The metabolome of the bellyache bush

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  • Published: May 15, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: The metabolome of the bellyache bush

Design of experiments: optimising output

Metabolites have a wide range of functions, including catalysis, defense and growth. Glucose, amino acids, pigments, antibiotics, vitamins and antioxidants are all examples of metabolites.

Metabolites – the small molecules produced during chemical processes – have a wide range of functions, including catalysis, defense and growth. Glucose, amino acids, pigments, antibiotics, vitamins and antioxidants are all examples of metabolites. Together, all the metabolites within a system are known as the metabolome, and the study of all the metabolites in a sample (such as a cell, tissue or organism) is called metabolomics.

Just as different species have different genetic compositions, they also have different metabolomes. In 2007, the Human Metabolome Database was released – an open access database containing detailed information on over 40,000 metabolites found in the body, and there are equivalent databases for yeast, E. coli, and Arabidopsis plants.

Analysing metabolites is useful for understanding what makes organisms different, and in the plant sciences has been used to investigate the relationships between species, the function of genes and to monitor the quality of herbal medicines.

Plant metabolomes contain a wide range of chemicals from inorganic compounds not built from living matter to fundamental biomolecules like carbohydrates and amino acids. Their very different physical properties are vital for plant function, but also present a major challenge to analytical techniques.

A range of techniques have been proposed to extract metabolites, but organic solvent-based methods remain the number one choice due to their ability to work alongside the major analytical platforms, such as liquid chromatography-mass spectrometry (LC-MS).

So far, most extraction strategies have been developed using ‘one variable at a time’ optimisation methods, in which one experimental input is changed at a time until all variables have been tested and an optimal set of conditions have been determined. This approach, while useful, does not account for the interactive effects between variables, not to mention its high costs and time requirements. An alternative, called ‘design of experiments’ or DoE, systematically arranges variables to estimate the interactions between them and predict optimal parameters. This method allows extraction of lots of reliable information but requires only a small number of experiments.

The ‘bellyache bush’

In a study newly published in the Journal of Separation Science, researchers from Brazil used the DoE approach to develop an optimal extraction and separation strategy for Jatropha gossypiifolia, better known to locals as bellyache bush (so named because it can be poisonous if eaten raw).

This plant is used for medicinal purposes throughout Africa and America for a huge range of ailments, from fevers and infections to high blood pressure and diabetes. Scientific reports have confirmed its beneficial properties, prompting the Brazilian Ministry of Health to place the species on the National List of Medicinal Plants of Interest to the Brazilian Public Health System – a list of plants that could be used to generate pharmaceuticals. As well as being a potential source of new drugs, the plant is a useful model system for metabolome analysis because it contains a diverse range of chemicals, including antifungals, antibiotics, analgesics and anti-inflammatories.

The researchers developed a sequential strategy for extracting and separating the metabolites from the leaves of the plant, using DoE and partial least squares (PLS) regression. PLS is a statistical method for building predictive models and was used here to predict which variables would lead to the most effective extraction and separation.

Proliferating peaks

The researchers tested the effect of 14 different solvents on the extraction process, matching the physicochemical properties of the solvents with the metabolites detected by LC. Under the initial extraction conditions (methanol as solvent, 20 litre sample volume, 3 ml/min flow rate and 25°C column temperature), 107 chromatographic peaks could be observed.

Statistical analysis showed that certain chemical properties of solvents, such as hydrogen bonds and the presence of a proton acceptor/donor, are beneficial for extraction. Surprisingly, this meant the chloroform-methanol mixture so commonly used in extraction protocols had a negative effect on the extraction of metabolites. Column temperature and flow rate were shown to be important for the separation of metabolites.

After optimisation, this time using propanol and chloroform as the extraction solvent as well as reducing the flow rate and raising the sample volume and column temperature, over 30% more peaks could be observed (a total of 140).

As well as improving understanding of this medicinal plant, these findings show how choosing the right optimisation strategy can enhance extraction and separation and achieve the best possible metabolite detection. “Our research verifies that DoE combined with PLS-DA is critical for the improvement of major steps in plant metabolomics, particularly when optimising sample extraction and enhancing chromatographic separation towards detection of metabolites in complex samples,” concludes Professor Ian Castro-Gamboa of Sao Paulo State University.

Related Links

J. Sep. Sci., 2016, 39, 1023–1030. Pilon et al., Partial least squares model and design of experiments toward the analysis of the metabolome of Jatropha gossypifolia leaves: Extraction and chromatographic fingerprint optimization.

Optimizing Organic Reactions with Design of Experiments and Principal Component Analysis

Wiki: Metabolomics

Mass spectrometry-based plant metabolomics: Metabolite responses to abiotic stress

Anti-inflammatory activity of leaves of Jatropha gossypifolia L. by hrbc membrane stabilization method

Jatropha gossypiifolia L. (Euphorbiaceae): A Review of Traditional Uses, Phytochemistry, Pharmacology, and Toxicology of This Medicinal Plant

The Human Metabolome Database

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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