Discovery and molecular elucidation of the anti-influenza material basis of Banlangen granules based on biological activities and ultra-high performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a wide range of applications, including human healthcare-associated treatments and bioactive compound discovery. However, complex chemical systems present a significant challenge for chemical-material-based research and quality control. For instance, Banlangen (BLG) granules is a well-acknowledged TCM preparation widely used in clinical treatment of virus infection. However, its chemical basis of anti-influenza efficacy remains unclear. AIM OF THE STUDY: In the present study, a systematic discovery strategy for identifying anti-influenza molecules based on biological activities and chemical analysis was established to contribute to the molecular elucidation of the anti-influenza material basis of Banlangen granules. MATERIALS AND METHODS: Hemagglutinase inhibition (HAI) and neuraminidase inhibition (NAI) assays were used to compare the anti-influenza activities of different fractions of BLG granules against H1N1, H5N1 and H7N9 viruses. A comparative qualitative analysis of the chemical constituents in BLG granules and their fractions was performed using ultra-high-performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap MS), in which a multiple mass spectrometry database platform and three compound identification strategies were used. The association between anti-influenza activities and chemical constituent characteristics was analyzed using multiple stoichiometries and data comparison strategies. RESULTS: The results showed that the chromatography fractions F3 and F4 of the BLG granules had the highest anti-influenza activity. A total of 88 compounds were identified in the BLG granules, including 31 alkaloids, 16 organic acids, 10 nucleosides, 8 phenylpropanoids, 6 sulfur-containing compounds, 5 amino acids, 4 aromatic compounds, 3 aldehydes and ketones, 2 flavonoids, 1 alcohol, 1 carbohydrate, and 1 aliphatic compound. Out of these, 31 characteristic compounds were identified in fractions F3-F4 as candidate compounds with anti-influenza activity. Additionally, 6-methoxyquinoline and 4-guanidinobutanal were identified in BLG granules and its raw material (Isatidis Radix) for the first time. CONCLUSION: In this study, we proposed a systematic discovery strategy to thoroughly investigate the anti-influenza activity, chemical identification, and constituents-activity relationship of BLG granules. These data not only provided a deeper understanding of the molecular mechanism of the activity of BLG granules, but also presented a basis for the discovery of potential novel drug candidates and quality evaluation and control of BLG granules.

Microscopic Mass Spectrometry Imaging Reveals the Distribution of Phytochemicals in the Dried Root of Isatis tinctoria.

The dried root of Isatis tinctoria L. (Brassicaceae) is one of the most popular traditional Chinese medicines with well-recognized prevention and treatment effects against viral infections. Above 300 components have been isolated from this herb, but their spatial distribution in the root tissue remains unknown. In recent years, mass spectrometry imaging (MSI) has become a booming technology for capturing the spatial accumulation and localization of molecules in fresh plants, animal, or human tissues. However, few studies were conducted on the dried herbal materials due to the obstacles in cryosectioning. In this study, distribution of phytochemicals in the dried root of Isatis tinctoria was revealed by microscopic mass spectrometry imaging, with application of atmospheric pressure-matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap-time-of-flight mass spectrometry (IT-TOF/MS). After optimization of the slice preparation and matrix application, 118 ions were identified without extraction and isolation, and the locations of some metabolites in the dried root of Isatis tinctoria were comprehensively visualized for the first time. Combining with partial least square (PLS) regression, samples collected from four habitats were differentiated unambiguously based on their mass spectrometry imaging.