In vivo antimalarial activity of Keetia leucantha twigs extracts and in vitro antiplasmodial effect of their constituents.

The West African tree Keetia leucantha (Rubiaceae) is used in traditional medicine in Benin to treat malaria. The twigs dichloromethane extract was previously shown to inhibit in vitro Plasmodium falciparum growth with no cytotoxicity (>100µg/ml on human normal fibroblasts). The dichloromethane and aqueous extracts of twigs of K. leucantha were evaluated in vivo against Plasmodium berghei NK 173 by the 4-day suppressive test and in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity. Bioguided fractionations were realized and compounds were structurally elucidated using extensive spectroscopic analysis. The in vivo antimalarial activity of K. leucantha dichloromethane and aqueous twigs extracts were assessed in mice at the dose of 200mg/kg/day. Both extracts exhibited significant effect in inhibiting parasite growth by 56.8% and 53.0% (p<0.0001) on day 7-postinfection. An LC-MS analysis and bioguided fractionations on the twigs dichloromethane extract led to the isolation and structural determination of scopoletin (1), stigmasterol (2), three phenolic compounds: vanillin (3), hydroxybenzaldehyde (4) and ferulaldehyde (5), eight triterpenic esters (6-13), oleanolic acid and ursolic acid. The antiplasmodial activity of the mixture of the eight triterpenic esters showed an antiplasmodial activity of 1.66 ± 0.54 µg/ml on the 3D7 strain, and the same range of activity was observed for isolated isomers mixtures. This is the first report on the in vivo activity of K. leucantha extracts, the isolation of thirteen compounds and analysis of their antiplasmodial activity. The results obtained may partially justify the traditional use of K. leucantha to treat malaria in Benin.

Bero J ，Hérent MF ，Schmeda-Hirschmann G ，Frédérich M ，Quetin-Leclercq J ... -  《-》

In vitro antiplasmodial activity of plants used in Benin in traditional medicine to treat malaria.

Bero J ，Ganfon H ，Jonville MC ，Frédérich M ，Gbaguidi F ，DeMol P ，Moudachirou M ，Quetin-Leclercq J ... -  《-》

Antiparasitic activities of two sesquiterpenic lactones isolated from Acanthospermum hispidum D.C.

Aerial parts of Acanthospermum hispidum D.C. are often used by traditional healers in Benin for various diseases and especially for malaria. To identify active compounds from extracts of Acanthospermum hispidum D.CV. leaves previously shown to possess antimalarial properties and analyse in vivo activity and toxicity of crude extracts. Compounds were isolated from aerial part of Acanthospermum hispidum D.C. and structurally elucidated using extensive spectroscopic analysis. Antiplasmodial activity was evaluated in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity and in vivo against Plasmodium berghei berghei by the 4-day suppressive test. Selectivity of extract and purified compounds on Plasmodium parasites were evaluated by using MTT test on J774 macrophage like murine cells and WI38 human normal fibroblasts and also against two other parasites: Trypanosoma brucei brucei and Leishmania mexicana mexicana. Acute and sub-acute toxicities of a crude extract were evaluated on mice. Two known sesquiterpenic lactones were isolated: 1 (15-acetoxy-8β-[(2-methylbutyryloxy)]-14-oxo-4,5-cis-acanthospermolide) and 2 (9α-acetoxy-15-hydroxy-8β-(2-methylbutyryloxy)-14-oxo-4,5-trans-acanthospermolide). 1 and 2 showed in vitro antiplasmodial activity against the chloroquine-sensitive strain (3D7) with IC(50) of 2.9±0.5 and 2.23±0.09μM respectively. Only 2 showed a high selectivity index (SI: 18.4) on Plasmodium compared to cytotoxicity against human fibroblasts cell line (WI38). 1 and 2 also showed interesting antiparasitic activities in vitro against Trypanosoma brucei brucei (IC(50) of 2.45±0.49 and 6.36±1.42μM respectively) and Leishmania mexicana mexicana (IC(50) of 0.94±0.05 and 2.54±0.19μM respectively). Furthermore, crude acidic water extract and fractions containing one of the two isolated compounds displayed a weak in vivo antimalarial activity against Plasmodium berghei berghei with a long half-life causing a delayed effect. In vivo acute (2000mg/kg) and sub-acute (1000mg/kg) toxicity tests on the crude acidic water extract did not show toxicity. Crude acidic water extract, fractions and pure isolated compounds from Acanthospermum hispidum showed promising in vitro antiplasmodial activity. Despite our study did not show in vivo acute and subacute toxicities of the crude acidic water extract, its weak in vivo antimalarial activity and the in vitro cytotoxicity of pure compounds and enriched extracts containing 1 and 2 indicate that the aerial parts of Acanthospermum hispidum should be used with caution for malaria treatments.

Ganfon H ，Bero J ，Tchinda AT ，Gbaguidi F ，Gbenou J ，Moudachirou M ，Frédérich M ，Quetin-Leclercq J ... -  《-》

In vivo anti-malarial activity and toxicity studies of triterpenic esters isolated form Keetia leucantha and crude extracts.

Beaufay C ，Hérent MF ，Quetin-Leclercq J ，Bero J ... -  《MALARIA JOURNAL》

Antiplasmodial activity of Heinsia crinita (Rubiaceae) and identification of new iridoids.

Heinsia crinita is used in traditional medicine for the treatment of febrile illness and erectile dysfunction. Its stem bark powder is found in some peripheral markets in the Democratic Republic of the Congo (DRC) as a remedy against malaria. Investigations were conducted on crude extracts of leaves, fruits and stem barks in view to validate their use and to determine which plant part possesses the best antiplasmodial properties. Different plant parts were extracted with methanol, ethanol and dichloromethane. Based on the preliminary assays, the dichloromethane extract of the stem bark was subjected to fractionation using preparative HPLC system and column chromatography. This step led to the isolation of two new iridoids which had their structures elucidated by NMR, UV, MS and FT-IR spectroscopic techniques. Extracts and pure compounds were tested in vitro against the 3D7 strain of Plasmodium falciparum. The inhibition of the parasite growth was evaluated in vitro by colorimetric method (p-LDH assay) and their cytotoxicity evaluated in vitro against the human non-cancer fibroblast cell line (WI38) through WST1 assay. The in vivo antiplasmodial activity was assessed by the inhibition of Plasmodium berghei growth in infected mice treated with the ethanol extract of H. crinita stem bark at the concentrations of 200 and 300mg/Kg/day per os, using a protocol based on the 4-d suppressive test of Peters and compared to a non-treated negative control group of mice (growth =100%). Finally the antioxidant activity of the same extract was evaluated using ABTS, DPPH and cell-based assays. A moderate in vitro antiplasmodial activity was observed for the dichloromethane extract of the stem bark of H. crinita (IC50 =29.2±1.39µg/mL) and for the two new iridoids, lamalbide 6, 7, 8- triacetate (IC50 =16.39±0.43µg/mL) as well as for its aglycone lamiridosin 6, 7, 8-triacetate (IC50 =0.44.56±1.12µg/mL). The ethanolic stem bark extract (200 and 300mg/kg/day, oral route) showed a moderate in vivo antimalarial activity in Plasmodium berghei-infected mice with 27.84±2.75% and 48.54±3.76% of inhibition of the parasite growth, respectively (p<0.01).). This extract displayed high cellular antioxidant activity using dichlorofluorescein-diacetate (DCFDA) on HL-60 monocytes. These crude extracts and pure compounds tested at the higher concentration of 100µg/mL did not show any cytotoxicity against WI38 cells. The results showed that H. crinita extracts possess antimalarial activity and contain some unusual iridoids with moderate antiplasmodial activity, therefore justifying to some extent its traditional use by the local population in DRC for this purpose. This is the first report of the isolation and antiplasmodial activity of these two new iridoids.

Tshisekedi Tshibangu P ，Mutwale Kapepula P ，Kabongo Kapinga MJ ，Tujibikila Mukuta A ，Kalenda DT ，Tchinda AT ，Mouithys-Mickalad AA ，Jansen O ，Cieckiewicz E ，Tits M ，Angenot L ，Frédérich M ... -  《-》