بررسی پایداری درون‌پوشانی و خواص آنتی‌اکسیدانی لوتئین در هیدروژل یوتا-کاراگینان-اینولین

Introduction
Lutein is a lipophilic carotenoid belonging to the xanthophyll family, abundantly found in dark green leafy vegetables (e.g., spinach, kale), egg yolk, and certain fruits. Due to its polyene structure with conjugated double bonds, lutein exhibits potent antioxidant activity and plays a critical physiological role in protecting the macula lutea of the retina from blue light-induced oxidative damage. Epidemiological and clinical studies have consistently demonstrated that adequate dietary intake of lutein reduces the risk of age-related macular degeneration (AMD) and cataracts, two leading causes of blindness worldwide. Beyond ocular health, lutein also contributes to systemic antioxidant defense, skin photoprotection, and cognitive health. However, the commercial application of lutein in functional foods, pharmaceuticals, and cosmetics is severely hampered by its high sensitivity to light, heat, oxygen, and acidic pH, as well as its poor water solubility and low bioavailability. Conventional surfactant-based delivery systems (e.g., Tween 80 micelles) fail to provide adequate physical barriers against pro-oxidant agents, leading to rapid degradation and loss of bioactivity. Therefore, there is an urgent need to develop advanced encapsulation systems that can protect lutein, enhance its stability, and enable controlled release. In this context, polysaccharide-based hydrogels have emerged as promising carriers due to their biocompatibility, biodegradability, and ability to form three-dimensional hydrophilic networks capable of entrapping lipophilic bioactives after appropriate stabilization.

Iota-carrageenan (ι-carrageenan) is a sulfated linear polysaccharide extracted from red seaweeds (Rhodophyta). In the presence of divalent cations such as Ca²⁺, ι-carrageenan undergoes a conformational transition from random coils to helical structures, followed by aggregation into a three-dimensional elastic, transparent, and thermally reversible gel network. Despite its excellent gelling properties, pure ι-carrageenan hydrogels suffer from limited mechanical strength, high syneresis, and insufficient barrier properties against oxygen diffusion—limitations that restrict their use in long-term protection of highly oxidizable compounds. Inulin, a natural fructan-type soluble dietary fiber derived from chicory roots, Jerusalem artichoke, garlic, and onion, offers a complementary functionality. In addition to its well-established prebiotic effects (stimulating beneficial gut microbiota such as Bifidobacteria and Lactobacilli), inulin increases total solid content, promotes the formation of microcrystalline domains upon cooling, and enhances gel network density. When combined with ι-carrageenan, inulin is expected to produce a hybrid hydrogel with improved compactness, reduced oxygen permeability, and enhanced encapsulation efficiency for lutein. Despite extensive research on carrageenan or inulin individually, no systematic study has yet investigated the ι-carrageenan-inulin hybrid hydrogel as a carrier for lutein. Therefore, the present study aimed to: (1) fabricate ι-carrageenan-inulin hybrid hydrogels containing three different inulin concentrations (15, 20, and 25% w/w); (2) characterize the structural, morphological, and physicochemical properties of the hydrogels using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM); (3) evaluate the encapsulation efficiency (EE%) and storage stability (30 days) of lutein in the hydrogel systems compared to a non-encapsulated control (lutein in Tween 80); and (4) assess the concentration-dependent antioxidant activity of lutein using the DPPH radical scavenging assay and determine the IC50 value.

Materials and Methods:
Iota-carrageenan (Sigma-Aldrich, Germany), inulin (≥90% purity from chicory, Sigma-Aldrich, USA), lutein (≥90%, Extrasynthese, France), Tween 80, n-hexane, absolute ethanol, calcium chloride (CaCl₂), and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Merck (Germany). Hydrogels were prepared by dissolving 3% (w/w) ι-carrageenan in deionized water at 70°C under magnetic stirring (500 rpm, 30 min). Inulin was added at 15%, 20%, or 25% (w/w) and stirring continued until complete dissolution and transparency. The solution was cooled to 50°C, and CaCl₂ was added to a final concentration of 20 mM to induce gelation. For lutein loading, 0.1 g lutein was dispersed in 4 g Tween 80 (30 min stirring at 25°C), then incorporated into the polymer solution prior to CaCl₂ addition. The mixtures were stored at 4°C for 24 h to complete gelation. A control sample (lutein in Tween 80 without hydrogel) was also prepared. For EE% determination, 1 g of hydrogel was mixed with 9 mL n-hexane, vortexed (2 min), centrifuged (4000 rpm, 10 min, 4°C), and the free lutein in the supernatant was quantified spectrophotometrically at 441 nm. Stability was evaluated over 30 days at 25°C under ambient light by measuring residual lutein at 5-day intervals. XRD patterns were recorded using a Siemens D500 diffractometer (Cu-Kα radiation, λ=0.154 nm, 40 kV, 30 mA, 2θ=5-70°, step size 0.05°, scan rate 1°/min). FTIR spectra were obtained on a Bruker Tensor 27 spectrometer (KBr pellets, 400-4000 cm⁻¹, resolution 4 cm⁻¹). SEM micrographs were taken on a TESCAN MIRA3 FEG-SEM after gold sputtering. DPPH assay was performed by mixing different lutein concentrations (10–50 mg/mL in ethanol) with 0.1 mM DPPH solution (1:3 v/v), incubating in darkness for 30 min, and measuring absorbance at 517 nm. Percentage inhibition was calculated, and IC50 was determined by linear regression. All experiments were performed in triplicate. Statistical analysis was conducted using SPSS version 26 (one-way ANOVA followed by Tukey’s post hoc test, significance level p < 0.05).
Results and Discussion:
Encapsulation Efficiency (EE%): Increasing inulin concentration from 15% to 25% significantly improved EE%, with values of 71.2 ± 1.2%, 76.5 ± 1.5%, and 82.1 ± 1.2% for 15%, 20%, and 25% inulin, respectively (p < 0.05). This enhancement is attributed to increased viscosity of the continuous phase, greater polymer chain entanglement, and formation of a denser gel network with reduced mesh size at higher inulin levels, which physically entraps lutein more effectively and limits its diffusion out of the matrix.
XRD Analysis: The XRD pattern of the control hydrogel (without lutein, H) exhibited a broad halo between 2θ = 18–25°, characteristic of predominantly amorphous polysaccharide matrices with limited semi-crystalline domains originating from local chain ordering through hydrogen bonding. After lutein incorporation (H+L sample), the diffraction intensity decreased significantly and the peak broadened, indicating a reduction in crystallinity and an increase in the amorphous fraction. This behavior suggests that lutein molecules intercalate between carrageenan and inulin chains, disrupting their regular packing and hydrogen-bonding network. Importantly, no sharp peaks corresponding to crystalline lutein appeared in the H+L pattern, confirming that lutein was molecularly dispersed or present in an amorphous state within the hydrogel matrix—a desirable feature for controlled release applications.
FTIR Analysis: FTIR spectra revealed a broad band at 3200–3400 cm⁻¹ assigned to O–H stretching vibrations of hydrogen-bonded hydroxyl groups. The intensity of this band was higher in the H sample compared to pure carrageenan or inulin, indicating strong inter-polysaccharide hydrogen bond formation. In the H+L sample, the intensity decreased, suggesting partial disruption of the hydrogen-bonded network due to lutein-polysaccharide hydrophobic interactions and/or steric effects. Characteristic bands for sulfate groups (S=O asymmetric stretching at 1220–1260 cm⁻¹), C–O–C stretching (1030 cm⁻¹), and carboxylate (–COO⁻) symmetric/asymmetric stretching (1410 and 1600 cm⁻¹) were retained but exhibited reduced intensities in H+L, implying that lutein interacts with polar groups of the polysaccharide matrix. The absence of new peaks further supports the molecular dispersion of lutein without crystalline phase separation.
SEM Morphology: SEM micrographs showed that the lutein-free hydrogel (H) possessed a compact, smooth, and relatively uniform structure with finely dispersed small pores, indicative of a stable and well-organized gel network. In contrast, the lutein-loaded hydrogel (H+L) displayed a significantly rougher, more heterogeneous, and highly porous microstructure with larger voids and thinner pore walls. This morphological transformation is attributed to the disorganizing effect of lutein on chain packing and the reduction of inter-chain interactions, leading to a more open and permeable network—a structure that can facilitate diffusion-controlled release of the encapsulated bioactive.
Storage Stability: In the control sample (lutein/Tween 80), approximately 50% of lutein degraded after 30 days, confirming the inability of conventional micelles to provide an effective barrier against oxygen and light. In contrast, all hydrogel formulations significantly retarded lutein degradation. After 30 days, residual lutein was 65% for the 15% inulin hydrogel, 72% for the 20% inulin hydrogel, and 78% for the 25% inulin hydrogel. The superior protection offered by the 25% inulin formulation is attributed to the highest network density, smallest pore size, lowest oxygen diffusivity, and formation of glassy or microcrystalline domains at high solid content, which collectively restrict molecular mobility and reduce oxidative attack. These results demonstrate that increasing inulin concentration directly and proportionally enhances the protective capacity of the hydrogel.

Antioxidant Activity (DPPH Assay): Lutein exhibited a clear concentration-dependent DPPH radical scavenging activity. As lutein concentration increased from 10 to 50 mg/mL, the percentage inhibition rose from approximately 17% to 69%. The calculated IC50 value was 32.8 mg/mL, indicating moderate-to-strong antioxidant potency. This activity originates from the conjugated polyene backbone of lutein, which enables electron or hydrogen donation to stabilize DPPH radicals, and from the terminal hydroxyl groups that further stabilize the resulting lutein radical. The obtained IC50 falls within the range previously reported for natural lutein from various botanical sources.

Conclusion:
This study successfully demonstrated that the ι-carrageenan-inulin hybrid hydrogel, particularly at 25% inulin concentration, serves as an effective encapsulation system for lutein, offering significantly enhanced encapsulation efficiency and long-term chemical stability compared to conventional surfactant-based delivery systems. Structural analyses (XRD, FTIR, SEM) confirmed that lutein was molecularly dispersed within the predominantly amorphous, porous, and hydrogen-bonded polysaccharide network without forming crystalline aggregates. Increasing inulin concentration from 15% to 25% systematically improved both EE% (from 71% to 82%) and residual lutein after 30-day storage (from 65% to 78%), owing to enhanced network compactness, reduced oxygen permeability, and restricted molecular mobility. Lutein retained its intrinsic concentration-dependent antioxidant activity (IC50 = 32.8 mg/mL) after encapsulation. These findings establish the ι-carrageenan-inulin (25% inulin) hydrogel as a scalable, biocompatible, and biodegradable carrier with strong potential for incorporation into functional food matrices (e.g., fortified beverages, dairy desserts), pharmaceutical ophthalmic formulations, and cosmetic anti-aging/sunscreen products. Future studies should investigate the rheological behavior, digestibility, and in vivo bioavailability of lutein-loaded hydrogels under simulated gastrointestinal conditions, as well as the potential synergistic effects with other lipophilic bioactive compounds.