Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos
Abstract
:1. Introduction
2. Results
2.1. Optimization of Extraction of P. cocos Reflux
2.2. Screening and Evaluation of Potential AChE Inhibitors in P. cocos
2.2.1. Screening of Potential AChEIs in P. cocos by UF-LC-MS
2.2.2. Molecular Docking Simulation of Active Compounds and AChE
2.3. Isolation of Active Compounds from P. cocos
2.3.1. HSCCC Separation of Potential AChE Inhibitors from P. cocos
2.3.2. Semi−Preparative HPLC Separation of Potential AChEIs from P. cocos
2.4. Structural Identification of Active Compounds
2.4.1. Identification of Potential AChEIs Using UPLC−Q−Exactive−MS
2.4.2. Identification of Potential AChEIs Using 1H-NMR and 13C−NMR Spectroscopy
2.5. Study on Enzymatic Reaction Kinetics of the Inhibition of AChE by Active Compounds
2.5.1. Inhibitory Types of Active Compounds in P. cocos on AChE
2.5.2. Inhibition Kinetics Analysis of Active Compounds in P. cocos on AChE
3. Discussion
4. Materials and Methods
4.1. Apparatus
4.2. Reagents and Materials
4.3. Screening for Potential AChEIs
4.4. HPLC Conditions
4.5. UPLC-Q-Exactive Conditions
4.6. Molecular Docking Simulation Verification of Active Compounds and AChE
4.7. Separation of Active Ingredients from P. cocos
4.7.1. HSCCC Separation Conditions
4.7.2. Semi−Preparative HPLC Separation Conditions
4.8. Study on Enzymatic Reaction Kinetics of AChE Inhibitors
4.8.1. Solution Preparation
4.8.2. Electrophoresis Method
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
References
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Peak No. | tR (min) | MS (Primary Mass Spectrometry) (neg.) m/z | MS2 (Secondary Mass Spectrometry) (neg.) m/z | Compound | Formula |
---|---|---|---|---|---|
1 | 12.137 | 485.2734 | 469.3123,467.2641, 439.1472, 423.2659, 337.0428 | Tumulosic acid | C31H50O4 |
2 | 15.683 | 481.3371 | 463.4434, 437.2927, 421.3485, 403.3042 | Polyporenic acid C | C31H45O4 |
3 | 17.562 | 483.7835 | 467.0731, 465.2334, 439.1454, 437.2517, 421.2265, 405.1832, 337.1517 | 3-Epidehydrotumulosic acid | C31H48O4 |
4 | 22.461 | 527.3791 | 509.1385, 481.2173, 482.8624, 467.2475, 465.1022, 449.1631 | Pachymic acid | C33H52O5 |
5 | 26.330 | 453.3451 | 435.2734, 393.1805, 371.0163 | Dehydrotrametenolic acid | C30H46O3 |
No. | Solvent System | v/v/v/v | K1 | K2 | K3 | K4 | K5 |
---|---|---|---|---|---|---|---|
1 | HEX:EtOAc:MeOH:H2O | 3.0:6.0:4.0:2.0 | 0.53 | 0.92 | 0.68 | 1.43 | 0.81 |
2 | HEX:EtOAc:MeOH:H2O | 5.0:1.0:5.0:1.0 | —— | 0.01 | —— | 0.04 | —— |
3 | HEX:EtOAc:MeOH:H2O | 6.0:2.0:5.0:1.0 | 0.02 | 0.06 | 0.03 | 1.35 | 0.02 |
4 | HEX:EtOAc:MeOH:H2O | 7.0:3.0:2.0:8.0 | 0.30 | 0.36 | 0.33 | 0.39 | 0.20 |
5 | PET:EtOAc:MeOH:H2O | 1.5:0.5:1.3:0.8 | 0.25 | 0.62 | 0.35 | 0.94 | 1.76 |
6 | PET:EtOAc:MeOH:H2O | 1.0:0.5:1.3:0.8 | 0.16 | 0.56 | 0.24 | 1.30 | 0.95 |
7 | PET:EtOAc:MeOH:H2O | 4.0:1.0:3.0:2.0 | 0.63 | 0.70 | 0.58 | 0.92 | 1.31 |
8 | PET:EtOAc:MeOH:H2O | 5.0:1.0:5.0:2.0 | 0.01 | 0.03 | 0.01 | 0.09 | 0.04 |
9 | HEX:MeOH:H2O | 2.0:1.0:1.0 | 0.04 | —— | 0.03 | 0.06 | 0.07 |
10 | HEX:MeOH:H2O | 3.0:2.0:4.0 | 0.02 | 0.03 | 0.02 | 0.02 | 0.13 |
11 | HEX:Ethanol:H2O | 2.0:1.0:5.0 | 0.91 | 0.88 | 0.99 | 1.03 | 0.94 |
12 | HEX:Ethanol:H2O | 3.0:3.0:5.0 | —— | 0.03 | 0.01 | 0.02 | 0.03 |
13 | EtOAc:MeOH:n-BuOH:H2O | 5.0:2.0:5.0:15.0 | 11.56 | 12.25 | 12.56 | 10.79 | —— |
14 | EtOAc:MeOH:n-BuOH:H2O | 5.0:2.0:2.5:12.0 | —— | —— | —— | —— | —— |
15 | EtOAc:MeOH:n-BuOH:H2O | 6.0:2.0:1.0:10.0 | 15.38 | 22.49 | 18.06 | 18.65 | —— |
16 | EtOAc:n-BuOH:H2O | 5.0:1.0:8.0 | 7.56 | 10.47 | 8.25 | 12.69 | 6.99 |
Time (min) | Event | Value | Duration | Inlet Vial | Outlet Vial | Summary | |
---|---|---|---|---|---|---|---|
1 | Rinse-Pressure | 20.0 psi | 3.00 min | BI1:A13 | BO2:A24 | forward | |
2 | Inject-Pressure | 0.5 psi | 5.0 s | BI:E15 | BO:A2 | Override, forward | |
3 | Inject-Pressure | 0.5 psi | 5.0 s | BI:F26 | BO:A2 | No override, forward | |
4 | 0.00 | Separate-Voltage | 10.0 kV | 20.00 min | BI:A1 | BO:A1 | 10.00 Min ramp, normal polarity |
5 | 20.00 | End |
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Wu, T.; Hou, W.; Liu, C.; Li, S.; Zhang, Y. Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos. Molecules 2023, 28, 1228. https://doi.org/10.3390/molecules28031228
Wu T, Hou W, Liu C, Li S, Zhang Y. Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos. Molecules. 2023; 28(3):1228. https://doi.org/10.3390/molecules28031228
Chicago/Turabian StyleWu, Tong, Wanchao Hou, Chunming Liu, Sainan Li, and Yuchi Zhang. 2023. "Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos" Molecules 28, no. 3: 1228. https://doi.org/10.3390/molecules28031228
APA StyleWu, T., Hou, W., Liu, C., Li, S., & Zhang, Y. (2023). Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos. Molecules, 28(3), 1228. https://doi.org/10.3390/molecules28031228