Isolation of gibberellin precursors from heavily pigmented tissues

The kauranoid precursors of gibberellins are difficult to isolate from heavily pigmented plant tissues. In this paper, we describe relatively simple and efficient procedures for the purification of these compounds from tissues containing chlorophyll and other high molecular weight pigments. Extracts of shoots from Thlaspi arvense L. were subjected first to size exclusion chromatography using ethyl acetate as the eluting solvent. This procedure resulted in the separation of kauranoids as a class of compounds from chlorophyll. Typically, a 90% reduction in mass of the kauranoid enriched-fraction was observed. This fraction was subjected to reverse phase high performance liquid chromatography and individual fractions analyzed by combined gas chromatography-mass spectrometry. Five kauranoids were identified in shoot extracts of T. arvense: ent-kaur-16-ene, ent-kaur-16-en-19-ol, ent-kaur-16-en-19-oic acid, trachylobanoic acid, and 7beta, 13-dihydroxykaurenolide. The metabolic relationships of these compounds to the gibberellins previously identified in this species (JD Metzger, MC Mardaus [1986] Plant Physiol 80: 396-402) are discussed. In addition, the utility of size exclusion chromatography in preparative situations is demonstrated by the purification of ent-kaurenoic acid in milligram quantities from the florets of Helianthus annuus L.