Dissertation committee:
Βασίλειος Ρούσσης, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Μαρία Ζερβού, Ερευνήτρια Β, Ινστιτουτο Βιολογίας, Φαρμακευτικής Χημείας και Βιοτεχνολογίας, Εθνικό Ίδρυμα Ερευνών
Ευσταθία Ιωάννου, Επίκουρη Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Όλγα Τζάκου, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Ευάγγελος Γκίκας, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Θωμάς Μαυρομούστακος, Καθηγητής, Τμήμα Χημείας, ΕΚΠΑ
Παναγιώτης Ζουμπουλάκης, Ερευνητής Β, Ινστιτουτο Βιολογίας, Φαρμακευτικής Χημείας και Βιοτεχνολογίας, Εθνικό Ίδρυμα Ερευνών
Summary:
Natural products, originating from terrestrial or marine organisms, constitute a prolific
source for pharmaceutically relevant products providing unique structural
chemodiversity. The marine environment, covering approximately 70% of the Earth’s
surface hosts a largely unexplored biodiversity, offering a huge potential for the
discovery of novel compounds to target incurable diseases or overcome drug resistance.
Due to the wide range of competitive ecosystems they survive in, marine organisms
have developed unique defense strategies and bioactive compounds that, in some cases,
are unparalleled by their terrestrial counterparts.
Red algae of the genus Laurencia are considered as one of the richest sources of new
secondary metabolites. The genus Laurencia currently encompasses 146 taxonomically
accepted species which are mainly found in tropical, subtropical, and temperate coastal
waters, including the Mediterranean Sea. Although intensively investigated for the last
50 years, new metabolites are still being isolated from Laurencia species. Displaying
more than 1,200 records in MarinLit database, Laurencia species biosynthesize a wide
spectrum of secondary metabolites, including sesquiterpenes, diterpenes, triterpenes and
C15 acetogenins that are frequently characterized by the presence of halogen atoms. An
array of the isolated metabolites exhibit important activities, such as antibacterial,
antifungal, insecticidal and cytotoxic, as reviewed in Chapter 1.
Recent reports comment on the complexity of the taxonomy of red algae of the genus
Laurencia which is related to the extended morphological plasticity along with the
chemical variation influenced to a significant degree by environmental and genetic
factors. Past attempts towards species identification based on characteristic metabolites
have proven unsuccessful and more importantly efforts to re-isolate bioactive
compounds were severely impeded.
Metabolomics consists an emerging technology with applications expanding in different
areas, such as health, natural products, biotechnological applications and nutrition.
Metabolomics platforms have been only applied to a limited number of projects relating
to marine organisms. Metabolomics studies require the application of high-throughput
analytical techniques, such as NMR spectroscopy and mass spectrometry (MS), in
combination with software (s/w) tools and databases. The high number of compounds
with unknown structures and the limited information retrieved from databases generated
for marine organisms reflect the increased difficulty in assessing the complex
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metabolome of the marine ecosystem. Applications of metabolomics in marine
organisms and research challenges are reviewed in Chapter 2.
The current study addressed the global secondary metabolic profile of Laurencia crude
extracts by applying state-of-the-art holistic techniques, i.e. UHPLC–PDA–HRMS and
HR NMR spectroscopy, along with software tools, databases (MarinLit and those
developed in-house) and multivariate data analysis tools.
The developed pipeline described in Chapter 4 was applied as a screening strategy for
dereplication purposes and for pinpointing the presence of new metabolites already
from the very early stages of a chemical investigation and prioritize selected Laurencia
crude extracts to be further subjected to the long and laborious procedure of in-depth
phytochemical analysis. This integrated approach has led to:
a) The construction of an ‘in-house’ Laurencia-focused NMR database, incorporating
experimental and / or literature NMR data of Laurencia and Aplysia metabolites. The
database is implemented in the interface of ACD/Labs s/w and facilitates the
interpretation of 2D HSQC data based on simulation algorithms enhancing the confident
discrimination of the secondary metabolites present in the sample. The database
includes approx. 300 records comprising structurally diverse compounds from the
classes of C15 acetogenins, sesquiterpenes and diterpenes.
b) The development of an 'in-house’ Laurencia-focused LC-MS library comprising
metabolites isolated in the past from Laurencia in our laboratory
c) The rapid detection of two new C15 bromoallene acetogenins in a crude extract of
Laurencia chondrioides collected from Kefalonia (marilzallene B and chondrioallene).
The obtained results have been published in Kokkotou et al. Phytochemistry, 2014, 108,
208–219.
Subsequently, in Chapter 5, an exploratory study comprising 103 Laurencia samples
collected across the Greek coastlines investigated the potential of LC-MS/PCA and 1H
NMR/PCA untargeted profiling to discriminate patterns among the various Laurencia
populations. The major findings of this extended study are summarized in the following:
a) The emergence of outlier samples probed to the presence of new natural products
b) Two major metabotypes of Greek Laurencia specimens were revealed, metabotype A
comprising an array of C15 acetogenins and metabotype B which is mainly
sesquiterpenes-oriented.
c) Moreover, the study revealed the influence of the marine eco-region (Ionian and
Aegean Sea) relatively to the expression of the two metabotypes on Laurencia obtusa
specimens.
To the best of our knowledge, this is the first untargeted metabolomics study performed
on a high number of Laurencia populations.
In Chapter 6, a targeted LC-HRMS approach was applied to the collection of Laurencia
populations screening for bioactive diterpenes and specifically the diterpene
neorogioltriol (NRG) exhibiting strong anti-inflammatory activity which was initially
isolated from a Laurencia glandulifera population from Kefalonia, the structural analog
neorogioldiol, as well as the congeners neorogioldiol B and prevezols A-E, isolated
from a Greek L. obtusa specimen from the coasts of Preveza. These compounds have
been proposed to belong to the same biogenetic pathway and thus this LC-HRMS
screening targeted to trace any of them or even related analogues. The presence of the
target compounds was identified in a population of Laurencia collected from Vatsa bay
(south-west coastal region of Kefalonia). Subsequently, collection of individual
specimens of the particular Laurencia population allowed for an intra-population study.
LC-MS/PCA analysis clearly revealed intra-population variability and discriminated
two clusters from the average population, one associated with an array of diterpenes and
another one comprising mainly sesquiterpenes. A reverse “V” shaped PCA map and the
allocation of the clusters also implied an interplay between the diterpenes and
sesquiterpenes abundance, suggesting a cross-talk between the two pathways related to
terpenes biosynthesis, namely MEP (methylerythritol) and MVA (mevalonic acid). This
cross-talk could account for the favorable biosynthesis of the bioactive diterpenes at the
expense of sesquiterpenes.
The seasonal variation of Laurencia secondary metabolome was also demonstrated from
this study, possibly driven by the changing of environmental conditions or the exposure
to pathogens, epiphytes or herbivores with an impact to the production of bioactive
diterpenes or sesquiterpenes.
