Screening Of Xanthine Oxidase Inhibitors Research Articles

Bioaffinity ultrafiltration combined with UPLC-ESI-QTrap-MS/MS for screening of xanthine oxidase inhibitors from Paederia foetida L. leaves

Paederia foetida L. leaf (PFL) is recognized for its uric acid-lowering. However, the key compounds and their mechanisms of countering hyperuricemia remain unclear. In this study, bioaffinity ultrafiltration in combination with UPLC-ESI-QTrap-MS/MS was used to screen and identify potential xanthine oxidase (XO) inhibitors in the purified PFL extract, and the possible mechanisms of their interaction were investigated by molecular docking. The experimental results showed that the purified PFL extract exhibited excellent antioxidant and anti-XO effect. Further the results of enzyme inhibition experiments in vitro showed that luteolin-7-O-glucoside (IC50 = 61.41 μg/mL), astragalin (IC50 = 114.89 μg/mL), quercitrin (IC50 = 124.50 μg/mL), dihydromyricetin (IC50 = 296.32 μg/mL), cyanidin-3-O-rutinoside (IC50 = 373.82 μg/mL), kaempferol-3-O-rutinoside (IC50 = 450.10 μg/mL) and (-)-epigallocatechin (IC50 = 610.48 μg/mL) in purified PFL extract were the major contributors to the XO inhibitory activity, and confirmed this finding. Molecular docking revealed these inhibitors interact with critical XO residues through hydrogen bonding, hydrophobic, and electrostatic interactions, highlighting flavonoids in purified PFL extract as potent XO inhibitors. This work provides insight into flavonoids in the purified PFL extract as potent XO inhibitors.

Screening of Xanthine Oxidase Inhibitors by Liquid Crystal-Based Assay Assisted with Enzyme Catalysis-Induced Aptamer Release.

Small-molecule drugs play an important role in the treatment of various diseases. The screening of enzyme inhibitors is one of the most important means in developing therapeutic drugs. Herein, we demonstrate a liquid crystal (LC)-based screening assay assisted with enzyme catalysis-induced aptamer release for screening xanthine oxidase (XOD) inhibitors. The oxidation of xanthine by XOD prevents the specific binding of xanthine and its aptamer, which induces a bright image of LCs. However, when XOD is inhibited, xanthine specifically binds to the aptamer. Correspondingly, LCs display a dark image. Three compounds are identified as potent XOD inhibitors by screening a small library of triazole derivatives using this method. Molecular docking verifies the occupation of the active site by the inhibitor, which also exhibits excellent biocompatibility to HEK293 cells and HeLa cells. This strategy takes advantages of the unique aptamer-target binding, specific enzymatic reaction, and simple LC-based screening assay, which allows high-throughput and label-free screening of inhibitors with high sensitivity and remarkable accuracy. Overall, this study provides a competent and promising approach to facilitate the screening of enzyme inhibitors using the LC-based assay assisted with the enzyme catalysis-induced aptamer release.

On-Flow LC-MS/MS method for screening of xanthine oxidase inhibitors

The screening of compounds is the initial step in research for the development of new drugs. For this reason, the availability of fast and reliable tools for the screening of a large number of compounds becomes essential. Among the therapeutic targets, the enzyme xanthine oxidase (XO) is of great interest for its importance as a biological source of superoxide radicals, which contribute to the oxidative stress on organisms and are involved in many pathological processes. In the present study, we validated a new method using an immobilized capillary enzyme reactor in an LC system directly coupled to triple quadrupole mass spectrometry to screen for XO ligands. The use of mass spectrometry provided selectivity and speed to the system, eliminating the analytical separation step. The Michaelis-Menten constant (KM) value determined for the immobilized enzyme was 14.5 ± 0.4 μmol L−1, which is consistent with the value previously reported for the XO-ICER with UV detection in a 2D LC method. The on-line approach was successfully applied to assay the XO inhibitory activities of thirty isolated compounds from different classes of natural products and provided greater productivity (288 analysis/day) than 2D LC method (84 analysis/day) of screened samples.

New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis.

Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side effects, thereby necessitating the identification of new inhibitors. During the screening of XO inhibitors from various mushroom extracts, we found that a methanolic extract of the fruiting bodies of Tyromyces fissilis, an inedible and non-toxic fungus, showed inhibitory activity. Both n-hexane and ethyl acetate layers, obtained by partitioning this extract exhibited XO inhibitory activity. Subsequently, using an activity-guided separation method, eight active compounds (1-8) were isolated. The structures of five of the new compounds, 2-4,6, and 7, were elucidated by spectral analysis and chemical derivatization. All compounds had a salicylic acid moiety with an aliphatic group at the C-6 position. Notably, 2-hydroxy-6-pentadecylbenzoic acid (1) showed the highest level of XO noncompetitive inhibition (58.9±2.2% at 25µM).

Screening the anti-gout traditional herbs from TCM using an in vitro method

The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation and xanthine oxidase (XOD) activity. The anti-inflammation effects of the herbal extracts were detected by an in vitro cell model, which was established by stimulating human umbilical vein endothelial cells (HUVEC) using sodium urate (MSU). In this model, the intercellular adhesion molecule-1 (ICAM-1) and interleukin-1 beta (IL-1β) were expressed, and the anti-inflammation effects of herbal extracts were evaluated by detecting the content changes of ICAM-1 and IL-1β in cell lysates and cell culture supernates using an enzyme-linked immunosorbent assay (ELISA). Moreover, an ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) method was used for the detection of XOD activity and the screening of XOD inhibitors in this research. The amount of uric acid from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid level could be reduced via the addition of an inhibitor. Results indicated that Salviae Miltiorrhizae Radix et Rhizome, Rhei Radix et Rhizoma, Polygoni Cuspidati Rhizoma et Radix, Selaginellae Herba, Paeoniae Radix Rubra, especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD activity. The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated with their bioactive components. And the differences between the herbal extracts were correlated with the amount of flavonoid and anthraquinone components. In our study, we have investigated the potential anti-inflammation bioactivity of 33 herbal extracts in vitro, which could provide a reference for further in vivo research in the prevention and treatment of gout.

Amperometric biosensor based on prussian blue and nafion modified screen-printed electrode for screening of potential xanthine oxidase inhibitors from medicinal plants

A simple and sensitive amperometric biosensor was developed for the screening of potential xanthine oxidase inhibitors from medicinal plants. This biosensor was prepared by immobilization of xanthine oxidase on the surface of prussian blue modified screen-printed electrodes using nafion and glutaraldehyde.The developed biosensor showed a linear amperometric response at an applied potential of +0.05V toward the detection of hypoxanthine from 5μM to 45μM with a detection limit of 0.4μM (S/N=3) and its sensitivity was found to be 600mAM−1cm−2. In addition, the biosensor exhibited a good storage stability.The inhibition of xanthine oxidase by allopurinol was studied under the optimized conditions. The linear range of allopurinol concentration is obtained up to 2.5μM with an estimated 50% of inhibitionI50=1.8μM.The developed biosensor was successfully applied to the screening of xanthine oxidase inhibitors from 13 medicinal plants belonging to different families. Indeed, Moroccan people traditionally use these plants as infusion for the treatment of gout and its related symptoms. For this purpose, water extracts obtained from the infusion of these plants were used for the experiments. In this work, 13 extracts were assayed and several of them demonstrated xanthine oxidase inhibitory effect, with an inhibition greater than 50% compared to spectrophotometry measurements that only few extracts showed an inhibition greater than 50%.

Practical method for the rapid screening of xanthine oxidase inhibitors in herbal extracts by high-performance liquid chromatography based on on-line precolumn enzymatic reaction.

A high-performance liquid chromatography method with on-line precolumn enzymatic reaction for the screening of xanthine oxidase inhibitors in natural extracts was developed. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, after which the reaction product, uric acid, was separated and detected by liquid chromatography using ultraviolet absorption at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of uric acid in comparison to a reference chromatogram obtained in the absence of any inhibitor. In the present study, the availability of on-line precolumn enzymatic reaction with ultraviolet detection was firstly evaluated by determining the inhibitory mechanism and IC50 values of allopurinol, a commercially available positive drug. Then, the newly developed method was applied to screening of ten natural extracts from traditional Chinese medicine and as a result, the extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim was found to be most positive for xanthine oxidase inhibition. The results obtained were compared with those obtained by offline enzyme assay and the effectiveness of the present method was confirmed. A rapid, low-cost, and fully automated method for xanthine oxidase inhibitor screening was proposed.

High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture

Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O2•−) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures.

Screening of xanthine oxidase inhibitors in complex mixtures using online HPLC coupled with postcolumn fluorescence‐based biochemical detection

Xanthine oxidase (XO) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction and/or underexcretion of which could cause the incidence of hyperuricemia such as gout. Herein, the inhibition of XO is recognized as one of the therapeutic approaches to treat gout. In the present study, an off-line fluorescence-based microplate method was first developed for an XO assay in which the enzyme converted pterin to its fluorescent metabolite isoxanthopterin. Then, a postcolumn continuous XO assay as a means of bioactivity assessment was coupled to HPLC separation to establish the online HPLC with diode array detection, biochemical detection, and MS/MS system for the screening of XO inhibitors. The availability of the online system was first tested with a positive drug, allopurinol, a well-known XO inhibitor, and subsequent analysis of Scutellaria baicalensis extract showed that two main bioactive compounds with XO inhibitory activities were observed, indicating that the developed online system was applicable to complex mixtures.

CE Method with Partial Filling Techniques for Screening of Xanthine Oxidase Inhibitor in Traditional Chinese Medicine

A combination of electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique was developed for screening of xanthine oxidase (XOD) inhibitors in substances used in traditional Chinese medicine (TCM). In order to achieve sufficient separation, a micellar electrokinetic chromatography (MEKC) method was employed for the separation. The enzyme activity was determined by the quantification of the peak area of the product, uric acid (UA), at 295 nm. Enzyme inhibition can be read out directly from the reduced peak area of UA in comparison to a reference electropherogram obtained in the absence of any inhibitor. The method was validated using a commercially available XOD inhibitor, 4-aminopyrazolo[3,4-d]pyrimidine, and the IC50 value was determined to be 29.90 ± 0.26 μM. Fifteen natural extracts from TCM were screened, and Cortex Phellodendri (the dried bark of Phellodendron chinense) extract was found to be positive for XOD inhibition.