Evaluation of Gastroprotective Effect of Betalain-Rich Ethanol Extract from Opuntia stricta var . Dillenii Employing an In Vivo Rat Model

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Introduction
Gastric ulcer disease is characterized by lesions and alterations in the gastric mucosa. Ulcers are characterized by anatomical destruction of the mucosa, which is immersed in gastric acid. Tis disease occurs as a result of many factors, such as a reduction in the mucus secretion associated with prolonged ingestion of anti-infammatory drugs, infection with Helicobacter pylori, and others. Gastric ulcers are characterized by many symptoms such as gastric pain, vomiting, stress gastrointestinal symptoms, abdominal discomfort, and fatulence. In patients, with gastric ulcers, the treatment is performed using synthetic drugs such as proton pump inhibitors, antiacids, and H 2 receptor antagonists. Nevertheless, these synthetic drugs cause many toxic efects such as cardiac arrest and liver-kidney toxicities [1][2][3][4][5][6][7][8]. Accordingly, recent research has focused on the search for efective plant-derived substances, which can be good sources for food additives as colorant, sweetener, preservative, and aroma [9][10][11][12][13][14][15][16]. Currently, there is a signifcant increase in the use of plant-derived colorants with a protective action against various perturbations and diseases such as diabetes, cancers, osteoporosis, and hyperlipidemia [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. It is notable that the ingestion of O. stricta prickly pears also improves platelet function and hemostatic balance, thus contributing to prevent atherosclerotic risks [33,34]. In this context, previous studies have reported that Opuntia fcusindica consumption protects from gastric diseases and perturbation [8,[35][36][37]. Of special interest, betalains are plant water-soluble pigments that can be used as natural colorants in sweets, juices, ice creams, and beverages without toxic efects [6,7]. In fact, betalains have interesting technological properties such as solubility in water and food color intensity. Previous studies have reported that betalains prevent the oxidation of low-density lipoproteins (LDL) and therefore help reduce patients' cardiovascular disease risk. Moreover, betalains prevent infammation in various organs such as the kidney, liver, and stomach [38,39]. Previous studies have reported that betalains exhibit cancer proliferation in the human colon, ovaries, and cervical epithelium [39][40][41][42][43][44]. No study has, however, been provided on the antiulcer efect of betalain-rich extract. Accordingly, the present study evaluated the action of the administration of O. stricta betalain-rich extract from the pulp and peel to rats with ethanol-induced gastric ulcers by the gastric gavage route.

O. stricta Collection.
Matured feshy fruit Opuntia stricta was collected during the month of November 2021 without damage and were laved and skinned manually. Te pulp or peel was blended and the seeds were removed and the juice was lyophilized.

Preparation of Betalain-Rich Extracts. Te betalains from
O. stricta (bar code ID: 000003165) pulp and peel were extracted according to the protocol of Sawicki et al. [45]. In brief, 20 g of pulp or peel from O. stricta and 10 mL of absolute methanol were mixed. Te mixture was homogenized for 5 min. After, 1 g of sodium citrate, 0.01 g of disodium citrate, 1 g sodium chloride, and 4 g of magnesium sulfate were added. Te mixture was homogenized for 5 min and then centrifuged for 15 min/3500 × g. Ten, the supernatant was mixed with 0.15 g strong anion exchanger (SAX) sorbent and 1 g magnesium sulfate. Te samples were homogenized for 3 min and centrifuged for 5 min/3500 × g. Finally, the supernatant was lyophilized, and two powders were obtained from the pulp and peel with betalain levels 94% and 96%, respectively.

Antiulcer Activity
Absolute ethanol was administered by the gastric gavage route to rats, 1 hour after the BRE or omeprazole ingestion. 90 min later, the rats were sacrifced and the stomach was detached from rats and was opened along the greater curvature. Te stomachs were gently rinsed with iced cold phosphate bufer for cleaning. Te stomachs were strained and photos were taken to examine the gastric lesions. Parts of the stomach of the diferent groups of rats were crushed in phosphate bufer, after centrifugation at 5000 rpm, and the samples were stored at −80°C until used for the biochemical analysis. Te area of gastric lesion was measured using ImageJ program.

Gastric Ulcerative Lesions and Ulceration Index
Determinations. Te total ulcer area for cleansed stomach was measured using an inverted microscope with a digital camera. Te stomach ulcer area was calculated by the ImageJ software (version 1.51J8) with digital calculable distance (mm) by means of an e-ruler. Te ulcer index (UI) was determined following the formula: ulcer index � ((ulcer area)/(total mucosa surface area)) × 100. Te curative ratio was determined following the formula: curative ratio � (US control − US treated)/(US control) [50].

Analytical Methods.
Te gastric volume was calculated by the methods described by [51]. Gastric juice was collected by centrifugation of gastric mucus at 3500 * g/ 5 min/4°C to remove insoluble mucosal gastric. Te gastric juice was calculated using graduate tubes. Te stomach lipid peroxidation was estimated by the determination of the rate of thiobarbituric acid reactive substances (TBARS) by the technique described by Buege and Aust [52]. Te superoxide dismutase activity was determined by the process of Marklund and Marklund [53]. Te glutathione peroxidase (GPx) activity was determined according to the protocol of Pagila and Valentine [54]; and the catalase (CAT) activity was measured according to the protocol of Aebi [55]. Te protein level was assayed by the determination of the level of proteins reacting with Cu 2+ in the alkaline solution (Kits Biolabo, France, ref. 95010). Te pH of the gastric juice was determined by a digital pH meter. Te histological study was performed according to the process described in our previous studies [56].

Statistical Analysis.
Tables and fgures are presented as means ± standard deviation. Te diferences between all groups were evaluated using ANOVA and Fisher's test, and the signifcance variation was considered at p ≤ 0.02.

Acute Toxicity Study.
Rats supplemented with BRE at quantities 0.8 and 1.6 g/kg were reserved for 7 days. Rats supplemented with BRE at a dose that varies from 0.2 to 1.6 mg/kg were characterized by the absence of any signs of toxicity and abnormalities such as behavioral changes, body weight changes, and biochemical analysis in blood changes.

Efects of BRE on Ethanol-Induced Macroscopic Lesions in
Rats' Stomach. Tis study showed that BRE exerts a therapeutic efect against gastric ulcers induced by absolute ethanol. In fact, the administration of BRE from the pulp or peel at a dose of 800 mg/kg bw is protective against gastric macroscopic injury, which was shown by the presence of hemorrhagic ulceration and the decrease in UI by 41% and 68% (p � 0.001 and p � 0.0008) as compared to untreated ulcer rats. Furthermore, the animals treated with BRE at 800 mg/kg were protected from gastric mucosa injury and damage induced by ethanol, with a very similar aspect to that of the normal rats. In addition, the supplementation of BRE from the pulp induced a signifcant decrease in the ulcer score by 32%, 38%, and 41% at doses 200, 400, and 800 mg/ kg, respectively (p � 0.007), as compared to untreated gastric ulcer rats. In gastric ulcer rats treated with BRE from peel, the reduction in ulcer score was 47%, 62%, and 68% (p � 0.005 and p � 0.003) after the administration of doses at 200, 400, and 800 mg/kg, respectively (

Efects of BRE on VGS in Ethanol-Provoked Gastric Ulcer in Rats.
Our study revealed that the gastric ulceration by ethanol induced an important rise in the VGS by 76% (p � 0.005) as compared to normal rats. Te supplementation of BRE from pulp at doses 200, 400, and 800 mg/kg, however, decreased the gastric mucus secretion by 16%, 21%, and 24% (p � 0.02, p � 0.002, and p � 0.001) at doses 200, 400, and 800 mg/kg, respectively (p � 0.02), as compared to gastric ulcer untreated rats. In addition, the administration of BRE from peel augmented the VGS secretion by 21%, 27%, and 35% (p � 0.01, p � 0.002, and p � 0.0009) at doses 200, 400, and 800 mg/kg as compared to gastric ulcer in untreated ulcer rats (Table 1).

Efects of BRE on Gastric Juice pH and Total Acidity and in
Ethanol-Induced Gastric Ulcer in Rats. Te present study revealed that the administration of the BRE from the peel and pulp at diferent doses (200, 400, and 800 mg/kg) stopped the rise of the total acidity and increased the pH near normal rats. In addition, the highest doses of BRE from the pulp and peel have better antisecretory activity as evidenced by the augmentation of the pH by 33% and 44% (p � 0.01) and reduction in total acidity by 25% and 28% in pulp and peel BRE treatment as compared to the gastric ulcer in untreated rats (Table 1).

Stomach Antioxidant Enzyme Activities and Cell Index
Toxicity in Ethanol-Provoked Gastric Ulcer. Table 2 indicates that ulcers caused a signifcant reduction in the SOD, CAT, and GPx activities by 35%, 78%, and 70% (p � 0.009 and p � 0.005), respectively, as compared to normal rats. In addition, the LDH activity and TBARS level in the stomach gastric ulcer in rats increased by 35% and 83% (p � 0.01 and p � 0.008), respectively. Te BRE pulp and peel administration at 800 mg/kg, however, increased the stomach SOD by 55% and 102% (p � 0.009 and p � 0.007), CAT by 75% and 100% (p � 0.007 and p � 0.008), and GPx activities by 58% and 104% (p � 0.007 and p � 0.02), respectively, as compared to untreated gastric ulcers in rats. Moreover, the stomach LDH activity and TBARS level were noted to decrease by 17% and 23% (p � 0.01 and p � 0.02) in gastric ulcers in rats treated with BRE from the pulp and by 22% and 41% (p � 0.01; p � 0.007) in gastric ulcers in rats treated with BRE from the peel, respectively.

Efects of BRE on Histological Evaluation of Gastric
Damage. Tis study showed that supplementation of ethanol by the gastric gavage route caused various lesions in the stomach of rats such as severe disruption of the gastric mucosa, gastric mucosa fattening, and necrotic lesions penetrating deeply into the mucosal and submucosal layers as compared to the gastric mucosa of normal rats (Figure 3). In addition, we showed that the administration of BRE from the pulp and peel or omeprazole prevented the gastric mucosal ulcer, the fattening of gastric mucosa, and necrotic lesions in the stomachs of rats.

Discussion
Previous studies reported that O. stricta betalains characterized by the presence of four bioactive molecules, which are betanin, isobetanin and betanidin, and sorhamnetin-3-O-rutinoside in the pulp and peel [57]. It seems that treatment with betalains and betalain-rich diets is not only nontoxic but could also prove to be a promising alternative to supplement therapies in oxidative stress-, infammation-, and dyslipidemia-related diseases such as stenosis of the arteries, atherosclerosis, hypertension, and cancer, among others [58]. Te results of this study showed that feeding absolute ethanol to rats via gastric gavage caused acute stress in the stomach as evidenced by SOD, CAT, GPX, and LDH activities' depletion, as well as a signifcant increase in RBARS levels, resulting in gastric mucosal damage and alteration, hemorrhagic ulceration, and gastric mucosal fattening as gastric ulceration. In fact, this is a very common disease that causes mucosal damage and ulceration, as well as a decrease in mucus content, total acidity, progressive lesions, and damaged areas [17][18][19][20][21][22][23][24][25]. Furthermore, this study is the frst to report that administration of natural food coloring such as betalain (200, 400, and 800 mg/kg) to ethanol-induced gastric ulcers in rats protected the gastric mucosa from injury, bleeding ulcers, gastric ulcers, and fattening of gastric mucosa. Furthermore, our study showed that 800 mg/kg BRE had the lowest UI compared to ulcerated rats treated with 200 and 400 BRE. Similarly, the results showed that gastric mucosal damage and histological changes were signifcantly reduced in rats treated with a BRE dose of 800 mg/kg bw compared to 200 and 400 mg/kg bw. Tis dose efect was also mentioned at UI, VGS, and TA levels. Tis study showed that taking 800 mg/kg body weight of BRE seemed to be better than lower doses. Onyeka et al.
Journal of Food Quality [36] showed that ingestion of the Opuntia fcus-indica extract in ethanol-treated rats protected against ethanolinduced gastric injury, ulceration, and histological changes in the stomach, and the best protective efect was observed at a dose of 800 mg/kg body weight. Tis protective efect of BRE against ulcer and gastric damage is in  accordance with Babitha et al. [59], who showed that betalain protected against gastric mucosal lesions by inhibiting neutrophil infltration and gastric ulceration. Kaur et al. [60] reported that betalains reduced neutrophil infltration and protected the gastric mucosa from infammation. Similarly, Hijazi et al. [61] reported that betalains increased NO levels and reduced proinfammatory mediators, thereby protecting the gastric tissue from damage. BRE protects the gastric mucosa from injury and bleeding ulcers leading to a reduction in the increase in total acidity, as indicated by an increase in the pH of gastric juice. Oxidative stress is thought to play a major role in gastric ulcers and lesions. In this study, in rats with ethanol-induced C U U+200 U+400 U+800 U+ OmepZ , an observable protective efect from ulceration of gastric mucosa and only a few and small lesions were showed as compared to the injuries seen in the ulcer control rat. In omeprazole gastric ulcer rats (U + OmepZ), a partial protective action from the ulcerations with hemorrhagic of gastric mucosa was showed. gastric ulcer, the activities of SOD, CAT, and GPx in the stomach of rats with gastric ulcer were signifcantly decreased, while the levels of TBARS and LDH were increased. However, administration of BRE to ethanol-induced gastric ulcer in rats stimulated the activities of gastric antioxidant enzymes such as SOD, CAT, and GPX. Tese enzymes are present in the human digestive tract and thus protect the gastric mucosa from damage and lesions, as showed by the increase in lipid peroxidation [36]. BRE stimulates the antioxidant system and protects the stomach from damage and lesions, and VGS was increased by 27% and 35% in gastric ulcers in rats treated with O. stricta pulp and peel natural pigments, respectively. Te results of this study showed that gastric ulcers caused an increase in VGS compared with normal rats. In fact, one of the most important gastric ulcers is the induction of gastric mucus secretion, an increase that may be a consequence of increased gastric acidity [62][63][64].
Tese results are in agreement with Hijazi et al. [61], who reported that beetroot betalains prevents mucosal damage by inhibiting neutrophil infltration into the ulcerated gastric tissue. Similarly, Byeon et al. [65] studied the gastroprotective efect of beetroot pigment on various gastric ulcer models in rats. Tey showed that dose-dependent inhibition of UI and improvement of CR resulted in ethanol-induced gastric ulceration in rats after administration of BRE. Histological evaluation of the stomach revealed that gavage of absolute ethanol supplementation in rats resulted in severe gastric mucosal disruption, fattening of the gastric mucosa, and lesions deep into the mucosal and submucosal layers compared with normal rats. Te anti-infammatory activity of betalains has been reported to reduce infammatory mediators and increase gastric anti-infammatory cytokines [66]. Tus, betalains can be used in the prevention and treatment of gastric ulcers and injuries. In fact, natural pigments prevent tissue damage caused by ethanol and protect the mucosal layer from damage caused by ulcers [49,67,68]. Our results are in accordance with Ofusori et al. [50] who reported that Celosia trigyna pigments are protective against ethanol-induced gastric lesions and ulcers in mice. In addition, Martelli et al. [69] showed that natural food coloring as anthocyanins is a promising functional food for the treatment of gastric ulcers, gastrointestinal infection conditions, and upper gastrointestinal dyspepsia. Furthermore, Lakshmi [70] reported that feeding natural pigments derived from plants at a dose of 800 mg/kg bw reduced gastric ulcer surface area, as shown by the decrease of UI and the increase of CR; improved gastric ulcer-related biochemical indices such a VGS, TA, and pH; stimulated activity of gastric antioxidant enzymes such as SOD, CAT, and GPx; and reduced the lipid peroxidation. In another study, mice were supplemented with 100 mg/kg anthocyanin-rich food by the gastric gavage route to protect from mucosal gastric ulceration and damage, as compared to untreated rats [71]. In ulcerated rats, the administration of Lycium barbarum C-phycocyanin signifcantly reduced the gastric cyclooxygenase-1, prostaglandin E2, and total nitrites and nitrate rates [68]. Galati et al. [72] evaluated the intestinal barrier protective activity of O. stricta juice against ulcers and gastric lesions. Previous studies reported that the administration of Opuntia fcus-indica juice to ethanolinduced gastric ulcer in rats increased gastric mucus production and protection of gastric mucosa against ulcers and lesions. Similarly, Kim et al. [73] showed that pigments from Opuntia fcus-indica at doses 800-1600 mg/kg signifcantly protect against gastric mucosal damage caused by stress.

Conclusion
Te results of this study showed that O. stricta betalains from the pulp and peel protect against ethanol-induced macroscopic hemorrhagic damage in gastric mucosa, gastric mucosal decrease, and ulcer index UI. In addition, the supplementation prevented gastric mucosal damage as shown by an important increase in the gastric antioxidant  Tere is severe disruption of the gastric mucosa, fattening of gastric mucosa, and necrotic lesions penetrating deeply into mucosal and submucosal layers in ulcer rats as compared to the gastric mucosa of normal rats (H&E stain, 10x). U + 800 mg BRE from pulp and U + 800 mg BRE from peel: gastric tissues are normal, mucus without ulceration, and only small and few lesions in gastric mucosa were observed. Stomach histological section of rats treated with OmepZ at dose 30 mg/kg bw: mild disruption to the submucosal layer.
activity. In addition, this study suggested that the consumption of BRE at a dose of 800 mg/kg seemed to be better than the lower doses in order to prevent gastric mucosal damage, alteration, and infammation caused by ethanol.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
All animals were used according to the guidelines of the Tunisian Society for the Care and Use of Laboratory Animals, and the study was approved by the University of Tunisia Ethical Committee (Code: 86/609/EEC). Te Ethics and animal Experimentation Committee is made up of 30 members and chaired by Dr. Besma Bel Hadj Jrad (e-mail: bbhj2002@yahoo.fr).

Conflicts of Interest
Te authors declare that there are no conficts of interest.