Abstracts abstract_book - First Brazilian Workshop on Bioinformatics

• 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
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.
M.Sc. Rafael Renatino Canevarolo
• Assoc. Brasileira de Bioinformática e Biologia Computacional - AB3C:
Meire Tarlá
2015 First Brazilian Workshop on Bioinformatics/ Chemometrics for
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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
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
Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1 Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
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
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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 . . . . . . . . . . . . . . . . . . . . . . . .
3 Poster Section . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Poster Section 01 .
Poster Section 02 .
Poster Section 03 .
Poster Section 04 .
March 13
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
Chemoinformatics/chemometrics on Brazil: an
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
André Fujita
Jane Finzi – Waters
March 14
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
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
1.1. Data sharing and standards in metabolomics: what we can learn from other “omics”
Data sharing and standards in metabolomics:
what we can learn from other “omics”
Reza Salek
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.
1. Lectures
Metabolomics: a powerful tool for elucidating plant growth regulation
Camila Caldana
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
Chemoinformatics on Brazil:
an overview
Fernando Batista da Costa
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
Metabolic fingerprint and biomarker discovery
Ieda Spacino Scarminio
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.
1.5. Statistical methods to analyze biological networks
Statistical methods to analyze biological networks
André Fujita
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.
1. Lectures
Metabolite identification strategies
using NMR
Jan Schripsema
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
Telling metabolic stories to explore metabolomics data: a case study on the yeast response
to cadmium exposure
Paulo Vieira Milreu
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.
1. Lectures
Application of MALDI in metabolomics: from
extract analysis to the imaging of tissue
Denise Brentan da Silva
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
1.9. The fascinating World from Senna spectabilis’s rizosphere: exploring molecular
diversity through the use of metabolomics and multiway analysis
The fascinating World from Senna spectabilis ’s rizosphere: exploring molecular diversity through the use of metabolomics and multiway analysis
Ian Castro-Gamboa
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.
1. Lectures
Classification models proposition
using Chemometrics: potential applications
in metabolomics
Edenir Rodrigues Pereira Filho
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.
2.1. Metabolomics research infrastructure available at Núcleo de Pesquisas em Produtos
Naturais e Sintéticos (NPPNS)
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.
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
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
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.
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
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
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.
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,
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
2.4. The role of secondary metabolites in plants diversification and evolution: a
remarkable example from the northern Andes
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.
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
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
Mixture design to optimize Baccharis genus
species extraction in untargeted metabolomic
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,
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
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
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
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).
2. Abstracts
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.
(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
Analysis of sugarcane transcriptome and metabolome changes along the plant maturation
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.
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
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.
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
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,
2.9. Xanthan gum removal for 1 H-NMR analysis of the intracellular metabolome of the
bacteria Xanthomonas axonopodis pv. citri 306
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
E-Mails: [email protected], [email protected] cnpem.br, [email protected], [email protected] cnpem.br,
[email protected]
2. Abstracts
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.
(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.
2.11. Differential metabolomic responses of Histoplasma capsulatum in biofilms and
planktonic yeasts
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.
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]
2. Abstracts
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.
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.
2.13. Serum and urine metabolic profile in response to renal ischemia/ reperfusion in a
swine model
Serum and urine metabolic profile in response
to renal ischemia/ reperfusion in a swine
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.
(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.
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
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.
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
E-Mails: [email protected], [email protected]
Financial Support: CNPq, Universal 2013.
2. Abstracts
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.
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
2.15. Metabolomic profiles of caffeine metabolization after coffee intake
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
Metabolomics, proteomics and chemometrics approaches for the infraspecific chemical variability study of a native Brazilian plant
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,
‡Institut für Biologie und Biotechnologie der Pflanzen - IBBP, Westfälische Wilhelms-Universität Münster.
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
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
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
†Center of Imaging and Spectroscopy by Magnetic Resonance (CIERMag) – IFSC, São Carlos Institute of Physics – USP – São Paulo University,
São Carlos.
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
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.
(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.
2.19. Metabolic fingerprinting of Eucalyptus grandis during the infection by Puccinia
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.
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.
2. Abstracts
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.
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
2.20. Metabolomics applied in a Brazilian population-based study
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
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,
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
2.21. Progress on metabolomics studies of Brazilian Cerrado plant species developed in
different aluminum availabilities
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
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
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
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
Financial Support: Financial support: FAPESP and CAPES.
2. Abstracts
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.
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
Dereplication of natural products based on
H ratio analysis nuclear magnetic resonance
spectroscopy and HPLC-DAD-ESI-QToF-MS/MS
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.
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
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)
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.
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
used for PHB bioprocess.
E-Mail: [email protected]
2. Abstracts
2.26. Differentiation of essential oils from Citrus fruits by multivariate analysis
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.
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
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]
Poster Section
Poster Section 01
Abstracts numbers 2.1, 2.5, 2.9, 2.13, 2.17, 2.21, 2.25.
Poster Section 02
Abstracts numbers 2.2, 2.6, 2.10, 2.14, 2.18, 2.22, 2.26.
Poster Section 03
Abstracts numbers 2.3, 2.7, 2.11, 2.15, 2.19, 2.23.
Poster Section 04
Abstracts numbers 2.4, 2.8, 2.12, 2.16, 2.20, 2.24.