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This method describes the extraction of coumarins and furanocoumarins from leaves of Ruta graveolens (a natural furanocoumarin) producer, and Nicotiana benthamiana.

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Extraction of Coumarins from Leaves, Petioles, Stems and Roots of Ruta graveolens and Nicotiana benthamiana
芸香和本氏烟草叶、叶柄、茎及根中香豆素的提取

植物科学 > 植物生物化学 > 其它化合物
作者: Alain Hehn
Alain HehnAffiliation: UMR1121 Laboratoire Agronomie et Environnement, Université de Lorraine-INRA, Vandoeuvre les Nancy, France
For correspondence: alain.hehn@univ-lorraine.fr
Bio-protocol author page: a153
Guilhem Vialart
Guilhem VialartAffiliation: UMR1121 Laboratoire Agronomie et Environnement, Université de Lorraine-INRA, Vandoeuvre les Nancy, France
Bio-protocol author page: a154
Alexandre Olry
Alexandre OlryAffiliation: UMR1121 Laboratoire Agronomie et Environnement, Université de Lorraine-INRA, Vandoeuvre les Nancy, France
Bio-protocol author page: a155
 and Frederic Bourgaud
Frederic BourgaudAffiliation: UMR1121 Laboratoire Agronomie et Environnement, Université de Lorraine-INRA, Vandoeuvre les Nancy, France
Bio-protocol author page: a156
Vol 2, Iss 22, 11/20/2012, 4330 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.288

[Abstract] This method describes the extraction of coumarins and furanocoumarins from leaves of Ruta graveolens (a natural furanocoumarin) producer, and Nicotiana benthamiana.

[Abstract] 该实验方案的中文版正在准备中...

Materials and Reagents

  1. Ruta graveolens or Nicotiana benthamiana plants
  2. Liquid nitrogen
  3. Ethanol
  4. Methanol
  5. (+)- Taxifolin (Extrasynthèse, catalog number: 1036 http://www.extrasynthese.com)

Equipment

  1. 2 ml microtubes
  2. Centrifuge for 2 ml tubes
  3. Pestle and mortar
  4. Bench top blendar (Polytron PT2100, Kinematica, http://www.kinematica ch)
  5. Vacuum concentrator (RC10.10 speed vacuum,  http://www.thermoscientific.com)

Procedure

  1. Harvest 200 mg fresh material of each organ which is supposed to be analyzed.
  2. Freeze it immediately in liquid nitrogen. Avoid thawing.
  3. Grind the material in a cooled mortar. Keep the material frozen during the grinding process.
  4. Add 2 ml of 80% ethanol and 50 μl of taxifolin 2 mg/ml to the mixture and homogenize with a bench top blender for 1 min (taxifolin will be an external control to compare the efficiency of the extraction between two samples. As the amount of taxifolin is the same in all the samples, final results can be compared to each other on the basis of the amount taxifolin detected).
  5. Transfer the crushed material into a 2 ml microtube.
  6. Centrifuge at 16,000 x g for 10 min at room temperature.
  7. Harvest the supernatant and transfert the sample in a fresh tube.
  8. Evaporate the sample overnight with a vacuum concentrator.
  9. Resuspend the pellet in 200 μl methanol prior to analyses by HPLC (analyses are performed between 300 and 350 nm wavelength. Identification of furanocoumarin is done by comparison with standard molecules).

Acknowledgments

The protocol described here is adapted from one reported previously (Vialart et al., 2012).

References

  1. Vialart, G., Hehn, A., Olry, A., Ito, K., Krieger, C., Larbat, R., Paris, C., Shimizu, B., Sugimoto, Y., Mizutani, M. and Bourgaud, F. (2012). A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants. Plant J 70(3): 460-470.

材料和试剂

  1. Ruta graveolens 或本尼岛烟草植物
  2. 液氮
  3. 乙醇
  4. 甲醇
  5. (+) - 紫杉叶素(Extrasynthèse,目录号:1036 http://www.extrasynthese.com

设备

  1. 2 ml微量管
  2. 离心2ml管
  3. 杵和臼
  4. 台式搅拌机(Polytron PT2100,Kinematica, http://www.kinematica ch
  5. 真空浓缩机(RC10.10高速真空, http://www.thermoscientific.com

程序

  1. 收获200毫克新鲜的材料的每个器官,应该进行分析
  2. 立即在液氮中冷冻。 避免解冻。
  3. 在冷却的砂浆中研磨材料。 在研磨过程中保持材料冻结。
  4. 添加2ml的80%乙醇和50μl紫杉叶素2mg/ml到混合物中,并用台式搅拌器匀化1分钟(紫杉叶素将是外部对照以比较两个样品之间的提取效率, 的紫杉叶素在所有样品中是相同的,最终结果可以基于检测到的紫杉叶素的量相互比较)。
  5. 将粉碎的材料转移到2ml微量管中
  6. 在室温下以16,000×g离心10分钟
  7. 收获上清液,并将样品转移到新管中
  8. 用真空浓缩器将样品蒸发过夜
  9. 将沉淀重悬在200μl甲醇中,然后通过HPLC分析(在300和350nm波长之间进行分析,通过与标准分子比较来鉴定呋喃香豆素)。

致谢

这里描述的方案改变自先前报道的(Vialart等人,2012)。

参考文献

  1. Vialart,G.,Hehn,A.,Olry,A.,Ito,K.,Krieger,C.,Larbat,R.,Paris,C.,Shimizu,B.,Sugimoto,Y.,Mizutani, Bourgaud,F。(2012)。 来自Ruta graveolens L.的2-氧代戊二酸依赖性双加氧酶。展示p - 椰油酰CoA 2'-羟化酶活性(C2'H):植物中伞形酮合成中缺失的步骤。植物J(70)(3):460-470。
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How to cite this protocol: Hehn, A., Vialart, G., Olry, A. and Bourgaud, F. (2012). Extraction of Coumarins from Leaves, Petioles, Stems and Roots of Ruta graveolens and Nicotiana benthamiana. Bio-protocol 2(22): e288. DOI: 10.21769/BioProtoc.288; Full Text



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