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.
Poria cocos (Schw.) Wolf (P. cocos) is a traditional Chinese medicinal materia that medicine food homologyl. In this study, based on the β-Amyloid deposition hypothesis of AD, a fast and efficient methods that ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) and molecular docking were developed for the rapid screening and identification of anti-5-lipoxygenase (anti-5-LOX) enzyme active ingredient of P. cocos ethyl acetate extract. Continuous counter-current chromatography (CCC) combined with semi-preparative high performance liquid chromatography (Semi-preparative HPLC) was developed for targeted separation of active components. Five potential 5-LOX inhibitors were screened by ultrafiltration affinity assay in P. cocos. The molecular docking simulation results are consistent with the ultrafiltration experimental results, which further verifies the accuracy of the experiment. Subsequently, five high-purity active ingredients (Tumulosic acid, Polyporenic acid C, 3-Epi-dehydrotumulosic acid, Pachymic acid and Dehydrotrametenolic acid) could be isolated by the established separation method, with the purities of 95.86%, 96.35%, 97.22%, 98.43% and 99.00%, respectively. The established continuous countercurrent chromatography can effectively improve the yield and purity of low content active ingredients in traditional Chinese medicine, and provide ideas and methods for the targeted separation of active ingredients. The methods of screening and isolating active ingredients established in this paper will be helpful for the development of new therapeutic drugs for Alzheimer's disease (AD).
The herbal pairing of Huangqi and Dangshen (HD) is traditional Chinese herbal medicine and has been widely used in China, especially to treat myasthenia gravis (MG). However, the mechanism of HD on MG is unclear. Aim of the Study. This study aims to investigate HD’s possible role in MG treatment. Materials and Methods. The TCMSP database was used to identify the active chemicals and their targets. The GeneCards, DisGeNET, and OMIM databases were used to search for MG-related targets. The STRING database was employed in order to identify the common PPI network targets. We next utilised Cytoscape 3.8.2 for target identification and the DAVID database for gene ontology (GO) function analysis as well as Encyclopaedia of Genomes (KEGG) pathway enrichment analysis on the selected targets. The AutoDock Vina software was used to test the affinity of essential components with the hub gene before concluding that the primary targets were corrected through molecular docking. Results. 41 active compounds were screened from HD, and the number of putative-identified target genes screened from HD was 112. There were 21 target genes that overlapped with the targets of MG, which were postulated to be potential treatment targets. Through further analysis, the results showed that the active compounds from HD (such as 7-methoxy-2-methylisoflavone, quercetin, luteolin, Kaempferol, and isorhamnetin) may achieve the purpose of treating MG by acting on some core targets and related pathways (such as EGFR, FOS, ESR2, MYC, ESR1, CASP3, and IL-6). Molecular docking findings demonstrated that these active molecules have a near-perfect ability to attach to the primary targets. Conclusion. Through network pharmacology, the findings in this study provide light on the coordinated action of several HD formula components, targets, and pathways. It provided a theoretical basis for further study of HD pharmacological action.
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