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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 (IC50 26 - 27 µM). 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 IC50 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.
Hyptis suaveolens (L.) Poit.
(Lamiaceae) is native of tropical
We found that the residue obtained by ethanol extraction of the aerial parts of H. suaveolens, evaporated in high vacuum in order to remove volatile constituents, inhibited growth of Plasmodium falciparum 3D7 strain (IC50 < 25 μg/ml). Bioactivity-guided fractionation of the extract afforded an abietane-type diterpene identified as dehydroabietinol (1) on the basis of 1H- and 13C-NMR spectra [8], [9] in conjunction with optical rotation data [10]. This is the first report on the presence of 1 in the genus Hyptis Jacq.
Dehydroabietinol (1) is a long-known product of reduction of dehydroabietic acid [10], which defines its stereochemistry at C-4, C-5 and C-10 [11]. The same compound was more recently considered new when isolated from Salvia pomifera and named pomiferin A [12]; usage of the later name should thus be discontinued [13].
The IC50 value determined for 1 with the chloroquine-sensitive P. falciparum 3D7 strain was 7.3 ± 0.3 μg/ml (25.6 ± 1.0 μM). Practically the same toxicity was observed with a chloroquine-resistant Dd2 strain (IC50 7.9 ± 0.8 μg/ml or 27.4 ± 2.7 μM) and with two human carcinoma cell lines (IC50 28.0 ± 0.7 μM and 30.2 ± 0.7 μM for KB-3-1 and KB-V1 cells, respectively).
Since malaria parasites are cultured inside erythrocytes and depend on the presence of nutrient uptake channels [14], their growth is likely to be affected by alterations of the erythrocyte membrane. We have recently demonstrated that amphiphiles causing transformation of erythrocytes into stomatocyte forms generally behave as apparent, moderately potent antiplasmodial agents in vitro [15]. Stomatocytes are produced when amphiphiles present at sublytic concentrations incorporate into the erythrocyte membrane and cause expansion of the inner membrane leaflet [16]. The changes of the erythrocyte membrane at concentrations around IC50 values could be observed in detail by electron microscopy [15]. Modifications of the erythrocyte membrane have previously been demonstrated to interfere with parasite invasion and growth [17], [18], [19]. Thus, before a compound can be accepted as a true antiplasmodial agent, it should be demonstrated that it does not cause erythrocyte membrane changes that correlate with the inhibition of parasite growth [15].
Effect of dehydroabietinol (1) on erythrocyte shape was therefore studied in the concentration range 0.78 - 400 μg/ml (2.7 μM - 1.4 mM) using light and transmission electron microscopy (Fig. [1]), which showed concentration-dependent changes of the erythrocyte shape. At 50 μg/ml and above, lysis of erythrocytes and formation of ghost cells was observed, the lysis being complete at 400 μg/ml. Stomatocytic changes were apparent at lower concentrations. At 1.56 - 3.13 μg/ml swollen discocytes and type 1 stomatocytes [20] were observed. At 6.25 - 12.5 μg/ml stomatocytes of type 1 dominated, but stomatocytes of type 2 were also apparent. The latter dominated in the concentration range 25 - 50 μg/ml. Even at the lowest concentration tested (ten-fold lower than IC50), the shape of erythrocytes was altered towards increased volume (Fig. [1]). Concurrently with the stomatocytosis, endovesiculation was observed, although the endovesicles were smaller and less in number than previously described for lupeol [15].
In conclusion, dehydroabietinol (1) alters the erythrocyte membrane causing formation of stomatocytes at concentrations which correlate well with the apparent IC50 value for the inhibition of P. falciparum growth. The observed, weak in vitro antiplasmodial activity of 1 is thus presumably an indirect effect on the host cell. The same may actually be the case with a number of other potentially membrane-active natural products reported to possess antimalarial activity [21], including fatty acids, saponins, octadecyl caffeate and possibly long-chain alkamides. Assessment of the antimalarial activity of especially apolar and amphiphilic natural products should therefore take into consideration their possible membrane effects at sublytic concentrations. Ideally, an antiplasmodial compound should not alter erythrocyte membrane shape at inhibitory concentrations, as shown for chloroquine [15]. Thus, we believe that microscopic studies should be an inherent part of antimalarial drug discovery programs in order to help to identify genuine antiplasmodial agents that affect the parasite biochemistry itself.
Fig. 1 Transmission electron micrographs showing effects of dehydroabietinol (1) on human erythrocyte shape. (A): Control (magnification 2 000 times). (B) and (C): Erythrocytes treated with 50 μg/ml of 1 (magnification 2 000 and 1 000 times, respectively); formation of spherostomatocytes and loss of cell color is apparent. (D): Erythrocytes treated with 1.56 μg/ml of 1 (magnification 4 000 times); formation of vesicles is apparent. (E): Erythrocytes treated with 0.78 μg/ml of 1 (magnification 1 000 times); the cells show perturbed shape.
Aerial parts of H. suaveolens
(L.) Poit.
were collected outside
In vitro growth inhibition assays with chloroquine-sensitive P. falciparum strain 3D7 (initial parasitemia 3 %) and chloroquine-resistant strain Dd2 (initial parasitemia 1.5 %) were carried out essentially as previously described [15], [22]. Inhibition curves were obtained with eight different concentrations of the test compound used in duplicate, and the reported IC50 values are averages of three independent determinations. Reference IC50 values obtained with chloroquine diphosphate were 42.5 ± 3.2 ng/ml (82.3 ± 6.2 nM) and 239 ± 15 ng/ml (463 ± 30 nM) for the 3D7 and Dd2 strain, respectively. For membrane shape studies, normal human erythrocytes were incubated for 48 h with 1 (0.78 - 400 μg/ml or 2.7 μM - 1.4 mM) and investigated by light microscopy and transmission electron microscopy similarly as previously described [15]. Assay for cytotoxicity with drug sensitive KB-3-1 cells and multidrug resistant KB-V1 cells were performed as detailed elsewhere [22], [23]; inhibition curves were constructed with six different concentrations, each in quadruplicate, and the reported IC50 values are averages of three independent determinations. Reference IC50 values for rhodamin 123 were 3.65 ± 0.86 μM and above 400 μM for KB-3-1 and KB-V1 cells, respectively. For chloroquine, the respective values were 76 ± 15 μM and 105 ± 5 μM.
Technical assistance of Ms. Dorte Brix (
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Prof. Jerzy W. Jaroszewski
Department of
Universitetsparken 2
DK-2100
Email: jj@dfh.dk
Fax: +45 3530 6040
