Methods On Discrimination And Identification Of Active Constituents From TCMs By Ultrafiltration-LC-MS

ObjectiverBased on the theory that TCMs express their bioactivities by special characteristics of synergistic action of multiple components and multiple acting sites in vivo,the bioactive compounds of TCMs were elucidated by characterizing the physiological disposition in vivo of TCMs, including absorption, distribution, metabolism and the ability to combine with peculiar receptor or cells. The mechanism of pharmacological activities and material basisof TCMs is unclear.To illustrate Chinese medicine efficacy material base in the body, methods innovation should be taken. Ultrafiltration mass spectrometry has been developed as a new method for drug screening because of its high speed, high sensitivity, and its high throughput screening ability. To promote the application of the ultrafiltration mass spectrometry in the interaction between small drug molecule and biological target, the potential ?-glucosidase inhibitors and cyclooxygenase-2 inhibitors were screened from Scilla bulbus and Toad venom by using ultrafiltration mass spectrometry respectively.Method:The ?-glucosidase inhibition assay was performed based on the positive contrast drug of acarbose and the substrate of 4-nitrobenzene-?-D-pyran glycosidase. The 95% EtOH extraction of Scilla bulbus were passed through an HPD-100 column chromatography. Then the inhibition rates of different fractions were determined. The significant inhibition of the active fraction was selected for screening the potential ?-glucosidase inhibitors. The ultrafiltration assay was developed for screening candidates of ?-glucosidase inhibitors. The interesting compounds were identified by UPLC-qTOF/MS. The cyclooxygenase-2 inhibitors were screened by liquid chromatography-tandemmass spectrometry (LC-MS-MS) based on the ultrafiltration assay. The extract of Toad venom and inhibitors were assayed for inhibition of cyclooxygenase-2 using LC-MS-MS. The IC50 value was determined by comparing the addition of PGE2 and PGD2.The inhibition actiVity was evaluated by the contrast of positive contrast drug of celecoxib.Results:The EtOAC extract and 95% eluate of Scilla bulbus were determined as the significant inhibition of the active fractions.Sixteen compounds,2-hydroxy-7-methoxy scillascillin,scillascillin,5,7-dihydroxy-3',4'-dimethoxy spiro{2H-1-b enzopyran-7'-bi cycl o[4.2.0]octa[1,3,5]-trien }-4-one,solinone,scillavone A,3,9-dihydroe ucomnalin,3-(3-hydroxy-4-methoxybenzyl)-5,7-dihydroxychroman-4-one,(3R)?5,7,3'?trihydroxy?4'-methoxy spiro{2H?1-benzopyran-7'-bicyclo[4,2,0]octa[1,3,5]-trien}-4-one, 2-hydroxy scillascillin,3-(4-hydroxybenzyl)-5,7-dihydro xychroman-4-one, 3-(4-hydroxybenzylidene)-5,7-dihydroxychrom an-4-one,3-(4-hydroxyb enzyl)-5-hydroxy-7,8-dimethoxychroman-4-one,3-(4-hydroxybenzyl)-5-hydroxy-6,8-dimethoxychroman-4-one,4-methylre sveratrol,(E)-resveratr ol and scillabene A that might bind to ?-glucosidase were screened and identified.The IC50 of 3-(4?hydroxybenzyl)-5,7-dihydroxychroman-4-one and 3-(4-hydroxybenzylidene)-5,7-dihydroxychroman-4-one was 180.3 ?M and 62.79 ?M determined by the ?-glucosidase inhibition assay. The alcohol extract of Toad venom for cyclooxygenase-2 ligands were screened by using ultrafiltration liquid chtomatography mass spectrometry and cinobufagin,resibufogenin bound to the enzyme active site.Conclusion:The potential ?-glucosidase inhibitiors and cyclooxygenase-2 inhibitors were screened and identified by using ultrafiltration mass spectrometry.The results laid the foundation for screening enzyme inhibitors rapidly from complex system of traditional Chinese medicine and for the further research.