In vitro α-glucosidase inhibitory activity of Tamarix nilotica shoot extracts and fractions

Abstract

α-glucosidase inhibitors represent an important class of type 2 antidiabetic drugs and they act by lowering postprandial hyperglycemia. Today, only three synthetic inhibitors exist on the market, and there is a need for novel, natural and more efficient molecules exhibiting this activity. In this study, we investigated the ability of Tamarix nilotica ethanolic and aqueous shoot extracts, as well as methanolic fractions prepared from aqueous crude extracts to inhibit α-glucosidase. Both, 50% ethanol and aqueous extracts inhibited α-glucosidase in a concentration-dependent manner, with IC50 values of 12.5 μg/mL and 24.8 μg/mL, respectively. Importantly, α-glucosidase inhibitory activity observed in the T. nilotica crude extracts was considerably higher than pure acarbose (IC50 = 151.1 μg/mL), the most highly prescribed α-glucosidase inhibitor on the market. When T. nilotica crude extracts were fractionated using methanol, enhanced α-glucosidase inhibitory activity was observed in general, with the highest observed α-glucosidase inhibitory activity in the 30% methanol fraction (IC50 = 5.21 μg/mL). Kinetic studies further revealed a competitive reversible mechanism of inhibition by the plant extract. The phytochemical profiles of 50% ethanol extracts, aqueous extracts, and the methanolic fractions were investigated and compared using a metabolomics approach. Statistical analysis revealed significant differences in the contents of the crude extracts and fractions and potentially identified the molecules that were most responsible for these observed variations. Higher α-glucosidase inhibitory activity was associated with an enrichment of terpenoids, fatty acids, and flavonoids. Among the identified molecules, active compounds with known α-glucosidase inhibitory activity were detected, including unsaturated fatty acids, triterpenoids, and flavonoid glycosides. These results put forward T. nilotica as a therapeutic plant for type 2 diabetes and a source of α-glucosidase inhibitors.

Fig 1. α-Glucosidase inhibitory activity of TN extracts.

(a) α-Glucosidase inhibitory effect of different TN extracts compared to acarbose. A concentration of 10 μg/mL of TN extracts or acarbose was used in this assay. Results are expressed as percentage inhibition relative to the control without inhibitor. Mean values are presented and bars represent standard deviations (n = 3). (b) Concentration-dependent inhibition of TNW, TN50E, TNW30M, and TNW40M compared to acarbose.

Fig 2. Lineweaver-Burk plot of TN extracts inhibitory activity.

Lineweaver-Burk plot in the absence (black) and presence of IC₅₀ (μg/mL) equivalent of Acarbose (green), TNW (orange) and TN50E extract (red). V₀ represents the initial rate of the reaction and [S] the concentration of pNPG in the reaction.

Fig 3. Reversibility of α-glucosidase inhibition and effect of pre-incubation.

(a) Reversibility of α-glucosidase inhibition determined by rapid dilution assay. Produced pNP (mM) was determined after incubation of the enzyme in the absence (no inhibitor) or presence of TN50E extract. Initial rates were measured for 15 min after dilution of the enzyme-inhibitor complex with substrate to initiate the reaction. (b) α-glucosidase inhibition in the presence of IC₅₀, IC₅₀/2 and IC₅₀/4 (μg/mL) of TN50E extract or acarbose before and after 1 h of pre-incubation with inhibitor. Mean values are presented and bars represent standard deviations.

Fig 4. Thermal and pH stability of TN extracts.

(a) pH stability of TN extracts (TNW and TN50E) compared to acarbose. Extracts were incubated at pH 3, 7.5 and 8 for 4 h at 37°C and their inhibitory effect was determined in the standard assay (pH 6.8). (b) Thermal stability of TN extracts (TNW and TN50E) compared to acarbose. Extracts were incubated at room temperature (RT), 37°C and 50°C for 4 h their inhibitory effect was determined in the standard assay (37°C). Mean values are presented and bars represent standard deviations.

Fig 5. Multivariate analysis of phytochemicals identified in TNW, TN50E extracts and methanolic fractions.

(a) Score plot of PCA analysis comparing TNW (green) and TN50E (red) extracts. (b) Score plot of PCA analysis comparing TNW methanolic fractions. MetaboAnalyst software was used to perform the analysis.

Fig 6. Molecules contributing to the difference between TNW and TN50E.

Identified phytoconstituents whose relative abundance are significantly different between TNW and TN50E extracts (p value < 0.01). The chemical classifications and sub-classifications are presented for each metabolite.