Study On The In Vitro Metabolism Of AKBA And KBA Of Boswellia Serrata

Objective: In this paper,we study the metabolism of 3-Acetyl-11-keto-β-boswellic acid(AKBA)and 11-keto-β-boswellic acid(KBA),the main active constituents of Boswellia Serrata.The main purpose is to study the metabolic characteristics,reveal the metabolic site of substrates,the major enzyme involved in the metabolism,the kinetic models and parameters of various metabolites in different biological samples,the metabolism differences among seven species.In addition,value the anti-inflammatory activities of the main metabolites,study the anti-inflammatory mechanism of metabolites.Methods:(1)Preparing the metabolites using the biosynthesis method,isolate and purify the metabolites with the phytochemistry method,identified the metabolites by spectral methods including two-dimensional NMR.(2)Investigation of the metabolism of AKBA and KBA in different biology samples including HLM,HIM,recombinant isoforms and liver microsomes from different speicies.(3)The anti-inflammatory activity and mechanism of the parent drugs and their metabolites were evaluated by griess reagent,MTT,western blotting and confocal laser immunoassay.Results: In this study,we systemically characterized both phase I and II metabolism of AKBA and KBA in vitro.In total,four major metabolites were firstly biosynthesized and identified using 1D and 2D NMR spectroscopy.The results showed that four metabolites were 21,16,and 30,20 hydroxylated products,respectively.Among them,three metabolites were novel.The kinetic parameters(Km,Vmax,CLint,and Ki)were also analyzed systematically in various biological samples.Finally,the deacetylation of AKBA and hydroxylation of KBA were confirmed to be the major metabolic pathways based on their large CLint and the high yields of KBA(46.7%)and hydroxylated KBA(50.8%)along with a low amount of AKBA(2.50%)in human primary hepatocytes.Human carboxylesterase 2(CE2)selectively catalyzed the deacetylation of AKBA to form KBA.Although CYP3A4,CYP3A5,and CYP3A7 catalyzed the metabolism of KBA,CYP3A4 played a predominant role in the hydroxylation reaction of KBA in human.Notably,deacetylation and regioselective hydroxylation exhibited considerable species differences.Deacetylation was only observed in human liver microsomes and primary human hepatocytes;21-and 20-mono-hydroxylation of KBA were primarily observed in human,monkey;and 16-and 30-mono-hydroxylation were observed in other species.More importantly,all four mono-hydroxylation metabolites exhibited a moderate anti-inflammatory activity.The 21-and 20-hydroxylation metabolites inhibited the expression of i NOS,the LPS-induced activation of Ik Bα and p65 phosphorylation,and suppressed p65 nuclear translocation in RAW264.7 cells.Conclusions:In this paper,we firstly determined AKBA and KBA metabolism in vitro.The kinetic models and parameters were determined,which providing the useful information for traditional Chinese medicine and its main components AKBA and KBA.Additionally,significant species differences were observed in the metabolism of AKBA and KBA.Meantime,the four major hydroxylation metabolites also exhibited a moderate anti-inflammatory activity compared with the parent compounds.These findings should provide some vital guidance in the selection of animal models for preclinical studies and in developments of new anti-inflammatory agents from AKBA and KBA.