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Yindan Xinnaotong soft capsule (YDXNT) is a clinically effective herbal prescription used for the treatment of cardiovascular and cerebrovascular diseases. Since Chinese medicines (CMs) exert their effects via a "multiple-components and multiple-targets" mode, discovery of the active compounds with interactive effects may contribute to reveal their mechanisms of action.This study aimed to establish an image-based fingerprint-efficacy screening strategy to identify active compounds with interaction effects from CM prescription, using YDXNT to inhibit microglia-mediated neuroinflammation as an instance.A multi-component random content-oriented chemical library of YDXNT was constructed by uniform design, and their chemical fingerprint was profiled by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) methods. Then the neuroinflammation activities of chemical library members of YDXNT were determined by image-based dual phenotypic quantification. Subsequently, fingerprint-efficacy correlation and random forest analysis were applied to predict the potentially active compounds with interactive effects. Finally, the interactive effects among the active compounds were confirmed by quantitative polymerase chain reaction (qPCR) and apoptosis analysis, and network pharmacology was applied to explore the possible mechanisms.Image-based fingerprint-efficacy correlation analysis revealed that six tanshinones (TNs) and four flavonoids (FAs) were potential anti-neuroinflammatory compounds. The inter-family of TNs and FAs possessed obvious interactive effects (combination index ≤ 0.825). Moreover, the combination of scutellarein and tanshinone I (2:1, w/w) was discovered as the possible interactive combinatorial components, which, comparing with individual scutellarein or tanshinone I, shown more powerful effects on anti-inflammatory and anti-apoptotic effects in lipopolysaccharide (LPS)-induced BV2 cells. Network pharmacology showed that the active compounds might suppress microglia-mediated neuroinflammation via multiple targets in the T cell receptor, Jak-STAT, and Toll-like receptor signaling pathways.The image-based fingerprint-efficacy strategy simplifies the screening process of efficacious component combinations in CMs for complex diseases, which also offers a promising approach to explore the integrative therapeutic mechanisms of CMs.

Discovering effective combinational components (ECCs) and quality control markers of TCMs is still facing challenges because the holistic healing system comprises hundreds of compounds. Here, taking Yindan Xinnaotong soft capsule (YDXNT), a TCMs preparation composed by 8 herbs, as a case, a strategy that integrated multiple chromatographic analysis and bioactivity assay was proposed for potential ECCs of neuroprotection discovery. Firstly, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF MS) and gas chromatography-mass spectrometry (GC-MS) were applied for comprehensive profiling of the chemical constituents in YDXNT. Given the fact that the complex matrix interference makes it more difficult to identify potentially active compounds, we proposed a structure-diagnostic ions-oriented strategy to remove interference ions from the raw UHPLC-MS data. The proposed strategy consisted of different filtering methods, including diagnostic fragment ions filtering (DFIF), mass defect filtering (MDF) and neutral loss (NL). Using this strategy, a total of 124 compounds were rapidly identified. Among them, 62 non-volatile and 5 volatile constituents in 30 batches of YDXNT were quantified by UHPLC tandem triple quadrupole mass spectrometry (QQQ-MS) and GC-MS methods, respectively. In order to facilitate the quality control of YDXNT, candidate ECCs were selected based on the threshold setting of absolute -contents, and their neuroprotective effects were examined. Finally, a combination of 16 compounds, accounts for 2.80% (w/w) of original YDXNT, was identified as its potential ECCs, which could be considered for the improvement of quality standardization of YDXNT.

A rapid ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ MS/MS) approach with high sensitivity and selectivity was developed for the quantification of twenty compounds, including 9 saponins, 8 flavonoids, 2 oligosaccharide esters and 1 phenolic acid, in rat plasma and brain, which was administrated intragastrically with Jia-Wei-Qi-Fu-Yin (JWQFY), Mass spectrometric detection was operated under multiple reaction monitoring (MRM) mode. All calibration curves possessed good linearity with correlation coefficients ( r2) higher than 0.9916 in their respective linear ranges. For intra- and inter-day precision, all the relative standard deviations (RSDs) at different levels were less than 14.68 %. Based on the UHPLC-QqQ MS/MS quantitative results, pharmacokinetic study and brain distribution of multiple components in JWQFY was then successfully performed. As a result, constituents with discrepancy structures showed diverse pharmacokinetic and distribution characteristics. For instance, ferulic acid (phenolic acid), 3, 6′-disinapoyl sucrose and tenuifoliside A (oligosaccharide esters) showed short Tmax ( 4 h). Besides, ferulic acid, epimedin C, icariin, glycyrrhizin, ginsenoside Rb1 and ginsenoside Rg1 were considered as the potential effective ingredients of JWQFY because of their relatively high exposure to blood and brain. Our study would provide relevant information for discovery of pharmacodynamic ingredients, as well as further action mechanisms investigations of JWQFY.

Jia-Wei-Qi-Fu-Yin (JWQFY) is a newly developed anti-Alzheimer's disease (AD) prescription modified from a classical traditional Chinese medicine formula, Qi-Fu-Yin (QFY). However, a systematic understanding of its chemical constituents and molecular mechanisms is still elusive. To address this problem, comprehensive chemical profiling followed by network pharmacology-based analysis of JWQFY was performed. Firstly, a total of 136 compounds were characterized by high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF MS), 17 of them were specifically identified in JWQFY comparing with QFY. Seventy compounds were further quantified via a validated HPLC coupled with triple quadrupole tandem mass spectrometry (QQQ MS) method. Then the protein targets of the seventy compounds were gathered from public databases for network construction. As a result, fifty-seven compounds were filtered, which interacted with 655 targets. Thirty-four of them were mapped into the KEGG pathway of AD, indicating JWQFY might exert anti-AD effects by anti-inflammation, neuronal apoptosis intervening, Aβ production inhibition and phosphorylating tau protein moderating. Furthermore, in the compound-target-AD network, a list of hub compounds and hub targets was identified based on their topological features, including the degree, node betweenness and closeness. Four of the hub compounds were specifically originated from JWQFY, supporting the modification rationality of this formula. This study provided a scientific basis for understanding the bioactive compounds and the multi-target mechanism of JWQFY.

The present study aims to investigate the roles of herb pairs containing Angelicae Sinensis Radix (Danggui) in Xin-Sheng-Hua Granule (XSHG) on hemolytic and aplastic anemia (HAA) mice. HAA model mice were induced by acetyl phenylhydrazine and cyclophosphamide; then the samples of XSHG and its decomposed recipes (DY, DC, DT, DH, DJ, and DZ) were orally administrated to these mice. Indicators of peripheral blood routine, organ index, and ATPase activities were tested. Moreover, the main effective components in these samples were also analyzed by UHPLC-TQ-MS/MS. Clear separation between the control and model groups from score plot of principal component analysis (PCA) was easily seen, indicating that HAA model was successfully conducted. Afterwards, relative distance calculation method between dose groups and control group from PCA score plot was adopted to evaluate the integrated effects of hematinic function of different samples. And the orders of hematinic effects were as follows: XHSG > DJ > DT > DZ > DH > DC > DY. Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicated changes when herb pairs containing Danggui were excluded from XSHG, respectively. Compared with XSHG, the vast majority of active compounds in sample DY (formula minus herb pair Danggui-Yimucao) decreased significantly, which could partly explain why herb pair Danggui-Yimucao made great contribution to XSHG. These findings showed that withdrawal analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG, which could lay foundation to reveal the compatibility rules of this formula.

Alkaloids, the principal constituents in the caulis of Sinomenium acutum, have gained an increasing interest over the past decades since they are widely employed as a clinical treatment for rheumatoid arthritis. In the present study, an integrated characterization strategy by combining mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension (DFIBE) was firstly proposed for rapid profiling of alkaloids in Sinomenii Caulis (SC) via ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The rectangular MDFSC window could more accurately screen the target alkaloids of different types, and the DFIBE could facilitate the acquisition of characteristic fragment ions for structural elucidation of alkaloids. High-performance liquid chromatography (HPLC) fingerprints with principal component analysis (PCA) and hierarchical clustering analysis (HCA) was established for identifying the chemical markers and simultaneous determination of sinomenine, magnoflorine, menisperine, stepharanine and ehydrodiscretine. A total of 91 alkaloids, including 82 targeted ones (26 morphinans, 22 aporphines, 20 protoberberines and 14 benzylisoquinolines) were unambiguously identified or tentatively characterized in SC, and 14 of them were reported for the first time. Sinomenine and magnoflorine could be selected as chemical markers to evaluate the quality of SC from different localities. In conclusion, the proposed method provided a potential approach for chemical profiling and holistic quality control of herbal medicines.

Ethnopharmacological relevance Monascus-fermented rice product (MFRP) has been regarded as a dietary supplement and traditional medicine with circulation-promoting effects in China and other countries for centuries. Aim of the study This study was carried out to profile the chemical components in MFRP, and provide available information for elucidating the potential lipid-lowering compounds other than monacolins. Materials and methods High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) and gas chromatography coupled with mass spectrometry (GC-MS) methods were applied to comprehensive analysis of chemical components in MFRP. Potential small molecules were identified by comparing with reference standards, or tentatively characterized by comparing their retention time and high-resolution mass spectral data with previous literature. The lipid-lowering properties of ten major non-monacolin compounds were evaluated in cholesterol-fed zebrafish larvae. And one with optimum lipid-lowering activity was subsequently evaluated in high fat diet-fed C57BL/6 J mice, with the dyslipidemia and ectopic lipid deposition being investigated. Results A total of 99 compounds were characterized in MFRP, including 38 monacolins, 5 decalins, 6 isoflavones, 13 pigments, 8 azaphilonoids, 11 amino acids, 4 nucleosides, 9 lipid acids, 4 phytosterols and glycerol. The preliminary screening showed that ergosterol remarkably reduced cholesterol levels in zebrafish larvae. Moreover, ergosterol delayed body weight gain and decreased circulating total cholesterol, triglyceride, low density lipoprotein cholesterol levels in high fat diet-fed mice. Ectopic lipid accumulation was also ameliorated in the liver and heart of obese mice. Conclusion Global analysis of chemical components and screening of lipid-lowering non-monacolin compounds in MFRP have improved our understanding of its therapeutic material basis.

As a well-known traditional Chinese medicine (TCM) prescription, Xin-Sheng-Hua Granule (XSHG) has been applied in China for more than 30 years to treat postpartum diseases, especially anemia. However, underlying therapeutic mechanisms of XSHG for anemia were still unclear. In this study, plasma metabolomics profiling with UHPLC-QTOF/MS and multivariate data method was firstly analyzed to discover the potential regulation mechanisms of XSHG on anemia rats induced by bleeding from the orbit. Afterward, the compound-target-pathway network of XSHG was constructed by the use of network pharmacology, thus anemia-relevant signaling pathways were dissected. Finally, the crucial targets in the shared pathways of metabolomics and network pharmacology were experimentally validated by ELISA and Western Blot analysis. The results showed that XSHG could exert excellent effects on anemia probably through regulating coenzyme A biosynthesis, sphingolipids metabolism and HIF-1α pathways, which was reflected by the increased levels of EPOR, F2, COASY, as well as the reduced protein expression of HIF-1α, SPHK1, and S1PR1. Our work successfully explained the polypharmcological mechanisms underlying the efficiency of XSHG on treating anemia, and meanwhile, it probed into the potential treatment strategies for anemia from TCM prescription.