Valérie Jullian

A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or beta-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-microM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC(50) of chloroquine phosphate (28microM) and quinine base (324microM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC(50) 38 and 87microM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.

Biological activities of nitidine, a potential anti- malarial lead compound

BackgroundNitidine is thought to be the main active ingredient in several traditional anti-malarial remedies used in different parts of the world. The widespread use of these therapies stresses the importance of studying this molecule in the context of malaria control. However, little is known about its potential as an anti-plasmodial drug, as well as its mechanism of action.MethodsIn this study, the anti-malarial potential of nitidine was evaluated in vitro on CQ-sensitive and -resistant strains. The nitidines selectivity index compared with cancerous and non-cancerous cell lines was then determined. In vivo assays were then performed, using the four-day Peters test methodology. To gain information about nitidines possible mode of action, its moment of action on the parasite cell cycle was studied, and its localization inside the parasite was determined using confocal microscopy. The in vitro abilities of nitidine to bind haem and to inhibit β-haematin formation were also demonstrated.ResultsNitidine showed similar in vitro activity in CQ-sensitive and resistant strains, and also a satisfying selectivity index (> 10) when compared with a non-cancerous cells line. Its in vivo activity was moderate; however, no sign of acute toxicity was observed during treatment. Nitidines moment of action on the parasite cycle showed that it could not interfere with DNA replication; this was consistent with the observation that nitidine did not localize in the nucleus, but rather in the cytoplasm of the parasite. Nitidine was able to form a 1-1 complex with haem in vitro and also inhibited β-haematin formation with the same potency as chloroquine.ConclusionNitidine can be considered a potential anti-malarial lead compound. Its ability to complex haem and inhibit β-haematin formation suggests a mechanism of action similar to that of chloroquine. The anti-malarial activity of nitidine could therefore be improved by structural modification of this molecule to increase its penetration of the digestive vacuole in the parasite, where haemoglobin metabolization takes place.

Hot and cold: Medicinal plant uses in Quechua speaking communities in the high Andes (Callejón de Huaylas, Ancash, Perú)

ETHNO-PHARMACOLOGICAL RELEVANCEnAn ethnopharmacological survey has been set up in high altitude Quechua speaking communities dwelling in Callejón de Huaylas (Ancash department, Peru) and in medicinal plant markets in order to document the medicinal plants use of 178 species within the frame of a traditional Andean medicinal system.nnnMATERIALS AND METHODSnA sound ethnopharmacological survey was performed during the years 2011, 2012 and 2013 in different places along Callejón de Huaylas valley in the peruvian Andes, were Quechua speaking communities dwell. Two different methodologies were used: first, plant uses were recorded during walks with informants and in medicinal plant markets held on a regular bases in the area (Huaraz, Carhuaz, Yungay). Secondly, traditional healers (curanderas, curanderos) were interviewed about their practices and healing sessions were observed, in order to understand better the traditional medicinal system as a whole (disease aetiology, diagnosis, treatments, healers).nnnRESULTSnAltogether, 178 medicinal species were collected. Most of the plants found on the market were also found in the wild and vice-versa. Medicinal plant trade is exclusively held by women, selling their merchandise to local people or to big retailer. Plants are classified according their hot or cold virtues, this in accordance with the local conception of the body physiology and disease aetiology, based on a hot-cold polarity. Main use notified for medicinal plants is (bath) against cold, a prophylactic measure against diseases of cold nature. Other uses include culture bound illnesses i.e. susto, aire, nervios, or heart pain, commonly cited in South America. Regarding symptoms, rheumatic/arthritic pain, musculoskeletal traumas, cough, pulmonary and respiratory problems, gastritis and stomach ache, were the most frequently cited. Diagnosis and treatment are intrinsically linked together and mainly based upon divination techniques using egg and cuy (Cavia porcellus L., Caviidae).nnnDISCUSSION AND CONCLUSIONnMedicinal plants use and traditional medicinal practices are still very vivid in Callejón de Huaylas as highlighted by the abundance of medicinal plants traded in the markets. In this business, women have a key position as healers at the family and community level. Medicinal uses of the majority of the species presented here are reported for the first time. Because medicinal plants sold on the market are collected from the wild and also because high altitude medicinal plants are generally small herbaceous species pulled out with their roots, there is a serious risk of over exploitation and extinction of endemic species.

Anti-Leishmanial Lindenane Sesquiterpenes from Hedyosmum angustifolium

The aim of this work is the isolation of anti-leishmanial compounds from the ethyl acetate extracts of the bark of HEDYOSMUM ANGUSTIFOLIUM. We have successfully isolated and characterized five sesquiterpenes: one new compound (oxyonoseriolide, 1), one compound isolated for the first time from a natural source (hedyosmone, 2), and three known sesquiterpenes (onoseriolide, 3; chloranthalactone A, 4; and spathulenol, 5) that had not been previously isolated from H. ANGUSTIFOLIUM. The biological activities of 1- 5 showed that onoseriolide ( 3) was the most active compound against axenic amastigotes from LEISHMANIA AMAZONENSIS and L. INFANTUM. Moreover, it was still active on the intramacrophagic amastigotes of L. INFANTUM. The isolated compounds have also been tested on PLASMODIUM FALCIPARUM and against various mammalian cell lines.

New Antiplasmodial Bromotyrosine Derivatives from Suberea ianthelliformis Lendenfeld, 1888

Four samples of Suberea ianthelliformis were investigated and furnished five new and 13 known brominated tyrosine‐derived compounds. Two of the new compounds were identified as araplysillin N20‐formamide and its N‐oxide derivative. Three other new compounds, araplysillins IV, V, and VI, were isolated and identified as analogs of araplysillin II. Most of these compounds exhibit moderate inhibitory activities against chloroquine‐resistant and ‐sensitive strains of Plasmodium falciparum, and were investigated for their PFTase inhibitory properties. The chemical content of the investigated sponges is correlated with their molecular phylogeny.

Antiplasmodial activity of New Caledonia and Vanuatu traditional medicines

Context: With the emergence of strains multiresistant to antimalarial drugs, the search for new active molecules remains a priority. Ethnopharmacology appears to be a good method of selection in such investigations. Objective: The aim of this research work is to select plants used in Melanesian traditional medicine, in New Caledonia and Vanuatu, which should be a promising source for the isolation of new antimalarial drugs. Materials and methods: Forty-seven plant extracts belonging to 12 families, traditionally used by the Melanesian people or belonging to an antimalarial known genus, were screened in vitro for antimalarial activity on Plasmodium falciparum chloroquine (CQ)-resistant (FcB1) and CQ-sensitive (HB3) strains. They were also tested for their inhibitory effects on a protein kinase (Pfnek) and their cytotoxicity on human breast adenocarcinoma (MCF7) cells. Results: Among all extracts, four displayed strong in vitro activities against P. falciparum: Gardenia urvillei Montrouzier, Scleria polycarpa Boeckeler, Terminalia catappa L. and Acronychia laevis J.R. & J.G. Forster, the latter being also toxic on MCF7 cells. Except for the extracts of S. polycarpa, all others that were active on P. falciparum, also possess an inhibitory effect on Pfnek. Discussion and conclusion: These results confirm that ethnopharmacology is an excellent approach for such investigations. The two countries considered clearly present advantages in the field. Indeed, local populations keep their traditional knowledge alive, and their flora is exceptionally rich. In New Caledonia, the high endemicity rate (74%) ranks the island as one of the world’s biodiversity hotspots. As a consequence, chances to discover new active natural compounds are also high.

New findings on Simalikalactone D, an antimalarial compound from Quassia amara L. (Simaroubaceae)

Quassia amara L. (Simaroubaceae) is a species widely used as tonic and is claimed to be an efficient antimalarial all over the Northern part of the Amazon basin. Quassinoid compound Simalikalactone D (SkD) has been shown to be one of the molecules responsible for the antiplasmodial activity of a watery preparation made out of juvenile fresh leaves of this plant. Because of its strong antimalarial activity, we decided to have a further insight of SkD pharmacological properties, alone or in association with classical antimalarials. At concentrations of up to 200μM, we showed herein that SkD did not exert any apoptotic or necrotic activities in vitro on lymphoblastic cells. However, an antiproliferative effect was evident at concentrations higher than 45nM. SkD was inefficient at inhibiting heme biomineralization and the new permeability pathways induced by the parasite in the host erythrocyte membrane. With respect to Plasmodium falciparum erythrocytic stages, SkD was almost inactive on earlier and later parasite stages, but potently active at the 30th h of parasite cycle when DNA replicates in mature trophozoites. In vitro combination studies with conventional antimalarial drugs showed that SkD synergizes with atovaquone (ATO). The activity of ATO on the Plasmodium mitochondrial membrane potential was enhanced by SkD, which on its own had a poor effect on this cellular parameter.

The living library of The Cotapata National Park in Bolivia: an example of application of Bolivian law on the access to genetic resources

Developing countries with a rich biodiversity want to control the use of this natural patrimony, especially in the research of natural compounds of pharmaceutical interest. Here we present the organization of six permanent plots in a mountain tropical forest on the east side of the Andean Cordillera in Bolivia, and their role in the discovery of plants with antiplasmodial or antileishmanial activities. Permanent plots are widely used in ecological survey, but rarely in bioprospecting. This set-up allows Bolivian authorities to control the bioprospecting, and facilitates further chemical studies on the bioactive plants.