Molino Ralph John Emerson J, Junio Hiyas A
SMPL Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, 1101, Philippines.
Rapid Commun Mass Spectrom. 2021 Apr 15;35(7):e9037. doi: 10.1002/rcm.9037.
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). MS 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.
高通量液相色谱/质谱（LC/MS）分析为天然染料研究提供了一个有趣的平台。一个具体的例子是对菲律宾木蓝发酵混合物的动态变化进行评估，以及对使用传统方法和优化方法制备的市售靛蓝进行研究。
对木蓝和苏门答腊木蓝的叶子进行甲醇提取和水发酵48小时。对经过2天和15天发酵制备的靛蓝粉末也使用超高效液相色谱/四极杆飞行时间质谱（UHPLC/QTOF-MS）进行分析。通过在社区策划的全球天然产物社会分子网络（GNPS）中进行库搜索来注释质谱图。在GNPS中没有库匹配的光谱通过分析其裂解途径进行注释。
基于UHPLC/MS的检测和裂解分析导致在木蓝叶发酵提取物中鉴定出无色靛蓝和未报道的色胺酮中间体5a-羟基-5,5a-二氢吲哚并[2,1-b]喹唑啉-6,12-二酮。拉古纳省的一个木蓝标本中不存在与靛蓝相关的代谢物，这解释了为什么它不产生蓝色染料。本地生产的靛蓝富含靛蓝素和靛玉红，根据具有相应预测裂解模式的产物离子进行区分。然而，靛蓝素的相对强度随着延长发酵以生产靛蓝的传统工艺而降低。
该研究首次展示了基于质谱同时分析木蓝活性转化发酵提取物中的反应中间体、靛蓝素染料、副产物和分解代谢物。新的结果包括无色靛蓝的注释质谱图，以及未报道的5a-羟基-5,5a-二氢吲哚并[2,1-b]喹唑啉-6,12-二酮的注释质谱图，它可能是色胺酮合成中的中间体。所提出的裂解方案可以指导复杂混合物中类似化合物的注释。
