Our researches may be divided in four parts:
- Crude extracts and pure compounds evaluations
- Isolation and structure identification of bioactive natural compounds
- Identification of target(s) and structure-activity relationships
- Quality control and analytical validated methods development
- Development of improved formulations for oral delivery
Plants used in traditional medicine in different countries are obtained through research collaborations (for example: Marocco, Benin, Congo Democratic Republic, Rwanda, Madagascar, Mauritius in Africa, Pakistant and Vietnam in Asia, Peru, Bolivia and Brazil in South America). The first step is the selection on an ethnopharmacological basis and a literature survey. Different extracts are prepared and pharmacologically evaluated according to their traditional use(s). Several properties are analysed in our lab or in collaboration with other teams who developed suitable pharmacological tests (LDRI, other UCLouvain or Belgian partners): in the last years we mainly focused on antimicrobial and antiparasitic activities, but two new projects were developed dealing with antidiabetic and anti-inflammatory activities.
Crude extracts are first evaluated by in vitro tests and their cytoxicity assessed on cancer and non-cancer cell lines.
The originality of our works is that we do not just realise screenings. The most promising extracts are also tested in vivo to assess their activity and eventual toxicity. The mode of administration is chosen according to the nature of the extract but most of them are given by oral route.
Several extracts possessing biological activities at low concentrations in vitro were identified (cfr publications).
The activities of the most interesting ones as well as purified compounds were also analysed in vivo. Acute and sub-acute toxicity tests are realised on rodents or using zebrafish (collaboration with Prof. Frédérich, University of Liège). Results indicate that extracts of Croton zambesicus, Ajuga iva and Marrubium vulgare showed, in vivo, antihypertensive properties but some extracts of Croton zambesicus also showed toxicities. Extracts from i.e. Keetia leucantha, Vitellaria paradoxa and Acanthospermum hispidum as well as isolated triterpenic esters proved to have antimalarial activities on mice infected by Plasmodium berghei.
Efficient extracts and pure compounds on mice infected with Trypanosoma brucei were also identified and their highest tolerated dose determined.
Our works on antimicrobial plants allowed us to identify some promising plant extracts and natural compounds reducing the resistance of methicillin resistant Staphylococcus aureus in vitro (collaboration with F. Van Bambeke) and in vivo (collaboration with Prof. Niset, San Diego, USA). Other collaborations allow us to identify extracts improving fish resistance to microbial infection (collaboration with P. Kestomont, UNamur) or possessing antihelminthic properties for cattle (collaboration with UAC Benin).
Other extracts were shown to reduce the cytokines production of LPS activated macrophages (collaboration with Prof. Muccioli) and possess anti-diabetic properties (collaboration with Prof. A. Belocqui). We also collaborate with this team to and develop formulations to improve oral bioavailability of natural compounds.
Plant extracts having interesting in vitro and/or in vivo activities are subject to bio and chimio- guided fractionations to identify active components which could constitute new leads for further developments.
Fractions obtained by different chromatographic methods are evaluated and active ones analysed by LC-MS to identify well known compounds (based on retention times and MSn spectra, collaboration with MASSMET platform) and determine those which should be further purified (unidentified substances). The use of molecular networks will also help identifying known compounds. Structural identification is based on UV, IR, SM, 1D and 2DNMR spectra.
In addition to known compounds, we identified several new molecules which are found for the first time in plants. Among them, we can point out diterpenes isolated from Croton zambesicus. Some of these diterpenes have been shown by our team to possess cytotoxic and pro-apoptotic properties but others relax significantly rat aorta contracted by KCl. Comparison of the cytotoxic and vasorelaxant activities of isolated molecules and synthetic analogues indicates that both effects are not linked. We can also cite several promising specific antiparasitic terpenic derivatives isolated form Keetia leucantha, Ocimum basilicum, Vitellaria paradoxa or Cymbopogon species or essential oil components from Vietnamese plants.
Identifications of antimalarial compounds is also guided by supervised metabolomics studies of crude extracts (collaboration with Prof. Choi, Leiden) while collaborations with LIST (Luxembourg Institute of Science and Technology, Dr André) allowed us to identify and produce potential bioactive compounds from in vitro plant cell cultures.
Once structures are identified, we realise further experiments in collaboration with specialised teams to determine their targets and modes of actions and compare their activities with related natural or (semi)-synthetic compounds to assess structure-activity relationships.
Researches on pure isolated compounds allowed us to determine some structure-activity relationships for the vasorelaxant effect of trachylobanes diterpenes (collaboration with N. Morel, IREC). Targets were identified as voltage dependent calcium channels.
Structure-activity relationships for the vasorelaxant activity of trachylobanes
Alkaloids inhibiting topoisomerase I were identified in Cassytha filiformis. Synthetic derivatives were prepared in Spain and were also shown to possess antimalarial properties with a high selectivity index. Structure-activity relationships have been studied.
In the antiparasitic domain, we identified several antitrypanosomal terpenic compounds, some of them inhibiting trypanosomal GAPDH activity, a key enzyme of glycolysis, a process vital for trypanosoma development during its human cycle. We also collaborate with the teams of Prof. J. Palermo (University of Buenos Aires), Profs. J. Poupaert and R. Frédérick (LDRI-CMFA) and Profs. G. Acrombessi and F. Gbaguidi (UAC-Bénin) for the evaluation of the antiparasitic activities of (semi)synthetic compounds and establishment of structure-activity relationships. Some semi-synthetic compounds showed very promising antiplamsodial in vitro activity, in the same range as artemisinin.
Their effects on parasitic cells are now studied by metabolomics using LC-MS and NMR data (in collaboration with M. Frédérch and P. De Tullio from ULiège and B. Govaerts and SMCS from UClouvain) to determine the biochemical pathways modified by these natural pure compounds and identify their targets.
The physico-chemical interactions of natural saponins with cholesterol and biological membranes were studied in collaboration with the team of M.P. Mingeot (TFAR-FACM/LDRI) and new results were obtained which could explain several activities of this class of compounds. We also analyse with Prof. Mingeot the interaction of terpenic compounds with parasites membrane models.
The last part of our research is to develop and validate analytical methods to identify and quantify natural compounds in complex media (crude extracts, cells, biological fluids…).
Analytical methods are useful:
- To control the quality of plant preparations
- To increase the yields and/or the quality of productions by studying the effects of growth/cultivating/harvesting conditions on the active molecules contents of plants.
- To analyse the mode of action, resorption and/or metabolisation of natural substances or derivatives
- To find methods to eliminate toxic compounds and find less toxic accessions.
Methods to identify by LC-MS and quantify several types of bio active molecules by GCFID, GC-MS, LC-UV or LC-MS in crude extracts (particularly alkaloids, mono-, di-, triterpenes, steroids, rotenoids and flavonoids) were developed and validated in collaboration, for LC-MS, with MASSMET platform. We also developed a model to predict and analyse metabolic stability, identify metabolites from pure compounds and quantify anti-angiogenic hemisynthethic products in blood.
The laboratory is also officially agreed (by the Federal Agency for Medicine and Health Products) for the quality control of drugs.
After identifying promising natural molecules or derivatives, formations are developed in collaboration with Prof. A Belocqui to improve their oral bioavailability.