Cocoa extract inhibits in vitro α-glucosidase activity: The role of polyphenols and melanoidins D. Tagliazucchi, A. Bellesia, A. Conte Department of Life Sciences, University of Modena and Reggio Emilia Introduction Studies on the health benefits of cocoa have recently focused on the anti-diabetic potential of cocoa extract. The intake of cocoa has been related to a reduction in the post-prandial levels of blood glucose in diabetic rats [1]. A possible explanation for these observations is that cocoa extract possesses the ability to inhibit enzymes involved in the digestion of carbohydrate, attenuating the post-prandial increase in glycemia. Methods Two grams of cocoa powder were added to 100 mL of boiling water, followed by heating for 5 minutes below boiling point. The cocoa extract was fractionated following the scheme reported in figure 1. All the obtained fractions were characterized for their ability to inhibit the activity of rat intestinal α-glucosidase and for their content in polyphenolic compounds. The enzyme α-glucosidase was extracted from rat intestinal acetone powder and assayed according to Oki et al. [2]. The rate of release of p-nitrophenol (PNP) from PNP-gluc was measured at 37 °C after incubation for 20 min in presence of 0.01 U/ml of rat intestinal α-glucosidase (one unit of α-glucosidase is defined as the quantity of enzyme that release 1.0 μmol of PNP from PNP-gluc per minute at pH 6.8 at 37°C) and variable amounts of buffer or different concentrations of inhibitor diluted in buffer. The IC50 is defined as the concentration of inhibitor required to inhibit 50% of the enzymatic activity. Total polyphenols were determined using the Folin-Ciocalteu assay. Results The inhibitory effect of cocoa extract against α-glucosidase was dose-dependent with an IC50 value of 7.87 mg/mL (Figure 2). After ultrafiltration, the majority of the brew was found to be LMW material (Table 1). The value of Kmix (defined as the absorption at a specific wavelength of a solution of cocoa extract or fractions at a concentration of 1 g/L) provides information on the relative amount of melanoidins (Kmix 405nm) and other cocoa compounds like proteins and flavanols (Kmix 280nm). The highest Kmix 405nm value was for the IMW fraction, suggesting that this fraction is relatively rich in melanoidins. IMW fraction also showed the highest Kmix 280nm value, which can be explained with the high content in phenolic groups (Table 1). The Kmix 280nm to Kmix 405nm ratio gives an estimation of the relative purity of the melanoidins in a fraction. The LMW fraction had the highest ratio, suggesting that this fraction is poor of melanoidins and relatively rich of UV absorbing compounds, such as flavanols. All the three fractions showed α-glucosidase inhibitory activity with the IMW fraction that was the most active (Figure 2). Both the HMW and IMW fractions were further subjected to ethanol precipitation (Figure 1). Only the fractions HMW EP50, HMW EP75, IMW EP25, and IMW EP75 were found to be active against α-glucosidase (Figure 2). The fraction with the highest inhibitory activity was IMW EP75 which showed an IC50 value of 0.87 mg/mL. This fraction is rich in melanoidins (high Kmix 405nm value) and is the richest in phenolic compounds (Table 2 and 3). LMW fraction was further fractionated with Sephadex LH-20 columns to separate un-bound low molecular weight flavanols (eluted with 50% ethanol) from bound tannins (eluted with 70% acetone). All the inhibitory activity was found in the un-bound fraction. This fraction was subjected to HPLC with C18 columns. By comparison with the retention times of external pure standards, catechin, procyanidin B1, procyanidin B2 and theobromine were identified (Figure 3). Procyanidin B2 was found to have the lowest IC50 value (Table 4). Cocoa extract Ultrafiltration LMW (<10KDa) IMW (10-30KDa) HMW (>30KDa) Ethanol precipitation EP25 EP50 EP75 ES75 Sephadex LH-20 Elution with EOH 50% Un-bound fraction (flavonols) Elution with Acetone 70% Bound fraction (tannins) Figure 1. Scheme for the isolation of various cocoa fractions from brew. Cocoa extract was firstly fractionated by ultrafiltration yielding three fractions named low molecular weight (LMW <10KDa), intermediate molecular weight (IMW 10-30KDa) and high molecular weight (HMW >30KDa) fractions. The melanoidins-rich fractions (IMW and HMW) were further subjected to ethanol precipitation. Absolute ethanol was slowly added to the fractions under continuous stirring until an ethanol concentration of 25% (v/v) was reached. The solution was left for precipitation overnight at 4 °C and then centrifuged for 20 min at 18900g. The supernatant and the residue were separated and the supernatant was subjected to further ethanol precipitation steps in which the ethanol concentration was increased in steps to 50 and a final concentration of 75% (v/v) following the same procedure. The samples were lyophilized, yielding “EP25”, “EP50”, and “EP75” for the cocoa fractions that precipitated at 25, 50, and 75% (v/v) ethanol, respectively. The lyophilized supernatant of 75% (v/v) ethanol was coded “ES75”. The LMW fraction was further fractionated with Sephadex LH-20 columns to separate tannins from low molecular weight flavanols. Table 1. Yield, Kmix 405nm, Kmix 280 nm, Kmix 280/405 ratio, and level of phenolic groups of cocoa fractions. Table 2. Yield, Kmix 405nm, Kmix 280 nm, Kmix 280/405 ratio, and level of phenolic groups of cocoa HMW fractions. Table 3. Yield, Kmix 405nm, Kmix 280 nm, Kmix 280/405 ratio, and level of phenolic groups of cocoa IMW fractions. Table 4. IC50 values of flavanols identified in the Sephadex LH20 un-bound fraction 2 4 6 8 10 12 Cocoa HMW IMW LMW EP50 EP75 EP25 IC50 (mg/mL) theobromine Figure 2. IC50 values of cocoa extract and fractions against rat intestinal α-glucosidase procyanidin B1 catechin procyanidin B2 Conclusion In summary, the present study provides the first evidence that cocoa extract and fractions are potent inhibitors of α-glucosidase in vitro. Using a combination of ultrafiltration and ethanol precipitation techniques we were able to isolate a fraction rich in melanoidins and phenolic compounds which exhibited the highest inhibitory activity (IMW EP75). Further in vivo studies are needed to examine whether cocoa extract and/or cocoa components can inhibit α-glucosidase in vivo. un-known Figure 3. HPLC separation of Sephadex un-bound fraction. References [1] Jalil, A.M.M. et al. Effect of cocoa extract on glucometabolism, oxidative stress, and antioxidant enzymes in obese-diabetic (ob-db) rats. J Agric Food Chem 2008, 56, 7877–7884. [2] Oki T. et al. Inhibitory effect of alpha-glucosidase inhibitor varies according to its origin. J Agric Food Chem 1999, 47, 550–553.