Studies on specialized metabolites like phenolics are of immense interest owing to their significance to agriculture, nutrition and health. In plants, phenolics accumulate and exhibit spatial and temporal regulations in response to growth conditions. Robust methodologies aimed at efficient extraction of plant phenolics, their qualitative and quantitative analysis is desired. We optimized the analytical and experimental bottlenecks that captured free, ester, glycoside and wall-bound phenolics after acid or alkali treatments of the tissue extracts and subsequent GC-MS analysis. Higher recovery of phenolics from the methanolic extracts was achieved through (a) Ultrasonication assisted extraction along with Methyl tert-butyl ether (MTBE) enrichment (b) nitrogen gas drying and (c) their derivatization using MSTFA for GC-MS analysis. The optimized protocol was tested on Arabidopsis rosette exposed to UV-B radiation (280-315 nm) which triggered enhanced levels of 11 monophenols and might be attributed to photoprotection and other physiological roles. Interestingly, coumaric acid (308 m/z) and caffeic acid (396 m/z) levels were enhanced by 12-14 folds under UV-B. Other phenolics such as cinnamic acid (220 m/z), hydroxybenzoic acid (282 m/z), vanillic acid (312 m/z, gallic acid (458 m/z), ferulic acid (338 m/z), benzoic acid (194 m/z), sinapinic acid (368 m/z) and protocatechuic acid (370 m/z) also showed elevated levels by about 1 to 4 folds. The protocol also comprehensively captured the variations in the levels of ester, glycoside and wall-bounded phenolics with high reproducibility and sensitivity. The robust method of extraction and GC-MS analysis can readily be adopted for studying phenolics in plant systems.
Supplementary information: The online version contains supplementary material available at 10.1007/s12298-022-01150-2.
Keywords: Arabidopsis; GC-MS; MTBE extraction; Metabolite profiling; Monophenols; Plant Phenolics; UV-B.
© Prof. H.S. Srivastava Foundation for Science and Society 2022.