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%.