柑橘汁胞中呋喃香豆素单体的提取及UPLC检测   

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Original research article

A brief version of this protocol appeared in:
Rice Protocol e-book

实验目的:提取并检测柑橘汁胞中4种呋喃香豆素单体物质。

关键词: 柑橘汁胞, 呋喃香豆素单体, 超高效液相色谱 (UPLC)

材料与试剂

  1. 离心管 (50 ml、10 ml)
  2. 一次性注射器
  3. 0.22 μm微孔滤膜
  4. 柑橘成熟果实
  5. 液氮
  6. 乙腈:色谱级 (Fisher Scientific, USA)
  7. 香柠檬亭,佛手苷,6',7'-二羟基香柠檬亭标准品:色谱级 (Sigma-Aldrich, USA)
  8. 环氧香柠檬亭标准品:色谱级 (Chromdex, USA)
  9. 乙酸乙酯:分析纯 (国药集团化学试剂有限公司,北京)
  10. 超纯水

仪器设备

  1. 研钵
  2. 天平
  3. 洁盟JP-100S超声波萃取仪 (洁盟,中国)
  4. Avanti J-20 XP型离心机 (Beckman Counter, USA)
  5. 5301型旋转蒸发仪 (Eppendorf, German)
  6. Waters PRO PLUS型超纯水制备仪 (Labconco, USA)
  7. Waters 超高效液相色谱 (UPLC):二极管阵列检测器 (Photodiode Array Detector, PDA),C18色谱柱 (规格为2.1 x 50 mm,粒径1.7 μm) (WATERS, USA)

实验步骤

  1. 分离取出柑橘成熟果实汁胞,用液氮在研钵中磨成粉状。
  2. 准确称量5 g粉状物,加入到50 ml的离心管中,加入15 ml提取试剂乙酸乙酯。
  3. 充分振荡5 min,将离心管置于超声波萃取仪中萃取30 min。
  4. 将离心管置于离心机中,转速为3,913 x g,离心5 min,收集上层有机相于新的离心管中。
  5. 向50 ml离心管的残留物再加入10 ml乙酸乙酯,重复步骤3-4一次。
  6. 重复步骤5一次。
  7. 将收集的有机相分装到10 ml离心管中,通过旋转蒸发仪在30 °C条件下蒸干浓缩,用乙腈定容至1 ml,用一次性注射器吸取上清经0.22 µm微孔滤膜过滤后进行UPLC检测。
  8. UPLC检测色谱条件:流动相A相乙腈溶液,B相超纯水,梯度洗脱条件见表1;流速0.4 ml/min;检测波长310 nm;进样量2 µl;样品室温度20 °C;色谱柱温度30 °C。

    表1. 呋喃香豆素单体物质梯度洗脱方法

结果与分析

4种呋喃香豆素单体标准品和梦能早柚的UPLC色谱图见图1。


图1. 四种呋喃香豆素单体标准品 (A) 和梦能早柚 (B) 的UPLC色谱图. RT指保留时间;1为佛手苷;2为6',7'-二羟基香柠檬亭;3为环氧香柠檬亭;4为香柠檬亭。

致谢

该实验方案改编自先前发表的文章Xu et al. (2015).

参考文献

  1. Xu, J., Ma, L., Jiang, D., Zhu, S., Yan, F., Xie, Y., Xie, Z., Guo, W. and Deng, X. (2015). Content evaluation of 4 furanocoumarin monomers in various citrus germplasms. Food Chem 187: 75-81.
  2. Widmer, W. and Haun, C. (2010). Variation in furanocoumarin content and new furanocoumarin dimers in commercial grapefruit (Citrus paradisi Macf.) juices. Journal of Food Science 70(4): C307-C312.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:田静, 马丽丽, 徐娟. (2018). 柑橘汁胞中呋喃香豆素单体的提取及UPLC检测. Bio-101: e1010215. DOI: 10.21769/BioProtoc.1010215.
How to cite: Tian, J., Ma, L. L. and Xu, J.  (2018). Extraction and UPLC-detection of Furan Coumarin Monomer from Citrus Juice Sacs . Bio-101: e1010215. DOI: 10.21769/BioProtoc.1010215.
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