Comprehensive characterization of anthraquinones in Damnacanthus indicus using mass spectrometry molecular networking and metabolomics-based herb discrimination†

Damnacanthus indicus is a widely used folk medicine in China, renowned for its various bioactivities. The key active components, anthraquinones, have not been comprehensively profiled due to their complex chemical nature. Establishing a high-throughput strategy to systematically characterize these anthraquinones is essential. Additionally, the cultivation of D. indicus across various provinces results in significant quality differences in the harvested herbs. Thus, developing an effective strategy to distinguish herbs from different regions and identify characteristic chemical markers for quality evaluation and control is crucial. In this study, a strategy based on ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was employed to systematically characterize the chemical composition of D. indicus. Mass spectrometry molecular networking was utilized to rapidly recognize and identify anthraquinones. Principal component analysis (PCA) was applied to cluster the herbs from different habitats, while partial least square discriminant analysis (PLS-DA) was used to screen for chemical markers distinguishing herb origins. The result showed that a total of 112 anthraquinones and 66 non-anthraquinone compounds were identified in D. indicus. The biosynthetic pathways of anthraquinones in this herb were proposed. PCA grouped 15 batches of herbs from different origins into three clusters, corresponding to the climate types of their habitats. PLS-DA identified 27 significant chemical markers that could robustly distinguish the geographical origins of the herbs. This study provides a valuable reference for the quality evaluation and control of D. indicus and offers a scientific basis for the pharmacological research and rational utilization of these medicinal resources.