Activity of neolignans isolated from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck against Trypanosoma cruzi

Article abstract of DOI:10.1248/bpb.29.2126

The in vitro antiproliferative effects of 4 neolignans purified from the ethyl-acetate extract from leaves of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck against Trypanosoma cruzi were investigated. These isolated compounds were identified through spectral analyses of UV, EI-MS, ¹H-, ¹³C-NMR, H-H COSY, gNOE, HETCOR, and HMBC. The compounds eupomatenoid-5, eupomatenoid-6, and conocarpan showed considerable activity against epimastigote forms of T. cruzi, with 50% inhibition concentrations (IC₅₀) of 7.0, 7.5, and 8.0 μg/ml respectively. After methylation, these compounds showed a lessened inhibitory activity to the growth of the protozoan, suggesting that loss of the hydroxyl group from their molecules reduces the activity. The compound eupomatenoid-3 showed lower activity than the hexane fraction. Eupomatenoid-5 was significantly more active than benznidazole, the antiparasitic drug of choice for treatment of Chagas' disease. The crude extract, hexane fraction, and eupomatenoid-5 caused no lysis in sheep blood at concentrations which inhibit the growth of epimastigote forms. The compound eupomatenoid-5 showed low cytotoxic effects against Vero cells. These results provide new perspectives on the development of novel drugs obtained from natural products with trypanocidal activity. However, the extracts and active compound isolated from P. regnellii var. pallescens should be further studied in animal models for in vivo efficacy.

Full text of DOI:10.1248/bpb.29.2126

2126
Notes
Biol. Pharm. Bull. 29(10) 2126—2130 (2006)
Vol. 29, No. 10
Activity of Neolignans Isolated from Piper regnellii (MIQ.) C. DC. var.
pallescens (C. DC.) YUNCK against Trypanosoma cruzi
Patrícia Shima LUIZE,a Tânia UEDA-NAKAMURA,b Benedito Prado Dias FILHO,b
c
,b
*
Diógenes Aparício Garcia CORTEZ, and Celso Vataru NAKAMURA
a Mestranda do Programa de Pós-graduação em Ciências Farmacêuticas; b Laboratório de Microbiologia Aplicada aos
c
Produtos Naturais e Sintéticos, Departamento de Análises Clínicas; and Laboratório de Farmacognosia, Departamento
de Farmácia e Farmacologia, Universidade Estadual de Maringá; Maringá, av. Colombo, 5790, 87020–900 Maringá,
Paraná, Brazil. Received January 17, 2006; accepted July 10, 2006
The in vitro antiproliferative effects of 4 neolignans purified from the ethyl-acetate extract from leaves of
Piper regnellii (MIQ.) C. DC. var. pallescens (C. DC.) YUNCK against Trypanosoma cruzi were investigated. These
isolated compounds were identified through spectral analyses of UV, EI-MS, 1H-, 13C-NMR, H–H COSY, gNOE,
HETCOR, and HMBC. The compounds eupomatenoid-5, eupomatenoid-6, and conocarpan showed considerable
activity against epimastigote forms of T. cruzi, with 50% inhibition concentrations (IC50) of 7.0, 7.5, and
8.0 mg/ml respectively. After methylation, these compounds showed a lessened inhibitory activity to the growth of
the protozoan, suggesting that loss of the hydroxyl group from their molecules reduces the activity. The com-
pound eupomatenoid-3 showed lower activity than the hexane fraction. Eupomatenoid-5 was significantly more
active than benznidazole, the antiparasitic drug of choice for treatment of Chagas’ disease. The crude extract,
hexane fraction, and eupomatenoid-5 caused no lysis in sheep blood at concentrations which inhibit the growth
of epimastigote forms. The compound eupomatenoid-5 showed low cytotoxic effects against Vero cells. These re-
sults provide new perspectives on the development of novel drugs obtained from natural products with try-
panocidal activity. However, the extracts and active compound isolated from P. regnellii var. pallescens should be
further studied in animal models for in vivo efficacy.
Key words anti-trypanosoma; neolignan; Piper regnellii; Trypanosoma cruzi
Chagas’ disease is an important medical problem in Latin the forms of crude extracts, infusions, or plasters to treat
America, affecting about 18 million people and causing the wounds and reduce swelling and skin irritations.9—11) Holetz
deaths of 45000 patients annually. The etiologic agent Try- et al.12) screened 13 Brazilian medicinal plants, and reported
panosoma cruzi is transmitted to the vertebrate host by a antimicrobial activity of the hydroethanolic extract of leaves
hematophagous reduviid bug vector. The initial acute phase of P. regnellii against the bacteria Staphylococcus aureus and
has a low mortality (ꢀ10%) and is often asymptomatic, Bacillus subtilis, and against the yeasts Candida krusei and
whereas the chronic phase typically occurs 10 to 20 years Candida tropicalis.
after the parasite is contracted, and affects 30 to 40% of those
Various species of Piper have been shown to have interest-
infected. Cardiac and gastrointestinal pathology are the most ing biological activities.13) Phytochemical study of P. regnel-
common manifestations of chronic disease.1) Currently avail- lii roots has demonstrated the accumulation of phenyl-
able chemotherapy, based on benznidazole and nifurtimox, is propanoids and dihydrobenzofuran neolignans.14) Several
unsatisfactory because of limited efficacy in the prevalent compounds of this class (benzofuran neolignans) have been
chronic stage of the disease and because of the frequent toxic isolated from species of Piperaceae, and show a variety of bi-
side effects.2) It is necessary to develop alternative, powerful ological activity including anti-PAF, antifungical, and insecti-
trypanocidal drugs with no side effects.
cidal.15) In the present study, we describe the anti-Try-
In spite of the great advances in modern medicine in re- panosoma cruzi activity of extracts and neolignans isolated
cent decades, plants still make an important contribution to from P. regnellii var. pallescens.
health care. Marked growth in the worldwide phytotherapeu-
tic market has occurred over the last 15 years.3) Plants pro- MATERIALS AND METHODS
vide chemical diversity and bioactivity, which has led to the
development of hundreds of pharmaceutical drugs. Natural
Plant Material The leaves of P. regnellii var. pallescens
products represent an unparalleled source of molecular diver- were collected in July 2002 in the Profa. Irenice Silva Garden
sity for drug discovery and development.4) Several studies of Medicinal Plants on the campus of the State University of
have demonstrated that plants show activity against T. Maringá. The plant material was identified through morpho-
cruzi.5—7) The idea that herbal drugs are safe and free of side logical analysis, by Marilia Borgo of the Botanical Depart-
effects is false. Plants contain hundreds of constituents and ment of the Federal University of Paraná, and a voucher
some of them are very toxic.3) Medicinal plants are distrib- specimen (N° HUM 8392) is deposited at the Herbarium of
uted worldwide, but they are most numerous in tropical the State University of Maringá, Paraná, Brazil.
countries.
General Experimental Procedures The NMR spectra
Piper regnellii (MIQ.) C. DC. var. pallescens (C. DC.) were obtained in a BRUKER ARX400 (9.4 T) and VARIAN
YUNCK (Piperaceae) is an herbaceous plant popularly known GEMINI 300 (7.05 T), using deuterated solvent for field
in Brazil as “Pariparoba” and grows in tropical and subtropi- homogeneity, TMS as the internal standard, and a constant
cal regions of the world.8) The leaves and roots are used in temperature of 298 K. IR: film NaCl plates; ES-MS were
∗ To whom correspondence should be addressed. e-mail: cvnakamura@uem.br
© 2006 Pharmaceutical Society of Japan

October 2006
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recorded on a Micromass Quattro LC, HR-MS: Autospec-
Micromass EBE and EI-MS on a CG/EM-SHIMADZU QP
2000 A. CC: silica gel 60 (70—230 and 230—400 mesh);
TLC: silica gel plates F254 (0.25 mm in thickness).
Isolation of the Constituents Dried leaves (200 g) of P.
regnellii var. pallescens were extracted with ethanol : water
(9 : 1) at room temperature. The solvent was removed under
vacuum at 40 °C to give an aqueous extract and a dark-green
residue. The aqueous extract was lyophilised (20.3 g) and the
(water-insoluble) residue in a glass bottle was washed with
ethyl acetate. The organic solvent was removed to yield the
ethyl-acetate extract (13.9 g). The aqueous and acetyl-acetate
extracts were assayed against the epimastigote forms of T.
cruzi.
The active ethyl-acetate extract (13.9 g) was submitted to
vacuum-column chromatography (silica gel 150 g) and eluted
with hexane, chloroform, chloroform/ethyl acetate 19 : 1,
chloroform/ethyl acetate 9 : 1, chloroform/ethyl acetate 1 : 1,
ethyl acetate, acetone, methanol, and methanol/water 9 : 1.
Each fraction (hexane, F1; chloroform, F2; chloroform/ethyl
acetate 19 : 1, F3; chloroform/ethyl acetate 9 : 1, F4; chloro-
form/ethyl acetate 1 : 1, F5; ethyl acetate, F6; acetone, F7;
methanol, F8; and methanol/water 9 : 1, F9) was assayed for
trypanocidal activity. The majority active hexane fraction—
F1 (7.0 g) was chromatographed by column chromatography
on silica gel 60 (70—230 mesh) eluted with hexane, hexane/
dichloromethane (49 : 1, 19 : 1, 9 : 1, 1 : 1), dichloromethane,
chloroform, ethyl acetate, acetone, and methanol, and
yielded 137 fractions. Thin-layer chromatography compari-
son of these fractions led to combination of four large frac-
tions (F24, F42, F51, F53). Fraction F53 (1.53 g) was chro-
matographed on silica gel 60 (230—400 mesh) with the mo-
bile phase hexane : dichloromethane : acetyl acetate (12 : 7 : 1)
to separate 23 fractions (F53-1 to F53-23). Fractions F24,
F42, F51, and F53-4 were identified by spectroscopic analy-
sis, and this procedure allowed the isolation of 4 pure
Chart 1. Isolation Scheme of Neolignans: Eupomatenoid-3 (1), Eupo-
matenoid-5 (2), Eupomatenoid-6 (3), and Conocarpan (4) from Hydroalco-
holic Extracts of Piper regnellii var. pallescens Leaves
compounds: 2-(3,4-methylenedioxyphenyl)-3-methyl-5(E)- forms of Trypanosoma cruzi Y strain was used as test organ-
propenyl benzofuran or eupomatenoid-3 (0.12 g), [2-(4ꢁ-hy- isms. The protozoans were grown in liver-infusion tryptone
droxy-5ꢁ-methoxyphenyl)-3-methyl-5(E)-propenyl benzofu- broth-LIT17) supplemented with 10% fetal bovine serum
ran] or eupomatenoid-5 (1.13 g), [2-(4ꢁ-hydroxyphenyl)-3- (Gibco Invitrogen Corporation, New York, U.S.A.).
methyl-5(E)-pro-penyl benzofuran] or eupomatenoid-6
Trypanocidal Activity in Vitro The epimastigote form
(0.11 g), and (2R,3R)-2,3-dihydro-2-(4ꢁ-hydroxyphenyl)-3- of T. cruzi was assayed with crude extract ethyl-acetate
methyl-5(E)-propenyl benzofuran or conocarpan (0.83 g) re- phase, hexane fraction, and isolated compounds. The extracts
spectively (Flowchart). Their structures were established by to be tested were dissolved in dimethyl sulfoxide (DMSO).
1
the chemical and spectroscopic methods UV, EI-MS, H-, The final concentration of DMSO did not exceed 1% and for
13C-NMR, H–H COSY, gNOE, HETCOR, and HMBC, and each experiment there was a growth control with and without
by comparison with literature data.14,16) The isolated com- DMSO.18) The experiments were performed in 24-well plates
pounds were tested against T. cruzi.
containing 1 ml of diluted compound at different concentra-
Methylation of Compounds To obtain diazomethane, tions. The starting inoculum consisted of 106 parasites per
5.0 g of diazo was dissolved in 45.0 ml of ether. The solution well in logarithmic growth phase. The cells were incubated
was cooled in an ice bath and added to a solution of potas- at 28 °C and the growth was determined by counting the
sium hydroxide (0.8 g) in 96% ethanol (20 ml). The mixture parasites with a Neubauer haemocytometer every 24 h over a
was stirred vigorously, and after 5 min in the ice bath, distil- 7-d period. Benznidazole (N-benzyl-2-nitro-1-imidazolacet-
lation was begun, using the Aldrich® Diazomethane Kit. The amide, Roche Pharmaceuticals, Rio de Janeiro, Brazil) was
isolated compounds eupomatenoid-5, eupomatenoid-6, and used as the reference drug. The assays were performed in du-
conocarpan (10 mg) were soluble in CHCl3 (2 ml) and were plicate on separate occasions.
methylated with diazomethane (CH2N2–Et2O) to give methyl
Red Blood Cell (RBC) Lysis Assay A 4% suspension
derivatives, respectively. The solvent was evaporated, and of freshly defibrinised sheep blood was prepared by adding
these compounds were then assayed against the epimastigote 2.0 ml of blood to 48 ml of 5% glucose solution. Stock solu-
forms of T. cruzi.
tions of crude extract ethyl-acetate phase, hexane fraction,
Microorganisms and Growth Conditions Epimastigote and eupomatenoid-5 solubilised in DMSO were further di-

2128
Vol. 29, No. 10
luted in 5% glucose at different concentrations. One millilitre
of RBC suspension was added and gently mixed, and the
tubes were incubated at 37 °C. Samples were centrifuged at
1000ꢂg for 10 min. Absorbance of the supernatant was de-
termined at 540 nm. The inhibition of haemolysis was calcu-
lated according to the equation: haemolysis inhibition (%)ꢃ
(ApꢄAs)/(ApꢄAc)ꢂ100; where Ap, As, and Ac are the
absorbance of the positive control (Triton X-114), test sam-
ple, and negative control respectively. These tests were per-
formed in duplicate on separate occasions.
Cellular Toxicity The Vero cells were seeded onto 96-
well microtitre plates at a concentration of 2.5ꢂ104 cells per
well, and allowed to proliferate for 24 h in DMEM contain-
ing 5% FBS. Different concentrations of eupomatenoid-5
were applied to the cell monolayer in triplicate. DMSO was
used as a negative control. After incubation at 37 °C with 5%
CO2 for 72 h, the cell growth was evaluated by the sulforho-
damine B assay.19) Data were calculated as the percentage of
inhibition. The concentration of 50% cellular toxicity (CC50)
was defined as the concentration which reduces the OD530 of
treated cells to 50% of that of untreated cells.
Fig. 1. Effect of Crude Extract Ethyl-Acetate Phase (CE) and Fractions
(F1—F9) at 50 mg/ml Obtained from Piper regnellii var. pallescens on
Growth of the Epimastigote Form of Trypanosoma cruzi after 96 h of Incu-
bation
RESULTS AND DISCUSSION
The 50% inhibition concentration (IC50) for T. cruzi, of the
crude extract ethyl-acetate phase obtained from leaves of P.
regnellii var. pallescens, was 54 mg/ml, whereas the aqueous
phase was inactive (IC50 ꢅ500 mg/ml). On the basis of this
finding, the ethyl-acetate extract was fractionated on silica
gel into nine fractions (F1—F9). The F1, F2, F3, and F4
fractions at a concentration of 50 mg/ml, showed a growth in-
hibition of 92.2, 86.9, 75.0, and 64.0% respectively (Fig. 1).
The other fractions (F5 to F9) showed an activity lower than
that of the crude extract ethyl-acetate phase in the growth of
T. cruzi.
The pure compounds isolated from the hexane fraction
(F1) were assayed against the epimastigote forms of T. cruzi
(Fig. 2). As illustrated in Figs. 2B, C, and D, T. cruzi showed
similar sensitivity when eupomatenoid-5, eupomatenoid-6,
and conocarpan were assayed at different concentrations. At
50 mg/ml these compounds inhibited 100% of the growth of
T. cruzi after 96 h of incubation. Eupomatenoid-3 showed
less activity than the hexane fraction (Fig. 2A). Compounds
1, 2, 3, and 4 were identified as eupomatenoid-3, eupo-
matenoid-5, eupomatenoid-6, and conocarpan respectively
(Flowchart). Their 1H-, 13C-NMR spectral data were identical
to those of published values.14,16)
Eupomatenoid-5, eupomatenoid-6, and conocarpan were
methylated and their antiproliferative effects were estimated.
Table 1 shows the values of IC50 of isolated and methylated
compounds obtained from the hexane fraction of P. regnellii
var. pallescens against the epimastigote forms of T. cruzi.
While the IC50 of eupomatenoid-3 against T. cruzi epimastig-
otes was 26.3 mg/ml, eupomatenoid-5, eupomatenoid-6 and
conocarpan showed similar IC50 levels of 7.0, 7.5, and
8.0 mg/ml, respectively. When these purified compounds
were methylated, the IC50 increased to 29.2, 21.8, and
Fig. 2. Effect of Compounds Isolated from Piper regnellii var. pallescens
on Growth of the Epimastigote Form of Trypanosoma cruzi: (A) Eupo-
matenoid-3, (B) Eupomatenoid-5, (C) Eupomatenoid-6, (D) Conocarpan
ꢀ, control; ꢁ, 50 mg/ml; ꢂ, 10 mg/ml; ꢃ, 5 mg/ml; ∗, 1 mg/ml.
24.5 mg/ml, respectively. After methylation, these compounds sesses no phenolic hydroxyl group. The phenolic hydroxyl
showed lowered inhibitory activity on the growth of the pro- present in the active structure is apparently able, through hy-
tozoans, suggesting that the loss of the hydroxyl group from drogen-bond links with the specific molecular structures on
their molecules reduces the activity. Eupomatenoid-3 pos- the cell surface, to inactivate essential metabolic pathways.20)

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2129
Table 1. Concentration of 50% Growth Inhibition (IC50) of the Epimastig-
ote Form of Trypanosoma cruzi after Treatment for 96 h with Compounds
Isolated from the Hexane Fraction of Piper regnellii var. pallescens, before
and after Methylation
IC50
(mg/ml)
IC50
(mg/ml)
Isolated compouds
Methylated compounds
26.3ꢆ5.4
7.0ꢆ1.3
7.5ꢆ0.5
8.0ꢆ0.5
—
—
Fig. 4. Effect of Amphotericin B (ꢀ), Crude Extract Ethyl-Acetate Phase
(ꢂ), Hexane Fraction (ꢃ), and Eupomatenoid-5 (ꢁ) from Piper regnellii
var. pallescens, in Sheep Blood after 2 h of Incubation at 37 °C
which showed 78% growth inhibition. The medium contain-
ing 1% DMSO did not affect the growth of the protozoans
(data not shown). Benznidazole, the most important antipara-
sitic drug for treatment of Chagas’ disease, has limited effi-
cacy in the chronic phase of the disease. Disparities in the
susceptibility of T. cruzi strains to drugs may explain, in part,
differences in drug efficacy in the treatment of vertebrate
hosts.23) Yong et al.18) showed that the values of IC50 of ben-
znidazole against T. cruzi Dm28 strains resistant and sensi-
tive to synthetic inhibitors of cysteine-protease, were 1.56
and 1.30 mg/ml respectively.
An important criterion in the search for compounds active
against T. cruzi with therapeutic potential is that they have no
toxic effects on mammalian host cells. For this purpose, a
test of cytotoxicity to sheep erythrocytes was performed in
order to determine the ratio of selectivity to biological activ-
ity. Figure 4 shows the effect of the crude extract ethyl-ac-
etate phase, hexane fraction, and eupomatenoid-5 isolated
from P. regnellii var. pallescens and amphotericin B in sheep
blood after 2 h of incubation at 37 °C. Neither the crude ex-
tract, hexane fraction, nor eupomatenoid-5 affected red blood
cell integrity at concentrations which inhibit the growth of
epimastigote forms of T. cruzi. At 100 mg/ml the percentage
of haemolysis varied from 1 to 10%. On the other hand, the
drug amphotericin B, which has a potent effect against T.
cruzi but is toxic to host cells, shows 78% haemolysis. As re-
ported by Lee et al.,24) amphotericin B showed strong
haemolytic activity with a minimum lytic concentration
(MLC) of 6.25 mg/ml within 60 min, whereas 1% DMSO did
not cause lysis.
29.2ꢆ0.6
21.8ꢆ3.5
24.5ꢆ2.1
Fig. 3. Percentage of Growth Inhibition of the Epimastigote Forms of Try-
panosoma cruzi after 96 h of Incubation with 10 mg/ml of Crude Extract
Ethyl-Acetate Phase (ꢄ), Hexane Fraction ( ), and Eupomatenoid-5 (ꢁ)
from Piper regnellii var. pallescens and Benznidazole (
)
Statistical analysis was performed using ANOVA (pꢀ0.01).
In the experiment used to evaluate cytotoxicity for mam-
malian cells, Vero cells were treated with the pure isolated
Abe et al.21) isolated 4 neolignans (eupomatenoid-1 and 7, compound, and after 72 h the viability was checked by the
licarin A and B) and 2 lignans (austrobailignan-7 and fra- sulforhodamine B assay. When Vero cells were treated with
gransin E1) from roots of Aristolochia taliscana active eupomatenoid-5, the 50% cytotoxic concentration (CC50)
against epimastigote forms of T. cruzi. They suggested that was 250 mg/ml. The toxicity for Vero cells and the activity
loss of the hydroxyl group reduces activity. Pessini et al.22) against the protozoans were compared by using the selectiv-
demonstrated the antifungal activity of the compound cono- ity index (SI) ratio (CC50 for Vero cells/IC50 for protozoans).
carpan isolated from P. regnellii var. pallescens, and reported A value greater than 1 is considered more selective for activ-
that the activity could be related to the presence of the phe- ity against the parasite. Eupomatenoid-5 was more selective
nolic hydroxyl.
against the parasites than the Vero cells, with an SI ratio of
Figure 3 shows the percentage of growth inhibition of epi- 35.7.
mastigote forms of T. cruzi at 10 mg/ml of crude extract,
These data revealed that leaves of P. regnellii var.
hexane fraction, eupomatenoid-5, and benznidazole. At this pallescens contain compounds with anti-trypanosomal activ-
concentration, the crude extract showed 30% inhibition, the ity, clearly indicating that our results provide preliminary sci-
hexane fraction 48%, and the pure compound 100%. Eupo- entific validation for the medicinal use of this plant. How-
matenoid-5 was significantly more active than benznidazole, ever, the extracts and active compounds isolated from P. r e g -

2130
Vol. 29, No. 10
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