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.

No abstract

Despite recent demonstration of the power of HPLC-PDA-MS-SPE-NMR (high-performance liquid chromatography-photodiode-array detection-mass spectrometry-solid-phase extraction-nuclear magnetic resonance) in structure determination of natural products directly from minute amounts of crude extracts, this technique leaves chirality of the compounds uncharacterized. In this work we demonstrate that postcolumn SPE is a useful method of analyte concentration and accumulation not only for NMR but also for CD (circular dichroism) spectroscopy. Thus, use of HPLC-PDA-MS-SPE-NMR in combination with CD allowed rapid detection of ( R)-(-)-gossypol [( R)- 1] in Thespesia danis, providing a very rare example of the predominance of the levorotatory enantiomer of gossypol. Enantioselectivity of the in vitro antiplasmodial activity of gossypol was also demonstrated; the IC50 value of ( R)- 1 was 4.5 +/- 0.2 microM, with the eudismic ratio of about 2.5. No gossypol was detected in Gossypioides kirkii.

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.Licochalcone A (Fig. 1) was originally isolated (39, 44) as a constituent of roots and rhizomes of various species of Glycyrrhiza L. (licorice root), and its structure was confirmed by synthesis (20,22,44). Subsequent studies resulted in reports of a variety of biological effects of licochalcone A, notably antibacterial (13-15, 17, 18, 36, 43), antileishmanial (3, 4, 8, 34, 45, 46), and antiplasmodial (6, 7, 23) ones. Thus, the compound inhibited growth of Plasmodium falciparum strain 3D7 parasites in vitro with 50% inhibitory concentrations (IC 50 s) of 5.6 Ϯ 0.6 M (35) and reduced parasitemia in mice infected with Plasmodium yoelii (7,23). Compounds related to licochalcone A exhibited comparable antiplasmodial activity in vitro (5, 35).The licochalcone A used in the present work was synthesized as previously described (22,33,44). The purity of the compound was assessed by high-performance liquid chromatography (C 18 column, acetonitrile gradient in water) and 600-MHz 1 H nuclear magnetic resonance and was higher than 99.9%. Determination of the in vitro IC 50 (Fig. 2) for P. falciparum strain 3D7 (initial parasitemia, 1.5%) was performed as previously described (49) using 12 concentrations (each used in triplicate) in the range from 0.098 to 25.0 g/ml (0.29 to 73.9 M); an average IC 50 calculated from three independent determinations was 2.10 Ϯ 0.56 g/ml (6.21 Ϯ 1.65 M), which was practically identical to the reported value (35). Microscopic examination of the cultures showed the presence of dead merozoites and ruptured schizonts at concentrations of 1.56 g/ml and above, and no internal parasites beyond the ring stage were observed.However, simultaneously with the growth inhibition, the exposure of the parasite cultures to licochalcone A caused pronounced membrane perturbations of the erythrocytes in which the parasites grow. The effect of licochalcone A on erythrocytes was the same regardless of whether parasitized or nonparasitized erythrocytes were used and was observed in the same concentration range as that of the in vitro antiplasmodial activity. In order to assess these membrane effects systematically, nonparasitized erythrocytes were incubated with licochalcone A at five different concentrations (0.098, 1.56, 3.13, 5.0, and 25.0 g/ml) for 48 h as in the growth inhibition assay. The deformations of the cell shape were investigated by light microscopy after fixation to glass slides with methanol followed by staining with Giemsa as well as by the hangi...

Dehydroabietinol isolated from Hyptis suaveolens (L.) Poit. was found to inhibit growth of chloroquine-sensitive as well as chloroquine-resistant strains of Plasmodium falciparum cultivated in erythrocytes in vitro (IC 50 26-27 microM). However, erythrocytes exposed to dehydroabietinol were transformed in a dose-dependent manner towards spherostomatocytic forms with concomitant formation of endovesicles, as disclosed by transmission electron microscopy. The erythrocyte shape alterations caused by dehydroabietinol correlated well with its apparent IC 50 value. Thus, dehydroabietinol incorporates into the erythrocyte membrane, and since invasion and survival of Plasmodium parasites is known to depend on the function of the erythrocyte membrane, the observed antiplasmodial effect of dehydroabietinol is presumably an indirect effect on the host cell. Because of these findings, microscopic investigations should be generally used to support claims of antimalarial effects of apolar natural products.