| Objective:On the basis of the previous research,this study used UPLC-Q-TOF-MS/MS technology to analyze and identify the whole components of malt.At the same time,the material basis and mechanism of malt treatment for hyperprolactinemia were discussed based on network pharmacology.Total component analysis of malt total alkaloids was carried out to compare the content changes of characteristic components in different germinating cycles and different processing products,and the pharmacodynamic effects of characteristic components on DRD1 and DRD2 were investigated by cell experiments in vitro.The preparation and quality control of malt syrup were preliminarily carried out.Methods:(1)AB SCIEX Triple TOF? 4600 High Resolution Mass Spectrometry and Waters Cortecs UPLC?C18(2.1×100mm,1.6μm)1290 UPLC were used for the gradient elution using acetonitrile-2mmol /L formic acid aqueous solution as mobile phase.The flow rate was 0.3m L/min,the detection wavelength was set at 254 nm,the column temperature was 25℃,and the sample volume was5μL.The electrospray ion source was used for Mass spectrometry,and the positive and negative ion modes were collected.Agilent Mass Hunter was used for qualitative analysis B.04.00 for data collection and processing.(2)The literature related to malt composition analysis was collected and the TCMSP database was used to summarize the compounds with high content analyzed by UPLC-Q-TOF-MS/MS,and the active ingredients of malt were screened out and the corresponding targets were predicted.HPRL-related targets were searched in OMIM and Genecards databases,and then crossed with malt targets to obtain key targets.The STRING database was used for analysis and their protein-protein interaction(PPI)network was constructed.The GO function and KEGG signaling pathway enrichment analysis of the key targets were carried out by using David database.Cytoscape 3.6.0 software was used to establish the malt active ingredient-target pathway model.(3)Extraction of total alkaloids from malt,UPLC-Q-TOF-MS/MS method was used to select suitable mobile phase,gradient elution conditions,mass spectrometry conditions,flow rate,detection wavelength,column temperature for total component analysis of malt alkaloids.The total malt alkaloids were prepared with different germinating cycles(1-5 days)and different processed malt alkaloids.The contents of three alkaloids,namely malt alkaloids,N-methyltyramine and arrowine,were compared in different germinating cycles and different processed malt alkaloids,and the pharmacological effects of these three alkaloids on DRD1 and DRD2 were investigated by in vitro cell experiments.(4)preparation of malt syrup,determine the sample appearance,and main ingredients hordenine as indicators,using thin layer chromatography(TLC)in the same silica G thin layer plate on the qualitative identification of malt syrup cuhk alkali is studied,at the same time establish a high performance liquid chromatography(HPLC)method,with hordenine as indicators,to quality control of syrup,and methodology validation of the method.Results:(1)By comparing with the database and referring to references,33 chemical components were detected in the data obtained from UPLC-Q-TOF-MS/MS,14 of which were alkaloids(malt pharmacodynamic substances).The cracking rules of some alkaloids and flavonoids were selected and analyzed.(2)Eighteen chemical components and 268 targets of malt were obtained after screening and removing the repeated products.There were 221HPRL-related targets,22 of which were targets of malt treatment for HPRL.PPI network analysis suggested that these 22 targets(such as DRD1,DRD2 and DRD3)might be key targets for malt treatment of HPRL.Through GO functional enrichment analysis(P<0.05),143 items related to dopamine binding,drug response,dopamine neurotransmitter receptor activity,positive transcriptional regulation of RNA polymerase II promoter,and negative regulation of protein secretion were obtained.KEGG signaling pathway enrichment analysis revealed 35 signaling pathways,including dopaminergic synapses,interactions in stimulating nerve tissue,calcium signaling pathway,gap binding,c AMP signaling pathway,etc.(3)By comparing with the database and referring to the references,the data obtained from UPLC-Q-TOF-MS/MS finally detected that the total malt alkaloids contained 33 chemical components.Comparing different germination period and different processed products of malt total alkaloid cuhk malt alkali,N-methyl tyramine,gramine three kinds of alkaloid content,in 3 batches of samples,the results showed that N-methyl tyramine the highest peak,most abundant,with the extension of germination period,the N-methyl tyramine gradually increased with the content of hordenine,germination 4 days,detected gramine,After 5 days of germination,the content of maltine was almost the same as that of N-methyltyramine.From the point of view of the whole process of germination,the growth rate of malt base is much higher than that of other substances,and from the structure of malt base,it is found that it has the same parent nucleus structure with N-methyltyramine,so it is inferred that the two alkaloids may be transformed into each other during the process of barley germination.Among the untreated raw malt total base extracts,the contents of malt and N-methyltyramine were the highest,and the peak of arrowidine could also be observed.The contents of the three alkaloids were significantly reduced after the processing of stir-fried malt,while the contents of the three alkaloids were very small compared with the contents of the unprocessed malt when the processing of coke malt continued.Competent receptor ligand binding experiments showed that maltine and N-methyltyramine had affinity with dopamine D1 receptor,but the affinity between ardostine and dopamine D1 receptor was weak,and all the three drugs had affinity with dopamine D2 receptor.Testing the drug to the D1 and D2 receptors excited or inhibition,the results show that the high and low doses of three drugs to dopamine D1 receptors had no effect,hordenine high,middle dose group of dopamine D2 receptors have excited effect,and low dose of hordenine without this function,N-methyl tyramine high and low dose of dopamine D2 receptors have excited effect,High and low doses of alodostine had no effect on dopamine D2 receptor.(4)According to the inspection results of the syrup sample,it is determined that the product is a thick liquid from brown to brown,with a sweet taste.Three batches of samples were taken to identify the malt in the syrup.The prepared test solution was sampled and determined according to the established chromatograms.The results showed that the average content of malt was 11.49 mg·g-1,which accounted for 1.15% of the malt syrup.Conclusions:(1)According to the results of UPLC-Q-TOF-MS/MS analysis of malt and its total alkaloid compounds,it can be seen that the chemical components of malt and the contents of some alkaloids change with the change of germination cycle.The changes of these components explored in this paper indicate that malt is closely related to clinical efficacy.(2)Using network pharmacology to explore the mechanism of malt in the treatment of hyperprolactinemia,it can be found that the active components in malt can act on the same target,but also can act on different targets,suggesting that malt plays a role in the treatment of hyperprolactinemia through the synergistic regulation of multi-component,multi-target and multi-pathway.(3)When the malt germinated for 5 days,its alkaloid composition and content reached the maximum,and it was the best harvest time.It is necessary to monitor the change of alkaloids by liquid/mass coupling(LC-MS)closely during malt processing because of the changes of chemical chemistry during malt processing.The pharmacodynamics of the three alkaloids monomers in malt total alkaloids were studied.It was found that malt and N-methyltyramine had an agonizing effect on DRD2 and could exert the bromocriptine-like effect. |
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