Original research article

The authors used this protocol in:
May 2012

Navigate this Article


 

Extraction of Coumarins from Leaves, Petioles, Stems and Roots of Ruta graveolens and Nicotiana benthamiana    

How to cite Favorites Q&A Share your feedback Cited by

Abstract

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

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.
Please login or register for free to view full text
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.
Q&A
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at eb@bio-protocol.org.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.