Double-enzymatic Preparation, Antioxidation And Stability Of Procyanidins Of Lotus Seed Pods

Lotus Seed Pod is ripened receptacle of Nelumbo nucifera Gaertn., has scattered stasis、hemostatic、dewetting effect.Research now suggests the main active substances in Lotus Seed Pod is procyanidins,which have strong physical activity. Procyanidins will have a wide applitcation prospect in pharmaceutical, food and cosmetics field.In this study, the procyanidins from Lotus Seed Pod were extracted and purified in optimal conditions.Then, established a new method of analytical antioxidative activity of LSPC, Finally the study has systematically tested stability of procyanidins.The results of this research are follows:1. To select a more accurate and convenient method of determining Procyanidins from Lotus Seed Pods, Three methods:n-butyl alcohol-hydrochloric acid method, UV spectrophotometry and vanillin-sulfuric acid method were used to analyze the content of the procyanidins of Lotus Seed Pods, and their sensitivity, stability, precision and recovery rate were study. Then the Three methods were compared and analyzed according to the reaction principles. As a result, UV spectrophotometry is simple and quick but the method applies only to high purity product. the vanillin-sulfuric acid method shows a broad linearity range and high precision, but the results of determination is total content of flavan-3-ols and procyanidins, so the result will be somewhat high. The products measure by n-butyl alcohol-hydrochloric acid method are of high quality, but the method is complicated operation and the reaction condition difficult to be controlled led to the result with low precision, besides, normal butanol is an expensive reagent with great toxicity.2. Response surface methodology(RSM) and central composite design was used to investigate the optimization of the extraction of the Lotus Seed Pods Procyanidins(LSPC). Experimental data were analyzed by solving the regression equation with SAS software. The results showed that the optimal technological conditions were:enzymatic hydrolysis temperature was 53.07℃, enzymatic hydrolysis time was 1.56 hours, pH was4.8, the ratio of pectinase to cellulase was 1:1.08, the yield of procyanidins was 5.28%.Comparing with single extraction with alcohol, the Procyanidins yield of the technic after optimize increase by 35.8%. The inoxidability of samples has been compared by DPPH, the result indicate that The procyanidins of double-enzymatic method have strong scavenging effects to DPPH radicals.3. Compare the adsorption and desorption rate of AB-8、HP-20、ADS-17、D101. Choose AB-8 and HP-20 to do adsorption dynamics research,which have higher adsorption and desorption rate, the results showed that the AB-8 is the best resin. the optimum technological conditions of AB-8 type macroporous resin is:the sample liquid concentration is 10.0 mg/mL, the sample flow velocity is 1mL/min, elution concentration is 60% alcohol, elution volume is 3.5 BV, under the conditions the purity of procyanidins is 102.39%4. Based on the static chemiluminescence method, the flow injection chemiluminescence analysis improved reproducibility and selectivity, and the results of the method are stable and reproducible. Determined the best reaction conditions of flow injection chemiluminescence analysis was:Luminol concentration is 0.5 mmol/L, pyrogallol is:0.3 mmol/L, pH of Na2CO3-NaHCO3 buffer is:10.45. This method have good stability, high accuracy, the linear range is 0.02-0.50 mg/mL.The method has been used for the determination the antioxidative activity of LSPC, the results show that the antioxidative activity of LSPC is greater than Vc, the results obtained with this method agree with the DPPH method.5. Stability and thermal degradation kinetics of the Lotus Seed Pods Procyanidins(LSPC) was studied. The results showed that LSPC was stable in low temperature, without illumination, and weak acid conditions. There were no significant effects of food materials such as NaCl, sucrose and glucose and preservative on stability of LSPC; H2O2 has a destructive effect to the stability of LSPC,while NaHSO3 is protective to LSPC. Fe3+、Fe2+、Pb2+ and Al3+ have significant destructive effects on the stability of LSPC, other metals had weak effects. There were different effects of four sterilizing processes on the stability of LSPC, of which pasteurization had the least effect, while high pressure steam sterilization had the highest effects. Degradation of LSPC is followed first-order reaction kinetics,and the thermal degradation activation energy of LSPC is 43.10 kJ/mol,and the rate constant(k0) is 1.51×105.The theoretical value and experimental value are propinquity,which indicate that the model is appropriate.