Rapid chemical characterisation of stilbenes in the root bark of Norway spruce by off-line HPLC/DAD-NMR.
BACKGROUND
Stilbenes are plant secondary metabolites that have shown promising and varied biological activities. Stilbenes are presently actively studied for the exploitation of this primary raw material resource, involving the concept of biorefining. Methods for the rapid discovery of new and known stilbene structures from various plant sources are thus keenly sought.


OBJECTIVE
To establish a simple and rapid technique of off-line HPLC with a diode-array detector (DAD) and NMR for the unambiguous structural elucidation of stilbene structures in the root bark of Norway spruce [Picea abies (L.) Karst.].


METHODS
The stilbene containing fraction was extracted from the plant bark with an ethanol:water mixture (95:5, v/v) preceded by defatting of hydrophobic compounds with n-hexane using the accelerated solvent extraction technique. A portion of the ethanol-water soluble extract was hydrolysed with β-glucosidase to prepare stilbene aglycones. The extracts were further purified and enriched using a polymeric adsorbent. Stilbene-enriched extracts were directly characterised by off-line HPLC/DAD-NMR in conjunction with HPLC/DAD and HPLC/DAD with electrospray ionisation MS(n).


RESULTS
Trans-isorhapontin and trans-astringin were identified as the major, and trans-piceid as a minor, stilbene glucosides of the bark of roots of Picea abies. Not only stilbene glucosides but also the corresponding stilbene aglycones, such as trans-resveratrol, trans-piceatannol and trans-isorhapontigenin, were rapidly identified from the hydrolysed extract. The acquired heteronuclear single-quantum coherence and heteronuclear multiple bond correlation spectra were used to assign the complete carbon NMR chemical shifts of trans-isorhapontin and trans-astringin without the need of acquiring a (13)C-NMR spectrum.


CONCLUSIONS
The off-line HPLC/DAD-NMR method is expedient for the unambiguous identication of structurally similar stilbenes in plant extracts.
Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014, Helsinki, Finland.
Daniel Girma
DG
Mulat
Harri
H
Latva-Mäenpää
Harri
H
Koskela
Pekka
P
Saranpää
Kristiina
K
Wähälä
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005960:Glucosides / Q000737:chemistry* / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D028222:Picea / Q000737:chemistry*; D024301:Plant Bark / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry* / Q000302:isolation & purification; D018517:Plant Roots / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013267:Stilbenes / Q000737:chemistry* / Q000302:isolation & purification
D005960:Glucosides; D010936:Plant Extracts; D013267:Stilbenes; C121481:astringin; C571114:isorhapontin
HPLC/DAD-ESI/MSn; HPLC/DAD-NMR; Norway spruce; Picea abies; on-line techniques; root bark; stilbenes; structural identification