Essential oils bearing specialized metabolites with potential hypoglycemic activity: a bio-guided fractionation approach driven by in vitro (porcine, human and fungal) α-amylase inhibition assays

Objective: Essential oils (EOs) are extensively studied for their biological properties due to their complex composition and use in various fields (e.g., cosmetics, food, pharmaceuticals). Among the biological activities, the inhibition of enzymes is studied in depth, especially enzymes involved in important human diseases. Type 2 diabetes mellitus (T2DM), a metabolic disorder characterized by uneven maintenance of blood glucose levels, can be treated by inhibition of α-amylase to reduce postprandial hyperglycemia. Acarbose and voglibose are inhibitors actually used in clinical practice. However, these drugs are associated with unpleasant gastrointestinal side effects. The aims of this study were to 1) investigate the use of homologous enzymes as models for human α-amylase by comparing human salivary α-amylase, Aspergillus oryzae α-amylase, and porcine pancreatic α-amylase, and 2) explore potential new α-amylase inhibitors derived from EOs and their constituents. Methods: Human salivary α-amylase, Aspergillus oryzae α-amylase, and porcine pancreatic α-amylase were compared using a combination of in vitro and in silico approaches. Enzyme sequences were aligned and structures superimposed, while kinetics were studied spectroscopically. Sixty EOs obtained by steam distillation or hydrodistillation from different plant species and botanical families were subjected to α-amylase in vitro enzyme assay and chemically characterized by gas chromatography coupled with mass spectrometry. Acarbose was used as a positive control. A bio-guided fractionation approach was adopted to isolate and identify the active fractions/compounds of the most active EOs. Results: The three α-amylase enzymes show strikingly different activities mediated specifically by different ions, despite relevant structural homology. These differences must be carefully considered when using α-amylases from different organisms as models for the human enzymes, which requires appropriate experimental conditions. The most active EOs for both human and porcine α-amylase were juniper (Juniperus communis L.), black pepper (Piper nigrum L.), and ginger (Zingiber officinale Roscoe), whereas different results were obtained for the fungal enzyme, against which each of the above EOs was inactive. The bio-guided fractionation approach showed that the α-amylase inhibitory activity of the EOs is the result of various interactions (antagonistic, additive, or synergistic) between their components and led to the identification of some bioactive compounds. Conclusions: This study confirmed that porcine pancreatic α-amylase can be used as a cheaper substitute for the human enzyme because they show similar activity behaviour, which is clearly related to greater structural similarity. In contrast, α-amylase from Aspergillus oryzae markedly differs from the human enzyme, making it a poorer choice. The above screening showed that some EOs are active towards α-amylase. These results are very promising and deserve further in-depth studies in view of developing a complementary treatment to conventional therapy.