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We present a simple and efficient method based on ultrafiltration high-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and identification of ligands obtainable from the extract of Scutellaria baicalensis. Five major compounds (chrysin-6-C-arabinosyl-8-C-glucoside, chrysin-6-C-glucosyl-8-C-arabinoside, baicalin, oroxylin A-7-O-glucuronide, and wogonoside) were identified as potentially effective inhibitors of lipoxidase and superoxide dismutase. Subsequently, specific binding ligands were separated by high-speed countercurrent chromatography, using ethyl acetate/ethyl alcohol/water acetate (0.1%) (1.0:0.1:1.0, v/v/v) as the solvent system. To the best of our knowledge, this is the first report of S. baicalensis extracts containing potent lipoxidase and superoxide dismutase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from the n-butyl alcohol layer of S. baicalensis guided by ultrafiltration high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could also be employed for the identification and isolation of other enzyme inhibitors., (© 2021 Wiley-VCH GmbH.)

Fried centipede, a popular food in southern China and Vietnam, is well known for its notable health benefits owing to its unique chemical composition. This study focused on acetylcholinesterase (AChE) inhibitor screening, bioactivity evaluation, and the extraction of bioactive compounds derived from fried centipedes. To determine the accuracy of in vitro AChE inhibitor screening, receptor-ligand affinity ultrafiltration and enzymatic reaction kinetics technologies were employed for the rapid screening and structural identification of bioactive compounds and to verify the enzyme inhibition activity and mechanism of the compounds. Moreover, molecular docking and dynamic simulations were utilized to assess the effectiveness of the target, and the integration of experimental and computational approaches facilitated a comprehensive analysis of the active compounds and their activities at four levels. To address the issues of low content and low extraction and separation efficiencies of bioactive compounds derived from centipedes, an activity-oriented complex preparation method (consecutive ultrasonic-assisted centrifugal extraction coupled online with UNIFAC countercurrent chromatography and semi-high-performance liquid chromatography (HPLC) was developed for efficient extraction, online concentration, and complex separation of the identified AChE ligands. As a result, six active compounds, including cytidine, guanine, uracil, hypoxanthine, xanthine, and β-thymidine demonstrated significant binding affinity to the AChE active sites within 3 h. This study provided valuable insights into the active compounds of fried centipedes and their potential anti-Alzheimer's disease properties. These findings contribute to the advancement of animal food preparation methodologies by combining cutting-edge extraction techniques, biological activity screening, and the development of AChE inhibitors from fried centipedes. Our method serves as a valuable reference for exploring and developing functional animal materials from diverse medicinal resources. • A strategy to enhance the efficiency of preparing medicine was established. • Molecular docking and dynamics simulations supported the results. • Six high-quality AChEIs were rapidly screened and isolated. • Consecutive extraction and online isolation technique were established. • The parameters for semi-HPLC were optimized by a UNIFAC mathematical model. [ABSTRACT FROM AUTHOR]

Poria cocos (Schw.) Wolf (P. cocos) has been widely used as medical plant in East Asia with remarkable anti-Alzheimer's disease (anti-AD) activity. However, the underlying mechanisms are still confused. In this study, based on the β-Amyloid deposition hypothesis of AD, an integrated analysis was conducted to screen and separation 5-lipoxygenase (5-LOX) inhibitors from triterpenoids of P. cocos and investigate the anti-AD mechanisms, containing bioaffinity ultrafiltration UPLC-Q-Exactive, molecular docking, and multiple complex networks. Five triterpenoids were identified as potential 5-LOX inhibitors, including Tumulosic acid, Polyporenic acid C, 3- Epi -dehydrotumulosic acid, Pachymic acid and Dehydrotrametenolic acid. 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. The commercial 5-LOX inhibitor that Zileuton was used as a positive control to evaluate the inhibitory effect of active ingredients on 5-LOX. Subsequently, the established separation method allowed the five active ingredients (Pachymic acid, 3- Epi -dehydrotumulosic acid, Dehydrotrametenolic acid, Tumulosic acid and Polyporenic acid C) with high purity to be isolated. Targeting network pharmacology analysis showed that five active ingredients correspond to a total of 286 targets. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis found that target cells were mainly enriched in Pathways in cancer, Lipid and atherosclerosis. Our results indicate that P. cocos extract has the potential to be used in the prevention and treatment of neurodegenerative diseases. This will help elucidate the mechanisms of action of various medicinal plants at the molecular level and provide more opportunities for the discovery and development of new potential treatments from health food resources. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Medicago sativa contains flavonoids, saponins, coumarins, sterols, monoterpenes, and organic acids, with flavonoids being the main active constituents. Flavonoids naturally contain a 2‐phenylchromone structure with antioxidant, free radical scavenging, cardiovascular, and trace estrogen‐like effects. Objective: Screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from M. sativa via ultrafiltration‐liquid chromatography‐mass spectrometry (UF‐LC–MS) combined with stepwise flow rate counter‐current chromatography (CCC). Method: Utilising the medicinal plants M. sativa as the research objects and UF‐LC–MS was used for activity screening followed by isolation and purification of the inhibitors by stepwise flow rate CCC. Finally, identification of the three active compounds was achieved by MS and nuclear magnetic resonance. Results: Three major compounds, viz. quercetin, genistein, and formononetin, were identified as potent neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors, respectively. A two‐phase solvent system of ethyl acetate/methanol/n‐butanol/water (5.0:1.5:5.0:10; v/v/v/v) was subsequently selected for separation by stepwise flow rate CCC. Conclusion: This novel approach based on UF‐LC–MS and stepwise flow rate CCC represents a powerful tool for the screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from complex matrices. Therefore, a useful platform for the large‐scale production of bioactive and nutraceutical ingredients was developed herein. An efficient method for the rapid screening and purification of potential NA, LDH, and LOX inhibitors from the M. sativa was established using UF‐LC‐MS combined with stepwise flow rate CCC.Three flavonoid compounds were successfully separated and their structures determined by MS and nuclear magnetic resonance (NMR).To the best of our knowledge, this is the first report of this technique for the screening and isolation of flavonoids from M. sativa [ABSTRACT FROM AUTHOR]

Given the abundance and intricate nature of the chemical components found in natural food ingredients, functional food research has long grappled with two significant technical challenges: rapid and precise screening of active substances and targeted preparation. This study outlines a novel methodology to enhance the efficiency of preparing natural products, presenting new extraction processes, biological activity screening, and the preparation of acetylcholinesterase inhibitors from Ganoderma amboinense. acetylcholinesterase inhibitors were rapidly screened using ultrafiltration coupled with liquid chromatography and mass spectrometry. The inhibitory effect of active compounds on acetylcholinesterase was investigated via enzymatic reaction kinetics, with the mechanism also explored. Additionally, molecular docking and molecular dynamics simulations were used to verify the anti-alzheimer's disease activity of active compounds. Following this, the biological activity of ligands identified were separated and purified using complex chromatography (consecutive-CCC and semi-preparative HPLC). We used n -hexane: ethyl acetate: methanol: water (4.0:6.0:4.0:6.0, v/v/v/v), which optimized by a universal quasichemical functional group activity coefficient-based mathematical model and semi-preparative HPLC, 25–60% A (0–50 min), 60–100% A (50–60 min). Twelve active ingredients viz. ganoderic acids I, C 2 , G, B, K, A, D, F, J, ganoderenic acid A, deacetyl ganoderic acid F, and lucidenic acid F with the binding affinity for AChE were identified and isolated from natural foods. We enhance the efficiency and purity of active compounds preparation by incorporating enzyme inhibition assays and computerized virtual screening with compound chromatography. With this approach, we seamlessly incorporate fast discovery, screening, and focused preparation of active ingredients, as well as delving into the mechanisms of action of active monomers and proteases, and the molecular-level interactions between active ingredients and proteases. This approach effectively mitigates the occurrence of false positives attributable to a single technique, thereby providing valuable data to underpin future advances in enzyme inhibitor screening and isolation. • Coupled application of enzyme inhibition assays and computerized virtual screening. • Application of complex chromatography separation. • Ganoderic acids I, G, K, J and lucidenic acid F conform to inhibition type MT. • Ganoderic acids C 2 , B, A, D, F, et al. conform to inhibition type C. • It provided a new promising platform for the production of functional food. [ABSTRACT FROM AUTHOR]