RESUMO
BACKGROUND: Baphicacanthis Cusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products. METHODS: Ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples. RESULTS: A total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%. CONCLUSION: This study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.
RESUMO
Untargeted mass spectrometry analysis is one of the most challenging and meaningful steps in the rapid structural elucidation of the highly complex and diverse constituents of traditional Chinese medicine. Specifically, it is a laborious and time-consuming way to identify unknown compounds. Herein, a workflow was proposed to expedite the annotations of the chemical structures in Pheretima aspergillum (E. Perrier) (Di-Long, DL). First, ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) was performed to obtain the untargeted mass spectral data. Then, the spectral data were uploaded to the Global Natural Products Social Molecular Networking (GNPS) platform to create a network and extract the Mass2Motifs (co-occurring fragments and neutral losses) using unsupervised substructure annotation topic modeling (MS2LDA). Finally, a structural analysis was performed using the proposed workflow of MS2LDA in combination with mass spectral molecular networking and in silico fragmentation prediction. As a result, a total of 124 compounds from DL were effectively characterized, of which 89 (7 furan sulfonic acids, 57 phospholipids and 25 carboxamides) were identified as potentially new compounds from DL. The results presented in this article significantly improve the understanding of the chemical composition of DL and provide a solid scientific basis for the future study of the quality control, underlying pharmacology and mechanism of DL. Moreover, the proposed workflow was used for the first time to accelerate the annotations of unknown molecules from TCM. Furthermore, this workflow will increase the efficiency of characterizing the 'unknown knowns' and elucidation of the 'unknown unknowns' from TCM, which are crucial steps of discovering the natural product drugs in TCM.