Gastroprotective and ulcer healing effects of essential oil from Hyptis spicigera Lam. (Lamiaceae)

Journal of Ethnopharmacology 135 (2011) 147–155

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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: [email protected] (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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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 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). 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

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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. 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). 3.2. Antiulcerogenic activity of essential oil (OEH) of Hyptis spicigera 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.

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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 )

Ethanol

Vehicle Lansoprazole OEH

– 30 12.5 25 50 100

149.90 43.10 75.96 89.24 74.35 4.28

NSAID

Vehicle Cimetidine OEH

– 100 100

** ***

± ± ± ± ± ±

Protection (%)

29.79 5.48** 24.41 13.89 20.84 2.03***

– 71.2 49.3 40.4 50.4 97.1

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

Kidneys ± ± ± ±

0.32 0.13 0.39 0.17

4.80 4.92 5.11 5.31

± ± ± ±

0.09 0.01 0.03 0.05

Liver 10.94 11.27 11.16 10.37

± ± ± ±

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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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 (Mitic´ Culafi c´ 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 ´ ´ Culafi mutagenic or genotoxic effects, indicating its safety (Miticc´ 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

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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.

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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

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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 (Coordenac¸ão de Aperfeic¸oamento Pessoal de Nível Superior) and FAPESP (Fundac¸ão de Amparo a Pesquisa do Estado de São Paulo). References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publish Corporation, Carol Stream. Adorjan, B., Buchbauer, G., 2010. Biological properties of essential oils: an updated review. Flavour and Fragrance Journal 25, 407–426. Aihara, T., Nakamura, E., Amagase, K., Tomita, K., Fujishika, T., Furutani, K., Okabe, S., 2003. Pharmacological control of gastric acid secretion for the treatment of acidrelated peptic disease: past, present, and future. Pharmacology and Therapeutics 98, 109–127. Allen, A., Flemströn, G., 2005. Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin. American Journal of Physiology, Cell Physiology 288, 1–19. Arrieta, J., Benitez, J., Flores, E., Castilho, C., Navarrete, A., 2003. Purification of gastroprotective triterpenoids from stem bark of Amphipterygium adstringens; roles of prostaglandins, sulphidryls, nitric oxide and capsaicin neurons. Planta Medica 69, 905–909. Avila, J.R., de La Lastra, C.A., Martin, M.J., Motilva, V., Luque, I., Delgado, D., Esteban, J., Herrerias, J., 1996. Role of endogenous sulphydryls and neutrophil infiltration in the pathogenesis of gastric mucosal injury induced by piroxicam in rats. Inflammation Research 45, 83–88. Bakkali, F., Averbeck, S., Averbeck, D., Waomar, M., 2008. Biological effects of essential oils—a review. Food and Chemical Toxicology 46, 446–475. Barbosa, P.P.P., Ramos, C.P., 1992. Studies on the antiulcerogenic activity of the essential oil of Hyptis mutabilis Briq. in rats. Phytotherapy Research 6, 114–115. ˜ Bourdy, G., DeWalt, S.J., Chávez de Michel, L.R., Roca, A., Deharo, E., Munoz, V., Balderrama, L., Quenevo, C., Gimenez, A., 2000. Medicinal plants uses of the Tacana, an Amazonian Bolivian ethnic group. Journal of Ethnopharmacology 70, 87–109. Bradford, M.M, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Brown, J.F., Keats, A.C., Hanson, P.J., Whittle, B.J., 1993. Nitric oxide generators and cGMP stimulate mucus secretion by rat gastric mucosal cells. American Journal of Physiology 265, 418–422. Corrêa, M.P., 1931. Dicionário das plantas úteis e das tóxicas cultivadas. Imprensa Nacional, Rio de Janeiro/RJ, Brasil. Curtis, G.H., ManNaughton, W.K., Gall, D.G., Wallace, J.L., 1995. Intraluminal pH modulates gastric prostaglandin synthesis. Canadian Journal of Physiology and Pharmacology 73, 130–134.

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