Therapeutic potential of essential oil of Melaleuca quinquenervia (Myrtaceae) in a rat model of ethanol-induced peptic ulcer

Purpose: To evaluate the effects of M. quinquenervia extract on ethanol-induced peptic ulcer in rats. Methods: The following three groups of (n = 6) Sprague Dawley rats were included in this study: vehicle (C), ethanol-administered (E) and ethanol + M. quinquenervia-treated (MQ). MQ group rats received 100 µg/mL M. quinquenervia essential oil just before 96 % ethanol induction (1 ml/kg). One hour after ulcer induction, the animals were euthanized, and gastric and duodenal tissues were removed. Tissue samples were analysed for myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), myeloperoxidase (MPO) activity, malondialdehyde (MDA) and glutathione (GSH) levels, and histopathological examinations were performed by light microscopy. Results: Gastric and duodenal GSH levels that decreased in the ethanol-administered ulcer groups (p < 0.001), rose following MQ treatment (p < 0.5). Moreover, elevated MPO and MDA levels (p < 0.5) in gastric tissues decreased after MQ-treatment. Similarly, the MQ-treated group showed recovery and control-like morphology compared to the ethanol group in both gastric and duodenal tissues when examined by microscopy. Conclusion: The results indicate that M. quinquenervia extract has a positive impact on gastric injury in rats due to its antioxidant activity. Thus, the plant has a potential for the clinical management of gastric ulcer.


INTRODUCTION
Melaleuca quinquenervia is a small-to-mediumsized tree from the Myrtacae family. Its leaves are mostly used in the treatment of respiratory diseases [1]. The essential oil of this plant (Niaouli oil) is known for its medicinal properties in many tropical countries, including New Caledonia, Australia and Madagascar [2], and is often used to treat pain, dermatosis, influenza, rhinitis, sinusitis and pharyngitis in Africa [3,4].
Acute gastric injury is a very common problem of the gastrointestinal tract, affecting an average of 4 million people per year. Its treatment is still being investigated [5]. Although Helicobacter pylori has a significant effect on the etiology and treatment of the disease, there is an increase in the incidence of gastric ulcers because of stress, alcohol consumption, and non-steroidal antiinflammatory drugs [6]. On the other hand, while omeprazole and similar anti-acid drugs, H2 receptor blockers, that are a class of gastric acidsuppressors like ranitidine and anticholinergics are frequently used in gastric diseases, they have many side effects and are insufficient to treat ulcer effectively [7].
Besides drugs, plant-derived natural products constitute a great potential remedy for gastric ulcer [8]. Several researches have shown that many natural products possess gastroprotective effects [9].
The constituents of Melaleuca quinquenervia offer a lot of benefits, including free-radical scavenging, anti-inflammatory, and antioxidant properties. Therefore, this study investigates the anti-inflammatory and anti-oxidants effects of this medical plant on a rat model of ethanol-induced peptic ulcer.

EXPERIMENTAL Animals
Female Sprague-Dawley rats (200 -250 g) were obtained from Marmara University School of Medicine Animal House, and kept under normal circumstances (22 ± 1 •C, 65 -70 % humidity, 12 h light/dark cycle). The rats were fasted at night before the experiment day. All animal experiments were approved by the Marmara University School of Medicine Animal Care and Use Committee (approval no. 63.2019.mar), and the principles and guidelines developed by New York Academy of Sciences were followed [11].

Study design and treatments
Female Sprague-Dawley rats (n = 18) that were not fed one day before the experiment day, were left overnight. On the experiment day, under the ether anaesthesia, randomly selected rats (n = 6) received orogastric saline (vehicle), ethanol (96 %; 1 ml/kg) and M. quinquenervia extract (100 µg/mL) + ethanol. One hour after administration, animals were euthanized. The stomach and duodenal tissues were dissected via greater curvature. The collected tissue samples were kept under -80 °C for further assessments for myeloperoxidase (MPO) activity, malondialdehyde (MDA) and glutathione (GSH) levels, and also histopathological examinations at light microscopic level.

Determination of malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity
Malondialdehyde levels show the products of lipid peroxidation. It was measured using a spectrophotometer at 532 nm by monitoring thiobarbituric acid-reactive chromogenic reagent formation, and results were expressed in nmol/g [12]. Similar to MDA, GSH was determined spectrophotometrically with Ellman's reagent [13] Results were expressed as µmol GSH/g tissue. Azurophilic granules in the polymorphonuclear leukocytes (PMN) are found to be the primary site for myeloperoxidase (MPO) enzyme. Therefore, its activity is used for estimating neutrophil infiltration in inflammation. MPO activity was measured according to the spectrophotometric procedure reported by Hillegass et al [14]. The 460 nm absorbance was measured for 3 min, and results were expressed as U/g tissue.

Macroscopical evaluation of ulcerative regions in stomach tissues
Collected stomach tissue samples were examined in terms of the macroscopical appearance for ulcerative regions.

Histological analysis
After the decapitation of the animals, stomach and duodenum tissues were removed. For light microscopic examinations, the tissue samples were incubated in 10% formaldehyde solution for fixation, and then they underwent routine histological assessments (Leica, TP1020). Subsequently, fixed tissues dehydrated through ascending alcohol series (70, 90, 96, and 100%) and cleared in xylene, then embedded in paraffin (Leica EG1150H+C). Approximately 4 -5 μmthick paraffin sections were manually cut on glass slides with a rotary microtome (Leica, RM2125RT) from the paraffin blocks, which include the tissues. Sections were dewaxed using xylene and then stained with hematoxylin and eosin (H & E) for light microscopic imaging (Olympus DP72 attached to BX51, Tokyo, Japan). The stained sections were semiquantitatively scored using 0-3 scoring system (0: none, 1: mild, 2: moderate, 3: severe) according to the modified criteria from the previous study [18]. For the stomach tissues, desquamation of surface epithelium, haemorrhage/congestion, degeneration of glandular cells, inflammatory cell infiltration were used as evaluation criteria with the highest score of 12. Semi-quantitative score was also obtained for duodenal tissues, with the highest score being 15, due to the inclusion of the degeneration of the villus morphology analysis to the evaluation.

Statistical analysis
GraphPad Prism 8.1 (GraphPad Software, San Diego, CA) was used for statistical analysis. The results are expressed as mean ± SEM. Analysis of variance (ANOVA) followed by Tukey's multiple comparison tests was performed to determine statistically significant difference between the groups. Values of p < 0.05 were regarded as statistically significant.

Macroscopic and histological evaluations
The results of the macroscopic evaluation of the ulcerative regions show that while the ethanol group showed severe ulcerative areas in the stomach, the M. quinquenervia-treated group displayed normal macroscopic morphology ( Figure 2).
Light microscopic examination and semiquantitative scores of the stomach tissues showed that the vehicle group had regular gastric pits with normal epithelial lining and proper glandular structures (Figure 3a). In the ethanol group, severe vascular congestion, glandular cell degeneration, damaged epithelial lining, and inflammatory cell infiltration were seen as signs of the ulcer (Figure 3 b and c).   The duodenal tissues showed regular villi organisation with proper epithelial lining (Figure 5  a). For all the evaluation criteria, significant increases were seen in the ethanol group, compared to the vehicle group (p < 0.001) (Figure 5 b and c). Control-like histological appearance was noticed in the M. quinquenerviatreated group (Figure 5 d), significantly (p < 0.001) (Figure 4 b).

DISCUSSION
Gastric mucosa has many natural defence mechanisms for preventing the damage caused by noxious agents. Histological and biochemical findings of this present study revealed that oxidative stress injury induced by ethanol was reversed by Melaleuca quinquenervia treatment. Stomach GSH levels, which decreased in ethanol-treated rats, were preserved when the rats received Melaleuca quinquenervia treatment. Neutrophil accumulation, identified by MPO activity, decreased markedly when the animals were treated with Melaleuca quinquenervia. Similar to MPO, MDA levels also tended to decrease in Melaleuca quinquenervia treated animals. Concomitant to biochemical findings, gastric damage was also significantly diminished by Melaleuca quinquenervia treatment, as assessed via histological scores. All these findings suggest that Melaleuca quinquenervia treatment has potent antiinflammatory and anti-oxidative effects on the damaged gastric mucosa.
Since previous reports showed the role of reactive oxygen species (ROS) in the pathophysiology of gastric mucosal damage [16], natural antioxidants are found to be beneficial, by protecting the stomach or by expediting the healing of mucosal damage [17]. Chemical studies reported that Melaleuca quinquenervia leaves contain triterpenoids, flavanones and polyphenols [18]. Past reports indicated that triterpenoids [19], flavanones [20] and polyphenolics [21] have antioxidant and antiinflammatory effects. On the contrary, one report revealed that Melaleuca quinquenervia does not have an anti-inflammatory effect [22] In agreement previous reports, the results of the present study showed that Melaleuca quinquenervia treatment markedly inhibited GSH depletion, implying a reduction in lipid peroxidation that prevents gastric damage. GSH is the prime defence against oxidative damage [23]. Oxidative stress causes a fast depression of GSH content of the cell. The current findings demonstrate that GSH decrease was preserved by Melaleuca quinquenervia. Additionally, MPO activity, a marker of inflammation, mostly increases ulcerations [24] also diminished by Melaleuca quinquenervia treatment, while increase in the other inflammation marker, MDA, was limited.
Likewise, healing of ulcer histologically revealed the anti-inflammatory effect of the extract.

CONCLUSION
The findings of this study indicate that Melaleuca quinquenervia has gastro-protective effects in rats due to its anti-inflammatory and antioxidant properties. Thus, Melaleuca quinquenervia extract may hay also exhibit similar properties in humans. However, further investigations are required to understand the mechanism of the plant's antioxidant and anti-inflammatory properties.