0% READ
FULL TEXT
INTRODUCTION
Gastric ulcer disease remains a critical global health challenge characterized by mucosal barrier disruption due to an imbalance between aggressive factor accumulations (such as gastric acid and pepsin) and endogenous protective defense elements. The pylorus ligation method serves as a highly robust preclinical experimental design to simulate this pathology. Pyloric ligation induces a rapid accumulation of endogenous gastric juices and corrosive hydrochloric acid, directly triggering structural auto-digestion and tissue injury within the mucosal lining.1,3
Ficus exasperata (Moraceae), widely known across West Africa as the sandpaper tree, is a prominent ethnomedicinal plant traditionally utilized to treat broad-spectrum inflammatory conditions, infectious ailments, and gastrointestinal distress. Recent pharmacological screenings have confirmed its multi-target bioactivities, including distinct antimicrobial, wound-healing, and anxiolytic properties. The leaves of Ficus exasperata are rich in distinct secondary metabolite clusters, notably polyphenols, flavonoids, tannins, and saponins.4
These phytochemical groups act as powerful radical-scavenging agents that mitigate cellular membrane breakdown under extreme physiological stress. Chronic disease and aging have a common denominator: oxidative stress. Oxidative stress is defined as a shifting equilibrium between the creation and removal of free radicals towards the formation.5,6
During oxidative phosphorylation, electrons from the electron transport chain leak to molecular oxygen in the mitochondria, producing the superoxide radical, which is regarded as a major endogenous source of ROS.7,8
The core hypothesis of this study was developed by evaluating the cross-functional synergy between the established folkloric application of Ficus exasperata and its high-density antioxidant profile. Because pylorus ligation directly induces gastric damage via a dual pathway of localized acid hypersecretion and intensive oxidative tissue breakdown, it was hypothesized that a methanolic leaf extract would provide comprehensive dual-action healing. It was anticipated that the extract's chemical compounds would act directly as an anti-secretory barrier agent while simultaneously upregulating systemic cellular enzymes to protect tissue integrity.
This study is deemed important because it bridges the gap between traditional herbal practices and rigorous, evidence-based preclinical pharmacology. While crude extracts of this plant have been broadly noted in ethnobotanical records, there is a critical shortage of precise scientific literature detailing how this plant explicitly alters micro-level enzymatic pathways inside damaged gastric tissue. data regarding the plant's mechanism of action. Ultimately, establishing these parameters validates Ficus exasperata as a viable, safe, and cost-effective natural template for future gastroprotective drug formulation.9,10
Therefore, the primary aim of this study is to investigate the In vitro antioxidant dynamics and preclinical in vivo gastroprotective potential of Ficus exasperata methanolic leaf extract using a pylorus ligation-induced mucosal injury model, with specific reference to its modulatory effects on critical volume of gastric content, acid neutralizing index (pH) and total acidity of treated stomach.
MATERIALS AND METHODS
Plant collection, Identification and Authentication
Leaves of Ficus exasperata were collected from the Konkan region, of Maharashtra. A plant taxonomist at the Sree Narayana College, Kollam (affiliated with the University of Kerala) recognized the species. The aforementioned centre’s herbarium received voucher specimen no 5804 (Figure 1).
Plant Ficus exasperata leaves morphological studies
Extract Preparation
Leaves of the Ficus exasperata were dried under the shade for 2 weeks and mechanically pounded into fine particles using mortar and pestle. 4000 mL of methanol was used to macerate 100 g of plant material for 72 hr at room temperature. After that mixture was filtered and the solvents were evaporated.11 Extractive Yield was calculated as 13.24% w/w.
Based on the extractive yield solvent methanol was selected for the extraction of plant material.
UV spectrometry of Flavonoids
The total flavonoid content was assessed using the modified UV-spectrophotometric technique for determining flavonoids. Ficus exasperata standard plot was created by calculating the absorbance of aqueous solutions containing 0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, and 1.4 μg/mL of the compound. At 340 nm, the absorbance of each solution was measured. The standard equation and regression coefficient were derived from triplicate measurements of the mean absorbance at each concentration during the experimentation. The absorbance values for solutions are shown in Table 1, and the concentration and absorbance value are graphically shown in Figures 2 and 3 the linear graph in figure had a regression coefficient of 0.9934.
Observations of evaluation of Total Flavonoid Content using (TFC) UV spectrometric method
In vitro Antioxidant Assays
DPPH Assay of Ficus exasperata Extracts
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to evaluate the free radical scavenging ability of antioxidants in plant extracts of Ficus exasperata. The assay was executed in a 96-well microplate format. Briefly, 100 µL of different concentrations of the plant extract solution were dispensed in designated wells. Subsequently 100 µL of freshly prepared 0.1 mM DPPH solution to each well to initiate the radical reactions, Solvent methanol was used as the blank control for calibration in the spectrophotometer. Different dilution (10-50 µg/mL) of plant extract was treated with DPPH Addition. For the control, added 100 µL of methanol instead of the plant extract. Serial dilutions of the standard antioxidant ascorbic acid were processed simultaneously to serve as the positive reference standard. The micro plates were gently agitated to ensure the homogenization and subsequently incubated in the dark place at ambient room temperature (25±2ºC) for 40 min. Following incubation the purple color of the solution turned to yellow. Reduced form was quantified by measuring the absorbances at 517 nm using a microplate reader the percentage of radical scavenging activity was calculated relative to control, and the corresponding experimental data are presented in Table 2 and Figure 4.
DPPH Radical Scavenging Assay
Ferric Reducing Antioxidant Power (FRAP) assay
The ferric reducing capacity of the Ficus exasperata extracts was determined using the Ferric Reducing Antioxidant Power (FRAP) assay. The working FRAP reagent was prepared freshly by mixing acetate buffer, 2,4,6-Tri(2-Pyridyl)-S-Triazine (TPTZ), and Ferric Chloride (FeCl3) in a volume ratio of 10:1:1, respectively. The reaction was initiated by adding 100 µL of the plant extract to test tubes containing 3 mL of the prepared working FRAP reagent. Trolox was employed as the reference standard and prepared in serial dilutions to establish a calibration curve. For baseline calibration of the UV-vis spectrophotometer, a reagent blank was prepared by mixing 3 mL of FRAP reagent with 100 µL of the methanol as a solvent vehicle. The reaction mixtures were vortexed thoroughly to ensure homogeneity and subsequently incubated in a water bath at 37ºC for 30 min. Following the incubation period, the analytical response driven by the reduction of the Ferric-Tripyridyl Triazine (Fe³⁺-TPTZ) complex to its intense blue ferrous (Fe²⁺-TPTZ) form was monitored. The absorbance of each sample was recorded at 593 nm. The antioxidant potential was quantified against the Trolox standard curve, and the quantitative findings are detailed in Tables 3 and 4, and visually represented in Figures 5 and 6.12,13
Ferric Reducing Antioxidant Power (FRAP) assay
In vivo pharmacological activity evaluation
Acute oral toxicity Study
No treatment-related mortality was observed at the maximum limit dose of 5000 mg/kg body weight during the 14-day post-administration period. Consequently, the median Lethal Dose (LD₅₀) of Ficus Exasperata via oral gavage was determined to be greater than 5000 mg/kg. According to the Globally Harmonized System (GHS) for chemical classification, the test substance falls under Unclassified, indicating low acute toxicity.
Pylorus Ligation-induced ulcer model
Healthy male adult Albino Wistar rats weighing 200-250 g were selected for this procedure. All the animals were kept in the standard laboratory environment (temperature: 24±2ºC, humidity: 65-70%, and a 12-hr light/dark cycle). Fasted the rats for 24 hr before the experiment to ensure an empty stomach, with access to water ad libitum. The study followed ethical guidelines for animal care and use, The study protocol was reviewed and approved by the Institutional animal ethical committee of Karpagam College of Pharmacy, Coimbatore, India. Protocol approval number KU/IAEC/Ph.D./324. All procedures were performed in accordance with the ethical committee.
Animals were divided in to six experimental groups.
• Normal Control Group:* Vehicle-treated (normal saline).
• Negative Control Group:* No treatment, pylorus ligation only.
• Ficus exasperata Low Dose:* (50 mg/kg).
• Ficus exasperata Medium Dose:* (100 mg/kg).
• Ficus exasperata High Dose:* (200 mg/kg).
• Positive Control Group:* Standard drug Ranitidine.
An oral gavage of Ficus exasperata extract and standard drug ranitidine was given to the animals 1 hr before to the pylorus ligation. (Distilled Water is used to make the dosing, prior to oral gavage.)
Surgical Procedure for pylorus ligation
To minimize intraoperative discomfort, the animals were anesthetized via an intraperitoneal (i.p.) injection of a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg). Once surgical anesthesia was achieved, each rat was secured in a supine position. A minimal midline abdominal incision (approximately 1-2 cm) was executed to expose the stomach. The pylorus was carefully isolated and ligated using a silk suture thread, taking strict precautions to prevent structural injury to the surrounding blood vessels and adjacent mesenteric tissues. Following successful ligation, the stomach was anatomically repositioned within the abdominal cavity, and the abdominal wall was closed sequentially using surgical sutures. The animals were monitored closely during the post-anesthetic recovery phase.
Sample Collection and Ulcer Assessment
During the post-ligation observation period, the animals were maintained in a fasting state without access to food or water for 4 hr to permit the accumulation of gastric secretions. At the conclusion of this period, the rats were humanely euthanized via cervical dislocation. The abdominal cavity was re-opened, and the stomach was excised rapidly. The gastric contents were collected into graduated centrifuge tubes for subsequent biochemical analysis. The stomach was then opened along the greater curvature, rinsed gently with normal saline, and the gastric mucosa was exposed. The degree of mucosal injury was evaluated macroscopically, and the gastric ulcer index was calculated by scoring the total number of lesions and grading their individual severity.
Biochemical Analysis of Gastric Secretions
Gastric Juice Volume and pH Determination
The collected gastric contents were transferred into graduated centrifuge tubes and centrifuged at 3000 rpm for 15 min to separate any solid debris. The total volume of the supernatant, representing the gastric juice volume, was measured and expressed in milliliters (mL). Subsequently, the hydrogen ion concentration (pH) of the clear gastric supernatant was quantified using a digital pH meter calibrated with standard buffer solutions.
Total Acidity Titration
To determine total acidity, a 1 mL aliquot of the centrifuged gastric juice was diluted with 9 mL of distilled water. The mixture was titrated against 0.01 N Sodium Hydroxide (NaOH) using two to three drops of phenolphthalein as an indicator. The titration endpoint was marked by the appearance of a persistent light pink coloration. Acidity was calculated and expressed in Milliequivalents per Liter (mEq/L) per 100 g of body weight.
Histopathological Examination
For microscopic evaluation, tissue samples from the stomach were harvested from both the control and high-dose treatment groups. The excised tissues were immediately fixed in 10% neutral buffered formalin for 24 to 48 hr. Following fixation, the specimens underwent automated dehydration through a graded series of ethanol, clearing in xylene, and embedding in paraffin wax blocks. Serial sections of approximately 4-5 µm thickness were cut using a rotary microtome. The sections were mounted on glass slides, stained with Hematoxylin and Eosin (H&E), and examined under a light microscope to assess structural alterations, inflammatory cell infiltration, and mucosal erosions.
Statistical Analysis
All experimental data were expressed as Mean±Standard Error of the Mean (SEM) for groups of six animals (n=6). Statistical significance and multi-group variations were analyzed using one-way Analysis of Variance (ANOVA), followed by Tukey's post-hoc multiple comparison test using GraphPad Prism software. A value of p<0.05 was considered statistically significant.
| Concentration (μg/mL) | Absorbance (nm) |
|---|---|
| 0 | 0.007 |
| 0.2 | 0.015 |
| 0.4 | 0.022 |
| 0.6 | 0.030 |
| 0.8 | 0.037 |
| 1 | 0.045 |
| 1.2 | 0.058 |
| 1.4 | 0.065 |
| Absorbance measurement data | |||||||
|---|---|---|---|---|---|---|---|
| Conc. μg/mL | Plant extract | Control | Standard | % RSA of PE | IC50 of PE | % RSA of Std | IC50 of Std |
| 10 | 0.710 | 0.800 | 0.500 | 11.25% | 40.11 μg/mL | 37.5% | 15.00 μg/mL |
| 20 | 0.600 | 0.800 | 0.300 | 25% | 40.11 μg/mL | 62.5% | 15.00 μg/mL |
| 30 | 0.521 | 0.800 | 0.200 | 34.87% | 40.11 μg/mL | 75% | 15.00 μg/mL |
| 40 | 0.401 | 0.800 | -- | 48.75% | 40.11 μg/mL | -- | 15.00 μg/mL |
| 50 | 0.312 | 0.800 | -- | 61% | 40.11 μg/mL | -- | 15.00 μg/mL |
| Concentration of Ascorbic Acid (μg/mL) | Absorbance (at 593 nm) |
|---|---|
| 20 | 0.205 |
| 40 | 0.400 |
| 60 | 0.612 |
| 80 | 0.802 |
| 100 | 1.100 |
RESULTS
Observations of evaluation of Total Flavonoid Content using (TFC) UV spectrometric method
Flavonoids are important secondary plant metabolites reported for their radical scavenging activity and capability to stabilize cellular membranes against lipid peroxidation. Spectroscopic analysis revealed a significant presence of flavonoids in the methanolic extract of plant Ficus exasperata the total flavonoid content was calculated to be 13.167 mg QE/gm. (Table 4 and Figures 2 and 3).
In vitro antioxidant Activity
DPPH Radical Scavenging Assay
The DPPH assay evaluated the hydrogen-donating capacity of Ficus exasperata extracts to neutralize free radicals. The extract demonstrated a concentration-dependent radical scavenging profile (Table 5 and Figure 4). At the lowest tested concentration of 10 µg/mL, the inhibition was 11.25%, which significantly increased to 61% at the maximum concentration of 50 µg/mL. The calculated half-maximal inhibitory concentration (IC50) of the Ficus exasperata extract was found to be 40.11µg/mL, whereas the standard ascorbic acid exhibited an IC50 value of 15.00 µg/mL.
Pharmacological Screening Antiulcer Activity of Extracts
Pylorus Ligation Induced Peptic Ulcers
The DPPH radical contains an odd electron that exhibits a strong absorption band at 517 nm, giving it a deep purple color in solution. When this electron becomes paired in the presence of a hydrogen-donating antioxidant, the radical is reduced to DPPH-H, resulting in decolorization to a yellow complex. The low IC50 value observed for Ficus exasperata underscores its potent radical-scavenging properties, which are likely mediated by the high concentration of secondary metabolites, such as polyphenols and flavonoids, present within the extract. Phenolic hydroxyl group’s act as excellent hydrogen donors, effectively terminating radical chain reactions before oxidative damage propagates (Table 5 and Figure 4).
Ferric Reducing Antioxidant Power (FRAP) assay
The reducing capacity of Ficus exasperata extract was quantified based on its ability to reduce a Ferric (Fe³⁺-TPTZ) complex to its intensely blue Ferrous (Fe²⁺-TPTZ) form at low pH. The extract showed a significant, concentration-dependent increase in reducing power (Figures 5 and 6). The FRAP assay serves as a reliable indicator of single-electron transfer capacity. The significant reduction of the Fe³⁺ complex indicates that Ficus exasperata extract is rich in antioxidants. The antioxidants act by breaking free radical chains via the donation of a hydrogen atom. This electron-donating ability confirms that the extract possesses strong reductive capacity, supporting its potential to mitigate intracellular oxidative stress in biological systems.
The linear increase suggests a strong correlation between extract concentration and reducing power. These findings highlight Ficus exasperata as a promising source of natural antioxidants, warranting further exploration to evaluate pharmacological potential and understand their mechanisms of action (Figures 5 and 6).
Pharmacological Screening Antiulcer Activity of Extracts
Pylorus Ligation Induced Peptic Ulcers
Effect on Gastric Secretion Volume, pH, and Acidity Pylorus ligation in the control and vehicle groups led to accumulation of gastric juice, severe mucosal damage, and hyperacidity. Oral pre-treatment with Ficus exasperata extract for 1 hr significantly and dose-dependently countered these pathological changes. The negative control group produced a substantial gastric juice volume of 4.2±0.3 mL, with a highly acidic pH of 2.4±0.1. This group also exhibited elevated total acidity 120±5.2mEq/L.
Pre-treatment with the high dose of Ficus exasperata extract 200 mg/kg significantly reduced the gastric juice volume to 2.8±0.1mL (p<0.05) and elevated the pH to 5.6±0.3. Concurrently, total acidity was suppressed to 50±3.6 mEq/L. These results were comparable to the standard anti-ulcer drug, ranitidine (50 mg/kg), which maintained a gastric volume of 2.5±0.1mL and a pH of 6.2±0.3.
Macroscopic examination of the stomach mucosa revealed severe hemorrhagic streaks and deep erosions in the control groups. This correlated with a high Ulcer Index score of 15.0±1.2. In contrast, animals treated with 200 mg/kg of Ficus exasperata extract showed a strong gastroprotective effect. They achieved an ulcer inhibition rate of 79.3 %, which closely rivaled the 83.3% protection shown by the ranitidine group. This was a significant increase compared to the vehicle group (p<0.05).
Histopathological Findings of stomach control and treated
Histopathological analysis of stomach sections from the control groups showed extensive mucosal erosion, deep ulceration, disrupted epithelial lines, and significant inflammatory cell infiltration. In contrast, stomach sections from the high-dose group (200 mg/kg) revealed a well-preserved mucosal architecture. The epithelial surface remained intact, and there was absence of deep mucosal hemorrhages and minimal inflammatory cell infiltration. This demonstrates that the extract is safe and free from systemic organ toxicity at the tested pharmacological concentrations (Figures 7-11).
Histopathological Findings of stomach control and treated
| Concentration of | Absorbance (at 593 nm) | Ascorbic acid equivalent antioxidant capacity |
|---|---|---|
| 50 | 0.450 | 26.36 μg/mL |
| 100 | 0.650 | 51.82 μg/mL |
| 200 | 0.900 | 94.55 μg/mL |
| Sl. No. | Group | Ulcer index (Mean ±SD) | Inhibition of Ulcer (%) | Volume mL | pH | Total acidity (mEq/L) |
|---|---|---|---|---|---|---|
| 1. | Normal Control | 0 | 0 | 2.1±0.2 | 5.8±0.2 | 39±2.4 |
| 2. | Negative Control vehicle treated | 15.0±1.2 | 0 | 4.2±0.3 | 2.4±0.1 | 120±5.2 |
| 3. | 9.8±0.8 | 34.7 | 3.6±0.2 | 3.2±0.1 | 95±4.8 | |
| 4. | 6.4±0.6 | 57.3 | 3.1±0.2 | 4.5±0.2 | 70±4.2 | |
| 5. | 3.1±0.5 | 79.3 | 2.8±0.1 | 5.6±0.3 | 50±3.6 | |
| 6. | Ranitidine (50 mg/kg) | 2.5±0.3 | 83.3 | 2.5±0.1 | 6.2±0.3 | 42±3.4 |
DISCUSSION
In the present study it is clearly found that the plant Ficus exasperata has reported quantity of flavonoids or antioxidants. Which has been evaluated by using In vitro antioxidant widely accepted DPPH and FRAP assay methods. Findings of the assay revealed that Ficus exasperata shows significant antioxidant activity by the results i.e. at the highest concentration 50 μg/mL in DPPH and 100 μg/mL in FRAP assay.
In the pharmacological in vivo antiulcer study there were notable variations between the groups, according to the morphological analysis of the stomach tissues. Compared to pylorus ligated group, animals treated with a low dose of Ficus exasperata (50 mg/kg) showed fewer gastrointestinal lesions, while some mucosal damage remained. These findings scientifically validate the traditional medicinal use of Ficus exasperata and highlight its strong potential as a natural therapeutic candidate for the management of gastric ulcer and oxidative stress-related ailments.
These results imply that at low dosages, Ficus exasperata offers modest efficacy on gastroprotection. While high dosage of Ficus exasperata (200 mg/kg) considerably demonstrate high potential and reduces mucosal damage and elicited a strong gastroprotective action. Treatment with ranitidine successfully shielded the stomach, resulting in little ulcers and maintained mucosal integrity. As we compare the activity of plant Ficus exasperata with standard ranitidine, the plant complies with the potential of standard.
Some other assessments of gastric parameters were experimented and the results founded as there is significant decrease in the volume of gastric juice, increase in pH and decrease in the total acidity at the highest dosage of Ficus exasperata (200mg/kg). it is reported that plant is having significant antioxidant and antiulcer activity.
CONCLUSION
Ficus exasperata, a medicinal plant widely acknowledged for its traditional therapeutic applications, demonstrates significant antioxidant and antiulcer activities. The findings of the present study reveal that
the pronounced antiulcer activity, evidenced by an impressive 79.3% inhibition, highlights the plant’s gastroprotective potential. Flavonoids, well-known for their ability to strengthen gastric mucosa, play a pivotal role in this effect by reducing oxidative damage, suppressing inflammatory mediators, and promoting mucosal defense mechanisms. Tannins and saponins further enhance the antiulcer efficacy by forming a protective layer over the gastric lining and improving tissue repair. Alkaloids and terpenoids complement these effects through their analgesic, anti- inflammatory, and cytoprotective properties.
Further research is recommended to elucidate the specific molecular mechanisms underlying its pharmacological effects and to explore its potential through clinical trials. The integration of Ficus exasperata into therapeutic regimens could offer a safe and effective alternative for managing gastric disorders and oxidative stress-related conditions.
