二氢杨梅素对酪氨酸酶抑制作用研究

研究了二氢杨梅素对酪氨酸酶的单酚酶和二酚酶抑制作用和机理。结果表明,二氢杨梅素对单酚酶、二酚酶的抑制率分别为95.87%、69.01%;二氢杨梅素对单酚酶的抑制作用表现为酶催化反应的迟滞时间延长;二氢杨梅素对二酚酶的抑制作用表现为可逆混合型抑制,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150μmol.L-1和83μmol.L-1。二氢杨梅素可作为天然的酪氨酸酶抑制剂。     

α-熊果苷对酪氨酸酶二酚酶激活动力学的研究

采用酶动力学方法,以L-多巴为底物,通过酪氨酸酶二酚酶催化反应体系,研究α-熊果苷对二酚酶活性的影响。实验结果表明:α-熊果苷对酪氨酸酶的二酚酶催化反应产生激活效应,反应过程没有迟滞时间;由Lineweaver-Burk双倒数作图得知,在一定范围内,随着催化反应系统中α-熊果苷浓度的增大,动力学参数Km逐渐减小,而最大反应速率Vmax逐渐增大,表明α-熊果苷对酪氨酸酶二酚酶的激活作用为竞争及非竞争混合型;并且从酶的构象变化及减少酶的自杀性失活的角度,阐述了α-熊果苷对酪氨酸酶二酚酶的激活机制。     

气相色谱法测定鱼塘水中2,4-二氯-6-硝基苯酚和五氯酚含量

鱼塘水中残留的2种除草剂(2,4-二氯-6-硝基苯酚和五氯酚)经GDX201固相萃取柱富集后,用二氯甲烷洗脱,洗脱液用N-(叔丁基二甲硅烷基)-N甲基三氟乙酰胺衍生化,所得衍生化产物溶于乙酸乙酯中,用气相色谱法测定,从而获得上述2种除草剂的含量。2,4-二氯-6-硝基苯酚和五氯酚的峰面积与其质量浓度均在10～200μg.L-1内呈线性关系,检出限(3S/N)分别为0.09μg.L-1和0.16μg.L-1。以鱼塘水为基体进行加标回收试验,测得回收率依次为98.3%,98.6%;测定值的相对标准偏差(n=5)分别为3.6%和4.3%。     

稀土及其配合物对蛇毒磷脂酶A_2活性的影响

分别研究了三价稀土离子 (La3 + ,Eu3 + ,Dy3 + ,Yb3 + )、二乙三胺五乙酸及其衍生物二乙三胺五乙酸 -双二甲酰胺 ,二乙三胺五乙酸 -双 (异烟肼 )与稀土离子的配合物以及Tb -谷氨酰胺配合物对蛇毒磷脂酶A2 活性的影响 .浓度低于 <3μmol/L的稀土离子可以激活磷脂酶A2 ,浓度大于 5 μmol/L后稀土离子对酶活性表现出抑制作用 ;外源Ca2 + 离子的加入可以缓解稀土离子对酶活性的抑制作用 ,表明稀土离子和钙离子是竞争性地结合在酶的活性部位 ;稀土离子和二乙三胺五乙酸及其衍生物的配合物对酶活性没有明显影响 ;Tb -谷氨酰胺在浓度大于 10 μmol/L后开始抑制酶的活性     

欧芹酚甲醚腙类衍生物的合成及抗乙酰胆碱酯酶活性研究

以香豆素类化合物欧芹酚甲醚为先导化合物,设计合成了15个欧芹酚甲醚腙类衍生物,所有目标化合物经熔点、¹H NMR和MS进行结构确证。体外抑制乙酰胆碱酯酶活性结果表明,在100μmol/L浓度下,目标化合物3d对乙酰胆碱酯酶具有较强的抑制活性,其抑制率达到66.1%。初步构效关系表明,在欧芹酚甲醚腙类化合物的苯环上引入斥电子基能提高对乙酰胆碱酯酶的抑制活性。分子对接表明化合物3d可以和乙酰胆碱酯酶的催化活性中心部位显著结合。     

富勒烯膦酸衍生物纳米水悬液对DNA限制性内切酶的抑制作用及其机理

富勒烯纳米颗粒水悬液的生物学效应正在引起人们的极大关注.采用具有EcoRⅠ、BamHⅠ、Cfr9Ⅰ和XmaⅠ单一酶切位点的pEGFP-N1超螺旋型质粒为底物,检测了单加成亚甲基富勒烯[60]二膦酸四乙酯（mono-methanophosphonate fullerene,MMPF）和二加成亚甲基富勒烯[60]二膦酸四乙酯（bis-methanophosphonate fullerene,BMPF）的纳米水悬液（分别表示为n-MMPF和n-BMPF）对各种DNA限制性内切酶的抑制作用.实验发现,n-MMPF和n-BMPF均对EcoRⅠ有抑制作用,但后者的作用更强些.加入n-BMPF后,对BamHⅠ酶切反应的半抑制浓度IC50值大于30μmol/L,而对EcoRⅠ的半抑制浓度IC50值仅为4.3μmol/L,对同工酶Cfr9Ⅰ和XmaⅠ的半抑制浓度IC50值分别为11.7和8.3μmol/L.当EcoRⅠ酶切反应完全被n-BMPF抑制时,在反应体系中增加底物pEGFP-N1的量,并不能观察到酶切产物线型质粒,但是在酶切体系中增加酶的量则能拮抗n-BMPF的作用.两种活性氧清除剂甘露醇和叠氮化钠在浓度为2-10mmol/L时,不能拮抗n-BMPF的作用,说明n-BMPF的抑制活性与活性氧无关.这些结果首次表明,富勒烯纳米颗粒水悬液具有作为DNA限制性内切酶抑制剂的生物活性.     

基于三辛胺缔合作用的草酸及柠檬酸电极的制备

草酸、柠檬酸可分别与三辛胺(TOA)生成稳定的缔合物,化学分析和红外光谱表明缔合比为1∶1。以此缔合物为活性物质制作的PVC膜电极,测定溶液中的草酸盐、柠檬酸盐的线性范围分别为1.0×10-4~1.0×10-1mol.L-1和1.0×10-5~1.0×10-2mol.L-1。该电极用于简单模拟体系中草酸和柠檬酸浓度的测定,效果良好。     

葡萄糖浓度的酶联荧光毛细分析法测定

该文以葡萄糖氧化酶和辣根过氧化物酶为催化剂,使含有对甲基酚的葡萄糖溶液体系通过酶偶联催化反应生成荧光物质,从而实现对葡萄糖浓度的测定。优化的实验条件为:反应时间20 min;NH3-NH4Cl缓冲溶液(pH 10.4);对甲基酚浓度30.0μmol/L。分别采用荧光毛细分析法和荧光光谱分析法测定了相同浓度的系列葡萄糖溶液的荧光强度。在5.0~500.0μmol/L范围内,两种方法测得的荧光强度均与葡萄糖浓度的对数成正比。通过对比测试结果分析了两种方法的优缺点。     

丹皮酚在APTS与离子液体复合修饰碳糊电极上的电化学性质及电分析方法

研究了丹皮酚(PN)在3-氨基丙基三氧基硅烷(APTS)与离子液体([BnMIM]PF6)复合修饰碳糊电极(APTS-[BnMIM]PF6/CPE)上的电化学行为和电化学动力学性质,并用循环伏安法(CV)及计时电流法(CA)测得PN在APTS-[BnMIM]PF6/CPE上的电极反应动力学参数。实验结果表明,PN在APTS-[BnMIM]PF6/CPE上发生了受扩散控制的不可逆电化学氧化过程。用方波伏安法(SWV)测得PN氧化峰电流与其浓度在9.0×10-7~2.0×10-4mol·L-1和3.0×10-4~1.5×10-3mol·L-1范围内呈良好线性关系,检出限(LOD,S/N=3)和定量下限(LOQ,S/N=10)分别为3.5×10-8mol·L-1和1.2×10-7mol·L-1。同时运用该方法对丹皮酚注射液中PN含量进行了电化学定量测定,其RSD为0.58%~2.4%,加标回收率为96.0%~102.0%。     

丹皮酚在APTS与离子液体复合修饰碳糊电极上的电化学性质及电分析方法

研究了丹皮酚(PN)在3-氨基丙基三氧基硅烷(APTS)与离子液体(［BnMIM］PF6)复合修饰碳糊电极(APTS-［BnMIM］PF6/CPE)上的电化学行为和电化学动力学性质,并用循环伏安法(CV)及计时电流法(CA)测得PN在APTS-［BnMIM］PF6/CPE上的电极反应动力学参数。实验结果表明,PN在APTS-［BnMIM］PF6/CPE上发生了受扩散控制的不可逆电化学氧化过程。用方波伏安法(SWV)测得PN氧化峰电流与其浓度在9.0×10-7~2.0×10-4 mol?L-1和3.0×10-4~1.5×10-3 mol?L-1范围内呈良好线性关系,检出限(LOD,S/N=3)和定量下限(LOQ,S/N=10)分别为3.5×10-8 mol?L-1和1.2×10-7 mol?L-1。同时运用该方法对丹皮酚注射液中PN含量进行了电化学定量测定,其RSD为0.58%~2.4%,加标回收率为96.0%~102.0%。     

Inhibitory Effects of Fatty Acids on the Activity of Mushroom Tyrosinase

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.     

Inhibition of α/β-K6P2W18O62·10H2O on the Activity of Mushroom Tyrosinase and Its Antimicrobial Effects

Dawson-type phosphotungstic polyoxometalate α/β-K₆P₂W₁₈O₆₂·10H₂O(P₂W₁₈) was synthesized and its inhibitory effect on the mushroom tyrosinase was investigated. It could inhibit diphenolase activity of mushroom tyrosinase as an irreversible inhibitor. When the concentration of the enzyme reached 0.0176 mg/mL, the concentration of P₂W₁₈ leading to 50% activity lost(IC₅₀) was 0.05 mmol/L for monophenolase and 0.64 mmol/L for diphenolase. In addition, the antimicrobial activity of P₂W₁₈ was evaluated by zone of inhibition test. The results show that P₂W₁₈ possesses effective antimicrobial ability against Escherichia coli, Bacillus subtilis, yeast, especially Escherichia coli and yeast.     

A Novel Heptapeptide with Tyrosinase Inhibitory Activity Identified from a Phage Display Library

Peptidic inhibition of the enzyme tyrosinase, responsible for skin pigmentation and food browning, would be extremely useful for the food, cosmetics, and pharmaceutical industries. In order to identify novel inhibitory peptides, a library of short sequence oligopeptides was screened to reveal direct interaction with the tyrosinase. A phage displaying heptapeptide (IQSPHFF) was found to bind most strongly to tyrosinase. The inhibitory activity of the heptapeptide was evaluated using mushroom tyrosinase. The results showed that the peptide inhibited both the monophenolase and diphenolase activities of mushroom tyrosinase with IC₅₀ values of 1.7 and 4.0 mM, respectively. The heptapeptide is thought to be a reversible competitive inhibitor of diphenolase with the inhibition constants (Ki) of 0.765 mM. To further investigate how the heptapeptide exerts its inhibitory effect, a docking study between tyrosinase and heptapeptide was performed. The simulation showed that the heptapeptide binds in the active site of the enzyme near the catalytically active Cu ions and forms hydrogen bonds with five histidine residues on the active site. Phage display technology is thus a useful approach for the screening of potential tyrosinase inhibitors and could be widely applicable to a much wider range of enzymes.     

SARS 3CL蛋白酶同源二聚化与底物结合互为别构调控因素

研究了严重急性呼吸系统综合症(SARS)冠状病毒3C-Like蛋白酶(3CLpro)在存在底物或抑制剂时的二聚体形成情况. 通过测定酶活性随酶浓度的变化, 拟合出在底物存在下酶二聚体的解离常数约为0.94 μmol·L-1, 小于纯蛋白酶的二聚体解离常数(14.0 μmol·L-1), 表明底物对二聚体的形成具有增强作用. 选用与底物具有类似结合方式的靛红类抑制剂N-萘甲基靛红-5-甲酰胺(5f), 利用超速离心沉降速率方法定量测定了SARS 3CL蛋白酶单体和二聚体在不同浓度5f时的含量, 发现5f同样具有诱导二聚体形成的能力. 在3 μmol·L-1蛋白酶浓度下测定得到诱导二聚的EC50 值(半数有效浓度)约为1 μmol·L-1, 说明二聚体中只有一个单体与抑制剂结合. 研究结果表明, 随着底物浓度的升高, SARS 3CL蛋白酶会形成更多的二聚体, 而二聚体含量的提高又反过来提高酶的活性, 这种双向别构调控机制有可能是病毒用来调控多聚蛋白水解速率和组装时机的一种方法.     

甲氧苄啶修饰玻碳电极安培法对过氧化氢的测定

在0.1 mol·L~(-1) KNO_3底液中,研究了甲氧苄啶在玻碳电极上的电化学性质,发现在0.140、0.177 V处出现1对可逆的氧化还原峰.进一步用电化学沉积的方法将甲氧苄啶修饰在玻碳电极上,考察了各种实验条件对修饰电极性能的影响,并通过电子扫描显微镜和电化学阻抗谱对修饰电极的表面性质进行了表征,所制得的修饰电极对过氧化氢的还原有很好的催化作用.在-0.3 V的工作电位下,用计时安培法对过氧化氢进行了测定,过氧化氢的浓度在1.96×10~(-5) ～1.10×10~(-3) mol·L~(-1)范围内与响应电流呈线性关系,检出限为4.0 μmol·L~(-1).该修饰电极制作简单、使用寿命长,实际试样的回收率为97% ～104%.     

FeS阳极过程电化学研究

采用高温合成法由光谱纯的Fe和S制备FeS,并应用电位扫描和电位阶跃法研究FeS在0.5mol·L-1H2SO4+0.5mol·L-1K2SO4溶液中的阳极电化学行为.考察酸度对FeS阳极极化的影响,测定阳极过程的传递系数β、交换电流I0和Fe2+在FeS中的固相扩散系数DFe以及不同电极电位下阳极电化学反应活化能ΔEa.     

Tyrosinase inhibition kinetic studies of standardized extract of Berberis aristata

The stem bark and wood of Berberis aristata DC (Daruharidra) are one of the principal ingredients of traditional skin lighting and exfoliating scrub preparation in India. The standardised extract of B. aristata was screened to evaluate their in vitro antityrosinase activity and inhibition kinetics. Phytochemical and pharmacological studies were carried out with different solvent fractions of the methanol extract of B. aristata (MEBA). RP-HPLC analysis was used to determine the berberine content in extract and fractions of B. aristata. MEBA showed maximum berberine content. Extract and fractions of B. aristata contain the maximum amount of alkaloids than other constituents. In tyrosinase inhibition assay, MEBA was found to possess highest dose-dependent monophenolase and moderate diphenolase activity. The enzyme kinetic study revealed that MEBA possessed mixed type inhibition of monophenolase activity of tyrosinase. These bioactivities indicate that the MEBA has antihyperpigmentation potential in human skin.     

莠去津分子印迹聚合物的合成及其吸附性能

以莠去津(1)为模板分子,甲基丙烯酸(MAA)为功能单体,乙二醇二甲基丙烯酸酯(EDMA)为交联剂,在偶氮二异丁腈(AIBN)的引发下,于65℃聚合17 h合成了对1有特异识别性能的分子印迹聚合物(2)。用紫外分光光度法探索了1与MAA的最佳比例,研究了2的吸附性能力,并利用高效液相色谱法对2的选择性进行了考察。用Scatchard法分析表明,2通过氢键作用力结合,存在两种结合位点,对1的吸附存在两种形态,最大表观吸附量(Qmax,1)为130.9 nmol.g-1,平衡离解常数(Kd,1)为30.8 nmol.L-1,Qmax,2为540.5 nmol.g-1,Kd,2为450.5 nmol.L-1。与西玛津相比,2对1显示出一定的选择性。以2作为填料制备出具有莠去津分子印迹的固相萃取柱,可对水质中2×10-8mol.L-1以下的待测物进行富集和分离,回收率近90%。     

The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of “Substrate‐Guiding Residues” for Enzymatic Specificity

Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono‐ and o‐diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono‐ and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed.     

High-resolution monophenolase/diphenolase/radical scavenging profiling for rapid screening of natural whitening candidates from Rosa rugosa cv. ‘Plena’

Antioxidants and tyrosinase inhibitory components were successfully screened and separated from Rosa rugosa cv. ‘Plena’ by high-performance liquid chromatography microfractionation bioactive screening combined with several separation and purification methods. Ethyl acetate extract of Rosa rugosa cv. ‘Plena’ showed high antioxidant activity and tyrosinase inhibitory activity. High-speed countercurrent chromatography, silica gel column chromatography, and semi-preparative high-performance liquid chromatography were used for the preparative separation of four bioactive components from ethyl acetate extract. Two tyrosinase-inhibiting active substances, flavogallonic acid, and N¹-N⁵-N¹⁰-tri-4-p-coumaroylspermidine, were isolated from Rosa rugosa cv. ‘Plena’, and they showed great monophenolase inhibition activity (half-maximal inhibitory concentration: 664.60 and 23.77 μg/ml, respectively) and excellent diphenolase inhibition activity (half-maximal inhibitory concentration: 23 614.61 and 16.80 μg/ml, respectively). Meanwhile, gallic acid, flavogallonic acid, and ellagic acid were shown to have excellent 1,1-diphenyl-2-picryl-hydrazyl antioxidant activity (half maximal inhibitory concentration: 6.66, 20.17, and 13.45 μg/ml), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant activity (half maximal inhibitory concentration: 3.53, 3.83, and 2.78 μg/ml). Molecular docking revealed that flavogallonic acid and N¹-N⁵-N¹⁰-tri-4-p-coumaroylspermidine had a strong binding affinity (–9.3 and –10 kcal/mol, respectively) to tyrosinase through hydrogen bonding and hydrophobic interactions.