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Article

Gastroprophylactic Effects of p-Cymene in Ethanol-Induced Gastric Ulcer in Rats

by
Suhayla H. Shareef
1,2,
Morteta H. Al-Medhtiy
3,
Ibrahim Abdel Aziz Ibrahim
4,
Abdullah R. Alzahrani
4,
Ahmed Aj. Jabbar
5,*,
Yaseen Galali
6,
Nabaz Fisal Shakir Agha
7,
Peshawa Y. Aziz
8,
Muthanna A. Thabit
9,
Derin N. F. Agha
10,
Nur Ain Salehen
11,
Zeena M. Ameen
12 and
Mahmood A. Abdulla
13
1
Department of Medical Biochemical Analysis, College of Health Technology, Cihan University-Erbil, Erbil 44001, Kurdistan Region, Iraq
2
Department of Biology, College of Education, Salahaddin University-Erbil, Erbil 44001, Kurdistan Region, Iraq
3
Department of Anatomy and Histology, Faculty of Veterinary Medicine, University of Kufa, Kufa 540011, Najaf Region, Iraq
4
Department of Pharmacology and Toxicology, Faculty of Medicine, Umm Al-Qura University, Makkah 77207, Saudi Arabia
5
Department of Medical Laboratory Technology, Erbil Technical Health and Medical College, Erbil Polytechnic University, Erbil 44001, Kurdistan Region, Iraq
6
Department of Food Technology, College of Agricultural Engineering Sciences, Salaheddin University-Erbil, Erbil 44002, Kurdistan Region, Iraq
7
Department of Anesthesia, Erbil Medical Technical Institute, Erbil Polytechnic University, Erbil 44001, Kurdistan Region, Iraq
8
Department of Medical Laboratory Science, Technical College of Applied Science, Sulaimani Polytechnic University, Sulaymaniyah P.O. Box 394, Kurdistan Region, Iraq
9
Cambridge International School-Erbil, Erbil 44001, Kurdistan Region, Iraq
10
Department of Pharmacology, Erbil Medical Technical Institute, Erbil Polytechnical University, Erbil 44001, Kurdistan Region, Iraq
11
Department of Biomedical Sciences, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
12
Department of Biomedical Sciences, College of Sciences, Cihan University-Erbil, Erbil 44001, Kurdistan Region, Iraq
13
Department of Medical Microbiology, College of Science, Cihan University-Erbil, Erbil 44001, Kurdistan Region, Iraq
 
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Author to whom correspondence should be addressed.
Processes 2022, 10(7), 1314; https://doi.org/10.3390/pr10071314
Submission received: 17 June 2022 / Revised: 29 June 2022 / Accepted: 30 June 2022 / Published: 4 July 2022
(This article belongs to the Special Issue Bioactive Compounds from Natural Plants)
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Abstract

:
The prevalence of gastric ulcers has increased in recent years, mainly because of non-steroidal anti-inflammatory drug utilization. Therefore, the current study investigates the gastroprotective effect of p-Cymene on absolute ethanol-induced acute gastric mucosal hemorrhagic lesions in rats. Thirty Sprague Dawley rats were randomly separated into five groups: normal control, ulcer control, reference, and two experimental groups. The normal and ulcer control groups were orally fed with 0.5% carboxymethylcellulose (CMC). The reference group was fed orally with 20 mg/kg omeprazole. The experimental groups were fed with 30 mg/kg and 60 mg/kg p-Cymene, respectively. After one hour, the normal group was fed with 0.5% CMC, and groups 2–5 were given absolute alcohol. After another hour all rats were sacrificed. The ulcer control group showed severe superficial hemorrhagic gastric mucosal lesions with decreased gastric mucus secretion and pH of gastric content. p-Cymene significantly reduced ethanol-induced gastric lesions, as evidenced by increased mucus and pH of gastric content, decreased ulcer area, reduced or absence of edema, and leucocyte infiltration of the subcutaneous layer. In gastric mucosal homogenate, p-Cymene displayed a significant increase in superoxide dismutase (SOD), catalase (CAT) activities, prostaglandin E2 (PGE2), and significantly reduced the malondialdehyde (MDA) level. In addition, p-Cymene increased the intensity of periodic acid–Schiff (PAS) stain of the gastric epithelium, and produced up-regulation of the HSP 70 protein and down-regulation of the Bax protein of the stomach epithelium, as well as a reduction in the levels of tumor necrotic factor-alpha and interleukin-6, while the level of interleukin-10 was increased. p-Cymene decreased the level of TNF-a and IL-6, and increased the level of IL-10. Acute toxicity with a higher dose of 500 mg/kg p-Cymene did not manifest any toxicological signs in rats and could enhance defensive mechanisms against gastric mucosal lesions. p-Cymene showed gastroprotective effects that could be attributed to its antioxidant nature, or its ability to increase mucus secretion, increase endogenous enzymes (SOD, CAT, PGE2), reduce MDA level, up-regulate HSP 70 protein, down-regulate Bax protein, and modulate inflammatory cytokines.

1. Introduction

A peptic ulcer is the greatest digestive system disorder worldwide. Several studies by peptic ulcer researchers have exposed that this syndrome is caused by an imbalance between mucosal invasive factors, for example, Helicobacter pylori infection, consumption of non-steroidal anti-inflammatory medications, pressure, cigarettes, and malnutrition, resulting in the disturbance of the defensive gastric mucosal barrier, leading to peptic ulcers, and defensive aspects of the stomach mucosa barrier which include mucus secretion, increased prostaglandin levels, and endogenous antioxidant enzyme activity [1,2,3].
Ethanol is a necrotizing agent that induces oxidative stress and stomach mucosal ulceration through the generation of highly cytotoxic free radicals [4,5]. Oxygen-derived free radical species induced by ethanol administration are among the mucosal invasive factors responsible for peptic ulcers by causing oxidative damage to gastric mucosal cells [6,7]. The exogenous and endogenous active oxygen and oxidants (superoxide dismutase and single oxygen peroxidase) can easily cause damage to gastric mucosal linings. A weak acid such as ethanol increases superoxide anion and hydroxyl radical production and lipid peroxidation in the gastric mucosa [8,9]. However, in addition, ethanol interrupts stomach mucus activity, changes cell permeability, and diminishes gastric mucus secretion, making gastric mucosal cells more susceptible to free radicals [10]. Several studies have discovered that ethanol administration induced functional and morphological changes in gastric mucosa, such as abnormal gastric hypersecretion, decreased mucus secretion and hemorrhagic necrotic foci that occurred in the gastric mucosa of experimental animals [3,11,12,13]. Of note, those experiments used a reference drug to compare with the tested medicinal plant, and the most common standard drug is omeprazole, scientifically known as a proton pump inhibitor (PPI), utilized worldwide for the treatment of peptic ulcer diseases. In the literature, studies that used omeprazole as a standard drug for the prevention of gastric ulcers on ethanol-induced gastric ulcers have been reported by huge numbers of academics [14,15]. Despite the anti-ulcer efficacy of this chemically synthetic drug (omeprazole), it has some downsides, with risk factors for asymptomatic hypomagnesemia, hypocalcemia, and hypernatremia [16]. Therefore, researchers are racing against the time to find a natural compound or medicinal herb to be a substitute for those chemically synthetic PPIs [17,18,19].
Medicinal plants and their active ingredients have been established to protect a variety of cells and tissues via initiation, and the rapid elevation of heat shock protein HSP 70 to protect against numerous necrotizing agents inducing gastric injury in rats has been reported by many researchers [20,21].
In the systematic literature, huge traditional medicinal plants or herbs and their active ingredients have been used as the primary therapeutic agents for peptic ulcers, as reported by many researchers [3,22,23,24].
The compound p-Cymene [1-methylethyl-4-(1-methylethyl)-benzen] has been known as a natural compound and a monoterpene that has toluene substituted by an isopropyl repositioning at the fourth sub-group (Figure 1). p-Cymene is an important monoterpene compound present in essential volatile oils of various aromatic medicinal plants and occurs naturally in innumerable foods [25]; moreover, it shows a wide spectrum of biological activities and is used in traditional medicines to convey analgesic [26], antioxidant [27], anti-inflammatory, anti-nociceptive, anxiolytic, anticancer, antimicrobial [28], and anti-tumor [29] actions, in addition to influencing the cardiovascular scheme [30] and central nervous scheme [31].
Furthermore, p-Cymene is also an active molecule remote from numerous therapeutic plants conventionally used for the treatment of an extensive variety of chronic and/or communicable illnesses connected to pain, inflammation, and oxidative pressure, and can scavenge reactive oxygen species (ROS) avoiding oxidation of biomolecules and inducing pain and inflammation [26]. Free radicals and oxidative stress have been correlated with numerous human health issues. p-Cymene upsurges activity of antioxidant enzymes and reduces oxidative stress in vivo [32]. p-Cymene is a significant intermediate used in pharmaceutical industries and for the manufacture of fungicides, pesticides, and as a flavoring agent [33]. A search of the literature has revealed no previous study on the effect of p-Cymene against gastric ulcers. Therefore, the current research is considered as the first project evaluating the gastroprotective role of the p-Cymene via analysis of histopathological examinations and measurement of anti-inflammatory cytokine and antioxidant enzymes in ethanol-induced gastric ulcer rats.

2. Materials and Methods

2.1. Chemicals and Reagents

All reagents and compounds were acquired from Sigma-Aldrich Chemical Co. (Darmstadt, Germany), except for omeprazole which was bought from a pharmacy. Omeprazole was dissolved in 0.5% CMC and delivered via gavage by mouth to laboratory animals at a dose of 20 mg/kg (5 mL/kg), as suggested by several co-researchers [34].

2.2. Acute Toxicity Study and Trial Rats

Thirty six (18 males and 18 females) healthy rats (6–7 weeks old, weighing between 180–210 g) were acquired from the Animal House Unit, Cihan University-Erbil. Rats were given usual rat pellets and tap water ad libitum, and placed individual cages with wide-mesh wire bottoms to avoid coprophagia. Rats were kept in cages for one week for adaptation. Acute toxicity training was utilized to determine a safe dose for p-Cymene. Rats were allocated similarly into 3 groups: vehicle (0.5% CMC, 5 mL/kg), 250 mg/kg, and 500 mg/kg p-Cymene. Preceding supplementation and treatment, overnight fasting was applied to all animal groups (food but not water). Food was removed for about 3–4 h after treatment. The rats’ experiences 24–48 h after management of p-Cymene were monitored for the start of scientific or toxicologic signs. The death rate was counted for over two weeks. Rats underwent injection of high xylazine and ketamine anesthesia on the 15th day. The collection of blood specimens from intracardial perforation separated serum specimens were taken for biochemical analysis. Histology and serum biochemistry parameters determined the subsequent normal procedures [12].

2.3. Investigational Animals for Stomach Ulcer

Rats (210–250 g) were purchased from the Experimental Animal House Unit, (Ethical No. BIO/13/09/2021/MAA), College of Science, Cihan University. All experimental rats were kept in accordance with human maintenance conferring standards from the “Director Care Usage Research Laboratory Animals”, as part of the “Nationwide Conservatory Knowledges”, available via “Nationwide Institution Healthiness (New York, NY, USA)”.

2.4. Initiation of Stomach Ulceration

Rats were reserved individually in special cages with a widespread net base to evade coprophagia. Rats were fed standard pellet food and allowed access to tap water. Experimental rats were placed randomly into 5 groups of 6 animals. Rats were kept in an animal house for one week for adaptation. The fasting scheme was as follows:
Group 1 and 2 received 0.5% carboxymethylcellulose CMC by mouth;
Group 3 received oral gavage of omeprazole 20 mg/kg in CMC, as a reference group;
Group 4 and 5 were fed with p-Cymene 30 mg and 60 mg/kg in CMC, respectively [35].
After one hour, all the groups except Group 1 underwent gavage by mouth of absolute alcohol (5 mL/kg). Sixty minutes later, all rodents were sacrificed by overdose of ketamine and xylazine, and blood specimens gathered via intracardial perforation serum were collected for biochemical analysis [36].

2.5. Gross Evaluations of Stomachs

The rat’s stomach was opened along the greater curvature, and then gastric tissues were rinsed with ice-cold buffered saline. Stomach lesions on the gastric epithelium appeared as extended groups of red injuries. Ulcers usually seemed similar to the long axis of the stomach; ulcers were photographed, and the stomach ulcer area was determined using ImageJ software.
Inhibition percentage (I%) was estimated by the following formula [37]:
I% = UAcontrol − UAtreated/UAcontrol × 100

2.6. Measurement of Stomach pH

Samples of stomach fillings were gathered, and hydrogen ion concentration in the stomach fluid was estimated by pH meter (PHS-25, Shanghai, China) titration via 0.1 N NaOH solutions, utilizing numerical pH meter acidity measured by mEq/L [38].

2.7. Measurement of Stomach Mucus Content

Stomachs were washed with ice-cold phosphate-buffered saline (PBS). The gastric mucosa was softly scrubbed off using clean glass slide, and mucus weight was quantified using an accurate electrical balance [39].

2.8. Formulating Stomach Tissue Homogenate

A small portion of each rat’s glandular stomach was washed carefully with ice-cold PBS. Using a homogenizer, a section of gastric wall was homogenized (10% w/v) with cold PBS, comprising mammalian protease inhibitor cocktail. Stomach homogenates were spun at 1000× g for 10 min at 4 °C. Clear fluid was measured for the amount of superoxide dismutase (SOD), catalase (CAT), prostaglandin E2 (PGE2), and malondialdehyde (MDA). Evaluation was performed according to the corresponding manufacturer’s instructions (Cayman, Ann Arbor, MI, USA) [40].

2.9. Measurements of Endogenous Antioxidant Enzymes

SOD, CAT, and PGE2 concentrations within stomach homogenate were quantified utilizing marketable standard kits (Elabscience, Wuhan, China). The manufacturer’s procedures were used to measure the quantities of homogenate supernatant [41].

2.10. Quantities of Lipid Peroxidation (MDA) Level of Gastric Homogenate

The MDA (malondialdehyde tetrabutylammonium salt) and thiobarbituric acid reactive substances (TBARS) tests were used to determine the amount of glandular stomach homogenate via profitable kits (Elabscience, Wuhan, China), according to the manufacturer’s method [42].

2.11. Histological Evaluation

Small slices (1–2 cm) of each stomach glandular epithelium were fixed immediately in 10% buffered formalin at room temperature for 24 h. They were then shadowed via tissue dehydration by ethanol and clearance with xylene penetration by paraffin using a tissue processing machine. Then, each tissue biopsy was embedded in paraffin, and sliced into 5 μm sections for placement onto slides (Leica Rotation Microtome) [12,38].

2.12. Hematoxylin and Eosin Stain

Sectioned slides were routinely stained with hematoxylin and eosin stain for histopathological investigation using a compound microscope [43,44].

2.13. Periodic acid Schiff Stain (PAS)

Sections of the glandular layer within the gastric tissue were stained with PAS to differentiate the acidic and basic glycoproteins level in the mucus. To evaluate the mucus secretion of the stomach’s glandular epithelium, sections of 5 μm thickness, stained with PAS, were used to visualize the gastric mucus and variations of acidic and basic glycoproteins after the construction method, which followed the manufacturer’s instructions (Sigma Periodic AcidSchiff (PAS) Kit, Merk, Darmstadt, Germany). Photographed stomach sections stained with PAS were evaluated using Image J software [45].

2.14. Immunohistochemical Staining

Stomach sections of 5 μm thickness, stained with immunostaining using the Animal Research Kit (Elabscience, Wuhan, China), were used to detect the immunohistochemical localized HSP 70 (1:100) and Bax (1:50) proteins. The mentioned proteins were bought from “Santa Cruz USA” [41].
The immunohistochemical stain-positive cell examination was conducted by enumeration of positive cells utilizing a microscope (AFS Model F108, Ningbo Beilun Aofusen Instrument Co., Ltd., Ningbo, China). Microscopic images were evaluated using Image J software to quantity strength of staining. Then, the percentage of positive cells was estimated.

2.15. Measurement Provocative Cytokines (TNF-α IL-6 and IL-10)

Determination of TNF-α, IL-6, and IL-10 serum was achieved by utilizing the desirable ELISA kit (MyBioSource, San Diego, CA, USA). This was assessed by following the manufacturer’s instructions, stated in the Rat TNF-α ELISA Kit (MBS267737) and the Rat IL-6 ELISA Kit (MBS355410). Cytokine strength measurement was conducted via “normal sanitized recombinant cytokines”.

2.16. Statistical Analysis

Statistical analysis was accomplished with IBM SPSS statistics windows version 24, using the one-way analysis of variance (ANOVA) procedure followed by Turkey’s post hoc test. Normality testing was performed using the Kolmogorov–Smirnov test. Data were presented as mean ± SEM. A value of p < 0.05 indicated statistical significance.

3. Results

3.1. Acute Toxicity Study

The acute toxicity test was carried out on 36 rats, in which rats were divided similarly into three groups: G1, with no treatment as a vehicle (0.5% CMC, 5 mL/kg); G2, treated with a single dose of 250 mg/kg; and G3, treated with a single dose of 500 mg/kg p-Cymene. The rats were kept under observation for 14 days. The test results found that all the rats survived, and none experienced any significant visible signs of toxicity at these doses. In addition, continuous observation revealed no abnormal signs and symptoms, and no changes in the behavior, body weight, or water and feed intake of the rats.
The acute toxicity test did not demonstrate any sign of toxicity. There was no histopathological sign of hepatic or renal toxicity (Figure 2). Furthermore, the blood biochemistry examination appeared normal (data not shown, but is available upon request).

3.2. Effect of p-Cymene on Gross Assessment of Stomach

Rodents fed with p-Cymene exhibited significant decreases in ulcerated gastric areas in comparison with the ulcerated control group (Figure 3). A reduction of stomach epithelial surface mucosa damage was also detected in the gross evaluation of the stomachs of experimental rats that were given p-Cymene, in comparison with the ulcer control group (Figure 3).

3.3. Influence of p-Cymene on Gastric Mucus Content

Experimental animals fed with p-Cymene showed a substantial upsurge in gastric mucus secretion compared to the ulcerated control group (Table 1).

3.4. Effect of p-Cymene on pH of the Stomach

The animals that received ethanol alone showed the lowest gastric pH. and there was a dose dependent increase in gastric pH for p-Cymene-treated rats. Furthermore, administration of 30 mg/kg p-Cymene caused an increase in the pH level, but this was not as significant as the administration of 60 mg/kg p-Cymene. In other words, the animals that received ethanol had the highest total acidity. Additionally, administration of 30 mg/kg p-Cymene reduced the acidity, but not as significantly as the administration of 60 mg/kg p-Cymene. Moreover, the gastric acidity of rats receiving the omeprazole (G3) was shown to significantly decrease compared to the ulcer control (G2); however, these pH changes were not significant in p-Cymene-treated rats (G4 and G5). Experimental rodents pretreated with the p-Cymene before administration of absolute ethanol had significantly increased gastric pH in comparison with the ulcerated control group (Table 1).

3.5. H&E Stain

The ulcerated control group presented severe injury to the stomach epithelium; deep lacerations of the stomach mucosal epithelium with edema leukocyte infiltration of the subcutaneous coat were observed. Rodents in the experimental groups fed with p-Cymene showed comparatively improved defense of the gastric epithelium and a reduced ulcerated area, as well as reduction in edema inflammatory cell permeation of the submucosal layer (Figure 4).

3.6. PAS Stain

Experimental rodents fed with p-Cymene displayed comparatively increased PAS staining intensity of glycoprotein in the gastric epithelium (magenta color), in comparison with the ulcerated control group (Figure 5).

3.7. Immunohistochemical Staining

Expression of HSP 70 and Bax Proteins

The appearance of HSP 70 and Bax proteins could be observed in the stained stomach sections. HSP 70 protein displayed down-regulation in the ulcerated control group and up-regulation in the omeprazole or p-Cymene groups. The appearance of the HSP 70 protein stain was provided by an intense brown color-stained antigen in rats pre-treated with omeprazole or p-Cymene (Figure 6). Similarly, the Bax protein was significantly up-regulated in the ulcer control group and down-regulated in the omeprazole and p-Cymene pre-treated groups. We measured the expression of HSP 70 and Bax proteins in tissue sections of gastric ulcers using ImageJ software. HSP 70 protein expression was down-regulated in the ulcer control group and up-regulated in the omeprazole and p-Cymene groups. However, Bax protein expression was up-regulated in the ulcerated control group and down-regulated in the omeprazole or p-Cymene groups (Figure 7).

3.8. Effects p-Cymene Endogenous Antioxidant Enzymes of Gastric Tissue Homogenate

The ulcerated control group exhibited suggestively inferior SOD, CAT, and PGE2 activity in comparison to the normal group (Figure 8). Experimental groups fed p-Cymene presented significantly restored SOD, CAT, and PGE2 levels, reaching the normal values. Rats treated with p-Cymene 500 mg/kg had significantly higher SOD (47.301 ± 2.167) and CAT (39.34 ± 1.52) values than that of p-Cymene 250 mg/kg (43.531 ± 1.00 and 35.52 ± 2.16) and ulcer control groups (18.361 ± 0.51 and 16.355 ± 1.44), respectively. The MDA levels were significantly lower (23.645 ± 2.19) in rats treated with p-Cymene 500 mg/kg when compared to the values of 29.45 ± 3.17 and 88.816 ± 6.04 of G2 ulcer control and G4 p-Cymene groups, respectively. The normal control and omeprazole-treated rats showed non-significant changes in their SOD, CAT, and MDA profiles. The prostaglandin E2 was significantly higher (32 ± 2.36) in the G5 group compared to 11.83 ± 1.32, 7.16 ± 1.16, and 22 ± 2.60 in the G1, G2, and G4 groups, respectively (Figure 8).

3.9. Effects of p-Cymene on Malondialdehyde (MDA) in Stomach Epithelial Homogenate

Rats in the ulcer control group exhibited a substantial increase in the MDA level of stomach epithelial homogenate in comparison to the normal group (Figure 8). The MDA of stomach homogenate was significantly reduced in rats fed with p-Cymene. MDA is used as an indicator of lipid peroxidation (Figure 8).

3.10. Effect of p-Cymene on Cytokines Level in Blood

The outcomes of inflammatory cytokine examination are presented in the Figure 9. The TNF-α, IL-6, and IL-10 were found in high levels in the ulcerated control group in comparison to the rats treated with omeprazole or p-Cymene. However, rats treated with omeprazole or p-Cymene had significantly reduced TNF-α and IL-6, whereas they had increased IL-10 compared to the ulcer control (Figure 9).

4. Discussion

Medicinal plants and their active ingredients have been used in traditional medicine for stomach ulcer remedies for a long time. Numerous studies by several peptic ulcer researchers have reported on using medicinal plants and their active compounds for anti-ulcer effects in rats [2,3,23,46]. Inducing acute hemorrhagic ulcers in a rat’s stomach by absolute alcohol administration was an easy and simple method of assessing anti-ulcer action traditional medicinal active compounds because absolute ethanol simply infiltrates the stomach epithelium, thus producing stomach injuries [4,47].
Absolute ethanol administration caused extensive disruption of the stomach mucus barrier, along with declined mucus secretion and decreased endogenous enzyme levels. Additionally, ethanol augmented the microvascular permeability and increased lipid peroxidation. Moreover, ethanol improved the free-radical production abilities of the stomach epithelium, and subsequently caused severe stomach epithelial damage [12,48]. Stomach mucus might perform an important role in the epithelial defense in contradiction to ethanol [1,49,50]. The results confirmed that p-Cymene defends the mucosa via concealing extra mucous contrary to extensive gastric destruction.
In our study, experimental groups fed with p-Cymene showed flattening of gastric folds and increased epithelial surface area, and reduction of stomach injury compared to the ulcer control cluster. Similarly, several academics using medicinal plants or their active ingredients reported a flatting of the gastric mucosa and the anti-ulcer effects of stomach mucosa in rats; indeed, a previous study has shown the anti-ulcer effects of Celosia trigyna in 50 mg/kg dosage against ethanol-induced ulcers in rats. Furthermore, another research team have demonstrated Monolluma quadrangula to be an anti-ulcer medicinal plant after showing its efficacy in 150 or 300 mg/kg dosage against ethanol-induced ulcers in rats [7,50].
The gastroprotective activity of p-Cymene reduced ethanol-induced stomach damage, and was also important in lessening inflammatory cell permeation in the submucosal layer. A previous study by Formiga et al. also reported the anti-inflammatory and anti-lesion activity of p-Cymene at a dosage 200 mg/kg, and have linked these actions with the p-Cymene efficiency in the cytoprotection of the intestinal barrier, preserving the mucus layer, and improving communicating junctions, also via positive effects on the endogenous antioxidants and immunomodulatory systems [30]. The antioxidant efficiency of p-Cymene via down-regulation of up-regulated antioxidant enzymes (SOD and CAT) and down-regulation of lipid peroxidation enzymes (MDA) has also been reported by several research articles [25,51].
As displayed in the results of the histological examination of the stomachs in the ulcer control group, there were severe hemorrhagic stomach injuries with increased leucocyte (neutrophil) penetration causing edema of the submucosal coating. Conversely, experimental animal groups fed with p-Cymene before absolute ethanol administration exhibited gastroprotective effects, as evidenced by comparison of the stomach mucosa [5,52].
Similar to these results, several investigators using various medicinal plants or their active isolated compounds have reported on the gastroprotection of rats stomachs; for example, previous phytochemical and biological studies on Gynura procumbens, Morus alba, and Rafflesia hasseltii have revealed the anti-ulcer efficacy of these medicinal plants against ethanol-induced ulcers in rats, and they have correlated this plant efficiency with their ability to decrease ulcer areas, the reduction or absence of edema and leucocyte infiltration of the submucosal layer via an increase of gastric PH and endogenous antioxidant enzymes, and their positive impact on immunohistochemistry in general [53,54,55].
Our finding revealed that experimental rats fed with p-Cymene exhibited an increased concentration of PAS staining in stomach sections in comparison to the ulcer control group. Similarly, several investigators using different medicinal plants have reported an increased intensity of PAS staining in stomach sections of experimental rodents [22,56].
The outcomes of the existing study show that experimental animal groups with significantly condensed MDA had raised SOD, CAT and PGE2 stomach tissue homogenate levels in response to oxidative pressure employed by the ethanol gavage. Similarly, a huge number of studies have been reported by many investigators using medicinal plants or synthetic compounds, which reveal a rise in endogenous enzyme levels (SOD, CAT, and PGE2) and a decrease in MDA levels in stomach tissue homogenate [11,46]. Endogenous enzymes are important for mucosal self-protective structures, and mitigate the oxidative pressure that encourage stomach ulcers, thereby reducing ROS ethanol-induced stomach damage. High levels of stomach endogenous enzymes bind and dispose of free radicals formed by the ethanol [20].
The present results suggest that p-Cymene applies strong anti-inflammatory activity in the stomach epithelium, and offers important protection of stomach content with PGE2. PGE2 is the most copious PG formed in the body, and so has been extensively considered in animal studies. PGE2 moderates all types of inflammation and is accountable for augmented prostaglandin construction in inflamed tissue [35,57].
In the present research, p-Cymene conserved stomach endogenous enzymes by avoiding lipid peroxidation. p-Cymene might yield gastroprotective activity and reduce lipid peroxidation by producing malondialdehyde (MDA) [2,12,58]. Results presented in this study regarding the repair of exhausted SOD, CAT, and reduced MDA, suggest the gastroprotective effect of p-Cymene instruction on antioxidant pathways.
In the current study, absolute ethanol augmented the production of reactive oxygen species (ROS), which inhibited the appearance of HSP 70 protein and increased the presence of proapoptotic proteins. Oxidative stress injures lipids, proteins, and DNA, and causes lipid peroxidation, cellular death, and tissue impairment [1]. HSP 70 proteins defend gastric epithelium oxidative stress produced by absolute alcohol. HSP 70 proteins prevent partially denaturized proteins from gathering or refolding. Up-regulation of HSP 70 in experimental groups fed with p-Cymene led to the protection of stomach epithelia. These results are consistent with the findings of other studies; several investigators have described that the up-regulation of HSP 70 is related to stomach defense against absolute ethanol, perhaps via the weakening of ROS-mediated stomach oxidative pressure [1,20,52].
The additional significant result of our study was the capability of HSP 70 to prevent the production of oxidative stress by ethanol. Important gastroprotective influences of HSP 70 have been described by several investigators [5,59]. HSP 70 is mostly initiated in the cytoplasm, nucleus, mitochondria, cell membranes, and in the extracellular space. Its appearance arises at areas of cellular tension, and it defends against injury. In stomach ulcers, HSP 70 defense involves encouraging normal protein construction through the elimination of injured proteins [4,50,60].
The Bax protein is pro-apoptotic, and is a participant of the Bcl-2 family, connected with the regulation of programmed cell death in mitochondrial injuries [61]. Absolute ethanol can cause the initiation of programmed cell death in the stomach epithelia via the over-expression pro-apoptotic proteins; in these cases, Bax induces down-expression of anti-apoptotic bodies, such as Bcl-2 [62]. Bax protein was found to be down-regulated and HSP70 protein was up-regulated in stomach tissue sections in rats fed with p-Cymene, in comparison with the ulcer control group. These results are consistent with the outcomes of previous research, which demonstrated that the initiation of HSP70 protein accompanied by the destruction of Bax protein in rats can lead to the defense of the stomach mucosa against injuries encouraged by absolute ethanol [21,46].
TNF-α, IL-6 and IL-10, concealed by macrophages, are described as playing a significant part in ethanol-induced stomach damage by polymorphonuclear neutrophil penetration in the submucosal layer [1]. In the present study, p-Cymene reduced the level of TNF-α and IL-6, while augmenting IL-10 in blood plasma. Pre-treatment with p-Cymene significantly reduced the TNF-α, IL-6, and augmented IL-10 levels, in comparison to omeprazole. As previously stated, IL-6 stimulates neutrophils, monocytes, and lymphocytes at the site of inflammation [63]. IL-6 induces the construction of most acute phase proteins in inflammatory reactions. p-Cymene exerts a cytoprotective outcome via an anti-inflammatory mechanism.
The mechanism of action of p-Cymene might be through free radical scavenging and quenching of the formation of single oxygen, which protects the stomach against oxidative stress, and stimulates gastric repair and anti-inflammatory mechanisms.

5. Conclusions

Based on the results of the current study, p-Cymene revealed a significantly gastroprotective effect on absolute ethanol-induced stomach injury in experimental rats, as established by gross and histopathological examination. It caused a significant upsurge in stomach mucus secretion, flattening of the gastric epithelium, an increase in pH stomach content, and decreased edema and inflammatory cell penetration of the submucosal coat of the stomach wall. In stomach epithelial homogenate, p-Cymene significantly increases SOD, CAT, and PGE2 levels, and substantially reduces the MDA level. Furthermore, p-Cymene causes an up-regulation of HSP 70 protein and down-regulation of Bax protein of stomach mucosa sections in experimental rats. Preventive consequences are largely due to antioxidant and anti-inflammatory mediators in the stomach mucosa. The mechanism of action of p-Cymene might be through free radical scavenging and the quenching of the formation of single oxygen, thereby defending the gastric mucosa against the oxidative stress of ethanol, and encouraging gastric mucosal restoration and anti-inflammatory mechanisms. The current study had several limitations including a small animal house, sample size, and facility shortage. Therefore, further studies using a larger sample size and an advanced laboratory are required for more confirmations of the mentioned biological activities of p-Cymene.

Author Contributions

S.H.S. and M.A.A. structured, designed, and conducted the methodology. M.H.A.-M., I.A.A.I., A.R.A. and A.A.J. analyzed the results. M.A.A., Y.G., N.F.S.A., P.Y.A., M.A.T. and D.N.F.A. participated in writing the article with the guidance of all authors. N.A.S. performed the laboratory tests. Z.M.A. did grammatical correction. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The current animal study approved by the ethics committee in Cihan university-Erbil (Ethical No. BIO/13/09/2021/MAA).

Data Availability Statement

The details regarding data supporting the reported results are available on request.

Acknowledgments

The authors would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work [Grant Code: 22UQU4331277DSR01].

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Chemical structure and chemical properties of p-Cymene.
Figure 1. Chemical structure and chemical properties of p-Cymene.
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Figure 2. Histological sections of kidney (first column) and liver (second column) in the acute toxicity test (H&E staining, 40×). Rats treated with 5 mL/kg vehicle (0.5% CMC) (A,B); rats treated with 250 mg/kg (5 mL/kg) (C,D); rats treated with 500 g/kg (5 mL/kg) (E,F). No significant changes were observed in the structures of livers and kidneys between the treated and control groups (H&E staining, 40×).
Figure 2. Histological sections of kidney (first column) and liver (second column) in the acute toxicity test (H&E staining, 40×). Rats treated with 5 mL/kg vehicle (0.5% CMC) (A,B); rats treated with 250 mg/kg (5 mL/kg) (C,D); rats treated with 500 g/kg (5 mL/kg) (E,F). No significant changes were observed in the structures of livers and kidneys between the treated and control groups (H&E staining, 40×).
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Figure 3. Effects of p-Cymene on the macroscopic appearance of gastric mucosa in ethanol-induced gastric mucosal damage in rats. The normal control group (A); the ulcer control group (B); the reference group (omeprazole, 20 mg/kg) (C); rats that receive 30 mg/kg of p-Cymene (D); and rats that received 60 mg/kg of p-Cymene (E).
Figure 3. Effects of p-Cymene on the macroscopic appearance of gastric mucosa in ethanol-induced gastric mucosal damage in rats. The normal control group (A); the ulcer control group (B); the reference group (omeprazole, 20 mg/kg) (C); rats that receive 30 mg/kg of p-Cymene (D); and rats that received 60 mg/kg of p-Cymene (E).
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Figure 4. Effect of p-Cymene on microscopic appearance of the gastric mucosa in ethanol-induced gastric ulcers in rats. (A) The normal control group displayed normal microscopic structure of the gastric mucosa. (B) The ulcer control group presented with severe structural injuries of the gastric mucosa with edema and leucocyte infiltration of the submucosal layer. (C) The omeprazole group exhibited mild damage of the gastric mucosa. (D) The group given p-Cymene 30 mg/kg presented moderate damage of the gastric mucosa. (E) The group given p-Cymene 60 mg/kg showed relatively mild damage of the gastric mucosa (H&E stain, magnification 10×).
Figure 4. Effect of p-Cymene on microscopic appearance of the gastric mucosa in ethanol-induced gastric ulcers in rats. (A) The normal control group displayed normal microscopic structure of the gastric mucosa. (B) The ulcer control group presented with severe structural injuries of the gastric mucosa with edema and leucocyte infiltration of the submucosal layer. (C) The omeprazole group exhibited mild damage of the gastric mucosa. (D) The group given p-Cymene 30 mg/kg presented moderate damage of the gastric mucosa. (E) The group given p-Cymene 60 mg/kg showed relatively mild damage of the gastric mucosa (H&E stain, magnification 10×).
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Figure 5. Effect of p-Cymene on microscopical observation of PAS glycoprotein staining (A–E) of the gastric mucosa in ethanol-induced gastric ulcers in rats. (A) Normal group—normal gastric mucosal structure and no signs of damage; (B) ulcer control group—severe damage to gastric mucosa and mild PAS stain (magenta color); (C) omeprazole group—insignificant damage and intensive PAS staining of gastric mucosa; (D) p-Cymene 30 mg/kg—mild to moderate damage of gastric mucosa with mild PAS stain of gastric mucosa; (E) p-Cymene 60 mg/kg—mild damage of gastric mucosa and with moderate PAS stain of gastric mucosa (PAS stain, magnification 20×).
Figure 5. Effect of p-Cymene on microscopical observation of PAS glycoprotein staining (A–E) of the gastric mucosa in ethanol-induced gastric ulcers in rats. (A) Normal group—normal gastric mucosal structure and no signs of damage; (B) ulcer control group—severe damage to gastric mucosa and mild PAS stain (magenta color); (C) omeprazole group—insignificant damage and intensive PAS staining of gastric mucosa; (D) p-Cymene 30 mg/kg—mild to moderate damage of gastric mucosa with mild PAS stain of gastric mucosa; (E) p-Cymene 60 mg/kg—mild damage of gastric mucosa and with moderate PAS stain of gastric mucosa (PAS stain, magnification 20×).
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Figure 6. Effects of p-Cymene on microscopical observation of HSP 70 protein expression (A–E) and quantitative analysis (F) of HSP 70 protein expression of gastric mucosa in ethanol-induced gastric ulcers in rats. Normal control group displayed normal gastric mucosal structure and very weak expression of HSP 70 protein in the gastric mucosa (A,F). Ulcer control group presented severe injury of the gastric mucosa and down-regulation of HSP 70 protein expression in ethanol-induced gastric ulcers in rats (B,F). Omeprazole group exhibited mild damage of the gastric mucosa and up-regulation of HSP 70 protein expression of gastric mucosa in ethanol-induced gastric ulcers in rats (C,F). p-Cymene group given 30 mg/kg showed moderate injury of the gastric mucosa and up-regulation of HSP 70 protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (D,F). p-Cymene group given 60 mg/kg displayed mild damage of the gastric mucosa and up-regulation of HSP 70 protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (E,F). (HSP 70 stain, magnification 20×). The data are presented as means ± SED. The antigen site appears as a brown color. There was significant (****) differentiation between experimental groups in HSP 70% protein expressions with the highest expressions recorded for the C and E rat groups. ns, non-significant; *, p < 0.05; ****, p < 0.0001.
Figure 6. Effects of p-Cymene on microscopical observation of HSP 70 protein expression (A–E) and quantitative analysis (F) of HSP 70 protein expression of gastric mucosa in ethanol-induced gastric ulcers in rats. Normal control group displayed normal gastric mucosal structure and very weak expression of HSP 70 protein in the gastric mucosa (A,F). Ulcer control group presented severe injury of the gastric mucosa and down-regulation of HSP 70 protein expression in ethanol-induced gastric ulcers in rats (B,F). Omeprazole group exhibited mild damage of the gastric mucosa and up-regulation of HSP 70 protein expression of gastric mucosa in ethanol-induced gastric ulcers in rats (C,F). p-Cymene group given 30 mg/kg showed moderate injury of the gastric mucosa and up-regulation of HSP 70 protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (D,F). p-Cymene group given 60 mg/kg displayed mild damage of the gastric mucosa and up-regulation of HSP 70 protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (E,F). (HSP 70 stain, magnification 20×). The data are presented as means ± SED. The antigen site appears as a brown color. There was significant (****) differentiation between experimental groups in HSP 70% protein expressions with the highest expressions recorded for the C and E rat groups. ns, non-significant; *, p < 0.05; ****, p < 0.0001.
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Figure 7. Effects of p-Cymene on microscopical observation of Bax protein expression (A–E) and quantitative analysis (F) of Bax protein expression of the gastric mucosa in ethanol-induced gastric ulcers in rats. Normal control group displayed normal gastric mucosal structure and very weak expression of Bax protein in the gastric mucosa (A,F). Ulcer control group presented severe damage of the gastric mucosa and up-regulation of Bax protein expression in ethanol-induced gastric ulcers in rats (B,F). Omeprazole group exhibited mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in etha-nol-induced gastric ulcers in rats (C,F). p-Cymene group given 30 mg/kg showed moderate injury of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (D,F). p-Cymene group given 60 mg/kg displayed mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (E,F). (Bax stain, magnification 20×). The data are presented as means ± SED. The antigen site ap-pears as a brown color. There was significant (****) differentiation between experimental groups in HSP 70% protein expressions with the highest expressions recorded for the B rat group. ns, non-significant; ** p < 0.001; **** p < 0.0001.
Figure 7. Effects of p-Cymene on microscopical observation of Bax protein expression (A–E) and quantitative analysis (F) of Bax protein expression of the gastric mucosa in ethanol-induced gastric ulcers in rats. Normal control group displayed normal gastric mucosal structure and very weak expression of Bax protein in the gastric mucosa (A,F). Ulcer control group presented severe damage of the gastric mucosa and up-regulation of Bax protein expression in ethanol-induced gastric ulcers in rats (B,F). Omeprazole group exhibited mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in etha-nol-induced gastric ulcers in rats (C,F). p-Cymene group given 30 mg/kg showed moderate injury of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (D,F). p-Cymene group given 60 mg/kg displayed mild damage of the gastric mucosa and down-regulation of Bax protein expression of the gastric mucosa compared to the ulcer control group in ethanol-induced gastric ulcers in rats (E,F). (Bax stain, magnification 20×). The data are presented as means ± SED. The antigen site ap-pears as a brown color. There was significant (****) differentiation between experimental groups in HSP 70% protein expressions with the highest expressions recorded for the B rat group. ns, non-significant; ** p < 0.001; **** p < 0.0001.
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Figure 8. The effect of p-Cymene on PGE2, MDA, CAT, and SOD in ethanol-induced gastric ulcers in rats. Pre-treatment groups: normal control 0.5% CMC (G1), ulcer control 0.5% CMC (G2), omeprazole 20 mg/kg (G3), p-Cymene 30 mg/kg (G4), p-Cymene 60 mg/kg (G5). Values presented as means (n = 6). ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Figure 8. The effect of p-Cymene on PGE2, MDA, CAT, and SOD in ethanol-induced gastric ulcers in rats. Pre-treatment groups: normal control 0.5% CMC (G1), ulcer control 0.5% CMC (G2), omeprazole 20 mg/kg (G3), p-Cymene 30 mg/kg (G4), p-Cymene 60 mg/kg (G5). Values presented as means (n = 6). ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Figure 9. The effect of p-Cymene on TNF-α, IL-6 and IL-10 in ethanol-induced gastric ulcers in rats. Pre-treatment groups: normal control 0.5% CMC (G1), ulcer control 0.5% CMC (G2), omeprazole 20 mg/kg (G3), p-Cymene 30 mg/kg (G4), p-Cymene 60 mg/kg (G5). Values presented as means (n = 6). ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
Figure 9. The effect of p-Cymene on TNF-α, IL-6 and IL-10 in ethanol-induced gastric ulcers in rats. Pre-treatment groups: normal control 0.5% CMC (G1), ulcer control 0.5% CMC (G2), omeprazole 20 mg/kg (G3), p-Cymene 30 mg/kg (G4), p-Cymene 60 mg/kg (G5). Values presented as means (n = 6). ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Table
Table 1. Effect of the p-Cymene on mucus weight, pH of stomach, ulcer area, and inhibition percentage of ulcer area in ethanol-induced gastric ulcer rats.
Table 1. Effect of the p-Cymene on mucus weight, pH of stomach, ulcer area, and inhibition percentage of ulcer area in ethanol-induced gastric ulcer rats.
Animal GroupsPre-Feeding
(5 mL/kg)		Mucus Weight (g)pHUlcer Area
(mm)2		Inhibition
(%)
G1—Normal control0.5 CMC1.8967 ± 0.65 a6.116 ± 0.53 a--
G2—Ulcer control0.5% CMC0.695 ± 0.32 b2.86 ± 0.07 b675.66 ± 9.66 a-
G3—Omeprazole20 mg/kg omeprazole1.8917 ± 0.47 a6.11 ± 0.50 a98.33 ± 4.45 b84.26% a
G4—p-Cymene (low dose)30 mg/kg
p-Cymene		1.5117 ± 0.39 c5.14 ± 0.05 a150.66 ± 49 c77.70% b
G5—p-Cymene (high dose)60 mg/kg
p-Cymene		1.7917 ± 0.61 d5.67 ± 0.53 a109.166 ± 4.62 d83.84% c
Mean value ± standard deviation (n = 6). Values indicated by different superscript letters within the same column are significantly different according to Tukey’s honestly significant difference test at a 5% significance level.
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Shareef, S.H.; Al-Medhtiy, M.H.; Ibrahim, I.A.A.; Alzahrani, A.R.; Jabbar, A.A.; Galali, Y.; Agha, N.F.S.; Aziz, P.Y.; Thabit, M.A.; Agha, D.N.F.; et al. Gastroprophylactic Effects of p-Cymene in Ethanol-Induced Gastric Ulcer in Rats. Processes 2022, 10, 1314. https://doi.org/10.3390/pr10071314

AMA Style

Shareef SH, Al-Medhtiy MH, Ibrahim IAA, Alzahrani AR, Jabbar AA, Galali Y, Agha NFS, Aziz PY, Thabit MA, Agha DNF, et al. Gastroprophylactic Effects of p-Cymene in Ethanol-Induced Gastric Ulcer in Rats. Processes. 2022; 10(7):1314. https://doi.org/10.3390/pr10071314

Chicago/Turabian Style

Shareef, Suhayla H., Morteta H. Al-Medhtiy, Ibrahim Abdel Aziz Ibrahim, Abdullah R. Alzahrani, Ahmed Aj. Jabbar, Yaseen Galali, Nabaz Fisal Shakir Agha, Peshawa Y. Aziz, Muthanna A. Thabit, Derin N. F. Agha, and et al. 2022. "Gastroprophylactic Effects of p-Cymene in Ethanol-Induced Gastric Ulcer in Rats" Processes 10, no. 7: 1314. https://doi.org/10.3390/pr10071314

APA Style

Shareef, S. H., Al-Medhtiy, M. H., Ibrahim, I. A. A., Alzahrani, A. R., Jabbar, A. A., Galali, Y., Agha, N. F. S., Aziz, P. Y., Thabit, M. A., Agha, D. N. F., Salehen, N. A., Ameen, Z. M., & Abdulla, M. A. (2022). Gastroprophylactic Effects of p-Cymene in Ethanol-Induced Gastric Ulcer in Rats. Processes, 10(7), 1314. https://doi.org/10.3390/pr10071314

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