Proteolytic activity in bovine milk xanthine oxidase preparations.

Five glycosyl-transferases have been found present in purified hepatocyte nuclei of the rat (mannosyl-, galactosyl-, N-acetyl-glucosaminyl-, N-acetyl-galactosaminyl- and sialyl-transferases); these are capable of fixing specific carbohydrates on to endogenous or exogenous protein acceptors.     

Differential in vitro inhibition studies of some cerium vanadate derivatives on xanthine oxidase

Abstract

In this preliminary study, a new series of some cerium vanadate derivatives have been investigated as new type of inhibitors of xanthine oxidase (XO; E.C 1.17.3.2). XO is a superoxide-producing enzyme found normally in serum and the lungs, and its activity is concerned with several important health problems such as gout, severe liver damage, vascular dysfunction and injury, oxidative eye injury and renal failure. In this study, we present a critical overview of the effects of these novel type agents on XO with comparing the efficacy and safety profiles of allopurinol, the efficient classical inhibitor of XO.     

Antioxidant,anti‐inflammatory,and enzyme inhibitory activity of natural plant flavonoids and their synthesized derivatives

The synthesized flavonoid derivatives were examined for their antioxidant, anti‐inflammatory, xanthine oxidase (XO), urease inhibitory activity, and cytotoxicity. Except few, all the flavonoids under this study showed significant antioxidant activity (45.6%–85.5%, 32.6%–70.6%, and 24.9%–65.5% inhibition by DPPH, ferric reducing/antioxidant power, and oxygen radical absorption capacity assays) with promising TNF‐α inhibitory activity (42%–73% at 10 μM) and IL‐6 inhibitory activity (54%–81% at 10 μM) compared with that of control dexamethasone. The flavonoids luteolin, apigenin, diosmetin, chrysin, O^3?, O⁷‐dihexyl diosmetin, O^4?, O⁷‐dihexyl apigenin, and O⁷‐hexyl chrysin, showed an inhibition with IC₅₀ values (4.5‐8.1 μg/mL), more than allopurinol (8.5 μg/mL) at 5 μM against XO and showing more than 50% inhibition at a final concentration (5 mM) with an IC₅₀ value of ranging from 4.8 to 7.2 (μg/mL) in comparison with the positive control thiourea (5.8 μg/mL) for urease inhibition. Thus, the flavonoid derivatives may be considered as potential antioxidant and antigout agents.     

Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza

In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in β-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC₅₀ of 29.8 μg/mL and 4.06 μg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.     

拐枣中总黄酮提取工艺优化及其抗黄嘌呤氧化酶活性研究

为了研究拐枣总黄酮(TF)最佳提取工艺及其体外抗黄嘌呤氧化酶(xanthine oxidase,XO)活性。在单因素试验基础上,实验选择料液比、提取时间和乙醇浓度为自变量,应用Box-Benhnken中心组合法进行3因素3水平试验设计,以拐枣总黄酮得率为响应值,进行响应面分析,并采用D101大孔树脂对总黄酮进行进一步富集纯化,在此基础上研究拐枣总黄酮体外抗XO活性。结果表明,拐枣总黄酮的最佳提取条件为料液比15.0 mL/g、提取时间1.85 h、乙醇浓度53.0%,在此条件下,拐枣总黄酮的平均提取率可达到2.56%。体外活性结果表明,我们首次发现拐枣总黄酮具有显著的抗XO活性,其IC_(50 )为18.63±1.67μg/mL。本研究可为从拐枣中寻找和发现天然抗XO活性分子提供参考,同时可为拐枣的开发利用提供思路。     

齿孔酸对高尿酸血症黄嘌呤氧化酶活性的影响

采用紫外分光光度法检测齿孔酸在体外对黄嘌呤氧化酶的作用,并进行动力学研究探讨其作用机制;采用酵母联合氧嗪酸钾诱导高尿酸血症小鼠模型,观察齿孔酸对高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性及血糖血脂的影响。研究发现,齿孔酸体在外能抑制黄嘌呤氧化酶活性,降低高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性,同时明显降低空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平,升高高密度脂蛋白胆固醇水平,提高口服糖耐受量。结果表明,齿孔酸是黄嘌呤氧化酶竞争性抑制剂,还能缓解高尿酸血症小鼠糖脂代谢紊乱,对高尿酸血症及痛风的防治具有潜在意义。     

齿孔酸对高尿酸血症黄嘌呤氧化酶活性的影响

采用紫外分光光度法检测齿孔酸在体外对黄嘌呤氧化酶的作用,并进行动力学研究探讨其作用机制;采用酵母联合氧嗪酸钾诱导高尿酸血症小鼠模型,观察齿孔酸对高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性及血糖血脂的影响。研究发现,齿孔酸体在外能抑制黄嘌呤氧化酶活性,降低高尿酸血症小鼠血清尿酸水平、血清黄嘌呤氧化酶活性、肝脏黄嘌呤氧化酶活性,同时明显降低空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白胆固醇水平,升高高密度脂蛋白胆固醇水平,提高口服糖耐受量。结果表明,齿孔酸是黄嘌呤氧化酶竞争性抑制剂,还能缓解高尿酸血症小鼠糖脂代谢紊乱,对高尿酸血症及痛风的防治具有潜在意义。     

Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.     

Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase

The aim of this study was to evaluate new natural inhibitor sources for the enzymes urease and xanthine oxidase (XO). Chestnut, oak and polyfloral honey extracts were used to determine inhibition effects of both enzymes. In addition to investigate inhibition, the antioxidant capacities of these honeys were determined using total phenolic content (TPC), ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity assays. Due to their high phenolic content, chestnut and oak honeys are found to be a powerful source for inhibition of both enzymes. Especially, oak honeys were efficient for urease inhibition with 0.012–0.021?g/mL IC₅₀ values, and also chestnut honeys were powerful for XO inhibition with 0.028–0.039?g/mL IC₅₀ values. Regular daily consumption of these honeys can prevent gastric ulcers deriving from Helicobacter pylori and pathological disorders mediated by reactive oxygen species.     

Catalytic activity and stability of xanthine oxidase in aqueous-organic mixtures

In the present study, bovine milk xanthine oxidase activity in various aqueous-organic mixtures and the effects of pH, temperature, and lyophilization on the enzyme activity have been investigated. The enzyme was incubated with xan-thine as the substrate in Sorenson’s phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and the activity was determined spectrophotometrically in the absence and presence of different fractions of nine water-miscible organic solvents at 27–50°C and at different pH values ranging from 6 to 9. The organic solvents reduced the enzyme activity to different extents. In spite of these inhibitory effects, the enzyme showed relatively good stability in the aqueous-organic mixtures compared with the aqueous medium. A significant increase in the activity of the lyophilized enzyme was observed in pure organic solvents. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 124–130.     

Kinetic study on the inhibition of xanthine oxidase by acylated derivatives of flavonoids synthesised enzymatically

Studies have reported that flavonoids inhibit xanthine oxidase (XO) activity; however, poor solubility and stability in lipophilic media limit their bioavailability and applications. This study evaluated the kinetic parameters of XO inhibition and partition coefficients of flavonoid esters biosynthesised from hesperidin, naringin, and rutin via enzymatic acylation with hexanoic, octanoic, decanoic, lauric, and oleic acids catalysed by Candida antarctica lipase B (CALB). Quantitative determination by ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS) showed higher conversion yields (%) for naringin and rutin esters using acyl donors with 8C and 10C. Rutin decanoate had higher partition coefficients (0.95), and naringin octanoate and naringin decanoate showed greater inhibitory effects on XO (IC₅₀ of 110.35 and 117.51?μM, respectively). Kinetic analysis showed significant differences (p?K_m values, whereas the values for V_max were the same, implying the competitive nature of XO inhibition.     

The molybdoenzymes xanthine oxidase and aldehyde oxidase contain fast- and slow-DTNB reacting sulphydryl groups

The reactivities with an excess of 5-5-dithiobis (2-nitrobenzoic) acid (DTNB) of sulphydryl residues present in xanthine oxidase and aldehyde oxidase were studied and compared. The results show that two classes of sulphydryl groups with quite different reactivities exist in both enzymes either native or denatured. Some of the available sulphydryl residues thus react instantaneously with the DTNB, whereas the others react very slowly following pseudo-first-order kinetics. The number of sulphydryl residues of each class and the rate constant of slowly reacting groups are, respectively, 1.7 and 0.8 in native xanthine oxidase and 1.6 and 1.7 in native aldehyde oxidase. In denatured enzymes, the number of fast- and slow-reacting sulphydryl residues obtained are, respectively, 13.9 and 7.9 in xanthine oxidase and 5.7 and 5.4 in aldehyde oxidase. Analogously, the rate constant for the slowly reacting groups is similar for the two native enzymes, but in denatured aldehyde oxidase it is double that of denatured xanthine oxidase.     

Tryptophan-containing milk protein-derived dipeptides inhibit xanthine oxidase

Of twelve dipeptides tested, only the Trp containing peptides Val-Trp and its reverse peptide Trp-Val showed a xanthine oxidase (XO) inhibitory activity. Studies with Val and Trp revealed that XO inhibition was mainly attributed to the Trp residue. No significant difference (P ≥ 0.05) was found for the XO inhibitory potency (IC₅₀) values for Trp, Val-Trp and Trp-Val, which were about 200 times higher than that for Allopurinol. Lineweaver and Burke analysis demonstrated that Trp, Val-Trp and Trp-Val were non-competitive inhibitors while Allopurinol was a competitive inhibitor. Of the different milk-protein substrates hydrolyzed with gastro-intestinal enzyme activities, only lactoferrin (LF) hydrolyzates displayed XO inhibition. Peptides present in a LF hydrolyzate (GLF-240 min) were adsorbed onto activated carbon followed by subsequent desorption with stepwise elution using acetonitrile (ACN). Separation and detection of Trp containing peptides within the different fractions were achieved using RP-HPLC coupled with fluorescence detection. The desorbed fractions displayed different XO inhibitory properties, with no inhibition in the unbound fraction and highest inhibition in fractions eluted with 30, 40 and 70% ACN. The fraction eluting at 40% ACN was significantly more potent (19.1 ± 2.3% inhibition at 1.25 mg mL⁻¹) than the GLF-240 min hydrolyzate (13.4 ± 0.4% inhibition at 1.25 mg mL⁻¹), showing the potential for enrichment of the bioactive peptides on fractionation with activated carbon.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

Liver aldehyde oxidase and xanthine oxidase genetics in the mouse

A 'null' activity variant for the major liver isozyme of aldehyde oxidase (AOX-1) in adult male mice and an electrophoretically distinct, high activity variant of the second liver isozyme (AOX-2) were used to examine the segregation of the genetic loci encoding these enzymes (Aox-1 and Aox-2 respectively) in breeding studies. A single recombinant between these loci was observed among the 147 backcross progeny examined, which confirms a previous report (Holmes, 1979) for close linkage and genetic distinctness of the two loci. An activity variant for mouse liver xanthine oxidase (XOX) is also reported which behaved as though controlled by codominant alleles at a single locus (designated Xox-1 ). Genetic analyses showed that the Xox-1 locus segregated independently of the multiple- A ox loci.     

细菌黄嘌呤氧化酶的稳定性

研究pH、温度、金属离子和一些添加剂对黄嘌呤氧化酶稳定性的影响。结果表明：黄嘌呤氧化酶在pH4．5～7．5的范围内较稳定;反应最适温度为37℃。在常温25～35℃该酶比较稳定,经45℃处理2h．可保持50％左右,不同种类、不同浓度的金属离子对黄嘌呤氧化酶活性表现出程度不同的激活或抑制作用;添加谷氨酸和天门冬氨酸,能有效提高黄嘌呤氧化酶的存放稳定性.     

Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae

Results on the screening of species of the lichen family Graphidaceae for superoxide-scavenging activity (SSA) and xanthine-oxidase inhibitory (IXO) activity have been presented. The potential of the extracts for scavenging of superoxide and inhibition of xanthine-oxidase under various physiological conditions has been evaluated. The methanolic extracts of the species of family Graphidaceae showed inhibitory properties of xanthine oxidase (IC50 = 2.0 to 5.26 microg/ml) with an additional superoxide scavenging capacity (IC50 = 3.63 to 13.88 microg/ml). The potential of the methanolic extracts for scavenging of superoxide and inhibition of xanthine oxidase remained stable at 4 degrees C. Thus the extracts can be maintained for longer periods for their therapeutic uses.     

Insights into the binding of ferulic acid to the thermally treated xanthine oxidase

Ferulic acid (FA) is a biologically active compound used as an additive in the food industry, and possesses a wide range of therapeutic effects for treating different health problems. The interaction between FA and bovine xanthine oxidase (XOD) has been investigated by means of fluorescence spectroscopy methods. The numbers of binding sites and the binding constants were estimated at various temperatures and the results indicated the existence of one specific FA binding site of XOD. Detailed information on the interaction between molecules gathered after performing in silico molecular docking indicated the accommodation of the FA molecule in a XOD binding pocket, in close vicinity to the active site residues. The formation of the XOD–FA complex causes the quenching of protein fluorescence. The process followed a static mechanism at lower temperatures, and a dynamic mechanism at higher temperatures. The thermodynamic parameters calculated on the basis of different temperatures revealed that the association between FA and XOD is a spontaneous process driven by enthalpy and dominated by hydrogen bonding and van der Waals interaction. The results of synchronous fluorescence and 3D fluorescence spectra showed that the conformation of protein was altered in the presence of FA. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of crypto- and neochlorogenic lactones as potent xanthine oxidase inhibitors in roasted coffee beans

Xanthine oxidase (XO) inhibitory activity has been found in boiling water extracts from roasted coffee beans. Therefore, assay-guided purification of the extracts was performed using size-exclusion column chromatography, and subsequently with reversed phase HPLC to afford lactone derivatives of chlorogenic acids. Among the tested lactones, crypto- and neochlorogenic lactones showed potent XO inhibitory activities compared with three major chlorogenic acids found in coffee beans. These XO inhibitory lactones may ameliorate gout and hyperuricemia in humans who drink coffee.     

In-vitro evaluation of selected chalcones for antioxidant activity

Synthetic chalcones (SCs) having different side chains on the 1-(2-Hydroxy-3-(2-hydroxy-cyclohexyl)-4,6 dimethoxy-phenyl(-methanone structure were examined in-vitro for their antioxidant abilities by DPPH (2,2-diphenyl-1-picryl hydrazine) radical scavenging activity, reducing ability, OH radical scavenging activity, inhibition of polyphenol oxidase (PPO) and formation of diene conjugates. Overall, with few exceptions, all the SCs showed moderate biological activity in all the parameters examined. The SCs were found to be reactive towards DPPH radical and had considerable reducing ability. With few exceptions, all the test compounds under study were found to possess moderate to poor OH radical scavenging activity and inhibited PPO significantly and all were found to be effective inhibitors of hydroperoxide formation. These findings suggest that these SCs can be considered as potential antioxidant agents which might be further explored for the design of lead antioxidant drug candidates.