1122 高等学校化学学报 Vol DTNB Sigma-Aldrich γ- APS Alfa Aesar Milli-Q Tecan infinite 200 HZP-150 KQ-400KDE R201 Milli-Q 18 2 MΩ cm Millipore Z383K HE

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1 Vol 34 高等学校化学学报 No CHEMICAL JOURNAL OF CHINESE UNIVERSITIES 1121 ~ 1126 doi /cjcu 陈晨 1,2, 史倩 1, 陈军辉 1, 张茹潭 1, 李鑫 1, 郑立 1 1,3, 王小如 AChE AChE AChE AChE 55 μl 5 mmol /L 6% AChE AChE AChE O652 1 A AChE AD 1 AChE AD AChE 2 AChE AChE 3 AChE AChE AChE 4 ~ Ellman s ESI- MS 10 AChE 11 ~ 13 AChE AChE AChE 16 ~ AChE AChE 18 AChE AChE AChE Hu 19 Liu AChE AChE Davisil grade ~ nm ATChI AA jhchen@ fio org cn

2 1122 高等学校化学学报 Vol DTNB Sigma-Aldrich γ- APS Alfa Aesar Milli-Q Tecan infinite 200 HZP-150 KQ-400KDE R201 Milli-Q 18 2 MΩ cm Millipore Z383K HERMLE mmol /L ph = % 1 3 AChE 21 AChE 1 4 AChE AChE ATChI DTNB 405 nm Thio-2-nitrobenzoic acid 405 nm AChE 4 AChE 0 1 mol /L ph = μl 40 μl 80 μl 0 5 mmol /L DTNB min 80 μl ATChI min r /min 1 min AChE 405 nm A sample 40 μl 50% A contrast AChE A blank Inhibition efficiency % = A contrast - A blank - A sample - A blank 100% 1 A contrast - A blank AchE 55 μl 5 mmol /L 50% mg /L AChE IC S /B SW S /N CV Z' g 25 ml 20 ml 30 min 4 50% AchE 1000 mg /L AChE 1000 mg /L AChE

3 No 5 陈晨等 : 基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立 ph AChE ph AChE 3 25 AChE AChE AChE μl 1 A AChE 10 ~ 55 μl 55 μl AChE 55 μl AChE 1 B mmol /L AChE 5 ~ 20 mmol /L 5 mmol /L ATChI Fig 1 Comparison results of different dose of immobilized AChE A and different concentrations of ATChI B DMSO 26 3 AChE 2 A 3 5% 10% 20% 30% 40% 50% AChE AChE DMSO > > DMSO DMSO 10% DMSO 1% ~ 10% AChE 2 B DMSO 2% ~ 7% AChE 8% 10% 27 6% DMSO Fig 2 Effects of methanol a ethanol b and DMSO c solutions A and low concentrations of DMSO B on the activity of immobilized AChE The error bar indicates standard deviation n = 3

4 1124 高等学校化学学报 Vol AChE AChE 3 10 mg /L AChE 28 IC μmol /L Ellman s AChE IC 50 IC nmol /L 29 Fig 3 Plots of the inhibition efficiencies of tacrine IC μmol /L 30 4 a and eserine b on immobilized AChE AChE n = S /B S S 3 S /B 31 SW SW 1 51 S /N S S /N AChE AChE CV 0 CV CV 1 88% CV 10% 32 Z' Z' factor 2 Z' 0 93 Z' 0 4 AChE AChE 34 AChE Table 1 AChE inhibition activity of different crude extracts of natural product Inhibition efficiency % Sample 1000 mg /L 1 Methanol Ethanol Ethyl acetate Styela clava AChE Hemicentrotus pulcherrimus Ellman s Cortex phellodendri chinensis AChE Coptis chinensis franch AChE Ulva lactuca L Enteromorpha prolifra mg /L 2 10 mg /L AChE 5 mg /L 10 mg /L AChE

5 No 5 陈晨等 : 基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立 Table 2 AChE inhibition activity of tacrine and eserine in 1000 mg /L different crude extracts of natural product Inhibition efficiency % Sample Solvent / Crude extract 5 mg /L 10 mg /L 50% Tacrine 50% Methanol Physostigmine 50% Methanol mg /L 0 2 Caffeic acid Hemicentrotus pulcherrimus Thymidine Hemicentrotus pulcherrimus mg /L Tacrine Hemicentrotus pulcherrimus % Tacrine Ulva lactuca L Physostigmine Hemicentrotus pulcherrimus Physostigmine Ulva lactuca L AChE Table 3 AChE inhibition activities of low concentrations of tacrine and eserine in crude extracts of natural product Added sample Inhibition efficiency % 50% Methanol 1000 mg / L Hemicentrotus pulcherrimus crude extract 1000 mg / L Ulva lactuca L crude extract 0 1 mg /L Physostigmine mg /L Physostigmine mg /L Tacrine AChE AChE Z' AChE AChE AChE AChE 1 Wang Q M Jiang H L Chen K X Chem J Chinese Universities Reale M Iarlori C Gambi F Feliciani C Isabella L Gambi D Neuropharmacol Mukherjee P K Kumar V Mal M Houghton P J Phytomedicine Jarvinen P Vuorela P Hatakka A Anal Biochem Ellman G L Courtney K D Andres V Featherstone R M Pharmacol Dong J Fan X Z Qiao F M Ai S Y Xin H Analytica Chimica Acta Carpinella M C Andrione D G Ruiz G Sara M Phytotherapy Res Brlihlmann C Marston A Hostettmann K Carrupt P A Testa B Chem Biodiversity Funeriu D P Eppinger J Denizot L Miyake M Miyake J Nat Biotechnol Liesener A Perchuc A M Schoni R Pure Appl Chem Ke B B Wan L S Huang X J Xu Z K Chem Res Chinese Universities Luckarift H R Johnson G R Jim C J Chromatogr B Zou M J Yang R Wang D Y Pestic Biochem Physiol Sun C Y Li H K Ping H Chem J Chinese Universities Xue R Kang T F Lu L P Cheng S Y Appl Surf Sci Forsberg E M James R Green A Brennan J D Anal Chem Tang Z M Wang T D Kang J W Electrophoresis Girelli A M Mattei E M J Chromatogr B Hu F L Zhang H Y Lin H Q Deng C H Zhang X M J Am Soc Mass Spectrom Liu Y Li Y Liu J Y Deng C H Zhang X M J Am Soc Mass Spectrom

6 1126 高等学校化学学报 Vol Chen J H Shi Q Chen C Li X Cao W Zheng L Wang X R Acta Chim Sinica Fallareroa A Pharmacol Res Si S Y Zhang Y Q Drug Screening Methods and Practice Chemical Industry Press Beijing Najera A I Renobales M D Barron L R Food Chem Jeffrey Y S Kenneth B Int J Bhmhem Cell Biol Santos N C Coelho J F Silva J M Saldanha C Biochem Pharmacol Sun Q Y Yang F M Chin Pharmacol Bull Zhao Q Tang X C Eur J Pharmacol Giacobini E Pharmacol Res Karczmar A Neuro Chem Int / Liu J Zhang L Y Chin Pharmacol Bull Wang Y Hao X Q Gao P Yang Y H Xiao X L Chin J Antibiot Zhang Y Feng Y Li X M Wang B G Oceanologia et Limnologia Sinica Yin S W Huang F C Zhang Y Y Xing N N Liu L P Nat Prod Res Dev Establishment of a New Model for in Vitro Screening of Acetylcholinesterase Inhibitors Based on Immobilized Enzyme CHEN Chen 1 2 SHI Qian 1 CHEN Jun-Hui 1* ZHANG Ru-Tan 1 LI Xin 1 ZHENG Li 1 WANG Xiao-Ru Key Lab of Analytical Technology Development and Standardization of Chinese Medicines The First Institute of Oceanography State Oceanic Administration Qingdao China 2 College of Fisheries and Life Science Shanghai Ocean University Shanghai China 3 College of Chemistry and Chemical Engineering Xiamen University Xiamen China Abstract An economical model for rapid in vitro screening of acetylcholinesterase AChE inhibitors was developed which used Ellman s reaction-based assay with reusable immobilized AChE AChE-immobilized aminated silica gel microspheres were applied to develop new screening method Firstly the experimental conditions were optimized And then inhibitory activity on immobilized AChE of tacrine and eserine positive controls was tested to validate the effectiveness of the new model Multiple technical parameters of this new model were also evaluated Finally the inhibitory activity on AChE of monomeric compounds and natural products was evaluated by the proposed new model The results show that the optimal conditions included the followings 55 μl of immobilized AChE as the appropriate dose 5 mmol /L acetylthiocholine iodide as substrate methanol and ethanol as well as dimethyl sulfoxide which was under 6% as sample solvent The obtained results also demonstrated that this new model was sensitive and specific for rapid screening of AChE inhibitors Good model validation and multiple parameters evaluation results were obtained The proposed model has many advantages good applicability continuous reuse of the immobilized AChE with reliable results etc This model is a powerful tool for identification of AChE inhibitors from natural products or chemical libraries produced by combinatorial chemistry Keywords Immobilized enzyme Acetylcholinesterase inhibitor Target enzyme inhibitor screening ( Ed : N,V,K)

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇