Characterisation of high molecular weight hop proanthocyanidins using Analytical Ultracentrifugation

Arthur Gadon,Robert Linforth,David Cook,Stephen E Harding

doi:10.1038/s41598-019-49171-4

Arthur Gadon, Robert Linforth + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-49171-4

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Abstract

We report the novel application of Analytical Ultracentrifugation (AUCF) to characterise the polymeric proanthocyanidin fraction of hops. Extraction of hop samples with 70% acetone (aq) followed by a C-18 Solid Phase Extraction yielded polyphenolic fractions for AUCF analysis. Sedimentation velocity experiments demonstrated the presence of discrete molecular weight bands of proanthocyanidins, as opposed to a continuous distribution of molecular weights. There were 4 such bands for Saaz hop (0.15, 1.1, 2.7 and 4.4S) and 3 bands for Magnum (0.15, 1.6 and 3.0S). The method resulted in a reproducible size (sedimentation coefficient) distribution for replicate runs of the same extract and for extracts prepared from different samples of the same hop variety. Sedimentation equilibrium experiments were then used to fit molecular weight distributions using the new SEDFIT-MSTAR method for the same samples. Thus we report for the first time polymeric proanthocyanidins in hops with molecular weights of up to 100 kDa in Saaz hop (or up to 56 kDa in Magnum). This represents the first application of AUCF to characterise complex fractions of polyphenolics extracted from botanical sources and the methodology developed should find wider application in the study of this diverse and bioactive class of compounds.

Highlights

Polyphenolic molecules are broadly distributed across the plant kingdom and represent an important part of the human diet
Hydrolysis of proanthocyanidins under these conditions produces anthocyanidins which were measured in a spectrophotometer by absorbance at 550 nm
This study has reported for the first time the application of Analytical Ultracentrifugation (AUCF) to the characterisation of polyphenolic extracts of hops, and shown the presence of high molecular weight compounds with an upwards size limit in the region of 100 kDa for Saaz

Summary

Introduction

Polyphenolic molecules are broadly distributed across the plant kingdom and represent an important part of the human diet. In an attempt to characterise the molecular size of the proanthocyanidins fractions extracted from hops, Linforth et al.[4] used a series of molecular weight cut-off spin filters to fractionate extracts. This indicated the presence of molecules greater in size than 30 kDa (equivalent to a DP of >100). Monagas et al.[8] commented that MALDI-TOF-MS is not well-suited to the analysis of highly polydisperse proanthocyanidins, due to the generation of multiply charged ions which makes spectral interpretation difficult They concluded that high molecular weight polymers were challenging with regard to suppression of ionisation, reduction of desorption and in-source fragmentation to form non-covalent ion clusters. Improved analytical methodologies will help to increase our understanding of the biological roles of these complex compounds[13]

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