Gastric ulcer is a painful lesion of the gastric mucosa which can be disabling, or even more very serious in the case of a perforation of the stomach and internal hemorrhage. Traditional pharmacopeias have shown the efficacy of various plant extracts in the treatment of this pathology. Some extracts from
Gastric ulcer is considered one of the most common human gastroduodenal disorders in the world. It is a disease characterized by a painful lesional excoriation, extending at least to the muscular mucosa of the gastric and/or duodenal wall, and more rarely of the lower portion of the esophagus close to the
The mucous membranes are contiguous to the skin which constitute the external protective barrier of the human body. They ensure the protection of the body’s internal cavities which are in relation to the external environment (respiratory, digestive, urinary, and reproductive systems). If the skin is protected by the squamous layer composed of dead keratinocytes, the internal mucous membranes are protected by mucus since the cells covering them are all living cells and are highly permeable, much more than the skin.
It is well admitted that the aim of healing skin injuries of full-thickness excisions, incisions, burns, erosions, and ulcers is to promptly close the wounds by full reepithelialization, in order to avoid microbial infections and to restore the elasticity and functionality of the extracellular dermal matrix, as well as the reformation of the skin appendages. The healing of gastric ulcers is also intended to restore the gastric epithelium and to reconstruct the extracellular matrix in order to close the wounds and also to restore all the glandular and other structures that constitute the gastric wall.
Some of the methods used for the assessment of the gastrodefensive effects of plant extracts or synthetic drugs are the pylorus ligation model [
Some fixed vegetable oils extracted by various processes (solvent, supercritical CO2, and cold pressure) have been proven to heal skin wounds [
The current study was devoted to the investigation of the gastroprotective as well as healing potential of OFI oil as a therapeutic against ethanol-induced peptic ulcer in rat model.
Fruits of prickly pear
The extra virgin oil of mature prickly pears seeds was naturally extracted by first cold pressing using a mechanical machine in order to preserve the quality of its components. The oil was filtered and stored in anti-UV hermetic bottles at room temperature.
Absolute ethanol was purchased from VWR Chemicals, Prolabo (France); sucralfate (Ulcar, 1 g; Sanofi-Aventis, France) and ranitidine (Azantac, 75 mg; GlaxoSmithKline, France) were acquired from a local pharmacy. The drug doses were prepared immediately before administration to the rats.
The analyses were performed at the laboratories of the National Oil Office of Tunisia (ONH).
The analyses were carried out according to the official methods of AOAC (American Oil Chemists’ Society, International). Saponification index was determined according to the Norm ISO 3657: 2013. Peroxide value was estimated in meq O2/kg of oil (NF T60-2201998). Refractive index was measured at 20° with an Abbe refractometer with temperature adjustment. Density was determined at 20° by gravimetry. Acid index and iodine value (g I2/100g) were calculated according to NF ISO 660-1996 and AOAC official method 940.28 (2013), respectively.
The identification and the quantification of the free fatty acids in the
The calculated oxidizability (Cox) value of the oil was calculated applying the formula based on the percentage of unsaturated C18 fatty acids, proposed by Fatemi and Hammond [
The analysis of triacylglycerol (TAG) fractions in OFI oil was carried out by the official qualitative and quantitative chromatographic method of the Equivalent Carbon Number (ECN 42) [
The OFI oil sterol identification and quantification were performed by Gas Chromatography-Flame Ionization Detection (GC-FID) [
The identification and quantification of the tocopherol fractions were carried out by high-pressure liquid chromatography (HPLC) [
OFI oil (1.5 g) was dissolved in 5 mL cyclohexane. Chlorophyll and carotenoid amounts were determined by the colorimetric method described in [
Chemical structures of pheophytin and lutein fractions. Images depict the chemical structures of major chlorophyll pigments in OI oil.
Total phenolic content in OFI oil was quantified using the Folin-Ciocalteu method as described in [
The determination of the flavonoid content in OFI oil was carried out as described in [
The scavenging activity towards the DPPH radical of OFI oil was evaluated using the method described in [
The percentage of inhibition (%) of the free radical DPPH was calculated as follows:
The results were expressed as Vit. C eq/g oil.
An ABTS assay was carried out as described in [
Adult male albino Wistar rats purchased from SIPHAT (Tunis, Tunisia) weighing from 180 to 190 g were acclimated for 2 weeks in the laboratory under environmentally controlled conditions: temperature of
A total of 30 adult rats were divided into three groups (
The antiulcerogenic effects of OFI oil were investigated using the ethanol-induced ulcer model in rats as described in [ Group 1: negative control group, was administered by intragastric gavage ( Group 2: positive control group, was administered by Group 3: sucralfate group, pretreated by Group 4: ranitidine group, pretreated by Group 5: dose 1 oil group, pretreated by Group 6: dose 2 oil group, pretreated by
After one hour to ethanol exposure, the stomachs were removed from each rat, cut along the great curvature, and internally inspected. Gastric ulcerated area surface, ulcer index, percentage of inhibition of ulceration, gastric mucus weight, and volume and pH of the gastric juice were determined.
The gastric ulcerated area surface was calculated in mm2 by planimetry, tracing all the ulcerated areas on a transparent graph paper, then summing them. The ulcer index (UI) was estimated as described in [
The mucus covering the gastric mucosa of each rat was gently scraped using a clean glass slide, then weighed using a sensitive balance, and the gastric volume measured using a graduated test tube as described in [
The induction of peptic ulcer was undertaken using the absolute ethanol Group 1: positive control group, not treated Group 2: sucralfate group, treated by Group 3: ranitidine group, treated Group 4: dose 1 oil group, treated Group 5: dose 2 oil group, treated
Every day 10 animals from each group were sacrificed. Their stomachs were quickly removed, opened along the great curvature, and the ulcerated areas measured as described above. Microphotographs of the scars were taken with a microscope. The remaining animals from each group were treated with their respective treatments until the next day, and so on until the end of the treatment period which was of five days till the complete healing of the mucosa ulcer of one of the groups.
Tissue specimens taken from 3 parts of the stomachs (cardia, fundus, and antrum) from the experimental animal groups were immersed in neutral-buffered formalin solution (10%) and dehydrated through a series of alcohol-water solutions using a Shandon tissue processor (Citadel 2000). After being cleared, the tissues were embedded in paraffin wax. The sections (5
Statistical data analysis was performed using SPSS statistical package (version 20.0), followed by
Table
Physicochemical characteristics of OFI oil.
Parameters | |
---|---|
Physical state at room temperature | Liquid |
Color | Greenish yellow |
Odor | Slightly fruity |
Property | Dry oil |
Texture | Noncomedogenic |
Density at 20°C (mass/volume) | |
Density by gravimetry at 20°C | |
Refractive index at 20°C | |
Acid index | |
Peroxide index (meq O2/kg of oil) | |
Iodine index (g I2/100 g of oil) | |
Saponification index (mg of KOH/g oil) |
The chromatography profile of the free fatty acid composition in OFI oil extracted by cold pressing (Figure
Chromatographic profile spectra of the fatty acids in the OFI oil. Data analysis of the chromatographic profile of free acids in OFI oil indicates the following composition according to their appearance on the graph: 1—C14 : 0; 2—C16 : 0; 3—C16 : 1; 4—C17 : 0; 5—C17 : 1; 6—C18 : 0; 7—C18 : 1; 8—C18 : 2; 9—C18 : 3
Free fatty acid composition of OFI oil.
Content (g/100 g of total fatty acids) | |
---|---|
Fatty acids | |
C14 : 0 | |
C16 : 0 | |
C16 : 1 |
|
C17 : 0 | |
C17 : 1 |
|
C18 : 0 | |
C18 : 1 |
|
C18 : 2 |
|
C 18 : 3 |
|
C20 : 0 | |
C22 : 0 | |
Fatty acid groups | |
SFA | |
UFA | |
UFA/SFA | |
PUFA | |
MUFA | |
PUFA/UFA (%) | |
Cox value |
SFA: saturated fatty acids; UFA: unsaturated fatty acids; PUFA: polyunsaturated fatty acids; MUFA: monounsaturated fatty acids; UFA/SFA: unsaturated fatty acid to saturated fatty acid ratio; PUFA/UFA: polyunsaturated fatty acid to unsaturated fatty acid ratio; values given are the means of three measurements±standard error.
The different categories of TAGs in OFI oil analyzed on the basis of the equivalent carbon number method (ECN 42) are presented in Table
Triacylglycerol (TAG) composition and positional distribution of fatty acids in OFI oil.
ECN (42) | TAG (g/100 g of total TAG) | ||
---|---|---|---|
ECN 42 | LLL | ||
PoLL | |||
ECN 44 | OLL | ||
PLL | |||
ECN 46 | OOL | ||
PoOO | |||
SLL | |||
ECN 48 | PLP | ||
OOO | |||
SOL | |||
POO | |||
ECN 50 | POP | ||
SOO | |||
AOO |
Figure
Chromatographic profile spectra of the sterols in the OFI oil obtained by GC-FID. GC-FID chromatographic profile indicates the following OFI oil composition in phytosterols according to their appearance on the graph: 1—cholesterol; 2—brassicasterol; 3—campesterol; 4—stigmasterol; 5—clerosterol; 6—
Phytosterol composition of OFI oil.
Sterol fractions | Content (% of total sterols) |
---|---|
Campesterol | |
Stigmasterol | |
Clerosterol | |
Cholesterol | |
Brassicasterol |
Vitamin E tocopherol fractions were estimated in mg/kg oil as follows:
Vitamin E tocopherol composition in OFI oil.
Tocopherol fractions | Content (mg/kg oil) | % total tocopherols |
---|---|---|
1.33 | ||
92.42 | ||
6.24 | ||
Total tocopherols | — |
Our findings indicated that OFI oil has a total phenolic content of 26.5 Gallic acid eq/g oil, flavonoid content of 3.1 mg Quercetin eq/g oil, carotenoid content of 10.520 mg/kg oil, and total chlorophyll content of 4.57 mg/kg oil (Table
Total phenolics, flavonoids, carotenoids, and total chlorophyll contents.
Compounds | Content |
---|---|
Total phenolics (Gallic acid eq/g oil) | |
Flavonoid mg Quercetin eq/g oil | |
Carotenoid (mg/kg) | |
Total chlorophylls (mg/kg) |
Table
Scavenging activity against free radical DPPH and ABTS of OFI oil.
Free radical | % inhibition (Vit. C eq/g oil) |
---|---|
DPPH | |
ABTS |
In this study, we did not notice any toxicity symptoms or mortality in the orally treated animals neither with the dose of 3.5 mL nor with dose of 7 mL of OFI oil/kg/bw over the five days of the experimental period.
Figure
Effects of the pretreatments on ulcer (a, b) and gastric parameters (c, d, and e) in ethanol-induced gastric ulcers in rats. Graphs represent the impact of absolute ethanol on ulcer and gastric parameters and the effects of the pretreatments in preventing gastric mucosae damages.
Our data indicated that absolute ethanol intake (positive control) provoked a wide range of ulceration in gastric mucosa. Sucralfate pretreatment significantly reduced ulcer areas (
Absolute ethanol intake caused a significant reduction of mucus secretion (
Ethanol intragastric gavage significantly enhanced (
Macroscopic assessment of the pretreatment on gastric mucosa ethanol-induced ulcer: negative control (a), positive control (b), sucralfate pretreated (c), ranitidine pretreated (d), dose 1 oil pretreated (e), and dose 2 oil pretreated (f). Scale bars on the photos indicate 10 mm. Images show gross assessment of the stomachs of the different experimental groups. Ulcer degree induced by ethanol was alleviated in a crescent mode by sucralfate, ranitidine, OFI oil dose 1, and OFI oil dose 2.
Microscopic assessment of the upper surface of ethanol-ulcerated gastric mucosae symptoms (
Treatment with absolute alcohol in the positive group caused severe vascular congestion of the lamina propria at the esophageal-cardiac junction, cellular desquamation, and a discrete surface erosion of the mucosa with moderate infiltration of neutrophils. At the fundic level, the vascular congestion was more significant as it reached the submucosal and subserosal vessels. In addition, treatment with absolute alcohol caused significant acidophilic necrosis of the crypt layer. A desquamative degeneration of the cells of the deep glands has been observed. At the antral level, a vascular congestion caused by alcohol was as severe as that observed at the fundus level. Necrosis with extensive disbonding of the crypto layer has been caused, reaching even the muscular layer in some areas, associated with the degeneration of glandular cells Pretreatment with sucralfate prior to the induction of gastric ulcer by absolute ethanol reduced the symptoms observed at the three levels mentioned above. At the esophageal-cardiac junction, sucralfate provided moderate mucosal protection. Superficial desquamation and slight vascular congestion were noted. At the fundic level, we observed superficial erosion of the surface epithelium, massive desquamation of cells at the glands and crypts, and discrete inflammatory infiltration by neutrophil polymorphs of the submucosa. At the antral level, we recorded clear desquamation of the glands as well as significant vascular congestions caused by absolute ethanol and infiltration with polymorphonuclear neutrophils and macrophages At the esophageal-cardial junction level, the force-feeding of the rats with absolute ethanol after pretreatment of the gastric mucosa with ranitidine caused less damage than that in the negative control and sucralfate group. Minimal cardiac desquamation, low neutrophil infiltration, and slight vascular congestion have been nevertheless observed. At the fundic level, massive cellular desquamation, some superficial exulcerations, reduced vascular congestion, and moderate polynuclear infiltration were noted. At the antral level, we noticed some neutrophil infiltrates. However, the glands were preserved. Histopathological examination of the H&E-stained biopsies collected from gastric mucosae from rats pretreated with OFI oil indicated that symptoms of ethanol-induced damages were minimal. Dose 2 oil (7 mL/kg/bw) was more efficient than dose 1 oil (3.5 mL/kg/bw). At the esophageal-cardial junction, epithelial cellular desquamations and neutrophil leukocyte infiltrations in the chorion were much reduced. At the fundic and antral levels, submucosal vessel congestion was reduced. A very low epithelial cellular desquamation accompanied by some superficial erosions have been observed. Some of these observations were presented in Figure
Microscopic assessment of H&E staining biopsies from gastric mucosa of the different experimental groups. M.er: mucosal erosion; N: necrosis; U: ulcer; Vc: vascular congestion. Images depict microscopic assessment of H&E staining biopsies collected from gastric mucosa from the experimental groups. Symptoms like mucosal ulcer, necrosis, and vascular congestion are indicated on the photographs.
Figure
Healing rate evolution of the ulcerated areas during the five days postulcer induction in the different experimental groups. Image represents the evolution of the healing rate of the ulcerated surface of gastric mucosa over five days postulcer induction. Comparison between the different groups indicates that OFI oil dose 2 is the most efficient treatment to speed-up the healing process compared to OFI oil dose 1, ranitidine, and sucralfate.
Gastric ulcer is a disease that is spreading all over the world causing enhanced morbidity and mortality rates in humans. Its etiology is due to an imbalance between the protective and aggressive factors of the gastric mucosa. Allopathic medicines used in the treatment of this pathology have many side effects which can be very harmful. Therapeutics used in the treatment of gastric ulcer are based mainly on the use of proton pump inhibitors (PPIs) as well as drugs that reduce HCl production by acting on histamine H2-receptors and those that stimulate mucus synthesis or act as gastric dressings [
The current study highlighted the antiulcerative protective and healing efficiency of OFI oil extracted by cold pressing against acute peptic ulcer induced by absolute ethanol, in comparison to two allopathic reference drugs, sucralfate and ranitidine. Our data have shown that OFI oil has significantly reduced the severe mucosal damages (lesions, ulcers, bleeding, and necrosis) observed in several areas in the gastric wall of starved rats then orally treated with absolute ethanol (positive control). We noted a major protection with dose 2 (7 mL/kg/bw) compared to dose 1 (3.5 mL/kg/bw) as it may give sufficient amounts of active biocompounds to be efficient. Gastric parameters were better normalized (
Pretreatment with the reference drugs had reduced the surface of the lesions and the necrosis areas in the gastric mucosa but the ulcerogenic effect of ethanol remained relatively strong. Ranitidine is a drug commonly administered to cure peptic ulcer and the gastroesophageal reflux disease (GERD) [
Our results are consistent with previous studies that have reported the ulcerative effect of orally applied ethanol on gastric mucosa [
The linear and punctual lesions inflicted to gastric mucosa by absolute ethanol intake could be due to a direct damaging effect on mucosal cell membranes and to indirect effects by the stimulation of inflammatory pathways with an upregulation of gene expression of proinflammatory cytokines such as TNF-
Furthermore, ethanol evoked an overproduction of HCl by parietal cells, thus reducing gastric juice pH and enhancing gastric juice volume. However, OFI oil pretreatment especially with dose 2 significantly reduced gastric juice volume and acidity, thus minimizing acid aggression of the mucosa. HCl secretion takes place in the parietal cells of the oxyntic glands following stimulation by gastrin, produced by the endocrine G cells of the glands. Histamine secreted by ECL (enterochromaffin-like) cells from the fundic glands contributes to the stimulation of mucosal parietal cells [
The significant increase in pH of gastric juice and mucus weight (
On one hand, our current study has demonstrated that OFI oil exhibited a strong healing effect of ethanol-induced gastric ulcer. The comparison between all experimental groups showed that gastric ulcer could heal naturally without treatment but in a slow mode. The healing process has been accelerated in ascending mode by ranitidine then sucralfate, OFI oil dose 1, and finally by OFI oil dose 2. The evolution of the ulcerated area during five days after inducing ulcer indicated a speeding-up of the healing process compared to the controls and to the reference drug-treated rats. In a previous work [
It should be remembered that the healing process is a very important phenomenon in restoring the integrity of the external (skin) and internal (mucous membranes) barriers of the human body. Any injury can be a gateway to germs and certain harmful or toxic substances.
The gastric ulcer healing process is an innate, genetically programmed injury repair of gastrointestinal mucosae. It includes overlapping phases: stopping blood loss if bleeding has occurred, inflammation, cell proliferation, reepithelialization and angiogenesis, granulation tissue formation, and extracellular matrix reconstitution and remodeling, as well as the reestablishment of gastric glands and other mucosal components [
Cellular and molecular events occur in the ulcer margin (mainly in epithelial cells), while other events occur in its base (mesenchymal cells and extracellular matrix). For instance, EGF-R, c-fos, c-jun, egr-1, Sp-1, TFF-2/SP, PDGF, EGF, VEGF, HGF, bFGF, KGF, and TGF-
OFI oil in sufficient amounts would on one hand have accelerated the ulcer healing rate by ensuring a protective lipid layer to the wound site against dehydration (like a dressing covering the mucosal crypts in a mimetic way such as sucralfate), thus promoting cytokine, growth, and transforming factor actions and on the other hand positively affected signaling pathways to restore the balance between aggressive and protective agents to regenerate mucosal components. This could be attributed to OFI oil antioxidants previously mentioned. The anti-inflammatory activity of phytosterols, especially
Our current findings suggest that
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this article.
Special thanks are due to Dr. Abdelkhalek Ben Rajeb (Anatomopathological Explorations Laboratory, Ennasr, Tunis) for his assistance in histological gastric biopsy slide interpretation and to Dr. Kamel Ben Ammar (National Oil Office, Tunis) for facilitating of OFI oil analysis in their laboratory.