Journal of Ethnopharmacology 170 (2015) 167–174

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Evaluation of antiplasmodial and antileishmanial activities of herbal

medicine Pseudelephantopus spiralis (Less.) Cronquist and isolated
hirsutinolide-type sesquiterpenoids
Cynthia Girardi a,b, Nicolas Fabre a,b, Lucie Paloque a,b, Arba Pramundita Ramadani c,d,
Françoise Benoit-Vical c,d, German González-Aspajo a,b, Mohamed Haddad a,b, Elsa Rengifo e,
Valérie Jullian a,f,n
a
Université de Toulouse, UPS, UMR 152 Pharma-DEV, Université Toulouse 3, Faculté des Sciences Pharmaceutiques, F-31062 Toulouse Cedex 09, France
b
Institut de Recherche pour le Développement (IRD), UMR 152 Pharma-DEV, F-31062 Toulouse Cedex 09, France
c
CNRS, LCC (Laboratoire de Chimie de Coordination) UPR 8241, 31077 Toulouse Cedex 4, France
d
Université de Toulouse, UPS, INPT, 31077 Toulouse Cedex 4, France
e
Programa de Investigación en Biodiversidad Amazónica (PIBA). Instituto de Investigaciones de la Amazonía Peruana-IIAP, Av. Abelardo Quiñones km 4.5,
Iquitos, Peru
f
Institut de Recherche pour le Développement (IRD), UMR 152 Pharma-DEV, Mission IRD, Casilla 18-1209, Lima, Peru

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Pseudelephantopus spiralis (Less.) Cronquist is distributed in the Car-
Received 11 February 2015 ibbean, Mesoamerica and Latin America. Preparations of the plant are traditionally used in Latin America
Received in revised form for the treatment of various diseases including fever, malaria, and spleen or liver inflammations.
5 May 2015
Materials and methods: Aerial parts of P. spiralis were extracted with either ethanol or distilled water.
Accepted 5 May 2015
Available online 14 May 2015
Seven hirsutinolide-type sesquiterpenoids were isolated: 8-acetyl-13-ethoxypiptocarphol (1), diacetyl-
piptocarphol (2), piptocarphins A (3), F (4) and D (5), (1S*,4R*,8S*,10R*)-1,4-epoxy-13-ethoxy-1,8,10-
Keywords: trihydroxygermacra-5E,7(11)-dien-6,12-olide (6), and piptocarphol (7). Extracts and isolated compounds
Pseudelephantopus spiralis (2, 3, 5–7) were screened for their in vitro antiplasmodial activity against the chloroquine-resistant
Sesquiterpene lactones
Plasmodium falciparum strain FcM29-Cameroon and antileishmanial activity against three stages of
Hirsutinolides
Leishmania infantum. Their cytotoxicities were also evaluated against healthy VERO cell lines and J774A.1
Malaria
Leishmaniasis macrophages, the host cells of the Leishmania parasites in humans.
Results: Aqueous extracts showed a greater inhibitory effect than alcoholic extracts, with IC50 on P.
falciparum of 3.0 mg/mL versus 21.1 mg/mL, and on L. infantum of 13.4 mg/mL versus 4 50 mg/mL. Both
extracts were found to be cytotoxic to VERO cells (CC50 o 3 mg/mL). Sesquiterpene lactones 2 and 3
showed the best activity against both parasites but failed in selectivity. Carbon 8 hydroxylated
hirsutinolides 5–7 presented the particularity of exhibiting two conformers observed in solution during
extensive NMR analyses in CD3OD and UHPLC-MS. The presence of a hydroxyl function at C-8 decreased
the activity of 5–7 on the two parasites and also on VERO cells.
Conclusion: The antiplasmodial activity displayed by the aqueous extract explains the traditional use of P.
spiralis in the treatment of malaria. This activity seems to be attributable to the presence of sesquiterpene
lactones 2 and 3, the most active against P. falciparum. Aqueous extract and compounds 2, 3 and 6 were
also active against L. infantum but lacked in selectivity due to their cytotoxicity towards macrophages.
Exploring the safety and antiplasmodial efficacy of this traditional remedy will require further
toxicological and in vivo studies in the light of the cytotoxicity towards healthy cell lines displayed by
the aqueous extract and compounds 2 and 3.
& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Tropical protozoan diseases are currently a major public health

n
problem throughout the world. Malaria, caused by Plasmodium
Corresponding author at: LID, Universidad Peruana Cayetano Heredia, Av.
species is the most devastating parasitic disease with 198 million
Honorio Delgado 430, San Martin de Porres, Lima, Peru. Tel.: +51 1 481 17 94.
E-mail address: valerie.jullian@ird.fr (V. Jullian). cases estimated in 2013 and about 600,000 deaths mainly due to

http://dx.doi.org/10.1016/j.jep.2015.05.014
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
168 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174

Plasmodium falciparum (WHO | Malaria Fact sheet N 94, 2014). the Reserva Allpahuayo-Mishana, Carretera Iquitos-Nauta, kilometre
Leishmaniasis, identified as a “Neglected Tropical Disease” by the 28, Loreto, Peru, in May 2012 and May 2013. Plant authentication
World Health Organization, is endemic in 98 countries and was confirmed during the collection by Elsa Rengifo, botanist at
territories, with a number of new cases estimated at 1.3 million the Instituto de Investigaciones de la Amazonía Peruana (IIAP).
per year (WHO | Leishmaniasis Fact sheet N 375, 2014). Leishmania A voucher is kept as reference in Université Paul Sabatier (Toulouse,
infantum, the agent responsible for the lethal visceral form of France), faculty of pharmaceutical science, department of pharma-
Leishmaniasis (Romero and Boelaert, 2010) is present in Latin cognosy under the number CG111.
America and in the Old World (WHO Expert Committee on the
Control of the Leishmaniases and Meeting, 2010). Many plants are 2.2. General experimental procedure
traditionally used in South American folk medicine for the treat-
ment of these two important parasitoses (Domingues Passero The 1H NMR and 13C NMR spectra (Figs. S1–S19 in the
et al., 2014; Kvist et al., 2006; Valadeau et al., 2009; Willcox and supplementary data) were recorded on Bruker Avance 300 MHz
Bodeker, 2004). Most of the drugs marketed for use against or 500 MHz instrument with samples diluted in CDCl3 or CD3OD.
malaria are directly or indirectly derived from natural products Mass spectra were acquired using a LTQ Orbitrap XL mass spectro-
(Cragg and Newman, 2013), and plant secondary metabolites still meter (Thermo Fisher Scientific). The system was equipped with
constitute a promising source of new antiprotozoal leads and/or an electrospray source operating in the positive ion mode. The
drugs (Fournet and Munoz, 2002; Schmidt et al., 2012a, 2012b; apparatus was controlled by Xcalibur software version 2.0.7.
Singh et al., 2014). Chromatography columns were filled with Merck silica gel 60A
Pseudelephantopus spiralis (Less.) Cronquist belongs to the (40–63 μm). Reverse phase separations were performed on Agi-
Asteraceae family and is one of the two species that make up the lent C18 Bond Elut SPE cartridge using a Varian Vac Elut SPS 24
genus Pseudelephantopus (Pruski, 2011). The plant is distributed Manifold system, and a ILMVAC LVS 101 Pump. Analytical TLC
in the Caribbean, Mesoamerica and Latin America (Macbride, were achieved on precoated Kieselgel 60 F254 20
20 cm2 plates
1936), and is considered a weed (Canizales et al., 2010; Ochoa (Merck, 0.5 mm thin) using UV 254 nm and 1% vanillin/10%
and Andrade, 2003; Tye, 2001). The species, named Mata pasto sulphuric acid reagent in EtOH for visualization. Chlorophylls were
or Pasto mula, is traditionally used in Peru to treat menstrual removed on Fisher Chemical activated charcoal. Freeze drying was
cramps and as liver depurative (Internal report: base de datos de performed on a Labconco™ Lyoph-Lock 6™ 7753001 lyophilizer.
las plantas medicinales 2010, IIAP, 2014). In southern Equator, Preparative HPLC were carried out on a LaChrom Merck Hitachi
the whole plant is cooked and used by oral administration to system, consisting of a LaChrom L-7100 Pump, a L-7455 DAD, a D-
treat fever, high blood pressure, shivering fits and malaria 7000 Interface and using a Phenomenex Luna 5 m C18(2) 100 Å,
(Bussmann and Sharon, 2006). In Colombia, infusions of roots 250
10 mm2 column.
or whole plant were dispensed as a beverage or in baths, in
combination with other plants, to treat malaria, spleen and liver
inflammations (Blair Trujillo and Madrigal, 2005). Moreover 2.3. Drugs or reagents
Pseudelephantopus spicatus (Juss. ex Aubl.) C.F. Baker, the second
and only other species in the Pseudelephantopus genus, is also Solvents CH2Cl2, CH3CN, and MeOH (HPLC grade) were
used in Peruvian Amazonia as a traditional antileishmanial purchased from Fisher Chemical (Fischer Scientific, France).
remedy (Odonne et al., 2013, 2011). A previous phytochemical Toluene, ethyl acetate and petroleum ether were analytical
investigation of this latter species led to the isolation of two reagent grade from Fisher Chemical (Fisher Scientific, France).
hirsutinolide-type sesquiterpene lactones exhibiting strong Formic acid (99  100%), 95% ethanol technical grade were from
antileishmanial activities (Odonne et al., 2011). Based on this Prolabo (VWR Chemicals, France). Sulphuric acid 96% and
result, P. spiralis was expected to synthesize structurally related vanillin 99% used for TLC development were from Acros Organ-
active compounds hence we decided to conduct the present ics (Fisher Scientific, France). Extraction and separation used
phytochemical investigation of P. spiralis to evaluate its anti- distilled water. High-purity water (18.2 MΩ cm; total organic
plasmodial and antileishmanial potential and thus confirm the carbon: 2 ppb) was obtained from a Milli-Q water purification
relevance of its traditional use. In this context, aqueous and system (Millipore, Merck, France) and used for analytical and
ethanolic extracts of the aerial parts of P. spiralis were prepared preparative HPLC. DMSO used for biological assays was from
and bio-guided assay led to the isolation and identification of Fisher Bioreagents (Fischer Scientific, France). Reference drugs
seven hirsutinolide-type sesquiterpene lactones. They were amphotericin B, miltefosin, pentamidine, doxorubicin, chloro-
identified as 8-acetyl-13-ethoxypiptocarphol (1), diacetylpipto- quine, artemisinin and antibiotics penicillin, streptomycin and
carphol (2), piptocarphins A (3), F (4) and D (5), geneticin as MTT reagent ( 4 97.5%) used for the biological
(1S*,4R*,8S*,10R*)-1,4-epoxy-13-ethoxy-1,8,10-trihydroxygerma- assays were purchased from Sigma-Aldrich (France). Media
cra-5E,7(11)-dien-6,12-olide (6), and piptocarphol (7). The RPMI 1640 and MAA-20, foetal calf serum (FCS) and L-glutamine
extracts and pure compounds were tested for their in vitro used for the biological assays were purchased from Gibco
antiprotozoal activities against two parasite species: a (Fischer Scientific, France).
chloroquine-resistant strain of P. falciparum and the promasti-
gote and axenic amastigote stages of L. infantum. The samples 2.4. Preparation of the crude extracts
were also assessed for their cytotoxicity on VERO cells and
macrophages in order to determine their selectivity indices. Aerial parts of the two batches of P. spiralis were dried in the
dark, at room temperature before being reduced to powder.

2. Materials and methods Preparation of the aqueous extract: Powdered aerial parts
(353 g) were extracted with boiling distilled water (4 L) for
2.1. Plant material 10 min. The aqueous extract was then frozen at – 20 1C and
lyophilized for 230 h to give 27.0 g of residue (yield 7.6% w/w).
Two batches of P. spiralis (Less.) Cronquist aerial parts were The extract was then stored in the dark in sterile universal
collected from a permanent plot in the Medicinal plant garden of tubes until use.
 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174 169

Preparation of the ethanolic extract: Powdered aerial parts (MHOM/MA/67/ITMAP-263, CNR Leishmania, Montpellier, France)
(2.1 g) were extracted under stirring with 95% EtOH were assessed using promastigotes expressing luciferase activity.
(80 mL) for 24 h at room temperature. Solvent was removed Briefly, promastigotes in log-phase in RPMI 1640 medium
under reduced pressure at 40 1C to give 71 mg of dry residue supplemented with 10% foetal calf serum (FCS), 2 mM
(3.4% w/w). The extract was then stored as described above. L-glutamine and antibiotics (100 U/mL penicillin, 100 mg/mL
streptomycin and 50 mg/mL geneticin), were incubated at an
average density of 106 parasites/mL in sterile 96-well plates with
2.5. Isolation and identification of pure compounds various concentrations of compounds dissolved in DMSO (final
concentration less than 0.5% v/v), in triplicate. Appropriate
Procedure A: isolation of compounds 1, 3, 4, 5, 6, 7: Powdered controls treated by DMSO and amphotericin B, miltefosine and
aerial parts (374 g) were extracted with EtOH (5
3 L) at room pentamidine, reference drugs purchased from SigmaAldrich, were
temperature for 48 h to give 30.7 g of dry residue (yield 8.2% w/w). added to each set of experiments. After a 72 h incubation period at
The residue was suspended in 800 mL of a H2O–MeOH 3:1 mixture 24 1C, each plate well was microscope-examined to detect the
and extracted with petroleum ether (6
600 mL). The lower phase possible formation of precipitate. To estimate the luciferase
was discarded and evaporation under reduced pressure of the activity of promastigotes, 80 ml of each well were transferred to
upper phase gave rise to 4.32 g of residue which was chromato- white 96-well plates, Steady Glow reagent (Promega) was added
graphed on a silica gel (200 g) column using a toluene gradient according to the manufacturer's instructions, and the plates were
containing increasing amounts of ethyl acetate (0–100%) followed incubated for 2 min. The luminescence was then measured in a
by ethyl acetate–MeOH 1:1 as eluting solvents. This afforded 12 Microbeta Luminescence Counter (PerkinElmer). Inhibitory
fractions. In order to eliminate chlorophylls and other pigments, concentration 50% (IC50) was defined as the concentration of
fractions 7–10 (80% toluene to 20%) were pooled (968.2 mg) and drug required to inhibit by 50% of the metabolic activity of L.
suspended in 500 mL MeOH containing 4 g of active charcoal for infantum promastigotes compared to the control.
1 h. The suspension was filtered on 0.45 mm nylon filter and a
chlorophyll free residue (322 mg) was obtained after evaporation
2.6.1.2. Antileishmanial activity on axenic amastigotes. L. infantum
of the methanol. It was suspended in 2 mL of MeOH–H2O 1:1 and
promastigotes in log phase were centrifuged at 900g 10 min, cell
subjected to SPE C18 cartridge (10 g) under vacuum (P¼5 mm Hg)
medium was then replaced by MAA-20 medium and maintained at
then eluted with increasing amounts of MeOH in H2O (50–100%).
37 1C, 5% CO2 to induce transformation into axenic amastigote forms
This afforded 13 fractions (A–M). Fraction A (113 mg) was chro-
(Sereno and Lemesre, 1997). Axenic amastigotes were incubated at an
matographed on SPE C18 cartridge (10 g) with H2O and increasing
average density of 4.106 cells/mL in sterile 96-well plates with various
amounts of MeOH to give 15 fractions (A1–A15). Fraction A3 was
concentrations of compounds dissolved in DMSO (final concentration
identified as 7 (0.5 mg). Fractions A7 and A9 were identified as 5
1% v/v), in duplicate. Appropriate controls treated by DMSO and
(4.5 mg) and 6 (6.5 mg), respectively. Semi-preparative HPLC
amphotericin B, miltefosine and pentamidine were added to each set
(MeCN-0.1% HCOOH in H2O, 1:1, 3 mL/min) of the pooled fractions
of experiments. After a 72 h incubation period at 37 1C, 5% CO2, the
A12 (10.2 mg) and A13 (3.9 mg) afforded 2.3 mg of a mixture
effects of the tested compounds were evaluated by estimation of the
containing 2 (Rt ¼7.8 min) and 4.7 mg of 1 (Rt ¼9.9 min). Semi-
luciferase activity of the axenic amastigotes. Eighty microliters of
preparative HPLC (MeCN-0.1% HCOOH in H2O, 11:9, 3 mL/min)
each well were transferred to white 96-well plates, Steady Glow
performed on pooled fractions B (6.8 mg) and C (21.7 mg) gave 3
reagent (Promega) was added according to the manufacturer's
(7.2 mg, Rt ¼ 20.4 min) and a fraction containing 4 as a main
instructions, and the plates incubated for 2 min. The luminescence
compound. Finally, the latter fraction (3.2 mg) was pooled with
was measured in a Microbeta Luminescence Counter (PerkinElmer).
fractions D (3.1 mg) and A15 (1.4 mg) and separated by semi-
Inhibitory concentration 50% (IC50) was defined as the concentration
preparative HPLC (MeCN-0.1% HCOOH in H2O, 3:2, 3 mL/min) to
of drug required to inhibit 50% of the metabolic activity of L. infantum
yield 4 (2.8 mg, Rt ¼12.5 min).
amastigotes compared to the control.
Procedure B: isolation of 2 and 3: The entire crude aqueous
extract (27.0 g) was suspended in H2O (200 mL) and extracted
by CH2Cl2 (6
200 mL). Evaporation of the CH2Cl2 layer under 2.6.1.3. Antileishmanial activity on intramacrophagic amastigotes. The
reduced pressure gave rise to 0.62 g of residue which was effects of the compounds tested on the growth of L. infantum
chromatographed on SPE C18 cartridge (10 g) under vacuum intracellular amastigotes were assessed in the following way. One
(P ¼5 mm Hg) with H2O and increasing amounts of MeCN to hundred microlitre of J774A.1 cells were seeded in 96-well plates at an
give 11 fractions (F1–F11). Fraction F8 (95 mg) was chromato- average density of 2.105 cells/mL and incubated for 24 h at 37 1C under
graphed on a 30
1.5 cm column, containing silica gel (10 g) 5% CO2. L. Infantum promastigotes were centrifuged at 900g for 10 min
and eluted with CH2Cl2 with increasing amounts of MeOH and the supernatant replaced by the same volume of RPMI 1640, 10%
(0–100%). This afforded 7 fractions (F81–F87). Fraction F81 FCS, pH 5.4 and incubated for 24 h at 27 1C. Then, half the macrophage
(38 mg) was pure and contained 3. Fractions F6 (185 mg) medium was removed, J774A.1 cells were then infected by 100 ml of
and F7 (135 mg) were pooled and chromatographed on a acidified promastigotes at an average density of 2.106 cells/mL (10:1
30
1.5 cm silica gel (10 g) column eluted with CH2Cl2 with ratio) and plates incubated for 24 h at 37 1C. Half of the infected
increasing amounts of MeOH (0–100%) to give 8 fractions macrophage medium was removed and medium containing various
(I–VIII). Fraction III (125 mg) was re-chromatographed on the concentrations of test compounds was added in duplicate. Appropriate
same column and gave 6 sub-fractions (III-1 to III-6). Fraction controls treated with or without solvent (DMSO), and various
III-4 was identified as compound 2 (50.8 mg). concentrations of amphotericin B were added to each set of
experiments. After 120 h incubation at 37 1C and 5% CO2, 100 ml of
well supernatant was removed and Steady Glow reagent (Promega)
2.6. Biological tests was added according to the manufacter's instructions. The plates were
incubated for 3 min. Hundred of each well were transfe-
2.6.1. Antileishmanial evaluation rred to white 96-well plates and the luminescence was measured
2.6.1.1. Antileishmanial activity on promastigotes. The effects of the in a Microbeta Luminescence Counter (PerkinElmer). Inhibitory con-
various samples on the growth of promastigotes of L. infantum centration 50% (IC50) was defined as the concentration of drug
170 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174

required to inhibit by 50% the metabolic activity of L. Infantum Desjardins et al. (Desjardins et al., 1979) and modified as follows.
intracellular amastigotes compared to the control. IC50 were Extract dilutions and compounds were tested 3 times indepen-
calculated by non-linear regression analysis processed on dose– dently, each dilution in triplicate, in 96-well plates with cultures at
response curves, using Table Curve 2D V5 software. IC50 values a parasitaemia of 1% and a haematocrit of 1%. For each test, the
represent the means calculated from three independent experiments. plates of parasite culture were incubated with products for 48 h
and tritiated hypoxanthine (PerkinElmer, France) was added to the
medium 24 h after the beginning of incubation (Benoit-Vical et al.,
2.6.2. Cytotoxicity evaluation on macrophages and VERO cell lines
2007). The parasite culture control (with solvent only) was
The evaluation of cytotoxicity by MTT assay on the J774A.1 cell line
referred to as 100% growth. Parasite growth was estimated by
(mouse macrophage cell line, Sigma-Aldrich) and VERO cells (monkey
[3H]-hypoxanthine incorporation. Inhibitory concentration 50%
epithelial cell line, Sigma-Aldrich) was done according to Mosmann
(IC50) was defined as the concentration of drug required to inhibit
(1983) with slight modifications. Briefly, cells (5.104 cells/mL) in
50% of the metabolic activity of P. falciparum compared to the
100 mL of complete medium, [RPMI 1640 supplemented with 10%
control.
foetal calf serum (FCS), 2 mM L-glutamine and antibiotics (100 U/mL
penicillin and 100 mg/mL streptomycin) for J774A.1 cell line and MEM
with 10% foetal calf serum (FCS), 2 mM L-glutamine and antibiotics 2.6.4. IC50, CC50 and selectivity index (SI) calculation
(100 U/mL penicillin and 100 mg/mL streptomycin), NEAA 1X for IC50 on L. infantum and CC50 on macrophages and VERO cells
VERO cell line] were seeded into each well of 96-well plates and were calculated by non-linear regression analysis processed on
incubated at 37 1C, 5% CO2. After 24 h incubation, 100 mL of medium dose–response curves, using Table Curve 2D V5 software. IC50
with various product concentrations and appropriate controls (DMSO values represent the mean value calculated from three indepen-
and doxorubicin) were added and the plates were incubated for 72 h dent experiments. Results were expressed as means followed by
at 37 1C, 5% CO2. Each plate well was then microscope-examined to standard deviation. The selectivity index (SI) value allowed the
detect possible precipitate formation before the medium was pipetted comparison of the toxicity of the extracts or compounds against
out of the wells. One hundred microliters of MTT solution (0.5 mg/mL normal cells compared to the activity against the parasites in order
in RPMI 1640) was then added to each well and the cells incubated to assess of their selectivity as antiparasitic. The SI on L. infantum
2 h at 37 1C. After this time, the MTT solution was removed and DMSO was calculated as the ratio between the CC50 values against
(100 mL/well) was added to dissolve the resulting formazan crystals. macrophages and IC50 values against promastigote forms. The SI
Plates were shaken vigorously for 5 min. Absorbance was measured at on P. falciparum was calculated as the ratio between the CC50
570 nm with a microplate spectrophotometer (EON). Inhibitory con- values against VERO cells and IC50 values against P. falciparum. In
centration 50% (IC50) was defined as the concentration of drug both cases, SI¼ CC50/IC50.
inducing 50% death of macrophages J774A.1 or VERO cells compared
to the control.
3. Results

2.6.3. Antiplasmodial evaluation 3.1. Structural elucidation of isolated compounds by NMR analysis
The antiplasmodial activity was assessed on the chloroquine-
resistant P. falciparum strain FcM29-Cameroon, cultured continu- Hirsutinolides 1–7 (Fig. 1) were clearly identified mainly on the
ously according to the method of Trager and Jensen (1976), in a 5% basis of their 1D (1H NMR, 13C NMR) and 2D (HSQC and HMBC)
CO2 atmosphere at 37 1C as previously described (Benoit-Vical NMR parameters, in CDCl3. They were compared with NMR data
et al., 2007). Briefly, the parasites were maintained in vitro in published in the literature in the same solvent. This permitted the
human red blood cells and diluted in RPMI 1640 medium, identification of 8-acetyl-13-ethoxypiptocarphol (1) ((Catalán
supplemented with 25 mM HEPES, 2.05 mM L-glutamine and et al., 1986), corrected in (Catalán et al., 1988)), diacetylpiptocar-
completed with 5% human serum (French Blood Bank, EFS). The phol (2) ((Catalán et al., 1986), corrected in (Bardón et al., 1993)),
antiplasmodial activity was assessed as previously reported by piptocarphin A (3), piptocarphin F (4), piptocarphin D (5) ((Cowall

14 14
OH OH
HO R 9 HO R 9
2 10 O 2 10 O
S 8 S R2 S 8 S H
O O
R R
3 11 3 11 13
13
15 5 15 5 12
12
O R1 O O R1
O
O O

R1 R2
R1
1 CH2CH3 COCH3
5 COCH3
2 COCH3 COCH3
6 CH2CH3
3 COCH3 COC(CH3)=CH2
7 H
4 CH2CH3 COC(CH3)=CH2
Fig. 1. Isolated compounds from Pseudelephantopus spiralis (Less.) Cronquist
 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174 171

et al., 1981), corrected in (Catalán et al., 1988)), (1S*,4R*,8S*,10R*)- by Borkosky and collaborators were the absence of a hydroxyl at
1,4-epoxy-13-ethoxy-1,8,10-trihydroxygermacra-5E,7 (11)-dien-6, C-10 and the α-orientation of the methyl at C-10. Moreover, it can
12-olide (6) (Kotowicz et al., 1998), and piptocarphol (7) (Bardón be noted that a unique conformer was described for other C-8
et al., 1993). In the course of our search for the improvement of hydroxylated hirsutinolides with an α-oriented C-10 methyl when
NMR spectra of some hirsutinolides presenting enlargements of 1H NMR analyses were recorded in CD3OD (Youn et al., 2012). This
NMR signals (Fig. S2 Supplementary data), NMR analyses were suggests that the presence of hydroxyl groups at C-8 and C-10 was
run in CD3OD while CDCl3 and C6D6 were generally the solvents responsible for the intramolecular bond occurring in compounds
prevailing in the literature (Bardón et al., 1993; Catalán et al., 1988; 5–7. Jakupovic and colleagues reported that the quaternization of
Kotowicz et al., 1998). In such conditions, it appeared that C-8 carbon 10 by an oxygenated group in hirsutinolides led to the β-
hydroxylated compounds 5–7 exhibited peaks distinctly split orientation of the methyl group at C-10 (Jakupovic et al., 1985).
while a unique form remained in CDCl3 for these three compounds From this assumption, the H-bond may occur between 8-OH and
(Figs. S6–S19 in Supplementary data). As far as we know, this is the 10-OH or with the oxygen of the ether bridge because of a
first time that such a phenomenon has been described for conformational change induced by the presence of the α-oriented
hirsutinolides. Heteronuclear 2D NMR experiments HSQC and hydroxyl group at C-10. Our hypothesis is therefore that this
HMBC recorded in CD3OD confirmed the existence of two forms hydrogen bond is partially disrupted by CD3OD, a polar protic
of the same compound. The fact that a unique form is present in solvent, and led to a mixture of two conformers.
CDCl3, even after a previous analysis in CD3OD, suggests the
reversibility of the reaction, and the involvement of the deuterated
NMR solvents. Moreover, compounds 5–7 were analysed by
UHPLC-MS under acidic conditions, and two main peaks display- 3.2. Biological activities
ing the same mass appeared in the chromatograms confirming the
presence of two forms. It is worth noting that this phenomenon 3.2.1. Antiplasmodial, antileishmanial and cytotoxicity properties of
only appeared for the C-8 hydroxylated derivatives 5–7. For C-8 ethanolic and aqueous extracts of P. spiralis
esterified analogues 1–4, only one form appeared whatever con- The antiplasmodial activities against P. falciparum of extracts
ditions suggesting the involvement of the hydroxyl function in a obtained from the leaves of P. spiralis are displayed in Table 1. The
hydrogen bond. 1H NMR spectra of compounds 5–7 recorded in aqueous extract exhibited the highest antiplasmodial activity with
CDCl3 exhibited a doublet signal close to δ 6 ppm with a coupling an IC50 value of 3.0 mg/mL. This corresponds to a good activity
constant of J ¼12 Hz corresponding to the non-exchangeable according to the standard antiplasmodial score (Willcox et al.,
hydroxyl proton 8-OH coupled with H-8 close to 5.5 ppm. There- 2011). The ethanolic extract with an IC50 of 21.1 mg/mL, showed a
fore, the NMR signal of H-8 was a ddd (J8/9a ¼ 11 Hz, J8/9b ¼2 Hz, J8/ low activity. The cytoxicity was evaluated on mammalian VERO
8-OH ¼12 Hz), or appeared as a doublet of doublets (J8/9a ¼ 11 Hz, J8/ cell lines. Both extracts displayed high cytotoxicity against this
8-OH ¼12 Hz) given the low value of the J8/9b coupling constant. healthy cell line with median cytotoxicity concentrations of
This large coupling constant suggests the presence of an intramo- 1.7 and 2.5 mg/mL for aqueous and ethanolic extracts, respectively.
lecular hydrogen bond occurring between the 8-OH and most Both extracts were also tested against the promastigote stage of
likely the ether bridge oxygen as suggested before for a similar L. infantum (Table 1). The aqueous extract was the more active
hirsutinolide (Yang et al., 2007). Borkosky et al. (1997) isolated a with a good efficacy, the IC50 value (IC50 ¼13.4 mg/mL) being
hirsutinolide structurally similar to 5 (referred as compound 7f in between 10 and 50 mg/mL (Osorio et al., 2007) while the ethanolic
their publication). The NMR signal of H-8 for this compound, in extract was inactive (IC50 450 mg/mL). The cytotoxicities of
CDCl3, appeared as a doublet of doublets (J8/9a ¼2 Hz and extracts were also assessed on macrophages as they are the host
J8/9b ¼6 Hz) indicating no intramolecular hydrogen bond. The only cells of Leishmania. The aqueous extract was shown to be cytotoxic
differences between compound 5 and the hirstunolide described with a selectivity index of less than 1.

Table 1
Antileishmanial and antiplasmodial activities of P. spiralis crude extracts and isolated compounds (2, 3, 5, 6 and 7), and their cytotoxicity on macrophages and VERO cell lines.
Inhibitory Concentration 50% (IC50) ¼ sample concentration inhibiting 50% of metabolic activity of parasites. Cytotoxic Concentration 50% (CC50) ¼ sample concentration
providing 50% death of macrophages J774A.1 or VERO cells.

Extracts/ IC50 Promastigotes L. infantum IC50 Axenic amastigotes L. infantum CC50 SIa IC50 P. falciparum FcM 29 CC50 VERO SIb
compounds Macrophages

H2O extract 13.4 7 2.6 mg/mL – 1.0 7 0.2 mg/mL 0.07 3.0 70.6 mg/mL 1.7 7 0.8 mg/ 0.6
mL
EtOH extract 450 mg/mL – ND ND 21.17 1.2 mg/mL 2.5 71.0 mg/ 0.1
mL
2 24.17 4.5 mM 4.7 7 1.8 mM 1.4 7 0.08 mM 0.06 7.8 7 1.2 mM 3.7 71.5 mM 0.5
3 9.5 70.2 mM 2.0 7 1.4 mM 0.9 7 0.07 mM 0.09 6.9 70.9 mM 1.8 7 0.07 mM 0.3
5 86.3 713 mM 17.0 70.6 mM 5.5 7 1.9 mM 0.06 50 mM (n¼ 1) 17.6 7 8.0 mM 0.4
6 31.5 7 3 mM 5.4 7 4.4 mM 3.1 71.0 mM 0.10 54.9 73.4 mM 75.2 737 mM 1.4
7 4100 mM 68 7 14 mM ND ND Not stable 907 40 mM ND
Amphotericin B 0.03 70.01 mM 0.34 7 0.2 mM 2.5 7 0.2 83 – 12.2 7 2.8 mM –
Miltefosine 8.8 72.8 mM – 155.3 7 15.2 mM 18 – – –
Pentamidine 0.5 70.3 mM – 0.53 7 0.57 mM 1 – – –
Doxorubicin – – 0.067 0.04 mM – – – –
Chloroquine – – – – 549.27 68 nM – –
Artemisinin
– – – – 13.9 7 7.7 nM – –

a
Selectivity index (SI) ¼CC50(macrophages)/IC50(promastigotes).
b
SI¼ CC50(VERO)/IC50(Plasmodium). 7 standard deviation.
172 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174

3.2.2. Biological activities of isolated compounds inferences about stereochemistry from NOE (Catalán et al., 1988).
The in vitro antileishmanial and antiplasmodial activities of The stereochemical determination of hirsutinolides was thus
isolated sesquiterpene lactones 2, 3, 5, 6 and 7 are presented in strewed with a number of missassignments over the years
Table 1. Compounds 1 and 4 could not be tested because of their regarding the chiral positions at C-8 (Catalán et al., 1988) and
instability. C-10 (Cowall et al., 1981; Herz and Kulanthaivel, 1983; Jakupovic
et al., 1985). Those errors were partially resolved thanks to X-ray
3.2.2.1. Antiplasmodial activity and cytotoxicity of isolated analysis made possible for 8α-angeloyloxyhirsutinolide 13-O-
compounds. The sesquiterpene lactones 2 and 3 were the most acetate (Catalán et al., 1988; Jakupovic et al., 1985). However,
active compounds with IC50 values of 7.8 mM and 6.9 mM, some uncertainties still remain for various molecules of the family
respectively. Compounds 5 and 6 were inactive against the (Bardón et al., 1988; Pollora et al., 2000). In this work, the
parasite (IC50 450 mM) and 7 could not be tested against malaria configuration was established by hypothesizing and comparing
because of its rapid degradation. In parallel, it appeared that 2 and the chemical shifts and the coupling constants of the compounds.
3 were strongly cytotoxic towards VERO cells with CC50 values of Knowing that two conformers coexist in CD3OD solutions of C-8
3.7 and 1.8 mM, respectively. Although compound 5 was less hydroxylated hirsutinolides facilitates the understanding of com-
cytotoxic (CC50 ¼17.6 mM) than 2 or 3, its antiplasmodial activity plex NMR and HPLC data, making their isolation easier and
was still not selective. Compounds 6 (CC50 ¼ 75.2 mM) and 7 avoiding errors in their identification. Isolated compound 7 has
(CC50 ¼ 90 mM) exhibited no cytotoxicity. been described before as an isomeric mixture of 8α and 8β
diastereoisomers (Issa et al., 2006). The 8S* configuration appears
3.2.2.2. Antileishmanial activity and cytotoxicity on macrophages of to be generally accepted now concerning all hirsutinolides, i.e.
isolated compounds. Compounds 2, 3, 5, 6 and 7 were tested with H-8 in the β position (Catalán et al., 1988; Jakupovic et al.,
against the promastigote and axenic amastigote stages of L. 1985). There have been no reports as far as we know about an 8R*
infantum. Generally, all the tested compounds are more active configuration of such compounds in the literature (except early
against the amastigote stage than the promastigote form of the reports that have since been revised (Bohlmann et al., 1979, 1978;
parasite. Hirsutinolide 3 displayed the strongest activity against Catalán et al., 1986; Cowall et al., 1981)). In this context, it is fully
the promastigotes and axenic amastigotes of L. infantum with IC50 justified to have doubts about the stereochemistry described for α-
values of 9.5 and 2.0 mM respectively. Note that the IC50 of 9.5 mM H-8 piptocarphol by Issa et al. (2006) (referred to as compound
against promastigotes is close to that of the reference miltefosine 4 in their publication). In their article, the NMR data of the two
(8.8 mM). Concerning the activities of the other compounds against isomers were not actually published but it can be suggested that
the two stages of the parasite, they can be ranked by decreasing there are in fact two conformers, as some of the NMR analyses
the order of efficiency: 2 46 45 47, 7 being totally inactive seemed to be carried out in CD3OD. This kind of phenomenon
against L. infantum. Concerning their cytotoxicity towards might also occur for other germacranolides exhibiting an ether
macrophages, the most interesting compound, 3, appeared much bridge spatially close to a hydroxyl function.
more cytotoxic than miltefosine with CC50 values of 0.9 and
155.3 mM, respectively. The other products 2, 3, 5 and 6 also 4.2. Biological analysis
exhibited strong cytotoxicities with CC50 between 1.4 and
5.5 mM. Compounds 2, 3, 5 and 6 were also tested on The results showed that the traditional preparation (aqueous
intramacrophagic amastigotes of L. infantum, and all compounds extract) of P. spiralis, exhibited a good antiplasmodial activity against
were found to be inactive at concentrations lower than their CC50 P. falciparum whereas the ethanolic extract was only weekly active.
on macrophages. This result validates the traditional use of this herb in the treatment
of malaria or associated symptoms in South America. Concerning the
antileishmanial activity against L. infantum, while the ethanolic
4. Discussion extract was inactive, the aqueous extract again presented a good
activity. From the seven hirsutinolide-type sesquiterpene lactones
4.1. Structural identification isolated from P. spiralis, five were tested for their antiprotozoal
activities. Compounds 2 and 3 were the most active molecules
One of the main problems encountered during the identifica- against both parasites but also the most toxic on both VERO cells
tion of the compounds isolated in this work concerned the C-8 and macrophages. Furthermore, they had no effect on intramacro-
hydroxylated hirsutinolides 5–7 that exhibited two conformers phagic amastigotes at concentrations non-toxic for the macrophage.
when NMR analyses were recorded in CD3OD. The hard process of Numerous sesquiterpene lactones are known to be bioactive as they
NMR structural elucidation of hirsutinolides has long baffled interact through Michael type additions with various nucleophilic
researchers who have faced it. Indeed, the complex structural core biological targets (Ghantous et al., 2010; Kupchan et al., 1971;
of these germacrane-type sesquiterpene lactones and the presence Merfort, 2011; Schmidt, 2006). This confers them a large spectrum
of four chiral carbons have generated various representations in of biological activities, including antiplasmodial and antileishmanial
the plan of their formula in the literature. Consequently, this led to activities (Lavault et al., 2005; Tiuman et al., 2005; Toyang et al.,
confusions about their stereochemistry (Catalán et al., 1988), 2013) also reported for hirsutinolides (Chea et al., 2006; Odonne et
finally resolved by the rigorous application of the conventional al., 2011; Pillay et al., 2007). It has been suggested by Pillay et al.
representation rules published by Rogers et al. (1972) for germa- (2007) that the antiplasmodial activity of hirsutinolides is due to
cranolide sesquiterpenes. Moreover, due to the conformational their 2(5H) furanone moiety. More generally, Schmidt et al. (2009)
flexibility of the 10-membered ring, the NMR spectra of hirsuti- demonstrated that the α,β-unsaturated carbonyl moiety was the key
nolides exhibited peak enlargement implying poorly resolved pharmacophore, not only for antiprotozoal activity, but also for the
signals, attributed to the existence of several conformers in cytotoxic properties of sesquiterpene lactones (Schmidt, 2006).
equilibrium. Analyses often have to be recorded at high tempera- Thereby, it is not surprising that compound 3, the only compound
ture in C6D6 to limit the broadening of peaks, even so several of tested here displaying a methacrylate ester in its structure, was also
the signals remained broad leading to difficulties in structural the most active molecule. Hirsutinolides 5–7, with a free OH on C-8,
elucidation (Bardón et al., 1993, 1992). This, along with the high were found to be the least active compounds suggesting that the
number of quaternary carbons in the structure, precludes drawing etherification of this hydroxyl group is important for activity and
 C. Girardi et al. / Journal of Ethnopharmacology 170 (2015) 167–174 173

cytotoxicity. Two of the isolated hirsutinolides (1 and 2) have already Toppan from the NMR platform of the Institut de Chimie de Toulouse
been isolated before from the traditional antileishmanial herb P. (ICT) for NMR analyses. The French Ministry of Higher Education and
spicatus, and tested for their in vitro antileishmanial activities against Research is greatly acknowledged for financial support (PhD grant,
axenic amastigotes of Leishmania amazonensis, one of the cutaneous contract number 2011-75).
Leishmaniasis species. Both were reported to be strongly active with
IC50 values of 0.37 mM (1) and 0.20 mM (2), close to the activity
displayed by Amphotericin B (IC50 ¼ 0.41 mM). Hirsutinolide 1 was Appendix A. Supporting information
not tested by our team but the activity of compound 2 against
amastigotes of L. infantum (IC50 ¼4.7 mM) appeared to be more than Supplementary data associated with this article can be found in
20 times less than against amastigotes of L. amazonensis. This could the online version at http://dx.doi.org/10.1016/j.jep.2015.05.014.
be explained by inter-species Leishmania variability regarding to the
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