Alpha-Amylase and Alpha-Glucosidase Enzyme Inhibition and Antioxidant Potential of 3-Oxolupenal and Katononic Acid Isolated from Nuxia oppositifolia

Abstract

Nuxia oppositifolia is traditionally used in diabetes treatment in many Arabian countries; however, scientific evidence is lacking. Hence, the present study explored the antidiabetic and antioxidant activities of the plant extracts and their purified compounds. The methanolic crude extract of N. oppositifolia was partitioned using a two-solvent system. The n-hexane fraction was purified by silica gel column chromatography to yield several compounds including katononic acid and 3-oxolupenal. Antidiabetic activities were assessed by α-amylase and α-glucosidase enzyme inhibition. Antioxidant capacities were examined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging assays. Further, the interaction between enzymes (α-amylase and α-glucosidase) and ligands (3-oxolupenal and katononic acid) was followed by fluorescence quenching and molecular docking studies. 3-oxolupenal and katononic acid showed IC₅₀ values of 46.2 μg/mL (101.6 µM) and 52.4 μg/mL (119.3 µM), respectively against the amylase inhibition. 3-oxolupenal (62.3 µg/mL or 141.9 μM) exhibited more potent inhibition against α-glucosidases compared to katononic acid (88.6 µg/mL or 194.8 μM). In terms of antioxidant activity, the relatively polar crude extract and n-butanol fraction showed the greatest DPPH and ABTS scavenging activity. However, the antioxidant activities of the purified compounds were in the low to moderate range. Molecular docking studies confirmed that 3-oxolupenal and katononic acid interacted strongly with the active site residues of both α-amylase and α-glucosidase. Fluorescence quenching results also suggest that 3-oxolupenal and katononic acid have a good affinity towards both α-amylase and α-glucosidase enzymes. This study provides preliminary data for the plant's use in the treatment of type 2 diabetes mellitus.

Keywords: 3-oxolupenal; ABTS; DPPH; Nuxia oppositifolia; amylase; antioxidant; docking; glucosidase; katononic acid.

Figure 1

Structures of (A) 3-oxolupenal, and (B) katononic acid.

Figure 2

Inhibitory effect of N. oppositifolia crude extract and different fractions against (A) pancreatic α-amylase, and (B) pancreatic α-glucosidase at 100 µg/mL concentration. Data are presented as mean ± standard deviation values of triplicate determinations. Different superscripts letters (a–c) for a given value within the figure are significantly different from each other (Duncan’s multiple range test (p < 0.05).

Figure 3

Comparison of (A) 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, and (B) 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging activity of N. oppositifolia crude extract and different fractions at 100 µg/mL concentration. Data are presented as mean ± standard deviation values of triplicate determinations. Different superscripts letters (a–c) for a given value within the figure are significantly different from each other using Duncan’s multiple range test (p < 0.05).

Figure 4

Interaction of α-amylase with 3-oxolupenal and katononic acid. Quenching in the fluorescence intensity of α-amylase in the presence of (A) 3-oxolupenal and (B) katononic acid. The binding parameters were deduced from (C) Stern–Volmer and (D) modified Stern–Volmer plots.

Figure 5

Interaction of α-glucosidase with 3-oxolupenal and katononic acid. Quenching in the fluorescence intensity of α-glucosidase in the presence of (A) 3-oxolupenal and (B) katononic acid. The binding parameters were deduced from (C) Stern–Volmer and (D) modified Stern–Volmer plots.

Figure 6

Molecular docking of 3-oxolupenal and katononic acid with α-amylase. (A) Binding of 3-oxolupenal with α-amylase, (B) amino acid residues and various interactions involved in 3-oxolupenal-α-amylase complex, (C) binding of katononic acid with α-amylase, and (D) amino acid residues and various interactions involved in katononic acid-α-amylase complex.

Figure 7

Molecular docking of 3-oxolupenal and katononic acid with α-glucosidase. (A) Binding of 3-oxolupenal with α-glucosidase, (B) amino acid residues and various interactions involved in 3-oxolupenal-α-glucosidase complex, (C) binding of katononic acid with α-glucosidase, and (D) amino acid residues and various interactions involved in katononic acid-α-glucosidase complex.