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Serge et al. 2285
Table 1. Relative content of alkaloids and tannins on roots and leaves of the seven selected plants (nd: not
determined).
Medicinal plants
(number of extractions)
Roots Leaves
Alkaloids (%) Tannins (%) Alkaloids (%) Tannins (%)
A. leiocarpus (n = 3) 0.02 13 0.1 20
B. rufescens (n = 2) 0.03 13 0.04 4
C. sieberiana (n = 2) 0.01 11 0.02 14
M. inermis (n = 6) 0.2 5 0.6 7
N. latifolia (n = 3) 0.3 2 nd nd
P.biglobosa (n = 2) 0.06 12 0.04 10
T. roka (n = 2) 0.1 5 0.2 6
Benth.)), two Rubiaceae (Mitragyna inermis (Willd., O.Ktze) and
Nauclea latifolia Sm).
The plants were collected during the wet season around the town
of Ouagadougou. They were identified by the Department of
Botany, Ouagadougou University, where voucher specimens have
been deposited.
Preparation of crude extracts
The air dried plants were powdered using a semi-industrial crusher.
Alkaloids were extracted according to Lannuzel et al. (2002),
modified as follows: After humidification with NH4OH (12.5%),
alkaloids were extracted using petroleum ether and treated with
H2SO4 (5%). The aqueous acid solution was washed with hexane,
alkalised with Na2CO3 (10%), and extracted with chloroform. The
organic solution was washed with water, dried over Na2SO4, and
evaporated. Alkaloid bases were dissolved in acetone with
Hydrochloric acid (HCl) vapour. The presence of hydrochlorate
alkaloid residue was confirmed using the Mayer and Draggendorf
test.
The powder was delipided and tannins were extracted using
acetone. After elimination of pigments, the acetone was evaporated
and the tannin precipitates were characterised by FeCl3 in
accordance with CEE recommendations (Bruneton, 1993).
Quantitative studies were conducted using gravimetric analysis. For
in vitro antiplasmodial activity, stock solutions of alkaloids and
tannins at 5.000 µg/ml were prepared after filtration using 0.22 mm
Millipore membrane. A chloroquine stock was prepared at 1.000
µg/ml.
Antiplasmodial activity
P. falciparum strains were isolated from infected children (4 to 7
years) at Barogo village, north of Ouagadougou. Thin blood films
were prepared and stained with Giemsa. The selected subjects
showed a blood parasite density ranging from 300 to 10.000
infected red blood cells per µl. Before treatment, venous blood was
collected in ethylenediametetraacetic acid (EDTA) Vacutainer®
tubes and immediately transported to the laboratory. Seven isolates
were used for antiplasmodial assay. Standard protocols of P.
falciparum in vitro cultures (Trager and Jensen, 1976) were used
(RPMI 1640 with 10% human serum, human erythrocytes O+,
incubated at 37°C with 5% CO2).
Continuous in vitro culture of the 3D7 chloroquine sensitive strain
and W2 chloroquine resistant strain was maintained in the
laboratory. Quantitative assessment of in vitro antiplasmodial
activity was determined using four concentrations of each extract.
The initial highest concentration of each plant extract was 1250
µg/ml, and the lowest was 1.25 µg/ml. All tests were made in
duplicate for 24 and 48 h, in a 96 well flat-bottom culture plate
(Costar, UK). Chloroquine was used as a positive control and drugfree
medium as a negative control. After incubation, thin blood films
were prepared and stained with Giemsa, for each well; the
percentage of infected red blood cells was determined
independently by two readers. In case of discrepancy a third
reading was made. Percentage inhibition curves = f (-log [plants
extract]) following a two order polynomial regression. IC50 were
classified according to Deharo et al. (2001): If it is less than 5 µg/ml,
the extract was considered highly active, 5 to10 µg/ml moderately
active, and over 10 µg/ml inactive. Each assay was run in duplicate
and the values given are averages of the two independent assays.
All field isolates were chloroquine sensitive (IC50 < 15 ng/ml).
Haemolytic activity
We determined the in vitro haemolytic activity of extracts using
human red blood cells O+; haemoglobin rate was determined at
550 nm (Tietz and Fiereck, 1973).
RESULTS AND DISCUSSION
Twenty-six extracts were prepared from the seven plants.
A very unequal distribution of alkaloids and tannins in
leaves and roots of the plants was observed. Qualitative
analysis showed that leaves and roots of A. leiocarpus,
C. sieberiana and P. biglobosa are rich in tannins and
poor in alkaloids. Conversely, M. inermis and N. latifolia
contain many alkaloids but few tannins. These raw
results were confirmed by quantitative study using
gravimetric analysis. A. leiocarpus, C. sieberiana, and P.
biglobosa contain 13, 11 and 12% tannins respectively in
the roots, and 20, 14 and 10% in the leaves (Table 1). M.
inermis, T. roka and N. latifolia contain 0.2, 0.1 and 0.3%
alkaloids, respectively in the roots and 0.6, 0.2 (M.
inermis and T. roka) in the leaves.
In preliminary studies, chloroquine and all 26 extracts
(alkaloids and tannins) were screened at four
concentrations on 3D7, W2 and on the seven strains
isolated from infected patients. Only a few extracts
showed an IC50 inferior to 10 µg/ml. Considerable