|BROWN, RON - University Of Reading|
|MUELLER-HARVEY, IRENE - University Of Reading|
|STRINGANO, ELISABETTA - University Of Reading|
|GEA, AN - University Of Reading|
|DRAKE, CHRISTOPHER - University Of Reading|
|ROPIAK, HONORATA - University Of Reading|
|FRYGANAS, CHRISTOS - University Of Reading|
|RAMSAY, AINA - University Of Reading|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2017
Publication Date: 8/16/2017
Citation: Brown, R.H., Mueller-Harvey, I., Zeller, W.E., Reinhardt, L.A., Stringano, E., Gea, A., Drake, C., Ropiak, H.M., Fryganas, C., Ramsay, A., Hardcastle, E.E. 2017. Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition. Journal of Agricultural and Food Chemistry. 65:8072-8082.
Interpretive Summary: Condensed tannins (CT) are members of the polyphenol class of plant secondary metabolites and are contained in some common forages such as sainfoin and birdsfoot trefoil. The consumption of CT-containing plant materials by ruminants (as pasture plants, contained in silage, or as amendments to total mixed rations) can exhibit agricultural, economic, and environmental benefits. These benefits include improving nitrogen utilization by the ruminant, mitigation of enteric methane and ammonia production, and control of nematode parasites. These benefits arise from interaction of CT with forage and microbial proteins. Studies on the molecular level of CT-protein interactions require highly pure and well-characterized CT. This communication compiles the results of several studies in an effort to provide researchers with a choice of easy-to-use CT purification methods. This paper outlines purification protocols used to obtain highly pure and well-characterized CT from 11 different plant species. The methods developed offer a rapid purification process relative to existing methods and provide specific details. This information will allow researchers to project, based on needs, the quantity of dried plant material needed for extraction and purification to obtain targeted amounts of CT for their proposed studies. In addition, the use of proton nuclear magnetic resonance spectroscopy was shown to provide a rapid and non-destructive method for qualitative assessment of purities of CT fractions.
Technical Abstract: Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts, and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP), and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD), but required a prepurification step, needed more time for large-scale purifications, and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146-2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1H-NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation toward repurification or 1H-13C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples.