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Background: American ginseng has an obvious anti-fatigue effect, but the effective material basis is still unclear. The spectrum-effect relationship is a scientific method that studies the correlations between chemical spectra and pharmacological effect. Objective: To reveal the real bioactive compounds in American ginseng saponin (AGS) based on a study of the underlying correlations between these compounds' occurrence in rat serum after their intake of AGS and the anti-fatigue effect of AGS. Methods: We utilized ultra-performance liquid chromatography (UPLC) with quadrupole and time-of-flight mass spectrometry (Q-TOF-MS) to analyze the extract of AGS and its constituents in serum after oral administration in rats. The anti-fatigue effect of AGS in rats was measured using the time weight-bearing swimming technique, the content of blood urea nitrogen, hepatic glycogen, and blood lactic acid. The relationship between the peak area values in fingerprints from rat serum and pharmacodynamic parameters of AGS was established using correlation analysis with partial least square regression (PLSR) method and gray correlation method. Results: We detected and identified 22 compounds from extract, and 8 prototype components from serum. Through PLSR and gray correlation method, it was found that the ginsenosides Re, Rb1, and Rb2 were significantly positively related to the pharmacodynamic data. Conclusions: Based on the spectrum-effect relationship, PLSR and gray correlation method can be used to screen for the anti-fatigue components available in AGS. Such an approach is of practical significance as it provides an effective means for exploring the material basis for the efficacy of American ginseng, particularly as an anti-fatigue agent.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a common difficult disease with a high disability rate. Siegesbeckia orientalis L. (SO), a Chinese medicinal herb that is commonly used for treating RA in clinical practice. While, the anti-RA effect and the mechanisms of action of SO, as well as its active compound(s) have not been elucidated clearly. AIM OF THE STUDY: We aim to explore the molecular mechanism of SO against RA by using network pharmacology analysis, as well as the in vitro and in vivo experimental validations, and to explore the potential bioactive compound(s) in SO. METHODS: Network pharmacology is an advanced technology that provides us an efficient way to study the therapeutic actions of herbs with the underlying mechanisms of action delineated. Here, we used this approach to explore the anti-RA effects of SO, and then the molecular biological approaches were used to verify the prediction. We first established a drug-ingredient-target-disease network and a protein-protein interaction (PPI) network of SO-related RA targets, followed by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Further, we used lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and vascular endothelial growth factor-A (VEGFA)-induced human umbilical vein endothelial cell (HUVEC) models, as well as adjuvant-induced arthritis (AIA) rat model to validate the anti-RA effects of SO. The chemical profile of SO was also determined by using the UHPLC-TOF-MS/MS analysis. RESULTS: Network pharmacology analysis highlighted inflammatory- and angiogenesis-related signaling pathways as promising pathways that mediate the anti-RA effects of SO. Further, in both in vivo and in vitro models, we found that the anti-RA effect of SO is at least partially due to the inhibition of toll like receptor 4 (TLR4) signaling. Molecular docking analysis revealed that luteolin, an active compound in SO, shows the highest degree of connections in compound-target network; moreover, it has a direct binding to the TLR4/MD-2 complex, which is confirmed in cell models. Besides, more than forty compounds including luteolin, darutoside and kaempferol corresponding to their individual peaks were identified tentatively via matching with the empirical molecular formulae and their mass fragments. CONCLUSION: We found that SO and its active compound luteolin exhibit anti-RA activities and potently inhibit TLR4 signaling both in vitro and in vivo. These findings not only indicate the advantage of network pharmacology in the discovery of herb-based therapeutics for treating diseases, but also suggest that SO and its active compound(s) could be developed as potential anti-RA therapeutic drugs.
