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In Vitro Antimalarial Activity and Chioroquine Potentiating Action of Two Bisbenzylisoquinoline Enantiomer Alkaloids Isolated from Strychnopsis thouarsii and Spirospermum pendul(florum S. Ratsimamanga-Urverg"2, P. Rasoanaivo1, L. Ramiaramanana2, R. Milijaona', H. Rafatro', F. Verdier3, A. Rakoto-Ratsimamanga , and J. Le Bras4'5 1 2

'I

Institut Malgache de Recherches Appliquees, B. P. 3833, Antananarivo, Madagascar Ecole pratique des Hautes Etudes, Laboratoire de Nutrition tropicale. Institut de Médecine et d'Epidemiologie Africaines et Tropicales (IMEAT), F-75944, Paris, France INSERM U13/IMEAT, F-75944, Paris, France Centre National de Référence Chimiosensibilité du Paludisme (IMEAT) and Laboratoire de Parasitologie, Université R. Descartes, F-75270 Paris, France Address for correspondence

Abstract The bisbenzylisoquinolines 7-O-demethyltetrandrine and limacine, respectively, isolated from

Strychnopsis thouarsii Bail, and Spirosperrnum pen-

duliflorum Thou. were evaluated for their intrinsic antimalarial activity in vitro and chloroquine potentiating action against the chloroquine-resistant Plasmodium falciparum FCM 29 originating from Cameroon. They both showed significant antiplasmodial potency in vitro with very similar IC50 values of respectively, 740 nM and 789 nM (IC50 = 214 nM for chloroquine used as standard

because of its onset of action, good tolerability, and low cost (2). However, a menacing problem in the chemotherapy of malaria occurs since 1960 with the spread of chloroquine resistance of Plasmodiumfalciparum. At present, despite the development of a few effective natural (artemisinine) and synthetic (mefloquine, halofantrine) antimalarial compounds, malaria still remains a major endemic disease as a result of the increasing development of multi-

drug resistance (3) ofPlasmodiumfalciparum.

Since 1987, a potentiating drug combination has constituted a new trend in malaria chemotherapy (4). As examples, the phenylalkylamine verapamil as well

drug), which demonstrated that the stereochemistry of the C-i and C-i' configuration likely plays a role in the chioroquine potentiating effect of these drugs. If confirmed in vivo, these results may account for the traditional use of the two plants as antimalarials and adju-

as cyproheptadine and several synthetic tricydic antidepressant drugs have been shown to reverse the chloroquine resistance of Plasmodium falciparum strains

vant to chloroquine in Madagascan folklore remedies.

ces (8). In our continuing research program on natural antimalarials, we investigated two medicinal plants used

Key words

Strychnopsis thouarsii. Spirospermum penduliflorum, Menispermaceae, 7-O-demethyltetrandrine, limacine, in vitro tests, Plasinodiumfalciparuin.

(5— 7). However, little

data have been collected on the chloroquine potentiating action of drugs from vegetable sour-

as an adjuvant to chloroquine in the Madagascan traditional herbal remedies: Strychnopsis thouarsii Baill. and Spirospermum penduliflorum Thou., both Menispermaceae endemic to Madagascar. We report here the in vitro intrin-

sic antimalarial effect and the chloroquine potentiating

Introduction Each

'CH3

year, despite the great efforts en-

couraged by WHO to control malaria (1), million peoples

from the tropical regions are infected by Plasmodium falciparwn malaria that sometimes leads to death. Alter the isolation of the bioactive components ofthe "fever tree" Cinchona, great effort was made to synthesise alternative

compounds with antimalarial action. Among these, chloroquine has been the most effective and widely used drug

limacine: R H, 1R, 1'R

7-O-demethyltetrandrine: R H, iS, 1'S tetrandrine: R CH3, 15, 1'S pheenanthine: R CH3 1R i'R Fig. 1 Chemical structures of BBI alkaloids.

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Received: November 17, 1991

In Vitro AntirnalarialActicity and C/sloroquine Potent/at/ny Action of Two Bisbenzylisoquinolines

ne and limacine (Fig. 1), respectively isolated from S. thouarsii and S. penduliflorum.

tions of the molecules were tested ranging from 8 to 0.062 pg/mI with each concentration in triplicate. For drug interaction studies, eight serial dilutions of chloroquine (12.5 to 3.200nM) with each concentration in trIplicate were tested with 4 fixed subinhibitory concentrations of each molecule.

Materials and Methods Plant ,naterials

The semi-micro in citro drug sensitivity test (11) was carried out in 24 flat bottomed well plates. 700 p1 of inoculum,

Plant materials known under the vernacular

parasitemia 0.5—1.0%), RPMI 1640 medium (Gibco), 10% pooled human serum, 25 nM of HEPES (Gibco), and 25 nM of NaHCO5, were distributed in each well. The plates were incubated with test compounds for the total of 42 hours. 0.5 pCi/well of lG-3H]-hypo-

names "amborasahamaintso" and "amborasaha-lavaravioa" were

collected in the eastern rain forest of Madagascar, Ampanotoamaizina region, in August 1989 and identified respectively as Strychnopsis thoearsii Baill. and Spirospermem pendul(florum Thou, by comparison with authentic specimens deposited at the Department of Botany, Parc Botanique et Zoologique de Tsimbazaza. Voucher specimens were kept as reference (RR 088909 and RR 088910) at the Institut Malgache de Recherches Appliquées.

Extraction 800 g of air dried and powdered plant material (leaves for Strychno ps/s thoaarsii and stem roots for Spirospermum penduliflorum) were exhaustively extracted by repeated maceration with 95 % ethanol to afford a crude extract which was

partitioned between chloroform and 2% aqueous hydrochloric acid. The organic layer was washed with distilled water, dried over anhydrous sodium sulfate, and evaporated to dryness under reduced pressure. The acidic aqueous phase was made alkaline

consisting of infected erythrocytes (haematocrit 2.5%, starting

xanthine (Amersham, France) was added 18 hours after incubation to assess parasite growth. The contents in each well were harvested onto glass filter discs. Discs were washed, dried, and placed into scintillation tubes containing 1.5 ml of xylene-based organic counting scintillant (Amersham). Radioactivity was measured on an Intertechnique 5L30 liquid scintillation spectrometer. 50% in-

hibitory concentration (IC50) and 90% inhibitory concentration (IC90) were calculated from the plot of the probit of chloroquine ac-

tivity and logarithm of drug concentration by linear regression analysis. Activity enhancement index (AEI) was calculated by dividing the IC90 for chloroquine alone by the IC95 for chloroquine plus molecules. In the study "potentiation" of chloroquine was defined as AEI equal to or greater than 1.5. "Complete reversal" of chloroquine resistance was defined as diminution of the IC55 values for chloroquine to less than or equal to 70 nM (similar to the IC55 va-

lues in chloroquine-sensitive parasites), and "partial reversal" of

with sodium bicarbonate and extracted three times with chloroform. The combined chloroform extracts were washed with water, dried over anhydrous sodium sulfate, and then filtered. Removal of chloroform phase under reduced pressure provided the crude tertiary alkaloids. The exhausted aqueous phase was then acidified with 2% hydrochloric acid to pH 2. A solution of

chloroquine resistance as diminution of the IC55 values between 70 and 120 nM. The cut-off point for in citro chloroquine resistance using the semi-microtest is 90—120 nM.

picric acid was added to the acidic solution in order to precipitate the quaternary salts as picrates.

molecules of BBIQ (12). ChloroquIne fractional IC55 was calculated by dividing the IC55 of chloroquine combined with the molecules by

lsobolograms were constructed by plotting a

pair of fractional IC55 for each combination of chloroquine and the IC55 of chloroquine alone and plotted on the horizontal axis.

Separation Counter current distribution (CCD) of the crude tertiary alkaloids of Strychno ps/s thouars/i leaves was performed

in a Craig apparatus using chloroform as stationary phase and phosphate/citric acid buffer at discontinuously decreasing p1-1 ranging from 7 to 3 as mobile phase. 200 transfers were accomplished for each buffer. Collection of fractions was monitored by TLC on silica gel using butanol-acetic acid-water (4/1/5; upper phase) and chloroform-methanol (95/5) as solvent systems. 7-0Demethyltetrandrine crystallized from acetone/hexane: m.p. 232°C; [a] + 221° (chloroform, c 0.1).

Tertiary alkaloid components from Spirospermum pendu4florum roots were submitted to silica gel column chromatography using chloroform and increasing amounts of methanol as eluent. Collection of fractions was monitored by TLC on silica gel using chloroform-methanol (96/4) as solvent system.

Limacine crystalhzed from in methanol/ether: m.p. 156°C; [a] —216° (chloroform, c 0.1).

In vitro antimalarial test protocols A chloroquine resistant strain of Plasmodium falciparum FCM 29 which was originally obtained from Cameroon

and cloned by the dilution method was used throughout. The parasites were maintained in continuous culture using the method described by Trager and Jensen (10), in a candle jar atmosphere at 37°C. Stock solutions of chloroquine sulfate (Specia), 7-0-domethyltetrandrine and limacine were prepared and subsequently diluted in sterile distilled water after dissolution in methanol (the concentration of methanol in the test never exceeded 0.1 %). For the determination of intrinsic antimalarial activity, eight 2-fold dilu-

The corresponding BBIQ fractional IC55 was calculated by dividing its fixed concentration by the IC55 of molecules alone and plotted on the vertical axis. A curve was then traced through the resulting

pairs of fractions from the ends of both axises on the graph. "Synergism", "additivity", and "antagonism", classical terms used to interpret isobolograms, were defined solely on the basis of the form of the curve: concave in synergism, convex in antagonism, and following the diagonal in additivity.

Results

_____________________

From Strychnopsis thouarsii, four aporphine alkaloids namely isocorydine, predicentrine, hriotulipiferine, N-methyllindcarpine, the morphinan alkaloid sinoacutine, and the BBIQ alkaloid 7-0-demethyltetrandrine were isolated (13). As for Spirospermum penduliforum the known alkaloid limacine was identified as the only tertiary alkaloid isolated from stem roots (Fig. 1). No

traces of qiiatenary salts were identified both in S. thonarsii leaves and S. pendulUlorum roots. Chemical structures were established largely by conventional spectral methods assist-

ed by the performance of the 'H-1H and 1H-'5C heteronuclear chemical shift correlated NMR experiments and by comparison of melting points and optical rotations with those published in the literature (14), because no authentic samples were available for direct comparison. With regard to the antimalarial results, IC55 and IC95 values obtained for chloroquine, 7-0-demethyltetrandrine, limacirie as weli as the combinations chloro-

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action in a resistant strain of Plasmodiumfalciparum of the two bisbenzylisoquinolines (BBIQ) 7-O-demethyltetrandri-

Planta Mcd. 58(1992) 541

542 Planta Med. 58(1992)

S. Ratsiinamanga-Urverg et al.

effective in reducing the IC90 of chloroquine than limacine. Isobolograms (Fig. 2) constructed from the data presented in Table 1 showed a concave shape of the curves, illustrating a synergistic drug action between chloroquine and 7O-demethyltetrandrine as well as chioroquine and limacine. However, it appeared that limacine produced a signfficantly slighter synergistic effect than 7-O-demethyltetrandrine

(P