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Final Report Summary - PLANTMEDS (Potential Antimalarial and Anticancer Lead Compound Discovery from Cameroonian Medicinal Plants)

II. Research results
II.1. Collection of plant material
Plants were located in the field according to information on sample sites and plant description documented at the Limbé Botanic Garden and the National Herbarium in Yaoundé. Healthy plants were selected together with the botanist of our research group, and 1- 4 kg collected from parts of plants applied traditionally.
II.2. Preparation of extracts
Air dried and powdered plant samples were macerated each in methanol and water at room temperature for 72 h and extracted (3 x 3000 mL, 24 h each). After evaporation under reduced pressure, the obtained crude extracts were suspended in 2000 mL aqueous MeOH (methanol, 9:1 v/v) and extracted with petroleum ether (3 x 500 mL). The aqueous layers were diluted with 60% v/v MeOH and extracted with dichloromethane (DCM) (3 x 500 mL). Work was done at the University of Douala / Cameroon.
II.3. Biological assays
Crude extracts were submitted to bioassays employing P. falciparum, provided by the biologist of the research group. Test results were used to select those extracts indicating the presence of active secondary metabolite.
II.3.1. Isolation of compounds
Work was carried out by column chromatography assisted by preparative thin layer chromatography using anisaldehyde/sulphuric acid spray as indicator of promising compounds, received from colour reaction induced by hot air gun treatment. Purification of compounds was completed.
III. Transfer of knowledge gained from FP7 Project 629482 and further actions
The FP7 Project 629482 resulted in a wealth of new knowledge concerning bioassays, structure elucidation and new spectroscopic methodologies. This new experience was and still is handed to Master, PhD, postdoctoral and coworkers of my research group by practical demonstration in the laboratory and two presentations given during our monthly seminar. New knowledge was as well profoundly discussed during informal meetings in the laboratory.
Since bioassays play an important role in natural product chemistry, steps are currently discussed to upgrade facilities within our laboratory that will allow us to test crude extracts and fractions from plants under work.
III.1. Spectroscopic Techniques Training and structure elucidation
Spectroscopy is an analytical techniques which has found acceptance in almost each and every analytical laboratory globally. The NMR, MS, UV and IR have contributed to qualification, identification, quantification and characterization studies on materials and molecules.
III.1.1. NMR technique
Among the analytical tools available today, NMR is probably one of the most valuable. It has not only allowed chemists to identify their molecules much more easily than before, or to characterize those extracted from nature in order to develop new drugs, it’s used in quality control and reserach for determining the content and purity of a sample and also to obtain a non-invasive and non-ionizing method. Study of the human body (Magnetic Resonance Imaging, MRI, which initially made it possible to obtain images of the soft parts, previously inaccessible by conventional methods, and now, dynamic images, allowing to control for example the blood flow in different regions of the brain).
We propose in this file to lay the theoretical bases of this tool and to sketch its use in chemistry by first exposing the nuclear magnetism before presenting the main factors useful to the chemists to finish on a description of the apparatus used.
The main objective of this training was to show, by presenting some usual applications of NMR spectrometry, from which experimental data it was possible to build the model of the NMR; and to see how, on the contrary, by relying on this model, one can then give a coherent interpretation of the experimental facts, that is to say the spectra obtained. The validity of a theoretical model lies in its ability to predict new experimental facts and, to predict what will be the spectrum of an organic molecule of known structure; we will use at the same time another model: the model of the Quantum mechanics. This successful application of quantum mechanics to NMR spectrometry will constitute a validation of the two proposed models.
In training, we hope to have shown the principles of the NMR, as well as its use for the chemist and finally the technical part which is often masked. We have opted for a description that does not use the mathematical tool and is based on the experience of similar phenomena. However, it should be noted that not everything is as immediate, but that a rigorous study, as simple as it is, requires mathematical tools and physical concepts far removed from everyday life.

III.2. Training phase concerning MIC assays with multi-resistant bacterial strains and cytotoxicity assay (monolayer assay) employing normal human body cells
While the antimicrobial activity of chemical constituents will be evaluated by MIC determination in micro titer plates by the broth microdilution method according to a standard protocol using drug resistant / multi drug resistant strains and cytotoxicity towards normal human body cells will be determined with the Vero cell line, both actions to be carried out under S2 safety conditions at the University of Bielefeld /Germany, a training phase 1st to 21st April 2017 is currently carried out by the team member Dr. Akono, the microbiologist of the group covering the following training steps:
• A lecture and two seminars on the methods of MIC assay (bioactivity, cytotoxicity) after a standard protocol published by National Committee for Clinical Laboratory Standards, Villanova, PA, 1997 as well as on safety standards
• Practical training in the microbiological laboratory at the University of Douala, Cameroon covering work under sterile conditions, preparation of fluid / agar media, sterilization by auto lavation, sub culturing of bacterial strains using clean bench and incubator, macro dilution assay for determination of MIC using non pathogen model bacteria, reading of test results
• To carry out MIC assays with isolated pure compounds by macro dilution method using model bacteria
This training will enable all PhD students of my research group to do preliminary biological screening on crude extracts, fractions and isolated compounds of current plant extracts under work. Positive results received from model bacteria will help to preselect suitable compounds for later investigation using multi-drug resistant bacterial strains.
V. Future work
Work on Cameroonian rainforest plants will be extended towards further chemically and pharmacologically not yet investigated species from the Rutaceae as well as Simaroubaceae family. Since members of these families are widely used in Cameroonian health care against a multitude of conditions, new leads are expected against bacterial infections, cancers, hypertension, diabetes, inflammation and malaria. Most research steps - including semi synthesis and drug preformulation - will be carried out in the Douala research laboratory; however, cooperation with European research partners will be continued on spectroscopic measurements and anticancer assays.
The project promote an intensified scientific cooperation between the University of Douala, Cameroon and the Liverpool John Moores University, UK. All master's degree students and the PhD were trained and supervised by partners´ workgroups. These students will be multiplicators for knowledge transfer of new approaches and methods applied, and help to develop and spread new scientific knowledge between Cameroonian natural products work groups. In addition, lectures and seminars given by the applicants to support students involved into the planned research project with background knowledge and theoretical training. Manuscripts of lectures and seminars together with additional literature will be distributed via intranet recently installed. This will develop the potential of e-learning for Higher Education, thereby widening the access to scientific knowledge.
The characterization of active fractions will allow producing pre-processed standardized fractions of medicinal plant extracts (“crude drugs”) to help the local population of Cameroon and other developing countries to fight against bacterial infections. This will improve the healthcare and socio-economic situation of the population who earn less than 50 cent/day, and simply cannot afford to access modern drugs and treatments.

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