Highlights

Skin pigmentation disorders are common issues linked to melanocytes. Alpha arbutin inhibits melanogenesis by blocking the tyrosinase enzyme. Its hydrophilic nature and log P value of -1.49 hinder its penetration into the stratum corneum, resulting in less than 1% reaching the melanocytes.

Alpha arbutin can be formulated as ethosomes. Ethanol acts as a permeation enhancer, and the nanosized particles further facilitate the drug’s permeation into the dermis and epidermis, with better retention compared to marketed products. Particle size and zeta potential values indicate the formation of a stable formulation.

The dermatokinetic study revealed better permeation and retention of alpha arbutin in the dermis and epidermis compared to the marketed formulation.

Introduction

Melanocyte-related disorders of skin pigmentation (hypopigmentaion and hyperpigmentation) are the most prevalent conditions. The majority of women have abnormal face pigmentation due to a combination of external and endogenous causes. Skin discoloration patients suffer from emotional and cognitive issues that affect their psychosocial health¹. There are numerous causes of hyperpigmentation which negatively impacts an individual’s quality of life. Addressing hyperpigmentation can be a challenging and discouraging process, particularly for women. The primary cause is the increase in melanin content in the dermis or epidermis².The process by which melanocytes produce melanin is known as melanogenesis. One important enzyme in the formation of melanin is tyrosinase. Tyrosine is converted to melanin by tyrosinase in a few steps. Melanin pigment’s primary function is to absorb ultraviolet (UV) radiation (UVA and UVB), thereby protecting the skin from UV radiation. Hyperpigmentation is the result of an excess of melanin produced by UV exposure. Therefore, one of the main strategies for avoiding skin hyperpigmentation is melanin inhibition ³. The most common treatment for hyperpigmentation is skin brightening ⁴. The sources of skin-whitening chemicals are both synthetic and natural. The primary obstacle of whitening chemicals is its poor stability. As a result, encapsulating these whitening agents can enhance their concentration at specific places and physicochemical stability ⁵.

Hydroquinone inhibits the tyrosinase enzyme and halts the melanogenesis process, making it a depigmenting agent. However, it can lead to several negative effects, such as mutagenicity in African populations and an increased incidence of ochronosis ^6,7.As such, the Food and Drug Administration (FDA) in the United States (US) and the European Union (EU) has prohibited the use of it in any over-the-counter preparation. The less cytotoxic derivatives of hydroquinone are utilized to achieve the whitening effects of the original drug. The derivative alpha arbutin is one of them. It is mostly made from hydroquinone through enzymatic synthesis. Alpha-arbutin is 4-hydroxyphenyl a-D-glucopyranoside structurally ⁸. Beta-arbutin, or 4-hydroxyphenyl b-D-glucopyranoside, is another derivative ⁶. When alpha-arbutin and its optical isomer, 4-hydroxyphenyl b-D-glucopyranoside, were tested for their ability to block tyrosinase, alpha-arbutin had a greater inhibitory action than beta-arbutin ⁸.Alpha-arbutin is used cosmetically, but it also has medicinal uses, including the treatment of urinary tract infections, anticancer activity, and antioxidant and anti-inflammatory qualities ⁶. Bearberry extract naturally contains arbutin, but it can also be produced by glucosidation from hydroquinone ⁷.Alpha arbutin is hydrophilic and at 20 ± 5 °C, it has a water solubility of 151 g/L. The Scientific Committee on Consumer Safety (SCCS) recommends using up to 2% w/w and 0.5% w/w of alpha arbutin in face creams and body lotions, respectively ⁹. Arbutin’s high hydrophilicity (log P value of -1.49) and poor penetration through the stratum corneum restrict its use in topical preparations, despite the fact that it effectively inhibits tyrosinase to produce whitening effects. As a result, conventional skin formulations like lotions and creams fail to produce sufficient skin deposition ¹⁰.

These hurdles can be resolved by ethosomes, which effectively deliver drugs of all types (amphiphilic, lipophilic, and hydrophilic) to the epidermis as well as deeper into the skin by enhancing drug permeation^11,12. Hence, in the present study, an attempt was made to develop an ethosomal suspension to encapsulate the drug in soy lecithin and incorporate it into a gel for easier application on the skin. Through this approach, drug penetration and retention in the deeper layers of the skin are improved.

Materials and Methods

Alpha arbutin was gifted by Barentz (Mumbai, India), Propylene glycol (PG) was obtained from Thomas Beaker (Mumbai, India), Soy Lecithin was purchased from HiMedia (Mumbai, India), Ethanol was obtained from SD Fine Chemicals (Mumbai, India) and Sepineo P 600 was gifted by Blue Cross Laboratories (Nashik, India). All chemicals used were pharmaceutical grade.

Preparation of ethosomal suspension

Alpha arbutin ethosomal suspensions were prepared using the cold method, as described by Touitou et al. ¹⁴. An ethanol solution with soy lecithin was made using a magnetic stirrer (REMI, Mumbai, India). PG was added to this ethanol-lipid mixture, referred to as the organic phase. Separately, a 2 % w/w drug solution was prepared in water, known as the aqueous phase. The system was maintained at 30 °C. The aqueous phase was added to the organic phase by means of syringe with continuous stirring at 700 rpm for 30 minutes ¹⁵. The mixture was then sonicated using probe sonicator (Athena Technology, Electro lab, India) for 25-30 minutes to reduce particle size, then homiginized and refrigerated overnight to obtain stable ethosomes ¹⁶. On next day, the mixture was centrifuged using cooling centrifugation at 10 ˚C, 8000 rpm for 10 minutes. Then, supernatant was collected as final ethosomal suspension. This suspension stored in refrigerator for further use. Various formulations with different lipid and ethanol concentrations were tested, as shown in Table 1. The optimal formulation was selected based on size analysis, zeta potential, polydispersity index (PDI), and % entrapment efficiency (% EE). The optimized ethosomal formulation was used to create a gel for further characterization.

Results

Vesicle size, PDI, and %EE:

Alpha arbutin ethosomal formulations exhibited entrapment efficiencies (EE) from 93.14 % to 94.99 %. The highest EE was achieved with F3, which had 20 % w/w ethanol and 4 % w/w soy lecithin (Table 2). Arbutin ethosomes ranged in size from 138.1 to 350.1 nm. The vesicle size was significantly influenced by the proportions of ethanol and soy lecithin. The smallest vesicles (138.1 ± 6.85 nm) were obtained with 20% w/w ethanol and 4 % w/w soy lecithin, while the largest vesicles were with 40 % w/w ethanol and 1 % w/w soy lecithin. Each formulation had a PDI ranging from 0. 383 to 0.498. Zeta potential, another key stability measure, reflects ethosome stability, with higher values indicating better stability. Ethanol imparts a negative charge to the vesicles, preventing aggregation. For batch F3, the zeta potential was -48.0 mV.

Table 2: % EE, vesicle size and PDI 

Formulation Batches	% EE	Vesicle size (nm)	PDI
F1	93.14±0.03	289.4±10.65	0.498
F2	94.06±0.01	292.6±9.13	0.432
F3	94.99±1.52	138.1±6.85	0.406
F4	94.39±0.03	323.8±7.27	0.443
F5	94.56±0.03	138.6±8.12	0.476
F6	94.22±0.72	199.0±7.52	0.383
F7	94.36±0.05	350.1±12.63	0.406
F8	94.12±0.03	264±10.34	0.393
F9	93.58±0.84	240.2±9.52	0.420

In vitro drug diffusion of ethosomal suspension

The % CDR for each batch was evaluated up to 90 minutes, with batch F3 showing the highest % CDR at 106.63±4.23 % (Figure 1).

Figure 1: % CDR of ethosomal suspension at the end of 90 m. 

FTIR analysis of drug and excipients:

The FTIR spectra of alpha arbutin, soy lecithin, and their mixture were analyzed in the 400-4000 cm^-1 range (Figure 2). Key peaks for alpha arbutin included 3400-3600 cm^-1 (OH), 1514.12 cm^-1 (phenyl ring), 2924.09 cm^-1 (C-H), and 1217.02 cm^-1 (C-O). For soy lecithin, significant peaks were observed at 3529.15 cm^-1 (OH), 2827.72 cm^-1 (symmetric C-H), 1739.79 cm^-1 (ester C=O stretch), and 721.38 cm^-1 (C=C bending). In the mixture, major peaks included 3529.75 cm^-1 (OH), 2924.09 cm^-1 (C-H), 1739.79 cm^-1 (C=O), 1514.12 cm^-1 (phenyl ring), and 721.38 cm^-1 (C=C).

Figure 2: IR spectra of A. Alpha arbutin B. Soy lecithin and C. Alpha arbutin + Soy lecithin 

FTIR of optimized ethosomal suspension:

FTIR spectrum of optimized formulation is shown in Figure 3. Key features include an -OH stretch at 3338.86 cm^-1, a C-H stretch at 2979 cm^-1, and an aromatic ring peak at 1508 cm^-1. Sharp peaks for P-O₂ and P-O-C shifted from 1062 cm^-1 to 1041 cm^-1.

Figure 3: IR spectra of optimized batch. 

Comparative study by in vitro diffusion with marketed formulation:

In vitro drug diffusion study between prepared ethosomal gel and marketed product shown significant difference at the end of 8 h (Figure 4)

Figure 4: %CDR of ethosomal gel and marketed cream at 8 h. 

Discussion

Increasing soy lecithin concentration from 1 % to 4 % w/w initially increased EE, but further increases in lecithin resulted in decreased EE likely due to reduced vesicle deformability. Vesicle size increased with higher ethanol concentrations up to 40 % w/w but decreased with higher soy lecithin concentration. A low PDI value implies the formation of bit polydisperse particles. The zeta potential value indicates that the prepared formulation has sufficient stability. The F3 formulation was selected as the optimized formulation based on the characterization results. FTIR data demonstrate that the drug and selected excipients are compatible. The pH of the formulated gel was comparable to that of human skin. The sufficiently large spreadability value suggests that the prepared gel can be easily applied to the skin and will reach to greater surface of skin. The in vitro study showed drug release from the prepared ethosomal gel was slower compared to the marketed product. The dermatokinetic study demonstrated that ethanol enhances the permeation of the drug, and the small particle size of ethosomes further facilitates the drug’s penetration and better retention into the epidermis and dermis layers over marketed formulation. In vitro drug release from the prepared ethosomal gel was comparatively slow over marketed product. The formulation was found to be physically stable, and the in vitro drug release showed no significant difference after 3 months.

Conclusion

In present study, ethosomes was successfully prepared and incorporated into a gel using Sepineo P 600. Batch F3 exhibited the smallest vesicle size, acceptable PDI, and zeta potential value for stability; therefore, it is considered the optimized batch. The release of alpha arbutin from prepared ethosomal gel was a bit slow when compared to marketed product. The dermatokinetic study demonstrates better retention of the drug, with the MRT of the ethosomal gel increasing 3.45 and 2.87 folds over the marketed product in the epidermis and dermis layers, respectively. This study demonstrates that ethosomes have the potential to serve as an effective vehicle for topical delivery of alpha arbutin. In the current research, an alpha-arbutin-loaded ethosomal gel was successfully developed and evaluated.