| Bletillae Rhizoma is the rhizoma of Bletilla striata(Thunb.)Reichb.f.,the name in Chinese is Bai Ji,with the function of lung repair,hemostasis,air dehumidification,tongqiao pain relief,swelling and pus discharge,muscular,sore sores.Lots of articles suggested that there is 30 percent to 50 percent of polysaccharide in Bletillae Rhizoma,which had contributed to the pharmacological effects of the herb.However,in recent years,some new compounds were found with the in-depth study of chemical components in Bletillae Rhizoma,which further promote the development of the new efficacy and pharmacological action of Bletillae Rhizoma.Especially the new compound named Militarine was found with the content up to 2%~3% in Bletillae Rhizoma,which provides a basis for quality control and related pharmacological research for Bletillae Rhizoma.The chemical name of Militarine is 1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate,which belongs to 2-isobutyl malate glucooxybenzyl ester compound.At present,the efficacy of Militarine is mainly reflected in the central nervous system,which has neuroprotective effects,can improve cognitive dysfunction and promote mental retardation and delay aging.Militarine contains 2 molecules of gastrodin in its molecular structure.Militarine and its similar structure of 2-isobutyl malate glucooxybenzyl ester compounds such as dactylorhin B,loroglossin,and dactylorhin A all have a clear central nervous effect,which may be related to the presence of a gastrodin structure in its molecular structure.Gastrodin is the main functional component of Chinese herbal medicine Gastrodia elata.Gastrodin and its aglycone phydroxybenzyl alcohol have been fully confirmed to have a definitive central nervous effect such as significant sedative and hypnotic effects,analgesic effects,treatment of anti-anxiety,anti-depression and other psychiatric symptoms.It also has a significant effect on Alzheimer's disease(AD)and Parkinson's disease.Therefore,the study of metabolites and metabolic pathways of Militarine in vivo help to elucidate the pharmacological mechanisms of action of Militarine and related compounds.Through the pharmacokinetic study of t Militarine in Bletillae Rhizoma is beneficial to the rational use of materials in Bletillae Rhizoma and related Chinese patent medicines.It also can provide a basis for in-depth pharmacological studies of Bletillae Rhizoma and related Chinese patent medicines.Identification of 2-isobutyl malate glucosylbenzyl ester compounds in Bletillae Rhizoma lays a solid foundation for the quality control of white and medicinal materials and related proprietary Chinese medicines.In this study,33 compounds belong to 2-isobutyl maltol gluconate ester compound were identified from the extract of Bletillae Rhizoma by QTOF high resolution mass spectrometry.Twelve major metabolites were identified by in vitro metabolism studies of Militarine.Further metabolism studies in rats identified 53 metabolites,suggesting metabolic pathways in Militaline rats and metabolic profiles in plasma,urine,feces,bile and cerebrospinal fluid,and clarifying the pharmacodynamic basis of Militarine.A new method of UPLC-MS/MS was established to study the pharmacokinetics,tissue distribution and excretion of Militarine in Bletillae Rhizoma and its main metabolites in rats.The established method for determining the content of Militarine in Bletillae Rhizoma,and measuring a plurality of batches of herbs,provides a reference for the quality control of Bletillae Rhizoma.The research of this paper mainly includes five parts:1.Identification of 2-isobutyl malate glucoxybenzyl ester compounds in Bletillae Rhizoma:Using the secondary mass spectrometry cleavage law and specific fragment ion information of Militarine,33 2-isobutyl malate glucosylbenzyl ester compounds were identified from the alcohol extraction site of Bletillae Rhizoma,7 of which are known compounds(Militarine),Gymnoside ?,Gymnoside ?,Gymnoside ?I,Dactylorhin A,Dactylorhin C,Dactylorhin E),25 are unknown compounds,containing multiple isomers.Seven of them are known compounds(Militarine,Gymnoside ?,Gymnoside ?,Gymnoside ?I,Dactylorhin A,Dactylorhin C,Dactylorhin E),and 25 are unknown compounds containing multiple isomers.2.The study of Militarine metabolism in vitro and in vivoAfter incubation with kidney,liver and duodenal homogenate,Militarine can be metabolized to produce gastrodin and the homoisomers of Gymnoside ? and Gymnoside ?.After administration of Militarine by intragastric administration,54 Militarine metabolites were detected from rats,including 13 metabolites in one phase and 41metabolites in two phases.40 metabolites were not reported in the literature.The main metabolic pathway of Militarine is hydrolysis to produce gastrodin.The oxidation reaction is manifested in the oxidation of gastrodin into the corresponding aldehyde and acid,or the removal of glucose to form aglycon,which can continue to be oxidized to aldehydes and acids.The two-phase metabolic reaction includes glucuronic acid conjugate,sulfuric acid conjugate,and glycine conjugate,wherein the glucose binding reaction can form multiple isomers of disaccharides and trisaccharides.The metabolites of Militarine are concentrated in urine and feces.Metabolites identified in cerebrospinal fluid,including gastrodin,gastrodin,phydroxybenzaldehyde,p-hydroxybenzyl alcohol sulfate conjugate and glucuronic acid conjugate,which may be the material basis for the action of Militarine central nervous system.3.The Pharmacokinetics study on Militaline of Bletillae Rhizoma in ratsThe new method of LC\MS\MS for the simultaneous determination of Militarine and its 9 metabolites in plasma was established and validated,and applied to the pharmacokinetic study of Militarine from Bletillae Rhizoma.Militarine from Bletillae Rhizoma is rapidly metabolized in rats to produce gastrodin.When extract of Bletillae Rhizoma are administered,the half-life of gastrodin in plasma is equivalent to 5 times that when administered alone,which is beneficial to increase and prolong the efficacy of gastrodin.4.The tissue distribution and excretion study on Militaline of Bletillae Rhizoma in ratsThe duodenum is the main absorption site and metabolic site of Militarine.The kidney is the main metabolic organ and excretory organ of Militarine and Gymnoside ?,Gymnoside ?.Gastrodin and p-hydroxybenzaldehyde detected in brain tissue may be the material basis for the central role of Militarine.Militarine is mainly excreted from bile,and the metabolite gastrodin is mainly excreted in urine.The Gymnoside ? and Gymnoside ? detected in the feces are the material basis for the large number of glycosylated metabolites in the identification of metabolites in Militarine.5.The Determination the content of Militarine in Bletillae RhizomaA method for determining the content of militarine in Bletillae Rhizoma was established,and the content of various batches of Bletillae Rhizoma materials was determined,which provide a basis for quality control of Bletillae Rhizoma and related products.In summary,through the identification of the metabolites and metabolic pathways of Militarine in vitro and in vivo,it was clarified that gastrodin is the main metabolite of Militarine.The pharmacokinetic study,tissue distribution and excretion studies further improved the in vivo process of Militarine in Bletillae Rhizoma,and confirmed the presence of gastrodin and gastrodin metabolites in the brain,which reveals the material basis of the central role of Militarine.The results of this study have important reference value for predicting the in vivo processes of other glycosyloxybenzyl esters with similar structures. |
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