Development of a process for obtaining non-mutagenic madder root (Rubia tinctorum) extract for textile dyeing

Madder, used as a red dye, contains anthraquinones, and some of these can react with DNA possibly causing mutagenic effects. This is especially true for 1,3-dihydroxyanthraquinones with a hydroxymethyl (e.g., lucidin 6, ibericin 10) or methyl group (e.g., rubiadin 9) at C-2. In this research, a new process was developed through which the concentration of mutagenic compounds is minimized by adapting extraction and fractionation parameters. The process was tested on lab scale but also on 5000 L industrial scale. The first step is the key biotechnological step. Roots are stirred in water and the concentration of lucidin 6 is reduced to (near) zero by endogenous enzymes. When lucidin 6 is absent, the formation of mutagenic ibericin 10 by a reaction with ethanol as extraction solvent, is not possible. Mutagenicity can be further reduced by heat treatment, which is common in industrial downstream processing, e.g., in spray drying. Removal of rubiadin 9 is possible by flash chromatography. All madder root fractions were tested in the Salmonella microsome assay (Ames test, TA100) for mutagenicity, which was correlated with the anthraquinone concentration.