Potatoes are the fourth most important crop in the world and the most consumed vegetable in the UK. Chlorogenic acid (5-caffeoylquinic acid, 5-CQA) is the predominant phenolic acid in potatoes⁽Reference Mattila and Hellstrom¹⁾ and it has been shown to inhibit the activity of alpha amylase when tested on artificial substrates⁽Reference Narita and Inouye²⁾. The aim of this project was to investigate the inhibition of porcine pancreatic alpha amylase by 5-CQA at levels expected in potato tissue and also using potato starch as a substrate. The results could support the hypothesis that potato varieties with high 5-CQA levels would have lower glycaemic effects than those with lower levels.

The inhibitory effects of different concentrations of 5-CQA (0−2 mg mL⁻¹) on the activity of porcine pancreatic α- amylase (0·33 units mL⁻¹) on potato starch (0·83–6·6 mg mL⁻¹) was examined. 5-CQA was added to the enzyme-substrate mixture at the start of the reaction or was pre-incubated for 10 minutes with the enzyme prior to addition of the substrate. Reducing sugar released was measured using the 3,5-dinitrosalicylic (DNS) colorimetric method at two reaction times (5 and 20 min). Reactions were carried out at 37°C in 20 mM sodium phosphate buffer pH 6·9 containing 6·7 mM NaCl. All reactions were carried out in triplicate.

The percent of inhibition for various concentrations of 5-CQA (2, 1·5 and 1 mg/ml) without pre-incubation were 45·69%, 25·15% and 3·47% respectively after 5 min of enzymatic reaction. Fig. 1 shows that the inhibition was more pronounced at 5 min compared to 20 min reaction time. Pre-incubation of the enzyme with 5-CQA (at 1·5 mg mL⁻¹) led to a small but significant decrease in activity, observed at both reaction times (P < 0·05).

Fig. 1. Effect of 5-CQA (1.5 mg mL-1) on amylase activity; n = 3, error bars = SD, a,b,cp < 0.05 and d,e,f p < 0.05 at same time point and different treatments .

The mechanism of inhibition was investigated by kinetic analyses. The Hans-Woolf plot (Fig. 2) and kinetic parameters (table below) show that 5-CQA decreased the V_max and increased the K_m of pancreatic alpha amylase, indicating a mixed-type inhibition mechanism when using potato starch as a substrate.

Fig. 2. Hans-Woolf plot at various 5-CQA concentrations.

The results of this study provide evidence that 5-CQA at concentrations found in food could reduce digestibility of potato starch with potential consequences on carbohydrate availability.