Study On The Detection Of Some Active Substances In Dachuanxiong Fang And The Preparation Of Standard Product

Dachuanxiong prescription is composed of chuanxiong Rhizoma:gastrodiae Rhizoma in a ratio of 4:1.It is a traditional Chinese medicine compound commonly used to treat migraine.The chemical components isolated and identified in gastrodiae Rhizoma are mainly phenols,organic acids,polysaccharides and steroids.In addition to gastrodin,Parishin components in gastrodia elata were also reported to have good neuroprotective effects.chuanxiong Rhizoma mainly contains phthalide,phenolic acid and alkaloid etc.Based on the previous findings of the research group,it was found that the hydroxybenzyl alcohol ester in gastrodiae Rhizoma was metabolized into gastrodin by 99%after entering the body,and Parishin was the prodrug of gastrodin in the body.However,gastrodin was difficult to pass the blood-brain barrier,and the active substance in the body has always been questioned.chuanxiong Rhizoma is mainly composed of phthalide in vivo and is unstable and easily oxidized in vivo,so it is speculated that these phthalides entering the plasma may be pharmacodynamic components.Therefore,it is important to further clarify the quality of gastrodiae Rhizoma and the active ingredients of gastrodin and chuanxiong Rhizoma.In this paper,the quality of gastrodiae Rhizoma,the identification of the metabolites in gastrodin,the electrochemical and mass spectrometry techniques and the separation and preparation techniques were used to prepare the metabolites of gastrodin and the components of ligustrum chuanxiong Hort phthalide.The main research contents are as follows:(1)In this study,A high-sensitivity fluorescence detection method was established for simultaneous quantitative analysis of gastrodin,p-hydroxybenzenyl alcohol,Parishin A,Parishin B and Parishin C in 28 batches of gastrodiae Rhizoma from different origins.The results showed that the quantitative lower limit of gastrodin and p-hydroxybenzyl alcohol was 3 ng·ml-1,and the quantitative lower limit of Parishin A,Parishin B and Parishin C was 10 ng·ml-1,with good precision(RSD<5.0%)and good repeatability(RSD<5.0%),and the average recovery(n=5)was 97.4%,96.9%,99.1%,98.5%and 96.3%,respectively.The contents of the above five components in 28 batches of samples were 0.48～20.01,0.08～2.54,0.49～15.44,1.65～8.20,0.73～2.12 mg·g-1,respectively.(2)In this study,UPLC-Q-TOF-MS was used to comprehensively identify the metabolites of gastrodin in blood,liver and brain tissues of rats.Results show that through the metabolites of precise molecular mass,mass spectrometry cracking rule and characteristic fragment ions,according to the diagnostic ion binding,neutral loss and Agilent Metabolite ID software,select 12 gastrodin metabolites in the body,its the main metabolic reactions such as oxidation,glucose aldehyde acid,sulfuric acid ester.(3)This study established the electrochemical and mass spectrometry technology preparation of gastrodin phase I metabolites.The results showed that,under the optimal electrochemical conditions,the phase I oxidation products of gastrodin were obtained by on-line electrochemical and mass spectrometry(EC-MS)simulation and electrochemical preparation electrode enrichment,and then 77 mg phase I oxidation products of gastrodin were obtained by preparative high performance liquid chromatography separation and purification.(4)In this study,a variety of atmospheric pressure chromatographic columns were used to separate the components of the chuanxiong Rhizoma.The results showed that 9 chemical components were obtained from 50%ethanol:ferulic acid,senkyunolide I,senkyunolide A,senkyunolide N,levistilide A.compounds 6 and 7 could not achieve the baseline separation in the high performance liquid phase,the separation in the preparative liquid phase could not be realized,and the monomer structure could not be confirmed.In this experiment,SFC method was established to separate compounds 6 and 7 respectively.Two monomers of compounds 6 and 7 reached the baseline separation.In the later stage,SFC was used to enrich and separate four monomers and confirm their structures.Conclusion:The fluorescence detection method with high sensitivity established in this study has the advantages of less impurity interference,good separation degree,high sensitivity and wide linear range,which is suitable for gastrodia elata composition analysis and quality control of medicinal materials,and provides reference for further improving the quality standard evaluation method of gastrodiae Rhizoma.Twelve metabolites of gastrodin in blood,liver and brain tissues were screened by UPLC-Q-TOF-MS/MS,and the main metabolic reactions were oxidation,gluconaldehyde acidification and sulfuric acid esterification.The established electrochemical simulation system can be combined with mass spectrometry to detect the generation of metabolites online,which has the advantages of less interferences,fast speed and no need for many animals,etc.thus opening up new ideas and methods for the preparation of metabolites of traditional Chinese medicine.The separation of 9 chemical components from chuanxiong Rhizoma by various normal pressure chromatographic columns has laid a foundation for the search of active components of chuanxiong Rhizoma.