four a-type procyanidin trimers are main flavanol ... cinnposter1.pdf · polymerization) on a ymc...
TRANSCRIPT
ABSTRACT:
Cinnamon spice is derived from bark of several Cassia and Cinnamomum genera. Depending on the
origin, there are distinctive differences between composition of the water-soluble procyanidins, which
are mainly consisting of A-type procyanidins, attributed with anti-diabetic activity. Trimers are dominant
group, while tetramers are at ca. 20% of the trimer content and other oligomeric DPs contributing
smaller pools. A Normal and Reverse Phase HPLC analysis of the bark of C. burmannii revealed a
pattern of four trimers. The presence of cinnamtannin B-1 (1) and D-1 (2) have been previously
documented1. Two other trimers were isolated by the use of Centrifugal Partition Chromatography
(Kromaton FCPC) followed by preparative HPLC and identified for the first time as aesculitannin B3,4
(3) and lindetannin2 (4). All contain one A-type interflavan bond between top unit being epicatechin
and the middle unit. Typically, some steric hindrance exists in these structures preventing free rotation
around B-type bond producing different sets of signals detected by NMR, which complicates
interpretation of the NMR spectra. The ratios of rotational isomers in 1-4 are different for each and are
strongly solvent depent. In methanol the ratios are equal to 1:3.7, 1:4.2, 1:45, and 1:91, while in DMSO
they are equal to 1:1.4, 1:2.5, 1:9, and 1:7.5, respectively. These differences are explained using DFT
optimization for different conformers showing two minima with energy barrier calculated from 1H
NMR temperature spectra as well as 13CNMR shielding constants and TD-DFT calculations.
Four A-type procyanidin trimers are main flavanol components of Cinnamomum burmanii Jan A. Gliński1, Alan Wong1, Peter Kinkade1, Vitold B. Glinski1, Sławomir Kaźmierski2, and Marta K. Jamróz3*(correspond), 1Planta Analytica LLC, 39 Rose Street, Danbury, CT 06810, USA, 2The Centre of
Molecular and Macromolecular Studies PAS, Sienkiewicza 112, Lodz, Poland, c Physical Chemistry Department, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02097 Warsaw, Poland
Conclusions: 1. The rotamers ratio for the trimeric procyanidins depends on the energy barrier between the two lo-west-energy conformers (higher barrier – higher amount of the second conformer).
2. The barrier is a result of steric hindrances caused either by the aromatic rings (B, E or H) in case of trimers with ent-catechin as a middle unit (lindetannin, aesculitannin B) or by C3-OH hydroxyl group in case of the other trimers (cinnamtannin B-1 and D-1).
3. Aromatic ring hindrance is easier to overcome than C3-OH, since the location of the ring can be ad-justed, whereas C3-OH hydroxyl group is bound in the pyran ring.
For each molecule conformational search with MMFF method was employed. Then, the torsion angle
(C9”-C8”-C4’-C3’) in the lowest energy conformers were changed by 30° and for each of the so
prepared geometries the conformational search was repeated with the dihedral frozen to established
value. So obtained geometries were subdued to partial optimization with DFT calculations (B3LYP/6-
31G**). Their structures were established to be well-known cinnamtannins B-1 and D-1, as well as
aesculitannin B and lindetannin. The compounds differ only in stereochemistry of the middle and/or
lower units, however each of them display different content of rotational conformers (rotamers),
identifiable in the 1H NMR spectra. In order to explain the occurrence of those rotamers and their
ratio, as well as to establish exact conformations of the rotamers DFT calculations were carried out.
1H NMR spectra of compounds 1-4. Signals originating
from low-abundant rotamers are circled.
3D structure of cinnamtannin B-1 with
rotatable torsion angle (C9”-C8”-C4’-C3’) –
marked as green.
Energy barrier calculationcs (B3LYP/6-31G**)
Normal phase HLPC separation of cinnamon
procyanidins according to their size (degree of
polymerization) on a YMC PVA column.
Reverse phase HPLC of cinnamon procyanidin
trimers (DP 3) on a C18 column.
Cinnamtannin B-1
Cinnamtannin D-1
Aesculitannin B
Lindetannin
Acknowledgment:
DFT Calculations were performed at the Interdisciplinary Centre for Mathematical and Computational Modeling, Warsaw Poland under
the computational grant G14-6.
Literature:
1. KB Killday et al., J.Nat.Prod. (2011) 74, 1833
2. CF Zhang et al, Chin. Chem. Lett. (2003) 14, 1033-1036
3. H-C Lin and S-S Lee, J.Nat.Prod. (2010) 73, 1375-1380
4. S Morimoto et al, Chem.Pharm.Bull. (1987) 35, 4717-4729
Purification Steps
Extraction: Cinnamon bark powder soaked in 70% acetone – 30% water for 18 h and the extract
evaporated to dryness.
Fractionation of the extract: Performed on a Centrifugal Partition Chromatograph, Kromaton
FCPC, equipped in a 1 L rotor, using EtOAc-H2O solvent system in ascending mode.
Final purification of trimeric procyanidins: Enriched CPC fractions were subjected to preparative
HPLC on a 50 x 250 mm YMC AQ column in a MeCN/water gradient. Achieved purities were in the
range of 97-99%.