Characterization of Natural and Alkaline-Oxidized Proanthocyanidins in Plant Extracts by Ultrahigh-Resolution UHPLC-MS/MS

Abstract

In this study, we analyzed the proanthocyanidin (PA) composition of 55 plant extracts before and after alkaline oxidation by ultrahigh-resolution UHPLC-MS/MS. We characterized the natural PA structures in detail and studied the sophisticated changes in the modified PA structures and the typical patterns and models of reactions within different PA classes due to the oxidation. The natural PAs were A- and B-type PCs, PDs and PC/PD mixtures. In addition, we detected galloylated PAs. B-type PCs in different plant extracts were rather stable and showed no or minor modification due to the alkaline oxidation. For some samples, we detected the intramolecular reactions of PCs producing A-type ether linkages. A-type PCs were also rather stable with no or minor modification, but in some plants, the formation of additional ether linkages was detected. PAs containing PD units were more reactive. After alkaline oxidation, these PAs or their oxidation products were no longer detected by MS even though a different type and/or delayed PA hump was still detected by UV at 280 nm. Galloylated PAs were rather stable under alkaline oxidation if they were PC-based, but we detected the intramolecular conversion from B-type to A-type. Galloylated PDs were more reactive and reacted similarly to nongalloylated PDs.

Keywords: UHPLC-DAD-MS/MS; high-resolution mass spectrometry; orbitrap; oxidation; tannins.

Figure 1

(A) A model structure for oligomeric B-type proanthocyanidins (PAs) with C4→C8 linkages: R₁ = H, procyanidin, R₁ = OH, prodelphinidin. B-type PAs can also be linked by C4→C6 bonds. A-type PAs have an additional C2→O→C7 or C2→O→C5 ether bond. The hydroxyl groups can also be substituted, for example, galloylated or glycosylated. (B) A galloyl group.

Figure 2

(A) UV chromatogram at 280 nm, (B) total ion chromatogram, (C) extracted ion chromatogram (EIC) at m/z 441, (D) EIC at m/z 729, and (E) EIC at m/z 1017 of the leaf extract of Ruprechtia salicifolia. PA = proanthocyanidin, NL = normalized intensity.

Figure 3

Characteristic fragmentation pathway and the MS/MS of B-type procyanidin dimer in the leaf extract of Cunninghamia lanceolata. The mechanisms are heterocyclic ring fission (HRF), retro-Diels‒Alder (RDA) fragmentation and quinone methide (QM) cleavage [31,32,33].

Figure 4

Characteristic fragmentation pathway and the MS/MS of A-type procyanidin dimer in the leaf extract of Aglaonema crispum. The mechanisms are heterocyclic ring fission (HRF), retro-Diels–Alder (RDA) fragmentation and quinone methide (QM) cleavage [26,34]. The ion at m/z 407 is a tentative suggestion for the RDA fragmentation and the sequential water elimination of the extension unit.

Figure 5

Characteristic fragmentation pathway and the MS/MS of A-type procyanidin trimer in the leaflet extract of Tectaria macrodonta. The mechanisms suggested are heterocyclic ring fission (HRF), retro-Diels‒Alder (RDA) fragmentation and quinone methide (QM) cleavage.

Figure 6

Total mass spectra of non-oxidized (A) and oxidized (B) leaf extract of Begonia bowerae “Nigra” and of non-oxidized (C) and oxidized (D) leaflet extract of Cyperus owanii. The exact masses of main ions of proanthocyanidins are listed in Tables S1 and S2.

Figure 7

The suggested mechanism for the conversion of B-type procyanidin dimer to A-type dimer according to [15,37].

Figure 8

Extracted ion chromatograms (EICs) of the non-oxidized leaflet extract of Microgramma mauritiana (A) the ions at m/z 577.11–577.17 showing the presence of B-type procyanidin dimers, (B) the ions at m/z 575.09–575.15 and of the oxidized extract of Microgramma mauritiana (C) the ions at m/z 577.11–577.17 and (D) the ions at m/z 575.09–575.15 showing the presence of A-type procyanidin dimers. (*) the ion corresponds to the isotopic signal of a doubly charged molecular ion for a tetrameric procyanidin. (**) the ion has the same elemental composition as A-type PC dimers but different fragment ions, NL = normalized intensity. Note that this study was qualitative, and the intensities cannot be compared as such.

Figure 9

Molecular ions with corresponding exact masses and molecular formulae for procyanidin oligomers with one or more A-type linkages from the leaflet extract of Tectaria macrodonta: (A) trimers, (B) tetramers, (C) pentamers, and (D) hexamers. The first ion of each oligomer (having one ether bond) is taken from the non-oxidized extract, and the other ions of the oligomer (having two or more ether bonds) from the oxidized extract. For A-type procyanidin hexamer with three ether bonds, the doubly charged ion was more abundant.

Figure 10

The mass spectra of small mixed B-type oligomeric procyanidins (PCs) and prodelphinidins (PDs) in the non-oxidized (A) and oxidized (B) leaf extract of Podocarpus macrophyllus. The exact masses of main ions are listed in Table S4. The abundances of the ions are fixed to normalized intensity of 6.9 × 10⁶.

Figure 11

Characteristic fragmentation pathway and the MS/MS of a galloylated procyanidin dimer in the leaf extract of Ruprechtia salicifolia. The position of the galloyl group is only indicative and could be any free OH group in the terminal unit. The mechanisms are heterocyclic ring fission (HRF), retro-Diels‒Alder (RDA) fragmentation and quinone methide (QM) cleavage.