对羟基肉桂酸对酪氨酸酶催化反应的抑制机理

本文研究了对羟基肉桂酸(HCA)对酪氨酸酶催化单酚底物L-酪氨酸和催化二酚底物L-多巴的抑制能力,并利用紫外-可见光谱、荧光光谱以及分子对接技术探究了其抑制机理。结果表明对羟基肉桂酸对酪氨酸酶催化单酚底物L-酪氨酸比催化二酚底物L-多巴具有更强的抑制作用,半抑制浓度分别为0.096 mmol/L和0.500 mmol/L;紫外-可见分析发现对羟基肉桂酸能与Cu2+发生螯合,使光谱发生明显红移。进一步通过荧光光谱分析得到,对羟基肉桂酸在酪氨酸酶溶液中并没有出现荧光淬灭反而随着对羟基肉桂酸浓度的增大荧光强度变强,说明对羟基肉桂酸被酪氨酸酶催化氧化成对应的醌类物质。利用分子对接技术揭示了对羟基肉桂酸通过氢键和疏水作用竞争性地占据了单酚和二酚底物的空间位置,并与酪氨酸酶中双核铜离子螯合,从而抑制酪氨酸酶催化L-酪氨酸和L-多巴氧化的活性机理。     

EGCG和异鼠李素的细胞抗氧化协同作用

目的：探究不同比例的食源性类黄酮表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)和异鼠李素(isorhamnetin)细胞抗氧化协同效应，为食源性类黄酮功能性食品的开发提供理论依据。方法：采用ChouTalalay联合指数法(Combination Index,CI)评价不同比例EGCG和异鼠李素组合对细胞抗氧化活性的影响。结果：EGCG和异鼠李素(6:4,c/c)联合作用时，EC₅₀值为5.01±0.1μg/mL，联合作用指数CI_avg值为0.76，表现出较强的协同作用。进一步细胞抗氧化酶实验发现，在EGCG+异鼠李素(1.5+1、3+2和6+4μg/mL)浓度梯度下，超氧化物歧化酶(Superoxide dismutase,SOD)活性相较于2,2’-偶氮二异丁基脒二盐酸盐[2,2-Azobis(2-amidinopropane)dihydrochloride solution,AAPH]处理组分别提高了5.2%、21.1%和49.1%；谷胱甘肽过氧化物酶(Glutathione peroxidase...     

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。     

香芹酚抗氧化和抑制酪氨酸酶活性的研究

采用清除DPPH、ABTS、羟自由基和超氧自由基,抑制脂质过氧化活性和抑制酪氨酸酶活性的方法,检测香芹酚的抗氧化活性和抑制酪氨酸酶活性。实验结果显示,香芹酚具有明显的抗氧化活性和抑制酪氨酸酶活性,呈浓度依赖性效应。在清除DPPH、ABTS、羟自由基、超氧自由基,抑制脂质过氧化活性和抑制酪氨酸酶活性试验中,香芹酚的体系终浓度IC50分别为100μmol/L、18.18μmol/L、16μmol/L、16μmol/L、12.5μmol/L和6.67μmol/L。作为阳性对照药物的山奈酚的体系终浓度IC50分别为80μmol/L、9.09μmol/L、12μmol/L、12μmol/L、8.33μmol/L和3.33μmol/L。香芹酚具有1个酚羟基,山奈酚具有3个酚羟基,分子结构的不同,导致山奈酚和香芹酚抗氧化活性和抑制酪氨酸酶活性不同。研究表明香芹酚具有明显的抗氧化活性和抑制酪氨酸酶活性,是值得进一步研究的抗氧化防衰老类候选药物、化妆品原料和食品添加剂。     

莲房原花青素对酪氨酸酶活力和黑色素生物合成影响的初步研究

目的：探讨莲房原花青素(LSPC)对酪氨酸酶及其黑色素生物合成的抑制机理。方法：采用酪氨酸酶多巴速率氧化法体外测定药物干预前后酪氨酸酶活性，求出酪氨酸酶抑制率，并作出相应Lineweaver-Burk曲线，推断其抑制类型。结果：莲房原花青素能显著抑制酪氨酸酶活性，其半抑制浓度IC50为1．15g/L。并能有效抑制形成黑色素的中间产物L-多巴色素向黑色素转化其半抑制浓度IC50为1．750g/L。结论 莲房原花青素可抑制酪氨酸酶活性，且属于酪氨酸酶竞争性抑制剂。     

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。     

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。     

沙棘黄酮对酪氨酸酶活力抑制作用的研究

研究沙棘黄酮对酪氨酸酶活性的抑制作用。实验测定了沙棘黄酮分别在温度、pH、时间、底物浓度、酶液加入量等因素条件下对酪氨酸酶活力的影响。结果显示,沙棘黄酮对酪氨酸酶活性的抑制率随着时间的延长,底物浓度的增加,酶量的增加而增强,而且在35℃及pH7.1时沙棘黄酮对酪氨酸酶活力的抑制作用最强可达到47.45%。说明沙棘黄酮对酪氨酸酶的活性具有较强的抑制作用。     

酪氨酸酶特异性卵黄抗体Fab’片段的制备及其对酶活力的抑制性能

针对导致黑色素沉淀的酪氨酸酶,制备了特异性卵黄抗体Fab’片段,研究其对酶活力的抑制效果并从动力学角度进行分析。结果表明,胃蛋白酶法制备Fab’片段的最适条件为：pH?4.0、反应比（卵黄抗体-胃蛋白酶）1∶150（mg/U）、反应时间8?h。经免疫亲和柱纯化后可得理化性质稳定、纯度大于95%的Fab’片段;该片段通过非竞争性结合抑制酪氨酸酶活力,IC50为11.16?μmol/L,结合常数为5.79×105,抑制效果相比完整的卵黄抗体分子大大提高。本研究可为卵黄抗体Fab’片段作为潜在的酶活力抑制剂在食品、化妆品和医药等行业中应用提供理论参考。     

柿叶黄酮对酪氨酸酶抑制机理的初步探讨

以柿叶为原料,用L-酪氨酸为底物测定柿叶黄酮对酪氨酸酶的抑制作用,并对柿叶黄酮进行了抗氧化试验和络合Cu2＋试验。试验结果表明：柿叶黄酮对酪氨酸酶的抑制效果明显,柿叶黄酮对DPPH.、超氧阴离子自由基（O2-..）和羟自由基（.OH）有较好的清除率,对Cu2＋有较强的络合能力,柿叶黄酮对酪氨酸酶的抑制与其较强的抗氧化能力及其络合金属离子的能力有关。     

Inhibitory effects of salicylic acid family compounds on the diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones that form brown or black pigments. In the present paper, inhibitory effects on the diphenolase activity of 10 compounds of the salicylic acid-family on the diphenolase activity of mushroom tyrosinase have been studied. The results show that some of these compounds behave as reversible inhibitors. Salicylic acid is a competitive inhibitor while 4-methoxysalicylic acid is non-competitive, 5-methoxysalicylic acid is mixed-I type and 4-methylsalicylic acid and 5-methylsalicylic acid are mixed-II type. The inhibition constants of these five compounds were evaluated. The inhibition strength follows the order: 4-methylsalicylic acid > 5-methylsalicylic acid > 4-methoxysalicylic acid > salicylic acid > 5-methoxysalicylic acid. Models of the interaction between the enzyme and the inhibitors are further discussed and compared.     

Inhibition of the activity of mushroom tyrosinase by alkylbenzoic acids

The inhibition kinetics of alkylbenzoic acids on the diphenolase activity of mushroom tyrosinase have been investigated. The results show that the alkylbenzoic acids assayed can lead to reversible inhibition of the enzyme; furthermore, o-toluic acid and m-toluic acid are mixed-type inhibitors and p-alkylbenzoic acids are uncompetitive inhibitors. The inhibition constants have been determined. For these p-alkylbenzoic acids, the inhibition strength follows the order: p-toluic acid < p-ethylbenzoic acid < p-propylbenzoic acid < p-isopropylbenzoic acid < p-tert-butylbenzoic acid < p-butylbenzoic acid < p-pentylbenzoic acid < p-hexylbenzoic acid < p-heptylbenzoic acid < p-octylbenzoic acid, indicating that the hydrophobic p-alkyl group played an important role in the inhibition of the enzyme. The inhibitory effects were potentiated with increasing lengths of the hydrocarbon chains. The inhibitory effects of o-toluic acid and p-isopropylbenzoic acid on the monophenolase activity have also been studied. The results show that both o-toluic acid and p-isopropylbenzoic acid can lengthen the lag time and decrease the steady-state activity of the enzyme.     

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.     

Inhibitory effects of substrate analogues on enzyme activity and substrate specificities of mushroom tyrosinase

Tyrosinase can catalyze the oxidation of o-diphenols to o-quinones. In this paper, some o-diphenols were used as mushroom tyrosinase substrates to study the catalyzed specificity of the enzyme. The enzyme kinetic analysis of substrate specificities and the substrate analogues towards mushroom tyrosinase has been investigated. Taking l-3,4-dihydroxyphenylalanine (I), 3,4-dihydroxyhydrocinnamic acid (II), 3,4-dihydroxycinnamic acid (III) and 1,2,4-benzenetriol (IV) as substrates, the results of specificity studies showed that the oxidation reaction of tested o-diphenols by mushroom tyrosinase followed Michaelis–Menten kinetics. The Michaelis–Menten constants for these four substrates were determined to be 0.615, 1.238, 0.331 and 1.886 mM, respectively. The values of V_m/K_m, which denotes the affinity of the enzyme to the substrate, were determined and compared, and the results showed that the affinity of the enzyme to these substrates followed the order: compound IV > III > I > II. Furthermore, mushroom tyrosinase cannot catalyze the oxidation of 3,4-dihydroxybenzonitrile (a), 3,4-dihydroxybenzaldehyde (b), 3,4-dihydroxybenzoic acid (c) and 2,3-dihydroxybenzoic acid (d). On the contrary, compounds a, b and c can inhibit the activity of tyrosinase for the oxidation of DOPA, while compound d had no effects on enzyme activity. The results show that compounds a and b are reversible non-competitive inhibitors.     

Inhibitory effects of the water extracts of Lavendula sp. on mushroom tyrosinase activity

This study aimed to evaluate the inhibition properties of six lavender species, including Lavendula angustifolia, Lavandula angustifolia “Vera”, Lavendula X allardii, Lavendula stoechas, Lavendula viridis and Lavendula X heterophylla, toward the activity of mushroom tyrosinase. When using l-3,4-dihydroxyphenylalanine (l-Dopa) as the substrate for mushroom tyrosinase, the water extracts of leaves and stems from L. stoechas and L. angustifolia “Vera” showed strong inhibitory effects against the activity of mushroom tyrosinase (70% and 66.4% inhibition, respectively). Oven-drying the leaves and stems or free-drying the water extracts significantly decreased the inhibitory abilities of the water extracts from all lavender species. The water extract from L. stoechas decreased the V_max values when using l-Dopa, catechol and 3,4-dihydroxyphenylacetic acid (DHPAA) as the substrates. It increased the value of K_m when l-Dopa and catechol were the substrates but it decreased the K_m when DHPAA was used. It behaved as a mixed-type inhibitor toward mushroom tyrosinase.     

Inhibitory effects on mushroom tyrosinase by p-alkoxybenzoic acids

The inhibitory kinetics of the diphenolase of mushroom tyrosinase by seven p-alkoxybenzoic acids has been studied. The results show that these derivatives of benzoic acid behave as reversible inhibitors. Among them, p-hydroxybenzoic acid is competitive, while p-methoxybenzoic acid is non-competitive, p-ethoxybenzoic acid is mixed-II type, and the rest all behave as classical uncompetitive inhibitors. The inhibition constants of all of the seven compounds assayed, characterizing the inhibition, were evaluated. The models of the interactions between the enzyme and the inhibitors are compared.     

龙井乙醇提取物对酪氨酸酶活性的抑制作用

以龙井、柿叶、贡菊和银杏叶为原料,用L-酪氨酸和L-DOPA为底物测定各原料的乙醇提取物对酪氨酸酶的抑制作用。结果表明,四种原料的乙醇提取物对酪氨酸酶均有一定的抑制作用,以龙井的抑制率最高,当浓度达到4mg·mL-1（以生药计）时,抑制率为30.36%,其IC50值为7.5mg/mL。龙井乙醇提取物对酪氨酸酶的抑制作用属于可逆过程,其抑制类型为混合型,KI为3.1,KIS为9.3。      

红酒对酪氨酸酶抑制作用的研究

通过研究红酒对酪氨酸酶的抑制作用,发现红酒对该酶具有良好的抑制作用.红酒中的多酚成分是抑制酪氨酸酶的重要活性物质,对酪氨陵酶的半抑制浓度(IC50)为0.14mg/L～0.18mg/L,抑制动力学类型为非竞争型.同时,通过测量红酒面膜原液中多酚含量及对酪氨酸酶的抑制活性,对市售红酒面膜产品的真伪及功效进行了鉴别和评价.