沙棘汁总黄酮测定方法的对比分析
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摘要
为确立适合沙棘汁总黄酮测定的方法,更好地开发沙棘汁的营养功效,以沙棘汁为试验材料,研究沙棘汁总黄酮NaNO2-Al(NO3)3-NaOH显色法、AlCl3-CH4O显色法以及紫外分光光度法3种检测方法的准确度与加标回收率,并采用高效液相法对检测结果进行验证,找出最佳方法。结果表明:NaNO2-Al(NO3)3-NaOH显色法、AlCl3-CH4O显色法以及紫外分光光度法检测波长分别为510,420和350nm。芦丁可作为3种检测方法的标准品。在浓度范围内,每个方法的标准曲线线性关系良好,相关系数高;测出的沙棘汁总黄酮含量分别为1.19,0.42和2.47mg·m L-1,RSD值分别为7%,25%和7%,加标回收率平均值分别为97%,147%和99.6%。本试验确立的高效液相色谱条件,可以较好地分离芦丁、槲皮素、异鼠李素3种对照品;沙棘汁中分离出多个黄酮组分,黄酮苷元在沙棘中相对含量较少,大部分以黄酮糖苷形式存在。芦丁可作为高效液相法的标准品,标准曲线的相关系数R2为0.9999,浓度在0～160μg·mL-1时存在良好的线性关系,测定沙棘汁中总黄酮含量为2.49mg·mL-1。可见,NaNO2-Al(NO3)3-NaOH显色法、AlCl3-CH4O显色法测定沙棘汁总黄酮的结果准确度较低,与高效液相验证结果比较偏差较大,都在50%以上,且需要额外添加化学试剂;紫外分光光度法结果准确度高,偏差只有1%,该方法无需额外添加化学试剂,对环境污染少,操作简便,是测定沙棘汁总黄酮含量的理想方法,该方法为沙棘总黄酮测定与功能性食品开发提供一定参考。
The object of this study is to confirm the ideal method of determining total flavonoids in sea buckthorn juice to develop its nutrition effects. The accuracy and recovery rates of NaNO2-Al(NO3)3-NaOH coloration method, AlCl3-CH4 O coloration method and UV spectroscopy method for determining total flavonoids were explored by using sea buckthorn juice as material, and their determining results were tested with HPLC determination result to confirm the ideal method. The result indicated that the determining wavelengths were 510 nm, 420 nm and 350 nm in the NaNO2-Al(NO3)3-NaOH coloration method, AlCl3-CH4 O coloration method and UV spectroscopy method, respectively. Rutin can act as the standard in three methods, and the standard curves have good linearity and correlation coefficient in reasonable concentration. The determining results of the total flavonoids were 1.19,0.42 and 2.47mg·mL-1; the RSD values were 7%, 25% and 7%; the average recovery rate were 97%, 147% and 99.6%. The standards of rutin, quercetin, and isorhamnetin could be separated by HPLC under the confirmed conditions. Many constituents were separated from the sea buckthorn juice with HPLC under the same conditions. There were many flavonoid glycosides but little aglycones. Rutin can act as standard control in HPLC method. The correlation coefficient of the standard curve was 0.9999.The linearity was very good between the concentration 0-160μg·mL-1. The determining result of total flavonoids was 2.49mg·mL-1with HPLC. It could be concluded that the results of NaNO2-Al(NO3)3-NaOH coloration method and AlCl3-CH4 O coloration method were not accurate comparing with the result of UV spectroscopy method, the deviation got to over 50%, these two methods needed additional chemical reagents. The result accuracy of the UV spectroscopy method was very high, the deviation was only 1%. What's more, t his method could be operated easily, used little additional chemical reagents, which was anenvironment-friendly method. So it was the ideal method of determining total flavonoids in sea buckthorn juice.
The object of this study is to confirm the ideal method of determining total flavonoids in sea buckthorn juice to develop its nutrition effects. The accuracy and recovery rates of NaNO2-Al(NO3)3-NaOH coloration method, AlCl3-CH4 O coloration method and UV spectroscopy method for determining total flavonoids were explored by using sea buckthorn juice as material, and their determining results were tested with HPLC determination result to confirm the ideal method. The result indicated that the determining wavelengths were 510 nm, 420 nm and 350 nm in the NaNO2-Al(NO3)3-NaOH coloration method, AlCl3-CH4 O coloration method and UV spectroscopy method, respectively. Rutin can act as the standard in three methods, and the standard curves have good linearity and correlation coefficient in reasonable concentration. The determining results of the total flavonoids were 1.19,0.42 and 2.47mg·mL-1; the RSD values were 7%, 25% and 7%; the average recovery rate were 97%, 147% and 99.6%. The standards of rutin, quercetin, and isorhamnetin could be separated by HPLC under the confirmed conditions. Many constituents were separated from the sea buckthorn juice with HPLC under the same conditions. There were many flavonoid glycosides but little aglycones. Rutin can act as standard control in HPLC method. The correlation coefficient of the standard curve was 0.9999.The linearity was very good between the concentration 0-160μg·mL-1. The determining result of total flavonoids was 2.49mg·mL-1with HPLC. It could be concluded that the results of NaNO2-Al(NO3)3-NaOH coloration method and AlCl3-CH4 O coloration method were not accurate comparing with the result of UV spectroscopy method, the deviation got to over 50%, these two methods needed additional chemical reagents. The result accuracy of the UV spectroscopy method was very high, the deviation was only 1%. What's more, t his method could be operated easily, used little additional chemical reagents, which was anenvironment-friendly method. So it was the ideal method of determining total flavonoids in sea buckthorn juice.
引文
[1]冯智鹏,冶贵生,袁海,等.青海野生西藏沙棘果渣黄酮的提取工艺[J].北方园艺,2017(1):142-147.
[2] POP R M,WEESEPOEL Y,SOCACIU C,et al.Carotenoid composition of berries and leaves from six Romanian sea buckthorn(Hippophae rhamnoides L.)varieties[J].Food Chemistry,2014,147(6):1-9.
[3] GUO R X,GUO X B,LI T,et al.Comparative assessment of phytochemical profiles,antioxidant and antiproliferative activities of Sea buckthorn(Hippophae rhamnoides L.)berries[J].Food Chemistry,2017,221:997-1003.
[4]白生文,汤超,田京,等.沙棘果渣总黄酮提取工艺及抗氧化活性分析[J].食品科学,2015,36(10):59-64.
[5] KIM J S,KWON Y S,SA Y J,et al.Isolation and identification of sea buckthorn(Hippophae rhamnoides)phenolics with antioxidant activity and glucosidase inhibitory effect[J].Agricultural and Food Chemistry,2011,59:138-144.
[6] FATIMA T,KESARI V,WATT I,et al.Metabolite profiling and expression analysis of flavonoid,vitamin C and tocoph erol biosynthesis genes in the antioxidant-rich sea buckthorn(Hippophae rhamnoides L.)[J].Phytochemistry,2015,118:181-191.
[7] GUO X F,YANG B,CAI W W,et al.Effect of sea buckthorn(Hippophae rhamnoides L.)on blood lipid profiles:a systematic review and meta-analysis from 11 independent randomized controlled trials[J].Trends in Food Science&T echnology,2017,61:1-10.
[8] OLAS B.Sea buckthorn as a source of important bioactive compounds in cardiovascular diseases[J].Food and Chemical Toxicology,2016,97:199-204.
[9]林赫杰,陈钰.沙棘研究现状、开发利用及发展前景[J].天津农业科学,2010,16(2):128-130.
[10]李清花,弓子敬,侯立功.沙棘功效研究及产业发展措施探索[J].种子科技,2017,35(12):80-81.
[11]杨霞,冯锋,刘荔贞,等.高效毛细管区带电泳法同时检测3个产地沙棘果粉中的活性物质[J].食品科学,2017,38(14):144-150.
[12]钱学射,金敬红.沙棘的药用研究与开发[J].中国野生植物资源,2015(6):68-72.
[13]臧茜茜,邓乾春,从仁怀,等.沙棘油功效成分及药理功能研究进展[J].中国油脂,2015,40(5):76-81.
[14]陶波,方梅,张嘉男,等.沙冬青种子总黄酮测定方法及纯化工艺研究[J].生物技术通报,2017,33(5):63-70.
[15]裘纪莹,陈相艳,刘孝永,等.银杏花粉及其发酵饮料的总黄酮含量及总黄酮测定方法比较[J].江苏农业科学,2015(10):371-373.
[16]刘锡建,刘则华,肖稳发,等.沙棘总黄酮的测定及其抗氧化性能[J].上海工程技术大学学报,2006,20(2):135-139.
[17]回瑞华,侯冬岩,关崇新,等.三波长-光谱法测定沙棘果汁中黄酮的含量[J].光谱学与光谱分析,2005,25(2):266-269.
[18]孟晓,余蓉,李永盛,等.沙棘枝叶总黄酮混合提取工艺研究[J].轻工科技,2018,34(1):13-16,141.
[19]吕敏.正交试验法优选沙棘叶总黄酮提取工艺研究[J].山西中医学院学报,2015,16(3):33-34,37.
[20]张慧娟,陈佳佳,苏琴琴,等.沙棘果肉黄酮与沙棘籽黄酮之槲皮素、山奈酚和异鼠李素含量的比较[J].中国中医药科技,2016,23(5):554-556,562.
[21]董怡,林恋竹,赵谋明.光果甘草叶总黄酮测定方法[J].食品科学,2013,34(6):195-198.
[22] FAWBUSH F,NOCK J F,WATKINS C B.Antioxidant contents and activity of 1-methyleyclopropene(1-MCP)-treated Empire apples in air and controlled atmosphere storage[J].Postharvest Biology and Technology,2009,52:30-37
[23]李红,张元湖.苹果果实中总黄酮的提取方法优化研究[J].山东农业大学学报(自然科学版),2003,34(4):471-474.
[24]王建华,韩雅珊,戴蕴青.果蔬中黄酮类化合物含量的测定[J].中国果品研究,1994(4):23-25.
[25] ECCLESTON C,BAORU Y,TAHVONEM R,et al.Effects of an antioxidant-rich juice(sea buckthorn)on risk factors for coronary heart disease in humans[J].Nutrition Biochemistry,2002,13:346-354.
[26]陈辉,黄仁录,邸科前,等.类黄酮化合物在动物营养中的研究进展[J].饲料工业,2006(6):9-11.
[27]张雪,丁长河.紫外分光光度法检测山楂总黄酮方法的建立[J].食品工业科技,2007(7):200-202.
[2] POP R M,WEESEPOEL Y,SOCACIU C,et al.Carotenoid composition of berries and leaves from six Romanian sea buckthorn(Hippophae rhamnoides L.)varieties[J].Food Chemistry,2014,147(6):1-9.
[3] GUO R X,GUO X B,LI T,et al.Comparative assessment of phytochemical profiles,antioxidant and antiproliferative activities of Sea buckthorn(Hippophae rhamnoides L.)berries[J].Food Chemistry,2017,221:997-1003.
[4]白生文,汤超,田京,等.沙棘果渣总黄酮提取工艺及抗氧化活性分析[J].食品科学,2015,36(10):59-64.
[5] KIM J S,KWON Y S,SA Y J,et al.Isolation and identification of sea buckthorn(Hippophae rhamnoides)phenolics with antioxidant activity and glucosidase inhibitory effect[J].Agricultural and Food Chemistry,2011,59:138-144.
[6] FATIMA T,KESARI V,WATT I,et al.Metabolite profiling and expression analysis of flavonoid,vitamin C and tocoph erol biosynthesis genes in the antioxidant-rich sea buckthorn(Hippophae rhamnoides L.)[J].Phytochemistry,2015,118:181-191.
[7] GUO X F,YANG B,CAI W W,et al.Effect of sea buckthorn(Hippophae rhamnoides L.)on blood lipid profiles:a systematic review and meta-analysis from 11 independent randomized controlled trials[J].Trends in Food Science&T echnology,2017,61:1-10.
[8] OLAS B.Sea buckthorn as a source of important bioactive compounds in cardiovascular diseases[J].Food and Chemical Toxicology,2016,97:199-204.
[9]林赫杰,陈钰.沙棘研究现状、开发利用及发展前景[J].天津农业科学,2010,16(2):128-130.
[10]李清花,弓子敬,侯立功.沙棘功效研究及产业发展措施探索[J].种子科技,2017,35(12):80-81.
[11]杨霞,冯锋,刘荔贞,等.高效毛细管区带电泳法同时检测3个产地沙棘果粉中的活性物质[J].食品科学,2017,38(14):144-150.
[12]钱学射,金敬红.沙棘的药用研究与开发[J].中国野生植物资源,2015(6):68-72.
[13]臧茜茜,邓乾春,从仁怀,等.沙棘油功效成分及药理功能研究进展[J].中国油脂,2015,40(5):76-81.
[14]陶波,方梅,张嘉男,等.沙冬青种子总黄酮测定方法及纯化工艺研究[J].生物技术通报,2017,33(5):63-70.
[15]裘纪莹,陈相艳,刘孝永,等.银杏花粉及其发酵饮料的总黄酮含量及总黄酮测定方法比较[J].江苏农业科学,2015(10):371-373.
[16]刘锡建,刘则华,肖稳发,等.沙棘总黄酮的测定及其抗氧化性能[J].上海工程技术大学学报,2006,20(2):135-139.
[17]回瑞华,侯冬岩,关崇新,等.三波长-光谱法测定沙棘果汁中黄酮的含量[J].光谱学与光谱分析,2005,25(2):266-269.
[18]孟晓,余蓉,李永盛,等.沙棘枝叶总黄酮混合提取工艺研究[J].轻工科技,2018,34(1):13-16,141.
[19]吕敏.正交试验法优选沙棘叶总黄酮提取工艺研究[J].山西中医学院学报,2015,16(3):33-34,37.
[20]张慧娟,陈佳佳,苏琴琴,等.沙棘果肉黄酮与沙棘籽黄酮之槲皮素、山奈酚和异鼠李素含量的比较[J].中国中医药科技,2016,23(5):554-556,562.
[21]董怡,林恋竹,赵谋明.光果甘草叶总黄酮测定方法[J].食品科学,2013,34(6):195-198.
[22] FAWBUSH F,NOCK J F,WATKINS C B.Antioxidant contents and activity of 1-methyleyclopropene(1-MCP)-treated Empire apples in air and controlled atmosphere storage[J].Postharvest Biology and Technology,2009,52:30-37
[23]李红,张元湖.苹果果实中总黄酮的提取方法优化研究[J].山东农业大学学报(自然科学版),2003,34(4):471-474.
[24]王建华,韩雅珊,戴蕴青.果蔬中黄酮类化合物含量的测定[J].中国果品研究,1994(4):23-25.
[25] ECCLESTON C,BAORU Y,TAHVONEM R,et al.Effects of an antioxidant-rich juice(sea buckthorn)on risk factors for coronary heart disease in humans[J].Nutrition Biochemistry,2002,13:346-354.
[26]陈辉,黄仁录,邸科前,等.类黄酮化合物在动物营养中的研究进展[J].饲料工业,2006(6):9-11.
[27]张雪,丁长河.紫外分光光度法检测山楂总黄酮方法的建立[J].食品工业科技,2007(7):200-202.