INTRODUCTION:

A worldwide public health concern, diabetes mellitus (DM) will affect 536 million people in 2021 and an estimated 783 million by 2045. Chronic vascular difficulties as well as financial, social, and medical pressures are brought on by it. One frequent consequence that may lead to poor quality of life, restricted mobility, and mental discomfort is diabetic foot¹. To enhance the psychological well-being and quality of life of patients with diabetic foot ulcers (DFUs), which are very dangerous and may result in 2.5 times as many mortality events as diabetic patients without foot problems, disease perception has been presented. The yearly prevalence of diabetic foot ulcers is between five and six percent and treatment options include amputation or prolonged antibiotic therapy. Greater ulcers or exposed bones increase the danger. The pace at which DFU heals varies and is influenced by several variables, including unloading, infection control, vascular health, and cardiometabolic condition^2,3. After three months, only 35–40% of healed ulcers remain, and after a year, only 47–50% do. The rates of bone infection in DFU are elevated by the extended period of compromised skin barrier. Regarding whether the length of DFU is a separate risk factor for the emergence of DF, the findings of earlier research are contradictory.

More than twenty plants, including broccoli, berries, cherries, citrus fruits, grapes, and onion, contain the powerful antioxidant bioflavonoid quercetin. Quercetin is well-known for its protective properties and targets a variety of signaling pathways. The anti-inflammatory, antidiabetic, free-radical scavenging, antihyperlipidemic, antiviral, anti-inflammatory, immunomodulatory, hypoglycemic, and lipid-lowering properties of quercetin's extracts and phytoconstituents have been shown. Plants and plant components are utilized for their taste, aroma, and medicinal qualities. Numerous cells and tissues, including those in diabetes, have been discovered to be protected by quercetin from the damaging effects of oxidative stress. It has been discovered to protect the kidneys, heart, and islets of the pancreas^4-6. Its impact on diabetic seminal vesicles, however, is seldom documented. Furthermore, its impact on diabetic seminal vesicles, however, is seldom documented. Given that oxidative stress is connected to the abnormalities of seminal vesicles in diabetes, quercetin could potentially provide some protection. Because quercetin is poorly soluble in water and has trouble being absorbed, metabolized, and eliminated, its use as a polyphenolic molecule is restricted. Because it cannot pass across the blood-brain barrier, it cannot be utilized to treat disorders of the central nervous system and may even be harmful if taken often or in high dosages⁷.

Recent developments in drug delivery, nanomedicine, and nanotechnology have been investigated to boost quercetin bioavailability. Zero-dimensional nanomaterials known as drug nanoparticles have drawn interest as a means of producing poorly soluble pharmaceuticals. By making quercetin more soluble and bioavailable, quercetin nanoparticles may be used as a strong antioxidant ability in the cytoplasm to combat reactive oxygen species^8-10. In 2023, Sobia Abid et al investigated the use of Phenyl Boronic Acid Functionalized-Quercetin nanoparticles (PBA-Qt NPs) in diabetic wound healing. In diabetic rats, PBA-Qt NPs demonstrated improved wound healing, better DPPH scavenging, and antibacterial activities against both Gram-positive and Gram-negative bacteria. Additionally, they improved wound closure by promoting angiogenesis, tissue repair, and regeneration. According to the study's findings, PBA-Qt nanoparticles show promise in the treatment of chronic diabetic wounds¹¹. The effect of quercetin on cell apoptosis in seminal vesicles in type 1 diabetic rats was studied in 2022 by Bingzheng Dong et al. In diabetic mice, quercetin was given in three dosages over four months. The results showed reduced levels of T-AOC and Nrf2, increased MDA levels, increased cleaved Caspase-3, and decreased Bax to BCL-2 ratio. Except for fasting blood glucose, all indices were reversed after quercetin administration. According to the research, quercetin may be able to stop type 1 diabetes-related seminal vesicle lesions¹².

Topical therapies are advantageous for wound care due to their ease of application and potential to accelerate the healing process. They should be seen as a supplement to systemic and surgical treatment, nevertheless¹³. Diabetes-related foot sores may be treated using a variety of topical treatments and equipment. Atraumatic dressings impregnated with silicon, foam, hydrogels, hydrocolloids, alginates, growth factors, vacuum-assisted devices, and hyperbaric oxygen therapy are a few of them. A patient's health, the wound's healing process, the environment around the wound, the dressing's characteristics, accessibility, cost, and availability should all be considered while selecting a regimen^14-16. Even while topical applications like ointments are very important, topical therapy and patient adherence are often associated with them. While phytochemicals have healing properties, their extraction and synthesis may be challenging; yet, if done correctly, they can provide excellent healing properties in diabetes-induced wounds. Additionally, conventional ointments and medication substances are not able to treat the diabetic wound properly^17,18.

One in three Americans will have diabetes by 2050, according to a new report, and up to 34% of diabetic people may have diabetic foot ulcers (DFU). Debridement, pressure unloading, moist wound environments, and infection management are among the current therapies. But a lot of DFUs don't heal, and they cost more than $31 billion a year. Topical biomaterials have been created to enhance healing and target certain DFU-related deficits¹⁷. The goal of the current research was to create and assess an ointment loaded with quercetin nanoparticles for the successful treatment of diabetic foot wounds. In this investigation, quercetin was isolated from recently harvested onions.

MATERIALS AND METHODS:

Quercetin, hydroxypropyl methylcellulose (HPMC), and poloxamer 407 were purchased from Sigma Aldrich. Onions were purchased from a vegetable market in Madurai. Hard paraffin, mineral oil, and benzoic acid were collected from S.D. fine chem. Mumbai. Hydroxy propyl methylcellulose was purchased from Vijaya Scientific Madurai.

Preformulation study:

Fourier Transform Infrared (FTIR) Spectroscopic Analysis:

To evaluate potential interactions between extract residue and polymer, FTIR analysis was conducted. Using a Perkin-Elmer FTIR spectrometer operating in the wave number range of 4000 - 400 cm^-1, Fourier transform infrared (FTIR) spectra of the extracted residue, quercetin, and nanoparticle were acquired¹⁹.

Preparation of onion powder:

The onion was collected and sliced into small pieces. Then they were kept in sun shade for the drying process. After drying they are made into powder by the grinding process.

Isolation of onion powder:

Five grams of onion powder were obtained, and the extraction was carried out using a cold maceration procedure for a week, using several solvents such as methanol, DMF, and DMSO. The mixes that were extracted with DMF had a higher concentration of quercetin. By using a calibration graph of standard quercetin (standard reference quercetin was acquired from Sigma Aldrich), the amount of quercetin in the extracted residue was determined.

Identification of quercetin by chemical test:

One ml of the stock solution was made and put into a test tube. When a few drops of dilute sodium hydroxide solution were added, a bright yellow color emerged. Once again, a few drops of dilute hydrochloric acid were added, and when the solution became colorless, flavonoids were present.

Preparation of Quercetin nanoparticle by solvent evaporation technique:

Using a magnetic stirrer, a small quantity of water was added to a beaker containing the weighed amount of HPMC, and the mixture was stirred for a short while. After weighing the extracted residue and adding methanol to it in another beaker, the mixture was transferred dropwise into the beaker holding the HPMC. It was then allowed to agitate for an h and sonicated using a probe sonicator. A rotatory flask evaporator was then used to transfer the resultant solution, and after 80 rpm at 60ºC, the concentrated residue was extracted. Drying and collecting the nanoparticles followed the solvent's full evaporation. Various experiments were constructed with varying concentrations of HPMC using the afore mentioned methodology.

Table 1. Composition of quercetin nanoparticles.

Formulation Code	QNPs-1	QNPs-2	QNPs-3	QNPs-4	QNPs-5
Extract d Residue (mg)	500	500	500	500	500
HPMC K4 (mg)	100	200	300	400	500
Poloxamer 407 (mg)	100	100	100	100	100

Evaluation of quercetin-loaded nanoparticles:

Particle size and zeta potential:

Using the multi-angle Malvern Zetasizer (by laser light scattering technique - Mastersizer-2000; and Malvern, UK), the particle size and zeta potential of quercetin-loaded nanoparticles were measured. To determine the particle size and zeta potential, 1 milliliter of the diluted nanoparticles was sonicated for 30 sec and then added to the master-size cell.

Determination of Drug Loading and Encapsulation Efficiency:

A UV-visible spectrophotometer (Shimadzu UV-2600, Japan) was used to measure the quantity of quercetin present in the developed nanoparticles. Centrifuging the extracted residue that was not entrapped for 15 min at 10,000 rpm separated it from the prepared quercetin nanoparticles. The supernatant was then collected and filtered through a syringe filter (0.22 microns), and the volume was increased to 10mL using a citric-phosphate buffer. The extracted residue was measured at 257 nm²⁰.

In-vitro dissolution study:

A dissolving chamber filled with citric-phosphate buffer (pH 6.8) was used to hold 50mg of drug-equivalent nanoparticles, which were then securely bound up in a dialysis membrane. At regular time intervals, 2mL of the sample was withdrawn and the same volume of fresh citric-phosphate buffer was. Further, the samples were diluted and analyzed at 257 nm using an ultra-violet spectrophotometer (Shimadzu UV-2600, Japan)²¹.

Optimization of developed nanoparticles:

A variety of experiments were conducted, and each nanoparticle was optimized based on factors such as particle size, polydispersibility index, and in-vitro dissolution studies.

X-ray Diffraction analysis

X-ray diffraction (XRD) is a traditional method used to identify and characterize crystalline materials. The Bruker D8 Advance Eco diffractometer was used to examine the crystalline structure of residue and developed nanoparticles. The diffraction pattern of the samples from X-rays at 2θ was recorded, spanning from 5° to 80°. An acceleration voltage of 40 kV and a tube current of 40 mA were used to run the device.

Differential Scanning Calorimetry (DSC):

The crystalline condition of extract residue and quercetin nanoparticles were analyzed by DSC using the model Q-2000 Thermal Analyzer apparatus. A sealed aluminum pan containing a sample of 2 mg was heated to a temperature of 10°C each min underflow of nitrogen (10 ml/min), ranging from 200 to 400ºC.

Scanning electron microscopic analysis:

A scanning electron microscope (SEM) (Zeiss, Model EVO 18) was used to examine the morphology of quercetin nanoparticles. The required quantity of quercetin nanoparticles was applied to a copper grid that had been coated with carbon to create the SEM sample, which was then visible. The SEM experiment was conducted with an accelerating voltage of 10kv throughout, and the analysis was carried out at a high vacuum of 300mTorr²².

Formulation of quercetin nanoparticles loaded ointment:

Quercetin nanoparticles loaded ointment (QNPsOT) prepared by trituration method. Trituration-based ointment filled with quercetin nanoparticles. Hard paraffin was melted in a china dish at 60 °C, then liquid paraffin was poured, followed by benzoic acid to preserve it. The generated developed quercetin nanoparticles were then added to the molten liquid and well-mixed. The molten liquid was gently put on the porcelain slab and continuously triturated with an ointment spatula. The triturated molten was allowed to cool to room temperature, resulting in a fine texture of QNPsOT.

Table 2. Composition of quercetin nanoparticles loaded ointment.

Ingredients	Quantity
Quercetin nanoparticles	5.0 gm
Liquid paraffin	4.0 ml
Hard paraffin	1.0 gm
Benzoic acid	100 mg

*555 mg of nano particle contains 10 mg quercetin.

Evaluation of quercetin nanoparticles loaded ointment:

Physical appearance and organoleptic properties:

The formulated QNPsOT was assessed visually for color, appearance, texture, homogeneity, and initial skin sensation. These organoleptic qualities also included physical appearance. For rheological investigations, a Brookfield Synchro-Lectric Viscometer (Model RVT) with Helipath Stand was used. After putting the 10 g QNPsOT into a beaker and letting it acclimate for five min, the dial reading was taken using a T-D spindle (no. 6) set to 10, 20, 30, 50, 60, and 100rpm. The viscometer's matching dial reading was recorded at each speed. The dial reading that corresponded to each decrease in spindle speed was recorded. The measurements were taken. The unit of measurement for viscosity is centipoises²³.

pH determination:

Each formulation was measured out to be around 2.5g in a 100ml dry beaker, to which 50ml of distilled water was poured and kept for 3 h. A pH meter (pH Tutor, Eutech Instruments) was used to measure the pH of prepared ointments. The averages of the three measurements were recorded after the determinations were made in triplicate.

Rheological studies:

For rheological investigations, a Brookfield Synchro-Lectric Viscometer (Model RVT) with Helipath Stand was used. After putting the 10g QNPsOT into a beaker and letting it acclimate for five min, the dial reading was taken using a T-D spindle (no. 6) set to 10, 20, 30, 50, 60, and 100rpm. The viscometer's matching dial reading was recorded at each speed. The dial reading that corresponded to each decrease in spindle speed was recorded. The measurements were taken. The unit of measurement for viscosity is centipoises.

Spreadability and skin irritation study:

One gram of prepared ointment was placed on a glass slide measuring 10 x 7cm followed by the same size on another slide was placed it. The experiment was conducted thrice, measuring the diameter of the distributed ointment after a one-kilogram weight was applied to the slide assembly for one minute. The investigation on skin irritation was conducted on Wistar albino rats. When the ointment was administered topically to animal skin throughout the study period, the signs of edema or erythema were evaluated.

Ex-vivo skin Permeation study:

Goat skin was used to test QNPsOT ex-vivo skin permeation. The goat skin was measured in the necessary dimensions for this evaluation. In addition, the skin was removed, and double-distilled water was used to cleanse the muscles and lipids. The cylinder was put on a platform after the cleansed skin was tied into one end of the open-ended cylinder. The goat skin that had been removed was then covered with 200mcg of QNPsOT. The cylinder was then submerged in a beaker filled with 100ml of citrate buffer, shaken at 100 rpm, and kept at 37°C. At various intervals of time (15, 30, 45, 60, 90, 120, 150, 180, 210, and 240min), an aliquot (1ml) was taken and replaced with 1 ml of new citrate buffer. Using an ultraviolet spectrophotometer (Shimadzu UV-2600, Japan), the amount of quercetin that penetrated the skin was measured. Three runs of the trials were conducted^24,25.

In-vitro antibacterial activity:

To determine QNPsOT's antibacterial activity, the Agar disk diffusion technique (also known Kirby-Bauer antibiotic testing) was used²⁶. After being made, nutrient agar was transferred into a conical flask and sterilized in an autoclave. Within the laminar air flow chamber, the sterilized medium was put onto a petri dish and allowed to solidify for 30min. Following solidification, the bacteria (Escherichia coli and Staphylococcus aureus) were switched around in the medium and left undisturbed for an h. The well was filled with 10mcg of QNPsOT, and the plates were left at room temperature for two h to enable any antimicrobials that were created to diffuse. At 37ºC, the plates were incubated. Every experiment was run through three times. Antimicrobial activity was ascertained by measuring the antibacterial zone's diameter and comparing it to the normal quercetin zone of inhibition.

RESULT AND DISCUSSION:

Fourier Transform Infrared (FT-IR) spectroscopy analysis:

The FTIR spectrum of purchased standard quercetin exhibited N-H stretching vibration at 3666.68 cm^-1, an O-H stretching band at 3363.86 cm^-1, C=C aromatic ring stretch bands at 1651.07 cm^-1 and 1514.12 cm^-1, a cyclo-benzene stretching vibration at 1431.18 cm^-1, =C-O-H of the phenolic group at 1313.52 cm^-1, an aryl ether ring C–O stretching vibration at 1217.08 cm^-1, C-CO-C bending and bending vibration in ketone (carbon position at 4) at 1105.21 cm^-1, and aromatic out-of-plane C-H bending vibrations at 883.41 cm^-1. The quercetin extract residue's FTIR spectrum revealed N-H stretching vibration at 3776.62 cm^-1, an O-H stretching band at 3406.29 cm^-1, C=C aromatic ring stretch bands at 1610.56 cm^-1 and 1527.62 cm^-1, a cyclo-benzene stretching vibration at 1450.57 cm^-1, =C-O-H of the phenolic group at 1346.31 cm^-1, an aryl ether ring C–O stretching vibration at 1205.51 cm^-1, C-CO-C bending and bending vibration in ketone (carbon position at 4) at 1031.92 cm^-1, and aromatic out-of-plane C-H bending vibrations at 871.72 cm^-1(Figure 3). The quercetin nanoparticles' FTIR spectrum revealed quercetin's characteristic peaks at 3776.62, 3431.36, 1610.56, 1479.40, 1209.37, 1055.06, and 879.54 cm^-1.

Identification of quercetin by chemical test:

Two ml of extract was mixed with three drops of sodium hydroxide to form an instant yellow color. Followed by adding dilute hydrochloric acid the solution turned colorless, which indicates the presence of flavonoids in the extract residue.

Preparation of Quercetin nanoparticle by solvent evaporation technique:

Using the extracted residue that was obtained from onion, the solvent evaporation process was used for developing the nanoparticle. A different experiment was conducted using varying concentrations of HPMC K4. Developed formulations were characterized by particle size, zeta potential, drug content, entrapment efficiency, and in-vitro dissolution studies, which were used to optimize the nanoparticle formulation.

Evaluation of quercetin-loaded nanoparticles:

Determination of Particle size and Zeta potential:

The process of fabricating QNPs was optimized by experimenting with different ratios of HPMC K4. Formulation QNPs-5 was found to have an average diameter of 185.5±0.38nm (Figure), whereas the other formulations (QNPs-1 to QNPs-4) showed diameters that ranged from 241.13±1.03 to 685.2±2.41nm (Figure 4). According to the results, QNP particle size decreases when HPMC K4 concentration rises to 100mg and 500 mg. The fairly low Polydispersity index of QNPs reflects the homogenous distribution of quercetin in the developed formulation. As is well known, the polydispersibility index is a metric used to characterize the particle size distribution of NPs. Nanoparticles made using non-ionic stabilizing agents (poloxamer 407) have negative zeta-potential values that vary from -12.46 ± 1.84 to 9.27±1.36 mV (QNPs-1 to QNPs-5), which may be attributed to the presence of polymer terminal carboxylic groups. Poloxamer 407 and HPMC K4 may interact to cause differences in the zeta potential in QNPs. Overall, the outcome shows that QNPs are more stable.

Determination of Drug Loading and Encapsulation Efficiency:

The drug loading and entrapment efficiency for QNPs was estimated by treating QNPs with methanol further diluted with citric-phosphate buffer pH 6.8 and quantified by UV-visible spectrophotometer (Table). The drug loading as well as % entrapment efficiency of developed QNPs-1 to QNPs-5 formulations was observed to vary between 17.8±1.56±1.06 to 27.84±1.60 % and 60.1±2.11 to 85.1±1.63%, respectively. It has been observed that QNPs-5 showed higher drug loading 27.84±1.60% and percent entrapment 85.1±1.63%, respectively, compared to other formulations.

In-vitro dissolution study:

QNPs were tested for their in vitro release behavior utilizing a dialysis membrane in citrate buffer (pH 6.8). Varied release patterns were seen in the dissolution profile of QNPs when polymer and surfactant were added in varied ratios (HPMC K4 and poloxamer 407). All of the formulations, QNPs-1 to QNPs-5, had an in vitro release profile that varied from 70.1±2.65 to 98.90 ±2.74% (Figure 5). The rapid drug diffusion from nanocarriers is responsible for the quicker release at 240 min. In all QNPs formulations, as seen in Figure 4, over 70% of the quercetin was released in less than 240 min. Compared to other QNPs formulations, the QNPs-5 had the greatest release profile, measuring 98.90±2.74% within 240 min.

Figure 5. In vitro release patterns of QNPs.

Optimization of developed nanoparticles:

Based on the particle size, drug loading, percent entrapment efficiency, and in-vitro release reports, the formulation QNPs-5 was selected for further analysis. The QNPs-5 showed better results in particle size nm (185.5±0.38), drug loading (27.84±1.60%), percent entrapment efficiency (85.1±1.63%), and in-vitro release (98.90±2.74%) compared to other QNPs formulations, and QNPs-5 was selected for further evaluation and ointment preparation.

Characterization of quercetin-loaded nanoparticles:

Differential Scanning Calorimetry analysis:

DSC is a crucial metric for evaluating a drug's compatibility with other excipients used in the formulation. The DSC thermogram of quercetin and QNPs, which were scanned between 200 and 400°C, is shown in Figure 6. A prominent endothermic peak was seen on the quercetin curve at 328.37°C (Onset-324.25 °C; End- 329.79°C; Peak Height-7.6007mW). Instead of a distinct endothermic peak, the QNPs' DSC thermogram showed a broad peak at 317.29 °C.

Figure 6. DSC graph of quercetin extract residue and QNPs-5.

X-ray Diffraction analysis:

The solid states and surface alterations of extract residue and QNPs-5 were examined using an XRD analyzer. The X-ray diffraction analysis of the extract residue's XRD spectrum revealed the presence of multiple distinct peaks, suggesting that the high crystalline form of quercetin with sharp distinct peaks showed up at diffraction angles of 2̟ at 8.9°, 9.2°, 10.1°, 11.6°, 11.8°, 16.7°, 17.9°, 19.1°, 20.2°, 22.3°, 22.9°, 23.2°, 27.9°, 28.7°, 29.9°, 31.5°, and 36.9° (Figure 7). Furthermore, at 16.7°, 22.9°, and 19.1°, respectively, the XRD spectra of extract residue revealed distinct peaks with high relative intensities of 100.0%, 90.8%, and 46.2%. Furthermore, at 16.7°, 22.9°, and 19.1°, respectively, the XRD spectra of extract residue revealed distinct peaks with high relative intensities of 100.0%, 90.8%, and 46.2%. Additionally, these spectra showed that the quercetin extract residue was 1.9% amorphous and 100% crystalline in nature.

Figure 7. XRD graph of extract residue and QNPs-5.

Scanning electron microscopic analysis:

The produced QNPs' morphology was investigated using a scanning electron microscope. It was discovered that the improved QNPs-5 lacked the strip-like quercetin structure and were instead smaller, spherical, smooth, and angular (Figure 8). The size of the adjusted QNPs-5 was confirmed by the SEM pictures, which agreed well with the Particle Size Analyzer. The SEM image of QNPs-5 at different magnifications is illustrated in the figure.

Figure 8. SEM images of quercetin nanoparticles (QNPs-5).

Evaluation of quercetin nanoparticles loaded ointment:

Physical appearance and organoleptic properties:

The physical characteristics of ointment formulations, including their color, texture, homogeneity, and skin feel, were assessed in the research to assess their organoleptic features. The findings included a pleasing look, a silky texture, no phase separation, a yellowish-brown color, and an aromatic odor (Table 4).

Table 4. Evaluation of quercetin nanoparticles loaded ointment.

Parameters	QNPsOT
Appearance	Smooth consistency
Color	Yellowish -brown
Texture	No grittiness and greasiness
pH	7.02 ± 0.174
Viscosity (Poise)	2460 ± 0.5
Skin irritation	No erythema
Spreadability	Even greater spreadability

pH determination:

The pH of QNPsOT was found to be 7.02±0.174, which is within the Indian Pharmacopoeia's range. The pH values measured are appropriate for human skin and do not irritate.

Rheological studies:

The measured viscosity of quercetin QNPsOT, which denotes pseudoplastic flow, was determined to be in the range 2460±0.5. The standard deviation (n=3) was determined by computing the average of the three measurements.

Spreadability and skin irritation study:

When the ointment was applied to human skin, QNPsOT had a higher spreading ability. For the investigation of skin irritation, Wistar albino rats were used. Applied topically to animals, and the formulated QNPsOT did not exhibit any signs of erythema or edema after three h of observation on the rat's skin.

Ex vivo Permeation study:

When topically administered to goat skin in an open-ended cylinder, the ex vivo skin permeation capacity of QNPs-5 was examined. The results showed that quercetin penetrated the skin immediately after 30 min, indicating that there was no delay in the drug's penetration of the skin. Here, poloxamer 407 emulsifies with the sebum to serve as a permeation enhancer and raise quercetin's thermodynamic coefficient. At min 60, 29.95 ± 3.31% of the medication was absorbed via the skin; the permeation profile continued for another 240 min. At 240 min, a maximum of 95.30 ± 4.08% quercetin was absorbed via the skin (Figure 9). This implies that the created QNPs-5 may be used topically to provide loaded quercetin for efficient treatment since they can readily penetrate the skin.

Figure 9. The skin permeation profile of QNPs-5.

(a) (b)

(c) (d)

Figure 10. Antibacterial activity of standard quercetin (a and b) and QNPsOT (c and d).

In-vitro antibacterial activity:

In this work, nutrient agar that had been autoclave-sterilized was used to compare the bactericidal activity of regular quercetin and QNPsOT. After sterilizing, the medium was put in a petri dish and given time to harden. After that, the medium was supplemented with microorganisms (Escherichia coli and Staphylococcus aureus), which were kept undisturbed for an h. Both standard quercetin and QNPsOT showed zones of inhibition of 0.8 cm and 1.2 cm, respectively, when the zones of inhibition were assessed (Figure 10).

CONCLUSION:

In the present work, solvent evaporation was used to develop quercetin extract residue-loaded nanoparticles (QNPs-1 to QNPs-5), and trituration techniques were used to prepare QNPs-loaded ointment. When compared to other QNPs formulations, the QNPs-5 formulation performed better in terms of particle size, drug loading, entrapment efficiency, and in-vitro release. An ex vivo investigation showed that developed QNPsOT increased quercetin permeation in goat skin. Furthermore, compared to standard quercetin, the QNPsOT containing extracted quercetin exhibited greater antibacterial activity. The results indicate that the ointment loaded with QNPs is appropriate for delivering quercetin with enhanced solubility, and also exhibited that the developed formulation could be suitable for topical administration with enhanced therapeutic efficacy.

REFERENCES:

1. Ogurtsova K, Guariguata L, Barengo NC, Ruiz PL, Sacre JW, Karuranga S, et al. IDF Diabetes Atlas: Global estimates of undiagnosed diabetes in adults for 2021. Diabetes Research and Clinical Practice. 2022 Jan 1; 183: 109118. doi: 10.1016/j.diabres.2021.109118.

2. Armstrong DG, Tan TW, Boulton AJM, Bus SA. Diabetic foot ulcers: a review. Journal of the American Medical Association. 2023 Jul 1; 330(1): 62-75. doi: 10.1001/jama.2023.10578.

3. Jaroenarpornwatana A, Koonalinthip N, Chawaltanpipat S, Janchai S. Is the duration of diabetic foot ulcers an independent risk factor for developing diabetic foot osteomyelitis? Foot. 2023 Jul 1;56:102000. doi: 10.1016/j.foot.2023.102000.

4. Anand David AV, Arulmoli R, Parasuraman S. Overviews of biological importance of quercetin: A bioactive flavonoid. Pharmacognosy Reviews. 2016 Jul 1; 10(20): 84-89. doi: 10.4103/0973-7847.194044.

5. Shen N, Wang T, Gan Q, Liu S, Wang L, Jin B. Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. Food Chemistry. 2022 Jul 1; 383: 132531. doi: 10.1016/j.foodchem.2022.132531.

6. Wang G, Wang Y, Yao L, Gu W, Zhao S, Shen Z, Lin Z, Liu W, Yan T. Pharmacological activity of quercetin: an updated review. Journal of Evidence-Based Complementary and Alternative Medicine. 2022 Jul 1;2022:3997190. doi: 10.1155/2022/3997190.

7. Sanad SM, Farouk R, Nassar SE, Alshahrani MY, Suliman M, Ezzat Ahmed A, Eid Elesawi I. The neuroprotective effect of quercetin nanoparticles in the therapy of neuronal damage stimulated by acrolein. Saudi Journal of Biological Sciences. 2023 Nov 1; 30(11): 103792. doi: 10.1016/j.sjbs.2023.103792.

8. Mascarenhas-Melo F, Gonçalves MBS, Peixoto D, Pawar KD, Bell V, Chavda VP, Zafar H, Raza F, Paiva-Santos AC. Application of nanotechnology in management and treatment of diabetic wounds. Journal of Drug Targeting. 2022 Oct 1; 30(10): 1034-1054. doi: 10.1080/1061186X.2022.2092624.

9. Sajid M, Płotka-Wasylka J. Nanoparticles: synthesis, characteristics, and applications in analytical and other sciences. Microchemical Journal. 2020 Jul 1; 154: 104623-104629. doi: 10.1016/j.microc.2020.104623.

10. Mascarenhas-Melo F, Gonçalves MBS, Peixoto D, Pawar KD, Bell V, Chavda VP, Zafar H, Raza F, Paiva-Santos AC. Application of nanotechnology in management and treatment of diabetic wounds. Journal of Drug Targeting. 2022 Oct 1; 30(10): 1034-1054.

11. Abid S, Sial N, Hanif M, Usman Abid HM, Ismail A, Tahir H. Unlocking the potential of phenyl boronic acid functionalized-quercetin nanoparticles: Advancing antibacterial efficacy and diabetic wound healing. Heliyon. 2023 Jan 1; 10(1): e23452. doi: 10.1016/j.heliyon.2023.e23452.

12. Dong B, Shi Z, Dong Y, Chen J, Wu ZX, Wu W, Chen ZS, Han C. Quercetin ameliorates oxidative stress induced cell apoptosis of seminal vesicles via activating Nrf2 in type 1 diabetic rats. Biomedicine and Pharmacotherapy. 2022 Jul 1; 151: 113108. doi: 10.1016/j.biopha.2022.113108.

13. Kavitha KV, Tiwari S, Purandare VB, Khedkar S, Bhosale SS, Unnikrishnan AG. Choice of wound care in diabetic foot ulcer: A practical approach. World Journal of Diabetes. 2014 Jul 1; 5(4): 546-556. doi: 10.4239/wjd.v5.i4.546.

14. Sood A, Granick MS, Tomaselli NL. Wound dressings and comparative effectiveness data. Advances in Wound Care. 2014 Aug 1; 3(8): 511-529. doi: 10.1089/wound.2012.0401.

15. Shi C, Wang C, Liu H, Li Q, Li R, Zhang Y, Liu Y, Shao Y, Wang J. Selection of appropriate wound dressing for various wounds. Frontiers in Bioengineering and Biotechnology. 2020 Jul 1; 8: 182. doi: 10.3389/fbioe.2020.00182.

16. Faraji N, Parizad N, Goli R, Nikkhah F, Golhkar M. Fighting diabetic foot ulcer by combination therapy, including larva therapy, Medi honey ointment, and silver alginate dressings: A case report. International Journal of Surgery Case Reports. 2023 Jul 1; 113: 109055. doi: 10.1016/j.ijscr.2023.109055.

17. Saghazadeh S, Rinoldi C, Schot M, Kashaf SS, Sharifi F, Jalilian E, Nuutila K, Giatsidis G, Mostafalu P, Derakhshandeh H, Yue K, Swieszkowski W, Memic A, Tamayol A, Khademhosseini A. Drug delivery systems and materials for wound healing applications. Advanced Drug Delivery Reviews. 2018 Jul 1; 127: 138-166. doi: 10.1016/j.addr.2018.04.008.

18. Wang Y, Han G, Guo B, Huang J. Hyaluronan oligosaccharides promote diabetic wound healing by increasing angiogenesis. Pharmacological Reports. 2016 Dec 1; 68(6): 1126-1132. doi: 10.1016/j.pharep.2016.07.001.

19. Selvakumar M, Venkateshwaran K, Ruckmani K, Shanmugarathinam A. Potentiating the solubility of BCS class II drug zaltoprofen using nanodispersion technology. Journal of Dispersion Science and Technology. 2023 Jul 1; 1-10. doi: 10.1080/01932691.2023.2173224.

20. Ciardi M, Ianni F, Sardella R, Di Bona S, Cossignani L, Germani R, Tiecco M, Clementi C. Effective and selective extraction of quercetin from onion (Allium cepa L.) skin waste using water dilutions of acid-based deep eutectic solvents. Materials. 2021 Nov 1; 14(21): 6465. doi: 10.3390/ma14216465.

21. Mandic L, Matkovic M, Baranovic G, Segota S. The increased release kinetics of quercetin from superparamagnetic nanocarriers in dialysis. Antioxidants. 2023 Mar 1; 12(3): 732. doi: 10.3390/antiox12030732.

22. Pandian SRK, Kunjiappan S, Ravishankar V, Sundarapandian V. Synthesis of quercetin-functionalized silver nanoparticles by rapid one-pot approach. Biotechnologia. 2021 Mar 1; 102(1): 75-84. doi: 10.5114/bta.2021.103764.

23. Manian M, Jain P, Vora D, Banga AK. Formulation and evaluation of the in vitro performance of topical dermatological products containing diclofenac sodium. Pharmaceutics. 2022 Sep 1; 14(9): 1892. doi: 10.3390/pharmaceutics14091892.

24. Gupta V, Trivedi P. Ex vivo localization and permeation of cisplatin from novel topical formulations through excised pig, goat, and mice skin and in vitro characterization for effective management of skin-cited malignancies. Artificial Cells, Nanomedicine, and Biotechnology. 2015 Jun 1; 43(6): 373-382. doi: 10.3109/21691401.2014.893523.

25. Ruela ALM, Perissinato AG, de Sousa Lino ME, Mudrik PS, Pereira GR. Evaluation of skin absorption of drugs from topical and transdermal formulations. Brazilian Journal of Pharmaceutical Sciences. 2016 Jul 1; 52(3): 527-543.

26. Wang S, Yao J, Zhou B, Yang J, Chaudry MT, Wang M, Xiao F, Li Y, Yin W. Bacteriostatic effect of quercetin as an antibiotic alternative in vivo and its antibacterial mechanism in vitro. Journal of Food Protection. 2018 Jan 1; 81(1): 68-78. doi: 10.4315/0362-028X.JFP-17-214.

Received on 13.04.2025 Revised on 23.05.2025

Accepted on 30.06.2025 Published on 06.10.2025

Available online from October 13, 2025

Asian J. Pharm. Res. 2025; 15(4):387-395.

DOI: 10.52711/2231-5691.2025.00061

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formulation Code	Particle size (nm)	PDI	Zeta Potential(mV)	Drug loading (mg)	Entrapment efficiency (%)
QNPs-1	685.3 ± 2.41	0.280± 0.01	-11.03 ± 2.05	17.8 ± 1.56	62.3 ± 1.95
QNPs-2	542.6 ± 3.22	0.476 ± 0.13	-10.83 ± 1.53	20.2 ± 1.87	60.1 ± 2.11
QNPs-3	386.5 ± 1.46	0.520 ± 0.07	-12.46 ± 1.84	21.48 ± 1.73	72.2 ± 2.53
QNPs-4	241.7 ± 1.83	0.472 ± 0.01	-11.52 ± 2.31	23.51 ± 2.03	82.3 ± 1.86
QNPs-5	185.5 ± 0.38	0.347 ± 0.14	-9.27 ± 1.36	27.84 ± 1.60	85.1 ± 1.63