Abstract

2,4-Di tert-butylphenol (2,4-DTNB) has been identified as an allelopathic substance present in plants. The biological activity of 2,4-DTNB relevant to human health has been explored, but evidence linking it to Alzheimer's disease (AD) remains lacking. Here, we evaluated the inhibitory potential and kinetic analysis of 2,4-DTNB based on fluorescence analysis in vitro, in silico. Moreover, the study also uses HPLC analysis to investigate the content of 2,4-DTNB in the Thai rice cultivars. The 2,4-DTNB contained in rice samples was approximately 38.40 ± 0.30 to 290.73 ± 2.48 μg/kg. The effect of all rice extracts has the potential to inhibit AChE. Specifically, the 2,4-DTNB has inhibitory potential against the AChE was 52.05 ± 2.43 (%). These results demonstrated that the AChE inhibition by rice extracts was independent of both the content and inhibitory potential of 2,4-DTNB. The 2,4-DTNB inhibited the AChE via a mixed-type inhibition mechanism with the K<inf>i</inf> of 100.00 μM. Fluorescence analysis indicated that the 2,4-DTNB was bound with aromatic amino acids such as TRP84, PHE330, PHE331, and TYR334, and by the concentration-dependent enhancement of intrinsic fluorescence and synchronous fluorescence changes of aromatic residues, particularly tyrosine, consistent with ligand-induced conformational alterations. The docking results also supported that the 2,4-DTNB was surrounded by amino acids at the midway between PAS and anionic subsite in the active site of AChE. These findings suggest that 2,4-DTNB, an allelopathic substance found in rice, is a potential candidate as an AChE inhibitor for AD treatment.