Screening Of Enzyme Inhibitors Research Articles

Liquid-capped encoded microcapsules for multiplex assays.

Although droplet microfludics is a promising technology for handling a number of liquids of a single type of analyte, it has limitations in handling thousands of different types of analytes for multiplex assay. Here, we present a novel "liquid-capped encoded microcapsule", which is applicable to various liquid format assays. Various liquid drops can be graphically encoded and arrayed without repeated dispensing processes, evaporation, and the risk of cross-contamination. Millions of nanoliter-scale liquids are encapsulated within encoded microcapsules and self-assembled in microwells in a single dispensing process. The graphical code on the microcapsule enables identification of randomly assembled microcapsules in each microwell. We conducted various liquid phase assays including enzyme inhibitor screening, virus transduction, and drug-induced apoptosis tests. The results showed that our liquid handling technology can be utilized widely for various solution phase assays.

Fabrication of enzyme reactor utilizing magnetic porous polymer membrane for screening D-Amino acid oxidase inhibitors

In this work, a unique D-amino acid oxidase reactor for enhanced enzymolysis efficiency is presented. A kind of magnetic polymer matrices, composed of iron oxide nanoparticles and porous polymer membrane (poly styrene-co-maleic anhydride), was prepared. With covalent bonding D-Amino acid oxidase on the surface of the matrices and characterization of scanning electron microscope and vibrating sample magnetometer, it demonstrated that the membrane enzyme reactor was successfully constructed. The enzymolysis efficiency of the enzyme reactor was evaluated and the apparent Michaelis-Menten constants of D-Amino acid oxidase were determined (Km was 1.10mM, Vmax was 23.8mMmin−1) by a chiral ligand exchange capillary electrophoresis protocol with methionine as the substrate. The results indicated that the enzyme reactor could exhibit good stability and excellent reusability. Importantly, because the enzyme and the substrate could be confined into the pores of the matrices, the enzyme reactor displayed the improved enzymolysis efficiency due to the confinement effect. Further, the prepared enzyme reactor was applied for D-Amino acid oxidase inhibitors screening. It has displayed that the proposed protocol could pave a new way for fabrication of novel porous polymer membrane based enzyme reactors to screen enzyme inhibitors.

Screening of enzyme inhibitors from traditional Chinese medicine by magnetic immobilized α-glucosidase coupled with capillary electrophoresis

As the close correlation between α-glucosidase inhibitors and the treatment of diabetes, in combination with capillary electrophoresis (CE), a method was developed to screen α-glucosidase inhibitors from traditional Chinese medicines (TCMs) by immobilizing α-glucosidase on magnetic nanoparticles. Such a magnetic immobilization would be beneficial for enzyme reusability, stability and separation. In this work, Fe3O4 nanoparticles were synthesized by solvothermal method. And the prepared nanoparticles were characterized by transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), size and zeta potential analysis. With the modification of chitosan (CS) and glutaraldehyde (GA), α-glucosidase was successfully immobilized on the magnetic nanoparticles. The pH and temperature endurance, storage stability and reusability of the immobilized α-glucosidase were studied and compared with those of the free one. With the magnetic immobilized α-glucosidase, the Michaelis-Menten constant (Km) was calculated to be 0.85mM, and the inhibition constant (Ki) and half-maximal inhibitory concentration (IC50) for acarbose were determined to be 7.37 and 13.69μM, respectively. Finally, the developed method was applied to screen α-glucosidase inhibitors from 18 TCMs.

Colorimetric Logic Gate for Pyrophosphate and Pyrophosphatase via Regulating the Catalytic Capability of Horseradish Peroxidase.

By regulating the catalytic capability of horseradish peroxidase (HRP), an artful colorimetric assay platform for pyrophosphate (PPi) and pyrophosphatase (PPase) was unprecedentedly designed. In this work, Cu(I), generated by reducing Cu(II) in the presence of ascorbate, could inhibit HRP's catalytic capability of transforming colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The robust coordination between PPi and Cu(II) is able to discourage the reduction of Cu(II) to Cu(I) effectively, thus restoring the original catalytic capability of HRP and regenerating blue-colored oxTMB. Upon PPase introduction, PPi would be hydrolyzed into orthophosphate, which could release Cu(II) free from the Cu(II)-PPi complex, and thus in turn allows the catalytic capability of HRP to be inhibited by Cu(I). HRP was activated or deactivated to different degrees depending on PPi or PPase levels, which could be indicated by using HRP-triggered catalytic system as a signal amplifier, thus paving a way for PPi and PPase sensing. Based on the colorimetric sensor for PPi and PPase, an "INH" logic gate was rationally constructed. With the merits of high sensitivity and selectivity, cost-effectiveness, and simplification, our proposed analytical system has also been verified to have potential to be utilized for enzyme inhibitor screening and diagnosis of PPase-related diseases.

DNA-linked Inhibitor Antibody Assay (DIANA) for sensitive and selective enzyme detection and inhibitor screening.

Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.

Online coupling solid-phase ligand-fishing with high-performance liquid chromatography-diode array detector-tandem mass spectrometry for rapid screening and identification of xanthine oxidase inhibitors in natural products.

Screening and analysis of bioactive compounds from natural products is challenging work due to their complexity. This study presents the first report on hyphenation of solid-phase ligand-fishing using immobilized xanthine oxidase microcolumn (IXOM) and high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) for screening and identification of XO inhibitors from complex mixtures. Solid-phase ligand-fishing system was hyphenated withthe HPLC system via four-port switching valve and a six-port injection valve as an interface for transferring effluents from IXOM to HPLC, and collecting chromatograms from LFMC (ligand-fishing microextraction column) and C18 column in a run by only one DAD. Mixtures containing allopurinol (positive control) and tryptophane (negative control) were analyzed in order to verify the specificity and reproducibilityof the approach. Subsequently, the newly developed system was applied to screening and identification of XO inhibitors from L. macranthoides and its human microsomal metabolites. Six prototype compounds (3-caffeoylquinic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid) and three metabolites (3-caffeoyl-epi-quinic acid, 5-caffeoyl-epi-quinic acid, 4-caffeoyl-epi-quinic acid) with XO binding affinities were identified. The XO inhibition activities of six prototype compounds were evaluated and confirmed using in vitro enzymatic assay. With the online system developed here, we present a feasible, selective, and effective strategy for rapid screening and identification of enzyme inhibitors from complex mixtures.

Protein-Labs on Separative Analytical Scale in Medicinal Chemistry: from the Proof of Concept to Applications

Coupling of a macromolecule such as an enzyme or a receptor to a chromatographic support has revealed a powerful analytical tool to face different aspects related to drug discovery. Chromatographic systems with immobilized macromolecules have been developed to be used for different applications in pharmaceutical analysis including enzyme inhibitor screening, biopharmaceutics structural analysis, chiral separations and binding studies. These areas of research are very challenging both for the technological and for the applicative aspects. This review offers an overview of general strategies and references that can be considered in order to develop innovative and reliable “protein-labs” for specific separative analytical applications of pharmaceutical interest. Keywords: Protein immobilization, bio-affinity chromatography, immobilized enzyme reactors, chiral stationary phases, drug binding, enantioseparation, inhibitor potency determination.

A simpler sampling interface of venturi easy ambient sonic-spray ionization mass spectrometry for high-throughput screening enzyme inhibitors

High-throughput screening (HTS) is often required in enzyme inhibitor drugs screening. Mass spectrometry (MS) provides a powerful method for high-throughput screening enzyme inhibitors because its high speed, sensitivity and property of lable free. However, most of the MS methods need complicated sampling interface system. Overall throughput was limited by sample loading in these cases. In this study, we develop a simple interface which coupled droplet segmented system to a venturi easy ambient sonic-spray ionization mass spectrometer. It is fabricated by using a single capillary to act as both sampling probe and the emitter, which simplifies the construction, reduces the cost and shorten the sampling time. Samples sucked by venturi effect are segmented to nanoliter plugs by air, then the plugs can be detected by MS directly. This system eliminated the need for flow injection which was popular used in classic scheme. The new system is applied to screen angiotensin converting enzyme inhibitors. High-throughput was achieved in analyzing 96 samples at 1.6 s per sample. The plugs formation was at 0.5s per sample. Carry-over between samples was less than 5%, the peak height RSD was 2.92% (n = 15). Dose-response curves of 3 known inhibitors were also measured to validate its potential in drug discovery. The calculated IC50 agreed well with reported values.

Screening of minor bioactive compounds from herbal medicines by in silico docking and the trace peak exposure methods

Screening of high potent enzyme inhibitors from herbal medicines is always lacking of efficiency due to the complexity of chemicals. The constituents responsible for the holistic effect may be trace-level chemicals, but these chemicals were covered by highly abundant compositions. To challenge this bottleneck, a strategy for screening minor bioactive compounds was proposed. It generally included four steps, (1) preliminarily find the enzyme binders by ultrafiltration; (2) optimise and predict the potential inhibitors by docking analysis; (3) selectively identify and prepare trace compounds by segment and exposure approach; (4) validate the activity and summarize the structure-activity relationship. As a case study, α-glucosidase (AGH) and Ginkgo biloba extract were used as the experimental materials. By comprehensive screening, 11 trace flavones were screened out and identified as strong AGH inhibitors. Among them, AGH inhibitory activities of syringetin and sciadopitysin were reported for the first time. Their IC50 values were 36.80 and 8.29μM, respectively, which were lower than that of a positive control acarbose. In addition, the AGH inhibitory activities of the flavonoids could be ranked, based on a decreased order, as biflavone, flavone, flavone glycoside, flavone biglycoside. The strategy is expected to be practical and useful for screening bioactive molecules from herbal medicines, especially for the minor compounds, which will definitely accelerate the discovery of new drug candidates.

False HDAC Inhibition by Aurone Compound.

Fluorescence assays are useful tools for estimating enzymatic activity. Their simplicity and manageability make them suitable for screening enzyme inhibitors in drug discovery studies. However, researchers need to pay attention to compounds that show auto-fluorescence and quench fluorescence, because such compounds lower the accuracy of the fluorescence assay systems by producing false-positive or negative results. In this study, we found that aurone compound 7, which has been reported as a histone deacetylase (HDAC) inhibitor, gave false-positive results. Although compound 7 was identified by an in vitro HDAC fluorescence assay, it did not show HDAC inhibitory activity in a cell-based assay, leading us to suspect its in vitro HDAC inhibitory activity. As a result of verification experiments, we found that compound 7 interferes with the HDAC fluorescence assay by quenching the HDAC fluorescence signal. Our findings underscore the faults of fluorescence assays and call attention to careless interpretation.

Peroxidase-like activity of the Co3O4 nanoparticles used for biodetection and evaluation of antioxidant behavior.

Nanostructured enzyme mimics are of great interest as promising alternatives to artificial enzymes for biomedical and catalytic applications. Studying the chemical interactions between antioxidants and nano-enzymes may result in a better understanding of the antioxidant capability of antioxidants and may help improve the function of artificial enzymes to better mimic natural enzymes. In this study, using Co3O4 nanoparticles (NPs) as peroxidase mimics to catalyze the oxidation of chromophoric substrates by H2O2, we developed a platform that acts as a biosensor for hydrogen peroxide and glucose and that can study the inhibitory effects of natural antioxidants on peroxidase mimics. This method can be applied specifically to glucose detection in real samples. Three natural antioxidants, gallic acid (GA), tannic acid (TA), and ascorbic acid (AA), were compared for their antioxidant capabilities. We found that these three antioxidants efficiently inhibit peroxidase-like activity with concentration dependence. The antioxidants showed different efficiencies, in the following order: tannic acid > gallic acid > ascorbic acid. They also showed distinct modes of inhibition based on different interaction mechanisms. This study serves as a proof-of-concept that nano-enzyme mimics can be used to evaluate antioxidant capabilities and to screen enzyme inhibitors.

Prussian blue nanoparticles encapsulated inside a metal–organic framework via in situ growth as promising peroxidase mimetics for enzyme inhibitor screening

Artificial enzyme mimics are of current research interest owing to their remarkable advantages over natural enzymes. Herein, as a novel peroxidase mimic material, MIL-101(Cr)@PB was fabricated by encapsulating Prussian blue (PB) nanoparticles into the host matrix of MIL-101(Cr) via a facile and mild in situ growth synthetic strategy. The crystallographic characteristics, morphology, and porosity of the as-synthesized MIL-101(Cr)@PB composites were carefully studied using XRD, SEM, TEM, TGA, and BET. The results show that the synthesized MIL-101(Cr)@PB possesses a reproducible and impressive intrinsic peroxidase-like activity even under extreme conditions. Exploiting this, a colorimetric platform for screening xanthine oxidase inhibitors was constructed. We hope that this work will elucidate the applications of metal-organic frameworks as carriers for enzyme mimics and enable a wider application in drug screening.

Application of Capillary Electrophoresis in Enzyme Inhibitors Screening

Abstract Capillary electrophoresis plays an irreplaceable role in the field of enzyme inhibitors screening with unique features of high-efficiency separation, short analysis time, minimal sample consumption, high sensitivity, ease to realize automatization etc. This review gives an overview of the applications of capillary electrophoresis in the field of enzyme inhibitors screening, and summarizes both the widely used off-line (pre-capillary) enzyme assay and on-line (in-capillary) enzyme assay. Finally, some future trends are briefly addressed.

Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures

Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography–mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96μM, respectively. The ligand–enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures.

Chemometrics Optimized Extraction Procedures, Phytosynergistic Blending and in vitro Screening of Natural Enzyme Inhibitors Amongst Leaves of Tulsi, Banyan and Jamun.

Background:Tulsi, Banyan, and Jamun are popular Indian medicinal plants with notable hypoglycemic potentials. Now the work reports chemo-profiling of the three species with in-vitro screening approach for natural enzyme inhibitors (NEIs) against enzymes pathogenic for type 2 diabetes. Further along with the chemometrics optimized extraction process technology, phyto-synergistic studies of the composite polyherbal blends have also been reported.Objective:Chemometrically optimized extraction procedures, ratios of polyherbal composites to achieve phyto-synergistic actions, and in-vitro screening of NEIs amongst leaves of Tulsi, Banyan, and Jamun.Materials and Methods:The extraction process parameters of the leaves of three plant species (Ficus benghalensis, Syzigium cumini and Ocimum sanctum) were optimized by rotatable central composite design of chemometrics so as to get maximal yield of bio-actives. Phyto-blends of three species were prepared so as to achieve synergistic antidiabetic and antioxidant potentials and the ratios were optimized by chemometrics. Next, for in vitro screening of natural enzyme inhibitors the individual leaf extracts as well as composite blends were subjected to assay procedures to see their inhibitory potentials against the enzymes pathogenic in type 2 diabetes. The antioxidant potentials were also estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay.Results:Considering response surface methodology studies and from the solutions obtained using desirability function, it was found that hydro-ethanolic or methanolic solvent ratio of 52.46 ± 1.6 and at a temperature of 20.17 ± 0.6 gave an optimum yield of polyphenols with minimal chlorophyll leaching. The species also showed the presence of glycosides, alkaloids, and saponins. Composites in the ratios of 1:1:1 and 1:1:2 gave synergistic effects in terms of polyphenol yield and anti-oxidant potentials. All composites (1:1:1, 1:2:1, 2:1:1, 1:1:2) showed synergistic anti-oxidant actions. Inhibitory activities against the targeted enzymes expressed in terms of IC50 values have shown that hydro-ethanolic extracts in all cases whether individual species or composites in varying ratios gave higher IC50 values thus showing greater effectivity.Conclusion:Current research provides the state-of-the-art of search of NEIs amongst three species by in-vitro assays which can be further utilized for bioactivity-guided isolations of such enzyme inhibitors. Further, it reports the optimized phyto-blend ratios so as to achieve synergistic anti-oxidative actions.SUMMARY The current research work focuses on the optimization of the extraction process parameters and the ratios of phyto-synergistic blends of the leaves of three common medicinal plants viz. banyan, jamun and tulsi by chemometrics. Qualitative and quantitative chemo profiling of the extracts were done by different phytochemical tests and UV spectrophotometric methods. Enzymes like alpha amylase, alpha glucosidase, aldose reductase, dipeptidyl peptidase 4, angiotensin converting enzymes are found to be pathogenic in type 2 diabetes. In vitro screening of natural enzyme inhibitors amongst individual extracts and composite blends were carried out by different assay procedures and the potency expressed in terms of IC50 values. Antioxidant potentials were estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay. Hydroalcoholic solvent (50:50) gave maximal yield of bio-actives with minimal chlorophyll leaching. Hydroethanolic extract of tulsi showed maximal antioxidant effect. Though all composites showed synergism, maximal effects were shown by the composite (1:1:2) in terms of polyphenol yield, antioxidant effect and inhibitory actions against the targeted enzymes. Abbreviations used: DPP4- dipeptidyl peptidase 4; AR- aldose reductase; ACE- angiotensin converting enzyme; PPAR-γ- peroxisome proliferator activated receptor-γ; NEIs- natural enzyme inhibitors; BE- binding energy; GLP-1- Glucagon like peptide -1; ROS- Reactive oxygen species; CAT- catalase; GSH-Px- glutathione per-oxidase; SOD- superoxide dismutase; pNPG- para-nitro phenyl-α-D-gluco-pyranoside solution; DPPH- 1,1-diphenyl-2-picrylhydrazyl; RSM- Response surface methodology; CCD- central composite design; DMSO- dimethyl sulfoxide; HHL- hippuryl-L-histidyl-L-leucine; GPN-Tos- Gly-Pro p-nitroanilide toluenesulfonate salt; ESC- experimental scavenging capacity; TSC- theoretical scavenging capacity; FRAP- Ferric Reducing Assay Procedure; ABTS- 2, 2’- azinobis (3-ethylbenzothiazoline-6 – sulfonic acid.

A strategy for screening of high-quality enzyme inhibitors from herbal medicines based on ultrafiltration LC-MS and in silico molecular docking.

A novel strategy of ultrafiltration LC-MS and in silico molecular docking was proposed to discover high-quality enzyme inhibitors from herbal medicines. Using this strategy, two compounds were predicted and finally demonstrated as potent xanthine oxidase inhibitors, whose in vitro IC50 values were lower than that of a positive control allopurinol.

Development of a capillary electrophoresis system with Mn(ii) complexes and β-cyclodextrin as the dual chiral selectors for enantioseparation of dansyl amino acids and its application in screening enzyme inhibitors

A novel CLE-CE system with Mn(ii)–[BMIm][l-Ala] complexes and β-CD as the dual chiral selectors based on the synergistic effect was successfully constructed for enantioseparation of Dns-d,l-AAs and applied in screening the tyrosinase inhibitors.

Poly β-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

A new chiral ligand exchange capillary electrophoresis system based on Zn(ii)–l-leucine complexes coordinating with β-cyclodextrin and its application in screening tyrosinase inhibitors

Tyrosinase plays a key role in melanin formation, and it is closely related to hyper pigmentation in animals and enzymatic browning in food. Thus, it is of great significance to screen inhibitors of tyrosinase. In this work, a new chiral ligand exchange-capillary electrophoresis (CLE-CE) system based on the coordination effect of Zn(II)–L-leucine complexes and β-cyclodextrin (β-CD) was developed for screening the inhibitors of tyrosinase. The effects of the concentration of β-CD, buffer pH, the ratio of L-leucine to Zn(II), and the complex concentration were investigated with Dns-D,L-tyrosine, Dns-D,L-valine and Dns-D,L-phenylalanine as the tested analytes. The optimum buffer conditions were composed of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II), 6.0 mM L-leucine and 4.0 mM β-CD at pH 8.2. It has been found that six pairs of Dns-D,L-AAs could be baseline-separated and five pairs of Dns-D,L-AAs were partly enantioseparated. Then the quantitative analysis of L-tyrosine was conducted and good linearity (r2 = 0.992) was obtained with a concentration ranging from 12.95 μM to 413.3 μM. A kinetics study of tyrosinase was realized, and the Km and Vmax were 636 μM and 312 μmol min−1 mg−1. Moreover, the proposed method was applied in screening the inhibitors of tyrosinase with four kinds of chalcones as the model inhibitors. The results demonstrated that the developed CLE-CE system was favorable for screening enzyme inhibitors, and showed great potential in related drugs discovery and clinical analysis in the future.

Rapid screening and detection of XOD inhibitors from S. tamariscina by ultrafiltration LC-PDA-ESI-MS combined with HPCCC.

Xanthine oxidase (XOD) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction of which could cause hyperuricemia, a risk factor for gout. Inhibition of XOD is a major treatment for gout, and biflavonoids have been found to act as XOD-inhibitory compounds. In this study, ultrafiltration liquid chromatography with photodiode-array detection coupled to electrospray-ionization tandem mass spectrometry (UF-LC-PDA-ESI-MS) was used to screen and identify XOD inhibitors from S. tamariscina. High-performance counter-current chromatography (HPCCC) was used to separate and isolate the active constituents of these XOD inhibitors. Furthermore, ultrahigh-performance liquid chromatography (UPLC) and triple-quadrupole mass spectrometry (TQ-MS) was used to determine the XOD-inhibitory activity of the obtained XOD inhibitors, and enzyme kinetics was performed with Lineweaver-Burk (LB) plots using xanthine as the substrate. As a result, two compounds in S. tamariscina were screened as XOD inhibitors: 65.31mg amentoflavone and 0.76mg robustaflavone were isolated from approximately 2.5g S. tamariscina by use of HPCCC. The purities of the two compounds obtained were over 98% and 95%, respectively, as determined by high-performance liquid chromatography (HPLC). Lineweaver-Burk plot analysis indicated that amentoflavone and robustaflavone were non-competitive inhibitors of XOD, and the IC 50 values of amentoflavone and robustaflavone for XOD inhibition were 16.26μgmL(-1) (30.22μmolL(-1)) and 11.98μgmL(-1) (22.27μmolL(-1)), respectively. The IC 50 value of allopurinol, used as the standard, was 7.49μgmL(-1) (46.23μmolL(-1)). The results reveal that the method for systematic screening, identification, and isolation of bioactive components in S. tamariscina and for detecting their inhibitory activity using ultrafiltration LC-ESI-MS, HPCCC, and UPLC-TQ-MS is feasible and efficient, and could be expected to extend to screening and separation of other enzyme inhibitors.