An integrated strategy for anti-inflammatory quality markers screening of traditional Chinese herbal medicine Mume Fructus based on phytochemical analysis and anti-colitis activity

Mume Fructus (MF) is used in traditional Chinese herbal medicine (TCM) to treat chronic cough, prolonged diarrhea, and other inflammation-related diseases. It is processed from Prunus mume fruit (PM) by drying at low temperature according to the Chinese Pharmacopoeia. The standard quality control method includes measurement of citric acid content, which is not sufficient to determine its clinical efficacy. In addition, the quality markers, that would ensure consistent drug composition and stability during extraction and processing of the drug, are currently not available.This study sought to determine and analyze the bioactive compounds in MF and to establish the quality maker evaluation system, which would enable accurate assessment of different processing and extraction approaches for MF preparation.First, a UPLC-QTOF-MS/MS method was established to identify the chemical constituents of PM and MF. Second, the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated rats were used to assess anti-inflammatory activity of water and ethanol extracts of PM and MF. Third, correlation analysis and multivariate statistical analysis was used to seek the candidate quality markers of MF. Fourth, molecular docking was used to predict the potential mechanism of identified compounds for the anti-inflammatory activity. Finally, a UPLC method was established to quantify the selected quality markers in MF products, that were prepared by different drying processes.99 components (28 newly reported) were identified from PM and MF. During the drying process several changes in the composition were observed; caffeoylquinic acids were degraded to p-coumaric acid, caffeic acid, protocatechuic acid, or p-hydroxybenzoic acid; multi-acetyl p-coumaroyl sucroses were degraded to mumeose R and p-coumaroyl-3-O-sucrose. On the other hand, contents of mumefural and amygdalin increased after drying process. In colitis rats, MF reduced more NO levels to greater extent in comparison to PM, which could be attributed to the presence of caffeic acid, p-coumaric acid, protocatechuic acid, p-hydroxybenzoic acid, mumefural, p-coumaroyl-3-O-sucrose, mumeose R, and amygdalin in MF. Moreover, water extracts were better than ethanol extracts in alleviating the IL-1β, IL-6, and IL-17 levels, possibly on account of citric acid and caffeoylquinic acids. The predicted mechanism of action could be through inhibition of the production of NLRP3, TLR4, and NF-κB proteins. Finally, 7 compounds (citric acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, caffeic acid, protocatechuic acid, and p-coumaric acid) were selected as quality markers of MF that could be used for the process quality control.This study revealed the material basis of PM and MF for anti-colitis activity and discovered the quality markers of MF which could reflect the anti-inflammatory activity and the processing process of MF.