This study aimed to explore the inhibitory effect and mechanism of the total alkaloids of Dendrobium officinale Kimura et Migo (DENA) against cholesterol esterase (CE). DENA was characterised by SEM, 1H NMR, and X-ray diffraction (XRD). The inhibitory effect and mechanism of DENA against CE were investigated through fluorescence chromatography, circular dichroism, and molecular docking. DENA inhibited CE activity (IC50 = 1.08 ± 0.09 mg mL−1), characterised by a non-competitive inhibition mechanism. Furthermore, DENA induced a fluorescence quenching in CE, causing a blue shift in the λmax. This coincided with a transition in the secondary structure of CE from a layered to a helical structure by circular dichroism, indicating a significant reduction in its stability. Moreover, molecular docking confirmed that DENA binds to amino acid residues in the enzyme through hydrogen bonds and hydrophobic interactions, leading to structural changes and reduced enzyme activity. These results suggest DENA has the potential to lower blood lipid levels by inhibiting CE activity.
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