INTRODUCTION:

Ultraviolet (UV) radiation is always needed to protect against its negative effects and to prevent wrinkles, premature aging, sunburns, and cancer. Sunscreens are used to shield the skin from UV rays from the sun and to support the body's natural defensive systems.

Its capacity to absorb, reflect, or scatter solar radiation is what determines how it works. Sun protection factor (SPF) is used to gauge how well a topical preparation protects against UVB rays. By comparing the amount of time required to create sunburn on skin protected by sunscreen to the amount of time required to cause sunburn on unprotected skin, the SPF of a sunscreen is determined. Sunscreens with a higher SPF provide more protection from sunburn.^1-4

Allium canadense, a polyphenolic phytoalexin, has been shown to heal skin wounds and strengthen anti-oxidant defenses against UVB-mediated damage. It contains gallic and ellagic acids, epicatechins, and peroxyl radical scavenging properties, which protect cells from oxidative damage.^5-7

MATERIALS AND METHODS:

Allium canadense extract was obtained from Vyankatesh Naturaals Pvt. Ltd, Chindwara, whereas Eudragit RS100 and S100 were obtained as a gift sample from Evonik India Pvt. Ltd. White soft paraffin, liquid paraffin, and hard paraffin were obtained from S.D. Fine Chem, Pvt. Ltd. Polyvinyl alcohol was obtained from Loba Chem Pvt.Ltd in Mumbai. All additional reagents and chemicals used were of analytical quality and were used as received.

Standard Calibration Curve:

Accurately weighed 10 mg of Allium canadense was dissolved in a tiny amount of methanol, and the volume was increased to 100 ml using methanol. The stock solution was prepared at a concentration of 100μg/mL. Aliquots of 0.8, 1.6, 2.4, and up to 5.6 mL were pipetted into 10 mL volumetric flasks and filled with methanol to achieve concentrations of 8, 16, 24, and 56 μg/mL, respectively. The solutions were evaluated at wavelength maxima λ max 304 nm using a UV-visible spectrophotometer.^8-11

Drug-Excipients Interaction Study:

Drug polymer interactions were investigated using FT-IR spectroscopy. A 1:1 ratio of drug and polymer was used, and the physical mixture of samples was weighed and thoroughly mixed with potassium bromide to create a homogeneous combination. A little amount of powder was compacted into a small pellet by applying pressure. The IR spectra of the beads from 400-4000cm-1 was recorded and compared to a reference to investigate potential interference.^12-14

Preparation of Microspheres:

The process involved weighing polymers, adding ethanol and dichloromethane to create an organic phase, and creating an inorganic phase by combining 2% Polyvinyl Alcohol with distilled water. Microspheres were formed by adding the organic solution to the inorganic solvent, agitating for 3 hours, and maintaining the heat to speed up evaporation. The microspheres were then filtered, rinsed, and dried on a petri plate for 24 hours. The dried microspheres were stored in amber-colored glass vials for later assessment.^15-16(Table-1).

Table 1: Formulation Chart of Allium canadense Microspheres

Batch	*Allium canadense* (mg)	Eudragit RS100 (mg)	Eudragit S100 (mg)	Ethanol: Dichloromethane
A1	250	100	100	1:1
A2	250	100	200	1:1
A3	250	100	300	1:1
A4	250	200	100	1:1
A5	250	200	200	1:1
A6	250	200	300	1:1
A7	250	300	100	1:1
A8	250	300	200	1:1
A9	250	300	300	1:1

Preparation of Cream Base:

All of the materials were carefully weighed individually in a beaker and melted in the sequence in which the paraffin's melting range decreased. Stir them thoroughly, then add the wool alcohol and continue to stir until the temperature stabilizes. To produce a homogenous formulation, add perfume after cooling to 35°C and stirring.^17-18(Table-2).

Table 2: Formula for Preparation of Plain Cream Base

Sr. No.	Ingredients	Quantity prescribed (gm)
1	Wool alcohols	0.6
2	Hard paraffin	2.4
3	White soft paraffin	1.0
4	Liquid paraffin	6.0
5	Perfume	q.s
6	Antioxidant	q.s

Evaluation of Microspheres:^19-21

Micromeretics Study:

The produced microspheres were tested for micromeretic characteristics such as tap density, bulk density, and angle of repose.

Tap Density:

Tapped density of produced microspheres was assessed using the tapping technique. An accurately weighed quantity of microspheres was transferred to a 10 mL measuring cylinder. After watching the predominant volume of microspheres, tapping was continued on a rough surface until no progressive variation in the volume was recorded, and therefore the tapped density was calculated.

Mass of Microspheres

Tap Density: –––––––––––––––––––––––––––––––––

Volume of Microspheres after tapping

Bulk Density:

It is the quantitative relationship between a particular mass of a powder and its total volume. The bulk density of the formed microspheres was estimated by pouring about 2 g of the microspheres into a clean measuring cylinder and measuring the starting volume. The bulk density was determined using the following equation:

Mass of Microspheres in gram

Bulk Density: –––––––––––––––––––––––––––

Volume of Microspheres in cm³

Angle of Repose:

Typically, the angle of repose of floating microspheres is used to assess their flow properties. The angle of repose of microspheres was calculated using a fixed funnel on a burette stand and the fixed funnel technique. The microspheres were allowed to freely fall down the fixed funnel until the apex of the conical pile produced met the funnel's tip. The angle of repose (θ) was found using the following formula:

Height of Pile

Tan θ = ––––––––––––––

Radius of Pile

Particle Size Measurement and Percent Drug Entrapment:

The study assessed particle size and distribution of microsphere formulations using optical microscopes, estimating mean size and volume mean diameter, and determining entrapment efficiency by crushing 100mg of each batch. The produced microspheres were ground in a mortar pestle and dissolved in 10 mL of methanol in a 100 mL volumetric flask. The capacity was then made up with methanol. The resultant solution was then filtered through a Buchner funnel using Whatman filter paper, diluted appropriately, and the absorbance was measured at 304nm. The proportion of drug entrapment was computed as follows:

Actual amount of Drug Found

% Drug Entrapment = –––––––––––––––––––––– × 100

Theoretical Amount of Drug

In-Vitro Drug Release:

The USP dissolving apparatus Type I was used to study drug release characteristics of pure drugs and microspheres. The drug was placed in a basket containing pH 6.8 solution for 12 hours, then diluted with buffer 6.8 solution. The diluted samples were then examined at 304nm using a UV spectrophotometer. The dissolution medium was phosphate buffer pH 6.8.^22-24

Ex-vivo Study:

The freshly removed goat skin was purchased from the local abattoir and meticulously shaved to avoid harming the skin tissues. Tissue samples were placed into Franz diffusion cells with a permeation area of 3.14 cm2. The acceptor compartment received 25 ml of phosphate buffer with a pH of 6.6. The temperature was maintained at 340 degrees Celsius. After 20 minutes of pre-incubation, a formulation corresponding to 1gm of prepared cream was put in the donor chamber. At specified time intervals, 1ml samples were extracted from the acceptor compartment, and the sampled volume was replaced with phosphate buffer pH 6.6 after each sampling, for 12 hours.^25-27

Histopathology Study:

Freshly acquired skin tissue samples were used in a histopathological investigation. The skin tissues were meticulously separated from the skin of a goat purchased from the nearby abattoir. The skin was thoroughly cleansed and shaved using depilatories. Skin tissue of 2 cm² was carefully extracted and utilized for testing. Two samples of skin tissue were utilized, one with the optimized cream and the other without the cream. Then, both samples were exposed to midday sunshine for two hours. Following this, both samples were subjected to microscopic examination to evaluate the harmful effect of UV radiation on the skin tissue samples.²⁸

Sunscreen Efficacy Testing:

A sodium nitroprusside solution was applied to petridish, covered with a cellophane membrane, and exposed to sunlight for 2 hours. The samples were then analyzed for Allium canadense content using a UV spectrophotometer method.^29-31

Determination of in vitro SPF:

The optimized cream was diluted with ethanol and water, ultrasonicated, and collected. Absorption values were determined from 290 nm to 320 nm at 5 minutes. The study was repeated three times, making decisions in average. The detection values were multiplied by EE (λ) values and repeated to obtain SPF values.^31-33

Photo Stability Determination:

2mg/cm² of three highly oily sunscreen lotions are weighed and equally distributed between two quartz silica-coated plates (5 mm thick and 25 mm wide). Use a thin coating to prevent absorption distortion. AUC for UVA, UVA1 (340-400 nm), UVA2 (320-340 nm), and UVB were assessed before (AUC front) and after (AUC rear) UV artificial (980 kJ / m²) UVA with 12 kJ / m² UV radiation (including UVB), as well as before and after natural UV. If AUCI (AUCI = AUC in the background / AUC in front) was more than 0.80, sunscreen was considered stable33. The AUC is determined using the following equation.³⁴

A is absorption, and λ is wavelength. For UVA, λmax = 400 nm and λmin = 320 nm. The same measurement was done for each UV range separately, before and after UV artificial, and before and after UV natural.

Kinetics Study:

The drug release data were fitted to zero order (cumulative% drug release versus time), first order (log of cumulative% drug retained versus time), Higuchi models (% cumulative drug released versus square root of time), and Korsmeyer-Peppas models (log% of cumulative drug release Vs log of time) to calculate the kinetics of drug release and determine the release mechanism of the drug from the prepared floating microspheres.³⁵

Surface Morphology:

The surface morphology of the produced microspheres was evaluated using scanning electron microscopy (SEM). SEM samples were made by scattering microspheres over a stub with double adhesive tape. The stubs were then coated with platinum in an argon environment, using a gold sputter module in a very high vacuum evaporator.³⁶

Stability Study:

The need for extensive testing to test the substance and provide evidence to demonstrate that the quality of a drug or drug product varies over time in response to various environmental factors such as temperature, light, and humidity, as well as elevated storage conditions, re-test times, and shelf lives, will be established. Stability tests were conducted at room temperature (250C ± 20C / 60% RH ± 5% RH) and accelerated testing (400C ± 20C/ 75% RH ± 5% RH) after 3 months of building preparation. The improved formulation was evaluated for percent drug content, percent cumulative drug release, and sunscreen effectiveness tests.^37-38

RESULTS AND DISCUSSION:

Standard Calibration Curve:

The findings of the standard calibration curve indicated that it follows the Beer's lamberts law as the equation produced was linear with the values of y =0.014x + 0.005 and the regression value of R2 = 0.999.

Figure 1: Standard Calibration Curve of Alliumcanadense

Drug – Excipient Interaction Study:

FTIR analysis of pure Allium Canadense revealed band characteristics at 3309cm^-1, 1458, 1504, 1597cm^-1, and 995cm^-1. Compared to the standard, the pure drug had the same peaks, confirming its purity. The optimized formulation was matched with the pure drug peaks, revealing no new formation, disappearance, or mismatching of peaks, indicating no physical or chemical interaction between the drug and excipients used.

Figure 2: FTIR of a) Pure Drug b) Optimized Formulation

Micromeretics Study: The micromeretics investigation of the produced microspheres revealed that they have good flow properties. The tap density ranged from 0.713±0.36-0.929±0.07gm/cm³, whereas the bulk density ranged from 0.798±0.29-0.878±0.17 gm/cm³. The carr's index and hausner's ratio were 4.8-7.2 and 1.03-1.08, respectively. The angle of repose ranged from 13.37 ± 0.32-21.25 ± 0.71. (Table-3).

Particle Size Measurement and Percent Drug Entrapment:

The study of percent drug content indicated that the percent entrapment was determined to be in the range of 46.18 ± 1.55% to 58.07 ± 1.02%, with batch A5 showing the greatest drug entrapment effectiveness. The particle size of the produced batches ranged from 33.12±2.50μm to 43.78±1.68 μm.

In-Vitro Drug Release:

The percent cumulative drug release of the produced microspheres ranged from 1.203% to 99.08% during a 12-hour period. It can be shown that the eudragit ratio of 1:1 (200:200) had the highest drug content entrapment and released the most amount of drug in the expected time period. The results show that batch A5 was the ideal mix for the microspheres. This batch was further investigated for the kinetics and stability investigation.

Table 4: Evaluation of Particle Size and Drug Entrapment Efficiency

Batch	Particle Size(μm)	Drug Entrapped (%)
A1	33.12± 2.50	46.18 ± 1.55
A2	36.44± 3.16	55.15 ± 2.17
A3	38.50± 2.09	55.72 ± 3.01
A4	40.56± 1.22	51.60 ± 3.61
A5	43.78± 1.68	58.07 ± 1.02
A6	41.07± 2.19	57.01 ± 1.26
A7	38.46± 2.38	55.70 ± 2.50
A8	35.61± 3.16	54.12 ± 2.40
A9	36.16± 2.01	55.70 ± 2.50

Figure 3: Percent Cumulative Drug Release of the Prepared Formulations

Ex-vivo Study:

The molecular permeability of the biological edge presents a difficulty to the multistep process. Many variables, including chemical composition, body composition, and biological interactions, can influence molecular saturation. A study of drug penetration via the skin indicated that drug withdrawal ranged from 1.134% to 99.98% during a 12-hour period. The A2 formulation had the maximum drug release (99.98%), although medication withdrawal lasted 10 hours. However, batch A5 demonstrated 98.47% drug withdrawal within the projected 12-hour timeframe. So the A5 collection remained an upgraded selection.

Figure 4: Ex-vivo Study of the Prepared Formulations

Histopathological Study:

The histopathological analysis was undertaken to assess the safety of the manufactured sunscreen lotion containing Allium canadense microspheres. The safety was examined by identifying any anomalies, such as tissue damage. The first image (a) showed that tissues were destroyed as a result of UV light exposure, whereas the second figure (b) showed that the manufactured sunscreen protected the skin from damaging UV rays. These findings imply that sunscreen containing Allium canadense microspheres might be considered safe for topical application.

Figure 5: Histopathological Study of the Skin a) Untreated b) Treated

Sunscreen Efficacy Testing:

The UV ray absorbance of all formulations was measured in order to determine which batch could absorb the most UV radiations. The analysis revealed that the batch of A5 had the highest absorbance of 2.432nm. Based on these data, it was considered that batch A5 was optimal.

Table 5: Absorbance of sodium nitroprusside solution (0.05% w/v) at 304 nm after exposed to sunlight for 2 hrs

Sr. No.	Petri Plates Covered With Formulation	Absorbance of Sodium Nitroprusside Solution (nm)
1	A1	2.011
2	A2	1.112
3	A3	2.077
4	A4	1.107
5	A5	2.432
6	A6	0.252
7.	A7	1.487
8.	A8	1.987
9.	A9	0.872

Table 6: In-Vitro SPF Value of Cream Formulation Measured Under DifferentWavelength

S. No.	Wavelength (λ nm)	EE×I (normalized)	Absorbance×CF×EE×I	SPF=∑EE (λ)×I (λ)×Absorbance (A)×10
1	290	0.0150	0.06405±0.002	3.909257
2	295	0.0817	0.339055±0.002
3	300	0.2874	1.155348±0.001
4	305	0.3278	1.27842±0.003
5	310	0.1864	0.702728±0.001
6	315	0.0839	0.305396±0.002
7	320	0.018	0.062640±0.002

Table 7: Results of Photo Stability Evaluation of Sunscreen Cream Batches

			After Natural UV Exposure		After Artificial UV Exposure
Formulation	Exposure Time(min)	UVA Radiation(kJ/m2)	UVA	UVB	UVA	UVB
A3	30	55	0.65	0.68	0.72	0.72
	90	165	0.68	0.63	0.65	0.69
	120	235	0.59	0.61	0.71	0.77
A5	30	62	0.75	0.70	0.60	0.81
	90	155	0.79	0.72	0.65	0.80
	120	242	0.84	0.87	0.90	0.82
A6	30	58	0.45	0.59	0.65	0.73
	90	160	0.85	0.88	0.78	0.81
	120	230	0.65	0.69	0.82	0.87

Table 8: Model fitting data for in-vitro releases kinetic parameters

Formulation	Zero order	Korsmeyer-Peppas	Higuchi Diffusion	Best Fit Model	Value of ‘n’
A5	0.944	0.963	0.966	Korsmeyer Peppas	0.491

Determination of in vitro SPF:

The SPF value obtained from the optimized cream formulation was 3.909257, indicating that the created microspheres loaded cream will have the property to block about 73% of UV rays, reflecting the cream's overall sunscreen performance. (Table-6).

Photo Stability Determination:

All of the components in the sunscreen mixture demonstrated high stability. The samples on the plate had the same width before and after 20 minutes of heating at 50°C. Less stable sunscreens begin to disintegrate as soon as they are exposed to sunlight. After 120 minutes of UV exposure, AUCI identified the A3 and A6 sunscreens as unstable. While the A5 formulation exposure demonstrated a change in length to short range, it was determined to be stable with all UV activity once and for all, including natural UV. As a result, the A5 can offer superior UV protection when used with a stable sunscreen. All of the cream has demonstrated good firmness and may be regarded an antioxidant environment. (Table-7).

Kinetics Study:

The study used various kinetic models to analyze drug release from microbeads, including zero order, first order, Higuchi, and Korsmeyer-Peppas. The results indicated that the prepared microspheres exhibited zero order kinetics, followed by super case-II transport, with a regression coefficient of 1.0. (Table-8).

Surface Morphology:

The surface morphology of prepared microspheres was studied at 5K and 25K magnification. The microspheres were spherical with a rough surface, few cracks, and holes due to in situ drying. The rate of solvent removal from the microspheres influenced the end product's morphology. A porous structure was observed on the shell's surface, suggesting a porous surface could enhance drug release from the prepared microspheres. (Figure-6).

Figure 6: Scanning Electron Microscopy of the optimized formulation at a) 5K and b) 25K

Stability Study:

The stability study of an optimized cream loaded with Allium canadense microspheres showed a 0.27% decrease in drug content, no changes in drug release behavior, and a change in UV absorption for sunscreen efficiency. During the accelerated study, there was a 0.51% difference in drug content, 0.18% increase in drug release, and a 0.245nm difference in sunscreen efficiency. The study concluded that the formulation was stable during the study process.

Table 9: Stability Study at Room Temperature (25°C ± 2°C / 60 % RH ± 5% RH)

Day	Drug Content (%)	Drug Release (%)	Sunscreen Efficiency (nm)
0	58.07 ± 1.02	99.08	2.432
15	58.07 ± 1.02	99.08	2.432
30	58.07 ± 0.58	99.08	2.412
60	57.87 ± 0.15	99.08	2.380
90	57.80 ± 1.85	99.08	2.372

Table 10: Stability Study at Accelerated testing (40°C ± 2°C / 75 % RH ± 5% RH)

Day	Drug Content (%)	Drug Release (%)	Sunscreen Efficiency (nm)
0	58.07 ± 1.02	99.08	2.432
15	58.07 ± 1.02	99.08	2.432
30	57.97 ± 1.47	99.08	2.358
60	57.71 ± 1.85	99.18	2.241
90	57.56 ± 1.27	99.26	2.187

CONCLUSION:

The microspheres-loaded cream formulation including Allium canadense was created in order to assess its effectiveness against UV radiation for sunscreen protection. The A5 batch was found to be optimal among the created batches since it had the highest SPF factor and so could absorb the most UV radiation when compared to the other batches. The photosensitivity of the created batches demonstrated that they are extremely stable, and the stability analysis of the prepared optimized batch yielded similar findings. Based on the findings of the study, it is possible to infer that Allium canadense microspheres are a superior and natural alternative to synthetic sunscreens.

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Received on 15.10.2024 Revised on 10.02.2025

Accepted on 05.04.2025 Published on 03.05.2025

Available online from May 05, 2025

Asian J. Pharm. Res. 2025; 15(2):141-148.

DOI: 10.52711/2231-5691.2025.00023

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Batch No.	Tap Density (gm/cm³)	Bulk Density (gm/cm³)	Carr’s Index	Hauser’s Ratio	Angle of Repose
Pure Drug	0.803±0.36	0.741±0.04	7.7	1.08	24.56 +0.22
A1	0.713±0.36	0.752±0.04	6.7	1.07	19.60⁰±0.25
A2	0.797±0.02	0.786±0.36	4.8	1.03	21.25⁰±0.71
A3	0.833±0.06	0.878±0.17	7.2	1.04	16.87⁰±0.06
A4	0.883±0.58	0.784±0.29	6.3	1.08	19.58⁰±0.02
A5	0.922±0.12	0.862±0.07	4.3	1.03	18.84⁰±0.08
A6	0.929±0.07	0.902±0.03	6.1	1.05	15.88⁰±0.47
A7	0.843±0.58	0.798±0.29	5.3	1.05	19.98⁰±0.18
A8	0.872±0.12	0.825±0.07	5.3	1.05	13.37⁰+0.32
A9	0.909±0.07	0.862±0.03	5.1	1.05	20.18⁰±0.22