Phillygenin (Phi) is one of the main chemical constituents of the fruit of Forsythia suspensa (Thunb.) Vahl. It has various bioactivities, including anti-inflammatory, anti-obesity and antipyretic activities. However, its exact targets and molecular mechanism are still poorly understood. Bioinformatics tools were used to explore the potential targets of Phi, and 8 predicted targets, 4 primary pathways (MAPK, PI3K-Akt, T-cell receptor and m-TOR signaling pathway) related to the inflammatory response, and Akt as an important node was mentioned. Moreover, a Phi alkylated molecular probe was synthesized and used to capture the target proteins Akt. Then Akt and its downstream signaling pathway were verified by molecular docking, intracellular enzyme activity evaluation, and accurate pathway analysis. The results indicated that Phi targets an allosteric inhibit pocket on Akt; reduces Akt phosphorylation; alleviates multiple inflammatory-associated downstream signal transduction pathways, including IKKα/β and NF-κB; and influences glucose metabolic parameters associated with the downstream GSK3β protein and glucose uptake. The results suggest that Phi could reduce inflammatory responses and influence glucose metabolic parameters by inhibiting Akt phosphorylation. Moreover, these findings suggest a potential application for Phi in respiratory and metabolic diseases therapy.

Cimicifuga rhizomes (CR) are used in the treatment of respiratory and cardiovascular diseases in traditional Chinese medicine, but their key effective components and mechanism of action have not yet been reported. In this study, the cardiac, antipyretic and sudorific effects of CR were evaluated using the toad heart failure in vitro model and mice fever and sweating in vivo models. Moreover, the UPLC/Q-TOF-MS-integrated β2-AR luciferase reporter gene assay system was used to screen the bioactive ingredients from CR extract, and the activity of this ingredient were verified using the above-mentioned in vitro and vivo models. Our results showed that CR had anti-heart failure, antipyretic and sweating effects, which could be antagonized by propranolol. On the other hand, cimicifugamide was screened as β2-AR agonist from CR and cimicifugamide could activate β1, 2-ARs more significantly than β3-AR in β-ARs selectivity assessment. The results not only revealed the key effective components and mechanism of CR in traditional use but also supplied a characteristic complementary ingredient for quality control of CR.Download high-res image (165KB)Download full-size image

BackgroundCirsium setosum (CS) is the aboveground part of Cephalanoplos segetum Kitam. Although it has been used as a hemostatic treatment for thousands of years and is still in use today, the mechanism of CS on regulating ARs is still not clear.PurposeIn this study, we aimed to clarify the mechanism of CS on regulating ARs.MethodsWe developed a simple method based on UPLC/Q-TOF MS combined adrenergic receptor dual-luciferase reporter assay systems for the rapid determination of active constituents in CS. The mechanism of tyramine, the main active component for regulating ARs, was further investigated by an in vitro norepinephrine biotransformation test and in vivo vaso activity tests.ResultsTwo phenethylamine ARs regulators (tyramine and N-methyltyramine) in CS were characterized, and it was found that tyramine could induce vasoconstriction via regulation of α1-ARs by mediating norepinephrine synthesis.ConclusionThe hemostatic effect of CS is associated with tyramine and N-methyltyramine, via regulation of α1-ARs, and the mechanism of tyramine is related to mediating norepinephrine synthesis by enzyme catalysis.Download high-res image (123KB)Download full-size image

BackgroundShufengjiedu Capsule (SFJD) is a type of Chinese traditional medicine compound for the treatment of acute upper respiratory tract infection. The present work aims to decipher the mechanism of SFJD.MethodsIn this study, we used target prediction and RNA sequence (RNA-Seq) based on transcriptome analysis to clarify the inflammation–eliminating mechanism of SFJD. Firstly, Pseudomonas aeruginosa (PAK) was used to induce acute lung injury in KM mice. After being treated by SFJD, the differently expressed genes were analyzed by RNA-Seq. Secondly, the chemical constituents of SFJD were identified by ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) and submitted to PharmMapper to predict targets. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and String 9.1 websites were employed to establish the interaction network of inflammation of these targets.ResultsThe results indicated that SFJD alleviated PAK induced lung injury in KM mice. We infer that the mechanism is a complex network containing 15 pathways related to inflammation regulated by 16 types of components from six types of herbs via 29 proteins. The ERK signaling pathway was a key pathway among them, which was predicted to be regulated by 14 types of components in SFJD. Phillyrin, emodin, and verbenalin were screened out by binding capacity, and the synergistic effect of them was further confirmed.ConclusionsVarious components of SFJD ameliorated PAK induced upper respiratory tract infection via multiple targets, of which ERK phosphorylation might be the key event regulated specifically by verbenalin, phillyrin and emodin.Download high-res image (134KB)Download full-size image

•UPLC/Q-TOF-MS and virtual docking were integrated to identify inhibitors of multiple human α-glucosidases.•The system solved the issue of trace quantities limiting the biological study.•Distinct selectivity of acarviostatins was clarified based on structural biology.•Acarviostatin I0-1 was revealed to be strong inhibitor of MGAM-N.We propose a strategy that integrates ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) and virtual docking to identify inhibitors of multiple human α-glucosidases. UPLC yielded AIB656, an acarviostatin-containing complex, which was analyzed by Q-TOF-MS to acquire structural information and was tested for inhibition of N-terminal (MGAM-N), C-terminal (MGAM-C) catalytic domain of maltase-glucoamylase, and human pancreatic α-amylase (HPA). A systematic computational study was performed to evaluate the inhibition activity for 51 identified acarviostatins with various sizes, including trace or difficult-to-prepare ingredients. We evaluated the selectivities of three α-glucosidases to acarviostatins and revealed the strong inhibition of MGAM-N by acarviostatin I0-1. The high accuracy of the dual-screening was validated using enzyme inhibition assays, and docking was suggested as a possible mechanism for the strong inhibition of MGAM-N by acarviostatin I0-1 and of MGAM-C by acarbose (acarviostatin I01). No compound in AIB656 was suitable for inhibiting all three α-glucosidases. Compared with conventional chromatographic separation and inhibitory activity detection, integrating UPLC/Q-TOF-MS identification and virtual validation was more convenient and more reliable. This strategy clearly demonstrates that MS data-based fingerprinting is a meaningful tool not only in identifying constituents in complex matrix but also in directly screening for powerful trace ingredients in natural products.

•UPLC-Q/TOF MS, PCA and the Hotmap were integrated to clarify the difference of chemical constituents from five BM species.•The antimuscarinic and anti-inflammatory effects of different BM species were firstly compared using our bio-assay.•ANN analysis were used to clarify the active ingredients for each bio-activity.•The emphasis on the bio-activities of various BM species could guild their use in TCM.Many species of Bulbus fritillariae are used as traditional medicines for thousands of years; however, their application is not standardized. To clarify the differences and homologies, the antimuscarinic and anti-inflammatory effects of five BM species were firstly tested and compared at cellular level. With an integrated strategy combining UPLC-Q/TOF MS, PCA and ANN analysis, the active ingredients among 28 different chemical markers were predicted and identified. SB and QB extracts showed the best antimuscarinic effects and several steroidal alkaloids, such as solanidine, contributed to this effects. However, ZB was superior to reduce the inflammatory response. Another five components were responsible by decreasing the expression of NF-κB, including puqiedine, zhepeiresinol, 2-monopalmitin, N-demethylpuqietinone, and isoverticine. More novelty, a new cluster of five BM species based on active ingredients as potential quality markers was depicted to illustrate their functions. These results of the study could make a reference for the medicinal application of BM species in clinic; and the integrated strategy provided an effective method to obtain the quality markers from medical herbs, which was helpful for the quality control of traditional medicinal products.