INTRODUCTION:

Wound healing is a complicated and dynamic system that involves the restoration of skin and tissue following an injury. Wound healing is expressed in several phases of physical assets that comprise the post-trauma recovery cycle. Wound healing is a three way process in which an inflammatory reaction leads to proliferative phase which results in tissue repair and finally tissue remodeling¹.

All of these three steps must be functional in order for optimal wound healing to occur in an orderly fashion. Wound healing can be influenced by a variety of variables, including oxygenation, age, nutrition, hormones, medicines, diabetes, dietary habits, smoking, and alcoholism.

As a result, it is equally important that such circumstances be thoroughly recognised in order to guide the wound healing therapy².

Nanotechnology has introduced novel technologies and procedures that will completely transform the medical and pharmaceutical industries. It has been demonstrated that structures made using nanoparticles are substantially stronger. Several fields of medical treatment are already benefiting from nanotechnology's advantages. Silver nanoparticles have lately sparked clinical attention due to their possible biological features, such as antibacterial activity, anti-inflammatory effects, and wound healing effectiveness, which might be used to build better wound and ulcer dressings³. Chemical procedures include electrochemical and photochemical reduction processes that, like physical techniques, include evaporation-condensation and laser ablation. Surfactants, polymers, biomolecules, and other forms of stabilizers are employed to stabilize and regulate the size of nanoparticles⁴.

Silver nanoparticles are the most extensively utilised nanoparticles and may be called one of the most significant nanoparticles. The nanoparticles range in size from 1Nm to 100Nm. Ag-NPs are widely used in a variety of industries because of its unique chemical and Physical properties, as well as their Antibacterial potential, which distinguishes them from bulk materials. Ag-NPs are now widely used in pharmaceuticals, women's grooming products, food processing, lighting, cosmetics, medical equipment, sunscreen, biosensors, textiles, and appliances. Ag-NPs have qualities that are important in their usage as biocides as well as their use to clean the environment³. It has been demonstrated that when Silver Nanoparticles come into contact with Bacteria, they generate Free Radicals which has the power to damage the cell membrane and make it more permeable which ultimately leads into cell death⁵.

Extensive study over the last five years has revealed curcumin is a potent physiologically therapeutic agent. Turmeric molecule with a wide variety of favourable pharmacological qualities such as anti-inflammatory, anti-oxidant, anti-microbial, and anti-cancer activity. Curcumin has been effectively created as an amorphous Nano solid dispersion, and it has been found to be favourably released from gelatin-based biomimetic NMs that could be given topically to experimental wounds⁶. Blood plasma and bile secretion measurements indicated that curcumin was poorly absorbed in the intestine and that the amount of curcumin that penetrated across the intestine into tissues was pharmacologically modest.

Curcumin is encapsulated using liposomes. Chitosan with bovine serum albumin, cyclodextrin and phospholipids can also be used for making complex with other materials. These all agents when formulated as matrix improve the poor solubility of the drug and also the bioavailability of the drug.⁷

Honey, the world's oldest food source, is a wonderful food with high energy and nutritional content. Apismellifera produces it from plant nectar, secretions, and excretions⁸. Honey functions as a probiotic agent and produces proteins in amounts ranging from 0.1 to 0.5%, depending on the shape and provenance of honey. Honey also contains a lot of B2 vitamins like riboflavin, B6, pantothenic acid, and nicotinic acid, as well as C and B1 vitamins (thiamine). Each of these minor components is known to have distinct nutritional or therapeutic benefits, and their combination accounts for the vast spectrum of natural honey applications⁹.

MATERIALS AND METHODS:

Preparation of Silver nanoparticles:

Preparation of 0.001M Silver nitrate: Dissolve 17mg of Silver nitrate _into0.1L distilled water

Preparation of 0.002M Sodium Borohydride: Dissolve 18.9mg of Sodium Borohydride into 0.250L distilled water.

Procedure of Preparation of Silver nanoparticles:

Add 100ml of 0.002M solution of NaBH₄ to flask. Magnetic bead was added the flask was kept in an ice bath on a stir plate. After 20min, drop 6.6mL of AgNO₃ solution approximately 1 drop/sec as soon as AgNO₃is added.

Preparation of Silver nanoparticles hydrogel:

The formulation is prepared as shown in Table 1. Silver Nano composites, various amounts of Carbopol 934 at different concentrations (1, 2, 3%) were weighed accurately and put in water for 3 days later these hydrogels were transferred to beakers. Accurately weighted Silver Nano composites were applied to the stirring hydrogel, along with the inclusion of Curcumin and Honey at doses as shown in the table1. The hydrogel was eventually neutralized using Triethanolamine. Sodium benzoate has been added to the composition as a preservative⁴.

Silver nanoparticles Characterization:

UV visible spectroscopy has characterized silver nanoparticles supervised by sampling the aqueous component. The visible UV spectra of the sample was analysed by Speactroquant Prove 600 spectrometer, which showed a spectra range of 300-600 Nm. Distilled water has been used to change the benchmark¹⁰.

Scanning Electron Microscope (SEM):

In an ion coater, the powdered Ag-NPs was evenly dispersed and then coated with platinum for 120 sec before being inspected by SEM (JEOL-JSM 6360 MODEL, JAPAN). The Energy-Dispersive X-ray (EDX) is the detector linked to the SEM instrument was used to conduct elemental analysis of the powdered Ag-NPs¹¹.

Analysis of silver nanoparticles by X-RAY diffraction:

The structure of the generated silver nanoparticles was evaluated using XRD analysis. The XRD patterns of the sample generated utilising the present reduction procedure are shown in Figure 2. The XRD examination demonstrates that nanometre-sized silver particles are produced. Based on this research, the average particle size is approximated using the Debye–Scherrer formula

0.9l

D= -------------

W cos ϴ

Where l is the X-ray wavelength (0.1541 Nm), W is the Full Width at Half Maximum (FWHM), ϴ is the diffraction angle, and D is particle diameter (size). At 45.1 and 64.7 degrees, the instrumental broadening is 0.00632 and 0.00912 respectively. After accounting for these factors, the average particle size is determined to be 15 2NM, with a measurement error of 13%¹².

EVALUATION:

Organoleptic properties:

Organoleptic relates to the measurement of the sensory organs which involves the macroscopic presence of the product, the scent and its taste. The null formulation (formulation without API) is opposed to the API formulation. Changes in properties imply decomposition. Homogeneity and stiffness wasmeasured by rubbing the gel between the thumb and index finger. Immediate feeling after application was used to check stiffness, greasiness, greasiness, etc.

Spreadability:

The therapeutic efficacy of the gel depends on the spread of the gel. The spread of the gel helps to apply the gel evenly to the skin, so formulated gels have good spread ability and satisfy the ideal quality of the topical application.

This was measured by placing 1g of the sample in between two horizontal slides of glass (10* 20cm) by holding a certain weight at the top and by measuring the diameter of the sample distributed in the slide of glass after one minute.

Extrudability:

It is the power required to push or force something out of something. Compression-extrusion testing involves applying force to a product until it flows through a test cell outlet, which might be one or more slots or holes. It is important to know that the force required to extrude from the tube. Evaluation of the gel formulation extrudability was based was based on the percentage of gel extruded from the lacquered aluminum collapsible tube on applying weight in grams to extrude at least 0.5cm ribbon of gel in 10 sec¹³.

More the amount of gel extruded, the better is the extrudability. The measurement was done in triplicate and average is calculated by using the formula,

Applied weight to extrude gel from the tube (gm)

E = -------------------------------------------------------------

Area

Viscosity:

It is defined as measurement of afluid's resistance to flow. This word describes the internal friction of a flowing fluid. A fluid with a high viscosity resists motion due to its molecular structure, which has a lot of internal friction. This was measured by using Viscometer. The instrument used here is Brookfield Viscometer LV DV-2+PRO with spindle number. 62. Level the viscometer by changing the legs and spinning the viscometer until the spindle is positioned in the middle. The sample was taken in the beaker. The viscometer speed was set at 5rpm and 10rpm. For each measurement of viscosity, 3 trials were performed¹⁴.

pH:

The pH was measured with a pH meter. A small amount of sample is taken and the bulb is dipped in the sample so that it does not touch the carton and wait until the reading is shown. For each sample, 3 readings were taken. For each period, the pH meter should be wiped using tissue paper.

Anti-microbial activity:

Using the disc diffusion technique, the antibacterial activity of the produced AgNPs hydrogel was tested. Mueller-Hinton agar was used to subculture pure cultures of the Staphylococcus aureus bacterium. Using sterile cotton swabs, the colony was swabbed evenly onto separate agar plates. With the help of sterile cork borer holes were made on agar plates with uniform diameter prepared hydrogel was placed inside the holes and a standard of Silver sulfadiazine marketed brand gel was also placed in the standard hole. All the plates were incubated at 35°C for 18–24 hr¹⁵.

Burn wound healing activity:

In this experiment, seven to nine-week-old male Wistar rats were recruited. The animal experiments were carried out in accordance with IAEC-approved guidelines. .Animal ethical certificate number NGSMIPS/IAEC/NOV-2019/165. The rats were anaesthetized with anaesthetic ether before to procedure. After anaesthesia, the rats' back hair was shaved, and circular burns 10mm in diameter were made on the dorsum of each rat using an aluminium bar heated in 100°C water for 30 seconds. The bar was put on the back in each spot for 10 seconds without any pressure to generate a burn wound. After 48 hous, the necrotic region was punctured and the therapies were applied to the wounds. The rats were housed in separate cages until 14 days.The wounds were categorised into three categories as follows:

Group I: There was no medicine given to the wounds.

Group II: A 1% silver sulfadiazine solution was applied topically to the wounds.

Group III: The wounds were treated topically with a Silver nanoparticles hydrogel mixture.

The wound closure % was calculated using the following formula to determine the pace of healing:

Wound closure (%) = [(wound area on day 0 – wound area on indicated day)/ wound area on day 0] ×100¹⁶.

RESULTS AND DISCUSSIONS:

The UV visible spectroscopy:

The following absorption spectrum at wavelength range 300-600 Nm was showed by the synthesized Silver Nanoparticles. The surface Plasmon resonance peak around the surface confirmed the formation of Silver Nanoparticles as shown in the Figure 1. Maximum peak absorption was recorded at 385.2nm which was ideal as per literature referred.

Figure 1: Spectrum of Silver nanoparticle.

SEM:

Figure 2: Image of Silver nanoparticle.

In SEM (Figure 2) the presence of manufactured nanoparticles is plainly visible. The nanoparticles were oval in form and spherical in shape. The majority of the nanoparticles were aggregated, although a few solitary particles were also seen. From the images the size of the Silver nanoparticles were in the range of 100Nm.

X-RAY diffraction analysis:

The synthesized nanoparticles have been structurally characterized by X-ray diffraction. There were no peaks of other “impurity” crystalline phases in the XRD pattern, which fit the standard values of the face-cantered-cubic form of metallic silver well.

Figure 3: X-RAY diffraction images of nanoparticles.

Evaluation and characterization of hydrogel

The hydrogel formulation were tested for the color, homogeneity, texture were done for physical evaluation. The results are given below Table 2. All formulations were found to be of good consistency, smooth appearance, and non-sticky and yellowish white in color.

Table 3: Evaluation parameters of formulated gel.

Samples	Spread ability	pH	Homogeneity
F1	51mm	9.6	Good
F2	45mm	6.7	Good
F3	58mm	7.3	Good
F4	50mm	6.3	Good
F5	55mm	8.0	Good
F6	51mm	6.3	Good

From the above Table 3 the results of spreadability indicates that all the hydrogels was spreaded easily by applying a little of pressure. All the sample formulations were homogeneous in nature. pH measurement is quite important parameter because the pH value of formulation should not deviate much from the skin pH. pH was measured using pH meter. All the formulations were tested for the spreadability and it was found to be in the range of 40-60mm. The highest and the good spreadability of gel formulation is 45mm which was for the F2 and the pH was 6.7 which is good and homogeneity was also found to be excellent.

Table 4: Evaluation of viscosity.

RPM	5	10
F1	1138cps	531.3 cps
F2	1171.66 cps	595 cps
F3	1144.33 cps	596 cps
F4	1125.16 cps	532 cps
F5	1164 cps	584 cps
F6	1153.91 cps	566.3 cps

Viscosity is one of the most important requirements for an in-situ gelling systems. The formulation must have the right viscosity to readily inject at the afflicted spot. It was found from the above Table 4 F2 formulation showed good results at both 5 and 10 rpm.

The Anti-microbial evaluation:

Zone of inhibition was measured in mm. The best formulation F2 from the above evaluation parameters was taken for the test and marketed the brand of Silver sulfadiazine gel was taken as standard. From the Table 5 and figure 4, F2 formulation showed a good zone of inhibition of 33.5mm as compared to the standard which showed 35.9mm zone of inhibition. The prepared gel can be definitely used for burn wound healing.

Figure 4: The Zoneof inhibition of silver nanoparticle gel and Silver sulfadiazine gel.

Table 5: Evaluation of Antimicrobial activity.

Formulation	Zone of inhibition in mm
F2	33.4
Silver sulfadiazine gel	35.9

Burn wound healing:

A rat model was used to explore the efficacy of silver nanoparticles hydrogel in burn wound healing. Table 6 reveals that the silver nanoparticles hydrogel was more successful in burn wound healing than the commercially available brand of Silver sulfadiazine gel (standard). Three groups were made each containing wistar rats. The wounds were examined for redness, inflammation, and hyperemia after seven and fourteen days. When comparing the silver nanoparticle hydrogel treated groups to the standard, On the 7th and 14th post-operative days, no signs of inflammation were found; however, On the 7th and 14th post-operative days, rashes and inflammation at the wound site in the negative control group was observed. Furthermore, wound shrinking in the AgNPshydrogel group was much faster than in the Standard gel group. After 14 days, the conventional groups had minor redness, but the formulation-treated group had no redness, hyperaemia, or inflammation.

Table 6: Burn wound healing study

Group number	Redness	Inflammation	Hyperemia
1 (control)	Mild redness	Severe	Severe
2 (standard )	None	Mild	Mild
3 (test )	None	None	None

DISCUSSION:

The surface Plasmon resonance peak surrounding the surface verified the development of PNSs in UV spectroscopy. According to literature, the maximum peak absorption was observed at 385.2nm, which was optimal. The nanoparticles were oval and spherical in shape, according to SEM data. The bulk of the nanoparticles were aggregated, although there were a few lone particles as well. There were no peaks in the XRD pattern of additional “impurity” crystalline phases that meet the conventional values of the face-cantered-cubic form of metallic silver, indicating that the produced nanoparticles were structurally similar to the relevant literature results.The characterisation and assessment of prepared hydrogels revealed that all formulations performed well. Furthermore, in terms of pH and viscosity, the F2 formulation performed better than the other formulations, i.e., at both 5 and 10 rpm. When compared to normal silver sulfadiazine, the zone of inhibition of the F2 formulation demonstrated exceptional results. After 14 days of study on Wistar rats, the conventional groups showed minimal redness, while the formulation-treated group had no redness, hyperemia, or inflammation.

CONCLUSION:

From the present study, it can be concluded that the preparation of SNPs embedded with Curcumin and Honey is found to be a new and successful approach.

The viscosity and spreadability of the formulation F2 was found to be very good as compared to all other formulation. The maximum zone of inhibition is obtained as compared to standard. Curcumin and Honey embedded gels showed emollient and enhanced the antimicrobial activity. The silver nanoparticles hydrogel found to be more effective in burn wound healing than the commercially available Silver sulfadiazine gel. Furthermore, wound shrinking in the AgNPs hydrogel group was much faster than in the Standard gel group. All the evaluation study showed good activity but formulation F2 was found to be the optimum and a very good antimicrobial agent.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors are thankful to the authorities of the NGSM the Institute of Pharmaceutical Sciences, Nitte (Deemed to be University), Mangaluru for providing all the necessary facilities.

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Received on 11.03.2023 Modified on 07.06.2023

Research J. Pharm. and Tech 2024; 17(6):2813-2818.

DOI: 10.52711/0974-360X.2024.00441

Ingredients	F1	F2	F3	F4	F5	F6
Silver	1mg (oxide)	2ml (solution)	1mg (oxide)	2ml (solution)	1mg (oxide)	2ml (solution)
Carbopol	50g(1%)	50g (1%)	50g (2%)	50g (2%)	50g (3%)	50g (3%)
Curcumin	10mg	10mg	10mg	10mg	10mg	10mg
Honey	2ml	2ml	2ml	2ml	2ml	2ml
TEA	1 to 2 drops	1 to 2 drops	1 to 2 drops	1to2 drops	1 to 2 drops	1 to 2 drops

Properties formulation	Color	Odor	Consistency	Appearance	Grittiness	Stickiness
F1	Yellow	Characteristic	Good	“Smooth”	No	None
F2	Yellow	Characteristic	Good	“Smooth”	No	None
F3	Yellowish white	Characteristic	Good	“Smooth”	No	None
F4	Yellowish white	Characteristic	Good	“Smooth”	No	None
F5	Yellowish white	Characteristic	Good	“Smooth”	No	None
F6	Yellowish white	Characteristic	Good	“Smooth”	No	None