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Antitrypanosomal Cycloartane Glycosides from Astragalus baibutensis
:
by Ihsan alıs¸* a ), Semra Koyunog˘lu b ), Akg l Yes¸ilada b ), Reto Brun c ), Peter R edi d ), and Deniz Tas¸demir e ) a
) Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara (phone: þ 90 312 3051089; fax: þ 90 312 311 4777; e-mail:
[email protected]) b ) Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara c ) Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel d ) Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Z:rich e ) Centre for Pharmacy and Phytotherapy, School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, U.K.
Baibutoside (5), a new cycloartane-type triterpene glycoside, has been isolated from the roots of Astragalus baibutensis along with four known glycosides, acetylastragaloside I (1), and astragalosides I, II, and IV (2 – 4, resp.). The structure elucidation of the compounds were achieved by a combination of one- and two-dimensional NMR techniques (DQF-COSY, HSQC, HMBC, and ROESY), and mass spectrometry (ESI-MS), where all the compounds were shown to have cycloastragenol ( ¼ (20R,24S)3b,6a,16b,25-tetrahydroxy-20,24-epoxy-9,19-cyclolanostane) as aglycone. All compounds were tested for in vitro antiprotozoal activity. Compounds 1 – 4 displayed notable activity vs. Trypanosoma brucei rhodesiense, with acetylastragaloside I (1) being the most potent (IC50 9.5 mg/ml). Acetylastragaloside I (1) was also lethal to T. cruzi (IC50 5.0 mg/ml), and it is the first cycloartane-type triterpene with remarkable trypanocidal activity against both T. brucei rhodesiense and T. cruzi. However, it exhibits some cytotoxicity on mammalian cells.
1. Introduction. – In the course of our structural studies on metabolites from Astragalus species growing in Turkey, a number of cycloartane-type triterpene glycosides were isolated, which possess diverse biological activities. Some cycloartane glycosides have been shown to have antitumor and AIDS antiviral activity [1]. The immunostimulant effects of several cycloartane-type triterpene glycosides on macrophage activation and expression of inflammatory cytokines were investigated [2]. Some other cycloartane glycosides obtained from Astragalus species act as modulators of lymphocyte proliferation [3 – 5]. Recently, we reported the effects of cycloartane glycosides on in vitro cytokine release [6] and the first antiprotozoal cycloartane glycosides from A. oleifolius [7]. As part of our continuing search for structurally and pharmacologically interesting cycloartane glycosides from the genus Astragalus, we now investigated the roots of an endemic species, A. baibutensis. Herein, we report the isolation, structure elucidation, and antiparasitic activity of a new cycloartane-type glycoside, baibutoside (5), and four known glycosides acetylastragaloside I (1), and astragalosides I, II, and IV (2 – 4, resp.). J 2006 Verlag Helvetica Chimica Acta AG, Z:rich
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2. Results and Discussion. – The crude extract was fractionated by VLC. The fraction rich in saponins was subjected to silica-gel open column chromatography with CH2Cl/MeOH/H2O mixtures as eluents to yield nine fractions. Further chromatographic separations of these fractions afforded a new cycloartane-type glycoside named, baibutoside (5), in addition to four known glycosides, acetylastragaloside I (1), and astragalosides I, II, and IV (2 – 4, resp.). All glycosides were cycloastragenol ( ¼ (20R,24S)-3b,6a,16b,25-tetrahydroxy-20,24-epoxy-9,19-cyclolanostane) derivatives [8]. Baibutoside (5) is a rare example of apiose containing cycloartane glycosides. The molecular formula of compound 5 was determined as C46H76O18 by electrospray-ionization mass spectrometry (ESI-MS), which exhibited positive and negative ion peaks for [M þ Na] þ and [M Na] at m/z 939.6 and 915.5, respectively. The NMR data of 5 (Table 1) were consistent for the presence of a cycloartane-type triglycosidic structure. All NMR assignments (Table 1) were based on DQF-COSY, HSQC, HMBC, and ROESY experiments. Taking into account the result of our comprehensive 1H- and 13 C-NMR studies, and previous knowledge derived from metabolites isolated from the genus Astragalus, the main features of a cycloartane-type triterpene were evident from the characteristic signals due to cyclopropane-ring CH2 H-atoms as AX system (d(H) 0.18 and 0.58 ppm, JAX ¼ 4.3 Hz, CH2(19)). Additionally, seven tertiary Me resonances were observed at d(H) 0.97, 1.30, 1.31, 1.41, 1.44, 1.59, and 1.99 ppm (each 3 H, s, Me(30), Me(27), Me(21), Me(18), Me(29), Me(26), and Me(28), resp.). The 13 C-NMR spectrum exhibited signals for 46 C-atoms, 30 of which were assigned to the aglycone moiety, while the remaining were due to one hexose and two pentose units. The NMR data attributed to the sapogenol moiety were in good agreement with those of cycloastragenol [8]. However, the 13C resonances attributed to C(3) (d(C) 88.5) and C(6) (d(C) 79.3) were found to be shifted downfield as þ 8 – 9 ppm, in comparison to those of cycloastragenol. This suggested C(3) and C(6) to be the sites of glycosidations on the sapogenol moiety, thus confirming the bidesmosidic structure of 5. The signals for three anomeric H-atoms at d(H) 4.82 (d, J ¼ 7.7 Hz, HC(1’)), 4.89 (d, J ¼ 7.8 Hz, HC(1’’)), and 6.53 (d, J ¼ 1.7 Hz, HC(1’’’)) indicated the presence of a triglycosidic structure. The corresponding anomeric 13C resonances were observed at
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Table 1.
13
C- and 1H-NMR Data for Baibutoside (5), and Significant HMBC Correlations (( D5 )pyridine, 13 C: 150 MHz, 1H: 600 MHz; d in ppm, J in Hz) d(C )
Aglycone: CH2(1) CH2(2) HC(3) C(4) HC(5) HC(6) CH2(7) HC(8) C(9) C(10) CH2(11) CH2(12) C(13) C(14) CH2(15) HC(16) HC(17) Me(18) CH2(19) C(20) Me(21) CH2(22) CH2(23) HC(24) C(25) Me(26) Me(27) Me(28) Me(29) Me(30) Xylose: HC(1’) HC(2’) HC(3’) HC(4’) CH2(5’) Apiose: HC(1’’) HC(2’’) C(3’’) CH2(4’’) CH2(5’’) Glucose: HC(1’’’) HC(2’’’) HC(3’’’) HC(4’’’) HC(5’’’) CH2(6’’’) a
925
32.2 (t) 30.2 (t) 88.5 (d) 42.7 (s) 52.6 (d) 79.3 (d) 34.5 (t) 45.4 (d) 21.1 (s) 28.9 (s) 26.2 (t) 33.4 (t) 46.3 (s) 45.1 (s) 46.1 (t) 73.4 (d) 58.2 (d) 21.0 (q) 28.5 (t) 87.3 (s) 28.6 (q) 34.9 (t) 26.5 (t) 81.7 (d) 71.3 (s) 28.2 (q) 27.1 (q) 28.3 (q) 16.6 (q) 19.8 (q)
d( H )
HMBC ( 13C ! 1H )
1.56 (m), 1.22 (m) 2.33 (m), 1.97 (m) 3.45 (dd, J ¼ 11.7, 4.4) – 1.92 (d, J ¼ 6.9) 3.78 (ddd, J ¼ 8.3, 8.3, 4.1) 2.27 (m), 1.87 (m) 1.96 (dd, J ¼ 10.0, 4.6) – – 1.80 (m), 1.29 (m) 1.62 (m), 1.54 (m) – – 2.36 (dd, J ¼ 13.8, 8.0), 1.85 a ) 4.97 a ) 2.53 (d, J ¼ 7.8) 1.41 (s) 0.58 (d, J ¼ 4.3), 0.18 (d, J ¼ 4.3) – 1.31 (s) 3.13 (m), 1.67 (m) 2.32 (m), 2.04 (m) 3.88 (dd, J ¼ 9.0, 5.3) – 1.59 (s) 1.30 (s) 1.99 (s) 1.44 (s) 0.97 (s)
CH2(19) HC(1’), Me(28), Me(29) Me(28), Me(29) Me(29) HC(1’’’), HC(5) Me(29) Me(29) Me(29)
Me(18), Me(30) Me(18), Me(30) Me(30) Me(18) HC(17) HC(17), Me(21), CH2(22)
Me(26), Me(27) Me(26), Me(27) Me(27) Me(26) HC(3), HC(5), Me(29) HC(3), HC(5), Me(28)
106.2 (d) 79.1 (d) 78.5 (d) 71.3 (d) 66.8 (t)
4.82 (d, J ¼ 7.7) 4.15 a ) 4.13 a ) 4.18 a ) 4.28 (dd, J ¼ 11.0, 4.6), 3.60 (dd, J ¼ 11.0, 9.8)
HC(3), HC(3’), CH2(5’) HC(1’’) CH2(5’) CH2(5’)
111.2 (d) 78.3 (d) 80.7 (s) 75.8 (t) 66.2 (t)
6.53 (d, J ¼ 1.7) 4.91 (d, J ¼ 1.7) – 4.81 (d, J ¼ 9.3), 4.40 (d, J ¼ 9.3) 4.30 (br. s, 2 H )
HC(2’), CH2(5’’) CH2(5’’) HC(1’’), CH2(5’’), HC(2’’) HC(1’’), HC(2’’) CH2(4’’)
105.3 (d) 75.7 (d) 79.1 (d) 71.9 (d) 78.1 (d) 63.2 (t)
4.89 (d, J ¼ 7.8) 4.03 (dd, J ¼ 7.8, 8.2) 4.19 a ) 4.12 a ) 3.89 a ) 4.48 (br. d, J ¼ 12.0), 4.31 a )
HC(6) HC(3’’’) HC(5’’’) HC(3’’’) HC(1’’’)
) Signal pattern unclear due to overlapping.
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d(C) 106.2, 105.3, and 111.2, respectively. The chemical shifts and coupling constants of the signals assigned to the sugar moieties revealed the presence of a xylose, a glucose, and an apiose unit. The relatively large J values of the anomeric H-atoms indicated borientations for xylose and glucose units. The configuration of the apiose moiety was established as b-d from the difference ( 224.78) between the molecular rotation of 5 ( 648) and that of 4 ( þ 160.78) (astragaloside IV (4): [a] 20 D ¼ þ 20.5 (c ¼ 0.2, MeOH)) [9]. The 13C-NMR resonances assigned to the sugar units showed that apiose and glucose units were terminal sugars. The third glycosidation site was evident from the signal shifted downfield at d 79.1 which was assigned to C(2’) of the xylose unit. These results showed that one of the two sugar moieties of the bidesmosidic structure is diglycosidic. The position of each sugar residue was unambigously determined by the HMBC experiment which showed long-range correlations between C(3) (d 88.5) of cycloastragenol and HC(1’) (d 4.45) of the xylose unit, C(2’) (d 79.1) of xylose and HC(1’’) (d 6.53) of the apiose unit, and C(6) (d 79.3) of cycloastragenol and HC(1’’’) (d 3.78) of the glucose unit. All these observations were supported by a ROESY experiment which showed correlations between HC(1’) of xylose (d(H) 4.82) and HC(3) of the aglycone (d(H) 3.45), HC(’’) of apiose (d(H) 4.91) and HC(2’) of xylose (d(H) 4.15), and finally, HC(1’’’) of glucose (d(H) 4.89) and HC(6) of the aglycone (d(H) 3.78). Further ROE correlations between HC(3)/HC(5), H(3)/ Me(28), HC(6)/HC(8), HC(6)/CH2(19), HC(8)/Me(18), HC(16)/Me(30), HC(17)/Me(30), HC(17)/Me(21) supported the presence of cycloastragenol as aglycone moiety. Thus, compound 5 was determined as (20R,24S)-3-O-[b-d-apiofuranosyl-(1 ! 2)-b-d-xylopyranosyl]-6-O-b-d-glucopyranosyl-3b,6a,16b,25-tetrahydroxy20,24-epoxycycloartane, and named baibutoside. Acetylastragaloside I (1), and astragalosides I, II, and IV (2 – 4, resp.) were also isolated from the aerial parts of A. trojanus and identified on the basis of their HR-ESIMS and NMR (1H and 13C) data, in comparison with literatures values [8] [10]. The antiprotozoal activities of the compounds 1 – 5 were assessed on a panel of parasites, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum. All compounds were also tested on primary L6 mammalian cells (rat skeletal myoblasts), in order to determine their selective cytotoxicity. As shown in Table 2, all compounds were devoid of activity against L. Table 2. In vitro Antiprotozoal Activity Results for Compounds 1 – 5. IC50 Values in mg/ml. Selectivity indices of the active compounds 1 – 4 were calculated ( IC50 value for L6 cell cytotoxicity/IC50 value for antiparasitic activity) and shown in parentheses. Compound
T. brucei rhodesiense
T. cruzi
L. donovani
P. falciparum
L6 Cell cytotoxicity
Standard (ref. compound) Acetylastragaloside I (1) Astragaloside I (2) Astragaloside II (3) Astragaloside IV (4) Baibutoside (5)
0.00295 a ) 9.5 (2.5) 57.6 ( > 1.6) 44.8 ( > 2.0) 46.9 ( > 1.9) > 90
0.31 b ) 5.0 (4.8) > 30 > 30 > 30 > 30
0.152 c ) > 30 > 30 > 30 > 30 > 30
0.002 d ) > 20 > 20 > 20 > 20 > 20
0.00186 e ) 24.2 > 90 > 90 > 90 > 90
a
) Melarsoprol. b ) Benznidazole. c ) Miltefosine. d ) Artemisinin. e ) Phodophyllotoxin.
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donovani and P. falciparum. Except for 5, all pure metabolites displayed some growthinhibitory effect on Trypanosoma brucei rhodesiense, with acetylastragaloside I (1) being the most active one (IC50 9.5 mg/ml). Compound 1 also revealed promising activity against T. cruzi, with an IC50 value of 5.0 mg/ml. On the other hand, acetylastragaloside I (1) possessed cytotoxic effect on mammalian cells, leading to narrow selectivity index values of 2.5 and 4.8. Very recently, we reported cyclocanthogenin-type glycosides from Astragalus oleifolius with remarkable leishmanicidal (L. donovani) and weak trypanocidal (T. brucei rhodesiense) activities [7]. To our knowledge, these were the very first cycloartane-type glycosides with antiprotozoal activity. Here, we also reported the first cycloartane glycoside (aglycone cycloastragenol) that inhibits both Trypanosoma cruzi and T. brucei rhodesiense in vitro. It is noteworthy that the dual activity was obtained only with the most apolar glycoside, acetylastragaloside I (1), bearing three Ac groups, one of which is on the the xylopyranosyl moiety. Although 1 appears as a promising trypanocidal agent, its cytotoxic potential on mammalian cells already presents an obstacle for the development of this compound as a lead compound. Experimental Part General. Column chromatography (CC) was carried out over silica gel (70 – 230 mesh, Merck). MPLC separations were performed on a Labomatic glass column (2.6 46 cm, i.d.), packed with LiChroprep RP-18, using a B0chi 681 chromatography pump. TLC was carried out on precoated silica gel 60 F 254 (Merck) with CH2Cl2/MeOH/H2O 61 : 32 : 7 and 80 : 20 : 2 mixtures. Compounds were visualized by spraying with 1% vanillin in conc. H2SO4 , followed by heating at 1058 for 1 – 2 min. Optical rotations were recorded with a Rudolph Autopol IV polarimeter with MeOH as solvent at 208. The 1D- and 2DNMR spectra were obtained on a Bruker Avance DRX-600 FT spectrometer operating at 600 (1H) and 150 (13C) MHz. The chemical-shift values are reported in ppm relative to TMS, and the coupling constants are in Hz. For the 13C-NMR spectra, multiplicities were determined by a distortionless enhancement by polarization transfer (DEPT) experiment. 1H,1H-DQF-COSY (double-filtered direct chemical-shift correlation spectroscopy). Inverse-detected 1H,13C-HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond connectivity), and ROESY were obtained by employing the conventional pulse sequences as described previously. ESI Mass spectra were recorded on a Bruker Esquire-LC-MS (ESI mode) spectrometer. Plant Material. Astragalus baibutensis Bunge was collected from Ahlatlıbel, Ankara, Central Anatolia, Turkey, in May, 1994. A voucher specimen (94102) has been deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey. Extraction and Isolation of the Compounds. The air-dried powdered roots (250 g) were extracted with MeOH under reflux. The solvent was removed by rotary evaporation to yield 20.1 g of extract (yield 8%). The MeOH extract (20.1) was fractionated by open CC (silica gel (150 g); CH2Cl2/MeOH/H2O 90 : 10 : 0.5 (300 ml), 90 : 10 : 1 (300 ml), 85 : 15 : 1.5 (300 ml), and 80 : 20 : 2 (600 ml)) to give 84 fractions (30 ml/fraction), which were combined to nine main groups (A – I) based on their TLC profiles: A (Frs. 32 – 39; 131 mg); B (Frs. 40 – 42; 88 mg); C (Frs. 43 – 47; 407 mg); D (Frs. 48 – 52; 504 mg); E (Frs. 53 – 59; 657 mg); F (Frs. 60 – 69; 1130 mg); G (Frs. 70 – 77; 1000 mg); H (Frs. 78 and 79; 3070 mg); I (Frs. 80 – 84; 1000 mg). Fr. C (407 mg) was first subjected to normal-phase silica-gel (50 g) CC with CH2Cl2/MeOH/H2O mixtures with increasing polarity (90 : 10 : 1, 85 : 15 : 1.5, and 80 : 20 : 2; each 200 ml) yielding three main fractions (C1: 30, C2: 121, and C3: 220 mg). Frs. C2 and C3 were separately applied to normal-phase silica gel CC (each 20 g) with CH2Cl2/MeOH mixtures with increasing polarity (98 : 2, 96 : 4, 94 : 6, 92.8 and 90 : 10; each 100 ml) to yield compound 1 (23 and 7 mg, resp.). Fr. D (504 mg) was first subjected to a normal-phase silica-gel (50 g) CC with CH2Cl2/MeOH/H2O mixtures with increasing polarity (80 : 20 : 1 and 80 : 20 : 2) to yield three main Frs. (D1: 83, D2: 263, and D3: 114 mg). Fr. D2 was
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further subjected to normal-phase silica gel (50 g) CC with CH2Cl2/MeOH mixtures (90 : 1, 85 : 15, and 80 : 20) to yield compound 2 (60 mg). Fr. F (1130 mg) was first subjected to reversed-phase MPLC (silica gel LiChrosorb C-18; column dimensions: 26 460 mm; stepwise MeCN/H2O gradient (40% MeCN, 500 ml, 20 ml/fraction)) to give 52 fractions. Frs. 23 – 27 (260 mg) were subjected to a normal-phase silicagel (50 g) CC with CHCl3/MeOH/H2O mixtures (90 : 10 : 1 (300 ml), 87.5 : 12.5 : 2 (200 ml), and 85 : 15.1 (200 ml)) to yield compound 3 (47 mg). An aliquot of Fr. H (830 mg) was subjected to MPLC on reversed-phase silica gel (LiChrosorb C-18; column dimensions: 3 24 mm) with stepwise MeOH/H2O gradient (70% MeOH (200 ml), 75% (200 ml), 80% (200 ml), 85% (200 ml), 20 ml/fraction) to yield the compound 4 (484 mg). Fr. I (1000 mg) was subjected to a silica-gel (100 g) CC with CH2Cl2/MeOH/H2O gradient (80 : 20 : 2 (400 ml), 75 : 25 : 2.5 (200 ml), and 70 : 30 : 3 (200 ml), 10 ml/fraction) to give compound 4 (133 mg), and a mixture of compounds 4 and 5 (407 mg). This mixture was further subjected to MPLC (column dimensions: 26 460 mm), with LiChroprep C-18 as stationary phase and MeOH/H2O mixtures (70 – 80% MeOH in H2O, (500 ml); 20 ml/fraction) to afford compound 5 (16 mg), and a mixture of 5 and 4 (54 mg). This mixture was further subjected to a silica-gel (25 g) CC with CH2Cl2/MeOH/H2O gradient (80 : 20 : 2, 75 : 25 : 2.5, and 70 : 30 : 3; each 200 ml; 12 ml/fraction) to give compound 4 (8 mg) and 5 (28 mg), resp. Baibutoside (5). Amorphous white powder. [a] 20 D ¼ 7.0 (c ¼ 0.1 MeOH); IR (KBr): 3400 (OH), 2933 (CH), 1166, 1077, 1042. 1H-NMR (500 MHz, (D5 )pyridine) and 13C-NMR (125 MHz, (D5 )pyridine): see Table 1. Positive- and negative-ion ESI-MS: 939.6 [M þ Na] þ , 915.5 [M Na] , resp. Biological Assays. Trypanosoma brucei rhodesiense and Cytotoxicity Assays. Minimum essential medium (50 ml) supplemented according to [11] with 2-sulfanylethanol and 15% heat-inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions were prepared covering a range from 90 to 0.123 mg/ml. Then, 104 bloodstream forms of Trypanosoma brucei rhodesiense STIB 900 in 50 ml were added to each well, and the plate was incubated at 378 under a 5% CO2 atmosphere for 72 h. Alamar Blue (10 ml; 12.5 mg resazurin dissolved in 100 ml of dist. H2O) was then added to each well, and incubation continued for a further 2 – 4 h. The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices Corporation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm [12]. Fluorescence development was expressed as percentage of the control, and IC50 values were determined. Cytotoxicity was assessed using the same assay and rat skeletal myoblasts (L6 cells). Trypanosoma cruzi. Rat skeletal myoblasts (L6 cells) were seeded in 96-well microtiter plates at 2000 cells/well in 100 ml of RPMI 1640 medium with 10% FBS and 2 mm l-glutamine. After 24 h, the medium was removed and replaced by 100 ml per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the b-galactosidase (Lac Z) gene. After 48 h, the medium was removed from the wells and replaced by 100 ml of fresh medium with or without a serial drug dilution. Seven threefold dilutions were used covering a range from 90 mg/ml to 0.123 mg/ml. After 96 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then, the substrate CPRG/Nonidet (50 ml) was added to all wells. A color reaction developed within 2 – 6 h and could be read photometrically at 540 nm. Data were transferred into a graphic programme (e.g., EXCEL), sigmoidal inhibition curves were determined, and IC50 values were calculated. Leishmania donovani. Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 378 in SM medium [13] at pH 5.4, supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO2 in air. The culture medium (100 ml) with 105 amastigotes from axenic culture with or without a serial drug dilution was seeded in 96-well microtiter plates. Seven threefold dilutions were used covering a range from 30 mg/ml to 0.041 mg/ml. After 72 h of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Alamar Blue (10 ml; 12.5 mg resazurin dissolved in 100 ml of dist. H2O) was then added to each well, and the plates were incubated for another 2 h. Then, the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analyzed with the software Softmax Pro (Molecular Devices Corporation, Sunnyvale, CA, USA). Decrease of fluorescence ( ¼ inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations. From the sigmoidal inhibition curves, the IC50 values were calculated.
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Plasmodium falciparum. Antiplasmodial activity was determined using the NF 54 (chloroquinesensitive) strain of P. falciparum. A modification of the [ 3H]hypoxanthine incorporation assay was used [14]. Briefly, infected human red blood cells in RPMI 1640 medium with 5% Albumax were exposed to serial drug dilutions in microtiter plates. After 48 h of incubation at 378 in a reduced O2 atmosphere, 0.5 mCi [ 3H]hypoxanthine was added to each well. Cultures were incubated for a further 24 h before they were harvested onto glass-fiber filters and washed with dist. H2O. The radioactivity was counted with a BetaplateTM liquid scintillation counter (Wallac, CH-Zurich). The results were recorded as counts per min (CPM) per well at each drug concentration and expressed as percentage of the untreated controls. From the sigmoidal inhibition curves, the IC50 values were calculated. The authors thank Dr. Engelbert Zass (ETH-Zurich) for performing Chemical Abstract searches for the new compound, and Prof. Dr. Zeki AytaÅ (Gazi University) for the authentification of the plant specimen.
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