Identification of two new phenathrenones and a saponin as antiprotozoal constituents of Drypetes gerrardii

Article abstract of DOI:10.1016/j.phytol.2014.05.005

In an in vitro screen of 206 extracts from South African plants, the CH₂Cl₂/MeOH (1:1) stem extract of Drypetes gerrardii Hutch. var gerrardii (Putranjivaceae) inhibited Plasmodium falciparum and Leishmania donovani (IC₅₀s of 0.50 and 7.31 μg/ml, respectively). In addition, the CH₂Cl₂/MeOH (1:1) extract of the leaves showed activity against Trypanosoma brucei rhodesiense (IC₅₀ of 12.1 μg/ml). The active constituents were tracked by HPLC-based activity profiling, and isolated by preparative and semi-preparative RP-HPLC chromatography. Their structures were established by HRESIMS, and 1D and 2D NMR (¹H, ¹³C, COSY, HMBC, HSQC, and NOESY). From the stem extract, a new phenanthrenone derivative, drypetenone D (1), and a phenanthrenone heterodimer, drypetenone E (2), were isolated. Compound 1 showed potent in vitro activity against P. falciparum (IC₅₀ of 0.9 μM) with a selectivity index (SI) of 71, as calculated from cytotoxicity data for L-6 cells. These data qualified 1 for in vivo assessment in the Plasmodium berghei mouse model, but the compound turned out to be inactive. Compound 2 also exhibited good in vitro antiplasmodial activity (IC₅₀ of 2.0 μM) and selectivity (SI 31). From the leaf extract, the saponin putranoside A (3) was isolated and identified. Compound 3 showed weak in vitro trypanocidal activity, with an IC₅₀ of 18.0 μM, and a SI of 4.

Full text of DOI:10.1016/j.phytol.2014.05.005

G Model
PHYTOL-711; No. of Pages 8
Phytochemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Identification of two new phenathrenones and a saponin as
antiprotozoal constituents of Drypetes gerrardii
Yoshie Hata a,d,1, Maria De Mieri a,1, Samad Nejad Ebrahimi a,e, Tsholofelo Mokoka c,
Gerda Fouche c, Marcel Kaiser b, Reto Brun b, Olivier Potterat a, Matthias Hamburger a,
*
a Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
b Swiss Tropical and Public Health Institute and University of Basel, Socinstrasse 57, CH-4002 Basel, Switzerland
c Biosciences, Council for Scientific and Industrial Research (CSIR), P.O. Box 395, Pretoria 0001, South Africa
d Department of Pharmacy, Faculty of Sciences, National University of Colombia, Carrera 30 No. 45-03, Bogota, DC 111321, Colombia
e Department of Phytochemistry, Medicinal Plant and Drugs Research Institute, Shahid Beheshti University, G.C., Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article history:
In an in vitro screen of 206 extracts from South African plants, the CH2Cl2/MeOH (1:1) stem extract of
Drypetes gerrardii Hutch. var gerrardii (Putranjivaceae) inhibited Plasmodium falciparum and Leishmania
Received 26 March 2014
Received in revised form 25 April 2014
Accepted 6 May 2014
Available online xxx
donovani (IC50s of 0.50 and 7.31
mg/ml, respectively). In addition, the CH2Cl2/MeOH (1:1) extract of the
leaves showed activity against Trypanosoma brucei rhodesiense (IC50 of 12.1
mg/ml). The active
constituents were tracked by HPLC-based activity profiling, and isolated by preparative and semi-
preparative RP-HPLC chromatography. Their structures were established by HRESIMS, and 1D and 2D
NMR (1H, 13C, COSY, HMBC, HSQC, and NOESY). From the stem extract, a new phenanthrenone derivative,
drypetenone D (1), and a phenanthrenone heterodimer, drypetenone E (2), were isolated. Compound 1
Keywords:
Antiprotozoal
Drypetes gerrardii
Putranjivaceae
showed potent in vitro activity against P. falciparum (IC50 of 0.9
mM) with a selectivity index (SI) of 71, as
calculated from cytotoxicity data for L-6 cells. These data qualified 1 for in vivo assessment in the
Plasmodium berghei mouse model, but the compound turned out to be inactive. Compound 2 also
Phenanthrenones
HPLC-based activity profiling
exhibited good in vitro antiplasmodial activity (IC50 of 2.0
extract, the saponin putranoside A (3) was isolated and identified. Compound 3 showed weak in vitro
trypanocidal activity, with an IC50 of 18.0 M, and a SI of 4.
mM) and selectivity (SI 31). From the leaf
m
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
Glossina) transmit the disease. The number of reported cases was
7197 in 2012. Trypanosoma brucei gambiense accounts for more
Tropical parasitic diseases have devastating social and eco-
nomic effects for Third World countries (Renslo and McKerrow,
2006). According to WHO estimations, 207 million cases of malaria
infections and 627,000 malaria deaths occurred worldwide in
2012. Malaria is transmitted by mosquitoes of the genus Anopheles
and caused by five Plasmodium species. Artemisinin derivatives are
the most recent drugs, but artemisinin-resistant Plasmodium
falciparum strains have already emerged in Asia (Miller et al.,
2013; WHO, 2013a,b). Human African trypanosomiasis (HAT) is
confined to sub-Saharan African countries where tsetse flies (genus
than 98% of the reported cases of HAT and Trypanosoma brucei
rhodesiense for the rest. Drugs in current use show varying degrees
of toxicity and efficacy, and all require parenteral administration
(WHO, June, 2013; Brun et al., 2011). There are more than 21
species of Leishmania sp. that cause various forms of leishmaniasis
ranging from the cutaneous form to the lethal, if untreated, visceral
form (VL). VL has an estimated global incidence of 200,000–
500,000 cases and a prevalence of 5 million cases worldwide.
Antileishmanial drugs are available, but suffer from limited
efficacy, long treatments, or high costs (Stuart et al., 2008;
WHO, 2010). In view of the unsatisfactory therapeutic options
currently available, there is an urgent need for new drugs to treat
these diseases (Renslo and McKerrow, 2006; Stuart et al., 2008).
Natural products constitute an invaluable source of new drug
scaffolds, particularly in the area of infectious diseases (Hannaert,
2011; Renslo, 2013). In the search for new antiprotozoal
compounds, we screened a library of South African medicinal
*
Corresponding author at: Division of Pharmaceutical Biology, University of
Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. Tel.: +41 61 267 14 25;
fax: +41 61 267 14 74.
E-mail address: matthias.hamburger@unibas.ch (M. Hamburger).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2014.05.005
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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PHYTOL-711; No. of Pages 8
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Y. Hata et al. / Phytochemistry Letters xxx (2014) xxx–xxx
plants for antiprotozoal activity (Hata et al., 2011, 2013; Mokoka
et al., 2011, 2013). A stem extract from Drypetes gerrardii Hutch.
var. gerrardii (Putranjivaceae) showed pronounced antiplasmodial
and antileishmanial activities. In addition, a leaf extract of the
same plant exhibited trypanocidal properties (Mokoka et al.,
2013). There are few reports about the phytochemistry of D.
gerrardii. So far, triterpenes belonging to the friedelane, hopane
and lupane series, and flavone dimers, amentoflavone and
drypetdimer A, have been isolated (Ng’ang’a et al., 2008, 2011).
The antiplasmodial activity of the dimeric flavonoids was
evaluated against the K1 strain of P. falciparum (Ng’ang’a et al.,
2012). We report here on the identification of the antiprotozoal
constituents from stem and leaf extracts, and on the biological
assessment of the isolated compounds.
obtained. The structures of 1 and 2 were determined by HRESIMS
and NMR analyses (1H NMR, 13C NMR, HMBC, HSQC, and NOESY).
Compound 1 had a molecular formula of C17H16O2 HRESIMS m/
z = 275.1067 [M+Na] + (calcd. for C17H16NaO2, 275.1043), thus
requiring 108 of indices of hydrogen deficiency. The 1H and 13C
NMR spectra displayed resonances in the low field region of the
spectrum and were indicative of a highly substituted aromatic ring
system. The 1H NMR spectrum in CDCl3 displayed an AX olefinic
spin system at
ortho coupled aromatic protons at
J = 8.5 Hz), two aromatic protons as singlets at
aliphatic geminal dimethyl group H 1.51 (6H, s), and an aromatic
d
H 8.20 (d, J = 10.1 Hz) and 6.29 (d, J = 10.1 Hz), two
H 7.75 (d, J = 8.5 Hz) and 7.39 (d,
H 7.59 and 7.53, an
d
d
d
methyl group at dH 2.42. The 13C NMR spectrum displayed
seventeen signals consistent with three methyls, six methines,
eight quaternary carbons including a carbonyl (dC 205.5), one
oxygenated aromatic carbon (dC 154.7) and a quaternary carbon
bearing the gem-dimethyl group (dC 48.1). A careful analysis of
HMBC and NOESY correlations (Fig. 4) suggested that 1 possessed a
phenanthrenone skeleton similar to trigohowilol A (4) (Tang et al.,
2012) except for the absence of the hydroxyl group at C-9 and of
the O-methyl substituent at C-6 in 1. The location of the scalar
2. Results and discussion
The CH2Cl2/MeOH 1:1 extract of the stems of D. gerrardii
inhibited P. falciparum and L. donovani with IC50 values of 0.50
mg/
ml and 7.31 g/ml, respectively. Activity in the extract was tracked
m
by HPLC-based activity profiling (Fig. 1). The chromatogram
exhibited a strong UV-absorbing peak at tr = 19.0 min which
correlated with 71% inhibition of L. donovani, and 95% inhibition of
P. falciparum. The compound was isolated by flash chromatography
of the CH2Cl2/MeOH (1:1) extract, followed by semi-preparative
RP-HPLC. In addition, a structurally related compound 2 was
coupled doublets (dH 7.75 and 7.39, J = 8.5 Hz) at positions C-9 and
C-10, respectively, was inferred from HMBC correlations from H-9
to C-8, C-4b and C-10a, and from H-10 to C-4a and C-8a. This
structural assignment was corroborated by a NOESY correlation
between H-9 and H-8, and between H-10 and the gem-dimethyl
group at C-1. Thus, the structure of 1 was established as shown in
Fig. 3 and the compound was named drypetenone D.
The molecular formula of compound 2 was determined as
C34H30O4
HRESIMS
m/z = 525.2063
[M+Na] + (calcd.
for
C34H30NaO4, 525.2036), with 20 indices of hydrogen deficiency.
The 1H and 13C spectra showed the presence of two sets of signals
resembling those found for compound 1, thus suggesting a
heterodimeric structure for 2. The 1H NMR spectrum displayed
the resonances of: (1) two gem-dimethyl groups, Me-1 (
and Me-10 (
H 1.45, s); (2) two aromatic methyl groups CH3-7 (dH
2.76, s), CH3-70 (
H 2.51, s); (3) two sets of AX olefinic doublets H-3
dH 6.03) and H-30 (dH 5.96), H-4 (dH 7.40) and H-40 (dH 8.70); (4)
two sets of ortho coupled aromatic protons H-9 (
H 7.78) and H-90
H 7.88), H-10 ( H 7.49); (5) three uncoupled
H 7.43) and H-100 (
dH 1.43, s)
d
d
(
d
(
d
d
d
aromatic protons H-5 (dH 6.93), H-8 (dH 7.77) and H-80 (dH 7.66).
The 13C NMR spectrum showed the signals for the aforemen-
tioned protonated carbons (see Table 1) and additionally: (1) two
carbonyl C-atoms resonating at
(2) three oxygen bearing aromatic carbons
(C-60) and C 134.2 (C-50); (3) two aliphatic quaternary carbons dC
48.0 (C-1), dC 48.6 (C-10); (4) ten aromatic quaternary carbons dC
d
C 204.5 (C-2) and
d
C 203.9 (C-20);
d
C 154.6 (C-6),
d
C 147.7
d
122.2 (C-4a) and
d
C 121.7 (C-4a0), 130.3 (C-4b) and
d
C 123.5 (C-4b0),
127.0 (C-7) and
d
C 127.6 (C-70), 129.0 (C-8a) and dC 128.7 (C-8a0),
147.5 (C-10a) and dC 149.2 (C-10a0). Based on HMBC and NOESY
correlations the two substructures A and B (Fig. 5) were identified.
Substructure A corresponded to compound 1, while in substruc-
ture B position C-50 was hydroxylated. In substructure B, the
substitution of the aromatic C ring unit was established on the
basis of the NOESY correlations between H-80 and H-90 and
between H-80 and CH3-70. The positions C-60 (dC 147.7) and C-50
(134.2) of ring C were oxygenated; C-60 chemical shift was
assigned to be d
C 147.7 by the 3JH–C HMBC correlation from 70-CH3
while the remaining upfield shifted 13C resonance was assigned to
position C-50. Considering the substituents of the substructures A
and B it became obvious that they were connected through an
ether linkage between position C-6 of substructure A and positions
C-50 or C-60 of substructure B. Unfortunately, no cross peaks were
detected in the HMBC spectrum from H-5 to substructure B. In a
further effort to discriminate between the two possible regioi-
somers, a long-range optimized (4 Hz) HMBC spectrum was
Fig. 1. HPLC-based activity profiling of the CH2Cl2/MeOH (1:1) stem extract of D.
gerrardii for antiprotozoal activity. HPLC-UV chromatogram (210–700 nm) (A), and
inhibition (in %) of L. donovani (B) and P. falciparum (C) by microfractions.
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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3
Table 1
1H and 13C NMR spectroscopic data of compounds 1 and 2 in CDCl3.
Position
1
2a
HMBC (H ! C)
b
c
dH (J in Hz)
dC
dH (J in Hz)
dC
1
–
48.1
205.5 5
123.6 6
139.9
104.22
154.7
126.8
130.2
130.2
121.8
121.6
131.0
127.9
147.1
28.0
–
48.0
204.5
124.0
138.8
103.0
154.6
127.0
131.3
130.0
123.0
122.2
130.3
129.0
147.5
27.8
2
–
–
3
6.29 (d, 10.1)
6.03 (d, 10.4 Hz)
C-1, C-4a
4
8.20 (d, 10.1)
7.40 (d, 10.4 Hz)
C-2, C-4b, C-10a
C-6, C-7, C-4a, C-8a
5
7.53 (s)
6.93 (s)
6
–
–
7
–
–
8
7.59 (s)
7.77 (s)
C-6
9
7.75 (d, 8.5)
7.78 (d, 8.7 Hz) 13,139.0
C-4b, C-10a
C-1, C-4a, C-8a
10
7.39 (d, 8.5)
7.43 (d, 8.7 Hz)
4a
–
–
4b
–
–
8a
–
–
10a
–
–
1-Me ꢀ 2
1.51 (s)
2.42 (s)
1.43
C-1, C-2, C-10a
C-6, C-7, C-8
7-Me
16.4
2.76
17.2
10
–
48.6
20
–
203.9
123.0 CC-10
141.8
134.2
147.7
127.6
128.0
131.0
122.9
121.7
123.5
128.7
149.2
27.8
30
5.96 (d, 10.6 Hz)
C-10, C-4a0
C-20, C-4b0, C-10a0
40
8.70 (d, 10.6 Hz)
50
–
60
–
70
–
80
7.66 (s)
C-60, C-90, C-4b0
C-4b0, C-10a0
C-1, C4a0, C-8a0
90
7.88 (d, 8.6 Hz) 13
100
7.49 (d, 8.6 Hz)
4a0
–
4b0
–
8a0
–
10a0
10-Me ꢀ 2
70-Me
–
1.45
2.51
C-10, C-20, C-10a0
C-60, C-70, C-80
16.5
a
The numbering does not follow the IUPAC nomenclature.
b
c
125.77 MHz.
150.95 MHz.
measured. No correlations between the C rings of the two
substructures were detected. However, position C-50 could be
unequivocally assigned by the 4JH–C HMBC observed with H-90 and
H-80. It was identified as the point of attachment of the ether
linkage on substructure B considering the diagnostic upfield shift
of dC C-50 (dC 134.2), with respect to C-60 (dC 147.7). A further
support to this structure assignment came from the NOESY
spectrum that showed a strong dipolar coupling between CH3-7
perpendicular and the distance between CH3-7 and H-40 was
˚
3.45 A for both conformers, thus matching the observed dipolar
coupling. The tridimensional rearrangement of drypetenone E (2)
could also provide an explanation of the significant differences in
the 1H chemical shift between drypetenone D (1) and substructure
A of 2.
There are few reports of naturally occurring phenanthrenones.
Such compounds have been isolated from a marine bacterium,
Pseudomonas stutzeri (Uzair et al., 2008), and from terrestrial plants
of the genera Strophioblachia (Seephonkai et al., 2013a,b;
Seephonkai et al., 2009) and Trigonostemon (Hu et al., 2009;
Kokpol et al., 1990; Tang et al., 2012; Zhu et al., 2010), which
belong to the Euphorbiaceae family. Interestingly, the genus
Drypetes was formerly included in this family.
(d dH 8.70). On the basis of these data the structure
H 2.76) and H-40 (
of 2 was assigned as reported in Fig. 3 and the new compound
named drypetenone E.
A conformational analysis of 2 was performed, followed by
geometrical optimization for the conformers found within 1 kcal/
mol from the corresponding global minimum energy. Two iso-
energetic conformers were found (Fig. 6). DFT-optimized struc-
tures showed that the two substructures A and B were almost
Compound 1 showed low in vitro activity against L. donovani
(Table 2). In contrast, 1 remarkably inhibited P. falciparum (IC50 of
Table 2
In vitro antiprotozoal activities of 1–3 against P. falciparum, T. b. rhodesiense, L. donovani, and cytotoxic activity against L-6 cells.
Compound
P. falciparum
IC50
M)a
T. b. rhodesiense
IC50
M)a
L. donovani
Cytotoxicity
IC50
M)a
(
m
SI
(
m
SI
IC50
(
m
M)a
SI
(m
1
0.96 ꢁ 0.25
71.4
31.4
6.0 ꢁ 2.7
11.5
–
14.0
–
4.9
–
68.4 ꢁ 4.6
2
2.04 ꢁ 0.15
–
64.0 ꢁ 22.4
3
inactiveb
18.0 ꢁ 2.6
3.8
–
7.8
–
8.8
–
68.2 ꢁ 3.9
Chloroquinec
Melarsoprolc
Miltefosinec
Podophyllotoxinc
0.004 ꢁ 0.001
–
–
–
–
–
–
–
–
–
0.003 ꢁ 0.001
–
–
–
–
–
–
–
0.552 ꢁ 0.051
–
–
–
–
-
0.019 ꢁ 0.006
SI (selectivity index): Quotient of IC50 for L-6 cells and IC50 against parasite.
a
Values are expressed as mean ꢁ standard error of the mean.
b
No activity observed at the highest test concentration of 90
Reference drugs.
mg/ml, which corresponds to a molar test concentration of 115.7 mM.
c
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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PHYTOL-711; No. of Pages 8
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Y. Hata et al. / Phytochemistry Letters xxx (2014) xxx–xxx
0.96
of 68
m
m
M) and displayed low cytotoxicity for the L-6 cell line (IC50
M). The selectivity index (SI) calculated as the quotient of
IC50 for L-6 cells and IC50 against parasites gave a value of 71.
Compound 2 displayed good in vitro activity against P. falciparum
(IC50 of 2.0
mM), even though the selectivity (SI = 31) was not as
high as the one of 1. The low yield of the compound probably
explains that its activity was not detected in the HPLC-based
activity profiling. These results granted in vivo assessment of the
antimalarial efficacy of 1 in a mouse model. The full suppressive
four-day test was performed on female NMRI mice infected with a
GFP-transfected Plasmodium berghei strain ANKA. Animals treated
with compound 1 showed no parasitemia reduction on day 4
compared to untreated control mice. The absence of activity in this
model can be due either to absorption problems or unfavourable
PK.
Molecular properties associated with drug-likeness and toxicity
are essential in early compound selection. Descriptors, such us
Lipinski’s ‘‘rule of 5’’ (molecular weight, log P, sum of H-donors/
acceptors), rotatable bonds, and polar surface area (PSA) can
predict drug-likeness. Compound 1 had a molecular weight of
252 Da, log P of 3.96, sum of H-donors/acceptors of 2, 0 rotatable
2
˚
bonds and a PSA of 37.3 A , as calculated by Molinspiration
Cheminformatics software (Cheminformatics, 2014). All these
values fulfil established drug-likeness criteria (Lipinski and
Hopkins, 2004; Pajouhesh and Lenz, 2005). Accordingly, oral
absorption and blood-brain barrier permeability can be expected
for compound 1. Oral availability is a desired characteristic for an
ideal antimalarial drug (Fidock et al., 2004) while blood-brain
barrier permeability is prerequisite for an advanced HAT infection
(Brun et al., 2011).
Fig. 2. HPLC-based activity profiling of the CH2Cl2/MeOH (1:1) leaf extract of D.
gerrardii for antitrypanosomal activity. HPLC-ELSD chromatogram (A) and
inhibition (in %) of T. b. rhodesiense (B) by microfractions.
It is noteworthy that the antiplasmodial triterpenoids which
were previously reported from the leaves and stems of D. gerradii,
including the most active lupane-type derivative resinone (IC50 of
0.09 mg/ml, SI 942) (Ng’ang’a et al., 2011, 2012), were not detected
by HPLC-MS analysis in the course of our study.
An early in silico toxicity prediction can help to anticipate
potential ‘‘off-target’’ activity.
There are a series of proteins involved in development of
adverse effects. When the ability of a compound to bind to these
proteins is simulated and quantified in silico, it is possible to obtain
an estimated toxic potential (TP). By using the virtual technology
‘‘Virtual ToxLab’’ we estimated a TP of endocrine and/or metabolic
disruption, carcinogenicity, and cardiotoxicity (Vedani et al.,
2012). Binding affinities of 1 towards 16 target proteins, were
calculated to obtain a TP of 0.52. This TP value categorizes 1 as a
class I compound (in a scale of 0 to IV, being IV the most toxic). The
binding affinity to critical proteins such as hERG, a potassium ion
3. Materials and methods
3.1. General experimental procedures
HPLC-grade methanol (MeOH), acetonitrile (MeCN) (Scharlau
Chemie S.A.), and water (obtained by an EASY-pure II from
Barnstead water purification system, Dubuque) were used for
HPLC separations. HPLC solvents contained 0.1% HCO2H (Sigma)
for analytical separations. CDCl3 (100 atom% D), DMSO-d6 (100
atom% D), and pyridine-d5 (100 atom% D) for NMR were purchased
from Armar Chemicals (Switzerland). Solvents used for extraction
were of analytical grade (Romil Pure Chemistry). Reference drugs
for bioassays were chloroquine (>98%, Sigma–Aldrich), melarso-
prol (purity > 95%, Sanofi-Aventis), miltefosine (purity > 95%,
VWR), and podophyllotoxin (purity > 95%, Sigma–Aldrich).
HPLC-PDA-MS analyses were performed on an Agilent 1100
system consisting of a degasser, a quaternary pump, a column
oven, a PDA detector and to an Esquire 3000 plus ion trap mass
spectrometer (Bruker Daltonics). Data acquisition and processing
were performed using HyStar 3.0 software (Bruker Daltonics).
HPLC-PDA-ELSD analyses were carried out on an Alliance 2695
HPLC system (Waters) equipped with a 996 PDA (Waters) and an
Alltech 2000ES ELSD. ELSD parameters were set as follows: N2
flow = 3.2 l/min, temp. = 115 8C, gain = 8, impactor = off. Empower
Pro software (Waters) was used to acquire and process data. Sugar
analysis of compound 3 was performed with a HP 5890 Series II gas
chromatograph equipped with a HP 5971 mass detector. He was
used as a carrier gas. Flash chromatography was carried out on a
chromatography system PuriFlash1 4100 (Interchim), controlled
with InterSoft V5.0 software. Semi-preparative HPLC was per-
formed on an Agilent 1100 series instrument equipped with a PDA
channel related with cardiotoxicity (IC50 of 40.2
mM), was low.
Nevertheless, 1 bound the progesterone receptor with high affinity
(IC50 of 46.8 nM). In case the compound progresses through the
drug discovery pipeline, this aspect would require further
attention since this receptor has a fundamental role during
pregnancy, and since antimalarial drugs are expected to be
administered also to pregnant women (Fidock et al., 2004).
The second active extract, the CH2Cl2/MeOH 1:1 leaf extract,
showed inhibition against T. b. rhodesiense with an IC50 of 12.1
mg/
ml. HPLC-based activity profiling revealed strong inhibition in the
time window of 19 – 20 min which matched with an intense peak
in the HPLC-ELSD chromatogram (Fig. 2). Targeted isolation of this
compound by a combination of flash chromatography and open
column chromatography afforded 3. Acid hydrolysis afforded two
sugar moieties which were identified by GC–MS analysis after
derivatization with cysteine methyl ester and silylation (Severi
et al., 2010) as L-rhamnose and D-glucuronic acid. The compound
was identified by means of HRESIMS, extensive NMR analyses (1H
NMR, 13C NMR, HMBC, HSQC, and NOESY) and comparison with
literature data as 3-O-[
a
-
L
-rhamnopyranosyl-(1 ! 3)-
b-D-glucur-
opyranosyl] oleanolic acid, known as putranoside A (Fig. 3) (Borel
and Hostettmann, 1987; Hariharan, 1973, 1974; Melek et al.,
2003). Compound 3 showed weak in vitro trypanocidal (IC50 of
18.0
mM) and antileishmanial activities (IC50 of 7.8 mM).
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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5
Fig. 3. Structures of compounds 1–4.
detector. Data acquisition and processing were carried out using
HyStar 3.2 software (Bruker Daltonics). For HRESIMS a micrOTOF
ESI-MS system (Bruker Daltonics) was used. Mass calibration was
performed with a solution of formic acid 0.1% in 2-PrOH/H2O (1:1)
containing 5 mM NaOH. Mass spectra were recorded in the range
of m/z 150–1500 in positive ion mode with the aid of micrOTOF
control software 1.1 (Bruker Daltonics). NMR spectra were
recorded at 18 8C on a Bruker AVANCE IIITM 500 MHz spectrometer
operating at 500.13 (1H) and 125.77 MHz equipped with a 1 mm
TXI microprobe (1H and 2D NMR) or a 5 mm BBO probe (13C NMR)
(Bruker BioSpin). The 13C spectrum of 2 was recorded on a Bruker
at room temperature for 1 h with occasional stirring. The extract
was filtered, and the residual plant material further extracted
overnight with 2 l CH2Cl2/MeOH (1:1), followed by filtration.
Finally, a third extraction of the pulp was carried out with 2 l
solvent for 1 h with filtration. The filtrates were combined and
concentrated using a rotary vacuum evaporator at a temp. below
45 8C, and the residue dried in vacuo at room temperature for
24 h. The dried extract of 35.0 g (4.4%, w/w) was stored at
ꢂ20 8C.
The dried, ground leaves (115 g) of the plant were extracted in a
similar way as described above to yield 6.31 g (5.5%, w/w) of
extract.
AscendTM III 600 MHz spectrometer equipped with
cryoprobe. Chemical shifts are reported as values (ppm) with
a QCI
d
the residual solvent signal as internal reference, J in Hz. Standard
3.4. Microfractionation and activity profiling
pulse sequences from Topspin 3.0 software package were used.
HPLC-based activity profiling was performed on a SunFireTM
3.2. Plant material
C18 column (3.5
m
m, 3.0 mm ꢀ 150 mm, Waters) equipped with a
precolumn (3 mm ꢀ 10 mm) as previously described (Adams et al.,
2009). H2O (solvent A) and MeCN (solvent B) were used as solvents,
and the following gradient was applied: 10% ! 100% B in 30 min,
then 100% B for 1 min. The flow rate was 0.5 ml/min. Aliquots
Samples of the stems of D. gerrardii were collected at Hlatikulu
Forest on top of the Lebombo mountains in Gwalewemi, KwaZulu
Natal Province in South Africa in April 2012 by a botanist, Mr. Hans
Vahrmeijer. A plant specimen was deposited at the South African
National Biodiversity Institute (SANBI), and the plant identified as
D. gerrardii Hutch. var. gerrardii (Putranjivaceae) (voucher speci-
men number PRE 45954).
(35
ml) of extracts (10 mg/ml in DMSO) were injected, and 32 one-
minute fractions collected into a 96 deep well plate (Eppendorf)
with the aid of a F204 fraction collector (Gilson). After solvent
removal in an Evaporex 96 channel N2-evaporator (Apricot
Designs), the dried fractions were re-dissolved in methanol
3.3. Extraction
(100 ml) and transferred to 96 v-well plates (Thermo Scientific),
dried again, and stored in a refrigerator (2–8 8C) until use for
Stems and leaves were collected, separated and dried
separately in an oven at 30–60 8C. Dried plant material was
ground to a coarse powder using a hammer mill, and stored at
ambient temp. prior to extraction. 800 g of dried, ground stems
were extracted with 4 l of a mixture of CH2Cl2 and MeOH (1:1)
bioassay.
3.5. Preparative isolation
The CH2Cl2/MeOH (1:1) stem extract of D. gerrardii (3.4 g) was
fractionated by flash chromatography on a silica gel 60 column
(15–40
m
M, 35 mm ꢀ 430 mm, 180 g) with a gradient of 100–0%
hexane in EtOAc in 3 h, using a flow rate of 40 ml/min. Fractions
(22 ml each) were collected and compared by TLC analysis [silica
gel; hexane/EtOAc (9:1 and 7:3); anisaldehyde/sulphuric acid
spraying reagent, heated at 110 8C]. Fractions 10 (121.4 mg) and 11
(34.8 mg) were found to contain phenanthrenones. They were
purified by semi-preparative RP-HPLC using a SunFireTM C18
column (3.5
m
M, 10 mm ꢀ 150 mm; Waters) equipped with a
precolumn (10 mm ꢀ 10 mm). H2O (Solvent A) and MeCN (Solvent
B) were used as solvents and the following gradient was applied:
40% ! 60% B in 5 min, then 60% ! 70% B in 25 min. The flow rate
was 4 ml/min. The fractions were dissolved in DMSO (Fr.10:
10 mg/ml; Fr. 11: 20 mg/ml) and injected in aliquots of 300
ml.
Fig. 4. Key HMBC and NOESY correlations of compound 1.
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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Y. Hata et al. / Phytochemistry Letters xxx (2014) xxx–xxx
Fig. 5. Key HMBC and NOESY correlations for substructures A and B of compound 2.
Compound 1 (24.8 mg, tR 7.7 min) was isolated from Fr. 10 and Fr.
11. Compound 2 (1.4 mg, tR 24.0 min) was obtained from Fr. 10.
The CH2Cl2/MeOH (1:1) leaf extract (355 mg) was separated by
flash chromatography on a Sepacore1 system (Bu¨chi) by using a
3-O-[
a
-
L
-Rhamnopyranosyl-(1 ! 3)-
b-D-glucuropyranosyl]
oleanolic acid (putranoside A, 3) [37–40]: White amorphous
substance; 1H and 13C NMR data see Table 1S Supplementary
content;
HRESIMS:
m/z = 801.4396
[M+Na] + (calcd.
for
pre-packed silica gel 60 cartridge (40–63
m
m, 40 mm ꢀ 150 mm)
C42H66NaO13, 801.4396).
with a gradient of 0–60% EtOAc in MeOH, over 2 h. The flow rate
was 15 ml/min. Fractions (20 ml; each) were combined based on
TLC analysis [silica gel; CHCl3/MeOH/H2O (65:30:5); vanillin/
sulphuric acid reagent, heated at 110 8C]. Similar fractions were
pooled into four fractions (Fr. A–D). Fr. B (148.7 mg) was further
3.6. Conformational analysis and geometrical optimization
Conformational analysis of compound 2 was accomplished with
Schro¨dinger MacroModel 9.1 software employing the OPLS 2005
(optimized potential for liquid simulations) force field in H2O.
Conformers within a 1 kcal/mol energy window from the global
minimum were picked for geometrical optimization and energy
calculation applying DFT with the B3LYP functional using the 6-
31G (d,p) basis set in the gas phase with the Gaussian 09
programme package (Frisch et al., 2009). Vibrational evaluation
was done at the same level to confirm minima.
purified by CC on silica gel (40–63
m
m, 8 mm ꢀ 160 mm, 12.1 g)
using a step gradient of CHCl3 and MeOH (100:0, 95:5, 92.5:7.5,
90:10 87.5:12.5, 85:15, 80:20, 70:30, 60:40, 50:50; 50 ml each) to
yield 9 sub-fractions (Fr. B1–B9). Subfraction Fr. B4 eluted with
CHCl3/MeOH (80:20) afforded pure compound 3 (31.3 mg).
Purity of compounds 1 and 3 was >95%, and that of 2 >90%, as
determined by 1H NMR (Figs. 1S, 5S, 9S, and 15S of Supplementary
content).
6-Hydroxy-1,1,7-trimethylphenanthren-2(1H)-one (drypete-
none D, 1): Yellow amorphous substance; 1H and 13C NMR data
in CDCl3 and DMSO-d6, see Table 1; HRESIMS: m/z = 275.1067
[M+Na] + (calcd. for C17H16Na O2, 275.1043).
6-Hydroxy-1,1,7-trimethyl-5-((2,8,8-trimethyl-7-oxo-7,8-
dihydrophenanthren-3-yl)oxy)phenanthren-2(1H)-one (drypete-
none E, 2): Yellow amorphous substance; 1H and 13C NMR data
in CDCl3, see Table 1; HRESIMS: m/z = 525.2063 [M+Na] + (calcd.
for C34H30NaO4, 525.2036).
3.7. Bioassays
Extracts were tested in vitro against P. falciparum (NF54 strain),
T. b. rhodesiense (STIB 900 strain), and Leishmania donovani (strain
MHOM/ET/67/L82), in 96-well microtiter plates at concentrations
of 10.0
mg/ml and 2.0 mg/ml. IC50s were subsequently determined
for active extracts. HPLC microfractions from the CH2Cl2/MeOH
(1:1) stem extract of D. gerrardii were assayed against P. falciparum
and L. donovani. Those from the CH2Cl2/MeOH (1:1) leaf extract
Fig. 6. DFT optimized conformers of compound 2. Atoms CH3-7 and H-40 showing a key NOESY correlation between the 2 substructures. Intramolecular distance between
CH3-7 and H-40 was 3.45 A in both conformers.
˚
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

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Borel, C., Hostettmann, K., 1987. Molluscicidal saponins from Swartzia madagascar-
iensis Desvaux. Helv. Chim. Acta 70, 570–576.
Brun, R., Don, R., Jacobs, R.T., Wang, M.Z., Barrett, M.P., 2011. Development of novel
drugs for human African trypanosomiasis. Future Microbiol. 6, 677–691.
Cheminformatics, M., 2014. Molinspiration. http://molinspiration.com/, Slovak
Republic (accessed January 2014).
were tested on T. b. rhodesiense. Compounds 1and 3 were evaluated
for their antiprotozoal activity against the three parasites. Due to
the scarcity of isolated material, compound 2 was tested only
against P. falciparum. Additionally, for pure compounds, cytotox-
icity was determined by using a rat skeletal myoblast cell line (L-6
cells). For the assays DMSO stock solutions (10 mg/ml) of extracts
and purified compounds were freshly diluted in medium (final
DMSO concentration in assay <1%). Assays were performed in two
independent replicates. In vivo antimalarial activity was assessed
as previously described (Peters, 1987) and conducted according to
the rules and regulations for the protection of animal rights
(‘Tierschutzverordnung’) of the Swiss ‘Bundesamt fu¨r Veterina¨r-
wesen’. They were approved by the veterinary office of the Canton
Basel-Stadt, Switzerland. The animal experiment approval number
for in vivo testing against P. berghei is 1731. Details on assay
protocols are provided as Supplementary content.
Fidock, D.A., Rosenthal, P.J., Croft, S.L., Brun, R., Nwaka, S., 2004. Antimalarial drug dis-
covery: efficacy models for compound screening. Nat. Rev. Drug Discov. 3, 509–520.
Frisch, M., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R.,
Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M.,
Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada,
M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T.,
Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J.A., Peralta, J.E., Ogliaro,
F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi,
R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J.,
Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E., Cross, J.B., Bakken, V.,
Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J.,
Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K., Zakrzewski,
V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas,
O., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J., 2009. Gaussian 09, Revision
A02 ed. Gaussian Inc., Wallingford, CT.
Hannaert, V., 2011. Sleeping sickness pathogen (Trypanosoma brucei) and natural
products: therapeutic targets and screening systems. Planta Med. 77, 586–597.
Hariharan, V., 1973. Saponins of seed-coats of Putranjiva roxburghii. Indian J. Chem.
11, 830–831.
Author’s contributions
Hariharan, V., 1974. Structures of putranosides from the seed coats of Putranjiva
roxburghii. Indian J. Chem. 12, 447–449.
Yoshie Hata, HPLC microfractionation of the extracts of D.
gerrardii, preparative isolation of compounds, recording and inter-
pretation of analytical data for structure elucidation (HPLC-PDA-ESI-
MS, TOF-MS, 1D and 2D NMR), preparation of manuscript draft.
Maria De Mieri, measurement and interpretation of NMR data,
manuscript preparation.
Hata, Y., Raith, M., Ebrahimi, S.N., Zimmermann, S., Mokoka, T., Naidoo, D., Fouche,
G., Maharaj, V., Kaiser, M., Brun, R., Hamburger, M., 2013. Antiprotozoal iso-
flavan quinones from Abrus precatorius ssp. africanus. Planta Med. 79, 492–498.
Hata, Y., Zimmermann, S., Quitschau, M., Kaiser, M., Hamburger, M., Adams, M.,
2011. Antiplasmodial and antitrypanosomal activity of pyrethrins and pyre-
throids. J. Agric. Food Chem. 59, 9172–9176.
Hu, X.-J., Wang, Y.-H., Kong, L.-Y., He, H.-P., Gao, S., Liu, H.-Y., Ding, J., Xie, H., Di, Y.-T.,
Hao, X.-J., 2009. New phenanthrenes from Trigonostemon lii Y.T. Chang. Tetra-
hedron Lett. 50, 2917–2919.
Samad Nejad Ebrahimi, contributed to isolation, and manu-
script preparation.
Kokpol, U., Thebpatiphat, S., Boonyaratavej, S., Chedchuskulcai, V., Ni, C.Z., Clardy, J.,
Chaichantipyuth, C., Chittawong, V., Miles, D.H., 1990. Structure of trigonoste-
mone, a new phenanthrenone from the Thai plant Trigonostemon reidioides. J.
Nat. Prod. 53, 1148–1151.
Tsholofelo Mokoka, processing of plant material, extraction.
Gerda Fouche, manuscript preparation, responsible for the
project at the CSIR.
Marcel Kaiser, performed in vitro and in vivo assays.
Reto Brun, writing manuscript, responsible for the project at the
Swiss TPH.
Lipinski, C., Hopkins, A., 2004. Navigating chemical space for biology and medicine.
Nature 432, 855–861.
Melek, F.R., Miyase, T., Khalik, S.M.A., El-Gindi, M.R., 2003. Triterpenoid saponins
from Schefflera arboricola. Phytochemistry 63, 401–407.
Olivier Potterat, data analysis and interpretation, manuscript
preparation.
Matthias Hamburger, analysis and interpretation of data,
manuscript preparation, and overall responsibility for the project.
All authors have approved the final version of the manuscript.
Miller, L.H., Ackerman, H.C., Su, X.-z., Wellems, T.E., 2013. Malaria biology and
disease pathogenesis: insights for new treatments. Nat. Med. 19, 156–167.
Mokoka, T.A., Zimmermann, S., Julianti, T., Hata, Y., Moodley, N., Cal, M., Adams, M.,
Kaiser, M., Brun, R., Koorbanally, N., Hamburger, M., 2011. In vitro screening of
traditional South African malaria remedies against Trypanosoma brucei rhode-
siense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum.
Planta Med. 77, 1663–1667.
Mokoka, T.A., Peter, X.K., Fouche, G., Moodley, N., Adams, M., Hamburger, M., Kaiser,
M., Brun, R., Maharaj, V., Koorbanally, N., Zimmermann, S., Hata, Y., 2013.
Antiprotozoal screening of 60 South African plants, and the identification of
the antitrypanosomal eudesmanolides schkurin I and II. Planta Med. 79, 1380.
Ng’ang’a, M.M., Chhabra, S., Langat-Thoruwa, C., Hussain, H., Krohn, K., 2008.
Chemical constituents from the leaves of Drypetes gerrardii. Biochem. Syst.
Ecol. 36, 320–322.
Ng’ang’a, M.M., Hussain, H., Chhabra, S., Langat-Thoruwa, C., Irungu, B.N., Al-
Harrasi, A., Riaz, M., Krohn, K., 2012. Antiplasmodial activity of compounds
from Drypetes gerrardii. Chem. Nat. Compd. 48, 339–340.
Ng’ang’a, M.M., Hussain, H., Chhabra, S., Langat-Thoruwa, C., Riaz, M., Krohn, K.,
2011. Drypetdimer A: a new flavone dimer from Drypetes gerrardii. Nat. Prod.
Commun. 6, 1115–1116.
Pajouhesh, H., Lenz, G.R., 2005. Medicinal chemical properties of successful central
nervous system drugs. NeuroRx 2, 541–553.
Peters, W., 1987. Chemotherapy and Drug Resistance in Malaria. Academic Press,
Inc., New York, pp. 145–273.
Renslo, A.R., 2013. Antimalarial drug discovery: from quinine to the dream of
eradication. Chem. Lett. 4, 1126–1128.
Renslo, A.R., McKerrow, J.H., 2006. Drug discovery and development for neglected
parasitic diseases. Nat. Chem. Biol. 2, 701–710.
Seephonkai, P., Pyne, S.G., Willis, A.C., Lie, W., 2013a. Bioactive compounds from the
roots of Strophioblachia fimbricalyx. J. Nat. Prod. 76, 1358–1364.
Seephonkai, P., Pyne, S.G., Willis, A.C., Lie, W., 2013b. Fimbricalyx A, a novel
phenanthrenone derivative having a rare 2H-benz[e]inden-2-one substructure.
Tetrahedron Lett. 54, 2085–2088.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank the Council for Scientific and Industrial
Research (CSIR) (grant SSAJRP 03), and the Swiss Confederation for
financial support under the Swiss South African Joint Research
Programme (grant JRP 03). Y. Hata gratefully acknowledges a Ph.D.
stipend from Administrative Department of Science and Technology
from Colombia (Colciencias) managed by LASPAU. We thank Dr. D.
Haussinger (Department of Chemistry, University of Basel) for
recording the 13C NMR spectrum of 2, O. Fertig for GC–MS analysis,
and Dr. M. Raith for her support in the structure elucidation. Thanks
are also due to Mrs. M. Rogalski for proof-reading the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2014.05.005.
Seephonkai, P., Sangdee, A., Bunchalee, P., Pyne, S.G., 2009. Cytotoxic and anti-
plasmodial compounds from the roots of Strophioblachia fimbricalyx. J. Nat. Prod.
72, 1892–1894.
Severi, J.A., Fertig, O., Plitzko, I., Vilegas, W., Hamburger, M., Potterat, O., 2010.
Oleanane Saponins and Glycerogalactolipids from the Leaves of Guapira gracili-
flora. Helv. Chim. Acta 93, 1058–1066.
References
Stuart, K., Brun, R., Croft, S., Fairlamb, A., Gurtler, R.E., McKerrow, J., Reed, S.,
Tarleton, R., 2008. Kinetoplastids: related protozoan pathogens, different dis-
eases. J. Clin. Invest. 118, 1301–1310.
Adams, M., Zimmermann, S., Kaiser, M., Brun, R., Hamburger, M., 2009. A protocol
for HPLC-based activity profiling for natural products with activities against
tropical parasites. Nat. Prod. Commun. 4, 1377–1381.
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005

G Model
PHYTOL-711; No. of Pages 8
8
Y. Hata et al. / Phytochemistry Letters xxx (2014) xxx–xxx
Tang, G.-H., Zhang, Y., Yuan, C.-M., Li, Y., Gu, Y.-C., Di, Y.-T., Wang, Y.-H., Zuo, G.-Y., Li,
S.-F., Li, S.-L., He, H.-P., Hao, X.-J., 2012. Trigohowilols A-G, degraded diterpe-
noids from the stems of Trigonostemon howii. J. Nat. Prod. 75, 1962–1966.
Uzair, B., Ahmed, N., Ahmad, V.U., Mohammad, F.V., Edwards, D.H., 2008. The
isolation, purification and biological activity of a novel antibacterial compound
produced by Pseudomonas stutzeri. FEMS Microbiol. Lett. 279, 243–250.
Vedani, A., Dobler, M., Smiesko, M., 2012. VirtualToxLab – a platform for estimating
the toxic potential of drugs, chemicals and natural products. Toxicol. Appl.
Pharmacol. 261, 142–153.
WHO, 2010. Working to Overcome the Global Impact of Neglected Tropical Dis-
eases: First WHO Report on Neglected Tropical Diseases. World Health Organi-
zation, Geneva, pp. 91–95.
WHO, 2013a. World Malaria Report 2013. World Health Organization, Geneva, pp.
v–xiv.
WHO, 2013b. Trypanosomiasis, Human African (sleeping sickness). Fact sheet
N8259 World Health Organization, Geneva.
Zhu, Q., Tang, C.-P., Ke, C.-Q., Li, X.-Q., Liu, J., Gan, L.-S., Weiss, H.-C., Gesing, E.-R., Ye,
Y., 2010. Constituents of Trigonostemon chinensis. J. Nat. Prod. 73, 40–44.
Please cite this article in press as: Hata, Y., et al., Identification of two new phenathrenones and a saponin as antiprotozoal constituents
of Drypetes gerrardii. Phytochem. Lett. (2014), http://dx.doi.org/10.1016/j.phytol.2014.05.005