乙胺硫脲抑制蘑菇酪氨酸酶活性动力学

研究了乙胺硫脲对蘑菇酪氨酸酶单酚酶和二酚酶活性的抑制效应.结果表明,乙胺硫脲对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,导致单酚酶活力和二酚酶活力下降50%的乙胺硫脲浓度(IC50)分别为14.8和45.2μmol/L.在降低酪氨酸酶酶活的同时,乙胺硫脲能明显延长单酚酶和二酚酶的延滞时间.探讨了二酚酶延滞时间产生的原因:...     

L-半胱氨酸对酪氨酸酶的抑制动力学研究

用酶动力学方法考察了L-半胱氨酸对酪氨酸酶单酚酶和二酚酶活性的抑制效应。结果表明,L-半胱氨酸对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,导致单酚酶活力和二酚酶活力下降50%的L-半胱氨酸浓度(IC50)分别为20.3μmol/L和52.0μmol/L。在降低蘑菇酪氨酸酶酶活的同时,L-半胱氨酸能明显延长单酚酶和二酚酶的延滞时间。探讨了二酚酶延滞时间产生的原因:L-半胱氨酸与酪氨酸酶催化氧化产物多巴醌反应,形成了无色的L-DOPA的L-半胱氨酸衍生物,从而阻断了多巴色素的形成,直至体系中的L-半胱氨酸反应完全后,多巴醌才开始转化为多巴色素。L-半胱氨酸对二酚酶的抑制作用表现为不可逆的竞争性抑制。     

茯苓提取物对酪氨酸酶抑制动力学及刺激性研究

研究了茯苓提取物对酪氨酸酶单酚酶和二酚酶活力的影响及其抑制动力学,测试结果表明:茯苓提取物对酪氨酸酶单酚酶和二酚酶的活力均有抑制作用,单酚酶和二酚酶活力下降50%的茯苓提取物质量浓度(IC_(50))分别为0.008和0.040 g/L。酪氨酸酶单酚酶的催化反应存在迟滞时间,茯苓提取物对酪氨酸酶二酚酶活力的抑制作用表现为可逆效应。Lineweaver-Burk图显示茯苓提取物对二酚酶的作用表现为非竞争性与竞争性混合的抑制类型。在此基础上采用红细胞溶血实验初步评估茯苓提取物对皮肤的刺激性,结果显示含2.000 g/L茯苓提取物的样品无刺激性,质量浓度远大于单酚酶和二酚酶活力下降50%的茯苓提取物质量浓度IC_(50),茯苓提取物在0.200~0.800 g/L的质量浓度范围内可保证其安全添加且具有美白功效。     

绿原酸对酪氨酸酶活性影响及动力学研究

测试了绿原酸对酪氨酸酶单酚酶和二酚酶活力的影响。实验结果表明,绿原酸对酪氨酸酶单酚酶和二酚酶活性均有激活作用,使单酚酶和二酚酶相对酶活力增加50%的绿原酸质量浓度(IC50)分别为10.5 mg/L和1.1 g/L。绿原酸能消除单酚酶的迟滞时间,对二酚酶的激活作用表现为竞争性激活,激活常数KI=1.91 g/L。     

羟基甲氧基苯甲醛在化妆品中起增白作用的研究

采用酶学动力学方法研究2-羟基-4-甲氧基苯甲醛(HMB)对酪氨酸酶的单酚酶和二酚酶活力的抑制效应.结果表明,HMB对酪氨酸酶单酚酶的效应只表现对稳态活力有明显的抑制作用,但对酶作用的迟滞时间没有影响;随着抑制剂浓度增大,单酚酶活力呈指数下降,导致酶活力下降50%的抑制剂浓度(IC50)为0.352 mmol·L-1.HMB对二酚酶的抑制作用表现为可逆过程,IC50为0.164 mmol·L-1;抑制作用机理研究表明HMB是一种混合型抑制剂,其抑制常数KI和KIS分别为0.131 mmol·L-1和0.253 mmol·L-1.通过对HMB抑制酪氨酸酶机理的研究,阐明HMB作为化妆品添加剂的增白作用机理.     

鹰嘴豆异黄酮对酪氨酸酶的抑制动力学

采用酶动力学方法研究了鹰嘴豆异黄酮对酪氨酸酶单酚酶和二酚酶活力的抑制动力学效应.结果表明,鹰嘴豆异黄酮对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,使单酚酶和二酚酶活力下降50%的鹰嘴豆异黄酮质量浓度(IC50)分别为0.07 mg/mL和0.03 mg/mL.在降低酪氨酸酶酶活的同时,鹰嘴豆异黄酮能明显延长单酚酶的迟滞时间,质量浓度为0.08 mg/mL的鹰嘴豆异黄酮能使其迟滞时间由1.26 min延长至2.62 min.Lineweaver-Burk图显示鹰嘴豆异黄酮对二酚酶的抑制作用表现为可逆的竞争性抑制.     

荔枝壳提取物对酪氨酸酶的抑制作用

通过对酪氨酸酶催化L-多巴氧化速率的测定,研究了荔枝壳粉的乙醇萃取液对体外酪氨酸酶活性的抑制作用。结果表明,酶抑制率达到50%(酪氨酸酶活力下降50%)时所需的澄清液中多酚的质量浓度为0.052 mg/mL。复合抑制剂的酶抑制作用的Lineweaver-Burk线特点的分析结果显示,荔枝壳粉提取物对酪氨酸酶的抑制作用不是单一成分的抑制作用,而可能是多种抑制成分共同作用的结果。     

香草酸对酪氨酸酶催化活性的抑制作用

采用酶动力学方法研究了香草酸对酪氨酸酶单酚酶和二酚酶活力的抑制效应。结果表明,香草酸对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,导致单酚酶活力和二酚酶活力下降50%的香草酸浓度(IC50)约分别为1.3 mmol/L和2.6 mmol/L。香草酸能明显延长单酚酶的迟滞时间,2 mmol/L香草酸能使迟滞时间由1.1 m in延长至4.7 m in。香草酸对二酚酶的抑制作用表现为混合型抑制,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1.76 mmol/L和8.57 mmol/L。     

奥扎格雷对蘑菇酪氨酸酶的抑制机理研究

以L-DOPA为底物,抗血小板凝聚药奥扎格雷对酪氨酸酶具有很强抑制性,导致酶活力下降50%的奥扎格雷浓度(IC50)为3.45 mmol/L。奥扎格雷对酪氨酸酶抑制作用表现为可逆的竞争性和反竞争性混合型的抑制。按邹承鲁[1]所介绍的酶失活过程中的底物反应动力学方法测定奥扎格雷对酪氨酸酶的抑制作用动力学,1.0 mmol/L的奥扎格雷对酪氨酸酶和酪氨酸酶-底物复合物的抑制作用动力学常数分别为87.28和66.07μmol/L。     

当归提取物对酪氨酸酶的抑制作用

以乙醇为提取剂,用高压均质技术提取当归,采用酶动力学方法研究了当归提取物对酪氨酸酶活力的抑制效应。结果表明,当归提取物对酪氨酸酶单酚酶和二酚酶活性均有抑制作用,相对抑制率达到50%的当归提取物质量浓度(IC50)分别约为0.32 g/L和0.58 g/L,比熊果苷抑制二酚酶活性的IC50值1.44 g/L有效得多。Lineweaver-Burk图显示当归提取物对二酚酶的抑制作用表现为竞争性抑制,对酪氨酸酶的抑制常数(KI)为0.24 g/L。     

Repositioning of Thiourea-Containing Drugs as Tyrosinase Inhibitors

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC₅₀ of 14 and 15 μM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 μM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.     

家蝇酪氨酸酶生物化学性质及农药对其抑制作用

采用时间动力学法对家蝇(Muscadomestica)酪氨酸酶的最适反应条件、对抑制剂的敏感度以及发育期变化规律进行了研究。结果表明:家蝇酪氨酸酶反应的最适pH值为7.4,反应线性时间为0~2min。不同发育阶段家蝇酪氨酸酶活性的大小依次为3龄>2龄>1龄>成虫>蛹。苯基硫脲(phenylthiourea,PTU)对家蝇的抑制中浓度(I50)值为0.15mmol/L,为酪氨酸酶特异性抑制剂;对硫磷、辛硫磷、灭多威、残杀威、吡虫啉及阿维菌素对家蝇酪氨酸酶有不明显抑制作用。     

Antioxidative Characteristics of Anisomeles indica Extract and Inhibitory Effect of Ovatodiolide on Melanogenesis

The purpose of the study was to investigate the antioxidant characteristics of Anisomeles indica methanol extract and the inhibitory effect of ovatodiolide on melanogenesis. In the study, the antioxidant capacities of A. indica methanol extract such as DPPH assay, ABTS radical scavenging assay, reducing capacity and metal ion chelating capacity as well as total phenolic content of the extract were investigated. In addition, the inhibitory effects of ovatodiolide on mushroom tyrosinase, B16F10 intracellular tyrosinase and melanin content were determined spectrophotometrically. Our results revealed that the antioxidant capacities of A. indica methanol extract increased in a dose-dependent pattern. The purified ovatodiolide inhibited mushroom tyrosinase activity (IC(50) = 0.253 mM), the compound also effectively suppressed intracellular tyrosinase activity (IC(50) = 0.469 mM) and decreased the amount of melanin (IC(50) = 0.435 mM) in a dose-dependent manner in B16F10 cells. Our results concluded that A. indica methanol extract displays antioxidant capacities and ovatodiolide purified from the extract inhibited melanogenesis in B16F10 cells. Hence, A. indica methanol extract and ovatodiolide could be applied as a type of dermatological whitening agent in skin care products.     

对羟基苯乙酸在酪氨酸酶催化中的动力学研究

采用酶动力学方法,研究了对羟基苯乙酸对蘑菇酪氨酸酶单酚酶和二酚酶酶活的动力学。实验结果表明,对羟基苯乙酸对酪氨酸酶单酚酶具有抑制作用,对单酚酶酶活的相对抑制率达到50%的对羟基苯乙酸浓度(IC50)为0.79mmol/L,并能明显延长单酚酶的延滞时间,0.8mmol/L的对羟基苯乙酸能使延滞时间由95s延长至182s。对于二酚酶活性,对羟基苯乙酸激活浓度为0.8mmol/L,其对二酚酶酶活的激活率达108%。Lineweaver-Burk图显示对羟基苯乙酸对二酚酶的激活作用表现为竞争性和反竞争性的混合型激活,对游离酶的激活常数和对酶-底物配合物的激活常数分别为-0.16和-0.19mmol/L。     

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety

We previously reported (E)-β-phenyl-α,β-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the β-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,β-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC₅₀ = 18.09 ± 0.25 μM), 1h (IC₅₀ = 4.14 ± 0.10 μM), and 2a (IC₅₀ = 15.69 ± 0.40 μM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC₅₀ = 48.62 ± 3.38 μM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.     

Depigmenting effect of <Emphasis Type="Italic">Cinnamomum cassia</Emphasis> Presl in B16F10 melanoma cells

To find efficient depigmenting agents, we examined several Chinese herbs for melanogenesis inhibition and toxicity. Cinnamomum cassia Presl exhibited low cytotoxicity at even high concentration (200 μg/ml). The effects on melanogenesis of cultured B16 melanoma cells, mushroom tyrosinase activity, and free radical scavenging activity were further assessed. The methanol extracts of this plant showed the suppression of melanin synthesis. Melanin content was dose-dependently decreased by this herb extract as compared with control cells. It also showed good anti-oxidative activity (IC₅₀=3.7 μg/ml) but no inhibition of mushroom tyrosinase activity. This result showed that Cinnamomum cassia Presl extract might be useful and safe as a new whitening agent in cosmetics.     

Melanogenesis inhibitory effect of dehydroevodiamine isolated from fruits of <Emphasis Type="Italic">Evodia rutaecarpa</Emphasis>

Dehydroevodiamine, an alkaloid, was isolated from the fruit of Evodia rutaecarpa and melanin production, and tyrosinase inhibition in B16F10 melanoma cells treated with the isolated dehydroevodiamine was investigated. The compound decreased melanin synthesis significantly without promoting cytotoxicity. The IC₅₀ value of dehydroevodiamine for melanogenesis and cell viability were 59.8 μM and 90.0 μM, respectively. The L-dopa oxidase activity of mushroom tyrosinase was reduced after dehydroevodiamine treatment by about 22.4% at a concentration of 33.2 μM. However, there was no effect on cellular tyrosinase activity. These results indicate that the observed decrease in melanin content after treatment with dehydroevodiamine was attributed to the direct inhibition of tyrosinase activity, rather than the suppression of tyrosinase gene expression. Dehydroevodiamine may be a promising new agent for use in cosmeceutical application.     

阿魏酸对蘑菇酪氨酸酶的抑制效应

The inhibitory effect of ferulic acid on the diphenolase activity of mushroom tyrosinase and the kinetic behavior were studied with L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The inhibitor concentration leading to 50% relative activity lost （IC50） was estimated to be 0.15 mmol·L^-1. The inhibition mechanism obtained from Lineweaver-Burk plots shows that ferulic acid is a competitive inhibitor and the inhibition of tyrosinase by ferulic acid is a reversible reaction. The equilibrium constant for ferulic acid binding with the tyrosinase was determined to be 0.25 mmol·L^-1 for diphenolase. Keywords tyrosinase, ferulic acid, kinetics, inhibition, L-DOPA, diphenolase