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OBJECTIVES: Diabetes mellitus (DM) is a worldwide increasing problem, associated with development of hyperlipidemia, coronary heart disease, hypertension, and other chronic diseases. Decreasing of glucose absorption by inhibition of α-amylase is one of the therapeutic approaches to retard diabetes type 2. Pancreatic α-amylase (PA) inhibition widely studied mechanism for determination of potential of natural compounds as antidiabetic agents. The aim of this work was identification of inhibitory secondary metabolites produced by Aspergillus egypticus, isolated from Helianthus tuberosus. MATERIALS AND METHODS: The PA inhibitory activity of the secondary metabolites determined using iodometric method. Isolation of inhibitory compounds was carried out by column chromatography, thin layer chromatography and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: It was found that the inhibitory concentration of a compound, K-10 (Rf : 0.74), isolated from metanolic extract of A. egypticus was 4.82 mg/mL. LC-MS/MS analysis of K-10 showed polymethoxylated flavones (PMF). CONCLUSION: The fungal endophyte A. egypticus-HT166S can be considered a source of PMF as potential agents for developing new PA inhibitors.