Organization • Núcleo de Pesquisas em Produtos Naturais e Sintéticos: Prof. Ph.D. Norberto Peporine Lopes Ph.D. Ricardo R. da Silva Camila Capel Godinho • AsterBioChem Research Group: Prof. Ph.D. Fernando Batista da Costa Ph.D. Ricardo Pereira Rodrigues M.Sc. Rosana Casoti M.Sc. Tiago Branquinho Oliveira M.Sc. Guillermo Federico Padilla Gonzalez M.Sc. Lucas Apolinário Chibli Annylory Lima Rosa • EMBL-EBI: Ph.D. Reza Salek • Centro Nacional de Pesquisa em Energia e Materiais, Laboratório Nacional de Biociências: Ph.D. Marcos Rodrigo Alborghetti • Centro Infantil de Investigações Hematológicas Dr. Domingos A. Boldrini: M.Sc. Rafael Renatino Canevarolo • Assoc. Brasileira de Bioinformática e Biologia Computacional - AB3C: Meire Tarlá 2015 First Brazilian Workshop on Bioinformatics/ Chemometrics for Metabolomics This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit <http://creativecommons.org/licenses/by/4.0/>. Acknowledgements Dear I-BWBM Participant, On behalf of the Organization committee, it is our pleasure to welcome you to Ribeirão Preto and to the First Brazilian Workshop on Bioinformatics/Chemometrics for Metabolomics. For this first year, we have a diverse range of topics from different analytical platforms, target organisms and scientific questions. We have participants from three countries, seven Brazilian states, with different knowledge backgrounds and experience. During the past ten years, we have witnessed the great increase of Metabolomics practitioners. One of the main aims of this Workshop is to stimulate the reflection on where Brazilian scientific community is positioned in this growing international community and how a national articulation could benefit the development of the national metabolomics research. As part of our reflection, and hopefully, of the discussion stimulated during the event, we want to share our ambitions to grow and serve our community. The activities suggested by the participants during this event will be shared at our portal (<http://nppns.fcfrp.usp.br/>) and we hope we can create a portfolio of benefits to the mail list subscribers, such as discussion forums, courses, advertising of job opportunities and so on. This event was only possible because of you, and we are grateful for your contributions through your subscription fee. We also want to express our gratitude to our speakers who will discuss their current research in metabolomics and promote the integration of the growing Brazilian community. The Organization also wishes to thank our funding agency, FAPESP and our sponsors; the Workshop would not be financially feasible without their support. Best wishes to everyone, and we hope you have a great workshop! Organization committee Contents Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.1 Data sharing and standards in metabolomics: what we can learn from other “omics” . . . . . . . . . . . . . . . . 1.2 Metabolomics: a powerful tool for elucidating plant growth regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Chemoinformatics on Brazil: an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Metabolic fingerprint and biomarker discovery . . . . . . 1.5 Statistical methods to analyze biological networks . . . . 1.6 Metabolite identification strategies using NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Telling metabolic stories to explore metabolomics data: a case study on the yeast response to cadmium exposure 1.8 Application of MALDI in metabolomics: from extract analysis to the imaging of tissue . . . . . . . . . . . . . . . 1.9 The fascinating World from Senna spectabilis’s rizosphere: exploring molecular diversity through the use of metabolomics and multiway analysis . . . . . . . . . . . . . . . 1.10 Classification models proposition using Chemometrics: potential applications in metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 14 15 16 17 18 19 20 21 22 2 Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.1 2.2 2.3 2.4 Metabolomics research infrastructure available at Núcleo de Pesquisas em Produtos Naturais e Sintéticos (NPPNS) 25 AsterBioChem: chemoinformatic tools applied for chemical and biological studies of Asteraceae . . . . . . . . . . 27 Metabolomic analysis and bioactivity assays in COX and LOX pathways-dependent of plants from subtribe Lychnophorinae . . . . . . . . . . . . . . . . . . . . . . . . . 29 The role of secondary metabolites in plants diversification and evolution: a remarkable example from the northern Andes . . . . . . . . . . . . . . . . . . . . . . . . 31 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21 Mixture design to optimize Baccharis genus species extraction in untargeted metabolomic studies . . . . . . . . Metabolomics assited by chemometrics for targeted phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . Potential metabolic biomarkers for chronic graft-versushost disease . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of sugarcane transcriptome and metabolome changes along the plant maturation cycle . . . . . . . . . Xanthan gum removal for 1 H-NMR analysis of the intracellular metabolome of the bacteria Xanthomonas axonopodis pv. citri 306 . . . . . . . . . . . . . . . . . . . . Impact of water deficit on sugarcane leaf secondary metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differential metabolomic responses of Histoplasma capsulatum in biofilms and planktonic yeasts . . . . . . . . . Identification of cuticular wax lipids from Eucalyptus grandis leaves and their relationship with rust disease . Serum and urine metabolic profile in response to renal ischemia/ reperfusion in a swine model . . . . . . . . . . Investigating the metabolic network of developing soybean seedlings . . . . . . . . . . . . . . . . . . . . . . . . . Metabolomic profiles of caffeine metabolization after coffee intake . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolomics, proteomics and chemometrics approaches for the infraspecific chemical variability study of a native Brazilian plant species . . . . . . . . . . . . . . . . . . . . Metabolomic studies of fungi isolated from the rhizosphere of Senna spectabilis . . . . . . . . . . . . . . . . . . Metabolomics and proteomics data integration during sugarcane development . . . . . . . . . . . . . . . . . . . Metabolic fingerprinting of Eucalyptus grandis during the infection by Puccinia psidii . . . . . . . . . . . . . . . Metabolomics applied in a Brazilian population-based study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Progress on metabolomics studies of Brazilian Cerrado plant species developed in different aluminum availabilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 35 36 37 38 40 41 42 43 45 46 48 50 52 53 54 56 2.22 Effect of the environment on the metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants . . . . . . . . . . . . . . . . . . . . . . . . 2.23 Metabolic profiling of Byrsonima species with antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . 2.24 Dereplication of natural products based on 1 H ratio analysis nuclear magnetic resonance spectroscopy and HPLCDAD-ESI-QToF-MS/MS . . . . . . . . . . . . . . . . . . . . 2.25 Metabolite profile of a biopolymer producer strain, by gas chromatography coupled to mass spectrometry (GCMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.26 Differentiation of essential oils from Citrus fruits by multivariate analysis . . . . . . . . . . . . . . . . . . . . . . . . 58 60 61 63 65 3 Poster Section . . . . . . . . . . . . . . . . . . . . . . . . . . 67 3.1 3.2 3.3 3.4 Poster Section 01 . Poster Section 02 . Poster Section 03 . Poster Section 04 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 67 67 67 Program 9 Time Topic Speaker March 13 08:00 09:15 09:30 10:30 11:00 12:00 14:00 15:00 16:00 16:30 17:30 18:30 Registration Opening Section Data sharing and standards in metabolomics: What we can learn from other “omics” Coffee break/ Poster section 01 Metabolomics: a powerful tool for elucidating plant growth regulation Lunch Chemoinformatics/chemometrics on Brazil: an overview Metabolic Fingerprint and biomarker discovery Coffee break/ Poster section 02 Statistical methods to analyze biological networks Advanced Analytical Tools in Mass Spectrometry and Data Processing: Meeting the challenges of Metabolomics Studies Conference Dinner Reza Salek Camila Caldana Fernando Batista da Costa Ieda Spacino Scarminio André Fujita Jane Finzi – Waters March 14 09:30 10:30 11:00 12:00 14:00 15:00 16:00 16:30 17:30 18:00 Metabolite identification strategies using NMR Coffee break / Poster section 03 Telling metabolic stories to explore metabolomics data: a case study on the yeast response to cadmium exposure Lunch Application of MALDI in metabolomics: from extract analysis to the imaging of tissue The fascinating World from Senna spectabilis’s rizosphere: exploring molecular diversity through the use of metabolomics and multiway analysis Coffee break/ Poster section 04 Classification models proposition using Chemometrics: potential applications in metabolomics Closing Section USP’s choir presentation Jan Schripsema Paulo Vieira Milreu Denise Brentan da Silva Ian CastroGamboa Edenir drigues Filho RoPereira 1 Lectures 1.1. Data sharing and standards in metabolomics: what we can learn from other “omics” 13 1.1 Data sharing and standards in metabolomics: what we can learn from other “omics” Reza Salek O aspect in any scientific result is “to be reproducible”. Commonly this is done by publications, but even better and in parallel is to make the results, dataset and techniques open and accessible without any restriction ideally via a data repository. In addition, reported data needs to follow standard reporting guidelines (known as minimum information) supported with computer readable control vocabulary. Following earlier genomics and proteomics communities’ success, metabolomics saw establishments of repositories for metabolomics data with both MetaboLights (<http://www.ebi.ac.uk/>) at the European Bioinformatics Institute and the Metabolomics Work bench by the NIH (<http://www.metabolomicsworkbench.org/>) in US and many more specialized repositories. Ideally, data sets (and results) should be stored in open exchange formats to make it as widely accessible as possible, vendor independent, enriched with terminological artifacts to ease exchange and query metabolomics experiments. I will try to introduce and give a descriptive overview of the standards effort up to the current EU coordination action for developing metabolomics standards with a worldwide participation, called COordination of Standards in MetabOlomicS - COSMOS (<http://cosmos-fp7.eu/>). One of the COSMOS aims is to develop and maintain exchange formats for raw data and processed information (identification, quantification), building on experience from standards developed within the Metabolomics Standards IniMSI) and Proteomics Standards InitiPSI). Also, the role that metabolomics repositories can play in adoption of such standards and making metabolomics results more extensively accessible. NE KEY 1. Lectures 14 1.2 Metabolomics: a powerful tool for elucidating plant growth regulation Camila Caldana M ETABOLOMICS , which represents the chemical composition in a cell, is a powerful tool in deciphering metabolism and bridging the phenotype–genotype. Despite significant advancement in analytic tools, complete coverage of the metabolome will always be constrained by polarity, stability, dynamic range and biological properties of metabolites. Therefore, the optimal choice for an analytical technology will largely depend on the goal of each study and is usually a compromise between selectivity and speed. The main goal of our group is to elucidate the processes underlying plant growth and production of biomass. As plant growth is closely linked to central metabolism network, we use gas chromatography coupled to mass spectrometry (GC-MS) technology for a comprehensive coverage of primary metabolism pathways (e.g. organic and amino acids, sugars, sugar alcohols, phosphorylated intermediates and lipophilic compounds). In my presentation, I will provide examples of the potential of metabolite profiles to predict plant performance as biomarkers and integral component of plant systems biology. 1.3. Chemoinformatics on Brazil: an overview 1.3 15 Chemoinformatics on Brazil: an overview Fernando Batista da Costa C HEMOINFORMATICS ( OR CHEMINFORMATICS ) is a relatively new discipline that combines concepts and topics from older disciplines, like chemometrics. Although it is younger than its sister bioinformatics, which actually has a different focus, chemoinformatics, which deals with the use of informatics methods in chemistry, is well established and has several interesting applications. For example, it is involved from drug discovery to property prediction of organic compounds or from chemical databases to structure search. In United States and Central Europe chemoinformatics has evolved considerably in the last decade and is now consolidated in both academia and industry. In Brazil, although it is popular in medicinal chemistry and related areas, chemoinformatics grows slower and most of its applications are still unknown to the scientific community. However, it appears that it is now starting to integrate itself with other fields, like pharmacognosy and natural products research, which, in turn, are somewhat connected with metabolomics and become an emerging new field to be better explored. Thus, with the fast growing of metabolomic applications in natural products-related fields, there is an urgent need to develop new and integrated applications that improve productivity, like databases and tools for property prediction and compound dereplication, so that a new and solid growth opportunity for chemoinformatics become possible. 1. Lectures 16 1.4 Metabolic ﬁngerprint and biomarker discovery Ieda Spacino Scarminio S of metabolites in the past have been limited to the analysis of a restricted number of molecules but currently with recent analytical developments several hundred analytes in a single sample can be detected. There are three major approaches used in metabolomics studies; (1) targeted analysis, (2) metabolite profiling and (3) metabolic fingerprint. Metabolomics are applied in different fields such as disease diagnosis, toxicology, plant science, pharmaceutical and environmental research. In metabolomic analysis, large amounts of data are produced in order to characterize samples. The use of chemometric methods is a commonly used strategy for the analysis these data. In this lecture strategies for the search of metabolic fingerprints and data analyses that are useful in biomarker discovery will be discussed. TUDIES 1.5. Statistical methods to analyze biological networks 1.5 17 Statistical methods to analyze biological networks André Fujita T regulatory, metabolic, and protein-protein interaction networks are examples of different layers of biological systems that share some features of complex networks, such as highly connected nodes, modularity, and small-world topology. Some studies indicate that changes in the topological structure of these networks may be associated with diseases. Therefore, methods to discriminate the processes that generate the different classes of networks (e.g., normal and disease) might be essential for the diagnosis, prognosis, and development of drug targets. It is known that several topological properties of a network (graph) can be described by the distribution of the set of eigenvalues (spectrum) of its adjacency matrix. Moreover, large networks generated by the same random process have the same spectrum distribution, i.e., a “fingerprint” that characterizes the network. Based on this relationship, I will present: (i) a definition of entropy of a graph spectrum to measure the “uncertainty” of a random graph; and (ii) concepts of “distance” between graphs based on the Kullback-Leibler and Jensen-Shannon divergences between graph spectra. Statistical methods for model selection and network model parameter estimation, as well as a statistical procedure to test the nullity of divergence between two classes of complex networks will be presented. The usefulness of these methods will be illustrated in applications of protein-protein interaction networks of different species and in networks derived from typically developing children compared to ones diagnosed with Attention Deficit Hyperactivity Disorder (ADHD). A user-friendly toolbox will be shown. HE GENE 1. Lectures 18 1.6 Metabolite identiﬁcation strategies using NMR Jan Schripsema M ETABOLOMICS has been defined as “the area of research which strives to obtain complete metabolic fingerprints, to detect differences between them and to provide hypothesis to explain those differences”. In the explanation of the differences, peaks need to be assigned to specific metabolites. Different approaches are available to reach this goal, among which 2D-NMR, the use of chromatographic systems, or the use of databases. 1.7. Telling metabolic stories to explore metabolomics data: a case study on the yeast response to cadmium exposure 1.7 19 Telling metabolic stories to explore metabolomics data: a case study on the yeast response to cadmium exposure Paulo Vieira Milreu G metabolic network reconstructions for a variety of organisms are available from the last decade onwards and some well-curated metabolic networks allow very precise in-silico simulations of cell growth and environmental adaptability. Nowadays, the application of metabolic network reconstruction to symbiotic systems and the possibility to obtain precise measurements of metabolite concentrations through metabolomics experiments give rise to new questions, such as identifying what possible pathways (or reaction chains) explains the concentration changes observed or identifying all possible minimum environment in which a given organism is able to produce some target metabolite. In this talk I will present our efforts to address such kind of biological problems through enumeration algorithms for different metabolic reaction sets that perform a special role in the network. Namely, metabolic stories are reaction sets that uncover possible explanations for a metabolomic experiment dataset. Factories are reaction sets that allow the production of a given metabolic target from a initial precursor set. Chemical organizations are reaction sets representing all steady or growing states of a metabolic system. ENOME - SCALE 1. Lectures 20 1.8 Application of MALDI in metabolomics: from extract analysis to the imaging of tissue Denise Brentan da Silva P LANT METABOLOMICS is a relatively recent research field that has gained increasing interest in the past few years. A technique widely applied in this field is electrospray ionization (ESI) mass spectrometry together with liquid chromatography. As a possible alternative to ESI, matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) can be useful, but there are many points that need to be explored for small compounds yet, including the data acquisition and processing. Recently, a protocol for metabolic fingerprinting from plant extracts by MALDI-TOF MS and subsequent multivariate data analysis by inhouse algorithms implemented in the R environment was developed for the taxonomic classification of them from different genera, families and orders. In addition, MALDI imaging is a promising technique that combines molecular mass analysis and spatial information in tissues, besides the data processing must be carefully applied, such as alignment, normalization and others, which are important to improve the quality of results. Despite to be a technique recently applied to plant tissue, the results could be extremely useful due to the problem of metabolite compartmentalization. 1.9. The fascinating World from Senna spectabilis’s rizosphere: exploring molecular diversity through the use of metabolomics and multiway analysis 1.9 21 The fascinating World from Senna spectabilis ’s rizosphere: exploring molecular diversity through the use of metabolomics and multiway analysis Ian Castro-Gamboa T of new and innovative analytical methods that may shed information towards the composition of complex natural mixtures is critical on bioprospection programs. Our research group “NuBBE” has incorporated the use of molecular virtual design using Nuclear Magnetic Resonance (NMR) aiming to increase the understanding of molecular relationships on dynamic natural matrixes and synergism effects of highly active crude extracts, previously screened using in vitro human cell lineages such as HL60 (Leukemia), MDAMB435 (Melanoma), HCT8 (Colon) and SF295 (Glioblastoma). Our research group has a library of extracts from previous Cerrado and Atlantic Rainforest bioprospection research programs, as well as endophytic fungi and microorganisms derived from rizhosphere habitats. Additionally, to speed up the selection of promising biologically active molecules, we have incorporated the use of chemometric and multivariate analysis towards dereplication. HE ESTABLISHMENT 1. Lectures 22 1.10 Classiﬁcation models proposition using Chemometrics: potential applications in metabolomics Edenir Rodrigues Pereira Filho C is a discipline that combines knowledge from mathematics, statistics and chemistry. In this presentation, I would like to show some examples highlighting the potentialities of Chemometrics for data analyses. The examples will describe the use of classification models in chromatographic fingerprints, biodiesel production and metals profile investigation in inorganic samples. HEMOMETRICS 2 Abstracts 2.1. Metabolomics research infrastructure available at Núcleo de Pesquisas em Produtos Naturais e Sintéticos (NPPNS) 2.1 25 Metabolomics research infrastructure available at Núcleo de Pesquisas em Produtos Naturais e Sintéticos (NPPNS) Silva, R. R.*; Lopes, N. P.* *Núcleo de Pesquisa em Produtos Naturais e Sintéticos, Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, Brazil. R S UPPORT C ENTERS (NAPs) are USP’s research integration structures, established with the objective of bringing together experts from one or more units around interdisciplinary research and/or instrumental research support. The Research Support Center in Natural and Synthetic Products (NPPNS) is integrating metabolomics research into its broad spectrum of phytochemical research. With a strong infrastructure on Mass Spectrometry analysis the NPPNS recognizes the central role of Bioinformatics/Chemometrics for metabolomics research and promotes, with its partners the I-BWBM. Here we briefly highlight some of our structure in four working projects on metabolomics, and invite the community to share our structure and knowledge, both on Mass Spectrometry and Bioinformatics. Project 1: It is essential to have a standardized database police to improve information reuse and faster knowledge growth. The NPPNS stores its spectrum on Global Natural Products Social Molecular Networking (GnPS – <http://gnps.ucsd.edu/>), an innovative initiative on spectral database and fragmentation analysis. Until the present date we have contributed with 413 spectrum depositions to GnPS, these spectrum will now be readily available to NPPNS as well as for all international scientific community for free. Project 2: Currently MALDI (Matrix-Assisted Laser Desorption/Ionization) ionization has been broadly used on imaging mass spectrometry, as a spatially resolved technique for direct analysis of biological samples. To be able to correctly separate matrix from sample peaks, a peak alignment is necessary. We present a simple design to an open source and user friendly software that allows inspection of multiple spectra, alignment, image comparison and spatial image exploration. Project 3: We have used in house R scripts to incorporate cosine similarity from MSMS data processed with XCMS2 to the probability ranking provided by ProbMetab package. The putatively ESEARCH 26 2. Abstracts annotated fumagillin exact mass, was highly correlated to unknown compounds. This mass was also linked to compounds, possibly sharing substructures, as for example, the floctafenic acid, with a high cosine score from massbank batch search. Project 4: We had previously shown that compiled chemical information provides better insights about the metabolic state of a biological sample. The grouped information can contribute to highlight biological patterns explaining sample grouping. In this project we highlight closer to geographical origin grouping as we merge different chemical profiles (LC-HRMS, LC-Ion trap-MS, GCMS), by merging presence/absence ion matrices. Bioinformatics has a central role on omics sciences. To make sense of multidimensional datasets it is necessary to have a physical infrastructure as well as methodological framework, to store and extract as much information as possible from these datasets. Here we highlighted our growing structure, where, from different metabolomics applications we use different statistical techniques focusing on data integration, and global data view, instead of being limited to few metabolites our targets are the metabolite interactions and network responses. E-mail: [email protected] 2.2. AsterBioChem: chemoinformatic tools applied for chemical and biological studies of Asteraceae 2.2 27 AsterBioChem: chemoinformatic tools applied for chemical and biological studies of Asteraceae Chibli, L. A.*; Da Costa, F. B.* *AsterBioChem Research Team, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil. A STER B IO C HEM RESEARCH TEAM (leader: Prof. Dr. Fernando B. Da Costa) is focused on studying different aspects of the chemistry and biology of the plant family Asteraceae or Compositae, one of the largest among the angiosperms with ca. 25,000 species spread in several countries, showing taxonomic, ecological, economic and medicinal interest. The research in chemistry of Asteraceae is focused on phytochemistry (isolation and identification of natural products - NP), metabolomics (metabolite fingerprinting through LC- MS) and chemoinformatics (chemometric and data mining tools), while the biology is basically concentrated on small-scale bioassays, i.e. high-throughput screening (HTS), using different models (e.g. enzymes and cells). The development of HTS evidenced the lack of efficiency and sensitivity of the classical phytochemical studies of plant extracts, once it only covers the major specialized metabolites. The association of HTS, metabolomics and MSA (multivariate statistical analysis) brings forth a non-reductionist approach that can determine which metabolites, either isolated or in mixtures, may be correlated with a certain biological activity. Herein we give an overview of AsterBioChem research field in metabolomics, highlighting a specific project entitled “Screening of inhibitors of the trypanosomatids’ enzymes DHODHs of Asteraceae using metabolomics allied to in silico methods”. Methods: Hidroalcoholic extracts from ca. 300 Asteraceae species will be submitted to: 1) Bioassays: in vitro screening for inhibitory activity of the enzyme dihydroorotate dehydrogenase from T. cruzi (TcDHODH) and L. major (LmDHODH); 2) Metabolomics approach: the metabolite fingerprints will be obtained by UHPLC-UV- HRFTMS (Thermo Scientific Exactive PlusT M equipped with OrbitrapT M technology (Thermo Fisher Scientific©); 3) Chemoinformatics analysis: the big set of data obtained will be 28 2. Abstracts explored and classified through chemometric (data mining) tools, such as MSA (unsupervised and supervised), ANN (artificial neural network) and Decision trees. Some of the software used for these analyses are the R, Weka (University of Waikato) and MZmine©. Results: The present project is still in progress and only some previous and inconclusive results will be presented. Conclusion: This approach constitutes a massive NP screening, being one of the most fast and efficient tools for drug discovery. Therefor, this project has its relevance and originality highlighted, as its potential for the discovery of new bioactive compounds for the treatment of Tropical Neglected Diseases. E-mail: [email protected], [email protected] Financial Support: FAPESP/Process number: 2010/51454-3, 2014/01 4436; CNPq/Process number: 306652/2010-2 2.3. Metabolomic analysis and bioactivity assays in COX and LOX pathways-dependent of plants from subtribe Lychnophorinae 2.3 29 Metabolomic analysis and bioactivity assays in COX and LOX pathways-dependent of plants from subtribe Lychnophorinae Godinho, C. C.*; Martucci, M. E. P.*; Casoti, R.†; Gobbo-Neto, L.* *Núcleo de Pesquiza em Produtos Naturais e Sintéticos – NPPNS, Departament of Physics and Chemistry, School of Pharmaceutical Science of Ribeirão Preto – FCFRP/USP. Do Café Avenue, S/N, 14040-903, Ribeirão Preto - SP, Brasil. †AsterBioChem Research Team, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, 14040-903, Ribeirão Preto, SP, Brazil. T LYCHNOPHORINAE SUBTRIBE, which belongs to Astearaceae family, is represented by 11 genera and 81 species, distributed in rupestrian fields and the Cerrado of Minas Gerais, of the Plateau Central. Many of the compounds in Lychnophorinae species, specially the sesquiterpene lactones and the phenolic compounds, are reported as inhibitors of mediators in the cascade of inflammatory process. Two enzymes are essential in the metabolism of arachidonic acid formed in the inflammatory process: cyclooxygenase (COX) and lipoxygenase (LOX), promoting the production of eicosanoids such as prostaglandins, prostacyclins and thromboxanes in COX-dependent pathway, and the production of leukotriene in LOX-dependent pathway. In the search for compounds with pharmacological activities, is evident the need for studies using more sensitive, rapid and effective analytical methodologies. One example is the metabolomics analysis, which produces a metabolic fingerprint that provides full information of the object in the study, and it can be used for various purpose. Through the data produced in the metabolomic analysis, it’s possible to determinate metabolic differences in samples by using statistical tools, and correlate these data with others for different applicability, by using in silico methods. This project aims to analyze the pharmacological potential of species of subtribe Lychnophorinae using metabolomics studies as subsidies, linking them with anti-inflammatory in vitro inhibition of cyclooxygenase and lipoxygenase-dependent pathways through in silico correlation. So far, HE 30 2. Abstracts the analysis of extracts of the Lychnophorinae species were made in UHPLC-UV-(DAD)-MS (Orbitrap), producing a metabolic fingerprint for each extract. Also the in vitro inhibition screening assays of COX and LOX-dependent pathways were conducted, revealing great pharmacological potential of the subtribe in both paths. In parallel, is being developed a database of the substances found in the subtribe, according to the literature. Financial Suport: FAPESP (process number 2014/01707-3) and CAPES. Contact: [email protected]; [email protected]; gobboneto @gmail.com. 2.4. The role of secondary metabolites in plants diversification and evolution: a remarkable example from the northern Andes 2.4 31 The role of secondary metabolites in plants diversiﬁcation and evolution: a remarkable example from the northern Andes Padilla-González, G. F.*; Diazgranados, M.†; da Costa, F. B.* *AsterBioChem Research Team, Laboratório de Farmacognosia, FCFRPUSP, Ribeirão Preto, SP, Brazil. †Jardín Botánico de Bogotá José Celestino Mutis, Bogotá, Colombia. T of the tropical Andes in South America was subjected to a series of climatic fluctuations as a consequence of the recurrent periods of glaciations and inter-glaciations that occurred in the late Pliocene and early Pleistocene, two to five million years ago. This led to the development of a new ecosystem called Páramo, which is characterized by a series of climatic and vegetation conditions that make it unique, among which stand out the “Frailejones”, a group of ca. 143 species grouped in eight genera that constitute the subtribe Espeletiinae (Asteraceae, Millerieae). The Frailejones are considered a classic example for studying rapid adaptive radiations and speciation mechanisms in plants, due to their remarkable morphological variation and recent speciation. Therefore, the evolutionary history and taxonomy of this group have been the focus of numerous studies in recent years. Chemically, recent studies demonstrated a possible correlation between the species metabolic fingerprints and their geographical origin, which is in accordance with their current phylogeny based on molecular markers. This study aims to investigate all species of Espeletiinae through a metabolomic approach by UHPLC-UV-MS and multivariate statistical methods, in order to establish correlations between species chemical composition and their taxonomy, morphology and biogeography. Additionally, we intend to perform phylogenetic analyzes with DNA sequences from the literature with the aim of mapping chemical characters into the molecular phylogenies in order to determine ancestral states, contributing to the understanding of the evolutionary processes that have shaped the current distribution and diversity of secondary metabolites in Espeletiinae. Preliminary results demonstrate that phylogenetic reconstructions based on a concatenated matrix composed of molecular and chemical markers led to more resolved tree topologies and improved inter-specific HE NORTHERN REGION 32 2. Abstracts relationships. Thus, secondary metabolites may play an important role in the diversification and adaptive success of a classic group to study rapid adaptive radiations in plants. 2.5. Mixture design to optimize Baccharis genus species extraction in untargeted metabolomic studies 2.5 33 Mixture design to optimize Baccharis genus species extraction in untargeted metabolomic studies Casoti, R.*; Bueno, P. C. P.†; Da Costa, F. B.* *University of São Paulo, School of Pharmaceutical Sciences of Ribeirão Preto – USP/FCFRP, Brazil. †Institute of Chemistry, São Paulo State University - UNESP, Araraquara/SP, Brazil. T Baccharis GENUS belongs the largest Asterae tribe of Asteraceae family. This genus is composed of approximately 400 species spread in America, which 178 species there are in the Brazil. In Baccharis species is composed of phenolics and terpenoids compounds that are associated with many medicinal activities or chemotaxonomic marker. The aim of the study was to compare the extraction power to both MeOH and EtOH solvents to samples of Baccharis genus by mixture design and LC/MS data. 2. Methods The stems and leafs were grinded with liquid nitrogen to 386 samples Baccharis. The extraction was made by sonication at 25°C in 20 min from the mix of all samples. For the extraction procedure of the metabolites, three factors have been taken into account: ethanol (EtOH), H2 O and methanol (MeOH) acidified HCO2 H 0.08%. These three factors were used to develop experimental design (simplex-centroid mixture design) with the software Statistic 8.0 (StatSoft®), which resulted in ten experimental conditions. In samples of approximately 10 mg, 2 mL of organic solvent were added according to the conditions previously established. After maceration, the plant material was centrifuged for 3min at 13,000 rpm and the extracts were partitioned with 400 µL of n-hexane. The extracts were further analyzed by UHPLC-ESI-HRFTMS using the following chromatographic conditions: ACE column (150x3 mm, 3µm) and gradient MeCN-H2 O (2 to 100% MeCN) in 30 min. For mass spectrometry (positive and negative modes), electrospray (ESI) source was used with range detection of 100 to 1200m/z, temperature at 300°C, spray voltage at 3.6 kV (positive mode) and 3.2 kV (negative mode) in a Exactive Plus analyzer (Thermo Scientific®). Deconvolution and peak alignment were carried out using the software MZmine 2.10 (free software). To determine the best extractive condition of Baccharis genus HE 34 2. Abstracts samples that is able to extract a higher diversity of substances of interest, the mixture design was based on yield (grams), peak area and number of extracted peaks (from m/z) of each sample both in the positive and negative ionization mode. These values are considered the dependent variables of the model. The LC/MS data were deconvoluted and aligned (MZmine 2.10), and then statistically analyzed (Statistica 8). The results showed that EtOH solvent was the best extractor solvent to Baccharis genus samples. The desirability surface showed a range concentration from 40 to 80% EtOH in solution aqueous to desirable maximum values. We determined that EtOH:H2 O (7:3) extraction by sonication (20 min) at 25°C is one between the best conditions of extraction of Baccharis genus for untargeted metabolomic studies. This methodology has been chosen how extraction protocol of secondary metabolites in untargeted metabolomic studies of the Baccharis genus. E-Mail: [email protected] Financial Support: FAPESP/Process number: 2012/14397-7. 2.6. Metabolomics assited by chemometrics for targeted phytochemistry 2.6 35 Metabolomics assited by chemometrics for targeted phytochemistry Dos Santos, F. A.*; Faleiro, D. P. V.*; Chagas-Paula, D. A.*; Da Costa, F. B.* *Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo U is an approach used for a greater detection of any secondary metabolite, a study aimed at providing a better chance for identifying probable active substances. However, this approach requires the use of chemometrics for the treatment of chemical data and identification of possible active metabolites by means of a discriminant analysis, such as OPLS-DA. Recent work has shown the application of this modern approach. Therefore, our purpose was to use chemometrics applied to metabolomics to point the possible inhibitory substances of pro-inflammatory enzymes COX-1 and 5-LOX. We have used HPLC-UV-TOF-MS and UHPLC-UV-Orbitrap-MS to generate chemical data of 17 hydroalcoholic extracts obtained from leaves of Aldama La Llave, which showed bioactivity against COX-1 and 5-LOX. The chemical data were analysed by the software and MZmine and dereplication was performed using multiple reaction monitoring (MRM) generated by HPLC-UV-IonTrap, UV data and exact mass of 313 metabolites from Aldama La Llave from literature. The OPLS-DA analysis allowed us to choose the likely COX-1 and 5-LOX inhibitors and subsequently was performed dereplication of these metabolites. Finally, we held the targeted phytochemistry of such metabolites by Sephadex column LH-20 and HPLC-semi-prep. The likely active and isolated compounds were elucidated by H1 NMR. We are in the final stage of structural elucidation, but dereplication obtained to date suggests that the substances are kaempferol-3-O- glucuronideo, quercetina-3-O-metil-7-glucuronideo e kaempferol-3-O-(6”-malonil- glucosideo). NTARGETED METABOLOMICS 2. Abstracts 36 2.7 Potential metabolic biomarkers for chronic graft-versus-host disease Alborghetti, M. R.*; Correa, M. E. P.†; Da Silva, A. A.†; Miranda, E. C. M. †; Mauricio Luis Sforca, M. L.*; Zeri, A. C. M.* *Brazilian Biosciences National Laboratory (LNBio)/ Brazilian Center for Research in Energy and Materials (CNPEM), Campinas-SP, Brazil. †Hematology and Hemotherapy Center, University of Campinas/ Hemocentro - Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, Brazil. G RAFT- VERSUS - HOST (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Whereas biomarker panels have been developed for the acute form of the disease, there is a lack of information about the chronic form (cGVHD). Using nuclear magnetic resonance, our study prospectively exploited the serum metabolome of 18 patients who had undergone allogeneic HSCT. We identified and quantified 63 metabolites and performed the metabolomic profiling on days -7 (D-7), D=0, D+10, D+100, relative to transplantation day. From 18 patients analyzed, 6 developed cGVHD and four metabolites were important to cluster cGVHD-free versus cGVHD groups, with predictive value for cGVHD: cystine, leucine (D+10 and D+100), phenylalanine and hypoxanthine (D+10). Cystine was more associated to cGVHD development than other metabolites (p = 0.004 two-way Anova, p=0.004 and p=0.03 t-test for day +10 and +100, respectively). Cystine, more than a biomarker, may be involved in the etiology of cGVHD. Cystine, essential for T cells activation and proliferation via dendritic cells, had its concentration increased in cGVHD group. Our results demonstrate the importance of biomarkers panels for predicting and understanding cGVHD, with the feasibility of prediction in early stages of disease, even where clinical symptoms are not yet evident, allowing therapeutic interventions even before tissue damage. 2.8. Analysis of sugarcane transcriptome and metabolome changes along the plant maturation cycle 2.8 37 Analysis of sugarcane transcriptome and metabolome changes along the plant maturation cycle Inada, D. T.* ; Lembke, C. G.* ; Nishiyama, M. Y.* ; Zambotti-Villela, L.* ; Silva, R. R.†; Souza, G. M.* *Institute of Chemistry, USP, São Paulo, SP. †Pharmaceutical School, USP ,Ribeirão Preto, SP. S UGARCANE is an important crop whose contribution for brazilian econ- omy has been growing. It is used for sugar production and also as an alternative fuel source, with great potential to overcome the challenges related to fossil fuels. Some molecular studies using sugarcane plants have been performed so far and a highlight must be given for the sequencing of EST’s (Expressed Sequence Tags) by the SUCEST project. Furthermore, studies of gene expression and metabolome of sugarcane were also conducted in order to get a better understanding of the process of sucrose accumulation in different environmental conditions. Despite that, for sugarcane breeding studies, many mechanisms still require a better comprehension. This work has the aim to develop a joint analysis of transcriptome and metabolome data obtained from samples of a commercial sugarcane variety (SP80-3280) during some points of maturation along the production cycle using the microarray methodology for transcriptome analysis and the LC-MS platform for metabolome analysis. For the metabolomic data, we have the interest in adopting bioinformatics tools for identifying the metabolites and analyze altered pathways in different tissues and experimental points. We also expect to perform an integration of transcriptome and metabolome data in order to relate the genes expressed in different plant tissues with the metabolites levels and identify their respective metabolic pathways. These analyses might help in defining major target networks or genes to be manipulated for improving plant productivity and increase sucrose content. E-mails: [email protected], [email protected], yutakajr @gmail.com, [email protected], [email protected], [email protected] 2. Abstracts 38 2.9 Xanthan gum removal for 1 H-NMR analysis of the intracellular metabolome of the bacteria Xanthomonas axonopodis pv. citri 306 Pegos, V. R.*†‡¶; Canevarolo, R. R.*†‡¶; Sampaio, A.*; Balan, A.* ; Zeri, A. C. M.* *Brazilian Biosciences National Laboratory, Rua Giuseppe Máximo Scolfaro, 10.000 – Polo II de Alta Tecnologia – Caixa Postal 6192 – CEP: 13083970 – Campinas/SP, Brazil. †Boldrini Children Hospital, Rua Dr. Gabriel Porto, 1270, Cidade Universitária – CEP: 13083-210 – Campinas/SP – Brazil. ‡Universidade Estadual de Campinas (UNICAMP), Instituto de Biologia (IB), Dep. Genética e Biologia Molecular. Cidade Universitária. Rua Monteiro Lobato, 255, CEP 13083-970, Campinas/SP, Brazil. ¶These authors contributed equally to this work and should be considered co-first authors. X is a genus of phytopathogenic bacteria that produces a slimy, polysaccharide matrix known as “xanthan gum”, which involves, protects and helps the bacteria during its process of host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industry, the xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In an 1 H-NMR-based metabolomic study of Xanthomonas, the spectum peak corresponding to the internal reference of concentration was abnormally shortened, indicating a cross reaction between the reference compound and the xantham gum which could compromise metabolic quantification. Aiming at an efficient gum extraction protocol, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolome observation: 3 washing cycles; 5-washing cycles, ultracentrifugation; standards protocol. Lower limits for bacterial pellet volumes for extraction were also probed (62,5 µL, 125 µL and 500 µL), and a strategy is illustrated with an initial analysis of X. citri’s metabolism by 1 H-NMR spectroscopy. The standard protocol on the literature showed a high concentration of xanthan gum and did not allow performing the metabolome identification and quantification. Next, the washing cycle 3 or 5 leads a low metabolome identification, ANTHOMONAS 2.9. Xanthan gum removal for 1 H-NMR analysis of the intracellular metabolome of the bacteria Xanthomonas axonopodis pv. citri 306 39 high trehalose concentration and decrease of the cell number on the sample. When 5 washing cycle was applied the number of the cell loosed was higher and poor spectrum quality was observed. The ultracentrifugation intervention leads a high number of metabolite identified and quantified and also the spectrum quality was acceptable. The ultracentrifugation protocol was optimized to best bacterial pellet volume and 125 µL and 500 µL are best volume, 62,5 µL could be usable on difficult samples. E-Mails: [email protected], [email protected] cnpem.br, [email protected], [email protected] cnpem.br, [email protected] 2. Abstracts 40 2.10 Impact of water deﬁcit on sugarcane leaf secondary metabolism Budzinski, I. G. F.* ; Regiani, T.*; Moraes, F. E.*; Labate, C. A.* *Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP. S (Saccharum spp.) is an important source for the production of sucrose and ethanol in many tropical and sub-tropical regions. Water deficit is one of the main factors that affect sugarcane growth, metabolism and yield. Irrigation is one alternative to mitigate water deficit in crops, however water is limited is some regions and equipment costs make this strategy expensive. The capacity of monitoring a set of metabolites could improve the understanding of mechanisms involved in plant responses to water deficit. Besides, differences in metabolite content can also represent good predictors for drought tolerant phenotypes. A LC-MS-based approach was performed to investigate changes in the leaf metabolite profile of a sugarcane drought tolerant variety (CTC-15) under three irrigation regimes (control, moderate and severe stress). Leaf (+1) metabolites were extracted from 50 mg of powder tissue. Samples were analyzed by UPLC-QTOF-MS. Reverse-phased chromatography was performed using the following gradient condition: 95% A (H2 O + 0.1% HCOOH) and 5% B (ACN + 0.1% HCOOH) for 6 minutes, 25% A and 75% B for 6 minutes, 5% A and 95% B for 1 minute. The range of the mass scan was 100-2000 m/z, in positive mode. Data processing and multivariate analysis were performed in MarkerLynx and MetaboAnalyst softwares. Metabolites were identified based on MS/MS fragmentation at different collision energies. Fragmented ion patterns were searched in the HMDB databank. We were able to discriminate samples and observe leaf metabolite changes in response to water deficit. PLS-DA model demonstrated a clear separation between treatments (Q2 >0.72). Metabolites features were ranked according to their contribution to groups separation, by the “variable importance in the projection” (VIP). Based on VIP results, the highly abundant metabolites were selected to MS-MS fragmentation and identification. Financial Support: FAPESP/Process number: 2012/22227-4. UGARCANE 2.11. Differential metabolomic responses of Histoplasma capsulatum in biofilms and planktonic yeasts 2.11 41 Differential metabolomic responses of Histoplasma capsulatum in bioﬁlms and planktonic yeasts Pitangui, N. S.*; Gomes, P. C.*; Sardi, J. C. O.*; Rodríguez-Arellanes, G.†; Taylor, M. L.†; Mendes-Giannini, M. J. S.*; Fusco-Almeida, A. M.* * Laboratório de Micologia Clínica, Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista (UNESP), Araraquara SP, 14801-902, Brasil. †Laboratorio de Inmunologia de Hongos, Departamento de Microbiologia y Parasitologia, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México DF, 04510, Mexico. H var. capsulatum is a dimorphic fungi pathogen that causes a major systemic mycosis, named histoplasmosis. The pathogenesis of histoplasmosis occurs as a result of inhalation of microconidia from mycelial phase affecting primarily the lung where occurs the yeast differentiation, which subsequently induces pulmonary infection and dissemination to other organs, particularly in immunocompromised individuals. Recently, a correlation between the H. capsulatum infection and biofilms formation has been established, structures characterized as complex three-dimensional network which induce, among others, antifungal resistance. Therefore, it is emerging the identification of new targets that may be selected in order to establish a new therapeutics for histoplasmosis. In this sense, this study aims to analyze the H. capsulatum differential metabolome in biofilms and planktonic growth. To address this purpose, a untargeted metabolomics approach will be performed such that the separation and detection of metabolites will be achieved by a method of liquid chromatography coupled with mass spectrometry (LC/MS). Through this approach aims to identify a set of differentially expressed metabolites in order to design a biofilm marker in infection to the host cell and/or therapeutic target for histoplasmosis. E-Mail: [email protected] ISTOPLASMA CAPSULATUM 2. Abstracts 42 2.12 Identiﬁcation of cuticular wax lipids from Eucalyptus grandis leaves and their relationship with rust disease Bini, A. P.*; Cataldi, T. R.*; Verdi, M. C. Q.*; Labate, C. A.* *Laboratório Max Feffer de Genética de Plantas, ESALQ-USP, Piracicaba, SP, Brasil. T is covered by a cuticle membrane composed by cutin associated with lipids, called cuticular waxes. The cuticle is the first interface between pathogen and plant, and some compounds in this surface can influence on pathogen adhesion and recognition. Therefore, when uredospores from biotrophic pathogens get in contact with the plant, they encounter the cuticle membrane that provides important signals to the pathogen. These signals trigger the early events of the infection process, such as fungal germination and differentiation, which is fundamental to plant-pathogen interactions. Thus, the main goal of the present study is to identify the lipids of the cuticular wax from Eucalyptus grandis leaves (resistant and susceptible to rust), as an attempt to identify possible chemical signals that could be related with the germination of uredospores of P. psidii, the causal agent of rust in Myrtaceae. For that, lipid profiles obtained by mass spectrometry from eucalyptus cuticular waxes will be compared with the lipid profile of olive oil, substance that promotes germination of P. psidii in vitro. This work aims to contribute to a better understanding of the processes involved in the early events of the interaction between E. grandis x P. psidii. Until now, the wax extraction and analytical parameters involved in the analysis by MALDI TOF/TOF have been tested and optimized for resistant and susceptible plants of E. grandis. The first mass spectra of the three study groups (susceptible and resistant plants, and olive oil) were obtained successfully and the next steps are the multivariate statistical analysis of data. E-Mails: [email protected], [email protected] Financial Support: FAPESP/Process number: 2013/07596-6. HE SURFACE OF PLANTS 2.13. Serum and urine metabolic profile in response to renal ischemia/ reperfusion in a swine model 2.13 43 Serum and urine metabolic proﬁle in response to renal ischemia/ reperfusion in a swine model Malagrino, P. A.*; Venturini, G.*; Schneider, P. Y.*; Dariolli, R.*; Padilha, K.*; Kiers, B.*; Gois, T. C.*; Motta-Leal-Filho, J. M.†; Takimura, C. K.*; Girardi, A. C. C.*; Carnevale, F. C.†; Canevarolo, R.‡; Malheiros, D. M. A. C.¶; Zeri, A. C. M.‡; Krieger, J. E.*; Pereira, A. C.*. *Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo Medical School, São Paulo, SP, Brazil. †Interventional Radiology Unit, Radiology Institute, Hospital das Clínicas, University of São Paulo Medical School, São Paulo, SP, Brazil. ‡Biosciences National Laboratory, LNBio, Campinas, SP, Brazil. ¶Department of Pathology, University of São Paulo Medical School, São Paulo, SP, Brazil. A (AKI) is a serious complication in hospitalized patients mainly caused by ischemia/reperfusion. AKI is defined as the abrupt decrease in kidney function based on acute alterations in serum creatinine or urine output. Nevertheless, changes in serum creatinine are late and vary with muscular mass, age, gender, metabolism and hydration of individuals. In this sense, new biomarkers for the accurate and early diagnosis are needed. Therefore, this study aimed characterized changes in the serum and urinary metabolomic profile during renal ischemia and reperfusion in a swine model. To study, a swine controlled, single-insult model of unilateral renal I/R without contralateral nephrectomy was used. Five animals underwent renal ischemia by balloon catheter placed and inflated into the right renal artery for 120 minutes and reperfusion over 24 hours. Serum of vena cava and urine of bladder sampling was collected before (pre-ischemia), during (ischemia) and after (reperfusion) ischemia following of analyzed by 1 H Nuclear Magnetic Resonance 600Hz. Chenomx NMR suite was used for metabolites identification and quantification. Trough of profile observation and PLS-DA analysis we select eight increased serum metabolites (L-glutamate, L-serine, N-isovaleroylglicine, L-methionine, L-proline, 2aminobutyrated, choline and creatinine) able to separate pre-ischemia and ischemia, recovering to basal levels after 11 hours of reperfusion. CUTE KIDNEY INJURY 44 2. Abstracts In addition, we select eighteen urinary metabolites able to distinguish the three periods: pre ischemia, ischemia and reperfusion. To understand the role these metabolites in pathophysiology of disease, systems biology analysis ware performed to all these metabolites by MetaCore and Ingenuity Pathway. This analysis resulted in involved of these select metabolites in cell death, lipids concentration, molecules transport, reactive oxygen species synthesis, cells proliferation and inflammation. Through network analysis we found changes in H+ , Na+ and Cl- ion transport pathways and association with Nuclear Factor-KappaB, calcineurin and proinsulin, pathways common in AKI. Interesting, through these metabolites we identified a new pathway (Huntingtin pathway) that had not been previously implicated in renal I/R. Therefore, the metabolomic profile found on the renal I/R enabled the identification of candidates to identify disease biomarkers and a new pathway related to renal injury. E-Mail: [email protected] 2.14. Investigating the metabolic network of developing soybean seedlings 2.14 45 Investigating the metabolic network of developing soybean seedlings Moreira, T. B.*; Williams, T. C. R.* *Universidade de Brasília, Departamento de Botânica, Campus Darcy Ribeiro, Brasília 70910-900. T HE POST- GERMINATIVE GROWTH of seedlings depends upon the mobilisation of reserves encountered in the seed, including proteins, lipids and carbohydrates. Whilst the metabolic pathways responsible for the breakdown of these reserves have been the object of intense study questions remain about how their use is integrated at the systems level to supply the metabolites and energy necessary for survival and growth of the young plant. In order to respond to these questions we have conducted an in depth analysis of the biochemical composition of cotyledons and hypocotyl/root of soybean (Glycine max) seedlings during post-germinative heterotrophic growth. Measurement of dry mass, protein, fatty acids, cell wall and carbohydrates over a four day period were used to calculate the rates of synthesis and breakdown of these biomass components. In addition a combination of HPLC and GC-MS was used to profile primary metabolites and raffinose series sugars. These latter measurements allow us to explore the behaviour of the seedling metabolic network, indicating which metabolic pathways are active during different stages of growth and suggesting how carbon and nitrogen may be transported between the two organs under study. The biomass composition data will now be integrated into a genome scale stoichiometric model of soybean metabolism, and flux balance analysis used to investigate the how metabolism operates during seedling growth with a focus on identifying how different seed storage reserves contribute to the carbon, nitrogen and energy balance of the growing seedling. The performance of the model will be validated by comparison with our metabolite profiling data and physiological gas exchange measurements. E-Mails: [email protected], [email protected] Financial Support: CNPq, Universal 2013. 2. Abstracts 46 2.15 Metabolomic proﬁles of caffeine metabolization after coffee intake Gois, T. C.*; Venturini, G.*; Padilha, K.*; Malagrino, P. A.*; Mendes, M. A.†; Grasselli, C.‡; Santos, P. C. Jr. L.*; Pereira, A. C.* *Laboratory of Genetics and Molecular Cardiology, Heart Institute – Medical School, University of São Paulo, São Paulo, Brazil. †CEPEMA-POLI, University of São Paulo, São Paulo, Brazil. ‡Nutrition School, Federal University of Alfenas, Minas Gerais, Brazil. C is one of the most consumed beverages in the world since the fifteenth century, especially in Brazil. However, there is controversy if the different physiological responses owing to coffee intake are beneficial or prejudicial to health. Caffeine (1,3,7-trimethylxanthine) is the main coffee component that has stimulating effects on the cardiovascular and central nervous systems. The specific effects in these systems change between individuals due to caffeine sensitivity be determined by genetic, environmental and lifestyle factors. Studies involving the main mediator enzyme of caffeine biotransformation (cytochrome P450 isoform 1A2) showed that there are two metabolic profiles between the usual coffee consumers: fast and slow metabolism. These and others differences in metabolism of caffeine may influence the physiological responses. In this study, we aimed to evaluate the differences between the metabolic profiles produced by fast and slow metabolizers and determine if these metabolites are associated with the physiological effects caused by coffee ingestion. 30 healthy men were recruited and subjected to a diet with low polyphenols content (36 hours prior to the end of the study) and 12 hour fasting. Blood and urine samples were collected before coffee ingestion. Following, the participants drank 150 mL of coffee prepared by infusion and filtration (6.8g coffee 100% Arabica Prima Qualitá®) followed by serum and urine samples collection during periods of 6, 12 and 24 hours after intake. All samples were stored at -80ºC. Evaluation of serum metabolomic profile was performed using chemical derivatization protocol (methoxylamine + pyridine [20mg/mL]; MSTFA + 1% TCMS) followed by GC-MS method (GC-MS QP 2010 Plus Shimadzu®). Partial results showed that through metabolomics analysis was possible identify two patterns metabolism of caffeine. Eighteen OFFEE 2.15. Metabolomic profiles of caffeine metabolization after coffee intake 47 subjects had caffeine until 12 hours and were grouped and classified as slow metabolizers, while seven individuals did not show any caffeine in the same period, which were grouped and classified as fast metabolizers. Subsequently, these profiles will be compared to metabolic profiles of the other metabolites. In addition, urine sample will be analysis for comparison between caffeine intake and caffeine excreted. E-Mail: [email protected] 2. Abstracts 48 2.16 Metabolomics, proteomics and chemometrics approaches for the infraspeciﬁc chemical variability study of a native Brazilian plant species Bueno, P. C. P.*; Pereira, F. M. V.*; Torres, R. B.†; Hippler, M.‡; Cavalheiro, A. J.* *Institute of Chemistry, São Paulo State University - UNESP, Araraquara/ SP, Brazil. †Agronomic Institute of Campinas, Herbarium - IAC, Campinas/ SP, Brazil. ‡Institut für Biologie und Biotechnologie der Pflanzen - IBBP, Westfälische Wilhelms-Universität Münster. T is an attempting to join metabolomics, proteomics and chemometrics to solve a biologycal question concerning plant chemical variability and phenotypic plasticity. The species Casearia sylvestris Sw. is a notable representative from the Salicaceae family because of its great pharmacological, economic and ecological relevance. Due to its high adaptative capacity, C. sylvestris is spread throughout latin America. In Brazil, this species occurs in practically all biomes covering a wide range of ecosystems and ecotones and, depending on the region where it grows, it exhibits considerable morphological variability. Accounting for the external morphological differences, it can be recognized two varieties: C. sylvestris var. sylvestris, which are generally trees that can be found in humid and dense forests, and C. sylvestris var. lingua, which is mainly found as shrubs and inhabits open and xeric habitats such as savannahs. Intermediate forms can be found beyond these two varieties, which makes delimiting the taxa difficult. To the chemical point of view, in variety lingua occur predominantly the phenolic compounds, while in variety sylvestris the clerodane-type diterpenes predominate. Considering this background, we hypothesize that the secondary metabolites composition is related to and/or conditioned by biomes, and mainly associated to their predominant morphotypes. To evaluate this hypothesis, we collected 140 C. sylvestris individuals from the Cerrado, Caatinga, Atlantic Forest, Pampa and Pantanal Brazilian biomes and applied (i) design of experiments (DOE) to developed an analytical methodology usHIS STUDY 2.16. Metabolomics, proteomics and chemometrics approaches for the infraspecific chemical variability study of a native Brazilian plant species 49 ing UHPLC-DAD for the simultaneous analysis of phenolic compounds and clerodane-type diterpenes, (ii) principal component analysis (PCA) for the infraspecific chemical variability study, (iii) metabolomic approaches for dereplication studies and (iv) high throughput proteomics using LC-MS/MS and bioinformatics, for the differencial proteomic study. The combination of DOE for the analytical method development with chemometrics allowed the assessment of important information about the distribution of C. sylvestris in Brazil. Results provided variables discrimination, showing an interesting distribution pattern of the two varieties according to their original biomes/ecosystems and metabolic profile. The proteomic approach allowed the annotation of 547 proteins in the two C. sylvestris varieties. From the photosynthetic point of view, there is an up-regulation o Rubisco followed by a dow-regulation of LHCII proteins in variety lingua when compared to variety sylvestris; also, in varitey lingua, the expression of enzymes from the secondary metabolism is deviated to the biosynthesis of phenolic compounds instead of clerodane-type diterpenes, characteristic of variety sylvestris. Results suggest an important mechanism of C. sylvestris adaptation to light in the level of protein expression, which directly reflects on the biosynthesis of its secondary metabolites. E-Mail: [email protected] 2. Abstracts 50 2.17 Metabolomic studies of fungi isolated from the rhizosphere of Senna spectabilis Selegato, D. M.*; Vieira, N. C.*; Freire, R. T.†; Pavani, V. D.*; Pilon, A. C.*; Castro-Gamboa, I.* *Nuclei of Bioassays, Ecophysiology and Biosynthesis of Natural Products (NuBBE), IQ/Ar, Chemistry Institute – UNESP – State University São Paulo. †Center of Imaging and Spectroscopy by Magnetic Resonance (CIERMag) – IFSC, São Carlos Institute of Physics – USP – São Paulo University, São Carlos. T N ATURAL P RODUCTS (NP) has been responsible for the increased use and incorporation of analytical tools that allows a rapid and efficient overview of the molecular composition from convoluted natural matrixes. Among the vast variety of dereplication techniques, mass spectrometry (MS) and Nuclear Magnetic Resonance (NMR) are the preferred spectroscopic state of the art technologies, primarily because of its reproducibility. NMR has been devised as one of the most promising techniques due to the broad coverage of metabolite detection, signal robustness, easiness of data handling for statistic treatment and short time analysis. Recently, NMR was integrated among high performance liquid chromatography (HPLC), allowing the analysis of complex plants and microorganisms extracts and, therefore, assisting on chemical composition studies of known bioactive chemotypes helping to selec those with novel molecular characteristics for further separation and purification. This work aims to study previously selected biologically promising extracts of fungi isolated from Senna spectabilis using statistical analysis (HCA, PCA and PLS) of the data generated through coupled spectroscopic technology, as well as in vitro bioassays, such as acetylcholinesterase inhibition, cytotoxicity and antifungal. For this analysis, new methodologies and dereplication techniques were created and applied using MATLAB® software version R2014a, allowing us to generate scripts that enables a rapid analysis of spectroscopic and chromatographic data of high complexity. Overall, 127 fungi were analyzed, 19 of which showed a viable growth and were submitted to NMR, LC-DAD/MS and bioassays. After applying the chemioHE SEARCH FOR NEW SOURCES OF 2.17. Metabolomic studies of fungi isolated from the rhizosphere of Senna spectabilis 51 metric’s methods developed on MATLAB, the fungi CSP-R18, CSP- R19, CSP-19a, CSP-19b, CSP-46 and CSP-37b showed very promising data, with a strong antifungal, cytotoxic and acetylcholinesterase inhibition potential, exemplifying the chemodiversity of symbiont microorganisms and the need for future studies both isolated and in co-culture. E-Mail: [email protected] 2. Abstracts 52 2.18 Metabolomics and proteomics data integration during sugarcane development Moraes, F. E.*; Budzinski, I. G. F.*; Regiani, T.*; Labate, C. A.* *Escola Superior de Agricultura Luiz de Queiroz, USP, São Paulo, SP. S (Saccharum spp.) is one of the most important cultivated grasses of the world and Brazil is the largest producer, which has as its main representative the São Paulo state, concentrating more than half of the area for this crop. Thus, it is necessary a high understanding of the mechanisms that regulate the sucrose accumulation in this plant. The genetic mechanisms that control sugarcane sucrose accumulation have been studied at various levels, such as gene identification and localization, identification of quantitative trait locus controlling, transcriptome, proteome, characterization and metabolite identification. With all these studies it is evident the necessity of a holistic approach to globally understand the plant during the sucrose accumulation. Thus, this work aims to characterize metabolites and proteins during plant development and sucrose accumulation, using bioinformatics to link these results by Bayesian networks in a systems biology approach. Therefore, it will be possible to create a gene regulation network, identify new gene functions, pathways and phenotypes related to the sugarcane sucrose accumulation. All information generated in this project will be integrated into SUCEST-FUN (<http://sucest-fun.org/>) servers, allowing users to crosslink between the data from transcriptomics, metabolomics, proteomics, molecular markers (such as E-QTLs and QTLs ) or any other service available on the server. E-Mails: [email protected], [email protected] Financial Support: FAPESP/Process number: 2012/12521-2. UGARCANE 2.19. Metabolic fingerprinting of Eucalyptus grandis during the infection by Puccinia psidii 2.19 53 Metabolic ﬁngerprinting of Eucalyptus grandis during the infection by Puccinia psidii Marques, F. G.*; Budzinski, I. G. F.*; Cataldi, T. R.*; De Moraes, F. E.*; Labate, Carlos A.* *Laboratório Max Feffer de Genética de Plantas, ESALQ-USP, Piracicaba, SP, Brazil. D wide range of application - pulp and paper production, construction or use in energy generation - Eucalyptus is one of the most important crops of the country, generating millions of direct and indirect jobs and moving important part of the national GDP with its various derivatives. However, as with many others exotic species planted on a large scale, the Eucalyptus, especially the Eucalyptus grandis species and its hybrids, are infected by native pathogens, of which the most important is the rust fungus, Puccinia psidii. The pathogen attacks clonal gardens and nursery seedlings up to two years old, causing losses of up to 40% of timber volume. Intending to characterize the changing metabolite profile of Eucalyptus grandis during the infection of P. psidii, a metabolomics approach will be carried out to detect differences between susceptible and resistant half-sibling plants, both mock-inoculated and inoculated with the fungus, at different time points after inoculation. The project aims the discovery of possible biomarkers of the disease, triggered by the interaction between host and pathogen, and also the modulation of the plant’s metabolism under biotic stress. UE TO ITS 2. Abstracts 54 2.20 Metabolomics applied in a Brazilian populationbased study Padilha, K.*; Venturini, G.*; Pires, T.*; Gois, T. C.*;Malagrino, P. A.*; Blatt, C.†; Pereira, A.* *Heart Institute, Sao Paulo, Brazil. †Agilent Technologies Brasil Ltda., Life Sciences &, São Paulo, SP, Brazil. M have been recently used in different studies to identify metabolic signatures of Type 2 Diabetes Mellitus (T2DM), metabolites associated with disease progression and response to treatment. In this population-based metabolomics study, we aimed to analyze metabolites from T2DM and healthy subjects from the general Brazilian population, using GC/MS. We also identified different serum metabolic patterns between groups and association of these metabolites with molecular pathways changed by the disease. 893 subjects participants of “the Baependi Hearts Study” participated in this study. Subjects were split into two groups: diabetic (91 individuals, glucose 126mg/dL) and nondiabetic (802 individuals). Serum samples were deproteinized with methanol, followed by lyophilization and derivatization. The extract was spiked with 50µL methoxyamine in pyridine solution. This methoximation reaction was performed at RT, followed by trimethylsilylation. adding MSTFA/TMCS. For each samples was add 5µL of internal standard solution (d27-myristic acid). 1µL of this derivative was used for GC/MS analysis by GC-MS (Agilent 5977A Series GC/MSD). Analysis was performed with NIST 11 and FiehnA. 01.00 compound library using Unknowns - Agilent MassHunter Workstation Quantitative Analysis. Data were normalized using QC by LOESS method according to injection order and analysis day. We found with our population study the same pattern of diabetes prevalence of the total Brazilian population. By GC-MS technique, normalization and statistical analysis were possible to identify differentially changed metabolites in serum of diabetics and non-diabetics. 156 metabolites upregulated in T2DM subjects compared to heathy individuals, were found. According to system biology analysis, these changed metabolites are related to Amino acids metabolism and its regulation, Metabolic disease, Obesity, Vitamin and cofactor metabolism, Protein degradation, Energy metabolism and its regulation and ETABOLOMICS PROFILES 2.20. Metabolomics applied in a Brazilian population-based study 55 Calcium regulation. Our data suggested these metabolic pathways are changed in diabetic subjects. Some metabolites identified such as BCAA are well discussed in diabetic´s literature and are related to insulin resistance. Analyzing just the patients from T2DM group, there were possible to separate the patients with controlled glucose and using antidiabetic drug (Metformin) from non-treated patients according their metabolites profile. We identified maltose, tyrosine, alanine, glutamine, 2-piperine carboxylic acid, eicosenoic acid, asparagine as upregulated in treated subjects; and ornithine, glycine, hipotaurine, tryptophan, cadaverine, tocopherol, tetradecanoic acid and butanedioic acid uregulated in nontreated diabetic. The metabolic profile from healthy subjects were closer to treated patients than non-treated patients. Aiming to identify metabolites related to early T2DM, we compared metabolic profile of healthy subjects with high level fasting glucose (between 100 and 125mg/dL) and low glucose (less than 100mg/dL). 150 metabolites were different between these groups. These metabolites could be useful as biomarkers in early T2DM diagnosis and monitoring of treatment. 2. Abstracts 56 2.21 Progress on metabolomics studies of Brazilian Cerrado plant species developed in different aluminum availabilities De Souza, M. C.*; Do Amaral, C. L.†; Alves, P. L. C. A.†; Da Costa, F. B.* *AsterBioChem Team, Laboratory of Pharmacognosy, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil. †Faculdade de Ciências Agrárias e Veterinárias – UNESP, Jaboticabal, Brazil. T C ERRADO comprises the second largest biome concerning plant diversity in Brazil, occupying 21% of the national territory in the past. Internationally, the Cerrados are considered an unlimited source of natural drugs; however, these species have been neglected in relation to the production of secondary metabolites and resistance to Al toxicity. Because of its abundance and toxicity, Al is considered one of the most limiting factors for plant development, damaging root systems and interfering on the production of secondary metabolites. Aiming at a sustainable use of these species, a massive investment in researches evaluating the production of secondary metabolites, root development, mineral nutrition as well as Al resistance/dependence becomes necessary. In this project we are proposing the evaluation of Al toxicity or benefits on the initial root development of four folk medicinal species: Vochysia tucanorum (Vochysiaceae – cerrado species, Al-accumulator), Solanum lycocarpum (Solanaceae – cerrado species, Al-non-accumulator), Helianthus annuus (Asteraceae – crop, cerrado alien species, high sensitive to Al toxicity) and Bidens pilosa (Asteracea – weed, cerrado alien species, low sensitive to Al toxicity). In order to accomplish our proposal, metabolomic variations, biometric and morphological changes on root system will be evaluated for seedlings cultivated on 10% of Hoagland’s nutrient solution, in acid pH (4.0), with five concentrations of Al (0, 100, 200, 400, 800 and 1600 mM) during 0, 24 and 48 h of root contact with Al. Root seedlings will be extracted in ethanol and analyzed by ultra-highperformance liquid chromatography coupled to high resolution mass spectrometry. After data treatment, principal component analysis (PCA) and multivariate analysis of variance (MANOVA) will be performed to HE 2.21. Progress on metabolomics studies of Brazilian Cerrado plant species developed in different aluminum availabilities 57 check variations between species and Al availabilities. The results of this project will contribute to: 1 – better understanding of Al toxicity and benefits on cerrado’s native species and alien species; 2 – subsidy for breeding plants of Al sensitive species, therefore reducing costs of production and also avoiding the expansion of agricultural fields on cerrado’s remains; 3 – regarding to highlights of ecological and agronomic uses, a better understanding about the metabolism and dependence/resistance of the cerrado’s species to the Al effects; moreover, the results of this project may contribute, in the future, to the conscious exploitation of secondary metabolites in cerrado’s species with pharmacological potential. E-Mail: [email protected] 2. Abstracts 58 2.22 Effect of the environment on the metabolic proﬁle of Tithonia diversifolia : a model for environmental metabolomics of plants Sampaio, B. L.*; Edrada-Ebel, R.†; Da Costa, F. B.* *AsterBioChem Team, Laboratory of Pharmacognosy, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Av. do Café s/n, Monte Alegre, 14040-903, Ribeirão Preto, SP, Brazil. †Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde. 161 Cathedral Street, Glasgow, G4 0RE, Scotland, United Kingdom. T and their surrounding environment is usually mediated by metabolites and this process is often related to variations in the production of plant metabolites. The species Tithonia diversifolia (Hemls.) A. Gray (Asteraceae), known as Mexican sunflower, is native both from Mexico and Central America, being encountered in both the tropical and sub-tropical areas of the Americas, Africa and Asia. It is described as an invasive weed in different ecosystems, mainly in Africa and China, once it is able to adapt to different climate and soil conditions. It was proposed to study the influence of environmental factors on the variation of the metabolites of T. diversifolia by an environmental metabolomics approach using UHPLC-DAD-(ESI)-MS and NMR techniques together with chemoinformatics methods. A study was carried out with samples of leaves, stems, roots and inflorescences of T. diversifolia obtained from specimens that grow in the states of Goiás and São Paulo, collected at different seasons throughout a 24 months period. A specific metabolomic approach was developed to determine the environment-metabolism relationship by the comparison of analytical data obtained by UHPLC-DAD-(ESI)-MS and NMR (J-resolved) analysis with environmental data (climate and soil). Unsupervised (HCA and PCA) and supervised (OPLS-DA) statistical methods were used for data analysis. Groups for each part of the plant were proposed from the HCA and PCA results, performed with the data obtained by UHPLC-DAD-(ESI)-MS, considering the environmental data. The grouping proposal showed seasonality for leaves and stems metabolites related to the variation of the rainfall, humidity, temHE CHEMICAL INTERACTION BETWEEN PLANTS 2.22. Effect of the environment on the metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants 59 perature and solar radiation levels, while the samples of inflorescences and roots were grouped in a pattern related to availability of soil nutrients (macronutrients: Ca, Mg, P and K; and the micronutrients Fe and Cu), with the solar radiation affecting the inflorescences. The results of the PCA and OPLS-DA performed with the NMR data corroborate the grouping proposal, with the loading plots showing the signals of chemical shifts for the major classes of metabolites used as discriminants, e.g. the signals from 5 to 6 ppm (double bonds present in the carbon skeletons) and from 2 to 3 ppm (hydrogens attached to saturated carbon near carbonyl groups), typical of unsaturated fatty acids and sesquiterpene lactones, present in samples of leaves and stems rich in these substances. Considering all the results presented herein, we can highlight the relationships between environment and the metabolic profile of T. diversifolia and also that the set of analytical techniques selected for this work combined with proper multivariate analysis allowed to obtain reliable data that contribute to a holistic understanding of the role of the metabolism in the adaptation of the species to the different environments and how each part of the plant is affected by the environment. Financial Support: Financial support: FAPESP and CAPES. 2. Abstracts 60 2.23 Metabolic proﬁling of Byrsonima species with antioxidant activity Fraige, K.*; Zeraik, M. L.*; Bolzani, V. S.* *NuBBE - Universidade Estadual Paulista Júlio de Mesquita Filho – Instituto de Química de Araraquara. T HE USE OF NATURAL PRODUCTS date from antiquity, and they are known on the treatment of various diseases, being considered one of the main sources of drugs due to present a wide range of chemical compounds and biological functions. The improvement of analytical techniques, metabolomics approaches and dereplication, which allow distinguishing new compounds from that already described, have increased the number of studied organisms and compounds identified, being the plants a main focus with good results. Malpighiaceae is a tropical family of plants occurring in the most part of Brazil, and known for its antimicrobial activity. Byrsonima is the largest genus of this family, and comprises species of medicinal importance, ornamental plants, and producers of edible fruits. This work involves the chromatographic analysis and the evaluation of the antioxidant capacity by DPPH and piranine based methods of six methanolic extracts from different species of Byrsonima (B. intermedia, B. coccolobifolia, B. verbascifolia and B. sericea). The extracts were prepared with solvents of increasing polarity (hexane, ethyl acetate and methanol), and the methanol extracts were subjected to bioassays. All extracts presented antioxidant capacity, ranging from 13.8% to 43.8% of DPPH radical scavenging at 4 µg mL−1 . The percentage of scavenged peroxyl radical by piranine based procedure varied between 64.4% and 100.4% at 3 µg mL−1 , and the highest antioxidant activity was observed for a B. intermedia extract (EC50 of 1.54 µg mL−1 ). The extracts were subjected to microfractioning and the same antioxidant assays, and the fractions responsible for the activity were analysed by LC-DAD-MS. HPLC-DAD analysis showed the presence of high amounts of flavonoids, which may be responsible for the antioxidant activity. Financial Support: FAPESP. 2.24. Dereplication of natural products based on 1 H ratio analysis nuclear magnetic resonance spectroscopy and HPLC-DAD-ESI-QToF-MS/MS 2.24 61 Dereplication of natural products based on H ratio analysis nuclear magnetic resonance spectroscopy and HPLC-DAD-ESI-QToF-MS/MS 1 Carnevale Neto, F.*†; Pilon, A. C.*; Gu, H.‡; Freire, R. T.*; Raftery, D.‡¶; Castro-Gamboa, I.* ; Lopes, N. P.† *Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais - NuBBE - UNESP - Instituto de Química - Departamento de Química Orgânica, Araraquara, São Paulo, Brazil. †Núcleo de Pesquisa em Produtos Naturais e Sintéticos - NPPNS, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo - FCFRP-USP -, Ribeirão Preto, São Paulo, Brazil. ‡Northwest Metabolomics Research Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, Washington, United States. ¶Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States. D has been extensively applied in plant metabolomics for fast in situ detection of known compounds in complex matrices. Current dereplication methods require separation techniques prior to detection due to the convoluted nature of the crude plant extracts. Although the most common hyphenated methods employed in dereplication as HPLC-DAD-MS and HPLC-MS-MS/MS, have proven to be very efficient for in situ detection, those techniques have serious limitations, including low chromatographic resolution, long run times, and lack of NMR confirmatory data. In order to improve the in situ identification, we propose a new approach for dereplication of natural products based on ratio analysis NMR spectroscopy (RANSY) of complex 1H NMR data directly from plant crude extracts, in association with HPLC-DADESI-QToF-MS/MS. RANSY is based on the fact that the ratio between two NMR peak intensities and/or areas from the same metabolite in a specific sample should be nearly constant. Therefore, the standard deviation of these ratios across different samples will in theory be zero, or very close to zero. On the other hand, if the two peaks originate from unrelated metabolites, the corresponding standard deviation will be typically large, except in those rare cases where the metabolites of interEREPLICATION 62 2. Abstracts est are highly correlated. In addition, RANSY offers a great potential to elucidation of unknowns by the ratio between signals of a spin system. To illustrate application of RANSY, fourteen Jatropha multifida (Euphorbiaceae) leaves extracts were prepared by maceration using different solvents according to the Snyder solvent selectivity triangle. The extracts were analyzed by HPLC-DAD-ESI-QToF-MS/MS and a 14.1 T NMR. 1 H RANSY was applied for identification of metabolites on the basis of the ratio between 1 H NMR peak intensities. Selective extraction generated a variation of the metabolite concentrations between samples, mandatory for RANSY application. The 1 H NMR spectrum generated by RANSY (spin-correlated system), was integrated with UV and MS data and led to dereplication of C-glucosylflavonoids, already reported from Jatropha. E-Mail: [email protected] 2.25. Metabolite profile of a biopolymer producer strain, by gas chromatography coupled to mass spectrometry (GC-MS) 2.25 63 Metabolite proﬁle of a biopolymer producer strain, by gas chromatography coupled to mass spectrometry (GC-MS) Matsubara, R. M. S. *; Piccoli, R. A. M.*; Rodrigues, M. F. A. *; Marques, T. O. *; Mello, C. M. *; Porgete, D S.† *Institute for Technological Research of São Paulo State - IPT. †Supporting Foundation for Institute for Technological Research of São Paulo State – FIPTl. M ETABOLOMICS , a comprehensive platform that involves analysis of thousands of intra and extracellular metabolites, emerges as a powerful alternative to improve the quality of development of biotechnological research. Samples were collected in the exponential growth phase of the polyhydroxybutyrate (PHB) fed-batch fermentations using the bacteria Cupriavidus necator. Two quenching and extraction procedures were evaluated: (i) Smart et al. (2010), Villas-Bôas & Bruheim (2007) (designate “VILLAS-BÔAS”), and (ii) Canelas et al. (2009) (designate “CANELAS”). The metabolites were analyzed in a GC-MS Agilent 7890A coupled to an Agilent 5975 MS Detector. The deconvolution and identification of compounds were made by Automatic Mass Spectral Deconvolution and Identification System (AMDIS), fitted with an in-house library of metabolites. R Project with Metab 4.0 package (Aggio, Villas-Bôas, & Ruggiero, 2011) was performed to process the responses of the MCF derivatives identified by GC-MS. T-test and Principal Component Analysis (PCA) were employed to evaluate differences between the two methods. The extracellular metabolites had closed profiles, lactic acid, caprilic acid, glutamic acid, succinic acid, proline, threonine and valine were significantly correlated (p < 0.05). Glutamic acid was the extracellular metabolite of greatest abundance. The majority of intracellular metabolite profiles presented similarities between the two methods, although the PCs showed that they could be distinguished. Palmitic acid was the most abundant intracellular metabolite. “CANELAS” method showed greater repeatability based on intracellular metabolites profiles in relation to “VILLAS-BÔAS”. Both quenching and extraction procedures evaluated are recommended for analysis of intracellular metabolites of Cupriavidus necator strain 64 used for PHB bioprocess. E-Mail: [email protected] 2. Abstracts 2.26. Differentiation of essential oils from Citrus fruits by multivariate analysis 2.26 65 Differentiation of essential oils from Citrus fruits by multivariate analysis Simas, D. L. R.*; Da Silva, A. J. R.* *Instituto de Pesquisas de Produtos Naturais IPPN-UFRJ. T C ITRUS GENUS is a rich source of essential oils (EO). The classical methods of chemical composition analysis and quality control of lemons essential oils has been GC-FID and GC-MS. Attenuated Total Reflectance (ATR) infrared spectroscopy can be used to evaluate the quality of commercial essential oils. In the present study we compare the results obtained when Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) are applied to data generated by Citrus fruits EO analysis by ATR/FT-IR and by GC-FID and GC/MS, to help discriminating samples of different Citrus EO. Citrus samples were acquired randomly in commercial establishments in Rio de Janeiro - Brazil. A total of 13 samples were analyzed: 5 of Citrus latifolia, Tanaka (var. “limão tahiti”), 4 Citrus limon, Burm. (var. “limão siciliano”), 4 Citrus aurantifolia, Swingle (var. “limão galego”). The fruit peels (150.0 g) were manually removed, homogenized with water and submitted to hydrodistillation in a modified Clevenger apparatus for 2 hours. After extraction the EO were dried over Na2 SO4 and stored at -18 ºC. Analysis by GC-FID and CG/EM were performed to determine the chemical composition of oils. FT-IR analysis were made in ATR mode. For each interferogram, in absorbance mode, 40 scans were accumulated at a resolution of 2.0 cm−1 and a Happ Genzel apodization function was applied. For PCA (NIPALS algorithms) and HCA (Ward’s method using Squared Euclidean Distance) the region between 1400-700 cm−1 (absorbance x wavelength) in ATR/FT-IR spectra was used and the percentage composition data obtained by GC-FID and GC/MS. The multivariate analyses were performed with the software Unscrambler X 10.2. PCA (5 PCs, weight 1 for all variables, cross validation with 1 sample per segment) were applied to both data set and in both analysis there was discrimination in three distinct groups. From scores of PCA was made a HCA. We have observed that PCA of ATR/FT-IR spectral data has the same discrimination power as the PCA based on the table composition obtained from GC-FID and GC/MS analysis. These PCA discriminated the samples in three distinct groups. However, the HE 66 2. Abstracts HCA of ATR/FT-IR spectral data failed to discriminate the EO in three different groups (ATR/FT-IR did not separate one sample of C. aurantifolia of the Citrus limon group) when compared to the HCA on GC/MS or GCMS-FID analysis data, which was observed separation into three different groups. Our results reveals a new possibility for performing quality control testing of commercial Citrus essential oils. E-Mail: [email protected] 3 Poster Section 3.1 Poster Section 01 Abstracts numbers 2.1, 2.5, 2.9, 2.13, 2.17, 2.21, 2.25. 3.2 Poster Section 02 Abstracts numbers 2.2, 2.6, 2.10, 2.14, 2.18, 2.22, 2.26. 3.3 Poster Section 03 Abstracts numbers 2.3, 2.7, 2.11, 2.15, 2.19, 2.23. 3.4 Poster Section 04 Abstracts numbers 2.4, 2.8, 2.12, 2.16, 2.20, 2.24.
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