Optimization of Enzymatic Production of Isoquercetin and Possible Existence of β-D-glucosidase Inhibitor in Jujube Leaf Extract

Abstract

Recently, quercetin and its glucosides including isoquercetin (quercetin-3-β-D-glucoside, Q3G) and rutin (quercetin-3-rutinoside) draw attention due to healthful bioactivities such as antiproliferative, antioxidant, anti-iflammatory. However, isoquercetin exhibits the highest bioavailability, which varies according to the type of sugar moiety. Rutin is the most common flavonoid in plant resources, especially in jujube leaves which are the waste of food industry. To increase the availability of isoquercetin, the enzymatic biotransformation of isoquercetin from rutin in the jujube leaf extract using hesperidinase from Aspergillus niger was optimized. Hesperidinase is an enzyme complex containing α-L-rhamnosidase and β-D-glucosidase activities. Employing response surface methodology (RSM), the isoquercetin yield was optimized concerning the temperature (40~60°C, X1) time (24~72 h, X2) and pH (2~6, X3). The second-order polynominal model was developed by experiments based on Boc-behnken design containing 15 experimental runs with three replicates at the center point. The coefficient (R2) and p-value of response surface regression equation for isoquercetin yield were 0.93 and 0.01, respectively. The results showed the statistical significance. Consequently, the optimum condition was predicted at stationary point as to produce 2.65 mg/mL of isoquercetin by processing at 47.3°C, 52.16 h, and under pH 5.31. The verification of the model was carried out at the optimal conditions. The experimental value of 2.57 mg/mL of isoquercetin showed good agreement with the predicted one. These results might provide important information for utilization of jujube leaf as a waste in the food industry. From the optimization study, one interesting phenomenon was observed. Under various treatment conditions, the bioconversion of isoquercetin from rutin (α-L-rhamnosidase activity) was well-performed but the accumulation of quercitin (β-D-glucosidase activity) was not. A further study was carried out to ensure the cause of the loss of β-D-glucosidase activity of hesperidinase whether it is affected by enzyme kinetics or possible enzyme inhibitor. Firstly, to examine the effect of enzyme kinetics, enzymatic biotransformation was conducted at 40°C and pH 3.8 with enzyme concentration ranging from 0.03125 mg/mL to 160.0 mg/mL for 24, 48 and 72 h. Regardless of reaction time, quercetin was not produced in the concentration of enzyme below 5 mg/mL, while the activity of rhamnosidase was observed. The esculin hydrolysis test was conducted by phenolic compounds to appraise whether it is competitive inhibition or not. Secondly, to evaluate the existence of possible inhibitors, jujube leaf extracts with various concentrations (0 – 20%) were mixed with 0.5 mM of 4-nitrophenyl β-D-glucospyranoside solution containing 0.3 unit/mL of β-D-glucosidase from Almonds. The Hanes-Woolf plot showed non-competitive inhibition. Therefore, these results strongly suggested that certain components in the jujube leaf extract act like an inhibitor of β-d-glucosidase. Further study is necessary to identify and confirm the possible inhibitor.