苹果渣中多酚物质的提取、分离及其抗氧化活性研究
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摘要
多酚物质是存在于植物组织中的次级代谢产物,目前对于其生物活性方面的研究已经十分广泛和深入,国际上也已经在2003年和2005年分别在法国和美国召开了第一届、第二届“多酚物质与人类健康国际大会”,多酚物质对人类健康方面的独特作用已经得到人们的普遍认可。苹果的果皮、果芯中均含有较多的多酚物质,在苹果榨汁过程中,大部分多酚物质随果皮和果芯而转移到果渣中,因此苹果渣中含有较多的多酚物质。我国苹果渣资源十分丰富且相对集中,年产湿苹果渣超过90万吨,利用苹果渣生产具有独特生理活性的多酚物质具有广阔的市场前景。
本文主要从以下几个方面对苹果渣中的多酚物质进行了试验研究:
一是确定总多酚含量的测定方法,研究其稳定性、精密度和加样回收率;
二是选择提取苹果渣多酚的最佳溶剂,研究苹果渣多酚的提取方法;
三是以树脂吸附法分离苹果渣多酚,并对多酚物质的吸附、解析特性及其影响因素进行研究;
四是以高效液相色谱法分析提取液中多酚经过大孔吸附树脂吸附前后组成的变化,以及不同浓度乙醇溶液洗脱树脂柱时洗脱液中多酚的组成差异;
五是研究苹果渣多酚的体外抗氧化活性,并将其与BHT和TBHQ进行对比。
研究结果表明:
1.用FC法测定结果稳定,当以单宁酸为标准品时,在0～9mg/L浓度范围内线性较好,标准曲线方程为Y_(A760)=0.0933X+0.0127,其R~2值为0.9994;FC法精密度高、重现性好,6次重复测定的RSD值仅为0.813%;该法回收率较高,在6次加样回收率试验中,其平均加样回收率达到99.60%,RSD值为0.500%。
2.甲醇、丙酮、乙酸乙酯和乙醇四种溶剂对苹果渣中多酚的提取能力大小顺序为:丙酮>乙醇>甲醇>乙酸乙酯。乙醇的提取能力略低于丙酮,综合考虑成本与溶剂毒性,选择乙醇为提取溶剂。
3.基于乙醇直接提取的二次回归正交旋转组合设计试验结果表明:乙醇溶液浓度、料液比、提取温度、提取时间4个因素对多酚提取效果的影响达到极显著水平,且乙醇溶液浓度与温度之间的交互作用显著;4个因素对多酚提取效果影响顺序为:温度>料液比>浸提时间>乙醇浓度;通过DPS软件的分析,得到了本试验最佳提取技术参数为:浸提温度70℃、料液比1∶14、提取时间6h、乙醇溶液浓度60%,以此条件进行试验,得到提取量平均值为3.75g/kg,与DPS软件给出
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, and
it'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 tested
concentrations;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.
本文主要从以下几个方面对苹果渣中的多酚物质进行了试验研究:
一是确定总多酚含量的测定方法,研究其稳定性、精密度和加样回收率;
二是选择提取苹果渣多酚的最佳溶剂,研究苹果渣多酚的提取方法;
三是以树脂吸附法分离苹果渣多酚,并对多酚物质的吸附、解析特性及其影响因素进行研究;
四是以高效液相色谱法分析提取液中多酚经过大孔吸附树脂吸附前后组成的变化,以及不同浓度乙醇溶液洗脱树脂柱时洗脱液中多酚的组成差异;
五是研究苹果渣多酚的体外抗氧化活性,并将其与BHT和TBHQ进行对比。
研究结果表明:
1.用FC法测定结果稳定,当以单宁酸为标准品时,在0～9mg/L浓度范围内线性较好,标准曲线方程为Y_(A760)=0.0933X+0.0127,其R~2值为0.9994;FC法精密度高、重现性好,6次重复测定的RSD值仅为0.813%;该法回收率较高,在6次加样回收率试验中,其平均加样回收率达到99.60%,RSD值为0.500%。
2.甲醇、丙酮、乙酸乙酯和乙醇四种溶剂对苹果渣中多酚的提取能力大小顺序为:丙酮>乙醇>甲醇>乙酸乙酯。乙醇的提取能力略低于丙酮,综合考虑成本与溶剂毒性,选择乙醇为提取溶剂。
3.基于乙醇直接提取的二次回归正交旋转组合设计试验结果表明:乙醇溶液浓度、料液比、提取温度、提取时间4个因素对多酚提取效果的影响达到极显著水平,且乙醇溶液浓度与温度之间的交互作用显著;4个因素对多酚提取效果影响顺序为:温度>料液比>浸提时间>乙醇浓度;通过DPS软件的分析,得到了本试验最佳提取技术参数为:浸提温度70℃、料液比1∶14、提取时间6h、乙醇溶液浓度60%,以此条件进行试验,得到提取量平均值为3.75g/kg,与DPS软件给出
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, and
it'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 tested
concentrations;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.
引文
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