阿魏酸异辛酯抑制酪氨酸酶活性的动力学研究

在30℃,pH=6.8的Na2HPO4-NaH2PO4缓冲体系中,采用酶动力学方法研究阿魏酸异辛酯对酪氨酸酶单酚酶和二酚酶活力的抑制动力学。实验结果表明,阿魏酸异辛酯对酪氨酸酶单酚酶和二酚酶活性均有良好抑制作用,对单酚酶和二酚酶活力的相对抑制率达到50%的阿魏酸异辛酯浓度(IC50)约分别为0.24 mmol/L和0.45 mmol/L,比熊果苷抑制二酚酶活性的IC50值5.3 mmol/L小得多。阿魏酸异辛酯能明显延长单酚酶的迟滞时间,0.4 mmol/L阿魏酸异辛酯能使迟滞时间由1.1 min延长至3.6 min。Lineweaver-Burk图显示阿魏酸异辛酯对二酚酶的抑制作用表现为竞争性抑制,抑制常数(K)I为0.20 mmol/L。     

菊苣酸抗氧化性及对抑制酪氨酸酶催化反应的动力学研究

以DPPH、·OH的清除效果和铁还原力为指标,采用酶动力学方法研究菊苣酸的抗氧化活性和对酪氨酸酶单酚酶和二酚酶的影响及抑制作用。结果表明:菊苣酸对DPPH、·OH自由基的清除率和铁还原力与菊苣酸浓度呈线性量效关系,IC_(50)值分别为13.63,14.26和13.78 mg/L。菊苣酸对酪氨酸酶单酚酶和二酚酶均具有较好的抑制作用,对单酚酶和二酚酶活性抑制的IC_(50)值分别为0.26 mmol/L和0.71 mmol/L,显著小于熊果苷对二酚酶活性抑制的IC_(50)值(5.3 mmol/L)。菊苣酸能明显延长单酚酶的迟滞时间。在浓度为0.40 mmol/L时,菊苣酸可使迟滞时间由134 s延长至383 s,延长2.87倍。Lineweaver-Burk菊苣酸对二酚酶的抑制表现为可逆竞争型抑制,最大反应速率(vm)为83.30μmol/L,抑制常数(KI)为0.14 mmol/L。     

肉桂腈抑制酪氨酸酶活性的动力学研究

在30℃、pH6.8的Na2HPO4-NaH2PO4缓冲体系中,采用酶动力学方法研究了肉桂腈对酪氨酸酶单酚酶和二酚酶活力的抑制动力学。实验结果表明,肉桂腈对酪氨酸酶单酚酶和二酚酶活性均有良好抑制作用,对单酚酶和二酚酶活力的相对抑制率达到50%的肉桂腈浓度(IC50)分别为0.13mmol/L和0.62mmol/L。肉桂腈能延长单酚酶的迟滞时间,0.2mmol/L肉桂腈能使迟滞时间由2min延长至2.7min。Lineweaver-Burk图显示肉桂腈对二酚酶的抑制作用表现为非竞争性可逆抑制,表观米氏常数(Km)为0.90mmol/L,抑制常数(KI)为0.64mmol/L。     

3-羟基-4-甲氧基肉桂酸抑制酪氨酸酶催化反应的动力学研究

在30℃,p H=6.8的Na2HPO4-Na H2PO4缓冲体系中,采用酶动力学方法研究了3-羟基-4-甲氧基肉桂酸对酪氨酸酶单酚酶和二酚酶活力的影响和抑制动力学。实验结果表明,3-羟基-4-甲氧基肉桂酸对酪氨酸酶单酚酶和二酚酶活性均有良好的抑制作用。对单酚酶和二酚酶活力的相对抑制率达到50%的3-羟基-4-甲氧基肉桂酸浓度(IC50)约分别为0.13 mmol/L和0.39 mmol/L,比熊果苷抑制二酚酶活性的IC50值5.3 mmol/L小得多。3-羟基-4-甲氧基肉桂酸能明显延长单酚酶的迟滞时间,0.2 mmol/L3-羟基-4-甲氧基肉桂酸能使迟滞时间由1.1 min延长至4.3 min。对二酚酶的抑制作用表现为可逆效应,说明其是通过抑制酶活力而导致催化效率的降低,而不是通过减少有效的酶量导致酶活力的下降。Lineweaver-Burk图显示3-羟基-4-甲氧基肉桂酸对二酚酶的抑制作用表现为竞争性抑制,最大反应速率(vm)为64.5μmol/min,抑制常数(KI)为0.11 mmol/L。     

咖啡酸激活酪氨酸酶催化反应的动力学研究

在30℃,pH6.8的Na2HPO4-NaH2PO4缓冲体系中,采用酶动力学方法研究了咖啡酸对酪氨酸酶单酚酶和二酚酶活力的激活效应.实验结果表明,咖啡酸对酪氨酸酶单酚酶和二酚酶活性均有激活作用,对单酚酶和二酚酶活力的相对激活率达到50%的咖啡酸浓度(IC50)分别为0.27mmol/L和1.35mmol/L.咖啡酸能消除单酚酶催化反应的迟滞时间,对二酚酶的激活作用表现为混合性激活,当咖啡酸浓度为0、0.50、1.0、1.50mmol/L时,米氏常数Km分别为0.31、0.25、0.20、0.15mmol/L.     

白藜芦醇对酪氨酸酶体外抑制活性的动力学研究

在30℃,pH 6.8的Na2HPO4-NaH2PO4的缓冲体系中,采用酶促动力学方法,研究了白藜芦醇对酪氨酸酶单酚酶和二酚酶的抑制作用。结果表明,白藜芦醇对酪氨酸酶、单酚酶和二酚酶均有抑制作用,对单酚酶抑制活性的IC50值(抑制率达到50%时的白藜芦醇质量浓度)约为5.1mg/mL,对二酚酶抑制活性的IC50值约为5.6 mg/mL。此外,白藜芦醇可延长单酚酶的迟滞效应,8 mg/mL的白藜芦醇能使迟滞时间从22 s延长至62 s,而对二酚酶则无此迟滞作用。Lineweav-er-Burk图分析表明,白藜芦醇对酪氨酸酶的抑制作用为混合型抑制,对游离酶的抑制常数(KI)和对酶-底物络合物的抑制常数(KIS)分别为3.4 mg/mL和35.98 mg/mL。     

螺旋藻中香豆酸的提取及其生物活性研究

试验选取螺旋藻粉为试验材料,探究了螺旋藻中活性物质香豆酸的最佳提取条件。通过核磁共振分析确定该提取物为香豆酸,化学式为C_9H_8O_3。采用紫外分光光度法和酶动力学的方法研究香豆酸对蘑菇酪氨酸酶的抑制效果、抑制机理及抑制类型,并检测其对DPPH自由基的清除能力。结果表明:香豆酸最佳提取工艺为:料液比1:20(g/mL),乙醇体积分数65%,浸提时间3 h,溶液pH 6;香豆酸对蘑菇酪氨酸酶有明显的抑制作用,其IC_(50)为55mmol·L~(-1),对酪氨酸酶的抑制过程是可逆的混合型抑制,抑制常数KI=0.221 1 mg·mL~(-1);香豆酸对DPPH自由基具有较好的清除作用,IC_(50)为0.155 g·L~(-1)。     

龙眼核多酚提取物对酪氨酸酶抑制作用的研究

研究龙眼核多酚提取物对酪氨酸酶活性的抑制作用。用龙眼核多酚提取物,分别以L-酪氨酸、L-多巴为底物,在475 nm处测定溶液吸光度和吸光度的变化,观察龙眼核多酚对单酚酶、双酚酶活性的影响。结果表明龙眼核多酚对酪氨酸单酚酶、二酚酶均有抑制作用,半抑制浓度IC_(50)分别为12.68、11.81 mg/m L。龙眼核多酚为酪氨酸酶可逆竞争性抑制剂,抑制常数(KI)为0.538 mg/m L。     

曲酸与阿魏酸对酪氨酸酶的抑制作用研究

本实验采用Lineweaver-Burk双倒数法探讨了曲酸与阿魏酸对蘑菇酪氨酸酶催化L-DOPA氧化的抑制作用并推测其抑制机理。通过对曲酸与阿魏酸的IC50与抑制常数KI的测定可知,曲酸对酪氨酸酶的抑制作用明显高于阿魏酸。分别采用分光光度法和ImageJ图像分析与处理软件定量地测定曲酸与阿魏酸对虾血淋巴和苹果褐变的抑制作用。曲酸对虾血淋巴褐变的抑制作用优于阿魏酸;对于苹果褐变,阿魏酸的抑制作用则高于曲酸。     

庚酸-3,4-二羟基苯酯和3,4-二羟基苯甲酸己酯对蘑菇酪氨酸酶抑制效应

目的:通过研究庚酸-3,4-二羟基苯酯和3,4-二羟基苯甲酸己酯对蘑菇酪氨酸酶二酚酶活力的影响,探讨其抑制机制和动力学,为其开发和利用奠定基础.方法:运用酶动力学研究方法,探讨效应物对蘑菇酪氨酸酶的抑制效应、抑制机理和抑制类型,并测定抑制常数.结果:庚酸-3,4-二羟基苯酯和3,4-二羟基苯甲酸己酯对蘑菇酪氨酸酶具有可逆抑制作用,庚酸-3,4-二羟基苯酯的抑制效应显著大于3,4-二羟基苯甲酸己酯,前者IC50为305 μmol/L,而后者测不到IC50.庚酸-3,4-二羟基苯酯对蘑菇酪氨酸酶二酚酶具有竞争抑制作用,其抑制常数KI为171.4μmol/L,而3,4-二羟基苯甲酸己酯对蘑菇酪氨酸酶二酚酶活力具有反竞争抑制作用,抑制常数KIS为389μmol/L.结论:庚酸-3,4-二羟基苯酯和3,4-二羟基苯甲酸己酯对蘑菇酪氨酸酶二酚酶的作用效应截然不同,显示了酶抑制剂的构象关系.     

Inhibitory effects of α-cyano-4-hydroxycinnamic acid on the activity of mushroom tyrosinase

The effects of α-cyano-4-hydroxycinnamic acid (HCCA) on the activity of mushroom tyrosinase have been studied. Results showed that HCCA could inhibit both the monophenolase activity and diphenolase activity of mushroom tyrosinase. For the monophenolase activity, the lag phase was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. When the concentration of HCCA reached to 80 μM, the lag time was lengthened from 20 s to 150 s and the steady-state activity was lost by about 75%. The IC₅₀ value was estimated to be 48 μM. For the diphenolase activity, the inhibitory effect of HCCA was also dose-dependent and the IC₅₀ value was estimated to be 2.17 mM. The kinetic analyses showed that the inhibition of HCCA on the diphenolase activity was reversible and competitive with the inhibition constants (K_I) determined to be 1.24 mM.     

Inhibitory activities of hydroxyphenolic acid–amino acid conjugates on tyrosinase

The inhibitory effects of hydroxyphenolic acid–aromatic amino acid conjugates on the activity of mushroom tyrosinase were investigated. Amongst the hydroxyphenolic acid–amino acid derivatives, protocatechuic acid–amino acid amide (PA-AA-NH₂) showed highly increased tyrosinase inhibitory activity. The results show that it could strongly inhibit both the monophenolase and diphenolase activities of tyrosinase. The IC₅₀ values, inhibition type, and K_I values of these hydroxyphenolic acid derivatives (PA-F-NH₂, PA-W-NH₂, PA-Y-NH₂) were evaluated and compared. Kinetic analyses of PA-AA-NH₂ as a tyrosinase inhibitor revealed that it acted as a reversible mixed-? type inhibitor, possibly by chelating copper at the active site of tyrosinase. These results suggest that aromatic amino acid conjugation assisted PA in binding to the active site of mushroom tyrosinase where it interrupted access to the substrate.     

Inhibitory effects of 4-chlorosalicylic acid on mushroom tyrosinase and its antimicrobial activities

The inhibitory effects of 4-chlorosalicylic acid on the activity of mushroom tyrosinase have been investigated. The results showed that 4-chlorosalicylic acid could strongly inhibit both monophenolase activity and diphenolase activity. The IC₅₀ values were estimated as 1.89 mM and 1.10 mM for monophenolase and diphenolase activities, respectively. For the monophenolase activity, 4-chlorosalicylic acid could not only lengthen the lag time, but also decrease the steady-state rate. For the diphenolase activity, kinetic analyses showed that the inhibition by 4-chlorosalicylic acid was reversible and its mechanism was mixed-II type, which is different from salicylic acid. The inhibition constants (K_I and K_IS) were determined to be 1.51 mM and 0.82 mM, respectively. Furthermore, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Aspergillus niger and Candida boidinii were investigated. The results showed that 4-chlorosalicylic acid was the most effective against E. coli with the MIC of 250 μg/ml and with the MBC of 500 μg/ml.     

Effects of nicotinic acid derivatives on tyrosinase inhibitory and antioxidant activities

The tyrosinase inhibitory and antioxidant activities of four nicotinic acid derivatives, namely, nicotinamide, methyl nicotinate, nicotinic acid hydroxamate (NAH), N-methyl nicotinic acid hydroxamate (NAH-M), and kojic acid, were studied. The tyrosinase inhibitory activity was found to follow the trend: NAH > kojic acid > NAH-M > methyl nicotinate > nicotinamide. The concentrations of half-inhibition (IC₅₀) of NAH against monophenolase and diphenolase activity were 2 and 1 μM, respectively; the inhibition mode was a mixed-type in the former and an uncompetitive type in the latter. In the antioxidant activity assays, NAH and NAH-M, but not nicotinamide or methyl nicotinate, showed dose-dependent DPPH radical scavenging activity, and their corresponding IC₅₀ values were 65.81 and 125 μM, respectively. NAH and NAH-M also showed hydroxyl radical scavenging activity and anti-low-density-lipoprotein peroxidation. These results indicate that NAH and NAH-M have the potential to be useful in cosmetics or food processing, and therefore, they should be investigated further.     

A Novel Inhibitor Against Mushroom Tyrosinase with a Double Action Mode and Its Application in Controlling the Browning of Potato

In order to search for a new method for the anti-browning of food products, a novel hydroxypyridinone (HPO) derivative with a formyl group was evaluated for its anti-tyrosinase property. This compound was found to exhibit potent tyrosinase inhibition on the monophenolase activity of mushroom tyrosinase with an IC₅₀ value of 1.33 μM, indicating that this HPO derivative was 12-fold stronger than kojic acid (IC₅₀ 15.89 μM). This molecule can inhibit tyrosinase via two action modes, namely copper reduction and chelation, and the formation of a Schiff’s base with the amino group at the active site of the enzyme. A synergistic effect of these two action modes to enhance the inhibitory activity was observed. This compound was also investigated for the inhibitory effect on diphenolase activity of mushroom; the inhibitory mechanism was found to be reversible and of competitive-uncompetitive mixed-type inhibition. This hydroxypyridinone was demonstrated to effectively control the browning of vegetable products.     

Effects of protein hydrolysate from chicken feather meal on tyrosinase activity and melanin formation in B16F10 murine melanoma cells

Tyrosinase is a copper-containing enzyme that controls mammalian melanogenesis. Tyrosinase inhibitors are important for their potential application in cosmetic products. Chicken feather meal is a rich source of amino acids, which have been linked with tyrosinase inhibition activity. This study investigated the tyrosinase inhibitory properties of protein hydrolysates prepared from chicken feather meal. Protein hydrolysates prepared by pepsin-pancreatin with MW <3 kDa exhibited strong tyrosinase inhibition activity for both monophenolase (IC₅₀ 5.780 ± 0.188 µg/mL) and diphenolase activities (IC₅₀ 0.040 ± 0.024 µg/mL) in a cell-free mushroom tyrosinase system. These samples were uncompetitive inhibitors with Ki values of 18.149 and 27.189 µg/mL in monophenolase and diphenolase activities, respectively. A cell culture model showed that this hydrolysate had the strongest inhibition on the viability of B16F10 cells (IC₅₀ 1.124 ± 0.288 µg/mL) and 0.210 µg/mL of the sample exhibited inhibition of tyrosinase activity by 50.493% and melanin synthesis by 14.680% compared to the control.     

Inhibitory effects of cinnamic acid and its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase

The effects of cinnamic acid and its derivatives (2-hydroxycinnamic acid, 4-hydroxycinnamic acid and 4-methoxycinnamic acid) on the activity of mushroom tyrosinase have been studied. Results showed that cinnamic acid, 4-hydroxycinnamic acid and 4-methoxycinnamic acid strongly inhibited the diphenolase activity of mushroom tyrosinase and the inhibition was reversible. The IC₅₀ values were estimated to be 2.10, 0.50 and 0.42 mM, respectively. 2-Hydroxycinnamic acid had no inhibitory effect on the diphenolase activity of the enzyme. Kinetic analyses showed that the inhibition type of cinnamic acid and 4-methoxycinnamic acid was noncompetitive with the constants (K_I) determined to be 1.994 and 0.458 mM, respectively. The inhibition type of 4-hydroxycinnamic acid was competitive, with the inhibition constant (K_I) was 0.244 mM.     

Mushroom tyrosinase inhibitory effects of isoflavones isolated from soygerm koji fermented with Aspergillus oryzae BCRC 32288

The inhibition of mushroom tyrosinase in soygerm koji, fermented with Aspergillus oryzae BCRC 32288, was investigated. A methanol extract of the soygerm koji was partitioned into hexane, ethyl acetate and water. The ethyl acetate extract showed potent anti-tyrosinase activity with an IC₅₀ value of 0.19 mg/ml. The active compounds were isolated by activity-guided silica gel column chromatography and high-performance liquid chromatography (HPLC) methods. Seven tyrosinase inhibitors were purified and identified as 6,7,4′-trihydroxyisoflavone, 7,8,4′-trihydroxyisoflavone, 5,7,8,4′-tetrahydroxyisoflavone, 7,4′-dihydroxyisoflavone (daidzein), 6-methoxy-7,4′-dihydroxyisoflavone (glycitein), 4′-hydroxyisoflavone-7-O-glucoside (daidzin), and 5,4′-dihydroxyisoflavone-7-O-glucoside (genistin) by comparing their mass, ¹H NMR, and ¹³C NMR spectral data with those in the literature. The purified seven isoflavones from fermented soygerm koji were divided into two groups, based on their inhibitory effects on mushroom tyrosinase. Five isolated isoflavones showed inhibitory activity against monophenolase activity of mushroom tyrosinase only, with IC₅₀ values of 0.009 ± 0.001 (6,7,4′-trihydroxyisoflavone), 0.203 ± 0.018 (daidzein), 0.218 ± 0.007 (glycitein), 0.267 ± 0.008 (daidzin), and 0.343 ± 0.013 (genistin) mM. The kinetic study indicated that the five inhibitors significantly lengthened the lag time of the monophenolase activity of tyrosinase and acted competitively for the l-tyrosine binding site of the enzyme. So, the five isoflavones were competitive inhibitors for the monophenolase activity of tyrosinase. The other two isoflavones, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone, inhibited both monophenolase and diphenolase activities of tyrosinase. Moreover, pre-incubation of each of the two isoflavones with tyrosinase resulted in total irreversible inhibition of the enzyme activity, even at concentrations as low as of 10 μM. Hence, 7,8,4′-trihydroxyisoflavone and 5,7,8,4′-tetrahydroxyisoflavone were irreversible inhibitors of mushroom tyrosinase.     

雀嘴茶中三大酚类成分的抗氧化活性和酪氨酸酶抑制活性分析

为深入挖掘雀嘴茶的利用价值,本文以其中含量丰富的6’-O-咖啡酰熊果苷(CA)、β-熊果苷和绿原酸三大酚类成分为研究对象,对其抗氧化活性和酪氨酸酶抑制活性进行分析。结果表明,雀嘴茶三大酚类成分均表现出较好的抗氧化活性,6’-O-咖啡酰熊果苷(CA)、β-熊果苷和绿原酸清除DPPH自由基的IC₅₀值分别为13.56±0.14、104.41±6.52和8.42±0.21μg/mL,清除ABTS⁺自由基的IC₅₀值分别为18.01±0.06、50.60±1.25和26.93±0.38μg/mL,清除OH自由基的IC₅₀值为2.64±0.06、>10.00和<1.00 mg/mL,对铁离子总还原能力的强度依次为绿原酸>CA>β-熊果苷;雀嘴茶三大酚类成分对酪氨酸酶的抑制活性差异较大,CA对酪氨酸酶兼具单酚酶和二酚酶抑制作用,其IC₅₀值分别为1.114±0.035和95.198±1.117μmol/L,β-熊果苷仅有单酚酶抑制作用,IC₅₀...