Antiplasmodial phenolic compounds from Piptadenia pervillei

Article abstract of DOI:10.1055/s-2008-1034328

Piptadenia pervillei Vatke (Fabaceae) was selected from a screening programme devoted to the search of naturally-occuring antimalarial compounds from plants of Madagascar. Bioassay-guided fractionation of the ethyl acetate extract of the leaves led to the isolation of four phenolic compounds, (+)-catechin (1), (+)-catechin 5-gallate (2), (+)-catechin 3-gallate (3) and ethyl gallate (4). Structures were determined by NMR and mass spectroscopy. Compounds 2 and 3 displayed the highest in vitro activity against the chloroquine-resistant strain FcB1 of Plasmodium falciparum with IC₅₀ values of 1.2 μM and 1.0 μM, respectively, and no significant cytotoxicity against the human embryonic lung cells MRC-5 was measured (IC₅₀ values > 75 μM). Five analogues (5-9) of (+)-catechin 5-gallate (2) were synthesized and evaluated for their antiplasmodial activity. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2008-1034328

Original Paper 417
Antiplasmodial Phenolic Compounds from Piptadenia
pervillei
Author
Voahangy Ramanandraibe1, Philippe Grellier2, Marie-Thérèse Martin3, Alexandre Deville4, Roger Joyeau4,
David Ramanitrahasimbola1, Elisabeth Mouray2, Philippe Rasoanaivo1, Lengo Mambu4
Affiliation
Affiliation addresses are listed at the end of the article
Key words
Abstract
and mass spectroscopy. Compounds 2 and 3 dis-
played the highest in vitro activity against the
chloroquine-resistant strain FcB1 of Plasmodium
falciparum with IC50 values of 1.2 μM and 1.0 μM,
respectively, and no significant cytotoxicity
against the human embryonic lung cells MRC-5
was measured (IC50 values > 75 μM). Five ana-
logues (5–9) of (+)-catechin 5-gallate (2) were
synthesized and evaluated for their antiplasmo-
dial activity.
"
● Piptadenia pervillei
!
"
● Fabaceae
Piptadenia pervillei Vatke (Fabaceae) was select-
ed from a screening programme devoted to the
search of naturally-occuring antimalarial com-
pounds from plants of Madagascar. Bioassay-guid-
ed fractionation of the ethyl acetate extract of
the leaves led to the isolation of four phenolic
compounds, (+)-catechin (1), (+)-catechin 5-gal-
late (2), (+)-catechin 3-gallate (3) and ethyl gal-
late (4). Structures were determined by NMR
"
● antiplasmodial activity
"
● (+)-catechin gallate
Introduction
was selected. This plant is endemic to Madagas-
car [6] and no traditional uses were reported.
The ethyl acetate extract of the leaves was found
!
Drug-resistant Plasmodium falciparum malaria is
a major killer and one of the most important ob-
stacles to world health, especially in the develop-
ing countries of Africa. Parasites have developed
resistance to all currently used antimalarial ther-
apeutics, and this has been a driving force and
challenge for much work devoted to the elucida-
tion of the mechanisms of this resistance [1]. In
the absence of effective vaccines, impregnated
bednets and chemotherapy will remain the two
most relevant tools for controlling malaria. Refer-
ring to chemotherapy, the development of a new
class of drug is an urgent matter. To this end,
screening of plant extracts for antiplasmodial ac-
tivity is a useful way for discovering new leads,
and the combination of structure-based design,
mechanism-based design and combinatorial syn-
thesis would be one appropriate strategy to bring
lead candidates into drug development [1], [2],
[3], [4].
to exhibit a high antiplasmodial activity (IC50
=
0.5 μg/mL on the chloroquine-resistant strain
FcM29 of P. falciparum) as well as a cytotoxic ef-
fect on murine P388 leukemia cells (IC50 = 8.5 μg/
mL) [5]. The resulting selectivity index value,
SI = 17, defined as the ratio of cytotoxicity over
the antiplasmodial activity, prompted us to fur-
ther investigate this species. A literature search
revealed no prior phytochemical or pharmaco-
logical studies on Piptadenia pervillei. The EtOAc
extract of this plant was submitted to a series of
bioassay-directed fractionations and the most
potent antiplasmodial fractions were rechroma-
tographed leading to the isolation of four phenol-
received September 11, 2007
revised January 25, 2008
accepted February 6, 2008
Bibliography
DOI 10.1055/s-2008-1034328
Planta Med 2008; 74: 417–421
© Georg Thieme Verlag KG
Stuttgart · New York
Published online March 11,
2008
ISSN 0032-0943
Correspondence
Dr. Lengo Mambu
USM 0502-UMR 5154
CNRS Chimie et Biochimie des
Substances Naturelles
Département Régulations,
Développement et Diversité
Moléculaire
Muséum National d'Histoire
Naturelle
CP 54, 57 rue Cuvier
75231 Paris Cedex 05
France
"
ic compounds 1–4 (● Fig. 1). In this paper, we
report the isolation and structure elucidation of
these molecules as well as their in vitro antiplas-
modial and cytotoxicity activities. To attempt an
investigation of the structure activity-relation-
ship, some analogues of compound 2 were syn-
thesized and evaluated for their antiplasmodial
and cytotoxicity activities.
As part of our search for novel antimalarial
agents from plants, a screening programme was
undertaken on plants of Madagascar on the basis
of their traditional uses as antimalarials or their
endemicity in ecologically different regions [5].
In our preliminary screen, Piptadenia pervillei
Vatke, renamed Entada pervillei Vatke (R.Vig.)
Tel.: +33-1-4079-5607
Fax: +33-1-4079-3135
mambu@mnhn.fr
Ramanandraibe Vet al. Antiplasmodial Phenolic Compounds… Planta Med 2008; 74: 417–421

418 Original Paper
Plant material
Leaves of Piptadenia pervillei were collected in the eastern forest
of Madagascar in August 2000. The plant material was identified
by Armand Rakotozafy by comparison with an authentic speci-
men deposited at the Department of Botany of the Parc Botani-
que et Zoologique de Tsimbazaza, Antananarivo. A voucher
specimen has been kept at the Institut Malgache de Recherches
Appliquées under the collection ID AR/MORA-014.
Extraction and bioassay-guided isolation
The air-dried powdered leaves of P. pervillei (200 g) were extract-
ed three times with ethanol (EtOH) (500 mL, 3×24 hours) at
room temperature, and the pooled ethanolic solutions were
evaporated to dryness under reduced pressure to afford a crude
extract. Partitioning of this extract between water and ethyl ace-
tate (EtOAc) (3×500 mL) yielded a soluble organic extract,
which displayed in vitro inhibitory activity against the chloro-
quine-resistant strain FcB1 of P. falciparum with an IC50 value of
3.7 μg/mL, whereas the water-soluble extract was devoid of anti-
plasmodial activity. Silica gel column chromatography of this ex-
tract (5 g) over 150 g (Ø column 40 mm, 25 mL fractions) with
the solvent gradient CH2Cl2-MeOH gave 7 fractions. Three frac-
tions, 3 (419 mg), 4 (850 mg) and 5 (1.83 g) showed significant
antiplasmodial activities with IC50 values of 1.8 μg/mL, 1.4 μg/
mL and 0.9 μg/mL, respectively. Purification of fraction 5 by col-
umn chromatography over silica gel (70 g) with CH2Cl2-MeOH-
H2O (80:20:5, 500 mL) yielded the active subfraction 53 with
an IC50 value of 0.6 μg/mL (173 mg) which was subjected to col-
umn chromatography over Sephadex LH-20 eluted with MeOH-
H2O (70:30, 300 mL). Further separation of subfraction 532
(17 mg) on Sephadex LH-20 (MeOH-H2O 85:15, 40 mL) furnish-
ed compounds 2 (5 mg) and 3 (1.5 mg) and on TLC, they gave
both a strong blue dark spot with FeCl3.
Fig. 1 Structures of compounds 1 – 3.
Materials and Methods
!
General
The 1H- and 13C-NMR spectra were recorded at 599.915 and
150.93 MHz, respectively, on a Bruker AVANCE-600 spectrome-
ter or at 400.13 and 100.61 MHz, respectively, on a Bruker
AVANCE-400 spectrometer at 298 K, equipped with a 1H-broad-
band reverse gradient probehead. Temperature was controlled
by a Bruker BCU-05 refrigeration unit and a BVT 3000 control
unit. Chemical shifts (δH) and (δC) are expressed in ppm relative
toTMS with coupling constants (J) given in Hz. Mass spectra data
were recorded with an electrospray time of flight mass spec-
trometer (ESI-TOF-MS) operating in the positive and negative
mode (QSTAR Pulsar I; Applied Biosystems). Optical rotations
were conducted on a Perkin-Elmer 341 polarimeter at 20 °C. An-
alytical and preparative TLC were carried out on precoated Kie-
selgel 60 F254 plates (Merck). Spots were detected under UV
(254 and 366 nm) before spraying with anisaldehyde-sulfuric
acid solution in EtOH followed by heating the plate at 110 °C or
with 2% ethanolic ferric chloride reagent. Column chromatogra-
phy was performed on 230–400 mesh silica gel 60 (Merck) and
on Sephadex LH-20 (25–100 μm; Pharmacia Biotech Ltd). Prep-
arative medium-pressure liquid chromatography (MPLC) was
carried with a pump K-120 (Knauer) and Flasmart cartridges
(silica gel 20–40 μm; AIT).
Successive treatment of fraction 4 on silica gel (34 g) with
CH2Cl2-MeOH (93:7, 250 mL) and Sephadex LH-20 (MeOH-H2O
70:30, 80 mL) afforded compound 1 (5 mg). Compound 4
(33 mg) was obtained from fraction 3 by column chromatogra-
phy over silica gel (20 g) with cyclohexane-EtOAc (60:40,
210 mL).
Fig. 2 Synthesis of the hemisynthetic derivatives. Reagents and conditions: (i) BrCH2Cl, Cs2CO3, DMF, reflux, 33%; (ii) (C6H5)2CCl2, Et3N, CH3CN, 0°C to r.t.,
20 h, 20%; (iii) tri-O-benzylgalloyl chloride, DMAP, pyridine, r.t., 24–48 h, 10–17%; (iv) H2, 10% Pd-C, MeOH or EtoAc, 12–24 h.
Ramanandraibe Vet al. Antiplasmodial Phenolic Compounds… Planta Med 2008; 74: 417–421

Original Paper 419
mean from 3 independent experiments. Camptothecin (Sigma
Aldrich Chimie) was used for comparison as standard drug of cy-
totoxic activity.
Table 1 Hemisynthetic derivativesa
Compounds
R
R1
R2
(+)-Catechin 5-O-gallate (2): 1H-NMR (600 MHz, CD3OD): δ = 7.16
(s, 2H, H2′′, H6′′), 6.81 (d, J = 1.6, H-6′), 6.73 (d, J = 8 Hz, 1H, H-5′),
6.70 (dd, J = 2, 8 Hz, H-2′), 6.23 (d, J = 2.2 Hz, 1H, H-8), 6.20 (d,
J = 2.2 Hz, 1H, H-6), 4.61 (d, J = 7.4 Hz, 1H, H-2), 3.95 (m, 1H, H-
3), 2.72 (dd, J = 8.2, 16 Hz, 1H, H-4b), 2.46 (dd, J = 5.2, 16 Hz, 1H,
H-4a); 13C NMR (150.75 MHz, CD3OD): δ = 166.6 (CO), 158.3 (C-
7), 157.2 (C-9), 151.9 (C-5), 146.9 (C-5′′, C-3′′), 146.5 (C-3′, C-4′),
140.1 (C-4′′), 131.9 (C-1′), 120.4 (C-1′′), 120.1 (C-6′), 116.2 (C-5′),
115.3 (C-2′), 110.7 (C-2′′, C-6′′), 106.6 (C-10), 103.6 (C-6), 101.7
(C-8), 83.2 (C-2), 68.3 (C-3), 28.8 (C-4).
11a
12a
13a
11b
12b
13b
5
H
H
Gp
H
H
Gp
Gp
H
H
Gp
Gp
H
C6H5
C6H5
Gp
Gp
H
C6H5
Gp
Ga
H
–
–
–
–
–
6
Ga
Ga
Ga
Ga
7
Ga
H
8
9
Ga
a
"
●
■
Results and Discussion
!
By bioassay-guided fractionation, four phenolic compounds 1–4
were isolated. Compound 1 was identified as (+)-catechin by
comparison of its spectral data with those published in the liter-
ature [11].
Preparation of compounds 5–9
The analogues of compound 2 were synthesized from commer-
Compound 2 isolated as an amorphous powder exhibited a qua-
si-molecular ion [M + H]+ at m/z = 443 in the ESI-TOF-MS spec-
trum in agreement with the molecular formula C22H18O10. Inter-
pretation of the COSY, HMQC and HMBC spectra demonstrated 2
to be a derivative of catechin 1. The chemical shift pattern of 7
additional 13C signals with reference to compound 1, together
with the presence of the singlet at δ = 7.16 accounting for two
protons, indicated the presence of a galloyl unit in compound 2.
The upfield shift of the C-5 signal and the downfield shift of C-6,
C-8 and C-10 signals with respect to compound 1 strongly sug-
gested that the galloyl moiety was linked to C-5. Long-range cor-
relations between H-6 and H-4 and C-5 were in agreement with
our hypothesis. The typical downfield shifts due to esterification
indicated the position of the carbon linked with the galloyl
group. However, the NMR spectral data of compound 2 were
also similar to those of (+)-catechin7-O-gallate isolated from
Sanguisorba officinalis with inversion at C-5 and C-7 [12]. In this
previous work, the structure was elucidated only by using 1 D
NMR data and chemical modification. To definitively establish
the position of the galloyl group, NMR spectra were performed
in deuterated DMF at 230 K, which, unlike those performed in
DMSO-d6, allowed us to distinguish a free hydroxy group (OH-
3) at δ = 5.69 and three free phenolic hydroxy groups at δ = 9.70,
10.24 and 10.65, respectively. The observation of a correlation
between the phenolic hydroxy group at δ = 10.65 and H-8 in the
NOESY spectrum confirmed its presence in position 7, and sub-
sequently allowed us to assign the galloyl group to position 5.
On the basis of all the above evidence, compound 2 was deter-
mined to be (+)-catechin 5-O-gallate known as (+)-catechin 5-
gallate [13].
"
cially available (+)-catechin (Sigma) as outlined in Fig. ● 2 (and
"
Table ● 1). The catechol function was protected either as the
methylene acetal with bromochloromethane (series a) and pro-
vided 10a in 33% yield, or with the diphenylmethylene ketal
group by treatment of 1 with dichlorodiphenylmethane (series
b) affording 10b in 20% yield [7], [8]. The reagent necessary for
galloylation was obtained from gallic acid by benzylation with
benzyl bromide followed by treatment with oxalyl chloride. Es-
terification of protected catechin (10a, 10b) by tri-O-benzylgal-
loyl chloride in pyridine and in the presence of DMAP gave a
mixture of three compounds for each series, 11, 12, 13, in 10–
17% yields [9]. The separation of these compounds was per-
formed by flash chromatography on silica gel or by MPLC and
the structure of each one was determined by 2 D NMR and MS
analysis. Removal of the benzyl and diphenylmethylene groups
was achieved by hydrogenolysis using 10% palladium on actived
charcoal. Deprotection of 11b gave a derivative of which the
spectral data were identical to those of compound 2. Compounds
5–7 were obtained without need of purification while com-
pounds 8 and 9 had to be purified on Sephadex LH-20 to remove
diphenylmethane generated during the reaction. All final com-
pounds displayed spectral data (NMR, MS) which were consis-
tent with the assigned structures.
In vitro antiplasmodial test
The in vitro antiplasmodial tests, based on the inhibition of [3H]-
hypoxanthine uptake by Plasmodium falciparum cultured in hu-
man blood, were performed as previously described [10]. Chlor-
oquine diphosphate (Sigma Aldrich Chimie SARL) was used as
positive control of the antiplasmodial activity.
Preliminary interpretation of the NMR data of compound 3 indi-
cated that it was an isomer of compound 2 and was a flavonoid
closely related to catechin 1. Thus, when 1H-NMR spectrum of
compound 3 was compared to that of compound 1, there was a
downfield shilft of H-3 (+δ 1.39), along with the appearance of a
singlet accounting for two protons at δ = 6.95. These preliminary
observations suggested that compound 3 might be a catechin
derivative esterified by gallic acid at C-3. The structure of com-
pound 3 was definitively assigned as (+)-catechin 3-O-gallate
known as (+)-catechin 3-gallate on the basis of comparison of
its spectroscopic data with those given in literature [11].
In vitro cytotoxicity test on mammalian cells
Cytotoxicity was evaluated using human diploid embryonic lung
cell line MRC-5. Cells were obtained from ATCC. They were
maintained for 5 days in culture in the presence of drug and the
cytotoxicity was determined using the colorimetric MTT assay
according to the manufacturer's recommendations (cell prolifer-
ation kit I, Roche Applied Science). The concentration causing
50% of growth inhibition (IC50) was obtained from the drug con-
centration-response curve, and the results were expressed as the
Ramanandraibe Vet al. Antiplasmodial Phenolic Compounds… Planta Med 2008; 74: 417–421

420 Original Paper
Table 2 In vitro antiplasmodial and cytotoxicity activities of compounds 1–9
Compound
Antiplasmodial test, IC50 SD (μM)a
Cytotoxicity test, IC50 (μM)b
Selectivity Index (SI)
1
75.9 4.6
1.2 0.2
1.0 0.2
8.1 0.6
6.4 1.1
18.7 2.2
6.6 0.5
16.3 2.3
10.2 1.5
0.12 0.04
ND
> 75
> 1
2
> 75
> 62
> 75
> 9
3
> 75
4
> 75
5
ND
ND
ND
8.4
2.7
1.9
ND
ND
6
ND
7
55.3 3.6
43.4 7.9
19.0 3.9
ND
8
9
Chloroquine
Camptothecin
0.0035 0.0009
a Chloroquine-resistant strain FcB1 was used for the antiplasmodial test.
b The cytotoxicity test was evaluated against MRC-5 cells.
ND: not determined. All experiments were realized in triplicate. Selectivity index is defined as the ratio of IC50 value determined on MRC-5 cells over the IC50 on
P. falciparum.
Compound 4, an amorphous powder, exhibited an [M + H]+ ion
peak at m/z = 199 compatible with the molecular formula
C9H10O5 as established by the positive ion TOF-MS. This com-
pound was unambiguously assigned as ethyl gallate. It was prob-
ably an artifact produced by esterification of gallic acid, which
occurs with alcoholic solvents during the extraction process
[14].
synthesis (FAS-II) pathway of P. falciparum, with IC50 values on
these enzymes in the range of 0.2–1.1 μM [22]. Among these cat-
echin derivatives, (–)-catechin 3-gallate was the most active in
vitro against P. falciparum with IC50 values of 0.4 and 3.2 μM, re-
spectively, against the chloroquine-sensitive strain NF54 and the
chloroquine-resistant strain K1. A similar range of inhibition was
observed for this compound on the chloroquine-resistant strain
FcB1 (IC50 value of 1μM) in the present study. The other C-3 gal-
late ester derivatives showed moderate antiplasmodial activity
(IC50s between 6.2 and 40 μM). Finally, epigallocatechin gallate
and epicatechin gallate have been found to potentiate the action
of the antimalarial drug artemisinin [23].
To investigate the structure activity-relationship, hemisynthetic
derivatives 5–9 (analogues of 2) were prepared and their anti-
plasmodial activity evaluated in vitro. These compounds dis-
played moderate to weak activity against P. falciparum with IC50
values between 6.4 to 18.7 μM and weak to no significant activi-
ty against MRC-5 cells. Protection of 3′,4′-hydroxy groups (com-
pound 5) reduced the activity in comparison to compound 2.
Surprisingly, compounds 6, 8 and 9 with the galloyl moiety in
position 7 were less active than those with it in position 3 or 5
(compounds 2, 3, and 5) suggesting probably that the proximity
with a free hydroxyl or phenolic group may be favourable to the
activity.
The in vitro antiplasmodial activity of compounds 1–4 are
"
shown in Table ● 2. (+)-Catechin 3-gallate (3) and (+)-catechin
5-gallate (2) were found to be the most active against the chlor-
oquine-resistant strain FcB1 of P. falciparum with IC50 values of
1.2 μM and 1.0 μM, respectively. Ethyl gallate (4) was less active
with an IC50 value of 8.1 μM. Catechin 1 showed no significant
activity. The antiplasmodial activity of compounds 2, 3 and 4 ap-
pears thus associated with the presence of a gallate ester which
plays an important role in inhibition against P. falciparum. Com-
pounds 2 and 3 exhibited comparable potency and the position
of the galloyl group either at 3 or 5 does not seem to influence
the activity. No significant cytotoxicity was measured on the hu-
man embryoic lung cells MRC-5 for these compounds (IC50
>
75 μM) and interesting selectivity towards P. falciparum was re-
corded (SI > 60). Work on phenolic compounds has shown that
gallic acid esters and also catechin gallate esters had interesting
anticancer properties, which seem to be caused by their antiox-
idant character [15], [16]. Some authors suggested that the for-
mation of reactive oxygen species might be involved in the gallic
acid induced apoptotic cell death but the mechanism of action
remains uncertain [17]. Their ability to inhibit growth of other
parasites was also tested and high activity was observed for eth-
yl gallate and catechin 3-gallate against Trypanosoma cruzi and
for catechin 3-gallate against Leishmania donovani [18], [19].
A previous investigation on catechin 1 had shown that it was
devoid of activity against P. falciparum and this was confirmed
in this work [20]. Catechin and propyl gallate had only been re-
ported to suppress the erythrocytic oxidative damage generated
during infection by Plasmodium berghei on mice thanks to their
antioxydant properties [21].
In summary, we have isolated and characterized four com-
pounds including two catechin gallates (2 and 3), catechin (1)
and ethyl gallate (4) from the leaves of Piptadenia pervillei by
means of bioassay-guided fractionation. Access to further cate-
chin gallate derivatives by synthesis showed that the best anti-
plasmodial activity was displayed when the catechol group is
free and a galloyl moiety is present in the 3 or 5 positions.
Acknowledgements
!
We thank the International Foundation for Science for giving
us a research grant (Dr Ramanandraibe, No. F/3423-1). This
work was supported by the CNRS and the French Ministry of
Research.
More recently, four catechins esterified with gallic acid at posi-
tion C-3, namely (–)-epicatechin gallate, (–)-epigallocatechin
gallate, (–)-catechin 3-gallate and (–)-gallocatechin gallate
were reported to be potent inhibitors of β-ketoacyl-ACP-reduc-
tase (FabG), β-hydroxacyl-ACP-dehydratase (FabZ), and enoyl-
ACP-reductase, enzymes involved in the type-II fatty acid bio-
Affiliation
1
Laboratoire de Bio-thérapeutique, Institut Malgache de Recherches Appli-
quées, Antananarivo, Madagascar
Ramanandraibe Vet al. Antiplasmodial Phenolic Compounds… Planta Med 2008; 74: 417–421

Original Paper 421
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