摘要
通过碱性甲醇溶液超声提取,结合杏仁来源β-葡萄糖苷酶水解及聚酰胺分散式固相萃取步骤,建立了超高效液相色谱测定大豆异黄酮活性总量的方法。样品中的丙二酰及乙酰类大豆异黄酮苷在碱性条件下水解成基本型苷,并在β-葡萄糖苷酶的作用下进一步脱去糖基转变为相应的苷元。样品中12种不同形式的大豆异黄酮转变为仅含3种大豆异黄酮苷元(大豆苷元、黄豆黄素、染料木素)后,以聚酰胺粉进行分散式固相萃取,C_(18)反相色谱柱(2.1 mm i.d.×50 mm,1.8μm)分离。结果显示,大豆苷元、黄豆黄素及染料木素在3 min内实现基线分离,3种异黄酮苷元的标准曲线相关系数(r^(2))均大于0.999,总异黄酮的回收率为94.3%~102%,相对标准偏差(RSD,n=6)小于5.0%,具有较高的准确度和精密度。该方法通过检测样品中的全部苷元来计算异黄酮总量,有助于降低异黄酮检测在分离度、准确度及检测成本上带来的挑战;考虑异黄酮在体内的转化吸收机制和生理效应,以苷元总量计能更科学地反映实际的异黄酮活性水平,避免因通过苷和苷元的简单加和而造成对异黄酮含量水平的高估。
To determine the total isoflavones(ISF),most analytical methods have focused on the three aglycone ISFs and three basic glucoside ISFs due to the unavailability of reference standards for malonyl-and acetyl-glucosides,and resulted in bias.Hence,an ultra performance liquid chroma⁃tography UPLC)method was developed that allows the quantitation of all 12 ISFs using only 3 isofla⁃vone standards.All glucoside ISFs,including daidzin,malonyl and acetyl daidzin,glycitin,malo⁃nyl and acetyl glycitin,genistin,malonyl and acetyl genistin,were converted into only 3 aglycone ISFs via alkaline methanol extraction and enzymatic hydrolysis.The malonyl glucosides and acetyl glucosides in sample were firstly hydrolyzed to their corresponding glucoside forms in alkaline metha⁃nol,the produced glucosides were further hydrolyzed by aβ-glucosidase from almonds,and the sug⁃ar moieties from glucoside were removed.The released and original existed aglycone units were con⁃centrated by polyamide through a dispersive solid-phase extraction,and determined by UPLC.All 3 aglycone ISFs(daidzein,glycitein and genistein)were separated in a reversed-phase C_(18)column(2.1 mm i.d.×50 mm,1.8μm).The mobile phase,mixed acetonitrile and phosphoric acid aqueous so⁃lution was employed to enable a 3 min of isocratic elution.All linearity correlation coefficients(r^(2))were higher than 0.999,and the recoveries were between 94.3%and 102%with relative standard de⁃viations(RSD,n=6)less than 5.0%,that demonstrated precise and accurate results in the determination of total ISFs.To determine the total ISFs by simply summing the concentrations of the aglycon ISF will mitigate the pressure from interference,deviation and cost on test.In addition,based on the mechanisms of absorption and metabolism,using aglycone(free)forms to quantitate total ISFs is ex⁃pected to present actual bioactivity of the phytoestrogens,this will avoid overestimate isoflavone lev⁃els or intake by simply adding of free and glucoside forms of isoflavone concentrations.
作者
顾文
黎晨曼
孙震
庞月红
冯永巍
沈晓芳
GU Wen;LI Chen-man;SUN Zhen;PANG Yue-hong;FENG Yong-wei;SHEN Xiao-fang(State Key Laboratory of Food Science and Resources,Jiangnan University,Wuxi 214122,China;Technology Innovation Center of Special Food for State Market Regulation,Wuxi Food Safety Inspection and Test Center,Wuxi 214142,China)
出处
《分析测试学报》
CAS
北大核心
2025年第1期106-115,共10页
Journal of Instrumental Analysis
基金
江苏省市场监督管理局科技计划项目(KJ2024070)。
