Intestinal responsive mucus adhesion-permeation conversion type oral delivery system as well as preparation method and application thereof

Abstract

The invention discloses an intestinal responsive mucus adhesion-permeation conversion type oral delivery system as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out self-crosslinking on succinylated soy isolate protein and hydroxyethylated chitosan, and then adding pterostilbene to carry out a co-precipitation reaction, so as to prepare pterostilbene-loaded mucus permeable nanoparticles; and carrying out surface modification on the pterostilbene-loaded mucus permeable nano-particles by adopting high methoxyl pectin, so as to prepare the intestinal tract responsive mucus adhesion-permeation conversion type oral delivery system. According to the oral delivery system provided by the invention, ingenious combination and intelligent conversion of gastric environment resistance, intestinal responsive release, intestinal mucus adhesion and permeability are realized, and the loading rate of pterostilbene, gastrointestinal tract resistance and mucus permeability are effectively improved; therefore, the intestinal epithelial cell uptake efficiency and intracellular antioxidant and anti-inflammatory activity of the free pterostilbene through oral administration are improved, and the preparation method has a good application prospect in the field of oral delivery of bioactive substances.

Description

Technical Field

[0001] The present invention relates to an intestinal-responsive mucus adhesion-permeation conversion type oral delivery system, its preparation method and application, and belongs to the technical field of oral delivery of natural active substances. Background Art

[0002] Due to their unique biocompatibility, safety, and multiple biological activities such as antioxidant and anti-inflammatory properties, natural active substances provide a solution with both safety and biological activity for the functional food field. However, the natural barrier function of the intestinal mucus layer severely limits the effective absorption of bioactive substances. The mucus layer is a viscous barrier composed of mucins that covers the intestinal epithelial cells. Traditional oral delivery carriers are easily intercepted by the dynamic network structure of the mucus layer, resulting in the rapid clearance or enzymatic inactivation of active substances on the mucus surface, ultimately leading to a decrease in bioavailability. Therefore, developing a delivery system with high mucus penetration ability has become the core technical strategy to improve the oral absorption efficiency of active substances.

[0003] Inspired by the natural mucus penetration mechanism of viruses, having a hydrophilic and electrically neutral surface property, and a sufficiently small particle size are important prerequisites for an oral delivery carrier to penetrate the mucus barrier. Currently, traditional strategies for improving the mucus permeability of delivery carriers include hydrophilic PEG coating and modification with mucus-penetrating peptides (such as MPPs). However, overuse of PEGylated formulations may induce the body to produce anti-PEG antibodies, reducing long-term efficacy and even causing severe allergic reactions in some people. Moreover, traditional PEG is a non-degradable polymer, and long-term accumulation may cause organ (such as liver, spleen) toxicity. In addition, the mucus-penetrating peptide modification strategy involves large-scale peptide synthesis and high purification costs, which limit its clinical translation. Therefore, based on the concept of virus biomimetic functionalization, an oral nanocarrier with both natural biological safety and low cost may be an optimized technical strategy to achieve high mucus permeability.

[0004] Furthermore, during oral delivery, the strong acidic environment (pH value of 1-3) of gastric juice and pepsin are important reasons for the degradation and premature release of natural active substances. The dynamic environment of the intestine also continuously promotes the peristalsis of intestinal contents, resulting in a shortened residence time of orally delivered substances, which has a very adverse impact on the local high-concentration delivery and absorption of active substances. Therefore, an oral delivery carrier also needs to have the characteristics of gastric environment resistance and intestinal mucus adhesion to optimize the delivery performance. By protecting the easily degradable active substances through gastric environment resistance and delivering them intact to the intestine, and by intestinal mucus adhesion to prolong the residence time of the carrier in the intestine and enhance the contact with the absorption interface, thereby improving the bioavailability of the loaded drug. However, so far, the design of how to combine the advantages of gastric environment resistance, intestinal response release, intestinal mucus adhesion, and permeability in an oral delivery carrier is still a technical difficulty to be solved urgently. Summary of the Invention

[0005] The main object of the present invention is to provide an intestinal-responsive mucus adhesion-permeation conversion oral delivery system, its preparation method and application, so as to overcome the deficiencies in the prior art.

[0006] To achieve the foregoing invention object, the technical solutions adopted by the present invention include:

[0007] The embodiment of the present invention provides a preparation method of an intestinal-responsive mucus adhesion-permeation conversion oral delivery system, which includes:

[0008] Provide succinylated soy protein isolate and hydroxyethylated chitosan;

[0009] Perform self-crosslinking of the succinylated soy protein isolate and the hydroxyethylated chitosan, and then add pterostilbene for coprecipitation reaction to obtain mucus-permeable nanoparticles loaded with pterostilbene;

[0010] And, use high-methoxyl pectin to modify the surface of the mucus-permeable nanoparticles loaded with pterostilbene to obtain an intestinal-responsive mucus adhesion-permeation conversion oral delivery system.

[0011] The embodiment of the present invention also provides an intestinal-responsive mucus adhesion-permeation conversion oral delivery system prepared by the foregoing preparation method.

[0012] The embodiment of the present invention also provides a pterostilbene oral product with anti-inflammatory and antioxidant effects, which at least includes the foregoing intestinal-responsive mucus adhesion-permeation conversion oral delivery system.

[0013] The embodiment of the present invention also provides the use of the foregoing intestinal-responsive mucus adhesion-permeation conversion oral delivery system in the preparation of intracellular antioxidant and anti-inflammatory active products.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) The present invention uses a strategy of low cost and high biosafety. Through the self-crosslinking of double-modified natural biodegradable biopolymers, the pterostilbene loading rate is increased, and nanoparticles with hydrophilic and electro-neutral surface properties are obtained, which can efficiently penetrate the intestinal mucus layer by loading pterostilbene;

[0016] (2) The present invention is based on pterostilbene-loaded mucoadhesive nanoparticles. Utilizing the intestinal enzyme-responsive degradation and intestinal mucus adhesion properties of pectin, through the simple encapsulation of high-methoxyl pectin, as the outer shell, it endows the delivery system with excellent resistance to the gastric environment. After successfully reaching the intestine, it adheres to the mucus layer through the carboxyl group of pectin and is gradually degraded in response to intestinal pectinase, thereby slowly releasing the internal pterostilbene-loaded mucoadhesive nanoparticles; in addition, due to the relatively high degree of methyl esterification of high-methoxyl pectin, its degradation rate is slower than that of low-methoxyl pectin, enabling a longer sustained-release effect to further increase the local drug concentration; moreover, compared with chemically adhesive materials, natural mucus-adhesive pectin is more environmentally friendly and biocompatible;

[0017] (3) The oral delivery system prepared by the present invention can achieve an ingenious combination and intelligent conversion of gastric environment resistance - intestinal response release - intestinal mucus adhesion - permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a comparative diagram of the surface hydrophilicity of SPI-CS in Comparative Example 1 of the present invention, SPI-HECS in Comparative Example 2, SSPI-CS in Comparative Example 3, and SSPI-HECS prepared in step (3) of Example 4;

[0020] Figure 2 It is a particle size distribution diagram of SSPI-HECS prepared in step (3) of Example 4 of the present invention;

[0021] Figure 3 It is a comparative diagram of the pterostilbene loading rate in SPI-CS in Comparative Example 1 of the present invention, SPI-HECS in Comparative Example 2, SSPI-CS in Comparative Example 3, and SSPI-HECS prepared in step (3) of Example 4 (samples marked with different letters indicate significant differences, p < 0.5);

[0022] Figure 4 It is a diagram of the change in the surface potential value of the nanoparticles prepared by crosslinking SSPI and HECS in different ratios in step (3) of Example 4 of the present invention;

[0023] Figure 5 It is a potential value diagram of SPI-CS in Comparative Example 1 of the present invention, SPI-HECS in Comparative Example 2, and SSPI-CS in Comparative Example 3;

[0024] Figure 6 It is a comparison chart of the mucus permeability of SSPI-HECS prepared in step (3) of Example 4 of the present invention and SPI-CS of Comparative Example 1;

[0025] Figures 7a - 7b It is a comparison chart of the mucus adhesion force and adhesion time of SSPI-HECS prepared in step (3) of Example 4 of the present invention and PE-SSPI-HECS prepared in step (4) (****p < 0.0001);

[0026] Figure 8 It is a chart showing the protective effect on pterostilbene loading of SSPI-HECS prepared in step (3) of Example 4 of the present invention and PE-SSPI-HECS prepared in step (4) during in vitro simulated digestion;

[0027] Figure 9 It is a chart of the intestinal epithelial cell uptake of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of the present invention after in vitro digestion and mucus penetration;

[0028] Figures 10a - 10d It is a chart of the cell inflammation regulation ability of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of the present invention after in vitro digestion and mucus penetration (samples marked with different letters indicate significant differences, p < 0.5);

[0029] Figures 11a - 11d It is a chart of the cell oxidative stress regulation ability of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of the present invention after in vitro digestion and mucus penetration (samples marked with different letters indicate significant differences, p < 0.5). Detailed implementation manners

[0030] In view of the deficiencies of the prior art, the inventors of this case have, through long-term research and a large number of practices, been able to propose the technical solution of the present invention. For the convenience of understanding this application, the following will describe this application in more detail. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] In the present invention, first, the isoelectric point of soy protein isolate is reduced by succinylation to block amino groups and introduce additional carboxyl groups (-COO - ), to provide more negatively charged carboxyl reaction sites for electrostatic interaction and cross-linking with the positively charged amino groups (-NH2 + ) of chitosan, thereby increasing the pterostilbene loading rate and achieving the preparation of a carrier with an electrically neutral surface property. Secondly, the chitosan used for cross-linking in the present invention is pre-modified by hydroxyethylation to introduce hydrophilic hydroxyethyl groups (-CH2CH2OH), while reducing the close packing of the chitosan molecular chains, thereby greatly improving the hydrophilicity of chitosan without changing the number of amino groups. In addition, the hydrophilicity of succinylated soy protein isolate is also significantly enhanced due to the increase in polar groups and the encapsulation of hydrophobic amino acids. Therefore, through the self-cross-linking of the above-mentioned modified natural proteins and polysaccharides, a mucus-permeable carrier with both electrically neutral and hydrophilic surface properties is prepared. Then, in the present invention, the hydrophobic active substance pterostilbene is loaded into the above-mentioned mucus-permeable carrier by the method of nano-coprecipitation, and the delivery system prepared in this way has a nano-scale particle size and is easier to pass through the mucus layer compared with micron particles. Finally, based on the above-mentioned pterostilbene-loaded mucus-permeable nanoparticles, using the intestinal enzyme-responsive degradation property and intestinal mucus adhesion property of pectin, through the simple encapsulation of high-methoxyl pectin, it is used as an outer shell to endow the delivery system with excellent gastric environment resistance. After successfully reaching the intestine, it adheres to the mucus layer through the carboxyl group of pectin and is gradually degraded in response to intestinal pectinase, thereby slowly releasing the internal pterostilbene-loaded mucus-permeable nanoparticles. The nanoparticles, relying on their biomimetic surface properties and nano-scale particle size, quickly penetrate the mucus layer and are efficiently taken up by epithelial cells, enabling pterostilbene to effectively exert antioxidant and anti-inflammatory effects intracellularly. It should also be noted here that due to the higher degree of methyl esterification of high-methoxyl pectin, its degradation rate is slower than that of low-methoxyl pectin, and a longer sustained-release effect can be achieved to further increase the local nanoparticle concentration. And, compared with chemically adhesive materials, natural mucus-adhesive pectin is more environmentally friendly and biocompatible. Therefore, the biomimetic intelligent oral delivery system prepared by the present invention can achieve the ingenious combination and intelligent conversion of gastric environment resistance-intestinal response release-intestinal mucus adhesion-permeability, significantly improving the cellular uptake rate of free pterostilbene and its intracellular antioxidant and anti-inflammatory biological activities, while also possessing the advantages of high cost-effectiveness and biological safety.

[0032] Specifically, as an aspect of the technical solution of the present invention, a preparation method of an intestinal-responsive mucus adhesion-permeation conversion type oral delivery system includes:

[0033] Providing succinylated soy protein isolate and hydroxyethylated chitosan;

[0034] Subjecting the succinylated soy protein isolate and hydroxyethylated chitosan to self-crosslinking, and then adding pterostilbene for coprecipitation reaction to obtain mucus-permeable nanoparticles loaded with pterostilbene;

[0035] And, surface-modifying the mucus-permeable nanoparticles loaded with pterostilbene with high-methoxyl pectin to obtain an intestinal-responsive mucus adhesion-permeation conversion type oral delivery system.

[0036] In some preferred embodiments, the preparation method specifically includes: dissolving soy protein isolate in water to form a soy protein solution, then adding succinic anhydride and stirring and reacting in an alkaline environment at a temperature of 25-40 °C for 1-4 h, and then performing dialysis and freeze-drying treatments to obtain succinylated soy protein isolate;

[0037] Further, the mass-volume concentration of the soy protein solution is 0.05-0.1 g / mL.

[0038] Further, the mass ratio of succinic anhydride to soy protein isolate is 0.05-0.2:1.

[0039] Further, the pH value of the alkaline environment is 8.0-8.5.

[0040] Further, the cut-off molecular weight of the dialysis bag used for dialysis is 15-30 kDa.

[0041] Further, the dialysis treatment time is 24-48 h.

[0042] In some preferred embodiments, the preparation method specifically includes:

[0043] Dissolving chitosan in an acetic acid solution to form a chitosan acetic acid solution, then adjusting the pH value of the chitosan acetic acid solution to neutral, and then performing filtration, washing, and drying treatments to obtain pretreated chitosan;

[0044] Mixing the pretreated chitosan with an alkaline solution and stirring at room temperature to obtain alkalized chitosan;

[0045] And, the alkalized chitosan is mixed with an isopropanol-water mixed solvent, ethylene oxide is added at 4°C, and then the reaction is carried out at 40-60°C for 8-24 h. At the same time, the pH value of the reaction system is maintained at 11 with an alkaline solution, and then the pH value of the reaction system is adjusted to neutral with an acidic solution to terminate the reaction. Then, it is centrifuged, washed, dialyzed, and freeze-dried to obtain hydroxyethylated chitosan.

[0046] Further, the mass-volume concentration of chitosan in the chitosan acetic acid solution is 0.01-0.1 g / mL.

[0047] Further, the alkaline solution includes any one or a combination of sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, etc., and is not limited thereto.

[0048] Further, the concentration of the alkaline substance in the alkalized chitosan is 1-4 mol / L.

[0049] Further, the mass-volume concentration of the pretreated chitosan in the alkalized chitosan is 0.02-0.05 g / mL.

[0050] Further, the volume ratio of isopropanol to water in the isopropanol-water mixed solvent is 1:1-1:5.

[0051] Further, the molar ratio of chitosan units to ethylene oxide in the alkalized chitosan is 1:2-1:5.

[0052] Further, the acidic solution includes any one or a combination of dilute hydrochloric acid, dilute sulfuric acid, dilute acetic acid, etc., and is not limited thereto.

[0053] Further, the number of times of the washing treatment is 1-3 times.

[0054] Further, the cut-off molecular weight of the dialysis bag used for dialysis is 5-15 kDa.

[0055] In some preferred embodiments, the preparation method specifically includes:

[0056] Disperse succinylated soy protein isolate and hydroxyethylated chitosan in water respectively to form a succinylated soy protein isolate suspension and a hydroxyethylated chitosan suspension;

[0057] Mix the succinylated soy protein isolate suspension and the hydroxyethylated chitosan suspension and disperse them at a high speed of 5000-15000 rpm for 1-10 min to form a neutral hydrophilic cross-linked system of succinylated soy protein isolate-hydroxyethylated chitosan;

[0058] Dissolve pterostilbene in ethanol to form a pterostilbene ethanol solution. Then, add the pterostilbene ethanol solution to an electro-neutral hydrophilic cross-linking system and stir at 200 - 1000 rpm for 0.5 - 2 h. Then, perform centrifugation and washing to obtain mucus-permeable nanoparticles loaded with pterostilbene.

[0059] Further, the mass-volume concentration of the succinylated soy protein isolate suspension is 0.1 - 0.3 g / mL.

[0060] Further, the mass-volume concentration of the hydroxyethylated chitosan suspension is 0.1 - 0.5 g / mL.

[0061] Further, the mass ratio of the succinylated soy protein isolate suspension to the hydroxyethylated chitosan suspension is 4:1 - 1:4.

[0062] Further, the concentration of the pterostilbene ethanol solution is 1 - 5 mg / mL.

[0063] In some preferred embodiments, the preparation method specifically includes: dissolving high-methoxyl pectin in deionized water at 60 - 80 °C to form a high-methoxyl pectin solution. Then, add the mucus-permeable nanoparticles loaded with pterostilbene and stir at 200 - 1000 rpm for 2 - 10 h. Then, perform centrifugation to obtain mucus-permeable nanoparticles loaded with pterostilbene with a pectin layer on the surface, that is, the intestinal-responsive mucus adhesion-permeation conversion type oral delivery system.

[0064] Further, the degree of esterification (DE) of the high-methoxyl pectin ≥ 50%.

[0065] Further, the methoxy content in the high-methoxyl pectin is 7% - 12%;

[0066] Further, the concentration of the high-methoxyl pectin solution is 0.5 - 2 mg / ml; the mass-volume ratio of the mucus-permeable nanoparticles loaded with pterostilbene to water is 100 - 500 mg:100 mL.

[0067] Further, the centrifugation speed used for the centrifugation treatment is 5000 - 20000 × g, and the centrifugation time is 20 - 60 min.

[0068] Further, the particle size of the mucus-permeable nanoparticles loaded with pterostilbene with a pectin layer on the surface is 106 - 255 nm.

[0069] In some more specific embodiments, the preparation method of the intestinal-responsive mucus adhesion-permeation conversion type oral delivery system includes the following steps:

[0070] (1) Preparation of succinylated soy protein isolate: Dissolve soy protein isolate in deionized water, and stir magnetically at room temperature until completely dissolved to form a soy protein solution. Dropwise add succinic anhydride while maintaining an alkaline environment in the system, continuously stir and react at 25 °C, adjust the reaction solution to neutral to terminate the reaction, dialyze the reaction solution with deionized water, change the solution at fixed intervals, and then freeze-dry to obtain succinylated soy protein isolate powder.

[0071] (2) Preparation of hydroxyethylated chitosan: Dissolve chitosan in 1% acetic acid solution, stir magnetically until completely dissolved to form a chitosan acetic acid solution, adjust the pH of the solution to neutral with NaOH solution, filter and collect the gel-like precipitate, wash it with deionized water, and then freeze-dry to obtain pretreated chitosan for standby. Add the dried pretreated chitosan to NaOH solution and stir at room temperature to prepare alkalized chitosan to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. Transfer the alkalized chitosan to an isopropanol-water mixed solution, vortex thoroughly to disperse it, and add ethylene oxide solution dropwise to the mixed solution at 4 °C for reaction. During the reaction, maintain the alkaline pH = 11 by adjusting with NaOH solution, adjust the reaction solution to neutral with dilute hydrochloric acid to terminate the reaction, then pour the reaction solution into absolute ethanol, centrifuge to collect the precipitate, and wash it 3 times with absolute ethanol. Place the precipitate product in deionized water for dialysis for 3 days, and then freeze-dry to obtain light yellow powdery hydroxyethylated chitosan.

[0072] (3) Preparation of pterostilbene-loaded mucoadhesive nanoparticles: Dissolve succinylated soy protein isolate powder in deionized water and stir magnetically until completely dissolved to prepare a succinylated soy protein isolate suspension. Dissolve hydroxyethylated chitosan in deionized water and stir magnetically until completely dissolved to prepare a hydroxyethylated chitosan suspension. Mix the above two solutions, homogenize them with a high-speed disperser, and then continue to stir magnetically to obtain an electro-neutral hydrophilic cross-linked system of succinylated soy protein isolate-hydroxyethylated chitosan. Dissolve pterostilbene in absolute ethanol to form a pterostilbene ethanol solution, dropwise add it to the above cross-linked system solution, stir magnetically, and then collect the nano-precipitate particles by centrifugation. Wash the precipitate with deionized water to obtain pterostilbene-loaded mucoadhesive nanoparticles.

[0073] (4) Preparation of an intestinal-responsive mucoadhesive-permeation conversion-type oral delivery system: Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved. Add the pterostilbene-loaded mucoadhesive nanoparticles prepared in step (3) to the above solution and stir magnetically. Then, collect the precipitate particles by centrifugation to obtain pterostilbene-loaded mucoadhesive nanoparticles modified on the surface of the pectin layer, that is, an intestinal-responsive mucoadhesive-permeation conversion-type oral delivery system.

[0074] Preferably, the mass-volume concentration of the soy protein solution in step (1) is 0.05 - 0.1 g / mL.

[0075] Preferably, the proportion of succinic anhydride added in step (1) is 0.05 - 0.2 g / g / g soy protein isolate.

[0076] Preferably, the continuous stirring time in step (1) is 1 - 4 h.

[0077] Preferably, the dialysis time in step (1) is 24 - 48 h.

[0078] Preferably, the molecular weight cut-off of the dialysis bag in step (1) is 15 - 30 kDa.

[0079] Preferably, the mass-volume concentration of the chitosan acetate solution in step (2) is 0.01 - 0.1 g / mL.

[0080] Preferably, the number of times of washing with deionized water in step (2) is 1 - 3 times.

[0081] Preferably, the concentration of NaOH in the alkalized chitosan in step (2) is 1 - 4 M.

[0082] Preferably, the mass-volume concentration of chitosan in the alkalized chitosan in step (2) is 0.02 - 0.05 g / mL.

[0083] Preferably, in the isopropanol-water mixed solution in step (2), the ratio of isopropanol / water is 1:1 - 1:5 (v / v).

[0084] Preferably, in the ethylene oxide solution in step (2), the ratio of chitosan unit / ethylene oxide is 1:2 - 1:5 (mol / mol).

[0085] Preferably, after dropping the ethylene oxide solution in step (2), the reaction temperature is 40 - 60 °C and the reaction time is 8 - 24 h.

[0086] Preferably, the molecular weight cut-off of the dialysis bag in step (2) is 5 - 15 kDa.

[0087] Preferably, the mass-volume concentration of the succinylated soy protein isolate suspension in step (3) is 0.1 - 0.3 g / mL.

[0088] Preferably, the mass-volume concentration of the hydroxyethylated chitosan suspension in step (3) is 0.1 - 0.5 g / mL.

[0089] Preferably, in step (3), the mixing ratio of the two solutions is succinylated soy protein isolate suspension: hydroxyethylated chitosan suspension = 4:1 - 1:4 (w / w).

[0090] Preferably, in step (3), the high-speed dispersion speed is 5000 - 15000 rpm.

[0091] Preferably, in step (3), the high-speed dispersion time is 1 - 10 min.

[0092] Preferably, in step (3), the concentration of the pterostilbene ethanol solution is 1 - 5 mg / mL.

[0093] Preferably, in step (3), the magnetic stirring speed is 200 - 1000 rpm.

[0094] Preferably, in step (3), the magnetic stirring time is 0.5 - 2 h.

[0095] Preferably, in step (4), the concentration of the high-methoxyl pectin is 0.5 - 2 mg / ml.

[0096] Preferably, in step (4), the addition amount of the pterostilbene mucus-permeable nanoparticles is 100 - 500 mg.

[0097] Preferably, in step (4), the magnetic stirring speed is 200 - 1000 rpm.

[0098] Preferably, in step (4), the magnetic stirring time is 2 - 10 h.

[0099] Preferably, in step (4), the centrifugation speed is 5000 - 20000 × g.

[0100] Preferably, in step (4), the centrifugation time is 20 - 60 min.

[0101] The present invention uses a low-cost and high-biosafety strategy to prepare an intelligent oral delivery system that achieves an ingenious combination and intelligent conversion of gastric environment resistance - intestinal response release - intestinal mucus adhesion - permeability, effectively improving the intestinal epithelial cell uptake efficiency of free pterostilbene via the oral route and its intracellular antioxidant and anti-inflammatory activities, and providing an oral carrier platform with both high-efficiency delivery and intelligent regulation functions in the field of oral delivery of bioactive substances.

[0102] As another aspect of the technical solution of the present invention, it relates to an oral delivery system with intestinal response mucus adhesion - permeability conversion prepared by the aforementioned preparation method.

[0103] As another aspect of the technical solution of the present invention, it also relates to a pterostilbene oral product having anti-inflammatory and antioxidant effects, which at least includes the aforementioned intestinal-responsive mucus adhesion-permeation conversion type oral delivery system.

[0104] As another aspect of the technical solution of the present invention, it also relates to the use of the aforementioned intestinal-responsive mucus adhesion-permeation conversion type oral delivery system in the preparation of intracellular antioxidant and anti-inflammatory active products.

[0105] The present invention is further illustrated by the following examples: According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0106] Unless otherwise specified, the various raw materials, reaction equipment, test equipment and test methods used in the following examples are well-known in the art.

[0107] Example 1

[0108] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL), and stir magnetically at room temperature until fully dissolved. Add succinic anhydride dropwise at a ratio of 0.1 g / g protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (molecular weight cut-off 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 hours, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0109] (2) Preparation of hydroxyethylated chitosan (HECS): Chitosan was dissolved in 1 wt% acetic acid solution (0.05 g / mL), and magnetically stirred until completely dissolved. The pH of the solution was adjusted to neutral with NaOH solution, and the gel-like precipitate was collected by filtration, washed 3 times with deionized water, and then freeze-dried to obtain pretreated chitosan for standby. The dried pretreated chitosan was added to 2M NaOH solution and stirred at room temperature to prepare alkalized chitosan to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. The alkalized chitosan was transferred to an isopropanol-water mixed solution (isopropanol∶water = 1∶5, v / v), and vortexed sufficiently to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶4 (mol / mol), ethylene oxide solution was added dropwise to the mixed solution at 4 °C, and the reaction was carried out at 45 °C for 10 h. During this period, the alkaline pH = 11 was maintained by adjusting with NaOH solution. The reaction solution was adjusted to neutral with dilute hydrochloric acid to terminate the reaction, and then the reaction solution was poured into absolute ethanol, and the precipitate was collected by centrifugation and washed 3 times with absolute ethanol. The precipitate product was placed in a dialysis bag (cut-off molecular weight 10 kDa) and dialyzed with deionized water for 3 days, and then freeze-dried to obtain pale yellow powdery hydroxyethylated chitosan (HECS).

[0110] (3) Preparation of pterostilbene-loaded mucoadhesive nanoparticles: Succinylated soy protein isolate powder was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. Hydroxyethylated chitosan was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. The above two solutions were mixed according to the ratio of succinylated soy protein isolate∶hydroxyethylated chitosan = 4∶1 (w / w), homogenized at a speed of 10000 rpm for 5 min by a high-speed disperser, and then continuously magnetically stirred at a speed of 600 rpm for 1 h to obtain an electro-neutral hydrophilic system composed of succinylated soy protein isolate-hydroxyethylated chitosan. Pterostilbene was dissolved in absolute ethanol (4 mg / mL), and was added dropwise to the above cross-linked system solution, and magnetically stirred at a speed of 600 rpm for 1 h, and then the nano-precipitate particles were collected by centrifugation. The precipitate was washed with deionized water to obtain pterostilbene-loaded mucoadhesive nanoparticles (SSPI-HECS).

[0111] (4) Preparation of an intestinal-responsive mucus adhesion-permeation conversion oral delivery system (PE-SSPI-HECS): Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved (0.5 mg / ml). Add the SSPI-HECS (300 mg) prepared in step (3) to the above solution and stir magnetically at a speed of 800 rpm for 5 h. Then, collect the precipitated particles by centrifugation at 10,000×g for 20 min to obtain SSPI-HECS modified with a pectin layer on the surface, that is, an intestinal-responsive mucus adhesion-permeation conversion oral delivery system (PE-SSPI-HECS).

[0112] Example 2

[0113] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL) and stir magnetically at room temperature until fully dissolved. Add succinic anhydride dropwise at a ratio of 0.1 g / g of protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (molecular weight cut-off 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 h, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0114] (2) Preparation of hydroxyethylated chitosan (HECS): Dissolve chitosan in a 1 wt% acetic acid solution (0.05 g / mL) and stir magnetically until completely dissolved. Adjust the pH of the solution to neutral with NaOH solution, filter and collect the gel-like precipitate, wash it 3 times with deionized water, and then freeze-dry to obtain pretreated chitosan for standby. Add the dried pretreated chitosan to 2 M NaOH solution and stir at room temperature to prepare alkalized chitosan to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. Transfer the alkalized chitosan to an isopropanol-water mixed solution (isopropanol∶water = 1∶5, v / v), vortex thoroughly to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶4 (mol / mol), add ethylene oxide solution dropwise to the mixed solution at 4 °C, place it at 45 °C and react for 10 h, and adjust and maintain the alkaline pH = 11 with NaOH solution during this period. Adjust the reaction solution to neutral with dilute hydrochloric acid to terminate the reaction, then pour the reaction solution into absolute ethanol, centrifuge to collect the precipitate, and wash it 3 times with absolute ethanol. Place the precipitate product in a dialysis bag (molecular weight cut-off 10 kDa) and dialyze with deionized water for 3 days, and then freeze-dry to obtain light yellow powdery hydroxyethylated chitosan (HECS).

[0115] (3) Preparation of pterostilbene-loaded muco-permeable nanoparticles (SSPI-HECS): Dissolve succinylated soy protein isolate powder in deionized water (0.02 g / mL), and stir magnetically until completely dissolved to prepare a suspension. Dissolve hydroxyethylated chitosan in deionized water (0.02 g / mL), and stir magnetically until completely dissolved to prepare a suspension. Mix the above two solutions in a ratio of succinylated soy protein isolate:hydroxyethylated chitosan = 2:1 (w / w), homogenize with a high-speed disperser at a speed of 10,000 rpm for 5 min, and then continue to stir magnetically at a speed of 600 rpm for 1 h to obtain a neutral hydrophilic system composed of succinylated soy protein isolate-hydroxyethylated chitosan complex. Dissolve pterostilbene in absolute ethanol (4 mg / mL), and add it dropwise to the above cross-linked system solution, stir magnetically at a speed of 600 rpm for 1 h, and then collect the nano-precipitation particles by centrifugation. Wash the precipitate with deionized water to obtain pterostilbene-loaded muco-permeable nanoparticles (SSPI-HECS).

[0116] (4) Preparation of an intestine-responsive mucoadhesive-permeation conversion oral delivery system (PE-SSPI-HECS): Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved (0.5 mg / ml). Add the SSPI-HECS (300 mg) prepared in step (3) to the above solution, and stir magnetically at a speed of 800 rpm for 5 h. Then, collect the precipitate particles by centrifugation at 10,000×g for 20 min to obtain SSPI-HECS modified with a pectin layer on the surface, that is, an intestine-responsive mucoadhesive-permeation conversion oral delivery system (PE-SSPI-HECS).

[0117] Example 3

[0118] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL), and stir magnetically at room temperature until fully dissolved. Add succinic anhydride dropwise at a ratio of 0.1 g / g protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (molecular weight cut-off 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 h, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0119] (2) Preparation of hydroxyethylated chitosan (HECS): Chitosan was dissolved in 1 wt% acetic acid solution (0.05 g / mL), and magnetically stirred until completely dissolved. The pH of the solution was adjusted to neutral with NaOH solution, and the gel-like precipitate was collected by filtration, washed 3 times with deionized water, and then freeze-dried to obtain pretreated chitosan for standby. The dried pretreated chitosan was added to 2M NaOH solution, and stirred at room temperature to prepare alkalized chitosan to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. The alkalized chitosan was transferred to a mixed solution of isopropanol-water (isopropanol∶water = 1∶5, v / v), and vortexed thoroughly to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶4 (mol / mol), ethylene oxide solution was added dropwise to the mixed solution at 4 °C, and the reaction was carried out at 45 °C for 10 h. During this period, the alkaline pH = 11 was maintained by adjusting with NaOH solution. The reaction solution was adjusted to neutral with dilute hydrochloric acid to terminate the reaction, and then the reaction solution was poured into absolute ethanol, and the precipitate was collected by centrifugation and washed 3 times with absolute ethanol. The precipitate product was placed in a dialysis bag (cut-off molecular weight 10 kDa) and dialyzed with deionized water for 3 days, and then freeze-dried to obtain pale yellow powdery hydroxyethylated chitosan (HECS).

[0120] (3) Preparation of pterostilbene-loaded mucoadhesive nanoparticles (SSPI-HECS): Succinylated soy protein isolate powder was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. Hydroxyethylated chitosan was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. The above two solutions were mixed according to the ratio of succinylated soy protein isolate∶hydroxyethylated chitosan = 1∶1 (w / w), homogenized at a speed of 10000 rpm for 5 min by a high-speed disperser, and then continuously magnetically stirred at a speed of 600 rpm for 1 h to obtain a neutral hydrophilic system composed of succinylated soy protein isolate-hydroxyethylated chitosan complex. Pterostilbene was dissolved in absolute ethanol (4 mg / mL), and added dropwise to the above cross-linked system solution, and magnetically stirred at a speed of 600 rpm for 1 h, and then the nano-precipitated particles were collected by centrifugation. The precipitate was washed with deionized water to obtain pterostilbene-loaded mucoadhesive nanoparticles (SSPI-HECS).

[0121] (4) Preparation of an intestinal-responsive mucus adhesion-permeation conversion oral delivery system (PE-SSPI-HECS): Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved (0.5 mg / ml). Add the SSPI-HECS (300 mg) prepared in step (3) to the above solution and stir magnetically at a speed of 800 rpm for 5 h. Then, collect the precipitated particles by centrifugation at 10,000×g for 20 min to obtain SSPI-HECS modified with a pectin layer surface, that is, an intestinal-responsive mucus adhesion-permeation conversion oral delivery system (PE-SSPI-HECS).

[0122] Example 4

[0123] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL) and stir magnetically at room temperature until fully dissolved. Dropwise add succinic anhydride in a ratio of 0.1 g / g protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (cut-off molecular weight 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 h, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0124] (2) Preparation of hydroxyethylated chitosan (HECS): Dissolve chitosan in a 1 wt% acetic acid solution (0.05 g / mL) and stir magnetically until completely dissolved. Adjust the pH of the solution to neutral with NaOH solution, filter and collect the gel-like precipitate, wash it 3 times with deionized water, and then freeze-dry to obtain pretreated chitosan for standby. Add the dried pretreated chitosan to 2M NaOH solution and stir at room temperature to prepare alkalized chitosan to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. Transfer the alkalized chitosan to an isopropanol-water mixed solution (isopropanol∶water = 1∶5, v / v), vortex thoroughly to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶(mol / mol), add ethylene oxide solution dropwise to the mixed solution at 4 °C, place it at 45 °C and react for 10 h, and adjust and maintain the alkaline pH = 11 with NaOH solution during this period. Adjust the reaction solution to neutral with dilute hydrochloric acid to terminate the reaction, then pour the reaction solution into absolute ethanol, centrifuge to collect the precipitate, and wash it 3 times with absolute ethanol. Place the precipitate product in a dialysis bag (cut-off molecular weight 10 kDa) and dialyze with deionized water for 3 days, and then freeze-dry to obtain light yellow powdery hydroxyethylated chitosan (HECS).

[0125] (3) Preparation of pterostilbene-loaded muco-permeable nanoparticles (SSPI-HECS): Dissolve succinylated soy protein isolate powder in deionized water (0.02 g / mL), and stir magnetically until completely dissolved to prepare a suspension. Dissolve hydroxyethylated chitosan in deionized water (0.02 g / mL), and stir magnetically until completely dissolved to prepare a suspension. Mix the above two solutions according to the ratio of succinylated soy protein isolate:hydroxyethylated chitosan = 1:2 (w / w), homogenize with a high-speed disperser at a speed of 10,000 rpm for 5 min, and then continue to stir magnetically at a speed of 600 rpm for 1 h to obtain a neutral hydrophilic system composed of succinylated soy protein isolate-hydroxyethylated chitosan complex. Dissolve pterostilbene in absolute ethanol (4 mg / mL), and add it dropwise to the above cross-linked system solution, stir magnetically at a speed of 600 rpm for 1 h, and then collect the nano-precipitation particles by centrifugation. Wash the precipitate with deionized water to obtain pterostilbene-loaded muco-permeable nanoparticles (SSPI-HECS).

[0126] (4) Preparation of an intestine-responsive mucoadhesive-permeation conversion oral delivery system (PE-SSPI-HECS): Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved (0.5 mg / ml). Add the SSPI-HECS (300 mg) prepared in step (3) to the above solution, and stir magnetically at a speed of 800 rpm for 5 h. Then, collect the precipitate particles by centrifugation at 10,000×g for 20 min to obtain SSPI-HECS modified with a pectin layer on the surface, that is, an intestine-responsive mucoadhesive-permeation conversion oral delivery system (PE-SSPI-HECS).

[0127] Example 5

[0128] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL), and stir magnetically at room temperature until fully dissolved. Add succinic anhydride dropwise according to the ratio of 0.1 g / g protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (cut-off molecular weight 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 h, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0129] (2) Preparation of hydroxyethylated chitosan (HECS): Chitosan was dissolved in 1 wt% acetic acid solution (0.05 g / mL), and magnetically stirred until completely dissolved. The pH of the solution was adjusted to neutral with NaOH solution, and the gel-like precipitate was collected by filtration, washed 3 times with deionized water, and then freeze-dried to obtain pretreated chitosan for later use. The dried pretreated chitosan was added to 2M NaOH solution, and stirred at room temperature to prepare alkalized chitosan, so as to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. The alkalized chitosan was transferred to an isopropanol-water mixed solution (isopropanol∶water = 1∶5, v / v), and vortexed sufficiently to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶4 (mol / mol), ethylene oxide solution was added dropwise to the mixed solution at 4 °C, and the reaction was carried out at 45 °C for 10 h. During this period, the alkaline pH = 11 was maintained by adjusting with NaOH solution. The reaction solution was adjusted to neutral with dilute hydrochloric acid to terminate the reaction, and then the reaction solution was poured into absolute ethanol, and the precipitate was collected by centrifugation and washed 3 times with absolute ethanol. The precipitate product was placed in a dialysis bag (cut-off molecular weight 10 kDa) and dialyzed with deionized water for 3 days, and then freeze-dried to obtain pale yellow powdery hydroxyethylated chitosan (HECS).

[0130] (3) Preparation of pterostilbene-loaded mucoadhesive nanoparticles (SSPI-HECS): Succinylated soy protein isolate powder was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. Hydroxyethylated chitosan was dissolved in deionized water (0.02 g / mL), and magnetically stirred until completely dissolved to prepare a suspension. The above two solutions were mixed according to the ratio of succinylated soy protein isolate∶hydroxyethylated chitosan = 1∶4 (w / w), homogenized at a speed of 10000 rpm for 5 min by a high-speed disperser, and then continuously magnetically stirred at a speed of 600 rpm for 1 h to obtain an electro-neutral hydrophilic system composed of succinylated soy protein isolate-hydroxyethylated chitosan. Pterostilbene was dissolved in absolute ethanol (4 mg / mL), and was added dropwise to the above cross-linked system solution, and magnetically stirred at a speed of 600 rpm for 1 h, and then the nano-precipitate particles were collected by centrifugation. The precipitate was washed with deionized water to obtain pterostilbene-loaded mucoadhesive nanoparticles (SSPI-HECS).

[0131] (4) Preparation of an orally administered delivery system (PE-SSPI-HECS) with intestinal-responsive mucus adhesion-permeation conversion property: Add high-methoxyl pectin to 100 mL of deionized water at 70 °C and stir magnetically until completely dissolved (0.5 mg / ml). Add the SSPI-HECS (300 mg) prepared in step (3) to the above solution and stir magnetically at a speed of 800 rpm for 5 h. Then, collect the precipitated particles by centrifugation at 10,000×g for 20 min to obtain SSPI-HECS modified with a pectin layer on the surface, that is, an orally administered delivery system with intestinal-responsive mucus adhesion-permeation conversion property (PE-SSPI-HECS).

[0132] Comparative Example 1

[0133] Preparation of nanoparticles (SPI-CS) loaded with pterostilbene using a soy protein isolate-chitosan composite system: Dissolve soy protein isolate powder in deionized water (0.02 g / mL) and stir magnetically until completely dissolved to prepare a suspension. Dissolve chitosan in a 1 wt% acetic acid solution (0.02 g / mL) and stir magnetically until completely dissolved to prepare a suspension. Mix the above two solutions in a ratio of soy protein isolate:chitosan = 1:2 (w / w), homogenize at a speed of 10,000 rpm for 5 min using a high-speed disperser, and then continue to stir magnetically at a speed of 600 rpm for 1 h to obtain a soy protein isolate-chitosan composite system. Dissolve pterostilbene in absolute ethanol (4 mg / mL) and add it dropwise to the above crosslinked system solution, stir magnetically at a speed of 600 rpm for 1 h, and then collect the nano-precipitated particles by centrifugation. Wash the precipitate with deionized water to obtain nanoparticles loaded with hydrophobic pterostilbene using a soy protein isolate-chitosan composite system, SPI-CS.

[0134] Comparative Example 2

[0135] (1) Preparation of hydroxyethylated chitosan (HECS): Chitosan was dissolved in 1 wt% acetic acid solution (0.05 g / mL), and magnetically stirred until completely dissolved. The pH of the solution was adjusted to neutral with NaOH solution, and the gel-like precipitate was collected by filtration, washed 3 times with deionized water, and then freeze-dried to obtain pretreated chitosan for standby. The dried pretreated chitosan was added to 2 M NaOH solution and stirred at room temperature to prepare alkalized chitosan, so as to enhance the nucleophilicity of hydroxyl (-OH) and amino (-NH2) groups and promote subsequent reactions. The alkalized chitosan was transferred to an isopropanol-water mixed solution (isopropanol∶water = 1∶5, v / v) and vortexed thoroughly to disperse. According to the ratio of chitosan unit∶ethylene oxide = 1∶4 (mol / mol), ethylene oxide solution was added dropwise to the mixed solution at 4 °C, and the reaction was carried out at 45 °C for 10 h. During this period, the alkaline pH = 11 was maintained by adjusting with NaOH solution. The reaction solution was adjusted to neutral with dilute hydrochloric acid to terminate the reaction, and then the reaction solution was poured into absolute ethanol, and the precipitate was collected by centrifugation and washed 3 times with absolute ethanol. The precipitate product was placed in a dialysis bag (cut-off molecular weight 10 kDa) and dialyzed with deionized water for 3 days, and then freeze-dried to obtain pale yellow powdery hydroxyethylated chitosan (HECS).

[0136] (2) Preparation of nanoparticles (SPI-HECS) loaded with pterostilbene using soy protein isolate-hydroxyethylated chitosan composite system: Soy protein isolate powder was dissolved in deionized water (0.02 g / mL) and magnetically stirred until completely dissolved to prepare a suspension. Hydroxyethylated chitosan was dissolved in deionized water (0.02 g / mL) and magnetically stirred until completely dissolved to prepare a suspension. The above two solutions were mixed according to the ratio of soy protein isolate∶hydroxyethylated chitosan = 1∶2 (w / w), homogenized at a speed of 10000 rpm for 5 min by a high-speed disperser, and then continuously magnetically stirred at a speed of 600 rpm for 1 h to obtain a soy protein isolate-hydroxyethylated chitosan composite system. Pterostilbene was dissolved in absolute ethanol (4 mg / mL) and added dropwise to the above cross-linked system solution, and magnetically stirred at a speed of 600 rpm for 1 h. Then, the nano-precipitate particles were collected by centrifugation. The precipitate was washed with deionized water to obtain nanoparticles (SPI-HECS) loaded with hydrophobic pterostilbene using the soy protein isolate-hydroxyethylated chitosan composite system.

[0137] Comparative Example 3

[0138] (1) Preparation of succinylated soy protein isolate (SSPI): Dissolve soy protein isolate in deionized water (0.06 g / mL) and stir magnetically at room temperature until fully dissolved. Add succinic anhydride dropwise at a ratio of 0.1 g / g protein, maintain the system pH = 8.0, and continuously stir and react at 25 °C for 4 h. Adjust the reaction solution to neutral to terminate the reaction. Place the reaction solution in a dialysis bag (molecular weight cut-off 20 kDa) and dialyze with deionized water for 24 h, changing the solution every 6 h, and then freeze-dry to obtain succinylated soy protein isolate (SSPI).

[0139] (2) Preparation of nanoparticles (SSPI-CS) loaded with pterostilbene using a succinylated soy protein isolate-chitosan composite system: Dissolve succinylated soy protein isolate powder in deionized water (0.02 g / mL) and stir magnetically until completely dissolved to prepare a suspension. Dissolve chitosan in 1 wt% acetic acid solution (0.02 g / mL) and stir magnetically until completely dissolved to prepare a suspension. Mix the above two solutions in a ratio of succinylated soy protein isolate:chitosan = 1:2 (w / w), homogenize at a speed of 10,000 rpm for 5 min using a high-speed disperser, and then continue to stir magnetically at a speed of 600 rpm for 1 h to obtain a succinylated soy protein isolate-chitosan composite system. Dissolve pterostilbene in absolute ethanol (4 mg / mL) and add it dropwise to the above cross-linked system solution, stir magnetically at a speed of 600 rpm for 1 h, and then collect the nano-precipitation particles by centrifugation. Wash the precipitate with deionized water to obtain nanoparticles loaded with hydrophobic pterostilbene using a succinylated soy protein isolate-chitosan composite system, SSPI-CS.

[0140] Performance characterization:

[0141] The surface hydrophilicity comparison diagrams of SPI-CS in Comparative Example 1, SPI-HECS in Comparative Example 2, SSPI-CS in Comparative Example 3, and SSPI-HECS prepared in step (3) of Example 4 of the present invention are as Figure 1 shown. It can be seen that the double modification of succinylation of soy protein isolate and hydroxyethylation of chitosan significantly improves the surface hydrophilicity of the composite nanoparticles SSPI-HECS, thus showing a lower water contact angle than the unmodified SPI-CS composite particles.

[0142] The particle size distribution diagram of SSPI-HECS prepared in step (3) of Example 4 of the present invention is as Figure 2 shown. By loading pterostilbene into the succinylated soy protein isolate and hydroxyethylated chitosan composite cross-linked system through a co-precipitation method, SSPI-HECS with uniform nano-particle size is successfully obtained, mainly distributed at 166 nm.

[0143] The comparison chart of pterostilbene loading rates in SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, and SSPI-HECS prepared in step (3) of Example 4 of the present invention is as follows Figure 3 As shown, in the present invention, the isoelectric point of soy protein isolate is reduced by succinylation, the amino groups are blocked and additional carboxyl groups are introduced to provide more negative charge reaction sites for electrostatic interaction with the amino groups of chitosan, thereby increasing the structural compactness of the cross-linking system and significantly improving the pterostilbene loading rate.

[0144] The change chart of the surface potential values of the nanoparticles prepared by cross-linking SSPI and HECS in different ratios in step (3) of Example 4 of the present invention is as follows Figure 4 As shown, during the preparation of SSPI-HECS, when the proportion of SSPI is high, the nanoparticles are dominated by carboxyl groups and exhibit negative charges. As the amount of HECS increases, due to the introduction of more electrostatic cross-linking between amino groups and carboxyl groups, the surface charge value of the nanoparticles gradually increases. When SSPI∶HECS = 1∶2, the nanoparticles reach the electrically neutral state.

[0145] The potential value charts of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, and SSPI-CS of Comparative Example 3 of the present invention are as follows Figure 5 As shown, with the same ratio of protein:chitosan = 1:2, the Zeta potential values of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, and SSPI-CS of Comparative Example 1 are 17.49, 12.35, and 10.35 respectively, indicating that none of them reach the surface electrically neutral state.

[0146] The comparison chart of the mucus permeability of SSPI-HECS prepared in step (3) of Example 4 of the present invention and SPI-CS of Comparative Example 1 is as follows Figure 6 As shown, the red fluorescence represents the FITC-labeled nanoparticles, and the green fluorescence represents the WGA-labeled mucus. The layer scanning images of CLSM show that the unmodified SPI-CS only penetrates to about 30 μm in the mucus layer, while the double-modified composite cross-linking system SSPI-HECS is still clearly visible at a depth of >50 μm in the mucus layer, indicating that SSPI-HECS has excellent mucus permeability.

[0147] The comparison chart of the mucus adhesion force and adhesion time of SSPI-HECS prepared in step (3) of Example 4 of the invention and PE-SSPI-HECS prepared in step (4) is as follows Figures 7a - 7b , The composite system PE-SSPI-HECS modified with high-methoxyl pectin significantly improves the mucus adhesion force and adhesion time of the nanoparticles SSPI-HECS.

[0148] The protective effect diagrams of SSPI-HECS prepared in step (3) of Example 4 of the present invention and PE-SSPI-HECS prepared in step (4) on pterostilbene loading during in vitro simulated digestion are as follows Figure 8 shown. The composite system PE-SSPI-HECS modified by high-methoxyl pectin significantly improves the gastrointestinal resistance of the nanoparticles SSPI-HECS, effectively protects the pterostilbene loading from premature release and destruction in the gastric environment, and increases the retention rate of pterostilbene in the nanoparticles.

[0149] The diagrams of the intestinal epithelial cell uptake of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of Example 4 of the present invention after in vitro digestion and mucus penetration are as follows Figure 9 shown. After sequential gastrointestinal digestion and mucus penetration, the uptake rates of SPI-CS, SPI-HECS, SSPI-CS, SSSPI-HECS, and PE-SSPI-HEC recovered by ultrafiltration in epithelial cells show significant differences. Due to the gastric protection of the high-methoxyl pectin layer and the high-efficiency mucus permeability of SSPI-HECS, PE-SSPI-HECS can effectively overcome the harsh gastrointestinal conditions and the capture of viscoelastic mucus, and thus be efficiently taken up by epithelial cells.

[0150] The diagrams of the cell inflammation regulation ability of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of Example 4 of the present invention after in vitro digestion and mucus penetration are as follows Figures 10a - 10d shown. Due to its excellent oral delivery performance and cell uptake efficiency, PE-SSPI-HECS has a significantly higher cell inflammation regulation ability than SPI-CS, SPI-HECS, SSPI-CS, and SSPI-HECS, which is specifically manifested as a significant decrease in pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, and a significant increase in the anti-inflammatory cytokine IL-10.

[0151] The diagrams of the cell oxidative stress regulation ability of SPI-CS of Comparative Example 1, SPI-HECS of Comparative Example 2, SSPI-CS of Comparative Example 3, SSPI-HECS prepared in step (3) of Example 4, and PE-SSPI-HECS prepared in step (4) of Example 4 of the present invention after in vitro digestion and mucus penetration are as follows Figures 11a - 11dAs shown, PE-SSPI-HECS has significantly more effective oral barrier overcoming performance and higher cell oxidative stress regulation ability than SPI-CS, SPI-HECS, SSPI-CS, and SSPI-HECS, manifested as a significant decrease in the lipid peroxidation product MDA and a significant increase in the antioxidant coenzymes SOD, GSH, and CAT.

[0152] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0153] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for preparing an intestinal responsive mucus adhesion-permeation conversion oral delivery system, characterized in that: include: Provide succinylated soy protein isolate and hydroxyethylated chitosan; The succinylated soy protein isolate and hydroxyethylated chitosan are self-crosslinked, and then pterostilbene is added for coprecipitation reaction to obtain mucus permeable nanoparticles loaded with pterostilbene; Furthermore, the mucus permeable nanoparticles loaded with pterostilbene are surface modified by using high methoxyl pectin to prepare an intestinal responsive mucus adhesion-permeation conversion type oral delivery system.

2. The preparation method according to claim 1, characterized in that: Specifically include: Dissolving soy protein isolate in water to form a soy protein solution, then adding succinic anhydride and stirring the solution in an alkaline environment at 25-40° C. for 1-4 hours, and then dialyzing and freeze-drying the solution to obtain succinylated soy protein isolate; Preferably, the concentration of the soy protein solution is 0.05-0.1 g / mL; Preferably, the mass ratio of succinic anhydride to soy protein isolate is 0.05-0.2:1; Preferably, the pH value of the alkaline environment is 8.0-8.5; Preferably, the molecular weight cutoff of the dialysis bag used for the dialysis is 15-30 kDa; preferably, the dialysis treatment time is 24-48 hours.

3. The preparation method according to claim 1, characterized in that: Specifically include: Dissolving chitosan in an acetic acid solution to form a chitosan acetic acid solution, adjusting the pH value of the chitosan acetic acid solution to neutral, and then filtering, washing, and drying the solution to obtain pretreated chitosan; The pretreated chitosan is mixed with an alkaline solution and stirred at room temperature to obtain an alkaline chitosan; and mixing the alkalized chitosan with an isopropanol-water mixed solvent, adding ethylene oxide at 4° C., reacting at 40-60° C. for 8-24 hours, while maintaining the pH value of the reaction system at 11, and then adjusting the pH value of the reaction system to neutral with an acidic solution to terminate the reaction, and then subjecting the mixture to centrifugation, washing, dialysis, and freeze-drying treatment to obtain hydroxyethylated chitosan; Preferably, the concentration of chitosan in the chitosan acetic acid solution is 0.01-0.1 g / mL; Preferably, the alkaline solution includes any one or more combinations of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution; Preferably, the concentration of the alkaline substance in the alkalized chitosan is 1-4 mol / L; Preferably, the concentration of pretreated chitosan in the alkalized chitosan is 0.02-0.05 g / mL; Preferably, the volume ratio of isopropanol to water in the isopropanol-water mixed solvent is 1:1-1:5; Preferably, the molar ratio of chitosan units to ethylene oxide in the alkalized chitosan is 1:2-1:5; Preferably, the acidic solution includes any one or more combinations of dilute hydrochloric acid, dilute sulfuric acid, and dilute acetic acid; Preferably, the dialysis bag used for the dialysis has a molecular weight cutoff of 5-15 kDa.

4. The preparation method according to claim 1, characterized in that: Specifically include: Dispersing succinylated soybean protein isolate and hydroxyethylated chitosan in water respectively to form succinylated soybean protein isolate suspension and hydroxyethylated chitosan suspension; The succinylated soy protein isolate suspension and the hydroxyethylated chitosan suspension are mixed and dispersed at a high speed of 5000-15000 rpm for 1-10 minutes to form a succinylated soy protein isolate-hydroxyethylated chitosan composite electrically neutral hydrophilic cross-linking system; Pterostilbene is dissolved in ethanol to form a pterostilbene ethanol solution, and then the pterostilbene ethanol solution is added to the electrically neutral hydrophilic cross-linking system and stirred at 200-1000 rpm for 0.5-2 hours, and then centrifuged and washed to obtain mucus permeable nanoparticles loaded with pterostilbene.

5. The preparation method according to claim 4, characterized in that: The concentration of the succinylated soy protein isolate suspension is 0.1-0.3 g / mL; and / or, the concentration of the hydroxyethylated chitosan suspension is 0.1-0.5 g / mL; And / or, the mass ratio of the succinylated soy protein isolate suspension to the hydroxyethylated chitosan suspension is 4:1-1:4; And / or, the concentration of the pterostilbene ethanol solution is 1-5 mg / mL.

6. The preparation method according to claim 1, characterized in that: Specifically include: High methoxy pectin is dissolved in deionized water at 60-80°C to form a high methoxy pectin solution, and then the mucus permeable nanoparticles loaded with pterostilbene are added and stirred at 200-1000rpm for 2-10h, and then centrifuged to obtain mucus permeable nanoparticles loaded with pectin layer surface modified, that is, the intestinal responsive mucus adhesion-permeation conversion type oral delivery system.

7. The preparation method according to claim 6, characterized in that: The esterification degree of the high methoxy pectin is ≥50%; and / or the content of methoxy groups in the high methoxy pectin is 7%-12%; and / or, the concentration of the high methoxy pectin solution is 0.5-2 mg / ml; the mass volume ratio of the mucus permeable nanoparticles loaded with pterostilbene to water is 100-500 mg:100 mL; And / or, the centrifugal treatment adopts a centrifugal speed of 5000-20000×g and a centrifugal time of 20-60 min; And / or, the particle size of the pterostilbene-loaded mucus-permeable nanoparticles modified on the surface of the pectin layer is 106-255 nm.

8. An intestinal responsive mucus adhesion-permeation conversion oral delivery system prepared by the preparation method according to any one of claims 1 to 7.

9. An oral product of pterostilbene with anti-inflammatory and antioxidant effects, characterized in that: An oral delivery system comprising at least the intestinal responsive mucoadhesion-permeation switching type according to claim 8.

10. Use of the intestinal responsive mucus adhesion-permeation conversion oral delivery system of claim 8 in the preparation of intracellular antioxidant and anti-inflammatory active products.

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