Journal of Ethnopharmacology 135 (2011) 147–155
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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Gastroprotective and ulcer healing effects of essential oil from Hyptis spicigera
Lam. (Lamiaceae)
Christiane Takayama a,∗ , Felipe Meira de-Faria b , Ana Cristina Alves de Almeida a ,
Deborah de Arantes e Oliveira Valim-Araújo b , Camilla Souza Rehen a , Ricardo José Dunder b ,
Eduardo Augusto Rabelo Socca a , Luis Paulo Manzo b , Ariane Leite Rozza e , Marcos José Salvador c ,
Claúdia Helena Pellizzon e , Clélia Akiko Hiruma-Lima d , Anderson Luiz-Ferreira a ,
Alba Regina Monteiro Souza-Brito a
a
Anatomy, Cell Biology and Physiology and Biophysics Department, Biology Institute, Campinas State University-UNICAMP, Campinas, SP, Brazil
Pharmacology Department, Faculty of Medical Sciences, Campinas State University-UNICAMP, Campinas, SP, Brazil
c
Plant Physiology Department, Biology Institute, Campinas State University-UNICAMP, Campinas, SP, Brazil
d
Physiology Department, Biosciences Institute, São Paulo State University-UNESP, Botucatu, SP, Brazil
e
Morphology Department, Biosciences Institute, São Paulo State University-UNESP, Botucatu, SP, Brazil
b
a r t i c l e
i n f o
Article history:
Received 1 September 2010
Received in revised form 11 January 2011
Accepted 1 March 2011
Available online 9 March 2011
Keywords:
Hyptis spicigera Lam.
Essential oil
Gastric protection
Healing action
a b s t r a c t
Ethnopharmacological relevance: Hyptis Jacq. (Lamiaceae) is being used in traditional medicine to treat
fever, inflammation and gastric disturbances. Hyptis spicigera Lam. is a native plant distributed across the
central region of Brazil. The essential oil extracted from this plant is used in folk medicine as antipyretic.
Aim of the study: The effects of the essential oil obtained from the aerial parts of Hyptis spicigera (OEH)
were evaluated for their gastroprotective and healing activities.
Materials and methods: OEH chemical composition was analyzed by gas chromatography–mass spectrometry (GC–MS). The gastroprotective action of the OEH was evaluated in rodent experimental models
(ethanol and NSAID). To elucidate mechanisms of action, the antisecretory action and involvements of
NO, SH, mucus and PGE2 were evaluated. The acetic acid-induced gastric ulcer model and Western Blot
assay (COX-2 and EGF) were also used to evaluate the OEH healing capacity.
Results: GC–MS analysis of OEH indicated three monoterpenes as major compounds: alpha-pinene
(50.8%), cineole (20.3%) and beta-pinene (18.3%) and, at the dose of 100 mg/Kg, p.o., OEH provided effective gastroprotection against lesions induced by absolute ethanol (97%) and NSAID (84%) in rats. OEH do
not interfere with H+ secretion in gastric mucosa and its gastric protection does not depend on nitric
oxide (NO) and sulfhydryl compounds (SH). The gastroprotective action of OEH occurs due to an increase
in the gastric mucus production (28%) induced by PGE2 levels. Furthermore, OEH demonstrated a great
healing capacity with 87% of reduction in ulcerative lesion area. It accelerated the healing of acetic acidinduced gastric lesions due to an increase in COX-2 (75%) and EGF (115%) expression in gastric mucosa.
No sign of toxicity was observed in this study, considering the analyzed parameters.
Conclusions: All these results suggest the efficacy and safety of Hyptis spicigera in combating and healing
gastric ulcer. Considering the results, it is suggested that the OEH could probably be a good therapeutic
agent for the development of new phytotherapeutic medicine for the treatment of gastric ulcer.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Gastric ulcer is one of the major gastrointestinal disorders,
which occurs due to an imbalance between the offensive (gastric
∗ Corresponding author. Tel.: +55 01935216192; fax: +55 1935216185.
E-mail address: christiane.takayama@gmail.com (C. Takayama).
0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.03.002
acid secretion) and defensive (gastric mucosal integrity) factors
(Laine et al., 2008). Stress, smoking, Helicobacter pylori and ingestion of non-steroidal anti-inflammatory drugs (NSAID) augment
the peptic ulcer incidences (Vonkeman et al., 2007). The literature reveals a great variety of chemical compounds isolated from
medicinal plants with antiulcer activity (Schmeda-Hirschmann and
Yesilada, 2005). Several plants are used in folk medicine for its
antiulcer properties. Therefore, the investigation of compounds
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C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
bearing antiulcer effects in medicinal plants may lead to the discovery of new potential antiulcer drugs (Lima et al., 2006). Among
these medicinal plants, the aromatic have been widely used since
ancient times (Edris, 2007). The pharmaceutical properties of these
plants are partially attributed to essential oils (Edris, 2007).
Among the aromatic plants, Hyptis Jacq. (Lamiaceae) is being
used in traditional medicine because of their pharmacological
activities (Falcão and Menezes, 2003). This genus, with way over
300 species, exhibits a major morphological diversity in the Brazilian Cerrado region (Harley, 1988). It has yielded a great number of
important medicinal species that are frequently used as remedies
in the treatment of gastrointestinal disturbances (Corrêa, 1931;
Pereda-Miranda, 1995).
The essential oils obtained from aerial parts and roots of Hyptis
mutabilis (Richard) Briquet possess a place in some Bolivian ethnic groups. According to ethnopharmacological studies, decoction
from its aerial parts and roots is used for fever and cystitis respectively (Bourdy et al., 2000). Another species from the same genus,
Hyptis spicigera Lam., has been used in folk medicine in northern
Nigeria as an antipyretic drug (Onayade et al., 1990). This plant
has a pantropical distribution and is used in traditional Mexican
medicine for the treatment of gastrointestinal disturbances, skin
infections, as well as wounds and insect bites (Kini et al., 1993). In
Brazilian folk medicine essential oils from Hyptis pectinata L. Poit.
are used to treat ache and inflammation (Adorjan and Buchbauer,
2010).
Regarding the genus Hyptis importance to folk medicine worldwide, growing interest has flourished, specially its pharmacological
aspects. Pharmacological studies on essential oil from Hyptis mutabilis (Richard) Briquet showed antiulcerogenic activity (Barbosa
and Ramos, 1992).
Based on popular indications of this genus and since the essential oil from another species presents antiulcerogenic activity, the
present work aimed on characterizing the effects of the essential
oil of Hyptis spicigera (OEH) on the gastric mucosa of animals challenged with different ulcerogenic agents.
2. Materials and methods
2.1. Animals
Male Unib: WH rats (n = 7, 150–250 g) from Central Animal
House of the Universidade Estadual de Campinas (CEMIBUNICAMP; São Paulo, Brazil) were used. The animals were fed a
certified Nuvilab® (Nuvital) diet with free access to tap water under
standard conditions of 12 h dark—12 h light, humidity (60 ± 1.0%)
and temperature (21 ± 1 ◦ C). Fasting was used prior to all assays
because standard drugs or essential oil treatment were always
administered orally (by gavage) or intraduodenally. Moreover, the
animals were kept in cages with raised floors of wide mesh to prevent coprophagy. The experimental protocols were approved by the
Institutional Animal Care and Use Committee (CEEA/IB/UNICAMP,
no. 1537-1).
2.2. Essential oil
The essential oil of Hyptis spicigera (OEH) was purchased from
Laszlo aromaterapia Ltda. Plants were collected in Distrito de
Caatinga (João Pinheiro, MG, Brazil), a Cerrado region. OEH was
isolated from inflorescences, leaves and stems from this specie by
steam distillation. A flowered “voucher” was identified by Jorge
Yoshio Tamashiro of Universidade Estadual de Campinas (UNICAMP) and deposited under the number 150422 at UEC herbarium
(Campinas, SP, Brazil).
2.3. Identification of essential oil constituents
The OEH samples were analyzed in a gas chromatographer coupled to an electronic (70 eV) mass spectrometer (GC–MS, Shimadzu,
GC-2010) equipped with a capillary column of fused silica (DB-5;
5.30 m × 0.32 mm × 0.25 m), helium as carrier gas (1.52 mL/min,
White Martins, 99.9%), injector at 250 ◦ C, detector at 250 ◦ C and split
injection mode. Mass spectrum acquisition was performed at the
mass range from 40 to 600 m/z. The essential oil (10 L) was diluted
in chloroform to produce 1 mL of chromatographic grade solvent,
1 L of which was injected as sample at the split ratio of 1:30.
The column temperature was heated to 60 ◦ C and programmed at
5 ◦ C/min to 220 ◦ C. The identification of substances was realized by
comparing its mass spectra with the GC–MS system database (NIST
62 lib.), the literature and with the Kovats retention indices (Adams,
1995).
2.4. Drugs and chemicals
The following drugs were used: lansoprazole (Medley, Campinas, Brazil), Tween 80® and acetic acid (Sinth, SP, Brazil), absolute
ethanol (© Merk KGaA, Darmstadt, Germany); cimetidine, carbenoxolone, indomethacin, L-NAME (NG -nitro-l-arginine methyl
ester), NEM (N-ethylmaleimide), Alcian Blue and NaCl were from
Sigma Chemical Co. (St. Louis, USA). The chemicals used in the
buffers and other solutions were all of analytical grade. All drugs
and reagents were prepared immediately before use.
2.5. Antiulcerogenic activity
Based on their respective specifications, the groups under each
experimental model included positive (lansoprazole, carbenoxolone, or cimetidine) and negative (vehicle-Tween 80 at 12%)
controls. After each experiment the animals were killed; the stomachs were opened along the greater curvature, pressed onto a glass
plate, and scanned. So that the lesions could be counted aided by
the AVSoft program. The results were expressed as total ulcerated
area (mm2 ). The antiulcerogenic activity of OEH was assessed on
two experimentally induced gastric ulcer models:
2.5.1. Ethanol-induced ulcer
After fasting for 24 h, the experimental groups were submitted to the treatments (p.o.) with vehicle, lansoprazole (30 mg/Kg),
OEH (12.5; 25; 50 or 100 mg/Kg) 1 h before induction of gastric
injury by absolute ethanol. Animals were killed, by CO2 gas, 1 h
after ethanol administration, the stomachs were removed, opened
along the greater curvature and the injuries calculated as described
previously (Morimoto et al., 1991).
2.5.2. NSAID-induced ulcer
Animals were fasted for 24 h. The gastric injuries were induced
by subcutaneous administration of indomethacin 30 mg/Kg in male
rats. The treatments (p.o.) with vehicle, cimetidine (100 mg/Kg),
OEH (100 mg/Kg) were carried out 30 min before administration
of the NSAID. Four hours after the NSAID administration the animals were killed by CO2 gas and the stomachs removed for lesion
quantification (Hayden et al., 1978).
2.6. Evaluation of mucosal protective factors
2.6.1. Evaluation of the gastric juice parameters
Animals were fasted for 24 h with free access to water. One hour
after oral treatment or immediately after intraduodenal administration of OEH (100 mg/Kg), cimetidine (100 mg/Kg) or vehicle,
pylorus ligature was performed (Shay et al., 1945). Four hours later
the animals were sacrificed by CO2 gas, the stomach was removed,
C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
inspected internally, and its contents drained into a graduated centrifuge tube and centrifuged at 2000 × g for 10 min. The supernatant
volume and pH were recorded with a digital pH meter (PA 200,
Marconi S.A., Piracicaba, Brazil).
2.6.2. Determination of mucus adhering to gastric wall
After 24 h of fasting the rats, under anesthesia (50 mg/Kg of
ketamine and 10 mg/Kg of xylazine), were submitted to longitudinal incision slightly below the xiphoid apophysis for the pylorus
ligature. The administration (p.o.) of the vehicle, carbenoxolone
(200 mg/Kg) and OEH (100 mg/Kg) was performed 1 h before the
ligature. Four hours after the ligature, animals were killed by CO2
gas, the glandular portion of the stomach was separated, weighed
and immersed in Alcian Blue solution for the mucus quantification
procedure. The absorbencies were measured in a spectrometer at
598 nm and the results expressed as g of Alcian Blue/g of tissue
(Rafatullah et al., 1990).
2.6.3. Determination of prostaglandin (PGE2 ) levels
Animals were fasted for 24 h and divided randomly into the
groups sham, vehicle + NSAID and OEH + NSAID. First, NSAID was
administered – indomethacin (dissolved in 5% sodium bicarbonate solution) 30 mg/Kg, s.c. – and 30 min after, the animals were
treated (p.o.) with vehicle or OEH (100 mg/Kg). Thirty minutes after
the oral treatment, the rats were killed by CO2 gas, the stomachs
removed, weighed and then placed in 1 mL of sodium phosphate
buffer (10 mM, pH 7.4). The tissue was finely minced and then incubated at 37 ◦ C for 20 min. The prostaglandin E2 level was quantified
with an immune-enzymatic dosage kit from R&D Systems (USA).
The methodology was according to Curtis et al. (1995).
2.6.4. Determination of the role of nitric oxide (NO) and
sulfhydryl compounds (SH) in gastric protection
Male rats were divided into 6 groups and pretreated (i.p.) with
saline, L-NAME (N-nitro-l-arginine methyl ester, 70 mg/Kg) an
inhibitor of the NO synthesis or NEM (N-ethylmaleimide, 10 mg/Kg)
a blocker of SH compounds. Thirty minutes after the pretreatment the animals were administered (p.o.) vehicle, carbenoxolone
(100 mg/Kg) or OEH (100 mg/Kg). After 60 min all the groups
received absolute ethanol (10 mL/Kg) to induce gastric ulcers. One
hour after receiving ethanol the rats were killed by CO2 gas for the
determination of gastric lesions (Arrieta et al., 2003).
2.7. Healing action
149
the essential oil on their individual weights: heart, lungs, liver and
kidneys.
2.7.3. Western Blot assay
Frozen glandular stomachs samples were homogenized in 1 mL
of ice cold buffer (PB 0.1 M, pH 7.4 and protease inhibitor 1%).
Homogenates were centrifuged (12,000 × g, 15 min, 4 ◦ C) and the
supernatants were collected and stored at −80 ◦ C. Protein concentration of the homogenate was determined following Bradford’s
colorimetric method (1976). Then, samples were treated with
Laemmili buffer (PB buffer 0.5 M, pH 6.8; glycerol, sodium dodecyl sulfate (SDS) 10%, bromophenol 0.1%, -mercaptoethanol) in a
1:1 proportion. Equal amounts of protein from samples (100 g)
were separated on 10% acrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the next step, proteins
were electrophoretically transferred onto a nitrocellulose membrane and incubated with specific primary antibodies: EGF (Santa
Cruz Biotechnology, Inc., USA) and COX-2 (Cayman Chemical, USA)
at dilution of 1:500. Each membrane was washed three times for
10 min and incubated with anti-goat immunoglobulin G antibody
(Zymed Laboratories, USA) for EGF and with anti-rabbit (Zymed
Laboratories, USA) for COX-2, both at dilution of 1:5000. To prove
equal loading, the blots were analyzed for -actin expression
using an anti--actin antibody (Sigma–Aldrich, MO, USA). Immunodetection was performed using enhanced chemiluminiscence
light-detecting kit (SuperSignal® West Femto Chemiluminescent
Substrate, Pierce, IL, USA). Densiometric data were performed following normalization to the control (housekeeping gene) by AVSoft
program.
2.8. Histological analysis
After 14 days of treatment in acetic acid-induced gastric ulcer
model, the animals were killed and their stomachs were removed
and opened throughout the great curvature. These samples were
fixed in ALFAC and enclosed in paraplast for histological analyses
by PAS (Vacca, 1985) staining.
2.9. Statistical analysis
Results were expressed as mean ± S.E.M. and statistical significance was determined by one-way analysis of variance followed
by Dunnett’s or Tukey’s test with P < 0.05 defined as significant.
2.7.1. Acetic acid-induced gastric ulcer
On this gastric ulcer induction, a cicatrisation model, rats
were not fasted. Anesthesia (50 mg/Kg of ketamine and 10 mg/Kg
of xylazine) was administered for the application of 100 L of
absolute acetic acid into the subserosal stomach layer of each
animal. Two days after surgery, treatments (p.o.) with vehicle,
cimetidine (100 mg/Kg) and OEH (100 mg/Kg) were administered once daily for 14 consecutive days. The animals were
sacrificed on the 15th day by CO2 gas and then the stomachs were removed for lesion quantification and processed for
Western Blotting and histological analysis (Okabe and Amagase,
2005).
3.2. Antiulcerogenic activity of essential oil (OEH) of Hyptis
spicigera
2.7.2. Toxicity evaluation
The toxicological parameters were set according to the method
of Souza-Brito (1994). Toxicity in the animals submitted to OEH
(100 mg/Kg) treatment, under the cicatrisation model described
above, was evaluated. For a period of 14 days, OEH effects were
observed daily (body weight progression, hair and mucosal alteration). The following organs were weighed to detect any effect of
In the absolute ethanol-induced gastric ulcer model, four doses
of OEH were tested: 12.5; 25; 50 and 100 mg/Kg. OEH provided significant gastric protection at dose of 100 mg/Kg which was chosen
for further studies to clarify the mechanisms underlying its gastroprotective activity. In the NSAID-induced gastric ulcer model, OEH
at the dose of 100 mg/Kg also demonstrated gastric protection. Both
model results are shown in Table 2.
3. Results
3.1. Chemical analysis of the essential oil
Gas chromatography–mass spectrometry (GC–MS) analysis of
OEH indicated fourteen compounds of which the major compounds
are three monoterpenes: alpha-pinene (50.8%), cineole (20.3%) and
beta-pinene (18.3%) (Table 1).
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C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
Peak
Compound
Composition (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Alpha-thujene
Alpha-pinene
sabinene
Beta-pinene
Beta-myrcene
Alpha-phellandrene
Beta-cymene
Cineole
Copaene
Beta-bourbonene
Caryophyllene
Humulene
Germacrene D
Caryophyllene oxide
0.18
50.78
1.10
18.30
0.13
0.30
0.23
20.31
0.24
0.29
7.01
0.38
0.16
0.59
Gastric mucus (μg / g)
Table 1
Chemical composition of the essential oil of Hyptis spicigera.
150
**
**
Carbenoxolone
OEH
100
50
0
Vehicle
Fig. 1. Quantification of adherent mucus in gastric mucosa of rats treated with
essential oil (OEH) from Hyptis spicigera. Results are presented as mean ± S.E.M.
ANOVA followed by Dunnet’s test, **P < 0.01 significantly different from negative
control group treated with vehicle.
Table 2
Effect of OEH under models of gastric ulcer induced by absolute ethanol and NSAID
in rats. Ulcer areas are presented as mean ± S.E.M. ANOVA followed by Dunnet’s test.
Experimental
models
Treatments
(p.o.)
Dose
(mg/Kg)
U.A. (mm2 )
Protection
(%)
Ethanol
Vehicle
Lansoprazole
OEH
–
30
12.5
25
50
100
149.90
43.10
75.96
89.24
74.35
4.28
29.79
5.48**
24.41
13.89
20.84
2.03***
–
71.2
49.3
40.4
50.4
97.1
NSAID
Vehicle
Cimetidine
OEH
–
100
100
20.67 ± 5.07
0.68 ± 0.17***
3.28 ± 1.06***
–
96.7
84.1
**
***
±
±
±
±
±
±
P < 0.01 significantly different from negative control group treated with vehicle.
P < 0.001 significantly different from negative control group treated with vehicle.
Fig. 2. Quantification of PGE2 levels in gastric mucosa of rats treated with essential oil (OEH) from Hyptis spicigera. Results are presented as mean ± S.E.M. ANOVA
followed by Tukey’s test. Different letters (a, b or c) represent intergroup statistical
differences (b and c: P < 0.05; a and b, and a and c: P < 0.001).
3.3. Effect of Hyptis spicigera essential oil (OEH) on gastric juice
parameters
The gastric juice parameters of the rats submitted to the
treatment with the essential oil administered by different routes
(Table 3) demonstrated that neither oral nor the systemic evaluation of the intraduodenal OEH administration altered the gastric
juice pH and volume.
3.5. Effect of Hyptis spicigera essential oil (OEH) on gastric
prostaglandin (PGE2 ) levels
OEH, concomitantly administered with NSAID (indomethacin),
a cyclooxygenase inhibitor, could not maintain PGE2 levels similar to the sham. But, when compared to the vehicle + NSAID, OEH
maintained high PGE2 levels (Fig. 2).
3.4. Effect of Hyptis spicigera essential oil (OEH) on gastric mucus
production
Fig. 1 shows that the animals treated with OEH augmented in
28.3% the amount of mucus adhering to the gastric mucosa, compared to vehicle control group, thus demonstrating a cytoprotective
activity on gastroprotection of OEH at the dose of 100 mg/Kg.
Table 3
Effects of essential oil (OEH) obtained from Hyptis spicigera on gastric juice parameters in rats submitted to pylorus ligature. Data are presented as mean ± S.E.M.
ANOVA followed by Dunnet’s test.
Route
Treatments
Dose
(mg/Kg)
Gastric juice
volume (mL)
pH
(units)
Intraduodenal
Vehicle
Cimetidine
OEH
–
100
100
0.94 ± 0.07
0.49 ± 0.07*
1.02 ± 0.17
1.62 ± 0.07
1.60 ± 0.06
1.41 ± 0.07
Vehicle
Cimetidine
OEH
–
100
100
0.82 ± 0.15
1.68 ± 0.18**
1.02 ± 0.25
2.44 ± 0.18
5.10 ± 0.08***
2.07 ± 0.20
Oral
*
**
***
P < 0.05 significantly different from negative control group treated with vehicle.
P < 0.01 significantly different from negative control group treated with vehicle.
P < 0.001 significantly different from negative control group treated with vehicle.
Table 4
Effect of oral Hyptis spicigera essential oil (OEH) treatment, under the ethanolinduced gastric lesion model, on rats pretreated with L-NAME or NEM. Data are
presented as mean ± S.E.M. ANOVA followed by Dunnet’s test.
Pretreatment
Treatment
Dose
(mg/kg,
p.o.)
Ulcer area (mm2 )
Inhibition
(%)
Saline
Vehicle
Carbenoxolone
OEH
–
100
100
292.50 ± 13.18
13.52 ± 1.76***
14.59 ± 6.58***
–
95.3
95.0
L-NAME (i.p.)
Vehicle
Carbenoxolone
OEH
–
100
100
870.80 ± 48.70
737.90 ± 29.16*
81.30 ± 28.61***
–
15.2
90.6
Saline
Vehicle
Carbenoxolone
OEH
–
100
100
229.70 ± 25.66
15.49 ± 4.97***
13.36 ± 5.52***
–
93.2
94.2
NEM (i.p.)
Vehicle
Carbenoxolone
OEH
–
100
100
364.30 ± 18.46
256.60 ± 21.35*
103.30 ± 33.49***
–
29.5
71.6
*
***
P < 0.05 significantly different from negative control group treated with vehicle.
P < 0.001 significantly different from negative control group treated with vehicle.
C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
151
Ulcer area (mm2)
30
20
10
***
***
***
Cimetidine
OEH
Sham
0
Vehicle
Fig. 3. Effect of oral administration, for 14 consecutive days, of Hyptis spicigera
essential oil (OEH, 100 mg/Kg) on the healing ulcer in rats with chronic ulcer induced
by 0.1 mL of absolute acetic acid. Data are expressed as mean ± S.E.M. ANOVA followed by Dunnet’s test, ***P < 0.001 significantly different from negative control
group treated with vehicle.
3.6. Role of nitric oxide (NO) and sulfhydryl compounds (SH) in
gastric protection in rats treated with essential oil of Hyptis
spicigera (OEH)
Table 4 shows that when the rats were pretreated with L-NAME,
a NO-synthase inhibitor, the OEH continued exerting its gastroprotective effect without the action of NO-synthase, thereby showing
that its activity does not depend on NO. The same was observed
in animals pretreated with NEM, a sulfhydryl (SH) inhibitor. The
essential oil maintained its protective action without SH compounds, demonstrating that the gastroprotective action of OEH
does not depend on SH either.
3.7. Healing action of essential oil of Hyptis spicigera (OEH)
3.7.1. Effect of essential oil of Hyptis spicigera (OEH) on acetic
acid-induced gastric ulcer model
In the acetic acid model, oral treatment with OEH for 14 consecutive days demonstrated that the essential oil of Hyptis spicigera
accelerates the healing of chronic gastric ulcer in rats, as can be seen
in Fig. 3. OEH at the dose of 100 mg/Kg significantly decreased the
main area of the lesion in 87.5% compared to the negative control
group treated with vehicle (P < 0.001).
3.7.2. Effect of Hyptis spicigera essential oil (OEH) treatment on
toxicological parameters
This experimental model can also provide the toxicological
parameters. There was no significant difference in body weight
development (data not shown) or in organ weights (Table 5) for all
groups. No macroscopic abnormalities were detected in the examined organs. Nor was mortality observed in any treatment group
during the 14-day study.
Table 5
Relation organs weight/body weight of rats after oral treatment with vehicle,
cimetidine (100 mg/Kg) or essential oil of Hyptis spicigera (OEH, 100 mg/Kg) for
14 consecutive days. The values were transformed into arc sine. Results are
mean ± S.E.M. ANOVA followed by Dunnet’s test, P > 0.05.
Treatment
Heart
Vehicle
Cimetidine
OEH
Sham
3.23
3.21
3.26
3.95
±
±
±
±
Lung
0.01
0.07
0.03
0.12
5.15
4.73
4.61
4.53
±
±
±
±
0.32
0.13
0.39
0.17
Kidneys
Liver
4.80
4.92
5.11
5.31
10.94
11.27
11.16
10.37
±
±
±
±
0.09
0.01
0.03
0.05
±
±
±
±
0.06
0.33
0.19
0.12
Fig. 4. Effects of oral administration during 14 days of essential oil of Hyptis spicigera (OEH) on cycloxygenase-2 (COX-2) (A) and epidermal growth factor (EGF) (B)
expression in gastric mucosa of rats submitted to gastric ulcer induced by acetic
acid. Densitometry was made following normalization to the control (housekeeping gene). Data are expressed as mean ± S.E.M. ANOVA followed by Dunnet’s test,
**P < 0.01 and ***P < 0.001 significantly different from Sham.
3.7.3. Effect of essential oil of Hyptis spicigera (OEH) on gastric
expression of COX-2 and EGF in acetic acid-induced gastric ulcer
model
Analysis of Western Blotting revealed that oral treatment with
OEH for 14 consecutive days after gastric exposure to absolute
acetic acid caused high levels of COX-2 and EGF (Fig. 4A and B
respectively) in the gastric mucosa of rats submitted to the chronic
ulcer model. OEH treatment augmented the enzyme COX-2 expression by 75% and by 115% the EGF expression, both compared to
Sham group.
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C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
Fig. 5. Histological slices of the stomachs from rats submitted to acetic acid induced gastric ulcer method. PAS staining. Note the large secretion of mucus in treatment group
(arrows). (A) vehicle group; (B) cimetidine (100 mg/Kg); (C) OEH (100 mg/Kg).
3.8. Histological analysis
Fig. 5 shows, by PAS method, that animals treated with OEH
at the dose of 100 mg/Kg during 14 consecutive days augmented
the amount of mucus production demonstrated by more positive
stained cells, compared to vehicle control group. This figure also
shows the mucosal integrity of treated animals. This data corroborates the effect of OEH on gastric mucus production showed in
Fig. 1.
4. Discussion
Aromatic plants have been used since ancient times for
their preservative and medicinal properties which are partially
attributed to essential oils (Edris, 2007). Essential oils are volatile,
natural, complex compounds characterized by a strong odor. Since
the middle ages, it has been widely used for the bactericidal,
virucidal, fungicidal, antiparasitical, insecticidal, medicinal and
cosmetic applications, especially nowadays in pharmaceutical, sanitary, cosmetic, agricultural and food industries (Bakkali et al.,
2008). Early reports indicated that essential oil components, especially monoterpenes, have multiple pharmacological effects (Edris,
2007).
Major components identified in the essential oil of Hyptis spicigera are alpha-pinene (50.8%), cineole (20.3%) and beta-pinene
(18.3%) (Table 1). This composition is very similar to that pointed
in a symposium: alpha-pinene (26.4%), cineole (21.5%), beta-pinene
(13,8%) and bicyclogermacrene (18.3%) (Maia and Andrade, 2009).
Generally, the major components are found to reflect quite well the
biophysical and biological features of the essential oil from which
they were isolated, the amplitude of their effects being just dependent on their concentration when they were tested individually.
However, it is possible that the activity of the main component
is modulated by other minor components, with synergy between
the compounds. In that sense, for biological purposes, it is more
informative to study the entire oil rather than some of its components because the concept of synergism appears to be meaningful
(Bakkali et al., 2008). Therefore, scientific research is generally
related to the whole compounds rather than the isolated compound
(Mercier et al., 2009).
Alpha-pinene, the major component of OEH, has been credited
with a series of pharmacological properties when alone or in synergy with other pinenes that include anti-inflammatory (Neves
et al., 2010), lipophilic, bactericidal, fungicidal, insecticidal, pesticidal, anticarcinogenic, diuretic, antioxidant, immunostimulant,
anti-convulsive, sedative, anti-stress, hypoglycaemic, capable of
expelling xenobiotics and anticholinesterase activity. The effects
of ␣-pinenes vary depending on the composition of monoterpenes
and sesquiterpenes (Mercier et al., 2009).
According to literature reports, -pinenes generally accompany ␣-pinenes in smaller quantities in the volatile extracts,
essential oleoresins and oils. Some specific studies show that
-pinenes, along with ␣-pinenes and other terpenes, are cytotoxic, lipophilic, bactericidal, fungicidal, insecticidal, acting against
osteoclasts, anticarcinogenic, pesticidal, antioxidant and sedative.
When ␣- and -pinenes are the major constituents of an essential oil, they warrant the anti-inflammatory and analgesic activity.
When administered alone, -pinene exhibit moderate antimicrobial activity (Mercier et al., 2009).
Cineole (1,8-cineole) also known as eucalyptol or cajeputol is a
terpene oxide present in many essential oils and is often employed
by the pharmaceutical industry in drug formulations as a percutaneous penetration enhancer and decongestant (Levison et al.,
1994). It has been systemically shown that cineole exerts antiinflammatory, analgesic, gastroprotective and hepatoprotective
effects (Santos et al., 2004), moderate antioxidant activity (MitićĆulafić et al., 2009), used for the treatment of bronchitis, sinusitis
and rheumatism (Santos and Rao, 2000) and has antimicrobial
activity alone, but not with the same efficacy when compared to the
entire essential oil (Hendry et al., 2009). Besides, it does not have
mutagenic or genotoxic effects, indicating its safety (Mitić-Ćulafić
et al., 2009).
The functional integrity of gastric mucosa depends on a balance
between aggressive factors and protective mechanisms. Thus, the
success of gastric pharmacological treatment relies not only on the
blockage of acid secretion, but also on augmentation of the protective factors of the gastric mucosa (Dajani and Klamut, 2000).
The mucosal protective agents consist of three functional factors:
mucus secretion, microcirculation and motility (Ueki et al., 1998);
two humoral factors: prostaglandins and nitric oxide (Whittle
et al., 1990); as well as neuronal sensitivity to capsaicin (Holzer,
1998). This ability of certain endogenous factors to protect the
gastric mucosa against damage to the gastric epithelium through
mechanisms not related to acid secretion inhibition was first
denominated “cytoprotection” and then characterized as “gastroprotection” (Szabo and Goldberg, 1990; Martin and Wallace, 2006).
The genesis of ethanol-induced gastric lesions has a multifactorial origin that includes oxidative stress, DNA damage
and a decrease in total glutathione content in gastric mucosal
cells as some of the involved factors, producing necrosis and
hemorrhage-like gastric tissue (La Casa et al., 2000). Furthermore,
the ulcerogenic activity of ethanol is driven by its capacity to
dissolve the constituent gastric mucus while concomitantly diminishing the transmucosal action potential, thus increasing the flow of
Na+ and H+ in the lumen and stimulating the secretion of histamine,
pepsin and H+ ions (Szabo and Brown, 1987). Considering that the
OEH (100 mg/Kg) exerted 97.1% of protection of the gastric mucosa,
it is undeniable that this substance exerts substantial protective
action on the gastric mucosa (Table 2). This result also indicates
a possible cytoprotective activity, since ethanol acts directly on
gastric mucosal cells. In addition, it is well known that ethanolinduced gastric ulcers are not inhibited by antisecretory agents
C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
such as cimetidine. However, agents that enhance mucosal defensive factors including prostaglandin E2 inhibit ethanol-induced
ulcers (Toma et al., 2002).
The aggressive properties of non-steroidal anti-inflammatory
drugs (NSAID) in the gastrointestinal tract continue to be the greatest impediment of their use in the treatment of inflammatory
illnesses such as rheumatoid arthritis (Avila et al., 1996). The inhibition of prostaglandin synthesis is known to be the main ulcerogenic
mechanism of the NSAID, besides provoking damage to the vascular
endothelium, reduction of the blood flow, formation of obstructive micro-thrombi and activation of neutrophils (Guth, 1992). In
the experimental induction model of gastric ulcers by a NSAID, the
essential oil of Hyptis spicigera (OEH) presented gastroprotection at
the dose of 100 mg/Kg (Table 2).
Given that the ulcerogenic properties of NSAID and ethanol are
due to the fact that they diminish the protective factors of the
mucosa such as prostaglandin and mucus (Konturek et al., 2005), it
can be affirmed that the antiulcerogenic activity of OEH observed in
these models may be attributed to these mucosal protective factors.
Some pharmacological agents that inhibit the H+ /K+ -ATPase
receptor, including histaminergic and cholinergic antagonists, act
in an antiulcer manner to reduce acid secretion in the stomach
(Aihara et al., 2003). Considering this fact, the present work evaluated the antisecretory action of the OEH for local (p.o.) or systemic
action (i.d.) though the pylorus ligature model. In both experiments
OEH did not show modifications in gastric acid juice parameters.
These results indicate therefore that the OEH does not exert antiulcerogenic action in an antisecretory manner (Table 3). This result
is significant to the ongoing search for an antiulcerogenic therapy
since the long term use of proton-pump inhibitors and H2 blockers can provoke serious side effects including hypergastrinemia by
means of augmented pH in the gastric lumen (Orlando et al., 2007).
Gastric mucus is the first line of defense against acid and adheres
together with bicarbonate secreted by the epithelium to serve as
a barrier against self-digestion (Allen and Flemströn, 2005). The
results obtained in the present work show a significant increase
(28.3%) in the amount of adherent mucus in the animals treated
with OEH thus justifying the previously observed gastroprotective
action (Figs. 1 and 5). Mucus is a viscous, elastic, adherent and transparent gel composed by 95% water and 5% glycoprotein. It is also
an important protective factor for the gastric mucosa because of
glycoprotein which can act as antioxidant, reducing damage in the
mucosa provoked by free radicals (Repetto and Llesuy, 2002).
Among the existing humoral factors in the mucosa, the
prostaglandin PGE2 plays an important role in protecting it by stimulating the secretion of mucus and bicarbonate, maintaining the
local blood flow and increasing the resistance of epithelial cells
against potential damage by cytotoxins (Hawkey and Rampton,
1985). Fig. 2 demonstrates that even with the administration of
a non-selective COX inhibitor (indomethacin), which consequently
caused a decrease in PGE2 levels, OEH were able to increase gastric
mucosa PGE2 at levels above those found in vehicle group which
was also treated with indomethacin. Although the augmented levels of PGE2 in rats treated with OEH when compared to vehicle
group, OEH was not able to maintain PGE2 at basal levels similar to
those found in normal rats. Thus we cannot assert that OEH is capable of stimulating PGE2 synthesis, but it can maintain at sufficient
levels to protect gastric mucosa against injuries. Considering that
gastric mucus synthesis is controlled by PGE2 , the action of OEH on
PGE2 levels explains the fact that this treatment augmented gastric mucus secretion by increasing the gastric mucosal protection,
confirming the gastroprotective action promoted by the OEH.
It was demonstrated that ulcer induction by ethanol is associated with reduced levels of SH compounds, especially intracellular
glutathione (GSH). In light of this, the present study evaluated the
role of SH compounds in the gastric protection promoted by OEH.
153
SH limits the production of free radicals, thus protecting the cell
(La Casa et al., 2000). On that basis, the animals were pretreated
with NEM, an SH inhibitor, to evaluate the interference of this protection mechanism in the OEH action. We can conclude that OEH
gastroprotective activity does not dependent on these compounds,
because OEH maintained the gastric protection in these animals
pretreated with ethanol instead of SH inhibition (Table 4). Besides
SH, another substance involved in the gastroprotection is nitric
oxide (NO), which is synthesized by NO-synthase (NOs) and plays
an important role in modulating the defense of gastric mucosa by
regulating mucus secretion (Brown et al., 1993), enhancing blood
flow (Wallace and Miller, 2000) and inhibiting neutrophil aggregation (Wallace et al., 1997). The evaluation of NO participation in the
gastroprotection promoted by OEH demonstrated that despite the
inhibition of NO by the action of the L-NAME-blocking NOs, OEH
continued exerting its effect (Table 4). Thus, it can be concluded
that OEH’s protective mechanism is not related to NO synthesis.
Historically, our understanding of the pathophysiology of peptic
ulcer disease focused on abnormalities in the secretion of gastric
acid and pepsin, and on the suppression of acid (e.g. H2 receptor
antagonists, proto-pump inhibitors) as a treatment strategy (Yuan
et al., 2006). Although suppressors of acid secretion have been
a mainstay to the promotion of ulcer healing for three decades,
there has been an increasing interest in recent years in the mechanisms through which ulcers heal, and the possibility that both
the speed and quality of healing may be pharmacologically modulated (Wallace, 2008). The so-called acetic acid ulcer model has
been developed to examine the healing process of peptic ulcers.
This model highly resembles human ulcers in both pathological
features and healing mechanisms since they are difficult to treat
and require a long time to heal (Okabe and Amagase, 2005). OEH
demonstrated to be able in accelerating the healing of chronic gastric ulcer in rats, showing a strong cicatrisation activity (Fig. 3).
Through this experimental model we can also state that OEH does
not present toxicological action at the dose of 100 mg/Kg in the
analyzed parameters, as can be observed in Table 5.
Ulcer healing, a genetically programmed repair process,
includes inflammation, cell proliferation, reepithelialization, formation of granulation tissue, angiogenesis, interactions between
various cells and the matrix and tissue remodeling, all resulting in
scar formation. The capacity to accelerate the ulcer healing process depends on many factors, like the epidermal growth factor
(EGF), fibroblast growth factor (bFGF), vascular endothelial growth
factor (vEGF), trefoil peptides and COX-2 in a well synchronized
spatial and temporal manner (Tarnawski, 2005). On what regards
the COX-2 and EGF expression, Western Blotting analysis in the
present study shows a great quantity of both in the gastric mucosa
of animals treated with OEH—COX-2 expression augmented 1.75
folds and EGF 2.15 folds, both in comparison to sham group (Fig. 4A
and B, respectively).
COX-2 plays an important role in the healing of gastric ulcers
whereas its inhibition delays ulcer healing (Peskar, 2005). At the
site of ulceration COX-2 appears to be the primary contributor to
prostaglandin synthesis and represents the second line of defense,
which is activated during ulcer healing to compensate the temporary loss of COX-1 occurring in the mucosa adjacent to the ulcer
and assisting COX-1 in safeguarding gastric mucosal integrity. Its
expression is up-regulated by various growth factors and cytokines
(Halter et al., 2001).
Growth factors and their receptors also play important roles in
cell proliferation and migration, repair of the tissue injury and ulcer
healing. The major growth factor receptor expressed in gastric progenitor cells, which controls cell proliferation, is epidermal growth
factor receptor (EGF-R) (Laine et al., 2008). Several authors associate the antiulcerogenic process with healing of chronic ulcers and
the participation of EGF (Konturek et al., 1992). The great increase
154
C. Takayama et al. / Journal of Ethnopharmacology 135 (2011) 147–155
of COX-2 (75%) and EGF (115%) expressions in restoring the gastric mucosa, in rats treated with OEH, indicates a strong healing
capacity of this essential oil.
Besides the popular use for treating muscular pain, luxation and
gastric disorders, the essential oil from Hyptis spicigera presents
substantial antiulcerogenic, gastroprotective and healing actions
that can be regarded as a promising target for the development of
a new drug for the prevention of gastric ulcer.
5. Conclusions
According to the results of this study we can conclude that
the antiulcerogenic and gastroprotective actions promoted by the
essential oil of Hyptis spicigera (OEH) are due to an increase in
the gastric production of mucus related to PGE2 in gastric mucosa.
These results indicate that OEH constitute an interesting adjuvant
to NSAID in the treatment of chronic inflammatory illnesses, with
the prospect of annulling the aggressive gastric effect of these drugs
on gastric mucosa without promoting alterations in physiological
functions of the stomach. Besides, the OEH demonstrated a strong
cicatrisation action modulated by the augmented expression of
COX-2 (75%) and EGF (115%) in the gastric mucosa.
Conflicts of interest
There is no conflict of interest.
Acknowledgements
We are grateful to Adriano Galvão de Carvalho, from Raizando
Óleos Essenciais, for donating Hyptis spicigera “voucher”. This work
was supported by CAPES (Coordenação de Aperfeiçoamento Pessoal
de Nível Superior) and FAPESP (Fundação de Amparo a Pesquisa do
Estado de São Paulo).
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