Covalent Gelatin‐Cyclodextrin Nanocarrier Systems for Pistacia atlantica Essential Oil: Physicochemical Characterization, Antioxidant Activity, and Molecular Docking Studies

ABSTRACT

Essential oil from Pistacia atlantica gum is rich in bioactive terpenes but limited by volatility and poor stability. Here, a chemically integrated nanogel system was developed by covalent conjugation of carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) to gelatin (CNG), followed by supramolecular stabilization with Tween 20 (CNGT) for essential oil encapsulation (CNGTO) using O/W emulsification and freeze‐drying. Two formulations with different loadings were prepared: CNGTO(1) (46.5%) and CNGTO(2) (56.5%). Dynamic light scattering revealed mean particle sizes ranging from 176.3 nm (CNG) to 693 nm (CNGTO(2)), depending on formulation and oil content. Encapsulation efficiencies reached 77.5% ± 1.4% and 81.4% ± 1.7% for CNGTO(1) and CNGTO(2), respectively. Encapsulation reduced the release rate compared to free oil, showing a biphasic release profile over 96 h. Although total antioxidant capacity of encapsulated samples (128.4–145.2 µg ascorbic acid equivalents g⁻¹) was slightly lower than free oil (166.8 µg g⁻¹), DPPH radical scavenging activity was enhanced. Total phenolic content reflected contributions from both oil and matrix. Four‐week storage studies demonstrated improved physicochemical and antioxidant stability. Molecular docking analysis using α‐pinene (33.65%, GC‐MS) provided mechanistic insight into terpene–cyclodextrin interactions within the hybrid matrix and the structural role of Tween 20 in modulating inclusion behavior.

1. Introduction

The pharmaceutical industry is undergoing a transformation, with increasing recognition of the therapeutic potential of natural products, particularly vegetable oils derived from diverse plant sources. Among these, Pistacia atlantica, a member of the Anacardiaceae family native to wide regions of Iran, is notable for its rich oil content and long‐standing role in traditional medicine. This fast‐growing species has traditionally been used to treat a variety of ailments, including joint pain, gastrointestinal disorders, and inflammatory conditions. The oleoresin of P. atlantica is widely utilized in both food and medicinal applications, underscoring its versatility and significance [1, 2]. Despite the numerous health benefits attributed to its oleoresin, several limitations hinder its broader application, including poor water solubility, undesirable taste, and the instability of bioactive compounds in aqueous systems [3]. Nanoencapsulation represents a promising strategy to overcome these limitations by enhancing the stability of active constituents and enabling controlled release. This approach can improve the bioavailability of lipophilic compounds, such as phenolics and terpenes, while addressing solubility constraints that restrict their use in pharmaceuticals and food formulations [4, 5].

Among various encapsulation techniques, emulsification is a widely adopted physicochemical method for entrapping lipophilic compounds within oil‐in‐water (O/W) systems. These emulsions can subsequently be converted into stable powders through drying techniques such as freeze‐drying, thereby broadening their applicability [6]. The selection of coating material plays a critical role in encapsulation efficiency and stability [7].

Gelatin, a protein obtained by controlled hydrolysis of collagen, is particularly advantageous due to its amphoteric nature, which facilitates emulsification and enhances the stabilization of sensitive compounds [8, 9]. Moreover, conjugation of gelatin with cyclodextrins (cyclic oligosaccharides capable of forming host–guest inclusion complexes) provides a synergistic approach for encapsulating volatile essential oils and improving their solubility and stability [10, 11, 12]. Nevertheless, emulsions stabilized solely by gelatin and β‐cyclodextrin may exhibit limited colloidal stability, including coalescence and flocculation, which can compromise functionality [13]. Incorporation of nonionic surfactants such as Tween 20 has been reported to improve emulsion stability and facilitate encapsulation of hydrophobic compounds by enhancing interfacial organization [14].

Despite the reported use of gelatin–cyclodextrin systems for essential oil encapsulation [12, 14], the molecular‐level interactions governing terpene inclusion and stabilization within such hybrid matrices remain insufficiently elucidated. In this context, the present study describes the preparation and characterization of type A gelatin covalently modified with β‐cyclodextrin (CNG) and further stabilized with Tween 20 (CNGT) as a nanostructured encapsulation matrix for P. atlantica essential oil (CNGTO). The physicochemical properties, antioxidant activity, and release behavior of the formulations were systematically evaluated. By integrating experimental characterization with molecular docking analysis, this work provides structural insight into terpene–cyclodextrin interactions within a chemically integrated biopolymeric network. These findings contribute to a deeper understanding of cyclodextrin‐functionalized gelatin systems for stabilizing terpen‐rich natural products.

2. Experimental Section

2.1. Materials

The oleoresin gum of P. atlantica Desf. (Anacardiaceae), locally known as “Saghez,” was commercially obtained from a traditional herbal supplier in Iran. The resin material originates from wild‐growing trees in mountainous region of Noudan, Fars Province, Iran (29°48'05' N, 51°4'‘39'' E). The botanical identity of the species was authenticated by Iranian Biological Resource Center (IBRC), National Institute of Genetic and Biological Resources of Iran. A voucher specimen is deposited in the IBRC Herbarium under the code IBRC P1011670. The purchased resin was verified by comparison with the authenticated voucher specimen to ensure taxonomic consistency. The resin essential oil was extracted using Soxhlet extraction with cyclohexane as solvent and concentrated by rotary evaporation. Detailed extraction parameters are provided in the Supporting Information (SI). All chemicals were of analytical grade and purchased from Sigma‐Aldrich (USA) or Merck (Germany).

2.2. Methods

2.2.1. Oleoresin Oil Extraction

The essential oil was extracted from the oleoresin gum by Soxhlet extraction, concentrated under reduced pressure using a rotary evaporator, and stored in dark glass bottle at 4°C until further use. Detailed experimental conditions are described in the SI.

2.2.2. GC‐MS Analysis

The chemical composition of the essential oil was analyzed by gas chromatography‐mass spectrometry (GC‐MS) using an Agilent system equipped with HP‐5MS capillary column. Compounds were identified by comparison with reference standards and spectral databases. Detailed analytical conditions are provided in the Supporting Information.

2.2.3. Preparation of Gelatin Modified With β‐Cyclodextrin (CNG)

Gelatin was chemically modified with carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) via carbodiimide‐mediated coupling to obtain a nanostructured hydrogel (CNG) [15, 16, 17]. Detailed reaction conditions, purification steps and characterization procedures are provided in the SI (Scheme 1A).

SCHEME 1.

(A) Schematic illustration of the synthesis of carboxymethyl‐β‐cyclodextrin (CM‐β‐CD) and the preparation of the β‐cyclodextrin‐containing gelatin hydrogel (CNG). (B) Schematic representation of essential oil (EO) encapsulation within the nanogel matrix.

2.2.4. Encapsulation of the Essential Oil (EO)

Encapsulation was carried out using the O/W emulsification method adapted from Medeiros et al. [18]. Gelatin modified with β‐cyclodextrin and stabilized with Tween 20 (CNGT) was used as the carrier matrix (Scheme 1B). The resulting emulsions (CNGTO(1) and CNGTO(2)) were subjected to ultrasonication and subsequently freeze‐dried for storage. Detailed formulation compositions and processing parameters (Table S1) are provided in the Supporting Information.

2.2.5. Characterization of the Prepared Samples

The structural and physicochemical properties of the prepared samples were analyzed using scanning electron microscopy (SEM), dynamic light scattering (DLS), zeta potential measurements, thermogravimetric analysis (TGA), UV–vis spectroscopy, and attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy.

2.2.6. Antioxidant Assays

The antioxidant activity of encapsulated and free EO was evaluated using the DPPH radical scavenging assay and phosphomolybdenum method. Radical scavenging activity was calculated as a percentage reduction in DPPH absorbance at 517 nm (Equation S1), while total antioxidant capacity was expressed as microgram ascorbic acid equivalents per gram of dry sample [19, 20, 21]. Detailed procedures are described in the Supporting Information.

2.2.7. Determination of Total Phenolic Content

The total phenolic content of encapsulated and free EO was determined using the Folin–Ciocalteu method, with gallic acid as the calibration standard [22]. Detailed procedures are provided in the Supporting Information.

2.2.8. Docking Studies

Molecular docking simulations were performed using the Molecular Operating Environment (MOE) software to investigate the interaction between α‐pinene, the major constituent of the EO, and the CNGT nanogel system. The molecular structures of gelatin [23], CM‐β‐CD, and Tween 20 were constructed and energy‐minimized using the MMFF94x force field (gradient threshold 0.01 kcal mol⁻¹ Å⁻¹). Their conjugation was simulated prior to docking to generate the integrated carrier framework. Because the polymeric carrier does not possess a predefined‐binding pocket, a blind‐docking approach was employed. In this approach, the entire receptor structure was defined as the search space to allow unbiased exploration of potential interaction regions. The docking grid was automatically generated to encompass the molecular assembly with sufficient spatial extension to ensure complete ligand sampling. Ligand placement was carried out using the Triangle Matcher algorithm. Initial scoring of generated poses was carried out using the London dG scoring function to estimate binding free energy based on enthalpic contributions. The top‐ranked poses were subsequently refined using force field‐based energy minimization, and final rescoring was conducted with the GBVI/WSA dG scoring function. This implicit solvation model estimates binding free energy by accounting for van der Waals interactions, electrostatic contributions, and solvation effects. Docking scores (kcal mol⁻¹) were used as relative indicators of binding affinity and interaction stability. To validate the docking protocol, simulations were repeated under identical parameters, and consistency of the top‐ranked binding poses was evaluated. The resulting conformations exhibited similar spatial orientations with low root‐mean‐square deviation (RMSD < 2.0 Å) between repeated runs, confirming reproducibility of ligand placement. Additionally, convergence of docking scores across independent simulations indicated that the predicted interactions correspond to energetically favorable regions within the CNGT matrix rather than random placements. All detailed computational parameters, including force field settings, and docking protocol specifications, are provided in the Supporting Information.

3. Results and Discussions

3.1. Essential Oil Content of Pistacia atlantica Gum

The essential oil of P. atlantica gum was extracted using Soxhlet extraction, yielding a viscous, light‐yellow liquid. The extraction yield exceeded 70% of the dry weight of the gum (Equation 1).

$$\mathrm{Essential}\mathrm{oil}(\%)=\frac{\mathrm{Extracted}\mathrm{oil}(\mathrm{g})}{\mathrm{Total}\mathrm{gum}\mathrm{used}\mathrm{in}\mathrm{the}\mathrm{process}(\mathrm{g})}\times 100$$ (1)

3.2. Chemical Composition of the Gum EO

The chemical constituents of P. atlantica gum essential oil were identified based on their elution order in the GC‐MS chromatogram (Figure S1). A total of 22 volatile compounds were detected, accounting for 50.90% of the total composition (Table S2). The predominant component was α‐pinene (33.65%), a concentration relatively higher than that reported for other plant parts, such as leaves and fruits [24, 25]. This high monoterpene content highlights the hydrophobic nature of the oil and justifies the need for an efficient encapsulation strategy.

3.3. FT‐IR Spectrophotometry

The FT‐IR spectra of nano‐gelatin, β‐cyclodextrin (β‐CD), and CNG are presented in Figure 1A. The results confirm successful conjugation of β‐CD to nano‐gelatin during the modification process. In the gelatin spectrum, the broad band at 3300 cm⁻¹ corresponds to N–H stretching vibrations associated with hydrogen bonding. The peaks at 1630, 1530 and 1243 cm⁻¹ are attributed to the amide I band (C═O stretching), amide II (N─H bending and C─N stretching), and amide III (N─H and C─N in‐plane stretching) bands, respectively. Bands in the range of 1388–1446 cm⁻¹ are assigned to C─O bending, C─N stretching, and C─H bending vibrations [26, 27, 28]. In the β‐CD spectrum, the bands at 3300 and 2925 cm⁻¹ correspond to O‐H and C─H stretching vibrations, respectively. The peaks at 1641, 1151, 1018 and 941 cm⁻¹ are attributed to H─O─H bending and C─O/C─O─C stretching vibrations of glucose units, and β‐CD ring structure [29, 30]. For CNG, in addition to the characteristic gelatin peaks, new absorption bands appeared at 1159, 1078 and 1027 cm⁻¹, corresponding to β‐CD‐related vibrations, confirming successful conjugation (Figure 1A). Upon incorporation of Tween 20 (CNGT, Figure 1B), the O─H stretching band shifted from 3475 cm⁻¹ to 3456 cm⁻¹ with reduced intensity indicating hydrogen‐bond interactions. The amide II band remained at 1530 cm⁻¹, while increased intensity at 1637 and 1251 cm⁻¹ decreased intensity at 1092 cm⁻¹ further suggest intermolecular interactions between Tween 20 and CNG [31]. The FT‐IR spectrum of the extracted EO (Figure 1B), exhibited characteristic absorption peaks at 3293, 2973, 1735, 1643, 1373, and 1063 cm⁻¹, corresponding to ─OH, C─H, C═O, C═C, ─CH₃, and C─O functional groups, respectively. No significant shifts or new peaks were observed in the CNGTO spectrum compared to CNGT, suggesting that encapsulation occurred predominantly through non‐covalent interactions without chemical modification of the EO [32].

FIGURE 1.

FT‐IR spectra of (A) nano‐gelatin, β‐cyclodextrin (β‐CD), and CNG, (B) Tween 20, CNGT, CNGTO, and EO.

3.4. Scanning Electron Microscopy

SEM analysis was performed to examine the surface morphology, physical size, and homogeneity of CNG, CNGT, and CNGTO forms. At 140.0 K× magnification (Figure 2A), CNG exhibited predominantly spherical and oval‐shaped nanoparticles that tended to form small aggregates (<100 nm). At lower magnification (40.0 K×, Figure 2B), particles appeared as stacked layers forming relatively compact surfaces. In the presence of Tween 20, CNGT displayed a smoother and more uniform surface without visible cracks (Figure 2C). The β‐CD moieties conjugated to gelatin appeared as raised domains on the nanogel surface, suggesting structural heterogeneity induced by functionalization. The SEM image of CNGTO (Figure 2D) revealed embedded and partially oil droplets distributed within the CNGT matrix. Higher density regions were observed around β‐CD bonding sites, indicating preferential localization of hydrophobic components. Considering the hydrophobic nature of essential oils, the presence of tween 20 likely facilitated more uniform distribution of oil droplets within the β‐CD‐functionalized nanogel network, improving structural homogeneity.

FIGURE 2.

SEM images of (A) CNG at 140.0 K× magnification (B) CNG at 40.0 K× magnification (C) CNGT and (D) CNGTO.

3.5. Particle Size Distribution and Zeta Potential

Particle size and zeta potential are key physicochemical parameters governing the applicability and stability of colloidal delivery systems in food and pharmaceutical formulations [33]. Figure 3 presents the DLS profiles obtained during the synthesis of the coating matrix and subsequent encapsulation process. CNG nanoparticles (Figure 3A) exhibited a mean hydrodynamic diameter of 176.3 nm, with 79.6% of particles ≤ 225.0 nm, indicating a relatively narrow size distribution. Upon incorporation of Tween 20 (CNGT, Figure 3B), the mean particle size increased to 284.0 nm, with 70.0% of particles ≤ 355.0 nm. This increase in hydrodynamic diameter can be attributed to surfactant adsorption at the nanogel interface, which forms a hydrated steric layer around the particles. Although the mean diameter increased, SEM analysis confirmed improved surface uniformity and reduced formation of large irregular aggregates, suggesting enhanced structural homogeneity. Encapsulation of the essential oil resulted in a marked shift toward larger particle sizes. The mean diameters of CNGTO(1) and CNGTO(2) were 436.6 and 693.0 nm, respectively (Figure 3C,D). The size is consistent with the incorporation of hydrophobic oil droplets within the nanogel network, leading to expansion of the hydrodynamic radius. As expected CNGTO(2), containing a higher EO loading, exhibited a larger mean diameter, indicating a direct correlation between oil content and particle size. Similar size enlargement trends have been reported in essential oil‐loaded biopolymeric nanocarriers [34].

FIGURE 3.

Particle size distribution and zeta potential of (A) CNG, (B) CNGT, (C) CNGTO (1), and (D) CNGTO (2).

Zeta potential measurements provide insight into surface charge and colloidal stability [35]. The mean zeta potentials of CNG, CNGT, CNGTO(1), and CNGTO(2) were −2.66, −2.79, −6.99, and −9.76 mV, respectively (Figure 3). All formulations exhibited slightly negative surface charges, primarily arising from residual ionizable functional groups within the conjugated gelatin–cyclodextrin network. As Tween 20 is a nonionic surfactant, the negligible difference between CNG and CNGT confirms that surfactant incorporation does not significantly alter electrostatic surface properties. Interestingly, encapsulation of the essential oil resulted in a progressive increase in negative zeta potential magnitude. This behavior may be attributed to conformational rearrangement of the polymeric matrix and increased exposure of ionizable groups at the particle–water interface upon oil loading.

From a colloidal stability standpoint, absolute zeta potential values between −10 and +10 mV generally indicate limited electrostatic stabilization. Therefore, despite the slightly increased negative charge in EO‐loaded formulations, the system remains within the low electrostatic repulsion regime. These findings suggest that dispersion stability is predominantly governed by steric stabilization provided by Tween 20 and β‐CD moieties rather than electrostatic repulsion.

3.6. Thermogravimetric Analysis

The thermal degradation behavior of CNGT, CNGTO(1), CNGTO(2), and CNGO was evaluated by TGA, and the corresponding thermograms are shown in Figure 4. The degradation temperatures (T _d) and weight losses (∆W) are summarized in Table 1. CNGT and EO‐loaded samples (CNGTO(1) and CNGTO(2)) exhibited three main stages of mass loss (Figure 4A–C). The first stage occurred between 49.5°C to 58.5°C (Td₁), with weight losses ranging from 3.3% to 5.4% (∆W ₁). This initial mass reduction is attributed to the evaporation of physically adsorbed moisture and volatile low‐molecular‐weight components. The slightly lower T _d1 values observed for EO‐loaded samples (49.5°C–52.7°C) compared to CNGT (57.3°C) may reflect the presence of volatile terpene fractions within the matrix. The second degradation stage occurred between 307.3°C and 329.5°C (T _d2), with ∆W ₂ values between 35.5% and 36.9%. This stage corresponds primarily to thermal decomposition of the gelatin backbone and partial degradation of β‐CD framework. Notably, the EO‐loaded samples exhibited the higher T _d2 value (320.7°C–329.5°C), suggesting that EO incorporation may contribute to delayed polymer degradation, possibly through hydrophobic interactions that restrict chain mobility. The third degradation stage, observed between 405.3°C and 413.5°C (T _d3), showed ∆W ₃ values of 67.0%–71.8%. This high‐temperature event is associated with decomposition of Tween 20 and carbonaceous residues formed during earlier degradation steps. The presence of this third stage exclusively in CNGT and CNGTO samples confirms the incorporation of Tween 20 into the nanogel matrix. Furthermore, the slightly higher T _d3 values in EO‐loaded samples (410.7°C–413.5°C) compared to CNGT (405.3°C) suggest enhanced thermal resistance in the presence of encapsulated oil, potentially due to increased matrix compactness and intermolecular interactions. To further elucidate the contribution of Tween 20, CNGO (without surfactant) was analyzed (Figure 4D). Unlike CNGT and CNGTO, CNGO displayed only two degradation stages, occurring at 58.5°C (∆W ₁ = 6.1%) and 316.5°C (∆W ₂ = 41.6%). The higher weight loss in the second stage compared to EO‐loaded samples indicates a less thermally stable polymer network in the absence of surfactant‐mediated stabilization. Importantly, the absence of a third high‐temperature degradation stage in CNGO confirms that this thermal event is directly related to Tween 20 incorporation. Overall, the comparative TGA data demonstrate that incorporation of Tween 20 and essential oil modifies the thermal degradation profile of the nanogel system. While the primary degradation temperature of the gelatin backbone remains within a similar range, EO loading and surfactant inclusion slightly shift high‐temperature decomposition toward higher Td values, indicating modest improvement in thermal stability.

FIGURE 4.

The thermograms of (A) CNGT, (B) CNGTO(1), (C) CNGTO(2), and (D) CNGO.

TABLE 1.

The degradation temperatures and weight loss of CNGT, CNGTO(1), CNGTO(2), and CNGO.

Sample	∆1		∆2		∆3
	T d1 (°C)	∆W 1 (%)	T d2 (°C)	∆W 2 (%)	T d3 (°C)	∆W 3 (%)
CNGT	57.3	4.4	307.3	35.5	405.3	67.0
CNGTO(1)	49.5	5.4	329.5	36.9	413.5	69.7
CNGTO(2)	52.7	3.3	320.7	35.5	410.7	71.8
CNGO	58.5	6.1	316.5	41.6	—	—

3.7. Determination of Encapsulation Efficiency

To quantify the amount of essential oil encapsulated in CNGTO(1) and CNGTO(2), the samples were first dispersed in distilled water and transferred to a separatory funnel. n‐Hexane was then added as the extraction solvent, and the organic phase was separated from the aqueous phase. After solvent evaporation under reduced pressure, the recovered essential oil was weighed. The encapsulation efficiency (EE%) was calculated according to Equation (2).

$$\mathrm{EE}\%=\frac{\mathrm{Extracted}\mathrm{essential}\mathrm{oil}(\mathrm{g})}{\mathrm{Total}\mathrm{essential}\mathrm{oil}\mathrm{used}\mathrm{in}\mathrm{the}\mathrm{process}(\mathrm{g})}\times 100$$ (2)

Encapsulation efficiencies, calculated from three independent experiments, were 77.5% ± 1.4% for CNGTO(1) and 81.4% ± 1.7% for CNGTO(2). These relatively high encapsulation efficiency values indicate that the CNGT nanogel matrix provides an efficient platform for entrapping the essential oil. The slightly higher efficiency observed for CNGTO(2) suggests improved loading capacity at increased oil content, possibly due to enhanced hydrophobic interactions within the gelatin–cyclodextrin network.

3.8. Investigation of EO Release From Encapsulated Samples

The release behavior of EO from the encapsulated samples was evaluated using a dialysis method. Briefly, 1.0 mL of the sample solution (0.001 g mL⁻¹) was placed in a dialysis bag and immersed in 50.0 mL of distilled water at room temperature. At predetermined time intervals, 1.0 mL of the release medium was withdrawn and replaced with an equal volume of fresh distilled water to maintain sink conditions.

The concentration of released EO was quantified indirectly based on its interaction with the stable radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH). DPPH exhibits strong absorbance at 517 nm, which decreases upon reaction with antioxidant compounds such as ascorbic acid (Figure 5A).

FIGURE 5.

(A) Variation in the visible absorption spectra of the DPPH solution (0.16 mM) in the presence of different concentrations of ascorbic acid (0.5–5.0 µg mL⁻¹) as a standard antioxidant. (B) Linear calibration curve of absorbance at 517 nm derived from the spectra shown in Figure 5A. (C) Cumulative percentage release of EO from free and encapsulated samples (CNGTO(1) and CNGTO(2)).

A calibration curve was constructed using ascorbic acid standard solutions (0.5–5.0 µg mL⁻¹) reacted with DPPH showing good linearity (A ₅₁₇ = −0.0327 (ascorbic acid) (µg mL⁻¹) + 0.7228, R ² = 0.989) (Figure 5B). Under these conditions, the concentration of released EO was expressed as ascorbic acid equivalents (µg mL⁻¹). To validate, the applicability of DPPH as an indirect release indicator, a control experiment was performed using blank CNGT (without EO) under identical dialysis conditions. Although CNGT exhibits antioxidant activity (Section 3.9) and DPPH radical scavenging activity (Section 3.10), the nanogel matrix remained confined within the dialysis bag due to its macromolecular structure and therefore did not diffuse into the external release medium. As shown in Figure 5C, the accumulated release calculated for the blank formulation remained below 5% throughout 96 h, confirming negligible contribution of the carrier system to the DPPH response. This indicates that the decrease in absorbance at 517 nm predominantly arises from EO molecules diffusing through the dialysis membrane rather than from the encapsulating matrix. Accordingly, the method is suitable for comparative of EO release under the applied conditions.

The cumulative release percentage was calculated according to Equation (3):

$$\mathrm{Cumulative}\mathrm{release}\mathrm{of}\mathrm{EO}(\%)=\frac{{\mathrm{V}}_{\mathrm{e}}{\sum }_1^{\mathrm{n}-1}{\mathrm{C}}_{\mathrm{i}}+{\mathrm{V}}_0{\mathrm{C}}_{\mathrm{n}}}{{\mathrm{m}}_0}\times 100\%$$ (3)

where, V _e is the volume withdrawn at each sampling point (1.0 mL), V ₀ is the total volume of the release medium (50.0 mL), C _i is the concentration of EO released at each time point, and m ₀ is the mass of the encapsulated EO. All release experiments were performed in triplicate under identical conditions and data are presented as mean ± standard deviation.

As shown in Figure 5C, the cumulative release of EO increased progressively with time. A rapid initial increase in release rate was observed for pure EO, CNGTO(1), and CNGTO(2) followed by a slower release after approximately 33 h. After 96 h, cumulative release reached 54% for pure EO, 30% for CNGTO(1), and 47% for CNGTO(2). It should be noted that the release values for encapsulated samples were normalized based on their respective encapsulation efficiencies (see Section 3.7). Overall, encapsulation significantly reduced the release rate compared to free EO, confirming the protective effect of the nanogel matrix.

The release profiles suggest a biphasic release mechanism: an initial burst phase attributed to diffusion of EO molecules located near or weakly associated with the nanogel surface, followed by a sustained release phase corresponding to diffusion of EO entrapped within the CNGT network. The reduced release percentages observed for CNGTO(1) and CNGTO(2) compared with free EO demonstrate the effective barrier function of the nanogel matrix against rapid diffusion. Furthermore, the relatively higher release from CNGTO(2) compared to CNGTO(1) can be attributed to its increased EO loading, which enhances the concentration gradient across the dialysis membrane and consequently facilitates faster diffusion.

3.9. Evaluation of Total Antioxidant Activity

To evaluate the total antioxidant activity of the encapsulated samples, a calibration curve was first constructed by measuring the absorbance of ascorbic acid reacting with the phosphomolybdenum reagent at 695 nm (Figure 6A). According to the obtained regression equation (A ₆₉₅ = 0.008 × (ascorbic acid) (µg mL⁻¹) – 0.0064, R ² = 0.997), (Figure 6B), the antioxidant activity of the samples was calculated and expressed as microgram ascorbic acid equivalents per gram of dry sample (Figure 6C).

FIGURE 6.

(A) Visible absorption spectra of solutions containing ammonium molybdate (4.0 mM), sulfuric acid (0.6 M), and sodium phosphate (28.0 mM) with varying concentrations of ascorbic acid (0.0–63.0 µg mL⁻¹). (B) Calibration curve for ascorbic acid based on absorbance at 695 nm derived from the Figure 6A. (C) Total antioxidant activity of EO, CNGT, CNGTO(1), and CNGTO(2).

The CNGT sample exhibited an antioxidant activity of approximately 124.9 µg g⁻¹ (ascorbic acid equivalents). The antioxidant activity of the encapsulated samples, CNGTO(1) (128.4 µg g⁻¹) and CNGTO(2) (145.2 µg g⁻¹) increased with increasing EO content. Pure EO, demonstrated the highest antioxidant activity (166.8 µg g⁻¹), which was greater than that of CNGT, CNGTO(1), and CNGTO(2).

These results indicate that although the nanogel matrix itself exhibits to a measurable antioxidant activity, encapsulation leads to a moderate reduction in the apparent antioxidant capacity of EO. This reduction may be attributed to restricted diffusion of EO molecules from within the matrix and possible interactions between EO constituents and the encapsulating agents (gelatin, β‐CD, and Tween 20), which may limit accessibility of active compounds to the assay reagent. From an application standpoint, while encapsulation slightly decreases the immediate antioxidant capacity, it provides advantages in terms of controlled release and improved physicochemical stability. Therefore, t the encapsulated system may be more suitable for applications requiring sustained antioxidant performance, such as food preservation or controlled delivery systems.

3.10. Evaluation of DPPH Radical Scavenging Activity

The free radical scavenging activity of the samples was evaluated using the DPPH assay, and the results are presented in Figure 7A,B. Pure EO and CNGT exhibited scavenging activities of 49.1% and 46.0%, respectively. In contrast, encapsulated samples, CNGTO(1) and CNGTO(2), showed significantly higher scavenging activities of 62.5% and 64.3%, respectively. Notably, the slightly higher scavenging activity observed for CNGTO(2) compared to CNGTO(1) is consistent with its greater EO loading, suggesting a positive relationship between EO content and radical scavenging performance within the nanogel system.

FIGURE 7.

(A) Visible absorption spectra of DPPH solution (0.16 mM) alone and in the presence of CNGT, CNGTO(1), CNGTO(2), and EO. (B) Column graph of t percentage free radical scavenging activity derived from Figure 7A.

3.11. Analysis of Total Phenolic Content

The total phenolic content (TPC) of the samples was determined using the Folin–Ciocalteu method, with gallic acid as the reference standard. Figure 8A,B display a typical standard addition curve for CNGTO(1), obtained from absorbance measurement at 729 nm. Similar calibration behavior was obtained for all tested samples. As shown in Figure 8C, CNGT exhibited a phenolic content of approximately 33.8 mg gallic acid equivalents (GAE) per gram of dry sample, indicating that the encapsulating matrix itself contributes substantially to the measured phenolic content. The phenolic content of the encapsulated samples increased with EO loading, reaching 35.4 and 42.6 mg GAE g⁻¹ for CNGTO(1) and CNGTO(2), respectively. For comparison, the EO amount corresponding to CNGTO(1) exhibited a phenolic content of 15.6 GAE g⁻¹. These findings demonstrate that both the encapsulating matrix (CNGT) and the incorporated EO contribute to the overall phenolic content of the system. The observed increase in TPC with higher EO loading further indicates effective incorporation of EO phenolic constituents within the nanogel matrix. This combined contribution from the carrier and the encapsulated oil enhances the functional potential of the system for antioxidant‐related applications.

FIGURE 8.

(A) Visible absorption spectra of gallic acid (0.0–10.0 µg mL⁻¹) in the presence of 50.0% Folin‐Ciocalteu. (B) Calibration curve derived from absorbance at 729 nm shown in Figure 8A. (C) Column graph of total phenolic content for CNGT, CNGTO(1), CNGTO(2), and EO.

3.12. Stability of the Encapsulated Samples

The stability of the encapsulated samples was evaluated from two perspectives: physical stability and antioxidant stability. In the first experiment, changes in particle size of CNGTO(1) solution were monitored over a 4‐week storage period in a dark glass bottle at 4°C using DLS (Figure 9A). The mean particle size gradually increased from 436.6 nm to 741.2 nm over time. This increase may be attributed to partial aggregation and structural rearrangements within the nanogel matrix during storage.

FIGURE 9.

(A) Changes in the sample size of the CNGTO(1) solution over 4 weeks measured by DLS. (B) Comparative column graph showing variation in radical scavenging activity of CNGTO(1) and free EO during 4 weeks storage.

In the second experiment, the antioxidant stability of EO was assessed by monitoring the radical scavenging activity of both CNGTO(1) and free EO over 4 weeks. As shown in Figure 9B, the radical scavenging activity of free EO decreased markedly over time, reflecting volatility and oxidative degradation of its active constituents. In contrast, CNGTO(1) exhibited a significantly slower decline in radical scavenging activity, indicating that encapsulation within the nanogel matrix effectively protected EO against degradation and preserved its antioxidant functionality.

Overall, these findings demonstrate that nanogel encapsulation improves both the physical stability (by moderating particle aggregation) and chemical stability (by protecting bioactive constituents) of EO during storage.

While the above results demonstrate the functional performance of the developed nanogel system, a comparison with previously reported EO encapsulation platforms is necessary to better position the present findings within the existing literature. Reported encapsulation efficiencies (EE%) for β‐cyclodextrin‐based inclusion complexes and gelatin‐derived carriers generally range between 60% and 90%, depending on oil composition, carrier architecture, and preparation strategy [36, 37, 38]. In this context, the EE% values obtained in the present study (77.5% for CNGTO(1) and 81.4% for CNGTO(2)) are positioned at the upper end of this reported range. This observation suggests that covalent CM‐β‐CD integration within the gelatin framework enhances structural organization and promotes more effective host–guest interactions compared to systems based solely on physical blending [12, 34].

Regarding colloidal properties, EO‐loaded biopolymeric nanoparticles stabilized predominantly through electrostatic repulsion often exhibits zeta potential values between −10 mV and −30 mV [39, 40, 41]. In contrast, the relatively low zeta potentials observed in the present system (−2.6 to −9.7 mV) indicate that electrostatic stabilization plays a limited role. Instead, steric stabilization provided by grafted β‐CD moieties and Tween 20 appears to be the dominant mechanism. Similar steric stabilization behavior has been reported in nonionic surfactant‐assisted encapsulation systems, where colloidal stability is maintained despite weak surface charge [42, 43]. This mechanistic distinction differentiates the present nanogel from conventional charged nanocarriers.

In terms of antioxidant stability, previous studies commonly report substantial losses in radical scavenging activity for free EO during storage due to oxidative degradation and volatility [44, 45]. Consistent with these findings, free EO in the present study exhibited a pronounced decline in antioxidant activity over time, whereas encapsulated EO retained a significantly higher proportion of its activity. Comparable protective effects have been observed in β‐CD inclusion systems and polymeric nanogels designed to reduce oxygen exposure and diffusion rates [46]. However, the present covalently integrated CNGTO systems demonstrates competitive antioxidant retention while simultaneously providing controlled release behavior.

Collectively, these comparisons indicate that the developed nanogel platform performs competitively with previously reported EO delivery systems, while offering a structurally integrated host–surfactant–polymer network that supports efficient encapsulation, distinct steric stabilization, and improved functional stability.

To further rationalize the molecular basis of these experimentally observed behaviors, computational modeling was performed to elucidate the interaction mechanisms governing α‐pinene inclusion and surfactant‐mediated structural organization.

3.13. Computational Modeling of CNGTO

Molecular docking simulations were performed to elucidate the structural basis of interactions between the CNGT nanogel system and α‐pinene, the major terpene component of the essential oil (33.65%, GC‐MS), in order to rationalize the experimentally observed encapsulation efficacy and release behavior. A representative fragment of Type A gelatin was constructed based on its characteristic amino acid composition enriched in Gly‐Pro‐Hyp sequences. Type A gelatin, derived from acid‐treated collagen, typically retains partial sequence features of the parent triple‐helix but exists predominantly in a denatured, random‐coil conformation. In agreement with this structural nature, the modeled gelatin fragment adopted a flexible coil‐like arrangement (Figure 10A). The limited chain length, incomplete Gly‐Pro‐Hyp repetition, and absence of interchain hydrogen bonding prevented stable triple‐helix formation.

FIGURE 10.

(A) Designed primary structure of the gelatin model consisting of a 40‐residue sequence and the modeled triple‐chain gelatin covalently conjugated to three CM‐β‐CD molecules. (B) CNGO system: gelatin‐CM‐β‐CD complex docked with α‐pinene in the absence of Tween 20. (C) CNGTO system: gelatin‐CM‐β‐CD complex docked with 10 Tween 20 followed by α‐pinene docking. (D) Close‐up view of α‐pinene docked in CNGO system, highlighting surface alignment and key interactions. (E) Close‐up view α‐pinene docked in CNGTO system showing deeper penetration into the CM‐β‐CD cavity and enhanced stabilization. Hydrogen bonds within the host matrix are depicted as dashed lines. Host–guest contact distances are shown as green lines and annotated with the corresponding values (Å), illustrating α‐pinene accommodation within the CM‐β‐CD cavity (color scheme: gelatin shown in random‐coil representation (white); Tween 20 molecules in stick representation (yellow); CM‐β‐CD in stick representation (blue); and α‐pinene in green stick representation).

The presence of reactive residues such as Lys, Glu, and Asp provides potential anchoring points for covalent conjugation, Accordingly, CM‐β‐CD was covalently grafted onto the gelatin backbone in the model. This conjugation introduced localized steric constraints and modified hydrogen‐bonding patterns, leading a reduced conformational flexibility and partial structural rearrangement. The incorporation of polar CM‐β‐CD increased the hydrophilic surface character of the matrix, while its hydrophobic cavity created potential inclusion sites for nonpolar guest molecules such as α‐pinene. For modeling purposes, three CM‐β‐CD units were attached to the gelatin fragment, balancing structural realism and computational tractability (Figure 10). To simulate cooperative interfacial effects, 10 Tween 20 molecules were docked onto energetically favorable regions of the conjugated matrix prior to α‐pinene docking (Figure 10B,C). The surfactant molecules formed an amphiphilic shell stabilized by hydrogen bonding with polar residues (e.g., Arg, Glu, and Asp) and hydrophobic contacts with Pro, Hyp, and Gly residues Figures 10A and S2. This organization is consistent with experimental observations of gelatin–surfactant interactions and supports the formation of a sterically stabilized nanogel interface.

Docking of α‐pinene to the CNGT system yielded binding energies in the range of −5.68 to −4.73 kcal mol⁻¹ for the matrix components and −3.64 to −3.70 kcal mol⁻¹ for the final host–guest complexes. Although these values indicate moderate interaction strength, such energy ranges are typical for non‐covalent host–guest inclusion systems dominated by van der Waals and hydrophobic interactions. In polymer‐based encapsulation matrices, stabilization arises from the cumulative effect of multiple weak, reversible interactions rather than a single high‐affinity binding event [47, 48, 49]. Therefore, binding energies in this range are sufficient to retain volatile terpenes within the carrier while maintaining the dynamic reversibility required for controlled release. Importantly, the binding mode of α‐pinene differed depending on the presence of Tween 20. In the absence of surfactant, α‐pinene preferentially aligned along the external surface of CM‐β‐CD, suggesting limited cavity accessibility (Figure 10D). In contrast, in the Tween 20‐containing system, α‐pinene penetrated more deeply into the hydrophobic CM‐β‐CD cavity (Figure 10E), indicating improved topological accommodation. This difference occurred despite similar overall docking scores, suggesting that Tween 20 primarily modulates binding geometry rather than intrinsic binding thermodynamics. Because α‐pinene is a nonpolar hydrocarbon lacking hydrogen‐bond donor or acceptor groups, no direct hydrogen bonds were observed between α‐pinene and the host structure. Instead, stabilization was governed by non‐directional van der Waals and hydrophobic interactions within the CM‐β‐CD cavity. In Figure 10E, hydrogen bonds within the host matrix (e.g., between cyclodextrin rim hydroxyl groups and gelatin residues) are highlighted as dashed lines to illustrate structural stabilization of the carrier. For α‐pinene inclusion, host–guest distances ranging from 2.8 Å to 4.9 Å were measured, consistent with typical van der Waals contact distances expected for physically driven encapsulation.

Collectively, the computational results indicate that Tween 20 does not substantially increase binding free energy but instead reorganizes the interfacial topology of the nanogel matrix to facilitate deeper guest inclusion. This mechanistic interpretation is consistent with experimental observations of higher encapsulation efficiency and improved retention in CNGTO systems compared to non‐surfactant formulations. The cooperative interplay between covalently grafted β‐CD cavities and surfactant‐mediated structural modulation provides a molecular‐level explanation for the enhanced stability and functional performance of the developed nanogel platform [50, 51, 52].

4. Conclusions

This study demonstrated that hydrogel nanoparticles prepared via O/W emulsification followed by freeze‐drying provide an effective strategy for encapsulating essential oils derived from P. atlantica. Covalent integration of CM‐β‐CD into the gelatin network, combined with Tween 20‐mediated interfacial modulation, resulted in uniform particle formation, reduced aggregation, and high encapsulation efficiency within the upper range reported for comparable EO delivery systems. Encapsulation significantly improved oxidative stability and preserved radical scavenging activity during storage compared to free EO, while enabling controlled release behavior. The DPPH‐based release monitoring approach provided a practical and reproducible method for indirect quantification of released bioactive compounds, supporting its applicability in laboratories with limited chromatographic facilities. Computational modeling further elucidated the molecular basis of encapsulation. Docking simulations indicated that α‐pinene inclusion within β‐CD cavities is governed by cumulative weak non‐covalent interactions typical of cyclodextrin‐based host–guest systems. Although binding energies were moderate, they were consistent with reversible stabilization suitable for controlled release. Importantly, Tween 20 was shown to modulate binding topology, facilitating deeper guest accommodation without substantially altering binding thermodynamics. This mechanistic insight establishes a clear structure–interaction–function relationship within the hybrid nanogel system. Overall, the developed protein–cyclodextrin–surfactant platform represents a structurally integrated and functionally stable carrier for hydrophobic terpenes. The combined experimental and computational approach provides a rational framework for designing next‐generation nanocarriers for food, pharmaceutical applications where improved stability, controlled release, and functional retention are required.

Author Contributions

Kamalluddin Zahedi: methodology, investigation, and writing – original draft preparation. Sedigheh Hashemnia: conceptualization, methodology, data curation, visualization, writing – review and editing and supervision. Zaynab Mokhtari: methodology, data curation, visualization, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71131‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.