A colon-targeted slow-release oryzanol nano-delivery carrier and a preparation method thereof

Abstract

This invention belongs to the field of biotechnology and discloses a colon-targeted sustained-release oryzanol nanocarrier and its preparation method. A composite nano-delivery system of oryzanol is constructed using *Ulva prolifera* polysaccharide and zein. The nanoparticles loaded with ORY using this carrier have a small particle size and good system stability; the PDI is less than 0.3, the absolute value of the zeta potential is greater than 40 mV, and the encapsulation efficiency and loading rate of ORY are as high as 73.3% and 4.46%, respectively. This carrier significantly improves the stability of ORY under environmental factors such as temperature, ion concentration, and light. It not only has gastrointestinal sustained-release activity and colon-targeting potential but also exhibits high affinity for macrophages, significantly downregulating the expression of inflammatory factors. This provides a targeted delivery strategy for improving the bioavailability of ORY and enabling it to exert its effects at colonic lesion sites.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to a colon-targeted sustained-release oryzanol nanocarrier and its preparation method. Background Technology

[0002] Oryzanol (ORY) is a complex mixture of phytosterols or triterpenoids and ferulic acid, primarily found in brown rice bran. Current research confirms that ORY possesses antioxidant, anti-inflammatory, cholesterol-lowering, hyperglycemic, and anti-tumor effects. Animal models of colitis have shown that ORY can downregulate the expression of inflammatory signaling pathways, inhibit the secretion of inflammatory factors, alleviate crypt rupture, and reduce goblet cell loss. However, its low water solubility, poor stability, short retention time in the gastrointestinal tract, and low bioavailability make it difficult for orally administered ORY to achieve specific enrichment at colonic lesions to exert a stronger therapeutic effect. Therefore, protecting the stability of ORY under environmental factors such as temperature, ion concentration, and light, and reducing its degradation in the acidic environment of the gastrointestinal tract and the presence of digestive enzymes, in order to enable ORY to exert its effects at colonic lesion sites, is a significant challenge that needs to be addressed to promote the intestinal health benefits of ORY.

[0003] Current research techniques often use proteins or polysaccharides as carriers to deliver hydrophobic active substances. For example, zein alone can self-assemble with ORY to form nanoparticles, but this delivery system suffers from problems such as low encapsulation efficiency, large particle size, and low bioavailability (Food Science & Nutrition, 2021, 9(2), 616-624.). While adding a chitosan coating to the ORY nanocarrier can increase the bioavailability of ORY, chitosan's stability in simulated gastrointestinal fluid is insufficient, and its effect as a carrier for sustained release and intestinal targeted delivery of ORY remains inadequate (International Journal of Pharmaceutics, 2023, 631, 122482.). Currently, research on ORY delivery is mostly limited to single proteins or polysaccharides, and there are no reports on the preparation of colon-targeted sustained-release delivery systems for ORY using the intermolecular interactions between proteins and polysaccharides. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, and addressing the issues of low water solubility, poor stability, short retention time in the gastrointestinal tract, low bioavailability, and difficulty in achieving specific enrichment at colonic lesions for ORY, as well as the poor stability and low encapsulation and loading rates of Zein as a single delivery carrier, this invention provides a colon-targeted sustained-release ORY nanocarrier and its preparation method. This invention utilizes *Ulva prolifera* polysaccharide (… Enteromorpha proliferaPolysaccharide (EP) and Zein were used to construct a composite nanodelivery system for ORY (ZEO). Compared with existing technologies, the encapsulation efficiency and loading rate of ORY increased to 73.30% and 4.46%, respectively. After encapsulation, the stability of the composite nanodelivery system ZEO was improved under different pH values, salt ion concentrations, heating, and UV irradiation conditions. In environments with pH values ​​from 2.0 to 8.0 or sodium chloride solutions (0-50 mM), the ZEO particle size remained below 300 nm, and the PDI was below 0.3. After 1 h of treatment at different temperatures (50-90 °C), the retention rate of ORY in ZEO reached over 90%. After 5 h of UV irradiation, the retention rate of ORY in ZEO reached over 75%, which is much higher than the retention rate of free ORY (17.13%). After 6 hours of gastrointestinal digestion, the release rate of ORY in the ZEO composite nanosystem was only 70.55%, significantly lower than the release rate of ORY monomer (86.21%). The bioavailability of ORY in ZEO reached over 30%, far exceeding that of free ORY (9.03%), solving the problem of short retention time and low bioavailability of ORY in the gastrointestinal tract. ZEO significantly enhanced the cellular uptake of ORY by macrophages, thereby significantly inhibiting the secretion of inflammatory factors TNF-α, IL-6, and IL-1β. After simulating gastrointestinal digestion in vitro, the zeta potential of the ZEO composite nanodelivery system changed from -41 mV to -17 mV, while maintaining electronegativity. It can accumulate in positively charged inflammatory areas through electrostatic adsorption, thereby enhancing its targeting effect on lesion sites. In a mouse model of colitis, 24 hours after oral administration, the colon of the mice continued to produce fluorescent signals. The ZEO composite nanosystem significantly prolonged the residence time of ORY in inflamed colon tissue, solving the problem of difficulty in achieving specific enrichment of ORY at colonic lesion sites.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: A method for preparing a colon-targeted sustained-release oryzanol nanocarrier includes the following steps: S1. Disperse Zein in ethanol and stir thoroughly at room temperature to obtain a Zein solution; S2. Disperse EP in deionized water and stir thoroughly for 8-12 h to obtain an EP solution; S3. Under stirring conditions, the Zein solution prepared in step S1 is added dropwise to the EP solution prepared in step S2 to obtain an EP-Zein mixed solution; S4. Under vacuum conditions, rotary evaporation was used to remove ethanol, centrifugation was used to remove insoluble large particles, and freeze-drying was performed to obtain the EP-Zein composite nanocarrier.

[0006] Furthermore, in step S1, the volume fraction of the ethanol solution is 65%-80%.

[0007] Furthermore, in step S1, the concentration of Zein is 5-20 mg / mL.

[0008] Furthermore, in step S2, the concentration of EP is 0.3-1.7 mg / mL.

[0009] Furthermore, in step S2, the stirring conditions are: stirring temperature of 25-45 ℃ and stirring speed of 200-600 rpm / min.

[0010] Furthermore, in step S2, the mass ratio of EP to Zein is 1:2-1:8.

[0011] Furthermore, in step S3, the stirring conditions are: stirring temperature of 25-30 ℃ and stirring speed of 200-600 rpm / min.

[0012] Furthermore, in step S4, the centrifugation conditions are: centrifugation speed of 3000-5000 rpm and time of 20-40 min.

[0013] This invention also claims protection for the colon-targeted sustained-release oryzanol nanocarrier prepared by the above preparation method, wherein EP and Zein are combined through electrostatic interaction and hydrogen bonding to form a nanocarrier for loading and delivering ORY; the mass ratio of ORY to Zein is 1:40.

[0014] This invention also claims protection for the application of the colon-targeted sustained-release oryzanol nanocarrier prepared by the above-described method in the delivery of ORY. Specifically, the application is the delivery of ORY in the treatment of colitis.

[0015] The advantages of this invention compared to the prior art are: This invention provides a colon-targeted sustained-release oryzanol nanocarrier and its preparation method. The specifically defined EP to zein ratio and preparation method improve the encapsulation efficiency, loading rate, environmental stability, gastrointestinal stability, and bioavailability of ORY. Simultaneously, this system also exhibits good anti-inflammatory properties and colon-targeting activity. The raw materials used in this invention are widely available and the technical solution is simple and easy to implement, laying a solid foundation for expanding the application of ORY in oral targeted delivery.

[0016] This invention utilizes EP and Zein to prepare a nanodelivery system for loading ORY. The nanoparticles formed using this carrier have a small particle size (less than 250 nm), exhibit good system stability (PDI less than 0.3, absolute zeta potential greater than 40 mV), and achieve encapsulation efficiency and loading rate of 73.3% and 4.46% for ORY, respectively. This carrier significantly improves the stability of ORY under environmental factors such as temperature, ion concentration, and light. It not only possesses gastrointestinal sustained-release activity and colon-targeting potential but also exhibits high affinity for macrophages, significantly downregulating the expression of inflammatory factors. This provides a targeted delivery strategy for improving the bioavailability of ORY and enabling it to exert its effects at colonic lesion sites. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0018] Figure 1 The figures show the characterization of the nano-delivery carriers under different Zein to EP mass ratios in Examples 1-5. Figure A shows the average particle size and polydispersity index (PDI) of different nano-delivery carriers, Figure B shows the zeta potential, and Figure C shows the turbidity.

[0019] Figure 2 The loading rate and encapsulation rate of ORY under different Zein to EP mass ratios in Examples 1-5.

[0020] Figure 3 This is a schematic diagram showing the encapsulation rate and loading rate of Examples 2, 3, Comparative Example 1, and Comparative Example 2.

[0021] Figure 4 This is a schematic diagram showing the encapsulation rate and loading rate of Examples 2, 3, 3, and 4.

[0022] Figure 5 The structure of the ZEO nanosystem prepared in Example 1 is characterized. Figure A shows the FTIR spectrum, and Figure B shows the X-ray diffraction pattern.

[0023] Figure 6 Figure 1 shows the effect of different environmental factors on the stability of the ZEO nanosystem prepared in Example 1. Figure A shows the pH stability analysis results, Figure B shows the ionic strength stability analysis results, Figure C shows the thermal stability analysis results, and Figure D shows the photostability analysis results.

[0024] Figure 7 Figure 1 shows the results of in vitro digestion and bioaccessibility analysis of the ZEO nanosystem prepared in Example 1. Figure A shows the results of in vitro simulated digestion analysis, and Figure B shows the results of bioaccessibility analysis.

[0025] Figure 8Figure 1 shows the uptake results of the ZEO nanosystem prepared for macrophages in Example 1. Figure A shows the cellular uptake results of ORY, ZO, and ZEO nanosystems by LPS-induced or uninduced macrophages; Figure B shows the effect of different time points on the uptake of ZEO nanosystems by LPS-induced macrophages; Figure C shows the quantitative graph of the relative fluorescence intensity of the cellular uptake of ORY, ZO, and ZEO nanosystems by LPS-induced or uninduced macrophages; and Figure D shows the quantitative graph of the relative fluorescence intensity of the effect of different time points on the uptake of ZEO nanosystems by LPS-induced macrophages.

[0026] Figure 9 Figure 1 shows the in vitro anti-inflammatory activity analysis of the ZEO nanosystem prepared in Example 1. Figure A shows the effect on the content of the inflammatory factor TNFα, Figure B shows the effect on the content of the inflammatory factor IL-1β, and Figure C shows the effect on the content of the inflammatory factor IL-6.

[0027] Figure 10 Figure 1 shows the colon targeting analysis of the ZEO nanosystem prepared in Example 1. Figure A shows the zeta potential of the ZEO nanosystem before and after simulated digestion in vitro. Figure B shows the fluorescence intensity of colitis tissue treated with the ZEO nanosystem at different time intervals. Figure C shows the relative fluorescence intensity quantification of colitis tissue treated with the ZEO nanosystem at different time intervals. Detailed Implementation

[0028] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0029] Example 1 Preparation of Zein nanocarriers: 1 g of Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. 4 mL of the Zein solution was added dropwise to 16 mL of ultrapure aqueous solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Then, the ethanol was removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the Zein nanocarrier.

[0030] Example 2 Preparation of EP-Zein nanocarriers: 0.1 g EP was dispersed in 300 mL of deionized water and stirred overnight at 600 rpm to obtain an EP solution. Then, 1 g Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. 4 mL of Zein solution was added dropwise to 16 mL of EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Ethanol was then removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the EP-Zein nanocarrier.

[0031] Example 3 Preparation of EP-Zein nanocarriers: 0.2 g of EP was dispersed in 300 mL of deionized water and stirred overnight at 600 rpm to obtain an EP solution. Then, 1 g of Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. 4 mL of the Zein solution was added dropwise to 16 mL of the EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. The ethanol was then removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the EP-Zein nanocarrier.

[0032] Example 4 Preparation of EP-Zein nanocarriers: 0.25 g of EP was dispersed in 300 mL of deionized water and stirred overnight at 600 rpm to obtain an EP solution. Then, 1 g of Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. 4 mL of Zein solution was added dropwise to 16 mL of EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Ethanol was then removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the EP-Zein nanocarrier.

[0033] Example 5 Preparation of EP-Zein nanocarriers: 0.5 g of EP was dispersed in 300 mL of deionized water and stirred at 600 rpm for 12 h to obtain an EP solution. Then, 1 g of Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. 4 mL of Zein solution was added dropwise to 16 mL of EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Ethanol was then removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the EP-Zein nanocarrier.

[0034] Application Example 1 Preparation of ORY-loaded EP-Zein composite nanodelivery system: The EP-Zein nanodelivery carrier obtained in Example 2 was followed in detail. 0.1 g EP was dispersed in 300 mL of deionized water and stirred overnight at 600 rpm to obtain an EP solution. Then, 1 g Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. ORY was added at a mass ratio of 1:40 to Zein, and the mixture was stirred at 600 rpm for 1 h to obtain an ORY-Zein composite solution. 4 mL of the ORY-Zein composite solution was added dropwise to 16 mL of the EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Then, the ethanol was removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to 20 mL to replenish the evaporated ethanol volume. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the ORY-loaded EP-Zein composite nanodelivery system.

[0035] Application Example 2 Preparation of ORY-loaded EP-Zein composite nanodelivery system: The EP-Zein nanodelivery carrier obtained in Example 3 was used. Details are as follows: 0.2 g of EP was dispersed in 300 mL of deionized water and stirred overnight at 600 rpm to obtain an EP solution. Then, 1 g of Zein was dispersed in 100 mL of 75% ethanol aqueous solution and stirred at 600 rpm for 1 h to obtain a Zein solution. ORY was added at a mass ratio of 1:40 to Zein, and the mixture was stirred at 600 rpm for 1 h to obtain an ORY-Zein composite solution. 4 mL of the ORY-Zein composite solution was added dropwise to 16 mL of EP solution at a rate of 1 mL / min using a syringe, and the mixture was stirred at 600 rpm for 0.5 h. Then, the ethanol was removed by rotary evaporation in a 40 °C water bath for 20 min. Deionized water was then added to a final volume of 20 mL to replenish the evaporated ethanol. The mixture was centrifuged at 3000 rpm for 15 min to remove insoluble large particles, and the supernatant was freeze-dried to obtain the ORY-loaded EP-Zein composite nanodelivery system.

[0036] Comparative Example 1 Preparation of EP-Zein composite nanodelivery system loaded with ferulic acid (FA): FA was added at a mass ratio of 1:40 to FA, and the mixture was stirred at 600 rpm for 1 h to obtain an ORY-Zein composite solution. Other operating conditions were the same as in Example 2 to obtain an FA-loaded EP-Zein composite nanodelivery system.

[0037] Comparative Example 2 Preparation of EP-Zein composite nanodelivery system loaded with ferulic acid (FA): FA was added at a mass ratio of 1:40 to FA, and the mixture was stirred at 600 rpm for 1 h to obtain an ORY-Zein composite solution. Other operating conditions were the same as in Example 3 to obtain an FA-loaded EP-Zein composite nanodelivery system.

[0038] Comparative Example 3 Preparation of ORY-loaded carrageenan (CA)-Zein composite nanodelivery system: 0.1 g CA was dispersed in 300 mL of deionized water and stirred at 600 rpm for 12 h to obtain a CA solution. Other operating conditions were the same as in Application Example 1 to obtain a CA-Zein composite nanodelivery system loaded with ORY.

[0039] Comparative Example 4 Preparation of ORY-loaded CA-Zein composite nanodelivery system: 0.2 g of CA was dispersed in 300 mL of deionized water and stirred at 600 rpm for 12 h to obtain a CA solution. Other operating conditions were the same as in Application Example 1 to obtain a CA-Zein composite nanodelivery system loaded with ORY.

[0040] Research Test Example 1 The particle size, polydispersity index (PDI), and zeta potential of the carrier in the above composite nanodelivery system were determined using a Malvern dynamic light scattering system (Malvern Zetasizer Nano ZS90), and the turbidity was analyzed using a UV spectrophotometer.

[0041] The embedding efficiency (EE) and loading rate (LC) of ORY are determined as follows: The 10 mg lyophilized EP-Zein composite nanodelivery system loaded with ORY prepared in Example 1 was dissolved in 10 mL of ethanol solution and sonicated at 500 W for 30 min to destroy the nanodelivery system and release ORY. The precipitate was removed by centrifugation at 8000 rpm for 10 min.

[0042] The absorbance of the supernatant was measured at 327 nm using a UV spectrophotometer, and the result was calculated according to the standard curve (y = 0.063x + 0.0545, R0). 2 = 0.993) Calculate the ORY concentration, and calculate EE and LC according to the following formulas: EE = (Total ORY added - Free ORY content) / Total ORY added × 100%; LC = (Total ORY addition - Free ORY content) / Total nanoparticle addition × 100%; like Figure 1 As shown, with the addition of EP, the zeta potential changed from positive to negative and its absolute value increased. The particle size gradually increased, the turbidity gradually increased, and the PDI decreased to below 0.2. These results indicate that the EP-Zein nanocarrier is a relatively stable nanocarrier. Compared with Examples 3-5, Example 2 had the smallest particle size (208.34 nm), a PDI of 0.19, a zeta potential of -40.63 mV, and a turbidity of 1.07. After loading ORY, as... Figure 2 As shown, the embedding rate and loading rate are at their highest at this time, at 73.28% and 4.46%, respectively.

[0043] Study Test Example 2 The methods for determining the embedding rate and loading rate of FA are as follows: The 10 mg lyophilized FA-loaded EP-Zein composite nanodelivery systems prepared in Comparative Examples 1 and 2 were dissolved in 10 mL of ethanol solution and sonicated at 500 W for 30 min to disrupt the nanodelivery system and release ORY. The precipitate was removed by centrifugation at 8000 rpm for 10 min.

[0044] The absorbance of the supernatant was measured at 318 nm using a UV spectrophotometer, and the result was calculated according to the standard curve (y = 0.118x + 0.0625, R0). 2 = 0.999) Calculate the FA concentration, and calculate the encapsulation rate and loading rate of Examples 2, 3, Comparative Example 1 and Comparative Example 2 according to the following formula: EE = (Total FA Added - Free FA Content) / Total FA Added × 100%; LC = (Total FA addition - Free FA content) / Total nanoparticle addition × 100%; like Figure 3 As shown, when the mass ratio of EP to Zein is 1:8 or 1:4, the encapsulation rate of FA can reach as high as 77.07% or more, but the loading rate drops to below 2.5%. This indicates that there is a lack of sufficient electrostatic attraction or other non-covalent interaction between FA and the carrier material, which means that although FA is physically encapsulated, the actual binding is not tight, resulting in a low loading rate of FA.

[0045] Study Test Example 3 The encapsulation rate and loading rate of Examples 2, 3, 3 and 4 were determined using a UV spectrophotometer.

[0046] like Figure 4 As shown, when the mass ratio of EP to Zein is 1:8 or 1:4, the loading rate of ORY can reach more than 3%. However, the loading rate of ORY for the nanocarrier with CA as the wall material drops to less than 1%, indicating that the internal space of the CA-Zein carrier is not fully utilized, resulting in a low loading rate of ORY.

[0047] Study Test Example 4 Structural characterization of the ORY-loaded EP-Zein composite nanodelivery system; Fourier Transform Infrared Spectroscopy (FTIR) Analysis To clarify the intermolecular forces in the composite nanodelivery system, Fourier transform infrared (FTIR) spectra were acquired using a Thermo Fisher Scientific Nicolet iS20 (USA). The lyophilized powder prepared in Example 2 was mixed with potassium bromide powder at a mass ratio of 1:100, and the spectral data were obtained in the spectral range of 400 to 4000 cm⁻¹. -1The sample was scanned 64 times under the given conditions, with a resolution set to 4 cm. -1 .

[0048] like Figure 5 As shown in Figure A, EP exhibits a series of typical characteristic absorption peaks of acidic polysaccharides, including the O–H vibration of the hydroxyl group (3421 cm⁻¹). -1 C=O vibration (1645.95 cm) -1 ) and C–O vibration (1432.85). zein showed amide I (1680.66 cm⁻¹) and C–O vibration (1432.85). -1 ) and amide II (1515.78 cm -1 The characteristic absorption peaks of C=O and C–O in the carboxyl group of ORY almost disappeared after the formation of the EP-Zein nanocarrier. The peaks of amides I and II were retained but shifted to lower wavenumbers, indicating an electrostatic interaction between EP and Zein. Simultaneously, the characteristic O–H absorption peak shifted to lower wavenumbers, indicating the formation of hydrogen bonds between EP and Zein. Furthermore, the C–O peak of ORY (1273.27 cm⁻¹) showed a significant absorption peak. -1 1175.4 cm -1 C=C (1634.86 cm) -1 The disappearance of the characteristic absorption peak indicates that ORY was successfully encapsulated in the EP-Zein nanocarrier.

[0049] X-ray diffraction (XRD) analysis To analyze the crystal structure of the composite nanosystem, Example 2 was tested using an X-ray diffraction analyzer (Rigaku Ultima IV, Japan). The detection conditions were: operating voltage of 40 kV, scan rate of 2 ° / min, and scan range of 10 °–60 °.

[0050] like Figure 5 As shown in Figure B, EP is in an amorphous state, while Zein is in a crystalline state. After the formation of the nanocarrier, the diffraction peak intensity of Zein decreased. This is because during the preparation of nanoparticles using the solvent return precipitation method, the hydrophobic region of Zein shrank, disrupting the crystal structure. ORY possesses a unique crystal structure, and after being encapsulated in the EP-Zein composite nanocarrier, the X-ray diffraction pattern of ORY completely disappeared, indicating that ORY was successfully encapsulated in the EP-Zein composite nanocarrier.

[0051] Study Test Example 3 Environmental stability analysis pH stability analysis To evaluate the tolerance of the composite nanosystem under different pH conditions, the pH of the dispersion of the nanosystem to be tested was adjusted to the range of 2.0 to 8.0 using 1.0 M hydrochloric acid or sodium hydroxide solution. After standing for 1 h, the particle size and PDI of Application Example 1 and Comparative Example 3 were analyzed using Malvern dynamic light scattering system.

[0052] like Figure 6 In Figure A, at pH 2.0, the particle size of Application Example 1 was the largest, reaching 265.87 nm, which was significantly higher than the particle size at pH 3.0-8.0. P The pH value was < 0.05, which is due to the decrease in surface charge density of the composite nanosystem caused by the low pH environment, resulting in a weakening of electrostatic repulsion between nanoparticles. Under the influence of van der Waals forces, the nanoparticles aggregated, leading to an increase in particle size. When the pH increased from 5 to 6, the particle size increased from 156.72 nm to 214.9 nm, mainly due to particle aggregation caused by proximity to the isoelectric point (6.2) of Zein. In the pH range of 2.0-8.0, the PDI of Example 2 was 0.23-0.15, indicating uniform dispersion of the nanosystem and demonstrating the good pH stability of the EP-Zein composite nanosystem. Figure 6 As shown in Figure B, when the pH is 2.0-3.0, Comparative Example 3 produced precipitation, and the particle size could not be measured, indicating that the CA-Zein composite nanosystem has poor stability under acidic conditions.

[0053] Ion strength stability analysis To evaluate the stability of the composite nanosystem under different ionic intensities, the dispersion of the nanosystem to be tested was thoroughly mixed with sodium chloride solutions of different concentrations (0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM), allowed to stand for 30 min, and then the particle size and PDI of Example 2 and Comparative Example 3 were analyzed using a Malvern dynamic light scattering system.

[0054] like Figure 6 As shown in Figures C and D, the particle size of both Example 2 and Comparative Example 3 increases with increasing salt ion concentration. This is because the electrostatic interaction between sodium chloride and the nanosystem weakens the electrostatic repulsion between the composite nanosystems, thereby intensifying the aggregation of the nanosystems and causing the particle size to increase. However, the particle size variation in Example 2 is relatively small (126.26-243.56 nm), while the particle size variation in Comparative Example 3 is relatively large (198.34-645.01 nm). This indicates that the EP-Zein composite nanosystem maintains good stability under high ionic strength, while the stability of the CA-Zein composite nanosystem is easily affected by the salt ion strength.

[0055] Thermal stability analysis To evaluate the stability of the composite nanosystem at different temperatures, the dispersion of the nanosystem to be tested was placed in a constant temperature water bath at 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for 1 h. After cooling to room temperature, the retention rate of ORY in Example 2 was measured.

[0056] ORY retention rate = (ORY content in the treated composite nanoparticles / ORY content in the original composite nanoparticle system) × 100% like Figure 6 As shown in Figure E, ZO and ZEO exhibit significant protective effects on ORY compared to free ORY. Notably, after treatment at 50 ℃, 60 ℃, 70 ℃, 80 ℃, and 90 ℃ for 1 h, the retention rate of ORY in the ZEO composite nanosystem reached over 96.14%, significantly higher than the retention rate of ORY in ZO under the same temperature conditions. This indicates that the composite nanocarrier formed by EP and Zein can significantly improve the thermal stability of ORY.

[0057] Light stability analysis To evaluate the stability of the composite nanosystem under ultraviolet light, the nanoparticle dispersion was irradiated under an ultraviolet lamp (365 nm, 30 W) for 5 h, and samples were taken every 1 h to detect the retention rate of ORY.

[0058] ORY retention rate = (ORY content in composite nanoparticles at a specific treatment time point / ORY content in composite nanoparticles before treatment) × 100% like Figure 4 As shown in Figure F, free ORY degrades rapidly under ultraviolet irradiation, with a retention rate of only 19.98% after 1 hour of irradiation. However, after encapsulation with nanocarriers and 5 hours of UV irradiation, the ORY retention rates in ZEO and ZO were 76.62% and 74.64%, respectively, which were much higher than those of free ORY (17.13%).

[0059] Study Test Example 4 In vitro simulated digestion analysis of ORY-loaded EP-Zein composite nanodelivery system The sustained-release characteristics of the ORY-loaded EP-Zein composite nanodelivery system in the human gastrointestinal environment were determined. 5 mL of the ORY-loaded EP-Zein composite nanosystem was uniformly mixed with an equal volume of simulated gastric fluid (SGF, pH=1.2). The pH of the reaction system was then adjusted to 6.8, and the mixture was incubated with 10 mL of simulated intestinal fluid (SIF) for 4 h. 1 mL of sample was taken every 30 min, centrifuged at 10000 rpm for 20 min, and the supernatant was used to measure the ORY content and calculate the release rate. Bioavailability was calculated using the following formula.

[0060] The bioavailability of ORY = ORY content in digestive fluid after in vitro simulated gastrointestinal digestion / ORY content in the composite nanodelivery system before in vitro simulated gastrointestinal digestion × 100% like Figure 7 As shown in Figure A, during a 6-hour simulated gastrointestinal digestion process, the ZEO composite nanosystem released ORY at a slower rate compared to the ORY monomer and ZO nanosystems. Specifically, in SGF at pH 1.2, only 35.81% of ORY was released from the EP-zein composite nanosystem in the first 2 hours, lower than the release rates of ORY monomer (45.81%) and ZO nanosystem (46.78%). In SIF at pH 6.8, 70.55% of ORY was released from the ZEO composite nanosystem after 6 hours of digestion, lower than the release rates of ORY monomer (86.21%) and ZO nanosystem (80.39%). Furthermore, as... Figure 7 As shown in Figure B, after 6 hours of gastrointestinal digestion, the bioavailability of ORY in the ZEO nanosystem was approximately 31.58%, significantly higher than that of free ORY monomers (9.03%). These results indicate that the EP-Zein composite nanocarrier can reduce the rapid release of ORY in the gastrointestinal environment, providing better protection for ORY and demonstrating its potential as a colonic delivery carrier for ORY.

[0061] Study Test Example 5 In vitro anti-inflammatory activity analysis of ORY-loaded EP-Zein composite nanodelivery system Macrophage uptake of composite nanoparticles Macrophages are considered important target cells for the treatment of colitis. Therefore, efficient uptake of ORY by macrophages by the EP-Zein composite nanodelivery system is crucial for ORY to exert its anti-inflammatory effect. RAW264.7 macrophages were loaded with ORY at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in 24-well plates and cultured overnight at 37 °C. Then, the cells were co-incubated with coumarin-6 (C6)-labeled composite nanoparticles for 4 h, followed by washing three times with phosphate-buffered saline (PBS) and staining with DAPI fluorescent dye for 15 min to label the nuclei. Excess DAPI dye was then washed away with PBS, and cell uptake was observed using a confocal laser scanning microscope (CLSM). For time-dependent assays, cells were harvested at specific time intervals (0, 1, 2, 3, and 4 h) and the above procedures were performed.

[0062] like Figure 8As shown in Figures A and C, LPS-activated cells took up the fluorescein C6-labeled composite nanodelivery system more efficiently than unactivated cells. Furthermore, regardless of cell activation status, the uptake of the C6-labeled composite nanodelivery system was significantly higher than that of free C6. In addition, with prolonged co-incubation time, the intracellular C6 fluorescence intensity gradually increased (…). Figure 8 (Figures B and D in the figure) These results indicate that the EP-Zein composite nanodelivery system has a high affinity for macrophages.

[0063] Anti-inflammatory effect evaluation Macrophages at 1×10 5 The nanoparticles were seeded at a density of 1 / well in 6-well plates and pre-cultured for 24 h. Then, they were co-cultured with 5 μg / mL LORY, ZO, or ZEO nanoparticles for 24 h, followed by treatment with 1 μg / mL LPS for 24 h. Subsequently, the levels of inflammatory factors (TNF-α, IL-6, and IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA) and biochemical assay kits.

[0064] like Figure 9 As shown, in LPS-stimulated macrophages, the EP-Zein composite nanodelivery system loaded with ORY significantly inhibited the production of inflammatory factors TNF-α, IL-6 and IL-1β. This inhibitory effect was significantly higher than that of free ORY, indicating that the EP-Zein composite nanocarrier synergistically enhanced the anti-inflammatory activity of ORY.

[0065] Study Test Example 6 Colon-targeting analysis of ORY-loaded EP-Zein composite nanodelivery system Changes in zeta potential of the composite nanodelivery system before and after gastrointestinal digestion in vitro During colonic inflammation, intestinal epithelial cells release positively charged proteins. Negatively charged nanodelivery systems accumulate in positively charged inflamed areas through electrostatic adsorption, thereby enhancing targeting of the lesion site. Therefore, to evaluate the potential of the ORY-loaded EP-Zein composite nanodelivery system to target inflammatory colonic tissue, we first assessed whether the ORY-loaded EP-Zein composite nanodelivery system retained its negative charge after gastrointestinal digestion. The ORY-loaded EP-Zein composite nanodelivery system dispersion was incubated in simulated gastric juice (SGF, pH=1.2) for 2 h, followed by incubation in simulated intestinal juice (SIF) for 4 h. The zeta potential was then measured to assess the charge change before and after digestion.

[0066] like Figure 10As shown in Figure A, after simulating gastrointestinal digestion in vitro, the zeta potential of the ORY-loaded EP-zein composite nanodelivery system changed from -41 mV to -17 mV, while still maintaining electronegativity, indicating its potential to target sites of colonic inflammation.

[0067] Colon targeting of composite nanodelivery systems To visualize the composite nanoparticles in the gut, fluorescein isothiocyanate (FITC) was used for labeling, and its distribution was monitored using an in vivo imaging system (IVIS). Fluorescence signals of the composite nanoparticle system were detected via the FITC channel (excitation wavelength: 488 nm, emission wavelength: 525 nm). Specifically, a colitis mouse model was established using dextran sulfate sodium (DSS). Healthy and colitis mice were fasted for 12 h, and then orally administered a FITC-labeled composite nanoparticle dispersion. Mice were anesthetized at specified time points (3, 6, 12, and 24 h), and changes in fluorescence intensity were detected using IVIS. After imaging, the entire gastrointestinal tract was removed from each mouse to assess the distribution and intensity of the fluorescence signal.

[0068] like Figure 8 As shown in Figure B, the composite nanodelivery system was present in the intestine, and at each time point, the fluorescence intensity of colitis mice (DSS group) was significantly stronger than that of healthy mice (control group), which showed weak fluorescence. In contrast, in the colitis model mice, the fluorescence signal of the orally administered composite nanodelivery system gradually moved along the digestive tract to the colon, reaching a peak at 12 h. Notably, even after 24 h, fluorescence signals continued to be generated, indicating that the composite nanodelivery system significantly prolonged the residence time of ORY in inflamed colonic tissue, further confirming the colon-targeting ability of the composite nanodelivery system.

[0069] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a colon-targeted sustained-release oryzanol nanocarrier, characterized in that, Includes the following steps: S1. Disperse Zein in ethanol and stir thoroughly at room temperature to obtain a Zein solution; S2. Disperse EP in deionized water and stir thoroughly for 8-12 h to obtain an EP solution; S3. Under stirring conditions, the Zein solution prepared in step S1 is added dropwise to the EP solution prepared in step S2 to obtain an EP-Zein mixed solution; S4. Under vacuum conditions, rotary evaporation was used to remove ethanol, centrifugation was used to remove insoluble large particles, and freeze-drying was performed to obtain the EP-Zein composite nanocarrier.

2. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S1, the volume fraction of the ethanol solution is 65%-80%.

3. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S1, the concentration of Zein is 5-20 mg / mL.

4. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S2, the concentration of EP is 0.3-1.7 mg / mL.

5. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S2, the stirring conditions are: stirring temperature of 25-45 ℃ and stirring speed of 200-600 rpm / min.

6. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S2, the mass ratio of EP to Zein is 1:2-1:

8.

7. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S3, the stirring conditions are: stirring temperature of 25-30 ℃ and stirring speed of 200-600 rpm / min.

8. The method for preparing a colon-targeted sustained-release oryzanol nanocarrier as described in claim 1, characterized in that, In step S4, the centrifugation conditions are: centrifugation speed of 3000-5000 rpm and time of 20-40 min.

9. The colon-targeted sustained-release oryzanol nanocarrier prepared by the preparation method according to any one of claims 1-8, characterized in that, EP and Zein form a nanocarrier through electrostatic interactions and hydrogen bonding to deliver ORY.

10. The application of the colon-targeted sustained-release oryzanol nanocarrier prepared by the preparation method according to any one of claims 1-8 in the delivery of ORY.

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