NAR-loaded nano cellulose-pectin Pickering emulsion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of Pickering emulsions, and particularly relates to an NAR-loaded nano cellulose-pectin Pickering emulsion as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, forming a PCNFs-PP particle stabilizer with a stable net structure from nanocellulose and pectin by virtue of a high-pressure homogenization method under various acting forces such as hydrogen bonds and the like, then preparing an NAR-loaded Pickering emulsion system by virtue of the high-pressure homogenization method, and showing that the Pickering emulsion system is spherical, good in dispersity and extremely high in encapsulation efficiency by virtue of TEM (Transmission Electron Microscope). Antioxidant activity evaluation shows that compared with free NAR, the free radical scavenging capacity of NAR-PE is improved by more than two times; in-vitro release and simulated digestion experiments show that the NAR-PE realizes the slow release effect of the NAR and improves the bioavailability of the NAR in gastrointestinal tracts; cell experiments show that the NAR-PE has no toxicity to HaCaT cells and has good biocompatibility.

Description

Technical Field

[0001] The invention belongs to the technical field of Pickering emulsions, and particularly relates to a NAR-loaded nanocellulose-pectin Pickering emulsion, a preparation method and an application thereof. Background Art

[0002] Naringenin (NAR) is a flavonoid compound that is widely found in citrus fruits such as grapefruit and lemon. Naringenin plays a significant protective and beneficial role in human health, such as lowering blood lipids, anti-inflammatory, antioxidant, and anti-cancer effects. Due to the powerful biological activity of naringenin, the biological and pharmaceutical industries have gradually increased their research interest in naringenin in recent years. However, naringenin also has some obvious disadvantages, such as poor water solubility, chemical instability, and low bioavailability. These factors greatly limit its potential application in health promotion. To overcome these problems, researchers usually load naringenin into different carrier systems to improve the bioavailability of naringenin.

[0003] Pickering emulsions (PE), surfactant-free emulsion systems that stabilize the oil-water interface through solid particles, have been widely used in food, cosmetics, and drug delivery. Compared to traditional surfactant-based emulsions, Pickering emulsions offer superior stability and sustained-release properties in formulations and drug delivery. In recent years, natural biopolymers have garnered significant attention and demonstrated great potential as stabilizers for Pickering emulsions.

[0004] Pectin (PP), a biopolymer, possesses numerous biological activities, including antioxidant, hypoglycemic, and anticancer effects. Furthermore, studies have shown that pectin is readily degraded by pectinases produced by colonic microbiota, facilitating targeted release of pharmaceutical agents within the intestine, making it an ideal candidate for biomedical applications and drug delivery. However, pectin's high hydrophilicity due to its abundance of functional groups, such as hydroxyl and carboxyl groups, results in poor stability in Pickering emulsions. Cellulose nanofibers (PCNFs), a nanomaterial derived from natural cellulose, possess excellent biocompatibility, biodegradability, and low toxicity, making them one of the most valuable biopolymers for drug delivery systems. Furthermore, due to their natural amphiphilicity and excellent mechanical strength, PCNFs can form a stable emulsion layer at the oil-water interface in Pickering emulsions through physical adsorption and interfacial interactions. By combining with pectin, PCNFs not only improve the physical stability of the emulsion but also achieve synergistic drug release in the intestine, further enhancing the bioavailability of naringenin. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to provide a NAR-loaded nanocellulose-pectin Pickering emulsion and its preparation method and application.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides a method for preparing a NAR-loaded nanocellulose-pectin Pickering emulsion, comprising the following steps:

[0008] 1) Preparation of grapefruit peel cellulose nanofibers

[0009] The pomelo peel sponge layer is dried and crushed, and sieved to obtain pomelo peel powder;

[0010] Grapefruit peel powder was dissolved in a hydrochloric acid aqueous solution, heated to remove pectin, allowed to cool, centrifuged, and the precipitate was freeze-dried to obtain crude cellulose. The crude cellulose was dissolved in a NaOH solution and mixed, heated to remove hemicellulose and lignin, and the precipitate was collected and freeze-dried to obtain cellulose. The cellulose was dispersed in deionized water and subjected to high-pressure homogenization to obtain grapefruit peel cellulose nanofibers (PCNFs).

[0011] The pH of the hydrochloric acid aqueous solution is 1 to 2;

[0012] The heating temperature is 70-90° C. and the heating time is 1-3 hours;

[0013] The concentration of NaOH is 8-10% (w / v);

[0014] The mass ratio of the cellulose to deionized water is 1:(150-250);

[0015] The pressure of the high-pressure homogenization is 300-500 MPa;

[0016] 2) Preparation of grapefruit peel pectin

[0017] The pomelo peel powder was mixed with NaOH solution and subjected to ultrahigh pressure treatment, followed by centrifugation. The supernatant was added with 95% ethanol and allowed to stand overnight. The precipitate was washed with 95% ethanol and freeze-dried to obtain pomelo peel pectin PP.

[0018] The mass ratio of the pomelo peel powder to the NaOH solution is 1:(35-60);

[0019] The concentration of the NaOH solution is 0.01M;

[0020] The pressure of the ultra-high pressure treatment is 500-600 MPa;

[0021] 3) Preparation of Nanocellulose-Pectin Pickering Emulsion

[0022] The grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP obtained in step 1) are dissolved and mixed, and subjected to high-pressure homogenization to obtain a nanocellulose-pectin Pickering emulsion;

[0023] The concentrations of the grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP are 0.1 to 0.9 wt % respectively, dissolved in ultrapure water, preferably 0.5 wt %;

[0024] The mass ratio of the grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP after dissolution is (1-3): (1-3);

[0025] The pressure of the high-pressure homogenization treatment is 50-80 MPa;

[0026] 4) Preparation of NAR-loaded nanocellulose-pectin Pickering emulsion

[0027] Naringenin NAR was dissolved in the oil phase, and nanocellulose-pectin Pickering emulsion was added, and high-speed homogenization and high-pressure homogenization were performed successively to obtain NAR-loaded nanocellulose-pectin Pickering emulsion (NAR-PE);

[0028] The oil phase includes soybean oil;

[0029] The rotation speed of the high-speed homogenizer is 10000-20000 r / min;

[0030] The pressure of the high-pressure homogenization is 300-1200 bar.

[0031] The present invention also provides a NAR-loaded nanocellulose-pectin Pickering emulsion prepared by the above method, which is in the form of spherical particles. In the structure, the NAR load is embedded in a network structure formed by the nanocellulose and pectin.

[0032] The present invention also provides an application of a nanocellulose-pectin Pickering emulsion as a carrier for delivering NAR or other hydrophobic bioactive substances.

[0033] The present invention also provides an application of a NAR-loaded nanocellulose-pectin Pickering emulsion in the preparation of sustained-release drugs.

[0034] The beneficial effects of the present invention are as follows:

[0035] The NAR-loaded nanocellulose-pectin Pickering emulsion of the present invention first forms a PCNFs-PP particle stabilizer with a stable network structure by using a high-pressure homogenization method to form nanocellulose PCNFs and pectin PP through multiple forces such as hydrogen bonds. The PCNFs can form a 3D network structure and aggregates connecting emulsion droplets at the oil-water interface, and synergistically act with the multi-layer network structure formed after adding PP to further reduce the emulsion particle size and improve the stability of the system. Then, a NAR-loaded Pickering emulsion system (NAR-PE) is prepared by the high-pressure homogenization method. TEM shows that the NAR-PE is spherical and has good dispersibility. The NAR-PE shows an extremely high encapsulation rate. The antioxidant activity evaluation showed that the free radical scavenging ability of NAR-PE was more than 2 times higher than that of free NAR; in vitro release and simulated digestion experiments showed that NAR-PE achieved a sustained-release effect of NAR and improved the bioavailability of NAR in the gastrointestinal tract; cell experiments showed that NAR-PE was non-toxic to HaCaT cells and had good biocompatibility. The above results also showed that the prepared PCNFs-PP polysaccharide-based composite particles stabilized Pickering emulsion is an effective carrier for delivering NAR and other hydrophobic bioactive substances. The present invention provides a theoretical basis and new strategy for the development of efficient carriers of natural active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Appearance of Pickering emulsions stabilized with different addition orders after 2 hours and 30 days (a) and emulsification index of Pickering emulsions stabilized with different addition orders after 30 days of storage (b);

[0037] Figure 2 Appearance of Pickering emulsions stabilized with different addition ratios after 2h and 30d (a) and emulsification index of Pickering emulsions stabilized with different addition ratios after 30d storage (b);

[0038] Figure 3 Appearance of Pickering emulsions stabilized with different addition concentrations after 2 hours and 30 days (a) and emulsification index of Pickering emulsions stabilized with different addition concentrations after 30 days of storage (b);

[0039] Figure 4 Appearance of emulsions with different high-pressure homogenization pressures and oil phase fractions after 2 hours (a) and 30 days (b);

[0040] Figure 5 Optical microscopy images of the emulsions at different homogenization pressures (a-e) and the changes in particle size and Zeta potential of the Pickering emulsion after storage for 30 days (f);

[0041] Note: a~e correspond to homogenization pressure p values of 300~1100 bar respectively; scale length is 50μm, Figure 6 、 Figures 8 to 10 same;

[0042] Figure 6 Optical microscopic structures of emulsions with different oil phase fractions (a-e) and changes in particle size and Zeta potential of Pickering emulsion after storage for 30 days (f);

[0043] Figure 7 The viscosity curve of the emulsion under different influencing factors and the frequency dependence curves of the storage modulus G' and loss modulus G";

[0044] Note: a, b represent different homogenization pressures (φ=50%); c, d represent different oil phase fractions (p=500 bar);

[0045] Figure 8 Appearance of emulsions at different ionic strengths after 2 h and 30 d (a), changes in particle size and zeta potential of Pickering emulsions after 30 d of storage (b), and optical microscope images (c);

[0046] Figure 9 Appearance of the emulsion after 2 h and 30 d at different temperatures (a), changes in particle size and Zeta potential of the Pickering emulsion after 30 d of storage (b), and optical microscope image (c);

[0047] Figure 10 Appearance of the emulsion at different pH values after 2 hours and 30 days (a), changes in particle size and Zeta potential of the Pickering emulsion after 30 days of storage (b), and optical microscope image (c);

[0048] Figure 11 Confocal laser scanning microscopy images (a–c) and cryo-scanning electron microscopy images (d–f) of Pickering emulsions stabilized by different particles;

[0049] Note: Figures d to f are Pickering emulsions stabilized by different particles, where Figure d is PP particles, Figure e is PCNFs particles, and Figure f is PCNFs-PP particles; the scale bars in Figures a to c are 100 μm; the scale bars in Figures d to f are 20 μm;

[0050] Figure 12 XRD patterns of PCNFs, PP and PCNFs-PP (A) and the effect of the mass ratio of PCNFs to PP on the EE and LC of NAR-PE (B);

[0051] Figure 13 FTIR spectra of PCNFs, PP and PCNFs-PP;

[0052] Figure 14FTIR spectra of NAR, PE and NAR-PE;

[0053] Figure 15 SEM images of PCNFs (CD), PP (AB), and PCNFs-PP (EF);

[0054] Figure 16 Particle size distribution of NAR-PE on the 0th day of storage (A), particle size distribution of NAR-PE on the 30th day of storage (B), and TEM images of NAR-PE (CD);

[0055] Note: Samples 1-5 correspond to PCNFs to PP mass ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively;

[0056] Figure 17 Degradation curves of naringenin at different PCNFs to PP mass ratios under light (A), dark (B), ultraviolet (C), and high temperature (50°C) (D) conditions;

[0057] Note: Samples a, b, c, d, and e correspond to PCNFs to PP mass ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively;

[0058] Figure 18 Figure 3 shows the results of the DPPH free radical scavenging ability (A) and ABTS free radical scavenging ability (B) of NAR-PE and PE at different PCNFs to PP mass ratios, where the antioxidant capacity of free NAR corresponds to the encapsulated NAR content at different mass ratios;

[0059] Figure 19 The content of naringenin in the release medium during in vitro release (A), the in vitro release curves of naringenin in different systems (B), the effect of simulated gastric fluid on the retention rate of naringenin (C), and the effect of simulated intestinal fluid on the retention rate of naringenin (D);

[0060] Figure 20 The graph shows the viability (37°C) of HaCat cells after incubation with NAR-PE and NAR for 24 hours. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0062] The present invention adopts:

[0063] Dried pomelo spongy tissue (Citrus maxima (Burm.)) from Shatian pomelo, a by-product of pomelo processing, was obtained from a local producer (Meizhou, Guangdong, China). The pomelo used was ripe, healthy, and disease-free.

[0064] Soybean oil was purchased from DR Food Store (Beijing, China);

[0065] Naringenin (4′,5,7-trihydroxyflavanone) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China);

[0066] HaCaT cells were purchased from Beina Chuanglian Biotechnology Co., Ltd. (China);

[0067] Other chemical reagents were of analytical grade.

[0068] Example 1

[0069] Preparation method of NAR-loaded nanocellulose-pectin Pickering emulsion

[0070] 1) Preparation of grapefruit peel cellulose nanofibers

[0071] The pomelo peel sponge layer (dried pomelo sponge-like tissue) was dried and then pulverized and passed through a 60-mesh sieve.

[0072] The powder was dissolved in a hydrochloric acid aqueous solution with a pH of 1.7 at a ratio of 1:20, heated at 80°C for 2 hours to remove pectin, allowed to cool, and centrifuged at 5000 rpm for 10 minutes. The precipitate was freeze-dried to obtain crude cellulose. The crude fiber was dissolved in an 8% NaOH (w / v) solution at a ratio of 1:20 and mixed. The mixture was heated at 70°C for 1.5 hours to remove hemicellulose and lignin. The precipitate was collected and freeze-dried to obtain cellulose. The cellulose was dispersed in deionized water and homogenized at 140 MPa for 10 cycles to obtain grapefruit peel cellulose nanofibers (PCNFs).

[0073] 2) Preparation of grapefruit peel pectin

[0074] The pomelo peel powder was mixed with a 0.01M NaOH solution at a ratio of 1:40 and placed in a vacuum sealed bag. The mixture was then treated with an ultrahigh pressure device (500 MPa, 10 min). The mixture was collected and centrifuged at 5000 r / min for 15 min. The supernatant was added with 95% ethanol at a ratio of 1:2 and allowed to stand overnight. The precipitate was washed three times with 95% ethanol to remove impurities. The ethanol was removed by rotary evaporation. The mixture was collected and freeze-dried to obtain pomelo peel pectin PP.

[0075] 3) Preparation of Nanocellulose-Pectin Pickering Emulsion

[0076] Grapefruit peel cellulose nanofibers (PCNFs) and grapefruit peel pectin (PP) were redissolved in deionized water, with the concentration of PCNFs and PP controlled at 0.5 wt%. PCNFs and PP were mixed at different mass ratios of 3:1, 2:1, 1:1, 1:2, and 1:3. PCNFs-PP were homogenized in a high-pressure homogenizer at 50 MPa for two cycles to obtain PCNFs-PP. The samples were freeze-dried and stored in a refrigerator at 4°C until use.

[0077] 4) Preparation of NAR-loaded nanocellulose-pectin Pickering emulsion

[0078] Naringenin NAR was dissolved in 50 mL of soybean oil and stirred at 600 rpm to obtain an oil phase containing NAR at a concentration of 4 mg / mL;

[0079] 50 mL of PCNFs-PP solution with different mass ratios was added to the oil phase. The mixture was T18 (IKA, Germany) was homogenized at a constant speed of 20000 r / min for 2 min to obtain a crude emulsion. The crude emulsion was homogenized twice by a high-pressure homogenizer at a pressure of 300 bar to obtain NAR-PE (NAR 0.2% w / v). The sample was stored in a refrigerator at 4°C.

[0080] In the following data analysis, all experiments were performed three times, and the results are expressed as mean ± standard deviation. Statistical differences were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple comparisons. Differences were considered statistically significant when the p value was less than 0.05.

[0081] NAR concentration was determined using HPLC (LC-20AT, Japan). The chromatographic column was a COSMOSIL 5C18-AR-II column (4.6 mm × 150 mm, 5 μm) stored at 30°C. The mobile phase consisted of methanol and 0.05% phosphoric acid solution (70:30, v / v) at a flow rate of 0.7 mL / min. The injection volume was 20 μL, and the detection wavelength was 289 nm.

[0082] Test Example 1

[0083] Properties of PCNFs-PP emulsion

[0084] Preparation of Nanocellulose-Pectin (PCNFs-PP) Emulsion: Pectin obtained by freeze drying was dispersed in ultrapure water to obtain a pectin solution with a concentration of 0.1-0.9 wt%. At the same time, a cellulose nanofiber solution with a concentration of 0.1-0.9 wt% was prepared and stored in a refrigerator at 4°C for later use. Homogenization was performed using T18 (IKA, Germany) with a fixed speed of 10000 r / min for 1 min.

[0085] The optimal emulsion preparation parameters were determined by measuring the emulsion zeta potential, particle size, emulsification index (EI), emulsification activity index (EAI), emulsification stability index (ESI), emulsion appearance, and emulsification index using the order of addition of emulsifier and oil phase, the ratio of emulsifier addition, and emulsifier concentration as variables. The test method is as follows.

[0086] Particle size and potential: Particle size was determined using a Mastersizer 3000 laser particle size analyzer. The zeta potential of the emulsions was measured using a Zatasizer Nano ZS 90 nanoparticle size analyzer. Samples were diluted 400-fold with deionized water before testing to avoid multiple scattering effects. Both measurements were performed at 25°C. The D3,2 value (surface-weighted mean diameter) was used to characterize the emulsion particle size. The D3,2 value (surface-weighted mean diameter) was calculated as follows:

[0087] D3,2=∑ i n i d i 3 / ∑ i n i d i 2 ;

[0088] Where: n i is the number of particles with the same diameter, d i is the diameter. All experiments were performed three times and the results are shown as the average value.

[0089] Emulsification activity index (EAI) and emulsion stability index (ESI): Add 50 μL of fresh emulsion to 5 mL of 0.1% SDS solution and mix thoroughly. Then, measure the absorbance at 500 nm using a UV spectrophotometer. After 10 minutes, repeat the above steps with 50 μL of the emulsion and read the absorbance. Use 0.1% SDS solution as a reference. The formulas for calculating the emulsification activity index (EAI) and emulsion stability index (ESI) are as follows:

[0090] EAI(m 2 / g) = 2 × 2.303 × A × DF / 1 × c × θ × φ;

[0091] ESI(min)=A0×△T / (A0-A 10 );

[0092] Where: A is the absorbance at 500nm; DF is the dilution factor, 100; c is 10000g / m 3; θ is the volume fraction of the oil phase of the emulsion, 0.5; φ is the length of the cuvette, 1 cm; A0 and A 10 The absorbance of the diluted emulsion at 0 and 10 minutes respectively; Δt is 10 minutes.

[0093] Emulsion appearance and creaming index: 5 mL of emulsion was pipetted into a test tube and stored at room temperature for 14 days. The effects of different high-pressure homogenization pressures and oil phase fractions on the storage stability of the emulsion were characterized by visual observation and measurement of the creaming index (CI). The creaming index was calculated using the following formula:

[0094] CI (%) = H2 × 100% / H1;

[0095] Where: H2 is the height of the whey layer and H1 is the total height of the emulsion.

[0096] 1) Effect of addition order on emulsion properties

[0097] With both PCNFs and PP concentrations fixed at 0.5 wt%, 2.5 mL of PP was homogenized with 5 mL of soybean oil for 1 minute. 2.5 mL of PCNFs was then added to the mixture and homogenized for 1 minute. The resulting Pickering emulsion was transferred to a glass bottle and designated A1. The order of PCNFs and PP addition was changed and the above steps were repeated. The resulting emulsion was designated A2. A mixture of 2.5 mL of PCNFs and 2.5 mL of PP was homogenized for 1 minute. 5 mL of soybean oil was then added to the mixture and homogenized for 1 minute. The resulting emulsion was designated A3.

[0098] Table 1 Zeta potential, particle size and emulsification properties of Pickering emulsions stabilized by different addition orders

[0099]

[0100] Zeta potential is an important indicator of system stability; its magnitude determines whether a dispersed system maintains good stability in its application environment. It is generally believed that when the absolute value of the zeta potential exceeds 30 mV, strong electrostatic repulsion prevents droplet aggregation and helps form a stable emulsion. In Table 1, the absolute values of the zeta potential for all samples remained stable above 33 mV with no significant differences, indicating that changing the order of adding the particle stabilizers had no significant effect on the zeta potential.

[0101] It is well known that smaller droplet size helps enhance the physical stability of the emulsion. Table 1 shows that the order of addition has a significant effect on droplet size. The emulsion stabilized by adding PCNFs first and then PP (sample A2) has the smallest D3,2 value of 11.8 ± 0.2 μm, which is significantly lower than that of the other samples. This may be because the rod-shaped PCNFs first combine with the oil to form a Pickering emulsion, and then the addition of PP forms an interfacial coating. These morphological changes affect the adsorption and accumulation behavior of the composite particles, thereby reducing the droplet size.

[0102] Emulsification activity index (EAI) and emulsion stability index (ESI) are important indicators for characterizing the emulsion's ability to stabilize and resist separation and maintain dispersion. In Table 1, changing the order of adding the particle stabilizer has no significant effect on EAI. However, the order of addition has a certain effect on ESI. The ESI value of sample A3 is lower, and there is no significant difference in the ESI values of other samples. The effect of different addition orders on the emulsification index (CI) of the emulsion is shown in Figure 1. Figure 1 As shown, all emulsions separated rapidly within the first 2 hours, followed by fat buoyancy within 3-4 days, reaching equilibrium. Samples A1 and A2 had higher emulsification indices than sample A3. Ultimately, the order of adding PCNFs first, followed by PP, was chosen as the particle stabilizer addition order in the Pickering emulsion system.

[0103] 2) Effect of addition ratio on emulsion properties

[0104] The PCNFs and PP concentrations were fixed at 0.5 wt %, and the total amount of PCNFs and PP added was 5 mL. PP and PCNFs were mixed and homogenized at a v / v ratio of 1:0, 1:1, 1:3, 1:5, and 1:7, respectively, for 1 minute. 5 mL of soybean oil was then added and homogenized for 1 minute. The resulting emulsions were transferred to glass bottles for later use. Samples were designated B1, B2, B3, B4, and B5, respectively.

[0105] Table 2 Zeta potential, particle size and emulsification properties of Pickering emulsions stabilized by different addition ratios

[0106]

[0107] The effects of different addition ratios of particle stabilizers on the properties of the emulsion are shown in Table 2. Changing the addition ratio has a significant effect on the particle size and Zeta potential of the emulsion. As the addition ratio of PCNFs increases, the absolute value of the Zeta potential of the emulsion shows a gradual upward trend, which may be attributed to the presence of a large number of carboxylate units on PCNFs. As the proportion of PCNFs increases, the Zeta potential value of the emulsion will further decrease. It is worth noting that the Zeta potential of sample B1 is only -17.5±2.1mV, indicating that the stability of the single pectin emulsion system is poor, which is also reflected in the emulsification index ( Figure 2Furthermore, as the proportion of PCNFs increased, the particle size of the emulsion decreased and then increased, indicating that adding a certain amount of PCNFs helped form a more stable emulsion. The EAI and ESI values of sample B2 were higher than those of the other samples to varying degrees.

[0108] Effects of different particle stabilizer addition ratios on emulsification index Figure 2 As shown in the figure, all samples quickly separated in the first two hours and reached equilibrium in 2 to 4 days. Among them, sample B1 broke after 14 days of storage, and the oil and water phases completely precipitated, indicating that single pectin may not be able to effectively stabilize the Pickering emulsion during storage. As the addition ratio of PCNFs gradually increased, the emulsification index of the emulsion showed a trend of first increasing and then decreasing. Except for samples B1 and B5, the CI values of the remaining samples remained at 80% or above after two weeks of storage. Finally, the addition ratio corresponding to sample B2 (PP: PCNFs = 1:1) was selected as the addition ratio of the particle stabilizer in the Pickering emulsion.

[0109] 3) Effect of addition concentration on emulsion properties

[0110] The PCNFs concentration was fixed at 0.5wt%, and the PCNFs and PP addition amounts were both 2.5mL. The PP concentrations were controlled at 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%. PCNFs and PP solutions of varying concentrations were homogenized for 1 minute. 5mL of soybean oil was then added and homogenized for 1 minute. The resulting emulsions were transferred to glass bottles for later use. These samples were designated C1, C2, C3, C4, and C5.

[0111] Table 3 Zeta potential, particle size and emulsification properties of Pickering emulsions stabilized by different addition concentrations

[0112]

[0113] The effects of different particle stabilizer addition concentrations on the emulsion properties are shown in Table 3. Due to the presence of carboxyl groups in pectin, pectin is considered a polyelectrolyte and is negatively charged under neutral conditions. As the PP addition concentration increases, the Zeta potential of the sample shows a gradual downward trend. Changes in addition concentration have a significant effect on the particle size of the emulsion. As the PP addition concentration increases, the particle size of the emulsion shows a trend of first decreasing and then increasing. The particle size of sample C3 is significantly lower than that of the other samples. Observing Table 3, it can be found that changing the addition ratio has little effect on the EAI value of the emulsion, but has a significant effect on the ESI value of the emulsion. The ESI value of sample C3 is significantly higher than that of the other samples.

[0114] Effects of different particle stabilizer addition ratios on emulsification index Figure 3As shown, all samples rapidly separated within the first two hours and reached equilibrium within three days. As the PP concentration increased, the emulsification index of the emulsions initially increased and then decreased. With the exception of samples C1 and C5, the emulsification index of all other samples was 80% or above. The concentration corresponding to sample C3 (0.5 wt% PP, 0.5 wt% PCNFs) was ultimately selected as the particle stabilizer concentration for the Pickering emulsion.

[0115] 4) Emulsion microstructure observation

[0116] After that, cellulose nanofibers at a concentration of 0.5 wt% were mixed with soybean oil and the mixture was homogenized. Then, a pectin solution (0.5 wt%) of the same volume as the cellulose nanofibers was added to the mixture and homogenized to obtain a crude emulsion. The crude emulsion was homogenized twice by a high-pressure homogenizer to obtain different high-pressure homogenization pressures (p = 300, 500, 700, 900, 1200 bar, ) and different oil phase fractions ( 30%, 40%, 50%, 60%, p = 500 bar) Pickering emulsions were prepared, and ProClean 300 (0.1%, v / v) was added to the emulsions to inhibit microbial growth. The formed Pickering emulsions were transferred to glass bottles and stored in a refrigerator at 4°C until use.

[0117] The microstructure of the emulsion was observed using an optical microscope with a 20x objective. To investigate the emulsion's stability mechanism, the emulsion was observed using confocal laser scanning microscopy (CLSM). Before observation, 20 μL of Nile Red and Fluorescent White (1 mg / mL) were added to 1 mL of the emulsion and incubated overnight in the dark at room temperature. Then, 20 μL of the emulsion was deposited onto a concave confocal microscope slide, covered with a clean coverslip, and the emulsion droplets were observed using a 20x objective. Excitation wavelengths were 514 nm and 405 nm, respectively. Cryo-scanning electron microscopy (cryo-SEM) was used to further investigate the distribution of PCNFs-PP within the emulsion droplets. A drop of the emulsion was deposited onto a sample stage, which was then rapidly frozen in liquid nitrogen slush for 30 seconds. The stage was then transferred to a sample preparation chamber under vacuum using a cryo-preparation transfer system for sublimation gold coating. The sample was sublimated at -90°C for 10 min and then sputtered with gold for 60 s at a current of 10 mA. The sample was then placed in the scanning electron microscope sample chamber for observation with a cold stage temperature of -140°C and an accelerating voltage of 5 kV.

[0118] In order to investigate whether high-pressure homogenization pressure (p) affects the properties of emulsions, emulsions with different high-pressure homogenization pressures (p = 300, 500, 700, 900, 1100 bar) were prepared, and the appearance of the emulsions at different storage times was photographed ( Figure 4 The results show that when the high-pressure homogenization pressure increases from 300 bar to 1100 bar, the appearance of the emulsion does not change significantly, and some samples show slight water precipitation after 30 days of storage. Excessive high-pressure homogenization pressure will lead to an increase in the emulsion particle size and a decrease in the absolute value of the potential ( Figure 5 d,e,f), which is similar to the research results of Ren et al. [Ren ZY,Chen ZZ,Zhang YY,et al.Characteristics and rheological behavior of Pickering emulsionsstabilized by tea water-insoluble protein nanoparticles via high-pressurehomogenization[J],International Journal of Biological Macromolecules 2020,151:247-256.], indicating that high-pressure homogenization can significantly reduce the particle size of droplets within a certain pressure range, and exceeding a certain pressure will lead to aggregation of droplets.

[0119] In addition, the oil phase ratio The effect on the stability of the emulsion is crucial. Figure 4 For different oil phase ratios ( As shown in the storage appearance diagram of the samples with different oil phase ratios (30, 40, 50, and 60%), when the oil phase ratio increases from 20% to 60%, the emulsion emulsion layer ratio gradually increases until it is completely emulsified. Although the emulsion layer ratio of some samples decreases slightly after 30 days of storage, they still show extremely high storage stability compared with other samples. Figure 6It was found that changes in the oil phase ratio can significantly affect the particle size of the emulsion. The results show that as the oil phase volume fraction increases, the emulsion layer ratio gradually increases and the droplet size gradually increases. Badar et al. also reported a similar situation [Badar IH, Wang ZY, Sun FD, et al. Influence of varying oil phase volume fractions on the characteristics of flaxseed-deriveddiglyceride-based Pickering emulsions stabilized by modified soy protein isolate [J], Food Research International 2024, 175: 113812.], when the oil-water volume ratio increased from 20% to 80%, the droplet size increased from about 7μm to 23μm, and the emulsification index also gradually increased from 30% to 100%. This may be attributed to the fact that as the oil phase ratio increases, the particles in the system are not sufficient to effectively stabilize all the oil droplets, and eventually aggregate to form larger droplets.

[0120] 5) Rheological properties

[0121] The rheological properties of the emulsion were characterized using a modular intelligent advanced rheometer equipped with a 40 mm parallel plate. The gap value was set to 1.0 mm and the temperature was 25 °C. For the dynamic viscoelastic test, a strain sweep was performed at a fixed frequency of 10 Hz (strain variation range of 0.1 to 10%) to determine the linear viscoelastic region. 0.5% was selected as the specific strain value for the frequency sweep, and the storage modulus (G') and loss modulus (G") were recorded as the frequency varied from 0.1 Hz to 10 Hz. For the steady-state shear viscosity analysis, the shear rate was set to 0.1 to 100 s -1 The apparent viscosity (η) of the sample is recorded as a function of shear rate over a range of .

[0122] In order to illustrate the effects of different high-pressure homogenization pressures and oil phase addition ratios on the properties of the emulsion, the rheological properties of the emulsion at different homogenization pressures and different oil phase ratios were evaluated using a rheometer. Figure 7 As shown in Figures a and c, the PCNFs-PP-stabilized Pickering emulsion exhibits typical shear-thinning behavior, with the apparent viscosity decreasing with increasing shear rate, demonstrating typical emulsion characteristics. The viscosity of the emulsion increases slightly with increasing homogenization pressure. Furthermore, within the dynamic frequency sweep range, higher homogenization pressure induces higher moduli. Therefore, the rheological properties of PCNFs-PP-based Pickering emulsions can be controlled by varying the homogenization pressure.

[0123] Figure 7 Figures b and d characterize the frequency-modulus curves of the emulsions at different homogenization pressures and different oil phase ratios. The results show that the storage modulus (G') of all emulsion samples is greater than the loss modulus (G"), which indicates that the Pickering emulsion stabilized by PCNFs-PP exhibits elastic gel-like behavior within a specific frequency range (0.1-10 Hz). With the increase of the oil phase volume fraction, the viscosity and modulus of the emulsion increase accordingly. Similar to the results of previous studies, this can be attributed to the close packing of oil droplets at high oil phase volume fractions, which leads to an increase in the viscosity and elasticity of the emulsion. When the oil phase volume fraction reaches 60%, the viscosity and modulus of the emulsion are the largest, which means that the system has greater plasticity and a stronger support structure. These results indicate that the formation of a three-dimensional network structure by PCNFs-PP plays an important role in the stability of the Pickering emulsion system.

[0124] 6) Stability

[0125] The effects of different pH, ionic strength and temperature on the stability of the emulsion were investigated. First, the concentrations of PP and PCNFs were fixed at 0.5wt%, the addition ratio was 1:1, the high-pressure homogenization condition was 500 bar, and the cycle was repeated twice. When investigating the effect of ionic strength on the stability of the emulsion, NaCl was used to adjust the ionic strength of the emulsion to: 0mM, 40mM, 80mM, 120mM, 160mM, 200mM. When investigating the effect of temperature on the stability of the emulsion, the storage temperature was controlled at 4°C, 25°C, 50°C, and 80°C. When investigating the effect of different pH values on the stability of the emulsion, 1M NaOH or 1M HCl was used to adjust the pH value of the sample to: 3, 5, 7, 9, 11. All samples were stored for 24 hours under different conditions.

[0126] During the application of Pickering emulsions, high temperature, high salt concentrations, and acidic and alkaline conditions are common process environments. To adapt Pickering emulsions to various drug delivery or food systems, the emulsion's stability needs to be evaluated in different external environments. The effects of varying ionic strength (40–200 mM), temperature (4, 25, 50, and 80°C), and pH (3.0–11.0) on the emulsion's properties were investigated.

[0127] In the practical application of Pickering emulsion, the influence of ionic strength on the stability of the emulsion cannot be ignored. The experiment explored the effect of different ionic strengths on the stability of Pickering emulsion. Figure 8 As shown in Figure 2, when the added NaCl concentration increased from 40 mM to 200 mM, some emulsion samples underwent slight stratification after storage for 30 days ( Figure 8 In addition, with the increase of ionic strength, the particle size of the emulsion increases from the initial 11.9 μm to 13.7 μm, while the absolute value of the Zeta potential of the emulsion decreases gradually ( Figure 8 This may be attributed to the electrostatic shielding effect after adding different concentrations of NaCl, which causes the droplets of the emulsion to aggregate to varying degrees. The optical microscope image ( Figure 8 The results of the study show that the Pickering emulsion stabilized by PCNFs-PP has good stability against ionic strength.

[0128] Effects of different storage temperatures on emulsion properties Figure 9 As shown in Figure 2, after 30 days of storage, the emulsion did not show any flocculation or stratification. Figure 9 (a) When the temperature increases from 4°C to 50°C, the absolute value of the emulsion's Zeta potential decreases from 44.93 mV to 43.13 mV. The absolute value of the Zeta potential remains greater than 30 mV, indicating that the emulsion system remains highly stable. The emulsion particle size increases from 9.45 μm to 11.9 μm (4-25°C), which may be attributed to the slowing of the thermal motion of the droplets at low temperatures, resulting in weakened interactions between the particles and a reduced tendency for the droplets to aggregate ( Figure 9 b). When the ambient temperature reached 80°C, the particle size of the emulsion increased significantly (24.3 μm, Figure 9 As the droplets aggregate, the zeta potential of the emulsion changes accordingly, decreasing from an initial 44.93 mV to 21.5 mV in Figures b and c). This is due to the thermodynamic instability of the emulsion, which gradually destabilizes under long-term high temperature conditions (80°C).

[0129] The results showed that the PCNF-PP-stabilized Pickering emulsion exhibited no significant change in droplet size (9.45-11.9 μm) after treatment at different temperatures (4-50°C), and the emulsion showed no obvious phase separation after long-term storage. This suggests that the PCNFs-PP synergistically stabilized Pickering emulsion can maintain long-term storage stability within a certain temperature range.

[0130] Effects of different pH values on emulsion properties Figure 10 As shown in Figure 2, similar to previous reports, the absolute value of the Zeta potential of the Pickering emulsion under acidic conditions (pH 3-5) is low ( Figure 10 b), which may be attributed to the protonation of some carboxylic acid groups on the surface of PP and PCNFs under acidic conditions, resulting in relatively weak electrostatic repulsion between droplets, and ultimately causing a slight stratification of the emulsion ( Figure 10 Under alkaline conditions, as the environmental pH increases, PP and PCNFs gradually deprotonate, and the absolute value of Zeta potential gradually increases ( Figure 10 b). It is worth noting that under acidic conditions, the emulsion exhibits a slight water precipitation phenomenon ( Figure 10 In a), as the environmental pH increases (pH 7-11), the water layer of the emulsion gradually disappears. This may be because citrus fiber has a higher swelling capacity under alkaline conditions, which allows water to enter it. Another study showed that the hydroxyl groups on the cellulose surface can form hydrogen bonds with water molecules, allowing water molecules to bind to the fiber surface, thereby reducing the precipitation of water. In addition, the particle size of the emulsion remained between 11.9 and 14.1 μm under different pH conditions, and the size of the emulsion droplets did not change significantly under an optical microscope ( Figure 10 c), indicating that the PCNFs-PP stabilized Pickering emulsion has strong pH stability.

[0131] All emulsions were stained with fluorescent dyes, and their morphologies were observed under CLSM. Figure 11 The CLSM image of the Pickering emulsion stabilized by PCNFs-PP is depicted. It can be seen that the oil phase ( Figure 11 a) is the PCNFs-PP aqueous phase showing blue fluorescence ( Figure 11 This demonstrates that the Pickering emulsion stabilized by PCNFs-PP is an oil-in-water (O / W) emulsion.

[0132] The distribution of particles in the emulsion can be directly observed in the cryo-scanning electron microscopy images ( Figure 11 ).observe Figure 11 It can be found in the d that PP presents a multilayer network structure in the emulsion, which stabilizes the emulsion by capturing the emulsion droplets in the layered structure. By observing the distribution of PCNFs in the emulsion droplets ( Figure 11 e), PCNFs were found to form 3D network structures and aggregates connecting the emulsion droplets.

[0133] After the addition of PP, the emulsion droplets were captured and stabilized in the 3D network structure formed by PCNFs-PP ( Figure 11 f) and wrap it with a multi-layer network structure formed by pectin, while observing Figure 11 It can be found from the df in that under the same homogenization conditions, the droplet size of the Pickering emulsion stabilized by PCNFs-PP is uniform, and the particle size is significantly smaller than that of the emulsion stabilized by a single particle.

[0134] 7) X-ray diffraction (XRD)

[0135] X-ray diffraction (XRD) patterns were recorded using a Rigaku MiniFlex600 X-ray diffractometer (Japan) at 40 kV and 30 mA, with a 2θ range of 5° to 60° and a scan rate of 2° / min.

[0136] The crystallinity diagrams of PCNFs, PP and PCNFs-PP were analyzed by XRD. Figure 12 As shown. All samples exhibited a distinct diffraction peak near 22.2°, attributed to the 1β structure of cellulose. Furthermore, samples containing PCNFs exhibited diffraction peaks at 16.6°, 22.2°, and 34.8°, corresponding to the 004, 200, and 110 planes of cellulose I, respectively, indicating that high-pressure homogenization did not destroy the crystalline structure of cellulose. For the pectin sample, a broad, diffuse peak appeared between 15° and 25°, indicating the presence of a non-crystalline structure and amorphous characteristics in the pectin. The crystallinity of PCNFs, PP, and PCNFs-PP calculated by the peak area method was 21.27%, 9.08%, and 15.88%, respectively. The results showed that PCNFs-PP had a lower degree of crystallinity than PCNFs, a phenomenon attributed to the addition of amorphous PP to PCNFs.

[0137] 8) Fourier transform infrared spectroscopy (FT-IR)

[0138] The FTIR spectra of the samples were detected using a Fourier transform infrared spectrometer (IRTracer 100, Shimadzu, Japan). Before the test, the samples were thoroughly mixed with dry KBr powder in a mortar at a ratio of 1:100 (w / w), then pressed into thin slices and placed in the optical path for scanning at a wavelength range of 400 cm -1 ~4000cm -1 .

[0139] The potential interactions during the formation of PCNFs-PP were analyzed by FTIR. Figure 13 As shown, PCNFs and PP are at 3282 cm -1 and 3277cm -1 The characteristic peak of PCNFs-PP is shifted to 3334 cm-1 when the two are mixed together. -1 , indicating that hydrogen bonds were formed between PCNFs and PP. -1 There is also a characteristic peak at 1739cm, which is related to the stretching vibration of the CH group. -1 A characteristic peak was observed at 1602 cm -1 The characteristic peaks related to C=O were observed at 1014-1018 cm -1 The characteristic peaks were observed to be related to the COC vibration of the glycosidic bond. In addition, the peak at 1739 cm -1 and 1602cm-1 The ratio of the peak areas at reflects the degree of methoxylation (DM) of pectin. FTIR results show that PP undergoes demethoxylation, resulting in a decrease in the DM value. According to the degree of methylation, pectin is divided into low methoxy (DM < 50%) or high methoxy (DM > 50%) types. Obviously, PP belongs to low methoxy pectin, which has stronger gelling properties compared with high methoxy pectin. Related research results show that ultra-high pressure assisted alkaline extraction will cause the HG region of pectin to be destroyed, and a large number of neutral sugar units will be attached to the RG-Ⅰ skeleton. The increase in the RG-Ⅰ domain helps to improve the viscosity, thermal stability, antioxidant and gelling properties of pectin.

[0140] In order to understand the interaction between NAR and PE, FTIR tests were performed on NAR, PE and NAR-PE. The results are shown in the figure below. Figure 14 The characteristic peaks of naringenin spectrum appear at 3113 cm -1 、2831cm -1 、1633cm -1 、1082cm -1 and 1600cm -1 3335cm -1 There is an obvious characteristic peak at 3331cm, which is the same as the characteristic peak of PCNFs-PP. It is a typical OH characteristic peak. After adding NAR, the characteristic peak was observed to shift to 3331cm in NAR-PE. -1 This may be related to the hydrogen bond between naringenin and PCNFs-PP. In addition, the characteristic peak in the figure is from 1647 cm -1 (PE) shifted to 1635cm -1 The presence of hydrophobic interactions (NAR-PE) was confirmed. More importantly, the absence of some key characteristic peaks of NAR in NAR-PE indicated that NAR was successfully encapsulated in PE. Similar observations were made for other lipophilic compounds. These results suggest that the formation of NAR-PE is the result of multiple interactions.

[0141] 9) Scanning electron microscopy (SEM)

[0142] The morphology of PCNFs, PP and PCNFs-PP was observed by scanning electron microscopy. The powdered pectin samples were pasted on the double tape of the SEM aluminum stub. By using a sputtering coater, the stub was then coated with approximately All samples were examined under a scanning electron microscope (JSM-6100, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV and a magnification of 100-2000X.

[0143] The microscopic surface morphologies of freeze-dried PP, PCNFs, and PCNFs-PP were observed by scanning electron microscopy (SEM). Figure 15 Compared with other samples, sample PP ( Figure 15 A, B) show a smoother and more regular surface, which may be because PP is prepared by a relatively mild treatment method. Studies have shown that a smooth layered structure can provide higher gel strength. In addition, the cross-section of the sample PP presents a multi-layer network structure with dense pores and cracks, which greatly increases the surface area and deformation resistance of PP. The microstructure of citrus pectin affects its various physical and chemical properties, such as hydration capacity and cation exchange capacity. Citrus pectin can rely on its large surface area to provide more binding sites through hydrogen bonding or dipole interactions, thereby effectively adsorbing water molecules. The surface morphology of the sample PCNFs was observed by SEM as follows Figure 15 As shown in C and D in the figure, it can be seen that the surface of PCNFs is rough and wrinkled. When magnified, the sample shows a network structure of cellulose nanofibrils. Studies have shown that because PCNFs have a high surface area and can provide abundant exposed hydroxyl groups, they can significantly improve the mechanical strength of the material. Figure 15 As can be seen in Figures E and F, the PCNFs-PP sample has a smooth surface and a dense structure, indicating good compatibility between the two components. The PCNFs-PP sample possesses both the multilayer structure of PP and the dense cellulose nanofibril network of PCNFs, resulting in a high surface area and enhanced mechanical properties.

[0144] Test Example 2

[0145] Characterization of NAR-PE

[0146] 1) Encapsulation efficiency (EE) and load factor (LC)

[0147] The sample was diluted 20-fold with anhydrous ethanol and sonicated for 20 minutes to extract naringenin. The sample was centrifuged at 10,000 rpm for 15 minutes. The supernatant was filtered through a 0.2 μm syringe filter and the naringenin content was determined by HPLC. EE and LC were calculated as follows:

[0148] EE (%) = (total NAR amount - free NAR amount in supernatant) × 100% / total NAR amount;

[0149] LC (%) = (total NAR amount - free NAR amount in the supernatant) × 100% / total amount of NAR-loaded PCNFs-PP;

[0150] In order to determine the ideal PCNFs-PP ratio for Pickering emulsion encapsulation of naringenin, PCNFs-PP were prepared using the same method, but the PCNFs-PP mass ratio was changed to 3:1, 2:1, 1:1, 1:2, and 1:3. The EE and LC of NAR-PE at different mass ratios were determined. The results are shown in Figure 2. Figure 12 As shown in Figure B, all samples maintained EE values above 99%. When the PCNFs:PP ratio was 2:1, the LC value of NAR-PE reached a maximum of 57.46 ± 0.7%. This is because excessively high PP content can reduce the mechanical strength of the system. A proper addition ratio ensures more stable encapsulation of the droplets, preventing leakage of naringenin.

[0151] 2) Particle size

[0152] Dynamic light scattering (DLS) was used to measure the particle size distribution of the emulsions over 30 days of storage using a Mastersizer 3000 laser particle size analyzer (Malvern Instruments Ltd., Malvern, UK). Samples were dispersed in ultrapure water for DLS measurements. Each measurement was performed at 25°C in triplicate.

[0153] In order to evaluate the storage stability of NAR-PE, the samples were stored at room temperature for 30 days. Figure 16 As shown in the figure, during storage, the particle size distribution of all samples showed a unimodal distribution. Droplet size is an important indicator for evaluating the emulsification ability of a particle stabilizer. Under the same conditions, the emulsification ability of a particle stabilizer can be characterized by the surface area of the emulsion droplets. With the increase of the PP addition ratio, the particle size of NAR-PE first decreased from 12±0.21μm (sample 1) to 10.4±0.06μm (sample 3) and then increased to 11.1±0.1μm (sample 5), indicating that sample 3 has stronger emulsification ability. At all PCNFs-PP addition ratios, the hydrodynamic diameter of the samples remained unchanged within 30 days, indicating that NAR-PE has strong stability against droplet aggregation.

[0154] 3) Transmission electron microscopy (TEM)

[0155] The morphology of the NPs was observed using an HT-7700 instrument (Hitachi, Japan). The sample was diluted to an appropriate concentration and dropped onto a carbon-coated copper grid. Excess dispersion was then absorbed with filter paper. The dried sample was placed in a sample holder for observation.

[0156] The effects of PCNFs-PP on the morphology and interfacial structure of NAR-PE droplets were analyzed by transmission electron microscopy. Figure 16As shown in Figures C and D, the NAR-PE droplets exhibit a spherical shape, indicating that the oil droplets formed during the emulsification process are relatively regular. The droplet edges are also clearly observed, indicating that the PCNFs-PP interact and adsorb at the oil-water interface, forming a dense protective layer. Fibrous structures can be observed extending outward from the droplets. These fibers, due to their high mechanical strength and large surface area, adsorb to the oil droplet interface, forming a physical barrier that prevents droplet aggregation. As a hydrophilic dietary fiber, PP binds well to the aqueous phase and hydrogen bonds with PCNFs through its hydroxyl and carboxyl groups, enhancing the fiber's adsorption capacity. PP and PCNFs also work synergistically, with the introduction of PP filling the gaps between the fibers and forming a distinct network structure around the droplets, making the entire interface denser and further enhancing interfacial stability. Furthermore, naringenin, a hydrophobic flavonoid with a certain degree of lipid solubility, can be stably encapsulated in the core-shell structure of the PCNFs-PP oil droplets.

[0157] 4) Physical and chemical stability

[0158] NAR-PE was stored for two months under various conditions: darkness, visible light, UV light, and elevated temperature. For storage in darkness, NAR-PE samples were stored at room temperature. To investigate the photodegradation of NAR, NAR-PE samples were exposed to visible light (λ = 400-700 nm) and UV light (λ = 100-400 nm) at room temperature. The NAR-PE samples were placed in a 50°C oven in darkness to determine the thermal sensitivity of NAR. The NAR content in the NAR-PE samples was regularly measured under different storage conditions.

[0159] NAR is easily affected by light and easily degraded. Considering that the emulsion is easily exposed to light during processing and transportation, it is extremely important to evaluate its light and UV stability. Figure 17 Center A shows the degradation curve of NAR during two months of storage under visible light conditions, with the degradation rate slightly lower than that under dark conditions. When the mass ratio of PCNFs to PP decreased from 3:1 to 1:3, the NAR retention increased from 91.99 ± 0.06% to 93.29 ± 0.24%, and then decreased to 88.58 ± 0.32%. Figure 17Figure C shows the degradation curve of NAR under UV irradiation. After two months of storage, the NAR retention rate was the lowest at 87.21±0.41%, indicating that only 22.79% of the NAR had been degraded during storage. This indicates that the NAR-PE system possesses excellent stability. Because the NAR is encapsulated within the core of the NAR-PE core-shell structure, the shell formed by the PCNFs-PP effectively blocks UV radiation, mitigating NAR degradation. Samples a and b exhibit the highest NAR retention rates, at 92.75±0.29% and 93.02±0.16%, respectively. This indicates that the PCNFs-PP mass ratio in samples a and b provides greater stability, thereby enhancing the encapsulation and protection of the NAR. Furthermore, the PP in the NAR-PE system contains abundant reactive groups, such as hydroxyl and carboxyl groups, which act as antioxidants, thereby inhibiting the oxidation and degradation of the NAR.

[0160] In a dark environment, the NAR degradation curves under different PCNFs-PP mass ratios are shown in Figure 2. Figure 17 As shown in Figure 2B, after two months of storage, the retention of NAR in all samples ranged from 90.4 ± 0.36% to 94.63 ± 0.41%. Because NAR is susceptible to oxidation and photodegradation, the degradation pathway for NAR under dark conditions is primarily through slow oxidative degradation. Compared to conditions exposed to light and UV light, the degradation rate of NAR is significantly reduced. Furthermore, a suitable PCNFs-PP mass ratio can form a denser interfacial protective layer on the droplet surface, reducing oxygen diffusion into the oil droplet and thus slowing the oxidative degradation of naringenin.

[0161] The thermal stability of active ingredients in drug delivery systems is crucial for further processing and utilization, so the retention of NAR during storage at 50°C for two months was determined. Figure 17 As shown in Figure D, NAR retention significantly decreases under high temperature conditions, with a clear downward trend observed in all samples after two months of storage. This phenomenon can be explained by the Arrhenius rate law. Generally, the reaction rate depends on the value of the rate constant, while the effect of temperature is exponentially related to the rate constant, meaning that the NAR degradation rate increases with increasing temperature. Notably, the NAR retention of sample e was only 86.38 ± 0.1%, and the emulsion sample exhibited significant stratification. This result is attributed to the higher mechanical strength of PCNFs, which plays a dominant role in the PCNFs-PP stabilization of PE, maintaining the oil-water interface at high temperatures and reducing NAR leakage. PP generally stabilizes oil-in-water emulsions by increasing the viscosity of the aqueous phase. With increasing PP addition and rising storage temperature, the stability of the NAR-PE system decreases, weakening its ability to protect the interface.

[0162] 5) Evaluation of antioxidant activity

[0163] A.DPPH free radical scavenging experiment

[0164] The antioxidant activity was evaluated by DPPH free radical scavenging activity. The sample was diluted 50-fold with anhydrous ethanol, and 4 mL of the sample (NAR-PE, PE, and free NAR) was thoroughly mixed with 4 mL of DPPH-ethanol solution (0.1 mM). After 30 minutes, the absorbance was measured at 517 nm using a UV-1900i spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The DPPH free radical scavenging activity (%) was calculated by the following formula:

[0165] DPPH radical scavenging activity (%) = [1-(A0-A1) / A2] × 100%;

[0166] Where A0 is the absorbance of the sample, A1 is the absorbance of ethanol, and A2 is the absorbance of a mixed solution of 4 ml deionized water and 4 ml DPPH-ethanol.

[0167] B. ABTS free radical scavenging activity

[0168] The ABTS free radical scavenging activity was determined and its antioxidant activity was evaluated. The sample was diluted 50 times with anhydrous ethanol before measurement. Among them, the ABTS solution was prepared with equal volumes of 7.4mM ABTS and 2.6mM potassium persulfate and reacted in the dark for 16 hours. The resulting solution was diluted with anhydrous ethanol and had an absorbance of 0.700 at 734nm, which was a working solution. Subsequently, 40 microliters of sample (NAR-PE, PE and free NAR) was mixed with 6mL of working solution for 5 minutes and measured at 734nm. The ABTS free radical scavenging activity (%) was calculated by the following formula:

[0169] ABTS free radical scavenging activity (%) = (Ac-At) × 100% / Ac;

[0170] where Ac and At are the absorbances of the working solution and sample, respectively.

[0171] As a flavonoid compound, NAR contains multiple phenolic hydroxyl groups and has significant antioxidant capacity. The DPPH and ABTS free radical scavenging abilities of NAR-PE, PE, and free NAR (with concentrations corresponding to different drug loadings in NAR-PE) were measured and compared. Figure 18As shown, the DPPH and ABTS radical scavenging activities of free NAR exhibited a concentration-dependent behavior. The highest drug loading was achieved at a PCNFs-PP mass ratio of 1:1, and the corresponding NAR concentration exhibited the highest DPPH and ABTS radical scavenging activities, reaching 18.47±0.86% and 20.25±0.9%, respectively. At the highest drug loading, the DPPH and ABTS radical scavenging rates of NAR-PE were 44.79±0.88% and 45.6±1.69%, respectively. Compared to free NAR, the DPPH and ABTS radical scavenging rates of NAR-PE were increased by 2.43 and 2.25 times, respectively. Clearly, the antioxidant capacity of NAR-PE was significantly enhanced compared to free NAR due to the increased specific surface area, solubility, and dispersibility.

[0172] The GalA backbone and neutral sugar side chains in PP are rich in carboxyl and hydroxyl groups, which react with free radicals and effectively neutralize them through hydrogen atom transfer or electron transfer mechanisms, thereby blocking the chain reaction initiated by free radicals and exhibiting strong antioxidant activity. PCNFs themselves lack antioxidant activity, so the antioxidant activity of PE increases with increasing PP addition ratios. Notably, the DPPH and ABTS free radical scavenging rates of NAR-PE were 44.79% and 45.6%, respectively (PCNFs:PP = 1:1), lower than the theoretical sum of the free radical scavenging rates of PE and NAR. This suggests that the binding of PE and NAR may mask their antioxidant binding sites, resulting in the slightly higher combined free radical scavenging rates of PE and NAR compared to NAR-PE.

[0173] 6) Drug release kinetics

[0174] The in vitro release profiles of NAR and NAR-PE were investigated by dialysis. 10 mL of each of NAR and NAR-PE (200 μg / mL) at the same concentration was placed in a dialysis bag (molecular weight cutoff, 1 kDa) in triplicate. Drug release was investigated at 37°C and 100 rpm. The release medium consisted of 100 mL of phosphate buffer (pH 7.4). Samples of 1 mL were taken at 0.5, 1, 2, 4, 8, 12, and 24 h, diluted with anhydrous ethanol, and the naringenin content was determined by HPLC. Phosphate buffer was then added at the same volume and temperature. The cumulative release percentage was calculated, and the in vitro release profile of naringenin was plotted.

[0175] The release characteristics of naringenin from free NAR and NAR-PE in PBS were evaluated using a dialysis bag method at pH 7.4. Within 5 hours of release, the release of naringenin from the sample NAR increased rapidly, showing that more than 80% of naringenin was released, indicating that the release of free naringenin was completely dependent on the solubility of the drug, and the release rate was fast and uncontrollable. The cumulative release rate of naringenin from NAR-PE within the first 5 hours was only about 20%. After 24 hours of release, the release rates of naringenin from free NAR and NAR-PE were 78.93±3.15% and 40.43±0.58%, respectively. Figure 19 It can be seen that the encapsulation of NAR-PE significantly reduced the release rate of naringenin, effectively increased the sustained release time of naringenin, and achieved a significant sustained release effect ( Figure 19 A and B in the figure). Previous studies have found that Peng et al. loaded solid lipid nanoparticles with naringenin, and the release rate of naringenin exceeded 70% after 24 hours in vitro release [Wu C, Ji P, Yu T, et al. Naringenin-loaded solid lipid nanoparticles: preparation, controlled delivery, cellular uptake, and pulmonary pharmacokinetics [J], Drug Design, Development and Therapy 2016.]. Under the same conditions, Manjit et al. released about 50% of naringenin loaded on gelatin-coated polycaprolactone nanofiber scaffolds after 24 hours [Manjit M, Kumar K, Kumar M, et al. Fabrication of gelatin coated polycaprolactone nanofiber scaffolds co-loaded with luliconazole and naringenin for treatment of Candida-infected diabetic wounds [J], International Journal of Biological Macromolecules 2024, 261.]. Compared with these studies, the slow and sustained release of naringenin from NAR-PE could maintain effective drug concentrations in the blood and reduce the need for frequent dosing.

[0176] Table 4 shows various kinetic models commonly used to simulate drug release mechanisms. The obtained drug release curves can be correlated with various kinetic models to select the best-fitting function. Four common kinetic models were used to evaluate the release mechanism of NAR from Pickering emulsions. According to R 2 and the value of n, the most suitable model was selected. As can be seen from Table 4, in the formula, Q t , Q0 and Q t / Q ∞ represent the cumulative release amount of NAR at time t, the initial release amount of NAR, and the fractional release amount of NAR, respectively. K0, K1, K H , K kp are the release rate constants of the corresponding equations, and n is the diffusion exponent. In the Korsmeyer-Peppas model, the release mechanism depends on the value of n. In the samples (spherical) of the present invention, n = 0.43, 0.43 < n < 0.85, and n = 0.85 are related to Fick diffusion, non-Fick diffusion, and erosion release (second type of mass transfer), respectively.

[0177] Table 4 Drug release kinetic models

[0178]

[0179] The zero-order, first-order, Higuchi, and Korsmeyer-Peppas models were used to further evaluate the effects of different systems on the release behavior of naringenin. Table 5 shows the kinetic release parameters and correlation coefficients of the in vitro release curves of naringenin in NAR and NAR-PE. Sample NAR and sample NAR-PE had the highest degree of fit with the first-order release kinetic model, that is, the release of the drug was driven by the concentration gradient. The R 2 in the Higuchi model for NAR-PE was 0.9579, indicating that the release of naringenin in the emulsion was mainly controlled by the diffusion mechanism. In addition, the Korsmeyer-Peppas model was the best model for fitting the release behavior of the naringenin-loaded system, and the release mechanism of naringenin in NAR-PE could be determined by the parameter n in the Korsmeyer-Peppas model. NAR-PE could be regarded as spherical particles, and the parameter n = 0.4535 indicated that the release behavior of naringenin at this time was between Fick diffusion and relaxation transport. It was shown that the release of the drug at this time was affected by the diffusion mechanism and the interfacial barrier formed by the particle stabilizer. Generally speaking, the release of naringenin in NAR was mainly controlled by solubility, showing rapid dissolution and diffusion release, and long-term release could not be achieved. The release of naringenin in NAR-PE was affected by the combined action of diffusion and interface control, with highly controllable release characteristics and the characteristics of long-term action.

[0180] Table 5 Kinetic release rate constants and correlation coefficients of naringenin in vitro release in different systems

[0181]

[0182]

[0183] 7) Bioaccessibility

[0184] In vitro simulated digestion was performed using simulated gastric fluid (SGF) at pH 1.2 supplemented with 0.32% (w / v) pepsin and 0.2% (w / v) sodium chloride; simulated intestinal fluid (SIF) at pH 6.8 supplemented with 20 mM phosphate buffer, 0.5% (w / v) trypsin, and 0.2% (w / v) sodium chloride. Briefly, samples were mixed with SGF / SIF in a 37°C incubator at 100 rpm for 3 h.

[0185] Samples were taken at 0.5, 1, 1.5, 2, 2.5, and 3 h to determine the naringenin content in the emulsion. The naringenin retention rate (%) was calculated as follows:

[0186] Naringenin retention rate (%) = (1-m1 / m2) × 100%;

[0187] Where m1 and m2 are the total content of naringenin in NAR-PE and the content of naringenin in digestive fluid, respectively.

[0188] Bioaccessibility refers to the effective part of a bioactive substance or drug that can be absorbed after digestion. Since NAR is almost insoluble in water, the bioaccessibility of free NAR in simulated gastrointestinal digestion is poor. Studies have shown that the digestibility of NAR in the gastrointestinal tract is less than 10%. However, since NAR has a certain fat solubility, its solubility can be increased by encapsulation in an O / W type Pickering emulsion. Another study showed that water-soluble polysaccharide PP can be coupled with NAR to improve the water solubility of NAR and its bioavailability in gastrointestinal digestion. The bioaccessibility of NAR-PE in simulated gastrointestinal fluid is as follows: Figure 19 As shown in Figures C and D, after 3 hours of simulated gastrointestinal digestion, the retention rates of NAR in NAR-PE were 82.1±0.6% and 57.6±0.4%, respectively. Approximately 17.9% and 42.4% of NAR were digested and absorbed in gastric and intestinal fluids, respectively. After 1.5 hours of gastrointestinal digestion, the digestion rate of NAR in NAR-PE leveled off. These results indicate that NAR is more bioaccessible in the intestine and that encapsulation and loading of NAR-PE can significantly improve NAR bioavailability, enabling slow and sustained release of NAR in gastrointestinal fluids.

[0189] 8) Cytotoxicity assay

[0190] Cell viability was measured to evaluate safety. Briefly, HaCaT cells were cultured at 2×10 4 Cells were seeded in 96-well plates at a density of 10 cells / well. After adherence, the cells were treated with 200 μL of DMEM solution containing different concentrations of NAR-PE or free NAR for 24 hours. The culture medium was then removed, and a 10% (v / v) CKK8 solution was added in a volume of 100 μL and incubated in the dark for 4 hours. Background subtraction was performed at 630 nm, and absorbance was measured at 550 nm using a microplate reader (Infinite M2000PRO, Switzerland). Cell viability was calculated by the percentage of absorbance between the experimental cells and the control cells (treated with DMEM alone).

[0191] The safety of NAR and NAR-PE was evaluated by measuring the cytotoxicity of different concentrations on HaCaT cells. Figure 20 As shown, when the concentration of naringenin was within the range of 0-100 μg / mL, the survival rate of cells in each group treated with NAR-PE and NAR remained above 95%, indicating that PE-based NAR-PE is non-cytotoxic and has good biocompatibility.

Claims

1. A method for preparing a NAR-loaded nanocellulose-pectin Pickering emulsion, characterized in that: The steps include: 1) Preparation of grapefruit peel cellulose nanofibers The pomelo peel sponge layer is dried and crushed, and sieved to obtain pomelo peel powder; Grapefruit peel powder was dissolved in a hydrochloric acid aqueous solution, heated to remove pectin, allowed to cool, centrifuged, and the precipitate was freeze-dried to obtain crude cellulose. The crude cellulose was dissolved in a NaOH solution and mixed, heated to remove hemicellulose and lignin, and the precipitate was collected and freeze-dried to obtain cellulose. The cellulose was dispersed in deionized water and subjected to high-pressure homogenization to obtain grapefruit peel cellulose nanofibers (PCNFs). The pH of the hydrochloric acid aqueous solution is 1 to 2; The heating temperature is 70-90° C. and the heating time is 1-3 hours; The pressure of the high-pressure homogenization is 300-500 MPa; 2) Preparation of grapefruit peel pectin The pomelo peel powder was mixed with NaOH solution and subjected to ultrahigh pressure treatment, centrifuged, the supernatant was added with 95% ethanol, and allowed to stand overnight, the precipitate was washed with 95% ethanol, and freeze-dried to obtain pomelo peel pectin PP; The pressure of the ultra-high pressure treatment is 500-600 MPa; 3) Preparation of Nanocellulose-Pectin Pickering Emulsion The grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP obtained in step 1) are dissolved and mixed, and subjected to high-pressure homogenization to obtain a nanocellulose-pectin Pickering emulsion; The pressure of the high-pressure homogenization treatment is 50-80 MPa; 4) Preparation of NAR-loaded nanocellulose-pectin Pickering emulsion Naringenin NAR was dissolved in the oil phase, and nanocellulose-pectin Pickering emulsion was added, and high-speed homogenization and high-pressure homogenization were performed successively to obtain NAR-loaded nanocellulose-pectin Pickering emulsion NAR-PE. The rotation speed of the high-speed homogenizer is 10000-20000 r / min; The pressure of the high-pressure homogenization is 300-1200 bar.

2. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, wherein Step 1) The concentration of the NaOH solution is 8-10% (w / v).

3. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, wherein In step 1), the mass ratio of the cellulose to deionized water is 1:(150-250).

4. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, wherein In step 1), the mass ratio of the pomelo peel powder to the NaOH solution is 1:(35-60).

5. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, characterized in that: Step 2) The concentration of the NaOH solution is 0.01M.

6. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, characterized in that: Step 3) The grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP are dissolved in ultrapure water at a concentration of 0.1 to 0.9 wt %, respectively.

7. The method for preparing the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 1, characterized in that: Step 3) The mass ratio of the grapefruit peel cellulose nanofibers PCNFs and grapefruit peel pectin PP after dissolution is (1-3): (1-3).

8. A NAR-loaded nanocellulose-pectin Pickering emulsion obtained by the preparation method according to any one of claims 1 to 7, characterized in that: It is a spherical particle in which the NAR load is embedded in a network structure formed by nanocellulose and pectin.

9. Use of the nanocellulose-pectin Pickering emulsion according to claim 1 as a carrier for delivering NAR or other hydrophobic bioactive substances.

10. Use of the NAR-loaded nanocellulose-pectin Pickering emulsion according to claim 8 in the preparation of sustained-release drugs.

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