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Rhei Radix et Rhizoma is common traditional Chinese medicine with multiple original plants. The content and proportion of the active components in Rhei Radix et Rhizoma from different plant species were compared to accurately evaluate the medicine qua-lity and provide a theoretical basis for precise use of this medicine in clinical practice. In this study, fresh Rhei Radix et Rhizoma samples were collected from the four-year-old plants of Rheum palmatum, R. tanguticum, and R. officinale. The relative content of 220 anthraquinones, anthrones, and tannins in the samples were determined by pseudo-targeted metabolomics, and the differential components were screened by multivariate statistical methods. The principal component analysis classified the samples into three clusters according to the original plants. The orthogonal partial least squares-discriminant analysis(OPLS-DA) screened out 117 differential components, including 8 free anthraquinones, 18 anthraquinone glycosides, 80 anthrones, and 11 tannins. Twenty-eight components had the highest content in R. tanguticum, mainly including sennosides, anthraquinone glycosides, and procyanidins. Thirty-five components showed the highest content in R. officinale, mainly including free anthraquinones and catechines. Fifty-four components showed the highest content in R. palmatum, mainly including dianthrones, while the structures of most of them cannot be determined temporarily. The content distribution of differential components in the three original plants indicates that R. tanguticum has the strongest effect of purging, while R. officinale has the strongest effect of clearing heat and purging fire, and both have stronger effects of resolvong stasis and dredging meridians than R. palmatum.

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Chinese patent medicine preparations containing Epimedii Folium and Psoraleae Fructus have been associated with the occurrence of idiosyncratic drug-induced liver injury(IDILI). However, the specific toxic biomarkers and mechanisms underlying these effects remain unclear. This study aimed to comprehensively assess the impact of bavachin and epimedin B, two principal consti-tuents found in Psoraleae Fructus and Epimedii Folium, on an IDILI model induced by tumor necrosis factor-α(TNF-α) treatment, both in vitro and in vivo. To evaluate the extent of liver injury, various parameters were assessed. Lactate dehydrogenase(LDH) release in the cell culture supernatant, as well as the levels of alanine aminotransferase(ALT) and aspartate transaminase(AST) in mouse plasma were measured. Additionally, histological analysis employing hematoxylin-eosin staining was performed to observe liver tissue changes indicative of the severity of liver injury. Furthermore, a pseudo-targeted metabolomics approach was employed, followed by multivariate analysis, to identify differential metabolites. These identified metabolites were subsequently subjected to Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. The results showed that at the cellular level, after 2 hours of TNF-α stimulation, bavachin significantly increased the release of LDH in HepG2 cells compared to the normal group and the group treated alone; after the combination of bavachin and epimedin B, the release of LDH further significantly increased on the original basis. Similarly, although the individual or combination treatments of bavachin and epimedin B did not induce liver injury in normal mice, the combination of both drugs induced marked liver injury in TNF-α treated mice, leading to a significant elevation in plasma AST and ALT levels and substantial infiltration of inflammatory immune cells in the liver tissue. Pseudo-targeted metabolomics analysis identified seven common differential metabolites. Among these, D-glucosamine-6-phosphate, N1-methyl-2-pyridone-5-carboxamide, 17beta-nitro-5a-androstane, irisolidone-7-O-glucuronide, and N-(1-deoxy-1-fructosyl) valine emerged as potential biomarkers, with an area under the curve(AUC) exceeding 0.9. Furthermore, our results suggest that the metabolism of nicotinic acid and nicotinamide, as well as the linoleic acid metabolic pathway, may play pivotal roles in bavachin and epimedin B-induced IDILI. In conclusion, within an immune-stressed environment mediated by TNF-α, bavachin and epimedin B appear to induce IDILI through disruptions in metabolic processes.

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The fermentation-driving ability of Daqu has been widely reported, while the potential influence of substances in Daqu on Baijiu flavor formation has attracted increasing interest. Pseudo-targeted metabolomics integrated proteomics combined with sensory evaluation strategy was applied to investigate the correlation between flavor characteristics and metabolic profiling of Daqu, and the mechanism of flavor formation was also elucidated. The 4-hydroxy-2,5-dimethylfuran-3-one (3.5 mg kg-1) and 2,3-dihydro-1 h-inden-5-ol (894.3 µg kg-1) were identified as the unique substances in qingcha qu, which were vital for raspberry flavor formation and associated with the up-regulation of amino acid metabolism. The dec-9-enoic acid (37.4 mg kg-1) was screened out as the substance related to the formation of cream flavor in hongxin qu produced through the shortening of fatty acid carbon chains and unsaturated modification of long chain fatty and acceleration of carbon metabolism in hongxin qu mediated by filamentous Aspergillus spp. was related to the smoky aroma enhancement.

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Isotope tracing assisted metabolic analysis is becoming a unique tool to understand metabolic regulation in cell biology and biomedical research. Targeted mass spectrometry analysis based on selected reaction monitoring (SRM) has been widely applied in isotope tracing experiment with the advantages of high sensitivity and broad linearity. However, its application for new pathway discovery is largely restrained by molecular coverage. To overcome this limitation, we describe a strategy called pseudo-targeted profiling of isotopic metabolomics (PtPIM) to expand the analysis of isotope labeled metabolites beyond the limit of known pathways and chemical standards. Pseudo-targeted metabolomics was first established with ion transitions and retention times transformed from high resolution (orbitrap) mass spectrometry. Isotope labeled MRM transitions were then generated according to chemical formulas of fragments, which were derived from accurate ion masses acquired by HRMS. An in-house software "PseudoIsoMRM" was developed to simulate isotope labeled ion transitions in batch mode and correct the interference of natural isotopologues. This PtPIM strategy was successfully applied to study 13C6-glucose traced HepG2 cells. As 313 molecules determined as analysis targets, a total of 4104 ion transitions were simulated to monitor 13C labeled metabolites in positive-negative switching mode of QQQ mass spectrometer with minimum dwell time of 0.3 ms achieved. A total of 68 metabolites covering glycolysis, TCA cycle, nucleotide biosynthesis, one-carbon metabolism and related derivatives were found to be labeled (> 2%) in HepG2 cells. Active pentose phosphate pathway was observed with diverse labeling status of glycolysis intermediates. Meanwhile, our PtPIM strategy revealed that rotenone severely suppressed mitochondrial function e.g. oxidative phosphorylation and fatty acid beta-oxidation. In this case, anaerobic respiration became the major source of energy metabolism by producing abundant lactate. Conclusively, the simulation based PtPIM method demonstrates a strategy to broaden metabolite coverage in isotope tracing analysis independent of standard chemicals.

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Astragali Radix is widely used because of its dual use in medicine and food, and its quality evaluation is of great importance. In this study, a pseudo-targeted metabolomics approach based on scheduled multiple reaction monitoring was developed, and a total of 114 compounds with good linearity, sensitivity, and reproducibility were selected for relative quantification, and the chemical differences between Astragali Radix of different growth patterns were further compared by chemometric analysis. With the help of multivariate and univariate analysis, 26 differential compounds between wild/semi-wild Astragali Radix and cultivated Astragali Radix were determined. Then five marker compounds were screened out by lasso regression, and further verified by systematic clustering, random forest, support vector machine, and logistic regression. In addition, malonyl-substituted flavonoids showed relatively higher content in wild/semi-wild Astragali Radix. Thus, the malonyl substitution was characteristic for flavonoids in wild/semi-wild Astragali Radix. In conclusion, the application of pseudo-targeted metabolomics and various statistical methods could offer multi-dimensional information for the holistic quality evaluation of Astragali Radix.

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Selenium (Se)-enriched green tea (Se-Te) has been recognized as a possible source of Se supplements, while the effect of Se enrichment on function of polyphenols in green tea is still unclear. In this study, a pseudo-targeted metabolomics strategy was carried out to reveal the regulatory mechanism of polyphenols extracted from Se-Te and regular green tea (Re-Te) on inflammatory response at cellular level. A novel analysis strategy using UHPLC/ESI Q-Orbitrap combined with MS-IOP was applied to profile the dynamic changes of metabolites in LPS-stimulated RAW264.7 macrophages during polyphenols incubation. A total of 128 characteristic variables (VIP > 1, p < 0.05) were screened in Se-Te group and the results of bioinformatics analysis and quantitative research indicated that in addition to the 6 conventional immune protective pathways involved in tea polyphenols, Se-enriched polyphenols were also participated in 3 unique antioxidant enzyme activation pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and pantothenate and CoA biosynthesis. The result of weight calculation based on topological analysis indicated that the promoting synthesis of antioxidant enzymes was the main mechanism of Se-Te polyphenols to inhibit inflammation. However, compared with Re-Te group, the intracellular B vitamin pathway in Se-Te group was disturbed, which is related to the fact that Se supplementation can promote the synthesis of selenoprotein and catalyze the reduction of thioredoxin by NADPH, thus blocking the signaling pathways of B vitamins. This study comprehensively explored the immune protective mechanism of polyphenols extracted from Se-Te and Re-Te under natural growth conditions, which could give a better understanding of the potential nutritional value of Se-Te as a widely used Se supplement.

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Pimacao is a traditional Chinese folk medicine and is the main component of the famous Chinese herbal remedy "Yunnan Baiyao" for its significant analgesic activity in the treatment of wounds. Due to increases in consumption, its wild population is now difficult to find, and adulterant from the same genus has occurred. However, this is challenging to distinguish the species of Veratrum in Pimacao using dried roots and rhizomes or medicinal powder. ITS2 sequences and steroidal alkaloids by the non-targeted and pseudo-targeted metabolomics methods were taken advantage of establishing an effective identification method. Based on the ITS2 sequence, metabolite profiling of steroidal alkaloids and morphological characteristics, the classification of two distinct subspecies in V. mengzeanum has been reinforced. In addition, the new subspecies V. mengzeanum subsp. phuwae was collected in China for the first time. The ITS2 sequence could be used in the identification of V. taliense, V. mengtzeanum, V. stenophyllum, and V. nigrum, but is insufficient for intraspecific identification. Simultaneously, 147 variables were labeled by non-targeted analysis accomplished utilizing an ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UPLC-ESI-QE-Orbitrap-MS) system consisting of an Orbitrap QE HF-X. Followed by a pseudo-targeted analysis method developed for the Qtrap 6500-plus mass spectrometry system coupled with an ESI source, 29 labeled steroidal alkaloids detected by the MRM mode could distinguish between four species. Notably, 25 labeled steroidal alkaloids could distinguish between three closely related species. These have the potential to be used as markers for identification. Furthermore, there were several variables with statistical differences between two subspecies of V. mengtzeanum and populations of V. taliense, V. mengtzeanum, and V. stenophyllum.

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INTRODUCTION: Breast cancer (BC) has become the most commonly diagnosed cancer worldwide. It is very critical for the differential diagnosis between BC and benign breast diseases (BBD). The characteristics of serum bile acids (BAs) profiling in patients with BBD and BC was elucidated so that potential biomarkers could be find out for the differential diagnosis of BC and BBD. METHODS: A pseudo-targeted approach was used to perform BAs metabolomics analysis in serum of 29 patients with BBD and 47 patients with BC by ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Partial least squares-discriminant analysis (PLS-DA) was used to establish a differential diagnostic model for BC, and the receiver operating characteristic (ROC) curve and logistic regression analysis were used to screen out bile acids as biomarkers for the differential diagnosis of BC and BBD. RESULTS: The serum BAs profile in BC group was quite different from that in BBD group. Compared with the BBD group, BC group had higher level of chenodeoxycholic acid (CDCA), while they had lower levels of dihydroxy tauro-conjugated BA (Tdi-1) and sulfated dihydroxy glyco-conjugated BA (Gdi-S-1). The sensitivity and specificity of PLS-DA model for patients classification were 100% and 92.3%, respectively. The combined biomarker, CDCA and Tdi-1, had high efficacy for the differential diagnosis (area under the curve was 0.954, 95% CI: 0.880-1.000) of BC. Besides, the performance was superior to traditional biomarkers in the differential diagnosis of BC with or without comorbidities. CONCLUSION: The profile of serum BAs in women with BC was quite different from that in patients with BBD. Serum BAs profiling analysis could be used as an effective tool for the differential diagnosis of BC and BBD.

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