Polymer-Concentration–Dependent Diffusion and In-Vitro Biological Evaluation of a Rutin-Loaded Hydrogel for Periorbital Application

Periorbital hyperpigmentation (POH), also known as "dark circles," is a common cosmetic dermatological diagnosis that includes discoloration below the eyes caused by a number of factors, such as too much melanin buildup, vascular congestion, thinning of the skin, and post-inflammatory hyperpigmentation [1–3].  The dermis in the periorbital area is much thinner (about 0.5 mm) than in other parts of the face. This makes the blood vessels and hemosiderin deposits below more visible [1]. When capillaries become more porous and microcirculation slows down, vascular pooling happens. This changes the skin colour to blue or brown [2]. POH is mostly caused by oxidative stress. Reactive oxygen species (ROS) turn on tyrosinase, which is the enzyme that controls how fast melanin is made. This speeds up the production of melanin [4]. Tyrosinase turns L-tyrosine into L-DOPA, which then turns into dopaquinone, which makes melanin. There is a direct connection between hyperpigmentary diseases and higher levels of tyrosinase activity [5]. Because of this, things that can both stop tyrosinase and act as antioxidants are thought to be good options for treating POH. Changes in pigmentation can make the area around the eyes swell. Localized swelling, poor lymphatic drainage, and the weakening of the orbital septum are all to blame for this. These things let fluid build up and infraorbital fat pads stick out [3]. The blood vessels are weak and the capillaries are more open, which makes this problem worse. Because of this, medicines that protect blood vessels and lower inflammation may have two health benefits. Rutin, also known as quercetin-3-O-rutinoside, is a type of flavonoid glycoside that can be found in a lot of plants. Many sources have said that it has strong antioxidant, anti-inflammatory, and capillary-stabilizing effects [6]. Rutin is an antioxidant because it stops lipid peroxidation, binds to metal ions, and gets rid of superoxide radicals [6,7]. It also strengthens and makes blood vessels less permeable, which helps microcirculation work better [6]. A lot of studies have shown that flavonoids like rutin and its aglycone quercetin can stop tyrosinase activity and the production of melanin [5,8]. These qualities make rutin a great choice for treating POH and the swelling that comes with it. Rutin is very important for medicine, but it doesn't dissolve well in water (less than 0.1 mg/mL) and doesn't easily pass through the skin because it has a high molecular weight and loves water [6]. Because of these physicochemical limits, it is harder for the body to absorb when used in regular topical products like creams and lotions.

Carbopol 940, is a cross linking polyacrylic acid polymer that helps make hydrogel systems which exhibits, increased permeation of drug through the skin [9]. When you neutralize Carbopol, it pushes away other molecules with electricity and makes its chains longer. This makes it thicker and gives it a gel-like shape. Fickian principles say that drug diffusion through polymeric matrices is affected by the amount of polymer, the density of crosslinks, and the porosity of the matrix [10]. Higuchi was the first to explain the idea of drug release from homogeneous matrices that is controlled by diffusion. He showed that the total amount of medicine released is directly related to the square root of the time under controlled diffusion conditions [11]. More research made it easier to understand how the Higuchi model works with hydrophilic polymer systems and the ideas behind it [10,12].  It's very important to find the right balance between long-lasting drug release, good spreadability, and cosmetic acceptability in skin care products that go around the eyes. Hydrogels are a good way to give out medicines that don't make things greasy, cold, or ugly. They might also help reduce swelling by making the skin cooler and more moist [9]. Ruthin has several medicinal applications, but little is known about how it functions when slowly released from a hydrogel matrix for ocular usage. In-vitro diffusion and tyrosinase inhibition anti-pigmentation tests demonstrate delivery and biological effect. The research question of this study was whether modulation of Carbopol 940 concentration could systematically control diffusion behavior of Rutin while maintaining anti-pigmentation activity in a hydrogel system. It was hypothesised that raising the concentration of Carbopol 940 would lower the diffusion rate of rutin in proportion to the increase in matrix density, while keeping its anti-melanogenic effect. The study was set up to explore this idea by systematically making the formulation and testing it in vitro.

2. MATERIALS AND METHODS

2.1 Materials

The active medicinal ingredient was rutin (quercetin-3-O-rutinoside, ≥98% purity). Gelling agent was carbopol 940. Propylene glycol co-solvent and penetration enhancer. Glycerin humectant. Tween 80 was used as a surfactant to improve medication solubility. Preservatives included phenoxyethanol and ethylhexylglycerin. Triethanolamine neutralized and adjusted pH. Potassium dihydrogen and disodium hydrogen phosphate made pH 7.4 phosphate buffer. In-vitro tyrosinase inhibition research employed mushroom tyrosinase and L-DOPA. Studies on in-vitro diffusion employed a 0.45 µm pore size cellulose acetate membrane. Double-distilled water was used throughout the study.

All chemicals and reagents were analytical and used as supplied.

2.2 Formulation Composition (15 g Batch)

2.3 In-Vitro Drug Release

In-vitro Rutin drug release from hydrogels was assessed using a Franz diffusion cell equipment, a classic semisolid topical formulation test [13]. A cellulose acetate membrane (0.45 µm pore size) was placed between the donor and receptor compartments (diffusion area = 2.54 cm²). To mimic physiological circumstances, the receptor compartment was filled with phosphate buffer (pH 7.4) and kept at 37 ± 0.5°C with continuous stirring (50 rpm). A precisely weighed 5 mg Rutin gel was inserted in the donor compartment. Aliquots were removed from the receptor medium and replaced with buffer at 1, 2, 4, 6, 8, and 12 h to maintain sink conditions. Samples were evaluated at 359 nm spectrophotometrically.

The cumulative percentage of drug release was computed, and data were shown as mean ± SD in triplicate trials (n = 3). Zero-order, first-order, Higuchi, and Korsmeyer–Peppas models evaluated release kinetics. The release mechanism was best described by the model with the greatest correlation coefficient (R²) [11, 12].

2.4 Tyrosinase Inhibition Assay (Anti-Pigmentation Study)

The optimised Rutin hydrogel was tested for anti-pigmentation using a mushroom tyrosinase inhibition assay, a classic melanogenesis inhibitor screening technique [4, 5].

In phosphate buffer (pH 6.8), mushroom tyrosinase enzyme solution was produced. In this case, L-DOPA was the substrate. Samples of gel were diluted to 25, 50, 75, and 100 µg/mL. For 20 minutes, enzyme, substrate, and sample solution were incubated at 37°C. The 475 nm spectrophotometric measurement of dopachrome production.

Equation for percentage inhibition:

% Inhibition = (Ac - As) x 100

Where, Ac is the control absorbance and

As is the sample absorbance

Results were given as mean ± SD for all studies completed in triplicate (n = 3).

Tyrosinase catalyses the rate-limiting step in melanogenesis, hence inhibiting it suppresses melanin production [4, 6]. Rutin and similar flavonoids reduce tyrosinase activity due to competitive and antioxidant processes [6].

3. RESULT

3.1 Physicochemical Evaluation (Optimized F4)

3.2 In-Vitro Drug Release

Figure 1 Cumulative % Drug release v/s Time Plots of formulation F1-F6

Figure 2 Comparative In-Vitro drug release

Cumulative release at 12 h:

Increasing polymer concentration significantly reduced drug release (p < 0.01).

3.3 Higuchi Kinetics

R² (Higuchi) = 0.991 → diffusion-controlled release [5].

Figure 3 Zero order and First order Kinetics for F4 formulation

Figure 4 Higuchi Diffusion Plot for F4 formulation

3.4 Tyrosinase Inhibition

Figure 5 In-Vitro Tyrosinase Inhibition for F4 formulation