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Journal of Ethnopharmacology 114 (2007) 153–160

The aerial parts of Guazuma ulmifolia Lam. protect against

NSAID-induced gastric lesions
B. Berenguer a , C. Trabadela a , S. Sánchez-Fidalgo a , A. Quı́lez a ,
P. Miño b , R. De la Puerta a , M.J. Martı́n-Calero a,∗
a Department of Pharmacology, Faculty of Pharmacy, University of Seville, Professor Garcı́a González Street 2, 41012 Sevilla, Spain
b Department of Chemistry and Pharmacy, Central University of Ecuador, Quito, Ecuador

Received 8 March 2007; received in revised form 2 July 2007; accepted 18 July 2007
Available online 22 July 2007

Abstract
Guazuma ulmifolia Lam., a member of the Sterculiaceae family, is used in folk medicine because of its antioxidant, antimicrobial and antihy-
pertensive properties. Most of the research work carried out on this plant has focused on the bark because of its high concentration of antioxidant
proanthocyanidins. The flowers and leaves of Guazuma ulmifolia, though less studied, are also used as a remedy for different conditions, such as
kidney and gastrointestinal diseases, fever and diabetes. The aim of this study was to assess the gastroprotective effects of an aqueous suspension
of the ethanolic extract from leaves and flowers of Guazuma ulmifolia in a model of acute gastric ulcer induced by diclofenac as ulcerogenic agent,
using the proton pump inhibitor omeprazole as a protection reference. Therefore, the extract was administered two times orally to three groups of
Wistar rats at doses of 500, 250 and 125 mg/kg, with a 24-h interval between doses. Diclofenac (100 mg/kg) was given 1 h after the last administra-
tion of the extract. Pretreatment with Guazuma ulmifolia or omeprazole decreased the ulcerated area in a dose-dependent way. Myeloperoxidase
activity as a marker of neutrophil infiltration was slightly reduced in vivo, whereas in vitro, anti-inflammatory activity was clearly inhibited in
a dose-dependent way. The lowest doses of the extract significantly decreased the levels of lipoperoxides, and superoxide dismuthase activity
increased to a similar extent as with omeprazole (P < 0.001). Examination of glutathione metabolism reflected a significant rise in glutathione
peroxidase activity at the highest dose of Guazuma ulmifolia. Finally, there was a faint elevation in prostaglandin E2 levels with all doses, though
the depletion induced by diclofenac could not be reverted. We conclude that the aerial parts of Guazuma ulmifolia protect gastric mucosa against
the injurious effect of NSAIDs mainly by anti-inflammatory and radical-scavenging mechanisms.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Guazuma ulmifolia; Gastroprotection; NSAID-induced gastric ulcer; Anti-inflammatory effect

1. Introduction betes, and externally as an ailment for wounds, skin eruptions

Guazuma ulmifolia Lam., commonly known as “guacimo”
or “mutamba”, is a member of the Sterculiaceae family that
grows in Ecuador, Panama and other Latin American coun-
tries from bush to tree size. In traditional medicine, the bark
of Guazuma ulmifolia is used in the treatment of diarrhea,
hemorrhages, fever, coughs, bronchitis, asthma, gastrointestinal
pain and hypertension, and as stimulant for uterine contractions
(Caballero-George et al., 2001; Domı́nguez and Alcorn, 1985).
Dried leaves are brewed into tea in some countries and used for
kidney and gastrointestinal diseases, fever, dysentery and dia-
∗ Corresponding author. Tel.: +34 95 4556722; fax: +34 95 4233765.
E-mail address: calero@us.es (M.J. Martı́n-Calero).
and even baldness. Guazuma ulmifolia leaves are also tradition-
ally boiled as a treatment for diabetes and this method has been
experimentally proven to decrease hyperglycaemia in rabbits
(Alarcon-Aguilara et al., 1998).
Currently, many countries with important biodiversity
resources are developing and using non-toxic preparations from
traditional medicinal plants for controlling various diseases, pro-
viding relief of symptoms comparable to that obtained from
allopathic medicine (Bandyopadhyay et al., 2004; Heinrich,
2003). Among the conditions that can possibly profit from tradi-
tional medicine are gastrointestinal diseases, particularly gastric
and duodenal ulcers. A number of plants have been proven to
possess gastroprotective and ulcer-healing properties in gastric
lesions induced by noxious agents, such as non-steroidal anti-
inflammatory drugs (NSAIDs) (Berenguer et al., 2006; Grover

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2007.07.019
154 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160
and Yadav, 2004; Toma et al., 2005), that inhibit cyclooxy-
genase (COX)-1 enzyme, thereby suppressing prostaglandin
(PG)-mediated beneficial effects on gastric mucosa (Wallace,
2005).
As most of the studies on Guazuma ulmifolia have been car-
ried out on the extract of the bark, the aim of this study was
to assess the gastroprotective effect of the ethanolic extract of
the leaves and flowers and to elicit the underlying mechanism.
For this purpose, we administered the extract to rats in a model
of diclofenac-induced gastric ulcer in comparison to the proton
pump inhibitor omeprazole. The role of Guazuma ulmifolia in
oxidative stress was analyzed by measuring changes in super-
oxide dismuthase (SOD) activity, levels of lipoperoxides and
glutathione metabolism: content of non-protein (NP-SH) and
total sulphydryls and glutathione peroxidase activity (GSH-Px).
PGE2 levels in gastric mucosa were also evaluated. Moreover,
we studied myeloperoxidase (MPO) activity in vivo as a marker
of neutrophil infiltration and in vitro as a test for enzyme inhibi-
tion and finally we investigated scavenging of hydrogen peroxide
in order to assess the mechanism involved in MPO inhibition.
2. Materials and methods
2.1. Plant material
The leaves and flowers of Guazuma ulmifolia (Sterculiaceae)
were collected in Ecuador in the province of Pichincha in the
central region of Ecuador. The Department of Botany, Biology
Faculty of the Central University of Ecuador authenticated the
botanical identity of the plants and voucher specimens are kept
under reference 188.
2.2. Preparation of the extract
Fresh leaves and flowers were dried at room temperature
for 10–14 days. Afterwards, they were pulverized and stored at
−10 ◦ C until use. A 1 kg quantity of the powder was extracted
with ethanol 96◦ during 24–48 h. The suspension was filtered,
and the residue percolated with ethanol used for the maceration.
This percolated residue was concentrated at 45 ◦ C in a rotary
evaporator. The yield of the concentrated was 5.96%.
2.3. Animals
Male and female Wistar rats (180–200 g) kept in standard
laboratory conditions were kept fasting overnight in single wire-
net floor cages with free access to tap water, and were randomly
assigned to groups of 10–12 animals. All experiments followed
a protocol approved by the local animal Ethics Committee and
the Local Government and were in accordance with the recom-
mendations of the European Union.
2.4. In vivo experimental protocols
2.4.1. Dose selection and administration route
Three different doses of the ethanolic extract: 125, 250 and
500 mg/kg body weight, were used in the present study. The
aqueous suspension of the above-mentioned extract of Guazuma
ulmifolia (in a volume of 1 mL/100 g body weight) was prepared
freshly each time and administered two times each to the dif-
ferent animal groups at 24 h interval by oral gavage. The doses
were selected according to previous experiments of our group
(data not shown).
2.4.2. Ulcer induction
Diclofenac (Sigma Chemical Co.) was used as ulcerogenic
agent at a dose of 100 mg/kg. One hour after the last administra-
tion of the plant extract animals received diclofenac 100 mg/kg
by the same oral route. Rats were sacrificed by cervical dislo-
cation 6 h after NSAID administration. Vehicle-treated (sham)
and diclofenac-treated rats were included as controls in all exper-
iments. Experimental times were selected according to former
experiments of our group that reflect that damage induced by the
assayed NSAID peaks 6 h after oral administration (Sánchez et
al., 2002).
2.4.3. Ulcer assessment
The stomach was harvested and opened along the greater cur-
vature, and the mucosa was exposed for macroscopic evaluation.
The ulcerated area was assessed using planimetry by a person
unaware of the type of treatment received by the animals, and
the Ulcer Index (U.I., mm2 ) was calculated as the arithmetic
mean for each treatment. Following the analysis, the mucosa
layer was blotted dry, and scraped off the underlying muscu-
laris externa and serosa. A homogeneous mixture of mucosa,
damaged, and macroscopically healthy tissue was snap-frozen in
liquid nitrogen, and stored at −70 ◦ C before biochemical studies.
2.4.4. Myeloperoxidase activity
MPO activity was assessed as a marker of neutrophil infil-
tration according to the method of Grisham et al. (1990). The
tissue was homogenized in phosphate-buffered saline (PBS),
pH 7.4 and centrifuged, and the pellet was again homogenized
in PBS, pH 6.0, containing hexadecyl-trimethylammonium
bromide (HETAB) and ethylendiamine tetraacetic acid. This
homogenate was subjected to one cycle of freezing/thawing and
a brief period of sonication. A sample of homogenate (0.5 ␮L)
was added to a 0.5 mL reaction volume containing PBS, pH 5.4,
HETAB and 3,3 ,5,5 -tetramethylbenzidine. The mixture was
incubated at 37 ◦ C. The reaction was started by the addition of
H2 O2 , and was terminated by the sequential addition of cata-
lase and sodium acetate, pH 3.0. The changes in absorbance
at 655 nm were measured with a microplate reader (Labsystem
Multiskan Ex). One unit of MPO activity was defined as the
amount of enzyme present that produced a change in absorbance
of 1.0 U/min at 37 ◦ C, and the results were expressed as U/mg
tissue.
2.4.5. Lipid peroxidation
The levels of thiobarbituric acid reactants (TBARS) in the
gastric mucosa as index of lipoperoxides production were mea-
sured according to the modified method of Okawa et al. (1979).
The mucosa was scraped with glass slides, weighed, and homog-
enized in 10 mL KCl (10%). The homogenate was supplemented
 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160
with 8.1% sodium lauryl sulphate, 20% acetic acid, and 0.8%
TBA, and boiled at 100 ◦ C for 1 h. After cooling, the reactants
were supplemented with 2.5 mL n-butanol, shaken vigorously
for 1 min, and centrifuged for 10 min at 2600 × g. Absorbance
was measured in a spectro-photometer Perkin-Elmer Lambda 3
at 532 nm, and the results were expressed as nmol of malonyl-
dialdehyde (MDA)/g tissue.
2.4.6. Superoxide dismuthase (SOD) activity
The enzymatic activity of SOD is based on the inhibition
of the reduction of cytochrome c according to the method of
McCord and Fridovich (1969). Samples of gastric mucosa were
homogenized (1:150) in a mixture of 50 mM PBS and 100 ␮M
EDTA (pH 7.8). The homogenate was supplemented with 0.1%
Triton. The assay method used 10 ␮M ferricytochrome c, 50 ␮M
xanthine, as source of O2 •− , and sufficient milk xanthine oxidase
(5 nM) to give a rate of increase in absorbance of 0.025 min−1
at pH 7.8 and 25 ◦ C. The reaction kinetic was measured in a
spectrophotometer at 550 nm at a rate of 0–70 s. Results were
expressed as U SOD/mg tissue. One unit of SOD is defined as
the amount of enzyme that causes 50% inhibition of cytochrome
c reduction.
2.4.7. Prostaglandin E2 (PGE2 ) levels
Gastric mucosa from the different animal groups was excised
and rapidly rinsed with ice-cold saline. The tissue was weighed
and homogenized in 6 mL TEAP buffer (pH 3.24) which
contained a cyclooxygenase inhibitor, lysine acetyl salicitate
(Inyesprin® , Grünenthal). The homogenate was centrifuged
(1500 × g, 10 min, 4 ◦ C) and the supernatant was removed and
passed through a reverse-phase octadecylsilica C18 Sep Pak car-
tridge which was washed with 10 mL distilled water, 10 mL 15%
ethanol, 10 mL hexane and 10 mL ethylacetate, and the eluate
collected. Each fraction with ethylacetate was evaporated, and
the dry residue redissolved in ethanol. PGE2 was determined
by a competitive enzyme immunoassay kit (Cayman® , Cat. No.
514010). Results were expressed as pg PGE2 /mg tissue.
2.5. Glutathione metabolism
2.5.1. Involvement of endogenous sulphydryl compounds:
total and non-protein SH contents
The amount of mucosal sulphydryls was measured in the
gastric mucosa of rats according to the method described by
Garg et al. (1991). The mucosal scrapings were suspended
in 1 mL sodium phosphate buffer, homogenized, made up to
2 mL with buffer and centrifuged at 1800 × g for 10 min at
4 ◦ C. The supernatant sulphydryl content was determined by
means of colorimetric reaction with Ellman’s reagent [5,5 -
dithio-bis-(2-nitrobenzoic acid)]. The protein sulphydryl levels
were obtained by subtracting the non-protein sulphydryl values
from that of the total sulphydryls. Light absorbance at 412 nm,
against a reagent blank, was measured with a spectrophotometer
(Perkin-Elmer Lambda 3). Sulphydryl concentration was calcu-
lated from freshly prepared standard curves of glutathione (GSH,
Sigma) and the results were expressed as nmol/mg tissue.
155
2.5.2. Glutathione peroxidase activity
Glutathione peroxidase activity was quantified by the method
of Yoshikawa et al. (1993). The reaction mixture consisted of
50 mM PBS, pH 7, 1 mM EDTA, 1 mM NaN3 , 0.2 mM NADPH,
1 unit/mL oxidized GSSG-Rd in PBS buffer, pH 7.8, 1 mM GSH
and 0.25 mM H2 O2 . Samples were added to 0.8 mL of the above
mixture and incubated for 5 min at 25 ◦ C before initiating the
reaction with the addition of peroxide solution. A sample of
supernatant fluid with 10% homogenate solution and 1.15% KCl
was prepared by centrifugation at 4000 × g for 10 min at 4 ◦ C.
The absorbance at 340 nm was recorded for 5 min. The activity
was the slope of the lines as micromoles of NADPH oxidized
per minute. The blank datum (the enzyme was replaced with
distilled water) was subtracted from each value. Results were
expressed as nmol/min/mg tissue.
2.6. In vitro experimental protocols
2.6.1. Leukocyte isolation
A suspension of leukocytes containing approximately 85%
polymorph nuclear leukocytes (PMNs) and 15 % mononuclear
cells was obtained from male Wistar rats (200–300 g) by an
i.p. injection of 10 mL of a solution of 6% oyster glycogen in
saline followed 16–20 h later by 60 mL ice-cold modified Hank’s
balanced salt solution (HBSS) free of Ca2+ and Mg2+ (Moroney
et al., 1988).The mixed peritoneal leukocytes were resuspended
in complete HBSS at 2.5 × 106 cells/mL containing 1.26 mM
Ca2+ and 0.9 mM Mg2+ . Cell viability based on Trypan blue
exclusion was greater than 95%.
2.6.2. Myeloperoxidase released from stimulated rat
peritoneal leukocytes
Triplicate aliquots of 0.5 mL leukocytes were preincubated
at 37 ◦ C for 10 min with 5 ␮L of an ethanolic plant extract (the
assayed doses were 50, 25 and 12.5 ␮g/␮L) or an equivalent
volume of the vehicle, 5 ␮L of calcium ionophore A23187 was
added in DMSO (1 ␮M) for a further 10 min of incubation. The
cells were pelleted by centrifugation at 2500 × g for 10 min at
4 ◦ C, and the supernatants were used to measure the release
of MPO. MPO release was determined by measuring the rate
of oxidation of o-dianisidine in a microplate assay (Bradley et
al., 1982). For this, the following reagents were added to the
wells of a 96 plate in the order stated: 50 ␮L supernatant, 50 ␮L
phosphate buffer (pH 6.0) containing 0.5% HETAB, 50 ␮L of
0.68 mg/mL o-dianisidine in distilled water and, to start the reac-
tion, 50 ␮L freshly prepared 0.003% hydrogen peroxide. The
optical density (OD) at 450 nm was read immediately and there-
after at 5-min intervals with intermittent shaking. The amount of
enzyme in the samples was obtained by comparison of the rate of
reaction with that in wells containing supernatants from the con-
trol group which consisted of leukocytes-treated only with the
calcium ionophore A23187. The release of MPO was expressed
as enzymatic activity (mU) compared with the maximum level
(control treated with A23187).
Any direct inhibitory effect of the extract on MPO activity
was assessed by adding of 50 ␮L of freeze–thaw disrupted cells
supernatant (as enzyme source) adding 50 ␮L of an appropri-
156 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160

ate dilution of the extract or their vehicle and proceeding with

the assay by adding dianisidine, detergent buffer and H2 O2 as
above.

2.6.3. Scavenging of hydrogen peroxide

The scavenging of hydrogen peroxide was measured by
adding 100 ␮L H2 O2 1 mM and 900 ␮L buffer NaCl/Tris HCl
pH 7.4–10 ␮L of the stock solution of the compounds, in
triplicate. After incubating the mixture for 30 min at room tem-
perature, 50 ␮L of a guaiacol solution (0.2%, v/v, in distilled
water), and 10 ␮L of horseradish peroxidase (1 mg dissolved in
200 ␮L of the pH 7.4 buffer) was added. After a further 10 min,
0.2 mL aliquots of the samples were transferred to a 96-well
plate and the OD450 values were read in a microplate reader (De Fig. 2. Effects of pretreatment with ethanolic extract of Guazuma ulmifolia
la Puerta et al., 1999). (Gz, 125, 250 and 500 mg/kg) and omeprazole (Om, 20 mg/kg) followed by
diclofenac treatment (Dc, 100 mg/kg) on myeloperoxidase activity. * P < 0.05
vs. sham.
2.7. Statistical analysis

Data from 8 to 10 experiments were pooled and expressed
as arithmetic means ± S.E.M. The data were evaluated
using ANOVA’s test followed by Tukey’s test for paired
data and the non-parametric Mann–Whitney U-test (Ulcer
Index determination). P-values were considered significant at
P < 0.05.
3. Results
3.2. Effects of diclofenac and Guazuma ulmifolia on MPO
activity
At the tested dose, diclofenac augmented MPO activity from
0.810 ± 0.09 U/mg tissue (sham level) up to 1.641 ± 0.272 U/mg
tissue (P < 0.05 vs. sham). All preventive treatments produced a
slight reversion of this inflammatory response, but this effect was
not statistically significant in comparison to the NSAID (Fig. 2).

3.1. Effects of diclofenac and Guazuma ulmifolia on gastric
mucosa Ulcer Index
As shown in Fig. 1, diclofenac caused important damage
on the glandular mucosa (21.84 ± 17.04 mm2 ). By con-
trast, pretreatment with Guazuma ulmifolia decreased the
ulcerated area in a dose-dependent way, though not to
the same extent as omeprazole. The gastroprotective effect
was highly significant at the doses of 250 and 500 mg/kg
(11.43 ± 2.12 and 9.08 ± 1.74 mm2 , respectively) (P < 0.01 vs.
diclofenac).
3.3. Inhibition of MPO activity in vitro
Activation of rat leukocytes with the calcium ionophore
A23187 causes calcium-dependent secretion of the contents of
the specific and azurophil granules. This can be measured after
pelleting the cells by assaying the amount of MPO enzyme in
the supernatant. In rat peritoneal neutrophils stimulated with
A23187, Guazuma ulmifolia dose-dependently decreased MPO
release (P < 0.001 and 0.05 vs. cells stimulated with A23187)
at the doses of 50 and 25 ␮g/mL. The highest dose produced a
descent comparable to that obtained with the positive controls
caffeic acid and indomethacin (Fig. 3).

Fig. 1. Effects of pretreatment with different doses of ethanolic extract of

Guazuma ulmifolia (Gz, 125, 250 and 500 mg/kg) and omeprazole (Om,
20 mg/kg) on gastric damage induced by diclofenac treatment (Dc, 100 mg/kg). Fig. 3. Inhibitory effect of ethanolic extract of Guazuma ulmifolia (Gz, 50, 25,
Data are expressed as the means ± S.E.M. ++ P < 0.01, +++ P < 0.001 vs. 12.5 ␮g/mL) on myeloperoxidase activity in vitro. * P < 0.05 and *** P < 0.001
diclofenac group. vs. cells stimulated with calcium ionophore A23187.
 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160 157

Fig. 5. Effects of pretreatment with ethanolic extract of Guazuma ulmifo-

Fig. 4. Effects of pretreatment with ethanolic extract of Guazuma ulmifolia lia (Gz, 125, 250 and 500 mg/kg) and omeprazole (Om, 20 mg/kg) followed
(Gz, 125, 250 and 500 mg/kg) and omeprazole (Om, 20 mg/kg) followed by by diclofenac treatment (Dc, 100 mg/kg) on superoxide dismuthase activity.
diclofenac treatment (Dc, 100 mg/kg) on lipid peroxidation. * P < 0.05 vs. sham; * P < 0.05 vs. sham; ++ P < 0.01 and +++ P < 0.001 vs. diclofenac 100 mg/kg.
+ P < 0.05 and ++ P < 0.01 vs. diclofenac 100 mg/kg.

The subsequent experiment proved that the extract did not which however could not revert the strong depletion caused by
show a direct inhibition of the enzyme activity and, on the other the NSAID.
hand, did not scavenge H2 O2 , which is the substrate for the
enzymatic reaction (data not shown). Thus, it appears that the
plant extract affects the pro-inflammatory secretory process by 3.7. Effects of diclofenac and Guazuma ulmifolia on
preventing MPO release from neutrophils. glutathione metabolism

Diclofenac caused a marked descent in the mucosal non-

3.4. Effects of diclofenac and Guazuma ulmifolia on lipid
protein SH content, from 1.93 ± 0.22 to 0.91 ± 0.10 nmol/mg
peroxidation levels
tissue, and in the total GSH content, from 17.93 ± 1.51
to 9.23 ± 0.82 nmol/mg tissue (P < 0.05 vs. sham). Admin-
As shown in Fig. 4, diclofenac treatment elevated
istration of Guazuma ulmifolia did not significantly affect
the concentration of MDA above that of the sham
the amount of mucosal GSH compared with the NSAID-
group (sham 101.26 ± 17.96 nmol MDA/g tissue, diclofenac
treated group. Omeprazole not only reverted the depletion
181.65 ± 15.68 nmol MDA/g tissue, P < 0.05). The two lowest
caused by diclofenac, but even enhanced non-protein SH
doses of Guazuma ulmifolia reverted the levels of lipoperoxides
and total GSH content (P < 0.01 vs. diclofenac). Further-
in gastric mucosa to sham values, though not as significantly as
more, diclofenac produced a non-significant descent of
omeprazole (P < 0.001 vs. diclofenac 100 mg/kg).
GSH-Px from sham (7.52 ± 1.23 nmol/min/mg tissue vs.
9.17 ± 1.34 nmol/min/mg tissue). The extract at the highest
3.5. Effects of diclofenac and Guazuma ulmifolia on SOD dose and the proton pump inhibitor induced a signifi-
activity cant increase in enzymatic levels (P < 0.05 vs. diclofenac)
(Table 1).
SOD activity in the sham group was 1452.57 ± 146.41 U/mg
tissue, whereas in the diclofenac group it descended to
741.4 ± 75.2 U/mg tissue. Pretreatment with the extract at the
three tested doses as well as omeprazole increased SOD activ-
ity levels in a very significant way (P < 0.01 and <0.001 vs.
diclofenac group). The groups pretreated with Guazuma ulmifo-
lia 250 mg/kg and omeprazole reflected the highest antioxidant
effect; these values were even significant vs. sham levels
(P < 0.05) (Fig. 5).

3.6. Effects of diclofenac and Guazuma ulmifolia on

mucosal PGE2 content

As shown in Fig. 6, basal PGE2 content in gastric mucosa was

339.06 ± 99.56 pg/mg tissue and diclofenac induced a marked
depletion (33.34 ± 2.77 pg/mg tissue; P < 0.001 vs. the sham
group). Pretreatment with Guazuma ulmifolia extract at all doses
as well as omeprazole produced a faint increase in PG levels,
Fig. 6. Effects of pretreatment with ethanolic extract of Guazuma ulmifolia
(Gz, 125, 250 and 500 mg/kg) and omeprazole (Om, 20 mg/kg) followed by
diclofenac treatment (Dc, 100 mg/kg) on prostaglandin E2 levels. *** P < 0.001
vs. sham.
158 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160
Table 1
Effects of pretreatment with ethanolic extract of Guazuma ulmifolia (Gz, 125, 250 and 500 mg/kg) and omeprazole (Om, 20 mg/kg) followed by diclofenac treatment
(Dc, 100 mg/kg) on non-protein SH content, total SH content and glutathione peroxidase activity
Treatment Non-protein SH (nmol/mg tissue)
Sham 1.93 ± 0.22
Dc, 100 mg/kg 0.91 ± 0.10*
Gz, 125 mg/kg 0.96 ± 0.15*
Gz, 250 mg/kg 1.03 ± 0.21*
Gz, 500 mg/kg 1.15 ± 0.17
Om, 20 mg/kg 2.05 ± 0.31++
+ P < 0.05 and ++ P < 0.01 vs. diclofenac 100 mg/kg.
* P < 0.05 vs. sham.
4. Discussion
The present study shows that pretreatment with aqueous
suspension of the ethanolic extract of leaves and flowers of
Guazuma ulmifolia exerts a protective action on diclofenac-
induced gastric ulcers in rats, as evidenced by the reduction in
the ulcerated area and the decrease of oxidative stress parameters
in gastric mucosa.
A number of studies have confirmed the antioxidant (Navarro
et al., 2003), antibacterial (Caceres et al., 1990; Camporese et
al., 2003), antiviral (Felipe et al., 2006), antisecretory (Hoer
et al., 1995, 1996) and antihypertensive properties (Caballero-
George et al., 2001, 2002) of Guazuma ulmifolia bark, mainly
attributable to its content in polymeric proanthocyanidins, which
consist of (−)-epicatechin units.
Proanthocyanidins are condensed tannins found predomi-
nantly in grape (Vitis vinifera) seed extract and other plant
material and are used industrially as antioxidants for edible fats,
oils and cosmetics. These polyphenols render antioxidant and
antisecretory properties to the bark extract of Guazuma ulmifo-
lia and completely inhibit cholera toxin, which explains the use
of this species as an antidiarrheic drug in developing countries
(Hoer et al., 1995).
Nevertheless, only a few studies have been conducted on the
extract of the leaves and flowers of this species and none of them
focused on its gastroprotective effect, which however had been
reported in traditional medicine.
Phytochemical screenings of Guazuma ulmifolia show that
the leaves contain tannins and other phenolic compounds (Choi
and Hwang, 2005; González-Gómez et al., 2006). Polyphe-
nols, especially tannins, are phytochemicals with antioxidant
and radical-scavenging properties. These compounds have also
shown antiulcerogenic properties due to their protein precipitat-
ing and vasoconstricting effects (Berenguer et al., 2006).
Another active constituent of the leaves is the alkaloid
caffeine, which exhibits antioxidant properties (Filip et al.,
2007). Beta-sitosterol, a phytosterol with anti-inflammatory
and immunomodulator activity, is also present (Delporte et
al., 2005). Furthermore, the essential oil in the leaf is rich
in beta-caryophyllene (13.7%) (Arriaga et al., 1997). This
anti-inflammatory sesquiterpene has been proven to inhibit
gastric mucosal lesions in rats when administered orally,
before the necrotizing agents absolute ethanol and 0.6 N
HCl, without affecting gastric acid secretion. However, beta-
Total SH (nmol/mg tissue) GSH-Px (nmol/min/mg tissue)
17.93 ± 1.51 9.17 ± 1.34
9.23 ± 0.82* 7.52 ± 1.23
12.41 ± 2.13 11.33 ± 1.24
13.50 ± 1.41 12.80 ± 1.21
13.72 ± 1.54 13.41 ± 1.22+
20.19 ± 2.73++ 13.72 ± 1.44+
caryophyllene failed to protect gastric mucosa when the lesions
were induced by stress, water immersion or s.c. administration
of indomethacin. For this reason, the antiulcerogenic effect of
beta-caryophyllene is supposed to be topical and attributable to
the enhancement of defensive factors via cytoprotection (Tambe
et al., 1996).
The damage on the gastrointestinal mucosa induced by
acute toxicity is associated with the activation of neutrophils
that adhere to microvascular endothelium and are a potential
source of oxygen free radicals (Hawkey, 2000). For experimen-
tal purposes, the determination of MPO concentration in gastric
mucosa is an index of neutrophil infiltration. In our in vivo
model, the extract of Guazuma ulmifolia showed a certain ten-
dency to counteract the increase in MPO concentration caused by
the NSAID. In vitro, however, Guazuma ulmifolia significantly
inhibited MPO release from stimulated neutrophils.
As for the flowers of Guazuma ulmifolia, these contain
flavonoids such as quercetin, kaempferol and kaempferitrin,
which possess anti-inflammatory, antiviral (Lyu et al., 2005) and
free radical-scavenging activity (Jung et al., 2006).
The antiulcerogenic pharmacological effect of many plants
is related to their flavonoid content, as these antioxidants inhibit
lipid peroxidation and other free radical-mediated processes that
lead to gastric damage (Alanko et al., 1999; La Casa et al.,
2000).
The process of lipid peroxidation is mediated by the
interaction of hydroxyl radicals with the cell membrane; subse-
quently producing lipid-derived free radicals such as conjugated
dienes and lipid hydroperoxides that cause oxidative damage
(Bagchi et al., 1998). The MDA levels, which indicate lipid
peroxidation of the membranes, were significantly increased
after diclofenac treatment, which is closely related to tis-
sue damage. Guazuma ulmifolia at the two lowest doses, and
omeprazole as well, reverted the content of TBA-reactive sub-
stances almost to sham levels. Preventive antioxidants such
as SOD and catalase enzymes are the first line of defence
against reactive oxygen species. In our study, the adminis-
tration of Guazuma ulmifolia combated diclofenac-induced
gastric lesions by a potent increase in SOD activity. This
indicates that Guazuma ulmifolia protects the stomach by pre-
serving antioxidant enzyme activity in the mucosa exposed to
damage.
GSH is an important constituent of intracellular protective
mechanisms against a number of noxious stimuli, and it is known
 B. Berenguer et al. / Journal of Ethnopharmacology 114 (2007) 153–160
to be a major low molecular weight scavenger of free radicals in
cytoplasm. The GSH redox cycle catalyzed by the endogenous
antioxidant enzyme GSH-Px reduces H2 O2 , thus breaking the
chain reaction leading from O2 − to the highly reactive • OH. At
the same time, the antioxidant activity of GSH-Px is coupled
with the oxidation of GSH to GSSG, which can subsequently be
reduced by glutathione reductase with NADPH as the reducing
agent (Villegas et al., 2000). Depletion of GSH enhances lipid
peroxidation, which in turn can cause increased GSH consump-
tion. In contrast, an increase in gastric non-protein SH content
limits the production of oxygen-derived free radicals, and could
be related to gastric protection against NSAIDs (La Casa et
al., 2000). Our results reflected changes in GSH metabolism:
although GSH-Px activity was not statistically decreased by
diclofenac, total GSH content was significantly decreased,
probably due to its consumption during oxidative stress. The
protective effect of Guazuma ulmifolia was not accompanied by
a significant increase in GSH levels. On the contrary, omepra-
zole elicited a marked elevation, which is in accordance with
former studies on proton-pump inhibitors (Natale et al., 2004).
However, the results revealed an increase in GSH-Px activity
due to the pretreatment with the plant extract. This enhance-
ment indicates that the anti-ulcerogenic effect of Guazuma
ulmifolia may appear, at least in part, through glutathione
metabolism.
Mucus is capable of acting as an antioxidant, and thus can
reduce mucosal injury mediated by oxygen free radicals. When
cells containing mucus are damaged by extracellular oxygen
radicals, intracellular mucus may be released into the gastric
tissue and prevent additional damage by scavenging these radi-
cals (Seno et al., 1995). PGE2 promotes the production of gastric
mucus and therefore protects the stomach epithelium from ROS
(Repetto and Llesuy, 2002). The slight increase in PG levels we
observed was not important enough to consider the involvement
of PG-dependent mechanisms.
Another humoral factor that contributes to mucosal protec-
tion during inflammatory reactions is nitric oxide (NO), which
is produced by nitric oxide synthase (iNOS) in macrophages
and other cells (Gyires, 2005). However, when excessively pro-
duced, NO may react with superoxide anion radical, giving rise
to the strong oxidant peroxynitrite and damaging functional tis-
sues. Therefore, a beneficial therapeutic strategy consists in the
inhibition of NO accumulation at inflammatory sites (Hobbs et
al., 1999). Recently, it has been demonstrated that the extract
of the leaves of Guazuma ulmifolia significantly inhibits the
release of NO in a culture supernatant of macrophages (Choi
and Hwang, 2005). This anti-inflammatory mechanism proba-
bly contributes to the global gastroprotective effect observed in
our study.
We conclude that pretreatment of rats with aqueous suspen-
sion of the ethanolic extract of leaves and flowers of Guazuma
ulmifolia protects the gastric mucosa against the injurious
effect of NSAID administration, mainly by anti-inflammatory
and radical-scavenging mechanisms. However, a detailed study
should be carried out in order to continue elucidating the phyto-
chemical compounds of the aerial parts and further evaluate its
gastroprotective effect in this context.
159
Acknowledgment
This work is included in a scientific cooperation programme
of the Education and Science Ministry of Spain with Latin Amer-
ica (CYTED, Proyecto X.10).
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