Study On The Active Components In Magnolia Officinalis And Saposhnikovia Divaricate

The present thesis aims to analyze the interactions between two lignin components in Magnolia officinalis Rehd.et Wils.(Magnolia officinalis)and rat liver microsomes,and identify the in vitro metabolites and pathways.In addition,the chromone components in the Saposhnikovia divaricate(Turcz)Schischk(S.divaricate)were separated and purified,and then the activity of S.divaricate was evaluated.Base on the in vitro metabolic system of rat liver microsomes(CYP450),through the cocktail hybrid probe drug method and the multi-reaction monitoring(MRM)scanning mode of ultra-performance liquid chromatography-triple quadrupole-MS(UPLC-QQQ-MS),to explore the interaction between the two lignin components in Magnolia officinalis(magnolol and honokiol)and seven CYP450 subtype enzymes(CYP2C,CYP2A6,CYP2D6,CYP2E1,CYP3 A,CYP1A2,CYP2B6).Ultra performance liquid chromatography-high resolution mass spectrometry(UPLC-HRMS)technology combined with specific inhibitors of seven CYP450 subtype enzymes were used to identify the metabolites and metabolic pathways.Using activity screening technology,ultrafiltration-liquid chromatography-mass spectrometry(UF-LC-MS),the lipoxygenase(LOX)was applied as a biological target molecule to rapidly screen lipoxygenase inhibitors from S.divaricate extracts.Using High-speed countercurrent chromatography(HSCCC)and Semi-preparative high performance liquid chromatography(Semi-preparative HPLC)were used to separate the active components in the S.divaricate extract.UPLC-HRMS technology combined with specific inhibitors of seven CYP450 subtype enzymes were used to identify the metabolites and metabolicpathways of the chromone components in S.divaricate under rat liver microsomes(CYP450)in vitro metabolic system.It was determined that the inhibition mechanism of the two lignin compounds in Magnolia officinalis on CYP2 C,CYP2D6,CYP2E1 and CYP2B6 subtype enzymes was an enhanced mechanical inhibition with preincubation time prolonging and increased substrate concentration.The metabolic processes of the five magnolol metabolites and the four honokiol metabolites were identified as follows: the CYP2E1 subtype enzyme was dominant power in the oxidation reaction of magnolol and honokiol terminal double bond.CYP3A4 subtype enzyme was main power for further hydrolytic metabolism.CYP1A2 subtype enzyme promoted carbonyl detachment.The CYP2A6 subtype enzyme only was in charge of the hydrogenation of magnolol.Five lipoxygenase inhibitors in the S.divaricate extracts were separated by HSCCC(Prim-O-glucosylcimifugin,95.28%.5-O-Methylvisammioside,97.89%.Sec-O-glucosyl hamaudol,59.88%.Cimifugin,92.33%)and Semi-preparative HPLC(Prim-O-glucosylcimifugin,95.68%.5-O-Methylvisammioside,91.43%.Sec-O-glucosyl hamaudol,86.12%.Hamaudol,90.69%).The main metabolic pathways of S.divaricate chromones in rat liver microsomes were deglycosylation,demethylation,decarbonylation and hydroxylation.Base on the in vitro metabolic system of rat liver microsomes(CYP450),combined with the cocktail hybrid probe drug method and the specific inhibitor method was an accurate,quick and simple method to explore the clinical effects of medicine.UF-LC-MS combined with HSCCC and Semi-preparative HPLC could rapidly screen and purify lipoxygenase inhibitors from complex Chinese medicine samples,and achieve the simultaneous separation and evaluate the chemical constituents of traditional Chinese medicine.As an efficient and convenient detection method,UPLC-HRMS would provide abundant and accurate data for the separation andidentification of active ingredients of Chinese medicine,metabolomics and pharmacokinetic evaluation.The systematic study of Magnolia officinalis and S.divaricate in this thesis provided a solid experimental basis for medicine development and clinical application of this two traditional Chinese medicines.