Sennoside A (SA), a typical prodrug, exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases. Hydrolases have been identified, but reductases remain unknown. By linking a photoreactive group to the SA scaffold, we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling (ABPP). From lysates of an active strain, Bifidobacterium pseudocatenulatum (B. pseudocatenulatum), 397 proteins were enriched and subsequently identified using mass spectrometry (MS). Among these proteins, chromate reductase/nicotinamide adenine dinucleotide (NADH) phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase/oxygen-insensitive NADPH nitroreductase (nfrA) was identified as a potent SA reductase through further bioinformatic analysis and The Universal Protein Resource (UniProt) database screening. We also determined that recombinant nfrA could reduce SA. Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.

OBJECTIVE To evaluate the pharmacodynamics of Wujiashen Gejiejing on rats with deficiency of lung qi. METHODS The rat model of deficiency of lung qi was established by sawdust fumigation. By comparing the general activity state, blood acid-alkali indexes, biochemical indexes related to chronic bronchitis and airway histological characteristics of rats in each group, the pharmacodynamics of Wujiashen Gejiejing on rats with deficiency of lung qi was evaluated. RESULTS Compared with model group, after the Wujiashen Gejiejing intervention, the body weight of the rats significantly increased; the levels of p(O2) and SaO2 in blood were significantly increased(P<0.001), p(CO2) was significantly decreased(P<0.05), and pH had a tendency to increase; the levels of endothelin(ET) and IL-1β in serum were significantly decreased(P<0.01 or P<0.05), and the levels of TNF-α in serum had a decreasing trend. The damaged lung structures were significantly improved. CONCLUSION Wujiashen Gejiejing can significantly improve the activity state and improve hypoxemia and hypercapnia of lung qi deficiency syndrome, improve the damaged lung structure and the function of lung ventilation, and has obvious anti-inflammatory effect. Its mechanism may be related to regulating the expression of inflammatory cytokines IL-1β, TNF-α and ET.

The comprehensive detection and identification of active ingredients in complex matrices is a crucial challenge.Liquid chromatography coupled with high-resolution mass spectrometry(LC-HRMS)is the most prominent analytical platform for the exploration of novel active compounds from complex matrices.However,the LC-HRMS-based analysis workflow suffers from several bottleneck issues,such as trace content of target compounds,limited acquisition for fragment information,and uncertainty in interpreting relevant MS2 spectra.Lycibarbarspermidines are vital antioxidant active ingredients in Lycii Fructus,while the reported structures are merely focused on dicaffeoylspermidines due to their low content.To comprehensively detect the new structures of lycibarbarspermidine derivatives,a"depict"strategy was developed in this study.First,potential new lycibarbarspermidine derivatives were designed according to the biosynthetic pathway,and a comprehensive database was established,which enlarged the coverage of lycibarbarspermidine derivatives.Second,the polarity-oriented sample prep-aration of potential new compounds increased the concentration of the target compounds.Third,the construction of the molecular network based on the fragmentation pathway of lycibarbarspermidine derivatives broadened the comprehensiveness of identification.Finally,the weak response signals were captured by data-dependent scanning(DDA)followed by parallel reaction monitoring(PRM),and the efficiency of acquiring MS2 fragment ions of target compounds was significantly improved.Based on the integrated strategy above,210 lycibarbarspermidine derivatives were detected and identified from Lycii Fructus,and in particular,170 potential new compounds were structurally characterized.The integrated strategy improved the sensitivity of detection and the coverage of low-response components,and it is expected to be a promising pipeline for discovering new compounds.