Gastroprotective properties of Lupeol-derived ester: Pre-clinical evidences of Lupeol-stearate as a potent antiulcer agent

Article abstract of DOI:10.1016/j.cbi.2020.108964

Lupeol (1) was isolated from hexane branch extract of Maytenus salicifolia and the Lupeol stearate (2), Lupeol palmitate (3), Lupeol myristate (4), Lupeol laurate (5) and Lupeol caprylate (6) were obtained reacting 1 with an adequate carboxylic acid. Swiss mice were treated with vehicle, carbenoxolone or Lupeol esters before administration of ethanol/HCl or indomethacin. Additionally, the involvement of nitric oxide (NO), sulfhydryl compounds (NP-SH), α-2 adrenergic receptors (α2-AR) and prostaglandins (PGE) in antiulcer effects was investigated using appropriate inhibitors or antagonist. Oxidative and inflammatory parameters were measured after euthanasia and anti-secretory effects was evaluated in pylorus-ligated rats. Ethanol/HCl ulcerated the gastric mucosa by 64.45 ± 6.58 mm², which the oral treatment with 1, 4 and 6 (10 mg/kg), and 3 and 5 (30 mg/kg) reduced the lesion area. Interestingly, 2 reduced the gastric ulcer by oral route in a potent and dose-dependent manner (ED₅₀ = 0.40 mg/kg), which was accompanied by the increase in reduced glutathione levels and by the reduction of lipids peroxidation and myeloperoxidase and superoxide dismutase activities. Moreover, 2 (0.1 mg/kg) also prevented the ulcerogenesis by intraperitoneal route. The participation of NO, NP-SH, α2-AR and PGE in 2-mediated gastroprotection was confirmed. In indomethacin-induced ulcer, 2 (1 mg/kg, p.o) also reduced the ulcer area and increased the PGE₂ levels. However, 2 did not alter the gastric acid secretion. Therefore, these findings indicate that the obtention of 2 potentiated the antiulcer activity of 1 and that this compound can elicit gastroprotective action due a diversified mode of action.

Full text of DOI:10.1016/j.cbi.2020.108964

Journal Pre-proof
Gastroprotective properties of Lupeol-derived ester: Pre-clinical evidences of Lupeol-
stearate as a potent antiulcer agent
Lincon Bordignon Somensi, Philipe Costa, Thaise Boeing, Luísa Nathália Bolda
Mariano, Bruna Longo, Cássia Gonçalves Magalhães, Priscila de Souza, Sérgio
Faloni de Andrade, Luisa Mota da Silva
PII:
S0009-2797(19)31180-9
https://doi.org/10.1016/j.cbi.2020.108964
CBI 108964
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 9 July 2019
Revised Date: 17 January 2020
Accepted Date: 24 January 2020
Please cite this article as: L.B. Somensi, P. Costa, T. Boeing, Luí.Nathá. Bolda Mariano, B. Longo,
Cá.Gonç. Magalhães, P. de Souza, Sé. Faloni de Andrade, L. Mota da Silva, Gastroprotective properties
of Lupeol-derived ester: Pre-clinical evidences of Lupeol-stearate as a potent antiulcer agent, Chemico-
Biological Interactions (2020), doi: https://doi.org/10.1016/j.cbi.2020.108964.
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Gastroprotective properties of Lupeol-derived ester: pre-clinical evidences of
Lupeol-stearate as a potent antiulcer agent
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Lincon Bordignon Somensia; Philipe Costaa; Thaise Boeinga; Luísa Nathália Bolda
Marianoa; Bruna Longoa; Cássia Gonçalves Magalhãesb; Priscila de Souzaa; Sérgio
Faloni de Andradea; Luisa Mota da Silvaa*
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aPrograma de Pós-Graduação em Ciências Farmacêuticas (PPGCF), Núcleo de
Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí
(UNIVALI), Rua Uruguai, 458, Centro, 88302-202 Itajaí, SC, Brasil
bDepartamento de Química, Centro de Ciências Exatas e Naturais, Universidade
Estadual de Ponta Grossa, Ponta Grossa, Paraná, Brasil
* Corresponding author:
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e-mail: luisa@univali.br
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Abstract
Lupeol (1) was isolated from hexane branch extract of Maytenus salicifolia and the
Lupeol stearate (2), Lupeol palmitate (3), Lupeol myristate (4), Lupeol laurate (5) and
Lupeol caprylate (6) were obtained reacting 1 with an adequate carboxylic acid. Swiss
mice were treated with vehicle, carbenoxolone or Lupeol esters before administration of
ethanol/HCl or indomethacin. Additionally, the involvement of nitric oxide (NO),
sulfhydryl compounds (NP-SH), α-2 adrenergic receptors (α2-AR) and prostaglandins
(PGE) in antiulcer effects was investigated using appropriate inhibitors or antagonist.
Oxidative and inflammatory parameters were measured after euthanasia and anti-
secretory effects was evaluated in pylorus-ligated rats. Ethanol/HCl ulcerated the gastric
mucosa by 64.45 ± 6.58 mm2, which the oral treatment with 1, 4 and 6 (10 mg/kg), and
3 and 5 (30 mg/kg) reduced the lesion area. Interestingly, 2 reduced the gastric ulcer by
oral route in a potent and dose-dependent manner (ED50= 0.40 mg/kg), which was
accompanied by the increase in reduced glutathione levels and by the reduction of lipids
peroxidation and myeloperoxidase and superoxide dismutase activities. Moreover, 2
(0.1 mg/kg) also prevented the ulcerogenesis by intraperitoneal route. The participation
of NO, NP-SH, α2-AR and PGE in 2-mediated gastroprotection was confirmed. In
indomethacin-induced ulcer, 2 (1 mg/kg, p.o) also reduced the ulcer area and increased
the PGE2 levels. However, 2 did not alter the gastric acid secretion. Therefore, these
findings indicate that the obtention of 2 potentiated the antiulcer activity of 1 and that
this compound can elicit gastroprotective action due a diversified mode of action.
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Keywords: gastric healing; oxidative stress; esterification; semisynthetic compounds.

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1.
INTRODUCTION
The gastric ulcer is a global pathology characterized by a decrease in the
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protective factors of the gastric mucosa, including the mucus, bicarbonate, blood
circulation, and antioxidants); and/or an increase in aggressive factors, such as alcohol,
nonsteroidal anti-inflammatory drugs (NSAIDs), reactive oxygen species (ROS),
pepsinogen activation [1]. The treatment for this disease has been based on anti-
secretory drugs such as histamine type 2 receptor antagonists (ranitidine and congeners)
or proton pump inhibitors, such as omeprazole. However, the discontinuation of these
treatments may lead to ulcer recurrence, whereas a prolonged treatment period has been
associated with several adverse effects [2].
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Lupeol is a pentacyclic triterpene found in several plants including Maytenus
salicifolia Reissek (Celastraceae) [3]. The gastroprotective activity of this compound
has already been described by Lira et al. [4] at a dose of 3 mg/kg by the oral route.
Besides, this triterpene presents many biological activities, including anti-inflammatory,
antiarthritic, antimutagenic, antitumor, hepatoprotective and antioxidant properties [5, 6,
7, 8, 9]. In addition to these biological activities, several recent studies show the
therapeutic potential of lupeol in different experimental models. Zingue et al. [10] have
demonstrated that lupeol has estrogenic properties in ovariectomized rats, an effect that
may be attributed to estrogen receptor transcriptional activity. Moreover, Pereira
Beserra et al. [11] have described, by using in vitro wound healing assays and human
neonatal foreskin keratinocytes and fibroblasts, the therapeutic potential of lupeol for
accelerating wound healing and tissue repair. Extending this knowledge about the
healing process, this same group of authors also demonstrated the wound healing
activity of lupeol in streptozotocin-induced hyperglycemic rats, highlighting the
potential of this triterpene in healing processes and tissue repair [12].

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The structural modification of natural compounds has been an interesting tool to
obtain analogous more effective and safer, which can lead to the identification of the
pharmacophoric group [13]. This practice is already used for pentacyclic triterpenes [14,
15]. In this context, Silva et al. [15] described the synthesis of Lupeol esters; and in
continuity, this study evaluated the gastroprotective and anti-secretory effects of Lupeol
esters and investigated the mode of action of the most potent derivative.
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2.
MATERIAL AND METHODS
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2.1 Esters obtaining
Lupeol (1) was isolated from the hexane extract of Maytenus salicifolia and the
esters: Lupeol stearate (2), Lupeol palmitate (3), Lupeol myristate (4), Lupeol laurate
(5) and Lupeol caprylate (6) were obtained reacting 1 with an adequate carboxylic acid,
as described early by Silva et al. [15].
2.2 Animals
Wistar rats (200-250 g) and Swiss mice (25-30g) were obtained from the central
laboratory of the Universidade do Vale do Itajaí (UNIVALI) and kept in polypropylene
boxes at 22 ± 2ºC in 12 hours light/dark cycles with free access to water and feed. The
animals were deprived of food eight hours prior to the experiments. All protocols were
approved by the Institutional Animal Ethics Committee on UNIVALI
(CEUA/UNIVALI, approval number 056/2017) and were carried out in accordance with
the International Standards and the Ethical Guidelines on Animal Welfare.
2.3 Ethanol-HCl induced-gastric ulcer in mice

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The mice were randomly separated into groups (n = 6) and treated with vehicle
(1% DMSO, 10 mL/kg, p.o), carbenoxolone (positive control, 200 mg/kg, p.o) or
Lupeol derivatives (0.1-30 mg/kg, p.o) before administration of ethanol-HCl (10 mL/kg,
p.o). After 1 hour of the treatments, 60% ethanol/0.3 M HCl (0.1 mL/10g) was given to
the induction of gastric ulcer, as described by Hara and Okabe [16]. Further, the animals
were euthanized in the CO2 chamber after 1 hour of ulcerogenic agent intake, the
stomachs were removed and opened by the greater curvature and the lesion area was
quantified by the EARP® program.
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In another set of experiments, mice were divided into groups (n = 6) and treated with
vehicle (1% DMSO, 10 ml/Kg, p.o), carbenoxolone (200, mg/kg p.o) and compound 2
(0.1 mg/kg, i.p). After 30 min from intraperitoneal or 1 hour from oral administrations,
the mice received ethanol-HCl as described above. The animals were euthanized after 1
hour in a CO2 chamber, the stomachs were removed, and the lesion area was quantified
as described above.
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2.4 Indomethacin induced-gastric ulcer in mice
Indomethacin induced-gastric ulcer was performed according to Fornai et al.
[17]. The animals received vehicle (1% DMSO, 10mL/kg, p.o), carbenoxolone (200
mg/kg, p.o) or ester 2 (0.3 - 3 mg/kg, p.o). After 1 hour, indomethacin (100 mg/kg, p.o)
was given and after 6 hours the animals were euthanized in a CO2 chamber, the
stomachs removed, opened by the greater curvature and ulcers were analyzed using the
EARP® program.
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2.5 Ethanol-HCl induced-gastric ulcer in mice pretreated with N-Ethylmaleimide
(NEM), N-Ω-Nitro-L-Arginine Methyl Ester (L-NAME), Indomethacin or
Yohimbine

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This experiment followed the protocol previously described by Matsuda and
Yoshikawa [18], Leite [19], and Arrieta et al. [20]. The mice were pretreated with
antagonists or inhibitors: NEM (10 mg/kg, s.c), L-NAME (70 mg/kg, i.p), indomethacin
(10 mg/kg, i.p), yohimbine (2 mg/kg, i.p) or saline (1 ml/kg i.p). After 30 min., the
animals received vehicle (1% DMSO, 10 mL/kg, p.o) or 2 (1 mg/kg p.o). Then, the
ethanol-HCl (Ethanol 60% + HCl 0.3 M, 10 mL/kg p.o) was given 1 hour later. After
another 1 hour, the mice were euthanized in a CO2 chamber, the stomachs removed,
opened by the greater curvature and analyzed as previously described.
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2.6 Pylorus ligature in rats
Rats were randomly distributed into experimental groups (n = 6) and
anesthetized with xylazine (10 mg/kg, i.p) and ketamine (50 mg/kg, i.p). A laparotomy
was performed subsequently, where the pylorus was sampled and ligated. Further,
vehicle (1% DMSO, 10 mL/kg) or 2 (1 mg/kg) were administered by intraduodenal
route, while the positive control group received omeprazole (20 mg/kg, p.o) 30 minutes
before the ligature. Subsequently, the abdominal wall was sutured. After 4 hours, the
animals were euthanized in a CO2 chamber, the stomach was removed and contents
were collected. The volume of gastric juice (mL) was measured using a graduated
cylinder after centrifugation (1500 × g, 15 min, 4 ° C), the pH was determined with a
pH meter and total acidity (mEq/ L/ 4 h) per titration with 10 mM sodium hydroxide
following the protocol described by [21].
2.7 Measurement of peptic activity
As described by Anson [22], 100 µL of gastric juice from pylorus ligated rats
was incubated with 500 µL of bovine albumin (5 mg/mL prepared in 60 µM HCl) at 37

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°C for 10 min. Then, 1 N Folin reagent was added and incubated at 25 °C for 30 min.
The absorbance of each sample was inferred at 660 nm and the results expressed in µM/
mL/ 4 h of tyrosine interpolating individual values on a standard tyrosine curve (30-
1000 mmol/mL).
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2.8 Preparation of the homogenate and protein analysis
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The gastric mucosa was homogenized with 200 mM potassium phosphate buffer
(pH 6.5). The homogenate was used to measure the levels of reduced glutathione (GSH)
and lipoperoxides (LOOH). Thereafter, the homogenate was centrifuged at 9000 ×g by
20 minutes and the supernatant was used to assess the activities of glutathione-S-
transferase (GST), superoxide dismutase (SOD) and catalase (CAT), while the
precipitated was used to measure myeloperoxidase (MPO) activity.
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Protein concentrations were determined in all samples using Bradford's reagent
and bovine albumin as standard following the manufacturer's instructions (Bio-rad®,
Hercules, CA, USA).
2.9 Quantification of the GSH and LOOH levels
As described by Sedlak and Lindsay [23], 50 µl of homogenate was added to 40
µl of 12.5 % trichloroacetic acid, then the material was centrifuged at 1.4 × g/ 15 min.
After, 20 µl of the supernatant was added to 270 µl of TRIS buffer (pH 8.9) and 10 µl of
5,5' dithiobis-2-nitrobenzoic acid (DTNB). The absorbance was measured after 5 min at
415 nm and the values were interpolated on a standard curve of GSH (1.25-10.00
µg/mL). Results are expressed in µg /mg of tissue.
To evaluate the LOOH amount, the method described by Jiang et al. [24] was
performed. Thus, 100 µl of methanol was added into 100 µl of homogenate and

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centrifuged at 9000 × g during 20 min at 4°C. Afterward, 30 µl of the supernatant was
added to 270 µl of the reaction medium containing 4 mM butylated hydroxytoluene, 250
mM FeSO4, 25 mM H2SO4 and 100 mM xylenol orange and incubated for 30 min at
25°C. Absorbances were recorded at 560 nm and the results expressed in mmol/mg of
tissue using the extinction coefficient of 46.6 µM/cm.
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2.10 Determination of SOD, CAT and GST activities
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The SOD activity was quantified as described by Marklund and Marklund [25].
Briefly, samples were incubated with 200 mM Tris-HCl-EDTA (pH 8.5) and 1 mM
pyrogallol for 20 min. Subsequently, absorbance was measured at 405 nm and SOD
activity was expressed as U/mg protein.
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The CAT activity was measured adding 5 µl of supernatant to 295 µl of reaction
medium (200 mM Tris-HCl-EDTA, pH 8.5, 47.35 mL of ultrapure water and 172.5 µl
of H2O2). The absorbance was measured at 240 nm and results expressed as
µmol/min/mg of protein, according to Aebi [26].
The GST activity was measured according to Habig et al. [27], where 50 µl of
the sample and 250 µl of the reaction medium (0.1 M buffer phosphate, 1-Chloro-
2,4dinitrobenzene (CDMB), and reduced glutathione (GSH)) were added. The
absorbance was measured at 340 nm and results expressed as mmol/min/mg of protein.
2.11 Determination of MPO activity
To determine MPO activity, the precipitate obtained as described above was
resuspended in 80 mM potassium phosphate buffer (pH 5.4) containing 0.5% hexadecyl
trimethyl ammonium bromide and centrifuged at 11,000 × g for 20 min at 4 ° C. The
MPO activity in the supernatant was determined at 620 nm with H2O2 and 3,3’,5,5’-

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tetramethylbenzidine and expressed in units of mili optical density (mO.D)/ mg protein
as described by Bradley et al. [28] and De Young et al. [29].
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Assessement of PGE2 levels
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Prostaglandin E2 (PGE2) concentration was determined in indomethacin-
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ulcerated
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mucosa
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using commercial Kit for enzyme immunoassay, following the manufacturer's
instructions, Cayman Chemical (Ann Arbor, Michigan, USA). For this determination
the indomethacin-ulcerated tissue was homogenized with 200 mM potassium phosphate
buffer (pH 6.5) and then centrifuged at 9000 × g for 20 minutes. The supernatant was
used to evaluate the levels of this eicosanoid.
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2.13 Statistical analysis
The results were expressed as means ± standard error o means (S.E.M). One or
two-way analysis of variance (ANOVA) followed by the Bonferroni’s test was used to
determinate the difference between the means using GraphPadPrism 5® Software
(GraphPad Software, La Jolla, CA, USA). A value of P<0.05 was considered significant
in all experiments.
3.
RESULTS
3.1 Compound 2 decreased ethanol/HCl-induced gastric ulcer in mice
As shown in figure 1, the acidified ethanol ulcerated the gastric mucosa by 64.45
± 6.58 mm2 in the vehicle-treated only group. As expected, the treatment with

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carbenoxolone (200 mg/kg, p.o), the positive control group, reduced the lesion area in
83.84%. The pretreatment with 1 (Figure 1A), 4 (Figure 1C) and 6 (Figure 1E)
diminished the lesion area by 60.4, 67.4% and 52.2%, respectively, both at a dose of 10
mg/kg (p.o). In addition, compounds 3 (Figure 1B) and 5 (Figure 1D), at the dose of 30
mg/kg p.o, reduced the lesion area by 95% and 70%, respectively, when compared to
vehicle-treated group.
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Interestingly, the pretreatment with 2 (Figure 2A) reduced the gastric ulcer by
oral route in a dose-dependent manner (ED50= 0.40 mg/kg, with 95% confidence
interval = 0.18 to 0.89 mg/kg). Additionally, representative images from these results
are depicted in figure 2B. Moreover, the intraperitoneal administration of 2, at a dose of
0.1 mg/kg, decreased the gastric injury by 55.3% (Figure 3).
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3.2 Compound 2 decreased the ulcer area and increased PGE2 in indomethacin-
induced ulcer model
As show in figure 4A, indomethacin-induced gastric lesions in an extension
equal to 2.40 ± 0.39 mm2, while the compound 2, at the doses of 0.3, 1 and 3 mg/kg,
decreased the lesion area by 67%, 77% and 78%, respectively, when compared to the
vehicle group. Additionally, carbenoxolone (200 mg/kg, p.o) decreased the gastric
lesions by 92%, in comparison with the vehicle group. In parallel, oral admnistration of
2 (1 mg/kg) increased the PGE2 amount in gastric tissue from mice exposed to
indomethacin-induced ulcer model, compared to vehicle-treated group (p<0.01, figure
4B). In a similar manner, carbenoxolone (200 mg/kg) also increased the PGE2 in
ulcerated tissues when compared to vehicle-treated group (p<0.01, figure 4B).

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3.3 Gastroprotective effects of compound 2 in L-NAME, NEM, indomethacin or
yohimbine-pretreated mice in the model of ethanol/HCl-induced ulcer
The pretreatment with L-NAME (Figure 5A) and NEM (Figure 5B) augmented
the ulcer area by 116% and 212%, respectively, when compared to the group pretreated
with saline only (46.57 ± 7.94 mm2). However, the pretreatment with Indomethacin
(Figure 5C) or Yohimbine (Figure 5D) did not alter the ulcerated area in comparison
with the saline group. In addition, the pretreatment with L-NAME (Figure 4A), NEM
(Figure 4B), Indomethacin (Figure 4C), and Yohimbine (Figure 4D) abolished the
gastroprotective effect of compound 2.
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3.4 Compound 2 did not change gastric secretion parameters
The volume of gastric juice in the vehicle group was 3.9 ± 0.4 mL; whereas in
the same group, the pH was equal to 3.16 ± 0.63, reaching a total acidity of 17.75 ± 2.01
Eq[H+]/mL/4 hours and a peptic activity of 1.96 ± 0.09 µM of tyrosine/4 hours. The
administration of 2 (1 mg/kg, i.d) did not change the volume, pH, acidity or peptic
activity when compared to the vehicle group. As expected, the administration of
omeprazole (20 mg/kg) was able to reduce the acidity and peptic activity in 62.3% and
24.5%, respectively. Moreover, the pH of the gastric medium in the group treated with
omeprazole was 6.69, as shown in table 1.
3.5 Compound 2 increases GSH and restores LOOH levels in ethanol/HCl-induced
gastric ulcer
As shown in table 2, the ulcerated group treated with vehicle presented GSH
levels equal to 124.7 ± 6.46 µg/mg of tissue, whereas the non-ulcerated group,
presented the GSH amount equal to 173 ± 8.91 µg/mg of tissue. The treatment with 2 (1

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mg/kg) or carbenoxolone (200 mg/kg) increased the GSH values by 63% and 42%,
respectively (Table 2).
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The LOOH content was increased by 13% in the vehicle group, compared to the
naive group (non-ulcerated group: 1.80 ± 0.03 mmol/mg of tissue). In contrast,
carbenoxolone (200 mg/kg) and 2 (1 mg/kg) reduced the LOOH levels by 12% and 10%
respectively, compared to the vehicle group (2.04 ± 0.04 mmol/ mg tissue) (Table 2).
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3.6 Compound 2 decreases the activity of SOD but does not change CAT or GST
activity
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The acidified ethanol increased the SOD activity in the vehicle-treated group by
24%, related to basal levels found in non-ulcerated mice (Naive: 7.02 ± 0.1 U SOD/mg
of protein). On the other hand, the administration of 2 reduced the SOD activity by
44%, compared to the vehicle group (Table 2). However, the administration of 2 did not
change the CAT or GST activity, compared to the vehicle group.
3.7 Compound 2 reduced the MPO activity
Expectedly, the MPO activity increased by 953% in ulcerated tissue, when
compared to non-ulcerated group (Naïve: 1.7 ± 0.20 mD.O/mg of protein). Oppositely,
2 (1 mg/kg) and carbenoxolone (200 mg/kg) were able to reduce this parameter by 78%
and 77%, respectively, compared to the vehicle group (Table 2).
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4.
DISCUSSION
The gastroprotective effect of Lupeol has been previously described by [4], as
well as some gastroprotective mode of action. In continuity to these studies, this
research evaluated the gastroprotective activity of the 1 and their esters (2, 3, 4, 5 and 6)

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obtained by Silva et al. [15] through structural modifications in the Lupeol molecule
aiming identify if those alterations altered and/or improved the anti-ulcer potential of
Lupeol.
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The ethanol-induced gastric ulcer is a classical model employed in
gastroprotective studies because ethanol enters the gastric mucosa causing an intense
vascular injury, decreasing the blood flow causing tissue necrosis and ROS generation
[30, 31]. Acidified ethanol undoubtedly caused lesions in the gastric mucosa that were
reversed by the action of compound 2 by oral and intraperitoneal treatments, suggesting
a systemic effect and not just a topical action by due to the oral route. This data
corroborates with Navarrete et al. [32] and Liby et al. [33], which described
gastroprotective actions to other triterpenes. Similarly, Da Rosa et al. [34] reported the
gastroprotective effect of the triterpenes maslinic and ursolic acids against acidified
ethanol-induced lesions. The estering is a method used to improve the biological effect,
to decrease side effects or to improve the absorption of a molecule. In fact, 2 (1 mg/kg)
demonstrated a superior anti-ulcer effect than Lupeol (3 mg/kg p.o) already described
by Lira et al. [4]. Similarly, Urban et al. [35] demonstrated that esterification in ring A
of lupane group may increase or decrease cytotoxic action.
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Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) lacking
specificity for both cyclooxygenase 1 or 2 (COX-1 or 2), however its use is related to
the appearance of gastric lesions due to the inhibition of COX-1 which in turn,
decreases the production of endogenous prostanoids, such as PGE2, a factor that is
related to the protection of the gastric mucosa [36]. The pretreatment with indomethacin
was able to reduce the gastroprotective effect of ester 2 demonstrating that its
gastroprotective effect also depends on the effect of prostaglandins. Corroborating with
our results, Lira et al. [4] demonstrated that Lupeol also has its effect depleted when

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pretreated with indomethacin. Interestingly, Geetha and Varalakshmi [37] suggested
that Lupeol exerts anti-inflammatory actions, but in a different manner compared to
NSAIDs, and unlike indomethacin, did not demonstrate the ulcerogenic effect in long
term treatment.
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Giving a continuity, we evaluated the role of NO and nonprotein sulfhydryl (NP-
SH) compounds as contributors to the gastroprotective activity displayed by 2. In the
ulcer genesis, ROS can cause depletion of NP-SH groups and NO, leading to the
damage in the gastric mucosa due to oxidative stress and poor blood circulation [38, 39,
40]. Indeed, it was evidenced that the gastroprotective effect of 2 is abolished in mice
pretreated with an inhibitor of NO synthase (i.e. L-NAME) or with an NP-SH blocker
(i.e. NEM), suggesting that an adequate blood flow and the bioavailability of endogens
antioxidants is crucial to antiulcer events elicited by 2. As expected, our results
corroborate with Lira et al. [4], which demonstrated that the gastroprotective effect of
Lupeol was also abolished in mice pretreated with L-NAME and NEM.
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9
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According to Gyires et al. [41], the α2 adrenoceptors are involved in gastric acid
secretion and possess crucial roles in other responses in the gastrointestinal tract.
Yohimbine is classified as a selective α2 adrenergic receptor antagonist [42] and in this
study was employed to analyze the participation of this receptor in the gastroprotection
exerted by 2. In this experiment, it was observed that the antiulcer effect of 2 was
abolished in mice pretreated with Yohimbine, suggesting that α2 - adrenergic receptors
participate directly in the gastroprotection action exerted by this ester. Confirming our
results, Lira et al. [4] showed that the effect of lupeol was also abolished in the presence
of Yohimbine.
Besides the mode of actions already discussed herein, it is important to
understand the antioxidants mechanisms involved in the gastroprotective action,

15
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because this mechanism occurs at the cellular level. Ethanol increases oxidative damage
in the gastric mucosa by decreasing blood flow, elevating lipid, hydroxyl and
superoxide peroxidation [43, 44]. The initial stage of cellular damage caused by ROS is
the cell membrane peroxidation [45]. As expected, LOOH levels were elevated in the
vehicle-treated group, while these levels were reestablished in the mice treated with 2 (1
mg/kg), inferring that oxidative damage was minimized by the action of this ester.
Furthermore, 2 were also able to raise GSH levels to values greater than those found in
the vehicle-treated group. GSH is a tripeptide present within the cell and plays a key
role in both non-enzymatic and enzymatic antioxidant pathways [46]. In this way, ester
2 is able to restore the oxidative balance.
3
4
5
6
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In parallel, the enzymatic antioxidant defense system includes SOD, an enzyme
that promotes the dismutation of superoxide anion; and CAT, which realizes the
conversion of hydrogen peroxide to water and oxygen. Moreover, the detoxifying
enzyme GST catalyzes the GSH conjugation with various endogenous and exogenous
electrophilic compounds [45, 47]. In this way, ester 2 reduced the SOD activity, but
CAT and GST activities were not altered in groups treated with 2, demonstrating that 2
does not require these oxidative pathways to exert its gastroprotective effect.
The activity of MPO is classically verified as a marker of neutrophil infiltration
in tissues because this enzyme is found in the azurophil granules of these inflammatory
cells [48, 49]. As expected, the contribution of the neutrophils to the genesis of the
gastric lesion was confirmed by the increase in the levels of MPO activity at the ulcer
site in the vehicle-treated group. In contrast, the treatment with 2 reduced this parameter
in ethanol/HCl-ulcerated tissue. Therefore, we can also infer that ester 2 avoided the
ulcerogenic process, at least in part, by the reduction of the inflammatory process
mediated by neutrophil migration.

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Finally, considering the results obtained, we evaluated the gastric anti-secretory
activity of the 2. The suppression of gastric acid is the main therapy used for the gastric
ulcer treatment. Despite of this, the compound 2 was not able to decrease the volume of
secretion, total acidity or peptic activity in the gastric juice, suggesting that the
mechanism of action of the compound 2, as explored in this study, differs from the
actions elicited by omeprazole, a classical standard drug used in the clinic due to its
inhibitory action of the proton pump.
3
4
5
6
7
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9
5.
Conclusion
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Together, our results confirmed that the esters 3, 4, 5 and 6 were able to reduce
the area of the ulcer lesion; however, ester 2 was able to reduce the ethanol acidified-
and indomethacin-induced gastric ulcer in lower doses, evidencing that the stearate
group enhanced the gastroprotective potency of Lupeol. Regarding mode of actions, the
participation of NP-SH, NO, PGE2 and α2-adrenoceptors directly participate in the
gastroprotective effect of this compound. Antioxidant properties include the increase in
GSH availability and the decrease of LOOH content, as well as a reduction in neutrophil
migration. Finally, the chemical modification on the Lupeol structure that provided
compound 2 increased its pharmacological action.
Conflict of interest
The authors have no conflict of interest.
Acknowledgments
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001. We also thank the support

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received from Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Universidade do Vale do Itajaí (UNIVALI).
3
4
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Legends for figures
3
Fig. 1 Effect of Lupeol and esters 3, 4, 5 and 6 on the acute gastric ulcer induced
by ethanol/HCl. Panel A - E: The animals received vehicle (Veh: DMSO 1%, 1 ml/kg,
p.o), carbenoxolone (Cbx: 200 mg/kg, p.o), Lupeol and its esters 3, 4, 5 and 6 (1 - 30
mg/kg, p.o). Results are expressed as means ± S.E.M. (n=6). One-way ANOVA
followed by Bonferronis’s test. *P<0.05; ** P<0.01; *** P<0.001 vs. the vehicle-
treated group.
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Fig. 2 Effect of compound 2 (lupeol stearate) on the acute gastric ulcer induced by
ethanol/HCl. Panel A: The animals received vehicle (Veh: DMSO 1%, 1 ml/kg, p.o),
carbenoxolone (Cbx: 200 mg/kg, p.o) or 2 (0.1 - 3 mg/kg, p.o). Panel B: Representative
images of the different experimental groups. Results are expressed as means ± S.E.M.
(n=6). One-way ANOVA followed by Bonferronis’s test. ** P<0.01 and *** P<0.001
vs. the vehicle-treated group.
Fig. 3 Gastroprotective effect of lupeol stearate (2) given by intraperitoneal rout on
the ethanol/HCl-induced gastric ulcer in mice. The animals received vehicle (Veh:
DMSO 1%, 1 ml/ kg), carbenoxolone (Cbx: 200 mg/kg, p.o) and 2 (0.1 mg/kg, ip).
Results are expressed as the means ± S.E.M. (n=6). One-way ANOVA followed by the
Bonferroni’s test. ** P<0.01 and P<0.001 vs. vehicle-treated group.
Fig. 4 Effect of Lupeol stearate (2) on ulcer area (A) and in the PGE2 levels (B) of
ulcerated tissues from indomethacin-induced gastric ulcer in mice. The animals
were orally treated with vehicle (Veh: DMSO 1%, 1 ml/kg), carbenoxolone (Cbx: 200

25
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2
mg/kg) or 2 (0.3 - 3 mg/kg). Results are expressed as the means ± S.E.M. (n=6). One-
way ANOVA followed by Bonferroni's test. ** P<0.01 and P<0.001 vs. the vehicle-
treated group.
3
4
5
Fig. 5 Effects of NEM, L-NAME, Yohimbine, and Indomethacin on the
gastroprotective effect of Lupeol stearate (2) against Ethanol/HCl-induced ulcer in
mice. The animals were treated with saline (10 ml/kg, i.p), NEM (10 mg/kg, i.p), L-
NAME (70 mg/kg, i.p), yohimbine (2 mg/kg, i.p) or indomethacin (10 mg/kg, i.p) 30
min prior to vehicle (Veh: DMSO 1%, 1 ml/kg) or compound 2 (C 2, 1 mg/kg, p.o)
administration. Results are expressed as the means ± S.E.M. (n=6). Two-way ANOVA
followed by Bonferroni’s test. * P<0.05 vs. vehicle-saline group. # P<0.05 vs. C 2-
saline group.
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Table 1. Effects of compound 2 on gastric acid secretion.
Volume
Acidity
pH
Peptic activity
Vehicle
3.99 ± 0.45
17.75 ± 2.01
3.16 ± 0.63
1.96 ± 0.09
Omeprazole
Compound 2
2.70 ± 0.30
3.30 ± 0.19
6.69 ± 1.60a
5.59 ± 0.65a
1.48 ± 0.06a
16.63 ± 1.87
3.50 ± 0.40
1.85 ± 0.09
Volume (mL); Acidity (mEq [H+]/mL); Peptic activity (mmol of tyrosine/4 hours/mL). Values are
expressed as means ± S.E.M (n=6). One-way analysis of variance (ANOVA) followed by
Bonferroni's multiple comparisons test. ap ˂ 0.05 when compared with the vehicle-treated group.

Table 2. Effects of compound 2 on oxidative and inflammatory parameters of ulcerated gastric tissue.
MPO
GSH
LOOH
SOD
CAT
GST
Naive
Vehicle
1.7 ± 0.20
17.9 ± 5.34a
4.1 ± 0.95b
3.9 ± 0.84b
173.9 ± 8.91
124.7 ± 6.46a
177.2 ± 14.91b
203.4 ± 9.49b
1.80 ± 0.03
2.04 ± 0.04a
1.80 ± 0.04b
1.84 ± 0.03b
7.02 ± 0.10
8.69 ± 0.45
7.92 ± 0.18
4.85 ± 1.05b
461.4 ± 63.56
292.2 ± 37.85a
445.9 ± 171.10
468.7 ± 46.12
979.0 ± 13.03
736.0 ± 92.70
1100.0 ± 206.10
719.2 ± 177.3
Carbenoxolone
Compound 2
Myeloperoxidase (MPO, mD.O/mg of protein); Reduced glutathione (GSH, µg/mg of tissue); Hydroperoxides lipids (LOOH, mmol/
mg of tissue); Superoxide dismutase (SOD, U/mg of protein); Catalase (CAT, µmol/min/mg of protein) and Glutathione S-
transferase (GST, mmol/min/mg of protein). Values are expressed as means ± S.E.M (n=6). One-way analysis of variance
a
b
(ANOVA) followed by Bonferroni's multiple comparisons test. p ˂ 0.05 when compared with the naive group. p ˂ 0.05 when
compared to the vehicle-treated group.