title: Bioassay-guided supercritical fluid extraction of cyclooxygenase-2 inhibiting substances in Plantago major L.
abstract: BACKGROUND
  Selective extraction of plant materials is advantageous for obtaining extracts enriched with desired constituents, thereby reducing the need for subsequent chromatography purification. Such compounds include three cyclooxygenase-2 (COX-2) inhibitory substances in Plantago major L. targeted in this investigation: α-linolenic acid (α-LNA) (18:3 ω-3) and the triterpenic acids ursolic acid and oleanolic acid.
  
  
  OBJECTIVE
  To investigate the scope for tuning the selectivity of supercritical fluid extraction (SFE) using bioassay guidance, and Soxhlet extraction with dichloromethane as solvent as a reference technique, to optimise yields of these substances.
  
  
  METHODS
  Extraction parameters were varied to optimise extracts' COX-2/COX-1 inhibitory effect ratios. The crude extracts were purified initially using a solid phase extraction (SPE) clean-up procedure and the target compounds were identified with GC-MS, LC-ESI-MS and LC-ESI-MS² using GC-FID for quantification.
  
  
  RESULTS
  α-LNA was preferentially extracted in dynamic mode using unmodified carbon dioxide at 40°C and 172 bar, at a 0.04% (w/w) yield with a COX-2/COX-1 inhibitory effect ratio of 1.5. Ursolic and oleanolic acids were dynamically extracted at 0.25% and 0.06% yields, respectively, with no traces of (α-LNA) and a COX-2/COX-1-inhibitory effect ratio of 1.1 using 10% (v/v) ethanol as polar modifier at 75°C and 483 bar. The Soxhlet extracts had ursolic acid, oleanolic acid and αLNA yields up to 1.36%, 0.34% and 0.15%, respectively, with a COX-2/COX-1 inhibitory effect ratio of 1.2.
  
  
  CONCLUSIONS
  The target substances can be extracted selectively by bioassay guided optimisation of SFE conditions.
authors.affiliation: High Throughput and Analysis, R&D, GE Healthcare, Björkgatan 30, S-751 84 Uppsala, Sweden. ake.stenholm@ge.com
authors.forename: A
authors.initials: A
authors.lastname: Stenholm
authors.forename: U
authors.initials: U
authors.lastname: Göransson
authors.forename: L
authors.initials: L
authors.lastname: Bohlin
mesh_terms: D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002245:Carbon Dioxide / Q000737:chemistry; D002417:Cattle; D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D010943:Plantago / Q000737:chemistry*; D011453:Prostaglandins / Q000096:biosynthesis; D015203:Reproducibility of Results; D012756:Sheep; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D011453:Prostaglandins; D002245:Carbon Dioxide; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2