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Studies on medicinal plants of Ivory Coast: investigation of Sida acuta for in vitro antiplasmodial activities and identification of an active constituent.

Summary

Sida acuta Burm. (Malvaceae) originating from Ivory Coast was selected after an ethnobotanical survey: traditional healers of malaria commonly used this plant for the treatment. Extracts were tested on two strains of Plasmodium falciparum: FcM29-Cameroon (chloroquine-resistant strain) and a Nigerian chloroquine-sensitive strain. Extracts were obtained by preparing decoction in water of the powdered plant, the technique used by most of the traditional healers. An ethanol extract was then made and tested. The I[C.sub.50] values obtained for these extracts ranged from 3.9 to -5.4 [micro]g/ml. Purification of this active fraction led to the identification of cryptolepine as the active antiplasmodial constituent of the plant.

Key words: Sida acuta, cryptolepine, antiplasmodial, activity

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Introduction

Traditional treatments to cure malaria are under investigation by numerous teams. In Africa, indigenous plants play an important role in the treatment of a variety of diseases as shown by Phillipson (1995). A part of our research program consists of the analysis of the antiplasmodial activities of plants used by traditional healers to treat and cure malaria symptoms (Benoit et al. 1995, 1996; Valentin et al. 1997; Mustofa et al. 2000). Such a strategy allows the selection of plants which are known to be used and obviates the limitations of blind screening. The more interesting plants are then selected in view to determine their active constituents. In this context, we had previously described Alchornea cordifolia, originating from Ivory Coast, as a good candidate on account of its antiplasmodial activities against a range of choroquine-sensitive and chloroquine-resistant strains of Plasmodum falciparum, and identified the active constituent of the plant: ellagic acid (Banzouzi et al. 2002). In this report, we present the results of similar investigations for Sida acuta (Malvaceae). This plant is used by traditional healers as a complementary treatment for malaria or other febrile illnesses (Kerharo and Adam, 1974). By bio-guided purification, we have identified an alkaloid: cryptolepine, as the principal active constituent of the plant.

Material and Methods

Plant extracts

The plants were collected in Ivory Coast and were identified by Professor Ake Assi by comparison with authentic specimens. A voucher specimen was deposited at the "Centre National de Floristique" of the University of Cocody, Ivory Coast. The part of the plant tested was the aerial part which was air dried (10 days at 33 [degrees]C) and powdered. Extracts were then prepared by decoction (powder) according to the following procedure: 100 g of dried powder was added 500 ml of boiling distilled water and kept boiling for 10 minutes. The preparation was then cooled to 4 [degrees]C, filtered (0.22 [micro]m, Gelman Sciences SA, France) and referred to as the mother solution. Three 1 ml aliquots of this mother solution were then dried at 120 [degrees]C until completely desiccated. The remaining dried part was weighed and the mass obtained gave the mother solution concentration (F1). The remaining extract was then tested for antiplasmodial activity either as fresh, frozen (-20 [degrees]C for 2-12 months) and lyophilized (from the frozen extract) forms. The powdered dried leaves were also submitted to extraction for 8 h with ethanol (100 g for 11 ethanol). Three consecutive extractions were made on the same batch, the 3 l of ethanol were evaporated, and gave 17.2 g (F2) of dry residue.

Bio-assay directed fractionation

Purification of the crude ethanolic extract (F2) was conducted by chromatography on silica gel (column [empty set] 8 cm, h leight 10 cm) successively eluted with ether (6L:F3), ethyl acetate (5 L : F4), methanol (8 L : F5). Cryptolepine was purified from F5 by additional chromatographies on silica gel. Degrees of purity at the different steps of purification were evaluated by HPLC from analytical samples. A C18 column eluted with a gradient of acetonitrile/water (from 5%/95% to 95%/5%) with 0.1% of trifluoroacetic acid was used, and detection of cryptolepine was done by UV at 254 nm.

Structural analysis

The structure of cryptolepine (Fig. 1) was determinated by mass spectrometry, and NMR spectroscopy. Mass spectra were recorded on a LKB 2091 spectrometer at 15 eV [[tau] (source) = 180 [degrees]C]. [.sup.1]H-NMR, [.sup.13]C-NMR, HSQC and HMBC experiments were recorded on a Bruker AC 360 instrument in DMSO-[D.sub.6] at 305 K; all chemical shifts as well as all the correlations observed were in accord with those already described (Tackie et al. 1991).

[FIGURE 1 OMITTED]

Parasite strains and in vitro culture

The FcM29-Cameroon strain of Plasmodium falciparum, chloroquine-resistant (with I[C.sub.50] for chloroquine at 230ng/ml), and a Nigerian chloroquine-sensitive strain (chloroquine I[C.sub.50]:41 ng/ml) were cultured continuously according to Trager & Jensen (1976), with modifications described by Van Huyssen and Rieckmann (1993). The I[C.sub.50] values of chloroquine were checked every two months and we observed no significant variations. The parasites were maintained in vitro in human red blood cells (O[+ or -]), diluted to 1% hematocrit in RPMI 1640 medium (GIBCO BRL, Paisley, Scotland) supplemented with 25 mM Hepes and 30 mM NaHC[O.sub.3] (GIBCO BRL, Paisley, Scotland) and complemented with 5% human AB+ serum. Parasite cultures were synchronized by a D-sorbitol lysis (5% of D-sorbitol Merck, Darmstadt, Germany in sterile water) as reported by Lambros and Vanderberg (1979).

Assay for antiplasmodial activity

The antiplasmodial activity of plant extracts was evaluated by a radioactive micromethod similar to that described by Desjardins et al. (1979) with modifications reported by (Valentin et al. 1997). Extract testing was performed three times in triplicate in 96-well culture plates (TPP, Switzerland) with cultures mostly at ring stages at 0.5-1% parasitemia (hematocrit: 1%). Parasite culture was incubated with each extract for two time intervals: 24 h and 72 h. These two incubation times allowed the following of the drug effect on the trophozoite-schizont transition (24 h) and on the second cycle (re-invasion and trophozoite maturation (72 h)). The 10-fold dilutions ranged between 100 [micro]g/ml to 0.01 [micro]g/m. Parasite growth was estimated by [[.sup.3]H]-hypoxanthine incorporation. Control consisted of parasite cultures free from any extracts which were referred to as 100% growth. I[C.sub.50] values were determined graphically in concentration versus percent inhibition curves.

Results and Discussion

The I[C.sub.50] values obtained with various fractions of plant on P. falciparum culture with the radioactive method are summarized in Table 1. Data are expressed as the mean [+ or -] standard deviation of the I[C.sub.50] of at least three independent experiments on different days for each strain. The purpose of these bio-directed assays was to identify the active(s) compound(s) of the plant. The biological activities of the traditional preparation (F1) was first assessed in vitro. Then the ethanolic extract (F2), which exhibited a better antiplasmodial activity than did the decoction was further investigated. The F2 fraction was submitted to chromatography with successive elutions with ether (F3), ethyl acetate (F4) and methanol (F5). The F3 and F4 fractions showed no significant activities while the methanolic fraction (F5) exhibited the best activity. This F5 extract was then fractionated on silica gel eluted with ethanol: 10 fractions were collected (5F1 to 5F10). 100 mg of the dried active fraction (5F6) were first washed with hot chloro-form and filtered off to give a yellow precipitate 5F6p which was inactive and a filtrate 5F6f which corresponded to the active part (I[C.sub.50]~0.03 mg/ml). An analytic sample of the filtrate (5F6f) was submitted to an HPLC test showing that it was constituted of one compound and a set of minor impurities. In the ultimate step, the filtrate was concentrated and 40 mg of the residue were chromatographed on silica gel eluted with a mixture of dichloromethane/methanol (90/10 vol/vol) leading to pure cryptolepine which exhibited same biological activities that 5F6f.

Among African medicines, plants are often used by healers to treat diseases identified as malaria (Sofoworo, 1980; Omulokoli et al. 1997). However, their utilisation are always limited to traditional preparations, and rationalisation of their use is necessary to improve the utilisation of such plants. In this context, determination of their active constituents appears to be of potential medical interest and may permit the elaboration of optimized simple preparations. In this work, we have confirmed the antiplasmodial activities of traditionnal preparations of S. acuta. This plant appeared as an excellent example for the elaboration of new simple preparations since its principal active constituent is due to one compound: cryptolepine. In addition, this alkaloid is known to exhibit various pharmacological activities like antitrypanosomal and antifungal effects (Ablordeppey et al. 1999; Arzel et al. 2001), cytotoxic effect on cancerous cell lines probably by a direct DNA-intercalating effect (Lisgarten et al. 2002) and finally an antihyperglycemic activity (Bierer et al. 1998). In conclusion, this study confirmed the occurrence of Sida acuta in traditional medicines, since its use is now unambiguously related to a known alkaloid in this field (Arzel et al. 2001).
Table 1. In vitro antiplasmodial activities of the different fractions
of S. acuta on Nigerian chloroquine-sensitive strain and on Fcm29-
Cameroon chloroquine-resistant strain.

 I[C.sub.50] ([micro]g/ml) on the Nigerian
 strain
 24 h 72 h

Decoction F1 50 41 [+ or -] 1.0
Ethanol F2 5.4 [+ or -] 1.4 4.8 [+ or -] 0.6
F3 (ether) >50 >50
F4 (AcOEt) >50 >50
F5 (methanol) 0.4 [+ or -] 0.03 0.5 [+ or -] 0.1
5F1-5F4 >50 >50
5F5 14.0 [+ or -] 1.5 15.0 [+ or -] 3.0
5F6 0.2 [+ or -] 0.05 0.2 [+ or -] 0.01
5F7 20 [+ or -] 2.0 16.0 [+ or -] 1.0
5F7-5F10 >50 >50
6F6p >10 >10
5F6f 0.03 [+ or -] 0.001 0.04 [+ or -] 0.01
Cryptolepine ([micro]M) 0.13 [+ or -] 0.04 0.17 [+ or -] 0.04
Chloroquine* ([micro]M) 0.076 0.076

 I[C.sub.50] ([micro]g/ml) on Fcm29 strain
 24 h 72 h

Decoction F1 >50 >50
Ethanol F2 5.0 [+ or -] 1.1 3.9 [+ or -] 1.2
F3 (ether) >50 >50
F4 (AcOEt) >50 >50
F5 (methanol) 0.4 [+ or -] 0.03 0.8 [+ or -] 0.01
5F1-5F4 >50 >50
5F5 >10 >10
5F6 0.3 [+ or -] 0.04 0.2 [+ or -] 0.01
5F7 10.0 [+ or -] 2.0 8.25 [+ or -] 2.0
5F7-5F10 >50 >50
6F6p >10 >10
5F6f 0.04 [+ or -] 0.08 0.04 [+ or -] 0.0011
Cryptolepine ([micro]M) 0.17 [+ or -] 0.08 0.17 [+ or -] 0.02
Chloroquine* ([micro]M) 0.21 0.21

* values checked every two months


Acknowledgements

The author wish to thank Mrs. Dominique Castel for excellent technical assistance. This work received financial support from the CAMPUS project (grant n[degrees] 97 342 186), from the Programme VIHPAL 2000 (Ministere de la Recherche). H. Menan is granted by the AUPELF.

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J-T. Banzouzi (1,2), R. Prado (3), H. Menan (4), A. Valentin (4), C. Roumestan (3), M. Mallie (4), Y. Pelissier (2), and Y. Blache (1)

(1) Laboratoire de Chimie Organique Pharmaceutique, E.A. 2414, Faculte de Pharmacie, Montpellier cedex, France

(2) Laboratoire de Pharmacognosie, UFR des Sciences Pharmaceutiques et Biologiques, Montpellier cedex, France

(3) Centre de Biochimie Structurale, Montpellier cedex, France

(4) Laboratoire d'Immunologie et Parasitologie, UFR des Sciences Pharmaceutiques et Biologiques, Montpellier cedex, France

Address

Y. Blache, Laboratoire de Chimie Organique Pharmaceutique, E.A. 2414, 15 Avenue Charles Flahault, Faculte de Pharmacie, 34060 Montpellier cedex 2, France

Tel.: 004 67 54 86 40.
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Article Details
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Author:Banzouzi, J.-T.; Prado, R.; Menan, H.; Valentin, A.; Roumestan, C.; Mallie, M.; Pelissier, Y.; Blach
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUFR
Date:Apr 1, 2004
Words:2416
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