橘色双冠丽鱼TYR基因的克隆及其发育时序和组织表达分析

酪氨酸酶(tyrosinase,TYR)是动物体内黑色素合成信号通路中起关键性作用的限速酶,黑色素生成的速度和种类取决于TYR基因的表达量和活性水平.为了解色素相关基因与体色变化的关系,采用cDNA末端快速克隆技术(rapid-amplification of cDNA ends,RACE)技术获得橘色双冠丽鱼(Amphilophus citrinellus) TYR基因cDNA全序列,并利用qRT-PCR分析了橘色双冠丽鱼体色变化不同时期和不同组织中TYR基因的表达差异.实验获得cDNA全长序列2 683 bp,其中阅读框1 620bp,编码540个氨基酸,5'UTR区246 bp,3'UTR区817 bp.氨基酸序列和系统发育分析表明,相比与脊椎动物间的保守性,TYR蛋白序列在鱼类间的保守性更高.qRT-PCR结果表明,TYR基因在胚胎各期均有表达,受精期表达量最低,血液循环期开始急速上升;在“黑色-灰色-亮黄色”三个典型体色褪黑过程中,各组织TYR基因表达量均在黑色期呈现最高值,显著高于灰色和亮黄色期,灰色到亮黄色时期鳞片和尾鳍的TYR基因表达量逐渐降低,但差异不显著(P＞0.05),而皮肤组织的TYR基因表达量在三个体色褪黑期均显著降低(P＜0.05);亮黄色时期,TYR基因在心、肾、脑、鳔、性腺、尾鳍和眼等组织均有表达,其中眼部表达量最高,其次是鳔和尾鳍,皮肤和性腺表达量最低.橘色双冠丽鱼TYR基因在三个典型体色褪黑期表达逐渐降低,可能与红色素细胞和黄色素细胞逐渐增多以及黑色素细胞数量和分布比例发生变化相关.本研究通过了解体色变异的分子基础,为鱼类体色遗传和体色改良研究积累资料.     

Screening of extracts of Japanese woods for melanin biosynthesis inhibition

The effects of 120 methanol extracts prepared from bark and heartwood of 69 types of Japanese wood on the melanin production of B16 melanoma cells were examined. The melanin content of B16 melanoma cells was determined spectrophotometrically at 405nm. The extracts were also examined for their effects on cell viability. We found that the methanol extracts of Fagus crenata (buna, wood, 100μg/ml), Sapium sebiferum (Nankinhaze, wood, bark, 10μg/ml), and Zelkova serrata (keyaki, wood, 10μg/ml) greatly inhibited the melanin production of B16 melanoma cells without significant cytotoxicity. However, these extracts did not inhibit tyrosinase activity at the concentration of 100μg/ml. These findings indicate that the depigmenting mechanism of these extracts involves the suppression of some pigmenting signals in stimulating melanogenesis rather than the inhibition of tyrosinase activity. Part of this study was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, Japan, March 2003     

酪氨酸酶的提取及其催化活性研究

［目的］进一步研究影响酪氨酸催化活性的因素，减少黑色素在生物体内的生成。［方法］以pH值7.2的Na2HPO4-HCl缓冲溶液为体系, 用分光光度计在480 nm处测定马铃薯提取液的吸光度,以吸光度对时间的变化率（ΔA/Δt） 为反应速率，建立多巴溶液的转换动力学曲线，从而可计算出酪氨酸酶的活性。［结果］结果显示，在480 nm波长下, 以pH值7.2的Na2HPO4-HCl缓冲溶液为体系，对测定体系干扰小, 所测数据波动小,酪氨酸酶的活性稳定性高，为进一步确定和研究影响酪氨酸酶活性的因素提供了理论依据。［结论］ 试验以缓冲溶液为底物研究和测定酪氨酸酶的催化活性，准确度高，效果佳。     

响应曲面法优化美拉德反应以制备蘑菇酪氨酸酶活性抑制组分

MRPs作为天然抗褐变剂,可抑制引起果蔬酶促褐变的酪氨酸酶的酶活。为进一步优化美拉德反应以制备酪氨酸酶活性抑制组分,本研究采用响应面法对MRPs制备工艺进行研究。在单因素实验基础上,确定美拉德反应初始pH、反应温度、反应时间、底物浓度比(糖∶氨基酸)4个因素,设计4因素5水平的二次回归中心组合试验。经响应面法分析,为方便实际操作,确定最佳工艺条件为:初始pH为2.94,反应时间为1.9 h,反应温度为110.09℃,底物浓度比(糖∶氨基酸)为0.57。经实验验证,得出结果与理论值相对误差较小,故采用二次回归中心组合设计得到的最优MRPs制备条件参数准确可靠。     

高良姜多糖对酪氨酸酶抑制作用的研究

[目的]研究高良姜多糖对酪氨酸酶活性的抑制作用。[方法]以新鲜的高良姜和马铃薯为原料,通过比色法、色差仪法、Photoshop图像灰度值分析等测定高良姜多糖对马铃薯酪氨酸酶活性的抑制率,以及对马铃薯匀浆褐变度和反应体系颜色值变化的影响。[结果]试验得出,高良姜多糖对酪氨酸酶的活力有显著的抑制作用,其IC50为0.315 mg/m L,能显著降低反应体系的褐变度。[结论]高良姜多糖具有较强的酪氨酸酶抑制活性,可望用于美白护肤、果汁护色、鲜切果蔬保鲜等领域,值得进一步研究。     

茶叶提取物的美白作用及其机理研究进展

茶叶是一种可以保健和预防人体疾病的健康食品,具有广泛的生物活性成分。整理归纳了近几年国内外茶叶美白功效的文献,从黑色素的合成过程、美白作用调节机理和茶叶提取物的美白作用三方面进行了综述,提出动物实验、病理学及毒理学等方面研究是未来开发茶叶提取物美白产品的研究方向,展望了茶叶美白产品的前景。     

酶的提取及其催化活性研究

[目的]酪氨酸酶是一种多酚氧化酶,具有广泛用途,因此对酪氨酸酶的研究受到广泛重视。[方法]试验以 pH=7.2的 Na2HPO4-HCl缓冲溶液为体系,用分光光度计在480nm处测定土豆提取液的吸光度,以吸光度对时间的变化率(ΔA/Δt)为反应速率,建立多巴溶液的转换动力学曲线,从而可计算出酪氨酸酶的活性。[结果]结果显示在此波长下,以 pH=7.2的Na2HPO4-HCl缓冲溶液为体系,对测定体系干扰小,所测数据波动小,酪氨酸酶的活性稳定性高,为进一步确定和研究影响酪氨酸酶活性的因素提供理论依据。[结论]该试验以缓冲溶液为底物研究和测定酪氨酸酶的催化活性,准确度高,效果佳。     

Phosphorus ameliorates crop productivity,photosynthetic efficiency,nitrogen-fixation,activities of the enzymes and content of nutraceuticals of Lablab purpureus L.

Hyacinth bean (Lablab purpureus L.) serves as a good source of vegetable proteins in human diet. Its seeds and pods contain as much as 20–28% protein. Besides, it contains tyrosinase enzyme, which has potential use in the treatment of hypertension. Phosphorus deficiency causes a serious yield and quality constraint of beans at Aligarh, Western Uttar Pradesh, India. To address the problem, a pot experiment was conducted to study the effect of basal phosphorus application on the agricultural performance of this medicinal legume. The plants were grown in pots containing soil supplied with five levels of phosphorus viz. 0, 25, 50, 75 and 100 mg P kg⁻¹ soil as potassium dihydrogen orthophosphate (KH₂PO₄). The growth and other physiological attributes, leaf nutrient contents, nodule-nitrogen and leghemoglobin content were studied at 60, 90 and 120 days after sowing (DAS), photosynthesis and other related parameters were measured at 90 DAS and yield and quality attributes were recorded at harvest (150 DAS). Nitrate reductase and carbonic anhydrate activities, leaf-N, -P, -K and -Ca contents and nodule-nitrogen and leghemoglobin contents reached the maximum extent at 60 DAS. At 90 and 120 DAS, the values decreased significantly. Chlorophyll content, carotenoids content, and photosynthesis were at maximum level at 90 DAS. At various growth stages, phosphorus application at 75 mg P kg⁻¹ soil resulted in maximum amelioration of most of the parameters studied. It increased the seed-yield by 38.3%, seed-protein content by 14.9% and seed-carbohydrate content by 5.0%, relative to the control. It was concluded that there was a hidden hunger of hyacinth bean for phosphorus owing to soil-phosphorus deficiency that was ameliorated effectively by its basal dressing at 75 mg P kg⁻¹ soil.     

天麻素对B16黑素瘤细胞黑色素形成的影响

[目的]探讨天麻素对体外培养的B16鼠黑素瘤细胞黑素合成的影响及其作用机制。[方法]选择不同浓度的天麻素作用于体外培养的小鼠B16黑素瘤细胞,测定细胞黑素生成量,通过测定天麻素对蘑菇酪氨酸酶活性的影响,初步探讨天麻素影响黑色素形成的机制。[结果]当浓度大于15 mmol/L时,天麻素能抑制B16黑素瘤细胞黑色素形成和酪氨酸酶活性,且呈浓度依赖性。天麻素对蘑菇酪氨酸酶的半抑制浓度(IC50)为(69.37±9.22)mmol/L,对酪氨酸酶的抑制作用表现为混合型可逆抑制,抑制常数Ki=(123.8±20.2)mmol/L,提示天麻素可能通过抑制酪氨酸酶活性抑制B16细胞黑色素的形成。[结论]天麻素可作为一种潜在的色素障碍性疾病治疗药物。     

Betula pendula leaves: polyphenolic characterization and potential innovative use in skin whitening products

The research of new tyrosinase inhibitors is currently important for the development of skin whitening agents; particularly, birch leaves extracts are included in many skin cosmetic products. In this study, the potential ability of Betula pendula leaves ethanolic extract (BE) was evaluated on mushroom tyrosinase activity. Results showed that BE was capable to inhibit dose-dependently l-DOPA oxidation catalyzed by tyrosinase. The inhibition kinetics, analyzed by Lineweaver-Burk plots, showed a noncompetitive inhibition of BE towards the enzyme, using l-DOPA as substrate. The inhibitory mechanism of BE as studied by spectrophotometric analysis, demonstrated its ability to chelate copper ion in the active site of tyrosinase. In addition, BE exhibited Fe(2+)-chelating ability (IC(50)=614.12±2.14 μg/mL), reducing power and radical-scavenging properties (IC(50)=137.22±1.98 μg/mL). These results suggest the usefulness of birch leaves extracts in cosmetic and pharmaceutical industries for their skin-whitening and antioxidant effects. Determination of the polyphenolic compounds in BE extracts was afterward achieved by means of high-performance liquid chromatography (HPLC) with photodiode array (PDA) and mass spectrometry (MS) detection. A total of 25 compounds were positively identified, through the complementary analytical information, and are reported in such a matrix for the first time. Knowledge on the qualitative composition and contents of these natural sources in fact represents mandatory information, for rational consumption and correlation of the beneficial effects to the specific amounts.