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DOI: 10.1055/s-0037-1608210
Simple non-specific enzymatic oxidation method for studying the bioactivity of phenolic compounds in plants
Publication History
Publication Date:
24 October 2017 (online)
A new oxidation method was developed to reveal species- and tissue-level bioactivity of plants. The method combines an existing alkaline oxidation method [1] with an in situ enzymatic oxidation step. Plant samples are incubated in an oven after freezing, enabling polyphenol oxidase (PPO) and other oxidizing enzymes getting into contact with phenolic compounds in the plant, resulting in oxidation of the compounds. As the results are analyzed using total phenolics assay on well plate reader and ultrahigh-performance liquid chromatography coupled to a diode array detector and mass spectrometer (UHPLC-DAD-MS), alterations in the levels of total phenolics and individual compounds can be observed. Depending on the available equipment in a laboratory, either of the analysis methods or both can be utilized.
Oxidation of individual compounds during in vitro alkaline oxidation tended to be consistent, whereas in situ enzymatic oxidation resulted in varying levels of oxidation of the same compound depending on the species and plant tissues. For example, a well-known PPO substrate 5-O-caffeoylquinic acid (5-CQA) all but disappeared from Menyanthes trifoliata L, but in Prunus padus it remained (Fig. 1), indicating that P. padus does not contain much PPO at least in active state.
The method provides a new way to inspect bioactivity of plants and the phenolic compounds they contain. The method is physiologically relevant, since the plant's own phenolics are oxidized by the plant's own enzymes, instead of phenolic extracts being oxidized by commercial enzymes, or extracted enzymes presented with substrates not necessarily present in the plant.
[1] Vihakas M, Pälijärvi M, Karonen M, Roininen H, Salminen J-P. Phytochemistry 2014; 103: 76 – 84.