Antiulcerogenic effect and cytotoxic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

Article abstract of DOI:10.1016/S0014-2999(03)01909-5

Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which, however, is accompanied by toxic effects. On the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin, expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays. To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl, stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC₅₀=500 μM in the neutral red uptake and MTT reduction tests and an IC ₅₀=200 μM in the nucleic acid content test. With regard to hepatocyte viability after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC₅₀=10-500 μM in the nucleic acid content and MTT reduction tests and an IC₅₀=120 μM in the neutral red uptake test. In another experiment, V79 cells were incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h incubation, semi-synthetic crotonin had an IC₅₀=500 μM in the MTT reduction and neutral red uptake tests and an IC₅₀=370 μM in nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated hepatocytes were in culture.

Full text of DOI:10.1016/S0014-2999(03)01909-5

European Journal of Pharmacology 472 (2003) 205–212
www.elsevier.com/locate/ejphar
Antiulcerogenic effect and cytotoxic activity of semi-synthetic
crotonin obtained from Croton cajucara Benth.
a,
Ana Beatriz Albino de Almeida , Patricia S. Melo , Clelia A. Hiruma-Lima ,
b
c
*
d
e
e
Juliano S. Gracioso , Ligia Carli , Domingos S. Nunes ,
b
Marcela Haun , Alba R.M. Souza Brito
d
a
Departamento de Farmacologia, Faculdade de Ci eˆ ncias M e´ dicas, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil
b
Departamento de Bioqu ´ı mica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil
c
Departamento de Fisiologia, Instituto de Bioci eˆ ncias, Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil
d
Departamento de Fisiologia e Biof ´ı sica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil
e
Departamento de Qu ´ı mica, Universidade Estadual de Ponta Grossa (UEPG), Ponta Grossa, PR, Brazil
Received 21 May 2003; accepted 22 May 2003
Abstract
Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which,
however, is accompanied by toxic effects. On the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to
determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated
from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin,
expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for
cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays.
To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the
antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl,
stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC50 = 500 AM in the
neutral red uptake and MTT reduction tests and an IC50 = 200 AM in the nucleic acid content test. With regard to hepatocyte viability
after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC50 = 10–500 AM in the nucleic
acid content and MTT reduction tests and an IC50 = 120 AM in the neutral red uptake test. In another experiment, V79 cells were
incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h
incubation, semi-synthetic crotonin had an IC50 = 500 AM in the MTT reduction and neutral red uptake tests and an IC50 = 370 AM in
nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a
possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also
evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant
differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with
semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also
show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated
hepatocytes were in culture.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Crotonin; Semi-synthetic; V79 cell; Cytotoxicity; Hepatocyte toxicity; Gastric ulcer
1. Introduction
*
Corresponding author. Departamento de Fisiologia e Biof ´ı sica,
Croton cajucara Benth. occurs widely in the Amazon
Instituto de Biologia, Cidade Universit a´ ria ‘‘Zeferino Vaz’’, UNICAMP,
C.P. 6109, Distrito de Bar a˜ o Geraldo, CEP. 13083-970, Campinas, SP,
Brazil. Tel.: +55-19-37886192; fax: +55-19-37886185.
region of northern Brazil, where it is popularly known as
‘sacaca’’. This plant has a history of safe use in folk
medicine in the form of a tea for ailments such as diarrhea,
‘
E-mail address: anabia5@yahoo.com.br (A.B. Albino de Almeida).
0014-2999/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0014-2999(03)01909-5

206
A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
diabetes, gastrointestinal disorders and inflammation of the
liver (Di Stasi et al., 1989). Previous studies with ‘‘sacaca’’
have shown that the major component of the bark is t-
dehydrocrotonin (Itokawa et al., 1989, 1990; Ichihara et al.,
acetate (75/25), using an air pressure of 4 psi to maintain
eluent flow at about 30 ml/min; 33 fractions of 50 ml were
collected. Pure trans-crotonin was isolated from fractions 3
to 13. It eluted as a single spot under several thin layer
chomatography conditions and showed very good agree-
ment with the published physicochemical properties of the
natural diterpene (Itokawa et al., 1989). The semi-synthetic
crotonin was named 4SRC (Fig. 1).
1
992), and among the minor constituents is clerodane t-
crotonin dehydrocrotonin (Itokawa et al., 1989, 1990). The
nor-clerodane diterpene trans-dehydrocrotonin is present in
sacaca bark tea, suggesting an important role for these
compounds in the traditional preparations (Souza-Brito et
al., 1998). Souza-Brito et al. (1998) have shown that
dehydrocrotonin has significant antiulcerogenic activity,
probably related to its antisecretory and gastroprotective
actions (Hiruma-Lima et al., 1999). However, it was also
demonstrated that dehydrocrotonin has cytotoxic effects
2.3. V79 fibroblast cultures
The cytotoxic effect of semi-synthetic crotonin, ex-
pressed as cell viability, was assessed in a lung fibroblast
cell line (V79) derived from Chinese hamsters. A cell line
that is commonly used for cytotoxicity studies (Souza-Brito
et al., 1998; Rodriguez and Haun, 1999). The V79 fibro-
blasts were grown as monolayers in Dulbecco’s modified
Eagle’s medium (DMEM), supplemented with 10% heat-
inactivated fetal calf serum, 100 IU penicillin/ml and 100 Ag
streptomycin/ml in a humidified incubator with a 5% CO2
atmosphere at 37 jC. The cells were plated onto 96-well
(
Rodriguez and Haun, 1999). The natural crotonin, present
in the bark, has a chemical structure similar to that of
dehydrocrotonin. However, it was not used because only a
small amount was isolated, which it was tested in the
ethanol/HCl ulcer induced model that presented significa-
tive protection (Hiruma-Lima et al., 2002). For this reason,
semi-synthetic crotonin, named here 4SRC, was prepared to
determine whether it has the antiulcerogenic activity of
dehydrocrotonin combined with diminished cytotoxicity.
4
plates at a density of 3 Â 10 per ml. The medium was
removed 48 h after cell seeding and replaced with one
containing semi-synthetic crotonin (80–400 AM), initially
dissolved in methanol and then diluted in DMEM. The final
concentration of methanol in the test and control media was
2
. Materials and methods
1%. The cells were exposed for 24 h to the test medium with
or without semi-synthetic crotonin (control). Each drug
2
.1. Animals
Male Wistar rats (150–200 g) and male Swiss mice (30–
5 g), all obtained from the Central Animal House of the
3
State University of Campinas (CEMIB/UNICAMP), were
used in these experiments. Animals were fed normal rodent
chow (NUVILAB CR-aR), with free access to tap water.
They were fasted before the experiments because the drugs
or test substances were always administered orally, except
for the animals submitted to pylorus ligature ulcer. All
experimental protocols were approved by the Ethics Com-
mittee for Animal Experimentation (CEEA) of the Institute
of Biology (IB), State University of Campinas (UNI-
CAMP).
2
.2. Preparation of semi-synthetic crotonin
The bark of C. cajucara Benth. was collected from an
experimental plantation in Benfica, near Bel e´ m, state of
Par a´ , Brazil, and was identified by Dr. Nelson A. Rosa. A
voucher herbarium specimen was deposited in the herbari-
um of the Museu Paraense Em ´ı lio Goeldi (accession number
2
47). Dehydrocrotonin was isolated from the bark as de-
scribed by Souza-Brito et al. (1998). To obtain trans-
crotonin, 12 g dehydrocrotonin was submitted to reduction
at room temperature for 12 h using 4 atm H , CHCl as
2
3
solvent and paladium over carbon (10%) as the catalyst. The
product of this reaction was purified through a silicagel 60
column (50 Â 3 cm) eluted with a mixture of hexane/ethyl
Fig. 1. Chemical structure of dehydrocrotonin (DHC) and crotonin (4SRC).

A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
207
concentration was tested in eight replicates and in three
experiments. At the end of incubation, three independent
endpoints for cytotoxicity, MTT reduction, total acid content
and neutral red uptake, were evaluated and each one is
presented in the form of IC .
free medium without semi-synthetic crotonin were used as
controls. At the end of the incubation period, three end-
points for cytotoxicity (MTT reduction, neutral red uptake
and nucleic acid content) were evaluated. The cytotoxicity
of semi-synthetic crotonin on ‘‘aged’’ hepatocyte cultures
was also studied. In this case, hepatocytes were plated onto
three 96-well plates and 4 h after seeding the medium was
replaced with serum-free medium and 24 h later the cells
were incubated with different concentrations of semi-syn-
thetic crotonin for 24 h. After treatment, MTT reduction,
nucleic acid content and neutral red uptake were determined
as described above.
5
0
2
.3.1. Nucleic acid content
The number of cells in control and treated wells was
estimated from their total nucleic acid content according to
Cingi et al. (1991). The cells were washed twice with cold
phosphate-buffered saline (PBS) and a soluble nucleotide
pool was extracted with cold ethanol. The cell monolayers
were then lysed by incubation in 0.5 M NaOH for 1 h at 37
jC. The absorbance of the NaOH fraction at 260 nm was
used as an index of cell number (Bianchi and Fortunat,
2.5. V79 fibroblasts cultured with conditioned medium from
rat hepatocytes
1
990).
The study was performed as described by Rodriguez and
Haun (1999). Rat hepatocytes were isolated and cultured as
described above. Four hours after plating, the cells were
incubated with serum-free medium containing different
concentrations of semi-synthetic crotonin. After 20 h, the
medium was removed and used to treat semiconfluent
cultures of V79 cells for 24 h as described above. After
treatment, cell viability was determined by the MTT reduc-
tion, neutral red uptake and nucleic acid content assays.
2.3.2. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide reduction (MTT reduction)
The tetrazolium reduction assay was performed using the
method of Denizot and Lang (1986). Briefly, fibroblasts
were washed once with phosphate-buffered saline (PBS)
before addition of 0.1 ml of serum-free medium containing
MTT (1 mg/ml) to each well. Following incubation for 4 h,
the supernatant was removed and the blue formazan product
obtained was dissolved in 1 ml of ethanol with stirring for
15 min in a microtitre plate shaker and absorbance was read
at 570 nm.
2.6. Acute gastric lesions
The antiulcerogenic activity of different doses of semi-
synthetic crotonin (50, 100 or 200 mg/kg) was assessed in
four experimental models of acute gastric ulcer. In all
experiments the doses of semi-synthetic crotonin were
diluted in 12% Tween 80 and a positive control group
was used. Semi-synthetic crotonin was orally administered
because it is the most common method of drug administra-
tion. After the animals were killed, the stomachs were
removed and opened along the greater curvature to deter-
mine the ulcerative index (UI) as described by Szelenyi and
Thiemer (1978).
2
.3.3. Neutral red uptake
The neutral red uptake assay was performed as described
by Borefreund and Puerner (1984). After 4 h of incubation
with serum-free medium containing 50 Ag of neutral red/ml,
the cells were washed quickly with PBS and then 0.1 ml of
an aqueous solution of 1% (v/v) acetic acid: 50% (v/v)
ethanol was added to each well to extract the dye. After
rapid shaking in a microtitre plate shaker, absorbance was
read at 540 nm.
2
.4. Primary culture of rat hepatocytes
2.6.1. Ethanol-induced ulcers
This ulcer assay was performed in groups of seven rats
Hepatocytes were isolated from male Wistar rats (200–
50 g) by the two-step collagenase perfusion method
2
each using the method of Morimoto et al. (1991). Rats were
randomly divided into two groups and fasted for 24 h before
the experiment. One milliliter of 99.5% ethanol was orally
administered to rats treated with semi-synthetic crotonin
(100 mg/kg) or with vehicle, 12% Tween 80 (10 ml/kg), 1
h before. One hour after the administration of ethanol, the
rats were killed, the stomachs were removed, opened and
the Lesion Index was determined as previously described.
described by Guguen-Guillouzo and Guillouzo (1986). Cell
viability measured by the Trypan blue exclusion test was
greater than 90%. The cells were seeded onto 96-well plates
5
at a density of 3 Â 10 cells/ml in Ham F-12 medium
supplemented with 10% fetal bovine serum, 0.2% bovine
À 6
serum albumin, 0.1 IU bovine insulin/ml, 10
M dexa-
methasone, 50 IU penicillin/ml and 50 Ag streptomycin/ml
and incubated at 37 jC in a humidified 5% CO atmosphere.
2
After 4 h for cell attachment, the medium was changed to
serum-free medium containing different concentrations of
semi-synthetic crotonin followed by incubation for 20 h;
each concentration was tested in eight replicates in three
different experiments. In all cases wells containing serum-
2.6.2. Hypothermal-restraint stress ulcers
The antiulcerogenic activity of semi-synthetic crotonin
was assessed in the hypothermic restraint stress-induced
gastric ulcer model in mice according to the method of
Levine (1971), with some modifications. Mice were fasted

208
A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
for 36 h and then received an oral dose of semi-synthetic
crotonin (50, 100 or 200 mg/kg), cimetidine (100 mg/kg) or
vehicle, 12% Tween 80 (10 ml/kg). One hour after the
treatments, gastric ulceration was induced by immobilizing
the animals in a closed cylindrical cage at 4 jC. After 3 h,
the mice were killed by cervical dislocation and the stom-
achs were removed and examined for ulcers as described
previously.
on the evaluated parameter). These values were calculated
by expressing the results as percentages of the controls and
then determining the values graphically from the dose–
response curves. The results of in vivo experiments are
expressed as the means F S.D. Statistical significance was
determined by Student’s t-test or by one-way analysis of
variance (ANOVA) followed by Dunnett’s test, with the
level of significance set at P < 0.05, depending on the num-
ber of groups used.
2.6.3. Nonsteroidal anti-inflammatory drugs-induced gas-
tric ulcers in cholinomimetic-treated mice
A total of 35 mice divided into five groups were fasted
for 24 h with free access to water. Thirty minutes after the
oral administration of semi-synthetic crotonin (50, 100 or
3. Results
A broad range of substance concentrations were used in
the viability assays, which measure the ‘‘killing capacity’’ or
the ‘‘metabolic capacity’’ of a given chemical, as is usual
when starting toxicity studies with an unknown compound
(Cingi et al., 1991). The cytotoxic effects of semi-synthetic
crotonin were measured by the MTT reduction, nucleic acid
content and neutral red uptake assays in V79 cells and
hepatocytes. The results indicated that there was a loss of
viability of V79 fibroblasts after 24 h of exposure to semi-
synthetic crotonin (Fig. 2). This loss of viability was dose-
dependent, causing cell detachment and death. Also, as
shown in Fig. 2, semi-synthetic crotonin toxicity was greater
in the nucleic acid content assay (IC50 = 200 AM) than in the
neutral red uptake assay (IC50 = 500 AM) assay and was
apparently absent in V79 fibroblasts as evaluated by MTT
reduction.
2
8
00 mg/kg), cimetidine (100 mg/kg) or vehicle, 12% Tween
0 (10 ml/kg), an indomethacin solution (30 mg/kg, dis-
solved in 5% sodium bicarbonate), was administered sub-
cutaneously to each group as described by Rainsford (1978).
Acetyl-h-methylcholine chloride (bethanechol) was admin-
istered intraperitoneally 15 min before indomethacin. The
animals were killed 4 h later, the stomachs were removed
and opened, and the gastric lesions were determined as
described above.
2
.6.4. Pylorus ligature
Male Swiss mice were fasted for 24 h. Thirty minutes
after intraduodenal treatment with cimetidine (100 mg/kg),
semi-synthetic crotonin (50, 100 or 200 mg/kg) or vehicle,
1
2% Tween 80 (10 ml/kg), the pylorus was ligated by the
method of Shay (1945). The intraduodenal route was used to
verify the systemic action of the substances. All animals
were killed 4 h later, the stomachs were removed and the
contents were drained into a graduated centrifuge tube via a
small incision. The volume of gastric fluid was recorded, the
pH was determined and the total acid output was calculated
by titrating the pH to 7.0 with 0.05 N NaOH.
To assess the involvement of cytochrome P450 in the
bioactivation of semi-synthetic crotonin, 24-h-old hepato-
cyte cultures were treated with different doses of semi-
synthetic crotonin for 20 h, as described by Hammond and
Fry (1996). The cytotoxicity observed in the MTT reduction
2
.6.5. Gastric wall mucus determination
Alcian blue binding to the gastric wall mucus was de-
termined by the method of Corne et al. (1974), with minor
modifications. The test drugs, semi-synthetic crotonin (50
and 100 mg/kg) or vehicle, 12% Tween 80 (10 ml/kg), were
given intraduodenally to pylorus-ligated mice. The mice
were killed 4 h after surgery and the glandular segments
from the stomachs were removed and weighed. Each
segment was transferred immediately to 0.1% Alcian blue
solution (0.16 M sucrose in 0.05 M sodium acetate, pH 5.8),
and incubated for 24 h. The segments were centrifuged at
3
000 rpm for 10 min and the absorbance of the supernatants
was measured at 615 nm. The amount of Alcian blue
extracted per gram of glandular tissue was then calculated.
2
.7. Statistical analysis
Fig. 2. Viability of V79 fibroblasts after treatment with semi-synthetic
crotonin for 24 h. Endpoints evaluated: neutral red uptake (NRU), nucleic
acid content (NAC) and MTT reduction (MTT). Each point represents the
mean F S.D. of three experiments in eight replicates.
The results of in vitro experiments are expressed as IC50
values (concentration that produced a 50% inhibitory effect

A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
209
(
IC50 = 10–500 AM) and nucleic acid content (IC50 = 10–
00 AM) tests in the 4-h rat hepatocyte culture was similar
5
to that observed in 24-h-old V79 fibroblasts after exposure
to semi-synthetic crotonin (Table 1). The IC50 was 10 AM in
the MTT reduction assays 500 AM in the nucleic acid
content assay, and 120 AM in the neutral red uptake assay.
A significant loss of cytotoxicity was also observed as the
hepatocytes aged.
To investigate whether or not the metabolites of semi-
synthetic crotonin are potentially toxic to other cell types, a
co-culture system of hepatocytes and V79 cells was used.
The co-culture technique is a useful tool for assessing the
toxicity of products resulting from hepatic bioactivation in
target cells (Fry et al., 1995). The medium from cultured
hepatocytes treated with semi-synthetic crotonin had the
same toxic effect on V79 fibroblasts as semi-synthetic
crotonin itself, as judged in the nucleic acid content and
neutral red uptake tests. The results of this experiment are
illustrated in Fig. 3. A slight stimulatory effect on the ability
of V79 cells to reduce MTT was observed after incubation
with the medium from hepatocytes cultured for 4 h.
To establish a general profile of the antiulcerogenic ac-
tivity of semi-synthetic crotonin, the compound was admin-
istered orally at three doses (50, 100 or 200 mg/kg) in
different gastric ulcer models in mice and rats. Table 2
presents the antiulcerogenic actions of semi-synthetic cro-
tonin, cimetidine or lanzoprazole used as positive control on
the gastric lesions induced by indomethacin–bethanechol,
hypothermic restraint stress, and absolute ethanol.
Fig. 3. Viability of V79 fibroblasts after treatment with conditioned medium
from rat hepatocytes cultured for 4 h. Endpoints evaluated: neutral red
uptake (NRU), nucleic acid content (NAC) and MTT reduction (MTT).
Each point represents the mean F S.D. of three experiments in eight
replicates.
100 mg/kg) did not modify the free mucus produced by the
gastric mucosa, as opposed to indomethacin, a classic cy-
clooxygenase inhibitor.
The next step was to evaluate semi-synthetic crotonin in
terms of its antisecretory activity. This effect was deter-
mined in hypothermic restraint stress-induced ulcer in mice.
Pretreatment with semi-synthetic crotonin at doses of 50,
1
00 or 200 mg/kg significantly inhibited the formation of
gastric lesions induced by hypothermic restraint-stress by
0%, 81.5% and 74%. These data can be seen in Table 2.
Pretreatment with a single dose of semi-synthetic croto-
nin (100 mg/kg) before absolute ethanol administration
significantly inhibited ulcer formation by 79%, confirming
the cytoprotective properties previously observed (Table 2).
When these results were compared with those obtained with
dehydrocrotonin in this model (Souza-Brito et al., 1998), we
observed a considerable decrease in the gastroprotective
effect of dehydrocrotonin (55%) against ethanol lesions.
We also investigated the possible gastroprotective activ-
ity of semi-synthetic crotonin in the NSAID-induced gastric
ulcer model (Table 2). In this model, semi-synthetic crotonin
6
Cimetidine (100 mg/kg), the reference antiulcerogenic
agent, significantly inhibited (66%) the ulceration induced
in this model.
Finally, we investigated the effects of semi-synthetic
crotonin on the biochemical parameters of gastric acid
Table 2
Effects of semi-synthetic crotonin (4SRC) on models of gastric lesions
induced in mice and rats
(
50, 100 and 200 mg/kg) significantly inhibited ulcer
Method
Treatment
n
Dose
ILU
Inhibition
(%)
formation by 16%, 47% and 41%, respectively. In contrast,
dehydrocrotonin did not show antiulcerogenic activity when
tested against NSAIDs such as indomethacin (Souza-Brito
et al., 1998).
(mg/kg)
Ethanol (rats) Control
4SRC
6
5
9
–
100
–
30.3 F 13.3
6.2 F 6.9
8.44 F 2.2
3.7 F 1.8
7.1 F 2.6
4.5 F 2.1
5.0 F 1.6
8.75 F 1.6
3.0 F 0.81
–
a
79.6
–
Indomethacin Control
b
(
mice)
Cimetidine 10 100
56.2
16.0
46.7
40.8
–
In order to verify this point, we measured the gastric wall
mucus of rats submitted to the Shay method (Table 3). In
contrast to our expectations, semi-synthetic crotonin (50 or
4
SRC
11
50
100
200
–
c
1
0
9
8
6
8
8
7
c
Hypothermic
restraint
Control
Cimetidine
4SRC
b
100
50
65.7
60.0
81.5
74.0
Table 1
c
stress
3.5 F 2.1
1.62 F 0.99
2.28 F 1.3
Influence of hepatocyte culture age on the cytotoxicity of semi-synthetic
crotonin
b
(
mice)
100
200
b
Hepatocyte
age (h)
NAC (IC50
)
NRU (IC50
)
MTT (IC50)
The results are reported as means F S.D.
(AM)
(AM)
(AM)
a
P < 0.01 compared to control (Student’s test).
P V 0.001 compared to control (Dunnett’s test).
P < 0.01 compared to control (Dunnett’s test).
4
10–500
>500
120
10–500
>500
b
c
2
4
>500

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A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
Table 3
Effects of semi-synthetic crotonin (4SRC) on the biochemical parameters of gastric juice obtained from pylorus-ligated mice
Method
Treatment
Dose
pH
Total gastric acid
(mEq/ml/3 h)
Gastric juice
volume (ml)
Mucus
(mg/g)
(
mg/kg)
Pyloric ligation
Control
–
3.83 F 0.34
4.68 F 0.36
3.57 F 0.38
3.77 F 0.34
3.68 F 0.36
3.0 F 0.8
4.5 F 0.7
3.2 F 1.0
2.4 F 0.7
9.42 F 2.8
4.47 F 2.2
8.14 F 1.5
12.09 F 5.4
0.424 F 0.348
0.440 F 0.183
0.412 F 0.168
0.282 F 0.162
0.348 F 0.108
0.690 F 0.84
0.559 F 0.168
0.401 F 0.150
0.467 F 0.221
–
a
a
Cimetidine
100
50
–
4
SRC
–
1
2
00
00
–
–
9.08 F 4.6
–
Gastric wall mucus
Control
–
–
–
–
2.61 F 0.5
3.00 F 0.6
2.13 F 0.7
1.98 F 0.5
a
Cimetidine
100
50
4
SRC
1
00
The results are reported as means F S.D.
P < 0.05 compared to control (Dunnett’s test).
a
secretion (pH, total gastric acid, gastric juice volume) using
the pylorus ligature method (Shay et al., 1945). As shown in
Table 3, the volume, pH and total acid output of gastric
secretions were not significantly affected by semi-synthetic
crotonin (50, 100 and 200 mg/kg), whereas cimetidine (100
mg/kg) increased the pH and reduced the acid output of this
secretion.
ment of cytochrome P450 in the toxic effects of chemicals.
To assess the involvement of cytochrome P450 in the
bioactivation of semi-synthetic crotonin, 24-h-old hepato-
cyte cultures were treated with different doses of semi-
synthetic crotonin for 20 h as described by Hammond and
Fry (1996). The cytotoxic effect of semi-synthetic crotonin
in these assays suggests that semi-synthetic crotonin, like
dehydrocrotonin (Rodriguez and Haun, 1999), is bioacti-
vated by the cytochrome P450 microsomal system and
generates less toxic metabolites as a function of time.
The results obtained with co-culture system with hep-
atocytes and V79 cells indicate that the toxicity of the
metabolites was similar to that of semi-synthetic crotonin
itself. The hepatic metabolism of xenobiotics is known to
generate superoxide anion and free radicals, with the former
being able to reduce tetrazolium compounds such as MTT
(Andrews et al., 1997). Our findings support the presence of
MTT-reducing metabolites in the medium from hepatocytes
treated with semi-synthetic crotonin and confirm the results
obtained by Rodriguez and Haun (1999) with dehydrocro-
tonin.
4
. Discussion
The balance between the therapeutic versus the toxico-
logical effects of a compound is an important parameter for
the determination of its applicability as an antiulcer or any
other pharmacological agent. The toxicological studies
described here were conducted on cultured cells, which
can be used to evaluate cytotoxicity (Ekwall and Ekwall,
1
988) and target organ toxicity. In some cases, cultured cells
may also provide information about the lethal dose in vivo
Shrivastava et al., 1991). We used V79 fibroblasts in our
(
assays because this cell line is well characterized and
commonly used in mutagenicity and toxicity studies (Cingi
et al., 1991).
Pretreatment with semi-synthetic crotonin (100 mg/kg)
was more effective in inhibiting ulcer formation than dehy-
drocrotonin in the ethanol-induced ulcer model, suggesting
an increase in the gastroprotective effect. The intragastric
application of absolute ethanol consistently produced severe
lesions in all the control rats. However, diluted ethanol
produced no more than minute lesions in the control rats
(Sikiric et al., 1999). For this reason, absolute ethanol was
used. It has been proposed that ethanol is responsible for the
stimulation of gastric and oral secretion and gastrin and
histamine release in a reflex way through sensitive terminals
present in the gastric and oral mucosa, consequently stim-
ulating gastric secretion. Based on this mechanism of
ethanol injury, we suggest that semi-synthetic crotonin
may act on the gastric mucosa as an antisecretory agent.
Gastric acid is not a primary cause but plays a permissive
role in the gastric mucosal damage induced by ethanol
(Tarnawski et al., 1983). Previous studies have demonstrat-
ed that most antiulcer agents, such as proton pump inhib-
A broad range of concentrations were tested in the via-
bility assays, which measure the ‘‘killing capacity’’ or the
‘‘metabolic capacity’’ of a given chemical, as is usual when
starting toxicity studies of an unknown compound (Cingi et
al., 1991). The cytotoxic effects of semi-synthetic crotonin
were measured by the MTT reduction, nucleic acid content
and neutral red uptake assays in V79 cells and hepatocytes.
The results indicated that there was a loss of viability of V79
fibroblasts after 24 h of exposure to semi-synthetic crotonin.
The cytotoxic effect of semi-synthetic crotonin was greater
in the nucleic acid content assay than in the neutral red
uptake assay and was apparently absent in V79 fibroblasts,
as evaluated by MTT reduction.
Primary cultures of mammalian hepatocytes suffer a
rapid and gradual loss of cytochrome P450 content without
there being a severe initial decline in the levels of phase II
enzymes following isolation. Hepatocyte cultures of differ-
ent ages (24 and 72 h) are used to determine the involve-
itors (omeprazole, lanzoprazole) and histamine H receptor
2

A.B. Albino de Almeida et al. / European Journal of Pharmacology 472 (2003) 205–212
211
anatagonists (cimetidine), have antisecretory activity (Lars-
son et al., 1983; Yamamoto et al., 1984; Lindberf et al.,
Hiruma-Lima et al., 1999). However, it seems that the
mechanisms involved in the antiulcerogenic action are
different for semi-synthetic crotonin and dehydrocrotonin,
probably due to the different spatial configuration of the
compound under study. Further studies are necessary to
investigate other possible mechanism(s) involved in this
antiulcerogenic action of semi-synthetic crotonin and they
are now in progress in our laboratory.
1
990; Kinoshita et al., 1997). Another action promoted by
ethanol is its ability to damage the gastric mucosa by
mechanical injury. Thus, semi-synthetic crotonin may also
act as a cytoprotective agent because drugs like misoprostol
are also active in this model (Morimoto et al., 1991).
The presence of gastric acid, the overproduction of
leukotrienes, the inhibition of prostaglandin (PG) synthesis
and the consequent disruption of the gastric mucosal barrier
are the main factors involved in the pathogenesis of ulcer
induced by indomethacin, a non-selective cyclooxygenase
inhibitor (Dajani and Agrawal, 1995). In the indomethacin-
induced ulcer model, semi-synthetic crotonin significantly
inhibited ulcer formation at high doses. However, dehydroc-
rotonin did not show antiulcerogenic activity in this model
Acknowledgements
This study was financed by the Brazilian agency
FAPESP (no. 9803442-2).
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