Bioassay-guided Supercritical Fluid Extraction of Cyclooxygenase-2 Inhibiting Substances in Plantago major L.

• Introduction - 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: alpha-linolenic acid (alpha-LNA) (18:3 omega-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. Method - 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 - alpha-LNA was preferentially extracted in dynamic mode using unmodified carbon dioxide at 40 degrees 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 (alpha-LNA) and a COX-2/COX-1-inhibitory effect ratio of 1.1 using 10% (v/v) ethanol as polar modifier at 75 degrees C and 483 bar. The Soxhlet extracts had ursolic acid, oleanolic acid and alpha LNA yields up to 1.36%, 0.34% and 0.15%, respectively, with a COX-2/COX-1 inhibitory effect ratio of 1.2. Conclusion - The target substances can be extracted selectively by bioassay guided optimisation of SFE conditions.

Nyckelord

In vitro enzyme assay

Soxhlet

supercritical fluid extraction (SFE)

cyclooxygenase-2

Plantago major L.

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