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Mise au point d'un système chromatographique permettant de détecter en ligne la présence d’inhibiteurs de l’acétylcholinestérase et de comparer qualitativement leurs activités inhibitrices

Abstract : The project focuses on the development of a chromatographic system coupling high-performance liquid chromatography (HPLC), immobilized enzyme reactor (IMER) and mass spectrometry (MS) detection to detect acetylcholinesterase (AChE) inhibitors and compare their respective inhibitory activities. Using this device, the AChE inhibitors can be separated on-line and their inhibitory activities can be compared directly by the simple measurement of the chromatographic peaks of the residual acetylcholine (ACh) on MS whose hydrolysis was inhibited by the desired compounds. 3 wildly known standards of AChE inhibitors (Galanthamine-GL, Huperzine A-HuA and Tacrine-TA), were selected to develop the analytical conditions of this coupling. After having validated this method with the 3 AChE inhibiting standards, the alkaloid extracts of different plants were injected into the device in order to screen the new AChE inhibitor candidates. Among the injected extracts, some AChE inhibitors could be discovered thanks to the presence of ACh peaks. To compensate for the drift of the chromatographic baseline (m/z 146), due to the organic phase gradient (MeOH), a counter-gradient pump was added to the outlet of the HPLC column. The results show that this addition of a counter-gradient pump considerably improves the horizontality of the baseline but raises it to high values and reduces correspondingly the sensitivity of the device compared to the chromatographic method performed without counter-gradient. This study led to the development of an innovative method by coupling HPLC, IMER and MS detection for AChE inhibitor screening. Thanks to this method, the activity of the various inhibitors can be easily evaluated by measuring the area of the ACh peaks corresponding to the desired compounds. 3 known AChE inhibitors were chosen to validate this method and have clearly demonstrated a growing dose-response relationship that binds peaks of ACh according to the injected AChE inhibitor amounts. This method CLHP-IMER-SM was applied to screening for AChE inhibitors in plant extracts. Thanks to this method, a new AChE inhibitor has possibily been discovered in the Lycoris Radiata extract.
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Submitted on : Tuesday, October 13, 2020 - 12:25:10 PM
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  • HAL Id : tel-02965518, version 1

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Ye Yuan. Mise au point d'un système chromatographique permettant de détecter en ligne la présence d’inhibiteurs de l’acétylcholinestérase et de comparer qualitativement leurs activités inhibitrices. Chimie analytique. Université de Strasbourg, 2019. Français. ⟨NNT : 2019STRAF016⟩. ⟨tel-02965518⟩

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