Original Papers 1035

A New Naphthoquinone Isolated from the Bulbs

of Cipura paludosa and Pharmacological Activity
of Two Main Constituents

Authors Priscila Batista Tessele 1, Franco Delle Monache 1, Nara Lins Meira Quinto 1, Gislaine Francieli da Silva 1,
Lilian Wunsch Rocha 1, Greice M. R. S. Lucena 2, Vania M. M. Ferreira 2, Rui D. S. Prediger 3, Valdir Cechinel Filho 1

Affiliations The affiliations are listed at the end of the article

Key words Abstract and 63 1 %, respectively; p. o. inhibitions of

l
" Cipura paludosa
! 36 7 % and 58 14%, respectively). It was also
l
" Iridaceae
Cipura paludosa (Iridaceae) is a plant that is dis- suggested that the anti-inflammatory and anti-
l
" naphthalene derivatives
tributed in the north region of Brazil. Its bulbs hypernociceptive effects of eleutherine or iso-
l
" 11hydroxyeleutherine

are used in folk medicine to treat inflammation eleutherine partly depend on the interference
l
" inflammation

l
" hypernociception
received April 6, 2010
revised Nov. 4, 2010
accepted Dec. 17, 2010
Bibliography
DOI http://dx.doi.org/
10.1055/s-0030-1250745
Published online January 31,
2011
Planta Med 2011; 77:
10351043 Georg Thieme
Verlag KG Stuttgart New York
ISSN 00320943
Correspondence
Prof. Dr. Valdir Cechinel Filho
Ncleo de Investigaes
Qumico-Farmacuticas
(NIQFAR) and Programa
de Mestrado em Cincias
Farmacuticas
Universidade do Vale
do Itaja (UNIVALI)
Rua Uruguai, 458
88302-202 Itaja SC
Brazil
Phone: + 55 47 33 41 76 64
Fax: + 55 47 33 41 76 01
cechinel@univali.br
and pain. Four naphthalene derivatives have been
isolated from the bulbs of this plant. Three of
them have been identified as the known naphtha-
lene derivatives, eleutherine, iso-eleutherine, and
hongkonin. The structure of the fourth and new
component was determined as 11-hydroxyeleu-
therine by extensive NMR study. In addition, the
in vivo effect of the two major compounds, eleu-
therine and iso-eleutherine, was evaluated in car-
rageenan-induced hypernociception and inflam-
mation in mice. Eleutherine and iso-eleutherine
(1.0434.92 mol/kg), dosed intraperitoneally
(i. p.) or orally (p. o.), decreased the carrageenan-
induced paw oedema (i. p. inhibitions of 36
7 % and 58 14 %, respectively; p. o. inhibitions
of 36 7 % and 58 14 %, respectively). Iso-eleu-
therine, but not eleutherine, significantly reduced
(inhibitions of 39 4 %) the plasma extravasation
induced by intradermal (i. d.) injection of carra-
geenan. Likewise, eleutherine and iso-eleutherine
(1.0434.92 mol/kg, i. p. or p. o.) were also effec-
tive in preventing the carrageenan-induced hyper-
nociceptive response (i. p. inhibition of 59 4 %
Introduction
! lecular biology techniques [14].
Plants have been used by humankind since time
immemorial to cure and alleviate a variety of dis-
eases. In recent years, there has been increasing
interest in the study of plants recommended by
folk medicine. Natural products and their deriva-
tives are believed to be an important tool for find-
ing new drugs, or prototypes of new drugs, and
research into these has progressed significantly
in the past decade, through the use of modern
with the synthesis or activity of mast cell prod-
ucts, kinins, cytokine, chemokines, prostanoids,
or sympathetic amines. Our findings show that
two major compounds of C. paludosa contain
pharmacologically active constituents that pos-
sess antinociceptive and anti-inflammatory activ-
ity, justifying, at least in part, its popular thera-
peutic use for treating conditions associated with
pain.
Abbreviations
!
NSAID: nonsteroidal anti-inflammatory drug
PGE2: prostaglandin E2
LPS: lipopolysaccharide
BK: bradykinin
SP: substance P
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
pharmacology, biochemistry, toxicology, and mo-
Currently, progress is being made in the develop-
ment of pain therapy. However, there is still a
need to discover new, effective, and potent anal-
gesics, especially for the treatment of chronic
pain. In this regard, it has been widely shown that
many plant-derived compounds present signifi-
cant analgesic and/or anti-inflammatory effects
[57]. Cipura paludosa Aubl., a member of the Iri-
daceae family, may be an alternative treatment
for these kinds of health problems. This species,

Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043

1036 Original Papers

popularly known as batata roxa, alho do mato, or cebolinha-

do-campo is widely distributed in the Amazon rainforest in the
north of Brazil [8]. It has been used in traditional medicine to treat
several disorders such as inflammation, infection, and pain [9].
Previous experimental studies have verified the potential in vivo
protective actions of ethanol extract of C. paludosa bulbs against
MeHg-induced neurotoxicity and also its antinociceptive effects
when evaluated using different models of inflammatory hyper-
nociception in mice and rats [9, 10]. Recently, our group has dem-
onstrated that this herbal product contains certain substances,
especially naphthoquinones, which exhibit in vivo protective ef-
fects against long-term emotional deficits in adult rats born to
dams treated with MeHg [11]. However, to the best of our knowl-
edge, there is no report in the literature that investigates its
chemical composition and active products.
In the present study, we report the isolation of four naphthalene
derivatives from the bulbs of C. paludosa, three of which are Fig. 1 Isolated compounds from C. paludosa bulbs.
known compounds, and the structure determination of a new
metabolite. The study also demonstrates the in vivo effect of the
two major compounds in different models of hypernociception
and inflammation in mice.
Table 1 NMR data in CDCl3 of 11-hydroxyeleutherin (4).

Posi- Long range connectivities

Materials and Methods tion
! 13
C 1
H C-H H-C
Plant material and phytochemical analyses 1 186.2
Plant material was collected in Porto Velho, in the State of Rond- 2 149.1 H3-15
nia, Brazil, in August 2009. A voucher specimen was authenti- 3 138.7
cated by Maria de Ftima Figueiredo Melo (INPA Instituto Na- 4 182.7
5 119.1 7.74 dd (7.7; 1.5) H-7 C-2 C-7 C-9
cional de Pesquisas da Amaznia, Manaus, Brazil) and deposited
6 135.1 7.68 br t C-7 C-8 C-10
at the Dr. Ary Tupinamb Penna Pinheiro Herbarium (Porto Vel-
7 118.2 7.31 dd (8.1; 1.5) H-5 C-5 C-6 C-9
ho, Brazil), under number 1782. Fresh bulbs of C. paludosa
8 159.7 OMe
(1.9 kg) were cut into small pieces and macerated with methanol 9 119.5 H-7
at room temperature for approximately seven days. After filtra- 10 133.8
tion, the solvent was removed by evaporation under reduced 11 67.6 4.46 br d (8.0) C-2 C-3 C-12
pressure and the residue successively partitioned with dichloro- 12 67.1 3.89 m C-11
methane (DCM) and ethyl acetate (EA) to yield the respective di- 13 18.6 1.41 d (6.2) C-11 C-12 C-15
chloromethane (DCM; 2.13 g) and ethyl acetate (EA; 4.43 g) frac- 14 67.4 4.94 br q (6.8) H3-15 C-2 C-3 C-12
15 19.0 1.59 d (6.8) C-1 C-2 C- 14
tions. Part of the DCM (1.19 g) was fractionated over silica gel
OMe-8 56.5 4.01 s C-2
(45 g) on column chromatography (50.0 4.0 cm), eluting with a
OH-11 3.84 br d (0.9)
gradient of n-hexane and n-hexane : ethyl acetate with increasing
polarity (pure n-hexane, 300 mL; 95 : 05, 300 mL; 90 : 10, 300 mL;
85 : 15, 300 mL; 8 : 2, 300 mL; 75 : 25, 200 mL, and pure ethyl ace-
tate, 100 mL). Fractions of 10 mL were collected and evaluated by
TLC using n-hexane: ethyl acetate (85 : 15) as the mobile phase.
Spots were visualised by UV (254 nm) and reaction with sulphu- 250 (4.18), 260 (4.22), 316 (3.62). Molecular formula C16H16O5,
ric anisaldehyde heated at 100 C. Fractions 2331 exhibited only found 288.0862, calcd. 288.0997. EIMS, m/z: 288 (2), 270 (6), 255
one spot and were combined affording 5.0 mg (0.44 % yield from (9), 244 (100), 229 (34), 216 (35), 201 (50). NMR data in l
" Table 1.

DCM) of pure hongkonin (3). Fractions 5865, which exhibited

the same chromatographic profile by TLC, were combined, giving
192 mg (17 % yield from DCM) of a yellow solid identified as iso-
eleutherine (2) in a high grade of purity (~ 97 %). Fractions 7783
were purified in the same way, affording 74 mg (6.5% yield from
DCM) of pure eleutherine (1). Finally, fractions 101134 (57 mg)
were purified by flash chromatography (30 1.5 cm) eluted with
200 mL of n-hexane : acetone (8 : 2), furnishing 5 mg (0.44 % yield
from DCM) of the new compound (R)- 11 hydroxyeleutherine (4)
(l" Fig. 1). Compounds 13 (l " Fig. 1) were identified based on
their spectral data in comparison with those of the literature
[12, 13].
4-hydroxyeleutherine (4): Crystal from hexane, mp 109.5 C
(dec.), []D = 118.7 (CHCl3: c = 0.19). UV (EtOH), max (log ):
Pharmacological studies
Animals: Male and female Swiss mice (2530 g) obtained from
the University of Vale do Itaja (UNIVALI, Itaja, Brazil) were used
in this study. Female mice were chosen to investigate mechanical
hypernociception based on the literature data, which indicate
that females are more susceptible to developing chronic nocicep-
tion. The animals were housed under conditions of optimum
light, temperature, and humidity (12-h light-dark cycle, 22 1 C,
60 to 80 % humidity), with food and water provided ad libitum. All
procedures used in the present study followed the Principles of
Laboratory Animal Care of NIH publication No. 85-23 and were
approved by the Animal Ethics Committee of UNIVALI (protocol
numbers 530/2008 UNIVALI). The number of animals (5-8 per

Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043


group) and the intensity of noxious stimuli used were the mini-
mum necessary to demonstrate consistent effects.
Hypernociception tests mechanical sensitisation: To induce me-
chanical hypernociception, the mice received an i. d. injection of
50 L of carrageenan (300 g/paw), PGE2 (1 nmol/paw), epineph-
rine (3 nmol/paw), bradykinin (BK; 500 ng/paw), or LPS (100 ng/
paw) under the surface of the right hindpaw [14, 15]. To assess
the systemic effect of the drug treatment, the mice were previ-
ously dosed with eleutherine or iso-eleutherine (MW = 286.29;
1.0434.92 mol/kg, i. p.) or vehicle (10 mL/kg, 0.9 % NaCl solu-
tion), 30 min before carrageenan injection. The mechanical hy-
pernociception of all the groups was assessed by means of von
Frey hair (VFH) for up to 48 h after the administration of irritant
drugs, as described below. With the aim of evaluating the effect of
oral treatment, mice received eleutherine or iso-eleutherine
(34.98 mol/kg, p. o., 1 h) and were then submitted to the test. In-
domethacin (13.9 mol/kg, i. p.) was used as a positive control for
the carrageenan model.
Inflammation models paw oedema: The animals were pretreated
with eleutherine or iso-eleutherine (1.0434.92 mol/kg, i. p.) or
vehicle (10 mL/kg, 0.9 % NaCl solution), and after 30 min, they re-
ceived in one hindpaw (right paw) a 50 L i. d. injection of saline
0.9 % containing carrageenan (300 g/paw), histamine (10 nmol/
paw), substance P (SP; 30 nmol/paw), PGE2 (3 nmol/paw), or BK
(3 nmol/paw) [16]. The contralateral hindpaw (left paw) received
50 L of saline and was used as the control. Oedema was mea-
sured using a plethysmometer (Ugo Basile) at different time
points after phlogistics injection. Oedema is expressed in l as
the difference between the right and left paws. With the aim of
evaluating the effect of oral treatment, mice received eleutherine
or iso-eleutherine (34.98 mol/kg, p. o., 1 h) and were then sub-
mitted to phlogistics injection. Indomethacin (13.9 mol/kg, i. p.,
30 min; purity 95%) or dexamethasone (1.27 mol/kg, s. c., 4 h;
purity 95 %) were used as positive controls.
Measurement of plasma extravasations: To evaluate plasma ex-
travasations, the mice received Evans blue (25 mg/kg) intrave-
nously 24 h prior to the test. They were then pretreated with
eleutherine or iso-eleutherine (1.0434.92 mol/kg, i. p.) or ve-
hicle (10 mL/kg, 0.9 % NaCl solution) and after 30 min, the mice
received a 50 L carrageenan injection (300 g/paw). The mice
were sacrificed 2 h after carrageenan injection, and the subcuta-
neous tissue of the carrageenan-injected paw was removed and
immersed in 1 mL of formamide solution for 12 h at 60 C. The ex-
travasated Evans blue was measured by ELISA at 650 nm.
Von Frey hairs-induced hindpaw withdrawal response: To evaluate
mechanical hypernociception, the mice were placed individually
in clear Plexiglas boxes (9 7 11 cm) on elevated wire mesh
platforms, to allow access to the ventral surface of the right hind-
paw. The withdrawal response frequency was measured follow-
ing 10 applications (duration of 1 s each) of von Frey hairs (VFH;
Stoelting). The stimuli were delivered from below, to the plantar
surface of the right hindpaw. The animals were acclimatised for
30 min before behavioural testing, and mechanical hypernoci-
ception was evaluated at several time points. A VFH of 0.6 g pro-
duces a mean withdrawal frequency of about 15%, which is con-
sidered to be an adequate value for the measurement of mechan-
ical hypernociception [14]. Therefore, 0.6 g VFH was used
throughout this study. To determine the basal mechanical thresh-
olds, all the groups were evaluated before the test or surgical pro-
cedures.
Hot-plate test: The hot-plate test was used to measure the re-
sponse latencies, according to the method described previously
Original Papers 1037
[17]. In these experiments, the hot-plate was maintained at
56 1 C. Animals were placed into a glass cylinder, and the time
(s) between placement and shaking or licking of the paws or
jumping was recorded as the index of response latency. The reac-
tion time was recorded for animals pretreated with eleutherine
or iso-eleutherine (3.49 mol/kg, i. p.) or with morphine (13.3
mol/kg; s. c.; purity 95 %), which was used as a positive control.
Animals that remained on the apparatus for an average of 8 s were
selected 24 h previously based on their reactivity in the model. A
latency period (cutoff) of 30 s was defined as complete antinoci-
ception. Animals were treated with the compounds or with mor-
phine 30 min before. Control animals received the vehicle used to
dilute these drugs. The maximal percentage of effect (%MPE) of
glutamate-induced hyperalgesia was calculated as follows:
postdrug  predrug  100
%MPEs
30  predrug
Drugs and reagents
The following drugs and reagents were used: PGE2, substance P,
and carrageenan (Fluka Riedel-de Han); formamide (Merck do
Brasil); histamine, LPS, and Evans blue (Sigma-Aldrich); indo-
methacin (All Chemistry do Brasil Ltda.); morphine (Dimorf)
was kindly provided by Cristlia; retention index mixture of lin-
ear hydrocarbons for GC (Sigma-Aldrich Company). The vehicle
used for diluting the compounds and positive drugs was saline
solution 0.9 %.
Statistical analysis
The results are presented as the mean SEM of 5 to 8 animals in
each group, except for the ID50 values (i.e., the dose of eleutherine
that reduced the hypernociceptive responses by 50 % relative to
the control values) which are presented as means accompanied
by their respective 95 % confidence limits. The ID50 values and
the percentages of inhibition were based on AUC (area under
curve), calculated using the entire time course of each experi-
ment and reported as the mean SEM of inhibitions obtained
for each individual experiment. Statistical comparison of the data
was performed by two-way analysis of variance (ANOVA) fol-
lowed by Bonferronis post-test or one-way ANOVA followed by
Dunnetts post-test. P values lower than 0.05 (p < 0.05 or less)
were considered significant.
Supporting information
The physicochemical data of the known compounds are available
as Supporting Information.
Results
!
The methanol extract of the bulbs of C. paludosa was partitioned
with dichloromethane and ethyl acetate, and the first was sub-
mitted to a combination of column chromatography and prepar-
ative TLC to afford three known naphthalene derivatives and one
new one. The three known compounds were identified as
dimethyldihydropyrano-naphthoquinones eleutherine (1), iso-
eleutherine (2), and naphthodiol hongkonin (3), by comparing
their NMR data with those available in the literature [12, 13].
Conversely, the structure of (R)-11-hydroxyeleutherine was as-
signed to the new compound 4, C16H16O5 (M+, m/z 288.0862 in
the HRMS).
Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043
1038 Original Papers

Fig. 2 Effects of acute administration of eleutherine (1.0434.92 mol/kg, the mean of 5 to 8 animals, and the vertical lines indicate the SEM. * P < 0.05,
i. p., 30 min) on oedema formation (panels A and B) and Evans blue leakage significantly different from control values when compared with the control
(panel C) induced by carrageenan injection in mice. Each group represents group (one-way ANOVA followed by Dunnetts post hoc test).

The anti-inflammatory effect of the C. paludosa isolated com-

Table 2 Effect of eleutherine and iso-eleutherine in mouse paw oedema or
pounds was firstly evaluated in carrageenan-induced paw oede-
mechanical hypernociception induced by several agents.
ma, an experimental model that is extensively employed for
studying anti-inflammatory agents. As observed in l " Fig. 2 A Mediators Eleutherinea Iso-eleutherinea Dexamethasoneb
and B, the single systemic treatment of mice with eleutherine Paw oedema
(1.0434.92 mol/kg, i. p., 30 min) reduced carrageenan-induced Histamine 38 8 %* 7 4% 45 8%**
oedema formation (300 g/paw), an effect observed 60 and (10 nmol/paw)
SP (30 nmol/paw) 5 5% 7 6% 30 5%*
120 min after the administration of this phlogistic agent, with in-
PGE2 (3 nmol/paw) 34 7 %* 23 7% 46 7%**
hibition of 36 7%. Likewise, iso-eleutherine (1.0434.92 mol/
BK (3 nmol/paw) 20 5 % 12 3% 45 9%**
kg, i. p., 30 min) also significantly inhibited the oedematogenic
Mechanical
response evoked by carrageenan in mice, at time points 60 and hypernociception
120 min. The percentage of inhibition for this effect was PGE2 (1 nmol/paw) 75 10%** 87 7%**
58 14 % (l " Fig. 3 A and B). A similar result was obtained with
Epinephrine 69 5 %** 37 7%**
the animals treated with indomethacin (13.9 mol/kg, i. p.) with (100 ng/paw)
inhibition of 44 5 %. When administered orally, only iso-eleu- BK (500 ng/paw) 99 1 %** 89 7%**
therine (34.92 mol/kg, p. o.) presented significant inhibition of LPS (100 ng/paw) 52 11%** 97 2%**

the carrageenan-induced paw oedema, with inhibition of a

3.49 mol/kg, i. p., 30 min; b 1.27 mol/kg, s. c., 4 h; * p < 0.05 and ** p < 0.01
31 3 % (l " Fig. 5 A and B). Indomethacin, dosed orally (13.9

mol/kg, i. p.), was able to reduce the paw oedema (inhibition

% = 45 3 %; l" Fig. 5 A and B). Likewise, eleutherine (3.49 mol/

kg, i. p., 30 min beforehand) produced a significant decrease in found to be effective in significantly preventing the carra-
mouse paw oedema induced by histamine and PGE2 (l " Fig. 6 A geenan-induced hypernociceptive response, with maximum in-
and B). Otherwise, iso-eleutherine did not interfere with the hibitions of 59 4% and 63 1 %, respectively, for i. p. administra-
paw oedema induced by different inflammatory mediators tion (l " Fig. 4 AD) and 78 6 % and 99 1 %, respectively, for p. o.

(l
" Fig. 6 AH). The percentages of inhibition, based on the AUC treatment (l " Fig. 5 C and D). The animals pretreated i. p. or orally

of each group, are listed in l " Table 2.
In addition, the pretreatment of the animals with iso-eleutherine
significantly reduced (MI% = 39 4 %) the plasma extravasations
induced by i. d. injection of carrageenan (300 g/paw) when com-
pared with the vehicle-treated animals (l " Fig. 3 C). Apart from
the above, eleutherine slightly interfered with the plasma extrav-
with indomethacin (13.9 mol/kg) presented inhibition of
76 8 % and 95 4%, respectively. Furthermore, when the ani-
mals were submitted to injections of specific pain mediators,
both eleutherine and iso-eleutherine were capable of interfering
with the mechanical sensitisation induced by i. d. injection of
PGE2, epinephrine, BK, and LPS in mice (l " Fig. 7 AH). The per-

asations (l " Fig. 2 C). centages of inhibition, based on the AUC of each group, are listed
The results observed in l " Fig. 4 (A and C) demonstrate that i. d. in l" Table 2.

injection of carrageenan into the mouse hindpaw significantly re- l" Fig. 8 demonstrates that the pretreatment with the com-

duced the withdrawal threshold to mechanical stimulus pounds was not capable of interfering with the thermal with-
(p < 0.001), which lasts for up to 48 h. The treatment with eleu- drawal threshold, compared with the results for the mice treated
therine or iso-eleutherine (1.0434.92 mol/kg, i. p. or p. o.) was with vehicle. Otherwise, the animals treated with the opioid ago-

Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043

 Original Papers 1039

Fig. 3 Effects of acute administration of iso-eleutherine (1.0434.92 mol/ * P < 0.05 and ** p < 0.01, significantly different from control values when
kg, i. p., 30 min) on oedema formation (panels A and B) and Evans blue leak- compared with the control group (one-way ANOVA followed by Dunnetts
age (panel C) induced by i. d. injection of carrageenan in mice. Each group post hoc test).
represents the mean of 5 to 8 animals, and the vertical lines indicate the SEM.

Fig. 4 Effects of (panel A and B) eleutherine

(1.0434.92 mol/kg, i. p., 30 min) or (panel C and
D) iso-eleutherine (1.0434.92 mol/kg, i. p.,
30 min) on the carrageenan-induced mechanical
hypernociception in mice. Each group represents
the mean of 5 to 8 animals, and the vertical lines
indicate the SEM. * P < 0.05 and ** p < 0.01, signifi-
cantly different from control values when com-
pared with the control group; # p < 0.001, signifi-
cantly different from basal mechanical sensitivity
threshold (one-way ANOVA followed by Dunnetts
post hoc test).

Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043

1040 Original Papers
nist morphine presented a significant increase in the latency of
paw withdrawal when submitted to increased temperature
(p < 0.01).
Discussion
! lar, the irradiation of H-11 ( 4.46) gave enhancement of both
This study presents, for the first time, the chemical characterisa-
tion of C. paludosa extract and the isolation of four compounds,
one of which is not found in the literature. Furthermore, we have
also demonstrated, for the first time, the anti-inflammatory and
anti-hypernociceptive effects of two isolated compounds, eleu-
therine and iso-eleutherine, administered systemically, using
models of inflammation and hypernociception in mice.
The chemical studies led to the isolation of three known com-
pounds (13), 1 and 2 being the major components evidenced in
the plant, together with a new compound which is described
here for the first time.
The complete NMR data for the new compound, 4, are reported in
l" Table 1 and are the result of HETCOR spectra for protonated
carbons as well as long range HETCOR and selective INEPT ex-
periments which established the C-H and HC correlations, re-
spectively. Examination of the signals in the aromatic region sug-
gested the presence of a methoxy-naphthoquinone moiety com-
mon to other substances of the same plant. In addition, there are
signals which accounted for the presence of two secondary
methyl groups at 1.59 (d, J = 6.2 Hz) and 1.40 (d, J = 6.8 Hz),
each linked to an oxygen-bearing carbon whose hydrogen is at
4.94 and at 3.89, respectively. The last carbon, 67.6, is also an
oxygenated methine, and its proton ( 4.46) is coupled (J = 8.0 Hz)
Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043
Fig. 5 Effects of oral administration of eleutherine
or iso-eleutherine (34.92 mol/kg, 1 h) on (A, B)
paw oedema and (C, D) mechanical hypernocicep-
tion induced by i. d. injection of carrageenan
(300 g/paw) in mice. Each group represents the
mean of 5 to 8 animals, and the vertical lines indi-
cate the SEM. * P < 0.05 and ** p < 0.01, signifi-
cantly different from control values when com-
pared with the control group (one-way ANOVA fol-
lowed by Dunnetts post hoc test).
to the aforementioned signal at 3.89, as shown by a cross peak
in the COSY spectrum. Therefore, 4 is an 11-OH derivative of eleu-
therine (1) or of iso-eleutherine (2), which differ in terms of the
cis or trans relationship, respectively, of the two secondary meth-
yl groups. The orientation of the substituents of the dimethylpyr-
an ring was deduced by NOE difference experiments. In particu-
Me-13 and Me-15. Conversely, irradiating each of the methyl
groups, the NOE effect was observed or H-11, therefore they must
be on the same side of the molecule, and the relative stereostruc-
ture is as depicted in 4 (l " Fig. 1).
Naphthalene derivatives are the usual biologically active metab-
olites of Eleutherine, a genus of the same family of Cipura [12, 13,
1820]. Hara and coauthors [19] have described the inhibitory ef-
fects of eleutherine and iso-eleutherine on the HIV replication in
H9 lymphocytes. Furthermore, it was also shown a vasodepres-
sant activity in an isolated Guinea pig heart experiment [18]. It
was reported that tablets with a mixture of naphthalene deriva-
tives, called Hong-cong-su-piang, are effective on angina pecto-
ris, similar to the effect observed with Persantine (dipyrimadol)
in a clinical trial [13]. A recent study has demonstrated that the
antibacterial activities of these compounds present in the Eleu-
therine genus are a potential for their application as natural food
preservatives [21].
Our data reveal that systemic treatment with eleutherine slightly
reduced paw oedema induced by carrageenan injection, but
without any significant effect on the plasma extravasations.
Moreover, iso-eleutherine, administered at the same range of
dose, clearly inhibited the carrageenan-induced paw oedema,
similarly to indomethacin, an NSAID used to reduce inflamma-
 Original Papers 1041

Fig. 6 Effects of systemic administration of eleutherine or iso-eleutherine

(3.49 mol/kg, i. p., 30 min) on the mouse paw oedema induced by (A and
B) histamine (10 nmol/paw), (C and D) SP (30 nmol/paw), (E and F) PGE2
(3 nmol/paw), or (G and H) BK (3 nmol/paw). Dexamethasone (1.27 mol/
kg, s. c., 4 h) was used as a positive control. Each group represents the
mean of 5 to 8 animals, and the vertical lines indicate the SEM. * P < 0.05
and ** p < 0.01, significantly different from control values when compared
with the control group (one-way ANOVA followed by Dunnetts post hoc
test). AUC (area under curve).

Fig. 7 Effects of systemic administration of eleutherine or iso-eleutherine

tion and used as positive control in this study. This data was also
confirmed by the reduction of Evans blue extravasations. Inter-
estingly, both compounds investigated in the present study were
capable of interfering in the acute phase of carrageenan-induced
oedema in mice. Unfortunately, neither of the compounds caused
anti-inflammatory or anti-hypernociceptive effects in a dose-de-
pendent manner. It is important to note that the anti-inflamma-
tory effect of iso-eleutherine was also observed when the com-
pound was dosed orally in a single administration, suggesting a
long-lasting profile for the anti-inflammatory actions of iso-eleu-
therine. The same was not observed for the compound eleuther-
ine, suggesting a possible influence on the bioavailability related
to the stereochemical configuration.
(3.49 mol/kg, i. p., 30 min) on the mouse mechanical hypernociception
induced by (A and B) PGE2 (1 nmol/paw), (C and D) epinephrine (100 ng/
paw), (E and F) BK (500 ng/paw), or (G and H) LPS (100 ng/paw). Each
group represents the mean of 5 to 8 animals, and the vertical lines indicate
the SEM. ** P < 0.01, significantly different from control values when com-
pared with the control group; # p < 0.001, significantly different from basal
mechanical sensitivity threshold (one-way ANOVA followed by Dunnetts
post hoc test). AUC (area under curve).
It has been well characterized that carrageenan, when injected
into the mice hindpaw, induces a rapid release of several vasoac-
tive mediators such as histamine and serotonin, followed by BK.

Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043

1042 Original Papers
Fig. 8 Effects of mor-
phine (13.3 mol/kg,
s. c.), eleutherine, or
iso-eleutherine (3.49
mol/kg, i. p.) in the
paw withdrawal latency
in the hot-plate test in
mice. ** P < 0.01, sig-
nificantly different from
control values (one-way
ANOVA followed by
Dunnetts post hoc
test).
This acute inflammatory response is sustained by the production
of prostaglandins and nitric oxide (product of iNOS activation)
[22]. Extending the results obtained with the carrageenan profile,
the effects of eleutherine and iso-eleutherine on oedema forma-
tion induced by histamine, SP, PGE2, or BK were also assessed.
Therefore, we might suggest that the anti-oedematogenic effects
observed for eleutherine or iso-eleutherine are related to one or
more of these pathways. In fact, eleutherine has been found able
to significantly interfere with paw oedema induced by histamine
or PGE2. On the other hand, iso-eleutherine did not interfere with
the paw oedema induced by any of the inflammatory mediators
used.
Hong et al. [23] have provided clear evidence for the immuno-
modulatory activity of these compounds. The authors demon-
strate that both eleutherine and iso-eleutherine partially inhibit
T helper cell proliferation. In addition, the compounds cited
above increased apoptosis and cytokine IL-2 levels. Indeed, T cells
are responsible for coordinating the cellular response in the in-
flammation through the release of several cytokines. Mosmann
et al. [24] suggested that CD4 + T cells can be functionally categor-
ised into two groups based on their cytokine production. The Th1
subtype is defined by its restricted cytokine production of IL-2,
IFN-, and TNF- [25]. According to a recent report by Song et al.
[26], iso-eleutherine inhibits the production of NO in LPS-acti-
vated macrophages cells. This effect was correlated with the inhi-
bition of iNOS protein and mRNA. Despite the existing in vitro re-
sults that indicate the activity of the compounds, our study
shows, for the first time, the anti-inflammatory activity of eleu-
therine and iso-eleutherine using in vivo experimental models.
It is also known that -carrageenan, when injected into the mi-
ces hindpaws, induces a local inflammatory response character-
ised by paw oedema, neutrophil migration, and the release of
several mediators, such as cytokines, chemokines, prostaglan-
dins, and sympathetic amines, culminating in the sensitisation
of sensorial afferent fibres [27]. In addition, the kinin B2 receptor,
activated after the injection of carrageenan, mediates the inflam-
matory hypernociception dependent on prostanoids and sympa-
thetic amines, in a cytokine-independent mechanism [28]. This
neuronal sensitisation causes a decrease in the mechanical
threshold. Once again, eleutherine or iso-eleutherine, adminis-
tered intraperitoneally or orally, was able to enhance the me-
chanical threshold, reducing the number of hindpaw with-
drawals. Lucena and coauthors [9] have demonstrated the anti-
nociceptive effects of ethanol extract of C. paludosa bulbs in in-
flammatory models of pain behaviour in rodents. The extract,
dosed systemically, inhibited the nociception induced by acetic
acid and the hyperalgesic response induced by different media-
Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043
tors, such as PGE2, PMA, and BK. The C. paludosa extract also re-
duced the mechanical and thermal hypernociceptive response in-
duced by CFAwhen administered acutely or chronically in mice [9].
The effect was also investigated of eleutherine and iso-eleuther-
ine in mechanical hypernociception induced by isolated inflam-
matory mediators, such as PGE2, epinephrine, and BK. The results
of the present study demonstrate that both evaluated com-
pounds interfered with the mechanical sensitisation induced by
PGE2, epinephrine, or BK. Verri and coworkers [29] have shown
that the release of prostanoids and sympathetic amines during
the inflammatory process is preceded by a cascade of cytokines
and chemokines, such as IL-1, TNF, and KC/CINC1, culminat-
ing in sensory neuron sensitisation. On the other hand, the acti-
vation of the kinin B2 receptors was associated with direct activa-
tion of small-diameter afferent sensorial fibres, and it was also
found to induce the release of prostanoids and sympathetic
amines in a cytokine-independent mechanism [28].
We also demonstrated that eleutherine and iso-eleutherine were
capable of interfering in mechanical hypernociception induced
by LPS. It is well recognised that the inflammatory hypernocicep-
tion induced by LPS or antigen challenge involves the release of
several inflammatory mediators. Similar to carrageenan, the i. d.
injection of LPS also induces prostanoids and sympathetic amines
release that acts on their specific metabotropic receptors present
on the sensory neurons [30]. The release of these hypernocicep-
tive mediators is commonly preceded by the cytokines cascade
[27]. We cannot discard the interference of eleutherine and iso-
eleutherine in the inhibition of the antigen presentation and con-
sequently the immune cascade induced by LPS. It is important to
mention that eleutherine or iso-eleutherine appears to not inter-
fere with the opioid system, as no change was observed in the
hindpaw withdrawal latency in the hot-plate test when com-
pared with the animals that received morphine.
Taken together, the present data demonstrate that the two main
compounds of C. paludosa, administered systematically, elicited a
pronounced anti-inflammatory and anti-hypernociceptive ac-
tion against chemical and mechanical models of pain in mice. Ex-
periments are currently in progress in our laboratory to investi-
gate the mechanism responsible for the anti-inflammatory ef-
fects of these compounds.
Acknowledgements
!
This work was supported by grants from following Brazilian insti-
tutions, Conselho Nacional de Desenvolvimento Cientfico e Tec-
nolgico (CNPq), Coordenao de Aperfeioamento de Pessoal de
Nvel Superior (CAPES), and Fundao de Apoio a Pesquisa Cien-
tfica e Tecnolgica do Estado de Santa Catarina (FAPESC). P. B. T.
and G. F. S. are supported by scholarships from CNPq-Brazil.
N. L. M. Q., R. D. S. P., G. M. R. S. L. and V. C. F. are supported by re-
search fellowships from CNPq-Brazil.
Affiliations
1
Ncleo de Investigaes Qumico-Farmacuticas (NIQFAR)
and Programa de Mestrado em Cincias Farmacuticas,
Universidade do Vale do Itaja (UNIVALI), Itaja, SC, Brazil
2
Faculdade de Cincias da Sade, Curso de Cincias Farmacuticas,
Universidade de Braslia (UnB), Braslia, DF, Brazil
3
Departamento de Farmacologia, Centro de Cincias Biolgicas,
Universidade Federal de Santa Catarina (UFSC), Florianpolis, SC, Brazil

References
1 Calixto JB. Efficacy, safety, quality control, marketing and regulatory
guidelines for herbal medicines (phytotherapeutic agents). Braz J Med
Biol Res 2000; 33: 179189
2 Strohl W. The role of natural products in a modern drug discovery pro-
gram. Drug Discov Today 2000; 5: 3941
3 Molinari G. Natural products in drug discovery: present status and per-
spectives. Adv Exp Med Biol 2009; 655: 1327
4 Cheng KW, Wong CC, Wang M, He QY, Chen F. Identification and charac-
terization of molecular targets of natural products by mass spectrom-
etry. Mass Spectrom Rev 2010; 29: 126155
5 Calixto JB, Beirith A, Ferreira J, Santos AR, Cechinel Filho V, Yunes RA. Nat-
urally occurring antinociceptive substances from plants. Phytother Res
2000; 14: 401418
6 Quinto NL, da Silva GF, Antonialli CS, de Campos-Buzzi F, Corra R, Ce-
chinel Filho V. N-antipyrine-3,4-dichloromaleimide, an effective cyclic
imide for the treatment of chronic pain: the role of the glutamatergic
system. Anesth Analg 2010; 110: 942950
7 Ding Y, Liang C, Kim JH, Lee YM, Hyun JH, Kang HK, Kim JA, Min BS, Kim
YH. Triterpene compounds isolated from Acer mandshuricum and their
anti-inflammatory activity. Bioorg Med Chem Lett 2010; 20: 1528
1531
8 Goldblatt P. Iridaceae [with contributions from J.C. Manning and P. Ru-
dall]. In: Kubitski K, editor. Families and genera of flowering plants,
Vol. 2. Heidelberg: Springer Verlag; 1998: 295335
9 Lucena GM, Franco JL, Ribas CM, Azevedo MS, Meotti FC, Gadotti VM,
Dafre AL, Santos AR, Farina M. Cipura paludosa extract prevents methyl
mercury-induced neurotoxicity in mice. Basic Clin Pharmacol Toxicol
2007; 101: 127131
10 Lucena GM, Gadotti VM, Maffi LC, Silva GS, Azevedo MS, Santos AR. Anti-
nociceptive and anti-inflammatory properties from the bulbs of Cipura
paludosa Aubl. J Ethnopharmacol 2007; 112: 1925
11 Lucena GM, Porto FA, Campos EG, Azevedo MS, Cechinel Filho V, Prediger
RDS, Ferreira VM. Cipura paludosa attenuates long-term behavioral def-
icits in rats exposed to methylmercury during early development. Eco-
toxicol Environ Saf 2010; 73: 11501158
12 Shibuya H, Fukushima T, Ohashi K, Nakamura A, Riswan S, Kitaka I. In-
donesian medicinal plants. XX. Chemical structures of eleuthosides A,
B and C, three new aromatic glucodides from the bulbs of Eleutherine
palmifolia (Iridaceae). Chem Pharm Bull 1997; 45: 11301134
13 Cheng Z, Huizu H, Wang C, Li Y, Ding J, Sankawa U, Noguchi H, Yoichi LH.
Hongconin, a new naphthalene derivative from Hong-Cong, the rhi-
zome of Eleutherine americana (Iridaceae). Chem Pharm Bull 1986;
34: 27432746
14 Quinto NLM, Medeiros R, Santos ARS, Campos MM, Calixto JB. Effects of
diacerhein on mechanical allodynia in inflammatory and neuropathic
models of nociception in mice. Anesth Analg 2005; 101: 17631769
15 Santodomingo-Garzn T, Cunha TM, Verri Jr WA, Valrio DA, Parada CA,
Poole S, Ferreira SH, Cunha FQ. Atorvastatin inhibits inflammatory hy-
pernociception. Br J Pharmacol 2006; 149: 1422
16 Fernandes ES, Passos GF, Medeiros R, da Cunha FM, Ferreira J, Campos
MM, Pianowski LF, Calixto JB. Anti-inflammatory effects of compounds
alpha-humulene and ()-trans-caryophyllene isolated from the essen-
tial oil of Cordia verbenacea. Eur J Pharmacol 2007; 569: 228236
Original Papers 1043
17 Santos ARS, Miguel OG, Yunes RA, Calixto JB. Antinociceptive properties
of the new alkaloid, cis-8,10-di-N-propyllobelidiol hydrochloride di-
hydrate isolated from Siphocampylus verticillatus: evidence for the
mechanism of action. J Pharmacol Exp Ther 1999; 289: 417426
18 Komura H, Mizukawa K, Minakata H, Huizu H, Qin G, Xu R. New anthra-
quinones from Eleutherine americana. Chem Pharm Bull 1983; 31:
42064208
19 Hara H, Maruyama N, Yamashita S, Hayashi Y, Lee KH, Bastow KF,
Chairul RM, Imakura Y. Elecanacin, a novel naphthaquinone from the
bulb of Eleutherine americana. Chem Pharm Bull 1997; 45: 17141716
20 Han AR, Min HY, Nam JW, Lee NY, Wiryawan A, Suprapto W, Lee SK, Lee
KR, Seo EK. Identification of a new naphthalene and its derivatives from
the bulb of Eleutherine americana with inhibitory activity on lipopoly-
sacharide-induced nitric oxide production. Chem Pharm Bull 2008; 56:
13141316
21 Ifesan BO, Hamtasin C, Mahabusarakam W, Voravuthikunchai SP. As-
sessment of antistaphylococcal activity of partially purified fractions
and pure compounds from Eleutherine americana. J Food Prot 2009;
72: 354359
22 Di Rosa M, Giroud JP, Willoughby DA. Studies on the mediators of the
acute inflammatory response induced in rats in different sites by carra-
geenan and turpentine. J Pathol 1971; 104: 1529
23 Hong JH, Yu ES, Han AR, Nam JW, Seo EK, Hwang ES. Isoeleutherin and
eleutherinol, naturally occurring selective modulators of Th cell-medi-
ated immune responses. Biochem Biophys Res Commun 2008; 371:
278282
24 Mosmann TR, Bond MW, Coffman RL, Ohara J, Paul WE. T-cell and mast
cell lines respond to B-cell stimulatory factor 1. Proc Natl Acad Sci USA
1986; 8: 56545658
25 Cher DJ, Mosmann TR. Two types of murine helper T cell clone. II. De-
layed-type hypersensitivity is mediated by TH1 clones. J Immunol
1987; 138: 36883694
26 Song SH, Min HY, Han AR, Nam JW, Seo EK, Seoung WP, Sang HL, Sang KL.
Suppression of inducible nitric oxide synthase by ()-isoeleutherin
from the bulbs of Eleutherine americana through the regulation of NF-
kappaB activity. Int Immunopharmacol 2009; 9: 298302
27 Cunha TM, Verri Jr WA, Silva JS, Poole S, Cunha FQ, Ferreira SH. A cascade
of cytokines mediates mechanical inflammatory hypernociception in
mice. Proc Natl Acad Sci USA 2005; 102: 17551760
28 Cunha TM, Verri Jr WA, Fukada SY, Guerrero AT, Santodomingo-Garzn T,
Poole S, Parada CA, Ferreira SH, Cunha FQ. TNF-alpha and IL-1beta me-
diate inflammatory hypernociception in mice triggered by B1 but not
B2 kinin receptor. Eur J Pharmacol 2007; 573: 221229
29 Verri WA Jr, Cunha TM, Parada CA, Poole S, Cunha FQ, Ferreira SH. Hy-
pernociceptive role of cytokines and chemokines: targets for analgesic
drug development? Pharmacol Ther 2006; 112: 116138
30 Khasar SG, McCarter G, Levine JD. Epinephrine produces a beta-adre-
nergic receptor-mediated mechanical hyperalgesia and in vitro sensiti-
zation of rat nociceptors. J Neurophysiol 1999; 81: 11041112
Tessele PB et al. A New Naphthoquinone Planta Med 2011; 77: 10351043