不同波长和标准品对红三叶异黄酮含量测定的影响
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摘要
红三叶(Trifolium pratense)是在全世界广泛分布的重要豆科牧草,其异黄酮成分不仅具有多种药用功能,也是该植物环境适应和生态竞争的重要物质基础。鹰嘴豆素A(Biochanin A, B)、芒柄花素(Formononetin, F)、染料木素(Genistein, G)和大豆苷元(Daidzein, D)是红三叶异黄酮的主要成分,均分别被作为标准品应用在异黄酮含量检测中,但是不同标准品对测定结果是否会引起误差鲜见报道。本研究分别以这4种化合物为标准品,开展不同波长下红三叶异黄酮含量测定结果的比对研究。结果显示,标准品和波长的选择对含量测定有显著影响(P <0.05)。根据含量的测定结果,4种标准品可分为两组:芒柄花素和大豆苷元为FD组,鹰嘴豆素A和染料木素为BG组。不同标准品组间所测定的异黄酮含量差异显著(P <0.05),组内差异较小且不显著。另外,不论植物中异黄酮的含量高低,标准品对测定结果的含量影响不明显。鹰嘴豆素A和染料木素的紫外吸收曲线在275 nm处与红三叶提取物斜率较为接近,建议优先考虑选用BG组标准品在275 nm下进行测定。此外,本研究发现,如果仅对该植物异黄酮含量进行半定量或定性比较,不同标准品和波长影响较小。
Red clover(Trifolium pratense) isoflavones exhibit many medicinal and health food functions and are widely used as phytoestrogens in clinical practice. Accurate quantification of isoflavones is of great importance for their further study and application. The four major isoflavones are biochanin A, formononetin, genistein, and daidzein, which are usually used as standard products for quantitative determination of isoflavones. However, scientific selection of standard products and their effects on the determination results have not been reported. In this study, the effects of different wavelengths on isoflavone content in red clover were studied using these four chemical constituents as standard products. The experimental results showed highly significant differentiation of content under different wavelengths and by the use of different standards(P <0.05). Based on the results, the four isoflavones could be divided into two groups, the FD group(formononetin and daidzein)and the BG group(biochanin A and genistein). The inter-group differences were highly significant(P < 0.05), whereas the intra-group differences were small. The differences may be primarily caused by the compatibility of compounds with red clover UV absorption, and were not closely related to the content of compounds. The UV absorption curve for biochanin A and genistein was close to the slope of the red clover extract at 275 nm. Therefore, it is suggested that these two compounds can be used as standards to determine isoflavone content at 275 nm. In addition, this study showed that the difference in the content of isoflavones was so small that the four standards were all used in semi-quantitative or qualitative comparisons.
Red clover(Trifolium pratense) isoflavones exhibit many medicinal and health food functions and are widely used as phytoestrogens in clinical practice. Accurate quantification of isoflavones is of great importance for their further study and application. The four major isoflavones are biochanin A, formononetin, genistein, and daidzein, which are usually used as standard products for quantitative determination of isoflavones. However, scientific selection of standard products and their effects on the determination results have not been reported. In this study, the effects of different wavelengths on isoflavone content in red clover were studied using these four chemical constituents as standard products. The experimental results showed highly significant differentiation of content under different wavelengths and by the use of different standards(P <0.05). Based on the results, the four isoflavones could be divided into two groups, the FD group(formononetin and daidzein)and the BG group(biochanin A and genistein). The inter-group differences were highly significant(P < 0.05), whereas the intra-group differences were small. The differences may be primarily caused by the compatibility of compounds with red clover UV absorption, and were not closely related to the content of compounds. The UV absorption curve for biochanin A and genistein was close to the slope of the red clover extract at 275 nm. Therefore, it is suggested that these two compounds can be used as standards to determine isoflavone content at 275 nm. In addition, this study showed that the difference in the content of isoflavones was so small that the four standards were all used in semi-quantitative or qualitative comparisons.
引文
[1]SAVIRANTA N M,ANTTONEN M J,WRIGHT A V,KARJALAINEN R O.Red clover(Trifolium pratense L.)isoflavones:Determination of concentrations by plant stage,flower colour,plant part and cultivar.Journal of the Science of Food and Agriculture,2008,88(1):125-132.
[2]KAURINOVIC B,POPOVIC M,VLAISAVLJEVIC S,SCHWARTSOVA H,VOJINOVIC-MILORADOV M.Antioxidant profile of Trifolium pratense L.Molecules,2012,17(9):11156-11172.
[3]BOOTH N L,OVERK C R,YAO P,TOTURA S,DENG Y,HEDAYAT A S.Seasonal variation of red clover(Trifolium pratense L.,Fabaceae)isoflavones and estrogenic activity.Journal of Agricultural and Food Chemistry,2006,54(4):1277-1282.
[4]KROYER G T.Red clover extract as antioxidant active and functional food ingredient.Innovative Food Science and Emerging Technologies,2004,5(1):101-105.
[5]陈学福,史高峰.红三叶异黄酮的研究进展.中草药,2006,37(2):309-312.CHEN X F,SHI G F.Research progress of isoflavones in red clover.Chinese Traditional and Herbal Drugs,2006,37(2):309-312.
[6]常平,张颍,夏开元,金宗濂.红车轴草提取物中异黄酮成分的分析.食品科学,2007,28(9):449-452.CHANG P,ZHANG Y,XIA K Y,JIN Z L.Analysis of Isoflavone in Extract of Trifolium pratense L.Food Science,2007,28(9):449-452.
[7]RIJKE E D,AARDENBURG L,DIJK J V,ARIESE F,ERNST W H O,GOOIJER C,GOOIJER C,BRINKMAN U T.Changed isoflavone levels in red clover(Trifolium pratense L.)leaves with disturbed root nodulation in response to waterlogging.Journal of Chemical Ecology,2005,31(6):1285-1298.
[8]LIU Q,XU R,YAN Z,JIN H,CUI H,LU L,ZHANG D,QIN B.Phytotoxic allelochemicals from roots and root exudates of Trifolium pratense.Journal of Agricultural and Food Chemistry,2013,61(26):6321-6327.
[9]马强,周玉新,雷海民,王长海.HPLC测定红车轴草中大豆苷元、染料木素、芒柄花素和鹰嘴豆芽素A的含量.中成药,2005,27(10):1193-1195.MA Q,ZHOU Y X,LEI H M,WANG C H.Determination of daidzein,genistein,formononetin and biochanin A in Trifolium pratense L.by HPLC.Chinese Traditional Patent Medicine,2005,27(10):1193-1195.
[10]SIVESIND E,SEGUIN P.Effects of the environment,cultivar,maturity,and preservation method on red clover isoflavone concentration.Journal of Agricultural and Food Chemistry,2005,53(16):6397-6402.
[11]陈寒青,金征宇.大孔吸附树脂对红车轴草异黄酮吸附分离特性的研究.天然产物研究与开发,2006,8(2):187-192.CHEN H Q,JIN Z Y.Study on separation of Trifolium pratense isoflavones by macroporous resins.Natural Product Research and Development,2006,8(2):187-192.
[12]TSAO R,PAPADOPOULOS Y,YANG R,YOUNG J C,MCRAE K.Isoflavone profiles of red clovers and their distribution in different parts harvested at different growing stages.Journal of Agricultural and Food Chemistry,2006,54(16):5797-5805.
[13]DAEMS F,DECRUYENAERE V,AGNEESSENS R,LOGNAY G,ROMNEE J M,FROIDMONT E.Changes in the isoflavone concentration in red clover(Trifolium pratense L.)during ensiling and storage in laboratory-scale silos.Animal Feed Science and Technology,2016,217:36-44.
[14]朱贤森,黄志勤,杨庆春,程齐来.红车轴草总异黄酮粉剂中总异黄酮含量测定研究.时珍国医国药,2008,19(8):1961-1962.ZHU X S,HUANG Z Q,YANG Q C,CHENG Q L.Study on the Determination of the content of isoflavones in Trifolium pratense Fenji.Lishizhen Medicine and Materia Medica Research,2008,19(8):1961-1962.
[15]张晓松,于丽娜,张永忠,许晶.等吸光点紫外分光光度法测定红三叶草中总异黄酮含量的研究.食品安全质量检测学报,2015,6(1):260-264.ZHANG X S,YU L N,ZHANG Y Z,XU J.Isoabsorptive point determination of content of red clover isoflavones by ultraviolet spectrophotometry.Journal of Food Safety and Quality,2015,6(1):260-264.
[16]陈寒青,金征宇.三波长紫外分光光度法测定红车轴草异黄酮含量的研究.食品科学,2005,26(5):194-197.CHEN H Q,JIN Z Y.Study on determination of Trifolium pratense isoflavones by three-wavelength UV spectrophotometry.Food Science,2005,26(5):194-197.
[17]陈寒青,金征宇,陶冠军,秦昉.红车轴草不同部位中异黄酮含量的测定.食品科学,2004,25(6):150-153.CHEN H Q,JIN Z Y,TAO G J,QIN F.Determination of isoflavones in different parts of Trifolium pretense by high performance liquid chromatography.Food Science,2004,25(6):150-153.
[18]冯今,王炳煜,金加明.岷山红三叶异黄酮含量动态变化研究.中国草食动物科学,2013,33(3):42-44.FENG J,WANG B Y,JIN J M.Dynamics of isoflavone concentration of Minshan red clover.China Herbivore Science,2013,33(3):42-44.
[19]金加明,车爱霞,任春明.红三叶提取物中总异黄酮含量测定的研究.饲料工业,2009,30(16):45-46.JIN J M,CHE A X,REN C M.Determination of total isoflavones in red clover extract.Feed Industry,2009,30(16):45-46.
[20]陈寒青,金征宇.红车轴草异黄酮提取工艺的研究.食品科学,2005,26(4):156-159.CHEN H Q,JIN Z Y.Study on Extraction technology of Trifolium pratense isoflavones.Food Science,2005,26(4):156-159.
[21]吴三林,陈敏,任华英.红车轴草总异黄酮正交提取工艺和大孔树脂吸附分离性能的研究.时珍国医国药,2010,21(8):1957-1959.WU S L,CHEN M,REN H Y.Orthogonal extraction of total isoflavones from Trifolium pratense and adsorption and separation properties of macroporous resin.Lishizhen Medicine and Materia Medica Research,2010,21(8):1957-1959.
[22]陈封政,李书华,弓加文.红三叶中总异黄酮含量测定.乐山师范学院学报,2005,20(5):49-50.CHEN F Z,LI S H,GONG J W.Determination of isoflavones in herb of red clover.Journal of Leshan Teachers College,2005,20(5):49-50.
[23]刘宝剑,魏彦明.均匀设计优化微波-超声波法提取红车轴草总黄酮的研究.广东农业科学,2010,37(3):166-167.LIU B J,WEI Y M.Study on extracting red clover total flavonoids by microwave-ultrasonic method with uniform design.Guangdong Agricultural Sciences,2010,37(3):166-167.
[24]金汝城,赵国磊,李贵文,逄艳.微波辅助提取红车轴草异黄酮的工艺研究.食品科学,2007,28(9):165-168.JIN R C,ZHAO G L,LI G W,PANG Y.Study on microware-assisted extraction of isoflavones in Tifolium pratense.Food Science,2007,28(9):165-168.
[25]金汝城,赵国磊,谢伟雪,贾超.大孔树脂吸附纯化红车轴草异黄酮的研究.中成药,2008,30(11):1615-1619.JIN R C,ZHAO G L,XIE W X,JIA C.Adsorption and purification of isoflavones from Trifolium pratense by macroporous resin.Chinese Traditional Patent Medicine,2008,30(11):1615-1619.
[26]奚静芳,周健,李亚尊.大孔吸附树脂对红车轴草中总异黄酮的提取纯化工艺研究.中国现代中药,2008,10(5):21-23.XI J F,ZHOU J,LI Y Z.Study on extraction and purification of total isoflavones from Trifolium pratense L.by macroporous obsorption resin.Modern Chinese Medicine,2008,10(5):21-23.
[27]曹春凤,于洪静,孟祥凤.红车轴草中异黄酮成分的提取工艺研究.黑龙江医药,2009,22(4):486-488.CAO C F,YU H J,MENG X F.Study on extraction process of isoflavones from Trifolium pratense.Heilongjiang Medicine Journal,2009,22(4):486-488.
[28]李玲,何桂霞,裴刚.红车轴草中异黄酮成分含量测定方法的研究.江西中医学院学报,2004,16(6):46-55.LI L,HE G X,PEI G.Determination of isoflavones in Trifolium pratense.Journal of Jiangxi University of Traditional Chinese Medicine,2004,16(6):46-55.
[29]汪海波.红车轴草异黄酮的分离纯化工艺研究.食品科学,2007,28(9):266-270.WANG H B.Study on isolating and purifying of red clover isoflavone.Food Science,2007,28(9):266-270.
[30]胡冬华,梁久来.红车轴草中异黄酮成分提取工艺优化和方法学研究.吉林中医药,2007,27(12):61.HU D H,LIANG J L.Study on extraction process optimization and methodology of isoflavones from Trifolium pratense.Jilin Journal of Traditional Chinese Medicine,2007,27(12):61.
[2]KAURINOVIC B,POPOVIC M,VLAISAVLJEVIC S,SCHWARTSOVA H,VOJINOVIC-MILORADOV M.Antioxidant profile of Trifolium pratense L.Molecules,2012,17(9):11156-11172.
[3]BOOTH N L,OVERK C R,YAO P,TOTURA S,DENG Y,HEDAYAT A S.Seasonal variation of red clover(Trifolium pratense L.,Fabaceae)isoflavones and estrogenic activity.Journal of Agricultural and Food Chemistry,2006,54(4):1277-1282.
[4]KROYER G T.Red clover extract as antioxidant active and functional food ingredient.Innovative Food Science and Emerging Technologies,2004,5(1):101-105.
[5]陈学福,史高峰.红三叶异黄酮的研究进展.中草药,2006,37(2):309-312.CHEN X F,SHI G F.Research progress of isoflavones in red clover.Chinese Traditional and Herbal Drugs,2006,37(2):309-312.
[6]常平,张颍,夏开元,金宗濂.红车轴草提取物中异黄酮成分的分析.食品科学,2007,28(9):449-452.CHANG P,ZHANG Y,XIA K Y,JIN Z L.Analysis of Isoflavone in Extract of Trifolium pratense L.Food Science,2007,28(9):449-452.
[7]RIJKE E D,AARDENBURG L,DIJK J V,ARIESE F,ERNST W H O,GOOIJER C,GOOIJER C,BRINKMAN U T.Changed isoflavone levels in red clover(Trifolium pratense L.)leaves with disturbed root nodulation in response to waterlogging.Journal of Chemical Ecology,2005,31(6):1285-1298.
[8]LIU Q,XU R,YAN Z,JIN H,CUI H,LU L,ZHANG D,QIN B.Phytotoxic allelochemicals from roots and root exudates of Trifolium pratense.Journal of Agricultural and Food Chemistry,2013,61(26):6321-6327.
[9]马强,周玉新,雷海民,王长海.HPLC测定红车轴草中大豆苷元、染料木素、芒柄花素和鹰嘴豆芽素A的含量.中成药,2005,27(10):1193-1195.MA Q,ZHOU Y X,LEI H M,WANG C H.Determination of daidzein,genistein,formononetin and biochanin A in Trifolium pratense L.by HPLC.Chinese Traditional Patent Medicine,2005,27(10):1193-1195.
[10]SIVESIND E,SEGUIN P.Effects of the environment,cultivar,maturity,and preservation method on red clover isoflavone concentration.Journal of Agricultural and Food Chemistry,2005,53(16):6397-6402.
[11]陈寒青,金征宇.大孔吸附树脂对红车轴草异黄酮吸附分离特性的研究.天然产物研究与开发,2006,8(2):187-192.CHEN H Q,JIN Z Y.Study on separation of Trifolium pratense isoflavones by macroporous resins.Natural Product Research and Development,2006,8(2):187-192.
[12]TSAO R,PAPADOPOULOS Y,YANG R,YOUNG J C,MCRAE K.Isoflavone profiles of red clovers and their distribution in different parts harvested at different growing stages.Journal of Agricultural and Food Chemistry,2006,54(16):5797-5805.
[13]DAEMS F,DECRUYENAERE V,AGNEESSENS R,LOGNAY G,ROMNEE J M,FROIDMONT E.Changes in the isoflavone concentration in red clover(Trifolium pratense L.)during ensiling and storage in laboratory-scale silos.Animal Feed Science and Technology,2016,217:36-44.
[14]朱贤森,黄志勤,杨庆春,程齐来.红车轴草总异黄酮粉剂中总异黄酮含量测定研究.时珍国医国药,2008,19(8):1961-1962.ZHU X S,HUANG Z Q,YANG Q C,CHENG Q L.Study on the Determination of the content of isoflavones in Trifolium pratense Fenji.Lishizhen Medicine and Materia Medica Research,2008,19(8):1961-1962.
[15]张晓松,于丽娜,张永忠,许晶.等吸光点紫外分光光度法测定红三叶草中总异黄酮含量的研究.食品安全质量检测学报,2015,6(1):260-264.ZHANG X S,YU L N,ZHANG Y Z,XU J.Isoabsorptive point determination of content of red clover isoflavones by ultraviolet spectrophotometry.Journal of Food Safety and Quality,2015,6(1):260-264.
[16]陈寒青,金征宇.三波长紫外分光光度法测定红车轴草异黄酮含量的研究.食品科学,2005,26(5):194-197.CHEN H Q,JIN Z Y.Study on determination of Trifolium pratense isoflavones by three-wavelength UV spectrophotometry.Food Science,2005,26(5):194-197.
[17]陈寒青,金征宇,陶冠军,秦昉.红车轴草不同部位中异黄酮含量的测定.食品科学,2004,25(6):150-153.CHEN H Q,JIN Z Y,TAO G J,QIN F.Determination of isoflavones in different parts of Trifolium pretense by high performance liquid chromatography.Food Science,2004,25(6):150-153.
[18]冯今,王炳煜,金加明.岷山红三叶异黄酮含量动态变化研究.中国草食动物科学,2013,33(3):42-44.FENG J,WANG B Y,JIN J M.Dynamics of isoflavone concentration of Minshan red clover.China Herbivore Science,2013,33(3):42-44.
[19]金加明,车爱霞,任春明.红三叶提取物中总异黄酮含量测定的研究.饲料工业,2009,30(16):45-46.JIN J M,CHE A X,REN C M.Determination of total isoflavones in red clover extract.Feed Industry,2009,30(16):45-46.
[20]陈寒青,金征宇.红车轴草异黄酮提取工艺的研究.食品科学,2005,26(4):156-159.CHEN H Q,JIN Z Y.Study on Extraction technology of Trifolium pratense isoflavones.Food Science,2005,26(4):156-159.
[21]吴三林,陈敏,任华英.红车轴草总异黄酮正交提取工艺和大孔树脂吸附分离性能的研究.时珍国医国药,2010,21(8):1957-1959.WU S L,CHEN M,REN H Y.Orthogonal extraction of total isoflavones from Trifolium pratense and adsorption and separation properties of macroporous resin.Lishizhen Medicine and Materia Medica Research,2010,21(8):1957-1959.
[22]陈封政,李书华,弓加文.红三叶中总异黄酮含量测定.乐山师范学院学报,2005,20(5):49-50.CHEN F Z,LI S H,GONG J W.Determination of isoflavones in herb of red clover.Journal of Leshan Teachers College,2005,20(5):49-50.
[23]刘宝剑,魏彦明.均匀设计优化微波-超声波法提取红车轴草总黄酮的研究.广东农业科学,2010,37(3):166-167.LIU B J,WEI Y M.Study on extracting red clover total flavonoids by microwave-ultrasonic method with uniform design.Guangdong Agricultural Sciences,2010,37(3):166-167.
[24]金汝城,赵国磊,李贵文,逄艳.微波辅助提取红车轴草异黄酮的工艺研究.食品科学,2007,28(9):165-168.JIN R C,ZHAO G L,LI G W,PANG Y.Study on microware-assisted extraction of isoflavones in Tifolium pratense.Food Science,2007,28(9):165-168.
[25]金汝城,赵国磊,谢伟雪,贾超.大孔树脂吸附纯化红车轴草异黄酮的研究.中成药,2008,30(11):1615-1619.JIN R C,ZHAO G L,XIE W X,JIA C.Adsorption and purification of isoflavones from Trifolium pratense by macroporous resin.Chinese Traditional Patent Medicine,2008,30(11):1615-1619.
[26]奚静芳,周健,李亚尊.大孔吸附树脂对红车轴草中总异黄酮的提取纯化工艺研究.中国现代中药,2008,10(5):21-23.XI J F,ZHOU J,LI Y Z.Study on extraction and purification of total isoflavones from Trifolium pratense L.by macroporous obsorption resin.Modern Chinese Medicine,2008,10(5):21-23.
[27]曹春凤,于洪静,孟祥凤.红车轴草中异黄酮成分的提取工艺研究.黑龙江医药,2009,22(4):486-488.CAO C F,YU H J,MENG X F.Study on extraction process of isoflavones from Trifolium pratense.Heilongjiang Medicine Journal,2009,22(4):486-488.
[28]李玲,何桂霞,裴刚.红车轴草中异黄酮成分含量测定方法的研究.江西中医学院学报,2004,16(6):46-55.LI L,HE G X,PEI G.Determination of isoflavones in Trifolium pratense.Journal of Jiangxi University of Traditional Chinese Medicine,2004,16(6):46-55.
[29]汪海波.红车轴草异黄酮的分离纯化工艺研究.食品科学,2007,28(9):266-270.WANG H B.Study on isolating and purifying of red clover isoflavone.Food Science,2007,28(9):266-270.
[30]胡冬华,梁久来.红车轴草中异黄酮成分提取工艺优化和方法学研究.吉林中医药,2007,27(12):61.HU D H,LIANG J L.Study on extraction process optimization and methodology of isoflavones from Trifolium pratense.Jilin Journal of Traditional Chinese Medicine,2007,27(12):61.