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Research Infrastructure Wageningen NMR Centre: NMR in Agriculture, Food and Biology

Final Report Summary - WNMRC (Research Infrastructure Wageningen NMR Centre: NMR in Agriculture, Food and Biology)

Despite decades of studies, there are still fundamental unanswered questions related to phloem flow in natural and semi-natural ecosystems. Phloem flow in plants has hardly been studied due to lack of proper methods. It is important to answer these questions, however, in order to improve our knowledge of the carbon cycle, of relevance for eco-physiological plant studies in relation to climate and climate change.

The unique intact plant 3T-MRI system has been used for long distance transport studies in intact plants and trees. The effects of root and shoot anoxia on source and sink of water flow and solute transport in the transport phloem of Ricinus was studied to answer the question if phloem flux is determined by sink or source strength. The results give new insights in the regulation of phloem flow. MRI flow imaging has been used as a reference for flow measurements by a new non-invasive heat pulse method. The dynamics in flow conducting area could be studied by MRI and the consequences for flux quantification by the classical heat pulse method were revealed. Sensors based on heat pulse techniques are in use in eco-physiological investigations. Therefore, this is totally new information of importance for water management and tree functioning in response to global warming and water management.

A number of publications as a result of former user projects has been published directed towards the optimisation and application of hyphenated LC-NMR-MS techniques for the analysis of different extracts, e.g. with well know or potential medicinal use or in relation to plant metabolomics. This method has been used by a number of users, e.g. for flavonoid search. Flavonoids are important bioactive dietary compounds. The chemical nature of the grain pigments of different types of pigmented rices (differently colored) has been analysed. Complete metabolic profiles of extracts from five rice samples, corresponding to different pigmented types. In combination with genetic profiles of the rices (determined at the CRA-GPG, Italy), full characterisation of the rices has been obtained. In this way a better understanding of the regulation of the flavonoid pathway in rice has been achieved. Flavonoids induce electrophile-responsive element (EpRE)-mediated expression of enzymes, including major defence enzymes against electrophilic toxicants and oxidative stress. The pro-oxidant chemistry of flavonoids (anthocyanidins and catechin metabolites) has been studied.

New unknown functional compounds present at low concentrations in different plant extracts. Several species from the Teucrium genus are reported as neo-clerodane diterpene producers, being one of the richest sources of these secondary metabolites. This type of compounds is insect antifeedants, hepatotoxic, cytotoxic, and anti-inflammatory. Four unstudied species from this genus have been collected from Morocco, and pre-purified in order to determine the presence of neo-clerodane diterpenes. Finally, six peaks were isolated and identified, including some totally new compounds.

The identification of minor phenoloic compounds and their glycoside derivatives in methanolic and methanolic / water plant extracts of O. dictamnus was done. Sixteen different fractions could be isolated. The fractions usually contained one or two components in considerable quantity and smaller amount of other compounds. Fourteen compounds were identified. From these compounds, only two have previously been reported in the literature to exist in O. dictamnus extracts. All the other compounds were identified for the first time, three of which were identified as totally new and are derivatives of rosmarinic acid, a well known compound for its anti-HIV properties.

Green tea represents a promising source of health-protecting photochemicals. It contains the unique set of polyphenolic compounds. These compounds can be converted in electrophilic quinones which can be damaging to the cell. Glutathione can protect the cell by reacting with these quinones forming adducts. 1H NMR in combination with MS has been used to identify glutathione adducts resulting from pro-oxidant quinone chemistry. From the NMR data it can be concluded that GSH is preferentially reacting with the C2'/C6' carbon centres in all catechin studied. In the case of anthocyanins, the situation is more complicated. Here, hemi-acetal structures are formed as based on NMR data analysis. Based on the NMR results a reaction mechanism has been proposed.

For the first time disturbed metabolic pathways in dairy cows with abomasal displacement, the most important disease complex associated with the abomasum in cattle, was studied using a 1H NMR-based metabolomics approach to find biomarkers for abomasal displacement. The most striking metabolites found were keton bodies. There were clear keton bodies in cows with LDA in which ketosis could not be determined clinically. Metabolites such as choline, betaine and leucine were decreased in cows with LDA. Cows with RDA in which there was no clear ketosis had a little bit more lactate and glucose, and less lipoproteins compared to controls. It was concluded that NMR evaluation on the blood of dairy cows with abomasal displacement and ketosis will provide important mechanistic insight into cellular metabolism in steatotic liver disease.

During the project various spin traps were studied for conventional (Fenton) and target systems. In addition, a reliable method of interpretation of ESR spectra by a combination of simulation-based fitting and artificial neural network was developed. In this way, the types of free radicals detected by ESR and NMR spin trapping allowed to establish the chemical reaction, occurring in the system. The obtained results allowed to expand previous study and gave new insight into newly developed system. Along with the study of proposed free radical system, the main aspects of ESR and NMR spectroscopy along with fluorine containing spin trap (FDMPO) have been investigated. The study of FDMPO is very important due to outstanding properties of the spin trap, which can be used for study of complex mixtures and have potential application in free radical biology and anti-oxidant capacity of complex mixtures / plant extracts.

Results obtained via this project have been reported in over 40 publications in diverse peer reviewed scientific journals, serving a very broad audience. In addition, they have contributed to five PhD and one Habilitations theses. Results have been reported in oral and poster presentations in a large number of different scientific meetings / congresses covering diverse fields of applications, and will be followed by more full publications.

The Helmholtz Association of German research centres has granted an application for a Virtual Institute for Portable NMR (VIP-NMR; please see www.portable-nmr.eu online ). WNMRC is one of the partners because of the worldwide leading position and infrastructure for intact plant research. This VIP-NMR will expand the possibilities for portable NMR applications for the users of WNMRC, allowing in situ field measurements. On the basis of the contacts and results two new EU projects have been defined and submitted: 13 NMR for sustainable bioeconomy (4F NMR), a combination of collaborative project and coordination and support action for integrating activities, SPROBE-MQ, a small collaborative project in work program topic exploring the microstructures in foods (10 partners). Further, two new DFG grant applications have at least partly been based on the cooperation and results of this project.

By the newly developed HPLC-(SPE)-MS-NMR method new natural compounds have been found and the structure elucidated. Some of these are of interest for industry for medical applications, others as functional food components. A new method for soil characterisation has been developed, which has big potential for practical use.

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