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PhD student Gregorio Peron e-mail: gregorio.peron@studenti.unipd.it tel.

1 PhD student Gregorio Peron tel.: Proposed supervisor Dott. Stefano Dall Acqua tel.: Project title English: Antioxidant activity evaluation of phytochemicals in animal models through an innovative metabolomic approach Italian: Studio dell attività antiossidante di fitocomposti in modelli animali mediante un innovativo approccio metabolomico 1. State of the art In the recent years the consumers demand of both food and non-food products intended for the prevention of diseases, or for well-being, has increased. Most of these products, sold both in the form of pharmaceuticals or food supplements, are of natural origin. These are usually defined nutraceuticals, because they derived from food sources that are purported to provide extra health benefits, in addition to their nutritional value. Various epidemiological studies have related regular intake of vegetables with the lower incidence of age-related diseases and cancer. This protective effect has been attributed to specific classes of natural compounds (called phytochemicals), which can exhibit antioxidant properties that contribute to protect the body from oxidative stress. Nevertheless, the biochemical mechanisms underlying their beneficial activities have not been well defined yet, due to the multi targeting nature of these natural products as well as due to the complexity of their chemical structures and to the difficulties of studying the antioxidant activity in vivo. An emerging field of research is metabolomics, that is concerned with the high-throughput identification and quantification of small molecule (<1500 Da) in the metabolome and provides a useful systemic approach to observe changes in animals or humans metabolome in response to 1

2 pharmaceutic, nutritional, genetics and environment stimuli. Metabolic fingerprinting is a complex matrix profiling strategy which can be applied to a variety of sample matrices like biofluids (e.g. tissue extracts, urine, plasma). The variety of metabolomic fingerprinting strategies has increased in recent years: example techniques have included nuclear magnetic resonance (NMR), high-pressure liquid chromatography mass spectrometry (HPLC-MS) and gas chromatography mass spectrometry (GC-MS), coupled with multivariate data analysis to explore and interpret complex spectroscopic data derived from biofluid or tissue samples. 2. Objectives and expected results Phytochemicals and plant extracts are widely used as health-promoters, especially the ones showing antioxidant and anti-aging properties. Nevertheless studies related to their real in vivo antioxidant activities as well as on their effects on healthy subjects are still lacking. The aim of the research project here described is to evaluate the effect of the administration of natural extracts in animal models (i.e. rats) by the metabolomic approach. First, plant extracts will be selected on the basis of previously known antioxidant and antiinflammatory activities. As example some of the extracts that will be considered will be Curcuma longa, Vitis vinifera, Vaccinium macrocarpon, Boswellia serrata and Commiphora mhyrra. Such extracts due to their phytochemical composition possess different antioxidant properties that are considered important for their health promoting activity. For this reason accurate phytochemical characterization of the selected plant extracts will be performed in the first part of the project in order to establish the amount of potentially active phytoconstituents (curcumin, resveratrol, etc). Each extract will be analyzed from a quali-quantitative point of view to determine the exact amount of phytochemicals present using the more appropriate analytical technique. Later, established quantities of standardized herbal extracts will be administered daily to healthy animals, for predefined periods of time. These amounts will simulate the quali-quantitative compositions of nutraceuticals on the market, so as to verify the long-term health-promoting usefulness in healthy in vivo models. The first goals will be the evaluation of metabolic changes in urine of healthy rats treated with different types of natural antioxidants and the creation and comparison of metabolomic datasets for 2

3 the different phytochemicals studied. Variations in the amount of urinary metabolites will be compared with the control group (rats to which no extracts are administered). Targeted analysis will be also performed in order to measure specific metabolites such as urinary markers of oxidative stress. Analysis of faecal samples with attention to bile acid and short chain fatty acids will be also performed in order to establish potential relationships of the intake of phytochemicals and intestinal microbiota. Plasma samples will be collected at the end of the experiments in order to measure plasma GSH and GSSG levels. The results above will be the starting point for the third goal of the project, which will be the search for possible biomarkers useful for the evaluation of the antioxidant effect of specific classes of compounds studied. 3. Methods First of all, each standardized extract will be analyzed under a quali-quantitative point of view to determine the amount of each antioxidant. The qualitative analysis will be performed by the more appropriate analytical technique considering the phytochemical composition. Quali-quantitative phytochemical profiles will be in general obtained by HPLC-MS and GC-MS. The animal experiments will be performed with the different antioxidants extracts using rats as in vivo models. The rats will be divided in multiple groups, each of which will be treated with the different extracts studied. Groups receiving no supplementation as negative control will also be used. Urine will be collected using metabolic cages and it will be divided in three equal amounts, which will be analyzed by 1 H-NMR, LC-MS and GC-MS to create the metabolomic datasets. These techniques will allow the observation of different analytical patterns leading to a more complete observation of the metaboloma. Different analysis conditions for the chromatographic techniques will be established on the basis of the analyzed compound. The raw data will be transformed in matrices that will be used to make a statistical comparison to identify the relevant spectral features that distinguish sample classes. A multivariate statistical analysis (PCA, PLS-DA, etc) will be applied on both NMR data and MS data. In each case, as results of statistical analysis, a set of signals responsible of the separation of the sample will be found and the next work will be concentrated on the identification of the metabolites that originated these signals. Exploiting MS/MS fragmentation it will be possible to investigate the structure of the 3

4 metabolites detected. Analysis for the identification of unmodified antioxidant in the samples will be also performed. 4. Workplan During the first year of the research project the characterization of the selected extracts will be performed to determine the amounts of potentially active phytocompounds. Later, the extracts will be administered to small groups of healthy rats, in such quantities as to simulate a regular intake of nutraceuticals in humans. Metabolomic profiles of both treated and untreated in vivo models will be created from the analysis of biological samples. Much of the work will be then dedicated to the interpretation and statistical analysis of the spectral results obtained, from which shall be identified specific signals that can be attributed to different in vivo effects of the various antioxidants studied. During the beginning of the second year the work will be focused on the molecular characterization of the most relevant metabolites identified. Based on the results obtained, the entire in vivo study will be repeated by changing the dose of phytochemicals administered to healthy rats and comparing the metabolomic profiles obtained with the previous ones. During the last year of the project, the study of antioxidant activity of the extracts whose beneficial effects on healthy subjects were demonstrated may be extended to a larger number of animal subjects. In addition, the specific antioxidants may be tested in non-healthy animal models in which oxidative stress will be induced, in order to assess the effects of specific antioxidant phytochemicals in disease patterns and create new metabolic datasets. 5. Relevant references Kelsey NA, Wilkins HM, Linseman DA. Nutraceutical antioxidants as novel neuroprotective agents. Molecules, 2010; 15 (11): Fardet A et al. Metabolomics provide new insight on the metabolism of dietary phytochemicals in rats. J Nutr, 2008; 138 (7): An D, Zhang Q, Wu S, Wei J, Yang J, Dong F, Yan X, Guo C. Changes of metabolic profiles in urine after oral administration of quercetin in rats. Food Chem Toxicol, 2010; 48 (6):

5 Dall'Acqua S, Stocchero M, Clauser M, Boschiero I, Ndoum E, Schiavon M, Mammi S, Schievano E. Changes in urinary metabolic profile after oral administration of curcuma extract in rats. J Pharm Biomed Anal, 2014; 100:

Terminology. Metabolite: substance produced or used during metabolism such as lipids, sugars and amino acids

Terminology. Metabolite: substance produced or used during metabolism such as lipids, sugars and amino acids Terminology Metabolite: substance produced or used during metabolism such as lipids, sugars and amino acids Metabolome: the quantitative complement of all the low molecular weight molecules present in