Caracterizar os constituintes químicos do óleo essencial das folhas de Hyptis martiusii;
Avaliar a atividade antiulcerogênica, in vivo, do óleo essencial das folhas de Hyptis
martiusii, através de modelos agudos de úlcera induzidos por etanol; HCl/etanol e indometacina;
Investigar a atividade antissecretória ácida gástrica basal do óleo essencial das folhas de Hyptis martiusii, pelo método da ligadura pilórica;
Avaliar as alterações na motilidade gastrointestinal utilizando os métodos de esvaziamento gástrico e trânsito intestinal.
4. Artigo
Antiulcerogenic activity of the essential oil of Hyptis martiusii Benth. (Lamiaceae)
Germana Freire Rocha Caldasa, Juciene Bezerra Rodrigues da Silvaa, Rafaella Farias da Nóbregab, Iggor Macêdo do Amaral Costab, Fabíola Fernandes Galvão Rodriguesc, José
Galberto Martins da Costac, Almir Gonçalves Wanderleya,b*
aDepartment of Pharmaceutical Sciences, Federal University of Pernambuco,
Zip Code 50740-521, Recife - PE, Brazil
bDepartment of Physiology and Pharmacology, Federal University of Pernambuco,
Zip Code 50670-901, Recife - PE, Brazil
cLaboratory of Research in Natural Products, Department of Biological Chemistry, Regional
University of Cariri, Zip Code 63105-100, Crato - CE, Brazil
*Corresponding author at: Department of Physiology and Pharmacology, CCB, University Federal of Pernambuco, Av. Prof. Moraes Rego, s/n, CEP 50670-901 – Cidade Universitária, Recife, PE, Brazil – Tel.: +55 81 21268530; fax.: +55 81 21268976. E-mail address: almirgw@globo.com.br (A. G. Wanderley).
ABSTRACT
Ethnopharmacology relevance: Hyptis martiusii (Lamiaceae) grows in abundance in the northeastern Brazil where is popularly known as cidreira-do-mato or cidreira-brava. Its leaves and roots are traditionally used in folk medicine in the treatment of intestinal and stomachic diseases. Aim of the study: To characterize the chemical constituents, to evaluate the antiulcerogenic activity of essential oil from leaves of Hyptis martiusii (EOHM) in models of experimental ulcers in vivo in rodents. Materials and Methods: Were used models of acute gastric ulcer induced by oral administration of indomethacin, absolute ethanol and acidified ethanol. The volume, pH and total acidity of gastric secretion were determined by the pyloric ligature method and the gastrointestinal motility using gastric emptying and intestinal transit methods. Results: Chemical analysis revealed the presence of 24 constituents (mono and sesquiterpenes) representing 92% of essential oil. It was observed that EOHM showed significant dose-dependent gastroprotective effect in models of acute injury induced by absolute ethanol, acidified ethanol and indomethacin. In pylorus ligature, EOHM increased the pH and decreased the gastric total acidity only with the highest doses, however not changed the volume of gastric juice. The EOHM reduced the rate of gastric emptying with only the highest doses, but did not show influence on intestinal transit in neither of three doses. Conclusion: Thus, it is concluded that the essential oil of Hyptis martiusii has gastroprotective effect by a mechanism of action not yet unclear and that which probably this effect observed may be related to the presence of the terpenes.
1. Introduction
Peptic ulcer are lesions in the gastrointestinal tract that usually occur in the stomach and duodenum characterized by mucosal damage secondary to the aggressive action of pepsin and gastric acid secretion. The disease affects millions of people around the world, only in the United States approximately 500,000 people develop peptic ulcers each year. The direct costs or indirect care of the disease are estimated at about $ 10 billion (Ramakrishnan and Salinas, 2007).
Currently, it is known that several factors are involved in its appearance, which include Helicobacter pylori infection, chronic use of nonsteroidal anti-inflammatory drug (NSAID), use of other drugs such as corticosteroids, bisphosphonates, anticoagulants and chemotherapy, ischemia gastric mucosa, age, genetic factors and until the lifestyle that includes stress, alcohol abuse, smoking and dietary habits (Dembinski et al., 2005; Jainu and Devi, 2006; Stewart and Ackroyd, 2008).
The treatment of peptic ulcer disease in general is based on inhibition of gastric acid secretion by histaminergic H2-receptor antagonists, proton pump inhibitors, antimuscarinics, as well as acid-independent therapy provided by antimicrobials against H. pylori, sucralfate and bismuth (Bighetti et al., 2005). However, prolonged use of these drugs also produce adverse reactions (Chan and Leung, 2002), may also increase the risk of developing cancer (Raghunath et al., 2005).
Among the various sources of new pharmacologically active molecules, medicinal plants are considered an important source of molecules for the treatment of gastric ulcer (Schmeda-Hirschmann and Yesilada, 2005). The chemical composition of the family Lamiaceae, especially the genus Hyptis is remarkable variability, are present compounds such as terpenes, flavonoids, lactones, lignans, phenolic derivatives, steroids and others (Falcão and Menezes, 2003).
Hyptis martiusii Benth. is a small shrub that belongs to the Lamiaceae family, commonly found in the north, southeast and northeastern Brazil (Almeida and Albuquerque, 2002). This species grows in abundance in the northeastern where is popularly known as cidreira-do-mato or cidreira-brava (Araújo et al., 2003). According to ethnopharmacological studies, the relevant parts of this plant used for medicinal purposes by the population are the leaves and roots. The infusion or decoction of the leaves from Hyptis martiusii is used against intestinal diseases and stomachic, while the roots in decoction are used for inflammation of
the ovary (Agra et al., 2008); however, there are no pharmacological data to prove these indications.
Antimicrobial, antitumoral, cytotoxic and insecticidal activities were identified (Araújo et al., 2006; Araújo et al., 2003; Costa-Lotufo et al., 2004; Costa et al., 2005; Coutinho et al., 2008), but no antiulcerogenic activity has been reported according to a literature survey. The present work evaluated the antiulcerogenic activity of essential oil from leaves of Hyptis martiusii using models of acute ulcer induced by indomethacin, ethanol and acidified ethanol. Additionally were determined the parameters (volume, pH and total acidity) of gastric secretion by the method of pyloric ligature and gastrointestinal motility in rodents.
2. Materials and methods
2.1. Plant material and extraction of essential oil
Leaves of Hyptis martiusii Benth. (Lamiaceae) were colleted from Araripe Plateau, in the county of Crato, Ceará, Brazil. The botanical identification was performed by Edson P. Nunes and a voucher specimen deposited at Prisco Bezerra Herbarium, from the Department of Biology, Federal University of Ceará (UFC), under the registration no. 43038. The essential oil was extracted from the dried leaves (700 g) of Hyptis martiusii using steam distillation for 3 h. The mixture water/oil was collected in Clevenger type apparatus modified (Gottlieb and Magalhães, 1960), dried over anhydrous sodium sulfate and filtered. The essential oil obtained was stored amber bottle at 5°C until the accomplishment of the pharmacological experiments and phytochemical analyses.
2.2. Gas chromatography coupled to a mass spectrometry analysis
Oil analysis was performed in a gas chromatographer coupled to a mass spectrometer (GC-MS, SHIMADZU QP5050A) equipped with a DB-5HT capillary column (30 m x 0.251 mm, 0.1 µm film thickness) the following specifications: helium as carrier gas at 1.7 mL/min); injector temperature 270 ºC and detector temperature 290 ºC; linear velocity of 47.3 cm/sec; flow carrier of 24 mL/min; pressure of 107.8 kPa; column temperature 60 ºC (2 min) - 180 ºC (1 min) at 4 ºC/min, then 180 - 260 ºC a 10 ºC/min (10 min). The mass spectrometer was operated using 70 eV of ionization energy. Identification of individual components of the
essential oil was based in the interpretation on their mass spectral fragmentation using standards of computer library MS searches (Wiley 229), retention indices and comparison with mass spectral database and literature data (Adams, 2001; Alencar et al., 1990).
2.3. Animals
Male and female Wistar rats (Rattus norvegicus var. albinus) weighing between 180- 200 g and 150-180 g respectively, were utilized for antiulcer experiments and male Swiss mice (35-40 g) were utilized for toxicity experiment. The animals were obtained from the Department of Physiology and Pharmacology from Federal University of Pernambuco (UFPE) and Research Center Aggeu Magalhães (CPqAM/Fiocruz/UFPE), Pernambuco, Brazil. They were maintained under standard environmental conditions (12 h dark/light cycle) and temperature (22 ± 2 °C). Water and industrialized dry food (Labina®, Purina, Brazil) were available ad libitum. All the experimentals protocols were submitted and approved by the Animal Experimentation Ethics Committee of the Biological Science Center - UFPE, under license no. 007764/2009-94 in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
2.4. Acute toxicity
Acute toxicity studies were performed on male Swiss mice as described by Souza Brito (1994). The animals were randomly divided into two groups (n = 5/group) and deprived of feed for 12 h with access to water ad libitum. The treated group received EOHM at a single oral dose of 5.0 g/kg and the control group received distilled water (10 mL/kg). The observations were performed at 30, 60, 120, 180 and 240 min after the oral treatments and daily for 14 days after of treatment. Were recorded daily behavioral changes, clinical sings of toxicity and mortality, the body weight, consumption of food and water based on Hippocratic screening (Malone and Robichaud, 1977).
2.5. Antiulcerogenic activity
Each experimental model presented two control groups, positive (pantoprazole, 40 mg/kg) and negative (vehicle - 1% Tween 80® aqueous solution), and a group treated with
essential oil of Hyptis martiusii (EOHM) at doses of 100, 200 and 400 mg/kg. For the experimental use the essential oil was emulsified in Tween 80 at 1% before administration to the animals. The animals were kept in metabolic cages to prevent coprophagy and had with free access to water prior in the experiments. The period in which the animals were fasted before the experiment varied according to the methodology used in each protocol. In all experiments, the animals were killed with thiopental (140 mg/kg, i.p.). The stomachs were removed, opened along the greater curvature, the gastric contents unvalued and stomach mucosa gently washed with 0.9 % saline solution to evaluation of the lesions, with the exception of pyloric ligature and gastric emptying protocols, where the gastric contents was the subject of study.
2.5.1. Ethanol-induced gastric ulcer
After 16 h fasted, male Wistar rats (n = 6) were orally pretreated with the EOHM (100, 200 e 400 mg/kg), vehicle (Tween 80 at 1%, 1 mL/100 g) and pantoprazole (40 mg/kg), one hour before administration of ulcerogenic agent. Gastric lesions were induced with ethanol (70%, 0.5 mL/100 g body wt., p.o.) according to the method described by Robert et al. (1979). The animals were killed one hour after induction of gastric lesions, the stomachs excised and examined for quantification of the lesions. The gastric lesion area was measured by planimetry (mm²) in relation to total area of gastric corpus. The percentage of inhibition was calculated as described by Nguelefack et al. (2005).
2.5.2. HCl/ethanol-induced ulcer
The experiment was performed according to the method of Mizui and Douteuchi. (1983) with modifications. After 24 h of fasting, male Wistar rats (n = 6) were orally pretreated with the EOHM (100, 200 e 400 mg/kg), vehicle (Tween 80 at 1%, 1 mL/100 g) and pantoprazole (40 mg/kg). One hour after treatment, all the animals received orally 0,3 M HCl/etanol 60% solution (1 mL/150 g body wt., p.o.) to induce acute gastric lesions. The animals were killed one hour later, their stomachs were removed and examined for quantification of the lesions using the parameters previously described.
2.5.3. Indomethacin-induced gastric ulcer
After fasted 16 h, male Wistar rats (n = 6) were orally treated with the EOHM (100, 200 and 400 mg/kg), vehicle (Tween 80 at 1%, 1 mL/100 g) and pantoprazole (40 mg/kg). Gastric lesions were induced in rats by administration indomethacin (30 mg/kg, s.c.) according to the method described by Djahanguri (1969). The animals were killed 6 h after indomethacin injection, their stomachs removed and inspected under magnification to determine the gastric lesions produced. The results were expressed as lesions, ulcers and total index, which were obtained from scores determined by various alterations in the gastric mucosa, considering the color, edema and hemorrhage, loss of mucus, petechiae or damage of the mucosa folds, the number and size of lesions necro-hemorrhagic (Vela et al., 1997).
2.5.4. Evaluation of the gastric secretion parameters- Pyloric Ligature
This assay was used the method described by Visscher et al. (1954) with modifications. A total thirty-five female rats was randomly divided into five groups (n = 7) and fasted for 16 hours with free access to 5% glucose solution. For pyloric ligation the animals were anaesthetized (xilazine - 5 mg/kgand ketamine 60 mg/kg, i.p.), the abdomen was opened and the pyloric ligature, immediately after ligature, were administered intraduodenally the EOHM (100, 200 e 400 mg/kg), vehicle (Tween 80 at 1%, 1 mL/100 g) and pantoprazole (40 mg/kg). Four hours later, the animals were killed, the abdomen opened and their stomachs removed, the gastric juice collected and centrifuged at 176 × g for 30 min. The final volume (mL) was determined after washing the mucosal side of the stomach with 2 mL of distilled water, and pH values measured in digital pHmeter. Total acidity of gastric secretion was determined was titrated to pH 7.0 with 0.1 N NaOH using phenolphthalein 2% as indicator. The total concentration of acid was expressed as mEq [H+]/mL/4h.
2.6. Evaluation of gastrointestinal motility
2.6.1. Gastric emptying
The experiment was carried out according to the method of Gupta and Brans (1978) with some modifications. After 6 h of fasting, male Wistar rats (n = 6) were orally pretreated
with the EOHM (100, 200 e 400 mg/kg), vehicle (Tween 80 at 1%, 1 mL/100 g) and atropine subcutaneously (3 mg/kg), which was used as positive control for the test. After 1 hour or 30 min the administration of the treatments, each animal received orally 1.5 mL of phenol red (0.5 mg/mL). The zero time control group was killed immediately after the administration of marker and the others groups were killed 30 min later. The stomachs were removed, opened by the greater curvature and washed with 7 mL of distilled water. The gastric content collected was centrifuged at 176 × g for 15 min. After centrifugation, 1 mL aliquots of supernatants were added to 1 mL of 1 N NaOH and the absorbance of the solution read in spectrophotometer at 560 nm. The results were plotted in a standard curve of phenol red and expressed as the concentration (µg) of dye retained in the stomach in relation the control group.
2.6.2. Intestinal transit
After removal of the stomach of the rats in model of gastric emptying, small intestine was removed for the evaluation of intestinal transit. With the aid of a ruler, measured the total length of the small intestine of each animal (distance between the region gastropiloric to the ileocecal junction) and the distance traveled by the phenol red (until the last portion of the intestine that contained at least 1 cm continuous marker). The results of these measures were expressed as a percentage of the distance traveled by the marker in relation to the total length of small intestine (Stickney and Northup, 1959).
2.7. Statistical analysis
Results were expressed as mean ± standard error of media (S.E.M). Differences between means were determined by one way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test. The analysis was performed using the program for Windows, GraphPad Prism® version 5.0. The level of significance to reject the null hypothesis was set at 5% (p < 0.05).
3. Results
3.1 Chemical analysis
The yield of essential oil from dried leaves of Hyptis martiusii was 1.3%. Chemical analysis by GC coupled to a MS identified 24 constituents representing 92.13% of EOHM and indicated the existence of mono and sesquiterpenes. The Figure 1 represent the chromatographic profile of essential oil. The major components of the oil were identified as bicyclogermacrene (10.60%), trans-caryophyllene (9.21%), caryophyllene oxide (7.47%), 1,8- cineole (7.01%), δ-3-carene (6.88%) and ledene (5.41). Table 1 shows the constituents identified, percentage composition and their retention index (RI) values.
3.2 Acute toxicity
The acute toxicity in mice of the essential oil from Hyptis martiusii obtained from dried leaves was investigated. It was observed that the EOHM has induced depression of the central nervous system (sedation) in the animals during a period of until 4 h after administration the oil. However, the results indicated that single administration of EOHM by the oral route at dose of 5.0 g/kg did not produce sign of acute toxicity or death in the treated animals. No significant changes in intake of food and water or body weight in mice were observed during the 14 days of observation (data not shown). Therefore, the LD50 could not be estimated, and it is possibly higher than 5.0 g/kg.
3.3 Ethanol-induced gastric ulcer
The administration of EOHM inhibited the formation of ulcerative lesions induced by absolute ethanol. In animals pretreated with EOHM at doses of 100, 200 and 400 mg/kg, significantly reduced the lesion area to 175.8 ± 32.71, 93.20 ± 44.91 and 28.00 ± 10.73 mm2 as compared to the control group (332.5 ± 51.36 mm2), with a percentage of inhibition of 47.13, 65.15 and 89.53 % respectively. The animals that received pantoprazole (40 mg/kg, p.o.) had the area of gastric mucosal injury reduced to 93.50 ± 26.04 mm2 corresponding to 65.04 % inhibition of the lesions (Figure 2 - Table 2). In order to rule out the possibility that the protective effect of EOHM mucosa depends on a local action, in others words, in the
direct contact of oil with the mucosa forming a protective barrier to the action of ethanol, the oil was administered at the highest dose (400 mg/kg) by intraperitoneal route. By the route, the EOHM also significantly protected the gastric mucosa in 83.0% (data not shown).
3.4 HCl/ethanol-induced gastric ulcers
The administration of EOHM reduced the area of gastric lesions induced by acidified ethanol. The results show that pretreatment of animals with EOHM at doses of 100, 200 and 400 mg/kg orally presented antiulcerogenic significant effect, reducing the ulcerated area to 29.67 ± 14.80; 21.50 ± 12.59 and 14.67 ± 5.50 mm2, corresponding to a percentage of inhibition of 84.20, 88.50 and 92.21 % respectively, when compared to the control group (187.80 ± 32.56 mm2). The animals that received pantoprazole (40 mg/kg, p.o.), the ulcerative lesion were reduced to 97.33 ± 23.51 mm2 corresponding to 48.18% inhibition of the lesions (Figure 2 - Table 2).
3.5 Indomethacin-induced gastric ulcers
Subcutaneous administration of indomethacin (30 mg/kg) produced a gastric lesions index from 4.20 ± 0.37; ulcer index 20.80 ± 3.33 and total index of 25.00 ± 3.48 in the control group. Pretreatment of animals with EOHM at doses of 100, 200 and 400 mg/kg orally 1 hour before administration of indomethacin, significantly reduced all indices determined as shown in Figure 3 - Table 3. The dose of 100 mg/kg reduced the incidence of lesions, ulcers and total index in 85.71, 75.48 and 77.20 %, the dose of 200 mg/kg in 72.38, 80.38 and 79.00 %, and dose of 400 mg/kg in 88.10, 90.00 and 89.68 % respectively. Pantoprazole reduced the index of injuries, ulcers and the total index at 57.1, 84.10 and 79.60 % respectively when compared with the control group.
3.6 Evaluation of the gastric secretion parameters - Pyloric Ligature
After 4 hours of pylorus ligature it was observed that the EOHM at doses of 100, 200 and 400 mg/kg administered by intraduodenal route, did not reduce gastric volume secreted in when compared the control group (3.54 ± 0.80 mL). In relation to the pH of gastric juice, the doses of 200 and 400 mg/kg EOHM increased pH for 2.10 ± 0.18 and 2.22 ± 0.15
respectively, when compared to control (1.50 ± 0.10) and reduced the total acidity (H+ concentration) of acid secretion only at a dose of 400 mg/kg to 7.85 ± 1.37 mEq[H+]/mL/4 h compared to control (29.67 ± 7.22 mEq[H+]/mL/4h). Pantoprazole modified on three parameters, reducing the volume to 1.03 ± 0.13 mL and gastric acidity to 4.57 ± 0.65 mEq[H+]/mL/4 h, and increased pH of gastric juice to 3.01 ± 0.12 (Figure 4 - Table 4).
3.7 Gastric emptying
The concentration of phenol red present in the stomach after 30 minutes of its administration was 4.63 ± 1.20, 10.68 ± 1.77 and 11.62 ± 1.30 µg/mL in animals treated with EOHM at doses of 100, 200 and 400 mg/kg respectively. The animals treated with atropine (positive control test) had the concentration of phenol red increased to 11.84 ± 0.47 µg/mL, compared to animals treated with vehicle, which the concentration was 2.25 ± 0.78 µg/mL (Figure 5). The results indicate that the animals treated with doses of 200 and 400 mg/kg showed a reduction in the rate of gastric emptying of 15.98 and 8.58%, while gastric emptying rate the group treated with atropine (3 mg/kg) was 6.85% when compared to control zero time.
3.8 Intestinal transit
In control group animals receiving only vehicle, the intestinal transit, measured by distance traveled by phenol red in relation to the total length of the small intestine, was 93.19 ± 1.92%. In animals treated with EOHM (100, 200 and 400 mg/kg) had a percentage of 84.25 ± 2.50, 76.40 ± 4.37 and 76.16 ± 7.63 % respectively. None of the doses showed effect on intestinal transit when compared with the control group. Only the group treated with atropine reduced the percentage of intestinal transit of 50.01 ± 6.82% (Figure 6).
4. Discussion
The phytochemical characterization of essential oil from dried leaves of Hyptis
martiusii demonstrated the presence of mono and sesquiterpenes. In research previously carried out by Araújo et al. (2003) also was reported that the chemical composition of essential oil from fresh leaves of same species is composed of mono and sesquiterpenes, who
reported as the major components 1,8-cineole, δ-3-carene, bicyclogermacrene and β- caryophyllene. The results this study differs from Araújo et al. (2003), however this discrepancy may be due to the fact that the oil was obtained under different conditions (dried leaves).
This work is the first report of gastroprotective activity of essential oil from leaves of
Hyptis martiusii Benth. Leaves in infusion or decoction are traditionally used in folk medicine against intestinal and stomachic diseases (Agra et al., 2008), but were not found in the literature that report the effect this specie on the gastrointestinal tract. According to Maity et