米糠中阿魏酸的提取分离与测定研究
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摘要
米糠是稻谷加工中的副产品，我国每年拥有1000万吨以上的丰富资源。米糠含有多种生理功能的活性物质，其中包括经济价值较高的阿魏酸，如将米糠深度开发，可使其价值提高数十倍，因此成为近年来食品与营养学研究的热点之一。但由于在生产中存在缺乏产品快速检测方法，产品质量不稳定，生产周期长，综合利用率低等问题，米糠常常被用来充当动物饲料，利用价值很低。据此，本论文进行了下列研究：
1．根据阿魏酸结构和性质的特点，利用反相高效液相色谱(RP-HPLC)和荧光光谱法测定阿魏酸。高效液相色谱法中，对流动相组成、pH值和柱温各种影响因素进行了研究，得到阿魏酸的RP-HPLC分析条件为：色谱柱为C_(18)ODS(4.6 mm×150 mm)；流动相为甲醇-水-冰乙酸(35:65:0.9，v／v)；紫外检测波长λ=322nm；流速为1.0mL／min；柱温为25℃。上述条件下，阿魏酸在1.0～5.0μg／mL范围内线性关系良好，r=0.99928。该方法相对标准偏差在0.98％以内，平均回收率为99.7％。荧光光谱法中，对溶剂、静置时间的影响进行了考察。阿魏酸的激发波长为344nm，发射波长为362nm。阿魏酸在0.2～2.2μg／ml浓度范围内呈良好的线性关系，相关系数为0.99957，相对标准偏差为1.32％，平均回收率为99.0％。上述两种阿魏酸定量测定方法具有简便、快速、灵敏、准确的特点。
2．对从米糠中提取阿魏酸的工艺进行优化，以提取液中阿魏酸的含量为指标，考察了碱溶液提取法、乙醇提取法、超声提取法、甲醇-甲酸提取法、碱醇提取法的优劣，用正交实验法对碱浓度、温度、时间、碱醇比和固液比5个因素进行研究，对从米糠中提取阿魏酸的工艺进行优化，得到最佳工艺条件：1％氢氧化钠溶液与乙醇按4:1的比例混和，80℃下提取6h，固液比为1:8，加入0.2g/L的亚硫酸钠作为抗氧化剂。此条件下阿魏酸的提取量为2.239mg／g米糠。
3．利用阴离子树脂进行分离富集阿魏酸，对阿魏酸在树脂上的吸附、解吸特性和吸附动力学行为进行了研究，结果表明D201树脂具有较好的吸附解吸性能。实验对样品的上样浓度、流速、洗脱流速和洗脱剂的组成进行了考察，得到阿魏酸的最佳分离纯化工艺流程为：上样流速为1.0BV／h，浓度为1800～2300mg／L，洗脱流速为2.0BV／h(乙醇：水：盐酸=65:31:4，v／v)。洗脱液浓缩、干燥后得到白色样品，且样品与阿魏酸对照品红外光谱图一致。
4．采用Fenton反应体系，研究了阿魏酸对羟基自由基的清除作用。随着阿魏酸浓度增大，清除作用增强。其在低浓度时即有很好的清除率，且在浓度较低的时候清除效果要好于另两种优良的羟自由基清除剂抗坏血酸和硫脲。
Rice bran is the byproduct of rice, and our country produces morethan 10 million tons rice bran per year. Because it has many kinds ofactive substances, such as ferulic acid, which is valuable, race bran hasbecome one of the focus food and nutrition research during the pastseveral years. However, there are many problems existing in practice,such as no rapid product analytical and evaluation method, instability ofproduct, long production cycle, low utilization, etc. The following aspectshave been done in our research to solve the problems listed above:
1. According to the properties of Ferulic acid in structure andcharacter, RP-HPLC analytical method and Spectrofluorimetry weredeveloped for determination of Ferulic acid. Effects of mobile phaseconstitute, pH and temperature on retention behaviors of Ferulic acid incolumn were discussed. The optimal chromatographic conditionsincluded ODS-C_(18) column (4.6 mm×150 mm) and the mobile phaseconsisting of a mixture of methanol-water-acetic acid (35:65:0.9, volumeratio) at a flow rate of 1.0 mL/min at 25℃. The UV detection wavelengthwas at 322nm. There is a good linear relationship in the range of 1.0～5.0μg/mL for ferulic acid, and the correlative coefficient is 0.99928. Therelative standard deviation is 0.98% and the average recovery is 99.7%.For Spectrofluorimetry, the excitation and emission wavelength is 344nmand 362mn, respectively. Influences of different solution and time on thefluorescence intensity were studied. The average recoveries of the methodwere 99.0% and the coefficients of variation were 1.32%. The linearrange was from 0.2 to 2.2μg/mL, correlation coefficient was 0.99957.These simple, rapid, sensitive and accurate methods could be used inquantitative determination of Ferulic acid in rice bran.
2. Total content of ferulic acid was taken as index to compare fiveextraction methods, including hydrolysis, reflux, ultrasonic. Thetechnological conditions of extracting Ferulic acid from rice bran wereoptimized. Base on the orthogonal design, five factors were optimized: concentration of Sodium hydroxide, temperature, extraction time, Sodiumhydroxide/ethanol ratio and solid/liquid ratio. The optimized conditionsare as follows: Sodium hydroxide, 1%; temperature, 80℃; extractiontime, 6 h; Sodium hydroxide/ethanol ratio, 4:1; solid/liquid ratio, 1:8,adding 0.2g/L sodium sulfite as antioxidant. Extraction of Ferulic acid is2.239mg/g rice bran.
3. The adsorbing and desorbing properties of Ferulic acid ondifferent adsorbents was studied, it shows that D201 was better in staticadsorption and elution. The optimal purification conditions were asfollows: flow rate was 1.0BV/h, content of Ferulic acid was from1800mg/L to 2300rag/L, and 2.0BV/h was the best elutionrate(methanol/water/hydrochloric acid=65:31: 4, volume ratio). The IRspectrum of sample was consistent with that of the standard Ferulic acid.
4. The antioxidation of Ferulic acid was discussed by the Fentonreaction method. As content of Ferulic acid increasing, the effect becamestronger. It appeared better effect than vitamin C and thiourea at lowconcentration.
1．根据阿魏酸结构和性质的特点，利用反相高效液相色谱(RP-HPLC)和荧光光谱法测定阿魏酸。高效液相色谱法中，对流动相组成、pH值和柱温各种影响因素进行了研究，得到阿魏酸的RP-HPLC分析条件为：色谱柱为C_(18)ODS(4.6 mm×150 mm)；流动相为甲醇-水-冰乙酸(35:65:0.9，v／v)；紫外检测波长λ=322nm；流速为1.0mL／min；柱温为25℃。上述条件下，阿魏酸在1.0～5.0μg／mL范围内线性关系良好，r=0.99928。该方法相对标准偏差在0.98％以内，平均回收率为99.7％。荧光光谱法中，对溶剂、静置时间的影响进行了考察。阿魏酸的激发波长为344nm，发射波长为362nm。阿魏酸在0.2～2.2μg／ml浓度范围内呈良好的线性关系，相关系数为0.99957，相对标准偏差为1.32％，平均回收率为99.0％。上述两种阿魏酸定量测定方法具有简便、快速、灵敏、准确的特点。
2．对从米糠中提取阿魏酸的工艺进行优化，以提取液中阿魏酸的含量为指标，考察了碱溶液提取法、乙醇提取法、超声提取法、甲醇-甲酸提取法、碱醇提取法的优劣，用正交实验法对碱浓度、温度、时间、碱醇比和固液比5个因素进行研究，对从米糠中提取阿魏酸的工艺进行优化，得到最佳工艺条件：1％氢氧化钠溶液与乙醇按4:1的比例混和，80℃下提取6h，固液比为1:8，加入0.2g/L的亚硫酸钠作为抗氧化剂。此条件下阿魏酸的提取量为2.239mg／g米糠。
3．利用阴离子树脂进行分离富集阿魏酸，对阿魏酸在树脂上的吸附、解吸特性和吸附动力学行为进行了研究，结果表明D201树脂具有较好的吸附解吸性能。实验对样品的上样浓度、流速、洗脱流速和洗脱剂的组成进行了考察，得到阿魏酸的最佳分离纯化工艺流程为：上样流速为1.0BV／h，浓度为1800～2300mg／L，洗脱流速为2.0BV／h(乙醇：水：盐酸=65:31:4，v／v)。洗脱液浓缩、干燥后得到白色样品，且样品与阿魏酸对照品红外光谱图一致。
4．采用Fenton反应体系，研究了阿魏酸对羟基自由基的清除作用。随着阿魏酸浓度增大，清除作用增强。其在低浓度时即有很好的清除率，且在浓度较低的时候清除效果要好于另两种优良的羟自由基清除剂抗坏血酸和硫脲。
Rice bran is the byproduct of rice, and our country produces morethan 10 million tons rice bran per year. Because it has many kinds ofactive substances, such as ferulic acid, which is valuable, race bran hasbecome one of the focus food and nutrition research during the pastseveral years. However, there are many problems existing in practice,such as no rapid product analytical and evaluation method, instability ofproduct, long production cycle, low utilization, etc. The following aspectshave been done in our research to solve the problems listed above:
1. According to the properties of Ferulic acid in structure andcharacter, RP-HPLC analytical method and Spectrofluorimetry weredeveloped for determination of Ferulic acid. Effects of mobile phaseconstitute, pH and temperature on retention behaviors of Ferulic acid incolumn were discussed. The optimal chromatographic conditionsincluded ODS-C_(18) column (4.6 mm×150 mm) and the mobile phaseconsisting of a mixture of methanol-water-acetic acid (35:65:0.9, volumeratio) at a flow rate of 1.0 mL/min at 25℃. The UV detection wavelengthwas at 322nm. There is a good linear relationship in the range of 1.0～5.0μg/mL for ferulic acid, and the correlative coefficient is 0.99928. Therelative standard deviation is 0.98% and the average recovery is 99.7%.For Spectrofluorimetry, the excitation and emission wavelength is 344nmand 362mn, respectively. Influences of different solution and time on thefluorescence intensity were studied. The average recoveries of the methodwere 99.0% and the coefficients of variation were 1.32%. The linearrange was from 0.2 to 2.2μg/mL, correlation coefficient was 0.99957.These simple, rapid, sensitive and accurate methods could be used inquantitative determination of Ferulic acid in rice bran.
2. Total content of ferulic acid was taken as index to compare fiveextraction methods, including hydrolysis, reflux, ultrasonic. Thetechnological conditions of extracting Ferulic acid from rice bran wereoptimized. Base on the orthogonal design, five factors were optimized: concentration of Sodium hydroxide, temperature, extraction time, Sodiumhydroxide/ethanol ratio and solid/liquid ratio. The optimized conditionsare as follows: Sodium hydroxide, 1%; temperature, 80℃; extractiontime, 6 h; Sodium hydroxide/ethanol ratio, 4:1; solid/liquid ratio, 1:8,adding 0.2g/L sodium sulfite as antioxidant. Extraction of Ferulic acid is2.239mg/g rice bran.
3. The adsorbing and desorbing properties of Ferulic acid ondifferent adsorbents was studied, it shows that D201 was better in staticadsorption and elution. The optimal purification conditions were asfollows: flow rate was 1.0BV/h, content of Ferulic acid was from1800mg/L to 2300rag/L, and 2.0BV/h was the best elutionrate(methanol/water/hydrochloric acid=65:31: 4, volume ratio). The IRspectrum of sample was consistent with that of the standard Ferulic acid.
4. The antioxidation of Ferulic acid was discussed by the Fentonreaction method. As content of Ferulic acid increasing, the effect becamestronger. It appeared better effect than vitamin C and thiourea at lowconcentration.
引文
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