Design and synthesis of novel nitrothiazolacetamide conjugated to different thioquinazolinone derivatives as anti-urease agents.

The present article describes the design, synthesis, in vitro urease inhibition, and in silico molecular docking studies of a novel series of nitrothiazolacetamide conjugated to different thioquinazolinones. Fourteen nitrothiazolacetamide bearing thioquinazolinones derivatives (8a-n) were synthesized through the reaction of isatoic anhydride with different amine, followed by reaction with carbon disulfide and KOH in ethanol. The intermediates were then converted into final products by treating them with 2-chloro-N-(5-nitrothiazol-2-yl)acetamide in DMF. All derivatives were then characterized through different spectroscopic techniques (1H, 13C-NMR, MS, and FTIR). In vitro screening of these molecules against urease demonstrated the potent urease inhibitory potential of derivatives with IC50 values ranging between 2.22 ± 0.09 and 8.43 ± 0.61 µM when compared with the standard thiourea (IC50 = 22.50 ± 0.44 µM). Compound 8h as the most potent derivative exhibited an uncompetitive inhibition pattern against urease in the kinetic study. The high anti-ureolytic activity of 8h was confirmed against two urease-positive microorganisms. According to molecular docking study, 8h exhibited several hydrophobic interactions with Lys10, Leu11, Met44, Ala47, Ala85, Phe87, and Pro88 residues plus two hydrogen bound interactions with Thr86. According to the in silico assessment, the ADME-Toxicity and drug-likeness profile of synthesized compounds were in the acceptable range.

Effect of High Hydrostatic Pressure Treatment on Urease Activity and Inhibition of Fishy Smell in Mackerel (Scomber japonicus) during Storage.

In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel (Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.

Soil urease inhibition by various plant extracts.

Urea is the most popular and widely used nitrogenous fertilizer. High soil urease activity rapidly hydrolyses applied urea to ammonia which contributes to soil nitrogen (N) losses and reduces N use efficiency of crop plants. The ammonia losses can be minimized by the inhibition of soil urease activity which has been explored using various potential chemical inhibitors. However, the soil urease activity inhibition potential of plant extracts is rarely explored to date. In the present study, extracts of 35 plant materials were taken and evaluated against jack bean urease. Eleven extracts, showing >50% jack bean urease inhibition, were selected and further investigated in 13 soils collected from various districts of Punjab, Pakistan. Interestingly, except Capsicum annum, Melia azedarach, Citrus reticulata and Quercus infectoria, the plant extracts showed urease inhibition activities in soils, the extent of which was lower as compared to that observed in jack bean urease though. Maximum urea hydrolysis inhibition (70%) was noted with Vachellia nilotica which was 40% more than that of hydroquinone (50%) followed by that of Eucalyptus camaldulensis (24%). The extracts of V. nilotica and E. camaldulensis were coated on urea and applied to soil in the next step. At 21st day, 239% and 116% more urea-N was recovered from soil treated with V. nilotica and E. camaldulensis extracts coated urea, respectively, as compared to uncoated urea. Conclusively, these results indicated that the coating of V. nilotica and E. camaldulensis extracts on urea prills prolonged urea persistence in soil owing to minimum urea hydrolysis, probably, the extracts of V. nilotica and E. camaldulensis showed their urease inhibition potential. The results of this study provide a base line for the identification of new soil urease inhibitor compounds from plant materials in future.

Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus.

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.

Phytochemical Fingerprints and Bioactivities of Ripe Disseminules (Fruit-Seeds) of Seventeen Gundelia (Kenger-Kereng Dikeni) Species from Anatolia with Chemometric Approach.

Gundelia species are known as "Kenger-kereng dikeni" in Anatolia, and their aerial parts are consumed as food. Also, roots and seeds (disseminules) of the Gundelia species are used to prepare gum and coffee. The chemical contents of ethanol and hexane extracts of disseminules of 17 Gundelia species, 13 of them are endemic, were studied using LC/MS/MS and GC/MS. Additionally, their antioxidant potential and enzyme inhibitory capacity against acetyl- and butyryl-cholinesterase, urease, and tyrosinase were determined. The unsaturated fatty acid ratios of Gundelia species were higher than their saturated fatty acid ratio. The highest sum of oleic and linoleic acid was detected in G. tournefortii var. tenuisecta (70.42 %). ß-Sitosterol, α-amyrin, 3-acetyllupeol were identified in 17 Gundelia species by GC/MS, while chlorogenic acid and luteolin by LC/MS/MS as major compounds. The ethanol and hexane extracts of G. siirtica, G. rosea, and G. mesopotamica indicated good cholinesterase inhibitory activity. Among all species, ethanol extract of G. colemerikensis exhibited the best activity in ABTS (IC50 : 32.30±0.98 µg/mL), DPPH (IC50 : 59.91±0.89 µg/mL), and CUPRAC (A0.5 : 57.41±1.03 µg/mL) assays. Ethanol extract of G. colemerikensis also displayed the highest inhibitory activity against butyrylcholinesterase (51.14±0.25 % at 200 µg/mL), urease (51.71±1.75 % at 200 µg/mL), and tyrosinase (39.50±0.85 % at 200 µg/mL) enzymes. According to the chemometric analysis of fatty acids, four groups were observed. Therefore, it is suggested that G. colemerikensis can be used in the pharmaceutical, food, and cosmetic industries due to its antioxidant and enzyme inhibition properties.

New isolate from Salvinia molesta with antioxidant and urease inhibitory activity.

Urease plays a significant role in the pathogenesis of urolithiasis pyelonephritis, urinary catheter encrustation, hepatic coma, hepatic encephalopathy, and peptic acid duodenal ulcers. Salvinia molesta was explored to identify new bioactive compounds with particular emphasis on urease inhibitors. The aqueous methanol extract was fractionated using solvents of increasing polarity. A series of column chromatography and later HPLC were performed on butanol extract. The structures of the resulting pure compounds were resolved using NMR (1D and 2D), infrared, and mass spectroscopy. The novel isolate was evaluated for antioxidant activity (using DPPH, superoxide anion radical scavenging, oxidative burst, and Fe+2 chelation assays), anti-glycation behavior, anticancer activity, carbonic anhydrase inhibition, phosphodiesterase inhibition, and urease inhibition. One new glucopyranose derivative 6'-O-(3,4-dihydroxybenzoyl)-4'-O-(4-hydroxybenzoyl)-α/ß-D-glucopyranoside (1) and four known glycosides were identified. Glycoside 1 demonstrated promising antioxidant potential with IC50 values of 48.2 ± 0.3, 60.3 ± 0.6, and 42.1 ± 1.8 µM against DPPH, superoxide radical, and oxidative burst, respectively. Its IC50 in the Jack bean urease inhibition assay was 99.1 ± 0.8 µM. The mechanism-based kinetic studies presented that compound 1 is a mixed-type inhibitor of urease with a Ki value of 91.8 ± 0.1 µM. Finally, molecular dynamic simulations exploring the binding mode of compound 1 with urease provided quantitative agreement between estimated binding free energies and the experimental results. The studies corroborate the use of compound 1 as a lead for QSAR studies as an antioxidant and urease inhibitor. Moreover, it needs to be further evaluated through the animal model, that is, in vivo or tissue culture-based ex-vivo studies, to establish their therapeutic potential against oxidative stress phosphodiesterase-II and urease-induced pathologies.

Crotoliganfuran, a new clerodane-type furano-diterpenoid from Croton oligandrus Pierre ex Hutch.

The phytochemical investigation of the methanol extract of the bark of Croton oligandrus Pierre ex Hutch yielded a new clerodane-type diterpenoid crotoliganfuran (1) along with ten other compounds including 12-epicrotocorylifuran (2), lupeol (3), syringic acid (4), aleuritolic acid acetate (5), aleuritolic acid (6), scopoletin (7), geddic acid (8), ß-sitosterol (9), vanilic acid (10) and stigmastane-3,6-dione (11). Their structures were established by spectroscopic means. The extract and all the isolates were screened for their inhibitory properties against butyrylcholinesterase and urease enzymes, respectively. The extract and compounds 1, 4 and 7 displayed the most potent urease inhibitory properties with IC50 values, 22.2, 26.7 and 28.5 µM, respectively. Compound 9 was the most active of all the tested compounds against butyrylcholinesterase enzyme with an IC50 value of 36.3 µM.[Formula: see text].

Phenolic, flavonoid content and radical scavenging activity of Smilax china with its inhibitory potential against clinically important enzymes.

Smilax china (SC) is a medicinal plant that has been traditionally used for a number of pathological disorders. In current study, its various fractions were assessed for radical scavenging, phenolic, flavonoid content and enzyme inhibition. The methanolic extract (MSC) of SC was subjected to fractionation using different solvents including n-hexane, benzene, chloroform, ethyl acetate and n-butanol. Results revealed that ethyl acetate fraction showed maximum phenolic (101.81 ± 0.13 mg GAE/g) and flavonoid (96.80 ± 0.39 mg QE/g) content with maximum radical scavenging potential (82.51 ± 0.18%, IC50=104.45 µg/ml) as well as urease (82.63 ± 0.79%), tyrosinase (81.30 ± 0.41%) and Butyrylcholinesterase (BChE) (62.47 ± 0.76%) inhibition at 0.5 mg/ml. Whereas, maximum α-Glucosidase (87.56 ± 0.13%) and Acetylcholinesterase (AChE) inhibition (82.34 ± 0.64%) was exhibited by n-hexane and benzene fractions, respectively. Present study has revealed the promising radical scavenging, phenolic, flavonoid and enzyme inhibitory potential of various fractions of SC extract. Thus, the study is a step forward towards evidence-based phyto-medicine.

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.

A Novel Urease Inhibitor of Ruminal Microbiota Screened through Molecular Docking.

Inhibition of the urease activity of ruminal microbiota is not only beneficial for increasing dietary and endogenic urea-N utilization efficiency in ruminants but also might be applicable for the preservation of nitrogen fertilizer in soil and treatment of gastrointestinal and urinary tract infections caused by ureolytic bacteria. To discover urease inhibitors to efficiently target ruminal microbiota, the identified ruminal microbial metagenomic urease gene was used to construct a homology model to virtually screen urease inhibitors from the ChemDiv database by molecular docking. The GMQE and QMEAN values of the homology model were 0.85 and -0.37, respectively, indicating a good model quality. The inhibition effect of the screened urease inhibitor for ruminal urea degradation was assessed by ruminal microbial fermentation in vitro. The toxic effect of the candidate inhibitor was performed using gut Caco-2 cells in vitro. The results showed that compound 3-[1-[(aminocarbonyl)amino]-5-(4-methoxyphenyl)-1H-pyrrol-2-yl] propanoic acid (ChemDiv_ID: 6238-0047, IC50 = 65.86 µM) was found to be the most effective urease inhibitor among the candidate compounds. Compound 6238-0047 significantly lowered the amount of urea degradation and ammonia production in ruminal microbial fermentation. The 24 h degradation rate of compound 6238-0047 in ruminal microbial fermentation was 3.32%-16.00%. In addition, compound 6238-0047 (10-100 µM) had no significant adverse effect on the cell viability of Caco-2 cells. Molecular docking showed that compound 6238-0047 could interact with Asp359 in the active site and Cys318 in the flap region by the hydrogen bond and Pi-Alkyl interaction, respectively. Compound 6238-0047 could be used as a novel inhibitor for decreasing the urease activity of ruminal microbiota.

Targeting the Protein Tunnels of the Urease Accessory Complex: A Theoretical Investigation.

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein-protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein-protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

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.

The In Vitro Activity of Essential Oils against Helicobacter Pylori Growth and Urease Activity.

The anti-H. pylori properties of 26 different commercial essential oils were examined in vitro by MIC (minimal inhibitory concentration) determination for the reference strain H. pylori ATCC 43504. We selected 9 essential oils with different anti-Helicobacter activities and established their phytochemical composition and urease inhibition activities. Phytochemical analysis of the selected essential oils by GC-MS method and antioxidant activity were performed. The phenol red method was used to screen the effect of essential oils on urease activity expressed as IC50 (the half of maximal inhibitory concentration). The most active essential oils, with MIC = 15.6 mg/L, were thyme, lemongrass, cedarwood and lemon balm oils; MIC = 31.3 mg/L-oregano oil; MIC = 62.5 mg/L-tea tree oil; MIC = 125 mg/L-pine needle, lemon and silver fir oils with bactericidal effect. Urease activity was inhibited by these oils with IC50 ranged from 5.3 to > 1049.9 mg/L. The most active was cedarwood oil (IC50 = 5.3 mg/L), inhibiting urease at sub-MIC concentrations (MIC = 15.6 mg/L). The statistical principal component analysis allowed for the division of the oils into three phytochemical groups differing in their anti-H. pylori activity. To summarize, the activity in vitro of the five essential oils silver fir, pine needle, tea tree, lemongrass, and cedarwood oils against H. pylori was found in this paper for the first time. The most active against clinical strains of H. pylori were cedar wood and oregano oils. Moreover, cedarwood oil inhibited the urease activity at subinhibitory concentrations. This essential oil can be regarded as a useful component of the plant preparations supporting the eradication H. pylori therapy.

Urease inhibitory potential of extracts and active phytochemicals of Hypochaeris radicata (Asteraceae).

Urease inhibition potential of compound (1), guaiane-type sesquiterpene (2), confertin (3) and scopoletin (4) was carried out with high throughout mechanism-based assay. These compounds were isolated from Hypochaeris radicata L., an Asteraceae family member. The pure compounds were screened for their urease and carbonic anhydrase inhibitory activities. The ethyl acetate fractions were subjected to column chromatography, which resulted in the isolation and purification of four compounds (1-4). On evaluation, compounds (1-4) exhibited selective activity against urease enzyme with an IC50 value of 180.11 ± 2.00, 27.18 ± 0.80, 24.12 ± 0.2 and 30.12 ± 1.10 µM respectively. The compounds (1-4) were found to be inactive against carbonic anhydrase enzyme. Thiourea was used as standard inhibitor (21 ± 0.14 µM) of urease enzyme.

Chemical analysis of the semipurified extract of Paullinia cupana and evaluation of in vitro inhibitory effects against Helicobacter pylori.

Paullinia cupana Kunth, commonly known as guarana, is a native Brazilian plant species from the Amazon area that presents various biological effects, including antimicrobial action. The aim of this study was to chemically analyse the semipurified aqueous extract (AqF) of the plant and to evaluate the activity of crude (CE), ethyl-acetate (EAF), and AqF extracts against Helicobacter pylori. The chemical profile of AqF was determined based on solid analysis 13C-NMR, direct infusion mass spectrometry (ESI-MS), and MALDI-TOF. The 13C-NMR spectrum showed characteristics of flavan-3-ol and oligomeric proanthocyanidins. ESI-MS revealed the presence of procyanidin, caffeic acid and its derivatives. MALDI-TOF analysis detected procyanidins of up to 6 units and profisetinidins of up to 5 units. Whereas CE and EAF showed inhibitory activity against H. pylori, CE, EAF, and AqF presented not high inhibitory activity against urease. The results demonstrate the potential of P. cupana to control and prevent H. pylori infection.

Phytochemical profiling, antioxidant, enzyme inhibition and cytotoxic potential of Bougainvillea glabra flowers.

In this study, phytochemical composition, antioxidant, enzyme inhibition and cytotoxic activities of methanol and dichloromethane (DCM) extracts of Bougainvillea glabra (B. glabra) flowers were investigated. Methanol extract was found to have higher total bioactive contents and UHPLC-MS analysis of methanol extract revealed the presence of well-known phenolic and flavonoid compounds. Antioxidant activities were performed by radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC), phosphomolybdenum (TAC) and metal chelating assays. From our result, we observed that methanol extract had many antioxidant compounds. The DCM extract exhibited higher cholinesterases and α-glucosidase enzyme inhibition, while methanol extract showed significant urease inhibition. Both extracts exhibited strong to moderate cytotoxicity against MCF-7, MDA-MB-231, CaSki, DU-145 and SW-480 cancer cells with IC50 values ranging from 88.49 to 304.7 µg/mL. The findings showed the B. glabra to possess considerable antioxidant, enzyme inhibition and cytotoxic potentials and therefore has potential to discover novel bioactive molecules.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

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.

Effect of patchouli alcohol on macrophage mediated Helicobacter pylori digestion based on intracellular urease inhibition.

BACKGROUND: Helicobacter pylori infects almost half of the world population and is listed as a type I carcinoma factor since 1994. Pogostemon cablin (Blanco) Benth. (Labiatae) has been used to treat gastro-intestinal diseases for thousands of years in many east Asian countries, and the key ingredient, patchouli alcohol (PA), has been observed to exert anti-H. pylori and anti-urease activities. PURPOSE: We investigated the effect of PA on H. pylori urease and its subsequent influence on macrophage phagosome maturation and function. METHODS: In H. pylori experiment, the berthelot method and pH shock assay were adopted to evaluate the effect of PA on extracellular and intracellular H. pylori urease. And then, Q-PCR and Western blot were carried out to analyze the alterations in the expression of urease-related genes and proteins after PA treatment. In the H. pylori and macrophage cell (RAW264.7) co-culture experiment, the effects of PA on H. pylori-induced phagocytosis and intracellular killing of RAW264.7 were investigated using gentamycin protection assay, and the underlying mechanism was explored by immunofluorescence. RESULTS: PA at 25 and 50 µM inhibited intracellular H. pylori urease activity but not isolated urease by down-regulating the gene expression levels of ureB, ureE, ureI and nixA and reducing the protein expression level of UreB, thereby inhibiting the acid resistance of H. pylori. PA also recovered the function of macrophage bacterial digestion, and prior treatment with ammonium chloride inhibited the efficacy of PA. CONCLUSION: PA suppressed intracellular H. pylori urease function and maturation, which increased macrophage digestion ability.

In Silico and In Vitro Anti-Helicobacter Pylori Effects of Combinations of Phytochemicals and Antibiotics.

Helicobacter pylori infection is a WHO class 1 carcinogenic factor of gastric adenocarcinoma. In the past decades, many studies have demonstrated the increasing trend of antibiotic resistance and pointed out the necessity of new effective treatment. This study was aimed at identifying phytochemicals that can inhibit H. pylori and possibly serve as adjuvant treatments. Here, in silico molecular docking and drug-like properties analyses were performed to identify potential inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase. These three enzymes are targets of the treatment of H. pylori. Susceptibility and synergistic testing were performed on the selected phytochemicals and the positive control antibiotic, amoxicillin. The in-silico study revealed that oroxindin, rosmarinic acid and verbascoside are inhibitors of urease, shikimate kinase and aspartate-semialdehyde dehydrogenase, respectively, in which, oroxindin has the highest potency against H. pylori, indicated by a minimum inhibitory concentration (MIC) value of 50 µg/mL. A combination of oroxindin and amoxicillin demonstrated additive effects against H. pylori, as indicated by a fractional inhibitory concentration (FIC) value of 0.75. This study identified phytochemicals that deserve further investigation for the development of adjuvant therapeutic agents to current antibiotics against H. pylori.