Description:FIELD OF THE INVENTION
The present disclosure relates to a cream for burn wound, specifically to a process for preparing silver nitrate cream for burn wound.

BACKGROUND OF THE INVENTION
For over a century, silver and its compounds have been widely used as antimicrobial agents due to the broad-spectrum antibacterial properties of the ionic (active) form of silver. Silver nitrate solutions were initially employed for treating burn wounds, but faced significant limitations. The silver cations readily react with chloride ions present in wound exudate, precipitating as insoluble silver chloride and diminishing the antimicrobial effectiveness. To maintain therapeutic silver ion concentrations, frequent re-applications of silver nitrate solution-soaked dressings were required.
While silver chloride offers slower silver ion release kinetics, ensuring longer antimicrobial activity, its low solubility limits the initial burst of silver ions needed for immediate antimicrobial action upon application. Silver nitrate creams containing sodium chloride were developed to provide both an initial burst of free silver ions from unreacted silver nitrate, followed by sustained release from the precipitated silver chloride. However, the high salt concentration can lead to wound dehydration and further injury.
Additionally, silver nitrate and its creams undergo undesirable browning or blackening reactions over time, making them aesthetically unpleasing and potentially staining wounds and dressings. There remains a need for improved silver-based antimicrobial formulations that can provide an optimal balance of immediate and sustained silver ion release without excessive saltiness or discoloration issues.

Incorporating additional active ingredients that can synergistically enhance antimicrobial activity, promote wound healing, and provide further benefits are also desirable. Novel formulations addressing the aforementioned limitations of current silver-based products would represent a significant advancement in burn wound care.
In the view of the foregoing discussion, it is clearly portrayed that there is a need for a cream for burn wound, specifically to a process for preparing silver nitrate cream for burn wound.

SUMMARY OF THE INVENTION
The present disclosure relates to a silver nitrated based cream for burn wound, specifically to a process for preparing silver nitrate cream for burn wound. The present invention discloses a silver nitrate cream composition for burn wounds and a process for preparing the same. The cream composition comprises cetostearyl alcohol, liquid paraffin, chlorocresol, Cetrimide, allantoin, chlorhexidine gluconate solution, and silver nitrate in specific weight percentages. The process involves heating separate oily and aqueous phases, combining them to form an oil-in-water emulsion, and then incorporating the remaining ingredients in a specific sequence and conditions to achieve a homogeneous silver nitrate cream formulation.
The present disclosure seeks to provide a silver nitrate cream composition for burn wounds. The composition comprises: 10-13 wt. % of cetostearyl alcohol; 6-10 wt. % of liquid paraffin; 0.09-0.11 wt. % of chlorocresol; 0.90-1.20 wt. % of Cetrimide; 0.09-0.11 wt. % of allantoin; 0.18-0.22 wt. % of chlorhexidine gluconate solution; and 0.18-0.22 wt. % of silver nitrate.

In an embodiment, the weight amount of the cetostearyl alcohol, liquid paraffin, chlorocresol, Cetrimide, allantoin, chlorhexidine gluconate solution, and silver nitrate, is, 11.0%, 9.0%, 0.1%, 1.0%, 0.1%, 0.2%, and 0.2%, respectively.
The present disclosure also seeks to provide a process for preparing silver nitrate cream for burn wound. The process comprises: heating oily phase comprising 10-13 wt. % of cetostearyl alcohol, 6-10 wt. % of liquid paraffin, and 0.09-0.11 wt. % of preservative chlorocresol at a temperature of 60°C ± 5°C; heating aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C simultaneously; adding the heated oily phase to the aqueous phase with continuous mixing to form an oil-in-water emulsion; allowing the emulsion to mix for a duration of 5 to 10 minutes; adding 0.09-0.11 wt. % of allantoin into the emulsion one by one after 5 to 10 minutes; incorporating 0.18-0.22 wt. % of chlorhexidine gluconate solution into the emulsion at 40°C thereby mixing continuously until the emulsion cools to room temperature; and adding 0.18-0.22 wt. % of silver nitrate into the emulsion slowly in a dark place with continuous mixing until complete homogenization of silver nitrate cream is achieved.

In an embodiment, allantoin is dissolved in water separately at 60°C.

In an embodiment, the Oily phase consist of 13% cetostearyl alcohol, 9% liquid paraffin, and 0.1% preservative chlorocresol is heated up to 60°C ± 5°C.

In an embodiment, the aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C is heated simultaneously.

In an embodiment, the Oily phase is added to the aqueous phase by continuously mixing with the help of a stirrer and after 5 to 10 mins, 0.1% allantoin and 0.5% sodium chloride is added into the emulsion one by one, wherein the Allantoin dissolve separately in little water at 60°C and sodium chloridedissolve separately in little water at room temperature.

In an embodiment, at 40°C, 0.2% chlorhexidine gluconate solution is incorporated into the emulsion and the mixing is continued until the emulsion cooled to room temperature.

In an embodiment, 0.2% silver nitrate is slowly added to the emulsion in the dark place, and complete homogenization is achieved with continuously mixing, wherein the silver nitrate dissolve separately in little water at dark.
An objective of the present disclosure is to provide a cream for burn wound, specifically to a process for preparing silver nitrate cream for burn wound.
Another object of the present disclosure is to a silver nitrate cream composition for burn wounds that can effectively deliver antimicrobial silver ions while addressing the limitations of previously used silver-based formulations.
Another objective of the present disclosure is to develop a silver nitrate cream composition that can provide an optimal balance of immediate and sustained release of silver ions for effective antimicrobial activity against a broad spectrum of microorganisms.
Another objective of the present disclosure is to formulate a silver nitrate cream that minimizes excessive saltiness and dehydration effects on the wound, which were associated with previous silver nitrate creams containing high concentrations of sodium chloride.
Another objective of the present disclosure is to incorporate additional active ingredients, such as allantoin and chlorhexidine gluconate, that can synergistically enhance antimicrobial activity, promote wound healing, and provide further benefits in the silver nitrate cream composition.
Another objective of the present disclosure is to develop a stable silver nitrate cream formulation that does not undergo undesirable browning or blackening reactions over time, thereby maintaining its aesthetic appeal and preventing staining of wounds and dressings.
Yet, another objective of the present disclosure is to provide a detailed process for preparing the silver nitrate cream composition, ensuring consistent and reproducible manufacturing of the formulation with the desired properties and performance.
To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates a flow chart of a process for preparing silver nitrate cream for burn wound in accordance with an embodiment of the present disclosure;
Figure 2A and 2B illustrates the macroscopic images of the wound contraction in accordance with an embodiment of the present disclosure;
Figure 3A and 3B illustrates Histological images of the rat skin showing the injury evidence at 0 days and the re-epitelization in accordance with an embodiment of the present disclosure; and
Figure 4 illustrates a graph representing the Quantification of Collagenase in skin homogenates after the treatment with different formulations in accordance with an embodiment of the present disclosure.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.
The present invention relates to a silver nitrate cream for burn wound healing. The composition of said cream comprises: 10-13 wt. % of cetostearyl alcohol; 6-10 wt. % of liquid paraffin; 0.09-0.11 wt. % of chlorocresol; 0.90-1.20 wt. % of Cetrimide; 0.09-0.11 wt. % of allantoin; 0.18-0.22 wt. % of chlorhexidine gluconate solution; and 0.18-0.22 wt. % of silver nitrate.
In an embodiment, the weight amount of the cetostearyl alcohol, liquid paraffin, chlorocresol, Cetrimide, allantoin, chlorhexidine gluconate solution, and silver nitrate, is, 11.0%, 9.0%, 0.1%, 1.0%, 0.1%, 0.2%, and 0.2%, respectively.
Figure 1 illustrates a flow chart of a process (100) for preparing silver nitrate cream for burn wound in accordance with an embodiment of the present disclosure.
Referring to Figure 1, the process (100) includes plurality of steps as described below,
At step (102), the process (100) includes heating oily phase comprising 10-13 wt. % of cetostearyl alcohol, 6-10 wt. % of liquid paraffin, and 0.09-0.11 wt. % of preservative chlorocresol at a temperature of 60°C ± 5°C.
At step (104), the process (100) includes heating aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C simultaneously.
At step (106), the process (100) includes adding the heated oily phase to the aqueous phase with continuous mixing to form an oil-in-water emulsion.
At step (108), the process (100) includes allowing the emulsion to mix for a duration of 5 to 10 minutes.
At step (110), the process (100) includes adding 0.09-0.11 wt. % of allantoin into the emulsion one by one after 5 to 10 minutes, wherein the Allantoin dissolve separately in little water at 60°C and sodium chloridedissolve separately in little water at room temperature.
At step (112), the process (100) includes incorporating 0.18-0.22 wt. % of chlorhexidine gluconate solution into the emulsion at 40°C thereby mixing continuously until the emulsion cools to room temperature.
At step (114), the process (100) includes adding0.18-0.22 wt. % of silver nitrate into the emulsion slowly in a dark place with continuous mixing until complete homogenization of silver nitrate cream is achieved.
In an embodiment, allantoin is dissolved in water separately at 60°C.
In an embodiment, the Oily phase consist of 13% cetostearyl alcohol, 9% liquid paraffin, and 0.1% preservative chlorocresol is heated up to 60°C ± 5°C.
In an embodiment, the aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C is heated simultaneously.
In an embodiment, the Oily phase is added to the aqueous phase by continuously mixing with the help of a stirrer and after 5 to 10 mins, 0.1% allantoin and 0.5% sodium chloride is added into the emulsion one by one, wherein the Allantoin dissolve separately in little water at 60°C and sodium chloridedissolve separately in little water at room temperature .
In an embodiment, at 40°C, 0.2% chlorhexidine gluconate solution is incorporated into the emulsion and the mixing is continued until the emulsion cooled to room temperature.
In an embodiment, 0.2% silver nitrate is slowly added to the emulsion in the dark place, and complete homogenization is achieved with continuously mixing, wherein the silver nitrate dissolve separately in little water at dark.
The process further comprises determining the silver nitrate content in a sample comprising steps of weighing 3.5g sample of the material in platinum crucible. Then, burning the sample at a controlled temperature of 40oC to remove organic matter. Then, heating the burnt sample in a muffle furnace at a higher temperature of 600°C to decompose silver nitrate into silver metal. Then, cooling the sample after 1 hour and dissolving the residue with 5ml nitric acid and 1ml water into the crucible to dissolve the ppt and heat with the help of burner and poured it into the previously 30 ml water containing beaker to convert silver metal back to silver nitrate. Then, washing the crucible again with the mixture of nitric acid and water (5:1) and poured into the beaker. Then, titrating the solution with a 0.01 solution of ammonium thiocyanate using a suitable indicator, preferably 5ml ferric ammonium sulphate solution to determine the endpoint. Thereafter, calculating the silver nitrate content based on the volume of ammonium thiocyanate solution used.

The present invention relates to preparation of a silver nitrate cream for burn wound healing application, wherein the cream composition comprises cetostearyl alcohol, liquid paraffin, chlorocresol, Cetrimide, allantoin, chlorhexidine gluconate solution, and silver nitrate in specific weight percentages. The process involves heating separate oily and aqueous phases, combining them to form an oil-in-water emulsion, and then incorporating the remaining ingredients in a specific sequence and conditions to achieve a homogeneous silver nitrate cream formulation. A detailed description of the preparation of the silver nitrate cream is described below, followed by the results obtained from the analysis carried out on the prepared cream.
The preparation of the silver nitrate cream involves several detailed steps to ensure a stable and effective formulation. The process begins with the acquisition of silver nitrate, which has an assay of 99.657% w/w from Agarwal Drug Pvt. Ltd., Haridwar. The first step is to prepare the oily phase. This phase consists of cetostearyl alcohol (13%), liquid paraffin (9%), and the preservative chlorocresol (0.1%). These components are combined and heated to 60°C ± 5°C, ensuring they are thoroughly melted and mixed.
Simultaneously, the aqueous phase is prepared by dissolving the surfactant sodium lauryl sulphate (1.56%) in water and heating this solution to the same temperature of 60°C ± 5°C. Once both phases reach the desired temperature, the oily phase is gradually added to the aqueous phase with continuous mixing using a mechanical stirrer. This mixing is maintained for 5 to 10 minutes to form a uniform oil-in-water emulsion.
After the initial emulsion is formed, allantoin (0.1%) and sodium chloride (0.5%) are incorporated. Allantoin is first dissolved separately in water at 60°C. This solution, along with the sodium chloride solution, is then added to the emulsion one after the other, with continuous mixing to ensure homogeneity.
Next, the emulsion is allowed to cool to 40°C. At this point, chlorhexidine gluconate solution is incorporated into the mixture. Stirring is continued until the emulsion cools to room temperature, ensuring that all components are evenly distributed throughout the emulsion.
Finally, a 0.2% silver nitrate is prepared in a dark place to protect it from light exposure, which can cause decomposition. This solution is then slowly added to the emulsion with continuous mixing to achieve complete homogenization. The mixing is continued until the emulsion reaches room temperature and a uniform consistency is obtained.
Throughout the entire process, maintaining sterile conditions is crucial to avoid contamination. All equipment and containers used should be clean and dry before use. The careful control of temperature and continuous mixing are key to producing a stable and homogeneous silver nitrate cream with consistent quality and therapeutic efficacy.
During the development of the silver nitrate cream formulation using the above mentioned process, two significant problems were encountered.
Firstly, when equimolar or double molar amounts of sodium chloride were added, the formulation (T1a and T1b) turned black. It remained stable with 0.552% (T1d; 8 mol eqv.) and 1.104% (T1e; 16 mol eqv.) sodium chloride, but these concentrations were too high, potentially worsening the wound due to excessive sodium chloride.
Secondly, the analysis of silver nitrate posed challenges. The standard titration method as per the Indian Pharmacopoeia (IP) was ineffective, detecting only 0.0024% w/w silver nitrate in the Formulation (T1d). Alternative analysis using ICP-MS revealed 0.004% w/w, and ICP-OES showed 0.006% w/w. The raw material's assay was confirmed to be 99.66% by titration and 99.48% by ICP-OES. Market formulation (T03) also showed 0.004% w/w by ICP-OES. Attempts to use AAS techniques were ineffective due to the solubility issues of silver chloride. Whereas, allantoin and chlorhexidine gluconate solution assayed were found to be within the limits of 0.100 and 1.96%w/w respectively.
To address this, a new method was developed to convert silver chloride to silver, then to silver nitrate, for straightforward analysis by titration. This involved controlled heating and the use of nitric acid. However, this method failed because silver chloride and sodium ions persisted, not converting to free silver at 600°C or 800°C. One drop of 0.01 M ammonium thiocyanate would initially change the solution's color to red, which rapidly disappeared, requiring over 50 ml of ammonium thiocyanate, indicating persistent sodium ions.
The study inferred that silver chloride and sodium ions did not ignite at these temperatures. The presence of nitric acid converted sodium ions to sodium nitrate, which reacted with ferrous thiocyanate (forming a red complex), leading to sodium thiocyanate and ferric nitrate as shown in Reaction-1:
Reaction-1
Fe(SCN)3 + NaNO3 = NaSCN +Fe(NO3)3 (Red Colour)
This indicated that the method was ineffective for achieving accurate silver nitrate analysis due to the interactions involving sodium ions and silver chloride.
In an embodiment, to address the issues caused by silver chloride and sodium salts such as sodium lauryl sulphate and sodium chloride, as described above, a new formulation was developed that excludes these sodium-containing compounds. In this revised formulation, cetrimide was used as a replacement for sodium lauryl sulphate. This change provided three significant benefits. Firstly, cetrimide acts as a surfactant within the formulation, fulfilling the same role as sodium lauryl sulphate. Secondly, cetrimide contains bromide ions, which react with silver nitrate to form silver bromide instead of silver chloride. This reaction prevents the blackening of the formulation that was observed with silver chloride. Thirdly, cetrimide has antiseptic properties that are effective for minor cuts and wounds, providing an additive therapeutic effect when combined with silver nitrate.
The new formulation (T04) consists of the following ingredients and quantities:
1. Silver Nitrate: 0.2%
2. Chlorhexidine Gluconate Solution IP: 0.2%
3. Allantoin: 0.1%
4. Cetrimide: 1.0%
5. Cetostearyl Alcohol: 11.0%
6. Liquid Paraffin: 9.0%
7. Chlorocresol: 0.1%
8. Purified Water: Quantity sufficient to make up to 100%
In an embodiment, the preparation of the T04 formulation of silver nitrate cream closely follows the aforementioned process for preparation, but with two modifications: the use of cetrimide (1.0%) as the surfactant instead of sodium lauryl sulphate, and the exclusion of sodium chloride from the formulation.
In an embodiment, an evaluation of the prepared cream is carried out, wherein the allantoin 0.0963% w/w, chlorhexidine gluconate solution 0.1992% w/w and silver nitrate 2.074% w/w were obtained . A detailed description of the evaluation is as given under.
A) Allantoin IP 0.1%w/w (0.09%w/w to 0.11%w/w)
Chromatographic condition:
Mobile phase: pipette 0.5 ml of orthophosphoric acid and dilute with 700 ml water and maintain the pH 3.0 with the help of dilute sodium hydroxide, sonicate and filter.
Detector 220 nm
Flow rate- 0.7 ml/minute
Column- shimadzu, 5 µm, C18, 4.6X250 MM
Injection size: 5µL
Column temperature: 30°C
Dilute: mobile phase
Standard solution: Weight allantoin WS (about 0.1 gm) W2 to 100 ml with mobile phase, dilute 1 ml of the solution to 100 ml with mobile phase.
Test Solution: weight of the sample 1 g W3 to 100 ml with mobile phase

AT2 W2 1 100 P
------------ X --------- X -------- X ---------- X --------- X 100
AS2 100 100 W3 100

AT2- Average area of principle peak in test solution
AS2- Average area of principle peak in standard solution
B) Chlorhexidine Gluconate solution IP 0.2%w/w (0.18%w/w to 0.22%w/w)
Chromatographic condition:
Mobile phase: Dissolve 0.8 g of Octane-1-sulfonic acid sodium salt (anhydrous) in a mixture of 100 ml of glacial acetic acid, 135 ml of water and 365 of methanol, sonicate and filter
Detector 254 nm
Flow rate- 1 ml/minute
Column- shimadzu, 5 µm, C18, 4.6X250 MM
Injection size: 10µL
Column temperature: 30°C
Dilute: mobile phase
Standard solution: Dilute Chlorhexidine Gluconate solution WS 5ml V1 to 100 ml with mobile phase, dilute 10 ml of the solution to 100 ml with water and again dilute 2ml of the solution to 100 ml mobile phase.
Test Solution: weight of the sample 5 g W1 to 100 ml with mobile phase

AT2 V1 10 2 100 P
---------- X --------- X -------- X ---------- X --------- X ---------- X 100
AS2 100 100 100 W1 20

Silver nitrate:
1 ). Weight approximately 3.5 g sample in platinum crucible. Sample was slowly heated at 40°C until the complete burning. After burning, the sample kept into the muffle Furnace. Furnace temperature should be 600°C. After 1 hours, sample was cooled and concentrated 5 ml nitric acid and 1 ml water was added into the crucible to dissolve the ppt and heat with the help of burner and poured it into the previously 30 ml water containing beaker. The crucible was washed again with the mixture of nitric acid and water (5:1) and poured into the beaker. Add 5ml ferric ammonium sulphate solution as an indicator and titrated with 0.01 ammonium thiocyanate solution.
Titre value X Equivalent factor X M X 100
-------------------------------------------------------
Weight of sample X 0.01

In an embodiment, a comparative investigation for wound healing activity is carried out Using Burn Wound Model, wherein the detailed description of the evaluation is given below,
Study Design and Animal Housing
The study utilized healthy Sprague Dawley rats of either sex, weighing between 100-130 grams. The animals were housed in a controlled environment, maintaining a constant temperature of 25°C ± 1°C, with a 10-hour light and 14-hour dark cycle throughout the experimental period. They were kept in large, spacious, and hygienic cages and had free access to water and a standard pellet diet until the end of the study.
Experimental Groups
The animals were divided into six groups, each comprising six rats:
- Group 1: Negative Control
- Group 2: Treatment with the Placebo
- Group 3: Treatment with Silver Nitrate Solution (0.2% w/v)
- Group 4: Treatment with the Marketed Formulation(T03).
- Group 5: Treatment with the Test Formulation (T04).
Burn Wound Induction
The hair on the back skin of the animals was shaved to expose the skin surface. Each animal was anesthetized by administering an intramuscular injection of xylazine and ketamine at doses of 5 mg/kg and 40 mg/kg, respectively. To create the burn wound, a cylindrical stainless-steel rod with a radius of 10 mm was preheated to 100°C and applied to the exposed skin for 10 seconds. The animals were closely monitored for any signs of infection throughout the study period.
Wound Observation
The wounds were observed on days 1, 3, 5, 7, 9, 11, 13, and 15 post-injury. Observations focused on wound contraction, histological changes, and collagenase enzyme activity.
Histological Examination
For histological analysis, tissue samples from the wound sites were collected and fixed in 10% formalin. Thin sections of skin were prepared and stained with hematoxylin and eosin. The mounted slides were examined under a microscope to assess the healing pattern of wounds over different days across the various groups of animals.
Collagenase Activity
Collagenase activity in the tissue samples was measured using a Type I Collagenase Measurement Kit, following the manufacturer's instructions. The process involved accurately weighing the skin samples from all groups and homogenizing them in the buffer provided in the kit using a mortar and pestle. The homogenates were then centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was transferred to 96-well plates, and each sample was incubated with 50 µL of the standard solution provided in the kit. An additional 50 µL of assay reagent was added to each sample, and the mixture was incubated for 2 hours at 4°C. Absorbance was recorded at 600 nm using an ELISA reader. Collagenase activity was quantified in units per microgram of protein in the tissue samples.
This detailed approach ensured a comprehensive evaluation of the wound healing activity of the various treatments, focusing on wound contraction, histological healing patterns, and collagenase activity, thereby providing valuable insights into the efficacy of the test formulation compared to controls and other treatments.
Figure 2A and 2B illustrates the macroscopic images of the wound contraction in accordance with an embodiment of the present disclosure.
In an embodiment, a newly developed process was utilized to analyze the novel formulation of silver nitrate cream. Using this method, the assay of silver nitrate was determined to be 0.2074% w/w.
Wound Contraction Study
Following the analysis of all active ingredients, a comparative study was conducted to evaluate the burn wound healing efficacy of the formulation. In this study, Formulation T-04, marketed silver nitrate cream, placebo, and silver nitrate solution were applied three times daily to the affected areas. Observations were made on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days post-injury.
The data collected clearly highlighted differences between the control and treatment groups. On the 3rd day, all groups exhibited the highest levels of inflammation with no signs of recovery. By the 9th day, signs of wound infection, including hemorrhagic and scabbed wounds, were observed in the control and placebo groups. In contrast, the groups treated with silver nitrate solution, marketed formulation (T-03), and Formulation T-04 showed no such signs of infection.
A significant wound contraction was observed in the group treated with Formulation T-04 starting from the 5th day. This group exhibited the fastest rate of wound contraction, with complete healing and visible scabs by the 13th day. The group treated with the marketed formulation also showed complete healing by the 15th day. The rate of wound closure was evaluated by calculating the open wound area over time, as detailed in Table-1. Macroscopic images documenting the wound contraction process are presented in Figures 2A and 2B.
The comparative data indicate that Formulation T-04 significantly enhances wound healing compared to the control, placebo, and other treatment groups. The rapid healing observed in the T-04 treated group underscores the formulation's potential effectiveness in burn wound management.
Table 1: Wound contraction Rate (%)
No of Days Percentage wound contraction (%)
Control Placebo Silver Nitrate solution(0.2% w/v) Marketed Formulation(T-03) Test Formulation (T-04)
1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
3 5.38 ± 0.87 6.93 ± 1.03 8.96 ± 1.88 7.28 ± 0.93 9.96 ± 1.79
5 7.78 ± 0.79 7.27 ± 1.26 15.78 ± 2.04 20.78 ± 1.68 29.98 ± 2.64
7 10.78 ± 0.85 12.78 ± 1.86 20.78 ± 1.89 25.37 ± 1.85 36.84 ± 4.38
9 15.78 ± 1.84 16.49 ± 0.95 23.78 ± 2.74 35.98 ± 2.64 49.67 ± 5.86
11 19.87 ± 2.02 21.76 ± 1.69 38.27 ± 2.89 68.26 ± 5.28 79.26 ± 6.09
13 27.83 ± 2.38 26.89 ± 2.74 42.67 ± 2.59 84.27 ± 4.21 93.99 ± 7.58
15 44.28 ± 3.06 47.97 ± 2.96 61.67 ± 4.13 92.67 ± 8.94 95.73 ± 10.74

Figure 3A and 3B illustrates Histological images of the rat skin showing the injury evidence at 0 days and the re-epitelization in accordance with an embodiment of the present disclosure.
The healing pattern and the rate of epithelialization were assessed through histological analysis, as depicted in figures 3A and 3B. On the first day, histological images from all groups displayed clear signs of tissue injury, characterized by a disrupted and damaged epidermis. There was evident ulceration along with significant infiltration of inflammatory cells, including neutrophils, lymphocytes, and macrophages.
By day 15, there was a noticeable reduction in the presence of inflammatory cells across all groups, indicating that the inflammation associated with the injury was subsiding and recovery was underway. Furthermore, there were clear signs of re-epithelialization after 15 days. However, the degree of epithelialization varied depending on the treatment administered.
In the control and placebo groups, re-epithelialization was minimal and not statistically significant. In contrast, the group treated with T-04 exhibited the greatest thickness of the new epithelial layer, indicating a higher rate of re-epithelialization. This was followed by the group treated with the marketed formulation and then the group treated with the silver nitrate solution. These findings suggest that T-04 significantly enhances the healing process, promoting faster and more effective epithelialization compared to the other treatments.
Figure 4 illustrates a graph representing the Quantification of Collagenase in skin homogenates after the treatment with different formulations in accordance with an embodiment of the present disclosure.
Referring to graphs shown in Figure 4, the analysis of collagenase activity revealed noteworthy findings within the experimental groups. Specifically, the collagenase activity was significantly higher (P < 0.05) in both the group treated with T-04 and the marketed formulation when compared to the control group (Figure 3). This suggests that both T-04 and the marketed formulation (T-03) have a notable impact on collagenase activity, likely influencing the wound healing process.
Interestingly, when comparing the group treated withT-04 and the marketed formulation (T-03) directly, no significant differences were observed in collagenase activity. This indicates that both treatments exhibit similar effects on collagenase activity, implying comparable mechanisms or levels of efficacy in modulating this enzyme's activity.
These findings highlight the potential effectiveness ofT-04 in influencing collagenase activity, aligning with its proposed role in promoting wound healing. Moreover, the similarity in collagenase activity between T-04 and the marketed formulation suggests thatT-04 could be a promising alternative or comparable option for wound management, warranting further investigation and clinical evaluation.
The present invention of silver nitrate cream for burn wounds stands out for its innovative formulation featuring cetrimide as a surfactant and bromide ion supplier, effectively preventing blackening typically associated with silver nitrate formulations due to silver chloride formation. This cream combines silver nitrate, chlorhexidine gluconate, and allantoin, providing potent antimicrobial, antiseptic, and healing properties. Through a comparative study on wound healing activity using a burn wound model, the cream demonstrated efficacy in promoting wound contraction, histological healing, and modulating collagenase activity. Beyond burn wounds, its versatility extends to potential applications in dermatological conditions and surgical wound care, marking it as a novel and beneficial solution for medical settings requiring antimicrobial and wound healing interventions.
The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
Benefit s, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.
, Claims:1. A Silver nitrate cream composition, water, the composition comprises:
10-13 wt. % of cetostearyl alcohol;
6-10 wt. % of liquid paraffin;
0.09-0.11 wt% of chlorocresol;
0.90-1.20 wt. % of Cetrimide;
0.09-0.11 wt. % of allantoin;
0.18-0.22 wt.% of chlorhexidine gluconate solution; and
0.18-0.22 wt.% of silver nitrate solution.

2. The composition as claimed in claim 1, wherein the weight amount of the cetostearyl alcohol, liquid paraffin, chlorocresol, Cetrimide, allantoin, chlorhexidine gluconate solution, and silver nitrate solution, is, 11.0%, 9.0%, 0.1%, 1.0%, 0.1%, 0.2%, and 0.2%, respectively.

3. A process for preparing silver nitrate cream for burn wounds, the process comprises:
heating oily phase comprising 10-13 wt. % of cetostearyl alcohol, 6-10 wt. % of liquid paraffin, and 0.10-0.1 wt. % of preservative chlorocresol at a temperature of 60°C ± 5°C;
heating aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C simultaneously;
adding the heated oily phase to the aqueous phase with continuous mixing to form an oil-in-water emulsion;
allowing the emulsion to mix for a duration of 5 to 10 minutes;
adding 0.09-0.11 wt. % of allantoin into the emulsion one by one after 5 to 10 minutes;
incorporating 0.1-0.2 wt. % of chlorhexidine gluconate solution into the emulsion at 40°C thereby mixing continuously until the emulsion cools to room temperature; and
adding 0.1-0.2 wt. % of silver nitratedissolve separately in little water at dark into the emulsion slowly in a dark place with continuous mixing until complete homogenization of silver nitrate cream is achieved.

4. The process as claimed in claim 3, wherein allantoin is dissolved in water separately at 60°Cand at room temp respectively and sodium chloridedissolve separately in little water at room temperature.

5. The process as claimed in claim 3, wherein the Oily phase consist of 13% cetostearyl alcohol, 9% liquid paraffin, and 0.1% preservative chlorocresol is heated up to 60°C ± 5°C.

6. The process as claimed in claim 3, wherein the aqueous phase comprising 0.90-1.20 wt. % of a surfactant Cetrimide containing water at a temperature of 60°C ± 5°C is heated simultaneously.

7. The process as claimed in claim 3, wherein the Oily phase is added to the aqueous phase by continuously mixing with the help of a stirrer and after 5 to 10 mins, 0.1% allantoin and 0.5% sodium chloride is added into the emulsion one by one, wherein the Allantoin dissolve separately in little water at 60°C and sodium chloride dissolve separately in little water at room temperature .

8. The process as claimed in claim 3, wherein at 40°C, 0.2% chlorhexidine gluconate solution is incorporated into the emulsion and the mixing is continued until the emulsion cooled to room temperature.

9. The process as claimed in claim 3, wherein 0.2% silver nitrate is slowly added to the emulsion in the dark place, and complete homogenization is achieved with continuously mixing, wherein the silver nitrate dissolve separately in little water at dark.

10. The process as claimed in claim 3, further comprises determining the silver nitrate content in a sample comprising steps of:
weighing 3.5g sample of the material in platinum crucible;
burning the sample at a controlled temperature of 40oC to remove organic matter;
heating the burnt sample in a muffle furnace at a higher temperature of 600°C to decompose silver nitrate into silver metal;
cooling the sample after 1 hour and dissolving the residue with 5ml nitric acid and 1ml water into the crucible to dissolve the ppt and heat with the help of burner and poured it into the previously 30 ml water containing beaker to convert silver metal back to silver nitrate;
washing the crucible again with the mixture of nitric acid and water (5:1) and poured into the beaker;
titrating the solution with a 0.01 solution of ammonium thiocyanate using a suitable indicator, preferably 5ml ferric ammonium sulphate solution to determine the endpoint; and
calculating the silver nitrate content based on the volume of ammonium thiocyanate solution used.