A New Xanthone from the Bark Extract of Rheedia acuminata and Antiplasmodial Activity of Its Major Compounds

Bioassay-guided fractionation of the ethyl acetate bark extract of Rheedia acuminata led to the isolation of the new compound 1,5,6-trihydroxy-3-methoxy-7-geranyl-xanthone (1), together with four known compounds 2-5. These compounds were tested in vitro for their antiplasmodial activity on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) and for their cytotoxicity against the human diploid embryonic lung cell line MRC-5.


Introduction
In South America several species of Clusiaceae are widely used in the manufacture of hulls, and well known for quality of their wood and for the healing properties of their latex used traditionally used for their effectiveness against dermatoses [1]. One of these, Rheedia acuminata (Ruiz & Pavon) Planchon and Triana, a tree growing in Amazonian rainforest, possesses an abundant latex used for OPEN ACCESS various medicinal purposes by the Guianese Amerindians (Palikur) in the form of patches or breakdowns applied to the wrinkling muscle. In addition, Rheedia acuminata fruits are commonly consumed in South America [1].
In an effort to find new natural antimalarial drugs and as part of investigation of French Guiana plants, we found that the ethyl acetate extract of the bark of Rheedia acuminata showed antiplasmodial activity (92% of inhibitory growth of Plasmodium falciparum (FcB1) at 10 µg/mL), whereas leaves and fruits extracts showed no significant and weak activity respectively (28% and 50% of inhibitory growth at 10 µg/mL, respectively). We report here the bioassay-guided fractionation of this extract on the basis of this antiplasmodial activity.
The antiplasmodial activity against the chloroquine-resistant strain of P. falciparum FcB1 and the cytotoxicity upon the human diploid embryonic cell line MRC-5 of compounds 1-5 are summarized in Table 1. The three xanthones 1, 2 and 3 isolated from Rheedia acuminata bark have IC 50 s against Plasmodium falciparum exceeding 10 µM. Compounds 4 and 5 have shown the best activity with IC 50 s around 3 µM. The IC 50 values obtained on MRC-5 cell line indicated that PPAPs 4 and 5 exhibited significant cytotoxicity whereas xanthones (1 to 3) showed weak cytotoxicity. Several xanthones, which have been isolated from various species of the family Clusiaceae, showed cytotoxic and antiplasmodial activities 9,10,19,20 and structure-activity relationships have been proposed in this series. For example, Winter et al. have demonstrated that xanthones with a hydroxyl group in peri positions with regards to the carbonyl (such as 1, 2 and 3), possess weak antiplasmodial activity due to their low affinity for the heme 21,22. In conclusion, the antiplasmodial activity detected in the crude extract is probably due to the presence of high quantity of the two PPAPs 4 and 5.

Plant Material
Trunk bark of R. acuminata were collected in French Guiana. A voucher specimen (PMF-258) was deposited in the Herbarium of Cayenne (French Guiana), and identified by M-F. Prévost (IRD).

Biological Activities
The extracts and compounds were tested against the chloroquine-resistant FcB1/ Colombia strain of Plasmodium falciparum in 96-well plates by measuring [3H]-hypoxanthine incorporation by parasite as previously described [23]. The growth inhibition for each compound concentration was determined by comparing the radioactivity incorporated in the treated culture with that in the control culture maintained on the same plate. The concentrations causing 50% inhibition of parasite growth (IC50) were calculated from the drug concentration-response curves. Chloroquine ® was used as a control compound.
The human diploid embryonic lung cells MRC-5 were seeded into 96-well microplates at 2000 cells per well. The cytotoxicity assays were performed according to a published procedure [24]. Taxotere ® was used as a control compound.

Conclusions
A chemical investigation of Rheedia acuminata bark was carried out in the framework of a global investigation on French Guiana flora. This study showed that the bark contained a new xanthone, 1,5,6-trihydroxy-3-methoxy-7-geranylxanthone, together with 2-(1'-1'-dimethylprop-2'-enyl)-1,4,5trihydroxyxanthone. Pyrojacareubin, isogarcinol and 7-epi-isogarcinol were isolated from the Rheedia genus, for the first time. The two PPAPs isolated from Rheedia acuminata, which exhibited cytotoxic and antiplasmodial properties, were likely responsible of the biological activity found in the crude extract.