing the "herbs-components-targets-diseases" network,and perform GO biological function and KEGG pathway enrichment analysis to find potential bioactive ingredients,targets and mechanisms.Results:1?The efficient and simple UPLC and 1H-NMR method were established to obtain the fingerprints of DKD,and the quality of the compound can be characterized relatively comprehensively.The preparation steps and detection process in the UPLC method was efficient and simple,and 5-hydroxymethylfurfural and baicalin can be used as references for quality markers of DKD.In the 1H-NMR method,the preparation process of the two-phase extraction method can ensure comprehensive characterization of the compounds with low and high polarity ingredients.2?A total of 121 components,including amino acids,phenolic acids,lactones,terpenoids,alkaloids,saponins,flavonoids and other compounds were identified based on rhe UPLC-MS data by comparing with the standards,literature and database.Paeoniflorin,notoginsenoside R1,ginsenoside Rg1 and ginsenoside Rb1 can be used as references forquality markers of DKD.3?Through the network pharmacological analysis,77 potential active ingredients in DKD and 87 potential targets related to clinical applicability had been found.The 12 ginsenosides,4 notoginsenosides,crocin I,echinacoside,isoacteoside and verbascoside might be part of the potential bioactive ingredients in DKD.And the mechanism of its effect might be participating in estrogen and progesterone regulation,uterine embryo development,smooth muscle regulation,hematopoiesis and coagulation processes,and conventional immune and inflammatory responses.4? Among the potentional bioactive ingredients from the analysis,baicalin,paeoniflorin,crocin I,verbascoside,ginsenosides and notoginsenosides can be used as alternative references for quality standards of DKD.Conclusion: 1 ? The established UPLC and 1H-NMR methods for fingerprints' detection can relatively comprehensively characterize the primary and secondary metabolites of DKD,it can be used as reference for fingerprints standards of DKD.2?121 compounds and isomers of DKD were identified through UPLC-MS detection.Ginsenosides,notoginsenosides,baicalin,paeoniflorin,crocin I,and verbascoside can be used as alternative references for quality standards of DKD.3?DKD might be play the clinical role by participating in estrogen and progesterone regulation,uterine embryo development,smooth muscle regulation,hematopoiesis and coagulation processes,and conventional immune and inflammatory responses through the network pharmacology analysis.It can also provide a research direction for further study on the pharmacological mechanism of the formula.