https://orcid.org/0009-0006-3247-080X
Figures
Abstract
Peptic ulcer disease affects 10% of the global population, and current treatments have limitations. Vicia faba seeds are traditionally used in Ethiopia for gastric ulcers and gastritis, but scientific evidence is lacking. Seeds were extracted with 80% hydro-methanol to evaluate its antiulcer effect, and part of the extract was fractionated into n-hexane, ethyl acetate, and aqueous solvents. The antiulcer activity of the crude extract and solvent fractions was tested using pylorus ligation and ethanol-induced ulcer models. Repeated dose studies were also performed on both models. In a pylorus ligation-induced ulcer model, single-dose studies showed that 200 mg/kg significantly reduced ulcer number, score (P < 0.05), and index (P < 0.01), providing 39.83% protection. The 400 mg/kg, significantly reduced ulcer number, and score (P < 0.01), providing 55.80% protection. In a repeated-dose study, 100 mg/kg significantly reduced ulcer number and score (P < 0.05). The 200 mg/kg dose showed stronger effects (P < 0.01, for the above parameters, respectively). The highest dose (400 mg/kg) significantly reduced ulcer parameters (P < 0.001) and also lowered gastric juice volume (P < 0.01), acidity (P < 0.05), and raised pH (P < 0.01), providing 59.17% protection. In the ethanol-induced ulcer model, single-dose crude extract provided an ulcer index protection of 23.39%, 34.90%, and 54.04% at 100, 200, and 400 mg/kg, respectively. Repeated administration of the crude extract increased ulcer protection to 34.94%, 50.91%, and 65.72% at the same respective doses. All doses of the aqueous and the ethyl acetate fractions also showed antiulcer activity. This study confirmed that Vicia faba seeds crude extract and solvent fractions have significant antiulcer activity, providing scientific evidence supporting the traditional use of V. faba seeds as a remedy for gastric ulcers in Ethiopia.
Citation: Getachew D, Beyna AT, Abdelwuhab M, Asrie AB (2025) Evaluation of anti-ulcer activity of hydromethanol crude extract and solvent fractions of Vicia faba (Fabaceae) seeds in mice. PLoS One 20(10): e0334119. https://doi.org/10.1371/journal.pone.0334119
Editor: Awatif Abid Al-Judaibi, University of Jeddah, SAUDI ARABIA
Received: December 18, 2024; Accepted: September 10, 2025; Published: October 9, 2025
Copyright: © 2025 Getachew et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data used to conduct this study are provided within the manuscript.
Funding: Our research is funded by the University of Gondar, Ethiopia. The funder, the University of Gondar, had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.
Competing interests: The authors declare no competing interests.
Abbreviations: COX-1, Cyclooxygenase-1; COX-2, Cyclooxygenase-2; DW, Distilled Water; HCl, Hydrochloric acid; H.pylori, Helicobacter pylori; H + /K + ATPase, Hydrogen/potassium adenosine triphosphatase; NaOH, Sodium hydroxide; NSAIDs, Nonsteroidal anti-inflammatory drugs; PPI, Proton pump inhibitor; PUD, Peptic ulcer disease; SPSS, Statistical Package for the Social Sciences; OECD, Organization for Economic Co-operation and Development; V.faba, Vicia faba
Introduction
The word ‘peptic’ is derived from the Greek term peptikos, which means to digest [1]. Peptic ulcer disease (PUD) is a prevalent digestive system disorder characterized by damage to the digestive tract resulting in a mucosal break exceeding 3–5 mm, with visible depth extending into the submucosa [2–4]. The persistence of a peptic ulcer arises from an imbalance between the protective elements of the stomach lining (including mucus and bicarbonate secretion, sufficient blood circulation, prostaglandin E2, nitric oxide, sulfhydryl compounds, and antioxidant enzymes) and aggressive factors (acid and pepsin secretions) [5–7]. The primary factors leading to this imbalance are predominantly attributed to Helicobacter pylori (H. pylori) infection and the taking of non-steroidal anti-inflammatory drugs (NSAIDs) [8]. The third important element is stress, which can cause ulcers through various mechanisms, such as increased histamine release, which can raise acid production, and decreased stomach mucus volume and consistency [9].
The current management of PUD consists of proton pump inhibitors (PPIs), histamine H2 receptor antagonists, antacids, the muscarinic (M1) receptor antagonist pirenzepine, antimicrobial agents, and PGE2, along with its analog misoprostol [10,11]. However, these medications have several limitations. They are expensive and carry risks of treatment failure and relapse, drug interactions, and various side effects, including impotence, gynecomastia, galactorrhea, and gastrointestinal infections [10]. All these factors enforce the need to develop new drugs for treating ulcers and to explore unique compounds sourced from the natural drug reservoir, particularly herbal resources.
Traditionally, several prospective medicinal plants for peptic ulcer treatment have been studied and reported to have several advantages: they tend to be safer, more affordable, readily available, and culturally accepted in comparison to modern drugs [12,13]. Ethiopia boasts a wealth of medicinal plants with significant potential for preventing, treating, or managing PUD. Among these, Vicia faba L. (V. faba), a member of the Fabaceae family, is an annual legume [14] cultivated in various eco-geographical regions in Ethiopia, including the Arsi and Bale highlands, the Central Highlands (South-West, West, and North Showa), Tigray, North and South Wollo, North and South Gondar, East and West parts of Gojam, Wollega, Guji highlands, Hadia, Sidama, and Gamogofa [15]. It is commonly referred to as a fava bean, broad bean, or horse bean in English [16]. In Amharic and Sidama, it is known as ‘Bakella’ [17]. Various ethnobotanical studies showed that V. faba leaves, roots, sprouts, pods, and beans have been utilized in traditional medicine, either as infusions or dietary components, for the natural management of a range of chronic conditions, encompassing depression [18], Parkinson’s disease [19], allergies, diarrhea [20], and stomach ulcers [21]. The plant is recognized for its application in diabetes treatment, given its antioxidant content with free radical scavenging activity, aiding in the rejuvenation of pancreatic beta-cells [22]. Additionally, the plant’s fruit has exhibited anticancer properties in individuals with colon cancer [23].
In vitro, antimicrobial assays showed that V. faba pod extracts inhibited the growth of bacteria such as B. subtilis, Staph Aureus, E. coli, Pseudomonas aeruginosa, and the fungus Candida albicans [24]. The beneficial properties of this plant are attributed to bioactive molecules (secondary metabolites), including phenolic compounds, tannins, alkaloids, glycosides, sterols and triterpenes, saponins, and others it consists of [24,25]. In Ethiopia, Indigenous traditional healers have been utilizing V. faba seed to address various health issues such as body swelling, bronchitis, tapeworm infestations, skin boils, and wounds [26–29]. Additionally, dried seeds are chewed to remedy gastric ulcers [30] and gastritis [16,31,26]. However, there is no scientific evidence in this regard. Therefore, the current study aims to verify the claims made by traditional medicine practitioners regarding the anti-ulcer effect of V. faba by investigating its effect in mice. Furthermore, the results of this experimental study will provide valuable insights for the scientific community, encouraging further exploration of this potential medicinal plant. This includes conducting advanced research on drug formulations, exploring the molecular mechanisms of plant-derived treatments, and isolating specific anti-ulcer compounds with scientific support.
Materials and methods
Equipment, drugs, chemicals, and supplies
In this experiment, the following chemicals, equipment, and reagents were utilized:
Chemicals, drugs, and reagents.
Diazepam 10 mg/ml injection (Lab tech chemicals, Gland pharm limited, India), Tween 80 (Uni-chem, India,) Ethyl acetate (Blulux laboratories, Abron, India), Ketamine hydrochloride injection USP (Rotex Medica, Germany), methanol absolute (Alpha Chemika, India), n-hexane (Central drug house (P) Ltd, India), ethyl acetate (Central drug house (P) Ltd, India), distilled water (UOG, Medical Laboratory), chloroform (Nice Chemicals Lab, Kerala), sodium hydroxide (Central Drug House, India), concentrated hydrochloric acid, phenolphthalein (Rankem, India), absolute ethanol (Bululux Laboratories, India), omeprazole 30 mg tab, and misoprostol 200 mcg tablet (Jai Pharma Ltd., India), normal saline 0.9% (Sansheng Pharmaceutical PLC, Ethiopia), Wagner’s reagent, formalin 10% buffered solution (Yilmana Chemicals, Ethiopia), Glacial acetic acid (Lobe chemi, India),2% Ferrous and Ferric Chloride,10% sodium hydroxide, concentrated sulfuric acid, benzene, diluted Ammonia. All chemicals used were of analytical grade and were purchased from the Ethiopian Pharmaceutical supply services in Gondar, Ethiopia.
Equipment and materials.
sensitive digital weighing balance (Abron Exports, India), dry oven (Abron Exports, India), PH-meter, centrifuge machine, test tubes, dropper, burets, deep freezer, lyophilizer, vacuum pump, mortar and pestle, sharp sterilized scissors, forceps, surgical threads with curved needles, round bottom flask, beaker, surgical scalpel blade, aluminum foil, face mask, gauze, Buchner funnel, Whatman filter paper (№ 1) (Okhla industry, New Delhi), gloves, and permanent marker.
Collection and preparation of the plant material for extraction
The V. faba seeds were purchased from a local farmer in Kusquam kebele, Gondar town, 720 km from Addis Ababa [32] at a longitude and latitude of 37.4636° E, 12.6056° N with an altitude of 2,133 meters above sea level on March 15/2024. The plant material was identified and authenticated by Mr. Getenet Chekole (Assistant Professor of Botanical Science in the Department of Biology at the College of Natural and Computational Sciences, University of Gondar). Subsequently, specimens were deposited at the University of Gondar herbarium with voucher 215/07/2024. The required amount of V. faba seeds was purchased on March 21, 2024. Then seeds were washed immediately with tap water, dried at room temperature, mechanically powdered, and stored in airtight containers [33].
Experimental animals
Adult Swiss albino mice of either sex, weighing between 25 and 30 g, and aged 8–12 weeks, were employed in this investigation [34]. The animals came from the Department of Pharmacology’s animal houses at the University of Gondar, Ethiopia’s School of Pharmacy, College of Medicine and Health Sciences. They were kept at room temperature with a 12:12 h light-dark cycle in cages made of polypropylene plastic, bedding made of wood shavings, and fed a typical pellet diet along with water ad libitum [35]. Before the trial started, the mice were given a week to get used to the lab environment [36]. The established protocols for the use of laboratory animals were adhered to in the care of the animals [37].
Preparation of hydro-methanol seed extract
A. crude extract.
The extraction was conducted using an adequate amount of V. faba seed powder with 80% methanol in a 1:10 (w/v) dry weight to solvent ratio [38]. The cold maceration technique was employed by soaking the samples in the solvent for three consecutive days (72 h) at room temperature, with frequent manual shaking and stirring. The mixture was then filtered using a muslin cloth, followed by a Whatman (№ 1) filter paper. The residue/marc was re-extracted twice with fresh solvent [39]. The fluid extracts were combined and evaporated to dryness at 40°C using a rotary evaporator to remove methanol. The extract was then dried in a lyophilizer to eliminate any remaining methanol and water. Finally, the dried extract was stored at 4°C in the refrigerator until use.
B. Fractionation.
Sixty grams of the methanol crude extract were subjected to successive fractionation. The solvents used for the fractionation process were hexane, ethyl acetate, and distilled water, according to their increasing order of polarity [39]. The crude extract was suspended in distilled water in a 1:5 w/v ratio and poured into a separatory funnel. Then an equal volume of n-hexane was added, and the mixture was intermittently shaken to extract hexane-soluble components. The procedure was repeated three times using fresh n-hexane solvent. The n-hexane extract was collected in a beaker [40]. Then, ethyl acetate was added to the aqueous residue, and a similar procedure with n-hexane fractionation was carried out. Then the mixture was shaken and separated into another beaker. The extracts were dried in a rotary evaporator at 40°C, and the yields for each extract were calculated. The aqueous residue was placed in a lyophilizer, and the total aqueous fraction was determined. After drying, the solvent fractions were stored in airtight containers in the refrigerator until their antiulcer activity was evaluated.
Preliminary phytochemical screening
Preliminary qualitative phytochemical screening tests of secondary metabolites were conducted on the crude extract and the solvent fractions using standard screening guidelines and procedures [41–43].
Acute oral toxicity test
Following the extraction and fractionation procedures, an acute oral toxicity test was conducted on May 2, and follow-up continued till 17/2024. An acute oral toxicity test was conducted according to the limit test recommendations outlined in OECD Guideline No. 425 [44]. On the initial day of the test, one female Swiss albino mouse, which had fasted for 3 hours, received an oral dose of 2000 mg/kg of the plant extract. The mouse was closely monitored for physical or behavioral changes over the subsequent 24 h, with particular attention during the first 4 hours. No signs of toxicity were evident at this dose. After 24 h from the dosing of the first mice, the other four mice were sequentially treated with the same dose. These mice were monitored daily for any indications of adverse effects, such as changes in appetite, hair erection, lacrimation, tremors, convulsions, salivation, diarrhea, mortality, and other potential symptoms for 14 days [44].
Grouping and dosing of animals
To investigate the anti-ulcer potential of V. faba on two distinct ulcer models—pyloric ligation-induced and 100% ethanol-induced ulcers—120 mice were randomly assigned to six mouse groups between May 20 and June 15, 2024. The test doses were determined by taking 1/20th of the limit test of the extract as the low dose (100 mg/kg), 1/10th of the limit test of the extract as the middle dose (200 mg/kg), and 1/5th of the limit test of the extract as the third dose (400 mg/kg), according to OECD 425 guideline [45]. Negative control groups received just the solvents of the extracts or fractions [11], and positive control groups received treatment with omeprazole at 30 mg/kg [34] and misoprostol at 5 µg/kg as a reference standard for ulcers induced by pylorus ligation and 100% ethanol, respectively [46].
Pylorus ligated induced ulcer model
A. Single dose.
The Shay rat model, modified by previous studies [44,45], was employed for this investigation. Mice were randomly assigned to five groups, each consisting of six animals. Group 1 was assigned as the negative control and administered a single dose of distilled water (DW, 10 ml/kg). Group 2 was treated with 100 mg/kg of the crude extract, while Group 3 received 200 mg/kg. Group 4 was administered 400 mg/kg of the crude extract. Group 5 was assigned as the reference standard and was administered omeprazole at 30 mg/kg.
B. Repeated dose.
Mice were randomly divided into five groups, each comprising six animals. All groups were pretreated daily for 10 days. Group 1 was pretreated with DW (10 ml/kg) and served as the negative control. Group 2 was treated with 100 mg/kg of the crude extract, while Group 3 received 200 mg/kg. Group 4 was administered 400 mg/kg of the crude extract. Group 5 was assigned as the reference standard and was given omeprazole at 30 mg/kg.
For both single and repeated pretreatments, mice underwent a 24-h fasting period before the experiment, with access to water permitted until the last 4 h. An hour after the final drug treatment, the mice were anesthetized with 50 mg/kg of ketamine HCl combined with 5 mg/kg of diazepam administered intraperitoneally [47], and a small midline incision below the xiphoid process was made to open the abdomen. The pyloric portion of the stomach was carefully lifted and ligated to prevent traction or damage to the blood supply of the gastric mucosa. The stomach was then carefully repositioned, and the abdominal wall was closed with interrupted sutures using Meri silk no. 2 [46]. After four hours of pyloric ligation, the mice were euthanized using 200 mg/kg of ketamine HCl via intraperitoneal injection [48]. The abdomen was opened along the greater curvature, and its content was drained into a test tube. The gastric juice was collected and centrifuged at 2000 rpm for 10 min [47]. The volume of the resulting supernatant was recorded, and this supernatant was used to determine the total acidity and pH. The stomach mucosa of each animal was washed with saline and clean water, labeled, and preserved in sodium phosphate-buffered 10% formalin until examination for lesions using a hand lens (10×) with subsequent scoring [49].
Ethanol-induced ulcer model
A. Single dose.
Ulcers were induced by administering absolute ethanol, following the method outlined by Hollander D. et al. [50] and previous studies with some modifications to assess the anti-ulcer effects of repeated and single-dose administrations [45,46]. Mice were randomly allocated to five groups, each consisting of six animals. Group 1 administered a single dose of DW (10 ml/kg) and was assigned as the negative control. Group 2 was pretreated with 100 mg/kg of crude extract, while Group 3 was pretreated with 200 mg/kg. Group 4 was pretreated with 400 mg/kg of the crude extract. Group 5 was assigned as the reference standard and was pretreated with a single dose of misoprostol at 5 µg/kg.
B. Repeated dose.
Animals were randomly allocated into five groups, each consisting of six animals. All groups were pretreated daily for 10 days. Group 1 administered a single dose of DW (10 ml/kg) and was assigned as the negative control. Group 2 was pretreated with 100 mg/kg of crude extract. Group 3 was pretreated with 200 mg/kg of the crude extract. Group 4 was pretreated with 400 mg/kg of the crude extract. Group 5 was pretreated with a single dose of misoprostol at 5 µg/kg and assigned as the reference standard.
For both single and repeated dose treatments, respective pretreatments were administered orally to mice that fasted for 24 h. After one hour of pretreatments, an ulcer was induced by administering absolute ethanol (99.9% w/v) at 1 ml/200 g body weight for each mouse [46]. After one hour of ethanol administration, animals were euthanized using anesthetics with 200 mg/kg of ketamine via intraperitoneal injection [48]. The stomachs were incised along the greater curvature, and ulceration was scored using a method similar to that outlined by pylorus ligation [51]. Finally, randomly selected photographs of the opened stomach from each group (6 mice) showing visual differences in the severity of ulceration between the negative control, the graded dose of the crude extract, and the reference standard drug are displayed.
Solvent fraction antiulcer effect study.
After evaluating the effect of the crude extract in pylorus ligation and ethanol-induced ulcer models, as the crude extract was found to show greater ulcer protection in the ethanol-induced ulcer model, the model was selected to study the effect of the solvent fractions. This selection was based on a previous study [47]. Sixty-six mice were randomly allocated into 11 groups, each comprising six animals. Group 1 was assigned as the negative control, receiving only the vehicle (2% Tween 80). Groups 2, 3, and 4 were pretreated with 100 mg/kg, 200 mg/kg, and 400 mg/kg of the aqueous fraction, respectively. Groups 5, 6, and 7 were pretreated with 100 mg/kg, 200 mg/kg, and 400 mg/kg of the ethyl acetate fraction, respectively. Groups 8, 9, and 10 were pretreated with 100 mg/kg, 200 mg/kg, and 400 mg/kg of the n-hexane fraction, respectively. Group 11 was assigned as the positive control and pretreated with misoprostol at 5 µg/kg. Respective pretreatments were administered orally to mice that fasted for 24 h. One hour after the pretreatments, ulcers were induced by administering 1 ml of absolute ethanol (99.9% w/v) per 200 g of body weight to each mouse. After one hour of ethanol administration, the mice were then euthanized using anesthetics with 200 mg/kg of ketamine via intraperitoneal injection. The stomachs were incised along the greater curvature, and ulceration was scored using a method similar to that outlined by pylorus ligation.
Parameters for evaluation of antiulcer activity
Macroscopic evaluation of the stomach.
The stomachs were opened along the greater curvature and rinsed with saline and clean water to remove gastric contents and blood clots. The mucosa of each animal was labeled and placed in 10% formalin buffered with sodium phosphate for examination. The tissues were then assessed using a 10 × magnifier lens to evaluate ulcer formation, and the number of ulcers was counted. Ulcers were scored based on the method described by Kulkarni [52] as normal-colored stomach (0), red coloration (0.5), spot ulcer [1], hemorrhagic streaks (1.5), deep ulcers [2], and perforation [3]. The mean ulcer score for each animal was expressed as the ulcer index. The ulcer index (UI) was measured using the following formula:
First, the US was calculated by dividing the sum of the severity score by the number of ulcers per mouse.
UI is the ulcer index, UN is the average number of ulcers per animal, US is the average severity score, and UP is the percentage of animals with ulcers (US ≥ 0.5). The percentage inhibition of ulceration was calculated as follows:
Determination of volume of gastric secretion, pH, and total acidity.
Mice were sacrificed, and the contents were emptied into a test tube by opening the abdomen along the larger curvature. After collecting the gastric juice, it was centrifuged for 10 minutes at 2000 rpm. The resultant supernatant’s volume was noted. A digital pH meter was then used to determine the pH of the centrifuged stomach output. In a 50 ml conical flask, 0.25 ml of gastric output was diluted with 0.25 ml of distilled water to measure the overall acidity. After adding two drops of phenolphthalein indicator, 0.01 N NaOH was used to titrate the liquid until a persistent pink hue was seen. The volume of 0.01 N NaOH used was recorded. The total acidity was then calculated in milli-equivalents per liter (mEq/L) using the following formula [35]:
where N = normality of NaOH.
Data analysis
Version 27 of the Statistical Package for the Social Sciences (SPSS) was used for data entry, coding, result cleaning, and analysis. For every parameter, the results were displayed as mean ± SEM. Tukey’s post hoc multiple comparison tests were used after a one-way analysis of variance (ANOVA) to establish statistical significance. The difference in means between groups with a p-value of less than 0.05 was considered statistically significant.
Ethical consideration
Ethical clearance was obtained from the Research and Ethics Committee, Department of Pharmacology, University of Gondar, with the reference number SOP4/86/2016. All procedures performed on animals were conducted following the rules and regulations outlined in the Guide for the Care and Use of Laboratory Animals [53].
Results
Phytochemical screening test result
The phytochemical screening of the seed of V. faba hydromethanol crude extract showed the presence of alkaloids, flavonoids, glycosides, phenolic compounds, plant steroids, saponins, tannins, and terpenoids. Table 1 presents phytochemicals identified in crude extract, aqueous, ethyl acetate, and n-hexane fractions.
Acute oral toxicity study
An acute oral toxicity test was conducted on female mice per the OECD-425 guidelines (OECD, 2008). The study indicated that the hydromethanolic crude extract of V. faba did not result in any deaths at a dosage of 2000 mg/kg within the first 24 h or over the subsequent 14 days. Additionally, physical and behavioral assessments of the mice did not indicate any apparent signs of acute toxicity.
Effects of hydromethanolic crude extract of V.faba seeds on pylorus ligation-induced ulcer
Single dose study.
The ulcer number (P < 0.05), ulcer score (P < 0.05), and ulcer index (P < 0.01) were all considerably decreased in single-dose tests at 200 mg/kg, offering a 39.83% ulcer index protection in comparison to the negative control. Even more noticeable results were seen with the 400 mg/kg dose of crude extract, which significantly reduced the number of ulcers (P < 0.01), ulcer score (P < 0.01), and ulcer index (P < 0.001), offering 55.80% protection against the ulcer index when compared to the negative control. Omeprazole (30 mg/kg) provided 55.80% ulcer index protection and significantly decreased ulcer number, ulcer score, and ulcer index (P < 0.001) when compared to the negative control. In comparison to the negative control and all extract doses, it also markedly raised the pH of the stomach (P < 0.001) and lowered the volume and overall acidity of the stomach juice (P < 0.001). Table 2 summarizes these results.
Furthermore, Fig 1 shows visual differences in several lesions in opened stomachs between negative control, different test doses of the crude extract, and positive control.
Repeated dose study.
The crude extract at 100 mg/kg significantly decreased ulcer number (P < 0.05) and score (P < 0.05) in a study with repeated daily administration, whereas 200 mg/kg showed a stronger reduction in ulcer number (P < 0.01) and score (P < 0.01) and ulcer index (P < 0.01). The greatest reductions in ulcer number (P < 0.001), score (P < 0.001), index (P < 0.001), decreased gastric juice volume (P < 0.01), decreased overall acidity (P < 0.05), and higher pH (P < 0.01) were observed at the maximum dose of 400 mg/kg. At 100, 200, and 400 mg/kg, the extract reduced the ulcer index by 33.28%, 44.87%, and 59.17%, respectively. Omeprazole (30 mg/kg) demonstrated the highest anti-ulcer effect with a substantial decrease in ulcer parameters (P < 0.001), decreased gastric juice volume (P < 0.001), decreased total acidity (P < 0.001), and elevated pH (P < 0.001). These effects are summarized in Table 3.
Moreover, Fig 2 displays the crude extract’s treating effect, showing fewer ulcers among the group pretreated with the crude extract compared to the negative control.
Effects of hydromethanolic crude extract of V.faba seeds on ethanol-induced ulcer
Single dose study.
In a single-dose study, the crude extract at 200 mg/kg significantly reduced ulcer number (P < 0.01), score (P < 0.05), and index (P < 0.01). The 400 mg/kg dose showed even greater reductions in ulcer number (P < 0.001), score (P < 0.01), and index (P < 0.01), providing 54.04% protection compared to the negative control. Misoprostol (5 µg/kg) demonstrated the strongest anti-ulcer effects, significantly reducing all parameters at P < 0.001 and altering gastric juice metrics. These results are summarized in Table 4.
Fig 3 displays visual differences in the severity of ulceration between negative control, crude extract, and reference standard treated groups.
Repeated dose study.
At 100 mg/kg, the crude extract dramatically decreased ulcer score and number (p < 0.05). The extract decreased ulcer score (p < 0.001) and ulcer number (p < 0.01) at 200 mg/kg. The most beneficial dose was 400 mg/kg, which changed gastrointestinal parameters (pH, acidity) and dramatically reduced ulcer number (p < 0.01), score, and index (p < 0.001). At 100, 200, and 400 mg/kg, the crude extract offers 34.94%, 50.91%, and 65.72% ulcer index protection, respectively. Misoprostol also significantly improved all metrics (p < 0.001). The detailed results are illustrated in Table 5.
Fig 4 and Fig 5 below show the ulcer index protection of the crude extract in pylorus ligation and absolute ethanol-induced ulcer administered in single and repeated doses; and the severity and number of ulcers in the negative control, group treated with V.faba repeated crude dose extract, and groups treated with the standard drug respectively.
UIPSL: ulcer index protection on a single dose of crude extract on pylorus ligation-induced ulcer model; UIPRL: ulcer index protection on repeated daily doses of the crude extract on pylorus ligation-induced ulcer model; UIPSE: ulcer index protection on a single dose of the crude extract on ethanol-induced ulcer model; UIPRE: ulcer index protection on repeated daily doses of the crude extract on ethanol-induced ulcer model.
Effects of the repeated dose solvent fractions of V. faba Seed crude extract on ethanol-induced ulcer
The aqueous fraction of the crude extract showed significant ulcer inhibition, with 100 mg/kg reducing ulcer metrics (p < 0.05), 200 mg/kg further decreasing ulcer number and score (p < 0.01), and 400 mg/kg providing the strongest ulcer number and ulcer score reduction (p < 0.001). Ulcer index protection was 23.46%, 46.57%, and 62.67% at 100, 200, and 400 mg/kg, respectively. The ethyl acetate fraction also demonstrated efficacy, with 100 mg/kg showing moderate ulcer number and score reduction (p < 0.05), and 400 mg/kg significantly reducing ulcer number (p < 0.01), score (p < 0.05), and index (p < 0.001), along with gastric parameter alteration. Ulcer index protection for the ethyl acetate fraction was 21.25%, 40.86%, and 55.21% at 100, 200, and 400 mg/kg, respectively. The n-hexane fraction only reduced scores and index at 400 mg/kg (p < 0.05). Misoprostol showed the strongest overall anti-ulcer activity, significantly improving all parameters. The detailed results are presented in Table 6 below.
The comparative ulcer index protection effects of these solvent fractions are illustrated in Fig 6.
AQ, aqueous fraction; ET, ethyl acetate fraction; NH, n-hexane fraction.
Discussion
The findings of this study showed that the hydromethanolic crude extract of V. faba and its solvent fractions possess promising antiulcer activity in experimental mouse models, supporting its traditional use in Ethiopia. In the acute toxicity study, administration of the crude extract at a dose of 2000 mg/kg produced no signs of toxicity or mortality during the 14-day observation period. This suggests that V. faba extract is non-toxic, with a median lethal dose (LD50) value greater than 2000 mg/kg in mice, indicating it is relatively safe.
Most of the phytochemical constituents identified in this study were also identified in other pharmacological research conducted in Egypt [54]. However, this study identified plant steroids and terpenoids, which were not observed in the Egyptian study. The differences in chemical composition could be attributed to variations in soil or climatic conditions, or the specific plant parts used. This study used V. faba seeds, whereas the Egyptian research focused on V. faba bean pods.
In experimental ulcer studies, ulcers can be induced by various methods. In this study, ulcers were induced using pylorus ligation and ethanol induction methods. In both models, repeated dose administration of V. faba seed extract produced stronger gastroprotective effects than single doses, likely due to the single-dose administration resulting in a short duration during which the plasma concentration remains within the therapeutic window, potentially failing to achieve the minimum effective plasma concentration. In contrast, repeated doses persistently provide a sufficient concentration of bioactive phytochemicals at the site of action, allowing for sustained therapeutic effects [47]. Higher doses have a more pronounced effect compared to lower doses, likely because they deliver a greater amount of bioactive compounds, which may enhance the modulation of gastric secretions and provide more effective protection against ulcers. The cumulative effect of higher doses could also lead to a more robust and prolonged biological response. Additionally, the highest repeated dose likely produced more significant changes in gastric physiology, such as a reduction in gastric secretion volume, a decrease in total acidity, and an increase in pH. This shows that the antiulcer activity of V. faba is mostly attributed to its gastro-protective effect, with minimal anti-secretory effect. This effect is consistent across the study of crude extract in both above mentioned models and aqueous fraction, and ethyl acetate fraction in ethanol-induced ulcers. These findings align with prior studies on other Fabaceae members, such as Tamarindus indica and Calpurnia aurea seeds (Fabaceae members) in a model of pylorus ligation-induced ulcers [49,55] and Cassia sieberiana and Glycyrrhiza glabra L. (Fabaceae family members) in a model of ethanol-induced gastric ulcers [34,56], which also demonstrated enhanced protection with higher or repeated dosing.
In the study of solvent fractions, the lesser antiulcer and anti-secretory effect of the ethyl acetate fraction compared to the aqueous fraction could be due to the lower concentration of the bioactive molecules it consists of [47]. This indicates that most biomolecules were highly polar and more dissolved in the aqueous (polar) solvent. The lack of an anti-secretory effect and the need for higher doses of n-hexane fraction for antiulcer activity also could be explained by the lack of some of the bioactive molecules, including glycosides, terpenoids, flavonoids, and tannins that were identified in the crude extract [49].
As identified by phytochemical assay, the hydromethanolic extract of V. faba contains a range of bioactive components, including flavonoids, tannins, phenolic compounds, alkaloids, terpenoids, glycosides, plant steroids, and saponins. Various previous studies have shown that V.faba is rich in polyphenolic compounds [57–60]. These polyphenolic compounds are known to have antioxidant activity, possibly by inhibiting different enzymes that are responsible for the generation of various reactive oxygen species. The polyphenolic compounds found in V.faba were found to have xanthine oxidase inhibiting activity, an enzyme responsible for the generation of reactive oxygen species [61,62]. Tannins help form a protective barrier at ulcer sites, shielding against toxic substances and proteolytic enzymes [63]. Flavonoids act as free radical scavengers and possess cytoprotective and anti-ulcerogenic properties [10]. Moreover, previous studies have shown that V. faba has flavonoids with established antiulcer activity , namely kaempferol and quercetin [64]. Moreover, previous studies have shown that V. faba has flavonoids with established antiulcer activity, namely kaempferol and quercetin [65]. Kaempferol reduces myeloperoxidase activity, limiting neutrophil infiltration and minimizing gastric injury, thereby promoting healing and reducing inflammation in the gastric mucosa [66]. The reduction in acid secretion could be attributed to the flavonoids it consists of, which are known to lower histamine release from mast cells by blocking histidine decarboxylase and inhibiting the H + /K + -ATPase enzyme [10,63] and alkaloids, which exhibit an anti-secretory effect by acting as H2-receptor antagonists and through their anticholinergic action [63]. So, the antiulcer activity of V. faba crude extract and solvent fractions could be due to its antioxidant, anti-inflammatory activity, and minimal anti-secretory activity. Therefore, V. faba could be a promising candidate for treating PUD due to its antioxidant and anti-inflammatory properties, directly targeting the disease’s pathogenesis. So, it is crucial to preserve the genome stability of V. faba to maintain its therapeutic potential and genetic integrity. This is especially important given the widespread use of chemicals like pesticides and herbicides that could potentially alter the plant’s genetic makeup; thus, using organic and less toxic alternatives and regularly monitoring chemical application is advisable.
Strengths and limitations of the study
This study provides preliminary evidence for the antiulcer potential of V. faba seed extract using pylorus ligation and ethanol-induced ulcer models. Both single and repeated doses of the crude extract and solvent fractions were tested, highlighting the influence of dose and treatment duration. However, this study is not without limitations. The limitations of this study include that while acute toxicity tests suggest safety, long-term effects remain unexamined. Ulcer scoring via the Kulkarni method does not account for ulcer size or area, which can be seen as a limitation. The study also lacked quantitative phytochemical analysis. Furthermore, this study lacks histological (microscopic) analysis, which limits the ability to fully assess tissue-level healing and inflammatory changes, thereby adding greater depth to the interpretation of ulcer healing. Most importantly, as this study was limited to mouse models, the relevance of these results to humans remains uncertain. Advanced preclinical studies, including in higher-order models, and well-designed clinical trials are essential to confirm the therapeutic potential of V. faba seeds.
Conclusion
The present study showed that the hydromethanolic crude extract of V. faba and its solvent fractions generally have significant gastroprotective activity in mouse models of gastric ulcer at 400 mg/kg single dose and 200 and 400 mg/kg repeated doses, with repeated dosing proving a more effective treatment. These findings offer preliminary support for the traditional use of V. faba seeds against gastric ulcers, but they should be interpreted cautiously given the reliance on animal models. Further studies, including phytochemical characterization, chronic toxicity testing, histological analyses, and well-designed clinical trials, are needed to confirm their safety and efficacy in humans.
Supporting information
S1 File. Parameters and their abbreviations used for the evaluation of anti-ulcer activity of hydromethanol crude extract and solvent fractions of Vicia faba (Fabaceae) seeds in mice.
https://doi.org/10.1371/journal.pone.0334119.s001
(XLSX)
References
- 1. Suganya K, Lalitha S. Ijsrset151332. IJSRSET. 2015;1(2):218–22.
- 2. Xie X, Ren K, Zhou Z, Dang C, Zhang H. The global, regional and national burden of peptic ulcer disease from 1990 to 2019: a population-based study. BMC Gastroenterol. 2022;22(1):58. pmid:35144540
- 3. Wakodkar SB, Shuddalwar A, Baheti DJR. Herbal potentials for treatment of peptic ulcers: a review. Int J Pharm Sci Rev Res. 2021;68(2):124–31.
- 4. Shubham BS, Rajeshree AK, Baban SS, Asaram KR. A review on herbal potential for treatment of peptic ulcer. Int Res J Modern Engineering Technologies. 2023;7807(05):7807–17.
- 5. Périco LL, Emílio-Silva MT, Ohara R, Rodrigues VP, Bueno G, Barbosa-Filho JM, et al. Systematic Analysis of Monoterpenes: Advances and Challenges in the Treatment of Peptic Ulcer Diseases. Biomolecules. 2020;10(2):265. pmid:32050614
- 6. Gugliandolo E, Cordaro M, Fusco R, Peritore AF, Siracusa R, Genovese T, et al. Protective effect of snail secretion filtrate against ethanol-induced gastric ulcer in mice. Sci Rep [Internet]. 2021;11(1):1–12. Available from:
- 7. Jaiswal F, Rai AK, Wal P, Wal A, Singh SP. Peptic ulcer: a review on etiology, pathogenesis and treatment. Asian J Pharmaceutical Education and Research. 2021;10(4):1.
- 8. He Y, Koido M, Sutoh Y, Shi M, Otsuka-Yamasaki Y, Munter HM, et al. East Asian-specific and cross-ancestry genome-wide meta-analyses provide mechanistic insights into peptic ulcer disease. Nat Genet. 2023;55(12):2129–38. pmid:38036781
- 9.
Gupta H, Bhandari U. Peptic ulcer and role of a polyherbal formulation in pathogenesis of ulcer in Wistar rats. 2023;:41894–900.
- 10. Yismaw YE, Abdelwuhab M, Ambikar DB, Yismaw AE, Derebe D, Melkam W. Phytochemical and Antiulcer Activity Screening of Seed Extract of Cordia africana Lam (Boraginaceae) in Pyloric Ligated Rats. Clin Pharmacol. 2020;12:67–73. pmid:32636685
- 11.
Abebaw M, Mishra B, Asmelashe D. Evaluation of anti-ulcer activity of the leaf extract of Osyris quadripartita Decne. (Santalaceae) in rats. 2017;1–11.
- 12. Mustefa A, Nardos A, Hailu D, Deyno S. Phytochemistry, efficacy, and safety of medicinal plants used traditionally for the management of peptic ulcer diseases in Ethiopia: a systematic review. Clin Phytosci. 2023;9(1).
- 13. Mahato TK. Exploring antibacterial & antiulcer activity of aegle marmelos linn.: A review. Int J Pharm Chem Anal. 2020;7(3):107–12.
- 14. Gu B-J, Masli MDP, Ganjyal GM. Whole faba bean flour exhibits unique expansion characteristics relative to the whole flours of lima, pinto, and red kidney beans during extrusion. J Food Sci. 2020;85(2):404–13. pmid:31887250
- 15. Nurgi N, Tana T, Dechassa N, Alemayehu Y, Tesso B. On-farm diversity of faba bean (Vicia faba L.) farmers’ varieties in Eastern Hararghe Zone, Ethiopia. Genet Resour Crop Evol. 2022;70(2):549–70.
- 16.
Dhull SB, Rose PK, Kidwai MK, Noor R, Chawla P. A review of nutritional profile and processing of faba bean (Vicia faba L.). 2022;1:1–13.
- 17.
Busse H. Handbook of Sidama Traditional Medicinal Plants. 2013.
- 18. Salehi B, Abu-Reidah IM, Sharopov F, Karazhan N, Sharifi-Rad J, Akram M, et al. Vicia plants-A comprehensive review on chemical composition and phytopharmacology. Phytother Res. 2021;35(2):790–809. pmid:32930444
- 19. Ramírez-Moreno JM, Salguero Bodes I, Romaskevych O, Duran-Herrera MC. Broad bean (Vicia faba) consumption and Parkinson’s disease: a natural source of L-dopa to consider. Neurología (English Edition). 2015;30(6):375–6.
- 20. Jamila F, Mostafa E. Ethnobotanical survey of medicinal plants used by people in Oriental Morocco to manage various ailments. J Ethnopharmacol. 2014;154(1):76–87. pmid:24685583
- 21. Abbas Z, Khan SM, Alam J, Khan SW, Abbasi AM. Medicinal plants used by inhabitants of the Shigar Valley, Baltistan region of Karakorum range-Pakistan. J Ethnobiol Ethnomed [Internet]. 2017;13(1):53. Available from:
- 22.
Bangar SP, Kajla P. Introduction: Global status and production of faba-bean. Faba bean: chemistry, properties and functionality. 2022. p. 1–15.
- 23. Sathya Prabhu D, Devi Rajeswari V. Nutritional and biological properties of Vicia faba L.: A perspective review. Int Food Res J. 2018;25(4):1332–40.
- 24. Elbadrawy E, Mostafa MY. Antioxidant, anti-inflammatory, antimicrobial and anticancer activities of the pods of green broad bean (Vicia faba L.), in vitro. Springer Science and Business Media LLC. 2023.
- 25. Ali AK, Toliba AO, Rady AH. Effect of gamma radiation on phytochemical compounds in faba bean (Vicia faba L.). Zagazig J Agric Res. 2019;46(3):757–67.
- 26. Belay H, Wondimu T. Functional food plants in Debre Markos district, East Gojjam, Ethiopia. Asian J Ethnobiol. 2019;2(1):8–21.
- 27. Abdela G, Girma Z, Awas T. Ethnobotanical study of medicinal plant species in Nensebo District, south-eastern Ethiopia. Ethnobot Res Appl. 2022;24:1–25.
- 28. Megersa M, Woldetsadik S. Ethnobotanical study of medicinal plants used by local communities of Damot Woyde District, Wolaita Zone, Southern Ethiopia. Nusant Biosci. 2022;14(1):10–24.
- 29. Tigray S. Ethnobotanical study of medicinal plants in and around Alamata, Southern. Ethnobotanical Study of Medicinal Plants in and Around. 2014;2(December):338–44.
- 30. Megersa M, Asfaw Z, Kelbessa E, Beyene A, Woldeab B. An ethnobotanical study of medicinal plants in Wayu Tuka District, East Welega Zone of Oromia Regional State, West Ethiopia. J Ethnobiol Ethnomed. 2013;9(1):68. pmid:24295044
- 31. Jagessar RC. Phytochemical screening and chromatographic profile of the ethanolic and aqueous extract of Passiflora edulis and Vicia faba L. (Fabaceae). J Pharmacogn Phytochem. 2017;6(6):1714–21.
- 32. Tegegne AD, Negewo MA, Desta MK, Nedessa KG, Belaye HM. City Profile, Gondar. Univ Gondar. 2020;29.
- 33. Beyna AT, Mengesha AK, Yefter ET, Kahaliw W. Evaluation of wound healing and anti-inflammatory activity of hydro-alcoholic extract and solvent fractions of the leaves of Clerodendrum myricoides (Lamiaceae) in mice. PLoS ONE. 2024;19(7):e0306766.
- 34. Jalilzadeh-Amin G, Najarnezhad V, Anassori E, Mostafavi M, Keshipour H. Antiulcer properties of Glycyrrhiza glabra L. extract on experimental models of gastric ulcer in mice. Iran J Pharm Res. 2015;14(4):1163–70. pmid:26664383
- 35. Belayneh YM, Amare GG, Meharie BG, Kifle ZD. Evaluation of the antiulcerogenic activity of hydromethanol extracts of Solanum incanum L. (Solanaceae) leaves and roots in mice; single and repeated dose study. Metabol Open. 2021;11:100119. pmid:34485890
- 36. Buschmann J. The OECD guidelines for the testing of chemicals and pesticides. Methods Mol Biol. 2013;947:37–56. pmid:23138894
- 37.
Press TNA. Guide for the Care and Use of Laboratory Animals: Eighth Edition.
- 38. Peiris DSHS, Fernando DTK, Senadeera SPNN, Ranaweera CB. Phytochemical screening for medicinal plants: guide for extraction methods. Asian Plant Res J. 2023;11(4):13–34.
- 39.
H S. An overview of extraction techniques for medicinal and aromatic plants. 2008.
- 40. Ponnuchamy V, Gordobil O, Diaz RH, Sandak A, Sandak J. Fractionation of lignin using organic solvents: A combined experimental and theoretical study. Int J Biol Macromol. 2021;168:792–805. pmid:33242547
- 41. Shaikh JR, Patil M. Qualitative tests for preliminary phytochemical screening: An overview. Int J Chem Stud. 2020;8(2):603–8.
- 42.
Savithramma N, Rao ML, Ankanna S. Screening of traditional medicinal plants for secondary metabolites. 2011;2(4).
- 43. Khade OS, K S, Sonkar RM, Gade PS, Bhatt P. Plant secondary metabolites: Extraction, screening, analysis and their bioactivity. Int J Herb Med. 2023;11(2):01–17.
- 44.
Guidelines O. Test guideline no. 425 acute oral toxicity: up-and-down procedure. 425. 2022.
- 45. Andargie Y, Sisay W, Molla M, Adela M. Evaluation of In vivo antidiarrheal activity of hydro-methanolic extract of the root of Rumex nepalensis in Swiss Albino mice. Metabol Open. 2022;15:100197. pmid:35785136
- 46. Adane H, Atnafie SA, Kifle ZD, Ambikar D. Evaluation of In Vivo Antiulcer Activity of Hydro-Methanol Extract and Solvent Fractions of the Stem Bark of Ficus thonningii (Moraceae) on Rodent Models. Biomed Res Int. 2021;2021:6685395. pmid:33928161
- 47. Sisay Zewdu W, Jemere Aragaw T. Evaluation of the Anti-Ulcer Activity of Hydromethanolic Crude Extract and Solvent Fractions of the Root of Rumex nepalensis in Rats. J Exp Pharmacol. 2020;12:325–37. pmid:33061674
- 48.
Pharmacology E, Mageshwaran B, Deepak L, Shewade G, Marshall G. Introduction to Basics of Pharmacology and Toxicology. 2021.
- 49. Andargie Y, Sisay W, Molla M, Norahun A, Singh P. Evaluation of the antiulcer activity of methanolic extract and solvent fractions of the leaves of calpurnia aurea (Ait.) Benth. (Fabaceae) in Rats. Evid Based Complement Alternat Med. 2022;2022:4199284. pmid:35815284
- 50. Sheibani M, Nezamoleslami S, Rahimi N, Abbasi A, Dehpour AR. The protective effect of dapsone against ethanol, stress, and indomethacin-induced gastric erosion in rats. ACTA. 2021.
- 51. Mishra AP, Bajpai A, Chandra S. A Comprehensive Review on the Screening Models for the Pharmacological Assessment of Antiulcer Drugs. Curr Clin Pharmacol. 2019;14(3):175–96. pmid:30864527
- 52.
Dhadde SB. Handbook of Experimental Pharmacology. 2019.
- 53. Bayne K. Revised guide for the care and use of laboratory animals available. The Physiologist. 1996;39.
- 54.
Elbadrawy E, Mostafa MY. Properties of green broad bean pods (Vicia faba L.). 2024;12(2):308–18.
- 55. Kalra P, Sharma S, Suman S, Kumar S. Antiulcer effect of the methanolic extract of Tamarindus indica seeds in different experimental models. J Pharm Bioallied Sci. 2011;3(2):236–41. pmid:21687352
- 56. Nartey ET, Ofosuhene M, Kudzi W, Agbale CM. Antioxidant and gastric cytoprotective prostaglandins properties of Cassia sieberiana roots bark extract as an anti-ulcerogenic agent. BMC Complement Altern Med. 2012;12:65. pmid:22607580
- 57. Amarowicz R, Shahidi F. Antioxidant activity of faba bean extract and fractions thereof. J Food Bioact. 2018;2016:112–8.
- 58. Luo Y-W, Wang Q, Li J, Wang Y, Jin X-X, Hao Z-P. The impact of processing on antioxidant activity of faba bean (<em>Vicia faba</em> L.). AJFST. 2015;7(5):361–7.
- 59. Elbadrawy E, Mostafa MY. Antioxidant, anti-inflammatory, antimicrobial, and anticancer properties of green broad bean pods (Vicia faba L.). Foods and Raw Materials. 2024;12(2):308–18.
- 60. Chaieb N, González JL, López-Mesas M, Bouslama M, Valiente M. Polyphenols content and antioxidant capacity of thirteen faba bean (Vicia faba L.) genotypes cultivated in Tunisia. Food Research Int. 2011;44(4):970–7.
- 61. Azeem F, Bilal A, Rana MA, Muhammad AA, Habibullah N, Sabir H. Drought affects aquaporins gene expression in important pulse legume chickpea (Cicer arietinum L.). Pakistan J Bot. 2019;51(1):81–8.
- 62. Choudhary DK, Mishra A. In vitro and in silico interaction of faba bean (Vicia faba L.) seed extract with xanthine oxidase and evaluation of antioxidant activity as a therapeutic potential. Natural Product Res. 2018;33(18):2689–93.
- 63. Ahmed O, Nedi T, Yimer EM. Evaluation of anti-gastric ulcer activity of aqueous and 80% methanol leaf extracts of Urtica simensis in rats. Metabol Open. 2022;14:100172. pmid:35313530
- 64. Kowalczyk M, Rolnik A, Adach W, Kluska M, Juszczak M, Grabarczyk Ł. Multifunctional compounds in the extract from mature seeds of Vicia faba var. minor: phytochemical profiling, antioxidant activity and cellular safety in human selected blood cells in in vitro trials. Biomed Pharmacother. 2021;139(May).
- 65. Coşkun Ö, Kanter M, Armutçu F, Çetin K. Protective effects of quercetin, a flavonoid antioxidant, in absolute ethanol-induced acut gastric ulcer. Eur Gen Med. 2004;1(3):37–42.
- 66. Li Q, Hu X, Xuan Y, Ying J, Fei Y, Rong J, et al. Kaempferol protects ethanol-induced gastric ulcers in mice via pro-inflammatory cytokines and NO. Acta Biochim Biophys Sin (Shanghai). 2018;50(3):246–53. pmid:29415150