Screening Of Carboxylesterases Inhibitors In Traditional Chinese Medicine Extract And Study Its Therapeutic Effect Of Thalidomide On Irinotecan-induced Diarrhea On Mice

1. Carboxylesterases (CEs), a well conserved multigene family of a/β-hydrolase fold proteins, catalyze the hydrolysis of a vast number of structurally diverse of endogenous and exogenous substrates including fatty acids, environmental toxins and drugs. Discovery of a selective and potent inhibitor of hCE2 is very essential for clinical translational applications.Fluorescein Diacetate (FD), a selective fluorescence probe substrate of hCE2, was used for rapid screening of the inhibitory effects of hundred herbal extracts towards hCE2. After a high-throughput screening campaign against the extract of hundred herbals, we found that the ethanol extract of Fructus Psoraleae (Bol-gol-zhee or Bu-gu-zhi) displayed potent inhibitory effect towards hCE2. The extract was then separated by using UFLC-UV fingerprinting, while the fractions of LC eluent were collected at 30-second intervals. The residual activities of hCE2 in the presence of each fraction were assayed and recorded, which can be used to find out the potent hCE2 inhibitor in Fructus Psoraleae efficiently. All constitutes can potently inhibit FD hydrolysis in human liver microsomes (HLM) and result in decreasing the fluorescence intensity in a dose-dependent manner.Our results demonstrated that five major constitutes in Fructus Psoraleae displayed potent inhibitory effects towards hCE2. These constitutes were identified as Neobavaisoflavone, Isobavachalcone, Bavachinin, Corylifol A, Bakuchiol, respectively, by comparison of LC retention times, UV spectra and MS/MS spectra with the authentic standards. Both Lineweaver-Burk and Dixon plots demonstrated that inhibition types of these constitutes against hCE2 in HLM were non-competitive inhibition, with low Ki values ranging from 0.62μMto 3.89μM.This is the first report of the chemical constitutes in FP as potent inhibitors of hCE2, implying that the extract of Fructus Psoraleae or their major constitutes can be used to alleviate the CPT-11 induced delayed-type diarrhea.2. And on the basis of this work, we found that baicalein displayed potent inhibitory effects towards hCE2. Both Lineweaver-Burk and Dixon plots demonstrated that inhibition types of these constitutes against hCE2 in HLM were non-competitive inhibition, with low Ki values as 6.2μM. on animal experiments, baicalein can effectively alleviate the intestinal toxicity of CPT-11. And it also reduced CPT-11-induced TNF-α, IL-6å'ŒIL-1β.The Diarrhea score in baicalein group were better than diarrhea group (P<0.05). The histological changes were milder.The results of this study provided a new clinical application value of baicalein.