1. INTRODUCTION:

Peptic ulcer disease (PUD), a prevalent gastrointestinal disorder of the 21st century, is characterized by chronic lesions in the stomach or duodenum, affecting approximately 10% of individuals in the United States during their lifetime. Its etiology results from an imbalance between preventive mechanisms (such prostaglandins and mucosal blood flow) and aggressive forces (like stomach acid and pepsin).

Contributing factors such as Helicobacter pylori infection, NSAID use, alcohol consumption, and stress exacerbate this imbalance. Current therapies including proton pump inhibitors, antibiotics, and H2 receptor antagonists reduce mortality but are marred by adverse effects, underscoring the need for safer, cost-effective alternatives¹.

Medicinal plants, with their multifaceted bioactive compounds (flavonoids, tannins, alkaloids), offer promising gastroprotective effects by modulating acid secretion, enhancing mucosal defense, and mitigating oxidative damage. Mangifera indica (mango), a cornerstone of traditional medicine in Asia, is rich in polyphenols, flavonoids (rutin, quercetin), and triterpenes, exhibiting antioxidant, anti-inflammatory, and antimicrobial properties. Preclinical studies highlight its efficacy against H. pylori and gastrointestinal disorders, positioning it as a viable candidate for ulcer management.

Drug delivery systems further enhance herbal therapeutics. While bioadhesive systems enable localized release, they risk mucosal irritation. Floating systems, conversely, prolong gastric retention without direct mucosal contact, improving patient compliance. Floating microspheres, in particular, optimize bioavailability through sustained release and reduced dosing frequency

This study develops Mangifera indica leaf extract-loaded chitosan microspheres, optimized via Box-Behnken design, to evaluate polymer concentration, cross-linker volume, and stirring speed on yield, entrapment efficiency, and particle size. By combining herbal efficacy with advanced delivery technology, this approach aims to address PUD challenges while minimizing side effects¹.

2. MATERIALS AND PROCEDURES:

2.1. Plant Substance:

The Mangifera indica (MI) leaves were gathered from Pandit Bhagwat Dayal Sharma University of Health Sciences, Rohtak's herbal garden. The plant material was taxonomically identified and authenticated by Dr. S.K Yadav, a botanist, prior to the initiation of the research.

2.2. Chemicals and Reagents:

The study's numerous chemicals and reagents were from reliable vendors. Chitosan, ethanol, and acetic acid were acquired from a supplier based in the United States. Glutaraldehyde was obtained from a chemical manufacturer located in Rohtak, India. A surfactant (Span 80) and carboxy methyl cellulose were procured from suppliers in Panipat Haryana.

2.3. Preparation of the Extract:

For the extraction process, 20 grams of the powdered leaves were extracted using a Soxhlet apparatus with ethanol as the solvent for one hour, after concentrating the ethanol extract to eliminate any leftover solvent, the dried extract was stored for later use in an airtight container². Fresh leaves of Mangifera indica were carefully cleaned with distilled water to remove any dirt or contaminants, and then allowed to air-dry at room temperature. After drying, the leaves were ground into a fine powder and passed through a 20-mesh sieve to ensure uniformity.

2.4. Chitosan microsphere preparation:

A homogeneous solution was made by dissolving chitosan in 10 millilitres of 5% aqueous acetic acid. After adding the plant extract to this solution, 100 mL of light liquid paraffin was added to emulsify it. The emulsion was agitated for five minutes at different speeds to guarantee adequate mixing. The water-in-oil (w/o) emulsion was then cross-linked with varying concentrations of glutaraldehyde (GA). For two hours, the mixture was continuously spun to guarantee complete cross-linking. The resulting microspheres were collected by vacuum filtering to remove excess paraffin, unreacted GA, and any surfactants. Following a thorough cleaning with petroleum ether and distilled water, they were dried for 24 hours at 50°C and kept in a desiccator until they were needed again ³. The percentage yield of MIEM was calculated as:

Percentage yield = (practical amount / theoretical amount) × 100.

2.5. Particle Size Analysis and Entrapment Efficiency Determination:

The particle size of Mangifera indica extract-loaded microspheres (MIEM) was measured using micrometers to ensure size uniformity⁴. For entrapment efficiency (EE) analysis, 100 mg of MIEM was ground, dissolved in ethanol, filtered, and diluted. A validated RP-HPLC method (Shimadzu, Japan) was used to quantify the concentrations of rutin and quercetin. The formula for EE (%) was (quantity of quercetin or rutin in microspheres / amount in extract added) × 100, and the formula for drug loading (%) was (quantity of quercetin or rutin in microspheres / total microsphere weight) × 100.

2.6. Optimization of Microsphere Formulation Process:

The formulation was optimized using a Box-Behnken design (BBD) in conjunction with response surface methodology (RSM) after preliminary trials revealed that the three most important factors influencing the development of Mangifera indica extract-loaded microspheres (MIEM) were chitosan concentration, glutaraldehyde volume, and stirring speed (RPM). Three independent process variables were chosen for optimization: chitosan concentration (A), glutaraldehyde volume (B), and stirring speed (C, in RPM). These factors were chosen for optimization based on their effects on key responses: microsphere particle size (Y), entrapment efficiency of rutin (Ya) and quercetin (Yb), and percentage yield (Y). The actual levels of these Table 1 contains a list of independent variables.

Entire of 17 experimental runs were conducted as per the BBD, comprising Three replicates, eight factorial points, and six axial points at the centre point to ensure model reliability. The experimental data were analyzed using a second-order polynomial equation (Equation 4) to evaluate the linear, interaction, and quadratic effects of the selected variables on the measured responses⁵.

(Equation 4)

Statistical Analysis and Model Validation:

The experimental data were evaluated using ANOVA to ensure the model’s reliability and adequacy. Design-Expert software (v7.0.3, StatEase Inc., USA) was used for response surface plots and model validation. A desirability function approach was applied to optimize all responses like particle size, rutin and quercetin entrapment efficiency (EE), and % yield. The optimized MIEM formulation was prepared under predicted conditions, and experimental values were compared with predicted outcomes to validate the model’s accuracy⁶.

2.7. Evaluation of Developed Optimized Formulation:

The improved Mangifera indica extract-loaded microspheres (MIEM) were assessed for anti-ulcer effectiveness in experimental animal models, floating/buoyancy capability, in vitro release tests, and scanning electron microscopy (SEM). Prior to these evaluations, compatibility tests were conducted using Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction (XRD) to investigate MIEM, Mangifera indica extract (MI), and combinations of the extract and other excipients. In order to identify possible chemical interactions, materials were combined with potassium bromide (KBr) to create pellets, which were then examined using an FTIR spectrophotometer (Shimadzu, Japan) across a wavelength range of 4000–400 cm⁻¹.Using a DSC-60 Plus (Shimadzu, Japan), DSC analysis was performed by heating precisely weighed samples in sealed aluminum crimped cells between 30°C and 300°C at a rate of 10°C per minute while maintaining a 40 mL/min nitrogen supply to evaluate thermal behavior and any interactions. The crystalline structure of the samples was analyzed using an X-ray diffractometer (Thermo Fisher Scientific, India's ARL Equinox 100), with diffraction patternsrecorded with a voltage of 40 kV and a current of 30 mA across a 2θ range of 0° to 50° to determine the physical condition of the components. These studies ensured the chemical and physical stability of the MIEM formulation before proceeding with further in vitro and in vivo evaluations⁷.

2.8. SEM Analysis of MIEM:

After being dried for a full night, the optimized microspheres were inspected for surface morphology the JEOL JSM-6480LV, a scanning electron microscope made in Japan. Before imaging, MIEM samples were sputter-coated with a thin conductive layer (e.g., platinum or zirconium) to enhance conductivity. The coated microspheres were mounted on a holder, and a focused electron beam scanned the surface to detect secondary electrons, revealing details like texture, morphology, and particle uniformity⁸.

2.9. Floating/ Buoyancy Capacity of MIEM:

100 mg of MIEM and 250 mL of 0.1 N HCl were combined, and the mixture was whirled for 24 hours at 100 rpm to evaluate buoyancy. The floating microspheres were collected and filtered following incubation. The buoyancy capacity was determined using the following formula, which takes the weight ratio of floating microspheres to the total weight of floating and settled MIEM: % buoyancy = (weight of floating MIEM / (weight of floating MIEM + weight of settled MIEM)) × 100 ⁹.

2.10. In Vitro Drug Release Studies and Kinetics:

The in vitro drug release of MIEM was examined using a USP dissolving equipment I (basket type). 500 mg of MIEM capsules were placed in 500 mL of 0.1 N HCl, maintained at 37.5°C, and spun at 100 rpm. Two millilitres of samples were taken out, filtered, and subjected to HPLC analysis for quercetin and rutin at predetermined intervals. The percentage of drug released was calculated based on the initial drug content. The release data were analyzed by fitting them into various kinetic models to determine the best-fit model based on correlation coefficients and model parameters, characterizing the drug release profile from MIEM¹⁰.

Table 1. Actual values of different levels of independent variables

Independent variables

Unit

Actual values

-1 Level

Actual values

+1 Level

Chitosan concentration (A)

Volume of Glutaraldehyde (B)

Stirrer speed, RPM (C)

RPM

800

1200

2.11. Evaluation of Antimicrobial Potential:

The Helicobacter pylori strain (ATCC or clinical isolate) was utilized for this study.Culture mediums included Columbia agar and Brain Heart Infusion (BHI) agar supplemented with 5–10% sheep blood.The test sample consisted of Mangifera indica extract-loaded chitosan microspheres, prepared following standard protocols. For controls, pure Mangifera indica extract served as the herbal positive control, while Amoxicillin was used as the standard antibiotic control. DMSO or sterile water acted as the negative control. Additional materials included sterile paper discs (6 mm diameter), McFarland standard (0.5), sterile saline, Petri dishes, sterile forceps, and a Vernier caliper. Microaerophilic conditions (5% O2, 10% CO2, 85% N2) were used for the incubation, which was conducted at 37°C¹⁰.

2.11.1 Preparation of Helicobacter pylori Inoculum:

After being subcultured on BHI or Columbia agar enriched with 5–10% sheep blood, Helicobacter pylori was incubated for 48–72 hours at 37°C in a microaerophilic environment. Colonies of bacteria were collected and suspended in sterile saline with turbidity adjusted to 0.5 McFarland standard (~1.5 x 10⁸ CFU/ mL) to guarantee consistent inoculation¹⁰.

2.11.2 Preparation of Test and Control Samples:

Sample	Purpose	Concentration
Mangifera indica microspheres	Test Sample	50, 100, 200 µg/mL
Mangifera indica extract	Positive Control	100 µg/mL
Clarithromycin/Amoxicillin	Positive Control	Standard dose
DMSO or sterile water	Negative Control	—

2.11.3 Disk Diffusion Method:

BHI or Columbia agar plates supplemented with 5–10% sheep blood were prepared and allowed to solidify. The standardized Helicobacter pylori inoculum was uniformly spread over the agar surface using a sterile swab. Sterile paper discs were placed on the inoculated plates, and 20 µL of each test and control sample was pipetted onto the respective discs. Plates were left at room temperature for 30 minutes to allow sample absorption¹⁰.

2.11.4 Incubation and Measurement:

For 48–72 hours, the plates were incubated in a microaerophilic environment at 37°C. A Vernier caliper was used to quantify the zones of inhibition surrounding the discs after incubation. All measurements were recorded in millimeters and performed in triplicate to ensure accuracy¹¹.

2.12 Statistical Analysis:

The data was analyzed using one-way ANOVA, and statistical significance was determined using Tukey's post hoc test. A significant p-value was defined as less than 0.05.

3. RESULTS AND DISCUSSION:

Mangifera indica, (mango plant), is widely used across Asian countries for its medicinal properties, particularly in functional foods aimed at preventing and treating various diseases. This herb's antioxidant capacity is enhanced by a range of secondary metabolites found in its leaves,comprising phenolic acids such as caffeic acid, ferulic acid, and chlorogenic acid, as well as flavonols such as 3-O-rutinoside and quercetin 3-β-D glucoside. These substances are essential for preventing oxidative stress brought on by reactive oxygen species (ROS), which are linked to the etiology of diseases including stomach ulcers. In this study, we focused on extracting the leaves of Mangifera indica to evaluate their antiulcer activity. Prolonged gastric residency time is necessary for an efficient treatment of gastric ulcers in order to guarantee that the medication reaches the stomach's submucosal area for the best possible therapeutic impact. Conventional dose forms like tablets and capsules, because to their heavy weight, pass through the stomach fast, necessitating high dosages and sometimes resulting in poor patient compliance. Therefore, our research aimed to develop floating microspheres containing the leaf extract. These microspheres, being lightweight, can float in gastric fluid, providing sustained release of active plant compounds to the targeted mucosa over an extended period. The obtained sample was verified by a botanist based on morphological features to guarantee the validity of the plant material, and a specimen was stored for further use in the department. The leaves were then extracted using a chosen solvent, giving 29% (w/w) of extract. It's vital to remember that differences in the concentration of secondary metabolites might arise owing to varying geographical origins given the raw material's environmental circumstances¹¹. Before undergoing further processing to create microspheres, an airtight container was used to store the extract.

3.1. Extract Microsphere Preparation (MIEM):

The development of microspheres is influenced by factors like polymer concentration, surfactant, and stirrer speed. Chitosan was chosen for its biocompatibility and use in sustained-release drug delivery systems, offering advantages over synthetic polymers, especially under mild pH conditions^[1]. Particle size and drug loading were assessed in preliminary batches that varied in chitosan concentration (0.5–2.5%), surfactant (Span 80, 0.5–1.5%), glutaraldehyde (1–7 mL), and stirrer speed (700–1600 RPM). Results showed that higher surfactant concentrations led to larger particles (~59 µm) and lower drug loading (~0.18%) compared to other variables. This is in agreement with another study showing that increased surfactant concentrations result in smaller particles^[2]. Chitosan concentration, glutaraldehyde volume, and stirrer speed were found to significantly affect particle size and drug entrapment efficiency and were chosen for more optimization research.

3.2. Optimization and Statistical Analysis of MIEM Using Box-Behnken Design:

The Box-Behnken design was employed to optimize Mangifera indica extract-loaded microspheres (MIEM) by evaluating three independent variables: chitosan concentration (1–2%), glutaraldehyde volume (1–5 mL), and stirrer speed (800–1200 RPM). Seventeen experimental runs revealed a percentage yield (Y1) of 85.2–98.2%, with ten batches exceeding 85%, indicating robust process efficiency. Entrapment efficiency (EE) for rutin (Y2a: 73.62–93.46%) and quercetin (Y2b: 71.83–89.84%) demonstrated effective encapsulation of bioactive compounds, while particle size (Y3: 37.04–50.47 µm) reflected variable-dependent morphological control. Quadratic models generated via Design-Expert software (Eqs. 8–11) highlighted significant linear and quadratic effects of variables (<0.005), except for glutaraldehyde’s quadratic term (B²) in EE models. Interaction effects were minimal, though chitosan × RPM (AC) significantly influenced yield (p<0.001). ANOVA confirmed model validity (p<0.001) with high R² (>0.9) and adequate precision (>4), ensuring reliable predictions. Non-significant lack of fit (p>0.05) further validated model accuracy. Response surface plots illustrated how variable combinations affected responses, guiding parameter optimization. For instance, low RPM and moderate chitosan maximized yield, while high chitosan and low glutaraldehyde enhanced EE. These findings underscore the model’s robustness in predicting optimal MIEM formulations, balancing high yield, encapsulation efficiency, and controlled particle size for sustained anti-ulcer therapy¹².

Y₁=92.022 + 1.0675A + 0.79625B - 6.14625C- 0.070000000000014 AB + 2.08 AC + 0.3425BC - 2.96725A² - 1.10475B² + 1.37525C² (Eq -8)

Y_2a = 82.16 + 1.90A + 1.94B -4.73C – 0.6075AB + 0.1075AC + 0.2925BC – 3.21A² – 0.9188B² + 5.74C²(Eq -9)

Y_2b = 79.36 + 1.66A + 1.48B – 4.47C + 0.3700AB – 0.5200AC - 0.2600BC – 2.78A² – 1.11B²+ 5.66C²(Eq -10)

Y₃=38.75+2.46A–2.56B–0.7188C-0.6550AB-0.54AC+0.59BC+3.85A²+2.02B² +1.12C²(Eq -11)

Table 2: The Box-Behnken design suggests several runs under various experimental settings.

		Independent variable			Responses
Standard	Run	Chitosan (A)	Glutaraldehyde (B)	RPM (C)	Y₁(Yield)	Y_2a(%EE for rutin)	Y_2B(%EE for quercetin)	Particle Size (µm)
R1	5	1	1	1000	85.84	73.62	71.83	44.05
R2	15	2	1	1000	88.06	78.34	75.94	50.47
R3	6	1	5	1000	87.98	78.94	75.73	40.07
R4	4	2	5	1000	89.92	81.23	78.36	43.87
R5	9	1	3	800	97.55	87.25	84.71	39.28
R6	16	2	3	800	95.58	91.13	89.03	45.09
R7	1	1	3	1200	81.12	78.03	76.47	38.78
R8	2	2	3	1200	87.47	82.34	78.71	42.41
R9	11	1.5	1	800	98.2	90.4	86.58	43.28
R10	12	1.5	5	800	98.7	93.46	89.84	37.13
R11	8	1.5	1	1200	85.2	79.91	78.49	40.8
R12	3	1.5	5	1200	87.07	84.14	80.71	37.04
R13	17	1.5	3	1000	91.8	82.39	79.49	38.83
R14	14	1.5	3	1000	92.2	81.09	78.62	38.07
R15	10	1.5	3	1000	91.9	83.67	80.05	39.66
R16	13	1.5	3	1000	92.71	82.62	79.7	39.16
R17	7	1.5	3	1000	91.5	81.03	78.92	38.04

Table 3: ANOVA was used to determine the significance of the various answers.

Response	F Value	Probability > F (p value)	Adjusted R²	Predicted R²
Y₁Model Lack of fit	252.64 0.53	<0.0001 0.68	0.99	0.98
Y_2aModel Lack of fit	57.30 0.18	<0.0001 0.93	0.96	0.95
Y_2bModel Lack of fit	170.86 0.33	<0.0001 0.8165	0.9896	0.9805
Y³Model Lack of fit	70.84 0.12	<0.0001 0.9430	0.9752	0.97

3.3. Optimization & Validation of the Development Model:

The formulation of Mangifera indica extract-loaded microspheres (MIEM) was optimized with the use of a numerical optimization method. This method, guided by the desired outcomes—such as minimal particle size and maximum entrapment efficiency (EE) and percentage yield—generated multiple solutions for optimal formulation conditions. Based on these criteria, Design-Expert software recommended using 1.66% (w/w) chitosan, 4.7 mL of the glutaraldehyde, and a stirring speed of approximately 850 RPM for the ideal preparation of MIEM.

To confirm the accuracy of these optimized parameters, experimental trials were conducted under the suggested conditions. The results demonstrated that the measured responses for particle size, EE, and percentage yield were all within the 95% confidence interval, as outlined in Table S11. This successful validation confirms the reliability of the developed model in predicting the optimal formulation parameters for MIEM preparation.

3.4. Evaluation of the Optimized Formulation:

The optimized Mangifera indica extract-loaded microspheres (MIEM) were thoroughly characterized. The particle size was found to be 40.1 ± 0.07 µm, with entrapment efficiencies of 86.93 ± 0.55% for rutin and 85.09 ± 0.15% for quercetin. The loading capacities were 0.237 ± 0.09% for rutin & 0.249 ± 0.021% for quercetin. FTIR, DSC, and XRD analyses were conducted to assess potential interactions between the API and excipients. The FTIR spectra indicated functional group preservation, suggesting compatibility between the extract and excipients. The optimized formulation showed peaks at 3362.96 cm⁻¹ (O-H), 1646.77 cm⁻¹ (amines), and 1376.7 cm⁻¹ (C-N), confirming successful encapsulation and extract-excipient compatibility. DSC analysis showed shifts in thermal transitions, indicating structural modification of chitosan, which enhanced stability but lowered thermal resistance. The extract's endothermic peak (Tonset = 91.56°C, Tpeak = 103.26°C) was absent in the optimized formulation, suggesting conversion to an amorphous state. XRD analysis revealed the absence of crystalline peaks for the extract in the MIEM, confirming molecular dispersion or amorphous form^[13]. SEM analysis confirmed the smooth surface morphology, and the capacity to float and the in vitro release studies supported the potential of MIEM for gastro-retentive drug delivery in the treatment of Helicobacter pylori-induced gastric ulcers.

Figure 4: The FTIR spectra of (R1) Leaf extract, (R2) Chitosan, (R3) Glutaraldehyde, (R4) Span 80, (R5) Physical mixture (extract, chitosan, Glutaraldehyde, span 80, (R6) Optimized formulation.

Figure 5: DSC thermogram of (R1) Extract, (R2) Physical mixture (extract, chitosan, Glutaraldehyde, span 80), (R3) Optimized formulation.

Figure 6: X-ray diffraction of (R1) Extract, (R2) Physical mixture (extract, chitosan, Glutaraldehyde, Span 80), (R3) Optimized formulation.

3.5. SEM Analysis:

The surface characteristics of prepared microspheres were examined using SEM, as illustrated in Figure 7. The SEM images revealed that the microspheres exhibited a spherical shape with uniform geometry. The surface appeared smooth and well-formed, indicating successful formulation under optimized conditions. The enlarged images further confirmed the absence of surface irregularities, suggesting consistent particle formation throughout the batch¹⁴.

Figure 7: A SEM of an optimized formulation (MIEM).

3.6. Floating Ability of MIEM:

The buoyancy of the Mangifera indica extract-loaded microspheres (MIEM) was evaluated, showing a floating capacity of 86.19 ± 2.9%. This test determined the microspheres' ability to remain afloat in gastric fluid. The floating behavior was sustained for about 24 hours, thanks to the hollow structure of the chitosan-based microspheres, which improved their suspension in the gastric environment¹⁵. The hydration of gel formers, polysaccharides, and polymers in the microspheres upon contact with gastric fluid forms a colloidal gel barrier, controlling the rate of fluid uptake and drug release. The hydration of the hydrocolloid layer maintains the gel layer's integrity, and the trapped air in the inflated polymer reduces density, contributing to the microspheres' buoyancy⁵.

3.7. In vitro release studies:

The in-vitro release profiles of rutin and quercetin from both the mulberry leaf extract and the formulated MIEM were evaluated. It was observed that approximately 83% of the active compounds from the crude extract were released within the first two hours. In contrast, the MIEM exhibited a more controlled release, with a similar concentration of marker compounds detected only after eight hours (Figure 8). This sustained release pattern highlights the ability of the floating microspheres to prolong gastric residence time, demonstrating their potential as an effective system for the controlled delivery of Mangifera indica extract¹⁶.

Figure 8: Percentage release of rutin and quercetin from Mangifera indica extract& optimized formulation (MIEM).

3.8. Evaluation of Antimicrobial Potential:

The Mangifera indica extract-loaded chitosan microspheres demonstrated significant anti- Helicobacter pylori activity in the disk diffusion assay¹⁷. A dose-dependent increase in antimicrobial efficacy was observed, with the highest concentration (200 µg/mL) yielding the most substantial zone of inhibition in table 4.

Table 4: Zone of inhibition measurements

Sample	Concentration (µg/mL)	Zone of Inhibition (mm) (Mean ± SD)
Mangiferaindicamicrospheres	50	14.2 ± 0.4
Mangiferaindicamicrospheres	100	18.9 ± 0.5
Mangiferaindicamicrospheres	200	23.8 ± 0.6
Mangiferaindica extract	100	15.3 ± 0.4
Amoxicillin	Standard dose	28.6 ± 0.5
DMSO/Water	—	No inhibition

Figure 9: Zone of inhibition: A-Amoxicillin (Positive control),B- Mangifera indica microspheres 200µg/Ml,C-Mangifera indica microspheres 50 µg/Ml, D-Mangifera indica microspheres 100 µg/Ml. E- Mangifera indica extract 100 µg/Ml, F-Water (Negative control)

The sustained release nature of the gastroretentive chitosan microspheres contributed significantly to the enhanced antimicrobial activity observed. The controlled release mechanism allowed prolonged exposure of Helicobacter pylori to the active constituents, leading to larger zones of inhibition compared to the pure extract. This finding highlights the importance of sustained release systems in improving the bioavailability and therapeutic efficacy of herbal formulations.

Statistical analysis confirmed the significance of the results (p < 0.05), with the 200 µg/mL microsphere formulation demonstrating markedly improved activity compared to the pure extract. While the standard antibiotic exhibited the highest inhibition, the microsphere formulation presents a promising natural alternative with potential benefits, such as prolonged gastric retention and reduced risk of antibiotic resistance.

Toxicity Evaluation of Mangifera indica Leaf Extract:

In this study, Mangifera indica leaf extract was used to develop floating microspheres for anti-Helicobacter pylori activity, evaluated only through in vitro assays. No new animal testing was conducted. However, toxicity data from Severi et al. (2009) showed that an aqueous extract given orally at 5 g/kg to Swiss mice caused no toxicity, mortality, or organ damage, with no significant weight changes or pathological findings. According to Loomis and Hayes (1996), this dose is “practically non-toxic.” Thus, existing data confirm the extract’s safety, and no additional toxicity studies were necessary^18-23.

4. CONCLUSION:

Gastro-retentive floating microspheres of Mangifera indica leaf extract (MIEM) were successfully developed and optimized using a Box-Behnken design (BBD) for Helicobacter pylori-induced gastric ulcers. The optimized formulation, based on chitosan concentration, glutaraldehyde volume, and stirrer speed, achieved high yield, excellent entrapment effectiveness for quercetin and rutin, as well as regulated particle size. Physicochemical characterization showed no chemical interaction between the extract and excipients, with the bioactive compounds dispersed in the chitosan matrix enhancing solubility and bioavailability. SEM images confirmed smooth, spherical microspheres, and in vitro release studies indicated sustained release over 24 hours. Antimicrobial tests demonstrated dose-dependent efficacy against H. pylori, with a 200 µg/mL dose achieving inhibition zones comparable to amoxicillin. MIEM combines the natural benefits of i with gastro-retentive technology, offering a promising alternative to conventional therapies. Future studies should assess in vivo efficacy and long-term stability for clinical validation.

5. DATA AVAILABILITY:

The additional data will be made available to the corresponding authors upon reasonable request.

6. CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

7. ACKNOWLEDGMENTS:

The authors want to acknowledge the Director and technical staff of the Central Instrumentation Laboratory Baba Mastnath University, Rohtak, for providing the necessary facilities to conduct the present research.

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Received on 07.05.2025 Revised on 17.10.2025

Accepted on 24.11.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2319-2328.

DOI: 10.52711/0974-360X.2026.00333

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