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柑橘中黄烷酮糖苷和黄酮类的提取及HPLC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:提取并检测柑橘中含量最为丰富的黄烷酮糖苷类和聚甲氧基黄酮类。

关键词: 柑橘, 类黄酮, 黄烷酮, 黄酮

材料与试剂

  1. 离心管
  2. 一次性注射器
  3. 0.22 μm微孔滤膜
  4. 2 ml进样瓶
  5. 柑橘材料
  6. 液氮
  7. 甲醇
  8. 甲酸 (色谱纯)
  9. 乙腈 (色谱纯)
  10. 超纯水

仪器设备

  1. 研钵
  2. 离心机
  3. 低温冷冻干燥仪
  4. 旋转蒸发仪
  5. FS60超声萃取仪 (Fisher Scientific, Pittsburgh, PA)
  6. Milli-Q system (Millipore, Milford, MA, USA)
  7. HPLC系统 (1525双元梯度泵、717自动进样器联合2996 PDA检测器)
  8. C18 Hypersil GOLD色谱柱 (4.6 x 250 mm, 5 µm, Thermo scientific, USA)

实验步骤

  1. 在研钵中,加入适量液氮,将在低温 (-54 °C) 冷冻干燥仪冻干过的柑橘材料研磨成粉。
  2. 取0.1 g的冻干粉末,加入到10 ml的离心管中,加入5 ml 80%甲醇溶液。
  3. 将离心管置于FS60超声萃取仪中,40 °C恒温震荡60 min。
  4. 将离心管置于离心机中,转速为3,900 rcf,离心5 min,取上清液。如果所测材料中类黄酮含量低,则通过旋转蒸发仪蒸干上清液,然后用1 ml 80%甲醇进行溶解。
  5. 用一次性注射器吸取1 ml上清经0.22 μm微孔滤膜过滤后进行HPLC检测,流动相为A相0.15%甲酸水溶液,B相纯乙腈溶液 (Chen et al., 2015a; Chen et al., 2015b)。
  6. 检测波长为280 nm (用于黄烷酮糖苷检测,如图1A)和340 nm (用于聚甲氧基黄酮检测,如图1B)。


    图1. 标准品以及甜橙果实提取物280 nm (A) 和340 nm (B) 的HPLC色谱图. 圣草次苷(1)、柚皮芸香苷(2)、橙皮苷(3)、香风草苷(4)、甜橙黄酮(5)、川皮苷(6)、橘黄酮(7)以及去甲川陈皮素(8)。Flevedo: 黄皮层; Albedo: 白皮层; SM: 囊衣; JS: 汁胞。

注意事项

  1. 在超声提取仪中进行类黄酮提取时,应每隔20 min将离心管上下震荡混匀一次。
  2. 黄烷酮类的最佳紫外吸收波长为280 nm左右,而黄酮类则是340 nm左右,进行类黄酮检测及分析时,需要限定波长范围。

溶液配方

  1. 80%甲醇提取液
    甲醇:水=4:1,体积比
  2. 0.15%甲酸水溶液
    水:甲酸=1,997:3,体积比

参考文献

  1. Chen, J., Li, S., Xu, J., Ding, F., Wang, Z., Cheng, Y. and Deng, X. (2015a). Concentration and distribution of main bitter compounds in fruit tissues of ‘Oroblanco’ (Citrus grandis L. x Citrus paradisi Macf.). Scientia Horticulturae 193: 84-89.
  2. Chen, J., Zhang, H., Pang, Y., Cheng, Y., Deng, X. and Xu, J. (2015b). Comparative study of flavonoid production in lycopene-accumulated and blonde-flesh sweet oranges (Citrus sinensis) during fruit development. Food Chemistry 184: 238-246.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:陈嘉景, 彭昭欣, 徐娟. (2018). 柑橘中黄烷酮糖苷和黄酮类的提取及HPLC检测. Bio-101: e1010205. DOI: 10.21769/BioProtoc.1010205.
How to cite: Chen, J. J., Peng, Z. X. and Xu, J. (2018). Extraction and HPLC-detection of Flavanone Glycosides and Flavonoids from Citrus. Bio-101: e1010205. DOI: 10.21769/BioProtoc.1010205.
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