黄嘌呤氧化酶抑制剂研究进展及发展前景

别嘌呤醇是治疗高尿酸血症及痛风的主要药物之一,可是该药物不但会引发皮肤炎症,还引其发热和多器官受损,甚至有引起死亡的报道。其安全性受到质疑。天然产物来源的黄嘌呤氧化酶抑制剂毒性小,安全性高,不易引起过敏反应且来源广,有的甚至可以用于日常饮食。本文对黄嘌呤氧化酶具有抑制作用的天然产物的研究进展及发展前景进行综述。     

马鲛鱼黄嘌呤氧化酶抑制肽的制备 工艺优化及抗氧化活性研究

目的 探究马鲛鱼黄嘌呤氧化酶抑制肽制备的最佳条件及其抗氧化活性。方法 以马鲛鱼为原料,黄嘌呤氧化酶抑制肽采用超声辅助酶法制备,并筛选最佳反应用酶;以单因素实验为基础,通过Box-Behnken响应面分析法,以黄嘌呤氧化酶抑制活性为响应值,优化得到酶解最优条件。通过1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除等方法测定膜分离物的抗氧化活性;肌肽和鹅肌肽的含量测定采用了高效液相色谱法。结果 最佳反应用酶为复配的中性蛋白酶与复合蛋白酶,总加酶量0.3%（中性蛋白酶∶复合蛋白酶=1:1, m:m）。优化所得的最佳酶解条件为：酶解温度50℃,加酶量为0.2%,pH为7.0。在此条件下黄嘌呤氧化酶抑制率37.49%,与回归理论模型基本符合。3个组分的膜分离物中,相对分子量小于1 kDa的酶解产物的抗氧化性能力最强。马鲛鱼中鹅肌肽的含量为1.558 mg/g,而肌肽含量则为0.10 mg/g。结论 马鲛鱼制备及工艺优化所得的黄嘌呤氧化酶抑制肽对黄嘌呤氧化酶有较好的抑制作用,为工业化制备马鲛鱼黄嘌呤氧化酶抑制肽提供了数据基础和理论依据,肌肽和鹅肌肽的含量测定为分离纯化马鲛鱼黄嘌呤氧化酶抑制肽试验奠定了基础。。     

超声辅助酶解制备大米蛋白黄嘌呤氧化酶活性抑制肽的工艺条件优化

摘 要: 本文以大米蛋白为原料,研究了碱性蛋白酶酶解法制备黄嘌呤氧化酶（Xanthine Oxidase,XOD）活性抑制肽,并对其体外功能活性进行评价。研究了蛋白酶种类、底物浓度、酶解温度、加酶量、酶解pH、酶解时间对大米蛋白水解度（degree of hydrolysis,DH）及酶解产物对黄嘌呤氧化酶活性抑制率的影响,探讨了不同超声处理方式对酶解过程的影响,通过响应面法对实验条件进行了优化结果如下：采用碱性蛋白酶,底物浓度5 %,酶解温度为56 ℃,加酶量为8000 U/g,酶解pH为10,超声功率70 W,超声酶解60 min,对所制备的大米蛋白黄嘌呤氧化酶活性抑制肽的功能活性进行了评价。在最优条件下其黄嘌呤氧化酶抑制活性的IC50为1.55 mg/mL, ABTS自由基清除作用、羟基自由基清除作用、DPPH自由基清除作用的IC50值分别为0.49、12.48、3.88 mg/mL。     

Inhibition of xanthine oxidase activity by an oxathiolanone derivative of quercetin

An oxathiolanone derivative of rutin could be produced in the stomach after the ingestion of rutin containing foods, and the oxathiolanone derivative could be hydrolysed to an oxathiolanone derivative of quercetin (quercetin-oxathiolanone) in the intestine. Quercetin-oxathiolanone as well as quercetin inhibited xanthine oxidase. Approximately 0.05 μM quercetin-oxathiolanone inhibited the activity by 50%, whereas 50% inhibition by quercetin was observed at approximately 0.4 μM. The results suggested that quercetin-oxathiolanone can be used as an effective inhibitor of xanthine oxidase and that the ingestion of rutin-rich foods may be useful to prevent the increase in the blood concentration of uric acid.     

青占鱼黄嘌呤氧化酶抑制肽制备工艺优化

目的 本研究以青占鱼为原料,对黄嘌呤氧化酶(xanthine oxidase,XOD)抑制肽的制备条件进行优化。方法 选用五种蛋白酶对其水解制备黄嘌呤氧化酶抑制肽,测定XOD抑制率和水解度并用以评价效果,通过单因素和响应面分析相结合的实验方法优化酶解条件,最后对其分子量分布情况进行了检测分析。结果 结果表明复配酶制剂为最适酶,最佳酶解工艺为：加酶量4000U/g,酶解时间4h,酶解温度55℃,pH 7.0,料液比1:2;同时测得最优工艺下XOD抑制率和水解度的实际值分别为65.21%和18.5%,与理论值基本无差别。采用该法制备的青占鱼XOD抑制肽分子量分布情况为：5500~3000Da的肽段占比56.44%,3000~1500Da的肽段占比31.07%,＜1500Da的肽段占比12.49%。结论 本研究可为青占鱼制备XOD抑制肽的生产工艺与机理研究提供一定的技术参考与理论支持。     

Antioxidant activity of anacardic acids

Anacardic acids, 6-pentadec(en)ylsalicylic acids isolated from the cashew Anacardium occidentale L. (Anacardiaceae) nut and apple, were found to possess preventive antioxidant activity while salicylic acid did not show this activity. These anacardic acids prevent generation of superoxide radicals by inhibiting xanthine oxidase (EC 1.1.3.22, Grade IV) without radical-scavenging activity. Notably, the inhibition kinetics of anacardic acids do not follow hyperbolic dependence of enzyme inhibition on inhibitor contents (Michaelis–Menten equation) but follow the Hill equation instead. Anacardic acid (C_15:1) inhibited the soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) catalyzed oxidation of linoleic acid with an IC₅₀ of 6.8 μM. The inhibition is a slow and reversible reaction without residual enzyme activity. The inhibition kinetics indicate that anacardic acid (C_15:1) is a competitive inhibitor and the inhibition constant, K_I, was 2.8 μM. Anacardic acids act as antioxidants in a variety ways, including inhibition of various prooxidant enzymes involved in the production of the reactive oxygen species and chelate divalent metal ions such as Fe²⁺ or Cu²⁺, but do not quench reactive oxygen species. The C₁₅-alkenyl side chain is largely associated with the activity.     

Effect of luteolin on xanthine oxidase: Inhibition kinetics and interaction mechanism merging with docking simulation

Xanthine oxidase (XO) catalyses hypoxanthine and xanthine to uric acid in human metabolism. Overproduction of uric acid will lead to hyperuricemia and finally cause gout and other diseases. Luteolin is one of the major components of celery and green peppers, its inhibitory activity on XO and their interaction mechanism were evaluated by multispectroscopic methods, coupled with molecular simulation. It was found that luteolin reversibly inhibited XO in a competitive manner with inhibition constant (K_i) value of (2.38 ± 0.05) × 10⁻⁶ mol l⁻¹. Luteolin could bind to XO at a single binding site and the binding was driven mainly by hydrophobic interactions. Analysis of synchronous fluorescence and circular dichroism spectra demonstrated that the microenvironment and secondary structure of XO were altered upon interaction with luteolin. The molecular docking results revealed luteolin actually interacted with the primary amino acid residues located within the active site pocket of XO.     

Antioxidative and xanthine oxidase inhibitory activities and phytochemical screening of the hydro-alcoholic extract of mace,aril of Myristica fragrans: Implication as an adjuvant therapy in gout

Myristica fragrans derivatives are used as spices in cuisines and also serve as an important source of traditional medicine worldwide. The present study has evaluated the antioxidative potential of the hydro-methanolic extract of the fruit aril of M. fragrans, which is also referred to as mace. This study also includes a quantitative phytochemical assessment of the extract and focuses on the ability of the extract to inhibit xanthine oxidase, a molecular target involved in nucleic acid metabolism and the enzyme responsible for the development of gout disease. The extract was found to contain different classes of secondary metabolites, including flavonoids, phenolics, tannins, alkaloids, and saponins. Furthermore, the antioxidative potential of the extract was evaluated against the radicals 2,2-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt and 2,2-diphenyl-1-pycrylhydrazyl, and there was a progressive reduction of the radicals with increasing doses of the extract. Moreover, a ferric reducing power assay also showed the antioxidant activity of the crude extract as compared to ascorbic acid, a well-known antioxidant. Another significant finding of the study is that the mace extract also considerably inhibits the enzyme xanthine oxidase in a concentration-dependent manner. Our results partially support the use of mace in dietary regimens as a nutraceutical that could serve as a prophylactic strategy or an adjuvant for the prevention and therapy of gout disease.     

Electrochemical biosensor based on gold coated iron nanoparticles/chitosan composite bound xanthine oxidase for detection of xanthine in fish meat

A xanthine oxidase was immobilized covalently onto chitosan bound gold coated iron nanoparticles (CHIT/Fe-NPs@Au) electrodeposited on the surface of pencil graphite electrode (PGE). A xanthine biosensor was fabricated using XOD/CHIT/Fe-NPs@Au/PGE as working, Ag/AgCl as reference and Pt as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The biosensor exhibited optimum current response within 3 s at pH 7.4, 35 °C and working range 0.1–300 μM, when polarized at 0.5 V vs Ag/AgCl. The sensitivity of the biosensor was 0.001169 mAμ M^–1 cm^–2 with detection limit of 0.1 μM (S/N = 3). The biosensor showed only 25% loss in its initial activity after its 100 uses over 100 days, when stored at 4 °C.     

槲皮素、芦丁、没食子酸抑制黄嘌呤氧化酶的活性及动力学特性

为了解槲皮素、芦丁、没食子酸及其与维生素C联用对黄嘌呤氧化酶(XOD)的抑制特性,本文通过建立黄嘌呤氧化酶体外抑制模型,以别嘌呤醇为阳性对照,测定这三种天然化合物在不同浓度下对XOD的抑制效果及抑制动力学。结果表明：三种物质对黄嘌呤氧化酶均有抑制作用,且具有浓度依赖性,其抑制效果与结构和本身性质有明显相关性,抑制作用强弱次序为槲皮素>芦丁>没食子酸,半抑制浓度IC50值分别为(5.51±0.41) μmol/L、(48.66±0.49) μmol/L、>200 μmol/L;单一没食子酸抑制效果较弱,而没食子酸在维生素C的协同下,对XOD的抑制率从20.17%提高至40.96%,起到提高抑制效果和体系稳定性的双重作用;以上三种物质对XOD的抑制类型属于不同模式的非经典型混合抑制剂。本文对研究此类物质的抑制机理和应用有重要意义。     

Improvement in the remaining activity of freeze-dried xanthine oxidase with the addition of a disaccharide–polymer mixture

In order to improve the remaining activity of a practically important freeze-dried enzyme, xanthine oxidase (XOD), the effects of disaccharide (sucrose and trehalose), polymer (bovine serum albumin: BSA and dextran) and a mixture of them on the loss of XOD activity during freeze-drying and subsequent storage were investigated. All samples were amorphous solids and their glass transition temperatures (T_g) were evaluated by using differential scanning calorimetry. Although dextran showed no stabilizing effect on the freeze-dried XOD, the others protected XOD from the activity loss during freeze-drying to a certain extent. It was found that the mixture of disaccharide (sucrose or trehalose) and BSA improved the XOD activity synergistically. The XOD activity of the samples decreased gradually during storage at a temperature range of between 25 and 60 °C. Samples stored at temperatures below the T_g showed a lower loss of XOD activity than those stored at just the T_g.     

Antioxidant and Xanthine Oxidase Inhibitory Activities of Persicaria hydropiper

Five different polarity fractions of methanolic extract from Persicaria hydropiper, which are consumed as vegetables, were evaluated for its total phenolic content, and antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl radical scavenging, ferric thiocyanate, and xanthine oxidase inhibition assays. Particularly, higher phenolic content was exhibited by butanol and ethyl acetate fractions with the values of 224.38 and 68.95 mg GAE/100 g dry extract, respectively. Both butanol and ethyl acetate fractions exhibited higher 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with IC₅₀ values of 28.61 and 25.55 μg/ml. Meanwhile, both fractions also were shown to inhibit xanthine oxidase activity compared to other fractions with IC₅₀ values of 28.72 and 165.25 μg/ml. As for the ferric thiocyanate method, all the fractions except hexane fraction showed similar activity against lipid peroxidation and were comparable to butylated hydroxyl toluene, with percentage of inhibition from 95 to 98%.     

D-氨基酸氧化酶性质研究

D-氨基酸氧化酶（DAAO）是一种以黄素腺嘌呤二核苷酸（FAD）为辅基的典型黄素酶。它催化D-氨基酸生成相应的α-酮酸和氨。该文介绍了D-氨基酸氧化酶的研究现状及其特性，包括它的生理功能、基本特性、结构特性、光学动力学特性。     

Inhibition kinetics of polyphenol oxidase by glutamic acid

The inhibition of polyphenol oxidase (PPO) by glutamic acid was investigated. Application of different concentrations of glutamic acid to mushroom solution and Ocimum basilicum L. extract showed that glutamic acid appeared to be an effective browning inhibitor. Glutamic acid showed uncompetitive inhibition for mushroom and Ocimum basilicum L. polyphenol oxidases using 4-methylcatechol as a substrate, for mushroom PPO using catechol as a substrate and for Ocimum basilicum L. polyphenol oxidase using pyrogallol as a substrate; mixed-type inhibition for mushroom polyphenol oxidase using pyrogallol as a substrate; and non-competitive inhibition for Ocimum basilicum L. polyphenol oxidase using catechol as a substrate. Furthermore, sodium azide was used as an inhibitor for comparison with the inhibition potency of glutamic acid. It was found that glutamic acid was a more power inhibitor than sodium azide. The type of inhibition observed depended on the substrate, inhibitor and enzyme source used.     

Effect of food preservatives on the inhibitory activity of acidocin CH5 and bacteriocin D10

The effect of food preservatives on the activities of acidocin CH5 and bacteriocin D10 was evaluated. Lactobacillus delbrueckii subsp. lactis LTI30 was used as an indicator strain. Methyl paraben and propyl paraben at concentrations higher than 0.075 g/l enhanced the activity of acidocin CH5 up to 45% and 50% respectively. As regards bacteriocin D10, the synergistic effect was less pronounced. The inhibitory effect of EDTA applied alone at 0.1–0.2 mmol/l against the indicator atrain increased from 4% to 10%. EDTA (0.1–0.2 mmol/l) in combination with acidocin CH5 or bacteriocin D10 increased their inhibitory effects from 50% to 70% and from 10% to 50% respectively.     

肉桂醛缩氨基脲对黄嘌呤氧化酶的抑制作用研究

为研究肉桂醛缩氨基脲对黄嘌呤氧化酶(XOD)的抑制作用,以黄嘌呤为底物,测定肉桂醛缩氨基脲对黄嘌呤氧化酶活性的IC50、抑制类型和抑制常数.抑制类型由Lineweaver-Burk双倒数作图法来判断,并计算相应的抑制常数(KI和KIS).还研究了肉桂醛缩氨基脲对小鼠血尿酸质量浓度、血清XOD活性和肝脏XOD活性的影响....     

乳酸对多酚氧化酶活性与构象的影响

以蘑菇多酚氧化酶(PPO)作为原料,研究了不同浓度的乳酸对PPO的相对酶活性、巯基含量、内源荧光强度和紫外强度的影响。结果表明:PPO的相对酶活性随着乳酸处理的浓度增大而减小,当乳酸浓度达到40mmol/L时,其相对酶活性下降到原酶的1.20%,PPO活性得到基本抑制;随乳酸浓度的增大,巯基含量逐渐减小,当乳酸浓度为30mmol/L时,巯基含量为原酶的81.59%;PPO相对酶活性由100%变化到0.17%时,其相对巯基含量从100%降低为80.09%,二者呈现较好线性关系(R2=0.9861);从紫外吸收光谱和荧光发射光谱的变化可以看出:经不同浓度乳酸处理过后PPO分子发生部分去折叠化,导致酶活性的降低。      

阿魏酸对黄嘌呤氧化酶的抑制机理

为系统地探讨阿魏酸抑制黄嘌呤氧化酶(xanthine oxidase,XO)活性的分子机制,采用酶学和光谱学方法测定了阿魏酸对XO抑制可逆性、抑制动力学、结合性质及结构的影响,采用分子模拟技术预测了阿魏酸与XO的结合构象。结果表明:在混合竞争的方式下,阿魏酸能可逆地抑制XO活性,抑制常数为4.5μmol/L,与咖啡酸、对香豆酸、绿原酸和没食子酸相比,阿魏酸对XO的抑制能力最强,其半抑制浓度为116.2μmol/L。荧光滴定实验结果表明:阿魏酸通过动态静态混合猝灭方式使XO荧光减少,其中静态猝灭占主导,且与XO存在一个结合位点,结合常数为3.38×10⁴L/mol(298K);ΔG<0、ΔH<0和ΔS>0,表明该结合过程主要是以氢键和疏水作用力驱动且自发进行。分子对接结果揭示了阿魏酸进入了XO中黄素腺嘌呤二核苷酸活性区域,影响XO正常催化过程。同步荧光光谱和圆二色光谱实验表明:阿魏酸能改变XO的二级和三级结构,使XO中酪氨酸和色氨酸残基周围极性增加及疏水性降低,并且α-螺旋含量升高,β-折叠含量降低,使XO二级结构趋于紧密,这是影响XO活性的另一原因。研究以期为阿魏酸作为XO抑制剂改善高尿酸血症提供一定的理论依据。     

一种多金属氧酸盐对多酚氧化酶的抑制活性评价及在南美白对虾保鲜上的应用

目的:研究新型多酚氧化酶抑制剂对南美白对虾的保鲜效果。方法:通过紫外和红外光谱表征合成的多酚氧化酶抑制剂H₈[P₂Mo₁₇Ni(OH₂)O₆₁](P₂Mo₁₇Ni),研究其对蘑菇多酚氧化酶(PPO)相对酶活的影响及抑制机制和抑制类型;再测定经P₂Mo₁₇Ni处理后的南美白对虾在贮藏过程中的感官评分、色差(L^*)值、总色差(ΔE)值、菌落总数、酸碱度(pH)值和总挥发性盐基氮(TVB-N)值的变化情况,评价P₂Mo₁₇Ni在南美白对虾保鲜上的作用。结果:P₂Mo₁₇Ni对体外蘑菇PPO的活性具有抑制作用,其半抑制浓度(IC₅₀)为(0.377±0.004) mmol/L,且对PPO的抑制模式为竞争型可逆抑制,抑制常数K_I为0.185 mmol...     

多酚化合物对黄嘌呤氧化酶抑制作用的研究进展

文章主要对近5年来多酚化合物抑制黄嘌呤氧化酶活性、抑制类型及相互作用研究进行了综述,并对其构效关系、结构修饰及金属配合物的开发与应用进行了展望,以期为多酚化合物作为降尿酸食品功能因子和药物的研发提供参考.