Extraction Efficiency and Alpha-Glucosidase Inhibitory Activities of Green Tea Catechins by Different Infusion Methods

Abstract

Alpha-glucosidase is an important target for glycemic control with the aim of reducing the risk of type 2 diabetes (T2D). Green tea catechins have been reported to inhibit alpha-glucosidase activity as a potential beverage to control blood glucose levels. However, the effects of the daily infusion style of green tea on tea catechins and their activity remain unclear. In this study, the extraction efficiency of catechins was investigated for 12 green tea extracts (GTEs) infused with 70% ethanol (70% EtOH for 24 h, a favored solvent for catechin extraction), room temperature water infusion (RT H₂O for 24 h, an easy way to drink tea), and hot water infusion (Hot H₂O for 90 s, a standard way to drink tea). Eight catechins were quantified by HPLC, and the inhibitory effect of GTEs and their catechins on alpha-glucosidase was measured with both rat intestinal enzymes and human Caco-2 cells. The inhibitory mechanism was further analyzed in silico by docking catechins to human alpha-glucosidase using Molecular Operating Environment software. The results showed that total catechins and gallate catechins were efficiently extracted in the order of 70% EtOH, RT H₂O, and Hot H₂O, and the inhibitory activity against alpha-glucosidase also followed a similar order. Pearson correlation analysis indicated that the alpha-glucosidase inhibitory activity of GTEs was significantly positively correlated with the contents of total catechins, especially gallate catechins. Gallate catechins, such as EGCg and ECg, showed lower IC₅₀ values than free catechins for the enzyme in both rats and humans. In silico simulation revealed that gallate catechins were bound to the different sites with free catechins, and the docking energy of gallate catechins was lower than that of free catechins. Taken together, our data indicated that the daily infusion style of green tea significantly impacted the extraction efficiency and alpha-glucosidase inhibitory activities of catechins, which will give us insight into the use of green tea catechins for glycemic control through efficient infusion.

Keywords: alpha-glucosidase; catechins; gallate catechins; green tea; infusion methods.

Figure 1

(a) HPLC chromatogram profiles of eight catechins as standard reference materials. EGCg: (−)-epigallocatechin-3-gallate; EGC: (−)-epigallocatechin; ECg: (−)-epicatechin-3-gallate; EC: (−)-epicatechin; GCg: (−)-gallocatechin-3-gallate; GC: (−)-gallocatechin; Cg: (−)-catechin-3-gallate; and C: (+)-catechin. (b) Chemical structures of eight catechins tested in this study.

Figure 2

Total catechin content of 12 GTEs infused with (a) 70% EtOH (25 °C, 24 h), (b) RT H₂O (25 °C, 24 h), and (c) Hot H₂O (90 °C, 1.5 min). Columns with different letters significantly differ (p < 0.05).

Figure 3

Gallate catechins content of 12 GTEs infused with (a) 70% EtOH (25 °C, 24 h), (b) RT H₂O (25 °C, 24 h), and (c) Hot H₂O (90 °C, 1.5 min). Free catechins content of 12 GTEs infused with (d) 70% EtOH (25 °C, 24 h), (e) H₂O (25 °C, 24 h), and (f) H₂O (90 °C, 1.5 min). Columns with different letters significantly differ (p < 0.05).

Figure 3

Gallate catechins content of 12 GTEs infused with (a) 70% EtOH (25 °C, 24 h), (b) RT H₂O (25 °C, 24 h), and (c) Hot H₂O (90 °C, 1.5 min). Free catechins content of 12 GTEs infused with (d) 70% EtOH (25 °C, 24 h), (e) H₂O (25 °C, 24 h), and (f) H₂O (90 °C, 1.5 min). Columns with different letters significantly differ (p < 0.05).

Figure 4

Ratio of gallate catechins/free catechins (GCs/FCs ratio) of 12 GTEs infused with (a) 70% EtOH (25 °C, 24 h), (b) RT H₂O (25 °C, 24 h), and (c) Hot H₂O (90 °C, 1.5 min). Columns with different letters significantly differ (p < 0.05).

Figure 5

Rat alpha-glucosidase inhibitory activity of catechins. Acarbose was a positive control. (a) Inhibitory percentages by different concentrations of each catechin. (b) Inhibitory concentrations of the half-activity (IC₅₀) of each catechin. Columns with different letters in the catechins group significantly differ (p < 0.05).

Figure 6

The inhibitory activity of major catechins on human alpha-glucosidase. Acarbose was used as a positive control. (a) Inhibitory percentages by defined concentrations of catechins. (b) Inhibitory concentrations of the half-activity (IC₅₀) of major catechins. Columns with different letters in the major catechins group significantly differ (p < 0.05).

Figure 7

Inhibitory activities of reconstituted catechins and GTE on human alpha-glucosidase. “Yabukita” from the Nishinoomote area infused with Hot H₂O (90 °C, 1.5 min) was used as the GTE in this experiment. * p < 0.05 was significantly different in the same concentration of catechins. ns was not significantly different (p < 0.05).

Figure 8

In silico binding site of EC (green), EGC (blue), ECg (red), and EGCg (yellow) in the Nt-SI protein (PDB ID: 3LPP). Kotalanol (KTL) was used as a control.

Figure 9

Interaction of catechins with amino acid residues of proteins at binding sites. (a) EGCg and (b) ECg are represented by white and yellow (gallate group) skeletons, and (c) EGC and (d) EC are represented by white skeletons. The green and blue dot lines in each figure indicate H-bond interactions, and the green letters indicate bound distance.