Laboratory of Organic Chemistry and Biochemistry, University of ioannina, Greece
me01375@cc.uoi.gr
Vassiliki G. Kontogianni: Dr Kontogianni is a post-Doctoral researcher in the Laboratory of Organic Chemistry, Department of Organic Chemistry and Biochemistry of University of Ioannina, Greece. Dr Kontogianni graduated from Department of Chemistry, University of Ioannina, Greece (1999-2003) and gained her MSc. (2005) and PhD (2009) from Department of Chemistry, University of Ioannina, Greece in using state of the art analytical tools to determine metabolites in complex extracts. She is the author of 2 publications and has participated in more than 6 national and international conferences with oral or poster presentations. Dr Kontogianni will train 2 students that will participate in the current project.
Abstract: A novel method for the determination of the total phenolic content using 1H NMR spectroscopy in the
âOH spectral region is presented. The use of DMSO-d6, which is an aprotic and strongly hydrogen bonding
solvent, allows the âappearanceâ of the relative sharp resonances of phenolic hydroxyl protons in the
region of 8â14 ppm. The determination of the total phenolic âOH content requires three steps: (i) a 1D
1H NMR spectrum is obtained in DMSO-d6; (ii) a subsequent 1D 1H NMR spectrum is recorded with
irradiation of the residual water signal which results in the elimination or reduction of the phenolic
âOH groups, due to proton exchange; and (iii) 1D 1H NMR spectra are recorded with the addition of a
progressively increased amount of salt, NaHCO3, which results in extensive linebroadening of the COOH
resonances thus allowing the discrimination of the phenolic from the carboxylic acid signals. Integration,
with respect to the internal standard TSP-d4, of the signal resonances between 14 and 8ppm in spectrum
(i) which are either eliminated or reduced in intensity in steps (ii) and (iii) allows the quantitation of
the total phenolic content. The method was applied to model compounds, a mixture of them and several
extracts of natural products. The results of the proposed 1H NMR method were compared to the Folin-
Ciocalteu (FC) reagent method. Additionally, since 1H NMR refers to the total phenolic hydroxyl protons,
a reaction factor, Ae, is proposed that corresponds to the hydroxyl reactivity. The 1H NMR method is rapid
and accurate bearing the inherent advantages of the NMR spectroscopy and can be applied directly in
complex extracts. Furthermore, it can be applied in a wide range of matrixes from crude plant extracts
and food products to biological samples.
Abstract: A novel strategy for NMR analysis of mixtures of oleanolic and ursolic acids that occur in natural products
is described. These important phytochemicals have similar structure and their discrimination and
quantification is rather difficult. We report herein the combined use of protonâcarbon heteronuclear
single-quantum coherence (1Hâ13C HSQC) and protonâcarbon heteronuclear multiple-bond correlation
(1Hâ13C HMBC) NMR spectroscopy, in the identification and quantitation of oleanolic acid (OA) and ursolic
acid (UA)in plant extracts of the Lamiaceae and Oleaceae family. The combination of 1Hâ13C HSQC and
1Hâ13C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular
backbone resulting in the identification and, thus, discrimination of oleanolic and ursolic acid without
resorting to physicochemical separation of the components. The quantitative results provided by
2D 1Hâ13C HSQC NMR data were obtained within a short period of time (â¼14 min) and are in excellent
agreement with those obtained by HPLC, which support the efficiency of the suggested methodology.
