The content of indigo precursors in Isatis tinctoria leaves--a comparative study of selected accessions and post-harvest treatments

We recently clarified the nature of indigo precursors in woad (Isatis tinctoria L.), by identifying the major indoxyl glycoside as isatan A (indoxyl-3-O-(6'-O-malonyl-beta-D-ribohexo-3-ulopyranoside)), and by correcting the structure of the related isatan B (indoxyl-3-O-beta-D-ribohexo-3-ulopyranoside). A quantitative densitometric assay for isatans A and B, and indican, was established and validated. HPTLC separation on silica gel was followed by densitometric analysis of indigoid pigments formed after treatment with dilute acid or base. The seasonal variation of indoxyl glycosides in woad leaves was investigated with first-year plants (rosette stage) of five defined I. tinctoria L. and one I. indigotica L. accessions. Isatan A content reached up to 7.6% of dry weight in I. tinctoria, and up to 21.8% in I. indigotica. The influence of various post-harvest treatments was studied. High concentrations of isatans A and B were found in freeze-dried leaf samples, whereas the content of indican was lowest. Conventional drying at ambient or 40 degrees C led to complete disappearance of isatans A and B. The concentration of indican, in contrast, was 3- to 5-fold higher in leaf samples submitted to drying at ambient and 40 degrees C, respectively.