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

PEOPLE MARIE CURIE ACTIONS
International Incoming Fellowships (IIF). Call: FP7-PEOPLE-2013-IIF.

Project: PLANTMEDs N°629482
The objective of this project was to discovery potential anticancer and antimalarial lead compounds from Cameroonian rain forest plants and if possible to produce pre-processed fractions containing the active agents of medicinal plant extracts of Cameroon allowing the local populations to fight successfully against cancer and malaria. Thereby, selection of plants concentrated on species belonging to families with recorded effectivity in traditional Cameroonian medicinal use, based on the idea that these chemically and pharmacologically untapped members might hold the key to novel valuable leads for the pharmaceutical industry.
I. Materials and Methods
I.1. Plants Material
The plants selected for the project, namely Fagara claessensii, Fagara poggei and Citrus reticulata from the family of Rutaceae - the latter added in the course of the project - as well as Hannoa ferruginea and Pierreodendron grandifolium from the family of Simaroubaceae were taxonomically identified in the Cameroonian rainforest together with a botanist, and vouchers were deposited at the Herbarium Yaoundé, Cameroon. In order to follow the rules set up by the Rio Convention on biological diversity, for each of the above species ten plants were located in the field. Small samples from each part of each tree (bark, roots, leaves, fruits/seeds) were collected to make a total of 5 kg for each part of each plant.
I.2. Extraction and bioassay guided isolation
Soxhlet extraction of the air dried and powdered each plant yielded - after evaporation under reduced pressure three fractions (n-hexane (Hex), dichloromethane (DCM) and methanol (MeOH)). These extracts were submitted to the 3-(4,5-dimethylthiazol-2-yl)-2,5-dphenyltetrazolium bromide (MTT) cytotoxicity/viability assay employing cancer cell lines A549, MCF7, PC3 and PNT2 and heam polymerisation.
The most active extract against all three cancer cell lines and/or malaria assay were subjected to Vacuum Liquid Chromatography (VLC) on TLC grade silica gel as the stationary phase. Elution was performed with mixtures of n-hexane and ethyl acetate (EA), namely 20% of EA in Hex, 50% of EA in Hex and 70% of EA in Hex to give three fractions. The active fractions, which were then submitted to column chromatography over silica gel 60 using a gradient system of n-hexane, CH2Cl2, ethyl acetate and MeOH. Finally, some isolated compound were submitted to the MTT assay and heam polymersation to identify the active compound.
II. Results and discussion

II.1. Phytochemical investigation of Zanthoxylum claessensii (synonym Fagara claessensii)
One new derivative of tirucallane together with twenty known compounds, were isolated from the hexane and dichloromethane (DCM) extracts of the bark and roots of Zanthoxylum claessensii (De Wild.) P. G. Waterman. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR and EI- and ESI–MS) as: citracridone II, citracridone III, citracridone I, 5-hydroxynoracronycine, 5-methoxynoracronycine, medicacridone, 5-methoxypsoralene, 5,8-dimethoxypsoralene, scopoletin, 7-methoxy-6-prenylcoumarine, marmesin, atalantoflavone, 2-methoxy-7,8-dehydroruteacarpine, 3,4-secotirucalla-4(28),7,24-triene-3,21-dioic acid, 1β-hydroxy-3,4-secotirucalla-4(28)-7,24-trièn-3,21-dioic acid, butilinic acid, lupeol, stigmasterol, β-sitosterol, β-sitosterol-3-O-β-D-glucopyranoside and stigmastérol-3-O-β-D-glucopyranoside. Some isolated compounds displayed a moderate level of cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC50 values of 13.4 and 20.6 mM.
II.2. Phytochemical investigation of Zanthoxylum poggei (synonym Fagara poggei)
The stem bark of Zanthoxylum poggei (Engl.) P. G. Waterman, was extracted with CH2Cl2 and subjected to bioassay-guided fractionation based on its inhibitory activity against the oxidative burst of whole blood (90%). This extract was subjected to column chromatography (silica gel) and preparative TLC to afford two new and nine known compounds (Fig. 2). The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR and EI- and ESI–MS) as poggeicridone, citracridone III, citracridone I, 5-hydroxynoracronycine, 2-methoxy-7,8- dehydroruteacarpine, 5-methoxynoracronycine, 2-hydroxyruteacarpine, 2-methoxyruteacarpine, 5,8,13,14-tetrahydro-2-methoxy-14-methyl-5-oxo-7H-indolo[20,30:3,4]pyrido[2,1-b]quinazolin-6-ium chloride, lupeol and β-sitosterol. All isolated compounds exhibited strong suppressive effects on the phagocytosis response upon activation with serum opsonized zymosan in the in vitro oxidative burst studies using whole blood. The IC50 values were in the range of 12.0-25.9 μM. These compounds displayed a moderate level of cytotoxic activity against the human Caucasian prostate adenocarcinoma cell line PC-3, with IC50 values of 15.8 and 22.1 μM (the IC50 value of the positive control standard doxorubicin was IC50 0.9 μM).
II.3. Phytochemical investigation of Citrus reticulata Blanco
1.2 kg of bark material of Citrus reticulata Blanco was subjected to column chromatography (silica gel) and preparative TLC to afford seven known compounds identified as scoparone, xanthyletin, β-amyrin, lupeol, stigmasterol, β-sitosterol and palmitic acid. In vitro assays show that the stem bark of C. reticulata widely used in Cameroon against tumours is probably a good crude drug treatment against lung, breast and prostate cancer. 70% EA in Hex fraction of the stem bark of C. reticulata show good activity against lung, breast and prostate cancer and could be used for the development of new anticancer agents. Moreover, first assays display very low toxicity of a Hex extract fraction against a human normal prostate cell line.
II.4. Phytochemical investigation of Hannoa ferruginea
Soxhlet extraction of 3.2 kg of bark material of Hannoa ferruginea led - after evaporation under vacuum - to 24.5 g of Hex, 34.6 g of DCM and 345.2 g of MeOH extract. These extracts were tested against cancer cell lines A549, MCF7 and PC3 (in addition to the normal cell line PNT2) using the MTT assay. Etoposide was also used as the positive control. In the following, the 50% and 70% of EA in Hex fractions were subjected to column chromatography (silica gel) and preparative TLC to afford two new coumarinolignanes, two new canthin-6-one alkaloids, two new stiblenes and twelve known compounds, identified as ferrunolignane A and B , cleomiscosin A, furrunoide A and B, 1-methoxycanthin-6-one, 1-hydroxycanthin-6-one, 5-methylcanthin-6-one, canthin-6-one (39), 1-methoxycanthin-6-one-3-oxide, canthin-6-one-3-oxide, β-carboline-1-propionic acid, ferrunoside A and B. The structures of compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR and EI-MS) and by comparison with the reported data.
Conclusion
The results obtained during this project are interesting and confirm the use of these plants by the rural population for the treatment of parasitic diseases and tumors. The results of the antimalarial tests as well as the phytochemical and pharmacological studies of the other parts of Hannoa ferruginea are still in progress. The bark of Citrus reticulata widely used in Cameroon against tumors turns out to be probably a good crude drug treatment against lung, breast and prostate cancer when extracted with hexane or dichloromthane. First assays showed very low toxicity of a hexane extract fraction in vitro. However, further in vitro and in vivo tests will be necessary before any recommendation of this drug can be given. A positive outcome might initiate formulation studies for the development of a safer anticancer plant product from the bark of this plant, to be manufactured in developing countries.
The various techniques acquired during his stay here, will be transfered to PhD and Master Students of the University of Douala. This will enable them to develop their potentials.

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LIVERPOOL JOHN MOORES UNIVERSITY
United Kingdom

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Life Sciences
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