1050 Notes Biol. Pharm. Bull. 29(5) 1050—1052 (2006) Vol. 29, No.

Antimalarial Activity of Cassane- and Norcassane-Type Diterpenes from

Caesalpinia crista and Their Structure–Activity Relationship
Surya Kant KALAUNI,a Suresh AWALE,a Yasuhiro TEZUKA,a Arjun Hari BANSKOTA,a Thein Zaw LINN,a
Puji Budi Setia ASIH,b Din SYAFRUDDIN,b and Shigetoshi KADOTA*,a
a
Institute of Natural Medicine, University of Toyama; 2630 Sugitani, Toyama 930–0194, Japan: and b Eijkman Institute for
Molecular Biology; Jalan Diponegoro 69, Jakarta 10430, Indonesia.
Received November 29, 2005; accepted February 28, 2006; published online March 1, 2006

Malaria is one of the most life-threatening infectious diseases worldwide and claims the millions of peoples
life each year. The appearance of drug-resistance Plasmodium falciparum has made the treatment of malaria in-
creasingly problematic, and thus, it is a dire need to search the new alternatives of current drugs. In the present
study, 44 cassane- and norcassane-type diterpenes isolated from Caesalpinia crista of Myanmar and Indonesia
were evaluated for their antimalarial activity against the malaria parasite Plasmodium falciparum FCR-3/A2
clone in vitro. Most of the tested diterpenes displayed antimalarial activity, and norcaesalpinin E (28) showed the
most potent activity with an IC50 value of 0.090 m M, more potent than the clinically used drug chloroquine (IC50,
0.29 m M). Based on the observed results, a structure–activity relationship has been established.
Key words cassane-type diterpene; antimalarial activity; structure–activity relationship; norcassane-type diterpene; Caesalpinia
crista

Malaria remains the greatest human killer among parasitic
infections, despite the continuous worldwide efforts to com-
bat this disease and the attempts to eradicate the causative or-
ganisms.1) It is caused by the protozoan parasite of the genus
Plasmodium and transmitted by mosquitoes of the genus
Anopheles. Four species of Plasmodium, P. falciparum, P.
vivax, P. ovale, and P. malariae, can produce malaria in
human. Among the four species, P. falciparum is the most
widespread and dangerous. Malaria is an endemic disease in
many parts of Asia, Africa, Central and South America, the
Australian Continents, and some Caribbean islands. World
Health Organization (WHO) estimates that each year 200—
300 million people suffer from the case of malaria, resulting
in up to 2.7 million deaths.2) The appearance of drug-resist-
ance P. falciparum since 1960 has made the treatment of
malaria increasingly problematic, and apparently the battle
has not been successful.3) Since the historical discovery of
quinine from Cinchona tree and the recent discovery of
artemisinin from Artemisia annua L. (Asteraceae),4) there is
anticipation that new leads may emerge from the tropical
plant sources.
Caesalpinia crista LINN. (Fabaceae) is a famous medicinal
plant widely distributed in tropical and subtropical regions of
Southeast Asia. This plant is locally known as “Ka-Lain” in
Myanmar and its seeds are used as anthelmintic, antipyretic,
anti-inflammatory, and antimalarial agent.5) In Indonesia, it is
known as “Bagore” and the decoction of roots have been
used as a tonic and for the treatment of rheumatism and
backache, while its seed kernels have been used as antimalar-
ial and anthelmintic.6) In our preliminary study on the isola-
tion of antimalarial agents from natural sources, we observed
that the CH2Cl2 extract of seed kernels of C. crista collected
from Indonesia and Myanmar exhibited significant inhibition
of the growth of the malaria parasite P. falciparum FCR-3/A2
clone in vitro with IC50 values of 0.095 and 0.21 m g/ml, re-
spectively. Furthermore, the same CH2Cl2 extract of seed
kernels of Indonesian C. crista also displayed significant in
vivo antimalarial activity against the growth of P. berghi in
∗ To whom correspondence should be addressed. e-mail: kadota@ms.toyama-mpu.ac.jp
mice.7) Thus, we carried out the detailed phytochemical in-
vestigation of this plant and isolated 40 new cassane- and
norcassane-type diterpenes together with 18 known diter-
penes.7—11) In this paper, we report a biological profile on the
antimalarial activity of the isolated diterpenes and their
structure–activity relationships.
MATERIALS AND METHODS
Diterpenes Cassane- and norcassane-type diterpenes
(Chart 1) used in this study were isolated from the CH2Cl2
extract of C. crista collected from Myanmar10—12) and In-
donesia.7—9) The purity of each diterpene was checked by
TLC and 1H-NMR spectrum, which did not show the pres-
ence of any impurity. Caesalpinins MA (1), ME—MJ (2—7),
ML (8), and MO (9) and norcaesalpinins MC (10) and MD
(11) were new compounds isolated from C. crista of Myan-
mar. Caesalpinins C—F (12—15), H—K (16—19), and M—
P (20—23) and norcaesalpinins A—F (24—29) were new
compounds isolated from C. crista of Indonesia. On the other
hand, caesalmins B (30), C (31), E (32), and G (33); cae-
saldekarin e (34); 2-acetoxycaesaldekarin e (35); 2-acetoxy-
3-deacetoxycaesaldekarin e (36); 6-acetoxy-3-deacetoxycae-
saldekarin e (37); 14(17)-dehydrocaesalmin F (38); bondu-
cellpins B (39) and C (40); 7-acetoxybonducellpin C (41); 1-
deacetoxy-1-oxocaesalmin C (42); z -caesalpin (43); and 1-
deacetylcaesalmin C (44) were known compounds.
Materials The malaria parasite P. falciparum FCR-3/A2
clone was a gift from Dr. M. Suzuki, Gunma University,
Japan. RPMI-1640 medium, hypoxanthine, gentamycine,
chloroquine, and Giemsa stain were purchased from Sigma
Chemicals (St. Louis, MO, U.S.A.), while 2-[4-(2-Hydroxy-
ethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer was
from Gibco BRL Products (Grand Island, NY, U.S.A.). The
AB
human serum and human erythrocytes were donated by
healthy volunteers.
Parasite Culture The malaria parasite P. falciparum
FCR-3/A2 clone was routinely propagated according to pre-
© 2006 Pharmaceutical Society of Japan
May 2006
Chart 1. Structure of Cassane- and Norcassane-Type Diterpenes
Chart 2. Structure–Activity Relationship of Cassane- and Norcassane-
Type Diterpenes on Antimalarial Activity
viously published procedure.13) Briefly, the malaria parasite
cultures (erythrocytic stage) were propagated in tissue cul-
ture flask containing RPMI-1640 medium supplemented with
gentamycin 25 m g/ml, hypoxanthine 50 m g/ml, 25 mM
HEPES buffer, 25 mM sodium bicarbonate, 10% AB

human serum, 5% hematocrit, and human erythrocytes and
incubated at 37 °C under 5% CO2. In erythrocyte (red blood
cells), the parasites will undergo schizogonic developmental
stage from the merozoite stage to mature schizont, which
contains 12—36 daughter merozoites that eventually invades
new red blood cells, which requires 36—48 h to proceed. The
parasite growth was monitored by making blood smear,
stained with Giemsa after 48 h to see the percentage of the
red blood cells that were infected by the parasite.
Determination of the Antimalarial Activity Anti-
malarial activity of the isolated compounds was determined
by the previously described procedure by Budimulja et al.14)
In brief, each compound was separately dissolved in DMSO
to obtain 102 M stock solution and kept at 20 °C until
used. The different concentration of the compounds was
freshly made from the stock solution. The malarial parasite P.
1051
falciparum FCR-3/A2 clone was added to the 24-well culture
plate which already containing different concentration of the
compounds in RPMI-medium (total volume 1 ml having 1%
parasitemia and 5% hematocrit/well) and incubated for 72 h
at 37 °C under 5% CO2 with medium change at 48 h. The
growth of parasite was monitored by making a blood smear
fixed with methanol and stained with Giemsa stain and num-
ber of the infected red blood cells were counted under the
microscope. Chloroquine was used as a positive control. The
concentration response parasite growth data were analyzed
by a linear regression function using the Sigma-plot 2000
computer program to determine the 50% inhibitory concen-
tration (IC50). The IC50 value is defined as the concentration
of compound producing 50% parasite growth inhibition rela-
tive to untreated control.
RESULTS AND DISCUSSION
In the present study, 44 cassane- and norcassane-type
diterpenes from C. crista of Myanmar and Indonesia have
been tested for their inhibitory activity of the growth of the
malarial parasite Plasmodium falciparum FCR-3/A2 clone.
Majority of them exhibited various potencies of activity in a
concentration-dependent manner (Table 1). The activities of
diterpenes 21 (IC50, 0.12 m M), 25 (IC50, 0.26 m M), 28 (IC50,
0.090 m M), 29 (IC50, 0.14 m M), 36 (IC50, 0.098 m M), 38 (IC50,
0.20 m M), 39 (IC50, 0.24 m M), and 40 (IC50, 0.12 m M) were
more potent than well-known antimalarial drug, chloroquine
(IC50, 0.29 m M), and norcaesalpinin E (28) and 2-acetoxy-3-
deacetoxycaesaldekarin e (36) showed the most potent activ-
ity with an IC50 value of 0.090 and 0.098 m M, respectively.
1052 Vol. 29, No. 5

Table 1. Inhibitory Effects of Constituents from Caesalpinia crista on stituted ones (1110, 184, 4231).
Growth of Plasmodium falciparum FCR-3/A2 Clone in Vitro Effect of Substituents on Ring C The diterpenes iso-
Compounds IC50 (m M) Compounds IC50 (m M)
lated from C. crista showed wide range of structural varia-
tion and substitution pattern in ring C. In general, 17-norcas-
1 3.5 24 0.80 sane-type diterpenes were more potent than the correspon-
2 3.6 25 0.26 ding diterpenes having an aldehyde, methoxycarbonyl, or
3 4.1 26 5.0 methyl group at C-17 (2821
4019). Similarly, norcae-
4 2.5 27 2.0
5 7.0 28 0.090 salpinin B (25) was more potent than the diterpenes possess-
6 2.1 29 0.14 ing a methylene, methyl, or methoxycarbonyl group at C-17
7 1.9 30 0.80 (251213).
8 0.65 31 3.4
9 10 32 10
CONCLUSION
10 3.1 33 10
11 1.0 34 4.0
12 0.76 35 6.5 In the present study, we have reported the in vitro anti-
13 0.8 36 0.098 malarial activity of 44 cassane- and norcassane-type diter-
14 6.5 37 10 penes isolated from C. crista of Myanmar and Indonesia.
15 0.65 38 0.20
16 10 39 0.24
Most of the diterpenes displayed significant inhibition of
17 10 40 0.12 growth of Plasmodium falciparum FCR-3/A2 clone in vitro.
18 1.0 41 0.60 Among the diterpenes tested, norcaesalpinin E (28), a C-17-
19 0.40 42 2.9 norcassane-type diterpene with an acetoxyl group at C-1 and
20 10 43 10 a hydroxyl group at C-7, showed the most potent activity
21 0.12 44 10
22 10
(IC50, 0.090 m M) which is stronger than the well-known anti-
23 1.7 Chloroquine 0.29 malarial drug, chloroquine (IC50, 0.29 m M). From the ob-
served antimalarial activities, we concluded that the presence
of an acetoxyl group at C-1 and a hydroxyl group at C-7
The activities of these diterpenes were greatly dependent on plays the most important role for the antimalarial activity.
the nature of the substitution, and careful evaluation of the The inhibiory activity exhibited by these diterpenes could
IC50 values led to the correlation between structure and activ- support the traditional use of the seed kernels of C. crista as
ity. an antimalarial drug.
Effect of Hydroxyl Group The presence of a hydroxyl
group at C-7 plays crucial role for strong antimalarial activ- Acknowledgement A part of this work was supported
ity. The diterpenes having a hydroxyl group at C-7 showed by a Grant-in-Aid for International Scientific Research (No.
stronger activity than those with an acetoxyl substituent at C- 16406002) from the Ministry of Education, Culture, Sports,
7 (2911, 3918, 4041) or without substituent at C-7 Science and Technology, Japan.
(3915). The strong activity of caesalpinin N (21; IC50,
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