Preparation method and application of food-derived cellulose-based nanocomposite particles

Abstract

The application discloses a preparation method and application of food-derived cellulose-based nanocomposite particles, and belongs to the field of precise nutrition delivery. The preparation method of the food-derived cellulose-based nanocomposite particles comprises the following steps: preparing oxidized food-derived nanocellulose through a TEMPO oxidation method on the basis of food-derived cellulose; mixing a protein aqueous solution with the oxidized food-derived nanocellulose suspension; then adding an ethanol solution containing a hydrophobic active substance, homogenizing, and removing the ethanol to obtain the food-derived cellulose-based nanocomposite particles. The method increases the overall structural stability of the nanoparticles, improves the encapsulation efficiency of fucoxanthin and the stability of the fucoxanthin during storage, and the fucoxanthin encapsulated in the nanoparticles can be effectively absorbed by cells, significantly improves the bioavailability of the fucoxanthin, and effectively inhibits the formation of lipid droplets by activating the Nrf2 / HO-1 / NQO1 signal pathway to reduce oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the field of precise nutrition delivery, and specifically relates to a preparation method and application of food-derived cellulose-based nanocomposite particles. Background Art

[0002] Metabolic diseases, such as obesity and type 2 diabetes, are often associated with excessive fat accumulation in the body. These conditions are not only due to abnormal lipid metabolism, but are also closely related to oxidative stress, which can further aggravate cellular dysfunction and inflammatory response. In order to simulate the excessive accumulation of fatty acids in the human body and its effects on cellular metabolism and function, researchers often use free fatty acid (FFA) models for in vitro experiments. This model not only helps researchers explore how to regulate fat accumulation by simulating the effects of excess fatty acids on cells in the human body, but is also used to evaluate potential antioxidant treatment strategies. Effective antioxidants can reduce oxidative stress and may help restore the normal metabolic function of cells, thereby improving metabolic abnormalities caused by excessive lipid accumulation.

[0003] Fucoxanthin, a natural carotenoid compound primarily extracted from brown algae, has been shown in numerous studies to have potential therapeutic effects on metabolic diseases. This unique marine bioactive compound possesses significant antioxidant and anti-lipid accumulation properties, effectively reducing lipid accumulation in the body and enhancing energy metabolism. Fucoxanthin's regulatory effects on blood glucose and cholesterol levels also make it a promising natural intervention for the treatment of type 2 diabetes and cardiovascular disease. These properties hold great promise for its broad application in clinical practice and in functional foods. Despite its significant bioactivity, fucoxanthin faces several limitations in its practical application. One major challenge is its extremely poor water solubility, which significantly restricts its effective distribution and absorption in the body. Furthermore, fucoxanthin is sensitive to light and easily degrades under light conditions, compromising its long-term storage stability. These factors collectively limit the potential of fucoxanthin as a therapeutic agent, particularly in settings requiring long-term or stable administration. Therefore, nanoencapsulation of fucoxanthin offers a novel solution.

[0004] Cellulose, as a natural polymer material, has been widely used in drug delivery systems in recent years due to its excellent biodegradability and wide range of sources. For example, carboxymethyl cellulose (CMC) and hydroxypropyl cellulose (HPMC) have shown good controlled release and stability effects in certain oral controlled release tablets and sustained release injection preparations. However, it should be noted that when dealing with drugs with poor water solubility or easy degradation, these traditional cellulose derivatives may not fully exert their advantages in drug release control and long-term stability. In addition, existing research has mostly focused on non-food-derived materials, which may bring potential safety and biocompatibility issues. Summary of the Invention

[0005] To solve the above problems, the application provides a preparation method of food-derived cellulose-based nanocomposite particles, which uses TEMPO-oxidized kelp nanocellulose as a skeleton structure in a drug delivery system, can stabilize the overall structure of the nanoparticles, and improve the drug delivery efficiency and bioavailability through its superior physical and chemical properties.

[0006] The application provides a preparation method of food-derived cellulose-based nanocomposite particles, which comprises the following steps:

[0007] (1) Using food-derived cellulose as raw material and sodium hypochlorite as oxidant, TEMPO-mediated oxidation method is used to prepare oxidized food-derived nanocellulose;

[0008] (2) The oxidized food-derived nanocellulose is dispersed in water to obtain an oxidized food-derived nanocellulose suspension, and mixed with a protein aqueous solution to obtain a protein / oxidized food-derived nanocellulose mixture;

[0009] (3) An ethanol solution containing a hydrophobic active substance is added to the protein / oxidized food-derived nanocellulose mixture in step (2), and homogenized, ultrasonicated and ethanol-removed to obtain food-derived cellulose-based nanocomposite particles.

[0010] In an embodiment of the application, in step (1), the food-derived cellulose is nanocellulose or nanocrystalline cellulose of any source.

[0011] In an embodiment of the application, in step (1), the food-derived cellulose is kelp nanocellulose, and the preparation method of the kelp nanocellulose comprises the following steps: extracting kelp cellulose, then performing cellulase enzymolysis, and finally obtaining kelp nanocellulose through ultrasonic treatment.

[0012] In an embodiment of the application, in step (1), the specific preparation method of the oxidized food-derived nanocellulose is as follows: the food-derived cellulose is dispersed in ultrapure water, and under ice bath conditions, TEMPO, NaBr and NaClO solutions are added, during the reaction process, 0.1 mol / L NaOH and HCl are used to maintain the pH value in the range of 10±0.2, when the pH value of the suspension is constant, ethanol is added to terminate the oxidation reaction, and the suspension is ultrasonicated, centrifuged, washed, dialyzed and freeze-dried.

[0013] In one embodiment of the present application, in step (1), in the specific preparation method of the oxidized food-derived nanocellulose, the adding ratio of the food-derived cellulose to ultrapure water is 1:20-2000 g / mL; the adding amount of TEMPO, NaBr and NaClO solution is 0.01-0.08 g, 0.05-0.5 g and 3-30 mL, respectively.

[0014] In one embodiment of the present application, in step (2), the protein can be a protein with good water solubility.

[0015] In one embodiment of the present application, in step (2), the protein is sodium caseinate.

[0016] In one embodiment of the present application, in step (2), the mass concentration of the protein in the protein aqueous solution is 0.25-1%.

[0017] In one embodiment of the present application, in step (2), the mass concentration of the protein and the oxidized food-derived nanocellulose in the protein / oxidized food-derived nanocellulose mixture is 0.5-2%; the mass ratio of the oxidized food-derived nanocellulose to the protein is 1:0.3-3.

[0018] In one embodiment of the present application, in step (3), the hydrophobic active substance is fucoxanthin.

[0019] In one embodiment of the present application, in step (3), in the ethanol solution containing the hydrophobic active substance, the concentration of the hydrophobic active substance is 1-50 mg / mL.

[0020] In one embodiment of the present application, in step (3), the volume ratio of the ethanol solution containing the hydrophobic active substance to the protein / oxidized food-derived nanocellulose mixture is 1:1-50.

[0021] The present application provides the food-derived cellulose-based nanocomposite particles prepared by the above-mentioned method.

[0022] The present application provides the use of the above-mentioned food-derived cellulose-based nanocomposite particles in preparing an antioxidant substance for preventing or reducing the free fatty acid-induced oxidative stress-related diseases.

[0023] The present application also provides the use of the above-mentioned food-derived cellulose-based nanocomposite particles in preparing a lipid-lowering drug.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] (1) The present application adopts kelp nanocellulose as the skeleton structure of the drug delivery system, which not only increases the overall structural stability of the nanoparticles, but also has lower toxicity and better environmental friendliness to the human body due to its excellent biocompatibility and biodegradability;

[0026] (2) The present application adopts kelp nanocellulose and sodium caseinate composite material, which effectively improves the packaging efficiency of fucoxanthin and its stability during storage;

[0027] (3) The present application encapsulates fucoxanthin in nanoparticles which can be effectively absorbed by cells, significantly improves the bioavailability of fucoxanthin, and effectively inhibits lipid droplet formation by activating the Nrf2 / HO-1 / NQO1 signaling pathway to reduce oxidative stress. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Characterization and analysis of kelp cellulose, kelp nanocellulose and TEMPO-oxidized kelp nanocellulose of Example 1: appearance and TEM images (A), SEM images (B), Zeta potential distribution (C) and Fourier transform infrared spectra (D) of kelp cellulose, kelp nanocellulose and TEMPO-oxidized kelp nanocellulose;

[0029] Figure 2 Encapsulation efficiency of fucoxanthin (A) and retention rate of fucoxanthin after 14 days of storage (B) of TEMPO-oxidized kelp nanocellulose / sodium caseinate nanoparticles of Examples 1-3 and Comparative Examples 1-2;

[0030] Figure 3 Low-temperature cold field scanning electron microscope images of fucoxanthin-loaded TEMPO-oxidized kelp nanocellulose / sodium caseinate nanoparticles of Example 1 and nanoparticles of Comparative Examples 1-2;

[0031] Figure 4 Effect of different concentrations of fucoxanthin-loaded kelp nanocellulose / sodium caseinate nanoparticles of Example 1 on cell viability;

[0032] Figure 5 Malondialdehyde (MDA) (A) and glutathione (GSH) (B) levels and superoxide dismutase (SOD) (C) and catalase (CAT) (D) activities of cells in different treatment groups;

[0033] Figure 6Example 1, Comparative Examples 3~4 Nanoparticles alleviate oxidative stress through Nrf2 / HO-1 / NQO1 signaling pathway: Western blot analysis showed the expression of Nrf2, HO-1 and NQO1 proteins in cells of different treatment groups (A), relative protein expression levels (B) and relative mRNA expression levels of Nrf2 (C), HO-1 (D) and NQO1 (E) in different treatment groups, different letters represent significant difference (p<0.05);

[0034] Figure 7 Example 1, Comparative Examples 3~4 Nanoparticles in vitro anti-lipid accumulation ability: BODIPY493 / 503 (A) and oil red O staining (B) of HepG2 cells; absorbance of oil red O at 510 nm (C), total cholesterol (TC) (D) and triglyceride (TG) (E) content of cells in different treatment groups, different letters represent significant difference (p<0.05). DETAILED DESCRIPTION

[0035] The fucoxanthin crude product (content 70%) used in the following examples and comparative examples was purchased from Shandong Crystallization Group. 500 mg of fucoxanthin crude product was added to 50 mL of anhydrous ethanol and dissolved thoroughly under light-proof conditions. After complete dissolution, centrifugation was performed at 10000 rpm for 15 min to remove insoluble impurities. Subsequently, the supernatant containing fucoxanthin was taken and diluted to a concentration of 5 mg / mL for the preparation of fucoxanthin-loaded nanoparticles.

[0036] The preparation of the kelp nanocellulose mentioned in the examples is as follows: the washed kelp is placed in a 60°C oven until it is completely dried, the dried kelp is ground into powder, 20g of the powder is accurately weighed and dispersed in 1L of a 2wt% sodium hydroxide aqueous solution, and is reacted at 120°C for 2h, centrifuged, and the precipitate is thoroughly washed, and this step is repeated 2-3 times to remove hemicellulose, and the precipitate is further dispersed in 1L of an aqueous solution containing 3.5wt% NaOH, 150mL glacial acetic acid, and 16g NaClO, and is bleached at 80°C for 1h, and the precipitate is collected by centrifugation and thoroughly washed, and this step is repeated 2-3 times until the precipitate is white. The precipitate is suspended in 1L of an aqueous solution containing 2wt% NaOH, 0.8wt% Na3PO4·12H2O, and 0.8wt% Na2SiO3·9H2O, and is reacted at 100°C for 2h to remove lignin, and the precipitate is centrifuged and washed until the pH is neutral, and the kelp cellulose (KC) is collected by freeze-drying. 0.5g of the kelp cellulose is accurately weighed and dispersed in 250ml of deionized water, 50mg of cellulase is added, and the reaction is carried out at 60°C and pH=4.8 for 3h, after the reaction is completed, the excess cellulase is removed by centrifugation at 10000rpm for 10min, and the kelp nanocellulose (KNC) is collected by ultrasonic cell disruption at 450W for 10min and freeze-drying.

[0037] In the following examples, wt% refers to mass percentage, for example, "1wt% sodium caseinate" means that 100g of deionized water contains 1g of sodium caseinate.

[0038] In the following examples, the measurement methods are as follows:

[0039] (I): Characterization and analysis method of kelp nanocellulose: the appearance morphology of the cellulose suspension is photographed by a camera, and the micro-morphology of the cellulose is observed by transmission electron microscopy (TEM, JEM-2100UHR, Japan) and field emission scanning electron microscopy (FE-SEM, SM-7800F, Japan); the Zeta potential of the cellulose is detected by a Malvern particle potential instrument (ZETASIZER 3000HSA, UK) at 25°C, and the sample concentration is 1mg / mL; the functional groups of the sample are determined by a Fourier transform infrared spectrometer (FT-IR, Spectrum Two, USA), and the measurement range of the spectrum is from 4000cm -1 to 400cm -1 , and the resolution is 4cm -1 .

[0040] (II): Method for measuring the encapsulation efficiency and retention rate of fucoxanthin: 0.5 mL sample was mixed with 2 mL dichloromethane / methanol (volume ratio 2:1) mixed solution, vortexed well and then centrifuged at 8000 rpm for 10 min. The dichloromethane layer was collected, the absorbance at 450 nm was measured, and the content of Fx was calculated according to the standard curve y=0.1092x-0.011 (R 2 =0.9995). The encapsulation efficiency (EE) and retention rate of Fx were calculated using the following equation:

[0041]

[0042] (III): Microscopic morphology observation of fucoxanthin-loaded nanoparticles: The microscopic structure of the nanoparticles was observed by cryo-SEM (Cryo-SEM, Hitachi, Japan); 10 microliters of sample were rapidly frozen by liquid nitrogen and then subjected to gold spraying treatment to enhance the conductivity and image clarity of the electron microscope imaging; after gold spraying treatment, the sample was transferred to the cryo-SEM for observation.

[0043] (IV): Method for measuring the biocompatibility of nanoparticles: First, DMEM medium was used to prepare sample solutions loaded with Fx nanoparticles (concentrations were 0, 21, 42, 84, 126, 168, and 210 pg / mL, respectively). 100 pL of HepG2 cell suspension was inoculated in a 96-well microplate at a density of 5x10 3 cells per well, and incubated at 37°C and 5% CO2 for 24 h. Then the medium in the well plate was replaced with 100 pL of sample solution, and incubation was continued for 24 hours. Next, 20 pL of 5 mg / mL 3-(4,5)-dimethylthiazol(-z-y1)-3,5-di-phenytetrazoliumromide (MTT, Beyotime Biotechnology, ST1537-1 g) was added to each 96-well plate, and further incubated at 37°C for 4 h. After removing the medium, 150 pL of DMSO was added to each well, and finally the absorbance of the 96-well plate at 570 nm wavelength was measured and the cell viability was calculated.

[0044] (V): Method for measuring the antioxidant capacity of nanoparticles: 78 mM and 354 mM DMSO stock solutions of palmitic acid (PA) and oleic acid (OA) were prepared, and then the palmitic acid and oleic acid stock solutions were mixed in a molar ratio of 1:2 in DMEM medium to give a final free fatty acid concentration of 1 mM. HepG2 cells were inoculated in a 96-well microplate at a density of 1x10 5Cells were seeded at a density of 1 x 105cells / well in 12-well microplates and incubated at 37 °C under 5% CO2for 24 h. Subsequently, the medium in the well plates was replaced with 1 mL of DMEM medium containing samples (the concentration of fucoxanthin-loaded nanoparticles was 42 pg / mL, equivalent to 2 pg / mL free fucoxanthin) and incubated for 12 h. After 12 h, the medium containing samples was removed, and DMEM medium containing free fatty acids and samples was added and incubated for 24 h. The control group was treated with fresh medium only, and the model group was stimulated with free fatty acids only. The glutathione (GSH), total superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels of cells were determined using biochemical kits provided by Nanjing Jiancheng Bioengineering Institute.

[0045] (VI): Western blotting assay method: After the cells were first treated according to the method for determining the antioxidant capacity of nanoparticles, they were collected and lysed, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk solution for 2 h, the Tris-HCl buffer solution containing 0.05% Tween 20 was used to wash 3 times. Finally, the corresponding antibodies were used, and the protein bands were visualized using enhanced chemiluminescence reagents. The expression of proteins was quantitatively analyzed by using ImageJ software. The detailed antibody information is as follows: Nrf2 (A0674, ABclonal, 1:1000), HO-1 (A1346, ABclonal, 1:1000), NQO1 (A23486, ABclonal, 1:1000), β-actin (AC038, ABclonal, 1:60000).

[0046] (VII): Real-time quantitative PCR assay method: First, total RNA was extracted from HepG2 cells using Trizol. Subsequently, RNA was reverse transcribed into cDNA using the Evo M-MLV RT kit and gDNA Clean for qPCR II. The RT-PCR analysis cycle conditions are as follows: initial denaturation at 95 °C for 30 s, followed by 45 cycles, each cycle including 95 °C for 5 s and 60 °C for 30 s. The final extension phase is 95 °C for 15 s, then 60 °C for 15 s. The relative mRNA level was determined by 2 -ΔΔCtThe primer pairs were: 5′-TCAAGAAAGGGTGTAACGCAACTA-3′ and 5′-CGACAGGATGCAGAAGGAGAT-3′ of human β-actin, 5′-TACTCCCAGGTTGCCCACA-3′ and 5′-CATCTACAAACGGGAATGTCTGC-3′ of human Nrf2, 5′-CGGGCCAGCAACAAAGTG-3′ and 5′-AGTGTAAGGACCCATCGGAGAA-3′ of human HO-1, and 5′-GGGCAAGTCCATCCCAACTG-3′ and 5′-GCAAGTCAGGGAAGCCTGGA-3′ of human NQO1.

[0047] (8) Determination of the anti-lipid accumulation ability of nanoparticles in vitro: The cells were treated according to the method for determining the antioxidant ability of nanoparticles, and the anti-fat accumulation ability of the nanoparticles was visualized using an Oil Red O staining kit and a lipid droplet green fluorescence detection kit (BODIPY493 / 503). The total cholesterol (TC) and triglyceride (TG) levels of the cells were then detected using kits provided by Nanjing Jiancheng Bioengineering Research Institute.

[0048] Example 1

[0049] S1: 1g of kelp nanocellulose was dispersed in 500mL of ultrapure water and, under ice bath conditions, 0.016g of TEMPO, 0.1g of NaBr, and 7.2mL of NaClO solution were added. During the reaction, the pH value was maintained in the range of 10±0.2 by using 0.1mol / L of NaOH and HCl. When the pH value of the suspension was constant, the oxidation reaction was considered complete and terminated by adding 3mL of ethanol. The suspension was sonicated at 400W for 5 minutes and finally centrifuged at 10,000 rpm for 15 minutes. The precipitate was washed with deionized water several times, dialyzed, and freeze-dried to obtain oxidized kelp nanocellulose (TKNC).

[0050] S2: At 4°C, 1 g of sodium caseinate was dissolved in 100 mL of deionized water, and oxidized kelp nanocellulose was dispersed in deionized water to obtain an oxidized kelp nanocellulose suspension with a concentration of 10 mg / mL. The two solutions were mixed and homogenized at 8000 rpm for 30 s to form a uniform mixture to obtain a sodium caseinate / oxidized kelp nanocellulose mixture. The concentration of the total biopolymer was set to 1 wt% (i.e., the mass concentration of protein and oxidized food-derived nanocellulose in the protein / oxidized food-derived nanocellulose mixture was 1%), and the mass ratio of oxidized kelp nanocellulose to sodium caseinate was 1:3.

[0051] S3: 1 mL of fucoxanthin ethanol solution (5 mg / mL) was added dropwise to 10 mL of the mixture of S2 and homogenized at 10,000 rpm for 2 min to form a coarse emulsion;

[0052] S4: The crude emulsion was sonicated at 600 W in an ice bath for 10 min, and then the ethanol was evaporated to obtain the fucoxanthin-loaded nanoparticles (P3C1@Fx).

[0053] In this paper, TEMPO-oxidized kelp nanocellulose (TKNC) was successfully prepared by TEMPO oxidation method, and a detailed comparative analysis was conducted between it and kelp cellulose (KC) without enzymatic hydrolysis and ultrasonic treatment, as well as kelp nanocellulose (KNC) without oxidation treatment. The comprehensive characterization of these nanocelluloses can not only reveal their microstructure and chemical properties, but also evaluate their application potential as potential drug carrier materials. Figure 1 As shown in Figure A, larger fiber bundles of KC were observed under TEM. These fiber bundles had complete structures and were not modified on the surface, showing a natural fiber state. The fibers of the KNC sample were more dispersed and smaller in size, indicating that enzymatic hydrolysis and ultrasonic treatment effectively decomposed the fiber bundles and released smaller fibers. In contrast, TKNC not only showed higher dispersibility, but also formed small and uniform nanofibers. Such highly dispersed nanofibers can provide a larger surface area, help enhance the stability of the suspension, and may be beneficial for subsequent functionalization or drug adsorption processes. The surface microstructure of kelp cellulose of different samples was further observed by SEM, and the significant differences showed that the treatment method had a significant effect on the cellulose structure ( Figure 1 B). First, the fiber structure of the KC sample is relatively rough and highly aggregated, which reflects the characteristics of incomplete decomposition of the fiber bundles. Compared with the KC sample, the KNC sample showed a more open structure and significantly increased pores after enzymatic hydrolysis and ultrasonic treatment. Finally, the TKNC sample was further treated with TEMPO oxidation to present a highly porous and network structure, which not only increased the specific surface area of ​​the material, but also provided more space for drug loading. At the same time, this structure can effectively enhance the ability of material exchange. These characteristics make TKNC an ideal drug carrier material. Zeta potential is an important parameter for evaluating the stability of suspended particles. The Zeta potential of the KC sample is -5.24±1.31mV. This low negative value indicates that the fiber surface charge is small, resulting in insufficient repulsion between particles, which makes the suspension less stable. After enzymatic hydrolysis, ultrasonic treatment and TEMPO oxidation treatment, the potential of cellulose dropped significantly to -29.34±1.83mV. This is because the TEMPO oxidation process increases the carboxyl content on the fiber surface, thereby significantly increasing the surface negative charge ( Figure 1C). These additional negative charges enhance the electrostatic repulsion between fibers, which in turn enhances the stability of the suspension and the dispersibility of cellulose. FTIR spectra provide an in-depth understanding of the chemical structural changes of different treated samples of laminarin cellulose. As shown in Fig. 1 1, Figure 1 D, the hydroxyl (O-H) stretching vibration peak near 3354 cm -1 and the C-H stretching vibration peak near 2917 cm -1 are preserved for all samples, indicating that the basic structure of cellulose remains stable during the treatment process, and the hydrogen bonding between water molecules and hydroxyl groups and the structure of saturated aliphatic carbon chains do not change significantly. In addition, the characteristic peak of the glucose β-glycosidic bond in cellulose is observed near 904 cm -1 , which is preserved in all samples, indicating that the sugar ring structure of cellulose is not destroyed. However, an enhanced peak at 1605 cm -1 is observed in the KNC sample, which is usually associated with the absorption of water bound to cellulose, indicating that enzymatic and ultrasonic treatment can increase the hydration capacity of cellulose. Finally, the TKNC sample exhibits a distinct carboxyl (C=0) stretching vibration peak at 1734 cm -1 , which is introduced by TEMPO oxidation treatment. The presence of this peak demonstrates that the carboxyl content on the surface of cellulose is increased by TEMPO treatment, which positively affects the solubility of the fibers.

[0054] Comparative Example 1

[0055] Method for preparing fucoidan-loaded laminarin nanofiber nanoparticles P0C1@Fx:

[0056] S1 : 1 g of laminarin nanocellulose was dispersed in 500 mL of ultrapure water, and 0.016 g of TEMPO, 0.1 g of NaBr, and 7.2 mL of NaClO solution were added under ice bath conditions; during the reaction, the pH value was maintained in the range of 10 ± 0.2 by using 0.1 mol / L NaOH and HC1. When the pH value of the suspension is constant, it is considered that the oxidation reaction is complete; the oxidation reaction was terminated by adding 3 milliliters of ethanol; the suspension was treated with ultrasound at 400 W for 5 minutes, and finally centrifuged at a speed of 10,000 revolutions per minute for 15 minutes, and the precipitate was washed with deionized water several times and then dialyzed, and the oxidized laminarin nanocellulose was obtained after freeze-drying;

[0057] S2: The oxidized laminarin nanocellulose (concentration of 10 mg / mL) was suspended in deionized water at 4°C;

[0058] S3: 1 mL of fucoxanthin ethanol solution (5 mg / mL) was added dropwise into 10 mL of S2 oxidized kelp nanocellulose, and homogenized at a speed of 10000 rpm for 2 min to form a coarse emulsion;

[0059] S4: The coarse emulsion was ultrasonically treated in an ice bath at 600 W for 10 min, and then the ethanol was removed by rotary evaporation to obtain fucoxanthin-loaded nanoparticles (P0C1@Fx).

[0060] Comparative Example 2

[0061] Preparation method of fucoxanthin-loaded sodium caseinate nanoparticles (P1C0@Fx):

[0062] S1: 1 g of sodium caseinate was dissolved in 100 mL of deionized water at 4°C;

[0063] S2: 1 mL of fucoxanthin ethanol solution (5 mg / mL) was added dropwise into 10 mL of S1 sodium caseinate solution, and homogenized at a speed of 10000 rpm for 2 min to form a coarse emulsion;

[0064] S3: The coarse emulsion was ultrasonically treated in an ice bath at 600 W for 10 min, and then the ethanol was removed by rotary evaporation to obtain fucoxanthin-loaded nanoparticles (P1C0@Fx).

[0065] Example 2

[0066] The preparation method was consistent with that of Example 1, except that the total concentration of biopolymer was set to 1 wt%, and the mass ratio of oxidized kelp nanocellulose to sodium caseinate was 1:1, to obtain fucoxanthin-loaded nanoparticles (P1C1@Fx).

[0067] Example 3

[0068] The preparation method was consistent with that of Example 1, except that the total concentration of biopolymer was set to 1 wt%, and the mass ratio of oxidized kelp nanocellulose to sodium caseinate was 3:1, to obtain fucoxanthin-loaded nanoparticles (P1C3@Fx).

[0069] Comparative Example 3

[0070] Free fucoxanthin: Free fucoxanthin was directly dispersed in ultrapure water to obtain a fucoxanthin solution with a concentration of 5 mg / mL.

[0071] As Figure 2As shown in Figure B, the retention rate of fucoxanthin after 14 days was determined, and the retention rate of fucoxanthin loaded in the nanoparticles of Example 1 reached 56.12 ± 1.89%, which was significantly higher than the retention rate of fucoxanthin in Comparative Example 1 (33.93 ± 1.15%) and Comparative Example 2 (42.92 ± 0.15%). In addition, the retention rate of fucoxanthin in Example 2 was 50.43 ± 2.12%, the retention rate of fucoxanthin in Example 3 was 42.05 ± 0.77%, and the retention rate of free fucoxanthin in Comparative Example 3 was 22.12 ± 0.51%. The highest retention rate in the sample of Example 1 can be attributed to its higher sodium caseinate content, which not only improves the initial encapsulation efficiency, but also enhances the structural stability of the nanoparticles through the synergistic effect of protein and cellulose. TKNC, as a natural polymer with high dispersibility and porosity, can provide additional protection for fucoxanthin, reducing the risk of mechanical or chemical degradation of fucoxanthin.

[0072] To further investigate the stability of fucoxanthin-loaded nanoparticles under actual storage conditions, a 14-day storage experiment was conducted on these nanoparticles. As shown in Figure A, the retention rate of fucoxanthin after 14 days was determined, and the retention rate of fucoxanthin loaded in the nanoparticles of Example 1 reached 56.12 ± 1.89%, which was significantly higher than the retention rate of fucoxanthin in Comparative Example 1 (33.93 ± 1.15%) and Comparative Example 2 (42.92 ± 0.15%). In addition, the retention rate of fucoxanthin in Example 2 was 50.43 ± 2.12%, the retention rate of fucoxanthin in Example 3 was 42.05 ± 0.77%, and the retention rate of free fucoxanthin in Comparative Example 3 was 22.12 ± 0.51%. The highest retention rate in the sample of Example 1 can be attributed to its higher sodium caseinate content, which not only improves the initial encapsulation efficiency, but also enhances the structural stability of the nanoparticles through the synergistic effect of protein and cellulose. TKNC, as a natural polymer with high dispersibility and porosity, can provide additional protection for fucoxanthin, reducing the risk of mechanical or chemical degradation of fucoxanthin. Figure 2

[0073] Cryo-SEM images provide a detailed view of the microstructure of the nanoparticles (Figure C). The images show that the nanoparticles of Example 1 have a more complex and uniform structure compared to the other samples. This complex structure may provide more physical encapsulation space and chemical adsorption sites for fucoxanthin, thereby improving the encapsulation efficiency and stability of the nanoparticles. Figure 3 ​). In the presence of cellulose alone (Comparative Example 1), the micrographs revealed a regular and continuous network structure, with obvious and uniformly distributed pores. This indicates that cellulose can form a stable scaffold structure. In the pure protein group (Comparative Example 2), the images revealed a clear aggregation of particles. Without cellulose, the particles are difficult to form a stable network structure and are prone to aggregation, which can affect their application effect in drug delivery systems. When the two are combined (Example 1), it is observed that cellulose effectively prevents excessive aggregation of particles, ensuring uniform distribution of particles at the microscale. In summary, the addition of food-derived nanocellulose significantly affects the stability and encapsulation efficiency of nanoparticles, and its synergistic effect with proteins enhances the overall performance of nanoparticles.

[0074] Comparative Example 4

[0075] Method for preparing unloaded fucoxanthin nanoparticles P3C1:

[0076] S1 : 1 g of kelp nanocellulose was dispersed in 500 mL of ultrapure water, and 0.016 g of TEMPO, 0.1 g of NaBr, and 7.2 mL of NaClO solution were added under ice bath conditions. During the reaction, the pH value was maintained in the range of 10 ± 0.2 by using 0.1 mol / L NaOH and HCl. When the pH value of the suspension was constant, it was considered that the oxidation reaction was complete; the oxidation reaction was terminated by adding 3 mL of ethanol; the suspension was ultrasonically treated at 400 W for 5 minutes, and finally centrifuged at a speed of 10,000 rpm for 15 minutes, the precipitate was washed with deionized water several times and then dialyzed, and the oxidized kelp nanocellulose was obtained after freeze-drying;

[0077] S2: 1 g of sodium caseinate was dissolved in 100 mL of deionized water at 4°C, and the oxidized kelp nanocellulose (concentration of 10 mg / mL) was resuspended in deionized water, and a uniform mixture was formed by mixing the two solutions and homogenizing at a speed of 8000 rpm for 30 s, the total biopolymer concentration was set to 1 wt%, and the mass ratio of oxidized kelp nanocellulose to sodium caseinate was 1:3;

[0078] S3: 1 mL of ethanol solution was added dropwise to 10 mL of S3 mixture, and homogenized at a speed of 10000 rpm for 2 min to form a coarse emulsion;

[0079] S4: unloaded fucoxanthin nanoparticles (P3C1) were obtained by ultrasonically treating the coarse emulsion at 600 W for 10 minutes in an ice bath, and then removing the ethanol by rotary evaporation.

[0080] The safety of the nanoparticle material was verified by the results of MTT Figure 4) even at higher sample concentration (P3C1@Fx concentration of 210 pg / mL), the cell viability remained above 93%. In summary, the nanoparticles demonstrated extremely high biocompatibility and cell compatibility, which is particularly crucial in the field of drug delivery.

[0081] To further investigate the cellular response to oxidative stress, the efficacy of the cellular antioxidant defense system was evaluated and revealed by measuring key biochemical indicators. These indicators include the level of lipid peroxides (represented by malondialdehyde, MDA), the content of intracellular antioxidant glutathione (GSH), and the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). These assays not only reflect the stress state of cells under oxidative pressure, but also reveal the effectiveness of antioxidant interventions. MDA is the main product of lipid peroxidation of cell membranes and is widely used as an indicator to assess the degree of oxidative damage to cells. In this patent, the model group (OA+PA) treatment significantly increased the MDA content, indicating that the cells suffered severe oxidative stress. In contrast, after P3C1@Fx treatment, the MDA content of the cells was significantly reduced, and the difference between its level and that of the control group was not significant, showing that P3C1@Fx treatment not only significantly inhibited the lipid peroxidation reaction, but also its effect was sufficient to restore the oxidative damage to almost normal levels Figure 5 A). In further biochemical analysis, the activities of GSH, SOD and CAT were also evaluated, which are key indicators of intracellular antioxidant defense. The results showed that P3C1@Fx treatment significantly restored the GSH content, indicating that it effectively maintained the reducing environment of the cells and reduced the oxidative stress Figure 5 B). Similarly, the activities of SOD and CAT also showed significant improvement in the P3C1@Fx treatment group Figure 5 C, 5D), the synergistic effect of SOD and CAT further enhanced the cell's ability to scavenge free radicals, effectively preventing cell damage caused by oxidative stress. These results collectively demonstrate the high efficiency of P3C1@Fx in enhancing the antioxidant defense capacity of cells, demonstrating its potential application value in preventing and reducing oxidative stress-related diseases induced by free fatty acids.

[0082] To further understand how the nanoparticle P3C1@Fx (Example 1) enhances the antioxidant capacity of cells through molecular mechanisms, this study focuses on its activation effect on the Nrf2 signaling pathway. Through Western blot and RT-PCR techniques, the expression changes of Nrf2 and its downstream antioxidant response elements such as HO-1 and NQO1 under the condition of oxidative stress induced by free fatty acids are evaluated. Nrf2 is a key transcription factor that regulates the antioxidant response of cells, and its activation can promote the expression of a series of defensive genes, thereby enhancing the antioxidant capacity and survival rate of cells. This patent aims to reveal how P3C1@Fx regulates the Nrf2 pathway and thus provides an effective antioxidant strategy by quantifying the expression of these key proteins and genes. Figure 6 Figures 6A and 6B show the expression of Nrf2, HO-1, and NQO1 proteins. Under oxidative stress conditions, the expression of Nrf2 showed down-regulation, which may be due to the fact that oxidative stress exceeds the self-regulation ability of the cells. In contrast, P3C1@Fx treatment significantly up-regulated the expression of Nrf2, indicating that the nanoparticles enhance their antioxidant response by stabilizing and activating Nrf2. HO-1, as a downstream target protein of Nrf2, also showed a significant increase in expression under P3C1@Fx treatment. HO-1 is a stress protein with strong antioxidant and anti-inflammatory effects that can help cells alleviate damage caused by free radicals. Therefore, the activation of Nrf2 directly promotes the overexpression of HO-1, further enhancing the protective mechanisms of cells. Similarly, NQO1, as another downstream target protein of Nrf2, also showed a significant increase in expression under P3C1@Fx treatment. Further results by RT-PCR confirmed that the mRNA expression of Nrf2 and its downstream proteins HO-1 and NQO1 was consistent with the protein expression results. After P3C1@Fx treatment, the expression levels of Nrf2, HO-1, and NQO1 mRNA were significantly enhanced compared to the model group (OA+PA group), with an increase of 1.876±0.125 times, 1.710±0.190 times, and 2.014±0.230 times, respectively (Figures 6C, 6D, 6E). These results collectively verify the antioxidant effect of the P3C1@Fx group of Example 1 at the molecular level, which is significantly better than the free fucoxanthin of Comparative Example 3 and the unloaded fucoxanthin nanoparticle group of Comparative Example 4, revealing that it enhances the protective mechanisms of cells by enhancing the Nrf2 and its regulated antioxidant response pathway. Figure 6

[0083] ​The ability of this nanocarrier to enhance Fx inhibition of lipid droplet accumulation was evaluated in a HepG2 cell model. The effects of Example 1 and Comparative Examples 3 and 4 on lipid droplet accumulation were visually compared by oil red O and BODIPY 493 / 503 fluorescent staining techniques, thereby verifying the potential advantages of nanotechnology in enhancing its bioactivity. BODIPY 493 / 503 was used as a fluorescent probe to label neutral fats in cells, and this dye is weakly fluorescent in water and other polar solvents, but emits bright green fluorescence once it binds to triglycerides. As shown in Figure 7 A, the experiment successfully established a model of lipid accumulation induced by excess free fatty acids, and the OA+PA group showed a significant increase in lipid droplets compared to the control group, which was visible by the strong green fluorescence of the BODIPY 493 / 503 fluorescent probe. Based on this model, the effects of each treatment group were compared. The green fluorescence intensity of the Comparative Example 4 group did not show a significant difference compared to the model group, indicating that pure P3C1 had limited effect on inhibiting lipid accumulation. This may be because the nanoshell material itself lacks direct regulation of the lipid metabolism process. In contrast, free Fx (Comparative Example 3) showed a certain effect on reducing lipid droplets, which is consistent with its known anti-fat activity. However, when P3C1 successfully loaded Fx (P3C1@Fx, Example 1), it showed the most significant reduction in lipid droplets, indicating that the nanoparticles improved the intracellular delivery efficiency and bioavailability of Fx, thereby enhancing its effect on anti-lipid accumulation. Oil red O staining results also support this observation Figure 7 B, in order to quantitatively analyze the oil red O staining results, the oil red O after staining was extracted with isopropanol, and the absorbance was measured by spectrophotometer. As shown in Figure 7 C, the P3C1@Fx group showed lower absorbance (1.220 ± 0.021) compared to the free Fx and P3C1 groups, confirming its superior performance in inhibiting fat accumulation. The effects of each treatment group were further analyzed and discussed by measuring the triglyceride (TG) and total cholesterol (TC) content in the cells Figure 7D, 7E). TG and TC contents of OA+PA group were significantly higher than the control group, which was in line with the expected effect of inducing lipid accumulation by adding free fatty acids. Compared with other treatment groups, P3C1@Fx group had the lowest TG and TC contents, which were 0.366±0.011 and 1.021±0.028 mmol / g prot, respectively. Overall, by loading the hydrophobic active substance fucoxanthin into P3C1, its ability to inhibit fat accumulation in the HepG2 cell model was significantly improved. This finding was not only verified in the visualization of lipid droplet accumulation, but also further supported by the determination of TG and TC. P3C1@Fx group showed a significant reduction in lipid droplets and a significant decrease in TG and TC contents, highlighting the potential of nanotechnology in improving the bioavailability of active ingredients and their therapeutic efficacy. These results provide strong experimental evidence for the use of nanocarriers to improve the application of marine carotenoids in anti-obesity therapy, especially in the potential to inhibit lipid droplet formation.

[0084] The above provided examples are not intended to limit the scope of the present application, nor are the described steps intended to limit the order of their execution. Those skilled in the art, in combination with the existing common knowledge, make obvious improvements to the present application, which also fall within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing food-derived cellulose-based nanocomposite particles, characterized in that: The following steps are involved: (1) Oxidized food-derived nanocellulose was prepared by a 2,2,6,6-tetramethylpiperidin-1-oxyl-mediated oxidation method using food-derived cellulose as raw material and sodium hypochlorite as oxidant; (2) dispersing the oxidized food-derived nanocellulose in water to obtain an oxidized food-derived nanocellulose suspension, and mixing it with a protein aqueous solution to obtain a protein / oxidized food-derived nanocellulose mixture; the mass concentration of protein and oxidized food-derived nanocellulose in the protein / oxidized food-derived nanocellulose mixture is 0.5-2%; and the mass ratio of oxidized food-derived nanocellulose to protein is 1:3; (3) adding the ethanol solution containing the hydrophobic active substance to the protein / oxidized food-derived nanocellulose mixture in step (2), homogenizing and ultrasonicating, and removing the ethanol to obtain food-derived cellulose-based nanocomposite particles; The food-derived cellulose is kelp nanocellulose, and the preparation method of kelp nanocellulose is: extracting kelp cellulose, then performing cellulase enzymolysis on the kelp cellulose, and finally performing ultrasonic treatment to obtain kelp nanocellulose; The protein is sodium caseinate; The hydrophobic active substance is fucoxanthin.

2. The method for preparing food-derived cellulose-based nanocomposite particles according to claim 1, wherein: In step (1), the specific preparation method of oxidized food-derived nanocellulose is as follows: food-derived cellulose is dispersed in ultrapure water, and 2,2,6,6-tetramethylpiperidine-1-oxyl, NaBr, and NaClO solutions are added under ice bath conditions; during the reaction, the pH value is maintained in the range of 10±0.2 by using 0.1 mol / L NaOH and HCl; when the pH value of the suspension is constant, ethanol is added to terminate the oxidation reaction; then, the suspension is sonicated, centrifuged, washed, dialyzed, and freeze-dried to obtain the oxidized food-derived nanocellulose.

3. The method for preparing food-derived cellulose-based nanocomposite particles according to claim 1, wherein: In step (2), the mass concentration of protein in the protein aqueous solution is 0.25~1%.

4. The method for preparing food-derived cellulose-based nanocomposite particles according to claim 1, wherein: In step (3), the concentration of the hydrophobic active substance in the ethanol solution containing the hydrophobic active substance is 1-50 mg / mL; and the volume ratio of the ethanol solution containing the hydrophobic active substance to the protein / oxidized food-derived nanocellulose mixture is 1:1-50.

5. Food-derived cellulose-based nanocomposite particles prepared by the method according to any one of claims 1 to 4.

6. Use of the food-derived cellulose-based nanocomposite particles according to claim 5 in the preparation of antioxidant substances for preventing or alleviating free fatty acid-induced oxidative stress-related diseases.

7. Use of the food-derived cellulose-based nanocomposite particles according to claim 5 in the preparation of lipid-lowering drugs.

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