摘要
建立了一种快速、灵敏且高精度的流动注射-分光光度法用于研究天然产物对酪氨酸酶活性的影响。此方法基于一定介质条件下L-3,4-二羟基苯丙氨酸(L-DOPA)在酪氨酸酶的作用下,发生氧化生成棕褐色多巴醌,其最大吸收峰位于475 nm处,而酪氨酸酶抑制剂能降低酶促反应速度,减少多巴醌形成的量,从而降低475 nm下的吸光度值,形成倒峰。在优化实验条件下,半抑制浓度的曲酸抑制酪氨酸酶活性测定相对标准偏差(n=10)为0.036%,与分光光度法和酶标仪微量法相比,精密度提高了10倍以上。咖啡酸对酪氨酸酶有较强的激活作用,对二酚酶相对激活率达到50%时的浓度(IC50)为0.170 mmol/L;薰衣草花水相提取物具有较强的抑制活性,其IC50值为0.96 mg/m L。与分光光度法及酶标仪微量法相比,流动注射-分光光度法精密度高,重复性好,适用于天然产物对酪氨酸酶活性影响的测定。
A rapid,sensitive and highly precise method was developed for the direct and continuous determination of the inhibitory effect on mushroom tyrosinase activity of natural products. Based on ο-quinone,L-DOPA was catalyzed by tyrosinase into the corresponding brown product,which has the biggest absorption at 475 nm. The tyrosinase inhibitor can reduce the enzymatic reaction rate,and then make the OD475 lower into a negative peak. The RSD of injection 10 times on the IC50 of kojic acid is 0. 036%,and the precision of the approach was improved more than 10 times as compared with the spectrophotometric method and microtitration assay. By using the sample for further verification,the results showed that caffeic acid has a potent activating effect on diphenolase activities of tyrosinase,its concentration leading to 50% activation rate（ IC50） is 0. 170 mmol/L. The water-soluble extraction from Lavandula angustifolia has a potent inhibitory effect on diphenolase activities of tyrosinase,its IC50 value is 0. 96 mg / m L. Compared with the spectrophotometric method and microtitration assay,flow injection- spectrophotometric approach has a higher precision,better repeatability and is suitable for the determination of influence on tyrosinase activity from natural products.
出处
《分析测试学报》
CAS
CSCD
北大核心
2015年第4期407-413,共7页
Journal of Instrumental Analysis
基金
国家自然科学基金项目(21365018)
新疆生产建设兵团科技攻关项目(2011AB030)
关键词
流动注射-分光光度法
天然产物
酪氨酸酶活性
flow injection-spectrophotometric approach
natural products
tyrosinase activity
