Peptic ulcer disease (gastric and duodenal ulcers) is a serious and growing health problem around the world that is estimated to affect 10% of the world's population with an annual increase of 4 million people. Furthermore, it is the most common gastrointestinal disease, involving 40% of developed countries and 80% of developing countries because of overuse of NSAIDs and Helicobacter pylori infection . Current clinical treatment of gastric ulcer indicates a high recurrence rate and low recovery. Therefore, gastric ulcers treatment is still a major challenge that has made the development of new drugs and alternative therapies as a necessary and urgent need. Previous studies have reported the anti-ulcer properties of many medicinal plants as an excellent potential source for drugs production . In recent years, many attempts have been made to detect gastroprotective and anti-ulcer drugs . In this regard, several studies have been widely performed using ethanol-induced gastric ulcer model in animals to investigate the gastroprotective activity of plants and drugs. The effect of ethanol generated gastric ulcer begins with its rapid penetration into the gastric mucosa, thereby increasing mucosal permeability and secretion of vasoactive mediators such as endothelin-1, leukotrienes C4, and histamine. These vasoactive agents increase gastric mucosal lesions by reducing blood flow to the mucosal membranes.
Moreover, ethanol damages blood vessels by reducing mucus production (by damaging its constituent components) and increasing the production of ROS, which leads to bleeding, tissue necrosis, and ultimately disruption of the protective barrier. Previous studies suggest that ethanol-induced gastric mucosal lesions are significantly associated with increased oxidative stress and the free radicals derived from oxygen, reducing antioxidant enzymes' activity. In this context, the infiltration and activation of neutrophils directly contribute to the increased ROS production. Therefore, it is likely that the infiltration and accumulation of neutrophils in the gastric mucosa result in free radical formation, which damages cellular components, including lipids and proteins [39, 40]. PCO is involved in many diseases, and it can be used as a diagnostic marker for oxidative stress. Therefore, the detection and reduction of PCO play a pivotal role in recovering these diseases [41, 42]. Importantly, in our study, the pre-treatment of the ethanol-induced rats with EPE and ranitidine ameliorate the ROS levels and PCO production. These effects are likely through the strengthening the antioxidant defense system of gastric cells.
Several studies have reported that ethanol can stimulate lipid peroxidation in cell membranes through ROS production, resulting in cell damage. Many studies have shown that ethanol can stimulate lipid peroxidation in cell membranes via ROS . The increased lipid peroxidation by ethanol accelerates gastric ulcers by destroying membrane integrity and increasing cell permeability of gastric epithelial cells. These events lead to MDA production as the end product of the oxidation of unsaturated fatty acids in cell membranes. MDA is commonly used as a reliable indicator of lipid peroxidation, which its level can be used to estimate the extent of ethanol-induced gastric tissue damage. In agreement with previous studies, our data showed that ethanol gavage significantly increases MDA production in gastric tissue [44–46]. Significantly, pre-treatment of ethanol-induced rats by EPE at doses of 250 mg/kg and 500 mg/kg reduced MDA levels. Different studies have shown that the generation of hypochlorous acid (HOCL), as one of the dangerous species resulting from the enzyme myeloperoxidase (MPO), increased following the oxidation of chloride ions by hydrogen peroxide in neutrophils. At this time, antioxidant defenses of gastric cells are activated and destroy H2O2, with the purpose to suppress this process. The antioxidant enzyme CAT mediates the conversion of H2O2 into water and oxygen. GSH can also neutralize H2O2 by functioning as a cofactor for the glutathione peroxidase (GPx) enzyme. Many evidence shows the reduction of CAT activity and GSH content in gastric tissue resulted from ethanol-induced gastric mucosal damage. This reduction leads to elevated ROS levels, resulting in increased lipid peroxidation and PCO formation [47, 48]. In this context, Amaral et al.  have demonstrated that the ethanol-induced mucosal damage increases the production and accumulation of ROS and MDA. The authors further showed that this occurs by enhancing the MPO enzyme activity that resulted from decreased CAT activity and GSH levels in gastric tissue. As well, Liu et al.  have reported that ethanol administration, damages gastric tissue by increasing MDA and PCO levels. The results of our study showed that EPE has high antioxidant properties by reason of its relatively high levels of phenolic and flavonoid compounds, glycosides, coumarin derivatives, and naphthoquinones [51, 52]. Polyphenols, as one of the plant antioxidants, can terminate the chain reaction by reacting with free radicals so that their phenolic hydroxyl group reduces this radical by giving an electron to free radicals. As a result, the aromatic free radicals formed will be stable owing to the resonance effect. On the other hand, polyphenols can enter the lipid bilayer of the gastric mucosa, protecting the entire lipid layer from oxidation [53, 54]. Our results indicated that EPE has a gastroprotective effect against gastric ulcers ethanol-induced. Because phenolic compounds play a major role in plants' antioxidant activity, the EPE's anti-ulcer activity may be related to these compounds. With their synergistic effect with antioxidants such as GSH, CAT, etc., these compounds can inhibit the chain reaction of free radicals and increase the antioxidant level of gastric tissue to protect it against ethanol-induced damage.
Here, we used ranitidine, which inhibits gastric acid secretion by blocking H2 receptors. Further, ranitidine has antioxidant properties , and therefore, it functions more efficiently than EPE. We could show the effects of ranitidine by biochemical, macroscopic, and microscopic analysis. Further, ranitidine increased the pH of gastric juice, which led to a decrease in the ulcer index. Many evidence shows even after gastric mucosal injury, the stomach is stimulated; thus, gastric juice does not affect gastric mucosa. It secretes a large amount of stomach acid, which increases the volume of gastric juice and reduces its pH. These events worsen gastric mucosal damage and, as a result, intensify gastric injury . It is manifested in our results that EPE effectively reduces gastric juice volume and increases gastric juice pH in rats that receive alcohol, thereby protecting the gastric tissues and preventing ethanol-induced gastric injuries. Yet, herbal extracts might act as H2-receptor blockers and prevent histamine from binding to its receptor . Therefore, a mechanism of action of EPE is related to a further reduction of gastric juice pH. In addition, macroscopic and microscopic evaluations and the inhibition percentage of ulcers also confirmed this effect.
NO•, as a gaseous free radical, plays a role in physiological processes and in pathophysiological conditions. The level of NO• is increased due to the activity of inducible nitric oxide synthase (iNOS), anion superoxide (O2•−) resulting from ethanol metabolism and neutrophils [58, 59]. Thus, NO• is prone to react with O2•− that results in proxy nitrite (ONOO−). This later free radical is a species with cellular toxicity that can oxidize various cellular components, disrupt cellular processes, disrupt cellular signaling pathways, and induce cell death. Interestingly, ethanol consumption is associated with increased expression of iNOS, which leads to increased NO• levels.
Along with this evidence, in response to ethanol-induced gastric ulcer increased the NO• production. The increase probably is due to the increased induction of iNOS expression, which our results are in accordance to the studies of Yu et al.  and Li et al. .
On the other hand, NO• as a vasodilator factor with a short half-life regulates vascular and nutrient blood flow. It maintains the stomach and mucus barrier's epithelial integrity and, most importantly, has a pivotal role in angiogenesis, tissue regeneration, and ulcer healing . As a result, in the present study, its reduction in the gastric tissue of the ethanol group may be caused by increased ONOO− and oxidative damage and the decreased endothelial nitric oxide synthase (eNOS) expression as a protective factor. Besides, direct-ethanol damage on epithelial cells and sub-mucosal endothelial vessels might also contribute to the reduction in the gastric tissue. According to Zhang et al.,  this event leads to the rupture of blood capillaries and reduced blood flow in these vessels . Numerous studies have also confirmed that NO• level in rats' gastric tissue under ethanol gavage was significantly decreased [64–67]. In this study, EPE improves the antioxidant defense system (by participating in free radical scavenging). It probably leads to a change in the nitric oxide system (by reducing iNOS expression and increasing eNOS expression) during ethanol's oxidative damage.