Acute metabolic actions of the major polyphenols in chamomile: an in vitro mechanistic study on their potential to attenuate postprandial hyperglycaemia

Abstract

Transient hyperglycaemia is a risk factor for type 2 diabetes and endothelial dysfunction, especially in subjects with impaired glucose tolerance. Nutritional interventions and strategies for controlling postprandial overshoot of blood sugars are considered key in preventing progress to the disease state. We have identified apigenin-7-O-glucoside, apigenin, and (Z) and (E)-2-hydroxy-4-methoxycinnamic acid glucosides as the active (poly)phenols in Chamomile (Matricaria recutita) able to modulate carbohydrate digestion and absorption in vitro as assessed by inhibition of α-amylase and maltase activities. The latter two compounds previously mistakenly identified as ferulic acid hexosides were purified and characterised and studied for their contribution to the overall bioactivity of chamomile. Molecular docking studies revealed that apigenin and cinnamic acids present totally different poses in the active site of human α-amylase. In differentiated Caco-2/TC7 cell monolayers, apigenin-7-O-glucoside and apigenin strongly inhibited D-[U-14C]-glucose and D-[U-14C]-sucrose transport, and less effectively D-[U-14C]-fructose transport. Inhibition of D-[U-14C]-glucose transport by apigenin was stronger under Na+-depleted conditions, suggesting interaction with the GLUT2 transporter. Competitive binding studies with molecular probes indicate apigenin interacts primarily at the exofacial-binding site of GLUT2. Taken together, the individual components of Chamomile are promising agents for regulating carbohydrate digestion and sugar absorption at the site of the gastrointestinal tract.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

HPLC-DAD chromatogram of ChE extract. Targeted compounds are referred to according to the retention time sequence. For retention time, refer to Table 1.

Figure 2

Analysis of compound 2 after acid hydrolysis. (A) HPLC-DAD chromatogram of the hydrolysed fraction and comparison of the released compounds with ferulic acid standard. (B) UV-Vis spectra of ferulic acid standard and compounds resulting from acid hydrolysis. (C) Comparison of the resulting compounds after acid hydrolysis with standards of (E)-MCA and 7-methoxycoumarin. (D) HPLC-DAD chromatograms of PMP derivatives of standard monosaccharides: 1-D-mannose; 2-D-glucose; 3-D-galactose; 4-D-xylose.

Figure 3

(A) Chemical structures of isolated compounds (1 & 2). Key NOESY correlations for compound 2 are indicated with arrows and percentage enhancements. (B) Scheme showing the synthetic aglycone and the corresponding coumarin ring closure.

Figure 4

Inhibition of human α-amylase activity by ChE and component (poly)phenols. (A) Dose-dependent inhibition of ChE. IC50 for acarbose was obtained and used as positive control. (B) Dose-dependent inhibition by individual (poly)phenols. Inhibition is expressed as % compared to control incubations (no inhibitor; normalised to 100%). (C) Combined and individual inhibition of (poly)phenols at concentrations equivalent to those present in the ChE: (Z)-MCAG, 257 µM; (E)-MCAG, 200 µM; apigenin 7-O-glucoside (A7G), 148 µM; apigenin, 12 µM. (D,E) Dixon plot showing the kinetic analysis of apigenin and A7G against α-amylase. The intercept value represents –Ki. Data is expressed as mean ± SEM of three independent experiments with at least three technical replicates. When not visible, the error bars are smaller than the data point(s). (F–J) Charge surface representation (in blue and red) of α-amylase binding cavity with respective ligands displayed in stick format (in purple); (F) (Z)-MCAG, (G), (E)-MCAG, (I) apigenin and (J) A7G. Next to each 3D complex are schematic representations of the 2D interactions between each ligand and amino acid residues including the length of each interaction (in Å) as displayed by Discovery Studio software. (K) Close-up 3D view illustrating the H-bond and π-π stacking interactions of apigenin adjacent to amylase active site residues.

Figure 5

Inhibition of rat maltase activity by ChE and (poly)phenols. (A) Dose-dependent inhibition of ChE. IC50 for acarbose was obtained and used as positive control. (B) Dose-dependent inhibition of individual (poly)phenols. (C) Combined and individual inhibition effect of (poly)phenols at concentrations equivalent to those present in the ChE: (Z)-MCAG, 257 µM; (E)-MCAG, 200 µM; apigenin 7-O-glucoside (A7G), 148 µM; apigenin, 12 µM. Data is expressed as mean ± SEM of three independent experiments with at least three technical replicates.

Figure 6

Monosaccharide transport across Caco-2/TC7 cells monolayers and inhibition by ChE and its major (poly)phenol constituents. (A-1) Transport D-[U-14C]-glucose and D-[U-14C]-sucrose in the absence of inhibitors. (A-2) Dose-dependent inhibition of D-[U-14C]-glucose and D-[U-14C]-sucrose transport by ChE. (A-3) Inhibition of D-[U-14C]-fructose by ChE at a single concentration (1 mg/mL). For D-[U-14C]-glucose and D-[U-14C]-sucrose transport the IC50 values were calculated as 0.24 ± 0.02 and 0.91 ± 0.06 mg/mL respectively. For D-[U-14C]-fructose, the maximum inhibition at the concentration tested was 28%. (B-1) Concentration-dependent inhibition of D-[U-14C]-glucose and D-[U-14C]-sucrose by apigenin 7-O-glucoside (A7G). (B-2) Inhibition of D-[U-14C]-glucose and D-[U-14C]-sucrose by apigenin at a single concentration (50 µM). (B-3) Inhibition of D-[U-14C]-fructose by A7G and apigenin at a single concentration; A7G (200 µM), apigenin (50 µM). (B-4) Inhibition of monosaccharide transport by ChE and combined inhibition of apigenin and A7G; apigenin, 12 µM; A7G, 148 µM. (C-1) Concentration-dependent inhibition of ChE under Na+-free conditions, IC50 = 0.138 ± 0.018 mg/mL. (C-2) Concentration-dependent inhibition of apigenin and A7G under Na+-free conditions. (C-3) Chemical structures of apigenin and A7G and their respective IC50 values under Na+-free conditions. (D) Combined inhibition of apigenin and A7G under Na+-free conditions; apigenin, 12 µM; A7G, 148 µM. Each data point represents the mean ± SEM of three independent experiments with six technical replicates. Results are expressed as % compared to the transported sugars in control incubations (no inhibitor; normalised to 100%). When not visible, the error bars are smaller than the data point. In Fig (A2), (B1), (C1 and C2) statistical difference is indicated against the incremental concentrations of each inhibitor. *p < 0.05, **p < 0.05.

Figure 7

Characterisation of the binding site of apigenin and apigenin 7-O-glucoside (A7G) on GLUT2. Transport experiments were conducted under Na+-free conditions. (A) Immunofluorescence detection of GLUT2 in differentiated Caco-2/TC7 cell monolayers. Row 1 shows control cell layer incubated with 4–6-diamidino-2-phenylindole (DAPI) and Cy3-conjugated donkey anti-rabbit secondary antibody only. Cells in rows 2–4 were incubated with DAPI, membrane marker wheat germ agglutinin (WGA), GLUT2 primary antibody and Cy3-conjugated donkey anti-rabbit secondary antibody. GLUT2 is shown in red, appearing orange when co-localising with WGA shown in green. Nuclei are stained with DAPI (blue). Scale bars (10 µm) are shown in the lower left corner of DAPI images. Scale bar applies to all imagesin the row. Images are representative examples of three independent immunostaining experiments. (B) Asymmetric D-[U-14C]-glucose transport without inhibitor. (C) Asymmetric D-[U-14C]-glucose transport in the presence of cytochalasin B and inhibition constants. (D) Asymmetric D-[U-14C]-glucose transport in the presence of 4,6-ethylidene glucose and inhibition constants. (E) Inhibition of asymmetric D-[U-14C]-glucose transport by apigenin and A7G. Dotted line represents the inhibition values obtained at the maximum concentration tested when D-[U-14C]-glucose transport was assessed from apical (a) to basolateral (b) (data replotted from Fig. 6C-2). Data is expressed as mean ± SEM of three independent experiments with at least three technical replicates. In panel (B,C) results are expressed as % compared to the transported glucose in control incubations (no inhibitor; normalised to 100%). When not visible, the error bars are smaller than the data point. In panel (B) and (C), statistical difference is indicated against the incremental concentration of cytochalasin B and 4,6-ethylidene glucose respectively. **p < 0.05.