Humans have been using blue indigo dye (indigotin) for thousands of years. Plants containing indigotin such as Isatis tinctoria, Polygonum tinctorium, and Indigofera tinctoria have been domesticated for extracting blue indigo pigment from leaves. However, there are no scientific records on the presence of indigotin in Indigofera australis, which is an Australian native shrub widespread in southeastern Australia, despite anecdotal evidence of blue dye extraction having been reported by artisans.
Indigo exists in plants in the form of the precursor indican, which is an indoxyl bound to β-D-glucose. Enzyme hydrolysis followed by oxidation of the indoxyls results in the formation of the blue indigo pigment.
In this study, I. australis leaves were investigated as a possible source of blue indigo pigment employing various conditions anecdotally reported to yield the dye.
Indigofera australis leaves collected from nature strips around Wagga Wagga NSW were submerged in sterile water in sterilized clear glass preserving jars and incubated at various temperature regimes. The effect of leaf microbiome on fermentation was tested by surface disinfection of leaves with bleach, and sterilising leaves with dry-heat treatment.
Optimal blue dye production was observed with disinfected leaves incubated at 4°C for a week followed by 25°C for another week. The production of blue pigment was unreliable when I. australis leaves were not disinfected, and when sterilised. This indicates the likelihood of endophytic microbiome underpinning the chemical reactions, which needs further investigation. In addition, there was no apparent relationship between the production of blue pigment and pH.
The presence of indigotin and its precursor indican in I. australis extracts were confirmed via uHPLC-MS analysis and comparison against commercially available standards. This is the first report confirming the presence of indican in I. australis leaves and optimal fermentation conditions to extract indigotin from the leaves of Australian native indigo.