Postprandial Hyperglycemia Lowering Effect of the Isolated Compounds from Olive Mill Wastes - An Inhibitory Activity and Kinetics Studies on α-Glucosidase and α-Amylase Enzymes

Abstract

In the present study, we isolated seven compounds from olive mill wastes (OMW), one of them being novel, and investigated their antidiabetic potential through inhibition of α-glucosidase and α-amylase enzymes. To assist the possible characterization of the mechanisms involved, we analyzed the inhibitory kinetics of the active compounds. Oleanolic acid 1, maslinic acid 2, 1-acetoxypinoresinol 3, and luteolin-7-O-β-d-glucoside 6 exhibited stronger inhibitory activity against both enzymes, with IC₅₀ values less than or close to that of acarbose. Other compounds pinoresinol and hydroxytyrosol-containing compounds (hydroxytyrosol acetate 4, hydroxytyrosol 7, and the novel one, 3,4-dihydroxyphenyl-2-methoxyethanol 5) showed weak activity against both enzymes (IC₅₀ > 500 μM). Our findings show that, first, the esterification of C-1 of the furofuran ring is the key feature for the stronger activity of 1-acetoxypinoresinol 3 against both enzymes (IC₅₀ = 13.9 and 313 μM for α-amylase and α-glucosidase, respectively), as compared to pinoresinol; second, the oleanane skeletons of the triterpenes (1 and 2) are optimum for the α-glucosidase and α-amylase inhibitory activities, while the hydroxytyrosol moiety may be responsible for the weak activities of 4, 5, and 7. Additionally, kinetics analysis of 1, 6, and 3 revealed that they inhibit α-glucosidase in mixed-type, noncompetitive, and uncompetitive mechanisms, respectively. We confirmed their mechanisms by measuring their affinity for the enzyme (K _i), and they all (1, 6, and 3) had a higher affinity for the enzyme, K _i > 1. This work adds more value to OMW for further studies as a potential source of lead antidiabetic compounds for the prevention and/or treatment of type 2 diabetes.

Figure 1

Chemical structures of the OMW compounds. (A) Selected significant HMBC correlations (solid arrows) of compound 5; (B) structures of the isolated compounds from OMW (1–7) and (+)-pinoresinol.

Figure 2

Chromatograms of OMW total extract, 200 ppm. (A) Extracted ion chromatogram (EIC) scan and (B) DAD chromatogram (λ = 280 nm) scan. The compounds were identified as oleanolic acid 1, maslinic acid 2, 1-acetoxypinoresinol 3, hydroxytyrosol acetate 4, 3,4-dihydroxyphenyl-2-methoxyethanol 5, luteolin-7-O-β-d-glucoside 6, and hydroxytyrosol 7 by comparing their retention times, mass spectral, and DAD data of each peak to those of the isolated pure ones or authentic one (for the case of (+)-pinoresinol). Compounds 1 and 2 could not be seen at the DAD scan as they cannot absorb UV at a wavelength of 280 nm.

Figure 3

Inhibitory effects of the isolated compounds on the enzymatic activities of α-glucosidase and α-amylase enzymes – all compounds presented at a concentration of 150 μM. Effect on α-glucosidase inhibitory activity is presented with white bars, while the effect on α-amylase inhibitory activity is presented with bars with black dots. The α-glucosidase inhibitory activity was quantified by measuring the amount of glucose oxide formed at the end of the reaction, while inhibitory activity on α-amylase was measured by a quantitative starch-iodine method. In both cases, the experiments were done in triplicates n = 3 (results are expressed as mean ± SD): OA, oleanolic acid; MA, maslinic acid; ACX, 1-acetoxy pinoresinol; LUT-G, luteolin-7-O-β-d-glucoside; DHPM, 3,4-dihydroxyphenyl-2-methoxyethanol; HOTy, hydroxytyrosol, and HOTy Ac, hydroxytyrosol acetate.

Figure 4

Kinetics analysis of α-glucosidase inhibition. Lineweaver–Burk plots of the reaction of α-glucosidase in the presence of the active compounds. (A) Oleanolic acid 1, (B) luteolin-7-O-β-d-glucoside 6, and (C) 1-acetoxypinoresinol 3. The concentration of sucrose was measured in the absence or presence of inhibitors at different concentrations (I, inhibitor; dark circles, no inhibitor; dark triangles, low concentration; black-lined transparent circles, high concentration).