The Study On Bioactive Substance And Chromatographic Fingerprints Quality Evaluation Of Chuankezhi Injection

Traditional Chinese medicine(TCM)injection extensively use in clinical application beacause of its fast and strong effect.Recently,the safety and effectiveness of TCM injections are difficult to guarantee due to the outdated product technology,unclear material foundation,and low quality control level,etc.Chuankezhi injection,which is one of TCM injection,consists of Epimedii Folium(淫羊藿)and Morindae officinalis Radix(巴戟天).It has the effects of warming kidney and nourishing yang,relieving cough and asthma.In order to comprehensively improve the quality control and efficacy of Chuankezhi(CKZ)injection,this study focuses on the pharmacodynamic substances,quality evaluation and metabolic process of CKZ injection.The aim of this study is to establish a new quality evaluation model with strong practicability and high quality controllability by using chromatographic fingerprints technology combined with quantitative detection of main active ingredients,and to explore the metabolism and pharmacological effect of the main pharmacodynamics of CKZ injection in-vitro and in-vivo respectively.The study provides data support for the safety and efficacy of CKZ injection for clinical application.ObjectiveA quality evaluation method based on LC-MS fingerprint was established for CKZ injection,and its metabolism and biological activity were explored by in-vivo and in-vitro experiments repectively in order to improving the quality control standards and evaluation methods of CKZ injection.MethodsAccording to the established LC-MS fingerprinting method of CKZ injection,LC-MS fingerprint of 34 batches of CKZ injection were analyzed,and the fingerprints were overlayed by normalization using ChemPat t ern2017 fingerprint.analysis software to construct "common pattern" fingerprint for CKZ injection.Based on the common pattern,34 batches of samples were subjected to similarity and stoichiometry analysis.The chemical identity of each characteristic peak in t.he common pattern was recognized by using chemical reference,literature data,and mass spectrometry cleavage.Metabolism studies of Epimedin C and Epimedin A were performed by using SD rats.Rat.s were randomly divided into an oral administration(dose:50 mg/kg)and an intramuscular administration(dose:20 mg/kg)group.Fecal and urine samples of each group were collected at 12 h before administration(control),and 0-24 h and 24-48 h after administration.HPLC-series linear ion trap electrostatic field orbitrap mass spectrometer was used to investigate the metabolic transformation of Epimedin C and Epimedin A in rats.The metabolites and mass spectrometry behavior were identified and analyzed.Human alveolar epithelial carcinoma cell line A549 was cultured in vitro and different concentrations of CKZ injection and its active components and metabolites were added.The viability and apoptotic effect of A549 cells were detected by CellTitle-Glo and Caspase-Glo 3/7 testing kits.ResultsAfter establishing LC-MS fingerprints of 34 batches of CKZ injection,the"common pattern" was successfully constructed and 21 chemical characteristic peaks were drawn.The similarity analysis was performed on each samples by using the common pattern,the overall similarity between batches is high except sample 1,3,4,and 6(similarity is less than 0.95).The results of principal component analysis(PCA)and similarity are consistent except samples 1,3,4 and 6,It indicated that the difference of batches was small and the quality was stable.20 out of 21 chemical characteristic peaks in common pattern were successfully recognized.Epimedin C and Epimedin A at 0-24 h after administration,14 and 19 possible metabolites were detected from fecal samples respectively.All fecal and urine sample from control and 24-48 after administration,the original chemical and its metabolites were not detected.It indicated that the Epimedin C and Epimedin A were fast eliminated,but in different metabolic routes in rat.The administration method will not change the metabolic pathways of Epimedin C and Epimedin A.The metabolic routes of Epimedin C were deglycation,dehydrogenation,oxidation,glycan-binding,demethylation and acetylation;while Epimedin A were deglycation,glycan-binding,demethylated,hydrogenated,acetylated and hydration.In vitro experiments showed that CKZ injection,Epimedin C and 2"-0-rhamnose icariin II were significantly inhibited the viability of A549 cells compared with control.Epimedin C and 2"-0-rhamnose icariin II may be.related to up-regulation of caspase3/7 protein which associated with apoptosis.ConclusionSuccessfully constructed the"common pattern" LC-MS fingerprint for CKZ injection and 21 chemical characteristic peaks were drawn.Similarity andstoichiometry analysis showed that the overall similarity was high,the difference between batches was small and the quality was stable;this study successfully recognized 20 characteristic peaks.Epimedin C and Epimedin A at 0-24 h after administration,14 and 19 possible metabolites were detected from fecal samples respectively.All fecal and urine sample from control and 24-48 after administration,the original chemical and its metabolites were not detected.It indicated that the Epimedin C and Epimedin A were fast eliminated,but in different metabolic routes in rat.The administration method will not change the metabolic pathways of Epimedin C and Epimedin A.CKZ injection,Epimedin C and 2"-0-rhamnose icariin II were significantly inhibited the viability of A549 cells compared with control.Epimedin C and 2"-0-rhamnose icariin Ⅱ may be the bioactive ingredient of CKZ injection.