Journal of Ethnopharmacology 121 (2009) 425–432

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

The anti-ulcerogenic effects of Curatella americana L.

Clélia Akiko Hiruma-Lima a,∗ , Clenilson Martins Rodrigues b , Hélio Kushima a , Thiago Mello Moraes a ,
Solange de Fatima Lolis c , Shirley Barbosa Feitosa c , Luciana Pietro Magri d , Fernanda Rocha Soares d ,
Maira Miranda Cola d , Fábio Donizete Pezzutto Andrade b , Wagner Vilegas b ,
Alba Regina Monteiro Souza Brito d
a
São Paulo State University - Departamento de Fisiologia, Instituto de Biociências, cp 610, CEP 18618-000, UNESP, Botucatu, SP, Brazil
b
São Paulo State University - Departamento de Química Orgânica, Instituto de Química, cp 355, CEP 14801-970, UNESP, Araraquara, SP, Brazil
c
Tocantins Federal University – Instituto de Biologia e Saúde Pública, Rua Sete, Quadra 15, Jardim dos Ipês, CEP 77500-000, Porto Nacional, TO, Brazil
d
Campinas State University - Departamento de Fisiologia e Biofísica, Instituto de Biologia, cp 6109, CEP 13083-970, UNICAMP, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Curatella americana L. (Dilleneaceae) is a medicinal plant very frequently
Received 8 July 2008 cited as acting against gastrointestinal disorders in ethnopharmacological inventories of the Cerrado
Received in revised form 10 October 2008 region of Brazil.
Accepted 18 October 2008
Aim of the study: The ethanolic extract (CEB) and infusion (BI) of Curatella americana bark were investigated
Available online 30 October 2008
for their ability to prevent and heal ulceration of the gastric mucosa.
Materials and methods: The preventive and healing actions of Curatella americana were evaluated in
Keywords:
experimental in vivo models in rodents that simulated this disease in human gastric mucosa.
Curatella americana L.
Dilleneaceae
Results: CEB significantly decreased the severity of gastric damage formation induced by the combination
Anti-ulcer action of several gastroprotective models (HCl/ethanol, indomethacin/bethanecol, absolute ethanol, stress and
Prostaglandin E2 pylorus ligature). But, unlike CEB, the BI did not exert gastroprotective effect. The gastroprotective action
Somatostatin of CEB involved antisecretory action, augmentation of gastric mucus (48%) and participation of endoge-
Gastrin nous sulfhydryl compounds that increase efficacy of barrier mucosa against injurious agents. CEB also
presents effective healing action in chronic gastric disease (1.90 ± 0.55 vs. 6.86 ± 0.46 mm2 in the control)
and its action mechanisms consisted of increasing the PGE2 (40%) and somatostatin levels (269%) while
decreasing the gastrin level in rat plasma (79%).
Conclusions: The gastroprotective effect and healing action of Curatella americana involved modulation
of PGE2 , somatostatin and gastrin levels, probably due to the presence of oligomeric and polymeric
proanthocyanidins in the bark.
© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
An ethnopharmacological inventory accomplished in Tocantins
Cerrado, in central Brazil region (Silva et al., 2000), showed a
high number of medicinal plants utilized against gastric pain and
gastric disease in general. This ethnopharmacological informa-
tion provided the bases for several pharmacological studies that
have conclusively justified their popular use against gastrointesti-
nal disease, specifically in evaluating the anti-ulcer action. Based
on the most recent ethnopharmacological inventory on Brazilian
traditional medicine, we aimed to investigate the gastroprotec-
tive and healing activities of Curatella americana. C americana L.
is a small tree that is very common in the Cerrado region and
∗ Corresponding author. Tel.: +55 1438116077; fax: +55 1438153744.
E-mail address: hiruma@ibb.unesp.br (C.A. Hiruma-Lima).
belongs to the Dilleniaceae family. This species has a wide neotrop-
ical dispersion and frequently occurs in savannas, dry forest and
Cerrado (Pott and Pott, 1994). Despite its low initial grown, this
species is considered a pasture invader (Pott and Pott, 1994).
This plant is used extensively in folk medicine throughout its
range (Corrêa, 1984). Infusion from its leaves and stems is used
against arthritis, diabetes and high blood pressure (Macedo and
Ferreira, 2004), while its bark is utilized to wash cuts (Corrêa,
1984; Vila Verde et al., 2003) and to treat cancers (Luz, 2001),
anaemia (Pinto and Maduro, 2004) and inflammations (Franco
and Barros, 2006); its flowers are indicated against cough, bron-
chitis and flu, while its fruit is useful only for painting animal
skin (Silva et al., 2001). The latex from its bark is used against
migraine, sinusitis and headache whereas the root of this plant is
used against lung pain (Silva, 1998). But the main application of this
species is against gastric pain, gastritis or gastric ulcers (Silva et al.,
2001).

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.10.017
426 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432
Phytochemical analyses carried out by El-Azizi et al. (1980)
described the chemical constituents of Curatella americana leaves
as presenting flavonoids (flavonol glycolide avicularin and gallic
acid), terpenes, phenolic compounds, saponins and steroids. Phy-
tochemical screening of medicinal plant of Mato Grosso do Sul
(Brazil) indicated the presence of triterpenoids and steroids in
leaves, and dye compounds and tannins from wood bark, used in
cortege (Honda et al., 1990).
Some pharmacological studies on different parts of this
species have already corroborated some folk medicine indica-
tions. Guerrero et al. (2002) described the antihypertensive action
of Curatella americana while Alexandre-Moreira et al. (1999) had
already confirmed the anti-inflammatory and analgesic activities
presented by its bark. Our group recently evaluated the antibac-
terial and antifungal activities of ethanolic extract from stem bark
(Costa et al., 2008). Thus, to continue our pharmacological evalua-
tion of this species, the present study aims to further describe the
gastroprotective action and healing effect of infusion and ethanolic
crude extract from bark of Curatella americana.
2. Material and methods
2.1. Animals
All experiments were performed on male Swiss mice (25–40 g)
or male Wistar rats (150–250 g) obtained from CEMIB/UNICAMP,
Campinas, SP, Brazil and the UNESP Animal House, Botucatu, SP,
Brazil. The UNESP Institutional Animal Care and Use Committee
approved all of the employed protocols following the recommen-
dations of the Canadian Council on Animal Care (Olfert et al., 1993).
The animals were fed a certified Nuvilab CR-a® (Nuvital) diet with
free access to tap water under standard conditions of 12 h dark–12 h
light and temperature (21 ◦ C ± 1%). Fasting was used prior to all
assays because standard drugs and vegetable samples were always
administered orally (by gavage) using saline (10 ml/kg) as the vehi-
cle. Moreover, the animals were kept in cages with raised floors of
wide wire mesh to prevent coprophagy.
2.2. Vegetal material and preparation of extract and infusion
Bark of Curatella americana was obtained from periodic collec-
tions realized by Dr. Clelia A. Hiruma-Lima in a Cerrado region
called the Ipês Garden, Porto Nacional city, Tocantins state, Brazil.
A flowered voucher was identified by Prof. Solange F. Lolis of
Tocantins University, Porto Nacional, Tocantins, Brazil, and was
deposited under no. 1637 at the HTINS Herbarium. The powdered
bark of Curatella americana (300 g) was exhaustively extracted with
ethanol (48 h, three times, 3 l each), successively, at room tem-
perature. Solvents were permitted to evaporate at 40 ◦ C under
reduced pressure to provide the crude ethanolic extract from bark
of Curatella americana (CEB) with 3.3% yield (10 g). The infusion
was prepared with dried milled bark of Curatella americana (300 g)
infused with boiling water (3 l) for 10 min. The mixture was filtered
through filter paper to remove macroparticles, cooled to room tem-
perature and lyophilized to furnish 8 g of extractive infusion of bark
(BI) with 2.6% yield.
2.3. Chromatographic profiling
The chemical compositions of CEB and BI were investigated by
high performance liquid chromatography coupled to a photodi-
ode array detector (HPLC-PAD), using a Jasco (Tokyo, Japan) HPLC
equipped with a PU-2089 quaternary solvent pump, an MD-2010
PAD and an AS-2055 autosampler. The analytical column was a
Phenomenex Synergi Hydro RP18 (250 mm × 4.6 mm i.d.; 4 ␮m)
with a Phenomenex security guard column (4.0 mm × 2.0 mm i.d.)
in which different mobile phase compositions were employed. In
the determination of flavonoids, the system used was the same
as reported by Rodrigues et al. (2008). Phenolic acids, catechins
and proanthocyanidins were established using the mobile phase:
water (eluent A) and acetonitrile (eluent B), both containing 0.1%
trifluoroacetic acid (TFA) with the follow gradient program: 0–15%
B (15 min), 15–20% B (30 min), 20–100% B (25 min) and 100% B
isocratic (5 min). The flow rate was 1.0 ml/min and the total run
time was 75 min. EZChrom Elite Data System software (Chromatec,
Idstein, Germany) was used for both detector operation and data
processing. Identification of compounds was performed by reten-
tion time comparison, UV spectral analyses and by spiking with
commercial standards under the same conditions.
2.4. Acute toxicity
The acute toxicity studies were performed in male mice (n = 10).
A single dose of CEB or BI was administered orally to groups of ani-
mals after a 12-h fast. Animals receiving saline served as control.
The signs and symptoms associated with the CEB or BI administra-
tion (5 g/kg, p.o.) were observed at 0, 30, 60, 120, 180 and 240 min
after and then once a day for the next 14 days. At the end of the
period the number of survivors was recorded and the acute toxi-
cological effect was estimated by the method described by Souza
Brito (1995).
2.5. Gastroprotective effect against different ulcerogenic agents
HCl/ethanol-induced ulcer (Mizui and Doteuchi, 1983)—mice
were divided into groups (n = 5–10) that had each fasted for 24 h
prior to oral dosing with the vehicle (10 ml/kg), lansoprazole
(30 mg/kg) and CEB or BI (100, 250, 500 or 1000 mg/kg). Fifty
minutes after the treatments, all animals received orally 0.2 ml
of a 0.3 M HCl/60% EtOH solution. Animals were killed by CO2
gas 1 h after the administration of HCl/EtOH solution. The stom-
achs were removed, opened along the greater curvature and fixed
between two glass plates. Ulcerative lesion was calculated accord-
ing to the methodology described by Szelenyi and Thiemer (1978).
Indomethacin-induced gastric ulcers in cholinomimetic-treated
mice according to Rainsford (1978)—in this model, gastric lesions
were induced with indomethacin (30 mg/kg, s.c.) and bethanechol
(5 mg/kg, i.p.) administered to mice after a 24 h fast. The CEB or
BI (100, 250, 500 or 1000 mg/kg), cimetidine (100 mg/kg) or vehi-
cle was administered orally 30 min before the induction of gastric
ulcer. The animals were killed by CO2 gas 4 h after treatment with
the ulcerogenic agent. The stomachs were removed and ulcerative
lesion was calculated as described previously. Absolute-ethanol-
induced damage (Morimoto et al., 1991)—after a total of 24 h fasting,
three groups of rats (n = 5) received an oral administration of CEB
(500 mg/kg—the lowest dose that presents significant results in all
acute experiments), lansoprazole (30 mg/kg) or vehicle (10 ml/kg).
One hour after treatment, all rats received 1 ml of 99.5% ethanol
to induce gastric ulcer. The animals were killed 1 h after treatment
with the ulcerogenic agent and the stomachs removed to deter-
mine the lesion damage. Hypothermic restraint stress ulcer (Levine,
1971). After having fasted for 24 h, the animals (n = 9) received an
oral administration of CEB (500 mg/kg), cimetidine (100 mg/kg) or
vehicle (10 ml/kg). One hour after treatment, mice were immobi-
lized in a restraint cage at 4 ◦ C for 4 h to induce gastric ulcer. The
animals were killed and the stomachs removed and opened along
the greater curvature to determine the gastric lesion. Shay ulcer—all
male Swiss mice (n = 8–9) were randomly divided into three groups
and fasted for 24 h with free access to water. Thirty minutes after
oral administration of CEB (500 mg/kg), cimetidine (100 mg/kg) as
 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432
positive control or vehicle (10 ml/kg), pylorus ligation was per-
formed as described by Shay et al. (1945). Four hours later the
animals were killed, the abdomen opened and another ligature was
placed around the oesophagus close to the diaphragm. The stomach
was removed and inspected internally from which the gastric juice
volume and pH were recorded. Then the contents were drained into
a graduated centrifuge tube and centrifuged at 3000 × g for 10 min.
The total acid content of gastric secretion was also determined by
titration to pH 7.0 with 0.01N NaOH using a digital burette (E.M.,
Hirschmann Technicolor, Germany). Gastric lesions were evaluated
by examining the inner gastric surface whereas the mucosal lesions
were counted and scored as described above.
2.6. Gastroprotective action mechanism
2.6.1. Determination of gastric secretion
A total of 47 mice were randomly divided into three groups and
fasted for 24 h with free access to water. The assay was performed
by the method of Shay et al. (1945). Immediately after pylorus lig-
ature, CEB (500 mg/kg), cimetidine (100 mg/kg) as positive control
or vehicle (10 ml/kg) was administered intraduodenally. Four hours
later the animals were killed and all of the procedures described for
the Shay ulcer were followed. Evaluation of participation of NO and
SH groups.
2.6.2. Evaluation of participation of NO and SH groups
Rats were divided into groups of 4–10 animals each that fasted
24 h. They previously had been treated intraperitoneally with NEM
(N-ethylmaleimide, Sigma, U.S.A.) at a dose of 10 mg/kg, L-NAME
(NG -nitro-l-arginine methyl ester, U.S.A.) at a dose of 70 mg/kg or
saline. Thirty minutes later, the different groups received an oral
dose of the vehicle, carbenoxolone (100 mg/kg) or CEB (500 mg/kg).
After 60 min, all groups were treated orally with 1 ml of absolute
ethanol for gastric-ulcer induction (Arrieta et al., 2003). Animals
were killed 1 h after ethanol administration and the stomachs
excised and gastric damage determined as described above.
2.6.3. Determination of mucus in gastric content
After mice (n = 6–7) had fasted for 24 h, under anesthesia,
the abdomen was incised and the pylorus ligated. The vehi-
cle (saline), indomethacin (30 mg/kg), carbenoxolone (250 mg/kg)
or CEB (500 mg/kg) was administered intraduodenally after the
pylorus ligature. The animals were killed 4 h after the drug treat-
ments. The stomach content was immersed in 10 ml 0.02% Alcian
blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8, and incubated
for 24 h at 20 ◦ C. The Alcian blue binding extract was centrifuged
at 2000 × g for 10 min. The absorbency of supernatant was mea-
sured at 615 nm using a light spectrophotometer U/2000 (Hitachi,
Japan). The free mucus in the gastric content was calculated from
the amount of Alcian blue binding [mg/wt tissue (g)] (Sun et al.,
1991).
2.6.4. Blood collection
The heparinised blood samples of Sham rats and those treated
with vehicle, lansoprazole (30 mg/kg) or CEB (500 mg/kg) were
obtained from the cardiac puncture (Krous, 1980). Immediately
after the blood was removed, it was submitted to centrifuga-
tion (3000 × g, at 6 ◦ C) for 10 min. After centrifugation, the serum
obtained was stored at −20 ◦ C until the experiments. The Sham
group without treatment experienced the general conditions of the
experimental group.
2.6.5. Somatostatin concentration
After induction of gastric ulcer by ethanol, the somatostatin hor-
mone was measured in serum of Sham rats and those treated with
427
vehicle, lansoprazole or CEB (500 mg/kg) by somatostatin radioim-
munoassay kit (RB-306, EURO-Diagnostica) (Krous, 1980; Motegi
et al., 1998).
2.6.6. Gastrin concentration
After induction of gastric ulcer by ethanol, the gastrin hor-
mone was measured in serum of Sham animals and groups treated
with vehicle, lansoprazole and CEB (500 mg/kg) by gastrin radioim-
munoassay kit (CIS Bio International GASK-PR) (Mayer et al., 1974;
Motegi et al., 1998).
2.6.7. Determination of prostaglandin E2 synthesis
Thirty minutes after treatment with the vehicle, indomethacin
(30 mg/kg), vehicle (10 ml/kg) or CEB (500 mg/kg), the rats were
killed and the abdomen opened. The Sham group without treat-
ment experienced the general conditions of the experimental
groups. Samples of the corpus (full thickness) were excised,
weighed and suspended in 1 ml of 10 mM sodium phosphate buffer,
pH 7.4. The tissue was minced finely with scissors and then incu-
bated at 37 ◦ C for 20 min (Curtis et al., 1995). Prostaglandin in the
buffer was measured using an enzyme immunoassay (RPN 222-
Amersham).
2.7. Healing action
2.7.1. Acetic acid-induced gastric ulcers
Male Wistar rats (n = 5–7) after having fasted for 24 h were used
in this experiment. Under anesthesia, laparotomy was performed
on all animals through a midline epigastric incision. After expos-
ing the stomach, 0.05 ml (v/v) of a 30% acetic acid solution was
injected into the subserosal layer in the glandular part of the ante-
rior wall. The stomach was bathed with saline to avoid adherence
to the external surface of the ulcerated region. The abdomen was
then closed and the animals were fed normally. CEB (500 mg/kg),
cimetidine (100 mg/kg) or vehicle (10 ml/kg) was administered
orally once a day for 14 consecutive days beginning 1 day after
surgery. Body weight was recorded daily throughout the exper-
iments to evaluate the possible chronic toxicity induced by CEB.
On the day after the last drug administration, the rats were killed
and the stomachs were removed and the pH of the gastric juice
secretions was recorded with a pH meter (Marconi, Brazil). The
gastric lesions were evaluated by examining the inner gastric sur-
face with a dissecting magnifying glass. The ulcer area (mm2 ) and
curative ratio (%) were subsequently determined (Takagi et al.,
1969).
2.8. Statistical analysis
Results were expressed as the mean ± S.D. Student’s t-test was
employed to compare the two groups (acute toxicity), whereas one-
way analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s
test was used for comparing three or more groups. P values less
than 0.05 were considered significant.
3. Results and discussion
As part of the pharmacological evaluation of BI and CEB from
Curatella americana, we investigated their acute toxicity in male
Swiss mice. Alexandre-Moreira et al. (1999) reported the LD50 value
was 671.1 mg/kg for bark hydroalcoholic extract from Curatella
americana in mice. But this value was obtained by intraperitoneal
administration of this extract and not by the oral route. Our evalua-
tion shows that a single oral administration of BI or CEB (5 g/kg) did
not produce any sign or symptom of toxicity in the treated animals.
After 14 days of acute oral administration of BI or CEB, no animal
428 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432

Table 1
Effects of different doses of crude extract of bark (CEB) and bark infusion (BI) of Curatella americana in HCl/ethanol-induced and indomethacin/bethanechol-induced gastric
lesion in mice. Results are expressed as mean ± S.D.

Model Treatments (p.o.) Dose (mg/kg) N Ulcer index (mm) Inhibition (%)

HCl/ethanol Vehicle – 10 77.30 ± 9.64 –

Lansoprazole 30 10 21.81 ± 10.65 72
CEB 100 5 78.30 ± 8.37 –
CEB 250 5 54.80 ± 21.31* 29
CEB 500 7 23.0 ± 11.0** 70
CEB 1000 7 9.86 ± 3.63** 87
Vehicle – 7 32.90 ± 8.82 –
Lansoprazole 30 7 16.40 ± 4.93** 50
BI 250 7 25.90 ± 6.26 21
BI 500 7 20.0 ± 5.42* 40
BI 1000 7 14.90 ± 3.24** 55

Indomethacin/bethanecol Vehicle – 11 38.30 ± 8.40 –

Cimetidine 100 11 14.51 ± 3.30** 62
CEB 100 5 36.91 ± 6.89 4
CEB 250 5 34.0 ± 8.06 11
CEB 500 6 22.20 ± 7.14** 42
CEB 1000 5 18.20 ± 8.07** 52
Vehicle – 5 23.80 ± 1.06 –
Cimetidine 100 7 15.40 ± 2.36 35
BI 250 5 24.80 ± 4.0 –
BI 500 5 19.60 ± 6.11 18
BI 1000 6 12.40 ± 3.06 50

CEB-ANOVA F(5;38) = 53.71 in HCl/ethanol model; F(5;37) = 19.90 in indomethacin/bethanechol model; BI-ANOVA F(4;30) = 10.57 in HCl/ethanol model; F(5;23) = 12.60 in
indomethacin/bethanechol model.
*
Dunnett’s test: P < 0.05.
**
Dunnett’s test: P < 0.001.

had died and no significant changes in daily body or organ weight
were observed. There were no significant alterations in water or
food consumption. At autopsy, no significant change or lesion was
observed in the viscera of any animal (data not shown). Loomis and
Hayes (1996) described the classification of some chemical agents
into categories of toxicity in which the dose of 5 g/kg was catego-
rized as practically nontoxic. Accordingly, this result indicates that
both preparations from Curatella americana have no acute toxico-
logical effect when administered orally. These results demonstrated
the need for continuation of pharmacological studies on the oral
administration of this species and motivated us to continue with
the assays.

Fig. 1. HPLC fingerprinting of Curatella americana. (A) Chromatogram of CEB and (B) chromatogram of BI using the characteristic mobile phase for determination of phenolic
acid derivatives, catechins and proanthocyanidins. (C) Chromatogram of CEB and (D) chromatogram of BI using the characteristic mobile phase for determination of flavonoids.
(1) Gallic acid, (2) (+)-catechin and (3) (−)-epicatechin were identified spiking CEB or BI with reference standards solutions under the same conditions. (I) Phenolic acid
derivatives and oligomeric proanthocyanidins and (II) polymeric proanthocyanidins. For chromatographic conditions see Section 2.
 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432 429

Table 2
Effects of crude extract of bark (CEB) from Curatella americana on ethanol-induced gastric lesions in rats, on stress-induced gastric lesions and pylorus ligature-induced gastric
lesions in mice. Results are expressed as mean ± S.D.

Experimental model Treatments (p.o.) Dose (mg/kg) N Ulcer index (mm) Inhibition (%)

Absolute ethanol Control – 5 113.41 ± 9.60 –

Lansoprazole 30 5 68.62 ± 10.72* 40
CEB 500 5 6.0 ± 4.90* 95

Stress Control – 9 13.56 ± 1.93 –

Cimetidine 100 9 1.89 ± 1.33* 86
CEB 500 9 4.56 ± 1.72* 66

Pylorus ligature Control – 8 42.10 ± 7.81 –

Cimetidine 100 9 29.80 ± 6.92* 30
CEB 500 9 27.40 ± 10.0* 35

ANOVA-ethanol model F(2;12) = 11.90; stress model F(2;24) = 5.31; pylorus ligature F(2;23) = 4.68.
*
Dunnett’s test: P < 0.05.

Curatella americana is a medicinal plant used extensively as
a remedy in folk medicine, including gastric disorders. However,
there are no data addressing its pharmacological effect on the
gastrointestinal system. Alexandre-Moreira et al. (1999) already
described the popular use in folk medicine of Curatella ameri-
cana by infusion to treat inflammation and ulcer. But they only
evaluated the anti-inflammatory action of this species presented
by its hydroalcoholic extract. We commenced our evaluation of
the action of Curatella americana by two different preparations,
BI and CEB, on gastric ulcers induced by two agents commonly
damaging to the human stomach (HCl/ethanol and NSAIDs). To
varying degrees, both CEB and BI pre-treatment induced a signifi-
cant protective effect (Table 1). CEB commenced its gastroprotective
action against damaging agents at the dose of 250 mg/kg and pre-
sented a dose-dependent response curve. But the effective dose
of CEB that provided a gastroprotective effect in all experiments
was 500 mg/kg. No significant differences were observed between
the groups treated with CEB at doses of 500 and 1000 mg/kg
(P > 0.05). In almost all experiments, BI exerted weaker gastropro-
tective action than CEB. Analysis of both preparations by HPLC-PAD
shows that CEB presented major number of eluted peaks in the
retention time (Rt ) range of 15–35 min and that these peaks rep-
resent major concentration in comparison with the compounds
found in BI (Fig. 1A and B) corroborating with the best response
of the gastroprotective effect found in CEB. Each eluted peak from
the chromatographic analysis was monitored through PAD scan-
ning from 200 to 600 nm, thus obtaining the UV spectrum of each
peak associated with the class of secondary metabolites present in
each preparation. Flavonoids were selectively monitored at 360 nm
without verifying the presence of this class in CEB or BI (Fig. 1C
and D). Phenolic acid derivatives, catechins and proanthocyani-
dins were monitored at 230 nm (Fig. 1A and B). Proanthocyanidins
of lower polymerization degree, including monomeric catechins,
were resolved in the retention time (Rt ) range of 15–45 min while
the broad unresolved peak eluting at Rt = 54.2 min is characteris-
tic of a mixture of polymeric proanthocyanidins (Gu et al., 2003;
Rodrigues et al., 2007).
The aforementioned results indicate that further experiments
should be conducted using CEB at the dose of 500 mg/kg, which
presented significant results at lower doses in the evaluated mod-
els.
Our results show that crude ethanolic extract of Curatella amer-
icana also exerted gastroprotective action against other damaging
agents in gastric human mucosa such as stress, increase in gas-
tric juice production and absolute ethanol. The data reveal that
pre-treatment with CEB significantly protected (95% and 66%) the
gastric mucosa against absolute ethanol and hypothermic-restraint
stress-induced ulcers, respectively (Table 2). Oral pre-treatment
with CEB also exhibits a protective profile (35%) in gastric ulcers
induced by pylorus ligature (Table 2).
The gastroprotective action of CEB against two preventive mod-
els, specifically HCl/ethanol and NSAIDs-induced gastric lesions,
indicated the involvement of this extract in enhancing, respectively,
cytoprotection factors and defensive agents of gastric mucosa.
These hypotheses were reinforced by the gastroprotective action of
CEB against two other preventive assays, the hypothermic-restraint
stress model and the pylorus ligature model. Compounds that exert
their activity through a mechanism affecting gastric acid content
are known to be effective in these both models, i.e., an antise-
cretory activity may be hypothesized. We evaluated the action of
CEB in producing modification of gastric juice content by differ-
ent routes (oral and intraduodenal) (Table 3) and we observed that
CEB exerted antisecretory action (decreases [H+ ] and increases pH)
only when this extract had been administered intraduodenally, a
result which indicates that CEB produces antisecretory effect by
systemic action and not by local neutralization.Nevertheless, the
confirmation of gastroprotective action from CEB by several mod-
els does not imply that this same plant also has a healing effect
on gastric mucosa. The ulcer produced by the injection of acetic
acid into the stomach wall spreads over a relatively large area that

Table 3
Effects of crude extract (CEB) of bark of Curatella americana administered by oral or intraduodenal route, on gastric juice parameters in pylorus ligature-induced gastric lesions
in mice. Results are expressed as mean ± S.D.

Route Treatments Dose (mg/kg) N pH (unit) Gastric volume (ml) [H+ ] mEq/ml/4 h

Oral Control – 8 4.43 ± 2.15 0.49 ± 0.11 7.02 ± 4.96

Cimetidine 100 9 6.63 ± 1.42* 0.43 ± 0.16 3.54 ± 2.14*
CEB 500 9 3.30 ± 0.95 0.44 ± 0.12 7.72 ± 2.32

Intraduodenal Control – 7 3.43 ± 0.55 0.77 ± 0.13 10.69 ± 1.21

Cimetidine 100 7 5.86 ± 0.55* 0.73 ± 0.09 2.56 ± 1.41*
CEB 500 7 5.14 ± 0.53* 0.63 ± 0.17 4.12 ± 1.40*

Oral route: ANOVA F(2;23) = 8.06 for pH, 2.79 for gastric volume, 2.67 for [H+ ]; Intraduodenal route: ANOVA F(2;18) = 3.14 for pH, 1.68 for gastric volume 5.76 for [H+ ].
*
Dunnett’s test: P < 0.01.
430 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432

Table 4
Effect of crude ethanolic extract of Curatella americana (CEB) on healing of ulcers produced by the injection of a 30% acetic acid solution into the stomachs of rats. The
ulceration was scored on the 15th day after surgery.

Treatment (p.o.) N Dose (mg/kg) Lesion area (mm2 ) pH Curative ratio (%)

Control 7 – 6.86 ± 0.46 3.29 ± 0.39 –

Cimetidine 8 100 1.63 ± 0.44** 2.89 ± 0.37 76
CEB 5 500 1.90 ± 0.55** 1.80 ± 0.47 72

ANOVA: F(2;17) = 20.50.

**
Dunnett’s test P < 0.001.

In order to disclose the action mechanism of this CEB action,

we evaluated the involvement of endogenous NO, sulfhydryl com-
pounds, gastric mucus and PGE2 in the anti-ulcer action. We also
investigated the hormonal effect (somatostatin and gastrin) acti-
vated by CEB in gastric mucosa.
We observed that NO synthase inhibitor (L-NAME) altered the
cytoprotection induced by CEB (Table 5). Oral administration of
CEB to animals pre-treated with L-NAME produced a rise in gas-
tric damage when compared to CEB group pre-treated with saline.
Fig. 2. Body weight gain in rats treated orally with vehicle, cimetidine (100 mg/kg) However, the action of CEB became evident when we evaluated
or crude ethanolic extract from Curatella americana (CEB 500 mg/kg) for 14 days after the participation of sulfhydryl compounds in gastroprotection. Pre-
ulcer formation by acetic acid solution injected into the stomach.
treatment of animals with NEM (an SH-blocker) markedly reversed
the previously observed gastroprotective action of CEB (Table 5).
does not heal with time. This model was assumed to be similar to The gastroprotective effect exerted by CEB (59%) was completely
human chronic ulcer, since it is difficult to treat (Okabe et al., 1971; abolished by pre-treatment with SH-blocker. These results indi-
Okabe and Amagase, 2005). Studies on cimetidine and omepra- cate a strong involvement of endogenous sulfhydryl compounds
zole have shown that anti-ulcer drugs, that promoted inhibition in the gastroprotective effect of this extract. NP-SH compounds are
of acid secretion, were not able to heal ulcers due to poor vascular- involved by controlling the production and nature of mucus. Gastric
isation in the healing area (Ito et al., 1994; Villegas et al., 2004). Our mucus is an important protective factor for the gastric mucosa and
results demonstrated for the first time that oral treatment with CEB consists of a viscous, elastic, adherent and transparent gel formed
for 14 days accelerates the healing of chronic gastric ulcer in rats by water and glycoproteins that covers the entire gastrointestinal
(Table 4). In addition, CEB significantly decreased (72%) the mean mucosa. The protective properties of the mucus barrier depend not
area of chronic ulcer (1.90 ± 0.55 mm2 vs. 6.86 ± 0.46 mm2 in the only on the gel structure but also on the amount or thickness of the
control, P < 0.05). Another important finding that this experimen- layer covering the mucosal surface (Laine et al., 2008).
tal model can provide regards the toxicological parameters from In our experiment, we observed the effect of CEB on free mucus
animal treated with extract for two consecutive weeks. Specifi- production by gastric mucosa (Table 6). Pre-treatment with CEB-
cally among these parameters, the average body weight taken daily induced significant increase of gastric mucus production (49%), a
before the treatment indicated no sign of toxicity in the animals rise which is correlated with its anti-ulcer effect observed consis-
treated with CEB, cimetidine and/or saline for 14 days (Fig. 2). These tently throughout these studies.
data, in association with those observed on acute toxicity, corrob- Among the compounds that increase both mucus produc-
orated the absence of toxicity of this medicinal plant. tion and antisecretory action, PGE2 is an endogenous compound
that modulates the cytoprotection on gastric mucosa (Peskar and
Maricic, 1998). Skarstein (1979) suggested that, since prostaglandin
Table 5
causes vasodilatation, there is an increased change in mucosal
Effect of crude ethanolic extract of Curatella americana (CEB) on gastric lesions
induced by absolute ethanol in rats pre-treated with NEM (SH-blocker) or L-NAME
blood flow around the ulcer in experimental animals as well as aug-
(inhibitor of NO synthase). Results are expressed as mean ± S.D. mented prostaglandin concentration in ulcerative regions rather
than in other parts of the gastric mucosa. Guerrero et al. (2002)
Pre-treatment (i.p.) Treatment (p.o.) N Lesion index (mm) Inhibitiona (%)
reported vasorelaxant activity of Curatella americana that may be
Saline+ Saline 7 107.43 ± 20.27 – mediated by PGE2 . We observed that pre-treatment of rats with
Saline+ Carbenoxolone 10 43.80 ± 13.69* 59
indomethacin (inhibitor of PGE2 production) markedly reduced the
Saline+ CEB 7 2.86 ± 0.96** 97
gastric mucosal PGE2 content from 73.8 to 30.0 pg/mg (Table 7).
L-NAME+ Saline 7 217.28 ± 31.29 – On the other hand, pre-treatment of rats with CEB-induced drastic
L-NAME+ Carbenoxolone 7 102.0 ± 37.89** 53
L-NAME+ CEB 7 19.57 ± 4.56*** 91
Table 6
Saline+ Saline 5 71.20 ± 8.82 –
Effects of crude ethanolic extract of bark from Curatella americana (CEB) adminis-
Saline+ Carbenoxolone 5 24.60 ± 5.71* 65
tered intraduodenally on Alcian blue binding to free gastric mucous from pylorus
Saline+ CEB 4 29.0 ± 17.01* 59
ligature mice.
NEM+ Saline 4 133.75 ± 13.77 –
NEM+ Carbenoxolone 4 166.50 ± 24.25 – Treatments Dose N Volume (ml) Alcian blue bound
NEM+ CEB 4 89.75 ± 29.79 – (mg/kg) (mg/wt tissue (g))

L-NAME model F(5;39) = 12.63; NEM model F(2;12) = 3.04. Control – 7 0.40 ± 0.06 4.04 ± 0.44
a
Percentage of inhibition in relation to respective control group treated with Indomethacin 30 6 0.55 ± 0.08 2.14 ± 0.47*
vehicle (ANOVA followed by Dunnett’s test). Carbenoxolone 250 6 0.83 ± 0.07* 6.77 ± 0.56*
* CEB 100 6 0.42 ± 0.09 6.0 ± 0.51*
P < 0.05.
**
P < 0.001. ANOVA F(3;21) = 7.98 (P < 0.05) for Alcian blue bound; 2.47 (P < 0.05) for volume.
*** *
P < 0.0001. Dunnett’s test: P < 0.05.
 C.A. Hiruma-Lima et al. / Journal of Ethnopharmacology 121 (2009) 425–432 431

Table 7 rats treated with lansoprazole (P < 0.001). Katagiri et al. (2005) also
Effects of crude ethanolic extract of bark from Curatella americana (CEB) on level
observed the same effects in omeprazole. The increase for somato-
of somatostatin, gastrin and prostaglandin E2 in rats. Results are expressed as
mean ± S.D. statin levels probably contributes to diminishing the secretion of
plasma gastrin observed under CEB.
Treatment (p.o.) Dose (mg/kg) N Gastrin (␮U/ml) Decrease (%)
Wittschier et al. (2007) reported the potential prophylactic tools
Sham – 3 333.61 ± 15.10 – that proanthocyanidins present against specific bacterial infection
Vehicle 10 ml/kg 4 349.20 ± 22.20 – including Helicobacter pylori, Campylobacter jejuni, Porphyromonas
Lansoprazole 30 3 46.61 ± 7.01** 86
gingivalis and Candida albicans. Therefore, the phytochemical com-
CEB 500 4 68.72 ± 16.28** 79
position of Curatella americana, primarily its proanthocyanidins,
Treatment (p.o.) Dose (mg/kg) N Somatostatin (␮U/ml) Increase (%) may also contribute important prophylactic tools against bacterial
infection that should antagonize the adhesive interaction of these
Sham – 3 20.82 ± 1.53 –
Vehicle 10 ml/kg 4 20.02 ± 7.49 – bacteria to the gastrointestinal tract.
Lansoprazole 30 4 88.74 ± 11.07** 322
CEB 500 4 76.75 ± 50** 269
4. Conclusions
Treatment (p.o.) Dose (mg/kg) N Prostaglandin E2 (pg/mg) Increase (%)
In conclusion, all these results taken together suggest that the
Sham – 4 71.80 ± 15.90 –
effectiveness of Curatella americana as a gastroprotective and heal-
Vehicle 10 ml/kg 4 73.80 ± 7.32 –
Indomethacin 30 4 30.0 ± 8.52** −58 ing medicinal plant is based on its ability to strengthen defensive
CEB 500 5 100.40 ± 6.69* 40 factors such as by elevating PGE2 levels in addition to other gas-
ANOVA: F(3;11) = 0.27 (P < 0.05) for gastrin; F(3;11) = 28.60 (P < 0.05) for somatostatin; troprotective actions including a stimulant effect on somatostatin
F(3;13) = 20.05 (P < 0.05) for prostaglandin E2 . synthesis and an inhibitory effect on gastrin secretion. The gastro-
*
Dunnett’s test: P < 0.05. protective effect and healing action of Curatella americana resulted
**
Dunnett’s test: P < 0.001. from the presence of oligomeric and polymeric proanthocyani-
dins, in the bark composition of this medicinal plant. Therefore,
the results obtained from the administration of CEB of Curatella
increase of PGE2 levels compared to rats treated only with saline americana under in vivo models not only support the ethnophar-
(P < 0.05). Thus, the augmentation of PGE2 induced by CEB corrob- macological use of this species but also demonstrate its potential
orates the gastroprotective and healing actions of CEB. as a new anti-ulcerogenic drug.
Phytochemical analyses of CEB composition demonstrate the
presence of oligomeric and polymeric proanthocyanidins. Proan-
thocyanidins are oligomeric and polymeric end products of the Acknowledgements
flavonoid biosynthetic pathway. Iwasaki et al. (2004) reported
that this compound was shown to have a protective effect on We are grateful to Prof. Solange F. Lolis - UNITINS for species
the gastric mucosa. The mechanisms underlying the effect of identification, to James Welsh for English revision and to FAPESP
proanthocyanidins are thought to correlate with mucoprotective (Biota-Fapesp Program) and CNPq for fellowships and other finan-
properties, bestowed not only by elevated prostaglandins but also cial support.
by an anti-gastrin effect.
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