Profiling the Philippine Blue: Liquid chromatography/mass spectrometry‐based metabolomics study on Philippine Indigofera

다국어 초록 (Multilingual Abstract)

High‐throughput liquid chromatography/mass spectrometry (LC/MS) analysis presents an interesting platform for natural dyes research. A particular example is the assessment of the dynamic changes in fermentation mixtures of Philippine Indigofera, and in the investigation of commercially available indigo prepared using traditional and optimized methods.
Leaves from Indigofera tinctoria and Indigofera suffruticosa were subjected to methanolic extraction and aqueous fermentation for 48 h. Indigo powders prepared following 2‐day and 15‐day fermentation were also subjected to profiling using ultra‐high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UHPLC/QTOF‐MS). MS2 spectra were annotated through a library search in the community‐curated Global Natural Products Social Molecular Networking (GNPS). Spectra with no library hits in GNPS were annotated by analysis of their fragmentation pathways.
UHPLC/MS‐based detection and fragmentation analysis led to characterization of leucoindigo and the unreported tryptanthrin intermediate, 5a‐hydroxy‐5,5a‐dihydroindolo[2,1‐b]quinazoline‐6,12‐dione, in the fermentation extract of I. tinctoria leaves. Indigo‐associated metabolites were absent in an Indigofera specimen in Laguna Province, which explained why it did not produce blue dye. Locally produced indigo was abundant in indigotin and indirubin, differentiated based on product ions with the corresponding predicted fragmentation pattern. The relative intensity of indigotin, however, decreased with the traditional process of extended fermentation to produce indigo.
The study is the first to demonstrate simultaneous MS‐based analysis of reaction intermediates, indigotin dye, side products, and catabolites on actively transforming fermentation extracts of I. tinctoria. New results include annotated mass spectra for leucoindigo, and for the unreported 5a‐hydroxy‐5,5a‐dihydroindolo[2,1‐b]quinazoline‐6,12‐dione, which is probably an intermediate in tryptranthrin synthesis. The proposed fragmentation schemes could guide the annotation of analogous compounds in complex mixtures.