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Antiplasmodial Activity of Natural Products: Effect of Incorporation into Erythrocyte Membrane

Hanne Ziegler

The subject of this Ph.D. dissertation is the search for novel antiplasmodial agents from plants. Plant extracts were tested in vitro for their inhibition of proliferation of Plasmodium falciparum parasites grown in human erythrocytes, using incorporation of tritiated phenylalanine as the index of growth. Extract constituents responsible for the activity were isolated using chromatographic methods. Finally, the structure of the purified constituents was elucidated using modern spectroscopic methods, including NMR at 800 MHz. In this way, the antiplasmodial component of Rinorea ilicifolia (Violaceae) was shown to be lupeol. Lupeol is a common plant constituent previously reported to posses antiplasmodial activity in vitro.

However, it was discovered that the incubation with lupeol, of P. falciparum parasites grown in erythrocytes, caused pronounced changes of the curvature of erythrocyte membrane towards stomatocytes. The results correlated well with the observed IC50 value for inhibition of growth. The erythrocyte membrane changes were observed using transmission electron microscopy. Moreover, preincubation of erythrocytes with lupeol rendered the cells unsuitable for parasite growth. The observed in vitro antiplasmodial effect of lupeol is thus concluded to be an indirect effect related to the presence of lupeol in the erythrocyte membrane, and not to a toxic effect of the compound on the parasite itself. True antiplasmodial effect should not be associated with changes of erythrocyte membrane at active concentrations, as demonstrated to be the case with chloroquine.

The antiplasmodial effect was also observed with a long range of synthetic stomatocytogenic amphiphiles. The correlation of the antiplasmodial effects with membrane changes was also observed with betulinic acid, betulin, and three semi synthetic analogues (betulinic amide, methyl betulinate and betulinic aldehyde). As with lupeol, preincubation of erythrocytes with the compounds, followed by extensive washing, afforded modified erythrocytes unsuitable for parasite invasion and growth. In the betulinic acid series, structure-activity relationships of the antiplasmodial effect were observed, the type of erythrocyte shape changes being apparently determined by hydrogen bonding properties of the agent.

Thus, hydrogen bond donors caused formation of echinocytes, whereas hydrogen bond acceptors caused formation of stomatocytes. Because compounds that have potential of being incorporated into erythrocyte membrane, such as the pentacyclic triterpenes mentioned above, are common in plant extracts, these observation have pronounced consequences on the bioactivity guided isolation of potential antiplasmodial agents from plants. Accordingly, the antiplasmodial constituents of Hyptis suaveolens (Lamiaceae), shown to be dehydroabietinol, and those isolated from Caloncoba echinata (Flacourtiaceae), shown to be (11R,20R)-11,20-dihydroxy-24-dammaren-3-one and (17S, 20R, 24R)- and (17S, 20S, 24R)-17,25-dihydroxy-20,24-epoxy-14(18)-malabaricen-3-one, all caused transformation of discocytes into stomatocytes.

The structures of C. echinata terpenoides, which are novel natural products, were determined using high-field 2D NMR techniques (mainly NOESY and HMBC) supported by HRMS. Mosher's method was used for determination of absolute stereochemistry. Pronounced membrane effects were also observed with licochalcone A. It is concluded; that the antiplasmodial in vitro activities reported in the literature for many natural products is due to membrane effects. It is therefore proposed, that microscopic studies should be generally used as a means of validation of in vitro toxicity data, in order to help eliminate antiplasmodial compounds exhibiting indirect effects on the host cell.


Ingen Dansk version