| Objective: To study the changes of processing components of Gastrodia elata processed with Jiang from Jiangxi "Jianchangbang" and its effect mechanism on migraine.Methods:(1)Study on composition changes of Gastrodia elata before and after processing.The volatile components were studied.The chemical components of Gastrodia elata and Gastrodia elata were extracted by cold dip method.The ginger was extracted by pressing method,and then detected by GC-MS to compare the components of 3 samples.The non-volatile components were extracted by ultrasound and detected by UPLC-Q-TOF-MS.The composition changes of four different processed products were compared and analyzed by means of Peak View1.2 and Sim CA14.1.(2)With gastrodin,gastroaglycones,p-hydroxybenzoic acid,salicin A,B,C,and E and comprehensive score as evaluation indexes,after examining the single factors such as solid-liquid ratio,time,temperature,particle size,and solvent,the response surface design method was used to optimize and screen the interaction of solid-liquid ratio,temperature and solvent that had a greater impact on the extraction,and the optimal extraction process of Rhizoma Zingiberis Recens and Gastrodia elata was obtained.(3)The migraine model was established in rats using nitroglycerin.The contents of ET and CGRP in plasma,5-HT and DA in brain tissue and the like were determined by ELISA,and the content of NO in plasma was determined by biochemical method.The behavioral and biochemical indicators of animals in each group were compared to investigate the differences of migraine effects between different processed products of Gastrodia elata.(4)The potential migraine-related targets of the active components of Gastrodia elata and Rhizoma Zingiberis Recens were predicted by Swiss Target Prediction,TCMSP analysis platform and database resources such as Genecard,TTD,OMIM,and Drugbank,and the network relationship diagram among drugs,components and diseases was constructed in the experiment.Combining with the PPI network constructed by the matching results,we screened the core targets of migraine induced by drugs.Besides,the functional signaling pathway diagram of Rhizoma Zingiberis Recens and Gastrodia elata for the treatment of migraine was established by GO function and KEGG pathway enrichment analysis.Molecular docking verification of the active components and core targets was performed using Discovery Studio2016 software,the docking strength of drug component targets on migraine target was analyzed,and the possible mechanism of Gastrodia elata processed with ginger on migraine was summarized.Results:(1)GC-MS analysis of volatile components detected 27,17 and 30 chemical components in Gastrodia elata,Gastrodia elata Rhizoma Zingiberis Recens product and fresh Rhizoma Zingiberis Recens respectively after 150 d days’ standing.After Gastrodia elata was processed with ginger,the content of volatile components was lower than that of Gastrodia elata raw product and Rhizoma Zingiberis Recens under the same condition.Part of the volatile components after Gastrodia elata was processed with ginger were not detected in Gastrodia elata,such as 1-hexene-3-ol,ethyl palmitate,valeraldehyde,4-methyl-1,3-dioxane,2,4,5-Trimethyl-1,3-dioxolane,ethyl linoleate,and five volatile components in Rhizoma Zingiberis Recens were introduced.Under positive and negative ion mode,47 chemical components of Gastrodia elata processed products were detected and identified from non-volatile components,including 25 phenols,6 organic acids,7 alkaloids,9 esters and other components.The results of PCA and OPLS-DA indicated that there were significant differences in the content of chemical components from different processed products of Gastrodia elata,and 21 processing differential components with the differential VIP value greater than 1,such as salicin F,salicin A,and bis(4-hydroxyphenyl)ether,were identified,and 12 components from Rhizoma Zingiberis Recens were added.(2)On an Acquity UPLC BEH C18 column(100 mm×2.1,1.7μm),volume flow of 0.2 m L/min,and column temperature of 35 C;Mobile phase: acetonitrile(A)-0.05%phosphoric acid aqueous solution(B);Gradient elution: 0–14 min,3%–29%(A);The detection wavelength was 220 nm,and the optimal extraction process was obtained as follows: 70% ethanol,solid-to-liquid ratio of 1:15,temperature of 80℃,and ultrasound for 40 min.(3)Compared with the blank control group,the number of head scratching in the model group was significantly increased(P< 0.01);Plasma NO and CGRP were significantly increased(P < 0.01),while ET and 5-HT were decreased(P < 0.01),suggesting that the model was successfully established and replicated.Compared with the model group,the scratching times of rats in the positive control group in the 0–30 and 30–60min time zones were significantly reduced(P < 0.01),the contents of NO and CGRP were decreased(P < 0.01),the contents of ET and 5-HT were increased(P < 0.05),and AD was increased(P>0.05)but without statistical significance.In the raw Gastrodia elata and Rhizoma Zingiberis Recens Gastrodia elata groups,the number of scratching of the head was significantly reduced in two time zones of 0–30 and 30–60 min(P < 0.01),while ET and 5-HT were increased(P<0.05).The contents of CGRP and NO were decreased(P < 0.05),with the decrease in Gastrodia elata group being more significant.The AD content of each group was increased,but it was particularly significant in the Gastrodia elata group(P < 0.01),and the increase in the raw Gastrodia elata group was not statistically significant(P>0.05).(4)In network pharmacology research,27 active components such as vanillin and gastrodin in Gastrodia elata,and 4 active components such as β-sitosterol in Rhizoma Zingiberis Recens were screened out through drug-like properties and gastrointestinal absorption,469 active component action targets were found out through component targets,1,496 migraine disease targets,123 drug disease common targets,and 17 drug and disease core targets.ACE,ESR1,EGFR,CNR1,PTGS2,CASP3,CCR5,AGTR1,SLC6A4,SRC,PIK3 CA,APP,GRM5,F2,COMT,OPRM1,HTR2 A,respectively.The GO functional enrichment analysis revealed that the biological processes of Rhizoma Zingiberis Recens and Gastrodia elata were mainly metabolism,biological regulation,protein binding,and vasoconstriction regulation.The KEGG pathway analysis revealed 10 significant pathways,which were mainly involved in serotonergic synapses,human cytomegalovirus infection,and neural active ligand-receptor interaction.The core targets for molecular docking verification showed good binding to the active ingredients.Conclusion:(1)Gastrodia elata and Rhizoma Zingiberis Recens processed have effects on components,and the main changes involve component content and chemical properties.The changes of volatile components in ginger were mostly the active components of Gastrodia elata,which had potential medical value,indicating that the processing method of Gastrodia elata with ginger juice proposed by Jianchang Gang was reasonable.(2)Among the changes of components in different processed products of Gastrodia elata,the changes of barricade glycosides were dominant,and these components were closely related to the action of migraine.At the same time,in combination with response surface methodology,the optimal extraction process of the active components in Gastrodia elata was stable and feasible,which could be used for extraction and administration in subsequent pharmacological studies.(3)Pharmacodynamic experiments further showed that the treatment with Gastrodia elata and Rhizoma Zingiberis Recens had an enhanced effect on nitroglycerin-induced migraine in rats.(4)The results of network pharmacology analysis showed that the possible action paths of Gastrodia elata after ginger treatment on migraine were diverse.To sum up,the experimental results showed that the volatile and non-volatile components of Gastrodia elata significantly changed after ginger treatment.The pharmacological study showed that ginger treatment could improve its effect on migraine.The main mechanism may be that it can improve the symptoms of migraine by reducing NO and CGRP and increasing the content of ET,DA and 5-HT.Meanwhile,the changed components,such as bis(p-hydroxybenzyl)sulfide,4,4’-sulfinyl(methylene)diphenol,β-sitosterol and stigmasterol,can exert effects on ACE,ESR1,EGFR,CNR1 and other targets.So as to exert that anti-migraine effect.It indicated that the efficacy of Gastrodia elata was enhanced after ginger treatment,which provided the basis for further study of Gastrodia elata. |
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