用气相色谱-质谱联用比较牛耳草代谢物的提取方法

Comparison of metabolite extraction from Boea hygrometrica by gas chromatography/mass spectrometry

摘要:

通过比较不同的提取方法对牛耳草新鲜和脱水叶片中代谢物的提取效率，旨在建立一种可以有效鉴定并分析牛耳草脱水过程中关键小分子代谢物的种类和含量变化的方法，为研究植物耐脱水分子机制提供技术方法。本研究以气相色谱-质谱联用 (GC-MS) 为分析方法，对复苏植物牛耳草代谢物提取方法进行比较。从提取总色谱峰数目、提取效率、代谢物保留时间和提取效率稳定性等方面比较甲醇溶液 (A法) 和甲醇-氯仿-水溶液 (B法) 两种提取方法的提取效果。对牛耳草新鲜样品提取结果表明，B法提取的总色谱峰数目多于A法；对9种共有代谢物的提取效率比较结果表明，B法的提取效率高于A法；对10种色谱峰的保留时间和提取效率的方法学考察结果表明，两者保留时间RSD (相对标准偏差) 值均小于1%，A法提取效率的RSD值≤10%的比例为50%，B法的为100%。A法对干样的提取色谱峰数目远少于鲜样，而B法对干样的提取色谱峰数目和鲜样没有显著差异，保留时间RSD值均小于1%，提取效率的RSD值与鲜样没有差异，稳定性良好。

Abstract:

This study is aiming to establish an efficient metabolite extraction method for exploration of molecular mechanisms of desiccation tolerance of the resurrection plant Boea hygrometrica using a metabolomics approach. The extracts of metabolite in B. hygrometrica using methanol solution (method A) and methanol-chloroform-water solution (method B) were analyzed by gas chromatography-mass spectrometry (GC-MS). The total numbers of chromatographic peaks, extraction efficiency, retention time and the peak stability were compared. The results showed that for fresh materials, the total chromatographic peak number of method B is more than that of method A; the extraction efficiency of nine representative metabolites by method B is higher than that by method A; the comparison of 10 random chromatographic peaks revealed that the relative standard deviation (RSD) values of the retention time are less than 1% for both methods, whereas the RSD values of the extraction efficiency is different. The percentage of peaks that owned RSD values of the extraction efficiency higher than 10% is 50% for method A and 100% for method B. In addition, method B was also efficient for dry materials from B. hygrometrica. The number of chromatographic peaks, RSD value of retention time and extraction efficiency of dry materials was similar to that of fresh materials using method B, but decreased sharply using method A. Putting together, our study provided evidence that method B is an efficient extraction method for further analysis of metabolites from this resurrection species.