氨基葡萄糖－半胱氨酸美拉德反应产物对B16-F10的抑制作用

为评估氨基葡萄糖－半胱氨酸美拉德反应产物(GCMRPs)对小鼠皮肤黑色素瘤细胞B16-F10黑色素合成能力的影响,选择不同浓度 GCMRPs 作用于 B16-F10细胞,观察该产物对B16-F10细胞形态、增殖能力、酪氨酸酶酶活及黑色素含量的变化.结果显示： GCMRPs可明显抑制B16-F10黑色素合成,并表现出一定的细胞毒性.该产物在500μg·mL-1时,黑色素合成抑制率为42.31%.与曲酸相比,其毒性较大.因此, GCMRPs在皮肤美白上的应用有待进一步研究.     

一种具高活性的酪氨酸酶基因启动子片段的分离

利用大肠杆菌启动子探测质粒ｐＫＫ２３２－８为载体,经ＨｉｎｄⅢ－ＢａｍＨⅠ完全消化后与ＨｉｎｄⅢ－ＢａｍＨⅠ部分消化的嗜麦芽假单胞菌染色休ＤＮＡ进行体外连接,转化Ｅ．ｃｏｌｉＨＢ１０１感受态细胞,在含氨苄青霉素（Ａｐ）和氯霉素（Ｃｍ）的选择平板上筛选转化子．从随机挑选的５４株转化子中,获得一株抗氯霉素水平达１０００ｍｇ／Ｌ的转化子Ｅ．ｃｏｌｉＰＡＳＩ,所含重组质粒被命名为ｐＡＳＩ．经限制性酶切分析和杂交分析表明,ｐＡＳＩ质粒上插入了一段来源于嗜麦芽假单胞菌染色体的ＤＮＡ片段,其大小为１．４ｋｂ．ＳＤＳ－ＰＡＧＥ分析表明转化子Ｅ．ｃｏｌｉＰＡＳＩ在含Ｃｍ的培养基上,额外表达了一条２２×１０３的ＣＡＴ蛋白质带．将重组质粒ｐＷＳＹ上的嗜麦芽假单胞菌ｍｅｌ基因亚克隆到ｐＡＳＩ上１．４ｋｂ启动子功能片段下游,构建成Ｅ．ｃｏｌｉＡＷＳ工程菌株,使黑色素产率提高了７０．６％．     

Valonea Tannin: Tyrosinase Inhibition Activity,Structural Elucidation and Insights into the Inhibition Mechanism

Valonea tannin is a natural product readily extracted from acorn shells that has been suggested to have potential skin whitening properties. This study investigated the tyrosinase inhibition activity of extracted valonea tannin and the associated structure–function activity. Nuclear magnetic resonance spectroscopy and molecular weight analysis with gel permeation chromatography revealed that valonea tannin could be characterized as a hydrolysable tannin with galloyl, hexahydroxydiphenoyl and open formed-glucose moieties and an average molecular weight of 3042 ± 15 Da. Tyrosinase inhibition assays demonstrated that valonea tannin was 334 times more effective than gallic acid and 3.4 times more effective than tannic acid, which may relate to the larger molecular size. Kinetic studies of the inhibition reactions indicated that valonea tannin provided tyrosinase inhibition through mixed competitive–uncompetitive way. Stern–Volmer fitted fluorescence quenching analysis, isothermal titration calorimetry analysis and in silico molecule docking showed valonea tannin non-selectively bound to the surface of tyrosinase via hydrogen bonds and hydrophobic interactions. Inductively coupled plasma-optical emission spectroscopy and free radical scavenging assays indicated the valonea tannin had copper ion chelating and antioxidant ability, which may also contribute to inhibition activity. These results demonstrated the structure–function activity of valonea tannin as a highly effective natural tyrosinase inhibitor that may have commercial application in dermatological medicines or cosmetic products.     

Copper–Oxygen Dynamics in the Tyrosinase Mechanism

The dinuclear copper enzyme, tyrosinase, activates O₂ to form a (μ‐η²:η²‐peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X‐ray crystal structures of the active tyrosinases with substrate l ‐tyrosine. At their catalytic sites, CuA moved toward l ‐tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen‐bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O?O bond. Thus, the copper migration induced by the substrate‐binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ? carbon atom.     

Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

Tyrosinases mediate the ortho‐hydroxylation and two‐electron oxidation of monophenols to ortho‐quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.     

火棘黄酮的提取及美白功效研究

采用循环超声提取法提取火棘黄酮,通过正交试验确定其最佳工艺,用D101大孔树脂将其纯化,测定了火棘黄酮的马铃薯酪氨酸酶抑制率。并制备了火棘黄酮美白霜,通过人体实验,评价其美白功效。结果表明:火棘黄酮最佳的提取条件为乙醇体积分数为70%,料液比(g∶m L)为1∶15,提取温度60℃,提取时间为30min。火棘黄酮对马铃薯酪氨酸酶具有明显的抑制作用,抑制率达到50%时火棘黄酮浓度(IC50)为10.36 mg·m L-1,是熊果苷的2倍。火棘黄酮制成的美白霜有一定的美白能力,能显著降低皮肤黑色素含量和增加皮肤亮度而美白皮肤。结果说明火棘黄酮具有一定的美白功效,可以作为天然的美白化妆品添加剂。     

Isolation,Characterization, and Medicinal Potential of Polysaccharides of Morchella esculenta

Mushroom polysaccharides are active medicinal compounds that possess immune-modulatory and anticancer properties. Currently, the mushroom polysaccharides krestin, lentinan, and polysaccharopeptides are used as anticancer drugs. They are an unexplored source of natural products with huge potential in both the medicinal and nutraceutical industries. The northern parts of Pakistan have a rich biodiversity of mushrooms that grow during different seasons of the year. Here we selected an edible Morchella esculenta (true morels) of the Ascomycota group for polysaccharide isolation and characterization. Polysaccharopeptides and polysaccharides from this mushroom were isolated using the green chemistry, hot water treatment method. Fourier transform infrared spectroscopy revealed the sugar nature and possible beta-glucan type structure of these polysaccharides. Antioxidant assays showed that the deproteinized polysaccharides have moderate free radical scavenging activity. These isolated polysaccharides exhibited good acetylcholinesterase (AChE) and butyryl cholinesterase (BChE) inhibition activities. Therefore, these polysaccharides may be valuable for the treatment of Alzheimer’s and Parkinson’s diseases. Further bioassays are needed to discover the true potential of M. esculenta polysaccharides for medicinal purposes.     

Analysis of Kojic Acid Derivatives as Competitive Inhibitors of Tyrosinase: A Molecular Modeling Approach

Tyrosinases belong to the functional copper-containing proteins family, and their structure contains two copper atoms, in the active site, which are coordinated by three histidine residues. The biosynthesis of melanin in melanocytes has two stages depending on the actions of the natural substrates L-DOPA and L-tyrosine. The dysregulation of tyrosinase is involved in skin cancer initiation. In the present study, using molecular modeling tools, we analyzed the inhibition activity of tyrosinase activity using kojic acid (KA) derivatives designed from aromatic aldehydes and malononitrile. All derivatives showed conformational affinity to the enzyme active site, and a favorable distance to chelate the copper ion, which is essential for enzyme function. Molecular dynamics simulations revealed that the derivatives formed promising complexes, presenting stable conformations with deviations between 0.2 and 0.35 Å. In addition, the investigated KA derivatives showed favorable binding free energies. The most stable KA derivatives showed the following binding free energies: −17.65 kcal mol⁻¹ (D6), −18.07 kcal mol⁻¹ (D2), −18.13 (D5) kcal mol⁻¹, and −10.31 kcal mol⁻¹ (D4). Our results suggest that these derivatives could be potent competitive inhibitors of the natural substrates of L-DOPA (−12.84 kcal mol⁻¹) and L-tyrosine (−9.04 kcal mol⁻¹) in melanogenesis.     

Na_6PMo_(11)FeO_(40)对小鼠黑色素瘤B16细胞内黑色素合成抑制及抑菌活性研究

研究了过渡金属取代的Kiggen型结构的杂多酸盐Na_6PMo_(11)FeO_(40)对体外培养的小鼠黑色素瘤B16细胞的生物学效应,并分析了其对4种细菌的抗菌活性.结果显示:不同浓度的Na_6PMo_(11)FeO_(40)对B16细胞的细胞形态和数量有不同程度的影响,对B16细胞的增殖率、酪氨酸酶活性以及黑色素合成量的抑制呈现出明显的浓度效应.浓度为50~200μmol/L的Na_6PMo_(11)FeO_(40)能显著抑制B16细胞增殖(P0.05或P0.01);浓度为200μmol/L的Na_6PMo_(11)FeO_(40)对酪氨酸酶活性抑制极显著(P0.01),IC50为166.5μmol/L;Na_6PMo_(11)FeO_(40)对藤黄八叠球菌、金黄色葡萄球菌、枯草芽孢杆菌和大肠杆菌均有抗菌作用,且对球菌的抑制效果优于杆菌.实验结果表明,Na_6PMo_(11)FeO_(40)可作为具有防腐抑菌功能的新型酪氨酸酶抑制剂.