Inhibition And Its Mechanism Of Hydroxygenkwanin,Diosmetin And Deoxyshikonin On CYP Enzymes By The Cocktail Approach

Cytochrome P450（CYP）is the most improtant and crucial metabolizing enzymes in phase I reaction,which is involved in the metabolism of nearly 90%drugs in clinic.When the CYP enzyme is induced or inhibited,it may lead to reduced pharmacological efficacy or increased toxicity,resulting in the failure of many drug treatments.Therefore,the potential CYP inhibition of co-administered drugs or traditional Chinese medicine should be investigated,so as to avoid unnecessary loss in drug development.As a traditional medicine for treating diseases in China,TCM has a thousands of year history,and Chinese medicine monomer has attracted more and more attention in recent decades due to its good activity.Hydroxygenkwanin（HGK）is the main flavonoid component of Daphne genkwa Sieb.et Zucc.Diosmetin（DIOS）,identified from citrus fruits and legume leaves,also belongs to the flavonoid.Deoxyshikonin is a naphthoquinone compound extracted from Lithospermum erythrorhizon Sieb.et Zuc.All the Chinese medicine monomer mentioned above have anti-inflammatory,anti-bacterial and anti-cancer effects,especially in the anti-cancer field,it has great development value.However,the inhibitory effect of these Chinese medicine monomers on CYP remains unclear.The aim of this study was investigated the potential inhibitory effects of these Chinese medicine monomer on CYP1A2,CYP2B1/6,CYP2C9/11,CYP2D1/6,CYP2E1 and CYP3A2/4 enzymes in human and rat liver microsomes（HLMs and RLMs）by the cocktail probe substrate approach in vitro,which will evaluate the potential herbal-drug interactions and provide guidance for the safe use and development of drugs.The main contents and achievements are summarized as follow:（1）The cocktail probe substrate approach was optimized,and the standard curve of CYP enzyme metabolites in RLMs and HLMs was established.It was found that the linear range was good and R²>0.99.The precision and accuracy were verified by selecting three concentrations of high,medium and low.The results showed that the accuracy of high,medium and low concentrations of CYP enzyme metabolites were all within±15%,and the precision was all less than 15%,which indicated that this method was feasible.（2）HGK was a strong inhibitor of CYP1A2 and CYP2C11 in RLMs,with IC₅₀ values at 4.31±0.24μM and 15.38±0.51μM,respectively.Meanwhile,HGK potently inhibited CYP1A2 and CYP2C9 in HLMs,with IC₅₀ values at 3.07±0.07μM and 13.63±1.90μM, respectively.Enzyme inhibition kinetics indicated that HGK was not only a competitive inhibitor of CYP1A2 and CYP2C11 in RLMs,but also competitively inhibited the activity of CYP1A2 and CYP2C9 in HLMs,with K_ivalues were 2.68±0.32μM,8.35±0.31μM,0.84±0.03μM and 8.09±0.44μM,respectively.（3）DIOS presented potent inhibitory effects on CYP1A2,CYP2B1 and CYP2C11 in RLMs,with IC₅₀ values at 2.24±0.07μM,10.80±0.37μM and 7.49±0.32μM,respectively.In HLMs,DIOS potently inhibited CYP1A2 and CYP2C9 in HLMs,with IC₅₀ values at 2.01±0.37μM and 4.77±0.39μM,respectively.Enzyme inhibition kinetics indicated that DIOS was a competitive inhibitor of CYP1A2,CYP2B1 and CYP2C11 in RLMs,In HLMs,DIOS also competitively inhibited CYP1A2 and CYP2C9,with K_i values were 2.49±0.30μM,1.52±0.04μM,4.41±0.39μM,0.41±0.09μM and2.58±0.15μM,respectively.（4）Deoxyshikonin potently inhibited six CYP enzymes in RLMs and HLMs with IC₅₀values<15μM.Enzyme inhibition kinetics indicated that in RLMs,deoxyshikonin was a competitive inhibitor of CYP2B1,CYP2E1 and a mixed inhibitor of CYP1A2,CYP2C11,CYP2D1,CYP3A2,with K_ivalues were 0.36±0.08μM,2.93±0.07μM,0.26±0.04μM,1.83±0.11μM,3.26±0.12μM and 18.66±1.04μM,respectively.In HLMs,deoxyshikonin was a competitive inhibitor of CYP1A2,CYP2E1 and a mixed inhibitor of CYP2B6,CYP2C9,CYP2D6 and CYP3A4,with K_i values were 2.21±0.10μM,1.78±0.12μM,1.68±0.04μM,0.20±0.03μM,4.08±0.14μM and 0.44±0.05μM,respectively.In conclusion,HGK,DIOS and deoxyshikonin showed different inhibition intensity and inhibition type on CYP enzyme in RLMs and HLMs.These studies will provide guidance for the safe use and subsequent development of these Chinese medicine monomers.In addition,due to the differences of species and environment in vivo and in vitro,further experiments on rats and humans are needed to carry out in vivo,so as to accurately evaluate the inhibition mechanism of these Chinese medicine monomers on CYP enzyme and the potential drug drug drug interaction.