Biological evaluation of coumarin derivatives as mushroom tyrosinase inhibitors

A series of coumarin derivatives were synthesised and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesised compounds exhibited significant inhibitory activities. Especially, 2-(1-(coumarin-3-yl)ethylidene)hydrazinecarbothioamide bearing thiose-micarbazide group exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 3.44 μM. The inhibition mechanism analysis of 2-(1-(coumarin-3-yl)-ethylidene)hydrazinecarbothioamide and 2-(1-(6-chlorocoumarin-3-yl)ethylidene)-hydrazinecarbothioamide demonstrated that the inhibitory effects of the compounds on the tyrosinase were irreversible. Preliminary structure activity relationships’ (SARs) analysis suggested that further development of such compounds might be of interest.     

Phenolic content,antioxidant and physico‐chemical properties of Sardinian monofloral honeys

In this study, fifty‐one monofloral Sardinian honeys from ten various floral origins were screened for their phenolic content, antioxidant activity, colour and electrical conductivity. The total phenolic amounts have been evaluated by Folin–Ciocalteu method, whereas quantification of several phenolic compounds (phenolic acids and flavonoids) has been carried out by HPLC‐DAD technique. The richest sample in phenolic compounds resulted strawberry tree honey with about 40 mg GAE/100 g, as well FRAP test and DPPH˙ test confirm that antioxidant activity of strawberry tree honey extract exceed both honey extracts and synthetic antioxidants like BHA and BHT. Among the studied phenolic compounds a total of five phenolic acids (ferulic, syringic, trans‐cinnamic, chlorogenic and p‐hydroxycinnamic) and nine flavonoids (catechin, kaempferol, rutin, quercetin, luteolin, apigenin, galangin, pinocembrin and pinobanksin) were identified. Our results show good correlations between total polyphenol amount and antioxidant activity and between colour and electrical conductivity.     

Association of sulfhydryl oxidase and xanthine oxidase in bovine mammary tissue

The presence of antigenically active xanthine oxidase was indicated in various relatively purified preparations of sulfhydryl oxidase obtained from bovine milk. Evidence for formation of a complex of the two enzymes was obtained by double immunodiffusion. Furthermore, sodium dodecylsulfate-gel electrophoresis of sulfhydryl oxidase and xanthine oxidase model mixtures indicated that high molecular weight species were present that reacted with both antisulfhydryl oxidase and antixanthine oxidase. Similar gel electrophoretic patterns visualized by protein-dye binding methods revealed a distinct band (greater than 200 kdalton) was formed upon incubation of mixtures of the two enzymes, the presence of which was unaffected by reduction of protein disulfide bonds. Immunofluorescent staining techniques showed both enzymes in the apical plasma membrane. Because sulfhydryl oxidase previously has been shown to catalyze conversion of the dehydrogenase form of xanthine oxidase to the oxidase form, this conversion may occur when xanthine oxidase contacts sulfhydryl oxidase in the apical plasma membrane. This conversion and the resulting potential for production of active oxygen species could be significant to membranotropic processes, such as fat globule secretion, and to the oxidative stability of the milk fat globule membrane.     

Biological evaluations of novel vitamin C esters as mushroom tyrosinase inhibitors and antioxidants

The inhibitory effects of vitamin C esters 1 and 2 on the diphenolase activity of mushroom tyrosinase have been studied. The results showed that compounds 1 and 2 inhibited tyrosinase with IC₅₀ values of 0.58 and 0.16 mM, respectively. The dose–response curves demonstrated that compounds 1 and 2 not only lengthened the lag time, but also decreased the steady-state rate. The kinetic analyses showed that the inhibition by compound 2 was reversible and its mechanism was mixed type, which was different from compound 1 (irreversible inhibitor). Furthermore, the antioxidant activities of these compounds against hydroxyl radical scavenging, superoxide anion radical scavenging, and DPPH radical scavenging were also investigated. Compounds 1 and 2 exhibited potential antioxidant activities. In particular, compound 2 was found to be the most effective antioxidant, more potent than the well-known antioxidants vitamin C and TBHQ.     

Milk xanthine oxidase: Properties and physiological roles

Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme that occurs as a major protein component of the milk fat globule membrane surrounding lipid droplets in milk. The enzyme has broad substrate specificity and is capable of reducing oxygen to generate the reactive oxygen species (ROS), superoxide and hydrogen peroxide. It can also reduce nitrite, yielding reactive nitrogen species (RNS), such as nitric oxide and peroxynitrite. By virtue of its capacity to generate ROS and RNS, milk XOR may play an antimicrobial defensive role in the neonatal gut, complementing endogenous enzyme of the intestinal epithelium. XOR is also involved in secretion of milk fat globules in a process dependent on the enzyme protein rather than on its enzymic activity, which is known to vary greatly with time after parturition and also between species. Some potential hazards and benefits of ingestion of cows milk XOR are briefly discussed.     

Characterization of microorganisms in Argentinean honeys from different sources

Seventy polyfloral honeys including commercial samples obtained from supermarkets, harvested from apiaries and purchased in bulk were initially examined for total antibacterial activity. From each sample, numbers of aerobic mesophilic bacteria, total coliforms, moulds and yeasts were determined and the presence of Salmonella spp., Shigella spp., Clostridium sulfite-reducers, Paenibacillus larvae and Bacillus spp. was investigated. Moisture content, pH and total acidity were also determined for all samples. Any honey diluted to concentrations from 75% to 1% (w/v) of full-strength honey showed total antibacterial activity. The numbers of aerobic mesophilic bacteria, moulds and yeasts were less than 10(3) cfu/g for all 70 samples. Faecal coliforms, Escherichia coli, Salmonella spp., Shigella spp. and Clostridium sulfite-reducers were not detected but P. larvae subspp. larvae, Bacillus cereus, Bacillus pumilus and Bacillus laterosporus were found among samples. For commercial, apiary and bulk honey the mean values for moisture content, pH and acidity, respectively, were 17.50%, 17.40% and 17.50%; 4.60, 4.10 and 4.20; and 18.30, 20.60 and 21 meq NaOH/kg. P. larvae was recovered from 35% of apiaries including hives in which the bees did not display symptoms of American foulbrood disease.     

Distribution of xanthine oxidase and xanthine dehydrogenase activity in bovine milk: Physiological and technological implications

The aim of this study was to evaluate the distribution of xanthine oxidoreductase (XOR) and its two forms, xanthine oxidase (XO) and xanthine dehydrogenase (XD), in milk fractions. XOR associated with milk phospholipid membranes was found to be distributed among an intra-membranous pool in which it takes the form of a mixture of XO and XD, with a clear predominance of XD, and a free pool of XO, of which 33% is found in the outer surface of milk fat globule membrane, 20.5% in the outer surface of whey membrane particles, and the remaining 46.7% in apparent solution. The inner-membrane XOR may play a nonenzymatic role in fat secretion, whereas extra-membranous XO is freely available for a role in the innate gland immune system and may affect milk quality.     

牛乳中黄嘌呤氧化酶研究综述

综述了牛乳中黄嘌呤氧化酶的各种性质及其应用。其性质主要包括:酶的构成、酶的分子量、酶的活性部位、酶的最适pH与最适温度、酶所催化的反应、反应类型、反应底物及产物等。另外,还介绍了一些酶反应动力学、酶活力的评价方法和酶的抑制剂等方面的问题。     

Dietary spices as a natural effectors of lipoxygenase,xanthine oxidase,peroxidase and antioxidant agents

The aim of this study was to determine antioxidant activities of selected spices and their influence on the activity of peroxidase and some prooxidant enzymes. Extracts from basil and rosemary were the strongest activators of peroxidase activity (204.7% and 205.8%, respectively). The highest ability for lipoxygenase inhibition was exhibited by tarragon and oregano extracts (60.2% and 57.9%, respectively). In turn, the highest xanthine oxidase inhibition was found in the case of black pepper and basil extracts (70.9% and 67.0%, respectively); whereas the lowest in the case of the cinnamon extract (28.09%). Linoleic acid was the most effectively prevented by oregano and rosemary extracts. O₂^? scavenging activities of basil, thyme, rosemary, tarragon and cinnamon extracts ranged from 47.5% to 32.7%. The H₂O₂ scavenging abilities ranged from 42.8% for tarragon to 99.2% for black pepper extract. The results obtained suggest that spice condiments used in food preparations contain phenolic/flavonoid compounds that can significantly inhibit prooxidant enzymes (lipoxygenase and xanthine oxidase) and enhance antioxidant enzymatic and non-enzymatic defense system, hence diet supplementation with herbs may be helpful in preventing or slowing down the progress of lifestyle-related and chronic diseases.     

Isolation of tyrosinase inhibitors from Artocarpus heterophyllus and use of its extract as antibrowning agent

A new furanoflavone, 7-(2,4-dihydroxyphenyl)-4-hydroxy-2-(2-hydroxy propan-2-yl)-2, 3-dihydrofuro(3, 2-g)chromen-5-one (artocarpfuranol, 1), together with 14 known compounds, dihydromorin (2), steppogenin (3), norartocarpetin (4), artocarpanone (5), artocarpesin (6), artocarpin (7), cycloartocarpin (8), cycloartocarpesin (9), artocarpetin (10), brosimone I (11), cudraflavone B (12), carpachromene (13), isoartocarpesin (14), and cyanomaclurin (15) were isolated from the wood of Artocarpus heterophyllus. Their structures were identified by interpretation of MS,( 1)H-NMR,( 13)C-NMR, HMQC, and HMBC spectroscopic data. Among them, compounds 1-6 and 14 showed strong mushroom tyrosinase inhibitory activity with IC(50) values lower than 50 microM, more potent than kojic acid (IC(50) = 71.6 microM), a well-known tyrosinase inhibitor. In addition, extract of A. heterophyllus was evaluated for its antibrowning effect on fresh-cut apple slices. It was discovered that fresh-cut apple slices treated by dipping in solution of 0.03 or 0.05% of A. heterophyllus extract with 0.5% ascorbic acid did not undergo any substantial browning reaction after storage at room temperature for 24 h. The antibrowning effect was significantly better than samples treated with the extract (0.03 or 0.05%) or ascorbic acid (0.5%) alone. The results provide preliminary evidence supporting the potential of this natural extract as antibrowning agent in food systems.     

新鲜奶油中黄嘌呤氧化酶的纯化及其酶学性质

采用正丁醇处理、硫酸铵沉淀、凝胶过滤层析和阴离子交换层析等分离技术,从新鲜的稀奶油中纯化得到SDS-PAGE电泳纯的黄嘌呤氧化酶,其最终回收率为19.9%,纯化倍数为82.42,酶比活为8.65U/mg。该黄嘌呤氧化酶的相对分子质量约为280kDa,最适作用温度为40℃,在35℃以下比较稳定;最适pH值是8.5,在pH6.5～8.5时较稳定;Zn2+、Fe2+和K+对黄嘌呤氧化酶活性有促进作用,Ag+、Mn2+、Ca2+和SDS对黄嘌呤氧化酶活性有抑制作用。     

双孢菇酪氨酸酶测活方法的改进及其抑制剂的筛选

对传统的酪氨酸酶活性测定的分光光度法(以多巴为底物,在475nm处比色)加以改进,使其灵敏度得以明显提高,同时确认了其反应的最佳条件和特征常数:即以L-酪氨酸为底物,于317nm处比色,pH6.5,在30℃下反应,测得酪氨酸酶Km=0.543mmol/L,Vmax=41.5U/min。利用此改进方法考察了苯甲酸、异Vc、EDTA、CA、H2O2、NaCl、环糊精及其组合对酪氨酸酶的抑制效果。结果发现,柠檬酸(CA)、苯甲酸、异Vc均为优良的酪氨酸酶抑制剂,抑制率均高于53%,另外,包含异Vc的各种二、三元组合效果普遍良好,抑制率均在75%以上,其中又以0.55%异Vc+1.6%CA+5%NaCl组合最佳,其抑制率高达86.8%。     

Chemiluminescent determination of xanthine oxidase activity in milk.

A chemiluminescent method for determining xanthine oxidase (XOD) activity was developed and applied to the assay of milk enzyme activity using a photomultiplier luminometer. Various kinds of milk and cream samples were analysed for XOD content. In pasteurized milk, XOD activity depended on the fat content and in UHT milk it disappeared owing to the heat treatment. Milk sample preparation was very simple, requiring only homogenization at 40 degrees C followed by a 1:10 dilution with UHT ('XOD-free') milk. The assay was carried out at 25 degrees C. The response obtained from XOD standard solutions in milk was linear from 0.1 to 500 enzyme units (U) l-1, but for the actual milk samples values ranged only from 1 to 135 U l-1. The detection limit at 2 SD was 0.1 U l-1 in milk, while in buffer it was 100 times lower. The intra-assay and interassay CV for XOD activity in milk were 6-12%.     

Characteristics of purified cows' milk xanthine oxidase and its submolecular characteristics

Xanthine oxidase (EC 1.2.3.2) was purified from fresh cows' milk by differential centrifugation and hydroxylapatite chromatography in the absence of reducing agents and proteases. The purified isolate possessed an absorbance at 280 nm:absorbance at 450 nm ratio of 4.84; an absorbance (1 cm at 280 nm 1%) of 11.9; an activity:absorbance at 450 nm of 141, a specific activity of 3.59 units/mg; and detectable dehydrogenase activity. The enzyme preparation was obtained in a reversible oxidase form that could be partially converted to xanthine dehydrogenase in the presence of 10mM dithiothreitol or 1% mercaptoethanol. Amino acid analyses revealed that the enzyme was hydrophobic in nature and that lysine constituted its N-terminal residue. The protein contained 22 disulfide and 38 sulfhydryl groups, four of which were detectable in the undenatured protein complex. Discontinuous PAGE in the presence of selected dissociation agents did not result in further resolution. Sodium dodecyl sulfate-PAGE of the purified enzyme revealed a sharp zone with a molecular weight of 151,000 +/- 4000 (i.e., monomer). The purified enzyme exhibited oxidase activity in the presence of 6 M urea and following limited proteolysis by trypsin, chymotrypsin, plasmin, pancreatin, pepsin, and papain. Proteolyzed xanthine oxidase migrated as a single zone in polyacrylamide gels in the presence and absence of dissociating agents such as 1% mercaptoethanol and 6 M urea. Restricted digestion of xanthine oxidase by proteases was indicated by the presence of three major zones with molecular weights ranging from 85,000 to 100,000, 30,000 to 35,000, and 18,000 to 20,000 commonly observed in SDS gels. Amino acid profiles of the principal peptidyl fragments of trypsin-cleaved xanthine oxidase indicated their hydrophobic nature and lysine as the N-terminal residue for all fragments.     

带鱼黄嘌呤氧化酶抑制肽制备及工艺优化

目的 制备带鱼黄嘌呤氧化酶抑制肽,研究含肌肽类活性肽的带鱼提取物对黄嘌呤氧化酶的抑制作用。方法 以带鱼为原料,通过超声辅助酶法制备带鱼黄嘌呤氧化酶抑制肽,高效液相色谱法测定肌肽类活性肽的含量。结果 以肽粉得率和黄嘌呤氧化酶抑制率为指标,筛选得到碱性蛋白酶为最佳反应用酶。以单因素实验为基础,通过Box-Behnken响应面分析法,以水解度、黄嘌呤氧化酶抑制活性、肌肽及鹅肌肽含量为响应值,优化得到酶解最优条件:酶解时间5 h,超声时间21 min,酶解温度49℃。在此条件下,得出水解度21.90%、黄嘌呤氧化酶抑制率52.03%、鹅肌肽含量0.66%和肌肽含量为0.15%,与回归理论模型基本符合。在最优酶解方案的基础上,将酶解液进一步分成不同组分,可得到肌肽类活性肽含量升高,抑制活性变大。结论 肌肽类活性肽对黄嘌呤氧化酶活性的抑制有着积极的作用,这为工业化利用带鱼制备黄嘌呤氧化酶抑制肽提供理论及指导。     

Wheat bran globulins: Competitive inhibitors of mushroom tyrosinase

The inhibitory capacity of the globulin fraction of wheat bran against the diphenolase activity of mushroom tyrosinase, using l-tyrosine as substrate, was evaluated. Enzyme kinetics was monitored in the presence of globulin solutions by measuring the absorbance at 475nm. Lineweaver-Burk plots were drawn in order to determine V_max, K_m, and type of inhibition. Results showed that globulins from wheat bran competitively inhibited, the activity of mushroom tyrosinase with a K_I of 0.79%(w/v). The degree of inhibition was 24% at 2 mM of the substrate L-tyrosine.     

Free and membrane-bound xanthine oxidase in bovine milk during cooling and heating

The effect of cold storage (5 C, 24 h) and heat treatment (60 C, 5 min) of milk on activities of free and membrane-bound xanthine oxidase has been studied. Both treatments enhanced total xanthine oxidase activity in milk. Activity of membrane-bound xanthine oxidase increased and free xanthine oxidase decreased in buttermilk while it increased in skim milk on cold storage. Heat of milk increased free and membrane-bound xanthine oxidase activities in both buttermilk and skim milk. The state of xanthine oxidase activity in skim milk from reconstituted milk, which was prepared by mixing xanthine oxidase inactivated skim milk and fresh cream, showed that only the free enzyme migrated from the cream phase to skim milk on cold storage. Very little xanthine oxidase activity was detectable in skim milk on heat treatment of the reconstituted milk sample. The overall increased activity of xanthine oxidase in milk during cold storage or heat treatment may not be due to the release of fat globule membrane enzyme to skim milk.     

Phenolic tyrosinase inhibitors from the stems of Cudrania cochinchinensis

The phytochemcal profiles of Cudrania cochinchinensis leaf, twig, stem and root were compared by HPLC analysis. It was found that C. cochinchinensis stem extract contained some unknown natural products with potential tyrosinase inhibitory activities. Therefore, the chemical constitutes in extract (95% ethanol) of C. cochinchinensis stem were further investigated in this study. A new racemic mixture, (±)2,3-cis-dihydromorin, and fifteen known phenolic compounds, dihydrokaempferol 7-O-β-d-qlucopyranoside, skimmin, quercetin-7-O-β-d-glucoside, 2,3-dihydroquercetin 7-O-β-d-glucoside, kaempferol-7-O-β-glucopyranoside, quercetin-3,7-di-O-β-d-glucoside, morin-7-O-β-d-glucoside, 1,3,5,8-tetrahydroxyxanthen-9-one, 2,3-trans-dihydromorin, aromadendrin, oxyresveratrol, genistin, protocatechuic acid, kaempferol 3,7-di-O-β-glucopyranoside, and naringenin were isolated. Spectral techniques (MS, (1)H NMR and (13)C NMR) were utilized for their structural identification and their inhibitory activities on mushroom tyrosinase were also evaluated. The results showed that tyrosinase inhibitory activities of (±)2,3-cis-dihydromorin (IC(50) = 31.1 μM), 2,3-trans-dihydromorin (IC(50) = 21.1 μM), and oxyresveratrol (IC(50) = 2.33 μM), were more potent than that of kojic acid (IC(50) = 50.8 μM), a well-known tyrosinase inhibitor, indicating that Cudrania cochinchinensis stem will be a great potential agent for the development of effective natural tyrosinase inhibitors.