文章摘要
高效液相色谱-紫外/质谱评价海洋天然产物体外抑制环氧合酶2活性方法研究
Evaluation of cyclooxygenase 2 inhibitory activity of marine natural products in vitro based on high performance liquid chromatography-UV / mass spectrometry
投稿时间:2018-11-23  修订日期:2019-01-07
DOI:
中文关键词: 环氧合酶2  高效液相色谱紫外检测  高效液相色谱-多级离子阱质谱法  抑制剂筛选  海洋天然产物
英文关键词: cyclooxygenase 2  HPLC-UV detection  HPLC-IT-MS/MS  inhibitor screening  Marine Natural products
基金项目:国家自然科学青年基金(No. 20905017)资助。
作者单位E-mail
史倩 齐鲁工业大学(山东省科学院) srdwjm@sina.com 
陈军辉 国家海洋局第一海洋研究所  
张大海 中国海洋大学  
郑立 国家海洋局第一海洋研究所  
李先国 中国海洋大学  
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中文摘要:
      目的 建立基于高效液相色谱-紫外和高效液相色谱-多级离子阱质谱分析的海洋天然产物体外抑制COX-2酶的活性评价方法。方法 采用Agilent Poroshall 120 C18 色谱柱,以乙腈-0.05%磷酸水溶液为高效液相色谱流动相,二元等度洗脱,流速为0.4 mL?min-1,检测波长为210 nm,进样量10 μL;质谱条件下,流动相改为乙腈-0.05%乙酸水溶液,进样量为1 μL,选用ESI离子源,负离子电离模式,喷雾气压35 psi,干燥气(N2)流速12 L?min-1,干燥气温度350 ℃。结果 建立的高效液相色谱-紫外和高效液相色谱-多级离子阱质谱法精密度和稳定性良好,对比两者测定酶底物花生四烯酸的适用性显示,基于HPLC-UV的评价方法适用极性较大的天然产物粗提物;HPLC-IT-MS/MS法则因其抗干扰能力强适用于复杂体系中极性较弱的活性物质的检测。以建立的HPLC-UV和HPLC-IT-MS/MS方法对海洋天然产物的COX-2酶抑制活性进行评价,天然产物粗提物中黄海葵Anthopleura lanthogrammica berkly乙醇粗提物和弯孢菌Curvularia lunata strain L2744发酵液粗提物对COX-2酶抑制作用较为明显,抑制率分别为70.1%和25.48%。结论 建立的评价方法具有适用性强、结果可靠等优点,有望为天然产物粗提物中COX-2酶抑制剂体外筛选的提供技术参考。
英文摘要:
      Objective To establish methods for evaluating the cyclooxygenase 2 (COX-2) inhibitory activity of marine natural products in vitro based on HPLC-UV and HPLC-IT-MS/MS. Methods Arachidonic acid (AA) which was the substrate of COX-2 was determined by injecting 10 μL of the samples on an Agilent Poroshall 120 C18 column with the isocratic elution solvent system composed of 0.05% phosphoric acid aqueous solution and acetonitrile. The flow rate was 0.4 mL?min-1 and the signal was acquired at 210 nm. The mobile phase was changed into 0.05% acetic acid aqueous solution and acetonitrile in IT-MS/MS condition with injection volume of 1 μL. ESI was used as ion source in negative mode with ion spray pressure 35 psi, dry gas flow rate 12 L?min-1 and drying gas temperature 350 ℃. Results The established HPLC-UV and HPLC-IT-MS/MS methods had good precision and stability. The comparisons of different applicability of two methods showed that the evaluation method based on HPLC-UV was suitable for crude extracts of natural products with high polarity, and the HPLC-IT-MS/MS method was suitable for the detection of active substances with weak polarity in complex systems because of its strong anti-interference ability. The COX-2 inhibitory activity of marine natural products was evaluated using the established HPLC-UV and HPLC-IT-MS/MS methods. The ethanol crude extract of Anthopleura lanthogrammica Berkly and crude extract of fermentation broth of Curvularia lunata strain L2744 had obvious inhibitory effect on COX-2, and the inhibition rates were 70.1% and 25.48%, respectively. Conclusions The established evaluation methods had the advantages of strong applicability and reliable results. It is expected to provide a technical reference for in vitro screening of COX-2 inhibitors from crude extracts of natural products.
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