Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos.

Poria cocos (P. cocos) is a traditional Chinese medicinal product with the same origin as medicine and food. It has diuretic, anti-inflammatory and liver protection properties, and has been widely used in a Chinese medicine in the treatment of Alzheimer's disease (AD). This study was conducted to explore the activity screening, isolation of acetylcholinesterase inhibitors (AChEIs), and in vitro inhibiting effect of P. cocos. The aim was to develop a new extraction process optimization method based on the Matlab genetic algorithm combined with a traditional orthogonal experiment. Moreover, bio-affinity ultrafiltration combined with molecular docking was used to screen and evaluate the activity of the AChEIs, which were subsequently isolated and purified using high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (semi-preparative HPLC). The change in acetylcholinesterase (AChE) activity was tested using an enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on AChE and explore its mechanism of action. Five potential AChEIs were screened via bio-affinity ultrafiltration. Molecular docking results showed that they had good binding affinity for the active site of AChE. Meanwhile, the five active compounds had reversible inhibitory effects on AChE: Polyporenic acid C and Tumulosic acid were non-competitive inhibitors; 3-Epidehydrotumulosic acid was a mixed inhibitor; and Pachymic acid and Dehydrotrametenolic acid were competitive inhibitors. This study provided a basis for the comprehensive utilization of P. cocos and drug development for the treatment of AD.

Gold-copper-doped lanthanide luminescent metal-organic backbone induced self-enhanced molecularly imprinted ECL sensors for ultra-sensitive detection of chlorpyrifos.

Herein, a self-enhanced molecularly imprinted polymer luminescence (MIP-ECL) sensing platform based on gold-copper doped Tb-MOFs (Au@Cu:Tb-MOFs) was constructed for ultra-sensitive detection of chlorpyrifos (CPF). In this work, Au@Cu:Tb-MOFs as co-reaction promoters greatly improve the ECL emission signal, while Au@Cu:Tb-MOFs were used as cathode emitters. And chlorpyrifos and 4,7-bis(thiophene-2-yl)benzo [c][1,2,5] thiadiazole were electropolymerized on electrode surface to form MIP, where this films with thiophene derivatives could greatly improve ECL signal. Notably, the introduction of MIP as recognition elements enabled specific identification of target analytes, in which molecular docking technique validated target analyte and functional monomers are tightly bound through Pi-alkyl interaction. As the concentration of CPF increases, the ECL signal gradually decreases, showing a good linear relationship in the range of 0.1-106 pg/mL with a low detection limit (LOD) of 0.029 pg/mL. Moreover, actual sample testing experiment of this method displayed a special correlation in organophosphorus detection and development potential in actual sample analysis.

Molecular mechanism of Achyranthis bidentatae radix and Morindae officinalis radix in osteoporosis therapy: An investigation based on network pharmacology, molecular docking, and molecular dynamics simulations.

Objective: To investigate the targets and mechanism of Achyranthis bidentatae radix and Morindae officinalis radix (ABR-MOR) for the treatment of osteoporosis (OP) by utilizing network pharmacology, molecular docking technology (MDT) and molecular dynamics simulation (MDS). Methods: The main drug active ingredients (DAIs) and target genes of ABR-MOR were screened by the TCMSP database. The relevant targets of OP were obtained from GeneCards, DisGeNET, and CTD databases. Venny mapping is used to determine the potential target of ABR-MOR in the treatment of OP. The potential targets were analyzed using a protein‒protein interaction network and the MCODE module, and were subjected to GO and KEGG enrichment analysis. The binding sites and conditions of potential key DAIs and core targets were verified through MDT and MDS. Result: The 32 DAIs and 212 targets of ABR-MOR were screened; 1453 OP-related targets were obtained, and 118 targets were mapped. The results of GO and KEGG enrichment analysis showed that the targets of DAIs-OP were mainly enriched in biological processes such as response to hormones, peptides, oxygen levels and reactive oxygen species, and positive regulation of cell migration. The main signaling pathways enriched in the regulation of the immune-inflammatory response, cell proliferation, senescence, apoptosis, angiogenesis, and estrogen signaling pathway. Additionally, the targets were also enriched in bone metabolism-related cell differentiation biological processes and the osteoclast differentiation signaling pathway. MDT and MDS results showed that wogonin, beta-sitosterol, and americanin A, the core DAIs of ABR-MOR, were able to form good ligand‒protein complexes with key targets such as PTGS2, PTGS1, PRKACA, PGR, MAPK1, AKT1, and RELA. Conclusion: This study preliminarily investigated the key targets, biological processes, and signaling pathways involved in the combined application of ABR and MOR for the treatment of OP. The results revealed that ABR-MOR may play a therapeutic role mainly by regulating immune-inflammatory responses, cellular biological processes, and osteoblast differentiation, which provides a theoretical basis for further experimental validation and a new strategy for the treatment of OP.

Investigation of the inhibition effect of arachidonic acid on the core structure of the HIV-1 gp41.

The gp41 transmembrane domain of the envelope glycoprotein of the human immunodeficiency virus (HIV) modulates the conformation of the viral envelope spike. During the HIV fusion process, C-terminal heptad repeat (CHR, C34) wrap antiparallel to the N-terminal heptad repeat (NHR, N36) helices to form a stable six-helix bundle (6-HB) core structure, which brings the viral and cell membranes into close proximity for fusion. Therefore, inhibiting the formation of 6-HB is considered to be a key activity of an effective HIV-1 fusion inhibitor. The level of arachidonic acid (AA) is increased in HIV infected patients. Our study provides a new insight into the functional role of AA during the formation of HIV-1 gp41 6-HB. Native polyacrylamide gel electrophoresis (N-PAGE), enzyme-linked-immunosorbent serologic assay (ELISA) and circular dichroism (CD) spectroscopy were used to investigate the inhibition of AA for the formation of 6-HB. Molecular docking technique was adopted to explore the underlying mechanism. HIV-1 JR-FL (R5 strain) Envelope was adopted to determine the inhibition effect of AA. AA is shown to interfere with the formation of α-helical complexes of N36 and C34 by N-PAGE, ELISA and CD spectroscopy. The isotherm titration microcalorimetry (ITC) results indicate there is a single class of binding site on N36. ΔH and ΔS are -12.43 kJ mol-1 and 70.07 J mol-1 K-1, respectively, indicating hydrophobic interaction and electrostatic forces are the main acting forces. The molecular docking results manifest that AA interacts with the hydrophobic residues (Trp-571, Leu-568, Val-570 and Leu-576) and ionic interactions occur between Arg-579 and the -COOH of AA. The inhibitory activity of AA on HIV-1 JR-FL is quantified by 50% effective concentration (EC50) and 90% effective concentration (EC90), which are 31.42 ± 1.08 and 133.47 ± 18.10 µg mL-1, respectively. All the results indicate that AA is able to inhibit the formation of 6-HB but cannot disrupt the preformed 6-HB. Therefore, AA is a potential inhibitor for the viral fusion/entry.