The Study On Improving Quality Standard Of Jiawei Guilingji Jiu And Functional Ingredients

Background:Jiawei Guilingji Jiu is a medicinal liquor produced by Shanxi Guangyuyuan State Medicine Co.,Ltd..Its main ingredients include Guilingji powder,Rehmannia glutinosa,Cistanche,and menthol.The main function is strengthening the brain and kidney,strengthening the function and prolong life.It is used for improving qi deficiency and blood deficit,forgetfulness,insomnia,loss of appetite,backache,Yin deficiency and Yang weakness,impotence and premature ejaculation,cold palace,abdominal pain and postpartum deficiency.Jiawei Guilingji Jiu originated from Guilingji,the Imperial Holy Medicine,which was a state-secret compound preparation of traditional Chinese medicine,and was created 450 years ago in Jiajing period of Ming Dynasty.The existing quality standard of Jiawei Guilingji Jiu is the thin-layer identification,which inclding ginsenoside Rg₁,ginsenoside Re,ginsenoside Rb₁,psoralen and isopsoralen.And there is no content determination method and no fingerprint method.There is a great distance between the existing standards and the relatively perfect quality control standards in the same industry.It needs to be improved urgently.Therefore,this study intends to improve the quality standard of Jiawei Guilingji Jiu by establishing an effective component prediction method based on chemical component analysis and network pharmacology,and then establishing a fingerprint method to reflect the overall quality control,and increaseing the thin layer identification and content determination.Objective:In order to find the potential active components of Jiawei Guilingji Jiu by using LC-MS combined with network pharmacology.In order to improve the quality control standard of Jiawei Guilingji Jiu by establishing the thin layer chromatography and content determination method,and establishing the fingerprint detection method suitable for Jiawei Guilingji Jiu.Methods:（1）Ingredients of Jiawei Guilingji Jiu was Analyzed and identified by UPLC-Q-TOF-MS high-resolution mass spectrometry.By using 0.1%formic acid water-0.1%formic acid acetonitrile as the mobile phase gradient elution,and the flow rate was 0.3ml·min^-1.Compares it with related databases and related literature according to the retention time,molecular weight and fragment ion fragmentation rules,infers its possible chemical structure and qualifies unknown compounds.（2）A common target network was constructed for ingredients and diseases of the Jiawei Guilingji Jiu by using network pharmacology technology.And analyze the nodes and connection frequency in the network,and it is possible to predict the components with a higher frequency of connection with the target as the material basis.Gene enrichment analysis of common targets is used to predict the key pathways of traditional Chinese medicine in the treatment of diseases.Find out the more important active ingredients,predict the target and pathway of action in the body,and clarify the mechanism of action.（3）The TLC of verbascoside and echinacoside increased on the existing basis.A thin-layer identification method established by using methanol-acetic acid-water（2:1:7）as the developing agent,unfold,take out,dry,and put in UV Check under the light（365nm）.The content of verbascoside and echinacoside of Jiawei Guilingji Jiu was determined by high performance liquid chromatography.methanol-0.5%phosphoric acid water was used as the mobile phase gradient elution,and the contents of newly-built rehmandin D and 5-hydroxymethyl furfural were determined by high performance liquid chromatography.（4）19 batches of Jiawei Guilingji Jiu were detected by using high performance liquid chromatography with 0.1%formic acid water-0.1%formic acid acetonitrile as the mobile phase gradient elution.And then the liquid phase results were imported into the similarity of the fingerprints of traditional Chinese medicines.The data is processed in the software of the evaluation system,and the fingerprint spectrum method of the Jiawei Guilingji Jiu is established.Results:（1）The Jiawei Guilingji Jiu identified 36 compounds by LC-MS high-resolution mass spectrometry.The chemical components of qualitative analysis including phenethanol glycosides,iridoids,ginsenosides,and organic Acid compounds,coumarin compounds,nucleoside compounds,polysaccharide compounds,flavonoids and other compounds.Among them,the two components of verbascoside and echinacoside have a relatively high signal response in the phenethyl alcohol glycosides,and it is recommended that they be quantitatively analyzed.（2）The results of network pharmacology show that the 8-ramenic acid,rehmannia glycoside D,ginsenoside Ro,isoflavin glucoside,psoralen,psoralen,Cistanche glycoside F,ginsenoside Rh₁,icariin,glycyrrhizin,echinacoside,ginsenoside F₁ and verbascoside may be potential compounds for its medicinal effects.And can be used as potential active ingredients of Jiawei Guilingji Jiu Reference.Pathway enrichment analysis found that Jiawei Guilingji Jiu may play a role in delaying aging by participating in the pathways that regulate key cell responses and apoptosis.It may play an anti-inflammatory role by participating in the regulation of inflammatory response and environmental stress pathways.Improve the symptoms of Qi deficiency and blood deficiency by participating in the regulation of hypoxic stress response pathway.In addition,the Jiawei Guilingji Jiu also participates in the regulation of multiple cancer pathways,which may have the potential to prevent related cancers.According to the analysis of the prediction results,it is recommended to determine the content of the two components of verbascoside and echinacoside,which are high in content and have pharmacological activity.In addition,the content of 8-loganic acid,rehmanin D,psoralen and cistanche F is recommended.The pharmacological activity is significant.It is recommended to construct a fingerprint for overall quality control.（3）The TLC method for verbascoside and echinacoside was established,and the HPLC method for four components was established.In thin-layer identification,the test product chromatogram shows the same color fluorescent spots on the corresponding position of the reference chromatogram,and it is negative without interference.The thin-layer identification method for verbascoside and echinacoside was successfully established.Samples were prepared in HPLC content determination The method and detection process are efficient and simple.The methodological investigation complies with the regulations of the pharmacopoeia.The determination methods of verbascoside and echinacoside,as well as rehmandin D and 5-hydroxymethyl furfural have been successfully established.（4）The fingerprint of the Jiawei Guilingji Jiu was established by gradient elution with 0.1%formic acid water and 0.1%formic acid acetonitrile mobile phase.The methodological investigation complies with the regulations of the pharmacopoeia.A total of 27 common peaks were calibrated in the fingerprint spectrum.And the chemical components of the Jiawei Guilingji Jiu were qualitatively analyzed by comparison with the reference product and UPLC-MS,and finally 15 common peaks were identified.Including phenethanol glycosides,Iridoids,organic acids and coumarin compounds can reflect the six potential active ingredients predicted by network pharmacology.Conclusion:In this study,the quality standard of the Jiawei Guilingji Jiu was improved from the perspective of the material basis reflecting the efficacy.First,36 compounds were identified through liquid chromatography-mass spectrometry technology,and Twelve potential active ingredients were predicted through network pharmacology.According to the predictions The active ingredients of the Jiawei Guilingji Jiu’s thin-layer identification,content determination and fingerprinting method were established.Among them,the verbascum glycosides and echinacosides were thin-layered.Identification and content determination.The fingerprints of the components such as8-loganic acid,rehmannia glycoside D,psoralen,cistanche glycoside F,echinacoside,and verbascoside were controlled as a whole.From the point and surface,the point and surface were combined to establish a quality standard that could reflect the stability of the medicinal ingredients of the Jiawei Guilingji Jiu.The quality control of modern Chinese medicine provides scientific method basis.