title: Allium ursinum L.: bioassay-guided isolation and identification of a galactolipid and a phytosterol exerting antiaggregatory effects.
abstract: OBJECTIVE
  We wanted to investigate the possible antithrom botic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adenosine diphosphate(ADP)-induced aggregation of human platelets by wild garlic, Allium ursinum L.
  
  
  METHODS
  For this purpose, a bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. For the bioassay, blood was taken from healthy human volunteers and platelet-rich plasma was prepared for turbidimetric platelet aggregation tests. Platelet-rich plasma, stimulated with 20 μ mol/l of ADP, was treated with extracts of different polarities, fractions and isolated single compounds from A. ursinum. The extracts were investigated by thin-layer chromatography(TLC), HPLC, mass spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and 1/2-dimensional (1)H/(13) C-nuclear magnetic resonance (NMR) spectroscopic techniques.
  
  
  RESULTS
  Fresh A. ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited ADP-induced aggregation of human platelets. Thisethanolic extract was subjected to liquid-liquid partition. Whilst the aqueous phase, containing the moiety of cysteine sulphoxide and thiosulphinate derivatives, showed only weak activity on platelet aggregation, the ethyl-acetate and particularly the chloroform partitions showed the high estaggregation-inhibiting potency. Thus, in our bioassay, the effects of alliins/allicins could be neglected. The chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient-elution open column chromatography on silica gel. The most active fractions 11–17 were separated again, yielding 10 subfractions. This afforded 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol and β-sitosterol-3-O-β-D-glucopyranoside, the structures of which were determined by ESI-MS and 1/2-dimensional (1)H/(13) CNMR spectroscopic techniques. Furthermore, the minute amounts of volatile oil of A. ursinum leaves obtained by steam distillation according to Ph. Eur. could be evaluated asa third aggregation-inhibiting principle.
  
  
  CONCLUSIONS
  In our study, for the first time, 2 active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol,could be identified exhibiting inhibitory action on ADP-induced aggregation in human blood platelets. As a major constituent, the galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol, not yet found in Allium sp., appears as a new, highly useful marker substance for A. ursinum drugs, or their pharmaceutical or food preparations,as shown by our orientating TLC analyses.
authors.affiliation: King Hussein Medical Centre, Amman, Jordan. dhes@medizin.uni-leipzig.de
authors.forename: Dina
authors.initials: D
authors.lastname: Sabha
authors.forename: Bahi
authors.initials: B
authors.lastname: Hiyasat
authors.forename: Kristina
authors.initials: K
authors.lastname: Grötzinger
authors.forename: Lothar
authors.initials: L
authors.lastname: Hennig
authors.forename: Franziska
authors.initials: F
authors.lastname: Schlegel
authors.forename: Friedrich-Wilhelm
authors.initials: FW
authors.lastname: Mohr
authors.forename: Hans Wilhelm
authors.initials: HW
authors.lastname: Rauwald
authors.forename: Stefan
authors.initials: S
authors.lastname: Dhein
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000328:Adult; D000490:Allium; D001681:Biological Assay; D001792:Blood Platelets / Q000187:drug effects; D005260:Female; D005960:Glucosides / Q000032:analysis / Q000494:pharmacology*; D005990:Glycerol / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D006801:Humans; D008297:Male; D010936:Plant Extracts / Q000032:analysis / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D012855:Sitosterols / Q000494:pharmacology*
chemical_list: C419082:1,2-dilinolenoyl-3-galactopyranosylglycerol; D005960:Glucosides; D010936:Plant Extracts; D010975:Platelet Aggregation Inhibitors; D012855:Sitosterols; D000244:Adenosine Diphosphate; D005990:Glycerol; C011015:lyoniside