Extraction, Separation and in Vitro Antioxidative Effects of Polyphenols in Apple Pomace
|School||Shaanxi Normal University|
|Course||Of Food Science|
|Keywords||apple pomace apple polyphenol extraction and separation HPLC antioxidative activity|
Polyphenols are widely common secondary metabolites in different tissues of plants. At present, there are many researches on the bioavailability of polyphenols, and there has been a remarkable rate of progress. The interest in health effects of polyphenols is exemplified by the 1st and 2nd "International Conference on Polyphenols and Health" held in France and America. Polyphenols in apples are predominantly localized in the peels and cores, after juicing, most of polyphenols come into the pomace. So there are abundant polyphenols in apple pomace. China has an abundant resource of apple pomace, and every year can get more than 0.9 million tons apple pomace. There is a glorious prospect to produce polyphenols utilizing apple pomace.In this paper, the following contents were particularly studied:Firstly, the quantitative method for total polyphenols was researched. And then we detected the stability, precision and recovery of the method.Secondly, one suitable solvent was picked up from several organic solvents, and mainly focused on the extraction methods of polyphenols from apple pomace.Thirdly, the most important thing was to choose the most suitable kind of resin from a few kinds of macroporous resins which could absorb and separate polyphenols from the extraction solution, and then studied on their absorbing and separating properties.Fourthly, the compositive differences of polyphenol in the extraction solution and the solution which effused from the resin column were detected by high performance liquid chromatography (HPLC). Different concentration ethanol were used to elute the polyphenols from the resin column, and the effluent solution were collected, concentrated, and then were analyzed by HPLC.At last, mainly researched on the in vitro antioxidative activities of apple pomace polyphenols through several models, and compared with butylated hydroxytoluene (BHT) and tert-Butyl hydroquinone (TBHQ).Through a great deal of experiments, we can draw conclusions as follows:1) Total content of polyphenol is quantitatively determined by means of Folin-Ciocalteu reagent which forms a highly stable blue colour and with sufficient precision and ruggedness. In this paper, tannic acid was used as the standard substance, and there is a very good linearity when the concentration of tannic acid changing from lmg/L to 9mg/L. The standard curve equation is YA760=0.0933X + 0.0127, and the R2 is 0.9994. The RSDs of precision and recovery of the experiments are 0.831% and 0.500%.2) The extraction capacity of methanol, acetone, ethyl acetate and ethanol are: acetone > ethanol > methanol > acetone. Ethanol was selected as the best one for its low toxicity and strong extraction capacity of polyphenols.3) Quadratic regression orthogonal rotary experiments for ethanol extraction of polyphenols were used in this paper. And the results were analyzed by DPS. The results indicated that the 4 factors in the experiments, ethanol concentration, solid vs. liquid ratio, temperature and extraction time, have strong influences to the extraction effects. And the interaction between ethanol concentration and temperature is also very strong. The influencing extents of the 4 factors are: temperature > solid vs. liquid ratio > extraction time > ethanol concentration. The best extraction conditions are: temperature is 70 °C, solid vs. liquid ratio is 1:14, ethanol concentration is 60%, and extraction time is 6 hours. Using these conditions, we got 3.75g polyphenols from 1 kilogram apple pomace, and it’s about 3.60% lower than the theoretical calculation value. The equation which showed the relationship between the extraction effect and the 4 factors is: Y=2.81467 - 0.08062*1 + 0.13513Z2 + 0.22079*3 + 0.10604X4 - 0.11984Xi2 + 0.03891Z32 + 0.04594Z!X3.4) Quadratic regression orthogonal rotary experiments for ultrasonic-assisted extraction of polyphenols were used in this paper. And the results were analyzed by DPS. The results indicated that the 4 factors in the experiments, ethanol concentration, solid vs. liquid ratio, temperature and ultrasonic-assisted extraction time, have strong influences to the extraction effects. And the interaction between ethanol concentration and solid vs. liquid ratio is also strong. The influencing extents of the 4 factors are: temperature > solid vs. liquid ratio > extraction time > ethanol concentration. The best extraction conditions are: temperature is 70 °C , solid vs. liquid ratio is 1:24, ultrasonic-assisted extraction time is 30 minutes, and ethanol concentration is 50%. Using these conditions, we got 4.07g polyphenols from 1 kilogram apple pomace, andit’s about 6.00% lower than the theoretical calculation value. The equation which showed the relationship between the extraction effect and the 4 factors is: Y=3.40075 -0.04158*! + 0.13083*2 + 0.24700*3 + 0.08617X, - 0.08042*1* - 0.05563*i*2.5) By captive tests, we detected the absorbing and eluting capacities of 10 kinds of macroporous absorbent resins producing by two companies. The results indicated that D4020 resin was the best one with higher absorbing and eluting capacities than the other 9 kinds of resins.6) D4020 resin could absorb polyphenols from the extraction solution quickly within 2 hours. High concentration of ethanol had a strong eluting effect to the polyphenols absorbed on the D4020 resin. The eluting rate of polyphenols would arrive at 97% with 70% ethanol at room temperature within 2 hours. Also, high temperature could lead to high eluting rate. Within 30 minutes, 95% polyphenols would be eluted at 70°C using 70% ethanol.7) The absorbing process was an exothermic course, it belonged to physical absorption. The whole process accorded with Langmuir isothermal model and Freundlich isothermal model.8) The results of high performance liquid chromatography analysis showed that the D4020 macroporous absorbent resin could effectively absorb polyphenols from apple pomace extraction solution, and most of the apple pomace polyphenols (APPs) could be absorbed on the resin column. The composition of APPs was very complicated;just 11 polyphenols could be identified. These polyphenols were chlorogenic acid, (+)-catechin, (-)-epicatechin, rutin, trans-cinnamic acid, phloridzin, 4-hydroxybenzoic acid, p-coumaric acid, gallic acid, caffeic acid and ferulic acid. In these polyphenols, the content of phloridzin was more than the others’.9) In vitro antioxidative experiments indicated that apple pomace polyphenols (APPs) had strong antioxidative activity. APPs showed very good reducing power and better than BHT and TBHQ. At a low concentration level, APPs gave a high scavenging activity for superoxide anion radical, and its the 50% inhibitory concentration (IC50) was 49.8mg/L. APPs also showed more effective scavenging DPPH- activity than BHT and TBHQ, the IC5o was 15.8mg/L. Compared with BHT and TBHQ, the scavenging activity for hydroxyl radical of APPs was much higher, and the IC50 was 522.4mg/L. Furthermore, APPs revealed powerful inhibitory effects on peroxidation of polyunsaturated fatty acid from yelk lipoprotein induced by Fe2+ at all the testedconcentrations;the IC50 was 126.4mg/L. Besides this, APPs also displayed very good inhibitory effects on fi -carotene/linoleic acid assay system at all the tested concentration levels, and the IC50 was 25.2mg/L.