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Thrombin was immobilized on magnetic core‐shell mesoporous silica nanoparticles (Fe3O4@nSiO2@mSiO2) for the first time and used to screen enzyme inhibitors from extracts of Notopterygium incisum. The immobilized thrombin was characterized by Fourier‐transform infrared spectroscopy, transmission electronic microscopy, thermal gravimetric analysis, and vibrating sample magnetometry. The results demonstrated that the immobilized thrombin possessed good thermal stability and high‐temperature resistance for feasible storage and reuse. Two ligands, notopterol (1) and isoimperatorin (2) were fished out from the extract of N. incisum using the immobilized thrombin. They inhibited thrombin with IC50 values of 59.35 ± 1.420 and 107.8 ± 1.660 µM, respectively. This is the first report of thrombin‐inhibitory activity associated with notopterol and isoimperatorin. Meanwhile, the inhibition mechanism of both compounds was investigated by enzyme kinetic analysis and molecular docking. The method used enables rapid screening for thrombin inhibitors from medicinal plants, providing an efficient tool for the discovery of anti‐thrombotic active compounds. [ABSTRACT FROM AUTHOR]

Coreopsis tinctoria Nutt. is an important medicinal plant in traditional Uyghur medicine. The skin-lightening potential of the flower has been recognized recently; however, the active compounds responsible for that are not clear. In this work, tyrosinase, a target protein for regulating melanin synthesis, was immobilized on the Whatman paper for the first time to screen skin-lightening compounds present in the flower. Quercetagetin-7-O-glucoside (1), marein (2), and okanin (3) were found to be the enzyme inhibitors. The IC50 values of quercetagetin-7-O-glucoside (1) and okanin (3) were 79.06 ± 1.08 μM and 30.25 ± 1.11 μM, respectively, which is smaller than 100.21 ± 0.11 μM of the positive control kojic acid. Enzyme kinetic analysis and molecular docking were carried out to investigate their inhibition mechanism. Although marein (2) showed a weak inhibition effect in vitro, it inhibited the intracellular tyrosinase activity and diminished melanin production in melanoma B16 cells as did the other two inhibitors. The paper-based ligand fishing method developed in this work makes it effective to quickly screen tyrosinase inhibitors from natural products. This is the first report on the tyrosinase inhibitory effect of those three compounds, showing the promising potential of Coreopsis tinctoria for the development of herbal skin-lightening products. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to establish a hollow fiber ligand fishing method for screening γ- Aminobutyrate transaminase (GABA-T) inhibitory component from the volatile oil of Acorus tatarinowii. Methods: Based on optimizing the enzyme assisted extraction process of volatile oil from A. tatarinowii using response surface methodology, GABA-T solution was injected into the hollow fiber lumen and used as a "bait" to fish out the potential active components in A. tatarinowii volatile oil, and its GABA-T inhibitory activity was verified. Results: The results showed that the optimum extraction conditions of volatile oil from A. tatarinowii were as follows: cellulase content 3 165 U/g, enzymolysis time 1.8 h, enzymolysis temperature 45 ℃, and the yield of volatile oil from A. tatarinowii was 2.56%. Finally, 51 compounds were identified, mainly including olefins, alcohols, ethers, ketones, phenols, acids, and other compounds. 3 mg/mL of GABA-T solution was injected into the cavity of the U-shaped hollow fiber tube, incubated for 160 min, sealed, and placed in the volatile oil of A. tatarinowii for ultrasonic (60 kHz) fishing for 20 min, and then analyzed with LCMS-IT-TOF system. Successfully filtered out α-Asarone has a concentration-dependent inhibitory effect on GABA-T activity, with an IC50 of 57.9 μg/mL. Conclusion: The established hollow fiber ligand fishing method is stable, feasible, and easy to operate.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme's proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease's active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices. [ABSTRACT FROM AUTHOR]

Uremic Clearance Granule (UCG) is a traditional Chinese medicines (TCMs) that has been recognized as potentially effective treatment for diabetic nephropathy (DN). However, the chemical composition and the hypoglycemic ingredients of UCG remain unclear. In this experiment, a magnetic ligand fishing protocol combined with HPLC coupled Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR-MS) technology was developed to screen and identify α-Glucosidase inhibitors from UCG. First, constituents of UCG was identified by HPLC-FT-ICR-MS. Next, α-Glucosidase coated Fe3O4@SiO2@NH2 (Fe3O4@SiO2@NH2@α-Glucosidase) nano composites was synthesized. The essential parameters (concentration of Glutaraldehyde (GA), ratio of Fe3O4@SiO2@NH2 to enzyme, crosslinking time and immobilization time) were optimized to obtain maximum enzyme activity and the performance of immobilized enzyme was compared with that of free enzyme. The α-Glucosidase inhibitory activities were then evaluated using autodock and the 4-Nitrophenyl-α-D-glucopyranoside (pNPG) method. As a result, 142 compounds were identified were identified by comparing the retention time, molecular ions and fragmentation behaviors with the reference compounds or the in-house database. 20 possible α-Glucosidase activity inhibitory components were identified using HPLC-FT-ICR-MS. Molecular docking and inhibition studies showed that 4 compuunds including Limonexic acid, Sanggenol A, Glabrone, Matrine were more likely to stronger α-Glucosidase inhibitory activities than acarbose. In conclusion, the proposed approach, which combined highly specific screening with HPLC-FT-ICR-MS, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicines (TCMs).

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α-Amylase inhibitors constitute a category of compounds capable of diminishing α-amylase activity and hold significant promise for the development of novel drugs. This study focused on assessing the potential of Schisandra chinensis Baill extract to inhibit α-amylase. However, sieving and segregating inhibitors from intricate mixtures presents an obstacle. Consequently, this study devised and refined a methodology centered on immobilized α-amylase to identify and isolate natural inhibitors from S. chinensis. The immobilized α-amylase, featuring a load capacity of 254.18 ± 1.20 μg/mg and a specific activity of 2.69 ± 0.01 U/mg, was achieved through meticulous optimization of preparation conditions. Subsequently, this immobilized enzyme was used to isolate bioactive compounds from S. chinensis extract. The results revealed the presence of an active compound with an IC 50 of 324.12 ± 11.23 μg/mL. This compound was isolated via magnetic immobilized enzyme-based ligand fishing, employing high-speed countercurrent chromatography, and was conclusively identified as 5-hydroxymethylfurfural. The molecular docking analyses revealed its ability to interact with the active pocket of α-amylase through multiple hydrogen bonds. [Display omitted] • α-Amylase was immobilized on mesoporous silica particles for ligand fishing. • Main active component was screened from S. chinensis by immobilized α-amylase. • 5-Hydroxymenthyfurfural was the main active component in S. chinensis fruit. • The interaction between active compound and α-amylase was investigated. [ABSTRACT FROM AUTHOR]

Coreopsis tinctoria Nutt. is an important medicinal plant in traditional Uyghur medicine. The skin-lightening potential of the flower has been recognized recently; however, the active compounds responsible for that are not clear. In this work, tyrosinase, a target protein for regulating melanin synthesis, was immobilized on the Whatman paper for the first time to screen skin-lightening compounds present in the flower. Quercetagetin-7-O-glucoside (1), marein (2), and okanin (3) were found to be the enzyme inhibitors. The IC50 values of quercetagetin-7-O-glucoside (1) and okanin (3) were 79.06 ± 1.08 μM and 30.25 ± 1.11 μM, respectively, which is smaller than 100.21 ± 0.11 μM of the positive control kojic acid. Enzyme kinetic analysis and molecular docking were carried out to investigate their inhibition mechanism. Although marein (2) showed a weak inhibition effect in vitro, it inhibited the intracellular tyrosinase activity and diminished melanin production in melanoma B16 cells as did the other two inhibitors. The paper-based ligand fishing method developed in this work makes it effective to quickly screen tyrosinase inhibitors from natural products. This is the first report on the tyrosinase inhibitory effect of those three compounds, showing the promising potential of Coreopsis tinctoria for the development of herbal skin-lightening products.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme’s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease’s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.

Cancer is the second leading cause of death in the world and its incidence is expected to increase with the aging of the world's population and globalization of risk factors. Natural products and their derivatives have provided a significant number of approved anticancer drugs and the development of robust and selective screening assays for the identification of lead anticancer natural products are essential in the challenge of developing personalized targeted therapies tailored to the genetic and molecular characteristics of tumors. To this end, a ligand fishing assay is a remarkable tool to rapidly and rigorously screen complex matrices, such as plant extracts, for the isolation and identification of specific ligands that bind to relevant pharmacological targets. In this paper, we review the application of ligand fishing with cancer‐related targets to screen natural product extracts for the isolation and identification of selective ligands. We provide critical analysis of the system configurations, targets, and key phytochemical classes related to the field of anticancer research. Based on the data collected, ligand fishing emerges as a robust and powerful screening system for the rapid discovery of new anticancer drugs from natural resources. It is currently an underexplored strategy according to its considerable potential. [ABSTRACT FROM AUTHOR]

Biflavonoids are naturally occurring compounds consisting of two flavonoid moieties that have received substantial attention from researchers. Although many kinds of biflavonoids are typically distributed in Selaginella uncinata with hypoglycemic effect, their anti‐α‐glucosidase activities are not yet clear. In this study, a ligand fishing strategy for fast screening of α‐glucosidase inhibitors from S. uncinata was proposed. α‐Glucosidase was first immobilized on Fe3O4 magnetic nanoparticles (MNPs) and then the α‐glucosidase–functionalized MNPs were incubated with crude extracts of S. uncinata to fish out the ligands. Furthermore, considering the similarity and easy confusion of the structures of biflavonoids, the fragmentation patterns of different types of biflavonoids were studied. Based on this, 11 biflavonoids ligands with α‐glucosidase inhibitory activities were accurately and quickly identified from S. uncinata with ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight–tandem mass spectrometry. Furthermore, these ligands were confirmed to be potential inhibitors through the in vitro inhibitory assay and molecular docking. [ABSTRACT FROM AUTHOR]

Lipase and α-amylase play a very important role in the screening of anti-obesity and anti-diabetic drugs. Here, bienzyme microspheres containing lipase and α-amylase were synthesized for simultaneous affinity screening of anti-obesity and anti-diabetic component from pomegranate peel. Lipase was immobilized by functionalized multi-walled carbon nanotube (MWCNT) coated with polyamide dendrimers to obtain MWCNT-lipase. Subsequently, MWCNT-lipase and α-amylase was encapsulated by sodium alginate (SA) hydrogel microspheres to form MWCNT/SA-bienzymes. Under the optimized reaction conditions, the immobilization rate of lipase and α-amylase was 65.16% and 95.79%, and the activities were 85.76% and 88.11% of free enzymes, respectively. Moreover, the enzyme activity conditions of MWCNT/SA-bienzymes were also optimized. The MWCNT/SA-bienzymes were more stable than two free enzymes at room temperature and can be reused. More importantly, in application, a compound (ellagic acid) was selectively screened from pomegranate peel extract by MWCNT/SA-bienzymes. In vitro activity determination confirmed that ellagic acid with IC 50 of 369.83 ± 11.75 μg/mL and 672.81 ± 18.55 μg/mL can interact with two enzymes through non-competitive inhibition. The molecular docking showed that ellagic acid bound with α-amylase and lipase through hydrogen bonds, van der Waals forces, etc, resulting in the binding energies of − 8.54 kcal/mol and − 6.87 kcal/mol, respectively. [Display omitted] • An effective strategy was proposed for screening of bifunctional inhibitor. • Bienzyme microspheres containing lipase and α-amylase were synthesized. • Multi-walled carbon nanotubes and hydrogels were used as carriers. • Ellagic acid has the dual function of inhibiting lipase and α-amylase. [ABSTRACT FROM AUTHOR]

Diabetes is a chronic metabolic disease caused by insufficient insulin secretion and insulin resistance. Natural product is one of the most important resources for anti-diabetic drug. However, due to the extremely complex composition, this research is facing great challenges. After the advent of ligand fishing technology based on enzyme immobilization, the efficiency of screening anti-diabetic components has been greatly improved. In order to provide critical knowledge for future research in this field, the application progress of immobilized enzyme in screening anti-diabetic components from complex natural extracts in recent years was reviewed comprehensively, including novel preparation technologies and strategies of immobilized enzyme and its outstanding application prospect in many aspects. The basic principles and preparation steps of immobilized enzyme were briefly described, including entrapment, physical adsorption, covalent binding, affinity immobilization, multienzyme system and carrier-free immobilization. New formatted immobilized enzymes with different carriers, hollow fibers, magnetic materials, microreactors, metal organic frameworks, etc., were widely used to screen anti-diabetic compositions from various natural products, such as Ginkgo biloba, Morus alba, lotus leaves, Pueraria lobata, Prunella vulgaris, and Magnolia cortex. Furthermore, the challenges and future prospects in this field were put forward in this review. [ABSTRACT FROM AUTHOR]

Morus alba leaves are a natural product with great antidiabetic potential. However, the therapeutic efficacy of natural products is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi‐target therapeutic functions are more effective than single‐target enzymes. In this study, a bienzyme system was constructed by co‐immobilizing α‐amylase and α‐glucosidase onto Fe3O4 for affinity screening of dual‐target active components in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by high‐speed countercurrent chromatography using a solvent system of ethyl acetate–n‐butanol–water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α‐amylase and α‐glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrate the effectiveness of the polydopamine magnetically immobilized bienzyme system for dual‐target affinity screening of active substances. This study not only reveals the chemical basis of the antidiabetic activity of M. alba leaves from a dual‐target perspective, but also promotes the progress of multitarget affinity screening. [ABSTRACT FROM AUTHOR]

In this study, a ligand fishing method was developed to screen potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors from coffee extracts by immobilization of IDO1 enzyme on amino-modified magnetic nanoparticles combined with UHPLC-Q-TOF-MS/MS analysis. Parameters including enzyme concentration, immobilization time, the pH of glutaraldehyde and the amount of magnetic nanoparticles were optimized. The results indicated that immobilized IDO1 could be reused 5 times and was stable during storage for 7 days. Several IDO1 ligands were captured by incubating immobilized IDO1 with coffee extract, of which 10 showed an obvious difference comparing to non-conjugated bare nanoparticles. In vitro inhibitory activity was further performed by CE analysis, in which ferulic acid and chlorogenic acid had better IDO1 inhibitory activity, with IC50 value of 113.7 μM and 307.5 μM. These results demonstrate that this method provides an effective platform for identifying and screening IDO1 inhibitors from natural products.

Nigella glandulifera is a traditional medicinal plant used to treat seizures, insomnia, and mental disorders among the Tibetan and Xinjiang people of China. Recent pharmacological research indicates that the seeds of this plant have a neuroprotective effect; however, the chemical components responsible for this effect are unknown. Monoamine oxidase B (MAO-B) has been recognized as a target for developing anti-Parkinson's disease drugs. In this work, MAO-B functionalized magnetic nanoparticles were used to enrich the enzyme's ligands in extracts of N. glandulifera seeds for rapid screening of MAO-B inhibitors coupled with HPLC-MS. Tauroside E and thymoquinone were found to inhibit the enzyme with IC50 values of 35.85 μM and 25.54 μM, respectively. Both compounds exhibited neuroprotective effects on 6-OHDA-induced PC-12 cells by increasing the cell viability to 52% and 58%, respectively, compared to 50% of the injured cells. Finally, molecular docking indicated strong interactions of both inhibitors with the enzyme. This work shows that MAO-B functionalized magnetic nanoparticles are effective for rapid screening of anti-PD inhibitors from complex herbal mixtures and, at the same time, shows the promising potential of this plant's seeds in developing anti-PD drugs. [ABSTRACT FROM AUTHOR]

Natural products are an important source of drug development and have good prospects in the treatment of many diseases. However, the efficacy of many natural drugs is achieved through multi-targets, which poses a huge obstacle to their development. In this study, a bienzyme microcapsule system based on α-amylase and α-glucosidase was constructed for the ligand fishing of natural compounds. The microcapsules were formed on the surface of calcium carbonate particles by self-polymerization of dopamine, which physically encapsulated iron oxide and α-glucosidase. Then, α-amylase was immobilized on the surface of magnetic material with glutaraldehyde as crosslinking agent, and calcium carbonate template was removed by EDTA to form a bienzyme system for affinity screening of active components from Toona sinensis. The results showed that two compounds, 1,2,3,4,6-penta-O-galloyl-β- D -glucose and quercitrin were affinity screened from the extracts of Toona sinensis. In vitro activity experiment proved that they were α-amylase and α-glucosidase inhibitors, respectively. Moreover, both of them were mixed type inhibitors that can interact with active centers of corresponding enzymes through multiple hydrogen bonds, van der Waals forces, etc. This study not only proved the effectiveness of the bienzyme system, but also provided a successful example for multi-target screening of natural products. [Display omitted] • Bifunctional microcapsules of α-amylase and α-glucosidase was constructed. • Active components were dual-target affinity screened by microcapsules. • Interactions between targets and active compounds were studied by molecular docking. [ABSTRACT FROM AUTHOR]

Introduction: As a famous traditional Chinese medicine, roots of Platycodon grandiflorus (Jacq.) A.DC. have shown multiple effects against neurodegenerative diseases. To investigate the components against Parkinson's disease (PD), the roots of P. grandiflora were selected as the research subject. Objective: Screening and identifying of monoamine oxidase B (MAO‐B) inhibitors from the roots of P. grandiflorum via enzyme functionalised magnetic nanoparticles (MNPs)‐based ligand fishing combined with high‐performance liquid chromatography–mass spectrometry (HPLC–MS) analysis. Method: MAO‐B functionalised MNPs have been synthesised for screening MAO‐B inhibitors from the roots of P. grandiflorum. The ligands were identified by HPLC–MS and nuclear magnetic resonance (NMR) analysis, and their anti‐PD activity was evaluated via MAO‐B inhibition assay and cell viability assay in vitro. Results: Two MAO‐B inhibitors were fished out and identified by HPLC–MS as protocatechuic aldehyde (1) and coumarin (2), with the half maximal inhibitory concentrations of 28.54 ± 0.39 and 25.39 ± 0.29 μM, respectively. Among them, 1 could also significantly increase the viability of 6‐hydroxydopamine‐damaged PC12 cells. Conclusion: The results are helpful to elucidate the anti‐PD activity of the plant, and the ligand fishing method has shown good potential in discovery of MAO‐B inhibitors. Monoamine oxidase B (MAO‐B) was immobilized on magnetic nanoparticles for fast screening of MAO‐B inhibitors from roots of P. grandiflorum. Two inhibitors were fished out and identified as protocatechuic aldehyde and coumarin with IC50 value of 28.54 and 25.39 μM, respectively. Further, protocatechuic aldehyde protected PC12 cells against damage of 6‐hydroxydopamine. This work demonstrated the high efficiency of ligand fishing in screening of active natural compounds and indicated the anti‐Parkinson's disease potential of this medicinal plant. [ABSTRACT FROM AUTHOR]

Ophiocordyceps xuefengensis (O. xuefengensis), the sister taxon of Ophiocordyceps sinensis (O. sinensis), is consumed as a "tonic food" due to its health benefits. However, little is known regarding the chemistry and bioactivity of O. xuefengensis. In this study, we characterized 80 indole-based alkaloids in the ethyl acetate fraction of O. xuefengensis by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC–Q–TOF–MS/MS), of which 54 indole-based alkaloids were identified as possibly new compounds. Furthermore, 29 of these compounds were established as potential anti-cancer compounds by ligand fishing combined with HPLC–Q–TOF–MS/MS. Moreover, molecular docking identified the NH- and OH- groups of these compounds as the key active groups. The present study has expanded the knowledge on the characteristic indole-based alkaloids and anti-cancer activity of O. xuefengensis. [Display omitted] • Eighty indole-based alkaloids were identified in Ophiocordyceps xuefengensis. • Twenty-nine anti-cancer indole-based alkaloids were screened and identified. • The NH- and OH- groups of indole-based alkaloids were the key active groups. [ABSTRACT FROM AUTHOR]

Uremic Clearance Granule (UCG) is a traditional Chinese medicines (TCMs) that has been recognized as potentially effective treatment for diabetic nephropathy (DN). However, the chemical composition and the hypoglycemic ingredients of UCG remain unclear. In this experiment, a magnetic ligand fishing protocol combined with HPLC coupled Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR-MS) technology was developed to screen and identify α-Glucosidase inhibitors from UCG. First, constituents of UCG was identified by HPLC-FT-ICR-MS. Next, α-Glucosidase coated Fe 3 O 4 @SiO 2 @NH 2 (Fe 3 O 4 @SiO 2 @NH 2 @α-Glucosidase) nano composites was synthesized. The essential parameters (concentration of Glutaraldehyde (GA), ratio of Fe 3 O 4 @SiO 2 @NH 2 to enzyme, crosslinking time and immobilization time) were optimized to obtain maximum enzyme activity and the performance of immobilized enzyme was compared with that of free enzyme. The α-Glucosidase inhibitory activities were then evaluated using autodock and the 4-Nitrophenyl-α-D-glucopyranoside (pNPG) method. As a result, 142 compounds were identified were identified by comparing the retention time, molecular ions and fragmentation behaviors with the reference compounds or the in-house database. 20 possible α-Glucosidase activity inhibitory components were identified using HPLC-FT-ICR-MS. Molecular docking and inhibition studies showed that 4 compuunds including Limonexic acid, Sanggenol A, Glabrone, Matrine were more likely to stronger α-Glucosidase inhibitory activities than acarbose. In conclusion, the proposed approach, which combined highly specific screening with HPLC-FT-ICR-MS, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicines (TCMs). [ABSTRACT FROM AUTHOR]

In this study, tyrosinase was immobilized on carboxyl functionalized silica‐coated magnetic nanoparticles for the first time to be used for fishing of tyrosinase's ligands present in complex plant extract. The immobilized tyrosinase was characterized by transmission electron microscopy, vibrating sample magnetometry, Fourier transform infrared spectroscopy, thermo‐gravimetric analyzer, and atomic force microscopy. The reusability and thermostability of the immobilized tyrosinase were found significantly superior to its free counterpart. Two tyrosinase's ligands, that is, caffeic acid (1) and rosmarinic acid (2), were fished out from extract of the traditional Chinese medicine Prunellae Spica by the immobilized tyrosinase. Compound 1 was found to be an activator of the enzyme with the half maximal effective concentration value of 0.27 ± 0.06 mM, while compound 2 was an inhibitor with the half maximal inhibitory concentration value of 0.14 ± 0.03 mM. Taking advantage of the convenience of magnetic separation and specific extraction ability of ligand fishing, the proposed method exhibited great potential for screening of bioactive compounds from complex matrices. [ABSTRACT FROM AUTHOR]

Carbon nanomaterials have huge application prospects in the fields of materials science and biomedicine. Graphene oxide (GO) can be served as an excellent carrier with good loading capacity and biocompatibility. However, lamelayers of GO are prone to aggregation and difficult to stably disperse in solution. Only relying on the mesh graphene sheets as a carrier still cannot achieve a good loading efficiency. Therefore, oriented layered graphene oxide pad (OGO) is synthesized and used as a carrier for ligand fishing. The structure of OGO exhibits good reproducibility and recyclability, and its regular open channels and low pore curvature can effectively ensure a high density of fishing sites and flow speed, improving the loading capacity and storage stability. According to the amount of immobilized cell membranes protein (CMP), OGO can reach 1.5 to 2 times loading capacity of GO. The established CMP@OGO is stable within 48 h at 4 °C, and the reuse rate of OGO is 93.04 ± 0.67%. Finally, salvianolic acid B, deoxyshikonin, and shikonin were identified as potentially active compounds in comfrey combined with a high‐performance liquid chromatography‐hybrid linear ion trap/orbitrap mass spectrometer. It can provide a promising platform for OGO to screen active compounds in high throughput. [ABSTRACT FROM AUTHOR]

The traditional method for natural products discovering could be time-consuming and labor-intensive, thus ligand fishing has attracted a lot of attention as a fast-screening method. Nevertheless, ligand fishing often relied on immobilized enzyme with solid carriers, which can be expensive and complicated to prepare. In this study, a carrier-free enzyme immobilization technology, cross-linked enzyme aggregates (CLEAs), was introduced by preparing carbonic anhydrase cross-linked enzyme aggregates (CA-CLEAs), aiming to develop a method for ligand fishing that is simpler, faster and more cost-effective. CA-CLEAs were synthesized with optimal conditions, and were proven to have the ability to capture CA inhibitors and recycle. The crude extract of Salvia miltiorrhiza (SM) was determined to have inhibitory activity against CA, and two major components of SM, i.e., phenolic acids and diterpenoids, were prepared and incubated with CA-CLEAs, respectively. Seven potential inhibitors were fished out and identified using liquid chromatography-mass spectrometry (LC-MS), and all of them were verified to have inhibitory activity, with two novel inhibitors, salvianolic acid C and tanshinone IIA, being discovered. Molecular docking simulations revealed the different inhibitory mechanisms of phenolic acids and diterpenoids. Furthermore, combination effect analysis was carried out, and the synergistic effects were discovered between two components, as well as between their representative compounds, i.e., salvianolic acid B and tanshinone IIA. In this study, a ligand fishing method based on carrier-free immobilized enzyme was established and applied to discover CA inhibitors. A variety of inhibitors with different inhibitory mechanisms were identified successfully, which could accelerate the development of CA inhibitors, demonstrating the efficiency and potential of the method in new bioactive compounds discovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A novel strategy of performing ligand fishing with enzyme‐modified open tubular microchannel was proposed for screening bioactive components present in medicinal plants. Monoamine oxidase B was immobilized onto the surface of the microchannel for the first time to specifically extract its ligands when the plant's extracts solution flows through the channel. The thermal and the storage stability of immobilized monoamine oxidase B were significantly enhanced after immobilization. Crocin I and Ⅱ were extracted from Crocus sativus, and tiliroside was extracted from Edgeworthia gardneri. All the three compounds were inhibitors of the enzyme with the half‐maximal inhibitory concentration values of 26.70 ± 0.91, 19.88 ± 2.78, and 15.65 ± 0.85 μM, respectively. The enzyme inhibition kinetics and molecular docking were investigated. This is the first report on the inhibitory effects of tiliroside and crocin Ⅱ. The novel ligand fishing method proposed in this work possesses advantages of rapidness, high efficiency, and tiny sample consumption compared to routine ligand fishing, with promising potential for screening active natural products in complex mixtures. [ABSTRACT FROM AUTHOR]

As for ligand fishing, the current immobilization approaches have some potential drawbacks such as the small protein loading capacity and difficult recycle process. The core–shell metal–organic frameworks composite (Fe3O4-COOH@UiO-66-NH2), which exhibited both magnetic characteristics and large specific surface area, was herein fabricated and used as magnetic support for the covalent immobilization of porcine pancreatic lipase (PPL). The resultant composite Fe3O4-COOH@UiO-66-NH2@PPL manifested a high loading capacity (247.8 mg/g) and relative activity recovery (101.5%). In addition, PPL exhibited enhanced tolerance to temperature and pH after immobilization. Then, the composite Fe3O4-COOH@UiO-66-NH2@PPL was incubated with the extract of Scutellaria baicalensis to fish out the ligands. Eight lipase inhibitors were obtained and identified by UPLC-Q-TOF-MS/MS. The feasibility of the method was further confirmed through an in vitro inhibitory assay and molecular docking. The proposed ligand fishing technique based on Fe3O4-COOH@UiO-66-NH2@PPL provided a feasible, selective, and effective platform for discovering enzyme inhibitors from natural products. [ABSTRACT FROM AUTHOR]

Fangji Huangqi Decoction (FHD) is a classical Chinese compound formula for treating rheumatoid arthritis (RA) with satisfactory effects. FHD is reputed for its ability to tonify qi with strengthening exterior, and dispel wind while removing dampness, but its mechanisms and bioactive compounds for treating RA remain unclear. The aim of this study was to explore the key target and bioactive compounds that were responsible for FHD-mediated improvements in RA. Using network pharmacology, we discovered that cyclooxygenase-2 (COX-2) was the key target of FHD against RA. We utilized a ligand fishing technique with COX-2 immobilized magnetic beads to recognize the bioactive components that act on COX-2. Then we carried out an in vitro assay of COX-2 enzyme inhibition and in vivo assay of carrageenan-induced inflammation and collagen-induced arthritis (CIA) to validate the bioactive effects of these captured ingredients. In the CIA assay, micro-CT, hematoxylin‒eosin staining and safranin-O/fast green staining were employed to assess the influence of the captured ligand on bone damage, pathological injury and cartilage destruction, respectively. Immunohistochemistry (IHC) and enzyme-linked immunosorbent assays (ELISAs) were used to detect the expression of COX-2 target in the ankle joint. interleukin-6 (IL-6) levels in the serum were also detected by ELISA. Molecular docking was used to reveal the binding mechanism of the COX-2 protein and the captured ligand. Eleven ligands, including tetrandrine, fangchinoline, cyclanoline, licochalcone B, ononin, calycosin and liquiritin, were specifically bound to the COX-2 protein, as determined by ultrahigh-performance liquid chromatography–mass spectrometry (UPLC–MS), seven of which were present at high levels. One ligand, tetrandrine, not only had a great inhibitory effect on COX-2 enzyme activity but also significantly reduced carrageenan-induced inflammation. In the CIA assay, middle- and high-dose tetrandrine (25 and 50 mg/kg) had effects comparable to those of FHD and celecoxib on ameliorating RA symptoms in CIA mice via the COX-2 target. Furthermore, compared with the low-dose tetrandrine group (12.5 mg/kg), the FHD group exhibited significantly lower arthritis index scores and serum IL-6 expression, although the content of tetrandrine in FHD extract solution was approximately 0.1% of that in the low-dose tetrandrine group. Hence, we inferred that tetrandrine was the main bioactive component responsible for the effects of FHD against RA by suppressing the expression of the COX-2 protein and inhibiting the enzyme catalytic activity of COX-2. The reason for these effects may be that tetrandrine can interact with the residue Tyr385 of COX-2, the enzymatic catalytic site of COX-2 to transform arachidonic acid (AA) to prostaglandin E2 (PGE2), and thereby reduce the production of prostaglandins and inflammatory metabolites. Moreover, in addition to tetrandrine, FHD contains other compounds that could supplement the activity of tetrandrine when FHD was used to treat RA, which is manifested the "multi-component" characteristic of how Traditional Chinese Medicine formulas treat diseases. Tetrandrine was discovered as a specific ligand of COX-2 through a magnetic separation ligand fishing technique from Fangji Huangqi Decoction (FHD), a Chinese compound formula used for treating rheumatoid arthritis (RA) in the clinic. Trough in vivo validation using collagen-induced arthritis mice, tetrandrine was confirmed to be the main bioactive component of FHD that alleviates RA symptoms through core target COX-2. [Display omitted] • COX-2@MBs with specific adsorption property were synthesized and used to discover the bioactive components in treating RA. • Tetrandrine was validated as the bioactive integrant of FHD in the treatment of RA via targeting to COX-2. • The anti-RA effect of FHD is superior to that of tetrandrine, indicating other components acting supportively for tetrandrine. [ABSTRACT FROM AUTHOR]

Obesity has become an increasingly serious public health problem. Pancreatic lipase (PL) is identified as a ideal target for the prevention and treatment of obesity. Orlistat, the only approved PL inhibitor (PLI), is a powerful weight loss drug but has many side effects. Therefore, there is an urgent need to discover powerful PLIs with high safety. Protein hydrolysate has been demonstrated to be a treasure trove of PLIs, but recognizing responsible functional peptides from them is like looking for a needle in a haystack. In this work, we synthesized and optimized a PL ligand fishing model (PLLFM) using magnetic nanoparticles (MNPs), then PLLFM was used to quickly fish out potential PLIs from the Cod meat hydrolysate (CMH). Finally, two new PLIs, GSPPPSG and KLEGDLK were identified with IC50 of 0.60 and 1.08 mg/mL, respectively. The Lineweaver-Burk diagram showed that GSPPPSG is a non-competitively dominant mixed-type PLI, whereas KLEGDLK is a competitive inhibitory-type PLI. Moreover, molecular docking suggested that both peptides can stably bind to the key amino acid residues of the PL active site, mainly through hydrogen bonding, hydrophobic, and electrostatic interactions. In general, we not only established a method to rapidly fish out potential PLIs from protein hydrolysate, but also provided safe and efficient lead compounds for the development of novel diet foods or drugs.

[Display omitted] • MAO-B was successfully immobilized onto cellulose filter paper for the first time. • A new method for ligand fishing based on CFP-DA-MAO-B was developed. • Natural MAO-B inhibitors, including one never reported before, were isolated and identified from Tibetan strawberry. • Neuroprotective effects of the inhibitors were demonstrated. • Potential of Tibetan strawberry as neuroprotective functional food was suggested. Tibetan strawberry (Fragaria nubicola) is a wild medicinal and edible plant in Tibet possessing various health benefits such as neuroprotection and anti-oxidation. However, there has been little study reported on its chemical constituents. To investigate the inhibitors of monoamine oxidase B (MAO-B) in Tibetan strawberry, we immobilized the enzyme onto cellulose filter paper for the first time to develop a new screening method. Two known glycosides (compounds 1 and 2) and one new iridoid glucoside (Compound 3) were fished out by this method, which was found to effectively inhibit MAO-B with IC 50 values of 16.95 ± 0.93, 24.69 ± 0.20, and 46.77 ± 0.78 μM, respectively. Molecular docking and kinetic analysis were performed to reveal the inhibition mechanism of these compounds. Furthermore, compound 1 exhibited neuroprotective effects against 6-OHDA-induced injury on PC12 cells. The developed method exhibits the advantages of rapidness and effectiveness in screening of MAO-B inhibitors from complex herbal extracts. [ABSTRACT FROM AUTHOR]

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking. [Display omitted] • Preparation and characterization of thrombin immobilized on Fe 3 O 4 @SiO 2 -NH 2. • Detection of enzymatic properties of immobilized thrombin. • Six thrombin inhibitors fished out with Traditional Chinese medicine extracts. • The thrombin inhibitory activity was verified by molecular docking technique. [ABSTRACT FROM AUTHOR]

A simple and rapid method for screening of tyrosinase (TYR) inhibitors present in traditional Chinese medicines (TCMs) was developed by combining ligand fishing and the fluorescent enzymatic assay based on dopamine-functionalized carbon quantum dots (CQDs-Dopa). Ligands of the enzyme present in the TCM extractions were firstly adsorbed on the enzyme-modified magnetic beads, and then the beads were magnetically separated and subjected directly to the CQDs-Dopa-based fluorescent assay. Finally, compounds were desorbed from the "active" beads and identified with ultra-performance liquid chromatography-triple quadrupole mass spectrometry. A known natural TYR inhibitor quercetin was selected to assess the feasibility and quantification performance of this method, and good linearity in the range of 0.01–0.16 mM (R2 = 0.992) with a low detection limit of 0.004 mM was obtained. This method was then applied to screen TYR inhibitors present in Scutellaria baicalensis and Sophora flavescens. Six TYR inhibitors including baicalin (1), baicalein (2), wogonin (3), oroxylin A (4), kurarinone (5), and sophoraflavanone G (6) were found, among which 1–4 were firstly discovered in this work. This is the first report on the in situ assessment of the target compounds obtained by ligand fishing in the form of a mixture, which exhibited the combined advantages of specific extraction ability of ligand fishing and the high sensitivity of CQDs-based fluorescent assay, showing great potential for fast screening of enzyme inhibitors from TCMs. [ABSTRACT FROM AUTHOR]

The microfluidic platform is a versatile tool for screening and locating bioactive molecules from functional foods. Here, a layer-by-layer assembly approach was used to fabricate core-shell CdSSe@ZnS quantum dot encoded superparamagnetic iron oxide microspheres, which served as a carrier for matrix metalloproteinase-2. The matrix metalloproteinase-2 camouflaged magnetic microspheres was further incorporated into a homemade microfluidic platform and incubated with extracts of fruits of Rosa roxburghii. The flow rate of the microfluidic platform was tuned. The major influencing parameters on ligand binding, such as dissociate solvents, incubation pH, ion strength, temperature, and incubation time were also optimized by using ellagic acid as a model compound. The specific binding ligands were sent for structure elucidation by mass spectrometry. The absolute recovery of ellagic acid ranged from 101.14 to 102.40% in the extract of R. roxburghii under the optimal extraction conditions. The linearity was pretty well in the range of 0.009–1.00 mg·ml−1 (R2 = 0.9995). The limit of detection was 0.003 mg·ml−1. The relative SDs of within-day and between-day precision were

Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n-BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC–Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens, and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC50) values of 197.9 and 125.4 μmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin–Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 μmol/L.

Nigella glandulifera is a traditional medicinal plant used to treat seizures, insomnia, and mental disorders among the Tibetan and Xinjiang people of China. Recent pharmacological research indicates that the seeds of this plant have a neuroprotective effect; however, the chemical components responsible for this effect are unknown. Monoamine oxidase B (MAO-B) has been recognized as a target for developing anti-Parkinson’s disease drugs. In this work, MAO-B functionalized magnetic nanoparticles were used to enrich the enzyme’s ligands in extracts of N. glandulifera seeds for rapid screening of MAO-B inhibitors coupled with HPLC-MS. Tauroside E and thymoquinone were found to inhibit the enzyme with IC50 values of 35.85 μM and 25.54 μM, respectively. Both compounds exhibited neuroprotective effects on 6-OHDA-induced PC-12 cells by increasing the cell viability to 52% and 58%, respectively, compared to 50% of the injured cells. Finally, molecular docking indicated strong interactions of both inhibitors with the enzyme. This work shows that MAO-B functionalized magnetic nanoparticles are effective for rapid screening of anti-PD inhibitors from complex herbal mixtures and, at the same time, shows the promising potential of this plant’s seeds in developing anti-PD drugs.

Toona sinensis is a traditional vegetable with rich nutrition and wide biological activity. In our previous study, an active composition against α-amylase was screened from T. sinensis by ligand fishing. To investigate the effect of T. sinensis on other targets of antidiabetes, in this work, α-glucosidase was immobilized on polydopamine-coated magnetic nanoparticles to further screen the compounds against α-glucosidase by ligand fishing. The enzyme activity test showed that the 70% ethanol extract of T. sinensis (crude sample) had weak α-glucosidase inhibitory activity, but after extraction with different solvents, the ethyl acetate extract showed the highest α-glucosidase inhibitory activity. After immobilization of α-glucosidase on polydopamine (PDA)-coated magnetic nanoparticles, the properties of the enzyme were characterized and determined, and then quercetin-3-O-α-L-rhamnopyranoside was screened by ligand fishing and isolated by high-speed counter-current chromatography using the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v). The results of enzymes assay showed that the screened compound (IC50 value of 374.94 ± 4.35 μg/mL) possessed moderate α-glucosidase inhibitory activity. The presented results not only proved that ligand fishing based on polydopamine-coated magnetic α-glucosidase was an effective method for the rapid discovery of natural active substances, but also further strengthened the evidence of antidiabetic effect of T. sinensis. [ABSTRACT FROM AUTHOR]

Effect-directed analysis (EDA) has been increasingly used for screening toxic contaminants in the environment, but conventional EDA procedures are often time-consuming and labor-extensive. This challenges the use of EDA for toxicant identification in the scenarios when quick answers are demanded. Herein, a solid phase microextraction ligand fishing (SPME-LF) strategy has been proposed as a rapid EDA approach for identifying acetylcholinesterase (AChE) active compounds in water. The feasibility of ligand fishing techniques for screening AChE active chemicals from environmental mixtures was first verified by a membrane separation method. Then, SPME fibers were prepared through self-assembly of boronic acid groups with AChE via co-bonding and applied for SPME-LF. As AChE coated SPME fibers selectively enriched AChE-active compounds from water, comparing sorbing compounds by the SPME fibers with and without AChE coating can quickly distinguish AChE toxicants in mixtures. Compared with conventional EDA, SPME-LF does not require repeating sample separations and bioassays, endowing SPME-LF with the merits of low-cost, labor-saving, and user-friendly. It is believed that cost-efficient and easy-to-use SPME-LF strategy can potentially be a rapid EDA method for screening receptor-specific toxicants in aquatic environment, especially applicable in time-sensitive screening. [Display omitted] • Ligand fishing is potentially used as a rapid effect-based analytical method. • AChE was immobilized on SPME fibers by self-assembly of boronic acid groups. • SPME-based ligand fishing method selectively adsorbed AChE active contaminants in water. [ABSTRACT FROM AUTHOR]

• Monoamine oxidase A was immobilized on carboxyl terminated magnetic beads (MBs@COOH@MAOA) for the first time. • MBs@COOH@MAOA showed much better performance in ligand fishing than MBs@NH 2 @MAOA. • H. zeylanica was explored for screening MAOA inhibitors for the first time. • Six potential monoterpene indoles were fished out and identified as MAOA inhibitors. In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC 50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted. [ABSTRACT FROM AUTHOR]

In this study, a method for the screening and identification of α-glucosidase inhibitors from natural products was developed. The α-glucosidase was immobilized on carboxyl terminated magnetic beads to form a ligand fishing system to screen the potential inhibitors. A total of 9 compounds were fishing out from the crude Houttuynia cordata Thunb. extract. Meanwhile, ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) was used for the identification of the chemical structures, including 3 chlorogenic acid isomers, 2 flavone C-glycosides and 4 flavone O-glycosides. The combination of enzyme immobilization magnetic beads and UHPLC-QTOF MS could be used for the screening of bioactive multi-components from herbs with appropriate targets. Taking the advantage of the specificity of enzyme binding and the convenience of magnetic separation, the method has great potential for rapid screening of α-glucosidase inhibitors from complicated natural product extracts. A method for the screening and identification of α-glucosidase inhibitors from natural products was developed. The combination of enzyme immobilization magnetic beads and UPLC-QTOF MS were used to form a ligand fishing system and for the identification of the chemical structures. This method has great potential for rapid screening of α-glucosidase inhibitors from complicated natural product extracts. [Display omitted] • α-Glucosidase enzyme immobilized on magnetic nanoparticles was developed. • 9 potential α-glucosidase inhibitors fished out from Houttuynia cordata Thunb. • Chemical structures of the 9 inhibitors were identified by UHPLC-QTOF MS. [ABSTRACT FROM AUTHOR]

Modern pharmacology studies have demonstrated that α-glucosidase is closely associated with a variety of metabolic disorders. Natural herbs have emerged as possible sources of α-glucosidase inhibitors (AGIs) in recent years. Thus far, highly selective screening of biologically active chemicals in complicated mixtures using ligand fishing approaches has not been able to achieve biomedical imaging, which is critical for real-time identification. Hence, this paper presented a novel technique for screening potential AGIs in Scutellaria baicalensis by utilizing fluorescent carbon dots that have been immobilized with α-glucosidase (α-glu-NH 2 -CDs). The synthesized carbon dots materials exhibited excellent fluorescence properties, biocompatibility, and an abundance of functional groups, as demonstrated by the utilization of several techniques. The activity of α-glucosidase covalently linked to the material under ideal immobilization conditions was 86.81% of that of the free form. The immobilized α-glucosidase also exhibited great temperature stability, pH stability and reusability according to an examination of its enzymatic characteristics. As a result, three ligands were identified as potential AGIs, i.e., baicalein, wogonin and scullcapflavone II by UPLC-MS/MS. The molecular docking and enzyme inhibition test indicated that the isolated compounds exhibited significant α-glucosidase inhibitory activities. Moreover, the proliferation inhibitory activity of the screened ligands on HCT-116 cells was further verified morphologically using in situ imaging technique. These results strongly suggested that the established method, which combined ligand fishing with in situ visual imaging, not only screen and separate the potential AGIs in Scutellaria baicalensis quickly and effectively, but also provide an effective platform for screening bioactive compounds on different receptors. • Amino-functionalized solid-state fluorescent carbon dots was synthesized by a one-step hydrothermal method. • A fluorescent ligand fishing based on carbon dots was developed for screening bioactive molecules. • The effectiveness of this strategy was demonstrated using a model mixture. • Three ligands as potential AGIs were fished from Scutellaria baicalensis crude extracts. • The activities of fished ligands were verified by in situ imaging and in vitro enzyme inhibition assays. [ABSTRACT FROM AUTHOR]

Ligand fishing, also described as affinity-based assay, represents a convenient and efficient approach to separate potential ligands from complex matrixes or chemical libraries. This approach contributes to the identification of lead compounds that can bind to a specific target. In the context of COVID-19, the search for novel therapeutic agents is crucial. Small molecule-based antiviral drugs, such as Amaryllidaceae alkaloids, have been described as potential candidates because they can inhibit RNA viruses. Among various SARS-CoV-2 proteins, Nsp3, Nsp4, and Nsp6 play a crucial role in the pathogenicity of the virus and are attractive targets for developing COVID-19 treatments. These proteins are responsible for the replication/transcription complex (RTC) within double-membrane vesicles (DMVs), and their inhibition disrupts the virus's infectious cycle. Herein, we have successfully expressed and immobilized the SARS-CoV-2 Nsp4 protein on magnetic beads (Nsp4-MBs) and employed a ligand fishing assay to screen a collection of ten Amaryllidaceae-based alkaloids and applied to Hippeastrum aulicum extract. Remarkably, four out of ten alkaloids, namely 2-α-7-dimethoxyhomolycorine (6), haemanthamine (5), albomaculine (8), and tazettine (9), exhibited selective affinities for Nsp4. Albomaculine (8) and haemanthamine (5) were also identified from extract by the affinity assay. These findings highlight the potential of these alkaloids as model compounds for future drug discovery studies aimed at developing therapeutic interventions against SARS-CoV-2 infections. • The NSP4 SARS-CoV-2 was expressed, purified and immobilized on magnetic particles • Ten alkaloids from Amaryllidaceae were screened out by Ligand fishing • Identified four potential binding proteins for Nsp4. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fabrication of α-glucosidase immobilized paper-based bioreactor. • High catalytic activity for immobilized α-glucosidase with C-terminus external. • Simultaneous ligand screening and visual ratiometric evaluating inhibitory activity. • Successful applications for Duzhong leaves and navel orange peels. • Molecular docking verified interactions of screened compounds to α-glucosidase. It is integral to establish methods to investigate inhibitory activity and screen inhibitors to α-glucosidase (α-Glu) from complex natural products. Here, α-Glu immobilized paper-based bioreactor platform was firstly developed for simultaneous ligand screening and visual ratiometric evaluating inhibitory activity. N-terminus of α-Glu was anchored on paper with chitosan and glutaraldehyde as cross-linking reagents. Smaller Michaelis constant (K m) for immobilized α-Glu (10.64 mM) than free α-Glu (15.63 mM) indicated higher catalytic activity. Sample (5 μL) was dropped on α-Glu immobilized paper for incubation, potent inhibitors would complex with α-Glu, while unbound compounds could be washed off. By chromatographic analyzing eluents from α-Glu and inactive α-Glu immobilized paper, potent inhibitors could be screened out. Simultaneously, activity of immobilized α-Glu was evaluated by fabricating visual ratiometric fluorescent system using ascorbic acid, Cu2+, and o -phenylenediamine without the interference of complex matrix. The developed platform was validated by model mixtures (positive control acarbose, IC 50 0.88 mg/mL; and negative control gallic acid, negligible inhibition at 5 mg/mL), and applied for real crude extracts. α-Glu inhibitory activities were visually detected for Duzhong leaves and navel orange peels (43.6 % and 26.0 % inhibition at 10 mg/mL), and four flavonoids were screened, identified, and verified as α-Glu inhibitors. Further, molecular docking studies revealed the possible inhibitory mechanisms, which came from the significant affinity interaction by forming hydrogen bonds. Results suggested the convenience, specificity, reliability, and efficiency of the proposed method. [ABSTRACT FROM AUTHOR]

Abstract Background The discovery of bioactive compounds in traditional Chinese medicine (TCM) has become an important field in TCM modernization. Ligand fishing is a suitable method for discovery of bioactive compounds in complex mixtures such as TCM with high selectivity. Because of unique advantage of low cost and convenience, paper-based microdevices can be good carriers for enzyme immobilized ligand fishing. Methods As an important enzyme for glucose metabolism, α-glucosidase was immobilized on polycaprolactone–chitosan-modified paper to prepare the microdevice with unique microfluid structure generated by 3D printing technology, which can be easily applied to screen active compounds in herbal extracts. The preparation conditions of the paper microarray were optimized. The activity of immobilized α-glucosidase was verified by colorimetric reactions which can be easily monitored by cellphone. The paper microarray with α-glucosidase immobilized was used to screen active compounds in the water extracts of mulberry leaves and lotus leaves. Results Several key parameters including Na2CO3 solution concentration, Na2CO3 solution volume, glutaraldehyde concentration, crosslinking time of glutaraldehyde and time of α-glucosidase immobilization were optimized. The proposed paper-based microarray was successfully applied in screening active compounds in two herbal extracts. Four compounds including chlorogenic acid, quercetin-3-O-glucuronide, isoquercetin, and quercetin were identified as α-glucosidase inhibitors. The compounds with significant non-specific adsorption caused by chitosan, such as isoquercitrin, astragalin, quercetin, were also found to be active compounds. Conclusions An enzyme immobilized paper microarray was designed and fabricated in this work. Polycaprolactone and chitosan were used to modify filter paper to prepare paper microarrays. Parameters of paper device preparation were optimized. Our findings suggested that 3D-printing paper-based microarrays can be a simple and low-cost approach for discovery of active compounds of TCM.

Background: Lactate dehydrogenase (LDH) is one of the important enzyme systems for glycolysis and gluconeogenesis. It can catalyze the reduction and oxidation reaction between propionic acid and L-lactic acid, which is usually overexpressed in cancer cells. Therefore, inhibiting the activity of LDH is a promising way for the treatment of cancer. In this study, an effective method based on ligand fishing and ultra performance liquid chromatography-mass spectrum (UPLC-MS) was established to screen and identify active ingredients from Selaginella doederleinii with potential inhibitory activity for LDH. Methods: Firstly, LDH was immobilized on the magnetic nanoparticles (MNPs), three immobilization parameters including LDH concentration, immobilization time and pH were optimized by single factor and response surface methodology for maximum (max) immobilization yield. Then, a mixed model of galloflavin and chlorogenic acid (inhibitors and non-inhibitors of LDH) was used to verify the specificity of immobilized LDH ligand fishing, and the conditions of ligand fishing were further optimized. Finally, combined with UPLC-MS, immobilized LDH was used to simultaneously screen and identify potential LDH inhibitors from the ethyl acetate extract of Selaginella doederleinii. Results: The prepared fishing material was comprehensively characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared spectrometer (FT-IR). The optimal immobilization conditions were obtained as LDH concentration of 0.7 mg/mL, pH value of 4.5, and immobilization time of 3.5 h. Under these conditions, the max immobilization yield was (3.79 ± 0.08) × 103 U/g. The specificity analysis showed that immobilized LDH could recognize and capture ligands, and the optimal ligand fishing conditions included that the incubation time was 30 min, the elution time was 20 min, and the concentration of methanol as eluent was 80%. Finally, two LDH inhibitors, amentoflavone and robustaflavone, were screened by immobilized LDH from the ethyl acetate extract of Selaginella doederleinii. Conclusions: The study provided a meaningful evidence for discovering the bioactive constituents in ethyl acetate extract of Selaginella doederleinii related to cancer treatment, and this ligand fishing method was feasible for screening enzyme inhibitors from similar complex mixtures.

In the present study, an efficient method based on ligand fishing and high-speed counter-current chromatography (HSCCC) was established to screen, enrich and separate the active components with the α-amylase inhibitory activity from a traditional dish Toona sinensis. The active components were screened from T. sinensis by ligand fishing using the magnetic immobilized α-amylase prepared through solvothermal and crosslinking methods. HSCCC was used to separate the target compound according to the K value. As a result, a potential active compound 1,2,3,4,6-penta-O-galloyl-β- d -glucose and a non-target compound quercetin-3-O-α-L-rhamnopyranoside were separated and identified. In - vitro experiments indicated that 1,2,3,4,6-penta-O-galloyl-β- d -glucose had the activity against α-amylase and the IC 50 value was 93.49 ± 0.80 μg/mL which was higher than that of the non-target compound. The result further confirmed the molecular fishing effect of magnetic immobilized α-amylase. The present study can not only find and separate the hypoglycemic substances in T. sinensis quickly and effectively, but also can provide a new approach for the study of natural active components. • Magnetic immobilized α-amylase was prepared to screen active composition from Toona sinensis. • The potential active compound was prepared by high-speed counter-current chromatography. • The activity of isolated compound was proved in vitro. [ABSTRACT FROM AUTHOR]

As for ligand fishing, the current immobilization approaches have some potential drawbacks such as the small protein loading capacity and difficult recycle process. The core–shell metal–organic frameworks composite (Fe3O4-COOH@UiO-66-NH2), which exhibited both magnetic characteristics and large specific surface area, was herein fabricated and used as magnetic support for the covalent immobilization of porcine pancreatic lipase (PPL). The resultant composite Fe3O4-COOH@UiO-66-NH2@PPL manifested a high loading capacity (247.8 mg/g) and relative activity recovery (101.5%). In addition, PPL exhibited enhanced tolerance to temperature and pH after immobilization. Then, the composite Fe3O4-COOH@UiO-66-NH2@PPL was incubated with the extract of Scutellaria baicalensis to fish out the ligands. Eight lipase inhibitors were obtained and identified by UPLC-Q-TOF-MS/MS. The feasibility of the method was further confirmed through an in vitro inhibitory assay and molecular docking. The proposed ligand fishing technique based on Fe3O4-COOH@UiO-66-NH2@PPL provided a feasible, selective, and effective platform for discovering enzyme inhibitors from natural products.

Inhibition of α -glucosidase activity is an effective way for treatment of type 2 diabetes mellitus. Epimedii Folium is an important source of α -glucosidase inhibitors (AGIs), however bioactive compounds and pharmacological mechanisms remained unclear. In this study, a novel strategy was established, which harnessed α -glucosidase functionalized magnetic beads to fish out potential AGIs, followed by UPLC-MS/MS analysis for their identification. Furthermore, molecular docking was employed to predict binding patterns between the AGIs and the enzyme, and IC 50 values was estimated as well. After response surface methodology optimization, the highest activity of Fe 3 O 4 @ α -glucosidase has been achieved when 1.17 mg/mL of α -glucosidase was immobilized in phosphate buffer (pH 6.81) for 4.22 h. Moreover, eight flavonoids were fished out from the extract of Epimedii Folium , and then identified to be epimedin A, epimedin B, epimedin C, icariin, sagittatoside A, sagittatoside B, 2"-O-rhamnosyl icariside II and baohuoside I. All of them were further confirmed to be AGIs through in vitro inhibitory assay and molecular docking. Among those, baohuoside I and sagittatoside B possessed stronger inhibitory activity than acarbose. The approach has a significant prospect in conveniently screening bioactive compounds that target various receptors, which provided an efficient platform for new drug development from natural products. Unlabelled Image • Fe 4 O 3 @ α -glucosidase magnetic nanoparticles for ligand fishing were constructed. • The novel approach is promising to screen bioactive compounds from herbal products. • Eight flavonoids as α -glucosidase inhibitors were fished out from Epimedii Folium. • Baohuoside I and sagittatoside B exhibited higher inhibitory activity than acarbose. [ABSTRACT FROM AUTHOR]

Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n -BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC–Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens , and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC 50) values of 197.9 and 125.4 μmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin–Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 μmol/L. An efficient neuraminidase ligand fishing protocol was developed and verified to rapidly screen the neuraminidase inhibitors (NAIs) from natural sources. Twenty-four potential neuraminidase inhibitors were identified from Duchesnea indica as potential NAIs by HPLC–Q-TOF-MS. One compound can inhibit neuraminidase activities in both the oseltamivir sensitive and resistant virus strains. Image 1 • An efficient ligand fishing protocol was developed to rapidly screen the neuraminidase inhibitors from natural sources. • 24 potential neuraminidase inhibitors were identified from Duchesnea indica as potential NAIs by HPLC-Q-TOF-MS. • One compound can inhibit neuraminidase activities in both the oseltamivir sensitive and resistant virus strains. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an attractive target for new cholesterol-lowering drug development. Here, we developed a method integrating ligand fishing, HPLC-Q-TOF-MS and interdisciplinary assay, aiming to explore potential PCSK9 inhibitors from mixtures rapidly and accurately. PCSK9 was expressed and purified firstly, and then the recombined PCSK9 was coated on the surface of magnetic beads (MBs). The PCSK9-immobilized MBs (PCSK9-MBs) were used for ligand fishing combined with HPLC and Q-TOF-MS/MS. Ginkgo biloba leaves (GBL), an herbal medicine widely used in Asia and Europe with good efficacy in treatment of hypercholesterolemia, were chosen as an illustration for ligand fishing. Two PCSK9 ligands were discovered from GBL and identified as kaempferol- 3-O-rutinoside (1) and kaempferol 3-O-2''-(6'"-p-coumaroyl) glucosylrhamnoside (KCGR) (2). In order to verify fishing results and pick out more powerful PCSK9 inhibitors, molecular docking assay was further performed and KCGR was optimized to be an excellent PCSK9 inhibitor by the confirmation of affinity and activity bioassay. These results suggested that the developed approach could be applied to screen and analysis potential bioactive constituents from mixtures, which may improve the efficiency of drug discovery. Moreover, KCGR separated from GBLwas expected to be a potential candidate of PCSK9 inhibitors. [ABSTRACT FROM AUTHOR]

In terms of ligand fishing, the amount and the relative activity recovery of enzymes immobilized on magnetic particles and nanoparticles are not preeminent. Therefore, the metal-organic framework (MOF) UiO-66-NH 2 was synthesized to immobilize the porcine pancreatic lipase (PPL) via precipitation-cross-linking, and the resulting novel biological matrices named PPL@MOF manifested high PPL loading capacity (98.31 mg/g) and relative activity recovery (104.4%). Moreover, the novel enzyme-MOF composite was applied to screen lipase inhibitors from Prunella vulgaris L. to enrich and improve the techniques of ligand fishing. As a result, 13 lipase ligands were obtained, and 12 compounds were determined by HPLC-Q-TOF-MS/MS. All of these ligands were further confirmed to be potential inhibitors through the verification of the activity assay and molecular docking. The proposed approach based on PPL@MOF was superior in terms of abundant protein loading capacity, high enzyme catalytic activity and easy preparation process. Taken together, our newly developed method provided a new platform for efficient discovering bioactive molecules from natural herbs. Image 1 • Pancreatic lipase was successful immobilized onto the metal-organic framework via the precipitation-cross-linking method. • PPL@MOF manifested high PPL loading capacity (98.31 mg) and relative activity recovery (104.4%). • 13 compounds were harvested from Prunella vulgaris efficiently and conveniently by the method of ligand fishing based PPL@MOF. • MOF-enzyme composite was first used for obtaining bioactive compounds directly from natural herbs. [ABSTRACT FROM AUTHOR]

Multidrug resistance is recognized as one of the main reasons leading to the failure of chemotherapy. Studies have shown that glutathione S‐transferase inhibitors could be regarded as multidrug resistance reversal agents. Herein, a method of applying enzyme immobilization, molecular docking, and high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was employed to screen glutathione S‐transferase inhibitors from natural products. Magnetic mesoporous silica microspheres were synthesized and modified with a poly(dopamine) layer, which has a large quantity of amino, enabling further non‐covalent binding with glutathione S‐transferase. Moreover, the immobilization conditions, namely, potential of hydrogen, catalase concentration, reaction temperature and reaction time, were optimized. In total, six potential compounds were isolated and identified from Perilla frutescens (L.) Britt leaves and green tea and molecular docking was applied to identify the binding site. Rosmarinic acid, (−)‐epigallocatechin‐3‐O‐gallate and (−)‐epicatechin‐3‐gallate showed higher binding affinity than the compounds, and their half maximal inhibitory concentration values were further determined. The results suggested that this proposed method was effective and convenient for identifying glutathione S‐transferase inhibitors from natural products. [ABSTRACT FROM AUTHOR]