A resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, its preparation method and application

Abstract

This invention provides a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, its preparation method, and its applications, belonging to the field of nanomaterials technology. This invention uses sulfated Hericium erinaceus β-glucan and chitosan as nanocarriers, and prepares the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite through electrostatic interaction self-assembly. This significantly improves the solubility, chemical stability, and bioavailability of resveratrol, thereby enhancing its anti-inflammatory effect, and exhibits an in vitro sustained-release effect compared to free resveratrol. The results of the examples show that the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite of this invention can enter M1 macrophages through endocytosis, effectively inhibiting the pro-inflammatory capacity of M1 macrophages, realizing the polarization of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, and exerting an anti-inflammatory effect.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, its preparation method, and its application. Background Technology

[0002] Resveratrol is a naturally occurring lipophilic polyphenol widely found in plants such as veratrum, grapes, soybeans, cherries, blueberries, and peanuts. It possesses biological activities including antioxidant, anti-inflammatory, hepatoprotective, anticancer, and antidiabetic effects. However, the practical application of resveratrol is limited by several factors, such as poor water solubility, unstable chemical structure (degradation and isomerization under the influence of light, oxygen, and oxidases), and low oral bioavailability.

[0003] To address the aforementioned challenges, numerous studies have been conducted in the food and pharmaceutical fields to encapsulate resveratrol into nanodelivery systems, such as nanoparticles, nanoemulsions, nanoliposomes, and hydrogels. For example, nanoemulsions are referenced in: Zhu PP, He JB, Huang S. Setal. Encapsulation of resveratrol zein-polyglycerol conjugate stabilized O / W nanoemulsions: Chemical stability, inhibitory astrointestinal digestion, and antioxidant activity[J]. LWT, 2021, 149: 112049; nanoliposomes are referenced in: Ghorbanzade T, Jafari SM, Akhavan S, et al. Nano-encapsulation of shale oil in nano-liposomes and its application informatization of yogurt[J]. Food Chemistry, 2017, 216: 146-152; and nanogels are referenced in: Buosi FS, Alaimo A, DiSanto MC, et al. Resveratrol encapsulation in high molecular weight chitosan-based nanogels for applications inocular treatments: Impacton human ARPE-19 culture cells [J]. International Journal of Biological Macromolecules, 2020, 165: 804-821.

[0004] However, most of the above nanodelivery systems suffer from low encapsulation rates and low bioavailability. Summary of the Invention

[0005] In view of this, the present invention aims to provide a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, its preparation method, and its application. The resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite obtained by the present invention has good solubility, stability, and bioavailability, as well as good anti-inflammatory effects and high encapsulation efficiency.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, comprising the following steps:

[0008] An ethanol solution of resveratrol was first mixed with a sulfated Hericium erinaceus β-glucan solution to obtain a resveratrol-sulfated Hericium erinaceus β-glucan complex.

[0009] The chitosan solution was mixed with the resveratrol-sulfated Hericium erinaceus β-glucan complex to obtain the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite.

[0010] Preferably, the molecular weight M of the sulfated Hericium erinaceus β-glucan is... w =15000~30000g / mol, degree of substitution Ds=1.3~1.8.

[0011] Preferably, the mass ratio of sulfated hericium erinaceus β-glucan to resveratrol is 2 to 12:1.

[0012] Preferably, the molecular weight M of the chitosan is... w =30000g / mol.

[0013] Preferably, the method for preparing the chitosan solution includes the following steps:

[0014] Chitosan was mixed with sodium acetate buffer solution, and the pH of the resulting mixture was adjusted to 4-6 to obtain a chitosan solution.

[0015] The concentration of the chitosan solution is 0.5–3 mg / mL.

[0016] Preferably, the mass ratio of chitosan to resveratrol-sulfated Hericium erinaceus β-glucan complex is 1:3 to 9.

[0017] Preferably, the second mixing rate is 200 to 1000 r / min.

[0018] The present invention provides a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite prepared by the above preparation method, comprising resveratrol, sulfated Hericium erinaceus β-glucan and chitosan bound by electrostatic interaction.

[0019] Preferably, the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite has a particle size of 191.07±1.52 nm; and the resveratrol loading rate in the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite is 5.15-5.45 wt%.

[0020] This invention provides the application of the above-mentioned resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite in the preparation of anti-inflammatory drugs.

[0021] This invention provides a method for preparing resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite (abbreviated as DS-CS-RESNPs), comprising the following steps: firstly, mixing an ethanol solution of resveratrol with a sulfated Hericium erinaceus β-glucan solution to obtain the resveratrol-sulfated Hericium erinaceus β-glucan composite; secondly, mixing a chitosan solution with the resveratrol-sulfated Hericium erinaceus β-glucan composite to obtain the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite. Hericium erinaceus β-glucan can induce lymphocyte proliferation and has strong immunomodulatory biological activity. After sulfate modification, the water solubility of Hericium erinaceus β-glucan is improved, and its immunomodulatory activity is enhanced. Chitosan is a product of the natural polysaccharide chitin after partial acetyl group removal, and has physiological functions such as antibacterial, anticancer, lipid-lowering, and immune-enhancing effects. This invention utilizes sulfated Hericium erinaceus β-glucan and chitosan as nanocarriers to prepare resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposites via electrostatic interaction self-assembly. This significantly improves the solubility, chemical stability, and bioavailability of resveratrol, thereby enhancing its anti-inflammatory effect. Furthermore, it exhibits a sustained-release effect in vitro compared to free resveratrol. The results of the examples show that in the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposites of this invention, the encapsulation rate of resveratrol is 91.84% ± 2.16% (n = 3), and the loading rate is 5.3% ± 0.15% (n = 3). It can enter M1 macrophages through endocytosis, effectively inhibiting the pro-inflammatory capacity of M1 macrophages and polarizing pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, thus exerting an anti-inflammatory effect. The resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite provided by this invention can effectively inhibit cytotoxicity and has good biocompatibility. At the same time, compared with free resveratrol, the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite exhibits better anti-inflammatory effects by inhibiting the release of NO and inflammatory factors IL-6, IL-1β and TNF-α, and can be applied in the food and pharmaceutical industries.

[0022] Meanwhile, the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite provided by this invention has a simple and quick preparation process, is easy to operate, and is convenient for practical application. Attached Figure Description

[0023] Figure 1 Transmission electron microscope image of DS-CS-RESNPs;

[0024] Figure 2 FT-IR spectra of RES, DS-CSNPs, and DS-CS-RESNPs;

[0025] Figure 3X-ray diffraction spectra of DS-CSNPs, RES, RES / DS-CSNPs, and DS-CS-RESNPs;

[0026] Figure 4 The effect of NaCl concentration on the average particle size of DS-CSNPs and DS-CS-RESNPs;

[0027] Figure 5 The effect of pH on the average particle size of DS-CSNPs and DS-CS-RESNPs;

[0028] Figure 6 The particle size changes of resveratrol nanocomposites during storage at 4℃ and 25℃.

[0029] Figure 7 Release curves of resveratrol from free resveratrol and resveratrol nanocomplex during in vitro simulated digestion;

[0030] Figure 8 Fluorescence inverted microscope images of RAW264.7 macrophages incubated with resveratrol nanocomposite for 0–72 h;

[0031] Figure 9 The effects of DS-CSNPs, free RES, and DS-CS-RESNPs on the cell viability of RAW264.7 cells;

[0032] Figure 10 The inhibitory rates of DS-CSNPs, free RES, and DS-CS-RESNPs on NO release from LPS-stimulated RAW264.7 cells were determined.

[0033] Figure 11 The effects of DS-CSNPs, free RES, and DS-CS-RESNPs on the release of TNF-α (a), IL-6 (b), and IL-1β (c) from RAW264.7 cells stimulated by LPS;

[0034] Figure 12 These are the results of a flow cytometry experiment. Detailed Implementation

[0035] This invention provides a method for preparing resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, comprising the following steps:

[0036] An ethanol solution of resveratrol was first mixed with a sulfated Hericium erinaceus β-glucan solution to obtain a resveratrol-sulfated Hericium erinaceus β-glucan complex.

[0037] The chitosan solution was mixed with the resveratrol-sulfated Hericium erinaceus β-glucan complex to obtain the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite.

[0038] This invention involves a first mixing of an ethanol solution of resveratrol and a sulfated Hericium erinaceus β-glucan solution to obtain a resveratrol-sulfated Hericium erinaceus β-glucan complex. In this invention, the concentration of the ethanol solution of resveratrol is preferably 0.5–5 mg / mL.

[0039] In this invention, the sulfated Hericium erinaceus β-glucan is preferably sourced from commercially available sources or prepared in-house. When prepared in-house, the method for preparing sulfated Hericium erinaceus β-glucan is as follows: ZHANG Z, TANG QJ, WUD, et al. Regioselective sulfation of β-glucan from Ganoderma lucidum and structure-anticoagulant activity relationship of sulfated derivatives [J]. International Journal of Biological Macromolecules, 2020, 155: 470-478.

[0040] In this invention, the molecular weight M of the sulfated Hericium erinaceus β-glucan is... w The concentration is preferably 15,000–30,000 g / mol, more preferably 20,000 g / mol, and the degree of substitution Ds is preferably 1.3–1.8, more preferably 1.66. In this invention, the concentration of the sulfated Hericium erinaceus β-glucan solution is preferably 1–3 mg / mL, more preferably 2 mg / mL.

[0041] In this invention, the mass ratio of sulfated hericium erinaceus β-glucan to resveratrol is preferably 2 to 12:1, more preferably 5 to 10:1.

[0042] In this invention, the preferred method for the first mixing is to add resveratrol solution dropwise to sulfated Hericium erinaceus β-glucan solution.

[0043] After obtaining the resveratrol-sulfated Hericium erinaceus β-glucan complex, the present invention further mixes the chitosan solution with the resveratrol-sulfated Hericium erinaceus β-glucan complex to obtain a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite. In the present invention, the molecular weight M of the chitosan is... w The preferred value is 30000 g / mol.

[0044] In this invention, the method for preparing the chitosan solution preferably includes the following steps:

[0045] Chitosan was mixed with sodium acetate buffer solution, and the pH of the resulting mixture was adjusted to 4-6 to obtain a chitosan solution.

[0046] In this invention, the pH adjuster used to adjust the pH value is preferably acetic acid.

[0047] In this invention, the concentration of the chitosan solution is preferably 0.5 to 3 mg / mL, more preferably 1 to 2 mg / mL.

[0048] In this invention, the mass ratio of chitosan to resveratrol-sulfated Hericium erinaceus β-glucan complex is preferably 1:3 to 9, more preferably 1:4 to 8.

[0049] In this invention, the second mixing method is preferably: chitosan solution is added dropwise to the resveratrol-sulfated Hericium erinaceus β-glucan complex under stirring conditions.

[0050] In this invention, the stirring rate during the second mixing is preferably 200-1000 r / min, more preferably 400-800 r / min, and the time is preferably 20-60 min, more preferably 30 min; the present invention preferably uses a magnetic stirrer for the stirring.

[0051] This invention provides a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite prepared by the above-described method, comprising resveratrol, sulfated Hericium erinaceus β-glucan, and chitosan bound together by electrostatic interactions. In this invention, the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite contains Hericium erinaceus β-glucan-resveratrol coating the chitosan.

[0052] In this invention, the particle size of the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite is preferably 191.07±1.52 nm; the resveratrol loading rate in the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite is preferably 5.15-5.45 wt%, more preferably 5.3 wt%.

[0053] This invention provides the application of the above-mentioned resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite in the preparation of anti-inflammatory drugs.

[0054] The following detailed description, in conjunction with embodiments, illustrates the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, its preparation method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0055] Example 1: Preparation of resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite

[0056] (1) Chitosan (M w =30000g / mol) was dissolved in 25mM sodium acetate buffer solution to prepare a 1.51mg / mL solution, and the initial pH of the chitosan solution was adjusted to 4.75 with acetic acid; resveratrol was dissolved in ethanol to prepare a 2mg / mL solution.

[0057] (2) Add resveratrol solution dropwise to sulfated Hericium erinaceus β-glucan (M w In a solution containing 20000 g / mol (degree of substitution Ds = 1.66), the mass ratio of sulfated Hericium erinaceus β-glucan to resveratrol was 9:1. Then, under magnetic stirring at 800 r / min, chitosan solution was added dropwise to the above mixed solution to make the mass ratio of chitosan to sulfated Hericium erinaceus β-glucan 1:5. After adding the chitosan solution and stirring for 30 min, a resveratrol nanocomposite suspension was obtained. The suspension was then freeze-dried to obtain the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, hereinafter referred to as resveratrol nanocomposite.

[0058] Structural characterization

[0059] (1) Determination of morphology, particle size, zeta potential and polydispersity index of resveratrol nanocomposites

[0060] Resveratrol nanocomposites were prepared according to the method described in Example 1. The particle size, zeta potential, and polydispersity index of the polymer were determined using a laser particle size analyzer (Zetasizer Nano ZS90, Malvern, UK). The particle size of the resveratrol nanocomposites was 191.07 ± 1.52 nm (n = 3), the zeta potential was -22.46 ± 1.46 mV (n = 3), and the polydispersity index was 0.229 ± 0.002 (n = 3).

[0061] The surface morphology of the resveratrol nanocomposite prepared in Example 1 was observed using transmission electron microscopy (TEM). The TEM image of the resveratrol nanocomposite is shown below. Figure 1 As shown, the resveratrol nanocomposite is basically spherical or subspherical in shape, with relatively uniform size and very few particles aggregated.

[0062] (2) Determination of resveratrol encapsulation rate and loading rate

[0063] The encapsulation efficiency of the resveratrol nanocomposite was calculated based on the measured free resveratrol content in the supernatant after ultracentrifugation at 12000×g for 20 minutes. Detection was performed using high-performance liquid chromatography (HPLC) at 306 nm. The mobile phase consisted of methanol-water solution (80:20 v / v) with isocratic elution. The flow rate was 0.8 mL / min, and the injection volume was 10 μL. The encapsulation efficiency and loading were determined by equations ① and ②, respectively.

[0064] ① Encapsulation rate = (Total resveratrol content - Content of free resveratrol in supernatant) / Total resveratrol content × 100%

[0065] ② Loading rate = Total resveratrol content in nanoparticles / Mass of nanoparticles × 100%

[0066] The encapsulation rate of resveratrol in the resveratrol nanocomposite was determined to be 91.84% ± 2.16% (n = 3), and the loading rate was 5.3% ± 0.15% (n = 3).

[0067] (3) Fourier transform infrared spectroscopy analysis

[0068] 2.0 mg of lyophilized resveratrol nanocomposite was placed at a designated position on an infrared spectrometer for scanning, with the spectral acquisition range being 500–4000 cm⁻¹. -1 The resolution is set to 4cm. -1 The FT-IR spectra of RES, DS-CSNPs, and DS-CS-RESNPs are shown below. Figure 2 As shown, in the infrared spectra of resveratrol (RES), sulfated Hericium erinaceus β-glucan-chitosan nanoparticles (DS-CSNPs), and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposites (DS-CS-RESNPs), the concentrations are located in the 3200–3550 cm⁻¹ range. -1 The broad characteristic peak is attributed to the stretching vibration of OH, and this peak is relatively broad because it overlaps with the absorption band of the NH stretching vibration. Compared with DS-CSNPs, the peak in DS-CS-RESNPs shows a blue shift, from 3383.13 cm⁻¹. -1 Moved to 3283.17cm -1 This indicates that RES binds to DS-CSNPs via hydrogen bonds. Therefore, the hydrogen bonds between RES and DS-CSNPs may be one of the main forces promoting nanoparticle formation.

[0069] (4) X-ray diffraction spectroscopy analysis

[0070] The physical states of sulfated Hericium erinaceus β-glucan-chitosan nanoparticles (DS-CS NPs), resveratrol (RES), a mixture of resveratrol and sulfated Hericium erinaceus β-glucan-chitosan nanoparticles (RES / DS-CS NPs), and a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite (DS-CS-RES NPs) were determined by X-ray diffraction. The X-ray diffraction spectra of DS-CS NPs, RES, RES / DS-CS NPs, and DS-CS-RES NPs are shown below. Figure 3 As shown, RES exhibits a specific crystalline morphology, with strong characteristic diffraction peaks observed within a diffraction angle range of 10–30°, and the diffraction intensity is highest at 13.414°, 16.543°, 19.369°, 22.542°, 23.759°, 25.389°, and 28.453°. Similarly, sharp peaks are still observed in RES / DS-CSNPs, indicating that RES remains in a crystalline state. However, due to the very low RES content (8.9% w / w) in RES / DS-CSNPs, the diffraction peak intensity is much lower than that of pure RES powder. When the same amount of RES was embedded in DS-CS-RESNPs, no obvious characteristic RES peaks were observed within the aforementioned diffraction angle range, confirming that RES in DS-CS-RESNPs exists in an amorphous form.

[0071] Test Example 1: Stability and sustained-release properties of resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite

[0072] (1) Stability of resveratrol nanocomposites

[0073] ① Effect of ionic strength on the stability of resveratrol nanocomposites

[0074] Resveratrol nanocomposite suspensions were prepared according to the method in Example 1, and different amounts of sodium chloride were added to the suspensions to make the final sodium chloride concentrations 0, 10, 20, 30 and 40 mM, respectively, to evaluate the effect of ionic strength on the stability of resveratrol nanocomposites. Finally, after standing at room temperature for 1 h, the particle size was measured.

[0075] The effect of NaCl concentration on the average particle size of DS-CSNPs and DS-CS-RESNPs is as follows: Figure 4As shown, resveratrol nanocomposites exposed to different concentrations of NaCl solution exhibited increasing particle size and decreasing surface charge with increasing NaCl concentration compared to the sulfated Hericium erinaceus β-glucan-chitosan nanoparticles. When the NaCl solution concentration reached 20 mM, aggregation of both the sulfated Hericium erinaceus β-glucan-chitosan nanoparticles and the resveratrol nanocomposites began to occur. However, precipitation occurred when the NaCl solution concentration reached 40 mM. This aggregation suggests that the presence of high-concentration salts may reduce electrostatic repulsion between particles through ionic electrostatic shielding, potentially leading to the aggregation of some polysaccharide molecules and promoting bridging flocculation.

[0076] ②The effect of pH on the stability of resveratrol nanocomposites:

[0077] A resveratrol nanocomposite suspension was prepared according to the method in Example 1. The pH was adjusted to 2.0–8.0 with 0.1 mM HCl or 0.1 mM NaOH to evaluate the effect of pH on the stability of the resveratrol nanocomposite. Finally, after standing at room temperature for 1 hour, the particle size was measured.

[0078] After being ingested by the human body, nanoparticles, acting as delivery carriers for functional components, undergo drastic pH changes in the gastrointestinal tract. Therefore, assessing the pH stability of composite nanoparticles is crucial for their practical applications. The effect of pH on the average particle size of DS-CSNPs and DS-CS-RESNPs is shown below. Figure 5 As shown, the particle size of the nanoparticles initially increases and then decreases, with the smallest particle size appearing at pH 4. When pH is 2–5, the nanoparticles do not aggregate, and there is no significant difference in particle size between the sulfated Hericium erinaceus β-glucan-chitosan nanoparticles and the resveratrol nanocomposite, indicating that the ionic interaction between sulfated Hericium erinaceus β-glucan and chitosan is not affected by resveratrol. When pH is 6–8, the particle sizes of the sulfated Hericium erinaceus β-glucan-chitosan nanoparticles and the resveratrol nanocomposite are large and differ significantly, indicating that the ionic interaction between sulfated Hericium erinaceus β-glucan and chitosan is affected by resveratrol.

[0079] ③ Storage stability of resveratrol nanocomposites at 25℃ and 4℃:

[0080] Sulfated Hericium erinaceus β-glucan-chitosan nanoparticle suspensions and resveratrol nanocomposite suspensions were stored at 4℃ and 25℃ for 18 days, respectively, to evaluate the storage stability of the nanoparticles. During storage, 0.03% sodium azide (w / v) was used to prevent microbial spoilage. A portion of the nanoparticle suspension was taken for particle size determination.

[0081] The particle size changes of resveratrol nanocomposites during storage at 4℃ and 25℃ are as follows: Figure 6 As shown, under storage conditions of 25℃, the particle size of resveratrol nanocomposites gradually increases with storage time. On day 18, the particle size reaches 551.61 nm. This is because, with prolonged storage, different nanoparticles aggregate and combine, forming larger particles, thus increasing the particle size distribution range. The particle size variation trend of resveratrol nanocomposites under storage conditions of 4℃ is roughly the same as that under 25℃ conditions, except that the variation range is smaller, indicating that the resveratrol nanocomposites are more stable under 4℃ conditions.

[0082] (2) The sustained-release properties of resveratrol nanocomposites

[0083] Simulated gastric digestion stage

[0084] Preparation method for simulated gastric juice (refer to the pharmacopoeia): Transfer 1.64 mL of concentrated hydrochloric acid to a beaker, slowly add 20 mL of distilled water to dilute, and transfer the solution to a 100 mL volumetric flask. Take 1 g of pepsin in another beaker, add a small amount of water to dissolve, and transfer to the volumetric flask mentioned above. Add water to make up to volume. Adjust the pH of the digestive juice to 2.0 ± 0.1 with 1 mol / L sodium bicarbonate solution and store at 4 °C.

[0085] Simulated gastric digestion process: 1 mL of freshly prepared sample solution was added to a dialysis bag, which was then sealed and placed in a test tube containing 5 mL of gastric juice. The test tube was placed in a 37°C constant-temperature shaker to begin simulating gastric digestion. At 0 min, 15 min, 30 min, 60 min, 120 min, and 240 min, 0.5 mL of the simulated gastric juice sample was taken, with the same volume of simulated gastric juice added after each sample collection. The collected simulated gastric juice sample was then inactivated by boiling water for 15 min, cooled to room temperature, and stored at 4°C for later use. The resveratrol content was analyzed by HPLC, and the resveratrol release was calculated.

[0086] Simulated intestinal digestion stage

[0087] Preparation method for simulated intestinal fluid (refer to the pharmacopoeia): Weigh 0.65 g of potassium dihydrogen phosphate into a beaker and slowly add 50 mL of distilled water to dissolve. Adjust the pH of the solution to 6.8 ± 0.1 with 4% sodium hydroxide solution and transfer to a 100 mL volumetric flask. Dissolve 1 g of trypsin in another beaker with a small amount of water and transfer to the same volumetric flask. Make up to volume with water and store at 4 °C.

[0088] Simulated intestinal digestion process: 1 mL of freshly prepared sample solution was added to a dialysis bag, which was then sealed and placed in a test tube containing 10 mL of intestinal fluid. The test tube was placed in a 37°C constant-temperature shaker to begin simulating intestinal digestion. At 37°C for 0 min, 15 min, 30 min, 60 min, 120 min, 240 min, and 480 min, 0.5 mL of the simulated intestinal fluid sample was collected. The same volume of simulated intestinal fluid was replenished after each sample collection. The collected simulated intestinal fluid sample was then inactivated by boiling water for 15 min, cooled to room temperature, and stored at 4°C for later use. The RES content was analyzed by HPLC, and the RES release amount was calculated.

[0089] like Figure 7 As shown, the release curves of free resveratrol and resveratrol nanocomposites during simulated in vitro digestion are illustrated. Free resveratrol diffuses rapidly during simulated gastrointestinal digestion, with 39.19% of the resveratrol ultimately released into the gastric juice. Figure 7 In the middle (a), 59.61% of resveratrol was released into the intestinal fluid. Figure 7 (b) Free resveratrol is soluble in anhydrous ethanol and is not encapsulated in any carrier. Therefore, during the initial digestion process, it can diffuse freely and rapidly within the dialysis bag via osmosis. In contrast, when resveratrol is encapsulated in a nanocomposite, its release rate during the simulated gastric digestion phase is much lower than that of free resveratrol. At the end of the simulated gastric digestion, the release rate of resveratrol from the nanocomposite is 12.68%, and at the end of the simulated intestinal digestion, the release rate is 39.83%. The resveratrol nanocomposite shows a 26.51% and 19.78% lower release rate of resveratrol in gastric and intestinal fluids, respectively, compared to the release rate of free resveratrol. The results indicate that, under simulated gastrointestinal digestion, the release rate of resveratrol from the nanocomposite is significantly reduced compared to that of free resveratrol. This trend is due to the stable colloidal structure of the nanoparticles in simulated gastric juice. The outer layer of sulfated Hericium erinaceus β-glucan provides some protection for resveratrol in the simulated gastrointestinal environment, significantly reducing the release of resveratrol in the gastrointestinal tract. These results indicate that the resveratrol nanocomposite exhibits excellent sustained-release performance during in vitro simulated gastrointestinal digestion, effectively protecting resveratrol from structural degradation caused by the extreme gastrointestinal environment, thereby improving its bioavailability.

[0090] Test Example 2: Evaluation of cellular uptake of resveratrol nanocomplex by RAW264.7 macrophages

[0091] The method described in the reference (Jin MF, Li SY, Wu YH, et al. Construction of Chitosan / Alginate Nano-Drug Delivery System for Improving Dextran Sodium Sulfate-Induced Colitis in Mice [J]. Nanomaterials (Basel), 2021, 11(8), 1884) was used to prepare resveratrol nanocomposites labeled with Rhodamine B isothiocyanate (RBITC) with red fluorescein, so as to analyze the cellular uptake of nanoparticles by fluorescence microscopy imaging. 2 × 10⁻⁶ ppm was used per milliliter. 4 RAW264.7 cells were seeded in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, culture medium containing RBITC-labeled resveratrol nanocomplex was added, and the cells were incubated together for 24 h, 48 h, and 72 h. The culture medium was removed, and excess RBITC-labeled resveratrol nanocomplex was washed away with PBS. The cells were then fixed with 4% paraformaldehyde for approximately 15–20 min and stained with DAPI for 5 min. Observation was performed using a fluorescence inverted microscope, and images were acquired in the RBITC and DAPI channels.

[0092] Fluorescence inverted microscope images of RAW264.7 macrophages incubated with resveratrol nanocomposite for 0–72 h are shown below. Figure 8 As shown, when cells were treated with resveratrol nanocomplexes at 0 h, no overlap of red and blue fluorescence was observed, indicating that no resveratrol nanocomplexes entered the cells. After 24 h of co-incubation, red fluorescence was observed on the cell surface, and cells began to absorb the resveratrol nanocomplexes. As time increased to 48 h, red and blue fluorescence overlapped, mainly distributed at the cell nucleus periphery. At 72 h, the overlapped fluorescence continued to increase, showing higher internalization efficiency, indicating that the resveratrol nanocomplexes entered the cell nucleus. These data suggest that the resveratrol nanocomplexes are eventually taken up by macrophages to exert their effects, and the uptake efficiency becomes higher over time.

[0093] Test Example 3: Evaluation of the in vitro anti-inflammatory activity of resveratrol nanocomplex

[0094] (1) Cytotoxicity against RAW264.7 macrophages

[0095] The cytotoxicity of resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplex to RAW264.7 cells was determined using the Alamarblue assay. RAW264.7 cell suspensions were inoculated into 96-well plates at a cell count of 5 × 10⁶ cells / mL.5 180 μL of culture medium was collected per well and incubated at 37°C in a 5% CO2 incubator for 24 h. The cell culture medium in the wells was then removed and replaced with 200 μL of fresh culture medium containing different concentrations of resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplexes (final concentrations: 5, 10, 15, 25 μg / mL resveratrol, based on resveratrol loading). After incubation at 37°C for 48 h, 20 μL of AlamarBlue reagent was added to each well for further incubation. The effects of resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplexes on the viability of RAW264.7 cells within the selected concentration range were evaluated using the AlamarBlue method. Each sample was tested in triplicate.

[0096]

[0097] The effects of DS-CSNPs, free RES, and DS-CS-RESNPs on the cell viability of RAW264.7 cells were as follows: Figure 9 As shown, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles had no effect on cell viability and exhibited no cytotoxicity. Cell viability treated with free resveratrol at concentrations of 5, 10, 15, 25, and 50 μg / mL were 93.1%, 62.23%, 53.96%, 45.54%, and 41.51%, respectively, demonstrating significant cytotoxicity. Conversely, nanocomposites with the same resveratrol concentrations all achieved cell viability of approximately 80% or higher under the same experimental conditions, indicating that the cytotoxicity of resveratrol-concentrated nanocomposites was negligible. These results suggest that encapsulating resveratrol into nanoparticles can significantly reduce their cytotoxicity.

[0098] (2) Effects on NO release from LPS-stimulated RAW264.7 macrophages

[0099] RAW264.7 cells were diluted with colorless RPMI 1640 medium to a concentration of 5 × 10⁶ cells / mL. 5A suspension of cells was added to 160 μL of each well in a 96-well plate. Cells were cultured at 37°C until complete adherence. The culture medium was then removed from the wells and replaced with 200 μL of fresh medium containing different concentrations of resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposites (final concentrations: 5, 10, 15, 25 μg / mL resveratrol, based on resveratrol loading). RAW264.7 cells were stimulated with 1 μg / mL LPS, and a blank control group (PBS group) was set up. After culturing at 37°C for 48 h, 100 μL of the supernatant was added to 50 μL of Leriess reagent. The reaction was allowed to proceed for 10 min, and the absorbance at 543 nm was measured to determine the NO release and calculate the inhibition rate.

[0100]

[0101] The inhibitory rates of DS-CSNPs, free RES, and DS-CS-RESNPs on LPS-stimulated NO release from RAW264.7 cells are as follows: Figure 10 As shown, within the concentration range of 5–25 μg / mL, resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplexes all inhibited LPS-stimulated NO release from RAW264.7 in a concentration-dependent manner. At concentrations of 15 and 25 μg / mL, the inhibitory effect of the resveratrol nanocomplex was higher than that of free resveratrol.

[0102] (3) Effects on the release of inflammatory factors IL-6, IL-1β and TNF-α from RAW264.7 stimulated by LPS

[0103] Cells were cultured according to the method in Example 5(2), and LPS and samples were added. After culturing at 37°C for 48 hours, the supernatant was taken and centrifuged at 12000g for 10 minutes. The contents of inflammatory factors IL-6, IL-1β and TNF-α were strictly measured according to the test requirements of the ELISA kit.

[0104] The effects of DS-CSNPs, free RES, and DS-CS-RESNPs on the release of TNF-α(a), IL-6(b), and IL-1β(c) from RAW264.7 cells stimulated by LPS are as follows: Figure 11 As shown, compared with the PBS group, LPS stimulation of RAW264.7 cells significantly increased the levels of IL-6, IL-1β, and TNF-α. When the concentration of resveratrol was 5–25 μg / mL, free resveratrol was more effective than sulfated Hericium erinaceus β-glucan-chitosan nanoparticles and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplex in reducing the level of the pro-inflammatory factor TNF-α. Figure 11(a). Furthermore, both free resveratrol and its nanocomposite reduced the release of IL-6 and IL-1β. Figure 11 (b, c). The results showed that, without causing cytotoxicity, the resveratrol nanocomposite exhibited a good effect in inhibiting the release of TNF-α, IL-6 and IL-1β, and had a significant anti-inflammatory effect.

[0105] (4) Induction of macrophage M1 / M2 phenotype and flow cytometry analysis

[0106] RAW264.7 cells were administered at a rate of 5 × 10⁻⁶ cells per ml. 5 Cells were added to 6-well plates at a density of 100 μg / mL. After culturing for 12 h, the cells were then cultured for 24 h at 37 °C with LPS (5 μg / mL) and different concentrations of resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposites (final concentrations: 5, 15 μg / mL RES, based on RES loading). Cells were collected into 2 mL sterile centrifuge tubes, 1 μL of LFcRblock was added, and the cells were incubated at 4 °C for 10 min. Cells were then incubated with 1 μL of PE-conjugated anti-mouse CD86 at 4 °C in the dark for 30 min. After centrifugation at 150 g for 5 min, and washing twice with PBS, the cells were incubated with 500 μL of fixation buffer at room temperature in the dark for 30 min. The fixation buffer was then removed by centrifugation at 150 g for 5 min. The cells were resuspended in 2 mL of intracellular static osmosis wash buffer and centrifuged at 150 g for 5 min to remove the supernatant. Cells were resuspended in 100 μL of wash buffer and incubated with 0.5 μL of LPC-conjugated anti-mouse CD206 antibody at room temperature in the dark for 30 min. Excess antibody was removed by washing cells with wash buffer, and cells were collected by centrifugation and resuspended in 500 μL of wash buffer. Flow cytometry analysis was performed using a BDAccuri C6 flow cytometer, and data were analyzed using FlowJo software.

[0107] Macrophage polarization is important in the development, progression, and resolution of inflammation. Macrophages can polarize into pro-inflammatory M1 types and anti-inflammatory M2 types. M1 macrophages produce pro-inflammatory mediators, while M2 macrophages have anti-inflammatory effects. RAW264.7 macrophages were analyzed using CD86 (M1 marker) and the mannose receptor CD206 (M2 marker). Flow cytometry results are shown below. Figure 12 As shown, after adding resveratrol, sulfated Hericium erinaceus β-glucan-chitosan nanoparticles, and resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomplexes, CD206 + The proportion of cells increased in a dose-dependent manner, while CD86... +The cell ratio showed a decreasing trend. This indicates that the sample promoted the transformation of M1 cells into M2 cells. Simultaneously, the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite was found to be more effective than sulfated Hericium erinaceus β-glucan-chitosan nanoparticles and free resveratrol, suggesting that the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite can promote the transformation of M1 cells into M2 cells and exert an anti-inflammatory effect.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite, comprising the following steps: An ethanol solution of resveratrol was first mixed with a sulfated Hericium erinaceus β-glucan solution to obtain a resveratrol-sulfated Hericium erinaceus β-glucan complex; the mass ratio of sulfated Hericium erinaceus β-glucan to resveratrol was 2-12:

1. The chitosan solution was mixed with the resveratrol-sulfated Hericium erinaceus β-glucan complex to obtain a resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite; the mass ratio of chitosan to resveratrol-sulfated Hericium erinaceus β-glucan complex was 1:3 to 9. The molecular weight M of the chitosan w =30000g / mol; The molecular weight M of the sulfated Hericium erinaceus β-glucan w =15000~30000g / mol.

2. The preparation method according to claim 1, characterized in that, The degree of substitution Ds of the sulfated Hericium erinaceus β-glucan is 1.3 to 1.

8.

3. The preparation method according to claim 1, characterized in that, The method for preparing the chitosan solution includes the following steps: Chitosan was mixed with sodium acetate buffer solution, and the pH of the resulting mixture was adjusted to 4-6 to obtain a chitosan solution. The concentration of the chitosan solution is 0.5–3 mg / mL.

4. The preparation method according to claim 1, characterized in that, The second mixing rate is 200–1000 r / min.

5. The resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite prepared by the preparation method according to any one of claims 1 to 4 comprises resveratrol, sulfated Hericium erinaceus β-glucan and chitosan bound by electrostatic interaction.

6. The resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite according to claim 5, characterized in that, The resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite has a particle size of 191.07±1.52 nm; the resveratrol loading rate in the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite is 5.15-5.45 wt%.

7. The use of the resveratrol-sulfated Hericium erinaceus β-glucan-chitosan nanocomposite according to claim 5 or 6 in the preparation of anti-inflammatory drugs.

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