Background

Peptic ulcer is a multifactorial chronic disease, and Helicobacter pylori infection, use of nonsteroidal anti-inflammatory drug (NSAIDs), and alcohol abuse are the main causative agents. It can prejudice any part of the gastrointestinal tract, characterized by ulceration of the mucosa, affecting more often the gastric and duodenal mucosa [1, 2].

The use of medicinal plants has been encouraged in public health because of the large number of people affected by diseases that generate economic impact. Furthermore, most synthetic drugs for treatment are associated with undesirable side effects [3, 4]. Several studies indicate the efficacy of medicinal plants for the peptic ulcer treatment, because of the presence of bioactive phytochemicals such as flavonoids, alkaloids, terpenes, tannins, saponins, phenolic acids, and others [2, 5].

The species Myrcianthes pungens, native of Rio Grande do Sul, belonging to the Myrtaceae family and popularly known as “guabiju” [6, 7], is used in folk medicine for possessing antidiarrheal properties [8], and its leaves have been described as diuretic and for the ability to reduce stomach disorders [9].

M. pungens fruits have high dietary fiber content, sugar, and protein, with low acidity and sweet taste. It has anthocyanins and high level of carotenoids and polyphenols, with antioxidant capacity [10, 11]. In a recent study, the peel, pulp, and leaves of M. pungens presented a significant antinociceptive activity. The presence of triterpenes and flavonoids were evidenced and may be responsible for the antinociceptive activity [12]. The aim of this study was to evaluate the phytochemical profile and the gastroprotective activity of the methanolic extract of M. pungens whole fruit, peel, pulp, seeds, and leaves.

Methods

Plant material

M. pungens was collected at Urussanga, in the state of Santa Catarina (Brazil), and identified by Prof. Oscar B. Iza (UNIVALI). A voucher specimen was deposited at the Barbosa Rodrigues Herbarium (Itajaí-SC) under number VC Filho152.

Preparation of extracts and phytochemical analysis

Fresh parts (whole fruit, peel, pulp, seeds, and leaves) of M. pungens were cut into small pieces and macerated with methanol at room temperature for approximately 7 days, providing the methanol extract from each part after solvent evaporation.

To verify the presence of phenolic compounds, aliquots of methanolic extracts were examined, using authentic samples as a parameter, by thin layer chromatography (TLC). The solvents chloroform and methanol (90:10) were used as mobile phase, and ferric chloride was used as a specific reactive of phenolic compounds.

Gastroprotective activity in vivo

Female Swiss mice (20–35 g) were provided by the Central Animal House of the Universidade do Vale do Itajaí (UNIVALI) (Itajaí, SC, Brazil). The animals were housed in groups of five, in standard cages, at room temperature (22 ± 2 °C) with 12-h dark/12-h light cycles, and received food and water ad libitum.

Twelve hours prior to the experiments, they were transferred to the laboratory and given only water ad libitum. In all experiments, the animals were kept in cages with wide-mesh raised floors to prevent coprophagy. The animals used in the present study were housed and cared for in accordance with the Federal Government legislation on animal care. The experiments were authorized by the Ethical Committee for Animal Care of UNIVALI (process number 005/14).

Ethanol/HCl-induced ulcer

The experiment was performed according to the method described by Mizui and Doteuchi [13], with some modifications. After 12 h of fasting, the animals were randomly divided into different groups of six animals each and pre-treated orally with cimetidine (positive control—100 mg/kg), vehicle (negative control—distilled water), and the methanolic extracts from each part of the plant at doses of 50, 125, and 250 mg/kg.

One hour after treatment, all animals received 0.1 mL/10 g (body weight) of a 0.3 mol/L HCl in 60% ethanol solution (ethanol/HCl) to induce gastric ulcer. Another hour later, the animals were sacrificed by cervical dislocation, and the stomachs removed and opened along the greater curvature.

Nonsteroidal anti-inflammatory drug-induced ulcer

Experiments were carried out according to the method described in Rainsford [14], with a few modifications. After 12 h of fasting, the animals were randomly divided into different groups of six animals each and pre-treated orally with cimetidine (positive control—100 mg/kg), vehicle (negative control—distilled water), and the methanolic extracts from each part of the plant at doses of 50, 125, and 250 mg/kg.

One hour after treatment, they received indomethacin (100 mg/kg body weight) to induce gastric ulcer. Twelve hours after treatment with indomethacin, the animals were sacrificed by cervical dislocation, and the stomachs removed and opened along the greater curvature.

Evaluation of gastroprotective activity

After completion of the two methods described above, the stomachs stretched on glass plates were scanned and analyzed using image analysis software EARP to determine the number and size of the lesions. The results are expressed as total lesion area (mm2) and relative lesion area (%). The data are reported as mean ± standard error of the mean (SEM) and were compared using one-way analysis of variance (ANOVA), followed by Dunnett’s pairwise test, and p values <0.05 were considered significant. The GraphPad INSTAT software was used for statistical analysis.

Results

The results obtained by the phytochemical analysis by TLC indicated the presence of quercetin and quercitrin in the leaves, quercetin in the peel, and catechin and epicatechin in the leaves and seeds.

The methanolic extracts of peel and leaves at all concentrations and pulp at concentrations of 125 and 250 mg showed defense capability of the gastric mucosa against ethanol, reducing the relative lesion area significantly when compared to the negative control, as well as cimetidine, which proved its gastroprotective effect (Table 1).

Table 1 Effect of oral administration of cimetidine (100 mg/kg) and M. pungens extracts (50, 125, and 250 mg/kg) on ethanol/HCl-induced gastric ulcers in mice (n = 6)
Full size table

In relation to indomethacin model, all M. pungens extracts presented defense capability of the gastric mucosa, reducing significantly total and relative lesions areas compared to the negative control (Table 2).

Table 2 Effect of oral administration of cimetidine (100 mg/kg) and M. pungens extracts (50, 125, and 250 mg/kg) on the indomethacin-induced gastric ulcer in mice (n = 6)
Full size table

Discussion

This study evidenced the presence of phenolic compounds in different parts of M. pungens. Nesello et al. [12] verified the presence of terpenes/steroids and phenolic compounds at the same evaluated parts. Desoti et al. [15] evaluated the methanol crude extract of M. pungens fruits and identified tannins, flavonoids, steroids, and alkaloids in its composition. Andrade et al. [10] identified anthocyanins, polyphenols, and flavonoids and also showed antioxidant activity. Nora [11] identified anthocyanins and carotenoids (β-carotene), as well as antioxidant activity of methanolic fruit extract.

The reason why seed extracts (all concentrations) and the whole fruit (concentrations of 125 and 250 mg) extracts were not as effective as other extracts/concentrations was not fully investigated in this study. A possible explanation would be the presence of irritant compounds in seeds and/or because their primary mechanism of action is maintaining or increasing the production of prostaglandins, mucus, and bicarbonate [16, 17].

There are no reports in the literature regarding the gastroprotective potential of M. pungens, but a study by Desoti et al. [15] showed that ethyl acetate and hexane extracts of M. pungens fruits showed bactericidal properties. The same authors also identified antifungal activity of ethyl acetate and methanol extracts and low cytotoxic effect of these extracts. Silveira et al. [6] identified acetylcholinesterase activity related to the reduction of gastric secretion in the ethanol extracts of green and ripe fruits of M. pungens.

In a recent study, the peel, pulp, and leaves of M. pungens inhibited the acetic acid-induced contractions in mice. The leaves were also active in the formalin, capsaicin, and glutamate models. The triterpenes alpha-amyrin and beta-amyrin and the flavonoids quercitrin and rutin were the major components of M. pungens leaves and may be responsible for the antinociceptive activity evidenced. The results demonstrate the efficacy of M. pungens, specially the leaves, as a potentially novel and effective analgesic agent for pain control [12].

The mechanism for the gastroprotective action of M. pungens in this study may be related to the presence of flavonoids evidenced in phytochemical analysis, which have been considered the responsible for attenuating the oxidative effects induced by indomethacin [18]. According to Klein-Junior et al. [19], flavonoids demonstrate gastroprotective activity in the induction model with indomethacin, reducing gastric lesions caused by these NSAIDs. Brzozowski et al. [20] also reported gastroprotective activity, as well as antibacterial and antioxidant action against ethanol-induced damages, observing increasing of vasodilation and prostaglandin related to the activity of NO, possibly stimulated by the phytochemical compound in discussion.

The results obtained from both models demonstrated that the methanolic extracts of different parts of M. pungens have gastroprotective activity, which suggests that the phenolic compounds present in the extract favor anti-inflammatory and antioxidants mechanisms, possibly acting in the maintenance of prostaglandin and NO, favoring the protection mechanisms and injury repair [21, 22].

Conclusions

M. pungens seeds and whole fruit extracts demonstrated no gastroprotective activity on ulcer model induced by ethanol. The remaining extracts (peel, pulp, and leaves) showed gastroprotective effect which can be explained by the presence of various phenolic compounds evidence in phytochemical analysis.

Further studies are necessary to confirm these effects and to elucidate the mechanisms of action of gastroprotective activity. The phytochemical studies are in progress to isolate/identify other bioactive compounds.