Joint research project

Extraction and characterization of active metabolites from red algae of the Lebanese and Italian Coasts: Pharmaceutical, medical and agro-food applications

Project leaders
Antonio Evidente, Hussein Kanaan
LIBANO - CNRS-L - National Council for Scientific Research of Lebanon
CNR/CNRS-L 2012-2013
Life Sciences
Thematic area
Biomedical sciences
Status of the project

Research proposal

Plant, microrganisms, marine organisms are sources of an enormous variety of natural products with highly diverse structures.
Hundreds of new secondary metabolites were isolated and chemically and biologically characterized also for their potential practical application in agriculture and medicine.
The progress in development of new and advanced chromatographic and spectroscopic techniques allowed the more easy isolation and chemical characterization of the main metabolites but also those of metabolites present in very low amount and with a very complex structures.
So that metabolites belonging to different class of natural compounds (terpenoids, polyketides, aromatic compounds, macrolides, butanolides, amino acid etc.) were isolated and their biological activities characterized. This compounds also had represented a lead to realize their total enatioselective syntheses or to prepare derivatives to carry out structure-activity relationships study, aimed to find compound with increased activity and specificity. Some study were also carry out on their mode of action to identify their receptors.
So that new, phytotoxins, new herbicides fungicides, antibiotics etc, were discovered and their potential  practical application in different fields as agriculture and medicine was evaluated. (Evidente and Motta 2001, Evidente et al. 2002, Evidente et al. 2005, Evidente 2006, Evidente and Abouzeid 2006).
Some plant and microbial metabolites also showed noteworthy anticancer activity in vitro so that the interest around this metabolites was noteworthy increase (Osbourn and Lanzotti, 2009;  Lamoral -Theys et al., 2009; 2010; Van Goietsenoven et al. 2010a; 2010b; 2010c; Said et al. 2010; de Vries et al., 2010; bury et al., 2011)
However, the interest in the natural metabolites from different sources is increased so that thousand of thousand of these were discovered.
The red algae belonging to Asparagopsis and Haematococcus genera, object of this project and collected on Lebanese and Italian coasts, are very promising source of new bioactive metabolites  with original carbon skeleton and high potential application in different field as pharmaceutical, medical and agro-food application
The success or failure of the extraction process is monitored by an appropriate assay. For example, a well-known protocol for extraction involved the extraction of the air-dried material with a water-methanol mixture. This extract is then defatted with petrol or a similar solvent and then re-extracted with methylene chloride. Once extracted from the fungal culture, the bioactive metabolites  must then be separated from the co-extractives. Rarely, this may involve simple crystallization of the toxin from the crude extract, requiring only minor manipulation to yield pure metabolite. More usually, however, it will involve further solvent partition of coextractives and extensive chromatography, taking advantage of particular properties of the desired compound such as acidity, polarity and molecular size.
The structure determination of the bioactive metabolites will carry out using advanced spectroscopic (IR, UV, 1D and 2D 1H- and 13C-NMR and EI and ESI MS techniques) optical (computantional and experimental OR and ECD and VCD) and chemical methods by preparing suitable derivatives through the chemical modification of their functionalities (Evidente and Motta, 2001).
The pure bioactive metabolites will be used as standards to develop analytical methods for identification and quantification in complex samples (Evidente et al. 2005). These methods may be based on chromatographic techniques related to MS, as in HPLC-MS and GC-MS, or based on spectroscopic techniques. Also immunochemical and cytofluorometric methods can be developed using the bioactive compound (if it is a macromolecular compound), or the metabolite may be conjugated to a hapten to obtain specific antibodies. These methods could be applied to optimization of  metabolite production, or for detection and quantification of the metabolite  in vivo by. TLC and HPTLC methods (Evidente et al., 2005) and HPLC methods in association with MS
The present project is aimed to purify secondary bioactive metabolites of red algae collected on Lebanese and Italian cost and charcterized their biological activity and evaluate their potential practical application in different fields.
In the framework of the present Agreement on Scientific and Technological Cooperation between CNR (Italy) and CNRS-L (Lebanon), we would like to cooperate with the team of Prof.Hussein Kanaan of Facuof Pharmacy, Lebanese University, give his long experience in ........We are confident that with their collaboration we will be able to achieve the objectives of our project.
Literature cited

A. Evidente and A. Motta: "Phytotoxins from fungi pathogenic for agrarian, forestall and weedy plants", in Bioactive Compounds from Natural Sources: Isolation, Chracterization, and Biological Properties (C. Tringali, Ed.) Taylor & Francis Group, London (2001), 473-525.
A. Evidente and A. Motta: "Bioactive metabolites from phytopathogenic bacteria and plants", in Studies in Natural Products Chemistery (Atta-ur-Rahaman, Ed.), Volume 26-Bioactive Natural Products (Part G), Elsevier, Amsterdam, The Netherlands (2002), 581-628.
 A. Evidente, A. Andolfi, M.A. Abouzeid: "Analytic Methods of Bioactive Metabolites Produced by plants and Microorganisms", Curr. Pharm. Anal., 1, 329-348  (2005).
 A. Evidente and M.A. Abouzeid: "Characterisation of Phytotoxins from Phytopathogenic Fungi and their Potential Use as Herbicides in Integrated Crop Management" in Handbook of Sustainable Weed Management (H.P. Singh, D.R. Batish, R.K.Kohli Eds.) The Harworth Press, Inc., New York, Chapter 17, pp. 507-532 (2006).
 A. Evidente: "Chemical and Biological Characterization of Toxins Produced by Weed Pathogenic Fungi as Potential Natural Herbicides" In Natural Products for Pest Management, S.O. Duke and A. M: Rimando Eds., ACS Symposium Serie, Washington, DC, Chapetr 5, pp. 62-75 (2006).
 A.E. Osbourn and Lanzotti V. Plant derived Natural Products- Synthesis, Function, and Application, Springer, 2009.
D. Lamoral-Theys, A. Andolfi, G. Van Goietsenoven, A., Cimmino, B.,  Le Calve, N. Wauthoz, V., Megalizzi, T.,  Gras, Thierry, C.,  Bruyere, J., Dubois, V., Mathieu, Veronique, A., Kornienko, R., Kiss, Robert, A. Evidente.   Lycorine, the Main Phenanthridine Amaryllidaceae Alkaloid, Exhibits Significant Antitumor Activity in Cancer Cells That Display Resistance to Proapoptotic Stimuli: An Investigation of Structure-Activity Relationship and Mechanistic Insight.    Journal of Medicinal Chemistry  52,  6244-6256 (2009). 
Lamoral-Thesis D., Decaestecker C., Matheiu V., Dubois, J., Kornienko, A., Kiss, R., Evidente A., Pottier L. "Lipohilic  "Lycorine and its derivatives for anticanc er drug design". Mini-Reviews in Medicinal Chemistry, 10, 41-50 (2010).
Van Goietsenoven, G., Andolfi, A., Lallemmand, B., A. Cimmino, Lamoral-Theys, D., A. Andolfi, Gras, T. Abou-Donia, A., . Dubois, J. Lefranc, F., B Mathieu, J.V., . A. Kornienko, A., Kiss, R.,  Evidente. A. "Amaryllidacea alkaloids belonging to different structural sbgroup display activity against apotsosis-resistant cancer cells". Journal of Natural Products  73, 1223-1227 (2010a).
Saidu Balde, El, H., Andolfi, A., Bruyere, C., A. Cimmino, ., Lamoral-Theys, D., Vurro, M.,  Van Damme. M., Altomare, C., Mathieu, J.V.,  Kiss, R.,  Evidente. A. "Investigations of fungal secondary metabolites with potential anticancer activity". Journal of Natural Products 73, 969-971 (2010).
de Vries-van Leeuwen I.J., Kortekaas-Thijssen C., Mandouckou J.A.N., Kas S., Evidente A., de Boer A.H. Fusicoccin-A selectively induces apoptosis in tumor cells after interferon-alpha priming. Cancer Letters 293, 198-206 (2010).
Van Goietsenoven G., Hutton J., Becker J.P, Lallemand B., Robert F., Lefranc F., Pirker C.,  Vandenbussche G., Van Antwerpen P., Evidente A., Berger W., Prévost M., Pelletier J., Kiss R, Goss Kinzy T., Kornienko A., Mathieu V. "Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas" The FASEB Journal, doi: 10.1096/fj.10-162263, 2010b.
Van Goietsenoven, G., Mathieu, V., Andolfi, A., Cimmino,  A., Lefranc, F., Kiss, R., Evidente A. In vitro growth Inhibitory eefects of cytochalasins and derivatives in cancer cells. Planta Medica (2010c) DOI 10.1055/ss-0030-1250523.
Bury M., Punzo B., Berestetskyi A., Lallemand B., Dubois J., Lefranc  F., Véronique M., Andolfi A., Kiss K., Evidente A. Evaluation of Anticancer Activity of Alternethanoxins A and B, two Fungal Polycyclic Ethanones, and of Two of Their Derivatives. International Journal of Oncology 38, 227-232 (2011).

Research goals

The proposal project aims at achieving the following objectives
1. Extraction of bioactive metabolites from dried algae tissues including low molecular lipohilic compound and polysaccharides.
2. Structure determines of the bioactive metabolites by spectroscopic, optical and chemical methods.
3. Biological characterization of the bioactive metabolite taking into account their potential practical application in agro-food, pharmaceutical and medical field
4. Development of analytical methods for optimising the production of bioactive metabolites

The methodology being the following:
Usual and advanced methodology and instrumental implementation will be used for the extraction of the lypophilic bioactive metabolites. The organic extract will be purified by chromatographic (column, TLC and HPLC chromatography) methods. Suitable techniques will developed for the purification of the hydrophilic metabolites by fractionated precipitation combined with and suitable chromatographic methods (gel filtration and/or ionic exchange at normal pressure and with HPLC). For the structure determination of bioactive new metabolites will be used spectroscopic methods (IR, UV, mono and bi-dimensional proton and carbon NMR, EI, ES and FAB-MS, and CD), optical (OR and CD)
Chemical methods which, through functional group transformation, will allow confirming structures. The purification processes of bioactive metabolites will be checked with opportune biological assays. The same assays will be used for the biological activities characterisation of purified metabolites.
The simplest chemical method will be optimized to confirm the structure interesting isolated bioactive metabolites and to prepare some derivative for studies on structure/activity relationships.
Simple, rapid and convenient analytical methods will be developed to identify and quantify the bioactive metabolites to optimize their production based on HPLC, spectrophotometric, immunochemical and cytofluorometric methods.
Finalization of the products following the control results:
Publication on international scientific journals
Bioassay to evaluated practical potential application in agro-food and medicine

Last update: 27/11/2021