No abstract

Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC 50 ) of 27.7 ؎ 0.5 M, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC 50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay.

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH(2)OH, CONH(2)) caused formation of echinocytes, whereas those lacking this ability (CH(3), CHO, COOCH(3)) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC(50) values in the range 7-28 microM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.

Six labdanes (1-6) and four isopimaranes (7-10), including three new natural products (7, 9, and 10), were isolated from Platycladus orientalis, and their structures determined using 1D and 2D NMR methods, ion-cyclotron resonance HRMS, and optical rotation data. Relative configurations of all chiral centers in the isopimaranes were determined using NOESY experiments at 600 and 800 MHz. Specific optical rotation data were used to correlate absolute configurations. Compounds 1-9 and aframodial (11) were tested for their in vitro antiplasmodial activity and for their ability to induce changes of erythrocyte shape in order to obtain data about possible correlation between the two effects. All compounds tested exhibited weak (IC(50) > 25 microM) in vitro antiplasmodial effects against Plasmodium falciparum strain 3D7. At the same time, the compounds caused echinocytic or stomatocytic changes of the erythrocyte membrane curvature, indicative of their incorporation into the lipid bilayer, in the concentration region where the antiplasmodial activity was observed. The antiplasmodial effect of these compounds thus appears to be an indirect effect on the erythrocyte host cell. Weak or moderate antiplasmodial activity observed with many other apolar natural products, in particular those with amphiphilic structures, is also likely to be an indirect effect.