Serial extraction of eucalypt leaves for nuclear magnetic resonance analysis (NMR)



Protocol for the serial extraction of eucalypt leaves for nuclear magnetic resonance analysis.


Several factors make it difficult to understand interactions between animals and plants. The first is the complexity of the chemical profile in plants. Related to this is the need to consider intraspecific as well as temporal and spatial variation in their chemical makeup. This variation makes it hard to design specific assays for compounds that might be active. McIlwee (2001) suggested that spectra of plants obtained with near infrared reflectance spectroscopy (NIRS) could indicate how much animals eat of those plants. In this way, NIRS could be used to predict feeding in vertebrate browsers independent of a detailed understanding of the underlying chemistry. While that approach has been useful in eucalypts (see Foley et al. 1998; Wallis and Foley 2003), NIRS is not suitable for deciphering the underlying chemistry that explains spectral differences. Nonetheless, spectroscopic approaches are attractive because they give a more holistic view of plant chemistry than do assays of broad classes of compounds, such as measuring nitrogen or tannins. In contrast to NIRS, {SUP()}1{SUP}H nuclear magnetic resonance spectroscopy ({SUP()}1{SUP}H NMR) is a spectroscopic approach that can provide structural information about the underlying chemical differences between groups of plants. For instance, we might use NMR spectra and principal components analysis to identify chemical differences between two groups of plants, one that animals eat and one that animals do not. Researchers refer to this as a metabolomic approach. Along with gas chromatography-mass spectroscopy, NMR is one technique for identifying differences. The key advantage of a metabolomic approach is that it makes no prior assumptions about what might be causing a difference. The method described below was the one we used to identify chemical differences in leaves from the two main subgenera of eucalypts – ”Symphyomyrtus” and ”Eucalyptus” that may explain dietary niche separation in marsupial folivores (Tucker et al. ”In Press”/wp-content/uploads/files/31-Extraction-sonication-data-sheet.doc”Oecologia” 116:293-305.


McIlwee AM, Lawler IR, Cork SJ, Foley WJ. 2001. Coping with chemical complexity in mammal-plant interactions: near-infrared spectroscopy as a predictor of ”Eucalyptus” foliar nutrients and of the feeding rates of folivorous marsupials. ”Oecologia” 128:539-548.

Tucker DJ, Wallis IR, Bolton J, Marsh KJ, Rosser AA, Brereton IM, Nicolle D, Foley WJ. In Press. A Metabolomic approach to identifying chemical mediators of mammal-plant interactions ”J Chem Ecol.”

Wallis IR, Foley WJ. 2003. Independent validation of near-infrared reflectance spectroscopy as an estimator of potential food intake of ”Eucalyptus” foliage for folivorous marsupials. ”Aust J Zool” 51:95-98.

Health, safety & hazardous waste disposal considerations

Read MSDS for dichloromethane and methanol. Both are potentially dangerous. DCM is a chlorinated hydrocarbon and any waste must be disposed of appropriately: store DCM waste separately from other non-halogenated organic solvents.


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