超高效液相色谱-串联四极杆飞行时间质谱法检测牡丹根皮中抗氧化活性成分及其抗氧化能力  

Detection of Antioxidant Active Components and Their Antioxidant Ability from Moutan Cortex by Ultra-High Performance Liquid Chromatography-Tandem Quadrupole Time-of-Flight Mass Spectrometry

作　　者：谢炎福 押辉远 李月佳 刘沛 XIE Yanfu;YA Huiyuan;LI Yuejia;LIU Pei(School of Food and Drug,Luoyang Normal University,Luoyang 471934,China)

机构地区：[1]洛阳师范学院食品与药品学院,洛阳471934

出　　处：《理化检验（化学分册）》2024年第7期660-667,共8页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：国家自然科学基金项目(21801110);河南省高等学校重点科研项目(24B550011)。

摘　　要：为能准确检测复杂基质牡丹根皮样品中的抗氧化活性成分及其抗氧化能力,进行了题示研究,并推测了1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除过程的反应机理。采用70%(体积分数,下同)乙醇溶液超声提取样品2次,过滤后合并滤液,浓缩、离心、稀释,配制成200 g·L^(−1)样品溶液,并进一步稀释成质量浓度为8,40,100,200,500,1000,5000,10000,25000 mg·L^(−1)的样品溶液系列。取上述配制好的样品溶液系列各300μL(不添加样品的为样品对照组)和无水乙醇600μL,再加入50 mg·L^(−1)DPPH溶液600μL,涡旋1 min,避光反应30 min。用70%乙醇溶液稀释200倍,过0.22μm滤膜,采用超高效液相色谱-串联四极杆飞行时间质谱法(UHPLC-Q-TOF-MS)测量样品加入前后DPPH准分子离子峰面积P_(0)和P,利用100%×(P_(0)-P)/P_(0)计算DPPH自由基清除率。结合自身谱库、标准品谱图以及牡丹根皮相关文献鉴定各抗氧化活性成分。结果显示:样品的质量浓度在8~1000 mg·L^(−1)内与DPPH自由基清除率呈线性关系,较酶标仪法受样品基质和颜色干扰小,且DPPH自由基清除率结果和酶标仪法的基本一致;从牡丹根皮中显著识别出18种抗氧化活性成分,包括15种可鉴定成分(包含两对同分异构体)和3种未知成分,其中氧化芍药苷、丹皮酚、牡丹皮苷H、没食子酰芍药苷、没食子酸甲酯、牡丹皮苷F、对羟基苯甲酸是牡丹根皮中主要的抗氧化活性成分。In order to accurately detect the antioxidant active components and their antioxidant ability in Moutan Cortex samples with complex matrix,the title study was conducted,and the reaction mechanism of 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)free radical elimination process was speculated.Extraction was conducted twice on the sample by ultrasound with 70%(volume fraction,the same below)ethanol solution.After filtration,the filtrate was collected,concentrated,and centrifuged.The supernatant was diluted to prepare 200 g·L^(−1)sample solution,and further diluted to prepare sample solution series with mass concentrations of 8,40,100,200,500,1000,5000,10000,25000 mg·L^(−1).300μL of the prepared sample solution series mentioned above(sample control group without sample addition)and 600μL of anhydrous ethanol,together with 600μL of 50 mg·L^(−1)DPPH solution were added.The mixed solution was vortexed for 1 min,reacted in dark for 30 min,and diluted 200 times with 70%ethanol solution.The solution obtained was passed through a 0.22μm filtrate membrane,and the filtrate was analyzed by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOFMS),to measure the peak areas P_(0)and P of DPPH reference molecule ions before and after sample addition,and calculate the DPPH free radical scavenging rate using 100%×(P_(0)-P)/P_(0).Various antioxidant active components were identified by combining their own spectrum library,reference substance spectra,and relevant literatures on Moutan Cortex.It was shown that linear relationship between values of the mass concentration of the sample and the DPPH free radical scavenging rate was kept in the range of 8-1000 mg·L^(−1).The proposed method was less affected by the interference of the sample matrix and color compared to the enzyme-linked immunosorbent assay method.The results of the DPPH free radical scavenging rate were consistent with those given by the enzyme-linked immunosorbent assay method.18 antioxidant active comp

关 键 词：1 1-二苯基-2-三硝基苯肼(DPPH) 超高效液相色谱-串联四极杆飞行时间质谱法(UHPLC-Q-TOF-MS) 牡丹根皮 抗氧化 

分 类 号：O657.63[理学—分析化学] R285.5[理学—化学] R282.71[医药卫生—中药学]