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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, CH2OH, CONH2) caused formation of echinocytes, whereas those lacking this ability (CH3, CHO, COOCH3) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC50 values in the range 7–28 μM, 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.

The well-known antiparasitic compound licochalcone A is a potent membrane-active agent that transforms normal erythrocytes into echinocytes in parallel with the inhibition of growth of Plasmodium falciparum cultures, the in vitro antiplasmodial effect apparently being an indirect effect on the host cell. In vitro experiments with synchronous cultures demonstrate that inhibition of invasion is the principal mechanism of growth inhibition. The erythrocyte membrane-modifying effect was also transiently observed in vivo in mice after intravenous administration.

Four abietane-type terpenoids, including two known royleanones and two new, rearranged 20-norabietanes, were isolated from the roots of the Iranian medicinal plant Salvia hydrangea DC. ex Bentham (Lamiaceae), which is used as an anthelmintic and antileishmanial remedy. Their structures were established using COSY, NOESY, HSQC, and HMBC spectral data. The possible identity of one of the 20-norabietanes with demethylmulticauline, previously reported from a different Salvia species, is discussed. A moderate in vitro antiplasmodial effect of the extract of S. hydrangea flowers was found to be associated with the presence of large amounts of pentacyclic triterpenes, mainly oleanolic acid. The observed antiplasmodial activity of oleanolic acid is apparently due to its incorporation into the erythrocyte membrane, which adversely affects the growth of Plasmodium falciparum parasites. Thus, oleanolic acid caused transformation of erythrocytes into stomatocytes in the concentration range where the in vitro antiplasmodial activity was observed.

Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC 50 ) of 27.7 ± 0.5 μM, 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.