The Inhibitory Effects Of 4 Extracts And 21 Monomers Of Hebal Medicine On Carboxylesterase 2

Significant numbers of the general population in the world has used herbal medicines as dietary or pharmceutical supplements,the lack of understanding of herbal medicines’complex components makes it difficult to prevent and treat with herbal drug-drug interactions.Schisandrae chinensis fructus,Mori cortex,Glycyrrhizae radix et rhizoma and Draconis sanguis are edible traditional herbal which were widely used in the word.These hebal medicine always be used with the other drugs.Carboxylesterases（CES）are members of the serine hydrolase superfamily.As an important class of phase I drug metabolizing enzymes in mammals,CES play important roles in biotransformation of both endogenous and exogenous compounds to polar products to facilitate their elimination.In humans,the majority of CES belong to the CES1 and CES2 gene families.As the major isoform distributed in human intestine and tumor tissues,CES2 plays animportant role in the hydrolysis of large ethanol and small acetyl substrates,such as aspirin,procaine and irinotecan.It has been found that the expression level of CES2 in obese and diabetic patients was significantly increased in recent years.Discovery of specific CES2 inhibitors or leading compounds from hebal medicines,can provide some basis for the herb-drug metabolic interactions and offering an alternative treatment of CES2 related diseases（such as SN-38 accumulation induced delayed diarrhea,obesity and diabetes）Objective:Fluorescein diacetate（FD）was employed as a specific fluorescent probe CES2,theresidual activity of CES2 was detected in human liver microsomes after theintervention with Schisandrae chinensis fructus,Mori cortex,Glycyrrhizae radix et rhizomaand Draconis sanguis extract,by which we screened the ingredients that exhibit strong inhibitory effects towards CES2.Furthermore,the inhibition type was investigated.Finally,this study will supply the important theoretical foundation for the CES2-related risk of herb-drug interaction.Methods:1.The ingredients of Schisandrae chinensis fructus,Mori cortex,Glycyrrhizae radix et rhizome,Draconis sanguis extract samples were extracted and identified by HPLC.In the incubationsystem of human liver microsomes,fluorescein diacetate（FD）,the probe substrate of CES2 and the above extracted herbal ingredient were incubated at 37^oC.The group with out the inhibitor was regarded as the control group.The inhibitory intensity was assessed by the metabolite production of FDby using the fluorescent microplate reader,through the analysis of residual activity CES2.2.Assays of the dose-response relationship of herbal ingredients.The values of IC₅₀were further determined when the inhibitory activity of the ingredients towards CES2was less than 50%.3.The inhibition kinetics studies will be conducted when the inhibitionis strong(IC₅₀<5μM).Different concentrations of herbalingredientand different concentrations of probe substrate concentration were used to determine the inhibition kinetic parameters and the type of inhibition.Results:1.Four herbal medicines exhibited inhibitory effect on the activity of CES2.among them,Mori cortex extracs showed the strongest inhibitory effect on CES2,with a residual activity of 1.3%.The second is Draconis sanguis extra ct,with a residual activity of 25.3%on CES2.The extract of Schisandrae chine nsis fructus showed the weakest inhibitory effect on CES2,with a residual activ ity of 33.6%.2.The monomer components that have strong inhibitory activity including:Deoxyschizandrin 23.49%,Gomisin J 34.59%,Gomisin G 42.98%,S chisantherin E 34.14%;Sanggenon C（SC）3.88%,Sanggenone D（SD）5.24%,Kuwanon G（KG）7.92%;Liquiritigenin 5.62%,Licochalcone A 1.68%,Glycyrrh etinic acid Glycyrrhetic acid 43.25%,Luteolin 17.81%,Loureirin A22.12%,Lou reirin B 23.39%.The IC₅₀ values of Deoxyschizandrin,Schisantherin E,Gomisi n J,Gomisin G,SD,KG,SC,Licochalcone A,Liquiritigenin,Glycyrrhetic acid,Luteolin,Loureirin A and Loureirin B were 8.06μM,38.6μM,48.7μM,19.5μM,1.09μM,1.14μM,1.02μM,1.2μM,0.58μM,81.18μM,3.02μM,46.61μM,45.6μM,respe ctively.Sanggenone D 1.09μM,Kuwanon G,Sanggenone C 1.14μM 1.02μM,L icochalcone IC₅₀ 1.2μM,0.58μM Glycyrrhizin and Glycyrrhetinic acid 81.18μM;Luteolin 3.02μM of Loureirin A,46.61μM,45.6μM of Loureirin B.The inhib itory behaviors of SC,SD,KG,luteolin,licochalcone A and liquiritigenin were shown in a concentration-dependent mode,Lineweaver-Burk plot showed that the tntersection was located in the second quadrant,which suggested that inhibi tion types of the six monomer components to CES2 were non-competitive inhi bition.The Ki values of the six monomers were 0.58μM,0.83μM,0.76μM,0.97μM,1.09μM,6.90μM,respectively.Discussion:SC,SD,KG,Luteolin,Licochalcone A and Liquiritigenin showed strong inhibitory effects on CES2 in vitro(IC₅₀<5μM);Gomisin J,Gomisin G and SchisantherinE,Loureirin A and Loureirin B showed medium inhibitory effects on CES2(5μM<IC₅₀<50μM);Glycyrrhetinic acid showed a weak inhibitory effect on CES2(50μM<IC₅₀<100μM).In this study,we firstly reported that the six natural monomer compositions exhibited strong inhibitory effects on the activity of CES2,which provides basis for of the evaluation of herb-drug interaction risk,and supply the treatment scheme of CES-2-related diseases（such as SN-38 accumulation–related diarrhea,obesity and diabetes）.