Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts

Area of Science:

• Biochemistry
• Analytical Chemistry
• Natural Product Chemistry

Background:

• Affinity-based assays are crucial for identifying bioactive compounds in complex mixtures.
• Profiling natural product extracts requires methods that are specific and require minimal sample preparation.

Purpose of the Study:

• To develop and validate an affinity-based protein assay for profiling crude aqueous natural product extracts.
• To identify selective binders from Lippia gracilis Schauer using the developed assay.

Main Methods:

• Covalent linking of acetylcholinesterase to magnetic beads to create an affinity matrix.
• Utilizing Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) for chemical characterization of binders.
• Modulating the fishing assay with galanthamine and applying it to Lippia gracilis leaf extracts.

Main Results:

• The assay successfully identified eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside (accurate mass 449.1131 m/z) as a selective binder.
• Demonstrated high selectivity, as eriodictyol 7-O-glucoside was not detected despite its presence in the extract.
• The assay requires no sample pretreatment and exhibits high specificity.

Conclusions:

• The developed magnetic bead-based affinity assay is effective for profiling natural product extracts.
• This method offers a specific and efficient platform for identifying bioactive compounds from natural combinatorial libraries.
• The assay's simplicity makes it suitable for broad applications in natural product research.