Study On The Pharmacodynamic Substances Of Qi-yu-san-long Decoction In Inhibiting Lung Adenocarcinoma Based On The Correlation Analysis Of Endogenous And Exogenous Metabolites

Objective: This study based on the ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MSE)technology screening and identifying the prototype components and its metabolites of QYSLD in rat plasma.A rat model of lung adenocarcinoma was established to evaluate the inhibitory effect of QYSLD on lung adenocarcinoma by related pharmacodynamic indexes.The pharmacodynamic substances of QYSLD inhibition of lung adenocarcinoma have been explored using spectrum-effective relationship.Method: Based on UPLC-Q/TOF-MSE technique combined with four-step analysis method,the prototype components and their metabolites in rat plasma after QYSLD administration were identified,and plasma samples were collected at different time points to analyze their dynamic metabolic profiles in vivo.Wistar rats were used as experimental animals for the construction of lung adenocarcinoma model by intraperitoneal injection of urethane.The appearance changes of rat lung,organ index,pathological section of lung tissue and microstructure change of lung tissue were used to evaluate the efficacy of the drug,and the success of the model was judged.Using the endogenous metabolites regulated by drug intervention as the drug efficacy index,grey correlation analysis(GRA)and canonical correlation analysis(CCA)were used to analyze the correlation between the metabolites related to QYSLD into the blood as the "spectrum" and the efficacy index of endogenous metabolites as the "effect" to screen the drug efficacy substances of QYSLD inhibiting lung adenocarcinoma.Results: 1.In vivo metabolism of QYSLD: A total of 101 QYSLD compounds(including 41 prototype components and 60 metabolites)were identified,and the metabolic pathways and dynamic metabolic profiles of alkaloids,saponins,flavonoids,iridoids,anthraquinones and phenylpropanes in this formula were revealed.Flavonoids in QYSLD mainly underwent deglycosylation,demethylation,dehydroxylation,glucuronidation and acetylation;alkaloids and saponins in QYSLD mainly underwent deglycosylation,desaturation,oxidation,reduction and acetylation;deglycosylation and demethylation were the main metabolic reactions of iridoids in QYSLD;sulfation was the main metabolic reaction of the anthraquinones in QYSLD;methylation,hydroxylation,sulfation and glucuronidation were the main metabolic reactions of phenylpropanoids in QYSLD.Secondly,dynamic metabolic profiles at different time intervals can better understand the characteristics of biotransformation of QYSLD metabolite in rat plasma.In the plasma samples of the administration at 0.5 h,15 prototype components and 11 metabolites were detected,indicating that these metabolites could be absorbed and metabolized quickly.There are four prototype components and 10 metabolites that can be detected after 24 h,indicating that these metabolites resided in the body for a long time.4 prototype components and 10 metabolites have the phenomena of appear-disappear-reappear.2.Pharmacodynamic evaluation results: The confirmation of the lung adenocarcinoma model was based on observations of lung appearance,spleen and lung indexes,pathological characteristics,and ultrastructural changes before and after modeling.Compared with the model group,the lung cancer appearance in the QYSLD groups were reduced,the spleen index was increased,and the lung index was decreased.The results of H&E staining in QYSLD group showed that the alveolar structure was basically intact,and the apoptotic tumor cells were round,with dark nuclei and condensed cytoplasm.The results of transmission electron microscopy in the QYSLD group showed that microvilli disappeared on the surface of apoptotic cells,and nuclear chromatin condensation and edge collection were observed.3.Results of pharmacodynamic substance screening: The correlation degree between the peak areas of exogenous metabolites and endogenous metabolites in GRA was greater than 0.8 as a screening criterion,and 38 QYSLD-related metabolites were predicted to be potentially inhibitory substances in lung adenocarcinoma.The correlation coefficient between the peak areas of exogenous metabolites and endogenous metabolites in CCA was greater than 0.6 as a screening criterion,and 29 QYSLD-related metabolites were predicted to be potentially inhibitory substances in lung adenocarcinoma.Among them,GRA and CCA together predicted 24 pharmacodynamic substances,including peimisine(P’2),3-hydroxy-flavone(P’6),formononetin(P’8),2-hydroxy-3-methylanthraquinone(P’10),1-(11Z-octadecenoyl)-Phosphocholine(P’11),E-p-coumaric acid-sulfate conjugate(M’3),caffeic acid-O-glucuronide(M’6),ferulic acid-O-sulfate conjugate(M’5),calycosin-O-glucuronide(M’8),formononetin demethylated metabolite(M’15),sulfate conjugates after demethylation of formononetin(M’7),formononetin-O-glucuronide(M’10),E-6-O-p-coumaroyl scandoside methyl ester deglycosylation metabolite(M’12),curcolone desaturated metabolite(M’13),2-hydroxy-3-methylanthraquinone-sulfate conjugate(M’14),imperialine-acetylated conjugate(M’22),methylated metabolite of Astragaloside Ⅶ after deglycosylation(M’23),reduction metabolites of Astragaloside Ⅰ with one acetyl group removed(M’24),solanamine dehydrated metabolite and solanamine acetylation conjugate(M’25 and M’26),peimisine acetylation conjugate after newly reduced reaction(M’27),soladulcoside A deglycosylation metabolite(M’29),cyclogalegenol and its desaturated metabolite(M’33 and M’28).Conclusion: First,UPLC-Q/TOF-MSE was used to quickly identify the components of QYSLD in plasma.Second,the rat model of lung adenocarcinoma was successfully established.Compared with the model group,all the parameters of the QYSLD group were improved.Finally,GRA and CCA analysis methods were used to analyze the correlation between endogenous and exogenous metabolites.In GRA,the correlation degree between exogenous metabolite peak area and endogenous metabolite peak area was greater than 0.8 as the screening criterion,and 38 QYSLD-related metabolites were predicted to be potential pharmacological substances for inhibiting lung adenocarcinoma.In CCA,the correlation coefficient between exogenous metabolite peak area and endogenous metabolite peak area was greater than 0.6 as the screening criterion,and 29 QYSLD-related metabolites were predicted to be effective substances for inhibiting lung adenocarcinoma.Among them,GRA and CCA jointly predicted 24 QYSLD-related metabolites to be effective substances for inhibiting lung adenocarcinoma.