高效液相色谱法测定番石榴叶提取物中的游离鞣花酸

Determination of Free Ellagic Acid Content in Guava Leaves by HPLC

研究高效液相色谱法测定番石榴叶中鞣花酸的方法。以甲醇(含0.01%HCl)作为提取液,70℃加热回流;色谱条件为:Intersil ODS-SP色谱柱(250mm×4.5mm,5μm),柱温35℃,进样体积10μL,紫外检测波长254nm;流动相为3%冰乙酸(A)-纯甲醇(B);梯度洗脱程序:0～4min B由20%至45%,保持6min,10～15min由45%至70%,15～20min由70%至100%,保持1min。结果表明:本方法的相对标准偏差为0.6%,鞣花酸在0.5～200mg/L范围内与峰面积呈良好的线性关系,相关系数为0.9998,加标回收率为95.86%～98.31%。本方法用于测定鞣花酸是切实可行的;番石榴叶片中鞣花酸含量高低与叶片的成熟度有关,其中幼嫩叶含量为0.814mg/g、成熟叶含量为0.023mg/g,而老叶中未检出。

A high performance liquid chromatographic method was established for the quantitative determination of free ellagic acid content in guava leaves.Hot ethanol reflux（containing 0.01%HCl）extraction was applied for the extraction of samples.The analyte was separated on an inertsil ODS-SP column（250 mm×4.5 mm,5μm）held at 35℃using a mobile phase composed of 3%glacial acetic acid（phase A）and net methanol（phase B）with the following gradient elution program：0-4 min,20%45%B;4-10 min,45%B;10-15 min,45%-70%B;15-20 min 70%-100%B;20-21 min 100%B.The injection volume used was 10μL.The detection wavelength was set as 254 nm.The relative standard deviation of retention time for 5 replicate injections was 0.6%.Free ellagic acid content displayed an excellent linear relationship with pear area over the range of 0.5 to 200 mg/L,with a correlation coefficient of more than 0.9998.The spike recoveries for free ellagic acid were between 95.86%and 98.31%.This method was used to determine free ellagic acid content in the real samples of young,mature and aged guava leaves.The results proved its good feasibility and showed that young,mature and aged guava leaves contained 0.814,0.023 mg/g and 0 mg/g free ellagic acid,respectively,suggesting that free ellagic acid content in guava leaves is related to their maturity.