advances and pitfalls in developing natural products as antifoulantsin ... · advances and pitfalls...
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Advances and pitfalls in developing
Natural products as antifoulants in
Brazil
� Advances and pitfalls in developing natural products as antifoulants in Brazil.Ricardo Coutinho
� Advances and pitfalls in developing natural products as antifoulants in Brazil.� Advances and pitfalls in developing natural products as antifoulants in Brazil.
Ricardo Coutinho, Ph.D
Division ofDivision of BiofoulingBiofouling, , DepartmentDepartment ofof OcenographyOcenography, Instituto, Instituto
de Estudos do Mar Almirante Paulo Moreira, de Estudos do Mar Almirante Paulo Moreira, BrazilBrazil
GothenburgGothenburg. 19.11.2009. 19.11.2009
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SUMMARY
�The beginning
�The development
�Problems and alternatives
�Study case
�Future perspective�Future perspective
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TheThe beginning
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-The use of natural products as antifoulants
has been an active research area for the last
30 years. Nevertheless, little advances has
been achieve in order to produce an
antifouling paint with a natural substance in
their matrix.their matrix.
-Research in natural products in Brazil started
in 1995 in the Brazilian Navy and Fluminense
Federal University with emphasis in the field
and laboratory test of antifouling (AF) activity
extracted.
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Methodology
Field tests – In 1995, Mark Hay and Willian Fenical went to
Arraial do Cabo,Rio de Janeiro, Brazil to make a survey in the
species that had natural products used to defense against
herbivore. Several experiments were done in the field using herbivore. Several experiments were done in the field using
differents methods.
-Some of the methods were adapted to test antifouling products
from marine organisms.
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Extract of Marine organismsExtract of Marine organisms
were incorporated into were incorporated into PhytogelPhytogel,,
place in plastic dish and exposedplace in plastic dish and exposed
to the field. The extract was to the field. The extract was
liberated slowly, as it presumablyliberated slowly, as it presumably
occurs in live occurs in live organismsorganisms..
fundo
1,0 m
PR
OF
UN
D
ID
AD
E
superfície
distorcedor
bóia
CT1 T2 T3 T4 T5 T6
C - ControleT - Tratamentos
Suporte de alumínio
Peso Peso
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Rafts trials where
used in theses
tests.
Cabo Frio Island
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Laboratory Test – Based on Da Gama et. al. 2002jj
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Laurencia First Test
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1 2 3 4 5 6
0
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40
60
80
100
Bioincrustação ao longo das semanas
Controle
Laurencia sp1
Incru
sta
çã
o (
%)
Tempo (semanas)
1 2 3 4 5 6
0
20
40
60
80
100
120
Bioincrustação ao longo das semanas
Controle
L. obtusa
Incru
sta
çã
o (
%)
Tempo (semanas)
��CromatografiaCromatografia emem camadacamada delgadadelgada dasdas espéciesespécies estudadasestudadas dedeLaurenciaLaurencia,, eluídaeluída duasduas vezesvezes emem clorofórmio,clorofórmio, visualizadavisualizada ee reveladarevelada comcomsulfatosulfato céricocérico -- TT == todastodas;; LL..oo..== LaurenciaLaurencia obtusaobtusa;; LL..ff..== LL.. filiformisfiliformis;; LL..aa..==LL.. arbusculaarbuscula;; LL..ss..== LL.. scopariascoparia;; LL.. spsp22== LaurenciaLaurencia spsp22;; LL.. spsp11== LaurenciaLaurenciaspsp11;; LL..alal..== LL.. aldingensisaldingensis..
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Behavior of exploration and
seleciotion of the substrate
by Perna perna and controls.by Perna perna and controls.
0
20
40
60
80
100
* p < 0,05
Perna perna
N = 10 réplicas
Bioensaio com Perna perna
Ati
vid
ad
e A
nti
incru
sta
nte
(%
Co
ntr
ole
)
Espécies Estudadas
L. obtusa*
L. arbuscula*
L. aldingensis*
L. scoparia*
L. filiformis*
Laurencia sp2*
Laurencia sp1*
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The development
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Sudatti. et. al.2008
SurfaceSurface elatolelatol concentrationsconcentrations did not inhibit did not inhibit herbivoryherbivory
by sea by sea urchins,settlementurchins,settlement of barnacle larvae, of barnacle larvae,
or mussel attachment, but or mussel attachment, but thallusthallus did. did.
Corps in cerise (*) and many small
and large vesicles (arrows) inside
the cortical cells are seen. Scale bar,
6 um.
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Fig. 1. Differential interferential optical microscopy images of living cortical, medullar and tricoblast cells of Laurencia obtusa. (a)
Transverse section view of cortical and medullar cells. Note the presence of two CC (*) in a cortical cell; (b) image of tricoblast cells,
where it can be also observed the presence of CC. Note the presence of connections between the CC and the cell periphery (arrows);
(c and d) superficial view of cortical cells showing elongated structures connecting CC to cell periphery (bars = 10 lm).
Salgado et.al.2008
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Fig. 5. Optical microscopy images (DIC, fluorescence and overlaid images) of semiFig. 5. Optical microscopy images (DIC, fluorescence and overlaid images) of semi--thin transversal sections. (a) A DIC thin transversal sections. (a) A DIC
image where it can be observed the CC (CC), the cell walls (CW) and the chloroplasts (C). (b) An image revealing the image where it can be observed the CC (CC), the cell walls (CW) and the chloroplasts (C). (b) An image revealing the
typical autotypical auto--fluorescence observed in these cells. (c) An overlaid image composed by DIC and by fluorescence images, fluorescence observed in these cells. (c) An overlaid image composed by DIC and by fluorescence images,
respectively. Note the autorespectively. Note the auto--fluorescence emission in CC and also in many vesicles distributed along the fluorescence emission in CC and also in many vesicles distributed along the ii ntrantra--cellular cellular
space and in chloroplast (bars = 10 lm).space and in chloroplast (bars = 10 lm).
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Fig. 6. Optical microscopy images of a cortical cell obtained using video-microscopy analysis. Note, in the sequence
‘a–f’, the vesicles displacement (arrow and arrow heads) migrating from CC in direction to the cell periphery. *,
chloroplast. (bar = 3 lm).
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Fig. 9. TriFig. 9. Tri--dimensional reconstruction of the tubular membranous connections. (adimensional reconstruction of the tubular membranous connections. (a––c) TEM images of sections used in the 3c) TEM images of sections used in the 3--D D
reconstruction process where is possible observe the tubular membranous connections (arrows), a CC region (*) and a vesicle inside
the connections (arrow head); (d and e) 3-D reconstruction. Cell wall is represented in blue color; the CC is red color; the membrane of
tubular connections in yellow color; and transported vesicle in orange color (bars = 400 nm).
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Antifouling activity of natural products from Antifouling activity of natural products from
Brazilian Brazilian SeaweedsSeaweeds
-In a recent work, we tested crude organic extracts from 51 populations
comprising 42 species of macroalgae from eight locations along the
Brazilian coast against a relevant fouling organism in laboratory
bioassays, the brown mussel Perna perna. bioassays, the brown mussel Perna perna.
Da Gama et. al. 2008
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Despite the unbalanced number of macroalgae tested among different
localities, there seems to be no latitudinal trend of increased
antifouling activity towards lower latitudes, where fouling pressure is
presumed to be higher.
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There was a clear phylogenetic pattern in antifouling
activity, with red macroalgae having the highest
proportion (55%) of active species (moderate or strong
fouling Inhibition), followed by brown macroalgae (14%).
Green seaweeds never exhibited strong AF activity
These results suggest that research efforts should be
focused on tropical red macroalgae in the quest for new
antifoulants
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ProblemsProblems andand alternativesalternatives
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Problem : Synthesis of Elatol
Alternatives :
-Comercial Analogs already with permission to use.
-Synthesis of others compounds :-Synthesis of others compounds :
-Easy synthesis
-Enough material to incorporated in a paint
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Problem : Low investiments in research (in Brazil).
Alternatives : Join projects with Universities, Federal and
State agency and paint companies.
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StudyStudy casecase
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Objectives
To test all the process from synthesis to the incorporation
In the paint matrix and to test in the field.In the paint matrix and to test in the field.
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The synthesis
OurOur biocidebiocide werewere synthesizedsynthesized in in thethe Federal Federal UniversityUniversity ofof
Rio de Janeiro, Rio de Janeiro, basedbased onon a a chemistrychemistry structurestructure similar asimilar aRio de Janeiro, Rio de Janeiro, basedbased onon a a chemistrychemistry structurestructure similar asimilar a
substancesubstance producedproduced byby a a spongesponge andand incorporatedincorporated in ain a
paintpaint matrixmatrix. .
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Incorporation in the Paint’s
Matrix
TheThe experimental experimental paintpaint waswas mademade in a in a joinjoin projectproject withewithe
TheThe InternationalInternational PaintPaint CompanyCompany ((AkzoAkzo Nobel) Nobel) locatedlocated inin
Niterói, Rio de Janeiro.Niterói, Rio de Janeiro.
TheThe Rio de Janeiro Rio de Janeiro StateState UniversityUniversity (UERJ) (UERJ) alsoalso
participatedparticipated in in thethe laboratoylaboratoy testtest ofof thethe paintpaint..
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Guanabara Bay, Rio de
Janeiro�Cabo Frio Island
Field tests
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TreatmentsWere tested three coating (3 repliqueted each):
-Main biocide ( Copper) substitute by our “natural” biocide.
-Commercial paint
-Our “natural” biocide used as accessory biocide together
with copper. with copper.
-Control (without antifouling paint)
The experiments lasting by 1 (one) year.
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Sinopse taxonômica Tipo morfo-funcional/ Grupos
Reino Plantae
Divisão ChlorophytaClasse Chlorophyceae
Ordem UlvalesFamília Ulvaceae
Foliácea
Divisão Phaeophyta
Classe Phaeophyceae
Ordem Ectocarpales
Família Ectocarpaceae
Filamentosa parda
Divisão Rhodophyta Ordem Ceramiales
Família CeramiaceaeFilamentosa Vermelhas
Reino Animalia
Filo CnidariaClasse Hydrozoa
Ordem Hydroida
Família CampanulariidaeObelia dichotoma (Linnaeus, 1758)
Classe Anthozoa Anêmonas
List of species presents in the plates
Classe AnthozoaOrdem Actiniaria
Família Actiniidae
Anêmonas
Filo AnnelidaClasse Polychaeta
Ordem CanalipalpataFamília Serpulidae
Serpulídeos
Filo Arthropoda
Subclasse Cirripedia Ordem Thoracica
Família BalanidaeMegabalanus tintinnabulum (Linnaeus, 1758)Balanus trigonus
Balanus improvisus
Balanídeos
Filo EctoproctaClasse Gymnolemata
Ordem Cheilostomata
Família Bugulidae
Bugula neritina (Linnaeus, 1758)Outros briozoários arborescentes
Família SchizoporellidaeSchizoporella errata (Johnston, 1847)
Briozoários incrustantes
Filo ChordataClasse Ascidiacea
Ordem StolidobranchiaFamilia StyelidaeStyela plicata (Lesueur, 1823)
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Revestimento 1 - Matriz com Biocida Natural
0%
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1a 2a 3a 4a 5a 6a 8a 12a
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Treatment 1 –”natural biocide” (4 months)
1a 2a 3a 4a 5a 6a 8a 12a
Semanas
Biofilme Obelia dichotoma Balanídeos
Filamentosas Pardas Bugula neritina Styela plicata
Serpulídeos Tubos mucosos Espaço vazio
��aa ��bb ��cc
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Revestimento 2 - Matriz Comercial
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Treatment 2- Commercial paint
Semanas
Biofilme Obelia dichotoma Balanídeos
Filamentosas Pardas Bugula neritina Styela plicata
Serpulídeos Tubos mucosos Espaço vazio
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Revestimento 3 - Matriz com Biocida Natural de Acessório
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Treatment 3 – “natural biocide as accessory
Biofilme Obelia dichotoma Balanídeos
Filamentosas Pardas Bugula neritina Styela plicata
Serpulídeos Tubos mucosos Espaço vazio
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Controle
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Semanas
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ControlControl
Biofilme Obelia dichotoma Balanídeos
Filamentosas Pardas Bugula neritina Styela plicata
Serpulídeos Tubos mucosos Espaço vazio
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�� treattreat. 1. 1 treat. 3
��MayMay
��JuneJune
��JullyJully
treat. 2 control
��AugustAugust
��NovemberNovember
��JullyJully
��OctoberOctober
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•• New synthesis
•Biocide incorporation in paint matrix
•Investiments by companies
Future Perspectives
•Investiments by companies
•Field tests (plates and ships)
•Toxicity tests
•Patents
•Registration
•Production
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INSTITUTO DE ESTUDOS DO MAR ALMIRANTE PAULO MOREIRAArraial do Cabo, Rio de Janeiro
INSTITUTO DE ESTUDOS DO MAR ALMIRANTE PAULO MOREIRAArraial do Cabo, Rio de Janeiro
� Advances and pitfalls in developing natural products as antifoulants in Brazil.Ricardodo Mar Almirante Paulo Moreira, Brazil
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AcknowledgementsAcknowledgements
Hans Elwing
Renato Crespo Pereira - Fluminense Federal University (UFF)
Bernardo da Gama – Fluminense Federal University (UFF)
Daniela Sudatti – Fluminense Federal University (UFF)
Leonardo Salgado – Botanic Gardem-Rio de Janeiro (JB)
Gilberto Amado Filho-Botanica Garden-Rio de Janeiro (JB)
Claudio Lopes – Rio de Janeiro Federal University (UFRJ)
Rosangela Lopes -Rio de Janeiro Federal University of Rio(UFRJ)
Aricelso Limaverde –Rio de Janeiro State University (UERJ)
5 post-doc, 19 Ph.Ds, 23 MSc.
Akzo Nobel
Sponsor : Brazilian Navy, CNPq, FINEP, FAPERJ
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Questions ?
Thanks
Questions ?