Synthesis, Characterization and Crystal Structures of Schiff Base Copper Complexes with Urease Inhibitory Activity.

Urease inhibitors can inhibit the decomposition rate of urea, and decrease the air pollution caused by ammonia. In this paper, four new copper(II) complexes [CuL(ONO2)]n (1), [Cu2L2(µ1,3-N3)2] (2), [CuBrL] (3), and [CuClL] (4), where L = 5-bromo-2-(((2-methylamino)ethyl)imino)methyl)phenolate, have been synthesized and characterized. The complexes were characterized by elemental analyses, IR and UV-Vis spectroscopy, molar conductivity, and single crystal X-ray diffraction. X-ray analysis reveals that Cu atoms in complexes 1 and 2 are in square pyramidal coordination, and those in complexes 3 and 4 are in square planar coordination. The molecules of the complexes are linked through hydrogen bonds and π···π interactions. The inhibitory effects of the complexes on Jack bean urease were studied, which showed that the complexes have effective activity on urease.

Synthesis, Crystal Structures and Urease Inhibition of 4-Bromo-N'-(1-(pyridin-2-yl)ethylidene)benzohydrazide and Its Dinuclear Copper(II) Complex.

A new dinuclear copper(II) complex [Cu2(µ-Br)2L2]·0.5MeOH with the benzohydrazone ligand 4-bromo-N'-(1-(pyridin-2-yl)ethylidene)benzohydrazide (HL) has been synthesized and characterized by elemental analysis, IR and UV-Vis spectroscopic studies. Single crystal structures of the complex and the benzohydrazone compound were studied. The Cu atoms in the complex are coordinated by two benzohydrazone ligands and two Br bridging groups, forming square pyramidal coordination. The complex has good inhibitory activity on Jack bean urease, with IC50 value of 1.38 µmol·L-1.

Hybrid Multivalent Jack Bean α-Mannosidase Inhibitors: The First Example of Gold Nanoparticles Decorated with Deoxynojirimycin Inhitopes.

Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.

Switchable Biocatalytic Reactions Controlled by Interfacial pH Changes Produced by Orthogonal Biocatalytic Processes.

Enzymes immobilized on a nano-structured surface were used to switch the activity of one enzyme by a local pH change produced by another enzyme. Immobilized amyloglucosidase (AMG) and trypsin were studied as examples of the pH-dependent switchable "target enzymes." The reactions catalyzed by co-immobilized urease or esterase were increasing or decreasing the local pH, respectively, thus operating as "actuator enzymes." Both kinds of the enzymes, producing local pH changes and changing biocatalytic activity with the pH variation, were orthogonal in terms of the biocatalytic reactions; however, their operation was coupled with the local pH produced near the surface with the immobilized enzymes. The "target enzymes" (AMG and trypsin) were changed reversibly between the active and inactive states by applying input signals (urea or ester, substrates for the urease or esterase operating as the "actuator enzymes") and washing them out with a new portion of the background solution. The developed approach can potentially lead to switchable operation of several enzymes, while some of them are inhibited when the others are activated upon receiving external signals processed by the "actuator enzymes." More complex systems with branched biocatalytic cascades can be controlled by orthogonal biocatalytic reactions activating selected pathways and changing the final output.

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase.

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Drug-Free Enzyme-Based Bactericidal Nanomotors against Pathogenic Bacteria.

The low efficacy of current conventional treatments for bacterial infections increases mortality rates worldwide. To alleviate this global health problem, we propose drug-free enzyme-based nanomotors for the treatment of bacterial urinary-tract infections. We develop nanomotors consisting of mesoporous silica nanoparticles (MSNPs) that were functionalized with either urease (U-MSNPs), lysozyme (L-MSNPs), or urease and lysozyme (M-MSNPs), and use them against nonpathogenic planktonic Escherichia coli. U-MSNPs exhibited the highest bactericidal activity due to biocatalysis of urea into NaHCO3 and NH3, which also propels U-MSNPs. In addition, U-MSNPs in concentrations above 200 µg/mL were capable of successfully reducing 60% of the biofilm biomass of a uropathogenic E. coli strain. This study thus provides a proof-of-concept, demonstrating that enzyme-based nanomotors are capable of fighting infectious diseases. This approach could potentially be extended to other kinds of diseases by selecting appropriate biomolecules.

Kinetic and structural analysis of the inactivation of urease by mixed-ligand phosphine halide Ag(I) complexes.

Soft metal ions can inactivate urease, a Ni(II)-dependent enzyme whose hydrolytic activity has significant implications in agro-environmental science and human health. Kinetic and structural studies of the reaction of Canavalia ensiformis urease (JBU) and Sporosarcina pasteurii urease (SPU) with Ag(I) compounds of general formula [Ag(PEt3)X]4 (X = Cl, Br, I), and with the ionic species [Ag(PEt3)2]NO3, revealed the role of the Ag(I) ion and its ligands in modulating the metal-enzyme interaction. The activity of JBU is obliterated by the [Ag(PEt3)X]4 complexes, with IC50 values in the nanomolar range; the efficiency of the inhibition increases in the Cl- < Br- < I- order. The activity of JBU upon [Ag(PEt3)2]NO3 addition decreases to a plateau corresponding to ca. 60% of the original activity and decreases with time at a reduced rate. Synchrotron X-ray crystallography on single crystals obtained after the incubation of SPU with the Ag(I) complexes yielded high-resolution (1.63-1.97 Å) structures. The metal-protein adducts entail a dinuclear Ag(I) cluster bound to the conserved residues αCys322, αHis323, and αMet367, with a bridging cysteine thiolate atom, a weak Ag…Ag bond, and a quasi-linear Ag(I) coordination geometry. These observations suggest a mechanism that involves the initial substitution of the phosphine ligand, followed by a structural rearrangement to yield the dinuclear Ag(I) cluster. These findings indicate that urease, in addition to the active site dinuclear Ni(II) cluster, possesses a secondary metal binding site, located on the mobile flap domain, capable of recognizing pairs of soft metal ions and controlling catalysis.

Green synthesis of silver and gold nanoparticles using Crataegus oxyacantha extract and their urease inhibitory activities.

This study reports the green synthesis and urease inhibitory activities of Ag and Au nanoparticles (NPs) using Crataegus oxyacantha extract. The synthesized NPs were characterized by UV-visible, FT-IR spectroscopy, atomic force microscopy, and scanning electron microscopy. The obtained NPs were spherical in shape, and their size was around 85 nm. A strong correlation between the phytochemicals present in the extract and their capability for the synthesis of NPs was observed. Furthermore, the shape, size, stability, and bioactivity of the NPs were strongly influenced by the stabilizing phytochemicals. The experimental analysis suggested that these NPs have substantial stability in a diverse range of physiological conditions such as pH, salinity, and temperature. The NPs exhibited potent urease enzyme inhibitory activities with percent inhibition of 99.25 and IC50 value of 1.38 ± 0.3, comparable to the standard (thiourea percent inhibition, that is, 98.2% and IC50 value 5.3 ± 0.04). These results suggested that the proposed NPs could be used in the homeopathic and pharmaceutical industries for biomedical applications.

Hydrazine clubbed 1,3-thiazoles as potent urease inhibitors: design, synthesis and molecular docking studies.

Synthesis of a novel series of hydrazine clubbed 1,3-thiazoles (5a-m) has been described by reacting hydrazine-1-carbothioamides (3a-k) with α-chloro- or bromo-acetophenones (4a-d) in refluxing ethanol in good to excellent yields (65-86%). Structural confirmation was based upon spectroscopic techniques such as 1H-NMR, 13C-NMR, FT-IR and mass spectrometry. The biological application of these motifs has been demonstrated in terms of their strong urease inhibition activity. The results of in vitro study revealed that all the compounds are the potent inhibitors of urease. The IC50 (ranging in between 110 and 440 nM) values were higher as compared to that of standard, i.e., thiourea (IC50 = 490 ± 10 nM). The synthesized compounds were docked at the active sites of the Jack bean urease enzyme in order to explore the possible binding interactions of enzyme-ligand complexes; the results reinforced the in vitro biological activity results.

Probing sulphamethazine and sulphamethoxazole based Schiff bases as urease inhibitors; synthesis, characterization, molecular docking and ADME evaluation.

In the current study, a novel series of Schiff base derivatives of (E)-4-(benzylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (3a-3f) and (E)-4-(benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (3g-3q) were synthesize. The structures of synthetic compounds were elucidated by various spectroscopic techniques such as FTIR, NMR and spectrometric HRMS analysis. Synthetic derivatives were evaluated for their Jack Bean urease inhibitory activity using established in-vitro assay. It is worth mentioning here that most of our derivatives of both series displayed moderate to strong inhibitory activity, ranging between IC50 = 2.48 ± 0.78 µM and 35.63 ± 1.26 µM, as compared to standard thiourea (IC50 = 20.03 ± 2.03 µM). Further, structure activity relationship studies suggest that the presence of halogen at ortho and para positions on the aryl ring in (E)-4-(benzylideneamino)-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide derivatives and hydroxy and halogen in (E)-4-(benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide derivatives increased the urease inhibitory activity. Furthermore, molecular docking studies were carried out in order to investigate the binding mode of this class of compounds to urease. In order to evaluate drug likeness of compounds ADME evaluation was done, and the synthesized compounds were found to be non-toxic and present passive gastrointestinal absorption. The data suggests the synthesized sulphamethazine and sulphamethoxazole derivatives can serve as a novel scaffold to inhibit urease.

Flavonoid analogues as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME evaluation.

A series of novel flavonoid analogues were designed and synthesized. The aimed compounds for urease inhibitory activities were clearly superior to the control drug thiourea (more than 10 times). Among these compounds, L2 (IC50 = 1.343 µM) and L12 (IC50 = 1.207 µM) exhibited the most excellent urease inhibitory activity in vitro. The molecular dockings of L2, L12 and L22 into urease were performed to explore the binding modes and their structure-activity relationship. Furthermore, these aimed compounds showed good druggable properties.

Cross-linkage urease nanoparticles: a high-efficiency signal-generation tag for portable pH meter-based electrochemical immunoassay of lipocalin-2 protein diagnostics.

An innovative signal-transduction tag based on cross-linked urease nanoparticles (CLENP) was designed for the development of a pH meter-based immunoassay of lipocalin-2 (LCN2). The CLENP was synthesized with a typical desolvation method using ethanol as desolvation agent, followed by functionalization with polyaspartic acid. The carboxylated CLENP were used as the signal-generation tags for the labelling of secondary antibodies via the carbodiimide coupling. Upon target LCN2 introduction, a sandwich-type immune reaction was performed between capture antibody-coated plate and the labeled secondary antibody on the CLENP. The conjugated CLENP in the microplate hydrolyzed urea into ammonia (NH4+) and carbonate (CO32-), resulting in the pH change of solution, which was determined with a handheld pH meter. The pH variation was proportional to target concentration in the sample. By monitoring the pH variation of the urea solution, the level of LCN2 at a concentration as low as 5.2 pg mL-1 was evaluated. The pH meter-based electrochemical immunoassay can be utilized for mass production of miniaturized lab-on-a-chip devices with handheld pH meter, thereby opening new opportunities for protein diagnostics and biosecurity. Graphical abstract An innovative signal-transduction tag based on cross-linked urease nanoparticles was designed for high-efficiency immunoassay of lipocalin-2 with pH meter readout.

Inhibition of urease activity by different compounds provides insight into the modulation and association of bacterial nickel import and ureolysis.

The nickel-dependent urease enzyme is responsible for the hydrolysis of urea to ammonia and carbon dioxide. A number of bacteria produce urease (ureolytic bacteria) and are associated with various infectious diseases and ammonia emissions from agriculture. We report the first comprehensive comparison of the inhibition of urease activity by compounds analysed under the same conditions. Thus, 71 commercially available compounds were screened for their anti-ureolytic properties against both the ureolytic bacterium Klebsiella pneumoniae and purified jack bean urease. Of the tested compounds, 30 showed more than 25% inhibition of the ureolytic activity of Klebsiella pneumoniae or jack bean urease, and among these, carbon disulfide, N-phenylmaleimide, diethylenetriaminepentaacetic acid, sodium pyrrolidinedithiocarbamate, 1,2,4-butanetricarboxylic acid, tannic acid, and gallic acid have not previously been reported to possess anti-ureolytic properties. The diverse effects of metal ion chelators on ureolysis were investigated using a cellular nickel uptake assay. Ethylenediaminetetraacetic acid (EDTA) and dimethylglyoxime (DMG) clearly reduced the nickel import and ureolytic activity of cells, oxalic acid stimulated nickel import but reduced the ureolytic activity of cells, 1,2,4-butanetricarboxylic acid strongly stimulated nickel import and slightly increased the ureolytic activity of cells, while L-cysteine had no effect on nickel import but efficiently reduced the ureolytic activity of cells.

Synthesis, Biological Evaluation and Molecular Docking of Deferasirox and Substituted 1,2,4-Triazole Derivatives as Novel Potent Urease Inhibitors: Proposing Repositioning Candidate.

A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2-(2-hydroxyphenyl)-4H-benzo[e][1,3]oxazin-4-one. For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that of acetohydroxamic acid. Two compounds, bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate group), represented the most potent urease inhibitory activity with IC50 values of 1.268 and 3.254 µm, respectively. In silico docking studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the discovery of novel urease inhibitors.

Bioassay-guided isolation and identification of anti-ulcer ecdysteroids from the seeds of Sphenocentrum jollyanum Pierre (Menispermaceae).

Sphenocentrum jollyanum seeds (MeOH extract and n butanol fraction) exhibited urease inhibitory activity (IC50 40.0 ± 0.92, 28.6 ± 0.41). The Ethyl acetate (EtOAc) fraction gave significant antacid activity with an increase in the baseline pH value of 1.2 to 1.61 ± 0.00 and 1.53 ± 0.00 at 50 and 100 mg, respectively, compared to the antacid activity of sodium bicarbonate (1.53 ± 0.00, 1.47 ± 0.00). Five known ecdysteroid compounds isolated from S. jollyanum ethyl acetate and n butanol fractions are Pinnatasterone (1), Polypodine B (2), 20-hydroxyecdysone (3), 20, 26-dihydroxyecdysone, (4) and Atrotosterone A (5). The compounds' structures were determined using extensive 1D and 2D NMR experiments, and the molecular mass for each of the compounds was confirmed by FAB-MS. Compounds 1-5 were evaluated for their urease inhibitory and antacid activities. Fractions were active in comparison with the standard drug acetohydroxamic acid, and sodium bicarbonate, respectively. Compounds 2, 3 and 1 showed significant urease inhibitory activity (IC50 7.0 ± 0.56, 13.8 ± 0.49 and 14.1 ± 0.59), respectively. The activity of compounds 4 and 5 were moderate compared to that of acetohydroxamic acid (IC50 value 20.3 ± 0.43). Very few compounds have been isolated from this plant despite the numerous biological activities reported for it. The antacid and urease inhibitory activities of this plant and isolated compounds are described for the first time.

Robust therapeutic potential of carbazole-triazine hybrids as a new class of urease inhibitors: A distinctive combination of nitrogen-containing heterocycles.

The inhibition of urease enzyme is very important as it plays a key role in the treatment of several urinary and gastrointestinal tract infections. This enzyme provides a suitable environment for Helicobacter pylori at the low pH of the stomach, a causative agent of gastric and peptic ulcer that may lead to cancer. In agriculture, the high urease content causes environmental and economic problems. In this pursuit, given the well-established importance of integrated pharmacophores in medicinal chemistry and to explore new inhibitors of urease featuring two distinct heterocyclic functionalities, we herein report a facile synthesis of carbazole-triazine hybrids (3a-j). These new propeller-shaped chemical scaffolds were evaluated for their urease inhibitory potential in order to identify suitable leads. The initial structure-activity survey work guided through in vitro bioactivity results recognized 3e and 3f as new starting point hits incorporating bulky iodo (3e) and strong electron-withdrawing nitro (3f) groups at the para-position of aryl amine component. The potent compounds (3e &3f) exhibited the highest activity with IC50 values of 5.6 and 6.7 µM, respectively. In the molecular docking analysis, these compounds depicted excellent binding interactions with the active site residues. The key interactions observed include hydrogen bonding, π-π interactions, π-cation and nickel atom coordination to the triazine nitrogen of both inhibitors.

Enzyme Inhibitory Kinetics and Molecular Docking Studies of Halo-Substituted Mixed Ester/Amide-Based Derivatives as Jack Bean Urease Inhibitors.

A series of halo-substituted mixed ester/amide-based analogues 4a-l have been prepared as jack bean urease inhibitor, which showed good to excellent inhibition of enzyme activity. The role of halo-substituted benzoyl moieties and alkyl substituted anilines in urease inhibitory kinetics was also investigated. The alkyl-substituted anilines 1a-b reacted with chloroacetyl chloride to afford intermediates 2a-b, which were then reacted with different halo-substituted benzoic acids 3a-f to prepare the title compounds 4a-l. The chemical structures of final products 4a-l were ascertained by FTIR, 1H NMR, 13C NMR, and mass spectra. The compound 4b showed remarkable activity with IC501.6 ± 0.2 nM, better than the standard thiourea having IC50472.1 ± 135.1 nM. The 2-chloro-substituted phenyl ring on one side of compound 4b and 4-isopropyl-substituted benzene on the other side play an essential role in inhibition of urease activity. Lineweaver-Burk plots (kinetics study) indicated about 4b derivative as a mixed type of inhibitor. The virtual screening performed against urease enzyme (PDBID 4H9M) showed that compounds 4b and 4e have binding energies of -7.8 and -7.9 Kcal/mol, respectively. Based upon our results, it was found that derivative 4b is a highly potent urease inhibitor, better than the standard thiourea.

4-Aminocoumarin based Aroylthioureas as Potential Jack Bean Urease Inhibitors; Synthesis, Enzyme Inhibitory Kinetics and Docking Studies.

BACKGROUND: Urease enzyme catalyzes the hydrolysis of urea into ammonia and CO2, excess ammonia causes global warming and crop reduction. Ureases are also responsible for certain human diseases such as stomach cancer, peptic ulceration, pyelonephritis, and kidney stones. New urease inhibitors are developed to get rid of such problems. OBJECTIVE: This article describes the synthesis of a series of novel 1-aroyl-3-(2-oxo-2H-chromen-4- yl)thiourea derivatives (5a-j) as Jack bean urease inhibitors. METHODS: Freshly prepared aryl isothiocyanates were reacted with 4-aminocoumarin in the same pot in an anhydrous medium of acetone. The structures of the title thioureas (5a-j) were ascertained by their spectroscopic data. The inhibitory effects against jack bean urease were determined. RESULTS: It was found that compounds 5i and 5j showed excellent activity with IC50 values 0.0065 and 0.0293, µM respectively. Compound 5i bearing 4-methyl substituted phenyl ring plays a vital role in enzyme inhibitory activity. The kinetic mechanism analyzed by Lineweavere-Burk plots revealed that compound 5i inhibits the enzyme non-competitively. The Michaelis-Menten constant Km and inhibition constants Ki calculated from Lineweavere-Burk plots for compound 5i are 4.155mM and 0.00032µM, respectively. The antioxidant activity results displayed that compound 5j showed excellent radical scavenging activity. The cytotoxic effects determined against brine shrimp assay showed that all of the synthesized compounds are non-toxic to shrimp larvae. Molecular docking studies were performed against target protein (PDBID 4H9M) and it was determined that most of the synthesized compounds exhibited good binding affinity with the target protein. Molecular dynamics simulation (MDS) results revealed that compound 5i forms a stable complex with target protein showing little fluctuation. CONCLUSIONS: Based upon our investigations, it is proposed that 5i derivative may serve as a lead structure for devising more potent urease inhibitors.

Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Insight into the inhibitory effects of Zanthoxylum nitidum against Helicobacter pylori urease and jack bean urease: Kinetics and mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum nitidum (Roxb.) DC. is a traditional Chinese medicine characterised by anti-inflammatory and anti-Helicobacter pylori, which is widely used to treat H. pylori-induced gastric disease in China. However, the underlying mechanism related to its anti-H. pylori activity remains unclear. Urease plays a crucial role in the colonisation and survival of H. pylori. AIM OF THE STUDY: The root aqueous extract of Z. nitidum against H. pylori urease (HPU) and jack bean urease (JBU) was investigated to illuminate the inhibitory potency, kinetics and potential mechanism. MATERIALS AND METHODS: Z. nitidum components were determined by UPLC. The enzyme inhibitory effects of Z. nitidum were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. Urease inhibition kinetics were determined by Lineweaver-Burk plots. Sulfhydryl group reagents and Ni2+-binding inhibitors were used in the mechanism study. Moreover, the molecular docking technique was used to investigate the binding conformations of the main compounds of Z. nitidum on Urease. RESULTS: According to UPLC results, the major components of Z. nitidum were magnoflorine, sanguinarine, nitidine chloride, chelerythrine, skimmianine and L-Sesamin. Z. nitidum has higher enzyme inhibitory activity on HPU (IC50 = 1.29 ± 0.10 mg/mL) than on JBU (IC50 = 2.04 ± 0.27 mg/mL). Enzyme inhibitory kinetic analysis revealed that the type of Z. nitidum inhibition against HPU was a slow-binding and mixed-type, whereas a slow-binding and non-competitive type inhibited JBU. Further mechanism study indicated that the active site of sulfhydryl group might be the target of inhibition by Z. nitidum. The molecular docking study indicated that the above six main components of Z. nitidum exhibited stronger affinity to HPU than to JBU through interacting with the key amino acid residues located on the mobile flap or interacting with the active site Ni2+. Results indicated that these components are potential active ingredients directed against urease. CONCLUSIONS: Z. nitidum inactivated urease in a concentration-dependent manner through slow-binding inhibition and binding to the urease active site sulfhydryl group. Our investigation might provide experimental evidence for the traditional application of Z. nitidum in the treatment of H. pylori-associated gastric disorders.