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Title: 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis

item Zeller, Wayne
item RAMSAY, AINA - University Of Reading
item ROPIAK, HONORATA - University Of Reading
item FRYGANAS, CHRISTOS - University Of Reading
item MUELLER-HARVEY, IRENE - University Of Reading
item BROWN, RONALD - University Of Reading
item DRAKE, CHRIS - University Of Reading
item Grabber, John

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/28/2015
Publication Date: 1/28/2015
Publication URL:
Citation: Zeller, W.E., Ramsay, A., Ropiak, H.M., Fryganas, C., Mueller-Harvey, I., Brown, R.H., Drake, C., Grabber, J.H. 2015. 1H-13C HSQC NMR spectroscopy for estimating procyanidin/prodelphinidin and cis/trans flavan-3-ol ratios of condensed tannin samples: correlation with thiolysis. Journal of Agricultural and Food Chemistry. 63:1967-1973.

Interpretive Summary: Analytical techniques allowing for the rapid assessment of chemical structure and composition of components isolated from plant materials remain a high priority for agricultural researchers. One important class of plant components contained in some forages is condensed tannins. Condensed tannins (CTs) belong to a class of compounds called polyphenols and have been shown to help protect protein from degradation, both during the ensiling process and during rumen digestion. Both of these activities increase nitrogen use efficiency on the farm, either through providing higher protein content in silage or through better protein utilization during rumination. Condensed tannins consist of repeating chains of mixed monomer units with different atomic arrangements. It is suspected that the composition present in CTs may play a role in how effectively they protect forage protein. Identifying the best CT composition in plants for protein protection in the silo and the rumen would improve both the economic and environmental sustainability of ruminant farm operations. A new method was developed to analyze purified CTs, isolated from several different legumes and forages, using nuclear magnetic resonance (NMR) spectroscopy. This new method of analysis should help identify structural difference in CTs which provide the largest impact in protecting protein degradation during the ensiling process and rumen digestion. These results will help plant breeders with selection for tannin content and structure. They will also help identify plant varieties that are good candidates for genetic modification.

Technical Abstract: Studies with a diverse array of 22 condensed tannin (CT) fractions from 9 plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans flavan-3-ol ratios can be appraised by 1H-13C HSQC NMR. The method was developed from fractions containing 44 to ~100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-2’,6’ signals from PD and of H/C-6’ signals from PC yielded nuclei-adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R2 = 0.99). Cis/trans flavanol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R2 = 0.89). Overall, 1H-13C HSQC NMR appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT if precautions are taken to avoid integration of cross-peak contours of contaminants.