1. A phytosomal formulation (F1) comprising 2,4-di-tert-butylphenol and a phospholipid for the effective management of peptic ulcer, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid ranges from 1:1 to 1:5.

2. The formulation as claimed in claim 1, wherein the phospholipid is soya lecithin.

3. The formulation as claimed in claim 1, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid is 1:4.

4. The formulation as claimed in claim 1, wherein the uniform particle size is maintained in the range of 90–110 nm with a PDI in the range of 0.250-0.360.

5. The formulation as claimed in claim 1 may further comprise one or more pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, or coating agents.

6. The formulation as claimed in claim 1, wherein the composition is formulated as a powder, capsule, tablet, or suspension suitable for oral administration.

7. The formulation as claimed in claim 1, wherein the composition exhibits antioxidant, anti-inflammatory, and gastroprotective activity.

8. The formulation as claimed in claim 1, wherein administration of the composition results in modulation of oxidative stress biomarkers, characterized by increased catalase and superoxide dismutase (SOD) activity, enhanced glutathione (GSH) and nitric oxide (NO) levels, and decreased thio-barbituric acid reactive substances (TBARs), thereby preserving gastric mucosal integrity and reducing inflammatory damage.

9. The formulation as claimed in claim 1, wherein administration of the formulation provides gastroprotective activity, characterized by a percent protection against peptic ulcer in the range of 70–90% at low dose and 80–100% at high dose.

10. A method for preparing the phytosomal composition as claimed in claim 1, wherein the method comprising the steps of: i. Dissolving 2,4-DTBP in 15 ml of methanol and soya lecithin in 15 ml of chloroform; ii. Transferring both solutions into a round-bottom flask (RBF); iii. Refluxing the mixture at 30-50°C for 2 hours with continuous stirring; iv. Evaporating the solvent at 40 °C and 120 rpm using a rotary vacuum evaporator until a thin lipid film is formed on the flask wall; v. Placing the flask containing the lipid film in a desiccator overnight to ensure complete solvent removal; vi. Rehydrating the dried film using phosphate buffer saline (PBS, pH 7.4) to obtain a dispersion; vii. Subjecting the dispersion to probe sonication at 60% amplitude with 5-second on–off intervals to produce phytosomes of the desired particle size; and viii. Lyophilizing the final phytosomal dispersion to obtain the desired formulation.

11. A method of treating peptic ulcer disease in a subject in need thereof, comprising administering a pharmaceutically effective amount of the phytosomal composition as claimed in claim 1, thereby reducing gastric ulcer index and oxidative stress, and increasing gastric mucosal protection.

Description:FIELD OF INVENTION
The present invention relates to the field of pharmaceutical and nutraceutical compositions, more particularly to phytosomal formulations of plant-derived phenolic compounds. Specifically, the invention pertains to a phytosome complex of 2,4-di-tert-butylphenol with phospholipids (such as soya lecithin) for use in the prevention and treatment of peptic ulcers and other gastrointestinal disorders involving oxidative stress and inflammation.

BACKGROUND OF THE INVENTION
Peptic ulcer is a major health issue affecting 11.22% of people in India. Various factors like H. Pylori, use of NSAIDs, smoking, stress, alcohol, trauma, spicy food, and genetics cause peptic ulcers. The available conventional treatments, such as H2 RAs, PPIs, and antacids, show limited efficacy and significant adverse effects that create poor patient compliance. Ulcer is an issue associated with stress or the use of NSAIDs, which requires chronic therapies, leading to a high cost of treatment. Chronic exposure of NSAIDS results in predictable and unpredictable drug-drug interactions, complicating long-term management and patient safety.
Also, peptic ulcers and other gastrointestinal problems are commonly caused by excess gastric acid, infection with Helicobacter pylori, long-term use of painkillers, and oxidative stress. Current drugs, such as proton pump inhibitors and antibiotics, provide relief but often lead to side effects and recurrence.
Natural plant-derived compounds, including phenolic molecules, are known for their antioxidant and anti-inflammatory effects that may protect the stomach lining. However, these compounds usually suffer from poor solubility and low absorption when taken orally.
To improve delivery of bioactives, phytosome technology has been developed. In this approach, a plant compound is bound with phospholipids, creating a complex that is better absorbed in the body. Prior art EP0300282A1, such as phytosomal silybin soy from milk thistle, has already shown improved bioavailability and clinical benefits in liver and gastric protection.
Banerjee S, Mukhopadhyay S, Das A, Banerjee S, Bose S, Banerjee S, et al. Preparation, Characterisation, Anticancer Potential and Safety Evaluation of a Soy Lecithin Phytosome Delivery System Loaded with Constituents from Barleria lupulina. Journal of Microencapsulation.
Barani M, Sangiovanni E, Angarano M, Rajizadeh MA, Mehrabani M, Piazza S, Gangadharappa HV, Pardakhty A, Mehrbani M, Dell'Agli M, Nematollahi MH. Phytosomes as Innovative Delivery Systems for Phytochemicals: Int J Nanomedicine doi: 10.2147/IJN.S318416 discloses that phytosomes are a complex of phospholipids and natural active phytochemicals, bound in their structures, obtained by the reaction between phosphatidylcholine (or any hydrophilic polar head groups) and plant extracts in an aprotic solvent. These formulations exhibit improved pharmacological and pharmacokinetic properties as compared to prevalent preparations. The lipid-soluble phosphatidyl portion completely covers the hydrophilic phytoconstituent-choline complexes. Phytosomes have remarkable benefits, such as high drug encapsulation, and reveal a better stability profile.
US5684204A discloses sulfur-containing di-tert-butylphenol compounds beneficial as anti-inflammatory agents. This patent highlights that certain derivatives provided gastroprotective benefits, reducing the occurrence of stomach ulcers and erosions typically associated with conventional NSAIDs, particularly when induced by ethanol or long-term NSAID administration. However, it does not cover the parent 2,4-di-tert-butylphenol itself, nor does it mention phytosomal formulations or delivery with phospholipids.
Despite this progress, there is no disclosure of 2,4-di-tert-butylphenol in phytosomal form for the prevention or treatment of ulcers. There remains a need for effective, bioavailable formulations that combine the therapeutic activity of 2,4-DTBP with the absorption advantages of phytosome technology.
Although many anti-ulcer drugs such as proton pump inhibitors (PPIs), H₂ receptor antagonists, and antacids are currently available in the market, they suffer from significant drawbacks, including short duration of action, side effects, and high recurrence rates of peptic ulcer disease (PUD) upon discontinuation.
Therefore, the present invention aims to provide a novel formulation comprising 2,4-di-tert-butylphenol in phytosomal form with phospholipids such as soya lecithin, to overcome the limitations of poor solubility and low oral bioavailability of the free compound. The invention seeks to enhance the absorption, stability, and therapeutic efficacy of 2,4-di-tert-butylphenol, thereby offering an effective composition for the prevention and treatment of peptic ulcers and reduced recurrence of ulcer symptoms as compared to conventional therapies.

OBJECT OF THE INVENTION
Accordingly, the main object of the present invention is to provide a novel 2,4-di-tert-butylphenol (2,4-DTBP) phytosome (F1) formulation for the effective management of peptic ulcer.
Another object of the present invention is to provide a phytosomal formulation of 2,4-di-tert-butylphenol with phospholipids such as soya lecithin to overcome the limitations of poor solubility and low oral bioavailability.
Yet another object of the present invention is to provide a composition that exhibits gastroprotective activity, particularly in the management and prevention of peptic ulcer disease (PUD) and related gastrointestinal disorders.
Yet another object of the present invention is to provide a phytosome-based drug delivery approach that enhances the bioavailability, improves patient compliance, and provides better efficacy and fewer side effects.
Yet another object of the present invention is to develop a formulation with antioxidant and anti-inflammatory properties, thereby reducing mucosal injury associated with oxidative stress and inflammation.
Yet another object of the present invention is to provide a method for the preparation of the phytosomal complex of 2,4-di-tert-butylphenol and phospholipids.

SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel 2,4-di-tert-butylphenol (2,4-DTBP) phytosome (F1) formulation for the effective management of peptic ulcer.
In one aspect, the present invention provides a phytosomal formulation of 2,4-di-tert-butylphenol with phospholipids such as soya lecithin, both are present in a pre-determined ratio.
In yet another aspect, the present invention relates to a novel phytosomal formulation (F1) comprising 2,4-di-tert-butylphenol (2,4-DTBP) and phospholipids such as soya lecithin. The formulation is developed to overcome the limitations of poor solubility, low stability, and reduced oral bioavailability of free 2,4-DTBP, enhancing its therapeutic efficacy in the prevention and treatment of peptic ulcer disease (PUD) and reduced side effects.
In yet another aspect, the present invention provides a formulation, wherein administration of the composition results in modulation of oxidative stress biomarkers, characterized by increased catalase and superoxide dismutase (SOD) activity, enhanced glutathione (GSH) and nitric oxide (NO) levels, and decreased thio-barbituric acid reactive substances (TBARs), thereby preserving gastric mucosal integrity and reducing inflammatory damage.
In yet another aspect, the phytosomes are prepared using thin-film hydration, resulting in nanosized vesicles with uniform particle distribution. The developed phytosomes exhibit an average particle size in the range of 90-110nm with a polydispersity index (PDI) in the range of 0.250-0.360, ensuring stability and enhanced absorption.
In yet another aspect, the F1 formulation provides gastroprotective activity with 70-90% protection at low doses and 80-100 % protection at high doses, thereby offering an effective and safe composition for managing peptic ulcers.

BRIEF DESCRIPTIONS OF DRAWINGS:
Figure 1. illustrates overlay of FTIR spectra of soya lecithin, 2,4-DTBP, and F1.
Figure 2. illustrates overlay of PXRD spectra of soya lecithin, 2,4-DTBP, and F1.
Figure 3. illustrates TEM micrographs of F1 phytosomes.
Figure 4. illustrates particle size distribution of F1 phytosomes formulation.
Figure 5. illustrates Raman spectra of F1 phytosomes.
Figure 6. illustrates pH variation of 2,4-DTBP, Standard (Yashtimadhu®),
F1 with the addition of 0.1N HCl
Figure 7. illustrates gastric mucosal damage in different groups observed in the pyloric ligation-induced ulcer model.
Figure 8. illustrates indomethacin effects in different groups, highlighting spot ulcers, erythema, or hemorrhagic streaks.
Figure 9. illustrates a) Comparison of pH levels among various groups, including treatment, standard, normal, and control groups; b) Effect of different treatments on gastric volume.
Figure 10. illustrates a) Ulcer index and; b) % protection of different groups in pyloric ligation-induced ulcer model.
Figure 11. illustrates a) Ulcer index and b) % protection of different groups in the indomethacin-induced ulcer model.
Figure 12. illustrates estimation of a) Free acidity, and b) Total acidity among various groups.
Figure 13. illustrates histopathological analysis of stomach tissue in pyloric ligation-induced ulcer in rats from different groups: a) Normal control; b) Positive control; c) Standard; d) 2,4-DTBP, low dose; e) 2,4-DTBP, high dose; f) F1, low dose; g) F1, high dose.
Figure 14. illustrates histopathological analysis of stomach tissue in indomethacin-induced ulcer in rats from different groups: a) Normal control; b) Positive control; c) Standard; d) 2,4-DTBP, low dose; e) 2,4-DTBP, high dose; f) F1, low dose; g) F1, high dose.
Figure 15. illustrates Estimation of biochemical parameters, a) Catalase, b) SOD.
Figure 16. illustrates Estimation of biochemical parameters, a) GSH, b) NO
Figure 17. illustrates Estimation of biochemical parameters, a) TBARs, b) Total protein

DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a phytosomal formulation of 2,4-di-tert-butylphenol with phospholipids such as soya lecithin to overcome the limitations of poor solubility and low oral bioavailability.
The present disclosure can be understood more readily by reference to the following description, taken in conjunction with the accompanying Figures and Examples, all of which form a part of this disclosure.
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of the invention covered in the present disclosure. However, such a particular form is only an exemplary embodiment, and without intending to imply any limitation on the scope of the invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Before the present disclosure or methods of the present disclosure are described in greater detail, it is to be understood that the specific products, methods, processes, conditions, or parameters are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. For example, "about" can mean within one or more standard deviations, or within ± 30%, 25%, 20%, 15%, 10% or 5% of the stated value.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or composites/scaffolds.
Reference throughout this specification to “certain embodiments”, “further embodiments”, “specific embodiments”, “further specific embodiment”, “one embodiment”, “a non-limiting embodiment”, “an exemplary embodiment”, “some instances”, or “further instances”, means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure.
As used herein, the terms ‘include’, ‘have’, ‘comprise’, ‘contain’ etc. or any form of said terms such as ‘having’, ‘including’, ‘containing’, ‘comprising’ or ‘comprises’ are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed.
As used herein, the term “invention”, “present invention”, “disclosure” or “present disclosure” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification.
The terms “process(es)” and “method(s)” are considered interchangeable within this disclosure.
For convenience, certain terms used in the specification and examples are collected in this section below:
RBF: round bottom flask
PBS: phosphate buffer saline
DTBP: di-tert-butylphenol
FTIR: Fourier Transform Infrared spectroscopy
PXRD: Powder X-ray diffraction
TEM: Transmission Electron Microscopy
PDI: Poly dispersity index
F1: first formulation batch
PUD: peptic ulcer disease
TBARs: thio-barbituric acid reactive substances
GSH: Glutathione
NO: Nitric Oxide
SOD: Superoxide Dismutase
DTNB: 5,5′-dithiobis-(2-nitrobenzoic acid

In one specific embodiment, the present invention provides a novel 2,4-di-tert-butylphenol (2,4-DTBP) phytosome (F1) formulation for the effective management of peptic ulcer.
In another embodiment, the present invention provides a novel F1 phytosome with optimized physicochemical properties and improved therapeutic efficiency, thereby offering a potential advancement in peptic ulcer and fewer side effects.
In yet another embodiment, the present invention provides phytosomal formulation designated as F1, comprising 2,4-di-tert-butylphenol and soya lecithin as phospholipid, wherein administration of the formulation results in modulation of oxidative stress biomarkers, characterized by increased catalase and superoxide dismutase (SOD) activity, enhanced glutathione (GSH) and nitric oxide (NO) levels, and decreased thio-barbituric acid reactive substances (TBARs), thereby preserving gastric mucosal integrity and reducing inflammatory damage.
In yet another embodiment, the present invention discloses a phytosomal composition comprising 2,4-di-tert-butylphenol and a phospholipid for the effective management of peptic ulcer wherein said 2,4-di-tert-butylphenol is complexed with the phospholipid to enhance solubility, stability, bioavailability and to reduce side effects, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid ranges from 1:1 to 1:5.
In yet another embodiment, the phospholipids are to be selected from a list comprising of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE).
In yet another embodiment, the present invention provides that the main source of phosphatidylcholine is soya lecithin or soya bean.
In yet another embodiment, the present invention discloses that the molar ratio of 2,4-di-tert-butylphenol and soya lecithin is 1:4.
In yet another embodiment, the particle size is maintained in the range of 90–110 nm with a PDI in the range of 0.250-0.360.
In yet another embodiment, the present invention provides a formulation, wherein administration of the composition results in modulation of oxidative stress biomarkers, characterized by increased catalase and superoxide dismutase (SOD) activity, enhanced glutathione (GSH) and nitric oxide (NO) levels, and decreased thio-barbituric acid reactive substances (TBARs), thereby preserving gastric mucosal integrity and reducing inflammatory damage.
In yet another embodiment, the present invention provides a formulation, wherein administration of the formulation provides gastroprotective activity, characterized by a percent protection against peptic ulcer in the range of 70–90% at low dose and 80–100% at high dose.
In yet another embodiment, the present invention discloses that the F1phytosome is prepared using the thin-layer hydration method, resulting in a uniform particle size distribution (PDI) value of 0.352, enhancing the anti-ulcer effect.
In yet another embodiment, the present invention provides that thin-layer hydration method for preparing the phytosomal comprising the steps of:
Dissolving 2,4-DTBP in 15 ml of methanol and soya lecithin in 15 ml of chloroform;
Transferring both solutions into a round-bottom flask (RBF);
Refluxing the mixture at 30-50°C for 2 hours with continuous stirring;
Evaporating the solvent at 40 °C and 120 rpm using a rotary vacuum evaporator until a thin lipid film is formed on the flask wall;
Placing the flask containing the lipid film in a desiccator overnight to ensure complete solvent removal;
Rehydrating the dried film using phosphate buffer saline (PBS, pH 7.4) to obtain a dispersion;
Subjecting the dispersion to probe sonication at 60% amplitude with 5-second on–off intervals to produce phytosomes of the desired particle size; and
Lyophilizing the final phytosomal dispersion to obtain the desired formulation.

In yet another embodiment, the composition as claimed in claim 1, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid is 1:4.
In yet another embodiment, the formulation may further comprise one or more pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, or coating agents.
In yet another embodiment, the composition is formulated as a powder, capsule, tablet, or suspension suitable for oral administration.
In yet another embodiment, the composition exhibits antioxidant, anti-inflammatory, and gastroprotective activity.
In yet another embodiment, the F1phytosome is prepared using the thin layer hydration method, resulting in a uniform particle size distribution (PDI), enhancing the anti-ulcer effect in in vitro acid neutralizing test and in vivo pyloric ligation and indomethacin-induced gastric ulcer in rats. The present invention provides a better alternative that offers potential anti-ulcer activity.
In yet another embodiment, the phytosomal composition comprising 2,4-di-tert-butylphenol and soya lecithin prepared by an anti-solvent precipitation method, wherein the active compound and phospholipid are dissolved in a water-miscible organic solvent, followed by controlled addition into an aqueous phase to induce precipitation of nanosized phytosomes.
In yet another embodiment, the anti-solvent precipitation method comprises the steps of:
Weighing 100 mg of 2,4-di-tert-butylphenol (2,4-DTBP) and 200 mg of soya lecithin and dissolving them in 20 ml of dichloromethane in a 100 ml round-bottom flask (RBF).
Refluxing the mixture at 30-50 °C for 2 hours to ensure complete mixing and interaction between drug and phospholipid.
removing the assembly and concentrate the solution to 10–15 ml by partial solvent evaporation.
Adding 20 ml of n-hexane (anti-solvent) slowly into the concentrated solution under constant stirring to induce precipitation of phytosomes.
Allowing the precipitated phytosomes to settle, then collect by centrifugation or filtration.
Drying the obtained phytosomes under vacuum or lyophilization to remove residual solvents and obtain a stable powder formulation.
In yet another embodiment, the present invention discloses that a comparative study was carried out between the phytosomal formulation of 2,4-di-tert-butylphenol and Yashtimadhu, a known herbal anti-ulcer agent. The results demonstrated that the 2,4-DTBP phytosomes exhibited superior gastroprotective activity in experimental models of peptic ulcer disease (PUD) as compared to Yashtimadhu, with significantly improved antioxidant capacity, mucosal protection, and reduction in ulcer index.
It is noted that PUD is a gastrointestinal disorder that erodes the mucosa or submucosa, which occurs due to the imbalance between the aggressive and defensive factors. It remains a major worldwide problem that affects approximately 11.22% of people in India. Many anti-ulcer drugs are available in the market, but they have major drawbacks with recurrence of PUD is also a major concern. The present invention highlighted the development of novel 2,4-DTBP phytosomal formation and evaluated it against gastric ulcer and with no or less side effects.
In the present invention, the inventors have developed a strategy for combating peptic ulcer disease. For the very first time, F1 phytosomal formulation was developed by the thin layer hydration method and then characterized via FTIR, TEM, PXRD, PDI, and Raman spectroscopy. Further, anti-ulcer activity was determined with in vitro activity, such as acid neutralizing capacity test, and in vivo studies, such as pyloric-ligation induced gastric ulcer model and indomethacin-induced gastric ulcer in rats. The in vitro invention revealed that the 2,4-DTBP and F1 had a better neutralizing capacity than the standard herbal medicine, Yashtimadhu®. This highlighted the formulation's ability to buffer the excessive acid present.
After the successful establishment of the animal models, inventors evaluated various parameters like gross mucosal damage, gastric pH, total acid volume, ulcer index, % protection, free acidity, and total acidity. Histopathological studies and biochemical estimation were also done to determine the effectiveness of the 2,4-DTBP and F1. Gastric pH was significantly elevated in the treatment group as compared to the positive control and standard group, while the gastric volume, ulcer index, free and total acidity gradually reduced in the treatment group, compared to the positive control and standard group.
Also, the elevated % protection of gastric ulcer indicated the better effectiveness of the 2,4-DTBP and F1, better potential to act as an anti-ulcer agent. In contrast, 2,4-DTBP and F1 markedly elevated the level of catalase, GSH, SOD, and NO, indicating the antioxidant defense property and mucosal protection, while reducing the level of TBARs indicated the free-radical scavenging or antioxidant property, and a decrease in total protein indicated the reduced mucosal damage or better protective effect.
Furthermore, histopathology studies revealed that the 2,4-DTBP and F1 reduced the mucosal and sub-mucosal damage. This also maintains the integrity of the epithelial lining, minimum inflammatory infiltration, and regenerative glandular activity. Anti-ulcer activity was determined through an in vitro acid neutralizing test and in vivo pyloric ligation and indomethacin-induced gastric ulcer in rats. Treatment with F1 significantly reduced ulcer index, gastric acidity (free and total acidity), and oxidative stress markers while elevating the anti-oxidant defense markers.
Even though we have explained the invention of the present disclosure using specific examples, this explanation is not meant to limit how you understand it. People who are skilled in this field may think of various changes and different versions of the invention after reading this description. We expect that such changes can be made without straying from the main idea or purpose of the invention as defined in the claims.

The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

EXAMPLES
Example 1: Characterization of Developed Phytosomes
The prepared phytosomal formulation of 2,4-di-tert-butylphenol was subjected to various characterization studies to evaluate their physical, chemical, and morphological properties:
FTIR: FTIR was done to determine the chemical stability and structure, and interaction of the 2,4-DTBP and soya lecithin. FTIR spectra of soya lecithin, 2,4-DTBP, and F1 were examined using FTIR spectrophotometry (BRUKER FT-IR Alpha spectrophotometer). Scanning was carried out in the 400–4000 cm-1 regions. The FTIR spectrum of soya lecithin had the distinctive signals at 2922 cm-1, and 2852 cm-1, related to the C–H stretching found in the long fatty acid chain; 1736 cm-1 (C=O stretching of ester bonds); 1230 cm-1 (P=O stretching); 1052 cm-1 (C-O-P stretching). 2,4-DTBP shows 3517 cm-1 (O-H stretching), 1361 cm-1, and 1176-1205 cm-1 (C-O stretching of phenolic groups). The spectra of F1 show characteristic peaks of soya lecithin and 2,4-DTBP, with notable shifts observed at 2926 cm-1, 2858 cm-1, 1724 cm-1, 1198 cm-1, 1254 cm-1, 1019 cm-1-1081 cm-1 (phospholipid related peaks); and 3383 cm-1-3275 cm-1, 1647 cm-1, 1507 cm-1-1458 cm-1, 1368 cm-1, 1198 cm-1-1254 cm-1 (phenol related peaks). The spectra reflect a combination of characteristic peaks seen in soya lecithin and 2,4-DTBP, along with possible shifts due to interaction within the complex (Figure 1).
PXRD: A powder X-ray diffractometer was used to record the XRD analysis. The beam diffraction was scanned between 5°C and 90°C. Separate spectra were run for 2,4-DTBP, soya lecithin, and F1. The X-ray diffractograms of 2,4-DTBP, soya lecithin, and F1 are shown. F1 displayed intense, sharp peaks, indicating its crystalline nature. The diffractogram of the phospholipids displayed minimal sharp peaks and exhibited an amorphous state. F1 showed a greater reduction in peak intensity and showed a high degree of amorphization. The presence of a more dominant broad hump shows that 2,4-DTBP is effectively incorporated into the lecithin matrix (Figure 2).
TEM: A drop of F1 dispersion was put on a carbon-coated copper grid, stained with phosphotungstic acid, and excess stain was removed to undertake a morphological analysis of the optimized formulation using TEM. TEM was used to see the grid after it had air-dried. Liposome-like vesicles with an integrated structure, uniform size, and even dispersion were discovered by TEM investigations. No indications of particle aggregation were seen, while a spherical, translucent morphology and closed vesicles were seen. The surface morphology, shape, size, and structure of F1 show spherical vesicles formed after interaction with the phospholipids (Figure 3).
PDI: The particle size analyzer was used to assess the size distribution of the and F1 at 25°C in an aqueous environment. The PDI was evaluated with the DLS, commonly measured to identify the particle size distribution. A homogenous and monodispersion of the solution was indicated by the PDI values between 0 to 0.5; if more than 0.5 suggested non-homogeneity and polydispersity. In contrast, the phytosomal formulation, F1, had a PDI value of 0.352. Also, the F1 had a size of 95.32 nm and an intercept of 0.352 (Figure 4).
Raman Spectroscopy: Raman spectroscopy determines the chemical structure, phase and morphology, crystallinity, and molecular interactions by the interaction of light with chemical bonds within materials of F1. F1 shows a reduction in crystallinity-related peaks and an enhancement in the amorphization (Figure 5).

Example 2: In-Vitro Anti-Ulcer Activity of the developed Phytosome
The gastroprotective potential of the phytosomal formulation of 2,4-di-tert-butylphenol (F1) was evaluated using established in-vitro models.
Acid Neutralizing Capacity Test: The antacid capacity of 2,4-DTBP, Standard drug (Yashtimadhu®), and F1 was determined using an acid-base neutralization reaction. 5 mg of the compound dissolved in 30 ml of distilled water and hydrochloric acid (HCl) of 0.1 M is used for titration, where methyl orange was taken as an indicator. pH was measured by using a pH meter every 2 minutes, along with the amount of HCl used.
Figure 6 shows the antacid activity of the 2,4-DTBP and F1 by measuring pH changes with the addition of 0.1N HCl. pH of all three samples decreased as the HCl was added, where 2,4-DTBP had the highest pH starting around 10. When acid was added, the pH of the 2,4-DTBP declined gradually and showed slow resistance to acidification. F1 had better acid resistance and also better than the standard (Figure 6).
Animal Study and Ethical Approval: The present invention was performed using adult male Wistar Albino rats weighing about 200 g to 250 g. The invention was carried out following CCSEA guidelines and conducted after prior approval of the Institutional Animal Ethical Committee (IAEC) (IAEC No.: BV/IAEC/ December 2024/12) and acclimatized for 10 to 15 days before experimentation in normal animal house conditions: 25±2°C room temperature; 45 to 55 % relative humidity; 12 h dark and light cycle; provided water ad libitum before one week of experimentation.

Example 3: In-Vivo Anti-Ulcer Activity of the developed Phytosome
The anti-ulcer efficacy of the phytosomal formulation of 2,4-di-tert-butylphenol (2,4-DTBP) was evaluated using standard experimental ulcer models in Wistar rats.
Acute Toxicity Study: Acute oral toxicity of the 2,4-DTBP was conducted before pharmacological screening according to the OECD guideline 423. Four different doses (5, 50, 300, and 2000 mg/kg) of 2,4-DTBP were administered orally to the other groups of animals (n=3) and later observed for any signs of toxicity and mortality. No signs of toxicity and mortality were observed for the compound 2,4-DTBP, and it was found to be safe up to 2000mg/kg of the dose.
Animal Models (Pyloric Ligation and Indomethacin) Induced Gastric Ulcer in Rats: After successful establishment of the pyloric-ligation-induced ulcer and indomethacin-induced gastric ulcer model in albino Wistar rats, we evaluated various parameters like gross mucosal damage, gastric pH, gastric volume, ulcer index, % protection, free acidity, total acidity, histopathology, and biochemical estimation.
Pyloric Ligation-induced Ulcer Model: Adult male albino Wistar rats were fasted for 36 hours before undergoing pylorus ligation operation. Water was given ad libitum throughout the fasting, and 1 ml of normal saline was given to each rat, twice a day. Seven groups of animals are arranged, consisting of three rats in each group. Group I includes the normal control that received saline water for 7 days. Group II includes the positive control group (pylorus ligated), which was given saline water for 7 days leading up to the day of pylorus ligation. Group III contains the standard group, which was given Yashtimadhu® for 7 consecutive days. 2,4-DTBP and F1 were administered from Groups IV to VII, for seven days in a row, and underwent pylorus ligated surgery on the 8th day. A sham surgical operation was done on Group I.
The abdomen was opened under anesthesia (Ketamine 80 mg/kg, Xylazine 10 mg/kg) and the pyloric portion of the stomach was ligated, allowing the stomach to fill with acid, and the abdominal wall was sutured. Water was not given to the animals during the recovery period. After 6 hours of ligation, animals were sacrificed by cervical dislocation, the stomachs were isolated, the gastric content was collected in a tube, and different parameters were evaluated.

Indomethacin-Induced Ulcer Model
Seven groups of 21 rats, each consisting of three rats, were created.
Group I: Normal control, saline water given for 7 days by oral gavage, 1 ml/rat.
Group II: The ulcer group received saline water, 1ml/rat OD by oral gavage for 7 days, and on the 8th day, indomethacin was given.
Group III: The Standard (Yashtimadhu® treated) group, Yashtimadhu® (150mg/kg) was given OD orally for 7 days.
Group IV to Group VII: The test compounds (2,4-DTBP and F1) treated group received test compounds at a low dose (10 mg/kg) and high dose (20 mg/kg) OD orally for 7 days.
Group II to Group VII, after 16 hours of fasting with free access to water, a single dose of indomethacin at 40 mg/kg is given by oral gavage, and the Group I rats are directly sacrificed on the 8th day. Stomach tissues were collected and cut along the greater curvature of the stomach. The number of ulcers per stomach was noted.

Assessment of Gross Mucosal Damage
The stomach tissue of each rat was cleaned of blood and debris using 0.9% saline solution and dried with the help of absorbent filter papers. Once cleansed, the stomachs were fixed in a clear area and set up with pins. Subsequently, digital imaging was performed on the stomach tissues, allowing the evaluation of possible damage to the mucosal lining.
After successful surgical procedures in the pyloric-ligation model, the excised stomachs were cut along their larger curvature, and images were taken of all the groups. The stomach tissues were expanded with the help of pins, and distinct mucosal lesions were observed along the glandular portion, the area close to the pyloric end. The red arrow highlighted the spot ulcer characterized by mucosal erosion, while the blue arrow indicated the redness of the mucosal area, suggesting inflammation or erythema, as shown in Figure 7.
In the positive control group, there are both inflammation and spot ulcers, while in the standard group, only spot ulcers are observed. Treatment groups, 2,4-DTBP at low dose (10 mg/kg) had only one spot ulcer, and at high dose (20 mg/kg), only inflammation was shown. In the other treatment group, F1 at a low dose (10 mg/kg) and high dose (10 mg/kg) had a normal-like stomach with no spot ulcers and inflammation.
After the successful establishment of the indomethacin-induced ulcer model in rats, the stomachs were isolated, macroscopic images were captured, and the ulcerative lesions present on the gastric surface were evaluated. In Figure 8, the light green coloured arrow represented the haemorrhagic streaks, blue colour highlighted the spot ulcers, while the yellow colour showed inflammation or erythema. In the positive control group, many hemorrhagic streaks were seen, and spot ulcers also, while in the standard group, there was the presence of only spot ulcers. 2,4-DTBP at low dose had fewer spot ulcers, but inflammation was there, and at high dose, only inflammation was seen. F1 at low dose had only one spot ulcer, and at high dose, no inflammation, streaks, or spot ulcers were observed.

Estimation of Gastric pH and Volume
Gastric content pH was evaluated. Concisely, each rat’s stomach content was centrifuged for ten minutes at 5000 rpm. The resulting supernatant was collected, and the total volume of gastric juice present in the stomach was measured and stored in a glass vial for further analysis. A pH meter was used to measure the pH after 1 ml of supernatant and 1 ml of distilled water were added.
Gastric pH and gastric volume were determined immediately after the tissue isolation to determine the secretory parameters. In Figure 9a, the gastric pH of Groups II to VII revealed a significant difference, as the positive control group had the lowest pH, indicating an acidic environment compared to all other groups. 2,4-DTBP and F1 showed an increment in the pH of the gastric content; the standard had a moderate pH level (~6), while 2,4-DTBP at low dose and high dose had almost the same pH as the standard. The formulation F1 revealed a significantly higher pH (~7).
In contrast to all the treatment and standard groups, the positive control group had the highest volume of gastric acid. 2,4-DTBP and F1 had significantly less gastric volume. These findings suggest that the 2,4-DTBP and F1 at low dose and high dose are effective in the reduction of gastric volume, while the high dose exhibited better efficacy, as shown in Figure 9 b. Table 1 highlights the evaluated values of all parameters, such as ulcer index, % protection, gastric pH, and volume, free and total acidity.

Table 1: Effect of test compounds (2,4-DTBP and F1) on the gastric pH, gastric volume, ulcer index, % protection, free acidity, and total acidity
Groups Gastric pH Gastric volume (ml) Ulcer Index % Protection Free acidity (mEq/l) Total acidity (mEq/l)
Model-1 Model-2 Model-1 Model-2
Normal - - - - - - - -
Positive Control 2.6±0.36 8.23±0.25 5±0.5 7.5±0.5 - - 75.4±2.8 125.8±2.6
Standard 6.3±0.2 6.33±0.15 4.5±0.5 3±0.5 10 60 45.6±2.1 65.2±2.7
2,4-DTBP (LD) 5.4±0.15 4.4±0.17 1.16±0.28 2.3±0.28 76.6 68.88 36.01±3 47.1±1.51
2,4-DTBP (HD) 6.7±0.2 4.26±0.20 0.83±0.28 0.8±0.28 83.3 88.88 27.7±1.9 41.7±1.20
F1 (LD) 6.73±0.2 4.16±0.64 0.66±0.28 1.83±0.2 86.6 75.55 35.2±0.6 42.03±1.0
F1 (HD) 5.8±0.1 3.73±0.20 0.66±0.28 0.5±0.5 86.6 93.3 16.9±0.86 34.3±1.60
Results are expressed as mean±SD (n=3 rats/group); Model-1: Pyloric-ligation induced gastric ulcer model; Model-2: Indomethacin-induced gastric ulcer

Macroscopic Ulcer Analysis
The stomach was incised along its greater curvature and was cleaned with saline water to remove any blood clots and gastric content, then examined for ulcer formation with the help of a 10x magnifying lens. Consequently, the ulcer index was determined.
0 – Normal colored stomach
0.5 – Red coloration
1 – Spot ulcers
1.5 – Haemorrhagic streaks
2 – Ulcers > 3 but < 5
3 – Ulcers > 5

The ulcer index was calculated based on the average ulcer score for each group.
The formula for quantifying the Ulcer Protection:
% Ulcer Protection=(Ulcer Index in Control-Ulcer Index in Test)/(Ulcer Index in Control)×100

In the pyloric ligation-induced gastric ulcer model, the ulcer index was computed based on the extent and severity of stomach lesions, and the % ulcer protection was evaluated with the control group, as shown in Figure 10a. 2,4-DTBP and F1 at the low and high doses had significantly lower ulcer index, and the percentage of ulcer protection was enhanced as compared to the positive control group. The highest protection and greatest decrease in ulcer index were observed in the F1 at high dose. These findings revealed that the treatment was effective in minimizing gastric acid secretion and preserving mucosal integrity.
In the indomethacin-induced ulcer model, the control group had the highest ulcer index, while the treatment groups had a significantly lower ulcer index as compared to the control group. The % protection of gastric ulcer is highest in formulation F1 at high dose as compared to all other groups, as shown in Figure 11. It indicated that the novel formulation at high doses had better anti-ulcer activity. It indicated that the novel formulation at high doses had better anti-ulcer activity.

Determination of Free Acidity and Total Acidity
The volume of 0.01N NaOH solution used to neutralize the produced HCl was utilized to assess the free acidity. 1 ml of the separated supernatant was combined with a few drops of Topfer’s reagent to start the procedure. Following that, the resulting mixture was titrated using a 0.01 N NaOH solution. The titration process was continued until the mixture’s color changed from light red to canary yellow, signifying the process’s endpoint, and then free acidity was calculated using the following formula.
The data obtained from this process was used to determine the free acidity by formula:
Free Acidity=(Volume of NaOH used×N×100)/0.1

where, N= Normality of NaOH.

Total acidity is determined by the volume of 0.01N NaOH required to neutralize all of the acids present. A few drops of phenolphthalein indicator were added to the mixture mentioned above. Following the addition of phenolphthalein, we used 0.01 N NaOH to titrate the mixture. This titration procedure was carried out repeatedly until the mixture turned red. The amount of alkali used to produce this colour shift indicated the sample’s overall acidity.
Free acidity and total acidity were expressed as mEq/L.
The levels of free acidity and total acidity were calculated as essential indicators of gastric secretory responses, represented in Figure 12. The free and total acidity were highest in the positive control group, which exhibited enhanced acid secretion. The standard group highlighted the reduction in acidity, while 2,4-DTBP and F1 had the most significant effect, as the free and total acidity had the lowest values, indicating lesser acid secretion and proving to be an effective treatment.
Histopathology of Stomach Tissue: After the animals were sacrificed, stomach tissues were isolated and stored in 10% formalin solution. Isolated tissues were then dehydrated with different ethanol concentrations and fixed in paraffin wax. For the adhesion of tissue, the tissue segment was cut with a thickness of 5 µm and placed on a poly-L-lysine-coated slide. Hematoxylin and eosin (H&E) staining was done, and the slides were examined under a trinocular light microscope, and photomicrographs were captured and analyzed for the appearance of gastric tissue at 10x magnification.
Microscopic examination of the stomach tissue in the pyloric-ligation model, a normal control rat, indicated the normal histological structure with normal gastric mucosa and surface epithelium as shown in Figure 13. On the other hand, a section from a pyloric ligated rat showed disturbance in the integrity of cells in the mucosa, which looked disorganized and inflamed, while the standard group indicated disruption of the muscular layer. 2,4-DTBP and F1-treated groups with low and high doses suggested recovery of mucosa and sub-mucosal layer, with lesser inflammation than the positive control group.
Microscopic examination of the stomach tissue in the indomethacin-induced gastric ulcer in the rat’s stomach tissue section of different groups was analysed and revealed marked pathological alterations as shown in Figure 14.
The mucosa, sub-mucosa, and the muscular layer seemed to be normal in the normal control group, with no alterations in the pathology of stomach tissue. In contrast, the indomethacin-treated group had sub-mucosal edema, mucosal ulceration, dense infiltration of inflammatory cells, while the standard group, Yashtimadhu®-treated, revealed lesser alteration in the mucosa, mild epithelial loss, and recovery of mucosa and sub-mucosa was shown. 2,4-DTBP and F1-treated groups showed recovery of mucosal and muscular layers more effectively than the standard and positive controls. F1 at high dose highlighted the notable mucosal healing and anti-inflammatory activity.
Biochemical Estimation
The stomachs were removed, cleaned with ice-cold saline, and then minced in phosphate buffer using surgical blades. The minced tissue was then homogenized using a homogenizer in PBS of pH 7.4. The stomach 10% (w/ v) tissue homogenate was centrifuged at 5000g for 10 minutes to remove the debris using a homogenizer, and the supernatant was collected for the assessment of biochemical parameters.
All the biochemical parameters like catalase, SOD, GSH, NO, TBARs, and total protein were evaluated to determine the oxidative stress burden and gastroprotective efficacy, as shown in Table 2. In contrast to the positive control group, catalase and SOD activity are significantly increased in the 2,4-DTBP and the F1 at low and high doses, as shown in Figure 15, indicating reduced oxidative stress, preserved mucosal integrity, and decreased inflammatory damage.
Catalase Assay: The principle behind this test relies on the degradation of hydrogen peroxide in the presence of catalase. To 1.2 ml of 50 mM phosphate buffer (pH 7.0), 200 µl of the tissue homogenate was added, and then the reaction was initiated by adding 1.0 ml of 30 mM hydrogen peroxide (H2O2) solution. Absorbance was measured at 240 nm, and a blank enzyme was run in parallel with 1.0 ml of distilled water. The catalase activity is expressed as A moles of H2O2 decomposed/ min/ mg protein.
Superoxide Dismutase (SOD) Assay: To the tubes containing 0.75 ml ethanol and 0.15 ml ice-cold chloroform, 0.1 ml tissue homogenate was added and then centrifuged. 0.5 ml of supernatant was mixed with 0.5 ml ethylene diamine tetraacetic acid (EDTA) solution (0.6 mM) and 1 ml carbonate-bicarbonate (0.1 M) buffer solution. 05 ml epinephrine (1.8 mM) was added to activate the reaction, and absorbance was read at 480 nm. The enzyme activity is reported as 50 % inhibition of epinephrine auto-oxidation per minute.
Glutathione (GSH) Assay: 5 % trichloroacetic acid (TCA) was used to precipitate 0.1 ml of tissue homogenate. After thoroughly mixing both contents, the proteins are precipitated and then centrifuged. To a final amount of 4.0 ml, 2.0 ml of DTNB reagent (0.6 mM) and phosphate buffer (pH 8.6) were added to the aliquot of clear supernatant. Absorbance was recorded at 412 nm and in parallel with a blank that contained 5 % TCA. Glutathione levels are reported as mg/100g of wet tissue.
Nitric Oxide (NO) Assay: The Griess-Illosvay reaction method was used to measure NO’s scavenging activity. The reaction mixture that contains 1 ml of sodium nitroprusside (15 mM) in PBS (pH 7.3) was incubated for 60 min. After incubation, an equivalent volume of Griess reagent was added and kept in the dark for around half an hour. The absorbance was then recorded at 546 nm using a microplate reader.
Thiobarbituric Acid Reactive Substances (TBARs) Assay: In a test tube, 1 ml of suspension medium from 10 % tissue homogenate was mixed with 0.5 ml of 30 % aqueous TCA and then 0.5 ml of 8 % aqueous thiobarbituric acid (TBA) reagent. After that, the tube was wrapped in aluminium foil and shaken for 30 min at 80° C in a water bath. After being removed, the tube was centrifuged for 15 min at 3000 rpm and left in ice-cold water for 30 minutes. Absorbance was measured in comparison to a blank at 540 nm, and the standard was 1,1,3,3-tetramethoxypropane. Lipid peroxide levels were reported as mM of TBA reactants/100 g of moist tissue.
Estimation of Total Protein Content: The amount of protein was calculated. 0.9 ml of water and 1 ml of alkaline copper reagent were added to 0.1 ml of tissue homogenate, the mixture was allowed to sit at room temperature for ten minutes. The colour was produced after the addition of 0.5 ml of Folin’s reagent, and the amount of protein was measured at 750 nm after 30 min in mg/100 g of tissue.
Statistical Analysis: The results were expressed as mean±SEM, and an Ordinary one-way ANOVA analyze the data with multiple comparisons among the positive control group using GraphPad Prism 9.3.0. (463) software (GraphPad Software, Inc., San Diego, California, USA). The probability value, P > 0.05, is considered significant.
Table 2: Effect of test compounds on pyloric-ligation induced gastric ulcer in rat’s stomach tissue
Groups Catalase (µmol/U/mg) SOD (unit/mg protein) GSH (unit/min/mg protein) NO (mMol) TBARs (nM) Total protein (µg/ml)
Normal 6.28±0.01 0.26±0.03 1.73±0.02 2.17±0.001 0.54±0.003 5.25±0.03
Positive Control 1.125±0.01 0.065±0.01 0.46±0.003 1.85±0.0007 1.20±0.004 30.6±0.26
Standard 2.21±0.002 0.139±0.01 0.63±0.009 2.48±0.0007 0.66±0.003 22.62±0.004
2,4-DTBP (LD) 2.62±0.003 0.17±0.008 0.76±0.007 2.30±0.003 0.89±0.005 18.84±0.008
2,4-DTBP (HD) 3.78±0.004 0.218±0.009 0.88±0.002 2.70±0.003 0.71±0.003 13.18±0.005
F1 (LD) 2.93±0.007 0.15±0.01 0.94±0.004 2.66±0.002 0.81±0.002 17.18±0.003
F1 (HD) 4.75±0.10 0.23±0.018 1.20±0.009 2.75±0.002 0.64±0.014 10.92±0.005
All results are shown as mean±SD; n=3 rats/group
GSH and NO were significantly decreased in the positive control group, indicating increased oxidative stress, while in the treatment groups of test compounds, 2,4-DTBP and F1, F1 had significantly elevated levels of GSH and NO, representing the increased mucosal defence and lesser oxidative stress in the stomach tissue, as shown in Figure 16.
A notable decrease in the TBARs and total protein was determined in the treatment groups of test compounds, in contrast to the diseased group, as shown in Figure 17. A significant reduction of the TBARs level in the 2,4-DTBP and F1 groups indicated the suppression of lipid peroxidation and oxidative stress. Also, an elevation in the total protein was observed in the positive control group, indicating tissue injury, inflammation, or cell damage. The standard-treated group represented the moderate decrease in the total protein, while 2,4-DTBP and F1, at low and high doses, had significant reduction in the total protein levels, indicating restored mucosal integrity and reduced cellular leakage, as shown in Figure 17.

It will be appreciated by those skilled in the art that the embodiments of the present invention described herein are merely illustrative and not restrictive in nature. Various modifications, substitutions, or equivalents may be employed without departing from the scope and spirit of the invention. The examples and methods provided are intended to demonstrate the novelty and efficacy of the formulation, and should not be construed as limiting.
, Claims:We claim
1. A phytosomal formulation (F1) comprising 2,4-di-tert-butylphenol and a phospholipid for the effective management of peptic ulcer, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid ranges from 1:1 to 1:5.
2. The formulation as claimed in claim 1, wherein the phospholipid is soya lecithin.
3. The formulation as claimed in claim 1, wherein the molar ratio of 2,4-di-tert-butylphenol to phospholipid is 1:4.
4. The formulation as claimed in claim 1, wherein the uniform particle size is maintained in the range of 90–110 nm with a PDI in the range of 0.250-0.360.
5. The formulation as claimed in claim 1 may further comprise one or more pharmaceutically acceptable excipients selected from diluents, binders, disintegrants, lubricants, or coating agents.
6. The formulation as claimed in claim 1, wherein the composition is formulated as a powder, capsule, tablet, or suspension suitable for oral administration.
7. The formulation as claimed in claim 1, wherein the composition exhibits antioxidant, anti-inflammatory, and gastroprotective activity.
8. The formulation as claimed in claim 1, wherein administration of the composition results in modulation of oxidative stress biomarkers, characterized by increased catalase and superoxide dismutase (SOD) activity, enhanced glutathione (GSH) and nitric oxide (NO) levels, and decreased thio-barbituric acid reactive substances (TBARs), thereby preserving gastric mucosal integrity and reducing inflammatory damage.
9. The formulation as claimed in claim 1, wherein administration of the formulation provides gastroprotective activity, characterized by a percent protection against peptic ulcer in the range of 70–90% at low dose and 80–100% at high dose.
10. A method for preparing the phytosomal composition as claimed in claim 1, wherein the method comprising the steps of:
i. Dissolving 2,4-DTBP in 15 ml of methanol and soya lecithin in 15 ml of chloroform;
ii. Transferring both solutions into a round-bottom flask (RBF);
iii. Refluxing the mixture at 30-50°C for 2 hours with continuous stirring;
iv. Evaporating the solvent at 40 °C and 120 rpm using a rotary vacuum evaporator until a thin lipid film is formed on the flask wall;
v. Placing the flask containing the lipid film in a desiccator overnight to ensure complete solvent removal;
vi. Rehydrating the dried film using phosphate buffer saline (PBS, pH 7.4) to obtain a dispersion;
vii. Subjecting the dispersion to probe sonication at 60% amplitude with 5-second on–off intervals to produce phytosomes of the desired particle size; and
viii. Lyophilizing the final phytosomal dispersion to obtain the desired formulation.
11. A method of treating peptic ulcer disease in a subject in need thereof, comprising administering a pharmaceutically effective amount of the phytosomal composition as claimed in claim 1, thereby reducing gastric ulcer index and oxidative stress, and increasing gastric mucosal protection.