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Anal Bioanal Chem (2013) 405:58075816 DOI 10.1007/s00216-013-6987-x
RESEARCH PAPER
NMR assignments and the acidbase characterizationof the pomegranate ellagitannin punicalagin in the acidic pH-range
Mrta Kraszni & Attila Marosi & Cynthia K. Larive
Received: 3 March 2013 /Revised: 13 April 2013 /Accepted: 15 April 2013 /Published online: 9 May 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of - and -punicalagin were clearly identified in the 1H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the 1H and 13C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and 1H NMR detection carried out to characterize the acidbase properties of punicalagin over the pH range 28. Simultaneous fitting of all of the pH-sensitive 1H
NMR signals produced similar but significantly different pKa values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin - (pKa1=4.570.02, pKa2=5.630.03) and - (pKa1=4.360.01, pKa2=5.470.02) anomers. Equivalent pKa values, (:6.640.01, :6.630.01)
were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel
with the proton dissociation steps as the pH is raised such that -punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the / anomer equilibrium are likely the consequence of a conformational rearrangement.
Keywords Gallagic acid . Ellagic acid . pKa . Titration . Anomer equilibrium
Introduction
There is a growing interest in pomegranate (Punica granatum L.) as a nutritional and medicinal product [1, 2]. Consumption of pomegranate juice is associated with various health benefits including inhibition of prostate cancer in men [3], reduction of serum oxidative stress and retardation of atherosclerosis development in diabetic patients [4, 5], and potential protection against colon cancer [6]. These health benefits are attributed to polyphenols, the predominant class of phytochemicals of pomegranate fruits, mainly consisting of hydrolysable tannins. The...