GASTROPROTECTIVE ACTIVITY OF ETHYL ACETATE FRACTION OBTAINED FROM THE STEM BARK EXTRACT OF DESERT DATE (BALANITES AEGYPTIACA)

The study aimed to investigate the gastroprotective activity of the ethyl acetate fraction derived from the stem bark extract of the Desert date tree (EFDD) (Balanites aegyptiaca L. Delile; family Zygophyllaceae) using ethanol and indomethacin-induced ulcer models in Wistar rats. The study was conducted using Wistar rats and divided them into five groups (n=5). Group 1 received 10% Tween 20 (1 ml/kg), which served as the control group. Group 2 was administered the standard drug, omeprazole (20 mg/kg). Groups 3-5 were the extract groups and received doses of 125, 250, and 500 mg/kg, respectively, of EFDD. Two ulcer models were used in the study namely ethanol-induced ulcers and indomethacin-induced ulcers. After administration of the respective treatments, evaluation of the mean ulcer indices in each group and calculation of the percentage ulcer inhibition compared to the control group were carried out. The results indicated that the EFDD, at doses of 250 and 500 mg/kg, significantly reduced the mean ulcer indices in both the ethanol and indomethacin-induced ulcer models compared to the control group (p<0.05). This suggests that the EFDD possesses gastroprotective properties. In conclusion, the EFDD exhibits gastroprotective activity. This finding supports the traditional use of Desert date in folkloric medicine for the treatment of ulcers. However, further research is necessary to explore the underlying mechanisms responsible for the observed gastroprotective effects.


INTRODUCTION
Peptic ulcers are characterized by the presence of sores or lesions that extend throughout the gastrointestinal mucosa, including the muscularis mucosae. This condition involves various stages of increased oxidative stress, inflammation, necrosis, neutrophil infiltration, and reduction in blood flow (da Silva et al., 2013). The development of peptic ulcer disease occurs when there is an imbalance between defensive and destructive factors in the stomach, with mucosal destructive factors such as pepsin, hydrochloric acid, and nonsteroidal anti-inflammatory drugs (NSAIDs), and defensive factors like mucin secretion, bicarbonate barriers, prostaglandins, and mucosal blood flow (Tytgat, 2011;Proctor and Deans, 2014). When the destructive factors outweigh the defensive factors, ulcers may occur. This condition affects a significant portion of the global population, leading to increased morbidity and healthcare costs (Sharifi-Rad et al., 2018). While various drug treatments have been employed to combat peptic ulcer disease, including acid-reducing pharmacological agents and H. pylori eradication regimens, these therapeutic measures have drawbacks such as high incidence of relapse, side effects, and tolerance (Sharifi-Rad et al., 2018). Traditional medicines offer an alternative approach to overcome these limitations (Zewdu & Aragaw, 2020). Traditional medicines, including medicinal plants, have been used since ancient times for the treatment of diseases, and approximately 80% of the world's population relies on these traditional remedies for their healthcare needs (Hu et al., 2020). Numerous medicinal plants have been validated for their effectiveness in alleviating ulcer-related symptoms, including Crepis sancta, Papaya carica, Cordia africana, Ficus thonningii, Lactuca sativa, and Balanites aegyptiaca (Ebada et al., 2020;Oloyede et al., 2015;Yismaw et al., 2020;Adane et al., 2021;Maheswari et al., 2020;Ugwah et al., 2019).
Balanites aegyptiaca, commonly known as Desert date and scientifically identified as Balanites aegyptiaca L. Delile of the Zygophyllaceae family, is a perennial tree primarily found in desert areas (Kabo et al., 2020). Traditional medicine practitioners have utilized decoctions of the Desert date stem bark to treat stomach ulcers (Chothani & Vaghasiya, 2011). Previous scientific research has reported the antiulcer activity of the aqueous extract of this plant (Ugwah et al., 2019). Given the significant impact of peptic ulcer disease on global morbidity, it is crucial to identify affordable and effective treatments that can cure the disease with minimal side effects. Our previous study on the aqueous extract has validated the claims of native traditional medicine practitioners. To further investigate whether the ethyl acetate fraction of Desert date (EFDD) could provide superior results compared to the crude aqueous extract, we conducted this study. Therefore, the aim of this study was to investigate the gastroprotective activities of the ethyl acetate fraction derived from the Desert date tree (EFDD).
dryness to obtain the ethyl acetate fraction (Gandhi et al., 2003). The ethyl acetate fraction was used for the study. The percentage yield was calculated using this expression; % yield = W2/W1 x 100 W2 = weight of the extract in grams W1 = weight of plant material in grams

Experimental Animals
Male Wistar rats weighing between 150 and 200 g were procured from the animal house of the Department of Pharmacology and Toxicology at Usmanu Danfodiyo University in Sokoto. The rats were housed in wellconstructed cages and allowed to acclimate for a period of 2 weeks. During this acclimation and experiment periods, the rats had unrestricted access to standard commercial chow and drinking water. The experimental protocol followed in this study adhered to the established guidelines for the care and use of laboratory animals as outlined in the guide for care and use of laboratory animals (2011), which ensures the welfare and ethical treatment of animals in research. The study received approval from the animal ethics committee of the Department of Pharmacology and Toxicology under the reference number PTAC/Ba/OT/002-18.

Phytochemical screening of the EFDD
Standard procedures were employed to conduct phytochemical screening of the EFDD. The screening aimed to identify the presence of various phytochemical compounds, including alkaloids, tannins, flavonoids, saponins, carbohydrates, steroids, and glycosides. The methods used for the screening followed established protocols as described in the literature (El-Olemy et al., 1994).

Acute toxicity study
The oral acute toxicity assessment of EFDD was conducted using the 'Up-and-Down' method in accordance with the guidelines outlined by the Organization for Economic Development (OECD) 425 (OECD, 2001). A single dose of 3000 mg/kg was administered orally to five female rats in this study. The procedure involved selecting one animal at a time, determining its weight, and administering an oral dose of the EFDD dissolved in 10% Tween 20, proportional to its weight. Each animal was closely observed for any signs of behavioural toxicity immediately following the administration of the fraction. Subsequently, a monitoring period of 14 days was initiated to assess any potential longterm lethal outcomes.

Antiulcer evaluation Ethanol-induced gastric ulcer model
The rats were assigned randomly to five groups (n=5). Prior to the experiment, the animals underwent a fasting period of 24 hours, during which they had unrestricted access to water for up to 2 hours (Ugwah-Oguejiofor et al., 2017). Group 1 received 10% Tween 20 at a dosage of 1 ml/kg, while Group 2 received Omeprazole at a dosage of 20 mg/kg. Groups 3-5 were the EFDD groups and received dosages of 125, 250, and 500 mg/kg of the EFDD respectively. To induce oral gastric lesions, absolute ethanol was orally administered to the rats at a dosage of 8 ml/kg after one hour of treatment. Two hours after the administration of ethanol, the animals were euthanized by cervical dislocation. The stomachs were then excised and opened along the greater curvature. The ulcer index (UI), a measure of the severity of gastric lesions, was determined using established protocols (Arun & Asha, 2008).

Indomethacin-induced gastric model
The rats were assigned randomly to five groups (n=5). Prior to the study, the rats underwent the fasting protocol as previously described. In Group 1, the rats were orally pretreated with 10% Tween 20 at a dosage of 1 ml/kg. Group 2 received the standard drug, omeprazole, at a dosage of 20 mg/kg. Groups 3, 4, and 5 were pre-treated with the EFDD at dosages of 125, 250, and 500 mg/kg, respectively. According to Nwafor et al., (2000), after a one-hour interval, oral gastric lesions were induced by administering 100 mg/kg p.o of indomethacin to all the groups. Following a four-hour period, the animals were euthanized by cervical dislocation, and their stomachs were excised and opened according to the previously described method. The UI were then calculated.

Measurement of ulcer index (UI)
The excised stomachs were carefully cut along the greater curvature, and the mucosa was rinsed with cold saline solution to eliminate any blood contaminants. Using a transparent millimeter scale rule, the surface area of each ulcerated region in the stomach was measured (Magaji et al., 2007). The cumulative length of all the lesions present in each stomach was designated as the ulcer index (UI). To assess the percentage inhibition of ulcer formation, the following formula was utilized: % Inhibition = (UIC -UIT) / UIC × 100 Here, UIC represents the ulcer index in the control group, and UIT represents the ulcer index in the group of rats subjected to the test substance (Navarrete et al., 1998). The resulting percentage provides a quantification of the inhibitory effect on ulcer formation.

Statistical analysis
The results were presented as Mean ± SD (standard deviation). Statistical analysis was performed using a oneway analysis of variance (ANOVA), followed by Dunnett's ttest for multiple comparisons. The statistical calculations were conducted using GraphPad Prism 6 software (Graph Pad Software Inc., CA, USA). A significance level was set at p<0.05.

The yield of ethyl acetate fraction of Balanites aegyptiaca
The percentage yield of the EFDD was 2.6% w/w.

Phytochemical studies
The EFDD showed the presence of tannins, flavonoid, saponin and steroid (Table 1).

Acute toxicity study
The acute toxicity study was conducted using the Up and Down method, administering the EFDD at a maximum oral dosage of 3000 mg/kg. No observable signs of toxicity were detected, and no mortality was observed within the 48-hour period following administration. Furthermore, during the 14day long-term observation period, no adverse effects or mortality were recorded.

Antiulcer evaluation EFDD in ethanol-induced ulcer model
The EFDD at the doses of 250 and 500 mg/kg significantly (p<0.05) reduced the mean UI in the rats ( Table 2). The percentage inhibition was much lower than that of the standard drug group (Figure 1).

DISCUSSION
The study evaluated the gastroprotective activity of the EFDD using ethanol and indomethacin-induced ulcer models in Wistar rats. The data obtained showed that the fraction possessed gastroprotective property.
To study the cytoprotective activity of the EFDD, ethanolinduced gastric ulcer model was employed. Ethanol-induced ulcer formation occurs probably due to stasis in gastric blood flow which contributes to the development of the haemorrhage and necrotic aspects of tissue injury (Guth et al., 1984). It induces a severe damage in gastric mucosa (impairment of defensive factor) by promoting a decrease in secretion of bicarbonate and mucus production which ultimately leads to the development of haemorrhagic and gastric mucosal injury (Hiruma-Lima et al., 2009). The EFDD exhibited its gastroprotective ability by inhibiting the ulcer index in the rats. This result is consistent with other gastroprotective studies using ethyl acetate fractions (Sobreira et al., 2017;Wang et al., 2018;Abubakar et al., 2020). The use of indomethacin, an NSAID, is one of the principal risk factors in gastric ulcers. Through the inhibition of cyclooxygenase (COX-1) and (COX-2) activities, indomethacin hampers the functioning of prostaglandin synthase and directly causes cytotoxic effects on the epithelium. This process results in the suppression of prostaglandin synthesis and the induction of cellular damage within the epithelial layer (Moleiro et al., 2009). Prostaglandins E2 confers Cytoprotection on the mucosal layer by inhibiting hydrochloric acid and stimulating mucus and bicarbonate secretions. In our study, pre-treatment of the rats with the fraction offered gastroprotection to the gastric mucosa against ulceration. The gastroprotective effect of the fraction may therefore be through prostaglandin-mediated pathway since non-prostanoid are known to protect gastric mucosa by mobilising endogenous prostaglandins (Ugwah et al., 2019). Again this is consistent with other studies showing gastroprotective activity of agents (Ruiz-Hurtado et al., 2021). The phytochemical constituents of the EFDD detected were tannins, flavonoids, saponins and steroids. Tannins are astringent in nature. They form tannin-protein complex hence protect the mucosal membrane from gastric acid attack (Bigoniya et al., 2006). The tannin-protein complex layer apart from conferring protection to the stomach in the case of gastric ulcers by increasing chemical and mechanical resistance to injury (de Jesus et al., 2012), they also promote tissue repair and possess anti-Helicobacter pylori effects (Vasconcelos et al., 2008). The presence of flavonoids in plants has been associated with their ability to effectively protect the gastrointestinal tract against ulcerative and erosive lesions, primarily due to their antioxidant properties (Harborne & Williams, 2000). Flavonoids are known to act by increasing the mucosal prostaglandin content, decreasing histamine secretion from mast cells, inhibiting histidine decarboxylase and by the inhibition of Helicobacter pylori growth (Yadav et al., 2021). Saponins is another phytochemical which was identified in the fraction. It has been shown to protect the stomach mucosa from acid by selectively inhibiting prostaglandin F2, which causes vasoconstriction of mucosal blood vessels (Aguwa & Okunji, 1986). Plants rich in steroids have also been reported as antiulcer agent (Emmanuel et al., 2020). This suggests that the antiulcer activity of the fraction may be due to the presence of these phytochemicals. The oral acute toxicity (LD50) of the EFDD was found to be >3000 mg/kg, this implies that the fraction may be considered to have a high degree of relative safety (Ani et al., 2021). However, additional studies are necessary to determine its toxic effects due to repeated and chronic administrations.

CONCLUSION
The EFDD demonstrated significant protection against ulcers induced by ethanol and indomethacin in the rats. This beneficial effect can be attributed to the presence of tannins, flavonoids, saponins, and steroids within the fraction. Additionally, the LD50 (median lethal dose) of the fraction is estimated to be greater than 3000 mg/kg, indicating a relatively high safety margin. Further investigations are warranted to isolate and identify the specific active compound(s) responsible for the observed gastroprotective effects of the fraction.