Resveratrol Trimers from Seed Cake of Paeonia rockii

In the course of screening natural products for antibacterial activities, a total acetone extract of the seed cake of Paeonia rockii showed significant effects against bacterial strains. Bioactivity-guided fractionation of the EtOAc-soluble fraction of the total acetone extract resulted in the isolation and identification of five resveratrol trimers, including rockiiol C (1), gnetin H (2), suffruticosol A (3), suffruticosol B (4) and suffruticosol C (5). The relative configuration of these compounds was elucidated mainly by comprehensive 1D and 2D-NMR experiments. Compound 1 was a new compound. All isolated compounds exhibited strong antibacterial activities against Gram-positive bacteria.


Introduction
Paeonia rockii subsp.rockii cv "spp."(Paeoniaceae) is a shrub widely distributed in China.The seeds oil (peony seed oil) from seeds of Paeonia ostii or Paeonia rockii has been authorized as a new resource food by the Ministry of Health of the People's Republic of China (Bulletin of the Ministry of Health of the People's Republic of China announced, 2011, No. 9).The seed cake is the major by-product in the preparation procedure of crushing cooking oil from the seeds of Paeonia rockii.Though no reports were OPEN ACCESS found regarding the chemical constituents of the seed cake of P. rockii, studies on the seeds of other Paeonia genera have been performed elsewhere [1][2][3][4][5][6][7].
In the course of screening natural products for antibacterial activities, it was found that the acetone extract from the seed cake of P. rockii had significant inhibitory effects on bacterial strains.Further bioactivity-guided fractionation led to the isolation and identification of one new resveratrol trimer, rockiiol C (1), together with the four known ones.Their structures are depicted in Figure 1.Herein, we mainly describe the isolation and structural elucidation of the new resveratrol trimer on the basis of various 2D-NMR techniques, including HSQC, HMBC, 1 H-1 H COSY and NOESY experiments.

Purification and Characterization
The EtOAc-soluble fraction of the acetone extract from the seed cake of P. rockii was separated by silica-gel, gel permeation chromatography and pre-HPLC (ODS-A) to give one new resveratrol trimer, rockiiol C (1), and four known compounds (2-5) (Figure 1).

Structural Elucidation of Compound 1
Compound 1, was obtained as a brownish-white amorphous powder and showed a positive reaction with Gibbs reagent.The HRESI-MS data (pseudomolecular ion [M+H] + at m/z 679.1965 (calcd.679.1970)) together with the 13 C-NMR data (42 carbons) indicated the molecular formula C42H30O9, which suggested that 1 was a resveratrol trimer.The IR spectrum of Compound 1 revealed the presence of hydroxy (3336 cm −1 ) and aromatic rings (1632, 1515 and 1450 cm −1 ).The UV spectrum displayed an absorption maximum at 289 nm, which was consistent with one or more non-conjugated phenyl rings.

Antibacterial Activities
The in vitro anti-bacterial effects of the isolated compounds (1-5) were tested.It was found that all of these compounds exhibited strong antibacterial activities against Gram-positive bacteria.The MIC values against bacterial strains of Compounds 1-5 are listed in Table 2.

Pseudomonas aeruginosa
Penicillin

Plant Material
The seeds of Paeonia rockii were collected at Luoyang Tuqiao flower and seeding CO.LTD, Luoyang city, Henan province, and were verified by Hou Xiao-gai (College of Agricultural, Henan University of Science and Technology) in September, 2012.A voucher specimen has been deposited in the Specimens Hall of Natural resources of Funiu Mountains, Henan University of Science and Technology.The seed cake was obtained in the preparation procedure of crushing cooking oil from the seeds.

Extraction and Isolation
Air-dried seed cakes of P. rockii (7.5 kg) were extracted by acetone at room temperature, and 500 g of dry crude residues remained after solvent evaporation in vacuo.The residues were dissolved in methanol, and the methanol dissolved fraction (300 g) was then added into the mixtures (1000 g) of silica and diatomite (1:1, W/W).The whole sample was dried at room temperature and then was fractionated successively with EtOAc and methanol to produce 110-g and 150-g dried fractions, respectively.The EtOAc-soluble fraction of the acetone extract was further repeatedly chromatographed on a silica gel column using a gradient solvent system (petroleum ether (PE)-acetone (8:1, 4:1, 2:1, 1:1)) to obtain 10 fractions.