Oxidative and enzymatic activity of plant polyphenols revealed by UHPLC-MS

A rapid method for estimating the enzymatic activity of plant polyphenols was developed. With this method, combined together with an oxidation method developed earlier [1], phenolic compounds possessing enzymatic and/or alkaline oxidation activity can easily be found. Each sample is studied under three conditions: 1) unaltered sample, 2) after oxidation at pH 10, 3) after enzymatic oxidation. After treatment, the samples are analysed using ultra-high performance liquid chromatography coupled with a mass spectrometer (UHPLC-MS) using fingerprint analysis methods [2]2222. The MS data of the various samples are aligned and analysed using MetAlign and SIMCA-P+ statistical software. The hierarchical clustering analysis (HCA) plot of 290 unaltered samples is shown in the picture below. Below the plot is a bar plot of average amounts of polyphenol subgroups in each of four clusters. Clusters contains: 1) species rich in hydrolysable tannins (HT), 2) species rich in quinic acid derivatives, 3) species rich in proanthocyanidins (PA) and quinic acid, and 4) species with an overall low content of polyphenols. Samples exhibiting bioactivity can quickly be found out with subsequent metabolic comparison of sample types. Shown below is the UV chromatogram of Malus domestica and Salix pylicifolia samples. M. domestica contains enzymatically active flavanones that are not oxidised in pH 10, while the main compound in S. pylicifolia is not oxidized by the plant's own enzymes, but the polyphenol profile is greatly altered in alkaline conditions. Combination of our fingerprint analysis method, MetAlign and SIMCA-P+ enables clustering of the studied samples, and identification of each cluster's main type of polyphenols. The presented activity method is relevant for insect herbivory, since plant polyphenols are oxidized enzymatically only if there's polyphenol oxidase enzymes present in the plant itself. Polyphenols oxidized in alkaline conditions or by plant's enzymes are easy to pinpoint.

Acknowledgements: Anne Koivuniemi and Marica Engström are acknowledged for their assistance during sample collection and chemical analyses.

Keywords: UHPLC-QqQ-MS, oxidative activity, enzymatic activity.

References:

[1] Vihakas M, Pälijärvi M, Karonen M, Roininen H, Salminen JP. Rapid estimation of the oxidative activities of individual phenolics in crude plant extracts. Phytochemistry 2014; 103: 76 – 84

[2] Engström M, Pälijärvi M, Salminen JP. Rapid fingerprint analysis of plant extracts for ellagitannins, gallic acid, and quinic acid derivatives and quercetin-, kaempferol- and myricetin-based flavonol glycosides by UPLC-QqQ-MS/MS. J Agric Food Chem 2015; 63: 4068 – 4079