L'activité antiplasmodiale, in vitro, in vivo du Sergeolide, Quassinoide isolé de #Picrolemma pseudocoffea$ a été mesurée, in vitro, sur des cultures de #Plasmodium faliciparum$ et in vivo par le test classique de la mesure de l'activité schizonticide sur le paludisme du rongeur, à #Plasmodium berghei$. Le sergeolide manifeste une forte activité antiplasmodiale, aussi bien in vitro que in vivo, à des doses extrêmement faibles. Une concentration de 0,006 microg/ml inhibe complètement la croissance des cultures de diverses souches de #Plasmodium faliciparum$, sensibles et résistantes à la choroquine. Une dose de 0,26 mg/kg/j réduit considérablement la virulence du paludisme à #Plasmodium berghei$ chez la souris. Cependant, la forte toxicité du sergeolide (DL50 : 1,8 mg/kg) limite l'intérêt de ce composé dans le traitement curatif du paludisme. (Résumé d'auteur

Fandeur, T.

Moretti, Christian

Polonsky, J.

Horizon / Pleins textes

. ,, ' 20 
%, 
- 
Planta Medica 1985 
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In Vitro and in Vivo Assessement of the Antimalarial 
Activity of Sergedide 
Thierry Fandeur', Christian Moretti2 and Judith Polonsky3 
Received: May 28,1984; Accepted: September 30,1984 
Abstrad: The antimalarial activity of sergeolide (a quassinoid from 
Picrolemma pseudocoffea) was investigated both, in vitro on Plas- 
modium falciparum cultures and in vivo through a classical test of 
schizontocidal action against Plasmodium berghei in mice. Sergeolide 
showed a very strong antiplasmodial activity in vitro as well as in vivo. 
Low concentrations (0.006 pglml) were able to fully inhibit the in vitro 
growth of chloroquine-sensitive and resistant strains of P. falcipamm. 
Small amounts (0.26 mg/kg/day) markedly reduced the virulence of ex- 
perimentally induced P. berghei infection in mice. However, 
sergeolide, because of its high toxicity (LD 5 0  1.8 mgkg), does not 
seem, in its present form to be useful for malaria curative treatment. 
Introduction 
Prophylactic and curative malaria treatment still depend on the 
'use of classical drugs (chloroquine and its derivatives, 
pyrimethamine and sulphonamides). However, in numerous 
part of the world, resistance to these common antimalarial 
drugs has become so widespread and intense that the develop- 
ment of new compounds for malaria therapy is a real 
emergency (1). 
A recent study, interestingly, has shown that certain quas- 
sinoids (bitter principles of the plant family Simaroubaceae) (2) 
were capable of inhibiting the ìn vitro growth of chloroquine- 
resistant strain of P. falciparum (3). This latter work unfortu- 
nately, did not connect the in vitro antimalarial activity of quas- 
sinoids with the presence or absence of antimalaÍia1 effects in 
vivo in experimental animal models. 
Laboratoire d'immunologie parasitaire, Institut Pasteur de la 
Guyane Française, 97306, Cayenne Cedex, Guyane Française 
* Unité de recherche des substances naturelles à intérêt biologique, 
Office de la Recherche Scientifique et Technique d'Outre Mer, BP 
165, Cayenne, Guyane Française 
ICSN Gif sur Ivette, Lab. de Chemie. 
Our communication is concerned with a study carried out to 
determine the antimalerial activity in vitro and in vivo of a new 
quassinoid, sergeolide, recently isolated from the French 
Guinanan Simaroubaceae: Picrolemma pseudocoffea. Well 
known by the indigenous population for its antimalarial prop- 
erties, Picrolemma pseudocoffea has for a long time been em- 
ployed in traditional medicine for malaria therapy (4). 
Materials and Methods 
Drugs 
The extraction and purification of the sergeolide from the dried ground 
roots of Picrolemma pseudocoffea were carried out using a method al- 
ready described (5). The compound in pure crystallized state was pre- 
pared according to the method established by Trager et al. (3). Briefly, 
sergeolide was dissolved in 95 YO ethanol at a concentration of 3 mg/d. 
For the in vitro study, a stock solution was prepared by diluting the 
ethanol solution in the proportion of 1 : 10 with RPMI culture medium 
(without bicarbonate). The stock solution was sterilized through a0.22 
pm filter (Schleicher and Schiill), then, in order to obtain final concen- 
trations of sergeolide ranging from 0.002 to 0.5 &ml, diluted asepti- 
cally prior use in complete RPMI medium. 
To investigate in vivo the acute toxicity and the antimalarial activity 
of sergeolide, the original ethanol solution was diluted with normal 
saline to obtain solutions ranging from 0.0015 to 1.2 mg of sergeolide 
per ml. 
Chloroquine solutions were prepared in the same way from 
chloroquine disulfate but, in this case, the initial solubilization was di- 
rectly performed in saline (3 mg of chloroquine per ml of solution). 
Control solutions, containing equal amounts of ethanol to those in- 
troduced with the sergeolide, were also prepared either from a stock 
solution of 10 YO ethanol in RPMI culture medium for the in vitro study 
(range of dilutions 1 : 1,5000,000-1 : 1,OOO) or directly from 95 YO 
ethanol for the in vivo study (range of dilutions 1 : 200-1 : 2.5). 
ORSTOM Fonds Documentaire 
J 
1 
In Vitro and in Vivo Assessement of the Antimalarial Activity of Sergeolide 21 
In vitro measurement of the anrimalarial activity 
Parasite strains: four strains of P .  falciparum were used throughout this 
work. 
Originally obtained from R. J .  M. Wilson (London, U. K.), the 
chloroquine-sensitive strain Ouganda Palo Alto (FUP strain) has since 
been fully adapted to the squirrel monkey (Saimiri sciureus), without 
losing its infecting potential for human red blood cells (6). This strain 
routinely maintained on splenectomized animals, was isolated from a 
highly infected Saimiri (30 % parasitemia), then cultivated in human 
A+ erythrocytes for more than a month prior to be used for this study. 
Three strains of Brazilian origin, namely 95/83, 96/83 and 97/83, 
kindly provided by V. E. Do Rosario (Belem, Brazil), were brought to 
our laboratory as parasitized blood, directly isolated from patients. 
They have all since been adapted to continuous culture in human red 
blood cells. All three are chloroquine-restistant using Rieckmann’s 
method (7) (Rosario, personal communication). 
Culture procedure: The cultures were carried out in human A’ red 
blood cells, according to Trager and Jensen’s methodology (8). The 
culture medium used throughout was RPMI 1640 supplemented with 
25 mM HEPES and 10% human A+ serum. The complete culture 
medium with different concentrations or dulutions of the compound to 
be test or without drugs (for the controls), was distributed into 16 mm 
wells of a 24 well plate (0.5 dwell) .  Each well the received 50 pl of a 
parasitized suspension, obtained by mixing red blood cells suspended 
in RPMI with enough infected erythrocytes to give an initial 
parasitemia ranging from 0.35 to 0.70 YO. Parasitized red cells were 
shaken gently with a vortex mixer, before and during their distribution 
into the wells. The plates were then incubated at 37” C under a continu- 
ous flow of a humidified gas mixture containing 2 YO 02, 3 YO COz, and 
95 % N2. Giemsa stained thin blood films were prepared at times O, 24, 
and 48 hours. 
Measurement of the acute toxicily of sergeolide 
The lethal dose 50 (LD 50) of sergeolide was determined from the 
graph obtained by plotting the doses (logarithmic scale) against the 
percentages of martality (probit scale). 
0.5 ml volumes of increasing doses of sergeolide (0.3.2.2, 6.6 and 
19.8 mgkg) were randomly administrated by subcutaneous injections 
to groups of 10 Swiss mice (bred at the “Institut Pasteur de la Guyane 
Française”, average weight: 30 f 2 g). The mortality was noted after 72 
hours. 
In order to determine the ethanol toxicity, control groups of 10 mice 
were treated with different dilutions of ethanol (1 : 167, 1 : 23, 1 : 7.5, 
1:2.5) corresponding to those used with the different doses of 
sergeolide. 
In vivo measurement of the antimalarial activity 
The antimalarial activity of sergeolide was determined and compared 
with that of chloroquine by a classical 4 day suppressive test against P. 
berghei in mice (9). The P. berghei strain used was the NK 65 strain, ob- 
tained from J. P. Vandenberg (New York, U.S.A.). 
80 male Swiss mice (average weight: 29 f 1 g) were injected intra- 
peritoneally with lo7 parasitized cells in 0.2 ml normal saline. The day 
of infection was numbered day O (DO) and the following, D + 1, D + 
2, etc. . . . 
Groups of 10 mice were randomly injected subcutaneously from DO 
to D + 3 with different doses of sergeolide (0.26,O.M and 0.026 mglkgl 
day), chloroquine (1.3,0.4 and 0.13 mg/kg/day), or ethanol (1 : 200 di- 
lution) in 0.5 ml normal saline. The suppressive effects of the different 
compounds was estimated at D + 4 on Giemsa stained thin blood films, 
in comparison with a group of 10 mice treated with normal saline. 
Results 
Antimalarial activity in vitro 
The P. falciparum growth is strongly inhibited by sergeolide. 
As shown in Table I, 50 % inhibition could be observed, in the 
case of the chloroquine-sensitive FUP strain, for very weak 
concentrations (0.002 and 0.006 pglml), higher concentratiaons 
resulting in almost complete inhibition. However, with regard 
Table I. Effects of sergeolide in vitro on the chloroquine sensitive strain FUP of Plasmodium falciparum 
Parasitemia after 24 h culture** Parasitemia after 48 h culture** 
Concen- Inhibi- 
Drugs* trations Parasitemia Differential counts Parasitemia Differential counts tiom*** 
R T S R T S 
Cunlml YO % % YO % YO % % % 
None O 0.9 f 0.1 86 10 4 1.4 f 0.1 68 13 9 O 
0.5 0.16 f 0.02 100 O O 0.09 f 0.08 100 O O 94 
O. 17 0.09 f 0.02 83 11 6 0.10 f 0.01 100 O O 93 
0.05 0.12 f 0.02 88 6 6 0.08 f 0.02 100 O O 94 
SG 0.018 0.33 f 0.13 93 7 O 0.23 f 0.05 100 O O 84 
0.006 0.4 f 0.1 96 4 O 0.63 f 0.09 100 O O 55 
0.002 0.63 ? 0.07 93 5 2 0.8 f 0.2 66 27 7 43 
0.5 0.21 f 0.05 63 37 O 0.07 f 0.01 78 22 O 95 
O. 17 0.13 k 0.02 84 16 O 0.10 f 0.01 82 18 O 93 
0.05 0.29 f 0.02 95 3 2 0.23 f 0.04 79 21 O 84 
CL 0.018 0.51 f 0.03 94 5 1 0.35 f 0.03 81 17 2 75 
0.006 0.53 & 0.05 88 6 6 1.2 f 0.1 67 14 19 14 
0.002 0.63 f 0.09 88 8 4 1.2 f 0.1 67 11 22 14 - 
1 : 6,000 0.72 f 0.02 89 6 5 1.3 f 0.2 68 18 14 7 
1 : 18,Ooo 0.7 f 0.1 86 9 5 1.4 f 0.2 56 17 27 O 
1 : 54,000 0.81 f 0.07 84 12 4 1.3 rt 0.1 59 17 24 7 
EtOH 1:162,OOO 0.58 f 0.17 YO 7 3 1.49 k 0.02 50 25 25 O 
1 : 486,000 0.71 f 0.04 80 13 7 1.2 f 0.3 63 11 26 14 
1 : 1460,000 0.5 f 0.2 84 13 3 1.3 f 0.2 60 15 25 7 
* - SG, sergeolide; CL, chloroquine; EtOH, ethanol. 
* *  - Percentages of parasitemia are the mean values f SD calculated from triplicates experimments. At time “0” the count was; 0.35 f 0.03 YO 
- R (ring), 71; T (trophozoite), O; S (schizonte), 29. 
*** - Percentages of inhibition were calculated at time “48” against control culture samples without drugs. 
I 
î . 22 Planta Medica 1985 
to chloroquine, its antiplasmodial activity becomes significant 
from threshold concentration of 0.018 pg/ml. The culture sam- 
ples containing equal of ethanol as were introducted with 
segeolide reacted in the same way as control culture samples 
without drugs. Differential parasite counts revealed that 
sergeolide, like chloroquine, is simultaneously effective against 
all asexual blood stages. 
Coneming  the three chloroquine-resistant strains P.  fal- 
ciparum (including the 97/83 isolate which showed in vivo a re- 
sistance to the association quinelfansidar) their in vitro growth 
were equally inhibited by sergeolide (Fig. 1). The drug is highly 
active at concentrations 2 0.006 pg/ml, whereas an identical in- 
hibitory effect can only be obtained with chloroquine at a 100 
fold higher concentrations (0.5 pglml). 
'1 95.83 
O 24 48 
4 7  96.83 
24 48 
HOURS 
i 
0.05 
0,OlJE 
Antimalarial activity in vivo 
We determined the minimun effective dose of sergeolide able 
to reduced by half the virulence of P. berghei infection experi- 
mentally induced in mice (ED 50). 
Doses of sergeolide were chosen according to the LD 50 pre- 
viously established. In order to check the reliability and evolu- 
tion of the experiment, a dose-response curve was also plotted 
for a known drug, chloroquine. 
From Figure 2 the ED 50 are: 
a) sergeolide: approximately 0.2 mg/kg/day (range of value in dose, 
0.08 to 0.4) 
b) choroquine: approximately 1 mg/kg/day (range of value in dose, 
0.16 to 4). 
The group of 10 mice treated with 0.5 mi of a 1 : 200 ethanol dilution, 
which corresponds ot the quantity of ethanol introduced with the 
sergeolide at its highest dose (0.26 mgkg/day) developed at D + 4 an 
average parasitemia identical to that of the control group treated with 
normal saline. 
Fig. 2. Effects of sergeolide and chloroquine in vivo on the NK 65 
strain of Plasmodium berghei in mice. 
A, sergeolide; B, chloroquine. 
Daily doses of drugs are plotted on a logarithmic scale. 
0.05 
O 0 0 6  
Discussion 
05 
Fig. 1. Effects of sergeolide in vitro on three chloroquine-resistant 
strains of Plasmodium fakiparum. 
Each point is the mean value k SD calculated from triplicate experi- 
ments. 
Acute toxicity of sergeolide 
For the calculation of the LD 50, we did not take into account 
the results from the group of mice injected with 19.8 mgkg, be- 
cause in the control group treated with ethanol at a l : 2.5 dilu- 
tion, two animals died. The quantity of ethanol introduced for 
this dilution ( z  5 gkg)  corresponds in fact, to the LD 50 of 
ethanol already described (10). No deaths were observed in the 
group of mice treated with smaller doses of ethanol. The results 
of our test fixed the LD 50 of sergeolide at 1.8 mg/kg. 
I 
EO 110 " ED- 
MILI Domm rolma 
Earlier work showed that the antimalarial activity of quas- 
sinoids runs in palle1 to their antileukemic properties (3). 
Considering the original structure of sergeolide (first known 
example of quassinoid with a butenolide function attached to 
the ring A), (Fig. 3) and its highly inhibitory action against 
mouse leukemia P 388 cell line, it seemed of interest to check 
the antimalarial activity of this new molecule, in vitro and in 
vivo. 
SERGEOLIOE 
Fig. 3. Structural formula of sergeolide. 
Novel Chromone Alkaloids from Schumanniophyton magnificum 
As suspected from the preliminary tests on its toxicity (S), 
sergeolide possesses a high antimalarial activity. In vitro by a 48 
hour culture method, it showed a significant inhibitory effect 
against P. fafciparum at a concentration of 0.006 pg/ml (Fig. 1) 
and even at 0.002 pg/ml (Table I). A comparable inhibitory ac- 
tion can only be obtained with chloroquine at a concentration 3 
fold higher in the case of a chloroquine-sensitive strain (Table 
I) and 100 fold higher for chloroquine-restistant strains (Fig. 1). 
It is of interest to note that the strain 97/83 having shown in vivo 
a resistance to  the association quininelfansidar, is identically 
sensitive to the antiparasitical action of sergeolide. 
Sergeolide, although very active in vivo (ED 50 0.2mg/kg/ 
day) at lower concentrations than that chloroquine (ED 50 1 
mg/kg/day), presents unfortunately, little interest in the cura- 
tive treatment of rodent malaria (ED 90 = 1.7 mg/kg/day), be- 
cause of its high toxicity (LD 50: 1.8 mg/kg). 
Our results argue that the antimalarial activity of Pic- 
rofemma pseudocoffea could be imputed to the presence of 
sergeolide. 
Isobruceine B, another highly toxic quassinoid (LD 50 ap- 
proximately 5 mg/kg) isolated from this French, Guianan 
Simaroubaceae, seems, on account of its 5 fold lesser antimala- 
rial activity in vitro (results not shown here), take only a moder- 
ate part in the antimalarial plant activity. 
If, as we suggest, sergeolide is the principal antimalarial 
compound of Picrolemma pseudocoffea, it remains, taking its 
high toxicity into account,’ to clarify the effective utilization of 
Picrolemma pseudocoffea in traditional medicine. It will be rel- 
evant to collect more information concerning the direction for 
use of the plant in malaria therapy (preparation, doses, treat- 
ment duration), the immune status of treated patients, and fi- 
nally the possible side-effects of the treatment. 
7 
J.  23 
As already suggested concerning sergeolide, modifications 
of the molecule are necessary in order to attenuate its toxicity, 
while preserving its antimalarial properties (5). 
However, a search for new compounds of the quassinoid 
family appears relevant, because of possible isolation of similar 
active molecules possessing high antimalarial activity versus re- 
duced toxicity. 
Acknowledgements 
We thank Mrs. M. Ribal for skilful technical assistance, and Mr. P. 
Ridel and Dr. J. P. Dedet for reviewing this manuscript. 
References 
(1) World Health Organization: Chemotherapy of malaria and resis- 
tance to antimalarials. Report of a WHO scientific group (1973) 
Tech. Rep. Ser. No. 529. 
(2) Polonsky, J. (1973) Fortschr. Chem. Org. Naturst. 30,101. 
(3) Trager, W., Polonsky, J. (1981) Am. J. Trop. Med. Hyg. 30,531. 
(4) Grenan, P., Moretti, C., Jacquemin, H., Pharmacopées 
traditionnelles de Guyane. Edition de l’Office de la Recherche 
Scientifique et Technique d’Outre Mer. (in press). 
(5) Moretti, C., Polonsky, J., Vuilhorgne, M.,Pronge,T. (1982)Tet- 
rahedron lett. 23,647. 
(6) Gysin, J. ,  Fandeur, T. (1983) Am. J. Trop. Med. Hyg. 32,461. 
(7) Rieckmann, K. H., Sax, L. J., Campbell, G. H., Mrema, J. E. 
(8) Trager, W., Jensen, J. B. (1976) Science 193,673. 
(9) Peter, W. (1980) Chemotherapy of malaria. In “Malaria”. 
(10) Mortier, F., Bogaert, J. P., Jouany, J. M., Dixneuf, P., De- 
(1978) Lancet L, 22. 
Academic Press, New York, 1,145. 
laveau, P. (1976) PI. Méd. Phyt. 10,36. 
Novel Chromone Alkaloids 
from Schumanniophyton magnificum 
P. J. Houghton1-2 and Yang Hairong’ 
Received: June 7,1984; Accepted: September 10,1984 
Abstract: Six chromone alkaloids were isolated from the root bark of 
Schumanniophyton magnificum Harms. Three of these were identified 
as the knwn  alkaloids schumanniophytine, schumannificine and N- 
methylschumannificine. The other three alkaloids are novel and were 
named isoschumanniophytine, anhydroschumannificine and N- 
methylanhydroschumannificine. In addition it was seen that the N- 
methylschumannificine isolated consisted of a mixture of two isomers 
and an altemative structure for schumanniophytine has been pro- 
posed. 
’ Pharmacognosy Research Laboratories, Department of Pharmacy, 
Chelsea College, University of London, Manresa Road, London 
SW3 6LX, U.K. 
Present address: - NorthyWest Plateau Institute of Biology, Xinjing, 
Qinghai Province, People’s Republic of China 
’ Address for correspondence 
Introduction 
Schumanniophytum magnificum Harms. (Rubiaceae) is. found 
in Nigeria and its uses there have been reported by Okogun et 
al. (1). There is current scientific interest in the plant because 
of the traditional use of it as a remedy for snake bites. 
Little chemical work has been done on the genus. Schlittler 
and Spitaler (2) investigated S. probfematicum and isolated the 
chromone noreugenin 1 and three alkaloids, schum- 
maniophytine 2 and two unnamed piperidin-2-ones 3a, 3b. 
Okogun et al. (1) examined the root bark of S. magnificum and 
isolated noreugenin 1 and two alkaloids with a novel structure, 
schumannificine 4a and N-methylschumannificine 4b. They 
also mentioned the presence of other alkaloids which they had 
not yet characterised. 


In vitro and in vivo assessement of the antimalarial activity of sergeolide

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