| Objective: By establishing a low-temperature ginger preparation process,we investigated the effect of low-temperature ginger preparation on the protein components in the Bombyx Batryticatus.And according to the clinical application and pharmacological effect of the silkworm,the pharmacological effect of the protein in the ionic glutamate receptor abnormality was explored,and at the same time,the pharmacological effect was compared with that of the raw product in order to make it clear that the ginger method has changed the pharmacological effect of the silkworm,and at last,the cyclic peptide compounds in the silkworm,Paecilomyces albidus,were investigated,so that it could provide a new theoretical basis for the pharmacological effect of the Bombyx Batryticatus and the mechanism of its action.Finally,a new study on the pharmacological action of the cyclic peptide compound C.albicans in the silkworm was conducted to provide a new theoretical basis for the pharmacological material basis and mechanism of action of the ginger silkworm.Methods: 1.Taking total protein and BEA content as the indexes of investigation,the range of factors was determined through the single-factor investigation of infiltration time,drying temperature,drying time and excipient ratio,and orthogonal experiments were used to establish the concoction process of low-temperature ginger preparation of stibnium.The most red compared with other concoction methods to observe the effect of ginger preparation on total protein and BEA content.2.The range of factors for the extraction of total amino acids from ginger Bombyx Batryticatus was determined by a one-way study of the buffer system,extraction solvent,extraction reagents and material-liquid ratio,and orthogonal experiments were conducted to determine the optimal method for the extraction of amino acids from the Bombyx Batryticatus.According to the results of orthogonal experiments,the optimal extraction process was established for the extraction of total amino acids from ginger Bombyx Batryticatus,raw Bombyx Batryticatus,and three other commonly used concoctions of Bombyx Batryticatus,and the content of 23 essential amino acids contained in the extracts were determined by LCMS/MS and analyzed the differences between ginger Bombyx Batryticatus and other concoctions as compared with the raw products.3.Qualitative identification of the protein components in the ginger prepared Bombyx Batryticatus by nano-lc-ms/ms,and discovery of the effect of the ginger preparation method on the types and quantities of protein components in the Bombyx Batryticatus by comparing them with those of the raw Bombyx Batryticatus.4.By examining the separation and purification of the protein of the ginger silkworm by salting out,isoelectric point method and ultrafiltration method,the optimal purification method was selected to extract and purify the protein components of the raw and ginger Bombyx Batryticatus and used for the subsequent study of their pharmacological effects.An in vitro cell model of Glu-induced HT-22 was established to simulate the pathological characteristics of abnormal ionotropic glutamate receptors,and low,medium and high doses of raw and ginger extracts were administered to study the mechanisms of action,and the differences and similarities between raw and ginger extracts in terms of the concentration of administration and the mechanisms of action were also compared.Firstly,the half inhibitory concentration of the raw and ginger extracts was determined by cell viability,and the effectiveness of the protein was determined by cell viability,ca+,ROS,mitochondrial membrane potential assay,and apoptosis assay,and then the mechanism of action and potency concentration of the two extracts were analyzed by transcriptomics and metabolomics,and the potential signaling pathways and mechanisms of action were discovered by the two-ohomics joint analysis.We also analyzed the potential signaling pathway and mechanism of action through the combined dual-omics analysis.Finally,the molecular biology experiments were verified by western blot.5.The oxidative stress model was established by inducing PC-12 cells by H2O2,the antioxidant effect of BEA was determined by cell viability,ros assay,mitochondrial membrane potential assay,and the mechanism of action was analyzed by metabolomics,and finally,the mechanism was verified by wb.Results: 1.Taking the total protein and leukocidin content as evaluation indexes,the optimal concoction process of ginger-made Bombyx Batryticatus was determined as the ratio of auxiliaries 1:10,the smothering time 1.5h,the drying time 4h,and the drying temperature50°C.The total protein content and leukocidin content of ginger-made Bombyx Batryticatus were found not to be significantly affected.Comparison of other concoctions and raw products showed that the content of Paederin and total protein of ginger-made stiffworms were not significantly affected,while the total protein content of bran-fried method was significantly reduced,and the content of Paederin of rice slop water system and dense bran-fried method was significantly reduced.2.The optimal process for the extraction of total amino acids was 1:90 ratio of material to liquid,extraction time of 40 min,and extraction solvent of 60% methanol.The results of the determination of the amino acid content of the 23 amino acids in the ginger Bombyx Batryticatus and other concoctions were as follows: the total content of the 23 amino acids in the ginger Bombyx Batryticatus was the highest,which was 49,402.4 μg/g,and the amino acids with the highest content were glutamic acid,aspartic acid,alanine,arginine,etc.,of which acidic amino acids had the absolute dominant proportion.The other methods of concocting had some influence on the amino acid content,the total amount of 23 kinds of amino acids were reduced to different degrees than the raw products,and the stiff Bombyx Batryticatus of the two frying methods were accompanied by the disappearance of two kinds of amino acids.3.The results of protein identification of ginger-made stiffworms showed that after the stiffworms were ginger-made,the proteins of Coccidioides albicans origin increased significantly in quantity and content compared with the raw products,and the highest content of proteins in the ginger-made stiffworms were xdw,etc.,which contained a variety of enzyme components as well as immune proteins,and the contents of 3’(2’),5’-diphosphate ribonuclease and δ-aminolevulinic acid dehydrogenase were higher,and the immune proteins The content of3’(2’),5’-diphosphate nucleotidase and δ-aminolevulinic acid dehydratase is high,and the content of immunogenic proteins,such as geranylgeranyl protein,is high.4.Through the examination of three protein isolation and purification methods,the final method selected for the isolation and purification of ginger stiffworm protein was the combination of isoelectric point method and ultrafiltration method,and the purity of the extracted protein was 76.83%.The half inhibitory concentrations(IC50)of the protein extracts of ginger and raw B.pinnatifida were 218.3 μg/m L for ginger and 220.5 μg/m L for raw B.pinnatifida,and the results of the cell viability assay showed that the protein extracts of ginger were lower than those of raw B.pinnatifida in terms of the onset of action concentration.When the concentration exceeded 50 μg,the protein extract of the ginger stiffworm could exert its efficacy.In order to facilitate the subsequent comparative experiments as well as to refer to the IC50 concentration results,the concentrations of 100 for the low dose,150 for the middle dose,and 200 μg/m L for the high dose were finally selected for the subsequent pharmacological experiments.The calcium ion concentration assay revealed that ginger stilbene was superior to the raw product in terms of effective concentration,and PG-BBPs exerted a better modulation effect on intracellular calcium ions as early as the low dose,while N-BBPs had an obvious dose-dependence,and this tendency was also shown in the results of the ROS as well as MMP assays.In the apoptosis assay,PG-BBPs had an optimal anti-apoptotic effect at a medium dose,and the apoptosis rate was close to the level of the blank control group,which was superior to that of N-BBPs in apoptosis level.The results of transcriptomics showed that the main differential genes between PG-BBPs and N-BBPs were concentrated in the MAPK signaling pathway and the PI3K/AKT signaling pathway However,in the comparative analysis,it was found that PG-BBPs were better than N-BBPs in the regulation of calcium ions and other related functions,and the enrichment of differential genes in signaling related functions of PG-BBPs was significantly better than that of N-BBPs,and the analysis of the corresponding dose groups showed that the number of differential genes in the low-dose group was significantly higher than that of N-BBPs.metabolism The results of metabolomics analysis showed that PG-BBPs were superior in the regulation of fatty acid biosynthesis and glutathione metabolism,and the related metabolites were significantly better than N-BBPs.The final joint analysis showed that the differential genes and metabolites of PG-BBPs were significantly more enriched in GPCRrelated signaling pathways,indicating that PG-BBPs have a more significant role in the activities regulated by the G-protein-coupled receptor(GPCR).The experimental results of WB suggest that PG-BBPs can reduce cellular damage by decreasing the phosphorylation level of MEK-ERK proteins and thus have an advantage over N-BBPs in terms of dosage.5 The antioxidant effect of BEA was determined by cell viability assay at concentrations of 0.5 and 1 μM,which disappears with increasing concentration.In ROS as well as MMP assays,BEA effectively reduced the elevation of ROS caused by oxidative stress and could increase MMP levels.The results of metabolomics analysis showed that BEA was mainly involved in lipid-related metabolism,and could significantly improve the effect of related metabolite callback.The results of WB experiments confirmed that BEA could improve cell survival under oxidative stress mainly by participating in the PI3K/AKT/MTOR signaling pathway.Conclusion: The concoction process indicated that the low-temperature ginger preparation of stiff Bombyx Batryticatus was stable and reliable,and the ginger method had no significant effect on the total protein and leukocidin content.The 23 essential amino acids of ginger-made stiffworms were slightly elevated compared with those of raw products,and it was speculated that they might originate from the addition of ginger juice during the smothering and moisturizing process.The results of protein identification showed that among the protein species of the ginger Bombyx Batryticatus,the number of proteins of C.albicans origin increased significantly compared with that of the raw product,and most of the proteins with higher content belonged to enzyme components,which might be potential bioactive components.The low-temperature ginger stiffener protein extracts showed better modulation in ionotropic glutamate receptor anomalies than the raw product,mainly in terms of effective concentration.Finally,the present study confirmed that BEA has antioxidant effects and showed excellent antioxidant activity in in vitro experiments,and this result may provide a strong basis for BEA as an active ingredient in the Bombyx Batryticatus. |
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