Slow Release and Active Effects of Combined Quercetin and Resveratrol Carriers in Hummus During Simulated Digestion

The antioxidant activity of free quercetin:resveratrol mixtures (Q:R) and their corresponding niosomal formulations (QR-NIOs) was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging assays across five quercetin (Q) ratios (Figure 1). In the DPPH assay, the 100:0 ratio showed the highest activity in both the free and niosomal groups (3833.45 ± 131.60 and 3479.95 ± 183.11 µmol TE/g D.W., respectively); these two values differed significantly. In contrast, ABTS activity was highest at the 50:50 and 75:25 ratios in both groups, with no significant difference between these ratios. The 25:75 ratio showed the lowest ABTS activity in both the free (983.60 ± 204.56 µmol TE/g D.W.) and niosomal (941.89 ± 235.41 µmol TE/g D.W.) groups. Separate two-way analyses of variance for each assay identified a significant effect of Q ratio on both DPPH and ABTS activity (p < 0.001). Presentation as a free mixture or niosomal formulation had a significant overall effect on DPPH activity (p < 0.001), but not on ABTS activity (p = 0.550); the interaction between ratio and presentation was not significant in either assay. Thus, the 50:50 and 75:25 ratios provided the strongest ABTS responses irrespective of presentation, whereas the 100:0 ratio yielded the highest DPPH response.

These findings are consistent with Lu et al. [22], who reported substantial DPPH-scavenging activity of quercetin and showed that niosomal encapsulation improved its solubility and photostability. Similarly, Machado et al. [23] found that resveratrol-loaded niosomes maintained DPPH activity comparable to that of free resveratrol, whereas ABTS activity depended on bilayer composition, with 96% retention in dodecanol-stabilized niosomes. In the present study, the similarly high ABTS values of the free and niosomal 50:50 and 75:25 mixtures indicate that their antioxidant activity was largely preserved after encapsulation. By contrast, the 100:0 ratio produced the highest DPPH activity in both presentations, with a significantly higher value for free quercetin than for its niosomal formulation. Together, these assay-dependent patterns show that antioxidant responses vary with the Q:R ratio and the assay conditions, without establishing a general increase in radical-scavenging activity following encapsulation.

Erythroprotective activity differed significantly according to the quercetin-to-resveratrol ratio (Q:R) and ABO blood group and Rhesus D (RhD) antigen status (p < 0.001; Table 1). The 50:50 formulation produced the highest overall value in A RhD+ erythrocytes (100.45 ± 1.24%) and was also the most effective treatment in B RhD+ cells (87.54 ± 2.75%), significantly exceeding both single-compound formulations within these groups. In AB RhD+ erythrocytes, quercetin alone (100:0) provided the greatest protection (98.53 ± 2.75%), followed by resveratrol alone and the 50:50 formulation, which did not differ significantly from each other. Resveratrol alone (0:100) generated the highest response in O RhD+ cells (88.45 ± 1.67%), whereas quercetin alone showed the highest numerical value in O RhD− cells (81.87 ± 2.84%), without differing from the 75:25 formulation. Across groups, A RhD+ consistently showed high responses, while the 50:50 formulation displayed its lowest activity in O RhD− erythrocytes (64.73 ± 2.46%). Thus, formulation performance depended strongly on the specific blood-group phenotype.

Notably, the 25:75 formulation never produced the maximum response in any phenotype and yielded the lowest activity in A RhD+, B RhD+, and AB RhD+ erythrocytes. Conversely, the optimal treatment differed among groups, demonstrating a distinct formulation-by-phenotype response pattern and confirming that no quercetin–resveratrol ratio provided uniformly superior erythroprotection across samples. Overall, the 50:50 formulation performed best, achieving the highest erythroprotection and significantly surpassing both individual compounds in A RhD+ and B RhD+ erythrocytes.

Our blood-group-dependent pattern agrees with González-Vega et al. [24], who found that AAPH-induced erythroprotection by quercetin and antioxidants varied across ABO/RhD phenotypes. Likewise, Chen and Deuster [25] showed that quercetin markedly reduced AAPH-mediated hemolysis in human erythrocytes, supporting its membrane-protective contribution; however, neither study evaluated Q:R ratios or dual-loaded niosomes.

Light microscopy corroborated the erythroprotective activity of QR-NIOs against AAPH-mediated oxidative damage (Figure 2). Erythrocytes exposed only to AAPH, the positive control (Figure 2A), showed extensive hemolysis, dense aggregates, indistinct membranes, and abundant debris. In contrast, erythrocytes without AAPH-induced hemolysis, the negative control (Figure 2B), retained uniform size, central pallor, regular contours, and discocyte morphology. AAPH-treated A RhD+ erythrocytes incubated with 50:50 QR-NIOs (Figure 2C) remained intact and morphologically homogeneous, closely resembling the negative control and corresponding to 100.45 ± 1.24% hemolysis inhibition. Under the same treatment, O RhD− erythrocytes (Figure 2D) exhibited heterogeneous staining, irregular contours, clear intracellular spaces, and occasional deformation, consistent with partial protection and 64.73 ± 2.46% inhibition. Therefore, the micrographs corroborated the blood-group-dependent efficacy quantified by the antihemolytic assay.

Collectively, antihemolytic measurements and microscopy demonstrated that 50:50 QR-NIOs provided maximal protection in A RhD+ erythrocyte but only partial protection in O RhD− cells. This phenotype-dependent preservation agrees with González-Vega et al. [24], who reported ABO/RhD-dependent antioxidant protection against AAPH-induced erythrocyte damage.

Porcine pancreatic elastase (PPE) activity was assessed using N-succinyl-Ala-Ala-Ala-p-nitroanilide as the substrate (Figure 3). PPE-mediated hydrolysis releases p-nitroaniline; therefore, reduced p-nitroaniline formation relative to the enzyme control indicates inhibition of PPE activity. The 50:50 QR-NIO formulation showed the highest mean inhibition (57.86 ± 2.43%), followed by 25:75 (53.56 ± 1.36%), 75:25 (51.53 ± 2.63%), 100:0 (40.70 ± 1.35%), and 0:100 (18.42 ± 2.74%). The 50:50 formulation also showed the most persistent inhibition over the 60 min assay (Figure 3), supporting its selection for further evaluation. These findings are consistent with reports that polyphenols, including quercetin, can inhibit elastase activity [26,27]. However, the present assay does not establish the inhibition mechanism or demonstrate that co-loading directly prolongs a specific polyphenol–elastase interaction.

α-Amylase inhibition differed according to the quercetin–resveratrol ratio (Figure 4). The 50:50 QR-NIO formulation showed the highest mean inhibition (93.23 ± 2.18%) and was assigned to statistical group a. This response was significantly greater than those of the 0:100, 25:75, and 75:25 formulations, whose inhibition values ranged from approximately 82% to 85% and were assigned to statistical group b. Quercetin-only niosomes (100:0) showed high activity, approaching 91.24 ± 2.56%, but their assignment to group ab indicated no significant difference from either 50:50 or the lower formulations. Consequently, the balanced 50:50 formulation provided the clearest enhancement of α-amylase inhibition, although its activity did not significantly exceed quercetin alone under these assay conditions.

α-Glucosidase inhibition depended significantly on the quercetin–resveratrol ratio (Figure 5), but the response pattern differed from that observed for α-amylase. Resveratrol-only niosomes (0:100) and the balanced 50:50 formulation produced the highest activities, both reaching 88.63 ± 3.85% inhibition and sharing group a. The 25:75 and quercetin-only 100:0 formulations showed intermediate responses of 78 ± 2.68% and were assigned to group b, demonstrating significantly lower inhibition than the two leading formulations. The 75:25 formulation exhibited the weakest activity (71 ± 3.62%) and formed an independent statistical group c, differing significantly from all others. Thus, increasing the quercetin proportion did not progressively enhance α-glucosidase inhibition. Instead, maximal activity was retained when resveratrol was used alone or combined equally with quercetin. Across both enzyme assays, 50:50 was the only formulation positioned within the highest statistical group for α-amylase and α-glucosidase, identifying it as the most consistently active ratio against sequential carbohydrate-hydrolyzing enzymes under the tested conditions.

The dual-enzyme response of 50:50 QR-NIOs agrees with Wang et al. [28], whose slow-release quercetin emulsion inhibited α-amylase and α-glucosidase and delayed starch hydrolysis, supporting the importance of delivery systems for preserving polyphenol activity. Zhang et al. [29] likewise demonstrated that resveratrol inhibited mammalian intestinal α-glucosidase and reduced postprandial glycemia in mice. These studies support the contributions of quercetin to α-amylase inhibition and resveratrol to α-glucosidase inhibition observed here. However, because 50:50 did not significantly outperform the best single-compound formulation in either assay, its broader dual-enzyme activity should be interpreted as complementary rather than synergistic.

Interactions between quercetin and resveratrol were evaluated by comparing observed activity with an additive response calculated from each compound’s proportional contribution. The interaction index (II), defined as experimental activity divided by theoretical activity, classified formulations as synergistic (II > 1), additive (II ≈ 1), or antagonistic (II < 1), thereby identifying combinations with effects exceeding those predicted from individual activities.

Niosomes are vesicular nanocarriers composed of nonionic surfactants that typically exhibit spherical or ovoid morphology when assembled [30]. SEM revealed rounded-to-ellipsoidal QR-NIOs with nanometric dimensions (Figure 5). Panel A showed a particle measuring approximately 241 × 205 nm, whereas Panel B displayed an elongated particle of 242 × 134 nm and smaller structures measuring 63–70 nm, demonstrating clear dimensional heterogeneity.

The relatively smooth surfaces and limited aggregation support the formation of discrete nanoscale particles, whereas flattening and elongation may reflect dehydration and vacuum exposure during SEM preparation. The principal innovation is the development of a dual-loaded quercetin–resveratrol niosomal system with preserved nanoscale morphology; however, SEM alone cannot conclusively confirm co-encapsulation, which requires compound-specific encapsulation efficiencies and complementary independent spectroscopic evidence.

Physicochemical characterization demonstrated that QR-NIOs formed a relatively homogeneous nanoscale dispersion, characterized by nanometric size, narrow size distribution, high encapsulation efficiency, and sufficient surface charge to support initial colloidal stability [30]. The co-formulated niosomes had a reported overall encapsulation efficiency of 90.63 ± 3.63%. In separate single-compound preparations, the measured vesicle-associated concentrations were 1.863 mM for quercetin and 1.7092 mM for resveratrol, from an initial concentration of 2 mM each. Across three independent preparations, the mean encapsulation efficiencies were 93.35 ± 2.31% for quercetin and 85.64 ± 2.11% for resveratrol. The mean hydrodynamic diameter of 149 ± 13.32 nm confirmed their nanometric size. The low polydispersity index (0.21 ± 0.02) demonstrated a comparatively narrow size distribution, and the negative ζ-potential (−34 ± 2.0 mV) suggested sufficient electrostatic repulsion to limit vesicle aggregation. These parameters support the initial colloidal stability and suitability of QR-NIOs for incorporation into functional food matrices, although stability during storage and gastrointestinal digestion remains to be experimentally confirmed.

The intensity-weighted distribution displayed a single dominant nanometric population without clearly resolved secondary peaks (Figure 6A), supporting limited aggregation and agreement with the reported particle size and polydispersity index (PDI). The difference between the modal peak and the mean diameter may arise from intensity weighting and the cumulant analysis used by dynamic light scattering (DLS). Likewise, the narrow negative ζ-potential peak (Figure 6B) indicated similar interfacial charge among most vesicles and was broadly consistent with the reported mean value. Although the combined distributions demonstrate a uniform initial formulation, they do not establish long-term stability under the ionic strength, pH, protein interactions, or bile-salt exposure expected in hummus and gastrointestinal environments.

The release profiles differed markedly among formulations (Figure 7A, Table S1). Free QUE diffused fastest, increasing from 45.69% at 1 h to 97.52% at 24 h, followed by RES (32.06–86.74%) and the unencapsulated quercetin–resveratrol (Q–R) mixture (21.60–80.35%). QR-NIOs showed the lowest cumulative release throughout the experimental period, rising from only 3.38% to 51.63% over the same period, without reaching a plateau. This attenuated initial release and progressive diffusion indicate sustained polyphenol retention by the niosomal system. Nevertheless, dialysis measures membrane passage rather than gastrointestinal bioaccessibility. Similar retardation of quercetin release after niosomal encapsulation has been reported, supporting the carrier-mediated modulation observed here [22]. The Korsmeyer–Peppas model adequately fitted all profiles (R² = 0.979–0.991; Figure 7B). QR-NIOs exhibited the lowest kinetic constant (k = 0.082) and exponent (n = 0.318), supporting the slowest apparent release among formulations. The time points, release intervals, and fitted parameters for each formulation are reported in Table S2. Free QUE and RES showed higher k values (0.241 and 0.248), whereas Q–R was intermediate (k = 0.194), directly consistent with the cumulative release profiles observed experimentally.

These kinetic parameters reinforce the experimental release profiles: the lower apparent k of QR-NIOs corresponded with their attenuated initial release and 51.63% diffusion after 24 h. For spherical systems, n ≈ 0.43 represents the Fickian benchmark; therefore, n = 0.318 is compatible with quasi-Fickian transport [22,31]. However, mechanistic interpretation requires fitting only the initial 60% of release. Additionally, dialysis-membrane resistance means that the model supports sustained apparent release but cannot demonstrate that niosomal-bilayer diffusion was the sole transport-controlling process. Lu et al. [22] likewise observed that free quercetin was released more rapidly than quercetin-loaded niosomes, supporting the capacity of niosomal formulations to delay apparent release. This qualitative pattern agrees with our 24 h dialysis results, in which cumulative passage was 97.52% for free quercetin, 80.35% for the unencapsulated 50:50 mixture, and 51.63% for QR-NIOs. However, Lu et al. evaluated a single-compound formulation intended for skin delivery in a different release medium; therefore, their release rates cannot be directly compared with those of our dual-loaded formulation, nor do the two studies establish the same transport mechanism.

Niosomal formulations were prepared at each of the five Q:R molar ratios described in Section 4.3 using a modified ultrasound-assisted thin-film hydration method [58,59]. For each 20 mL batch, Span 60 (400 mg; 2.0% w/v), Tween 80 (200 mg; 1.0% w/v), and cholesterol (20 mg; 0.1% w/v) were dissolved in food-grade ethanol. Quercetin and resveratrol were added from their DMSO stock solutions in amounts corresponding to a total nominal polyphenol concentration of 4 mM: 100:0 (4:0 mM), 75:25 (3:1 mM), 50:50 (2:2 mM), 25:75 (1:3 mM), or 0:100 (0:4 mM). The mixture was stirred until homogeneous and subjected to rotary evaporation at 45 °C and 100 rpm under reduced pressure (250–300 mbar) for approximately 10 min, or until a uniform thin film formed. The film was further dried under vacuum, hydrated with phosphate-buffered saline (PBS, pH 7.4), and adjusted to a final suspension volume of 20 mL.

The hydrated vesicles were processed using a probe-type ultrasonic processor (Benchmark Scientific, Inc., Sayreville, NJ, USA) equipped with a 10 mm-diameter horn. Sonication was performed at a nominal frequency of 25 kHz and an amplitude setting of 65%, with alternating 2 s-on and 2 s-off pulses for a programmed time of 15 min. The recorded probe temperature was approximately 23 °C. The five niosomal formulations were evaluated in the ratio-screening assays alongside their corresponding free mixtures. Based on the combined results of the ratio-screening assays, the 50:50 niosomal formulation was selected for further study. Its biological activities were reassessed, and it was characterized by scanning electron microscopy (SEM), average particle size, polydispersity index (PDI), ζ-potential, and encapsulation efficiency measurements. This selected formulation was subsequently used to enrich the hummus.

The antioxidant interactions of dual-loaded quercetin–resveratrol niosomes (QR-NIOs) were investigated to identify the formulation exhibiting the greatest synergistic antioxidant effect and to determine the optimal quercetin–resveratrol ratio. Antioxidant activity was assessed using two complementary methods: DPPH and ABTS, which evaluate different mechanisms of antioxidant action, including free radical scavenging and reducing power. For the DPPH assay, 20 µL of each sample was mixed with 200 µL of DPPH solution adjusted to an absorbance of 0.70 ± 0.02 at 515 nm and incubated in darkness for 30 min. In the ABTS assay, 20 µL of sample was combined with 270 µL of ABTS•+ solution adjusted to an absorbance of 0.70 ± 0.02 at 734 nm and maintained in the dark for 30 min.

All measurements were performed in triplicate. Antioxidant activity was expressed as µmol Trolox equivalents per gram (µmol TE/g) for DPPH and ABTS assays. For the DPPH and ABTS assays, antioxidant activity was calculated using the following equation:(1)Inhibition(%)=Acontrol−ASampleAcontrol×100 where A_control is the measured absorbance of the control reaction without the test sample and A_sample is the measured absorbance of the reaction containing the test sample. Results were expressed as the percentage of elastase inhibition relative to the control.

The erythroprotective activity of different dual-loaded quercetin–resveratrol niosome (QR-NIO) formulations was evaluated using an AAPH-induced oxidative hemolysis assay to identify the formulation exhibiting the greatest membrane-protective and antioxidant effects. Quercetin–resveratrol mixtures (100:0, 75:25, 50:50, 25:75, and 0:100, w/w) were prepared at concentrations of 12.5, 25, 50, 100, and 200 μg/mL. Human erythrocytes obtained from healthy adult volunteers were washed three times with phosphate-buffered saline (PBS, pH 7.4) and adjusted to a final cell suspension of 2% (v/v).

The reaction mixture consisted of 100 μL of erythrocyte suspension, 100 μL of the corresponding quercetin–resveratrol combination, and 100 μL of freshly prepared AAPH solution (40 mM). Oxidative controls contained erythrocytes incubated with AAPH alone, whereas negative controls received PBS instead of AAPH. After incubation at 37 °C for 3 h under gentle agitation (45 rpm), samples were centrifuged (1500 rpm, 10 min), and hemolysis was quantified by measuring the absorbance of the supernatant at 540 nm. Erythroprotective activity was expressed as the percentage inhibition of AAPH-induced hemolysis relative to the oxidative control, and the combination showing the highest protective effect was selected for subsequent niosome formulation [60]. The inhibition of AAPH-induced oxidative damage in human erythrocytes was calculated using the following equation:(2)HemolysisInhibition%=ACpos−ATS−ACnegACpos×100 where A_Cpos is the absorbance of the positive control (erythrocytes exposed to AAPH without a test formulation), A_TS is the absorbance of erythrocytes exposed to AAPH in the presence of the test formulation, and A_Cneg is the absorbance of the negative control (erythrocytes not exposed to AAPH). Absorbance at 540 nm reflects hemoglobin released into the supernatant and was used as an indicator of hemolysis. Thus, lower hemoglobin release in the treated sample corresponds to greater inhibition of AAPH-induced hemolysis.

As a complementary study, erythrocyte morphology was microscopically evaluated to provide qualitative evidence of the protective effects of dual-loaded quercetin–resveratrol niosomes (QR-NIOs) against AAPH-induced oxidative damage through the preservation of erythrocyte membrane integrity. After incubation, erythrocytes were collected by centrifugation, and thin blood smears were prepared on clean glass slides, air-dried, and stained with Wright’s stain according to standard hematological procedures. The stained smears were examined using an Eclipse FN1 optical microscope (Nikon Instruments Inc., Tokyo, Japan) equipped with NIS-Elements imaging software (version 6.1) at 100× magnification under oil immersion. Representative micrographs were obtained for the positive control (AAPH-induced hemolysis), the negative control (untreated erythrocytes), and the QR-NIO formulation that demonstrated the greatest protective effect against AAPH-induced hemolysis among the evaluated quercetin–resveratrol combinations [22,24,60].

The anti-inflammatory activity of dual-loaded quercetin–resveratrol niosomes (QR-NIOs) was evaluated by measuring their inhibitory effect on porcine pancreatic elastase (PPE, Type IV, Sigma-Aldrich, St. Louis, MO, USA), following the method described by Lee et al. [61] with minor modifications. Elastase activity was determined using N-succinyl-Ala-Ala-Ala-p-nitroanilide (1.015 mM) as substrate prepared in Tris-HCl buffer (0.1 M, pH 8.0), while the PPE enzyme was prepared at 1.376 U mL⁻¹ in Tris-HCl buffer (0.2 M, pH 8.0). The reaction mixture consisted of 10 μL of the corresponding QR-NIO formulation, 40 μL of PPE solution, and 100 μL of substrate solution, giving a final reaction volume of 150 μL. Absorbance was monitored at 410 nm for 60 min at 28 °C using a microplate reader.

All determinations were performed in triplicate, and elastase inhibition was expressed as the percentage inhibition relative to the enzyme control. The QR-NIO formulation exhibiting the highest inhibitory activity was selected for subsequent physicochemical characterization and biological evaluation. Elastase inhibition was calculated according to the following equation:where A is the absorbance of the control and B is the absorbance in the presence of the extract. Results were expressed as percentage inhibition of elastase activity. (3) Inhibition % 1 − A B = × 100

The α-amylase and α-glucosidase inhibitory activities of dual-loaded quercetin–resveratrol niosomes (QR-NIOs) were assessed by evaluating their inhibitory activity against the carbohydrate-digesting enzymes α-amylase [62,63] and α-glucosidase [63,64]. For the α-amylase assay, 50 μL of each QR-NIO formulation was incubated with 50 μL of porcine pancreatic α-amylase (1 U/mL) prepared in 20 mM sodium phosphate buffer (pH 6.9) containing 6 mM NaCl at 37 °C for 10 min. Subsequently, 50 μL of 1% (w/v) soluble starch was added, and the reaction proceeded for 15 min before termination with 100 μL of 3,5-dinitrosalicylic acid (DNS) reagent. After heating at 95 °C for 5 min and cooling to room temperature, the mixtures were diluted with distilled water, and absorbance was measured at 540 nm. Acarbose and buffer served as positive and negative controls, respectively. The percentage of α-amylase inhibition was calculated using the following equation:(4)Inhibition%=1−ASampleAControl×100

The inhibitory effect of dual-loaded quercetin–resveratrol niosomes (QR-NIOs) against α-glucosidase was determined using p-nitrophenyl-α-D-glucopyranoside (pNPG) as the substrate. Briefly, 50 μL of each formulation was incubated with 50 μL of α-glucosidase solution (1 U/mL) prepared in 100 mM phosphate buffer (pH 6.8) at 37 °C for 10 min. The reaction was initiated by adding 50 μL of 5 mM pNPG and terminated after 20 min with 100 μL of 0.2 M sodium carbonate. Absorbance was recorded at 405 nm. Acarbose and buffer were used as positive and negative controls, respectively, and enzyme inhibition was expressed as a percentage using the following equation: (5) Inhibition % 1 100 − × A Sample A Control =

The synergistic interaction between quercetin and resveratrol was determined by comparing the experimentally observed inhibitory activity of each combination with the theoretical additive effect calculated from the activities of the individual compounds at the corresponding concentrations [65,66]. The expected inhibitory activity (Eexpected) was calculated as:(6)Eexpected=Fq−Eq+Fr−Er where FQ and FR represent the weight fractions of quercetin and resveratrol in each formulation, respectively, while EQ and ER correspond to the inhibitory activities of quercetin and resveratrol tested individually at the same total concentration. The interaction index (II) was subsequently calculated according to:(7)II=EexperimentalEexpected

Interaction index values greater than 1.0 were interpreted as synergistic effects, values close to 1.0 indicated additive interactions, and values lower than 1.0 suggested antagonistic behavior.

The morphology and surface characteristics of the selected dual-loaded quercetin–resveratrol niosomes (QR-NIOs) were examined by scanning electron microscopy (SEM), with adaptations from previously reported nanovesicle characterization procedures [67,68]. Before analysis, the niosomal dispersion was freeze-dried, and the resulting powder was mounted on aluminum stubs using conductive carbon tape. Samples were sputter-coated with a thin gold layer to minimize surface charging and improve image contrast. Micrographs were acquired using a JSM-6610LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) under high-vacuum conditions at an accelerating voltage of 20 kV and working distances of 9–10 mm. Representative images were recorded at ×70,000 and ×80,000 magnification to assess apparent particle shape, surface topography, dry-state dimensions, morphological heterogeneity, and aggregation. SEM observations were interpreted together with dynamic light-scattering measurements because freeze-drying, vacuum exposure, and conductive coating may alter vesicle dimensions and morphology.

The physicochemical properties of the selected 50:50 dual-loaded quercetin–resveratrol niosomes (QR-NIOs) were evaluated by measuring their average particle size and polydispersity index (PDI) by dynamic light scattering (DLS) and their ζ-potential by electrophoretic light scattering. Briefly, 1.0 mL of the niosomal suspension was diluted in 100 mL of phosphate-buffered saline (PBS, pH 7.4) and transferred to disposable cuvettes for analysis [69]. Measurements were performed at room temperature using a Nano-ZS90 analyzer (Malvern Instruments Ltd., Malvern, UK).

The encapsulation efficiency of quercetin and trans-resveratrol was determined by size-exclusion chromatography followed by multicomponent UV–Vis spectrophotometry. A 2.5 mL aliquot of the QR-NIO dispersion was applied to a PD-10 column packed with Sephadex G-50 and equilibrated with phosphate-buffered saline (PBS; 10 mmol/L, pH 7.4). Sixteen 0.5 mL fractions were collected. Vesicle-containing fractions were distinguished from those containing free compounds by light scattering at 600 nm and particle-size analysis; their elution windows were established using empty niosomes and free-polyphenol controls. Vesicle-associated compounds were extracted with methanol. Quercetin and resveratrol were then quantified using absorbance measurements at 370 and 306 nm, respectively, and simultaneous equations derived from separate calibration curves for both compounds. The encapsulation efficiency of each compound was calculated from its amount in the vesicle-containing fractions relative to its total amount in an unpurified aliquot, with mass recovery assessed across the collected fractions. Analyses were performed in triplicate under reduced lighting to limit resveratrol photoisomerization. Overall apparent encapsulation efficiency was calculated on a mass basis as:(8)EEApparentglobalencapsulationefficiency(%)=mQUEenc+ mRESencmQUEtotal+ mREStotal×100 where the apparent global encapsulation efficiency (%) represents the apparent percentage of the total polyphenol mass associated with the niosomal fraction; m_QUEenc is the mass of encapsulated quercetin recovered from the vesicle-associated fractions; m_RESenc is the mass of encapsulated resveratrol recovered from the vesicle-associated fractions; m_QUEtotal is the total mass of quercetin initially present in the unpurified niosomal formulation; m_REStotal is the total mass of resveratrol initially present in the unpurified niosomal formulation; and 100 is the conversion factor used to express the result as a percentage.

The apparent global encapsulation efficiency represents the combined mass fraction of quercetin and resveratrol associated with the niosomal population. This ensemble-level parameter represents the combined fraction of polyphenols associated with the niosomal population but does not demonstrate that both compounds are simultaneously encapsulated within each individual vesicle. All analyses were conducted in triplicate, and results were expressed as the mean ± standard deviation.

The release profile of the optimal synergistic dual-loaded quercetin–resveratrol niosomes (QR-NIOs) was evaluated using a dialysis membrane diffusion method [70]. Briefly, 10 mL of the niosomal suspension was placed into pre-hydrated dialysis membranes (Molecular-weight cutoff [MWCO] 8000–12,000 Daltons) and immersed in 100 mL of phosphate-buffered saline (PBS, 0.05 M, pH 7.4) at 37 °C under constant agitation (100 rpm). Aliquots were collected at predetermined intervals (0.5–24 h) and replaced with fresh PBS to maintain sink conditions. Free quercetin, free resveratrol, and an unencapsulated 50:50 quercetin–resveratrol mixture were prepared from individual DMSO (10%) stock solutions diluted in phosphate-buffered saline (PBS; 0.05 M, pH 7.4). These controls did not contain niosomes. For each control, 10 mL of solution was placed in a pre-hydrated dialysis membrane and tested under the same receiving-medium, temperature, agitation, and sampling conditions as the QR-NIO dispersion.

Released polyphenols were quantified by ultraviolet–visible (UV–Vis) spectrophotometry using calibration curves, and cumulative release was expressed as the percentage of quercetin and resveratrol released over time. Release data were fitted to the Korsmeyer–Peppas equation, Mt/M∞ = ktⁿ, where Mt/M∞ represents the released fraction at time t. The fit included only data points within the initial release region (Mt/M∞ ≤ 0.60), excluding the 0 h value. Release data for each time point are provided in Table S1, while the fitted rate constant (k), release exponent (n), and coefficient of determination (R²) are reported in Table S2.

Functional hummus was prepared using cooked chickpeas as the principal food matrix, with modifications from previously reported hummus formulations [71,72]. Cooked chickpeas, tahini, extra virgin olive oil, water, lemon juice, sodium chloride, and paprika were combined at the proportions specified in Table 10 and homogenized using a Waring Commercial Laboratory Blender (Model 7011HS, Waring Products, Stamford, CT, USA) at 18,000 rpm for 1 min until a smooth and uniform paste was obtained. The resulting base formulation was divided into equal portions. For the preparation of QR-NIO-enriched functional hummus (QRH), 2.0 g of the selected QR-NIO dispersion was incorporated per 100 g of product, replacing an equivalent amount of water.

The total polyphenol content was standardized to 100 µmol per 100 g of hummus. Based on molecular weights of 302.24 and 228.24 g/mol for quercetin and resveratrol, respectively, the 100:0 and 0:100 endpoints corresponded to 30.22 mg of quercetin and 22.82 mg of resveratrol per 100 g. Accordingly, the selected 50:50 molar formulation provided 15.11 mg of quercetin and 11.41 mg of resveratrol per 100 g of QRH. Intermediate compositions were calculated proportionally according to their respective molar fractions. After QR-NIO incorporation, QRH was mechanically mixed at 500 rpm for 5 min to promote uniform dispersion throughout the food matrix. Non-functionalized hummus (NFH), prepared without QR-NIOs, was processed under otherwise identical conditions. Commercial hummus (CH) was included as an external comparator where indicated. All formulations were aseptically portioned into sterile, airtight containers and stored at 4 ± 1 °C for up to 28 days before physicochemical, microbiological, sensory, antioxidant, and biological evaluations.

The microbiological stability of functional chickpea hummus enriched with synergistic dual-loaded quercetin–resveratrol niosomes (QR-NIOs) was evaluated over 28 days of refrigerated storage (4 ± 1 °C) at 0, 7, 14, 21, and 28 days [72]. Three independent production batches were prepared, and six biological replicates per batch (n = 6) were analyzed at each sampling point, yielding 18 samples per storage time. For microbiological analysis, 25 g of each sample was aseptically homogenized with 225 mL of sterile buffered peptone water, followed by serial decimal dilutions. Results were expressed as colony-forming units per gram (CFU/g) or as presence/absence in 25 g for pathogenic microorganisms. Microbiological quality was assessed by enumerating aerobic mesophilic bacteria, yeasts and molds, total and fecal coliforms, and Enterobacteriaceae, while Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Clostridium perfringens were detected using standardized selective culture and microbiological confirmation procedures.

All microbiological analyses were performed following the standardized analytical procedures established in NOM-210-SSA1-2014 for the detection and enumeration of foodborne microorganisms and in accordance with the hygienic manufacturing criteria described in NOM-251-SSA1-2009. The microbiological quality of the QR-NIO-enriched chickpea hummus throughout refrigerated storage was interpreted according to the corresponding Mexican microbiological specifications applicable to ready-to-eat foods.

Microbiological analyses were performed using the applicable detection and enumeration methods described in NOM-210-SSA1-2014 [73]. Sample preparation and handling followed the relevant hygienic practices described in NOM-251-SSA1-2009 [74]. Acceptance limits and microbiological thresholds were evaluated according to NOM-093-SSA1-1994 [75]. Results for each microorganism were reported across the 28-day refrigerated storage period.

The gastrointestinal stability and bioaccessibility of the functional hummus enriched with the selected synergistic dual-loaded quercetin–resveratrol niosomes (QR-NIOs) were evaluated using a static in vitro digestion model comprising oral, gastric, and intestinal phases [76,77]. Briefly, 5 g of hummus was mixed with simulated salivary fluid containing α-amylase (150 U/mL, pH 7.0) and incubated at 37 °C for 10 min under gentle agitation to reproduce oral digestion. Subsequently, the pH was adjusted to 2.0, simulated gastric fluid containing porcine pepsin (315 U/mL) was added, and samples were incubated at 37 °C for 2 h to mimic gastric conditions.

Following gastric digestion, the pH was adjusted to 7.0 with sodium bicarbonate and simulated intestinal fluid containing pancreatin (4 mg/mL), lipase (60 U/mL), trypsin (100 U/mL), and bile salts (10 mM) was incorporated. Digestion proceeded for 2 h at 37 °C under continuous agitation. The resulting digesta were transferred into pre-hydrated dialysis membranes (MWCO 12–14 kilodaltons) and immersed in phosphate buffer to separate dialyzable and non-dialyzable fractions. The dialyzable fraction was considered the potentially bioaccessible fraction, whereas the non-dialyzable fraction represented compounds remaining within the intestinal lumen. Bioaccessibility was calculated as the percentage of quercetin and resveratrol recovered in the dialyzable fraction relative to their initial content using the following equation: (9) Bioaccessibility % Polyphenol content in the dialysate fraction Initial Polyphenol content × 100

Aliquots were collected after the oral, gastric, and intestinal digestion stages and stored at −20 °C until analysis. Each fraction was subsequently evaluated for total phenolic and flavonoid contents, antioxidant, anti-inflammatory, and α-amylase and α-glucosidase inhibitory activities. All analyses were performed in triplicate to assess the protective effect of QR-NIO encapsulation during simulated gastrointestinal digestion.

Sensory acceptance of the hummus formulations was assessed using a hedonic test with 30 untrained volunteers aged 20–45 years from the university community, following previously reported procedures with minor modifications [32,78]. All participants provided informed consent. Each participant evaluated all three formulations: commercial hummus (CH), experimental hummus without niosomes (NFH), and experimental hummus enriched with 5% (w/w) of the selected 50:50 quercetin–resveratrol niosomal formulation (QRH). Thus, the product was a within-participant factor. Approximately 15 g of each sample was served at room temperature in identical containers labeled with three-digit codes. Presentation order was randomized among participants, and water and unsalted crackers were provided for palate cleansing between samples.

Participants rated appearance, color, aroma, flavor, bitterness, texture, mouthfeel, spreadability, and overall acceptability on a 10-point hedonic scale ranging from 0 (dislike extremely) to 9 (like extremely). Each participant’s score for each formulation and attribute was recorded, preserving the pairing of the three product evaluations. Scores were summarized as means ± standard deviations.