不同波长和标准品对红三叶异黄酮含量测定的影响

Effects of wavelengths and standard products on the analysis of isoflavone content in red clover

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.